key: cord- -ceatyj o authors: huang, yong; xing, na; wang, zengguo; zhang, xiujuan; zhao, xiaomin; du, qian; chang, lingling; tong, dewen title: ultrasensitive detection of rna and dna viruses simultaneously using duplex undp-pcr assay date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: ceatyj o mixed infection of multiple viruses is common in modern intensive pig rearing. however, there are no methods available to detect dna and rna viruses in the same reaction system in preclinical level. in this study, we aimed to develop a duplex ultrasensitive nanoparticle dna probe-based pcr assay (duplex undp-pcr) that was able to simultaneously detect dna and rna viruses in the same reaction system. pcv and tgev are selected as representatives of the two different types of viruses. pcv dna and tgev rna were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for tgev and pcv were added to form a sandwich-like complex with nucleic acids released from viruses. after magnetic separation, dna barcodes specific for pcv and tgev were eluted using dtt and characterized by specific pcr assay for specific dna barcodes subsequently. the duplex undp-pcr showed similar sensitivity as that of single undp-pcr and was able to detect copies each of pcv and tgev in the serum, showing approximately -fold more sensitivity than conventional duplex pcr/rt-pcr assays. no cross-reaction was observed with other viruses. the positive detection rate of single mmps- and duplex mmps-based duplex undp-pcr was identical, with . % for pcv , . % for tgev and . % for pcv and tgev mixed infection. this duplex undp-pcr assay could detect tgev (rna virus) and pcv (dna virus) from large-scale serum samples simultaneously without the need for dna/rna extraction, purification and reverse transcription of rna, and showed a significantly increased positive detection rate for pcv ( %) and tgev ( . %) preclinical infection than conventional duplex pcr/rt-pcr. therefore, the established duplex undp-pcr is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility. along with the development of large-scale and intensive swine production, mixed infections of multiple pathogens are increasingly becoming common in swine farms. porcine reproductive and respiratory syndrome virus (prrsv), porcine circovirus type (pcv ), classical swine fever virus (csfv), porcine pseudorabies virus (prv), transmissible gastroenteritis virus (tgev), porcine parvovirus (ppv) and porcine epidemic diarrhea virus (pedv) are major pathogens causing heavy economic losses in swine industry [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . on the basis of clinical signs, it is difficult to determine whether sick pigs are infected by single or multiple viruses [ ] . therefore, it is imperative to establish an effective and rapid method to detect multiple dna and rna viruses simultaneously in single sample. traditional diagnostic methods of dna and rna viruses are mainly dependent on detection of viral proteins and nucleic acids. currently, common methods for detecting viral antigen in solution is enzyme-linked immunosorbent assay (elisa), but elisa shows some shortcomings that are difficult to overcome. for example, elisa requires at least one highly specific antibody for a particular viral antigen, but a specific antibody is not able to detect all viral strains of a kind of virus due to genotype difference; a elisa kit is usually for one kind of virus, leading to that elisa detection are complicated, time-consuming and expensive, and it is difficult to achieve scale detection when we need to detect a variety of viruses at the same time. in addition, it is difficult to find viral infection by elisa when viral load is below a certain level because the sensitivity base line of elisa is relatively higher [ , ] . the nucleic acids-based detection methods include conventional pcr, rt-pcr, loop-mediated isothermal amplification (lamp) and real-time pcr, which have been used in the diagnosis of virus infection [ ] [ ] [ ] [ ] [ ] . although lamp and real-time pcr are more sensitive than conventional pcr or rt-pcr, lamp can only detect one pathogen at a time and lamp products are difficult to identify, while real-time pcr requires special or expensive instruments and easily shows false positive results [ , ] . however, more critical is that all the existing pcr-based assays need rna/dna extraction. it is known that the extraction and detection procedures of dna and rna are different from each other. rna is easily degradable as compared to dna, so in pcrbased methods of detecting rna viruses, viral genomic rna extracted from field samples should be utilized to synthesize complementary dna (cdna) first, which are time-consuming and labor-intensive. undp-pcr is an ultrasensitive nanoparticle dna probe-based pcr method, in which magnetic microparticles (mmps) coated with virus specific dna probes and gold nanoparticles (aunps) coated with virus specific oligonucleotides are used to enrich virus genomes from samples and form an aunp-rna/dna-mmp complex. then the specific oligonucleotides are released and characterized by pcr after the complex is washed. in the previous study, we established this method to detect dna virus pcv , which exhibited a detection limit of copies of pcv genomic dna and copies of pcv in serum that is -fold more sensitive than conventional pcr [ ] . however, it is still needed to test whether this method can be used in the detection of rna virus. in this study, we aimed to develop a method for simultaneous detection of preclinical dna and rna virus mixed infection in the same reaction system based on undp-pcr method. tgev and pcv , as the representatives of rna and dna viruses respectively chosen from a variety of viruses related to porcine diseases, were used to establish duplex undp-pcr assays. pcv , a dna virus with a circular genome of . kb, has been reported to cause wide infection throughout the world and serious damage to pig producers, while tgev, an enveloped virus with a positive-stranded rna genome, has been recognized as a principal causative agent of enteric disease [ , ] . firstly, the undp-pcr assay for tgev was developed and identified. then, single mmps-based or duplex mmps-based duplex undp-pcr assays for both pcv and tgev was developed. mmps coated with specific dna probes for either tgev or pcv (single mmps), or for both tgev and pcv (duplex mmps) were used to enrich tgev and pcv viral genomes from serum samples, and aunps coated with optimal oligonucleotides (oligo) specific for either tgev or pcv were used to magnify weak signals from very low level of tgev/pcv virus enriched by mmps from serum samples. the duplex undp-pcr assay is suitable for simultaneous detection of rna and dna viruses in early viral infection, providing an effective approach for diagnosis of swine diseases. viruses and cells tgev strain (genbank no. hq ), pcv strain (genbank no. eu ), ppv yl strain (genbank no. jn ), pedv strain (genbank no. af ) and prrsv shaanxi strain (genbank no. hq ) used in this study were isolated and purified previously by our team and stocked in our laboratory [ ] [ ] [ ] [ ] [ ] . the csfv shimen strain (genbank no. ay ) was provided kindly by professor yanming zhang [ ] . these virus strains were maintained at - °c and used as standard viruses for this study. tgev, pcv and ppv were propagated in pk- cells. csfv was propagated in st cells. pedv was propagated in vero cells. prrsv was propagated in marc- cells. four types of cells (pk- , st, vero, and marc- ) were maintained in dulbecco's modified eagle medium (dmem) (gibco, gaithersburg, md, usa) supplemented with % heat inactivated fetal calf serum (gibco). during the period from nov, to jun, , serum samples were collected from healthy pigs in pig-producing farms near xianyang and baoji of shannxi province, china. the pigs were humanely euthanized by injecting with mg/kg of ketamine in the jugular vein, then - ml of blood samples were collected from each pig by jugular venipuncture. the serum samples were tested using the conventional pcr/rt-pcr assay and single or duplex undp-pcr. the experiment was approved by the ethical committee for animal experiments of the northwest a&f university and performed according to the animal ethics procedures and guidelines of the people's republic of china. no other specific permissions were required for this study. purification of viral dna or rna viral rna/dna kit (omega, usa) was used to extract and purify viral genomic dna or rna from virus-infected cell cultures or serum samples according to the manufacture's protocol. then the extracted viral rna was reverse transcribed into the complementary dna (cdna) using the reverse transcriptase kit (takara corp., japan) according to manufacturer's instructions. in this study, specific tgev dna probes and oligonucleotides used in undp-pcr were designed via multiple sequence alignment of complete genomes of orf a from various tgev strains published on national center for biotechnology information using vector nti and dnastar software package. primers for conventional rt-pcr method of detecting tgev and pedv were designed using primer-blast software on the basis of tgev and pedv highly conserved region of orf a. primers for conventional pcr/rt-pcr detection of pcv , ppv, prrsv and csfv were designed as described in previous studies [ , ] . all the primers, probes and oligonucleotides designed for this experiment owned higher specificity to make sure diagnosis more precise. the primers, probes and oligonucleotides presented in table were synthesized by sangon (shanghai, china). preparation of mmps coated with tgev and / or pcv specific probes for undp-pcr assay in the presence of water-soluble n-ethyln - [ -dimethylaminopropyl] carbodiimide hydrochloride (edc), tgev and/ or pcv specific oligonucleotide probes modified with ' amino (nh ) were bond to the carboxylated-modified myone dynabeads to form a peptide bond. the detailed steps of the assay were according to manufacturer's instruction [ ] . then the functionalized mmps were resuspended in tris-edta (te) buffer to a concentration of mg/ml and stored at °c until required. preparation of aunps coated with tgev or pcv specific oligos aunps coated with tgev or pcv specific oligo were prepared as described in previous study [ ] , briefly, ml of nm-diameter aunps ( nmol/l) were washed and resuspended in μl sterile deionized water. then, the ' sulfydryl (sh)-modified tgev/pcv specific oligonucleotides were added and mixed with aunps to establish covalent au-s bond. in the binding process of thiolated aunps and sh-modified oligonucleotides, a final concentration of . m pbs ( . m nacl in . m of phosphate buffer, ph . ) was developed by adding . m pbs to the reaction tube three times to incubate for more than hours at room temperature. then, unbound dna probes were removed by washing twice with . m pbs. finally, the prepared functionalized aunps were stored in . m pbs at °c until used. viral serum samples were mixed with same volume of lysis buffer ( . m tris-hcl, mm edta, . m nacl, . % sds, ph . ), and then boiled for minutes to release viral rna or dna. the products were mixed with μl of probe-coated mmps and × hybridization buffer ( ×ssc, . % tween- and % sds in h o). the mixture of these components was incubated for minutes by stirring. subsequently, μl of functionalized aunps were added, followed by incubation at °c for minutes. the sandwich-like aunp-rna/dna-mmp complexes in the tube were separated using magnetic wells and washed twice with ml of hybridization buffer and twice with ml te buffer to remove remaining hybridization buffer and unbound probes-functionalized mmps and oligos-functionalized aunps. the oligonucleotides on the surface of gold nanoparticles were eluted using μl of elution buffer ( . m dtt, mm tris-hcl, mm edta, ph . ) for minutes at room temperature. then, the eluted oligonucleotides were precipitated with naac and absolute alcohol. the precipitated oligonucleotides were mixed with specific capture ssdna and detected by pcr using specific detect-pcr primers for pcv and/or tgev in table . the pcr assay was performed as described in the previous study [ ] . the pcr products were separated by electrophoresis through . % agarose gel stained with ethidium bromide and were photographed under uv light. the sensitivity, specificity, and reproducibility of the undp-pcr assay for tgev tgev genomic rna was extracted using rna kit and reverse transcribed to synthesize cdna. then, the part of tgev orf a gene was amplified from cdna using the primers ( '-cgtaatggtgacagccgat- '/ '-agcagcatcacggaaaccat- '). the bp amplified products were cloned into pmd- t simple vector (takara, japan) and sequenced. the concentration of the plasmid was measured by nanodrop spectrophotometer (thermo scientific, usa). the formula: amount (copies/μl) = . × (copies/mol) × concentration (g/μl)/mw (g/mol) was used to calculate the plasmid copy number. the viral copy number in samples per ml was tested by real-time pcr with serially diluted plasmid from to copies/ml. to test the sensitivity, the quantitative serum samples were diluted serially from to copies/ml, and then detected by undp-pcr and conventional rt-pcr. the specificity of undp-pcr was tested through comparing with pcv , ppv, prrsv, pedv and csfv. inter-assay and intra-assay were performed in three replicates by testing different concentrations of diluted serum samples ( × copies/ml, × copies/ml, copies/ml) for three consecutive days to test reproducibility of the undp-pcr assay for tgev. to test the sensitivity of duplex undp-pcr assay for tgev and pcv , the serum samples containing tgev or pcv were diluted serially from to copies/ml respectively. the diluted samples containing same viral copy numbers of tgev and pcv per ml were mixed, and then tested by single mmps-based and duplex mmps-based duplex undp-pcr, or conventional pcr/rt-pcr. the specificity and reproducibility of duplex undp-pcr assay for dna and rna viruses in the study of evaluating the specificity of duplex undp-pcr, ppv, pedv, prrsv and csfv were tested by the established method. the inter-assay and intra-assay tests were carried out in triplicate by detecting three different concentrations of mixed serum containing serial diluted tgev and pcv ( , , copies/ml) to evaluate the reproducibility of this assay. to find a rapid and ultrasensitive diagnosis method for preclinical mixed infection of dna and rna viruses, a series of related experiments were performed to establish two kinds of protocol for duplex undp-pcr assay as schematically depicted in fig . in the previous study, we have optimized and established a undp-pcr method for dna virus pcv , which can detect copies/ml of pcv serum sample and exhibited high specificity and reproducibility [ ] . in the present study, pcv and tgev are selected as representative dna and rna viruses, respectively. therefore, a undp-pcr method for rna virus tgev needs to be established first. tgev, an enveloped virus of the coronaviridae family, contains a single-stranded positivesense rna genome of . kb. the first two-thirds of the viral genome encodes two replicases and only exists in genomic rna, while the last third exists in both genomic rnas (grna) and subgenomic mrnas (sgmrnas) to encode structural and nonstructural proteins of virus [ , , ] . in this assay, we first quantified viral number using primers targeted to tgev grna and different sgmrnas and found that viral numbers were significantly different when the primers were targeted to tgev grna and different sgmrnas (data not shown). therefore, we designed probes targeted to replicase protein-encoding region orf a to quantify the amount of tgev grna. six targeted regions were selected from the ' end, middle and ' end of the orf a for designing probes to ( table ). all the targeted sequences of these probes are highly conserved in variety of tgev strains. probes to were coated to magnetic microparticles to prepare functional magnetic beads mmp-p , -p , -p , -p , -p and -p , respectively. to select the optimal probes for capturing of tgev genomic rna, capture efficiency of these designed probes were determined by conventional rt-pcr assay. the results showed that all six probes could capture tgev rna, however mmp-p and mmp-p exhibited higher capture ability for tgev rna (fig a) . next, we assessed capture efficiency of functionalized gold nanoparticles coated with oligonucleotides (oligo) to that shared same targeted sequence with probe to . the prepared functionalized au-nps were precipitated by centrifugation and were detected by rt-pcr assay. the results showed that oligo and -coated au-np possessed higher binding ability with tgev nucleic acid (fig b) . therefore, in the undp-pcr assay for tgev, probe -functionalized mmps and oligo -functionalized au-nps are optimal for capture of tgev rna and formation of sandwich complex. serial -fold dilutions of tgev in serum samples were tested to assess the sensitivity of undp-pcr for rna virus. tgev genomic rna was released by boiling with lysis buffer with rnase inhibitors and was used to form aunp-rna-mmp complexes, followed by magnetic separation and oligonucleotide elution. the oligonucleotides were then purified and detected by undp-pcr. as shown in fig a, visible targeted bands around bp could be seen in simultaneous detection of dna and rna virus by duplex undp-pcr lanes representing serum samples with viral concentrations ranging from copies/ml to copies/ml respectively, but it could not be detected in the negative control serum without tgev and the tgev serum sample below copies/ml, indicating that the detection limit of undp-pcr assay for tgev was copies/ml in serum sample. however, at least copies/ ml of tgev serum sample was able to be detected by conventional rt-pcr assay (fig b) , suggesting that the sensitivity of undp-pcr specific for tgev was -fold that of the conventional rt-pcr for tgev. the reproducibility of undp-pcr for tgev was estimated by three independent runs for three consecutive days, with triplicates of each concentration ( × copies/ml, × copies/ ml, copies/ml). consistent results of undp-pcr were obtained in the inter-assay and intra-assay test (fig a) . ppv, pcv , prrsv, csfv, pedv, tgev and the serum collected from healthy pigs were used to evaluate the specificity of the established undp-pcr for tgev. in the fig b, the undp-pcr detection only appeared to be positive with tgev, whereas no specific pcr products of bp were obtained from the assay using pcv , csfv, prrsv, pedv, ppv or healthy pigs serum as pending samples, which indicated that undp-pcr assay had high specificity for tgev and did not show cross-reactivity with pcv , csfv, prrsv, pedv and ppv. to compare the sensitivity of single mmps-based and duplex mmps-based duplex undp-pcr assay for simultaneous detection of tgev and pcv in same reaction assay system, qualified serum samples of tgev and pcv were diluted serially with the range from to copies/ml. then the diluted samples containing tgev and pcv were mixed as the template for testing the sensitivity of single mmps-based and duplex mmps-based duplex undp-pcr assay. as shown in fig a and b , the detection limits of single mmps-based and duplex mmps-based duplex undp-pcr were identical with copies/ml for tgev and pcv in serum. however, at least copies/ml of pcv and tgev serum sample was able to be detected by conventional duplex pcr/rt-pcr assay (fig c) . these results suggested that the sensitivity of duplex undp-pcr specific for pcv and tgev was -fold that of simultaneous detection of dna and rna virus by duplex undp-pcr the conventional duplex pcr/rt-pcr for these two viruses, and that mmps coated with one virus probe or two virus probes did not affect the capture efficiency of functionalized mmps and the sensitivity of undp-pcr assay. the specificity and reproducibility of duplex undp-pcr for both dna and rna viruses to evaluate the specificity of single mmps-based and duplex mmps-based duplex undp-pcr for both dna and rna viruses, magnetic beads coated with specific probes for tgev and pcv alone or together and gold nanoparticles coated with specific oligos for tgev and pcv alone were added to one single reaction tube. as shown in fig a and b , both assays were specific to the target viruses, producing specific amplified products ( bp for tgev and bp for pcv ). no amplicons were yielded with ppv, prrsv, csfv, pedv and samples from health pigs. three independent replicates assay of both assays gave highly consistent results respectively (fig c and d ). pre-clinical serum samples from epidemic farms without diseased pigs were detected for tgev and pcv using duplex undp-pcr assay and conventional duplex pcr/rt-pcr. conventional duplex pcr/rt-pcr detection showed that among samples, samples were pcv positive, samples were tgev positive, but none of samples were found to be positive for both pcv and tgev. whereas duplex undp-pcr assay showed that samples were pcv positive, samples were tgev positive, samples were positive for both pcv and tgev ( table ). the positive samples detected by the duplex undp-pcr method included all of the samples found to be positive by the conventional duplex pcr/rt-pcr. the positive detection rate of duplex undp-pcr was . % for pcv , . % for tgev and . % for pcv and tgev mixed infection, which increased % and . % for pcv and tgev preclinical infection than that of conventional duplex pcr/rt-pcr (p < . ). as shown in table , the results were consistent when single mmps-based and duplex mmps-based duplex undp-pcr were used to detect these samples, suggesting two kinds of duplex undp-pcr possessed same detection efficiency. next, the relative band intensities of the unknown samples were compared with that of standard virus samples ( , and copies/ml pcv and tgev) to evaluate the viral loads of pcv and tgev in all of positive samples. among pcv positive samples, samples were over copies/ml, samples were between and copies/ml, samples were between and copies/ml, and samples were below copies/ml in viral load (data not shown); among tgev positive samples, samples were over copies/ml, samples were between and copies/ml, samples were between and copies/ml, and samples were below copies/ml (fig a) . among pcv and tgev double positive samples, the pcv viral loads of samples were between and copies/ml, sample was between and copies/ml, and sample was below copies/ml, the tgev viral loads of samples were between and copies/ml, sample were between and copies/ml, and sample was below copies/ml (fig b) . taken together, this undp-pcr assay was found to be the best method for detecting dna and rna viruses in preclinical samples simultaneously. all the results indicated that undp-pcr-based assay was a rapid and economical approach with high specificity and high sensitivity. to date, traditional approaches to detect mixed infection of rna and dna viruses present some limitations, such as complex virus genome isolation procedures, limited sensitivity, narrow detection range and lack of specific antibodies for different viruses, which lacks the [ , ] . in addition, low virus titer in early stage of infection is not able to be detected by conventional pcr or rt-pcr, failing in timely diagnosis of infection. viruses are more likely to spread across piggeries and cause more sickness and death in piglets, which will bring huge threat to pig industry [ ] . with the development of nanotechnology, the undp-pcr based on nanoparticle and dna probe makes it possible to detect dna and rna viruses simultaneously especially in subclinical infection without the need for nucleic acid extraction separately [ ] . duplex undp-pcr assay gains more merits over established traditional approaches for rna/dna virus diagnosis. the first advantage that should be mentioned is that duplex undp-pcr is more time-saving and cost-effective than other routine pcr-based methods. in this study, field samples collected from pig-producing farms were boiled with lysis buffer for min to release viral genome, so there is no need to extract nucleic acid using dna/rna extraction kits. particularly, unlike other pcr-based assays, extracted and purified rna need to be reverse transcribed into cdna. hence, the whole detection process could be completed in short period of time. secondly, this assay could detect dna and/or rna virus in serum samples with an extreme low concentration in preclinical infection. accordingly, functionalized magnetic beads and gold nanoparticles which were coated with tgev and/or pcv specific probes and oligonucleotides targeting two distinct virus genomic sequences were added to form a sandwich complex. in each binding events, the gold nanoparticles carried with large number of oligonucleotides, which could be used as templates for subsequent pcr assay. thus, weak signals from extreme low concentration of samples were highly amplified. in addition, the duplex undp-pcr could detect dna and rna virus from large-scale serum samples simultaneously. in the reaction system, magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for tgev and pcv were added to capture and enrich viral nucleic acid, so this approach could detect infection of tgev and pcv alone or together in a single reaction tube. the results showed that duplex undp-pcr (single mmps-based and duplex mmps-based) developed in the study was able to detect copies for pcv and tgev. the sensitivity of duplex undp-pcr was approximately -fold that of conventional duplex pcr/rt-pcr. in terms of specificity, specific probes and oligonucleotides for tgev and pcv were assessed through testing capture efficiency and specificity of different probes-or oligonucleotidescoated magnetic beads or gold nanoparticles. as a consequence, duplex undp-pcr showed high specificity with tgev and pcv , yielding different size of pcr products ( bp and bp respectively). the results of detection for preclinical samples indicated that duplex undp-pcr assay ( . % for pcv , . % for tgev and . % for pcv and tgev mixed infection) described here was more sensitive than conventional detection methods ( . % for pcv , . % for tgev and % for pcv and tgev mixed infection). the duplex undp-pcr assay developed in this study provided a useful tool for simultaneous detection of rna (tgev) and dna viruses (pcv ) without the need for viral nucleic acid extraction, purification and reverse transcription. this assay could increase positive detection rates of virus infection and is useful to evaluate the viral loads in pre-clinically infected samples. in summary, the duplex undp-pcr assay is an economical and rapid 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coronavirus discontinuous rna synthesis establishment and application of a multiplex pcr for rapid and simultaneous detection of six viruses in swine a transmissible gastroenteritis in pigs nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins we thank professor yanming zhang (northwest a&f university) for providing csfv. we also would like to thank benxiang and hengda for providing preclinical samples. conceived and designed the experiments: yh nx dt. performed the experiments: nx zw xz qd xz lc. analyzed the data: yh nx. contributed reagents/materials/analysis tools: yh dt. wrote the paper: yh nx. key: cord- - tf oqa authors: wang, kai; ran, ling; yan, tao; niu, zheng; kan, zifei; zhang, yiling; yang, yang; xie, luyi; huang, shilei; yu, qiuhan; wu, di; song, zhenhui title: anti-tgev miller strain infection effect of lactobacillus plantarum supernatant based on the jak-stat signaling pathway date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: tf oqa transmissible gastroenteritis (tge), caused by transmissible gastroenteritis virus (tgev), is one many gastrointestinal inflections in piglets, characterized by diarrhea, and high mortality. probiotics are ubiquitous bacteria in animal intestines, which have many functions, such as promoting intestinal peristalsis and maintaining the intestinal balance. we found that the supernatant of the lp- strain of lactobacillus plantarum, isolated in our laboratory, and named lp- s had marked anti-tgev effect on ipec-j cells. lp- s could induce large amounts of interferon-β in ipec-j cells in the early stage ( h) of infection with tgev, and increased the level of phosphorylated signal transducer and activator of transcription and its nuclear translocation in the late stage ( – h) of infection. this resulted in upregulated expression of interferon-stimulated genes, and increased the transcription and protein expression of antiviral proteins, resulting in an anti-tgev effect. transmissible gastroenteritis virus (tgev) is the pathogenic agent of porcine transmissible gastroenteritis (tge), which causes vomiting, diarrhea, and high mortality in suckling piglets (masters, ) , resulting in heavy losses to the pig breeding industry (zhao et al., ) . in particular, viral diarrhea diseases are more serious because of limited treatment options. probiotics comprise microorganisms that have beneficial activities to the host, and mainly comprise clostridium butyricum, lactobacillus, bifidobacteria, actinomycetes, and yeasts. they usually occupy the human gut and reproductive system, and can improve the balance of the host microecology (fuller, ; maragkoudakis et al., ; da silva sabo et al., ; stofilova et al., ) . there is growing interest in the oral administration of appropriate probiotics to reduce the pressure in the intestines and produce an effective innate immune response (pollmann et al., ; maragkoudakis et al., ) . in recent years, probiotic animal feed supplements have been developed as viable alternatives to antibiotics because of the ban on antibiotics in feed (scharek et al., ) . the addition of probiotic feed can prevent the infection of pathogens causing intestinal diseases, directly benefiting the animal host (villena et al., ) , or can indirectly enhance the host's immune response by balancing the disordered microbiota (lee et al., ) . in addition, many basic and clinical studies have confirmed that probiotic strains have antiviral effects (lee et al., ; yuan et al., ) . studies have shown that lactobacillus plantarum can stimulate the body's innate and acquired immunity, and contributes to the production of inflammatory factors that inhibit the replication of the virus in the body. for example, l. plantarum strain yu (lpyu) not only has high interleukin (il)- -inducing activity mediated by tolllike receptor (tlr) in mouse peritoneal macrophages, but also communicates with natural killer cells (nk) in the spleen to stimulate the production of iga to enhance the body's anti-h n virus activity (kawashima et al., ) . in addition, l. plantarum l- can stimulate the production of type i interferon (ifn- ) to effectively inhibit the proliferation of h n (maeda et al., ) . tgev is an important gastrointestinal diarrhea virus; therefore, research and exploration into the antiviral mechanism of probiotics could lead to the development of oral probiotics to prevent and treat tgev infection. the body reacts rapidly to viral invasion by synthesizing and secreting type i interferon ifn- (ifn-α/β), which plays a key role in the host antiviral response. the binding of ifn- to its receptor (interferon α/β receptor, ifnar) leads to activation of the janus family kinase (jak) and subsequent signal transduction and transcriptional activator (stat) signaling cascade, resulting in the activation and upregulation of interferon-stimulating genes (isgs), ultimately activating ifn to exert its antiviral effects (zhao et al., ; chen et al., ) . a strain of l. plantarum was successfully isolated and named lp- (song han et al., ) . we wondered whether the ipec-j cells treated by lp- could induce an antiviral mechanism through the ifn-β/jaks/stat/isgs pathway after tgev infection. in the present study, we found that the supernatant of l. plantarum lp- (lp- s) could significantly inhibit tgev infection. by detecting the replication of tgev n gene in porcine intestinal epithelial cells treated with lp- s at different time points, we confirmed that lp- s had a preventive effect against tgev. then, by detecting the levels of ifn, p-stat and isgs, we further confirmed that lp- s exerts its anti-tgev role by upregulating the expression of ifn-β. porcine kidney cells (st) to amplify the virus and the experimental model pig jejunal cells (ipec-j cells) were both cultured in roswell park memorial institute (rpmi) medium (gibco, grand island, ny, united states) containing % fetal bovine serum (fbs, gibco), under • c and % co . ipec-j cells and st cells were purchased from the shanghai sur biotech co., ltd (shanghai, china). l. plantarum lp- was isolated and stored in our laboratory. its s ribosomal gene sequence has been submitted to genbank (mh ). lp- s was isolated from a culture of l. plantarum lp- after shaking for h at • c. the tgev miller strain was preserved in our laboratory. viral fluid was collected from st cells after replication for approximately h when the cells showed obvious cytopathic effects (cpes). the genbank sequences of the tgev n gene (gq- . ) coding sequence (cds) conserved region, the porcine mx cds (mx dynamin like gtpase ; ah . ), the mx cds (mx dynamin like gtpase ; ay . ), the isg cds (interferon-stimulated protein, kda; nm_ . ), the oasl cds ( - -oligoadenylate synthetase like; nm_ . ), the pkr cds (double stranded rna-dependent protein kinase; ab . ), the zap cds (zeta-chain associated protein; gu_ . ), and internal reference pig β-actin gene (actb; xm_ . ) were obtained and pairs of specific primers were designed using primer . software for quantitative realtime pcr [performed using sybr premix ex taq ii (takara, shiga, japan)] as follows: tgev-n (forward: -ttcaacccc ataaccctccaacaa- and reverse: -ggcccttcac cat gcgatagc- ), mx (forward: -atctgtaagcagg agaccatcaactt g- and reverse: -ctcgccacgtcca ctatcttgtc- ), mx (forward: -ttcactcgcatccgc acttcag- and reverse: -agctcctctgtcgcactc tgg- ), isg (forward: -ggcagcacagtcctgtt gatgg- and reverse: -tgcgtcagccagacctcat agg- ), oasl (forward: -cgttggtggtgg agacaca tacag- and reverse: -tcaggcgacaccttccagg atc- ), pkr (forward: -acaggacctgcacataact tgagg- and reverse: -tgctgtcggcagtgatgaaga ac- ), zap (forward: -gctcagtgcgaac acctgga tg- and reverse: -tgacagatgaaggcgtggag agg- ), and actb (forward: -ctcttccagc cctcct tcc- and reverse: -ggtccttg cggatgtcg- ). the designed primers were synthesized by shanghai shenggong biotechnology service co., ltd. (shanghai, china). after centrifugation of lp- with an od of . at rpm/min for min, the obtained supernatant was filtered through a . -µm filter, and then diluted with rpmi high sugar medium to six gradients at two times ratio, i.e., lp- s was diluted to obtain od values of . , . , . , . , . , and . , respectively. ipec-j cells were seeded at × /ml in -well plates and incubated overnight in % co at • c. the medium was discarded when the cells reached % confluence in the -well plate, and then µl of each gradient dilution of lp- s was added to each well. the medium in the control group was replaced with rpmi . after incubating for min, the supernatant was discarded, and the cells were washed twice with phosphate-buffered saline (pbs). culture was continued and the cytopathic effect (cpe) was observed daily. the maximum non-toxic dose of lp- s to the cells was detected using the -( , dimethylthiazol- -yl)- , -diphenyltetrazolium bromide (mtt) reagent (bbi, shanghai, china), when % of the cells in the negative control group were damaged. mtt assays for each dilution were repeated three times independently. lp- s was prepared as above and then diluted to an od of . , . , . , and . with rpmi high-sugar medium. ipec-j cells were seeded at . × /ml in -well plates and cultured in % co at • c. at % confluence, the medium was discarded, the cells were washed three times with pbs, ml of each gradient dilution of lp- s was added to each well, and the cells incubated at • c in % co for min. the medium in the control group was replaced with rpmi . for the other wells, the supernatant was discarded, tgev (multiplicity of infection (moi) = . ) in rpmi medium was added to lp- s-treated ipec-j , and incubated at • c in % co for . h. the supernatants were discarded and incubation continued in rpmi high glucose medium. after h of culture, proteins were extracted for western blotting to detect the levels of the tgev n protein after treatment with different concentrations of lp- s. ipec-j cells treated with lp- s for . h were exposed to tgev (moi = . ). the ipec-j cells treated with lp- s were sampled at h post infection (hpi), hpi, and hpi and then frozen and thawed three times to collect virus particles in the cells and supernatants. gradient dilution of ipec-j cells was performed from − and − , respectively. tgev titers of ipec-j cells treated with lp- s for different times were detected using st cells in -well plates. each dilution gradient was assayed in replicate wells. the tcid of the virus in the different groups was calculated by reed and muench methods. ipec-j cells were inoculated into -well plates at . × /ml. the ipec-j cells treated with lp- s for . h were exposed to tgev (moi = . ) for rna extraction and protein sampling. when the cells reached % confluence, rna was extracted and reverse transcribed into cdna and quantified at ng/ml. absolute fluorescence quantitative pcr was performed using fluorescence quantitative pcr. the reaction parameters were as follows: pre-denaturation at • c for min, followed by cycles of denaturation at • c for s, annealing at • c for s, and prolongation at • c for s. the reaction for each sample was repeated three times. the bio-rad cfx manager random matrix method was used to analyze the linear relationship between cycle threshold (ct) value and the copy number to calculate the copy number of the tgev-n gene. protein samples were also extracted at the same time point and detected using western blotting. the primary antibody was a mouse monoclonal antibody against tgev-n, and the secondary antibodies were horseradish peroxidase (hrp)-conjugated goat anti-mouse antibodies (proteintech,wuhan,china). the immunoreactive protein bands were visualized using a vilber fusion fx chemiluminescent imager. ipec-j cells were inoculated into -well plates at . × /ml. when they reached % confluence, three experimental groups were established: an lp- s optimal concentration treatment group infected tgev, a tgev single infection group, and the uninfected control group. cell samples at , , , and hpi were centrifuged at rpm for min, and the supernatant subjected to an enzyme linked immunosorbent assay (elisa) to detect ifn-β. transmissible gastroenteritis virus (moi = . ) was infected to ipec-j cells that had been treated with lp- s for . h, and then cell samples at , , and hpi were collected to produce protein lysates. in addition, tgev (moi = . ) was infected into ipec-j cells cultured in rpmi for . h as the tgev control group and the proteins were extracted at the same time points as the lp- s group. the protein concentration was determined using the bicinchoninic acid method and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. the separated proteins were transferred to a nitrocellulose membrane (bio-rad). the membranes were blocked using % skimmed milk and incubated with the following primary antibodies: anti-stat rabbit polyclonal antibodies (british biorbyt company), anti-phospho-(p)stat (tyr ) rabbit polyclonal antibodies (biorbyt), anti-zap rabbit polyclonal antibodies (abcam), anti-pkr rabbit polyclonal antibodies (abcam), anti-(p)pkr (t ) rabbit polyclonal antibodies (abcam), and anti-β-tubulin rabbit polyclonal antibodies (proteintech). secondary antibodies comprised goat anti-rabbit immunoglobulin (h + l). the immunoreactive protein bands were visualized using the vilber fusion fx imaging system (vilber), and the grayscale values of each band were analyzed by graphpad prism. ipec-j cells were seeded at a density of . × in cell slides in -well culture dishes. these cells were set as three experimental groups comprising an lp- s optimal concentration treatment group, a tgev alone infection group, and a blank (uninfected) control group, when they reached % confluence. the cells were sampled at , , and hpi; washed three times with pbs; fixed with % paraformaldehyde at • c for . h; washed three times with pbs; permeated by . % triton-x- for min; washed three times with pbs; and blocked by % bovine serum albumin for min at room temperature. primary antibodies comprising anti-p-stat protein rabbit polyclonal antibodies and anti-tgev-n mouse monoclonal antibodies were added and incubated overnight in • c in a wet box. the cells were then washed three times with pbs, proportionally added cy -goat anti-rabbit fluorescence and fluorescein isothiocyanate (fitc)-labeled goat anti-mouse fluorescent secondary antibodies were then added, the cells were incubated for h in a dark room at • c, incubated with -( -amidinophenyl)- h-indole- -carboxamidine (dapi) for min, rinsed with pbs. laser confocal microscopy was then used to observe p-stat , its nuclear translocation, and the tgev n protein. the protein levels were analyzed using the zen software. ipec-j cells were seeded at . × /ml in -well plates, grown to % confluence, and then divided into three groups: cells treated with lp- s for . h and then infected with tgev (moi = . ), tgev infection alone, and the blank control (uninfected cells). the ipec-j cells were sampled at , , and hpi for qrt-pcr. total rna was extracted using the rnaiso plus reagent (takara), and then single-stranded rna was isolated using an rna pcr (amv) ver . kit (takara). cdna was then synthesized via reverse transcription. quantitative real-time pcr was then performed using the sybr premix ex taq ii (takara) to detect the mrna levels of zap, pkr, oasl, and isg (fluorescent primers were synthesized by shanghai shenggong bioengineering technology service co., ltd.). small interfering rna (sirna) targeting stat , -ggaacagaaatacacctat- (produced by ribo, china) . transfected with the stat -specific sirna using lipofectaminetm (invitrogen, united states), according to the manufacturer's instructions, to the tgev-infected groups treated with lp- s and tgev infection alone groups, respectively, in ipec-j cells. consirna transfected as control groups. the collected samples were analyzed by western blotting using a rabbit pab recognizing stat as the primary antibody and hrp-conjugated goat anti-rabbit igg as the secondary antibody. all results were plotted and analyzed using graphpad prism software (graphpad inc., la jolla, ca, united states). the data were presented as the mean ± standard deviation (sd) of three independent experiments. data were statistically compared using the t test. a p value < . ( * p < . and * * p < . ) was considered statistically significant. the mtt assay (figure ) showed that the higher dilution ratio of lp- s, the higher the cell viability. the maximum non-toxic dose toward the cells was greater than % od ; therefore, the maximum non-toxic dose of lp- s to ipec-j cells is od . ( figure a) . the tgev inhibition rate of lp- s decreased with increasing dilution factor and was thus concentration dependent. the results showed that amount of tgev miller strain (moi = . ) was reduced by / -fold by lp- s, i.e., the od was . , and the treated ipec-j cells received the highest non-toxic dose of lp- s ( figure b) . the experimental data (figure ) showed that the tgev titers at , , and hpi after tgev infection of ipec-j cells treated with lp- s were . tcid /ml, . tcid /ml, and . tcid /ml, respectively. compared with the tgev control group, the amount of viral of lesions decreased by . , . , and . -fold at , , and hpi, respectively, in the lp- s group and no lesions appeared in the blank control group. therefore, lp- s has a significant anti-tgev effect ( * * p < . ) and is optimal for antiviral activity at hpi. we found that the tgev n gene copy number in ipec-j cells treated with lp- s was lower than that in cells infected with tgev only ( * p < . , * * p < . ). in addition, the viral n gene copy number in the tgev infection group was positively correlated with the infection duration ( figure a) . western blotting showed that the level of the tgev n protein in the lp- s group was lower than that in the tgev group at all three time points. no expression of tgev n protein was observed in the blank control group (figures b,c) . these results demonstrated that lp- s could inhibit the transcription and protein expression of tgev n. the results in figure indicate that ifn-β levels gradually increased in ipec-j cells during tgev infection. however, compared with the tgev-infected group, the ifn-β level of the tgev (moi = . )-infected group treated with lp- s increased significantly with infection time ( * * p < . ). therefore, lp- s can significantly increase the level of intracellular ifn-β during tgev infection. the results in figure show that there was almost no change in the total expression of stat in ipec-j cells under different treatments and at different time points; however, the amount of phosphorylated stat changed significantly. the amount of phosphorylated stat in the lp- s group was higher than that in the tgev group at the different time points ( * p < . and * * p < . ), while only a small amount of phosphorylated stat was detected in the blank control group at the different time points. therefore, although tgev infection could significantly increase the amount of phosphorylated stat , lp- s could further significantly increase the amount of phosphorylated stat in cells after tgev infection. the results in figure show that the amount of p-stat (red fluorescence) in the nuclei of tgev infected cells treated with lp- s at different time points was significantly higher than that in the nuclei of cells in tgev infected group. at the same time, the amount of p-stat correlated positively with the duration frontiers in microbiology | www.frontiersin.org figure | mtt cytotoxicity test and lp- s concentration screening. (a) results of lp- s cytotoxicity as detected using the mtt method on / , / , / , and / times dilutions of lp- s acting on ipec-j cells. the cell adherence state remained basically unchanged. according to the experimental data, the lp- s undiluted group, the / -fold dilution group and the / -fold dilution group showed significantly difference in cytotoxicity ( * * p < . ), and the / -fold dilution group showed no significant difference compared with that of the control group (p > . ). (b) the expression of the tgev n protein in ipec-j cells treated with lp- s at different dilutions was detected by western blotting. lane , infected tgev group after lp- s / dilution pretreatment; lane , infected tgev group after lp- s / dilution pretreatment; lane , infected tgev group after lp- s / dilution pretreatment; lane , infected tgev group after lp- s / dilution pretreatment; lane , tgev infection group; and lane , uninfected control cells (normal group). (c) grayscale analysis of the relative expression of tgev n in ipec-j cells infected with lp- s at different dilutions showing that tgev n protein levels were decreased in the / -fold dilution of lp- s treatment group after h, compared with the / -fold dilution and tgev infection group, there was a significant difference ( * * p < . ), which was significantly different from the control group ( * p < . ). of infection after lp- s-treatment of ipec-j cells infected with tgev. however, the amount of red fluorescence emitted by p-stat labeled with cy correlated negatively with the amount of green fluorescence emitted by fitc-labeled tgev n protein. we found that when tgev was directly infected into ipec-j cells, the red fluorescence of p-stat was mainly gathered around the cell wall and only a small amount appeared in the nucleus; the green fluorescence and red fluorescence of the blank control group were not obvious. meanwhile, the signal intensity of p-stat in the nuclei of the lp- s treatment group correlated positively with time ( figure ) . therefore, lp- s could significantly increase the level of p-stat and promoted its translocation into the nucleus, while simultaneously inhibiting the expression of tgev n. we found that the mrna expression levels of zap, mx , mx , pkr, oasl, and isg were significantly higher in the lp- s treated group than in the tgev infected group at various points after infection. the expression levels of zap, pkr, oasl, and isg in each experimental group increased with time. the expression levels of mx and mx peaked at h and then decreased at h (figures a-f) . the results showed that the best time to stat -sirna targeting gene silencing stat was h (figure g) , and the best interference fragment was stat -sirna ( figure h) . after gene silencing stat , we found that the expression of isgs decreased compared to the groups which no knock down stat , and the expression isgs of lp- s-treated tgev infected group was higher than that of tgev infected alone group (figures i-n) . the results showed that tgev infection of ipec-j cells treated with lp- s could stimulate the expression of isgs in cells to inhibit viral replication. as expected, the protein levels of zap, pkr, p-pkr (figures a-d) at , and hpi in the lp- s group were significantly higher than those in negative control group and tgev group (zap, * p < . and pkr, * * p < . ). there was figure e where the expression of this gene was knocked down using sirna is significantly lower than the level of unknocked down stat in figure a . after gene silencing stat , the protein levels of lp- s, lp- s pretreatment of ipec-j cells infected with tgev; tgev, cells directly infected with tgev; con, uninfected cells (normal group). there were significant differences in ifn-β levels between the tgev group and the lp- s group at different time points ( * * p < . ). e-h) at , , and hpi in the lp- s group were significantly higher than tgev group (p-stat /stat , * p < . ; zap, * p < . ; and p-pkr/pkr, * p < . ). the results showed that lp- s could there was no significant difference between the two groups (p > . ) at h; however, the difference was extremely significant at and h ( * * p < . ). lp- s, lp- s pretreatment of ipec-j cells infected with tgev; tgev, cells directly infected with tgev. showed that the level of phosphorylated stat was higher in the lp- s group than in the tgev group after h (p > . ); after h of lp- s treatment, the level of phosphorylated stat in the lp- s group was significantly higher than that in the tgev group ( * * p < . ); after h of lp- s treatment, the level of phosphorylated stat in the lp- s group was significantly higher than that in the tgev group ( * p < . ). the results showed that treatment of tgev-infected ipec-j cells with lp- s ( - h), induced increased of levels of phosphorylated intracellular stat . the nucleus was identified using zen blue software and the intensity of red fluorescence emitted by cy -labeled p-stat was analyzed. the statistical results showed that the fluorescence intensity of cy in ipec-j cells treated with lp- s was significantly higher than that in the tgev group after h ( * p < . ). in the late stage of tgev infection ( - h), the fluorescence signal intensity of p-stat in the nucleus of this group was significantly higher than that in tgev group ( * * p < . ). increase the expression of isgs and inhibit the replication of tgev in ipec-j cells, which was basically consistent with the expression trend of the pkr and zap genes. previous studies have found that many lactic acid bacteria can inhibit the infection of diarrhea-causing viruses (such as rv and pedv) in the host through a variety of methods (hou et al., ; kawakami et al., ) . some lactic acid bacteria can be recognized by toll like receptor (tlr)- or tlr- to enhance their response to stimulation by the interferon inducer poly (i: c) and induce a large amount of ifn-i (maeda et al., ; kanmani and kim, ; lee et al., ) . at the same time, they can also upregulate the transcription of il and tnfa (reyes-diaz et al., ) . in addition, some lactic acid bacteria can enhance the expression of surface molecules and cytokines in intestinal (a-f) relative mrna expression levels of mx , mx , pkr, zap, isg , and oasl, respectively. the expression levels of zap, mx , and mx in lp- s group after h were not significantly different from those in the tgev group (p > . ). the expression level of isg was significantly higher in the lp- s group than in the tgev group ( * p < . ). the expression levels of pkr and oasl were significantly higher in the lp- s group than in the tgev group ( * * p < . ). the expression levels of zap, mx , mx , pkr, oasl, and isg in the lp- s group after and h were significantly higher than (continued) figure | continued those in the tgev group ( * * p < . ). (g,h) screening of sirna targeting stat optimal treatment time and gene silencing efficiency fragment. as shown in the figure, the optimal time for sirna targeting stat is h, and the best gene silencing sirna fragment is sirna ( * p < . ). (i-n) relative mrna expression levels of mx , mx , pkr, zap, isg , and oasl, respectively, after targeting gene silencing stat . the expression levels of pkr and oasl in lp- s group after h were not significantly different from those in the tgev group (p > . ). the expression level of mx , pkr, zap, isg , and oasl was significantly higher in the lp- s group after h than in the tgev group ( * p < . and * * p < . ). the expression levels of zap, isg and oasl were significantly higher in the lp- s group after h than in the tgev group ( * p < . and * * p < . ). antigen presenting cells (apc), and enhance the molecular expression of mhc-ii and il- β (villena et al., ) . in addition, other lactic acid bacteria can also stimulate the response level of tlr- to poly (i: c) (hosoya et al., ) . they can also regulate the role of tlr- , tlr- , and tlr negative regulators in the immune response, further enhancing the production of ifn-i induced by cells, and upregulate the transcription level of related antiviral factors (such as mxa and oasl) (castillo et al., ) . other studies have shown that probiotics can inhibit tgev infection by adsorbing virus particles and stimulating cells to produce innate immunity (chai et al., ) . recent studies have shown that the main reasons why the body's interferon-beta (ifn-β) cannot fully exert its antiviral effect after tgev infection are as follows: first, the cells do not respond in time to the immune response because of the level of viral replication and the virus titer of tgev in the early stage of infection, resulting in lower levels of ifns, which is the main cause of the short burst of tgev latency (zhu et al., ) . second, ifn-β does not play a direct antiviral role. its antiviral function is produced by activating the ifn-mediated jak-stat signaling pathway to stimulate downstream interferonstimulating genes (isgs) (saha and pahan, ; li, ; proia et al., ) . and, activation of the jak-stat signaling pathway requires phosphorylated stat (tyr ) enters the nucleus to activate interferon-stimulating factors isgs (including mx , mx , pkr, oas, isg , and zap) (hovanessian, ; zhu et al., ; shi et al., ; goujon et al., ; amici et al., ; li et al., ; nigg and pavlovic, ) . zap can bind viral rna directly and prevent the accumulation of viral rna in the cytoplasm. it can also recruit rna exosomes to degrade target viral rna (li et al., ) . pkr-mediated inhibition of viral replication is activated by the formation of dsrna during the replication of single-stranded rna after viruses invade cells. the main reason is that the amino terminus of pkr can recognize the dsrna domain and the carboxyl terminus has the kinase domain. when the viral double-stranded rna is recognized, the inactive pkr protein located in the cytoplasm is phosphorylated. on the other hand, it can also regulate the cell immune response and autophagy caused by virus invasion to inhibit the virus. at the same time, pkr can activate the nuclear factor kappa b (nf-kb) signaling pathway via phosphorylation and further induce ifn production in cells (sudhakar et al., ; amici et al., ) . in the present study, we found that ipec-j cells still produced ifn-β after infection with tgev, which increased with time, (f) relative expression of p-stat /stat in lp- s treatment group was significantly higher than tgev infected alone at h ( * p < . ). (g) relative expression of zap in lp- s treatment group was significantly higher than tgev infected alone at h ( * p < . ). (h) relative expression of p-pkr/pkr in lp- s treatment group was significantly higher than tgev infected alone at h ( * p < . ). reaching a peak at h and no longer increased at h. at the corresponding time points, the level of infection, viral titer, and replication of tgev on ipec-j cells showed an increasing trend. after lp- s treatment, ipec-j cells produced a large amount of ifn-β at the early stage of tgev infection ( h), which was significantly higher than that of cells infected with tgev only. the induced level of ifn-β in lp- s-treated ipec-j cells was significantly different from that in the tgev infected group at the same time point. the induction level of ifn-β in the lp- s treated group was significantly higher than that in tgev infected group at the different time points. the level of ifn-β induction correlated positively with time, peaking at h before decreasing toward h. the early boost in ifn-β production in ipec-j cells treated with lp- s might be one of the reasons for its inhibition of tgev. in addition, although tgev delayed the expression of ifn-β in the early stage of infection, it promoted the expression of ifn-β at the peak of viral replication. the expression of ifn-β was parallel to the increase of viral rna replication level at - h, which demonstrated that tgev replication remained high in the late stage of infection when ifn-β was produced in large quantities. this might be caused by the inhibitory effect of tgev on ifn-β-mediated signaling pathways, resulting in the inability of ifn-β to regulate the transcription and expression of downstream target cytokines and exert an antiviral role. although there was no significant difference in the levels of p-stat between the lp- s group and the tgev group at hpi, the level of p-stat in the lp- s group was significantly higher than that in the tgev group at the later stages ( and h). the level of p-stat in the lp- s group correlated positively with time from - h. the results showed that ipec-j cells treated with lp- s could effectively increase stat phosphorylation in the late stage of virus infection. at the same time, the level of p-stat changed slightly from - h after infection with tgev. the results were quite different from those reported in previous studies on the infection of st cells by tgev. in addition, the level of p-stat in ipec-j cells infected with tgev from - h was significantly lower than that in st cells infected with tgev from - h, which might reflect the difference between the cell lines and viruses used. further experiments showed that the level p-stat nuclear translocation in the lp- s group was significantly higher than that in the tgev group at the same time points, and p-stat nuclear accumulation correlated positively with time. in tgevinfected cells, p-stat at the late stage of infection ( - h) accumulated in large amounts near the cell membrane and only a few nuclear translocations occurred. we speculated that the reason might be that the intracellular stat protein is activated by ifn-β after ipec-j cells are directly infected with tgev to form a homologous or heterodimer, and the virus interacts with its receptor irf . the interaction resulted in the inability of activated stat to undergo nuclear translocation through receptor-induced endocytosis, and could only dissociate around ifnar, which reduced the jak-stat signaling pathway cascade response and antagonized the antiviral effect of ifn-β. the fluorescence value of activated stat was significantly higher than that of blank control group. this indicated that tgev could not completely escape the immune mechanism of ifn-β figure | proposed mechanism of the anti-tgev effect of lp- s. lp- s increases ifn-β expression, and the binding of ifn-β to its receptor ifnar leads to activation of the janus family kinase (jak) and subsequent activation of signal transduction and transcriptional activator (stat ) signaling cascades. these signaling pathways upregulate downstream interferon-stimulated genes (isgs), including mx , mx , pkr, oas, isg , and zap), which produce the corresponding antiviral proteins, e.g., zap and pkr, ultimately activating ifn-β to exert an antiviral effect. production in ipec-j cells. at the same time, the fluorescence intensity of p-stat in the nucleus of the cells in the lp- s treatment group correlated positively with time, and the intensity of fitc-labeled tgev n protein was significantly lower than that in the tgev treatment group at the same time point. these results showed that ipec-j cells treated with lp- s could indeed activate the jak-stat signaling pathway when infected with tgev, and the intensity of jak-stat signaling pathway was significantly higher in the lp- s-treated cells than in the cells directly infected with tgev. as the signaling pathway cascade response increased, the replication level of tgev in ipec-j cells was further inhibited. finally, the transcriptional levels of isgs in the lp- s treatment group were different at different time points. mx and mx reached their peak at h, while the transcriptional levels of the two isgs decreased at h after tgev infection. the transcription levels of pkr, zap, oasl and isg were positively correlated in groups. we speculated that the decrease in mx and mx mrna transcription levels within h after lp- s treatment might be related to the cycle of infected cells. the expression of isgs of gene silenced stat decreased as a whole compared to the groups which no knock down stat , indicating that stat could affect downstream isgs. while the isgs of tgev infected group treated with lp- s showed an upward trend, which further confirmed that lp- s could activate downstream isgs. to further explore the difference in the intracellular response of lp- s treated ipec-j cells to tgev infection, we detected the changes in zap and pkr protein levels. the level of the zap protein in the lp- s and tgev groups was consistent with its transcription level at the time point. although the level of the zap protein in lp- s treated cells was significantly higher than that in the tgev infection group, the difference in the transcription level of zap was significantly lower than that in the tgev infection group. these results suggested that after lp- s treatment, the ifn-β and jak-stat signaling pathways induced in the cells are enhanced, and the expression of the zap protein is still limited, suggesting that other factors have an impact on the transcriptional regulation of zap. the protein level of pkr was consistent with its mrna transcription level. there was no significant difference in the p-pkr/pkr values between the two groups at and h; however, the p-pkr/β-tubulin protein values were in line with our expectations. the results showed that the p-pkr level in the lp- s group was significantly higher than that in the tgev group, and correlated positively with time. therefore, we believe that the increase in p-pkr is mainly determined by the expression of the pkr protein. based on the above results, lp- s plays an antiviral role by stimulating the ifn-β-mediated jak /stat pathway, resulting in upregulation interferon-stimulating genes, which induce the synthesis of antiviral proteins such as zap and pkr (figure ). the datasets generated for this study are available on request to the corresponding author. inhibition of viral protein translation by indomethacin in vesicular stomatitis virus infection: role of eif alpha kinase pkr oral administration of a probiotic lactobacillus modulates cytokine production and tlr expression improving the immune response against salmonella enterica serovar typhimurium infection in mice antiviral effects of a probiotic enterococcus faecium strain against transmissible gastroenteritis coronavirus matrix metalloproteinase facilitates hepatitis b virus replication through binding with type i interferon (ifn) receptor to repress ifn/jak/stat signaling inhibitory substances production by lactobacillus plantarum st pa cultured in hydrolyzed cheese whey supplemented with soybean flour and their antimicrobial efficiency as biopreservatives on fresh chicken meat probiotics in man and animals human mx is an interferon-induced post-entry inhibitor of hiv- infection immunobiotic lactic acid bacteria beneficially regulate immune response triggered by poly(i:c) in porcine intestinal epithelial cells surface-displayed porcine epidemic diarrhea viral (pedv) antigens on lactic acid bacteria interferon-induced and double-stranded rnaactivated enzymes: a specific protein kinase and , -oligoadenylate synthetases protective effects of lactic acid bacteria against tlr induced inflammatory response in hepatoma hepg cells through modulation of toll-like receptor negative regulators of mitogen-activated protein kinase and nf-kappab signaling feeding of lactic acid bacteria and yeast on growth and diarrhea of holstein calves lactobacillus plantarum strain yu from fermented foods activates th and protective immune responses enhanced protection against infection with transmissible gastroenteritis virus in piglets by oral co-administration of live attenuated salmonella enterica serovar typhimurium expressing swine interferon-alpha and interleukin- probiotics in human health and disease: from nutribiotics to pharmabiotics micronized and heat-treated lactobacillus plantarum lm stimulates host immune responses via the tlr- /mapk/nf-kappab signalling pathway in vitro and in vivo zinc finger antiviral protein inhibits coxsackievirus b virus replication and protects against viral myocarditis canonical and non-canonical jak-stat signaling oral administration of heat-killed lactobacillus plantarum l- enhances protection against influenza virus infection by stimulation of type i interferon production in mice lactic acid bacteria efficiently protect human and animal intestinal epithelial and immune cells from enteric virus infection the molecular biology of coronaviruses oligomerization and gtp-binding requirements of mxa for viral target recognition and antiviral activity against influenza a virus effects of a probiotic strain of enterococcus faecium on the rate of natural chlamydia infection in swine multifaceted intervention by the hsp inhibitor ganetespib (sta- ) in cancer cells with activated jak/stat signaling milk fermented by specific lactobacillus strains regulates the serum levels of il- , tnf-alpha and il- cytokines in a lps-stimulated murine model regulation of inducible nitric oxide synthase gene in glial cells impact of the probiotic bacteria enterococcus faecium ncimb (sf ) and bacillus cereus var. toyoi ncimb on the development of serum igg and faecal iga of sows and their piglets positive regulation of interferon regulatory factor activation by herc via isg modification identification of a intestinal probiotic strain from piglets and preliminary study on the strain against the proliferation of tgev in vitro cytokine production in vitro and in rat model of colitis in response to lactobacillus plantarum ls/ phosphorylation of serine in initiation factor alpha (eif alpha) promotes complex formation between eif alpha(p) and eif b and causes inhibition in the guanine nucleotide exchange activity of eif b immunobiotic lactobacillus rhamnosus strains differentially modulate antiviral immune response in porcine intestinal epithelial and antigen presenting cells egfr as a negative regulatory protein adjusts the activity and mobility of nhe in the cell membrane of ipec-j cells with tgev infection surfactin inhibits membrane fusion during invasion of epithelial cells by enveloped viruses effects of virulent and attenuated transmissible gastroenteritis virus on the ability of porcine dendritic cells to sample and present antigen endothelial cell proteomic response to rickettsia conorii infection reveals activation of the janus kinase (jak)-signal transducer and activator of transcription (stat)-interferon stimulated gene (isg) pathway and reprogramming plasma membrane integrin/cadherin signaling transmissible gastroenteritis virus does not suppress ifn-beta induction but is sensitive to ifn in ipec-j cells stat serine phosphorylation occurs independently of tyrosine phosphorylation and requires an activated jak kinase the authors gratefully acknowledge peng yuan, zhou yang, and other veterinary medicine students from the southwest university for their valuable suggestions and assistance. key: cord- -rmnjs t authors: welch, siao-kun wan; saif, linda j. title: monoclonal antibodies to a virulent strain of transmissible gastroenteritis virus: comparison of reactivity with virulent and attenuated virus date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: rmnjs t twelve hybridomas secreting monoclonal antibodies (mabs) against miller virulent strain of transmissible gastroenteritis virus (tgev) were generated and characterized. in a cell culture immunofluorescence (ccif) assay, three mabs directed against peplomer protein (e ) had perinuclear fluorescence and four unclassified mabs showed cell membrane fluorescence. six of these seven mabs neutralized both attenuated and virulent tgev, and the seventh (an unclassified mab) neutralized only the latter virus. two mabs able to bind the cell membrane of infected cells had low neutralizing antibody titers ( to ) but were able to distinguish between virulent and attenuated tgev ( - to -fold differences in neutralizing titers). two e -specific mabs had higher neutralizing antibody titers ( to , ) and showed - to -fold differences in titers against the attenuated and virulent tgev strains. five mabs which were specific for nucleocapsid (n) protein had cytoplasmic, particulate fluorescence in ccif, and did not neutralize tgev. comparison of ccif antibody titers of mabs to the virulent and attenuated strains of tgev indicated that differences existed in titers of most e and all n-specific mabs, with titers consistently higher against virulent tgev (homologous strain). hyperimmune antisera prepared in gnotobiotic pigs against the attenuated, virulent and a recent isolate of tgev immunoprecipitated the major structural proteins of both the attenuated and virulent tgev strains. relative mol. wt. differences in the e and e proteins between the two virus strains were revealed using either the hyperimmune pig sera or mabs. in addition to the k n protein, a k protein was coimmunoprecipitated by the hyperimmune sera and mabs, but mainly from lysates of attenuated tgev. miller virulent strain of transmissible gastroenteritis virus (tgev) were generated and characterized. in a cell culture immunofluorescence (ccif) assay, three mabs directed against peplomer protein (e ) had perinuclear fluorescence and four unclassified mabs showed cell membrane fluorescence. six of these seven mabs neutralized both attenuated and virulent tgev, and the seventh (an unclassified mab) neutralized only the latter virus. two mabs able to bind the cell membrane of infected cells had low neutralizing antibody titers ( to ) but were able to distinguish between virulent and attenuated tgev ( -to -fold differences in neutralizing titers). two e -specific mabs had higher neutralizing antibody titers ( to , ) and showed -to -fold differences in titers against the attenuated and virulent tgev strains. five mabs which were specific for nucleocapsid (n) protein had cytoplasmic, particulate fluorescence in ccif, and did not neutralize tgev. comparison of ccif antibody titers of mabs to the virulent and attenuated strains of tgev indicated that differences existed in titers of most e and all n-specific mabs, with titers consistently higher against virulent tgev (homologous strain). hyperimmune antisera prepared in gnotobiotic pigs against the attenuated, virulent and a recent isolate oftgev immunoprecipitated the major structural proteins of both the attenuated and virulent tgev strains. relative mot. wt. differences in the e and e proteins between the two virus strains were revealed using either the hyperimmune pig sera or mabs. in addition to the k n protein, a k protein was coimmunoprecipitated by the hyperimmune sera and mabs, but mainly from lysates of attenuated tgev. transmissible gastroenteritis virus (tgev) belongs to the genus coronavirus of the family coronaviridae [ ] . it causes enteric disease in swine, producing a usually fatal diarrhea in seronegative piglets less than weeks old [ ] . although there is only one known serotype, both attenuated and virulent strains of tgev have been described [ ] . despite development of inactivated or live attenuated vaccines, no safe, effective, practical prophylaxis is yet available [ ] . thus a need exists to further define the protective antigens of tgev and to evaluate possible antigenic differences between attenuated and virulent strains of tgev. transmissible gastroenteritis virus possesses club-shaped peplomers and is enveloped and pleomorphic, with a diameter of - nm [ ] . analysis of tgev revealed three major structural and two minor nonstructural proteins [ ] . the glycosylated peplomer protein (e ) has an apparent molecular weight (mol. wt.) of - k and elicits neutralizing antibodies [ ] . the nucleocapsid protein (n), associated with the rna genome, is phosphorylated and has a mol. wt. of - k [ ] . the matrix or transmembranous protein [e ] is also glycosylated and has a tool. wt. of - k [ ] . the two minor proteins whose functions are unknown have mol. wts. of k and . k. recently, a k intracellular protein was identified in cells infected with the purdue strain of tgev [ ] : its function is also unknown. in two previous studies, monoclonal antibodies (mabs) to the structural proteins of the attenuated (purdue) strain of tgev were described [ , ] . there are no published reports describing mabs to a virulent strain of tgev. in the present study, a panel of mabs specific for structural proteins of the virulent (miller) strain of tgev was produced. these mabs were further characterized in various comparative assays including cell culture immunofluorescence, virus neutralization, and radioimmunoprecipitation for reactivity against the miller virulent and purdue attenuated strains of tgev. primary porcine kidney (ppk) cells were used for propagation of tgev and a swine testicle (st) cell line was used in various assays (described below). both ppk and st cells were maintained in eagle's minimum essential medium (mem) (gibco, grand island, ny) supplemented with % fetal bovine serum (fbs) (gibco) and pg/ml of gentamicin (schering veterinary, kenilworth, ny). for virus neutralization (vn), cell culture immunofluorescence (ccif), and radioimmunoprecipitation assays, the purdue attenuated strain (p ) and low cell culture-passaged miller (m ) virulent strain of tgev were used. both virus stocks were prepared in ppk cells. viruses were harvested h post-infection by three cycles of freezing and thawing, and the viruses stored in aliquots at -- °c. the miller virulent strain (m c) of tgev has been maintained by five serial passages in gnotobiotic pigs and represents the reference challenge strain of tgev [ ] . the field zy isolate of tgev was obtained during an outbreak of tgev in from a day-old diarrheic pig. both the m c and m strains of tgev, and the recent field zy isolate of tgev produced clinical signs and lesions typical of virulent tgev in gnotobiotic pigs, including vomiting, diarrhea and villous atrophy. pools of intestinal contents from m c infected gnotobiotic piglets were purified as described in the following section. intestinal contents containing m c tgev or tgev negative intestinal contents (from noninfected gnotobiotic pigs) were diluted t : in tris-cac buffer . m tris-hc , . m nac , and mm caci ), ph . and sonicated for rain (biosonik iii, bronwill, rochester, ny) on ice. crude suspensions were clarified by low speed centrifugation at , x g for rain and supernatant fluids were layered onto discontinuous sucrose gradients of %, %, and % in tris-cac buffer. after centrifugation at , x g for h, the light-scattering bands at %/ % and %/ % interphases were collected separately and diluted : in tris-cac buffer. sucrose was removed by pelleting virus at , x g for h and virus pellets were resuspended in tris-cac buffer. for each fraction, virus integrity was assessed by immune electron microscopy (iem) [ ] and virus titers were determined by ccif (reciprocal of the endpoint dilution showing immunofluorescing cells). the fractions ( %/ % and %/ % interphases) with intact viral particles and virus titers > l s were used to immunize balb/c mice. confluent st cell monolayers in -well plates were infected with either m or p strains of tgev at a multiplicity of infection (m.o.i.) of . pfu/cell in eagle's mem. after incubation at °c for h, monolayers were rinsed with phosphate buffered saline (pbs), ph . and fixed with % acetone. the fixed cells were used for ccif immediately. one hundred microliter per well of undiluted cell culture fluids from fusion plates or from limiting dilution plates or serial two-fold dilutions of ascites were added. the plates were incubated at °c in a humid incubator for h, and then rinsed with pbs for minutes. goat anti-mouse igg + iga + igm conjugated to fluorescein isothiocyanate (fitc) (kirkegaard & perry, gaithersburg, ma) at a : dilution was added to each well. after h incubation at °c, plates were rinsed once with pbs, ph . and once with pbs, ph . for rain. following addition of a drop of mounting medium ( % glycerol in pbs, ph . ), cells were examined for immunofluorescence (indicative of the presence of tgev antibodies) using a fluorescence microscope (olympus im, japan). to screen hybridomas for virus neutralizing (vn) antibodies to tgev, a cytopathic effect (cpe) reduction assay was performed. briefly, gl of hybridoma cell culture fluids were transferred to each well of a -weu plate. an equal volume of tcids / gl of m or p strain of tgev was added. the mixture was incubated at °c for h and gl of cells/ml of an st cell suspension in eagle's mem supplemented with % fbs was added to each well. the plates were incubated at °c for h and neutralizing activity was determined by the absence of cpe. a plaque reduction assay was performed using -day old st cell monolayers in sixwell plates to determine virus neutralizing antibody titers. equal volumes of m or p strains of tgev containing to plaque forming units (pfu) in gl were added to serial fourfold dilutions of heat inactivated ( °c, rain) ascites fluids and incubated at °c for h. then, gl of inoculum were added to duplicate wells followed by an additional h incubation at °c. to each well, ml of . % noble agar and . % (of . % stock) neutral red in eagle's mem were added. the virus neutralizing antibody titers were expressed as the reciprocal of the highest sample dilution which produced an % reduction in plaques when compared to the virus control wells. seronegative gnotobiotic pigs were used for production of tgev hyperimmune sera. two pigs were inoculated with m c virulent tgev, one pig with zy isolate tgev and pig with p attenuated tgev. each pig was inoculated orally with - ml of tgev and subsequently hyperimmunized (both intramuscularly and subcutaneously), one time with virus mixed with an equal volume of freund's complete adjuvant at two weeks post-oral inoculation, and three times with virus mixed with freund's incomplete adjuvant, at weekly intervals. sera were collected days after the last injection. hybridomas were produced using modifications of procedures described previously [ ] . to obtain hybridomas secreting tgev specific mabs, female balb/c mice were hyperimmunized five times at weekly intervals with semi-purified m c strain of tgev [approx. fluorescent focus units (ffu)/mt]. spleen cells from these mice were fused with sp / myeloma cells at a ratio of : in the presence of % polyethylene glycol (mw , sigma, st. louis, mo). hybridomas secreting mabs to tgev were detected by vn and ccif tests two times at day intervals. selected clones which were positive by either test were subcloned at least two times by limiting dilution [ ] using conditioned medium which was prepared as follows: thymocytes and spleen cells from female balb/c mice were prepared using x cells/ ml and maintained in rpmi supplemented with % fbs at °c for days. the supernatant medium was then removed and stored at °c for use in limiting dilution. the isotype and subisotype of mabs were determined by using the ouchterlony immunodiffusion technique [ ] . monoclonal antibodies produced in culture medium were precipitated with % ammonium sulfate and resuspended to x the initial concentration. monospecific antiserum to each isotype or subisotype was purchased commercially (icn immunobiologicals, lisle, il). ascites fluids containing mabs were produced in pristane ( , , , -tetramethyl pentadecane, aldrich chemical co., milwaukee, wi) primed-mice as described previously [ ] . mice were primed at least to days prior to use. approximately to x t hybridoma cells were injected intraperitoneally. ascites was produced and the fluids harvested - days post-injection. tests were performed using modifications of procedures described by wesley and woods [ ] . briefly, seven-day old confluent st cell monolayers grown in cm ~ flasks were infected with m tgev at an m.o.i, of . pfu/cell or p tgev at an m.o.i, of . pfu/ cell. mock infections using tissue culture media were done concurrently for each virus. at h post inoculation (pi) mock and tgev infected monolayers were washed with and maintained in methionine-deprived eagle's mem supplemented with gg/ml of gentamicin. labeling experiments were repeated using l ~tg/ml of actinomycin d added at h pi for studying the effect of actinomycin d on viral protein synthesis and for reducing the amount of labeled host cell proteins. at h, pi, ~tci/ml of l- s-methionine (amersham, arlington heights, il) was added to each flask containing mi of methionine-deprived medium. the flasks were incubated at °c for an additional h with gentle agitation. the flasks were then rinsed with pbs (ph . ) containing mm phenylmethylsulphonyl fluoride (pmsf, sigma), and the cell monotayers were lysed in ml lysis buffer . m nac , . m kc , . ram mgci , ram tris-hc , ph . , % triton x- , and units/ml aprotinin). cells were lysed at room temperature for rain and cell debris and particulate material were removed by centrifugation at , x g for rain. the supernatant was stored in ~tl aliquots at -- °c. the rip assay was performed using modifications of methods described by wesley and woods [ ] . cell lysates were preabsorbed with pooled normal mouse sera bound onto protein-a sepharose (pharmacia, sweden) at °c for h. undiluted mouse ascites fluids ( gl) or hyperimmune porcine sera ( ~tl) were absorbed onto protein-a sepharose and then reacted with unlabeled, uninfected st cell lysate for h at °c. this treated asc~tes or sera bound to protein-a sepharose was mixed with to gl of preabsorbed cell lysate at °c for h and then at °c overnight. the sepharose was pelleted in a microfuge and washed four times with lysis buffer and one time with . m tris-hc (ph . ) buffer. the immune complexes bound to sepharose were pelleted and t gl of laemmli's sample buffer [ ] was added. the mixtures were heated at °c for min and the sepharose was pelleted in a microfuge. the resulting supernatants were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) in % stacking and % ( : -acrytamide : bis) running gels. the gels were treated with en hancer (new england nuclear, boston, ma), dried and exposed to x-ray films (kodak xrp- ) at -- °c for days [ ] . molecular weight standards used were c-methylated myosin ( , ), phosphorylase-b ( , ), bovine serum albumin ( , ), ovalbumin ( , ), carbonic anhydrase ( , ), and lysozyme ( , ) (amersham). negative controls including a mab against osu porcine rotavirus, sp / cell-induced ascites, and tge negative porcine and mouse sera were also analysed by rip. a panel of twelve m a b s against the virulent strain of t g e v was generated and their reactivities against virulent and attenuated t g e v characterized (table ) . their isotype and subisotype specificity was also determined ( table ) . protein profiles of p t and m infected cell lysates are shown in fig. . viral specific proteins (e , e , and n) were evident for both p and m infected cell lysates. n o differences in protein profiles were observed with or without the presence of actinomycin d (data not shown). a unique approx. k species of protein was revealed only in the p infected cell lysates (fig. ) . seven neutralizing and five non-neutralizing m a b s were selected for characterization of their viral protein specificity by r i p of s-methionine labeled p and m t g e v infected cell lysates (table ) . a faint b a n d in the k (e ) region was evident with m a b s d , a , h , and d (in fig. a represented by d ), but because of the low intensity of this reaction and t- fig. c represented by h ) . the k protein band was consistently more intense in immunoprecipitates of the p than of the m strain of tgev. a and processed for rip. resulting immune complexes were analysed on % sds-polyacrylamide gels and the gel autoradiographs were exposed for days. p p infected lysate; m m infected lysate; c mock infected control. molecular weight markers are shown in the far left lane. o origin, bb bromophenol blue dye marker, e membrane protein, e peplomer protein, n nucleocapsid protein band (possibly actin) with a mol. wt. of k as consistently resolved in immunoprecipitation of mock and tgev infected cell lysates ( fig. a, b, c) . tge viral specific proteins (e , e , and n) were not immunoprecipitated by negative control sera or ascites fluid (e.g., mabs against porcine osu rotavirus, sp / cells induced ascites fluid and tgev negative mouse and porcine sera) (data not shown). results of rip of p or m tge viral proteins using the four hyperimmune sera are shown in fig. . the viral proteins immunoprecipitated by these sera were compared with those recognized by the mabs. the three major tge viral proteins (e , e , n) from m or p infected cell lysates were immunoprecipitated by each of the hyperimmune sera prepared against the three tge strains (virulent, attenuated and field isolate). the relative mol. wt. of the e protein was consistently higher and the tool. wt. of the e protein was consistently lower for the m strain of tgev than for p tgev. sera produced against all three strains oftgev immunoprecipitated the k protein from p infected cell lysates: this viral protein was undetectable or of low intensity in lysates from m -infected cells (fig. ) . seven of the selected hybridornas produced tge virus neutralizing antibodies (three e -specific and four unclassified mabs) ( table ). four to -fold differences in neutralizing antibody titers against attenuated and virulent tgev were observed for unclassified mabs h and d , and h and e specific mab e ( table ). monoclonal antibody h did not neutralize the p attenuated strain of tgev (titer < ); whereas it has a titer of against the m virulent strain of tgev. monoclonal antibodies d and e (anti-e ) had to -fold higher neutralizing antibody titers against m than against the p strain of tge virus. both p and m tgev produced plaques larger than those in virus control wells (data not shown) in the presence of mab h and d in plaque reduction assays. only mabs h and c had higher neutralizing antibody titers ( to -fold) against p than against m tgev ( table ) . none of the anti-n mabs neutralized tgev. all twelve mabs reacted with tgev in a ccif test and five hybridomas produced tgev antibodies which were detected only by ccif. the antibody titers determined by ccif against both p and m tgev are summarized in table . using ccif, four mabs whose protein specificity was undetermined, showed cell membrane fluorescence (fig. a) . three mabs which reacted with e protein showed faint diffuse perinuclear fluorescence (fig. b) . those mabs which reacted with n protein produced bright particulate cytoplasmic fluorescence (fig. c ). all mabs except the four unclassified mabs had higher ccif titers against homologous (m ) tgev than heterologous (p ) tgev; fold differences were observed for mabs h , e , and f (anti-e and n, respectively) and -fold differences were noted for mab h (anti-n). no differences in fluorescence staining patterns of mabs directed against the same proteins were observed when either p or m was used to infect st cells. however differences in fluorescence intensity were observed using either p or m as the test antigen (data not shown). monoclonal antibody e and f produced stronger immunofluorescence with m infected st cells than p t infected st cells at all dilutions tested. monoclonal antibody g , a , and h at lower dilutions ( : to : , ) induced similar fluorescence intensity with either p or m infected st cells, but brightness diminished after : , to : , dilutions for p infected st cells. monoclonal antibodies against the virulent strain of tgev were generated, and viral protein specificities were determined for all but four mabs. anti-e mabs recognized e proteins with tool. wt. of - k in rip, as reported similarly by others for mabs to p tge [ , ] . anti-n mabs immunoprecipitated a k protein derived from both p and m infected cell lysates, and a k protein was co-immunoprecipitated mainly from p infected cell lysates. the same finding was noted when three of the four hyperimmune porcine sera were tested. this unique k protein has not been reported by other siao-kun wan welch and linda j. saif researchers. one explanation may be that the percentage of cross-linker in our gel system was different from others; therefore, the resolution for the k protein was better. our results suggest that although this k protein occurs in both p and m infected cells and was detected by hyperimmune sera and mabs produced against virulent tgev, it accumulates more in the p infected cells. the nature of this extra species recognized by n-specific mabs is unclear. it may be a precursor or cleavage product of the viral n protein. more conclusive explanations may be obtained if the complete gene coding sequence for the n protein becomes available, or the kinetics for n protein synthesis are explored. in addition, a minor k band detected in p infected cell lysates, but not in m cell lysates ( hpi), may be similar to the k protein described in p cell lysates by wesley and woods [ ] or to the k protein described by hu et al. [ ] . whether this k protein is an intrinsic protein for only purdue attenuated tgev or a cleavage product of e is not yet known. nevertheless, this protein was not immunoprecipitated by the mabs or the four hyperimmune swine sera. the four mabs which had low neutralizing antibody activity, showed high background and low intensity reactions in rip and were reported as unclassified mabs which may possibly be e -specific. additional tests are required to confirm their protein specificities using either more extensive absorption methods or a solid phase immunoisolation technique [ ] . epitopes which elicited tgev neutralizing antibodies resided on the e and e proteins [ ] . in contrast, anti-e mabs produced by jimenez et al. [ ] did not neutralize tgev. anti-e mabs prepared against a mixture of virulent miller and purdue attenuated strains of tgev had virus neutralizing antibody titers only in the presence of guinea pig, rabbit or swine complement [ ] . it is possible some anti-e mabs may inactivate tgev infectivity via complement-mediated virolysis. interestingly, all four unclassified mabs (which may react with e protein) produced in the present study had low to moderate virus neutralizing antibody titers, but the neutralizing activities were not enhanced in the presence of hemolytic units/ml of swine complement (data not shown). by comparison, woods et al. [ ] reported that their e -specific tgev mabs had vn activity only in the presence of to hemolytic units/ml of swine complement. the distinctive immunofluorescence patterns associated with the different tgev protein specificities, suggest that a panel of such mabs may be useful for studies of the morphogenesis of tgev. immunofluorescence patterns in tgev-infected cells reacted with e -specific mabs suggest that e proteins are produced mainly in the cell perinuclear regions. anti-n mabs produced bright cytplasmic particulate immunofluorescence in tgev-infected st cells suggesting that n proteins are abundant in cytoplasm and often in aggregates reflected by the particulate nature of the fluorescence. in the case of the four unclassified mabs, fewer immunofluorescent tgev-infected st cells were evident (although all cells were infected with the same m.o.i.) and fluorescence on the cell surface was more distinct around cell to cell junctions. laude et al. [ ] demonstrated similar fluorescence patterns. however the membrane fluorescence was shown in paraformaldehyde-fixed cells reacted with an anti-e mab and a fluorescence pattern for anti-e mabs was not identified. biochemical and biophysical differences among field isolates and cell cultureattenuated tgev have been reported [ , ] , but serologic differences were not identified [ , ] . using mabs to purdue tgev in competitive assays, delmas et al. [ ] demonstrated that the neutralization mediating determinants were highly conserved among tgev strains. furthermore, jimenez et al. [ ] showed that neutralizing epitopes on attenuated tge virus were conformational and highly conserved. our data suggest differences exist between a virulent and attenuated strain of tgev, perhaps in the density of certain epitopes expressed on the viruses, or the proteins they elicit in infected cells. first, neutralizing antibody titers of unclassified mabs h and d and anti-e mab e were , and -fold higher respectively, to m than p tgev. second, anti-e mab h had a -fold higher neutralizing antibody titer to p than to m . third, all anti-n and anti-e mabs had to -fold higher ccif titers to m than p . furthermore, fluorescence intensity was stronger against the m strain than p strain of tgev. finally, differences in the relative mol. wt. of e and e proteins and in the quantity of the k protein between the virulent and attenuated tgev strains, imply that phenotypic variation between the strains may exist. this has not been reported previously. further studies are needed to define and compare tgev epitopes on attenuated, virulent and field isolates of tgev. although major epitopes mediating neutralization were associated with e proteins and these were conserved, nonneutralizing mabs against e , e , or n proteins may be unique for certain isolates and could thus be used for differentiation of tgev strains as shown previously by laude et al. [ ] . it is important to continue characterizing critical epitopes of the virulent strain of tgev for future development of possible subunit or r d n a vaccines. monoclonal antibodies to tgev described in this study, as well as additional mabs, should also aid in development of better diagnostic reagents for detection and possible differentiation of field and vaccine strains of tgev. antibody responses in serum, colostrum and milk of swine after infection or vaccination with transmissible gastroenteritis virus a film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gel antigenic structure of transmissible gastroenteritis virus. ii. domains in the peplomer glycoprotein comparison of properties between virulent and attenuated strains of transmissible gastroenteritis virus the polypeptide structure of transmissible gastroenteritis virus / ) antigenicity of structural components from porcine transmissible gastroenteritis virus in vitro differentiation and 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dm (ed) handbook of experimental immunology transmissible gastroenteritis immune electron microscopy of transmissible gastroenteritis virus and rotavirus (reovirus-like agent) of swine isolation of molecules recognized by monoclonal antibodies and antisera: the solid phase immunoisolation technique identification of a , molecular weight antigenic polypeptide in transmissible gastroenteritis virus-infected cells neutralization of porcine transmissible gastroenteritis virus by complement-dependent monoclonal antibodies key: cord- -wtnf p authors: chen, xiaojuan; tu, chongzhi; qin, tao; zhu, liqi; yin, yinyan; yang, qian title: retinoic acid facilitates inactivated transmissible gastroenteritis virus induction of cd (+) t-cell migration to the porcine gut date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: wtnf p the digestive tract is the entry site for transmissible gastroenteritis virus (tgev). tgev transmission can be prevented if local immunity is established with increased lymphocytes. the current parenteral mode of vaccination stimulates systemic immunity well, but it does not induce sufficient mucosal immunity. retinoic acid (ra) plays an important role in the induction of cells that imprint gut-homing molecules. we examined whether ra assist parenteral vaccination of pigs could improve mucosal immunity. we demonstrated that elevated numbers of gut-homing cd (+) t cells (which express α β and ccr molecules) were presented in porcine inguinal lymph nodes and were recruited to the small intestine by ra. intestinal mucosal immunity (iga titre) and systemic immunity (serum igg titre) were enhanced by ra. therefore, we hypothesized that ra could induce dcs to form an immature mucosal phenotype and could recruit them to the small intestinal submucosa. porcine t-cells expressed β integrin and ccr receptors and migrated to ccl by a mechanism that was dependent of activation by ra-pretreated dcs, rather than direct activation by ra. together, our results provide powerful evidence that ra can assist whole inactivated tgev (wi-tgev) via subcutaneous (s.c.) immunization to generate intestinal immunity, and offer new vaccination strategies against tgev. transmissible gastroenteritis (tge), which is caused by transmissible gastroenteritis virus (tgev), is a highly contagious disease in newborn piglets . after entering the digestive tract, tgev can replicate in intestinal enterocytes and then induce enteritis and watery diarrhoea . both live and killed tgev vaccines (intramuscular route or subcutaneous injection) are currently available to control tge; however, they are not always successful . these vaccination strategies can stimulate systemic immunity well; however, they do not induce sufficient mucosal immunity, especially the induction of local, virus-specific siga antibodies . determining how to induce a mucosal immune response and improve local immunity in the intestine is important in preventing enteropathogen infection. excellent induction of mucosal immunity depends on the inductive and effector sites . the mucosal immune mechanism includes naive lymphocyte activation in classical inductive sites (such as intestinal peyer's patches), after which the sensitized lymphocytes migrate to the blood circulation before homing to effector sites (such as the intestinal epithelium or lamina propria) and differentiating into effector lymphocytes that contribute to immunity . effective viral clearance requires the rapid migration of effector t cells to the site of intestinal infection. intestinal lymphocyte homing includes lymphocytes selectively passing through the postcapillary venule and migrating directly to the intestinal epithelium or lamina propria. t cells migrating to the intestine require the expression of specific receptors, including homing receptors, such as α β -integrin and ccr , and their corresponding ligands (i.e., addressin-cell adhesion molecule , madcam ) on endothelial cells from intestinal postcapillary venules as well as ligands (such as ccl ) on the intestinal epithelium , . ccr /ccl interactions can induce the homing of effector t and b cells to the gut , . additionally, these interactions can guide plasmacytoid dendritic cells (dcs) to the intestine , . retinoic acid (ra), a vitamin a metabolite, has emerged as a critical factor in mucosal immune responses . ra induces intestinal cytokines generation , and iga responses , , , and ra supplementation reduces morbidity and mortality due to enteric infectious diseases . furthermore, ra was shown to stimulate t cell reagents. retinoic acid (ra), -(and )-carboxyfluorescein diacetate succinimidyl ester(cfda-se), bovine serum albumin (bsa), lps (from escherichia coli :b ), were purchased from sigma-aldrich, saint louis, usa. fitc-conjugated mouse anti-pig cd α ( - - ) monoclonal antibody (mabs), rat anti-mouse integrin β (na/le) mabs, were purchased from bd biosciences, usa. fitc-conjugated mouse anti-human cd (kd ) mabs, fitc or pe-conjugated mouse anti-pig swine workshop cluster a (swc a) ( - mabs, pe-conjugated mouse anti-pig cd ( - - ) mabs, rabbit anti-human ccr (e ) mabs, rabbit anti-human ccr mabs (extracellular domain), pe/cy -conjugated rat anti-mouse cd b (m / ) mabs, rabbit anti-human cd (sp ) mabs, ro - were purchased from abcam, hongkong. fitc-conjugated mouse anti-pig sla-dr ( e / ) mabs, pe-conjugated mouse anti-human hla-dp (hl- ) mabs were obtained from lifespan biosciences, usa. rabbit anti pig igg, goat anti pig iga antibody were purchased from bethy laboratories, usa. pe-conjugated goat anti-rat igg antibody was bought from santa cruz biotechnology, texas, usa. purified tgev s-ad protein . purified porcine ccl protein was generated in our lab. dylight -conjugated goat anti-rabbit igg antibody, dylight -conjugated goat anti-rabbit igg antibody, dylight -conjugated goat anti-rabbit igg antibody were purchased from multiscience, hangzhou, china. abc-based system (biotinylated goat anti rabbit igg antibody) was used as the secondary antibody with dab as a chromogen was (boster, wuhan, china). porcine intestinal epithelial cell line (ipec-j ) and swine testicle (st) cell lines were purchased from jennio biotech, guangzhou, china. vaccine. the tgev strain (shxb, wild-virulent strain) was supplied by the jiangsu academy of agricultural sciences (nanjing, china) . the viral % tissue culture infectious dose (tcid ) of the shxb tgev strain was × . tcid / ul. the viruses were inactivated by ultraviolet radiation (uv) for h and tested for complete loss of infectivity by inoculation into st cells for three passages . ra was dissolved with dimethyl sulfoxide (dmso) according to the manufacturer's instructions. an ra working solution was diluted with corn oil (changshouhua, shandong, china). immunization. piglets were randomly divided into groups, with pigs per group. the first group was immunized via the subcutanoues route with ml of corn oil into the right groin as a control, the second group was immunized via the s.c. route with ml of ra ( mg/ml in corn oil), the third group was immunized via the s.c. route with ml of wi-tgev ( × . tcid / ul), the fourth group was immunized via the s.c. route with ml of wi-tgev ( × . tcid / ul) combined with ml of ra ( mg/ml in corn oil), and the last group was orally immunized with ml of wi-tgev ( × . tcid / ul) ( table ). all of the piglets were immunized at the age of days and given a booster immunization at the age of days on the same side. all of the animals were anaesthetized and killed days after the booster immunization. sample collection. on days , , , , after the first vaccination, four pigs were sampled randomly from each group for determination of serum and faecal tgev-specific antibody. the serum was separated by centrifugation and stored at − °c for tgev-specific igg antibody detection. for tgev-specific iga antibody detection, . g faecal sample was resuspended with ml of pbs, and the supernatant was collected by centrifugation and stored at − °c for tgev-specific iga antibody detection. on days after first vaccination, pigs were exsanguinated, the ileum and the right side of the inguinal lymph nodes (ilns) were stored in pbs. and then cells from the ileum and ilns were isolated and analyzed by flow cytometry . a portion of the ileum was triturated with a mortar and liquid nitrogen, and the homogenate was weighed and dissolved in pbs at a dilution of mg per μl. the supernatants of these homogenates were collected after centrifugation for the detection of specific iga antibodies. after dilution, the homogenate supernatant protein concentrations were measured using a bca protein assay kit (thermo scientific pierce). the ileal tissue was either frozen in liquid nitrogen and stored at − °c for immunofluorescence detection or fixed in bouin's liquid for immunohistochemical detection. flow cytometry analyses. cells from the ileum and ilns were isolated as previously described . the cells were washed twice with cold pbs and stained with specific fluorescent antibodies at °c for . h per the manufacturer's guidelines. to determining the levels of homing receptor on cd + cells, cells were stained with antibodies against integrin β (bd, , . μg for cells), ccr (abcam, ab , . μg for cells) and cd α (bd, , μg for cells). to examination of tissue dc phenotypes, cells were stained with antibodies against cd b (abcam, ab , . μg for cells) and swc a (abcam, ab , . μg for cells), or cd (abcam, ab , μl for cells) and hla-dp (lsbio, ls-c / , μl for cells). to analysing bm-dc phenotypes, cells were stained with antibodies against cd a and swc a (abcam, ab , μg for cells), hla-dp and swc a, or integrin β , ccr and swc a. the respective isotype controls were used as negative controls. after three pbs washes, in each sample were acquired × cells by flow cytometry with a bd facs calibur (bd biosciences, us) , . data were analyzed using flowjo . (tree star). siga and cd + t lymphocyte detection. paraffin-embedded ileal sections were dewaxed in xylene and rehydrated in decreasing concentrations of ethanol . after being blocked with % bovine serum for min, the sections were incubated with the primary antibodies overnight at °c, followed by incubation with secondary antibodies at room temperature for h. the iga + cells were labelled with goat anti-pig iga followed by alexa fluor donkey anti-goat igg. the cd + cells were labelled with rabbit anti-human cd igg followed by biotinylated goat anti-rabbit igg, and then the sections were sealed with a coverslip for examination. the respective isotype controls were used as negative controls. the sections were visualized with an axioplan microscope (zeiss, oberkochen, germany), × magnification. specific igg and iga detection. the tgev-specific igg and tgev-specific secretory iga (siga) levels were measured with enzyme-linked immunosorbent assays (elisas) as previously described . briefly, elisa plates were coated with . μg of purified recombinant tgev s-ad protein (the major antigenic sites a and d in tgev)/well at °c overnight. following protein removal, the plates were blocked with . % (wt/vol) bovine serum albumin (bsa) in pbs for h at °c and then incubated with μl of samples for h at °c. after washing with pbst, μl of hrp-conjugated rabbit anti-pig igg or goat anti-pig iga antibody was added at a : , dilution and incubated for h at °c. the plates were washed times and incubated with , ′ , , ′ -tetramethylbenzidine (tmb). after minutes, the reaction was arrested with sulphuric acid ( m), and the absorbance was read at nm with a microplate reader. swine bm-dcs were isolated as per our advanced methods . briefly, bone marrow was extracted from the femurs of piglets and treated with red blood cell lysing buffer. the bone marrow cells were differentiated into dcs by resuspending the cells in complete medium (rpmi- (invitrogen) supplemented with % foetal bovine serum (fbs) (wisent, ca), % penicillin/streptomycin, ng ml − porcine granulocyte-macrophage colony-stimulating factor (gm-csf), and ng ml − porcine il- (prospec, ness ziona, israel) and plated at × cells per ml in -well plates. non-adherent granulocytes were removed by discarding the culture medium after h of culture. on day of culture, the clusters were harvested and subcultured overnight so that the adherent cells could be removed. non-adherent cells were collected after days of culture, washed, and used as immature dcs for subsequent studies. in vivo homing assay. three piglets were used in vivo homing assay. porcine peripheral blood mononuclear cells (pbmcs) were isolated from the blood of piglets by density centrifugation using histopaque ( . g l − ) (sigma). to isolate t cells, pbmcs were labelled with a mouse anti-cd antibody (abcam, hong kong) followed by incubation with rat anti-mouse igg microbeads (macs; miltenyi biotec, germany) . cd + t cells were cultured with or without ra for h. thereafter, the cells were divided into two parts, one part was detected with flow cytometry to determining the homing receptor levels on cd + cells, cells were stained with antibodies against integrin β , ccr and cd α , and the other part was harvested for the in vivo homing assay. for the in vivo homing assay, the cells treated with ra were labelled with cell tracker cm-dii (life technologies, eugene), and the cells treated without ra were labelled with cfda-se (invitrogen), respectively, according to the manufacturer's instructions. thereafter, the cells were centrifuged and extensively washed. then, million cells from each preparation were mixed and intravenously injected into recipient piglets (n = ). the recipients were construction of porcine ccl . the dna fragment of porcine ccl (nm_ . , life technologies, eugene) was amplified from porcine genome used the primers f-ggactcagatctcgagg ccaccatgaggccgtggctcctggcc and r-tctggaacatcgtatgggtatggtcct ggaatagctgttg, and inserted into the lentiviral vector plvx-acgfp . lentiviral production was achieved through calcium phosphate transfection of plp , plp , plp/vsvg (invitrogen) plasmids. lentivirus packaging referenced literature . for infection × ipec-j cells were plated per cm dish a day prior to infection. at the day of infection the viral supernatant was supplemented with polybrene ( mg/ml, life technologies, eugene) and added to the cells for h. the multiplicity of infection was set to to obtain single copy integration of the synthetic gene circuit. porcine ccl high-expressed clone was cultured to collect supernatant and activity was assayed by nanodrop uv-vis spectrophotometer (thermo scientific, usa). transwell migration assay. migration assays were performed as previously described using -well falcon cell culture inserts with μm pores (corning, new york, usa). cd + t cells were isolated from pmbcs with the same treatment as above. ra, ro - or ra plus ro - were added to the bm-dcs at different concentrations for h before use. following repeated washes, pre-treated bm-dcs were mixed together with t cells at × /well (dc/t-cell ratios of : ) for days. thereafter, the mixed cells were collected for transwell migration assays. to induce cell migration rpmi- medium ( μl) containing ccl ( ng/ml) was placed in the lower chamber, and rpmi- medium ( μl) containing × mixed cells (dcs mixed with t cells) was placed in the upper chamber. after h of incubation at °c, to determine the amount of cd + cells in the lower chamber, the migrated cells were collected, we counted the number of cd + cells by flow cytometry. statistical analysis. all of the data were expressed as the means ± s.d. and analyzed with spss . . the data were analyzed with non-parametric tests, the unpaired mann-whitney for the in vivo data and the paired wilcoxon for the comparisons between conditions with cells from the same piglet. a p value < . was considered statistically significant. in the intestinal mucosal immune system, cellular immune responses play an important central role in the outcome of several viral infections . virus-specific cd + t cells play a central role in controlling and eliminating most pathogen infections. to the best of our knowledge, there are limited reports of studies focused on the design of cd + t cells-based vaccines in mucosal immunity , . when given together with ra, exogenous antigens can effectively stimulate lymphocytes, which have an increased expression of gut-homing receptors and decrease the expression of skin-homing receptors on lymphocyte surfaces. these research studies indicated that the skin could replace peyer's patches as an outstanding mucosal immune inductive site . as we known, the area of abdominal skin was large and the temperature was constant, which was conducive to the vaccine absorption. furthermore, there was less nerves distribution in abdominal skin, which was beneficial to reduce the injection pain and stress. inguinal lymph node was located in the groin, so in our study the skin on the right groin was selected as an immune inductive site. we subcutaneously immunized piglets with corn oil, ra, or wi-tgev alone or in combination with ra, or we orally immunized piglets with wi-tgev alone. to analyze whether ra has the capability of generating gut-homing cells; therefore, cells were isolated from the right side of the inguinal lymph nodes (ilns) and ileum after immunization. intriguingly, the flow cytometry data from the ilns showed that the number of cd + cells that expressed β -integrin and ccr was significantly increased after ra administration compared with the control (fig. a ,b, p < . ). furthermore, the s.c. administration of ra plus tgev increased the number of gut-homing cd + cells expressing β -integrin and ccr compared with the s.c. administration of tgev alone in the ilns (fig. a ,b, p < . ). lymphocyte homing to the small intestine is believed to play a crucial role at mucosal immunity effector sites, and cd + effector t cells in intestinal epithelium can effectively prevent or directly limit infection at the entrance site of enteric viruses . furthermore, effector t cells maintain the epithelial barrier and tissue homeostasis . in our study, the ileum was selected as the mucosal immune effector site. additionally, we assessed the level of cd + cell homing to the ileum. notably, the flow cytometry data from the ileum showed that following the ra plus tgev s.c. and oral tgev treatments, the gut tropism receptor frequency on cd + cells was remarkably enhanced compared with the controls (fig. c ,d, p < . ). next, we further evaluated the number of cd + cells in the porcine ileum with immunohistochemistry. in the cross-sectional view, cd + t cells were gathered in the epithelial layer and villous lamina propria (fig. e, × magnification) . the ra plus tgev s.c. administration substantially expanded the areas with cd + t cells compared with the controls (fig. f , p < . ), although these levels were lower than those observed with the oral tgev treatment. next, to verify whether ra could assist wi-tgev in enhancing the mucosal and systemic immune responses, we determined the local secretion iga and systemic igg, immunization methods as above. our immunofluorescence results showed that the iga-secreting cells were mainly gathered in the villous lamina propria or around the ileal glands (fig. b-f) . in the ileum, the areas of iga-secreting cells of the ra plus tgev s.c. and oral tgev treatment were significantly increased than control (p < . ), and the ra plus tgev s.c. treatment significantly increased the areas containing iga-secreting cells compared with the tgev s.c. alone treatment (fig. g, p < . ) , however, this level was slightly less than the level observed following the oral tgev delivery. furthermore, we noted that the iga-secreting cells displayed iga and ccr co-expression in the villous lamina propria after the ra plus tgev challenge (fig. h-j) . a similar finding was observed in the faeces, in which the subcutaneous ra plus tgev immunization obviously increased the tgev-specific iga levels compared with the s.c. tgev alone treatment at days , and (elisa method) (fig. k, p < . ) , and this effect lasted longer than that of the oral tgev immunization. for the systemic immune response, in the serum, we found that the tgev-specific igg titres in the ra plus tgev s.c. treatment group remained at a high level during the entire immunity period ( - days) and were substantially greater than those in the other groups at day (fig. l, p < . ) . these observations demonstrated that ra-assisted tgev immunization could recruit iga-secreting cells to the ileum and induce a robust antigen-specific iga response in the faeces. dcs are the main cellular element that controls t lymphocyte activation and regulation , which is critical for subsequent immunity responses of the intestinal mucosa. porcine small intestine dcs are express mhcii (sla-dp), cd a (swc a), cd , cd r , or cd b , . to determine whether ra in the vaccine could recruit dcs to the small intestine, we isolated intestinal cells from piglets and determined the dc numbers in the lamina propria by flow cytometry. very few hla-dp + cd + dcs (fig. a,c) or swc a + cd b + dcs (fig. b,d) migrated to the small intestinal lamina propria following tgev s.c. treatment. nevertheless, the s.c. administration of ra plus tgev and oral tgev (positive control) significantly enhanced the frequency of these migrated dcs compared with tgev s.c. treatment (fig. c,d, p < . ) . these findings suggested that s.c. ra treatment could effectively facilitate tgev not only by increasing the cd + cell numbers (fig. c,d) but also by increasing the dc numbers in the lamina propria of the ileum. observed that ra-assisted tgev treatment increased the dc numbers in the porcine intestine; however, the mechanism impacts dcs migration is unclear. a previous study showed that ra induced gut-homing receptors on mice bm-dcs in a narrow time window and had a stringent dose response . thus, we examined whether ra could induce porcine bm-dcs to express gut-homing receptors. after we directly treated porcine bm-dcs with ra for or h in vitro, the expression levels of the gut-homing receptors integrin β and ccr were decreased in a dose-independent manner (fig. a,b) , nm ra treatment significantly reduced the expression of the gut-homing receptors on dcs compared with controls (p < . ). next, to test whether ra could affect porcine bm-dc maturation, we analyzed the expression of the phenotype markers cd a and hla-dr. after bm-dcs were incubated with ra for or h, the percentages of swc a + cd a + (fig. c,d) and swc a + hla-dr + (fig. e ,f) dcs were decreased in a dose-independent manner, but there was no significant difference. to better understand whether ra could directly imprint gut-homing-specific receptors on porcine t cells, unactivated t cells were directly treated with or without ra in vitro, and then the homing receptors β integrin and ccr were detected with flow cytometry. however, there was no significant difference between their levels of homing receptor levels on cd + cells with (up to nm) or without ra treatment (fig. a) . next, to evaluate whether ra-treated t cells could preferentially home to the small intestine, we performed competitive homing assay in vivo. t cells were isolated from a piglet and treated with or without ra, labelled with cm-dii (red, molecular probes) or cfse (green) for long-term cellular labeling. equal numbers of cells from the two populations were mixed and adoptively transferred into piglets via intravenous injection. twenty-four hours later, the mixed cells that homed to the ileal villi were detected by flow cytometry. however, the two populations homed equally into the ileum. there was no significant difference between the levels of homing cells in the piglets with or without ra-treated t cells (fig. b-f) . observations of the intestinal villi and peyer's patches (pps) in cryosections further confirmed the flow cytometry results of homing cells (fig. g,h) . these data of t-cell gut-homing further confirmed the above results that ra did not directly increase expression of gut-homing receptors on t cells. for a deficiency of gut-homing receptors on t cells, t cells could not recruited into ileum. thus, ra did not directly induce t-cell homing to the gut in the piglets. in an mlr assay, ra-treated dcs cocultured with cd t cells, flow cytometry analyses were performed by collecting the mixed cells after days incubation to determine t-cell proliferation and the homing receptor expression levels on t cells (fig. a) . the results showed that the bm-dcs treated by ra stimulated t-cell proliferation were increased in a dose-independent manner at bm-dc/t-cell ratios of : (fig. b) and : (fig. c) , but there was no significant difference compared with the untreated bm-dc group. additionally, the β integrin and ccr expression levels on t cells were detected with flow cytometry. the data showed that , nm ra-treated bm-dcs induced cd + cells to highly express β integrin and ccr compared with the untreated bm-dc group (fig. d , p < . , bm-dc/t-cell ratios of : ). at bm-dc/t-cell ratios of : , , nm ra-treated bm-dcs still induced cd + cells to highly express β integrin and ccr , but there was no significant difference compared with the untreated bm-dc group (fig. e) . to verify whether the porcine cells that expressed ccr could respond to ccl (ccr ligand), we evaluated the migration capacity of t cells towards ccl (fig. f) , which is constitutively expressed by intestinal epithelial cells . all of the t cells when activated by ra-activated dcs showed strong responsiveness to ccl , the number of migrated cells with an ra dose-independent increase (fig. g) . notably, the maximum responsiveness to ccl relied on exogenous ra ( , nm) conditioning. on the contrary, the ability of the t cells to migrate from the apical side to the basolateral side of the transwell filters was significantly reduced after ra receptor agonists (rars) (ro- - , , to , nm) were added with ra ( , nm) to the culture (fig. h) , the number of migrated cells among , or , nm ro- - plus ra or ra treatments was no significant difference, which was consistent with a previous observation . taken together, the cell migration assay results revealed a strong chemotactic response in t cells when activated by ra-activated dcs. these data demonstrated that ra-pretreated bmdcs could activate t cells to express high functional levels of the gut-homing receptor ccr , as well as to migrate towards the porcine chemokine ccl . current tgev vaccines are effective in protecting neonatal piglets from tgev infection. two types of tge vaccine are currently licensed for commercial use, including live attenuated vaccines, which are delivered orally, and inactivated vaccines, which are administered via parenteral inoculation to induce immunity . although live attenuated vaccines induce efficient protective immunity, live viruses are unsafe and can revert to virulence. in the development of a vaccine, safety and effectiveness are important considerations. in recent years, inactivated vaccines have been known for their safety and have been given higher priority in the pig industry, but inactivated vaccines require multiple booster immunizations and/or supplementation with adjuvants, because they are less immunogenic than live vaccines. the initial tgev infection occurs in the digestive tract. the oral route of vaccine delivery should be an effective method to prevent viral adhesion and colonization, thus decreasing viral shedding in the digestive tract . however, the oral route for vaccine delivery is the most challenging and difficult to achieve, particularly for inactivated vaccines. the efficacies of oral inactivated vaccines are currently poor, mainly because inactivated antigens must overcome a series of mucosal barriers, including mucus, digestive fluid, and compact epithelium, before they are captured by submucosal antigen-presenting cells (apcs) . thus, oral administration generally requires a large concentration of antigens to achieve effective immune protection levels . in this study, the antigen dose for the oral immunization (positive control) was times greater than that of the subcutaneous immunization, which significantly increased the production cost of the vaccines. an ideal tgev vaccine should induce both local mucosal and systemic immune responses. one effective strategy to improve local immunity involves inducing lymphocyte trafficking to the intestinal mucosa. ra is critical for inducing gut-homing receptors on t cells, which enhances their migration capacity to the intestinal mucosa . our data confirmed that ra plus tgev s.c. could generate a great number of gut-homing cd + t cells in the inguinal lymph nodes below the subcutaneous immunization site. this was consistent with a previous report that subcutaneous ra-assisted antigen treated mice had increased lymphocytes at the injection site lymph nodes and these cells had up-regulated gut-homing receptor levels and down-regulated skin-homing receptor levels . these results suggested that ra played an important role in immune induction. cd + effector t cells in the intestinal epithelium can effectively prevent or directly limit enteric virus infection , as well as maintain the epithelial barrier and tissue homeostasis . the in vivo assay in our study demonstrated that using ra plus tgev s.c. could greatly increase the number of cd + cells expressing β integrin and ccr in the ileum. however, the cd + cells in the s.c. tgev alone treated mice did not show significant changes, indicating that the ra was necessary to see cd + cell increase in β integrin and ccr . furthermore, it was recently reported that ccr expression on t cells may also regulate the localization and ability of mature cd α α intestinal intraepithelial lymphocytes in the small intestinal epithelium to influence mucosal immune responses. cd α α t cells of intraepithelial lymphocytes (iel) that likely function as regulatory t cells , these cd α α t cell subsets in gut maintain gastrointestinal homeostasis . in agreement with the migration of gut-homing cd + cells, our observations showed that after ra plus tgev s.c. treatment, many cd + t cells were recruited to the intestinal villi and lamina propria, and these data also showed that ra-assisted antigen s.c. can establish a stronger and faster cellular immune response to defend against foreign pathogens . siga plays an important role in reducing viral adhesion and capturing invasive viruses . thus, a large amount of iga-secreting cell homing to the gut is important to produce iga antibody against intestinal pathogens . ra is required to induce gut-tropic iga-secreting cells in mice and humans , , . in our study, iga-secreting cells were, remarkably, recruited to the intestinal lamina propria after the s.c. ra-tgev treatment, which was in line with the subsequent strongly initiated iga antibody response in the local porcine intestinal mucosa. furthermore, the ccr expression on the iga-secreting cells helped their cellular recruitment, and this was consistent with a previous report that ra was critical for the generation of gut-homing receptors on iga-secreting cells . interestingly, ra plus tgev immunization generated a long-lasting specific-siga response in the faeces, even though it was lower than the levels following the oral immunization, implying that an enhancement in siga secretion may be conducive to tgev eliminate and infection resistance in the gut. however, it is worth mentioning that the antigen dose in the oral immunization was times higher than that in the subcutaneous immunization in our study. thus, at equal amount of antigen, it is possible that ra addition increases iga secretion even above the secretion seen in response to the oral vaccine. therefore, the ra administration in the subcutaneous immunization not only improved tgev mucosal humoral immune responses but also was less expensive. moreover, serum igg plays a vital role in systemic immune responses, particularly for protecting against invasive pathogens that enter the bloodstream . ra combined with the s.c. tgev injection indeed induced a pronounced igg response, which could compensate for the disadvantages of the oral immunization. dcs have unique capacities to induce primary t-cell responses , , . ra has an important influence on dc differentiation, maturation and functions, such as the capacity for dcs to induce iga in peyer's patches . in our study, we found that ra could induce wi-tgev to increase the dc number in the intestinal lamina propria of piglets in vivo. the immature dcs (idcs) that are located beneath the intestinal epithelium play an important role in monitoring and capturing antigen . this implied that the dcs that were recruited following the ra-assisted tgev s.c. treatment had the characteristics of immature dcs, as they patrolled the intestinal tract, captured antigens in the intestinal lumina, quickly migrated to mlns for antigen presentation and improved the porcine mucosal immunity. dcs are derived from proliferating precursors in the bone marrow that migrate via the blood to lymphoid and non-lymphoid tissues . to investigate the mechanisms by which ra acted on dcs, we directly treated porcine bm-dcs with ra (in vitro), however, the ra treatment was unable to up-regulate the gut-homing-specific molecules as in mice , even with a wide range of doses. the possible reason for this may have been due to the species differences between porcine and mice. our results also showed that ra couldn't up-regulated the expression of maturation phenotype molecules on porcine bm-dcs in vitro, such as swc a, cd a, and hla-dr molecule expression; these findings were supported by a previous study showing that ra treatment endowed bm-dcs with immature phenotypes via the down-regulation of cd / , cd , and mhcii expression in mice . these observations suggest that ra inhibits porcine bm-dc maturation, and they may help explain our previous observation that the dcs recruited to the intestinal lamina propria following ra-tgev treatment were immature dcs that were conductive to capturing antigen and initiating a specific antibody response. given that ra failed to directly induce gut-homing receptors on dcs, we wondered whether ra could induce porcine lymphocytes to express the integrin β and ccr receptors. however, in the porcine in vitro and competitive homing experiments, ra was unable to directly induce unactivated t cells to express gut-homing-specific receptors, and it also failed in inducing t-cell migration to the small bowel. we wondered about the mechanisms that induce cd + t cells to express the integrin β and ccr receptors after ra-assisted tgev s.c. challenge scientific reports | : | doi: . /srep in vivo. some studies have suggested that dcs are crucial in regulating downstream activation and differentiation of t cells , . moreover, some evidence has demonstrated that cd + t cells and iga-secreting b cells express gut-homing receptors via intestinal dc interactions , . the above analysis tempts us to speculate that ra may regulate dcs in the porcine inguinal lymph nodes to imprint gut tropism receptors on lymphocytes. intriguingly, our data confirmed that ra cooperated with bm-dcs in promoting t cell proliferation, suggesting that ra-pretreated bm-dcs may have a powerful capacity to transfer antigens to t cells. our data further indicated that t cells expressed high gut-homing-specific receptor levels following incubation with ra-pretreated bm-dcs, implying that porcine ra-pretreated bm-dcs may have the ability to imprint integrin β and ccr receptor expression on lymphocytes. moreover, a cell migration assay was performed using transwell inserts, and the t cells that were treated with ra and dcs displayed a strong chemotactic response to porcine ccl , which was consistent with a recent study in mice that showed a powerful homing capacity for these cells. additionally, the capacity of ra-pretreated dcs to induce gut-homing receptors on responding t cells was blocked by an rar antagonist, and those t cells lost the ability to migrate, supporting the hypothesis that ra is indispensable for generating gut tropic t cells in piglets. in view of the in vitro study, the ra-pretreated bm-dcs showed a significant ability to activate t cells to express gut-homing receptors for migration. one possible mechanism for this, with regard to the ra-pretreated dcs, is that ra could act as a "mucosal adjuvant" to induce mucosal dc and lymphocyte activation , . furthermore, exogenous ra promotes intestinal lamina propria dcs to produce endogenous ra , which guarantees gut-homing α β and ccr expression by effector t cells . thus, another possibility is that the ra-pretreated dcs may also generate or combine with other cytokines to jointly promote gut-homing molecule expression, such as ra inducing mucosal dcs to secrete tgf β and il- , which enhance gut-homing receptors on lymphocytes . however, the mechanistic knowledge by which porcine intestinal dc subsets develop is still limited; therefore, further studies will be needed to obtain insights into these potential mechanisms. viral diarrhoea is the second major cause of death and a main reason for malnutrition in children under five years of age. compared to murine models, porcine models have substantial advantages, including physiological, anatomical and genetic similarities to the human intestine as well as defence mechanisms against disease similar to those of humans , , and these models have recently been widely applied to studies of intestinal immunity , gut microbiota , nosogenesis of rotavirus infection in newborns , and even organ transplantation . our research regarding s.c. ra-assisted antigen treatment in the piglet model may helpful to guide the prevention and control of viral diarrhoea in children. taken together, our results demonstrate that ra provides a great opportunity for novel vaccine designs. ra interacts with dcs to guide lymphocyte migration to the intestinal tract, which enhances both mucosal and systemic immunity and is thus beneficial in preventing tgev transmission between piglets and reducing the risk of diarrhoea. the molecular biology of coronaviruses effects of virulent and attenuated transmissible gastroenteritis virus on the ability of porcine dendritic cells to sample and present antigen immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants mucosal immunization and adjuvants the mucosal immune system: features of inductive and effector sites to consider in mucosal immunization and vaccine development cellular and molecular mechanisms for induction of mucosal immunity dendritic cells in intestinal immune regulation generation of 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in neonatal piglets initial in vivo experience of pig artery patch transplantation in baboons using mutant mhc (ciita-dn) pigs this work was supported by national natural science foundation of china ( ) and priority academic program development of jiangsu higher education institutions (papd). x.c. design and performed all the experiments, analyzed the data and drafted the manuscript, c.t. and l.z. performed the immunofluorescence histochemical and immunohistochemical, t.q. and y.y. did dendritic cell isolation and culture, performed flow cytometry analyses, participated in the statistical analyses and writing the manuscript, q.y. supervised the experiment and participated in the experiment designs, q.y. conceived and coordinated the study, participated in writing the manuscript. all the authors read and approved the final manuscript. the authors declare no competing financial interests. key: cord- -yyfgl x authors: guo, jinyue; li, fei; qian, shaoju; bi, dingren; he, qigai; jin, hui; luo, rui; li, shaowen; meng, xianrong; li, zili title: tgev infection up-regulates fcrn expression via activation of nf-κb signaling date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: yyfgl x it has been well characterized that the neonatal fc receptor (fcrn) transports maternal igg to a fetus or newborn and protects igg from degradation. we previously reported that fcrn is expressed in a model of normal porcine intestinal epithelial cells (ipec-j ). transmissible gastroenteritis is an acute enteric disease of swine that is caused by transmissible gastroenteritis virus (tgev). how porcine fcrn (pfcrn) expression is regulated by pathogenic infection remains unknown. our research shows that ipec-j cells infected with tgev had up-regulated pfcrn expression. in addition, the nf-κb signaling pathway was activated in ipec-j cells by tgev infection. furthermore, treatment of tgev-infected ipec-j cells with the nf-κb-specific inhibitor bay - resulted in down-regulation of pfcrn expression. transient transfection of pfcrn promoter luciferase report plasmids with overexpression of nf-κb p transcription factor enhanced the activation of the luciferase report plasmids. we identified four nf-κb transcription factor binding sites in the promoter region of this gene using luciferase reporter system, chromatin immunoprecipitation, electromobility shift assay, and supershift analysis. together, the data provide the first evidence that tgev infection up-regulates pfcrn expression via activation of nf-κb signaling. immunoglobulin g is a major ig isotype in mucosal secretions and is involved in host defense. it has now been years since the remarkable foresight by f.w.r. brambell, who described a saturable receptor that transports maternal igg to a fetus or newborn. a few years later, he put forth the hypothesis of the presence of a similar receptor that protected igg from degradation, eventually identified as the neonatal fc receptor (fcrn) . fcrn was originally isolated from the intestine of neonatal rodents and identified as the receptor responsible for the transmission of maternal antibodies from mother to pup [ ] [ ] [ ] [ ] . in recent decades, researchers have showed that fcrn is most closely structurally related to the major histocompatibility complex class i molecule and is composed of a heavy chain associated noncovalently with β -microglobulin (β m) , . fcrn was also shown to bind igg at the ch -ch interface in a ph-dependent way. binding occurs in acidic (ph ≤ . ) environments, and igg is released at neutral (ph ≥ . ) conditions , . fcrn is a transport receptor which mediated transfer of iggs across the human placental barrier or the rodents intestinal epithelial barrier to a fetus or newborn , , . fcrn, therefore, plays a major role in the passive acquisition of maternal immunity by newborn mammals. in addition, fcrn is capable of protecting igg from degradation and maintaining igg levels in the bloodstream . fcrn has been indicated to be expressed in a variety of mammalian species, including mouse, human, rat, sheep, cow, pig, possum and camel . the level of fcrn expression plays an important role in controlling igg levels in tissues and blood . some studies have shown that mice deficient in either β m or the heavy chain of fcrn fail to transport igg, meanwhile the serum half-life of igg is shortened , . more recently, several publications indicated that transgenic (tg) mice that over-express bovine fcrn in the mammary gland have increased igg levels in both milk and serum . meanwhile, some researchers have reported fcrn overexpression by tg modification in mice and rabbits not only prolongs the igg half-life but also enhances the humoral immune response of these animals [ ] [ ] [ ] . more specifically, these tg animals displayed significantly larger spleens containing a higher number of ag-specific b cells and plasma cells in response to immunization, increased antibody diversity and prolonged ag-specific igg half-life . this augmented immune response is also reflected in the ability of fcrn tg mice to produce high levels of ag-specific antibodies, b cells and plasma cells to weakly immunogenic targets or evade recognition by the immune system . nuclear factor-κ b (nf-κ b) is a family of transcription factors that mediates signal-induced expression of numerous genes involved in the innate and adaptive immune responses, inflammation, and autoimmune diseases . some articles have reported that nf-κ b signaling regulates functional expression and function of the human and bovine fcrn , . in the present study, we have analyzed the nf-κ b binding site in the promoter of pfcrn gene. transmissible gastroenteritis virus (tgev) is a member of the family coronaviridae in the order nidovirales . it replicates in the differentiated enterocytes covering the villi of the porcine small intestine and causes severe gastroenteritis in young tgev-seronegative pigs. diseased pigs often present with vomiting, dehydration, and severe diarrhea. consistent with in vivo pathological changes, tgev infection induces morphological and biochemical changes in host cells and some porcine cell lines in vitro. previous studies have reported that tgev infection induces apoptosis in pk- cells via activation of the p signaling pathway . quantitative proteomic analysis reveals that tgev infection activates the janus kinase signal transducer and activator of the transcription (jak-stat ) signaling pathway . several publications have also shown that tgev could infect ipec-j cells, and that it could also down-regulate proteins involved in tight and adherens junctions to alter the epithelial barrier integrity . these findings suggest that tgev infection induces a number of changes in host-cell biology that could influence cells function. in the present study, we investigated how tgev infection activated the nf-κ b pathway in vitro and up-regulated pfcrn expression in ipec-j cells. our studies showed that pfcrn expression can be triggered by tgev infection, through nf-κ b signal activation. an nf-κ b-specific inhibitor significantly down-regulated expression of pfcrn gene by tgev infection. furthermore, we identified the direct involvement of nf-κ b-specific binding sites by using several complementary strategies. these observations support the hypothesis that up-regulation of pfcrn expression following virus infection appears to be an innate immune response against invading pathogens that could help the host clear infection effectively. to determine whether tgev is capable of modulating pfcrn expression in ipec-j cells, infectious tgev or uv-inactivated virus was adsorbed to confluent monolayers of ipec-j cells at an moi of and pfcrn mrna was measured , , , , and h post-virus exposure by real-time rt-pcr. we found that the level of pfcrn mrna expression was -fold higher at , and h post-infection, compared to mock-infected cells (fig. a) . however uv-inactivated virus treatment only caused a . -fold up-regulation in pfcrn expression, compared to mock-infected cells (fig. a) . replicating virus had a more pronounced effect than uv-inactivated virus. to determine whether the increased mrna levels resulted in increased pfcrn protein production, ipec-j cells were cultured in the presence or absence of infectious virus at an moi of and pfcrn protein was measured in cell lysates by western blot (fig. b) . increased levels of pfcrn were detected by h and h tgev exposure (fig. b) . thus, the elevation in protein levels correlated with the increased mrna levels by h. tgev infection activates nf-κb signaling pathway. ipec-j cells were transiently transfected with p -egfp fusion expression plasmid and the subcellular localization of the fusion protein was analyzed using confocal laser microscopy. as shown in fig. a , p -egfp was located exclusively in the cytoplasm in mock-infected ipec-j cells (top panels), but it rapidly translocated to the nucleus when those cells were infected with tgev (bottom panels). cells were cotransfected with ng/well of nf-κ b luciferase reporter plasmid pnf-κ b-luc and ng/well of the renilla luciferase construct prl-tk which was served as an internal control. twenty-four hours later, cells were infected or control-infected with tgev. cells were harvested at the indicated times and luciferase activity was measured using a dual-luciferase assay system. data represent relative firefly luciferase activity normalized to renilla luciferase activity. nf-κ b-regulated luciferase expression was significantly enhanced during tgev infection at , , and h, compared to mock-infected samples (fig. b) . these two results demonstrated that the classical pathway of nf-κ b was activated in ipec-j cells by tgev infection. effect of nf-κb inhibition on pfcrn expression. inhibition of pfcrn induction by bay - , a small molecule that blocks the phosphorylation of iκ bα , suggested that the early proinflammatory response involved activation of the classical nf-κ b pathway. ipec-j cells were pretreated with bay - ( μ m) for min and subsequently infected by tgev. treatment with bay - significantly reduced tgev infected pfcrn mrna levels to that of the tgev infected cells without bay - treatment as assessed by real-time rt-pcr at h (fig. ). therefore inhibition of pfcrn induction by bay - suggested that early activation of the classical pathway of nf-κ b activation was critical for subsequent up-regulation of pfcrn mrna. software was used for searching putative transcription start sites in the ′ -flanking region of the pfcrn gene (minimum promoter score: . ). computational analysis revealed that the pfcrn gene contains two transcription start sites (fig. s ). transcription start site- has a higher score than the transcription start site- . furthermore, the alignment of pfcrn and human fcrn (hfcrn) upstream sequences of the start codon showed that regions near transcription start site- of pfcrn gene have a high homology with the transcription start site nucleotide sequence of hfcrn gene (fig. s ). in the study, the transcription start site- was considered as the transcription start site of pfcrn gene (fig. s ). scientific reports | : | doi: . /srep to study whether nf-κ b regulates pfcrn expression through a mechanism that involves direct binding to a putative regulatory nf-κ b binding sequence located in the pfcrn gene, we utilized luciferase reporter constructs of the pfcrn promoter that contained nf-κ b p binding sites. we created four luciferase reporter gene constructs with sequentially shortened fragments of the promoter region: pfcrn-luc- (− ~+ ), pfcrn-luc- (− ~+ ), pfcrn-luc- (− ~+ ) and pfcrn-luc- (− ~+ ). furthermore, transient transfection of the four luciferase reporter plasmids in ipec-j cells revealed that these plasmids significantly increased the basal promoter activity above that of the empty vector (fig. a ). next, we tested whether pfcrn promoter activity could be up-regulated by tgev infection. the results showed that luciferase activity was significantly enhanced using the pfcrn-luc- , pfcrn-luc- and pfcrn-luc- constructs during tgev infection compared with mock-infected ipec-j cells, while the luciferase reporter construct harboring the shortest fragment of pfcrn-luc- had not significant differences comparing with mock-infected ipec-j cells (fig. b ). to directly assess the involvement of nf-κ b in pfcrn expression, we investigated whether overexpression of nf-κ b p affects the promoter activity of pfcrn. we transiently transfected ipec-j cells with the p -tag b plasmid. we found that pfcrn-luc- , pfcrn-luc- and pfcrn-luc- constructs were induced in the presence of p , while pfcrn-luc- had not significant differences comparing with mock group (fig. c) . these results indicate that the overexpression of nf-κ b p is sufficient to up-regulate pfcrn expression. meanwhile the nf-κ b sensitive region is located in the sequence between − and − of the pfcrn promoter. screening for nf-κb binding sites adjacent to the pfcrn gene. the canonical nf-κ b dna binding sequence is a common -bp consensus dna element that has been identified as ′ -gggrnnyycc- ′ (where r is an a or g; n is any nucleotide; y is c or t) . we searched for putative nf-κ b-binding sequence(s) along this promoter region (− ~− ). computational inspection revealed that the promoter of the pfcrn gene contained sequences with a similarity to the nf-κ b consensus sequence (fig. a ). we used a chip assay to verify that these putative nf-κ b binding sequences had the capability to directly bind nf-κ b proteins in living cells. first, cross-linking the dna with bound nf-κ b proteins in situ in tgev infection verus mock-infected ipec-j cells, nf-κ b-dna complexes were co-incubated with p specific antibody. then, the purified dna fragments were measured by pcr. as shown in fig. b , pcr with specific primers (table ) produced a band from dna ) were separated by electrophoresis in a % sds-polyacrylamide gel, transferred to a pvdf membrane, and blotted with an pfcrn-specific (top panel) or a gapdh-specific ab (bottom panel). blots were then incubated with a hrpconjugated secondary ab and visualized using chemiluminescence. the bar graphs represented results of three independent experiments. intensities of proteins bands were calculated from the peak area of densitograms by using image software. coprecipitated with p (fig. b , − , − , − and − ), however the sequence (− ) failed to produced a band. in a negative control, immunoprecipitation with normal mouse igg did not generate any corresponding pcr products (fig. b, lane ) . the results suggest that p transcription factor interacted with the four nf-κ b binding sequences of pfcrn gene in ipec-j cells. emsa and supershift assays further confirmed the four nf-κ b binding sites that were identified from the chip assay. nuclear extracts co-incubated with oligonucleotides containing nf-κ b binding sequences (fig. a , − , − , − and − ). as shown in fig. a , a higher amount of complexes formed when bound to nuclear extracts from tgev-infected cells than when bound to nuclear extracts from mock-infected cells. these complexes were further shifted by anti-p monoclonal antibody, indicating that the complexes contained p transcription factor (fig. a, upper arrow, supershifted nf-κ b-specific complex, lane ) . to verify the binding specificity, a competition assay was performed. although the specific band could not be significantly inhibited by unlabeled oligonucleotides in − , − and − nf-κ b binding sequences (fig. a, lane ) . nonlabeled oligonucleotide was added to a -fold excess, the specific band was inhibited by unlabeled oligonucleotides in − , − and − nf-κ b binding sequences (fig. b, lanes , , ) . fcrn has more recently been shown to express in a variety of mammalian species . meanwhile, several studies have reported the distribution, function, and regulation of human and rodent fcrn expression , , , . our study demonstrated that pfcrn mediated bidirectional igg transport across polarized ipec-j cells that potentially provide the mucosal protection . some studies have showed that up-regulation of fcrn expression can augment the igg transport across the polarized epithelial cells . fcrn overexpression boosts humoral immune response in transgenic mice . tgev is an enveloped, non-segmented, single-stranded positive-sense rna virus . the ′ one-third of the genome encodes viral structural and accessory proteins, including the spike (s) glycoprotein, the membrane (m) glycoprotein, the small envelope (e) glycoprotein, the nucleocapsid (n) protein, and three accessory proteins a, b, and , meanwhile ′ -proximal two-thirds of the genome encode the viral replicase . our study is the first to show that tgev infection up-regulates pfcrn mrna and protein in ipec-j cells (fig. ) . levels of pfcrn mrna in uv-inactivated virus treatments are only . -fold lower compared to replicating virus infection. these results show that tgev-mediated fcrn up-regulation is mainly related to the replication of virus, meanwhile uv-inactivated tgev-mediated fcrn up-regulation maybe related to viral structural and accessory proteins. some researchers have shown that fasl-and mitochondria-mediated pathways were activated by tgev infection, which induces apoptosis in pk- cells . moreover, tgev infection promoted the activation of p signaling to induce cell apoptosis . our results show that tgev infection activates the nf-κ b signaling pathway, determined by a p nuclear translocation assay and nf-κ b luciferase report system assay (fig. ) . furthermore, pfcrn expression induced by tgev infection was strongly reduced by the nf-κ b-specific inhibitor bay - in ipec-j cells (fig. ) . this was corroborated by the overexpression of nf-κ b p , which up-regulated the activation of pfcrn promoter luciferase report plasmids. this complementary experiment lessens the concern of specificity or toxicity of the chemical inhibitor. the promoter regions for human and bovine fcrn have been analyzed, and the regulation of expression has been shown to be mediated with p transcription factors , . in this study, we analyzed the ′ -flanking region of the pfcrn gene by tess and tfsearch programs, and found there are five potential nf-κ b transcription factor p binding sites between position − and − . therefore, we constructed luciferase reporter plasmids with sequentially shortened fragments of the fcrn promoter region. transient transfection of the pfcrn promoter luciferase reporter plasmids revealed that pfcrn-luc-( - ) plasmids resulted in increased promoter activity in the presence of tgev infection (fig. b) , further demonstrating that tgev up-regulates pfcrn expression in ipec-j cells. pfcrn-luc-( - ) plasmids were also activated by overexpression of nf-κ b p plasmid (fig. c) . these results showed that the nf-κ b sensitive region of fcrn promoter is located in the sequence between − and − . purified dna fragments were subjected to pcr analysis using primer pairs (table ) . chip was performed at least three times. co-fcrn- ′ -atgtgccgtgggtgtggcccta- ′ ′ -aacttgcatccctgataaga- ′ co-fcrn- ′ -atggctgtggtataggctgata- ′ ′ -cctttttacagcaatacatgcc- ′ co-fcrn- ′ -tggttgatccagacaatagaat- ′ ′ -gccgcagcagtgatccca- ′ co-fcrn- ′ -ggcaccatattgtaggattcca- ′ ′ -caatcaccgatgtgtacgtt- ′ co-fcrn- ′ -catcggtgattgccagga- ′ ′ -tgccaccacggccacta- ′ we mapped four nf-κ b binding sequences in our chip experiment (fig. ). this result was further confirmed by emsa and supershift analysis (fig. ) . these results indicated strong and effective molecular interactions between nf-κ b p and the selected transcription binding sites of the pfcrn promoter. nf-κ b is a ubiquitous transcription factor that regulates the transcription of genes such as chemokines, cytokines, proinflammatory enzymes, adhesion molecules, proinflammatory transcription factors and so on. based on previous studies, nf-κ b is activated via two distinct kinase-dependent pathways, the classical nf-κ b pathway and the alternative nf-κ b pathway. while our research mainly concentrated on the classical nf-κ b pathway, its activity can be modulated significantly by additional factors that transfer into the nucleus and bind to nf-κ b binding sequences. in this regard, we analyzed the ′ -flanking region of the pfcrn gene. and found there are several other transcriptional factors in this region, such as ap- and sp . some studies have suggested that nf-κb dimers can act synergistically with ap- and sp transcriptional factors to influence gene regulation [ ] [ ] [ ] . these protein-protein interactions may be involved in mediating transcriptional regulation of the pfcrn gene in response to stimuli and can functionally cooperate to elicit maximal activation of the promoter. in summary, tgev infection up-regulates pfcrn expression in ipec-j cells, and activates the nf-κ b signaling pathway. furthermore, pfcrn expression was strongly reduced by inhibitor bay - treatment after tgev infection. this result deduced that the up-regulation of fcrn expression by tgev infection may be related to nf-κ b signaling pathway. we constructed fcrn promoter luciferase report plasmids by computational inspection. according to the analysis of the activation of pfcrn promoter luciferase report plasmids by tgev infection and overexpression p plasmid, the nf-κ b sensitive region of fcrn promoter is located in the sequence between − and − . there are four nf-κ b binding sites confirmed by chip and emsa experiments. in the study, upregulation of pfcrn expression following virus infection may play an important role in effectively protecting the mucosal surface from the pathogens invasion. and ) , and these complexes could be further shifted by p -specific abs (*supershifted κ b-specific complex, lane ). n.s, non-specific bands. (b) a competition assay was performed. nonlabeled oligonucleotide in − , − and − nf-κ b binding sequences was added to a -fold excess (fig. b, lanes , and ). distinct κ b-specific protein-dna complexes were detected using nuclear extracts (fig. b, lanes , and ). scientific reports | : | doi: . /srep cell lines, antibodies, virus and plasmids. ipec-j cells were cultured in dulbecco's modified eagle's media (dmem)/ham's f- ( : ) (hyclone, usa) with % fetal bovine serum (gibco, usa) and % penicillin/ streptomycin. all cells were grown in a humidified atmosphere of % co at °c. tgev strain wh- (genbank accession no. hq ), which was isolated in china, was propagated in pk- cells. affinity-purified rabbit anti-cytoplasmic tail of porcine fcrn (anti-pfcrn-ct) polyclonal antibody was prepared in our laboratory. horseradish peroxidase (hrp)-conjugated goat anti-rabbit and rabbit anti-mouse igg were purchased from thermo scientific pierce (usa). mouse anti-gapdh monoclonal antibody was purchased from boster (china). p -egfp fusion expression plasmid was prepared in our laboratory, p gene was pcr subcloned into pegfp-c vector. p -tag b plasmid encoding p was prepared in our laboratory, p gene was pcr subcloned into pcmv-tag b vector. quantitative real-time rt-pcr. confluent monolayers of ipec-j cells were inoculated with tgev at a multiplicity of infection (moi) of for , , , , , and h at °c. total rna was extracted from ipec-j cells with trizol reagent (invitrogen, usa). then, total rna was reverse-transcribed into cdna using reverse transcriptase (takara, china). real-time rt-pcr was performed in triplicate in three separate experiments using pfcrn primers ( ′ -ggcgacgagcaccactactg- ′ and ′ -agccgaccatgattccaacc- ′ ) and gapdh primers ( ′ -acatggcctccaaggagtaaga- ′ and ′ -gatcgagttggggctgtgact- ′ ) and the sybr premix ex taq ii (takara, china). all reactions were performed for cycles: s at °c and s at °c. pfcrn expression was calculated following normalization to gapdh levels by the comparative delta delta threshold cycle (Δ Δ c t ) method. the specific amplification reactions were confirmed by melt curve analysis. lysates from ipec-j cells were prepared as described previously . the total proteins were resolved on a % sds-polyacrylamide gels under reducing conditions and electrotransferred onto a polyvinylidene fluoride membrane (bio-rad, usa). affinity-purified rabbit anti-pfcrn-ct polyclonal antibody and mouse anti-gapdh monoclonal antibody were used as primary antibodies, then hrp-conjugated goat anti-rabbit igg or hrp-conjugated rabbit anti-mouse igg were used as secondary antibodies. western blot analysis was performed as described previously . confocal laser microscope assay. ipec-j cells were grown to approximately - % confluency on coverslips placed in -well plates. after transfection with . μ g of p -egfp fusion expression plasmid per well, cells were mock-infected or infected with tgev at a moi = . h after stimulation, cells were fixed with % paraformaldehyde, permeabilized with . % triton x- , and stained with dapi (invitrogen, usa) to detect nuclei. construction of reporter plasmids. four fragments of the ′ -flanking region of the pfcrn were pcr subcloned into the luciferase expression vector pgl (promega, usa) through sac i and hind iii digestion. pfcrn-luc- , containing sequences from − to + of the pfcrn gene promoter was amplified by the pcr primer pairs ( ′ -gcgagctcgctatag ctctgattcgacc- ′ and ′ -caagcttctgagcgggagacctgggg- ′ ); pfcrn-luc- , which contains the segment from − to+ of the pfcrn gene promoter was amplified using the reverse primers described above and a forward primer ( ′ -gcgagctcgcttcagctggacccgtagc- ′ ). plasmid pfcrn-luc- , which contains the segment from − to + of the pfcrn gene promoter, was generated using the reverse primers described above for pcr amplification and a forward primer ( ′ -gcgagctctacttaaaggggtacggggt- ′ ). plasmid pfcrn-luc- , which contains the segment from − to + of the pfcrn gene promoter and was generated using the reverse primers described above for pcr amplification and a forward primer ( ′ -gcgagctcgggggtgctgacgaggtaagaa- ′ ). lipofectamine (invitrogen, usa). cells were transfected with n g of pfcrn promoter luciferase report plasmids and n g of a renilla luciferase prl-tk control plasmid. twenty-four hours later, cells were infected with or without tgev (moi = ). cells were harvested at the indicated time and luciferase activity was measured using a dual-luciferase assay system (promega, usa). the values for firefly luciferase were normalized to the renilla luciferase activity and expressed as fold activation over the mock-infected group. in order to detect whether tgev infection activates nf-κ b signaling pathway, ipec-j cells were cotransfected with ng/well of nf-κ b luciferase reporter plasmid pnf-κ b-luc and ng/well of the renilla luciferase construct prl-tk. cells were infected or control-infected with tgev and were harvested at the indicated times and luciferase activity was measured using a dual-luciferase assay system. chromatin immunoprecipitation (chip). chip was performed according to the manufacturer's recommendations (epigentek, usa). in brief, ipec-j cells were infected with or without tgev (moi = ) for h. the cells were fixed with % formaldehyde. the nuclei were isolated and dna was sheared by sonication. chromatin was immunoprecipitated with μ g of ab specific for p or with μ g of normal igg as negative control for h at room temperature by an orbital shaker ( - rpm). the dna samples were amplified by pcr primers (table ) in optimized conditions. preparation of nuclear extracts, emsa and supershift assay. nuclear extracts were prepared using a nuclear and cytoplasmic extraction kit (cwbio, china). the single-strand oligonucleotides was labeled with biotin on the ′ end dna, annealed to form double-stranded oligonucleotides containing the tested nf-κ b sequences from the pfcrn promoter: pfcrn κ b- ( ′ -aaaaatggga gtttccatttccg- ′ ), pfcrn κ b- scientific reports | : | doi: . /srep ( ′ -gtagcctgggaacttccagat gcc- ′ ), pfcrn κ b- ( ′ -ccagaagaggcaaattcctagagac- ′ ) and pfcrn κ b- ( ′ -aaggggtacggggtctccttgggg- ′ ). for competition assays, a -fold excess of nonlabeled oligonucleotide was incubated during the preincubation time. for the supershift assays, μ g anti-p monoclonal antibody directed against nf-κ b p was preincubated with the nuclear extracts. the complexes were run on a % native polyacrylamide gel. emsa experiments were performed according to the lightshift chemiluminescent emsa kit (thermo scientific pierce, usa). data from three independent studies were analyzed using anova to identify significant changes between tgev-infected and mock-infected cells. all results are expressed as mean ± sem from three independent experiments. p values < . were considered significant (*p < . and **p < . ). interference by human and bovine serum and serum protein fractions with the absorption of antibodies by suckling rats and mice ph-dependent binding of immunoglobulins to intestinal cells of the neonatal rat the mechanism of intestinal uptake and transcellular transport of igg in the neonatal rat receptor-mediated transport of igg an fc receptor structurally related to mhc class i antigens crystal structure of the complex of rat neonatal fc receptor with fc isolation and characterization of an fc receptor from neonatal rat small intestine crystal structure and immunoglobulin g binding properties of the human major histocompatibility complex-related fc receptor(,) bidirectional fcrn-dependent igg transport in a polarized human intestinal epithelial cell line expression of functionally active fcrn and the differentiated bidirectional transport of igg in human placental endothelial cells neonatal fcr expression in bone marrow-derived cells functions to protect serum igg from catabolism chapter : multitasking by exploitation of intracellular transport functions the many faces of fcrn enhanced half-life of genetically engineered human igg antibodies in a humanized fcrn mouse model: potential application in humorally mediated autoimmune disease the major histocompatibility complex-related fc receptor for igg (fcrn) binds albumin and prolongs its lifespan the mhc class i-like igg receptor controls perinatal igg transport, igg homeostasis, and fate of igg-fccoupled drugs over-expression of the bovine fcrn in the mammary gland results in increased igg levels in both milk and serum of transgenic mice fcrn overexpression in transgenic mice results in augmented apc activity and robust immune response with increased diversity of induced antibodies characterization of the rabbit neonatal fc receptor (fcrn) and analyzing the immunophenotype of the transgenic rabbits that overexpresses fcrn transgenic expression of bovine neonatal fc receptor in mice boosts immune response and improves hybridoma production efficiency without any sign of autoimmunity neonatal fcr overexpression boosts humoral immune response in transgenic mice jankovics, i. & kacskovics, i. fcrn overexpression in mice results in potent humoral response against weakly immunogenic antigen nf-kappab regulation in the immune system nfkappab induces overexpression of bovine fcrn: a novel mechanism that further contributes to the enhanced immune response in genetically modified animals carrying extra copies of fcrn nf-kappab signaling regulates functional expression of the mhc class i-related neonatal fc receptor for igg via intronic binding sequences molecular biology of transmissible gastroenteritis virus transmissible gastroenteritis virus infection induces cell apoptosis via activation of p signalling quantitative proteomic analysis reveals that transmissible gastroenteritis virus activates the jak-stat signaling pathway transmissible gastroenteritis virus and porcine epidemic diarrhoea virus infection induces dramatic changes in the tight junctions and microfilaments of polarized ipec-j cells distribution of rat neonatal fc receptor in the principal organs of neonatal and pubertal rats characterization of the rat intestinal fc receptor (fcrn) promoter: transcriptional regulation of fcrn gene by the sp family of transcription factors neonatal fc receptor-mediated igg transport across porcine intestinal epithelial cells: potentially provide the mucosal protection molecular characterization and pathogenesis of transmissible gastroenteritis coronavirus (tgev) and porcine respiratory coronavirus (prcv) field isolates co-circulating in a swine herd strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model transmissible gastroenteritis virus infection induces apoptosis through fasl-and mitochondria-mediated pathways tgev nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p signaling communication between nf-kappa b and sp controls histone acetylation within the proximal promoter of the monocyte chemoattractant protein gene sp binding is critical for promoter assembly and activation of the mcp- gene by tumor necrosis factor cross-coupling of the nf-kappa b p and fos/jun transcription factors produces potentiated biological function transfer of igg in the female genital tract by mhc class i-related neonatal fc receptor (fcrn) confers protective immunity to vaginal infection this work was supported by national natural science foundation of china ( to z.l.) j.g. and z.l. conceived and initialed the study; j.g., f.l. and s.q. extracted the data set; j.g., d.b., q.h., h.j., r.l., s.l. and x.m. performed the analysis. j.g. and z.l. wrote the paper. key: cord- -clxzp ph authors: weber, olaf; schmidt, axel title: coronavirus infections in veterinary medicine date: journal: coronaviruses with special emphasis on first insights concerning sars doi: . / - - - _ sha: doc_id: cord_uid: clxzp ph nan tion is high frequency rna recombination [ ] . this strategy might be important for the crossing of species barriers for many important animal coronaviruses as well as the "sars-cov". the members of the nidovirales order, however, especially differ with respect to their envelopes and nucleocapsids [ ] . the biology of coronaviruses has been described in detail in other chapters. therefore, this chapter focuses on clinical and economical aspects of coronavirus infections in companion (pets), live-stock (farm) and laboratory animals. based on phylogenetic analysis and antigenic cross reactivity, three groups can be distinguished in the coronaviridae family. some important viruses that are discussed below belong to group i and include the canine enteric coronavirus (cecov), the transmissible gastroenteritis virus (tgev) of swine, the porcine epidemic diarrhoea virus (pedv), the porcine respiratory coronavirus (prcov) and the feline coronaviruses (fcovs). most coronaviruses are enzootic and/or endemic in the respective host species. the vast majority of infections occur with inapparent clinical signs. the virus usually infects and replicates in epithelia and outbreaks of gastrointestinal and/or respiratory diseases are often seasonal. clinical signs are in general more severe in younger animals. immunocompromised animals and infected adults also serve as a major virus reservoir. genomic recombination appears to be a very common event in vitro and in vivo [ ] in coronaviruses also relevant in animal health. therefore, interactions of different coronaviruses can lead to new types and novel clinical entities [ , ] . porcine coronaviruses include i) tgev; ii) prcov which is a mutant of tgev; iii) pedv i and ii; and iv) haemagglutinating encephalomyelitis virus (hev). the latter belongs to the coronavirus group ii. tgev and pedv are highly infectious and highly contagious enteric viruses of swine. pedv and tgev infections are considered difficult to distinguish clinically and also histopathologically [ ] [ ] [ ] but can be distinguished by using modern, up-to-date techniques such as pcr and/or specific sequencing [ ] . tge is an economically important disease that might result in high mortality and was first described in the s. while older animals generally recover by time, piglets under the age of three weeks usually die from the infection. the tgev genome consists of a single-stranded, positive-sense . -kb rna. the viral membrane contains three transmembrane proteins: the s protein ( kda), m protein ( to kda), and minor e protein ( -kda). the s surface protein initiates the infection by binding to the cell surface. it also mediates the subsequent fusion between the viral and cellular membranes. the s protein binds to aminopeptidase n and to sialic acid. aminopeptidase n binding is required for tgev to initiate the infection of cells [ ] . recognition of sialic acids appears to be important for both, the haemagglutinating activity and the enteropathogenicity of tgev [ ] . the enteric tropism of tgev presumably also requires the binding to a co-receptor that maps around amino acid of the s protein as well as other additional co-factors [ , ] . the importance of sialic acid binding for entropathogenicity is supported by the fact that the non-enteropathogenic prcov lacks sialic acid binding activity. this can be explained by a large deletion in the s gene that results in a truncated spike protein [ , ] . prcov causes a mostly mild and moderate epizootic respiratory disease and might have worked like a vaccine against tge in a number of swine populations. tgev is transmitted either directly or indirectly through contact with feces of infected pigs or via vector animals such as dogs and cats. like other coronaviruses, tgev is fragile and highly sensitive to disinfectants as further detailed in the chapter by wolff et al. epidemic peaks mostly occur during the cold season. after an incubation period of one to three days, symptoms of tgev-associated disease emerge that include watery diarrhea, typical foul smelling yellowish-green feces that often contains flecks of undigested milk, vomiting, and loss of appetite. while the mortality rate is high in suckling piglets -up to % in piglets under two weeks of ageclinical symptoms in older piglets and adults are often mild and these animals will survive if especially their hydration status is adequate and ensured. the mortality rate is generally lower in these animals and will largely depend on additional factors such as secondary bacterial infections, cardiovascular decompensation, chilling and dampness. outbreaks usually only last a couple of weeks with occurrence of specific neutralizing antibodies in mucosa, blood and milk. further, lactating sows are highly susceptible. clinical signs may include vomiting, severe diarrhea, malnutrition, and cease of lactation. in large herds the disease can persist for some time, often contributing to post-weaning diarrhea. the clinical symptoms of endemic/enzootic tge are usually less severe in the older pigs, making a clinical differentiation between tge and other infectious enteric diseases, like that caused by rotaviruses and/or clostridia, impossible. upon histopathological examination, villous atrophy is frequently found in both rotaviral and enzootic tge infections. mixed infections are possible and underscore the importance of a strict disease management and/or prevention regimen. tge represents a reportable disease also in eu member states. there is no specific treatment available yet, however, electrolytes, nursing and enhanced management of the piglets may reduce mortality and lethality. in smaller herds cross-suckling of affected piglets onto recovered sows would offer a biological treatment. it is critical to almost impossible to assess general hygiene measures in prevalently infected herds. modified live vaccines are available and immunization of pregnant swine is a common vaccination strategy. cecov is associated with moderate to severe enteritis in young puppies. the genome contains the open reading frames (orfs) a and b, encoding polyproteins leading to the viral replicase formation. downstream of orf b are orfs encoding the coronavirus structural proteins s (orf ), e (orf ), m (orf ) and the nucleocapsid (n) protein [ ] . the e protein has a function for virus assembly [ ] , the m protein is a type iii glycoprotein [ ] . orf encodes the spike (s) protein, a glycoprotein ranging from , to , amino acids (aa) in length [ ] . this large protein has three structural domains. the large external domain at the n-terminus is furthermore organized into two sub-domains s and s with the s sub-domain including the n-terminal half of the molecule and forming the globular portion of the spikes. s contains sequences responsible for binding specific cellular receptors. s sequences are extremely variable, and mutations in the s region have been associated with problems of altered antigenicity and pathogenicity/virulence. in contrast, s sequences are genetically much more conserved and contain two heptad repeat motifs that suggest a coiledcoil structure [ ] . sequence analyses of cecov detected in fecal samples that were collected from dogs with diarrhea showed mutations accumulating over the m gene [ ] . a genetic drift to fcov type ii was also observed in the sequence of cecov detected in the faeces of puppies infected naturally during the late stages of long-term viral shedding. infection by mixed populations of genetically different cecov and recombination in vivo might, therefore, be common events [ ] . the clinical signs of these coronavirus infections vary. they commonly include vomiting, diarrhea and "unspecific" symptoms such as depression, anorexia, and fever. puppies most obviously die from severe dehydration. the majority of dogs that are not severely affected recover without any treatment. animals with severe symptoms of dehydration need supportive care stabilizing the hydration status. antibiotic/antibacterial treatment may be indicated in order to prevent exacerbations of bacterial superinfections. although vaccination may be indicated in kennels, sanitation is also the economically most effective way to control these coronavirus infections and, therefore, should be maintained by keeping the kennels free of feces and cleaning the environment by an appropriate desinfection regimen. fcov is commonly associated with mild enteric infections but is also associated with feline infectious peritonitis (fip). fip is a routinely fatal disease in both wild and domestic felidae. fcov can be distinguished into two serotypes: i and ii, on the basis of a virus neutralization assay in vitro using both type-specific feline sera and monoclonal antibodies directed against the s protein [ ] [ ] [ ] . the prevalence of these two serotypes is uncertain; type ii fcovs may account for up to thirty percent of the fip cases in cats in japan [ ] . the s protein of the type ii fcov shares immunodominant neutralization epitopes with the s protein of canine coronavirus [ ] . the s proteins of type ii fcov strains show great amino acid sequence identity to those of cecov (approximately %) and tgev (approximately %) but not to several type i strains (approximately %) [ , ] . in the first phase of the infection the symptoms are extremely unspecific. a mild upper respiratory disease, as evidenced by watery eyes and sneezing, might be diagnosed. a high percentage of primarily infected cats clear the virus; some of them, however, become long-term virus carriers. only a small percentage of exposed cats -higher, up to twenty percent in kennels -develop fip, months or years after primary infection. it is still unclear whether an endogenous reactivation could also be responsible for this pathomechanism. the clinical signs of fip usually gradually increase in severity over a period of several months, starting with rather unspecific signs such as inappetence, depression, rough fur, weight loss and fever. the forms of the lethal fip may be effusive (wet) and/or non-effusive (dry and/or proliferative). combinations of both clinical manifestations are rather common. the most characteristic clinical sign of wet fip is ascites. other symptoms may be rather unspecific like swollen lymph nodes, ocular symptoms with conjunctivitis and/or corneal ulcers. as the name suggests, fluid accumulation is minimal in the dry form of fip. instead, other, rather unspecific symptoms dominate. the dry form progresses slowly, often making clinical diagnosis difficult. weight loss, depression, anemia, and fever, are frequently observed symptoms. signs of severe kidney and/or liver failure, pancreatic, neurological or ocular disorders are observed in various combinations. a characteristic granulomatous inflammation is mostly observed by biopsy or in pathological examinations if performed. a cure does not exist yet. the therapy should provide supportive care and to alleviate the self-destroying inflammatory response of the disease. short-term remissions in a small percentage of patients have been described. a combination of corticosteroids, cytostatic drugs and antibiotics may be helpful in some cases despite the often fatal overall prognosis pro vitam. virulence of fcov strains appears to correlate to their ability to infect macrophages [ ] . the clinical symptoms are induced by immune complex reactions. antibodies are not only not protective, they might even accelerate the onset and the course of the disease in form of an antibody-enhanced infection (aei) such as observed in dengue fever in humans [ ] . the pathogenesis of the lesions, however, is not yet fully understood in its complexity. on the one hand there is evidence that immune complexes and subsequent activation of complement factors play an important role in the pathogenesis of fip [ ] . on the other hand, abnormal cytokine or chemokine secretion patterns -i.e. in infected immunocompetent cellscould also play a pathogenic role in the development of typical fip lesions like in granulomas [ ] . bovine coronavirus (bcov) is an important cause of neonatal calf diarrhea [ ] but may also infect the respiratory tract and has been recognized as the causing agent especially for winter dysentery in adult cattle. enteric and respiratory virus strains are antigenically related [ ] but differ genetically [ ] . amino acid alterations in the s subunit of the s protein (e.g. residues , , , , , , and ) of respiratory isolates conferred significant changes to the structure of the protein compared with the bcov strains that cause winter dysentery and calf diarrhea. bcov was first reported in [ ] [ ] [ ] . the zoonotic potential of bcov remains to be determined although a case of transmission to humans has been reported [ ] . bcov possesses a single-stranded, enveloped, nonsegmented rna genome of positive polarity [ ] . the mature virion con-tains five major structural proteins -the nucleocapsid (n) protein, the transmembrane (m) protein, the haemagglutinin/esterase (he) glycoprotein, the spike (s) protein, and a small membrane (e) protein [ , ] . he fulfills receptor binding and detachment functions. the s glycoprotein also recognizes the -o-acetylated sialic acid, apparently with a higher affinity than he [ , ] . the s protein is proposed to be responsible for the primary attachment of bcov to other cell surface receptors [ ] . variations in the s glycoprotein are most likely responsible for host specificity and tissue tropism [ ] . bcov is distributed worldwide and antibodies can be detected in the vast majority of cattle [ , ] . bcov infects calves/cattle by both the oral and/or respiratory route. although the virus can be detected in healthy animals, the most common source of enteric infection is diarrhoeic faeces from other infected animals. the virus infection of the enteritic tract starts in the small intestine and spreads after an initial replication throughout the gastrointestinal (gi) tract. since the virus replicates in the surface distal villi of the epithelial cells of the gi tract, these cells will eventually be destroyed, leading to fusions of adjacent villi in the small intestine and to atrophy of the colonic ridges [ ] . the severity of clinical signs varies with the age and especially the immunological status. usually, a yellowish diarrhea is observed that lasts for about three to seven days. it is difficult to distinguish between rota-and coronavirus-associated infection based on clinical signs solely. if diarrhea is severe, calves become pyrexic and dehydrated. infections with respiratory bcov often appear after stress such as shipment and/or environmental disturbances. infected animals will develop clinical signs of respiratory distress including wheezing and nasal discharge three to four days after infection. bacterial superinfections often complicate the clinical status. respiratory disease was induced experimentally after oral inoculation in colostrum-deprived calves [ ] . as for other coronaviruses, seasonal changes in temperature, environmental factors but also the immune status play an important role in the transmission of the virus and the clinical outcome of the infection. different virus isolates have been reported to have differences in tissue tropism [ ] . these authors report that about % of the infections in calves involve the respiratory tract in parallel and the enteric tract, whereas each % only involve either the respiratory or the gi tract. as for other coronaviruses, diagnosis/diagnostics requires detection of specific nucleic acids. virus isolation may be difficult and mostly not practicable in all day diagnostics. alternatively, nasal swabs might be used for detection of bcov antigen by immunofluorescence tests or other appropriate immunological methods. interestingly, a fragment amplified from "sars-cov" (bni fragment) showed % homology with bcov and mouse hepatitis virus (mhv) at the amino acid level [ ] . infectious bronchitis virus (ibv) is a major cause of disease in domestic fowl and causes an acute, highly contagious disease of the respiration and sometimes also urogenital tract [ ] . the ibv genome consists of approximately kb [ ] and codes for the spike (s) glycoprotein, the membrane (m) glycoprotein, and the nucleocapsid (n) phosphoprotein [ ] . ibv is distributed worldwide, and different variants have been isolated [ ] [ ] [ ] [ ] [ ] [ ] . ibv strains within a geographic region might be unique and distinct, examples are europe, the usa, and australia, i.e. for avian ibv [ ] . the different antigenic types make the use and possible efficacy of a single vaccine extremely questionable. the natural hosts for ibv are chicken and pheasants. ibv infections represent an important economic threat for the poultry industry. infected animals of all ages show signs of an acute, highly contagious respiratory disease. it is characterized by coughing, sneezing, and a nasal discharge. the major production loss results from the reduction in egg production and inferior egg quality. in younger birds there may be a high death rate, weight losses, or problems in weight development. some virus strains may also induce primary infectious kidney lesions. the lethality in these animals may be up to %. as for other coronavirus infections, only a virological examination, mainly based on serological techniques, can lead to the appropriate diagnosis. other important respiratory diseases include newcastle disease (nd) and infectious laryngotracheitis (ilt). according to regulations of many eu member states, nd is a disease that requires immediate reporting and action by veterinary authorities. ilt is also a reportable animal disease. there is no specific treatment for infectious bronchitis. antibiotic treatment might help to prevent or reduce secondary, i.e. bacterial superinfections. strict hygienic management and/or isolation of the flock may help to interrupt the disease cycle. different live virus vaccines have been developed and are currently in use; however, the use of live vaccines complicates especially serological diagnostics. sequencing of the s glycoprotein gene is the method recommended by the oie (office international des epizooties) to discriminate between different ibv strains. turkey coronavirus (tcov) causes acute and highly contagious enteritis of significant economical importance in turkeys [ ] . the clinical signs usually appear at seven to days of age in turkeys under six weeks of age and consist of diarrhea, litter eating, decreased feed efficiency and decreased weight development. morbidity is high, although mortality might be low. tcov is difficult to eradicate. tcov-induced enteritis has been described to be minnesota's most costly turkey disease from to [ ] . tcov treatments of the disease are often unsuccessful and there are currently no effective vaccines and/or other medications to prevent this disease. the local immune system of the mucosa of the gastrointestinal tract plays a major role in the protection against an infection and modulates clinical signs as well. recent studies indicate that neutralizing, intestinal secretory mucosal iga antibodies to tcov are elicited in turkeys following infection with tcov and that local mucosal antibodies may provide protective immunity for infected turkeys to recover from tcov infection [ ] . as for other coronaviruses, mechanical vectors play an important role in the transmission of the virus: it was demonstrated recently that house flies can transmit tcov [ ] . tcov from intestinal contents of diarrheal poults could be propagated in a human adenocarcinoma line and one-day-old turkey poults inoculated orally with tissue culture-adapted tcov isolates developed mild to severe diarrhea [ ] . however, the passaging of tcov could not be reproduced by other investigators so far [ , ] . this viral enteritis is different from haemorrhagic enteritis (he), another economically important disease of turkeys that is caused by a type ii adenovirus (reviewed by sharma [ ] ). in contrast to bluecomb disease, turkeys younger than four weeks of age are clinically "resistant" to he. in addition to the enteritis, a pathological frequent finding is hepatosplenomegalia. bursectomy and/or splenectomy abrogate clinical he [ ] . mhv belongs to the coronaviridae family and represents one of the most important pathogens of the laboratory mouse. mhv is serologically related to other coronaviruses of rats, pigs, cattle, and also humans. it is a very well-studied virus, because of its adverse influence on several research approaches and consecutively also results. about different mhv strains have been reported so far. some strains are polytropic; they infect a variety of tissues and cause symptoms in various organs. other strains are more specifically organotropic, e.g. enterotropic, and cause villus attenuation, syncytia formation and mucosal necrosis of the terminal small intestine and the colon. mhv is very contagious; transmission occurs by aerosol, faeces and many other contacts/transmission routes. there are usually no clinical symptoms in infected adult mice. clinical signs such as weakness, diarrhea, wasting and weight loss are observed in young mice. the mortality rate varies but might be high. since laboratory animals almost always come from controlled breeding providers/animal environment (e.g. spf = specifically pathogen free), serology is a highly reliable method to detect the target infection. considering the costs of modern biomedical research, infection with mhv is an economically important disease in small laboratory animals. the effects of the mhv infection on immunocompromised mice include enhanced phagocytic activities of macrophages, rejection of xenograft tumors [ ] , abnormal tumor growth patterns [ ] , altered response to chemical carcinogens [ ] , and impaired liver regeneration. pharmcokinetics of test compounds might also be 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hemorrhagic enteritis virus in turkeys influence of the mouse hepatitis virus (mhv) infection on the growth of human tumors in the athymic mouse influence of mouse hepatitis virus on the growth of human melanoma in the peritoneal cavity of the athymic mouse research complications and state of knowledge of rodent coronaviruses the role of circulating interferon in the modifications of the immune responsiveness to mouse hepatitis virus (mhv- ) natural patogens of laboratory mice, rats and rabbits and their effects on research peritoneal and macrophage alterations caused by naturally occurring mouse hepatitis virus virus-receptor interactions and interspecies transfer of a mouse hepatitis virus key: cord- - ta lhnw authors: decaro, nicola title: alphacoronavirus(‡): coronaviridae date: journal: the springer index of viruses doi: . / - - - - _ sha: doc_id: cord_uid: ta lhnw nan year of event event references transmissible gastroenteritis virus (tgev) associated with enteritis in swine doyle and hutchings ( ) coronaviruses associated with common colds in humans tyrrell and bynoe ( ) radiolabeling (tgev) clarifies fundamental coronavirus protein composition (s, n, m proteins) garwes and pocock ( ) ictv approves coronaviridae family with one genus, coronavirus tyrrell et al ( tyrrell et al ( ) demonstration that antibodies to feline enteric coronavirus enhance feline infectious peritonitis pedersen and boyle ( ) alternative model for transcription (tgev): discontinuous transcription during negative strand synthesis sethna et al ( sethna et al ( ) amino peptidase n receptor for tgev and hcov- e delmas et al ( ) ictv recognises coronaviridae as containing genera: coronavirus and torovirus cavanagh et al ( ) year of event event references ictv recognises the order nidovirales containing families coronaviridae and arteriviridae cavanagh et al ( cavanagh et al ( ) recombinant tgev shows that s protein determines enteropathogenicity and virulence sanchez et al ( ) coronaviridae: the viruses and their replication the coronaviridae. plenum, new york sturman and holmes key: cord- - ic in i authors: hu, xiaoliang; li, nannan; tian, zhige; yin, xin; qu, liandong; qu, juanjuan title: molecular characterization and phylogenetic analysis of transmissible gastroenteritis virus hx strain isolated from china date: - - journal: bmc vet res doi: . /s - - - sha: doc_id: cord_uid: ic in i background: porcine transmissible gastroenteritis virus (tgev) is the major etiological agent of viral enteritis and severe diarrhea in suckling piglets. in china, tgev has caused great economic losses, but its role in epidemic diarrhea is unclear. this study aims to reveal the etiological role of tgev in piglet diarrhea via molecular characterization and phylogenetic analysis. results: a tgev-hx strain was isolated from china, and its complete genome was amplified, cloned, and sequenced. sequence analysis indicated that it was conserved in the ′ and ′-non-translated regions, and there were no insertions or deletions in nonstructural genes, such as orf a, orf b, orf a, orf b, and orf , as well as in genes encoding structural proteins, such as the envelope (e), membrane (m), and nucleoprotein (n) proteins. furthermore, the phylogenetic analysis indicated that the tgev-hx strain was more similar to the tgev purdue cluster than to the miller cluster. conclusions: the present study described the isolation and genetic characterization of a tgev-hx strain. the detailed analysis of the genetic variation of tgevs in china provides essential information for further understanding the evolution of tgevs. transmissible gastroenteritis virus (tgev) is the etiological agent of transmissible gastroenteritis (tge), and it can cause viral enteritis and severe diarrhea with high morbidity in pigs of all ages, as well as high mortality in suckling piglets [ ] . it occurs at swine-raising farms and results in significant economic losses [ , ] . tgev is an enveloped virus belonging to the coronaviridae (cov) family and the nidovirales order. it possesses a large . -kb single-stranded, positive-sense rna genome. about two-thirds of the entire rna comprises open reading frames (orfs) a and b, encoding rna replicase. the ′ one-third of the genome comprises genes encoding structural and non-structural proteins [ , ] . the genes of tgev are arranged in the order of ′-rep-s- a- b-e-m-n-orf - ′. the spike (s) gene of tgev encodes an approximately , -amino acid protein, with a molecular weight ranging from - kda without glycosylation and - kda after glycosylation. functionally, the s glycoprotein is the major target of neutralizing antibodies, and it is also related to host cell tropism [ ] , interaction with its cellular receptor, pathogenicity, fusion, and hemagglutination activity [ ] [ ] [ ] . orf encodes a small hydrophobic protein (hp) during viral replication. the intracellular localization of the hp suggests that it may play an important role in the process of membrane integrity during viral replication and/or virion assembly [ , ] . in this report, we isolated a tgev-hx strain of tgev from the feces of piglets in heilongjiang province in china. to better understand the molecular characteristics of this isolate, its complete genome sequence was obtained, and a phylogenetic tree was constructed based on the complete orf sequence of the s gene. the results provide molecular and phylogenetic information for a chinese isolate of tgev, which may assist in elucidating the genetic evolution of tgev in china. pigs used in this study were approved by the institutional animal care and use committee (iacuc) of the harbin veterinary research institute (hvri), the chinese academy of agricultural sciences. no animals were sacrificed specifically for this study. feces samples were collected at the farm. five fecal samples were collected from piglets with diarrhea in a suburb of harbin, the capital of heilongjiang province, p. r. china. as both tgev and pedv can cause diarrhea, two pairs of specific primers (tgev-nf; tgev-nr; pedv-nf, pedv-nr) were employed to identify the kinds of viruses in the above samples (table ). pcrs were conducted as below, and the cycling parameters for the pcr included °c for min, followed by cycles of °c for . min, °c for . min, and °c for min, and a final extension at °c for min. then, pcrpositive viral samples were inoculated into pk- cells, which were grown as a monolayer in dulbecco's modified eagle medium (dmem) (gibco, grand island, ny, usa) containing % fetal calf serum (gibco, grand island, ny, usa) and % co in air. viruses were passaged three times and were harvested by three cycles of freezing and thawing. cellular debris was removed by low speed centrifugation at , × g (eppendorf, hamburg, germany) for min, and the supernatant was aliquoted and stored at − °c. viral titers were determined using the reed-muench method [ ] . extraction of viral rna, reverse transcribed-polymerase chain reaction (rt-pcr) and complete genome sequencing viral rna was extracted from pk- cells infected with tgev-hx using the trizol reagent (invitrogen, carlsbad, ca, usa). cdna was generated by adding μl of rna to the following components: μl of × reverse transcription buffer, μl of dntps mixture ( . mm), . μl of rnase inhibitor, μl of random primer ( μm), . μl ( u) of amv reverse transcriptase, and μl of sterile water. the components were gently mixed in an eppendorf tube and incubated at room temperature for min, then transferred to a water incubator at °c for h prior to storage at − °c . the resulting cdna was amplified by pcr using la taq dna polymerase (takara, tokyo, japan). ten pairs of primers were designed based on conserved regions of tgev strain h (table ). pcrs were conducted in a total of volume of μl containing μl of × buffer, μl of dntps mixture ( . mm), μl of cdna, μl of forward primer ( μm), μl of reverse primer ( μm), u of la taq polymerase (takara, tokyo, japan), and μl of sterile water. the cycling parameters for the pcr included °c for min, followed by cycles of °c for min, °c for min and °c for min, and a final extension at °c for min. two primers (p-f, p-r) were employed to confirm the ′ and ′ ends of the viral genome by rapid amplification of cdna ends (race) using the race cdna amplification kit (invitrogen, carlsbad, ca, usa). the pcr products were run on agarose gels, and correctly sized amplicons were observed. then, the pcr products were purified using the axygen gel extraction kit (axygen, usa) and cloned into pmd -t (takara, tokyo, japan). three to five independent clones of each tgev amplicon were sequenced. the dna was sequenced using an abi xl sanger-based genetic analyzer (applied biosystems, waltham, ma, usda). pk- cells infected with tgev were harvested by freezing and thawing three times. one ml of cell culture was centrifuged for min at × g. the supernatant was transferred into a new microfuge tube and centrifuged for min at , × g. then, the pellet was negatively stained with % phosphotungstic acid and analyzed on a transmission electron microscope (h- , hitachi, tokyo, japan) [ ] . sequence data were assembled and analyzed using clustal x software ( . ) and dnastar. to determine the relationship between the tgev representative isolates and the hx strain, phylogenetic trees based on the s gene were constructed using molecular evolutionary genetics analysis (mega) software (version . ) using the neighbor-joining (nj) method. bootstrap values were estimated for , replicates. the sequences of the tgev reference strains were obtained from genbank, and the details are summarized in table . the sequences obtained in this study were assembled and submitted to genbank under the accession number kc . the pcr results confirmed that one of five samples was tgev-positive, designated tgev-hx; the other four samples were tgev-negative and all five samples were pedv-negative ( figure a ). after three passages, cytopathic effects (cpe) were found in the pk- cells, as evidenced by the cells rounding up and enlarging, the formation of syncytia, and the detachment of cells into the medium (figure b and c) . the median tissue culture infective dose (tcid ) of tgev-hx ( . / . ml) was measured using the reed-muench method. when observed by electron microscopy, the virus displayed a circular shape, and the surface projections were petal-shaped, with a diameter ranging from to nm, which is similar in size to known tgevs ( figure d ). the full-length genome sequence of the tgev-hx strain was deduced by combining the sequences of overlapping cdna fragments. the genome sequence of the tgev-hx strain was , nucleotides (nt) long, including the poly a tail. the ′ portion of the genome contained a -nt non-translated region (ntr), and orf a ( - , ) and orf b ( , the replicase genes were composed of orf a and orf b, which contained a -nt common region (nt , - , ) and a "slippery site" ( ′-uuuaaac- ′, nt , - , ). the orf a gene of tgev-hx was predicted to encode a protein of , amino acids (aa), while orf b was predicted to encode a , -aa protein. nucleotide sequence analysis indicated that there were no deletions or insertions in the orf ab region of the miller and purdue tgev strains. orf a and orf b of tgev-hx were predicted to encode -aa and -aa proteins, respectively. no deletions or insertions were found in the orf a or orf b genes of tgev-hx. the orf gene of tgev-hx was predicted to encode a -aa protein, which contained the common pp c-binding motif ′-rviflvi- ′ [ ] . no deletions or insertions were found in orf of tgev-hx. the nucleotide sequence of the s gene of tgev-hx was , nt in length, encoding a predicted protein of , aa. a -nt deletion was found in the s gene at nt , - , of the tgev-hx, sc-y, and wh- strains, which caused the s protein to be two amino acids shorter than those of the purdue, miller , ts, h and h strains (figure a) . amino acid of the purdue, miller , and ts strains was s, while in the tgev-hx, sc-y, wh- , h , and h strains, it was a ( figure b) . amino acids , , , , , , , , , , and , of tgev-hx were identical to those of strains sc-y and wh- , but different from those of strains ts, h , and h . sequence analysis revealed no deletions or insertions in the e and n genes of any of the tgevs. the predicted e, m, and n proteins were -, -, and -aa long, respectively (table ) . to investigate the homology of tgev-hx to other tgevs, the nucleotide and predicted amino acid sequences of the nonstructural and structural protein coding genes were compared ( table ). the results suggested that tgev-hx showed higher identity to strains sc-y, wh- , and purdue, and less identity to ts, miller , and h . phylogenetic analysis of the complete s gene showed that tgev strains could be divided into two groups ( figure ) . the tgev-hx strain had a close relationship with the purdue strain and is more distant evolutionarily from the miller strains group and strain isu- . as an enteropathogenic coronavirus, tgev is the major cause of viral enteritis and diarrhea in neonatal pigs, resulting in significant economic losses. currently, tgev occurs sporadically in parts of europe, north america, and asia. the fact that wild and domestic carnivores (foxes, dogs, cats, and possibly minks) seroconvert to tgev indicates that they are potential subclinical carriers of tgev. [ ] . in summation, tgev has become a new challenge for the pig farming industry. as few tgev genome sequences have been published, little is known about tgev evolution. the results of this study will provide necessary information for further understanding the evolution of tgev. a complete sequence analysis indicated that no deletions or insertions were found in the ′-and ′-ntr regions of tgev-hx, suggesting that its replication and transcription mechanism was not changed. a -nt (nt , - , ) deletion in the s gene was found in the tgev-hx, sc-y, and wh- strains, but not in the purdue, miller , ts, h , and h strains. it was showed that this deletion was observed in the attenuated purdue strains pur -c and pur -mad, and it was considered to play a role in viral attenuation [ ] . competition studies using monoclonal antibodies led to the prediction of at least four main antigenic sites, designated a, b, c, and d [ , ] . the a and b sites (aa - ) have been mapped, and they serve as the major antigenic sites, including the binding site for the viruses host receptor, aminopeptidase n (apn). single amino acid changes in the s protein can greatly impact its antigenicity [ ] . the serine to alanine mutation at amino acid may have a significant influence in receptor binding or neutralizing antibody interactions [ ] . five chinese strains, tgev-hx, sc-y, wh- , h , and h , ha and alanine residue at this position, while the purdue, miller , and ts strains had a serine residue, which suggested that the antigenicity of the s protein of the five chinese strains may be changed. furthermore, amino acids , , , , , , , , , , and , of tgev-hx were identical to those of strains sc-y and wh- , but different from those of strains ts, h , and h . the nucleotide and amino acid sequence homology analysis of the structural proteins and non-structural proteins indicated that tgev-hx was highly similar to the wh- , sc-y, and purdue strains, and had a lower sequence similarity to the miller , ts, h , and h strains. the phylogenetic analysis showed that tgev-hx was closely related to the sc-y, wh- , and purdue strains, which belonged to the purdue cluster, while the miller , ts, h , and h strains belonged to the miller cluster, which was consistent with the results of the homology comparison. the data obtained in this study indicated that hx had different ancestors than the early chinese strain h , and it might be derived from the same ancestor as the sc-y, wh- , and purdue strains. the present study provides the complete genome sequence of a tgev-hx strain from china. by comparing the s gene and protein with those of other tgev strains, we have gained a further understanding of the genetic structure, diversity, and evolution of the tgev-hx strain. our next work is to evaluate the characteristics of mutations in the s gene using a reverse genetic approach in animal experiments. development of protection against coronavirus induced diseases development of protection against coronavirus induced diseases genetic analysis of porcine respiratory coronavirus, an attenuated variant of transmissible gastroenteritis virus virus taxonomy: classification and nomenclature of viruses sequence comparison of porcine respiratory coronavirus isolates reveals heterogeneity in the s, , and - genes point mutations in the s protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus two amino acid changes at the n-terminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism four major antigenic sites of the coronavirus transmissible gastroenteritis virus are located on the amino-terminal half of spike glycoprotein s targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence transmissible gastroenteritis coronavirus gene is not essential but influences in vivo virus replication and virulence the -kda hydrophobic protein encoded at the ′ end of the porcine transmissible gastroenteritis coronavirus genome is membrane-associated a simple method of estimating fifty percent endpoints genetic characterization of a new insect flavivirus isolated from culex pipiens mosquito in japan coronavirus gene counteracts host defenses and modulates virus virulence transmissible gastroenteritis virus and porcine respiratory coronavirus complete genome sequence of transmissible gastroenteritis coronavirus pur -mad clone and evolution of the purdue virus cluster major receptor-binding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein antigenic structure of transmissible gastroenteritis virus. i. properties of monoclonal antibodies directed against virion proteins submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution abbreviations tgev: transmissible gastroenteritis virus; cov: coronaviridae; orf: open reading frames; s: spike; hp: hydrophobic protein. the authors declare that they have no competing interests.authors' contributions hxl participated in the study design and collection of samples, conducted all laboratory and statistical analyses, participated in the interpretation of analyses, and drafted the manuscript. lnn, yx, and tzg participated in the study, provided oversight of the laboratory analysis, participated in interpretation of analyses, and helped to draft and critically revise the manuscript. qjj and qld participated in the study design and in the interpretation of analyses, and helped to draft and critically revise the manuscript. all authors have read and approved the final manuscript. key: cord- -v tff gk authors: nguyen, t.d.; bottreau, e.; aynaud, j.m. title: transmissible gastroenteritis (tge) of swine: in vitro virus attachment and effects of polyanions and polycations date: - - journal: vet microbiol doi: . / - ( ) - sha: doc_id: cord_uid: v tff gk four transmissible gastroenteritis virus (tgev) strains (purdue- , d- , -sg and gep-ii) and two cell lines (swine testis-st and pig kidney-rpd) were used to study virus attachment and cell susceptibility. virus attachment was partially thermodependent and the rate varied, depending on the strain. identical tgev inocula produced a higher plaque number by plaque assay in the swine testis cell line (st) than in the pig kidney cell line (rpd) but [( )h]uridine-labèlled virus was found associated equally well with both cell lines. a field tgev strain (gep-ii), which was unable to multiply in cell cultures, appeared able to inhibit the attachment of radiolabelled cell-passaged virus. therefore, the susceptibility to tgev infection was apparently not determined at the virus-to-cell attachment stage. the attachment sites on the cell surface were specific, however, differences in tgev attachment determinant between strains were not observed. attachment of all the virus strains tested was enhanced by deae-dextran and inhibited by dextran sulfate, poly-l-lysine (pll), poly-l-α-ornithine (plo) and protamine sulfate. attachment of virus to the host cell plasma membrane is the first step of the virus replication cycle and has been suggested to be one of the major determinants of cell susceptibility to virus infection. in most cases, this process occurs when viral protein (s) bind specifically to cell surface receptors (tardieu et al., ) . it was also demonstrated that the degree of virus virulence can be determined at this level ( mak et al., ; mims and white, ) . transmissible gastroenteritis virus (tgev) is a member of the coronaviridae. in the natural host, it infects intestinal epithelial cells, causing severe diarrhea in newborn piglets, but only mild diarrhea in adult swine (bohl, ) . the virus replicates in vitro in several porcine cell culture types (bohl, ) . however, viral isolation in cell cultures from field outbreaks is not always successful (dulac et al., ) . in contrast to virulent infection, peroral vaccination of sows, using attenuated virus, resulted in poor immunity ( saif and bohl, ; bohl, ) . in the latter cases, it was not clear whether or not virus attached to target cells. in this paper, we report the results of experiments on tgev attachment in vitro using four virus strains and two cell culture types. the effect of polycations and polyanions on virus attachment was also examined. the four tgev strains: purdue- , d- , -sg and gep-ii and two cell lines: swine testis (st) and pig kidney (rpd) have been described elsewhere ( laude et al., ; aynaud et al., ) . all but the gep-ii strain can multiply in these two cell lines. strain -sg is a tgev derived from d- by passages in cell cultures and stomach juice of adult swine alternatively (aynaud et al., ) . minimal essential medium (mem) containing iu ml- of penicillin and pg ml-x of streptomycin sulfate was used. for cell growth, the medium was supplemented with % fetal calf serum. virus stock was produced by infecting confluent monolayers of rpd cells in -cm plastic tissue-culture flasks (falcon) with the cell-passaged strains and by inoculation of non-immune newborn piglets for the gep-ii strain. a plaque assay (aynaud et al., ) was used with the following modifications. a volume of . ml of virus suspension containing - plaque forming units (p.f.u.), was inoculated onto the cell monolayers ( ~ × ~ cells per well) in six-well plastic plates (costar). virus attachment was allowed to occur at ° c. at the times indicated in fig. , the inoculum was removed from the cell sheets by washing twice with mem and replaced with mem containing % agarose (indubiose). #ci ml- of [ - h] uridine (amersham, sp. act. ci tool-l). the virus harvested was then partially purified by pelleting in an r- rotor centrifuge (beckman) at ×g for h at ° c. the pellet was resuspended in mem by vortexing and diluted to give a titer of ~ s p.f.u. ml- , corresponding to c.p.m, ml- . the attachment of labelled virus was carried out at ° c. confluent monolayer cells ( ~ × cells per well) grown in -well plastic plates (costar) were incubated with the radiolabelled virus suspension ( . ml per well) for min with gentle rocking. following three washings with mem to remove the inoculum, the cells were solubilized with n naoh then neutralized with . n hc . the whole content of a well was transferred to a scintillation vial. the radioactivity was measured using acs scintillation liquid (amersham) in an lkb liquid scintillation counter. the unlabelled tge virus (strains purdue- , d- , -sg and gep-ii) was first inoculated onto the cell monolayers, grown in -well plastic plates, following incubation for h at °c, the virus was removed by extensively washing the cells with cold mem. the radiolabelled virus was then inoculated and further steps in the experiment were performed as described above. results are expressed as a/ao x , a being the radioactivity associated with the cells which were first inoculated with different amounts of unlabelled virus and ao the radioactivity bound to the control cells. the following products were used. polycations: deae-dextran (pharmacia, mol. wt. -- ), protamine sulfate ( sigma, salmonine, grade x), poly-l-lysine (sigma, mol. wt.= ), poly-l-~-ornithine (sigma, mol. wt. -- ). polyanions: heparin (choay-france), iu ml- and dextran sulfate ( sigma, mol. wt. = ) and an amorphous product: dextran t (pharmacia, mol. wt.= ). these were prepared immediately before use by dilution in mem. the minimum toxic concentration for the cells had been determined previously. the effect of these chemicals on virus attach- ment was tested by using the plaque assay as described above, except that the inoculum containing - p.f.u. was prepared in the presence of the chemical at appropriate concentrations and incubated with confluent monolayer cells for min at ° c. the inoculum was then removed by extensive washing and replaced with mem containing % agarose. the chemicals were omitted from control cultures. to test whether the chemicals had an effect on viral replication step (s) following attachment, after incubation with virus alone and removal of the inoculum, the cells were incubated with the chemicals for min at ° c. the cells were then washed free of the chemicals and mem containing % agarose was added. plaques were formed by infectious virus particles which were attached to the cells and not removed by washing. the percentage of infectious virus particles in the inoculum which attached to the cells, as a function of incubation time, is indicated in fig. . it can be seen that the attachment rate of tgev strain purdue- to the cells was much greater than that of other strains (d- and -sg). after h of incubation with the cells, most of the infectious virus particles ( > % ) of the purdue-ll strain were found attached to the cells, whereas attachment was only % in the case of the d- and -sg strains. results of attachment of [ h ] uridine-labelled virus to the st and rpd cells showed that for any strain tested, the radioactivity was found equally associated with both cell types (table i) . differences in susceptibility to tgev infection, indicated by plaque formation, between the st and rpd cells and effects of temperature on virus attachment were studied by incubating virus inoculum with the cells for min at either or °c. it was found (table ii) that at °c, the infectious virus particles attached to the cells better than at °c (p < . ). the plaque numbers observed also showed that the st cells were significantly (p < . ) more susceptible than the rpd cells. this difference was also observed in infected cells to which agar-overlay medium was added without removal of the inoculum (table iii) . the attachment of the labelled virus was reduced by pre-incubation of the cells with unlabelled virus. the reduction was inversely proportional to the m.o.i. (for purdue- , d- and -sg strains) and dilutions (for the gep-ii strain ) of the unlablled virus previously inoculated. figure shows a typical experiment using the labelled virus strain purdue-ll and the st cells. experiments using the rpd cells and other labelled virus strains (d- and -sg ) gave similar results (not shown). these. results suggested a specificity of tgev attachment sites on the cell surface. moreover, the strain gep-ii, which does not multiply in either st or rpd cells, was found to be able to attach to the cells and to inhibit the subsequent attachment of labelled virus. deae-dextran enhanced virus attachment (fig. ) , when tgev partially purified by pelleting was used. the curves reach a plateau beginning at a concentration of /zg ml- of deae-dextran. an inhibitory effect on virus attachment was obtained when poly-l-lysine, protamine sulfate and dextran sulfate were used (figs. , and ) . the effect of poly-l-~x-ornithine was similar to that of poly-l-lysine (data not shown). similar plaque numbers were observed in the control and in the pre-infected cells which were incubated with polyanions or polycations (for min at °c ) after the removal of inoculum. no effect of heparin and dextran was observed. the effect of all chemicals was consistent for all tgev strains tested. our determination of an incubation period ( min) that preceded maximal virus attachment allowed the effects of temperature, polyanions and polycations on virus attachment to be studied. the results shown in table i suggest that the attachment of tgev is partially thermodependent. differences in attachment rate between strains were also observed. the tgev strain purdue-ll attached to the cells much faster than the others. although both the tgev strains purdue- and -sg were established as high passage strains ( and passages, respectively), the attachment rates of these strains were different (fig. ) . the attachment rates of d- and -sg were found to be similar, however. differences betweeen these two strains have been described (aynaud et al., ) , -sg having higher resistance to acidity and digestive enzymes and smaller plaques in st cells. it is evident that these two characteristics are not related to attachment rate. no explanation is available for the difference in virus attachment rate between strains. interference with infectious virus attachment by varying numbers of defective particles which are present in the different virus inocula is unlikely. although the infectious virus titre of the -sg strain is lower (by log, aynaud et al., ) , the intensity of virus-antibody reaction in elisa is similar for the three virus strains (unpublished data), suggesting a -fold greater number of defective particles for the -sg strain compared to the others. the incubation of ~ - p.f.u. with x cells in a well ( for plaque formation ) would have rendered such an interference insignificant. all three virus strains produced a higher number of plaques in st cells than in rpd cells (table ii) , but the radioactively labelled virus was found to be similarly associated to both cell types. this strongly suggests that the difference in plaque number observed between the two cell lines is not determined at the level of virus attachment. also, the field tgev strain gep-ii, which was unable to multiply in cell cultures even in the presence of deae-dextran, was able to attach to the cells, thereby inhibiting the subsequent attachment of labelled virus. a higher synthesis of viral rna was observed for the three virus strains in st cells than in rpd cells (nguyen, ) . a defective multiplication of tgev may be an explanation for the low susceptibility of pig kidney cell lines which has been described by garwes et al. ( ) . according to these authors, the multiplication of tgev in llc-pk cells is stopped at the maturation step. their observations and our present findings tend to reinforce the statement that genetic susceptibility or resistance to coronavirus is not determined at the level of virus receptors ( shif and bang, ) . the results shown in fig. , on the other hand, demonstrate a specificity of the attachment sites of tgev on the cell surface. it is interesting to note that the difference in attachment site between virus strains which has been dem-onstrated for other viruses, e.g., reovirus (weiner et al., ) , was not observed in the case of tgev. moreover, until now only one antigenic determinant of tgev has been reported (bohl, ) and the present study suggests that this also applies to the attachment determinant. studies of foot and mouth disease virus showed that attachment and antigenicity determinants are different (meloen et al., ) . it is likely that tgev possesses the same characteristic as neutralizing polyclonal antibodies from infected swine were unable to inhibit virus attachment . since the absolute ratio of tgev particles to p.f.u. has not been established, we could not determine the number of virus attachment sites per cell. at an m.o.i, of ~ - p.f.u. per cell, the unlabelled virus strain purdue- inhibited attachment of nearly % of the same radiolabelled virus strain at a m.o.i, of ~ p.f.u. per cell. in agreement with sturman and holmes ( ) , who demonstrated that the coronavirus receptors/cell ratio was low ( ~ ), our results suggest that the ratio of tgev attachment sites per cell could not be high, in contrast to many other viruses, for which the host cell possesses as many as × attachment sites (armstrong et al., ) . effects of polyanions and polycations on the virus cycle as well as on cellular uptake have been studied. these substances are believed to change the electric charges of ligands or cell receptors by combining with them and in that way enhancing or inhibiting the macromolecule (virus)-cell interactions (ryser, ; toyoshima and vogt, ) . the infectivity of other coronaviruses has been reported to be enhanced by deae-dextran (bradburne and tyrrell, ; takayama and kirm, ; hirano et al., ; sato et al., ) . our results on tgev have identified another coronavirus member with this characteristic. the effects of deae-dextran (polycation, enhancer), dextran sulfate (polyanion, inhibitory) and dextran ( amorphous, no effect) are compatible with the explanation for the mechanism of action by electric charges of these molecules, but if the activity of polycations and polyanions relies only on their electric charges, contradictions can be seen in our results. among polycations used, deae-dextran had an enhancing effect whereas others (pll, plo, protamine sulfate) were inhibitory. such contradictions might be explained by the size of molecules used, since the effect of these molecules on cell uptake depends upon size as well as charge (ryser, ) . differences between strains in the level of enhancement or inhibition of virus infectivity by polyanions and polycations (figs. , and ) might be explained by differences in their rate of attachment to the cells. the most important observation was the one-way effect of these molecules on virus attachment with all the strains studied. this again leads to the conclusion that there is no difference in the nature of attachment sites between tgev strains. studies on reovirus receptor of l cells and comparison with reovirus receptor of erythrocytes transmissible gastroenteritis (tge) of swine: survivor selection of tge virus mutants in stomach juice of adult pigs transmissible gastroenteritis the propagation of coronavirus in tissue cultures isolement du virus de la gastroenterite transmissible du porc sur cultures cellulaires et comparaison antig~nique avec deux souches americaines defective replication of porcine tgev in a continuous cell line effect of diethyl aminoethane dextran on plaque formation of mouse hepatitis virus in vitro properties of low and high passaged strains of transmissible gastroenteritis coronavirus of swine studies of the early events of the replication cycle of three variants of mengo encephalomyelitis virus in mouse fibroblast cells the main antigenic determinant detected by neutralizing monoclonal antibodies on the intact foot and mouth disease virus particle is absent from isolated vp viral pathogenesis and immunology.v. determinant of viral virulence and host resistance caractgrisation in vitro du coronavirus de la gastroent~rite transmissible (get) et immunog~nicit~ d'un mutant att~nu~ ( -sg) neutralizing iga and igg do not inhibit attachment of transmissible gastroenteritis (tge) virus a membrane effect of basic polymers dependent on molecular size passive immunity in tge of swine: ig classes of milk antibodies after oral-intranasal inoculation of sows with a live low cell culture-passaged virus inducement of cytopathic changes and plaque formation by porcine encephalomyelitis virus in vitro interaction of mouse hepatitis virus and macrophages from genetically resistant mice i. adsorption of virus and growth curves molecular biology of coronaviruses improved method for titration of mouse hepatitis virus type in a mouse cell culture interaction of viruses with cell receptors enhancement and inhibition of avian sarcoma viruses by polyanions and polycations interaction of reovirus with cell surface receptor. i. murine and human lymphocytes have a receptor for haemagglutinin of reovirus type key: cord- - reu yz authors: reguera, juan; santiago, césar; mudgal, gaurav; ordoño, desiderio; enjuanes, luis; casasnovas, josé m. title: structural bases of coronavirus attachment to host aminopeptidase n and its inhibition by neutralizing antibodies date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: reu yz the coronaviruses (covs) are enveloped viruses of animals and humans associated mostly with enteric and respiratory diseases, such as the severe acute respiratory syndrome and – % of all common colds. a subset of covs uses the cell surface aminopeptidase n (apn), a membrane-bound metalloprotease, as a cell entry receptor. in these viruses, the envelope spike glycoprotein (s) mediates the attachment of the virus particles to apn and subsequent cell entry, which can be blocked by neutralizing antibodies. here we describe the crystal structures of the receptor-binding domains (rbds) of two closely related cov strains, transmissible gastroenteritis virus (tgev) and porcine respiratory cov (prcv), in complex with their receptor, porcine apn (papn), or with a neutralizing antibody. the data provide detailed information on the architecture of the dimeric papn ectodomain and its interaction with the cov s. we show that a protruding receptor-binding edge in the s determines virus-binding specificity for recessed glycan-containing surfaces in the membrane-distal region of the papn ectodomain. comparison of the rbds of tgev and prcv to those of other related covs, suggests that the conformation of the s receptor-binding region determines cell entry receptor specificity. moreover, the receptor-binding edge is a major antigenic determinant in the tgev envelope s that is targeted by neutralizing antibodies. our results provide a compelling view on cov cell entry and immune neutralization, and may aid the design of antivirals or cov vaccines. apn is also considered a target for cancer therapy and its structure, reported here, could facilitate the development of anti-cancer drugs. the coronaviridae is a large family of enveloped, plus-rna viruses. they are involved in respiratory, enteric, hepatic and neuronal infectious diseases in animals and humans that lead to important economic losses [ , ] , as well as to high mortality rates in severe acute respiratory syndrome cov (sars-cov) infections [ ] . the covs are a numerous group of coronaviridae. they have been clustered in the coronavirinae subfamily, which includes three approved genera, alpha-, betaand gammacoronavirus, as well as a tentative new genus, the deltacoronavirus [ ] . representative cov species in each genus are alphacoronavirus (comprising transmissible gastroenteritis virus (tgev), porcine respiratory cov (prcv) and related canine and feline covs), human coronavirus (hcov- e and hcov-nl , genus alphacoronavirus), murine coronavirus (including mouse hepatitis virus (mhv), genus betacoronavirus, cluster a), severe acute respiratory syndrome-related coronavirus (sars-related cov, genus betacoronavirus, cluster b), avian coronavirus (including infectious bronchitis virus (ibv), genus gammacoronavirus), and bulbul-cov (tentative genus deltacoronavirus) [ ] . cov particles display characteristic large surface projections or peplomers ( - nm) comprised of homotrimers of the spike glycoprotein (s), a type i membrane protein [ , ] . the peplomers have a globular portion connected by a protein stalk to the transmembrane domain [ ] . the globular region is formed by the n-terminal s region, whereas the stalk corresponds to the membrane-proximal s region, which mediates virus fusion to host cells and adopts a helical structure characteristic of class i virus fusion proteins [ ] . determinants of cov tropism locate at the s region [ , ] , which mediates attachment of cov particles to cell surface molecules, initiating virus entry into cells and infection. there is considerable variability in receptor usage among the covs. most alphacoronavirus such as tgev and hcov- e use apn [ , ] , whereas the related hcov-nl uses a distinct cell entry receptor, the human angiotensin converting enzyme (ace ) [ ] ; sars-cov also recognizes the ace receptor [ ] . sars and nl cov bind to common regions of the ace protein, although the structures of their receptor-binding domains (rbds) are quite distinct [ , ] . mhv uses the cell adhesion molecule ceacam a [ ] ; a recent crystal structure showed that the mhv rbd adopts a galectin-like fold [ ] . the use of alternative receptors that confer extended tropism has been described for sars-cov, mhv and tgev [ , ] . the mammalian apns (cd ) are type ii cell surface metalloproteases whose large glycosylated ectodomain has a zinc metal ion at the active site [ ] . apn is linked to many cell functions, leading it to be termed the ''moonlighting enzyme'' [ ] . animal models confirmed a role for this cell surface enzyme in angiogenesis [ ] . peptides and inhibitors that target apn showed a link between this protein and tumor growth and invasion [ , ] . apn is a target for cancer chemotherapies; drugs that bind this protein have been developed to treat tumors, some of which are in clinical trials [ ] . as mentioned above, apn is also a major cov cell entry receptor [ , , ] . cov recognition of apn is species-specific, and specificity is associated with n-linked glycosylations in the apn protein [ ] . cell tropism and immune neutralization have been extensively studied in some porcine alphacoronavirus, such as the enteropathogenic tgev and porcine respiratory cov (prcv), a nonenteropathogenic virus derived from tgev [ ] . both viruses use porcine apn (papn) for cell entry. the apn-binding domain in tgev, prcv and other alphacoronavirus locates at the c-terminal portion of the s region [ , , ] , which bears epitopes recognized by cov-neutralizing antibodies [ , , , ] . most tgev-neutralizing antibodies cluster at antigenic site a [ , ] , comprised within the rbd at the s region ( figure a ) [ ] ; the other antigenic sites defined in the tgev s region (b through d) are outside the rbd ( figure a ) [ ] . to date, there is no structural information available on antibody neutralization and apn recognition by alphacoronavirus. we determined crystal structures of the prcv rbd in complex with the papn ectodomain, and the tgev rbd in complex with the neutralizing monoclonal antibody (mab) af [ ] . the rbd adopts a b-barrel fold, with a distinct protruding tip engaged in papn recognition. the structures show how these porcine alphacoronavirus recognize its cell entry papn receptor and how immune neutralization of these covs is achieved by antibody targeting of receptorbinding residues in the s protein. the mechanisms used by tgev to escape immune neutralization and the evolution of receptor recognition in the cov family are discussed. apn-binding domain and epitopes for neutralizing mabs overlap in tgev and prcv s proteins apn receptor recognition and envelope s antigenicity are well documented in tgev and related prcv. the papn-binding figure . apn-binding domain and epitopes for neutralizing mabs overlap in tgev and prcv s proteins. a. scheme of tgev and prcv s proteins showing the s , s , transmembrane (t) and cytoplasmic (cy) regions. location of the c, b, d and a antigenic sites [ ] , and the papn rbd (bar with n and c-terminal residues) [ ] are shown. length is indicated for mature s regions. b. a short, soluble s protein variant containing the tgev rbd region binds to cell surface papn. binding of a bivalent sa-fc fusion protein (sa) to bhk-papn (open histograms), alone (left) or in the presence of the site a-specific ac mab (right), as analyzed in facs. filled histograms correspond to an unrelated fc fusion protein. c. binding of site a-specific tgevneutralizing mab to the sa protein. mab binding to plastic-bound sa protein, monitored by optical density (od). site a mabs are specific for the aa ( bb ), ab ( de ) and ac ( ac , af ) subsites [ ] . an anti-ha mab that binds to the ha tag in the sa protein was used as control. d. site a-specific mabs prevent sa protein binding to papn. binding of the sa protein to bhk-papn cells in panel b was monitored alone or in the presence of site a (shown in c) or site d-specific ( dg ) mab, and the binding ratio determined (see materials and methods). c, d. mean and standard deviation for three experiments. doi: . /journal.ppat. .g the cell surface aminopeptidase n (apn), a membranebound metalloprotease target for cancer therapy, is a major cell entry receptor for coronaviruses (covs), agents that cause important respiratory and enteric diseases. in some covs, the virus envelope spike glycoprotein (s) mediates attachment of the virus particles to the host apn protein and cell entry, which is blocked by antibodies that prevent cov infections. the crystal structures of the s proteins of two porcine cov in complex with the pig apn (papn) or with a neutralizing antibody shown here, reveal how some cov bind to its cell surface apn receptor and how antibodies prevent receptor binding and infection. the report uncovers a unique virus-receptor recognition mode that engages a glycan n-linked to the papn ectodomain, revealing structural determinants of the receptor-binding specificity in covs. neutralizing antibodies target viral residues used for binding to the apn receptor and entry into host cells, showing that efficient cov neutralization requires immune responses focused toward key receptor binding motifs in the virus envelope. these structural insights, together with the structure of the apn ectodomain, provide a compelling view of relevant cell membrane processes related to infectious diseases and cancer. domain was mapped within residues to of the mature tgev s polypeptide [ ] , whereas tgev mab-resistant (mar) mutants defined four antigenic sites (c, b, d and a) [ , ] ( figure a) . antigenic sites c and b are not present in the prcv s protein. antigenic site a determinants are located within the papn-binding domain at the c-terminal moiety of the tgev and prcv s regions ( figure a ) [ , ] . we recently reported the modular dissection of the n-terminal s region of tgev and prcv, and the preparation of soluble s length variants with single antigenic sites [ ] . we produced a recombinant short s protein fragment termed sa, which comprises only residues to of the tgev s protein that binds cell surface papn ( figure b ) and displays conformational epitopes for the three antigenic a subsites (aa, ab, and ac) ( figure c ). antibodies clustered at the aa ( bb ), ab ( de ) and ac ( af and ac ) subsites blocked binding of the soluble sa protein to papn ( figure d ). the sa protein therefore includes the papn-binding domain of tgev and epitopes for site aneutralizing mab. we applied x-ray crystallography to s protein variants containing the rbd of the related tgev and prcv, and have identified how these alphacoronavirus bind to the cell surface papn and its inhibition by neutralizing antibodies. we attempted crystallization of the soluble papn-binding sa protein derived from the tgev s, alone and in complex with several neutralizing mabs. crystals were prepared with the sa protein in complex with the fab fragment of the af mab [ ] ; the structure of the complex was determined and refined using diffraction data extending to . Å resolution (materials and methods; table ). the asymmetric unit of the crystals contains two antibody-rbd complexes, one of which is shown in figure . residues pro to val of the tgev s protein, previously identified as the papn-binding domain ( figure a ) [ ] , were well defined in the crystal structure. they folded in a single domain structure, the rbd of tgev (figure a) . the rbd adopts a bbarrel fold formed by two b-sheets with five b-strands each (scheme in figure s a ). n-and c-terminal ends are on the same side of the domain (terminal side), which presumably lies close to other s protein domains; at the opposite side, two b-turns (b -b and b -b ) form the tip of the barrel (figure a) , where the mab binds to the rbd. the immunoglobulin (ig) variable domains of the mab heavy (v h ) and light (v l ) chains contact the b -b , b -b and b -b regions of the tgev rbd ( figure b ), burying a virus protein surface of , Å . the buried surface of the af mab is , Å , with equal contribution by the v h ( %) and v l ( %) ig domains. complementarity determining regions (cdr) of the antibody heavy (h ) and light (l and l ) chains, the n-terminus of the light chain and the c, c and c b-strands of the v h domain contact the viral rbd tip ( figure b ). the cdr-h of the af mab is relatively long, with two-residue insertion (tyr h and asp h ) relative to other homologous h loops in reported mab structures (table s ). the rbd b -b hairpin with tyr at its tip is at the center of the interacting surface and penetrates between the v l and v h ig domains of the af mab ( figure b and c). similar antibodyantigen recognition is described for some peptides and is common for small hapten molecules [ , ] . the rbd b -b region contributed % of the rbd surface buried by the af mab, and docked between the af mab variable domains ( figure b ). the b-turn is fully buried between the mab ig domains ( figure c ), forming a contact network with mab residues ( figure d ). the rbd residue tyr at the bottom of the pocket contacts mab residues trp h and tyr h , whereas its hydroxyl group is hydrogen bonded to the side chain of gln l and main chain carbonyl of tyr h ( figure c and d). these structural findings on af recognition of the rbd b -b region correlate with af mab binding to peptides (mkrsgygqpia ) that include this hairpin region [ ] . the rbd b -b and b -b regions are at the periphery of the epitope ( figure b ); their contribution to interaction with af is smaller than that of the b -b region, representing respectively , % and % of the rbd surface buried by the mab. they contact either the v l or v h ig domains ( figure b ). rbd residues leu and trp at the b -b loop contact the n-terminus, cdr-l and cdr-l of the v l domain, whereas the b -b loop contacts the long cdr-h loop ( figure b and c). to characterize cov attachment to its apn receptor, we attempted crystallization of the papn ectodomain in complex with tgev and prcv s protein variants comprising their rbds (materials and methods). crystals were obtained only with a mixture of a prcv s protein (s h) and the papn. using these crystals, we determined the structure of the prcv rbd-papn complex by molecular replacement using previously solved structures of the tgev rbd shown in figure ( % sequence identity) and of the papn ectodomain (materials and methods and table ). the asymmetric unit of the crystals contained two macromolecular rbd-papn complexes ( figure a ). the prcv rbd adopts a b-barrel fold like the tgev rbd ( figure s ). each papn molecule was engaged by the tip of a single prcv rbd molecule, which bears two exposed aromatic residues (tyr and trp) ( figure a , in red), and they bound to a membrane-distal region of the papn ectodomain ( figure a ). the rbd n-and cterminal ends and the remaining cov s are also distant from the papn, and are unlikely to contact the receptor molecule. based on a cryo-em structure of the sars-cov s [ ] , the rbd must be also at the viral-membrane distal side of the s and therefore, the receptor binding edge must be accessible for cov binding to the apn receptor. the papn is a type ii membrane protein and the n-terminal end of the ectodomain must be near the cell membrane ( figure a ). the n-terminal residues of the crystallized papn ectodomain are largely disordered in the structure and they might form a flexible region close to the cell membrane. the papn ectodomain is composed of four domains ( figure a ). domain i (orange) is made of b-strands, domain ii (yellow) adopts a thermolysin-like fold bearing a zinc ion at the catalytic site, domain iii (red) is a small b-barrel domain, and the c-terminal domain iv (green) is composed of alpha-helices (domain boundaries are shown in figure s ). the papn molecule structure is closely related to that of the human endoplasmic reticulum aminopeptidase- [ , ] (root-mean-square deviation of . Å for residues sharing % sequence identity, based on dali server). domain ii bearing the enzyme active site is the most related domain ( % identity), whereas domain iv is the most distinct ( % identity). the zinc ion is coordinated to conserved residues at the papn active site in domain ii ( figure s ). the active site conformation is similar to that of other aminopeptidases ( figure s ). the papn crystallized in complex with the prcv rbd had an open conformation [ , , ] , in which domain iv was , - Å from domains i and ii; this creates a central cavity in which the zinc ion at the catalytic site is highly accessible ( figure a ). the mammalian apns are cell surface metalloproteases that form membrane-bound dimers [ ] . the crystallized papn ectodomain also behaved as a dimer in solution ( figure s ). the papn dimeric assembly showed in figure a buried a large accessible surface (, Å ) in each monomer. the dimerization surface comprises residues spread across domain iv, which are distinct from those recognized by cov ( figure s ). similar dimeric assemblies were observed in two crystal structures determined for the papn ectodomain alone (not shown), crystallized using distinct conditions. the papn molecular assembly shown here might thus be representative of the dimer described for mammalian apn on membrane surfaces [ ] . in the crystals of the prcv rbd-papn complex, the rbd tip contacts a membrane-distal region of the papn ectodomain ( figure a ). the conformations of the receptor-binding loops (b -b and b -b ) at the tips of the two prcv b-barrel domains in the structure are identical ( figure s b ), suggesting very similar rbd-papn interactions in both complexes of the asymmetric unit. the virus-receptor interaction buried , Å of the virus protein, % of which corresponded to the b -b region ( figure b ) and % to the b -b turn ( figure c ). the size of the papn surface buried by the rbd was similar (, Å ), and included papn residues ranging from alpha helix (a ) to (a ) in domain iv, and a few domain ii residues ( figure s , table s ). the end of the papn helix a and helix a contacted the b -b region of the rbd ( figure b ). the tyr side chain (tyr in tgev), which protrudes at the b-turn in prcv and tgev rbds ( figure b and d) , is almost fully buried in the complex, locating between the first n-acetyl glucosamine (nag ) linked to papn asn , the end of helix a , and the first half of helix a ( figure b ). the hydroxyl group of the rbd tyr was hydrogen bonded to side chains of papn residues glu and trp , and contributed to virus-receptor binding specificity. the preceding rbd gly residue was at the papn proximal side of the b-turn, hydrogen bonded to the papn asn main chain; at the opposite side, the rbd gln side chain formed a network of hydrogen bond interactions with papn nag and asn side chain ( figure b ). the n-acetyl moiety of the glycan also interacted with rbd residues at the b and b strands ( figure b , table s ). the papn n-linked glycan and surrounding residues that contact the cov rbd b -b region in the structure were identified as one of the apn determinants of the cov host range [ ] . the second relevant virus-receptor interacting region engaged a b-turn at the beginning of the rbd b -b loop ( figure c and d). the unique rbd trp residue, which protrudes at the turn, docked in a papn cavity formed by the coils that precede helices a in domain iv and a in domain ii ( figure c and s ). the bulky side chain of the rbd trp residue packed against papn residues his and pro , and its imino group was hydrogen bonded to the main chain carbonyl of asn ( figure c ). the rbd trp as well as the rbd tyr at the b-barrel tip in tgev and prcv appear to be central residues in the virus-receptor interaction, as they contact with many papn residues and contribute also to binding specificity by mediating polar interactions with the papn (table s ) . to confirm the contribution of the prcv or tgev rbd bbarrel tip in papn receptor recognition, we analyzed binding of wild type and mutant tgev rbd proteins to cell surfaceexpressed papn ( figure a ). mutations in the three regions (b -b , b -b and b -b ) that build the receptor binding edge of the b-barrel decreased rbd binding to papn, whereas mutations outside the receptor-binding region (v ngly) had no effect on receptor recognition. deletion of the papn asn glycosylation site also abolished tgev rbd binding to cell surface-expressed papn ( figure b ). deletion of the homologous glycan in feline apn similarly prevents cell infection by feline, canine and porcine covs, all of which share the glycan-binding tyr residue in the b -b turn (see below), whereas addition of this glycan to human apn is sufficient to render it a tgev receptor [ ] . we determined the crystal structures of the related tgev and prcv rbds bound to two distinct ligands. the rbds adopt bbarrel structures with small differences in the ligand binding loops (figures s ). in the rbd, each of the two highly twisted b-sheets that build the b-barrel is formed by five b-strands ( figure a ). the bent b-strand (b ) crosses both b-sheets and has a b-bulge at asn ( figure a , magenta). at one side of the b-barrel, all bstrands are antiparallel ( figure a, cyan) , whereas on the opposite a dali search of structural homologs showed the greatest similarity (z score of ) with the rbd of the ace receptorbinding hcov-nl (root-mean-square deviation of . Å for residues), the other alphacoronavirus rbd whose structure is known [ ] . the cores of the tgev and hcov-nl b-barrel domains are structurally similar, but the loops at the tips ( figure b and d). the tip region of the hcov-nl rbd is the ace receptor-binding edge and has a ''bowl''-shaped conformation ( figure c ) that differs from the tgev rbd protruding edge. aromatic residues protrude from the b-turns at the tip of the bbarrel in tgev, whereas they are partially buried at the center of the ''bowl''-shaped edge in hcov-nl ( figure b and c). the distinct rbd tip conformation in ace -binding hcov-nl and in apn-binding tgev might be a determinant of their distinct cell entry receptor specificities. the degree of sequence identity in the rbd region among members in the species alphacoronavirus (, % identity) suggests a structure closely related to that of tgev, including conforma-tion of the receptor-binding loops (b -b and b -b ) at the bbarrel tip ( figure ). therefore, tgev, prcv, ccov and fcov must recognize the apn receptor in similar fashion. in contrast, the receptor-binding loops at the tip appear to have a different conformation from tgev in the hcov- e rbd, which also binds to the apn. in this cov, the b -b region has two cys, as in hcov-nl , and lacks the apn-binding tyr residue in alphacoronavirus , although it preserves the two gly residues found in the tgev b-turn ( figure ). the b -b loop in hcov- e is markedly shorter than in tgev, but it also has a trp residue. sequence identities between the rbd of tgev and ibv (gammacoronavirus) or the bulbul-cov (tentative deltacoronavirus) are relatively large (, %), and similarities are found mostly in bstrands and at the rbd c-terminal half ( figure ) . these data indicate a conserved rbd fold between alphacoronavirus and gammaor deltacoronavirus. there is less sequence similarity between the alphaand betacoronavirus rbd regions (, %), which correlates with notable structural differences between their detail of the rbd b -b region with the exposed tyr residue interacting with the papn. side chains of rbd and papn residues engaged in the interaction are shown as sticks with carbons in magenta or green, respectively. nag glycan n-linked to papn asn is shown with carbons in yellow and the electron density map, determined without the glycan, shown as a blue mesh contoured at sigma. c. detail of the rbd b -b region with the trp residue interacting with the papn. in b and c, rbd residues are numbered following the tgev sequence shown in d, and intermolecular hydrogen bonds are shown as dashed red lines. d. structure-based sequence alignment of the tgev and prcv rbds. b-strands are marked with bars. tgev sequence is numbered. in red, af mab-(for tgev) and papn receptor-binding residues (for prcv) identified by the structures. residues absent in the rbd structures are in grey, and the thrombin recognition sequences at the end of recombinant porcine cov rbds are in lowercase letters. doi: . /journal.ppat. .g rbds [ , , ] . the rbds of the sars and mhv betacoronavirus adopt folds unrelated to the b-barrel shown for alphacoronavirus. the most tgev-neutralizing mabs, including af , recognize antigenic site a in the s protein, divided into the aa, ab and ac subsites [ ] . to further characterize site a antigenic determinants in the tgev rbd, we mutated rbd residues targeted by the af mab ( figure ) and some surrounding residues, and analyzed binding to other site a-specific mabs. the antigenicity of residues in the b -b region, in the center of the epitope for af ( figure c ), was determined by monitoring mab binding to rbd mutants with tgev residue substitutions gly (g d), tyr (y a) and gly (g d) ( figure a ). all three substitutions abolished rbd binding by the ac subsite-specific mabs af and ac . the y a rbd mutant was recognized by aa-( bb ) and ab-specific ( de ) mabs ( figure a) , and mab de also bound the g d mutant. in contrast to the antibody binding profile of the y a rbd mutant, ala substitution of the tgev trp residue (w a), a papn-binding residue in the b -b loop at the periphery of the rbd epitope for af ( figure c ), did not affect binding by the ac-specific mabs ( af and ac ), whereas rbd recognition by bb and de mabs was greatly reduced ( figure a ). deletion of the b -b turn (lwd a mutant) reduced ac mab binding to the rbd markedly, with a partial reduction in af binding ( figure a ); this indicates that mab ac recognizes a broader epitope, which correlates with its higher tgev neutralization activity [ ] . replacement with ala of rbd residues thr and asn at the b -b hairpin, which contacts the af mab in the rbd- af structure ( figure c) , reduced binding by all site a-specific mab ( figure a ). this might be a result of a conformational effect induced on the nearby b -b region of the rbd. results for antibody binding to rbd mutants showed that site a epitopes extend across the tgev rbd tip, although there are some differences among the three a subsites ( figure b ). the epitopes recognized by aa-and ab-specific mabs bear the exposed tgev trp residue at the b -b loop, whereas epitopes for the acspecific mabs center on tyr in the b -b turn. none of the mab tested simultaneously targeted the two aromatic side chains (tyr and trp) at the tip of the tgev rbd that bind to the papn. subsite-specific residues defined by mar mutants (lys for aa, arg for ab and gly for ac) might be located at the periphery of their respective epitopes ( figure b ). ab and ac subsites appear to be relatively far apart, with the aa epitope in an intermediate position. the rbd tip, shown here as the papn-binding edge of the domain (figure ) , is the main s protein determinant of antigenic site a, recognized by the most effective neutralizing antibodies of tgev and related cov infections [ , ] . here we show how a group of covs attaches to the cell surface apn metalloprotease for entry into host cells, and how some covneutralizing antibodies prevent infection. the rbd-receptor complex structures determined for alphacoronavirus indicate that the conformation of the receptor binding edge in the envelope s proteins probably determines their receptor-binding specificity. the cov that bind apn analyzed here have protruding receptorbinding motifs that engage recessed surfaces on the receptor. this mode of receptor recognition is essentially opposite to that reported for cov binding to the ace receptor, where recessed receptor-binding motifs in the viral rbd cradle exposed surfaces of the ace ectodomain [ , ] . in the case of papn, an nlinked glycan is also engaged in the virus-receptor interaction. the inherent flexibility of this glycan might facilitate the initial contact of the cov tyr residue with apn amino acids, and subsequent virus-receptor interactions could lock the bound tyr between the glycan and an a-helix ( figure b ). the glycan n-linked to asn in papn is also conserved in canine and feline apn proteins ( figure s ), as are the viral s protein residues that interact with this glycan in the rbd b -b and the b -b regions ( figure ). this unique glycan-virus interaction must thus be conserved among the different covs in the species alphacoronavirus , in accordance with the glycan requirement reported for cell infection by ccov, fcov, and tgev/prcv [ ] . the lack of this glycan in human apn ( figure s ) and the absence of the interacting tyr residue in the b -b region of hcov- e rbd ( figure ) imply distinct virus-apn local contacts in humans. as shown for the alphacoronavirus group, however, hcov- e probably has a protruding receptor-binding edge in the envelope s, responsible for its apn-binding specificity. the structure of the rbd- af complex, together with structure-guided rbd mutagenesis and mab binding data, demonstrated that the receptor-binding region is a major antigenic determinant in the envelope s protein of cov that bind apn. potent tgev-neutralizing antibodies, such as the ac mab [ ] , target key apn-binding residues in the s (figure ) , preventing infection. data from antibody neutralization-resistant tgev mar mutants nonetheless show that some substitutions can be accommodated in the receptor-binding region of alphacoronavirus, which confer the ability to escape immune neutralization, while preserving . substituted residues at the rbd tip that contact papn in the prcv rbd-papn structure are shown in figure , except for the v ngly mutant, with a glycan at rbd position in the b -b b loop, outside the rbd tip (see figure d) . b. tgev rbd binding to cell surface papn glycosylation mutants. relative binding of the sa protein and the anti-ha mab to ha-tagged papn proteins with (papn) or without the glycan linked to asn (n a and t v). mean and standard deviation for three experiments. doi: . /journal.ppat. .g the receptor-binding affinity necessary for cell entry [ , ] . our results thus demonstrate that the receptor-binding region in alphacoronavirus is under selective pressure from the immune system, as described for other viruses [ , , , ] . it is tempting to speculate that immune pressure on exposed receptor-binding residues in the cov s could lead to conformational changes in receptor-binding edges of cov rbds. this would result either in changes in the apn-recognition mode observed with hcov- e and tgev, or in conformational changes in the rbd tip that lead to a receptor specificity switch for cell entry, as observed for hcov-nl [ ] . virus use of recessed binding regions, as for hcov-nl , is a well-defined strategy for hiding conserved receptorbinding residues from antibodies [ , ] . like hcov-nl , sars-cov uses a recessed, although broader ace -binding surface, which can accommodate mutations that permit crossspecies receptor recognition [ ] . it remains to be understood why, despite major changes in the receptor-binding region, all these cov use metalloproteases as cell entry receptors. in the course of our studies, we also determined the crystal structure of the cell surface apn, an important target for cancer therapies. the domain architecture of apn resembles that of related aminopeptidases [ , , ] . here we show a unique dimer configuration for the apn, mediated by its domain iv, the most divergent domain among m aminopeptidases [ ] . the implication of these structural findings for apn biology will require further biochemical analysis. knowledge of the structure is leading to research on the mechanism of action of numerous anti-tumor compounds that target mammalian apn [ ] ; these studies will be fundamental for improving drug specificity. the detailed view of the apn-cov interaction shown here might also lead to development of small molecules to block cov infection. we have identified the receptor-binding region as the major antigenic site in the alphacoronavirus envelope s, which could guide the design of immunogens that boost cov-neutralizing immune responses to key motifs for virus cell entry. design of soluble s proteins variants of tgev and prcv has been described [ ] . the sa protein containing the rbd of tgev was derived from the sc strain, and contains residues b-strands are marked (bars) above or beneath their sequences. tgev sequence is numbered. ace receptor-binding residues reported for hcov-nl [ ] , as well as papn receptor-binding residues for tgev (supplementary table s ) are colored as in c. residues absent in the rbd structures are in grey, and the thrombin recognition sequence at the end of the tgev rbd is in lowercase letters. doi: . /journal.ppat. .g to of the tgev s, an n-terminal influenza hemagglutinin ha peptide, and either a flag mab epitope (monovalent sa-flag variant) or the human igg fc portion (bivalent sa-fc variant) at the c-terminal end. the engineered soluble papn contains residues to (ectodomain) of the cell surface protein fused to ha and flag tags at the n and c terminus, respectively [ ] . the soluble s protein crystallized in complex with the papn was derived from the prcv hol strain (s h in [ ] ), and contains the n-terminal residues of the prcv s protein and same c-terminus as the tgev-derived sa protein [ ] . a recombinant membrane bound papn with an ha tag at the cterminal end was engineered for cell surface expression. thrombin recognition sequences were introduced between the tags and the viral or papn protein sequences. proteins were produced in transiently transfected t or stably transfected cho-lec . . . (cho-lec) cells as described [ ] , and concentration in cell supernatants determined by elisa. proteins prepared in cho-lec cells were used in crystallization experiments. hybridoma cells secreting the tgev s mabs were grown in dmem supplemented with % fcs in roller bottles. proteins secreted to culture supernatants were initially purified by affinity chromatography. all protein samples were further purified by size exclusion chromatography in hepes-saline buffer ( mm hepes, mm nacl) ph . . the fab fragment of the af mab was prepared by papain digestion of the purified antibody. the reaction was terminated by the addition of e (sigma) and the fab fragment purified by size exclusion and ion exchange chromatography using hepes-saline buffer ph . . the polypeptide chains of the ig variable domains of the af mab were determined by sequencing of their cdna prepared from reverse transcribed mrna purified from hybridoma cells. binding of anti-tgev s or -ha (control) mab to wild type and mutant sa proteins was tested in -well plates, using purified mab or hybridoma supernatants. the sa-fc fusion proteins in serum-free (opti-mem, invitrogen) cell supernatants were bound to plastic, and mab binding monitored by optical density (od nm ). at least four sa-fc protein concentrations ranging from to mg/ml were used in duplicate and average binding determined in each experiment. binding ratios were determined after correction for background binding. apn binding assays were also carried out with the sa-fc fusion protein comprising the tgev rbd. bhk-papn cells constitutively expressing cell surface papn were used for binding experiments comparing wild type and mutant rbds, whereas transiently transfected t cells were used for analysis of rbd binding to papn glycosylation mutants. binding was monitored as the percentage of stained cells with the fc fusion proteins and fitc labeled anti-fc antibodies by fluorescence-activated cell sorting (facs), as shown in figure b . the percentage of cells stained was determined for each protein sample and corrected for background staining. papn binding ratios for wild type and mutant rbd proteins shown in figure a were determined from the percentage of bhk-papn cells stained with same concentration of wild type and mutant sa-fc proteins. the binding ratios for wild type and mutant papn glycosylation mutants shown in figure b were determined from the percentage of sa-fc stained t cells expressing similar amounts of ha-tagged papn proteins. cell surface expression of the papn-ha protein was determined with the ha ac mab. the tgev rbd in complex with the af fab fragment was crystallized using the size exclusion-purified complex of a table ). crystallization of the papn ectodomain in complex with porcine cov s proteins was carried out with mixtures of the receptor protein and several tgev and prcv protein variants comprising the receptor-binding region (sa, s h and s h in [ ] ). crystals appeared only in trials performed with an equimolar mixture of papn and the s h protein derived from the prcv s at a final protein concentration of mg/ml, and with a crystallization solution of % peg- k, . m lithium sulfate and . m tris buffer ph . . crystals were transferred to crystallization solution containing % ethylene glycol and frozen for diffraction data collection at the id beamline (prcv rbd-papn in table ). the structure of the tgev rbd- af fab fragment was initially determined by the molecular replacement (mr) method using the phaser program [ ] , and two search models having either the variable or constant regions of the pdb id aif mab structure. the af fab model structure was built manually following electron density maps determined from the mr solution, after improvement with the dm program [ ] . the af fab structure was refined with the program phenix.refine [ ] , which provided an excellent electron density map for building residues to of the tgev s, as well as four residues of a thrombin recognition site at the c-terminus. final structure refinement of the complex was carried out with data extending to . Å resolution (statistics in table ). three cycles of solvent correction, refinement of individual coordinates and atomic displacement parameters combined with tls were applied in each step of structure refinement with phenix.refine, which was alternated with manual adjustment of the model to the electron density maps. all residues are in allowed regions of the ramachandran plot. sa protein residues included in the structure of the tgev rbd are shown in figure d . the structure of the prcv rbd-papn complex was resolved by the mr method using the papn structure determined alone (manuscript in preparation) and the tgev rbd structure as search models. mr solutions were obtained for the two papn molecules (chains a and b) of the asymmetric unit and for one rbd molecule (chain e). the three molecules were adjusted manually and refined with the phenix.refine program. the second rbd molecule (chain f) bound to papn molecule b was built manually into the electron density map. the residues nterminal to the prcv rbd in the s h protein were largely disordered or degraded during crystallization, and are absent in the structure. the complex structure was refined with the program phenix.refine applying solvent correction, ncs, refinement of individual coordinates and atomic displacement parameters combined with tls ( table ). the current model comprises residues to of the papn ectodomain with a zinc metal ion at the papn enzyme active site, and residues to of the prcv s, homologous to the tgev s residues to that defined the tgev rbd structure ( figure d ). all the residues are in allowed regions of the ramachandran plot. coordinates and structure factors have been deposited in the protein data bank with id codes f m (tgev rbd- af ) and f c (prcv rbd-papn). buried surfaces and residues at the molecular complex interfaces were determined with the pisa server (http://www. ebi.ac.uk/msd-srv/prot_int/pistart.html). only residues with at least % of their surface buried at interfaces in the two independent molecules of the crystal asymmetric units are shown. figure d was prepared with ligplot (http://www.ebi.ac.uk/ thornton-srv/software/ligplot/), figure a with ribbons [ ] and the other structure representations with pymol (pymol.org). structural alignments were carried out with modeller using a gap penalty of [ ] . accession numbers of the alphacoronavirus s proteins mentioned are q pkz (tgev), q (ccov), p (fcov), p figure . determinants of tgev s antigenic site a. a. binding of tgev-neutralizing, site a-specific mabs to rbd mutants. relative binding (%) of mutants to wild type sa protein is shown for tgev sspecific mabs (top; described in figure c ) and a control anti-ha antibody (see materials and methods). rbd regions in which mutations locate are shown (bottom; see also figure d ). mean and standard deviation of data from at least three experiments. b. antigenic site a in the tgev rbd and epitopes for antibodies. surface and ribbon representation of the rbd with the af contact regions colored as in figure b . three antibody-binding residues (tyr , trp and asn ) in the loops at the rbd tip, as well as tgev lys , arg and gly residues associated with aa, ab and ac subsites [ ] , respectively. lines indicate epitopes for mabs specific for each of the three antigenic subsites: aa in yellow, ab in green and ac in red. doi: . /journal.ppat. .g (hcov- ), q q s (hcov-nl ), b vdw (bulbul-cov) and q q p (ibv). the prcv hol s protein sequence is reported in reference [ ] . sequence identities among s proteins were determined with psiblast (http://www.ebi.ac.uk/tools/sss/ psiblast/). accession number for the papn protein is p . figure s structures of tgev and prcv rbds. a. secondary structure elements of the rbd structures. b-strands are shown with arrows and colored in blue and cyan, a b-bulge at the b-strand is shown in magenta, helix with a red cylinder, coils with black lines, and disulphide bonds with green lines. b. stereo view of the superimposed asymmetric unit rbd structures of tgev (blue and cyan), complex with the af mab, and of prcv (green and red), complex with the papn protein. view as in figures a and a . locations of n and c terminal ends are indicated in lowercase letters. (tif) figure s mammalian apn ectodomains. sequence alignment of the porcine, canine, feline and human apn proteins with conserved residues highlighted in red. secondary structure elements of the papn structure determined in complex with the rbd of prcv are shown above the sequences. cov-binding residues and those engaged in papn dimerization are highlighted in blue and green, respectively, whereas those at the papn catalytic site are in yellow. residues coordinating the zinc ion are marked with an asterisk, and the n-linked glycosylation site recognized by cov is marked with a triangle at the papn asn . the beginning of each of the four apn domains is indicated. (tif) figure s aminopeptidases active site. side chains of residues at the catalytic site of four structurally aligned zinc aminopeptidases based on domain ii are shown with stick representation, and with the coordinated zinc ion as a cyan sphere. human erap- (pdb code xdt) is shown in green, aminopeptidase n of e. coli (pdb code hpt) in magenta, aminopeptidase n of neisseria meningitidis (pdb code gtq) in blue, and papn in yellow. the glutamic acid located in the gamen motif is labeled in blue and those located at the conserved hexxhx e motif are in red (sequence in figure s ). (tif) figure s dimerization of the papn ectodomain in solution. size exclusion chromatography of the soluble papn ectodomain. continuous line shows optical density (od) at nm for the elution volume. papn protein was run through a superdex / column (ge healthcare) with hepessaline buffer ph . . exclusion volume and size (kda) of molecular weight markers are indicated. determined molecular weight for the single recombinant glycosylated papn ectodomain is about kda, whereas the protein elutes with a volume corresponding to , kda. (tif) table s sequence of homologous cdr-h loops in known mab structures. sequence of homologous heavy chain cdr-h loops to that of the af mab, identified by a blast search among protein structures, whose pdb codes are shown. (tif) table s intermolecular contacts in the prcv rbd-papn complex structure. rbd and papn residues in close contact (# Å ) in the two complexes of the crystal asymmetric unit, computed with the program ncont [ ] . rbd residues from the b -b , b -b and b -b regions at the tip of the bbarrel domain are shown, with those engaged in hydrogen bonding in red. tgev/prcv numbering is given for the rbd residues. (tif) the molecular biology of coronaviruses encyclopedia of virology coronaviruses post-sars: update on replication and pathogenesis virus taxonomy: ninth report of the international committee on taxonomy of viruses assembly of coronavirus spike protein into trimers and its role in epitope expression architecture of the sars coronavirus prefusion spike the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor human aminopeptidase n is a receptor for human coronavirus e aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev crystal structure of nl respiratory coronavirus receptor-binding domain complexed with its human receptor angiotensinconverting enzyme is a functional receptor for the sars coronavirus structure of sars coronavirus spike receptor-binding domain complexed with receptor cloning of the mouse hepatitis virus (mhv) receptor: expression in human and hamster cell lines confers susceptibility to mhv the moonlighting enzyme cd : old and new functions to target impaired angiogenesis in aminopeptidase n-null mice the neovasculature homing motif ngr: more than meets the eye novel aminopeptidase n (apn/cd ) inhibitor f can suppress invasion of hepatocellular carcinoma cells as well as angiogenesis aminopeptidase n (cd ) as a target for cancer chemotherapy mutational analysis of aminopeptidase n, a receptor for several group coronaviruses, identifies key determinants of viral host range genetic evolution and tropism of transmissible gastroenteritis coronaviruses major receptor-binding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein identification of a receptor-binding domain of the spike glycoprotein of human coronavirus hcov- e residues involved in the antigenic sites of transmissible gastroenteritis coronavirus s glycoprotein mechanisms of transmissible gastroenteritis coronavirus neutralization four major antigenic sites of the coronavirus transmissible gastroenteritis virus are located on the amino-terminal half of spike glycoprotein s antigenic modules in the n-terminal s region of the transmissible gastroenteritis virus spike protein crystal structures of an antibody to a peptide and its complex with peptide antigen at . a crystal structures of a quorum-quenching antibody crystal structures of the endoplasmic reticulum aminopeptidase- (erap ) reveal the molecular basis for n-terminal peptide trimming structural basis for antigenic peptide precursor processing by the endoplasmic reticulum aminopeptidase erap structure of aminopeptidase n from escherichia coli suggests a compartmentalized, gated active site reconstitution of purified amphiphilic pig intestinal microvillus aminopeptidase. mode of membrane insertion and morphology the canyon hypothesis: hiding the host cell receptor attachment site on a viral surface from immune surveillance evolution subverting essentiality: dispensability of the cell attachment arg-gly-asp motif in multiply passaged foot-and-mouth disease virus structural basis of immune evasion at the site of cd attachment on hiv- gp structure of the measles virus hemagglutinin bound to the cd receptor pushing the boundaries of molecular replacement with maximum likelihood the ccp suite: programs for protein crystallography phenix: a comprehensive python-based system for macromolecular structure solution ribbon models of macromolecules comparative protein modelling by satisfaction of spatial restrains we thank the esrf for provision of synchrotron radiation facilities through bag-madrid projects, as well as the swiss-sls facility, n. cubells for technical help and c. mark for editorial assistance. key: cord- - td efjw authors: zhou, yanrong; wu, wei; xie, lilan; wang, dang; ke, qiyun; hou, zhenzhen; wu, xiaoli; fang, ying; chen, huanchun; xiao, shaobo; fang, liurong title: cellular rna helicase ddx is involved in transmissible gastroenteritis virus nsp -induced interferon-beta production date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: td efjw transmissible gastroenteritis virus (tgev), an enteropathogenic coronavirus (cov) of porcine, causes lethal watery diarrhea and severe dehydration in piglets and leads to severe economic losses in the swine industry. unlike most covs that antagonize type i interferon (ifn) production, previous studies showed that tgev infection induces ifn-i production both in vivo and in vitro. however, the underlying mechanism(s) remain largely unknown. in this study, we found that tgev infection significantly facilitated ifn-β production as well as activation of the transcription factors ifn regulatory factor (irf ) and nuclear factor-kappab (nf-κb) in porcine kidney (pk- ) cells. screening of tgev-encoded proteins demonstrated that non-structural protein (nsp ) was the most potent ifn-β inducer and induced ifn-β production mainly by activating nf-κb but not irf . further analysis showed that nsp interacted with ddx , a member of the dexd/h helicase family. knockdown of ddx by specific small interfering rna (sirna) significantly decreased nsp -induced ifn-β production and nf-κb activation. furthermore, tgev-induced ifn-β production and ifn-stimulated gene (isg) expression were decreased in cells transfected with ddx -specific sirna, indicating the vital role of ddx to tgev-induced ifn-β responses. in summary, our data revealed a potential coactivator role of host rna helicase ddx to the induction of ifn-β response initiated by tgev and demonstrated that nsp is an important ifn inducer among the tgev-encoded proteins. transmissible gastroenteritis virus (tgev), an enteropathogenic coronavirus (cov) of porcine, causes lethal watery diarrhea and severe dehydration in piglets and leads to severe economic losses in the swine industry. unlike most covs that antagonize type i interferon (ifn) production, previous studies showed that tgev infection induces ifn-i production both in vivo and in vitro. however, the underlying mechanism(s) remain largely unknown. in this study, we found that tgev infection significantly facilitated ifn-β production as well as activation of the transcription factors ifn regulatory factor (irf ) and nuclear factor-kappab (nf-κb) in porcine kidney (pk- ) cells. screening of tgev-encoded proteins demonstrated that non-structural protein (nsp ) was the most potent ifn-β inducer and induced ifn-β production mainly by activating nf-κb but not irf . further analysis showed that nsp interacted with ddx , a member of the dexd/h helicase family. knockdown of ddx by specific small interfering rna (sirna) significantly decreased nsp -induced ifn-β production and nf-κb activation. furthermore, tgev-induced ifn-β production and ifn-stimulated gene (isg) expression were decreased in cells transfected with ddx -specific sirna, indicating the vital role of ddx to tgev-induced ifn-β responses. in summary, our data revealed a potential coactivator role of host rna helicase ddx to the induction of ifn-β response initiated by tgev and demonstrated that nsp is an important ifn inducer among the tgevencoded proteins. keywords: transmissible gastroenteritis virus, non-structural protein , ddx , interferon-beta, innate immune response, pattern-recognition receptors introduction transmissible gastroenteritis virus (tgev) is a member of the alphacoronavirus genus within the family coronaviridae in the order nidovirales. tgev infection mainly causes acute enteric disease characterized by lethal watery diarrhea, severe dehydration, and high mortality in suckling piglets less than weeks old, which has led to severe economic losses in the global swine industry since gene name sirna sequence hddx (sense) ggagcuucugauaauuggagguguu hddx (anti-sense) aacaccuccaauuaucagaagcucc pddx (sense) gaaagaccuuggucuggcauuugaa pddx (anti-sense) uucaaaugccagaccaaggucuuuc the first outbreak in in ilinois, usa ( , ) . tgev contains a single-stranded, positive-sense rna genome of about . kb ( ) , including at least nine open reading frames (orfs). two slightly overlapping orfs, orf a and orf b, located at the ' two-thirds of the viral genome, encode a replicase complex that is proteolytically processed into non-structural proteins (nsp to ) ( ). structural proteins, nucleocapsid (n) protein, membrane (m) glycoprotein, spike (s) glycoprotein, a small envelope (e) glycoprotein, and accessory proteins a, b, and are encoded by genes located at the ′ end ( ) . as early as , the presence of high levels of type i interferon (ifn-i) activity in the digestive tract of tgev-infected newborn piglets was first observed ( ) . thereafter, charley et al. reported that tgev infection in human or bovine peripheral blood mononuclear cells also induced high ifn-α production ( ) . subsequently, bosworth et al. demonstrated that ′, ′-oligoadenylate synthetase (oas), a well-known ifn-stimulated gene (isg), was increased in tgev-infected pigs ( ) . our previous quantitative proteomics analysis revealed that tgev infection induced canonical ifn-i signaling through janus kinase signal transducer and activator of the transcription (jak-stat ) pathway, and eight tested isgs, including ifninduced protein with tetratricopeptide repeats (ifit ), ifit , ifit , oas , oas , mx , mx , and isg were upregulated after tgev infection ( ) . these early results indicated that tgev infection activated the ifn-i pathway in vitro and in vivo. however, the underlying mechanism(s) utilized by tgev to induce ifn-i, and especially which viral protein(s) contribute to it, remain largely unclear. the ifn-i response is a well-known innate immune reaction that occurs in response to virus infection and considered as an important bridge between innate and adaptive immunity. nuclear factor-kappab (nf-κb) and ifn regulatory factor (irf ) are two critical transcription factors for the regulation of ifn-i production. secreted ifn-i then stimulates the jak-stat signaling pathway to induce the expression of numerous isgs, which collaborate to regulate the replication of virus ( ) . many viruses antagonize ifn responses to benefit their propagation, and some viruses such as human immunodeficiency virus-type ( ), vesicular stomatitis virus (vsv) ( ), influenza a virus (iav) ( ) , encephalomyocarditis virus ( ), reovirus ( ), herpes simplex virus (hsv ) ( ), respiratory syncytial virus ( ), newcastle disease virus ( ) , and sendai virus (sev) ( ) initiate innate immune responses. different viruses employ different mechanisms to regulate innate immune responses. for example, hsv induces ifn-α/β production through toll-like receptor (tlr ), dna-dependent activator of ifn regulatory factors (dai), and ifn-inducible ( ) . for iav infection, at least tlr , tlr , retinoic acid-inducible gene i (rig-i), and pyrin domain-containing (nlrp ) are responsible for the detection of iav and subsequent innate immune responses ( ) . elucidating the mechanisms through which viruses regulate innate immune responses will help us understand the interactions between virus and host. this study sought to identify tgev-encoded protein(s) involved in the induction of ifn-β production. our results revealed that tgev nsp was the best inducer of the ifn-β pathway among the tgev-encoded proteins. mechanistically, nsp activates nf-κb but not irf , and it interacts with rna helicase ddx , which in turn activates ifn-β production. porcine kidney (pk- ) cells and hek- t cells were cultured in dulbecco's modified eagle's medium (invitrogen, carlsbad, ca, usa) supplemented with % fetal bovine serum in a humidified incubator with °c/ % co . tgev strain wh- (genbank accession no. hq ) was propagated and titered in pk- cells. recombinant vsv-expressing green fluorescent protein (vsv-gfp) was generously provided by prof. zhigao bu from the harbin veterinary research institute, china. rabbit polyclonal antibodies against p , irf , phosphorylated irf (p-irf ), and ddx were purchased from abclone (wuhan, china). rabbit polyclonal antibody against phosphorylated p (p-p ) was purchased from cell signaling technology (beverly, ma, usa). anti-β-actin antibody was purchased from beyotime (nantong, china). mouse monoclonal antibodies (mabs) against hemagglutinin (ha) and flag were purchased from medical and biological laboratories (mbl, nagoya, japan). mab against tgev n protein was prepared by our laboratory. horseradish peroxidase (hrp)-conjugated goat anti-rabbit antibody and hrp-conjugated goat anti-mouse antibody were purchased from mbl. alexa fluor -conjugated donkey anti-rabbit igg, alexa fluor -conjugated donkey anti-mouse igg, and alexa fluor -conjugated donkey antimouse igg were obtained from santa cruz biotechnology inc. (santa cruz, ca, usa). expression plasmids of tgev-encoded proteins used in this study were constructed by rt-pcr amplification from the genomic rna of tgev strain wh- and cloned into expression vector pcaggs-ha. the details of primers used for pcr clone are available on request. the p gene was derived from human rela cdna and cloned into pegfp-c vector. the ddx expression plasmid was constructed by rt-pcr amplification from the cdna of pk- cells and cloned into pcmv-tag b vector. luciferase reporter plasmids p -luc (ifn-β-luc), × prdiii/i-luc (referred to as irf -luc), × prdii-luc (referred to as nf-κb-luc), and the internal control plasmid prl-tk have been described previously ( ) . small interfering rna (sirna) targeting ddx or negative control sirna (sinc, invitrogen) were each transfected at a final concentration of nm. the sirna sequences used here are listed in table . np- , nm phenylmethylsulfonyl fluoride], and protein concentration was measured and adjusted. for each immunoprecipitation, µg of cell lysate protein was incubated with µg of indicated antibody and µl of protein a/g-agarose (beyotime) overnight at °c. after three washes with ml lysis buffer, precipitates were subjected to % sds-page and subsequently analyzed with immunoblot analysis using the indicated antibodies. porcine kidney (pk)- cells were fixed with % paraformaldehyde for min followed by permeabilization with pre-cooled methanol for min, blocking with % bovine serum albumin for min, incubated with the indicated primary antibodies for h, followed by staining with specific alexa fluor-conjugated secondary antibodies for h. the cells were subsequently stained with ', -diamidino- -phenylindole (beyotime) for min. after washing with pbs, fluorescent images were obtained using an olympus fv laser scanning confocal microscope (olympus, japan). total rnas were extracted using trizol reagent (invitrogen). real-time rt-qpcr was performed using sybr green real-time pcr master mix (toyobo biologics, osaka, japan) in the abi prism sequence detection system (applied biosystems). individual transcripts in each sample were assayed three times. the fold change in gene expression relative to normal was calculated using the delta-delta cycles to threshold (ΔΔct) method. primers ( table ) were designed using primer express software (version . ; applied biosystems, carlsbad, ca, usa). all experiments were performed at least three times with reproducible results. data are presented as the mean ± sd. statistical analysis was performed using one-way anova without interaction terms followed by dunnett's for multiple comparisons. all animal experiments were approved by the hubei administrative committee for laboratory animals (permission number ) and complied with the guidelines of hubei laboratory animal welfare and ethics of hubei administrative committee of laboratory animals. previous studies demonstrated that tgev infection potently induced ifn-α ( , , ) , as well as ifn-β in ipec-j and swine testicular (st) cells ( ) ( ) ( ) . however, whether tgev infection induces ifn-β in pk- cells remains unknown. to explore the effect of tgev on ifn-β, dual luciferase assays were performed. pk- cells were transfected with ifn-β-luc and prl-tk. after the induction of ifn-i is reliant on the co-regulation of transcription factors irf and nf-κb. to investigate the potential mechanism(s) involved in the ifn-β production by tgev infection, the effect of tgev on irf and nf-κb promoters were also tested. as displayed in figures c,d , tgev infection also upregulated irf and nf-κb promoter activity dosedependently, indicating that irf and nf-κb are involved in the ifn-β production by tgev infection. because of the high sensitivity of vsv-gfp to ifn, vsv-gfp expression is commonly monitored for ifn detection. to further evaluate the ifn-β response in tgev-infected cells, a vsv-gfp-based ifn detection assay was performed. pk- cells were infected or mock infected with sev or tgev (moi = . , . , . ). then, cell supernatants were collected, uv-irradiated, and then transferred onto fresh pk- cells. after h, cells were infected with vsv-gfp and observed under a fluorescence microscope at hpi. as a positive control, supernatants collected from sev-treated cells suppressed vsv-gfp replication prominently compared with the negative control group (mock). in accordance with the results of dual luciferase assays described above, tgev infection limited the replication of vsv-gfp in a dose-dependent manner (figure e) . these results suggested that tgev infection increased ifn-β production. transmissible gastroenteritis virus encodes non-structural proteins (nsp - ), four structural proteins (n, m, s, e), and three accessory proteins (orf , orf a, orf b) . to identify the key viral protein(s) involved in ifn-β induction, an ifn promoterreporter system was employed to screen tgev-encoded proteins for their relative capacities to activate the ifn-β promoter. hek- t cells were cotransfected with ifn-β-luc, prl-tk, and different tgev protein expression vectors. as shown in figure a , nsp was the most significant inducer of ifn-β production. furthermore, similar to sars-(coronavirus) cov ( ), tgev m glycoprotein also potently mediated ifn-β induction. because nf-κb and irf are necessary transcription factors for ifn-β production, we examined the effects of all tgev-encoded proteins on irf and nf-κb promoter activity. interestingly, nsp upregulated an approximately . -fold change in nf-κb promoter activity, but only an approximately . -fold change in irf promoter activity (figures c,e) . m glycoprotein induced a higher fold change in irf , but a lower fold change in nf-κb compared with nsp . because m glycoprotein has been investigated previously ( ), we focused on nsp . to confirm the ability of nsp to induce ifn-β production, large-scale screen experiment was also conducted in pk- cells, a permissive cell line of tgev infection. as shown in figures b,d,f, nsp was also a potent ifn inducer that mainly induced activation of nf-κb but not irf promoter in pk- cells. to confirm the above large-scale screen results, increasing doses ( . , . , . µg) of pcaggs-ha-nsp and ifn-β-luc, irf -luc or nf-κb-luc together with prl-tk were cotransfected into hek- t cells. in line with the results in figure , nsp enhanced the activation of ifn-β and nf-κb in a dose-dependent manner (figures a-c) . interestingly, nsp induced the activation of nf-κb to a greater extent than that of irf , indicating nf-κb has a fundamental role in nsp induced ifn-β activation. similar results were also obtained in pk- cells (figures d-f) . nf-κb and irf activation are characterized by the phosphorylation and subsequent translocation of p (nf-κb subunit) or irf to the nucleus, respectively ( ) ( ) ( ) . next, we investigated the role of nsp in the phosphorylation of p and irf . hek- t cells were transfected with increasing amounts of pcaggs-ha-nsp , and cell lysates were examined for the expression levels of p-p or p-irf and total p or irf at h post-transfection. as shown in figure g , nsp overexpression had no significant effect on the amount of p , irf , and p-irf ; however, markedly increased p phosphorylation levels, indicating the activation of nf-κb, rather than irf is associated with nsp -induced ifn-β production. therefore, the subcellular location of p was further investigated after nsp overexpression. as shown in figures h,i , ectopic expression of nsp resulted in the nuclear translocation of overexpressed p in pk- cells ( figure h ) and endogenous p in hek- t cells (figure i ). early studies suggested that nsp of cov ibv and sars-cov interacts with host protein ddx ( ) . therefore, we investigated whether tgev nsp also interacts with ddx by co-ip. hek- t cells were cotransfected with pcaggs-ha-nsp and pcmv-tag b-ddx . because ddx is a dexd/h-box helicase and is associated with rna metabolism, the lysates were treated with rnase to avoid the effect of rna on the co-ip experiment. as shown in figure a , flag-tagged ddx was coprecipitated by ha-tagged nsp , indicating the interaction between nsp and ddx . reversed ip with flag antibody further confirmed this interaction ( figure b) . to test whether the colocalization of nsp and ddx occurs, a prerequisite for the interaction, pk- cells were transfected with pcaggs-ha-nsp or empty vector and fixed at h posttransfection. ha-tagged nsp protein was detected with mouse anti-ha antibody, and ddx was detected with rabbit anti-ddx antibody. the results revealed that nsp was colocalized with ddx and distributed both in the cytoplasm and nucleus (figure c) , which further confirmed the interaction between tgev nsp and ddx . because nsp activates ifn-β and interacts with ddx , we investigated whether ddx is involved in nsp -induced ifn-β production. synthesized sirna targeting human ddx (hsiddx ), which efficiently decreases the expression of endogenous ddx mrna ( figure a ) and protein (figure b) , was selected. next, hek- t cells were transfected with hsiddx or sinc, followed by co-transfection of ifn-β-luc or nf-κb-luc, prl-tk, along with pcaggs-ha-nsp or empty vector. the results revealed that knockdown of ddx significantly decreased nsp -induced promoter activity of ifn-β ( figure c ) and nf-κb ( figure d) . moreover, mrna expression levels of nsp -induced ifn-β were downregulated values are the mean ± sd of three independent tests. **p < . or ***p < . compared with empty vector group. (g) hek- t cells were transfected with increasing quantities ( , , µg) of pcaggs-ha-nsp or empty vector for h, and then subjected to immunoblotting with antibodies specific for endogenous irf , phosphorylated irf (p-irf ), p , or p-p . anti-ha mouse antibody was used to confirm the expression of nsp . β-actin expression was used as a loading control. the ratio of phosphorylated/total p and phosphorylated/total irf was analyzed using imagej software. (h) pk- cells were cotransfected with plasmids encoding ha-tagged nsp protein or empty vector together with plasmids encoding egfp-tagged p protein. then, the cells were fixed and immunostained with anti-ha monoclonal antibodies (mabs) to observe the nuclear translocation of overexpressed p using confocal microscopy. (i) hek- t cells were transfected with plasmids encoding ha-tagged nsp protein or empty vector. then, the cells were fixed and immunostained with anti-p antibody to observe the nuclear translocation of endogenous p using confocal microscopy. by ddx deficiency (figure e) , suggesting the involvement of ddx in ifn-β induction by nsp . to determine whether ddx is involved in tgev-induced ifnβ activation, we designed three pairs of sirnas targeting porcine ddx (psiddx ) and selected one with the best knockdown efficiency as demonstrated by rt-qpcr ( figure a ) and western blot assay (figure b) , for subsequent experiments. pk- cells were cotransfected with ifn-β-luc or nf-κb-luc, prl-tk, together with psiddx or sinc. at h post-transfection, cells were infected with tgev for h, followed by dual luciferase assay. ddx depletion had no effect on the basal activity of ifn-β and nf-κb promoter, but significantly decreased tgev-induced activation of ifn-β and nf-κb (figures c,d) . we also detected the expression level of p-p in tgev-infected pk- cells when ddx was silenced. as shown in figure e , knockdown of ddx reduced tgev-induced p phosphorylation. these results suggested that ddx is associated with tgev-induced ifn-β and nf-κb activation. interferon-i initiates a series of signaling cascades through the jak/stat pathway, resulting in the expression of numerous isgs ( ) . furthermore, nf-κb activation plays a pivotal role in regulating the transcription and expression of many proinflammatory cytokines. because ddx is involved in tgev-induced ifn-β and nf-κb activation, theoretically, it should have an impact on tgev-induced isg and pro-inflammatory cytokine expression. the expression levels of some isgs (ifit , ifit , ifit ) and proinflammatory cytokines (il- , il- ) in ddx -knockdown cells were analyzed after tgev infection. as expected, ddx depletion inhibited the expression of ifit , ifit , ifit , as well as il- and il- to some degree, compared with that in cells transfected with sinc (figures f-j) . the innate immune response characterized by the synthesis of ifn and proinflammatory cytokines is the first line of antiviral defense. multiple studies have reported the involvement of covs in the regulation of innate immune responses. the majority of covs decreased dsrna-mediated ifn-β production, including porcine epidemic diarrhea virus (pedv) ( ), severe acute respiratory syndrome coronavirus (sars-cov) ( ) , and infectious bronchitis virus (ibv) ( ) . interestingly, mouse hepatitis virus (mhv)-induced ifn-α/β and established an antiviral state in plasmacytoid dendritic cells and macrophages, but failed to produce ifn in neurons, astrocytes, and hepatocytes ( , ) , indicating that mhv induction of ifn-α/β is cell type dependent. a previous study showed that tgev-induced ifn-α secretion in vitro and in vivo ( ) . here, we found that tgev infection increased the production of ifn-β in a dose-dependent manner in pk- cells, in line with previously reported data ( ) . the significant roles of many cov proteins in the regulation of innate immune response have been identified. for example, nsp , nsp , nsp , papain-like protease (plpro), orf b, orf , ( ) . this indicated the potential effect of tgev nsp on ifn-β induction, which is in line with our data and further confirms our conclusion using a reverse genetic system. although nsp was identified as a key ifn-β activator among tgev-encoded proteins, it remains unclear which prr(s) is involved in its detection and induction of ifn-β production. indeed, viral proteins, similar to viral nucleic acids or replication intermediates, can in some cases also function as pamps, specifically recognized by certain host prrs, such as tlr and tlr , to modulate the ifn responses during viral infection ( , ) . for example, the m glycoprotein of sars-cov has been reported to function as a novel cytosolic pamp to promote ifn-β production by activating a non-canonical tlr signaling cascade ( ) . in addition to the four major prr groups reported previously, including tlrs, rig-i-like receptors, nod-like receptors and cytoplasmic dna receptors ( ) , multiple dexd/h-box helicases, such as ddx , ddx , ddx , and ddx , were reported recently to act as prrs and sense viral pamps to activate the nf-κb signaling pathway and induce ifn-β production ( ) ( ) ( ) ( ) ( ) . previous study revealed that ddx , ddx , and dhx form a complex with the adaptor molecule trif to sense dsrna in dendritic cells ( ) . in this paper, ddx interacted with tgev nsp in a rna-independent manner and enhanced both tgevand nsp -induced activation of ifn-β responses. however, the direct interactions between nsp and ddx or dhx were not observed. we speculated that nsp may be sensed by ddx / ddx /dhx complex by interacting with ddx . these results suggest that nsp may be recognized as pamp by ddx , (e) pk- cells were transfected with psiddx or sinc, and h later, cells were mock infected or infected with tgev (moi = . ) for h. then, the cells were collected for western blot assay with specific antibodies against p , p-p , ddx , or tgev n protein, using β-actin expression as a loading control. the ratio of phosphorylated/total p was analyzed using imagej software. (f-j) pk- cells were treated as described for (e) and collected at hpi. cell rnas were extracted for rt-qpcr to examine the mrna expression levels of ifit (f), ifit (g), ifit (h), il- (i), and il- (j). the mrna expression levels were normalized to porcine gapdh transcripts. values are the mean ± sd of three independent tests. *p < . or **p < . compared with the sinc group. which triggers an antiviral response. further studies are required to investigate this in more detail. ddx is a dexd/h helicase family member composed of the dead-box and related deah, dexh, and dexd protein family and is involved in multiple cellular processes of rna metabolism ( , ) . besides these traditional roles, it appears that multiple proteins of the dexd/h-box helicase family are associated with viral components and/or have alternative effects on viral propagation ( ) ( ) ( ) . for instance, ddx shows antiviral functions against vaccinia virus, denv, and hbv ( - ), but is of benefit for hcv and hiv infection ( , ) . in addition, ddx interacts with the nsp of venezuelan equine encephalitis virus and enhances viral multiplication ( ) . the interaction of ddx with human immunodeficiency virus type rev protein is involved in the regulation of virus replication ( ) . in the present study, the knockdown and ectopic expression of ddx demonstrated that ddx had antiviral activity against tgev replication (data not shown). interestingly, earlier studies also showed an interaction between ddx with coronavirus (ibv and sars-cov) nsp , and in contrast to tgev, this interaction might enhance the replication of ibv ( ) . this difference suggests that ddx is not likely to be a general target against cov infection. furthermore, it should be noted that the effect of ddx on progeny tgev production was moderate (data not shown). difference from other covs, such as sars-cov and mers-cov which antagonize ifn-i, tgev infection induces ifn-i production, and a most recent paper showed that poly(i:c)induced ifn-i responses could only inhibit tgev replication in the early infection stage, but failed in the late infection stage ( ) . they also demonstrated that the activation of ifn-i responses by tgev infection cannot inhibit viral replication. our results are consistent with the conclusion proposed by zhu and colleagues. in addition, it is surprising that the expression levels of ifn-β are paralleled with the increase of viral rna during tgev infection. these may explain why the effect of ddx on tgev replication was moderate accompany with its significant role in ifn-β induction by tgev. however, more studies are required to investigate the complex interaction between tgev and ifns. in conclusion, our data demonstrate that tgev infection induces ifn-β production and nsp is the most significant ifn-β inducer among the tgev-encoded proteins. nsp interacts with cellular dexd/h helicase ddx to activate ifn-β in a nf-κb dependent manner, and ddx is associated with tgev-induced ifn-β production, revealing a potential coactivator role of host rna helicase ddx on virus and viral protein induced innate immune responses. all animal experiments were approved by the hubei administrative committee for laboratory animals (permission number ) and complied with the guidelines of hubei laboratory animal welfare and ethics of hubei administrative committee of laboratory animals. author contributions yz, ww, and lf designed research; yz, ww, and lx performed research; yz, yf, dw, and sx analyzed data; yz and lf wrote the first draft of the manuscript. hc, qk, zh, and xw contributed to modify the manuscript. all the authors read and approved the manuscript. acknowledgments simulation of the economic impact of transmissible gastroenteritis on commercial pig production in australia effects of virulent and attenuated transmissible gastroenteritis virus on the ability of porcine dendritic cells to sample and present antigen complete sequence ( kilobases) of the 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genome-wide, proteomic, and molecular studies ddx dead-box rna helicase inhibits hepatitis b virus reverse transcription by incorporation into nucleocapsids viral targeting of dead box protein reveals its role in tbk /ikkepsilon-mediated irf activation dead-box rna helicase ddx x inhibits denv replication via regulating type one interferon pathway understanding the interaction of hepatitis c virus with host dead-box rna helicases requirement of ddx dead box rna helicase for hiv- rev-rre export function venezuelan equine encephalitis virus non-structural protein (nsp ) interacts with rna helicases ddx and ddx in infected cells ddx is an rna-dependent atpase involved in hiv- rev function and virus replication key: cord- -adpn fb authors: pan, yongfei; tian, xiaoyan; qin, pan; wang, bin; zhao, pengwei; yang, yong-le; wang, lianxiang; wang, dongdong; song, yanhua; zhang, xiangbin; huang, yao-wei title: discovery of a novel swine enteric alphacoronavirus (seacov) in southern china date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: adpn fb outbreaks of diarrhea in newborn piglets without detection of transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv) and porcine deltacoronavirus (pdcov), have been recorded in a pig farm in southern china since february . isolation and propagation of the pathogen in cell culture resulted in discovery of a novel swine enteric alphacoronavirus (tentatively named seacov) related to the bat coronavirus hku identified in the same region a decade ago. specific fluorescence signal was detected in vero cells infected with seacov by using a positive sow serum collected in the same farm, but not by using tgev-, pedv- or pdcov-specific antibody. electron microscopy observation demonstrated that the virus particle with surface projections was – nm in diameter. complete genomic sequencing and analyses of seacov indicated that the extreme amino-terminal domain of the seacov spike (s) glycoprotein structurally similar to the domain of the alphacoronavirus nl , whereas the rest part of s structurally resembles domains b to d of the betacoronavirus. the seacov-s domain associated with enteric tropism had an extremely high variability, harboring -amino-acid (aa) substitutions and a -aa insertion, compared to that of hku , which is likely responsible for the extended host range or cross-species transmission. the isolated virus was infectious in pigs when inoculated orally into -day-old newborn piglets, leading to clinical signs of diarrhea and fecal virus shedding. these results confirmed that it is a novel swine enteric coronavirus representing the fifth porcine coronavirus. coronavirus (cov) is an enveloped, single-stranded, positive-sense rna virus of the order nidovirales, family coronaviridae, subfamily coronavirinae, which comprises four genera, alpha-, beta-, gamma-, and delta-cov. covs infect humans, other mammals, and birds, causing subclinical or respiratory and gastrointestinal diseases (de groot et al., ; woo et al., ) . as of date, three types of swine enteric covs (secovs): transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv) and porcine deltacoronavirus (pdcov), have been identified to induce clinical diarrhea in young pigs (jung et al., ; pensaert and de bouck, ) . in particular, emergences of variant pedv fatal to newborn piglets in china in late (pan et al., ) , and later in the united states in tian et al., ) , have posed a serious threat to the pork industry. most recently, several chimeric secov strains with a tgev genomic backbone replaced by a pedv spike (s) gene were identified from swine fecal samples in europe (akimkin et al., ; belsham et al., ; boniotti et al., ) , implying that novel secov pathogens could emerge by inter-cov recombination under co-infection. the s gene encodes a glycoprotein, forming trimer projections on the viral surface, which is a major structural protein critical for cov attachment and entry into the host cell (hulswit et al., ) . in addition to recombination events between two distinct covs, amino acid (aa) mutations in the s protein may alter the tropism of the virus. for example, -aa substitutions and a -aa insertion in the amino-terminal domain (ntd) of the s glycoprotein of a murine hepatitis cov (mhv) variant confer the ability to bind and in some cases infect cells of nonmurine species including swine cells (schickli et al., ) . in this study, we report the isolation and genetic characterization of a novel swine enteric alphacoronavirus (tentatively named seacov), related to a bat enteric coronavirus, from a pig farm that reported newborn-piglet diarrhea in southern china in . this is yet another example to corroborate that the extended host range of cov, here from bat to pig, is likely associated with aa substitutions at the ntd of the s glycoprotein. furthermore, we conducted a pilot experimental infection study with this novel seacov, confirming its infectivity and ability to induced clinical signs of diarrhea in piglets. baby hamster kidney fibroblast cell line bhk- (atcc ccl- ), swine testis cell line st (atcc crl- ), porcine kidney epithelial cell line llc-pk (atcc cl- ), and african green monkey kidney epithelial vero cell (atcc ccl- ) were individually grown in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs) and % antibiotics (penicillin, streptomycin, w/v). a vero cell line stably expressing the tgev receptor porcine aminopeptidase n (vero-papn) was cultured in dmem supplemented with μg/ml puromycin and antibiotics (unpublished data). all cells were grown at °c with % co . a pan-cov rt-pcr assay was used to detect the unknown pathogen with a pair of primers: cor-fw ( ′-acwcarhtvaayytnaartaygc- ′) and cor-rv ( ′-tcrcayttdggrtartccca- ′) as described (moes et al., ) . after the pathogen (seacov) was identified, specific primers targeting the seacov-nucleocapsid (n) gene (the forward primer seaf: ′-atggataaacctgaatggaagcg- ′, and the reverse primer sear: ′-caccatctcaacctcttcctcag- ′) were used for virus detection during isolation and subsequent passages. fecal specimens collected from diarrheic piglets and positive for seacov rna were homogenized in dmem containing antibiotics followed by centrifugation at × g for min. the supernatants was inoculated onto confluent monolayers of bhk- , st, llc-pk or vero cells cultured with the maintenance medium plus trypsin (mmt) at °c and % co . the mmt consisted of dmem supplemented with % fbs, % antibiotics and μg/ml trypsin (sigma). cells were observed daily to record the development of cytopathic effect (cpe) as described previously (pan et al., ) . the virus strain isolated in vero cells with mmt, designated as ch/ gd- / , was plaque-purified in the presence of trypsin using neutral red staining as described (qin et al., ) . it was passaged serially using the culture supernatant and the viral titer was determined by plaque assay. supernatant from purified seacov-infected cell cultures showing cpes was negatively-stained. grids were stained with % sodium phosphotungstic acid (ph . ) for . min and examined using a hitachi model h- tem. vero cells infected with seacov on -well plates were washed twice with phosphate-buffered saline (pbs) and fixed with acetone. one hundred and fifty microliters of the collected sow serum samples at a : dilution in pbs was added to the cells in each well and incubated for h at room temperature. cells were washed thrice with pbs followed by addition of μl fitc-labeled rabbit anti-pig igg (thermo fisher scientific, usa) at : dilution. after incubation for h at room temperature, the cells were washed with pbs, stained with μl ′, -diamidino- -phenylindole (dapi) at : dilution and visualized under a fluorescence microscope. for antibody cross-reactivity test, vero cells infected with seacov or pedv (zju/g / strain; genbank accession no. ku ), vero-papn cells infected with tgev (purdue strain; a gift from dr. rong ye at shanghai medical college of fudan university), and llc-pk cells infected with pdcov (hunan strain; genbank accession no. ky ) were stained with the anti-pedv-n, anti-tgev-n and anti-pdcov-n monoclonal antibody (purchased from medgene labs, brookings, sd, usa), respectively. the fitc-conjugated goat anti-mice igg (thermo fisher scientific, usa) was used as the secondary antibody followed by dapi staining. total rna was extracted from the isolated virus with trizol reagent, and cdnas were subsequently amplified by superscript ii with specific primers according to the manufacturer's instructions (thermo fisher scientific). a total of primer pairs based upon the bat cov hku strain gd - (genbank accession no. ef ; supplemental table s ) were designed to amplify the complete genome of seacov. pcr products were purified and cloned into a pcr-blunt vector (thermo fisher scientific). for each amplicon, three to five individual clones were sequenced to determine the consensus sequence. the sequences were assembled and analyzed using the dnastar program. multiple alignments of the full-length genomes, non-structural protein genes and s genes with representative cov sequences and phylogenetic analyses were performed using the neighbor-joining method in mega . , respectively. structure homology-modeling of seacov s glycoprotein was performed by the swiss-model server (https://www.swissmodel.expasy.org/). a pilot animal experiment was approved by the experimental animal ethics committee of zhejiang university (approval no. zju ). briefly, ten -day-old conventional piglets, free of seacov, pedv, tgev, and pdcov rna in the feces, were assigned into two groups with in each. piglets in each group were housed with their mothers (seacov rna and serum antibody negative as determined by ifa) without any artificially supplemental colostrum or milk. piglets in group one were each challenged orally with a seacov/ch/gd- / /p isolate at a dose of × plaque-forming units (pfu)/ml ( ml per pig), whereas piglets in group two each received ml of dmem orally as negative controls. all the piglets were monitored daily for any signs of illness. two piglets in each group were euthanized at days post-infection (dpi) while the remaining three in each group were necropsied at dpi. the duodenum, jejunum and ileum samples were subjected to histological examinations by hematoxylin and eosin (he) staining, respectively. the villous height (vh) and the crypt depth (cd) were measured on a minimum of eight different sites per small intestinal segment, and the ratios of vh to cd were then calculated to quantify the villous atrophy according to previously described (jung et al., ) . fecal swabs for viral rna detection were collected at , , , , and dpi from all five pigs until they were alive. beginning from february , a remarkable increase in outbreaks of newborn-piglet diarrhea occurred in a commercial pig farm located in guangdong province of southern china. clinical signs of affected pigs were characterized by acute vomiting and watery diarrhea (fig. a) . the mortality rate was over % in piglets less than days old during february-may . in addition, the small intestine of the diseased pigs y. pan et al. veterinary microbiology ( ) - displayed thin walls and contained yellow watery feces (fig. b) , which was indistinguishable from that of pedv infection described previously pan et al., ) . fecal and small intestinal samples collected from affected piglets in this farm were submitted to our labs at zhejiang university and hog production division of wen's foodstuffs group, respectively, for routine laboratory diagnostics. upon laboratory analysis by rt-pcr, rna of pedv, tgev, pdcov or porcine hemagglutinating encephalomyelitis virus (phev), was not detected in these samples (data not shown). other possibly known viral pathogens associated with piglet diarrhea such as porcine enterovirus, rotavirus or mammalian orthoreovirus (qin et al., ) also could not be detected. subsequently, samples were tested by a pan-cov rt-pcr assay designed to amplify a conserved region of -bp in the orf b gene for all cov members (moes et al., ) . this test was positive for all the selected samples collected during february to may (data not shown). sequencing of the pcr products revealed that they were % identical to the corresponding region (nucleotide [nt] positions - ) of four known bat enteric alphacoronavirus hku strains (genbank accession nos. ef to ef ) identified from guangdong province and hong kong in and (lau et al., ) . the prevalence rate of bat cov hku from these two regions was reported to be . % ( / ) and . % ( / ) in chinese horseshoe bats (rhinolophus sinicus), respectively (lau et al., ) . hku infection associated with the other animal species has never been investigated. the results from pan-cov rt-pcr detection indicated that an hku like viral pathogen might be responsible for outbreaks of diarrhea in the pig farm. in an effort to isolate the novel swine enteric hku -related cov (seacov), suspension supernatants of selected hku -positive samples were prepared and inoculated in a panel of bhk- , st, llc-pk and vero cell lines routinely used to isolate porcine covs. cultured supernatants from each inoculated cell line were blind-passaged serially. from vero cell culture, we successfully isolated one seacov strain with cpe characterized by syncytia formation at h post-infection, beginning from passage two (p ) and in the following passages after plaque purification (fig. c) . furthermore, viral antigens were demonstrated in seacov-infected vero cells by ifa, with a serum sample collected from a sow mothering the diseased piglets (fig. d ), but not with the specific monoclonal antibodies against the n protein of pedv, tgev or pdcov (fig. ) , suggesting that seacov are probably antigenetically distinct from the three known porcine covs. seacov antibody-negative sera from the same farm were also found, as staining with these sera in seacov-infected cells displayed no fluorescent signal (fig. e) . electron microscopy of a negatively stained sample from the supernatant of virus-infected vero cells demonstrated that the virus particle was to nm in diameter, and had surface projections typical of cov (fig. f) . seacov rna was detected in supernatants from all virus passages to date (p to p ) by rt-pcr with primers seaf and sear. the virus titer reached up to × pfu/ml at p . this isolated cov strain was designated as seacov/ch/gd- / . we next determined the complete genome of p of ch/gd- strain by rt-pcr amplification of regions covering the entire seacov, as described previously for pedv or pdcov genomic cloning wang et al., b) . the complete genome sequence of the ch/ gd- / /p strain has been deposited in genbank under accession no. mf . the genomic sequence of ch/gd- / /p is , nt in length, excluding the poly(a) tail. the genome organization is similar to those of the four hku strains and a bat cov identified in yunnan province in southwestern china (btrf-alphacov/yn , genbank no. kj ), with the typical gene order ′-orf a/ b (orf ab)-s-orf -e-m-n-ns a- ′ (fig. ) . the ch/gd- / /p strain is -nt longer than hku ( , nt), including a -nt (ttg) insertion at nt , - , (corresponding to the hku /gd sequence) in the nonstructural protein (nsp) region, a -nt (ggcctc) insertion at nt , - , in the s gene, and a -nt (gta) deletion at nt , - , in the m gene (fig. ) . however, these insertions/deletion are not unique for seacov since they are also present in the btrf-al-phacov/yn genome in comparison with hku . seacov shared . % nt sequence identity with the four hku strains, and exhibited . % nt identity with btrf-alphacov/yn . accordingly, seacov is phylogenetically located between hku and btrf-alphacov/yn , together forming a sublineage closely related to the proposed alphacov group- b lineage, including pedv and human covs nl and e, at the complete genome level (fig. a) . however, analysis of the phylogenetic tree constructed based on the s genes (fig. b ) indicated that these six hku -related cov strains along with a newly identified rat alphacov, lrnv (wang et al., a) , formed a separate lineage clustered within the betacovs. the previous studies have suggested that hku and the related lrnv probably resulted from an ancient recombination event with an alphacov genomic fig. . ifa results of vero cells infected with seacov or pedv, vero cells stably expressing porcine aminopeptidase n (vero-papn) infected with tgev, and llc-pk cells infected with pdcov at h post-infection. seacov-infected vero cells were stained with the anti-pedv-n, anti-tgev-n or anti-pdcov-n monoclonal antibody, respectively (left panels). cells infected with pedv, tgev or pdcov were stained with the respective virus-specific antibody as the controls (right panels). the fitc-conjugated goat anti-mice igg was used as the secondary ab in ifa. magnification = ×. fig. . schematic diagram of the genomic structure of seacov and the proposed domain organization of the seacov spike protein s subunits according to the structure similarity analysis with nl and mhv that are both structure available. numbers indicate amino acid positions in s glycoprotein of seacov, nl or mhv, respectively. see supplemental fig. s for the detailed sequence alignment. nucleotide insertion/deletion at three locations (nsp , s and m genes) in seacov compared to the consensus sequences of four bat-cov hku strains (genbank accession nos. ef to ef ) are marked by "*". y. pan et al. veterinary microbiology ( ) - backbone replaced by a betacov s gene (lau et al., ; wang et al., a) . furthermore, pairwise comparison of seacov genomic sequence with hku indicated that the most dissimilar region was in the s gene, particularly, in the extreme ntd (aa - ). the entire seacov s protein had . % aa identical with s of the hku /gd strain, but there was only a . % identity in the extreme ntd of the s protein (s-ntd) between seacov and hku . we identified a total of -aa substitutions plus a -aa insertion (resulting from a -nt insertion as mentioned above) within the seacov s-ntd compared to hku . in contrast, only aa substitutions were found in the remaining part of the s protein. the extreme ntd changes in seacov are likely to be associated with the extended host range, similar to a previously reported mhv variant that was able to expand nonmurine-species tropism, with the phenotype mapped to substitutions and a -aa insert in ntd of s subunit (schickli et al., ) . during the time of this manuscript preparation, a sequence of another hku -related seacov strain gds , identified in the same region, was reported online but it did not give in-depth analyses (gong et al., ) . it remains unknown if gds can be isolated in cell culture. moreover, neither detection of serum anti-seacov antibodies nor observation of virus morphology was demonstrated. nevertheless, comparative sequence analysis showed that the gds strain, having the same genomic size ( , nt), shared . % nt homology with gd- / /p at the complete genome level. however, the gds sequence was determined by the next generation sequencing, which should theoretically be less accurate than the gd- / /p sequence determined based upon the consensus sequences from different short rt-pcr fragments covering the full-length genome. the s-ntd of gds also contains -aa substitutions and a -aa insertion compared to that of hku . there are only three aa differences at the positions (d/g), (m/r) and (a/v) in the s-ntd between gds and gd- / /p . the corresponding aa in bat cov hku at these positions are g, m and m. for nonstructural protein genes analysis, the seacov gd- / /p exhibited . % and . % nt identities, . % and . % nt identities, or . % and . % nt identities with gds and hku based on the orf ab, the orf a, or the orf b genes, respectively. these sequence analyses suggested that the seacov strains gd- / and gds could have the same origin. the s glycoprotein of seacov or hku is unique and not related to any currently known betacovs at the aa sequence level. most recently, the structures of several cov s glycoprotein trimmers have been resolved (walls et al., a (walls et al., , b . the betacovs comprise of four domains (domains a-d) in the s subunit whereas human alphacov nl shows an additional domain, named domain (equivalent to the extreme ntd) compared to betacovs (fig. ) . therefore, a structure homology-modeling was performed in order to better understand the evolutionary origin of the s glycoprotein of seacov/hku in the protein structure level. surprisingly, the result suggested a hybrid structure of seacov-s: the extreme ntd (domain ) of seacov-s is structurally similar to that of nl , whereas the rest part in s structurally resembles domains b to d of the betacov mhv ( fig. and supplemental fig. s ). the s subunits of seacov and mhv also have a similar structure (supplemental fig. s ). the deduced structure of the linking region between domain and domain b of seacov is uncertain. we hypothesize that the domain a is likely not present, which may be a unique feature of seacov/hku s subunit. since domain and domain a are structurally similar and might come from a gene-duplication event (walls et al., b) , we also hypothesize that either of them is likely dispensable in the s of covs. in addition, the presence of domain in seacov/hku is in line with the enteric tropism of these viruses since pedv and tgev also possess this domain (hulswit et al., ) . future study on developing the seacov infectious clone and resolving the alphacov/betacov-hybrid seacov-s glycoprotein structure are warranted to confirm these findings. in order to test whether or not, seacov is able to infect pigs, we performed a pilot challenge experiment using the cell-cultured seacov/ch/gd- / isolate. as expected, the five piglets in dmem-inoculated group neither showed clinical sign nor shedding y. pan et al. veterinary microbiology ( ) - virus in the feces throughout the experimental period (data not shown). in contrast, clinical signs characterized by acute vomiting and watery diarrhea (similar to fig. a) were observed in the five seacov-infected piglets at to h post-infection, and thereafter lasted until necropsy. fecal virus shedding was detected in five seacov-infected pigs at , and dpi, and in three remaining pigs at and dpi by rt-pcr with the primers seaf and sear (data not shown). sequencing of the pcr products indicated that they were identical with the seacov n gene sequence, confirming that the infectious virus was originated from the seacov isolate. upon histopathological analysis, no intestinal lesions were observed in control pigs (fig. ) ; the mean duodenal, jejunal and ileal vh/cd were . ( ± . ), . ( ± . ) and . ( ± . ), respectively. typical microscopic lesions, showing gradual atrophy with significantly reduced vh/cd (the mean jejunal or ileal value was . [ ± . ] or . [ ± . ]), diminishing capillaries and central lacteals of the intestinal villous (fig. ) , were detected in the jejunum and ileum of seacov-infected piglets. the duodenal sections displayed only mild microscopic lesions (the mean duodenal vh/cd = . [ ± . ]) in all seacov-infected pigs (fig. ) . it was different from the result observed for the experimental infection using the virulent chinese pedv strain, in which marked microscopic lesions in all the three parts of the small intestine were found (zhang et al., ) . the results indicated that the seacov isolate is actually infectious and causes diarrhea in pigs. since the specific non-swine antibodies against the structural proteins of seacov are not available currently, further comprehensively pathological studies by immunohistochemistry and serological assays, which is not the scope of this study, are warranted to provide more information on seacov infection. in summary, we have isolated, sequenced and genetically characterized a novel swine enteric alphacoronavirus, which is probably distinct from pedv, tgev and pdcov antigenetically, from diarrheal samples in a pig farm of southern china in . the isolated seacov can actually infect and cause diarrhea in pigs, and should represent the fifth porcine coronavirus in addition to pedv, tgev (considering that porcine respiratory virus, prcv, is a variant of tgev), pdcov and phev. to our knowledge, this is also the first study describing seacov related to the bat coronavirus hku that could be isolated and propagated in cell culture. however, infection of vero cells (a monkey cell line) with seacov also raises concerns about its potential host range other than swine. we also identified that the extreme ntd (aa - ) of seacov spike protein consists of -aa substitutions and a -aa insertion compared to that of hku , which is likely to be responsible for the cross-species transmission. moreover, this region but not the other betacov-related domains of seacov s subunit is structurally similar to the alphacov domain , implying that these viruses gained enteric tropism through this domain. the results provide much needed information on seacov and hku evolution, and the availability of seacov in cell culture will guide future efforts to develop effective vaccines against seacov. . representative histological examinations of the duodenum, jejunum and ileum samples collected at days post-infection from piglets inoculated with seacov or dmem in the animal challenge experiment. sections of jejunum and ileum in the seacovinfected group showed scattered areas of villi atrophy, whereas the section of duodenum showed mild microscopic lesions as compared to the dmem control group. y. pan et al. veterinary microbiology ( ) - new chimeric porcine coronavirus in swine feces characterization of a novel chimeric swine enteric coronavirus from diseased pigs in central eastern europe in porcine epidemic diarrhea virus and discovery of a recombinant swine enteric coronavirus virus taxonomy: ninth report of the international committee on taxonomy of viruses a new bat-hku -like coronavirus in swine origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states coronavirus spike protein and tropism changes pathology of us porcine epidemic diarrhea virus strain pc a in gnotobiotic pigs porcine deltacoronavirus infection: etiology, cell culture for virus isolation and propagation, molecular epidemiology and pathogenesis complete genome sequence of bat coronavirus hku from chinese horseshoe bats revealed a much smaller spike gene with a different evolutionary lineage from the rest of the genome a novel pancoronavirus rt-pcr assay: frequent detection of human coronavirus nl in children hospitalized with respiratory tract infections in belgium isolation and characterization of a variant porcine epidemic diarrhea virus in china a new coronavirus-like particle associated with diarrhea in swine genetic and pathogenic characterization of a novel reassortant mammalian orthoreovirus (mrv ) from a diarrheic piglet and seroepidemiological survey of mrv in diarrheic pigs from east china the n-terminal region of the murine coronavirus spike glycoprotein is associated with the extended host range of viruses from persistently infected murine cells evidence of recombinant strains of porcine epidemic diarrhea virus cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy discovery, diversity and evolution of novel coronaviruses sampled from rodents in china complete genome sequence of porcine deltacoronavirus strain ch/sichuan/s / from mainland china discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus identification and pathogenicity of a variant porcine epidemic diarrhea virus field strain with reduced virulence this work was supported by the national key research and developmentprogram of china ( yfd and yfd ), and the national natural science foundation of china ( ). we thank dr. narayan paudyal for conducting english language review. supplementary data associated with this article can be found, in the online version, at https://doi.org/http://dx.doi.org/ . /j.vetmic. . . . key: cord- -aea d f authors: escribano, j.m.; borca, m.v. title: immunogenicity of a recombinant coronavirus spike glycoprotein expressed in transgenic plants date: journal: bundesgesundheitsblatt gesundheitsforschung gesundheitsschutz doi: . /s sha: doc_id: cord_uid: aea d f recently, it has been demonstrated that plants offer the possibility of producing low cost subunit vaccines that can be either parenterally or orally administered. here we review data we obtained on the immunological response elicited by two recombinant versions of the glycoprotein s from the swine transmissible gastroenteritis coronavirus (tgev) expressed in transgenic plants. arabidopsis or potato plants were genetically transformed with cdnas constructs encoding the n-terminal domain (aa residues – ) or the full-length glycoprotein s of tgev, responsible for the neutralizing antibody induction against the virus, under the control of the cauliflower mosaic virus s (camv s) promoter. genomic dna and mrna analysis of leave extracts from transformed plants demonstrated the incorporation of the foreign cdna into the plant genomes as well as their transcription. expression of recombinant polypeptides was observed in most transgenic plants by elisa using specific antibodies. mice immunized either parenterally with leave extracts from transgenic arabidopsis plants or, more interestingly, fed with potato tubers, developed antibodies that specifically reacted with tgev in elisa, immunoprecipitated the glycoprotein s and in some cases neutralized the virus infectivity. from the above results, we conclude that transgenic plants expressing glycoprotein s polypeptides may be potentially used as a source of recombinant antigen for vaccine production. recently, it has been demonstrated that plants offer the possibility of producing low cost subunit vaccines that can be either parenterally or orally administered.here we review data we obtained on the immunological response elicited by two recombinant versions of the glycoprotein s from the swine transmissible gastroenteritis coronavirus (tgev) expressed in transgenic plants.arabidopsis or potato plants were genetically transformed with cdnas constructs encoding the n-terminal domain (aa residues - ) or the full-length glycoprotein s of tgev, responsible for the neutralizing antibody induction against the virus, under the control of the cauliflower mosaic virus s (camv s) promoter.genomic dna and mrna analysis of leave extracts from transformed plants demonstrated the incorporation of the foreign cdna into the plant genomes as well as their transcription.expression of recombinant polypeptides was observed in most transgenic plants by elisa using specific antibodies.mice immunized either parenterally with leave extracts from transgenic arabidopsis plants or, more interestingly, fed with potato tubers, developed antibodies that specifically reacted with tgev in elisa, immunoprecipitated the glycoprotein s and in some cases neutralized the virus infectivity.from the above results, we conclude that transgenic plants expressing glycoprotein s polypeptides may be potentially used as a source of recombinant antigen for vaccine production. swine transmissible gastroenteritis virus (tgev) is the causative agent of acute diarrhea of newborn piglets that provokes high rate mortalities in affected farms. protective immunity against this disease has to be developed in pregnant sows in order to confer passive protection to the piglets trough colostrum and milk. neutralizing antibodies against the virus are directed mainly to glycoprotein s [ , ] , and relevant epitopes in neutralization have been mapped into the n-terminal domain of this protein [ ] . four major antigenic sites have been described in glycoprotein s, of which site a is the immunodominant [ , , ] . only vectors expressing the glycoprotein s with tropism that favored antigenic presentation in the mucosal surfaces have conferred protection against tgev in suckling piglets [ ] . the concept of vaccine production in transgenic plants was first introduced by mason and co-workers in [ ] . proteins involved in protective immune response can be produced at a low cost and easily purified from plant extracts for parenteral inoculation. in addition, oral immunization by edible vaccines produced in transgenic plants could stimulate immune responses at the portal of entrance for many pathogens, facilitating the design of large-scale immunization programs. the presence of specific antigens in plants, even at low levels, can raise immune reactions by the oral route comparable to conventional vaccines [ , , ] . today, hepatitis b surface antigen [ ] , e. coli heat-labile enterotoxin (lt-b) antigen [ ] , norwalk virus capsid protein [ ] , vp antigen from foot and mouth disease virus [ , ] , cholera toxin b subunit [ ] , glycoprotein s from swine transmissible gastroenteritis virus [ ] , and vp from rabbit hemorrhagic disease virus [ ] are vaccine antigens expressed in transgenic plants and tested for the immune response elicited in immunized animals. here, we review the data obtained about the feasibility of expressing the glycoprotein s from tgev in two different transgenic plants, as well as the antigenicity and immunogenicity of the plant-derived protein administered either parenterally or orally to mice. the protein s is an excellent model for developing oral vaccines against enteric pathogens of mammals because of its immunogenicity and resistance to degradation in the gut. the binary prok i and prok ii recombinant plasmids (fig. ) , carrying a cdna coding for the n-terminal region or the full-length glycoprotein s respectively, were obtained by subcloning the corresponding sequences from previously obtained constructs into prok plasmid [ ] . recombinant prok derived plasmids allow selection of transformants on media containing kanamycin and stable integration into nuclear chromosomal dna from the plant. prok uses the camv s promoter to drive nominally constitutive transcription of the cloned genes. plant transformation with prok i and ii was carried out as described [ , ] by a. tumefaciens-mediated transformation. potato plants were transformed only with prok i plasmid. the transgenic plants resistent to the selective medium appeared similar in morphology to non -transgenic plants. more than different lines of arabidopsis transformants containing each construct were self-pollinated to obtain f lines. fifteen different transformed potato clones were also obtained with prok i. all lines from arabidopsis and potato were positive when screened for the presence of the recombinant genes by pcr analysis (fig. , a and b ). most plants harboring recombinant genes showed specific transcription of foreign genes by rt-pcr analysis (fig. , a ). the presence of the recombinant polypeptides in the plants harboring and expressing the foreign genes was investigated by elisa and western blot using an anti-tgev polyclonal serum. results demonstrated that leave or potato ex-tracts from all analyzed plants resulted positive in elisa (fig. , a and b ). however, no specific reaction in western blot was detected in any of the plant extracts analyzed (data not shown), probably due to the low levels of recombinant protein expression and to the conformational nature of most of the immunodominant epitopes present in this protein. from a titration elisa using different virus dilutions and a monospecific anti-glycoprotein s antibody, we have found that about - µg of soluble leave or tuber protein contain a glycoprotein s antigenic mass equivalent to that contained in . µg of purified tgev. the percentage of the total soluble protein corresponding to recombinant glycoprotein s polypeptides accumulated in the tissues of plant transformants could represent between . to . % of the total soluble protein. leave extracts from transgenic arabidopsis plants expressing the n-terminal or full-length glycoprotein s, were used to immunize mice.a control mouse was immunized with a leave extract from a plant transformed with prok plasmid. after three immunization doses ( µg of total protein per animal per injection), the specificity of mice sera was tested by an elisa using purified tgev as antigen. figure (a ) shows that all sera reacted with the virus showing, as expected, different titers. mice developed specific antibodies against glycoprotein s after immunization doses (fig. , a ). sera from all immunized mice were tested in a tgev neutralization assay. both glycoprotein s polypeptides produced in transgenic plants elicited virus neutralizing antibodies (neutralization indexes between . and . ; fig. , a ). serum from a nonimmunized mouse or from the mouse immunized with the plant transformed with prok plasmid did not show virus neutralization activity (fig. , a ) . transgenic potato tuber ex- gentechnisch veränderte pflanze · vakzine · coronavirus · tgev n-terminal (mice to ) or the full-length glycoprotein s (mice to ), using purified tgev as antigen. mice and represent elisa titers of two mice immunized with a prok transformed plant extract.( ) immunoprecipitation of glycoprotein s induced by tgev by sera from an immunized mouse after one ( ) , two ( ) or three ( ) immunization doses with transgenic plant extracts.a pool of monoclonal antibodies against glycoprotein s was used as control (c+).( ) neutralization indexes of sera from immunized mice with plant extracts expressing the n-terminal (mice to ) or full-length glycoprotein s (mice to ).the neutralization index is defined as the ratio between the log of virus titer in the presence of a control mouse serum and sera from mice immunized with transgenic plants expressing the antigens ( to ), or transformed with prok (c-).a rabbit anti-tgev serum showing high neutralization titer was also used as positive control (c+).elisa and neutralization index values are the mean of three independent experiments.panel b, ( ) elisa titers of sera from mice inoculated intraperitoneally with potato tuber extracts expressing the nterminal domain of glycoprotein s (mice to ), using purified tgev as antigen.mice and represent elisa titers of two mice immunized with a prok transformed plant extract.( ) elisa titers of sera from orally immunized mice with transgenic potato tubers expressing the n-terminal domain of glycoprotein s (mice to ).mice and represent elisa titers of two mice immunized orally with a prok transformed potato.( ) immunoprecipitation of glycoprotein s expressed by a recombinant vaccinia virus by antibodies present in sera from orally immunized mice with potato tubers expressing the n-terminal domain of the glycoprotein.a pool of specific monoclonal antibodies was used as a control positive serum (c+) and a serum from a mouse immunized with potato tubers transformed with prok was used as a negative control (c-) tracts also induced specific antibodies in immunized mice using the same immunization protocol (fig. , b ) . finally, to analyze the oral immunogenicity of glycoprotein s expressed in potato plants, a group of mice were fed with transgenic potato tubers expressing the n-terminal domain of glycoprotein s. the animals were fed three times per week with two gram of transgenic potatoes during a two months period. sera from these animals were analyzed by elisa showing an specific antibody response against the plant-derived glycoprotein s, indicating the oral immunogenicity of this protein when expressed in plants (fig. , b ) . as expected, no antibody response was detected in mice fed with potato tubers not expressing the glycoprotein s. the specificity of sera reactivity in elisa with the virus-induced protein was confirmed by immunoprecipitation of glycoprotein s expressed by a recombinant vaccinia virus (fig. , b ). in this report we show that full-length or the globular part (n-terminal domain) of tgev spike protein (glycoprotein s) expressed in transgenic plants retained the antigenic properties and elicited neutralizing antibodies when used to immunize animals. expression in eukaryotic hosts is required for antigenic determinants that are dependent on glycosilation. of the three major antigenic sites defined on glycoprotein s involved in the induction of tgev-neutralizing antibodies, sites a and b are complex, conformational, and glycosilation dependent. site d can be represented by synthetic peptides, although glycosylation has a minor effect on its conformation [ ] . several genetically engineered vaccines using prokaryotic vectors have failed against tgev. glycoprotein s expressed at high levels in escherichia coli and used to inoculate animals did not induce neutralizing antibodies or confer protection in vivo [ ] . plant cells present differences in protein glycosilation with respect to animal cells that could determine the loss of antigenic determinants in antigens expressed in transgenic plants. glycosylation in plants may differ in the extent of glycosylation, processing, or both of n-linked oligosaccharide side chains [ ] . furthermore, the complex glycans of plants are often smaller than those of animals in part due to the absence of sialic acid [ ] . the only precedent of a glycoprotein expressed in plants for vaccine development is the glycoprotein g of rabies virus [ ] . this protein expressed in tomato plants showed a molecular mass about four and six kda less than that obtained from virus-infected cells but still larger than the unglycosylated polypeptide chain [ ] . the molecular mass of glycoprotein s expressed in arabidopsis thaliana could not be determined because we were not able to detect the recombinant protein in western blotting. however, antigenic determinants with strong dependence of glycosylation seem to be preserved since the plant-derived antigens induced neutralizing antibodies in immunized animals, indicating that critical antigenic sites are at least in part correctly glycosylated in plants. the above described work demonstrates the feasibility of expressing glycoprotein s polypeptides in plants. because the site of insertion of the transferred dna into the cellular chromosomal dna is random, different levels of protein expression in independent transformants are expected.we obtained expression levels similar to that described with equivalent constructs expressing hepatitis b surface antigen or rabies virus glycoprotein [ , ] . more recently, expression levels of norwalk virus capsid protein in tobacco have been shown to be higher than the above mentioned antigens (up to . % of total soluble protein) [ ] . we have not found significant differences in foreign antigen plant expression between the two forms of glycoprotein s studied or among the different plants transformed. the use of different promoters, the use of plant-derived leader sequences and signal peptides, and mainly the modification of the codon usage of this protein could improve expression levels in plants. the demonstration that many proteins from pathogens, including some expressed in transgenic plants [ , , ] , are immunogenic when administered orally, encourage the study of other antigens expressed in plants to develop edible vaccines. glycoprotein s from tgev is an interesting model since this protein is resistant, at least when incorporated into the viral particle, to the gut degradation. in addition, the protective immune responses against tgev have to be stimulated at the mucosal surfaces in order to induce secretory and lactogenic immunity [ , , , ] . in the above described work, we have demonstrated that the n-terminal domain of glycoprotein s expressed in potato tubers is immunogenic when mice were fed with the transgenic potatoes [ ] . in conclusion, these results make reasonable to extend similar immunological studies in pregnant sows, in order to test the lactogenic immunity conferred to suckling piglets by the plant-derived glycoprotein s. antigenicity of structural components from porcine transmissible gastroenteritis virus critical epitopes in transmissible gastroenteritis virus neutralization antigenic structure of e -glycoprotein of transmissible gastroenteritis coronavirus epitope specificity of protective lactogenic immunity against swine transmissible gastroenteritis virus four major antigenic sites of the coronavirus transmissible gastroenteritis virus are located on the amino-terminal half of spike glycoprotein s antigenic homology among coronaviruses related to transmissible gastroenteritis virus tropism of human adenovirus type -based vectors in swine and their ability to protect against transmissible gastroenteritis coronavirus expression of hepatitis b surface antigen in transgenic plants oral immunization with a recombinant bactíerial antigen produced in transgenic plants expression of norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice induction of a protective antibody response to foot and mouth disease virus in mice following oral or parental immunization with alfalfa transgenic plants expressing the viral structural protein vp immunogenicity of transgenic plant-derived hepatitis b surface antigen protective immune response to foot-and-mouth disease virus with vp expressed in transgenic plants efficacy of a food plant-based oral cholera toxin b subunit vaccine expression of immunogenic glycoprotein s polypeptides from transmissible gastroenteritis coronavirus in transgenic plants immunization with potato plants expressing vp o protein protects against rabbit hemorrhagic disease virus expression of biologically active viral satelite rna from nuclear genome of transformed plants agrobacterium mediated gene transfer by infiltration of adult arabidopsis thaliana plants. c r acadl sci paris sciences de la vie residues involved in the antigenic sites of transmissible gastroenteritis coronavirus s glycoprotein studies of tgev spike protein gp expressed in e. coli and by a tgev-vaccinia virus recombinant detection, biosynthesis and some functions of glycans n-linked to plant secreted proteins expression of the rabies virus glycoprotein in transgenic tomatoes characterization of the iga and subclass igg responses to neutralizing epitopes after infection of pregnant sows with the transmissible gastroenteritis virus or the antigenically related porcine respiratory coronavirus passive immunity in transmissible gastroenteritis of swine: immunoglobulin classes of milk antibodies after oral-intranasal inoculation of sows with a live low cell culture-passaged virus lack of protection in vivo with neutralizing monoclonal antibodies to transmissible gastroenteritis virus oral immunogenicity of glycoprotein s from transmissible gastroenteritis coronavirus expressed in transgenic plants key: cord- -eqzs au authors: britton, p.; cármenes, r. s.; page, k. w.; garwes, d. j.; parral, f. title: sequence of the nucleoprotein gene from a virulent british field isolate of transmissible gastroenteritis virus and its expression in saccharomyces cerevisiae date: - - journal: mol microbiol doi: . /j. - . .tb .x sha: doc_id: cord_uid: eqzs au subgenomic mrna from a virulent isolate of porcine transmissible gastroenteritis virus (tgev) was used to produce cdna which was sequenced. two non‐overlapping open reading frames (orfs) were identified. the largest, encoding a polypeptide of amino acids (relative molecular mass (m(r)) ), was shown to be the viral nucleoprotein gene. the second orf, found ’to the larger orf, encodes a polypeptide of amino acids (m(r) ) which has yet to be assigned to a viral product. the nucleoprotein gene was expressed in yeast cells under the control of two types of yeast promoters: the constitutive pgk promoter, and the inducible gal promoter. yeast cells containing recombinant plasmids, with the nucleoprotein gene in the correct orientation, produced a polypeptide of m, , identical to the viral product, that reacted with a specific monoclonal antibody. the coronaviruses comprise a large group of enveloped positive-stranded rna viruses and cause a variety of infections in a wide range of animal hosts (garwes, ; siddell et at., ) . transmissible gastroenteritis virus (tgev) is a coronavirus that causes gastroenteritis in pigs, resulting in a high mortality among neonates. tgev is composed of a capped polyadenylated single-stranded rna genome of m, . x ^ or kb in length (garwes et al.. ; dennis and brian, ) and three major structural polypeptides: a surface glycoprotein (spike or peplomer protein) with a monomeric m. , a giycosylated integral membrane protein observed as a series of potypeptides of m, - , and a basic phos-phorylated protein {the nucleoprotein) of m, associated with the viral rna (garwes and pocock, ) . infection of susceptible cells with tgev results in the synthesis of a 'nested" set of subgenomic mrna species with common ' termini but different extensions in the ' direction, a phenomenon also observed with other coronaviruses such as infectious bronchitis virus (ibv, stern and kennedy. ) , though the size and number of the mrna species vary with the virus. tgev appears to have, in addition to the genomic sized rna species, four major ( . . . , . and . kb) and two low abundant ( . and . kb) mrna species (britton etal., ; jacobs etal., ) . each coronavirus subgenomic mrna species is believed to direct the synthesis of a protein from the ' proximal region (sturman and holmes, : stern and sefton, ) . in vitro translation studies have shown that the mrna species of . kb directs the synthesis of the viral nucleoprotein (britton et al., ; jacobs ef al., ) . kapke and brian ( ) have sequenced the ' end of genomic rna from the avirulent purdue strain of tgev and identified two complete open reading frames within the first bp of the genome. one of the open reading frames encodes a polypeptide of m, and has properties similar to those found in the ibv and mouse hepatitis virus (fyihv) nucleoproteins. here we report the production of cdna from subgenomic mrna species isolated from tgev-infected cells using a virulent british isolate of tgev. £ coti cells are frequently unable to process eukaryotic proteins correctly, often producing the protein in the wrong conformational structure as a result of incorrect folding, leading to the degradation of foreign gene products. several heterologous proteins have been successfully expressed in saccharomyces cerevisiae, including eukaryotic proteins (calf chymosin, mellor ef al.. ) and viral antigens (hepatitis b surface antigen (hbsag), valenzuela etal., ; fviiyanohara etai, ) . s. cerevisiae cells are easy to grow and unlike e. coli cells have the advantage that they lack any pathogenicity, making them particularly useful as a source of large quantities of hormones and antigens of interest in human and veterinary medicine. we have expressed the nucleoprotein gene in yeast celts and the product was shown to be identical to the tgev nucleoprotein by immunoblotting using a specific monoclonal antibody. cloning from tgev mrna species tgev poly{a)-containing mrna species were fractionated on non-denaturing sucrose gradients and tgev cdna, derived from the . kb mrna species, was synthesized by the vector-primer system, and isolated as described in experimental procedures. out of colonies transformed with recombinant dna, contained dna that hybridized to ^^p-labelled tgev poly{a)-containing rna. three recombinant plasmids, shown to contain cdna inserts of . kbp and designated pts - , pts - and ptsi - , were labelled with p^s]-datp by nick translation and hybridized to glyoxylated tgev mrna species separated on % agarose gels as described in experimentat procedures. all three recombinant plasmids were found to hybridize to all tgev rna species. in contrast, other recombinant plasmids derived from different mrna species only reacted to one or more tgev rna species. this observation indicated that ptsi - , pts - and pts - contained regions of cdna complementary to regions present on all the tgev nested' mrna species. this phenomenon can only occur if the cdna was derived from the ' end of the rna and since the sizes of the cdna inserts were about . kbp, then most of the . kb mrna species was copied. plasmid pts - was purified and a restriction map constructed {fig. a). the end containing the poly(a) tail was confirmed by probing southern blots with ^^p-labelled oiigo(dt)i -i oligonucleotides. tgev poly(a)-containing mrna species isolated from infected cells were primed using a bp h/ndl!l-xtial fragment purified from the ' end of the tgev cdna cloned in plasmid pts - . the cdna generated was endrepaired and cloned into plasmid puc following the addition of fcori linkers. a recombinant plasmid, pf f- ( fig. ib) , was identified and found to contain a cdna insert of . kbp with restriction sites at one end identical to those at the ' end of plasmid pts - (fig. a) . indicating that the two cdna fragments were contiguous. the cdna from plasmids ptsi - and pf f- was subcloned into m mp vectors using either the shotgun method (bankier and barrell, ) or by using specific restriction fragments and sequencing entirely in both directions. the corresponding dna sequence bp from the ' end of the first open reading frame to the poly(a) tail is illustrated in fig. . the cdna was translated in all six reading frames, of which only the translation in the virussense strand revealed any open reading frames. the longest open reading frame, initiating from the atg at position , of bp overlapped both cdna inserts and encoded a potypeptide of amino acids, with an m^ of . the basic nature and size of the polypeptide indicated that it corresponded to the viral nucleoprotein which is observed to have m, and in tgev-infected cells and m, in virions (garwes et al., ) . the difference between the theoretical and predicted molecular weights of the protein may result from the degree of phosphorylation. garwes ef al. ( ) for tgev and stohlman and lai ( ) for mhv have shown that the nucleoproteins are phosphorylated on serine residues, which represent ( %) of the residues in strain fs / and ( %) in the purdue strain of tgev (kapke and kapke and bnan ( ) . the nucleoprotein (bases - ) and a hypothetical hydrophobic prote,r^bases,t )thactaaacdtgggr!s^^^^^^ .,,_ ,","" ^f^f'^^^';'""°"^*°'^'"j^'"'^^epurduestrain (kapkeandbrian, i ) .theboxedaminoacidsareidenticalresiduesfound between ibv boursnelle(a/.. )andf; hv(skinnerandsiddellj ).thebrokennnesrefertothetwomaiorand weaker areasof homology betwe^t hethreenudeoprate,n^s^d™dbydot-malrixanalysis (fig. ) .thesesequencedatahavebô pen reading frame, initiating from the atg at position , is composed of bp and encodes a polypeptide of amino acids of m, -initial analysis using a universal codon usage table (staden and mclachlan, ) , or a codon usage table constructed from the tgev nucleoprotein gene, suggested that the polypeptide might not be produced because of its poor codon usage. the smaller open reading frame should be present on an mrna species of . kb; the smallest mrna species detected in tgev-infected cells is the . kb low-abundance species (britton ef at., ) , showing that a polypeptide of m, could be produced by this mrna species. ). the polypeptide sequence has three areas of homology with the nucleoproteins from ibv (boursnell et al., ) and mhv (skinner and siddell, ) as analysed by dot matrix (fig. ). previous work (britton et at., ) showed that antibodies raised against a purified chimaeric protein, produced from the pts - h/ndlll fragment ( - kbp) fused to the ' end of the tacz gene, immunoprecipitated tgev nucleoprotein. this confirmed that the cdna carried on pts - contained most of the nucleoprotein gene. a second open reading frame, present only in ptsi - , is found bp ' to the nucleoprotein gene. tbis second a. ibv beaudette nucleoprotein (boursnell ef at-, ) and tgev fs / nucleoprotein. b. mhv jhm nucleopfotein (skinner and siddell, ) and tgev fs / nucleoprotein. c. tgev purdue nucleoprotein (kapke and brian, ) and tgev fs / nucleoprotein. the compariscmis used a window of residues with a stringency of . plasmids pts - and pf f- were digested with restriction endonucleases to produce fragments that did not overlap with puc fragments and the nucleoprotein gene vi/as constructed by cloning the fragments into the purified . kbp nde\-sma\ fragment from plasmid puc (fig. ) . a recombinant plasmid, ppbnio, was found to consist of the truncated puc vector with an insert consisting of the fragments from plasmids ptsi - and pf f- in the correct order. plasmid ppbnio contained the tgev nucleoprotein gene initiating bp ' from the wdel site. the plasmid also contained the smaller ' orf which initiates, in a different reading frame, bp ' from the end of the nucleoprotein gene and terminates bp ' to the drall sma\ combined site between the tgev cdna insert and the puc dna. the . kbp nde\-pvu\ fragment from ppbnio, containing the complete nucteoprotein gene, was purified, repaired using the klenow fragment of e. coii dna polymerase i, and samhi linkers were added (fig. ) . the resulting . kbp dna fragment was gel-purified and cloned into the samhi site of plasmid pbr . a recombinant plasmid, pbnp {fig. ), consisting of the . kbp samhi fragment in pbr , was used as a source of the nucieoprotein gene in a samhi cassette. under a constitutive promoter the . kbp samhi fragment from plasmid pbnp was ligated into the g/ll site of the yeast expression plasmid, pma , to position it downstream of the constitutive yeast pgk promoter. two recombinant plasmids were identified as having the . kbp samhi fragment in opposite orientations in pma . plasmid pynpi consisted of the tgev nucleoprotein in the correct orientation for expression from the yeast pgk, and pynp had the samhi fragment in the opposite orientation so that expression from the pgk promoter was prohibited. under an inducible promoter. the . kbp samhi fragment from plasmid pbnp was ligated into the samhi site of the yeast expression plasmid pb to position it downstream of the galactose inducible yeast gal promoter. a recombinant plasmid, pyngi, was found to contain the tgev nucleoprotein gene in the correct orientation for expression under the control of the yeast gal promoter. the expression plasmid pb does not contain a yeast terminator sequence, unlike plasmid pma , which is believed to improve the efficiency of expression. the . kbp g/ll-samhi fragment, containing the pgk terminator sequence, was purified from plasmid pma and ligated onto the . kbp samhi fragment from pbnp . the resulting mixture was electrophoresed into an agarose gel and a dna fragment corresponding to . kbp was purified and ligated into the samhi site of plasmid pbr . a recombinant plasmid, pbrnp , was isolated whioh contained the . kbp fragment in the correct orientation and fused onto the correct end of the tgev nucleoprotein gene-containing fragment. the . kbp samhi fragment was cloned into the samhi site of plasmid pb as described for the . kbp samhi fragment. two recombinant plasmids were isolated which had the . kbp fragment in opposite orientations. plasmid pyng (fig. ) consisted of the fragment in the correct orientation for expression from the yeast gal promoter but differed from plasmid pyngi in that it now had the pgk termination sequence spliced to the other end. plasmid pyng was similar to pyng except that the fragment was in the opposite orientation prohibiting expression of the tgev nucleoprotein gene from the gal promoter the recombinant plasmids pynpl, pynp . pyngi. pyng and pyng and expression vectors pma and pb were transformed into the s. cerevisiae strain bwg - a by the spheroplast method. yeast transformants were selected at °c for their ability to grow in the absence of leucine (pma , pynpi and pynp ) or uracil (pb , pyngi, pyng and pyng }. the generation time of the yeast strain before or after transformation with any of the plasmids was the same ( . h in synthetic media). the stability of the plasmids in the yeast strain was determined by growing the cells in complex non-selective media (ypd) and plating the cells on either synthetic selective media (sd) or on ypd. after seven generations in ypd, % of cells conserved pma and its derivatives pynp and pynp and % of cells conserved pb and its derivatives pyngi, pyng and pyng . in order to check that the yeast cells had not modified the recombinant plasmids in any way, plasmid dna was rescued from the yeast cells, amplified through dhl e coii cells and analysed by restriction endonuclease digestion. none of the plasmids rescued from the yeast cells appeared to be modified. yeast ceils transformed with the above piasmids were grown in the presence of % glucose or % galactose to show the production of any polypeptides under the control of tfie yeast promoters. to determine whether any tgevspecific polypeptides could be detected in transformed yeast the cells were disrupted with glass beads and the cell lysates were dot blotted onto nitrocellulose membranes and probed with a specific mouse monoclonal antibody as described in experimentai procedures. yeast cells transformed with pynp grown in the presence of % glucose or % galactose and cells transformed with pyngi and pyng grown in the presence of % galactose but not % glucose reacted with the monoclonal antibody. none of the other cell lysates reacted with the antibody. the nucleoprotein was also detected in yeast spheroplasts using this monoclonal antibody by immunofluorescence, and in cell extracts by enzyme-linked immunoabsorbance assay (elisa) (data not shown). cell lysates were prepared from cells transformed with pynpi and pynp grown in the presence of % glucose, and pyngi. pyng and pyng grown in the presence of % glucose or % galactose and analysed on % sds-polyacrylamide gels. the fractionated polypeptides were electrophoretically transferred onto nitrocellulose membranes which were probed as described. from fig, a it can be seen that the tgev nucleoprotein gene cloned in the correct orientation in plasmid pynpi and in plasmids pyng and pyng , in the presence of % galactose, produced a polypeptide of mr detected by the nucleoprotein monoclonal antibody da . the nucleoprotein gene cassette cloned in the opposite orientation did not produce the polypeptide of m, or any other polypeptide detectable with da under any conditions. expression from the gal promoter appeared to be more efficient than from the pgk promoter. the presence of the pgk termination sequence following the nucleoprotein gene under the control of the gal promoter did not seem to increase the efficiency of expression. there was no distinguishable difference in the sizes between fhe tgev nucleoprotein produced in the yeast cells and that present in purified viral particles (fig. b) . nucleoprotein degradation products, of which the main one had an m, of ( fig. a ), also reacted with the nucleoprotein monoclonal antibody. the complete nucleoprotein gene, from a british field isolate of tgev, was cloned over two cdna fragments. both cdna clones were sequenced and two potential open reading frames were identified in the viral sense strand. the initiation codons of both open reading frames were preceded by the heptameric sequence actaaac, which is similar to the sequences preceding fhe nucleoprotein, matrix, spike genes and other open reading frames (whose products have not yet been identified) in ibv and mhv genomes. the heptameric sequence contains the hexameric sequence, ctaaac, found by kapke and brian ( ) for the purdue strain of tgev. it has been suggested that this sequence is involved in the initiation of mrna synthesis for other coronaviruses {shteh ef ai., ). the sequence context of the initiation codon in the nucleoprotein gene, taaatgg, often occurs among functional eukaryotic initiator sequences, and the sequence at the initiation codon for the second open reading frame, gagatgg, is also favoured for eukaryotic initiation samples were probed with a specific monoclonal antibody as described in ihe lext. a. the cell-free extracts were as follows: lanes and are from the yeast recipient cell, lanes and cells transformed with pynpi and pynp : lanes and cells transformed with pyng . fanes and cells transformed wilh p yng ; and lanes and cells transformed with pyng . the celts used for lanes . , , , and were grown in the presence of glucose and cells used for lanes . . and were grown in the presence of galactose b. lanes and are extracts from cells translormed with pvng and pyng grown in rhe presertce of galactose. lane contained fractionated proteins from purified tgev virions. sequences (kozak, ) . both tgev genes terminate with the same single stop codon, taa. the -base sequence, tggaagagct, observed by kapke and brian ( ) , found bp from the poly(a) tail, is similar to that found in both ibv ( bp from the po!y(a) tail) and mhv ( bp from the poly(a) tail) and may be a recognitioti site or binding site involved in the synthesis of the negative rna strand (boursnell ef al.. ; kapke and brian, ) . knowledge of the amino acid sequences of gene products from different viral strains can help to locate domains in the proteins conserved for their structure and function. comparison of all the genes between virulent and aviruient strains may be useful for the identification of sequences involved in the pathogenicity of the virus. the amino acid sequence of the nucieoprotein from fs / and purdue strains of tgev differ by . % and most of the changes are relatively conservative. however, the change at base results in an amino acid substitution of cysteine for strain fs / to arginine for purdue, there are three areas of homology between the nucleoproteins of tgev (strains fs / or purdue), ibv (beaudette) and mhv (jhm) (figs. and ) , indicating that all three coronavirus subgroups share common ancestry. the areas of homology. shown in fig. , between tgev amino acid residues - and - appear to be present to a similar extent in the three viruses. however, the homology in the region between amino acid residues - is much less between ibv than mhv, indicating that tgev more closely resembles mhv. dot matrix analysis shows that this region between tgev strains fs / and purdue has multiple repetitions (fig. ) , probably resulting from a series of alignments of serine-arginine repeats, which could be a site for interaction with genomic rna. there is no detectable homology between the nucleotide sequences of the nucleoprotein genes of the three viruses. this suggests that the three regions of protein homology may be involved in either rna interaction or the formation of the correct conformation for interaction. there is no antigenic homoiogy between the nucleoproteins of tgev, ibv and mhv (pedersen et al., ) , suggesting that the antigenic sites are present in the non-homologous regions. motz ef al. ( ) have shown that major antigenic sites on the epstein-barr virus kd early protein occurred at hydrophilic p-turns. determination of the secondary structure of tgev nucleoprotein using the predict program and the hydrophilicity of the primary structure showed that the areas of non-homology with ibv and mhv contained potential hydrophilic p-turns, indicating that the areas responsible for antigenicity may fall within these regions. none of the amino acid substitutions between tgev fs / and purdue strains fall within the two main homology regions, except for a conservative serine/ threonine substitution at amino acid position , which indicates that the differences between the nucieoproteins of the two strains will probably not be responsible for the difference in the pathogenicity of the two strains, though there may be antigenic variations. the -bp open reading frames of the two tgev strains differed in amino acid sequence by . %. most of the changes observed are in the first base of the triplet, although they also resulted in conservative ammo acid substitutions, with the exception of the alanine (fs / ) to arginine (purdue) change at position that results from a double substitution at positions and of the triplet. substitution of asparagine for aspartate at position of the gene results in the introduction of a potential a/-giycosylation site in the fs / strain as compared to the purdue strain. to date, no product has been identified in either infected cells or virions that corresponds to this polypeptide. however, garwes (unpublished results) has detected a polypeptide of m, - , produced in infected cells, which does not appear to be incorporated into virions. the polypeptide is probably similar to the polypeptide of mr identified by wesley and woods ( ) and later sized as m, (wesley and woods, ) produced by the purdue strain of tgev. the presence of a potential glycosylation site at amino acid of fs / , which may add up to to the molecular weight of the polypeptide, and the hydrophobicity of the molecule may cause the difference in the molecular weight between the theoretical and observed values. three oligopeptides have been synthesized for the production of antibodies in order to probe infected cells and virions for the presence of the polypeptide of m, . the tgev nucleoprotein gene was constructed from the two cdna clones and sandwiched between samhi linkers to produce a gene cassette for transfer to suitable expression vectors. the gene cassette, inserted in the correct orientation in the yeast expression plasmids. produced a polypeptide under the control of the yeast promoters, identical in size to the nucleoprotein found in tgev virions and which reacted with a tgev nucleoprotein monoclonal antibody. this observation demonstrated that the nucleoprotein gene construction was correct. to our knowledge, this is the first coronavirus protein expressed in yeast cells. the tgev nucleoprotein gene or its product did not appear to be toxic to the yeast cells. the nucleoprotein gene inserted in the wrong orientation in the yeast expression vectors did not produce the tgev nucleoprotein, indicating that the viral cdna did not contain sequences capable of acting as a promoter in the yeast cells. the pgk (phosphoglycerate kinase) promoter has been shown to be very efficient (mellor ef a/., ) , which makes it useful for the expression of foreign proteins. expression of the tgev nucleoprotein gene under the control of the pgk promoter was constitutive, as observed for other foreign genes (mellor ef at., ) and to overcome any potential toxicity of the tgev nucleoprotein the gene cassette was also subcloned into a yeast expression vector under the control of the gal inducible promoter. this allowed selection of yeast cells containing the recombinant plasmids with the nucleoprotein gene but without expression from the gal promoter. the tgev nucleoprotein was expressed in the presence of galactose but not glucose and the product was the same size and reacted with the same monoclonal antibody as the product obtained under the control of the pgk promoter. the level of expression observed under the control of the gal promoter was higher than that observed under the control of the pgk promoter, although the latter is known to be one of the most efficient yeast promoters when acting with its natural gene (mellor ef al., ) . comparison of the yeast (sharp efa/., ) and the tgev nucleoprotein codon usage showed that there was little difference in codon usage (data not shown), which should not cause any reduction in efficiency of expression. the presence of yeast transcription termination sequences has been found to enhance the efficiency of expression. the expression plasmid containing the pgk promoter contains a termination site downstream of the cloning site used, whereas the plasmid containing the gal promoter contains no yeast termination sequence. no apparent increase in expression was observed between the products from the gal promoter in the presence or absence of the pgk terminator. in addition to the tgev nucleoprotein gene, the samhi cassette also encoded the second potential open reading frame. while several genes can be translated from polycistronic mrna in e. coli, no polycistronic mrna has been found in yeast. expression occurs only from the first open reading frame downstream of a promoter when an artificial or foreign gene is introduced into yeast cells as observed with other eukaryotic cells. thus it is unlikely that any expression will occur from the second open reading frame. the antibodies raised against one of the three synthetic oligopeptides have been used in an elisa test on yeast extracts for the presence of the potential product of this open reading frame but no product has been detected so far (data not shown). strains, plasmids and media e. coli strains dh , c and jm were used for routine plasmid construction. e. coli transformants were selected on lb plates containing ampicillin ( ^.g ml"'), sacctiaromyces cerevisiae strain bwg - . gal^ guarente ef al., ) was used as a host strain for the expression of recombinant plasmids. the constitutive pgk expression vector. pma , has been described by mellor et al. ( ) . the inducible gau-gal expression vector, pb , was derived from pcge by the addition of a samhi site to the gal side of the . -kbp gal -gal divergent promoter (boeke ef at., ) . yeast cells were grown in synthetic media containing % glucose or galactose and . % yeast nitrogen base (difco) supplemented with the required amino acids. tgev strain fs / was grown in llc-pk cells in the presence of [^h]-uracil and actinomycin d (garwes ef at., ) . messenger rna was isolated from the cells and purified from other rna species on poly(u) sepharose as described previously (britton ef al.. ) . the tgev mrna species were fractionated on - % (w/v) linear sucrose gradients in mm tris-hci (ph . ), mm lici. mm edta. and . % sds, for h at hooooxga^, and x using an mse preparative ultracentrifuge. standard reccmbinant dna methods were used (maniatis er al., ) with enzymes purchased from new england biolabs (cp laboratories. bishop's stortford. england) unless otherwise stated in the text. dna fragments were isolated from agarose gels by electroelution. ligation reactions were carried out as described by britton ef at. ( ) . e. coli cells were routinely transformed using the rbci method developed by v. simanis (hanahan. ) . vector dna was routinely treated with alkaline phosphatase prior to ligation. yeast cells were transformed using the spheroplast method described by rothstein ( ) . preparation of the oligo(dt) tailed vector primer. purified puc was digested using psfl, and homopolymer tails of about deoxythymidylate (dt) residues were added to the dna in a solution containing . m potassium cacodylate, mm tris. (okayama and berg, ) . the extent of tailing was analysed by digestion of the vector-primer with haell. an increase in the size of the two smallest fragments, compared to psfl-and haell-digested puc , indicated the addition of oligo(dt) tails. one of the dt homopolymer tails was removed by digestion with samhi and ecori and chromatography on cl- b sepharose (pharmacia ltd.). synthesis ot tirst-strand cdna. a sample of the oligo(dt)-tailed vector-primer was added to ^.g of rna solution, containing the . kb tgev mrna species (prepared as described above), previously denatured at °c for min. synthesis of first-strand cdna was carried out in a solution containing mm tris-hci, ph . synthesis of second-strand cdna synthesis. the second strand of cdn a was produced by a modification of the rna/dna replacement method of okayama and berg ( ) . the vector-primer containing the mrna/cdna hybrid was radissolved in fxl of mm tris-hci, ph . . mm mgcl , mm (nh ) so , mm kci, . mm nad^ ii.g nuclease-free bsa, tim dntps, jici [^^pj-dctp ( - . ci mmol"', new cloning using a specitic restriction fragment for priming cdna synthesis. the bp h/ndlll-xtial fragment (fig, ) from plasmid pts - was purified. a sample of the fragment was boiled for min and added to poly(a) mrna. isolated from tgev-infected llc-pkl cells, at °c. the mixture was incubated at x for min and coofed to °c, first-and second-strand cdna syntheses were carried out as described above. transformation of cdna clones into e. coli cdna was end-repaired using t dna polymerase as described by maniatis etat. ( ) . the cdna-vector-primer was self-ligated using t dna ligase at ^ for h followed by treatment with rna ligase (pharmacia ltd.) for a further h. the cdna derived from the restriction fragment primer was ligated to phosphorylated ecori linkers (pharmacia ltd.). cdna of kbp was purified and ligated into puc previously cut with fcori. the tigation mixtures were routinely diluted five-fold and used to transform e. coli cells. cdna derived using the vector-primer method was transformed into strain jm and cdna derived from the restriction fragment primer method was transformed into strain c using the method of hanahan ( ) . all ampicillin-resistant colonies were replica plated onto biodyne membranes (p/n bnng . j^m, pal. portsmouth. england) and the plasmid copy number was amplified by transferring the membranes onto lb plates containing chloramphenicol ( jig ml" ^ hanahan and meselson, ) . the cells were tysed according to the method of grunstein and hogness ( ) , but including the % sds step described by maniatis ef at. ( ) . the membranes were pre-hybridized in buffer containing formamide and poly(a) (boehringer) as described by maniatis et al. ( ) .^p -labelled tgev poly(a)-containing rna ( x ^ c.p.m.) was used to probe the cdna for h at "c, the membranes were then washed four times at room temperature in x ssc containing . % sds (xi ssc = . m naci. . m trisodium citrate ph , ), once in x ssc containing . % sds at "c for . h and autoradiographed. plasmid dna was prepared and purified from positive clones as described by britton ef al. ( ) . tgev poly(a)-containing rna was glyoxylated as described by fvlaniatis ef al. ( ) and separated on a % agarose gel. the rna was immediately transferred onto biodyne membranes in x ssc for h and baked at x for h. nick translation ot recombinant plasmid dna and hybridization to tgev rna were removed by chromatography through sephadex g- . labelled recombinant plasmid dna was used to probe the tgev mrna northern biots. random subclones of the cdna inserts frorth^tsi - andpf f- were generated by purifying h/ndlll. pvull or the haelll-pwll fragments from pts - and the £cori fragmentfrom pf f- and cloning sonicated {deininger, ) fragments into smal-cut, phosphatase-treated m mp (amersham international). in addition, various restriction endonuclease fragments, deduced from the restriction maps (fig. ) , were cloned into m mp-vectors to cover areas of sequence partially covered by random clones. m /di-deoxynucleotide sequencing (sanger e(a/., ; bankier and barren, ) was carried out using [a-^^s]-datp (amersham international) and sequencing reactions were analysed on buffer gradient gels (biggin e( at. ) . a sonic digitizer (graf/bar, science accessories corporation) was used to read the data, which were analysed on a vax / , using the programs of staden ( staden ( , and uwgcg (devereuxef a/., ). protein secondary structure was determined from its primary structure using predict, a program that compares eight different prediction algorithms (eliopoulos et al., ) . yeast cells were isolated at mid-exponential phase peiieted and resuspended in ml of phosphate-buffered saline (pbs. mm potassium phosphate. mm naci. ph . ) containing mm pmsf (phenyl methyl sulphonyi fluonde) and mm benzamidine. cell-free extracts were obtained by disruption of the cells with glass beads (diam. . mm) for min at ^ and clarified by centrifugation. samples containing |ig of protein were fractionated on % sds-polyacrylamide gels as described by britton ef a/. ( ) . following electrophoresis, the separated proteins were transferred onto nitrocellulose membranes (schleicher & schuelt. ba . pore size , p.m) using an eiectroblotting apparatus (biorad laboratories ltd.) at v for h in electrophoresis running buffer with . % sds and % methanol. nitrocellulose membranes containing the fractionated yeast proteins were washed several times with pbs containing % gelatine and . % tween . the presence of tgev nucleoprotein was determined using a monoclonal antibody (da : previously raised against tgev nucleoprotein in mice; garwes etat., ) , rabbit anti-mouse antibodies, and - n.c\ '^^l-protein a (amersham international pic, mci mg "' protein a. code no. im ) per blot. shotgun dna sequencing buffer gradient gels and ^^s label as an aid to rapid dna sequence determination ty elements transpose through an rna intermediate sequences of the nucleocapsrd genes from two strains of avian infectious bronchitis virus towards a genetically-engineered vaccine against porcine transmissible gastroenteritis virus expression of porcine transmissible gastroenteritis virus genes in e. coli as p-galactosidase chimaeric proteins location and direction of transcription of the ptsh and ptst genes on the escherichia coli ki genome phosphotransferase-mediated regulation of carbohydrate utilisation in escherichia coli k ; identification of the products of genes on the specialised transducing phages \iex(crr) and xgsrftgs) random subcloning of sonicated dna: application to shotgun dna sequence analysis coronavirus cell-associated rna-dependent rna polymerase a comprehensive set of sequence anaiysis programs for the vax a structural model for the chromophorebinding domain of ovine rhodopsin coronaviruses in animals the polypeptide structure of transmissible gastroenteritis virus defective replication of porcine transmissible gastroenteritis virus in a continuous cell line identification of heat-dissociable rna complexes in two porcine coronaviruses identification of epitopes of immunologicai importance on the peplomer of porcine transmissible gastroenteritis virus colony hybridization; a method for the isolation of cloned dnas that contain a specific gene a gau -cyc hybrid yeast promoter identifies the gal regulatory region as an upstream site studies on transformation of £scftencft/aco// with plasmids techniques for transformation of escrtench/a coli characterization and translation of transmissible gastroenteritis virus mrnas sequence analysis of the porcine transmissible gastroenteritis coronavirus nudeocapsid protein gene comparison of initiation of protein synthesis in prokaryotes, eukaryotes and organelles molecular cloning. a laboratory manual factors affecting heterologous gene expression in saccharomyces cerevisiae efficient synthesis of enzymatically active calf chymosin in saccharomyces cerevisiae expression of hepatitis bsurtace antigen gene in yeast expression of the epstein-barr virus kda eariy protein in escherichia coli ior use as antigen in diagnostic tests high-efficiency cioning of fulllength cdna antigenic relationship of the feiine infectious peritonitis virus to coronaviruses of other species cloning in yeast dna sequencing with chain terminating inhibitors codon usage in yeast: cluster analysis cleariy differentiates highly and lowly expressed genes the '-end sequence of the murine coronavirus genome: implications for muitiple fusion sites in leader-primed transcription the biology of coronaviruses coronavirus jhm, nucleotide sequence of the mrna that encodes nudeocapsid protein codon preference and its use in identifying protein coding regions in long dna sequences automation of the computer handling of gel reading data produced by the shotgun method of dna sequencing graphic methods to determine the function of nucieic acid sequences coronavirus multiplication strategy, i. identification and characterisation of virus-specif ied rna coronavirus multipiication: the locations of genes for the virion proteins on the avian infectious bronchitis virus genome phosphoproteins of murine hepatitis virus synthesis and assembly of hepatitis b virus surface antigen particles in yeast identification of a , molecular weight antigenic potypeptide in transmissible gastroenteritis virus-infected cells antibody response in swine to individual transmissible gastroenteritis virus (tgev) proteins this research was supported by the biomolecular engineehng programme of the commission of the european communities. contract no. gb - - -uk. r. s. carmenes (from departa-mento de bioquimica, universidad de oviedo, spain) was supported by an eec training grant. dr f. parra was supported by a nato fellowship. we wouid iike to thank dr r. serrano (embl, heidelberg, rfa) for supplying plasmid pb and yeast strain bwg - a. dr g. yarranton (celltech ltd.) for plasmid pma , and miss fiona stewart of this institute for the monoclonal antibody da , key: cord- - bj eh d authors: pensaert, m. b.; debouck, p.; reynolds, d. j. title: an immunoelectron microscopic and immunofluorescent study on the antigenic relationship between the coronavirus-like agent, cv , and several coronaviruses date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: bj eh d a possible antigenic relationship between the porcine enteropathogenic coronavirus-like agent (cvla) and known coronaviruses was examined by immunoelectron microscopy (iem) and by immunofluorescence (if). cvla did not show cross reactivity with infectious bronchitis virus, transmissible gastroenteritis virus (tgev), canine coronavirus (ccv) hemagglutinating encephalomyelitis virus (hev), neonatal calf diarrhea coronavirus (ncdcv) or feline infectious peritonitis virus (fipv). antigenic relationship was detected by iem between tgev and ccv, ncdcv and hev and by if between tgev and ccv, tgev and fipv, hev and ncdcv. in the third report of the international committee on taxonomy of viruses, the family coronaviridae is cited to contain definite members, probable members and possible members ( ) . the species of this monogenerie family are grouped mainly on physieo-chemieal characteristics. they are pleomorphie enveloped particles, averaging nm diameter, containing rna and essential lipid. they all bear unique definite projections. the eoronaviruses multiply in cytoplasm and mature by budding through endoplasmic retieulum ( ) . some eoronaviruses cause respiratory problems in birds and man, others are associated with enteritis in different species and some eoronaviruses affect multiple organs ( ) . antigenic relationships exist only between some members of this family (i ). in , a coronavirus-like agent (cvla) was isolated from an epizootic of diarrhea on belgian swine breeding farms ( ) . based on its morphologic characteristics, the cvla was suggested to be a tentative member of the family corona- - / t/ / /$ i. viridae ( ) . the cvla was shown by serologic cross protection studies to differ from the two knowm porcine coronaviruses, transmissible gastroenteritis virus (tgev) and hemagglntinating encephalomyelitis virus (hev) ( , ) . the purpose of the present report is to compare cvla antigenically with known coronaviruses. the demonstration of antigenic similarities to accepted coronaviruses would certainly contribute to definite classification of cvla within the family coronaviridae. this study was performed by means of immunoelectron microscopy (iem) and immunofluoreseence (if). both techniques have been used before to study serologic differences between virus species within, a virus family ( a, , ) . the coronaviruses selected for the present study were tgev, hev, neonatal calf diarrhea coronavirus (ncdcv), canine coronavirus (ccv), avian infectious bronchitis virus (ibv) and feline infectious peritonitis virus (fipv). origin of antigens the cvla was obtained by intestinal perfusion of a cesarean-derived colostrumdeprived (cdcd) piglet, previously inoculated orally with an intestinal homogenate containing the cv isolate ( ) . the cell culture-adapted purdue strain of tgev grown in sk- cells and the lth cell culture passage of the vw strain of t-iev ( ) grown in primary porcine kidney cells, were used as infected tissue culture fluid. the massachusetts- strain of ibv, in the form of allantoic fluid of inoculated embryonic eggs, was obtained from dr. s~'asoake, laboratory of avian pathology, state university of gent, belgium. ncdcv (norden laboratories vaccine strain) was grown in primary calf kidney ceils, then concentrated -fold by precipitation with per cent saturated ammonium sulphate. the american type culture collection strain - of ccv ( ) was grown in secondary dog kidney (dk/ ) cells. fipv was omitted from the antigens used for iem as starting material with satisfactory morphology" could not be obtained. preparation of specific antisera a monospeeific hyperimmune serum against the cv isolate of cvla was prepared in a cdcd pig. in the indirect fluorescent antibody technique using small intestinal cryostat sections of an experimentally inoculated pig, this antiserum bad a titer of : ( ). hyperimmune serum against a virulent belgian strain of tgev was raised in a conventionally reared pig. this hyperimmune serum had a virus neutralizing titer of : when tested against the cell culture adapted purdue strain of tgev. this serum had no detectable antibodies against the cvla and hev when tested by the indirect fluorescent antibody technique and by the seroneutralization test respectively. a monospecific hyperimmune serum against the vw strain of i-iev was prepared in a cdcd pig ( ). this serum had a virus neutralizing titer of l : t , in a microplate neutralization test with the cell culture adapted vw strain grown in pk-t cells ( ) . chicken ibv antiserum was provided by dr. si'a~oo~e, laboratory of avian pathology, state university of gent, belgium. this serum was raised in a spf chicken and had a hemagglutinationinhibition titer of : when tested against the massachusetts- strain. the ncdcv (british isolate) antiserum was obtained from a gnotobiotic calf and had a titer of : , in an indirect immunofluorescence test. ccv convalescent serum was obtained from a normal dog in the open population. this serum had a titer of : in a mierotiter neutralization test using tcid~ of ccv in dk/ cells. the iem test virus-containing fluids were clarified at ×g, at °c for minutes. the supernatant was sonified for seconds and clarified again at , × g, at ° c for minutes. antisera were inactivated at. °c for minutes. one hundred ~xl of antigen was mixed with ~ of an appropriate serum dilution which was determined in the homologous systems. the mixture was held at °c for hour and at °c overnight. one drop of the mixture was then placed on mesh formvar-eoated grids and stained with percent k-phosphotungstate, pit . i. grids were examined using a zciss em s- electron microscope. micrographs were taken at an instrumental magnification of , × az~d photographically enlarged to , ×. preparation of the antigens frozen sections from jejunum of a cdcd pig, experimentally infected with the cv isolate, were used us the source of cvla antigen ( ) . the preparation of the sections has been previously described (i ). frozen sections from the jejunum of a piglet, naturally infected with tgev, were used as the source of tgev antigen. these sections were shown to be free from cvla, hev and rotavirus. the vw strain of hev was cultivated in pk- cells grown on leighton coverslips. primary calf kidney celt monolayers were grown in mierotiter trays (stei~lin, england) and infected with ncdcv (norden laboratories). after hours the cell sheets were fixed with per cent acetone and used as antigen. coverslip cultures of dk/ cells, infected with ccv - were harvested after hours incubation. brain smears from fipv infected mice were obtained from dr. osteri~aus, national institute of public health, bilthoven, the netherlands. they were used as the source of fipv antigen. the propagation of fipv in mouse brain has been previously described ( ) . due to non-specific staining reactions, the ibv system could not be used for the present immunofluorescent studies. the globulin fraction of the hyperimmune scra against cvla, tgev and hev, mentioned above, was conjugated with fluoreseein isothioeyanate (fitc). dilutions of the conjugated antisera were tested for optimal fluorescence in their homologous system. the optimal dilution, usually : , was then used in the heterologous systems. the antisera against ncdcv and ccv, mentioned above, were used in an indirect if staining technique. in the homologous system, the ncdcv antiserum produeed bright fluorescence at a dilution of : and the ccv antiserum at a dilution of : . fitc conjugated anti-bovine and anti-dog globulins were produced in rabbits and obtained from nordic immunological laboratories, maidenhead, berks, u.k. the aseitie fluid from a eat naturmly infected with fipv was obtained from dr. pastoret, laboratory of virology, state university of ligge, belgium. this ascitie fluid had a virus-neutralizing titer of > when tested against the cell culture adapted purdue strain of tgev. the globulin fraction of this fluid was conjugated with fitc ~. the undiluted conjugate produced bright fluorescence in fipv-infected mouse brain smears. fluorescent antibody staining procedure indirect fluorescent antibody staining was carried out as follows. antigen-containing substrates were treated with the optimal dilution of antiserum. after an incubation period of minutes in a moistened chamber at ° c, the substrates were washed in changes of phosphat.e buffered saline solution (pbs) for minutes each. they were subsequently stained with the fitc eonjugatex] antiglobulins. after an incubation time of minutes at ° c in a moistened chamber, the substrates were washed as described above. finmly they were rinsed in distilled water for minute and dried in a warm air stream. all substrates were mounted with per cent glycerol in pbs except for the mierotiter trays which were read unmounted. direct fluorescent antibody staining was carried out by treating antigen-containing substr~ges with an optimum dilution of fitc-labelled antiviral serum as described above. the results of the i e m are shown in table . i m m u n e aggregates were observed in all homologous systems. t h e y were recognized by aggregation of widely spaced virus particles, surrounded by a fuzzy rim of antibodies. figure shows an example of a positive homologous and two negative heterologous reactions. cvla did not show detectable antigenic cross-reactivity with ibv, t g e v , ccv, t t e v or ncdcv. t g e v antiserum coated ccv antigen but not vice versa. an antigenic relationship was observed between ncdcv and h e v (fig. ) . a n t i s e r u m a g a i n s t a n t i g e n s c v l a the results of the immunofluoreseence tests are shown in table . conjugated antiserum to cvla reacted only with the homologous antigen, and cvla viral antigen was not detected by any of the other antisera. antigenic cross-reactivity was shown between tgev and ccv and between tgev and fipv. antiserum to tgev reacted with ccv, but did not show detectable antigenic cross-reactivity with fipv. on the other side, antiserum to ccv and to fipv both reacted with tgev. the antigenic relation between ccv and fipv was not studied. a "two= way" antigenic relationship between hev and ncdcv was also detected. the earlier results in which cvla was found to be antigenically unrelated to the known porcine coronavfi'uses, tgev and i-iev ( , t ) , are hereby confirmed. the present study did not reveal any evidence for a "one-way" or "two-way" cross-reactivity between the cvla and non-porcine eoronaviruses. since cvla cannot be cultivated in in vitro systems, only serologic tests such as iem and if could be used for examining cross-reactivity with coronaviruses. the results of the present study are expressed either as positive or negative because only one pai~icular serum dilution was used in each of the tests. the dilution of the serum was kept as low as possible in order to assure a maximal degree of sensitivity. even a low degree of cross-reactivity would, therefore, most likely have been detected. the present results suggests that cvla may represent a serologically distinct coronavirus species. such a feature is not unique within the family coronaviridae since the prototype species, ibv, does not appear to be related to other coronaviruses ( , ) . since the present study did not reveal further evidence for a more definite classification of the cvla, its morphological appearance remains the only feature for a tentative grouping within the coronavirus family. the existence of an antigenic relationship between hev and ncdcv has earlier been reported using virus neutralization ( ) , if ( ) and enzyme-linked immunosorbent assay (elisa) ( ). it is further corroborated by the present work using iem. the immunofluorescent cross reactivity of tgev antiserum with ccv, reported by pederse~ et al. ( ) , is confirmed by the present study. the relationship between these viruses can also be shown by iem. in contrast to the findings of pedersen et al. ( ) , ccv antiserum was found in the present experiments to cross-react in the indirect if with tgev. while the existence of different seretypes of ccv cannot be ruled out, other points should be taken in consideration in trying to explain these contradictory results. the antibody titer of the serum used may be important, particularly when the antigenic relationship between coronaviruses is examined. similar observations were made by i~eynolds and gagwes ( ) in examining the tgev-fipv relationship. furthermore, it cannot be excluded that the dog serum, used in the present study, contained homologous tge antibodies. the serum had been randomly collected. it is known that dogs can be naturally infected with tgev ( ). the present finding in which the dog serum, used in the if test, failed to aggregate tgev in the iem test is somewhat unexpected and is difficult to explain. in heterologous iem test, hyperimmune sera may be necessary to obtain positive reactions. the antibody concentration in the convalescent dog serum, used in the present study may have been too low to establish aggregation of tgev. the results of our if studies comparing fipv with tgev, are in agreement with the "one way" antigenic relationship existing between these viruses as earlier reported ( , t ). indeed, tgev antiserum failed to react with fipv. the "two way" antigenic cross reactivity between fipv and tgev, described by pederse~n et al. ( ) , could not be confirmed in the present studies. we gratefully acknowledge the financial support from the institute for encouragement of scientific research in industry and agriculture (iwonl), brussels, belgium. the technical assistance of miss chantal vanmaercke is gratefully appreciated. virus isolation and immunofluorescence in different organs of pign infected with hemagglutinating encephalomyelitis virus recovery" and characterisation of a coronavirus from military dogs with diarrhoea antigenic relationships amongst coronaviruses serological crossreactivity within the picoronaviruses as studied by electron microscopy. canad experimental infection of pigs with a new porcine enteric coronavirus, cv diagnosis of bovine coronavirus infections with hemadsorption-etution-hemagglutination assay (heha) and with enzyme-linked-immunosorbent assay (elisa) feline infectious peritonitis : a coronavirus disease of cats transmissible gastroenteritis in dogs: experimental intestinal infection with transmissible gastroenteritis virus classification and nomenclature of viruses feline infectious peritonitis {fip) virus. iv. propagation in suckling mouse brains antigenic relationship of the feline infectious peritonitis virus to coronaviruses of other species diagnosis of transmissible gastroenteritis in pigs by means of immunofluorescence characteristics of a corouavirus causing vomition and wasting in pigs tile corona, viruses: clinical and structural aspects with some practical implications a new coronavirus-like particle associated with diarrhea in swine a seroepizootiologie study of vomi$-ing and wasting disease virus in pigs virus isolation and serum antibody responses after infection of cats with transmissible gastroenteritis virus characterization of a calf diarrheal coronavirus [:h]tersuchungen fiber die antigenverwandtschaft dcr viren der felinen iufekti ser peritonitis und der transmissiblen gastroenteritis des schweines morphological and antigenic relationships between viruses (rotaviruses) from acute gastroenteritis of children, calves, piglets, mice and foals received september , key: cord- -grw s pf authors: lai, michael m.c.; cavanagh, david title: the molecular biology of coronaviruses date: - - journal: advances in virus research doi: . /s - ( ) - sha: doc_id: cord_uid: grw s pf publisher summary this chapter discusses the manipulation of clones of coronavirus and of complementary dnas (cdnas) of defective-interfering (di) rnas to study coronavirus rna replication, transcription, recombination, processing and transport of proteins, virion assembly, identification of cell receptors for coronaviruses, and processing of the polymerase. the nature of the coronavirus genome is nonsegmented, single-stranded, and positive-sense rna. its size ranges from to kb, which is significantly larger when compared with other rna viruses. the gene encoding the large surface glycoprotein is up to . kb, encoding an imposing trimeric, highly glycosylated protein. this soars some nm above the virion envelope, giving the virus the appearance-with a little imagination-of a crown or coronet. coronavirus research has contributed to the understanding of many aspects of molecular biology in general, such as the mechanism of rna synthesis, translational control, and protein transport and processing. it remains a treasure capable of generating unexpected insights. viruses. the coronavirus polymerase gene alone ( - kb) is about the same size as the whole of the picornavirus (- kb) and vesicular stomatitis virus (- kb) genomes added together. the gene encoding the large surface glycoprotein is up to . kb, encoding an imposing trimeric, highly glycosylated protein. this soars some nm above the virion envelope, giving the virus the appearance-with a little imagination-of a crown or coronet (latin corona, hence the name of the genus). coronaviruses are responsible for a number of economically important diseases. avian infectious bronchitis virus (ibv) was the first coronavirus to be isolated, from the domestic fowl, and propagated in the s. in addition to respiratory disease, which can predispose chickens to possibly lethal secondary bacterial infections, some strains also cause nephritis (king and cavanagh, ; cook and mockett, ) . porcine transmissible gastroenteritis virus (tgev) causes devastating disease in newborn pigs, with mortality often approaching % (enjuanes and van der zeijst, ) . intriguingly, there are also naturally occurring mutants [i.e., porcine respiratory coronavirus (prcv)] of tgev which cause only mild respiratory disease and no enteritis. several other coronaviruses also cause enteritis: bovine coronavirus (bcv), turkey coronavirus (tcv; bluecomb virus), feline coronavirus (fcv), canine coronavirus (ccv) and porcine epidemic diarrhea virus (pedv). fcv may also cause feline infectious peritonitis. an fcv has been isolated from a cheetah and bcvs from wild sambar deer and waterbuck (tsunemitsu et al., ) . these bcvs caused enteritis when inoculated into domestic calves. humans are known to suffer from two very different coronaviruses, human coronavirus (hcv) oc and hcv , both of which are a cause of the common cold. there is evidence for the presence of coronaviruses in tissues taken from multiple sclerosis (ms) patients (reviewed by cavanagh and macnaughton, ) . this inflammatory, demyelinating neurological disease is associated with autoreactive t lymphocytes sensitized to myelin components of the central nervous system. recently, talbot and colleagues ( ) have demonstrated that many cd ' t-cell lines derived from ms patients showed a human leukocyte antigen-(hla)-dr-restricted, crossreactive pattern of antigen activation after in vitro selection of either myelin basic protein or hcv- e proteins, suggesting that molecular mimicry between hcv and myelin may be a n immunopathological mechanism in ms. other coronaviruses [some strains of murine hepatitis virus (mhv) and porcine hemagglutinating encephalomyelitis virus (hev)] are well-known causes of neurological diseases, and mhv has been studied for many years in this context (dales and anderson, , although many mhv strains cause primarily hepatitis. the s and early s was the period in which coronavirus virion proteins and nested-set arrangements of mrnas were identified and the discontinuous nature of coronavirus transcription was initially demonstrated. the first published sequence of a coronavirus gene appeared in , starting an era in which the whole of the genomes of four coronaviruses were clonedin piecesand sequenced. this decade has seen the manipulation of these clones, and of complementary dnas (cdnas) of defective-interfering (di) rnas, to study coronavirus rna replication, transcription, recombination, processing and transport of proteins, virion assembly, identification of cell receptors for coronaviruses, and processing of the polymerase. this review is largely concerned with these areas. some topics are notable by their absence, space not permitting their inclusion. for example, the elucidation of the molecular basis of the antigenic properties of the large surface (spike) glycoprotein and its role in tissue tropism has been omitted. for these topics and all others both within and without the compass of our review for which a concurrently comprehensive and in-depth treatise is desired, the reader is referred to the book edited by siddell ( a) . individual chapters in that book will be referenced a t the appropriate places in this review. all coronaviruses belong to one genus, coronavirus, within the family coronaviridae . initially, serological analysis was used to differentiate coronavirus species and showed that they could be divided into four antigenic groups (holmes, ) . the species and group divisions were subsequently refined by monoclonal antibody analysis and nucleotide sequencing, which revealed the close relatedness between tcv and bcv, resulting in the current classification of three antigenic groups (table i) . the same groupings emerge regardless of which structural protein sequences are compared (siddell, b) . within group , tgev, fcv, and ccv are particularly closely related, all the members of group being tightly clustered. the sole member of group , ibv, not only differs extensively from all other coronaviruses but also exhibits extensive variation within the species. the coronaviridae had remained a monogeneric family for a quarter of a century, until an accumulation of observations which showed that many of the features thought to be characteristic of the coronaviridae applied equally well to the genus torovirus, which had not been officially assigned to a family (figs. and , table ). therefore, in , the international committee for the taxonomy of viruses (ictv) formally expanded the coronaviridae to include torovirus . the bringing together of coronavirus and torovirus was not the end of the taxonomic story; another family, arteriviridae, shared important characteristics in relation to the genome, structure, and strategies of transcription and translation (table i ) (plagemann and moennig, ; snijder and spaan, ) . however, the distinct morphology of the arteriviruses (fig. , and their underlying differences from the coronaviruses in the size of the genome (fig. ) and structural proteins (table ii) , precluded their inclusion in the coronaviridae. the common features uniting the two families (table ) are at the heart of a proposal that an order be created to contain coronaviridae and arteriviridae to reflect their common features and, probably, their evolutionary relationships. the name nidovirales, from the latin nidus, meaning nest, has been designated for the order, as all members produce mrnas in an extensive nested-set arrangement. the remainder of this review is restricted largely to the coronaviruses. coronaviruses are enveloped, more or less spherical, approximately nm in diameter, with a prominent fringe of -nm-long petalshaped surface projections (spikes) composed of a heavily glycosylated type i glycoprotein, spike protein ( s ) (fig. ) . a subset of the coronaviruses (table i ) has an additional layer of short spikes (caul and egglestone, ; dea and tijssen, ) , which consist of hemagglutininesterase (he) protein, also a type i glycoprotein. these small spikes are not essential for viral infectivity. both the large and small spikes are anchored in the envelope, which is a lipid bilayer formed by virus budding from intracellular membranes. the envelope is associated with, in addition to the s and he proteins, a smaller type i integral membrane protein (m), which spans the envelope three times. an even smaller protein [envelope (e) or small membrane (sm) protein] has recently been shown to be an integral membrane protein of the viral envelope. inside the envelope is a ribonucleoprotein (rnp) core, which comprises the rna genome and a single species of nucleocapsid protein n. electron microscopic observation of viral rnp showed a long helix of to nm (macnaughton et al., ; sturman and holmes, ) . a very recent study of intact and detergent-treated tgev virions (risco et al., ) by negative-staining, ultrathin sectioning, freezefracture, immunogold mapping and cryoelectron microscopy showed a surprising new feature of coronavirus particles, namely, a spherical, probably icosahedral, core inside the virion (fig. ) . these internal cores comprise not only the n protein and rna but also the m protein, m being the major core shell component. disruption of the cores released helical nucleocapsids. the presence of an icosahedral core in the coronavirus virion had heretofore been unsuspected. this core structure was also detected with mhv virion (risco et al., ) . this surprising new finding gives us cause to reconsider our view of coronavirus architecture. thus, the precise structure of the core and rnp inside the virion is not certain. toroviruses and coronaviruses have a similar morphology and virion composition ( fig. , table ) but are distinguishable in a number of ways (table ) (weiss and horzinek, ; horzinek, , ; koopmans and horzinek, ) , necessitating their inclusion in separate genera. the morphology of the arteriviruses is substantially different from that of coronaviruses and toroviruses, particularly in having an icosahedral rnp core ( fig. ) ; hence, a separate family is maintained for arteriviruses. however, the recent discovery of the icosahedral core for coronavirus (risco et al., ) may have blurred this distinction. the s glycoprotein is the outermost component of the virion, and is responsible for the attachment of the virus to cells (collins et al., ; godet et al., ; kubo et al., ) and for instigating the fusion of the virus envelope with cell membranes. it is the primary target for the host's immune responses; neutralizing antibodies are induced mainly by s (collins et al., , and immunization in animals with s alone can induce protection from some coronaviruses (ignjatovic and galli, ; torres et al., ) . within a coronavirus species, sequence variation is usually exhibited more by s than by any other structural proteins; the variation of the s protein sequence probably confers a selective advantage in immune animals. these and other aspects have recently been reviewed in detail . the s protein is large, ranging from some (ibv) to amino acids (fcv). there are many potential n-linked glycosylation sites ( to , most of which have glycans attached. the s preproprotein has a n-terminal signal sequence and a membrane-anchoring sequence near the c terminus (fig. ) . the s protein may be cleaved into s and s subunits; the extent of its cleavage varies greatly among the species (cavanagh, ) . a high proportion, up to loo%, of the s protein is cleaved in some coronaviruses (ibv, mhv, bcv, tcv, pedv) (cavanagh, a) ; none is cleaved in others (tgev, fcv, ccv) (garwes and pocock, ) ; and very little of the s protein of hcv- and hcv-oc is cleaved, although the s of oc is completely fig . models of the virions of a coronavirus, a torovirus, and an arterivirus. the he protein is present only in antigenic group coronaviruses (see table i ). reproduced with permission from cavanagh et al. ( ) . present in only a subset of coronaviruses (table i) . may have an isometric core in addition (risco et al., ) . no such protein described. table i ) and with respect to the number and position of nonstructural protein genes. the polymerase genes encode two orfs, l a and lb, which overlap. l, leader sequence; he, hemagglutinin-esterase; s, spike; e, small membrane protein; m, integral membrane protein; n, nucleocapsid protein; an, poly(a) tail; g, and gl, small and large glycoproteins, respectively. risco et al. ( ) for tgev. this model illustrates the observation that internal cores (ic), possibly icosahedral, were observed inside virions of tgev. the cores comprise the helical ribonucleoprotein (nc) (genome rna + n protein) and the m protein. reproduced with permission from risco et al. ( ). cleaved if trypsin is present (hogue and brian, ) . the extent of s cleavage depends on the cell type (frana et al., ) . cleavage generates two glycopolypeptides, n-terminal s and c-terminal s , the latter being acylated . s is probably linked to the s subunits by noncovalent linkage: trypsin treatment of mhv virions caused cleavage of all s proteins without disrupting the spikes ; however, s can be released from virion by either urea or mild alkali treatment (cavanagh and davis, ; weismiller et al., ) . among the coronavirus genus as a whole, the s polypeptide is much more conserved than s . regions of up to % amino acid identity (particularly in the transmembrane domain) exist between the s polypeptides of coronaviruses in the different antigenic groups, whereas and (b) (schmidtet al., ) . the amino acid numbering has been normalized with respect to that of the longest known mhv s protein, that of mhv (jhm) ( s . e. . (a) the protein has an amino-terminal signal peptide (sp) and a transmembrane (tm) sequence near the c terminus. the glycosylated propolypeptide is cleaved a t a basic connecting peptide (cp) to yield glycopolypeptides s and s . the locations shown are those of three mutations present in mutants of mhv recovered from a persistently infected neural cell line, the mutants requiring a ph of . - . for membrane fusion (gallagher et al., ) . (b) s of another mhv-jhm (schmidt et al., ) , which has a -amino acid deletion with respect to (a). bacterial expression products containing residues - and - bound mab f and g, respectively, both of which neutralize virus infectivity and inhibit membrane fusion. the arrow indicates the positions of amino acid substitutions in jhm mab f-resistant mutants (grosse and siddell, ) . a peptide comprising residues - bound another mab that neutralized virus and inhibited fusion (luytjes et al., ). there is almost no conservation of the s sequence. furthermore, comparison of s sequences among strains of a given species, or between species of a given group, reveals hypervariable regions, which include frequent deletions, mutations, or recombination (cavanagh et al., ; s. e. parker et al., ; banner et al., ; gallagher et al., , suggesting that this region is externally exposed and not essential for the structure. the s polypeptide has two regions with a seven-residue periodicity, forming heptad repeats (fig. ) indicative of a coiled-coil structure (de groot et al., ) . indeed, current evidence suggests that the mature s protein forms an oligomer; for tgev, it is probably a trimer (delmas and laude, ). however, a dimer structure has been proposed for ibv s protein (cavanagh, ~) . therefore, the oligomeric s protein is envisaged as being anchored in the membrane by a n a-helical region near to the c terminus of s . just beyond the outer membrane surface is the shorter (minor) repeat structure predicted to be an a! helix of - nm. the major repeat indicates a helix of - nm, which may form the narrow stalk of the spikes (de groot et al., ) . all coronavirus s proteins have a highly conserved eight-residue sequence kwpww/yvwl, the last five residues of which probably form the beginning of the membrane-spanning domain. terminating residues upstream of kwp is a leucine-zipper motif, the length varying from three to five heptad repeats (britton, ) . the highly conserved sequences of s may play a role in forming the stalk, which has a more rigid structure. in contrast, the s domain is predicted to form the globular portion of the spikes, consistent with its highly variable nature. the s protein has two important biological activities for the virus: a. induction of membrane fusion. this activity may be required for viral entry into cells or for cytopathic effects. expression of the recombinant s gene has provided unequivocal evidence that the s protein alone is sufficient to cause membrane fusion, as shown by syncytium formation (de groot et al., ; pfleiderer et al., ; yo et al., ; taguchi, ) . several regions of the s protein, widely separated in a linear sense, have been implicated in the membrane fusion process by the following observations: ( ) s of bcv expressed in insect cells caused fusion (yo et al., ) . ( ) a monoclonal antibody that inhibited cell fusion was shown to bind to the s domain of mhv ( fig. ) (luytjes et al., ) . ( ) changes at three s residues ( , , and in the mhv s protein; fig. ) were associated with a change from a requirement for a neutral ph to an acidic ph for fusion (gallagher et al., ) . ( ) two bacterial expression products containing residues - ) and - ) ofthe jhm strain ofmhvinducedmonoclonal antibodies f and g, respectively, both of which inhibited fusion ( fig. ) . ( ) chemical modifications of the cysteine residues, specifically residue in the ectodomain of s , reduced the fusion activity of the jhm strain of mhv (gallagher, ) . this result also suggests strain-specific differences in the conformation of the s protein, since the fusion activity of the a strain of mhv was not affected by this modification. ( ) some mutations to cysteine residues within the transmembrane domain of s adversely affected fusion, suggesting that the transmembrane domain is involved in conformational changes that are associated with fusion activity (bos et al., ) . these results combined suggest that the s ectodomain contains the major determinants for membrane fusion; however, s does not contain hydrophobic domains typical of fusion proteins. thus, several disparate regions, including some in the s , may contribute to the fusion activity, probably because some of these regions are juxtaposed in the threedimensional structure or can affect the overall conformation of the spikes. interestingly, monoclonal antibody-resistant mutants of the jhm strain ofmhvselected with antibody f had mutations not a t the antibody-binding site (residues - of s l ) , but at a distant site, i.e., residues - in the s domain (grosse and siddell, ) (fig. ) , suggesting that s is folded such that regions which are widely separated in the linear sense are juxtaposed to form functional domains. early studies of coronvirus-induced cell-cell fusion suggested that only cleaved s was able to promote cell fusion . more recent studies in which mhv s proteins with mutated s connecting peptides were expressed have shown that cleavage is not essential for fusogenic activity, although cell-cell fusion is more efficient when the s protein is cleaved (stauber et al., ; taguchi, ; bos et al., ) . furthermore, naturally occurring mutants of mhv, derived from persistently infected mouse cells, which are defective in s cleavage, have delayed fusion activity (gombold et al., ) . expression of the feline infectious peritonitis virus (fipv) s protein, which is not cleaved a t all, also resulted in syncytia formation (de groot et al., ) . these results indicate that s protein cleavage is not required for but can enhance membrane fusion. whether membrane fusion activity, as manifested by syncytia formation, is required for viral infectivity has not been established. there are mhv strains (e.g., mhv- ) that do not cause syncytia formation in cultured cells; however, these viruses may be able to cause virus-cell membrane fusion within the infected cells. b. receptor binding. monoclonal antibodies (mab) against the s protein of most coronaviruses can neutralize viral infectivity; thus, it is assumed that the s protein mediates virus binding to the receptors on target cells. indeed, the s protein or a portion of it can bind to the viral receptor molecules in uitro. this has been demonstrated for mhv and tgev s proteins (godet et al., ; kubo et al., ) . the binding domain has been mapped to the n-terminal amino acids of mhv s protein. site-directed mutagenesis of this region showed that mutations of the residues at position and positions , , and abolished the binding of the protein to the receptor (suzuki and taguchi, , suggesting that the receptor-binding site might comprise discontiguous regions in the linear sense. the s subunit is not involved in receptor binding (taguchi, ) . the receptor-binding sites of tgev s protein have been mapped to a -residue region (aa - ) of the s (godet et al., , which overlaps with a n epitope for a neutralizing mab. this neutralizing mab was able to block the binding of the -residue polypeptide to the receptor; conversely, the receptor did not block the binding of the mab to this polypeptide, suggesting that the receptor-binding determinants and the neutralizing epitopes are distinct and are part of a domain of s whose configuration is independent of the remainder of the s protein. s proteins of bcv and hcv-oc bind to -o-acetylneuraminic acid (schultze et al., a) ; this binding is required for viral infection. the significance of this binding will be discussed in section v,b on virus attachment. intriguingly, several coronavirus s proteins share some sequence identity with the receptor for the fc fragment of mammalian immunoglobulins (fcy receptor). thus, mab to the fcy receptor could immunoprecipitate s protein from the mhv-infected cells, and s could bind to the fc fragment of immunoglobulin. this molecular mimicry was first demonstrated for mhv and, more recently, for bcv and tgev as well (oleszak and leibowitz, ; oleszaket al., oleszaket al., , . it may play a role in modulating viral pathogenicity. this potential function is significant because expression of the s protein in the infected cells induces not only humoral antibodies but cellular immunity as well (welsh et al., ) ; the potential binding of s to the fc fragment of immunoglobulin may modulate these immune responses. the m protein is one of only two of the structural proteins [the other being the e protein (see below)] that are essential for the production of coronavirus-like particles. the sequence of the m protein reveals that the m polypeptides comprise - amino acids, except for some members of the tgev group, which have an additional or so residues a t the amino terminus, forming a cleavable membrane insertion signal. the amino-terminal or so residues of the mature m protein of all the coronaviruses are hydrophilic, exposed at the virion surface, and have a small number of glycosylation sites. glycans are of the n-linked type for ibv and the tgev group and o-linked for the mhv group (rottier, ) . the remainder of the n-terminal half of the molecule forms three helical membrane-spanning domains, although a mutant m protein which lacked all three of the membrane-spanning domains did associate with membranes in uitro (mayer et al., ) . the structure of the c-terminal half is uncertain, but it is believed to be largely situated on the inside of the viral envelope, based on protease susceptibility cavanagh et al., b) and sequence-based predictions (armstronget al., ; rottier et al., ) . however, some m molecules of tgev virions have the c terminus exposed a t the virion surface (laviada et al., ; risco et al., ) . moreover, mab specific for the c-terminal amino acids of m neutralized tgev virions in the presence of complement and caused antibody-mediated, complementdependent cytolysis of tgev-infected cells (risco et al., ) . studies with mutant mhv m proteins expressed from vaccinia virus recombinants had shown that some had the n terminus and others the c terminus a t the luminal side of the endoplasmic reticulum, equivalent to the outer surface of virions (locker et al., ) . some molecules of one mutant m protein had both termini at the luminal surface, and other molecules had both termini at the cytoplasmic surface (locker et al., , ) . thus, the precise topology and the structural role of the m protein are still not certain. recent studies have shown that some m proteins are also associated with the rnp core of tgev and constitute the outer shell of the internal core (risco et al., ) . this core-associated m can be clearly separated from the viral envelope. therefore, m may play a dual structural role in forming both the envelope and the internal core of the virion. several properties of the m protein suggest that it is involved in virus particle assembly: ( ) the m protein binds to the purified nucleocapsid in uitro (sturman et al., ) . ( ) when the m protein was expressed alone, it was localized in the golgi complex, near the location where virus particles bud (tooze et al., ; tooze and tooze, ) . however, recent studies showed that the site of m protein retention in the golgi was slightly different from that for viral particle budding (klumperman et al., , suggesting that additional factors are involved in virus particle assembly. this will be discussed in section v,h on virus assembly. the m protein of tgev has a n additional biological activity: induction of a-interferon (charley and laude, ; laude et al., ) . thus, it may play a role in viral pathogenesis. monoclonal antibodies against the m protein do not neutralize viral infectivity, suggesting that m is not involved in receptor binding. however, some of these antibodies can neutralize viral infectivity in the presence of complement (collins et al., ; laviada et al., ) , indicating that part of the m protein is exposed on the virion surface. the he glycoprotein-or perhaps one should say the he gene-of coronaviruses is something of a n enigma. only coronaviruses belonging to the mhv group possess the he gene (table i) . even there, not all virus strains within a species express the he protein (luytjes et al., ; . as with many of the so-called nonstructural protein genes of coronaviruses, the product of the he gene is not essential for viral replication, certainly not in the cell types used in the laboratory. the he protein was first detected in bcv (king et al., ) and some mhv strains; however, acceptance of it as a legitimate virus-encoded protein was delayed because in one of the mhv strains studied most thoroughly, a , virions lacked he. the he gene of a was later shown to lack the initiation codon of the he open reading frame (orf); thus, the he gene is a pseudogene in this (luytjes et az., ) and several other mhv strains . a complete, functional he gene was subsequently identified in the jhm strain and several others . the he glycoprotein, of approximately kda ( amino acids in bcv), has been detected in virions of hev, mhv, hcv-oc , bcv, and tcv. when analyzed under nonreducing conditions, the he protein migrates as a dimer of approximately kda (king et al., ) . the mature protein is believed to exist in the virion as a dimer, anchored by the c terminus, forming a fringe of short spikes visualized by electron microscopy (caul and egglestone, ; dea and tijssen, ) . it is not known whether each spike consists of more than one he dimer. those coronaviruses which contain he in their virions cause hemagglutination much more efficiently than those that do not. similar to the s protein, he alone can mediate hemagglutination and hemadsorption (king et al., ; hogue and brian, ; vlasak et al., b; deregt et al., ; pfleiderer et al., ; schultze et al., a) ; however, he seems to have weaker activity than s (schultze et al., a) . he binds to -o-acetylated neuraminic acid (vlasak et al., ; schultze et al., a) , which is also a target for s binding. some he-specific mab can neutralize bcv infectivity (deregt and babiuk, ; deregt et al., ) . thus, he protein of bcv may participate in virus binding to the receptor. the relative importance of he and s in hemagglutination and tissue tropism of bcv is not known. as its name implies, the he protein also has esterase activity; specifically, it is a neuraminate-o-acetylesterase. it hydrolyzes the - acetylated sialic acid on erythrocytes, thereby reversing hemagglutination induced by the he or s protein; thus, he is considered a receptordestroying enzyme (vlasaket az., a,b; yokomori et az., ; . the putative esterase active site is fgds, encoded by amino acids - of the mature he polypeptide of bcv (m. d. parker et al., ; kienzle et al., ) . in these respects, it resembles the hemagglutinin-esterase-fusion (hef) glycoprotein of influenza c viruses, which also has hemagglutinating and -o-acetylated sialic acidhydrolyzing esterase activities. moreover, the he protein of coronavi-ruses shares some % amino acid identity with the hef of influenza c virus, including conservation of the position of the putative esteraseactive site fgds and many cysteine residues (luytjes et al., ; s. e. parkeret al., ; kienzle et al., ; zhanget al., ) . unlike the hef protein of influenza c virus, which is cleaved into two subunits (nakada et al., , the coronavirus he protein is not cleaved and lacks most of the c-terminal subunit of the hef of influenza c virus. because of the close relatedness between the coronavirus he protein and the influenza c virus hef protein, and because the he gene is present in only one coronavirus group, it was proposed that the he gene was acquired by a coronavirus as a result of recombination between an ancestral coronavirus and influenza c virus (luytjes et al., ) . interestingly, the torovirus berne virus also has an he pseudogene (gene ; fig. ) (snijder and horzinek, ) , the amino acid sequence of which has approximately % identity with the c-terminal part of the coronavirus he. the functional significance of he for coronaviruses is not known. among coronaviruses, only bcv requires he for infectivity; however, the presence of he may affect the pathogenicity of some coronaviruses, as evidenced by the findings that passive administration of he-specific mab in mice altered mhv pathogenicity and that mhvs with an he have different neuropathogenicity from those without he . conceivably, the presence of he in an mhv may allow the virus to utilize an alternative receptor independently of the s protein. however, this is not the case, as evidenced by the finding that an mab specific for the murine biliary glycoprotein molecule, which is the major mhv receptor recognized by s, inhibited the infectivity of an he-containing mhv (gagneten et al., ) . thus, the he protein does not enable a virus to bypass the primary mhv receptor and may provide only an auxiliary function for virus binding to target cells. until recently it was thought that coronaviruses possessed three (s, n, m) or four (including he) structural proteins. it is now clear that coronaviruses, but not toroviruses, possess an additional virion protein, the e protein. it plays an essential role in virion assembly. it has been shown that the e and m proteins are the only two viral proteins absolutely required for virion assembly (bos et al., ; vennema et al., ) . this protein has been demonstrated for ibv (smith et al., ; liu et al., , tgev (godet et al., ) and mhv (yu et al., ) . when the deduced e proteins of the other coronaviruses are taken into account, it transpires that the e proteins vary from t o amino acids, corresponding to molecular weights of to , (siddell, ~) . siddell has highlighted a number of features common to all the e proteins, namely, a hydrophobic region of some two dozen residues, starting near the n terminus; a cysteine-rich region immediately downstream from this; a conserved proline residue in the middle of the molecule, and otherwise very low amino acid identity in the genus as a whole; and an abundance of charged residues in the cterminal half of the protein (siddell, ~) . it is now well established that this protein is associated with highly purified virion preparations (liu et al., ; godet et al., ; yu et al., ) . liu and inglis calculated the ratio of s:n:m:e proteins in virions of ibv-beaudette strain to be : : : , indicating an amount of e protein similar to that of s protein (liu et al., ) . in contrast, godet et al. estimated that the s:m:e protein ratio in virions of tgev was : : (godet et al., , and vennema et al. ( ) have suggested an m:e ratio of approximately oo:l for virions of mhv. it is not clear why there is such a wide range of variations. the e protein in the cells is localized in the perinuclear region, with some migrating to the cell surface (godet et al., ; yu et az., ) . experimental evidence suggests that the e protein is anchored in the membrane by sequence in the n-terminal half of the molecule. thus antibodies specific for epitopes in the c-terminal half of the tgev e protein produced cell-surface fluorescence in paraformaldehyde-fixed, tgev-infected cells (godet et al., ) , but the precise topology of the protein has not been elucidated. the role of the e protein in virion assembly will be discussed in section v,h on virus assembly and release. the e proteins of ibv and mhv are translated from the third and second orfs, respectively, of mrnas and of the respective viruses. both of these are polycistronic mrnas (see figs. and and section v,g, ). in contrast, in all other viruses, the e protein is derived from a monocistronic mrna. the mechanism of translation of the ibv and mhv e proteins is discussed in section v,g. the n protein is a -to -kda phosphoprotein which, together with the genomic rna, forms a helical nucleocapsid (rnp). the rnp of coronaviruses have been reported variously as being from - to - nm in diameter (see laude and masters, , for references) . the n protein in rnp provides only limited protection to the rna genome against ribonucleases. the n proteins vary from to amino acids in length, are highly basic, and have a high ( - %) serine content, potential targets for phosphorylation. sequence conservation within the genus is low. thus, the n proteins of ibv and tgev have only % identity with that of bcv. even within the mhv group, the n proteins of mhv and bcv share only % identity, whereas the m proteins of these two viruses have % identity (lapps et al., ) . based on sequence comparison, three structural domains in the n protein have been identified (parker and masters, ) . the middle domain is an rna-binding domain (masters, ; nelson and stohlman, ) which binds to both coronaviral and nonviral rna sequences in uitro (robbins et al., ; stohlman et al., ; masters, ) ; however, it does not contain any motifs characteristic of other rna-binding proteins. under specific binding conditions, the mhv n protein binds to the leader rna sequence, particularly nucleotides - . furthermore, an anti-n mab immunoprecipitated all of the mhv rna molecules which had the leader sequence . the n protein of ibv also bound to the ' untranslated region of the ibv rna in uitro (zhou et al., ) . these rna-binding properties are consistent with the fact that the n protein interacts with the viral genomic rna to form nucleocapsid. this interaction is necessary for the formation of virus particles, as n alone cannot be incorporated into virus particles, whereas the n-rna complex can (bos et al., ; vennema et az., ) . however, the specificity of the rna-n protein interaction required for nucleocapsid formation has not been elucidated. the n protein also binds to membranes and phospholipid (anderson and wong, ) . this may be another property which facilitates the formation of virus particles. the finding that the n protein binds to the ' and ' ends of viral rna suggests that the n protein may also modulate viral rna synthesis because the ends of the rna are likely involved in the regulation of rna synthesis. in an in uitro rna replication system, the addition of mhv n-specific antibodies inhibited viral rna synthesis (compton et al., ) , suggesting that the n protein is a component of the rnasynthesizing machinery. the ability of n to bind to the membrane (anderson and wong, ) may enable the formation of the rna replication or transcription complex, in view of the fact that viral rna synthesis occurs in the membrane fraction of infected cells dennis and brian, ) . the three structural domains of the n protein are separated by spacer regions, which are not conserved (masters, ) . the functions of the n-and c-terminal conserved domains are not yet clear. using a targeted recombination approach (koetzner et al., ; masters et al., ) to generate recombinant viruses that have a chimeric n gene containing parts of bcv and mhv sequences, peng et al. ( a) have shown that there is strict sequence specificity within the conserved structural domains for viable recombinants. since the n protein constitutes the nucleocapsid, mutations within the n protein will likely affect the stability or viability of the virus. indeed, several temperaturesensitive and thermolabile mutants of mhy have deletions or mutations within the n protein (koetzner et al., ; peng et al., b) . viruses with site-specific mutations of the n gene have been generated by targeted recombination techniques; interestingly, revertants of these mutants often have second-site mutations located a t different domains, suggesting that there are interactions between different domains of the n protein (peng et al., ) . the role of phosphorylation in the n protein has not been elucidated. the coronavirus contains a positive-sense, single-stranded rna genome, which is the largest viral rna genome known, ranging from . to kb. the large size of the viral rna requires the virus to develop special mechanisms of rna synthesis to counter the deleterious effects of the possible errors during rna synthesis. the virion rna functions as an mrna and is infectious. it contains approximately - functional genes, or of which encode structural proteins. the genes are arranged in the order '-polymerase-(he)-s-e-m-n- ', with a variable number of other, mostly nonstructural and largely nonessential, genes interspersed among them (fig. ) . this gene arrangement also applies to toroviruses and arteriviruses (fig. ) . the ' terminus of the coronavirus genome is capped, and the rna starts with a leader sequence of - nucleotides, which is also present a t the ' end of mrnas, followed by a -to -nucleotide untranslated region (utr). at the other end of the genome is a ' utr of - nucleotides followed by a poly(a) tail. almost two-thirds of the entire rna is occupied by the polymerase gene, which comprises two overlapping orfs, la and lb. at the overlap region is a specific seven-nucleotide "slippery" sequence and a pseudoknot structure, characteristic of the ribosomal frameshifting signal (brierley et al., (brierley et al., , lee et al., ; , which is required for the translation of orf lb. the architecture of the nonstructural protein genes interspersed between the known structural protein genes varies significantly among different coronavirus species (fig. ) . for example, in hcv- e, gene contains two orfs, whereas in the related virus pedv these two orfs are fused (duarte et al., ) . in hcv-oc , gene is missing altogether (mounir and talbot, ) . finally, in ibv, two orfs are inserted between m and n genes. the variability of gene structure indicates the plasticity of coronavirus rna and the frequent occurrence of recombination and also suggests that there is no strong conservation pressure on these nonstructural proteins. there is a stretch of consensus sequence, ucuaaac (for mhv), or a related bcv (hcv-oc ) fig . comparative genome structure of the different coronaviruses. the complete sequences are available for mhv, ibv, tgev, and hcv- e. the gene sequences of the remaining viruses have not been completed. gene sequences are interrupted and shortened to highlight the remaining genes. the vertical lines represent mrna start sites; thus, each region between two vertical lines represents a separate gene ("transcription unit"). the structural protein genes are marked by various symbols, and nonstructural protein genes are represented by unfilled boxes. the gene arrangements of ns protein genes and e protein gene are very heterogeneous in terms of transcription unit and the relative size and position among different strains of the same virus species; only the representative one is presented. the numbering system for the genes of hcv- e deviates from the published one to be consistent with the other viruses. hcv-oc does not have a gene . sequence, a t sites immediately upstream of most of the genes. these sequences represent signals for transcription of subgenomic mrnas (see section v,e). finally, a pseudoknot structure has been shown to be present a t the ' end of the coronaviral rna (williams et al., ) . a characteristic feature of the coronaviridae, and of the arteriviridae as well, is that all known member species generate a '-coterminal nested set of five or more mrnas (see fig. ). each coronavirus and arterivirus subgenomic mrna has the leader sequence a t its ' end. curiously, no leader rna sequence is present in the torovirus rnas (fig. ) . in , the coronavirus study group published its recommendations for the nomenclature of coronavirus genes, mrnas, and proteins (cavanagh et al., ) . at that time it was reluctant to apply the term "nonstructural" t o the potential products of genes which were suspected of not being structural proteins. this caution was a consequence of our lack of knowledge of those gene products, a situation which has improved greatly in the last years or so. this has resulted in the term "nonstructural (nsl" being applied more widely to several gene products. every gene that encodes the ns proteins has been deleted in a t least some naturally occurring virus isolates; thus, most of the ns genes are not essential for viral replication. however, some of the ns proteins may play a role in viral tissue tropism or pathogenicity. the polymerase is encoded by gene , which accounts for approximately two-thirds of the genome (fig. ) . the complete polymerase gene of four coronaviruses (ibv, mhv, hcv- e, and tgev) covering each of the three coronavirus groups has been sequenced lee et al., ; bonilla et al., ; eleouet et al., ) . although the polymerase genes vary in size from approximately to kb, the encoded proteins have many structural features in common. the degree of amino acid identity for this gene product is greater than is observed for any other coronavirus gene product. the polymerase gene is predicted to encode a protein of approximately - kda. proteins of this size have not been detected in coronavirus-infected cells, in part because of co-translational polypro-tein processing. the pol gene encodes two orfs, l a and lb, which overlap by a few dozen nucleotides (figs. and ). the second, orf lb, is in the - reading frame with respect to the upstream orf l a and is translated following ribosomal frameshifting in the overlap region. this will be examined in more detail in section v,g. there is greater amino acid identity among the l b than the l a orfs. for example, l a and l b of ibv, the least typical coronavirus in terms of protein sequences, have amino acid identityhimilarity of approximately / % and / %, respectively, compared with those of mhv, hcv, and tgev. it is the l a orf which accounts for the mhv polymerase gene being approximately - kb longer than those of ibv, hcv, and tgev. a number of functional domains within pol have been predicted following computer-based motif analyses hodgman, ; gorbalenya et al., a,b; lee et al., ) ; some of these functional domains have been confirmed by experimental analysis. the location of these motifs is illustrated in fig. . three motifs have been identified in orf l a , indicating the presence of one or two papain-like cysteine proteases (plp): a chymotrypsinlpicornaviral c-like protease (lee et al., ) . (a) the polymerase gene comprises two orfs, l a and lb, which overlap, the l b orf being translated after ribosomal frameshifting. (b) the positions of motifs: plp and , papain-like protease; x domain, highly conserved between ibv and mhv; clp, picornavirus- c-like protease; md, membrane-associated domain; gfl, growth factorlike; pol, rna-dependent rna polymerase; mb, metal-binding motif; hel, helicase. (c) genetic complementation groups fu and baric, ) . (d) processing scheme for part of the l a orf (denison et al., ( clp) and a cysteine-rich growth factor-related protein (gfl). mhv, hcv- e, and tgev have two plp domains ( and , with plpb corresponding to the single plp domain of ibv. sequence corresponding to a cysteine protease of streptococcus pneumoniae has been identified in l a of ibv. upsteam of plpb is a region termed the x domain, a region of particularly high conservation between ibv and mhv and similar to one near the thiol protease of alpha-and rubiviruses . there is no functional evidence so far to link the gfl with known growth factors, but the predictions of most of the protease domains have been confirmed by experimental analysis. the first plp domain of mhv is responsible for the cleavage of p /p and p from the n terminus of the mhv orf l a polyprotein ( fig. ) (baker et al., (baker et al., , bonilla et al., bonilla et al., , . this plp was inhibited by zinc chloride but not by leupeptin (baker et al., ; denison et al., ) . deletion analysis defined this proteinase domain to be within the sequence encoded by the . - . -kb region from the ' end of the genome. site-directed mutagenesis showed that residues cys- and his- were essential for protease activity (baker et al., ) . some amino acid sequences between the p cleavage site and the plp domain were also essential for the cis cleavage that generates p (baker et al., ; bonilla et al., ) . the function of plp has not been demonstrated. the clp domain extends for approximately amino acids and is homologous to proteases encoded by picornaviruses and several other virus genera. the putative clp domain of hcv- e has been expressed as a /?-galactosidase fusion protein in escherichia coli and shown to have autocatalytic proteolytic activity, releasing an active clp protein (ziebuhr et al., ) . an antiserum against this fusion protein immunoprecipitated a -kda protein from hcv- e-infected cells. similar activity has been demonstrated for the clps of mhv and ibv (tibbles et al., ) . this protease cleaves not only its own boundaries but also several downstream sites within orf l a and orf lb, probably both in cis and in trans. computer analysis predicted that the catalytic center of the ibv clp would include cys- , his- , and glu- (gorbalenya et al., a,b) . site-directed mutagenesis confirmed the role of the cys and his residues but showed that the glu residue was not essential (liu and brown, ) . the same approach confirmed that the predicted qs(g) dipeptide bonds in the l b orf are the targets for the protease activity of the clp of ibv (discussed further in section v,g). similar conclusions were reached for clp activity of mhv and hcv- e grotzinger et al., ) . the importance of cys- in the clp of mhv has been demonstrated (seybert et al., ) . in uitro transcription and translation of a cdna containing the putative clp of mhv produced polypeptides of and kda, which were subsequently processed to products of and kda . the -kda protein possesses the c-like protease activity (lu et al., ) . the clp domain is flanked by predicted membrane-spanning domains, which may be important for the proteolytic activity (tibbles et al., ) (fig. ) . poor expression of the ibv clp protein in vitro led to the discovery that this protease was ubiquinated and subsequently degraded by an adenosine triphosphate (atp)-dependent protease present in reticulocyte lysate (tibbles et al., ) . this is the third example of a viral protein subject to turnover in this manner and involves a different virus class from the previously reported examples, in a picornavirus (oberst et al., ) and an alphavirus . the ubiquitin-mediated, atp-dependent proteolytic pathway is a major cellular, nonlysosomal, protein degradation system, which may cause rapid turnover of the coronaviral polymerase. the functional domains associated with rna synthesis are located within the more conserved l b orf. these include domains for an rnadependent rna polymerase, a nucleoside triphosphate (ntp)-binding/ helicase domain, and a zinc-finger nucleic acid-binding domain (metal binding domain) (fig. ) . computer analyses identified the polymerase domain h o d p a n , ; gorbalenya et al., a,b) . unlike the gdd motif present in many viruses, the corresponding sequence in coronaviruses is sdd. whether the polymerase gene products contain activities other than proteases and polymerases is not known. the coronaviruses exhibit great heterogeneity with respect to the number and genome location of ns protein genes and in regard to the number of orfs within a gene (fig. ) . the functions of these ns proteins are still unknown. there are two genes located between the polymerase and s genes of these viruses (fig. ) . gene - encodes the he protein, while gene encodes an ns protein of unknown function. the gene protein com-prises approximately amino acids ( kda) (luytjes et al., ; shieh et al., ; labonte et al., ) . the bcv and mhv homologs share % amino acid identity, while the homolog of hcv-oc has % identity with that of bcv. this gene product has been detected in the cytoplasm of mhv-, bcv-, and hcv-oc -infected cells and may be phosphorylated (bredenbeek et al., ; zoltick et al., ; coxet al., ; labonte et al., ) . computer analysis of its sequence suggested the presence of a nucleotide-binding site (luytjes et al., ) . however, no function has been assigned to this protein, and it is not required for virus replication in culture (schwarz et al., ) . interestingly, the c terminus of the torovirus orf l a product (polymerase) has - % sequence identity with the gene product of mhv (snijder et al., ) . this evolutionary relationship between coronavirus and torovirus suggests that the gene product is probably involved in viral rna synthesis, since it is expressed as part of the torovirus polymerases. there are two to three orfs in this region, and their structure and the mechanism of expression of gene products vary markedly among different coronavirus species. they can be expressed as two different genes, i.e., expressed from two separate mrnas (e.g., mrnas and of mhv and bcv and mrnas and - of tgev) or localized in one gene, thus requiring internal initiation of translation from a single polycistronic mrna (e.g., mrna of the ibv and fcv groups). in ibv, it contains three orfs ( a, b, and c); orf c encodes the e protein, which is a viral structural protein, while a and b encode ns proteins. the gene products of both orfs a and b (approximately kda) have been detected in small quantities in virus-infected cells (liu et al., ) . in tgev, this region contains two orfs, being separated from the e protein gene. these two orfs are encoded by mrnas and - , respectively, the predicted protein products being approximately and kda, respectively. in a related nonenterogenic strain, prcv, however, there are multiple deletions in this region, essentially inactivating one or both of the orfs (rasschaert et al., ; wesley et al., ) . it has been suggested that the absence of the a product, in addition to a shorter s protein, might be associated with their lack of enteropathogenicity. however, vaughn et al. ( ) have recently described two prcv strains which have an intact a gene (vaughn et al., ) . canine coronavirus has gene orfs equivalent to those of tgev, exhibiting high amino acid identity (> %), although the second orf is truncated by a stop codon (horsburgh et al., ) . two other members of the tgev group exhibit a variation on the same theme. pedv and hcv- e lack a homolg of orf a of tgev and ccv. pedv has an orf corresponding to b of tgev, while hcv- e has two orfs corresponding to the single orf of pedv (duarte et al., ) . members of the group i coronaviruses also exhibit great heterogeneity in this region. mhv-jhm produces mrna , which encodes a -kda protein. this protein has been detected in virus-infected cells (ebner et al., ) . in contrast, hcv-oc contains only amino acids in this region (mounir and talbot, ) . gene of mhv has two orfs, a and b. the latter encodes the structural e protein and is the predominant product made from mrna . it is not clear whether orf a is translated at all. at least one strain of mhv lacks the a orf ; also, hcv-oc has the a orf but is unable to produce a corresponding mrna (mounir and talbot, ) . in summary, there is great heterogeneity with respect to the number, size, and mechanism of expression of orfs between the s and e genes. these ns proteins probably are not required for viral replication. the lack of necessary function may account for the heterogeneity which arose during evolution. ibv is unique in that it has two orfs ( a and b), which encode proteins of . and . kda, respectively. these proteins have been detected in very small amounts in virus-infected cells (liu and inglis, a) . the function of these orfs is not clear. tgev has an additional gene , which encodes a . -kda protein (garwes et al., ; tung et al., ) , in the region corresponding to the ' end untranslated region of other viruses (fig. ) . this protein is hydrophobic and is associated with the endoplasmic reticulum and cell surface membranes (tung et al., ) , but its nuclear localization has also been reported (garwes et al., ) . fcvs and ccv have two orfs in the same region, the first being analogous to the single orf of tgev. the second ( b) orf encodes a -kda soluble protein containing the sequence ktel (vennema et al., , which is similar to the endoplasmic reticulum retention signal, kdel. the protein is partially retained in the endoplasmic reticulum but is also slowly secreted out of the cells. the functions of these proteins are not known. coronaviruses have relatively restricted host ranges, infecting only their natural hosts and closely related animal species. occasionally, cross-species infection of coronaviruses occurs, such as the experimental infection of monkey by mhv, which causes central nervous system demyelination (murray et al., ; cabirac et al., ) , and the occasional infection of humans by bcv, which causes diarrhea. bcv also infects turkeys and tgev infects dogs, suggesting some flexibility in their host range. the expansion of viral host range can be achieved by passing the coronavirus in a heterologous cell line, as demonstrated by the emergence of an mhy variant with the ability to infect originally nonpermissive cell lines, such as human cells, after serial passages (baric et al., ) . in animals, coronaviruses have restricted tissue tropism; for example, most hcv strains cause only respiratory infections. different strains of a coronavirus may have distinct tissue specificity; for example, tgev infects both the gastrointestinal tract, causing fatal diarrhea, and respiratory tract tissues without causing primary respiratory symptoms, whereas prcv, which is closely related to tgev, infects the respiratory tract of pigs but replicates poorly in the intestinal tract (cox et al., ) . the species and tissue specificity of a coronavirus infection is a t least partially dictated by the nature and distribution of cellular receptors and other related molecules that regulate virus entry, as evidenced by the viral replication that results when viral rna is directly introduced into cell types of other animal species. thus, coronaviruses have the potential to replicate in many cell types. the complete coronavirus replication cycle takes place in the cytoplasm. it has been shown that mhv can replicate in enucleated cells and in the presence of actinomycin d and a-amanitin, suggesting that nuclear functions are not required for viral replication wilhelmsen et al., ) . there are, however, reports of the inhibition of replication by actinomycin d of some coronaviruses, including feline enteric coronavirus (lewis et al., , ibv (evans and simpson, , hcv- e (kennedy and johnson-lussenberg, , and mhv in some cell lines (dupuy and lamontagne, ) . thus, nuclear functions may be required for viral replication under certain conditions. this issue has not been resolved. the first step in viral infection is the binding of the virus to target cells. hemagglutination and hemadsorption have been used as assays for studying virus-cell interaction, although the erythrocyte itself is not a target cell for coronavirus infection. several coronaviruses, including hev, ibv, bcv, and some strains of mhv and hcv, can cause hemagglutination (sugiyama and amano, ; schultze et al., ; zhang et al., a) . the binding residue on the cell surface is a -o-acetylated neuraminic acid of glycoproteins or glycolipids (schultze et al., , although different coronaviruses may prefer different structural isoforms of -o-acetylated neuraminic acid. for bcv, the virus binding to erythrocytes is mediated through either the s or he protein, both of which have hemagglutinating activities, the s protein having the stronger activity (king et al., ; schultze et al., a,b) . the he protein of bcv and hev also recognizes -o-acetylated neuraminic acid, and its esterase activity is also specific for this molecule; thus, he protein has both receptor-binding and receptor-destroying activities (vlasak et al., a,b; schultze et al., b) . expression of the he protein of mhv on the cell surface conferred a hemadsorption activity ; however, even viruses that lack he protein (e.g., ibv) can cause hemagglutination, suggesting the role of s protein in hemagglutination. thus, the he and s proteins of various coronaviruses may have comparable functions, enabling the virus to bind the sialic acid residues; however, only the he protein confers the receptordestroying activity. the residue necessary for hemagglutination by ibv is a , -linked n-acetylneuraminic acid . curiously, the hemagglutinating activity of ibv is not evident until the virus particle is treated with neuraminidase, suggesting that the s protein itself is covered by sialic acid. although virus binding to erythrocytes provides a good model system for studying virus-cell interactions, it may not necessarily reflect the actual mechanism of virus attachment to target cells. the classical study of virus attachment to target cells involved the in uitro binding of mhv to macrophages from genetically susceptible m i c w l m. c. lai and david cavanagh and resistant mouse strains (shif and bang, ) . this study showed that mhv bound equally well to cells from resistant and susceptible mice, even though macrophages from resistant mice were resistant to virus infection. similar observations have been made on splenic lymphocytes, thymocytes (krzystyniak and dupuy, , and glial cells (wilson and dales, ) ; thus, it appears that genetic resistance is not exerted at the level of virus binding in these cases. similarly, established tissue culture cell lines, including murine and primate cells, irrespective of their degree of susceptibility or resistance to mhv, bound mhv to the same extent (van dinter and flintoff, ; kooi et al., kooi et al., , . thus, virus may bind to a ubiquitous molecule on the cell surface, which, however, may not lead to virus infection. whether this ubiquitous molecule is a sialic acid-containing glycoprotein has not been established. the binding of bcv to its target cells, such as mdck cells, is mediated by -o-acetylneuraminic acid residues similar to those on erythrocytes. removal of the sialic acid by neuraminidase abolished virus attachment, while resialization restored it . hcv-oc binds to a similar sialic acid residue but prefers a form slightly different from that for bcv (kunkel and herrler, ) . the he protein of bcv can also mediate virus binding to target cells, and this binding may be required for viral infection, as suggested by the finding that mab against he inhibited bcv infectivity (deregt and babiuk, ; deregt et al., ) . one inhibitor of the esterase activity of he protein, diisopropylfluorophosphate, also inhibited bcv infection (vlasak et al., a) . the s protein of bcv probably also participates in virus binding to target cells, as suggested by the finding that the mab against s protein can neutralize bcv infectivity . the relative importance of s and he proteins is not clear. in contrast, none of the mab against the he protein of mhv inhibited mhv infection . despite the finding that the binding of he and s proteins to target cells is necessary for bcv infection, the binding of bcv or hcv-oc to n-acetylneuraminic acid in itself is not likely the basis of viral cell tropism because sialic acid is a common cell surface carbohydrate residue; thus, an additional, more cell type-specific molecule is probably required for viral infection. the finding that mhv and other coronaviruses bound to resistant as well as susceptible cells indicates that this binding may represent an initial step in the virus attachment process, which is not sufficient for viral infection. it is likely that a more specific binding between virus and cells is required for the establishment of viral infection. this binding involves a specific virus receptor molecule on the cell surface. a. mhv receptor. the mhv receptor was the first coronavirus receptor to be identified. it is the murine homolog of a member of the carcinoembryonic antigen (cea) family (dveksler et al., ; williams et al., ) and belongs to the biliary glycoprotein (bgp) subfamily. the terminology of mhv receptors in the literature is somewhat controversial, the following terms being used interchangeably: mmcgm , mhvr- , and bgpa. it has an immunoglobulin-like structure, consisting of four immunoglubulin-like loops, the n-terminal loop being the virus-binding domain (dveksler et al., b) . the sequence of the c terminus (cytoplasmic domain) of the receptor is not essential. glycosylation of the protein also is not necessary for its receptor function in uiuo (dveksler et al., ) . the functional significance of the receptor in viral infection in uiuo was demonstrated by the finding that an mab against the mhv receptor inhibited viral infection in mice (smith et al., ) . subsequently, several additional members of cea family were found to serve as mhv receptors, including an mmcgm -like protein (also termed mhvr- and bgpb), which is the product of an alternatively spliced form of mmcgml rna and is expressed in both the liver and brain, in contrast to the liver-specific expression of mmcgml (yokomori and dveksler et al., a) ; an allelic gene product of the bgp gene in sjl mice, a mouse strain resistant to mhv infection (yokomori and lai, ; dveksler et al., a) ; bgp- , which is the product of a new member of the murine bgp gene (nedellec et al., ) ; and a novel pregnancy-specific glycoprotein (psg)-like protein, which is expressed in the mouse brain, in contrast to placenta-specific expression of other psg molecules . all these molecules contain a consensus motif in the virus-binding domain (n-terminal loop). thus, several different cea family members, which are differentially expressed in different cells and tissues, can potentially serve as an mhv receptor. different strains of mhv may use different cearelated molecules as receptors at different efficiencies (compton, ; . the prototype mhv receptors (mhvr- ) are expressed in the liver, gastrointestinal tract, b cells, macrophages, and endothelial cells but not in t cells (coutelier et al., ; godfraind et al., ) , consistent with the target cell specificity of mhv. however, the mhv receptor is also expressed in other tissues, e.g., kidney, which are not targets for mhv infection. also, sjl mice express a functional mhv receptor (yokomori and lai, ; dveksler et al., a) but are resistant to mhv infection (knobler et al., ) . thus, receptor expression is not sufficient for viral infection. it is not yet clear which molecules are used by mhv as receptors in cross-species infection (e.g., rats and monkeys) (murray et al., ; cabirac et al., ) . recently it was shown that bgp and cea molecules of human origin could serve as receptors for some mhv strains . the expression of the receptor molecules on the cell surface is necessary for virus infection, and the expression level of the receptor may determine the relative susceptibility or resistance to viral infection in some cells. during persistent viral infection of cultured murine cells, the expression level of the receptor is offen reduced, resulting in the relative resistance of the cells to viral superinfection, which could be overcome by the expression of an exogenous receptor (sawicki et al., ; chen and baric, ) . thus, there is a rough correlation between receptor expression and the susceptibility of a cell type to virus infection. under certain circumstances, virus may infect cells by a receptorindependent mechanism; for example, mhv-infected murine cells may fuse with human cells, which do not have mhv receptors, and cause the latter cells to become infected (gallagher et al., ) . it has been shown that mhv infects polarized epithelial cells through the apical, but not the basolateral, surface (rossen et al., a (rossen et al., , . it is not clear whether the virus receptor is differentially expressed on the two different surfaces. b. receptors for tgevand hcv- e. the receptors for tgev and hcv- e have been identified as aminopeptidase n (apn) of the porcine and human species, respectively (delmas et al., ; yeager et al., ) . prcv also uses porcine apn as a receptor; thus, virus binding to the receptor is not sufficient to explain the differences in tissue tropism between tgev and prcv. apn is a member of the membrane-bound metallopeptidase family and is widely distributed on diverse cell types; it is highly expressed on the brush border membrane of enterocytes. some of the antibodies against human apn can block hcv- e binding (yeager et al., ) ; however, the catalytic site of the protease activity of apn is not required for receptor function, and the inhibitors of apn do not block viral infection . similar to mhv, tgev infects polarized cells through the apical, but not the basolateral, surface (rossen et al., ) . again, it is not clear whether this is restricted by the differential expression of apn on the different sides of the cells. tgev has also been shown to bind to a -kda protein on the surface of the enterocytes on the villi of the small intestine (weingartl and derbyshire, ) . pcrv does not bind to this molecule. both the temporal expression (mainly in the newborn) and spatial distribution patterns (on the villi of the gastrointestinal tract) of the -kda protein correspond to the pattern of susceptibility of piglets to tgev infection. thus, the expression pattern of this molecule appears to have a better correlation than the porcine a€" with the tissue tropism of tgev. this -kda molecule may be an alternative receptor used by tgev. the relative functional significance of this molecule and a€" as tgev receptors is not yet clear. the fipv strains of fcv and canine coronavirus apparently utilize the a€" of feline and canine species, respectively, as receptors (benbacer et al., ) . cross-species utilization of feline a€" by coronaviruses of different species (canine, feline, and human) has also been reported (tresnan et al., ) . fipv, however, is unique among coronaviruses in that it causes an antibody-dependent enhancement (ade) phenomenon (weiss and scott, , which is the result of the binding of the virus-antibody complex to fc receptors on the surface of macrophages, leading to enhanced virus uptake and spread. this ade phenomenon has been attributed to the s protein-antibody complex (vennema et al., b; corapi et al., ; olsen et al., ) . the fc receptor may be a co-factor or an alternative receptor for fipv entry into macrophages. in this regard, the s protein of mhv has been shown to have limited sequence homology with the murine fc receptor and to have the ability to bind to the fc fragment of immunoglobulin oleszak et al., ) . whether the fc receptor plays a role in mhv infection is not clear. however, mhv does not exhibit ade. c. receptors for other coronaviruses. sialic acid (n-acetyl- - acetylneuraminic acid)-containing glycoproteins are probably a component of the cell surface molecules required for bcv and hcv-oc infection because the removal of sialic acids inhibits bcv infection and resialylation restores virus infectivity ; however, it is unlikely that it is the primary receptor molecule used by these viruses since the distribution of these molecules is more widespread than the susceptible target cells. the identity of the specific receptor for these viruses has not been determined. for hcv-oc , it has been shown that the virus binds to a major histocompatibility complex class i molecule (collins, ) . however, the receptor function of this molecule has not been established. the mechanism of coronavirus entry into target cells has been controversial. early electron microscopic studies visualized virus (mhv and ibv) particles inside lysosome-like vesicles near plasma membranes, suggesting that virus enters cells by endocytosis ("viropexis") (david-ferreira and manaker, ) ; however, other studies suggested that virus enters cells by direct fusion between virions and the plasma membrane (doughri et al., ) . lysosomotropic drugs, such as ammonium chloride and chloroquine, inhibited mhv- virus entry (krzystyniak and dupuy, ) . also, mhv-specific antibodies did not lyse virusinfected cells during the virus-entry process, as would be the case if the virus fused with the cell membrane (krzystyniak and dupuy, ) . these results suggested that mhv- enters cells by an endocytotic pathway. similar studies using the a strain of mhv, however, showed that ammonium chloride delayed, but did not inhibit, the viral infection of l- cells (mizzen et al., ) . the effects of ammonium chloride were interpreted to be inhibiting virus uncoating in this case. recent studies by the same group have further shown that only a small proportion of adsorbed virus enters cells by the endocytotic pathway since ammonium chloride, chloroquine, and dansylcadaverine, all of which inhibit receptor-mediated endocytosis, did not have significant effects on mhv entry (kooi et al., ) . the majority of mhv particles enter cells by virus-cell fusion at the plasma membrane. this interpretation is consistent with the finding that the optimum ph for mhvinduced cell fusion is . (weismiller et al., ; kooi et al., , rather than the acidic ph expected for a virus that enters cells by an endocytotic pathway (e.g., vesicular stomatitis virus). the optimum ph for bcv-and ibv-induced cell fusion is also neutral (payne and storz, ; li and cavanagh, ) . these findings suggest that coronavirus enters cells by virus-cell fusion at the plasma membrane. on the other hand, virus internalization by endocytosis may be a nonproductive mechanism which does not depend on virus-receptor interaction, since some mhv-resistant cell lines can internalize mhv particles as efficiently as susceptible cell lines (kooi et al., ) . most surprisingly, even vero cells, an african monkey kidney cell line which presumably does not have an mhv receptor, can internalize virus (kooi et al., ) . therefore, it is likely that mhv enters cells by both acidic-phdependent (endocytosis) and -nondependent pathways (kooi et al., ) . the exact mechanism of virus entry may depend on cell types and virus strains. interestingly, an mhv variant which has mutations in the s protein has an acidic optimum ph of . - . , in contrast to the ph of . for the parental virus (gallagher et al., ) . this virus variant probably enters cells by an endocytic pathway, a fact supported by the finding that infection of this variant virus is inhibited by ammonium chloride or chloroquine. what triggers virus internalization after virus-receptor binding is not clear. it has been shown that a conformational change in the s protein could be induced at ph . and incubation at °c . whether this represents the expected conformational change following virus-receptor binding is not clear. irrespective of the mechanism of virus internalization, fusion between the viral envelope and cell membrane must occur, either at the cell surface or in the endosome, for viral infection to take place. virus-induced cell-cell fusion has been used to investigate the ability of a virus to induce fusion. early studies with mhv indicated that virus-induced fusion from without (caused by virions at the cell surface) or fusion from within (caused by de ~o u o synthesized s protein on the cell surface) required cleavage of the s protein (frana et al., ; sturman et al., ) . work on bcv supported this view (payne and storz, ; storz et al., ) . however, more recent experiments involving the expression of s protein (de groot et al., ; stauber et al., ; taguchi, ) and studies of mhv fusion mutants (gombold et al., ) have indicated that uncleaved s can cause syncytium formation, though less efficiently than the cleaved s. of course, coronaviruses such as tgev, which have no cleaved s protein, are infectious, in fact, highly so. since fusion of the virion envelope with a cell membrane is an essential part of the infection process, these results suggest that tgev must be able to cause viruscell fusion. thus, virus-cell fusion and cell-cell fusion may have different requirements, and, for at least some coronaviruses, s cleavage is not required for the fusion of a virion with a cell membrane. nevertheless, cleaved s may be more efficient at inducing fusion for some coronaviruses. the concentration of s at the surface of a virion may be higher than at the cell surface, such that even the uncleaved s can induce virion-cell fusion, even though it cannot cause cell-cell fusion. virusreceptor interaction may also trigger a signal transduction pathway to facilitate the internalization of the virus-receptor complex. one study showed that tyrosine kinase is activated in macrophages immediately following mhv- infection (dackiw et al., ) . it is not yet known whether this is required for virus entry. the mechanism of virus uncoating, i.e., the release of virion rna from the nucleocapsid, after the virus has been internalized remains unclear. one study suggested that virus uncoating may involve an endosomal neutral phosphatase, which preferentially dephosphorylates the nucleocapsid protein (mohandas and dales, ) . furthermore, while immature oligodendrocytes were sensitive to jhm virus infection, differentiated oligodendrocytes were resistant, probably due to a block in virion uncoating (beushausen et al., ) . the factors responsible for the differences in these two types of cells may involve protein kinases (wilson et al., ) . additional cellular factors may be required for viral penetration and uncoating. various murine cell lines, all of which express virus receptors, show different degrees of susceptibility to infection by different mhv strains (kooi et al., ; asanaka and lai, ; yokomori et al., ) . cell-cell and virus-cell fusion studies indicated that virus infection is blocked at different stages of virus entry, including penetration and uncoating, in different cell lines (van dinter and flintoff, ; asanaka and lai, ) . these cell lines could be grouped into at least three complementation groups with respect to the virus entry process (flintoff, ; asanaka and lai, ) . thus, virus penetration and uncoating appear to require separate cellular factors. it has been suggested from the studies using recombinant viruses between the a and jhm strains of mhv that viral s protein may interact with these cellular factors . the nature of these factors is not yet clear. following virus uncoating, the first macromolecular synthetic event is predicted to be the synthesis of an rna-dependent rna polymerase(s) from the incoming viral genomic rna, as is the case for all positive-strand rna viruses. the polymerase is translated from gene at the ' end of the genomic rna, most likely directly from the incoming genomic rna. the process of primary translation has not been observed experimentally. however, inhibitors of protein synthesis applied early in the infection blocked rna transcription (mahy et al., ; perlman et al., ; sawicki and sawicki, , indicating that protein synthesis, most likely the translation of a viral polymerase, is necessary for viral rna synthesis. this virus-specific polymerase is responsible for the synthesis of negative-strand rna from the incoming genomic rna and subsequent transcription of mrnas from the negative-strand template. the nature of polymerase is discussed in section iv,a. since the genomic-sized rna is used for both packaging into virus particles to become virion rna and as an mrna for protein translation, the distinction between rna transcription and rna replication is often blurred. in this review, we will use the term "transcription" to describe the synthesis of subgenomic mrnas as well as genomic rna used for translation; the term "replication" will be used to describe the synthesis of the genomic rna destined to be packaged into virions. coronavirus rna synthesis occurs via an rna-dependent rna transcription process; thus, rna synthesis can occur in the presence of actinomycin d (with the exception of some coronaviruses, as discussed in section v,a). the majority of the virus-specific rnas in the cells are mrnas, which are transcribed from a negative-strand rna template. for clarity of discussion, the structure of the mrnas will be discussed first. coronavirus mrnas consist of six to eight species of different sizes, depending on the coronavirus species and strains (lai, ) . the largest mrna is equivalent to the genomic rna, and the remainder are subgenomic in size. these rnas are designated mrnas through , in order of decreasing size, according to the recommendations of the coronavirus study group in (cavanagh et al., ) . some mrnas have been given a hyphenated name, e.g., mrna - , because they were discovered after the original set of mrnas was named. they have a nested-set structure, and all of them contain sequences starting at the ' terminus and extending to various distances toward the ' end (stern and kennedy, b; lai et al., ; leibowitz et al., ) . the smallest mrna contains only the ' terminal orf, while each next larger mrna contains one additional orf. the structure of the mrnas in relation to the genome structure is shown in fig. . thus, except for the smallest mrna, all of the mrnas are structurally poly- cistronic. in general, each orf in the genome is represented by an mrna, whose sequence starts from a consensus signal upstream of the orf, and only the ' most orf of each mrna can be translated; thus, each mrna is functionally monocistronic. however, there are exceptions: some mrnas, e.g., mrna of mhv and mrna of ibv, are translated into two or three proteins by different mechanisms (see section v,g). several additional minor mrna species have been detected, some of which could only be detected by reverse transcription-polymerase chain reaction (la monica et al., ; schaad and baric, ) . these minor rnas probably represent rna transcripts from weak or atypical mrna start signals (see below). most do not contain a complete orf at the ' end; thus, they are probably not functional. furthermore, in mhv, several mrnas, e.g., mrnas - , - , and - , are transcribed only in some virus strains la monica et al., ) . the syntheses of these mrnas appear to be differentially regulated by the sequence at the ' end of the viral genome la monica et al., ) . coronavirus mrnas have another unique structural feature: their ' ends have a leader sequence of approximately - nucleotides, which is derived from the ' end of the genomic rna (lai et al., , spaan et al., ) . the leader sequences of all the mrnas are identical for a given strain of virus, except for slight variations at some of the leader-mrna fusion sites, and are identical to the sequence present at the ' end of the genomic rna. at the mrna start sites on the viral genomic rna, there is a short stretch of sequence that is nearly homologous to the ' end of the leader rna (budzilowicz et az., ) . this sequence constitutes part of the signal for subgenomic mrna transcription (makino et al., ) . sequence comparison of viral genomic and mrnas suggests that subgenomic mrnas are derived by fusion of the ' end genomic rna sequence (leader) to the mrna start sites on the viral genomic rna. the mrna start sites are usually located between the genes; hence, they are termed intergenic (ig) sequences. however, some of the igs may overlap the coding region of the upstream gene. the core sequence of the ig for mhv is ucuaaac or a slightly variant form of this sequence at various ig sites (joo and makino, ) . other virus species also have similar ig sequences. the leader sequence of mhv ranges in length from to nucleotides, the variation resulting from the heterogeneity of the ' end sequence, which contains two to four copies of a pentanucleotide (ucuaa) repeat. the homologous nucleotides (ucuaa) at the ' end of the leader and ig sites serve as fusion sites for the leader and mrnas. some of the mhv mrnas are heterogeneous, consisting of several subspecies, each containing different copy numbers of the ucuaa repeat . this fact suggests that the fusion between the leader rna and the mrnas is not very precise. the length and sequence of the leader rna in other coronaviruses vary. however, the ' end of the leader sequence always contains a pentanucleotide ucuaa or a closely related sequence. mrnas of coronaviruses other than mhv are usually homogeneous in their structure, probably a reflection of the fact that leader rna at the ' end of the genome and ig sites in these viruses contain only a single copy of the the ucuaa-like sequence (hofmann et al., a) . the copy number of this pentanucleotide repeat apparently plays an important role in the regulation of mrna transcription. coronavirus rna synthesis is mediated by rna-dependent rna synthesis via a negative-strand rna intermediate (complementary to the genomic rna). coronavirus negative-strand rna represents no more than - % of the total intracellular virus-specific rna sawicki and sawicki, ) . bothgenome-sized and subgenomic negative-strand rnas, which correspond in number of species and size to those of the virus-specific mrnas, have been detected (sethna et al., ; hofmann et al., ) . the relative molar ratios of the various subgenomic negative-strand rna species are comparable to those of the positive-strand subgenomic mrnas. the ' end of the negativestrand rna contains poly(u) sequences, which are shorter than the poly(a) sequences present on the positive-strand rnas . at the ' end of the negative-strand rna is the complementary sequence of the leader rna (anti-leader) (sethna et al., ) . structurally speaking, the subgenomic negative-strand rnas appear to be mirror images of the positive-strand subgenomic mrnas. all of the negative-strand rnas in the infected cells are present in the form of double-stranded rna, no free negative-strand rna is detected . in virus-infected cells, virus-specific mrna synthesis can usually be detected a few hours after infection and throughout most of the viral replication cycle (stern and kennedy, a; leibowitz et al., ; keck et al., a) . the molar amounts of the different mrna species vary; smaller mrnas are generally more abundant than larger ones, but this rule does not always hold true. nevertheless, the relative ratio of different subgenomic mrna species remains constant throughout, suggesting that the synthesis of the various subgenomic mrna species is regulated coordinately. some viruses may show slight variations in the amounts of individual mrna species present during infection (hiscox et al., a) . later in infection, there appears to be an enhanced synthesis of the genomic-sized rna (keck et al., a) . the kinetics of negative-strand rna synthesis follows a pattern similar to that of positive-strand mrna synthesis; however, the peak of negative-strand rna synthesis appears to occur earlier than for positive-strand rna sawicki and sawicki, ) . thereafter, negative-strand rna synthesis drops significantly, in contrast to that of positive-strand rna synthesis, and negative-strand rna appears to be stable sawicki and sawicki, ) . a similar pattern of kinetics of negative-strand rna synthesis is also seen in the accumulation of the negative-strand rna of a di rna, which very rapidly reaches a steady-state level after transfection . therefore, the negative-strand rna probably functions as a template for multiple rounds of positive-strand rna synthesis. this conclusion is supported by the study of a ts mutant defective in negative-strand rna synthesis (schaad and baric, ) . however, the ability to synthesize negative-strand rna seems to be maintained throughout the viral life cycle, as evidenced by the finding that a transfected di rna can replicate even when transfected late in the infection (jeong and makino, ) . since all subgenomic rnas consist of a leader rna derived from the ' end of the genome and a body sequence derived from various downstream sequences, they must be synthesized by fusion of two discontiguous sequences either during or after transcription. an early study showed that the leader sequence of each mrna can be exchanged freely between two coinfecting viruses, suggesting that the leader rna and mrnas are transcribed independently and can conjoin in a random fashion (makino et al., b) . more recent studies using di rna constructs that contain an inserted mrna start signal (see below) established that the leader rna and mrnas are usually derived from two separate rna molecules (jeong and makino, ; zhang et al., b) . these studies unequivocally showed that coronaviral mrna synthesis is carried out by either a discontinuous transcription or transsplicing process, which fuses sequences from two different rna molecules. several transcription models have been proposed, each of which is consistent with some of the experimental data. these models are not mutually exclusive, as components of each model may operate at different stages of the viral replication cycle. before presenting these models, we will discuss several findings pertinent to coronaviral rna transcription. . coronavirus replication takes place entirely in the cytoplasm. nuclear functions are believed not to be required for rna synthesis wilhelmsen et al., ) ; thus, viral rna transcription does not involve the conventional rna splicing machinery present in the nucleus. . early ultraviolet (w) transcriptional mapping studies indicated that in the late stage of viral replication, the w target size of each subgenomic and genomic mrna is approximately equivalent to the physical size of the respective mrna (jacobs et al., ; stern and sefton, a) ; thus, each mrna is transcribed independently rather than derived by the processing of a large precursor rna. however, early in infection, the w target sizes of the subgenomic mrnas were found to be equivalent to that of the genomic rna (yokomori et al., ) ; thus, at least early in infection, the synthesis of a genomic-length rna is required for subgenomic mrna synthesis, although it is not clear whether this requirement is for a positive-or a negative-stranded, full-length rna. a more recent analysis of the uv target sizes of subgenomic mrnas of mhv suggested that, even late in the infection, the w target sizes of some subgenomic mrnas are slightly larger than their physical lengths but smaller than genomic size (den boon et al., ) . similar observations were made for equine arteritis virus (an arterivirus). this recent result is consistent with either of two interpretations: (a) the subgenomic mrnas are derived from a slightly longer rna template or (b) they are derived from a mixture of templates of different sizes (genomic as well as subgenomic). the difference in w target size between the early and late stages of viral rna replication suggests that different mechanisms of rna synthesis may operate at the different stages of the viral replication cycle. . the molar ratios of different subgenomic mrna species and those of subgenomic negative-strand rnas are similar (sethna et al., ; hofmann et al., ) , suggesting that subgenomic mrnas and subgenomic negative-strand rnas are derived from each other or under the same transcriptional regulation. . the leader rna at the ' end of each mrna is identical in each mrna and to the leader rna at the ' end of genomic rna. furthermore, there is sequence homology between the ' end of the leader rna and the mrna start sites on the genomic rna (budzilowicz et al., , where the leader sequence is fused to the mrnas. there is some sequence divergence between the leader rna and some of the mrna start sites; in these cases, the leader rna of the resulting mrnas usually mimics the sequence of the mrna start site rather than the leader at the ' end of the genome. this finding was used to suggest the possible presence of rna proofreading activity during coronavirus transcription (lai, (lai, , ; van der most et al., ). the following transcriptional models (fig. ) address the possible mechanism of fusion between the leader sequence and mrnas. most of the experimental evidence came from mhv studies. the exceptions will be noted. a. leader-primed transcription. this model proposes that the virion genomic rna is first transcribed into a genomic-length, negativestrand rna, which, in turn, becomes the template for subsequent subgenomic mrna synthesis. the leader rna is transcribed from the ' end of the negative-strand rna and dissociated from the template. the free rna subsequently associates with the template rna at various mrna start sites and serves as a primer for transcription of mrnas. it is proposed that the discontinuous transcription step takes place during positive-strand rna synthesis. several pieces of evidence are compatible with this model: detected in the cytoplasm of mhv-infected cells (baric et al., ) . some of these are dissociated from the template rna and, thus, may serve as a potential source of primers in this transcription model. these rnas have distinct sizes which are reproducible from cell to cell (baric et al., ) ; however, they are not exactly the same size as the leader sequence present in the subgenomic mrnas. thus, these free leader rnas must be processed before they are incorporated into mrnas. a temperature-sensitive mutant of mhv, which synthesizes leader rna but not mrnas at the nonpermissive temperature, has been isolated . the isolation of this mutant suggests that mhv mrna synthesis is discontinuous, requiring different viral proteins for the synthesis of leader rna and mrnas. thus, a distinction can be made between leader rna synthesis and mrna synthesis. of the viruses is derived from the other coinfecting virus (makino et al., b) . this result suggests that the leader sequence and body sequence of each mrna are derived from two separate pools. this phenomenon is reminiscent of the rna reassortment that occurs in rna viruses with segmented rna genomes. this result is best explained by the possibility that free leader rnas participate in viral rna synthesis. in an in vitro transcription system utilizing cytoplasmic extracts from mhv-infected cells, exogenous leader rnas can be utilized for mrna synthesis (baker and lai, ) . the exogenous leader rna was incorporated into the subgenomic mrnas at a site that matched precisely that of the endogenous leader rna present in the viral subgenomic mrnas, regardless of the length of the exogenous leader rna used, suggesting that the exogenous leader rna sequence was processed before being incorporated into mrnas. furthermore, the truncated leader rna which lacked the ' end ucuaa sequence could not be incorporated into mrnas, suggesting the importance of this sequence in transcription (baker and lai, ) . . the leader rna sequence, specifically the copy number of the ucuaa repeats at the ' end of the leader rna, can affect the transcription of some viral subgenomic mrnas. for example, whereas an mhv strain containing two ucuaa repeats transcribes mrna - , a strain with three ucuaa repeats does not, despite identical sequences in the mrna start sites of these two viruses la monica et al., ) . this finding suggests that the leader rna plays an essential role in the regulation of mrna transcription. according to this model, the free leader rna binds to the mrna start site (ig) of the full-length negative-strand template via the complementary sequences between the ' end of the leader (positive-strand) and the ig site of the template rna (negative-strand) and serves as the primer for rna transcription. the free leader rna (primer) may be longer than the leader sequence in the subgenomic mrnas. there are certain mismatched nucleotides between the leader and template at some mrna start sites; in the latter case, sequences in the mature mrnas usually match those of the template instead of the leader. therefore, the free leader rna probably undergoes ' end cleavage before transcription starts to remove the leader nucleotides that are not complementary to the template rna (lai, (lai, , van der most et al., ) . transcription is then initiated from the ' end of the processed leader rna. this model is consistent with most of the sequence data of mrnas. it also explains the curious finding that some mrnas of mhv are heterogeneous in the copy number (from two to four) of the pentanucleotide (ucuaa) repeats at the leader-mrna fusion site . this heterogeneity is best explained by the imprecise binding between the leader rna and template rna due to the presence of multiple copies of ucuaa (lai, ) . indeed, bcv, which contains only one copy of ucuaa in both the ' leader and ig sites, does not show this type of heterogeneity in its mrnas (hofmann et al., a) . some recent data, however, cannot be explained by this rna sequence-homology-driven transcription model. a particular mhv strain (mhv-bc), which has four copies of the ucuaa in the leader rna, synthesizes some subgenomic mrnas that are very heterogeneous in length and in leader-mrna fusion sites . the sequence data of its mrnas showed that the leader rna of this virus is randomly fused to sites where no sequence homology exists between the leader and fusion sites (zhang et al., ) . a similar though less conspicuous heterogeneity in the leader-mrna fusion sites has also been observed in another mhv strain in a di rna-based transcription system (see section v,e, ) (van der most et al., ) . these findings suggest that the sequence complementarity between the leader rna and ig sites may not be the driving force for mrna transcription. thus, a modified version of the leader-primed transcription model proposes that the ucuaaac sequence provides a recognition signal for viral polymerases and viral or cellular transcription factors. these proteins bind to the leader and ig sites of the template rna, and the subsequent rna-protein and protein-protein interactions result in the formation of a transcription complex to initiate mrna transcription and effect leader-mrna fusion zhang and lai, ) . the salient feature of this model is that the discontinuous transcription step occurs during positive-strand rna synthesis; thus, transcriptional regulation is exerted mainly during positive-strand rna synthesis. this is consistent with current knowledge of the regulation of mhv rna synthesis. it has been shown that mhv mrna transcription requires multiple cis-acting rna sequences (see section v,e, ). in contrast, the initiation of negative-strand rna synthesis requires only the ' end -nt plus poly(a) . thus, most of the regulatory elements appear to regulate positive-strand rna synthesis. since the free leader rna is the centerpiece of this transcription model, it readily explains why the leader rna from a different virus can be utilized freely in trans during mixed infections (makino et al., b) . however, this model does not explain the finding that subgenomic replicative-intermediates (ri) and replicative-form (rf) rnas were detected and were functional during viral rna synthesis (sawicki and sawicki, ; schaad and baric, ) (see section v,e, ,b). it is possible that the subgenomic mrnas synthesized can be transcribed into subgenomic negative-strand rnas, which, in turn, become the templates for mrna transcription at a later stage in the viral replication cycle. this would explain why the uv target sizes for mrnas are nearly equivalent to the physical sizes of mrnas late in the infection and yet are equivalent to the genomic-sized rna early in the infection (yokomori et al., b) . in contrast to the leader-primed transcription model, this model proposes that the discontinuous transcription step occurs during negative-strand rna synthesis, generating subgenomic negativestrand rnas, which then serve as templates for subgenomic mrnas in uninterrupted transcription. this model was proposed to account for the detection of subgenomic negative-strand rnas (sethna et al., ; hofmann et al., ) and subgenomic ris (sawicki and sawicki, ) in virus-infected cells. in this model, ig (mrna start site) sequences on the genomic rna serve as termination or pausing signals for negative-strand synthesis (konings et al., , and the nascent subgenomic negative-strand rna then jumps to the leader rna sequence at the ' end of the genomic rna by an unknown mechanism to continue rna synthesis. as a result, the nascent negative-strand subgenomic rna fuses with the negative-strand leader sequence, generating a subgenomic negative-strand rna that contains an anti-leader sequence at its ' end and a poly(u) sequence at its ' end sethna et al., ) . structurally, these negativestrand rnas are mirror images of the subgenomic mrnas and, thus, can potentially serve as a template for uninterrupted transcription of subgenomic mrnas. in this model, the regulation of subgenomic mrna transcription would be exerted on negative-strand instead of positive-strand rna synthesis. this model is consistent with the following observations: . subgenomic negative-strand rnas have been detected in virusinfected cells (sethna et al., ; hofmannet al., ) . these rnas have structures that are mirror images of those of the completed subgenomic mrnas. the relative molar ratios of the different subgenomic negative-strand rnas are similar to those of the corresponding viral mrnas (sethna et al., ; hofmann et al., ). in the infection (sawicki and sawicki, ) . the smaller ris were precursors of the smaller mrnas and the larger ris generated the larger mrnas, suggesting that each subgenomic mrna was transcribed from the corresponding subgenomic-sized negative-strand template (sawicki and sawicki, ) . another study, which analyzed the subgenomic rfs of a temperature-sensitive mutant of m w , also suggested that subgenomic negative-strand rnas are functional (schaad and baric, ) ; although, in this study, ris were not directly examined. . the uv targets for subgenomic mrna synthesis at the later stage of viral replication are subgenomic in length (jacobs et al., ; stern and sefton, a; yokomori et al., b) , roughly corresponding to the physical lengths of each subgenomic mrna, suggesting that the templates for these mrnas are subgenomic. . in di rna systems (see section v,e, ), when multiple ig sequences were present, the sequences in the ' end often had a higher transcription efficiency than those at the ' end, consistent with the proposal that igs serve as transcriptional termination sites, which impede the elongation of the negative-strand rnas (van marle et al., ; krishnan et al., ) . however, in some cases, the higher transcription efficiency of the ' proximal ig was observed only when the neighboring igs were very close together, suggesting a spatial constraint rather than sequential interference (joo and makino, ) . this model, however, cannot explain why the w targets for subgenomic mrna synthesis early in infection are of genomic size (yokomori et al., b) and why, later in the infection, the targets for these same mrnas are still larger than the respective subgenomic mrnas but not longer than genomic size (den boon et al., ) . it also cannot explain why the nature of the leader sequence can regulate differential transcription of various mrna species, such as mrna - of mhv, inasmuch as the leader sequence on the template rna is localized downstream of the transcription termination site for negative-strand rna synthesis. finally, it is difficult to explain why the leader rnas are derived in trans. c. trans-splicing of nascent rna transcripts. this model proposes that the fdl-length positive-or negative-strand rnas are spliced posttranscriptionally to generate subgenomic rnas. it was initially considered unlikely because of the findings that coronavirus replicates in the cytoplasm rather than in the nucleus wilhelmsen et al., ) , where the splicing machinery is present, and that uv target sizes of subgenomic mrnas are equivalent to the physical sizes of subgenomic mrnas (jacobs et al., ) . furthermore, there are no consensus splicing donor and acceptor sequences in the coronavirus genomic rnas. however, the trans-splicing model is compatible with recent findings that early in infection, the w targets for subgenomic mrna synthesis are of genomic length (yokomori et al., b) , and that both the leader rna and ig sequence of mhv negative-strand rna bind to a cellular factor, heterogeneous nuclear rnp (hnrnp) al, which is involved in alternative rna splicing h.-p. li and m. m. c. lai, unpublished observation) . a modified splicing model thus can be proposed as follows: a full-length negative-strand rna is first synthesized. components of the splicing machinery derived from the nucleus or cytoplasm then bind to the leader sequence and ig sites on the negative-strand rna and form a splicing complex. the leader and ig can be derived from different rna molecules. splicing between the leader and ig generates a subgenomic negative-strand rna. once the spliced subgenomic negative-strand rnas are generated, they are used as templates for subsequent mrna synthesis. later in infection, even the subgenomic negative-strand rnas may be able to participate in rna splicing to generate smaller subgenomic negativestrand rnas because they themselves also contain the leader and ig sequences. this model may thus explain why the w target for mrna transcription is of genomic length early in infection (yokomori et al., b) and may shed light on the recent puzzling finding that later in infection, the w target sizes are still larger than the actual sizes of the subgenomic mrnas (den boon et al., ) . it also explains the functional roles of subgenomic ris (sawicki and sawicki, ) . this potential splicing, however, must be different from conventional rna splicing because it occurs in the cytoplasm, and the splicing donor and acceptor sequences must also be different from the conventional ones. since some of the splicing factors are probably derived from the nucleus, this model predicts that nuclear functions are involved in mhv rna transcription. based on the findings that some coronaviruses, including bcv, tgev, and ibv (sethna et al., ; hofmann et al., ; zhao et al., ) , contain subgenomic mrnas in the virion, probably as a result of nonspecific rna packaging, it was proposed that these virion-associated subgenomic mrnas can be used directly as templates for the synthesis of subgenomic negative-strand rnas, which, in turn, serve as templates for the synthesis of additional subgenomic mrnas (sethna et al., ) . this model may explain the presence of subgenomic negative-strand rnas and ris in the infected cells, but it cannot explain the genomiclength nature of the w target sizes for mrna synthesis early in infection (yokomori et al., b) , nor can it explain how leader rnas from different virus strains can be randomly incorporated into mrnas of a different virus. furthermore, the virion-associated subgenomic mrnas have not been detected in all coronavirus species. the available data cannot unequivocally rule out any of the proposed transcription models. the primary difficulty in experimental analysis is that once the subgenomic mrnas are synthesized, by whatever mechanism, they are transcribed into negative-strand rnas because the cis-acting signal for negative-strand rna synthesis in mhv resides in the nucleotides at the ' end plus poly(a) (lin et al., ) , which is present in every subgenomic rna. thus, it is difficult to separate the primary and secondary events of transcription. it is possible that these transcription models are not mutually exclusive. for example, early in infection, a leader-primed transcription or trans-splicing mechanism may operate, generating subgenomic mrnas, which are then amplified into subgenomic negative-strand rnas; the latter serve as templates for further amplification of subgenomic mrnas thereafter. the subgenomic negative-strand rna can be used for either uninterrupted transcription or leader-primed transcription to generate positive-strand subgenomic rnas. a combination of these models would be consistent with most of the experimental data. this twostep model of primary and secondary transcription (jeong and makino, ) may explain the apparent differences in the possible mechanism of transcription between early and late stages of viral infection. because of the large size of coronavirus rna, no infectious cdna or rna clones are now available for reverse genetics studies. this difficulty has hampered progress in the study of the molecular biology of coronaviruses. di rnas of several coronaviruses (see section vi,e) have been molecularly cloned and used as a substitute for the genomic rna to study the cis-and trans-acting signals involved in viral rna synthesis. although natural di rnas do not contain an mrna start signal and, consequently, cannot transcribe an mrna, the insertion of such a signal into the di rna allows an mrna to be transcribed from the transfected di rna in the virus-infected cells, thus enabling studies of the regulatory sequences for transcription. following is a summary of information that has been obtained using this approach. it should be cautioned, however, that regulation of rna transcription probably depends on overall rna conformation and that the cis-acting sequence required for rna synthesis very often varies with the di rna vector used; therefore, the results obtained from di rna studies may not be directly applicable to the viral genome. a full understanding of the regulation of viral rna synthesis still awaits the development of an infectious cdna clone. the following cis-acting signals for coronavirus rna transcription have been determined primarily from mhv di rna studies (with some from bcv di) (fig. ) . a. ig sequence. the ig sequence can be considered to be the promoter element for transcription. it also serves as the mrna start site and the site of fusion between the leader rna and body sequence of mrnas. a seven-nucleotide core sequence, ucuamc, is sufficient to initiate mrna synthesis (makino et al., ) . extensive site-specific mutagenesis studies have shown that most of the single-nucleotide mutations within this core sequence could be tolerated, although the transcription efficiency of some of these mutants was lower (joo and makino, ; van der most et al., ) . these seven nucleotides represent the minimum promoter; deletion of a nucleotide results in complete ablation of mrna transcription. the effects of the sequences near the promoter on transcription are contradictory: in certain situa- tions, the nature of the neighboring sequences did not affect transcription (makino and joo, , but under other circumstances, it did (jeong et al., ) . thus, the strength of the promoter appears to depend on the context of the overall rna sequence and structure. the relative flexibility of sequence requirement of the promoter sequence in the di rna system appears to differ significantly from that seen in the viral genomic rna. in the mhv genome, there are more than stretches of sequence resembling the ucuaaac sequence, in addition to the six promoters for the known subgenomic mrnas (joo and makino, ). yet, most of these did not promote mrna synthesis from the viral rna genome to any appreciable extent, in contrast to their ability to promote transcription in the di rna vector system (joo and makino, ) . in the viral genome, the single-nucleotide substitution of a g residue in the core promoter sequence completely abolished mrna synthesis (shieh et al., , whereas this is tolerated in the di rna (joo and makino, ) . thus, there appear to be significant differences between the sequence requirement for mrna synthesis in the di rna and in the natural viral genomic rna. when there are multiple ig sequences in the di rna, the order of the ig sequences may influence transcriptional efficiency. an ig located at the ' end generally has an advantage in initiating mrna synthesis (van marle et al., ; krishnan et al., ) . the sequences near the igs may suppress transcription (jeong et al., ) . b. the leader sequence at the ' end of the dz rna. the leader sequence at the ' end of the viral genomic rna becomes the leader sequence of subgenomic mrnas; thus, it fills a structural role for mrna synthesis. however, the leader rna of the subgenomic mrnas is not derived exclusively from the leader rna of the same (di) rna; in fact, most are derived in trans from a separate rna molecule, such as helper virus rna (jeong and makino, ; liao and lai, ; zhang et al., ) . nevertheless, mrna transcription from an ig site in the di rna still requires the presence of a leader rna sequence at the ' end of the di rna as a cis-acting sequence . deletion of this cis-acting leader abolished transcription. furthermore, the sequence of this leader rna, particularly its ' end sequence, can affect the efficiency of transcription from certain ig sequences on the di rna (zhang et al., ) . for example, the leader rna containing two pentanucleotide (ucuaa) repeats transcribes an mrna from the ig - site more efficiently than the leader rna with three ucuaa repeats. thus, the cis-acting leader rna plays a role similar to that of an enhancer. these findings suggest that the leader rna serves two functions : ( ) it supplies the leader rna to the subgenomic mrnas, and ( ) it serves as an enhancer-like sequence to regulate transcription. this finding also suggests that there is either a direct or an indirect interaction between the leader and ig sequences. some additional sequences downstream of the leader may also enhance transcription from an ig site in the di rna ; however, the precise sequence requirement is not known. this sequence requirement shows some virus sequence specificity, since it cannot be replaced with other viral rna sequences . it may be needed to maintain overall rna conformation for the recognition of the ig sequence. c. the ' utr. in an mhv di rna construct, partial deletion of the ' utr completely abolished transcription from an upstream ig site in the di rna (lin et al., ) . this stretch of ' utr is probably involved in positive-strand rna synthesis, since the length of this required sequence ( nt) is significantly longer than that required for negative-strand rna synthesis ( nt). the ' utr requirement for mrna transcription is surprising, since positive-strand rna synthesis starts from the ' end; thus, the ' end sequence is the last to be transcribed. this ' utr sequence requirement is similar to that for rna replication (kim et al., b; lin and lai, ) (see section v,f). this finding suggests that the ' end may interact with the ' end and possibly with ig sequences during transcription. a nine-nucleotide sequence, uuuauaaac. this sequence, located immediately downstream from the ucuaa repeats at the ' end of the leader rna in the viral genome, plays a significant role in rna transcription. it is deleted from the genome of one of the mhv strains and is often deleted in naturally occurring di rnas . in this particular mhv strain (mhv- , the leader-mrna fusion sites are very heterogeneous and do not always occur at the usual ucuaaac sites (zhang et al., ) . this nine-nucleotide sequence can serve as an mrna start signal, allowing transcription of an almost genomic-length mrna . in the di rnas, the presence or absence of this nine-nucleotide sequence influences transcription efficiency from the downstream ig site and, most importantly, affects the source of the leader rna incorporated into subgenomic mrnas (zhang et al., b) . when this nine-nucleotide sequence is present, the leader sequence in the subgenomic mrnas is contributed both from the di rna in cis and from helper virus rna in trans. when this sequence is missing, the leader rna is derived exclusively from the helper virus rna (zhang et al., b) . thus, this nine-nucleotide sequence appears to regulate the mechanism by which the leader rna is fused t o the subgenomic mrnas. these results combined suggest that multiple rna regions are involved in the regulation of mrna transcription. however, a recent study appears to contradict the need of cis-acting sequences other than the igs for mrna transcription. when a negative-strand rna containing only an ig sequence of tgev and a reporter gene was transcribed in situ from a transfected cdna by using a recombinant vaccinia virus-t rna polymerase expression system, this rna was transcribed in the presence of tgev, generating an mrna with a correctly fused tgev leader sequence (hiscox et al., b) . the leadercontaining mrna could have been generated by either of the transcription mechanisms described (section v,e, ,a or section v,e, ,b) above. this study suggests that this negative-strand ig site is sufficient for transcription. however, it is possible that this activity represents a basal level of transcription and that other cis-acting sequences may enhance the efficiency of transcription. the application of inhibitors of protein synthesis at any time during the viral life cycle inhibits viral rna synthesis, suggesting that continuous protein synthesis is required for rna synthesis sawicki and sawicki, ) . a similar observation has been made using an inhibitor of cysteine proteases, which inhibits a specific step of the processing of gene l a products of mhv (see section v,g, ), suggesting that continuous production of polymerase is required for viral rna synthesis. the precise nature of the viral proteins involved has yet to be determined. temperature-sensitive mutants of mhv that are defective in rna synthesis at the nonpermissive temperature have been divided into at least five complementation groups, indicating that at least five proteins are involved in viral rna synthesis (leibowitz et al., a; baric et al., ) (see fig. ). all of these complementation groups are mapped within the gene region (including both l a and lb). sequence analysis showed that gene l b contains an rna polymerase motif (gorbalenya et al., ; lee et al., ) . polymerase activities have been demonstrated in membrane fractions of bcv-and mhv-infected cells dennis and brian, ) , and several in vitro rna synthesis systems have been reported (compton et al., ; baker and lai, ) ; however, the nature of polymerases in these systems has not been identified. in one study, it was demonstrated that the antibodies against the n protein could inhibit rna synthesis, suggesting that n protein may be involved in rna synthesis (compton et al., ) . in addition to viral proteins, cellular factors may also be involved in rna synthesis. several cellular proteins have been shown to bind to the regulatory elements of mhv rna, including the ' and ' ends of the genomic rna and the ' end of the negative-strand rna and ig sites yu and leibowitz, a,b; zhang and lai, ) . the binding sites for the cellular proteins at the ' end of genomic rna and the ' end of negative-strand rna are complementary . the protein p , which binds to the negative-strand leader sequence and the ig site, is particularly interesting. site-specific mutations of the ig site affected the binding of this protein and the efficiency of rna transcription to the same extent, suggesting that the binding of this protein is required for rna transcription . this protein recently has been identified as hnrnp a (h.-p. li and m. m. c. lai, unpublished observation) . the mutations at the ' end of the viral genomic rna that abolished the binding of cellular proteins also inhibited both negative-strand and positive-strand rna synthesis, although the correlation between protein binding and rna replication was not absolute (yu and leibowitz, a) . thus, cellular proteins probably play a significant role in viral rna replication and transcription. curiously, viral proteins in the infected cell extract could not be cross-linked to the viral rna in vitro, suggesting that viral proteins may interact with viral rna only indirectly through cellular proteins. this is in contrast to the finding that the purified n protein can bind to the leader rna sequence in vitro stohlman et al., ) . the reason for this discrepancy is not clear. the genomic-sized rna in coronavirus-infected cells theoretically consists of two populations: the messenger rna (mrna l), which is translated to yield gene l a and l b products, and the genomic rna, which is destined to be packaged into virion. early studies demonstrated that, late in the infection, most ( %) of the genomic-sized rna in the cells was associated with the viral nucleocapsid, while the remainder ( %) was present in polysomes perlman et al., ) . presumably, early in infection, most of the genomic-sized rna would be associated with polysomes to serve as mrnas for the synthesis of polymerase; however, this has not been demonstrated. it is not clear whether there is any difference in structure and mechanism of synthesis between these two rna populations. since genomic rna requires uninterrupted synthesis from the fulllength template, whereas mrnas involve discontinuous transcription, the two types of genomic-sized rna (mrna and virion genome rna) may be synthesized by two different mechanisms. a recent study suggests that at least some of the mhv genomic-sized rnas are indeed synthesized by a discontinuous transcription, using the u c u m repeat in the leader rna and the nine-nucleotide uuuauaaac immediately downstream of the leader rna as the transcription start site . this raised the possibility that mrna and virion genomic rna are distinguishable. however, it cannot be inferred from this study that the fate of the genomic-sized rna products derived from discontinuous transcription is different from the fate of those derived from uninterrupted rna synthesis. the possible involvement of discontinuous transcription in generating genomic-sized rna may explain several interesting findings regarding mhv genomic rna . the copy number of the ucuaa repeat in the leader sequence of the genomic rna, which ranges from two to four copies in different mhv strains, rapidly evolves during virus passage (makino and lai, a; la monica et al., ) . starting with a pure virus population, the copy number in the viral genomic rna rapidly becomes heterogeneous during serial passages in tissue culture, and a new virus population with a different copy number of ucuaa repeats emerges (makino and lai, a) . since this sequence variation is seen in the leader region but not in the ig regions, where uninterrupted rna synthesis probably occurs, this finding is best explained by the discontinuous transcription mechanism involving the ' leader region. the imprecise fusion of the leader rna to the mrna start sites would result in heterogeneity of the copy number of the ucuaa repeats lai, ) . such heterogeneity is not observed when the virus, e.g., bcv, contains only one ucuaa copy in the leader rna (hofmann et al., a) . the ucuaa region at the ' end of the genomic rna is a hot spot of rna recombination during mixed infection of mhvs, resulting in recombinant mhvs with a crossover site at the ' end of the leader rna sequence . this result is best explained by the discontinuous rna synthesis at the ' end of the genomic rna. . if the generation of di rnas is viewed as an anomaly of rna replication, the structure of naturally occurring di rnas reveals an insight into the mechanism of rna replication. most of the naturally occurring mhv di rnas have a copy number of the ucuaa repeat different from that of the parental virus, and most lack a ninenucleotide sequence downstream of the ucuaa repeats . as discussed above, this is a reflection of discontinuous transcription in the region. the understanding of the mechanism of rna replication has been aided by the use of in uitro-transcribed di rna generated from cloned cdna. when di rna was transfected into virus-infected cells, the leader rna was rapidly replaced by that of the helper virus (makino and lai, b; . this leader exchange is dependent on the presence of the nine-nucleotide sequence (uuuauaaac) in the di rna (makino and lai, b) , consistent with the finding that this sequence serves as an mrna start signal for discontinuous transcription . the use of the cloned di rna also allowed the determination of the cis-acting signals for rna replication (kim et al., ; lin and lai, ) . it was shown that more than nucleotides at both the ' and ' ends of the di rna are required for rna replication, and that some mhv di rnas also required a stretch ( nt) of internal sequence in the gene region for rna replication; however, the requirement for the internal sequence was not observed in other mhv or bcv di rna constructs (chang et al., ; luytjes et al., ) . thus, this internal sequence probably plays a role in maintaining the overall rna conformation for some di rnas (y. n. . again, the requirement of a ' end sequence ( nt) that is longer than that required for negative-strand rna synthesis ( nt) is a surprise. these ' end sequences are probably required for positive-strand rna synthesis during rna replication. this finding is reminiscent of the sequence requirement for rna transcription discussed above and suggests that there is a direct or indirect rna-rna interaction between the ' and ' ends during rna replication. these di rna studies also showed that replication of di rna is inhibited when an mrna is transcribed from an ig site within the same di rna, and that the mechanism of inhibition is due not to competition for the same transcription machinery (jeong and makino, , but most likely to the overlap of the cis-acting signals for these two different processes. however, the sequence requirements for replication and transcription are different, indicating that these two processes are distinguishable. the mrna transcription and genomic rna replication may be regulated by the same mechanism throughout most of the viral replication cycle. however, the ratio between the genomic rna and subgenomic rnas, as detected by radioactive uridine incorporation, increases during the late stages of the bcv replication cycle (keck et al., a) , suggesting a possible switching mechanism from transcription to replication. it has been shown that genomic rna replication is coupled to encapsidation, since no free genomic rna is found . since the encapsidation of rna requires the n protein, this protein may participate in the regulation of switching between transcription and replication. the sequences of coronavirus mrnas usually start from a site immediately upstream of a gene. these mrnas, except for the smallest mrna, are structurally polycistronic, containing multiple orfs. only the ' most orf in the mrnas is translatable; the remaining orfs are usually functionally silent. thus, most of these mrnas are functionally monocistronic (see fig. ) . the s, he, m, and n proteins, and in most coronaviruses the e protein, are translated from separate mrnas by this mechanism; initiation of their translation is unremarkable, utilizing a cap-dependent translation mechanism. many ns proteins, however, are translated from truly polycistronic mrnas, i.e., two or three proteins are translated from the same mrna. for these mrnas, the first orf, e.g., a of ibv or a of mhv, is probably also translated by the same mechanism as the structural protein genes. for internal orfs, e.g., e protein of ibv and mhv, an alternative mechanism must be employed to initiate translation internally. one characteristic of coronavirus mrnas is the presence of the leader rna sequence at the ' end, which not only participates in rna transcription, but also regulates the efficiency of translation. it has been shown that the presence of the mhv leader sequence on a heterologous mrna in a chimeric rna construct can enhance its translation in virus-infected cell lysates but not in uninfected cell lysates (tahara et al., ) . this effect conceivably will enable the efficient translation of viral mrnas in the face of shutoff of translation of cellular mrnas in the infected cells (siddell et al., ; hilton et al., ) . the mechanism of translational enhancement by the leader rna has not been determined. it has been shown that during persistent infection of bcv, the leader rna sequence underwent frequent mutations (hofmann et al., b) . one of these mutants had an intraleader short orf and a lower translation efficiency, indicating that the leader sequence in-deed can modulate translation. another region which can potentially regulate the translation of coronavirus mrnas is the ' utr (other than the leader sequence) of mrnas. the genomic rna (mrna ) has a particularly long ' . an mhv with a specific point mutation within the ' utr was selected during persistent infection in uitro (chen and baric, ) . this mutant had a significantly higher translation efficiency than the wild-type virus. different subgenomic mrnas had ' utr of various lengths, which may also affect their translation. for the translation of internal orfs, several different mechanisms are used by coronaviruses: a. ribosomal frameshifing within the polymerase gene. all of the coronavirus genes (polymerase) sequenced so far contain two overlapping orfs. several features of the ibv polymerase gene sequence , coupled with the absence of a distinct mrna for orf lb, suggested that translation of orf l b involved ribosomal frameshifting from orf la, thus synthesizing a large polyprotein containing both l a and l b sequences. subsequently, a highly efficient ( % frequency) - frameshift was demonstrated experimentally in uitro (brierley et al., ; somogyi et al., ) and in uiuo (brierly et al., ) . this mechanism has been shown to operate in gene of mhv, hcv- e, and tgev as well (bredenbeek et al., ; lee et al., ; eleouet et al., ) . in all cases, the mechanism involves two essential elements: a slippery site followed by an rna pseudoknot (brierley et al., ) . the site at which the ribosome slips backward has the sequence uuuaaac. the pseudoknots of ibv and mhv are similar, comprising two base-paired regions stacked coaxially in a quasi-continuous manner and connected by two singlestrand loop regions. the hcv- e pseudoknot is more complex . it is the overall shape and stability of the pseudoknot that are important, not the nucleotide sequence per se. two reasons have been put forward to explain why coronaviruses should employ ribosomal frameshifting to translate orf l b (brown and brierly, ) . one reason is that this is done primarily to control the relative amounts of the l a and l b products. that could be achieved in other ways, of course, e.g., by translating orf l b from a separate mrna, this will require that the transcription of l a and l b mrnas is tightly regulated. the other reason may be to avoid making a l b mrna. such an mrna might be packaged into virions in competition with genomic rna, as the rna region corresponding to the l b orf of mhv contains a sequence that is essential for packaging into virions (fosmire et al., ) (see section vi,e,l). mrna. the e proteins of ibv and mhv are encoded by the third and second orf, respectively, of the corresponding genes and (fig, ) . cells infected with ibv contain the products of all three of the gene orfs (liu et al., ) . both of the mhv gene orfs are translated in vitro (budzilowicz and weiss, , but only the b orf product has been detected in virus-infected cells . experiments have shown that the e protein orf of both ibv and mhv mrnas is translated by a cap-independent, internal ribosomal entry mechanism (liu and inglis, b; thiel and siddell, ) . furthermore, if the a and b orfs were eliminated from the ibv mrna, translation of the c (e) orf did not occur (liu and inglis, b) . this suggested that the d b region contains an internal ribosome entry site (ires) for the e protein orf. le and colleagues have predicted the existence of secondary structures in the d b region of ibv which resemble the ires elements of picornaviruses (le et al., ) . they predicted a -nucleotide sequence in d b which would fold into five stem-loops, forming a compact structure by the interaction of two pseudoknots. c. translation of nonstructural proteins. in addition to the ns proteins encoded from the '-most orfs of mrnas, several other ns proteins are encoded from an internal orf of some viral mrnas, e.g., b of ibv and hcv- e, b of bcv, and b of fcv (fig. ) . most of these products have been detected in virus-infected cells; however, the mechanism of the internal initiation of translation has not been elucidated. bcv and mhv rna contains an additional internal orf within the n protein gene. this orf (termed i) is in a different reading frame from that of n protein and encodes a hydrophobic protein (senanayake et al., ; fischer et al., ) . this protein is translated in virusinfected cells by a leaky ribosomal scanning mechanism from the bicistronic mrna of n gene (senanayake et al., ) . it is a nonessential gene. the mechanism of its regulation is not yet clear. the gene product is predicted to be nearly - kda. it is probably processed into multiple proteins posttranslationally by its own proteases. the processing pathway has just begun to be explored. remarkably, the protease domains and potential cleavage sites predicted by computer analysis (gorbalenya et al., ; lee et al., ) have largely been confirmed by experimental data. initially, in vitro translation of virion rna of mhv revealed several polypeptides of more than kda (leibowitz et al., b; denison and perlman, ) . in addition, a -kda product was detected and shown to have originated by cleavage from the n terminus of a precursor (denison and perlman ; soe et al., ) , now known to be the beginning of the orf l a polyprotein (fig. ) . the cleavage which generates p is carried out by plp (fig. ) . it cleaves between residues gly- and val- , mutation of either residue resulting in almost no cleavage (dong and baker, ; hughes et al., ) . in addition to p , the mhv orf l a encodes a protein of more than kda, which is cleaved to a -kda product, which, in turn, is cleaved to produce a -kda and a -kda product (denison et al., ) (fig. ) . another protein of kda is derived from sequence immediately downstream of the p -encoding region, thus representing the n-terminal part of the large polyprotein initially found in in uitro translation (probably more than kda) (fig. ) . the cleavage of p from the polyprotein was also carried out by plpl (bonilla et al., , . inhibition of the c-terminal cleavage of p by e d, an irreversible inhibitor of cysteine (thio) proteinases, inhibited mhv replication . in addition, the clp domain is cleaved from the polyprotein by the autocatalytic cleavage activity of clp itself to generate a - kda protein, which contains both the trans-and cis-acting proteolytic activities (lu et al., , liu and brown, ; ziebuhr et al., ) . e d also inhibited the clp protease activity. the processing pathway of the l b protein sequence is less clear. there is experimental evidence with ibv and hcv- e that the l b polyprotein is cleaved in trans by the clp encoded by orf l a (liu et al., ; ziebuhr et al., ; grotzinger et al., ) . a polypeptide of approximately kda, representing the extreme c terminus of orf l a and the n terminus of the frame shifted orf lb, was immunoprecipitated from ibv-infected cells. the cleavage sites of the -kda protein appear to be at the q/s sites, as predicted from the computer analysis and consistent with the known substrate specificity of the picornavirus c protease. a similar observation was recently made with hcv- e (grotzinger et al., ) . this -kda protein contains the putative rna polymerase motif and thus may represent the coronavirus polymerase. the coding region for this protein belongs to complementation group d, which has been shown to effect mrna transcription (fig. ) . b. processing of the structural proteins the s protein is co-translationally glycosylated with nlinked glycans. conversion of the high mannose (simple) glycans of the s protein to complex ones is a slow process, the half-life being one to several hours (vennema et al., a) . the s protein undergoes multiple disulfide linkages to fold into a more complex structure (opstelten et al., ) and oligomerize into a trimer in the golgi complex (delmas and laude, ) . the s prepropolypeptide is converted to a propolypeptide by removal of the n-terminal signal peptide. whether the propolypeptide is cleaved to generate s and s depends on the virus species and strain and, to some extent, on the cell type in which the virus is grown (frana et al., ) . the sl-s cleavage site in ibv and mhv is adjacent to several basic residues (cavanagh et al., a; luytjes et al., ) . those coronaviruses whose s protein is not cleaved, e.g., fcv, tgev, and ccv, have no such pairs of basic residues. cleavage of the mhv s protein occurred after conversion of the glycans from simple to complex forms (vennema et al., a) . after cleavage, the s and s subunits are held together by noncovalent linkages (cavanagh et al., b; sturman et al., ) . the s protein of mhv is acylated, possibly involving some of the many cysteine residues in the c-terminal, hydrophilic tail of s (schmidt, ; sturman et al., ; van berlo et al., ) . the processing of s proteins is reviewed in greater detail by cavanagh . modification of the m protein depends greatly on the virus species. the major modification is glycosylation. the oligosaccharides of ibv and the tgev group are of the co-translationally added n-linked glycans (stern and sefton, b) . the conversion of the high mannose to complex glycans is not very efficient. in contrast, viruses of the mhv group have o-linked glycans which are added posttranslationally (holmes et al., ; niemann et al., ; ; tooze et al., ; locker et al., a; krijnse-locker et al., ) . the m protein of tgev is also sulfated (garwes et al., , but whether this is linked directly to the polypeptide or to glycans is unknown. unlike the m proteins of ibv and the mhv group, which have an internal membrane insertion sequence, those of the tgev group have an n-terminal membrane insertion sequence that is absent from the mature m protein (laude et al., ) . this signal sequence, however, is not an essential requirement for the membrane insertion of the m protein (kapke et al., ; vennema et al., ) . . he protein. the he glycoprotein has n-linked glycans which are converted to complex ones in the golgi complex. the n-terminal signal sequence is cleaved from the mature protein, which then forms dimers by disulfide bonds (king et al., ; hogue et al., ; kienzle et al., ; yo et al., ) . of mhv e protein (yu et al., ) . however, this was not observed for the e protein of tgev when expressed in insect cells (godet et al., ) . . nprotein. the n protein is phosphorylated, the phosphate linkage being exclusively to serine residues (stohlman and lai, ) . the role of phosphorylation is unknown. in virus-infected cells, the assembly of virus particles presumably starts with the formation of rnp, which interacts with the components of viral envelope proteins to form enveloped virus particles and bud into the endoplasmic reticulum (er) and golgi complex. several recent advances shed light on this process: early studies have shown that the s proteins are not necessary for virus particle formation; thus, denuded virus particles without spikes can be formed in the virus-infected cells treated with tunicamycin, which inhibits n-glycosylation and transport of the s and he proteins (holmes et al., ) . further, recent studies have shown that the minimum requirement for the formation of viruslike particles (vlp), i.e., empty virus particles, is the m and e proteins (bos et al., ; vennema et al., ) ; . the sites of virus budding are in the er and golgi, near the sites of accumulation of the m protein (dubois-dalcq et al., ; tooze et al., ; tooze and tooze, ; klumperman et al., ) ; thus, the interaction between the m and e proteins appears to be the key event for virus particle assembly. the incorporation of the nucleocapsids and s and he proteins into virus particles may involve subsequent interactions of these components with the m-e complex. the virus assembly and release process has been studied in most detail for mhv (j. tooze et al., tooze et al., , tooze and tooze, ; s. a. tooze et al., ; krijnse-locker et al., ) , and the gross features have recently been confirmed for ibv, tgev, and fipv . recently, an ultrastructural study of the replication of ibv in renal ductotubular epithelial cells of infected chicks has also been very informative (chen and itakura, ) . the first virions form in the perinuclear region, in small, smooth vesicles/ tubules between the rough er and the cis side of the golgi stack. later, the rough er becomes the major site of virion assembly, extending beyond the perinuclear region. virions then proceed through the golgi complex, at the trans side of which they are collected into vesicles of the constitutive exocytic pathway and subsequently released from the cell. the major determining factor for the site of virus assembly appears to be the site of localization of the m protein, which is in the golgi complex. there are some points of difference among the coronaviruses. when the m protein of mhv was expressed, it accumulated in the trans-golgi membranes, consistent with its o-linked glycosylation, which occurs efficiently (locker et al., a; klumperman et al., ) . in contrast, expression of the ibv m protein from cdna resulted in its accumulation in cis-golgi membranes; consequently the highmannose n-linked glycans of the m protein were not efficiently converted to complex ones (machamer et al., ; klumperman et al., ) , in agreement with the properties of the m protein in the ibv virions. glycosylation of the coronavirus m protein is not essential for its translocation or for virus particle formation. the m protein exists as monomers in the er, but it oligomerizes to form variously sized complexes during transport through the golgi and trans-golgi network (locker et al., ) . it is likely that the m molecules in the virus particles are in complexed form. the sequence requirements for insertion of the nascent m polypeptide into the rough er have not been precisely defined. with the exception of the tgev group, the coronavirus m proteins do not have an amino-terminal signal peptide. even in the case of the tgev group, the signal peptide is not essential for membrane insertion of the m protein (kapke et al., ; vennema et al., ) . rather, one of the three transmembrane sequences of the m protein is responsible for the insertion of m into the er and its final localization in the golgi complex (machamer and rose, ; mayer et al., ; armstrong et al., ; locker et al., ) . different domains of the m protein of ibv and mhv have been identified as the sequences responsible for the final localization of the protein. the first membrane-spanning domain of the ibv m protein performs this function, the m protein being concentrated in the cis-golgi membranes (machamer and rose, ; machamer et al., swift and machamer, ) . in contrast, the carboxyterminal domain of the mhv m protein, probably in combination with a middle domain, directs the protein to the trans-golgi (armstrong and patel, ; weisz et al., ; krijnse-locker et al., ) . it should be borne in mind, however, that the major site ofvirus particle formation is proximal to either of the golgi compartments, namely, in an intermediate compartment between the er and the golgi complex . thus, it is proximal to the major site of m accumulation. what is responsible for that? the answer would appear to be that the s glycoprotein and the nucleocapsid interact with the m protein molecules before the m proteins have migrated to the golgi, precipitating virus particle formation. it has been shown that the coronavirus m protein can interact with nucleocapsids (sturman et al., ) . this interaction requires the presence of viral rna, since the n protein alone cannot be incorporated into the vlps (bos et al., ; vennema et al., , suggesting either that m interacts with viral rna, or that rna-n protein binding induces a conformational change in the n protein, enabling it to interact with m. interaction between the m and s proteins has also been demonstrated. the m and s proteins co-sediment under certain ionic conditions after dissolution of virions with mild detergents (cavanagh, b) , and cell-associated complexes containing m and s have been detected (opsteltenet al., ) . the s protein undergoes certain conformational changes induced by disulfide linkage before it is able to interact with m (opstelten et al., . inhibition of correct oligomerization of s by dithiothreitol prevented interaction of s with m and, as a result, the rate of transport of the m protein to the trans-golgi increased (opstelten et al., ) . this result suggests that s-m interaction can retard the transport of the m protein. the ability of the s or he protein to interact with the m protein appears to be a prerequisite for their incorporation into virus particles. in this regard, it is interesting to note that mhv ts mutants with a deletion in the ectodomain of the s protein or those with defects in oligomerization of the s protein do not incorporate the s protein (ricard et al., ; luytjes et al., ) . also, partial deletions in the ectodomain of the he protein prevent its incorporation into virus particles . these results suggest that the interaction of s or he with m occurs through the ectodomain or requires the correct protein conformation in the ectodomain. the formation of the s-m complex occurs in the pre-golgi complex, whereas the s-m complex progresses until the golgi complex, indicating that this interaction is not sufficient to localize it in the pre-golgi complex, the ultimate site of virion budding . thus, m-nucleocapsid interaction may also contribute to the determination of the site of virus assembly. in this regard, it is important to note that the recent discovery that m is present in the viral rnp core, as well as in the envelope (risco et al., ) may further indicate the crucial role of the m protein in the virus assembly process. only the m and e proteins are required for the production of vlps (bos et al., ; vennema et al., ) . these particles were formed when the m and e proteins were expressed from transfected plasmids. s protein was incorporated into the vlps if expressed. in the absence of viral rna, the n protein also was not incorporated. when all the structural proteins were expressed from plasmids in the presence of an mhv di rna, which contains a packaging signal, and in the absence of helper virus, the vlps contained the di rna (bos et al., ) . moreover, these vlp were "infectious," i.e., on transfer of the released vlps to a new cell culture, they were able to infect the cells, as revealed by the rescue of the di rna by helper virus. these results show that n is dispensable for the formation of vlps but the packaging of rna into virion requires an interaction between m and the n-containing ribonucleoprotein, as previously demonstrated (sturman et al., ) . the expression of the m protein alone in the cells did not lead to vlp formation or induction of curvature in the m-containing intracellular membranes. the presence of the e protein together with the m protein triggered both events, but the ratio of m:e in virions was as high as oo:l (vennema et al., ) . this has led to the suggestion that e does not have frequent, regular positions in the lattice formed by m but rather occupies strategic positions within the lattice to cause membrane curvature. alternatively, its role may be to close the neck of the virus particle as it pinches off from the membrane in the final stage of budding. what determines the site of virion budding? it is possible that the e protein dictates the site of budding, since this protein is also localized in the perinuclear region and associated with membrane (godet et al., ; yu et al., ) . alternatively, it may be the interaction of the rnp-nucleocapsid with the s-m complexes which halts the migration of the latter and promotes budding. relevant to this notion is the observation that the nucleocapsids and free n protein have affinity for membranes (anderson and wong, ) . it should be remembered, however, that in the absence of s, he, and nucleocapsids, the e and m proteins alone can induce budding to form vlps (bos et al., ; vennema et al., ) . it is not yet clear whether the budding site of vlp containing only m and e is the same as that for the complete virion. empty virus particles have previously been isolated from ibv, which were grown in embryonated fowl eggs (macnaughton and davies, ) . this supports the view that even during natural infection, virus budding can be induced without involvement of the viral nucleocapsid. parallels have been drawn between the e protein of coronaviruses, the m protein of orthomyxoviruses, and the k protein of alphaviruses. all are minor envelope proteins that play a role in virus assembly. once the virus particles bud into the pre-golgi compartment, they are transported through the golgi complex. whether the golgiassociated posttranslational modifications occur before or after incorporation of the proteins into virus particles is not known. retrograde transport of the proteins may be required for some steps of the virus assembly process. finally, the release of virus particles from the cells appears to be restricted to certain areas of cells. tgev grown in polarized llc-pk cells both enter and exit by the apical surface (rossen et al., ) , whereas mhv-a enters polarized murine kidney cells (mtal) by the apical surface but is released via the basolateral surface (rossen et al., a) . however, the site of virus release varies with different cell lines (rossen et al., ) . the factors governing this process are not known (rossen et al., b) . probably because of the large size of their rna genomes, coronaviruses have developed a variety of genetic mechanisms, among which are rna recombination and generation of di rna, to maintain their genetic stability and, as a side product, generate diversity. coronaviruses also readily undergo genetic mutation, a characteristic common to all rna viruses. thus, they evolve rapidly and are heterogeneous. these genetic phenomena provide virologists with useful tools for understanding coronavirus biology, particularly because reverse genetics studies for coronaviruses are not yet feasible. using a variety of chemical mutagens, several laboratories have isolated mhv temperature-sensitive (ts) mutants which cannot produce infectious virus particles or cause different plaque morphology at the nonpermissive temperature (haspel et al., ; robb et al., ; wege et al., ; koolen et al., ; schaad et al., ) . some of these mutants have been characterized with respect to their ability to synthesize rna and have been grouped into at least seven complementation groups (leibowitz et al., a , five of which have the rna (- phenotype (i.e., cannot synthesize rna at the nonpermissive temperature) (leibowitz et al., a; schaad et al., ) (see fig. ). with the use of recombination analysis (see below), the possible genetic defects of the mutants were mapped on the rna genome fu and baric, ) . it appears that all of the rna (-) mutants have genetic defects within gene , suggesting that gene encodes rna polymerase and other proteins involved directly or indirectly in viral rna synthesis. the genetic defects of some of these mutants have been confirmed by rna sequence analysis of the mutants and their revertants . these five different complementation groups have been demonstrated to affect different steps of rna synthesis, including the synthesis of leader rna, negative-strand rna, and positive-strand rna (fig. ) , suggesting that different steps of rna synthesis require different viral proteins . it is still not possible, however, to correlate the genetic defects definitively with the known processed products of the gene polyprotein. among the rna (+) mutants, two complementation groups have been assigned to the gene encoding the s protein fu and baric, , but the phenotype of these mutants has not been well characterized. another rna (+) mutant, alb , has a single amino acid substitution in the n-terminal domain of s protein and cannot incorporate s protein into the virus particles (ricard et al., ) . still another group of rna (+) mutants have a defective n protein (koetzner et al., ; masters et al., ; peng et al., a) and produce smaller plaques at the nonpermissive temperature; several of these mutants have a deletion in the n gene (masters, ) and are defective in rna-binding activity (peng et al., a) . most wildtype revertants have a second-site mutation in the n protein and restored rna-binding activity (peng et al., a) . another class of viral mutants was obtained by a specific selection scheme, e.g., by treating viruses with neutralizing mab and selecting mutant viruses resistant to neutralization. since neutralizing antibodies are usually directed against the s protein, all of the neutralizationescape mutants were presumed to have defects in the s gene. this was indeed the case (reviewed by . depending on the neutralizing mab used for selection, the mutants obtained had either deletions or point mutations in the neutralization epitopes of the s protein (gallagher et al., ; wang et al., ) . these mutants generally retain growth properties very similar to those of the parental virus but often have significantly different pathogenic properties with altered tissue tropism (dalziel et al., ; fleming et al., fleming et al., , wege et al., ) . during serial virus passages in tissue culture or in animals, coronaviruses often undergo various deletions or substitutions even in the absence of experimentally applied selection pressure. these genetic changes probably provide the emerging virus variants with evolutionary advantages under experimental conditions or in natural infection. the deletions occur most frequently within the s gene, particularly within a hypervariable region encoding the s subunit (s. e. wang et al., ) . in fact, some natural isolates of mhv have a deletion of - nucleotides in this region (fig. ) . similar deletions have been detected in virus variants during central nervous system (cns) infections of rats . in persistent infections of cultured cells of cns origin, viruses with point mutations or deletions in the gene encoding s protein are frequently selected (gallagher et al., ; gombold et al., ; rowe et al., ) . these viruses often have altered cell fusion and pathogenic properties. the most striking effect of deletions during natural virus infection is illustrated by the emergence of prcv from tgev. tgev causes epizootic enteric infection in pigs, resulting in a very high mortality rate in newborn pigs. an attenuated virus strain that is related t o tgev but infects only respiratory tissues was isolated in western europe in the early s (pensaertet al., ). an independent isolate of prcv was subsequently obtained in the united states (wesley et al., ) . both of these prcv isolates have similar extents of deletion in the n terminus of the s protein, in addition to smaller deletions in gene , which eliminates its expression (rasschaert et al., ; wesley et al., ; laude, ) . although it is not yet possible to link the changes in viral pathogenicity to the deletions in the s gene or gene , the tgev-prcv evolution illustrates the power of deletions in coronavirus evolution. different ts mutants with defects in different coronaviral genes have been demonstrated to complement each other. the available ts mutants of mhv have been divided into at least seven complementation groups, five of which have an rna (-phenotype (leibowitz et al., a) (fig. ) . it is worth noting that these five rna (-) complementation groups have been mapped in gene , which is translated into a polyprotein. the existence of five complementation groups within this gene indicates that this polyprotein is processed into at least five different proteins that function independently. it is not possible, however, to complement the genetic defects of a virus by expressing a wildtype viral protein from an exogenous vector. mixed infection with mhv and murine leukemia virus in tissue culture cells yielded a pseudotype mhv which contained a murine leukemia virus envelope protein and was neutralized by antibodies against both murine leukemia virus and mhv (yoshikura and taguchi, ) . this phenotypic mixing of viral proteins suggests the lack of a stringent requirement for a virus-specific spike protein for the formation of coronavirus particles. pseudotype formation of virus particles has also been achieved by expressing a viral protein, e.g., he protein, from a di rna vector (see section vi, e), which was incorporated into virus particles . one unique genetic feature of coronaviruses is their ability to undergo rna recombination at a very high frequency; this is particularly true of mhv, in which recombinant viruses containing parts of the genomic sequences of both parental viruses could be isolated at high frequency when two strains of mhv with defined genetic markers were co-infected into culture cells or animals. this genetic phenomenon was first discovered using two ts mutants of mhv . subsequently, many different recombinant mhvs were isolated (keck et al., , b makino et al., ) using a combination of selection markers, such as ts markers, resistance to neutralizing antibodies, and cytopathic effects (the ability of the virus to cause fusion). based on the distribution of the crossover sites on the viral rna genome, it appears that recombination can occur practically anywhere on the viral genome, although some combinations of virus strains favor selection of viruses with certain recombination sites (lai, ) . for example, between the mhv a and jhm strains, recombination occurs mostly at the ' end of the genome and rarely at the ' end. in contrast, recombination between the mhv- and jhm strains occurs readily a t the ' end . the most surprising finding with regard to mhv recombination is the extremely high frequency of recombination, which has been calculated to be nearly % for the entire mhv genome . this high frequency of recombination is reminiscent of the reassortment of segmented rna genomes in viruses such as influenza virus and reovirus. the recombination map for mhv is nearly linear, suggesting the random occurrence of recombination ; however, more careful analysis of the recombination frequency showed that there is an increasing gradient of recombination frequency (in the direction of '+ ') across the genome, suggesting that subgenomic mrnas, which represent preferentially the ' end sequences, may participate in rna recombination baric, , ) . recombination has now been demonstrated experimentally for ibv (kotier et al., ) and tgev (ballesteros et al., ) in embryonated eggs or tissue culture; however, the recombination frequency for these viruses has not been determined. recombination can provide a powerful tool for virus evolution. for example, in a study in which ts mutants of the a strain of mhv were co-infected with a wild-type jhm strain, the majority of the progeny viruses after a single passage were recombinants which contained the ' end of the a genome (makino et al., a) , suggesting that this recombinant virus has evolutionary advantages. recombination has also been demonstrated during virus infection in animals (keck et al., ) . similar to the situation in other rna viruses, coronavirus recombination probably occurs by a copy-choice mechanism (lai, ) . it has been shown that mhv rna synthesis normally pauses at certain sites on the rna genome (baric et al., ) . the nascent, incomplete rna transcripts may dissociate from the template rna and then rebind to the template to resume rna synthesis. when the nascent rna binds to a different template, the resumed rna synthesis will result in a recombinant rna. whether coronavirus recombination occurs more frequently at certain rna sites with more complex secondary structure is not yet known. when rna recombination was examined under nonselective conditions (by reverse transcription-polymerase chain reaction detection of the intracellular rna from virus-infected cells), recombination sites appeared to be random; only after serial passages did "hot spots" of rna recombination become apparent . this finding indicates that the recombination hot spots may be the result of selection. recombination has been detected during natural infections of coronaviruses, most notably ibv. sequence analysis of natural ibv strains has provided convincing evidence that some ibv strains are recombinants between different ibv strains; recombination sites have been detected so far in the ' half of the s gene and at the ' end of viral rna (kusters et al., ; cavanagh and davis, ; wang et al., wang et al., , jia et al., ) . thus, recombination is a natural evolutionary strategy for coronaviruses. rna recombination may also explain the difference in genome structure among different coronaviruses. for example, ibv contains an additional gene, gene (a nonstructural protein gene) inserted between gene m and gene n (fig. ) . this insertion could be the result of a recombination mechanism involving the consensus ig sequence, which provides a favored recombination site. since all of the coronavirus genes are flanked by consensus ig sequences, each gene can be considered a gene "cassette," which can be rearranged by homologous recombination involving the consensus ig sequence. a nonhomologous recombination event between coronavirus rnas and other virus or cellular rnas may also explain the gene insertions in some coronaviruses. for example, mhv and bcv contain an additional gene, he, which is similar in sequence to the he gene of influenza c virus (luytjes et al., ) . this gene may have been derived by recombination between a coronavirus and influenza c virus. comparison between genome structures of coronavirus and torovirus also suggests that several recombination events may have been involved in rearranging the order of several genes during the evolution of these viruses (snijder et al., ) . recombination has been demonstrated to occur between viral rna and a transfected rna fragment derived from the viral genome (koetzner et al., ; liao and lai, ) . since transfection of both the positive-and negative-strand rna fragments led to recombination, these results suggested that recombination can occur during both positive-and negative-strand rna synthesis (liao and lai, ) . recombination can also take place between di rnas and viral rna reciprocally, i.e., the viral rna sequence can be incorporated into di rna, and vice versa, during viral rna replication. the incorporation of a helper viral rna sequence into di rna accounts at least partially for the continuous evolution of mhv di rna species during serial passages in cultured cells (see the next section). this phenomenon also explains why some genetic markers in the di rna were rapidly replaced by the helper viral rna sequences during di rna replication kim et al., a) . on the other hand, the incorporation of di rna sequences into viral rna by recombination provides an important tool to introduce desired sequences into the viral genome. for example, when an mrna or di rna containing the n gene of mhv was transfected into cells infected with an mhv ts mutant containing a defective n protein, recombination occurred between the di rna and the wild-type viral rna, resulting in recombinant viruses which had a wild-type rna sequence derived from the transfected rna in place of the defective n gene (koetzner et al., ; van der most et al., ; masters et al., ; peng et al., a ). an mhv recombinant containing a chimeric n protein of bcv and mhv has also been derived by this rna recombination strategy (peng et al., ) . this targeted rna recombination promises to be a powerful tool. recombination is thus one of the most unique aspects of coronavirus biology. it can potentially provide a genetic mechanism by which coronaviruses maintain their sequence integrity. in view of the large size of the coronavirus rna, it is predictable that most of the viral rna molecules would contain mutations due to the high error frequencies of rna polymerases; recombination may provide a repair mechanism for the virus (lai, ). similar to most rna viruses, coronaviruses can readily generate di particles when viruses are passaged in tissue culture at a high multiplicity of infection. this has been demonstrated for mhv, ibv, and tgev. when mhv was serially passaged, different types of di rna appeared at different passage levels, suggesting that di rnas continue to evolve and that new dis have a selective advantage under the evolving cellular conditions . however, the ibv and tgev dis appear to be more stable (penzes et al., ; mendez et al., ) . the generation of di rnas is probably caused by polymerase jumping during rna replication or nonhomologous rna recombination. although no sequence homology exists at the fusion sites of different rna regions within the di rna, a high degree of potential secondary structure does exist at some of its rna fusion sites (makino et al., b) , which may have facilitated the pausing and template switching of rna polymerase during synthesis. if nonhomologous recombination is involved in generating di rna, it probably occurs between two different rna molecules because di rnas are generated only at high multiplicity of infection. recombination between an existing di rna and helper virus rna has been shown to contribute to the evolution of mhv di rnas during virus passages . the coronavirus di rnas can be grouped into three types. the first type is of nearly genomic size and is typified by dissa rna of mhv . this di rna is efficiently packaged into virus particles and contains several deletions in the viral genome, but it contains a functional gene , which encodes rna polymerase, and a functional gene , which encodes n protein. these two functional gene products are sufficient to support di rna replication (k. h. ; thus, this type of di rna can replicate without a helper virus (makino et al., a; k. h. kim and makino, ) . by definition, it is not a di rna, inasmuch as it is not defective in replication; however, because it is smaller than the genomic rna and is produced at a high multiplicity of infection, it is classified as a di rna. this type of di is unique to coronavirus. a -kb di rna has been described for tgev (mendez et al., , but whether it can replicate in the absence of a helper virus has not been examined. the second type di rna is typified by disse of mhv (makino et al., ) . this di rna is truly defective and can replicate only in the presence of helper viruses. it replicates very efficiently, but is poorly packaged into virus particles because it lacks a specific rna-packaging signal. this type of di rna typically contains both the ' and ' ends of the wild-type viral rna and one or several discontiguous regions of the wild-type rnas. because of the high efficiency of replication, this type of di can still be serially passaged in tissue culture for at least several passages, probably because a small amount of di rna can be nonspecifically packaged into the virion. the third type of di rna is represented by dissf of mhv-jhm (makino et al., ) and di-a of mhv a (van der most et al., ). it is similar to the second type but contains an rna-packaging signal and is thus packaged efficiently into virus particles. this type of di rna has been detected in ibv (penzes et al., ) and tgev (mendez et al., ) . a small di rna ( . kb) of bcv may also belong to this type , but whether this di rna can be specifically packaged into virion is not certain. all three types of di rnas contain an orf, which encodes a protein fused from two different viral proteins. this orf is not required for the replication of mhv di rna (liao and lai, ' ) ; nevertheless, mhv di rnas with a functional orf usually have an evolutionary advantage over those without one or with a smaller orf kim et al., a) . therefore, a di rna containing a short orf was often rapidly replaced by di rnas containing a longer orf that had been generated by recombination or mutation kim et al., a) . the translatability of the orf may be more important than the nature of the actual protein translated from this orf (van der most et al., ) , suggesting that translation of rna may facilitate rna replication. reduction of the orf of a n ibv di rna to just amino acids did not diminish its capacity to be replicated or packaged (penzes et al., ) . however, it has been shown for a bcv di rna that a bcv-specific n protein translated from the di orf (a cis-acting protein) is required for efficient di rna replication . the variation in the sequence requirement for rna replication of these di rnas may be related to their overall rna conformation. the significance of di rna in the biology and natural evolution of coronaviruses is not known. di rnas provide useful tools for studying the sequence and structural requirements for various functions of viral genomic rna. as they contain cis-acting signals for rna replication, they are mini-versions of the viral genomic rna. however, it should be cautioned again that because of the small size of the di rna compared to the genomic rna, the structural requirements for various rna functions, as determined from the use of di rna constructs, may be different from those of the whole viral genome. the following cis-acting signals for various rna functions have been determined using various di rnas: . rna-packaging signal. in a comparison of mhv di rnas that are efficiently and inefficiently packaged, it was determined that the packaging signal for mhv di rna is localized near the ' end of gene (in the l b region, approximately kb from the ' end) (makino et al., ; van der most et al., ; fosmire et al., ) . this packaging signal forms a stem-loop structure which may be required for the rna-packaging activity (fosmire et al., ) . it is necessary and sufficient for the packaging of di rna or a heterologous rna into the virions (woo et al., ) . the fact that this packaging signal is localized in gene , which is present in genomic but not subgenomic rnas, is consistent with the packaging of genomic but not subgenomic rnas in virus particles. the packaging signal for di rnas of other coronaviruses has not been determined. however, some coronaviruses have been shown to package subgenomic mrnas at low efficiency (sethna et al., ; hofmann et al., ; zhao et al., ) . these are probably packaged nonspecifically; however, the possibility that these viruses may have a different rna packaging signal cannot yet be ruled out. similarly, di rnas that do not contain this packaging signal, such as disse rna of mhv (makino et al., ) and di rna of bcv chang et al., , can be packaged at low efficiency, thereby maintaining themselves for at least several passages in tissue culture. for mhv di rna, it has been shown that only nucleotides at the ' end plus a stretch of poly(a) sequence are required for negative-strand rna synthesis ; no specific upstream rna sequences are required. however, when an mrna is transcribed from an ig site in the same di rna, the negative-strand rna synthesis from this di rna is inhibited, suggesting a common element involved in mrna transcription and negative-strand rna synthesis . one unanswered question is whether or not the sequence requirements for the synthesis of genomic and subgenomic negative-strand rna are identical. . replication signal. sequential deletion analysis has shown that the replication (i.e., complete cycles of negative-and positive-strand rna synthesis) of mhv disse or dissf rnas requires approximately - nucleotides from both the ' and ' ends. the minimum sequence requirement for rna replication may vary with different di rnas. these issues have been discussed in section v,f. . transcriptional signal. di rnas normally do not transcribe subgenomic mrnas because they do not have ig sequences. thus, natural di rnas can synthesize only the full-sized di rna. however, by introduction of the consensus ig sequences into di rna (makino et al., , it has been possible to use di rna as a vector for determining the sequence requirement for subgenomic rna transcription. the cis-and trans-acting signals for transcription have been described in section v,e. . recombination. di rnas of mhv have been demonstrated to undergo a high frequency of recombination with helper virus rna. as discussed above, this accounts for the evolution of mhv di rna species during serial passages of viruses . furthermore, mhv di rnas with a smaller orf are frequently replaced by a di rna with a larger orf by recombination with the helper virus rna (de groot et al., ; kim et al., a) , suggesting that recombination between di rnas and helper virus rnas occurs readily. the reciprocal recombination between di rna and helper virus rna, i.e., the transfer of di rna sequences to the helper virus rna, also has been observed. as a result, the genetic markers on the di rna can be incorporated into the helper virus rna (koetzner et al., ) . recombination between two di rnas, however, has not been described. sequence requirements for rna recombination also have not been studied. bcv di rnas also undergo frequent recombination . however, di rnas of ibv and tgev appear to be more stable. coronavirus research has made tremendous progress in the last decade. the virus family has grown in size, and many of the features thought to be unique to coronaviruses have now been found to be shared by some other viruses. since the last time this serial publication published the first comprehensive review of the molecular biology of coronaviruses (sturman and holmes, ) , the literature on this virus has grown to exceed anyone's ability to do a comprehensive review of every topic relating to coronaviruses. in this review, we have concentrated on areas which have shown the most progress and which present the most challenges. our choice of literature was meant to be representative but is by no means comprehensive. notably missing from this review are the molecular studies related to viral pathogenesis and the interactions between the virus and cells. coronavirus research has contributed to the understanding of many aspects of molecular biology in general, such as the mechanism of rna synthesis, translational control, and protein transport and processing. it remains a treasure capable of generating unexpected insights. despite two decades of studies on the molecular biology of this virus, there are still many problems to be solved: . with regard to the mechanism of rna transcription, many conflicting data remain. coronavirus undoubtedly utilizes a unique, discontinuous transcription mechanism, but how it acts is a subject of debate. an in uitro rna transcription system, so necessary for an understanding of rna synthesis, is still in its infancy. related to this question is the nature of rna polymerase. the sheer size of the polymerase gene presents a daunting task. the availability of the cdna clones and expression vectors for this gene has just begun to allow this black box to be cracked open. this will undoubtedly be a fruitful area of future research. . the last two years have seen the unraveling of the mechanism of coronavirus assembly, which, as it turns out, involves a littlecharacterized e protein. how the various viral structural proteins interact with each other in the various subcellular compartments to form a complete virus particle is an exciting frontier. . after more than years since the first coronavirus was seen under electron microscope, an unexpected new feature of the virus, namely, an icosahedral core with a helical nucleoprotein, was recently uncovered. this structure places coronavirus in a unique position among rna viruses because it takes on the characteristics of positive-, negative-and double-strand rna viruses in morphology. this recent finding challenges us to reevaluate the structure of coronaviruses. . the ability to perform reverse genetic studies of coronavirus is still very limited. expression of individual viral genes and targeted recombination of very limited rna regions are the only available genetic means for examining the structure and function of the coronavirus genome. perhaps it is an unrealistic dream, but progress in polymerase chain reaction technology may one day allow an infectious cdna for coronavirus rna to be made. the early events of viral replication have so far been largely ignored. identification of the cellular receptors for the viruses may finally provide penetrating molecular tools to allow these issues to be examined. it will not be a surprise to discover that virus penetration and uncoating play defining roles in the cellular tropism of viruses. . are nonstructural protein genes really unnecessary? even if they are auxiliary genes, they may prove to play significant roles in the biology of the virus. . finally, what of the potential interaction between the virus and host, which has been one of the major themes of virology in recent years? it may be a little premature to conclude that cellular factors play major roles in coronavirus replication, but there is little doubt that cells are playing more active roles than was previously suspected. is the nucleus contributing to the coronavirus replication? this may require reexamination. these are but some of the exciting challenges for the coronavirologists to tackle. the next decade should bring us an even better understanding of the various aspects of the molecular biology of coronaviruses. membrane and 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is investigator of the howard hughes medical institute. key: cord- -r akpce authors: wang, jingjing; deng, feng; ye, gang; dong, wanyu; zheng, anjun; he, qigai; peng, guiqing title: comparison of lentiviruses pseudotyped with s proteins from coronaviruses and cell tropisms of porcine coronaviruses date: - - journal: virol sin doi: . /s - - - sha: doc_id: cord_uid: r akpce the surface glycoproteins of coronaviruses play an important role in receptor binding and cell entry. different coronaviruses interact with their specific receptors to enter host cells. lentiviruses pseudotyped with their spike proteins (s) were compared to analyze the entry efficiency of various coronaviruses. our results indicated that s proteins from different coronaviruses displayed varied abilities to mediate pseudotyped virus infection. furthermore, the cell tropisms of porcine epidemic diarrhea virus (pedv) and transmissible gastroenteritis virus (tgev) have been characterized by live and pseudotyped viruses. both live and pseudoviruses could infected vero- ccl- (monkey kidney), huh- (human liver), and pk- (pig kidney) cells efficiently. ccl (cat kidney) cells could be infected efficiently by tgev but not pedv. overall, our study provides new insights into the mechanisms of viral entry and forms a basis for antiviral drug screening. [image: see text] coronaviruses (covs) are important infectious pathogens that are associated closely with respiratory and enteric diseases in humans and animals (perlman and netland, ; belouzard et al., ; li, ) . covs have a single-strand positive sense rna genome and consist of four groups: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus (table ) . viral entry into cells is highly dependent on the interaction between viral particles and the host cells. covs use a variety of cellular receptors and co-receptors, including proteins and sugars, to facilitate their entry into cells. infection begins with an interaction between the virus and its specific receptors (li, ) . severe acute respiratory syndrome coronavirus (sars-cov) of group beta uses a type i integral membrane protein, angiotensin-converting enzyme (ace ), as a receptor . there is no structural homology between sars-cov and the human coronavirus nl (hcov-nl ) receptor-binding domain (rbd), but they recognize the same receptor, ace (wu et al., ) . although the rbd crystal structures of transmissible gastroenteritis virus (tgev) and hcov-nl are similar, they use different receptors (reguera et al., ) . aminopeptidase n (apn), also known as cd , is a type ii transmembrane protein (tusell et al., ) . tgev and porcine epidemic diarrhea virus (pedv) infect cells through interaction with porcine (p) apn (li et al., ; nam and lee, ) . human (h) apn is a receptor for hcov- e (belouzard et al., ) . in the same beta group, the receptors for mouse hepatitis virus (mhv) and bovine coronavirus (bcov) are carcinoembryonic antigen-related cell adhesion molecule (ceacam ) and a sugar, respectively, despite their high sequence homology peng et al., ) . ceacam , the first identified coronavirus receptor (dveksler et al., ) , is a type i transmembrane multifunctional protein of the immunoglobulin superfamily (de haan et al., ) . cell entry and interspecies transmission of covs are mediated by the spike protein (s). cov s is a class i fusion protein (bosch et al., ) . in covs, s is a significant surface protein and plays an important role in mediating infection of virions (schwegmann-wessels et al., ) . s is also responsible for receptor binding and the fusion of viral and cellular membranes. all cov s share the same functional component in two domains: an nterminal domain called s that is responsible for receptor binding, and a c-terminal s domain that is responsible for fusion. s contains two subdomains, an n-terminal domain and a c-terminal domain ( figure a ). both function as rbds and bind a variety of proteins and sugars (belouzard et al., ) . because some covs have strong pathogenicity, the lentiviral pseudotype system is a reliable tool to study the proteins of highly pathogenic viruses under conventional biosafety conditions. hiv-luc is an hiv- based lentiviral vector bearing the luciferase reporter gene and has been used in the production of pseudotyped viruses lu et al., ; tang et al., ) . pseudovirus entry efficiency is characterized based on the luciferase activity. viral particles pseudotyped by various s proteins have been described for several viruses. in our study, we compared the efficiency of pseudotyped viruses with s proteins from different groups of covs. furthermore, the cell tropisms of tgev and pedv were characterized by live and pseudotyped viruses. t cells were cultured and maintained in rpmi medium (invitrogen, carlsbad, ca, usa) supplemented with % fetal bovine serum (fbs) at °c in an atmosphere of % co . pk- , huh- , vero-ccl- , mdbk, ccl , bsr, and mdck cells were cultured and maintained in dulbecco's modified eagle's modium (dmem) (invitrogen) supplemented with % fetal bovine serum (fbs) at °c in an atmosphere of % co . the pedv and tgev strains (genbank accession numbers: kt . for pedv, hq . for tgev) were isolated from a suckling piglet. rabbit anti-pedv n protein and rabbit anti-tgev n protein polyclonal antibodies are stored by the lab. the s protein sequences of different groups of covs (genbank accession numbers: abd . for sars-cov-s, aar . for mhv-s, np_ . for hcov- e-s, abg . for tgev-s, np_ . for pedv-s, aat . for hcov-oc -s, abm . for bcov-s, and np_ . for avian infectious bronchitis virus (ibv) s) were codon-optimized and synthesized (genscript, nanjing, china). the s fragments were cloned into the pcdna . (+) vector with a c-terminal his-tag. on the day before transfection, t cells were seeded in six-well plates. the next day, the cells were transfected with s plasmids plus the lentiviral vector hiv-luc lentiviruses pseudotyped with s proteins were solubilized by boiling in sodium dodecyl sulfate sample buffer. the s proteins were fractionated by sodium dodecyl sulfate- % polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. the nitrocellulose membrane was incubated with a : dilution of a mouse anti-his antibody (proteintech group, chicago, il, usa). goat anti-mouse igg (boster, wuhan, china) was used as the secondary antibody. a plasmid (vector pcdna . (+)) with each viral receptor gene (ace , ceacam , hapn, and papn) was transfected into t cells with lipofectamine . after incubation for - h at °c, the supernatant was replaced by dmem containing % fbs. after h, the cells were infected by lentiviruses pseudotyped with the s proteins from covs of different groups. for analysis of the lentiviruses pseudotyped with the s proteins from covs of different groups, different cell lines were seeded in -well plates. t cells expressing receptors were infected by the same lentiviruses and incubated at °c for h. dmem ( µl) containing % fbs was added subsequently to all cells and incub-ated for an additional - h. the pseudovirus entry efficiency was characterized based on luciferase activity. pedv and tgev were used to infect various cell lines from different species, including pk- (pig kidney), huh- (human liver), vero-ccl- (monkey kidney), mdbk (bovine kidney), ccl (cat kidney), bsr (hamster kidney), and mdck (canine kidney) cells at a multiplicity of infection of . . trypsin ( µg/ml) was included in the cell culture medium to facilitate live virus infections. cells infected with viruses were fixed with . % (v/v) paraformaldehyde at or h post-inoculation. pedv or tgev was detected with fluorescein isothiocyanate-labeled rabbit anti-pedv or anti-tgev n protein antibodies, respectively, and observed under a fluorescence microscope. all experiments were done at least twice (in most cases three or more times). data were analyzed statistically using two-tailed student's t-tests for comparison of pcdna . and cov s pseudovirus. the pseudovirus entry efficiency is reported as the luciferase activity. statistical analyses were performed to compare the treatment groups: ***p < . ; ** . < p < . . cov diversity is reflected in the variable s proteins (heald-sargent and gallagher, ). therefore, we compared the s protein amino acid sequences of covs from different groups, including sars-cov, mhv, hcov- e, tgev, pedv, hcov-oc , bcov, and ibv (gen-bank accession numbers abd . , aar . , np_ . , abg . , np_ . , aat . , abm . , and np_ . , respectively) using clustalw ( figure b ). because s is responsible for receptor binding, the amino acid sequences of the s domain were aligned ( figure c ). the s proteins from covs of the same group shared sequence similarities of greater than %, with some similarities of up to %. the s sequence similarities of covs from different groups were less than %. to determine the success of pseudovirus construction, the s proteins of pseudoviruses were confirmed by western blotting (figure a ). the pseudoviruses were quantified with an hiv- p eia kit and used to infect cells expressing the corresponding receptor. lentiviruses pseudotyped with sars-cov s protein could efficiently infect t cells expressing ace , and the pseudovirus level after entry reached relative light units (rlu) ( figure b ). the s proteins of hcov- e, tgev, and pedv exhibit high homology. however, their pseudovirus infection efficiency varies, and they are approximately -fold less efficient than the sars-cov-s pseudovirus. the tgev-s and pedv-s pseudoviruses showed similar infection efficiencies. although hcov-oc , bcov, and ibv utilize a sugar as their receptors, their pseudovirus infection efficiency was not clear. tgev-s and pedv-s pseudoviruses were used to infect pk- , huh- , vero-ccl- , mdbk, ccl , bsr, and mdck cells ( figure c ). the tgev-s and pedv-s pseudoviruses could enter pk- , huh- , and vero-ccl- cells with a similar efficiency. however, there was an intriguing finding that ccl cells could be infected efficiently by tgev-s but not pedv-s pseudoviruses. to further study the cellular entry of covs, we used live pedv and tgev to infect different cell lines. pedv efficiently infected vero-ccl- (monkey kidney), huh- (human liver), and pk- (pig kidney) cells (figure ) . infection was not evident in mdbk (bovine kidney), ccl (cat kidney), bsr (hamster kidney), or mdck (canine kidney) cells. this is consistent with the data in figure c . tgev efficiently infected cells from pigs (pk- ), humans (huh- ), monkeys (vero-ccl- ), and felines (ccl ) (figure ) . overall, our study demonstrates that pedv and tgev can infect cells from different species. covs recognize a variety of cell-surface molecules as their host receptors, including proteins, sugars, and heparan sulfate (li, ) . the cell entry mechanisms of these viruses are mediated by the viral s proteins that bind cellular receptors and mediate the fusion of viral and cellular membranes (heald-sargent and gallagher, ) . we demonstrated that s proteins from covs of different groups played an important role in mediating viral infection at different efficiencies. sars-cov-s pseudoviruses efficiently entered cells expressing ace . this result is consistent with previous reports demonstrating that ace is a receptor for sars-cov (schwegmann-wessels et al., ; wu et al., ) . hcov- e-s and mhv-s pseudoviruses showed strong infectivity, in part because they use proteins as a receptor belouzard et al., ) . moreover, pedv-s and tgev-s pseudoviruses had similar infection efficiencies in pk- , huh- , or vero-ccl- cells, and tgev-s pseudovirus-infected ccl cells. the sugar moiety -n-acetyl- -o-acetylneuraminic acid (neu , ac ), found on cell-surface glycoproteins or glycolipids, is recognized by bcov and hcov-oc . in addition, two other types of sugars, -n-glycolylneuraminic acid (neu gc) and -n-acetylneuraminic acid (neu ac), can serve as receptors or co-receptors for some alpha-and gamma-covs (cavanagh and davis, ; krempl et al., ; peng et al., ; shahwan et al., ) . the s-to-sugar binding affinity is lower than the s-protein interaction, which partially explains why the bcov and ibv pseudoviruses have low entry efficiency. although pedv and tgev use the same receptor (papn) and the s proteins present high homology, live pedv and tgev prefer different cell lines; pedv prefers vero-ccl- , huh- , and pk- cells. this is consistent with a previous report (liu et al., ) . however, tgev prefers vero-ccl- , huh- , pk- , and ccl cells. this indicates that pedv and tgev may have different species preferences. these results are consistent with the observation that their receptor-binding domains are located in non-homologous regions (belouzard et al., ) . however, the mechanism underlying different cell tropisms requires further investigation. the s protein plays a crucial role in entry into host cells by mediating receptor binding and membrane fusion. covs use a variety of receptors and triggers to ac- tivate fusion (belouzard et al., ) . the first and most important step of virus infection is the interaction between the virus and its cellular receptor. infection of lentiviruses pseudotyped shows that s proteins from different covs have varied abilities to mediate pseudotyped virus infection in different cell types from different tissues. these findings further our understanding of the mechanisms underlying viral invasion and contribute to the development of drugs against covs. this work was supported by the national natural science foundation of china (grant no. ). this article does not contain any studies with human or animal subjects performed by any of the authors. there is no competing interest in this research. g.p. and j.w. designed the experiments and wrote the paper. j.w. performed the experiments. f.d. and q.h. contributed to critical discussions of the data. all authors approved the final manuscript. mechanisms of coronavirus cell entry mediated by the viral spike protein the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex coronavirus ibv: removal of spike glycopolypeptide s by urea abolishes infectivity and haemagglutination but not attachment to cells murine coronavirus with an extended host range uses heparan sulfate as an entry receptor cloning of the mouse hepatitis 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identifies key determinants of viral host range a virus-binding hot spot on human angiotensin-converting enzyme is critical for binding of two different coronaviruses key: cord- -fxhkfjl authors: have, p.; moving, v.; svansson, v.; uttenthal, Å.; bloch, b. title: coronavirus infection in mink (mustela vision). serological evidence of infection with a coronavirus related to transmissible gastroenteritis virus and porcine epidemic diarrhea virus date: - - journal: veterinary microbiology doi: . / - ( ) -g sha: doc_id: cord_uid: fxhkfjl abstract antibodies to a transmissible gastroenteritis virus (tgev)-related coronavirus have been demonstrated in mink sera by indirect immunofluorescence, peroxidase-linked antibody assays and immunoblotting. this is the first serological evidence of a specific coronavirus infection in mink. the putative mink coronavirus (mcv) seems to be widespread in the danish mink population with a prevalence approaching %. analysis by immunoblotting has shown that mcv is closely related to tgev by the spike (s), matrix (m) and nucleoprotein (n) polypeptides. furthermore, antibodies to mcv also cross-reacted with n and m polypeptides of porcine epidemic diarrhea virus (pedv). thus mcv may occupy an intermediate position between the tgev group of coronaviruses and pedv. the possibility that mcv may be associated with syndromes of acute enteritis in preweaning mink is discussed. coronaviruses infect a number of mammalian and avian species and the mammalian viruses are usually placed in two major serological groups represented by mouse hepatitis virus (mhv) and transmissible gastroenteritis virus (tgev) (sturman and holmes, , siddell et al., ) . the mhv group includes bovine coronavirus (bcv), porcine hemagglutinating encephalomyelitis virus (hev) and human coronavirus (hcv) oc- . the tgev group includes porcine respiratory coronavirus (prcv) (pensaert et al., (pensaert et al., , s~inchez et al., , canine coronavirus (ccv), feline infectious peritonitis virus (fipv) and feline enteric coronavirus (fecv) . mature virions of coronaviridae contain three major structural proteins (cavanagh et al., ) of which the spike (s) glycoprotein and transmembrane matrix (m) glycoprotein are glycosylated. the nucleoprotein (n) is associated with the positive-stranded rna genome. coronaviruses are responsible for various disease manifestations in their host species, depending on virus tropism (e.g. pneumotropic, enterotropic or neurotropic viruses ). porcine epidemic diarrhea coronavirus (pedv) (pensaert et al., ) , causing a gastroenteritis similar to tge in pigs, is as yet unclassified, although a slight serological relationship with fipv has been demonstrated (yaling et al., ) . until recently, no known cell culture system was available for propagation of pedv in vitro but this has now been accomplished using vero cells and medium including proteases such as trypsin (hofmann and wyler, ) . the apparent mr values of pedv structural proteins have been found to be - k for the s protein, for the n protein and - for the m protein . this serological study on mink coronavirus was initiated by our observations in electron microscopy of coronavirus-like particles in stool samples from neonatal mink kits suffering from diarrhea. specific coronavirus infections have so far not been described in mink but coronavirus-like particles in feces have recently been associated with epizootic catarrhal gastroenteritis in mink (gorham et al., ) . the present paper provides the first serological evidence for infection in mink with a coronavirus (here designated mcv) serologically related to tgev and pedv. tgev (purdue strain) and prcv (danish isolate dk / , have, ) were grown in a swine testicle (st) cell line (mcclurkin and norman, ) . ccv (strain - ) was grown in a- cells (atcc crl ). fipv (strain df , atcc vr- ) and fecv (strain wsu - , atcc vr ) were grown in crandell feline kidney (crfk) cells. pedv (strain / ) was kindly provided by dr. s. cartwright, weybridge, as an intestinal passage of pedv-infected colostrum-deprived newborn piglets. after a further two passages in piglets the virus was adapted to grow in vero cells by the method of hofmann and wyler (hofmann and wyler, ) . four groups of mink sera were selected for evaluation of mcv antibodies: a, mink sera were obtained during from the danish fur breeders association as part of routine sampling for plasmacytosis testing (aleutian disease, adv). b, sera were obtained in from a mink farm showing a high level of neonatal mortality. c, sera were obtained from mink kept for experimental purposes. d, mink sera collected during - were examined in order to trace the history of coronavirus infection in mink. hyperimmune or convalescent porcine, canine and feline sera against tgev, prcv, ccv, fipv and pedv were included as controls. porcine sera were produced by experimental oronasal inoculation of spf pigs with tgev, prcv or pedv, respectively. canine antiserum to ccv was obtained from a recovered dog in a kennel having an acute outbreak of ccv gastroenteritis. fipv antiserum was from a cat (field case) with clinically diagnosed fip. monolayers for ifat were prepared by infecting secondary porcine kidney cells with tgev and acetone fixation after h. sera were examined at a dilution of / in pbs using acetone-fixed monolayers of virus-infected cells on multitest slides. following incubation in a moist chamber for h at ° c, the slides were rinsed three times in pbs and incubated with fitc-conjugated protein-a (pharmacia) at a dilution of / in pbs. after incubation for min at °c slides were rinsed and mounted for microscopy. virus-infected cells were prepared in -well tissue culture microplates by mixing cell suspensions with an appropriate dilution of virus. following incubation for h cells were fixed in % acetone in pbs for min, air dried at °c for h and stored at - °c or used immediately (jensen, ) . monolayers were incubated with dilutions of test sera in pbs- . % tween (pbst) for h at °c. following washing in pbst, peroxidase-conjugated protein a (zymed) or peroxidase-conjugated anti-swine igg (dako), both diluted / in pbst, were added and incubated for min at °c. finally, the substrate -amino- -ethylcarbazole was added and left for min. serum titres were recorded as the highest dilution causing specific cytoplasmic staining. neutralization tests were carried out in -well microplates. sera were heatinactivated and two-fold dilutions were mixed with tcidso of virus and incubated for h at ° c. following incubation, the appropriate cell suspension was added, plates were sealed and incubated at °c in a % co -air mixture. neutralizing titres were evaluated after - days using the method of reed and muench ( ) . tgev, ccv or pedv culture supernatants were clarified at g in a ja rotor for min followed by pelleting at g for h in the same rotor. pelleted virus was separated by sds-page on a % gel after solubilisation at °c for min in sample buffer ( . m tris-hc , % glycerol, % sds, ph . ) under non-reducing conditions. electrophoresis was performed using laemmli buffers (laemmli, ) . proteins were transferred electrophoretically ( ma, min ) to nitrocellulose or polyvinylidene difluoride sheets by semi-dry blotting using tris-glycine-methanol buffer ( mm glycine, mm tris base, . % (w/v) sds, % methanol) ph . . strips were blocked by incubation for h with % (w/v) skimmed milk powder in pbst. sera were diluted / in the same buffer and applied for h on a tilt shaker. peroxidase-conjugated protein a (zymed) was diluted / in pbst and applied for min. specific bands were revealed using the substrate o-dianisidine dihydrochloride (sigma, . mg/ml in pbs). initially, mink sera were examined in ifat using acetone fixed monolayers of secondary swine kidney cells infected with tgev as substrate and fitcconjugated protein-a to detect specific binding of antibodies to tgev. of sera collected during for routine testing of adv antibodies, were found to contain tgev cross-reacting antibodies in ifat at a dilution of / . the pattern of fluorescence was most often diffusely cytoplasmic and very similar to that seen with sera from fipv-infected cats. the serological data on mink sera have been summarized in table . all of sera from a farm showing high neonatal mortality contained tgev cross-reacting antibodies in ifat at a dilution of / . these sera represented adult females as well as months old male and female kits. also, all of sera from experimental animals were positive in ifat for tgev cross-reacting antibodies. sera obtained during - were titrated in tgev pla and of samples were positive with titres ranging from to . positive samples were evenly distributed over the years with no obvious time-clustering. due to cytotoxicity some of the sera included in this study could not be examined in neutralizatioti test. all of the sera originating from experimental animals were tested in neutralization tests against tgev and prcv (initial dilution / ), ccv, fipv and fecv (initial dilution / ) and none of the sera neutralized any of these viruses. furthermore, sera ( / positive by pla) tested against tgev and prcv and sera ( / positive by pla or ifat) tested against fipv and fecv were also negative for neutralizing antibodies. a high-titered mink antiserum and representative high-titered antisera against tgev, prcv, ccv, fipv and pedv were selected for further studies of the serological relationship of the putative mink coronavirus to other coronaviruses. these sera were used to develop western blots of tgev, ccv and pedv. it should be noted, that antisera to ccv and fipv are field sera and not homologous to the strains applied in this study. it is apparent, that antisera against tgev, prcv, ccv and mcv all react strongly with all three major structural proteins s ( k), m ( k) and n ( k) of tgev (fig. ) . a fourth polypeptide ( - k) is revealed by antiserum to tgev only. in contrast, fipv antiserum reacts primarily with n and m of tgev. antiserum against pedv reacts weakly with s and n of tgev. a similar pattern was found when using ccv instead of tgev as antigen (fig. ) except that staining of the s protein was only evident with ccv antiserum. in parallel experiments weak staining of the s protein of ccv has been observed with tgev, prcv and mcv antisera (not shown). antiserum against pedv reacts with s, m ( k) and n ( k) of pedv whereas antisera against tgev, prcv, ccv and fipv barely reacts with n of pedv (fig. ) . mink antiserum occupies an intermediate position in that there is a distinct reaction with both m and n of pedv. by cross-neutralization tgev, prcv, ccv, fipv and fecv were found to be very similar, all being neutralized by antisera to tgev, prcv and ccv (table ) . on the other hand antisera to fipv, pedv and mcv did not neutralize any of the viruses. in particular, antiserum to fipv did not neutralize the strains of fipv and fecv applied in this study. neutralization of pedv was not attempted, due to the extreme dependance of replication on trypsin. the reactivity of these sera was also tested in pla against tgev, fecv/ fipv and pedv (table ). the titres of antisera against tgev, prcv and ccv are higher against tgev and fecv/fipv than against pedv. antiserum against fipv and mcv are broadly reacting with tgev, fecv/fipv and pedv whereas antiserum against pedv reacts strongly with the homologous virus and only weakly with fecv. examination of mink sera by ifat has shown a high prevalence of antibodies reacting with tgev. these results indicate that one or more coronaviruses related to tgev are widespread in the danish mink population and that such viruses have been present at least since . the high prevalence of seropositive animals over several years points towards an enzootic coronavirus infection in balance with its host population. at present, it is unknown whether this coronavirus infection causes any disease but if so, it may be expected that the virus would manifest itself primarily in young animals either as a consequence of waning maternal antibodies or inadequate lactogenic immunity. a number of clinical syndromes which might be associated with coronavirus infection have been described in mink. epizootic catarrhal gastroenteritis (ecg) (larsen and gorham ) occurs primarily in mink four months and older exhibiting a mucoid diarrhea over - days. coronavirus-like particles have recently been identified by negative contrast electron microscopy in feces of animals with ecg (gorham et al., ) . a similar observation has been made by one of us (bloch, unpublished results), who found coronavirus-like particles in stool samples from neonatal mink kits. these mink kits originated from farms with a high neonatal mortality. the mink kits were suffering from diarrhea, referred to by farmers as "wet mink kits". the epizootiology of this syndrom is still unclear (henriksen ) . fip-like symptoms (pyogranulomas, vasculitis and immune-complex disease ) have not been described in mink except for adv-associated immune-complex disease (porter et al., ) but such symptoms should be kept in mind when looking for possible effects of coronavirus infection. from the data presented here it is clear that there is a close serological relationship between the putative mcv and both tgev and ccv. the results of western blots show that antibodies to mcv react with all three major structural proteins of tgev and ccv. however, none of the mink sera neutralized any of these viruses, indicating that the antigenic domains responsible for neutralization in the spike (s) protein of tgev and ccv are probably absent from mcv. in this respect mcv may resemble field strains of fipv/ fecv as it has been found that feline field sera having ifat and pla titres of or higher against tgev generally do not neutralize tgev or the laboratory strains of fipv and fecv used in this study (have, unpublished data) . it would thus be interesting to have more recent isolates of feline coronavirus and analyze their relationship to mcv. no clear relationship has been found between pedv and any other known coronavirus (pensaert et al., ) except for a weak cross-reaction with the n protein of fipv (yaling et al., ) . the present results have confirmed this cross-reaction and further shown that it extends to tgev, prcv, ccv and mcv. in addition, the m proteins of pedv and mcv are clearly related as seen by the reaction of mink antiserum with the m protein of pedv. the present study has shown that infection with a coronavirus serologically related to tgev and pedv is common in the danish mink population. at present, nothing is known of the pathogenic potential of mink coronavirus, if any, nor the preferred site of replication in the host. studies aimed at answering these questions as well as attempts to isolate the virus have been initiated. recommendations of the coronavirus study group for the nomenclature of the structural proteins, messenger rnas, and genes of coronaviruses characterization of the structural proteins of porcine epizootic diarrhea virus, strain cv detection of coronavirus-like particles from mink with epizootic catarrhal gastroenteritis serologic differentiation from transmissible gastroenteritis virus using monoclonal antibodies so-called "wet mink kits" -acute enteritis in pre-weaning mink propagation of the virus of porcine epidemic diarrhea in cell culture detection of antibodies against hog cholera virus and bovine viral diarrhea virus in porcine serum cleavage of structural proteins during the assembly of the head of bacteriophage t a new mink enteritis: an initial report studies on transmissible gastroenteritis of swine. ii. selected characteristics of a cytopathogenic virus common to five isolates of transmissible gastroenteritis an immunelectron microscopic and immunofluorescent study on the antigenic relationship between the coronavirus-like agent, cv , and several coronaviruses isolation of a porcine respiratory, non-enteric coronavirus related to transmissible gastroenteritis aleutian disease of mink a simple method of estimating fifty percent endpoints antigenic homology among coronaviruses related to transmissible gastroenteritis virus the biology ofcoronaviruses the molecular biology of coronaviruses porcine epidemic diarrhea virus (cv ) and feline infectious peritonitis virus (fipv) are antigenically related key: cord- -kllqjn authors: woods, roger d.; wesley, ronald d. title: cultivation techniques for animal coronaviruses: emphasis on feline infectious peritonitis virus, canine coronavirus, transmissible gastroenteritis virus, and porcine hemagglutinating encephalomyelitis virus date: journal: j tissue cult methods doi: . /bf sha: doc_id: cord_uid: kllqjn techniques are described for the growth and characterization of some mammalian coronaviruses. because of the fastidious nature of their growth requirements, most will replicate only in cells derived from the natural host or a closely related species. fetal cat cells are used to grow fipv, and porcine cells are used to grow tgev and hev. however, ccv will replicate in both feline and canine cells. although all four of these viruses prefer to replicate in a cell in the stationary phase of growth, fipv is able to replicate in an actively growing cell. each virus causes a cytopathic effect in monolayer cell cultures under agar or media to h postinfection. primary isolation of each virus from field specimens is difficult, although most can usually be isolated after to blind passages in the cell culture. the coronaviridae family of viruses has a worldwide distribution. these viruses cause economically important diseases in man and in domestic and laboratory animals { ). at the present time the group consists of recognized viruses and or more unclassified isolates. the viruses and their abbreviations are listed in table . these viruses comprise four distinct antigenic groups plus a miscellaneous group ( ) . generally these viruses infect epithelial cells of the respiratory tract [human coronavirus (hcv), infectious bronchitis virus (ibv), rat coronavirus (rcv), porcine respiratory coronavirus (pcv)] and epithelial cells of the gastrointestinal tract [bovine coronavirus (bcv), canine eoronavirus (ccv), transmissible gastroenteritis virus {tgev), turkey coronavirus (tcv), feline enteric coronavirus (fecv), human enteric coronavirus (hecv)]. in addition, feline infectious peritonitis virus tfipv), mouse hepatitis virus (mhv), rabbit coronavirus (rbcv), and hemagglutinating encephalomyelitis virus (hev) exhibit other tissue tropisms and alternate pathogenic mechanisms ( ) . the feline, murine, and avian coronaviruses will usually cause a mild or inapparent infection in adults, hut usually a severe diarrheal disease and often death in neonatal animals ( ) . basic studies on these viruses have been limited because of their fastidious growth in cell culture ( ) . most coronaviruses will grow only in cells derived from the natural host animal or in cells from a closely related species ( , ) . growth of hcv, hecv, and rbcv in cell culture is difficult, whereas (fig. c, d) . after cpe is observed, freeze each flask at -- ° c. when grown under these conditions, tgev will have a plaque titer of approximately x pfu/ml to h after inoculation, and hev will have a plaque titer of approximately x pfu/ml to h after inoculation. to study the pathogenesis and molecular biology of coronaviruses, it is beneficial to identify cell lines in which the viruses will grow to titers greater than x s pfu/ml, thus providing sufficient viral mass for molecular studies. at the present time, most of the animal coronaviruses have been adapted to cell culture ( , , }. the methods presented in this report are easy to reproduce and could be adapted by laboratories worldwide for in vitro growth of fipv, ccv, hev, and tgev. such procedures have been used to grow these viruses to titers ranging from about x pfu/ml for fipv ( ) to x pfu/ml for tgev ~ l although most coronaviruses can now be grown in cell culture, their primary isolation from field specimens is still difficult i }. no one isolation technique or method can be used for the entire group. probably the most successful isolation method is to blindly passage . -pm-filtered fluids to times in a host organ cell line known to support growth of suspected virus, until a cpe is observed. the use of monospecific fluorescent antibodies is recommended to follow in vitro growth of the virus and to confirm virus identity. several techniques have been used to enhance or improve the chances of isolating these viruses. some of these techniques include addition of pancreatin . %}, trypsin ( pg/ml}, or deae-dextran to cell culture media at the time of initial inoculation, maintaining a slightly acidic ph in the culture media, the use of primary cell cultures instead of secondary cultures, and incorporation of high titer hyperimmune antisera in the culture media to suppress the growth of contaminating viruses. coronavirus-induced cpe is dependent on the virus, cell line, and isolate. the cpe ranges from an inap-parent infection in persistently infected cell lines to nearly complete cell disruption with several cell cultureadapted viruses , ). in a typical coronavirusinfected cell culture the first sign of cpe will be the appearance of granular and refractile cells, followed by formation of enlarged rounded cells, ballooned cells, and finally the detachment of infected cells from the culture flask ). when grown under the conditions described, optimal viral titers are usually obtained when approximately % of the cell sheet is observed with cpe. both fipv and ccv induce a similar cpe in crfk ceils. thirty-six to h postinfection (pi), fipvinfected crfk cells are granular and refractile. over the next h small multinucleated ~ to nuclei~ cells are formed by fusion and then they detach from the flask ~. the cpe is focal and under . % agar-mem- % fbs, distinct plaques up to mm in diameter form in the cell sheet in to h. initial cpe in ccv-infected crfk appears within h and consist of granular, refractile, and amorphorus multinucleated tgreater than nuclei) cells. during the next to h the cpe will spread over the entire sheet producing numerous ballooned cells and finally infected cells detaching from the flask. under . % agar-mem, ccv will form distinct plaques . mm diameter within h ~ ). initial cpe produced by attenuated and virulent tgev in st cells is similar to that observed with fipv and ccv in crfk cells, whereas cpe produced by hev in spth cells is slightly different. sixteen to h pi, tgev infected st cells are granular, refractile, and greatly enlarged. over the next to h the infection will spread over the cell sheet, and with advancing infection the round cells become ballooned and detach from the flask. under . % agar-mem, attenuated tgev produces clear uniform plaques up to mm in diameter within h, whereas the virulent virus produces diffuse, irregular plaques approximately . to mm in diameter within h i , ). in to h pi. hev infected spth ceils the cpe will show small areas of syncytia, which are easily visible with an inverted microscope. over the next h the syncytia degenerates, producing syncytial debris in the culture medium and clear holes with an opaque irregular shape in the cell sheet ( ) . after disruption of the syncytia, ballooned structures appear and the formation of new syncytia is seldom observed. a hemadsorption plaque assay has been developed for titration of infectivity of hev t l the crfk cell line has been used for the primary isolation and growth of fipv. isolation of fipv from clinical specimens is very difficult using cell lines. however, virus can frequently be recovered if crfk cells are inoculated and blindly passaged to times in the presence of trypsin ( i. the incorporation of trypsin into the fipv growth medium enhances both the isolation and growth of fipv. the reason for this enhancement is unknown, but a similar observation has been recorded for bcv, ibv, and mhv t , ~. in addition to crfk cells, a fetal cat whole fibroblast (fcwf) cell line will support the growth of fipv, as well as fecv, ccv, and tgev ( , t, and corn-journal of tissue culture methods vol. , no. , parison of antigenic and serologic relatedness of the enteric coronaviruses was done in this cell line ~ ). one to three cell culture passages of virulent fipv in crfk or fcwf cells do not destroy its virulence for susceptible cats. a vaccine produced with a crfk cell culture attenuated strain was unable to protect cats against the original virulent strain, although vaccinated cats do develop a neutralizing antibody response t ). using cdna clones of cell-culture-adapted fipv, the complete nucleotide sequence of the fipv peplomer protein has been determined ( }. the crfk and a- ( ) cell lines can be used for the isolation and propagation of ccv. this virus will grow to a titer of x pfu/ml or higher in either cell line. in addition, ccv can be adapted to grow in fcwf and st cells. however, neither ccv nor fipv will grow on primary isolation in st cells, and this property may allow one to biologically differentiate these antigenically related coronaviruses. a single passage of ccv in crfk cells does not decrease the infectivity for the natural host; however, prolonged serial passage in any cell line may result in an attenuated virus that grows well in cell culture but is avirutent for dogs. a cellculture-adapted ccv vaccine has been prepared and provides dogs vaccinated either i.m. or s.c. with neutralizing antibodies that are protective against a virulent ccv challenge ( ) . the spth cell line can be used for the primaryisolation of virus from clinical specimens and for the in vitro growth of hev ( ~. although virus grown in cell culture is still infective for pigs, it is less virulent than field strains (w. l. mengeling, ames, ia, personal communication). no vaccine is currently available for this virus. the st cell line has been used for more than yr to grow tgev ). this cell line can be used for primary isolation of virus from clinical specimens and in vitro growth of tgev it }. virus from clinically positive animals will usually have a titer near )< s pfu/ml, and after or passages in st cells it may have a titer of to x l pfu/ml. passage of the virus in serum-free media in -d-old st cells at an moi of . produces a maximum virus titer to h postinoculation. subpassage of the virus under the same conditions in st cells that are less than d old or more than d old will produce a lower virus titer t }. cell-culture-adapted tgev is still infective for pigs after passages in st cells, but pig virulence is usually reduced after only l to passages in st cells. three tissue-cnlture-adapted, modified-live tgev vaccine strains are sold for use against virulent tgev in pregnant swine. two of the vaccine viruses are grown in porcine cell lines, and the source of the third vaccine virus is unknown. although the safety and ability of these vaccines to elicit virusneutralizing antibodies in pregnant swine has been proven, their efficacy has been questioned , ) . the complete nucleotide sequence of the three major structural proteins of tgev has been determined on cdna clones of attenuated virus grown in st cell culture ( , , , l o isolation of transmissible enteritis agent of turkeys in avian embryos propagation of hemagglutinating encephalomyelitis virus in porcine cell culture. zbi coronavirus and gastroenteritis in foals characterization of the virus of sialodaeryoadenitis of rats: a member of the coronavirus group establishment of a canine cell line: derivation, characterization and viral spectrum vaccination against transmissible gastroenteritis itge}: pros and cons the complete nueleotide sequence of avian infectious bronchitis virus: analysis of the polymerase-coding region structural polypeptides of coronavirus ibv development, characterization and viral susceptibility of a feline {fells catus~ renal cell line crfk) comparison of the morphology of three coronaviruses evaluating a canine coronavirus vaccine through antigen extinction and challenge studies local and systemic cell mediated immunity against transmissible gastroenteritis; an intestinal infection of swine characterization of a new coronavirus-like agent isolated from parrots replication of coronaviruses the nucleotide sequence of the peplomer gene of porcine transmissible gastroenteritis virus {tgev); comparison with the sequence of the peplomer protein of feline infectious peritonitis virus {fipv) critical epitopes in transmissible gastroenteritis virus neutralization sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsm protein nucleotide sequence of the porcine transmissible gastroenterltis coronavirus matrix protein gene isolation of transmissible gastroenteritis virus from pharyngeal swabs obtained from sows at slaughter overview: molecular biology of coronaviruses the genome of human coronavirus strain e studies on transmissible gastroenteritis of swine. ii. selected characteristics of a cytopathogenic virus common to five isolates from transmissible gastroenteritis neonatal ca[f diarrhoea; propagation, attenuation, and characterization of a coronavirus-like agent hemadsorption plaque assay for hemagglutinating encephalomyelitis virus characteristics of a coronavirus tstrain n} of pigs comparison of tgev vaccines an eight-year study of the viral agents of acute gastroenteritis in humans: ultrastructural observations and seasonal distribution with a major emphasis on coronavirus attempted immunization of cats against feline infectious peritonitis; using avirulent live virus or sublethal amounts of virulent virus infection studies in kittens using feline infectious peritonitis virus propagated in cell culture antigenic relationship of the feline infectious peritonitis virus to eoronaviruses of other species isolation of a porcine respiratory, non-enteric coronavirus related to transmissible gastroenteritis the predicted primary structure o| the peplomer protein e of the porcine coronavirus transmissible gastroenteritis virus comprehensive virology, rot. plaque assay and improved yield of human coronavirus in a human rhabdosarcoma cell line the structure and replication of coronaviruses the biology of coronaviruses relatedness of rabbit coronavirus to other coronaviruses transmissible gastroenteritis {tgei o! swine: effect of age of swine testes cell culture monolayers on plaque assays of tge virus enhancement of plaque formation and cell fusion of an enteric pathogenic coronavirus by trypsin treatment the molecular biology of eoronaviruses the biology and pathogenesis of coronaviruees small plaque variant transmissible gastroenteritis virus studies of enteric coronaviruses in a feline cell line key: cord- -yfjme q authors: magtoto, ronaldo; poonsuk, korakrit; baum, david; zhang, jianqiang; chen, qi; ji, ju; piñeyro, pablo; zimmerman, jeffrey; giménez-lirola, luis g. title: evaluation of the serologic cross-reactivity between transmissible gastroenteritis coronavirus and porcine respiratory coronavirus using commercial blocking enzyme-linked immunosorbent assay kits date: - - journal: msphere doi: . /msphere. - sha: doc_id: cord_uid: yfjme q this study compared the performances of three commercial transmissible gastroenteritis virus/porcine respiratory coronavirus (tgev/prcv) blocking enzyme-linked immunosorbent assays (elisas) using serum samples (n = ) collected over a -day observation period from pigs inoculated with tgev strain purdue (n = ), tgev strain miller (n = ), prcv (n = ), or with virus-free culture medium (n = ). elisa results were evaluated both with “suspect” results interpreted as positive and then as negative. all commercial kits showed excellent diagnostic specificity ( to %) when testing samples from pigs inoculated with virus-free culture medium. however, analyses revealed differences between the kits in diagnostic sensitivity (percent tgev- or prcv-seropositive pigs), and all kits showed significant (p < . ) cross-reactivity between tgev and prcv serum antibodies, particularly during early stages of the infections. serologic cross-reactivity between tgev and prcv seemed to be tgev strain dependent, with a higher percentage of prcv-false-positive results for pigs inoculated with tgev purdue than for tgev miller. moreover, the overall proportion of false positives was higher when suspect results were interpreted as positive, regardless of the elisa kit evaluated. importance current measures to prevent tgev from entering a naive herd include quarantine and testing for tgev-seronegative animals. however, tgev serology is complicated due to the cross-reactivity with prcv, which circulates subclinically in most swine herds worldwide. conventional serological tests cannot distinguish between tgev and prcv antibodies; however, blocking elisas using antigen containing a large deletion in the amino terminus of the prcv s protein permit differentiation of prcv and tgev antibodies. several commercial tgev/prcv blocking elisas are available, but performance comparisons have not been reported in recent research. this study demonstrates that the serologic cross-reactivity between tgev and prcv affects the accuracy of commercial blocking elisas. individual test results must be interpreted with caution, particularly in the event of suspect results. therefore, commercial tgev/prcv blocking elisas should only be applied on a herd basis. tality in piglets in tgev/prcv-naive herds. the virus was first described by doyle and hutchings in in the united states and subsequently reported worldwide ( ) ( ) ( ) . porcine respiratory coronavirus is a naturally occurring spike gene deletion ( to kda) mutant of tgev first isolated in belgium in ( ) . it infects the upper respiratory tract, tonsils, or lungs, with limited intestinal replication ( , ) . porcine respiratory coronavirus itself does not appear to be an important primary pathogen, with the exception of its contribution to the porcine respiratory disease complex ( ) . tgev and prcv share biological and molecular features but differ in their epidemiology, clinical presentation, and pathogenesis. real-time reverse transcription-pcr (rrt-pcr) and multiplex microarray hybridization using primers targeting the = region of the s gene spanning the deletion region in pcrv strain are commonly used for the diagnosis of tgev and differentiation of tgev and prcv ( ) ( ) ( ) ( ) . serum antibodies provide serological evidence of tgev or prcv infection, but prcv-infected pigs produce antibodies that cross-react and cross-neutralize tgev, i.e., conventional serological tests cannot differentiate between tgev-and prcv-infected animals. this presents a complication for tgev seroprevalence studies and serological surveys of sows or slaughterhouse swine tested for international trade ( ) . to address the issue of cross-reactivity, monoclonal antibodies targeting antigenic regions of tgev that have been deleted from the prcv s protein ( ) ( ) ( ) ( ) ( ) ( ) ( ) have been used to develop blocking enzyme-linked immunosorbent assays (elisas) for tgev/ prcv differential serodiagnosis ( ) ( ) ( ) ( ) . several commercial tgev/prcv blocking elisas are available, but comparative test performances have not been reported in recent publications. in this study, the diagnostic test performances of three commercial tgev/prcv blocking elisa kits were evaluated using serum samples of precisely known porcine coronavirus immune status. clinical observations and virus shedding. mild watery diarrhea was observed in pigs inoculated with tgev miller strain between to days postinoculation (dpi). no clinical signs were observed in pigs from the negative-control, tgev purdue, and prcv-inoculated groups throughout the experiment. all animals survived until the end of the study ( dpi). all fecal samples collected from pigs in the tgev strain purdue, tgev strain miller, prcv-inoculated, and negative-control groups were tested by rrt-pcr and found to be tgev and prcv negative prior to the inoculations. likewise, all oral fluid samples, with the exception of one false-positive (threshold cycle [c t ], . ) result in the tgev purdue group, were negative before inoculation. the detection of tgev (s and n genes) and prcv (n gene) in pen-based feces and oral fluids by rrt-pcr is shown in fig. . transmissible gastroenteritis virus was detected in feces between and dpi by rrt-pcr in pigs inoculated with tgev strain miller ( fig. a and c) . no significant fecal shedding was detected in pigs inoculated with tgev strain purdue or prcv throughout the study ( fig. a and c). viral shedding was specifically detected by rrt-pcr in oral fluid samples collected from pigs inoculated with tgev strain purdue ( to dpi), tgev strain miller ( to dpi), and prcv ( to dpi) ( fig. b and d) . tgev antibody response over the course of the experimental inoculation. serum samples collected from all groups of pigs prior to inoculation were antibody negative for porcine coronaviruses (tgev, prcv, porcine epidemic diarrhea virus [pedv] , and porcine delta coronavirus [pdcov]). all pigs in the negative-control group remained tgev and prcv seronegative throughout the monitoring period when tested with any of the three tgev/prcv differential blocking elisa kits evaluated in this study ( the percentages of tgev antibody-positive serum samples reported by the three commercial elisa kits evaluated over the -day study period for pigs inoculated with tgev strains purdue and miller are presented in fig. a to f, respectively. suspect results are presented as positives or negatives. the first tgev-specific antibody detection was reported between and dpi in both tgev-inoculated groups (strains purdue and miller). the number of tgev antibody-positive pigs detected increased through the study, regardless of the elisa kit used. for the tgev purdue inoculation group, no significant differences (p Ͼ . ) were found in the percentages of tgev-seropositive pigs reported by the three elisas regardless of the time postinoculation and interpretation of suspect results. in contrast, for the tgev miller inoculation group, we found that a significantly higher (p Ͻ . ) percentage of tgev-seropositive animals was detected by the swinecheck tgev/prcv recombinant elisa than by both the ingezim corona diferencial elisa (dpi ) and svanovir tgev/prcv-ab elisa ( , , and dpi) only when suspect results were interpreted as negative. a nonspecific tgev antibody response to prcv-inoculated pigs was reported by the three elisa kits between and dpi ( fig. g to i). no significant differences in the percentages of tgev false positives were found between elisas over the monitoring period when tgev suspect results were interpreted as negative (p Ͼ . ). however, the overall proportion of false positives was higher when suspect results were interpreted as positive, regardless of the elisa kit evaluated. moreover, the percentage of tgev-false-positive results reported by the svanovir tgev/prcv-ab elisa was significantly greater (p Ͻ . ) than those obtained with the swinecheck tgev/prcv recombinant elisa ( , , and dpi) and ingezim corona diferencial elisa ( to dpi). prcv antibody response over the course of the experimental inoculation. with the exception of one false-positive result reported ( dpi) by the ingezim corona diferencial elisa kit, the negative-control group remained seronegative for prcv by the three commercial elisas throughout the study. on prcv-inoculated animals, an early prcv-specific antibody response was first detected at to dpi by the three commercial elisas and lasted through dpi ( fig. g to i ). an analysis of the proportion of prcv-seropositive animals showed significant differences between elisa kits over time following prcv inoculation, regardless of the interpretation of suspect results (p Ͻ . ). the percentage of prcvseropositive animals detected by the svanovir tgev/prcv-ab elisa was significantly lower than those with both the swinecheck tgev/prcv recombinant elisa (dpi to ) and ingezim corona diferencial elisa (dpi to and ) (p Ͻ . ). no differences were found between the swinecheck tgev/prcv recombinant and ingezim corona diferencial elisas. on tgev-inoculated animals, a nonspecific prcv serum antibody response was detected by the three commercial elisas. this serologic cross-reactivity seemed to be tgev strain dependent, with a higher percentage of prcv-false-positive results in the tgev strain purdue-inoculated group ( fig. a to c) than in the tgev strain miller group ( fig. d to f). the cross-reactivity appeared more marked at early stages postexposure ( to dpi). no differences were found between elisa kits over the course of the study, except at dpi when the proportion of prcv-false-positive animals detected by the svanovir tgev/prcv-ab elisa was significantly greater (p Ͻ . ) than that by the ingezim corona diferencial elisa regardless of the interpretation of suspect results. comparative diagnostic performance of the tgev/prcv differential elisas. the analytical specificity and diagnostic sensitivity and specificity of the three commercial differential tgev/prcv elisa kits evaluated in this study are presented in table . diagnostic parameters are presented relative to the interpretation of suspect results as positive or negative. overall, the diagnostic sensitivity for the tgev miller inoculation group was higher than for the tgev purdue group, independent of the commercial elisa kit evaluated. the swinecheck tgev/prcv recombinant elisa kit showed the highest diagnostic sensitivity for antibody detection in pigs inoculated with tgev strain miller ( %), regardless of the interpretation of suspect results. the diagnostic sensitivity for the tgev strain purdue-inoculated group was higher with the svanovir tgev/prcv-ab elisa kit ( %) when suspect results were considered positive, while it was slightly higher with the ingezim corona diferencial elisa kit ( %) when suspect results were interpreted as negative. the diagnostic sensitivities for prcv antibody detection obtained with both swinecheck tgev/prcv recombinant elisa ( %) and ingezim corona diferencial elisa ( to %) were greater than that of the svanovir tgev/prcv-ab elisa kit ( %). with the exception of the ingezim corona diferencial elisa, the diagnostic sensitivity calculated for each elisa kit was not affected by the interpretation of suspect results (positive versus negative). the three commercial elisas showed % diagnostic specificity for both tgev and prcv detection, with the exception for one prcv-false-positive result reported by the ingezim corona diferencial elisa, which resulted in a diagnostic specificity of . % for prcv detection. the diagnostic specificity of any of the evaluated elisa kits was unaffected by the interpretation of suspect results. the ingezim corona diferencial elisa kit showed the highest analytical specificity (less cross-reactivity) for tgev-specific antibody detection ( . %). for all three commercial elisas, we found higher cross-reactivity to prcv within the tgev purdue inoculation group than in the tgev miller group. the highest analytical specificity for prcv was obtained with the ingezim corona diferencial elisa ( %) when suspect results were interpreted as positive or with the svanovir tgev/prcv-ab elisa kit ( %) when suspect results were interpreted as negative; however, the svanovir tgev/ prcv-ab elisa kit showed the lowest analytical specificity ( %) of the kits when suspect results were interpreted as positive. the greatest risk of introducing tgev is through the importation of pigs from endemically infected countries. seronegative pigs of all ages are susceptible to infection with tgev ( , , ) , with the route of infection typically being oral or oral-nasal ( , ) . to prevent tgev from entering a naive herd, it is important to introduce only animals from tgev-free and serologically negative herds, along with disciplined biosecurity. therefore, quarantine and testing for tgev-seronegative animals are requirements for export/import into a herd. moreover, the detection of tgev antibodies, particularly in reproductive animals, can assist in diagnosis and control of the disease. however, tgev serology is complicated by cross-reactivity with prcv ( ), an s protein deletion mutant of tgev with altered tissue tropism (nonenteropathogenic but respiratory) that circulates subclinically in most swine herds worldwide ( , , ) . this cross-reactivity may explain why a region endemic for prcv has less tgev-associated disease ( ) . porcine respiratory coronavirus does not cause significant losses in swine except for its contribution to the porcine respiratory disease complex. historically, prcv diagnosis was based on recognition of respiratory disease in growing pigs that have high antibody titers to tgev but have had no clinical enteric disease or lesions of atrophic enteritis. conventional serological tests, such as virus neutralization, indirect fluorescent antibody, agar-gel precipitation, indirect immunoperoxidase, radioimmunoprecipitation, and some elisas based on polyclonal tgev antibodies, cannot be used for differentiation between tgev and prcv, because the two viruses share antigenic determinants located on the spike (s), membrane (m), nucleoprotein (n), and envelope (e) structural proteins ( , ) . the exception is a large deletion ( amino acids in length) in the amino terminus of the prcv s protein that is responsible for the loss of hemagglutination activity ( ) . this deletion provided the opportunity to develop blocking enzyme-linked immunosorbent assays for differentiation of prcv and tgev infections and laid the basis for the commercial tgev/prcv differential elisas that are currently available ( ) ( ) ( ) ( ) . few field reports have described the use of tgev/prcv blocking elisas to differentiate antibodies to tgev and prcv and their utility at the herd level ( , , ( , , ) . specimens primarily used for tgev virus detection include feces and intestinal contents, and those for prcv detection include nasal swabs or lung homogenates. in this study, the verification and monitoring of the tgev or prcv viral shedding status were performed using rrt-pcr testing of pen-based feces and oral fluids. this process provided further information on the dynamics of viral shedding in feces versus oral fluids. viral shedding was detected in both feces and oral fluids. however, for pigs inoculated with prcv and, interestingly, for tgev purdue, virus shedding was only detected in oral fluids. this could be related to differences in pathogenicity and tissue tropism, which were beyond the scope of this study. these results indicated that oral fluids may replace feces as a more suitable specimen for direct detection and surveillance of tgev/prcv by rrt-pcr. overall, we observed differences in the test performances of the three commercial elisa kits evaluated in this study. however, the differences were more marked for the svanovir tgev/prcv-ab elisa kit, compared to either the swinecheck tgev/prcv recombinant elisa or the ingezim corona diferencial elisa, for which test performance was comparable. this could be due to differences/similarities in assay design. for instance, both the swinecheck tgev/prcv recombinant elisa and the ingezim corona diferencial elisa use a tgev recombinant s protein antigen on a plate, while the svanovir tgev/prcv-ab elisa claims to use noninfectious tgev antigen of an unknown source, which could detect antibodies to a variety of viral proteins. the way the antigen is presented may also affect assay performance. the antigen can either be bound directly to the plate wells (as in the swinecheck tgev/prcv recombinant elisa and svanovir tgev/prcv-ab elisa) or may be captured by antigen-specific antibodies immobilized on the plate (as in the ingezim corona diferencial elisa). in addition, the svanovir tgev/prcv-ab elisa is the only kit using unlabeled tgev and tgev/prcv mouse monoclonal antibodies (mabs), which requires an additional incubation step with anti-mouse horseradish peroxidase (hrp)-conjugated secondary antibody. undisclosed differences in buffer composition could also be involved in the differences in assay performance among the kits. all three elisa kits showed higher diagnostic sensitivity in the detection of anti-tgev antibodies in pigs inoculated with tgev strain miller ( . to . %) than in pigs inoculated with tgev strain purdue ( . to . %), regardless of the kit used. the differences in diagnostic sensitivity among tgev-inoculated groups could be due to strain-related differences in virulence, which may have impacted the magnitude of the immune response. indeed, in this study, pigs inoculated with tgev strain miller showed mild watery diarrhea between and dpi, while no clinical signs or viral shedding in feces were observed in the pigs inoculated with tgev purdue. the diagnostic specificity of the three commercial elisa kits was evaluated on pigs of precisely known negative porcine coronavirus immune status (i.e., the porcine coronavirus negative-control group) to rule out potential cross-reactivity with other porcine coronaviruses (i.e., pigs virologically and serologically negative for pedv, pdcov, porcine hemagglutinating encephalomyelitis virus [phev], tgev, and prcv). all three kits evaluated in this study showed excellent diagnostic specificity, ranging from % (svanovir tgev/prcv-ab elisa) to % (swinecheck tgev/prcv recombinant elisa and ingezim corona diferencial elisa). these results were consistent with previous reports in the field where a tgev/prcv blocking elisa (i.e., swinecheck and biovet) showed a diagnostic specificity of % in wild boar populations historically free from coronavirus disease ( ) . with the final goal of maximizing both diagnostic sensitivity and specificity (but understanding that there is a balance between the two parameters), diagnostic specificity is of paramount importance during tgev/prcv screening due to the impact of false-positive results on global pig trade operations. the false-positive rate of any diagnostic test is a function of the specificity of the test and the prevalence of the disease. the assessment of the selectivity of the antigen-antibody response (analytical specificity) was conducted on each of the three commercial blocking elisas using a panel of heterologous monotypic known-status-positive sera generated under experimental conditions. specifically, in this study, the analysis of the analytical specificity was circumscribed to the cross-reactivity between tgev (strains purdue and miller) and prcv. however, the potential cross-reactivity between tgev/prcv against other swine coronaviruses has previously been reported ( ) . a test was considered analytically specific for tgev or prcv when it did not react against heterologous positive sera. as with the diagnostic sensitivity, the overall analytical specificity varied among and across kits and groups of inoculation, even with intrakit variations depending upon the interpretation of suspect results. serological cross-reactivity between tgev and prcv was previously reported by use of an immunoblotting assay based on tgev/prcv structural proteins (s, m, and n) and polyclonal antisera ( ) . this study further confirms that the serological cross-reactivity between tgev and prcv is significant even when using tgev/prcv differential blocking elisas. overall, in this study, we observed a poor analytical specificity (i.e., high cross-reactivity) for prcv antibody detection in pigs exposed to tgev strain purdue ( to %), with no significant differences among kits. however, with the exception of the svanovir tgev/prcv-ab elisa ( to %), we found an acceptable analytical specificity for prcv antibody detection in pigs exposed to tgev miller using the swinecheck tgev/prcv recombinant elisa ( to %) and the ingezim corona diferencial elisa ( %). current tgev/prcv differential immunoassays are based on a blocking elisa format, which is inherently more specific than indirect elisas. in the blocking elisa format, the degree to which specific antibodies in the test serum sample prevent binding of an agent-specific mab is measured. therefore, the higher the levels of specific antibodies are in a serum sample, the lower the likelihood for cross-reactivity. in fact, the overall higher tgev antibody detection rate observed within the group inoculated with tgev strain miller correlates with the lower cross-reactivity against prcv. that would also explain the overall higher rate of serologic cross-reactivity (regardless of the elisa kit used) reported during the first few weeks postinoculation, when specific antibody levels are still low. previous reports indicated that the accuracy of the commercial tgev/prcv blocking elisas for differentiating between u.s. tgev and prcv strains was low ( , , ) . individual test results must be interpreted with caution, particularly in the event of "suspect" results. we have demonstrated that interpretation of suspect results can impact significantly the diagnostic performance of any of the commercial kits evaluated in this study, with differences in their robustness in response to changes in the interpretation of suspect results. while analytical specificity improved overall when suspect results were interpreted as negative, this often comes at the cost of diagnostic sensitivity. in the event of suspect, undetermined, or unexpected positive results, it is recommended that serology testing on paired serum samples be conducted, in which a second sample should be drawn to days after the first sample collection. paired serum samples should be tested together to maximize the diagnostic value of test results. moreover, false-positive results reported at early stages postexposure (when animals are actively shedding virus) could be confirmed by rrt-pcr on oral fluid specimens, as evidenced in this study. the presence of tgev remains a barrier to international livestock trade ( ) . the blocking elisa format alone was determined to be useful in large swine populations for the detection of tgev-infected herds. moreover, the ability to specifically detect prcv antibodies minimized the probability of false-positive tgev results and subsequent exclusion of those herds for trading. however, this study demonstrates that the serologic cross-reactivity between tgev and prcv at the individual pig level affects the accuracy of commercial blocking elisas. therefore, it is important to remember that the tgev/prcv blocking elisas should only be applied on a herd basis ( , , , ) . experimental design. "known-status" serum samples (n ϭ ) collected from pigs experimentally inoculated with tgev purdue (n ϭ ), tgev miller (n ϭ ), prcv (n ϭ ), or with culture medium (minimum essential medium [mem] life technologies, carlsbad, ca) (negative control; n ϭ ) were used to evaluate the diagnostic performance (diagnostic sensitivity and specificity) and antibody crossreactivity (analytical specificity) of the following three commercial tgev/prcv blocking elisas: . at % confluence of the cell monolayer, the maintenance medium was decanted, and the monolayer was washed twice with maintenance medium. thereafter, each flask of cells was inoculated with l of each virus mixed with postinoculation medium, i.e., mem supplemented with . % tryptose phosphate broth (sigma-aldrich) and . % yeast extract (sigma-aldrich). the cells were incubated at °c with % co for h to allow virus adsorption. after incubation, ml of postinoculation medium was added to each flask without removing viral inoculum. the flasks were incubated at °c with % co and subjected to one freeze-thaw cycle at Ϫ °c when % cytopathic effect (cpe) was observed. cell debris was removed by centrifugation at , ϫ g for min at °c, and the virus content was harvested by collecting the supernatant. virus titration was performed on confluent st cell monolayers grown in -well plates (costar; corning) using a method described elsewhere ( ) . animals. the animal study was conducted at the iowa state university (isu) livestock infection disease isolation facility (lidif) with the approval of the iowa state university office for responsible research. forty-eight -week-old conventional pigs were acquired from a commercial wean-to-finish farm with no history of porcine coronavirus infection. during the herd prescreening process and prior to the beginning of the study, fecal and nasal swabs from all animals were tested for tgev, prcv, porcine hemagglutinating encephalomyelitis virus (phev), porcine epidemic diarrhea virus (pedv), and porcine delta coronavirus (pdcov) by real-time reverse transcription-pcr (rrt-pcr) assays. serum samples were tested for all of the pathogens listed above using antibody-based methods, as described elsewhere ( ) ( ) ( ) . upon arrival, all animals were ear-tagged and randomly assigned to one of four inoculation groups (n ϭ per group in the tgev miller, tgev purdue, prcv, and negative-control groups). pigs within each group were housed in pens of pigs in the same room. each pen was equipped with nipple drinkers, and pigs were fed twice daily with an antibiotic-free commercial diet (heartland co-op, west des moines, ia, usa). details regarding viral dose and route of inoculation for each group are presented in table . the infectious dose used for each virus was previously determined in a pilot study (data not shown) with the objective of stimulating a humoral response effectively. pigs were evaluated clinically twice daily throughout the study. the tgev and prcv infectious status of every pig was established and monitored throughout the study by rrt-pcr tests on fecal and oral fluid samples and elisa-based tests on serum samples. sample collection. blood samples were collected on Ϫ , , , , , , , , , , and days postinoculation (dpi) from the jugular vein or cranial vena cava using a single-use blood collection system (becton dickinson, franklin lakes, nj) and serum separation tubes (kendall, mansfield, ma). serum was separated by centrifugation at , ϫ g for min, aliquoted into -ml cryogenic tubes (greiner bio-one gmbh, frickenhausen, germany), and stored at Ϫ °c until use. floor fecal samples were collected daily from each pen ( pigs per pen) within each inoculation group ( pens per group) from Ϫ to dpi. approximately ml of feces was placed in a -ml cryogenic tube (bd falcon). fecal samples ( l) from each group on the same day postinoculation were pooled into a -ml cryogenic tube (bd falcon) at the end of the study for rrt-pcr testing. oral fluid samples were collected from each pen within each inoculation group twice a day ( : a.m. and : p.m.) from Ϫ to dpi. in brief, -strand . -cm % cotton rope (web rigging supply, inc., carrollton, ga) was hung from a bracket fixed to one side of each pen for min, during which time the pigs chewed on and interacted with the rope. after min, the wet end of the rope was severed, sealed in a plastic bag, and then passed through a clothes wringer (dyna-jet, overland park, ks). the oral fluid accumulated in the bottom of the bag was decanted into -ml conical tubes (corning), aliquoted into -ml cryogenic tubes (greiner bio-one gmbh), and stored at Ϫ °c. real-time reverse transcription-pcr. feces and oral fluid samples were submitted to the isu veterinary diagnostic library (vdl) for porcine coronavirus screening by rrt-pcr. transmissible gastroenteritis virus-specific spike (s) and tgev/prcv-specific nucleocapsid (n) genes were targeted and amplified by dually labeled probe-based rrt-pcr. in brief, sample rna was extracted and eluted using the ambion magmax viral rna isolation kit (life technologies) and a kingfisher magnetic particle processor (thermo fisher scientific) following the procedures provided by the manufacturers. the tgev s gene and the tgev/prcv n gene-based rrt-pcr were modified from previous procedures ( ) and performed routinely at the isu-vdl using the taqman fast -step mastermix (thermo fisher scientific). the rt-pcrs were conducted on an abi fast instrument (life technologies) as follows: °c for min, °c for s, °c for s ( cycles), and °c for s. the rrt-pcr results were analyzed using an automatic baseline, with a threshold value of . . a quantification cycle (c t ) value of Ͻ was considered positive for both s and n gene-based rrt-pcr. samples were considered positive for tgev when both s and n genes were rrt-pcr positive. for prcv, samples were considered positive when the s gene rrt-pcr was negative and the n gene rrt-pcr was positive. transmissible gastroenteritis virus and prcv c t data were reported as "adjusted c t ," calculated as shown in the following equation: adjusted c t ϭ ͑ cutoff c t value Ϫ sample c t value ͒ tgev/prcv differential blocking elisas. serum samples were tested for tgev-and prcv-specific antibodies using the following three commercially available tgev/prcv differential blocking elisa kits: (i) swinecheck tgev/prcv recombinant (biovet, canada), (ii) ingezim corona diferencial (ingenasa, spain), and (iii) svanovir tgev/prcv-ab (svanova, sweden). all assays were performed according to the manufacturers' instructions. the pig serum samples and controls were first added to the tgev antigencoated wells for the first incubation step. in the ingezim corona diferencial elisa, plate wells are coated with a recombinant tgev s protein which is captured by a specific monoclonal antibody (mab). the swinecheck tgev/prcv recombinant elisa is also based on a recombinant tgev s protein but directly coated on plate wells, while the svanovir tgev/prcv-ab elisa uses noninfectious tgev antigen (source unknown) as a coating antigen. if anti-tgev or anti-prcv antibodies are present in the test sample, they will bind to the viral (tgev) antigen in the wells and block the antigenic sites. in contrast, if anti-tgev or anti-prcv antibodies are absent in the test sample, these sites will remain free. then, after a washing step to eliminate unbound antibodies, a horseradish peroxidase (hrp)-conjugated mouse anti-tgev mab targeting the n-terminal region of the s glycoprotein that is deleted in prcv, or an anti-tgev/prcv mab that binds to conserved regions of both tgev and prcv, is added into the first well (odd column) or second well (even column), respectively, and attaches to specific free sites of the virus. thus, when antibodies in the test sample occupy the binding sites on the antigen, the binding of the conjugated mabs is blocked. if anti-tgev or anti-prcv antibodies are absent in the test sample, these sites will remain free. the amount of antibody in the test sample that is bound to the tgev antigen is inversely proportional to the intensity of the color. antibody elisa results were expressed as the percentage of inhibition (swinecheck tgev/prcv and svanovir tgev/prcv-ab) or optical density (ingezim corona diferencial) and interpreted (positive, negative, or suspect/inconclusive) for tgev and prcv according to the manufacturers' instructions. the diagnostic sensitivity, diagnostic specificity, and analytical specificity of the three commercial elisas were calculated based on the true status of the samples compared to the specific detection of tgev (miller and/or purdue) and/or prcv antibodies. specifically, porcine coronavirus-negative serum samples (n ϭ ) were used to estimate diagnostic specificity for the three elisa kits. serum samples positive for tgev (purdue and miller strains) (n ϭ ) collected between and dpi and prcv-positive samples (n ϭ ) collected between and dpi were used to calculate the time of detection, antibody detection over time, and the overall diagnostic sensitivity of the three elisa kits for tgev and prcv ab detection, respectively. a transmissible gastroenteritis in pigs molecular basis of transmissible gastroenteritis virus epidemiology porcine coronaviruses isolation of a porcine respiratory, non-enteric coronavirus related to transmissible gastroenteritis porcine respiratory coronavirus related to transmissible gastroenteritis virus polymicrobial respiratory disease in pigs respiratory and fecal shedding of porcine respiratory coronavirus (prcv) in sentinel weaned pigs and sequence of the partial s-gene of the prcv isolates development of a reverse transcription-nested polymerase chain reaction assay for differential diagnosis of transmissible gastroenteritis virus and porcine respiratory coronavirus from feces and nasal swabs of infected pigs molecular characterization and pathogenesis of transmissible gastroenteritis coronavirus (tgev) and 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hybridization technique for the detection of swine enteric and respiratory coronaviruses, transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus (prcv), in formalin-fixed paraffin-embedded tissues prevalence of and risk factors associated with viral and bacterial pathogens in farmed european wild boar antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains simulation of the economic impact of transmissible gastroenteritis on commercial pig production in australia a simple method of estimating fifty per cent endpoints pathogenesis of porcine epidemic diarrhea virus isolate (us/iowa/ / ) in -week-old weaned pigs reactivity of porcine epidemic diarrhea virus structural proteins to antibodies against porcine enteric coronaviruses: diagnostic implications multiplex real-time rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus this work was funded in part through the iowa pork producers association through the national pork board (pork checkoff funds, grant - ; des moines, ia) and the iowa state university veterinary diagnostic laboratory (ames, ia). data analysis. analyses were conducted using commercial statistical software (sas version . . ; microsoft corporation, usa). the percentage of results for tgev/prcv elisa were interpreted (positive, negative, or suspect) according to the manufacturer's recommendations. the percentage of seropositive animals per day postinoculation detected by each kit were analyzed in two ways, by considering suspect results to be positive or by considering suspect results to be negative. significant differences (p Ͻ . ) in elisa-positive rates relative to tgev (purdue or miller strains) and prcv were calculated using pearson's chi-square test or fisher's exact test. the diagnostic sensitivity and specificity of the three commercial elisas were evaluated based on experimental serum samples of precisely known porcine coronavirus immune status (i.e., samples collected on Ͻ dpi were negative, and serum samples collected on Ͼ dpi were positive). analytical specificity (bidirectional cross-reactivity) between tgev versus prcv was evaluated using samples (n ϭ ) collected between and dpi from animals inoculated with tgev strain purdue (n ϭ ), tgev strain miller (n ϭ ), and prcv (n ϭ ) in each given case. figures were created using commercial graphics software (sigmaplot version . ; systat software, inc.). the funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication.we declare no conflicting interests with respect to authorship or the publication of this article. key: cord- -nt jcjm authors: garwes, d.j.; lucas, m.h.; higgins, d.a.; pike, b.v.; cartwright, s.f. title: antigenicity of structural components from porcine transmissible gastroenteritis virus date: - - journal: vet microbiol doi: . / - ( ) - sha: doc_id: cord_uid: nt jcjm pregnant sows were inoculated with inactivated transmissible gastroenteritis virus and with preparations of virus surface projections and subviral particles derived by detergent treatment of the virus. neutralising antibody was demonstrated in serum and colostrum from animals that received whole virus or preparations of surface projections whereas subviral particles failed to stimulate neutralising antibody formation. similar results were obtained with serum from rabbits inoculated with whole virus and structural components. all three preparations stimulated the formation of agglutinating antibodies, as demonstrated by sedimentation analysis and filtration studies with radiolabelled virus. the immunoglobulin classes responsible for neutralising antibody activity in sows inoculated by the intramammary route were examined. in each case where the immunoglobulin class was determined, igg was found. one sow that received surface projections also had iga with neutralising activity in her colostrum. in contrast, infection of sows with live whole virus resulted in neutralising antibody of the igg, igm and iga classes. infection of pigs with the coronavirus causing transmissible gastroenteritis (tgev) results in disease that is most severe, frequently fatal, in young animals and of less severity in animals over weeks old. following recovery of the animals from infection, neutralising antibody can be detected in serum and secretions; transfer of this antibody, predominantly iga, in the colostrum and milk from a convalescent dam to her offspring confers reasonable protection against disease in her piglets (bohl et al., b; saif et al., ; stone et al., ) . parenteral administration of inactivated virus stimulates circulating antibody of the igg class saif et al., ; lucas et al., ) but, as little or no specific iga antibody is formed, the low level of igg in the milk affords little protection to newborn piglets during the period that they are most susceptible. work is in progress to investigate how an iga response can be stimulated by inactivated virus but more information is required about the nature of the viral antigen. we have previously demonstrated that inactivated purified tgev is capable of inducing a primary neutralising antibody response in pigs , suggesting that a virus structural element is the antigen responsible for stimulating the neutralising antibody. there is evidence, however, that at least one soluble component from tgev-infected swine testis cells can stimulate neutralising antibody production . following development of techniques to separate and purify the surface profections (sp) from the internal proteins of a subviral particle (svp) (garwes et al., ) it has been possible to determine which of these components are involved in the neutralising antibody response. this report describes the immune response in swine and rabbits inoculated with inactivated tgev and purified subviral components. antigen preparation the fs / isolate of tgev was used after six passes through gnotobiotic piglets aged -- days. a % (w/v) suspension of infected small intestine was clarified by centrifugation at × g for min and was then inoculated onto secondary pig thyroid (apt/ ) cells as monolayer cultures in medium containing galactose . virus was harvested following freezing and thawing the cultures at h after infection and was purified as previously described (garwes and pocock, b) . isolation of purified sp and svp antigen preparations was achieved by differential centrifugation after treatment of purified virus with % nonidet-p (garwes et al., ) . each antigen preparation (whole virus, sp and/or svp) was diluted : with growth medium, formaldehyde was added to . % and the preparations were held at °c for h. following this treatment, no infectivity could be detected in any of the samples. the preparations were stored at °c until used. each preparation of antigen for immunisation studies was derived from -- × pfu (sows) or × l s pfu (rabbits) of tgev. virus used for radio-immune assay was prepared from apt/ cells infected with tgev fs / passaged times through primary pig kidney cells. the medium contained h-uridine (specific activity ci/ mmole; the radiochemical centre, amersham) at pci/ml and the virus was harvested h after infection and purified as described (garwes and pocock, b) . infectivity was titrated by plaque assay. virus neutralisation test sera, colostra and milk samples were tested as previously described (cartwright, ) . purified tgev that had been grown in the presence of h-uridine was mixed with equal volumes of sera or % bovine serum albumin, and incubated at °c for i h. a sample was removed for titration of infectivity and the remainder was layered on to a linear -- % (w/w) sucrose gradient and centrifuged at × g for h. the gradient was fractionated by siphon into aliquots, material that had sedimented to the bottom of the tube was resuspended in distilled water and a sample was removed from each fraction for determination of radioactivity using a toluene-triton x based scintillant. measurement of virus agglutination by antibody was accomplished by incubating # samples of h-uridine labelled tgev with equal volumes of test sera diluted in isotonic saline at °c for h. to each reaction mixture was then added ul of rabbit anti-porcine igg serum (miles laboratories ltd., stoke poges) and incubation continued for h at °c followed by storage overnight at ° c. the samples were passed through cellulose acetate membrane filters of nm mean pore diameter (type eg, millipore (u.k.) ltd., london), the filters were washed with four changes of . ml distilled water and their radioactivity was determined by liquid scintillation. earlier attempts to use membrane filters composed of mixed esters of cellulose (mf filters, millipore (u.k.) ltd., london) were not successful as purified tgev is bound irreversibly to them (unpublished observations). this problem was overcome by using the cellulose acetate membranes to which the virus did not bind. solutions of serum igm and igg and colostral iga were obtained by gel filtration and ion-exchange chromatography (porter, ; vaerman and heremans, ) . antisera were raised in rabbits by injection of precipitin lines made by immunoelectrophoresis of the immunoglobulin solutions against rabbit antiserum to pig serum (goudie et al., ; higgins, ) . sera were made class specific by absorption with glutaraldehyde cross-linked immunoglobulin solutions (avrameas and ternynck, ) . specificity was assessed in immunoelectrophoresis and immunodiffusion tests against serum, colostrum and immunoglobulin solutions. sera and colostral wheys were examined by gel filtration in sephadex g- (pharmacia) in . m tris-hcl, ph . , containing . m naci and mm edta. wheys were also examined by ion-exchange chromatography on deae-cellulose (de- , whatman), using the six step buffer system described by stone et al. ( b) . the eluate obtained with each buffer was treated as a single fraction. presence of igm, igg and iga in the fractions was observed by microimmunodiffusion against specific antisera. fractions were concentrated about five-fold by dialysis against polyvinylpyrrolidone and examined for neutralising antibodies to tgev. the allocation of antibody activity to an immunoglobulin class was made by comparison of the distribution of antibody activity and immunoglobulins in the fractions. twelve pregnant sows were inoculated by the intramammary route: one with live, whole virus, three with formalin-inactivated virus, two with a mixture of purified sp and svp, four with sp alone and two with svp only. in addition one sow was given live virus orally and two sows were kept as uninoculated controls. for the intramammary inoculations, . ml of the antigen preparation was injected through the skin near the teat into three mammary glands of each sow three times. the same glands were inoculated at approximately , and week before farrowing. for the first inoculation, the antigen was emulsified in complete freund's adjuvant. colostrum samples were taken from inoculated and uninoculated glands separately at farrowing and milk samples were taken for several days after that. blood samples were removed before the first inoculation and at the intervals shown in table i . three rabbits were inoculated with tgev antigen: one with inactivated whole virus, one with sp and one with svp. each rabbit received two subcutaneous inoculations of antigen emulsified in complete freund's adjuvant at an interval of weeks, followed by three intramuscular inoculations of aqueous antigen at weekly intervals. one rabbit was kept as an uninoculated control. blood samples were taken from each animal before the first inoculation and at intervals up to months. p/o = orally; i/mam = by the intramammary route. --= not detected. those not tested are indicated by blank spaces. piglets born to the experimental sows were challenged at days of age by oral administration of ml of a suspension of tgev-infected intestine diluted to contain approximately x tcids per ml (equivalent to approximately x lds per ml in piglets reared away from the sows). the suspension was free, as far as it was possible to determine, from moulds, bacteria and other viruses. the results of neutralisation tests for serum and colostrum are shown in table i . after intramammary inoculation of sows, antibody was detected at the time of farrowing in serum and colostrum from all of three sows given whole killed virus, from out of given surface projections with subviral particles, from out of given surface projections only and from neither of the animals given subviral particles alone. neutralising antibody in colostrum only was detected in out of the given purified surface projections. there was little or no difference between antibody titres of colostrum from inoculated and uninoculated glands. the levels of neutralising antibody in milk from sows inoculated with inactivated whole virus or viral components fell to undetectable levels between to days after farrowing. neutralising antibody titres in sera of all sows, including the uninoculated controls, rose during the first weeks after farrowing. this reflects the immune response to infection of the dam by live virus administered to her piglets days after farrowing. the results are shown in table ii . the immune response to infection with live virus (sow no. ) resulted in neutralising antibodies in the three immunoglobulin classes reported previously (ristic and abou-youssef, ; bohl et al., b; salf et al., ; lucas et al., ) although in the example given igg antibody was detected in serum but not in colostrum. samples from sows that received intramammary inoculation contained neutralising antibodies of the igg class in all cases where the specific imunoglobulin could be characterised. no igm antibodies were detected in any sample and iga antibodies were demonstrated in the colostrum from only out of sows that received sp antigen. the only litter that survived challenge was that of sow no. which had been inoculated with sp antigen and had produced neutralising iga antibodies in the colostrum. the other litters experiences - % mortality and all piglets from the control uninoculated sows died. serum samples taken from the rabbit that had been inoculated with inactivated whole tgev showed neutralising antibodies rising to a maximum titre of l/ . neutralising antibodies were detected also in the serum of the rabbit that received sp antigen; the rate of antibody production was slower, however, and the peak titre reached was l/ . no neutralising antibody was found in serum from the rabbits inoculated with svp antigen and the uninoculated animal remained seronegative throughout the course of the experiment. the sedimentation profiles of h-uridine tgev after incubation with bovine serum albumin or with sera from sows that had been inoculated with viral antigen preparations are illustrated in fig. . virus incubated with either bovine albumin or normal pig serum sedimented as a sharp, homogenous band of radioactivity with very little material collecting at the bottom of the gradient. in contrast, however, virus incubated with sera from animals inoculated with viral antigen moved through the gradient as a broad band, suggesting heterogeneity of size and density, with a large proportion of the total radioactivity sedimented at the table i. bottom of the tube. there were no obvious differences between the profiles obtained after treatment of virus with sera that contained neutralising antibodies (sows no. , and ) or with serum from an animal that received svp antigen and which produced no neutralising antibodies (sow no. ). incubation of h-uridine tgev with sera from sows inoculated with viral antigen resulted in the formation of immune complexes that were retained by membrane filters (fig. ) . after incubation with serum from uninoculated animals approximately % of the virus was retained on the filter, probably due to aggregation that occurs in purified tgev preparations. sera from sows inoculated with whole virus, sp antigen or svp antigen preparations produced virus complexes that were retained most efficient samples of h-uridine labelled tgev containing ct/min were incubated for h at °c in dilutions of test sera followed by incubation with rabbit anti-porcine igg serum. after filtration through cellulose acetate membrane filters and repeated washing with isotonic saline, the retained radioactivity was determined by scintillation counting. test sera from: sow no. -" -', sow no. ~ e, sow no. = -', uninoculated sow d d. ly at a serum dilution of / . at higher serum dilutions there was reduced retention, implying that the level of antibodies was limiting. serum dilutions lower than / gave suboptimal retention due, probably, to an excess of antibodies and the formation of smaller complexes. omission of the rabbit anti-porcine igg serum from the test resulted in poorer retention of radioactive virus at all dilutions of experimental serum and a decline in the reproducibility of the test. discussion we report the immune response of sows and rabbits to tgev preparations containing whole virus, isolated surface projections or subviral particles produced by removal of the surface projections and envelope lipid from the virus by detergent treatment. sows receiving whole virus, isolated sp antigen and mixtures of sp and subviral particles produced antibody capable of neutralising virus infectivity; sows that received subviral particles alone, however, did not. similarly rabbits immunised with purified whole virus or surface projections produced neutralising antibody to tgev while administration of subviral particles produced no detectable neutralising activity. these data suggest that neutralising antibody raised during infection of swine with tgev is directed against a structural antigen of the virion and that this antigen is associated with the surface projection, a similar finding to that obtained with other lipid enveloped viruses (webster and laver, ; cartwright et al., ; jennings et al., ; hunsmann et al., ) . the fact that no neutralising antibody was produced in the pigs and rabbits that received subviral particles alone strongly suggests that the structural elements of these particles are not involved in virus neutralisation; conclusive proof of this, however, would necessitate the use of more animals than were available in the present study. the possibility existed that the tgev subviral particles were not antigenic in the animals used. this seemed unlikely as we had previously shown that these structures of nm diameter, readily visualised by electron microscopy, contained all the structural proteins except for the sulphated glycoprotein of the sp (garwes et al., ) . standard serological tests, such as complement fixation and radial immunodiffusion, are not sensitive enough to detect the levels of antibodies present in the experimental sera. the radioimmune assay techniques described above indicated, however, that antibody directed against the surface of the virus was produced in response to inoculation of sows with whole virus or either of the structural moieties. the sedimentation analyses showed that these sera caused an increase in the sedimentation coefficient of the virus, presumably by coating the particles with antibody molecules, and brought about sufficient aggregation to pellet radioactivity to the bottom of the gradient. application of this serum-induced clumping of radiolabelled virus to filtration studies demonstrated that sows receiving svp antigen were capable of producing antibodies to surface components at a similar level to those animals receiving whole virus or sp antigen. the use of this technique to quantitate agglutinating antibody is complicated by the need to incorporate rabbit anti-porcine igg serum into the test. as discussed in the text, purified tgev adsorbs irreversibly to membrane filters containing cellulose nitrate. this necessitated using cellulose acetate filters, for which the smallest pore diameter available is nm. assuming that virus aggregates are approximately spherical and composed of particles -- nm in diameter, the smallest aggregate that sould be retained reproducibly by such filters would need to comprise -- virions. the secondary antiserum to porcine immunoglobulin is required to achieve complexes of this size. the low level of retention of virus incubated with low dilutions of immune sera presumably reflects the solubility of antigen--antibody complexes in the presence of large excesses of either of the two reactants. analysis of the immunoglobulin classes responsible for neutralising activity after inoculation of sows by the intramammary route showed that igg was the predominant antibody species in both serum and colostrum, consistent with previous findings with inactivated tgev lucas et al., ) but differing from the stimulation of iga to ferritin reported by bourne et al. ( ) . whether this discrepancy is due to differences in the types of antigen used is not known. it is of interest that sow no. , which received sp antigen, produced neutralising antibody of the iga class in the colostrum and was the only animal capable of protecting her piglets against challenge with live virus. the possibility that this sow encountered live tgev prior to farrowing and that the experimental inoculation generated a secondary immune response cannot be totally ruled out, since infection with the live virus stimulates igm and iga antibody production (sow no. above; lucas et al., ; stone et al., ) and may result in limited protection of the litter. there was no other reason to believe that prior infection with tgev had occurred, however. all the control sows remained sero-negative throughout the experimental period. the apparent lack of neutralising antibody of the igm class in the pigs that received inactivated viral material may have resulted from the materials used and the route of inoculation; the times at which the serum samples were taken may have been too late, however, to have detected a transitory igm response. most of the sows showed rising neutralising antibody titres after challenge of the piglets, probably in response to cross-infection. those inoculated with sp antigen that had no detectable neutralising antibody at farrowing nevertheless developed titres by to weeks, comparable with those that were sero-positive at farrowing. the two sows inoculated with svp antigen alone, however, as well as having no detectable serum neutralising antibody at farrowing, developed lower titres that were more comparable with those achieved by the uninoculated controls. it has been reported that a soluble antigen from tgev-infected swine testis cells could induce neutralising antibody formation when injected into rabbits. from the data presented above we should anticipate that the soluble antigen concerned is likely to comprise viral sp either free in solution or bound to small fragments of membrane. since the viral sp is characterised mainly by the molecular weight of its component glycopolypeptide and has neither haemagglutinin nor enzyme activity associated with it, the task of recognising the presence of sp in crude tissue extracts is difficult. it should be possible, however, to develop a radioimmune assay using specific antibody to purified components. such a test would facilitate the preparation of potent antigen suspensions and work is in progress to this end. the cross-linking of proteins with glutaraldehyde and its use for the preparation of immunoadsorbents a. immunology of transmissible grastroenteritis antibody responses in serum, colostrum and milk in swine after infection or vaccination with tge virus the influence of route of vaccination on the systemic and local immune response in the pig surface structure of vesicular stomatitis virus a cytopathic virus causing a transmissible gastroenteritis in swine. ii. biological and serological studies effect of precipitation with methanol on antigenic potency of tge virus the polypeptide structure of transmissible gastroenteritis virus isolation of subviral components from transmissible gastroenteritis virus a simple method for producing antibody to a single selected diffusible antigen. the lancet fractionation of fowl immunoglobulins properties of mouse leukaemia viruses. ix. active and passive immunisation of mice against friend leukaemia with isolated viral gpt~ glycoprotein and its corresponding antiserum the immune response of hamsters to purified haemagglutinins and whole virus vaccines following live influenza virus infections attempts to vaccinate sows against tge the influence of ph on the growth and stability of transmissible gastroenteritis virus in vitro transfer of immunoglobulins igg, iga and igm to lacteal secretions in the parturient sow and their absorption by the neonatal piglets comments on the immunology of transmissible gastroenteritis isolation of porcine immunoglobulins and determination of the immunoglobulin classes of transmissible gastroenteritis viral antibodies transmissible gastroenteritis in neonatal pigs: intestinal transfer of colostral immunoglobulins containing specific antibodies a. partial characterisation of the principal soluble antigens associated with the coronavirus of transmissible gastroenteritis by complement fixation and immunodiffusion chromatographic separation of gram quantities of immunoglobulins from porcine colostrum against transmissible gastroenteritis virus immunoglobulin a in the pig. i. preliminary characterisation of normal pig serum iga influenza virus subunit vaccines: immunogenicity and lack of toxicity for rabbits of ether-and detergent-disrupted virus key: cord- -nmkilkcx authors: reynolds, d. j.; garwes, d. j. title: virus isolation and serum antibody responses after infection of cats with transmissible gastroenteritis virus date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: nmkilkcx transmissible gastroenteritis virus was administered orally to cats. no clinical disease resulted but infectious virus was isolated from faeces for up to days after infection and serum antibody was detected by neutralisation and immunofluorescence tests. transmissible gastroenteritis virus was administered orally to cats. no clinical disease resulted but infectious virus was isolated from faeces for up to days after infection and serum antibody was detected by neutralisation and immunofluorescence tests. . the spread of transmissible gastroenteritis virus (tgev) from infected pig herds and the recrudescence of infection after long periods have not been adequately explained. experiments on the host range of the virus have indicated that nonporcine hosts could have a role in the epidemiology of tge ( , , , , , t ) . indirect immunofluorescence (iif) and virus neutralisation (v n!) tests have been used recently to demonstrate antibody to tgev in cats. in holland and great britain samples from cats with clinical feline infectious peritonitis (fip) frequently have antibody to tgev in the i i f test ( ). however, neutralising antibody has not been found in sera from dutch cats with fip. american and dutch studies have recently demonstrated that sera from cats experimentally infected with f i p will cross-react, at low titre, with tgev-infected pig cells by immunofluoreseence but will not neutralise tgev in vitro ( , ) . this evidence has been used to suggest that an antigenic relationship exists between tgev and f i p virus. the potential of the cat to excrete tgev was studied by virus reisolation and the serological responses to tgev and f i p were monitored; thus the possible epidemiological role of the cat in tge could be assessed. in addition information was obtained on the possible origin and relationship of vn and i i f antibodies to tgev which occur naturally in cats, often at high titres ( , ). tgev strain kn , used as inoculum in this experiment, was prepared as a clarified per cent suspension of small intestine from an infected gnotobiotic piglet. i t had a titre of i. × pfu/ml. - / / / /$ . four twelve-week old kittens were obtained from an spf cat colony with no evidence of neutrmising antibody to tgev and no clinical history of fip. the kittens were housed separately in plastic isolators ( ). three animals (a, b and c) were given ml of virulent tgev orally, the fourth (d) was given ml phosphate buffered saline and retained as an uninfected control. animal a was sacrificed days after infection in an attempt to detect evidence of tgev replication in the tissues, whilst b and c were retained for days. animal d was given oral tgev days after the commencement of the experiment and days later (day ) it was hyperimmunized with . ml tgev viral antigen produced from cell cultured virus ( ), injected intraperitoneally. this dose was repeated days later (day ) and after another days (day ) the eat was killed and exsanguinated. control and infected animals were tested for tgev excretion by attempted isolation of virus from faeces for up to days after infection; occasionally rectal swabs were used to obtain fresh samples. cell growth medium was used to prepare per cent faecal suspensions and elute material from rectal swabs. both types of preparation were clarified by centrifugation at x g for minutes and . ml of the supernate was inoculated onto monolayer cultures of secondary adult pig thyroid (apt/ ) cells on glass eoverslips. the medium was changed after hours and days later any viral eytopathic effect was recorded, the medium was removed and passaged in fresh apt/ cultures and the eoverslips were stained to demonstrate virm antigen by indirect immunofluorescence, using paired sera from a gnotobiotic piglet before and after tgev infection, followed by fitcconjugated rabbit anti-swine globulin (nordic immunological laboratories, london). cultures with evidence of tgev antigen were passaged again in the presence of inhibitory levels of specific antiserum. recovery of tgev was judged successful if cells showed a characteristic cytopathic effect, produced cytoplasmic fluorescence with monospecific tgev antiserum alone and the infectivity could be neutralized by specific tgev antiserum. tgev was not isolated from the faeces of any animal before infection or from the uninfected control. uninfected eoverslip cultures, set up concurrently with infected samples, remained free of tgev. tgev was isolated from the faeces of animal b on days , , and after infection and from animal c on days , , and after infection. virus was not isolated from faeces of animal a in the days after infection nor from animal d either before or after oral administration of tgev. there were no signs of disease in the eats after infection. reisolation of tgev was attempted from animal a, killed days after infection and animals b and c killed days after infection. tissue suspensions were prepared and treated as described for faecal suspensions. tonsil, stomach, upper, middle and lower jejunum, ileum, caecum, colon, rectum, kidney, liver, spleen, lung, brain and mesenteric lymph node were the tissues used. pooled tissue suspensions from b and c were administered orally to -day old spf piglets and were also inoculated onto apt/ monolayers for antigen detection by the i i f test. the piglets showed no sig as of tge nor was there a detectable serological response. the coverslip cultures remained negative by immunofluoreseenee. the survival of infectious tgev in the inoculum was studied at ° and ° c to give an indication of the probable time course of viral inactivation in the host and in faeces. viral antigen was demonstrated by immunofluorcseence after infection of coverslip cultures by the method described for faecal virus reisolation. infectivity titres of tgev were determined by plaque assay on apt/ ceils in mm plastic petri dishes. infectivity was still present after but not days at ° c and was lost within hours at ° c. virus could be demonstrated by both plaque assay and immunofluorescence in infectious samples. blood was taken from the cephalic vein of animals b, c and d before and regularly after infection to monitor the serum for vn and i i f antibodies to tgev. a microtitre test ( ) was used to assay tgev neutralising antibody levels in serum and the responses of cats b, c and d are presented in figure as the toglo of the reciproem of the end-point dilution, calculated by kxrbeg's method ( ) . antibody could not be detected prior to infection and animal a remained negative for the three days prior to sacrifice. the uninfected control also remained negative. in animals b, c and d vn antibody was first detected approximately i week after exposure to tgev and the titres rose to maximum values of - -- .a. twenty-one days after first hyperimmunization cat d had developed a marked secondary response, with a vn antibody level of . which persisted until death. antibody to tgev was not detected in the i i f test before infection. following experimental infection with tgev serum antibody, detectable in the i i f test, developed and persisted similarly in the three cats tested (fig. ) . the i i f test titres were consistently lower than vn test titres. after hyperimmunization a marked increase in the i i f test titre was detected in d. this animal also developed antibody to apt/ cell antigens in the i i f test, although the titre was four-fold lower than that of specific antiviral antibody. antibody to f i p was assayed in an i i f test, by dr. n. c. pedersen~ university of california, davis, u.s.a. ( ) and in a suckling mouse brain-adapted f i p neutralisation test ( ) by drs. a. d. m. e. ost~r~avs and m. c. horzi~k, university of utrecht, the netherlands. no fip-specific antibody was demonstrable in the sera before or after oral infection with tgev. antibody was detected by the i i f test in cat d within days of the first hyperimmunization, however, reaching a titre of . which remained constant for the following days (fig. ) but this serum failed to neutralise f i p infectivity in the mouse brain system. this experiment demonstrates that cats exposed to tgev can excrete the virus for a period significantly longer than would be expected from passive transit of ingested virus. the eat could therefore constitute a practical epidemiological hazard to susceptible pigs. the ability of tgev excreted by the eat to infect pigs or other cats was not investigated but similar work ( ) demonstrated the ability of dogs and foxes to excrete tgev which could infect piglets. hence, contact between these carnivores and infected pigs should be prevented. a study of the replication site of tgev in the cat was not possible with the limited number of animals. virus isolation from only two of the animals tested may indicate some variation in host. susceptibility to infection although the serological responses were similar in all animals. f i p and tge, two antigenically-related viruses, are now known to infect eats experimentally but result in differing immune responses. following a single exposure to tgev, cats developed homologous antibodies, detectable by vn and i i f tests. antibody to f i p was detected only following hyperimmunization, which resulted in extremely high anti-tgev titres. experimental f i p results in no tgev neutralising antibody but antibody to both viruses is found using i i f tests with f i p titres exceeding tgev titres. these results provide serological evidence of a distant antigenic relationship between tgev and fip, perhaps due to an internal group antigen not involved in virus neutralisation. this situation can be compared to the closer relationship of the canine coronavirus to tge, agents which cross-react in vn and :[if tests ( , ) . hyperimmunization may be important in stimulating antibody to heterologous viruses. it is probable that infection with one or more tgev-related viruses could result in the complex serological picture in f i p cases previously described ( ) . f i p virus itself may have more than one antigenic type, which would also result in the differing responses found in f i p cases in the u.k., u.s.a. and the netherlands. we recovery and characterization of a coronavirus from military dogs with diarrhoea epizootiology of porcine transmissible gastroenteritis (tge) effect of precipitation with methanol on antigenic potency of tge virus epidemiological studies of transmissible gastroenteritis in swine feline infectious peritonitis (fip) virus. iii. studies on the multiplication of fip virus in the suckling mouse beitrag zur kollektiven behandlung pharmakologischer reihenversuche the effect of propagation of transmissfble gastroenteritis (tge) virus in pups and the lungs of baby pigs on the immunologic properties of the virus transmissible gastroenteritis in the neonatal dog infection of cats with tgev transmissible gastroen~eritis (tge) of swine. tile possible role of dogs in the epizootiology of tge seroepidemiology of feline infectious peritonitis virus infections using transmissible gastroenteritis virus as antigen serological studies of naturally occurring feline infectious peritonitis antigenic relationship of the feline infectious peritonitis virus to eoronaviruses of other species detection of transmissible gastroenteritis virus neutralising antibody in cats the production of gnotobiotic piglets and calves by hysterotomy under general anaesthesia micro-color test for assay of transmissible gastroenteritis virusneutralizing antibodies untersuchungen fiber die antigenverwandtschaft der viren der felinen infekti sen peritonitis (fip) und der transmissiblen gastroenteritis (tge) des sehweines authors' address" mrs. i). j. t~eynolds, institute for research on animal diseases, compton, newbury, berkshire :rg nn, england.i~eceived july t , key: cord- -c kctjz authors: dai, lei; hu, wei wei; xia, lu; xia, mi; yang, qian title: transmissible gastroenteritis virus infection enhances sglt and glut expression to increase glucose uptake date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: c kctjz transmissible gastroenteritis virus (tgev) is a coronavirus that causes villus atrophy, followed by crypt hyperplasia, reduces the activities of intestinal digestive enzymes, and disrupts the absorption of intestinal nutrients. in vivo, tgev primarily targets and infects intestinal epithelial cells, which play an important role in glucose absorption via the apical and basolateral transporters na(+)-dependent glucose transporter (sglt ) and facilitative glucose transporter (glut ), respectively. in this study, we therefore sought to evaluate the effects of tgev infection on glucose uptake and sglt and glut expression. our data demonstrate that infection with tgev resulted in increased glucose uptake and augmented expression of egfr, sglt and glut . moreover, inhibition studies showed that egfr modulated glucose uptake in control and tgev infected cells. finally, high glucose absorption was subsequently found to promote tgev replication. transmissible gastroenteritis (tge) is a highly contagious infectious disease of pigs that is characterized by vomiting, diarrhea, and dehydration. notably, the mortality rate of this disease in seronegative suckling piglets can reach up to % [ ] . tge is caused by the tge virus (tgev), which infects the gastrointestinal tract, causing villus atrophy and crypt hyperplasia, and disrupting intestinal nutrition absorption [ , ] . glucose is among the nutrients absorbed in the intestinal epithelium, and glucose uptake in epithelial cells depends on two types of glucose transporters, the apically expressed na + -dependent glucose transporter (sglt ) and basolaterally expressed facilitative glucose transporter (glut ). specifically, sglt mediates the na+/glucose cotransport function of the kidney and intestine as a secondary active transporter, while glut serves as a facilitated diffusion system for transport through lipid bilayers [ ] [ ] [ ] . the epidermal growth factor receptor (egfr) was previously reported to transiently increase glucose transport [ , ] . moreover, a recently study suggested that egfr may act as another receptor for tgev, in addition to porcine aminopeptidase (papn) [ ] . egfr-dependent regulation of glucose uptake has been observed in tumor cells, and egfr has been shown to prevent autophagic cell death by maintaining intracellular glucose levels through interaction with and stabilization of sglt [ ] . however, the involvement of egfr in virus-induced effects on glucose uptake has yet to be evaluated. therefore, in the study, we aimed to examine the in vitro effects of tgev infection on glucose uptake and the expression of sglt and glut in porcine intestinal columnar epithelial (ipec-j ) cells, which have been shown to offer a practical model for studying tgev infection [ , ] . cell lines ipec-j cells, which are porcine intestinal columnar epithelial cells that were originally isolated from the middle jejunum of neonatal piglets, were purchased from dsmz (braunschweig, germany), while hek t cells were purchased from the american type culture collection (atcc; manassas, va, usa). ipec-j and hek t cells were maintained in roswell park memorial institute medium (rpmi) and dulbecco's modified eagle's medium (dmem) with high glucose, respectively, supplemented with hepes, % fetal bovine serum (fbs; gibco, grand island, ny, usa), and % penicillin-streptomycin (invitrogen, carlsbad, ca, usa) at ˚c in a % co incubator (thermo fisher scientific, waltham, ma, usa). tgev strain shxb was isolated in shanghai, china. the complete genome sequence for this virus is available at the genbank database (id number: kp . ) [ ] . for experimental assays, cells were incubated with tgev at a multiplicity of infection (moi) of for h at ˚c in serum-free medium and washed with phosphate-buffered saline (pbs; ph . ) at ˚c three times to remove unbound virus. cells were then cultured in medium containing % serum. total rna was extracted from ipec-j cells infected with tgev using trizol reagent (invitrogen), according to the manufacturer's instructions. cdna was generated by reverse transcription using hiscript qrt supermix for qpcr (vazyme biotech, nanjing, china), according to the manufacturer's instructions. tgev release was assessed by measuring the levels of viral nucleoprotein (n) gene expression via quantitative rt-pcr using a takara sybr green qpcr kit (takara, shiga, japan). the primer sequences were as follows: n-f (sense), '-caattcccgtggtc ggaaga- ', n-r (antisense), '-tttacgttggcccttcacca- '. pcr products were purified using a gel extraction kit (omega bio-tek, inc., norcross, ga, usa) and cloned into the pjet . vector (thermo fisher). plasmids were serially diluted and used as standards for quantitative analysis. the initial copy number of the tgev n gene was calculated using the following formula: x = -k log ct + b, where x is the initial copy number and k, ct, and b refer to the slope rate, cycle threshold, and constant, respectively. quantitative real-time pcr was performed with an abi prism detection system (applied biosystems, foster city, ca, usa). at the indicated time points post-infection, cells were washed with pbs and lysed in radioimmunoprecipitation assay (ripa) buffer (thermo scientific) containing a phosphatase inhibitor and protease inhibitor (thermo scientific), according to the manufacturer's instructions. the protein concentrations of the resulting lysates were determined using a pierce bca protein assay kit based on the bicinchoninic acid spectrophotometric method (thermo scientific). after centrifugation at , × g for min, proteins in the supernatant ( - μg protein) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) on - % gradient gels, and transferred to polyvinylidene fluoride (pvdf) membranes (merck millipore, darmstadt, germany). membranes were blocked for h in tris-buffered saline (tbs) containing % nonfat dry milk, and probed with the indicated primary antibodies at ˚c overnight, according to the manufacturer's instructions. the following antibodies were used in this study: anti-p-egfr antibody (d a ; cell signaling technology, danvers, ma, usa), anti-egfr (c ; cell signaling technology, danvers, ma, usa), anti-sglt (ab ; abcam, cambridge, uk), anti-glut (sc- ; santa cruz biotechnology, dallas, tx, usa), anti-βtubulin (e - ; enogene biotech, new york, ny, usa). membranes were then exposed to species-specific horseradish peroxidase (hrp)-conjugated secondary antibodies (dilution: : , ), and proteins were detected by enhanced chemiluminescence (ecl; thermo scientific) and autoradiography. the resulting bands were quantified using quantity one -d analysis software ( - ; bio-rad, hercules, ca, usa). the density of each band was measured and normalized to that of β-tubulin expression. all data were expressed as means ± standard deviations (sd) of the results of three independent experiments. plvx-shrna is a human immunodeficiency virus type (hiv- )-based lentiviral expression vector designed to express a small hairpin rna (shrna) for rnai studies (clontech laboratories, inc., mountain view, ca, usa). the best silencing efficiencies were observed with clones nm_ (porcine egfr) and nm- . (porcine sglt ). the shrna for egfr, three shrnas for sglt , and overexpression plasmid for egfr were designated as shegfr, shsglt - , shsglt - , shsglt - , and plvx-egfr, respectively. hek t cells were transfected with μg of specific expression plasmid per cells using the x-tremegene hp dna transfection reagent (roche, basel, switzerland), according to the manufacturer's instructions, diluted in opti-mem (invitrogen) in a t- cell culture flasks. lentiviral particles (moi = ) were subsequently added to the transfected ipec-j cells and gently mixed. after comparing amino acid sequences of egfr, sglt and glut from ncbi (table ) , we respectively selected ag , phlorizin as the inhibitor of egfr and sglt . glucose uptake experiments -[ h]deoxyglucose ( -dg) uptake experiments were carried out according to the method described by henriksen et al., but with modifications [ ] . briefly, the culture medium was removed by aspiration, and cells were gently washed twice with uptake buffer ( mm nacl, mm kcl, mm kh po , mm mgcl , mm cacl , mm glucose, mm l-alanine, nm indomethacin, and mm hepes-tris; ph . ). after washing, cells were incubated in uptake buffer containing -dg at ˚c for min. at the end of the incubation period, cells were washed three times with ice-cold uptake buffer and dissolved in ml of . % sds. the level of intracellular -dg uptake was determined by measuring the radioactivity of a -μl aliquot of each sample using a liquid scintillation counter (ls ; beckman instruments, fullerton, ca, usa). the remainder of each sample was then used to determine protein expression levels of glucose transporters. the radioactivity counts of each sample were normalized with respect to the protein and corrected for time uptake per milligram of protein. all uptake measurements were carried out in triplicate. glucose uptake was calculated as follows: glucose uptake (nm/ cells) = (glucose concentration of the control group-glucose concentration of the experimental group)/cell number. cfse stocks ( mm in dmso; invitrogen, merelbeke, belgium) were stored at - ˚c prior to use, thawed, and diluted in phosphate-buffered saline (pbs) to the desired working concentrations. ipec-j cells were resuspended in pbs ( . % bsa) at × cells/ml and incubated with cfse (final concentration: μm) for min at ˚c. cells were washed and resuspended in culture medium for min to stabilize cfse staining, and cfse-labeled cells were analyzed by flow cytometry. the viability of raw . cells was determined using a cell counting kit- assay kit (beyotime biotechnology, beijing, china), as previously reported. briefly, raw . cells were plated at a density of × cells/well in -well plates in ml roswell park memorial institute medium and incubated for h. twenty microliters of cell counting kit- reagent was then added to each microwell, and plates were incubated for h at ˚c. the absorbance of the colored solution was measured using a microplate reader (bio-rad laboratories) at a test wavelength of nm and a reference wavelength of nm. all results are presented as means ± sd of the results of three independent experiments. significant differences between control and experimental groups were analyzed using student's ttest. p-values < . were considered statistically significant. as shown as in fig a, tgev infection triggered a significant increase in glucose uptake in ipec-j cells at and h post-infection (hpi). in addition, during late-stage infection, tgev-infected cells were able to continue transporting glucose, even when the concentration of glucose in the cell culture medium was low. these data indicate that tgev infection promotes glucose uptake in ipec-j cells. while fig b- f show the mrna and protein levels of sglt and glut at various time points. notably, while there were no marked changes in the mrna expression of the sglt and glut genes after tgev infection, there were significant increases in the protein expression of both sglt and glut at and hpi. to exclude the possibility that cell viability and proliferation may affect glucose uptake, cfse-labeled mock-infected (control), tgevinfected (tgev), and carbonyl cyanide m-chlorophenyl hydrazone (cccp)-treated (cccp) cells were subjected to flow cytometric analysis. cccp is known to reduce cell viability and proliferation, and was therefore utilized as a positive control [ ] . as shown in fig g- i , previous studies have shown that the expression of egfr is closely related to glucose transport, especially phosphorylation region of egfr. egfr kinase activity regulates the peak glucose uptake and total egfr may likely be maintaining basal glucose uptake [ , ] . therefore, we first examined whether egfr expression was stimulated by tgev infection. western blot analysis showed that tgev infection not only increased total egfr expression (fig a) but also revealed that more phosphorylated egfr was present in tgev infected relative to control cells (fig b) . taken together, these results indicated that tgev infection increased the total and phosphorylation protein expression of egfr at hpi. together, these results indicate that tgev infection promoted the total and phosphorylation protein expression of egfr at hpi. to explore whether egfr contributes to the observed tgev infection increase in glucose uptake, we explored changes in glucose uptake and the expression of glucose transport molecules by inhibiting or overexpressing egfr in mock-infected cells. we choose ag as egfr inhibition because it can inhibit the egfr tyrosine kinase activity and total egfr [ , ] . as shown in fig a- c , treatment with the egfr inhibitor ag suppressed glucose uptake and markedly decreased egfr and p-egfr protein expression, indicating that ag regulated glucose uptake in mocked-infected cells. furthermore, cells treated with ag exhibited markedly lower levels of sglt protein expression than the control cells; in contrast, there was no effect on glut protein expression. thus, we concluded that ag modulated glucose uptake in mock-infected cells via downregulation of egfr, which resulted in reduced sglt expression. as shown in fig d- f , we sought to then strengthen these findings via modulation of egfr expression. transfection with shegfr resulted in significant downregulation of egfr β-tubulin was also analyzed. doi: . /journal.pone. .g and p-egfr protein expression and decreased glucose uptake compared with the cells transfected with the control shrna. conversely, plvx-egfr transfection resulted in significantly increased egfr and p-egfr protein expression and glucose uptake. these data demonstrate that modulation of egfr and p-egfr expression affects glucose uptake in the absence of tgev infection. furthermore, transfection with shegfr and plvx-egfr resulted in decreased and increased sglt protein expression, respectively; in contrast, glut protein expression was unaffected by either treatment. together, these results indicate that egfr and p-egfr regulates glucose uptake in mock-infected ipec-j cells by modulation of sglt protein expression. because sglt is involved in mediating the functions of egfr in hek- t cells [ ] , we explored the relationship between egfr and sglt expression in ipec-j cells. as shown in fig h and j- l , ipec-j cells transfected with three sglt -specific shrnas for h exhibited reduced sglt expression and reduced egfr protein expression. furthermore, we choose phlorizin as sglt inhibitor because phlorizin's principal pharmacological action is to block intestinal glucose absorption through inhibition of the sodium-glucose symporters located inmucosa of the small intestine [ , ] . ipec-j cells treated with phlorizin for h showed downregulation of sglt expression without inducing cell death, as demonstrated by cck assay analyses (data not shown). similarly, western blot analysis showed that ipec-j cells treated with phlorizin exhibited lower egfr expression than dmso-treated cells. in conclusion, inhibition of sglt by shrna or phlorizin treatment disrupted egfr expression. notably, these treatments also resulted in decreased glucose uptake (fig g and j) . consistent with these findings, ipec-j cells transfected with shegfr or treated with ag also exhibited lower sglt expression (fig b and e) . these data indicate that there is an association between egfr and sglt expression in ipec-j cells. to further investigate the role of tgev infection-enhanced egfr in the regulation of glucose uptake, we examined the effects of tgev infection after treatment or transfection with ag , shegfr, or plvx-egfr on glucose uptake. as shown in fig a and d , transfection with shegfr and treatment with ag significantly inhibited glucose uptake, whereas transfection with plvx-egfr enhanced glucose uptake. therefore, we concluded that egfr mediates glucose uptake in tgev-infected ipec-j cells. moreover, as shown in fig b, c , e and f, after tgev infection, western blot analysis showed that transfection with shegfr and treatment with ag resulted in lower protein expression of sglt and glut than that observed in the control group, whereas transfection with plvx-egfr resulted in increased sglt and glut protein expression. together, these results indicate that egfr influences glucose uptake in tgev-infected cells by promoting both sglt and glut expression. previous studies have suggested that the intracellular glucose concentration is closely linked with viral infections, particularly for double-stranded dna viruses [ ] [ ] [ ] . consistent with this conclusion, we found that tgev enhanced glucose uptake in ipec-j cells. because glucose is the main energy source for cellular metabolism, tgev replication in ipec-j cells requires large amounts of glucose/energy. thus, we explored whether high glucose uptake in tgev-infected cells affected tgev replication. tgev contains a . - . -kb singlestranded, positive-sense rna genome; the virion rna functions as an mrna and is infectious. the n protein, in particular, is a critical component of the replication-transcription complex [ ] [ ] [ ] . thus, we treated tgev-infected cells with medium containing . , , , or mm glucose and harvested the virus at hpi. as shown in fig a, glucose absorption concentrations were . , , . , and . mm for medium containing . , , , and mm glucose, respectively. furthermore, western blot analysis showed that tgev n protein was more abundant when tgev-infected cells absorbed more glucose, particularly in medium containing or mm glucose (fig b) . consistent with this finding, there was a concurrent increase in the copy number of the tgev n gene as glucose absorption levels increased (fig c) . as described above, in tgev-infected ipec-j cells, higher glucose concentrations promoted tgev replication. to exclude the possibility that cell viability and proliferation affect tgev replication, flow cytometric analysis of cfse-labeled cells was performed in the presence of . , , , and mm glucose. as shown in fig d- f , high glucose concentrations did not significantly affect proliferation and viability. as recently reported, viral infection may result in increased or decreased glucose uptake. for example, the hepatitis b virus x protein regulates hepatic glucose homeostasis via nitric oxide synthase [ , ] . additionally, human cytomegalovirus (hcmv) enhances glucose uptake during the first h after infection, even when a high level of glucose is constantly present in the medium [ ] . hepatitis c virus (hcv) replication suppresses cellular glucose uptake through downregulation of cell surface glucose transporters [ ] , while rotavirus infection impairs rabbit intestinal brush-border membrane na + -solute cotransport activities [ ] . however, the effects of coronaviruses (including tgev) on glucose uptake have not been reported. we found that tgev infection increased glucose uptake of ipec-j cells and the protein expression of sglt , glut after hpi (fig ) , while tgev infection in ipec-j cells reached a peak at hpi [ ] , we just provided foresight to explore the interaction between tgev infection and glucose uptake, which is still worth exploring. most of the glucose absorbed in the intestine is transformed to glutamine [ ] , which was reported to promote the replication of many viruses, such as porcine circovirus (pcv) and hcmv, in vitro [ , ] . because viral replication requires a lot of energy, we examined the effects of glucose absorption on tgev propagation. our results demonstrate that increased glucose absorption enhanced tgev replication, suggesting that glucose may be transformed into glutamine in infected cells. in this study, increases in sglt and glut protein expression directly stimulated glucose uptake in vitro. although the role of other transporters cannot be ruled out, as sglt and glut dominate intestinal glucose transport, they may play important effect on glucose uptake in tgev infection. however, no significant changes were observed in sglt or glut mrna expression levels, indicating that tgev infection primarily affects the protein expression of sglt and glut . these findings were consistent with previous reports demonstrating that tgev infection influences protein translation. additionally, sglt and glut have been shown to mediate intestinal glucose uptake transmissible gastroenteritis virus promotes increased glucose uptake through de novo synthesis of mrna and protein [ ] . within the last decade, the mechanisms of passive or diffusive components of intestinal glucose uptake have come under debate [ ] . the current paradigm describing intestinal uptake is that glucose enters the absorptive cell through sglt in the brush-border membrane and exits into the blood through glut , a member of the facilitative glucose transporter family, located in the basolateral membrane. we have now provided evidence for an alternative mechanism for the passive component of absorption, i.e., rapid trafficking of glut to the brush-border membrane was found to be controlled by the sglt -dependent activation of a protein kinase c (pkc)-dependent pathway and also by mitogen-activated protein kinase (mapk) intracellular signaling pathways [ ] [ ] [ ] . glut exhibits a > -fold-higher glucose transport capacity than sglt and provides a major route of glucose absorption with rapid absorptive capacity [ , ] . glut plays an important role in glucose absorption across the brush border membrane in normal jejunum [ ] . although our experiments did not directly demonstrate the rapid trafficking of glut , we found that tgev infection significantly increased glucose uptake at h, indicating accelerated glucose absorption. thus, tgev infection might promote glucose trafficking through glut . moreover, because the tgev increased the protein expression of p-egfr ( fig b) and tgev spike protein is capable of binding to egfr, thereby activating the downstream pkc-dependent and mapk intracellular signaling pathways [ ] , the trafficking of glut to the brush-border membrane could be stimulated, allowing rapid transport of glucose. previous studies have shown that glucose transport plays an important role in viral invasion. glut -mediated glucose transport regulates hiv infection [ ] and is a receptor for human t-cell leukemia virus (htlv). additionally, perturbations in glucose metabolism resulting from interactions between htlv envelope proteins and glut are likely to contribute to htlv-associated disorders [ ] ; thus, analysis of the relationship between viral infection and glucose uptake is critical. notably, in mock-infected cells, egfr mediate sglt protein expression. indeed, previous literatures reported that egfr maintains intracellular glucose levels through interaction with and stabilization of sglt [ ] [ ] . importantly, we found that egfr modulated sglt expression during infection, but it cannot be concluded unfortunately that the cellular effect of tgev infection on sglt is mediated via egfr. because the drug treatment and knock down/overexpression against sglt had similar effects with egfr (figs and ) . in other words, it could also be direct effect on sglt , which in results in similar effect on egfr. in fact, egfr and sglt are co-expressed in many other type cells [ , ] . moreover, previous studies have shown that egfr is a target receptor for viruses and bacteria, including tgev and escherichia coli [ , ] . thus, because egfr also interacts with sglt , this protein might comprise another ubiquitous target receptor. egfr has been reported to increased glucose uptake which is critical for the cell [ ] , we found the egfr and p-egfr both promote glucose uptake in ipec-j cells during tgev infection. previous literatures reported that egfr kinase activity regulates the peak glucose uptake and total egfr may likely be maintaining basal glucose uptake [ , ] . in our study, the ipec-j cells have been in a rich glucose medium during tgev infection, we also did not explore the respective role of egfr and p-egfr in glucose uptake. the mechanism through which tgev causes diarrhea has not been elucidated. on the other hand, glucose uptake has been shown to cause diarrhea [ , ] . for example, enteropathogenic e. coli rapidly inactivates sglt through multiple mechanisms. indeed, the finding that one mechanism occurs more rapidly than microvilli effacement provides a plausible explanation for the rapid onset of severe watery diarrhea, given the crucial role of sglt in the daily uptake of large amounts of fluids from the normal intestine. in contrast, our data indicate that tgev infection resulted in increased sglt expression. however, tgev infection did lead to rapid glucose uptake, which in turn supplied glucose to the virus and promoted long-term infection by tgev in the intestine. prevalence of porcine epidemic diarrhoea virus and transmissible gastroenteritis virus infection in korean pigs. the veterinary record culture to form protein using nutrients, isolation, culture of cells for expression of recombitant protein. google patents increased litter survival rates, reduced clinical illness and better lactogenic immunity against tgev in gilts that were primed as neonates with porcine respiratory coronavirus (prcv). veterinary microbiology na+-d-glucose cotransporter sglt is pivotal for intestinal glucose absorption and glucose-dependent incretin secretion the role of sglt and glut in intestinal glucose transport and sensing american journal of physiology endocrinology and metabolism egf-induced inhibition of glucose transport is mediated by pkc and mapk signal pathways in primary cultured chicken hepatocytes stimulation of glucose uptake by transforming growth factor beta: evidence for the requirement of epidermal growth factor-receptor activation the epidermal growth factor receptor regulates cofilin activity and promotes transmissible gastroenteritis virus entry into intestinal epithelial cells survival of cancer cells is maintained by egfr independent of its kinase activity 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and reviews phlorizin, a competitive inhibitor of glucose transport, facilitates memory storage in mice. neurobiology of learning and memory host cell glucose metabolism during abortive infection by adenovirus type early enhanced glucose uptake in human cytomegalovirus-infected cells enhanced regional uptake of -deoxy-d-[ c] glucose in focal herpes simplex type encephalitis autoradiographic study in the rat complete genome sequence of transmissible gastroenteritis coronavirus pur -mad clone and evolution of the purdue virus cluster virus taxonomy. classification and nomenclature of viruses-coronaviridae the coronavirus nucleocapsid protein. the coronaviridae: springer hepatitis b virus x protein regulates hepatic glucose homeostasis via activation of inducible nitric oxide synthase systems-level metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy hcv replication suppresses cellular glucose uptake through down-regulation of cell surface expression of glucose transporters rotavirus infection impairs intestinal brush-border membrane na+-solute cotransport activities in young rabbits transmissible gastroenteritis virus and porcine epidemic diarrhoea virus infection induces dramatic changes in the tight junctions and microfilaments of polarized ipec-j cells glucose and glutamine provide similar proportions of energy to mucosal cells of rat small intestine glutamine starvation enhances pcv replication via the phosphorylation of p mapk, as promoted by reducing glutathione levels glutamine metabolism is essential for human cytomegalovirus infection dietary regulation of the intestinal sodium-dependent glucose cotransporter (sglti) the facilitated component of intestinal glucose absorption regulation of glut , glut and intestinal brushborder fructose absorption by the extracellular signal-regulated kinase, p mitogen-activated kinase and phosphatidylinositol -kinase intracellular signalling pathways: implications for adaptation to diabetes stimulation of fructose transport across the intestinal brush-border membrane by pma is mediated by glut and dynamically regulated by protein kinase c the diffusive component of intestinal glucose absorption is mediated by the glucose-induced recruitment of glut to the brush-border membrane the slc family of facilitated hexose and polyol transporters apical glut : a major pathway of intestinal sugar absorption the facilitated component of intestinal glucose absorption glut -mediated glucose transport regulates hiv infection the ubiquitous glucose transporter glut- is a receptor for htlv localization of the na+-d-glucose cotransporter sglt in the blood-brain barrier a protein kinase involved in the regulation of inflammatory cytokine biosynthesis retention of native-like oligomerization states in transmembrane segment peptides: application to the escherichia coli aspartate receptor potent diarrheagenic mechanism mediated by the cooperative action of three enteropathogenic escherichia coli-injected effector proteins. proceedings of the national academy of sciences of the united states of america diarrhea caused by carbohydrate malabsorption key: cord- -bv dh eu authors: möstl, karin title: coronaviridae, pathogenetic and clinical aspects: an update date: - - journal: comparative immunology, microbiology and infectious diseases doi: . / - ( ) - sha: doc_id: cord_uid: bv dh eu abstract a review is given about pathogenetic and clinical aspects of the well-known as well as of recently detected members of the family coronaviridae. special attention is paid to coronavirus infections of domestic cattle and pets, whereas avian, murine, rat and human coronaviruses are summarized briefly. coronaviruses represent a large family of mammalian and avian pathogens, first described in [ ] . as several members of this virus group are known to cause economically important diseases, much effort was put into research during the last years. special reviews cover the replication strategy of coronaviruses [ ] , their glycoproteins [ ] and their structure and genome expression [ ] . the recent detection of previously unknown coronaviruses or mutants, like the "porcine epidemic diarrhea"-virus (pedv) and the tge-like "porcine respiratory coronavirus" (prcv) on one hand and new knowledge about pathogenetic mechanisms, for example in fipv-infections, on the other hand are the basis for this review article. coronaviruses are pleomorphic particles with a diameter of - nm, possessing typical club-shaped spikes. their physicochemical and biological properties and antigenic relationships were reviewed by wege et al. [ ] . antigenically they are grouped into classes: however, highly sensitive techniques like immunoblotting revealed some discrepancies. an antigenic relationship was detected between a mhv strain and hcv- e, a mhv strain and ibv and between fipv and hev (see ref. [ ] ). while the antigenic classification of pedv still remains unclear, an antigenic cross-reaction at the nucleocapsid level between pedv and fipv was detected [ ] . there are three known porcine coronaviruses: transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv) and hemagglutinating encephalomyelitis virus (hev). recently, a virus antigenically related to tgev spread through the european swine population. it infects only the respiratory tract and is therefore called "porcine respiratory coronavirus" (prcv) or "respiratory variant of tgev". transmissible gastroenteritis (tge) was first described by doyle and hutchings [ ] . the classical enteric variant of tgev is an enteropathogenic agent causing severe diarrhea. although swine of all ages are susceptible to infection, the most severe clinical symptoms are observed in newborn piglets reaching a mortality rate of about %. with increasing age the mortality rate declines due to age resistance and is very low in pigs over weeks of age. the virus usually enters by the oral route. it is able to resist the low ph of the gastric juices, reaches the small intestine and destroys the villous enterocytes causing atrophy of the villi. the resulting acute malabsorption syndrome is the consequence of a reduced enzymatic activity and cellular transport of nutrients and electrolytes of the damaged villous enterocytes. the replacement of villous epithelium in the small intestine is markedly accelerated in animals of weeks and older compared to newborn pigs. the young villous cells produce less virus resulting in an age-dependent resistance to tge [ ] . furthermore, an impaired lymphocyte cytotoxicity was found in newborn piglets and was accounted for the high susceptibility to tgev during the first weeks of life [ ] . the clinical signs in piglets usually are vomiting, followed by a watery yellowish diarrhea, loss in weight and dehydration. for details see pedersen [ ] . in animals younger than days death occurs about -- days after the onset of disease. in older pigs inappetence and diarrhea of short duration are observed, but subclinical infections also occur. sometimes lactating sows show severe clinical signs including vomiting, diarrhea, fever and agalactia. the incubation period varies between h and days. in a susceptible herd tgev spreads very rapidly to animals of all ages. this epizootic form usually lasts a few weeks, but may persist in an enzootic form, if susceptible animals are continually present [ ] . most sows of such herds are infected and thereafter provide passive immunity to their suckling piglets, which are protected to a variable degree. under such conditions diarrhea especially occurs after weaning [ ] . colostrum and milk of immune sows contain high titres of antibodies especially of the iga class (see ref. [ ] ), which protect piglets provided that the antibodies are in continual contact with the enterocytes of the gut mucosa. the term "lactogenic immunity" was coined by haelterman [ ] . active immunity is only acquired by gut infection resulting in the production of iga. sensitized lymphocytes migrate from the gut to the mammary gland leading to local iga production in lactating sows (gut-mammary immunologic link). after parenteral inoculation of tgev only antibodies of the igg class are produced, which mediate merely poor protection. in order to induce maternal immunity sows may be infected with virulent tgev at least weeks ante partum (planned infection). gut material of piglets which died in the acute phase of tge is fed to sows. as this procedure includes some disadvantages such as the possible transmission of other pathogenic agents, much effort has been made in order to develop other effective vaccination procedures in pregnant sows. none has proved satisfactory (see ref. [ ] ). however, oral application of live, nonattenuated virus resulted in elevated titres of protective milk-antibodies for days after farrowing [ ] . for diagnosis tgev antigen can be detected by immunofluorescence in the small intestine of piglets at an early stage of disease, by virus isolation in tissue culture or by elisa. for serological investigations the neutralization test is frequently used because of its sensitivity and reliability. in a variant of tgev was isolated in belgium [ ] and great britain [ ] . as it causes a respiratory infection and does not replicate in the enteric tract, it was named "respiratory variant" of tgev [ ] and recently "porcine respiratory coronavirus". it spreads quickly in the swine population, probably by an aerogenic route, replicates in the nasal mucosa, trachea and lungs and usually does not cause clinical signs [ , ] . recent studies, however, demonstrated that the virus can cause pneumonia [ ] . antibodies produced in infected animals cannot be differentiated from antibodies against the classical tgev by serum neutralization test. this fact causes problems in the sectors of import and export certificates. recently, using monoclonal antibodies an antigenic difference was demonstrated between tgev and prcv concerning an alteration of the e protein portion [ ] . it is the basis for an elisa distinguishing between antibodies against the two variants of tgev. according to hooyberghs et al. [ ] prcv-seropositive sows do not mediate protection to their piglets against tgev-field infection, whereas bernard et al. [ ] described such a protection under experimental challenge conditions. diarrhea in pigs very similar to tge was described in great britain [ ] . a "coronavirus-like" agent was identified as causative virus in [ , ] , that was different from the other known porcine coronaviruses. it was named pedv, in germany the name "epizootische virusdiarrhoe" (evd) was proposed [ ] , in france vannier and debouck [ ] called it "diarrh e pid mique porcine" (d.e.p.). the pathogenesis of ped is very similar to that of tge. the epithelial cells both of the small and partly of the large intestine are destroyed resulting in villous shortening. after an incubation period of h the clinical signs consisting of diarrhea, vomiting and dehydration appear and are hard to differentiate from those of tge [ ] . contrarily to tge, weaned and feeder pigs are the most affected groups [ ] . in previously uninfected herds, however, ped may result in severe clinical disease in suckling piglets and mortality rates of up to %. in general, tge spreads more quickly in infected herds causing a more severe syndrome [ ] , but heinritzi et al. [ ] described that during the last years cases of tge, they often showed an altered clinical course, more and more similar to ped. very similar pathogenetic mechanisms of ped and tge cause the same immunological situation. protection against ped is based on intestinal mucosal immunity, which is limited to a short period after infection. lactogenic immunity, but not circulating antibodies are protective for suckling piglets. there are no vaccines available. for diagnosis virus antigen is detected by immunofluorescence (see ref. [ ] ) or by a blocking-elisa [ ] . hev was first isolated from the brain of a suckling piglet with encephalomyelitis [ ] . later a virus was detected in animals with vwd that was antigenically related to hev [ ] . mengeling and cutlip [ ] identified hev as a responsible agent for the encephalomyelitis as well as for vwd. the virus is probably transmitted through nasal secretions [ ] . it first replicates in the upper respiratory tract, in general without inducing clinical symptoms. subsequently it spreads via peripheral nerves to the central nervous system causing either encephalomyelitis or vwd. severe clinical signs are seen almost exclusively in piglets younger than weeks. after an incubation period of - days they show repeated vomiting, are listless and pale and huddle together. andries [ ] postulated that vomiting is caused by viral replication in the vagal sensory ganglion or by infected neurons affecting the vomiting center. in the course of encephalomyelitis generalized muscle tremors and hyperesthesia are commonly observed, sometimes blindness, opisthotonus and nystagmus occur. weakness is followed by coma and death is observed in most of the young animals. the wasting was assumed to be caused by a disturbance of stomach emptying [ ] . although hev is distributed worldwide among pig populations, clinical disease occurs seldom. as most sows are seropositive, their piglets are protected by maternal antibodies till an age-dependent resistance has developed. during the phase of passive immunity subclinical infections occur inducing an active immunity in pigs - weeks of age (see ref. [ ] ). in order to avoid clinical signs in piglets it is favorable to obtain immune sows at the time of farrowing. for diagnosis virus may be isolated from the tonsils, the brain and lungs of diseased piglets by cultivation in several tissue culture systems. for serological investigations the hemagglutination-inhibition-or seroneutralization-test are commonly used. a coronavirus-tike agent was detected in the feces of a calf with diarrhea [ , ] and characterized as coronavirus [ ] . bcv was recognized as an economically important infectious agent producing diarrhea in newborn calves. later it was identified to have a second tropism in being involved in respiratory disease in older calves [ ] . after peroral incorporation bcv infects villous and to some extent crypt enterocytes. following experimental inoculation lesions in the small intestine were observed similar to those described for tge in pigs [ ] . after an incubation period of - h severe, often watery diarrhea is observed [ ] . in some cases an extensive loss of water and ions causes death. in some investigations the highest incidence of coronavirus-induced diarrhea was found during the second and third weeks of life, whereas other authors observed the most severe cases during the first week of life (see ref. [ ] ). as a typical enteric viral infection, bovine coronavirus infection can be prevented by passive local immunity (lactogenic immunity), whereas no or limited protection is mediated by serum antibodies. the mammary secretions of seropositive dams contain specific coronavirus-antibodies, which, contrary to the swine, belong to igg as the major isotype [ ] . for effective protection these antibodies have to be continuously present in the calf's gut lumen. but contrary to monogastric animals in cattle elevated antibody titres are only present in the colostrum and decrease sharply upon transition to milk. in parallel, protection against enteric infections declines. many attempts were made to enhance lactogenic immunity against coronavirus as well as rotavirus, a pathogenetically very similarly acting enteric viral pathogen. in order to prolong the protective effect of colostral antibodies first-day colostrum of seropositive cows was fed to calves, which proved beneficial if originating from vaccinated dams [ ] , but not from field-infected cows [ ] . most efficacious, however, is active vaccination of cows late in pregnancy. as on premises with enzootic infections almost all cows possess antibodies, a booster reaction occurs after parenteral vaccination resulting in a more effective and longerlasting lactogenic immunity. the route and time of immunization, the adjuvant used and the viral dose and form (live or inactivated) are of remarkable influence (reviewed in ref. [ ] ). some authors demonstrated significantly enhanced antibody-titres in mammary secretions of intramuscularly vaccinated cows, whilst other authors did not (see ref. [ ] ). in our investigations significant increases were not found either, although remarkable protective effects were shown by clinical and virological parameters [ ] . attempts to immunize calves actively by the oral route were unsuccessful under field conditions because of interference with maternally-derived antibodies [ , ] . the involvement of bcv in respiratory tract affection was first described by thomas et al. [ ] . coronaviruses were isolated in calves with non-febrile rhinitis and cough [ ] as well as in cases of severe pneumonia [ ] . in the course of two outbreaks of respiratory disease both syndromes occurred in an enzootically infected herd [ ] . remarkably, calves with persisting maternal antibodies were not affected, whereas older calves whose titres had just decreased fell sick. investigations performed by reynolds et al [ ] indicated that enteric as well as respiratory infection was caused by a monotypic bcv. so it was assumed that active vaccination of older calves might prevent clinical disease on premises affected by the respiratory form of bcv [ ] . since enteric viral agents are difficult to propagate in cell cultures, diagnosis first was performed by electron microscopy. the development of an elisa permitted detection of rota-and coronavirus antigen in fecal samples [ ] . a disease syndrome consisting of inflammation of the visceral serosa and exudation into ihe body cavities, called feline infectious peritonitis (fip), was first described by wolfe and griesemer [ ] . montali and strandberg [ ] observed a second form, characterized by granulomatous inflammation in several parenchymatous organs. in order to differentiate the two forms the first one was called "wet" or "effusive", the second one "dry" or "noneffusive". a viral agent morphologically similar to other coronaviruses was demonstrated and later characterized as a member of the family coronaviridae (see ref. [ ] ). many years later a feline coronavirus was isolated from mild enteral infections and was called feline enteric coronavirus (fecv). it was shown to be very closely related to fipv [ ] . fipv is distributed worldwide, but compared to the high percentage of seropositive cats, clinical disease is observed relatively rarely. strains of feline coronaviruses vary greatly in infectivity and virulence and may either induce fip or enteritis. strains causing enteritis are very infectious and therefore widely distributed. they are of low pathogenicity but induce the production of circulating antibodies. fipv strains, however, vary extremely in infectivity and virulence and only some of them cause typical signs of fip [ ] . by using monoclonal antibodies two antigenic types of feline coronaviruses with marked difference in the peplomer (e ) glycoproteins were found [ ] . these two types correlated well with virulent or avirulent isolates. although there are a few cases described with evidence for in utero infection (see ref. [ ] ), the most frequent route of infection is probably the oral ingestion, followed by virus replication in the intestinal epithelium. whereas fecv strains do not spread wider than into the lymph nodes, fip-causing virus strains have a tropism for phagocytic cells. they replicate in macrophages and are disseminated by them. pedersen [ ] assumed that not only the properties of the infecting virus strain were responsible for the outcome of the disease, but that also the immunologic situation of the host and the type and degree of developing immunity may be of great importance. while humoral antibodies were shown to enhance the development of clinical disease (see ref. [ ] ), immunity to fip seems to be cell-mediated. pedersen [ ] proposed a scheme for the pathogenesis of fip: a strong humoral, but lacking cellular immunity is assumed to be responsible for development of effusive fip. strong humoral and weak cellular immunity may lead to noneffusive fip. a strong cellular immunity, however, may inhibit the development of disease. weiss and cox [ ] demonstrated delayed-type hypersensitivity-like reactions to fipv in the skin of cats which had remained asymptomatic after a previous challenge-exposure with fipv. the assumption is supported by the fact that cats having recovered from fipv infections and harboring the virus in a latent form often develop clinical fip after a coinfection with feline leukemia virus (felv), that mainly depresses the cellular immunity. in general a high percentage of cats with naturally occurring fip is coinfected with felv. after an incubation period of some weeks to months the clinical signs of fip-disease start with chronic fever, accompanied by a progressive decline in the general condition. effusive fip is characterized by peritonitis and ascites and/or pleuritis causing pleural effusion and dyspnea. the peritoneal and pleural fluids as well as the blood serum contain very high levels of protein, especially of the beta and gamma globulins (see ref. [ ] ), the visceral serosa of the abdomen and the thorax show pyogranulomatous lesions. in cats with noneffusive fip the clinical signs are variable depending on the affected organ systems, where typical granulomatous lesions have developed. frequently ocular and cns signs are observed. clinical illness lasts for - weeks (in cats with noneffusive form also for a longer period) and usually leads to death. contrary, infection with fecv remains subclinical in most cats or induces only mild diarrhea. pedersen et al. [ ] assumed that many cats are virus carriers shedding fecv in their feces and that often kittens are infected after weaning. after infection with feline coronaviruses antibodies appear in the serum of clinically ill cats as well as of animals remaining healthy. frequently, but not always, there are very high anti-fipv-titres in the diseased cats. these as well as non virus-specific globulines lead to a characteristic hypergammaglobulinemia. antibodies play an important role in the pathogenesis of fip by leading to the development of circulating immune complexes which are deposited in several organs. such immune complexes were shown to consist of viral antigen, igg and complement (see ref. [ ] ). horzinek and osterhaus [ ] justly interpreted fip as an immune-complex disease. the role of humoral antibodies in fip pathogenesis and the unknown mechanism of immunity result in the lack of an effective vaccine. immunization with attenuated as well as inactivated virus strains does not mediate protection but on the contrary enhances clinical disease following virus exposure. heterotypic vaccination with for example tgev was not protective against fipv challenge (see ref. [ ] ). recently, however, christianson et al. [ ] characterized a temperature sensitive fipv, which propagates at °c, but not at 'c. it is avirulent, replicates only in the upper respiratory tract and the authors described that this mutant is able to stimulate protective immune responses without inducing sensitization of the cat. furthermore, consistent vaccination against felv is expected to reduce the occurrence of clinical fip by eliminating the suppression of the cat's immune response towards fip caused by felv infections. although presently most of the cats which have developed clinical signs of fip will die, sometimes spontaneous remission is observed. there is no consistently effective treatment available. in some cats therapeutic success may be achieved by application of glucocorticosteroids because of their immunosuppresive effect, but in most cases the fatal course of the disease is only prolonged [ ] . as a diagnostic aid serological tests are used. a heterologous indirect immunofluorescence assay was developed by osterhaus et al. [ ] using tgev-infected cells as antigen, later elisas became commercially available. however, neither test allows differentiation between cats infected with fipv or fecv and between virus shedders and non-shedders. the test result often is helpful in ascertaining diagnosis in clinically sick cats, as animals with acute fip usually develop very high titres. on the other hand a significant decrease of titre may be observed in preterminal stages. unfortunately, relatively high titres may also occur in healthy cats with subclinical or latent infections. because of these difficulties interpretation of serological tests offers limited value to the clinician. the tests are, however, helpful for screening purposes and epidemiological investigations. recently, walter et al. [ ] reported a modified avidin-biotin-peroxidase-complexmethod in order to demonstrate viral antigens in paraffin sections of tissues of fipvinfected carcasses. epizootics caused by ccv were reported in germany [ ] and in u.s.a. [ ] . in most cases a mild gastroenteritis was the only symptom recorded, but especially in younger pups more severe signs of disease were observed. as indicated by serological investigations ccv is widespread within canine populations. in germany a seroprevalence of % was described [ ] , but only in % of diarrheic feces coronaviruses were detected [ ] . distress and several infectious agents aggravate clinical disease by contributing to the "canine enteritis complex" [ ] . after oral inoculation of ccv the enterocytes of the intestinal villi are infected leading to villous atrophy. as the infection does not spread further than to the mesenteric lymph nodes, no viremia occurs. after an incubation period of days clinical signs consist of diarrhea, vomiting, depression, anorexia and dehydration [ ] . similar to other typical enteric viral infections the local immunity of the gut appears to be responsible for protection [ ] . after infection with ccv low serum antibody titres develop that do not mediate immunity. a killed ccv vaccine is currently under investigation for its effectiveness (see ref. [ ] ). many different murine coronavirus strains have been described, the first one was isolated from a spontaneously paralyzed mouse [ ] . infections with mhv often take a subclinical or inapparent course. depending on the virus strain, the age of the affected animal, its genetic background and immunological situation, disease occurs resulting in acute fatal hepatitis or encephalomyelitis or enteritis. the infection of the nervous system takes place by invasion via the olfactory nerve after an initial replication in the nasal mucosa and is characterized by an acute or chronic demyelinating disease. enteropathogenic strains of mhv cause an acute intestinal disease with a high mortality rate during the first weeks of life. the aspects of infections with mhv were reviewed by wege et al. [ ] . two virus strains have been reported, the first infecting the respiratory tract and causing respiratory disease, especially during the first h of life. in animals older than -- days the course of the disease is mild or clinically inapparent [ ] . the second virus strain affects the salivary and lacrimal glands leading to sialodacryoadenitis with rhinitis [ ] . infectious bronchitis virus (ibv) is an economically important pathogen for the chicken raising industry. it causes respiratory disease, which was already described in [ ] and is distributed worldwide. at least serotypes of ibv have been described. after infection of the respiratory tract, usually by aerogenic spreading, the virus also replicates in the kidneys, the ovaries and oviduct. the clinical signs consist of tracheitis, bronchitis, decrease of egg production and egg quality. sometimes nephritis is observed. the most severe disease occurs in chickens up to an age of weeks with a mortality of - %, whereas in older animals ibv infection takes a milder course. for prophylactic purposes modified live vaccines are applicated by drinking-water. turkey coronavirus (tcv) was identified as the aetiologic agent of the transmissible enteritis of turkeys [ ] . while the infection takes a mild course in older animals, turkeys between and weeks of age develop depression, loss of appetite and weight and watery diarrhea. ensuing circulation disorders mediate the typical syndrome "bluecomb disease". the pathogenetic mechanisms are very similar to other enteric coronavirus infections resulting in villous atrophy. protection is only achieved by local immunity. for a comprehensive review of clinical signs, pathogenesis and immunology of avian coronaviruses see wege et al. [ ] . human coronaviruses are distributed worldwide. they infect the respiratory tract and are responsible for common colds [ ] , in children pneumonia may be observed. additionally, coronavirus-like particles were detected in diarrheal stool specimens from humans [ ] and primates [ ] . in the u.s.a. coronavirus-like particles were demonstrated in the feces of foals suffering from severe diarrhea [ ] . huang et al. [ ] isolated a coronavirus-like agent from horses suffering from acute equine diarrhea syndrome, called "potomac fever". horses of all ages were affected showing fever, inappetence and diarrhea followed by death in about % of the cases. coronaviruses show different organ tropisms, mainly resulting in three disease complexes: gastrointestinal disease, respiratory disease and generalized disease. the different pathogenetic mechanisms implicate distinct immunological situations. the important role of the local immunity in enteric infections has been well documented. in newborn animals protection by lactogenic immunity can be enhanced by vaccination of the dam quite successfully in the bovine and to a lesser extent in the sow. fipv-infections, however, are crucial at present with regard to prognosis as well as immunoprophylaxis. maybe virus mutants with specific tropisms will offer new aspects for vaccination, as for example prcv against tge and the ts-mutant against fip. coronavirus leader-rna-primed transcription: an alternative mechanism to rna splicing the novel glycoproteins ofcoronaviruses coronaviruses: structure and genome expression the biology and pathogenesis ofcoronaviruses porcine epidemic diarrhea virus (cv ) and feline infectious peritonitis virus (fipv) are antigenically related a transmissible gastroenteritis in pigs age-dependent resistance to transmissible gastroenteritis of swine antibody-dependent and spontaneous cell-mediated cytotoxicity against transmissible gastroenteritis virus infected cells by lymphocytes from sows, fetuses and neonatal piglets. ('an transmissible gastroenteritis virus (classical enteric variant) neue aspekte im klinischen verlauf der coronavirusinfektion der schweine lactogenic immunity to transmissible gastroenteritis of swine relationship among transmissible gastroenteritis virus antibody titers in serum, colostrum, and milk from vaccinated sows, and protection in their suckling pigs isolation of a porcine respiratory, non-enteric, coronavirus related to transmissible gastroenteritis new porcine coronavirus? transmissible gastroenteritis virus (respiratory variant) isolement, identification et pouvoir pathogene chez le pore d'un coronavirus apparent~ au virus de la gastro-enterite transmissible antigenic differentiation between transmissible gastroenteritis virus of swine and a related porcine respiratory coronavirus transmissible gastroenteritis: outbreaks in swine herds previously infected with a tgev-like porcine respiratory coronavirus natural infection with the porcine respiratory coronavirus induces 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antigenen bei der infektion der katze durch ein coronavirus (fip) und der lnfektion des hundes durch das parvovirus-typ recovery and characterization of a coronavirus from military dogs with diarrhea canine viral enteritis. i. status report on corona-and parvo-like viral enteritides elektronenmikroskopische nachweisrate enteraler viren bei durchfallkrankheiten von hund, katze, kalb, schwein und fohlen im jahr --elektronenmikroskopische untersuchungsbefunde update on canine coronavirus infections and interactions with other enteric pathogens of the dog canine coronavirus a murine virus ohm) causing disseminated encephalomyelitis with extensive destruction of myelin. i. isolation and biological properties of the virus rat coronavirus (rcv): a prevalent, naturally occurring pneumotropic virus of rats. arch. ges characterization of the virus of sialodacryoadenitis of rats: a member of the coronavirus group an apparently new respiratory disease of baby chicks electron microscopy of coronavirus-like particles characteristic of turkey bluecomb disease seroepidemiologic studies of coronavirus infection in adults and children coronavirus particles in faeces from patients with gastroenteritis enteric coronaviruses in primates coronavirus and gastroenteritis in foals isolation of coronavirus-like agent from horses suffering from acute equine diarrhoea syndrome key: cord- -i tkdgy authors: suo, siqingaowa; wang, xue; zarlenga, dante; bu, ri-e; ren, yudong; ren, xiaofeng title: phage display for identifying peptides that bind the spike protein of transmissible gastroenteritis virus and possess diagnostic potential date: - - journal: virus genes doi: . /s - - - sha: doc_id: cord_uid: i tkdgy the spike (s) protein of porcine transmissible gastroenteritis virus (tgev) is located within the viral envelope and is the only structural protein that possesses epitopes capable of inducing virus-neutralizing antibodies. among the four n-terminal antigenic sites a, b, c, and d, site a and to a lesser extent site d (s-ad) induce key neutralizing antibodies. recently, we expressed s-ad (rs-ad) in recombinant form. in the current study, we used the rs-ad as an immobilized target to identify peptides from a phage-display library with application for diagnosis. among the phages selected that specifically bound to rs-ad, the phage bearing the peptide tlnmhlfpfhtg bound with the highest affinity and was subsequently used to develop a phage-based elisa for tgev. when compared with conventional antibody-based elisa, phage-mediated elisa was more sensitive; however, it did not perform better than semi-quantitative rt-pcr, though phage-mediated elisa was quicker and easier to set up. transmissible gastroenteritis virus (tgev) is a member of the coronaviridae family and is a major cause of enteric disease in pigs where it threatens swine production and triggers substantial economic losses in the industry [ ] [ ] [ ] [ ] . its genome is composed of positive-stranded rna approximately . -kb in length. the virus consists of four structural proteins: envelope (e), membrane (m), spike (s), and nucleocapsid (n) proteins [ , , ] . non-structural proteins, which comprise two-thirds of the -proximal end, are encoded by open reading frames a and ab as well as the replicase. in contrast, the end of the genome encodes both non-structural and structural proteins ( -s- a- b-e-m-n- - ) [ ] . the s protein, which induces neutralizing antibodies, is important in the initiation of infection [ ] [ ] [ ] and has been further delineated into four antigenic sites a, b, c, and d which are located within the n-terminal region of the s protein [ ] . among these, only site a and to a lesser extent site d (herein defined as s-ad) are involved in eliciting neutralizing antibodies. recent work demonstrated that recombinant s-ad (rs-ad) was able to induce antibodies capable of neutralizing tgev infection in vitro [ ] . edited attenuated or inactivated tgev vaccines are less than optimal because they are capable of reverting back to virulent phenotypes and generally do not prevent viral shedding. therefore, effective diagnostic tests have become important in virus management and control. phage display is a proven technology for identifying small peptide ligands that can bind specific target proteins [ ] [ ] [ ] [ ] . it has been utilized in antibody engineering [ ] , drug discovery [ ] , vaccine development [ ] , and molecular diagnosis. in virology, phage display has been used to identify peptides that interact with several viruses such as bovine rotavirus [ ] , adenovirus type [ ], andes virus [ ] , sin nombre virus [ ] , coronavirus [ ] , and avian h n virus [ ] . herein, we use similar technology and advance previous work by using the rs-ad as an immobilizing target to select phages from a peptide display library, with diagnostic potential for tgev. our results indicate that phages bearing peptide ligands that bind rs-ad can be used to develop a phage-mediated elisa with high sensitivity and specificity to distinguish tgev from other common swine viruses. biopanning swine testis (st) cells were purchased from atcc and used to propagate tgev strain pur -mad [ ] . the rs-ad was produced and purified as described elsewhere [ ] . a -mer phage-display library was purchased from new england biolabs for panning according to published protocols [ , , ] using the rs-ad as a target at a concentration of lg/well. the -well plates coated with rs-ad, were initially incubated with the phage library ( . pfu/ml; ll/well) suspended in tbst ( mm tris-hcl [ph . ], mm nacl, . % tween- ) for min. subsequent pannings , , and were performed using incrementally higher concentrations of tween- . the phage titers of the input, output (elution), and amplified phages were determined as defined by the manufacturer. indirect elisa was used to assess the phages that remained after four rounds of biopanning. either tgev ( . mg/ml) or rs-ad ( lg/well) in . m nahco ph . was used to coat -well plates at °c for h. the next day, the plates were blocked with % bovine serum albumin (bsa) in tbs (tbsb) for h, washed ( ) with tbst, and then incubated with phage ( . pfu/ml in . m nahco , ph . ; lg/well) for h at °c. the plates were again washed with tbst, then incubated for h at °c with rabbit anti-m antibody ( : in tbsb; abcam), followed by horseradish peroxidase (hrp)-conjugated goat anti-rabbit igg antibody (garp; : in tbsb, sigma). the od nm was determined in triplicate as previously described [ ] . ten phages with the highest affinity for binding rs-ad as determined by elisa were amplified, precipitated with polyethylene glycol-nacl, and then used for dna extraction according to the manufacturer's instructions (new england biolabs). amplification of the genes encoding the exogenous peptides was performed using sense ( -tcacctcgaaagcaagctga) and anti-sense ( -ccctcatagttagcgtaacg) m primers followed by dna sequencing [ , ] . the pcr conditions were as follows: °c for min, cycles of °c for s, °c for s, °c for s, and a final extension at °c for min. to compare the sensitivities of phage-mediated elisa to antibody-mediated elisa, tgev serially diluted in . m nahco (ph . ) was coated onto duplicate elisa plates overnight at °c followed by blocking with % skim milk for h at rt. the selected phages or unbound phage complexes (negative control) diluted in pbs ( . pfu/ml) were added to one set of plates, followed by anti-m antibody ( : in pbs ? bsa). to the second set of duplicate plates, rabbit anti-tgev polyclonal antiserum serially diluted in pbs ? bsa, and normal rabbit serum were added as the primary and control antibodies, respectively. after incubating both sets of plates for h at °c followed by extensive washing, garp ( : ) was added as described above. the od values were read on all plates; od ratios where od (sample-negative standard) (p)/od (positive control-negative standard) (n) [ were judged as positive. all experiments were performed in triplicate. the tcid of tgev was determined using the reed-muench method, and tgev was adjusted to . mg/ml in pbs. total rna was extracted from ll of virus (fastgene, china) and dissolved in ll of sterile water. reverse transcription was performed in ll using ll of rna ( ng/ll), oligo dt as primer, and m-mlv reverse transcriptase as recommended by the manufacturer (takara, china). the resulting cdna ( ll) was used as a template for pcr in ll which included . ll of easy taq polymerase (takara, china), ll of dntp ( . mm), pcr buffer ( ll), and . ll each of sense ( -cttagtagtaatattttgcatac) and antisense ( -tatagcagatgatagaattaaca) primers. amplification conditions were as follows: °c for min, then cycles of °c s, . °c s, and °c s followed by a final extension at °c for min. the amplified fragment was confirmed by dna sequencing. phage specificity was evaluated against the following panel of porcine viruses: tgev, strain hr/dn [ ] , porcine epidemic diarrhea virus (pedv; strain hljby) [ ] , porcine reproductive and respiratory syndrome virus (prrsv; strain jilintn ) [ ] , porcine circovirus type ii (pcv ; strain pcv -ljr) [ ] , porcine parvovirus (ppv; strain ppv ) [ ] , porcine pseudorabies virus (prv; strain kaplan) [ , ] , and porcine rotavirus (prov; isolate dn ) [ ] . all viruses were initially coated at lg/ml then serially diluted in . m nahco (ph . ) and subjected to phage-elisa as described above. average od values were obtained from three independent experiments. data were collated and the mean ± sd values were determined. arithmetic means were compared between treatment groups using anova (spss . ; spss inc., chicago, illinois, usa) followed by duncan's multiplerange test. values of p \ . and p \ . were defined as statistically significant (''*'') or highly significant (''**''), respectively. in this study, we used phage display to select -mer peptides that bind rs-ad [ ] and that may function for diagnosing tgev infections. after four rounds of panning, rs-ad-specific phages increased from . in the first round to . in the fourth round (table ) . following the last screen, we selected phage clones from the original that bound both rs-ad and tgev. this subset was characterized by elisa with respect to their binding efficiencies (fig. ) . pcr amplification and sequencing indicated that nine distinct -mer peptides were identified among the phages that were selected ( table ). in contrast to previous reports [ , , ] , these peptides exhibited substantial sequence diversity in the number of peptides that bound to rs-ad. it is not known if this relates to the length of the target protein or to changes made in the panning process to enhance binding specificity. as shown in fig. , we selected four (phtgev-sad- , phtgev-sad / , phtgev-sad , phtgev-sad ) of the ten phages with the highest binding affinity to tgev for further testing. the lowest detectable quantity of tgev for the above defined phages was . , . , . , and . mg, respectively, suggesting that phtgev-sad was the most sensitive when used in a phage-based elisa. binding directly to tgev was uncharacteristically better than binding to the rs-ad used in the selection process (figs. , ) . this is likely attributable to more complete folding of the native protein or to better accessibility of the binding epitope in the native form. the minimum quantity of tgev required for detection via antibody-based elisa was . lg (p/n value [ ) (fig. ) , whereas the minimum quantity of tgev required for phtgev-sad -based elisa was . lg. this is consistent with the phage-mediated elisa being more sensitive than conventional antibody elisa. a number of elisa-based assays have been developed over the years for detecting tgev, many of which have been directed at differentiating tgev from prcv-infected animals. among the earlier ones, sestak et al. [ ] targeted the s glycoprotein of tgev in a competition elisa where recombinant s protein was coated onto plates and used to capture host antibodies. using a monoclonal ab to epitope d and which is specific for tgev, the investigators were able to differentiate the infectious agents. liu et al. [ ] cloned and expressed the nucleoprotein (n) to develop an elisa. compared to the virus neutralization assay, they demonstrated % sensitivity and specificity; however, they did not characterize or address the lower level of sensitivity in vitro or in vivo. in , elia et al. [ ] used the recombinant s protein to develop an elisa to assess swine-like tgev coronaviruses in canine hosts. given the novelty of the virus, they were unable to compare it to other assays currently in use. zou et al. [ ] use techniques similar to those developed here, i.e., peptide display, to target the m protein of tgev in developing an elisa-based diagnostic test. in this case, the sensitivity of the elisa exceeded that when the phage-mediated elisa and antibody elisa were compared to rt-pcr which targeted a -base pair fragment of the s gene, the rt-pcr was most sensitive of all assays tested. this is not unexpected given the higher sensitivity of pcr assays in general. pcr amplification was positive using cdna equivalents of . lg of tgev (data not shown). real-time pcr and/or nested pcr would clearly have generated even more sensitive results. in addition, phages expressing peptide that bind to tgev s-ad did not bind to other selected viruses (fig. ) . table sequences of tgev rs-ad peptides. predicted amino acid sequences were generated for ten selected phages in summary, we identified peptides that specifically bind to tgev and can form the basis of new diagnostic tests where the sensitivity of phtgev-sad was . lg of tgev. this sensitivity fared quite well when compared to the antibody-mediated elisa which had a sensitivity of . lg but fell short of the sensitivity of rt-pcr; however, phtgev-sad- provides a quicker and less costly alternative to rt-pcr. diseases of swine th ed the authors declare no conflicts of interest. key: cord- -mmdeyph authors: paton, d. j.; brown, i. h. title: sows infected in pregnancy with porcine respiratory coronavirus show no evidence of protecting their sucking piglets against transmissible gastroenteritis date: journal: vet res commun doi: . /bf sha: doc_id: cord_uid: mmdeyph eighteen litters of sucking piglets were challenged with one of two strains of transmissible gastroenteritis virus (tgev). during pregnancy, their seronegative dams had been either inoculated intranasally with porcine respiratory coronavirus (prcv), inoculated orally with tgev or left untreated. on the basis of weight gain, clinical signs and survival, no differences in response to challenge was detected when piglets suckled by prcv inoculated sows were compared with those suckled by uninoculated sows. such a difference was evident when the litters of sows successfully pre-immunized with tgev were compared with those of unicoculated or prcv-inoculated sows. the possibility of transplacental transmission of prcv was investigated in two litters born to sows that had been inoculated with this virus in late pregnancy. all sixteen live-born piglets were seronegative for the virus at birth and prcv was not isolated from tissues taken from two stillborn piglets. on the basis of weight gain, clinical signs and survival, no differences in response to challenge was detected when piglets suckled by prcv inoculated sows were compared with those suckled by uninoculated sows. such a difference was evident when the litters of sows successfully pm-immunized with tgev were compared with those of uninoculated or prcv-inoculated sows. the possibility of transplacental transmission of prcv was investigated in two litters born to sows that had been inoculated with this virus in late pregnancy. all sixteen live-born piglets were seronegative for the virus at birth and prcv was not isolated from tissues taken from two stillborn piglets. keywords: epidemiology, pigs, pregnancy, protection, respiratory coronavirus, transmissible gastroenteritis virus transmissible gastroenteritis virus (tgev) is a coronavirus which can affect all ages of pigs, although resistance to the disease increases with age. sucking piglets are the most susceptible and the disease in these animals is characterized by vomiting, watery diarrhoea, dehydration and a high mortality. pigs that recover from tge develop an immunity that protects them against re-infection. immune sows can also passively protect their sucking piglets against tge (bay et al., ) by antibodies present in milk (haelterman, % ). tgev has been isolated from a number of organs, including the respiratory tract. however, the major target organ is the gut, where multiplication of the virus leads to villous atrophy, gastroenteritis and viral dissemination in faeces. porcine respiratory coronavirus (prcv) is a new variant of tgev with an altered pathogenesis and epidemiology (pensaert er af., ): it multiplies mainly in the respiratory tract and spreads between pig herds aerogenically. there are no enteric symptoms and, in experimentally infected pigs, even the respiratory tract infection is usually asymptomatic, although an exception to this has been reported by van nieuwstadt and pol ( ) . despite the in vivo differences in the behaviour of tgev and prcv, the two viruses are closely related antigenically and antibodies raised against either virus neutralize the heterologous virus equally effectively in in vitro tests. apparent associations between the dissemination of prcv and reduction in the incidence of tgev (jestin et az., ) raise the question of whether or not this crossneutralization may have in vivo sign&axe. this study was undertaken to investigate whether or not previous exposure of sows to the now widespread but relatively avirulent prcv would cross-protect their sucking litters against the less common but more virulent disease of tge. the possibility of transplacental transmission of prcv was investigated because of anecdotal reports of herd reproductive problems associated with prcv seroconversion and because of interest in the prcv status of hysterectomy-derived piglets. and methods viral inocula prcv inocula were prepared from a uk field isolate of the virus (stopps). a study of the pathogenesis of this isolate has already been reported by o'toole et al. ( ) . the virus was shown to multiply mainly in the respiratory tract, with only isolated foci of intestinal infection. a lyophilized stock of the virus which had been passaged three times in primary pig kidney monolayers (ppkm) was passaged once more, either in ppkm or in a five-day-old colostrum-deprived piglet, to provide the virus for inoculation of the pregnant sows. the ppkm virus had a titre of lde tcid ,, ml-'. the colostrum-deprived piglet was killed h after prcv infection. a % ?ung homogenate (lh) was made in phosphate buffered saline containin units penicillin ml-', pg streptomycin ml-' and kl units mycostatin ml -? (pbsa). this had a virus titre of * tcid f ml-' in ppkm. sows received two ml doses of ppkm virus or a single ml dose o lh virus. the tgev strains used were derived from a uk field isolate ps / ( ) and the miller strain of us origin. the virus had been passaged times through secondary pig thyroid monolayers, whilst the miller virus had not been tissue culture adapted. a gut homogenate of each was prepared by the oral inoculation of one-day-old colostrum-deprived piglets. twenty-two hours after infection, the piglets were killed and the small intestine of each was removed and homogenized in pbsa. aliquots of these stocks were stored at - °c and thawed immediately before use as inocula. a % lethal dose (ld ) for neonatal pigs was determined for each gut homogenate by orally inoculating a series of tenfold dilutions into three-day-old piglets housed in isolators. six piglets were used in this way for the titration of each stock. thereafter, a ml dose of ld was used to challenge neonates. pregnant sows were given a ml dose of ld or were fed on half a small intestine from a nine-day-old piglet infected one day before with ld of the miller stock homogenate. the pigs in this study came from herds known to be free from tgev and prcv infection. before entering the experiments, sera from all pigs were shown to be free of neutralizing antibodies to tgev and prcv when tested at a : dilution in an in vitro virus neutralization (vn) test as described by paton ( ) . serum dilutions were incubated in microtitre plates at °c for h with tcid,, of tissue culture adapted tgev, strain ps / . the plates were then seeded with a dog rectal tumour cell line (a ). the neutralization titre was the highest dilution of serum which completely inhibited viral cytopathic effect after five days. the piglets used for preparing and titrating virus stocks came from the laboratory's own closed herd. eighteen pregnant sows were obtained from commercial farms, from one herd, and (sows and ) from another (table i) . they were brought to the laboratory approximately eight weeks before their expected farrowing dates and were housed in isolation thereafter. in an attempt to synchronize farrowings, pg of the luteolytic agent cloprostenol ('planate': coopers) were given by intramuscular injection to most of the sows at between and days of gestation (table ii) . sows farrowed in crates, in strawed cubicles, with piglet heat lamps to one side. two days before tgev challenge, all piglets had their teeth clipped and were given iron injections. piglet water troughs were provided from the day of challenge onwards. pregnant sows were inoculated with prcv or tgev or left untreated (table i) . five sows were given second inoculations with prcv to see if this would boost their immune responses. sows and were given virus passaged in another pig in order to more closely mimic the tgev inoculations. sows , and were given second doses of tgev in an attempt to increase the chances of their being successfully immunized. prcv inoculations were given intranasahy, whilst tgev inoculations were given intraorally after overnight fasting. for both purposes, a syringe attached to a short length of flexible plastic tubing was used. all piglets were dosed with tgev orally with a syringe. age at challenge and strain of virus given are shown in table ii . sow seroconversions were monitored by regular blood sampling. precolostral blood was collected from the umbilical cords of newborn piglets from the litters of three sows to test for in utero seroconversion. two of these sows had received prcv in pregnancy (nos. and ), whilst the other (no. ) was an uninoculated control. samples of tonsil, trachea, lung, pulmonary lymph node, submandibular lymph node, duodenum, jejunum, ileum and mesenteric lymph node were taken from two pigs which were stillborn in the litter of sow for attempted virus isolation. tissues were prepared as % homogenates in pbsa, incubated for min at room temperature, clarified at g for min and then the resulting supernatants were inoculated onto ppkm. a single passage was made after seven days. all cultures were observed daily for cytopathic effects. cultures were initially grown in a hank's based medium containing % bovine serum and antibiotics. the maintenance medium was earle's containing % bovine serum and antibiotics. all piglets were bled from the jugular vein or anterior vena cava on the day before being challenged with tgev. colostrum and milk were collected from sows, where necessary, with the aid of an intramuscular injection of iu of oxytocin ('oxytocin-s': intervet). serum, colostrum and milk were examined for prcv/tgev antibodies by the vn test (paton, ) . all the piglets were weighed daily from soon after birth. they were examined at least twice daily for signs of illness, including diarrhoea, and assigned a daily clinical score of to , based on physical appearance and demeanour (see clinical scoring criteria: table iii ). an index of illness for each litter was calculated by averaging the worst clinical score achieved by each of that litter's piglets (average worst clinical score per litter). an attempt was also made to quantify the duration of diarrhoea for each litter by calculating the percentage of piglet days on which diarrhoea was observed from to days post-challenge (percentage piglet diarrhoea days). piglet faeces samples were collected regularly and examined for tgev by elisa. the elisa method, which has been described by paton ( ) , employed a solid phase, double antibody sandwich, incorporating a monoclonal capture antibody and a peroxidase labelled polyclonal indicator antibody. any piglets that became exceptionally weak or moribund were killed humanely. the mann-whitney test with one-sided probabilities was used to compare data from different groups of sows. the serum antibody titres in the sows two days before far-rowing are shown in table i . the antibody titres in colostrum, in prechallenge piglet sera and in milk collected on the day of piglet challenge are shown in table ii . some samples could not be assayed at dilutions of less than : or : because of cytotoxicity. none of the sows inoculated with prcv showed any signs of illness but all had seroconverted prior to farrowing. where given, the second inoculation of prcv did not appear to affect the antibody response. of the three sows inoculated in pregnancy with virus, only sow showed signs of illness (anorexia and diarrhoea on days and post-inoculation) and this sow was also the only one to seroconvert. sows and , inoculated with the miller strain of tgev, showed no illness, but both seroconverted. the three tgev seropositive sows transferred broadly similar amounts of colostral prcv/tgev neutralizing antibody to their piglets, as did their prcv seropositive counterparts. reciprocal titres of neutralizing antibody levels in milk on the day of challenge of the piglets ranged from to for the three tgev seropositive sows' litters and from to for the seven prcv seropositive sows' litters. the piglets from all three litters blood sampled before ingestion of colostrum had no serum neutralizing antibodies to tgev/prcv. the two stillborn pigs from sow appeared grossly normal post mortem apart from unexpanded lungs. virus was not isolated from any of the tissues sampled. four sows (nos. , , and ) farrowed earlier than expected and before receiving cloprostenol. consequently their piglets were slightly older at the point of challenge (table ii) . following infection of the piglets with tgev, a number of nursing sows became ill. a variety of clinical signs were observed, including anorexia, pyrexia, diarrhoea and agalactia. data showing the effects of challenge on piglets and on sows are given in table iii . tgev excretion in piglet faeces was confirmed by elba for all litters except that of sow no. , where there was no diarrhoea. sow serum antibody titres at the end of the experiments are shown in table i . three sows remained seronegative, even though they had been suckling piglets challenged with the strain of tgev - days previously. results obtained for both challenge strains of tgev appeared to be similar and, for comparative purposes, they have been grouped together. the three litters born to tgev inoculated sows which had seroconverted had lower values than any of the six litters born to uninoculated sows or any of the seven litters born to prcv inoculated sows in respect of proportion of piglets scouring, percentage piglet diarrhoea days and average worst clinical score. these differences are statistically significant, with p = . for the difference between tgev seropositive and uninoculated sows andp = . for the difference between tgev seropositive and prcv inoculated sows. the proportion of piglets dying was zero in all the tgev seropositive sow litters and this was lower than in any of the prcv litters. this again is significant with p = . . one uninoculated sow litter also had no deaths, so the p value is . for the difference between tgev seropositive and uninoculated sows. in respect of all these measures, the litters born to prcv inoculated and to uninoculated sows were similar and no significant differences were found, all probabilities exceeding . . the sows infected with prcv in late pregnancy farrowed apparently normal piglets. precolostral sera from two of these litters were all prcv seronegative and the virus could not be isolated from two stillborn littermates. thus there was no evidence that transplacental transfer of prcv had occurred. the virus had a low infectivity for sows. this might have resulted from its passage in tissue culture. the variation in mortality amongst piglets within similar treatment groups might in part have been due to variable sow illness and agalactia. although tgev was excreted by most of the challenged piglets, the challenge doses were evidently low, since many piglets sucking unimmunized sows survived. the doses had been determined by titrations carried out in piglets removed from their dams and it would therefore appear that such animals are much more susceptible to tge than naturally nursed ones. one benefit of a low challenge dose should be a high sensitivity for detection of low levels of lactogenic protection which might otherwise be swamped. no evidence for cross-protection between immunity to prcv and tgev was found in this study but the number of litters was too small to be certain that none exists whatsoever. however, statistically significant protection was demonstrated in the successfully tgev inoculated group, despite this comprising only three litters, relative to both other groups, while no differences could be found between these other groups, which were both larger. thus if cross-protection does occur it is clearly of a much lesser order than that provided by the homologous virus. this study used a single strain of prcv and two strains of tgev. other strains exist and could give different results. the present association in the uk and other european countries between a high prevalence of prcv and a low incidence of tge cannot in itself be taken as conclusive evidence of cross-protection, since tge incidence has been known to fluctuate widely in the past. attenuation of tgev by tissue culture passage can produce viruses which retain their respiratory tropism, but lose their entero-pathogenic&y (furuuchi et al., ) . in this respect, they are similar to prcv. such attenuated viruses have been extensively investigated for use as possible tge vaccines, but have generally been found to be not fully effective (saif and bohl, ) . bernard et al. ( ) gave tgev to piglets sucking sows that had previously been naturally infected with prcv. they concluded that these sows did provide some lactogenic protection to their litters, although this was less than that provided by sows immunized with virulent tgev. hooyberghs et al. ( ) reported outbreaks of tge affecting sucking piglets in herds previously infected with prcv. the piglets did not seem to be protected, although recovery on a herd basis was possibly more rapid. van nieuwstadt ef al. ( ) , using experimental animals, found no evidence that prior infection of young pigs with prcv protected them from a later challenge with tgev. the lack of cross-protection observed in this study was in spite of similar levels of prcv/tgev neutralizing antibody in the serum, colostrum and milk of prcv immunized and tgev immunized sows. previous studies have shown that iga in milk is of paramount importance in protection of sucking piglets against tge (saif and bohl, ) . further work is therefore in progress to characterize with respect to class the anti-prcv/tgev antibodies from the sows in this experiment. this study failed to demonstrate any evidence that previous infection of sows with a uk prcv isolate could provide lactogenic protection against tge caused by the or miller strains of tgev. prcv infection of two sows in the last third of gestation did not result in detectable fetal infections. transmissible gastroenteritis in swine. a study of immunity we are very grateful for the assistance provided to us by mr b.n.j. parker and colleagues, the staff at grange farm and our own colleagues in the porcine section of the virology department.mr m. richards and mr a.r. sayers produced the statistical analyses and mrs r. pearce typed the manuscript. key: cord- -yr dq authors: hulkower, rachel l.; casanova, lisa m.; rutala, william a.; weber, david j.; sobsey, mark d. title: inactivation of surrogate coronaviruses on hard surfaces by health care germicides date: - - journal: american journal of infection control doi: . /j.ajic. . . sha: doc_id: cord_uid: yr dq background in the severe acute respiratory syndrome outbreak, finding viral nucleic acids on hospital surfaces suggested surfaces could play a role in spread in health care environments. surface disinfection may interrupt transmission, but few data exist on the effectiveness of health care germicides against coronaviruses on surfaces. methods the efficacy of health care germicides against surrogate coronaviruses, mouse hepatitis virus (mhv) and transmissible gastroenteritis virus (tgev), was tested using the quantitative carrier method on stainless steel surfaces. germicides were o-phenylphenol/p-tertiary amylphenol) (a phenolic), % ethanol, : sodium hypochlorite, ortho-phthalaldehyde (opa), instant hand sanitizer ( % ethanol), and hand sanitizing spray ( % ethanol). results after -minute contact time, for tgev, there was a log reduction factor of . for % ethanol, . for phenolic, . for opa, . for : hypochlorite, . for % ethanol, and . for % ethanol. for mhv, log reduction factors were . for % ethanol, . for phenolic, . for opa, . for : hypochlorite, . for % ethanol, and . for % ethanol. conclusion only ethanol reduced infectivity of the coronaviruses by > -log after minute. germicides must be chosen carefully to ensure they are effective against viruses such as severe acute respiratory syndrome coronavirus. health care-associated infections are responsible for thousands of deaths worldwide each year. approximately % of all nosocomial infections are because of viral exposure, and, in pediatric wards, viruses account for at least % of health care-associated infections. studies have shown viruses to be common in health care environments and capable of surviving for extended periods of time on environmental surfaces. in these settings, health care workers, medical devices, and environmental surfaces can act as both a reservoir for infection and a mode of transmission of infection to patients and staff. , in , the nosocomial transmission of viral disease proved to be a major contributor to a worldwide outbreak of severe acute respiratory syndrome (sars), caused by a novel human coronavirus (cov) (sars-cov). outbreaks of sars occurred in multiple health care facilities, infecting patients, staff, visitors, and volunteers. sars-cov was also found on environmental surfaces in hospitals where outbreaks occurred, and studies demonstrated that it could survive on surfaces for to hours. airborne transmission was the main route of spread; however, rabenau et al observed that ''there are a number of instances when transmission occurred through other means that are often still not well defined, '' and other studies of outbreak settings showed that providing handwashing facilities reduced transmission in hospitals, suggesting that hands and surfaces could have played a role in transmission. the outbreak highlighted the need for effective and quick evaluation of means for controlling the spread of nosocomial infection. disinfection of hospital surfaces is an effective measure for reducing the risk of exposure for health care workers and patients; appropriate disinfection of contaminated surfaces and equipment is crucial in interrupting the spread of viruses such as sars-cov. , [ ] [ ] [ ] however, to assist in the selection of appropriate germicidal agents for use against coronaviruses on hospital surfaces and equipment, data are needed on the effectiveness of commonly used hospital germicides against coronaviruses. these data must accurately reflect disinfectant efficacy against viruses under the conditions in which they occur on surfaces, such as desiccation and embedding in proteinaceous matrices. many previous disinfection studies have used liquid suspension methods for testing germicide efficacy. [ ] [ ] [ ] these studies report greater efficacy against viruses than studies performed with carrier methods. viruses may be more resistant on surfaces than in suspension because they can adsorb to the surface or become embedded in organic material and may be more difficult to inactivate with chemical germicides than viruses suspended in liquid. thus, it is possible that suspension tests overestimate the level of antimicrobial activity of germicides against viruses on surfaces. carrier-based methods may more closely resemble real environmental conditions in which viruses contaminate surfaces and provide a more conservative estimate of germicide activity against viruses that are dried onto environmental surfaces. this study was undertaken using the carrier method to evaluate chemical germicides commonly used in health care settings for their efficacy in reducing infectivity of coronaviruses on environmental surfaces. the germicides selected were surface germicides and hand sanitizers. although hand sanitizers are not used for surface disinfection, the quantitative carrier test can help determine whether or not the active ingredients are effective against coronaviruses. germicide evaluation was done using non-human coronaviruses as surrogates for the coronaviridae family and pathogenic human coronavirus such as sars-cov. the family coronaviridae is divided into groups. groups i and ii include human, mammalian, and avian coronaviruses, and group iii consists of avian coronaviruses. although sars is thought to be related to the group coronaviruses, and phylogenetic analyses have indicated it may be closely related to mouse hepatitis virus (mhv), there is still disagreement about the exact placement of sars-cov within the coronavirus family. based on this uncertainty, representative of each group of mammalian coronaviruses was included in the study to determine whether there was any difference in their survival and persistence in water. the viruses included in the study were transmissible gastroenteritis virus (tgev), a diarrheal pathogen of swine and a member of the group i coronaviruses, and mouse hepatitis virus (mhv), a pathogen of laboratory mice and a member of the group ii coronaviruses. mhv and tgev were kindly provided by r. baric, university of north carolina, chapel hill. tgev was grown in swine testicular cell cultures. mhv was grown in delayed brain tumor cell cultures. viral stocks were propagated by infecting confluent layers of host cell cultures in flasks, harvesting cell lysates, clarifying by centrifugation ( , g, minutes, c), and storing resulting supernatants as virus stock at c. viral titers were determined by the plaque assay method on confluent host cell layers in -mm petri dishes with overlay medium consisting of % agarose, eagle's minimum essential medium, % bovine serum replacement (fetal clone ii; hyclone, logan, ut), % lactalbumin hydrolysate, and gentamicin ( . mg/ml)/ kanamycin ( . mg/ml). cell layers were stained with a second overlay containing % neutral red at hours postinfection, and plaques were visualized at hours postinfection. hard water was prepared according to the usepa opp microbiology laboratory standard operating procedure for disinfectant sample preparation for hard water preparation ml of solution a and ml of solution b were added to a volumetric flask and brought up to l with sterile deionized water. this solution was diluted with additional liters of sterile deionized water. final solution was adjusted to ph . to . by drop wise addition of sodium hydroxide or citric acid. a hardness testing kit (hach model -ep mg/l no. - ; hach corp, loveland, co) was used to confirm that hardness of the prepared water was to mg/l caco . six hospital-grade germicides were tested. the germicide types, active ingredients, and use-dilutions are summarized in table . germicides requiring dilution were prepared on the day of the experiment, using hard water as the diluent. all germicides were used by the manufacturer's expiration date. neutralizing solutions were used to inactivate germicide activity after the experimental contact time. vesphene iise (steris corp, mentor, oh), % ethanol, clorox anywhere spray (clorox co, oakland, ca), and purell sanitizing hand gel (johnson & johnson inc, new brunswick, nj) were neutralized using % glycine. chlorine bleach was neutralized with . % thiosulfate and cidex-opa (johnson & johnson inc, new brunswick, nj) with . % sodium bisulfite. to prevent cytotoxicity in cell culture assays, each neutralizing solution, with the exception of % glycine, was prepared as a stock solution and diluted to the use concentration with cell culture medium. glycine was prepared by adding . ml cell culture medium to . ml of a % glycine solution. sodium thiosulfate was prepared as a % (wt/vol) stock solution and diluted with cell culture medium to a use concentration of . %. sodium bisulfite was prepared as a % stock solution and diluted in cell culture medium to a use concentration of . %. test surfaces were -cm stainless steel carriers with a no. polish. the quantitative carrier test method used was adapted from sattar et al. each germicide experiment used control carriers (no germicide applied) and test carriers (germicide applied). each experiment was performed in duplicate. each carrier was placed in a -well plate, and ml of virus suspension was applied. the virus was allowed to dry for hours. after drying, ml of use-dilution germicide was placed on the dried virus suspension on test carriers, and ml of cell culture medium was placed on control carriers. after -minute contact time, ml of neutralizing solution was added to the test carriers to halt virucidal activity, and ml of cell culture medium was added to the control carriers. to elute viruses from carriers, ml of % beef extract (ph . ) was then added to all carriers. carriers were agitated on a shaking platform ( rpm) for minutes. the liquid from each well was then recovered, diluted, and assayed for virus infectivity as previously described. to determine the reduction in virus infectivity, the concentration of virus per -ml sample volume was calculated. reduction in viral titer was calculated using difference in virus concentration between test carriers and control carriers. log reductions were calculated for each germicide based on independent exposure trials. statistical analysis was performed using sas . ( ; sas institute inc, cary, nc). a -way analysis of variance (anova) was used to compare the log reduction among germicides. additionally, a -way anova was performed to compare the efficacy of all surface germicides between the virus types. reductions of mhv and tgev infectivity on surfaces by hospital germicides are shown in table . for mhv, only % ethanol and purell hand gel ( % ethanol) produced a log infectious virus titer reduction factor . . after -minute contact time. hypochlorite ( : use dilution) was least effective, producing a log reduction factor , . hypochlorite, vesphene iise, cidex-opa, and clorox anywhere spray each produced a log reduction factor of , . , with log reduction factors of . , . , . , and . , respectively. statistical analysis using -way anova showed that the mean log reduction factors for the germicides differed significantly (p , . ). for tgev, infectivity reduction factors of . -log were observed for % ethanol ( . ) , purell hand gel ( . ), and clorox anywhere spray ( . ). vesphene and cidex against tgev produced intermediate log infectivity reduction factors of . and . , respectively. as seen with mhv, hypochlorite exposure resulted in an infectious tgev titer log reduction factor , ( . ). statistical analysis using -way anova showed that the mean log reduction factors were significantly different (p , . ) among the germicides. analysis of the mean log reductions of mhv and tgev by germicides was done using the tukey multiple comparison test to determine whether reductions by individual germicides significantly differed from one another (p , . ) ( table ). in addition, -way anova was used to compare independent variables, germicide and virus type, and their influence on the log virus reduction factor. this analysis aids in determining how much of the variability in reduction is explained by each of these variables, as well as potential interaction between them. using -way anova, infectivity reduction results are statistically significant (p , . ). a type iii sum of squares test was used to examine variation among mean reduction results. germicide had a greater influence on log viral reduction (p , . ) than did the virus type (tgev vs mhv) (p . ). additionally, when interaction between germicide and virus type was evaluated using the anova test, it was determined that there is a statistically significant interaction between virus type and germicide type (p . ). health care-associated transmission can play an important role in the spread of coronavirus infection, and coronavirus nucleic acids have been found on hospital surfaces in outbreak settings. data from surrogate coronaviruses suggest that these viruses can survive for long periods on hard surfaces, potentially posing a continued risk of infection if health care surfaces are not adequately disinfected. the efficacy of hospital surface germicides was tested against coronaviruses, mhv and tgev, used as surrogates for sars-cov. these findings expand the available data on disinfection beyond what has been previously studied using other surrogates such as human coronavirus e. although e shares some tissue tropism with sars, there are important differences. studies of sars patients and outbreaks demonstrated that sars-cov is also a fecally shed virus, with intestinal tissue tropism; e lacks this. this may indicate important differences in resistance to environmental stressors because fecally shed viruses must be able to survive the conditions in the gastrointestinal tract, including extremes of ph, abundance of other microbes, and bile salts. this gastrointestinal tropism has played an important role in at least major outbreak; this suggests that surrogates such as mhv (a virus with multiple tropisms) and tgev (an enteric virus) that reflect this diversity in tissue tropism are necessary. a log viral reduction factor of . has been previously suggested as a benchmark for effective virucidal activity against coronaviruses and other viruses on surfaces. , , , the results of this study show that, of the commonly used hospital germicides tested, only the ethanol-based germicides were able to achieve this level of reduction of infectious virus after minute of contact time. for mhv, the ethanol-based germicides ( %, %, and % ethanol) gave the greatest reduction in infectivity, with log reduction factors ranging from . to . . this was greater than the reductions observed with hypochlorite, phenolic, and orthophthalaldehyde based germicides. these same performance trends are evident in tests of these germicides against tgev. the ethanol-based germicides gave log infectivity reduction factors ranging from . to . , greater than those observed for hypochlorite, phenolic, and orthophthalaldehyde germicides. the phenolic and orthophthalaldehyde germicides had greater virucidal activity against tgev than against mhv. however, only the reductions in infectivity by orthophthalaldehyde were significantly different between mhv and tgev. statistical analysis indicates that mean log viral reductions differed significantly based on both the type of germicide and the type of virus tested and that there are interaction effects between germicide and virus type. hence, both the selection of germicide and the type of virus will influence the resultant magnitude of reduction of virus infectivity titer. several previous studies have determined that surface disinfection is an important method for preventing viral transmission from surfaces to humans. the risk of acquiring infection decreases proportionately to the amount of viral agent present on surfaces. this study shows that ethanol-based germicides achieve the greatest reduction in viral titer on surfaces. these findings are consistent with previous studies of coronavirus disinfection, but this study provides more precise estimates of inactivation on surfaces than have been previously observed with other human coronaviruses, such as e. some previous studies of chemical disinfection of e have been limited by cytotoxicity problems that limited the ability to measure infectious virus reduction. sattar et al found that % ethanol reduced human coronavirus e dried onto a stainless steel surface by . . %. this study shows that the actual reduction is slightly greater than -log ( . for tgev and . for mhv). the available data on disinfection of sars-cov itself are not extensive, partly because of the challenges of working with sars. much of the available data focuses on alcohols. seventy percent ethanol was found in study to inactivate sars by . log . rabenau et al reported a log reduction factor of . after -second contact time using % ethanol and % propoanol, a reduction that was actually greater than what they observed with other germicides classified as chemical steriliants. these results support the findings of this study that ethanol-based disinfectants are efficacious against coronaviruses. however, flammability limits the use of ethanol for spills or contamination events involving large surface areas. other studies show that disinfectant efficacy can vary by virus type, with nonenveloped viruses such as adenovirus differing from enveloped viruses such as the coronaviruses. studies of hospital germicide efficacy against adenovirus using the same carrier-based method with -minute contact times found greater log reduction factors by opa ( . ) than were observed in this study for tgev ( . ) and mhv ( . ). in contrast, vesphene iise reduced adenovirus by a log reduction factor of only . , compared with . for mhv and . for tgev in this study. reduction of adenovirus by % ethanol was similar to that observed for coronavirus in this study, but . % hypochlorite reduced adenovirus approximately -log greater than reductions observed for coronaviruses with . % hypochlorite. this suggests that disinfection efficacy may differ greatly by virus type and that nonenveloped viruses may not be appropriate surrogates for predicting the effects of disinfectants on enveloped viruses such as coronavirus and influenza. hypochlorite demonstrated a log reduction factor , after minute for both tgev and mhv when applied at the : ( . %) use-dilution prescribed by the manufacturer. previous studies of coronavirus disinfection have found higher reductions with concentrations of hypochlorite greater than the recommended usedilution, suggesting these results are consistent with a concentration-dependent effect. sattar et al reported . % reduction of viral titer for human coronavirus e when . % and . % sodium hypochlorite solutions were tested with -minute contact times. commercial marketers of sodium hypochlorite recommend contact time of minutes. in actual use, it is likely that contact times are shorter than this, and increases in concentration may be necessary to offset the use of shorter contact times. the results of this study suggest that the : use-dilution should not be recommended for use on surfaces with suspected contamination by coronaviruses. hypochlorite is an important environmental surface disinfectant in health care; without the flammability and rapid evaporation of ethanol, it is suitable for large surface area spills. increases in both sodium hypochlorite concentration and contact time should be evaluated to determine whether these factors would improve virucidal activity of hypochlorite on hard surfaces to achieve a . -log reduction performance target. organic matter may play an important role in the poor performance of hypochlorite on surfaces observed in this study. a concentration of mg/l on a surface produced a log reduction factor , of mhv and tgev in this study. the poor performance of hypochlorite against viruses dried into surfaces may be due to the high oxidant demand exerted by the proteinaceous cell culture medium matrix in which the viruses were suspended. this results in consumption of available hypochlorite by the proteins and other organic compounds (eg, amino acids) present in the matrix, rendering it unavailable for disinfection. use of viruses suspended in a proteinaceous matrix simulates the real world conditions under which viruses shed by human hosts occur in health care environments. viruses are not shed by an infected host as single purified particles; they occur as aggregates, surrounded by membranes and embedded in feces, mucus, and other proteinaceous matrices. together, these results suggest that changing the recommended use-dilution of hypochlorite may address the problem of oxidant demand exerted by proteinaceous material such as body fluids and result in effective inactivation of coronaviruses shed from human hosts. hypochlorite, phenolic, and opa disinfectants tested reduced infectious viral titer by , log after -minute contact time. opa is used as a high-level disinfectant for semicritical equipment such as endoscopes; viruses can be deposited on the surfaces of semicritical equipment items during patient care. these results suggest that sufficient contact time is crucial to ensure that inactivation of viruses on the surfaces of semicritical equipment items takes place. according to the manufacturer, the phenolic disinfectant tested demonstrated a -to -log reduction in viral titer after minutes contact time when tested against another surrogate www.ajicjournal.org vol. no. coronavirus, avian infectious bronchitis virus. the results from this study, using -minute contact time, suggest that virucidal efficacy is greatly compromised if germicides are not used according to label directions and contact time is shorter than manufacturer recommended contact times. enveloped viruses of great nosocomial importance and pandemic potential, such as sars cov and avian influenza, are extremely challenging to work with in the laboratory. in the event of re-emergence of sars-cov, and in the context of pandemic influenza control, data are needed to guide decisions on appropriate disinfection of health care surfaces for control of viral transmission. tgev and mhv are nonpathogenic to humans and easily propagated and assayed in cell culture by plaque and quantal mpn assays. , this study shows that these types of surrogate viruses can help expand our knowledge of practical aspects of virus control, such as inactivation by disinfectants, for viruses of public health importance. it also increases the available data both for disinfectants that have previously been studied with sars co-v and disinfectants that have not. previous studies using sars have evaluated benzalkonium chloride and magnesium monoperphthalate-based products, povidone iodine, formalin, and glutaraldehyde. this current study included sodium hypochlorite and a phenolic, which have not been evaluated in previous studies. previous investigators have tested several types and concentrations of alcohols, including isopropanol, propanol, and ethanol, for which varying results have been observed in studies of sars. the data from this study can add to existing knowledge to help clarify how effective alcohols are against coronaviruses on surfaces. four of the germicides tested showed greater reductions of tgev compared with mhv. this suggests that mhv is potentially a more conservative surrogate for evaluating disinfectant efficacy against sars-cov. these studies should be replicated using sars-cov to determine which surrogate virus is a more suitable model for the response of sars to these germicides. there is still an important role to be played by surrogate viruses, especially for the evaluation of new disinfectants or the re-evaluation of use of current disinfectants (such as changes in dose and contact time); the available data suggest that both tgev and mhv may serve as conservative surrogates for modeling control of sars-cov by health care germicides in worst case scenarios. learning from sars: preparing for the next disease outbreak nosocomial spread of viral disease microbicides and the environmental control of nosocomial viral 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inactivation of sars coronavirus by means of povidone-iodine, physical conditions and chemical reagents apic guideline for selection and use of disinfectants replication and plaque formation of mouse hepatitis virus (mhv- ) in mouse cell line dbt culture studies on transmissible gastroenteritis of swine: ii. selected characteristics of a cytopathogenic virus common to five isolates from transmissible gastroenteritis inactivation of the coronavirus that induces severe acute respiratory syndrome, sars-cov key: cord- -melttpiq authors: yu, tian-fei; shao, shu-li; xu, xing-jun; lv, jian-wei; li, ming title: express transmissible gastroenteritis virus spike gene b and c antigen sites in multiple expression systems date: journal: information technology and agricultural engineering doi: . / - - - - _ sha: doc_id: cord_uid: melttpiq in order to illuminate the antigenicity of porcine transmissible gastroenteritis virus (tgev) spike protein b and c antigen sites, the truncated spike gene including b and c antigen sites of chinese isolate th- was expressed respectively in e.coli, baculovirus and pichia pastoris expression systems. dot enzyme-linked immunosorbent assays (dot-elisa) based on these three recombinant proteins were developed preliminarily. ten sera obtained correspondingly from ten piglets two months old which showed up clinical symptom were used for examination. the study indicates that the assays are rapid, reliable and sensitive and it has the potential for use as serological methods for tgev diagnosis. transmissible gastroenteritis (tge) is a highly contagious viral disease of swine characterized by vomiting, diarrhea, and dehydration. its causative agent is transmissible gastroenteritis virus (tgev), considered the principal etiologic agent responsible for dramatic outbreaks of diarrhea and high mortality of newborn pigs, in which mortality approaches % [ ] [ ] . porcine respiratory coronavirus (prcv) is believed to be a mutant of tgev, as it has been shown to be genetically related to tgev but has a selective tropism for respiratory tissue with very little to no replication in the intestinal tissue of infected swine [ ] . tgev and prcv share high homology in genome and can generate the full cross-reaction neutralizing antibody [ ] [ ] [ ] , so it is difficult to discriminate tge in clinical diagnosis with traditional serological methods. the truncated spike gene including b and c antigen sites which is absent in prcv was expressed in e.coli, baculovirus and pichia pastoris in this research. dot-elisa assays based on these three recombinant proteins were developed to detect tgev antibodies and could avoid antibody cross-reaction from prcv theoretically. tgev strain named th- was isolated from a suburb of harbin, heilongjiang province, p.r. china, and swine testicle (st) cell line was grown as monolayer in dulbecco's modified eagle medium (dmem) (gibco, usa) containing % fetal calf serum (gibco, usa) and % co in air. viruses were harvested by three cycles of freezing and thawing, cellular debris was removed by low speed centrifugation at . ×l g (hitachi cr e, japan) at ℃ for min, and virions in supernatant were pelleted by centrifugation at . × g at ℃ for . h (hitachi cr e, japan). all the three pairs of primers were prepared according to the sequence of tgev strain th from a data base, genbank (accession no.af ). pgu/pgl contained ecorⅠ or salⅠ restriction enzyme site respectively, phu/phl contained pstⅠ or salⅠ restriction enzyme site respectively, and pyu/pyl contained ecorⅠ or notⅠ restriction enzyme site respectively. all the primers contained artifical start codon or termination codon (table. ). rna was extracted as described by yin et al [ ] , meanwhile the st cell rna was also extracted as the negative control. extracted rna μl was added into the below components: ×reverse transcription buffer μl, dntp mixture ( . mm) μl, rnase inhibitor . μl, primer pyl μl, amv reverse transcriptase μl ( u), sterile water . μl, gently mixed in an eppendorf tube and incubated at room temperature for min, then transferred to a water incubator at ℃ for l h prior to stored at - ℃ until use in pcr. pcr amplification was performed using pe pcr equipment (usa). pcr was in μl volumes, using μl of ×buffer ( mm kc , mm tris-cl ph . , mm mgcl , . % gelate, μl dntp mixture ( mm), μl cdna, μl of primers and lμl extaq polymerase ( . u takara, dalian, china). for the pcr the mixture was submitted to cycles of amplification involving heating at ℃ for s, ℃ (e.coli expression), . ℃ (baculovirus expression), ℃ (pichia pastoris expression) for s, and ℃ for s, there was then a final extension time of min at ℃. a μl aliquot of pcr product was visualized by agarose gel electrophoresis ( % agarose, vs for min, . μg/ ml ethidium bromide included in gel) and subsequent u.v transillumination. the purified pcr product was named ts, tg or ty respectively. the amplified s gene (ts) dna was digested with ecorⅠ, salⅠ and the resulting fragment was inserted into the ecorⅠ, salⅠ sites of the vector pgex- p- (pharmacia biotech, inc., usa) to place the cdna under the control of the tac promoter. the amplified s gene (tg) dna was digested with pstⅠ, salⅠ and the resulting fragment was inserted into the pstⅠ, salⅠ sites of the transfer vector pbluebachis a (invitrogen co., usa) to place the cdna under the control of the polyhedrin promoter. the amplified s (ty) gene dna was digested with ecorⅠ, notⅠ and the resulting fragment was inserted into the ecorⅠ, notⅠ sites of the transfer vector ppic k (invitrogen co., usa). the three recombinant vectors were sequenced by sangon bio-company, shanghai, china. expression of b and c antigen sites in e.coli and recombinant proteins purification was followed the procedures offered by the description of glutathione s-transferase (gst) gene fusion system (amersham pharmacia biotech, third edition, revision ). expression of b and c antigen sites in baculovirus and pichia pastoris were followed manual of bac-n-blue transfection and manual of expression guide and methods for expression of recombinant proteins in pichia pastoris (invitrogen co., usa). twelve percent sds polyacrylamide gel was used to analyze the three recombinant proteins. after electrophoresis, one of the gels was stained with coomassie brilliant blue r to visualize the protein bands. the proteins of the other gel were transferred onto the nc membrane for the western blot analysis. the diluted protein samples were spotted on the nc membrane for the dot-elisa. the membrane was blocked with % non-fat dried milk powder in pbs with . %tween (blocking solution). the membrane was probed with the tgev immunized rabbit serum, in blocking solution ( : ). a peroxidase conjugated sheep anti-rabbit igg (promega, usa) was used as the secondary antibody ( : ) and the signal was detected with h o and -chlor- -naphthol as a chromogenic substrate. Ⅰ illustrate). this was consistent with the expected molecular mass of the fusion protein of pgex- p-ts which consisted of the gst ( kd) and s gene b and c antigen sites subunit ( kd). two different mois, and were tested. seed two -well plates with cells in each well. the final volume in each well should be - . ml. the harvest (post infection) was, hours, hours, hours, and hours. as shown in fig. . illustrate, recombinant baculovirus expressed kd protein corresponded to the molecular weight of tg of tgev at hours post-infection and the protein was accumulated in high amount till hours post-infection. no specific band was detected in the culture medium by coomassie brilliant blue staining. sf cells were inoculated recombinant virus with mois, . infected cells were harvested after h. the cell sediments were resuspended in pbs (ph . ). after interruptible ultrasonic treatment, the lysate and supernatant were analyzed by sds-page. the result showed that recombinant protein was soluble bulk in supernatant (the result was not given). the recombinant b and c antigen sites protein expressed into the yeast culture supernatant was identified on the bases of its molecular weight. numerous bands were observed in the - kd molecular mass range. a sharp band was observed at molecular masses of approximately kd by sds-page. the recombinant protein is the major protein component observed in the culture supernatant ( fig. . Ⅳ illustrate). the antigenicity of the three recombinant proteins was analyzed by dot-elisa assay. when the amount of spotting is ng, the recombinant protein expressed in prokaryotic system shows the positive reaction in contrast with gst ( fig. . Ⅰ illustrate). when the amount of spotting is ng, the ultrasonic lysis supernatant of sf cell infected by recombinant baculovirus show the positive reaction in contrast with ultrasonic lysis supernatant of sf cell (fig. . Ⅱ illustrate). when the amount of spotting is ng, the recombinant b and c antigen sites protein expressed into the yeast culture supernatant show the positive reaction in contrast with the gs cells transformed with ppic k plasmids (fig. . Ⅲ illustrate). . analysis of the antigenicity of recombinant protein expressed in pichia pastoris expression system. dots a ( ng), a ( ng), a ( ng), a ( . ng) were the positive yeast culture supernatant; dots b ( ng), b ( ng), b ( ng), b ( . ng) were the gs cells supernatant transformed with ppic k plasmids. ten field sera obtained from ten piglets about two months old which showed typical signs of epizootic tge used for examination and the ten corresponding sera were positive detected by dot-elisa (fig. ) . the spike protein of tgev has been shown to contain four major antigenic sites (a, b, c, and d). site a is the main inducer of neutralizing antibodies and has been previously subdivided into the three subsites aa, ab, and ac. site a contains the residues , , and , which are essential in the formation of subsites aa, ab, and ac, respectively. the peptide -mksgygqpia- represents, at least partially, subsite ac which is highly conserved among coronaviruses. this site is relevant for diagnosis and could be of interest for protection. other residues contribute to site b (residues and ), site c (residues and ), and site d (residue ). site c can be represented by the peptide -p-p/s-n-s-d/e- but is not exposed on the surface of native virus [ ] . site b is dependent on intracellular glycosylation and is complex and conformation-dependent. this site is formed by at least three epitopes. although site b is conformation dependent, mabs specific for this site can bind tgev spike protein by immunoblotting providing that the samples were not treated with -mercaptoethanol. most probably, renaturation of spike protein occurs during the blotting of the protein to nitrocellulose paper [ ] . site c is linear and continuous. it is recognized by mabs in western blot analysis after treatment of the virus with . % sds and % -mercaptoethanol; it is represented by synthetic nonapeptides derived from tgev spike protein [ ] ; it is present in recombinant products expressed in bacteria, which do not reconstitute the native spike protein [ ] ; and it is formed in the absence of glycosylation [ ] . in addition, because binding and sequencing studies indicate that site c is not present in the respiratory variants of tgev, this peptide could be useful to discriminate serum from tgev or prcv infected animals. the purified recombinant protein expressed in prokaryotic system in this research, can be recognized specially by polyclonal antibody according to the research of yang et al [ ] . although, the three recombinant proteins shared the same antigen sites, the quantity of amino acids of the former exceed the latter (the recombinant protein expressed in eucaryotae has amino acids, the recombinant protein expressed in prokaryosyte has amino acids). if this difference has an effect on antigenicity between them or how deeply effect on antigenicity need more experiment to identify. recently, some new ways to detection tgev were developed, such as real-time rt-pcr [ ] [ ] , but these ways need expensive equipment and reagent. it seems to unsuitable applied in open country, especially in the third world countries. so we developed the simple ways to detecting antibody induced by tgev, and the results seem more reliable. the study indicates that the assay reported above is rapid, reliable and sensitive and it has the potential for use as serological method for tgev diagnosis. transmissible gastroenteritis virus infection: a vanishing spectre complete sequence ( kilobases) of the polyprotein-encoding gene of transmissible gastroenteritis virus isolation of a porcine respiratory, non-enteric coronavirus related to transmissible gastroenteritis antigenic variation among transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus strains detected with monoclonal antibodies to the s protein of tgev detection transmissible gastroenteritis virus by rt-pcr and differentiation from porcine respiratory coronavirus field isolates of transmissible gastroenteritis virus differ at the molecular level from the miller and purdue virulent and attenuated strains and from porcine respiratory coronavirus molecular cloning and phylogenetic analysis of orf region of chinese isolate th- from transmissible gastroenteritis virus residues involved in the antigenic sites of transmissible gastroenteritis virus s glycoprotein localization of antigenic sites of the e glycoprotein of transmissible gastroenteritis coronavirus comparsion of antigenicity between expressed proteins of the fragment including s gene whole antigenic sites and the deleted fragment in porcine respiratory coronavirus of transmissible gastroeritis virus development of a novel real-time rt-pcr assay with lux primer for the detection of swine transmissible gastroenteritis virus a real-time taqman rt-pcr assay with an internal amplification control for rapid detection of transmissible gastroenteritis virus in swine fecal samples key: cord- - ix c he authors: rajan, v. title: an oral vaccine for tgev immunization of pigs date: - - journal: commercial plant-produced recombinant protein products doi: . / - - - - _ sha: doc_id: cord_uid: ix c he transmissible gastroenteritis virus (tgev) is a commercially important pathogen of hog farms and causes contagious, lethal diarrhea in piglets. while orally and parenterally administered vaccines made from inactivated or attenuated tgev are commercially available, they require individual administration to piglets, which is time and labor intensive, and run the risk of reversion to pathogenicity. also, parenteral vaccines produce neutralizing serum antibodies which may be less effective against an orally transmitted pathogen, compared to an oral vaccine that would induce the production of mucosal antibodies. there has been an effort to produce subunit vaccines in an edible form in plants for convenient administration through feed. these efforts towards the expression of the s-antigen of tgev in maize seed, its effectiveness at inducing neutralizing antibody production in the colostrum of gilts, and its efficacy in protecting piglets against challenge by virulent tgev are summarized here. epidemic diarrhea virus (pedv), and porcine hemagglutinating encephalomyelitis virus (hev) (sestak and saif ) . it is pertinent to examine the structure of tgev and related viruses to explain the choices of epitopes available for the production of effective subunit vaccines. all coronaviruses have an envelope with radiating structures composed of trimers of the kda spike glycoprotein (s, also referred to as peplomer e ), as well as the smaller membrane glycoprotein (m, - kda) and envelope protein (e , kda) ( fig. . ). the m protein interacts with the nucleocapsid n protein and the viral rna to form the icosahedral nucleoprotein core (masters ) . the s-protein, specifically the n-terminal domain between amino acids - , binds aminopeptidase n receptor in the epithelium of the small intestine and mediates the fusion of the host and viral membranes and uptake (belouzard et al. ). the surface and subviral structural components of the virus have been assessed for antigenicity, and neutralizing antibodies were found to be associated with the surface components which function in recognition and binding with subviral and host proteins (garwes et al. ) . the m protein induces interferon production and also binds neutralizing antibodies (laude et al. ). the s (or e ) protein has been found to be the most effective epitope at inducing neutralizing antibodies. the s-protein has four major antigenic sites at the n-terminal: a, b, c, and d; a and d are involved in antigen neutralization (reguera et al. ; correa et al. ; fig. . schematic of the coronavirus virion, with the minimal set of structural proteins. reproduced with permission from masters ( ) jiménez et al. ). induction of cross-protection with the use of a related virus has been attempted. pcrv shows tropism towards respiratory tissues and differs from tgev in having a deletion of the n-terminal - amino acids (containing antigenic sites a and d) of the s-protein, indicating that the a and d sites may be involved in tissue specificity. immunization with pcrv was partially protective under the same conditions- % of piglets survived challenge (de diego et al. ) . the s-antigen was therefore selected as the most efficient immunogen for a subunit vaccine. high-level expression of heterologous eukaryotic proteins has been successfully attempted in both prokaryotes and eukaryotes. however, many eukaryotic proteins require posttranslational modifications such as glycosylation, which are not carried out by prokaryotes, and so eukaryotic systems are sometimes necessary to obtain functional end products. the benefits of plants as production systems are primarily the low cost of production and rapid scale-up that make it more responsive than other methods to high production targets. traits introduced by transgenic techniques into separate lines can be combined by traditional breeding methods to produce lines with a combination of these traits. the production of bulk plant material requires only space, fertile soil, and sunlight, which are less onerous to provide than sterile fermentation chambers and animal care. plants do not harbor animal pathogens, and the concern for contamination of products with animal pathogens, such as prions or viruses, is alleviated. plant material has been shown to serve to bioencapsulate proteins, permitting slow release in the gut (kong et al. ; verma et al. ; bailey ) . thus, there may be a degree of dosage flexibility with oral antigens administered in plant tissue, and the feed can be administered over a larger window to accommodate time frames for induction of secretory and humoral immunoglobulins (bailey ; daniell et al. ) . important considerations before attempting expression of heterologous eukaryotic proteins in plants are the requirements for posttranslational protein modifications, such as glycosylation, and the presence of allergens and toxins. glycosylation moieties and patterns differ between animals and plants which has raised concerns over the potential for nonfunctional proteins or an allergic reaction to the plant glycosylated protein. these differences, however, do not appear to be significant functionally (ma et al. ; suzuki et al. ) , and plant carbohydrate-specific iges in allergic patients were shown to be clinically irrelevant when the protein is orally administered (mari et al. ; bosch and schots ) . this lack of allergenicity may be partly explained by tolerance to plant glycosylation induced by oral exposure (see ghaderi et al. for an overview of glycosylation of biotherapeutic proteins). potential concerns about glycosylation-induced allergenicity may also be overcome by the removal of plant glycosylation sites, inactivation of plant glycosylases, and the expression of plant deglycosylases and mammalian-type glycosylases (sethuraman and stadheim ; desai et al. ; mamedov et al. ) . host plants should also be screened for toxic or allergenic metabolites that may co-purify or be co-administered with the target protein. edible tissues such as grain from plants with generally recognized as safe (gras) status may be directly used for feed-based vaccines (naqvi et al. ) . seeds are a natural, desiccated, storage system lacking proteases, and recombinant proteins produced in seed have been stable at room temperature for extended periods of time (naqvi et al. ) . selection and backcrossing into parent seed and hybrid lines can establish uniform concentrations of antigen expression (hood et al. ) and standard processing and formulation methods can reliably produce consistent concentrations of antigens for administration, which is not straightforward with perishable edible tissues such as lettuce leaves and bananas. bioencapsulation of the antigen in plant tissue prevents premature degradation resulting in the antigen persisting in the intestine, a key factor for the induction of a protective siga response, important for protection against ingested pathogens. a system that exemplifies the gras system is maize (or corn) grain which was used in the work described here. its structure and specific advantages are described below. the maize kernel contains three main regions: the embryo, the endosperm, and the pericarp (fig. . ). the pericarp includes the seed coat and fruit layers. the pericarp originates from the ovary wall and functions to protect the seed. the aleurone layer is the outermost layer of the endosperm and lies directly below the pericarp. aleurone grains contain enzymes involved in the breakdown of starch and proteins in the germinating seed. the embryo and endosperm are well suited to heterologous protein accumulation as both are rich in protein and have associated tissue-specific promoters. the volume of the endosperm is greater than that of the embryo allowing, in theory, more protein accumulation but, in practice, embryo-preferred promoters have been used to produce some of the highest recorded concentrations of heterologous proteins (hood et al. ; egelkrout et al. ) . desiccation of the seed provides an environment that protects proteins from enzymatic degradation (ma et al. ) , and maize seed has cystatins (yamada et al. ; massonneau et al. ) and other protease inhibitors (jongsma and bolter ) as a defense against proteolysis by insect pathogens. the ideal vaccine for tgev will provide mucosal, lactogenic, and systemic immunity, while being simple to administer to droves which may number in the many thousands. potentially, the methods to protect naïve piglets at highest risk from tgev infection are to provide immune colostrum by vaccinating sows and an oral/nasal vaccine to boost secretory neutralizing antibody levels. since tgev is an enteric disease, the induction of siga is inferred to be more protective. it has been observed that the mucosal immunoglobulin, iga, is more efficiently induced through gut-associated lymphoid tissue (galt) if the antigen persists in the intestine (foss and murtaugh ) . induction of passive immunity transmitted through colostrum can be mediated by either of the soluble immunoglobulins igg or iga. initially, igg is more abundant and protects against systemic infection, whereas igas in colostrum provide protection to the gut lumen where the enterocytes in which tgev replicates reside. iga production and mucosal immunity is thus the response desired. there have been three main routes taken to effect protection in young piglets: ( ) parenteral immunization with inactivated virus, ( ) oral/nasal administration of attenuated virus, and ( ) oral administration of viral subunit antigens in feed. parenteral immunization of young piglets with inactivated virus is ineffective as it does not induce the local immune response in the gut, nor are piglets at this age capable of mounting an immune response. however, parenteral immunization of pregnant sows can induce production of protective immunoglobulins in colostrum and milk. the disadvantage of this approach is the necessity of individually vaccinating sows, which can be time-consuming and expensive. immunization must also be done on a schedule prior to birth that maximizes immunoglobulin presence in the colostrum, and that can be difficult, especially with large numbers. nasal administration of attenuated viruses does overcome this difficulty, but each pregnant animal still needs to be isolated and the vaccine delivered separately. as with parenteral injection, the vaccination schedule is important because, while the attenuated virus does replicate in the gut, thus stimulating antibody production over an extended period, viral replication is suboptimal. while the oral vaccine can be more easily administered, the frequency of inoculation has to be modulated to maximize colostrum immunoglobulin timed with birth. the option of using the oral route to deliver viral subunit antigens in feed is fairly straightforward in terms of administration: the food is put out in feeding troughs and animals are monitored by observation to ensure all animals have had access to the food. however, dosage for orally administered vaccines in feed is particularly significant, because antigens could be ingested at greater or lesser doses depending on the amount of feed consumed, and some of the antigens would be expected to be destroyed by the digestive process. the typical vaccine dosage in feed may be up to times greater than that used for parenteral administration, but this may be reduced by the use of suitable targeting or carrier molecules (carter and langridge ) . still, common consensus is that a precise dose, taking into account degradation in the gut, must be given to all animals. interestingly, verma et al. ( ) have shown that the response from a -fold range of antigen levels given encapsulated in plant materials is comparable. finally, with any vaccine, oral or parenteral, there is a concern over the potential induction of tolerance, which has been shown to occur for orally administered antigens with repeated doses of antigens over a long term and is mediated by regulatory t-cells. a single large dose has also been shown to induce anergy-a mechanism of adaptive tolerance that cause t-cells, in an environment presumably low in co-stimulators, to become unresponsive to antigens (weiner ; weiner et al. ) . therefore, prior to widespread vaccination, tests must be conducted to ensure this is not an issue. with these caveats addressed, plants have proven to be efficient hosts for heterologous protein production. de diego et al. ( ) showed that oral immunization with tgev was more effective at inducing the presence of neutralizing antibodies in colostrum and milk compared to intranasal immunization with pcrv. they attributed this difference to galt being more effective than bronchus-associated lymphoid tissue (balt), going even to the extent of questioning if balt was indeed an integral mammalian structure (pabst ) . pigs have been shown to not have balt constitutively (delventhal et al. ; pabst and gehrke ) . thus, the use of intranasal stimulation by pcrv may have been less effective for this reason. peyer's patches in the gut have long been known to be associated with galt, and oral immunization has been shown to be effective in producing iga and igg globulins in milk and was % effective in protecting - day piglets obtained from seronegative sows against challenge. this experiment (de diego et al. ) established that persistence in the gut and therefore a longer-term mucosal response in older animals can be achieved by using an attenuated live vaccine which replicates in the gut. a variety of viruses have been used as vectors for expression of full length and truncated versions of s-antigen. baculovirus-infected cells containing the n-terminal fragment of the s-antigen induced neutralizing serum antibodies in piglets (tuboly et al. ) . furthermore, oral immunization of piglets using s-antigen-recombinant porcine adenovirus induced both neutralizing antibodies in sera and mucosal antibodies in the intestine (tuboly et al. ) . fusions of the s-protein expressed in e. coli were immunogenic and produced cognate antibodies but did not neutralize the virus. s-protein expressed in vaccinia virus produced neutralizing antibodies when injected into animals (hu et al. ) . the expression of s-antigen in nuclear polyhedrosis virus and its expression in insect s cells allowed the production of a secreted version which was immunogenic in rats despite lack of proper glycosylation (godet et al. ) . mucosal and serum antibodies were also elicited in rabbits orally inoculated with an attenuated strain of salmonella typhimurium expressing a fusion of the d-epitope of the s-antigen to e. coli heat-labile toxin b-subunit (lt-b) (smerdou et al. ) . various tgev antigens have been produced in plants. gomez et al. ( ) expressed either the n-terminal residues, or the entire s-protein, under the control of the camv s promoter in arabidopsis. the antigen was purified from leaves and administered to mice. the mice produced antibodies that reacted specifically with tgev and neutralized infective particles. subsequently, the same group expressed the n-terminal domain in potatoes and obtained a serum response to both intraperitoneally delivered tuber extracts and orally administered tubers. immunoprecipitation and elisa results showed that antibodies were detecting the native protein; however, the sera did not neutralize the virus in vitro (gomez et al. ) . tuboly et al. successfully produced neutralizing serum antibodies in piglets using tobacco plants expressing various permutations of the s gene under the control of a synthetic super promoter . in an early experiment using s-antigen, gomez et al. ( ) used a camv s promoter to drive expression of an un-optimized sequence in arabidopsis. while the protein produced was immunogenic, its levels were too low to be visualized by sds-page and could only be detected by western blotting. subsequently, used plant-optimized s-antigen sequences driven by a super promoter and increased levels to . - . % total soluble protein (tsp) s-antigen expression in tobacco. using codon-optimized sequences for maize fused to the barley alpha-amylase signal sequence (baass), lamphear et al. ( ) achieved levels of s-antigen expression in maize seed of mg/kg. baass targets protein to the cell wall and is later cleaved-a process that permits higher levels of accumulation in the apoplast than is possible in the cytoplasm. using standard plant techniques, f lines of maize that had high levels of expression were selected and backcrossed to commercial maize lines to obtain stable, increased levels of protein expression. since the protein was produced in maize, a gras plant, further purification before oral administration was not necessary. lamphear et al. ( ) studied the levels of serum neutralizing antibodies induced by administration of tgev-s corn alone in piglets. - -day-old tgev seronegative piglets were divided into three groups (normal rations, control corn, tgev-s corn) of four piglets each, with mg of the antigen administered in ground corn mixed with medicated milk replacer. days after the last antigen administration, the piglets were challenged with ml of orally administered virulent tgev. serum was collected and neutralizing antibody levels measured (fig. . a) . piglets fed tgev-s corn showed more than tenfold greater neutralizing serum response as well as milder symptoms (a geometric mean titer of . in tgev-s-corn fed piglets compared to titers of in the control groups). while the presence of serum antibodies is significant, greater protection will potentially be afforded by mucosal antibodies. pregnant gilts received the following treatments: group a (oral corn vaccine on days À to À and À to À ), group b (oral corn vaccine on days À to À and À to À ), group c (oral corn vaccine on days À and À ), group d (oral corn placebo on days À to À ), group e (intramuscular live vaccine on days À and À ), and group f (oral corn vaccine on days À to À ), where the "À" sign represents days before farrowing. reproduced with permission from lamphear et al. ( ) . see text (sect. . . ) for discussion the induction of neutralizing antibodies in both serum and colostrum was examined in gilts following the administration of oral tgev vaccine in maize comprising a subunit vaccine of the s-protein expressed in corn (lamphear et al. ) . two kg doses of corn containing mg of antigen were administered to gilts previously sensitized with three doses of modified live vaccine (mlv-tgev, intervet) with two oral administrations and days before farrowing and one intramuscular injection days before farrowing. subsequently, they were divided into six groups for testing (a-f; see fig. . b) and administered oral tgev-s corn or control corn at various schedules. tgev-s corn was given to group a (oral corn vaccine on days À to À and À to À ), group b (oral corn vaccine on days À to À and À to À ), group c (oral corn vaccine on days À and À ), group d (oral corn placebo on days À to À ), group e (intramuscular live vaccine on days À and À ), and group f (oral corn vaccine on days À to À ), where the "À" sign represents days before farrowing. serum antibody levels dropped by up to half at the time of farrowing but were still relatively high compared to control (data not shown). colostrum obtained on the day of farrowing showed neutralizing antibodies were present. antibody levels in animals in group c administered test corn for days were almost . times higher than animals administered intramuscular injection with modified live virus, indicating that corn was more effective at inducing secreted antibody than parenteral vaccine and that the window of administration may be important. that secretory antibodies were produced in colostrum is a clear indication of the stimulation of a mucosal response. because it was not clear if the antibodies induced by oral administration were protective against infection, a trial to test protection against challenge was carried out with - -day-old, specific pathogen-free piglets lamphear et al. ) . piglets were fed tgev-s corn, control placebo corn, and orally administered mlv-tgev controls. all piglets were challenged with virulent tgev on day and monitored twice daily for symptoms for days until the end of the study. following challenge, piglets were scored twice daily for signs of diarrhea (normal ¼ , creamy ¼ , watery ¼ ) and other symptoms (dehydration and depression, anorexia ¼ , vomitus ¼ , moribund or death ¼ ) to give a total clinical score. clinical symptoms for each study group were scored as follows: percent morbidity incidence [(number of animals with clinical signs scoring ! divided by total number of animals) Â ], percent morbidity incidence and duration [(total number of clinical observations ! divided by the product of the total number of pigs and days scored) Â ], or clinical severity index (total clinical score divided by the product of the total number of pigs and days scored). half of the control group and about % of the mlv-tgev group showed morbidity, compared to % of the tgev-s corn ( -day administration) group. morbidity duration and clinical severity were highest in control corn, as expected, but tgev-s corn showed better protection even than mlv-tgev. increased duration of administration showed slightly higher morbidity levels (fig. . ) , but piglets administered tgev-s corn for days showed no symptoms. this unexpected finding suggests the possible induction of oral tolerance to extended exposure of antigen for longer periods than days. the levels of humoral and secreted (serum and stool) antibodies in these piglets were not monitored, but it seems likely that the levels were diminished by the extended exposure. this is an important result, as it indicates that a shorter duration of administration of oral vaccine is more effective and more economical as well. s-antigen expression in maize seed was directed by a constitutive polyubiquitin promoter and the protein was present in endosperm, aleurone, and embryonic tissues. whole seed expressed antigen at a level of μg/g. fractionation to determine the sites of protein accumulation within the seed showed that the embryo (germ tissue) accumulated the highest concentration of recombinant protein of μg/g-double the concentration of whole grain. the levels in bran (aleurone and pericarp layers) were very low, and endosperm contained levels in between those in grain and pericarp (lamphear et al. ) . stability of antigen was examined in whole grain stored for months at ambient temperature without humidity monitoring, at c in a seed storage facility at % humidity, and in corn meal stored at c ( fig. . ). levels of antigen in stored grain were compared to freshly harvested grain, and no difference was found. this important result indicates that antigen is stable even under uncontrolled conditions at ambient temperature, making it less onerous for storage and transport over long periods and permitting elimination of the cold chain for transport and delivery. tgev-s antigen was not a novel target for expression, but several hurdles that limited its efficacy were overcome. maize, as the bioencapsulating agent, presumably prolonged exposure to galt tissues. high levels of expression and stability were achieved using a constitutive promoter and an apoplast targeted baass sequence. the antigen was stable in stored grain, overcoming a major economic hurdle. reproduced with permission from lamphear et al. ( ) in this work, it was also unexpectedly discovered that the smallest dose regimens, administered purely by the oral route, were also the most effective. significantly, when young pigs were fed a constant diet of the antigen for weeks, protection was reduced, an indication that tolerance may develop with long-term exposure (lamphear et al. ) . the duration of protection for piglets following antigen administration in feed was not tested. administration of antigen over a -day period gave the highest levels of protection against live challengeeven higher than oral vaccination with a modified live virus (fig. . ; see sect. . . ). in gilts, as well, the shortest oral regimen of two boosters showed the highest colostrum levels of serum neutralizing antibodies. thus, if passive immunity transferred by colostrum is protective in suckling pigs, then this regimen would be more economical as well as less onerous. in gilts, colostrum levels of antibodies persisted for about - days following piglet birth (lamphear et al. ) , which provides passive immunity while the piglets developed their gut flora and become suitable subjects for the administration of vaccines in feed. fig. . antigen stability in tissues from transgenic corn seed: measurement of extracted tgev-s antigen as mg antigen per g extracted soluble protein from grain or grain meal stored for months at either c, c, or at ambient temperature in a grain storage facility in iowa. values represent mean ae one standard deviation. figure reproduced with permission from lamphear et al. ( ) regulatory approval must be obtained for each step of the process, including compliance for growing transgenic plants to licensure of the vaccine product. plant-based vaccines have already been approved for use with livestock and with humans. in , the usda approved a plant-produced vaccine against newcastle disease virus for administration to fowl by dow agrosciences. in , fda approved the first pharmaceutical product produced in plants, elelyso™ (taliglucerase alfa), to protalix biotherapeutics and pfizer. thus, precedents now exist for both human and animal pharmaceuticals produced in plants. the existence of these plant-based vaccines smooths the path for the development and regulatory approval of future plant-based vaccines. some essential regulatory procedure for growth of transgenic plants and approval of veterinary vaccines in the usa are described below. similar to the requirement for other vaccines produced in yeast or eggs, the plant crop must not inadvertently be intermixed with commodity food. one important feature of the plant production system is that field-grown plants have the potential to pollinate other food or feed crops. the aphis arm of the usda issues permits for the growth of transgenic corn. these include a number of growing restrictions to prevent intermixing of the crop inadvertently with other food and feed crops. to this point, the usda has developed a highly restrictive set of isolation conditions for growing the crop, over and above the standard conditions for producing vaccines, including a one-mile isolation corridor from other corn. as maize pollen is relatively heavy, it normally only pollinates other corn plants within a few meters (luna et al. ) making this an extreme precaution. nevertheless, as the growing acreage needed for such a product is low (~ , acres, or . % of the total acreage of corn in the usa to produce two doses of mg of vaccine at mg antigen/kg corn for each of the estimated million swine and assuming a yield of bushels/acre), it is quite reasonable to find isolated areas to grow the crop. after harvesting, the corn can be processed into corn meal, blended to obtain precise dosing, and formulated to the final product. production of a vaccine is only the first step in the process towards licensing. the vaccine must be safe and effective in order to obtain usda approval for use through the veterinary biologics program. guidelines are set out by the usda under the virus-serum-toxin act ( u.s.c. - ). the cost of production is always a potential limitation for any product to be commercialized. in this case, since the product is produced in corn which is a feed source for pigs, it does not need to be purified thereby reducing a major expense. furthermore, the corn itself has value as a feed product, further lowering the effective cost of the vaccine. the actual cost of the vaccine can therefore be calculated by the cost to keep the grain segregated from food and feed crops and loss in yield over commodity crops. both of these costs are further reduced by having high levels of antigen in maize, thereby limiting the amount of corn needed to be grown. in theory and in practice, the cost should be lower than the traditional injected vaccines. the indirect benefit of not having to physically inject the animals provides another value proposition making this even more desirable. the first group that needs to accept this product is the swine producers. this is largely dependent on their perceived need for added protection from the disease. once this hurdle is overcome, there is always the concern over trying any new product. acceptance by swine farmers, however, should be relatively straightforward since this product should cost less to administer than traditional vaccines. even though farmers may accept this relatively readily, the general public is leery of transgenic crops. on a rational basis, this product would not pose a threat to the general public for several reasons: ( ) the product will be quickly digested and will not persist in the animal at the time of slaughter; ( ) the protein itself is present in the food chain when animals are infected by the virus, and the use of vaccines limits its presence; ( ) the protein itself poses no threat as it has no toxicity or enzymatic activity; ( ) as a protein, the degradation products are amino acids that are common to all living organisms; ( ) as pork is cooked, any protein would be quickly denatured prior to human consumption; and ( ) growing maize expressing s-antigen will involve very small acreage, thus a nationwide concern would not be triggered in the event of an inadvertent exposure. unfortunately, logical arguments are not always sufficient; undoubtedly a subset of the general public will fear the product for reasons other than safety arguments. this can be due to a general fear of transgenic crops, vaccines, fear of new products, aversion to technology, or other fears. while all of these are considerable hurdles, they can be overcome if the benefits of this product outweigh the risks. in this case, the benefits can be determined by how much of a threat is perceived by the disease and the cost and efficiency of this approach can eliminate the threat. while the goal of most vaccine production in plants is commercialization, the dearth of products on the market speaks to the many barriers that must be crossed before the goal is achieved: ( ) the product must be made at a level in plants that makes it commercially viable; ( ) for scale-up with plants such as corn, adequate barriers to dissemination must be incorporated and aphis approval obtained; ( ) the product must be tested in the target animal to rule out negative side effects, and approval for marketing must be obtained; and ( ) finally, a large investment to scale-up and market the product is needed. for large companies such as dow agrosciences, which take a product from the lab to the market, this is less formidable than for small biotechnology companies which need to move to market at speed lest funding wane while the process is still underway. protection with a subunit antigen expressed in corn, exclusively by the oral route, is shown for the first time to be effective in piglets, the target species for immunization. this demonstration, using corn-encapsulated s-antigen administered orally as both primer and booster, could circumvent the need for parenteral vaccinations or oral immunizations with modified live virus, making the process of vaccinating large herds much more economical and less time-consuming than using injected vaccines. this work has three significant outcomes: ( ) the demonstrated use of an oral subunit vaccine in production of neutralizing antibodies in both adults and young pigs, ( ) neutralizing antibodies from both active and passive immunity being protective to a direct challenge with live virus, and ( ) short duration of oral exposure of antigen ( days) being sufficient to develop complete protection from challenge. the use of maize seed that can be administered directly through feed clearly shows that this approach provides protection that can be as good if not better than injectable products. using antigen-expressing corn as a top dressing on feed has the additional advantage of also bioencapsulating the antigen, which extends its contact with galt in the gut and provides a greater immune response. storage stability has also been demonstrated, with both the whole seed and meal, at room temperature and with refrigeration. in short, this approach demonstrates that a practical, low-cost, heat-stable, orally delivered vaccine is achievable. production of subunit antigens in well-tolerated, edible plant tissue opens the door to a lot of possibilities. several antigens to different diseases can be combined into a single vaccine by standard breeding techniques. since antigens expressed in maize seed are tolerant to storage over long periods at ambient temperature, they can be stockpiled against zoonotic outbreaks. the elimination of the cold chain can be highly significant in rural areas. the use of the edible vaccine as both primary and booster increases convenience and lowers duration of administration to animals. adjuvants can be co-expressed as needed to improve immunogenicity. clearly, there is ample room for improvement of this technology. the use of tgev-s protein clearly shows commercial potential as described above. using more recent technology, improvements could be made to produce a higher concentration of the antigen or having it targeted specifically to the embryo. this study also represents one of the first clear demonstrations of providing protection against a pathogen in animals, paving the way for other vaccine antigens to be tested. a model system for edible vaccination using recombinant avidin produced in corn seed mechanisms of coronavirus cell entry mediated by the viral spike protein plant glycans: friend or 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diseases oral tolerance a cysteine protease from maize isolated in a complex with cystatin key: cord- -fvdq yes authors: wang, jinfeng; wang, jianchang; zhang, ruoxi; liu, libing; shi, ruihan; han, qingan; yuan, wanzhe title: rapid detection of transmissible gastroenteritis virus in swine small intestine samples using real-time reverse transcription recombinase polymerase amplification date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: fvdq yes a rapid and specific real-time reverse-transcription recombinase polymerase amplification assay (rt-rpa) was developed to detect the transmissible gastroenteritis virus (tgev) in this study. the primers and exo probe were designed to be specific for a portion of spike (s) gene conserved in tgev, but absent in the closely related porcine respiratory coronavirus (prcv). the amplification was performed at °c for min. the assay could only detect the tgev, and there was no cross-reaction with other pathogens tested. using the in vitro transcribed tgev rna as template, the limit of detection of the developed rt-rpa was copies per reaction. the assay performance was evaluated by testing clinical samples by rt-rpa and a real-time rt-pcr. fourteen samples were tgev rna positive in rt-rpa ( . %, / ), which were also positive in the real-time rt-pcr. the diagnostic agreement between the two assays was % ( / ). the r( ) value of rt-rpa and real-time rt-pcr was . by linear regression analysis. the developed rt-rpa assay provides a useful alternative tool for rapid, simple and reliable detection of tgev in resource-limited diagnostic laboratories and on-site facilities. transmissible gastroenteritis (tge) is an acute enteric viral disease of pigs resulting in vomiting and diarrhea in all ages of pigs and with high mortality rate in piglets (garwes, ) . the disease is caused by tge virus (tgev), which is an enveloped, single-stranded, positivesense rna virus belonging to the genus alphacoronavirus of the family coronaviridae (saif and wesley, ) . tge was first reported in in the united states, and tgev was first isolated and identified in (doyle and hutchings, ) . since then, tgev has spread throughout the world including america, europe and asia, and has caused significant economic loss in the pig industry (kim et al., a; stevenson et al., ) . since the mid s, a variant respiratory form of the tgev known as porcine respiratory coronavirus (prcv) has become common in pigs (laude et al., ) . compared to tgev, prcv has a deletion of between and nucleotides near the ′ end of the spike (s) gene (laude et al., ) . although prcv replicates predominantly in the respiratory tract, some pigs infected with prcv could also shed the virus in their feces (costantini et al., ; saif and wesley, ) . therefore, any diagnostic assay for tgev should be able to differentiate it from prcv. rapid and specific detection of tgev would be extremely important in the prevention and control of tge. the conventional methods for detection of tgev are time-consuming, laborious, and requiring welltrained technicians, such as virus isolation, immunofluorescence assay, electron microscopy and elisa (carman et al., ; dulac et al., ; van nieuwstadt et al., ) . a series of developed rt-pcr assays for pedv require the high-precision, sophisticated and expensive instruments, well-trained technicians and good laboratory circumstance, thus unsuitable for being applied in under-equipped laboratories and on-site applications, such as rt-pcr (paton et al., ) , nanoparticle-assisted rt-pcr (zhu et al., ) and real-time rt-pcr (vemulapalli et al., ) . recently, rt-lamp assays have been developed for tgev detection (chen et al., ; li and ren, ) . six primers were needed in the rt-lamp assays for tgev, the reaction time was or min, and the results were visualized by agarose gel electrophoresis (chen et al., ; li and ren, t recombinase polymerase amplification (rpa) is an isothermal gene amplification technique that has been demonstrated to be a rapid, specific, sensitive, and cost-effective molecular diagnostic method (daher et al., ; piepenburg et al., ) . rpa employs three core enzymes: a recombinase, a single-stranded dna-binding protein (ssb) and a strand-displacing polymerase. the recombinase forms a nucleoprotein filament with primers and probes. this filament scans the double-stranded dna (dsdna) target searching for homologous sequences and invades the dsdna once homology is found. then a d-loop structure is formed, which is a local separation of dna strands in which the complementary strand is stabilized by ssb and the target strand is hybridized with primer. recombinase disassembly from the nucleoprotein filament, the strand-displacing dna polymerase adds bases to the ′-end of the primer and extension occurs (daher et al., ; piepenburg et al., ) . real time detection of rpa amplicons could be performed through adding exonuclease iii and exo probes to the reaction mixture. the exo probe typically consist of an oligonucleotide backbone that contains an abasic nucleotide analogue (tetrahydrofuran residue or thf) flanked by a dt-fluorophore and a corresponding dtquencher group. in a double-stranded context the thf residue presents a substrate for exonuclease iii, which will cleave the probe at the thf position, thereby separating the fluorophore and the quencher and generating a fluorescent signal. real-time rpa assays had been developed for the detection of a series of important viruses in swine (wang et al., a,b) . in this study, we developed and evaluated the userfriendly on-site detection platform integrating the real-time rpa technology and a field-deployable device (genie iii tube scanner) for the rapid detection of tgev. the genomic rna or dna of tgev (strain hb-yx), procine epidemic diarrhea virus (pedv, strain jscz ), porcine rotavirus (porv, strain hb-bd/ ), prcv, porcine deltacoronavirus (pdcov), lawsonia intracellularis (strain lx ), porcine circovirus (pcv , strain hb-mc ) and porcine parvovirus (ppv, strain bj- ) were kept in our laboratory. seventy-six small intestinal samples from the piglets with signs of severe watery diarrhea, dehydration were collected from eleven pig farms in hebei province between april and february . the piglets were - days old. the samples were homogenized with phosphate-buffered saline (pbs, ph . ) as a % (w/v) suspension and centrifuged for min at g at °c. two hundreds microliter of the supernatant was used for rna extraction using the trizol reagent. all the sample rna were quantified using nd- c spectrophotometer (nanodrop, wilmington, usa), and were stored at − °c until use. the bp rt-pcr product encompassing the tgev-specific s gene was generated from viral genomic rna extracted from strain hb-yx using f and r primers (paton et al., ) . the resulting fragment was ligated into a pgem-t easy vector and transformed into e.coli dh α chemically competent cells according to standard procedures. the in vitro transcribed tgev standard rna was produced with ribomax large scale rna production system-t (promega, madison, usa). the length from the t promoter region to the ndei cut site of the pgem-t easy vector is nucleotides, generating a total transcript of nucleotides in length. the in vitro transcripts were quantified using nd- c and the copy number of rna molecules was calculated by the following formula: amount (copies/μl) = [rna concentration (g/ μl) /(transcript length in nucleotides × )] × . × . the in vitro transcribed rna was diluted in ten-fold serial dilutions to achieve rna concentrations ranging from . × to . × °copies/μl, which were used as the standard rna for tgev rt-rpa assay. according to the s gene nucleotides of different tgevs (accession numbers: af ; af ; af ; ay ; af ; af ; hq ; m ; m ; s ; z ) and prcvs (accession numbers: kr ; m ; m ; m ; m ) available in genbank, the rt-rpa primers and exo probe were designed to be specific to a portion of the s gene sequences conserved in tgev but absent in prcv. the forward primer was: ´-ttc agaggcaaattgtggtaatatgctgtatggc- ´; the exo probe was: ´-gcagatgaggttgttgcttatttacatgg(rox-dt)g(thf)(bhq -dt)agttaccgtattag-c spacer- ´; the reverse primer was ´-acg catatcaccaaatgtgacagtgccagacca- ´. the primers and probe were synthesized by a commercial company (sangon, shanghai, china), and the length of the amplicon was bp. rt-rpa reactions were performed in a total volume of μl containing . μl rehydration buffer and . μl magnesium acetate ( mm) from the twistamp tm rt exo kit (twistdx, cambridge, uk). other components included nm each rpa primer, nm exo probe, and μl of viral rna or μl sample rna. the rt-rpa reactions were performed at °c for min in the genie iii scanner device. ten nanograms of the different pathogen genomic rna or dna were used as template in the rt-rpa, and only the tgev rna were detected by rt-rpa while the other pathogens were not detected (fig. ) . no cross detections were observed in five independent reactions, demonstrating the high specificity of rt-rpa assay for the detection of tgev. in the analytical sensitivity analysis, the limit of detection (lod) of the rt-rpa was . × copies per reaction, which was the same as the realtime rt-pcr (fig. ) . the two-fold serial dilutions of the in vitro transcribed tgev rna were made from . × to . × copies, and from . × to . × copies, respectively. the above two-fold serial dilutions of the tgev rna, and . × copies in vitro transcribed tgev rna were further used in the rt-rpa, and the lod of the assay still was copies per reaction. the threshold time (tt) values were . min and . min for copies and copies, while there were no fluorescence amplification curves for . copies and copies. the rt-rpa assay was performed five times on the quantitative rna with similar results. for evaluating the potential applicability of the developed rt-rpa assay, clinical samples had rna extracted and were tested by the rt-rpa and real-time rt-pcr (vemulapalli et al., ) . fourteen samples ( . %, / ) were tgev rna positive in the rt-rpa, which were also positive in the real-time rt-pcr. the overall agreement between the rt-rpa and the real-time rt-pcr was % ( / ). the basic rpa assay were also performed for the tgev rna positive samples using the twistamptm rt basic kit (twistdx, cambridge, uk), and the reaction system was the same as rt-rpa except the exo porbe was not used. the rpa amplification products were sequenced by a commercial company (sangon, shanghai, china), then were blasted in the genbank. the blast results demonstrated that all the sequences showed more than . % homology to tgev strains in genbank, which confirmed the good specificity of the rt-rpa assay for tgev. for the positive samples, it took − min in the rt-rpa to see amplification, while it took - min in the real-time rt-pcr with the ct values ranging from . to . . the performance of the rt-rpa was comparable to real-time rt-pcr, and the former is much faster. the threshold time (tt) and cycle threshold (ct) values of the two assays were respectively well at an r value of . (fig. ) . the molecular diagnostic assays, which could be suitable for deployment closer to suspect cases of tge, would be of significant importance in the tge controlling. recent advances of the rpa technology on the portable tube scanner device provide a simple, convenient and inexpensive method for the point-of-care (poc) detection of pathogens in clinical samples (abd el wahed et al., a,b) . in this study, we developed a rt-rpa platform integrating the real-time rpa technology and a tube scanner genie iii for the rapid and sensitive detection of tgev. considering the close relationship between tgev and prcv, multiple alignment of the tgev and prcv s gene sequences were made, and a nucleotide-long region corresponding to sequence between nucleotides and of the tgev s gene orf, which is absent from all prcv strains, was set as the target of rt-rpa. other tgev strains were not included in the assay except for the isolated field strain hb-yx, which is the deficiency of this study. the rpa is tolerant to - mismatches in primer and probe showing no influence on the performance of the assay (abd el wahed et al., ; daher et al., ) , and there were only - mismatches in this study with other circulating tgev strains available in genbank. it is assumed the assay would detect all the circulating strains of tgev, based on the facts that the rt-rpa assay targeted the conserved s gene of tgev. the tube scanner genie iii weighs only . kg and incorporates a rechargeable battery that could support operation for a whole day. it is especially important for tgev detection in under-equipped laboratories and in the field. the user-friendly on-site detection platform offers an excellent tool for initial screening of clinical specimens for tgev, which should make valuable contributions to the rapid diagnosis of tge. the real-time rt-pcr required the expensive thermal cycler (vemulapalli et al., ) , and the rt-lamp were inconvenient to perform due to the agarose gel electrophoresis (chen et al., ; li and ren, ) . the developed rt-rpa assay needed no complicated apparatus, was performed easily and completed within min, showing distinct advantages in terms of equipment requirements, convenience and detection time. in conclusion, a user-friendly on-site detection platform for tgev was developed. the good analytical specificity and sensitivity, simple and easy operation protocol make the platform ideal for the rapid detection of tgev in under-equipped laboratories and the promising tool for poc detection of tgev, especially in the resource-limited settings. all authors declare that they have no conflicts of interest. a portable reverse transcription recombinase polymerase amplification assay for rapid detection of foot-and-mouth disease virus recombinase polymerase amplification assay for rapid diagnostics of dengue infection diagnostics-in-a-suitcase: development of a portable and rapid assay for the detection of the emerging avian influenza a (h n ) virus field 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interactions reverse transcription loop-mediated isothermal amplification for rapid detection of transmissible gastroenteritis virus detection of transmissible gastroenteritis virus by rt-pcr and differentiation from porcine respiratory coronavirus dna detection using recombination proteins transmissible gastroenteritis and porcine respiratory coronavirus emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences solid phase immune electron microscopy for diagnosis of transmissible gastroenteritis in pigs a real-time taqman rt-pcr assay with an internal amplification control for rapid detection of transmissible gastroenteritis virus in swine fecal samples an exo probe-based recombinase polymerase amplification assay for the rapid detection of porcine parvovirus reverse transcription recombinase polymerase amplification assay for the rapid detection of type porcine reproductive and respiratory syndrome virus a sensitive duplex nanoparticle-assisted pcr assay for identifying porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus from clinical specimens this work was supported by natural science foundation youth project of hebei province (c ) and science and technology project foundation of hebei province ( d) and partially funded by the fund for one-hundred outstanding innovative talents from hebei institution of higher learning (slrc ). key: cord- -mwgccr a authors: delmas, bernard; gelfi, jacqueline; l'haridon, rené; vogel; sjöström, hans; norén; laude, hubert title: aminopeptidase n is a major receptor for the enteropathogenic coronavirus tgev date: journal: nature doi: . / a sha: doc_id: cord_uid: mwgccr a coronaviruses, like many animal viruses, are characterized by a restricted host range and tissue tropism( ). transmissible gastroenteritis virus (tgev), a major pathogen causing a fatal diarrhoea in newborn pig, replicates selectively in the differentiated enterocytes covering the villi of the small intestine( ). to investigate the molecular determinants of the infection, we characterized the surface molecule used by the virus for binding and entry into host cells. here we report that aminopeptidase n, an ectoenzyme abundantly expressed at the apical membrane of the enterocytes, serves as a receptor for tgev. monoclonal antibodies were selected for their ability to block infection by tgev of porcine cell lines. they recognized a brush-border membrane protein of m(r), k, which was identified as aminopeptidase n by ammo-terminal sequencing. two lines of evidence supported the view that the peptidase itself acts as a receptor. first, virions bound specifically to aminopep-tidase n that was purified to homogeneity. second, recombinant expression of aminopeptidase n conferred infectivity by tgev to an otherwise non-permissive cell line. fast termination of pde activation observed under similar conditions . more recent work indicates that transducin gtpase can be faster under more physiologial conditions , - , but the mechanism of gtpase acceleration has remained unclear. the data of fig. show that pde itself serves as a gtpase-activating factor. the maximal gtpase rate observed in this reconstitution study (- . s-i) is still about lo-fold slower than the rate of the recovery from a photoresponse. but a more rapid rate (> . s -i) is observed in suspensions of disrupted rod outer segments l for the fast component of gtpase suppressed by micromolar concentrations of cgmp. our study allows us to conclude that this faster gtpase is a property of that transducin which activates pde, and thus the extent of pde-dependent gtpase acceleration is higher in rod outer segment suspensions than in reconstituted membranes. more recent data (v. y.a. et al., manuscript in preparation) shows that further concentration of rod outer segment suspensions (> flm rhodopsin) increases gtpase rates by at least twofold, close to the turn-off time of the photoresponse. the data shown in fig. indicate a feedback mechanism in retinal rods based on cgmp-dependent regulation of the lifetime of activated pde. such a mechanism might function during rod background adaptation, when the duration and light sensitivity of the photoresponse is diminished , . a reasonable model is that background light depletes intracellular cgmp levels, causing dissociation of cgmp from the non-catalytic binding sites on pde. this would accelerate the gtpase activity that terminates each pde activation event, leading to a faster and/or smaller photoresponse. such a mechanism might work in parallel with the known calcium feedback regulation of adaptation • the regulation of gtp-binding protein gtpase activity by an effector described here, although not previously described for a heterotrimeric g protein, has been documented extensively for several other classes of gtp-binding proteins (for example ref. ) . it is observed for elongation and initiation factors whose gtpase activity is enhanced by ribosomes. the class of small gtp-binding proteins including the product of proto-oncogene ras interact with gtpase-activating proteins (gaps) that may also be their effectors . the intrinsic gtpase of the heterotrimeric signal-transducing g proteins is considerably more rapid than that of the small gtp-binding proteins (for example refs - ), but still in several systems such as photoreception , , lfaction and muscarinic receptor-induced potassium channel regulation it has seemed to be too slow to explain the nature· vol . june letters to nature rapid on-off cycle of the relevant effectors. because acceleration has now been associated with the effector enzyme in the photoreceptor, it is relevant to search for similar mechanisms in other systems using heterotrimeric g proteins. aminopeptidase n is a major receptor for the enteropathogenic coronavirus tgev acts as a receptor. first, virions bound specifically to aminopeptidase n that was purified to homogeneity. second, recombinant expression of aminopeptidase n conferred infectivity by tgev to an otherwise non-permissive cell line. to obtain monoclonal antibodies against the tgev receptor, hybridomas were prepared from a mouse immunized with st cells, a swine testis cell line highly susceptible to tgev. several of the resulting antibodies exhibited, in three different porcine cell systems, a blocking activity comparable to that of a high-titre neutralizing anti-tgev antibody (fig. ). by contrast, no significant protection by the antibodies was observed after virus challenge in a feline cell system, or towards irrelevant viruses (group a bovine rotavirus or vesicular stomatitis virus; data not shown). therefore, the selected antibodies had the characteris- tics expected for antibodies recognizing a major tg ev receptor. the monoclonal antibodies all recognized a polypeptide of relative molecular mass is , ( s k) in st cell extracts, together with a faint band interpreted as the mannose-rich intracellular precursor (fig. , lane o. when using solubilized brush-border membranes from pig small intestine, three major species of ls k, sk and sok were coimmunoprecipitated (fig. , lane ) . the first amino acids of the sk species were detemined through n-terminal sequencing: nhz-ala-lys-gly-phe-tyr-i e-ser-lys-ala-leu-gly-i e-leu-gly-i e-leu-leu-gly-val-ala-ala-val-ala-thr-i e-i e-ala-leu-ser-val-cooh . this sequence was identical to the n-terminal sequence (minus the first met) of porcine aminopeptidase n, deduced from the exon i nucleotide sequence material immunoprecipitated with g antibody from the triton x- solubilized cell iysates was analysed by western blotting as described in further evidence that the anti-tgev-receptor antibodies recognized aminopeptidase n was obtained by showing that ( ) an antibody raised against rabbit aminopeptidase n reacted with the same three polypeptides in brush-border membrane preparations (fig. , lane ) : k and k, corresponding to the b (amino) and c (carboxy) subunits of the pig aminopeptidase, and k, uncleaved aminopeptidase ; ( ) the immunoprecipitated material hydrolysed leucine p-nitroanilide, a chromogenic substrate specific for aminopeptidase (ref. ; data not shown). two experiments were designed to demonstrate any direct association between aminopeptidase n and the virus. first, soluble aminopeptidase n was centrifuged after incubation in the presence of virions (fig. a) . aminopeptidase n-specific bands were recovered with pelleted tgev virions only. second, when the aminopeptidase was incubated in the presence of adsorbed virions (fig. b) , it bound to tgev and not the other enteric viruses. in both assays, earlier incubation with an antibody against aminopeptidase n reduced the binding considerably. because the two components were purified to homogeneity, it was concluded that the interaction between the aminopeptidase and tgev occurs in the absence of any other cellular protein. the gene encoding aminopeptidase n ( apn) was expressed in non-permissive cells to see whether this would confer them with the capacity to bind tgev. a pig intestine complementary dna library was screened by use of a homologous dna probe derived from the ' end of apn gene. a full-length cdna copy was . cloned and contained an open reading frame of , nucleotides encoding a polypeptide % identical to human aminopeptidase (data not shown). mdck cell clones stably transformed with the porcine apn cdna expressed a polypeptide of k which reacted with antibodies against aminopeptidase n (fig. a) . the aminopeptidase activitl of the transfected clones was about -fold higher compared with non-transfected clones. on viral challenge, all of the three independent clones tested seemed to be fairly susceptible to tgev infection, as proved by extensive destruction of the infected monolayers and synthesis of the viral structural polypeptides (fig. b, c) . earlier incubation with an antibody specific for aminopeptidase n prevented the appearance of viral cytopathic effect. these results show that aminopeptidase n was the only porcine protein necessary to confer susceptibility on canine kidney cells naturally resistant to tgev. moreover, the protease function of the molecule did not seem to be involved because it was blocked by bestatin, an inhibitor of aminopeptidase, without preventing the infection (fig. b) . so far, defined receptors include molecules that belong to the immunoglobulin superfamily, such as cd for hiv , icam-l for rhinovirus b , poliovirus receptor and a carcinoembryonic antigen for murine hepatitis coronavirus , and also an aminoacid transporter for murine leukaemia retroviruses l • our study provides strong evidence that porcine aminopeptidase n serves as a receptor for an enveloped rna virus, tgev. this emphasizes the diversity of the membrane-bound proteins that viruses subvert for gaining entry into cells. aminopeptidase n is a well documented ectoenzyme that binds to the membrane through an n-terminal segment , , . human aminopeptidase n is identical to cdb, a surface antigen of many myeloid cells , it is a zinc-binding protease that catalyses the removal of n-terminal, preferentially neutral residues from peptides, it is expressed in many tissues at different levels , the highest activity being found in the small intestinal mucosa, where the aminopeptidase represents about % of the protein content of the apical membrane of the differentiated enterocytes, and in the kidney brush border. it is also expressed to a lesser extent in liver, lung and colon, where the virus does replicate, but without causing the specific histopathological damage seen in the small intestine • in the intestine, the distibution of the receptor and the site of multiplication of tgev are thus strikingly correlated. this argues for a pivotal role of aminopeptidase n/cd in determining the tissue tropism of tgev, investigating the nature of the virus interaction with aminopeptidase n could provide a rationale for the design of an antiviral strategy against tgev and related infections. to develop a monoclonal antibody against the hcv- e receptor, we produced hybridomas against deoxycholatesolubilized membrane proteins of two hcv- e-susceptible human cell lines (wi lung fibroblasts and hl myeloid leukaemia cells). a monoclonal antibody designated rbs protected wi and rd human cell lines from hcv- e-induced cytopathic effects and protected wi cells from virus infection ( fig. la-c) . rbs pretreatment reduced the number of hcv- e-infected wi- cells at h post-infection by %, compared with cells pretreated with control mouse ascites. by contrast, rbs did not inhibit replication of hcv-oc in wi or rd cells, indicating that the receptor specificities of hcv-oc and hcv- e are different. susceptibility to hcv- e infection in mouse-human somatic cell hybrids depends on a gene located on human chromosome (ref. ) . a promising candidate for the hcv-ii to whom correspondence should be addressed. • this exopeptidase removes amino-terminal residues to complete the digestion of short peptides in the gut and helps break down neurotransmitter peptides in the brain , , , . hapn is identical to cd , a glycoprotein identified on granulocytes, monocytes and their bone marrow progenitors , o. porcine aminopeptidase n is a receptor for transmissible gastroenteritis virus, a porcine corona virus in the same serogroup as hcv- e (ref. ). because aminopeptidase from humans, pigs and other mammals are structurally similar , - , we investigated whether hcv- e and rbs would bind specifically to hapn and whether expression of hapn by murine cells would make them susceptible to infection with hcv- e. murine nih t cells transfected with hapn cdna in a retroviral vector (hapn- t ) and untransfected nih t cells were chauenged with hcv- e and hcv-oc to determine their susceptibility to virus infection. although the control nih t cells were resistant to hcv- e infection (fig. id) , the hapn-transfected mouse cells were susceptible to infection with this virus (fig. ie) . by contrast, hapn- t cells were no more susceptible than nih t cells to infection with hcv-oc (data not shown). thus, expression of hapn confers hcv- e susceptibility, but not hcv-oc susceptibility, on murine cells. we analysed binding of rbs to membrane preparations from hapn- t or parental nih t fibroblasts. the antibody bound to membranes of hapn- t but not to those of nih t cells (fig. a) , indicating that rbs recognized hapn. similarly, hcv- e virions bound more strongly to hapn- t membranes than to nih t membranes (fig. b) , and rbs competi- gen gray·keller for participating in the preliminary stage of this work we thank r. christon for microvillar preparations, j. c. huet for n-terminal sequencing acknowledges the support of the commission of the european communities. lk.v., h. . and d.n. are members of the biomembrane research center key: cord- -ts aiykg authors: ding, li; huang, yong; du, qian; dong, feng; zhao, xiaomin; zhang, wenlong; xu, xingang; tong, dewen title: tgev nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p signaling date: - - journal: biochemical and biophysical research communications doi: . /j.bbrc. . . sha: doc_id: cord_uid: ts aiykg abstract our previous studies showed that tgev infection could induce cell cycle arrest and apoptosis via activation of p signaling in cultured host cells. however, it is unclear which viral gene causes these effects. in this study, we investigated the effects of tgev nucleocapsid (n) protein on pk- cells. we found that tgev n protein suppressed cell proliferation by causing cell cycle arrest at the s and g /m phases and apoptosis. characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of n gene resulted in an accumulation of p and p , which suppressed cyclin b , cdc and cdk expression. moreover, the expression of tgev n gene promoted translocation of bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase- , resulting in cell apoptosis in the transfected pk- cells following cell cycle arrest. further studies showed that p inhibitor attenuated tgev n protein induced cell cycle arrest at s and g /m phases and apoptosis through reversing the expression changes of cdc , cdk and cyclin b and the translocation changes of bax and cytochrome c induced by tgev n protein. taken together, these results demonstrated that tgev n protein might play an important role in tgev infection-induced p activation and cell cycle arrest at the s and g /m phases and apoptosis occurrence. transmissible gastroenteritis virus (tgev) is an enveloped virus that contains a large, positive-sense, single-stranded rna genome [ ] , belonging to family coronaviridae [ ] . tgev, together with human coronavirus e (hcov- e), porcine epidemic diarrhea virus (pedv), canine coronaviruses (ccovs) belongs to alpha coronavirus [ ] . tgev replicates in enterocytes and provokes villous atrophy, resulting in lethal watery diarrhea and dehydration in piglets, which is considered to be a central event in the pathogenesis of tgev infection [ ] . the -two-thirds of the tgev genome encodes the replicasetranscription complex, rep a and rep b [ ] . during tgev replication, a -coterminal nested set of subgenomic mrnas, which encode other viral proteins. tgev has four major structural proteins: the spike (s), the integral membrane protein (m), the nucleocapsid protein (n) and a small envelope protein (sm) [ ] . tgev n protein, a multifunctional phosphoprotein, plays a primary structural role in packaging the rna genome into a helical ribonucleoprotein, as well as regulatory roles in viral rna synthesis (replication and transcription), translation, and modulation of host cell metabolism [ ] . tgev is known to cause cell cycle arrest and apoptosis via activation of p signaling in different host cell types including st and pk- cells [ , , ] . the evidence of induction of apoptosis by tgev implies an involvement of apoptosis in the pathogenesis of tge [ , ] . however, it remains to be known which tgev protein(s) induces cell cycle arrest and apoptosis in cells that support productive viral replication. current evidence indicates that tgev n protein might be involved in caspase-mediated proteolysis within the host cell [ ] . further studies revealed that the tgev n protein could locate nucleolar and might possess the function to disrupt cell division [ ] . however, whether tgev n protein could affect cell division and cell apoptosis and the associated molecular mechanisms are not clear. in the present study, we demonstrate that tgev n protein, when expressed within cells, can cause cell cycle arrest and apoptosis in pk- cells, in which p and p activation, cyclin b and cdc decrease, bax translocation to mitochondria, cytochrome c release and subsequent activation of caspase- are required. fied % co . the tgev shaanxi strain was used as previously described [ , ] . full-length n gene from tgev was pcr amplified, ligated into the pgem-t easy vector (promega, madison, usa), and then subcloned into between xhoi/ecori sites of eukaryotic expression vector pegfp-n (egfp) (clontech, palo alto, ca), to produce the recombinant his-tagged construct pegfp-n -tgev-n (n-egfp). this vector contains the cytomegalovirus promoter for the expression of n protein with a six-histidine tag. they were sequenced to confirm that no errors were introduced as a result of pcr amplification. in vitro expression of the n-egfp was tested in transient expression experiments using pk- cells. pk- cells, grown in mm plate, were transfected with egfp vector only, or n-egfp. after h, h and h of transfection, the expression of n was directly observed under a fluorescence microscopy and further confirmed by western blot analysis using his antibody (sigma-aldrich, usa). the effects of tgev n protein on cell viability were determined using the mtt assay. briefly, pk- cells, grown in -well culture plates, were transfected with egfp vector only, or n-egfp expression vector. after h, h and h of transfection, the cells were treated with ll of mg/ml mtt and the resulting formazan crystals were dissolved in di-methyl sulfoxide (dmso). the absorbance was measured by microplate spectrophotometer (bio-tek instruments, inc., winooski, usa) at nm. results were expressed as percentage of the controls, which were arbitrarily assigned % viability. to determine cell cycle status, nuclear dna content was measured by using propidium iodide staining. briefly, cells were fixed in % ethanol for min at °c. after washing with phosphatebuffered saline (pbs), the cells were stained with . % triton x- (sigma), lg/ml rnase a (sigma) and lg/ml propidium iodide (sigma) for min and analyzed using a facscan flow cytometer (beckman coulter, inc., fullerton, ca., usa). annexin v-fitc apoptosis kit (biovision, inc., ca., usa) was used for apoptosis detection according to the manufacturer's protocol. briefly, harvested cells were washed twice with pbs and resuspended in ll binding buffer, followed by adding ll of annexin v-fitc and ll of pi. after incubation in the dark for min at room temperature, a total of , cells were acquired and percentage of positive cells were analyzed by flow cytometry (beckman). pk- cells, grown in mm plate, were transfected with egfp vector only, or n-egfp. after transfection, cells were collected and lysed by ripa. isolation of mitochondrial and cytosolic proteins was performed using the mitochondria/cytosol fractionation kit (pierce, rockford, il, usa). protein concentrations were measured using bca protein assay reagent (pierce). equivalent amounts of proteins were loaded and electrophoresed on % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page). subsequently, proteins were transferred to polyvinylidene difluoride (pvdf) membranes (millipore corp, atlanta, ga., usa). the membranes were blocked with % nonfat dry milk at room temperature for h, and then incubated with indicated primary antibodies over night at °c, followed by hrp-conjugated secondary antibodies at room temperature for h. the signal was detected using ecl reagent (pierce, rockford, il., usa). caspase- activity was measured by colorimetric assay kit (bio-vision) according to the manufacture's instructions. briefly, cell lysates were prepared and protein concentrations were measured using bca protein assay reagent (pierce). then lg of protein in each sample was incubated with caspase- substrate at °c for h. samples were read at nm in microplate spectrophotometer (biotek instruments, inc., winooski, usa). the pk- cells were transfected with the n-egfp expression vector, and the expression of n protein was directly examined under the fluorescence microscopy and further confirmed by western blot using anti-his antibody to detect his-tagged n protein. after h of transfection, the n-egfp protein was dispersed throughout the cytoplasmic region, while the egfp control showed a diffuse distribution pattern (fig. a) . the expressing n-egfp proteins were detected as expected, while no signal was detected in the egfp control when detected using the anti-his antibody (fig. b) . these results suggested that the n protein of tgev was expressed in pk- cells. to determine the effect of tgev n protein on expressed pk- cells, cell viability was firstly tested by mtt assay. result showed that the cell viability of pk- cells transfected with n-egfp expression vector had significantly reduced at h of post-transfection (fig. c) . the apparent inhibition did not occur in the egfp empty vector-transfected cells. these data demonstrated that expression of tgev n gene in pk- cells inhibited cell viability. to determine whether the growth inhibition of tgev nexpressing cells was due to the arrest of cell cycle progression at a certain phase, at different times after transfection, cell cycle profiles were analyzed by flow cytometry (fig. a, upper panel) . the histograms were quantitatively analyzed by use of a curve-fitting program to determine the percentage of cells in each of the g / g , s, and g /m phases ( fig. a, lower panel) . results showed that the proportion of cells in s and g /m phases significantly increased at h of post-transfection, and further increased with transfection time in the cells transfected with n-egfp expression vector, compared to that in the cells transfected with egfp control vector ( fig. a) , which strongly indicated that cell cycle arrest was occurred in tgev-n-expressing cells. to further determine the effects of tgev n protein on cell progression through s and g /m phase, we investigated its effects on protein levels of the p , p , cyclin b , cdc , and cdk , which regulate transition through the s and g /m boundary checkpoint. fig. b showed that the levels of p significantly increased at h and h after cells transfected with n-egfp expression vector compared with egfp empty vector-transfected cells and nontransfected groups. the amounts of p were slightly increased at h, and significantly increased at h in the cells expressed tgev n gene. furthermore, the expression of cyclin b , cdc and cdk decreased in the cells expressed tgev n gene compared with the control cells (fig. b) , which coincided with the changes of these cycle-related molecules in tgev-infected cells. these results indicated that tgev n protein caused s and g /m-phase arrest through regulation of certain cell cycle factors to modulate the proceeding of s and g /m phases. to examine whether apoptosis was initiated in the cells expressed tgev n gene, pk- cells transfected with n-egfp expression vector or egfp empty vector, or untransfected cells were subjected to annexin v and pi staining and flow cytometry analysis. as shown in fig. a , annexin v positive cells increased about . %, % and . % in the cells transfected with n-egfp expression vector than that in egfp empty vector-transfected cells at h, h and h of post-transfection, respectively. whereas, annexin v positive cells increased about . %, . % and . % in the cells transfected with egfp empty vector than that un-transfected control cells at h, h and h of post-transfection, respectively. collectively, these data indicated that apoptosis was initiated in the pk- cells expressed tgev n gene. to further elucidate the mechanism of the tgev n-induced apoptosis, the translocation of bax and release of cytochrome c were analyzed by western blot. as shown in fig. b , bax was translocated from cytosol to mitochondria at h of post-transfection, and steadily increased in mitochondria with the transfection time. following bax translocation to mitochondria, cytochrome c released from mitochondria to cytosol in the cells expressed tgev n gene. the activity of caspase- significantly increased in the cells transfected n-egfp expression vector compared to un-transfected cell or egfp empty vector-transfected cells (fig. c) . taken together, the results demonstrate that tgev n protein triggered the efflux of cytochrome c into the cytosol and the activation of caspase- , thus leading to apoptosis occurrence in the cells. in order to investigate whether p pathway regulate tgev n protein-induced cell cycle arrest and apoptosis, we tested cell cycle profiles and apoptosis in tgev n protein expressed pk- cells with specific p inhibitor pifithrin-a (pft-a). results showed that pft-a attenuated tgev n protein-induced cell cycle arrest at s and g /m phases (fig. a ). in addition, the apoptotic rate induced by tgev n protein was slightly inhibited by pft-a (fig. b) . to further explore the roles of p , we investigated the effects of pft-a on the expression of cell cycle and apoptosis regulating factors in tgev n protein expressed pk- cells. as expected, incubation of pk- cells with pft-a, cdc , cdk and cyclin b expression slightly increased in tgev n protein expressed pk- cells with pft-a treatment compared to that without pft-a treatment (fig. c) . furthermore, the translocation of bax and cytochrome c was moderately reversed by pft-a treatment in tgev n protein expressed pk- cells (fig. c) . the activity of caspase- was reduced in tgev n protein expressed cells with pft-a treatment compared to the cells without pft-a treatment (fig. d) . taken together, these results suggest that p might play key roles in mediation of tgev n protein induced cell cycle arrest and apoptosis. several viruses-encoded proteins have been shown to activate the cell cycle or apoptotic process by interacting with various cellular components [ ] [ ] [ ] . virus-induced apoptosis of host cells can either facilitate virus dissemination or be part of the host defense response invoked to counteract viral infection [ ] . several in vitro studies have demonstrated that tgev infection induced cell cycle arrest and apoptosis through activation of p pathway in different host cell lines [ , ] . however, it is unclear which viral genes play a dominant role in induction of the cell cycle arrest and apoptosis. in this study, we have investigated the effects of tgev n protein on cell growth, cell cycle progression and cell apoptosis, as well as the mechanism that tgev n protein causes the cell cycle arrest and apoptosis. one of the common phenomena of viral infection is to induce the g /m-phase arrest in host cells. human immunodeficiency virus type (hiv- ) vpr activates cell cycle inhibitor p to prevent cell proliferation by causing arrest at the g /m phase of the cell cycle [ ] . human papillomavirus (hpv) type e protein induces g /m arrest through inhibition or delay in the activation of cdk /cyclin b kinase activity [ ] . chicken anemic virus (cav) apoptin protein and herpes simplex type (hsv- ) icp protein can interfere with kinetochores to block cell cycle progression at g /m phase [ , ] . previous studies have revealed that the tgev n could locate nucleolar and might function to disrupt cell division [ ] . here, our results showed that expression of tgev n protein activated p and up-regulated cell cycle inhibitor p , and then inhibited the activation of cdc /cyclin b , leading to g /m arrest, which was consistent with our previous results found in tgev-infected cells [ ] , suggesting that tgev n protein might play an important role in tgev-induced cell cycle arrest. in previous studies, we found tgev infection induces s and g /m arrest, which is aimed to establish a pseudo-s phase state for viruses to replicate of their genomes [ ] . the results presented in here also provided evidences for s phase arrest and following g /m arrest, suggesting that tgev n protein might play an important role in establishing a pseudo-s phase state for virus replication. the same phenomena were also found in other viruses [ ] . many viral proteins are involved in induction of apoptosis. infectious bursal disease virus vp [ ] , rubella virus capsid protein [ ] , hepatitis c virus core protein [ ] , and japanese encephalitis virus ns b-ns [ ] have been shown to induce apoptosis alone in cell culture and play an important role in viral pathogenesis. over the past few years, studies report multifaceted roles for the viral n protein in trafficking, translation and in modulation of immune signaling. previous studies have indicated that tgev n protein underwent caspase-mediated proteolysis during tgev-induced apoptosis [ ] . in this study, we further investigated its function in modulation of apoptosis and demon-strated that tgev n protein was capable of inducing apoptosis in the transfected pk- cells, which might be important for the pathogenesis of tgev. p is a key element in the induction of cell cycle arrest or apoptosis in viruses infected cells [ , ] . our previous studies have demonstrated that tgev infection could induce cell cycle arrest and apoptosis via activation of p signaling in cultured cells, suggesting that p might play important roles in tgev-induced cell cycle arrest and apoptosis [ ] . in the present study, we also found that p was activated to induce the change of apoptotic and cell cycle regulating factors such as cdc , cdk , cyclinb , bax and cyt c in tgev n protein expressed pk- cells. these studies further suggest p might play an important role in the pathogenesis of tgev. in conclusion, the present study demonstrates that the expression of tgev n gene induced s-phase and g /m-phase arrest, and subsequent apoptosis through the activation of p and p , deduction of cyclin b/cdc , promoting translocation of bax to mitochondria, followed by a release of cytochrome c into the cytosol, results in activation of the effector caspase- , thus leading to cellular destruction. li ding and yong huang designed the experiments, interpreted the data and wrote the article. li ding performed the experiments with assistance and advice from qian du, feng dong, xiaomin zhao, wenlong zhang and xingang xu. yong huang and dewen the activity of caspase- was measured by colorimetric assay kit. results are means ± sd. ⁄ p < . , ⁄⁄ p < . versus egfp empty vector-transfected cells. transmissible gastroenteritis coronavirus induces programmed cell death in infected cells through a caspase-dependent pathway ratification vote on taxonomic proposals to the international committee on taxonomy of viruses molecular basis of transmissible gastroenteritis virus epidemiology, the coronaviridae generation of a replicationcompetent, propagation-deficient virus vector based on the transmissible gastroenteritis coronavirus genome strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model transmissible gastroenteritis virus infection induces cell cycle arrest at s and g /m phases via p -dependent pathway transmissible gastroenteritis virus infection induces apoptosis through fasland mitochondria-mediated pathways the viral nucleocapsid protein of transmissible gastroenteritis coronavirus (tgev) is cleaved by caspase- and- during tgev-induced apoptosis localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division regulation of ros in transmissible gastroenteritis virus-activated apoptotic signaling pk- cells were transfected with n-egfp expression vector or egfp empty vector and treated with pft-a for h. cells were collected and stained with propidium iodide for facs analysis. (b) effect of pft-a on the apoptosis induced by tgev n protein. pk- cells were transfected with n-egfp expression vector or egfp empty vector and treated with pft-a for h. cells were collected and stained with annexin v and pi for facs analysis. values are mean ± sd. ⁄ p < . versus tgev n gene expressed cells without pft-a. (c) effects of pft-a on the expression of cell cycle and apoptosis regulating factors. pk- cells were transfected with n-egfp expression vector or egfp empty vector and treated with pft-a for h. cells were collected and subjected to western blot analysis. cox and actin were used as internal controls for the mitochondrial fractions and the cytosolic fractions, respectively. (d) effects of pft-a on caspase- activity. pk- cells were transfected with n-egfp expression vector or egfp empty vector and treated with pft-a for h. cells were collected and the activity of caspase- was measured by colorimetric assay kit avian influenza virus a/hk/ / (h n ) ns protein induces apoptosis in human airway epithelial cells protein kinase a phosphorylation activates vpr-induced cell cycle arrest during human immunodeficiency virus type infection g /m cell cycle arrest in the life cycle of viruses cell cycle arrest by vpr in hiv- virions and insensitivity to antiretroviral agents transmissible gastroenteritis virus infection induces cell apoptosis via activation of p signaling hiv- vpr activates cell cycle inhibitor p /waf /cip : a potential mechanism of g /m cell cycle arrest role for wee in inhibition of g -to-m transition through the cooperation of distinct human papillomavirus type e proteins the viral protein apoptin associates with the anaphase-promoting complex to induce g /m arrest and apoptosis in the absence of p degradation of nucleosome-associated centromeric histone h -like protein cenp-a induced by herpes simplex virus type protein icp the structural protein odv-ec of autographa californica nucleopolyhedrovirus is a multifunctional viral cyclin induction of apoptosis< i> in vitro</i> by the -kda nonstructural protein of infectious bursal disease virus: possible role in viral pathogenesis rubella virus capsid protein induces apoptosis in transfected rk cells hepatitis c virus core protein modulates trail-mediated apoptosis by enhancing bid cleavage and activation of mitochondria apoptosis signaling pathway japanese encephalitis virus ns b-ns protease induces caspase activation and mitochondria-mediated apoptosis in human medulloblastoma cells potential role for p in the permissive life cycle of human cytomegalovirus cross talk between pml and p during poliovirus infection: implications for antiviral defense tong revised the paper. all authors have read the paper and approved to submit it to your journal. there is no conflict of interest of any authors in relation to the submission. key: cord- -l dpv nx authors: o’toole, d.; brown, i.; bridges, a.; cartwright, s. f. title: pathogenicity of experimental infection with ‘pneumotropic’ porcine coronavirus date: - - journal: research in veterinary science doi: . /s - ( ) - sha: doc_id: cord_uid: l dpv nx virus localisation and lesions were studied in one-week-old piglets following combined intranasal-oral inoculation with a british isolate of ‘pneumotropic’ porcine coronavirus (pcv) and were compared with the effects of transmissible gastroenteritis virus (tgev) infection in five piglets. unlike tgev-infected piglets, all pcv-inoculated piglets remained clinically healthy. seroconversion was detected at seven days after inoculation. mild bronchointerstitial pneumonia involving terminal airways was consistently present at two days after infection and thereafter. both pcv and tgev infected bronchiolar epithelium and alveolar macrophages but, unlike tgev, replication by pcv in villous enterocytes was limited and did not cause villous atrophy. virus localisation and lesions were studied in oneweek-old piglets following combined intranasal-oral inoculation with a british isolate of 'pneumotropic' porcine coronavirus (pcv) and were compared with the effects of transmissible gastroenteritis virus (tgev) infection in five piglets. unlike tgev-infected piglets, all rcv-moculated piglets remained clinically healthy. seroconversion was detected at seven days after inoculation. mild bronchointerstitial pneumonia involving terminal airways was consistently present at two days after infection and thereafter. both pcv and tgev infected bronchiolar epithelium and alveolar macrophages but, unlike tgev, replication by rev in villous enterocytes was limited and did not cause villous atrophy. a new coronavirus of pigs which has a close antigenic relationship to transmissible gastroenteritis virus (tgev) has recently been identified as enz ' tic in great britain, france, belgium and hoiland (reviewed by jestin et al ) . conventional serological tests do not distinguish between 'pneumotropic' porcine coronavirus (rcvj-infected and tgevinfected pigs although a blocking elisa developed by garwes et al ( ) shows promise. the significance of this r-ev as a pathogen is unclear. studies in belgium indicated that po was non-pathogenic and infected respiratory but not enteric tracts (pensaert et al ) . several field reports have however linked rev with outbreaks of respiratory disease (jest in et al , veterinary investigation service . the aim of this study was to establish whether or not pcv caused lesions in susceptible piglets with particular attention to respiratory and digestive tracts, and to map the distribution of the virus in selected tissues using immunocytochemistry and virus tion. owing to its similarity to tgev, the changes in r-ev-infected piglets were compared with those in piglets foilowing tgev infection. twenty-four piglets from three litters of conven-tional breeds were used. they were hysterotomy derived one or two days before their due date and subsequently colostrum-deprived using a modification of the methods described by tavernor et al ( ) . the three parent sows were seronegative to r-evitgev and originated in herds with no previous history of transmissible gastroenteritis. piglets were reared individually in sterile aluminium anodised cages in plastic isolators kept at to °c with positive pressure ventilation. they were fed on a diet of sterilised evaporated milk (carnation foods) diluted with a mineral solution containing ferrous sulphate three, four or five times daily according to a schedule based on age. piglets were also given an oral vitamin supplement (adibec drops; parke, davis). as only single isolators with individual cages were available, the study was done in two periods one month apart. the two batches of piglets were maintained under identical conditions. the first batch consisted of monarch piglets aged six days, all from one litter. they were inoculated with r-ev (nine piglets) or were uninoculated controls (three). following sterilisation of cages and isolators, a second batch of piglets (large white cross landrace) aged seven days from two litters were inoculated with rev (five) or tgev (five) or were uninoculated controls (two). before use, empty cages and isolators were sterilised with ethylene oxide and formaldehyde. microbiological checks were carried out by swabbing the interior of isolators and placing swabs into tryptose broth at °c. clinical signs, feed intake and rectal temperatures were monitored daily. blood samples were collected for serology from the anterior vena cava of all piglets before inoculation and before euthanasia from the heart of piglets six days after inoculation and thereafter. apart from the one tgev-inoculated piglet which died as a result of transmissible gastroenteritis, piglets were killed in isolators by intracardiac sodium pentobarbitone at one to days after infection (table ) the rev isolate (stopps) was isolated from a nasal s~ab taken .fr~m one of several pigs with respiratory isolate was twice passaged in porcine kidney pnrnary cultures and the resultant virus stock was titrated before use. piglets received () ' tcid (?o per cent tissue culture infective dose) of r-ev in tissue culture fluid (i ml intranasally and ml orally). the tgev isolate (miller) was a gut homogenate prepared from experimentally infected piglets. piglets received - tcid of tgev in filtered gut homogenate (i ml intranasally and ml orally). . to confirm the specificity of the pcv and tgev inocula, extracts of each were inoculated on to porcine kidney primary cultures and stained at hours using a conventional direct fluorescent anti-o~y test and an indirect test employing a differentlat.mg monocl~nal antibody ( bi) (garwes et a ) which reacts with tgev alone using a murine fluorochrome conjugate. following euthanasia in isolators, piglets were removed and portions of turbinate, right cranial lobe of the lung, terminal ileum and ileal contents were taken aseptically for virus isolation. the extent of pneumonia, where present, was estimated subjectively. no tissues were taken for virus isolation from three of the five tgev-inoculated piglets. the following tissues were collected for immersion fixation in per cent neutral, phosphate buffered formalin and processed for light microscopy: oesophagus, stomach, small intestine (six levels), caecum, colon, liver, pancreas, snout -)vith nasal conchae, larynx, trachea, lymph, nodes (left tracheobronchial, cranial mediastinal and mesenteric), thymus, spleen, tonsil, kidney, bladder, gonad, thyroid, adrenal skin, m. semintendinosus, brain (seven levels) and spinal cord (three levels). the primary bronchus of the left lung was cannulated and lobes were inflated with sorensen's phosphate buffered per cent glutaraldehyde at room temperature. cranial, middle and caudal lobes were processed for light microscopy. only a limited range of tissues, chiefly from respiratory and digestive tracts, was taken from three of the five tgev-inoculated piglets. additional samples of nasal conchae mid-trachea right middle lung lobe and distal ileum~ere collected fresh and immersion fixed for hours in per cent neutral phosphate buffered formalin. tissues were dehydrated in ethanol, cleared in xylene, embedded in paraffin wax and sectioned at fam. sections were stained using a peroxidase-antiperoxidase (pap) technique following overnight incubation with a in , dilution of monoclonal antibody (da ) which reacts with nucleoprotein of tgev and pcv (garwes et al ) . sections were counterstained with mayer's haemalum. selected samples of liver, spleen and thymus fixed for varying lengths of time from control, tgev-and pcv-inoculated piglets were similarly stained. test sections were accompanied by three controls: a duplicate of each section stained as above but with the primary antibody step omitted; ileum from a piglet infected hours earlier with tgev (positive tgev control); and lung from a piglet infected four days earlier with r-ev (positive rev control). for ultrastructure, i mm? pieces of consolidated, glutaraldehyde-fixed lung were taken from one pcvinoculated piglet and one tgev-inoculated piglet, both at three days after infection. tissue was rinsed in buffer, post-fixed in per cent osmium tetroxide, dehydrated in ethanol followed by propylene oxide, and embedded in araldite. ultrathin sections were stained with uranyl acetate and lead citrate. virus was reisolated using porcine kidney cells in a hanks' medium containing per cent bovine serum, ' per cent sodium bicarbonate solution, units penicillin ml : j, fag streptomycin ml j and units mycostatin ml j. the maintenance medium was earle's containing i per cent fetal bovine serum, ' per cent bicarbonate solution and antibiotics. tissue collected at necropsy was prepared as per cent homogenates in phosphate buffered saline with antibiotics, incubated for minutes at room temperature, clarified at g for minutes and the resulting supernatant inoculated on to porcine kidney primary cultures. tissue culture fluid was passed on to fresh cell sheets at seven days and virus growth was tested by the tgev direct fluorescent antibody test. positive controls were r-cv-lntected cultures and negative controls were uninfected cultures. serum antibody was detected using the standard tgev microserum neutralisation test using a canine tumour cells (modified from witte ). all five tgev-inoculated piglets developed typical signs of transmissible gastroenteritis. anorexia, diarrhoea, dehydration and hypothermia began at two days after infection and resulted in the death of one piglet at three and a half days after infection. the two remaining roev-inoculated piglets at that time were killed as they were moribund. no clinical signs of pneumonia were evident. by contrast, r-evinoculated and uninoculated control piglets remained clinically healthy over the days of the study. for convenience and as there were no differences between the two batches of pcv-inoculated piglets, the descriptions of lesions in these animals are combined. all but one of the r-ev-inoculated piglets developed a cranioventral bronchointerstitial pneumonia involving from less than per cent to approximately per cent of lung parenchyma. grossly, red consolidated areas formed a sublobular or lobular mosaic in cranial and middle lobes. piglets killed at seven to days after infection also had moderately enlarged tracheobronchial lymph nodes. histologically, pulmonary lesions were mild or moderate and involved small calibre bronchioles, alveolar ducts and peribronchiolar alveoli. between two and six days after infection, acute changes were characterised by individual bronchiolar cells bulging into the lumen, ..; i'm immunocytochemistry r-ev antigen was identified in the epithelial cytoplasm of small and medium bronchioles in eight of piglets and its distribution was closely correlated with areas of pneumonia. typically, solitary cells or small groups of cells at different stages of detachment from the basement membrane were stained (fig ) . viral antigen was also present in degenerated intraluminal cells and in alveolar macrophages. three r-ev-infected piglets had widely separated positively stained villous enterocytes (fig ) and one had positively stained turbinate epithelial cells. antigen was absent in spleen, thymus and liver. d. o'toole. i. brown. a. bridges. s. f. cartwright thymic atrophy and multifocal pulmonary congestion. four of the tgev-inoculated piglets had bronchointerstitial pneumonia similar in histological character to the pcv-infected piglets. other lesions detected histologically were moderate thymic atrophy in four piglets, depletion of splenic periarteriolar sheaths in four piglets, mild multi focal hepatic necrosis in five piglets, mild rhinitis in two piglets, moderate ulcerative laryngitis in one piglet and multifocal renal tubular necrosis with dilatation in one piglet. no gross or microscopic lesions developed in the control group. two of the five piglets had occasional intra-alveolar syncytial cells. similar cells were present in some of the pcv and rcsv-intected piglets in otherwise normal areas of lung and were assumed to be an incidental finding (castleman et al ) . followed by degeneration and detachment. macrophages, fibrin and cellular debris accumulated in bronchioles, alveolar ducts and alveoli (fig ia) . the naked basement membrane of bronchioles was covered by attenuated epithelial cells. interalveolar septa were thickened. evidence of repair was present at seven days after infection and was a characteristic feature up to ii days after infection. there was increased mitotic activity with hyperplasia in the epithelium of medium and to a lesser extent small calibre bronchioles. rarely, multinucleated epithelial cells were present in hyperplastic bronchiolar epithelium (fig ) . modest cuffs of iymphoblasts, macrophages and plasma cells accumulated around bronchioles and peribronchiolar vessels. by ii days after infection, alveolitis had largely resolved. some bronchioles still lacked a complete epithelial lining at this time. other changes in the rcv-lnoculared piglets were lymphoid follicular hyperplasia in thoracic lymph nodes, mild rhinitis with disorganisation, hyperplasia and microcystic change in turbinate epithelium (five of piglets), ulcerative laryngitis (one of ), necrosis of laryngeal glands (one of ), multifocal tracheitis (one of ), multifocal necrosis of tracheal glands (one of ), mild ileitis (one of ) and mild multifocal hepatic necrosis (one of ). in addition to typical enteric lesions of transmissible gastroenteritis (pensaert et al ) , three of the five tgev-inoculated piglets had gross evidence of mild cranioventral pneumonia. other gross lesions were dehydration, congestion of mesenteric vessels, all five tgev-inoculated piglets had viral antigen in villous enterocytes associated with atrophic villi (fig ) . tgev antigen was detected in bronchiolar epithelial cells and in alveolar macrophages in areas of pneumonia in three piglets, and hepatocytes in necrotic foci also contained intracytoplasmic viral antigen in three piglets. no positive staining was found in thymus or spleen. tissues from the five uninoculated piglets were uniformly negative for pcv/tgev antigen. the range of cell types which contained coronavirus-like particles in the lung of the r-ev-infected piglet was identical to that in the rosv-infected piglet. viral particles were most commonly found near the apical plasmalemma of non-ciliated cuboidal bronchiolar cells. virus occurred in the apical cytoplasm of these cells in endoplasmic reticulum or in membrane bound vesicles and followed a maturation sequence similar to that of tgev in villous enterocytes (wagner et al ) (fig ) . some degenerating cells contained intracytoplasmic virus. virus also occurred on the' surface of alveolar macrophages and free in alveoli. some type i pneumocytes were degenerative but none contained virus. virus isolation results are summarised in table i . r-ev was recovered from the turbinates of nine piglets at two to eight days after infection, from the lungs of eight piglets at one to nine days after infection, from distal small intestine of eight piglets and intestinal contents of nine piglets at two to ii days after infection. the titre of virus in the intestine and intestinal contents of rcv-intected piglets was less than in tgev-infected piglets. virus was isolated from the lung of one of two tgev-inoculated piglets. all piglets were seronegative before virus inoculation. rev-inoculated piglets seroconverted at seven days after infection, with titres rising up to at ii days after infection. the uninoculated, control piglets remained seronegative throughout the study period. this study confirms a previous report (pensaert et al ) and many observations from the field that rev is a roav-iike coronavirus which replicates extensively in the respiratory tract but does not cause clinical disease. the bronchointerstitial pneumonia observed in the r-ev-infected piglets was similar in character to that in the 'rcav-infected piglets. the possibility of dual infection is unlikely as individual piglets were maintained in isolation and as neither control nor pcvinoculated piglets had clinical signs or intestinal lesions of transmissible gastroenteritis. the close similarity between pneumonia caused by each corona- virus is not surprising given the similarity of pcv to tgev and suggests pneumonia in each group had a similar pathogenesis. in the lung, replication of rev and tgev in non-ciliated cuboidal bronchiolar cells which are most numerous in terminal airways (baskerville ) accounts for pneumonia involving small calibre bronchioles and adjacent parenchyma. previous studies of the lungs of tgev-infected piglets found virus in bronchial and bronchiolar cells (underdahl et al ) and in pneumocytes (underdahl et a , mocsari and horvath ) . alveolar macrophages were proposed as a target cell population following infection in vitro by cell adapted but not wild-type tgev strains (laude et al ) . in the present study no virus was detected in alveolar epithelium. degeneration of these cells was either the result of a low concentration of virus in affected cells, unproductive infection or inadvertent destruction by the acute inflammatory process in terminal airways. the absence of clinical signs following r-ev infection of piglets confirms the results of a previous study that this coronavirus dies not cause diarrhoea or enteric lesions (pensaert et al ) . unlike that study, however, in this study r-ev was isolated from small intestine suggesting a limited capacity by this isolate to replicate in enterocytes. immunocytochemistry confirmed the presence of antigen-positive enterocytes. the paucity of these cells explains the absence of villous atrophy and the low titre of virus in gut compared with tgev. in many respects pcv behaved like a stable, low pathogenicity strain of tgev. non-enteropathogenic tgev strains retaining their capacity to infect respiratory tract tissues were produced in attempts to find a safe, effective vaccine to transmissible gastroenteritis (furuuchi et al ) . belgian isolates of r-ev do not, however, possess the characteristics of vaccinal strains of tgev (jestin et al ) . other differences between rev and tgev infection were the absence of renal tubular nephrosis (goodwin and jennings ) and the paucity of hepatic necrosis in pcv-infected piglets. there was no evidence of thymic atrophy or splenic lymphoid depletion in r-cv-inf'ected piglets, changes present in the tgev-infected group. the correlation between the results of pap staining of fixed tissue and virus isolation was good, although isolation was more sensitive perhaps because of its greater ability to detect small quantities of virus. the pap method was a reliable technique for identifying pcvitgev antigen in paraffin wax-embedded tissue and was compatible with formalin fixation unlike an earlier pap technique using polyclonal lapine anti-tgev serum which worked on acetone-fixed but not formalin-fixed tissues (chu et al ) . fixation time in formalin was not critical for our pap method and we have successfully stained tgev-infected porcine gut fixed in a variety of solutions (periodate-lysineparaformaldehyde-dichromate, formal sublimate, acetone and carnoy's), given that the pneumonia caused by pcv is nondescript with no morphological hallmarks other than the occasional syncytial bronchiolar cell to facilitate aetiological diagnosis, the pap method may have an application when rev is suspected of playing a role in field investigations of porcine pneumonia. to date conclusive evidence of such a role has not been established. research in veterinary science ii we are grateful to mr b. j. n. parker for performing hysterotomies and mr a. weller for animal care, dr d. j. garwes key: cord- - odgm hm authors: godet, murielle; l'haridon, rene; vautherot, jean-francois; laude, hubert title: tgev corona virus orf encodes a membrane protein that is incorporated into virions date: - - journal: virology doi: . / - ( ) -p sha: doc_id: cord_uid: odgm hm abstract the coding potential of the open reading frame orf ( amino acids) of transmissible gastroenteritis virus (tgev) has been confirmed by expression using a baculovirus vector. five monoclonal antibodies (mabs) raised against the k recombinant product immunoprecipitated a polypeptide of a similar size in tgev-infected cells. immunofluorescence assays performed both on insect and mammalian cells revealed that orf was a membrane-associated protein, a finding consistent with the prediction of a membrane-spanning segment in orf sequence. two epitopes were localized within the last c-terminal residues of the sequence through peptide scanning and analysis of the reactivity of a truncated orf recombinant protein. since the relevant mabs were found to induce a cell surface fluorescence, these data suggest that orf may be an integral membrane protein having a cexo-nendo orientation. anti-orf mabs were also used to show that orf polypeptide may be detected in tgev virion preparations, with an estimated number of molecules incorporated per particle. comparison of amino acid sequence data provided strong evidence that other coronaviruses encode a polypeptide homologous to tgev orf . our results led us to propose that orf represents a novel minor structural polypeptide, tentatively designated sm (small membrane protein). virology , - ( ) the coding potential of the open reading frame orf ( amino acids) of transmissible gastroenteritis virus (tgev) has been confirmed by expression using a baculovirus vector. five monoclonal antibodies (mabs) raised against the k recombinant product immunoprecipitated a polypeptide of a similar size in tgev-infected cells. immunofluorescence assays performed both on insect and mammalian cells revealed that orf was a membrane-associated protein, a finding consistent with the prediction of a membrane-spanning segment in orf sequence. two epitopes were localized within the last c-terminal residues of the sequence through peptide scanning and analysis of the reactivity of a truncated orf recombinant protein. since the relevant mabs were found to induce a cell surface fluorescence, these data suggest that orf may be an integral membrane protein having a cexo-nendo orientation. anti-orf mabs were also used to show that orf polypeptide may be detected in tgev virion preparations, with an estimated number of molecules incorporated per particle. comparison of amino acid sequence data provided strong evidence that other coronaviruses encode a polypeptide homologous to tgev orf . our results led us to propose that orf represents a novel minor structural polypeptide, tentatively designated sm (small membrane protein). transmissible gastroenteritis virus (tgev), an important pathogen of swine, is a member of the coronaviridae, a family of enveloped viruses with a large (- kb) continuous, positive rna genome. sequencing data have led to the identification of a number of large open reading frames (orfs). orfla and b, which account for the ' two-thirds of the coronavirus genome, are assumed to encode nonstructural proteins including the viral replicase/transcriptase. the seven to eight remaining orfs are expressed through a set of ' coterminal, subgenomic size mrnas of which only the unique region is translationally active. these include the orfs coding for the virion structural proteins, i.e., the nucleocapsid (n) and two or three envelope glycoproteins: the spike (s) and the membrane (m) proteins, and the hemagglutinin-esterase (he) present in a coronavirus subset. these orfs are distributed following the consensus gene order ' pol-(he)-s-m-n '. the other orfs are interspersed within the genome and their number and position differ among coronavirus members (reviewed by spaan et a/., ; and lai, ) . they have been shown to be expressed by functionally mono-, di-, or tricistronic mrnas and were generally assumed to encode nonstructural proteins, the function of which is still unknown or conjectural. ' to whom correspondence and reprint requests should be addressed. in tgev genome, four such orfs have been deduced. two of them are expressed by the same mrna (mrna ) in two out of the three virus strains sequenced (rasschaert et a/., ; kapke et a/., ; britton et al., ; wesley et al., ) . the predicted product of orf a is to codon long, with a variable c-terminal end. it appears to be dispensable for virus replication since it was found to be absent in a tgev variant strain sp (wesley et a/., ) as well as in the closely related porcine respiratory coronavirus (prcv) (rasschaert eta/., ) . orf b (expressed by a separate mrna species numbered -l in miller strain) has a constant length ( residues); however, one clone of purdue-l strain was reported to have an orf b which is shortened by codons at the ' end and in several cdna clones by codons at the ' end (rasschaert et al., ) . orf was predicted to encode a amino acid long hydrophobic polypeptide (rasschaer-t et a/., ; kapke et a/., ; britton et a/., ; wesley eta/., ) . so far, the putative products of the three above-mentioned orfs have not been identified in infected cells. the last orf, orf , is located downstream of the n gene (kapke and brian, ; rasschaert et al., ; britton et al., ) an unusual feature among coronaviruses. a polypeptide of m, k, reacting with antibodies produced against an orf synthetic peptide, has been characterized in tgev-infected cells (garwes et al., ) . in this study we report the identification of a product of one of these orfs (orf ) in infected cells and its preliminary characterization. in particular, we show evidence that orf represents a novel virion-associated polypeptide with a possible counterpart in other coronaviruses. autographa cahfornica nuclear polyhedrosis virus (acnpv) and recombinant baculoviruses were grown and assayed in confluent monolayers of spocfoptera frugiperda (sf ) cells in medium containing % (v/v) fetal bovine serum, according to the procedures described by brown and faulkner ( ) . propagation of the high cell passage purdue-l strain of tgev in swine cell lines pd or st was done as previously described (laude et al., ) . manipulations of plasmid dna were performed according to the procedures described by sambrook et a/. ( ) . restriction enzymes, t dna ligase, and calf intestine alkaline phosphatase (cip) were purchased from boehringer-mannheim. the baculovirus transfer plasmid containing the full-length cdna copy of tgev orf coding sequence was constructed following the general scheme outlined in fig. . the ndel-sspl fragment ( . kbp) derived from plasmid ptg - (rasschaert et al., ) was digested with the ddel restriction enzyme. ddel-sspl dna fragment was repaired with the klenow large fragment of dna polymerase i and cloned into the barnhi site of the pvl vector (luckow and summers, ) . the resulting plasmid, named pvlorf , contained a . kbp insert. a second plasmid, pvlorf a was constructed by inserting an orf gene in which the ' last nt were deleted through pcr mutagenesis @char-f et a/., ) on ptg - using the oligonucleotides ' g aagaagggatccatacctatgac and ' clta-tagggatcctaagcatg as ' and ' amplimers, respectively. these amplimers were designed to introduce an additional stop codon tag at the 'end of the gene and a barnhi cloning site at each end. the amplification product was digested by bamhl and ligated into the pvl cloning site. the orientation and sequence of the orf and orf a inserts relative to the acnpv polyhedrin leader were determined by restriction analysis and partial dna sequencing. in these constructs, the initiation codon of the orf and orf a sequences were positioned at and bp from the bamhl cloning site, respectively. transfer of the tgev orf gene into the acnpv genome was accomplished by transfection of sf cells using the calcium phosphate precipitation technique as described by summers and smith ( ) . recombinant baculoviruses were screened by dot blot hybridization using an orf specific [ p]-labeled dna fragment as a probe. four polyhedrin-negative clones were tested for orf expression. tgev orf a gene was introduced into a linear form of acnpv dna (kitts eta/., ) . circular acrpg-sc dna was linearized by digestion at the unique bsu site. two hundred nanograms of bsu or mock digest viral dna was mixed with pg of pvlorf a dna and transfected into sf cells using the lipofectin method (kitts et a/., personal communication) according to the procedure of the manufacturer (gibco-brl). after a -day incubation the culture supernatants were harvested and plated. a dozen well-isolated plaques were picked out and screened for orf a expression using [ s]methionine-labeled cultures. three balb/c mice were injected threefold intraperitoneally at a month interval with x ' acorf -infected cells (disrupted in freund complete adjuvant for the first injection). three days before fusion, the mice were boosted both intraperitoneally and subcutaneously with orf protein purified from x ' infected cells by % sds-page. splenocytes from one mouse that tested positive by immunoprecipitation assay were fused with sp,o myeloma cells. supernatants of hybrid clones were tested in a comparative immunofluorescence assay (see below) using acorf or acnpv-infected sf monolayers. subcloning of orf -specific antibody-producing hybridomas and ig isotyping were done as described elsewhere (l' haridon et al., ) . iggs purified from ascites fluids by ammonium sulfate precipitation and gel permeation on a sephacryl-s column were used in all experiments. screening of hybridoma was performed on sf cell monolayers established in -well microplates, infected with baculovirus (m.o.i. pfu) and fixed with acetone/ethanol (v/v) at hr p.i. for surface fluorescence analysis, aliquots of cells in suspension were stained with mabs at pglml in grace medium and then with fitc conjugate (each step hr at ") and spotted onto glass slides. alternatively, spotted cells were fixed with % paraformaldehyde and permeabil-ized or not with . % triton x-l before staining ( hr at "). similar experiments were performed on st cell monolayers infected by tgev at a m.o.i. of . pfu and fixed hr p.i. the procedures for metabolic labeling of insect and mammalian cells were as reported previously (godet et a/., ; . monolayers of x o sf cells or x o pd cells labeled with [ s]methionine were washed with pbs and lyzed in ml of pbs-triton (tris, mm, ph . , o/o triton x-l , o kallicrein units of aprotinin per milliliter). resulting cytosols of sf cells and pd cells were centrifuged min at , g or hr at , rpm in a ti rotor (beckman), respectively and stored in aliquots at - ". lmmunoprecipitation assay aliquots of radiolabeled cytosols or virions were adjusted to . ml with pbs-triton buffer containing the appropriate mab ( pg/ml) or ~ of ascites fluid from a feline infectious peritonitis virus-infected cat (used as a source of anti-tgev polyclonal antibodies) and protein a-sepharose beads ( ~ of a % suspension); after a -hr incubation at room temperature with agitation, the immune complexes were extensively washed with pbs-triton, then with . m nacl + mn/l tris (ph ). beads were treated for min at " in sample buffer. the immunoprecipitated material thus released was analyzed by % or - % sds-page. virions in the supernatant of cultures labeled as above were purified following a described procedure (laude et a/., ) . the material pelleted by ultracentrifugation at , rpm in a ti rotor was resuspended in distilled water and was applied to a linear sucrose gradient ( ml, to % sucrose w/v in distilled water). centrifugation was performed in an sw rotor for hr at , rpm and ". gradients were collected from top to bottom into -p. fractions. half of each fractions was run at , rpm for min and the resulting pellets were analyzed by sds-page electrophoresis on a - % gradient gel. the material remaining in the virus-containing fractions was pooled, pelleted as above, and split into three parts; one was analyzed directly by % sds-page; the two others were solubilized in pbs-triton, immunoprecipitated by a different mab each, and analyzed as above. in one experiment, virion-associated material was subjected to a second round of gradient purifica- laude et al., ) prior to gel analysis. peptides were synthesized on polyethylene pins (geysen eta/., ) by using a commercially available kit, according to the procedure given by the manufacturer (cambridge research biochemicals). immunoreactivity of the immobilized peptides was assayed by elisa using anti-orf mabs ( pglml) as primary antibody and an anti-mouse igg (h + l) peroxydase conjugate (biosys). insertion of the entire orf coding sequence into the genome of acnpv baculovirus was performed by using the transfer plasmid pvl (see methods and specific probe and a polyhedron-negative phenotype were selected, amplified, and tested for the expression of orf . among four selected orflf-expressing clones, one, designated acorf , was retained for subsequent studies. analysis of [ s]methioninelabeled acorf -infected cells revealed the presence of a major polypeptide of i'@ ok (fig. ) in good agreement with the predicted m.w. of orf product ( . k). the time course for its synthesis was found to be from to hr p.i. screening of positive hybridoma clones was achieved by comparative indirect immunofluorescence assay on acorf -or acnpv-infected cells. among hybridomas tested, positive clones, all producing mabs of iggl isotype, were subcloned and used for the production of ascites fluids. when assayed by immunoprecipitation of acorf -infected cells extracts, all mabs recognized a single species of m, ok, identical to the target protein (fig. a, lane ). an immunoreactive species comigrating with recombinant orf protein was detected in tgev-infected mammalian cells as well (lane ). analysis of the recombinant protein in nonreducing conditions revealed the existence of oligomeric forms, which were not observed with the authentic orf protein (fig. b) . orf product is a membrane-associated protein indirect immunofluorescence assays were performed to determine the subcellular location of orf protein. in acetone-fixed cultures of both acorf -infected sf and tgev-infected cells, all anti-orf mabs induced a strong fluorescence, which were polarized in a juxtanuclear region consistent with golgi localization (fig. a) . in addition, a bright fluorescence was observed on unfixed (fig. b) positively stained cells were consistently observed (fig. d) , whereasvirtually all the cells showed the presence of orf antigen after permeabilization (not shown). the fluorescence observed in nonpermeabilized cells was unlikely due to cell damage since only a few of them were stained positively with a mab directed against an intracellular antigen (tgev n protein; data not shown). these data were interpreted as reflecting a late accumulation of orf at the outer membrane of tgev-infected cells. these observations showing that orf protein is found in association with cellular membranes are consistent with the prediction of a membrane-spanning domain in its amino acid sequence (fig. ) . anti-orf monoclonal antibodies recognize the c-terminal domain of the protein assuming that orf was an integral membrane protein, it was of interest to determine whether exposed epitopes were located within the carboxy or amino do- main of the polypeptide chain. the peptide scanning method (geysen et al., ) was used since two anti-orf mabs were shown to be reactive toward denatured and reduced protein. a set of peptides encompassing all overlapping linear nonapeptides homologous with orf sequence was synthesized and tested against each of the five mabs. as shown in fig. , mab v strongly recognized a linear epitope centered on residues ayknf (positions to ; see fig. ). mab s gave comparable results, although a lesser reactivity was observed when compared to mab v . no reactivity was observed with the three remaining mabs, possibly because of a lower avidity. to test the possibility that these three anti-orf mabs may also recognize the carboxy-subterminal region of the molecule, a second recombinant baculovirus designated acorf a, which expressed a truncated form of orf lacking the c-terminal amino acids, was constructed via pcr mutagenesis. immunoprecipitation analysis revealed that a polypeptide of slightly reduced size was expressed by acorf ainfected cells and recognized by polyclonal antibodies but not by any of the anti-orf mabs (partial data in fig. ) . in order to determine whether the protein is incorporated within the virion as a possible envelope protein, labeled tgev particles were purified by centrifugation and analyzed by gel electrophoresis (fig. ) . five welldefined major bands were visible, which corresponds to the previously recognized virion-associated polypeptides: (i) a k band (s protein), (ii) a k band (n peptides fig. . epitope mapping of anti-orf mab v . a series of nonapeptides (overlapping ) spanning the length of the entire orf sequence was tested for elisa reactivity toward mab v . n-terminus is on the left. protein), and (iii) three bands corresponding to different species of m protein; - k identified as complex type glycosylated forms, k (high mannose form) and k (unglycosylated form) (delmas and laude, ) . a single additional minor band of m, ok, similar to that of orf polypeptide, was detected (fig. a) . the fact that very few, if any, other polypeptides copurified renders unlikely the possibility that the ok polypeptide remained nonspecifically associated to the sedimenting virus particles. true association of the k band with virions was confirmed by showing that the observed polypeptide pattern remained unchanged after an additional round of purification by isopycnic centrifugation. lmmunoprecipitation with mab v of detergent-dissociated material from virus-containing fractions confirmed the identity between the k virion-associated and orf polypeptides (fig. b) . densitometric tracing of autoradiograms at different times of exposure was performed to evaluate the relative amounts of the virion-associated polypeptides. the re- sulting values were corrected according to the predicted number of met residues in the respective sequences. this led to an estimated molar ratio of : : for orf , s, and m polypeptides. a common feature of coronavirus genomes appears to be the existence, immediately upstream from the membrane protein m gene, of an orf to nucleotide long, which is predicted to encode a hydrophobic polypeptide. furthermore, recent studies on mouse hepatitis virus (mhv), infectious bronchitis virus (ibv), and bovine (bcv) coronaviruses have shown that the products translated from these orfs were associated with the membrane of infected cells (leibowitz et a/., ; abraham et a/., ; smith et al., ) , as evidenced now for tgev orf . these observations prompted us to reexamine the relevant amino acid sequences for possible similarities that earlier comparative analysis by us and others failed to detect (except for the closely related mhv and bcv viruses). figure shows a tentative alignment of the orf -like sequences from coronavirus members, which was outlined using the program multalin (corpet, ) and refined manually. on the basis of the deduced consensus sequence, in which residues are conserved in at least out of the sequences aligned ( positions), we conclude that these proteins share significant similarities. as a striking feature, a - residue-long hydrophobic stretch, followed by a cluster of or cysteines is present to residues from the n terminus the c-terminal part of the polypeptides seems to be more distantly related; in particular, ibv protein extended the consensus sequence by to residues, depending of the strain (see liu et a/., for ibv orf c sequence data). in this study we have confirmed the coding potential of the orf encoded by tgev mrna and character- ized several properties of its translation product. baculovirus-vectored expression of orf resulted in the synthesis of a ok polypeptide. the same species was identified in tgev-infected cells by immunoprecipitation using monoclonal antibodies (mabs) raised against the recombinant polypeptide. examination of its subcellular localization by immunostaining showed that orf translation product is a membrane-associated polypeptide. this finding is consistent with the prediction in the second n-terminal quarter of a stretch of uncharged residues with proper-ties of a membrane-spanning domain (fig. ) . immunofluorescence data also suggested that orf may enter the exocytic pathway. in tgev-infected cells, however, orf could be detected in association with the cell surface only at a late stage of the infection. assuming that the observed surface fluorescence was related to externally exposed determinants, it was of interest to map the relevant epitopes on the amino acid sequence of the molecule. peptide scanning led to the identification of residues -ayknf- as the core sequence of the binding site of of the mabs (cavanagh et a/., ) . pairwise homologies are marked by dots and bold letters; gaps are indicated by dashes. bottom line, consensus sequence showing residues identical in at least three of the five sequences. sequence data from rasschaert et al., ; raabe and siddel, ; skinner et al., ; woloszyn et al., ; and boursnell et al., . the last n-terminal residues of ibv orf c (beaudette strain) are not shown. studied. furthermore, a recombinant orf protein truncated of its last c-terminal amino acids was no longer recognized by any of the mabs. these results support the view that an antigenic, possibly immunodominant site is expressed in the c-subterminal part of orf protein. in addition, they led us to speculate that the region of the molecule which is translocated across the membrane would correspond to its carboxy domain. recently, a striking correlation has been reported between the transmembrane orientation of eukaryotic proteins and the disposition of charged residues surrounding the most n-terminal membranespanning sequence. it has been proposed that the difference in the charge of the residues flanking the presumed anchor segment determines its orientation with the more positive portion facing the cytosol (hartmann et a/., ) . applying such a rule to tgev orf sequence gives charges of - and + for the n and c flanking segments, respectively, which predicts a nexo-cendo orientation, in contrast to the available experimental evidence. thus further experiments, including the production of antibodies directed to the n-terminal part of the protein, are needed for a definite assignment of the transmembrane orientation of orf . the possible role of the cysteine cluster immediately downstream the hydrophobic segment was also examined. gel analysis of recombinant orf protein under nonreducing conditions revealed the presence of multimeric, predominantly dimeric forms. in contrast, orf synthesized in tgev-infected cells could be detected in a monomeric form only, suggesting that the formation of disulfide-bridged species is an artifact, presumably linked to the high level of expression of orf in the insect cells (kiefhaber et al., ) . the possibility was tested that the cystein residues could serve as an acylation site, as demonstrated for coronavirus s protein (schmidt, ) . however, no incorporation of palmitic acid chains could be detected in recombinant orf protein (data not shown), as would be expected if the target residues belong to the orf ectodomain. an important implication of the above findings was that orf could represent a structural polypeptide present in the virus envelope. indeed, purified preparations of labeled tgev virions revealed the presence of a previously unrecognized ok polypeptide which was specifically immunoprecipitated by anti-orf mabs. based on the estimated molar ratio and assuming that coronavirions bear (roseto et a/., ) to spikes, each composed of s molecules (delmas and laude, ) it can be inferred that approximately - copies of orf protein are incorporated into tgev virions (purdue strain). such a small number of molecules in virus particles does not seem to reflect a selective exclusion since s and orf accumulated in infected cells at a ratio comparable to that found in virions (data not shown). these results lead us to conclude that orf may represent a minor structural polypeptide, which we propose to designate by the tentative acronym sm, standing for "small membrane" protein. several lines of evidence lend support to the view that a gene encoding an sm-like protein is a common feature of the coronavirus genomes: (i) an orf predicting a polypeptide with striking similarities to tgev orf was identified in the genome sequence of each of the coronaviruses examined (fig. ) and the fact that tgev sm was recognized by anti-fipv antibodies argues for the presence of a related gene also in feline infectious peritonitis virus genome; (ii) the product expressed from the relevant mhv, bcv, and ibv orfs was reported to have properties of a transmembrane polypeptide (leibowitz et a/., ; smith et al., ; abraham et a/., ) ; and (iii) although expressed through a mono-, di-, or tricistronic mrna (abraham et a/., ; budzilowicz and weiss, ; liu et al., ) the assumed sm-encoding genes are all located upstream and adjacent to the m protein gene. therefore, not only the sequences show significant similarities but the gene order '. . s sm/m . ' is conserved, as would be expected for a structural protein. finally, preliminary experiments in this laboratory allowed us to detect orfs-encoded polypeptide in association with bcv particles (n. woloszyn, p. boireau, and j. f. vautherot, unpublished results). small integral membrane proteins have been described in several other enveloped rna viruses, including sindbis and semliki forest togaviruses, influenza a and b viruses, simian virus , and respiratory syncitial paramyxoviruses (garoff et a/., ; welch and sefton, ; lamb et al., ; hiebert, ; olmsted and collins, ) . the influenza virus m protein ( k) and the alphavirus k protein are both acylated, nexo-cendo transmembrane polypeptides. m and k have been shown to represent minor structural polypeptides, with an estimated number of + and f molecules per virion, respectively (zebedee gaedigk-nitschko and schlesinger, ). m has been reported to form tetrameric channels within the membrane and to be a target of the antiviral drug amantadine and of the ctl response to influenza virus infection (hay et al., ; lamb et al., ; surgrue and hay, ) . site-directed mutagenesis studies on sindbis and semliki forest viruses have demonstrated that k protein is dispensable for virus production but exerts a role late in the assembly, possibly during virus budding (gaedigk-nitschko and schlesinger, ; liljestrom et a/., ). the sh protein of sv has been reported to be orientated in the mem-brane with its n-terminus domain exposed at the cytoplasmic face, as it might be the case for tgev sm. whether sh is incorporated into virions is still questioned, as well as its potential role (hiebert eta/., ) . the apparent conservation of sm gene in the coronavirus genome strongly implies that its product is essential for an efficient replication of the virus. based on its location and its low copy number in particles, we speculate that sm would more likely play a role in modulating assembly and/or release of the virion. thus, eluci-.dating the function of the coronavirus sm protein might contribute to a better understanding of an important aspect of the biology of enveloped viruses. finally, sm may be a potent surface antigen since murine antibodies recognized a domain of the protein possibly exposed on live infected cells. preliminary experiments indicated that anti-sm antibodies are readily detected in the serum from infected swines. therefore, the role of sm protein in humoral and cellular immune response to tgev infection should be worth investigating in the future. we thank j. gelfi for technical assistance, j. levin for revising the manuscript, and m. nezondb for the artwork, part of this work was carried out with the support of the e.e.c program eclair. note addedinproof. during the submission process of this article, a communication by (d. x. liu and s. c. lnglis ( , virology , - ) reported the association of ibv orf c protein with the virion envelope. this strengthens the view that sm-like proteins 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basis for the pathogenesis of transmissible gastroenteritis virus. . viral nucleotide sequence of the bovine enteric coronavirus becv f mrna and mrna unique regions influenza a virus m, protein: monoclonal antibody restriction of virus growth and detection of m, in virions key: cord- -k siwur authors: méchin, marie-claire; der vartanian, maurice; martin, christine title: the major subunit clpg of escherichia coli cs a fibrillae as an expression vector for different combinations of two tgev coronavirus epitopes date: - - journal: gene doi: . /s - ( ) - sha: doc_id: cord_uid: k siwur abstract previously, two b-cell epitopes from the entero-pathogenic transmissible gastroenteritis virus (tgev), namely the c epitope (tgev-c) amino acids (aa) – and the a epitope (tgev-a) aa – of the spike s protein (tgev-s), have been separately expressed on the cs a fibrillae at the surface of escherichia coli following insertion into a same region of clpg. however, the resulting chimeras induced a marginal tgev-neutralizing antibody (ab) response in mice. here, with the view to improving this response, we introduced tgev-c alone or in different tandem association with tgev-a (a::c or c::a) in twelve putatively exposed regions of clpg. among the resulting engineered proteins only , carrying up to extra aa, had not essentially disturbed the correct cs a fibrillae formation process. six partially permissive sites accepting only tgev-c and three highly permissive sites tolerating a::c or c::a tandem peptide, were identified throughout clpg. intact bacteria or extracted cs a hybrid fibrillae expressing tgev epitopes at any of the permissive sites, were recognized by ab directed against the foreign parent protein, providing a direct argument for exposure of the corresponding clpg region at the cell surface and for antigenicity of the epitopes in the polymeric cs a fibrillae context. the potential of cs a fibrillae as carriers of the tgev peptides indicates that there may be three positions (n terminus, aa – and – ) in clpg which may turn out to be important fusion sites and therefore be relevant for the eventual design of tgev vaccines. unexpectedly, tgev-a, whatever its position in clpg, mediated the partial proteolytic degradation of the hybrid proteins, suggesting that it functions as a substrate for a cellular protease, and thereby that its suitability as a vaccine antigen candidate is doubtful. epitope-based recombinant vaccine technologies offer the potential for oral or mucosal delivery, especially when the relevant epitope genetically fused to a carrier protein, is displayed as a heterologous peptide on the surface of a bacterial strain (rabinovich et al., ) . however, coupling foreign peptides to carriers can result in a poor immunogenicity of the chimeric antigens due to the local conformational restrictions imposed on epitopes by the embedding structure (benito et al., ) . therefore, approaches to enhancing immunogenicity are critical. since the conformation of a foreign sequence within a carrier varies widely depending on the flanking sequences (tishminetzky et al., ) , the insertion of an antigen into different exposed regions of a delivery protein, thus changing antigen conformation, could permit a strategy for finding appropriate environments for peptide presentation among the complex assortment of molecular contexts offered by the carrier. a second strategy to improving immunogenicity is the fusion of the one-letter code for aa designation is used. numbered vertical arrows above the nt sequence indicate the number and position of the twelve selected insertion sites. asterisks denote the nt modified by site-directed mutagenesis and the corresponding mutated aa residue. the resulting engineered restriction sites spei and bglli in plasmids ppsx s and ppsx s (bousquet et al., ) , coding for mutants clpg and cipg , are mentioned. (a-e) in panel c represent (a) predicted secondary structured, (b) hydrophilic (hatched box), (c) variable (black boxes) or conserved (open boxes) (girardeau et al., ; mrchin et al., ) (stanssens et al., ) , or performed on double-stranded dna using an adapted protocol from the methods of jung et al. ( ) and deng and nickoloff ( ) . mutations were verified by dna sequencing (sanger et al., ) . the oligos used in this study were synthesized and, when necessary, page-purified and '-phosphoryled (eurogentec, belgium); in the oligos shown below, names are indicated, numbers in parentheses refer to nt position in the coding strand of the clpg gene (see c), and bold characters represent mutations resulting of the spei site introduction: vn , ( )cttcaagcagtaactagtaaccctaacgcg( ); np , ( )caagcagtaaacactagtcctaacgcgggc( ); pn , ( )gcagtaaaccctactagtaacgcgggcaat( ); na , tandem peptides to immunogenic carrier proteins (broekhuijsen et al., ; martineau et al., ; khan et al., ) . a third strategy might be the insertion of epitopes in an accessible surface region previously observed as a natural immunodominant site on the native carrier molecule. here, we developed these different approaches simultaneously by using the major clpg subunit of the e. coli cs a fibrillae as the carrier protein (bousquet et al., ; der vartanian et al., ) , and two b-cell epitopes from tgev consisting of the site c (aa - ) and site a (aa - ) of tgev-s as the foreign antigenic determinants (delmas et al., ; gebauer et al., ) . the continuous site c elicits neutralizing ab and the site a is part of highly immunogenic conformational antigenic region (delmas et al., ; correa et al., ) . tgev-a or tgev-c inserted in the position aa - of cipg was previously shown to induce a low neutralizing ab response in mice (bousquet et al., ) . the aim of this work was to explore recombinant clpg::tgev proteins for the display of different combinations of the two tgev peptides in particular contexts that could result in improved epitope performance, and thus allow the design of vaccine antigens. for this purpose, we examine the permissivity (charbit et al., ) fig. . foreign sequences used in this study for the construction of the chimeric clpg genes. the six double-stranded synthetic oligos coding for tgev-c (oligos #l, # and # ) and tgev-a (oligos # , # and # ) of tgev-s of the porcine purdue-ll strain of tgev are shown. the bolded aa residues correspond to the residues - and - of tgev-s, spanning the sites c and a, respectively. restriction sites are underlined. introducing tgev-c alone or in tandem with tgev-a in these sites and by investigating the influence of the resulting modifications on the expression of cs a fibrillae. we discuss the nature of permissive sites and the positioning and tandem insertion effects of the epitopes. twelve sites within clpg were selected for insertion of tgev peptides (fig. ) . one of them is located at the n terminfis (fig. b) , and the others are distributed along the aa - region (fig. c) . this region contains a variable flexible loop structure (aa - ) carrying a hydrophilic domain and several accessible continuous immunodominant epitopes, one of which (aa - ) is exposed on the native clpg subunit at the surface of the polymeric cs a fibrillae (fig. c ). for these reasons, we hypothesized that the region aa - is naturally favorable to the presentation of the tgev epitopes. to construct insertion p]asmid vectors as a preliminary step for viral epitopes insertion in the selected regions aa - and - of clpg ( fig. c) , the corresponding clpg sequences were submitted to oligodeoxyribonucleotide (oligo) site-directed mutagenesis to create unique restriction sites at different positions within these sequences. thus, a spei site was independently engineered after each codon expressing every one of aa composing the region aa - ( fig. c) , resulting in the addition of the dipeptide threonine-serine (ts) (fig. ) . the aa - peptide-encoding sequence was y-ended by a spei site and y-ended by a bglli site after two rounds of mutagenesis which induced changes in clpg (fig. c , mutants cipg and clpg ); the first round allows insertions into the site aa - and the second the replacement of the region aa - . the strategy of clpg::tgev hybrids construction was to use these engineered restriction sites, and two naturally occurring sites, sphi (fig. b) and muni (fig. c unique xhoi site with spei-compafible ends into the spei site of ppsx s (see fig. and its legend); pgac and pgxc were constructed by inserting the oligo # ( fig. ) with xhol-compatible ends into the xhoi site of pgc , resulting in the addition of the oligo # in the correct and in-frame reverse orientations, respectively; pga (bousquet et al., ) was made by replacing the -bp spei-bglii fragment from ppsx s (see fig. and its legend) with oligo # (fig. ) having '-spei and bglli- ' compatible ends; pgca was constructed by inserting the oligo # containing unique xhoi site with spei-compatible ends into the spei site of pga ; pgisa was engineered by cloning the oligo # (fig. ) comprising unique xhoi site with sphi-flanked ends into the unique sphi site of pdsph ; pgac was obtained by inserting the oligo # with xhoi-compatible ends into the xhoi site of pgisa ; pgca was constructed in two steps from pdev : in the first step, the oligo # including unique nsii site with sphi-flanked ends was cloned into the sphi site of clpg (fig. b) and in the second step, the nsii site in oligo # allowed the insertion of oligo # (fig. ) with nsii compatible ends downstream of oligo # ; pco was made from pdev by inserting the oligo # ( fig. ) with muni-compatible ends into the muni site of clpg (fig. c) ; pvn , pnp , ppn , pna , pag , pgn , pnr and prg were constructed from pdev as follows: a spei site within clpg was introduced after each codon expressing every one of aa residues covering the region - of cipg (fig. c ) by in vitro mutagenesis using oligos vn , np , pn , na , ag , gn , nr and rg (fig. legend) ; pvn c, pnp c, ppn c, pna c, pag c, pgn c, pnr c and prg c were obtained by inserting the oligo # containing xhoi site with spei compatible ends into the spei site of pvn , pnp , ppn , pna , pag , pgn , pnr and prg respectively; pvn ac, ppn ac, pna ac, pgn ac and pnr ac were made by cloning the oligo # with xhoi-compatible ends into the xhoi site of pvn c, ppn c, pna c, pgn c and pnr c, respectively; pvn xc, pnp xc, ppn xc, pag xc and pgn xc were engineered from pvn c, pnp c, ppn c, pag c and pgn c, respectively, as indicated just above except that oligo # was inserted in-frame in the reverse orientation, as designated by the letter x; pdev ca was made by replacing a . -kb muni-saci fragment from pgca with the . -kb muni-saci fragment from pgcai . (c) the presence oltgev-c and -a is specified by the letters c and a respectively; ca or ac, (fig. ). all these constructions summarized in fig. are described in detail in the fig. legend. in total, mutants were obtained. twenty-eight of them contained at least one tgev peptide, among which had both tgev-c and -a as an a::c or c::a fusion. finally, modifications in clpg resulted in an insert of , , , , or aa in length (fig. ) . each of the mutant plasmids (fig. ) was transferred into e. coli dh ~ bearing the trans-complementing plasmid pdsph . the cell-surface location of the corresponding hybrid clpg subunits (fig. ) on cs a fibrillae was determined on intact cells by in situ colony immunoblotting and from isolated mutated cs a polymers by immunodot analysis using a cs a-specific polyclonal ab (pab), the tgev-c-specific b. mab and the tgev-a-specific af mab (not shown). out of recombinant cs a fibrillae carrying at least one tgev peptide only failed to react whatever ab. in contrast, the remaining hybrids were capable of exposing the tgev peptides at the cell surface on the correctly assembled cs a fibrillae. nine permissive were identified throughout clpg. the positions aa - / , - and - appeared to be the most permissive targets since the largest insertions ( or aa), did not interfere with the cs a fibrillae formation. even the hybrid clpg / -ca protein with an insert of aa long, resulting from the simultaneous tandem addition of the two tgev peptides in both positions aa - / and - , was incorporated in cs a polymer. in positions aa to less residues ( aa) was tolerated. positions aa - , - and - were nonpermissive since, except for clpg with only two extra aa, no hybrid was detected. all nonpermissible insertions were located in or near a predicted a-helix structure, and targeted conserved aa residues (fig. , aa - ) . by contrast, all permissive sites, excluding the clpg-n terminus, were included in a predicted loop (fig. , aa - ) that is more likely to be flexible enough to accommodate large inserts, as indicated by the proposed cipg topology (mrchin et al., ) . while the two fully permissive sites (aa - and - ) were located between the top and the end of the loop, the six partially permissive sites (aa to ) targeted region immediately beginning this loop. these data suggest a relationship between the permissivity of clpg and the local cipg structures into which the epitope was inserted. on the basis of these results, we conclude that clpg as a carrier is very flexible since insertions varying in length and nature can be made in different sites without affecting cs a formation. only hybrid clpg subunits displaying at least one tgev peptide on cs a were characterized by western immunoblot analysis (fig. ) . proteins cipg -c, clpg -c, clpg -c, clpg -c, clpg -c, clp g -c, clpg -xc, clpg -ca, clpg -c, glpg -ac and clpg -c migrated as a single band which corresponds to the expected full-length hybrid since revealed by anti-clpg pab, b, mab and af mab (fig. a, b, c) . mutants clpg -a, clpg -ca and cipg -ac showed two protein products, all reacting with anti-clpg (fig. a) ; their upper band detected by the three ab represents the whole fusion molecule which was more abundant in clpg -ca. in clpg a and clpg -ac, the major lower band was recognized by only anti-clpg while in clp-g -ca the minor lower band was additionally by b. mab (fig. b) . in clpg / -ca the two uppermost bands were lighted whatever ab (fig. a, b , c) and the two lowermost bands only with anti-clpg and b. ab (fig. a, b) . these observations indicated that most of the hybrids carrying tgev-a was subjected to an incomplete proteolysis which, however, did not prevent the cs a formation ( fig. and section . )). in a general way, clpg hybrids containing tgev-a reacted faintly with afi mab (fig. c) . peptides c and a in tandem; x, cryptic peptide encoded by oligo # (fig. ) inserted in-phase but in the reverse orientation. (d) aa changes resulting from the engineering of the tgev epitopes-encoding oligos (fig. ) in cipg: the numbers refer to the indicated first and last aa residues of the wild-type (wt) cipg sequence; residues from the original clpg protein are in small characters and additional residues are in large bold type. hatched boxes, tgev-c; black boxes, tgev-a; open boxes, cryptic peptide x: tqqadhsqiss. (e) total number of added aa with respect to wt cipg. (f) cs a fibrillae biogenesis: + and -, synthesis and no synthesis, respectively. methods: the production of hybrid cs a polymers was detected by in situ colony immunoblotting and immunodot analysis. for colony blots analysis, single colonies were streaked on a solid agar lb plate containing appropriate antibiotics. after overnight incubation at °c, a nitrocellulose filter (pore diameter, . ~tm; schleicher and schuell) was carefully applied on agar surface. blots were blocked and then washed with % bsa- . % tween in pbs until the bulk of bacteria was removed. the filters were further incubated with appropriate primary ab in % bsa in pbs. bound primary ab were detected by incubation of the filters with either horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary ab, and developed with h -~-chloronaphthol. for the immunodot analysis of the extracted mutated cs a fibrillae, bacteria growing overnight on lb agar medium with the appropriate antibiotics were carefully scraped and suspended in pbs. this suspension was then vigorously agitated for ][ min with a top mix shaker, and placed at °c for min (thermo-elution of the cs a polymer). after centrifugation at x g for min, the resulting supernatant was used for experiments. cs a fibrillae-specific rabbit antiserum (anti-cs la) was obtained as described by girardeau et al. ( ) . mab b. (delmas et al., ) and mab af (gebauer et al., ) raised, respectively, against the c and a sites of tgev-s on native coronavirus, were used. fig. legend) were mixed with an equal volume of x laemmli buffer, boiled for min, separated by % sds-page and semi-dry electrotransferred onto nitrocellulose sheets (towbin et al., ) . control, wt cs a fibrillae produced by e. coil dh e [pdev , pdsph ] . western blots were then treated as described in the fig. legend by using as primary ab either (a) anti-clpg pab, or (b) b. mab, or (c) af mab, or (d) anti-g q pab, or (e) anti-t p pab. clpg subunit-specific rabbit antiserum (anti-clpg pab) was obtained as described by girardeau et al. ( ) . the g q peptide (gqlqavnpnagnrgq) and the t p peptide (tftnpvvsttqwsap) correspond to the aa residues - and - of cipg, respectively. the synthetic g q and t p peptides were obtained from neosystem (strasbourg, france) and their purity was > % as determined by high-performance liquid chromatography. to prepare anti-g q and anti-tl p antisera, peptides g q and t p were coupled to bsa using glutaraldehyde (sigma); rabbits were primed by intradermic injection of gg of peptide emulsified with incomplete freund's adjuvant, then boosted and days later, and finally bled days after the last immunization. although we have not sequenced the n-and c-terminal parts of cleaved products from cipg::tgev hybrids, several lines of evidence suggest that a proteolytic event occurs within the tgev-a oligopeptide: ( ) no truncated products from any of the hybrids containing one copy of tgev-a was detected with af mab; ( ) no proteolysis happened with clpg alone or in association with tgev-c; ( ) clpg -xc chimera that differs from clpg -ac in only a tgev-a sequence in reverse orientation, was not cleaved; ( ) the immunoblot patterns of clpg / -ca, carrying two copies of tgev-a, are consistent with the presence of a cleavage site within each copy since four fragments were visualized. this implies that tgev-a fused to the n terminus of clpg was cleaved in a fashion similar to tgev-a in the c-terminal part of clpg. in this case, proteolytic cleavage would result in a n-terminal fragment too small (< aa) to be detected, that explaining why no truncated product was visualized from clpg -ca or clpg -ac (fig. ) . to probe more precisely the n-and c-terminal parts of the truncated proteins generated by clpg -a, clpg -ca and clpg -ac we used two additional specific pab, anti-g q and anti-t p, that recognize the peptides g q (aa - ) and t p (aa - ) in clpg, respectively; insertions in g q sequence abolished the binding of anti-g q (fig. d , mutants clpg -c to clpg -c). in these constructs, g q is placed aa upstream from the clpg::tgev epitope fusion junction and t p to aa downstream. western immunoblots indicated that the intact clpg -ca and cipg -ac molecules reacted with both anti-g q and anti-t p; in cipg -a the intact molecule is undetectable because too low in amount (fig. d, e) . by contrast, their degraded forms were probed by anti-g q but not by anti-t p (fig. d) . together with the facts that the cleaved form of clpg -ca was detected with b. mab (fig. b) but not with af mab (fig. c) , and that the truncated product of cipg -ac did not react with any of the two mab (fig. b, c) , these data suggest that protease cleavage occurs within tgev-a. furthermore, the uncleaved form of clp-g -ca was substantially more abundant than its truncated form (fig. a) . in contrast, cipg -a and clpg -ac showed a prominence in amount of the cleaved products, indicating that c::a fusion, rather than a::c fusion, significantly reduced cleavage process, probably due to the protective placement of the hydrophobic tgev-c motif at the n-terminal end of tgev-a. therefore, we speculate that the site of cleavage must be n-terminally located with respect to the tgev-a sequence. supporting this hypothesis, the two first aa residues of tgev-a consist of lysine (k) and arginine (r) which are often involved in the proteolytic maturation of viral envelope glycoproteins (moulard et al., ) . assistance, s. dutilloy for secretarial assistance, dr. l. enjuanes and dr. h. laude for providing mab af and mab b. , respectively. this work was supported by grants agre- -c from european economic community (eclair program). ( ) three nonpermissive sites (aa - , - and - ) , six partially permissive sites (aa to ) and three fully permissive sites (aa - / , - and - ) were identified throughout cipg. the latter sites appeared as the most versatile targets since the largest insertions consisting of ~ta residues did not interfere with cs a biogenesis. even an insert of aa long resulting of the addition of the tgev-c::tgev-a tandem peptide in both positions aa - / and - was normally incorporated in cs a polymer. the binding of the tgev-specific mab with hybrid fibrillae indicates that the tgev antigenic determinants expressed at any of the permissive sites are exposed on both clpg and cs a proteins at the e. coli cell-surface. we show here that the potential of clpg as a carrier for foreign peptides may be influenced by the general predicted properties of the local sequences. ( ) unlike tgev-c, tgev-a expressed at any permissive site of clpg rendered the hybrid proteins partially susceptible to proteolytic degradation, indicating that it was influential in, but not critical for, fibrillae integrity, presumably because providing a protease cleavage site. ( ) cs a fibrillum appears as a potent cell-surface presenting vector for tgev epitopes. such hybrid fibrillae might be valuable tools in the development as components of a subunit vaccine. whether these hybrid fibrillae induce specific ab is currently under investigation. the clpg exposure vector system described here provides a number of insertion sites and therefore a variety of flanking aa sequences for the expression of tgev epitopes. consequently, a further study of the immunogenicity of the tgev epitopes in different contexts of clpg will improve our understanding of the relationship between flanking sequences and the immunogenicity of the epitopes. improved mimicry of a foot-and-mouth disease virus antigenic site by a viral peptide displayed on fl-galactosidase surface cs a capsule-like antigen as an exposure vector for heterologous antigenic determinants synthesis of fusion proteins with multiple copies of an antiigenic determinant of footand-mouth disease virus permissive sites and topology of an outer membrane protein with a reporter epitope localization of antigenic sites of the e glycoprotein of transmissible gastroenteritis coronavirus four major antigenic sites of the coronavirus transmissible gastroenteritis virus are located on the amino-terminal half of spike protein site-directed mutagenesis of virtually any plasmid by eliminating a unique site permissible peptide insertions surrounding the signal peptide-mature protein junction of the cipg prepilin: cs a fimbriae of escherichia coli as carriers of foreign sequences residues involved in the formation of the antigenic sites of the s protein of transmissible gastroenteritis coronavirus sequence analysis of the clpg gene, which codes for surface antigen cs a subunit: evidence of an evolutionary relationship between cs a, k and f subunit genes cs a, a new k -related fimbrial antigen on bovine enterotoxigenic and septicemic escherichia coli strains a simple and efficient method for the oligodeoxyribonucleotide-directed mutagenesis of double-stranded plasmid dna construction, expression and immunogenicity of multiple tandem copies of the schistosoma mansoni peptide - of the p glutathione s-transferase expressed as c-terminal fusions to tetanus toxin fragment c in a live aro-attenuated vaccine strain of salmonella expression of heterologous peptides at two permissive sites of the male protein: antigenicity and immunogenicity of foreign b-cell and t-cell epitopes hydrophobic cluster analysis and secondary structure predictions revealed that major and minor structural subunits of k -related adhesins of escherichia coli share a common overall fold and differ structurally from other fimbrial subunits role of proteolytic processing of viral envelope glycoproteins vaccine technologies: view to the future dna sequencing with chain-terminating inhibitors efficiency oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex dna method using alternative selectable markers immunoreactivity of chimeric proteins carrying the hiv- epitope igpgraf: correlation between predicted conformation and antigenicity electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheet: procedure and some applications key: cord- -wm eyaam authors: becares, martina; sanchez, carlos m.; sola, isabel; enjuanes, luis; zuñiga, sonia title: antigenic structures stably expressed by recombinant tgev-derived vectors date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: wm eyaam coronaviruses (covs) are positive-stranded rna viruses with potential as immunization vectors, expressing high levels of heterologous genes and eliciting both secretory and systemic immune responses. nevertheless, its high recombination rate may result in the loss of the full-length foreign gene, limiting their use as vectors. transmissible gastroenteritis virus (tgev) was engineered to express porcine reproductive and respiratory syndrome virus (prrsv) small protein domains, as a strategy to improve heterologous gene stability. after serial passage in tissue cultures, stable expression of small prrsv protein antigenic domains was achieved. therefore, size reduction of the heterologous genes inserted in cov-derived vectors led to the stable expression of antigenic domains. immunization of piglets with these tgev vectors led to partial protection against a challenge with a virulent prrsv strain, as immunized animals showed reduced clinical signs and lung damage. further improvement of tgev-derived vectors will require the engineering of vectors with decreased recombination rate. the order nidovirales comprises enveloped single-stranded, positive-sense rna viruses. the nidovirales order includes the coronaviridae family that contains viruses with the largest known rna genome, of around kb (enjuanes et al., ) . coronavirus (covs) infect a wide range of mammalian and avian species. the development of efficient cov reverse genetics systems (almazan et al., (almazan et al., , (almazan et al., , (almazan et al., , casais et al., ; thiel et al., ; yount et al., yount et al., , makes them promising expression vectors, with several advantages over other viral expression systems. covs replicate in the cytoplasm without a dna intermediary, making integration of the virus genome into the host cell chromosome unlikely (lai and cavanagh, ) . in addition, these viruses have the largest rna virus genome and, in principle, have room for the insertion of large foreign genes (enjuanes et al., ; masters, ) . as covs in general infect both respiratory and enteric mucosal surfaces, they may be used to target the antigen to these areas, stimulating the mucosal immune system to induce a pleiotropic secretory immune response, including lactogenic immunity . in fact, it has been described that a pleiotropic secretory immune response is best induced by the stimulation of gut associated lymphoid tissues (saif, ) . moreover, the tropism of covs may be engineered by modifying the spike (s) gene (casais et al., ; sanchez et al., ) , and non-pathogenic cov strains infecting most species of interest (human, porcine, bovine, canine, feline, and avian) are available and therefore are suitable to develop safe virus vectors (cavanagh et al., ; ortego et al., ) . in fact, several studies have reported the construction of covderived viral vectors expressing high levels of heterologous proteins, including reporter and viral proteins (bentley et al., ; ribes et al., ; shen et al., shen et al., , . foreign gene expression levels can be regulated by the use of different transcription-regulating sequences (trss) ranging from intermediate to high gene expression levels (alonso et al., a) . in addition, our group has recently identified an optimized transcription-regulating motif, enhancing by -fold the mrna levels of a viral gene, which can be used in expression vectors based in cov genomes (mateos-gomez et al., ) . additionally, a combination of these trss could be used to drive the expression of two or three heterologous genes from just one infectious cdna (i.e., dicistronic or tricistronic vectors). genetic stability of a heterologous gene within the viral vector is essential for its development as a live immunization vector. in general, the stability of heterologous genes is high for dna viruses and negative rna viruses, in which the low level of recombination contributes to the maintenance of the inserted foreign genes (bukreyev et al., ) in contrast, positive rna viruses are highly prone to recombination, both homologous and non-homologous (alejska et al., ; figlerowicz et al., ) leading to the loss of the inserted genes and avoiding their expression over a long time period. covs are positive rna genomes with high recombination frequency (denison et al., ; lai, ; sanchez et al., ) . genetic instability leading to the loss of heterologous genes has been frequently reported in cov-derived vectors, both in vitro (bentley et al., ; cruz et al., ; sola et al., ) and in vivo (bentley et al., ) . in view of the frequent instability of cov-based vectors expressing proteins of large size, we explored whether the reduction of heterologous gene size was a useful strategy to increase insert stability, by reducing the probability of the presence of toxic domains in the inserted gene or protein. in fact, the expression of small protein domains is a common strategy used to reduce toxicity when toxic proteins are expressed in bacteria (edwards et al., ; samuelson, ) . tgev infects the enteric and respiratory tissues of newborn piglets resulting in a mortality of nearly % (saif and wesley, ) . interestingly, some non-enteric tgev variants with alterations in the s protein have a tropism restricted to the respiratory tract, and show attenuated phenotype (sanchez et al., ) . tgev-derived vectors have been successfully engineered for the expression of green fluorescent protein (gfp). the gfp gene was expressed by replacing the non-essential genes a and b, leading to very stable ( passages in tissue culture) high expression levels of the heterologous protein ( μg/ cells) . recombinant tgev (rtgev) vectors have been engineered for dicistronic expression of heterologous genes, such as porcine reproductive and respiratory syndrome virus (prrsv) gp and m proteins (cruz et al., ) , or rotavirus vp and vp , in which formation of rotavirus virus like particles (vlps) in the cytoplasm of rtgev infected cells was observed (enjuanes et al., ) . tgev has been previously used as an immunization vector to confer partial protection against prrsv infection (cruz et al., ) . in addition, an engineered rtgev in which the tropism was modified replacing the s protein by the homologous one from mouse hepatitis virus (mhv) was used to confer protection against rotavirus infections (ribes et al., ) . the engineered rtgev expressing rotavirus vp protein was then evaluated in the mouse model. the recombinant virus triggered a humoral response via systemic (serum igg and iga) and mucosal (intestinal iga) antibodies. in addition, partial protection against rotavirus-induced diarrhea was observed in % of the challenged animals. porcine reproductive and respiratory syndrome (prrs) is the most important infectious disease affecting swineherds worldwide. it is characterized by reproductive failure in sows, as well as severe pneumonia in piglets (lunney et al., ) . the causative agent of prrs is prrs virus (prrsv) that is included in arteriviridae family, in the order nidovirales. prrsv is an enveloped, singlestranded positive sense rna virus of approximately kb in length that contains open reading frames (orfs). orf a and orf b encode the replicase non-structural proteins, while orfs to encode structural proteins: the small envelope protein (e), the membrane protein (m), nucleocapsid protein (n) and the glycoproteins gp a, gp , gp , gp , and the recently identified protein a (dokland, ; firth et al., ; johnson et al., ) . currently, prrs causes huge economic losses in the swine industry, but commercially available vaccines are only partially effective (charerntantanakul, ) . prrsv infection induces a weak innate immune response, probably contributing to the reduced and delayed subsequent humoral and cellular immune responses, and also to virus persistence (kimman et al., ). this is probably due to the limited interferon alpha (ifn-α) elicited by prrsv (albina et al., ; calzada-nova et al., ) . the knowledge on prrsv correlates of protection is limited. neutralizing antibodies against prrsv are mainly directed to gp protein (kim and yoon, ; ostrowski et al., ) , although neutralizing antibodies recognizing gp and gp have also been described following prrsv infection (costers et al., ; oleksiewicz et al., ; vanhee et al., ) . prrsv m protein is a potent inducer of t-cell proliferation in piglets infected with prrsv, and may also play a role in protection (bautista et al., ; jeong et al., ) . current vaccines against prrsv have a limited efficacy. best results have been obtained using modified live vaccines, although they have several problems such as incomplete protection, virus shedding and possible reversion to virulence (charerntantanakul, ) . vector-based vaccines could represent an advantage to stimulate both humoral and cell immune responses against prrsv (cruz et al., ) . given the potential of cov-derived vectors, and the requirement of more efficient vaccines against prrsv, the work presented here is focused on the use of tgev as a vector for the expression of prrsv antigenic combinations. the expression of prrsv small domains containing the epitopes relevant for protection would lead to a significant increase in vector stability. previous work from our laboratory has shown that rtgevs coexpressing full-length prrsv gp (wild type or modified) and m proteins induced partial protection against prrsv (cruz et al., ) . the modest results obtained may be due to the instability of gp protein in the rtgev system, resulting in a significant loss of gp expression in - passages in tissue culture. expression of full-length prrsv gp or gp proteins was also toxic for rtgev leading to the loss of the heterologous gene sequence (m. becares, s. zuñiga and l. enjuanes, unpublished results) . in this work the stability of the expression of small domains of prrsv gp , gp and gp , previously described as potentially relevant in the induction of protection against prrsv has been studied, in comparison with the expression of full-length proteins, using rtgev vectors. our results showed that reduction of the heterologous genes size inserted in the cov-derived vector is a promising strategy to achieve stable expression. additionally, as prrsv m protein was stable in rtgev, several antigenic structures were engineered using this protein as scaffold for the expression of small antigenic domains, resulting in high stability. furthermore, immunization of piglets with these live attenuated rtgev vectors partially protected against prrsv, with reduction of clinical signs and lung damage as well as a faster viremia decrease. prrsv m protein is a long non-glycosilated membrane protein of around amino acids, which is the most highly conserved structural protein of prrsv (meng et al., ) and has been involved in the induction of t-cell response against prrsv (bautista et al., ) . a rtgev vector expressing prrsv m protein was generated encoding prrsv m gene in the location previously occupied by non-essential genes a and b. prrsv m gene expression was driven by the transcription-regulating sequence of gene a (trs a ) (fig. a) . a rtgev-s . -trs a -m was recovered, with a titer of pfu/ml, as expected for rtgev viruses. in order to test the stability of prrsv m protein expressed by this vector, cloned viruses were serially passaged in tissue culture and the maintenance of the heterologous gene was evaluated at different passages by the analysis of plaque-purified viral clones. the presence of the heterologous sequence in the viral genome was evaluated by rt-pcr, using specific primers flanking the insertion region. after and serial passages of the rtgev-s . -trs a -m, all the isolated viral clones still contained m gene sequence (fig. b) , with the expected size and sequence as revealed by pcr product sequencing. in addition, all the isolated clones expressed m protein mrna (fig. b) , confirming m gene stability in rtgev system. moreover, m protein expression was analyzed by immunofluorescence in st cells infected at moi . . m protein was expressed in % of the infected cells (fig. c) , with expression levels remaining constant through the passages (data not shown). altogether, these results indicated that expression of prrsv m protein in rtgev was fully stable. expression of small domains of prrsv gp protein using rtgev netralizing antibodies recognizing gp are considered the most relevant for protection, with the epitope responsible for the neutralization located in the ectodomain of gp protein (kim and yoon, ; ostrowski et al., ) . previous studies from our group indicated that the partial protection observed after immunization with rtgevs expressing full length gp protein, both wild type or glycosylation mutants, was probably due to the toxicity of this protein in rtgev system, leading to heterologous gene loss with passages (cruz et al., ) . as a consequence, we decided to evaluate the expression of small domains containing epitopes potentially relevant in protection, as the reduction of potential toxic domains could increase vector stability. in a first approach, a rtgev co-expressing gp ectodomain (gp ecto) and m protein was engineered. gp ecto transcription was driven by the trs a , and that of m protein by an optimized trs partially derived from gene n trs (trs n ) (alonso et al., a) . m protein was included in the rtgev construct because it was fully stable and we previously observed that it increased gp stability, probably by forming the heterodimer observed in the native virus (cruz et al., ) . in fact, we postulate that the expressed gp domains and m protein could form a heterodimer similar to the one observed in the virus, what may be important for its immunogenicity. gp ecto consisted in the most n-terminal amino acids of the olot gp protein, which according to bioinformatics predictions cover the ectodomain of the protein. this domain included the protein motifs relevant in protection, such as the immunodominant epitope and the epitope genomic rna (grna) and subgenomic mrna (mrna) encoding prrsv m protein were detected. the arrow indicates the expected size of the corresponding pcr product. numbers on the left indicate the molecular weight markers (mw) size in base pairs. (c) immunofluorescence analysis of st cells infected with the passage rtgev-s . -trs a -m at hpi. a polyclonal antibody specific for tgev and a secondary antibody staining red were used to identify virus-infected cells. expression of prrsv m protein was detected with a monoclonal antibody and a secondary antibody staining green. critical in neutralization as well as the glycosylation sites ( fig. a) . to allow gp ecto protein detection, a hemaglutinin (ha) tag was fused to gp protein domain ( fig. a ). this tag is small ( amino acids) and was previously used for cov protein tagging, without showing any toxicity (alvarez et al., ) . rtgev-s . -trs a -gp ecto-trs n -m was recovered with titers similar to those of wt virus. the stability of gp ecto domain with virus passages was analyzed by rt-pcr analysis of isolated clones. after passages in tissue culture, % of the isolated clones contained the gp ecto sequence ( fig. b ) and expressed the corresponding mrna (data not shown), representing a modest increase of stability compared with gp full-length ( % stable) (fig. b) . unfortunately, in both cases, the heterologous gp sequences were lost at passage (data not shown), indicating that gp ecto long-term stability did not represent a sufficient improvement as compared to full-length gp . similar conclusions were extracted from immunofluorescence analysis of protein expression (data not shown). in a second step, an additional reduction in gp size was designed, by eliminating the predicted signal peptide of gp , whose cleavage is controversial (thaa et al., ; wissink et al., ) . the resulting amino acid fragment of gp protein (gp fr) was inserted in rtgev, leading to rtgev-s . -trs a -gp fr-trs n -m virus. this small domain contained the gp epitope critical in neutralization, glycosylation sites and the cysteine residue involved in the gp -m heterodimer formation. for gp fr detection a flag tag was fused at the carboxi-terminus ( fig. a ). this flag tag has been successfully used in cov protein tagging (alvarez et al., ) . the additional size reduction of the gp fragment cloned in rtgev led to an improvement in heterologous gene stability after passages in tissue culture, with % of the independent clones containing gp fr sequence (fig. b ) and expressing gp fr mrna (data not shown). moreover, long-term stability was significantly improved, with up to % of the isolated clones stably maintaining gp fr after passages in tissue culture (data not shown). studies on prrsv immunobiology have revealed that prrsv neutralizing antibodies recognized epitopes within the minor structural glycoproteins gp a, gp and gp (costers et al., ; oleksiewicz et al., ) . rtgevs were engineered expressing these proteins, alone or in various combinations, including the tricistronic expression of gp a, gp and gp . none of those proteins was stably expressed by rtgev vectors, with prrsv gp protein resulting extremely toxic for rtgev system, leading to its expression loss in early stages (m. becares, s. zuñiga and l. enjuanes, unpublished results). the recent identification of antigenic, linear domains in gp and gp (costers et al., ; vanhee et al., ) allowed the application of the small domain expression strategy to these proteins. fusion domains including gp or gp epitopes critical in neutralization, flanked by a few amino acids (table ) , and preceded by the corresponding signal peptide were designed. these peptidic domains consisting of and amino acids of gp (gp fr) and gp (gp fr), respectively, were fused to the flag tag at their c terminus end (fig. a) . the gp and gp fragments were cloned in tgev genome in the location previously occupied by non-essential genes a and b and their transcription was driven by the trs a . recombinant viruses rtgev-s . -trs a -gp fr and rtgev-s . -trs a -gp fr were recovered with titers similar to those of the wt virus. the stability of the recombinant viruses was analyzed after and passages in tissue culture, by studying plaque-purified clones by rt-pcr. all the clones maintained the heterologous gene sequence, and (gp ecto), and gp fragment (gp fr) that comprises the ectodomain lacking the signal peptide (sp). immunodominant epitope (ide) and epitope critical in neutralization (ecn), n-glycosylation sites (yellow), and the cysteine involved in gp -m heterodimer formation (red) are also shown. gp ecto and gp fr included an ha or flag tag, respectively, for their detection (tg, blue). (b) rt-pcr analysis of ten clones from plaque-purified passage rtgev-s . -trs a -gp -trs n -m (gp ), rtgev-s . -trs a -gp ecto-trs n -m (gp ecto) and rtgev-s . -trs a -gp fr-trs n -m (gp fr) viruses. the arrow indicates the expected size of the corresponding pcr product. numbers on the left indicate the molecular weight markers (mw) size in base pairs. lower size bands (indicated by red asterisks) correspond to deletion products from heterologous gene, meaning genomic instability. numbers on the right indicate the overall stability of each construct. expressed the corresponding mrna (fig. b ), indicating that both gp fr and gp fr were fully stable in the rtgev vector. protein detection using anti-flag antibody failed for gp fr, gp fr and gp fr, both in immunofluorescence and western blot assays (data not shown). altogether, these data revealed that heterologous gene size reduction led to a drastic increase in the stability of rtgev vectors. m protein is the most conserved structural protein among prrsv strains (kapur et al., ; murtaugh et al., ) and it is the main inducer of virus-specific t-cell response (bautista et al., ; jeong et al., ) . our results indicated that m protein was fully stable in rtgev (see above). therefore, we postulated that m protein could be used as a scaffold for the expression of small antigenic domains. as a proof of principle, the gp epitope critical in neutralization (ecn) domain was selected for expression fused to m protein. two exposed locations into m protein were predicted using tmpred transmembrane topology prediction algorithm (hofmann and stoffel, ) : the n-terminus and a loop comprising amino acids to . gp ecn was inserted at these m protein locations, leading to chimeric structures, gp ep-ntermm and gp ep-mloop, respectively (fig. a) . these chimeric genes were cloned into rtgev vector, and recombinant viruses rtgev-s . -trs a -gp ep-ntermm and rtgev-s . -trs a -gp ep-mloop were rescued with titers similar to those of the parental virus. the stability of the recovered viruses was analyzed. after or passages in tissue culture independent clones were screened by rt-pcr. all the independent clones maintained the heterologous gene sequence (data not shown) after passages, whereas after passages % and % of rtgev-s . -trs a -gp ep-ntermm and rtgev-s . -trs a -gp ep-mloop, respectively, contained the heterologous gene sequence and expressed the corresponding mrna (fig. b) . in order to evaluate stability and expression levels of the chimeric proteins, double immunofluorescence was performed on cells infected with passage rtgev-s . -trs a -gp ep-ntermm and rtgev-s . -trs a -gp ep-mloop viruses. m protein scaffold was detected in % of infected cells in both cases (fig. c ). this detection level was similar to that observed in rtgev-s . -trs a -m expressing full-length wt m protein (see above). in contrast, flag epitope was detected in % and % of the rtgev-s . -trs a -gp ep-ntermm and rtgev-s . -trs a -gp ep-mloop infected cells, respectively (fig. c) . these results strongly suggested that m protein n-terminus was a more exposed location, and therefore better to present antigens. this data is in agreement with previous observations demonstrating that arterivirus m protein is tolerant to manipulations of its ectodomain (verheije et al., ) . in order to evaluate the protection provided by rtgevs expressing prrsv antigens, the rtgevs that showed an increased stability in cell culture were tested in vivo. for that purpose, rtgev-s . -trs a -m, rtgev-s . -trs a -gp fr-m, rtgev-s . -trs a -gp fr, rtgev-s . -trs a -gp fr, and rtgev-s . -trs a -gp ep-ntermm were selected for in vivo experiments. two groups of twelve days-old piglets were inoculated with  pfu/animal of each rtgev-s . -trs a -m, rtgev-s . -trs a -gp fr-m, rtgev-s . -trs a -gp fr, rtgev-s . -trs a -gp fr, and rtgev-s . -trs a -gp ep-ntermm (immunized group), or rtgev (non-immunized group), respectively, by three routes: oral, nasal and intragrastic. previous data from our group indicated that, in these conditions, the virus in the inoculum reached the target organs (respiratory and digestive tracts) and replicated to high titers (cruz et al., ; sanchez et al., ) . a boost was performed weeks after inoculation using the same conditions. two weeks later, a challenge was performed with  tcid of prrsv olot -like virulent strain. a control group was inoculated with  pfu/animal of rtgev and boosted two weeks later, but not challenged. pigs were monitored for clinical signs, focusing on respiratory symptoms such as tachypnoea, and abdominal breathing. prrsv infection resulted in moderate fever, depression and respiratory signs that persist from days to after challenge (fig. a) . the percentage of animals showing respiratory symptoms was significantly higher in the non-immunized group than in the immunized group (fig. a) . moreover, the average weight gain, which was reduced in challenged animals, was higher in immunized animals than in non-immunized animals between and days post-challenge (data not shown). lung damage was analyzed by histopathology of lungs from five randomly chosen piglets per group. lungs from challenged piglets exhibited features that are characteristic of prrsv infection such as pneumocyte hypertrophy and hyperplasia, and intra-alveolar accumulation of cell debris (fig. b, left panels) . the lungs from immunized animals showed a lower degree of lung damage than those from non-immunized piglets (fig. b, right panel) , indicating a certain degree of protection. the lower extent of lung inflammation observed in immunized animals was in agreement with the lower levels of proinflamatory cytokine il- observed in vaccinated animals' sera (fig. c) . immunized animals showed a moderate increase in il- by days post-challenge ( dpi, as shown in the figure), but levels rapidly returned to normal, while non-vaccinated animals showed a higher elevation of this cytokine, that continued at elevated levels during the experimental infection. altogether, these results suggested that rtgev vectors expressing prrsv antigens conferred partial protection against prrsv infection. to further analyze the protection conferred by rtgevs expressing prrsv antigens, prrsv viremia was analyzed by rt-qpcr at different times post-challenge. similar virus titers in serum were obtained in all challenged animals at the initial stages postchallenge ( - dpi) (fig. ) . interestingly, a significant reduction in virus titer was observed in the immunized group at - days post-challenge ( - dpi). in order to evaluate the potential of rtgevs stably expressing prrsv antigens as inducers of immunity against prrsv, the humoral response was analyzed at different times postinoculation. the antibody response against the tgev vector, prrsv virus and prrsv individual proteins expressed by rtgevs were determined by elisa. all the animals elicited a high humoral immune response against tgev indicating that the vector infected target tissues as expected, even though the piglets presented preexisting anti-tgev antibodies (data not shown). seroconversion against total prrsv was observed in all challenged animals by day after infection with the virulent virus, while for individual gp , gp and m protein it was detected by day after challenge. in all the above-mentioned cases, no differences were observed between immunized and non-immunized animals (data not shown). the humoral response against prrsv n protein followed identical pattern to that obtained for anti-prrsv antibodies (data not shown). these data indicated that anti-prrsv total antibodies response was most likely directed against n protein and did not play a role in protection, in agreement with previous reports showing an early non-neutralizing antibody response obtained after prrsv infection (mateu and diaz, ) . interestingly, a higher and faster antibody response against gp protein was found from day post-immunization in immunized animals as compared to the non-immunized ones (fig. a) . the neutralizing antibody response was evaluated in sera from immunized and non-immunized animals at , and days postchallenge. non-immunized animals showed higher levels of prrsv neutralizing antibodies than the immunized ones (fig. b ). this data, suggested a less effective prrsv infection in the immunized piglets, supporting partial protection against prrsv infection. in this study rtgev was engineered for the expression of small protein domains relevant in immune response against prrsv. previous results from our group indicated that instability of certain heterologous gene expression by rtgev might represent an important limitation for its use as an immunogenic vector. the expression of small protein domains was used as a strategy to improve heterologous gene stability. stable expression of protein antigenic domains contained in highly unstable full-length proteins was achieved. additionally, as full-length prrsv m protein was stably expressed by rtgev, it was used as a scaffold for the generation of chimeric proteins that exposed other prrsv antigens, resulting in highly stable expression. therefore, size reduction of the heterologous genes inserted in cov-derived vectors resulted in a promising strategy to achieve stable expression. protection experiments showed that rtgev, stably expressing prrsv antigenic structures, elicited partial protection against prrsv, with a reduction of clinical signs and lung damage in immunized piglets. the potential of cov-derived vectors as systems for gene delivery has been limited due to its restricted stability. in general, genetic stability is highly dependent on the nature of the foreign gene, with some inserts maintained at least twenty passages whereas others are lost at passage two (de haan et al., ; enjuanes et al., ; shen et al., ; sola et al., ) . in addition, other factors affect genetic stability of recombinant covs, such as the insert size (de haan et al., ) , and the genomic location in which it is inserted (bentley et al., ) . the maintenance of the inserted genes will also depend on the recombination rate, conditioned both by the insert size and the presence in the foreign sequence of regions showing homology with the virus genome, favoring homologous recombination (wang et al., ) . furthermore, other heterologous gene or protein characteristics may affect insert stability, leading to loss of the inserts harmful for the infected cell or virus replication. therefore, it is very difficult to predict the specific insert stability in advance, before a highly effort-consuming process to generate the cov-derived vectors expressing the heterologous antigen has been accomplished. prrsv gp and m genes have similar lengths ( and nucleotides, respectively) and small ectodomains exposed between three transmembrane domains. nevertheless, m protein was fully stable in rtgev vectors, while gp protein resulted toxic and its expression was lost after - passages of the virus in cell culture. interestingly, prrsv hydrophobic m protein resulted highly toxic in other expression systems, including bacteria and insect cells (jeong et al., ; plana-durán et al., ) . as deletion of the heterologous genes could be due to homologous recombination between the heterologous gene and tgev genome because of sequence identity, this possibility was analyzed. no statistically significant sequence identity was identified between prrsv gp sequence and tgev genome. in fact, analysis of the deletions observed in the unstable recovered viral clones did not show a common pattern of recombination. in constrast, random deletions were observed ranging from small deletions affecting trs, to larger ones covering most prrsv gp gene sequence (data not shown), that in all cases led to the loss of protein expression. additionally, the gp gene, when expressed alone in rtgev vectors, was lost at very early passages while it was stably maintained until passage when it was co-expressed with m protein, most likely due to the formation of gp -m heterodimers (cruz et al., ) . these data suggested that the instability was caused by protein toxicity affecting host cell viability or viral life cycle, rather than a negative effect of the heterologous gene sequences on virus genomic stability. this toxicity would confer selective advantage to those viral clones that did not express gp protein. in this work we showed that size reduction of the foreign insert significantly improved heterologous gene stability. even in the case of highly toxic inserts, such as prrsv gp protein, size reduction led to % stability. therefore, heterologous gene size reduction is a promising strategy to achieve stable expression in tgev-derived vectors and in general in covs. this effect could be due to a decrease of the probability of non-homologous recombination in shorter sequences, or to the elimination of protein domains toxically affecting the host cell or rtgev life cycle. this result is in agreement with previous studies showing that gene size affected foreign gene stability in cov vectors, as the larger firefly luciferase gene resulted less stable than the shorter renilla luciferase gene, when expressed by feline infectious peritonitis virus (fipv) vectors (de haan et al., ) . in this paper we used prrsv m protein, which we have shown that displays a high stability in rtgev vectors as a scaffold for the expression of small antigenic domains. this approach may be useful to modify the trafficking and accumulation of small protein domains expressed alone. in fact, protein detection using anti-flag antibody failed for gp fr, gp fr and gp fr, both in immunofluorescence and western blot assays (data not shown), probably due to low accumulation of those peptides inside the cell. interestingly, gp ep-ntermm was detected at high levels, indicating a higher accumulation in the infected cell of the chimeric protein. long-term stability of cov-derived vectors has not been systematically addressed. few proteins have been reported to be stably expressed by covs. among these, gfp was stable for more than or passages in tissue culture when expressed by mhv or tgev, respectively (sarma et al., ; sola et al., ) , but it showed instability in ibv-derived vectors (bentley et al., ) . prrsv m protein was also stably expressed by rtgev for more than passages (this manuscript). proteins such as luciferase expressed by mhv and ibv-derived vectors (bentley et al., ; de haan et al., ) , prrsv gp protein expressed using tgev virus vectors (cruz et al., ) or gus when expressed by tgev minigenomes (alonso et al., b) were lost at early passages. in our experience, using rtgev vectors, only around % of the heterologous genes were stably expressed for more than passages in tissue culture [(alonso et al., b; cruz et al., ; sola et al., ) , and unpublished results]. this instability is a key limiting factor in the use of cov-based vectors for the expression of full-length proteins. to improve the stability and efficacy of cov-derived vectors it would be essential to understand the factors that control the high recombination frequency of covs. to this end, a detailed analysis of cov proteins involved in genetic recombination is needed. several enzymes involved in cov rna synthesis, such as nsp (helicase), nsp (endonuclease), nsp (exonuclease), nsp and nsp (rna processivity components), or n protein could modulate recombination in covs. the engineering of recombination defective cov mutants by knocking-down one or several genes involved in the recombination process could be the first step to achieve stable expression of large heterologous genes. in this study, three prrsv protein domains from gp , gp and gp proteins, recognized by neutralizing antibodies were expressed by rtgev and used as immunogens (costers et al., ; plagemann, ; vanhee et al., ; wissink et al., ) , and the humoral response elicited by these rtgevs was measured. after challenge, a faster response against gp protein was observed in immunized piglets. in contrast, the response against gp and gp was similar in immunized and non-immunized piglets. these data suggested that gp fragment was immunogenic, while gp and gp domains were antigenic but had a reduced immunogenicity. immunization of piglets with a combination of rtgev expressing prrsv antigens led to a clear reduction of clinical symptoms after challenge, a lower degree of lung damage and a faster viremia reduction. these results represent an improvement over previous vaccination experiments using rtgev vectors expressing prrsv antigens (cruz et al., ) . prrsv correlates of protection remain to be identified (kimman et al., ) , what represents an additional limitation for the development of new vaccine candidates. neutralizing antibodies seem relevant for preventing prrsv infection (lopez and osorio, ) , but not enough to provide full protection (murtaugh and genzow, ) . t-cell responses seem also required in prrsv clearance (mateu and diaz, ) . in the present study, immunized animals showed a significant faster recall antibody response against gp protein, which is generally considered the main target of neutralizing antibodies (kim and yoon, ; ostrowski et al., ) . non-immunized animals developed a higher neutralizing response after challenge with a virulent prrsv strain, what is considered as an indication of higher infection, whereas the immunized animals were significantly, although partially, protected against prrsv infection. with the available data, it is not possible to determine whether the observed protection was due to an undetectable neutralizing antibody response before challenge [even commercial available vaccines have been reported to fail in the induction of detectable levels of neutralizing antibodies before challenge (geldhof et al., ) ] to immune cell responses [most likely directed to m protein present in the immunization cocktail], or both. the higher gp protein specific antibody response was observed from day post-challenge, while significant differences in neutralizing antibodies between immunized and non-immunized animals were observed between and days post-challenge, correlating with the differences in viremia, what suggests that the observed neutralizing response was due to a higher infection of nonimmunized swine. the relative contribution to protection of the humoral and cellular responses has not been determined. when a correlation between protection and induction of specific cytokines was analyzed, il- levels were significantly different between immunized and non-immunized piglets, with levels consistently higher in non-immunized animals. the exacerbated il- response elicited in non-immunized animals correlated with the higher lung damage observed in these animals. this result was in agreement with previous studies showing that piglets with more severe symptoms, including viremia and lung lesions, had a continuous elevation of il- in serum, while in animals with milder symptoms il- levels returned to normal by dpi (petry et al., ) . higher, but not significant, levels of ifn-α were also observed in immunized animals compared to non-immunized animals (data not shown), at day post-prrsv challenge. this result suggested that immunized animals developed a higher innate immune response, which nevertheless did not seem strong enough to induce a higher adaptative immune response. the construction of rtgevs expressing small antigenic domains has considerably improved the stability of the expression vectors. nevertheless, some of these small antigens may have limited immunogenicity. therefore, the expression of full-length antigens by engineering cov vectors with decreased recombination rate deserves further attention to definitely launch covs as efficacious vaccine vectors for animal and human health. experiments involving animals were performed in strict accordance with eu ( / /ue) and spanish (rd / and / ) guidelines. all the protocols were approved by the in site ethical review committee. baby hamster kidney (bhk- ) cells stably transformed with the gene coding for porcine aminopeptidase n (bhk-papn) (delmas et al., ) were grown in dulbecco's modified eagle's medium (dmem) supplemented with % fetal calf serum (fcs) and g ( . mg/ml) as a selection agent. recombinant tgev viruses obtained in this work were grown in swine testis (st) cells (mcclurkin and norman, ) . tissue culture adapted prrsv olot (genbank kc ) strain was grown and titrated in monkey kidney marc- cells (kim et al., ) . challenge was performed with a virulent prrsv strain homologous to prrsv olot (prrsv-olot -like). prrsv-olot -like was propagated in porcine macrophages differentiated from fresh peripheral blood mononuclear cells (pbmcs) as previously described (enjuanes et al., ) . briefly,  pbmcs isolated from fresh blood by centrifugation were seeded in -mmdiameter plates in roswell park memorial institute medium (rpmi) supplemented with % heat-inactivated swine serum. after h, non-adherent cells where removed and attached macrophages, that showed % of confluence, were infected with tcid of the parental prrsv-olot -like. at h postinfection (hpi), when cytopathic effect was clear, supernatant was collected and centrifuged. virus was titrated in porcine alveolar macrophages (pams) as previously described (duan et al., ) . fusion products gp fr, gp fr, gp fr gp ep-mloop and gp ep-ntermm were chemically synthesized and purchased from gen-eart (germany). the prrsv olot protein sequences forming the fusion products are summarized in table . gp ecto sequence was amplified by pcr using the forward primer ( -gcaggtcctatgtacccctacgacgtgcccgactacgccatgagatg-ttctcacaaattggggc- ) and the reverse primer ( -gcgctcagct-caggtctcgactgcccaatcaaaatg- ), which included ppumi and blpi restriction sites (underlined), respectively. m sequence was amplified using the forward primer ( -gcaggtcctatgggaagcc-tagacgatttttg- ) and reverse primer ( -gggctaagcttacc-ggccatacttgacgagg- ), which included ppumi and blpi restriction sites (underlined), respectively. in both cases, plasmid psl-trs a -orf -trs n -orf (cruz et al., ) was used as a template. prrsv sequences, both chemically synthesized or pcr amplified, were digested with restriction endonucleases ppumi and blpi and cloned into the same sites of plasmid psl-tgev-s . - ab including tgev genomic sequence from nt to . prrsv sequences replaced non-essential genes a and b, leading to intermediate plasmids psl-trs a -gp fr, psl-trs a -gp fr, psl-trs a -gp fr, psl-trs a -gp ep-mloop, psl-trs a -gp fr-ntermm, psl-trs a -gp ecto and psl-trs a -m. for the generation of the dicistronic vectors psl-trs a -gp ecto-trs n -m and psl-trs a -gp fr-trs n -m, the sequence of m protein preceded by the optimized synthetic trs n (alonso et al., a) was obtained from psl-trs a -orf -trs n -orf by digestion with restriction endonuclease blpi and cloned into the same site of psl-trs a -gp ecto and psl-trs a -gp fr. finally, all intermediate plasmids containing prrsv sequences were digested with avrii. the resulting fragments were cloned into the same sites of plasmid pbac-tgev-s . (c.m. sanchez, m. becares, s. zuñiga and l. enjuanes, unpublished results) . this plasmid was derived from the original pbac-tgev fl (almazan et al., ) , containing restriction sites paci and mlui flanking s gene (ortego et al., ) . cloning steps led to plasmids pbac-s . -trs a -gp fr, pbac-s . -trs a -gp fr, pbac-s . -trs a -m, pbac-s . -trs a -gp ep-ntermm, pbac-s . -trs a -gp ep-mloop, pbac-s . -trs a -gp ecto-trs n -m and pbac-s . -trs a -gp fr-trs n -m. all cloning steps were checked by sequencing of the pcr fragments and cloning junctions. transfection and recovery of infectious rtgevs from cdna clones bhk-papn cells were grown to % confluence in -mmdiameter plates and transfected with μg of the corresponding pbac and μl of lipofectamine (invitrogen), according to the manufacturer's specifications. after h of incubation at c, cells were trypsinized and plated over a confluent st monolayer grown in -mm-diameter plate. after a -day incubation period, the cell supernatants were harvested (passage ). rtgevs were cloned by three plaque purification steps. rtgevs were grown and titrated as previously described (jimenez et al., ) . two clones of each rtgev were serially passaged, independently, in st cells every h. at passage and ten viral clones were plaque purified. rna from recombinant viruses was purified from infected st cells grown to overconfluence on -well plates. total intracellular rna was extracted at hpi using the rneasy mini kit (qiagen) according to the manufacturer's recommendations. reverse transcription was performed with high capacity rna-to-cdna™ kit (life technologies) according to the manufacturer's instructions. pcrs were performed to analyze the size and sequence of viral genomic rna (grna), at the locus where the heterologous genes were inserted, and heterologous mrna size and sequence synthesis. the primers used and the expected pcr fragment sizes are shown in table . subconfluent st cells grown on glass coverslips were mock infected or infected at a multiplicity of infection (moi) of . with each rtgev. at hpi cells were washed with phosphate-buffered saline (pbs), fixed with % paraformaldehyde, permeabilized with . % triton x- in pbs and blocked in pbs with % fcs. monoclonal antibodies specific for flag (flag m , : , sigma), prrsv m protein (em e c , : , kindly provided by inge-nasa), or a polyclonal rabbit serum specific for tgev ( : ) were used. bound primary antibody was detected with a alexa fluor or -conjugated antibodies specific for mouse or rabbit, respectively ( : , invitrogen). cell nuclei were stained with , -diamidino- -phenylindole (dapi) ( : , sigma). confocal microscopy was performed using a leica sp laser scanning microscope, and images were collected and processed with las af software (leica, wetzlar, germany). the percentage of infected cells expressing prrsv antigens was estimated by the analysis of independent microscopy fields, which represent an average of more than cells. forty-five twelve days-old non-colostrum-deprived piglets, born from prrsv seronegative sows, were inoculated with rtgev by three different routes (oral, gastric and intranasal) following standard procedures (sanchez et al., ) . piglets were divided into three -animal groups. piglets of group were inoculated with a mix of  plaque forming units (pfu)/animal of each of rtgev-s . -trs a -gp fr, rtgev-s . -trs a -gp fr, rtgev-s . -trs a -gp fr-m, rtgev-s . -trs a -m and rtgev-s . -trs a -gp ep-ntermm. piglets of groups and were inoculated with  pfu/animal of rtgev-s . . two weeks after the first immunization, all piglets were boosted in the same conditions, and two weeks later piglets of groups and were challenged with tcid of prrsv-olot -like per animal by intranasal route. infected animals were monitored daily to detect symptoms of disease, and body weights were determined every days. blood samples were taken at days , , , , , , , and post-first inoculation. five and ten animals per group were euthanized and necropsied at days and , respectively. lung macroscopic lesions were evaluated, and lung samples were collected frozen and in % buffered-formalin. five piglets per group were randomly chosen for histopathological study. lung representative sections were fixed with % paraformaldehyde and stored in % ethanol at c. paraffin embedding, sectioning and hematoxylin-eosin staining were performed by the histology service in the national center of biotechnology (cnb-csic, spain). samples were examined with a zeiss axiophot fluorescence microscope. determination of the lung damage score was obtained from unbiased observation of microscopy fields per animal, scoring from to attending to interstitial, peribronchiolar, and perivascular inflammation (page et al., ) . quantification of porcine il- β, il- , ifn-α, ifnγ, tnfα, il- and il- in serum samples was carried out using swine cytokine magnetic -plex panel (life technologies, tm), and the luminex is analyzer, according to the manufacturer's instructions. three serum samples, corresponding to the same experimental group and the same date of extraction, were randomly pooled and analyzed in a single well. data were calculated by xponent software using a fiveparameter model derived from the known reference cytokine concentrations supplied by the manufacturer. the sensitivity of this assay allowed the detection of cytokine concentrations with the following limits of detection: il- β ( . pg/ml), il- ( . pg/ ml), ifn-α ( . pg/ml), ifnγ ( . pg/ml), tnfα ( . pg/ml), il- ( . pg/ml), il- ( . pg/ml). viral rna was isolated from μl of serum using magmax™ viral rna isolation kit (life technologies) according to the manufacturer's instructions. prrsv rna quantity was measured by rt-qpcr analysis using a custom taqman assay detecting prrsv n rna (taqman probe -fam-acggcttttaatcaaggc-mgb; forward primer -ttccctctgcttgcaatcg- ; reverse primer -ggatgaaagcgacgcagttc- ), and the agpath-id one-step rt-pcr kit (life technologies) according to the manufacturer's instructions. the data were acquired with an abi prism sequence detection system and analyzed with abi prism sds version . . software (applied biosystems). viremia levels were expressed as the rt-qpcr cycle threshold (ct) values. antibodies induced against tgev and prrsv viruses or prrsv purified proteins were detected by elisa as described before (sambrook and russell, ) . prrsv gp , gp , gp , m and n proteins were expressed using the baculovirus-insect cell system. recombinant proteins were purified to near homogeneity by metal chelate affinity chromatography using ni-nta agarose (sigma-aldrich, madrid, spain) as previously described (nogales et al., ) . elisas were performed using partially purified tgev ( . μg per well) and prrsv ( . μg per well) viruses, or prrsv purified proteins gp ( . μg per well), gp ( . μg per well), gp ( . μg per well), m ( . μg per well) and n ( . μg per well). antigens were bound to -well microplates, saturated with % bovine serum albumin (bsa) in pbs for h at c and incubated with serial dilutions of the serum sample in wash buffer ( . % bsa, . % tween in pbs) for min at c. microplates were washed three times with wash buffer. bound antibodies were detected by incubation with peroxidase-conjugated protein a (biorad) diluted : in pbs with . % bsa. elisa was developed with k-blue tmb substrate (neogen, lexington, ky) for min table analysis of rtgevs stability by rt-pcr. expected product size and primers used for the analysis of viral grna and heterologous mrna expressed by rtgevs. expected size (bp) grna a mrna b reverse primer ( - ) a pcr for grna analysis was performed with the forward primer ( -attacgaaccaattgaaaaagtgc- ) and the reverse primer ( -ccgcctga-gaaaaggctgcattg- ) in all cases. b in all cases, forward primer ( -gtgagtgtagcgtggctatatctcttc- ), complementary to the viral leader sequence was used. c mrna size shown in the table corresponds to gp fr or gp ecto. at room temperature. reactions were stopped with . m h so , and the absorbance was read at nm. the elisa values of the sera were expressed as sample to positive ratio [sp-ratio¼(od of sample À od of negative control)/(od of positive controlÀ od of negative control)]. serial dilutions of heat-inactivated serum were incubated for h at c in the presence of pfus of prrsv-olot in dmem containing % fcs. the mixtures were added to confluent marc- cells in -well plates. after one hour incubation at c medium was removed and ml of dmem containing % fcs and . % agar was added. after h, cells were fixed with % formaldehyde in pbs, stained with a crystal violet solution, and lysis plaques were counted. a positive control serum, obtained from a prrsv infected pig at dpi led to - % of virus neutralization at a sera dilution of : . in contrast, a non-immune control serum led to a neutralization of to % in the same experimental conditions. the neutralization index of each serum sample was expressed relative to the one obtained with the negative control serum in the experiment [neutralization index ¼ % À (pfus serum sample/pfus negative control)  ]. two-tailed, unpaired student t tests were used to analyze difference in mean values between groups. all results were expressed as means the standard deviations of the means. chisquare test was used to analyze statistical significance of differences in percentages of immunized and non-immunized groups. p values o . were considered significant (noymer, ) . interferon-alpha response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus two types of non-homologous rna recombination in brome mosaic virus construction of a sars-cov infectious cdna clone and a replicon to study coronavirus rna synthesis engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate engineering the largest rna virus genome as an infectious bacterial artificial chromosome coronavirus reverse genetic systems: 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persistence infectious rna transcribed in vitro from a cdna copy of the human coronavirus genome cloned in vaccinia virus characterization of antigenic regions in the porcine reproductive and respiratory syndrome virus by the use of peptide-specific serum antibodies chimeric arteriviruses generated by swapping of the m protein ectodomain rule out a role of this domain in viral targeting positional effect of gene insertion on genetic stability of a clover yellow vein virus-based expression vector the major envelope protein, gp , of a european porcine reproductive and respiratory syndrome virus contains a neutralization epitope in its n-terminal ectodomain reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus systematic assembly of a fulllength infectious cdna of mouse hepatitis virus strain a we thank h. nauwynck and m. vanhee for sharing information about prrsv epitopes and ingenasa for kindly providing us with anti-m protein monoclonal anitbodies. we also thank m. gonzález, s. ros and r. fernández for technical assistance.this study was supported by grants from the ministry of science and innovation of spain (bio - ), and the european community's seventh framework programme (fp / (fp / - key: cord- -o rkx z authors: kim, seung won; ramakrishnan, m. a.; raynor, peter c.; goyal, sagar m. title: effects of humidity and other factors on the generation and sampling of a coronavirus aerosol date: - - journal: aerobiologia (bologna) doi: . /s - - - sha: doc_id: cord_uid: o rkx z suspensions of transmissible gastroenteritis virus (tgev), a porcine coronavirus, were nebulized at rates of . – . ml/min into moving air using a collison nebulizer or a plastic medical nebulizer operating at pressures ranging from to psi. the airborne viruses were collected on heating, ventilating, and air conditioning (hvac) filters in an experimental apparatus and also sampled upstream of these test filters using agi- and biosampler impinger samplers. to study the effects of relative humidity (rh) on tgev collection by the filters and samplers, the virus was nebulized into air at , , , and % rh. there were no significant changes in virus titer in the nebulizer suspension before and after nebulization for either nebulizer at any of the pressures utilized. aerosolization efficiency – the ratio of viable virus sampled with impingers to the quantity of viable virus nebulized – decreased with increasing humidity. biosamplers detected more airborne virus than agi- samplers at all rh levels. this difference was statistically significant at and % rh. nebulizer type and pressure did not significantly affect the viability of the airborne virus. virus recovery from test filters relative to the concentration of virus in the nebulizer suspension was less than %. the most and the least virus were recovered from filter media at % and % rh, respectively. the results suggest that tgev, and perhaps other coronaviruses, remain viable longer in an airborne state and are sampled more effectively at low rh than at high humidity. non-clinical health care workers and workers handling animals. potential threats to the general public include bioterrorism, especially from the smallpox virus, and emerging infectious diseases, such as severe acute respiratory syndrome (sars) and avian influenza. thus, virtually everyone is at the risk of exposure to contagious viruses via inhalation. viruses differ from other microorganisms in that they can replicate only inside a host cell. however, they can survive in the environment and be transmitted through air in the absence of host cells (gerone et al. ) . viruses are - nm in size, and they usually travel in air carried by other materials, such as droplets of respiratory secretions or dust particles (otten and burge ) . epidemiological evidence suggests that the transmission of viral infections may occur inside and between neighboring buildings (donaldson ; riley et al. ) . blood and tissue aerosols generated during general and dental surgery may also contribute to the transmission of these viruses (reponen et al. ) . testing the ability of heating, ventilating, and air conditioning (hvac), respiratory protection, and other filters to capture biological aerosols is important to ensure that the filters perform as expected. viruses are more difficult and expensive to use as a test aerosol for filters than other biological particles, and considerable background knowledge and experience are required to handle them properly. particles the size of single viruses will be captured primarily by brownian diffusion (lee and liu ) . however, few studies have measured the collection efficiency of filters challenged by virus particles. although bacteriophages (e.g., ms ) have been used as a surrogate for human viruses in some studies research triangle institute ) , they cannot be expected to represent all types of human and animal viruses. hence, studies with these viruses are sorely needed. in this study, we used transmissible gastroenteritis virus (tgev) of pigs as a surrogate for sars virus. laboratory studies using tgev are safe to perform because the virus has no known adverse effect on humans. further, the data obtained using tgev should be applicable to the sars virus because both tgev and sars virus are enveloped, positive-stranded rna viruses belonging to the family coronaviridae (fauquet et al. ) and their physicochemical properties are similar. although tgev has been utilized previously as a sars virus surrogate for studies of their genome expression (thiel et al. ) , it has not been used in studies investigating the sampling and behavior of viral aerosols (spendlove and fannin ; tseng and li ) . the methodology for generating, storing, and collecting viral aerosols has been reviewed by a number of authors cox ; mitchell ; buttner et al. ; sattar and ijaz ) . factors affecting the viability of a virus during nebulization include nebulizer type, nebulizer air pressure, nebulization time, microorganism type, and humidity of the dilution air (adams et al. ; sattar and ijaz ) . marthi ( ) listed humidity, temperature, radiation, and open-air factors (cox et al. ) as the most important parameters influencing the viability of airborne microorganisms. the method of aerosol generation, composition of the generation fluid, sampling method, and collection medium were deemed to be secondary parameters. although some factors may be more important than others for a particular virus, all of them play a role in viral aerosol viability (spendlove and fannin ) . humidity in buildings can vary as a function of season and location; consequently, it is an important factor to study in terms of virus viability in the indoor environment. bacteriophages, influenza viruses, and reoviruses each exhibit a unique dependence on relative humidity (rh) (loosli et al. ; hemmes et al. ; adams et al. ; trouwborst and de jong ) . however, the influence of humidity on the viability of coronaviruses such as the sars virus and tgev has not been studied previously. all-glass impingers (agi) are commonly used for collecting viral aerosols (spendlove and fannin ; sattar and ijaz ; sattar and ijaz ; tseng and li ) . biosamplers were developed by willeke et al. ( ) , and their sampling performance for bacteria and fungi has been studied (lin et al. (lin et al. , . agi- samplers collect most particles larger than . lm in diameter (buttner and stetzenbach ) . the physical collection efficiency of the biosampler has been shown to be about , , , and % for . -, . -, -, and -lm particles, respectively (willeke et al. ). biosamplers yield equivalent or higher culturable counts of bacteria relative to agi- samplers under the same conditions (lin et al. ). the physical sampling efficiency for both the agi- and the biosampler is less than % for particles with a diameter of less than . lm and less than % for many particles with a diameter smaller than . lm (hogan et al. ) . these authors also showed that the recovery of airborne ms and t bacteriophages using an agi- sampler was greater than that using a biosampler. however, comparisons of biosampler and agi- performance have not been made for coronaviruses such as sars virus and tgev. the ability of samplers to collect airborne particles with submicrometer diameters is important because many particles produced by coughing and sneezing are less than a micrometer in diameter. gerone et al. ( ) found that % of the particles produced by sneezing and % of the particles produced by coughing were smaller than lm in diameter. nicas et al. ( ) correctly pointed out that most of the mass of particles produced by coughing and sneezing will initially be in particles larger than lm in diameter. however, the water portion of droplets smaller than about lm in diameter will evaporate almost instantaneously leaving much smaller virus-containing particles suspended in the air (musher ) . filtration is an important method for reducing concentrations of biological particles inside of buildings. the possibility of using hvac filters as a sampling system for airborne microorganisms has recently been proposed (farnsworth et al. ). in addition, due to the threats of bioterrorism and pandemic influenza, more consideration is being given to the virus removal efficiency of filters in hvac systems (hitchcock et al. ) . the objective of this study was to characterize factors affecting tgev nebulization and to select the best sampling procedure for measuring the airborne concentration of this coronavirus. characterization of factors affecting virus nebulization can be an important step towards the use of viruses as test aerosols. once we are able to produce reliable virus-containing aerosols, that knowledge can be used in many ways; for example, respirator and ventilation filters can be tested for their virus filtration efficiencies, and virologists and toxicologists can use these techniques in their health effects research. measurements of airborne tgev concentrations and the recovery of tgev from a filter medium were performed using an air filter testing apparatus. suspensions of tgev were placed in a nebulizer and aerosolized into air flowing through the apparatus. the tgev suspensions were titrated before and after nebulization on different occasions to evaluate the effects of nebulization on virus viability. the airborne virus particles were sampled at an rh of , , , and % using two different types of impinger samplers. the virus particles were also collected on filter media inserted into the apparatus. the tgev collected by the samplers and the tgev eluted from the test filters were quantified. each combination of the four humidity levels with the two samplers was repeated three times for a total of separate tests. using these data, we evaluated the tgev's airborne concentration and its recovery from test filters as a function of rh and sampler type. each of the steps in this process is described in more detail in the following sections. nebulization experiments were performed in a filter test apparatus (fig. ). this apparatus was used by mccullough et al. ( ) to study the penetration and loading of respirator filters with airborne fungi and bacteria. tgev was aerosolized into the apparatus using a nebulizer and passed through a custom-made charge neutralizer containing a polonium- alpha particle source to imbue the virus with an equilibrium charge distribution. this viral aerosol was then mixed thoroughly with filtered dilution air before it entered the test section. the rh of the dilution air was controlled to , , , or % (± % variation). the test section consisted of a -cm-diameter vertical duct and a pneumatically controlled filter holder. the total air flow rate in this section was l/min. the temperature during the experiments was approximately °c. swine testicular (st) cells (atcc crl ) were used for the propagation of the test virus. the cells were grown in eagle's minimum essential medium (mem) (mediatech, herndon, va.) containing iu/ml penicillin, lg/ml streptomycin, lg/ml neomycin, lg/ml fungizone, and % fetal calf serum. the purdue strain of tgev was propagated by infecting st cells grown to - % confluency. the virus was allowed to adsorb to cells at °c for h, followed by the addition of mem without fetal calf serum and incubation at °c. after the appearance of virus-induced cytopathic effects (cpe), generally - h post-infection, the cells were subjected to two cycles of freezing and thawing. the cell debris was removed by centrifugation at g for min, and the viral supernatant was aliquoted in small vials followed by storage at À °c until use. on the day of testing, ml of stock virus was thawed at ambient temperature and placed into a nebulizer. the tgev concentration in the -ml suspensions used during the experiments ranged from . · to . · tcid /ml ( % tissue culture infecting dose per milliliter) with a geometric mean of . · tcid /ml. the tgev titers utilized in this study were maximized to the best of our ability.. to determine the titers of tgev, serial tenfold dilutions of the nebulizer suspensions, the collection liquid from the samplers, and the filter eluates were prepared in maintenance medium, followed by inoculation in st cells grown in -well plates using four wells per dilution. after days of incubation at °c, inoculated cells were examined microscopically for the appearance of virus-specific cpe, and titers were calculated by the karber method (karber ). to determine the effect of nebulization parameters such as nebulizer type, nebulization time, and incoming air pressure on the virus, the titers of tgev before and after nebulization were measured. two nebulizers, a -jet collison nebulizer (bgi, waltham, mass.) and a plastic medical nebulizer (retec aerosol generator; cavitron corporation, englewood cliffs, n.j.), were utilized in initial tests. in subsequent tests, only the collison nebulizer was used. dry, filtered air was supplied to the nebulizers at pressures ranging from to psi. for tests during which impinger sampling and collection by filters were performed, air pressure to the collison nebulizer was fixed at psi. the liquid aerosolization rates for virus suspensions were between . and . ml/min depending on the type of nebulizer and the pressure. airborne virus concentrations were determined using agis (agi- ; ace glass, vineland, n.j.) and biosamplers (skc, eighty four, pa.) attached to sampling inlets inserted into the test apparatus. the inlets, with an inside diameter of . cm, were positioned about cm upstream from the mounting location of test filters. although the inlets were perpendicular to the flow direction, losses during sampling were minimal because the flow inside the apparatus was slow enough that a representative sample could be drawn (brockmann ) . the flow fig. schematic diagram of the filter test apparatus. hepa, high-efficiency particulate air rate for both samplers was . l/min, and sampling time was limited to min to minimize both possible stress on viruses and evaporation of the maintenance medium used as the collection liquid. no corrections were applied to account for incomplete physical collection of the airborne viruses or for possible inactivation of tgev during the sampling procedure. to examine the recovery of tgev from a filter, we selected an hvac filter medium. these filters camfil farr, rancho dominguez, calif) were made from a microfine fiberglass laminated to a reinforcing backing. according to the manufacturer's specifications, this class of filter is rated merv by the american society of heating, refrigerating and air-conditioning engineers (ashrae) standard . - - (ashrae . merv filters have been measured in laboratory tests to have efficiencies of - % for particles with diameters of . - . lm and % or greater for particles with diameters of . - lm. virus-laden particles with a diameter smaller than . lm would be expected to be collected with at least % efficiency by diffusion. test filter samples were cut from a regular-pleated hvac filter media and placed into a plastic holder that was sandwiched between gasketed flanges using the pneumatically controlled mounting system. the portion of the test filters exposed to the tgev aerosol was a circle (diameter: cm), leading to a face velocity at the filter of cm/s. during each of the separate tests, a filter was exposed to virusladen air for min. immediately after nebulization, the filter was removed and cut into small pieces that were eluted with ml % beef extract- . m glycine solution (ph . ) using a vortex mixer for min. the ph of the eluate was immediately adjusted to ph . with m hydrochloric acid. to assess the effect of nebulization time, the titers of tgev in the nebulizer suspensions after or min were compared with those of the original suspensions. these times were long enough for ml of a suspension to be circulated through the nebulizer many times. therefore, we can assume that almost all of the virus in the nebulizer experienced the stress of nebulization at least once. to compare titers before and after nebulization, the quantity c is defined here as where c a is the virus titer after nebulization and c b is the titer before nebulization. for min of sampling, pairs of virus titers were compared, all using the collison nebulizer. for min of sampling, pairs of virus titers were compared. four pairs were from the collison nebulizer and eight were from the plastic medical nebulizer. geometric means and % confidence intervals for c were calculated from these data. to measure the degree to which the nebulizers generated airborne viruses that could be sampled and remain viable, aerosolization efficiency, g a , was calculated in the same way as adams et al. ( ) : in which n a is the total number of viruses recovered from airborne sampling and n n is the total number of viruses aerosolized from the nebulizer during a test. aerosolization efficiencies smaller than % could be caused by inactivation of the tgev during transport through the air stream, inactivation due to sampling stresses, and incomplete physical sampling of the test virus. the mean and standard deviation of g a was calculated from the three replicates for each combination of rh and sampler. the recovery of tgev from the test filter can be calculated in two ways: ( ) relative to the airborne virus concentration, r a , and ( ) relative to the nebulizer suspension concentration, r n . the terms r a and r n can be calculated according to in which n f is the total number of viruses recovered from filter, v b is the volume of nebulizer suspension before nebulization, and v a is the volume of nebulizer suspension after nebulization. the amount of virus aerosolized from the nebulizer was the product of the volume of the nebulizer suspension consumed and the virus titer of the suspension. each combination of humidity and sampler had three replicates; a mean and a standard deviation for r a were calculated for replicates with non-zero virus titers in the collection liquid of the sampler. the mean and standard deviation for r n was determined for the six replicates at each rh. tseng and li ( ) suggested that r n is a better indicator for virus study than r a . the titers of virus before and after nebulization and values of c calculated from eq. are summarized in table for the separate tests. a c of means that the titer is the same after nebulization as before. the % confidence intervals in the table show that the geometric mean of c was not significantly different from for any of the pressures tested; nebulization did not affect the titers significantly. although the value of c decreased from . at psi to . at psi, changes in c with pressure were not significant (p = . ). the effects of nebulizer type and nebulization duration are confounded with pressure. nonetheless, there were no obvious effects due to these parameters (p = . for nebulizer type; p = . for nebulization time). the data indicate that neither the type of nebulizer, the nebulizer pressure, nor the nebulization time was an important factor affecting survival of tgev during nebulization. ijaz et al. ( ) found similar results after a -min nebulization of human coronaviruses, poliovirus type , rotavirus, and rhinovirus. these findings may be explained by the small size of viruses. because they are small, they have little inertia when moving, and therefore do not experience much physical stress due to acceleration or deceleration and impaction during nebulization. viruses with structures differing from those of tgev may show a different result. as shown in fig. , aerosolization efficiency, calculated using eq. , decreased as a function of rh. sampling with biosamplers led to higher aerosolization efficiency than sampling with agi- samplers. at and % rh, the difference was statistically significant at p = . and p < . , respectively. this suggests that, relative to agi- samplers, biosamplers may have a higher physical sampling efficiency for the tgev-containing particles and/or sample in a way that reduces inactivation of tgev. our finding of higher virus levels measured with the biosampler for the coronavirus tgev is, in general, opposite the results reported by hogan et al. ( ) showing higher airborne concentrations measured using the agi- for ms and t bacteriophages. however, our aerosolization efficiency was similar to those of reoviruses nebulized using a chicago atomizer without prehumidification of secondary air (adams et al. ) . these authors also found that aerosolization efficiencies increased by approximately tenfold following the prehumidifying of the secondary air in the chicago atomizer. two important processes may affect the measurements of aerosolization efficiency in our study: ( ) the viruses may have been damaged during sampling and ( ) some of them may have passed through the sampler without being collected. these processes were disregarded in the calculations, which would lead to underestimation of true aerosolization efficiencies. agranovski et al. ( ) developed and tested a bubbler for sampling airborne microorganisms and utilized it to sample vaccinia virus in laboratory studies with a recovery of about % over h of sampling. the authors were careful to note that vaccinia strains are more robust than many other viruses. when agranovski et al. ( ) utilized the bubbler to sample sars virus experimentally, they recovered more than % of the virus in h of sampling. a comparison of these data to the results from our study with tgev suggests that the bubbler may be a more suitable sampler for coronaviruses than the agi- and the biosampler. the two different recoveries calculated by eqs. and are summarized in table . because some of the measured airborne virus concentrations were zero, corresponding filter recoveries based on airborne virus concentration, r a , could not be calculated. r a estimated using agi- samplers showed higher values than r a from biosamplers. this could be explained if the sampling efficiency for agi- samplers were lower than that of the biosampler some r a showed more than % recovery. loss of survivability or infectivity during sampling could be the reason for these observations. both r a and r n showed large variations. table shows that no virus was found in the three agi- samples at , , or % rh. for the biosampler, virus was observed in only one of three samples at and % rh. these results indicate that the sampling of viable airborne tgev became more difficult as the rh increased and that the biosampler sampled tgev somewhat more effectively than the agi- . virus recovery according to eq. could not be evaluated for all conditions due to the inability of the samplers to yield sufficient viable tgev for quantification. if it were possible, higher titers in the nebulizer suspension might have yielded quantifiable concentrations of tgev in the samplers. however, our methods were optimized to produce titers that were as high as possible for tgev propagated in st cells. the inability to quantify tgev in the samples may be caused primarily by inactivation during travel through the air and during sampling. in addition, the recovery calculated in eq. may also be inaccurate because the size-selective sampling efficiency of agi- samplers and biosamplers is low for particles the size of individual viruses (hogan et al. ) fig. aerosolization efficiency, g a from eq. , as a function of relative humidity (rh) measured using the agi- all-glass impinger (agi) and the biosampler (bio). error bars represent ± sd from the mean for each condition aerobiologia ( ) : - and the size distribution of the tgev-containing particles being sampled is not known. figure shows the results for virus recovery according to eq. as a function of rh. overall recovery was less than %. potential reasons for recoveries less than % include ( ) inactivation during nebulization, ( ) inactivation during transport through the air, ( ) inactivation during collection and retention on the test filter, and ( ) penetration through the test filter. because nebulization losses were shown to be minimal and the filters tested in this study were fairly efficient, most losses are probably due to inactivation during transport or during collection and/or retention on the test filter. the results in table and fig. suggest that eq. is a more useful expression for evaluating tgev recovery from filters than eq. . our findings indicate that the survivability of tgev decreased substantially in the short time between aerosolization and sampling. in addition, the higher recovery of tgev at % rh suggests that this virus may survive longer in an airborne state and after sampling or collection at low rh than at high rh. akers ( ) has proposed that humiditydependent inactivation of aerosolized virus occurs immediately after they are sprayed and, once established, does not drastically changed with the aging of the aerosol. it is generally believed that lipidcontaining viruses, including tgev, survive better at low levels of rh and that the high rh levels are more conducive to the airborne survival of lipid-free viruses . many airborne viruses are affected greatly by the rh of the air. in this study, tgev exhibited a better survival at low rh than at high humidity. although the reasons for this behavior are uncertain, they may be related to tgev's structure. in general, influenza virus survives best at low rh and low temperature (loosli et al. ; hemmes et al. ) . reoviruses show the least airborne viability near % rh (adams et al. ) , while bacteriophage ms virus recovery from filters relative to nebulizer titers, r n from eq. , as a function of relative humidity (rh). error bars represent ± sd from the mean for each condition aerosolized from . m nacl was found to have the least relative recovery near % rh (trouwborst and de jong ) . unlike coronaviruses such as tgev and influenza viruses, neither reoviruses nor ms have an envelope. our results, combined with those just mentioned, suggest that enveloped viruses may be equipped to survive more effectively at low rh than at high rh, whereas non-enveloped viruses may behave differently. trouwborst and de jong ( ) suggested that the inactivation of airborne virus is related to the airwater interface and that this may be an important mechanism for lipid-containing viruses like tgev. many biological materials are hygroscopic and demonstrate hysteresis in their water sorption isotherms. following wet dissemination, the rate of desiccation depends on the prevailing rh. the most portable target molecules of rh stress are outer membrane phospholipids and proteins (cox ). this mechanism may partly explain the sensitivity of tgev to relative humidity. tgev was nebulized, then sampled using agi- impingers and biosamplers, and finally collected on an hvac test filter to measure the effects of nebulization stress and the recovery of viable virus from the filter. titration of tgev in an aqueous suspension before and after the suspension was nebulized indicated that the act of nebulization did not inactivate the virus. not surprisingly, our results indicate that neither the type of nebulizer, the pressure of the air supplied to the nebulizer, nor the duration of nebulization affected the viability of tgev significantly. virus recoveries calculated based on the amount of the tgev-containing suspension nebulized (r n ) showed a dependence on rh, with higher virus recovery at % rh than at higher humidities. the reason for this behavior is uncertain, but the results are consistent with those of previous investigations and suggest that enveloped viruses have a different relationship between virus recovery and humidity than non-enveloped viruses. aerosolization efficiencies were also higher at lower rh, and biosamplers collected airborne tgev viruses at a significantly higher efficiency than agi- samplers at and % rh. all of the results reported here are specific to tgev, and the extent to which these results with tgev apply to other coronaviruses like the sars virus has not yet been tested. however, tgev's behavior is likely to be comparable to that of other coronaviruses due to structural similarities. other viruses, particularly those from other genera, may behave differently. aerosol stability of infectious 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air cleaner assessment of experimental and natural viral aerosols virus survival as a seasonal factor in influenza and poliomyelitis improving performance of hvac systems to reduce exposure to aerosolized infectious agents in buildings: recommendations to reduce risks posed by biological attacks sampling methodologies and dosage assessment techniques for submicrometre and ultrafine virus aerosol particles development of methods to study the survival of airborne viruses % end-point calculation. archiv for experimentelle pathologie und pharmakologie theoretical study of aerosol filtration by fibrous filters long-term sampling of airborne bacteria and fungi into a non-evaporating liquid survival of airborne microorganisms during swirling aerosol collection experimental airborne influenza infection. i. influence of humidity on survival of virus in air resuscitation of microbial bioaerosols improved methods for generation, sampling, and recovery of biological aerosols in filter challenge tests aerosol generation for instrument calibration how contagious are common respiratory tract infections? toward understanding the risk of secondary airborne infection: emission of respirable pathogens viruses biological particle sampling environmental technology verification: test report of control of bioaerosols in hvac systems airborne spread of measles in a suburban elementary school spread of viral infections by aerosols airborne viruses methods of characterization of virus aerosols mechanisms and enzymes involved in sars coronavirus genome expression interaction of some factors in the mechanism of inactivation of bacteriophage ms in aerosols collection efficiencies of aerosol samplers for virus-containing aerosols improved aerosol collection by combined impaction and centrifugal motion acknowledgements this study was supported in part by a grant from the pilot project research training program of the niosh-sponsored midwest center for occupational health and safety at the university of minnesota, by minnesota medical foundation award - - , and by a nontenured faculty grant from m company. key: cord- -sd izamc authors: song, zhenhui; yang, yang; wang, li; wang, kai; ran, ling; xie, yilu; huang, leishi; yang, zhou; yuan, peng; yu, qiuhan title: eif a interacts with the membrane protein of transmissible gastroenteritis coronavirus and plays a role in virus replication date: - - journal: research in veterinary science doi: . /j.rvsc. . . sha: doc_id: cord_uid: sd izamc abstract transmissible gastroenteritis coronavirus (tgev) is enteropathogenic coronavirus that causes diarrhea in pigs, and is associated with high morbidity and mortality in sucking piglets. the tgev membrane (m) protein is a decisive protein for the proliferation of viral proteins, and is associated with virus assembly and budding. to identify the cellular proteins that interact with the tgev m protein, yeast two-hybrid screening was employed, and seven cellular proteins were identified m-binding partners. using the gst pull-down approach and a co-ip assay, the m protein was found to interact with porcine intestinal cells via eukaryotic translation initiation factor -alpha (eif a ), an essential component of the cellular translational machinery. additionally, confocal microscopy revealed that eif a and m were colocalized in the cytoplasm. furthermore, the function of eif a in intestinal cells during tgev infection was examined. a knockdown of eif a by sirna markedly decreased m protein proliferation and tgev replication in target cells. thus demonstrating that eif a plays a significant role in tgev replication. the present study provides mechanistic insight into the interaction between the tgev m protein and intestinal cells which contributes to the understanding of coronavirus replication and may be useful for the development of novel therapeutic strategies for tgev infection. porcine transmissible gastroenteritis (tge) is a highly contagious disease caused by transmissible gastroenteritis virus (tgev), particularly in the colder seasons of winter and early spring (piñeyro et al., ) . the clinical symptoms of tgev include acute diarrhea, vomiting, and dehydration. although all aged swines can be infected with this virus, suckling piglets are the most susceptible and have a mortality rate as high as % (mullan et al., ) . tgev is a positive single stranded rna virus with a full length of . kb genome, which have a typical cap structure at ′ end and a poly (a) tail at the ′ end, and encodes four structural proteins including spike [s] , membrane [m] , nucleocapsid [n] , and envelope [e] , as well as other five non structural proteins (lai and cavanagh, ; eleouet et al., ) . the m and e proteins might be thought to be involved in the formation of virus-like particles (vlps) (baudoux et al., ) . in addition, the gene of m protein is highly conserved and encoded a decisive protein for viral proliferation (zhenhui song et al., ) . as an important structural protein of tgev particles, the m protein is located in the lipid envelop and associates with the golgi complex in the cell, suggesting that the m protein plays a pivotal role in virus assembly and budding. additional evidence indicates that the m protein is an indispensable part for the replication of virus particles in host cells, which may also be used for virus-specific diagnostics and treatment (cologna and hogue, ; de haan et al., ; zou et al., ) . however, there have been few reports regarding the interaction between the tgev m protein and intestinal cells. the identification of the cellular ligands which interact with viral proteins is likely to provide a better understanding of the dynamics of viral replication, virus-mediated cellular modulation, and pathogenic mechanism. in the present study, using yeast two-hybrid screening, seven cellular proteins, including eukaryotic translation initiation factor -alpha (eif a ), were identified to be m-ligands. the eukaryotic translation initiation factor, eif a, is a number of rna helicase, which is required for the binding of mrna to s ribosomal subunits with function to unwind rna secondary structures in the ′-utr of mrna. there are three closely related eif a proteins in, eif a , eif a , and eif a , which not only participate in initiation and translationare but also https://doi.org/ . /j.rvsc. . . received may ; received in revised form december ; accepted december associated with multiple life processes such as embryogenesis (lu et al., ) . although both eif a and eif a display high protein homology and assemble into the eif f complex required for micrornamediated gene regulation, evidence suggests that eif a , but not eif a , plays a key role in this process of formation of the eif f complex (meijer, ) . eif a is localized in the nucleus, where it plays a role as an mrna clamp and is related to nonsense-mediated mrna decay (chan et al., ) . moreover, previous reports have shown that eif a interacts with vp of infectious bursal disease virus (ibd) and inhibits the rna polymerase activity of ibdv to reduce its replication in host cells (tacken et al., ; gao et al., ) . therefore, we aimed to elucidate whether eif a interacts with the m protein of tgev and impacts virus replication. in this study, we demonstrate that eif a in intestinal cells associates with the tgev m protein and further examined the specific role of eif a on tgev replication by knocking down eif a expression using small interfering rna (sirna). porcine intestinal epithelial cells (piecs) were developed at our institute. the cells were isolated from the jejunum of a newborn piglet, grown in dulbecco's modified eagle's medium (dmem)/f- medium (gibco) with % fetal bovine serum (fbs, gibco brl), and maintained in maintenance medium (dmem/f- supplemented with % fbs) in a % co incubator. the tgev chongqing (cq) strain was isolated from sick piglets with symptoms of diarrhea by our lab (zhenhui et al., ) . c-myc antibodies, gst-tag antibodies, and his-tag antibodies were purchased from sangon biotech, china. horseradish peroxidase (hrp)conjugated goat anti-rabbit igg was purchased from proteintech (usa). fitc-conjugated goat anti-rat igg (h + l) was purchased from bbi life sciences (usa), and cy -conjugated goat anti-rabbit igg (h + l) secondary antibody was purchased from boster, china. the beta tubulin rabbit polyclonal antibody (β-tubulin) and eif a rabbit polyclonal antibody were purchased from proteintech (usa). the mab to the tgev m protein was kindly donated by yu bai from wenzhou college of sciences and agriculture. y hgold and y were purchased from clontech, japan. the escherichia coli rosetta strain was provided by prof. ji xiang li. the plasmids applied in the yeast two-hybrid, pfastbac™-m was stored in the laboratory at − °c, and both pgbkt and pgbkt - were purchased from clontech, japan. pmd -t simple was purchased from takara, japan. the tgev m protein was amplified from pfastbac™-m by pcr that added the ecor i and sal i restriction enzyme sites, and was then cloned into a pmd -t simple vector. after verification by pcr, it underwent directional cloning into the bait vector, pgbkt , of the yeast two-hybrid system and was identified by pcr. y hgold yeast were transformed with either pgbkt -m or pgbkt (control). the expression product of y hgold (pgbkt -m) was tested by western blot using the c-myc tag antibody ( : ) as the primary antibody and hrp-conjugated goat anti-rabbit igg ( : ) as the secondary antibody. the yeast cultures were diluted / and / , plated onto sd/−trp plates, and incubated at °c for three to five days. the size and number of yeast colonies treated with pgbkt -m were compared to that of the control to test the bait for toxicity. the yeast cultures were diluted at / and / and μl and spread onto sd/−trp, sd/− trp/x-α-gal, and sd/−trp/x-α-gal/aba (aureobasidin a) to test whether the bait induced autoactivation. a yeast two-hybrid assay was performed according to the instructions of the matchmaker @ gold two-hybrid system (catalog no. ; clontech). briefly, the bait strain y hgold (pgbkt -m) and y (piecs cdna) were mixed and incubated at °c for h until a threelobed structure similar to a "mickey mouse" face was visible at × magnification under a microscope. the culture mixture was diluted at / , / , / , and / , and μl was spread onto sd/−trp/−leu/−ade. all monocolonies that grew were plated out onto higher stringency. sd/−trp/−leu/−ade/-his. finally, all of the white colonies from the second screening that grew on sd/−trp/−leu/−ade/-his/x-α-gal/aba. the positive colonies were selected and inoculated into sd/ −trp/−leu/−ade/-his liquid medium. the positive prey plasmids were rescued and their cdna inserts were sequenced to identify the candidate proteins. eif a appeared at a high frequency among the candidate proteins, which could be connected with virus proliferation and synthesis of viral proteins (jk ndzinu et al., ; gao et al., ) . therefore, the candidate protein, eif a , was selected as a protein of interest for further verification of its interaction with the tgev m protein. the gst-m fusion protein was expressed in e.coli rosetta under induction by mm isopropyl-b-d-thiogalactopyranoside (iptg), then immobilized on glutathione-sepharose b serving as a bait protein at °c for h, followed by the addition of purified his-eif a protein at °c for h. the isolated pull-down proteins were then analyzed by % sds-page analysis and western blot. gst protein expression was used as a control. the co-ip assay was carried out using ip kit kip- (protientech, china). piecs infected with tgev (moi = . ) and control cells were harvested at h and washed three times with cold-pbs. ip lysis buffer containing mm protease inhibitor was added for min on ice. after centrifugation at , ×g for min, the lysate supernatant which contaning - mg of total protein was incubated with rabbit pab to eif a ( μg) overnight at °c. then, μl resuspended protein a/g-agarose was added to this mixture and subjected to rotated incubation at °c for h. after washing five times with washing buffer containing mm protease inhibitor and centrifugation at ×g for s, μl elution buffer was added to the elute immune complex. the isolated bound proteins were analyzed by western blot. the tgev mock-infected piecs lysate was used as a control. piecs were cultured in six-well plates, and the cell monolayer was table primers for qrt-pcr. primer sequence grown to a confluence of %- %. transfection with the sirnas (three sirnas targeting eif a produced by ribo bio, china) was performed with lipofecatmine @ in accordance with the manufacturer's instructions. the three sirnas ( nm) were complexed with lipofecatmine @ by incubating them together at room temperature for min. after removing the cell culture supernatant, the complex was added and incubated for h prior to analysis. piecs were transfected with the best sirna targeting (sirna , ′-ggagactatatgggagcaa- ′) against eif a for h, then infected with tgev (moi = . ). the virus suspensions were collected at h, h, h, and h, serially diluted from − to − , and added to st cells in -well culture plates. each dilution was added to eight wells. the tcid was calculated using the reed and muench method. the mock-treated cells and cells treated with tgev at each time point served as controls. . . immunofluorescence assay piecs were infected with tgevfor h. the cells were washed three times with pbs, and fixed in paraformaldehyde ( %) for min at room temperature and permeabilized with . % triton x- for min. after blotting with % bsa (bovine serum albumin, bbi), the cells were incubated with tgev m protein ( : ) mab and rabbit pab to eif a ( : ) overnight at °c. the cells were then incubated with fitc-labeled goat anti-rat igg ( : ) and cy -labeled goat anti-rabbit igg ( : ). subsequently, the cells were stained with , -diamidino- phenylindole (dapi) for min and examined under a zeiss lsm with airyscan. piecs were transfected with sirna against eif a for h and then inoculated with tgev for h. total rna was extracted using the rnaiso plus (invitrogen, usa) reagent and subjected to reverse transcription with primerscript™ rt master mix (takara, japan) according to the manufacturer's instructions. quantitative pcr analysis was performed to amplify the membrane protein (m) gene and eif a using the cdna as the template and the β-actin gene as the internal standard. data analysis was based on the measurement of the cycle threshold (ct). the relative level of m gene and eif a expression was calculated using the -△△ct method. the primers are presented in table . . . western blotting piecs were transfected with sirna and infected with tgev for h. piecs were washed three times with cold-pbs and lysed in radioimmunoprecipitation assay (ripa, μl/well) buffer (beyotime, china) containing the protease inhibitor, pmsf ( . μl). the protein concentrations of the resulting lysates was determined using a pierce bca (beyotime, china). after centrifugation at , ×g for min, proteins in the supernatant ( μg protein) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) on % gradient gels, and transferred to polyvinylidene fluoride (pvdf) membranes (merck millipore, darmstadt, germany). the membranes were blocked for h in tris-buffered saline (tbs) containing % non-fat dry milk at room temperature, and incubated with the primary antibodies ( : ) at °c overnight. after washing three times with tbst, the membranes were incubated with hrp-conjugated goat anti-rabbit igg (sangon biotech) at °c for h and the proteins were visualized using , ′-diaminobenzidine and detected by enhanced chemiluminescence (ecl; thermo scientific) and autoradiography. all calculations were performed using graphpad prism . . all data are presented as the mean ± sd or with the standard errors of the mean from three independent experiments. a one-way analysis of variance (anova) and t-test were employed to determine the statistical differences between multiple groups. p-values < . were considered statistically significant (*p-value < . ; **p-value < . ;***p-value < . ). the bait vector, pgbkt , of the yeast two-hybrid system was constructed to screen the interactions between the tgev m protein and host proteins, and the recombinant plasmid was successfully transformed into y hgold yeast cells (fig. a) . yeast harboring pgbkt -m and those harboring pgbkt (control) were spread at two dilutions onto sd/trp plates, and the yeast size and number of colonies produced by pgbkt -m were compared with that produced by the control pgbkt . the results suggest that the bait vector displayed no toxicity toward the yeast (fig. b) . the yeast harboring pgbkt -m were spread at two dilutions on three different selective media plates. no colony growth was observed on the sd/−trp/x-α-gal/aba, indicating that there was no self-activation phenomenon to the reporter genes (fig. c) . a western blot showed that pgbkt -m produced an approximately kda fusion protein (fig. d) . these results indicate that the constructed pgbkt -m bait vector could be used for screening host proteins that interact with the tgev m protein using the yeast twohybrid system. tgev m protein-associated cellular proteins were identified using the yeast two-hybrid system. the zygotes typically displayed a threelobed structure similar to a "mickey mouse" face at. × magnification under the microscope ( fig. a) after the bait strain y hgold (pgbkt -m) and y (piecs cdna) were mixed and incubated. after three rounds of screening on different nutritionallydeficient medium plates, a total of blue clones that grew on the sd/ −trp/−leu/−ade/-his/x-α-gal/aba were visible and identified by pcr ( fig. b and c) . finally, a database search with sequence analysis (table ) . these proteins included the multifunctional protein, ade , regulator of g protein signaling, small nuclear ribonucleoprotein smd , p x purinergic receptor , rna polymerase ii transcription subunit ,eukaryotic initiation factor a (eif a ), and centromere-related protein e. since it has been previously reported that the host translation initiation factor, eif a , is involved in the promotion of virus proliferation and viral protein synthesis (ndzinu et al., ; li et al., ) . eif a was used as the candidate protein for further research and validation. next, the interaction between the m protein and candidate protein eif a was verified using a gst pull-down assay and co-ip. the his-eif a fusion protein solution was added to glutathione ™ b agarose beads adsorbed with the gst-m fusion protein at the same concentration. gst protein was used as a control to eliminate non-specific protein binding. as shown in fig. a , sds-page electrophoresis and the western blot results revealed that the eif a protein was present in the gst-m protein immobilized beads but not in the other gels and the western blot indicated that the size of the his-eif a fusion protein was approximately kda (fig. b.) . a co-ip assay was performed to further verify the interaction between eif a and tgev m in vitro.the western blot presented in fig. c shows that the m protein was precipitated by the antibody in the tgev-infected group but not in the mock-treated group. these results confirm the interaction between eif a and the m protein both in vitro and in vivo. indirect immunofluorescence was used to validate where eif a and m were localized in piecs following tgev infection. the results indicated that eif a was distributed in both the cytoplasm and nucleus. the green fluorescence of m protein and the red fluorescence of eif a were observed to be partially overlapping in the cytoplasm, indicating that eif a was co-localized with the m protein within piecs during tgev infection (fig. ) . to further study the role of eif a in tgev replication, the eif a protein of piecs was inhibited using sirna . at h post transfection, the cells were lysed with trizol and the level of eif a mrna expression was detected using rt-qpcr. at h post transfection, the cells were lysed for eif a protein expression by western blot. as shown in fig. a , sirna knocked down protein expression more efficiently than the other interference fragment, and quantitative analysis further confirmed a significant inhibitory effect of sirna against eif a (fig. b) . to investigate the role of eif a on tgev replication, eif a was knocked down in piecs with sirna , which were subsequently infected with tgev at an moi of . . the viral loads in the supernatants treated with sirna were significantly lower compared to those of the negative sirna-and mock-treated groups (p < . and p < . , respectively) (fig. ) . similarly, compared with the tgev group, the level of m protein and mrna expression decreased following the knockdown of eif a in piecs and infected with tgev for h (fig. a and b) . together, these results suggest that eif a interacts with the tgev m protein and impairs tgev replication. the culture supernatants of the cells and the virus-associated cells infected with tgev were collected at different points. the viral titers are presented as the averages from three independent samples. **p < . and ***p < . . tgev is a highly contagious disease in the coronavirus that can cause acute diarrhea in piglets at - weeks of age, and causing severe economic losses (duque and ospina, ) . thus, identification of host proteins that interact with tgev viral proteins is a vital for studying the mechanisms of viral pathogenesis. in this study, using a yeast two-hybrid system, we identified seven cellular proteins that can interact with the tgev m protein. these proteins are primarily related to gene transcription, protein folding, protein degradation and metabolism. of these, eif a was identified as a novel m ligand. however, eif a had not been identified in intestinal cells in previous study, likely due to the different cell types used to construct the cdna library. in vivo, tgev replicates in the intestinal epithelial cells of susceptible animals, inducing cell injury, villi atrophy, as well as reduced villous height and crypt depth (haelterman, ; schwegmann and herrler, ; xia et al., ) . therefore, it is important to identify host proteins in the natural target cells of tgev to study viral replication and pathogenesis. previous studies have found that viral nucleic acids and structural proteins are assembled into viral particles in the region between the endoplasmic reticulum (er)and golgi, and that the particles are subsequently transported into the extracellular by budding of vesicles (corse and machamer, ; kuo and masters, ) . our previous research demonstrated that infectious virions entered the intestinal cells by membrane fusion and mature viruses budded into vacuoles, which were gradually to the cell membrane before being released (song et al., ) . moreover, research into tgev has established that the m protein as a linker may contribute to initiate the viral particle assembly by interacting with genomic rna and nucleoproteins in pre-golgi compartments (risco et al., ) . therefore, this study provides evidence that host cellular proteins interacting with the tgev m protein will be serve to futher study of viral replication and pathogenesis. in the present study, eif a was identified to interact with the tgev m protein using a gst pull-down assay and co-ip. eif a is one of variants (eif a and eif a ) reported in mice and rabbits (nielsen and trachsel, ) and associates with eif a to form a complex (marintchev et al., ) . a previous study showed that eif a acts to limit ibdv replication in infected df cells, which is consistent with our findings that knocking down eif a expression can inhibit the replication of tgev in intestinal cells. furthermore, we identified the mechanism by which eif a reduced tgev replication in intestinal cells to be via the down-regulation of m expression. some studies have found that eif a plays an significant role in the virus infection cycle. for example, eif a reduces ibdv replication by inhibiting rna polymerase activity in host cells (li g et al., ) . similar findings have also been reported that human eif a was found to interact with the rdrp ns b of hepatitis c virus by using a yeast two-hybrid system, which may facilitates the genome rna synthesis of ns b protein (kyono et al., ) . in the present study, our results suggest that eif a may be required for tgev replication. therefore, eif a may potentially serve as new therapeutic target for controlling tgev infection. elucidating the interactions between host cell and viral components is essential for understanding coronavirus pathogenesis. in this study, the tgev m protein was shown to interact with the eukaryotic translation initiation factor, eif a . moreover, the knockdown of eif a expression with small interfering rna (sirna) led to the inhibition of m protein synthesis and decreased viral copy numbers. since such inhibition occurs during the later stages of the virus replication cycle, inhibition of m protein expression, which plays a key role in the morphological processes of coronaviruses, likely affects the assembly of viral particles, there by leading to decreased tgev titers released into the supernatant of piec. therefore, eif a can play a dual role by either forming the eif f complex to activate translation or binding to the ccr -not complex to facilitate mirna-mediated transcription repression (mathys et al., ) . furthermore, one study showed that cellular mirnas can inhibit viral infection and that the virus mutates to escape suppression by cellular mirnas (zheng et al., ) . therefore, since the underlying mechanism by which eif a /tgev affects tgev/ eif a replication is extremely intricate and may involve cellular mirnas, further research is required to confirm this mechanism. in summary, this study is the first to identify proteins involved in the porcine intestinal epithelial interaction with the tgev m protein using a yeast two-hybrid system. a total of seven cellular proteins were identified, which provided novel insight into the mechanism of coronavirus replication. the interaction between the cellular eif a and the tgev m protein was confirmed in tgev-infected piecs. moreover, the knockdown of eif a reduced tgev replication in piecs. therefore, our results may be useful in understanding the mechanisms involved in tgev replication and developing novel strategies for infection control. coronavirus pseudoparticles formed with recombinant m and e proteins induce alpha interferon synthesis by leukocytes eif a is a novel component of the exon junction complex coronavirus nucleocapsid protein rna interactions infectious bronchitis virus e protein is targeted to the golgi complex and directs release of virus-like particles coronavirus particles assembly: primary structure requirements of the 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microrna-mediated gene regulation simulation of the economic impact of transmissible gastroenteritis on commercial pig production in australia eif a is a host factor required for efficient hiv- replication the mouse protein synthesis initiation factor a gene family includes two related functional genes which are differentially expressed first retrospective studies with etiological confirmation of porcine transmissible gastroenteritis virus infection in argentina two types of virus-related particles are found during transmissible gastroenteritis virus morphogenesis sialic acids as receptor determinants for coronaviruses the assembly of virus-like particles of porcine transmissible gastroenteritis virus in vitro by baculovirus expression system morphogenesis and proliferative rule of porcine transmissible gastroenteritis virus in porcine intestinal epithelial cells vp , the rna-dependent rna polymerase and genome-linked protein of infectious bursal disease virus, interacts with the carboxy-terminal domain of translational eukaryotic initiation factor aii impact of tgev infection on the pig small intestine human microrna hsamir- - p suppresses enterovirus replication by targeting the viral genome isolation and identification of a new strain of porcine transmissible gastroenteritis virus from chongqing, southwestern china transmissible gastroenteritis virus: identification of m protein-binding peptide ligands with antiviral and diagnostic potential this work was supported by the graduate scientific research in chongqing, china. (no. cys ). the authors declare that there are no conflicts of interest. key: cord- -mmgb cxo authors: to, l. t.; bernard, s.; bottreau, e. title: transmissible gastroenteritis coronavirus: surface antigens induced by virulent and attenuated strains date: - - journal: research in virology doi: . /s - ( ) - sha: doc_id: cord_uid: mmgb cxo summary three strains of the transmissible gastroenteritis virus (tgev) possessing different degrees of pathogenicity for piglets were examined for their capacity to express m and s glycoproteins on the infected cell surface using a microwell immunoperoxidase test. these two viral glycoproteins were easily detected on the plasma membrane of . % paraformaldehyde-fixed swine testis (st) or pig kidney (rp.d) cells which were infected with high-passaged purdue- and low-passaged d- strains and a high-passaged attenuated ( -sg) mutant of tgev. no significant differences were found between attenuated and virulent strains with regard to the viral antigen expression on the membrane of infected cells over a -h period. transmissible gastroenteritis (tge) is a highly contagious enteric infection of swine caused by a transmissible gastroenteritis coronavirus (tgev) (woode, ) . the causative agent of tge belongs to the coronaviridae, a family of enveloped viruses possessing a single-stranded co-linear rna genome of positive polarity (for review, see sturman and holmes, ) . three major structural proteins have been described for all coronaviruses: a high tool. wt. ( kda) glycoprotein (s) which forms the characteristic peplomers of the "corona", a small ( kda) transmembrane glycoprotein (m) and a phosphorylated protein (n, - kda) associated with rna (garwes and pocock, ; garwes et al., ; horzinek et al., ; laude et al., ) . the peplomer glycoprotein is assumed to be involved in both virus adsorption to the cell and induction of virus-neutralizing antibody (garwes et al., ) . the transmembrane glycoprotein is postulated to play a key role in alpha-interferon induction (charley and laude, ) . tgev infection is followed by a very high mortality rate of up to % in piglets which are less than weeks old (haelterman, ) . sows that are naturally exposed to the virulent tgev produce immune milk, which passively protects newborn pigs (saif and bohl, ; bachman, ) . in contrast, attenuated tgev does not induce complete protection by lactogenic immunity (salf and bohl, ) . since the virulence of tgev has been shown to decrease by serial passages in tissue culture, many authors have tried to differentiate the highpassaged (hp) attenuated strains from the lowpassaged (lp) virulent strain by in vitro markers, such as the level of the thermosensitivity of replication (furuuchi et al., ; hess and bachman, ) , the resistance to digestive enzymes, low ph and temperature (laude et al., ) , and by comparing viral replication and synthesis of structural antigens (nguyen et al., ) . using an hp attenuated mutant of tgev ( -sg strain), which survives in the physicochemical environment of the digestive tract of adult pigs (aynaud et al., ) , to study passive protection against tgev infection in piglets, we found that this new tgev mutant was capable of inducing protective lactogenic immunity and that it could be considered as candidate for an oral tgev vaccine (bernard et al., ; aynaud et al., ) . however, the exact mechanism leading to the induction of protective immunity following oral immunization of sows with this mutant is still unknown. in mouse hepatitis virus (mhv), a well studied coronavirus, the m protein migrates to the golgi apparatus, but is not transported to the plasma membrane as readily as the s protein (sturman and holmes, ) . for porcine tgev, the presence of the virus envelope s antigen on the surface of infected cells was demonstrated by immunofluorescence , while the presence of the m antigen on the plasma membrane has only been suspected by unspecified monoclonal antibodies (mab) (welch and saif, ). there has not been any published report concerning the presence of n antigen on the plasma membrane of infected cells. however, our group and others (laviada et al., ) have recently demonstrated the presence not only of s but also of m viral antigens on the membrane of st cells infected with hp purdue- strain of tgev. the purpose of the present study was to determine whether the lp virulent d- strain and hp attenuated -sg mutant were capable of expressing their m and s glycoproteins on the infected cell membrane in a similar way to the hp purdue- strain. for the sake of comparison, the kinetics of expression of viral antigens on the plasma membrane and in the cytoplasm of st and rp.d cells infected by these strains of tgev was also studied with a view to discovering markers for differentiating hp attenuated strains from lp virulent strains. rp.d is a previously described pig kidney cell line (laude et al., ) . the mcclurkin swine testis (st) cell line was supplied by e.h. bohl (wooster, oh, usa). minimal essential medium (mem) supplemented with ° foetal calf serum, penicillin ( iu/ml) and streptomycin ( ~tg/ml) was used for cell growth. purdue- is an hp tgev strain (bohl et al., ) , d- is a virulent strain which was isolated from an acute case of tge (p. vannier, cneva, laboratory of porcine pathology, ploufragan, france) and passaged times in rp.tg cells (aynaud et al., ) and -sg is an hp attenuated mutant which was previously obtained in our laboratory through serial cycles of survivor selection in gastric juice (aynaud et al, ) . for the experiments with inactivated virus, a viral suspension of each of these strains was exposed to ultraviolet light ( s, mw/cm ) (charley et al., ) . subsequent titration by plaque assay showed that the tgev strains were fully inactivated following this treatment. three mab, anti-m ( / ), anti-s ( / ) (delmas et al., ) and anti-n ( - ), were prepared and used as ascitic fluids following injection of balb/c mice with the antibody-producing hybridomas . confluent monolayers of . x cells/cm in -well, flat-bottomed plastic plates (falcon , becton dickinson) were incubated with a volume of . ml of virus suspension at a multiplicity of infection (m.o.i.) of . after a -min incubation at °c under . co , the inoculum in each well was removed and the cells were washed twice with phosphate-buffered saline (pbs). the monolayers were then overlaid with . ml of mem containing heat-inactivated ( °c, min) normal calf serum and the plate was incubated at °c under . co . the cell culture supernatant was harvested at the indicated time intervals and kept at - c until titration. an ipt which had been previously developed for the detection of surface viral antigens induced by purdue- strain in infected st cells was used. briefly, the infected monolayers harvested at indicated times were washed twice with pbs and the cells fixed with . paraformaldehyde (prolabo-france) at °c for rain. after cell saturation with % skimmed milk in pbs without calcium and magnesium for min at room temperature, the monolayers were overlaid with . ml of each of abovementioned mab at working dilutions for min at oc. the reagents were removed from the plates by rinsing twice with tap-water and twice with pbs containing . tween- (scrva) and were then replaced with . ml/well of an optimal dilution of peroxidase-labelled goat anti-mouse fc serum (icn immunobiologicals, israel). after a further min of incubation at c, the plates were washed as before and the enzymatic reaction was developed by incubation at c for h with , 'azino-bis( -ethyl-benzthiazoline- -sulphonic acid) (abts; boehringer mannheim)/h substrate solution. the supernatant was transferred to another plate containing . ml of sodium dodecyl sulphate (sds) to stop the enzymatic reaction and to permit the reading of the plate. the peroxidase was quantified by measuring the optical density at nm with "titertek multiscan" (flow laboratories, irvine, scotland, uio. each antigen quantity, tested in quadruplicate, was expressed as the difference between the for the detection of virus-induced antigens in cytoplasm, the infected cells were fixed with % acetone at - °c for min and the ipt was applied as for surface antigens. a plaque assay (aynaud et al., ) was used to titrate the infectious virus in the cell culture supernatants sampled. briefly, to -day-old monolayer cultures of st cells were produced by seeding x cells per -mm container ( -well trays). the cultures were inoculated with an appropriate tgev dilution, and ml mem supplemented with calf serum and agarose (indubiose) were added. plaques were counted by neutral red staining following incubation at to °c in . % co for h. for the detection of m and s viral antigens in the culture supernatants, an elisa immunocapture technique (bernard et al., ) was used. briefly, -well microtitre plates (nunc-immunoplates, - ) precoated with anti-m, anti-s and anti-n mab, were incubated for h at °c in carbonate buffer (ph . ). after washing, the plates were blocked overnight at °c with ~/ skimmed milk in pbs. viral antigens were bound onto the precoated plates by incubating wells for h at °c with supernatants from st and rp.d cell cultures infected with either purdue- , d- or -sg strain. the peroxidase-labelled pig igg polyclonal antibodies (bernard and lantier, ) were added for the next h at °c. the enzymatic reactions were developed as mentioned above. tgev which was inactivated by ultraviolet irradiation failed to induce production of viral antigens while the infectious viruses did, as shown by ipt in infected st cells ( fig. la and b) . also, neither infectious virus particles nor structural viral antigens could be detected by plaque assay and elisa immunocapture in the cell culture supernatants sampled at the indicated time intervals. this experiment showed clearly that lp virulent d- strain and hp attenuated -sg mutant were also capable of expressing their glycoproteins on the plasmic membrane of infected st cells, as previously described for hp purdue-ll strain . strain. in contrast, rp.d cells infected with each of these tgev strains showed the same od values for m and s antigens at h p.i. we have recently developed a microwell ipt for detecting and quantifying the expression of viral s and m glycoproteins on the plasma membrane of st cells infected with purdue- strain of tgev . in the present study, this technique was used to demonstrate and compare the expression of surface viral antigens in st and rp.d cells infected with lp virulent d- , hp purdue- strains and hp attenuated -sg mutant. with this approach, we tried to find markers which would enable the differentiation of hp attenuated strains and lp virulent strains with regard to antigen expression on infected cell surface. of the mutant viruses tested, the purdue- is an hp attenuated strain ( passages in st-cell culture). however, undeg our experimental conditions, this strain was weakly virulent for newborn piglets (shirai et al., ) . the -sg is an attenuated mutant previously obtained in our laboratory from the virulent gep-ii strain by serial cycles of survivor selection in gastric juice of adult pigs (aynaud et al., i ) . this mutant survives in the physico-chemical environment of the digestive tract of adult pigs, is nonpathogenic for piglets (aynaud et al., ) and is capable of inducing lactogenic immunity in sows following oral immunization (bernard et al, ; aynaud et al., ) . the original virulent d- strain is a mutant obtained from the virulent gep-ii strain by passages in rp.tg cells (aynaud et al., ) and is pathogenic for newborn piglets (bernard, unpublished data) . unlike the gep-ii strain, the virulent d- strain could be grown in in vitro cell culture. no differences in the capacity to express surface viral glycoproteins ( fig. ) were found between the tgev strains, as the presence of m and s glycoproteins was determined easily in infected st and rp.d cells, while the presence of n antigen was not (data not shown). in contrast, the n (data not shown), m and s ( fig. ) antigens were easily detected by ipt in the cytoplasm of tgev-infected cells which were fixed with ° acetone. for the purpose of comparing the expression of viral antigens on the surface of infected cells, the anti-n mab was used as a marker to ensure that after pfa fixation, the cell membrane would remain intact and only the viral antigens expressed on the plasmic membrane of infected cells would be detected. experiments using inactivated virus have demonstrated that protein synthesis is a prerequisite for antigen expression on the cell membrane. our previous results indicated that the expression of m, s and n antigens appeared in multimodal patterns which peaks at , and h p.i. when st cells were infected with m.o.i. of purdue- virus . using the same m.o.i, of d- strain and -sg mutant, the patterns of expression of viral antigens in infected cells were also multimodal (data not shown). this phenomenon was due to incomplete infection of cell monolayers, which led to multi-cycle multiplication of virus. laude et al. ( ) found that about of st cells expressed s antigen at h p.i. when cells were infected at a m.o.i, of . x - pfu/cell of purdue- virus. in order to have glycoproteins appearing at the cell surface under single-cycle conditions of viral multiplication, a high m.o.i. ( pfu/celi) was chosen to ensure that all cells were infected. it is interesting to note that the levels of expression of surface m and s antigens of the virus strains were not significantly different when cells were infected with high m.o.i. ( pfu/cell) ( fig. ). this observation implies that the capacity to express glycoproteins on the cell membrane was not a marker for differentiating hp and lp tgev strains. for all mutant viruses used, surface virus antigen quantity was significantly lower with the rp.d cells than with the st cells. this could be explained by the influence of cell culture systems on virus replication and synthesis of viral antigens (nguyen et al., i ) . furthermore, the appearance of the m and s antigens on the outer membrane of the cells could depend on an antigen-processing system, as previously described for other viruses (long and jacobson, ; yewdell et al., ) . with all our different combinations of viruses and cells, a lag was seen between the cytoplasmic antigens which had decreased in quantity h p.i., while the surface antigens were still increasing. the decrease in cytoplasmic antigen expression can be explained since h p.i. is the moment at which the virus progeny begin to be released from the cytoplasm of infected cells. concerning the production of infectious viruses and synthesis of structural antigens ( fig. ) the infectious titres of hp purdue- and lp virulent d- (about pfu/ml) were higher than that of hp attenuated -sg mutant (about pfu/ml) whereas the quantities of viral m and s antigens in the culture supernatants and cytoplasm of cells infected with these viruses were similar. this experiment indicated clearly that the -sg mutant was characterized by a high structural antigen content and low infectivity in comparison with the other viruses, since a high quantity of s antigen was detected at time . these observations are consistent with our previous results (nguyen et al., ) of the comparison of viral replication and synthesis of structural antigens of these strains of tgev. the -sg mutant was characterized by low infectivity, delayed and restricted growth associated with low and delayed rna synthesis and a high structural antigen content. in contrast, purdue- and d- strains were characterized by high infectivity and a normal pattern of virus replication rna and structural antigen synthesis. in conclusion, no significant differences in in vitro expression of tge viral antigens on plasma membranes were observed between the virus strains and the cell lines used which could explain the major differences existing between the virulence and the immunogenicity conferred by the different virus strains (saif and bohl ; bernard et al., ; aynaud et al., ) . research on in vivo expression of tgev antigens on the surface of intestinal cells of infected sows, especially in those undergoing oral immunization by hp attenuated -sg mutant, should be carried out in order to answer this question. induction of lactogenic immunity to transmissible gastroenteritis coronavirus using an attenuated mutant able to survive in the physicochemical environment of digestive tract transmissible gastroenteritis (tge) of swine: survivor selection of tge virus mutants in stomach juice of adult pigs comparative aspects of pathogenesis and immunity in animals lactogenic immunity to tge of swine induced by the attenuated nouzilly strain of tge virus : passive protection of piglets and detection of serum and milk classes by elisa detection of transmissible gastroenteritis coronavirus by a sandwich elisa technique a new conjugate for the elisa quantitation of porcine iga antibody response in serum, coiostrnm and milk of swine after infection or vaccination with transmissible gastroenteritis virus myxovirus and coronavirus induces in vitro stimulation of spontaneous cell-mediated cytotoxicity by porcine blood leukocytes induction of alphainterferon by transmissible gastroenteritis virus : role of transmembrane glycoprotein el antigenic structure of transmissible gastroenteritis virus: --ii. domains in the pepiomer glycoprotein ), comparison of properties between virulent and attenuated strains of transmissible gastroenteritis the polypeptide structure of transmissible gastroemeritis virus ), isolation of subviral components from transmissible gastroenteritis virus antigenicity of structural components from transmissible gastroenteritis virus on the pathogenesis of transmissible gastroenteritis of swine antigenic relationship among homologous structural components from porcine, feline and canine coronaviruses in vitro differentiation and ph sensitivity of field and cell-cultureattenuated strains of transmissible gastroenteritis virus antigenic structure of transmissible gastroenteritis virus. --i. properties of monoclonal antibodies directed against virion proteins in vitro properties of low-and high-passaged strains of transmissible gastroenteritis coronavirus of swine expression of swine transmissible gastroenteritis virus envelope antigens on the surface of infected cells: epitopes externally exposed ), pathways of viral antigen processing to ctl etude compar~e de souches de coronavirus de la gastroent~rite transmissible: conditions de la r plication viraie de la synth se des anti-g nes structuraux passive immunity against enteritic viral infection lactogenic immunity to transmissible gastroenteritis (tge) of swine induced by the attenuated nouzilly strain of tge virus: neutralizing antibody classes and protection the molecular biology of coronaviruses fixed-cell immunoperoxidase technique for the study of surface antigens induced by the coronavirus of transmissible gastroentefitis (tgev) monoclonal antibodies to a virulent strain of transmissible gastroenteritis virus : comparison of reactivity with virulent and attenuated virus transmissible gastroenteritis expression of influenza a virus internal antigen on the surface of infected p cells we are grateful to dr jean-marie aynaud for helpful advice.we also wish to acknowledge the fondation marcel m~rieux for awarding a scholarship to the leading author. trois souches du virus de la gastroent rite transmissible (get) poss dant une pathog nicit diff rente pour les porcelets ont ~t examines, /t l'aide d'une technique d'immunop roxidase en microplaque, pour leur capacit d'expression des glycopro-t ines m et s/l la surface des cellules infect es. ces deux glycoprot ines sont facilement d tect es sur la membrane plasmique des cellules st (testicule du pore) et des cellules rp.d (rein de pore) infect es par trois souches diff&entes de virus de la get, et fix es a la paraformald~hyde. aucune difference d'expression des antig~nes viraux sur la membrane des cellules infect es sont observables en fonction des souches virales et des lign es cellulaires utilis~es.mots-cl~s: coronavirus, gastroent&ite transmissible, virulence, antig~nicit~; expression, souches hp et lp, immunoperoxidase, glycoprot ines met s. key: cord- -yxz nil authors: weingartl, h.m.; derbyshire, j.b. title: binding of porcine transmissible gastroenteritis virus by enterocytes from newborn and weaned piglets date: - - journal: vet microbiol doi: . / - ( ) -l sha: doc_id: cord_uid: yxz nil enterocytes were harvested by chelation in a series of seven fractions from the tips of the villi to the crypts of the jejunum of newborn or weaned piglets. binding of the low cell culture passaged miller- strain of transmissible gastroenteritis virus (tgev) to villous enterocytes from newborn piglets was at a high level, similar to that observed to culturedswine test is (st) cells. binding of the virus to cryptal enterocytes from newborn piglets or to villous or cryptal enterocytes from weaned piglets was significantly lower. in a competitive virus binding assay with radiolabelled virus, the binding of tgev to st cells was found to be saturable, while binding to mdbk cells, in which the virus fails to replicate, was at a lower level and was non-saturable. in the same assay, virus binding to the villous enterocytes from the jejunum of a newborn piglet was saturable, while binding to cryptal enterocytes from a newborn piglet, and to villous and cryptal enterocytes from a weaned piglet, was non-saturable. it was concluded that the high susceptibility of newborn piglets to tgev infection, and the tropism of the virus for villous enterocytes, may relate to the presence of specific, saturable binding sites on the plasma membrane of villous enterocytes. ) which infects the mature enterocytes of the small intestine, leading to villous atrophy, diarrhoea, malabsorption and death in newborn piglets (saif and heckert, ) . the virus does not infect the cryptal enterocytes, and there is also evidence that the enterocytes which replace those lost from the villi during the infection are also resistant to the virus (pensaert et al., ) . the tropism of the virus for mature villous enterocytes has not been explained, and a major objective of the present study was to determine whether this might relate to the ability of the virus to bind to these cells. a further feature of the pathogenesis of tge is that the severity of the disease is inversely related to the age of the piglet (moon et al., ) , and it has been postulated that the high sensitivity of the neonate may relate to the slow replacement rate of enterocytes in newborn piglets (moon, ) , to the facilitation of viral replication by deep cytoplasmic tubular invaginations in neonatal enterocytes (wagner et al., ) and by the lack of natural killer cell activity in newborn piglets (cepica and derbyshire, ) . the possibility that there might be a decline in enterocyte virus-binding activity with age has not been investigated. specific attachment sites for tgev have been demonstrated in porcine cell culture (nguyen et al., ) , and a to kda protein was subsequently identified as a receptor molecule on swine testis (st) cells (flakus et al., ) . recently, delmas et al. ( ) identified aminopeptidase n as a major receptor for tgev on both st cells and porcine enterocytes. aminopeptidase n is also a receptor for the human coronavirus e (yeager et al., ) while mouse hepatitis virus attaches to a glycoprotein of the carcinoembryonic antigen family on intestinal brush border and hepatocyte membranes (williams et al., ) . the s glycoprotein of the bovine coronavirus was shown recently to attach to a sialic acid receptor on the surface of red blood cells (schultze et al., ) , although this virus may also attach to the same receptor by means of the haemagglutinin-esterase glycoprotein (vlasak et al., ) , which is lacking in tgev. in this paper we describe the binding of tgev to susceptible porcine and resistant bovine cell cultures, and to enterocytes contained in a series of fractions collected from the tips of the villi to the crypts of the jejunum of neonatal and weaned piglets. as evidence of specific binding to these cells, the saturability of virus binding was determined in a competitive assay with radiolabelled virus. stable swine testis (st) cells (mcclurkin and norman, ) and madin-darby bovine kidney (mdbk) cells (american type culture collection) were cultivated by standard methods. enterocytes were harvested as described by weiser ( ) from the jejunum of eight newborn piglets at or days of age and from four weeks old weaned piglets which were killed with an intravenous overdose of sodium pentobarbitone. an approximately m length of jejunum was rinsed five times with . m nac , mm dithiothreitrol (dtt) and then closed at one end with surgical thread. the intestine was filled with citrate buffer ( . mm dtt, . mm kc , mm nac , mm sodium citrate, mm kh po , . mm na hpo , ph . ), closed with a clip and incubated in hanks' balanced salt solution (hbss) containing . mm dtt for min at °c. the contents of the intestine were then discarded and replaced with magnesium and calcium-free phosphate-buffered saline (pbs) containing . mm ethylenediamine tetraacetic acid (edta) and . mm dtt. the intestine was incubated for min at °c, when the released cells were collected by centrifugation at g for min and washed three times with pbs. the min cycles of chelation with edta were repeated six times to obtain seven fractions of enterocytes. samples of intestine were fixed and sections were stained with haematoxylin and eosin after each chelation cycle to monitor the progressive detachment of enterocytes. alkaline phosphatase activity was determined in each enterocyte fraction as described by weiser ( ) . a volume of . ml of packed cells was incubated at °c for min in . m tris-hc , ph . , . mm znc , mm hgc and . mm p-nitrophenyl phosphate. after incubation, . ml of . n naoh was added to the assay system, and the released p-nitrophenol was determined spectrophotometrically at nm and related to the protein content of the sample which was determined by the bradford ( ) method with the bio-rad protein assay kit ii (bio-rad, richmond, california). periodic acid-schiff staining of the cells was performed as described by gratecos etal. ( ) . the low cell culture passaged miller- strain of tgev (welch and saif, ) , kindly supplied by dr. l.j. saif, ohio agricultural research development center, wooster, ohio) was cultivated in st cells maintained in serum-free medium. plaque assays were also performed on st cells. for the preparation of radiolabelled virus, tgev was cultivated in st cells in the presence of /~ci ml- of s-methionine (amersham, oakville, canada ). the cell culture supernatant was clarified by centrifugation at × g for min at °c, and the labelled virus was pelleted through a % sucrose cushion in tes buffer ( . m tris-hc , mm edta, . m nac , ph . ) supplemented with . % tween , by centrifugation at × g for h at °c. enterocyte fractions i to vii, st cells or mdbk ceils were incubated at a concentration of l cells m - for min at °c with , or pfu ml- tgev. the cells were removed by centrifugation at × g for min, and the supernatants tested for residual activity by plaque assay in st cells. the % virus binding was calculated as pfu virus added -pfu residual virus~ ~ ~-~dadd~ ) × . each sample of ceils was tested in triplicate against each concentration of virus, and the mean % binding was calculated for each sample. the procedure was modified from that described by schlegel et al. ( ) . enterocytes harvested from the tips of the villi (fractions i and ii ) or from the crypts (fraction vii) of the jejunum of one newborn and one weaned piglet, st cells or mdbk cells, at a concentration of cells ml -~ were incubated with pfu m - of tgev, or with eagle's minimum essential medium (emem), for h at °c. to each sample, s-methionine labelled tgev was added, in concentrations ranging from - to pfu/cei , and incubation was continued for min at ° c. the cells in each sample were then pelleted by centrifugation at ×g for min, and the pellets resuspended in /tl of % triton x- (sigma chemical co., st. louis, missouri) in pbs. a portion ( /a) of the suspension was transferred into a scintillation vial with ml of universal scintillation cocktail (icn biomedicals, irvine, california) and the radioactivity measured as counts min-(cpm) in a liquid scintillatio~ counter. the assay was run in triplicate on each sample, and mean values obtained. the cpm obtained from the cells preincubated with emem represented total virus binding, while the counts obtained after preincubation with pfu ml-~ of unlabelled tgev represented non-saturable virus binding. saturable binding was obtained by subtracting the non-saturable binding cpm from the total binding cpm. virus binding to mdbk cells was less than % for each of the three concentrations of tgev, while for st cells the mean ___ sd % virus binding was + , + and +_ for concentrations of tgev of , and pfu ml- respectively. in the competitive virus binding assays, there was no evidence that the binding of tgev to mdbk cells was saturable, while for st cells a typical hyperbolic saturable binding curve was obtained (fig. ) , indicating the presence of saturable binding sites for tgev on these cells. histological examination of sections of intestine after each fraction was harvested revealed that the enterocytes were released progressively from the tips of the villi (fraction i) to the crypts (fraction vii) as chelation with edta proceeded. representative sections are shown in fig. remained in the jejunum of the newborn piglets after the harvesting of fraction vii, while in the weaned piglets, small numbers of cells remained deep in the crypts after the removal of this fraction. these could be released by chelation for a further min. the highest levels of alkaline phosphatase activity (mean ___ sd mu mg-l protein) were found in the enterocytes contained in fraction i from four piglets killed at days of age. the activity in the same fraction from the weaned piglets was + mu mg-~ protein. the remaining fractions, from both newborn and weaned piglets had lower levels of activity than fraction i, ranging from + mu to + mu in the newborn piglets and from ___ mu to _+ mu mg- protein in the weaned piglets, and there were no consistent differences in activity among fractions ii to vii. the periodic acid-schiff stained preparations of enterocytes showed a progressive reduction in staining intensity from fractions i to vii in both the newborn and weaned piglets. in the virus binding assays, similar results were obtained for each of the three concentrations of virus which were used, and the findings when pfu ml- of tgev were added to the enterocyte fractions are shown in fig. . high levels of virus binding were obtained with the enterocytes in fractions i and ii, derived from the tips of the villi of newborn piglets, with relatively low binding to enterocytes in the remaining fractions from the newborn piglets, and in all the enterocyte fractions from the weaned piglets. when analysed by the student's t-test, binding was significantly higher (p < . ) to enterocytes in fractions i and ii from the newborn piglets than in all other enterocyte fractions, among which significant differences in virus binding did not occur. in the competitive virus binding assays, st and mdbk cells were included ¢ . saturable binding of radiolabelled tgev to enterocytes harvested from the tips of the villi (•) or the crypts (•) of the jejunum from a newborn piglet. increasing amounts of labelled tgev were incubated with enterocytes and virus binding was determined as cpm for cells preincubated in emem (total binding) or in unlabelled virus (non-saturable binding). saturable binding was calculated by subtraction of non-saturable binding cpm from total binding cpm. in each assay as positive and negative controls for saturable virus binding, and the results obtained confirmed the findings reported above for these cells. of the enterocyte fractions harvested from the tips of the villi or the crypts of a newborn or a weaned piglet, and tested in the competitive virus binding assay, only the enterocytes harvested from the villi of the newborn piglet showed saturable binding of tgev (fig. ) , similar to that demonstrated for st cells. binding of tgev to the other three fractions, like the binding of the virus to mdbk cells, was non-saturable. binding of tgev to enterocytes from the crypts of the newborn piglet is shown in fig. , and similar results were obtained with enterocytes from the villi and crypts of the weaned piglet. studies on the attachment of tgev to st cells were first described by nguyen et al. ( ) , who concluded that the attachment was to specific sites on the cell surface since incubation of the cells with one strain of tgev inhibited the attachment of another strain of the virus. the present findings on the saturability of virus binding to st cells support this conclusion. binding of tgev to mdbk cells, in which the virus fails to replicate, was at a much lower level than to st cells, and since the binding to mdbk cells was nonsaturable, it is concluded that these cells lack specific receptors for tgev. the technique of release of enterocytes, by chelation, from the tips of the villi to the jejunal crypts, developed initially for use in rats (weiser, ) was successfully applied to piglets. histological examination of the intestine after each cycle of chelation confirmed the progressive release of enterocytes in each fraction. a high level of alkaline phosphatase activity was found only in the enterocytes harvested from the tips of the villi. this contrasted with findings in the rat (weiser, ) in which there was a progressive decline in enzyme activity from the villi to the crypts. however, in the present study there was a progressive decline in the intensity of periodic acid-schiff staining, from the villi to the crypts, as described in the rat (gratecos et al., ) , corresponding to the glycoprotein content of the plasma membrane, which increases with progressive differentiation of the enterocytes. binding of tgev to the villous enterocytes of newborn piglets occurred at a level comparable to that demonstrated for st cells, and significantly higher than virus binding to all other enterocyte fractions from both newborn and weaned piglets. the competitive binding assays indicated that saturable binding occurred only to the villous enterocytes of newborn piglets. since saturable binding is accepted as evidence of binding to specific receptors on the cell surface (tardieu et al., ) this finding would suggest that specific tgev receptors on enterocytes are restricted to neonatal villous enterocytes. this could be an important factor contributing to the susceptibility of villous, rather than cryptal enterocytes, to infection with tgev. the apparent lack of specific virus binding to villous enterocytes from weaned piglets was a somewhat puzzling finding since the virus is capable of infecting these cells in older piglets, although the latter are much less susceptible to infection than newborn piglets. our use of a cell culture adapted strain of tgev which is somewhat less virulent than some field strains of the virus may have contributed to this result, but it may be that specific receptors are far fewer in older piglets, and below the level of detection by the methods used in the present study. a low level of specific binding may have been masked by non-specific binding of the virus to the cell surface. it is also possible that the non-specific binding of tgev to villous enterocytes may lead to productive infection, but much less efficiently than binding to specific receptors. it has been shown that the dose of virus needed to infect an adult pig is times greater than that required to infect a neonate (witte and walther, ) and this may reflect the sparseness of receptors on the villous enterocytes of older pigs. it is concluded that the high susceptibility of newborn piglets to tgev, and the tropism of the virus, may be related to the presence of specific, saturable binding sites on the plasma membrane of the villous enterocytes in the neonate. shepherd et al. ( ) observed tge viral particles traversing the intact microvilli of mature enterocytes in the small intestine of an experimentally infected piglet, and postulated the synthesis of brush border receptors as the enterocytes differentiated during their migration from the crypts to the tips of the villi. the present findings provide the first evidence in support of such a hypothesis. the relationship of the aminopeptidase n receptor for tgev, demonstrated recently by delmas et al. ( ) , to our findings, remains to be explored by determining the distribution of this enzyme in different enterocyte fractions from newborn and weaned piglets. transmissible gastroenteritis virus (classical enteric variant) and transmissible gastroenteritis virus (respiratory variant) a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding antibody-dependent and spontaneous cell-mediated cytotoxicity against transmissible gastroenteritis virus infected ceils by lymphocytes from sows, fetuses and neonatal pigs aminopeptidase n is a major receptor for the enteropathogenic coronavirus tgev studies of transmissible gastroenteritis virus receptors on swine testicle cells plasma membranes from rat intestinal epithelial cells at different stages of maturation i. preparation and characterization of plasma membrane subfractions originating from crypt cells and from villous cells studies on transmissible gastroenteritis of swine ii. selected characteristics of a cytopathogenic virus common to five isolates of transmissible gastroenteritis epithelial cell migration in the alimentary mucosa of the suckling pig age dependent resistance to tge of swine. . clinical signs and some mucosal dimensions in the small intestine transmissible gastroenteritis (tge) of swine: in vitro virus attachment and effects of polyanions and polycations transmissible gastroenteritis of swine: virus-intestinal cell interactions i. immunofluorescence, histopathology and virus production in the small intestine through the course of infection enteropathogenic coronaviruses saturable binding sites for vesicular stomatitis virus on the suface of vero cells the s protein of bovine coronavirus is a hemagglutinin recognising - -acetylated sialic acid as a receptor determinant the mucosal lesion in viral enteritis. extent and dynamics of the epithelial response to virus invasion in transmissible gastroenteritis of piglets interaction of viruses with cell surface receptors human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza c viruses electron microscopy of intestinal epithelial cells of piglets infected with a transmissible gastroenteritis virus intestinal epithelial cell surface membrane glycoprotein synthesis i. an indicator of cell differentiation monoclonal antibodies to a virulent strain of transmissible gastroenteritis virus: comparison of reactivity with virulent and attenuated strains receptor for mouse hepatitis virus is a member of/he carcinoembryonic antigen family ofglycoproteins age-dependent susceptibility of pigs to infection with the virus of transmissible gastroenteritis human aminopeptidase n is a receptor for human coronavirus e this research was supported by the natural sciences and engineering research council of canada, and by the ontario ministry of agriculture and food. technical assistance was provided by marta dubisz, tricia horner and l~va varady. key: cord- -jjft den authors: rodák, l.; Šmíd, b.; nevoránková, z.; valíček, l.; smítalová, r. title: use of monoclonal antibodies in blocking elisa detection of transmissible gastroenteritis virus in faeces of piglets date: - - journal: j vet med b infect dis vet public health doi: . /j. - . . .x sha: doc_id: cord_uid: jjft den monoclonal antibodies (mab) to the transmissible gastroenteritis virus (tgev) nucleoprotein (n) and membrane protein (m) were prepared and used for the comparative assessment of three blocking elisa variants to detect tgev. the competitive blocking elisa format showed the highest sensitivity, allowing detection of ( ) tcid( ) tgev/ml in culture medium. ninety‐nine porcine field faecal samples obtained from herds affected with diarrhoea were examined, and various tgev levels were found in nine samples from six herds. however, only in three samples were significant tgev concentrations demonstrated. the relationship between incidence of tgev gastroenteritis and the spread of porcine respiratory coronavirus infection in pig farms is discussed. rotaviruses and coronaviruses rank, along with bacterial infections, among the most significant causal agents of gastroenteritis in pigs. the two coronaviruses causing transmissible gastroenteritis (tge) and porcine epidemic diarrhoea (ped) are morphologically identical, but they are antigenically different. tge gastroenteritis, occurring mostly in the enzootic form, is not such a problem in europe as it was in the past (sˇteˇpa´nek et al., ; pritchard et al., ) . although different results on the prevalence of tgev infection were given (kim and cho, ; chae et al., ) , this infection still may be topical in some countries. decreased incidence of tge gastroenteritis is largely influenced by the spread of porcine respiratory coronavirus (prcv) infection in pig herds (bernard et al., ; sˇesta´k et al., ) . high level of nucleotide sequence homology ( %) of the two viruses was proven by genome analysis (britton et al., ) with major deletions in genome coding s protein of prcv. therefore, it is presumed that prcv is a spike (s) gene deletion mutant of tgev, and minor alterations of the viral genome led to distinct viral tropism (rasschaert et al., ; sˇesta´k et al., ; ballesteros et al., ; constantini et al., ) . three major structural proteins occur in coronaviruses: the glycoprotein s (m r - k) found in the viral protrusions (corona), the nucleocapsid phosphoprotein n (m r - k) and the membrane protein m (m r - k) (saif, ; utiger et al., ) . while viral antigens n and m of tgev and prcv are identical, differences were demonstrated in glycoprotein s epitopes, which do not trigger production of neutralizing antibodies. monoclonal antibodies (mab) to those epitopes are currently used to distinguish between tgev and prcv antibodies (simkins et al., ) . however the situation is different when detection of the virus in faecal samples is considered. although the presence of prcv in faecal samples was demonstrated by extremely sensitive rt-pcr technique (constantini et al., ) , little if any intestinal multiplication of prcv was proven (cox et al., a,b,c) . it follows that the method of lower sensitivity based on the use of antibodies to both tgev and prcv antigens could be suitable for specific demonstration of tgev in faeces. therefore the objective of our study was to check such mab in modifications of blocking elisa method, and to implement the optimal one for tgev demonstration. the strain of tgev, capm v- (collection of animal pathogenic microorganisms, brno, czech republic) was propagated in the porcine kidney cell line (pk- ) in eagle-mem medium. after development of cytopathic effect and centrifugation ( g for min) of the medium, the pellet was resuspended in / of the original medium volume in phosphate-buffered saline (pbs) as crude viral antigen (v-ag). crude control antigen (c-ag) was prepared similarly from uninfected cells. purified v-ag was prepared from the supernatant of the infectious medium by ultracentrifugation on cushions of and % sucrose according to hofmann and wyler ( ) . antigens were kept at ) °c before use. culture media with known tissue culture infectious dose (tcid /ml) were used for the determination of blocking elisa method sensitivity. to exclude possible crossreactions with other agents, crude v-ag (tge, ped and rota a) were tested by all elisa methods used. the reactivities of mab with five tgev strains (v- , shizuoka, purdue, and two of our field isolates cz- and cz- ) were also examined. two hysterectomy derived, colostrum-deprived -day-old piglets were kept in sterile conditions, and orally infected with · . tcid tgev. diarrhoea appeared hpi in both piglets; samples of faeces were collected during seven consecutive days. seven weeks post-infection piglets were challenged with · . tcid tgev and killed days later by exsanguination under total anaesthesia. the titre of tgev antibodies in serum obtained (swspos.) was determined by indirect elisa ( ). tgev-negative swine blood serum (swsneg.) was obtained from -day-old uninfected piglet. the immunoglobulin fraction (swatgev) prepared from positive serum was used as a binding antibody in the blocking elisa methods. rota-and coronaviruses were detected by electron microscopic examination of faecal samples and culture media after negative staining with % ammonium molybdate solution in water, ph . (sˇmı´d et al., ) . monolayers of infected (tgev, pedv, rotavirus a) and uninfected cells were fixed with acetone min at °c. after inhibition of endogenous peroxidase (li et al., ) , the tgev was detected by mab by direct and indirect immunoperoxidase (ip) tests. the reactions were read after - min incubation in a substrate solution containing chromogen aec ( -amino- -ethylcarbazole; sigma, st louis, mo, usa). low molecular weight standard (lmw; pharmacia, uppsala, sweden) and purified tge v-ag were separated by discontinuous electrophoresis (laemmli, ) in % polyacrylamide gel (miniprotean cell; bio-rad labs, hercules, ca, usa). after transfer to a nitrocellulose (nc) membrane ( . lm; bio-rad) and separation of the nc lanes, the lane containing lmw was stained with colloid gold (moeremans et al., ) , and lanes with v-ag were used for western blot (wb) analysis. after incubation ( h/ °c) with the tested sera or mab incubations with peroxidase conjugates and subsequently in a chromogen solution dab ( , ¢-diaminobenzidine; sigma) followed. hybridomas producing mab to tgev (mabtgev) were prepared according to galfre`and milstein ( ) by fusion of splenic lymphoid cells of immunized mice of the line balb/c with cells of the myeloma line sp / . hybridomas producing mab with optimal results in indirect elisa were checked by wb and ip tests and selected mab were used for preparation of ascitic fluids. after purification by ion exchange chromatography, mabtgev obtained were stored at ) °c in % glycerol or used for the preparation of peroxidase conjugates. rabbit antibodies to swine and mouse immunoglobulins (raswig, ramoig) or swine antibodies to mouse immunoglobulins (swamoig) were purified from hyperimmune sera by affinity chromatography. these antibodies and mab were conjugated with horseradish peroxidase (hrpo, type vi-a; sigma) using the periodate method (boorsma and streefkerk, ) . stock conjugate solutions adjusted to mg of immunoglobulin/ml were further diluted for ip, wb and elisa tests · to · in pbs containing . % tween and . % lactalbuminhydrolysate (pbst-lah). indirect elisa tgev antibodies in tested sera, in culture media of hybridomas and in purified mabtgev preparations were assayed using the indirect elisa method. pairs of wells of microtitre plates (nunc-immunoplate; polysorp, roskilde, denmark), alternatingly precoated with tge crude v-ag and c-ag were incubated with diluted tested samples. after second incubation with conjugates (always h/ °c), the reactions were visualized in a substrate solution with chromogen tmb ( , ¢, , ¢tetramethyl-benzidine; sigma). after min, the reaction was stopped by addition of m h so , and absorbances were measured spectrophotometrically at nm. control wells filled with the diluent (blank) at the first incubation, or with control negative and positive serum were included in each examination. samples showing a difference in optical density of at least . (after subtraction of absorbances of blank wells) were classified as positive. three variants of elisa method with specificity checked by blocking test were compared by box titration using faeces of experimentally infected piglets hpi. two double antibody sandwich elisa variants (das-elisa) and competitive blocking elisa (cb-elisa) method were compared. microtitre plate wells (nunc-immuno plate; maxisorp, roskilde, denmark) precoated with binding antibodies was used. pairs of wells filled with ll of mixtures of faeces and swsneg. or swspos. fivefold diluted : to : or : to : , respectively, were examined. the second incubation was performed using the detection antibodies (conjugate). pbst-lah containing . m nacl and mg edta.na /ml for samples dilution at the first incubation and rinsing of wells four times with pbst between incubations at °c were used. pairs of wells filled with the diluent (blank) or the mixtures of crude v-ag (tgev, pedv, rotavirus a) and swsneg./pos. during the first incubation were included in each analysis. the following variants of the blocking elisa method were investigated. binding antibody mabtgev was used. the first incubation with antigen test samples was performed for h, the second incubation with the conjugate hrpo-mab-tgev for h. binding antibody swatgev was used. after h of incubation with the antigen test samples, the wells were supplemented with ll diluent containing . lg mabtgev/ml, and the incubation continued for another hour. the second h incubation was done using the conjugate hrpo-swamoig. binding antibody swatgev was used. the first incubation with the antigen test samples was performed for h and the second h incubation was conducted with the conjugate hrpo-mabtgev. the samples were regarded as positive if the net absorbance (na), i.e. the difference of average absorbances in the wells l. rodák et al. incubated with swsneg./pos. was > . , and the reactions were blocked by > % in the wells with swspos. blocking percentages were determined using the formula: %b ¼ ) [(a swspos. · ):(a swsneg. )]. antibodies to prcv and tgev were assessed and differentiated in blood serum samples of sows from randomly selected five herds. commercial kit (ingezim corona diferencial; ingenasa, madrid, spain) was used for examination. sample preparation and evaluation of the results was carried out according to the manufacturer's instructions. in total, faecal samples from piglets with diarrhoea on farms were examined. most samples were from piglets younger than days. after delivery, they were diluted in two to three volumes of earle's medium, centrifuged ( min, g), and the supernatants examined by electron microscopy and cb-elisa method. samples were kept at ) °c before elisa analysis. after verification of the specificity of hybridomas producing mabtgev by wb, elisa and ip tests, four preparations (d /g ; d /g ; b /f and b /f ) were selected for further use. all were of the isotype igg and reacted with the membrane protein m (d /g ) and nucleoprotein n (b /f ) in wb analysis. weak reactivity of mab d /g with epitopes present on protein m and nucleoprotein n were detected (fig. ) . conformational changes of viral antigens resulted in markedly decreased or even absent reactiveness of mab b /f in wb analysis. all mab used reacted specifically with native tgev strains in ip (fig. ) and elisa tests according to results given in table . indirect elisa titres of individual mabtgev solutions containing mg ig/ml ranged between · and · . cross reactivity of mab with other viral antigens (rotavirus a and pedv) could not be detected by any of methods used. the reactivity of individual mab with five tgev strains was checked by cb-elisa examination of infectious culture media. according to obtained positive na and %b values, the mab d /g and b /f react with all tgev strains, while mab d /g and b /f react with strains v- and cz- only. by the use of mab d /g alone or mixture of mab in cb-elisa examination of various tgev strains (table ) or positive field faecal samples (results not shown) the same results were obtained. only a little lower absorbance values with d /g were detected. therefore the mixture of equal concentrations (w/v) of all mab was selected for routine cb-elisa examination of field samples. sensitivity of the three blocking elisa variants was checked by box titration of mixtures of faces of an experimentally infected piglet hpi and swsneg./swspos. competitive blocking elisa variant (cb-elisa) was selected as optimal (table ). working dilutions of faecal samples : , swsneg./ pos. : , mabtgev mixture containing mgig/ml : and hrpo-swamoig : were used for routine cb-elisa examinations. high tgev concentration in the tested sample is connected with high absorbance values obtained, and the other way round. it follows also, that evaluation of samples with different tgev concentrations is influenced significantly both by sample and swsneg./pos. dilutions. in table results with swsneg./pos. diluted · and · only are given. the culture medium from pk- infected cells containing . tcid tgev/ml was analysed by box titration. mixtures of medium diluted : to : and porcine sera diluted : to : respectively, were examined. positive na and %b values were obtained at all dilutions of both medium and sws (results not shown). the sensitivity of the cb-elisa method was therefore estimated to exceed tcid tgev/ml. cb-elisa method specificity was confirmed by examination of crude v-ag (tgev, pedv, rotavirus a). positive results were obtained with tge v-ag only with average absorbances in the wells incubated with swsneg./pos. . / . (na ¼ . , %b ¼ . ). in the wells with crude pedv and rotavirus a the absorbances were ) . / . and . / . respectively. by examination of blood serum samples of sows from five herds, tgev antibodies were detected only in a single sample. prcv antibodies were detected in ( %) samples in all herds. percentage of prcv-positive animals in respective herds ranged between and % (results not shown). the tgev was detected by cb-elisa in all faecal samples, taken between and days after experimental infection of piglets. the propagation of tgev in the intestine thus exceeds dpi. according to absorbance values obtained, highest tgev concentrations were detected between and dpi (table ) . altogether field faecal samples from herds were examined by cb-elisa. various tgev concentrations were found in nine samples from six herds. however, high tgev concentrations confirmed by absorbances . - . (na ¼ . - . , %b ¼ . - . ) were detected in three samples only. the maximum absorbances in the remaining faecal samples indicating low tgev concentration reached . . the results obtained by em and cb-elisa were poorly correlated reaching the values of about %. similarly, as in our previous study concerning pevd detection (roda´k et al., ) corona-like particles without typical structures (corona) were frequently detected. gastroenteritis caused by tgev is less important at present in comparison with the s and s when mortality of piglets reached - % in affected herds (sˇteˇpa´nek et al., ) . in addition to possible decreased tgev virulence, it is explained by the spread of prcv infection in pig herds and by protection of piglets by crossreacting maternal antibodies (saif et al., ; lanza et al., ) . for diagnosis of gastroenteritis caused by coronaviruses elisa methods are used in addition to conventional virological procedures and molecular virology methods (paton et al., ; pritchard et al., ; kim et al., ) . the Ôdas-elisaÕ with specificity checked by blocking test has been used for both tgev and pedv demonstration in faeces (van nieuwstadt et al., ; carvajal et al., ) . therefore, the detection of tgev in faecal samples by blocking elisa method was the objective of our study. we supposed that mab against n and m antigens of various tgev strains could be useful for this purpose in spite of their crossreactivity with prcv. faecal shedding of prcv after infection was demonstrated by nested-rt-pcr (constantini et al., ) . however, the demonstration of ingested virus cannot be excluded. little if any enteric multiplication of prcv was demonstrated by immunofluorescence after experimental infection of -week-old piglets only, and it remained limited to a few unidentified cells located in or underneath the epithelial layer of the villi and/or crypts (cox et al., a,b,c) . the rt-pcr is extremely sensitive allowing rna detection of only occasional viral particles in the sample. although the sensitivity of tgev detection by cb-elisa in faecal samples is sufficient ( tcid tgev/ml), we suppose that the possibility to demonstrate traces of prcv is relatively low. nevertheless, this assumption will be further checked by application of rt-pcr in following experiments. the sensitivities of tgev detection by das-elisa and cb-elisa were compared and higher sensitivity and specificity of the last method was proven. while in das-elisa conjugated mab are used, cb-elisa method is based on the use of unconjugated mab. the results obtained also indicate (tables and ). this was confirmed by examination of field faecal samples; nine of them were tgev positive. however, high tgev concentrations, suggesting that the virus is an important causative agent of gastroenteritis, were detected in three samples only. moreover, in all tgevpositive samples, the presence of rotavirus a and/or pedv was demonstrated, indicating the importance of mixed enteric viral infections (results not shown). the demonstration of tgev antibodies in only one of randomly selected field sow serum samples indicates that the animals produce antibodies if the tgev antigenic stimulation is high enough to compete with the stimulation by other enteric pathogens. the results also correlate with the assumption that the immunity of sows to prcv leads to decreased occurrence of tgev gastroenteritis (bernard et al., ; lanza et al., ; sˇesta´k et al., ) . effectivity of virus detection in faecal samples may be affected by the time of sample collection after the first symptoms of diarrhoea had emerged and the conditions of their shipment. marked destruction of coronaviruses occurs in the gastric and intestinal contents (aynaud and bottreau, ) . it follows, that characteristic structures of coronaviruses are less frequent by em examination and cellular substructures may imitate Ôcorona-likeÕ particles. this can explain low correlation between em and cb-elisa results. the results described, confirm suitability and sufficient sensitivity of the cb-elisa for routine demonstration of tgev in field faecal samples. cb-elisa methods were also successfully applied in detection of group a rotavirus and pedv in faeces (roda´k et al., (roda´k et al., , . in spite of higher sensitivity of rt-pcr technique, the advantages of cb-elisa examinations are lower costs, the possibility to demonstrate simultaneously three most important causal agents of viral gastroenteritis by uniform method and to examine sufficient number ( - ) of samples. this can give information both on their incidence in pig herds and on the effectiveness of immunoprophylactic measures. transmissible gastroenteritis of swine: stability of coronavirus in gastric and intestinal contents two amino acid changes at the n-terminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism natural infection with the porcine respiratory coronavirus induces protective lactogenic 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pigs with or without maternal antibodies preparation of monoclonal antibodies: strategies and procedures enzyme-linked immunosorbent assay for the detection of porcine epidemic diarrhea coronavirus antibodies in swine sera transmissible gastroenteritis of pigs in southeastern korea in - : prevalence and factors associated with the infection differential detection of transmissible gastroenteritis virus and porcine epidemic diarrhea virus by duplex rt-pcr cleavage of structural proteins during the assembly of the head of bacteriophage t lactogenic immunity and milk antibody isotypes to transmissible gastroenteritis virus in sows exposed to porcine respiratory coronavirus during pregnancy use of azide and hydrogen peroxide as an inhibitor for endogeneous peroxidase in the immunoperoxidase method sensitive colloidal (gold or silver) staining of protein blots on nitrocellulose membranes comparison of two methods for detection of transmissible gastroenteritis virus in feces of pigs with experimentally induced infection detection of transmissible gastroenteritis virus by rt-pcr and differentiation from porcine respiratory coronavirus transmissible gastroenteritis and porcine epidemic diarrhoea in britain porcine respiratory coronavirus differs from transmissible gastroenteritis virus by a few genomic deletions verification of sensitivity and specificity of group a rotavirus detection in piglets faeces with monoclonal blocking elisa methods an elisa optimized for porcine epidemic diarrhoea virus detection in faeces coronavirus immunogens immunity to transmissible gastroenteritis virus and porcine respiratory coronavirus infections in swine contribution of passive immunity to porcine respiratory coronavirus to protection against transmissible gastroenteritis virus challenge exposure in suckling pigs competition elisa, using monoclonal antibodies to the transmissible gastroenteritis virus (tgev) s protein, for serologic differentiation of pigs infected with tgev or porcine respiratory coronavirus electron microscopic demonstration of porcine epidemic diarrhea virus in the czech republic physicochemical and biological properties of strains isolated in czechoslovakia identification of proteins specified by porcine epidemic diarrhoea virus this work was supported by projects qf and mze of the ministry of agriculture of the czech republic. the authors wish to thank mrs farnı´kova´, mrs lickova´, miss hoydenova´and miss bacˇinska´for their skilful technical assistance. key: cord- -ohs z m authors: ballesteros, m. l.; sánchez, c. m.; enjuanes, l. title: two amino acid changes at the n-terminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism date: - - journal: virology doi: . /viro. . sha: doc_id: cord_uid: ohs z m abstract to study the molecular basis of tgev tropism, a collection of recombinants between the pur -mad strain of transmissible gastroenteritis coronavirus (tgev) infecting the enteric and respiratory tracts and the ptv strain, which only infects the respiratory tract, was generated. the recombinant isolation frequency was about − recombinants per nucleotide and was . -fold higher at the ′-end of the s gene than in other areas of the genome. thirty recombinants were plaque purified and characterized phenotypically and genetically. all recombinant viruses had a single crossover and had inherited the ′- and ′-halves of their genome from the enteric and respiratory parents, respectively. recombinant viruses were classified into three groups, named to , according to the location of the crossover. group recombinants had the crossover in the s gene, while in groups and the crossovers were located in orf b and orf a, respectively. the tropism of the recombinants was studied. recombinants of group had enteric and respiratory tropism, while group recombinants infected the respiratory, but not the enteric, tract. viruses of both groups differed by two nucleotide changes at positions and . both changes may be in principle responsible for the loss of enteric tropism but only the change in nucleotide was specifically found in the respiratory isolates and most likely this single nucleotide change, which leads to a substitution in amino acid of the s protein, was responsible for the loss of enteric tropism in the closely related pur- isolates. the available data indicate that in order to infect enteric tract cells with tgev, two different domains of the s protein, mapping between amino acids and and around amino acid , respectively, are involved. the first domain binds to porcine aminopeptidase n, the cellular receptor for tgev. in the other domain maps a second factor of undefined nature but which may be the binding site for a coreceptor essential for the enteric tropism of tgev. to amino acids to of the spike protein were able to efficiently recognize the papn. transmissible gastroenteritis virus (tgev) is a mem-since the s protein is responsible for the virus binding ber of the coronaviridae family (cavanagh et al., , to the cell, it is expected that this protein would play an enjuanes and van der zeijst, ; siddell, ) . tgev essential role in the control of the dominant tropism of replicates in both the villus epithelial cells of the small tgev. accordingly, there are data suggesting a correlaintestine and in lung cells of newborn piglets, resulting tion between tropism and s protein structure. porcine in a mortality of nearly % (saif and wesley, ) . respiratory coronaviruses (prcvs) have been originated frequently these tgev strains are referred to as enteric, independently in europe (callebaut et al., ; pensaert as opposed to the respiratory strains which do not infect et al., ; sá nchez et al., ) and in north america the enteric tract. coronaviruses attach to host cells (vaughn et al., ; wesley et al., wesley et al., , b ) from through the spike (s) glycoprotein (cavanagh et al., ; enteric isolates (enjuanes and van der zeijst, ; sá n-holmes et al., ; sturman and holmes, ; suñé et chez et al., ) . prcvs replicate to high titers only in al ., ) , and tgev entry into swine testis (st) cells the respiratory tract (cox et al., ) and have a large is mediated through interactions between the virus s deletion at the end of the spike gene, in positions glycoprotein and the porcine aminopeptidase n (papn) ranging from nucleotides (nt) to (enjuanes and which serves as the cellular receptor (delmas et al., van der zeijst, ; sá nchez et al., ; vaughn et al., ) . the s glycoprotein domain recognized by the cel- ; wesley et al., ) . since this deletion is present lular receptor on st cells is thought to be located spain all independently derived prcvs it may be responsible tially close to the antigenic sites a and d (suñé et al., for their loss of enteric tropism. ). in fact, recent studies (godet et al., ) showed however, it can not be excluded that other viral genes, that baculovirus-expressed polypeptides corresponding apart from the s gene, could be involved in the determination of the tropism of tgev. in fact, changes in orf a have been associated with the loss of enteric tropism. nonical sequence cuaaac required for the leader auer et jimé nez et al., ) . the clone obtained was named ptv-ts-dmar c.c - d.e . the ptv-ts and primed transcription by the introduction of deletions and point mutations. these mutations lead to the lack of ptv-ts-dmar virus strains were grown at the permissive temperature ( Њ). orf a expression (britton et al., ; enjuanes and van der zeijst, ; laude et al., ; rasschaert et al., virus neutralization, temperature inactivation, and ; wesley et al., a) . purification interestingly, the purdue-type virus (ptv), which displays respiratory tropism, has an s gene with an identical the procedure for virus neutralization has been desize to that of the enteric isolates. the ptv s gene was scribed (correa et al., ; suñé et al., ) . the neusequenced and compared with the homologous setralization index (ni) was defined as the log of the ratio quence of several enteric isolates. only three nucleotide of the plaque-forming units (pfu) after incubating the differences, not observed in enteric isolates, were noted virus in the presence of medium or the indicated mab. to and all introduced amino acid substitutions. two of these analyze virus inactivation by temperature, viruses were changes were located at nucleotides and within grown at both the permissive ( Њ) and nonpermissive the area deleted in prcvs, while the other was outside, ( Њ) temperatures. the temperature inactivation index at nucleotide (sá nchez et al., ) . the nucleotide (tii) was calculated as the log of the ratio of the pfu change at position was also present in several enafter growing the virus at Њ or Њ. teric isolates. these data lead us to propose that alter-to partially purify tgev, st cells were grown in ations in the s gene around residue could affect cm roller bottles and infected with a multiplicity of infecenteric tropism (sá nchez et al., ) . tion (m.o.i.) of pfu/cell. supernatant was harvested in order to analyze the role of different viral genes in hr postinfection (h.p.i.) and clarified by centrifugation tropism, we have isolated recombinant tgevs by crossin a sorvall gsa rotor for min at rpm. virions ing the enteric pur strain and the respiratory strain were concentrated by centrifugation at rpm at Њ ptv-ts-dmar, a temperature-sensitive mutant (ts) resisin a kontron tst . rotor for hr through a % (w/v) tant to neutralization by monoclonal antibodies specific sucrose cushion. to clear the virus from the remaining for two different antigenic subsites (dmar). analysis of sucrose, the pellet was resuspended in ten ( mm the tropism of the recombinant isolates demonstrates tris-hcl, ph . , mm edta, m nacl) and sedimented that two changes at nucleotides and of the spike by centrifugation under the same conditions. the viral gene, leading to aspartic acid to asparagine and to alapellet was resuspended in ml of tne ( mm trisnine to serine amino acid changes, respectively, were hcl, ph . , mm nacl, mm edta). associated to the loss of enteric tropism in the ptv isolate. the binding of a large panel of mabs to purified virus cells and viruses was performed by ria as previously (correa et al., ; sá nchez et al., ) using optimum amounts of virus viruses were grown in swine testis (st) cells (mcclur-( . mg of protein per well). kin and norman, ). the virus strain pur -mad-cc has been described (sá nchez et al., ) . the rna isolation purdue virus strain ptv was previously named neb (sá nchez et al., ) ; however, due to sequence similar-genomic rna was extracted from partially purified virus as described previously (mendez et al., ) . briefly, ity to the pur strain, its name was changed to ptv (purdue-type virus). a ts mutant derived from ptv was st cells from roller bottles ( cm ) were infected with a m.o.i. of . medium was harvested at h.p.i. kindly provided by m. welter (ambico). this ts mutant was obtained after -fluorouridine mutagenesis using a virions were partially purified as described above. the viral pellet was dissociated by resuspending in ml previously described procedure (robb et al., ) and three cycles of plaque purification. the ts mutant growth of tne containing % sds and digested with ng of proteinase k (boehringer mannheim) for min at room was reduced ú -fold at the restrictive temperature ( Њ) and showed a reduced capacity for rna synthesis. temperature. rna was extracted twice with phenol-chloroform and precipitated with ethanol. two neutralizing monoclonal antibodies (mabs), c.c and d.e , that are specific for the antigenic subsites rna from tgev-infected st cells was obtained as described previously (mckittrick et al., ) . briefly, st aa and ab, respectively (correa et al., ) , were used to select the neutralization escape mutant (double mab-cells, grown in -cm wells, were infected with tgev at a m.o.i. of . at h.p.i., the cells were resuspended resistant mutant, dmar) using the ptv-ts strain. the procedure used to obtain the dmar mutant was identical to in ml of phosphate-buffered saline (pbs) and were incubated for min on ice with ml of mm vanadyl the one previously described (correa et al., ; geb- absence of neutralizing mabs. a significant proportion of virions resulting from this infection should have only the spike protein encoded by one virus genome. the super-m . the origin of the nucleotides in the recombinant natant was harvested at h.p.i., and neutralization with viruses, in the positions indicated by these molecular mabs c.c and d.e was performed to select recommarkers, were determined by sequencing rt-pcr-debinant viruses. potential recombinants were plaque isorived cdna fragments using the fmol dna sequencing lated at restrictive temperature in the presence of the two system (promega). the primers used to sequence the neutralizing mabs used in the selection. the surviving genetic markers m -m are described in table . isolates were phenotypically characterized by calculating their neutralization and temperature inactivation indices. virus tropism analysis in parallel, independent st cell cultures were infected with each of the two parental viruses and the same re-viral tropism was determined in nih miniswine (luncombinant selection procedure was attempted. ney et al., ; sachs et al., ) or in piglets derived from crossing belgium landrace and large white swine. two-to three-day-old conventional (i.e., non-colostrumdeprived) piglets were used. piglets were obtained from to identify nucleotide differences between the two pasows that were seronegative for tgev neutralizing antirental viruses, cdna fragments covering different rebodies by ria. inbred and outbred animals were oronagions of the genome were synthesized by rt-pcr. sally and intragastrically inoculated with doses of these regions included the first kb from the -end of and pfu, respectively, in a final volume of the genome, orf nucleotides to , the first ml of pbs supplemented with % of fetal calf serum. . kb of the s gene, and the most . kb end of ptv-groups of piglets inoculated with the same virus were ts-dmar. these cdnas were subcloned into pbluescript grouped and housed in isolation chambers located in a (sk ) (stratagene) or pgem-t (promega) vectors. plas-p level containment facility at Њ to Њ. animals were mid dna was purified using the flexiprep kit (pharmacia) fed three times per day with ml of milk formula for and sequenced with sequenase . (usb). sequence newborns (nidina -nestlé ). virus titers at , , , and data were compiled using the university of wisconsin days postinoculation were determined in tissue extracts genetic computer group (uwgcg) sequence analysis from jejunum and ileum and lungs. tissues were homogsoftware package and compared to previously published enized at Њ using a tissue homogenizer pro- (pro-pur virus strains (eleouet et al., ; kapke and scientific) . lungs and jejunum and ileum extracts were brian, ; mendez et al., ; rasschaert et al., ; obtained by homogenizing the whole organs in order to sá nchez et al., ) . mutations were confirmed by seobtain representative samples. quencing the viral rna of the two parental viruses pur -mad and ptv-ts-dmar. results rna was directly sequenced by oligodeoxynucleotide primer extension and dideoxynucleotide chain termina-generation and characterization of tgev mutant ptvtion procedures (sanger et al., ) , using a modified ts-dmar c.c , d.e protocol previously described (fichot and girard, ). nucleotide differences between the parental viruses to generate a panel of recombinants between enteric and respiratory strains of tgev, parental viruses that were used as genetic markers and were named m to facilitate the selection of recombinants were generated markers, the first kb from the end of the genome, and characterized. a double mar mutant virus (ptv-ts-orf nucleotides to , the first end . kb dmar c.c - d.e ) derived from the respiratory ptv-ts of the s gene, and the most end . kb were sestrain of tgev was isolated. the growth of ptv-ts and quenced in the ptv-ts-dmar isolate. these sequences ptv-ts-dmar viruses in st cells at Њ was at least were compared to the pur -par strain (eleouet et al., fold lower than at Њ, while the parental pur virus ; rasschaert et al., ) and to pur -mad (menstrain replicated similarly well at both temperatures (data dez et al., ; sá nchez et al., ) . nine nucleotide not shown). the antigenic characterization of the mutant differences between the two parental viruses were iden-ptv-ts-dmar and its ancestor ptv-ts by ria and neutraltified (fig. a) . these markers were named m to m in ization using mabs showed (fig. ) that the ptv-ts-dmar order from the -end of the genome. the ptv-ts-dmar mutant lacked the s protein antigenic subsite aa comvirus was derived from a ptv-ts isolate which originated pletely, and subsite ab partially, since some mabs spefrom ptv. these three related isolates all display respiracific for these subsites did not bind or neutralized this tory tropism. all of them differ with the enteric pur isolate. in addition, the s protein of the escaping mutant mad by three nucleotide substitutions in the s gene (m , was not bound by mabs b.f and f.c , suggesting m , and m ). in addition, ptv-ts-dmar differs by a fourth that the epitopes recognized by these mabs are located nucleotide change (m ), which is responsible for the in subsites aa or ab or in close association with these dmar mutation. two of the nine nucleotide differences subsites. sequencing of the s gene in the ptv-ts-dmar (m and m ) did not result in an amino acid change. m mutant showed that the loss of antigenic subsites aa was located in the intergenic region between orf a and and ab was due to a single point mutation at nucleotide orf b, and m in orf b. neither of these two areas , resulting in a change of aspartic acid to tyrosine are expressed in ptv or pur -mad strains. at position . analysis of the recombinant genome sequences showed that the recombinants originated by fusing sequences of the enteric parental virus to sequences in order to study the molecular basis of tgev tropism of the respiratory parental virus. using these molecular recombinant viruses were obtained by coinfecting st markers, the recombinants were classified into three cells with the enteric pur -mad and respiratory ptvdifferent groups according to the position of the crossts-dmar strains (fig. ) . st cell monolayers were infected over (fig. ) . group recombinants contained clones in parallel, with either pur -mad or ptv-ts-dmar and their crossover was located within the nucleostrains. to isolate recombinants, selective pressure tides spanning the genetic markers m and m (fig. ). based on virus inactivation at high temperature and mab group recombinants comprised isolates that had neutralization was used. the virus titer in the st cell culture coinfected with the two parental viruses was . the crossover located between m and m markers. the pfu/ml, while cells infected with the respiratory viruses included in groups and had the same seor enteric parental viruses contained and less than quence except for nucleotides and of the s pro-pfu/ml, respectively, indicating that recombinant viruses tein gene (genetic markers m and m ) that were derived resistant to the selective pressure were likely generated. from the enteric parent in group isolates, and from the the supernatant from the st cell culture coinfected respiratory parent in group isolates. group recombiwith the enteric and respiratory strains was used to nants included isolates that had recombined between plaque purify putative recombinant clones and the genetic markers m and m . progeny of the coinfection was phenotypically character-molecular marker m was sequenced in all recombiized under restrictive conditions. thirty of the clones nant viruses, since it was the nucleotide difference loanalyzed showed the recombinant phenotype (table ) . cated closest to the end. all recombinants inherited most of the progeny isolates ( %) showed the expected this marker from the pur -mad strain, indicating that selectable recombinant phenotype (sr), being resistant there was most likely only one crossover at the -half of to both mab neutralization and temperature inactivation. the genome. three nucleotide differences were observed among the recombinant viruses, % were dmar mutants from gene up to the end of the genome (fig. a) . only which were partially sensitive to the restrictive temperaone of these differences (genetic marker m , located ture (ts intermediate phenotype recombinants, ipr). no in orf ) led to an amino acid change. however, this virus was isolated with the phenotypic characteristics nucleotide difference was not present in the respiratory against which the selection had been performed (nonse-ptv strain, strongly suggesting that it was not involved lectable recombinants, nsr) ( table ). in the control of tgev tropism. the possibility of a second genotypic characterization of the recombinant crossover at the half of the genome was not analyzed, isolates because if a second crossover had taken place, it would have replaced a fragment by another one with an equiva-the identification of genetic markers was required to map the recombination sites. in order to identify such lent sequence. lar for the two viruses from each group. in addition, one isolate from group recombinants was studied. tropism the tropism of the three groups of recombinants was was studied in parallel in the parental viruses pur , next studied. two isolates from group and two from group were evaluated. the results obtained were simi-ptv-ts-dmar, and the ptv strain. each isolate was tested at least three times. all recombinants and the parental shown) indicated that the ts mutation mapped at the orf a of the respiratory isolate, between genetic markviruses were isolated from the lungs, but only the pur ers m and m (i.e., from nt to nt of orf ). strain and the recombinants of group could be isolated the dmar mutation was localized at position of s from the small intestine (fig. ) . maximum virus producgene (genetic marker m ). thus, the minimum distance tion in the lungs varied from to pfu/g tissue (fig. for recombination with the selective pressure used (high ). ptv-ts-dmar produced less infectious virus than the temperature and neutralization by mabs) was the interval other strains. maximum ptv-ts-dmar production was between markers m and m . the recombinant isolation about pfu/g of lung tissue, while its ancestor ptvfrequency was calculated as the ratio between the progwt could replicate to higher titers ( pfu/g tissue). eny virus titer with a recombinant genotype ( . pfu) and the titer ( pfu) of the parental viruses recombinant isolation frequency grown in parallel in the absence of selective pressure, the procedure used to isolate recombinant viruses divided by the distance between m and m . this frefavored the selection of viruses which had recombined quency was £ . recombinants per nucleotide between the two markers used in the selection, the ts for this interval. the recombinant frequency was also calculated for recombinants of groups , , and and and the dmar mutations. preliminary results (data not below this bar, the location of nine nucleotide differences (genetic markers m to m ) between the two parental viruses is indicated. recombinants were classified into three groups (named to ), and the origin of their genomes, whether derived from the enteric parental (dark bars) or from the respiratory parental (white bars) is indicated. in the bars corresponding to the parental viruses pur -mad and ptv-ts-dmar, the individual nucleotide differences are indicated. (b) summary of the genetic characterization of the groups of recombinant viruses. the two markers flanking each crossover and the distance expressed in nucleotides between the two markers are indicated in columns and , respectively. since the exact location of the ts mutation is not known, the crossover in group recombinants is indicated as a maximum distance. the number of recombinants included in each group and the percentage in relationship to the total recombinant virus population are shown in column . the last column shows the frequency of recombinants isolated in each group, as the ratio of the number of isolates in the group relative to the number of nucleotides between the molecular markers flanking the crossover site. were . , . , . , respectively residues and of the spike gene were responsible (fig. b ). since the exact location of the ts mutation is not for the loss of enteric tropism. known, the interval in which the crossover takes place in group recombinants can not be precisely defined and recombination among isolates of the tgev cluster the frequency provided for this group is the minimum. initial attempts to isolate tgev recombinant viruses the calculated data indicate that the frequency of recomusing selective pressure with neutralizing mabs specific binant isolation at the of the s gene was . -fold higher for a single epitope lead to the isolation of neutralization than that of group recombinants. escape mutants instead of recombinants (data not shown). to diminish the frequency of escape mutants, discussion selective pressure with two mabs specific for different epitopes of antigenic subsites aa and ab (gebauer et in order to determine the role of different viral genes in tgev tropism, a collection of recombinants was al., ) were used. this strategy, in fact, decreased the frequency of neutralization escape mutants, although the generated by coinfecting st cells with enteric (pur -mad) and respiratory (ptv-ts-dmar) strains of tgev. phe-selected dmar mutant used as a parental virus in the recombination (ptv-ts-dmar) had only a single nucleotide notypic, genotypic, and biological characterizations of the recombinants showed that two nucleotide changes at change, instead of two nucleotide changes that might to study tgev tropism, to -day-old non-colostrum-deprived piglets were individually inoculated with two isolates from group , with two isolates from group , and with isolate from group recombinants. recombinants were isolated by crossing the enteric strain pur and the respiratory strain ptv-ts-dmar. top horizontal thick bars indicate the genome and the origin of each recombinant, whether enteric (dark bar) or respiratory (white bar). the thin horizontal bar indicates the s gene. triangles indicate the positions of nucleotides and of the s gene and the origin of these nucleotides, whether enteric (dark triangle) or respiratory (white triangle). diagram is not at scale and the size of the s gene has been magnified. the recovery of infectious virus was determined in pfu per gram of tissue at the indicated time in h.p.i. each virus was tested at least three times. vertical thin bars indicate standard error of the mean. have been expected for a mar mutant escaping to the . -fold higher than that of group recombinants. this could be due to a selective advantage in their growth on simultaneous neutralization by two different mabs. isolation of tgev recombinants required the use of cell cultures or to a higher recombination frequency at the -end of s gene, between nucleotides and . selection pressure. using this procedure the frequency of recombination was estimated at £ . for tgev. in fact, extensive sequence variability has been observed in this region. during the isolation of tgev-defective in-in contrast, the isolation of the mhv recombinant does not require the use of selection pressure (makino et al., terfering viruses, a deletion is introduced at the beginning of the s gene, starting from nucleotides to and ). the recombination frequencies estimated for both coronaviruses are not directly comparable since the se-ending at orf (mendez et al., ) . in addition, during the generation of both european and american prcvs lection strategies were very different; nevertheless, from reported data it seems that the recombinant isolation in field conditions, four different deletions at the beginning of the s gene have been identified in positions rang-frequency is higher for mhv than for tgev. both recombination and the generation of defective interfering (di) ing from nucleotides to (sá nchez et al., ; vaughn et al., ; wesley et al., ) . these data genomes occurs at a lower frequency in tgev (mendez et al., ) than in mhv (lai, ) , possibly due to a suggest that the -half of the s gene is an area with an intrinsically high recombination frequency. although a higher accuracy in the replication of tgev rna. group recombinants were isolated at a frequency selective advantage for the recombinants could not be excluded, it seems unlikely because group and re-tine until the fourth day postinoculation, virus was never detected in the enteric tract in any of the more than combinants differ only in two nucleotide positions located at the -half of the s gene (nucleotides and piglets inoculated with a respiratory isolate. this indicates that the virus detected in the enteric tract was not ), and recombinants which had the same -half s gene as group recombinants grew as efficiently as due to residual virus from the inoculation, nor swallowed virus originating in the respiratory tract, but was the result pur . an increased recombination frequency in the s gene of mhv has also been described (fu and baric, of local virus replication in the intestine. all the isolated recombinants, including the ones lack- ). ing enteric tropism, were temperature resistant, indicating that the ts mutation was not responsible for the loss molecular basis of tgev tropism. of enteric tropism. studies on pur -par mar mutants also showed a only nine nucleotide differences were found between the enteric pur -mad and the respiratory ptv-ts-dmar correlation between the n-terminal half of the s protein and viral pathogenesis (bernard and laude, ) . nev-strains of tgev. four of them mapped in the s protein gene at nucleotides , , , and . the nucle-ertheless, these results did not differentiate between virus tropism and virulence, since only parameters such otide change at position of s protein gene, which is responsible for the neutralization escape phenotype, as death, or weight loss, caused by the virus mutants were studied, but not virus replication in enteric or respi-is not responsible of the loss of enteric tropism since it was not present in the respiratory isolate ptv which ratory tissues. coronavirus spike protein is involved in virus attach-lacks enteric tropism. in order to analyze which of the other three nucleotide ment to cells (cavanagh et al., ; holmes et al., ; sturman and holmes, ; suñé et al., ) . studies changes located in the s protein gene, at positions , , and , were involved in the control of the enteric on the inhibition of virus binding to cells indicated that the receptor binding site for tgev had to be located tropism, recombinant viruses containing one or the three nucleotide differences from the respiratory isolate were between antigenic sites d and a of the spike protein (suñé et al., ) , mapping between amino acids selected. these recombinants belong to groups and , respectively. group recombinants only infected lungs, and . in agreement with these data, it was shown that porcine apn, the receptor for tgev (delmas et al., while group replicated in the epithelial cells of both the enteric and respiratory tracts. the two nucleotide ), binds to s protein residues between aminoacids and (godet et al., ) . these sequences map changes between the enteric recombinants (group ) and the respiratory ones (group ) were at nucleotides to a distal area in relationship to amino acid of s protein, which, as shown in this paper, influences tgev and of the s protein gene, which caused amino acid changes from aspartic acid to asparagine at residue enteric tropism. since papn is a protein present in lung epithelium and in enterocytes (kenny and maroux, ; and from alanine to serine at residue . these results demonstrate that two amino acid changes at the noré n et al., ; semenza, ) , and the respiratory ptv isolate conserves the papn binding site previously n-terminus of the viral spike protein were associated to the loss of enteric tropism in the tgev cluster of viruses. described, the loss of enteric tropism in the ptv isolate should not be due to a failure in papn attachment. fur-the possibility that the loss of enteric tropism was a consequence of the addition of a nucleotide change at thermore, it has been demonstrated that prcv isolates attach to papn (delmas et al., ) , although they can-position of s gene to a preexisting change at nt cannot be completely ruled out. nevertheless, this not infect the enteric tract. this apparent discrepancy could be explained if an interaction between papn and possibility seems unlikely because most enteric viruses have the same nucleotide as ptv at position (sá n-two domains of s protein located at both areas (amino acids near residue and amino acids to ) are chez et al., ), indicating that most likely a single nucleotide change at position was responsible for required to infect the enteric tract. alternatively, a putative second factor, such as coreceptor, mapping around the loss of enteric tropism. nucleotides and are located within the area of the s gene which is deleted amino acid of the spike protein could be specifically required to infect the enteric tract and responsible for in prcvs, strongly suggesting that this deletion was responsible for the loss of enteric tropism in prcvs. in the loss of enteric tropism in ptv and prcv isolates. other explanations are also possible and the loss on human immunodeficiency and other virus systems it has also been shown that a single point mutation can alter enteric tropism could also be due to: (i) a decrease in the ph stability required to allow the virus passage tropism (takeuchi et al., ) . an intragastric inoculation route was employed to as-through the stomach, (ii) a decrease in virion resistance to bile salts and proteolytic enzymes in gut, and (iii) an sure that the inoculum of each isolate was introduced into the stomach, independently of their tropism. while alteration in the strength or affinity of the s/receptor interaction. recent studies also located the receptor binding viruses with enteric tropism have been found in the intes- cell biology of virus entry, ble gastroenteritis virus spike protein results in markedly reduced replication, and pathogenesis'' (r. w. compans, a. helenius, and pathogenicity critical epitopes in transmissible gastroenteritis virus neutralrespiratory coronaries: comparison with transmissible gastroenteriization sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene genetic evolution and tropism of transmissible hydrolases of kidney and intestine coronavirus-organization, replication and expresgastroenteritis coronaviruses bullido, sion of genome antigenic homology among coronaviruses recoronavirus-molecular features and virus host interactions the swine major histocompatibility complex: its structure and function. in ''swine in chain-terminating inhibitors anchoring and biosynthesis of stalked brush border membrane proteins: glycosidases and peptidases of enterocytes highfrequency rna recombination of murine coronaviruses a monoclonal antibody gastroenteritis of swine. ii. selected characteristics of a cytopathogenic virus common to five isolates from transmissible gastroenteri-siddell the molecular biology of corotis efficient naviruses mechanisms of transmissible gastroenteritis coronavirus neutralization analysis of the receptor binding site of murine coronavirus spike protein elsemodification of cell tropism by a single point mutation at the neutralization epitope in the env gene three new isolates of porcine respiratory coronavirus with various pathogenicities and gastroenteritis virus in swine: old and news. th int porcine respiratory coronavirus differs from transmissible gastroenteritis virus by a few for the pathogenesis of transmissible gastroenteritis virus genetic analysis of porcine respiratory coronavirus, an attenuated variant of transmispartial sequence of the genomic rna, its organization and expression pathogenic murine coronaviruses. iii. biological and biochemical characterization of for a porcine respiratory coronavirus, antigenically similar to transmissible gastroenteritis virus, in the united states spike protein-dependent cellular factor other than the viral receptor is required for mouse hepatitis virus entry transmissible gastroenteritis comparison of porcine transmissible gastroenteritis virus (tgev) with site of the jhm strain of mhv in rats on the s subunit porcine respiratory coronavirus. viiith international congress of virolof the spike protein (suzuki and taguchi, ) , and in ogy, pp. p - . iums, berlin.mhv it was suggested that a second cellular factor, apart callebaut, p., correa, i., pensaert, m., jimé nez, g., and enjuanes, l. from the cellular receptor which interacts with the s pro- ( ) . antigenic differentiation between transmissible gastroenteritis virus of swine and a related porcine respiratory coronavirus. j. tein, is involved in virus entry (yokomori et al., ) . gen. virol. , - the requirement of a coreceptor to infect cells has been cavanagh, d., brian, d. a., enjuanes, l., holmes, k. v., lai, m. m. c., described in human immunodeficiency virus and in polio- laude, h., siddell, s. g., spaan, w., taguchi, f., and talbot, p. ( ). virus (deng et al., ; dragic et al., ; feng et al., revision of the taxonomy of the coronavirus, torovirus, and arteri- ; shepley and racaniello, ) . orf a is not expressed in prcvs isolates, while it is coronavirus ibv: virus retaining spike glycopolypeptide s but not s expressed in enteric strains. thus, it has been proposed is unable to induce virus-neutralizing or haemagglutination-inhibiting that orf a plays an essential role in the control of virus antibody, or induce chicken tracheal protection. j. gen. virol. , enteropathogenecity (britton et al., ; laude et al., laude et al., - laude et al., . wesley et al., ) . rna sequence comparison of correa, i., jimé nez, g., suñé , c., bullido, m. j., and enjuanes, l. ( ) .antigenic structure of the e glycoprotein from transmissible gastro-the -half of the respiratory virus ptv-ts-dmar, from enteritis coronavirus. virus res. , - .orf a to -utr, with that of the enteric isolate pur - key: cord- -kh t kfz authors: o'connor, jennifer black; brian, david a. title: downstream ribosomal entry for translation of coronavirus tgev gene b date: - - journal: virology doi: . /viro. . sha: doc_id: cord_uid: kh t kfz abstract gene b (orf b) in porcine transmissible gastroenteritis coronavirus (tgev) encodes a putative nonstructural polypeptide of . kda with unknown function that during translation in vitro is capable of becoming a glycosylated integral membrane protein of kda. in the virulent miller strain of tgev, orf b is ′-terminal on mrna – and is presumably translated following ′ cap-dependent ribosomal entry. for three other strains of tgev, the virulent british fs / and taiwanese tfi and avirulent purdue- , mrna species – is not made and orf b is present as a non-overlapping second orf on mrna . orf b begins at base on mrna in purdue strain. in vitro expression of orf b from purdue mrna -like transcripts did not fully conform to a predicted leaky scanning pattern, suggesting ribosomes might also be entering internally. with mrna -like transcripts modified to carry large orfs upstream of orf a, it was demonstrated that ribosomes can reach orf b by entering at a distant downstream site in a manner resembling ribosomal shunting. deletion analysis failed to identify a postulated internal ribosomal entry structure (ires) within orf a. the results indicate that an internal entry mechanism, possibly in conjunction with leaky scanning, is used for the expression of orf b from tgev mrna . one possible consequence of this feature is that orf b might also be expressed from mrnas and . expression of coronavirus genes occurs through the synthesis of a Ј coterminal nested set of mrnas. although coronavirus mrnas are structurally polycistronic (the Ј-most mrna in many but not all viral species is monocistronic), evidence from studies of translation both in vitro and in vivo has suggested that most function as monocistronic messages. that is, despite their polycistronic configuration, usually only the Ј-terminal orf on each is abundantly translated (reviewed in lai and cavanagh, , and luytjes, ) . the location of some coronavirus genes, however, is not Ј-terminal on any mrna, which would require that the gene, if expressed, be translated by a mechanism allowing translation reinitiation, leaky scanning, frameshifting, or a downstream entry of ribosomes. examples of such genes include ( ) orf b on mrna , from which a polyprotein is synthesized following Ϫ ribosomal frameshifting (brierly et al., ; eleouet et al., ) ; ( ) orfs b and c on avian infectious bronchitis virus mrna (boursnell et al., ) , from which . -and . -kda proteins are synthesized following leaky scanning and internal ribosomal entry, respectively (liu et al., ; liu and inglis, ; le et al., ) ; ( ) orf b on mouse hepatitis virus mrna (skinner et al., ) , from which a . -kda protein(the e protein) is synthesized (budzilowicz and weiss, ; leibowitz et al., ) , by an apparent internal ribosomal entry mechanism (thiel and siddell, ) ; and ( ) the i orf in mrna of the bovine and mouse hepatitis coronaviruses, from which the i protein is made in the ϩ reading frame relative to n (senanayake et al., ; fischer et al., ) following ribosomal scanning (senanayake and brian, ) . in this study, we examine the mechanism by which gene b is expressed from mrna in the purdue strain of tgev and demonstrate that, surprisingly, it may be approached by ribosomes entering internally and not necessarily through a leaky scanning step as would be predicted from mrna sequence. gene b in tgev is unusual in that for one strain of virus, the virulent miller strain (wesley et al., ) , it is expressed as the Јterminal orf on mrna - , whereas in three other strains, the virulent british fs / and taiwanese tfi strains and the avirulent purdue- strain, it is expressed as the second orf on mrna (britton et al., ; chen et al., ; this study) (note mrna structures in fig. a ). the differences in transcription patterns appear to be a function of the canonical tgev ucuaaac intergenic sequence positioned nt upstream of gene b in the genome, which in the miller virus totally conforms to the canonical sequence but in the british fs / and purdue- strains is ucuaaau and in the taiwanese tfi strain is acaaaac. the nonconforming intergenic sequences apparently fail to promote synthe- sis of a subgenomic mrna. translation from orf b when it occurs as the second orf on mrna must, therefore, require either a reinitiation of translation after translation of the upstream orf, a leaky scanning by ribosomes over a long distance ( nt; fig. b ), or a downstream entry of ribosomes. because gene b is not intact in some strains of tgev (i.e., it is either severely truncated by frameshift mutations as in the purdue- strain [rasschaert et al., ] or by deletions as in the avirulent miller strain [wesley et al., [wesley et al., , ) and cannot produce a full-length product, it has been suggested that its product fulfills a specialized function, perhaps during animal infection (reviewed in enjuanes et al., ) , and is not required for virus replication. similar conclusions were reached after revelations of a truncated gene b in strains of the closely related porcine epidemic diarrhea virus (vaughn et al., ) . it might therefore be assumed that gene b is not translated when it occurs as a downstream orf as in mrna . however, a product from gene b is made in cells infected with the purdue- strain of virus (o'connor and brian, ) , indicating a mechanism must exist for its synthesis from mrna . here we report that, whereas mrna has a sequence predicting leaky scanning for the translation of orf b by the model of kozak ( ) , experimental results with mutant constructs suggested downstream entry of ribosomes might also be used. furthermore, deletion analysis indicated that the internal entry of ribosomes did not depend on an immediate upstream internal ribosomal entry structure (ires) and suggested ribosomes are entering very close to the orf b start site by a mechanism resembling shunting. mrna , but not mrna - , is made in cells infected with the purdue- strain of tgev northern analyses of purdue- virus-infected cells carried out previously in our laboratory with oligonucleotide probes specific for the Ј end of the genome (i.e., a sequence from within the Ј-proximal hp orf) had identified eight species of mrna, leading us to conclude that orfs encoding the . -kda (gene a) and . -kda (gene b) proteins are each Ј-terminal on separate mrna species (sethna et al., ) . to test this conclusion, separate northern analyses were done with probes specific for the -nt leader and for genes a and b. our rationale was that mrna species and - would be distinguishable with probes binding within orfs a and b because transcripts of and nt (or even and nt if they included poly a tails of nt in length) are resolvable on a gel of % agarose. northern analyses with the separate probes revealed bands with identical mobilities, indicating the presence of mrna , but not mrna - , in rna from purdue- virus-infected cells ( fig. a, lanes - ) . to test this conclusion by a second method, rt-pcr analysis was done with oligonucleotide primers specific to gene b and the minus strand of the leader. amplified products of and nt would be expected from mrnas - and , respectively. a product of nt northern blot analysis using gene-specific probes. (b) rt-pcr analysis of rna from purdue or miller virus-infected cells using a plus-strand-detecting oligonucleotide from within orf b as the first primer (for rt and subsequent amplification) and a minus-strand-detecting oligonucleotide from within the leader sequence for amplification. products were electrophoresed on a gel of % agarose and stained with etbr. might also be found from mrna , the mrna encoding the spike protein. from rt-pcr analysis only a single product of nt with the proper sequence as determined by cloning and sequencing was obtained (fig. b , lane ; sequencing data not shown), indicating the presence of mrna but not mrna - . to establish that the experimental protocol would have detected mrna - if present, rna was extracted from miller virus-infected cells and used in parallel. from this, the expected -nt mrna - -derived product was obtained (fig. b , lane ). no product was obtained with rna from uninfected cells (fig. b, lane ) . genes a and b, but not gene , are translated in vitro from synthetic mrna -like transcripts containing all three orfs to test by in vitro translation whether the . -and -kda gene b products (o'connor and brian, ) are synthesized when orf b is positioned downstream of orf a (beginning at base ) on synthetic transcripts, uncapped transcripts of porf a- b- dna linearized at the bamhi site nt downstream from the stop codon of gene (fig. c) were translated in either wheat germ extract or rabbit reticulocyte lysate. in both, products from genes a (the . -kda protein containing one methionine) and b (the . -kda form of the protein containing eight methionines and the -kda form presumably containing seven methionines), but not gene (the . -kda e protein containing four methionines), were obtained (results for wheat germ extract are shown in fig. a , lane ; note the marker positions in lanes - and the absence of endogenous product in lane ). because a nearly -kda protein was also synthesized from an endogenous transcript in (some) rabbit reticulocyte lysates and the presence of abundant globin protein in the lysate interfered with the resolution of small proteins (data not shown), all subsequent studies described were carried out in wheat germ extract. thus, from uncapped transcripts bearing similarity to mrna the first methionine codon in gene b (for synthesis of the . -kda protein) and also the second (assuming synthesis of the -kda protein initiates at the second methionine codon [o'connor and brian, ] ) are accessed for translation. on a molar basis, the amount of . -kda gene b product is approximately one-fifth of that from gene a (fig. b ). there was no evidence of a . -kda e protein from orf in this (fig. a , lane ) or in subsequent experiments, indicating ribosomal accessibility of orf b was probably not the result of template fragmentation. an upstream leader-containing sequence in transcripts of plorf a- b- ( fig. c) , although containing an additional nts not found on mrna , had only a small effect on the rate of translation from orf b relative to a (fig. a , lane , and fig. c ), indicating the leader se-quence may not strongly influence translation from the downstream orf; however, this needs confirmation with transcripts precisely mimicking the Ј end of mrna . as with transcripts of porf a- b- , no product was evident from orf . translation of gene b from mrna -like transcripts shows a pattern not fully consistent with a leaky scanning model from precedents in eukaryotic mrnas, it is unlikely that ribosomes would approach orf b on mrna by a mechanism of translation reinitiation, since three inframe strong stop codons follow orf a (fig. b) . however, an approach by leaky scanning according to the model of kozak ( kozak ( , a ,b) might be expected, since the initiator codon for orf a (ugua ugg, in the quantitation of products from the indicated constructs as determined from ambis radioanalytic imager scans of the gel shown in (a). note that only detected proteins are represented in the bar graph. * denotes a putative aggregate of the orf b product; † indicates plasmid dna was linearized at the scai site within orf b before transcription. same reading frame as orf b) and for three other potential small orfs within gene a (uaga ugc, caua ugc, and ucca ugc, all in the ϩ reading frame relative to orf b) are within contexts considered weak for initiation, whereas that for orf b (aaaa uga) is considered relatively strong. to test for ribosomal scanning on mrna -like transcripts, three approaches were taken. in the first, the effect of a Ј cap on the synthesis of a and b gene products was measured. increased synthesis from both would be expected if Ј cap-dependent entry followed by leaky scanning were used (kozak, (kozak, , a . increased synthesis from b might also be expected if a cap-dependent shunting mechanism were used (jackson, ; mathews, ) . as can be observed in fig. a , lanes and , and figs. b and c, enhanced translation of both orfs a and b resulted when capped transcripts of porf a- b- and plorf a- b- were translated. these results are therefore consistent with the mechanisms of leaky scanning and cap-enhanced ribosomal shunting. in the second approach, the competitive effect of a soluble cap analog on the translation of orfs a and b from capped transcripts of porf a- b- was measured. with either leaky scanning or cap-dependent shunting, but not with cap-independent internal entry, competitive inhibition of translation from both orfs would be expected (iizuka et al., ; jackson, ; mathews, ) . nearly the same rate of inhibition was found, - % with . mm and - % with mm cap analog (figs. a and c), indicating either mechanism of capdependent entry could be functioning in the translation for orf b. in the third approach, the sequence context surrounding the a start codon in transcripts of porf a- b- was modified to become strongly favorable for translation (gccgccatgg) (kozak, b) and the relative amounts of a and b gene products were measured. with leaky scanning, a diminished synthesis from b relative to a would be expected regardless of the capped status of the transcripts (kozak, b) , whereas with shunting a change in the relative amounts would not necessarily be expected. as can be noted in figs. a and b, whereas the accumulation of a product increased almost % relative to b with the improved kozak consensus for capped transcripts, no increase was observed with uncapped transcripts. intriguingly, the absolute amount of gene b product appeared nearly identical under all conditions of translation. these results, therefore, are not fully consistent with the leaky scanning model for orf b translation of orf b is not blocked by the upstream insertion of an -nt-long sequence containing three sequential orfs to test for an internal entry of ribosomes onto orf b, porf a- b- was modified to pscatorf a- b- by the placement of an -nt-long sequence containing three sequential orfs upstream of orf a (fig. c ) and the products of translation were quantitated. an internal entry of ribosomes, either directed by an ires element or by a shunting mechanism, would typically not be blocked by the presence of upstream orfs of this dimension (reviewed in jackson et al., ; jackson, ; mathews, ) . transcripts of pscatorf a- b- possessed a Ј utr of nt; a five-methionine-containing -nt scat orf beginning within an excellent kozak context (aaaatgg) at base ; a five-methionine-con-taining . -kda protein-encoding orf beginning within a fair kozak context (atcatgc) at base ; a one-methionine-containing . -kda protein-encoding orf beginning within a fair kozak context (cggatga) at base ; and orf a beginning at base , orf b beginning at base , and orf beginning at base . in addition, there is a -kda protein-encoding orf beginning within a fair kozak context (tccatga) at base within the scat orf (in the ϩ reading frame relative to scat). all in all, aug codons exist upstream of orf b. when transcripts of pscatorf a- b- were translated the following features were noted: . from uncapped transcripts, only products from the scat and b orfs, in a molar ratio of approximately : . , were obtained (fig. a, lane ; fig. d ), indicating that among the downstream orfs, translation from orf b had been a selective one and was probably not the result of initiation on fragmented transcripts. . from capped transcripts, an enhanced accumulation from the scat orf was observed but not from the b orf (fig. a, lane ; fig. d ), unless an inexplicable putative aggregated form of orf b (identified by an asterisk in fig. a , lane , and noted earlier [o'connor and brian, ] ) was included in the total. (the aggregate was not included in the bar in fig. d.) . from capped transcripts in the presence of soluble cap, a similar rate of inhibition was observed for products of both the scat and b orfs ( - % with . mm and - % with . mm cap analog; figs. b and d), mirroring the results with mrna -like transcripts of porf a- b- (figs. a and c). . no translation of the remaining five orfs was observed from either uncapped or capped transcripts. these results show that translation of orf b positioned nt downstream from the Ј terminus in the synthetic construct is initiated by some form of internal entry of ribosomes and not by scanning, is influenced by a cap, and is not the result of a fragmented template. although no universally identifying primary or secondary structural features of ires elements are known, certain secondary structural features do appear necessary for ires function (reviewed in jackson, ) . within tgev gene a, secondary structures can be predicted (figs. a and b) that share features with the putative ires element in ibv mrna (liu and inglis, ; le et al., ) , leading us to postulate that gene a might contain an ires. the predicted structures are five stemloops (i-v), four of which can be drawn as components of pseudoknots. the free energies of these are calculated to be, respectively, Ϫ . , ϩ . , Ϫ . , Ϫ . , and ϩ . are relatively unstable and suggest a low probability for their existence in viral rna. nevertheless, to test whether gene a might function as an ires for translation of orf b, deletions within it were prepared and tested. these were (mostly) bidirectional for distances of , , , , , , , and nt, and represented Ј-ward deletions of , , , , , , , and nt from the first nucleotide in gene a, respectively, for which the mutants were named. results shown in fig. c and summarized in fig. d indicate that the relative molar amounts of scat and the . -kda gene b products (ϳ : . ) remained essentially unchanged between wild-type and ⌬ . the only exception was for ⌬ , for which the molar ratio was : . along with an inexplicable enhancement of an uncharacterized band with an approximate molecular weight of kda. for ⌬ , which leaves only nt upstream of the orf b start codon, the relative amounts were surprisingly : . and not : , as expected. for ⌬ , there was abundant synthesis of the scat protein but no synthesis of the . -kda protein; however, there was synthesis of the -kda gene b product (o'connor and brian, ) (fig. d) . these results are not consistent with a mechanism of ribosomal entry within gene a but rather with one in which ribosomes enter within nt from the start codon of gene b. because the -kda gene b product is found with mutant ⌬ , the intriguing possibility exists that ribosomes are entering at or downstream of the gene b start site and are scanning in the upstream direction to reach the start codon. based on precedents in eukaryotes (reviewed in mathews, ) , four mechanistic possibilities should be considered as explanations for how ribosomes approach the downstream orf b on tgev mrna for translation: ribosomes could ( ) translate the upstream orf and then reinitiate synthesis on the downstream orf, ( ) scan through the upstream orf(s) without the act of translation in a manner known as leaky scanning, ( ) bypass the upstream orf(s) by using an internal ribosomal entry site similar to that used by picornaviruses and flaviviruses on genomic rna, or ( ) bypass the upstream orf(s) after first binding to the mrna in a cap-dependent manner and then undergo shunting to a downstream site on the mrna. among these, shunting is the most recently recognized and is exemplified by translation on pregenomic rna of the cauliflower mosaic virus (futterer et al., ) and rice tungro bacilliform virus (futterer et al., ) , both pararetroviruses, on adenovirus mrna (yueh and schneider, ) , and on sendai paramyxovirus mrna kolakovsky, , ; latorre et al., ) . we conclude that orf b is translated from mrna , and that the likelihood is high that an internal entry of ribosomes is used, possibly one with shuntlike features, and perhaps in conjunction with leaky ribosomal scanning through orf a. the relative contribution of each mechanism on mrna could not be established by the experiments performed here. however, an internal entry of ribosomes was demonstrated by the use of constructs, in which four extensive orfs within an -nt sequence were placed upstream of orf a and synthesis from orf b was shown to remain approximately one-eighth to one-fifth of that from the Ј-terminal orf. the internal entry showed some properties of shunting in that ( ) no ires element of the type directing internal entry in picornaviruses and togaviruses could be demonstrated within sequence upstream of gene b and ( ) translation of orf b in capped transcripts from the synthetic multicistronic pscatorf a- b- showed some inhibition by a competing soluble cap in the translation mix. that is, internal entry in the multicistronic transcript may follow a cap-dependent step as described for shunting in the adenoviruses, pararetroviruses, and paramyxoviruses. in general, the mechanistic features of ribosomal shunting, so far described for only viral mrnas (jackson, ; mathews, ) , remain to be clarified. in the case of adenovirus and pararetrovirus mrnas, an upstream donor structure appears necessary for the shunting step. in pararetrovirus, this appears to be a stable hairpin preceded by a short open reading frame (hemmings-mieszczak and hohn, ) . in the case of tgev orf b shunting reported here, a requirement for an upstream structure seems unlikely, since shunting took place in the presence of foreign sequence (pscatorf a- b- ) as well as (relatively) native sequence (porf a- b- ) at the Ј terminus. in this respect, the tgev orf b shunting pathway bears similarity to that in paramyxovirus mrna, for which no apparent requirement for a donor structure was found (latorre et al., ) . likewise, it is not clear what determines the landing site in a ribosomal shunt. certainly in the experiments reported here it is not apparent how ribosomes might have been directed to land so close to the b initiation codon in tgev mrna . it is clearly not the postulated secondary structures within gene a, because internal entry took place after these had been removed or disrupted (⌬ , figs. c and d ). one possibility is that ribosomes are directed to land at or near the start site of gene b by specific sequences or by higher-order structures situated very near the landing site. precedents for this are found in sendai virus, wherein sequences both upstream and downstream of the y orf are required for shunt landing (latorre et al., ) , and in hepatitis c virus, wherein sequences extending nt into the orf are required for ires-directed landing (reynolds et al., ) . curiously, such a landing site might require that ribosomes backscan to find the gene b start codon, a process postulated to explain the translation of certain sv and influenza virus transcripts (peabody et al., ; williams and lamb, ) . our findings were particularly intriguing because some evidence had suggested the existence of iresdirected, cap-independent translation for the third orf in tricistronic ibv mrna (liu and inglis, ; le et al., ) and for the second orf in the bicistronic mhv mrna (thiel and siddell, ) . the influence of the cap, however, was not examined in the mhv studies, and the possibility remains that a form of shunting might also be exhibited during the translation of these mrnas. the consequences of an internal ribosomal entry onto gene b for virus replication are not immediately apparent, but one might be that it enables a constitutive synthesis of b protein because, in principle, any of the viral mrnas containing gene b (mrnas , , and ) could serve as templates. cells and virus. the purdue- and miller strains of tgev were obtained from e. bohl, ohio state university. purdue- virus was plaque-purified from infectious genomic rna, grown on swine testicle (st) cells in medium containing % fetal calf serum (atlanta biologicals), and used within eight passages of plaque purification (brian et al., ; kapke and brian, ) . miller virus was similarly grown but was plaque-purified twice from infectious virus on st cells and used within passages of plaque purification. northern analysis of tgev mrnas. northern analyses were performed as described (sethna et al., ) and quantitation was done with the ambis photoanalytic imaging system (ambis, san diego, ca). cells were infected with tgev at a multiplicity of infection (m.o.i.) of and total rna was extracted at and h postinfection (hpi). blots were probed with radiolabeled synthetic oligodeoxynucleotide specific for the leader (oligonucleotide l ϩ, Јcgggatcctcgggtttagttcgagttggtg-tccgaagacaaaatctagcacaaggctagttaaagt-aaaagaagagatat Ј), gene a (oligonucleotide . , Јgttcgtcaagtacagcatctacgg Ј), or gene b (oligonucleotide , Јcttctcataaacggtgcagctct-gcc Ј). probes were radiolabeled to a specific activity of . to . ϫ cpm/pmol by the forward reaction. construction of plasmids. tgev purdue sequences used in this study have been published (kapke, et al., a; sethna et al., ) . the construction of porf b- ( fig. c) , formerly called porf , has been described (o'connor and brian, ) . porf b- carries genes b and and nt of gene in vector pgem- z (promega biotech) (a sequence obtained from cdna clone pft [ fig. a] ). porf (fig. c) was made from porf b- by first removing the -nt m-containing sphi fragment, religating, and then removing the -nt hindiii-bbsi fragment and religating after blunt-ending with mung bean nuclease. porf a- b- (fig. c) , which carries genes a, b, and downstream of the t rna polymerase promoter in pgem- z (promega biotech), was made in three steps. first, psp orf a, from which gene a and nt of gene b can be transcribed with rna polymerase sp , was created by ligating the bp nsii-psti fragment from clone pft (a clone containing nucleotides to from the genome Ј end [ fig. a ; tung et al., ] , prepared as described in kapke et al., a,b) into the psti site of pgem- z. second, psp orf a- b- was created by ligating the -bp bsrgi-ecori fragment from porf b- into the -nt vector-containing ecori-bsrgi-linearized fragment of psp orf a. third, the entire -nt sphi-hinfi insert from psp orf a- b- , after blunt-ending with t dna polymerase, was ligated in the reverse orientation into similarly blunt-ended ecori-saci-linearized pgem- z. to place the viral leader upstream of orf a, plorf a- b- ( fig. c ) was constructed by a previously published procedure (sethna et al., ; hofmann et al., ) . briefly, cdna was made from the Ј end of mrna with a primer specific to gene b (oligo (ϩ) [ Јcttct-cataaacggtgcagctctgcc Ј]), and amplified by pcr using oligo (ϩ) and oligo leadergac(Ϫ) ( Јgcgggc-ccgggacttttaaagtaaag , which binds to the minus-strand of the leader), to create a leader-containing fragment. the product was digested with smai and bsrgi, and the large fragment was ligated in a three-way ligation reaction with the -nt bsrgi-sphi fragment of porf a- b, and with pgem- z that had been linearized with hindiii, blunt-ended with t dna polymerase, and digested with sphi. to create a strong kozak context for the a start codon, thus creating porf a(k)- b- , an overlap pcr mutagenesis procedure (horton et al., ) was used. for this, complementary mutagenesis primers . kozak(ϩ) ( Јcaa-tgtcaatggtggccctgtaatgac Ј) and . kozak(Ϫ) ( Јgtcattacagggccaccatggacattg Ј), and primers oligo (ϩ) ( Јtgccaccatacaatgtgaca Ј, which binds to bases - within orf b) and pgem zf(Ϫ)ndei(Ϫ) ( Јgagagtgcaccatatgcggtgt Ј, which binds to bases - within pgem- z), were used together in the overlap procedure to amplify a -nt product from porf a- b- dna. after digestion with restriction enzymes nari and bsrgi, the -nt product was cloned into nari-bsrgi-linearized porf a- b- . to create pcatorf a- b- (fig. c) , the orf a- b- containing -nt sphi fragment from porf a- b- was placed into the sphi site of pcat (fig. c) , which was made by cloning the bamhi fragment from pcm (pharmacia) into the bamhi site of pgem- z. for better size resolution of the large proteins, the cat gene in pcatorf a- b- was truncated by nt on its Ј end by digestions with ncoi and nuclease bal , thus forming pscatorf a- b- (fig. c) . this, along with a frameshift, resulted in a total shortening of the cat protein (now called scat) by aa. the junctions of all constructs were confirmed by sequencing plasmid dna. preparation of nested deletions within gene a, the postulated internal ribosomal entry region. to obtain deletions within gene a, pscatorf a- b- dna was linearized at base of orf a with tth i, treated with bal and mung bean nucleases, purified by electrophoresis, and religated. transformants were screened for deletions by pcr and the sequenced constructs were named for the number of bases deleted downstream of the gene a start site (the total number of deleted bases is also noted). in vitro translation and analysis of products. in vitro transcription with t rna polymerase was carried out on linearized plasmid dnas as recommended (promega biotech). porf b- was linearized with ecori, porf a- b- with scai or bamhi, as indicated, and pscatorf a- b- with asei. the -nt dna fragment from scailinearized porf a- b- was purified by affinity chromatography (geneclean; bio ) to ensure transcription of only orf a. for preparation of capped rna transcripts, . mm m g( Ј)ppp( Ј)g and . mm gtp replaced . mm gtp in the transcription mix (promega biotech). each preparation of rna was purified by biospin column chromatography (bio-rad), quantitated by spectrophotometry, and monitored for degradation by agarose gel electrophoresis. in vitro translation was carried out in methionine-depleted wheat germ extracts or rabbit reticulocyte lysates as recommended by the manufacturers (promega biotech and ambion, inc.). in some preparations, translation products were treated with rnase a before electrophoresis as recommended by ambion, inc., to remove a -kda endogenous band caused by the binding of charged trna to proteins. fifty-microliter reaction volumes contained ci s-methionine ( ci/mmol; icn) and . g of rna transcript. to test for inhibition of translation by exogenous methylated cap analog, m g( Ј)ppp( Ј)g (new england biolabs) was added to the translation mix to final concentrations of . , . , . , and . mm. radioactivity in the separated products was quantitated by scanning dried gels with the ambis photoanalytic imaging system (san diego, ca) or by scanning autoradiograms of the gels with the bio-rad imaging spectrophotometer (bio-rad). each experiment depicted was done minimally on three separate preparations of transcript rna. standard deviation measurements were made from the results of three separate experiments. sequencing of coronavirus ibv genomic rna: three open reading frames in the Ј "unique" region of mrna d genome of porcine transmissible gastroenteritis virus an efficient ribosomal frame-shifting signal in the polymerase-encoding region of the coronavirus ibv sequence of the coding regions from the . kb and . kb mrna subgenomic species from a virulent isolate of transmissible gastroenteritis virus in vitro synthesis of two polypeptides from a nonstructural gene of coronavirus mouse hepatitis virus strain a cloning and sequencing of an . kb region from the Ј end of a taiwanese virulent field isolated of the coronavirus transmissible gastroenteritis virus (tgev) scanning independent ribosomal initiation of the sendai virus x protein scanning independent ribosomal initiation of the sendai virus y proteins in vitro and in vivo complete sequence ( kilobases) of the polyproteinencoding gene of transmissible gastroenteritis virus molecular basis of transmissible gastroenteritis coronavirus (tgev) epidemiology. in "the coronaviridae the internal open reading frame within the nucleocapsid gene of mouse hepatitis virus encodes a structural protein that is not essential for viral replication nonlinear ribosome migration on cauliflower mosaic virus s rna position-dependent att initiation during plant pararetrovirus rice tungro bacilliform virus translation a stable hairpin preceded by a short open reading frame promotes nonlinear ribosome migration on a synthetic mrna leader leader-mrna junction sequences are unique for each subgenomic mrna species in the bovine coronavirus and remain so throughout persistent infection gene splicing by overlap extension: tailor-made genes using polymerase chain reaction cap-dependent and cap-independent translation by internal initiation of mrnas in cell extracts prepared from saccharomyces cerevisiae a comparative view of initiation site selection mechanisms cap-dependent and cap independent translation: operational distinctions and mechanistic interpretations sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene nucleotide sequence between the peplomer and matrix protein genes of the porcine transmissible gastroenteritis coronavirus identifies three large open reading frames the amino-terminal signal peptide on the porcine transmissible gastroenteritis coronavirus matrix protein is not an absolute requirement for membrane translocation and glycosylation the scanning model for translation: an update an analysis of vertebrate mrna sequences: intimations of translational control structural features in eukaryotic mrnas that modulate the initiation of translation the molecular biology of coronaviruses sendai virus y proteins are initiated by a ribosomal shunt distinct structural elements and internal entry of ribosomes in mrna encode by infectious bronchitis virus detection of a murine coronavirus nonstructural protein encoded in a downstream open reading frame a polycistronic mrna specified by the coronavirus infectious bronchitis virus internal entry of ribosomes on a tricistronic mrna encoded by infectious bronchitis virus coronavirus gene expression: genome organization and protein synthesis interactions between viruses and the cellular machinery for protein synthesis the major product of porcine transmissible gastroenteritis coronavirus gene b is an integral membrane glycoprotein of kda effect of upstream reading frames on translation efficiency in simian virus recombinants enteric coronavirus tgev: partial sequence of the genomic rna, its organization and expression unique features of internal initiation of hepatitis c virus rna translation bovine coronavirus i protein synthesis follows ribosomal scanning on the bicistronic n mrna the nucleocapsid gene of bovine coronavirus is bicistronic minus-strand copies of replicating coronavirus mrnas contain antileaders coronavirus subgenomic minus-strand rna and the potential for mrna replicons coronavirus mhv-jhm mrna has a sequence arrangement which potentially allows translation of a second downstream open reading frame internal ribosomal entry in the coding region of murine hepatitis virus mrna improved estimation of secondary structure in ribonucleic acids the . kilodalton hydrophobic protein encoded at the Ј end of the porcine transmissible gastroenteritis coronavirus genome is membrane associated sequence comparison of porcine respiratory coronavirus isolates reveals heterogeneity in the s, , and - genes nucleotide sequence of coronavirus tgev genomic rna: evidence for mrna species between the peplomer and matrix protein genes genetic basis for the pathogenesis of transmissible gastroenteritis virus genetic analysis of porcine respiratory coronavirus, an attenuated variant of transmissible gastroenteritis virus effect of mutations and deletions in a bicistronic mrna on the synthesis of influenza b virus nb and na glycoproteins selective translation initiation by ribosome jumping in adenovirus-infected and heat-shocked cells we thank seulah ku and gwyn williams for the construction of pcat. this work was supported by public health service grant ai from the national institutes of health and grant - - from the u.s. department of agriculture, and in part with funds from the university of tennessee college of veterinary medicine center of excellence program for livestock diseases and human health. key: cord- - aka v authors: ortego, javier; ceriani, juan e.; patiño, cristina; plana, juan; enjuanes, luis title: absence of e protein arrests transmissible gastroenteritis coronavirus maturation in the secretory pathway date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: aka v a recombinant transmissible gastroenteritis coronavirus (rtgev) in which e gene was deleted (rtgev-Δe) has been engineered. this deletion mutant only grows in cells expressing e protein (e(+) cells) indicating that e was an essential gene for tgev replication. electron microscopy studies of rtgev-Δe infected bhk-papn-e(−) cells showed that only immature intracellular virions were assembled. these virions were non-infectious and not secreted to the extracellular medium in bhk-papn-e(−) cells. rna and protein composition analysis by rnase-gold and immunoelectron microscopy showed that rtgev-Δe virions contained rna and also all the structural tgev proteins, except the deleted e protein. nevertheless, full virion maturation was blocked. studies of the rtgev-Δe subcellular localization by confocal and immunoelectron microscopy in infected e(−) cells showed that in the absence of e protein virus trafficking was arrested in the intermediate compartment. therefore, the absence of e protein in tgev resulted in two actions, a blockade of virus trafficking in the membranes of the secretory pathway, and prevention of full virus maturation. since the identification of a novel coronavirus associated with severe acute respiratory syndrome (sars), and the discovery of new human coronaviruses such as hcov-nl and hcov-nh, associated with respiratory illnesses, the interest on coronaviruses has clearly increased (esper et al., a (esper et al., , b kuiken et al., ; van der hoek et al., ) . the design of antiviral drugs interfering with coronavirus replication (hertzig et al., ) and the development of replication-competent propagation-deficient virus vectors (ortego et al., ) are potential powerful tools to prevent and control coronavirus infections. transmissible gastroenteritis coronavirus (tgev) is a member of the coronaviridae family within the nidovirales order (enjuanes et al., b) . tgev is an enveloped virus with a single-stranded, positive-sense rna genome of . kb. about two-thirds of the genome encode the replicase gene, which comprises open reading frames a and b, the last one being expressed by ribosomal frameshifting (brierley et al., ; penzes et al., ) . the ′ one-third of the genome includes structural and nonstructural genes, in the order ′-s- a- b-e-m-n- - ′ (enjuanes et al., a) . in the coronaviridae family, the viral envelope contains at least three structural proteins. the most abundant is the membrane (m) protein, spanning the membrane three or four times and interacting with the nucleocapsid (n) and spike (s) proteins during assembly (escors et al., ; rottier, ) . the second most abundant is the s protein, a large type itransmembrane glycoprotein that forms peplomers and is responsible for cell receptor binding and membrane fusion (lewicki and gallagher, ; sui et al., ; suñé et al., ; suñé et al., ) . the third is the small envelope (e) protein, a transmembrane protein detected as a minor structural component (godet et al., ; liu and inglis, ; shen et al., ; yu et al., ) . another essential constituent of the virion is the n protein, an internal phosphoprotein that interacts with the genomic rna to form the viral nucleocapsid (escors et al., ; kapke and brian, ; narayanan and makino, ) . coronavirus maturation takes place at the cis-golgi network also known as endoplasmic reticulum-golgi intermediate compartment (ergic) (salanueva et al., ; tooze et al., tooze et al., , . the e protein plays an important role during virus budding and transiently resides in a pre-golgi compartment before progressing to the golgi apparatus (corse and machamer, ; maeda et al., ; raamsman et al., ) . it has been proposed that the e protein induces virus envelope curvature in pre-golgi membranes during mhv and sars-cov infections (arbely et al., ; kuo et al., ) . studies on the assembly of coronavirus structural proteins by heterologous mammalian expression systems have shown that coexpression of e and m proteins from bovine coronavirus (bcov), mhv, tgev, ibv, and sars-cov results in the formation of virus like-particles (vlps) that are morphologically identical to spikeless virions (baudoux et al., ; corse and machamer, ; hsieh et al., ; kuo et al., ; mortola and roy, ; vennema et al., ) . in addition, it has been described that both the mhv and ibv e proteins are sufficient for the generation of vlps (corse and machamer, ; maeda et al., ) . these observations suggested that neither the n nor the s proteins are needed for viral budding. in contrast, recent studies (huang et al., ) described that m and n proteins are necessary and sufficient for the formation of sars-cov pseudoparticles. therefore, the role of e protein in coronavirus morphogenesis requires additional studies. the construction of coronavirus full length cdna clones (almazán et al., ; casais et al., ; thiel et al., ; yount et al., yount et al., , or strategies of targeted recombination (koetzner et al., ; kuo et al., ; masters, ) have allowed the manipulation of viral genomes to study coronavirus morphogenesis. recently, a recombinant mhv virus with the entire gene e deleted was constructed (kuo and masters, ) . this virus replicates with a low infectious titer, indicating that e protein is critical, but not essential for mhv replication in vitro. at variance, our laboratory has described an essential role for the e protein during tgev replication. rtgev-Δe virus was rescued by complementation within e + packaging cell lines (ortego et al., ) . in this article, we confirm that e protein is essential for tgev replication in different cell lines, and report the first evidence that tgev virions containing rna are generated in absence of e protein, although virus maturation was arrested in the budding compartment, and immature virions were accumulated between the rough endoplasmic reticulum and cis-golgi. these results provide evidence of an essential role of the e protein during tgev morphogenesis and in the intracellular virus trafficking through the secretory pathway. a replication-competent propagation-deficient rtgev-Δe virus has been developed. this virus was rescued by complementation within e + packaging cell lines (ortego et al., ) . to determine whether tgev propagation deficiency was dependent on the infected cell line, a collection of e − cell lines permissive for tgev replication (st, crfk, bhk- papn, and llc-pk ) were infected with rtgev-Δe. virus production was undetectable in infected bhk-papn, llc-pk , st, and crfk e − cell lines either after h postinfection or during four consecutive cell passages. in contrast, infection with rtgev-Δe of e + cell lines produced infective virus, with titers up to pfu/ml in bhk-papn-e + and pfu/ml in llc-pk -e + . these data indicated that the lack of infectious rtgev-Δe virus production was independent of the cell line. to confirm the propagation deficiency of rtgev-Δe, st-e − cells were infected with this virus and the propagation analyzed by plaque assay and immunofluorescence microscopy. lysis plaques were observed in monolayers of st-e − cells infected with rtgev-wt at h post-infection, whereas no plaques were generated by rtgev-Δe in these cells up to h post-infection ( (risco et al., ) but with a higher diameter (larger than -nm). mature intracellular virions were not observed in rtgev-Δe infected bhk-papn-e − cells. to determine whether mature or immature virions were secreted from rtgev-Δe infected bhk-papn-e − cells, the presence of virions in the supernatants of infected bhk-papn-e + and e − cells was analyzed after virion purification by silverstaining sds-page and negative-staining electron microscopy ( fig. ) . no structural tgev proteins were detected in the supernatants of bhk-papn-e − cells infected with rtgev-Δe at h post-infection, whereas s, m, and n proteins were detected in the supernatants of infected cells expressing e protein (fig. b ). the absence of secreted virions in bhk-papn-e − cells infected with rtgev-Δe was confirmed by negative-staining electron microscopy on -fold concentrated supernatants, whereas virions were observed in supernatants of bhk-papn-e + infected cells (fig. c ). these observations demonstrated that rtgev-Δe infected bhk-papn-e − cells did not secrete mature or immature virions. the composition of the large immature viral particles assembled in bhk-papn-e − cells was determined by immunogold detection in sections of freeze-substituted samples. mabs specific for s, m, and n proteins provided a positive signal on the rtgev-wt and rtgev-Δe virions assembled on bhk-papn-e − cells, whereas only the rtgevwt virions bound the mab specific for e protein, as expected ( fig. a) . to study whether rtgev-Δe virions incorporated rna, bhk-papn-e − cells infected with rtgev-Δe or rtgev-wt were analyzed by rnase-gold electron microscopic cytochemistry. rnase-gold complex showed similar binding levels to virions assembled on bhk-papn-e − cells infected with rtgev-Δe or rtgev-wt ( fig. b (+)) indicating that the rtgev-Δe virions incorporated rna. binding of rnasegold to ribosomes of the rough endoplasmic reticulum (rer) was also clearly observed ( fig. b (+)), whereas the rnasetreated negative controls lacked any labeling either on ribosomes or on viral particles ( fig. b (−)), confirming binding specificity. the intracellular localization of tgev structural proteins was studied in bhk-papn-e − and e + cells infected with the rtgev-Δe by indirect immunofluorescence using mabs specific for tgev s, m, n, and e proteins. the immunofluorescent signal associated with the tgev structural proteins m, n, and s was similar in bhk-papn-e − and e + cells ( fig. ) with some significative differences. thus, proteins s and m were accumulated in the perinuclear region in infected bhk-papn-e − cells, whereas specific signals for these two proteins were extended to the cytoplasm in infected bhk-papn-e + cells. immunofluorescence specific for n protein was observed in the cytoplasm of bhk-papn-e − and e + cells but, in addition, a punctuate immunofluorescence pattern that corresponded to the accumulation of secretory vesicles in late infection stages was also observed in bhk-papn-e + cells (salanueva et al., ) . the changes observed in the subcellular localization of the tgev structural proteins, in the absence of e protein, suggested an arrest in tgev intracellular transport, whereas in bhk-papn-e + cells rtgev-Δe virions progressed through the golgi to release from the cell by exocytosis. previous studies (salanueva et al., ) suggested that tgev morphological transformation from large immature particles into more condensed mature virus particles takes place during its transport along the exocytic pathway. to determine the effect of e gene deletion on tgev infection of bhk-papn-e − cells, the subcellular localization of virus particles and endoplasmic reticulum (protein disulfide-isomerase, pdi), intermediate compartment and cis-golgi (kdel receptor), and trans-golgi (giantin) specific markers were compared by immunofluorescence laser scanning confocal microscopy in rtgev-wt and rtgev-Δe infected bhk-papn-e − and e + cells at different times post-infection ( , , , and h post-infection). the immunofluorescence pattern observed with endoplasmic reticulum markers was similar in bhk-papn-e + and e − cells infected with rtgev-wt or rtgev-Δe, and in non-infected cells, at all the time points analyzed, indicating that the overall distribution of this organelle in the cell cytoplasm was not significantly affected by any of these two viruses (fig. ). only the time points and h are shown for simplicity. kdel receptor immunofluorescence specific signal in the intermediate compartment and cis-golgi was almost undetectable in rtgev-wt infected bhk-papn-e − and e + cells (fig. ). in contrast, the kdel receptor specific signal in bhk-papn-e − cells infected with rtgev-Δe showed a progressive and strong accumulation at h post-infection (fig. ) . immunoblots were performed and an increase in the amount of kdel-r was observed in rtgev-Δe infected e − cells, consistent with the result obtained by confocal microscopy (data not shown). similar effect has been described (salanueva et al., ) when tgev infection is blocked with monensin, a drug that selectively affects the golgi complex interrupting the secretory pathway (data not shown). furthermore, the infection with rtgev-Δe in presence of e protein provided in trans showed no accumulation of kdel receptor (fig. ) . these data indicated that the accumulation of ergic vesicles could be a consequence of a membrane trafficking blockade in the secretory pathway leading to the inhibition of tgev morphogenesis in the absence of e protein. staining for the golgi complex in approximately % of rtgev-wt infected bhk-papn-e + and e − cells showed fragmentation and dispersion of golgi membranes at h post-infection (fig. ) , whereas this disruption of golgi was observed in only % of rtgev-Δe infected bhk-papn-e + cells at h post-infection probably due to the delay in the infection progress. in fact, later in the infection, the disruption golgi was up to % in rtgev-Δe infected bhk-papn-e + (data not shown). in contrast, disorganization of golgi was not observed in rtgev-Δe infected bhk-papn-e − cells at the analyzed times, indicating that the virus maturation did not progress from ergic to trans-golgi in the absence of e protein. taken together, these data indicate an arrest in the virus maturation in ergic, which is the tgev budding compartment (salanueva et al., ) . to confirm that the virus particles were blocked in the ergic in the absence of e protein, immunoelectron microscopy was developed to analyze the markers of the membranebound compartments containing virus particles. a different it has been shown that e protein is essential for tgev morphogenesis. in tgev-Δe infected bhk-papn-e − cells virions containing rna were assembled, virus transportation was arrested in the ergic and full virus maturation was blocked and mature infectious virus was not secreted to the extracellular medium. previous studies have shown that, in the group coronavirus mhv and sars-cov (dediego et al., ; kuo et al., ; kuo and masters, ) , e protein is not essential for virus replication, although Δe defective mutants have a reduced growth in cell culture and in animal models. in contrast, for group coronavirus tgev, we and others (curtis et al., ; ortego et al., ) have shown that cells transfected with an e gene-deleted tgev infectious cdna do not produce infectious virus. similarly, a gene e knockout mutant of the arterivirus equine arteritis virus (eav), is unable to produce infectious progeny (snijder et al., ) . kuo and masters have suggested that the apparent lethality of e gene knockouts in eav and tgev may reflect a slow infection kinetics of these viruses that would allow uninfected cells to overgrow infected cells and obscure the detection of Δe mutants of some nidovirus (kuo and masters, ) . our results clearly showed, in this and in a fig. . detection of kdel-r and rtgev in bhk-papn-e + and e − cells by immunofluorescence. tgev infection and the ergic marker kdel-r were monitored in bhk-papn-e + and e − cells infected with rtgev-wt and rtgev-Δe at and h p.i. by immunofluorescence microscopy using a rabbit polyclonal antibody specific for tgev and a kdel-r specific mab. previous publication (ortego et al., ) , that e protein is essential for tgev replication and that no infectious virus was produced at all. a simple explanation is that these viruses belong to different cov groups and have different assembly requirements. the generation of infectious rtgev-Δe virus was dependent on e protein expression and independent of the infected cell line. thus, rtgev-Δe infected cells did not secrete virus to the extracellular medium, and the virus did not spread to neighboring uninfected cells, and therefore did not generate plaques in infected st-e − cells up to h postinfection, confirming that rtgev-Δe is a propagation-deficient virus in the absence of e protein provided in trans. furthermore, no virus amplification was observed using supernatants or cell extracts from bhk-papn-e − cells infected with rtgev-Δe virus when used to infect fresh bhk-papn-e + cells, although these cells could have amplified minute amounts of rtgev-Δe virus. these data confirmed that e protein was essential for tgev virus replication. therefore, a different behavior is shown by coronavirus from groups and in the requirement for e protein during virus assembly. the requirement of e and m proteins for the formation of virus-like particles has been previously described (vennema et al., ) . co-infection with recombinant baculoviruses showed that the membrane and envelope proteins are sufficient for the efficient formation of coronavirus vlps (mortola and roy, ) . in contrast, huang and coworkers reported that two viral genes, encoding the sars-cov m and n proteins, are necessary and sufficient for formation of vlps by cotransfection of expression plasmids (huang et al., ) . this discrepancy could be explained by the different cell types used in these studies (insect sƒ and human t cells, respectively). in our study, electron microscopy analysis of bhk-papn-e − cells fig. . detection of giantin and rtgev in bhk-papn-e + and e − cells by immunofluorescence. tgev infection and the golgi apparatus marker giantin (gig) were monitored in bhk-papn-e + and e − cells infected with rtgev-wt and rtgev-Δe at and h p.i. by immunofluorescence microscopy using a rabbit polyclonal antibody tgev specific and a giantin specific mab. infected with rtgev-Δe demonstrated that only immature tgev virions were assembled in the absence of e protein. these virions contained rna, and also all the structural tgev proteins (s, m, and n) except the deleted e protein. these results indicate that the e protein is essential for the formation of mature tgev virions. in the absence of e protein, the assembled virions displayed a morphology different from the full-length virus, were non-infectious, accumulated in the cytoplasm of the infected cells, and were not released to the extracellular medium or transported to neighboring cells. tgev replication depends on the exocytic pathway to complete its morphogenesis (salanueva et al., ) . virions were assembled as large particles with annular morphology at perinuclear compartment of the cells, and changed their morphology into small dense virions during the transport along the exocytic route (risco et al., ; tooze et al., ) . in cells infected with rtgev-Δe, an accumulation of the kdel receptor and the lack of fragmentation and dispersion of golgi membranes were observed in absence of e protein. these effects and rtgev-Δe virus assembly into mature particles were recovered by providing e protein in trans. in addition, immunoelectron microscopy studies with an intermediate compartment specific marker confirmed that, in the absence of the e protein, the maturation of the virus is arrested in the ergic. a similar effect has been described when tgev infection is blocked with monensin, a drug that selectively affects the golgi complex interrupting the secretory pathway. monensin blocks the viral transport from ergic, leading to the accumulation of numerous large viral particles in dilated pre-golgi ergic elements of vacuolar morphology (salanueva et al., ) . the comparable deficiencies observed in viral morphogenesis in the absence of e protein and as a result of treatment with monensin suggest an aberrant assembly process due to the absence of e protein. in principle, e protein could act directly on the ergic and golgi compartments, on the virus itself, or in both. it has been postulated that e protein prepares the membrane or other viral proteins occupying strategic positions within the budding pathways (salanueva et al., ) , as proposed for small acylated glycoproteins of alphaviruses and orthomyxoviruses that also display ion channel activity (gaedigk-nitschko et al., ; ivanova et al., ; liao et al., ; wilson et al., ; zebedee and lamb, ) . the absence of e protein would inhibit the progression through the ergic-golgi compartments, resulting in the accumulation of virions in the ergic and of the ergic itself. e proteins from mhv and sars-cov have viroporin activity (liao et al., ; madan et al., ) . viroporins act at late stages of the viral cycle promoting the exit of new virus particles from the cell (gonzalez and carrasco, ) . the exact mechanism by which viroporins alter the permeability of the plasma membrane is unknown, and it is possible that this activity is displayed both at the plasma membrane and on cellular organelles (aldabe et al., ; van kuppeveld et al., ) . coronavirus e protein localizes in membranes of ergic, where it could modify their permeability, leading to the disruption of ion gradients at intracellular organelles (madan et al., ) , facilitating the transport of the virions along the exocytic pathway and their maturation. the absence of this viroporin activity mediated by the e protein possibly leads to the arrest of the transport of virions through the secretory pathway, the accumulation of non-mature virions in ergig, and the absence of release of new infectious virus from the cell. the construction of e protein mutants affecting e protein ion channel activity could assess whether this postulate is correct. e proteins from the different coronavirus groups show different channel selectivity. the e protein from the group coronavirus hcov- e is k + selective, whereas from the group mhv and sars-cov, and group coronavirus ibv, e protein is na + selective (wilson et al., (wilson et al., , . the diverse channel selectivity among the coronavirus groups could be translated in differences in the effect of e protein in the secretory pathway and may explain the discrepancies among the gene e-defective mutants from the three coronavirus groups, and why e protein is essential for replication of group viruses and non-essential for replication of group viruses. additional analysis of the gene e-defective mutants during assembly is required to shed light on the functions of the e protein in coronavirus morphogenesis in the three virus groups of the coronaviridae family. in conclusion, the arrest of tgev maturation and the accumulation of virions in the secretory pathway due to the absence of the e protein confirm that this protein is essential for tgev transportation and morphogenesis. the ability to generate tgev-Δe mutants that are replication-competent but propagation-deficient by complementation in packaging cell lines, supports the potential use of these mutants as vaccine vectors. recombinant rtgev-mlui-fsei (rtgev-wt) and rtgev-Δ abΔe (rtgev-Δe) (ortego et al., (ortego et al., , were grown as described (jiménez et al., ) . baby hamster cells (bhk- ) stably transformed with the gene coding for the porcine aminopeptidase n (bhk-papn) (delmas et al., ) were grown in dulbecco's modified eagle's medium (dmem) supplemented with % fetal calf serum (fcs) containing geneticin g ( . mg/ml) as a selector agent. porcine kidney cells, llc-pk (european collection of cell cultures no. ), were grown in medium supplemented with mm glutamine and % fcs. cat kidney cells, crfk (atcc ccl ), were grown in dmem supplemented with % fcs. bhk-papn-e + and llc-pk -e + cells were grown as described previously (ortego et al., ) . standard virus titrations were performed in porcine swine testis (st) cells. titrations of virus with the e gene deleted were performed in llc-pk cells expressing the e protein. the murine mabs d.g , b.b , and b.d , specific for tgev s, m, and n proteins, respectively, have been previously described (gebauer et al., ; risco et al., ; sánchez et al., ) . the murine mab q , specific for tgev e protein, was a kind gift of h. laude (inra, jouy-en josas, france). the rabbit serum specific for pdi and the murine mab specific for giantin was generously provided by a. nieto (centro nacional de biotecnología, csic, madrid, spain) and m. renz (institute of immunology and molecular genetics, karlsruhe, germany), respectively. the murine mab specific for kdel receptor and the rabbit polyclonal antibody specific for ergic- /p were purchased from stressgen biotechnologies corp. (victoria, bc, canada) and sigma-aldrich (st. louis. mo, usa), respectively. tgev virions were sedimented through a % sucrose cushion in ten (tris-hcl mm [ph . ], edta mm, nacl m) . % tween by centrifugation in an sw beckman rotor at , rpm for h at °c. sucrose was removed and the pellet was washed with ten. pellet was recovered by suspending it in ten- . % tween and sedimented by centrifugation in a sw beckman rotor for h at , rpm. virions were recovered by suspending the pellet in tne (tris-hcl mm [ph . ], edta mm, nacl mm) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and silver staining or negative staining and electron microscopy. monolayers of bhk-papn cells expressing tgev e protein and control cells were infected with rtgev-wt or rtgev-Δe virus. the cells were fixed in situ at , , and h post-infection with a mixture of % glutaraldehyde and % tannic acid in . m hepes buffer (ph . ) for h at room temperature. the fixed monolayers were removed from the dishes in the fixative and transferred to eppendorf tubes. after centrifugation in a microcentrifuge, the cell pellets were washed with hepes buffer and processed for embedding in eml- (taab laboratories, berkshire, united kingdom) as described (risco et al., ) . the cells were post-fixed with a mixture of % osmium tetroxide and . % potassium ferricyanide in distilled water for h at °c. after four washes with hepes buffer, samples were incubated with % uranyl acetate, washed again, and dehydrated in increasing concentrations of acetone ( , , , and %) for min each at °c. infiltration in the resin eml- was done at room temperature for day. polymerization of infiltrated samples was done at °c for days. ultrathin ( -to -nm-thick) sections of the samples were stained with saturated uranyl acetate and lead citrate by standard procedures. cultures of cells were subjected to a mild fixation with a solution of % paraformaldehyde containing . % glutaraldehyde in pbs at °c for min. small pellets of chemically fixed cells were cryoprotected with glycerol, applied to small pieces of filter paper, blotted, and quick frozen in liquid ethane. vitrified specimens were transferred to a reichert-jung afs freeze-substitution unit (leica, vienna, austria) and maintained for h at − °c in a mixture of methanol and . % (w/v) uranyl acetate. after freeze-substitution, samples were infiltrated in lowicryl k m (eml laboratories) at − °c and polymerized with uv light. ultrathin sections of the samples were either stained or processed for immunogold labeling. immunogold detection of tgev proteins on ultrathinsections of infected bhk-papn cells was performed at room temperature with mabs specific for m, n, s, e, and ergic- proteins by established procedures (risco et al., ) . sections collected on formvar coated gold electron microscopy grids were incubated for min with tris buffer-gelatin and then floated on drops of diluted primary antibodies for min. after jet washing with pbs, samples were incubated for min with secondary antibodies conjugated with -nm-diameter gold particles and washed again with pbs and distilled water. samples were then allowed to dry on filter paper before being stained with saturated uranyl acetate for min, followed by min with lead citrate. all samples were analyzed with a jeol ex ii electron microscope. for ultrastructural detection of rna, a complex of rnase and -nm-diameter colloidal gold (ey laboratories, san diego, ca) was used as described previously (risco et al., ) . ultrathin sections from epon-included samples were collected on gold electron microscopy (em) grids covered with collodion and incubated for min at room temperature with the gold conjugated rnase diluted : in pbs. after being washed with pbs and distilled water, samples were stained with uranyl acetate and lead citrate. to unmask viral rna molecules, sections were subjected to a treatment before incubation with rnase-gold consisting on min incubation at °c with proteinase k ( μg/ml in tris-edta), washing with te, min fixation with % paraformaldehyde in pbs, washing again with pbs, and incubation for min with . m nh cl. as a cytochemical control, some sections were pre-incubated for min at °c with a solution of non-conjugated rnase ( μg/ml) before being treated with the rnase-gold conjugated. virus from supernatants of bhk-papn-e + and e − cells infected with rtgev-Δe virus, concentrated -fold, were analyzed by negative staining as described (bremer et al., ) . briefly, samples were adsorbed to uv light-activated copper grids for min at room temperature. grids were washed two times in h o (escors et al., ) and stained with % uranyl acetate for min. samples were visualized in a jeol exii transmission electron microscope. cells were plated on glass coverslips. infections were performed at an m.o.i.= pfu/cell at °c in dmem containing % fcs. the inoculum was removed at min and the cells were maintained in dmem % fcs. at the times indicated, cells were washed with pbs and fixed by addition of % paraformaldehyde for min at room temperature. for dual-labeling experiments in which, one primary antibody was derived from mouse and the other one from rabbit, both antibodies were combined in a pbs-fcs % diluent containing . % saponin (superfos-biosector, vedback, denmark). antibodies were allowed to adsorb for min at room temperature, and washed three times with pbs-fcs %. cells were then incubated for min at room temperature with a mixture of anti-rabbit and anti-mouse secondary antibodies conjugated to alexa or alexa . the coverslips were washed five times with pbs-fcs %, mounted on glass slides, and analyzed with a laser scanning confocal system radiance (bio-rad) on a zeiss axiovert microscope. argon ion and he-ne lasers at and nm were employed as excitation sources. image acquisition was performed using lasersharp v. software. poliovirus protein bc increases cytosolic free calcium concentrations engineering the largest rna virus 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respiratory syndrome coronavirus mouse hepatitis virus gene b protein is a new virion envelope protein influenza a virus m protein: monoclonal antibody restriction of virus growth and detection of m in virions we thank s. zuñiga, i. sola, j.l. moreno, m.l dediego, and e. alvarez for critically reading the manuscript and helpful discussions. we are also grateful to d. dorado, and r. arranz for their excellent technical assistance. this work was supported by grants from the comisión interministerial de ciencia y tecnología (cicyt), the consejería de educación y cultura de la comunidad de madrid, fort dodge veterinaria, and the european communities (projects fmdv vaccine. qrlt- - and dissect, sp -ct- - ). key: cord- -zpeh pmc authors: huang, xin; chen, jianing; yao, gang; guo, qingyong; wang, jinquan; liu, guangliang title: a taqman-probe-based multiplex real-time rt-qpcr for simultaneous detection of porcine enteric coronaviruses date: - - journal: appl microbiol biotechnol doi: . /s - - - sha: doc_id: cord_uid: zpeh pmc swine enteric coronaviruses are a group of most significant pathogens causing diarrhea in piglets with similar clinical symptoms and pathological changes. to develop a simple, rapid, accurate, and high-throughput detection method for diagnosis and differential diagnosis on swine enteric coronaviruses, specific primers and probes were designed based on the highly conserved regions of transmissible gastroenteritis virus (tgev) n, porcine epidemic diarrhea virus (pedv) m, porcine deltacoronavirus (pdcov) m, and porcine enteric alphacoronavirus (peav) n genes respectively. a taqman-probe-based multiplex real-time rt-qpcr assay was developed and optimized to simultaneously detect these swine enteric coronaviruses. the results showed that the limit of detection can reach as low as copies in singular real-time rt-qpcr assays and copies in multiplex real-time rt-qpcr assay, with all correlation coefficients (r( )) at above . , and the amplification efficiency at between and %. this multiplex real-time rt-qpcr assay demonstrated high sensitivity, extreme specificity, and excellent repeatability. the multiplex real-time rt-qpcr assay was then employed to detect the swine enteric coronavirus from field diarrheal samples. the results manifested that tgev and pdcov were the main pathogens in these samples, accompanied by co-infections. this well-established multiplex real-time rt-qpcr assay provided a rapid, efficient, specific, and sensitive tool for detection of swine enteric coronaviruses. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. porcine enteric viruses, the pathogens of viral diarrhea in pigs, caused huge economic losses in the swine industry in recent years ). there were more than ten enteric viruses discovered from swine gut in the past, including but not limited to transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv), porcine deltacoronavirus within these swine enteric viruses, coronaviruses are the most devastating pathogens responsible for acute diarrhea, vomiting, dehydration, and high mortality in neonatal and suckling piglets. according to the genetic and antigenic characters, coronavirus was divided into four genera: alpha-, beta-, gamma-, and delta-cov (woo et al. ) . during the past years, some alphacoronaviruses (pedv, tgev, and peav) and a deltacoronavirus (pdcov) emerged or reemerged in the pig farms and resulted in severe diarrhea and mortality in the early stage of suckling piglets. the tgev and pedv were the traditional causal agents responsible for diarrhea in pigs for the past decades. however, the variant strains of pedv were discovered since and circulating in pig herds thereafter, showing up to % death rate for piglets younger than -week-old (lyoo et al. ; sun et al. ) . another swine enteric virus, porcine deltacoronavirus (pdcov), was firstly discovered from healthy pig herds by a electronic supplementary material the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users. research team of hong kong in when they did a molecular epidemiology study in mammals and birds (woo et al. ) . two years later, this virus was reported to cause severe diarrhea and/or vomiting and atrophic enteritis in the usa ) and later on in china (song et al. ) . subsequently, a novel porcine enteric alphacoronavirus peav was discovered in from some pig farms located in southern china, causing more than , piglets' death (gong et al. ) . this virus was also named as seacov (pan et al. ) or sads-cov (zhou et al. b ) by the other research groups. all these four swine enteric coronaviruses, causing similar clinical symptoms and pathological changes in piglets, circulate in the pig herds and result in huge economic losses across the world in recent years. to develop a simple, rapid, accurate, and high-throughput detection method for diagnosis and differential diagnosis on swine enteric coronaviruses, a taqman-probe-based multiplex real-time rt-qpcr assay was established to simultaneously detect tgev, pedv, peav, and pdcov from the same reaction vial. to our knowledge, this is the first multiplex real-time rt-qpcr method for swine enteric coronaviruses detection. the tgev was previously propagated and preserved in our laboratory. the pedv and pdcov were isolated from clinical samples and confirmed by conventional pcr and dna sequencing (genewiz, suzhou, china). the nucleoprotein gene of peav (genbank access number: mf ) was synthesized from genewiz biotech company (suzhou, china). field samples were collected from diarrheal piglets between and from liaoning, shandong, chongqing, shaanxi, ningxia, and gansu provinces. all samples were stored at − °c until use. the primers and taqman probes for real-time qpcr assay were designed by the software beacon designer (premier biosoft international, palo alto, ca, usa). the detailed information of primers and probes were listed in table . all clinical samples were resuspended with phosphatebuffer saline (pbs), vortexed and centrifuged at , ×g at °c for min. an aliquot of μl supernatant was applied for total rna extraction with rnaiso reagent (takara, dalian, china) following the manufacturer's instruction. the total rna in rnasefree water was reversely transcribed into cdna using revertaid first strand cdna synthesis kit (thermo scientific, waltham, usa). the cdna was subjected to real-time qpcr analysis with the multiplex rt-qpcr method established in this study. the m genes of pedvand pdcov, and n genes of tgevand peav were constructed into pet- a(+) vector. the recombinant plasmids were linearized by smai enzyme digestion and recovered by pcr purification kit. the purified recombinant plasmids were quantified by spectrophotometric analysis. the copy number of recombination plasmids was calculated by using the following formula (huang et al. ): to establish the standard curves for single coronavirus, each plasmid was diluted in a tenfold series, from copies/μl to copies/μl. for multiplex standard curves, each of the four linearization plasmids was adjusted to × copies/μl and pooled with equal volume to made × copies/μl of each plasmid. the pooled plasmid was then diluted serially by tenfold to establish multiplex standard curves. all real-time qpcr reaction systems were set to a volume of μl. for single qpcr amplifying tgev, pedv, and pdcov, μl × transstart probe qpcr supermix (transgene, beijing, china), nm primers and probe each, μl plasmid dna template, and . μl nuclease-free water were pooled and mixed. for peav amplification, μl × transstart probe qpcr supermix (transgene, beijing, china), nm each primer, nm probe, μl plasmid dna template, and . μl nuclease-free water were pooled and mixed. all reactions were amplified on a bio-rad cfx ™ real-time system (bio-rad, hercules, ca, usa) at °c for s, followed by cycles of °c for s and °c for s. for reaction system of multiplex real-time pcr, μl × transstart probe qpcr supermix combined with all primers, probes, templates, and nuclease-free water to a final volume of μl. the concentrations of each primer and probe of pedv, pdcov, tgev, and peav were optimized for better outputs. the amplifying cycles of multiplex qpcr were carried out as same as singular real-time qpcr. the cq value higher than was considered negative. all qpcr results were analyzed by cfx manager™ software. to analyze the sensitivity of established multiplex rt-qpcr, the linearization standard plasmids prepared above were diluted tenfold serially to a final concentration between . × copies/μl and . × copies/μl in nuclease-free water. the diluted standard plasmids were used as templates for realtime qpcr amplification. to estimate the specificity of this established multiplex rt-qpcr, standard dnas, or cdnas of major swine viruses, including porcine astrovirus (pastv), porcine kobuvirus (pkv), type o foot-and-mouth disease virus (fmdv-o), type a foot-and-mouth disease virus (fmdv-a), porcine reproductive and respiratory syndrome virus (prrsv), classical swine fever virus (csfv), porcine rotavirus (prv), porcine circovirus (pcv ), and pseudorabies virus (prv) were used as templates for amplification. the nuclease-free water was served as negative template control. to evaluate its repeatability, tenfold serially diluted standard template between . × copies/μl to . × copies/μl were used to test the coefficients of variation of real-time pcr. for intra-assay repeatability, all samples were triplicated. for inter-assay repeatability, the assays were repeated three times individually at different locations. a total of fecal samples were collected from diarrheal pig farms located in liaoning, shandong, chongqing, shaanxi, ningxia, and gansu provinces of china between and . all samples were diluted fivefold with sterile phosphate-buffered saline (pbs), vortexed, and centrifuged at ×g at °c for min. the supernatant was collected and used to extract viral rna with trizol reagent (invitrogen, carlsbad, ca, usa). the cdnas were generated by reverse transcript system (promega, madison, wi, usa) using extracted total rna as templates and hexamer random primers. all cdna from clinical samples were measured by the multiplex rt-qpcr assay developed in this study. to develop a multiplex real-time rt-qpcr, the single real-time rt-qpcrs for detection of the individual virus were firstly established with different fluorescence-labeled target probes, for details, fam for pedv m gene, cy for pdcov m gene, hex for tgev n gene, and texasred for peav n gene. the standard curves for each virus were generated using . × copies to . × copies of tenfold serially diluted linearized plasmids conceiving target genes. the results demonstrated that the single real-time rt-qpcr assays for each virus were successfully established at the limit of detection at approximately copies (fig. ). all the standard curves showed an excellent correlation coefficient and amplification efficacy, for instance, pedv (r = ; eff% = . ), pdcov (r = ; eff% = . ), tgev (r = . ; eff% = . ), and peav (r = . ; eff% = . ), indicating that the single real-time rt-qpcr for each virus was valid and reliable. to establish the multiplex real-time rt-qpcr method, all primer sets, probes, and serially diluted standard plasmids for detection of pedv, pdcov, tgev, and peav were mixed with × transstart probe qpcr supermix and nuclease-free water. the concentrations of each primer and probe were optimized for the optimum output. the optimal final concentrations of primers and probes were as follows: nm primer and nm probe for pedv, nm primer and nm probe for pdcov, nm primer and nm probe for tgev, and nm primer and c the amplification curves (top) and a standard curve (bottom) for detection of tgev n gene were generated. the probe was labeled with hex at ′-end and bhq at ′-end. d the amplification curves (top) and a standard curve (bottom) for detection of pedv n gene were generated. the probe was labeled with texasred at ′-end and bhq at ′-end nm probe for peav. the results demonstrated that the multiplex real-time rt-qpcr could detect all target genes of these four viruses efficiently with high correlation values (fig. ) . all the standard curves showed excellent correlation coefficient and amplification efficacy, for details, pedv (r = . ; eff% = . ), pdcov (r = . ; eff% = . ), tgev (r = . ; eff% = . ), and peav (r = ; eff% = . ) (fig. ) . the limit of detection of this multiplex rt-qpcr was approximately copies of each virus per reaction (fig. ) . the specificity of multiplex real-time rt-qpcr assay to evaluate the specificity of the multiplex rt-qpcr developed in this study for swine enteric coronavirus detection, the dnas/cdnas of nine other major swine viruses were used as templates for amplification with this multiplex system. the cdnas of pedv, pdcov, tgev, and peav were served as positive control while the nuclease-free water was used as a negative control. the results displayed that all swine enteric coronaviruses were successfully detected. however, there was no positive signal detected from other nine swine viruses, pastv, pkv, fmdv-o, fmdv-a, prrsv, csfv, prv, pcv , prv, and negative control using this multiplex rt-qpcr system (fig. ) , demonstrating that the taqmanprobe-based multiplex rt-qpcr system was highly specific. to determine the sensitivity of this multiplex real-time rt-qpcr, tenfold serial dilution of linearized plasmid mixtures were added to the amplification system. the results showed that . × copies of tgev, pedv, pdcov, and peav were detectable with the ct values at . , . , . , and . respectively ( table ). however, . × copies of each target were not detectable in the same amplification system (table ) . also, the amplification exhibited reliable and high efficacy, pedv (r = . ; eff% = . ), pdcov (r = . ; eff% = . ), tgev (r = . ; eff% = . ), and peav (r = . ; eff% = . ). this multiplex real-time rt-qpcr demonstrated the same sensitivity level when using viral rnas as templates (supplemental fig. s ). these results implied that the taqman fig. establishment of multiplex real-time rt-qpcr. a amplification curves and b standard curves of optimized multiplex taqman-probe-based realtime rt-qpcr for detection of pedv, pdcov, tgev, and peav were generated at the optimum amplification conditions. the correlation coefficient and amplification efficacy of the standards curves, pedv (r = . ; eff% = . ), pdcov (r = . ; eff% = . ), tgev (r = . ; eff% = . ), and peav (r = ; eff% = . ), were ideal for detecting the target genes enteric coronaviruses (pedv, tgev, pdcov, and peav) as less as to copies. to estimate the reproducibility of the multiplex real-time pcr, tenfold serial dilution of pooled linearization plasmids were in a triplicate manner for both intra-assay and inter-assay. for intra-assay, the standard plasmids were amplified three times simultaneously. for inter-assay, standard curves were done at three individual times and using a different batch of a standard substance. as shown in table , the coefficient of variation for pedv intra-assay was . - . % and interassay was . - . %. the pdcov amplification exhibited the coefficients of variation at . - . % and . - . % for inter-assay and intra-assay respectively. as for tgev, the coefficients of variation were . - . % and . - . % corresponding to intra-assay and inter-assay. the amplification of peav showed relative higher coefficients of variation, which was . - . % for intra-assay and . - . % for inter-assay. these results indicated that the taqman-probe-based multiplex real-time rt-qpcr assay established in this study was repeatable and reliable. the cdnas originated from clinical samples were subjected to amplification by this multiplex real-time rt-qpcr and confirmed by conventional rt-pcr (supplemental fig. s ). the results demonstrated that the samples from henan and shaanxi provinces were swine enteric coronavirusnegative while the gansu province suffered a high prevalence of coronaviruses, showing . % ( / ) tgev, . % ( / ) pdcov, and . % ( / ) pedv positive (table ). in ningxia province, tgev was also highly prevalent but the positive rates for the other three swine enteric coronaviruses were quite low or not detectable (table ). only pedv ( / , . %) and pdcov ( / , . %) among these four coronaviruses were detectable from clinical samples of chongqing. all these four swine enteric coronaviruses were detectable in samples of liaoning province, with tgev positive rate at . % ( / ) and the other three were less than % (table ) . overall, pdcov ( / , . %) and tgev ( / , . %) were the main coronaviruses detected from all these clinical samples. the positive rate for the newly emerged coronavirus in guangdong, china, peav, was rarely found from these provinces. we further analyzed the co-infection of coronaviruses for all these positive samples. the results elucidated that the main co-infection were caused by a dual coronavirus, for example, pdcov and tgev co-infection samples, five pedv and pdcov co-infection samples, and four pedv and tgev co-infection samples (fig. ) . some clinical samples were co-infected by three ( pedv, pdcov, and tgev co-infection) or all four coronaviruses (fig. ) . these results indicated the pathogens of viral diarrhea disease in chinese pig farms were complicated. porcine diarrheal disease is one of the most severe diseases in pig farms. swine enteric coronaviruses are the most significant pathogens causing diarrhea in piglets, especially for newborn piglets (butler et al. ). all swine enteric coronaviruses, including traditional pedv, tgev, and newly emerged pdcov, peav, could cause serious diarrhea, vomiting, dehydration, weight loss, and up to % death in suckling piglets (gong et al. ; hsu et al. ) . previously, some investigators developed a serial of elisa assays to detect pedv, tgev, or pdcov infections based on m, n proteins or whole virus (luo et al. ; ma et al. ; su et al. ). however, molecular diagnostic tools, rather than serological methods, for swine enteric coronaviruses are urgently needed due to their similar and high pathogenicity to suckling piglets, lacking a mature immune system. for swine enteric coronavirus detection, conventional rt-pcr, multiplex rt-qpcr, or pan-coronaviruses rt-pcr methods targeting m, n, s, or polymerase genes were developed in recent years (hsu et al. ; song et al. ) . additionally, zhou et al. ( a) recently reported a specific real-time pcr for the detection of peav. to our knowledge, there was no such method that could simultaneously detect and differentiate tgev, pedv, pdcov, and peav from the same detection vial. to solve this urgent and important issue, we developed this multiplex real-time rt-qpcr for the detection and differential diagnosis of the swine enteric coronaviruses circulating in pig herds. taqman-probe-based real-time qpcr is a rapid, high specificity, high sensitivity, and great reproducibility detection tool for identification of viruses (chang et al. ). among the seven open reading frames (orfs) and four structural protein genes, s (spike), e (envelope), m (membrane), n (nucleocapsid) of coronaviruses (su et al. ), the m and n genes are highly conserved within the same antigenic group but less homologous between each of these four swine enteric coronaviruses (yang et al. ) . to establish a high-specific multiplex real-time rt-qpcr for the detection and differential diagnosis of swine enteric coronaviruses, primer sets, and taqman probes were designed targeting the highly conserved regions of pedv or pdcov m genes and tgev or peav n genes, based on bioinformatics analysis of each virus. our results demonstrated that each primer and probe set can only detect target gene itself and could not bind with any other targets, indicating high specificity. our results showed that the singular real-time rt-qpcrs were capable to detect as less as copies of pedv, pdcov, tgev, and peav templates. however, the detection limit of each target gene in the multiplex real-time rt-qpcr was approximately copies, implying that the sensitivity of multiplex real-time rt-qpcr was lower than that of singular realtime rt-qpcr probably due to the competition between primers, probes, templates, and reagents. the analysis of clinical diarrhea samples using this developed multiplex rt-qpcr elucidated that the co-infection of swine enteric coronaviruses commonly existed in some pig farms. co-infection of swine enteric coronaviruses may cause recombination between co-infected viruses. during and , some new swine enteric coronaviruses were generated by recombination with pedv and tgev and spread across the central eastern european countries (akimkin et al. ; belsham et al. ; boniotti et al. ) . the recombination might create more virulent enteric virus strains or new viruses, leading to potential outbreaks or pandemics of swine viral diarrhea. additionally, co-infections may promote the evolution of non-pathogenic enteric viruses into high virulent and pathogenic viruses. a recent example showed the existence of pdcov in pig herds for many years in china mainland and hong kong but without observed clinical symptoms (pan et al. ; woo et al. ) . unfortunately, this virus caused severe outbreaks in some states of the usa since (ma et al. ; wang et al. ). this virulence change was most probably caused by the previous exposure to pedv or other co-infected swine enteric viruses. therefore, swine enteric coronaviruses co-infection may bring risks for the prevention and control of swine diarrhea. in summary, we developed a taqman-probe-based multiplex real-time rt-qpcr with high specificity and sensitivity for simultaneous detection and differential diagnosis of swine enteric coronaviruses, pedv, tgev, pdcov, and peav. this real-time rt-qpcr assay is of great significance for the prevention and control and epidemiological investigation of swine viral diarrhea. conflict of interest the authors declare that they have no competing interests. ethical statement this article does not contain any studies with human participants or animals performed by any of the authors. new chimeric porcine coronavirus in swine feces characterization of a novel chimeric swine enteric coronavirus from diseased pigs in central eastern europe in porcine epidemic diarrhea virus and discovery of a recombinant swine enteric coronavirus porcine reproductive 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of gammacoronavirus and deltacoronavirus generation, identification and functional analysis of monoclonal antibodies against porcine epidemic diarrhea virus nucleocapsid evaluation of two singleplex reverse transcription-insulated isothermal pcr tests and a duplex real-time rt-pcr test for the detection of porcine epidemic diarrhea virus and porcine deltacoronavirus development of a taqman-based real-time rt-pcr assay for the detection of sads-cov associated with severe diarrhea disease in pigs fatal swine acute diarrhoea syndrome caused by an hku -related coronavirus of bat origin publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -oilurica authors: cui, tingting; theuns, sebastiaan; xie, jiexiong; den broeck, wim van; nauwynck, hans j. title: role of porcine aminopeptidase n and sialic acids in porcine coronavirus infections in primary porcine enterocytes date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: oilurica porcine epidemic diarrhea virus (pedv) and transmissible gastroenteritis virus (tgev) have been reported to use aminopeptidase n (apn) as a cellular receptor. recently, the role of apn as a receptor for pedv has been questioned. in our study, the role of apn in pedv and tgev infections was studied in primary porcine enterocytes. after seven days of cultivation, % of enterocytes presented microvilli and showed a two- to five-fold higher susceptibility to pedv and tgev. a significant increase of pedv and tgev infection was correlated with a higher expression of apn, which was indicative that apn plays an important role in porcine coronavirus infections. however, pedv and tgev infected both apn positive and negative enterocytes. pedv and tgev miller showed a higher infectivity in apn positive cells than in apn negative cells. in contrast, tgev purdue replicated better in apn negative cells. these results show that an additional receptor exists, different from apn for porcine coronaviruses. subsequently, treatment of enterocytes with neuraminidase (na) had no effect on infection efficiency of tgev, implying that terminal cellular sialic acids (sas) are no receptor determinants for tgev. treatment of tgev with na significantly enhanced the infection which shows that tgev is masked by sas. coronaviruses are known as human and animal pathogens that mainly infect the epithelium of the respiratory or intestinal tract. porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev), and its variant porcine respiratory coronavirus (prcv) are classified as alphacoronavirus. they are enveloped viruses containing a single-stranded, positive-sense rna genome of approximately . kb. the positive ssrna serves as mrna for the generation of viral replicative proteins by translation of open reading frame (orf) a and orf b. the genome contains a untranslated region (utr), a utr, and at least seven orfs. orf a and orf b make up two-thirds of the viral genome and encode the non-structural replicase polyproteins (replicases a and b), which further guide the viral replication and translation, regulate cellular processes, and potentially fulfill other unknown functions. the remaining proximal third of the genome encodes four structural proteins (spike (s), envelope (e), membrane (m), and nucleocapsid (n) by orf , orf , orf , and orf , respectively). the s protein is a type i glycoprotein that projects from the virions surface forming in the present study, the co-culture system of primary porcine enterocytes that was established in our laboratory was used to study the role of apn in pedv and tgev infection in their target cells [ ] . further, it was investigated whether sialic acids are cellular receptors for tgev in primary enterocytes. primary porcine enterocytes were isolated from the ileum of three-day-old piglets and co-cultured with porcine myofibroblasts [ ] . euthanizing piglets was done in agreement with the european legislation on animal experiments. all experimental procedures were approved by the local ethical committee of the faculty of veterinary medicine, ghent university (ec / ), and all methods were carried out in accordance with the approved guidelines. the enterocytes were maintained with dulbecco's modified eagle's-f ham medium (dmem-f ). tgev purdue and miller grown on swine testicle (st) cells and pedv cv strain grown on vero cells were used in this study. pedv cv fecal suspension was collected from a three-day-old infected suckling piglet. a twenty percent fecal suspension was prepared in phosphate buffered saline (pbs) containing u/ml penicillin (continental pharma, puurs, belgium), mg/ml streptomycin (certa, braine l'alleud, belgium), mg/ml gentamicin (gibco brl, merelbeke, belgium), and . % v/v fungizone (bristol-myers squibb, braine l'alleud, belgium). hydrocortisone, spermidine, and wnt agonist were purchased from sigma-aldrich (sigma, st-louis, mo, usa). porcine insulin was purchased from protein specialists (prospec, rehovot, israel). the small intestinal contents (ic) were collected from the duodenum of a three-day-old suckling piglet. after euthanasia, a cm long segment of duodenum was closed by two surgical clamps and was removed from a piglet. then, one clamp was removed and the intestinal contents were released from the lumen into a ml centrifugation tube. in order to collect all the intestinal contents, the lumen was washed once by filling it with ml dulbecco's modified eagle's medium (dmem) containing u/ml penicillin, mg/ml streptomycin, mg/ml gentamicin, and . % v/v fungizone. then, the dmem was released from the lumen into the ml tube that already contained the intestinal contents. after centrifugation ( rpm, min at • c), the supernatant of the intestinal contents was collected and stored at − • c until use. three-and seven-day-old primary porcine enterocytes were fixed in hepes-buffered glutaric aldehyde (sigma, st-louis, mo, usa) for scanning electron microscopy as described previously [ ] . after h fixation, the samples were treated with % osmium tetroxide for h at room temperature (rt), followed by ascending grades of alcohol dehydration. in order to avoid the water vaporization obstructing the electron beam and interfering with image clarity, the dehydrated samples were transferred to a critical point drier (cpd, bal-tec, balzers, liechtenstein) for complete drying. finally, the dried samples were mounted on a metal stub and were sputter-coated with platinum. the microvilli of all the samples were acquired with a jeol jsm lv scanning electron microscope (jeol ltd., tokyo, japan). twenty-four h post co-cultivation, monolayers of enterocytes were cultured with medium containing hydrocortisone ( and µg/ml), spermidine ( and µm), insulin ( and µg/ml), wnt agonist ( . µm), or intestinal contents ( %) for h. the cells were fixed with % paraformaldehyde for min and immunofluorescence staining was conducted for apn expression analysis. cells were incubated with mouse monoclonal anti-porcine apn antibodies (imm ; kindly provided by prof. eric cox, ghent university) containing % normal goat serum for h at • c, followed by goat anti-mouse-igg fitc labeled antibodies for h at • c. nuclei were stained with hoechst for min at rt. the percentage of apn positive cells were analyzed by fluorescence microscopy (leica microsystems gmbh). at twenty-four h of co-cultivation, monolayers of enterocytes were treated with µg/ml hydrocortisone, µm spermidine, µg/ml insulin, . µm wnt agonist, or % intestinal contents for another h. then, the susceptibility of treated enterocytes to pedv and tgev was tested. cells were inoculated with µl of tgev miller at a multiplicity of infection (m.o.i.) of and µl of the pedv cv vero adapted strain at . tcid /ml or viral rna copies/ml of fecal suspension with µg/ml trypsin. after min of incubation at • c, unbound viral particles were removed by three washing steps with dmem. cells were further incubated in medium containing µg/ml hydrocortisone, µm spermidine, µg/ml insulin, . µm wnt agonist, or % intestinal contents for h ( • c, % co ) and fixed with % paraformaldehyde for immunofluorescence staining. to determine the co-localization of viral antigens and apn, co-cultured enterocytes were infected with tgev miller and purdue and pedv vero adapted and non-adapted strains. after h incubation, cells were fixed with % paraformaldehyde for min and immunofluorescence staining was performed. cells were incubated with mouse monoclonal anti-porcine apn antibodies containing % normal goat serum for h at • c, followed by goat anti-mouse-igg fitc labeled antibodies for h at • c. then, cells were permeabilized with . % triton x- for min at rt and incubated with mouse monoclonal anti-porcine pedv antibodies (kindly provided by prof. luis enjuanes, national center for biotechnology) or swine polyclonal anti-tgev antibodies [ ] containing % normal goat serum for h at • c, followed by goat anti-mouse igg a af labeled antibodies or goat anti-swine texas red labeled antibodies (molecular probes). nuclei were stained with hoechst for min at rt and the results were analyzed by a leica tcs spe laser scanning spectral confocal system linked to a dm b fluorescence microscope (leica microsystems). to remove sas from enterocytes, monolayers of co-cultured enterocytes were washed three times with warm dmem. then, cells were incubated with mu/ml na from vibrio cholerae (roche diagnostics, risch-rotkreuz, switzerland) at • c for h. cells that were mock-treated were incubated with dmem and underwent the same manipulations as na-treated cells. to remove sas from the virus, virus suspensions were incubated with mu/ml na from vibrio cholerae at • c for h. the mock-treated virus was incubated in dmem and underwent the same manipulations as the na-treated virus. afterwards, cells were inoculated with either na-treated or mock-treated virus (m.o.i of for tgev purdue and miller). after min incubation at • c, cells were washed three times with medium and further incubated in medium for h ( • c, % co ). then, cells were fixed with % paraformaldehyde for min at rt. immunofluorescence was performed to measure the percentage of infected cells. the cells were permeabilized with . % triton x- for min at rt. then, cells were incubated with swine polyclonal anti-tgev antibodies containing % normal goat serum for h at • c, followed by goat anti-swine-igg fitc labelled antibodies for h at • c. nuclei were stained with hoechst for min at rt. the percentages of infected cells were determined by fluorescence microscopy. to determine the co-localization of viral antigens and sas, co-cultured enterocytes were infected with tgev miller and purdue. after h incubation, cells were fixed with % paraformaldehyde for min and a double immunofluorescence staining was performed. cells were incubated with biotinylated maackia amurensis lectin ii (vector laboratories) for h at • c. the lectin was subsequently stained with streptavidin-fitc (invitrogen) for h at • c. then, cells were permeabilized with . % triton x- for min at rt and incubated with swine polyclonal anti-tgev antibodies containing % normal goat serum for h at • c, followed by goat anti-swine-igg texas red labeled antibodies. nuclei were stained with hoechst for min at rt and results were analyzed by a leica tcs spe laser scanning spectral confocal system linked to a dm b fluorescence microscope. to characterize the attachment of tgev to primary enterocytes, direct virus-binding studies were carried out with tgev particles. cells were chilled on ice for min and washed three times with dmem. then, cells were inoculated with tgev miller and purdue particles at a m.o.i. of for h at • c. unbound virus particles were removed by three washings with dmem. cells were fixed with % paraformaldehyde for min and a double immunofluorescence staining was performed. cells were incubated with biotinylated maackia amurensis lectin ii or mouse monoclonal anti-porcine apn antibodies for h at • c, followed by streptavidin-fitc or goat anti-mouse-igg fitc labeled antibodies for h at • c. then, cells were permeabilized with . % triton x- for min at rt and incubated with swine polyclonal anti tgev antibodies containing % goat serum for h at • c, followed by goat anti-swine-igg texas red labeled antibodies. nuclei were stained with hoechst for min at rt and the results were analyzed by a leica tcs spe laser scanning spectral confocal system linked to a dm b fluorescence microscope. data were statistically processed by spss (t-test). the data are represented as means with standard deviation (sd) of three independent experiments. results with p-values of < . were considered significant. the percentages of microvilli positive enterocytes were analyzed by scanning electron microscopy. a higher percentage of microvilli positive cells ( %) was observed at seven days cultivation compared to three days cultivation ( %). the expression of apn at seven days cultivation ( . ± . %) was significantly higher than at three days cultivation ( . ± . %; figure a ,b). the data suggest that primary enterocytes underwent a differentiation process in vitro. they terminally differentiate into mature enterocytes during the long cultivation time. next, enterocytes were inoculated with tgev and pedv at three and seven days of cultivation. the results showed that a significantly higher infection was detected in enterocytes at seven days cultivation (miller: . ± . %; purdue: . ± . %) than at three days cultivation (miller: . ± . %; purdue: . ± . %) for tgev. an increased trend of infection was detected in enterocytes at seven days cultivation ( infected cells per well) than at three days cultivation ( infected cells per well) for pedv but without significance (p = . ; figure c ). data are expressed as mean ± standard deviation (sd) of the results of three separate experiments. statistically significant differences in comparison with data from three days cultivation are presented as *p < . or **p < . . cells were treated with hydrocortisone, spermidine, porcine insulin, wnt agonist, or small intestinal contents to analyse their effects on enterocyte differentiation. cells were treated with the aforementioned products for h. afterwards, the differentiation marker apn was stained by immunofluorescence and the percentage of apn positive cells was counted ( figure a ). the results showed that without treatment, . ± . % of cells were apn positive. the treatment with and µg/ml hydrocortisone significantly increased the apn expression to ± . % and ± . %, respectively. the treatment with mm and mm spermidine significantly enhanced the apn expression to ± . % and ± . %, respectively. similarly, and µg/ml insulin treatment significantly increased the apn expression to ± . % and ± . %, respectively. since there was no dose-dependent enhancement, the lower concentration of each product ( µg/ml of hydrocortisone, µm of spermidine and µg/ml of porcine insulin) was used for the next cells were treated with hydrocortisone, spermidine, porcine insulin, wnt agonist, or small intestinal contents to analyse their effects on enterocyte differentiation. cells were treated with the aforementioned products for h. afterwards, the differentiation marker apn was stained by immunofluorescence and the percentage of apn positive cells was counted ( figure a ). the results showed that without treatment, . ± . % of cells were apn positive. the treatment with and µg/ml hydrocortisone significantly increased the apn expression to ± . % and ± . %, respectively. the treatment with mm and mm spermidine significantly enhanced the apn expression to ± . % and ± . %, respectively. similarly, and µg/ml insulin treatment significantly increased the apn expression to ± . % and ± . %, respectively. since there was no dose-dependent enhancement, the lower concentration of each product ( µg/ml of hydrocortisone, µm of spermidine and µg/ml of porcine insulin) was used for the next experiment. the treatment of wnt agonist and intestinal contents showed a trend of increased apn expression up to ± % (p = . ) and ± . % (p = . ), respectively ( figure b ). to determine the effect of apn on coronavirus replication, the enterocytes were precultured with µg/ml hydrocortisone, µm spermidine, µg/ml insulin, . µm wnt agonist, or % intestinal contents for h prior to inoculation with pedv cv vero adapted strain, cv fecal suspension, and tgev miller. for pedv cv vero adapted strain, only ± cells were infected per well without pretreatment. the highest infection ( ± infected cells per well) was observed in cells that were pretreated with intestinal contents. because of the variation between the three replicates, the treatment with intestinal contents was not significantly different from the mock treatment (p = . ). when cells were pretreated with wnt agonist, a significant increase of infection ( ± infected cells per well) was observed (p = . ). an increased trend of infection (but not significantly different) was observed after pretreatment with hydrocortisone ( ± infected cells per well, (p = . ), spermidine ( ± infected cells per well, p = . ), and porcine insulin ( ± infected cells per well, p = . ). for cv fecal suspension, the highest infection was observed when to determine the effect of apn on coronavirus replication, the enterocytes were precultured with µg/ml hydrocortisone, µm spermidine, µg/ml insulin, . µm wnt agonist, or % intestinal contents for h prior to inoculation with pedv cv vero adapted strain, cv fecal suspension, and tgev miller. for pedv cv vero adapted strain, only ± cells were infected per well without pretreatment. the highest infection ( ± infected cells per well) was observed in cells that were pretreated with intestinal contents. because of the variation between the three replicates, the treatment with intestinal contents was not significantly different from the mock treatment (p = . ). when cells were pretreated with wnt agonist, a significant increase of infection ( ± infected cells per well) was observed (p = . ). an increased trend of infection (but not significantly different) was observed after pretreatment with hydrocortisone ( ± infected cells per well, (p = . ), spermidine ( ± infected cells per well, p = . ), and porcine insulin ( ± infected cells per well, p = . ). for cv fecal suspension, the highest infection was observed when cells were pretreated with the wnt agonist ( ± infected cells per well), showing a four-fold higher infection compared to mock-treated cells ( ± infected cells per well). when cells were pretreated with intestinal contents, ± cells were infected. for tgev miller, incubation with intestinal contents, porcine insulin, and wnt agonist significantly increased the virus infection from . ± . % to . ± . %, . ± . % and . ± . %, respectively. hydrocortisone and spermidine increased the virus infection to . ± . % and . ± . % (figure ) . the results show that pretreatment of primary enterocytes with hydrocortisone, spermidine, porcine insulin, wnt agonist, and intestinal contents could stimulate the expression of apn and enhance the infection of pedv cv vero adapted and non-adapted strains and the tgev miller in the enterocytes. statistically significant differences in comparison with data from mock treatment are presented as *p < . or **p < . . to assess the role of apn in the replication of pedv and tgev, a double immunofluorescence staining of apn and virus was performed. for cv vero adapted strain, . ± . % of apn positive cells and . ± . % of apn negative cells were infected. for cv fecal suspension, . ± . % of apn positive cells and . ± . % of apn negative cells were infected. for tgev miller, . ± . % of apn positive cells and . ± % of apn negative cells were infected. for tgev purdue, more infection was found in apn negative cells ( . ± . %) than in apn positive cells ( . ± . %; figure ). the results suggest that for pedv and tgev miller, apn may be the predominant receptor, while tgev purdue mainly uses an additional receptor for virus infection. to assess the role of apn in the replication of pedv and tgev, a double immunofluorescence staining of apn and virus was performed. for cv vero adapted strain, . ± . % of apn positive cells and . ± . % of apn negative cells were infected. for cv fecal suspension, . ± . % of apn positive cells and . ± . % of apn negative cells were infected. for tgev miller, . ± . % of apn positive cells and . ± % of apn negative cells were infected. for tgev purdue, more infection was found in apn negative cells ( . ± . %) than in apn positive cells ( . ± . %; figure ). the results suggest that for pedv and tgev miller, apn may be the predominant receptor, while tgev purdue mainly uses an additional receptor for virus infection. to assess the role of sas as receptor determinants, enterocytes were pretreated with mu/ml na prior to inoculation with tgev miller and purdue. miller infected . ± . % of the na-treated enterocytes and . ± . % of mock-treated cells. for purdue, the percentage of infection was . ± . % and . ± . % for na-treated and mock-treated cells, respectively. this implies that tgev does not depend on terminal sa residues on the enterocytes surface for infection. since it has been reported that removal of sas on the surface of coronaviruses improves binding and infection [ ] , the replication of mock-treated, and na-treated viruses was compared in untreated epithelial cells. na pretreatment of virus significantly enhanced infection from . ± . % to . ± . % for miller. for purdue, na pretreatment of virus significantly increased infection from . ± . % to . ± . % ( figure ). these data show that removal of sa from tgev promotes binding and replication of tgev in enterocytes. viruses , , of to assess the role of sas as receptor determinants, enterocytes were pretreated with mu/ml na prior to inoculation with tgev miller and purdue. miller infected . ± . % of the na-treated enterocytes and . ± . % of mock-treated cells. for purdue, the percentage of infection was . ± . % and . ± . % for na-treated and mock-treated cells, respectively. this implies that tgev does not depend on terminal sa residues on the enterocytes surface for infection. since it has been reported that removal of sas on the surface of coronaviruses improves binding and infection [ ] , the replication of mock-treated, and na-treated viruses was compared in untreated epithelial cells. na pretreatment of virus significantly enhanced infection from . ± . % to . ± . % for miller. for purdue, na pretreatment of virus significantly increased infection from . ± . % to . ± . % ( figure ). these data show that removal of sa from tgev promotes binding and replication of tgev in enterocytes. data are expressed as mean ± sd of the results of three separate experiments. statistically significant differences in comparison with data from mock treatment are presented as *p < . . double immunofluorescence staining was further performed to assess the role of sa on tgev replication. tgev miller could infect both sa positive cells ( . ± . %) and sa negative cells ( . ± . %). the tgev purdue replicated slightly better in sas negative cells ( . ± . %) compared to sas positive cells ( . ± . %; figure ). double immunofluorescence staining was further performed to assess the role of sa on tgev replication. tgev miller could infect both sa positive cells ( . ± . %) and sa negative cells ( . ± . %). the tgev purdue replicated slightly better in sas negative cells ( . ± . %) compared to sas positive cells ( . ± . %; figure ). primary enterocytes were inoculated with tgev particles (m.o.i. = ) at °c. the binding of tgev to apn positive/negative and sas positive/negative cells was examined by double immunofluorescence staining. no significant differences were observed between the percentage of apn positive cells with bound tgev miller ( . ± . %) and the percentage of apn negative cells with bound tgev miller ( . ± . %; p = . ). the percentage of apn positive cells with bound tgev purdue ( . ± . %) was lower than the percentage of apn negative cells with bound tgev purdue ( . ± . %), but was not significantly different (p = . ). the percentage of miller particles that colocalized with apn ( ± %) was significantly higher than non-colocalized particles ( ± %). the percentage of purdue particles that colocalized with apn ( ± %) was significantly lower than non-colocalized particles ( ± %; figure ) . the percentage of sa negative cells with bound tgev miller ( . ± . %) and with bound tgev purdue ( . ± . %) was significantly higher than the percentage of sa positive cells with bound tgev miller ( . ± . %) and with bound tgev purdue ( . ± . %). the percentage of tgev miller particles ( ± %) and tgev purdue ( ± %) that colocalized with sa was significantly lower than the particles that did not colocalize, with miller at ± % and purdue at ± % (figure ). primary enterocytes were inoculated with tgev particles (m.o.i. = ) at • c. the binding of tgev to apn positive/negative and sas positive/negative cells was examined by double immunofluorescence staining. no significant differences were observed between the percentage of apn positive cells with bound tgev miller ( . ± . %) and the percentage of apn negative cells with bound tgev miller ( . ± . %; p = . ). the percentage of apn positive cells with bound tgev purdue ( . ± . %) was lower than the percentage of apn negative cells with bound tgev purdue ( . ± . %), but was not significantly different (p = . ). the percentage of miller particles that colocalized with apn ( ± %) was significantly higher than non-colocalized particles ( ± %). the percentage of purdue particles that colocalized with apn ( ± %) was significantly lower than non-colocalized particles ( ± %; figure ). the percentage of sa negative cells with bound tgev miller ( . ± . %) and with bound tgev purdue ( . ± . %) was significantly higher than the percentage of sa positive cells with bound tgev miller ( . ± . %) and with bound tgev purdue ( . ± . %). the percentage of tgev miller particles ( ± %) and tgev purdue ( ± %) that colocalized with sa was significantly lower than the particles that did not colocalize, with miller at ± % and purdue at ± % (figure ). porcine epithelial cells of the small intestines are the target cells for pedv and tgev in vivo. these cells show a high surface expression of apn, and apn has been reported to be the cellular receptor for pedv and tgev [ , ] . however, vero cells with undetectable apn expression were historically used for pedv propagation questioning the role of apn as a cellular receptor for pedv [ ] . in addition, overexpression of porcine apn in non-susceptible cells did not robustly support pedv infection, and knock-out of apn in susceptible cells did not abrogate pedv infection [ ] . the present study was performed to determine the role of apn in pedv and tgev infection in their target primary porcine enterocytes. we found that a higher infection of pedv and tgev was correlated with a higher apn expression. however, both pedv and tgev did not only infect apn porcine epithelial cells of the small intestines are the target cells for pedv and tgev in vivo. these cells show a high surface expression of apn, and apn has been reported to be the cellular receptor for pedv and tgev [ , ] . however, vero cells with undetectable apn expression were historically used for pedv propagation questioning the role of apn as a cellular receptor for pedv [ ] . in addition, overexpression of porcine apn in non-susceptible cells did not robustly support pedv infection, and knock-out of apn in susceptible cells did not abrogate pedv infection [ ] . the present study was performed to determine the role of apn in pedv and tgev infection in their target primary porcine enterocytes. we found that a higher infection of pedv and tgev was correlated with a higher apn expression. however, both pedv and tgev did not only infect apn positive enterocytes, but also apn negative cells. our results demonstrated that pedv and tgev may use another additional unknown receptor for entry in primary enterocytes. the epithelium of the small intestines is continuously and rapidly renewed in a process involving cell generation and migration from the multi-potent stem cells in the crypts to the differentiated cells at the tips of the villi within - days. in our study, after three days cultivation, the primary enterocytes were positive for sucrase and iso-maltase, which are considered as differentiation markers for epithelial cells [ ] . however, the expression of aminopeptidase n was only around %, indicating that the primary enterocytes are not fully mature at three days cultivation. therefore, we analyzed apn expression in enterocytes at a later time point (seven days cultivation). a significantly higher expression of apn was detected at seven days cultivation compared to three days cultivation. interestingly, the seven-day-cultured enterocytes with a higher apn expression showed significantly higher infection to tgev than that of enterocytes cultured for three days. this agrees with the fact that the virus mainly infects and destroys mature enterocytes lining the villi of small intestines [ ] . however, the infection efficiency of pedv in intestinal epithelial cells was still low, indicating that other factors than apn need to be considered for pedv infection. therefore, several positive enterocyte differentiation factors were tested. hydrocortisone plays an important role in the metabolism of proteins, lipids, and carbohydrates, and is a known promoter for differentiation of cultured cells. hydrocortisone was found to be a critical factor for the differentiation of skeletal muscle, osteoblasts, and endothelial cells [ ] . sorrell and colleagues demonstrated that hydrocortisone significantly upregulated the expression of apn in human dermal fibroblasts [ ] . besides, wnt signaling is required for the formation of normal crypt-villus units of intestines, and stimulates the differentiation of intestinal secretory epithelial cells [ ] . activated wnt signaling has also been shown to promote mesenchymal differentiation [ ] . the original rationale for including intestinal contents in our primary cell cultures was trying to mimic the in vivo situation in the intestinal tract. intestinal contents contain a large number of enzymes, such as: amylase, which digests carbohydrates to monosaccharides; pancreatic enzymes, which digest proteins into amino acids; and lipase which digests fats. it has been demonstrated that intestinal contents play an important role in virus infection. the proteases (trypsin) in the intestinal contents activate rotavirus infection by cleaving the outer capsid protein vp [ ] . the propagation of porcine enteric calicivirus (pec) on a cell line critically relies on the presence of intestinal contents in the culture medium [ ] . chang et al. demonstrated that the bile salts in intestinal contents are essential for growth of pec by inducing the protein kinase a (pka) signaling pathway [ ] . in our study, the intestinal contents collected from the upper duodenum promoted the expression of apn and enhanced the infection of both tgev and pedv, especially the cv vero adapted strain. further investigation is needed to determine which growth-promoting factor in the intestinal contents is responsible for the increase of coronavirus infection. to date, coronaviruses use four different proteins as cellular receptors. mouse hepatitis virus (mhv) initiates the infection by binding to the carcinoembryonic cell adhesion molecule on hepatocyte membranes and intestinal brush border membranes [ ] . next, apn was found to act as a receptor for porcine, feline, canine, and human coronaviruses [ ] . the receptors for the highly pathogenic human respiratory viruses sars-cov type and type as well as middle east respiratory syndrome coronavirus are angiotensin-converting enzyme (ace ) and dipeptidyl peptidase (dpp ) [ , ] . cong and colleagues proved that the porcine small intestine epithelial cell line was more susceptible to pedv when a high expression level of apn was present, and that interference with apn expression in epithelial cells inhibited pedv infection, demonstrating that apn serves as an essential receptor for pedv [ ] . in addition, a transgenic mouse model expressing porcine apn was proven to be susceptible to pedv, which confirmed that apn plays a role as the cellular receptor for pedv [ ] . in our study, we found that the higher infection of pedv in enterocytes is correlated with higher apn expression. both aged enterocytes and enterocytes treated with differentiation factors expressed more apn and were more susceptible to pedv, which indicates that apn may play role in pedv infection in enterocytes. moreover, we found that the apn was expressed at the apical surface of enterocytes and pedv infected times more enterocytes at the apical surface than at the basolateral surface (supplementary figure s ). these results were in agreement with the previous finding that pedv enters polarized cells via the apical membrane [ ] . our results indicate that apn facilitates the entry of pedv into primary enterocytes. shirato and colleagues indicated that apn may promote pedv replication in porcine kidney cell line, cpk cells via its protease activity [ ] . how apn facilitates pedv infection in enterocytes should be further investigated. however, due to the fact that other molecules besides apn will also be expressed at the apical plasma membrane during differentiation, they may also contribute to the higher susceptibility of differentiated enterocytes. recently, increasingly more data has been published that show that apn is not a cellular receptor for pedv. overexpression of apn in non-susceptible cells did not confer susceptibility of the cells to pedv and knocking out apn in susceptible cells did not abrogate pedv infection, which indicates that apn is not required as a cellular receptor for pedv in vitro [ , , ] . furthermore, apn knockout pigs retained their susceptibility to pedv confirming that pedv may use another additional receptor in pigs [ ] . in agreement with these findings, we found that pedv can infect apn negative primary enterocytes, which further confirmed that a cellular receptor different from apn exists in enterocytes for pedv replication. the primary enterocytes isolated and cultured in our study are not % positive for apn, as we not only get the villi epithelial cells, but also the crypt epithelial cells during our isolation procedure. by immunofluorescence staining of ileum tissue of a three-day-old piglet, we observed that the villi epithelial cells are apn positive, while the crypt epithelial cells are apn negative (data not shown). as pedv has been shown to infect goblet cells and crypt stem cells in addition to villous mature enterocytes [ ] , we believe that pedv may also use another cellular receptor besides apn to infect intestinal cells. taken together, our results indicate that although apn could significantly promote pedv infection in enterocytes, an additional cellular receptor exists in enterocytes for pedv replication. since ace is expressed in the gut epithelial cells, it will be tested in the near future if it can function as a pedv receptor. in addition to pedv, apn has been identified as a major cellular receptor for tgev. in the present study, it was shown that a higher expression of apn significantly increased the replication of tgev in enterocytes, indicating that apn plays an important role as a cellular receptor for tgev. whitworth and colleagues demonstrated that apn knockout pigs were resistant to tgev infection, indicating that apn is the sole functional receptor for tgev [ ] . however, we found that except apn positive enterocytes, tgev could also infect apn negative enterocytes, suggesting that an additional receptor also exists in enterocytes for tgev. the result obtained in our in vitro experiment may not fully reflect the in vivo experiments, as the in vivo situation is composed of a complex set of cells and tissues. the purdue strain used in our study has been passaged times in primary kidney cells and a nucleotide mutation (t to g at nucleotide position ), which causes a serine (s) to alanine (a) at aa [ , ] . this mutation may be correlated with the cell adaptation and also it may result in a broader cell tropism of the adapted strain, which may explain that the virus is able to grow more efficiently in apn negative cells. purdue has been proven to infect primary colon epithelial cells and porcine myofibroblasts, which are both negative for apn [ ] . in vivo, tgev shows a higher cell tropism to villous enterocytes of newborn piglets compared to older pigs and causes only high mortality in the early life of piglets. since apn is present on enterocytes from both newborn and older pigs, the age-dependent susceptibility to tgev infection may be caused by an additional receptor that is specifically present in newborn piglets. taken together, we hypothesize that apn is the determinant cellular receptor for tgev, but an additional receptor exists in young piglets. apart from apn, tgev also uses sa as an attachment mediator on the cells. shahwan and colleagues have shown that na treatment of jejunum epithelial cryosections did not reduce the tgev spike protein binding in vitro and were doubting on the role of sa during infection [ ] . in our study, removal of sa from intestinal cells had no effect on tgev infection, showing that terminal sa residues are not receptor determinants for tgev. removal of sa from tgev virions significantly enhanced the viral infectivity in vitro. this indicates that sa on the virion surface masks the binding site of the viral protein to cellular receptors. removal of sa from virions facilitates tgev to bind to its functional receptor on the enterocyte membrane. to confirm the role of apn and sa in tgev infection in primary enterocytes, a binding assay was performed in this study. the results demonstrated that both tgev miller and purdue could bind to apn positive and sa positive cells. meanwhile, miller and purdue could also bind to apn negative and sa negative enterocytes. furthermore, our study showed that apn and sa double-negative enterocytes could be infected by tgev miller and purdue (supplementary figure s ) , which suggests that besides apn and sa, tgev can use another cellular receptor for the replication in enterocytes. further investigation will focus on identifying this unknow receptor. a binding assay for pedv could not be conducted due to the low titer of the virus stock. based on our previously established primary enterocyte co-culture system, which is very relevant for the in vivo situation, it was shown that a higher expression of apn on the enterocytes resulted in a higher infectivity of tgev and pedv. however, tgev and pedv could also infect apn negative enterocytes, indicating that an additional receptor exists in enterocytes besides apn. tgev did not show binding activity on sas on the surface of enterocytes. these new insights stimulate the search for unknown receptors for pedv and tgev, which can assist further research on antiviral intervention. coronavirus spike proteins in viral entry and pathogenesis the coronavirus membrane glycoprotein mapping of the coronavirus membrane protein domains involved in interaction with the spike protein absence of e protein arrests transmissible gastroenteritis coronavirus maturation in the secretory pathway severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i 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aminopeptidase n and its susceptibility to porcine epidemic diarrhea virus resistance to coronavirus infection in amino peptidase n-deficient pigs goblet cell depletion in small intestinal villous and crypt epithelium of conventional nursing and weaned pigs infected with porcine epidemic diarrhea virus a competitive inhibition elisa for the differentiation of serum antibodies from pigs infected with transmissible gastroenteritis virus (tgev) or with the tgev-related porcine respiratory coronavirus complete genomic sequences, a key residue in the spike protein and deletions in nonstructural protein b of us strains of the virulent and attenuated coronaviruses, transmissible gastroenteritis virus and porcine respiratory coronavirus sialic acid binding properties of soluble coronavirus spike (s ) proteins: differences between infectious bronchitis virus and transmissible gastroenteritis virus we are grateful to e. cox for supplying the antibody against apn. we also thanks l. enjuanes for supplying the antibody against pedv. special thanks go to marthe pauwels for the excellent technical assistance. the authors declare no conflict of interest. key: cord- -o j d j authors: page, kevin w.; britton, paul; boursnell, michael e. g. title: sequence analysis of the leader rna of two porcine coronaviruses: transmissible gastroenteritis virus and porcine respiratory coronavirus date: journal: virus genes doi: . /bf sha: doc_id: cord_uid: o j d j the leader rna sequence was determined for two pig coronaviruses, tranmissible gastroenteritis virus (tgev), and porcine respiratory coronavirus (prcv). primer extension, of a synthetic oligonucleotide complementary to the ′ end of the nucleoprotein gene of tgev was used to produce a single-stranded dna copy of the leader rna from the nucleoprotein mrna species from tgev and prcv, the sequences of which were determined by maxam and gilbert cleavage. northern blot analysis, using a synthetic oligonucleotide complementary to the leader rna, showed that the leader rna sequence was present on all of the subgenomic mrna species. the porcine coronavirus leader rna sequences were compared to each other and to published coronavirus leader rna sequences. sequence homologies and secondary structure similarities were identified that may play a role in the biological function of these rna sequences. transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus (prcv) belong to the family coronaviridae, a large group of pleomorphic enveloped viruses with a positive-stranded rna genome. tgev causes gastroenteritis in pigs, resulting in a high mortality in neonates ( ) . prcv was isolated in several european countries between and ( - ), does not cause diarrhea, and has been shown to replicate in the respiratory tract with little or no clinical signs, but is very similar antigenically and serologically to tgev ( , ) . virions from both viruses contain two envelope glycoproteins of relative molecular mass (mr) , (spike) and m r , - , (membrane protein) and a phosphorylated nucleoprotein of m r , . cdna probes to the structural protein genes of tgev hybridized to the appropriate mrna species of prcv, suggesting a high degree of homology at the rna level (unpublished data). coronavirus proteins are expressed from a "nested" set of subgenomic mrnas with common ' termini but different ' extensions. the sequence of each mrna that is translated to produce viral proteins appears to correspond to the '-terminal region that is absent on the preceding smaller mrna species. it has been shown for the coronaviruses, mouse hepatitis virus (mhv) and infectious bronchitis virus (ibv), the subgenomic mrna species possess short "leader sequences" at their ' ends. these sequences are not transcribed as a contiguous mrna species, but are derived from the ' end of the genomic rna and are probably joined to the ' end of each mrna by a process of discontinuous transcription ( ) ( ) ( ) ( ) ( ) . the leader sequence appears to be produced by a mechanism termed leader-primed transcription, in which the leader rna is transcribed independently, dissociated from the template, and then binds to the template (negative-sense strand) at specific transcriptional start sites (i , ) . the mechanism appears to involve the recognition of consensus sequences identified on the genomic rna at those points corresponding to the ' ends of the subgenomic mrnas. these consensus sequences may act as a binding site for the rna polymeraseleader complex ( ) ( ) ( ) ( ) ( ) ( ) . it has been previously postulated that a heptameric sequence, actaaac ( ) ( ) ( ) , or a hexameric sequence, ctaaac ( ) ( ) ( ) , may be involved in the binding of the tgev rna polymerase leader. in this paper we describe the elucidation of the leader rna sequences from the porcine coronaviruses tgev and prcv, the first leader sequence to be described from the tgev serogroup of coronaviruses. comparison of the leader rnas of tgev and prcv with published leader rnas of other coronaviruses was used to identify areas of conserved sequence and potential secondary structure that may be involved in the transcription of coronavirus subgenomic mrna species. confluent cultures of a pig kidney cell line llc-pk were infected with a virulent british field isolate of tgev strain fs / or a british isolate of prcv strain / at a moi of - pfu per cell. after hr at ~ the inoculum was removed and replaced with medium containing ixg/ml actinomycin d to inhibit host-cell rna synthesis ( ) . after a further -hr incubation, r of [ , - h]uridine (amersham international plc, trk. , - ci/mm) was added per culture bottle and the cells were incubated for a further hr. the cells were lysed with guanidinium thiocyanate, the rna pelleted through . m cesium chloride and poly(a)-containing rna isolated by poly(u) sepharose affinity chromatography, as described previously ( ) . two oligonucleotides were synthesized by the phosphoramidite method using an applied biosystem a synthesizer. one oligonucleotide, oligo ( '-tggatt-catccccccaacta-y), was complementary to the nucleoprotein gene bp downstream from the initiation atg codon ( ) , as shown in fig. , and was used for primer extension. the second oligonucleotide, oligo ( '-agagata-tagccacgctacactcactttac-y), was complementary to the ' end of the leader rna ( fig. ) and was used for northern blot analysis of viral mrna. gel-purified oligo ( ng) was '-end-labeled ( ) using u of t polynucleotide kinase (gibco-brl, paisley) and ixci [~/- p]atp (amersham international plc, pb , ci/mm. poly(a)-containing rna ( . p~g) isolated from tgev-and prcv-infected cells was resuspended in water and heated at ~ for min. a further incubation was carried out using the two mrna preparations in p.l reaction volumes containing u of rnasin (promega biotec, liverpool), mm tris-hc (ph . ), mm mgc , mm kc , mm -mercaptoethanol, mm dithiothreitol, mm dntps, '-end-labeled oligo ( ng), and u of amv reverse transcriptase (super-rt, anglian biotech ltd, colchester) for min at ~ formamide dye ( % formamide, mm naoh, mm edta, . % xylene cylanol blue, . % bromophenol blue) was added and the mixture boiled for min and electrophoresed on a cm buffer gradient sequencing gel ( ) . the wet gel was autoradiographed for hr to locate the primerextended products, which were excised from the gel. the labeled fragments were eluted from the polyacrylamide gel and chemically cleaved ( ) . samples of the cleaved products from each of the primer extended products were electrophoresed on % polyacrylamide gels at w constant power for two different lengths of time. tgev and prcv poly(a)-containing rna was glyoxylated and separated on a % agarose gel ( ) . the rna was transferred onto biodyne a membranes (pall p/n bnng r . ~m, gallenkamp) in x ssc (x ssc = . m naci, . m trisodium citrate, ph . ) for hr and baked at ~ for hr. the membrane was boiled in mm tris-hcl ph . for min to remove glyoxal groups from the rna and prehybridized in the presence of % formamide for hr at ~ ( ) . the viral mrna species were hydribidized with p-labeled oligo in the presence of % formamide for hr at ~ the membrane was washed four times in x ssc containing . % nadodso for rain at room temperature and autoradiographed. following primer extension, using oligo at the ' end of the nucleoprotein gene from the porcine coronaviruses tgev and prcv, labelled fragments of approximately bases were produced and purified from gels. larger molecular weight species were also observed (data not shown) in minor amounts, presumably corresponding to read-through sequences upstream of the nucleoprotein gene primed from the larger mrna species. the nucleotide sequences of the two fragments, determined by chemical cleavage, were identical. the resulting nucleotide sequence of the tgev leader rna sequence is shown in relation to the tgev nucleoprotein gene in fig. . the leader rna sequence diverges from the genomic sequence bp upstream of the nucleoprotein gene, corresponding to the first nucleotide of the membrane protein gene stop codon ( ), indicating a length of nucleotides of unique sequence (fig. ). the nucleotide leader sequence of tgev and prcv has a low content of g ( %) and c ( %), and a high a ( %) and t ( %) content, with % of the t residues grouped in threeto four-nucleotide motifs (fig. ) . these values are similar to those observed from the tgev genome so far sequenced, except that the values for a ( . %) and t ( . %) are more similar on the genome than on the leader sequence. analysis of the tgev nucleoprotein nucleotide sequence ( ) revealed a potential rna polymerase-leader complex binding site. the site, actaaac, is seven nucleotides upstream of the nucleoprotein initiation codon and has also been found to precede all the tgev structural protein genes and two of the three potential genes shown to be at the ' end of mrna species ( ) ( ) ( ) . this consensus sequence is found two nucleotides downstream of the nucleotide where the leader rna and tgev genomic sequences diverge, indicating that this sequence is involved in the leader-primed transcription oftgev mrna molecules. as can be seen from fig. , of the mrna species from the fs / strain of tgev have the sequence aactaaac, of which the '-end adenosine residue is the next base down from the divergence point. in fact, the consensus sequence at the spike/orf -orf gene junction has the sequence gaactaaac and at the nuc/orf gene junction has the sequence cgaactaaac, indicating that the region of the leader sequence ' to the homology motif, actaaac, may vary between and nucleotides depending on the tgev gene. computer analysis has also detected a homology between the leader rna sequence and the ' end of the negative strand (i.e., the reverse complement of the noncoding region at the ' end of the positive strand). this is shown in fig. . the nucleotides on the leader rna sequence, bases - , and on the negative strand, bases to counting from the first base after the poly(a) tail, have an overall homology of % and include the sequenc~ ctaaac, which is part of the postulated tgev rna polymerase-leader complex binding site. this is very similar to the observation for ibv ( ) involving sequences present at the ' end of the ibv genome, and on the ibv leader rna sequences, with the ' end of the ibv negative strand. the homology observed included the sequence cttaac, which is part of the postulated ibv rna polymerase-leader complex binding site ct(t/g)aacaa. an oligonucleotide, oligo , was synthesised that was complementary to the ' end of the tgev and prcv leader rna sequences (fig. ) . the oligonucleotide was end-labeled and used to probe tgev and prcv mrna species that were northern blotted onto biodyne membranes. as can be seen from fig. , the labeled probe hybridized to all of the tgev and prcv mrna species. the intensity of the bands corresponding to labeled probe hybridized the spike mrna species, and genomic rna was lower than that observed for the smaller mrna species due to less of these larger species being isolated from the poly(u) sepharose column used in the isolation of mrna. the fact that the probe hybridized to all of the mrna species showed that the leader rna sequence was present on the other rna molecules of tgev and both strains of prcv was not unique to the nucleoprotein mrna species. the two porcine coronavirus leader sequences were identical, indicating that the two viruses probably use the same rna polymerase-leader complex binding site, actaaac, for the synthesis of subgenomic mrna species. the seqhp comparison program of the los alamos ( ) package was used to compare the leader rna sequences determined in this paper and those published for five other coronaviruses belonging to two different serogroups. the sequences were compared from the ' ends to the point of divergence from the genomic sequences. the percentage homologies, table , were expressed as the number of bases matched to the longer of the two sequences being compared. the homology of the leader sequences fell into three groups. leader rnas from coronaviruses belonging to different serological groups had homologies in the region of - %. serologically related viruses like human coronavirus (hcv) (strain oc ) and mhv (strains a and jhm) have about % homology. the third group involved different strains of mhv, a , and jhm, which showed a homology of %. this observation indicates that tgev and prcv, which have a homology of %, are probably different strains of the same virus or that prcv has very recently diverged from tgev. in order to identify common areas of homology, the leader rna sequences from seven coronaviruses were aligned. as can be seen from fig. , these fell into two groups. one group consists of mhv (strains a and jhm) with hcv (oc ), which have a fairly high degree of homology along their lengths. the other group consists of tgev and prcv (not shown on the diagram) with hcv ( e) and ibv, which have high homologies at their ' ends and areas of homology at their ' ends. there are good homologies towards the ' ends, involving the postulated rna polymerase-leader complex binding sites and sequences upstream of these sites, between the groups, but very little if any homology between the ' ends. ( ) and strain jhm ( ); avian, ibv strain beaudette ( , ). as seen from fig. simple alignment did not reveal very much information about the homologies of the leader rna sequences from the different coronaviruses, except at the ' ends involving the consensus sequences. in order to identify any potential similarities in these sequences, the secondary structure of the rna sequences in fig. were analyzed. potential secondary structures of the leader rna sequences were determined using the computer program fold ( ) from the uwgcg dna analysis programs ( ) . the coordinates determined by the fold program were displayed graphically using the uwgcg program squig-gles. the potential secondary structures obtained were compared and, as can be seen from fig. , the overall shape of these sequences are very similar, except for the avian coronavirus ibv. all the molecules appear to be composed of two stem-loop structures. the two mhv molecules are very similar in shape and, as seen from fig. and table , are very homologous, %, at base sequence. the secondary structures of the coronavirus leader rna sequences are probably influenced by their biological function, which results in the similarity of these potential structures. this paper presents evidence that the nucleoprotein mrna species of tgev and the closely related porcine respiratory variant of tgev, prcv, contain an identical leader rna sequence of about nucleotides. sequencing studies on tgev have shown that the heptameric sequence actaaac occurs on the genome upstream of the genes and is believed to be the binding site for the leader of the genomic rna. this mechanism has been termed leader-primed transcription and involves not only the leader rna primer, but also consensus sequences along the genome found upstream of the genes, which act as binding sites for the leader rna primer. comparison of tgev and prcv viral products has shown very little difference between the two coronaviruses, and until recently is was impossible to differentiate between the two viruses using antisera. prcv is fully neutralized by antisera prepared against tgev, and the majority of monoclonal antibodies (mabs) raised against tgev virion proteins cross-react with prcv. however, mabs, raised against antigenic determinants of the spike protein from either the virulent british isolate fs / ( ) or the avirulent purdue strain of tgev ( ) have been identified that do not recognize prcv. these observations and the fact that the leader rna sequences from tgev and prcv are identical supports the evidence that the two viruses are very similar and that prcv may have evolved as a tgev variant. comparison of the tgev leader rna sequence with the genomic sequence upstream of the nucleoprotein indicates that the length of the unique sequence of the leader sequence is nucleotides. the point of divergence is two bases upstream of the actaaac sequence, supporting the evidence that the tgev rna polymerase-leader complex binding site is actaaac. four out of the six mrna species from the fs / strain of tgev have the sequence aactaaac, and the '-end adenosine residue is the next base down from the divergence point in the nucleoprotein mrna (fig. ) . the differences in the homologies between the leader rna and sequences upstream of the consensus sequence on the genomic rna may play a role in the levels of transcription of a particular mrna species. the mrna species of . kb has been shown to have an open reading frame at the ' end encoding a potential polypeptide of m r ( ) . this particular mrna does not have the heptameric consensus sequence but has the hexameric ctaaac sequence, and it is interesting to note that it is the least abundant tgev mrna species (observed from tgev mrna in total cell lysates). hybridization of oligo to the . -kb mrna species showed that this species does contain the tgev leader rna, confirming that it is a true mrna species, even though it is the only tgev species not to have the heptameric consensus sequence. comparison of the seven coronavirus leader rna sequences against each other identified three groups (table ) : non-serologically related viruses had about - % homology; serologically related viruses had about % homology; viral strains had about - % homology. however, tgev and hcv ( e) have been placed in the same serological group, but have only % homology within their leader rna sequences, suggesting that the two viruses are not particularly related. tgev and hcv ( e) have been shown to have % homology at the amino acid level within their derived nucleoprotein sequences ( ) , whereas the homology between the derived nucleoprotein amino acid sequences for different viruses within the mhv serological group are between % and % homology. this indicates that the serological grouping of coronaviruses is not a particularly useful test, as similar epitopes may exist on the viral structural proteins. comparisons of nucleic and amino acid sequences from the viruses will provide a more accurate method for grouping the viruses. it will be interesting to compare the leader sequences of bovine coronavirus (bcv), which is serologically related to hcv (oc ) and mhv (a and jhm), with feline infectious peritonitis virus (fipv) and canine coronavirus (ccv), which are serologically related to tgev, once their sequences have been determined. the large variation in sequence length and content made the alignment of the different leader sequences difficult. however, alignment of the six different coronaviruses revealed that they fell into two groups. there appears to be some conservation of short sequence motifs between the seven leader sequences. toward the ' end of the sequences, a tag motif is conserved in all the leaders, followed by a string of ts. in five out of seven of the sequences, this motif is taganntt. about ten nucleotides downstream of this region is a conserved ct motif, which is followed by a series of nucleotides differing in number, depending on the coronavirus, followed by the postulated rna polymerase-leader complex binding site. the largest number of nucleotides between the ct motif and the consensus sequence are found on tgev and prcv, the shortest is found on hcv ( e) and ibv. it is interesting to note that there is a five-base insert in mhv strain jhm when compared to mhv strain a , which is also present in hcv (oc ) within this region. all the mammalian coronaviruses appear to have the motive ctaaac, except hcv (oc ), which has ctaaat. recent sequence data suggest that coronaviruses fipv and bcv have actaaac as their mrna consensus sequence. upstream of the tag motif there is an act motif occurring in six out of seven sequences. toward the ' end of the leader rna sequences, the homologies are patchy and limited to short matches, occurring only between pairs of sequences. the area upstream of the consensus sequence has been suggested to be involved in the binding of nucleoprotein to the leader rna sequence at nucleotides - in mhv ( ) . it was suggested that mrna species and genomic rna form a complex with the nucleoprotein by the protein binding to or near the leader sequence attached to the rna molecules ( ) . secondary structure analysis of the leader rna sequences showed that all the sequences except for ibv possess a putative double stem-loop structure (fig. ). in the case of the mammalian coronaviruses, the consensus sequences and upstream regions of homology are on the second stem-loop structure, leaving the possibility that the rna-dependent rna polymerase could interact with the first stem-loop structure. the ibv consensus sequence is present on the free ' end of the single stem-loop structure, possibly leaving the single stem-loop structure to interact with the polymerase. virus infections of vertebrates (eds) coronaviruses molecular cloning: a laboratory manual we thank miss k. mawditt, of this laboratory, for synthesizing oligos and and dr. s. f. cartwright, central veterinary laboratory, weybridge for prcv strains / and / . this work was supported by a research contract from the biomolecular engineering programme of the commission of the european communities, contract no. bap- -uk(hi). key: cord- -rfu bkag authors: gómez, n.; carrillo, c.; salinas, j.; parra, f.; borca, m. v.; escribano, j. m. title: expression of immunogenic glycoprotein s polypeptides from transmissible gastroenteritis coronavirus in transgenic plants date: - - journal: virology doi: . /viro. . sha: doc_id: cord_uid: rfu bkag abstract the use of transgenic plants as vaccine production systems was described recently. we report on the immunological response elicited by two recombinant versions of the glycoprotein s from the swine-transmissible gastroenteritis coronavirus (tgev) expressed in transgenic plants. arabidoposis plants were genetically transformed with cdnas constructs encoding either the n-terminal domain (amino acid residues – ) or the full-length glycoprotein s of tgev, responsible for the neutralizing antibody induction against the virus, under the control of the cauliflower mosaic virus s (camv s) promoter. genomic dna and mrna analyses of leaf extracts from transformed plants demonstrated the incorporation of the foreign cdna into the arabidopsis genome, as well as their transcription. expression of recombinant polypeptides were observed in most transgenic plants by elisa using specific antibodies. mice immunized with leaf extracts from transgenic plants developed antibodies that reacted specifically with tgev in elisa, immunoprecipitated the virus-induced protein, and neutralized the virus infectivity. from these results, we conclude that transgenic plants expressing glycoprotein s polypeptides may possibly be used as a source of recombinant antigen for vaccine production. swine-transmissible gastroenteritis virus (tgev) is the causative agent of acute diarrhea of newborn piglets that provokes high mortality rates in affected farms. protective immunity against this disease must be developed in pregnant sows to confer passive protection to the piglets through colostrum and milk. neutralizing antibodies against the virus are directed mainly to glycoprotein s (garwes et al., ; jimenez et al., ) , and relevant epitopes in neutralization have been mapped into the n-terminal domain of this protein . four major antigenic sites have been described in glycoprotein s, of which site a is the immunodominant (de diego et al., ; delmas et al., ; sa  nchez et al., ) . glycoprotein s from tgev has been expressed using different vectors with tropism that favored antigenic presentation in the mucosal surfaces (smerdou et al., ; torres et al., ) . these vaccination approaches promoted systemic and mucosal antibody induction and, in the case of adenovirus vector, conferred protection to suckling piglets (torres et al., ) . the development of genetic transformation technology in plants has made possible the expression of foreign genes in different plant species, making reasonable the idea of using plants as bioreactors to produce recombi-nant proteins. the concept of vaccine production in transgenic plants was first introduced by mason et al. in . proteins involved in protective immune response can be produced at a low cost and easily purified from plant extracts for parental inoculation. in addition, oral immunization by edible vaccines produced in transgenic plants could stimulate immune responses at the portal entrance of many pathogens, facilitating the design of large-scale immunization programs. the presence of specific antigens into plants, even at low levels, can raise by the oral route immune reactions comparable to those raised by conventional vaccines (haq et al., ; mason et al., ) . hepatitis b surface antigen (thanavala et al., ) , escherichia coli heat-labile enterotoxin (lt-b) antigen (haq et al., ; tacket et al., ) , norwalk virus capsid protein (mason et al., ) , vp antigen from foot and mouth disease virus (carrillo et al., ) , and cholera toxin b subunit (arakawa et al., ) are the vaccine antigens expressed in transgenic plants and tested for the immune response elicited in immunized animals. additionally, rabies virus glycoprotein was expressed in transgenic tomatoes, but the immune response induced by administration of these plants to animals was not tested (mcgarvey et al., ) . in the present study, we investigated the feasibility of expressing the glycoprotein s from tgev in transgenic plants, as well as the antigenicity and immunogenicity of the plant-derived protein. the s protein is an excellent model for developing oral vaccines against enteric pathogens of mammals because of its immunogenicity and resistance to degradation in the gut. the binary prok i and prok ii recombinant plasmids ( fig. ) , carrying a cdna coding for the n-terminal region or the full-length glycoprotein s respectively, were obtained by subcloning the corresponding sequences from previously obtained constructs. recombinant prok plasmids allow selection of transformants on media containing kanamycin and stable integration into nuclear chromosomal dna from the plant. prok uses the cauliflower mosaic virus s (camw s) promoter for nominally constitutive transcription of the cloned genes. plant transformation with prok i and ii was carried out as described in materials and methods by agrobacterium tumefaciens-mediated transformation. the transgenic plants resistant to the selective medium appeared similar in morphology to the nontransgenic arabidopsis plants. more than different lines of transformants containing each construct were obtained and self-pollinated to obtain f lines. all lines were positive when screened for the presence of the recombinant genes by polymerase chain reaction (pcr) analysis ( fig. a ). most plants harboring recombinant genes showed specific transcription of foreign genes by reverse transcription (rt)-pcr analysis (fig. b ). to rule out the possibility of amplification of contaminant dna sequences present in the rna preparations, we treated the purified rna with ribonuclease before foreign gene amplification by using taq polymerase. no amplified dna fragments were detectable under those conditions, assessing the rna dependence of the reaction (fig. b ). the presence of the recombinant polypeptides in the plants harboring and expressing the foreign genes was investigated in four plants of each construct, selected to be analyzed by elisa and western blotting using an anti-tgev polyclonal serum. results demonstrated that leaf extracts from all selected plants were positive on elisa (fig. ) . however, no specific reaction on western blotting was detected in any of the plant extracts analyzed (data not shown), probably due to the low levels of recombinant protein expression and to the conformational nature of most of the immunodominant epitopes present in this protein. from a titration elisa using different virus dilutions and a monospecific anti-glycoprotein s antibody, we found that ϳ ± g of soluble leaf protein contains a glycoprotein s antigenic mass equivalent to that contained in . g of purified tgev. the percentage of the total soluble protein corresponding to recombinant glycoprotein s polypeptides accumulated in the leaves of arabidopsis transformants could represent . ± . % of the total soluble leaf protein. leaf extracts from transgenic plants expressing the n-terminal (plants ± ) or full-length glycoprotein s (plants ± ) were used to immunize mice. a control mouse was immunized with a leaf extract from a plant transformed with prok plasmid. after three immunization doses, the specificity of mice sera was tested by an elisa using purified tgev as antigen. figure a shows that all sera reacted with the virus showing, as expected, different titers. a kinetic of antibody induction in an immunized mouse (number ) was studied by immunoprecipitation of glycoprotein s induced by tgev in infected st cells. this mouse serum immunoprecipitated specifically the virus protein after two immunizations (fig. b) . finally, sera from all immunized mice were tested in a tgev neutralization assay. both glycoprotein s polypeptides produced in transgenic plants elicited virus-neutralizing antibodies (neutralization indexes of . ± . ; fig. c ). serum from a nonimmunized mouse (not shown) or from the mouse immunized with the plant transformed with prok plasmid did not show virus neutralization activity (fig. c ). in this report, we show that full-length or the globular part (n-terminal domain) of tgev spike protein (glycoprotein s) expressed in transgenic plants retained the antigenic properties and elicited neutralizing antibodies when used to immunize animals. expression in eukaryotic hosts is required for antigenic determinants that are dependent on glycosylation. of the three major antigenic sites defined on glycoprotein s involved in the induction of tgev-neutralizing antibodies, sites a and b are complex, conformational, and glycosylation dependent. site d can be represented by synthetic peptides, although glycosylation has a minor effect on its conformation (gebauer et al., ) . several genetically engineered vaccines using prokaryotic vectors have failed against tgev. glycoprotein s expressed at high levels in escherichia coli and used to inoculate animals did not induce neutralizing antibodies or confer protection in vivo (hu et al., ) . plant cells present differences in protein glycosylation with respect to animal cells that could determine the lose of antigenic determinants in antigens expressed in transgenic plants. glycosylation in plants may differ in the extent of glycosylation, processing, or both of n-linked oligosaccharide side chains (faye et al., ) . furthermore, the complex glycans of plants are often smaller than those of animals, in part due to the absence of sialic acid (faye et al., ) . the only precedent of a glycoprotein expressed in plants for vaccine development is the glycoprotein g of rabies virus (mcgarvey et al., ) . this protein expressed in tomato plants showed a molecular mass ϳ and ϳ kda less than that obtained from virus-infected cells but still larger than the protein size predicted for the unglycosylated polypeptide chain (mcgarvey et al., ) . the molecular mass of glycoprotein s expressed in arabidopsis thaliana could not be determined because we were not able to detect the recombinant protein on western blotting. however, antigenic determinants with strong dependence of glycosylation seem to be preserved because the plant-derived antigens induced neutralizing antibodies in immunized animals, indicating that critical antigenic sites are at least in part correctly glycosylated in plants. this work demonstrates the feasibility of expressing glycoprotein s polypeptides in plants. because the site of insertion of the transferred dna into the cellular chromosomal dna is random, different levels of protein expression in independent transformants are expected. we obtained expression levels similar to that described with equivalent constructs expressing hepatitis b surface antigen or rabies virus glycoprotein (mason et al., ; mcgarvey et al., ) . more recently, expression levels of norwalk virus capsid protein in tobacco have been shown to be higher than the above mentioned antigens (up to . % of total soluble protein; mason et al., ) . we have not found significant differences in foreign antigen plant expression between the two forms of glycoprotein s studied. the use of different promoters, the use of plant-derived leader sequences and signal peptides, and mainly the modification of the codon usage of this protein could improve expression levels in plants. the demonstration that many proteins from pathogens, including some expressed in transgenic plants (haq et al., ; mason et al., ) , are immunogenic when administered orally, encourages the study of other antigens expressed in plants to develop edible vaccines. glycoprotein s from tgev is an interesting model because this protein is resistant, at least when incorporated into the viral particle, to gut degradation. in addition, the protective immune responses against tgev have to be stimulated at the mucosal surfaces to induce secretory and lactogenic immunity (de diego et al., saif and bohl, ; wesley et al., ) . once we have determined the feasibility of expressing immunological active polypeptides from tegv glycoprotein s in plants, studies on the immune response of plant-derived glycoprotein s polypeptides in pigs are necessary. seeds of arabidopsis thaliana (heynh, ecotype columbia) were sown in pots containing a mixture of universal substrate and vermiculite ( : ). to synchronize germination, pots were placed at °c for h in darkness and then transferred to a growth chamber at °c with a -h photoperiod. irrigation was carried out with distilled water and, occasionally, with a mineral nutrient solution (haughn et al., ) . a -pb cdna fragment (nucleotides ± ; fragment i) and a -pb cdna fragment (nucleotides ± ; fragment ii) encoding for the n-terminal and full-length glycoprotein s from tgev purdue strain, respectively, were amplified by rt-pcr from viral rna and cloned into pbacpak plasmid (clontech). the rt primers used were Ј-cccaactatggtaccatcaat aacagc- Ј (complementary primer to nucleotides ± ) and Ј-cgcgggatccttaatggacgtg-cactttttc- Ј (complementary primer to nucleotides ± ). then, the cdna was synthesized by using the primer Ј-gcgcggatccatgaaaaactatttgt-gg- Ј. subsequently, dna fragments i and ii were subcloned in the binary prok plasmid (baulcombe et al., ) under the control of the camw s promoter, yielding the recombinant plasmids prok i and prok ii, respectively (fig. ) . plasmids prok i and prok ii were used for arabidopsis plant transformation as described elsewhere (bechtold et al., ) with slight modifications. a. tumefaciens (c c strain) containing prok i or prok ii plasmids was grown in ml of lb medium con- taining g/ml kanamycin until an od value of was reached. after centrifugation, bacteria were resuspended in ml of . g/l murashige and skoog medium containing g/l -benzilaminopurine and % sucrose. the ± -week-old plants were immersed in the a. tumefaciens suspension by inversion of the pots, and vacuum infiltration was performed in a vacuum chamber at mb for min. infiltrated plants were rinsed with water and placed in the greenhouse until attaining maturity. transgenic t seeds were selected by germination in petri dishes containing gm [ . g/l murashige and skoog, % sucrose, . g/l -(n-morpholino)ethanesulfonic acid (mes), g/l agar, ph . ] and g/ml kanamycin. two-week-old transgenic plants were transplanted into soil and allowed to attain maturity. the plants were self-pollinated to obtain t plants and used for further analysis. the presence of the foreign cdna sequences in generated transgenic arabidopsis was detected by pcr. plant extracts were prepared by macerating leaves (ϳ mg) with pestle and mortar in l of a buffer containing mm tris±hcl, ph . , mm nacl, mm edta, and . % sds. the resulting extract was mixed with l of m ch coona, ph . , and incubated for min at Ϫ °c. then, samples were centrifuged, and the dna contained in the supernatant was precipitated and resuspended in l of te buffer. pcr was performed on . g of dna with a pair of primers that specifically amplify a -bp fragment of the glycoprotein s gene (sense primer, Ј-gcgcggatccatgaa-aaactatttgtgg- Ј; antisense primer, Ј-gcgcgg-tacccgatgtgaagctattg- Ј). glycoprotein s mrna in transgenic plants was analyzed by rt-pcr. total rna from the leaves of transformed plants was isolated using the fast rna kit (bio ) proteins from leaves were obtained by homogenization of leaves in a blender with liquid nitrogen, and the resulting powder was resuspended in buffer ( . g of fresh wt/ml) containing mm mes, ph , mm nacl, mm edta, . % triton x- , . m sucrose, . mm spermine, . mm spermidine, mm dtt, and mm phenylmethylsulfonyl fluoride. the extract was filtered and centrifuged min at , g, and the resulting supernatant was used for glycoprotein s polypeptides expression analyses. elisa plates were coated with l ( g/ml of pbs) of a mixture of two monoclonal antibodies, ac and dh (kindly provided by dr. l. enjuanes, centro nacional de biotechnologõ Âa, csic, spain), recognizing the antigenic sites a and d of the glycoprotein s, respectively . antibodies were incubated for h at °c, and then plates were washed and blocked h at °c with % fetal bovine serum in pbs containing . % tween . after washing the plates, leaf proteins from transgenic plants ( g of total soluble protein per well, diluted in l of pbs, ph ), containing full-length or the n-terminal domain of glycoprotein s, were added to react with the previously adsorbed antibodies in the microtiter elisa plates during h at °c. plates were then washed six times with . % tween in pbs, and l of rabbit anti-s protein, obtained after three immunization doses with the baculovirus-expressed n-terminal fragment of glycoprotein s and diluted at : in pbs containing . % tween , was added per well and left to react for h at °c. plates were washed again six times with pbs±tween buffer, and immunocomplexes were incubated with protein a±peroxidase (sigma) diluted : in pbs±tween for h at °c. finally, plates were washed again, and l of a freshly prepared solution of o-phenylenediamine dihydrochloride (sigma) and h o was added. reactions were stopped with n h so , and the absorbance was measured at nm. balb/c mice (one per arabidopsis plant) were immunized intramuscularly on days , , and with leaf extract in pbs ( g of total protein per animal per injection) in complete freund's adjuvant for the first inoculation and in incomplete adjuvant for the others. mice sera were evaluated for anti-glycoprotein s-specific antibodies by elisa using purified tgev as antigen. coated elisa plates with l of pbs, ph . , containing . g of virus were blocked as described above with % fetal bovine serum, and after washing of the plates six times, sera diluted : in pbs±tween were added ( l per well) and incubated for h at °c. then, plates were washed again to remove unbound antibodies, and goat anti-mouse antibodies ( : ) were added to reveal immunocomplexes. after being washed and developed with o-phenylenediamine dihydrochloride substrate as described above, reaction was stopped with n h so , and plates were read at nm. immunoprecipitation of glycoprotein s by sera from a mouse after different immunization doses was carried out essentially as previously described for mouse antibodies (bullido et al., ) . briefly, st cells infected with tgev (m.o.i. ) were incubated for h, pulse labeled for h with ci/ml of s-methionine ( ci/mmol; amersham international, amersham, england)/ml, and lysed with lysis buffer ( mm tris±hcl, mm nacl, mm edta, % nonidet p- , ph . , mg/ml bovine serum albumin, and mm phenylmethylsulfonyl fluoride). the lysate ( cpm) was incubated with a control mouse serum ( l) for h and precleared with a % (v/v) suspension of protein g±sepharose (pharmacia, sweden) in lysis buffer. the precleared s-labeled cell extract was incubated with mice sera ( l) for h at °c, and immunocomplexes were incubated with % suspension of protein g±sepharose for h with gentle mixing. beads were washed three times with lysis buffer and boiled in sds±electrophoresis buffer. the antigen± antibody complexes were analyzed in . % sds±page. a plaque reduction assay with sera from immunized mice was performed as described previously (jime  nez et al., ) . the neutralization index of each serum was expressed as the log of the ratio of the pfu/ml of virus obtained using a normal serum and that observed in the presence of a given anti-glycoprotein s mouse serum. efficacy of a food plant-based oral cholera toxin b subunit vaccine expression of biologically active viral satellite rna from nuclear genome of transformed plants agrobacterium mediated gene transfer by infiltration of adult arabidopsis thaliana plants monoclonal antibody f / recognizes the ␣ chain of the porcine ␤ integrin involved in adhesion and complement mediated phagocytosis protective immune response to foot-and-mouth disease virus with vp expressed in transgenic plants antigenic structure of e -glycoprotein of transmissible gastroenteritis coronavirus epitope specificity of protective lactogenic immunity against swine transmissible gastroenteritis virus characterization of the iga and subclass igg responses to neutralizing epitopes after infection of pregnant sows with the transmissible gastroenteritis virus or the antigenically related porcine respiratory coronavirus four major antigenic sites of the coronavirus transmissible gastroenteritis virus are located on the amino-terminal half of spike glycoprotein s detection, biosynthesis and some functions of glycans n-linked to plant secreted proteins antigenicity of structural components from porcine transmissible gastroenteritis virus residues involved in the antigenic sites of transmissible gastroenteritis coronavirus s glycoprotein oral immunization with a recombinant bacterial antigen produced in transgenic plants sulfonylurea-resistant mutants of arabidopsis thaliana studies of tgev spike protein gp expressed in e coli and by a tgevvaccinia virus recombinant critical epitopes in transmissible gastroenteritis virus neutralization expression of hepatitis b surface antigen in transgenic plants expression of norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice expression of the rabies virus glycoprotein in transgenic tomatoes passive immunity in transmissible gastroenteritis of swine: immunoglobulin classes of milk antibodies after oral-intranasal inoculation of sows with a live low cell culturepassaged virus antigenic homology among coronaviruses related to transmissible gastroenteritis virus characterization of transmissible gastroenteritis coronavirus s protein expression products in avirulent s. typhimurium ⌬cya ⌬crp: persistence, stability and immune response in swine immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato immunogenicity of transgenic plant-derived hepatitis b surface antigen tropism of human adenovirus type -based vectors in swine and their ability to protect against transmissible gastroenteritis coronavirus induction of antibodies protecting against transmissible gastroenteritis coronavirus (tgev) by recombinant adenovirus expressing tgev spike protein lack of protection in vivo with neutralizing monoclonal antibodies to transmissible gastroenteritis virus we thank j. c. oliveros, p. go  mez puertas, and a. brun for helpful discussions and suggestions and covadonga alonso for critical reading of the manuscript. this work was supported by grant bio ± from comision interministerial de ciencia y tecnologõ Âa of spain and by grant bid /oc-ar pid from secyt-conicet, rep. argentina. key: cord- -brdtsi authors: maragkoudakis, petros a.; chingwaru, walter; gradisnik, lidija; tsakalidou, effie; cencic, avrelija title: lactic acid bacteria efficiently protect human and animal intestinal epithelial and immune cells from enteric virus infection date: - - journal: int j food microbiol doi: . /j.ijfoodmicro. . . sha: doc_id: cord_uid: brdtsi this study aimed to examine the potential antiviral activity of lactic acid bacteria (lab) using animal and human intestinal and macrophage cell line models of non tumor origin. to this end, lab strains selected on the basis of previous in vitro trials were co-incubated with cell line monolayers, which were subsequently challenged with rotavirus (rv) and transmissible gastroenteritis virus (tgev). in order to elucidate the possible mechanism responsible for the antiviral activity, the induction of reactive oxygen species (ros) release as well as the attachment ability of lab on the cell lines was investigated. various strains were found to exhibit moderate to complete monolayer protection against viral rv or tgev disruption. highest protection effects were recorded with the known probiotics lactobacillus rhamnosus gg and lactobacillus casei shirota against both rv and tgev, while notable antiviral activity was also attributed to enterococcus faecium pck , lactobacillus fermentum aca-dc , lactobacillus pentosus pca and lactobacillus plantarum pca and pcs , depending on the cell line and virus combination used. a variable increase (of up to %) on the release of no(−) and h( )o( ) (ros) was obtained when lab strains were co-incubated with the cell lines, but the results were found to be lab strain and cell line specific, apart from a small number of strains which were able to induce strong ros release in more than one cell line. in contrast, the ability of the examined lab strains to attach to the cell line monolayers was lab strain but not cell line specific. highest attachment ability was observed with l. plantarum aca-dc , l. paracasei subsp. tolerans aca-dc and e. faecium pcd . clear indications on the nature of the antiviral effect were evident only in the case of the l. casei shirota against tgev and with l. plantarum pca againt both rv and tgev. in the rest of the cases, each interaction was lab-cell line–virus specific, barring general conclusions. however, it is probable that more than one mechanism is involved in the antiviral effect described here. further investigations are required to elucidate the underlying mode of action and to develop a cell line model as a system for selection of probiotic strains suited for farm animal applications. animal diarrheal diseases represent a serious threat to farm animal welfare but also to the economic viability of animal husbandry. since the ban of antibiotic growth promoters in the european union (e.u.) in (regulation /ec, , probiotic animal feed supplementation has risen as a viable alternative to antibiotics, as reported for several monogastric or ruminant farm animals (scharek et al., ; pollmann et al., ; stella et al., ; maragkoudakis et al., submitted) . probiotics (fuller, ; guarner and shaafsma, ) are comprised primarily of lactic acid bacteria which form part of the normal enteric flora of man and animals. probiotic feed supplementation may benefit the animal host directly, by preventing the infection and combating the causative agent of the intestinal disorder, or indirectly, by balancing the disrupted equilibrium of the enteric flora and augmenting the host's immune responses. apart from bacteria, however, viruses are often the causative agent of farm animal diarrheal disease, including among them the transmissible gastroenteritis virus (tgev) and rotavirus (rv). tgev can cause al disease that has very high mortality rates (often %) in young piglets and is characterized by vomiting and severe diarrhea, while the symptoms manifest in a milder way in adult pigs with lower severity and mortality rates (saif and heckert, ) . tgev belongs to the family of coronaviruses and can been encountered globally where intensive pork industry is practiced, causing great economic losses (sestak and saif, ) . rv on the other hand, a member of the reoviridae family, is better known as being the leading cause of infant and young children diarrhea (parashar et al., ) accounting for more than , deaths each year in children under five years (who epidemiological report, ) . however, rv is also a common cause of diarrhea in a variety of farm animals such as lambs, calves and pigs (holland, ; saif and heckert, ) , in which the infection international journal of food microbiology ( ) s -s severity may range from asymptomatic to fatal, having severe economic implication in animal husbandry. although various literature and clinical studies have confirmed the beneficial and alleviating effects of probiotic bacteria, such as lactobacillus rhamnosus gg, on the infection and symptoms of rotavirus diarrhea (isolauri, ; pant et al., ) , no data is available on field studies of probiotic administration against viral enteric disorders of farm animals. such studies can be limited by their complexity, as well as the ethical and economical implication of using valuable assets such as farm animals in viral diarrhea challenge studies. as an alternative, however, animal intestinal epithelial or macrophage cell line models can be developed and utilized, for studying the interaction between probiotics, viruses, and the host epithelium. very few attempts have been made only recently to study the impact and potential benefit of probiotic strains on animal cell lines. nissen et al. ( ) studied the gut health promoting activity of putative probiotic strains, using pig intestinal and macrophage cell lines. ivec et al. ( ) first reported a cell line model where probiotics were applied on pig epithelial cell lines to protect against vesicular stomatitis virus (vsv), followed by botić et al. ( ) who reported similar results using probiotics to protect a porcine macrophage cell line against vsv disruption. addressing the scarce relevant literature, the aim of this study was to investigate in vitro the applicability of potential and established probiotic lactic acid bacteria as protective agents against the farm animal diarrheal viruses tgev and rv. a pool of promising lab strains, studied in detail for their probiotic properties by the e.u. funded project pathogencombat (fp - ), has been included in this study. to assess the antiviral effects, various farm animal cell lines have been co-incubated with the lab strains prior to their challenge with rv and tgev viruses. additional functional properties that relate to the possible underlying mode of action of the antiviral effect have been also studied, including release of reactive oxygen species (ros) from the cell lines due to probiotic interaction, as well as the attachment ability of probiotic strains on the cell line monolayers. all lab strains were cultured for routine use in de man, rogosa and sharp broth (mrs, oxoid, u.k.) for h at or °c, depending on species. all strains (table ) belong to the collection of the european research project pathogencombat (fp - ) and were drawn from the collections of the agricultural university of athens, greece (pca/ aca-dc), danisco s/a, denmark (pcd), max rubner institute in karlsruhe, germany (pck), and the university of maribor, slovenia (pcs). mrs agar ( . % g/v, oxoid, u.k.) was used for growth on solid media while enumeration of the strains was carried out by the standard serial dilution method, at or °c, according to species, for h. all strains were stored at − °c in cryovials with mrs broth supplemented with % (v/v) of glycerol until use. the strains applied on intestinal epithelial and macrophage cell lines in this study were selected on the basis of previous in vitro and in vivo work (table ) dealing with their probiotic and protective properties such antimicrobial activity against food spoilage, food and clinical pathogens, survival in simulated gastrointestinal tract and food processing conditions, as well as attachment on epithelial cell lines and induction of cytokine release from human macrophage cell lines. the cell lines used comprised of intestinal epithelial and monocyte/ macrophage derived cell lines of farm animal and human origin ( table ) . all cell lines are available at the dept. of biochemistry, faculty of medicine, university of maribor, maribor, slovenia. cells were grown in dulbecco's modified eagle's medium (dmem, sigma-aldrich, missouri, usa), supplemented with % fetal calf serum (biowhittaker, maryland, usa), l-glutamine ( mmol/l, sigma-aldrich), penicillin ( units/ml, sigma-aldritch) and streptomycin ( mg/ml, sigma-aldrich) at °c in a humidified % co atmosphere in tissue culture flasks (corning, usa) until confluent. the cell culture medium was regularly changed. to perform biological assays, the cells were seeded in -well plates at a concentration of × cells/ml and incubated for - h as described above until confluency. just before use, the culture medium was removed and the monolayers were washed twice with dmem without phenol red and supplements. transmissible gastroenteritis coronavirus (tgev) and rotavirus rf strain (rv) were used in this study. both viruses were propagated in clab and psi cells in the presence of trypsin ( µg/ml of dmem) as described previously (botić et al., ) . supernatant containing the virus was collected from the flasks when cytopathic effect (cpe) was observed ( - h at °c, % co ) by microscopy and collected by centrifugation at g for min. virus was stored at − °c until used. tcid (tissue culture infective dose) was determined exactly as previously described (botić et al., ) . . . cytopathic effect (cpe) reduction assay the potential antiviral activity of probiotic bacteria was performed as described previously (botić et al., ; ivec et al., ) . initially, lab strains were applied on the confluent monolayers as already described (maragkoudakis et al., ) . briefly, overnight lab cultures were harvested ( g, min, °c) and washed twice with pbs buffer, ph . ., before resuspension in non-supplemented dmem to a concentration of cfu/ml. the growth medium of -well plates was removed and the monolayers were washed twice with pbs. subsequently, μl of bacterial dmem suspension was transferred onto the monolayers before incubation at °c in a % co atmosphere for min. afterwards the non-bound bacteria were washed off with non-supplemented dmem and tgev or rv were applied ( μl of . tcid /ml and . tcid /ml) on the monolayers. the plates were then incubated at the same conditions and monitored for signs of cytopathic effect (cpe), i.e. monolayer disruption, after h and h, as described previously (botić et al., ; ivec et al., ) . for ros determination, lab strains were applied on healthy cell line monolayers as described above. the no concentration was determined by measuring the accumulation of nitrate using a modified griess reagent (sigma), according to the griess reaction as previously described (ivec et al., ; pipenbaher et al., ) by absorbance measurement at nm the release of h o was determined by transferring μl of supernatant into a new -well plate and adding μl of . % peroxidase and μl of , ′, , ′-tetramethylbenzidine (tmb) solution (diluted with distilled water : ), according to the instructions of the supplier (pierce, rockford, usa). after min the reaction was stopped by addition of µl h po and the absorbance was measured at nm. lab strains were grown, applied on confluent cell monolayers in -well plates and incubated as described above. following incubation the medium supernatant with the non-attached bacteria was removed and the intestinal cell lines were washed twice with non supplemented dmem. μl of trypsin solution (botić et al., ) were then added to detach adhered bacteria, which were subsequently enumerated by the standard serial dilution method on mrs agar plates at or °c, according to species, for h, under microaerobic conditions (anaerocult a, merck, germany). experimental data were analysed with the statgraphics centurion xv software (statpoint inc., usa) using the anova multiple sample comparison. statistical significant effects (p b . ) were further analysed and means were compared by the tukey's hsd test. a selected number of lab strains were first applied on the clab porcine epithelial cell line. the application of rv resulted in the complete disruption of the monolayer, with a survival percentage of only %. however, the co-incubation of the clab cell line with specific lab strains resulted in increased survival percentages, from % up to % (fig. ) . after this preliminary assay, selected lab strains where then applied to four more intestinal epithelial and macrophage cell lines, which apart from rv, where also challenged with tgev. the application of rv (fig. a) and tgev (fig. b ) on all cell lines tested led to a heavy disruption of the monolayers, with survival percentages of approx. % of that of the healthy confluent cell lines. as with the preliminary assay on clab cells, the pretreatment with lab strains prior to viral challenge led from small to marked increased survival percentages and a protective effect against monolayer disruption. in both cases of rv and tgev, highest protection percentages were observed on all cases of viral challenge with l. casei shirota and l. rhamnosus gg. specifically looking at both the preliminary and subsequent antiviral assays, in the case of the cell line challenge with rv, l. rhamnosus gg conferred a notable protective effect on monolayers against viral disruption, with survival ranging from . ± . % (on h ) to . ± . % (on psi), i.e. complete protection. other notable protection effects (n % survival, i.e. four times higher than the survival obtained with the virus application) were observed with e. faecium pck , l. fermentum aca-dc and l. plantarum pcs (on clab), l. casei shirota (on clab and psi) and l. plantarum pca (on gie). similarly, the application of l. casei shirota conferred a protective effect against tgev monolayer disruption, with cell line survival ranging from . ± . % (on psi) to . ± . % (on gie), i.e. almost complete protection, compared to healthy confluent monolayers. notable protection effects (n % survival) were also obtained with l. rhamnosus gg (on tlt, gie and h ), l. fermentum aca-dc (on tlt), e. faecium pcd (on psi), l. plantarum pca (on gie) and l. pentosus pca (on h ). in all other cases, the application of lab against viral disruption of the cell line monolayers led to survival percentages less than b %, i.e. from no protection at all to up to three times better survival, compared with that of the viral challenge. in addition, the application of the lab strains on the examined cell lines did not lead to any detrimental effects on the cell line integrity (figs. and c) . generally, the survival of the cell lines with the applied lab strains ranged from - %, compared to healthy monolayers, with the main exception evident in the case of the psi cell line, that appears to be more sensitive to the addition of lactic acid bacteria, as a slightly lower survival percentage is observed ( %). apart from the psi cell line, similar slightly reduced survival can be observed in few additional cases, notably with gie cell line (with e. faecim pcd , l. plantarum pca and l.rhamnosus gg) and the clab cell line (with e. faecium pcd and pck ). however, statistical analysis revealed that significant differences (i.e. higher survival, p b . ) exist only in the case of tlt cell line compared with gie, clab and psi as well as with h compared with gie and psi. the release of no − and h o from the various cell lines, due to coincubation with lab can be seen in figs. and . as before, a preliminary screening with selected strains was done on the clab cell line, before proceeding to screen more lab strains on other cell lines. the results obtained are lab strain specific but also cell-line specific, as variable values are obtained when the same lab strains are applied to difference cell-lines. none of the lab strains adversely affected the release of no − in any of the cell lines tested. instead, there was an increase in the no − production, ranging from - %, which as previously, was lab strain and cell line dependent. however, some trend lines could be observed, such as the increased release of no − by the clab, gie, h and psi cell lines stimulated by l. plantarum pca . other strains, inducing an increased no − release (n %), include l. pentosus pca (on gie and h ), l. plantarum aca-dc (on clab and psi) as well as l. casei shirota (on h and tlt). results were even more variable in the case of h o , with secretion values that ranged from a slight decrease ( %) to a marked increase ( %) compared to the control. the only exception where a trend can be observed was in the case of the psi cell line, in which the application of all lab strains led to a decrease in the h o release, ranging from to %. as before, a trend could be observed in h o release induction with strains l. plantarum pca (on gie and tlt), l. plantarum aca-dc (on h and tlt), as well as l. casei shirota (h and tlt). nine lab strains were selected and applied on the intestinal epithelial cell lines h , clab, psi and gie (fig. ) . since no strain variability was observed in the no − and h o assays, the strains were selected on the basis of previously performed work. as can be observed, l. plantarum aca-dc , l. paracasei subsp. tolerans aca-dc and e. faecium pcd demonstrate increased attachment ability on four cell lines tested, ranging from to %, with the only exception being l. paracasei subsp. tolerans aca-dc on psi cells ( % attachment). although notable attachment ability was also demonstrated by l. fermentum aca-dc , the rest of the strains tested exhibit lower attachment ability, ranging from - %. in contrast to the no − and h o experiment, the results obtained from this assay are strain specific, as the attachment ability of the strains remained the same even on different intestinal epithelial cell lines, with no statistical differences observed on attachment of the strains between the different cell lines (p n . ). in humans, the alleviating effects of probiotic lactic acid bacteria on diarrheas associated with antibiotic therapy (arvola et al., ; vanderhoof et al., ) or acute rotavirus infection (isolauri, ; fig. . reactive oxygen species (ros) release from the clab intestinal epithelial cell line, co-incubated with selected lab strains. results are predented as a % of release of no − ( ) and h o ( ) from the clab cell line without the presence of lab, and expressed as mean ± standard deviation of two repetitions in triplicates. pant et al., ) diarrheas are nowadays well established and in fact constitute the major clinically proven health benefit of probiotics (isolauri, ) . in farm animals, some studies have demonstrated the efficacy of probiotic administration in reducing the incidence of diarrhea (taras et al., ; scharek et al., ) , which, however, have not been associated with viruses. taking the above into account, as well as the important economic losses in animal husbandry due to tgev and rv enteric disorders, this study aimed to examine directly the potential protective effect of selected lab on animal and human cell lines challenged with viruses. the intestinal and macrophage cell lines used in this study were not of tumor origin, in order to simulate more closely the host-probiotic interactions, as previously reported (pipenbaher et al., ). the cell lines were co-incubated with various lab strains and challenged subsequently with tgev and rv, in order to simulate a possible scenario of bacteria feed supplementation, where the probiotic organisms are already present in the intestinal lumen at the time of viral infection. for the preliminary antiviral assay, the intestinal porcine clab cell line was chosen, as it represented a good model for both viral infection and lab adhesion, based on previous laboratory data (unpublished) . a lab strain specific antiviral protective effect was observed in both preliminary clab assay and subsequent challenges of the other cell lines, with the known probiotics l. casei shirota and l. rhamnosus gg, as well as e. faecium pcd and pck , l. plantarum pcs and pca and l. fermentum aca-dc . although both of the known probiotics used in this study have been used extensively in the literature, few cases have been reported on their antiviral effects in animals (zhang et al., ) and, to the best of our knowledge, none have been reported on cell lines. in fact, very few data exists in general on the use of probiotics as viral infection deterrents on cell lines. recent studies (botić et al., ; ivec et al., ) reported the protective effect that various lactic acid bacteria, including the probiotic lactobacillus paracasei f , conferred upon porcine intestinal epithelial and macrophage cell line infected with vesicular stomatitis virus (vsv). in the above studies, the antiviral effect mechanism was attributed to a variety of factors, including possible competition for attachment sites between probiotic bacteria and the virus and stimulation of innate cell responses or pro-inflammatory responses from the cell lines. the elucidation of the underlying mechanism of action of the probiotic strains was attempted also in this study. to this end, we investigated the induction of reactive oxygen species no − and h o release (ros) by cell lines co-incubated with lactic acid bacteria. ros has a variety of defensive roles in the host, such as killing of intracellular pathogens, tumor cells, but also virus-infected cells (hibbs et al., ; keyaerts et al., ) . the antiviral effects of no − in particular have been previously reported (bi and reiss, ; paludan et al., ; ellermann-eriksen, ) . however, over expression of no − , especially chronic, could have toxic side-effects also for the host cells (brown, ) . another mechanism that could be involved in the anti viral effect is the competition for attachment sites on the epithelial cell surfaces between probiotic bacteria and virus particles. this protective mode of action has already been cited in cell line interactions between probiotic bacteria and intestinal pathogens (cocconier et al., ; ouwenhand et al., ; lievin-le moal et al., ; maragkoudakis et al., ) , and implied as a possible mechanism of protection against vsv cell line infection (botić et al., ; ivec et al., ) . in the present work the two strains that presented the most pronounced antiviral protection were l. casei shirota and l. rhamnosus gg. in the case of l. casei shirota, the high ros release in the tlt cell line coincides with a high level of protection against tgev in the same cell line. however, in all other cases with l. casei shirota, no correlations can be found between ros release and antiviral activity. in addition, the low attachment capacity of the strain, reported also elsewhere (juntunen et al., ; ouwenhand et al., ; maragkoudakis et al., ) does not allow for direct linking of attachment ability and antiviral effect. similar observations can be drawn with l. rhamnosus gg, which, in spite of a low ros induction and attachment ability in all cell lines, was found to exert a major antiviral effect in both tgev and rv, in all cell lines tested. on the other hand, l. plantarum pca seems to be a constitutive inducer of ros release, as it was able to stimulate ros release in, four out of five cell lines tested. in addition, on the goat epithelial cell line (gie) strain pca induced increase release of both no − and h o . this could explain the antiviral activity obtained on the gie monolayers with the particular lab strain, where a notable protective effect against both rv and tgev was observed. the attachment ability of the same strain however appears to be rather low (~ %), and so it is unclear if adhesion could contribute to the antiviral effect. the highattaching strain l. plantarum aca-dc , also able to induce increase ros release in h and tlt, exhibited only low antiviral potential. similarly, another strain with high-attachment ability, e. faecium pcd , but no particular ros release induction, exhibited only a mild antiviral effect on only once cell line (gie). in the present work, clear indications on the nature of the antiviral effect were evident only in the case of the l. casei shirota against tgev on the human macrophace cell line (tlt) and with l. plantarum pca againt both rv and tgev on the goat epithelial cell line (gie). each lab-cell line-virus interaction is specific, barring the drawing of general and definite conclusions. it is however evident from the obtained results that probably more than one mechanism may be involved in the observed antiviral effect, as previously hypothesized in similar studies (botić et al., ; ivec et al., ) . in addition, apart from ros and attachment, the release of pro-inflammatory cytokines as interleukins (il) and or the antiviral interferon-γ (ifn-γ), is an important mechanism by which probiotic bacteria may protect host cells (miettinen et al., ; hessle et al., ; maassen et al., ; ivec et al., ) . the mechanism of action of the antiviral effects reported in this study needs to be further investigated and so a more detailed, holistic approach would need to be adopted, focusing less on the screening of lab strains against various cell lines and viruses, and more on in depth studies of the phenomenon. to conclude, this study reported for the first time, to the best of our knowledge, a protective effect of lactic acid bacteria against tgev and rv on animal and human intestinal and macrophage cell lines of non tumor origin. although preliminary, the results presented here are of particular importance and merit further investigation, as they can lead to development of specific in vitro models for selection of probiotic strains with antiviral effects. carefully selected probiotics could then be applied as animal feed supplement in order to provide specific protection against subclinical or acute viral diarrheas or intestinal disorders in general in farm animals. prophylactic lactobacillus gg reduces antibiotic-associated diarrhea in children with respiratory infections: a randomized study inhibition of vesicular stomatitis virus infection by nitric oxide a novel eukaryotic cell culture model to study antiviral activity of potential probiotic bacteria cell biology. no says yes to mitochondria antagonistic activity of lactobacillus acidophilus lb against intracellular salmonella enterica serovar typhimurium infecting human enterocyte-like caco- /tc- cells macrophages and cytokines in the early defence against herpes simplex virus atypical genetic locus associated with constitutive production of enterocin b by enterococcus faecium bfe probiotics in man and animals lactobacilli from human gastrointestinal mucosa are strong stimulators of il- production nitric oxide: a cytotoxic activated macrophage effector molecule some infectious causes of diarrhoea in young farm animals probiotics in human disease interactions of macrophages with probiotic bacteria lead to increased antiviral response against vesicular stomatitis virus adherence of probiotic bacteria to human intestinal mucus in healthy infants during rotavirus infection inhibition of sars-coronavirus infection in vitro by s-nitroso-n-acetylpenicillamine, a nitric oxide donor compound lactobacillus acidophilus (strain lb) from resident human adult gastrointestinal microflora exerts activity against brush border damage promoted by a diarrhoeagenic escherichia coli in human enterocyte-like cells strain-dependent induction of cytokine profiles in the gut by orally administered lactobacillus strains probiotic potential of 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milk, as starters for the production of fermented milks nitric oxide (no) production in mammalian non-tumorigenic epithelial cells of the small intestine and macrophages induced by individual strains of lactobacilli and bifidobacteria effects of a probiotic strain of enterococcus faecium on the rate of natural chlamydia infection in swine regulation / (ec), of the european parliament and of the council of september on additives for use in animal nutrition enteropathic coronaviruses impact of the probiotic bacteria enterococcus faecium ncimb (sf ) and bacillus cereus var. toyoi ncimb on the development of serum igg and fecal iga of sows and their piglets inhibition of penicillium nordicum in mrs medium by lactic acid bacteria isolated from foods trends in emerging viral infections of swine effect of administration of live saccharomyces cerevisiae on milk production, milk composition, blood metabolites, and fecal flora in early lactating dairy goats performance, diarrhea incidence, and occurrence of escherichia coli virulence genes during long-term administration of a probiotic enterococcus faecium strain to sows and piglets lactobacillus gg in the prevention of antibiotic associated diarrhoea in children weekly epidemiological report molecular characterization, technological properties and safety aspects of enterococci from hussuwa, an african fermented sorghum product probiotic lactobacillus acidophilus enhances the immunogenicity of an oral rotavirus vaccine in gnotobiotic pigs lactobacillus fermentum aca-dc displays probiotic potential in vitro and protects against trinitrobenzene sulfonic acid (tnbs)-induced colitis and salmonella infection in murine models this work was performed in the framework of the e.u. funded project entitled «control and prevention of emerging and future pathogens at cellular and molecular level throughout the food chain» (pathogencombat, fp - ). key: cord- -vda gj authors: jin, yu-bei; yang, wen-tao; shi, chun-wei; feng, bo; huang, ke-yan; zhao, guang-xun; li, qiong-yan; xie, jing; huang, hai-bin; jiang, yan-long; wang, jian-zhong; wang, guan; kang, yuan-huan; yang, gui-lian; wang, chun-feng title: immune responses induced by recombinant lactobacillus plantarum expressing the spike protein derived from transmissible gastroenteritis virus in piglets date: - - journal: appl microbiol biotechnol doi: . /s - - - sha: doc_id: cord_uid: vda gj transmissible gastroenteritis coronavirus (tgev) is one of the most severe threats to the swine industry. in this study, we constructed a suite of recombinant lactobacillus plantarum with surface displaying the spike (s) protein coming from tgev and fused with dc cells targeting peptides (dcpep) to develop an effective, safe, and convenient vaccine against transmissible gastroenteritis. our research results found that the recombinant lactobacillus plantarum (nc -psip -pgsa-s-dcpep) group expressing s fused with dcpep could not only significantly increase the percentages of mhc-ii(+)cd (+) b cells and cd (+)cd (+) t cells but also the number of iga(+) b cells and cd (+)cd (+) t cells of ileum lamina propria, which elevated the specific secretory immunoglobulin a (siga) titers in feces and igg titers in serum. taken together, these results suggest that nc -psip -pgsa-s-dcpep expressing the s of tgev fused with dcpep could effectively induce immune responses and provide a feasible original strategy and approach for the design of tgev vaccines. transmissible gastroenteritis of pigs is a highly contagious disease caused by porcine transmissible gastroenteritis virus and has clinical symptoms of severe diarrhea, vomiting, dehydration, and high death rate for newborn piglets (peng et al. ) . in , doyle and hutchings for the first time reported tge in the united states (doyle and hutchings ) , and it has now become a worldwide swine disease and has caused great harm to the global pig industry . tgev is one of the main causes of piglet disease and death today. the spike (s) protein of transmissible gastroenteritis virus encoded by the s gene of tgev is located in the outermost organelles and the main antigen protein of tgev; it carries the major b lymphocyte epitopes and is the only structural protein that induces the body to produce neutralizing antibodies and provide immunoprotection (krimmling et al. ) . tgev is also a cell-dependent viral antigen, and t cells can respond to whole virus particles and cause a cellular immune response (chen et al. ) . lactobacillus is a group of bacteria that can ferment carbohydrates, producing large amounts of lactic acid (könig and fröhlich ) . they are long-term colonizers of the human or animal gut and have many benefits to the body, and they are recognized internationally as food-grade safety microbes (saad et al. ) . lactobacillus has become the best choice for the expression of heterologous proteins and live vector vaccines presenting antigens in the field of genetically engineered vaccines because of its many advantages, such as easy culture, simple operation, and high safety (trombert ) . the lactobacillus plantarum nc strain was a strain of lactobacillus that was isolated from grass silage in the s (axelsson et al. ) . now, it is used as a model strain in the development of genetic tools, for instance, transformation, conjugation, and expression vectors (yang et al. a) . compared with lactobacillus of animal origin, such as lactococcus lactis, due to its resistance to stress and suitability as a host bacterium for expressing foreign protein, lactobacillus plantarum has gained increasing attention (jiang et al. ) . dendritic cell peptides (dcpep) are derived from phage oligopeptides, which can significantly enhance the immune response (yang et al. b ). dcpep has been considered to be a bridge between adaptive and host immunity because of its potent antigen-presenting ability and important role in inactivating and modulating the immune response (mohamadzadeh et al. ) . it can effectively capture foreign antigens and autoantigens and then present them to t cells, which induce different immune responses according to the environment . poly-γ-glutamic acetase synthase a (pgsa) is a constituent protein of the polyglutamate synthetase (pga) system of bacillus subtilis that can act as a bacterial surface display component and stably anchor-related enzyme systems to the surface of the cell membrane (narita et al. ) . given the characteristics of the pgsa protein, it has been applied to a variety of prokaryotic protein surface displays. in particular, it has been successfully applied in gram-positive receptor strains such as lactic acid bacteria, which provide a theoretical basis for us to study how to anchor exogenous proteins on the cell wall surface of lactobacillus plantarum (lei et al. ; yang et al. c) . previously, we successfully constructed recombinant l. plantarum nc (nc -psip -pgsa-s-dcpep) to enable the s antigen to be effectively recognized by the dc in the intestinal mucosa and to be exhibited on the surface of l. plantarum, and recombinant nc -psip -pgsa-s-dcpep can successfully express pgsa-s-dcpep displayed on the cell wall surface of l. plantarum (data not published). in the present study, oral administration of nc -psip -pgsa-s-dcpep significantly increased the secretion of il- , ifn-γ, secretory immunoglobulin a (siga), and igg and the number of mhc-ii + cd + b cells and cd + cd + t cells in piglets, which provided a potential to protect against tgev challenge. this provides a strategy for the preparation of oral lactic acid bacteria vaccines to prevent transmissible gastroenteritis of pigs. the recombinant l. plantarum (for the delivery of s protein) used in this study was previously constructed by our laboratory. all the recombinant l. plantarum were cultured in man rogosa sharpe (mrs) medium at °c without shaking. when required, erythromycin was added to l. plantarum strain nc (ccug ) at μg/ml. a total of one-month-old tgev-seronegative crossbreed junmu white pigs were provided by jilin university pig farm and assigned to five groups (n = ) named saline, nc -psip -pgsa, nc -psip -pgsa-s-ctrlpep (the s gene fragment (genbank accession no. kt , source- , to , )/spike protein genbank accession no. amb , source- to and the control peptide (epihpettftnn)), nc -psip -pgsa-s-dcpep (dc peptide (fypsyhstpqrp)) (shao-hua et al. ) , and tgev inactivated vaccine. groups were housed in a specific pathogen-free (spf) environment. the piglets of the same group were placed in a uniform environment; however, they were separated into rooms and fed a balanced diet with free access to water. the piglets were administered orally with ml saline, cfu nc -psip -pgsa, cfu nc -psip -pgsa-s-ctrlpep, cfu nc -psip -pgsa-s-dcpep, and ml inactivated tgev, respectively, approximately twice at -day intervals. the feces and serum were collected every week after the initial immunity. the blood samples were incubated at °c overnight without shaking and then centrifuged at °c and rpm for min, and the serum was obtained and stored at °c for subsequent analysis. the feces were collected as previously published . the piglets were handled and maintained under strict ethical conditions according to international recommendations for animal welfare. all group piglets were euthanized at days after secondary immunization, and the intestinal segments were collected as previously described but with minor alterations (zhao et al. ) . in detail, upon washing with ice-cold pbs, the fixed tissues were immediately embedded in oct (embedding medium for frozen tissue specimens; sakura, usa) and then stored at − °c until further use. frozen tissue sections ( μm thick) were cut on a frozen slicer (leica cm s cryostat, germany) and transferred to poly-l-lysine-coated microscope slides. the slides were air-dried and stored for up to weeks at − °c before immunofluorescence staining. the animal management procedures and all laboratory procedures abided by the demands of the animal care and ethics committees of jilin agriculture university, china. the levels of tgev-specific siga in feces and igg in the sera were identified by enzyme-linked immunosorbent assay (elisa) according to previously published methods mou et al. ) . moreover, the levels of il- and il- in serum were determined by elisa according to the manufacturer's instructions (lvye biotechnology, china) with minor alterations. the difference between the original manufacturer's recommendations and the changed was only in the sample processing method. the changed was only that all samples were added at twofold dilutions from / to / in diluted buffer, and the other steps followed the instructions in the kits. finally, the absorbance at nm was read with a microplate reader (biotek, usa). single-cell suspensions were prepared from spleens, mesenteric lymph nodes (mlns), peyer's patches (pps), and ileum lamina propria (ilp) using modifications of previously published methods (rios et al. ) , and then stained with specific antibodies as described previously (sinkora and sinkorova ) . the percentages of cd + cd + t cells and mhc-ii + cd + igm + b cells were evaluated by facs (bd lsrfortessa, usa). mouse antipig mabs were used as primary immunoreagents. singlecell suspensions from spleens and pps were stained with specific antibodies for anti-igm (m , igg ), anti-mhc-ii (msa , igg a), and anti-cd (mem- , igg ) to analyze the percentages of mhc-ii + cd + igm + b cells. goat polyclonal abs specific for mouse ig subclasses labeled with allophycocyanin (apc), allophycocyanin/ cyanine tandem complex (apc-cy ), and peridininchlorophyll-protein complex (percp) were used as secondary antibodies. all fluorescent secondary antibodies were purchased from southern biotech (usa). the single-cell suspensions from mlns were stained with anti-cd (fitc) (bb - e - c ; bd pharmingen), anti-cd (apc) ( - - ; bd pharmingen), and anti-cd (pe) ( - - ; bd pharmingen) to analyze the percentages of cd + cd + t cells. mlns are mainly used to analyze the immunoprotection supplied by lactic acid bacteria vaccines in mucosal immunity. to examine the viability of t cells from the mlns, t-cell proliferation was performed. single-lymphocyte suspensions of each group of piglets (n = ) were prepared from the mlns when the piglets were euthanized as previously published (jiang et al. ) with slight changes. in detail, the lymphocytes were incubated in triplicate in -well plates at × cells/well in roswell park memorial institute medium (rpmi- ) containing % fetal calf serum (fcs) and stimulated with μg/ml phytohemagglutinin (pha) and culture medium as a negative control in a % co incubator (thermoscientific, usa) at °c for days. a thiazolyl blue (mts) solution was added to each well, and μl was added to each well to develop the color after days. the od values were then detected using a microplate reader (biotek, usa), and the value of the negative control wells was set to zero after h of incubation. the lymphocytes from the spleens, mlns, pps, and ilp were determined by real-time rt-pcr (qpcr) using a cfx tm real-time pcr detection system (bio-rad, usa). qpcr was used to quantify the messenger rna (mrna) of cd , cd , cd , tlr- , tlr- , il- , il- , ifn-γ, tgf-β, b-cell activating factor (baff), and a proliferationinducing ligand (april) in the total rna isolated from × lymphocytes of spleens, mlns, pps, and ileum lamina propria using an rna extraction kit according to the manufacturer's recommendations (takara, japan). total rna concentration was identified with biotek epoch microplate reader (biotek, usa). the total rna were reverse transcribed using the primescript™ rt reagent kit with gdna eraser (takara, japan) according to the manufacturer's instructions. the specific primer sequences are listed in table . in the end, the − ΔΔct method was utilized to calculate relative gene expression compared with the β-actin gene control (livak and schmittgen ) . the cryosections were incubated with anti-pig cd , antipig cd , anti-pig cd (three-antibody combination; bd biosciences, usa), or anti-pig iga (fitc) (abcam, uk) as described previously (subramaniam et al. ) but with some changes. briefly, phosphate-buffered saline (pbs) containing % pig serum was used to block fc receptors for min. combinations of cd , cd , cd , or iga mabs were added to the slides overnight at °c; subsequently, the slides were washed three times for min each time, with fresh changes of tbs-tween. the cell nuclei were then stained with , -diamidino- -phenylindole (dapi) solution (invitrogen, usa) for min and washed three times for min each time, with fresh changes of tbs-tween. the slides were imaged by confocal microscopy (zeiss lsm , germany), and the imaging results were analyzed using zeiss (blue edition). if not additionally declared, the data were presented as arithmetic mean values ± sem, and statistical analysis was performed by one-way analyses of variance (anovas) using graphpad prism . software. p < . was considered statistically significant. the rna of lymphocytes from spleens (sl), mlns (ml), pps (pl), and ilp (il) were extracted to analyze the expression of tlr- and tlr- by real-time rt-pcr (fig. ) . the tlr- and tlr- expression in ilp, pps, and mlns was higher in the nc -psip -pgsa-s-dcpep group than in the other groups compared with the saline, nc -psip -pgsa, nc -psip -pgsa-s-ctrlpep, and tgev inactivated vaccines (fig. ) . moreover, the results also showed that tlr- and tlr- of lymphocytes from spleens displayed the same trend in the nc -psip -pgsa-s-dcpep group than in the other groups (fig. ) . therefore, the results suggested that nc -psip -pgsa-s-dcpep could enhance innate immune responses in piglets. ( × cfu in ml), saline ( ml), or tgev inactivated vaccine ( ml), respectively, for each piglet on days - and - the rna of lymphocytes from ilp, pps, mlns, and spleens were extracted to analyze the expression of these costimulatory molecules cd , cd , and cd in b cells by real-time rt-pcr. furthermore, to assess the expression of cd and mhc-ii on the surface of b cells, we performed flow cytometry. all piglets were euthanized days after secondary immunization. the results showed that nc -psip -pgsa-s-dcpep significantly improved the expression of cd , cd , and cd in the b cells from ilp, pps, mlns, and spleens by real-time rt-pcr compared with the other groups ( fig. a-c) . in addition, the results found that nc -psip -pgsa-s-dcpep could also significantly increase the expression of cd and mhc-ii on the surface of b cells from pps compared with the groups of saline (p < . ), nc -psip -pgsa (p < . ), nc -psip -pgsa-s-ctrlpep (p < . ) (fig. d) , and spleens compared with the groups of saline (p < . ), nc -psip -pgsa (p < . ), and nc -psip -pgsa-s-ctrlpep (p < . ) by flow cytometry (fig. e) ; however, nc -psip -pgsa-s-dcpep was not significant compared with the group of tgev inactivated vaccine about increasing the expression of cd and mhc-ii on the surface of b cells from pps and spleens (fig. d, e) . in summary, the all data suggested that nc -psip -pgsa-s-dcpep could induce b-cell activation not only mucosally but also systemically in piglets. the number of iga + b cells in ilp coming from all groups of piglets was determined by immunofluorescence. the results showed that nc -psip -pgsa-s-dcpep could significantly increase the number of iga + b cells in ilp compared with the groups of saline (p < . ), nc -psip -pgsa (p < . ), nc -psip -pgsa-s-ctrlpep (p < . ), and tgev inactivated vaccine (p < . ) (fig. ). in addition, the titers of anti-tgev siga in feces and anti-tgev igg titers in the serum of piglets after oral immunization were determined by elisa. we found that the tgev-specific siga antibody titers in feces induced by nc -psip -pgsa-s-dcpep significantly increased compared to saline (p < . ) and nc -psip -pgsa (p < . ) from days to days and that the value reached the maximum at days after initial immunity (fig. a ). in addition, nc -psip -pgsa-s-dcpep did not significantly increase the tgev-specific siga antibody titers in feces compared to tgev inactivated vaccine (positive control) (p > . ) (fig. a) . the results also showed that nc -psip -pgsa-s-dcpep could also significantly induce specific igg antibody titers in serum compared with others ( fig. b) . however, there were no significant differences between nc -psip -pgsa-s-dcpep and tgev inactivated vaccine (p > . ) (fig. b) . therefore, the data suggested that nc -psip -pgsa-s-dcpep served as the vaccine to immunize the piglets and could induce local mucosal immune responses and systemic immune responses. in addition, to further demonstrate that the recombinant l. plantarum could influence cell-mediated immunity, we prepared a single-cell suspension of mlns lymphocytes coming from piglets immunized with saline, recombinant l. plantarum, and tgev inactivated vaccine groups days after the boost immunization and then carried out a cell proliferation test. the results showed that nc -psip -pgsa-s-dcpep resulted in significantly higher levels of t-cell proliferative responses than saline (p < . ) (fig. ) . however, there were no significant the mean values ± sem of three independent experiments are shown. *p < . , **p < . , ***p < . . ns, not significant. the error bars represent standard deviations differences compared with the nc -psip -pgsa, nc -psip -pgsa-s-ctrlpep, and tgev inactivated vaccine (fig. ). this result implied that nc -psip -pgsa-s-dcpep could enhance the viability of t cells of mlns in piglets. the single-cell suspensions from the mlns were stained with anti-cd antibody, anti-cd antibody and anti-cd antibody to detect by facs. as shown in fig. , nc -psip -pgsa-s-dcpep significantly increased the percentages of cd + cd + t cells in mlns compared to the groups of saline (p < . ), nc -psip -pgsa (p < . ), nc -psip -pgsa-s-ctrlpep (p < . ), and tgev inactivated vaccine (p < . ). moreover, the number of cd + cd + t cells in the ilp coming from all groups of piglets was determined by immunofluorescence. the results showed that nc -psip -pgsa-s-dcpep could significantly increase the number of cd + cd + t cells in ilp (fig. ) . these results indicated that oral immunization with nc -psip -pgsa-s-dcpep could effectively increase ileum local lymphocyte cells and induce local mucosal immune responses in piglets. the cytokine expression was also detected in the splenic lymphocytes (sl), mesenteric lymph node lymphocytes (ml), and ileum lamina propria lymphocytes (il) by qpcr or elisa in all piglets. in this study, the expressions of il- and il- in serum induced by nc -psip -pgsa-s-dcpep were higher than saline (p < . ), nc -psip -pgsa (p < . ), and nc -psip -pgsa-s-ctrlpep (p < . ) by elisa (fig. a, b) . in addition, the il- , il- , ifn-γ, and tgf-β expressions of sl, ml, and il induced by nc -psip -pgsa-s-dcpep were higher than those induced by the saline, nc -psip -pgsa, and nc -psip -pgsa-s-ctrlpep by qpcr (fig. c-f) ; however, nc - psip -pgsa-s-dcpep was not significantly different compared with the nc -psip -pgsa and nc -psip -pgsa-s-ctrlpep on the il- expression in il (fig. d) . besides, we also found that the expressions of baff and april in sl, ml, and il were the higher in the nc -psip -pgsa-s-dcpep group than in the other groups compared with the saline, nc -psip -pgsa, and nc -psip -pgsa-s-ctrlpep by qpcr (fig. g, h) . moreover, the baff and april expressions in the ml and il in the nc -psip -pgsa-s-dcpep group were significantly enhanced compared to the tgev inactivated vaccine; however, there was no significant difference in sl (fig. g, h) . the higher expression of baff and april suggested that nc -psip -pgsa-s-dcpep could effectively promote b-cell maturation and activation. lactobacillus plantarum is a well-characterized bacterium that is a particularly popular probiotic used to express heterogeneous proteins and deliver targeted antigens . in a previous research report, due to the use of the intracellular expression vector psip to express target antigens aiming to protect against newcastle disease virus (ndv), the research results did not achieve the expected results (jiang et al. ) . to overcome these hurdles, as a powerful technology, surface display may be a good choice to express heterogeneous peptides and proteins on cells, making use of natural functional components of microorganisms (raha et al. ; kuczkowska et al. ). poly-γ-glutamic acid synthetase a (pgsa), which is a constituent protein of the polyglutamate synthetase system (pga) of bacillus subtilis, has been widely used to anchor foreigner protective antigens on the surface of lactic acid bacteria (sewaki ; lei et al. ) . in previous studies, we successfully used surface display technology to express a number of targeted proteins, such as hemagglutinin subunit (ha ) of avian influenza virus (aiv), and murine il- and spike protein (s) of porcine epidemic diarrhea virus (pedv) (cai et al. ; huang et al. ; jiang et al. ). because dc targeting strategies could stimulate stronger and lasting immune responses, they have attracted the eyes of an increasing number of people. in the present study, to reduce the number of vaccinations of l. plantarum, which served as oral administration, the specific -mer dc-binding peptides were utilized to target dcs from the bacteriophage library. previous studies have found that anthrax pa or hepatitis c virus (hcv) ns , which genetically fused dc-binding peptides, could efficiently deliver antigens to dcs (shao-hua et al. ) . in previous reports, this vaccine strategy offered a variety of benefits in that the constructed l. plantarum expression of protein-fused dcpep could activate mucosal dcs, b cells, and t cells to induce the immunological response and could regulate inflammatory responses that took place in a mucosal microenvironment (steinman and idoyaga ) . our laboratory has previously studied yang et al. ) the effects of recombinant lactic acid bacteria expressing target antigens and dcpep on mhc-ii + cd + dcs; however, the impact of recombinant lactic acid bacteria cells has not been studied in mhc-ii + cd + b (such cells not only can secrete siga involved in mucosal immunity but also can act as antigen-presenting cells) (adler et al. ; mizoguchi and bhan ) . therefore, in this study, we mainly researched the effects of recombinant lactic acid bacteria nc -psip -pgsa-s-dcpep expressing dcpep and target antigen s protein on mhc-ii + cd + b cells, cd + cd + cells related to mucosal immune responses, and anti-tgevspecific antibodies. in this study, the results showed that the recombinant lactic acid bacteria nc -psip -pgsa-s-dcpep could raise not only the rate of mhc-ii + cd + b cells in pps and spleens by flow cytometry but also the number iga + b cells in ileum lamina propria by immunofluorescence and t cells in the mesentery by flow cytometry (figs. , , and ) . this research also showed that nc -psip -pgsa-s-dcpep could increase s-specific siga antibody titers in fecal matter to produce mucosal immune responses and s-specific igg antibody titers in serum to produce humoral immune responses by elisa analysis (fig. ) . the researchers found that immunization with antigenfused dc targeting peptides could notably induce cd + cd + t cells to expand and proliferate (lahoud et al. ; similarly, our results showed that nc -psip -pgsa-s-dcpep expressing s-dcpep also significantly boosted the number of cd + cd + t cells in mlns compared with s alone (fig. ) . it has been considered that cd + cd + t cells serving as a type of t-helper cells could promote other immune cells to mature and activate by secreting a variety of cytokines. the primary liability of the significant production of mucosal siga in the nc -psip -pgsa-s-dcpep group should be borne by observed t-cell expansion. this may be the cause for recombinant lactic acid bacteria nc -psip -pgsa-s-dcpep expressing dcpep and target antigens s protein being able to better assist recombinant antigen-induced dendritic cell proliferation and activation in piglets in vivo. dcs captured the dcpep binding targeted antigen s protein and then presented the antigens to t cells; by secreting cytokines, th cells activated the b cells . another reasonable explanation is that dcpep may also be recognized by b cells which some b cells also have antigenpresenting function, which subsequently presented the antigens to t cells with apc function. of course, the specific mechanism needs to be further studied. ifn-γ, which is a cytokine, could enhance phagocytic activity to efficiently kill pathogens and is produced by nk cells and t lymphocytes. it was reported that ifn-γ could cause th responses to protect against pathogen infection by adjusting chemotaxis and enhancing antigen presentation (schroder et al. ). il- promotes the expression of mhc-ii and cd in b cells and enhances the ability of b cells to present antigen so that the immune system can produce an immune response to antigen stimulation. the th and th cell responses are related to the secretion of ifn-γ and il- , respectively. as a consequence, in this study, the secretion levels of ifn-γ and il- were analyzed in the immunized animals to indirectly reflect the capacity of the vaccine to induce the th or th response. in addition, the balance between th and th responses was evaluated by the secretion levels of these cytokines (chen et al. ) . in this study, compared to the saline control group in the piglets, we found that nc -psip -pgsa-s-dcpep could significantly induce the secretion of ifn-γ and il- , indicating that nc -psip -pgsa-s-dcpep significantly heightened the immunogenicity of the tgev vaccine and triggered the immune response of th -and th -type cells (fig. ) . the results also suggested that nc -psip -pgsa-s-dcpep administered orally to piglets had a powerful potentiation influence on both humoral and cellular immunity. a previous study showed that oral administration of lactobacillus fermentum cect notably heightened not only the production of th -type cytokines in serum but also specific siga antibody responses to influenza (olivares et al. ). the increased percentages of il- -secreting th cells then possibly stimulated the differentiation of iga + b cells and increased production of mucosal siga antibodies. in addition, the primary function of th cells is to induce b-cell proliferation and produce antibodies that are related to humoral immunity. it was reported that il- has significant functions that participate in pro-and anti-inflammatory effects . previous research showed that recombinant lactobacillus induced the expression of il- in both systemic and mucosal immune responses to protect against tgev infection (jiang et al. ) . similarly, in this study, we found that piglets immunized with nc -psip -pgsa-s-dcpep could remarkably induce the expression of il- in spleen cells (systematic immune responses) and mesenteric lymph node cells (mucosal immune responses) compared with other groups (fig. ). regulatory t (treg) cells take part in the regulation of anti-inflammatory responses primarily through the secretion of cytokines such as il- and tgf-β (noack and miossec ) . previous studies have found that the oral administration of lactobacillus casei can increase the expression of tgf-β in blood, and this is essential for th differentiation in the spleen (jiang et al. ) . microbe-associated molecular patterns or soluble factors from probiotic bacterial genome dna, such as probiotic bacterial genome dna cpg, may regulate immunoregulatory effects and enhance iga. there was a report that compared with the vaccinated group, which was not colonized in piglets, the vaccinated probiotic colonized in piglets dramatically increased small intestinal tlr expression (vlasova et al. ) . tlr recognizes bacterial cpg motifs and the higher expression of tlr coming from mncs was very important for mucosal iga to participate in the immune response. a previous study showed that toll-like receptors (tlrs) play an important role in the activation of dcs using recombinant lactobacillus (kathania et al. ) . recombinant lactobacillus has been reported to serve as a vaccine to effectively inhibit the expression of tlr in piglets . however, in this study, we also evaluated the expression of tlr- and tlr- in piglets immunized with recombinant l. plantarum by real-time rt-pcr analysis and found that recombinant l. plantarum could increase the number of tlr- and tlr- expression in pigs to stimulate the host mucosal immune system (fig. ) . it is likely that lactobacilli induced the expression of tlr- and tlr- , which is related to the higher lactobacilli quantity (wen et al. ). it is well known that the expression levels of cytokines and tlr in splenic lymphocytes are indicators of systemic immunity, but the expression of cytokines and tlr in the cells coming from mlns is concerned with local and mucosal immune responses. baff and april are usually secreted by not only monocytes and intestinal epithelial cells but also t cells (fagarasan et al. ). baff has a strong b-cell chemotaxis and can induce activated b cells to secrete large amounts of igg, iga, and igm as costimulatory factors for b-cell proliferation and differentiation in vitro, which can help the immature b cells of peripheral blood survive and differentiate into mature b cells in vivo (boneparth and davidson ) . a previous study showed that piglets colonized by lactobacillus rhamnosus showed that probiotic treatment enhanced the expression of april in the gut but had no influence on the expression of baff in mncs compared with control groups (kandasamy et al. ) . in this study, we found that recombinant lactic acid bacteria nc -psip -pgsa-s-dcpep also boosted april and baff expression in the gut (fig. ) . a previous study reported that the cd -cd l and cd -cd pathways are essential for the activation of t cells and activation of polyclonal b cells (tokunaga et al. ) . this research also found that recombinant lactic acid bacteria nc -psip -pgsa-s-dcpep could improve the expression of cd and cd /cd (fig. ) . the above results provided a reason for why nc -psip -pgsa-s-dcpep could enhance the number of iga + b cells and cd + cd + t cells in the ilp. it is well known that the mucosal immune response is considered the first barrier function to neutralize viruses, including tgev. previous research studies have already demonstrated that siga participates in the response protecting against disease at mucosal surfaces (liu et al. ) . siga agglutinates and incapacitates pathogens, by which it inhibits pathogen adhesion to the mucosal surface and is easily cleared in the secretions. therefore, siga disenables pathogens to interact with epithelial cell receptors and inhibits the assembly of viral particles within the host cell cytoplasm (kurashima and kiyono ) . recombinant lactic acid bacteria can induce the production of siga in the intestines. in our research, we found that recombinant lactobacillus nc -psip -pgsa-s-dcpep could significantly increase the titer of anti-tgev siga in feces and anti-tgev igg titer in the serum of piglets after oral immunization, comparing the different versions of nc -psip -pgsa, nc -psip -pgsa-s-ctrlpep, and nc -psip -pgsa-s-dcpep (fig. ). in addition, it is worth noting that the levels of specific antibodies induced by nc -psip -pgsa-s-dcpep could continue days at a high level. this may be related to lactobacillus colonization in the intestinal tract. comparing the different versions of nc -psip -pgsa, nc -psip -pgsa-s-ctrlpep, nc -psip -pgsa-s-dcpep, and tgev inactivated vaccines, upon immunization, we also found that nc -psip -pgsa-s-dcpep triggered expected immune responses between b and t cells, and as expected, the expression of siga was higher and continual. in addition, it has been shown that lymphocyte proliferation occurs in the ileum lamina propria of piglets immunized with an oral dose of recombinant l. plantarum. it was further suggested that recombinant lactobacillus could induce mucosal immune responses in piglets. in summary, all results recommended that the nc -psip -pgsa-s-dcpep expressing the s of tgev fused with dcpep could effectively induce immune responses, including mucosal immune and systemic immune responses. in conclusion, this study suggested that immunized piglets with nc -psip -pgsa-s-dcpep could enhance the percentages of mhc-ii + cd + b cells and cd + cd + t cells and induce the expression of cytokines to initiate immune responses. furthermore, nc -psip -pgsa-s-dcpep could significantly raise not only the specific siga titers in feces but also igg titers in serum. the nc -psip -pgsa-s-dcpep provided a feasible original strategy and approach for the design of tgev vaccines. fig. analysis of the expression of cytokines. the levels of cytokines il- (a) and ifn-γ (b) in serum were detected by respective elisa kits. in addition, the rna of lymphocytes from spleens (sl), mlns (ml), and ilp (il) were extracted to analyze the expression of il- (c), il- (d), ifn-γ (e), tgf-β (f), baff (g), and april (h) by real-time rt-pcr. the mean values ± sem of three independent experiments are shown. *p < . , **p < . , ***p < . . ns, not significant. the error bars represent standard deviations conflicts of interest the authors declare that there are no competing interests. ethical approval all applicable international and national guidelines for the care and use of piglets were followed. the other function: class ii-restricted antigen presentation by b cells genome sequence of the naturally plasmid-free lactobacillus plantarum strain nc (ccug ) b-cell activating factor targeted therapy and lupus surface-displayed il- by recombinant lactobacillus plantarum reduces th responses of raw . cells stimulated with poly(i:c) or lps baculovirus as an avian influenza vaccine vector: differential immune responses elicited by different vector forms retinoic acid facilitates inactivated transmissible gastroenteritis virus induction of cd + t-cell migration to the porcine gut a transmissible gastroenteritis in pigs adaptive immune regulation in the gut: t cell-dependent and t cellindependent iga synthesis construction and immunogenicity analysis of lactobacillus plantarum expressing a porcine epidemic diarrhea virus s gene fused to a dc-targeting peptide upregulation of mdp and tuftsin gene expression in th and th cells as an adjuvant for an oral lactobacillus casei vaccine against anti-transmissible gastroenteritis virus construction and immunological evaluation of recombinant lactobacillus plantarum expressing hn of newcastle disease virus and dc-targeting peptide fusion protein a phase trial of the oral lactobacillus casei vaccine polarizes th cell immunity against transmissible gastroenteritis coronavirus infection molecular mechanisms underlying protection against h n influenza virus challenge in mice by recombinant lactobacillus plantarum with surface displayed ha -ltb expression and purification of swine rag in e. coli for production of porcine rag polyclonal antibodies lactobacilli and bifidobacteria enhance mucosal b cell responses and differentially modulate systemic antibody responses to an oral human rotavirus vaccine in a neonatal gnotobiotic pig disease model colonic immune stimulation by targeted oral vaccine biology of microorganisms on grapes, in must and in wine infection of porcine precision cut intestinal slices by transmissible gastroenteritis coronavirus demonstrates the importance of the spike protein for enterotropism of different virus strains immunogenic properties of lactobacillus plantarum producing surface-displayed mycobacterium tuberculosis antigens mucosal ecological network of epithelium and immune cells for gut homeostasis and tissue healing targeting antigen to mouse dendritic cells via clec a induces potent cd t cell responses biased toward a follicular helper phenotype lactococcus lactis displayed neuraminidase confers cross protective immunity against influenza a viruses in mice comparison of the immune responses induced by oral immunization of mice with lactobacillus casei-expressing porcine parvovirus vp and vp fused to escherichia coli heat-labile enterotoxin b subunit protein analysis of relative gene expression data using real-time quantitative pcr and the (−delta delta c(t)) method immunobiology of b cells in inflammatory bowel disease. crohn's disease and ulcerative colitis targeting mucosal dendritic cells with microbial antigens from probiotic lactic acid bacteria expression of major antigenic sites a and d in s gene of transmissible gastroenteritis virus of swine (tgev) in escherichia coli and development of indirect elisa for detection of the antibody against tgev display of active enzymes on the cell surface of escherichia coli using pgsa anchor protein and their application to bioconversion th and regulatory t cell balance in autoimmune and inflammatory diseases oral intake of lactobacillus fermentum cect enhances the effects of influenza vaccination porcine epidemic diarrhea. emerging and re-emerging infectious diseases of livestock cell surface display system for lactococcus lactis: a novel development for oral vaccine antigen sampling by intestinal m cells is the principal pathway initiating mucosal iga production to commensal enteric bacteria an overview of the last advances in probiotic and prebiotic field activation of b cells by a dendritic celltargeted oral vaccine interferon-gamma: an overview of signals, mechanisms and functions generation of mucosal vaccine utilizing lactobacillus display system lactobacillus plantarum vaccine vector expressing hemagglutinin provides protection against h n challenge infection new progress regarding the use of lactic acid bacteria as live delivery vectors, treatment of diseases and induction of immune responses in different host species focusing on lactobacillus species b cell lymphogenesis in swine is located in the bone marrow features of the dendritic cell lineage efficient priming of cd t cells by langerin-expressing dendritic cells targeted with porcine epidemic diarrhea virus spike protein domains in pigs down-regulation of cd and cd on b cells in patients with life-threatening systemic lupus erythematosus after successful treatment with rituximab recombinant lactic acid bacteria as delivery vectors of heterologous antigens: the future of vaccination? lactobacilli and bifidobacteria promote immune homeostasis by modulating innate immune responses to human rotavirus in neonatal gnotobiotic pigs porcine epidemic diarrhea in china toll-like receptor and innate cytokine responses induced by lactobacilli colonization and human rotavirus infection in gnotobiotic pigs cross-protective efficacy of dendritic cells targeting conserved influenza virus antigen expressed by lactobacillus plantarum construction and immunological evaluation of recombinant lactobacillus plantarum expressing so of eimeria tenella fusion dc-targeting peptide protection of chickens against h n avian influenza virus challenge with recombinant lactobacillus plantarum expressing conserved antigens recombinant lactobacillus plantarum expressing ha antigen elicits protective immunity against h n avian influenza virus in chickens effects of virulent and attenuated transmissible gastroenteritis virus on the ability of porcine dendritic cells to sample and present antigen key: cord- -r fb authors: torres, juan m.; sÁnchez, carlos; suÑÉ, carlos; smerdou, cristian; prevec, ludvik; graham, frank; enjuanes, luis title: induction of antibodies protecting against transmissible gastroenteritis coronavirus (tgev) by recombinant adenovirus expressing tgev spike protein date: - - journal: virology doi: . /viro. . sha: doc_id: cord_uid: r fb abstract ten recombinant adenoviruses expressing either fragments of , , or nt or the full-length spike gene of transmissible gastroenteritis coronavirus (tgev) have been constructed. these recombinants produce s polypeptides with apparent molecular masses of , , , and kda, respectively. expression of the recombinant antigen driven by ad promoters was inhibited by the insertion of an exogenous sv- promoter. most of the recombinant antigens remain intracytoplasmic in infected cells. all the recombinant-directed expression products contain functional antigenic sites c and b (gebaueret al., ,virology , – ). the recombinant antigen of kda and that of kda, which represents the whole spike protein, also contain antigenic sites d and a, which have previously been shown to be the major inducers of tgev-neutralizing antibodies. interestingly, here we show that recombinant s protein fragments expressing only sites c and b also induced tgev-neutralizing antibodies. the chimeric ad –tgev recombinants elicited lactogenic immunity in hamsters, including the production of tgev-neutralizing antibodies. the antisera induced in swine by the ad recombinants expressing the amino-terminal % of the spike protein (containing sites c and b) or the full-length spike protein, when mixed with a lethal dose of virus prior to administration to susceptible piglets, delayed or completely prevented the induction of symptoms of disease, respectively. immune response to coronaviruses enjuanes and van der zeijst, ) : the spike protein (s) transmissible gastroenteritis coronavirus (tgev) in- (buchmeier et al., ; cavanagh et al., ; daniel et fects the enteric and respiratory tissues of newborn pigal., ; daniel and talbot, ; koolen et al., ) , lets resulting in mortality of nearly % (saif and wesley, the membrane protein (fleming et al., ; . protection of newborn animals from tgev infecal., ; welch and saif, ) , and the nucleoprotein tion requires the induction of secretory iga in milk. previ-(buchmeier et al., ; ; lecomte et ous studies have shown that precursors of mucosal iga al., ; nakanaga et al., ; talbot et al., ; wesplasma cells originate in lymphoepithelial structures in seling et al., ) . the study of the induction of protecthe gastrointestinal and respiratory tracts. these precurtive immunity to tgev has focused on s protein because sor cells switch to iga production in gut-or bronchusit is the major inducer of tgev-neutralizing antibodies associated lymphoepithelial tissues and migrate to dis- jimé nez et al., ; laude et al., seminated mucosal effector sites, including gastrointesti- ) and it mediates binding of tgev to its cellular nal and upper respiratory tracts, as well as to exocrine receptor godet et al., ) . a correlatissues such as the mammary gland. recombinant hution between the antigenic and the physical structure of man adenovirus (ad ) has efficiently been used to in-s protein has been established ; duce protection against viral infections (berkner, ; jimé nez et al., ; suñé et al., ) . site a is also graham and prevec, ) . we have reported that ad involved in the induction of in vivo protection (de diego infects mucosal tissues of swine (torres et al., (torres et al., ), et al., , but the precise roles of the different antiindicating that recombinant adenoviruses might be used genic sites in eliciting resistance to tgev are unknown to induce mucosal immunity against tgev. helper-inde- (enjuanes and van der zeijst, ) . pendent ad -based vectors with the capacity to express in this paper we describe ad -tgev recombinants foreign genes of up to . kb have been developed (bett expressing either full-length tgev spike protein or three et al., ) . truncated amino-terminal fragments of this protein. these recombinants induced immune responses in hamsters and swine which neutralized tgev infectivity. in addition, we demonstrate that porcine serum from ad-tion. finally, we show that virus-neutralizing antibodies tains the -end of ad from the xhoi site at map units (m.u.) with a deletion of the xbai d fragment from are induced in the milk of ad-tgev-immune hamsters. . to . m.u. within the ad e coding region. plasmid pab also contains the -end of ad from map unit materials and methods to with a -nucleotide deletion in the e coding eukaryotic cells and viruses region. plasmid pfg contains a deletion of essential sequences to the left of e in the ad genome that renders the epithelial swine testicle (st) cell line (mcclurkin it unable to produce infectious ad (bett et al., ; and norman, ) and human cells which constitu-hanke et al., ; mittal et al., ) . tively express the -end % of the ad genome (graham et al., ) were used to grow the recombinant construction of recombinant vectors adenoviruses. pur -mad strain of tgev (sá nchez et al., ) was cloned, sequenced, and used as a source the general procedure followed to construct recombiof the s gene . neutralization of nant ad viruses expressing tgev s gene fragments tgev was performed by incubating serial -fold dilu-(ad-ts) is summarized in fig. . s gene sequences were tions of the virus with a / dilution of the antibody at flanked by sv- pr and polyadenylation sequences Њ for min, and the virus-antibody mixture was plated when indicated (fig. ) , by subcloning them into plasmid on st cells as previously described (correa et al., ) . psv x or psv x . cassettes with s gene sequences the neutralization index (ni) was defined as the log of were inserted into the unique xbai site of the partially the ratio of the pfu after incubating the virus in the deleted e gene on plasmid pfg k or pab , both presence of medium or the indicated antiserum. ni indiof which include the -end of ad . alternatively, s gene ces are determined rather than titers since in the first fragments were removed from the original plasmid or procedure virus-antibody mixtures are evaluated in the from psv x -ts vectors without sv- pr signal, or withplaque assay without further dilution of the antibody, proout both pr and polyadenylation sequences, using the viding highly reproducible results and information about restriction endonucleases indicated in fig. . in this case, the potency of the antibody (the titer reduction expressed fragment ends were blunted with klenow and t dna in logarithmic units rather than the ability of the serum polymerase and cloned into the xbai site of pfg k to neutralize a few pfu). or pab plasmids that were blunted and dephosphory-ad strain dl contains a small deletion from to lated according to standard procedures (maniatis et al., map units and an unknown substitution in the e ) . each of these plasmids is noninfectious by itself, region (jones and shenk, ) . pfg is an infectious but can generate infectious virus following cotransfection circularized form of ad dl carrying a . -kb dna of cells along with a plasmid, pfg , which coninsert (pmx ) encoding ampicillin resistance (apr) and a tains the -end of ad ( fig. ) (graham and prevec, bacterial origin of replication. plasmid pfg was used ; hitt et al., hitt et al., , . this results in the rescue as positive control for infectious ad dna (graham et of genes cloned into the e region of viral vectors. coal., ) . transfection was performed essentially as described using the calcium phosphate precipitate technique (gra-plasmids and bacteria ham and van der eb, ) . after to days, plaques were isolated and expanded, and viral dna was ana-the tgev s gene was cloned into bluescript (stralyzed by hindiii restriction enzyme digestion. viruses tagene) or pya plasmids (smerdou et al., ) as prewith the expected dna pattern were plaque purified viously described . escherichia coli three times and the junction of the constructs was se-dh or xl -blue cells (stratagene) were transformed quenced to verify the expected primary structure. recomwith newly constructed plasmids by electroporation binants ad-ts and ad-ts are identical to recombi- (dower et al., ) . plasmid dna was prepared by the nants ad-ts and ad-ts , respectively, except that the alkaline lysis method (birnboim and doly, ) and purifirst two were constructed using cloning vector pab fied by cscl-ethidium bromide density gradient centrifuwith the large deletion on e gene, while in the construcgation. s gene fragments or the full-length s gene were tion of the second pair of recombinants plasmid flanked either by sv- promoter (pr) alone or by both pfg k , with the smaller deletion on e , was used. pr and polyadenylation sequences, as indicated. s gene fragments were first subcloned into psv x or psv x immunoprecipitation of s antigens expressed by plasmids . the structures of the three recombinant ad-ts key plasmids (pfg k , pab , and pfg ) used in the construction of ad -tgev recombinants have been subconfluent cells grown in dulbecco's modified eagle medium with % horse serum (gibco europe) were reported previously (bett et al., ; mittal et al., ) . plasmid pfg k was derived from pfg (ghosh-infected with ad-ts recombinants at a multiplicity of infection (m.o.i.) of pfu per cell. after hr of virus choudhury et al., ) and as essential features con- (maniatis et al., ) . s gene sequences previously cloned into bluescript(sk ) (promega) or pya (smerdou et al., ) plasmids were excised using the indicated restriction endonucleases and subcloned into psv x or psv x , in which the s gene sequences were flanked by sv- pr, polyadenylation sequences, or both. to generate recombinants ad-ts , ad-ts , ad-ts , and ad-ts s gene sequences were cloned directly into plasmid pfg k or pab . s gene sequences either alone or flanked by sv- sequences were subcloned into the xbai site of pfg k or pab , or excised with the indicated restriction endonucleases, blunted using the klenow polymerase fragment, and cloned into blunted xbai unique site of these vectors. infectious ad-ts recombinants expressing s protein fragments were generated by cotransfecting cells with pfg k -ts or pab -ts (which carry s gene sequences from tgev and pfg plasmids). diagrams are not to scale. the origins of dna fragments flanking the s gene are indicated with squares filled with different motifs. numbers below the bar representing the ad genome (bottom) indicate map units. mcs, multicloning site; pr, promoter; an, polyadenylation signal; de , deletion in e gene; r.e., restriction endonuclease; ts refers to sequences derived from tgev spike gene. adsorption at Њ, fresh medium was added and cells dium, and refed with fresh medium containing mci/ml of pro-mix: l-[ s] in vitro methionine/cysteine labeling were incubated for hr at Њ. medium was then replaced by methionine-and cysteine-free medium con-mix ( ci/mmol, cod. no. sjq , amersham ibé rica). cell monolayers were incubated . hr, detached with a taining % dialyzed serum. cells were incubated for hr at Њ, washed with methionine-and cysteine-free me-rubber policeman, washed with cold phosphate-buffered saline, ph . (pbs), collected by centrifugation at standard) and ad-ts recombinants grown under the same conditions were immunoprecipitated in parallel. rpm for min at Њ in a microfuge, and lysed in ripa buffer ( mm tris-hcl buffer, ph . , mm nacl, % the same number of infected cells was analyzed for each recombinant. similar relative expression levels were ob-triton x- , % sodium dodecyl sulfate (sds), and . mm pmsf). viscosity was reduced by mixing the tubes tained in many (ú ) experiments. after protein resolution in polyacrylamide gel electrophoresis and autoradiogra-in a vortex mixer and passing the samples through a . -mm needle times. extracts were centrifuged at phy, the intensity of the immunoprecipitated bands from ad-ts extracts was compared with that of the reference , g for min at Њ in a microfuge. labeled proteins were immunoprecipitated with tgev-specific porcine se-[ s]tgev with known protein concentration (determined using bca protein assay reagent, pierce) to estimate rum which had been preadsorbed several times with cells infected with adenovirus ad dl . further the amount of s antigen. absorption of the antiserum did not eliminate the nonspecific bands. antigen-antibody complexes were bound immunofluorescence to protein a-sepharose by overnight incubation at Њ. sepharose beads were washed three times with ripa st cells at a density of approximately . cells/ cm in microslide culture chambers (miles scientific) buffer containing . % sds, and the final pellet was resuspended in electrophoresis sample buffer containing were infected with adenovirus ad which contains no s gene insert, or with ad-ts recombinants, at a m.o.i. . % sds and % -mercaptoethanol (laemmli, ) . samples were boiled for min, the beads were sedi-of pfu/cell. at hr postinfection, cell monolayers were washed and fixed either with methanol:acetone ( : ) at mented by low-speed centrifugation, and supernatants were analyzed by polyacrylamide gel electrophoresis Њ for min or with % paraformaldehyde in pbs for min at room temperature. cells were washed three and autoradiography. to estimate the amount of protein expressed by each recombinant different dilutions of su-times with pbs and once with . % bovine serum albumin (bsa) in pbs for min at room temperature. the cells crose gradient-purified s-labeled tgev (used as an were incubated with hybridoma supernatants containing highest dilution giving a binding threefold higher than background. a mixture of mabs d.b , b.h , and d.g (specific for s protein sites b, c, and d, respectively) or with mab detection of the different antigenic sites in the s protein fragments, encoded by recombinant ad-ts, was h - specific for a k ad antigen. after three additional washings with pbs, cells were covered with a carried out by cria using the antiserum elicited in hamsters by the different recombinants. the binding of i- : dilution of fluoresceinated goat anti-mouse immunoglobulins (cappel laboratories) in . % bsa in pbs, labeled mabs to purified tgev bound to microplates was performed as previously reported (correa et al., ) incubated for min at room temperature, washed five times for min each with pbs, and mounted on glyc-with some modifications. briefly, purified tgev ( . mg/ well) was plated, remaining binding sites were saturated erol-pbs ( : ). with % bsa in pbs, and i-labeled mabs (sp act . binding of i-labeled mabs to cells infected cpm/mg; cpm/well) were added and incuwith recombinant ad-ts bated for hr at Њ in the presence of fivefold dilutions of the competitor antiserum prepared in pbs with . % confluent st cell monolayers plated on -well mi-bsa. microplates were washed six times with . % bsa croplates were infected (m.o.i. pfu/cell) with recombiand . % tween- in pbs. well bottoms were cut and nant ad-ts viruses. at hr postinfection, cells were bound radioactivity was determined in a gamma counter. washed with pbs and fixed in methanol:acetone ( : ) for the percentage of radioactivity bound was determined min at Њ or in % paraformaldehyde in pbs for in relation to the radioactivity bound in the absence of min at room temperature. cells were washed three times competitor mab. purified homologous mabs were used with pbs and for hr with . % bsa in pbs. aliquots of as positive controls in the cria. . ml of i-labeled purified mabs ( cpm/well; . cpm/mg) (greenwood et al., ) protection of swine by immune serum . % bsa were added to each well and incubated for hr at room temperature, and the cell monolayers were the virulent tgev strain pur -sw -st ( washed six times with pbs. mab binding was deter-pfu/swine) was mixed with ml of the porcine antiserum mined by collecting the cells in . ml of . n naoh induced by recombinants ad-ts or ad-ts , incuand counting the radioactivity in a gamma counter. bated at Њ for min, and administered using a gastric tube to -day-old miniswine born from tgev-seronega-immunization of hamsters and swine tive sows. inoculated animals were fed three times per day with milk formula for newborns (nidina , nestlé ) eight-week-old golden syrian hamsters were immucontaining ml of the antiserum. control animals were nized with infectious ad-ts recombinants by three treated following the same procedure but using serum routes: oral ( pfu in . ml of pbs), nasal ( induced by wt ad . virus titers after , , and days in pfu/ . ml), and intraperitoneal ( pfu/ . ml). animals challenged with virus treated with control serum the virus was administered at days , , , and , and , , or days postinoculation in animals challenged and orbital plexus puncture bleedings were performed with tgev immune serum-treated virus were determined at days , , , , , and . females with highest in tissue extracts from jejunum and ileum, lungs, mesentiters of tgev-specific antibodies were crossed with nonteric, and mediastinal lymph nodes. tissue homogenizaimmune males, and days later another dose of the tion was performed at Њ using an omni homogehomologous ad-ts recombinant was administered. nizer (omni international). twenty-four hours after delivery, hamsters were subcutaneously administered iu of oxytocin. the milk was collected hr later by applying vacuum with a syringe. results milk was diluted fourfold in pbs and stored at Њ. ad -tgev recombinants one-month-old swine, from crossing large white and belgium landrace, were immunized three times at , , ten ad -tgev recombinants expressing tgev s and days, each time by three routes: oral ( gene fragments were constructed using vectors with dif-pfu), nasal ( pfu), and intraperitoneal ( ferent deletions on e gene or combinations of sv- pfu per dose). serum was collected days after the promoter and polyadenylation signals. using these relast immunization. combinants s protein fragments of four different sizes were expressed. the recombinants were obtained by radioimmunoassay (ria) and competitive ria (cria) replacing the e gene of the ad genome with s gene with i-labeled mabs sequences starting from nt and the first -end , , , or nt of the s gene. these recombinants ria was performed using purified tgev as antigen ( . mg/well) as previously described (jimé nez et al., code for fragments of , , , and amino acids (aa) extended from the amino-terminus (fig. ) . the ). titers in ria were defined as the inverse of the faint band (results not shown). recombinant products with apparent molecular masses of and kda (fig. , lanes c and e, respectively) were obtained for recombinant s protein fragments of and aa, respectively. recombinants ad-ts and ad-ts , both coding for polypeptides of aa, gave a main band of kda and a minor band of kda (lane d), which probably corresponds to an underglycosylated form of the antigen or to a degradation product. the difference between the expected and the apparent molecular mass of the recombinant products suggests that these are heavily glycosylated, as occurs during s protein synthesis after tgev serum using extracts from cells infected with the chimeric ad-ts viruses (fig. ) . the amount of s protein was based on the comparison of band intensity after last product represents the full-length spike protein. the immunoprecipitation and autoradiography of s-labeled constructs were obtained using either plasmid recombinant antigens and reference sucrose gradient-pfg k or plasmid pab (fig. ) , with deletions of purified s-labeled tgev with known protein concentra- . or . kb, respectively, in e (bett et al., ) . tion. both reference virus and recombinant antigens were recombinant plasmids were constructed as summarized labeled and analyzed in parallel using the same experi- (fig. ) . when indicated, the s gene fragments were mental conditions. since the distribution of the methioflanked by pr and polyadenylation signals (fig. ) by clonnine and cysteine in the different fragments was similar, ing them into vector psv x or psv x . inserts were no significant correction of band intensity was necessary subcloned into plasmid pfg k or pab containing in the analysis. the expression levels ranged from . the -end half of ad . human cells were cotransto mg of s protein per infected cells. maximum fected with one of these plasmids and pfg , which expression levels ( to mg/ cells) were obtained contains almost the entire ad genome with a lethal for recombinants ad-ts , ad-ts , and ad-ts , interdeletion across the e region. fully infectious ad-ts mediate levels ( to mg/ cells) for ad-ts , ad-ts , viruses were recovered following recombination in co-ad-ts , and ad-ts , and minimum (around . mg/ transfected cells. recombinant viruses were plaque cells) for recombinants ad-ts , ad-ts , and adpurified. the dna from all the recombinants gave the ts . relative expression levels were highly reproducpattern and sequence expected for each insert by hindiii ible in different experiments. all the recombinants, inrestriction endonuclease analysis and sequencing of cluding those expressing minimum amounts of antigen, dna junctions (results not shown). were also consistently positive in the immunofluores-after infection of cells with ad-ts recombinants, cence and i binding assays and in the induction of s protein antigens remained cell associated. tris buffer tgev-specific antibodies (see below). containing % sds was used to solubilize them. the when indicated, the s gene fragment cloned into ad estimated size of recombinant s antigen expressed by was flanked by pr and polyadenylation signals (fig. ) . comparison of the expression levels in constructs with and representative results are shown (fig. ) . s polypep-s gene fragments of the same size indicated that ad tides were detected with a polyclonal tgev-specific porrecombinants made using pfg k plasmids excine serum. good specific immunoprecipitation bands pressed higher levels of antigen than those based on were systematically obtained with all recombinants explasmid pab , although in some cases (i.e., recombinant ad-ts compared with ad-ts ) the level of ex-cept ad-ts , ad-ts , and ad-ts , which gave a pression was similar (results not shown). in recombinants with the same e deletion it was also observed that removal of sv- pr yielded ad-ts recombinants with higher expression levels (results not shown). to study the cellular location of recombinant s antigen, we used immunofluorescence analysis of st cells infected with four selected recombinants each coding for s fragments of different size: , , , and (full-length s protein) amino acids. a bright fluorescent signal was observed in the cytoplasm of methanol-acetone-fixed cells infected with recombinants ad-ts , ad-ts , and ad-ts (results not shown). highest fluorescence intensity was seen with tgev-infected cells and ts recombinants. when immunofluorescence was performed with a human ad -specific mab (which binds k protein) bright fluorescence was observed on discrete though amino acids to of s protein site d are areas of the nucleus, but not in the cytoplasm (results coded by recombinant ad-ts , this site was poorly recnot shown), in contrast to the cytoplasmic fluorescence ognized by mab d.g specific for d site on ad-ts observed with tgev-specific mabs. infected cells (fig. ) . the four antigenic sites were an estimation of the relative amount of s antigen loweakly detected in cells infected by recombinant adcated in the cytoplasm or accessible on the surface of ts , probably due to the low replication level of this ad -infected st cells was determined by studying the recombinant. binding of i-labeled mab d.b (site b-specific) to methanol-or paraformaldehyde-fixed cells (results not immunogenicity of the recombinants shown). this mab was selected because it recognizes an epitope present in all ad-ts recombinants. cells in-immune responses elicited by the different recombinants were studied by inoculating hamsters both orona-fected with recombinants ad-ts , ad-ts , and ad-ts permeabilized with methanol-acetone expressed the sally and intraperitoneally (fig. ) . seven of the ten recombinants summarized in fig. elicited titers in ria highest amount of s antigen, which ranged between and % of the amount expressed on st cells infected higher than and ni between and . the best inducers of tgev-neutralizing antibodies were recombi-with tgev. in cells infected with these recombinants the binding of site b-specific mab to exposed antigen was nants ad-ts , ad-ts , and ad-ts , expressing either the smallest protein fragment or the full-length protein around % of the binding to cytoplasmic s antigen of tgev-infected cells. that is, the amount of s antigen (fig. ) . four recombinants (ad-ts , ad-ts , ad-ts , and detected on the surface of the infected cells was at least sixfold lower than that seen in the cytoplasm. the recom-ad-ts ), each expressing s gene fragments of different lengths (fig. ) were selected to study the induction of binant products were not detected in the supernatants of infected cells, although the media were not concen-an immune response to sites a, b, and d by cria (fig. ). site c was not included in the study because the trated to detect small antigen amounts. proper folding of the s protein fragments expressed amino acid sequence pnsd recognized by mabs specific for this site is present in pro-by the four selected recombinants was evaluated by determining the amount of i-labeled mab specific for anti-teins of the immunoglobulin superfamily and other serum proteins (correa et al., ; i. correa and l. enjuanes, genic sites a, b, c, and d bound to infected st cells (fig. ) . all recombinants expressed sites c and b. recombi-unpublished results). recombinant ad-ts induced an immune response to antigenic sites b, d, and a (fig. ) . nant ad-ts , in addition, expressed sites d and a. al- fig. . immune response induced by ad-ts recombinants in hamsters. groups of four golden syrian hamsters were immunized at time and at times indicated by arrows (see materials and methods) with the indicated recombinants. sera collected at , , , , and, in some cases, at and days postinfection were evaluated by ria and neutralization against tgev. mean serum titers and standard deviation errors are represented for each time point. the titer by ria was defined as the inverse of the highest antibody dilution giving a binding three times higher than the background in the ria assay. the ni was defined as the log of the ratio of the pfu after incubating the virus in the presence of medium or the indicated antiserum. all recombinants induced a strong response to site b and milk was determined between days and during lactation (fig. ) . the three recombinants induced anti- (fig. a) which is conformation and glycosylation depenbodies in serum with titers in ria ranging from dent . as expected, site a was only to . and in milk from to (fig. reconstituted by recombinants ad-ts and ad-ts , a). serum and milk antibodies neutralized tgev with expressing the full-length s protein or the -kda s nis ranging from to and around , respectively (fig. antigen, but not by recombinants which do not include b). as expected, recombinants with no insert did not the residues implicated in this site (fig. c) . elicit tgev-specific antibodies. while antibody titers in induction of lactogenic immunity by ad-ts sera decreased with insert size, the ni increased, sugrecombinants gesting that antibodies to site a contributed significantly to the neutralization of tgev. induction of immune response in swine by ad-ts , ad-ts , and ad-ts were crossed with nonrecombinants ad-ts and ad-ts immune males and administered a third dose of the homologous ad-ts recombinant days before delivery. the ad-ts and ad-ts recombinants expressing the smallest insert and the full-length spike protein, re-the presence of tgev-specific antibodies in the sera and , respectively). to study the potential of these antisera for protection against tgev, sera induced by these recombinants were examined for the ability to prevent tgev infection. virulent tgev (pur -sw -st strain, pfu/dose) was mixed with the antibody induced by each recombinant, incubated at Њ for min, and administered to highly susceptible -day-old miniswine. virus titers were determined in jejunum and ileum, lungs, mesenteric, and mediastinal lymph nodes at , , , and days postinoculation. the results (fig. ) indicated that virus titers found in the enteric tissues were between and -fold lower when virus was premixed with antiserum induced by recombinant ad-ts (fig. d) , and very low titers (õ pfu/g of tissue) of infectious virus were detected in the small intestine of newborn pigs that were administered the antibody elicited by recombinant ad-ts (fig. f) . in contrast, titers ranging between and pfu/g of tissue were detected in the tissues of control animals to which serum induced by wt ad , used as a control, was administered (fig. b ). in addition, neither mortality nor clinical symptoms were observed in animals treated with serum induced by recombinant ad-ts (fig. e) , while control animals presented diarrhea - hr postinfection and died around day postinfection (fig. a) . ten ad -tgev recombinants have been constructed and screened for their ability to express spike protein fragments of tgev. four recombinants expressing the full-length spike protein or truncated fragments spanning different lengths of s protein from the amino-terminus have been selected, and their ability to induce virusneutralizing antibodies was determined. these ad-ts viruses induced lactogenic immunity in hamsters, and the recombinant expressing the full-length s protein elicited antisera that, when mixed with a lethal dose of virus prior to administration to susceptible piglets, prevented the induction of disease symptoms. helper-independent ad viruses with a deletion in the e gene have been constructed, and the s gene was inserted into the e gene. two types of ad recombinants interest to determine the comparative levels of expression in these two plasmids. expression levels were always higher using ad viruses with the smaller deletion spectively, were selected to study the induction of tgevneutralizing antibodies in swine. although the level of in e , independent of the insert size, suggesting that removal of the splicing acceptor site after the l gene recombinant antigen produced in st cells was high for recombinant ad-ts and low for ad-ts (figs. and might have reduced e gene expression. sequences inserted without an exogenous polyadenylation signal ), both recombinants induced high titers of tgev-specific antibodies in swine as determined by ria ( were successfully expressed, indicating that the polyadenylation signal of the e gene has probably been used. and , respectively) and by neutralization (ni of in general, recombinants with relatively small inserts low levels of s antigen and, accordingly, of all antigenic sites (a, b, c, and d), probably due to low replication ( , , and nt) expressed larger amounts of s polypeptide than those with larger ( -nt) inserts. levels. nevertheless, antigenic sites a and b were properly folded after infection with ad-ts virus since high the recombinants with smaller inserts gave ad titers in cell culture between and pfu/ml, while antibody levels against these sites were elicited in hamsters, as detected by cria (fig. ). s protein trimer forma-ad -ts virus with an insert of nt consistently gave titers lower than pfu/ml. thus, the level of expression tion easily explains the dichotomy between low expression levels and high efficiency in eliciting a high immune in these recombinants correlates well with their level of replication. the three recombinants (ad-ts , ad-ts , response. s protein trimers (the native form of the glycoprotein in the virus) probably are more stable and better and ad-ts ) with genome sizes lower than % of wt ad were stable after passages, while the recombi-represent the peplomer in the native virion. although recombinant ad-ts contains the sequences coding for nant with a genome size close to % of wt ad (ad-ts ) was unstable (results not shown). these results site d core (located in s protein from aa to ) lenstra et al., ; posthumus et are in line with previous work suggesting that the ad virion has the ability to package approximately % of al., ), it was very weakly detected by site d-specific mabs, while sites c and b, also encoded in this recombi-the wt genome length. this value is generally considered to be the maximum working capacity of the system nant, were well represented. site d may have been hidden by incorrect folding of the s protein in this area. (ghosh-choudhury et al., ; berkner, ; bett et al., ) . site a, the major inducer of tgev-neutralizing antibodies, was detected in larger amounts after infection by recom-viruses in which the inserted gene was flanked by an sv- pr always showed lower expression levels than binant ad-ts (expressing s protein without the membrane anchor domain) than by recombinant ad-ts those not flanked by this pr (fig. ) . this suggests that the sv- pr, in the context that has been used in this (which expresses the full-length s protein). this may be a consequence of the higher expression levels provided work, is inhibiting and transcription is probably driven from the nearby ad e pr. the transcription could also by ad-ts , since it has been previously shown (godet et al., ) that the full-length spike forms trimers and be driven from the major late protein pr that is located far to the left at m.u. . similar observations have been reconstitutes site a better than truncated s proteins missing the membrane anchor domain. in fact, one of the two made with other ad -based vectors containing analogous e substitutions (schneider et al., ; major inducers of tgev-neutralizing antibodies was ad-ts virus, in spite of the low amount of s protein pro-and both et al., ) . antigenic sites c, b, d, and a (starting from the amino-duced by this recombinant. seven of ten ad-ts recombinants expressing s frag-terminal end) have been defined on s protein (correa et al., ; gebauer et al., ) . sequences coding for ments induced tgev-neutralizing antibodies in hamsters. recombinant ad-ts , expressing a truncated form sites c and b were included in all recombinants and, in fact, s polypeptides with these two sites were detected of s protein spanning aa from the amino-terminus (which includes sites c and b but not site a), induced after infection with all ad-ts viruses. the recombinant coding for the full-length s protein (ad-ts ) expressed virus-neutralizing antibodies. since site c does not in- fig. . protection of swine with porcine sera elicited by ad-ts recombinants. tgev-specific swine antiserum was elicited by administration of wt ad virus, ad-ts , or ad-ts recombinants (see materials and methods). the number of swine surviving after the oral administration of pfu of the virulent strain pur -sw -st of tgev mixed with antisera induced by (a) wt ad or by the recombinants (c) ad-ts or (e) ad-ts expressing the amino-terminal nt or the full-length spike protein, respectively, is shown. the recovery of infectious virus was determined , , and or days postinfection (when the animals either died or were sacrificed) in the indicated tissue homogenates, in animals administered the virulent virus with serum from (b) ad , (d) ad-ts , or (f) ad-ts immune swine. three groups of five swine were used to follow the survival rate. the infectious virus was followed in three groups of three animals each. mean values have been represented. standard deviations were lower than % in all cases and are not shown. duce virus-neutralizing antibodies, site b, or neighboring and van der zeijst, ) or, alternatively, other factors similarly to those described in mouse hepatitis virus sys-antigenic domains involved in virus neutralization, have been reconstituted in a functional form. it has been pro-tem (fazakerley et al., ; yokomori et al., ) . recombinant adenoviruses expressing only the amino-posed that factors mapping in the s segment which has been deleted in the porcine respiratory coronavirus terminal residues of the s polypeptide (which are mostly deleted in prcv strains) provide partial protection (prcv) (from aa to ) (callebaut et al., ; sá nchez et al., ) , and more precisely alterations in amino against tgev. these data indicate that the amino-terminal s protein fragment might be relevant to confer enteric acid (or residues close to it) might be involved in the loss of enteric tropism (sá nchez et al., ) . these fac-tropism by complementing the binding of n-aminopeptidase (identified as a major tgev receptor) to an s protein tors might be the presence of a second receptor binding site recognized by a putative second receptor (enjuanes domain mapping close to antigenic site a (delmas et al., ; godet et al., ) . another observation supports with higher titers in swine than in hamsters, although both species were permissive to virus infection. s protein this hypothesis. ad vectors have been used to express the amino-terminal aa of prcv s protein, resulting has been previously expressed using e. coli (hu et al., (hu et al., , or poxviruses (pulford and britton, ) , but in production of tgev-neutralizing antibodies (callebaut et al., ) which did not protect against challenge with tgev-neutralizing antibodies were only elicited with recombinant poxviruses. expression of s antigenic site d, virulent tgev (callebaut and pensaert, ). by contrast, the ad-ts recombinant eliciting antiserum provid-as a fusion protein on the surface of e. coli led to induction of tgev-neutralizing antibodies when purified re-ing passive protection against challenge with virulent tgev carries s sequences derived from tgev instead combinant antigen was used as immunogen, but not when live vector was administered (bousquet et al., of prcv. the presence of aa (from residue to ) in recombinant ad-ts , which are deleted in the ). using salmonella typhimurium, site d has been expressed and tgev-neutralizing antibodies have been prcv, might have been critical to achieve the observed protection. this interpretation is in agreement with the elicited in serum and in mucosal areas using live recombinant bacteria (smerdou et al., ) but protection ex-partial protection seeing with the antiserum elicited in swine by recombinant ad-ts , which includes the se-periments using these systems have not been reported. ad vectors have a high probability of inducing effective quences deleted in prcv, but at the same time indicates that larger spike protein fragments (as those including mucosal immunity against tgev, since this virus showed tropism for mucosal tissues in pigs, and the animals site a) are needed to elicite full protection. protection by recombinant adenoviruses expressing s infected with this virus experienced neither respiratory nor intestinal disorders (callebaut and pensaert, ; protein fragments lacking site a extends the results recently reported (tulboly et al., ) on s protein expres- callebaut et al., ; torres et al., ) . sion using baculoviruses. these authors showed induction of tgev-neutralizing antibodies only with recombi-acknowledgments nants expressing s protein fragments spanning aa we thank granja cantoblanco de animales de laboratorio (hospital or more from the amino-terminus, that is, with s protein general g. marañó n, comunidad de madrid) and laboratorios sobrino cyanamid (olot, girona) for providing inbred and outbred swine, re-fragments including site a, but not with s protein frag- antigenic differentiation between transmissible gastroenteri-tgev expressed by recombinant baculovirus tgev coronavirus orf encodes a membrane protein that is incorporated callebaut major antigen of porcine respiratory coronavirus virus retaining spike glycopolypeptide s but not s adenovirus-based expression is unable to induce virus-neutralizing or haemagglutination-inhibiting vectors and recombinant vaccines a new technique for the antigenic structure of the e glycoprotein from transmissible gastroassay of infectivity of human adenovirus dna mcderinfection by affinity-purified spike glycoprotein of murine hepatitis mott charactermurine coronavirus spike glycoprotein and evidence that it forms istics of a human cell line transformed by dna from human adenovipart of a complex tridimensional structure the prepara-epitope specificity of protective lactogenic immunity against swine tion of i-labelled human growth hormone of high specific radioactransmissible gastroenteritis virus -herpes simplex virus igg fc receptor induced using recombinant . adenovirus vectors expressing glycoproteins e and i techniques for human adenovirus vector construction and character cell biology: a four major antigenic sites of the coronavirus transmissible gastroen-laboratory handbook high efficiency expression of the surface glycoprotein gp of porcine transmissitransformation of e. coli by high voltage electroporation. nucleic ble gastroenteritis virus studies of fazakerley tgev s protein gp expressed in e. coli and by a tge-vaccinia the v a . envelope glycoprotein deletion mutant of mouse hepativirus recombinant monoclonal antibodies to the matrix (e ) glycoprotein ( ). critical epitopes in transmissible gastroenteritis virus neutralof mouse hepatitis virus protect mice from encephalitis. virology , ization isolation of adenovirus type host range deletion mutants defective for transformation of rat embryo s missible gastroenteritis virus immunogenic peptide comprising a mouse hepatitis virus a b-cell epitope and an influenza virus t-cell epitope protects residues involved in the formation of the antigenic sites of the s against lethal infection human adenovirus cloning vectors based on infectious acid changes in the viral glycoprotein m affect induction of alpha bacterial plasmids processing and antigenicity of entire and anchor-free spike glycoprotein-s of coronavirus lecomte antigenic homology among coronaviruses retype -induced acute disease by an anti-nucleoprotein monoclonal antibody glycoprotein of vsv by infectious adenovirus vectors selection of mimotopes from a random sequence expression library by monoclonal antibodies against transmissible gastroentritis coro molecular cloning: tis coronavirus s protein fused to e. coli heat-labile toxin b subunit in attenuated salmonella for oral immunization. submitted for publi-a laboratory manual studies on transmissible gastroenteritis of swine. ii. selected characteristics of a cytopatho-in ''immunochemistry of viruses. ii. the basis for serodiagnosis and vaccines mechanisms of transmissible gastroenteritis coronavirus neutralization rus based vector using the firefly luciferase as a reporter gene protective effect of monoclonal antibodies on lethal mouse hepatitis virus infection hepatitis virus- (strain jhm): correlation with biological activities , and their use in protection against transmissible gastroenteritis virus immunogenicity of the s protein of transmissible gastroenteritis virus ex-f. l. ( ). a recombinant human adenovirus vaccine against rabies intracellular processing of the porcine coronavirus transmissible gastroenteritis virus spike protein strain of transmissible gastroenteritis virus: comparison of reactivity with virulent and attenuated virus mouse hepatitis ''diseases of swine iowa state univ. press, virus spike and nucleocapsid proteins expressed by adenovirus vector protect mice against a lethal infection a spike protein-dependent cellular factor other than the viral receptor is required for mouse hepatitis virus entry key: cord- - d aojh authors: zou, hao; zarlenga, dante s.; sestak, karol; suo, siqingaowa; ren, xiaofeng title: transmissible gastroenteritis virus: identification of m protein-binding peptide ligands with antiviral and diagnostic potential date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: d aojh the membrane (m) protein is one of the major structural proteins of coronavirus particles. in this study, the m protein of transmissible gastroenteritis virus (tgev) was used to biopan a -mer phage display random peptide library. three phages expressing tgev-m-binding peptides were identified and characterized in more depth. a phage-based immunosorbent assay (phage-elisa) capable of differentiating tgev from other coronaviruses was developed using one phage, phtgev-m , as antigen. when the phage-elisa was compared to conventional antibody-based elisa for detecting infections, phage-elisa exhibited greater sensitivity. a chemically synthesized, tgev-m peptide (peptgev-m ; haltpikyippg) was evaluated for antiviral activity. plaque-reduction assays revealed that peptgev-m was able to prevent tgev infection in vitro (p < . ) following pretreatment of the virus with the peptide. indirect immunofluorescence and real-time rt-pcr confirmed the inhibitory effects of the peptide. these results indicate that peptgev-m might be utilized for virus-specific diagnostics and treatment. transmissible gastroenteritis (tge) is a highly contagious disease of swine characterized by up to % mortality in suckling piglets (laude et al., ) . the clinical symptoms include acute diarrhea, vomiting and dehydration. pigs of all ages and categories are susceptible. in seronegative herds, tge can cause devastating economic losses (saif and wesley, ; yin et al., ) . the virus responsible for tge (tgev) has a glycoprotein surface envelope and positive-sense rna genome of approximately . kb (ortego et al., ) . the virus consists of four structural proteins: spike (s), small membrane (sm or e), membrane (m), and nucleocapsid (n) proteins (spaan et al., ; saif and wesley, ; penzes et al., ) . the s protein is a large transmembrane surface glycoprotein that induces virus-neutralizing (vn) antibodies (jiménez et al., ; laude et al., ; suñé et al., ) ; the n protein, together with genomic rna, forms the viral nucleocapsid cavanagh, ) ; and the sm protein regulates virion assembly and release (laude et al., ) . the m protein is the most abundant component of the coronavirus particle (rottier, ) . roughly one-third of the m protein assumes a topology where part of the endo domain constitutes the fourth transmembrane segment, thereby positioning the carboxy terminus on the exterior portion of the virion (risco et al., ; masters, ) . it has been suggested that the m protein induces innate immunity including interferon production (charley and laude, ; laude et al., ) . phage display is a powerful technology that has been applied to antibody engineering (hayden et al., ; cyranka-czaja and otlewski, ) , drug discovery and manufacturing (kay et al., ; harper et al., ) , ligand identification (ehrlich and bailon, ; ladner and ley, ; yi et al., ; beer and liu, ) , and development of new diagnostics gazarian et al., ) and vaccines (lesinski and westerink, ; samoylova et al., ) . phage display yields billions of heterologous fusion peptides that are expressed on the surfaces of filamentous bacteriophage (scott and smith, ) . inasmuch as each phage expresses only one fusion peptide, the technology permits high throughput panning of a phage library with the intent of identifying single phage particles with the capacity to bind a target protein. this permits epitope mapping and easy purification at marginal costs. in the current study, three tgev m-binding phage and the concomitant peptides were identified from a phage display library. results indicate the ability of these peptides to function as tgev-specific diagnostic reagents. one peptide was further studied for its potential as an inhibitor of tgev infectivity. swine testis (st) cells were grown in dulbecco's mem with % fetal bovine serum at °c, % co . tgev (pur -mad strain) (sánchez et al., ) was propagated in st cells in the absence of serum, followed by gradient ultracentrifugation purification (krempl and herrler, ) . cytopathic effect (cpe) assays were performed as described (ren et al., b) . briefly, st cells seeded onto -well tissue culture plates (costar, usa) were inoculated with -fold serially-diluted tgev. after incubation at °c for h, cpe was recorded at  magnification using the bds microscope (optec, china). total rna was isolated from tgev-infected st cells using a commercial protocol ( , fastgene, china). sense (p : -ggggggatccatgcgctattgtgctatg) and antisense (p : -ccccgaattcttataccatatgtaataa) primers were used in rt-pcr. the amplified m gene was inserted into the bamhi-ecori site of a prokaryotic expression vector, pgex (novagen, germany), resulting in the recombinant plasmid, pgex-tgev-m. after pgex-tgev-m was transformed into rosette escherichia coli bacteria by heat shock (bowyer, ) , expression of the glutathione s-transferase (gst) tagged-tgev-m fusion protein was induced with isopropyl-b-d-thiogalactoside (iptg) and visualized by % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page). the recombinant protein of interest (rtgev-m) was gel purified (ren et al., a) . in parallel, rgst from pgex, was expressed and purified as well. the concentration of rtgev-m was measured spectrophotometrically using a nanodrop spectrophotometer according to manufacturer's instructions. biopanning of the phage was performed using the ph.d™- phage display peptide library kit according to manufacturer's instructions (e sc, new england biolabs, usa) with minor modifications ren et al., a) . during the first round of binding the phage library to the rtgev-m, lg/well of rtgev-m was used. in the second round, rtgev-m was replaced by rgst as a negative control to remove non-specific binding to gst. the rd- th rounds were performed under the similar conditions except for gradually decreasing the concentration of rtgev-m ( , . , , . lg/well) and increasing the concentration of tween from . % to . % to increase the stringency of binding. the phage remaining after the last round of biopanning were titered and were selected for phage amplification and further study. phages interacting positively with rtgev-m were identified by indirect elisa. the -well plates (fep , jet bio, china) were coated either with rtgev-m in . m nahco (ph . ) or with purified tgev in dmem at lg/well at °c. plates were blocked with % bsa in tbs buffer ( mm tris-hcl, ph . , mm nacl) for h at °c then washed x with tbs-tween. the selected phages derived from the last round of biopanning were added separately to the wells at concentrations of .  plaque forming units (pfu)/ml in . m nahco (ph . ) and incubated at °c for h. the remaining steps were performed as described (ren et al., a) . plates were read at od with an elx (bio-tek, usa) elisa reader. . . virus detection using elisa ten tgev-positive phage clones were amplified using an e. coli expression system ( ). the dnas of interest were extracted, purified ( , qiagen, germany), pcr amplified and sequenced to corroborate the presence of tgev-m sequences ( ). deduced amino acid sequences were determined (wu et al., ) . phage elisa and antibody-elisa were compared for their abilities to detect the presence of the virus as described (wu et al., ) . for phage-eli-sa, tgev serially-diluted in dmem was coated onto elisa plates overnight at °c. then either the specific phage clone or a nonspecific phage complex (negative control) from the phage display library was diluted in pbs ( .  pfu/ ll) and added to the wells. next, the wells were incubated with commercially-available rabbit anti-m antibody ( : in pbs) followed by horseradish peroxidase-conjugated goat anti-rabbit antibody (garp) ( : in pbs). for antibody-mediated elisa, rabbits were immunized once with mg rtgev-m in freund's complete adjuvant followed by three additional immunizations at week intervals with the same amount of antigen in freund's incomplete adjuvant. animals were bled days after the final boost. tgev was coated onto elisa plates to which was added rabbit anti-tgev-m followed by garp secondary antibody ( : ). in all experiments, the concentrations of tgev were determined experimentally such that elisa values could be measured and where [od phage (p)/od negative control (n)]> was considered positive. the specificity of tgev-reactive phages for tgev infection was evaluated by phage-elisa. a panel of selected viruses prepared in our laboratory consisting of tgev (ren et al., ) , porcine epidemic diarrhea virus (pedv) strain hljby , porcine reproductive and respiratory syndrome virus (prrsv) strain jilintn (gao et al., ) , porcine rotavirus (prv) (ren et al., c) , porcine pseudorabies virus (prv) strain kaplan (sui et al., ) , porcine parvovirus (ppv) isolate ppv , and porcine circovirus type ii (pcv ) strain pcv -ljr (zhu and ren, ) were assembled for comparative studies. these viruses were diluted in dmem to final concentrations of lg/well and coated onto elisa plates at °c for h. elisa was performed according to standard protocols (wu et al., ) . the unpanned ph.d™- phage display peptide library (phage l) was used as a negative control. statistical significance of the od values among all viral antigens was evaluated where [od virus /od phage l]> was judged as positive and subsequently analyzed using the t-test. the amino acid sequence corresponding to the phage with the highest binding activity to tgev (peptgev-m ) was commercially synthesized (boshi, china). the peptide was diluted in sterile water to a final concentration of mg/ml. purified rtgev-m or control protein rtgev s-ad (meng et al., ) was coated onto elisa plates ( lg/well) at °c. the ll of serially-diluted peptgev-m ( , , or lg/ml) were mixed with ll of phtgev-m or ll of phtgev-s-ad ( .  pfu/ml) and incubated at °c for h. after washing, the wells were incubated with rabbit anti-m antibody ( : dilution; abcam, china) for h followed by a : dilution of garp for min; the od values were recorded. three experimental approaches were used to evaluate the antiviral activity of peptgev-m (miyazaki et al., ; ren et al., b) . first, to assess the ability of the peptide to bind tgev in vitro, tcid of tgev was pre-incubated with peptgev-m at , , , . or . lg/ml before infecting st cells. second, to determine any impact of peptgev-m on st cells, cells were treated with serially-diluted peptide at °c for h prior to tgev infection. finally, to determine the effect of peptgev-m on st cells after tgev infection, cells were first infected with tgev ( tcid ) at °c for h then washed and incubated with serially-diluted peptgev-m at °c for h. all the cells were grown to % confluency in -well plates overlaid with % methylcellulose then cultured for - h to maximize plaque development. to evaluate the effects of peptgev-m on tgev replication in vitro, indirect immunofluorescence assay (ifa) was performed. briefly, confluent st cells were incubated with peptide-treated tgev [ ll of serially diluted ( À - À ) peptide was incubated with ll of tgev ( tcid )] at °c for h then processed for ifa as previously described (meng et al., ) ; an unrelated peptide that binds the pedv-s protein, peppedv-s (diluted À ) was used as a negative control. the impact of peptgev-m on virus replication was evaluated by real time rt-pcr targeting a portion of the tgev-s gene. the tgev was treated with serially-diluted peptide ranging from to . lg/ml at °c for h; lg/ml of peppedv-s was used as negative control. after washing the st cells with pbs, confluent monolayers were infected with peptide treated tgev ( tcid ) at °c for h. then the virus-containing culture was frozen and thawed three times followed by the addition of an equal volume of % pge- at room temperature for min. the samples were centrifuged at , rpm for min and the pellets were suspended in rnase-free water. total rna was extracted according to the manufacturer's instructions (fastgene, china) and real-time rt-pcr was performed on cdna as described (ren et al., b) . it was anticipated that a detrimental effect of peptgev-m on viral infectivity would be reflected in significantly reduced numbers of tgev s gene copies. using the previously described techniques ren et al., a) , the rtgev-m protein was expressed in e. coli (fig. ) for use in biopanning the phage display library to identify peptides that bind the m protein of tgev. during the isolation process it was determined that rtgev-m was present within inclusion bodies. peak expression was observed at h post-iptg induction. the mass of the rtgev-m was $ kda (fig. ) . five rounds of biopanning were performed using the rtgev-m as the target and one additional round with gst as target. the amount of eluted phages increased between rounds and from .  to .  , respectively. ten different tgev-m protein-reactive phages were identified by elisa, when rtgev-m was used as the coating antigen (fig. a) . all ten phages exhibited better binding to rtgev-m than to other phage-expressed protein controls (p < . , fig. a ). phages m , m and m -m had higher binding affinity (p < . ) with tgev than with the other phages (fig. b) . among all ten reactive phages that bound rtgev-m with high affinity, phtgev-m exhibited the highest binding to tgev (p < . , fig. b ) followed by phtgev-m .and phtgev-m . rna samples isolated from tgev-m-reactive and control phages were amplified by rt-pcr. all samples yielded products approximately bp in length (data not shown). all deduced peptides ( aa in length) were unique (table ) . phages bearing the peptides ltfpvtttppav, mthnmhgpnsep and haltpikyippg were selected for their high-affinity binding with rtgev-m (fig. a) and tgev (fig. b) . these three peptides were named peptgev-m , peptgev-m and peptgev-m , respectively. the binding efficiencies of the selected tgev-m-reactive phages were examined by phage-elisa. virus concentrations between . - lg/well all exceeded the signal limitations of the assay. as illustrated in fig. a , titratable virus concentrations fell in the range < . ug/well. the lowest concentrations of virus detectable with phtgev-m , phtgev-m and phtgev-m where p/n p were . , . , and . lg/well, respectively. for antibody-based elisa, the rabbit antibody titers against rtgev-m where p/n p were determined to be : .  . when assaying the antibody binding to tgev, at virus concentrations serially-diluted tgev in dmem was coated into elisa plates followed by incubation with serially-diluted rabbit against tgev serum and antibody binding using garp; normal rabbit serum was used as negative control. od ratios where p/n > were considered positive. the experiment and determination of od values were derived from three independent assays. the concentration of the virus is indicated on the x axis. antibody and virus dilutions are as indicated. > . lg/ml, p/n values began to exceed the limits of detection. virus ( . - . lg/well) were reproducibly detected (p/n p ) with antibody dilutions less than / (fig. b) . the binding specificities of phtgev-m , phtgev-m , phtgev-m for non-tge viruses were examined (fig. ) . in all cases p/ n > for tgev only. demonstrable binding was not observed with any other viruses tested. further, our results showed that pept-gev-m bound to rtgev-m and did not bind other proteins (fig. ) . increasing the concentration of peptgev-m effectively competed with binding of phtgev-m for rtgev-m as evidenced by decreasing od values; however, peptgev-m did not compete with phage binding when a non-specific protein, rtgev-s-ad was used as the coating antigen (fig. ) . to determine the impact of peptgev-m on tgev infection, three separate approaches were used: ( ) tgev was treated with peptide before inoculation of st cells; ( ) st cells were treated with peptide prior to tgev inoculation, and; ( ) st cells were infected with tgev before being treated with peptide. as expected, the results showed no impact of peptgev-m on st cells already infected with tgev or on st cells pretreated with peptgev-m prior to incubation with the virus (data not shown). however, when tgev was pre-incubated with peptgev-m , its infectivity decreased in a dose-dependent manner: at lg/ml of peptgev-m , the drop in infectivity was nearly % whereas at lg/ml a % drop in virus infectivity was observed (table ) suggesting peak inhibition was reached. these data indicate that peptgev-m is able to interfere with the ability of the virus to infect st cells. the antiviral effects of peptgev-m -treated virus were further investigated by ifa. results demonstrated a dosedependent reduction in the infection of st cells in the presence of peptgev-m whereas the unrelated peptide, peppedv-s , had no effect on virus uptake (fig. ) . real-time rt-pcr was used to confirm and quantify the tgev inhibitory effects of peptgev-m (fig. ) . results showed that the amount of viral rna was lower (p < . ) when tgev was pretreated with peptgev-m than in peptide-untreated controls. at the highest concentrations ( lg/ml), virus rna levels dropped . %; however, significant (p < . ) and reproducible reductions ( %) were observed when . ug/ml of peptgev-m were used. these data corroborated the ifa studies. despite the availability of commercial vaccines, tge still poses a regional threat to the swine industry due to the high morbidity and mortality that tgev causes in suckling piglets under weeks of age (miyazaki et al., ) . although passive, lactogenic immunity induced by virulent or attenuated tgev vaccines can provide effective protection, sufficient risk remains. albeit safer, inactivated phtgev-m to rtgev m protein was monitored using anti-m antibody and garp secondary antibody which target phtgev-m only. in parallel, rtgev s-ad was screened as a negative control. concentrations of peptgev-m are shown on the y axis. statistical significance is indicated by '' ⁄ '' (p < . ) or, '' # '' ( . < p < . ) relative to controls. all experiments were performed in triplicate. vaccines provide less protection. as such, accurate diagnostic tests are needed to better effect tge prevention and therapy. phage display and biopanning are powerful methods for identifying key ligands within large target antigens that may be involved in protection and/or diagnosis (wu et al., ) . this technology has been used for identifying receptor binding domains (rbds) in tgev that may interact with its papn cellular receptor (ren et al., a) . it has also been used for identifying immunogenic proteins in pig sera from convalescent animals with a history of salmonella typhimurium infection (meyer et al., ) , and for targeting tissues like bone-marrow dendritic cells and kidney, liver, lung, spleen and visceral adipose tissues (jung et al., ) . since the m protein is one of the three major tgev structural proteins, it was used as a target in biopanning a -mer phage display peptide library to identify peptides that ultimately inhibit virus binding. to this end, the tgev-m protein was first expressed in e. coli as a gst fusion protein. given the possibility of identifying gst-binding phage, we included additional panning steps using purified recombinant gst as the target to remove phage that did not specifically target the rtgev-m protein. also, the stringency was systematically increased by panning with decreasing concentrations of rtgev-m and increasing concentrations of tween- . three of tgev-m phages identified in this study were evaluated by elisa for detecting tgev infection. results showed that the widely used tgev-m antibody-elisa and phage-elisa both could detect the virus successfully; however, phage-elsia is more costeffective because it does not involve animals and phages are in unlimited supply. further, even using serum from animals boosted peptide concentration(ug/ml) relative amplication peptgev-m peppedv-s fig. . inhibition of virus infectivity monitored by rt-pcr. real time rt-pcr was used to quantify tgev rna in st cells and therefore virus infectivity. pcr was performed on the tgev s-ad gene. tcid tgev virus was pre-incubated with -fold, serially-diluted ( - . lg/ml) peptide (peptgev-m ) then added to st cells for h. an un-related peptide peppedv-s at the maximal concentration was used as control. virus-derived cdna was amplified and dct values were measured in triplicate. the relative amplification of tgev s-ad in tgev-infected cells was normalized to beta-actin and calculated using À ct method. statistical significance is indicated by '' ⁄ '' (p < . ) relative to lg/ml peptide. x with rtgev-m, the phage-elisa appeared more sensitive. clearly, targeting the m protein rather than the whole virus could sacrifice sensitivity. although we demonstrated specificity in phage and peptide binding to rtgev-m, we have not determined how well-exposed the binding region is to the surface of the virus. as such, using the m protein as the biopanning agent is probably not as effective as using whole virus. also, this may have contributed to the lower than expected signal strength in the polyclonal ab-elisa especially when the ab titer using rtgev-m protein as antigen was so high. the use of antivirals represents one approach to treat coronavirus infections (ren et al., b) . in this study, a peptide encoded by phtgev-m that interacted with the rtgev-m protein was synthesized. the interaction was deemed specific in that it was titratable and similar results were not observed with interactions between peptgev-m and another tgev structural protein, rtgev s-ad (meng et al., ) . plaque-reduction assays showed that tgev infectivity decreased only when virus was pre-treated with the peptide. in contrast, when the peptgev-m was incubated with st cells alone or with tgev-infected st cells, no inhibitory effect was observed. this is an expected outcome given that rtgev-m was used to pan for bioreactive phage and this protein is not present on the surface of or within st cells. collectively the data presented here are consistent with peptgev-m binding to the surface of the virus and either interfering with the ability of the virus to invade the cell or generate progeny virus. neither incubating the peptide with st cells prior to or after adding virus had a demonstrable effect on replication. the virus-specific effects of peptgev-m were confirmed by ifa and rt-pcr. in summary, peptide sequences that recognize the tgev-m protein were identified in our study using phage display technology. phages bearing these peptides may be utilized in serology-based diagnosis of tge, peptgev-m had direct inhibitory effects on tgev infectivity in vitro. in future research, peptides identified in this study may be subjected to antiviral testing. panning of a phage display library against a synthetic capsule for peptide ligands that bind to the native capsule of bacillus anthracis dna-mediated transformation of fungi the coronaviridae study group of the international committee on taxonomy of viruses, revision of the taxonomy of the coronavirus, torovirus, and arterivirus genera induction of interferon alpha by transmissible gastroenteritis coronavirus: role of 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the entry process of transmissible gastroenteritis virus isolation, genome phylogenetic analysis and in vitro rescue of a newly emerging porcine circovirus type . pak the research work of the authors was supported by the program for new century excellent talents at the heilongjiang provincial university ( -ncet- ). key: cord- -gyb mjb authors: chai, weidong; burwinkel, michael; wang, zhenya; palissa, christiane; esch, bettina; twardziok, sven; rieger, juliane; wrede, paul; schmidt, michael f. g. title: antiviral effects of a probiotic enterococcus faecium strain against transmissible gastroenteritis coronavirus date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: gyb mjb the enteropathogenic coronavirus transmissible gastroenteritis virus (tgev) causes severe disease in young piglets. we have studied the protective effects of the probiotic enterococcus faecium ncimb (e. faecium), which is approved as a feed additive in the european union, against tgev infection. e. faecium was added to swine testicle (st) cells before, concomitantly with, or after tgev infection. viability assays revealed that e. faecium led to a dose-dependent rescue of viability of tgev-infected cells reaching nearly to complete protection. virus yields of the e. faecium–treated cultures were reduced by up to three log( ) units. western blot analysis of purified tgev revealed that the levels of all viral structural proteins were reduced after e. faecium treatment. using transmission electron microscopy, we observed attachment of tgev particles to the surface of e. faecium which might be a means to trap virus and to prevent infection. increased production of nitric oxide in the cells treated with e. faecium and elevated expression of interleukin and pointed to stimulated cellular defense as a mechanism to fight tgev infection. transmissible gastroenteritis virus (tgev) infects enteric and respiratory tissues and causes severe gastroenteritis with a mortality rate close to % in newborn piglets [ , ] . the appearance of the closely related tgev variant porcine respiratory coronavirus (prcov) has been found beneficial in preventing tgev infections, possibly through induction of neutralizing antibodies that can provide crossprotection against tgev infection [ , ] . however, tge prevalence is still being reported, and some tgev strains have been isolated from domestic pigs in different parts of the world [ ] . commercially available vaccines, either inactivated or attenuated, have failed to provide full protection to piglets [ ] . it is likely that the parentally applied inactivated viruses do not induce the local immune response in the small intestine that is required for protection. therefore, the discovery and development of new, highly potent anti-tgev agents and effective approaches for controlling the emergence of tgev infection remains an important mission. probiotics are defined as live microbial food supplements with health-promoting attributes. potent mechanisms of beneficial action include the production of antimicrobial agents, modulation of immune responses and promotion of host innate defense mechanisms [ , , , , ] . enterococcus faecium ncimb (e. faecium) is authorized in the eu for use as a probiotic feed additive for sows and piglets and several other farm animal species. beneficial effects of the probiotic e. faecium such as immune modulation and improvement of nutrient transport have previously been reported in several studies [ , , , , ] . in vitro studies have also demonstrated that e. faecium could reduce the rate of invasion of pathogens-for instance, salmonella in intestinal cell lines [ , ] . but detailed knowledge on the impact of e. faecium on viral infections in vivo and in vitro is lacking. the purpose of the present study was to establish an in vitro model to investigate the antiviral potential of e. faecium. we used an established swine testicle (st) cell line to assess the protective effects of e. faecium on tgev infection in terms of viral replication and cell survival. to gain insight into its possible mechanisms of action, the effects of e. faecium on viral protein synthesis as well as the induction of inducible nitric oxide synthase (inos) and selected cytokines were investigated. our results described here suggest that this probiotic e. faecium strain exhibits antiviral activity against tgev and may possibly serve as a useful antiviral agent against coronavirus infections in vivo. the epithelial swine testicle (st) cell line was maintained in dulbecco's modified eagle's medium (dmem, pan biotech) supplemented with % fetal bovine serum (hyclone), and % penicillin/streptomycin (biochrom), growing at °c in a % co humidified incubator. the tgev strain purdue -mad (kindly provided by dr. c. schwegmann-wessels, institut für virologie, tierärztliche hochschule hannover) was used in this study. stock virus was propagated in st cells to a titer of . e? pfu/ml. all infections were done at a multiplicity of infection of . . enterococcus faecium ncimb isolated from a commercial product used in animal nutrition (cylactin Ò , cerbios-pharma sa, lugano) was used in this study and cultivated in todd-hewitt-bouillon (thb, roth). the number of viable bacteria in ml of bacterial culture was determined by plating bacteria on agar. bacterial cultures were then centrifuged at rpm for min, and bacteria were washed twice to remove excess thb. finally, the viable e. faecium particles were resuspended in dmem to a stock concentration of . e? cfu/ml. heat inactivation of bacteria was performed by heat treatment with e. faecium ( . e? , . e? , . e? cfu/ml) in dmem in a water bath at °c for min. bacterial culture supernatants were obtained from growing bacterial cultures in thb. bacteria were removed by centrifugation at rpm for min, and supernatants were collected. assessment of cellular toxicity of e. faecium suspensions of ll containing different amounts of e. faecium ranging from . e? to . e? were added to st cell monolayers in a -well plate (greiner bio-one) for . h before washing away. at the end of the incubation period, a methylthiazolyl-diphenyl-tetrazolium bromide (mtt) viability assay was carried out as described previously [ ] . the cell survival rate was determined as bacteria average od value/control average od value. the % cytotoxic concentration (cc ) was defined as the concentration that inhibited cell proliferation by %, and a non-cytotoxic concentration of e. faecium was used for antiviral assays. four different experimental protocols were applied to investigate the antiviral activity of e. faecium. three setups focused on the effect of e. faecium on the cells by varying the treatment period in relation to infection with tgev. a fourth setup assessed the direct effect of the probiotic on virus particles. in brief, monolayers of st cells were treated with e. faecium for . h, which was washed away before infection with tgev for h (pretreatment assay), e. faecium and tgev were added to the cell layer together during the -h infection period (competition assay), or e. faecium was added for . h right after the infection period (post-infection treatment assay). after probiotic treatment as well as after infection with tgev, cells were washed twice and kept in medium containing % penicillin/streptomycin to kill any viable bacteria that were left. to assess direct effects of e. faecium on tgev without cells being involved, the virus was mixed with different concentrations of e. faecium and incubated for . h at °c. after centrifugation for min at rpm to sediment bacterial cells, the virus containing supernatants were used to infect st cells (cell-free preincubation assay). the antiviral effects of heat-inactivated e. faecium as well as serially diluted e. faecium supernatants were also tested in the competition assay. virus-infected st cells and cells without addition of e. faecium served as controls from which samples were collected at and h after infection (hpi) for the % tissue culture infective dose (tcid ) and the mtt viability assay, respectively. relative survival of cells was calculated as follows [ ] : percent viable cells = [(od value of e. faecium group -od value of infection control)/(od value of blank control -od value of infection control)] . transmission electron microscopy (tem) in order to examine possible direct binding of virus by e. faecium, the cell-free preincubation assay was performed by mixing e. faecium with tgev at a bacteria-tovirus ratio of for . h. after centrifugation for min at rpm to sediment bacterial cells, the pellet was resuspended in ml karnovsky's fixative. the samples were centrifuged for min at rpm and a drop ( ll) was taken from the bottom of the tube and negatively stained with % phosphotungstic acid for min. finally, the samples were evaluated with a transmission electron microscope (zeiss cr). virus yield reduction assay st cell monolayers were infected with tgev with or without probiotic bacteria treatment according to the experimental design. at hpi, aliquots of the supernatants were taken, and serial tenfold dilution steps were performed. infectivity was determined by endpoint dilution titration on st cells in a -well plate. the plate was incubated for hpi, and infectivity was determined by recording the virus-induced cytopathic effect (cpe). virus titer was calculated by the method of reed and muench, which is usually used for the calculation of ld [ ] and documented as tcid values. st cells were infected in culture dishes with -cm growth areas under competition assay conditions with e. faecium. at hpi, virus from cell culture fluids and cells were collected by ultracentrifugation in a l - ultracentrifuge (beckman coulter) at , rpm for . h using a sw rotor. the pellet containing virus particles, with equal amounts of total protein, underwent sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the separated proteins were electro-transferred to hybond lfp (pvdf) membranes (ge healthcare) using a feline anti-tge polyclonal antiserum (natutec) at a dilution of : and an anti-feline igg polyclonal antiserum (rockland) at a dilution of : , . antigenantibody complexes were detected using a western blotting substrate (pierce Ò ecl plus). immunodetectable protein bands on the membrane were visualized using the fusion sl imaging system (vilber lourmat), and protein amounts were estimated by densitometric analysis using the fusion-capt software (vilber lourmat). three independent experiments and appropriate gel exposures yielded very similar results for each treatment modality. detection of nitric oxide (no) release at hpi, in different assays, no release was determined by measuring the amount of released no using the griess-assay (promega) according to the manufacturer's protocol. lps ( . mg/ml)-stimulated cells were used as a positive control. samples from untreated cells with or without prior infection served to define basal values. total rna from st cells was isolated using a gene matrix rna purification kit (eurx) as described by the manufacturer. reverse transcription (rt) was performed using a revertaidtm first strand cdna synthesis kit (fermentas) according to the manufacturer's instructions. pcr reactions were performed in a total volume of ll in an icycler iq detection system (bio-rad laboratories). data analysis was based on the measurement of the cycle threshold (c t ). the differences in the ct values of untreated samples versus treated samples were calculated by using the delta-delta-ct method [ , ] . each sample was measured in triplicate from three independent experiments. the names of genes, the genbank accession number, the primer sequences, the annealing temperatures, and the sizes of the amplification products are listed in table . all calculations were performed with ibm spss . statistical analysis of virus titers and no detection was performed by two and one factorial anova, respectively, followed by scheffé's post hoc test. cytokine expression data analysis was performed by paired t-test. p-values less than . were considered statistically significant. all data are given as the mean ± sd. before the probiotic e. faecium can be used for interference studies, the concentration range in which its addition to cells is non-toxic was defined. the results from cell viability assays (fig. ) show that e. faecium was non-toxic at concentrations up to . e? cfu/ml. the viability rate of st cells was %, and no morphological differences were observed between bacteria-treated and mock-treated cells at this concentration. therefore, the highest concentration of e. faecium chosen for the interference study with tgev was . e? cfu/ml. the cc of e. faecium in st cells was calculated to be . e? cfu/ml. (fig. a) show that e. faecium provided protection from tgev infection in a dose-dependent manner. up to % protection was achieved at the highest concentration of e. faecium ( . e? cfu/ml) when the probiotic was added to the cells together with the virus during the infection period (competition assay). to find out whether e. faecium inactivates tgev particles by direct physical interaction with virus, a cell-free preincubation assay was performed. the results show that the infectivity of tgev was also reduced in a concentration-dependent manner. these results from the mtt assay ( fig. a) were confirmed by flow cytometry using propidium iodide staining in an independent experiment (data not shown). furthermore, as illustrated by electron microscopy (fig. ) , virus particles seemed to be bound by e. faecium and attached to the e. faecium surface. because the competition assay exhibited the most pronounced antiviral activity in terms of cell survival (fig. a) , the antiviral effect of heat-killed e. faecium and e. faecium supernatant in the competition assay was also tested. the results (figs. b, c) show that heat-killed e. faecium and e. faecium supernatant still had antiviral activity, but it was much less pronounced, suggesting that live e. faecium is necessary to exhibit the observed virusreducing effects. the anti-tgev activities of e. faecium were confirmed by measuring released infectious virus in the culture medium using a tcid assay. as expected, the results (fig. ) are consistent with those from the cell viability assays, since tgev yields were found to be reduced by treatment with e. faecium. again, the inhibition of virus production was most effective in the competition assay, when cells had been exposed to the highest concentration of e. faecium ( . e? cfu/ml), amounting to a three-log reduction. reduced virus titers were also found in the cell-free preincubation assay, which indicates that the probiotic e. faecium also has antiviral capacity at the level of direct physical interaction with virus particles. tgev produced on a large scale under e. faecium interference conditions (competition assay) was enriched by ultracentrifugation and subjected to sds-page followed by western blot analysis. protein assays revealed strongly reduced amounts of total viral protein when virus from probiotic treated cells was analyzed (fig. ) dilution of e.faecium metabolic products after h, an mtt assay was carried out. results are plotted as percent viability, with uninfected cells without e. faecium taken as %. results are given as mean ± standard deviation from at least three independent experiments the probiotic during the infection period, tgev protein levels were reduced by more than %. more importantly, after western blotting with antibodies raised against total tgev protein, densitometric inspection failed to show any major aberrations of the relative polypeptide compositions of the virus particles, indicating that the levels of all viral proteins were evenly reduced. in a first approach to elucidating the mechanism of the effect of probiotic treatment on tgev production, the synthesis of antiviral no was measured. as shown in table the levels of the positive control lps, which is a strong inducer of no release. cytokines are important components of cellular defense mechanisms against microbial infection. treatment of st cells with the probiotic could modulate the cellular expression patterns of cytokines and thereby reduce the efficiency of tgev multiplication. as a first step to test this hypothesis, we studied the production of selected cytokines under the influence of e. faecium in tgevand mock-infected st cells at h, h, h, h and hpi (competition assay). a clear increased expression of cytokines was observed, reaching the highest levels of expression at hpi. the results show that administration of . e? cfu/ml e. faecium together with the virus significantly increases mrna expression levels of the pro-inflammatory cytokines interleukin (il- ) and il- (an approximately -and -fold increase, respectively) when compared with tgev-infected st cells that had not been exposed to the probiotic (fig. ) . tumor necrosis factor-alpha (tnf-a), interferon a (ifn-a) and toll-like receptor- (tlr- ) mrna expression showed a less pronounced increase when compared with tgevinfected cells that had not been exposed to the probiotic. administration of . e? cfu/ml e. faecium alone increased similar mrna expression levels of those cytokines. il- b, il- and il- levels were apparently below the detection limit of the pcr assay applied in this study. to assess the potential prophylactic or therapeutic effect of the probiotic bacteria e. faecium on tgev infection, increasing concentrations of probiotic e. faecium bacteria were added to st cells before, concomitantly with, or after tgev infection for a short period of time, and cell viability as well as virus titers in the culture medium were quantitatively assessed later after long-term incubation. a low moi of . was chosen in order to allow multiple infection cycles, as this more closely reflects natural infection. by pre-treatment of st cells with e. faecium (pretreatment), the viability of tgev-infected cells was protected, and virus yields were reduced (figs. and ). it appears that e. faecium can interfere with virus attachment and/or entry into cells. several studies have demonstrated that probiotics can block viral attachment by competitive inhibition if they are able to bind viral receptors at the surface of cells. freitas and coworkers [ , ] reported that the lactobacillus casei strain dn and a strain of bacteroides thetaiotaomicron produce a soluble compound that partially protects epithelial cells from rotavirus infection in vitro by modulating the apical glycosylation pattern of the cells. the post-infection treatment assay suggested that the antiviral activity of e. faecium also contributed to the stimulation of pro-inflammatory factors (i.e., increased mrna expression levels of il- and il- ). pagnini and coworkers have shown that the multiple probiotic formulation vsl# could stimulate the epithelial production of tnf-a and activate nf-jb in vitro [ ] . probiotic bacteria may also indirectly interfere with virus by altering the state of cells, stimulating innate and/or adaptive immunity [ , ] . in this study, the expression of antiviral cytokines il- and il- may alter the state of cells, eventually leading to an antiviral response. in our cell-free pre-incubation assay, improved survival and a significant drop in virus titer were also observed ( figs. and ) . in theory, the virus could also fail to infect the host cells if it is trapped by adsorption to the bacterial surface, and from the tem result (fig. ) , we did observe that virus particles were trapped by e. faecium. there may be some molecular mimicry between a bacterial surface molecule (such as glycoprotein with sialic acid) and a eukaryotic cellular receptor used by a virus for attachment [ ] . in this study, the competition assay in which virus and probiotic bacteria are present in the culture medium side by side, exhibited the most pronounced antiviral activity in terms of cell survival (figs. and ) . this most pronounced antiviral activity most likely resulted from the sum of overlapping mechanisms at different time points before and after virus infection, as shown before, including the finding that the levels of all of the viral structural proteins were equivalently reduced after e. faecium treatment (fig. ) indicates that indeed fewer tgev particles were released from these infected cells. likewise, reduced synthesis of tgev proteins may decrease the amount of virus-induced damage and subsequently also ameliorate the cytopathic effect in virus-infected st cells, which logically must lead to a rescue of cell viability. as the competition assay was the most effective antiviral approach, we looked for possible direct mechanisms for the effect of probiotic treatment by looking for no release from infected cells. we found that e. faecium could significantly induce no release ( table ). the antiviral effects of no have been well studied for several viral infections [ , , [ ] [ ] [ ] . although no production is believed to be released mainly in macrophages, we did detect an increase of no release in st cells upon treatment with e. faecium and tgev, and this release of no was dose dependent. this indicates that an induction of inos could indeed play a role in the mechanisms for our observation that e. faecium inhibits tgev infection in st cells. for the competition assay, we also compared expression of the antiviral cytokines il- and il- . tgev infection of st cells without e. faecium did not significantly increased expression when compared to mock control. however, e. faecium treatment significantly increased the production of pro-inflammatory factors il- and il- in tgev-infected st cells. this result is consistent with those of other authors [ , , , , ] , who showed il- or il- production following the interaction of probiotics with the intestinal epithelium. because il- and il- responses in intestinal epithelial cells play important roles in the pathogenesis and immune defense against enteric pathogens [ ] , the increased level of those cytokines could also possibly indicate an enhanced innate response. according to scientific opinion, probiotic concentrations between and cfu/g of intestinal contents are required to elicit potential benefits to the host [ , ] . in feeding trials with piglets, concentrations of - cfu e. faecium/g digesta could be detected in the intestine [ ] . thus, although not directly comparable, the effective concentrations of e. faecium used in the present study are in a similar range, which adds to the relevance of these data. in conclusion, the results of the present study show that e. faecium inhibits tgev replication in st cells and that possibly overlapping mechanisms lead to the observed reduction of virus growth: direct interference with virus attachment, adsorptive trapping or inactivation of virus particles through surface components of the probiotic bacteria, and the stimulation of pro-inflammatory cytokines il- and il- as well as no production. the data suggest that e. faecium may serve as a useful antiviral agent against infection with tgev and possibly other viruses. challenge experiments with different porcine viruses in piglets are under way to substantiate this hypothesis. nitric oxide inhibits the replication cycle of severe acute respiratory syndrome coronavirus field evaluation of the efficacy of a probiotic containing bacillus licheniformis and bacillus subtilis spores, on the health status and performance of sows and their litters nidovirales: a new order comprising coronaviridae and arteriviridae development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic lactobacillus and 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impact of the probiotic bacteria enterococcus faecium ncimb (sf ) and bacillus cereus var. toyoi ncimb on the development of serum igg and faecal iga of sows and their piglets transmissible gastroenteritis virus infection: a vanishing specter an interdisciplinary study on the mode of action of probiotics in pigs transgenic mice secreting coronavirus neutralizing antibodies into the milk influence of a probiotic strain of enterococcus faecium on salmonella enterica serovar typhimurium dt infection in a porcine animal infection model immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants increased litter survival rates, reduced clinical illness and better lactogenic immunity against tgev in gilts that were primed as neonates with porcine respiratory coronavirus (prcv) alive and dead lactobacillus rhamnosus gg decrease tumor necrosis factor-alphainduced interleukin- production in caco- cells acknowledgements the study was funded by the deutsche forschungsgemeinschaft (dfg) through grant sfb / to sub-project a (mfgs). key: cord- - v ktj authors: wu, aimin; yu, bing; zhang, keying; xu, zhiwen; wu, de; he, jun; luo, junqiu; luo, yuheng; yu, jie; zheng, ping; che, lianqiang; mao, xiangbing; huang, zhiqing; wang, lan; zhao, jun; chen, daiwen title: transmissible gastroenteritis virus targets paneth cells to inhibit the self-renewal and differentiation of lgr intestinal stem cells via notch signaling date: - - journal: cell death dis doi: . /s - - - sha: doc_id: cord_uid: v ktj infection with transmissible gastroenteritis virus (tgev) has been associated with villous atrophy within h, which seriously disrupts intestinal homeostasis. however, the underlying mechanisms remain elusive. in this study, we found that tgev infection severely disrupted intestinal homeostasis via inhibition of self-renewal and differentiation in lgr intestinal stem cells (iscs). profoundly, tgev-encoded nsp /nsp protein complex-mediated the inactivation of notch signaling provided a mechanistic explanation for this phenomenon. initial invasions by tgev-targeted paneth cells through aminopeptidase n (apn) receptor, then inducing mitochondrial damage and ros generation in them, ultimately causing paneth cell decrease and loss of notch factors (dii and hes ), which are essential for lgr iscs self-renewal and differentiation. interestingly, loss of notch signaling induced goblet cells differentiation at the cost of absorptive enterocytes and promoted mucins secretion, which accelerated tgev replication. therefore, the more differentiation of goblet cells, the greater tgev infection in jejunum. these results provide a detailed mechanistic pathway by which villous atrophy sharply occurs in tgev-infected jejunum within h. thus, the pathogenesis of tgev can be described as a “bottom up scenario”, which is contrary to the traditional “top down” hypothesis. together, our findings provide a potential link between diarrheal virus infection and crypt cells response that regulates paneth cells function and lgr iscs fate and could be exploited for therapeutic application. the mammalian intestinal epithelium exhibits rapid self-renewal, with complete turnover of epithelial cell lining every - days , . in addition to its role in nutrient digestion and absorption, the intestinal epithelium also forms as a critical barrier against luminal pathogens to maintain intestinal homeostasis . however, this barrier function is known to be susceptible to irradiation, chemical injury or infection with pathogens . one of the central mechanisms to maintain intestinal barrier function is through activating crypt cells (paneth cells and intestinal stem cells, iscs) . whereas the mechanism by which irradiation or chemical toxins activate crypt cells response has been extensively studied , , far less is known about the response of crypt cells to diarrheal virus infection, such as tgev. tgev, together with the human coronaviruses (hcov-nl and hcov- e), belongs to the genus alphacoronavirus within the subfamily coronaviridae , which are single-stranded, positive-sense rna viruses relevant in animal and human health . generally, tgev causes transmissible gastroenteritis with high morbidity in pigs of all ages and as high as % mortality in newborn piglets, especially those within weeks of birth . most notably, tgev not only causes devastating impact on the global pig industry, but also is a potential threat to human health, as its infection suppresses protein translation in diverse human cells , . the economic and health implications of tgev have aroused significant concern worldwide . the typical symptomatic pathway of tgev infection is villous atrophy within h, followed by crypt hyperplasia, concomitant with lethal watery diarrhea, and ultimately severe dehydration in piglets until death . tgev infection causes intestinal barrier dysfunction and disrupts intestinal homeostasis , , which could affect intestinal epithelium renewal. the intestinal homeostasis and intestinal epithelium renewal are normally sustained by fast-cycling stem cells located around the base of the crypt . these internal cycling stem cells are distinguished by lgr expression, which is suggested to mediate cell proliferation in a number of tissues . small populations of lgr iscs regularly divide to produce highly proliferative progenitors known as transit-amplifying (ta) cells and terminally differentiate into the absorptive (enterocytes) or secretory (paneth cells, goblet cells and enteroendocrine cells) cell lineages while gradually migrating upwards toward the top of the villi . one exception to this migratory path is paneth cells, which follow a downward migratory path to the crypt bottom and intermingle with lgr iscs . the close interaction of lgr iscs and paneth cells is essential for maintaining lgr iscs fate , since paneth cells provide crucial niche factors (such as wnt a, bmp, and notch factors) for lgr iscs self-renewal and differentiation . of particular significance, notch factors regulate ta differentiation status and intestinal developmental pattern . paneth cells express notch ligands, which bind the notch receptor on lgr iscs to activate expression of downstream genes, such as hes and hes , . these notch target genes are essential for iscs homeostasis, as inhibition of the notch signaling results in complete conversion of epithelial cells into goblet cells both in vitro and in vivo , . in contrast, activation of the notch signaling promotes proliferation to the absorptive cell lineage with a concomitant loss in secretory cell lineage differentiation . among notch ligands, dii and dii are essential, and inhibition of both results in the loss of stem and progenitor cells . in this study, we explore how tgev infection targets paneth cells and through them disrupt lgr iscs and their ability to regenerate and differentiate, as well as adjacent effects on goblet cells and their role in maintaining the protective lining of the intestinal epithelium. we challenged the assumption that tgev disruption of the jejunum occurs via a "top down" pathway with direct effects on the crypt cells, and instead consider how tgev may function as a "bottom up" scenario with infection of paneth cells initiating a cascade of events that ultimately inhibit intestinal epithelial homeostasis. these findings revealed a previously unrecognized link between diarrheal virus and crypt cells response that modulates iscs homeostasis and could be exploited for therapeutic application. as tgev major targets organ is small intestine. therefore, we collected the duodenum, jejunum and ileum tissues for intestinal architecture analysis. histological analysis of these samples revealed that tgev significantly disrupted intestinal morphology in small intestine, especially in jejunum (fig. a) . the most striking features were villous atrophy. villous height decreased sharply, accompanying with enterocyte shedding, followed by crypt hyperplasia (fig. a, b) . subsequently, the representative tight junction proteins zo- , occludin, and claudin- were determined by using western blot (wb). the results indicated that tgev solely down-regulated zo- protein level in infected jejunum (fig. c, d) . moreover, we found that tgev infection robustly halted cell proliferation both in crypt and villous (fig. e , f), as assessed by ki staining. unlike cell proliferation, tgev induced cell apoptosis solely in crypt cells, as detected by tunel staining (fig. g, h) . these results also were confirmed in ipec-j cells (a jejunal epithelial cell line). tgev infection not only inhibited cell proliferation, but also induced cell apoptosis in h post infected cells ( supplementary fig. s ). together, these data strongly suggest that tgev infection inhibits epithelial selfrenewal and disrupts intestinal homeostasis. to test the hypothesis that tgev infection disruption intestinal homeostasis through inhibiting the self-renewal and differentiation of lgr iscs, wb, facs, fish and if staining were used to determine the self-renewal of lgr iscs in tgev-infected jejunum. we observed a significant decrease in lgr iscs number within crypt tissues for tgev-infected jejunum (fig. a, b) . similarly, olfm , another marker of iscs, was repressed ( fig. a-d) . in parallel, similar results were found in tgev-infected ipec-j cells ( supplementary fig. s a, b) . we then examined the differentiation pattern of lgr iscs in tgev-infected jejunum. the number of enterocytes was distinctly decreased in tgev-infected jejunum, as revealed by si protein expression (fig. c, d) . similar results were determined in paneth cells (fig. e, f) . moreover, tgev infection not only decreased the number of paneth cells in crypts, but also caused enteroendocrine cells loss in crypts and villi (fig. c , d, g, and h). in contrast, muc protein level and pas staining showed that tgev infection strongly increased the number of goblet cells both in crypts and villi (fig. c , d, i, and j). subsequently, we found that tgev infection not only halts cell proliferation and but also induces cell apoptosis in lgr iscs ( supplementary fig. s c, d) . together, tgev infection induces goblet cells differentiation at the cost of absorptive enterocytes in tgevinfected jejunum and severely alters lgr iscs fate. to uncover the potential mechanisms for lgr iscs loss and the impact of tgev infection on the niche signals for lgr iscs fate decision, we took advantage of tgevinfected ipec-j cells model. facs for cd demonstrated that tgev infection resulted in cd cell loss in tgev-infected ipec-j cells ( supplementary fig. s a ), and if staining results also revealed that tgev infection decreased the expression of cd in tgev-infected jejunum ( supplementary fig. s b, c) . subsequently, we found tgev infection caused paneth cell (cd + ssc high cells) and enteroendocrine cell (cd + ssc low cells) loss ( supplementary fig s d; fig. a , b), as was observed in tgev-infected jejunum ( fig. e-h) . we next examined apoptotic cell death and cell proliferation to determine potential mechanisms for paneth cells loss with tgev infection. either early cell apoptosis or late cell apoptosis was robustly induced by tgev in cd + ssc high cells ( supplementary fig. s a, b) . meanwhile, similar results were detected in cd + ssc low cells ( supplementary fig. s c , d), but no changes in cell apoptosis were found in cd − cells ( supplementary fig. s e , f). cell apoptosis mainly occurred in cd + cells. moreover, combining with facs analysis of paneth cells proliferation with ki staining, tgev infection inhibited the proliferation of paneth cell (cd + ssc high cells) and enteroendocrine cell (cd + ssc low cells) in tgev-infected ipec-j cells ( supplementary fig. s g, h) . subsequently, we detected the mitochondrial function and ros generation in tgev-infected ipec-j cells, which largely contribute to cell apoptosis and barrier functions of intestinal epithelial cells. as expected, tgev infection significantly induced mitochondria damage and ros production not only in paneth cells (cd + ssc high cells) but also in enteroendocrine (cd + ssc low cells) (fig. a, b) . since paneth cells provide various niche factors to support iscs self-renewal and differentiation. paneth cells loss seriously affects the number and function of iscs through reducing niche factor generation. to reveal the effect of tgev on niche factor production, we first detected the mrna expression of the representative niche factor markers in tgev-infected jejunum (fig. c ) and ipec-j cells (fig. d) . these results showed that tgev infection significantly inhibited notch ligand dii and notch effector hes mrna expression. for wnt (wnt ) and bmp (tgf-β) signaling, no significant changes in mrna level were observed in tgev-infected jejunum or ipec-j cells (fig. c, d) . then dii and hes protein expression was quantified in tgev-infected jejunum and ipec-j cells by using wb (fig. e-g) . infection by tgev disrupted the notch signaling for lgr iscs self-renewal and differentiation via down-regulating dii and hes protein expression both in in vivo (fig. e , f) and in vitro (fig. g ). in addition, tgev infection decreased si, cga, cd protein expression (fig. g) . alternatively, goblet cells (muc ) were up-regulated in tgev-infected ipec-j cells (fig. g) , with similar effect on goblet cells was detected in tgev-infected jejunum (fig. c , i, j). subsequently, we inhibited notch signaling in ipec-j cells by using n-s-phenyl-glycine-t-butyl ester (dapt), a γsecretase inhibitor, which turns proliferative cells in intestinal crypts and adenomas into goblet cells. as expected, dapt rapidly induced goblet cells differentiation at the cost of absorptive cells in ipec-j cells (data not shown). surprisingly, notch signaling inhibition strongly promoted tgev infection and replication in ipec-j cells (fig. h) . therefore, these results demonstrate that tgev infection alters lgr iscs fate via inducing paneth cells loss and disrupting notch signaling factors. moreover, goblet cells differentiation enhances tgev infection and replication. as noted earlier, tgev infection distinctly affects the number and function of paneth cells. to uncover the specific mechanism, we detected the infection proportion and replication of tgev in ipec-j cells. of interest, (see figure on previous page) fig. tgev infection destroys the integrity of intestinal architecture and disrupts intestinal homeostasis. a representative h&e stained crosssection showing villous atrophy and enterocytes shedding in tgev-infected intestine. scale bar, μm. b quantification of villous height (n = ) and crypt depth (n = ) in tgev-infected jejunum (n = ). c western blot for junction protein zo- , occludin, and claudin- of jejunum from control and tgev-infected piglets. actin serves as a control. d quantitation of bands to demonstrate the protein level of zo- . e, f jejunal cross-section stained with ki (scale bars, μm) and quantification of the proliferation cells per crypt (n = ) and villus (n = ). g crosssection of jejunum from control and tgev-infected piglets stained with tunel (the white arrows show cell apoptosis in crypt; scale bars, μm). h quantification of necrotic cells per crypt (n = ) and villus (n = ). tgev was mostly detected in cd + cells, which mainly include cd + ssc high cells (paneth cells) and cd + ssc low cells (enteroendocrine). in cd + ssc low cells the effect was most pronounced, with the percentage of tgev-positive cells nearly -fold as many as compared to other cell types in h post tgev-infected ipec-j cells. however, a few cells were infected by tgev in cd − cells, which are mostly absorptive enterocytes (fig. a ). although the difference of the infection proportion gradually narrowed in h post infection, cd + ssc low cells still contained more intracellular tgev. notably, less tgev was detected in h post infection paneth cells, which is opposite with the result of h post infection. these data suggest that tgev selectively targets cell types for infection and replication. we followed those observations by examining tgev infection rate over shorter time periods in several cell types. surprisingly, in contrast to results from and h incubation, after only h tgev infection was higher in cd + ssc high cells than cd + ssc low or cd + ssc − . about % of cd + ssc high cells carried tgev, but only % cells were infected with tgev in other cell types, such as all, cd − and cd + ssc low ipec-j cells (fig. b) . it suggests that tgev selectively targets to cd + ssc high cells for initial invasion. similar to other coronaviruses (covs), tgev utilizes apn (cd ) as its receptor for cell invasion (supplementary fig. s b ). since tgev mainly targets paneth cells for initial invasion, we detected tgev receptor apn expression in different cell types (fig. c) . facs results showed only about . % cells were cd positive in ipec-j cells. however, . % cd + ssc high cells and . % cd + ssc low cells expressed cd . notably, almost no cd was expressed in other ipec-j cells (cd − cells) (fig. c ). subsequently, removing cd + cells from ipec-j cells by using facs markedly decreased tgev infection by %. as expected, additional supplementation of cd + cells in ipec-j cells significantly promoted tgev infection (fig. d) . removing cd + cells didn't affect the number of cd + ssc high and cd + ssc low cells in cd + -removed ipec-j cells, even upon tgev infection ( supplementary fig. s a ) and rescued lgr iscs loss, which was induced by tgev ( supplementary fig. s b ). in brief, tgev initially invades paneth cells through apn (cd ) receptor. subsequently, we found that inhibition of tgev infection by apn gene knockout in ipec-j cells rescues the fate of lgr iscs (supplementary fig. s ). this event directly inhibited tgev infection and replication in supplementary fig. s a) , and proteins that cause cd cells loss (supplementary fig. s c) . moreover, there were tgev-encode proteins which down-regulated notch signaling ( supplementary fig. s b) . as represented via a venn diagram, five tgev encode proteins (nsp , nsp , nsp , nsp , and orf a) simultaneously affect cell proliferation, cd + cell number and notch signaling (dii and hes protein expression) (fig. a) . among these viral proteins, nsp was the most significant inhibitor of cell proliferation and notch signaling (fig. b, c) , and strongly decreased cd + ssc high and cd + ssc low cell number (fig. d ). in addition, nsp down-regulated si, cd and cga protein level, but significantly up-regulated muc protein expression (fig. b) . moreover, nsp decreases olfm expression in nsp stable cell lines (fig. b) . similar results were observed in tgev-infected jejunum (fig. c-j) . therefore, we focused on nsp , which mediated cell proliferation and lgr iscs fate decision. of note, nsp induces similar phenomenon to nsp in ipec-j cells ( fig. ; supplementary fig. s ). tgev-encoded nsp /nsp protein complex not only interact with dii but also alter dii promoter activity normally, nsp activity is regulated by nsp , and forms a protein complex with nsp to inhibit the translation and/or stability of host proteins in other covs, such as sars-cov and mers-cov. moreover, tgev and tgev-encoded nsp and nsp distinctly inhibit notch signaling factors (dii and hes ). therefore, we investigated whether tgev-encoded nsp and/or nsp alter notch signaling via interacting with these factors (dii and/or hes ). to explore this point, we performed ip experiments in nsp and nsp stable transfected ipec-j cell lines, and co-ip experiments in nsp and nsp transiently transfected hek t cells. anti-flag immunoprecipitation followed by anti-dii and anti-ha western blotting showed specific binding of nsp and nsp to dii (fig. a, b) , but no interactions were detected between nsp and hes as well as nsp and hes . to further test their interactions between nsp , nsp and dii , we utilized a duolink proximity ligation assay (pla), which can demonstrate proteinprotein interactions in situ by eliciting a fluorescent signal. pla signals were detected in dii -ha co-transfection with nsp -flag and/or nsp -flag cells, revealing the interaction of these proteins (fig. c) . we also tested for the interaction between tgev-encoded nsp and nsp by using pla. indeed, nsp was observed to bind to nsp (fig. c) . to test whether the colocalization of nsp , nsp and dii occurs, hek t cells were co-transfected with plvx-dsred-monomer-flag-nsp , plvx-acgfp -flag-nsp , plvx-mvenus-ha-dii and fixed at h post transfection. the results showed that nsp not only co-localized with nsp , but also co-localized with dii (fig. d) , which further confirmed the interaction between tgevencoded nsp , nsp and dii . these results suggest that tgev-encoded nsp and nsp form a complex in the host to alter dii protein level. to reveal the potential mechanism of nsp and nsp in reducing dii protein level, we cloned a fragment spanning − bp to + bp (p ) of the dii predicted promoter into the pgl -basic vector (fig. e) . hek t cells were co-transfected with p , prl-tk (renilla luciferase control reporter vectors), vector, nsp and/or nsp . we found that nsp robustly down-regulates dii promoter (p ) activity. however, nsp did not alter the transcriptional activity of dii promoter (fig. f) . subsequently, we divided dii promoter (p ) into three sections (fig. e) and detected the promoter activity of these fragments by using dualluciferase reporter system. nsp was observed to inhibit the promoter activity of three different dii promoter fragments by about - % (fig. g) . although nsp slightly enhanced the dii promoter (p ) activity by about %, nsp still inhibited the dii promoter (p ) activity even in the presence of nsp (fig. g ). it is now well established that intestinal crypt cells respond to damage induced by high-dose irradiation or chemicals by activation of reserve stem cells , [ ] [ ] [ ] . here, we reveal intestinal crypt cells exhibit a novel response to a diarrheal virus (fig. ) . in this study we found that tgev infection results in villous atrophy within h and inhibits intestinal epithelium renewal by halting the selfrenewal and differentiation of lgr iscs. as the epithelium of the intestine is the fastest renewing tissue, sustained by lgr iscs , once lgr iscs lose the ability of self-renewal and diferentiation, it will seriously affect intestinal epithelium turnover and perturb intestinal homeostasis. a recent report similarly showed that heligmosomoides polygyrus infection causes lgr iscs loss through activating ifn-γ generation and induces fetal-like reversion in the intestinal stem-cell niche iscs are apoptosis sensitive cells to different types of stresses (such as ros), so it is easy to be attacked . previous study demonstrated that tgev-encoded n protein induced ros generation, which contributes to cell apoptosis activation via p signaling in st cells . although tgev induces mildly ros production in tgev-infected ipec-j cells, it robustly promotes ros generation in lgr iscs (data not shown), which provides a potential explanation of lgr iscs loss and why more apoptosis cells were observed in crypt. except for cell apoptosis, tgev-mediated paneth cells loss also contributes to the developmental fate of lgr iscs. tgev directly invades paneth cells through the apn receptor, and then activates ros generation, which ultimately induces paneth cells apoptosis. paneth cell loss severely affects the niche factors secretion (such as wnt and notch factors) needed for lgr iscs self-renewal and differentiation . among these niche factors, notch factor is often linked to developmental patterning , intestinal stem-cell self-renewal and crypt homeostasis . although a few studies shown that paneth cells are dispensable for survival, proliferation, and stem-cell activity , . this because the other cell type severs the same function as paneth cells, such as mesenchymal cells , , . beyond stimulating lgr iscs cells with niche signals, paneth cells also provide essential nutrients to iscs . therefore, paneth cells are essential for lgr iscs. in our study tgev infection significantly down-regulates notch ligand dii and notch effector hes protein expression both in vitro and in vivo. inactivation of dii causes the loss of stem and progenitor cells . thus, paneth cells loss, affecting notch signaling activation, may be the foundational mechanism of tgev-mediated inhibition of lgr iscs self-renewal and differentiation. fig. tgev-encoded nsp /nsp protein complex not only interact with dii but also alter dii promoter activity. a ipec-j cells were stable transfected with empty vector, plvx-acgfp -flag-nsp and plvx-acgfp -flag-nsp . cell lysates were immunoprecipitated with anti-flag mabs followed by immunoblot of dii mabs to assess the interaction between nsp , nsp and dii protein. b hek- t cells were transfected with empty vector, plvx-dsred-monomer-flag-nsp , plvx-acgfp -flag-nsp and/or plvx-mvenus-ha-dii plasmids. cell lysates were coimmunoprecipitated with anti-flag® m antibody followed by immunoblot of dii using anti-ha mabs to assess the interaction between nsp , nsp and dii protein. notch signaling is a developmental switch for intestinal secretory cells and absorptive enterocytes , as its suppression leads to a block of differentiation of enterocytes and a dramatic increase in the number of goblet cells gastrointestinal tract, mucins have the ability to promote tgev infection. as mucins are rich in sialic acids , they are interaction partners for tgev and thus may help to penetrate the mucus layer to gain access to apn on the surface of the intestinal epithelial cells . thus, over time, the more tgev enters into intestinal epithelium, the more severe damage occurs in tgev-infected jejunum. a circle promoting infection is then gradually formed within h of tgev infection. this is, at least in part, reason for villous sharp shortness and high mortality of tgev infection. thus, the mucins are a "double-edged sword" that needs to be balanced gently. base on these results, it is not difficult to find that the disruption of tgev mainly occur in crypt, and villous atrophy is the ultimate result of crypt damage. this "from bottom to top" finding of tgev pathogenesis to intestine is quite novel to the traditional "top down" understanding of tgev infection that villus damage occurs first and damage to crypt next. together, our data suggests that the notch pathway might represent the cell developmental "switch" of lgr iscs and paneth cells in the case of tgev infection. therefore, notch signaling may be an attractive therapeutic target for tgev infection. the fact that notch signaling controls key steps of differentiation in most and has become therapeutic targets for many diseases . subsequently, we found that tgev-encoded nsp significantly attenuates notch signaling (dii and hes ) activation. nsp , a ′-o-methyltransferase ( ′-o-mtase) plays a crucial role for ′-o-methylation of viral mrna in capping of viral rna, which permits viral infection with reduced host recognition , . the absence of ′-o-methylation or nsp mutant activates a more robust type i ifn response that ablates viral infection and replication , . thus, capping of viral rna by nsp is an effective and successful strategy to disrupt host immune recognition. for tgev, its encoded nsp not only helps itself to evade host recognition, which promotes tgev infection and replication, but also inhibits notch signaling via inhibiting the dii promoter activity, and ultimately disrupts intestinal homeostasis. generally, covs nsp activity is strictly dependent on its interaction with nsp , , this is no exception for tgev. tgev-encoded nsp interaction with nsp to prevent virus infection by cell innate immunity mechanisms. inhibition of nsp /nsp mtase activities by a mtase specific inhibitor adohcy significantly inhibits tgev infection and replication (data not shown). additionally, nsp , nsp and their complex can interact with dii , which normally binds to notch receptors. this interaction may disturb notch signaling. notably, nsp not only binds to dii , but also down-regulates dii promoter activity, which provides a good explanation of tgev and its nsp and/or nsp -mediated dii mrna and protein decrease. therefore, nsp /nsp complex is likely another novel therapeutic target for tgev infection. in summary, our findings highlight a link between intestinal crypt cells response and virus infection that regulates paneth cells function and lgr iscs fate. tgev infection causes loss of notch signaling, which inhibits lgr iscs self-renewal and differentiation and causes villous atrophy. this phenomenon can also be regarded as an explanation of tgev pathogenicity, and possibly represents similar pathways used by other diarrheal virus (such as rotavirus) and bacteria, to disrupt intestinal homeostasis. given its central role in tgev infection, notch signaling is an attractive therapeutic target for tgev infection. future studies will explore how intestinal crypt regenerates the damaged gut with diarrheal virus infection. a total of -days-old dly male piglets were obtained from a swine herd at sichuan agricultural university and artificially fed with milk for three days. after that, all piglets were randomly divided into two groups, the tgev-infected group (tgev) and the control group (control) in this study (n = ), and then piglets were orally inoculated with either or × pfu tgev (tcid = − / μl) according to the previous assignment (control vs. tgev infected). each experimental group of piglets was housed in a separate room in a high-security isolation facility. piglets that developed significant diarrhea and lived two days after infection were used in the experiment. the experimental procedures used in this study were approved by the animal care advisory committee of sichuan agricultural university. intestinal tissue was collected and fixed overnight in % pfa in pbs and embedded in paraffin. in all, -μm sections were stained by he and pas for histological analysis and goblet cells staining. sample were embedded in oct and μm sections were performed for immunofluorescence staining by using the following primary antibodies: tunel (roche, ), anti-ki (bd; ), anti-cd (thermo fisher; ma - ), anti-muc (santa cruz; sc- ), anti-cga (immunostar; ). all primary antibodies were used as : dilutions. goat anti-mouse alexa fluor® (abcam, ab ) and goat anti-mouse alexa fluor® (ab ) secondary antibodies were used at : - : dilutions. for fluorescence in situ hybridizations (fish), tissues of jejunum were fixed overnight in % pfa, paraffin embedded, and sectioned at μm. the probe sequences targeting lgr , olfm and lys (lysome) was: lgr probe ( ′-fam-gacgacaggcggttggacgataggt-fa m- ′), olfm probe ( ′-fam-cactgacacctcgcc accattcca-fam- ′) and lys probe ( ′-cy -gca ccgatcatagaccttggcctgta- ′). the protocols used for in vitro transcription and in situ hybridization were previously described . all images were acquired and processed with zeiss axio-imager z with apotome or leica sp confocal microscope. ipec-j cells were obtained from atcc and cultured in dmem/f supplemented with % fbs, iu/ml of penicillin, mg/ml of streptomycin, mg/ml hegf and nm hepes, at °c in a % co atmosphere incubator. tgev was provided by prof. zhiwen xu. confluent ( %) ipec-j cells were inoculated with tgev at moi of for h at °c. the unattached virus were removed and the cells were washed one time with pbs. subsequently, fresh growth medium was added. the tgev genes were constructed by rt-pcr amplification from the genomic rna of tgev strain wh- and cloned into lentiviral vector plvx-acgfp -n . all plasmids were confirmed by sequencing and transfected into hek t packaging cells with plp/vsvg and pspax two plasmids packing system. stably transfected ipec-j cells were selected with . μg/ml puromycin selection for weeks. selected cells were tested for tgev genes mrna expression by reverse transcription-pcr (rt-pcr). flow cytometry (facs) experiments were performed with the following antibody: alexa fluor® -lgr (bd; ), fitc-cd (bd; ), pe-cy tm-cd (bd; ), percp-cytm . -cd (bd; ). facs measurements were performed using a bd-facs service device and analyzed with flowjo software (flowjo llc). mfi is defined as the difference in the signal intensity between an unstained control and a stained sample. for sorting, a facs aria sorp device (becton dickinson) was used. apoptotic cell death was detected by fitc-, alexa fluor® -conjugated annexin v with propidium iodide (pi) staining assay (biolegend; and ) after the manufacturer's protocols. briefly, ipec-j cells were harvested, then cells were resuspended in μl × binding buffer followed by incubation with μl / cells annex v per test for min on ice. subsequently, μl × binding buffer and μl pi ( mg/ml) was added to the sample and immediately analysis by facs. cell proliferation was determined by using facs. ipec-j cells infected with tgev were harvested and fixed with % formalin in pbs for min at room temperature. fixed cells were permeabilized with . % triton x- in pbs at room temperature for min. for intracellular ki (bd; ) staining, cells were incubated for h with ki antibody ( . μg/ cells), followed by secondary antibody staining with : dilution of goat anti-mouse alexa fluor® (abcam; ab ) for additional h. total rna was extracted from samples using trizol reagent (invitrogen) according to the manufacturer's protocol and cdna synthesised using the prime script tm rt reagent kit with gdna eraser (takara, rr a). relative gene expression was calculated with the ΔΔc t method, normalizing the results to the value for the gapdh gene. protein extraction and western blotting were performed as described previously, using primary antibodies against olfm (abcam; ab ), si (santa cruz; sc- ), cd (thermofisher; ma - ), muc (santa cruz; sc- ), cga (immunostar; ), hes (santa cruz; sc- ), dii (abcam; ab ) and β-actin (santa cruz; ). the dilution of primary antibodies was : . the second goat anti-rabbit and goat anti-mouse antibodyies conjugated to hpr (santa cruz; sc- and sc- ) was used ( : ). the porcine anp gene (gene id: ) was disrupted in ipec-j cells by using the crispr-cas technique. in brief, two guide rnas (grnas) targeting the first exon of the anp gene, ggtaggcggtaccggttcca (anp-ko- ) and gcgttgtgggtaggcggtac (anp-ko- ), and complementary oligonucleotides were designed. the annealed grna duplexes were cloned into the len-ticrisprv vector (addgene; ) by using the bsmbi restriction site. the grna-expressing vectors were transfected into hek t cells, and cell culture supernatants containing viral particles were harvested and used for infection of ipec-j cells. ipec-j cells were subsequently subjected to puromycin selection for weeks. selected clones were tested for anp mrna expression by reverse transcription-pcr (rt-pcr). a representative clone from each of the two crispr constructs was used in the experiments described here. immunoprecipitation (ip) and co-immunoprcipitation (co-ip) nsp and nsp stable ipec-j cells were harvested and lysed in lysis buffer. μg of cell lysate protein was incubated with . μg anti-flag® m antibody (cst; ) and μl of protein a magnetic beads (cst; ) overnight at °c. samples were washed three times with lysis buffer, resuspended in x sample buffer and analyzed with immunoblot analysis using dii antibody. for co-ip experiments, hek t cells expressing nsp -flag, nsp -flag and/or dii -ha constructs were harvested and lysed in lysis buffer. samples were then centrifuged to remove precipitated proteins and were incubated with anti-flag® m antibody overnight at °c. samples were washed three times with lysis buffer, resuspended in x sample buffer and analyzed with immunoblot analysis using the indicated antibodies. hek t cells were seed into -well plates at × cells/well and co-transfected with different dii predicted promoter plasmids, nsp and nsp . all cells were transfected with the renilla luciferase control plasmid prl-tk (promega, e ). luciferase activity was measured with a dual-luciferase assay kit (beyotime; rg ) with glomax microplate luminometer (promega) in luminometer mode following the manufacturer's protocol. the raw values of firefly luciferase were normalized to renilla luciferase. hek t cells expressing nsp -ha, nsp -flag or dii -ha construct were co-cultured on a cover slip. cells were fixed with % pfa for min at room temperature, blocked with . gelation-pbs, and stained with : rabbit anti-flag and : mouse anti-ha antibodies for h at room temperature. cells were then stained with : donkey anti-rabbit plus and donkey anti-mouse minus second antibodies. after washing with . % gelatin-pbs, proximity ligation assay (pla) was done according to manufacturer's protocol (sigma). each of the experiments described here was performed in at least three independent biological replicates. statistical analysis was performed by using graph pad prism software. all results were unpaired two-tailed student's t test and/or one-way analysis of variance (anova). p ≤ . were considered to be statistically significant (*p < . , **p < . , ***p < . ). interplay between metabolic identities in the intestinal crypt supports stem cell function the intestinal crypt, a prototype stem cell compartment inflammasome, inflammation, and tissue homeostasis parasitic helminths induce fetal-like reversion in the intestinal stem cell niche dll + secretory progenitor cells revert to stem cells upon crypt damage intestinal enteroendocrine lineage cells possess homeostatic and injury-inducible stem cell activity microrna- promotes transmissible gastroenteritis virus (tgev)-induced mitochondrial damage via targeting rb , upregulating interleukin- receptor accessory protein (il rap), and activating p mapk pathway in vitro the nucleoprotein is required for efficient coronavirus genome replication impact of tgev infection on the pig small intestine alphacoronavirus transmissible gastroenteritis virus nsp protein suppresses protein translation in mammalian cells and in cell-free hela cell extracts but not in rabbit reticulocyte lysate induction of alpha interferon by transmissible gastroenteritis coronavirus: role of transmembrane glycoprotein e cellular rna helicase ddx is involved in transmissible gastroenteritis virus nsp -induced interferon-beta production il- suppresses the infection of porcine enteric coronaviruses and rotavirus by activating stat signal pathway adult intestinal stem cells: critical drivers of epithelial homeostasis and regeneration identification of stem cells in small intestine and colon by marker gene lgr mini-gut organoids: reconstitution of the stem cell niche transcription factor achaete scute-like controls intestinal stem cell fate beyond growth signaling: paneth cells 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recombination in small intestinal stem cells utilizing the olfm locus stem cells and the impact of ros signaling tgev nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p signaling the canonical notch signaling pathway: unfolding the activation mechanism intact function of lgr receptorexpressing intestinal stem cells in the absence of paneth cells redundant sources of wnt regulate intestinal stem cells and promote formation of paneth cells tales from the crypt: new insights into intestinal stem cells wnt ligands secreted by subepithelial mesenchymal cells are essential for the survival of intestinal stem cells and gut homeostasis transmissible gastroenteritis of swine: virus-intestinal cell interactions. i. immunofluorescence, histopathology and virus production in the small intestine through the course of infection the gastrointestinal mucus system in health and disease the sialic acid binding activity of the s protein facilitates infection by porcine transmissible gastroenteritis coronavirus notch signaling in development, tissue homeostasis, and disease coronavirus non-structural protein : evasion, attenuation, and possible treatments '-o methylation of the viral mrna cap evades host restriction by ifit family members middle east respiratory syndrome coronavirus nonstructural protein is necessary for interferon resistance and viral pathogenesis coronavirus nonstructural protein is a cap- binding enzyme possessing (nucleoside- 'o)-methyltransferase activity expression pattern of wnt signaling components in the adult intestine the authors declare that they have no conflict of interest.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.supplementary information accompanies this paper at (https://doi.org/ . /s - - - ).received: september revised: january accepted: january key: cord- -d bxe c authors: yuan, xiaomin; lin, huixing; fan, hongjie title: efficacy and immunogenicity of recombinant swinepox virus expressing the a epitope of the tgev s protein date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: d bxe c to explore the possibility of developing a vaccine against transmissible gastroenteritis virus (tgev) infection, a recombinant swinepox virus (rspv-sa) expressing a tgev protective antigen has been constructed. immune responses and protection efficacy of the vaccination vector were assessed in both mice and pig models. an indirect elisa assay suggested that when mice were vaccinated with rspv-sa, the level of igg against tgev was enhanced dramatically. the cytokine assays were employed and the results indicated that both the th -type and th -type cytokine levels raised after vaccination with rspv-sa in mice models. results from the passive immunity protection test of new born piglets demonstrated that the recombinant live-vector vaccine, rspv-sa, could % protect piglets from the spv infection, and there was no significant clinical symptom in the rspv-sa treatment group during this experiment. the data suggest that the novel recombinant swinepox virus is a potential vaccine against tgev infection. transmissible gastroenteritis virus (tgev) is a member of coronaviridae, which is the etiological agent of transmissible gastroenteritis. although the virus is capable of infecting swine of all ages, suckling piglets are the most susceptible and have a mortality rate up to % [ ] . tgev is a pleomorphic enveloped virus containing a positive-stranded rna genome and four structural proteins: the spike (s) protein, the integral membrane (m) protein, the minor envelope (e) protein, and the nucleocapsid (n) protein. among which, the spike (s) protein, one of the key structural membrane proteins of coronaviruses, is an attractive target for generating neutralizing antibodies against the virus due to the critical role it plays in the host cell invasion [ , ] . precisely, the s protein mediates the attachment of virus particles to targets via binding of itself to the specific receptors. at the n terminus of the s protein, there are four antigenic sites, a, b, c, and d, which have been shown to be involved in the stimulation of neutralizing antibodies (fig. ) [ ] . previous studies have determined that the a site (which is fully dependent on glycosylation for proper folding) is predominantly responsible for stimulating neutralizing host antibodies [ ] [ ] [ ] [ ] [ ] [ ] . spv is a natural mild attenuated virus and has been widely applied as a vaccine. given that poxvirus-vectors can prevent a great deal of important diseases in both humans and animals it is not surprising that many of these vectors been licensed and used extensively [ ] [ ] [ ] . additionally, spv is a safe vaccine vector as there is no risk of cross-species infection [ ] . therefore, both for biological and clinical practicality, spv is regarded as an appropriate and promising veterinary vaccine for swine, owing to its ability to effectively express foreign genes, its large packaging capacity for recombinant dna, its low cost of delivery and its specific host restriction [ ] . the potential value of spv as a live vector vaccine is being studied extensively. because spv is able to pack large amounts of recombinant dna and to induce appropriate immune responses in vivo, it is a promising candidate for the development of a recombinant vaccine [ , ] . as of yet, pigs are the only known hosts of swinepox virus and therefore may be useful in developing a safe vaccine for clinical application [ , ] . in this study, we constructed a recombinant swinepox virus expressing s-a (a epitope of the s protein) of tgev and characterized recombinant virus replication and expression of the s protein in pk- cells. we further investigated the potential of this approach for use in the vaccination of pigs against tge. type culture collection. swine transmissible gastroenteritis virus (tgev, china strain, shxb) was purchased from the jiangsu academy of agricultural sciences, and the titer was determined as × pfu/ml st cell. tgev convalescent positive serum was purchased from the jiangsu academy of agricultural sciences, and the neutralizing antibodies were used at a dilution of : , . the pusz swinepox virus vector was generated previously [ ] . two primers, sa ( -gcgtcgacatgggtcttggtatga-agcgtag- ) and sa ( -cgggatcctta tagcgtcctgt-tagtttgtc- ) were used to amplify the s-a gene ( bp, km ) from the tgev genome, which was inserted into the pusz plasmid to construct pusz -sa subsequently. the recombinant swinepox virus, rspv-sa, was generated by homologous recombination of wtspv with pusz -s-a as previously described [ ] . pcr and indirect immunofluorescence were employed to analyze the s-a gene expression and the expression of s protein. the replication capacity and genetic stability of rspv-sa were also evaluated by. the generation and screening of the recombinant swinepox virus assays were performed as described previously [ ] . a subconfluent culture of pk- cells was infected with wtspv ( . moi) for h, and subsequently transfected with g of the pusz -sa plasmid using exfect tm transfection reagent (vazyme biotech co., ltd). after h, pk- cells were harvested and lysed by five rounds of freezing and thawing. subsequently, the lysate was used to infect pk- cells grown in a -well plate for further purification of recombinant viruses. . ml of medium with % lmp agarose (dingguo, beijing, china) was added to each well and incubation was continued for five days until plaques became visible under a light microscope. after - days, a second overlay medium containing x-gal was added. the plaques were resuspended in . ml of medium with % fbs. plaque isolation was repeated for - rounds until all plaques in a given well were stained blue. the recombinant spv bearing s-a of tgev was designated as rspv-sa. the rspv-sa genomic dna from the pk- cells infected with rspv-sa was extracted by sds-protease k-phenol. we utilized wtspv genomic dna from pk cells infected with wtspv as a negative control. pcr was performed for min at • c; followed by cycles of min at • c, min at • c, and min at • c. amplifications were performed with dna polymerase (promega, shanghai, china) using primers sa ( -gcgtcgacatgggtcttggtatgaagcgtag- ) and sa ( -cgggatccttatagcgtcctgttagtttgtc- ). indirect immunofluorescence assays (ifa) were performed as described previously [ ] . pk- cells were grown on a -well plate and infected with the wtspv and rspv-sa at × pfu/ml per well. pbs-treated cells were used as a negative control. at h post-infection, cells were washed three times in pbst and fixed with cold methanol for min at − • c. cells were then washed three times with pbst and blocked by pbst with % bsa. preparations were incubated for h at • c with tgev convalescent positive serum ( : in dilution buffer, pbst with % bsa). after three washes with pbst, cells were treated with the rhodamineconjugated secondary antibody (staphylococcal protein a-rhod, boshide, wuhan, china) at a : dilution (diluted in pbs) for min at • c. after a final wash with pbs, all wells were examined by fluorescence microscopy (zeiss, germany). nine six-week-old balb/c mice were randomly divided into three groups ( mice per group), and immunized three times at , , and days with rspv-sa ( × pfu/ml in . ml of pbs) or wtspv ( × pfu/ml in . ml of pbs), the control group injected with pbs. eight one-month-old swine (large white) were randomly divided into four groups ( pigs per group) and were immunized twice at and days with infectious rspv-sa ( × pfu/ml in ml of pbs), inactivated-tgev ( × pfu/ml in ml of pbs), wtspv ( × pfu/ml in ml of pbs) or pbs, each time via three routes: oral, nasal, and intraperitoneal. serum was collected days after the last immunization. twelve one-day-old pigs were randomly divided into four groups for passive immunization experiments ( pigs per group). high titers of antibodies were collected from piglets following the first immunization. mice and swine serum were incubated at • c min to complement inactivated. all experimental protocols involving mice or swine were approved by the laboratory animal monitoring committee of jiangsu province. pk- cell monolayers were infected with wtspv and rspv-sa (moi of ) and incubated for h at • c. extracts, representing approximately × cells, were electrophoresed through an sds- % polyacrylamide gel and the separated proteins were transferred onto a pvdf membrane. after a h transfer, the membrane was blocked with % skim milk in phosphate buffered saline with . % tween- (pbst) overnight at • c. the membrane was incubated with swine convalescent serum ( : dilution) containing tgev for h at • c and washed three times with pbst. immunodetection was performed with staphy-lococcal protein a-hrp at • c. following the secondary antibody probing, the membrane was washed four times with pbst. the membrane was then developed with , -diaminoben-zidine substrate until optimal color development was observed. serum was collected from mice and pigs, and detected the tgev-specific antibodies by indirect elisa. the purified tgev was resuspended in l pbs (ph . ), and used the best titer of virus for coating -well plates, which was determined by titration. samples were then incubated overnight at • c. this incubation was followed by three pbst washes, and blocking with % skim milk (in pbst) at • c for h. serum samples were serially diluted and incubated at • c for h. the samples were set up at the same time and divided into three groups: the tgev positive serum, the negative control serum (spv positive serum) and the blank control (without serum). after three pbst washes, horseradish peroxidase (hrp)-conjugated goat anti-spa igg ( : , diluted in pbst, signalway antibody) was added to each test well. the plates were then incubated at room temperature in the dark for min and then washed three times with pbst. the tmb microwell peroxidase substrate system (tiangen) was used to develop the reaction. samples were developed for min and the reaction terminated with . m sulphuric acid. all assays were performed in duplicate. a microplate reader (bio-rad) was used to measure the reaction product at an absorbance of nm [ ] . evaluation of cellular immunity was performed by detecting levels of ifn-␥ and il- . three mice were sacrificed at days after the first inoculation with wtspv ( × pfu/ml in . ml), pbs or rspv-sa ( × pfu/ml in . ml). the mouse spleen was removed aseptically. splenocytes were isolated, counted, and diluted to a density of × cells/ l. the evenly separated cells were aliquot into -well plates ( l/well). then, l/well of dmem with tcid / l tgev was added to each well. after a h incubation, the supernatants were collected and the mrna of ifn-␥ and il- were probed by rt-qpcr relative to ␤-actin as described previously [ , ] . an assay for neutralizing antibodies was performed as described previously [ , ] . to explore whether mice or swine generated tgev neutralizing antibodies, serum from the pbs, wtspv, inactivated-tgev and rspv-sa treated mice and pig were collected at , , , , days post-primary immunization ( : - : , dilution in a l volume). and sera were mixed with equal volume of tcid /ml tgev and incubated at • c. after . h incubation, we utilized sera treated viruses to infect st cells in -well plates and overlaid cells with agar at • c in a % co atmosphere. cells were monitored daily for three days to detect tgev-specific cpe. the virulent tgev strain shxb ( × pfu/ml in ml) was mixed with ml of the porcine antiserum induced by recombinants rspv-sa, inactivated tgev or pbs, incubated at • c for min, and administered using a gastric tube to -day-old swine born from tgev-seronegative sow. inoculated animals were fed three times per day with formula for newborns that contained ml of the antiserum. at three days following viral infection, small intestine tissue was collected from newborn pigs sectioned for histology. microstructure characteristics were analyzed under the microscope (olympus, cx fs , philippines) [ ] . all data were analyzed using one-way anova and values of p < . were considered significant. to analyse the recombinant virus and confirm the presence of the sa gene, two specific primers were designed to amplify the inserted sa gene. the gene encoding the neutralizing antigen epitopes of tgev is a . kb fragment and the specific fragments was detected in spv-sa as shown in fig. a . the recombinant spv-sa was also confirmed by observing blue foci in plaque assays. rspv-sa and wtspv were determined to be approximately × pfu/ml in ml for both. as shown in fig. b , the ifa demonstrated that the expression of the a epitope of the s protein was present in infected cells. western blot analysis showed a specific band of target protein with a size of kda in the cell lysates infected with rspv-sa (fig. e) . the kda molecular weight is consistent with the predicted size of the sa protein of tgev. according to these data, we suggested that the a epitope of the s protein was expressed efficiently by the rspv-sa virus (fig. ) . to monitor the s-specific antibody titers of mice vaccinated with rspv-sa, an elisa was used. after an initial boost days postvaccination, the s-a-specific antibody titers increased gradually in mice (fig. a) . the antibody titers in rspv-sa-immunized mice were higher at all-time points post-vaccination (p < . , n = ) compared to wtspv or pbs-treated mice. the p/n value of the positive group was greater than . . persistent high levels of neutralizing antibodies (fig. a) were detected in the rspv-sa group with a mean titer of : at days post-inoculation. in mice vaccinated with wtspv and rspv-sa at days postinoculation (fig. b) , the qpcr results showed a distinct variation of il- and ifn-␥ between wtspv and rspv-sa treatment groups. the relative quantity normalized by beta-actin of il- mrna in rspv-sa was . times higher compared to wtspv. the ifn-␥ mrna levels increased . fold in rspv-sa when compared to wtspv. the concentrations of il- and ifn-␥ in rspv-sa-vaccinated mice were significantly higher than the control groups. these results indicate that rspv-sa induces th -type and th -type cytokine responses during cellular immunity. during the vaccination procedure, several poxes of mmϕ could be observed around the injection position at five days post-inoculation in both rspv-sa and wtspv treatment groups, respectively, which was not observed in the inactivated-tgev and pbs treatment groups. this symptom could be disappeared spontaneously in the following days. no pigs developed further symptoms such as fever or severe inflammation, and the spiritual condition and appetite of both treatment groups was considered as good. these two group pigs recovered in days. all group pigs maintained rectal temperatures of . - . • c. from this experiment, we reveal that vaccination with rspv-sa and wtspv is well tolerated by pigs. rspv-sa induced a moderate level of tgev-specific igg as shown in fig. . persistent high levels of tgev-specific neutralizing antibodies are shown in fig. b . the second boost led to of the levels of tgev-specific neutralizing antibodies. persistent high levels of neutralizing antibodies were detected in the rspv-sa group at a mean titer of : in swine (p < . , n = ). all newborn piglets in different groups were fed with the mixture of tgev and the corresponding sera from pigs vaccinated with pbs, wtspv, inactivated-tgev, or rspv-sa. both pbs and wtspv treatment groups developed a very severe diarrhea symptom, significantly losing of weight and appetite, and the mortality rate was up to % in days. meanwhile, in the inactivated-tgev and rspv-sa treatment groups, there was no obvious clinical symptoms and % morality rate observed (fig. . and table ). after the sacrificing of all the piglets, the small intestine tissue from different groups were used for the pathological examination. histological samples from pbs and wtspv treatment groups showed prominent histopathological changes in the small intestine characterized by serious fracture of the small intestine mucosa, epithelial expansion, vacuolar degeneration, necrosis, shedding, and lamina propria congestive edema and hemorrhage. and such pathological changes were not observed during the examination of the samples from inactivated-tgev and rspv-sa treatment groups. all the results indicate that rspv-sa inoculation provides complete protection passive immunity against tgev challenge in pigs (fig. ) . in the present study, we have engineered the swinepox virus to express the a epitope of the tgev s protein. our data showed that this recombinant virus was not only able to induce a strong immune response against the a antigen of tgev in mice and pigs, but also had safety degree and protection efficacy against the virulent homologous tgev infection in pigs. the traditional way to protect swine from tgev infection is to immunize pregnant sows with inactivated or attenuated vaccine, resulting in the production of secretory iga in the colostrum. despite the protection that secretory iga offers piglets, this method does not provide protection after the cessation of breastfeeding. furthermore, inactivated or attenuated viral vaccines still maintain the ability of developing the viral toxicity [ ] . currently, there are various potential vaccines against tgev but many present certain challenges, such as difficulties in the vaccination process, high production cost, low immunogenicity, biological risk and carcinogenicity. the results from our experiments indicate that the rspv-sa vaccine is a very promising candidate in the safety and immunogenicity respect. however, it should be noticed that, as an alive viral vector vaccine, it is possible that the spv per se would interfere the vaccination. to evaluate this possibility, randomly sampled pig sera were tested via the agarose gel diffusion assay, and the results showed the spv positive rate was around %. moreover, there was no report of spv epidemic in these recent years. thus, together with gel diffusion assay, the interfering from spv per se could be considered as minimum. for the first time, we generated a recombinant spv that expresses the neutralizing epitopes (a epitope in protein s) of tgev, and we verified that the s-a was expressed efficiently in our system. the antigen induced neutralizing antibodies against tgev in st cells, and potentiated strong th -type and th -type cytokine responses in our mouse model. these results suggest that rspv-sa induces humoral and cellular immune responses effectively and efficiently. given the th cell secretion of il- , ifn-␥, ifn-␣, and tfn-␤, it is apparent that th cells play an important role in antiintracellular pathogenic infection. because th cells in our study secreted il- , il- , il- and il- , it is likely that these cells were able to effectively stimulate b cell proliferation and hence, igg and ige antibody production (relevant to humoral immunity). we defined ifn-␥ and il- to be representative of the th -type and th -type cytokine secretory capacity, respectively. our results indicate that in our hands, both th -type and th -type cytokine levels increased dramatically. due to the immature immune system, the maternal antibodies from breast milk account for the immune defensive ability of piglets. based on the feasibility and relevant published papers [ ] , we decided to mimic the breast milk from female pig by mixing the anti-serum generated by vaccinating the days old piglets with spv-sa and the normal cow milk, and use it for passive immunity protection test on the days old piglets. in the study, the recombinant spv vectors were capable of protecting neonatal piglets against mortality and severe disease after a challenge with virus. the rspv-sa vector induced high titers of antibodies in swine. however it is worth noting that rspv-sa may enhance the anti-tgev antibody titer in swine as well as the anti-spv antibody, which will likely affect immune efficiency. with respect to clinical value, it is imperative to study this multivalent vaccine. to summarize, we first report that spv can be used as a live vector vaccine when it expresses the s-a protein of tgev. we determined that not only b-cells, but also t-cells were induced successfully. thus, rspv-sa provides thorough protection against virulent tgev challenge in swine. lastly, our data indicate that rspv-sa is a promising vaccine to prevent tgev infection. transmissible gastroenteritis virus infection: a vanishing specter coronaviruses: structure and genome expression antigenic structure of transmissible gastroenteritis virus. ii. domains in the peplomer glycoprotein bacterial expression of antigenic sites a and d in the spike protein of transmissible gastroenteritis virus and evaluation of their inhibitory effects on viral infection evaluation on the efficacy and immunogenicity of recombinant dna plasmids expressing spike genes from porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus construction, safety and immunogenicity analysis of attenuated salmonella typhimurium harbouring tgev dna vaccine localization of antigenic sites of the e glycoprotein of transmissible gastroenteritis coronavirus expression of swine transmissible gastroenteritis virus envelope antigens on the surface of infected cells: epitopes externally exposed clinical evaluation of transmissible gastroenteritis virus vaccines and vaccination procedures for inducing lactogenic immunity in sows a monoclonal antibody against transmissible gastroenteritis virus generated via immunization of a dna plasmid bearing tgev s gene years and counting: centers for disease control and prevention identifies opportunities and challenges for diabetes prevention and control development of a recombinant vaccinia-rabies vaccine for oral vaccination of foxes against rabies recombinant fowlpox virus inducing protective immunity in non-avian species recombinant swinepox virus expressing betagalactosidase: investigation of viral host range and gene expression levels in cell culture construction of recombinant swinepox viruses and expression of the classical swine fever virus e protein replication and expression of a swinepox virus vector delivering feline leukemia virus gag and env to cell lines of swine and feline origin feline b . and b . proteins produced from swinepox virus vectors are natively processed and biologically active: potential for use as nonchemical adjuvants first insights into the protective effects of a recombinant swinepox virus expressing truncated mrp of streptococcus suis type in mice immune responses and protection efficacy of a recombinant swinepox virus expressing ha against swine h n influenza virus in mice and pigs a novel vaccine against streptococcus equi ssp. zooepidemicus infections: the recombinant swinepox virus expressing m-like protein joint production of prime/boost pairs of fowlpox virus and modified vaccinia ankara recombinants carrying the same transgene recombinant adenovirus encoding the ha gene from swine h n influenza virus partially protects mice from challenge with heterologous virus: a/hk/ / (h n ) co-expressing gp and m proteins under different promoters in recombinant modified vaccinia virus ankara (rmva)-based vaccine vector enhanced the humoral and cellular immune responses of porcine reproductive and respiratory syndrome virus (prrsv) parvovirus evades interferondependent viral control in primary mouse embryonic fibroblasts induction of antibodies protecting against transmissible gastroenteritis coronavirus (tgev) by recombinant adenovirus expressing tgev spike protein oral immunization of mice with recombinant lactococcus lactis expressing porcine transmissible gastroenteritis virus spike glycoprotein the authors of this paper have no financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. key: cord- -wm k qh authors: paton, d. j.; brown, i. h.; vaz, e. k. title: an elisa for the detection of serum antibodies to both transmissible gastroenteritis virus and porcine respiratory coronavirus date: - - journal: british veterinary journal doi: . / - ( ) -k sha: doc_id: cord_uid: wm k qh abstract a competition elisa utilizing a mab directed towards a peplomer protein epitope common to tgev, prcv and related feline and canine coronaviruses is described. transmissible gastroenteritis (tge) is a disease of pigs caused by a coronavirus and characterized by a watery diarhoea often fatal to sucking piglets . in britain, it has occurred as periodic epizootics, the last of which was in the winter of / . the virus may also persist within endemically infected herds (pritchard, ) and in these circumstances fatalities may be much less common . at this laboratory, large numbers of samples from scouring pigs have been examined for the presence of tgev over many years and using a variety of methods ; but very few positive identifications have been made in the last years . porcine respiratory coronavirus (prcv) is antigenically very similar to the virus of tge, but it spreads aerogenically and causes mainly inapparent respiratory infections (pensaert et al., ) . it is known to have been present in britain at least since (brown & cartwright, ) and it had already become widespread in the national herd by (brown & cartwright, unpublished survey results) . indirect elisas for tge serology were first reported before the appearance of prcv (nelson & kelling, ) . antibodies to tgev and prcv show complete crossneutralization and are therefore both detected by virus neutralization tests (vnts) using laboratory strains of tgev . such tests (paton, ) have been widely used, both for diagnosis in cases of respiratory or enteric disease and for export certification purposes . serological differentiation between tgev and prcv is now possible using competition elisas and monoclonal antibodies (mabs) to tgev-specific epitopes (callebaut el al., , van nieuwstadt & boonstra, . however, the vnt continues to be necessary for investigation of respiratory disease . furthermore, it is more sensitive than differential elisas examined at this laboratory and it has generally been retained as the preferred method for the export certification of individual animals . this communication describes a competition elisa utilizing a mab ( a .c ) directed towards a peplomer protein epitope common to tgev, prcv, and a number of related feline and canine coronaviruses, but not to other porcine coronaviruses (sanchez et al., ) . indirect fluorescent antibody tests using rabbit anti-mouse conjugate confirmed that the mab recognized all tgev/prcv strains tested . these comprised five british isolates of tgev and two of prcv, five tgevs from bulgaria, three tgevs from the netherlands and a single prcv isolate from the ussr. elisa antigens prepared by detergent treatment (octyl-b d-glucopyranoside : aldrich) of llcpki cells, infected with the purdue strain of tgev or mock-infected, were coated onto alternate wells of flexible polystyrene elisa plates (falcon) with . m bicarbonate buffer ph . . a full description of the preparation of these antigens is given in a report on experiences with a tgev-specific, serological test that distinguishes tgev from prcv and which utilizes the same antigens (brown & paton, ) . fifty microlitres of a in dilution of test serum were added to each of duplicate pairs of wells and left overnight at room temperature . the remainder of the test could be completed within an hour, all incubations being at °c . a / dilution of mab a .c in pbs ph . with . % tween (pbst) and % ox serum was dispensed into all wells without removing the test serum and left for min . after washing, bound a .c was detected by a goat antimouse peroxidase conjugate (nordic : / for min) and a tetramethyl benzidine/ hydrogen peroxide substrate ( min) . the substrate reaction was stopped with m sulphuric acid . pbst was used for washing the plates between stages and as the serum diluent . the conjugate diluent was pbst plus % negative pig serum . the elisa value was expressed as a percentage of the result obtained with a negative control serum calculated from net optical densities (optical density of positive antigen wells minus that of negative ones) . the threshold value for the test was established using vnt negative (titre < ) sera from farms, all of which had no history of tgev/ prcv infection . subtracting three standard deviations from the mean of these results (savigny & voller, ) gave a cut-off value of . % . vnt positive sera from field cases (n= ) and from pigs experimentally infected with tgev or prcv (n= ) all scored positive in elisa (values : - %) . despite the good qualitative agreement between elisa and vnt, no significant correlation was detected (coefficient= . ) between elisa values and vnt titres for positive sera . sera collected at abattoirs during , from sows, were examined by elisa and ( . %) were positive . a recent serological survey of british pigs by tgev-specific elisa indicated a seroprevalence of only . % (brown & paton, ) , suggesting that most tgev/prcv antibody positive samples reflect prcv infection and that such infections remain common . the elisa is very similar to the vnt with respect to sensitivity and specificity but is much quicker and cheaper to perform . export certification involves processing large numbers of sera concomitantly and the semi-automation that can be achieved using the elisa technique would be a considerable benefit . for differential diagnosis, this elisa could be used in conjunction with a tge-specific test and indeed with minimal modification both could be performed side by side on the same antigen coated plate . since the mab a .c is directed towards an epitope that is highly conserved amongst tgev/ prcvs, the risk of false negative results due to antigenic diversity amongst these viruses appears to be low . veterinary record manual of recommended diagnostic techniques and requirements for biological products for lists a and b diseases veterinary quarterly , virology , the mab a .c was supplied by dr enjuanes, universidad autonoma de madrid, spain . foreign virus isolates were given by dr valicek (brno, czechoslovakia) and dr van nieuwstadt (lelystad, the netherlands) . key: cord- -uhi unn authors: paton, david; ibata, georgina; sands, jenny; mcgoldrick, adrian title: detection of transmissible gastroenteritis virus by rt-pcr and differentiation from porcine respiratory coronavirus date: - - journal: journal of virological methods doi: . /s - ( ) - sha: doc_id: cord_uid: uhi unn abstract an rt-pcr method was developed that amplified genetic material from the ′ end of the s protein gene of both transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus (prcv), but discriminated between the two by the size of the product generated. a number of restriction endonuclease enzymes were assessed for recognition of the amplicons so produced. the assay was shown to detect viral rna from all of the different tgev and prcv isolates examined, covering a period from to . detection of tgev in clinical specimens was possible using a spin column method to extract rna and sensitivity was compared to virus isolation and antigen detection elisa. the method could provide a means of confirming positive results from immunological screening tests such as fat and elisa, reducing the need for virus isolation and convalescent serology. (tge) is a highly contagious pig disease that has been reported from many parts of the world including america, europe and asia. the causative virus, tgev, is a member of the coronaviridae, and has a large, single-strand, positive sense rna genome. since the mid os, a variant respiratory form of the tge virus known as porcine respiratory coronavirus (pro, has become common in pigs in europe and has more recently been reported from north america and asia (pensaert et al., ; wesley et al., ) . it generally causes mild disease under experimental conditions. in europe, the emergence of prcv has been associated with a reduction in the incidence and severity of cases - / /$ . elsevier science b.v. all rights reserved. pzi so - ( ) - of tge. compared to tgev, prcv has a deletion of between and nucleotides near the ' end of the s gene, resulting in the loss of some antigenic sites on the s protein (laude et al., ) , and of sialic acid binding activity (schultze et al., ) . rapid methods for the detection of tgev are important because of the highly contagious nature of the disease. since the virus is often difficult to adapt to growth in cell cultures, the most widely used techniques are immunological, principally a fluorescent antibody test (fat) for cryostat sections of intestine and an antigen elisa for detection of virus in faeces (paton, ) . differentiation of tgev from the closely related prcv is possible using anti-s protein monoclonal antibodies (mabs) directed against non-neutralising antigenic sites that are lacking in prcv (garwes et al., ; callebaut et al., ) . there are also reports of the use of dna probes and of rt-pcr as detectors of tgev rna (bae et al., ; vaughn et al., ; wesley et al., ; jackwood et al., ) but the methods were not shown to be suitable for the direct detection of virus in clinical samples. an in situ hybridization method for detection of tgev and prcv has been reported, but requires tissues rather than faeces (sirinarumitr et al., ) . here we report the development of an rt-pcr method for the detection and differentiation of tgev/prcv. a total of archived viruses were examined in this study, mainly european, but also including isolates from north america and japan (table ) . the viruses had been passaged to varying extents ( > in some cases) in a variety of cell cultures. to prepare stocks for this study, viruses were passaged in a pig kidney cell line (llcpkl, flow), maintained in modified eagle's medium with % foetal calf serum. samples used for rt-pcr were extracted from either cell lysates or cell culture supernatants. uninfected cell controls were always processed concomitantly. in the case of cell lysates, cultures were processed at - h post infection or mockinfection, depending on the extent of virus growth, and total rna was extracted by the acid-phenol method (stallcup and washington, ) . for cell culture supernatants, rna was recovered using commercially available spin columns (qiaamp hcv kits, qiagen). a pair of oligonucleotide primers was designed to amplify the ' end of the gene encoding the s protein of tgev/prcv. the target region straddles a large deletion ( - nucleotides) found exclusively in isolates of prcv, but not tgev. the forward primer (f , '-tatttgtg-gtyttggtygtaatgc) is equivalent to nucleotide l- of the s protein gene of tgev, and the reverse primer (r , '-ggctgt'itg-gtaactaatttrcca) is complementary to nucleotides - . the primers were chosen by analysis of an alignment of tgev/prcv sequences available in genbank. the predicted size of the amplified product is bp for tgev and - bp for prcv. reverse transcription was in ~ volumes, using ~ of sample, . ~ of random hexamers (pharmacia, pmol), ~ of dntps ( mm), ,ul of x rt buffer (promega), . ,ul of rnasin ( u, promega) and . ,ul of m-mlv reverse transcriptase ( u, promega). samples were incubated at °c for min and then at °c for min, before cooling to °c. pcr was in ,ul volumes, using ~ of x buffer ( mm kcl, mm tris-cl ph . , mm mgcl,, . % gelatin), ~ dntps ( mm), ~ triton xl (lo%), ~ cdna, . ~ of each primer ( pm) and . ~ of taq polymerase ( . u, promega). the temperature profile was cycles of s at °c min at °c and min at °c. there was then a final extension time of min at °c. a ~ aliquot of each pcr product was visualised by agarose gel electrophoresis ( % agarose, volts for min, . pug/ml ethidium bromide included in gel) and subsequent u.v. transillumination. an rt-pcr product of the size expected for tgev or prcv was amplified from total rna extracted from cells infected with each of the viruses examined in this way (fig. ). the large difference in the size of amplicon for tgev and prcv is obvious. the slightly larger size of amplicon obtained with american prcv isolates, compared to european ones was also evident if the amplicons were run on a polyacrylamide gel (data not shown). (wesley et al., ) *accession numbers at bmo collection of animal pathogenic microorganisms (original strain designation in parenthesis). tcvl, central veterinary laboratory (the authors' laboratory). @not known. the sensitivity of the rt-pcr for virus detection in culture fluids was compared with cell culture virus isolation. virus titres were calculated as % tissue culture infectious doses (tcid,,) by titrating virus stock:s in tenfold steps, and assaying for cell culture infectivity by viral cytopathic effect (cpe) and immunostaining of cultures fixed in % acteone (holnn jensen, ) . immunostaining used a polyclonal antiserum against tgev - , a rabbit anti-porcine peroxidase conjugate (dako) and the substrate -amino -ethyl carbazole. the supernatant fluids from cell cultures infected with tgev strains - and slagharen had cell culture infectivity titres of . and . per ~ , whilst with rt-pcr a visible band was detectable from samples diluted to w and -* respectively. the technique's application to clinical samples was evaluated on specimens obtained following investigation of a pig herd that showed typical signs of epizootic tge (jones and paton, ) . the tge virus isolated from the case was designated - . two faecal samples and three samples of small intestine were obtained from three-week-old pigs with diarrhoea. a sample of intestine was homogenised in pbs and fed to four five-day-old piglets held in isolation and fed on milk replacer. profuse diarrhoea began within h in all pigs and faeces samples were collected over the next h, after which the pigs were euthanased. faeces samples from the field case and the experimentally infected pigs were all tgev positive in a routinely used elisa (paton, ) . the dilution at which tgev could be detected in elisa was between w and e . fat on cryostat sections of intestine, using a commercial source of tgev antiserum (vmrd, inc.), confirmed tgev antigen in two out of three of the field samples and in all four pigs inoculated experimentally. clarified, % homogenates of intestine and faeces from both the field outbreak and the subsequent experimentallyinduced cases were used to inoculate cell cultures for attempted virus isolation as previously described (paton, ) . cell cultures used included secondary pig kidney, llcpkl , secondary canine kidney and a canine rectal tumour cell line (a ). in each case, except for the secondary pig kidney cultures, virus isolation was attempted with and without added trypsin ( pg/ml, with daily medium renewal). the - isolate grew very poorly in the cell types assessed. no cpe was evident in any culture after four blind passages, with or without added trypsin, but immunostaining revealed a small number of infected plaques in cells within the a and llcpkl cultures. tenfold dilution of the inoculum from the third cell culture pass lead to complete loss of detectable infectivity at the fourth passage. for rt-pcr, samples of faeces were diluted in in a disruption buffer containing mm tris-cl, ph . , with % pvp- , % peg , mm nacl, and . % tween (rowhani et al., ) , vortexed and left to stand at room temperature for min. earlier experiments had shown an increase in sensitivity of up to ten-fold, when disruption buffer was used instead of pbs (data not shown). the suspensions were clarified by centrifugation at g for min and ~ of supernatant was used in qiaamp spin-column kits, according to the manufacturer's recommendation. ten per cent intestinal homogenates in pbs plus antibiotics were clarified by centrifugation at g for min. thereafter, ~ aliquots were processed in qiaamp kits as above. all of the faeces samples were positive by rt-pcr, and the same two out of three pigs from the source outbreak for - were positive for tgev using both fat on cryostat sections of intestine and rt-pcr on gut homogenates. to assess the sensitivity of the rt-pcr, three positive faeces samples were diluted in ten fold steps in disruption buffer, prior to extraction of rna. the dilutions at which tgev could be detected in the three samples were lo-', w , and w for rt-pcr, compared to w , e , and e respectively for elisa. to confirm the authenticity of the amplified products restriction endonuclease (re) analysis was assessed using each of three enzymes (hueiii, hinji and suu ai) for tgev and the enzyme sau ai for prcv. these enzymes were predicted to cut the amplified region of the purdue strain of tgev. the dna was extracted from a -~ aliquot of each pcr reaction using wizard prep columns (promega) and a one tenth volume of this was digested for h in a -~ volume using the manufacturer's recommended digestion buffer (promega). half of the digest was then examined by agarose electrop:horesis, as above, except using % high resolution agarose (metaphor, flowgen) and electrophoresis at volts for min. the restriction enzyme ayaeiii produced the predicted cut into two fragments of approximately and bp for all tgelv isolates (fig. ) . the other enzymes did not g:ve a consistent pattern (data not shown). hinjl gave extra bands with two isolates ( - and - ) , whilst sau ai gave extra bands with - and did not cut - , nor the prcv isolate indiana . isolation of tgev in cell cultures is a slow and unreliable diagnostic method. the process may require multiple blind passages to be made before characteristic cpe is evident, although this period may be somewhat shortened by immunostaining of the cell monolayer with tgev-specific reagents. it has been observed that some isolates are more readily adapted to cell culture than others and in a large proportion of cases, success is very elusive (vaughn and paul, tious virus was present. attempts to improve yield by trypsin treatment, using a method modified from that of honda et al. ( ) had no beneficial effect. to avoid the difficulties of virus isolation in routine diagnosis, immunoassays such as fat and elisa have been developed for the detection of tgev antigens in gut sections or faeces (pensaert et al., ; bernard et al., ; van nieuwstadt et al., ) . however, in many european countries, tgev has been either absent or present at very low levels in the last ten years. in such circumstances, a positive diagnosis of tgev becomes more significant, and it may be necessary to confirm diagnoses made by antigen detection tests using an independent assay procedure. serology is an option, but suffers from two disadvantages. firstly, rapid diagnosis may not be possible, due to the need to allow time for convalescence and antibody development. secondly, it usually requires a repeat visit to the farm to be made, for the appropriate samples to be collected. there are reports of the development of nucleic acid probes for detection of tgev rna (bae et al., ; vaughn et al., ; wesley et al., ) , but the methods did not describe direct use on clinical samples and may lack sensitivity for such work. it would be desirable to assess the rt-pcr method on a larger number of clinical samples, including ones from older pigs. from a small number of titrations, the rt-pcr method appeared more sensitive than elisa, but slightly less sensitive than virus isolations. the sensitivity of the rt-pcr may have been reduced by using only two of ~ of extracted rna and again only two of ~ of the rt reaction mix. nevertheless, a recent field isolate ( - ) that was not readily grown in cell culture was detected with a strong rt-pcr signal, using faeces or intestine submitted from the field case. another tgev isolate ( - ) could not be recovered in cell culture from an archived frozen stock of liquid culture supernatant, but an rt-pcr product was amplified. furthermore, sequencing revealed that the amplicons obtained from each of these two viruses were unique (data not shown). differentiation of transmissible gastroenteritis virus from porcine respiratory coronavirus and other antigenically related coronaviruses by using cdna probes specific for the ' region of the s glycoprotein gene detection of transmissible gastroenteritis coronavirus antigens by a sandwich enzyme-linked immunosorbent assay technique antigenic differentiation between transmissible gastroenteritis of swine and a related porcine respiratory coronavirus a cytopathic virus causing a transmissible gastroenteritis in swine. i. isolation and properties identification of epitopes of immunological importance on the peplomer of porcine transmissible gastroenteritis virus differentiation of porcine coronavirus from transmissible gastroenteritis virus cytopathogenicity of transmissible gastroenteritis virus in pigs detection of antibodies against hog cholera virus and bovine viral diarrhoea virus in porcine serum. a comparative examination using cf, pla and npla assays the multiplication of transmissible gastroenteritis viruses in several cell lines originated from porcine kidney and effects of trypsin on the growth of the viruses molecular differentiation of transmissible gastroenteritis virus and porcine respiratory coronavirus strains classical transmissible gastroenteritis returns porcine respiratory coronavirus: molecular features and virus-host interactions pathogenicity of experimental infection with "pneumotropic" porcine coronavirus transmissible gastroenteritis diagnosis of transmissible gastroenteritis in pigs by means of immunofluorescence isolation of a porcine respiratory, non-enteric coronavirus related to transmissible gastroenteritis development of a detection system for woody plants based on pcr analysis of immobilized virions transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has a sialic acid (n-glycolylneuramic acid) binding activity in situ hybridization technique for the detection of swine enteric and respiratory coronaviruses, transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus (prcv), in formalin-fixed paraffin-embedded tissues region-specific initiation of mouse mammary tumour virus rna synthesis by endogenous rna polymerase ii in preparations of cell nuclei the isolation of the cytopathogenous strains of the originator of transmissible gastroenteritis of pigs in tissue cultures comparison of the antibody response to transmissible gastroenteritis virus and porcine respiratory coronavirus, using monoclonal antibodies to antigenic sites a and x of the s glycoprotein comparison of two methods for detection of transmissible gastroenteritis virus in feces of pigs with experimentally induced infection antigenic and biological diversity among transmissible gastroenetritis virus isolates of swine three new isolates of porcine respiratory coronavirus with various pathogenicities and qike (s) gene deletions use of non-radioactive dna probes to differentiate porcine respiratory coronavirus and transmissible gastroenteritis virus isolates evidence for a porcine respiratory coronavirus, antigenitally similar to transmissible gastroenteritis virus, in the united states differentiation between transmissible gastroenteritis virus and porcine respiratory using a cdna probe we are very grateful to all those who supplied us with viruses for use in this study. viruses were given by l. valicek, brno, czech republic; a. van nieuwstadt, lelystad, the netherlands; k. van reeth, gent, belgium and m. frey, ames, usa. our thanks to g. wibberley for tge antigen elisa testing, and to p. lowings for helpful discussions. this work was supported by the ministry of agriculture fisheries and food, uk. key: cord- -juj nc authors: pulford, david j.; britton, paul title: intracellular processing of the porcine coronavirus transmissible gastroenteritis virus spike protein expressed by recombinant vaccinia virus date: - - journal: virology doi: . / - ( ) -k sha: doc_id: cord_uid: juj nc abstract the spike (s) protein from a virulent british field isolate of porcine transmissible gastroenteritis virus (tgev) fs / was constructed from cdna and inserted into the vaccinia virus (vv) thymidine kinase gene locus under the control of the vv early/late gene p . k promoter. recombinant s protein was synthesized as an endo-β-n-acetylglucosamini-dase h (endo h)-sensitive glycoprotein with high mannose simple oligosaccharides (gp ) that underwent post-translational modification to an endo h-resistant glycoprotein with complex oligosaccharides (gp ). immunofluorescence analysis demonstrated that the majority of recombinant s protein was retained at the golgi but some s protein was expressed on the plasma membrane. monoclonal antibodies (mabs) raised against native s protein reacted with this recombinant s protein; also, mice infected with the recombinant vaccinia virus (rvv) expressing the s protein induced tgev neutralizing antibodies. a truncated s protein (sΔ) was also expressed in rvv-infected cells by introducing a deletion into the s protein cdna that removed amino acids from the c-terminus. the sΔ protein (gpl ) was shown to be antigenically similar to tgev s protein by immunofluorescence and immunoprecipitation tests but was retained in the endoplasmic reticulum and not expressed on the cell surface. transmissible gastroenteritis virus (tgev) belongs to the family coronaviridae, a large group of enveloped viruses with a positive-stranded rna genome. the virus causes gastroenteritis in neonatal pigs, resulting in a high mortality and morbidity. tgevvirions are composed of three structural proteins; a basic phosphorylated nucleoprotein (n) m, , was shown to associate with the viral genomic rna to form the nucleocapsid and interact with a glycosylated membrane protein (m) observed as a series of polypeptides n/r, - , , and the peplomer or spike (s), a surface h/l, , glycoprotein (garwes and pocock, ) . the tgev s protein has been shown to elicit a neutralizing antibody response (laude et a/., ; jimenez et al., ; gatwes et a/., ) capable of conferring some protection to suckling pigs (garwes eta/., / ) . by analogy to the s protein of mouse hepatitis virus (mhv), the tgev s protein may possess the cell receptor binding components (collins et a/., ) and virulence determinants of the virus (fleming et al., ) . the s protein of tgev and coronaviruses antigenically related to tgev such as feline infectious peritonitis ' present address: department of pathology and microbiology, school of medical sciences, university of bristol, university walk, bristol, ltd, uk. *to whom all correspondence and reprint requests should be addressed. virus (fipv), canine coronavirus, and porcine respiratory coronavirus, are not cleaved (for a review see spaan et al., ) . however, the s proteins from mhv, infectious bronchitis virus, bovine coronavirus, human coronavirus (oc and e), and porcine hemagglutinating encephalomyelitis virus which are not antigenically related to tgev are proteolytically cleaved into two subunits (spaan et a/., ) . some coronavirus s proteins have been demonstrated to induce cell fusion (collins et a/., ; sturman et al., ; de groot eta/., ) , generating multinucleated syncytia. the complete amino acid sequences for the s proteins of the virulent fs / strain ) and the avirulent purdue strain (jacobs et al., ; rasschaert and laude, ) have been published and compared with other coronaviruses. the fs / strain s protein has a -amino acid precursor polypeptide with potential n-linked glycosylation sites and % sequence homology at the nucleotide and the amino acid level with the purdue strain. from the deduced amino acid sequence the coronavirus s protein was shown to contain characteristic features: a -amino acid cleavable secretory signal; a heptad repeat sequence that forms a-helical structures which may interact with other subunits to form a coiled-coil oligomeric structure ; a hydrophobic sequence near the c-terminus that is probably responsible for anchoring the s protein to the virion envelope. this membrane anchor region is imme-diately followed by a cysteine-rich domain, a feature common to all other coronaviruses, that may stabilize protein-lipid interactions. in this paper we report the construction of a fs / cdna s gene and its expression by a recombinant vaccina virus (rvv) to study the antigenicity and cellular localization of the s protein. the role of the membrane anchor domain was investigated by the introduction of a c-terminal gene deletion downstream of the heptad repeat sequence. tgev, strain fs / , was cultivated in a porcine continuous cell line (llc-pkl) maintained with medium containing pg ml-' trypsin (hofmann and wyler, ) . cv- and human thymidine kinase negative (htk-) cells were grown in eagles mem medium (flow labs) containing % heat-inactivated fetal calf serum. transfections were performed on subconfluent monolayers of cv- cells previously infected with wild-type vaccinia virus (vv) (wr strain) using plasmid dna calcium phosphate precipitates (mackett et a/., ) . recombinant vaccinia viruses were cultivated in htk-cells in the presence of pg ml-' -bromodeoxyuridine (budr; sigma) as described by mackett et al. ( ) . of the tgev s and sa genes from cdna cloning procedures were as described by maniatis et a/. ( ) . enzymes were used according to manufacturers' instructions (new england biolabs, bethesda research labs). plasmids ptg , ptg , and ptg were used to reconstruct a dna copy of the tgev s gene; the procedure is outlined in fig. . essentially barnhi linkers (pcggatccg, no. biolabs) were added to the hoal site known to be bp upstream of the s gene initiation codon ) and the . kb hindill fragment was cloned into puc and excised as a . -kb xbal-barnhi fragment. the barnhi-sty fragment derived from ptg , the styl-kpnl fragment derived from ptg , the kpnl-xbal fragment derived from ptg , the . -kb xbal-barnhi fragment, and barnhi dephosphorylated pbr were mixed, ligated in a five-way reaction mixture, and transformed into dhl escherichia co/i cells. as a result of the different cohesive ends an insert of . kb would correspond only to the complete s gene. an aprtcs transformant was found to contain a plasmid, ppbp , with a . -kbp insert in pbr , corresponding to the tgev s barnhi gene cassette. the reconstructed gene was verified by sequencing the junction regions. the tgev cdna s gene was subcloned from ppbp and inserted into the barnhi cloning site of the vv plasmid insertion vector pgs (mackett et al., ) downstream of the w early/late p , k promoter. the recombinant plasmid, pgsp- , containing a correctly orientated s gene, was identified by restriction endonuclease digestion with sall. the s protein with a c-terminal deletion was generated by cloning a . -kbp sali fragment from pgsp- into the sali site of pucl . restriction endonuclease digestion of recombinant plasmids produced either a . -or . -kbp barnhi fragment and the latter was recloned back into barnhi-digested pgs . the recombinant plasmid insertion vector pgspa- was found to contain a correctly orientated sa gene by sryl restriction endonuclease digestion of plasmid dna. a synthetic oligonucleotide primer '-gtgtgcggctac-tataacta- ', that binds to the complementary dna strand bp downstream of the barnhi cloning site in pgs , verified the position of the sa protein stop codon in pgspa- by sequencing ( fig. a) . the rvvs, vts- and vtsa- , were generated with pgsp- and pgspa- using methods described by mackett et al. ( ) . thymidine kinase negative viruses were screened by dna dot blot and dna from vts- and vtsa- infected cells was analyzed by southern blot using a [ p]-labeled s gene cdna probe as described in . groups of three female balb/c mice were immunized by intraperitoneal (ip) inoculation with l- x o pfu/ mouse of partially purified recombinant or wildtype vv as described by . after weeks, one animal from each group was sacrificed to obtain convalescent serum, the remaining animals were hyperimmunized with homologous virus by ip inoculation and bled after a further weeks. mice initially inoculated with either phosphate-buffered saline (pbs) or wr strain vv were inoculated after weeks with a dose of vts- to establish if age or a previous w infection affected the stimulation of tgev neutralizing antibody. tgev neutralizing antibody was assessed by plaque reduction assay as described by garwes et a/. ( ) . viral proteins were routinely radiolabeled by incubating tgev-infected llc-pkl cells or rvv-infected htk-cells, at hr p.i. or hr p.i., respectively, in methionine-free medium for hr and then in the presence of -l &i ml-' l-[ s]methionine (amersham international; see figure legends for further details). after pulse labeling, cells were washed in pbs ( mm potassium phosphate, m/l/l naci, ph . ) and chased in medium supplemented with mm l-methionine. cells were washed in pbs before treating with test lysis buffer ( mm tris-hci, ph . , mm edta, mm naci, % triton x-l , . % aprotinin). nuclei and ceil debris were pelleted from infected cell lysates by centrifugation at , g using a beckman tla- ultracentrifuge. tissue culture medium was clarified by low speed centrifugation. immunoprecipitations were performed by mixing vol of cell lysate with l/l th vol of porcine tgev hyperimmune antiserum at ' for hr and the resulting immune complexes were incubated overnight with formalinfixed staphy/ococcus aureus cells (sac; brl) previously washed three times in test buffer. immune complexes were washed three times with test buffer and resuspended in laemmli sample buffer, and proteins separated on % sds-polyacrylamide gels (laemmli et a/., ) . c-methylated proteins (amersham, code cfa. ) were routinely used as molecular weight markers. proteins were detected by fluorography after immersing gels in . m sodium salicylate for min. pelleted immune complexes were washed as described above, resuspended in ~i mll/l tris-hci, ph . , containing . % sds, and boiled for min. the solubilized proteins were incubated in the presence or absence of mu endo h (boehringer-mannheim) in mmsodium citrate, ph . , at " for hr. the proteins were analyzed on % polyacrylamide gels and detected by fluorography. glass coverslips with subconfluent monolayer cultures of htk-or llc-pkl cells were infected with tgev and wild-type or rvvs. at hr p.i. cells were either fixed with cold % acetone and air dried or washed in cold pbs and maintained at " for surface staining. cells were incubated for hr with porcine tgev hyperimmune antiserum diluted :loo in pbs and then extensively washed with pbs. cells were then incubated for min with fluorescein isothiocyanate-conjugated rabbit anti-pig immunoglobulin g (nor-die immunology) diluted : in pbs. coverslips were washed, air dried, and mounted on glass slides with % glycerol. fluorescent cells were observed and photographed with a leitz wetzlar uv microscope. a . -kbp barnhi s gene cassette was constructed from tgev fs / cdna and used to generate ppbp (fig. ) . the -bp s gene was capable of encoding a precursor polypeptide of amino acids with a m, , . after cleavage of the n-terminal signal peptide, shown to be absent in virion-associated purdue strain s protein (rasschaert and laude, ) the protein would consist of amino acid residues with a ai, , . the s gene barnhi gene cassette was subcloned into the vv insetion vector pgs to give pgsp- as described under materials and methods. the sa gene, contained in pgspa- , was bp long, capable of encoding a -amino acid polypeptide, corresponding to a deletion of residues from the complete s protein and also included eight amino acid residues derived from the pucl polylinker and pgs sequences. the sa gene terminated in a new tga stop codon contained within the vv tk sequences (fig. ) . the predicted size of the precursor sa polypeptide was m, , , which when modified by the removal of the -amino acid n-terminal signal peptide was reduced to m, , . the c-terminal deletion also resulted in removal of eight potential nlinked glycosylation sites predicted for the s protein. the s gene initiation codon was bp away from the vv early promoter rna start site for both pgsp- and pgspa- insertion vectors. the tgev s genes were inserted into the vv genome by homologous recombination into the tk locus, and southern blot analysis was used to confirm that the resulting rvvs vts- and vtsa- contained the . -and . -kbp tgev barnhi gene fragment, respectively. two plaque-purified rvv clones, vts- and vts- , were used to immunize balb/c mice, and the level of tgev neutralizing antibodies was measured by a plaque reduction assay (table ). both recombinant virus clones induced tgev neutralizing antibodies that were boosted with a second inoculation. mice previously inoculated with the wild-type vv and inoculated with vts- produced a -fold lower level of tgev neutralizing antibody compared to the convalescent serum of vts- . a primary infection with wild-type vv could have stimulated the mouse immune system fig. . construction of the tgev s gene from the fs / cdna. the complete tgev s gene was generated by a four-way ligation into pbr . the thin lines represent tgev cdna and the thick lines vector dna sequence. the top line represents the three cdna clones used to generate specific cdna fragments. if more than one enzyme was used they are listed in a descending order next to the appropriate arrow. the . -bp hpal-pstl fragment from ptg had barnhi linkers added and was then digested with barnhi and sty to generate a . .kb fragment with a barnhi site upstream of the s gene initiation codon. the . .kb hindill-hindill fragment from ptg was initially cloned into puc and removed as a xbal-barnhi fragment, with the barnhi site derived from the puc polylinker sequence, to provide a barnhi site within the tgev orf- a gene 'to the end of the s gene. the relevant fragments were then ligated together with pbr such that only the correct alignment of the cohesive ends would give a fragment of . kb in pbr corresponding to the tgev s gene bamhl cassette. into producing a rapid clearance of a subsequent rw infection and prevented the expression of large amounts of s protein. mice given a single inoculation with vts- weeks after the other mouse group had a fourfold reduction in the level of tgev neutralizing antibody. this suggested that age may have a significant effect on the induction of tgev neutralizing antibodies, but further animal studies are needed to make any firm conclusions. expression of the recombinant spike antigens tgev gene products synthesized by vts- and vtsa- were analyzed by pulse labeling rvv-infected htk-cells with l-[ s]methionine in the presence or absence of pg ml-' tunicamycin for hr. tgev s protein was immunoprecipitated from cell lysates or tissue culture fluids using porcine tgev hyperimmune antiserum. this resulted in s polypeptides of aj , (gpl ) and aj , (gp ) being immunoprecipitated from vts- -infected cell lysates in the absence of tunicamycin (fig. , lane ) while in the presence of tunicamycin, a aa, , (~ ) precursor s polypeptide was synthesized (fig. , lane ) . cells infected with vtsa- expressed a m, , glycosylated polypeptide (gpl ) only (fig. , lane ) that was expressed as a aa, , (~ ) precursor protein (fig. , lane ) in the presence of tunicamycin. it was noted that the levels of s protein expressed were appreciably less in the presence of tunicamycin (fig. ) . comparison of the nonspecifically precipitated proteins in the presence or absence of tunicamycin (fig. ) indicated that the reduction in s protein synthesis was not due to inhibition of viral replication in the presence of tunicamycin. these observations suggested therefore that either glycosylation was required for efficient synthesis of the s protein or the majority of the anti-s antibodies were directed against the glycosylated form of the protein resulting in less efficient immunoprecipitation of the nonglycosylated form. although gp was immunoprecipitated from the vts- -infected cell culture medium in the absence of tunicamycin (fig. , lane ) no detectable extracellular sa protein (gpl ) was recovered from medium of vtsa-l-infected cells (fig. , lane ) suggesting that the membrane anchor is required for export out of cells. the lack of detectable ~ or ~ in the culture medium of vts-l-and vtsa-l-infected cells, respectively (fig. lanes and ) , could result from the levels being too low to be detected by the antibodies due to reduction in synthesis or the lack of glycosylation of the s protein in the presence of tunicamycin. the rate of s protein intracellular transport was compared by measuring the acquisition of endo h resis- note. tgev neutralizing antibody was titrated by a % plaque reduction assay and expressed as the reciprocal of the antiserum dilution. tance in tgev-, vts-i-, and vtsa- -infected cells. after a i-hr pulse, the tgev s protein consisted of a major gp component and a minor gp component (fig. a) . after a -hr chase, gpl and gp were present in approximately equal proportions but after a -hr chase, gp was the dominant s protein component and the majority was endo h resistant. tgev s protein steadily became endo h resistant with time such that after a -hr chase virtually all s protein had been modified in the golgi. s protein was also detected in the tissue culture medium of tgev-infected cells after a -hr chase and accumulated steadily with time. digestion of gp from extracellular or intracellular sources with endo h reduced the size of the s protein to m, , . this observation has also been made for the fipv s protein from fipv-infected feline cells (vennema eta/., ) and suggested that coronavirus s proteins still have high mannose or hybrid oligosaccharide structures following golgi processing. the recombinant s protein was completely endo h sensitive after a -hr pulse and after chasing became partially endo h resistant with approximately half of the protein observed as unresolved components of high molecular weight (fig. b ). extracellular s protein was not detected in the culture medium after chasing for hr because s protein transport from the rer through the golgi stack and out of the cell was significantly disrupted in the absence of coronaviral morphogenesis. infection of llc-pkl cells with vts- produced a discrete mr , endo h-resistant form of s protein (data not shown). this was in contrast to the partial endo h-resistant forms observed in vts- -infected htk-cells, implying that the tgev s protein may be post-translationally modified to a different extent depending on the origin of the cell line. the sa protein remained completely endo h sensitive, even after a -hr chase, demonstrating that the truncated s protein was glycosylated with high mannose oligosaccharides only and was not subject to golgi-specific modifications (fig. b ). the cellular location of the s and sa protein in rvvinfected cells was analyzed by indirect immunofluorescence microscopy (fig. ) . acetone-fixed tgev-infected llc-pkl cells, probed with anti-s-specific serum, had a granular appearance with occasional bright accumulations in localized parts of the cell (data not shown). in vts-l-infected htk- (fig. a ) and llc-pkl (fig. c) an intracellular compartment in all infected cells (represented with arrows), while vtsa- -infected cells had a uniform distribution of sa protein throughout the cytoplasm ( fig. b and d ). recombinant s protein was also observed on the cell surface of unfixed infected htk-cells (fig. e ), unlike sa protein (fig. f) , demonstrating that the full-length protein has all the intrinsic properties for cell surface transport and does not require the cooperative effect of other tgev proteins. the possibility that soluble s protein released from human cells may bind back to cell surface receptors and give the appearance of cell surface fluorescence can be disregarded as tgev s protein only binds porcine cell surface receptors. studies described in this paper with tgev recombinant s protein have shown that it is an n-linked glyco-protein with a m, - , . mice inoculated with two separate clones of vts demonstrated that the recombinant s protein was immunogenic and elicited tgev neutralizing antibodies. this was in contrast to previous studies with tgev n and m proteins expressed by rvvs that induced an immune response but no neutralizing antibodies . the tgev s protein neutralizing epitopes and major antigenic sites were retained by the sa protein but this protein was not transported to the plasma membrane or through the golgi stack and must therefore be accumulated in the endoplasmic reticulum. hu et al. ( ) constructed an incomplete tgev s protein gene that initiated bp downstream of a hpal site but ended bp upstream of a xbal site. this gene construct expressed an endo h sensitive m, , polypeptide that was not transported to the cell surface in rvv-infected cells (hu et al., ) . sequence analysis of the tgev purdue-l (jacobs et al., ; rasschaet-t et al., ) and fs / strains demonstrated that the xbal site was bp from the s protein initiation codon and within an orf of bp, implying that the s gene product expressed by hu et a/. ( ) was a truncated protein. coronavirus s proteins are known to be highly glycosylated with n-linked oligosaccharides and undergo modification with complex sugars at the medial compartment of the golgi stack during virus maturation (niemann et al., ) . simple high mannose and hybrid structures can be removed from glycoproteins by digestion with endo h but glycoproteins modified with complex sugar structures are resistant to cleavage with endo h (hubbard and ivatt, ; dunphy and rothman, ) . the rvv vts- produced two s protein species in human cells with different oligosaccharide composition, assigned gpl and gp , while the sa protein was expressed in human cells as a single glycoprotein species designated gp . the gp was partially resistant to endo h, suggesting that gpl was modified to gp by the addition of complex oligosaccharides at the golgi. the sa protein remained endo h sensitive even after extensive chasing, implying that it was not transported to the golgi stack. pulse-chase analysis of tgev-or vts- -infected cells demonstrated that s protein was initially synthesized as gpl but gradually accumulated as gp due to post-translational modifications. endo h digestion of recombinant s protein expressed in htk-produced a range (n/l, - , ) of partially resistant polypeptides. both of these observations were made for the fipv ( -l ) s protein expressed in a bovine papilloma virus transformed mouse cell line (de groot et a/., ) . however, the tgev s gene expressed by a rvv in porcine llc-pkl cells produced an endo h-resistant gp product with no partially resistant endo h intermediates. a similar observation was also made for the fipv s gene expressed by a rw in feline cells (vennema et al., ) , suggesting that expression of coronavirus s proteins in a cell line compatible with their origin may have profound effects upon the extent of post-translational processing of these proteins. the acquisition of endo h resistance was significantly retarded for recombinant s protein compared to s protein expressed during a tgev infection. retardation of coronavirus s protein intracellular transport has also been observed for the fipv and mhv s proteins expressed by rvvs (vennema et al., ) . in this study, retardation between the rer and the golgi in the absence of virus budding was interpreted as a transient accumulation of the s protein at or near the site of virus budding and suggested that the s protein may have a role in defining the site of virus budding, a func-tion previously ascribed to the m proteins of coronaviruses (booze et al., ) . lmmunofluorescence studies with vts-infected cells demonstrated that recombinant s protein accumulated in a polar perinuclear compartment of the cell, a similar observation was seen for the cellular localization of rvv expressed tgev m protein (pulford and britton, ) and at the cell surface. the presence of tgev gp in the culture medium of vts-infected cells suggested that gp may be released from the cell. proteins can be transported from the rer to the golgi by interacting with carrier proteins or following membrane incorporation, by the flow of membrane from the er to the cell surface. this membrane flow may be responsible for the cell surface presence of coronavirus s proteins. the sa protein was not processed in the golgi apparatus, nor found on the cell surface nor found to be exported into the extracellular medium, indicating that the c-terminal membrane anchor domain of tgev s protein may be required for all of these functions. puddington eta/. ( ) used a mutant vsv g protein to establish that the cytoplasmic tail of the g protein was essential for its transport through the golgi stack. in addition, bray eta/. ( ) used rws to demonstrate that a dengue virus envelope protein truncated at the c-terminus, unlike the wild-type protein, was not secreted into the extracellular medium. however, sveda et a/. ( ) found that c-terminal sequences of the influenza virus hemagglutinin (ha) were essential for cell surface expression and that mutants with a disrupted anchor domain were secreted, suggesting that influenza virus ha, unlike tgev, vsv, and dengue virus envelope proteins, underwent a different protein maturation pathway. evidence suggests that unless a membrane glycoprotein folds into a native or near-native conformation it will not be exported from the rer (lodish, ) . however, the general conformation and antigenicity of the sa protein appeared to be retained as it reacted with both polyclonal and monoclonal tgev s antisera in contrast to denatured s protein or s protein expressed in e. co/i cells (correa et a/., ; delmas et a/., ; pulford, unpublished observations) . the results presented in this paper suggest that the sa protein was retained in the rer possibly due to the removal of essential c-terminal transport signals. we would like to thank miss k. mawditt for synthesizing the 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assembly key: cord- -jb u xn authors: gerdts, volker; zakhartchouk, alexander title: vaccines for porcine epidemic diarrhea virus and other swine coronaviruses date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: jb u xn the recent introduction of the porcine epidemic diarrhea virus (pedv) into the north american swine herd has highlighted again the need for effective vaccines for swine coronaviruses. while vaccines for transmissible gastroenteritis virus (tgev) have been available to producers around the world for a long time, effective vaccines for pedv and deltacoronaviruses were only recently developed or are still in development. here, we review existing vaccine technologies for swine coronaviruses and highlight promising technologies which may help to control these important viruses in the future. coronaviruses were first described in the mid- s and subsequently isolated from a number of species including man, mice, swine and chicken. these viruses share a common morphological characteristic, a fringe of club-shaped projections, - nm long, around a pleomorphic - nm viral particle, having a resemblance to a solar corona (masters, ) . coronaviruses infect humans and various animal species, causing respiratory, gastrointestinal and neurological diseases as well as hepatitis. prominent examples include the severe acute respiratory syndrome virus (sars-cov), middle-eastern respiratory syndrome virus (mers-cov) and the feline infectious peritonitis virus (fipv), to name a few. swine coronaviruses can be divided into respiratory (prcov) and enteropathogenic coronaviruses such as transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv) and porcine deltacoronavirus (pdcov). the latter have similar epidemiological, clinical and pathological features. the family coronaviridae is currently divided into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus. tgev and pedv belong to the alphacoronavirus genus, whereas pdcov belongs to genus of deltacoronaviruses. coronaviruses are enveloped, single-stranded, positive-sense rna viruses with the largest rna genome of approximately kb reported to date. the genomic rna includes and untranslated regions (utr), and it is capped and polyadenylated. open reading frame (orf) a and orf ab occupy the two-thirds of the genome and encode two replicase polyproteins (pp a and pp ab). expression of pp ab protein requires a ribosomal frameshift during translation of the genomic rna. produced polyproteins are proteolytically cleaved into nonstructural proteins, nsp through nsp by the proteinase activity of nsp and nsp . the -proximal one-third of the genome encodes four structural proteins, including spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins. some betacoronaviruses have an additional membrane protein, hemagglutinin esterase (he). interspersed between these genes are genes encoding accessory proteins. the number of these genes varies between different coronaviruses. for instance, tgev has accessory genes, pdcov has , whereas pedv has only one (fig. ) . the viral rna genome is packaged by the n protein into a helical nucleocapsid. in addition to the structural role, the n protein prolongs s-phase cell cycle, induces endoplasmic reticulum stress, up-regulates interleukine- expression and antagonizes type i interferon production (ding et al., ; xu et al., b) . the s protein, which forms peplomers on the virion surface, mediates binding to host receptors and membrane fusion. it can be divided into s and s domains. in some coronaviruses the s protein is processed into s and s fragments by cellular proteases or trypsin (belouzard et al., ; wicht et al., ) . the s protein is a major target for virus neutralizing antibodies (chang et al., ; reguera et al., ) . the m protein is the most abundant virion component and also contains conserved linear b-cell epitopes (zhang et al., ) . the e protein is responsible for the assembly of virion, and it causes endoplasmic reticulum stress and interleukin- expression up-regulation (xu et al., a) . accessory genes are dispensable for virus growth in vitro, but they may play an important role in the virus survival in the infected host. indeed, the product of the tgev accessory gene orf reduces the expression of genes involved in the antiviral defense of the immune system, e.g. the interferon response, and inflammation (cruz et al., ) . the orf protein of pedv functions as an ion channel, and it is thought to be related with virulence of pedv (song et al., ; wang et al., ) . one of the pedv non-structural proteins, nsp , was shown to be a type i interferon suppressor (zhang et al., a) . interestingly, pdcov lacks the nsp gene. coronaviruses target predominantly type i and ii pneumocytes (prcov) or villous-and crypt enterocytes in the intestine (tgev, pedv and pdcov). pedv also infection goblet cells in the small intestine (jung and saif, ) . in addition, infection of alveolar macrophages and lamina propria macrophages has been shown for some but not all swine coronaviruses (laude et al., ; park and shin, ) . entrance of the virus into the target cells is mediated by a series of receptor ligand interactions including heparin sulfate (huan et al., ) and aminopeptidase n (apn) (chen et al., ; li et al., ) . importantly, the expression levels of aminopeptidase n appear to correlate with the level of infection, at least for pedv. the higher the expression levels the more severe is the infection (li et al., ; zhang and yoo, ) . as a result, it may be perceivable that piglets born with lower apn levels in the brush border may be more resistant to pedv than piglets with higher levels. enteric infections with tgev and pedv are characterized by severe diarrhea, vomiting and dehydration with high morbidity and mortality especially in piglets less than two weeks of age. in contrast, infections with respiratory coronaviruses cause very mild and transient disease in pigs of all ages, which often get unnoticed by the producer. unless complicated by concurrent infections, prcov infections are only short lasting with temporary phases of coughing and respiratory distress. however, prcov can become a more significant problem during co-infections with other pathogens such as the porcine reproductive and respiratory syndrome virus prrsv (jung et al., ) . infection of enterocytes with pedv results in villous atrophy which can lead to malabsorption, diarrhea and anorexia. within - h post infection, vomiting may occur, which typically does not last longer than - days post infection. diarrhea can be found within to h post infection depending on the dose of the virus and the age of the piglets. diarrhea typically lasts for about - days, but can last longer, and results in severe weight loss that often cannot be made up during the normal production cycle. viral shedding is highest between days - , but can last for days to weeks post infection. surviving piglets start to recover around - days post infection, typically around the same time when proliferation of the crypt epithelium and regeneration of the villi occurs. similarly, tgev infects villous enterocytes and causes disease that clinically is indistinguishable from pedv. mortality rates are highest in young piglets, often reaching about %. in contrast, infection with pdcov causes milder infections in piglets between and weeks of age. diarrhea, vomiting and anorexia can be found in infected animals. in general, infected animals display much milder signs compared to infections with pedv and tgev. the innate immune response to enteric coronaviruses in pigs is characterized by a rapid antiviral response in the intestine, including the release of interferons, nuclear factor kb and other antiviral molecules (chattha et al., ; jung and saif, ; sang et al., ) . pigs can produce three types of interferons (sang et al., ) : type i interferons include well known interferons such as interferon a and b (ifn-a/b) and in pigs are encoded by as many as different genes. the only type ii interferon in pigs is ifn-ɣ. type iii interferons include ifn-l (interleukin ; il- ), ifn-l (il- a), ifn-l (il- b) and ifn-l (kotenko et al., ; park et al., ; prokunina-olsson et al., ; sheppard et al., ; zhang and yoo, ) . their functions are unknown in pigs. especially type i and iii interferons are used by the host to counteract viral infections. in response, most viruses including pedv and tgev have developed strategies to evade and interfere with the interferon response. several viral proteins, including structural and non-structural proteins have been identified for pedv and tgev that can suppress the interferon response. for an excellent review on the evasion of immunity by porcine coronaviruses please see (zhang and yoo, ) . the adaptive immune response to swine enteric coronaviruses is based on secretory antibodies and cytotoxic t cells. these include secretory iga antibodies (siga) that are produced by antibodysecreting cells in the lamina propria of the mucosal tissues and systemic antibodies such as igg and igm are found in serum and interstitial tissues and some isotypes can be transsudated across the mucosal epithelium into the lumen (chattha et al., ; horton and vidarsson, ) . the cellular response to swine coronaviruses is characterized by t helper cells that are supporting the production of antibodies and cytotoxic t cells that are targeting virus infected epithelial cells. in pigs, these are predominantly ɣɗ- cells, most of which can be found within the intraepithelial layer (bonneville et al., ) . the majority of t cell epitopes are located in the spike and nucleoprotein of coronaviruses (channappanavar et al., ; saif, ; sestak et al., ) . additionally, cd t cell epitopes have been found in the membrane protein of the human sars-cov (yang et al., ) in neonatal piglets, the main mechanism of protection is mediated by lactogenic immunity. during lactation, siga, igg and igm are passively transferred to the piglet via colostrum and milk (bohl and saif, ; saif and bohl, , ; salmon et al., ) . colostrum contains predominantly igg, which is transudated from sow serum and absorbed by the piglet within the first - h of life. secretory iga is predominantly found in milk, after transitioning from colostrum to milk around - days of age (langel et al., ) . siga is produced by antibody secreting cells in the mammary gland, and it was shown many years ago by bohl and saif (bohl et al., a ) that these cells migrate from the gut to the mammary gland at the end of pregnancy. this was confirmed by others in a variety of species and chemokines such as ccl and others have been found responsible for recruiting these antibody secreting cells to the mammary gland (bourges et al., ; lazarus et al., ; meurens et al., meurens et al., , wilson and butcher, ) . thus, in order to enhance the level of maternal immunity the oral route seems to be the most obvious choice for vaccinating the sow. indeed, most vaccines for enteric coronaviruses are designed to induce lactogenic immunity by vaccinating the sow, however, most of them are administered via systemic injection. in the absence of effective vaccines for pedv, many producers are currently using a lock-down of the barn combined with feeding back infectious live virus to pregnant sows. however, the duration of immunity often does not extent more than a few years (table ) , depending on the type of vaccine being used with live vaccines typically providing longer lasting immunity. even after feed-back, immunity starts to wane after a relatively short period of time, often even less than a few months. for an excellent review of the role of lactogenic immunity for pedv see (langel et al., ) . in addition to antibodies, the colostrum also contains innate effector molecules such as defensins and antimicrobial peptides, interleukins and cytokines hlavova et al., ; mair et al., ; nechvatalova et al., ; salmon et al., ) . the level of cross-protection is somewhat unclear for coronaviruses. for pedv, goede et al. reported that -day old piglets born to sows that had been infected with a mild strain of pedv seven months previously, were protected against infection with a more virulent strain of pedv (goede et al., ) . in this experiment the sows were challenged with a more virulent pedv virus at day of gestation, and orally re-challenged when the piglets were three days old. none of the sows displayed significant clinical symptoms. the piglets were orally challenged with ml of mucus scrapings of the more virulent pedv. while mortality and morbidity rates varied significantly amongst the piglets in each group, the overall morbidity and mortality was reduced in piglets born to sows that had been pre-exposed to pedv. in the s, tgev was responsible for severe economic losses around the globe. several vaccine technologies were developed and commercialised. by administration to sows, the importance of lactogenic immunity was established (bohl et al., b; saif and bohl, ) . however, with a disappearance of the disease in many parts of the world, fewer vaccines are now commercially available in north america and europe (table ). most current commercial tgev vaccines are live attenuated vaccines that are given to the sow during gestation in order to provide lactogenic immunity to the newborn piglet. these vaccines are often available as bi-or trivalent vaccines combined with rotavirus, pedv and/or escherichia coli. experimental vaccines include novel dna vaccines, vectored vaccines and recombinant vaccines (table ) . for example, the porcine adenovirus was used to deliver the tgev spike protein (tuboly and nagy, ) . yuan et al. used the swine pox virus to express the a epitope of the spike protein (yuan et al., ) . dna plasmids were generated for both pedv and tgev for the development of a dna vaccine (meng et al., ) . recombinant proteins (spike and nucleocapsid) have been extensively evaluated as recombinant vaccine following expression in bacteria, yeast and plants. many of these are being assessed for their potential to mucosal immunity after oral administration. oral delivery has not been demonstrated. the first vaccine for pedv in the us was developed by harrisvaccines tm in iowa, and conditionally licensed in . the vaccine, initially called iped vaccine, was based on a truncated version of the pedv spike gene produced in the sirravax℠ rna particle technology platform , which is based on the a pvek replicon vector derived from the venezuelan equine encephalitis virus. the technology is a propagation defective, single cycle rna particle technology that is believed to target dendritic cells. a longer version of the spike genes was codon optimized and used in the second-generation vaccine called iped plus, which now is commercially available as porcine epidemic diarrhea vaccine, rna (ped rna). the vaccine induced immunity in young pigs after two doses given intramuscularly in a threeweek interval. vaccinated weaned pigs when challenged with homogenized gut tissue from a clinical isolate displayed significantly reduced severity of clinical signs (diarrhea) and reduced viral shedding for the first h (mogler et al., a) . vaccine efficacy was further tested in naïve sows. after three vaccinations at , , and weeks pre-farrowing piglets from both groups were challenged between and days of age with tcid (pedv/co/ ). average litter mortality in the control group was %, while average mortality in the vaccinated groups was % (crawford et al., ) . similar results were found in sows previously exposed to pedv. after oral challenge of piglets within the first week average litter mortality was reduced from % in the control group to % in the vaccinated group. finally, greiner et al. ( ) evaluated the vaccine in sows that had been previously exposed to pedv. vaccination induced higher titers against the s protein in the colostrum of vaccinated sows and reduced overall pig mortality by % (greiner et al., ) . a second vaccine for pedv in the us was developed by zoetis and made commercially available in under a conditional license. the vaccine consists of an inactivated whole virus formulated with an adjuvant. the vaccine was tested in pedv negative sows, which were vaccinated twice, weeks apart, with the vaccine (n = ) or an adjuvant placebo (n = ). the vaccine was safe and immunogenic and induced neutralizing antibodies to both the whole virus and the spike protein . the vaccine was also tested under field conditions in a pedv positive commercial herd. sows (n = ) were vaccinated at -and weeks pre-farrowing, control sows (n = ) received placebo. vaccination resulted in reduction of pre-weaning mortality due to pedv from . % in piglets born to control sows versus . % in piglets born to vaccinated sows. vaccination resulted in about times higher neutralization antibody titers compared to the control group, and an additional . pigs per sows survived . a third vaccine was recently developed by the vaccine and infectious disease organization-intervac in canada. the vaccine is based on inactivated virus formulated with an adjuvant. when administered to seronegative sows and weeks prior to farrowing, high levels of neutralizing antibodies against pedv were found in colostrum and milk, as well as in the serum of piglets born to vaccinated sows. piglets were orally infected at days of life with pfu of pedv isolate co . it was found that % of all piglets (n = ) born to vaccinated sows survived the infection and showed significantly reduced clinical symptoms, weight loss and viral shedding. in contrast, all piglets from unvaccinated sows displayed severe clinical symptoms including weight loss and dehydration, and % of these piglets died within days post infection. these results were confirmed in two additional clinical trials. a large field trial involving > sows was performed in three commercial units in saskatchewan, canada to assess the vaccine in different genetics, health statuses and management systems. the vaccine demonstrated to be completely safe to use; no adverse events including injection site reactions and reproductive complications were observed. vaccine efficacy was evaluated in % of these animals by transporting the pregnant sows a week before farrowing to the vido-intervac high containment facility. following oral challenge at days of age, survival was significantly higher in piglets born to vaccinated sows than those from control sows (berube et al., ) . the assessment of duration of immunity is currently ongoing. in addition, an affinity tagged pedv s protein was expressed in the hek system to be used as subunit vaccine. when administered to pregnant sows the vaccine partially protected newborn piglets against infection with pedv (makadiya et al., ) . since early s, pedv outbreaks have been reported in several asian countries, including china, japan, thailand, taiwan, the philippines, south korea and vietnam. in october , a largescale outbreak of severe pedv was reported in china (wang et al., ) . in late , pedv outbreaks occurred in japan, south korea and taiwan . phylogenetic analysis of pedv fulllength genomic sequences reveals that pedv can be genetically divided into groups: g (classical) and g (field epidemic or pandemic). each group can be further divided into two subgroups: a and b, a and b, respectively. it is also possible that the low to moderate effectiveness of current pedv vaccines are due to genetic differences between vaccine and field epidemic strains (kim et al., ) . for disease control, an inactivated bivalent tgev and pedv vaccine was introduced in china in . in march , a trivalent attenuated vaccine (pedv, tgev and porcine rotavirus) was also approved. all these vaccines were based on the classical cv (g -a) strain that can be grown to high titers in green monkey kidney vero cells. there are no data published on the efficacy of these vaccines. however, de arriba et al. (de arriba et al., ) orally inoculated -day-old conventionally reared piglets with two different doses of the attenuated cv- strain and challenged with the same virulent pedv strain three weeks later. the vaccinated pigs were partially protected against the challenge, and % of the low dose-and % of the high dose-exposed pigs did not shed virus after challenge. since winter of , china experienced severe ped outbreaks with devastating damage to the swine industry. these outbreaks can be explained by re-emergence of new pedv strains. to answer industry demand, chinese researchers have developed a bivalent (pedv and tgev) attenuated vaccine that contained the pedv strain zj (g -b) and a bivalent attenuated vaccine based on the aj strain (g -b). inactivated bivalent vaccine based on the g b strain has also been developed. these vaccines are currently under clinical evaluation (wang et al., ) . in japan, pedv strain p- (g -a) was attenuated after passages in vero cells (sato et al., ) . subsequently, this strain has been employed as an intramuscular (im) live attenuated vaccine (p- v) in japan and south korea. in addition, two south korean virulent pedv strains (sm - and dr- ) were attenuated by serial cell culture passages. the attenuated sm - strain has been used as an im live or killed vaccine, whereas dr- was used as an oral live vaccine. this oral vaccine was registered and commercialised in the philippines in . in south korea, the multiple dose vaccination program ( or im vaccinations in the following order: live-killed-killed or live-live-killed-killed, respectively) at -or -week intervals starting before farrowing is commonly recommended in pregnant sows (lee, ) . according to a south korean study, the administration of commercial vaccines increased the survival rate of piglets challenged with a virulent wild-type pedv from . % to over %. however, all vaccines did not significantly reduce morbidity rate and virus shedding (lee, ) . also, song et al. (song et al., ) reported % reduction of the mortality rate of the suckling piglets born to the sows that were im vaccinated with dr- pedv vaccine. despite of nationwide use of commercially available vaccines, south korea experienced a devastated ped epidemic in - . pedv g -b strains were responsible for recent severe ped epidemics in asian countries and north america. considering this fact, south korean researchers (baek et al., ) tested an inactivated vaccine based on serially cultured g -b strain kor/ knu- / . pregnant sows were immunized im with the inactivated adjuvanted vaccine at and weeks prior to farrowing. six-day old piglets were challenged with the homologous virulent virus. piglets born to vaccinated sows had reduced morbidity, mortality and quickly recovered daily weight gain. further studies are needed to evaluate efficacy of this vaccine in -or -day old piglets under field conditions. all commercially available vaccines that are in use in asia are traditional live attenuated or killed vaccines. however, researchers are working on the next-generation vaccines for pedv (table ) . for instance, hou et al. (hou et al., ) expressed the pedv n protein on the surface of lactobacillus. oral and intranasal inoculations of recombinant l. casei into pregnant sow and mice resulted in high levels of n-specific serum igg and mucosal iga. similarly, liu et al. (liu et al., ) expressed s and n protein in recombinant l. casei and reported enhanced mucosal and systemic immune responses after oral immunization of mice. meng et al. (meng et al., ) evaluated the immunogenicity of recombinant dna plasmids expressing s genes from pedv and tgev in mice. the results showed that the recombinant dna plasmids increased the proliferation of t lymphocytes and the number of cd + and cd + t lymphocytes. in addition, the dna vaccines induced a high level of ifn-g in the immunized mice. at days post-immunization, the recombinant dna plasmids bearing full-length s genes of tgev and pedv stimulated high levels of virus-neutralizing antibodies. zhang et al. (zhang et al., b) have also constructed bivalent dna vaccine co-expressing s genes of tgev and pedv. attenuated salmonella typhimurium was used for oral delivery this vaccine into pigs. vaccinated pigs developed tgev and pedvspecific cellular and humoral immune responses; however, challenge experiment was not conducted in this study. there have also been reports on expressing recombinant pedv s protein fragments in plants (bae et al., ) and in a cell line (oh et al., ) . feeding mice with transgenic tobacco plants that express the s protein fragment containing the pedv neutralizing epitope induced pedv-specific antibody and cell-mediated immune responses. in another study, oh et al. (oh et al., ) established porcine cell line stably expressing s fragment of the pedv spike protein. pregnant sows were immunized im times with the purified and adjuvanted protein , and weeks prior to farrowing. the sows developed pedv-specific neutralizing antibody response in serum and colostrum. one -to -day-old piglet was selected randomly from each farrowing sow for challenge with virulent pedv. the piglets born to vaccinated sows showed reduced morbidity, mortality and virus shedding. however, a low number of challenged piglets hamper the author's conclusion about efficient protection of neonatal piglets. with the disappearance of tgev around the world, the need for tgev vaccines has dropped over the last few years. for north america and europe, only two major international animal health companies continue to offer tgev vaccines. in contrast, many parts of asia including china and korea are still dealing with tgev outbreaks, and different types of vaccines are still available. the introduction of pedv into the north american herd in - , however, has reversed this picture and has highlighted the global need for effective vaccines. coronaviruses represent an important group of animal pathogens that can have devastating impacts in a variety of species. for an industry to rely on biosecurity alone seems somewhat risky and, in case of an emerging disease, can lead to major economic losses, as witnessed in north america over the last two years. indeed, the phylogeny of coronaviruses demonstrates a great deal of diversity in antigenic variants, which may lead to limited cross-protection against infection with different strains. thus, it is important to continue to survey novel pedv variants that may emerge locally or globally through antigenic drift (point mutations) or antigenic shift (recombination events). effective prevention and control of pedv and other coronaviruses can only be achieved through the use of vaccines. an ideal vaccine prevents mortality and clinical disease in newborn piglets, the age group most seriously affected by the disease, as well as viral shedding. as lactogenic immunity is a key mechanism of protection, efforts to enhance the levels of antibodies in the milk through formulation (adjuvants) and delivery (mucosal) are critical. however, time is also of essence when dealing with a new strain, as traditional manufacturing vaccine methods like virus isolation, inactivation or attenuation can be time-consuming. therefore, research on next-generation vaccines such as rna particle, dna, sub-unit and viral-vectored approaches is critical for the prevention of future outbreaks of emerging coronavirus diseases. induction of antigen-specific systemic and mucosal immune responses by feeding animals transgenic plants expressing the antigen efficacy of an inactivated genotype b porcine epidemic diarrhea virus vaccine in neonatal piglets colostral antibodymediated and cell-mediated immunity contributes to innate and 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diarrhea virus dr strain construction and characterization of recombinant porcine adenovirus serotype expressing the transmissible gastroenteritis virus spike gene pedv orf encodes an ion channel protein and regulates virus production porcine epidemic diarrhea virus variants with high pathogenicity porcine epidemic diarrhea in china proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture ccl controls immunoglobulin (ig)a plasma cell accumulation in the lactating mammary gland and iga antibody transfer to the neonate porcine epidemic diarrhea virus e protein causes endoplasmic reticulum stress and up-regulates interleukin- expression porcine epidemic diarrhea virus n protein prolongs s-phase cell cycle, induces endoplasmic reticulum stress, and up-regulates interleukin- expression long-lived effector/central memory t-cell responses to severe acute respiratory syndrome coronavirus (sars-cov) s antigen in recovered sars patients efficacy and immunogenicity of recombinant swinepox virus expressing the a epitope of the tgev s protein immune evasion of porcine enteric coronaviruses and viral modulation of antiviral innate signaling identification of a conserved linear b-cell epitope in the m protein of porcine epidemic diarrhea virus suppression of type i interferon production by porcine epidemic diarrhea virus and degradation of creb-binding protein by nsp construction of a bivalent dna vaccine co-expressing s genes of transmissible gastroenteritis virus and porcine epidemic diarrhea virus delivered by attenuated salmonella typhimurium mucosal and systemic isotypespecific antibody responses and protection in conventional pigs exposed to virulent or attenuated porcine epidemic diarrhoea virus the authors declare no conflict of interest. key: cord- -f htekhz authors: yu, meiling; wang, li; ma, sunting; wang, xiaona; wang, yusai; xiao, ya; jiang, yanping; qiao, xinyuan; tang, lijie; xu, yigang; li, yijing title: immunogenicity of egfp-marked recombinant lactobacillus casei against transmissible gastroenteritis virus and porcine epidemic diarrhea virus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: f htekhz porcine transmissible gastroenteritis virus (tgev) and porcine epidemic diarrhea virus (pedv) are the causative agents of highly fatal acute diarrhea in pigs, resulting in enormous losses in the pig industry worldwide. to develop an effective bivalent oral vaccine against tgev and pedv infection, the d antigenic site of the tgev spike (s) protein and the major antigen site (core neutralizing epitope—coe) of the pedv s protein were used as immunogens, and the enhanced green fluorescent protein (egfp) gene was used as a reporter to construct genetically engineered lactobacillus casei rlppg(f)-t g -egfp- d-coe. the expression of proteins of interest by the recombinant l. casei was confirmed by confocal laser scanning microscopy and a western blot assay, and the immunogenicity of rlppg(f)-t g -egfp- d-coe in orally immunized mice was evaluated. the results showed that levels of anti-pedv and anti-tgev serum immunoglobulin g (igg) and mucosal secreted immunoglobulin a (siga) antibodies obtained from the mice immunized with rlppg(f)-t g -egfp- d-coe, as well as the proliferation levels of lymphocytes, were significantly higher than those in mice orally administered phosphate-buffered saline (pbs) or rlppg-t g . moreover, the serum igg antibodies showed neutralizing effects against pedv and tgev. our data suggest that the antibiotic resistance-free genetically engineered l. casei bivalent oral vaccine provides a safe and promising strategy for vaccine development against pedv and tgev. larger-scale outbreak in the united states in . herds vaccinated with the cv -inactivated vaccine were also infected, resulting in tremendous losses to the swine industry [ ] [ ] [ ] . accumulating evidence indicates that this large-scale recurrence of ped was caused by highly virulent pedv variants [ ] [ ] [ ] [ ] . in addition, co-infection of tgev and pedv often causes higher morbidity and mortality in newborn piglets. therefore, the development of a safe and highly efficient vaccine against tge and ped would be of great importance. it is now clear that the gastrointestinal mucosas are the primary sites of pedv and tgev infection [ , ] and that mucosal immunization is a promising way to prevent pedv and tgev infection. however, the existing live attenuated/inactivated vaccines administered parenterally cannot effectively induce mucosal immunity against pedv and tgev infection [ ] [ ] [ ] [ ] . although some vaccines for tge and ped developed in china can effectively stimulate intestinal mucosal immunity through houhai acupoint injection, this immunization procedure is more time-consuming and labor-intensive. therefore, vaccines that can induce a mucosal immune response against pedv and tgev would be of great significance, in particular those demonstrated to be safe, inexpensive, easy to use, and effective. the spike (s) glycoproteins of tgev and pedv possess antigen epitopes, and previous reports have demonstrated that the d antigen site (amino acids - ) in the s protein of tgev and the core neutralizing epitope (coe; amino acids - ) in the s protein of pedv can elicit neutralizing antibodies against tgev and pedv infection, respectively. this suggests that these are promising candidate antigens for the development of a genetically engineered vaccine [ , , ] . lactobacillus casei (l. casei) is a potential delivery vehicle for oral vaccines, because it is a probiotic bacterium characterized by its safety and resistance to gastric acid and bile [ , ] . previous reports have shown that recombinant l. casei live vaccine is able to colonize the murine intestines for five days [ ] and the swine intestines for longer [ ] . moreover, we have previously constructed a recombinant l. casei live vaccine expressing the d antigen site of the tgev s glycoprotein combined with muramyl dipeptide and tuftsin as adjuvants, suggesting the possibility of a promising oral vaccine against tgev challenge [ ] . however, plasmid-mediated antibiotic resistance is commonly used as a selective marker for genetically engineered bacteria [ ] [ ] [ ] . this could result in potential biosafety issues due to the transfer of antibiotic resistance from genetically engineered bacteria to environmental pathogens. enhanced green fluorescent protein (egfp) is a luminescent jellyfish protein with the amino acid substitutions necessary to generate a strong fluorescence signal when excited by ultraviolet or blue light. it is widely used in biological research, including in studies of cell differentiation, gene tracking, and protein localization and operation in vivo [ , ] , providing a potential candidate for a screening marker to replace antibiotic resistance. in this study, a genetically engineered l. casei strain, rlppg f -t g -egfp- d-coe, was constructed using the tgev s protein d antigen site and pedv s protein-neutralizing antigen epitope region coe as immunogens, l. casei as an antigen delivery vehicle, and egfp as a selective marker, combined with a constitutive expression plasmid, ppg-t g . the immunogenicity of this strain when orally administered in mice was evaluated, suggesting a potential approach for the prevention of tgev and pedv infection. all applicable international and national guidelines for the care and use of animals were followed. approval ( nefu- , april ) was obtained from the institutional committee of northeast agricultural university for the animal experiments. tgev strain th and pedv strain hlj- were isolated by our laboratory from pedv/tgev-positive samples collected from pig farms in which a severe outbreak of acute diarrhea had been reported in piglets [ , ] . l. casei atcc was kindly provided by jos seegers (nizo institute, netherlands). african green monkey kidney cells (vero cells; atcc ccl- ) and swine testicle (st) cells were purchased from the china center for culture collection (wuhan, china) and were cultured in dulbecco's modified eagle medium (dmem; gibco, gaithersburg, md, usa) supplemented with % fetal bovine serum (fbs; gibco) at • c with % co . the details of all plasmids used in this study are listed in table . the primers used for amplifying genes encoding d (a peptide of the the d antigenic site of the tgev spike (s) protein was repeated six times), coe, and egfp are listed in table . the linker sequence (ggggs) was added in primers fs and re. a schematic diagram of the dna plasmid construction is shown in figure . in brief, the gene encoding d was inserted into plasmid pmd t-coe at saci and mlui sites, generating plasmid pmd t- d-coe; then, the fusion dna fragment d-coe, obtained from pmd t- d-coe by saci and apai digestion, was inserted into the corresponding sites of plasmid ppg-t g , generating recombinant plasmid ppg-t g - d-coe. next, the gene encoding egfp was inserted into ppg-t g - d-coe at saci and kpni sites, generating recombinant plasmid ppg-t g -egfp- d-coe; finally, a chloramphenicol resistance (cm r ) gene selective marker in ppg-t g -egfp- d-coe was deleted using restriction enzymes stui and ncoi, followed by blunt-end treatment and ligation, giving rise to recombinant plasmid ppg f -t g -egfp- d-coe. all recombinant plasmids were identified by restriction enzyme digestion and sequencing. for construction of the recombinant lactobacillus strain, l. casei competent cells were prepared according to a method previously described [ ] , followed by electroporation. briefly, ng of recombinant plasmid ppg f -t g -egfp- d-coe was gently mixed with µl of l. casei competent cells at • c for min; then, the mixture was transferred into a pre-cooled gene pulser (bio-rad, hercules, ca, usa) disposable cuvette (inter-electrode distance of . cm) and subjected to a single electric pulse ( . v; Ω; µf) with a gene pulser (bio-rad). after growth at • c for h, the recombinant lactobacillus strain with green fluorescence signal was collected through flow cytometry using a facscalibur (bd biosciences, san diego, ca, usa) at nm and was grown on an de man-rogosa-sharpe (mrs) plate at • c for h. this was followed by pcr confirmation and a chloramphenicol sensitivity assay, giving rise to recombinant strain rlppg f -t g -egfp- d-coe. the hereditary stability of recombinant l. casei strains was detected, and rlppg f -t g -egfp- d-coe was analyzed for stability by serially transferring the cultures after h of incubation into mrs medium at • c ( % inoculum; generations). plasmids were extracted from the cells, and pcr was used to confirm the presence of the gene egfp- d-coe in the plasmid using the primers fe and rs . plasmids ppg-t g - d-coe and ppg f -t g -egfp- d-coe were generated according to the steps indicated by the arrows. ① the gene encoding d was inserted into plasmid pmd -t-coe at saci and mlui sites, generating plasmid pmd -t- d-coe; ② fusion dna fragment d-coe, obtained from pmd -t- d-coe by saci and apai digestion, was inserted into the corresponding sites of plasmid ppg-t g , generating recombinant plasmid ppg-t g - d-coe; ③ the gene encoding enhanced green fluorescent protein (egfp) was inserted into ppg-t g - d-coe at saci and kpni sites, generating recombinant plasmid ppg-t g -egfp- d-coe; ④ the chloramphenicol resistance (cm r ) gene as a selective marker in ppg-t g -egfp- d-coe was deleted using restriction enzymes stui and ncoi, followed by blunt-end treatment and ligation, giving rise to recombinant plasmid ppg f -t g -egfp- d-coe. for analysis of the expression of proteins of interest by recombinant strains rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe, the bacterial strains were grown in basal mrs broth at °c for h (optical density of sample measured at a wavelength of nm (od ) ≈ . ) without shaking and were harvested by centrifugation at , × g for min. cells were washed twice with sterile phosphate-buffered saline (pbs; ph . ) and lysed using a mini-beadbeater (biospec, bartlesville, ok, usa). after centrifugation, the same quantity of total protein in the supernatants of each sample was isolated by sodium dodecyl sulfate % polyacrylamide gel figure . schematic drawing of the construction of dna plasmids. plasmids ppg-t g - d-coe and ppg f -t g -egfp- d-coe were generated according to the steps indicated by the arrows. the gene encoding d was inserted into plasmid pmd -t-coe at saci and mlui sites, generating plasmid pmd -t- d-coe; fusion dna fragment d-coe, obtained from pmd -t- d-coe by saci and apai digestion, was inserted into the corresponding sites of plasmid ppg-t g , generating recombinant plasmid ppg-t g - d-coe; the gene encoding enhanced green fluorescent protein (egfp) was inserted into ppg-t g - d-coe at saci and kpni sites, generating recombinant plasmid ppg-t g -egfp- d-coe; the chloramphenicol resistance (cm r ) gene as a selective marker in ppg-t g -egfp- d-coe was deleted using restriction enzymes stui and ncoi, followed by blunt-end treatment and ligation, giving rise to recombinant plasmid ppg f -t g -egfp- d-coe. for analysis of the expression of proteins of interest by recombinant strains rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe, the bacterial strains were grown in basal mrs broth at • c for h (optical density of sample measured at a wavelength of nm (od ) ≈ . ) without shaking and were harvested by centrifugation at , × g for min. cells were washed twice with sterile phosphate-buffered saline (pbs; ph . ) and lysed using a mini-beadbeater (biospec, bartlesville, ok, usa). after centrifugation, the same quantity of total protein in the supernatants of each sample was isolated by sodium dodecyl sulfate % polyacrylamide gel electrophoresis (sds-page) and was transferred onto polyvinylidene fluoride membranes, followed by development with mouse anti- d monoclonal antibody/rabbit anti-coe polyclonal antibody (diluted at : ) prepared in our lab, or mouse anti-egfp monoclonal antibody (zsgb-bio, beijing, china; diluted at : ). horseradish peroxidase (hrp)-conjugated goat anti-mouse/rabbit igg antibody (sigma, st. louis, mo, usa) was utilized as a secondary antibody, diluted at : . immunoblots were visualized with chemiluminescent substrate reagent (pierce, rockford, il, usa) according to the manufacturer's instructions. laser confocal microscopy was used to confirm the expression of fusion protein egfp- d-coe on the surface of rlppg f -t g -egfp- d-coe using rlppg-t g - d-coe as a control. briefly, recombinant strains were cultured in mrs medium at • c for h; then ml of culture was collected by centrifugation at × g for min. the pellets were washed three times with pbs, re-suspended in ml of pbs, and smeared on a microscope slide. images were viewed by laser confocal microscopy (zeiss, oberkochen, germany). in order to evaluate the immunogenicity of recombinant strain rlppg f -t g -egfp- d-coe used as an oral vaccine, -week-old female specific pathogen-free (spf) balb/c mice (derived from mus musculus) (n = ) were obtained from liaoning changsheng biotechnology co., ltd. (liaoning, china) and kept under spf conditions for one week with free access to a standard chow diet and water, in accordance with institutional guidelines. prior to oral administration, the recombinant lactobacillus strains were cultured for h in mrs medium without shaking, washed with sterile pbs, and re-suspended in pbs at a concentration of cfu ml − . spf balb/c mice were randomly divided into four groups ( mice per group): pbs, rlppg-t g , rlppg-t g - d-coe, and rlppg f -t g -egfp- d-coe. the immunization dosages are shown in table . the mice were immunized once a day for consecutive days and boosted twice at week intervals. after immunization, serum samples were collected from the immunized mice on days , , , , and , and were stored at − • c until they were required for use. mucosal lavage samples were obtained from the vaginas of the mice by washing with µl of sterile pbs (ph . ) and were stored at − • c until analysis. in addition, fecal samples were collected and treated according to a method previously described [ ] . briefly, a . g fecal pellet was suspended in µl of pbs containing mmol l − phenylmethylsulfonyl fluoride (sigma) and % bovine serum albumin (bsa) and was then incubated at • c for h. after centrifugation, the supernatants were stored at − • c until use. to detect tgev-and pedv-specific antibodies in the collected samples, polystyrene microtiter plates were coated with purified tgev/pedv for h at • c, using cultured st/vero cells as a negative antigen control. after blocking with % skim milk at • c for h and washing three times with pbs- . % tween (pbst), serum and mucus samples serially diluted in pbs- % bsa were added to wells in triplicate, and then plates were incubated for h at • c. after washing with pbst, hrp-conjugated goat anti-mouse igg or iga antibody (invitrogen, carlsbad, ca, usa) was added to each well ( : ) and incubated for an additional h at • c. the substrate o-phenylenediamine dihydrochloride (sigma) was used for color development, and the absorbance was measured at nm. the neutralizing capacities of serum antibodies obtained from the mice immunized with rlppg f -t g -egfp- d-coe were determined. briefly, the % tissue culture infective dose (tcid ) values of tgev and pedv were detected by the reed-muench method. serum antibodies collected from the vaccinated mice on day post-immunization were diluted at : - : (in a total of µl), mixed with an equal volume of pedv or tgev ( tcid per µl) and incubated at • c for h. then, the treated viruses were added to a confluent monolayer of vero and st cells cultured in -well plates. the cells were overlaid with % methylcellulose, and the plates were incubated at • c in a % co atmosphere and examined daily for five days for tgev-and pedv-specific cytopathic effects (cpe). on day post-immunization, splenocytes from three mice from each group were prepared for a lymphocyte proliferation assay as previously described [ ] . briefly, µl of splenocytes ( × cells ml − ) was suspended in roswell park memorial institute (rpmi) medium containing % fetal calf serum and then transferred to a -well flat-bottom plate; the cells were re-stimulated for h with . , . or µg ml − tgev- d protein and pedv-coe protein (produced by escherichia coli prepared in our lab), using . µg ml − concanavalin a (cona) and culture medium as positive and negative controls, respectively. the plates were supplemented with µl of -( , -dimethylthylthiazol- -yl)- , -diphenyltetrazoliumbromide (mtt) per well and incubated for an additional h, and then proliferation was measured using od values. the experiment was carried out in triplicate, and the lymphocyte proliferation index in the spleen (si) was calculated as the mean reading of triplicate antigen stimulation wells divided by the mean reading of triplicate negative control wells. data are shown as the means ± standard errors of three replicates per test in a single experiment repeated three times. tukey's multiple comparison tests were used to analyze differences among the groups. a p-value of < . was considered statistically significant, and p < . was considered highly significant. following flow cytometry screening and growth on antibiotic-free mrs plates, egfp-marked recombinant lactobacillus strain rlppg f -t g -egfp- d-coe was obtained and identified with colony pcr (figure a) . as shown in figure b , the strain rlppg f -t g -egfp- d-coe could not grow on mrs agar medium in the presence of chloramphenicol. the result of the hereditary stability of recombinant l. casei strains showed that rlppg f -t g -egfp- d-coe is stable for more than generations. for confirming the expression of the proteins of interest by the recombinant lactobacillus strain constructed in this study, the cell lysates of recombinant strains rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe were analyzed by a western blot assay. as shown in figure , the expected immunoblot bands of fusion protein pgsa- d-coe of kda expressed by rlppg-t g - d-coe (figure a ,b) and the fusion protein pgsa-egfp- d-coe of kda expressed by rlppg f -t g -egfp- d-coe were observed (figure a-c) , but these proteins were not expressed in rlppg-t g or l. casei. moreover, green fluorescence could be observed by laser confocal microscopy on the surface of rlppg f -t g -egfp- d-coe but not on rlppg-t g - d-coe (figure ) , indicating that egfp could successfully be used as a selective marker. for confirming the expression of the proteins of interest by the recombinant lactobacillus strain constructed in this study, the cell lysates of recombinant strains rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe were analyzed by a western blot assay. as shown in figure , the expected immunoblot bands of fusion protein pgsa- d-coe of kda expressed by rlppg-t g - d-coe (figure a ,b) and the fusion protein pgsa-egfp- d-coe of kda expressed by rlppg f -t g -egfp- d-coe were observed (figure a-c) , but these proteins were not expressed in rlppg-t g or l. casei. moreover, green fluorescence could be observed by laser confocal microscopy on the surface of rlppg f -t g -egfp- d-coe but not on rlppg-t g - d-coe (figure ) , indicating that egfp could successfully be used as a selective marker. for confirming the expression of the proteins of interest by the recombinant lactobacillus strain constructed in this study, the cell lysates of recombinant strains rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe were analyzed by a western blot assay. as shown in figure , the expected immunoblot bands of fusion protein pgsa- d-coe of kda expressed by rlppg-t g - d-coe (figure a ,b) and the fusion protein pgsa-egfp- d-coe of kda expressed by rlppg f -t g -egfp- d-coe were observed (figure a-c) , but these proteins were not expressed in rlppg-t g or l. casei. moreover, green fluorescence could be observed by laser confocal microscopy on the surface of rlppg f -t g -egfp- d-coe but not on rlppg-t g - d-coe (figure ) , indicating that egfp could successfully be used as a selective marker. anti-tgev/pedv-specific secreted iga (siga) and igg antibodies were assessed to evaluate the ability of rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe to induce mucosal and systemic immune responses using balb/c mice as a model. as shown in figure , anti-tgev/pedv-specific antibodies were detected at high levels days post-immunization and were significantly increased after the booster immunization, peaking at days post-immunization. the levels of anti-tgev/pedv-specific mucosal siga in mouse feces and vaginas, as well as the levels of anti-tgev/pedv-specific serum igg antibody in mice orally immunized with rlppg-t g - d-coe/rlppg f -t g -egfp- d-coe, were significantly higher (p < . ) than those in the pbs or rlppg-t g groups. in addition, the levels of anti-tgev/pedv siga in the vaginas of mice orally immunized with rlppg f -t g -egfp- d-coe days post-immunization were significantly higher (p < . ) than those in the rlppg-t g - d-coe group. there was no statistical difference (p > . ) observed in the pbs or rlppg-t g groups before and after immunization. anti-tgev/pedv-specific secreted iga (siga) and igg antibodies were assessed to evaluate the ability of rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe to induce mucosal and systemic immune responses using balb/c mice as a model. as shown in figure , anti-tgev/pedv-specific antibodies were detected at high levels days post-immunization and were significantly increased after the booster immunization, peaking at days post-immunization. the levels of anti-tgev/pedv-specific mucosal siga in mouse feces and vaginas, as well as the levels of anti-tgev/pedv-specific serum igg antibody in mice orally immunized with rlppg-t g - d-coe/rlppg f -t g -egfp- d-coe, were significantly higher (p < . ) than those in the pbs or rlppg-t g groups. in addition, the levels of anti-tgev/pedv siga in the vaginas of mice orally immunized with rlppg f -t g -egfp- d-coe days post-immunization were significantly higher (p < . ) than those in the rlppg-t g - d-coe group. there was no statistical difference (p > . ) observed in the pbs or rlppg-t g groups before and after immunization. the neutralizing capacities of the serum antibodies induced in mice orally immunized with recombinant strains against tgev (figure a ) and pedv (figure b ) showed inhibitory activity against viral infection. the anti-tgev neutralizing antibody titers were − ( : ) and − . ( : . ) and the anti-pedv neutralizing antibody titers were − . ( : . ) and − . ( : . ) in mice immunized with rlppg f -t g -egfp- d-coe and rlppg-t g - d-coe, respectively. these were significantly different from those obtained in the rlppgf-t g and pbs groups (p < . ). bars represent the mean ± standard error of each group. * p < . , ** p < . vs. phosphate-buffered saline (pbs) and vector control groups; # p < . , ## p < . vs. rlppg f -t g -egfp- d-coe group. the neutralizing capacities of the serum antibodies induced in mice orally immunized with recombinant strains against tgev (figure a ) and pedv (figure b ) showed inhibitory activity against viral infection. the anti-tgev neutralizing antibody titers were − ( : ) and − . ( : . ) and the anti-pedv neutralizing antibody titers were − . ( : . ) and − . ( : . ) in mice immunized with rlppg f -t g -egfp- d-coe and rlppg-t g - d-coe, respectively. these were significantly different from those obtained in the rlppgf-t g and pbs groups (p < . ). the proliferation of spleen lymphocytes upon stimulation with purified coe or d proteins was analyzed by an mtt assay. the results showed that the proliferation levels of spleen lymphocytes from mice orally immunized with rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe were significantly higher than those of spleen lymphocytes from mice in the rlppgf-t g and pbs control groups (p < . ). there was no significant difference (p > . ) observed between the rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe groups (figure ). over the past decades, large-scale outbreaks of diarrhea in swine caused by pedv and tgev have occurred in america, europe, and asia, resulting in considerable economic losses to the pig industry; in particular, pedv infection has been responsible for many of these outbreaks [ , ] . the pedv strain hlj- used in this study was isolated from an outbreak of acute diarrhea in piglets in hegang, heilongjiang province, china [ ] , and phylogenetic analysis based on the s gene revealed that hlj- shares high homology with u.s. pedv strains. both belong to the giia subgroup, which represents epidemic and pandemic field strains [ , ] . occasionally, changes in the antigenicity of pedv due to amino acid mutations have resulted in vaccination failure [ , ] . the the proliferation of spleen lymphocytes upon stimulation with purified coe or d proteins was analyzed by an mtt assay. the results showed that the proliferation levels of spleen lymphocytes from mice orally immunized with rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe were significantly higher than those of spleen lymphocytes from mice in the rlppgf-t g and pbs control groups (p < . ). there was no significant difference (p > . ) observed between the rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe groups (figure ) . the proliferation of spleen lymphocytes upon stimulation with purified coe or d proteins was analyzed by an mtt assay. the results showed that the proliferation levels of spleen lymphocytes from mice orally immunized with rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe were significantly higher than those of spleen lymphocytes from mice in the rlppgf-t g and pbs control groups (p < . ). there was no significant difference (p > . ) observed between the rlppg-t g - d-coe and rlppg f -t g -egfp- d-coe groups (figure ). over the past decades, large-scale outbreaks of diarrhea in swine caused by pedv and tgev have occurred in america, europe, and asia, resulting in considerable economic losses to the pig industry; in particular, pedv infection has been responsible for many of these outbreaks [ , ] . the pedv strain hlj- used in this study was isolated from an outbreak of acute diarrhea in piglets in hegang, heilongjiang province, china [ ] , and phylogenetic analysis based on the s gene revealed that hlj- shares high homology with u.s. pedv strains. both belong to the giia subgroup, which represents epidemic and pandemic field strains [ , ] . occasionally, changes in the antigenicity of pedv due to amino acid mutations have resulted in vaccination failure [ , ] . the over the past decades, large-scale outbreaks of diarrhea in swine caused by pedv and tgev have occurred in america, europe, and asia, resulting in considerable economic losses to the pig industry; in particular, pedv infection has been responsible for many of these outbreaks [ , ] . the pedv strain hlj- used in this study was isolated from an outbreak of acute diarrhea in piglets in hegang, heilongjiang province, china [ ] , and phylogenetic analysis based on the s gene revealed that hlj- shares high homology with u.s. pedv strains. both belong to the giia subgroup, which represents epidemic and pandemic field strains [ , ] . occasionally, changes in the antigenicity of pedv due to amino acid mutations have resulted in vaccination failure [ , ] . the s protein neutralizing antigenic epitopes of pedv have been reported to provide cross-protection against variant strains [ , ] . therefore, we selected the coe of the pedv s protein as an immunogen for developing an effective vaccine against pedv infection. pedv and tgev infections initially occur on mucosal surfaces, especially the intestinal mucosal epithelial surface. therefore, mucosal vaccination is an effective strategy for preventing viral diarrheal diseases [ ] . in this study, we used l. casei to deliver the tgev s protein protective d antigenic site (repeated six times) and the pedv s protein coe for developing an oral mucosal vaccine against pedv and tgev. our results suggested that the genetically engineered l. casei strain rlppg f -t g -egfp- d-coe can be used as a bivalent oral vaccine for pedv and tgev, eliciting mucosal and humoral immune responses against both tgev and pedv via oral immunization. this was evidenced by significantly higher levels of virus-neutralizing antibodies, anti-pedv/tgev serum igg, and mucosal siga in mice orally immunized with rlppg f -t g -egfp- d-coe, compared to the levels for the rlppg-t g or pbs groups. moreover, the genetically engineered l. casei vaccine is safe and easy to administer, making it practical and convenient. mucosal immunity plays an important role in preventing viral diarrheal diseases, and siga antibodies from durable lactogenic immunity are a good way for piglets to obtain passive immunoprotection, indicating the importance of the siga antibody in the control of viral infection. at the same time, the level of siga can reflect the status of viral infection and the protective efficacy of vaccines [ ] . in the present study, anti-tgev/pedv siga antibodies could be effectively induced at high levels in the feces and vaginas of mice orally treated with rlppg f -t g -egfp- d-coe. iga is reported to peak at weeks and decline at weeks in piglets [ ] ; thus, if durable immunity was present at days post-immunization, newborn pigs could obtain immune protection. therefore, we assessed the levels of the siga antibody for days post-immunization. our data showed that the levels of siga in the feces of mice orally immunized with rlppg f -t g -egfp- d-coe gradually increased and peaked at , and days post-immunization, indicating that a mucosal immune response can be effectively elicited by rlppg f -t g -egfp- d-coe after oral immunization. therefore, the developed vaccine provides a promising strategy for protecting piglets from pedv and tgev infection via oral immunization. moreover, the neutralizing activity of an antibody is an important index used for evaluating the immunoprotective efficacy of a vaccine. in this study, high levels of serum antibody were elicited following oral immunization with rlppg f -t g -egfp- d-coe. this could effectively neutralize the pedv/tgev infection, and higher antibody titers reflected higher neutralizing activities. as a comparison, the neutralizing antibody titer induced by the recombinant l. casei oral vaccine developed in this study was higher than that induced by a previously developed dna vaccine [ ] . therefore, oral immunization with genetically engineered l. casei strain rlppg f -t g -egfp- d-coe may provide effective protection for piglets against pedv and tgev infection. in addition, our results showed that the egfp-marked recombinant lactobacillus oral vaccine rlppg f -t g -egfp- d-coe (antibiotic-free selective marker vaccine) exhibited a similar immunogenicity to the antibiotic resistance marker vaccine rlppg-t g - d-coe, as the levels of antibodies induced by the rlppg f -t g -egfp- d-coe and rlppg-t g - d-coe vaccines were not significantly different (p > . ). notably, the use of egfp as a selective marker in rlppg f -t g -egfp- d-coe would avoid the main disadvantage of traditional plasmid expression systems by eliminating the use of antibiotic resistance genes as selective markers for genetically engineered bacteria [ , , ] . moreover, rlppg f -t g -egfp- d-coe was constructed with a constitutive expression plasmid developed by our lab, exhibiting a significant advantage to inducible gene expression systems that require the use of an inductive agent. additionally, a pgsa-derived anchoring matrix from bacillus subtilis [ , ] was used to express the fusion proteins, which were displayed on the bacterial surface, eliciting good immunogenicity. the improved plasmid expression system used in this study therefore provides a powerful tool for the development of recombinant lactobacillus oral vaccines. in conclusion, an egfp-marked recombinant lactobacillus oral vaccine, rlppg f -t g -egfp- d-coe, was constructed in this study and provides a promising strategy for the development of a bivalent oral vaccine against tgev and pedv infection. further investigations are underway to evaluate the immunogenicity of this vaccine following its oral 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cardiomyocytes ins-egfp transgenic pigs: a novel reporter system for studying maturation, growth and vascularisation of neonatal islet-like cell clusters genetic variation analysis of s genes of pedv endemic strains and immunogenicity detection of hlj- strain cloning and homology comparison of s gene for isolate th- of porcine transmissible gastroenteritis virus construction and characterization of lactobacillus pentosus expressing the d antigenic site of the spike protein of transmissible gastroenteritis virus virulence and serological studies of recombinant infectious hematopoietic necrosis virus (ihnv) in rainbow trout chromosomal insertions in the lactobacillus casei upp gene that are useful for vaccine expression up-regulation of mdp and tuftsin gene expression in th and th cells as an adjuvant for an oral lactobacillus casei vaccine against anti-transmissible gastroenteritis virus oral vaccination of mice against tetanus with recombinant lactococcus lactis novel approach for isolation and identification of porcine epidemic diarrhea virus (pedv) strain nj using porcine intestinal epithelial cells physiological and biochemical characteristics of poly gamma-glutamate synthetase complex of bacillus subtilis ter beek, a. rodz and pgsa play intertwined roles in membrane homeostasis of bacillus subtilis and resistance to weak organic acid stress the authors declare no conflict of interest. key: cord- - zyoddl authors: hu, xiaoliang; tian, jin; kang, hongtao; guo, dongchun; liu, jiasen; liu, dafei; jiang, qian; li, zhijie; qu, juanjuan; qu, liandong title: transmissible gastroenteritis virus papain-like protease antagonizes production of interferon-β through its deubiquitinase activity date: - - journal: biomed res int doi: . / / sha: doc_id: cord_uid: zyoddl coronaviruses (covs), such as human coronavirus nl (hcov-nl ), severe acute respiratory syndrome cov (sars-cov), murine hepatitis virus (mhv), porcine epidemic diarrhea virus (pedv), and middle east respiratory syndrome coronavirus (mers-cov), encode papain-like (pl) proteases that inhibit sendai virus- (sev-) induced interferon (ifn-β) production. recently, the crystal structure of transmissible gastroenteritis virus (tgev) pl has been solved, which was similar to that of sars-cov pl (pro), which may antagonize host innate immunity. however, very little is known about whether tgev pl can antagonize host innate immune response. here, we presented evidence that tgev pl encoded by the replicase gene could suppress the ifn-β expression and inhibit the nuclear translocation of interferon regulatory factor (irf ). the ability to antagonize ifn-β production was dependent on the intact catalytic activity of pl . furthermore, tgev pl exerted deubiquitinase (dub) activity which strongly inhibited the retinoic acid-induced gene i- (rig- -) and stimulator of interferon gene- (sting-) dependent ifn expression. our data collectively suggest that tgev pl can inhibit the ifn-β expression and interfere with rig- - and sting-mediated signaling through a viral dub activity. our study has yielded strong evidence for the tgev pl mechanisms that counteract the host innate immunity. the innate immune system is the first line of defense that protects the host against viral infection, and the induction of ifn-/ is a crucial antiviral mechanism of the innate immune system [ ] . the initiation of ifn expression is triggered by pathogen-associated molecular patterns (pamps) through host pattern recognition receptors (prrs) [ ] . after viral rnas are sensed by prrs, signals are transmitted to different downstream adaptor molecules (such as ifnpromoter stimulator (ips- )); and then i b kinase-(ikk-) related kinases are recruited. next, interferon regulatory factor (irf ), nuclear factor b (nf-b), and atf- /c-jun are activated by the kinase complexes and translocate to the nucleus and directly induce the expression of type i ifns [ ] . tgev is an enveloped virus belonging to the coronaviridae (cov) family and the nidovirales order. covs are positive-strand rna viruses that replicate in the cytoplasm of infected cells [ ] . covs encode two types of cysteine proteases, m pro , and papain-like proteases, pl and pl , which contained nonstructural protein (nsp ) and nsp , respectively. pl pro is served mainly as in processing of the replicase pp a and pp ab polypeptides [ ] . other than their role in replicase polyprotein processing, pl domains possess an additional but related enzymatic activity, in hcov-nl [ ] , mhv [ ] , sars-cov [ , ] , and mers-cov [ ] , through their deubiquitination (dub) enzymes, which play a key role in antagonizing ifn induction. however, tgev pl processes the nsp /nsp site and is capable of hydrolyzing isopeptide bonds in both lys -and biomed research international lys -linked polyubiquitin chains [ ] . whether tgev pl could antagonize the production of ifns was unknown. in the present study, we found that tgev pl encoded by the replicase gene could suppress the ifn-expression and inhibit the nuclear translocation of interferon regulatory factor (irf ) and exerted deubiquitinase (dub) activity which strongly inhibited the retinoic acid-induced gene i-(rig- -) and stimulator of interferon gene-(sting-) dependent ifn expression. cells and viruses. hek t cells and pk- cells were cultured in dulbecco's modified eagle's medium (hyclone, logan, usa) containing % (v/v) fetal calf serum supplemented with penicillin ( u ml − ) and streptomycin ( g ml − ). sendai virus (sev) was obtained from the centre of virus resource and information (wuhan institute of virology, chinese academy of sciences). plasmids and agents. ifn--luc, x prdiii/i-luc (referred to as irf -luc), x ap- -luc, and x nf-b-luc luciferase reporter plasmids were constructed according to an earlier protocol [ ] . accession numbers of sting, irf , and mavs were kc , kc , and kc , respectively. expression plasmids for rig- (p-flag-rig- ) and tbk- (p-flag-tbk- ) were generated with the following primers: rig- forward, -tttggatccatgacagca-gagcagcggcggaat- , rig- reverse -tttaag-cttcactcaaggttcgggattccctg- ; tbk- forward, -tttgaattcatgcagagcacttctaatcat-cttt- , tbk- reverse -tttagatcttaaagacag-tcaacattgcgaa- . to construct the dna expression vector, pmyc-pl , pflag-pl , and ppl -myc, encoding tgev pl , standard reverse transcription-(rt-) pcr was applied to amplify cdna of the total rna extracted from pk- cells infected with the tgev strain hx, using the following primers: pl -forward, -gtacaagaa-gctgaacaatttaa- ( - bp), pl reverse, -atcgtttttaggactttgaattt- ( - bp). all constructs were validated via dna sequencing. pdsred -mito was purchased from clontech (tokyo, japan). transfection agent was performed with x-tremegene hp (roche, switzerland) per the manufacturer's instructions. luciferase reporter gene assay. hek- t cells grown in -well plates were cotransfected with . g/well reporter plasmid, . g/well prl-tk plasmid (promega, madison, usa) as an internal control for normalization of transfection efficiency, and the indicated expression or empty control vector plasmid. where indicated, cells were also mockinfected or infected with sev ( hemagglutinating activity units/well) at h after cotransfection. cells were subsequently lysed, and firefly and renilla luciferase activities were determined with the dual-luciferase reporter assay system (promega, madison, usa), according to the manufacturer's protocol. data are presented as mean relative luciferase units ± standard deviation from triplicate samples. for statistical analysis, data were compared between empty vector-and tgev pl -transfected groups with the unpaired, two-tailed student's t-test using spss . software. values < . were considered statistically significant [ ] . elisa. cell supernatants of transfected pk- cells were centrifuged at , for min to remove cell debris and stored at − ∘ c until use. secreted ifn-in the cell supernatants was determined using commercial porcine ifn-(interferon beta) elisa kit (elabscience, china) according to the manufacturer's instructions. immunoblot analysis. hek t cells were cultured in well plates and mm dishes were transfected with the appropriate plasmids. after h, cells were harvested by the addition of lysis buffer and protein concentrations in whole cell extracts measured. equal amounts of samples were subjected to sds-page and analyzed for tgev pl , sting, tbk- , and irf proteins via immunoblotting using ha, flag, or gfp-tagged antibodies (sigma, st louis, usa). expression of p-irf , irf , and gapdh was detected with the rabbit-anti p-irf (ab ), irf (ab ) (abcam, cambridge, uk), and a mouse anti-gapdh monoclonal antibody (sigma, st louis, usa). assay of deubiquitinase activity in cultured cells. hek t cells were cotransfected with pcdna . -ha-ub plus the indicated amounts of tgev pl , p-flag-rig- , and p-flag-sting constructs. the effect of tgev pl on ubiquitinated proteins in cultured cells was assessed by immunoblot analysis. coimmunoprecipitation analysis. coimmunoprecipitation experiments were performed on hek t cells transfected with the indicated expression plasmids as described in an earlier report [ ] . immunofluorescence assay. hek t cells were plated on fibronectin-treated glass coverslips in -well plates. to evaluate the localization of tgev pl , cells were cotransfected with plasmid dna expressing flag-pl ( ng per well) and pdsred-mito ( ng per well) using x-treme gene hp, according to the manufacturer's protocol. hek t cells were cotransfected with irf -gfp ( ng per well) and empty vector ( ng per well) or irf -gfp ( ng per well) and flag-pl ( ng per well). h after transfection, cells were infected with sev ( hemagglutinating activity units/well) for h. next, cells were fixed with % paraformaldehyde for min and permeated with . % triton x- for min at room temperature. after three washes with pbs, cells were blocked with pbs containing % bovine serum albumin for h, followed by incubation with a mouse monoclonal antibody against flag ( : ) for h at room temperature. cells were treated with fluorescein isothiocyanate-labeled goat anti-mouse (sigma, st louis, usa) for h, and subsequently with , -diamidino- -phenylindole (dapi) for min at room temperature. samples were washed with pbs, and fluorescent images were acquired under a confocal laser scanning microscope (tcs sp ; leica, solms, germany). to assess the formation of sting dimers, hek t cells were transfected with flag-sting ( ng per well) and the lysates were subjected to western blot, as described earlier [ ] , with the indicated antibodies. is an ifn antagonist. the crystal structure of tgev pl has been determined [ ] . the structure of tgev pl is similar to that of sars-cov pl pro . in order to determine whether tgev pl is capable of blocking ifn-production, we assessed ifn-promoter activity in the presence of pl (figure (a) ). hek t cells were cotransfected with tgev pl and ifn-luciferase or renilla luciferase reporter plasmids for h and subsequently infected with sev to activate the rig- -dependent ifn-expression pathway. we observed the inhibition of sev-induced ifn-promoter activation in the presence of pl , similar to the antagonistic function of nl plp and porcine epidemic diarrhea virus (pedv) plp , clearly indicating that tgev pl could act as an interferon antagonist. to establish the mechanisms by which pl inhibits ifnexpression, transcriptional activities of nf-b, irf , and ap- were analyzed using the luciferase assay to identify the precise transcription factor involved. notably, the luciferase activities of all three transcription factors were significantly inhibited by tgev pl in a dose-dependent manner ( figures (b) , (c), and (d)). furthermore, flag-pl also significantly inhibited ifn-production in pk- cells at protein level ( figure (e) ), which was further confirmed with the result that tgev pl could block the production of interferon. to further establish whether tgev pl affects irf phosphorylation or migration from the cytoplasm to nucleus, hek t cells were transfected with tgev pl and/or irf -egfp. then the result was analyzed using western blot and confocal microscopy. in figure (f) , the level of p-irf was decreased significantly by tgev pl compared with that of sev-induced. furthermore, irf -egfp was located in the cytoplasm compared with mock-infected hek t cells but translocated to the nucleus when the cells were inoculated with sev. in contrast, after being inoculated with sev, it was found that irf -egfp was translocated from cytoplasm to nuclear in mock infected hek t cell, which was not observed in cells transfected with tgev pl (figure (g) ). our results collectively suggested that tgev pl suppressed ifn-transcription by interfering with nf-b-, irf -, and ap- signaling-mediated ifn expression. to determine whether tgev pl is capable of blocking stingmediated activation of the ifn-promoter, we assessed promoter activity in the presence of sting along with increasing amounts of tgev pl . stimulation of hek t cells with sting alone resulted in a robust increase in ifnpromoter activity. coexpression of sting and tgev pl induced a dose-dependent decrease in ifn-activity, clearly indicating antagonistic activity of tgev pl on stingmediated activation of the ifn-promoter (figure (a) ). sting dimerization is reduced in the presence of hcov-nl . sting dimmers are visualized as an kd band on sds-page. to further determine whether tgev pl inhibits sting-mediated signaling through disrupting the stability of sting dimers, hek t cells were cotransfected with plasmid dna expressing sting in the presence or absence of tgev pl and sev, and cell lysates were evaluated for dimmers via immunoblotting (figure (b) ). interestingly, the results indicated that sting dimerization was not affected by tgev pl . inhibiting ifn-expression. to determine whether catalytic activity is required for tgev pl -mediated inhibition of ifn-expression, hek t cells were cotransfected with alanine mutants of three conserved catalytic residues of tgev pl (c a, h a, and d a) with or without rig- , mavs, sting, or tbk- , and ifn--luc and prl-tk plasmids, followed by infection with sev to activate ifnpromoter activity. tgev pl mutation at two of the catalytic sites (c a and h a) led to almost complete loss of ifn antagonistic activity, relative to wild-type tgev pl , but the d a mutant showed a little inhibition for ifn-promoter activity (figures (a), (b) , (c), (d), and (e)). based on the results, we conclude that the intact catalytic triad of tgev pl is required to inhibit activation of the ifn-promoter driven by sting and tbk- . recent studies have revealed that sting acts as a scaffold protein for tbk- and irf and links them to the mavs complex in mitochondria upon viral infection [ ] . moreover, activation of sting is critical for stimulation of irf- activity. here, we observed that tgev pl protein inhibits sting-and tbk- -induced activation of ifn-. additional localization experiments showed that pl existed in mitochondria (figure (f) ). modification of signaling molecules by ubiquitin (ub) plays a critical role in activation of the ifn response. tgev pl has been shown to possess dub activity. here, we investigated the dub activities of tgev pl and its catalytic mutants. hek t cells were cotransfected with pcdna ha-ub and tgev pl , and the level of ubiquitinated proteins was assessed via western blot. the level of ub-conjugated proteins was reduced dramatically in cells transfected with wild-type tgev pl , while the ubiquitinated ub-ha level was not reduced in the presence of the c a, h a, and d a mutants (figure (a) ). next, we investigated whether tgev pl recognizes and deubiquitinates the key regulators, rig-i and sting, in the ifn signaling pathway. tgev is known to induce robust expression of ifn-at the late step of the replication and is distinct from covs [ , ] . moreover, tgev infection activates transcription factors nf-b, irf , and ap- in porcine kidney cells and a delayed activation of the ifn response in intestinal epithelial cells [ , ] . however, the mechanism of its evasion of the innate immune system has never been reported. the current study firstly showed antagonistic function of the tgev pl protein against the irf signaling pathway to inhibit ifninduction through its dub activity. to combat the host antiviral effects, coronaviruses likely take advantage of pl activity to escape from the host innate antiviral response. hcov-nl (pl -tm) and sars-cov (plpro-tm) inhibit sting-mediated activation of irf- nuclear translocation and induction of irf- -dependent promoters [ , ] . pl of mhv strongly inhibits cardif-, tbk -, and irf -mediated ifn-reporter activities and prevented nuclear translocation of irf [ ] . pedv plp negatively regulated rig-i and sting-mediated ifn-expression [ ] . moreover, tgev pl displays a similar structure to sars-cov pl [ ] and gives rise to the speculation that tgev pl may similarly act as an ifn antagonist. in the present study, we first found that overexpressed tgev pl inhibited sting-and tbk- -mediated ifntranscription and antagonized the type i ifn response stimulated by sev in pk- cells. the catalytic activity of tgev pl is essential for inhibiting ifn-transcription. furthermore, sting dimerization is reduced in the presence of hcov-nl pl -tm, which was not affected by tgev pl . these results suggested that tgev pl acted as an ifn antagonist to negatively regulate host antiviral innate immunity. ubiquitination and deubiquitination are critically involved in regulation of virus-induced type i ifn signaling pathways [ , ] . recently, dubs have been reported in a variety of viruses, such as foot-and-mouth disease virus, lpro [ ] , human cytomegalovirus, ul [ ] , herpes simplex virus type , ul [ ] , and porcine reproductive and respiratory syndrome virus, nsp [ , ] . interestingly, all covs have evolved to encode dub enzymes, which may contribute to modulation of the innate immune response. plp of hcov-nl , sars-cov, mhv, pedv, and mers-cov dramatically reduced the levels of ubiquitinated sting, rig-i, tbk , and irf- to negatively regulate host antiviral innate immunity. here, we showed that tgev pl interferes with and significantly inhibits ubiquitination of rig- and sting, which are essential activators of type i ifn signaling. then, the levels of phosphorylated irf- were reduced, which blocked nuclear translocation of irf to activate the transcript of ifns. three catalytically inactive mutants of tgev pl (c a, h a, and d a) found to be defective in dub activity failed to inhibit virus-induced inf-expression, indicating that the dub function of tgev pl is directly involved in inhibition of type i ifn induction. however, the membrane protein m and envelope protein e of tgev were translated at the late step of the replication as the major inducing component of ifns. further studies are required to establish the precise functions of pl protease/dub activity in coronavirus interactions with the host innate immune response. our results are the first report identifying tgev pl that is responsible for inhibiting the induction of ifn-. we found that tgev pl displayed ifn antagonist activity dependent on the intact catalytic triad (c , h , and d ) and interfered with rig- -and sting-mediated signaling through a viral dub activity. these characteristics of tgev pl served as a multifunctional protein with a critical regulatory role in tgev interactions with the host antiviral innate immune response. moreover, these findings contribute to our understanding of the molecular mechanisms of innate immunity evasion strategies utilized by tgev. antiviral signaling through pattern recognition receptors rna recognition and signal transduction by rig-i-like receptors rig-i like receptors and their signaling crosstalk in the regulation of antiviral immunity development of protection against coronavirus induced diseases: a review virus-encoded proteinases and proteolytic processing in the nidovirales deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases plp , a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production regulation of irf- -dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity mers-cov papain-like protease has deis-gylating and deubiquitinating activities papainlike protease from transmissible gastroenteritis virus: crystal structure and enzymatic activity toward viral and cellular substrates molecular cloning and functional characterization of porcine ifn-promoter stimulator (ips- ) replicates and repeatswhat is the difference and is it significant? a brief discussion of statistics and experimental design the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase eris, an endoplasmic reticulum ifn stimulator, activates innate immune signaling through dimerization the adaptor protein mita links virus-sensing receptors to irf transcription factor activation interferon-response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus coronavirus pseudoparticles formed with recombinant m and e proteins induce alpha interferon synthesis by leukocytes transmissible gastroenteritis virus infection induces nf-b activation through rlr-mediated signaling transmissible gastroenteritis virus does not suppress ifn-induction but is sensitive to ifn in ipec-j cells ubiquitylation in innate and adaptive immunity ubiquitination, ubiquitinlike modifiers, and deubiquitination in viral infection the leader proteinase of foot-andmouth disease virus negatively regulates the type i interferon pathway by acting as a viral deubiquitinase cleavage specificity of the ul deubiquitinating protease activity of human cytomegalovirus and the growth of an activesite mutant virus in cultured cells a deubiquitinating enzyme encoded by hsv- belongs to a family of cysteine proteases that is conserved across the family herpesviridae immunodominant epitopes in nsp of porcine reproductive and respiratory syndrome virus are dispensable for replication, but play an important role in modulation of the host immune response the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein possesses deubiquitinating and interferon antagonism functions this work was supported by the national key technology support program ( bak b - ), the state of international science and technology cooperation projects ( dfb ), and the national natural science foundation of china ( ). interferon regulatory factor dub:deubiquitinase rig- :retinoic acid-induced gene i sting:stimulator of interferon gene prr:pattern recognition receptors pamp:pathogen-associated molecular patterns mda :melanoma differentiation gene ips- :ifn-promoter stimulator ikk: i b kinase tbk :tank binding kinase nf-b:nuclear factor b nsp :nonstructural protein . the authors declare that they have no conflicts of interest regarding the publication of this paper. key: cord- -j suk b authors: wesley, ronald d.; woods, roger d. title: immunization of pregnant gilts with prcv induces lactogenic immunity for protection of nursing piglets from challenge with tgev date: - - journal: veterinary microbiology doi: . / - ( ) -g sha: doc_id: cord_uid: j suk b abstract the level of passive protection against transmissible gastroenteritis virus (tgev) was evaluated by experimentally infecting pregnant gilts with different doses of porcine respiratory coronavirus (prcv) and challenging their litters at days of age. an overall survival rate of % was found for piglets nursing the prcv-infected gilts, compared to a % survival rate for piglets of nine uninfected control gilts. six of the prcv-infected gilts had adequate levels of immunity to resist infection with tgev following the challenge of their litters. these six completely immuned gilts also solidly protected their litters from tgev as shown by a % piglet survival rate through weaning at weeks of age. the results suggest that respiratory infection with prcv induces a substantial degree of protective lactogenic immunity against tgev. . since then the virus has spread by contact and by aerosol throughout much of europe (s~tnchez et al., ; cox et al., ) . tgev challenge studies for piglets nursing sows immunized with prcv suggest widely varying results (bernard et al., ; paton and brown, ; de diego et al., ) . however, field observations indicate that the incidence of tgev has declined in european countries concomitantly with the spread of prcv. in , another prcv was isolated from three swine herds located at geographically distant sites in the midwest and the east coast of the united states . when compared with a european prcv strain, the u.s. prcv had a similar but non-identical deletion in the spike protein gene (rasschaert et al., ; wesley et al., ) . thus, the u.s. prcv is a new tgev variant and did not spread to the united states from europe. although genetically different, phenotypically, the u.s. and european strains of prcv are similar in that both have a tropism for respiratory tract tissue and both lack the ability to infect and destroy swine enterocytes and to cause enteric disease. however, unlike the rapid dissemination of prcv in europe, prcv in the united states has not spread widely as shown by a u.s. national swine survey (usda, aphis, ) . that survey indicated that % of u.s. swine herds were serologically positive for tgev antibody, a lower incidence than that found in a survey ofmidwestern swine in the early s (egan et al., ) . the purpose of this paper was to determine if the u.s. and european prcv strains induce comparable levels of passive protective immunity to tgev. in this study, pregnant gilts were vaccinated with the u.s. strain of prcv. the results are discussed and compared to the protective immunity induced by the european prcv strains. the prcv used in these studies was isolated originally from nasal swabs taken from weaned pigs. this isolate has been referred to as prcv-ind/ or isu- . the prcv was plaquepicked once on swine testicular (st) cells. prcv inocula were prepared in st cells by two passages at a low multiplicity of infection. usually, the inocula were concentrated by centdfugation at rpm, h, °c in a beckman sw rotor. the pelleted virus was resuspended in ml f- medium (gibco), titrated, and stored at - ° c. for some experiments, cell-free virus supernatant that had not been concentrated was used to inoculate gilts. twenty-one pregnant gilts, serologically negative for tgev antibody, were used in this study. each gilt was housed separately in an individual isolation room. nine gilts served as uninfected controls and gilts were vaccinated with prcv at the times and doses indicated in table . prcv was given to the pregnant gilts at , , and weeks before farrowing. the initial two doses were given orally/intranasally (o/in), while the third dose was divided --a portion given o/in and a portion emulsified with freund's incomplete adjuvant (difco) and given intramuscularly (im). group i gilts received the higher doses of prcv (table ) . gilts in groups ii and iii received approximately -fold less virus at each exposure and the piglets of group iii gilts also received × plaque forming units (pfu) of prcv intranasally (in) at day of age. no respiratory problems were observed in the prcv-inoculated group iii piglets. four-day-old piglets nursing either vaccinated or control gilts were challenged with approximately pig-lethal-doses of the virulent miller (p ) strain of tgev . the challenge virus, prepared as an intestinal homogenate, was briefly sonicated to dissociate aggregates and diluted in cold f- medium containing % fetal bovine serum. each piglet was challenged with ml of diluted tgev directly into the stomach via a tube. following challenge exposure, each piglet was weighed daily and the body temperature of each gilt was measured daily. clinical signs for both gilts and piglets were recorded twice daily. surviving piglets, for survival rate determinations, were those animals alive on day post challenge (pc). serum samples from blood collected during the vaccination protocol, colostrum collected within h post-farrowing, and milk collected on post-challenge day were tested for tgev/prcv neutralizing antibody. the virusneutralizing (vn) antibody titer is reported as the reciprocal of the serum dilution causing a tgev plaque reduction of % on st cells . survival rates, vn titers, and daily weight gains were each compared by the analysis of variance (the f-test). differences with p< . were considered to be significant. the serum neutralization (sn) titers of gilts following the vaccination protocol with prcv are shown in table . the sn titers at weeks following the primary exposure to prcv ranged from - with a geometric mean, ~= +_ . there was no correlation between the initial oronasal virus dose and the magnitude of the sn response. a second oronasal exposure to prcv did not stimulate sn antibody levels. however, the third vaccination with prcv, in which the dose was split o/in and im with freund's incomplete adjuvant, did increase the sn titer of each gilt, $= ___ (p= . ). litters from the prcv-vaccinated gilts (n = ) and the unvaccinated control gilts (n = ) were challenged with virulent tgev at days of age. the survival rate of litters from gilts given the higher prcv dose (group i) or approximately -fold less prcv (groups ii and iii) were similar (table ) . infecting baby piglets at day of age with prcv (group iii) and challenging with tgev at days of age did not improve the litter survival rate. the combined survival rate of litters from prcv-vaccinated gilts was % ( / piglets). in contrast, the survival rate of litters from seronegative control gilts was % ( / piglets). by day post-challenge (pc), litters of both the prcv-vaccinated gilts and the control gilts ceased gaining weight (table ). on average, litters of control gilts lost weight more rapidly than those of prcv-vaccinated gilts; however, only differences in the day pc weights were significant at the % confidence level (p= . ). all litters exposed to tgev developed clinical signs of vomiting and diarrhea within - days pc. following these early signs, piglets of prcv-vaccihated gilts often had a "downy", bloated appearance during days - pc, and most of these piglets survived. in contrast, most of the piglets of control gilts were dehydrated, gaunt, weak, and died between days - pc. % ( / ) qnitial prcv dose. one-day-old piglets of group iii gilts were exposed to prcv. all nine of the control gilts were secondarily infected with tgev following challenge of their litters because each gilt seroconverted by days pc (data not shown). no clinical signs of tgev infection, eg. soft feces or diarrhea, were observed in control gilts; however, seven of nine gilts had an elevated temperature ( ~> ° c) for one day between - days pc. half ( / ) of the prcv-vaccinated gilts did not become infected by exposure to tgev as shown by the lack of an anamnestic serological response (table ). these six completely immuned gilts ( # , , , , , ) also showed solid protection of their litters, % survival ( / piglets). two prcv-vaccinated gilts ( # ,p ) had soft stools pc, and gilt # had a pronounced diarrhea lasting for day. three prcv-vaccinated gilts ( # , , ) had an elevated temperature for day either on the second or third day pc. four of the prcv-vaccinated gilts showed a strong anamnestic response to tgev (table ) with a four-fold or greater increase in sn titer. two gilts had a less pronounced serological response to tgev exposure. low milk recovery at day pc suggested that gilt # may have become agalactic during the pc period, and thus, lost piglets to starvation rather than to tgev infection. colostral vn titers did not correlate with litter survival (table ) . also, the change in the vn titer between colostrum and -day milk sample was not as indicative of protection from challenge as were the serological changes described above. of the six protected gilts, five had lower vn titers in the day milk samples than in the colostral samples, and the vn titer of gilt # table serological response of prcv vaccinated gilts to tgev challenge of their litter discussion our results indicate that experimental vaccination of seronegative, naive gilts with prcv induces lactogenic immunity against tge. the use of firstlitter gilts in these experiments is the most stringent test for colostral immunity because the possibility of a previously undetected exposure to tgev is reduced. gilts were experimentally infected with different doses of prcv to determine the level of cross-protection, and their litters were uniformly challenged with tgev at days of age. to stimulate a maximum level of passive immunity, the gilts were immunized three times at , , and weeks prior to farrowing. the last vaccination was divided between the o/in and im routes, the latter emulsified with adjuvant to further stimulate immunity. passively acquired cross-protection to tgev by vaccination with prcv was significant (p= . ), but varied among the litters. the overall survival rate was % of piglets farrowed by prcv-vaccinated gilts, compared to a % survival rate of tgev challenged piglets from nine control gilts. within a litter, the survivors ranged from % ( litters) to % ( litter), although piglets from the litter with a % survival rate could possibly have died from starvation due to agalactia caused by secondary infection of the gilt with tgev. of the prcv-vaccinated gilts, six were completely immuned against secondary tgev infection. litters from these six immuned gilts had a survival rate of % ( / piglets). resistance to infection and passive protection were probably due to the fact that these gilts produced and transmitted to their suckling piglets the highest levels of protective antibody. the other six prcv-vaccinated gilts were infected with tgev, as shown by increased sn titers following challenge. two of the vaccinated gilts had soft stools following challenge and a third had a pronounced but transient diarrhea. thus, gilts vaccinated with the u.s. strain of prcv were not completely protected against tgev. similar observations were made by hooyberghs et al. ( ) in belgium where tgev outbreaks occurred in swine herds that were naturally exposed to the european isolate of prcv. the level of vn antibody in serum and colostrum that was induced by our prcv vaccination protocol did not correlate with piglet survivability. the lack of correlation between these parameters was reported previously for prcv (bernard et al., ) and has been recognized as a consistent feature of tgev vaccination and challenge experiments (saif and wesley, ) . this suggests that the immunodominant neutralizing epitopes for prcv and tgev are probably not the major contributors to passive protection. our results also show that the initial prcv dose, in the range of - × pfu, did not correlate with the magnitude of the primary immune response. thus, the degree to which prcv replicates in the respiratory tract and induces immunity is probably more dependent on host factors and the virus strain rather than on the initial virus dose. the second booster dose did not increase the humoral immune response, but the third dose did (p= . ), presumably due to the emulsification with adjuvant and the im route. this increase in immunity from the third dose by the im route may explain why antibody against the u.s. prcv protected piglets better than previously reported resuits with sows exposed to european prcv isolates. studies to measure the level of passive protection induced in sows with european prcv isolates have yielded variable results. in studies by paton and brown ( ) and by de diego et al. ( ) , seronegative sows were experimentally infected with either a low or high dose of prcv, respectively. in both instances, following two oronasal exposures to prcv, the serum and colostrum vn titers were approximately i -fold less than we report here following infection with the u.s. prcv. paton and brown ( ) , using a challenge tgev of low virulence, concluded that prcv-vaccinated sows transmitted no passive protection to their nursing piglets, whereas de diego et al. ( ) protected % ( / ) of the piglets from two prcv-vaccinated sows. bernard et al. ( ) also tested passive protection against tgev in sows naturally infected with prcv. serum titers in these sows were also low because of the > year time interval since exposure to prcv. with these naturally infected sows and using a virulent challenge virus, bernard et al. ( ) protected % of the piglets. the survival rate among these litters was highly variable; two sows protected all piglets in their litters and two sows did not protect any of their piglets. our results support the observations of bernard et al. ( ) and de diego et al. ( ) and show that there is a link between respiratory infection with prcv and secreted protective antibody in the mammary glands of post-parturient gilts. since the serum and colostral vn titers are lower following vaccination with european isolates, it is possible that the european prcvs could have lower infectivity for sows and gilts than the u.s. prcv isolate. in the united states, tge is still a significant problem. in europe, the spread of prcv has apparently eliminated tgev as a significant disease, probably by reducing the number of susceptible pigs. why prcv has not spread similarly in the united states is not known. the results of this study and the situation in europe indicate that inoculating swine with prcv would be beneficial in the control of tge. efforts to vaccinate with prcv alone or to combine prcv with attenuated enteric tgevs should help in eliminating tge as a disease problem. natural infection with the porcine respiratory coronavirus induces protective lactogenic immunity against transmissible gastroenteritis intestinal protection against challenge with transmissible gastroenteritis virus of pigs immune after infection with the porcine respiratory coronavirus. vaccine, i l epitope specificity of protective lactogenic immunity against swine transmissible gastroenteritis virus prevalence of swine dysentery, transmissible gastroenteritis, and pseudorabies in iowa, illinois and missouri swine porcine respiratory coronavirus isolated from two u.s. swine herds transmissible gastroenteritis: outbreaks in swine herds previously infected with a tgev-like porcine respiratory coronavirus i. morbidity/mortality and health management of swine in the united states. usda, animal and plant health inspections service sows infected in pregnancy with porcine respiratory coronavirus show no evidence of protecting their sucking piglets against transmissible gastroenteriris isolation of a porcine respiratory, non-enteric coronavirus related to transmissible gastroenteritis porcine respiratory coronavirus differs from transmissible gastroenteritis virus by a few genomic deletions transmissible gastroenteritis genetic evolution and tropism of transmissible gastroenteritis coronaviruses lack of protection in vivo with neutralizing monoclonal antibodies to transmissible gastroenteritis virus evidence for a porcine respiratory coronavirus, antigenically similar to transmissible gastroenteritis virus, in the united states genetic analysis of porcine respiratory coronavirus, an attenuated variant of transmissible gastroenteritis virus neutralization of porcine transmissible gastroenteritis virus by complement-dependent monoclonal antibodies we are grateful to david michael for excellent technical assistance. was unchanged. however, the vn titers also declined in the milk samples of three of the prcv-vaccinated gilts that became infected secondarily with tgev. key: cord- -oxyzndsj authors: ortego, javier; sola, isabel; almazán, fernando; ceriani, juan e; riquelme, cristina; balasch, monica; plana, juan; enjuanes, luis title: transmissible gastroenteritis coronavirus gene is not essential but influences in vivo virus replication and virulence date: - - journal: virology doi: . /s - ( ) -x sha: doc_id: cord_uid: oxyzndsj transmissible gastroenteritis coronavirus (tgev) contains eight overlapping genes that are expressed from a ′-coterminal nested set of leader-containing mrnas. to facilitate the genetic manipulation of the viral genome, genes were separated by duplication of transcription regulating sequences (trss) and introduction of unique restriction endonuclease sites at the ′ end of each gene using an infectious cdna clone. the recombinant tgev (rtgev) replicated in cell culture with similar efficiency to the wild-type virus and stably maintained the modifications introduced into the genome. in contrast, the rtgev replication level in the lungs and gut of infected piglets and virulence were significantly reduced. rtgev in which gene expression was abrogated (rtgev-Δ ) were recovered from cdna constructs, indicating that tgev gene was a nonessential gene for virus replication. interestingly, in vivo infections with rtgev-Δ showed an additional reduction in virus replication in the lung and gut, and in virulence, indicating that tgev gene influences virus pathogenesis. transmissible gastroenteritis coronavirus (tgev) is a member of the coronaviridae family, which, together with the arteriviridae and roniviridae families forms the nidovirales order (cowley and walker, ; enjuanes et al., a; mayo, ) . tgev replicates in both the villous epithelial cells of the small intestine and the lung cells of newborn piglets, resulting in a mortality of nearly % (saif and wesley, ) . the tgev genome contains a leader sequence at the Ј end and a poly(a) tail at the Ј end. genes are arranged in the order Ј-rep-s- a- b-e-m-n- - Ј (enjuanes et al., b; penzes et al., ) . the Ј end of the majority of tgev genes overlaps with the Ј terminus of the next gene (enjuanes et al., b) , complicating insertion of heterologous genes into the viral genome and deletion of different genes to determine whether they are essential. the tgev gene , located at the Ј end of the genome, encodes a amino acid hydrophobic protein that may play a role in membrane-associated replication complexes or in virion assembly (tung et al., ) . gene is a groupspecific gene (de haan et al., ) with homologous versions in group coronaviruses such as feline infectious peritonitis virus (fcov) and canine enteric coronavirus (ccov) (enjuanes et al., a; lai and cavanagh, ) . in contrast, group coronaviruses such as mouse hepatitis virus (mhv), bovine enteric coronavirus (bcov), or the human coronavirus (hcov) oc , and group coronaviruses such as avian infectious bronchitis virus (ibv), do not have a homologous gene (enjuanes et al., a; lai and cavanagh, ) . interestingly, the group coronavirus hcov- e does not have gene (herold et al., ) , which could indicate that this gene is nonessential for coronavirus replication, even in group coronaviruses. to study whether gene is dispensable for tgev replication, its deletion by reverse genetics would be required. we used a genomic tgev cdna clone assembled as a bacterial artificial chromosome (bac) (almazán et al., ; gonzález et al., ) to separate the overlapping genes by duplicating sequences at the Ј flank of each gene and by introducing unique restriction endonuclease sites between each gene pair. gene separation allowed the deletion of gene , showing that it is nonessential for virus replication. furthermore, we show that the accumulation of modifications in gene domains where the trss are located and insertion of restriction sites led to the generation of a collection of recombinant tgevs (rtgevs) with variable virulence, including a highly attenuated recombinant. these viruses could be the basis for coronavirus vector development. to facilitate genetic manipulation of the viral genome, full-length cdna clones were constructed by separating the contiguous genes and inserting unique restriction sites between each gene pair (fig. a ). cdna clones with the wild-type sequence or containing one [pbac-tgev-paci (p), pbac-tgev-mlui (m), pbac-tgev-fsei (f), and pbac-tgev-asci (a)], two [pbac-tgev-fsei-pmei (f-pm), pbac-tgev-pmei-asci (pm-a), and pbac-tgev-paci-mlui (p-m)], three [pbac-tgev-fsei-pmei-asci (f-pm-a)], or five [pbac-tgev-paci-mlui-fsei-pmei-asci (rs)] restriction endonuclease sites (fig. b) were transfected into bhk cells expressing the porcine amino peptidase n (papn) (bhk-papn cells). on the third day of transfection, a cytopathic effect was observed in cells transfected with each cdna, but not in mock-treated cells. virus production was amplified by passing the supernatants four times in cultured cells. after the fourth passage, viruses were cloned by three plaque-isolation steps, and their genomes were partially sequenced. all the rtgev viruses conserved the modifications engineered in the cdnas (data not shown), indicating that the orf separation and the insertion of unique endonuclease restriction sites between genes were stably maintained in the rtgev genomes. cloned rtgev viruses containing the unique restriction sites showed similar growth kinetics in cell culture to the parental rtgev-wt after infection at both low ( . ) and high ( ) m.o.i. (fig. ) , indicating that removal of the overlapping region between tgev genes and the insertion of endonuclease restriction sites did not significantly affect the in vitro virus replication. to analyze whether gene was essential for viral growth, recombinant virus genomes with gene deleted were generated from pbac-tgev-rs constructs, contain-ing either the s gene from the tgev strain pur-c (sánchez et al., ) able to infect both the enteric and the respiratory tracts (pbac-tgev-sc -rs-⌬ ) or the s gene from the strain ptv (sánchez et al., ) with an exclusive respiratory tropism (pbac-tgev-sptv-rs-⌬ ). the rtgev-⌬ contained a deletion spanning nucleotides upstream of the orf aug and the first nucleotides of this orf (fig. a) . bhk-papn cells were transfected with plasmids including five restriction endonuclease sites and carrying gene (pbac-tgev-sptv-rs and pbac-tgev-sc -rs), or without this gene (pbac-tgev-sptv-rs-⌬ and pbac-tgev-sc -rs-⌬ ). virus titers were determined in supernatants throughout four additional passages in cell culture ( fig. b and c). viruses were recovered from the cdnas containing the deletion of gene , and viral titers increased with passage, basically to the same extent as the viruses with gene (rtgev-sptv-rs and rtgev-sc -rs). as expected, no virus was recovered from the mock-transfected cultures. after four passages in cell cultures, the recombinant viruses were cloned by three plaque isolation steps. the cytopathic effect and plaque morphology produced by the rtgev-sptv-rs-⌬ and rtgev-sc -rs-⌬ were identical to those of the parental viruses containing the complete genome (data not shown). the isolate rtgev-sc -rs-⌬ induced the formation of large-size plaques ( -mm-diameter), whereas the virus rtgev-sptv-rs-⌬ induced smallsized plaques ( -mm-diameter). the cloned viruses with gene deleted showed standard growth kinetics after infection at an m.o.i. of ( fig. d and e). recombinant rtgev-sptv-rs-⌬ and rtgev-sc -rs-⌬ generated the highest virus titers (around ϫ and pfu/ml, respectively) at h postinfection, similar to those of the parental viruses rtgev-sptv-rs and rtgev-sc -rs. these data indicated that the protein encoded by gene was not essential for tgev replication in cell culture. to confirm that the subgenomic mrna (sgmrna) was not transcribed, bhk-papn cells were infected with rtgev-sptv-rs or rtgev-sptv-rs-⌬ viruses. total rna was extracted and analyzed by northern blot with a probe complementary to the Ј end of the tgev genome (fig. a ). the mobility and relative amount of the sgmrnas , , , , and were indistinguishable in both viruses. as expected, sgmrna was transcribed in rt-gev-sptv-rs-infected cells, but not in cells infected with rtgev-sptv-rs-⌬ . analysis of viral proteins at h p.i. by western blot showed that the amount of s, n, m, and e proteins was similar in rtgev-sptv-rs-⌬ -infected cells and in cells infected with the parental virus rtgev-sptv-rs, except for protein that was not detected in rtgev-sptv-rs-⌬ virus-infected cells (fig. b) , confirming that the partial deletion of gene prevented the synthesis of protein . identical results were obtained with rtgev-sc -rs and rtgev-sc -rs-⌬ (data not shown). in vivo growth of a selected set of rtgevs containing the unique endonuclease restriction sites, and the rtgev-rs-⌬ viruses, was determined by infecting newborn piglets. the animals were sacrificed at , , , and days p.i. recombinant viruses with a modification including a restriction endonuclease site Ј upstream of some genes, in general, showed lower titers than the wild-type virus, al- outlined sequences indicate the punctual mutations introduced to generate unique restriction sites. the core sequence (cs) is underlined. the atg start codon is shown in bold. duplicated sequences are indicated by dark boxes. tgev genes are indicated by light boxes. b, replicase b gene; s, spike gene; e, envelope gene; m, membrane gene; n, nucleoprotein gene. *, gene rep b termination codon (tga) and the initiation codon of gene s (atg) partially overlap. the sequence of the and nt located Ј upstream of genes s and a, respectively, are described in the full-length tgev genome sequence previously reported (penzes et al., ) . (b) schematic illustration of the full-length tgev cdna without (top bar) or with one (pbac-tgev-p, pbac-tgev-m, pbac-tgev-f, and pbac-tgev-a), two (pbac-tgev-f-pm, pbac-tgev-pm-a, and pbac-tgev-p-m), three (pbac-tgev-f-pm-a), or five (pbac-tgev-rs) restriction endonuclease sites. cmv, cytomegalovirus immediate-early promoter; rep, replicase; an, poly(a) tail of a residues; hdv, hepatitis delta virus ribozyme; bgh, bovine growth hormone termination and polyadenylation sequences. though there was some titer variability over the days postinfection ( fig. a and b) . alteration in domains Ј upstream of two or more genes did not lead to a significant decrease in virus titer in comparison with recombinants with a single modification. interestingly, analysis of viral growth in the gut of infected piglets showed a -to -fold reduction of recombinant viruses containing one or more restriction sites in relation to the rtgev-wt virus ( fig. d and e) . the rtgev-rs, that included modifications Ј upstream of five genes and insertion of endonuclease restriction sites between each contiguous gene, showed a titer decrease comparable with that of rtgevs with two (tgev-f-a) or three (tgev-f-pm-a) restriction endonuclease sites. these data show that modification of sequences Ј upstream of genes affected virus replication in the gut. recombinant viruses were isolated from the gut and sequenced. all the modifi-cations introduced were stably maintained in the tgev genome during in vivo infections (data not shown). the growth in lungs of gene deletion mutants (rtgev-sptv-rs-⌬ and rtgev-sc -rs ⌬ ) showed around fold reduction of virus titers compared with the parental viruses rtgevsptv-rs and rtgev-sc -rs (fig. c ). in contrast, in vivo growth of rtgev-sc -rs-⌬ in the gut showed slightly lower replication levels than rtgev-sc -rs ( ϫ pfu/g) probably because the introduction of modifications at the Ј upstream of five genes had already caused a significant titer reduction (fig. f ). as expected, the respiratory recombinants rtgev-sptv-rs and rtgev-sptv-rs-⌬ did not grow in the gut since the s gene was derived from the respiratory ptv strain. piglet survival after infection by rtgev viruses was compared with survival after infection with their virulent parental virus tgev-pur -c (tgev-sc -wt). rtgev viruses with one, two, three, or five unique restriction sites were highly attenuated (they produced mild enteritis and led to to % survival at days p.i.), except for rtgev-p and rtgev-m viruses, in which the restriction sites paci and mlui were introduced by point mutations, without introducing trs duplication. in these two recombinant viruses the survival was from to %. piglets infected by rtgev-sc -rs-⌬ showed % survival, although a very mild and transitory enteritis was observed in % of the animals. these data indicate that gene deletion further reduced virus virulence. in general, a good correlation be-tween growth of recombinants with a single restriction endonuclease insertion and virulence was not clear. this could be due to differences in the distribution of viral antigens and inflammatory responses in pigs infected with wild-type or each mutant. nevertheless, in viruses with two or more restriction endonuclease sites inserted there was a good correlation between virus titers in the gut and mortality. tgev genomes with all the genes separated by unique endonuclease restriction sites have been engineered. the separation of tgev genes implied the duplication of sequences Ј upstream of each gene, a sequence domain involved in regulating transcription, and affected in vivo virus growth and virulence. in this article, the first demonstration that tgev gene is nonessential for virus viability is provided. in addition, it has been demonstrated that gene deletion affects tgev replication and attenuates virus virulence in piglets, its natural host. interestingly, the intro- fig. . in vivo growth kinetics of rtgev viruses. two-to three-day-old non-colostrum-deprived swine were used to study the growth kinetics of rtgev viruses containing one (a and d) or more (b and e) endonuclease restriction sites, or partial deletion of gene (c and f). groups of to piglets were oronasally ( ϫ pfu/pig) and intragastrically ( ϫ pfu/pig) inoculated. virus titers at the indicated number of days postinoculation were determined in the indicated tissue extracts. the whole organs were homogenized to obtain representative samples. error bars represent standard deviations of the mean from four experiments. (g) number of surviving piglets at different days postinoculation. results from a representative experiment of two that gave similar results are shown. recombinant virus titers were compared with that of the wild-type virus by the kruskal-wallis test and, in general, were significant (p Ͻ . ) between viruses with gene deleted and the wild-type virus. western blot analysis of lysates from bhk-papn cells infected with either rtgev-sptv-rs or rtgev-sptv-rs-⌬ viruses. cell extracts were obtained at h p.i., resolved by to % gradient sds-page, transferred to nitrocellulose membranes, and immunoblotted with monoclonal antibodies specific for s, n, m, and e, and with an antiserum specific for a protein peptide (garwes et al., ). duction of one or more modifications into the tgev genome has led to the generation of a collection of tgev recombinants with a variable degree of virulence. overlapping of genes has been proposed as a mechanism by which nidoviruses preserve the genetic integrity of vital parts of their genomes (de vries et al., ) . nevertheless, in coronavirus we have generated viable and stable rtgev viruses in which genes have been separated by the insertion of unique restriction endonuclease sites and modification of the domains where the transcription-regulating sequences (trss) map. maximum titers of mutant rtgev viruses in cell culture were similar to those obtained for the parental virus, indicating that changes in the sequences between tgev genes had little effect on viral yield. in contrast, in vivo assays showed lower tgev mutant virus replication in the lungs and gut, and attenuation of virulence in viruses in which a trs duplication was introduced between genes. in arteriviruses, mutants that had the overlap between orfs and , or between orfs and removed, were also viable (de vries et al., ) . taken together, these data demonstrate that gene overlapping is not an obligatory requirement for nidovirales viability. viral attenuation resulting from gene separation was possibly due to modification of the trss affecting mrna transcription levels. in the case of nonsegmented negativestrand rna viruses it has also been shown that sequence alterations, such as restriction endonuclease site or heterologous gene insertion, and gene rearrangement may affect virus replication (wertz et al., ) . gene is a group specific gene, absent in genome of coronaviruses from groups and (armstrong et al., ; boursnell et al., ; kamahora et al., ; lapps et al., ; skinner and siddell, ) . interestingly, no mortality was observed in piglets infected with rtgev-⌬ . since these recombinants still replicate with titers between and pfu/g of tissue ( fig. c and f) , tgev-⌬ mutants could be good candidates as virus vectors. a relationship between gene and virulence has also been observed in the fcovs. the most Ј end genes of these viruses are genes a and b. deletions in fcov orf a, which is homologous to the tgev orf , have been reported in a natural infection of a cat population and correlated with a decrease in virulence (kennedy et al., ) . similarly, mutations in fcov orf b occur in vitro and have also been correlated with loss of virus virulence (herrewegh et al., ) . therefore, the most Ј end genes of group coronaviruses seem to influence in general virus pathogenesis. deletion of gene did not affect virus replication in cell culture. therefore, the reduction of in vivo virus replication and virulence was probably due to an effect on virus-host interaction. it has been suggested that coronavirus groupspecific genes, such as gene , may affect host immune response (de haan et al., ) . it would be of interest to determine whether the immune response to a heterologous gene inserted in recombinant viruses with and without gene is influenced by the presence of this gene. in an attempt to identify gene homologous protein sequences or motifs, a sequence search was performed in the databases using gene sequences without success. the high hydrophobicity of tgev protein (garwes et al., ) could facilitate its insertion in membranes providing a role in virus replication, since coronavirus replication complexes have been associated with membranes (dennison and sims, ; snijder et al., ) . similarly, the tentative location of protein within the nucleus (garwes et al., ) could be taken as an indication for a possible interference with the cell cycle, similarly to coronavirus nucleoprotein that seems to interact with cell nucleolus proteins and interfere with cell cycle (hiscox, ) . therefore, genes a, b, and of group coronaviruses are nonessential for replication. similarly, genes a/he and / a, and possibly gene e, are dispensable in mhv (de haan et al., ; kuo and masters, ) . manipulation of transcription-regulating sequences and deletion of nonessential genes, such as gene , will facilitate the study of the molecular basis of viral attenuation and provide an attractive approach to generate attenuated rtgevs with high potential as virus vectors. baby hamster kidney cells (bhk- ) stably transformed with the gene coding for the porcine aminopeptidase n (bhk-papn) (delmas et al., ) were grown in dmem supplemented with % fetal calf serum and geneticin g ( . mg/ml) as a selection agent. bhk-papn cells were used for all the experiments except for standard virus titrations that were performed in porcine swine testis (st) cells (mcclurkin and norman, ) . virus titers were compared by the kruskal-wallis test (motulsky, ) and, when significant (p Ͻ . ), it was indicated. rtgev viruses were generated from pbac-tgev constructs containing the s gene from the virulent tgev strain pur-c (sc ) as described (almazán et al., ; gonzalez et al. ) . viruses containing the s gene from the attenuated strain ptv (sptv) were derived from the corresponding pbac tgev vectors with gene sc by replacing this gene by that (sptv) of the respiratory strain. two different approaches were followed to introduce into pbac-tgev unique restriction endonuclease sites separating each gene of tgev genome (fig. a) , leading to pbac-tgev-rs. the first approach was the introduction of punctual mutations in the tgev genome to generate the restriction sites paci (between genes rep b and s) and mlui (between genes s and a) (fig. b) . due to overlapping in the tgev genome, the second approach involved the duplication of , , and nucleotides from the Ј transcription-regulating sequences of genes m, n, and (trs-m, trs-n, and trs- ) after the restriction sites fsei, pmei, and asci, respectively ( fig. a and b) . point mutations, duplications, and insertion of restriction endonuclease sites were generated by overlapping pcr amplification from the tgev genome as described (ortego et al., ) . the assembly of the full-length pbac-tgev constructs was performed as previously reported (almazán et al., ; gonzález et al., ) . to generate the deletion ⌬ , oligonucleotide primers Ј-asc- . -vs ( Ј-gaggcgcgcctgctgtatttat-tacag- Ј), including the restriction site asci (underlined) and the deletion of nucleotides from trs- plus the first nucleotides of orf , and bgh -rs ( Ј-cagatg-gctggcaactagaaggc- Ј) were used to generate a pcr product comprising from nt , to nt , of the tgev cdna clone. pcr product was digested with asci and bamhi and cloned into the asci-bamhi-digested pbac-tgev-sc -rs and pbac-tgev-sptv-rs, generating pbac-tgev-sc -rs-⌬ and pbac-tgev-sptv-rs-⌬ , respectively. bhk-papn cells were grown to % confluence on -mm-diameter plates and transfected with g of cdna plasmid and g of lipofectin (life technologies, gibco) according to the manufacturer's specifications. recovery and amplification of viruses were performed as described (ortego et al., ) . cell lysates were analyzed by to % gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page). proteins were transferred to a nitrocellulose membrane and analyzed as described (ortego et al., ) , using mabs specific for s ( b.h ), n ( d.c ), m ( d.b ), and e (v ) proteins (ortego et al., ) , and a swine polyclonal antibody specific for tgev protein (provided by p. britton, compton, uk). total rna was extracted by using an ultraspec rna isolation system (biotecx) according to the manufacturer's instructions and analyzed by northern blotting as described (ortego et al., ) . two-to three-day-old non-colostrum-deprived swine, from crossing large white and belgium landrace, were used to study in vivo growth kinetics of rtgev, as described (sanchez et al., ) . piglets were obtained from sows seronegative for tgev, as tested by radioimmunoassay. engineering the largest rna virus genome as an infectious bacterial artificial chromosome sequence of the nucleocapsid gene from murine coronavirus mhv-a sequences of the nucleocapsid genes from two strains of avian infectious bronchitis virus the complete genome sequence of gill-associated virus of penaeus monodon prawns indicates a gene organisation unique among nidoviruses the group-specific murine coronavirus genes are not essential, but their deletion, by reverse genetics, is attenuating in the natural host genetic manipulation of equine arteritis virus using full-length cdna clones: separation of overlapping genes and expression of a foreign epitope further characterization of aminopeptidase-n as a receptor for coronaviruses mhv-a gene proteins are associated with two distinct membrane populations coronaviridae nidovirales the polypeptide of mr of porcine transmissible gastroenteritis virus: gene assignment and intracellular location stabilization of a full-length infectious cdna clone of transmissible gastroenteritis coronavirus by the insertion of an intron nucleotide sequence of the human coronavirus e rna polymerase locus the molecular genetics of feline coronavirus comparative sequence analysis of the orf a/ b transcription unit of different biotypes the nucleolus-a gateway to viral infection? sequence analysis of nucleocapsid gene and leader rna of human coronavirus oc deletions in the a orf of feline coronavirus associated with an epidemic of feline infectious peritonitis genetic evidence for a structural interaction between the carboxy termini of the membrane and nucleocapsid proteins of mouse hepatitis virus the molecular biology of coronaviruses sequence analysis of the bovine coronavirus nucleocapsid and matrix protein genes a summary of taxonomic changes recently approved by ictv studies on transmissible gastroenteritis of swine. ii. selected characteristics of a cytopathogenic virus common to five isolates from transmissible gastroenteritis comparing two paired groups: paired t and wilcoxon tests generation of a replication competent, propagation-deficient virus vector based on the transmissible gastroenteritis coronavirus genome complete genome sequence of transmissible gastroenteritis coronavirus pur -mad clone and evolution of the purdue virus cluster transmissible gastroenteritis targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence coding sequence of coronavirus mhv-jhm mrna non-structural proteins and interact to modify host cell membranes during the formation of the arterivirus replication complex the -kda hydrophobic protein encoded at the Ј end of the porcine transmissible gastroenteritis coronavirus genome is membrane-associated adding genes to the rna genome of vesicular stomatitis virus: positional effects on stability of expression this work was supported by grants from the comisión interministerial de ciencia y tecnología (cicyt), la consejería de educación y cultura de la comunidad de madrid, fort dodge veterinaria, and the european union (frame v, key action , control of infectious disease projects qlrt- - , qlrt- - , and qlrt- . i.s. received postdoctoral fellowships from the community of madrid and the european union (frame v, key action , control of infectious disease projects). key: cord- -p ma akz authors: enjuanes, luis; almazán, fernando; ortego, javier title: virus-based vectors for gene expression in mammalian cells: coronavirus date: - - journal: new comprehensive biochemistry doi: . /s - ( ) -x sha: doc_id: cord_uid: p ma akz publisher summary the coronavirus and the torovirus genera form the coronaviridae family, which is closely related to the arteriviridae family. both families are included in the nidovirales order. recently, a new group of invertebrate viruses, the roniviridae, with a genetic structure and replication strategy similar to those of coronaviruses, has been described. this new virus family has been included within the nidovirales. coronaviruses have several advantages as vectors over other viral expression systems: ( ) coronaviruses are single-stranded rna viruses that replicate within the cytoplasm without a dna intermediary, making integration of the virus genome into the host cell chromosome unlikely, ( ) these viruses have the largest rna virus genome and, in principle, have room for the insertion of large foreign genes, ( ) a pleiotropic secretory immune response is best induced by the stimulation of gut-associated lymphoid tissues, ( ) the tropism of coronaviruses may be modified by manipulation of the spike (s) protein allowing engineering of the tropism of the vector, ( ) non-pathogenic coronavirus strains infecting most species of interest (human, porcine, bovine, canine, feline, and avian) are available to develop expression systems, and ( ) infectious coronavirus cdna clones are available to design expression systems. within the coronavirus two types of expression vectors have been developed: one requires two components (helper–dependent expression system) and the other a single genome that is modified either by targeted recombination or by engineering a cdna encoding an infectious rna. this chapter focuses on the advantages and limitations of these coronavirus expression systems, the attempts to increase their expression levels by studying the transcription-regulating sequences (trss), and the proven possibility of modifying their tissue and species-specificity. the coronavirus and the torovirus genera form the coronaviridae family, which is closely related to the arteriviridae family. both families are included in the nidovirales order [ , ] . recently, a new group of invertebrate viruses, the roniviridae, with a genetic structure and replication strategy similar to those of coronaviruses, has been described [ ] . this new virus family has been included within the nidovirales [ ] . coronaviruses have several advantages as vectors over other viral expression systems: (i) coronaviruses are single-stranded rna viruses that replicate within the cytoplasm without a dna intermediary, making integration of the virus genome into the host cell chromosome unlikely [ ] ; (ii) these viruses have the largest rna virus genome and, in principle, have room for the insertion of large foreign genes [ , ] ; (iii) a pleiotropic secretory immune response is best induced by the stimulation of gut associated lymphoid tissues. since coronaviruses in general infect the mucosal surfaces, both respiratory and enteric, they may be used to target the antigen to the enteric and respiratory areas to induce a strong secretory immune response; (iv) the tropism of coronaviruses may be modified by manipulation of the spike (s) protein allowing engineering of the tropism of the vector [ , ] ; (v) non-pathogenic coronavirus strains infecting most species of interest (human, porcine, bovine, canine, feline, and avian) are available to develop expression systems; and (vi) infectious coronavirus cdna clones are available to design expression systems. within the coronavirus two types of expression vectors have been developed (fig. ) , one requires two components (helper-dependent expression system) (fig. a ) and the other a single genome that is modified either by targeted recombination [ ] (fig. b. ) or by engineering a cdna encoding an infectious rna. infectious cdna clones are available for porcine [ , ] (fig. b. and b. ), human ( fig. b. ) [ ] , murine [ ] and avian (infectious bronchitis virus, ibv) coronavirus [ ] , and also for the arteriviruses equine infectious anemia virus (eav) [ ] , porcine respiratory and reproductive syndrome virus (prrsv) [ ] , and simian hemorrhagic fever virus (shfv) [ ] . the availability of these cdnas and the application of target recombination to coronaviruses [ ] have been essential for the development of vectors based on coronaviruses and arteriviruses. this review will focus on the advantages and limitations of these coronavirus expression systems, the attempts to increase their expression levels by studying the transcription-regulating sequences (trss), and the proven possibility of modifying their tissue and species-specificity. coronaviruses comprise a large family of viruses infecting a broad range of vertebrates, from mammalian to avian species. coronaviruses are associated mainly with respiratory, enteric, hepatic and central nervous system diseases. in humans and fowl, coronaviruses primarily cause upper respiratory tract infections, while porcine and bovine coronaviruses (bcovs) establish enteric infections that result in severe economical loss. human coronaviruses (hcov) are responsible for - % of all common colds, and have been implicated in gastroenteritis, high and low respiratory tract infections and rare cases of encephalitis. hcov have also been associated with infant necrotizing enterocolitis and are tentative candidates for multiple sclerosis. in march , a new group of hcovs has emerged as the ethiological agent of the severe acute pneumonia syndrome (sars) affecting thousands of people, mostly in china, singapore, and toronto. in addition, human infections by coronaviruses seem to be ubiquitous, as coronaviruses have been identified wherever they have been looked for, including north and south america, europe, and asia and no other human disease has been clearly associated with them with the exception of respiratory and enteric infections. virions contain a single molecule of linear, positive-sense, single-stranded rna (fig. ) . the coronavirus genome with a size ranging from . to . kb is the largest viral rna known. coronavirus rna has a terminal cap followed by a leader sequence of - nucleotides and an untranslated region of - nucleotides. at the end of the genome there is an untranslated region of - nucleotides followed by a poly(a) tail. the virion rna, which functions as an mrna and is infectious, contains approximately - functional genes, four or five of which encode structural proteins. the genes are arranged in the order -polymerase-(he)-s-e-m-n- , with a variable number of other genes that are believed to be non-structural and largely non-essential, at least in tissue culture [ ] . about two-thirds of the entire rna comprises the rep a and rep b genes. at the overlap between the rep a and b regions, there is a specific seven-nucleotide ''slippery'' sequence and a pseudoknot structure (ribosomal frameshifting signal), which are required for the translation of rep b as a single polyprotein (rep a/b). the third of the genome comprises the genes encoding the structural proteins and the other non-structural ones. coronavirus transcription occurs via an rna-dependent rna synthesis process in which mrnas are transcribed from negative-stranded templates. coronavirus mrnas consist of six to eight types of varying sizes, depending on the coronavirus strain and the host species. the largest mrna is the genomic rna that also serves as the mrna for rep a and b, the remainder are subgenomic mrnas (sgmrnas). the mrnas have a nested-set structure in relation to the genome structure (fig. b ). coronaviruses are enveloped viruses containing a core that includes the ribonucleoprotein formed by the rna and nucleoprotein n ( fig. a) . the core is formed by the genomic rna, the n protein and the membrane (m) protein carboxyterminus. most of the m protein is embedded within the membrane but its carboxyterminus is integrated within the core and seems essential to maintain the core structure [ , ] . at least in the transmissible gastroenteritis virus (tgev) the m protein presents two topologies. in one (m ), both the amino and the carboxyl termini face the outside of the virion, while in the other (m) the carboxy-terminus is inside [ ] . in addition, the virus envelope contains two or three other proteins, the spike (s) protein that is responsible for cell attachment, the small membrane protein (e) and, in some strains, the hemagglutinin-esterase (he) [ ] . the replicase gene encodes a protein of approximately - kda which is co-translationally processed. several domains within the replicase have predicted functions based on regions of nucleotide homology [ ]. the coronaviruses have been classified into three groups ( , and ) based on sequence analysis of a number of coronavirus genes [ ] . helper-dependent expression systems have been developed using members of the three groups of coronaviruses ( fig. ). group coronaviruses include porcine, canine, feline and hcovs. expression systems have been developed for the porcine and hcovs since minigenomes are only available for these two coronaviruses. one expression system has been developed using tgev-derived minigenomes [ ] . the tgev-derived rna minigenomes were successfully expressed in vitro using t polymerase and amplified after in vivo transfection using a helper virus. tgev-derived minigenomes of . , . and . kb were efficiently used for the expression of heterologous genes [ , ] . the smallest minigenome replicated by the helper virus and efficiently packaged was . kb in length [ ] . using m minigenome, a two-step amplification system was developed based on the cloning of a cdna copy of the minigenome after the immediate-early cytomegalovirus promoter (cmv) [ ] . minigenome rna is first amplified in the nucleus by the cellular rna pol ii and then, the rnas are translocated into the cytoplasm where they are amplified by the viral replicase of the helper virus. the -glucuronidase (gus) and a surface glycoprotein (orf ), that is the major protective antigen of the prssv, have been expressed using this vector [ ] . tgevderived helper expression systems have a limited stability and minigenomes without the foreign gene replicate about -fold more efficiently than those with the heterologous gene [ ] . expression of gus gene and prrsv orf with these minigenomes has been demonstrated in the epithelial cells of alveoli and in scattered pneumocytes of swine lungs, which led to the induction of a strong immune response to these antigens [ ] . the hcov- e has also been used to express new sgmrnas [ ] . it was demonstrated that a synthetic rna including nt from the end plus nt from the end was amplified by the helper virus. most of the work has been done with mouse hepatitis virus (mhv) defective rnas (minigenomes) [ , ] . three heterologous genes have been expressed using the mhv system, chloramphenicol acetyltransferase (cat), he, and interferon (ifn-). expression of cat or he was detected only in the first two passages because the minigenome used lacks the packaging signal [ ] . when virus vectors expressing cat and he were inoculated intracerebrally into mice, he-or cat-specific sgmrnas were only detected in the brains at days and , indicating that the genes in the minigenome were expressed only in the early stage of viral infection [ ] . a mhv minigenome rna was also developed as a vector for expressing ifn-. the murine ifn-gene was secreted into culture medium as early as h posttransfection and reached a peak level at h post-transfection. no inhibition of virus replication was detected when the cells were treated with ifn-produced by the minigenome rna, but infection of susceptible mice with a minigenome producing ifn-caused significantly milder disease, accompanied by less virus replication than that caused by virus containing a control vector [ , ] . ibv is an avian coronavirus with a single-stranded, positive-sense rna genome of , nt. a defective rna (cd- ) derived from the beaudette strain of the ibv virus was used as an rna vector for the expression of two reporter genes, luciferase and cat [ ] . helper-dependent expression systems have a limited stability probably due to the foreign gene since tgev minigenomes of . , . and . kb, in the absence of the heterologous gene, are amplified and efficiently packaged for at least passages, without generating new dominant subgenomic rnas [ ] . the expression of gus, prssv orf , or cat using tgev-or ibv-derived minigenomes in general increases until passages three or four, expression levels are maintained for about four additional passages, and steadily decrease during successive passages [ ] [ ] [ ] ] . using ibv minigenomes cat expression levels between and mg per cells have been described. the highest expression levels ( - mg of gus per cells) have been obtained using a two-step amplification system based on tgev derived minigenomes with optimized trss [ , ] . using minigenomes derived from tgev and ibv expression was highly dependent on the nature of the heterologous gene used. luciferase expression with tgev and ibv minigenomes was reduced to almost background levels, while expression of gus or cat was at least - -fold higher than background levels, respectively. the construction of cdna clones encoding full-length coronavirus rnas has considerably improved the genetic manipulation of coronaviruses. the enormous length of the coronavirus genome and the instability of plasmids carrying coronavirus replicase sequences have hampered, until recently, the construction of a full-length cdna clone. infectious coronavirus cdna clones have been described for coronaviruses [ , , ] and for arteriviruses [ , ] . the strategy used to clone tgev infectious cdna was based on three points [ ] : (i) the construction was started from a minigenome that was stably and efficiently replicated by the helper virus [ ] . during the filling in of minigenome deletions a cdna fragment that was toxic to the bacterial host was identified. this fragment was reintroduced into the cdna in the last cloning step; (ii) in order to express the long coronavirus genome and to add the cap required for tgev rna infectivity, a two-step amplification system that couples transcription in the nucleus from the cmv promoter, with a second amplification in the cytoplasm driven by the viral polymerase, was used; and (iii) to increase viral cdna stability within bacteria, the cdna was cloned as a bacterial artificial chromosome (bac), that produces a maximum of two plasmid copies per cell. a fully functional infectious tgev cdna clone, leading to a virulent virus infecting both the enteric and respiratory tract of swine was engineered. the stable propagation of a tgev full-length cdna in bacteria as a bac has been considerably improved by the insertion of an intron to disrupt a toxic region identified in the viral genome (fig. ) [ ] . the viral rna was expressed in the cell nucleus under the control of the cmv promoter and the intron was efficiently removed during translocation of this rna to the cytoplasm. intron insertion in two different positions (nt and ) allowed stable plasmid amplification for at least generations. infectious tgev was efficiently recovered from cells transfected with the modified cdnas. the great advantage of this system is that the performance of coronavirus reverse genetics only involves recombinant dna technologies carried out within the bacteria. using tgev cdna the green fluorescent protein (gfp) gene of . kb was cloned in two positions of the rna genome: either by replacing the non-essential a and b genes or between genes n and . the engineered genome was very stable (> passages in cultured cells) and led to the production of high expression levels ( mg/ cells) when the gfp replaced genes a/b but was unstable when cloned between genes n and [ ] . in this case, the gfp gene was eliminated by homologous recombination between preexisting trs sequences and those introduced to express gfp. using the most stable vector, the acquisition of immunity by newborn piglets breast fed by immunized sows (lactogenic immunity) was demonstrated [ ] . gus expression levels using coronavirus based vectors are similar (fig. ) to those described for vectors derived from other positive strand rna viruses such as sindbis virus ( mg per cells) [ ] . a second procedure to assemble a full-length infectious construct of tgev was based on the in vitro ligation of six adjoining cdna subclones that span the entire tgev genome [ ] . each clone was engineered with unique flanking interconnecting junctions that determine a precise assembly with only the adjacent cdna subclones, resulting in a tgev cdna. in vitro transcripts derived from the full-length tgev construct were infectious. using this construct, a recombinant tgev was assembled that replaced orf a with the gfp gene, leading to the production of a recombinant tgev that grew with titers of pfu/ml and expressed gfp in a high proportion of cells [ ] . an infectious cdna clone has also been constructed for hcov- e, another member of group coronaviruses [ ] . in this case, the system is based on the in vitro transcription of infectious rna from a cdna copy of the hcov- e genome that has been cloned and propagated in vaccinia virus (fig. ) . briefly, the full-length genomic cdna clone of hcov- e was assembled by in vitro ligation, and then cloned into the vaccinia virus dna under control of the t promoter. recombinant vaccinia viruses containing the hcov- e genome were recovered after transfection of the recombinant vaccinia virus dna into cells infected with fowlpox virus. in a second phase, the recombinant vaccinia virus dna was purified and used as a template for in vitro transcription of hcov- e genomic rna that was transfected into susceptible cells for the recovery of infectious recombinant coronavirus (fig. ) . a coronavirus replicon has been derived from the hcov genome using the same procedure described for the full-length genome construction [ ] . this replicon included the and ends of the hcov- e genome, the replicase gene of this virus and a single reporter gene coding for gfp located downstream of a trs element for coronavirus mrna transcription. when rna transcribed from this cdna was transfected into bhk- cells, only . % of the cells showed strong fluorescence. this data shows that the coronavirus replicase gene products suffice for discontinuous sgmrna transcription, in agreement with the requirements for the arterivirus replicase [ ] . the expression of a heterologous gene (gfp) by a tgev replicon was increased between -and -fold when tgev n protein was in cis co-expressed [ ] . in addition, expression from a tgev replicon was also observed when n protein was in trans co-expressed using the venezuelan equine encephalitis virus vector [ ] . furthermore, expression from hcov based vectors also was significantly increased by co-expression of n gene. therefore, it seems that n protein either stabilizes coronavirus replicons or increases their replication, transcription or translation. reverse genetics in this coronavirus group has been efficiently performed by targeted recombination between a helper virus and either non-replicative or replicative coronavirus-derived rnas (fig. b. ). this approach, developed by masters' group [ , ] , was first applied to the engineering of a five-nucleotide insertion into the untranslated region ( utr) of mhv [ ] . this approach was facilitated by the availability of an n gene mutant, designated alb , that was both temperaturesensitive and thermolabile. alb forms tiny plaques at restrictive temperature that are easily distinguishable from wild-type plaques. in addition, incubation of alb virions at non-permissive temperature results in a -fold greater loss of titer than for wild-type virions. these phenotypic traits allowed the selection of recombinant viruses generated by a single crossover event following cotransfection into mouse cells of alb genomic rna together with a synthetic copy of the smallest subgenomic rna (rna ) tagged with a marker in the utr. an improvement of the recombination frequency was obtained between the helper virus and replicative defective rnas as the donor species. whereas between replication competent mhv and non-replicative rnas a recombination frequency of the order of À was estimated, the use of replicative donor rna yielded recombinants at a rate of some three orders of magnitude higher [ ] . this higher efficiency made it possible to screen for recombinants even in the absence of selection. in this manner, the transfer of silent mutation in rep a gene of a minigenome to wild-type mhv at a frequency of about % was demonstrated. targeted recombination has been applied to the generation of mutants in most of the coronavirus genes. thus, two silent mutations have been created so far in gene a [ ] . the s protein has also been modified by targeted recombination. changes were introduced by one crossover event at the end of the s gene that modified mhv pathogenicity [ ] . targeted recombination mediated by two cross-overs allowed the replacement of the s gene of a respiratory strain of tgev by the s gene of enteric tgev strain pur-c leading to the isolation of viruses with a modified tropism and virulence [ ] . in this case the recombinants were selected in vivo using their new tropism in piglets. a new strategy for the selection of recombinants within the s gene, after promoting targeting recombination, was based on elimination of the parental replicative tgev by the simultaneous neutralization with two mabs (i. sola and l. enjuanes, unpublished results). mutations have also been introduced by targeted mutagenesis within the e and m genes. these mutants provided corroboration for the pivotal role of e protein in coronavirus assembly and identified the carboxyl terminus of the m molecule as crucial to assembly [ ] . targeted recombination was also used to express heterologous genes. for instance, the gene encoding gfp was inserted into mhv between genes s and e, resulting in the creation of the largest known rna viral genome [ ] . an infectious mhv cdna clone has recently been assembled in vitro. a method similar to the one developed to assemble an infectious tgev cdna clone based on the in vitro ligation of seven contiguous cdna subclones has been applied to the construction of a cdna that spanned the . kb of the mhv a strain [ ] . the ends of the cdnas were engineered with unique junctions, which were directed to assembly with only the adjacent cdnas subclone, resulting in an intact mhv-a cdna construct. the interconnecting restriction site junctions that are located at the ends of each cdna are systematically removed during the assembly of the complete full-length cdna product, allowing reassembly without the introduction of nucleotide changes. rna transcripts derived from the full-length mhv-a construct was infectious, although virus recovery was enhanced - -fold in the presence of rna transcripts encoding the nucleocapsid protein, n. the infectious ibv cdna clone was assembled using the same strategy reported for hcov- e with some modifications [ ] . similarly to hcov- e, the ibv genomic cdna was assembled downstream of the t promoter by in vitro ligation and cloned into the vaccinia virus dna. however, recovery of recombinant ibv was done after the in situ synthesis of infectious ibv rna by transfection of restricted recombinant vaccinia virus dna (containing the ibv genome) into primary chick kidney cells previously infected with a recombinant fowlpox expressing t rna polymerase. engineered cdnas are having an important impact on the study of mechanisms of coronavirus replication and transcription and provide an invaluable tool for the experimental investigation of virus-host interactions. replication-competent propagation-deficient virus vectors based on tgev genomes deficient in the essential gene e that are complemented in packaging cell lines have been developed [ , ] . two types of cell lines expressing tgev e protein have been established, one with transient expression using the non-cytopathic sindbis virus replicon psinrep (fig. ) and another stably expressing e gene under the cmv promoter. the rescue of recombinant tgev deficient in the non-essential a and b genes, and the essential e gene reached high titers (>  pfu/ml) in cells transiently expressing the tgev e protein, while this titer was up to  pfu/ml in packaging cell lines stably expressing protein e. interestingly, virus titers were related to protein e expression levels [ ] . recovered virions showed the same morphology and stability at different ph and temperatures than the wild type virus. a second strategy for the construction of replication-competent propagationdefective tgev genomes expressing heterologous genes, involves the assembly of an infectious cdna from six cdna fragments that are ligated in vitro [ ] . the defective virus with the essential e gene deleted was complemented by the expression of e gene using the venezuelan equine encephalitis replicon expression vector. however, titers of recombinant tgev-Áe expressing the gfp were at least -or -fold lower (around pfu/ml) than with the system using stably transformed cells or the sin vector to complement deletion of the e gene [ ] . coronavirus minigenomes have a theoretical cloning capacity close to kb, since their rna with a size of about kb is efficiently amplified and packaged by the helper virus and the virus genome has about kb. in contrast, the theoretical cloning capacity for an expression system based on a single coronavirus genome like tgev according to current available knowledge is between and . kb taking into account that: (i) the non-essential genes a ( . kb), b ( . kb), and most of gene [ ] have been deleted leading to a viable virus; (ii) the standard s gene can be replaced by the s gene of a porcine respiratory coronavirus (prcv) mutant with a deletion of . kb; and (iii) both dna and rna viruses may accept genomes with sizes up to % of the wild type genome. this cloning capacity will probably be enlarged by deleting non-essential domains of the replicase gene. these domains are being identified by comparing the arterivirus replicase gene (i.e., for eav .  nt) and that of coronavirus (i.e., for tgev .  nt) [ ] . differences in size between these two replicase genes could correspond to nonessential domains in the coronavirus replicase that may be dispensable. to optimize expression levels it is essential to improve virus vector replication levels without increasing virulence, to optimize the accumulation of total mrna levels, and to improve mrna translation. these results can only be achieved by determining the mechanism involved in these processes. a brief review of the mechanism of mrna transcription in coronavirus and arterivirus is described to help achieve this goal. coronavirus rna synthesis occurs in the cytoplasm via a negative-strand rna intermediate that contains short stretches of oligo(u) at the end. both genome-size and subgenomic negative-strand rnas, which correspond in number of species and size to those of the virus-specific mrnas have been detected [ , ] . coronavirus mrnas have a leader sequence at their ends. at the start site of every transcription unit on the viral genomic rna, there is a trs that includes a highly conserved core sequence (cs) that is nearly homologous to the end of the leader rna. this sequence constitutes part of the signal for sgmrna transcription. the common leader sequence is only found at the very terminus of the genome, which implies that the synthesis of sgmrnas involves fusion of non-contiguous sequences. the mechanism involved in this process is under debate. nevertheless, the discontinuous transcription during negative-strand rna synthesis model is compatible with most of the experimental evidence [ ] [ ] [ ] . because the leader-mrna junction occurs during the synthesis of the negative strand within the sequence complementary to the cs (ccs) the nature of the cs is considered crucial for mrna synthesis. transcription levels may be influenced by many factors. the three that we [ ] consider most relevant are: (i) potential base pairing between the leader end and sequences complementary to the trs located at the end of each nidovirus gene (ctrs), that guide the fusion between the nascent negative strand and the leader trs. a minimum complementarity is needed between the leader-trs and the ctrss of each gene. extension of this complementarity increases mrna synthesis up to a certain extent, beyond a certain extension addition of or cs flanking sequences does not help transcription [ , , ] ; (ii) proximity of a gene to the . since the trss act as signals to slow down or stop the replicase complex, the smaller mrnas should be the most abundant. although this has been shown to be the case in the mononegavirales [ ] and in coronaviruses shorter mrnas are in general more abundant, the relative abundance of coronavirus mrnas is not strictly related to their proximity to the end [ , ] ; and (iii) potential interaction of proteins with the trss rna, and protein-protein interactions that could regulate transcription levels. the reassociation of the nascent rna chain with the leader trs is probably mediated by approximation of the leader trs through rna-protein and protein-protein interactions. the three factors implicated in the control of mrna abundance assume a key role for the trs. hence, in order to engineer vectors with high expression levels, it seems relevant to define the characteristics of the trs, including the size of the and trs sequences flanking the cs. the cs of coronaviruses belonging to groups i (hexamer -cuaaac- ) and ii (heptamer -ucuaaac- ) share homology, whereas the cs of coronaviruses belonging to group iii, like that of ibv, have the most divergent sequence ( -cuuaacaa- ). also, arterivirus css have a sequence ( -ucaacu- ) that partially resembles that of ibv. thus, the css of different coronaviruses are quite similar though slightly different in length. this cs is essential for mrna synthesis, and can be considered to be a defined domain in the trs because it is particularly conserved within a nidovirus family, while the flanking sequences, both at the ( trss) and at the ( trss) have a unique composition for each gene even within the same virus. the influence of the cs in transcription has been analyzed in detail in the arteriviruses [ , ] . using an infectious cdna clone of eav it has been shown that sgmrna synthesis requires base pairing interaction between the leader trs and ctrs in the viral negative strand. the construction of double mutants in which a mutant leader cs was combined with the corresponding mutant rna body cs, resulting in the specific restoration of mrna synthesis, suggested that the sequence of the cs per se is not crucial, as long as the possibility for cs base pairing is maintained. nevertheless, it has been shown that other factors, besides leader-body base pairing, also play a role in sgmrna synthesis and that the primary sequence (or secondary structure) of trss may dictate strong base preferences at certain positions [ ] . in addition, detailed analysis of the trs used in the arteriviruses [ ] , mhv [ ] , bcov [ ] , and tgev [ ] indicate that non-canonical cs sequences may also be used for the switch during the discontinuous synthesis of the negative strand during transcription in the nidovirales. the promotion of transcription from a given cs is also a function of the cs flanking sequences. data from different laboratories working with different nidoviruses have shown that cs flanking sequences can critically influence the strength of a given fusion site [ , , , [ ] [ ] [ ] . although approximations to the definition of the trs have been made, the precise length of the trs requires further work to optimize accumulation of mrna levels. studies on coronavirus transcription were performed using more than one cs to express the same mrna. the accumulated amounts of sgmrna remained nearly the same for constructs with one to three css, and transcription preferentially occurred at the -most trs [ , [ ] [ ] [ ] . this observation is consistent with the coronavirus discontinuous transcription during the negative-strand synthesis model [ ] . driving vector expression to different tissues may be highly convenient in order to preferentially induce a specific type of immune response, i.e., mucosal immunity by targeting the expression to gut-associated lymph nodes. in addition, it seems useful to change the species specificity of the vector to expand its use. both tissue-and species-specificity have been modified using coronavirus genomes. group coronaviruses attach to host cells through the s glycoprotein by interactions with aminopeptidase n (apn) which is the cellular receptor [ , ] . group coronaviruses use the carcinoembryonic antigen-related cell adhesion molecules (ceacam) as receptors. engineering the s gene can lead to changes both in the tissue-and species-specificity [ , ] . tropism change in general leads to a change in virulence. certainly this is the case in porcine coronavirus with a virulence directly related to its ability to grow in the enteric tract [ ] . gene expression among the non-segmented negative-stranded rna viruses is controlled by the highly conserved order of genes relative to the single transcriptional promoter. rearrangement of the genes of vesicular stomatitis virus eliminates clinical disease in the natural host and is considered a new strategy for vaccine development [ ] . in coronavirus, genes closer to the end are in general expressed more abundantly than end distal ones and, in principle, gene order change can also lead to virus attenuation (p. rottier, personal communication). the arteriviridae include four members: eav, prrsv, shfv and lactate dehydrogenase-elevating virus of mice (ldhv). defective genomes of eav have been isolated and used to express a reporter gene (cat) in cell culture [ ] . more interestingly, infectious cdna clones have been obtained for eav [ ] , prrsv [ , ] and shfv [ ] creating the possibility of specifically altering their genomes for vector development and vaccine production. to insert genes in different positions a unique restriction endonuclease site has been introduced between consecutive eav genes [ ] . the viruses recovered expressed epitopes of nine amino acids from mhv within the ectodomain of the membrane (m) protein for at least three passages [ ] . foreign epitopes have also been expressed by using prrsv vectors [ ] . both helper-dependent expression systems, based on two components, and single genomes constructed by targeted recombination, or by using infectious cdnas, have been developed for coronaviruses. the sequences that regulate transcription have been characterized mainly using helper-dependent expression systems. these expression systems have the advantage of their large cloning capacity, in principle higher than kb, produce reasonable amounts of heterologous antigens ( - mg/ cells), show a limited stability (synthesis of heterologous gene is maintained for around passages), and elicit strong immune responses. in contrast, coronavirus vectors based on single genomes have at present a limited cloning capacity ( - . kb), expression levels of heterologous genes are -fold over those of helper dependent systems (> mg/ cells) and are very stable (> passages). furthermore, replication-competent propagation-deficient expression systems based on coronavirus genomes have been developed increasing the safety of these vectors. the possibility of expressing different genes under the control of trss with programmable strength, and engineering the tissue and species tropism indicate that coronavirus vectors are very flexible. thus, coronavirus-based vectors are emerging with a high potential for vaccine development and, possibly, for gene therapy. van regenmortel, m.h.v. virus taxonomy. classification and nomenclature of viruses virus taxonomy. classification and nomenclature of viruses proc. natl. acad. sci. usa the nidoviruses (coronaviruses and arteriviruses) proc. natl. acad. sci. usa proc. natl. acad. sci. usa proc. natl. acad. sci. usa this work has been supported by grants from the comisio´n interministerial de ciencia y tecnologı´a (cicyt), la consejerı´a de educacio´n y cultura de la comunidad de madrid, and fort dodge veterinaria from spain, and the european communities (life sciences program, key action : infectious diseases). key: cord- - bshqly authors: dong, wanyu; xie, wenting; liu, yunbo; sui, baokun; zhang, hao; liu, liran; tan, yubei; tong, xiaohan; fu, zhen f.; yin, ping; fang, liurong; peng, guiqing title: receptor tyrosine kinase inhibitors block proliferation of tgev mainly through p mitogen-activated protein kinase pathways date: - - journal: antiviral res doi: . /j.antiviral. . sha: doc_id: cord_uid: bshqly emerging coronaviruses (covs) primarily cause severe gastroenteric or respiratory diseases in humans and animals, and no approved therapeutics are currently available. here, a , a receptor tyrosine kinase inhibitor (rtki) of the tyrphostin class, is identified as a robust inhibitor of transmissible gastroenteritis virus (tgev) infection in cell-based assays. moreover, a exhibited potent antiviral activity against the replication of various covs, including murine hepatitis virus (mhv), porcine epidemic diarrhea virus (pedv) and feline infectious peritonitis virus (fipv). we further performed a comparative phosphoproteomic analysis to investigate the mechanism of action of a against tgev infection in vitro. we specifically identified p and jnk , which are the downstream molecules of receptor tyrosine kinases (rtks) required for efficient tgev replication, as a targets through plaque assays, qrt-pcr and western blotting assays. p and jnk inhibitors and rna interference further showed that the inhibitory activity of a against tgev infection was mainly mediated by the p mitogen-activated protein kinase (mapk) signaling pathway. all these findings indicated that the rtki a directly inhibits tgev replication and that its inhibitory activity against tgev replication mainly occurs by targeting p , which provides vital clues to the design of novel drugs against covs. coronaviruses (covs) are enveloped viruses possessing a singlestranded, positive-sense rna genome and belong to the family coronaviridae and the order nidovirales. the cov genome is currently the largest known viral rna genome, with a total length of - kb. covs consist of four genera: alpha-, beta-, gamma-, and a tentative new genus, deltacoronavirus (de groot et al., ) . covs commonly cause gastroenteric or respiratory diseases in animal hosts as well as in humans. given that there are currently no approved vaccines or antiviral strategies for many pathogenic covs (ramajayam et al., ) , it is increasingly important to identify broad-spectrum antiviral compounds. these compounds will promote quick responses to threats of new or changing pandemics, possibly even without accurate identification of the agents. transmissible gastroenteritis virus (tgev), the causative agent of porcine transmissible gastroenteritis, together with porcine epidemic diarrhea virus (pedv), human covs e (hcov- e) and canine covs (ccovs), belong to alpha coronavirus (carstens, ) . tgev causes fatal acute diarrhea, vomiting, and dehydration, with mortality rates of nearly % in suckling piglets less than weeks old (pritchard et al., ) , resulting in severe economic losses in the swine industry worldwide. approximately two-thirds of the ′-proximal region of the tgev genome encodes the replicase gene (rep), which contains two open reading frames (orf a and orf b). polyprotein a (pp a) and polyprotein ab (pp ab) are translated by rep (gorbalenya et al., ) and are proteolytically processed by virus-encoded proteases into non-structural proteins (nsps), nsps - , many of which have enzymatic activities, such as papain-like protease (plp or nsp ), c-like protease ( cl), rna-dependent rna polymerase (rdrp, nsp ) helicase (nsp ). these nsps along with putative cellular factors are believed to form replication/transcription complexes, which play an important role in cov rna transcription and replication (neuman et al., ) . as the crystal structures of a large number of viral nonstructural and structural proteins have been solved, targeted drug design has been attempted (tong, ) . unfortunately, such efforts have not led to advances in antiviral drugs beyond the preclinical stage (hilgenfeld and peiris, ) . overall, this target-based approach ignores other possible targets, including host cell signaling pathways or other host factors that are essential for cov replication. furthermore, rna viral genomes typically replicate with low fidelity and undergo rapid evolutionary changes. thus, targeting cellular factors involved in virus infection provides an excellent strategy for drug development because such treatment is not easily evaded by the high mutation rates in viral genomes (zhou et al., ) . protein kinases and phosphatases involve a wide variety of cellular functions. receptor tyrosine kinases (rtks) are a group of growth factor receptors and key components of the biological control networks that regulate many biological processes including cell proliferation and differentiation as well as survival (lemmon and schlessinger, ) . rtks also play an important role in transforming extracellular and intracellular signals and activating or linking them to downstream signaling pathways, such as the ras/mitogen-activated protein kinase (mapk), pi k/akt, and jak/stat pathways (pawson, ; schlessinger, ) . as rtks are both master regulators of normal cellular processes and play a vital role in the development and progression of various cancers, they have been extensively studied as targets for the treatment of many types of malignancies (roussidis and karamanos, ) . recently, a growing number of studies has shown that several rtks and other tyrosine kinases are involved in viral replication. for example, the receptor tyrosine kinase axl can function as an entry factor for dengue virus and zika virus (zikv) (meertens et al., ; meertens et al., ) . in addition, the protein tyrosine kinase inhibitor genistein was shown to block the replication of type- human immunodeficiency virus (hiv- ), herpes simplex virus type (hsv- ), and arenaviruses (stantchev et al., ; vela et al., ; yura et al., ) . ptp b, the protein tyrosine phosphatase was also shown to be a target for antiviral therapy (carbone et al., ) . imatinib, an abelson (abl) kinase inhibitor, was shown to be a potent inhibitor of both sars-cov and mers-cov in vitro (dyall et al., ) . two rtk inhibitors (rtkis), known as ag and tyrphostin a , can block multiple steps of influenza a virus replication, but both the underlying mechanism of this inhibitory effect and its target are unclear (kumar et al., a; kumar et al., b) . the raf/mek/erk pathways downstream of rtks are involved in murine hepatitis virus (mhv) rna synthesis (cai et al., ) , and a recent study indicated that epidermal growth factor receptor (egfr) is a promoter for tgev entry (hu et al., ) . in the current study, we used the model alpha-coronavirus tgev to screen inhibitors of cov replication using a high-throughput assay based on the pronounced cytopathic effect (cpe) caused by tgev infection in pk- cells. we identified tyrphostin a (a ), a specific rtki, as having a strong antiviral activity against tgev. in addition, a appears to be a broad-spectrum cov inhibitor, as it blocked the replication of mhv, pedv and feline infectious peritonitis virus (fipv) with comparable efficacy. furthermore, we employed both pharmacological inhibitors and rna interference to demonstrate that a dramatically suppresses viral replication and viral rna synthesis mainly through the p signaling pathway. our findings provide molecular insight into the potential role of host rtk signaling pathways in promoting the replication of covs and suggest that tyrphostin a could be developed as a potential anti-cov therapy. pk- , st, vero-ccl (african green monkey kidney epithelial cells) and ccl (cat kidney epithelial cells) were maintained at °c with a % co incubator in dulbecco's modified eagle's medium (dmem, invitrogen, carlsbad, ca, usa) supplemented with % fetal bovine serum (fbs) and penicillin-streptomycin ( units/ml). fipv - was purchased from american type culture collection (atcc®; manassas, va, usa). the pedv yn strain (genbank accession number: kt . ) and tgev wh- strain (genbank accession number: hq . ) were isolated from suckling piglets. pedv and fipv were propagated in vero-ccl and ccl cells, respectively. tgev was grown in pk- or st cells. the viral titers were determined by plaque assay on pk- (tgev), vero-ccl (pedv) or ccl (fipv) cells. infection of cultured cells with pedv was conducted in the presence of trypsin at a concentration of μg/ml with serum-free medium. library of pharmacologically active compounds (lopac ) was purchased from sigma-aldrich, each compound was dissolved to mm in dimethyl sulfoxide (dmso) and stored at − °c. tyrphostin a , ribavirin and flunarizine were purchased from sigma. the p inhibitor birb and the jnk inhibitor db were obtained from apexbio. ly was purchased from targetmol. a gapdh-specific monoclonal antibody was purchased from proteintech (chicago, il, usa). antibodies against p , jun-n-terminal kinases (jnks), phosphorylated p (p-p ) and phosphorylated jnks (p-jnks) were purchased from cell signaling technology (washington, dc, usa). the anti-phosphorylated p (p-p ) antibody is specific for y and t . the anti-tgev n polyclonal antibody was prepared in our laboratory. tyrphostin a , birb and db were stored as mm stock solutions in dmso. ly was stored as a mm stock solution in dmso. the cytotoxic effect of reagents on cell viability was measured in -well plates using celltiter-glo (promega) according to the manufacturer's protocol. cell viability was determined using a veritas microplate luminometer (promega) with values normalized to those of untreated cells. accordingly, throughout this work, no cytotoxicity was observed for a at μm, birb at μm, db at μm or ly at μm. the effects of a on the infection of tgev in pk- or st cells, pedv in vero cells, fipv in ccl- cells, and mhv in l cells were determined by comparing the levels of viral replication in target cells in the absence or presence of a . target cells were pretreated in triplicate with dmso or a ( μm) for h, followed by virus infection at a multiplicity of infection (moi) of . . cell supernatants were harvested at - h post infection (hpi), and viral titers were quantified by plaque formation assays. to assess the effect of the inhibitors of jnk and p on tgev, pk- cells were infected with tgev and treated with birb , db or dmso. the culture supernatants were collected at different time points ( , , , , and hpi) and stored at − °c. the tgev titer was determined by plaque assay using pk- cells as described previously and quantified as plaque-forming units (pfus) per ml. the antiviral mechanism of a was determined by time-of-addition assay as previously described (basu et al., ) ; the procedure is shown schematically in fig. a . pk- cells were infected with tgev at an moi of . for h ( h). a at μm was added to the cells at h preinfection (− h), during infection ( h), and h post-infection (+ h). to exclude a possible direct inactivating effect of a on tgev, the virus w. dong, et al. antiviral research ( ) was incubated with a at °c for h, and the mixtures were used to infect pk- cells for h. duplicate wells were used for each process. cell cultures treated with the drug vehicle (dmso) were used as a control. at hpi, the culture medium was harvested for virus titration. the samples used for quantitative proteomic analysis consisted of four groups: pk- cells infected at an moi of with tgev, uninfected cells that were treated with a , uninfected cells that were treated with dmso, and pk- cells infected with tgev and treated with a . at hpi, the cells were collected for protein extraction, digestion, and labeling. cell samples from four groups were collected with cell scrapers and washed twice with ice-cold phosphate-buffered saline (pbs) containing the protease inhibitor cocktail. the cells were lysed in μl of lysis buffer, and the remaining debris was removed by centrifugation at , ×g at °c for min. the supernatant was collected and the protein concentration was determined with a bicinchoninic acid (bca) kit according to the manufacturer's instructions. trypsin was added to the protein solution at a : trypsin-to-protein mass ratio for a first digestion overnight and at a : trypsin-to-protein mass ratio for a second h-digestion. the peptides were then labeled with different tmt tags and the labeled samples were mixed, desalted and dried by vacuum centrifugation. after fractionation by high-ph reverse-phase hplc using a thermo betasil c column, the tryptic peptides were dissolved in . % formic acid (solvent a) and directly loaded onto a homemade reverse-phase analytical column ( -cm length, μm i.d.). the gradient comprised an increase from % to % solvent b ( . % formic acid in % acetonitrile) over min, a further increase from % to % over min and a climb to % in min; the concentration was held at % for the last min. all steps were performed at a constant flow rate of nl/min with an easy-nlc ultra-performance liquid chromatography (uplc) system. the peptides were subjected to a nanoelectrospray ionization (nsi) source followed by tandem mass spectrometry (ms/ms) with a q exactive™ plus instrument (thermo) that was coupled online to the uplc. the applied electrospray voltage was . kv. the m/z scan range was to for full scans, and intact peptides were detected in the orbitrap at a resolution of , . peptides were then selected for ms/ms using a normalized collision energy (nce) setting of , and the fragments were detected in the orbitrap at a resolution of , . a data-dependent procedure that alternated between one ms scan and ms/ms scans with . s of dynamic exclusion was used. the automatic gain control (agc) was set at e . the fixed first mass was set as m/z. pk- cells were treated with birb , db or dmso for h prior to infection with tgev at . moi for h at °c. total rna from pk- cells was extracted using trizol reagent (invitrogen, grand island, ny) after , , , or hpi, respectively. cdna was generated using the revertra ace qpcr rt kit (toyobo, japan) according to the manufacturer's instructions. quantitative real-time pcr was performed using sybr green (bio-rad). n gene expression was calculated using a relative quantification ( −ΔΔct ) model and normalized to that of the internal control gapdh. the primers targeting the tgev n protein and gapdh were as follows: tgev n-specific primers (forward, ′-gagcagtgccaagcattaccc- '; reverse, ′-gacttctat ctggtc gccatcttc- ′) gapdh-specific primers (forward, ′-acatggcctcc aaggagtaaga- '; reverse, ′-gatcgagttggggctgtgact- ′). specific pig p sirna sequences were obtained from a published paper (gan et al., ) and synthesized by genepharma (shanghai, china). nc sirna was purchased from genepharma. the sequence of the p -specific sirna sequence was ′-gcaggagcugaacaagacatt- '. sirna transfection was performed with transfection reagent according to the manufacturer's instructions, and the efficiency of the sirna was characterized by western blotting. the proportion of the target protein was calculated using imagej software. pk- cells were transfected with p sirna or nc sirna; after h, the cells were infected with . moi tgev and treated with a for h or infected with . moi tgev only. cells treated with tgev and dmso at h post transfection were used as controls. virus yield in supernatants of the cultures was detected by the plaque assay. pk- cells were cultured in six-well plates and were mock-infected or infected with tgev at an moi of . . at the indicated post infection times, cells were harvested with lysis buffer (beyotime, china) containing protease inhibitors and were boiled for min with sample loading buffer (beyotime, china). the samples were fractionated by sds-page. gels were transferred onto polyvinylidene difluoride (pvdf) (merck millipore, usa), blocked in % bovine serum albumin or % dried skimmed milk in tbs ( mm tris-hcl [ph . ], mm nacl) at room temperature for h and then incubated with the indicated primary antibodies. after washing three times, the membranes were exposed to a species-specific horseradish peroxidase-conjugated secondary antibody for another h and washed three times followed by enhanced chemiluminescence (ecl, thermo scientific) detection by autoradiography (bio-rad). gapdh was used as a loading control. all experiments were performed in triplicate. data are presented as the mean ± standard deviation (sd). the differences in means were analyzed by student's t-test. p values less than . were considered statistically significant (*p < . , **p < . , and ***p < . ). to achieve a robust signal for high-throughput screening (hts), the pk- cell number, moi, and assay endpoint were optimized to meet the conditions of s/b > , z′ > . and cv < %. we then screened the library of pharmacologically active compounds for anti-tgev activity under the optimized conditions using a cpe-based hts assay. two compounds, flunarizine and a , exerted strong inhibitory effects on tgev replication in vitro and their structures are shown in fig. a . to eliminate cytotoxic effects, cell viability at various concentrations of flunarizine or a was evaluated using the mtt assay ( fig. b and d). ribavirin has previously been reported to inhibit mers-cov replication (falzarano et al., ) and was thus used as a positive control. compared to the dmso control, flunarizine and ribavirin were not cytotoxic in pk- cells at concentrations up to μm and μm, respectively (fig. b) . subsequent mtt assays showed no cytotoxicity up to μm a (fig. d ). to compare antiviral potencies, pk- cells were infected with tgev at an moi of . and then treated with dmso or compounds at various concentrations, and the virus yield in the supernatants was determined at hpi ( fig. c and e). compared to the vehicle control (dmso), both a and ribavirin displayed dose-dependent inhibitory activities against tgev in pk- cells. no inhibition was observed by treating cells with μm flunarizine, a concentration that was not cytotoxic. note that the strong viral inhibition produced by flunarizine at μm and a at μm might be due to cytotoxicity at those concentrations (fig. c) . based on the cytotoxicity and antiviral efficacy results, we chose a at μm for further evaluation in this study. taken together, these initial studies identified a as a specific rtki that can strongly suppress the tgev yield. to explore whether the antiviral activity of a is affected by the cell type or virus-to-cell ratio, viral inhibition assays were performed in epithelial and fibroblast derived cell types and at various mois. both pk- and st cells were treated with a or vehicle control (dmso) and infected with tgev at an moi of . , . , or . as peaks of tgev replication at different mois were observed at various time points, the viral yield in the supernatants was quantified at h (moi = . ), h (moi = . ), or h (moi = ) post infection. as shown in fig. a and b, a strongly inhibited tgev production in the two cell lines and at all tested virus-to-cell ratios, although some variation between the cell types was observed. in addition, a to > log decrease in infectious progeny was observed in the a treatment group compared to that of the dmso treatment control. taken together, the results demonstrate that the inhibition of tgev replication by a is not limited to a single cell type and is effective even with relatively high levels of input virus. compared with its behavior in st cells, a displayed greater inhibition against tgev in pk- cells. one explanation for this difference may be that a uptake differs per cell line. to determine the specific step(s) of the tgev life cycle inhibited by a , we performed time-of-addition assays. pk- cells were treated with a h before tgev infection (− h), at the same time as infection ( h) and h post-tgev infection ( h) (fig. c ). tgev infected pk- cells were treated with dmso as a control. the titer of infectious viral particles in the supernatant at hpi was measured by plaque assays. when added at either °c or °c during ( h) virus infection, a displayed relatively slight inhibitory activity against the tgev infection (fig. d ), suggesting that a has a low effect on virus binding and entry. in addition, there was no significant decrease in virus yield compared to the dmso-treated control when a was added before tgev attachment. interestingly, when a was added h post tgev infection, the virus yields were reduced by more than logs. these findings indicated that a mainly blocks the post-adsorption stage of the tgev life cycle. additionally, the inhibitory effect of a is likely exerted through cellular mechanisms rather than directly by reducing virion infectivity. to investigate whether the tgev inhibitor a is a potential broadspectrum cov inhibitor, we assessed its activity against three additional covs: the betacoronavirus mhv (strain a ) and the alphacoronaviruses fipv and pedv. the initial cell viability assay verified that a did not result in any signs of toxicity in vero-ccl , ccl- and a cells at concentrations up to μm (data not shown). cells were treated with a at μm or with dmso, followed by infection with virus at an moi of . . cell supernatants were harvested at hpi for mhv on a cells, hpi for fipv on ccl- cells or hpi for pedv on vero-ccl cells and titrated by the plaque assay (fig. ) . when an a dose of μm was used, pedv progeny titers were decreased by~ log. fipv and mhv production were reduced by a similar extent (~ and~ -log reduction, respectively). the overall results demonstrate that a displays cov inhibitory activity, inhibiting infectious pedv, mhv and fipv with similar potencies. to explore the downstream mechanism by which a might regulate a cellular anti-coronaviral response, we performed a comparative proteomics analysis to identify changes in the phosphorylation status using a tmt-labeled quantitative lc-ms/ms approach. pk- cells were subjected to four different experiments: mock control (untreated), a treated, tgev-infected pk- cells (tgev-infected), and tgev infection with a treatment (tgev/a ). after the three biological replicates were combined, a total of phosphosites corresponding to phosphorylated proteins were identified in the phosphoproteomic analysis among all groups (n = for each group), of which phosphorylated proteins were quantified (table s ). the proteins that met the criteria of a p < . and fold-change ratios ≥ . or ≤ . were considered differentially regulated phosphoproteins (drps). of the phosphoproteins, phosphoproteins were significantly upregulated and were downregulated in the tgev-infected group relative to those in the untreated control ( fig. s a and table s ). among the proteins with a > . -fold increase over the proteome background, . % are nuclear-encoded mitochondrial proteins ( fig. s b and table s ). moreover, the levels of phosphorylated mapk /p and mapk /jnk were significantly upregulated (by > -fold and by > -fold, respectively) without any change in overall protein expression in tgev infected samples compared with that in the mock control ( fig. s b and table s ). in contrast, a treatment reduced p and jnk phosphorylation in tgev-infected pk- cells (fig. s c and table s ). treatment with a alone did not affect the overall expression or phosphorylation status of p and jnk relative to the untreated control (table s ) . surprisingly, comparison of the a -treated group and mock control group showed that the phosphorylation statuses of multiple proteins markedly increased with a treatment, including elongation factor tu (tufm), s ribosomal protein s (mrps ), letm , ef-hand domain-containing protein and delta-like protein (jag ). in addition, the phospho-signaling of fourteen proteins decreased significantly in the a -treated pk- cells compared with that in the untreated control cells (fig. s a and table s ). overall, these results reveal a strong correlation between a and tgev, jnk and/or p . to gain insight into the biologically relevant functions of the changes in protein phosphorylation after tgev infection and subsequent a treatment, enrichment (with ≥ . -fold change in phosphorylation) of gene ontology (go) terms and functional categories the data represent averages from at least three independent experiments, with error bars indicating standard deviations. statistical analysis was performed using student's t-test (**, p < . ; ***, p < . ). was assessed. as shown in fig. s a and table s , proteins exhibiting differential phosphorylation after a treatment are associated with various biological processes, including cell growth, energy pathway, cell communication, protein metabolism and signal transduction. we then investigated the pathways affected by tgev infection and subsequent a treatment. the results indicated that the activity of several kinase-mediated signaling pathways was increased ( fig. s a and table s ); these pathways included egfr , il- -mediated signaling events and mapk pathways. we also utilized motif-x online software (version . , http://motif-x.med.harvard.edu/) to assess the enrichment of significant motif substrates after a treatment, which resulted in the identification of three tyrosine-based phosphorylation motif sequences: yxxxr, kxxxxy, and yxxxxk. according to the results, the most significantly phosphorylated amino acid is tyrosine followed by threonine, yxxxrxx is also the most significant kinase motif, which is consistent with the properties of a as an inhibitor of rtks ( fig. s b and table s ). furthermore, two phospho-motifs, yxxxr and kxxxxy, were enriched during tgev infection ( fig. s b and table s ), indicating that the most significantly phosphorylated amino acid during tgev infection is tyrosine. the motifs identified above are found in many protein kinase and mapk family substrates. in addition, tp is the only enriched threonine motif, and it is a substrate that commonly binds to the gsk- , erk , erk , and cdk kinases ( fig. s a and table s ). the motif analysis along with the results of overrepresentation of phosphoproteins predicted to be substrates of mapk support an increase in p and jnk activity during tgev infection. a protein network related to tgev/a -regulated phosphoproteins was assembled by ingenuity pathway analysis (ipa, versions . to . ), and of the drps were recognized and mapped in the predicted network ( fig. s c and table s ). mapk families, especially p (mapk ) and jnk (mapk ), were found to be important nodes that interact with many other proteins and were predicted to play important roles in the interaction network, and their phosphorylation levels were increased and then decreased during tgev infection and subsequent a treatment mentioned above. this interaction network offers clues for further elucidation of the pathogenic mechanism of tgev. to verify the drps identified in the phosphoproteomic analysis, we examined the lysates of tgev-infected pk- cells by western blotting with phospho-specific antibodies. in the tgev-infected cells, the levels of p and jnk phosphorylation increased from hpi to hpi, while no change was detected in the total amount of p and jnk during the progression of tgev infection. in uninfected cells, the phosphorylation statuses of p and jnk were weak and unchanged from hpi to hpi. in addition, the levels of total p and jnk proteins were consistent with those in tgev-infected cells, at a relatively high level. additionally, the phosphorylation status of p and jnk following tgev infection was in accordance with the results of the phosphoproteomic analysis. the results showed that infection of pk- cells with tgev activated p and jnk signaling, as evidenced by increased phosphorylation levels of p and jnk ( fig. a and b) . to investigate the relationships among a , p and/or jnk and tgev infection, we further assessed the phosphorylation and overall expression levels of p and jnk in tgev-infected cells and in cells receiving a treatment by western blotting. pk- cells were infected with tgev at an moi of . and treated with a or dmso, and cell lysates were collected at hpi for immunoblotting. as shown in fig. c and d, phosphorylation of p and jnk was upregulated in tgev-infected cells; dmso treatment did not alter the upregulated p and jnk phosphorylation induced by tgev infection. in contrast, the increase in p and jnk phosphorylation induced by tgev infection was dramatically reduced by a treatment. the total protein levels of p and jnk were equivalent in the different treatment groups. of these, we identified that p and jnk exhibited increased phosphorylation upon tgev infection, and treatment of pk- cells with a resulted in downregulation of p and jnk phosphorylation in tgevinfected cells. as above, we performed western blotting to further measure the phosphorylation and overall expression levels of p in tgev-infected cells (moi of ) and in cells receiving a treatment. as shown in fig. e , a dramatically reduced the phosphorylation of p induced by tgev infection. to explore whether a affects tgev replication by inhibiting downstream mediators of the p or jnk mapk pathways, we treated tgev-infected pk- cells with the specific inhibitor birb or db and determined the toxicity of inhibitors in pk- cells using the mtt assay. concentrations of birb less than μm and db less than μm were not toxic to the tested cells (data not shown). pk- cells were also pretreated with different concentrations of inhibitors for h prior to infection and then infected with tgev at an moi of . , and virus titers were determined at hpi by plaque assay. as shown in fig. a , the p inhibitor reduced tgev replication in a dose dependent manner compared with dmso treated control cells. the inhibitory effects of the two inhibitors were significantly different, with marked inhibition observed for birb at μm. in contrast, virus titers were only slightly reduced by treatment with db , even at μm, the concentration at which the maximal antiviral effect was observed without cytotoxicity. this finding suggests that db might not be as effective as birb at inhibiting tgev propagation. we further examined the kinetics of tgev growth in pk- cells in the presence of an inhibitor or dmso, and the results showed that the overall process of tgev replication was markedly delayed when cells were treated with birb or db at their respective optimal concentration (fig. b) . interestingly, we found that the amount of tgev released into the medium was significantly reduced from hpi to hpi by treatment with birb . in contrast, the virus yield was affected by db treatment between hpi and hpi, and the inhibitory effect of db on virus propagation was relatively weaker than that of birb , suggesting that p activation plays a more important role in the viral life cycle than does jnk . we further examined the effects of birb and db on viral rna synthesis and protein synthesis. tgev-infected pk- cells were cultured in the presence of birb , db or dmso. the cells were collected at different time points post infection and the levels of the n gene were quantified. as shown in fig. c , the mrna abundance was approximately -to -fold lower from to hpi in cells treated with birb than in cells treated with dmso alone, and the db treated group showed a reduction in tgev n mrna level by - fold. the results indicate that viral rna synthesis was significantly inhibited by birb and db . the amounts of viral structural protein n were also clearly reduced in both birb -treated and db treated cells compared to that in dmso-treated cells, with birb showing much greater effects, demonstrating that birb and db inhibited the synthesis of tgev proteins (fig. d) . these results are in accordance with the n mrna expression data. together, these findings suggest that the p mapk signaling pathway is required for efficient tgev replication. finally, the essential regulatory role of p in tgev infection was validated using a second p -specific inhibitor, ly . we determined the toxicity of ly in pk- cells using the mtt assay and found that ly at a concentration less than μm was not toxic to the tested cells (data not shown). tgev-infected pk- cells were treated with the p inhibitor ly , and we determined the virus titers by plaque assay at hpi. overall, the data demonstrate that ly significantly reduced tgev replication (fig. e ). we also evaluated tgev growth kinetics in pk- cells in the presence of ly , and the results were similar to the results reported above, with the overall process of tgev replication being markedly delayed when cells were treated with ly (fig. f ). we used sirna-mediated knockdown to further address whether a inhibits tgev replication by targeting the downstream mediator p . pk- cells were transiently transfected with sirna targeting p or with negative control (nc) sirna for h, and the sirna targeting p apparently reduced the expression of p , as determined by western blotting (fig. a) . next, pk- cells in -well plates were transfected with p sirna or nc sirna for h prior to infection with tgev (moi = . ), and the virus yield was determined by a plaque assay at different time points post infection. compared to nc sirna, the sirna targeting p markedly decreased the viral titer by~ . log (fig. b) at hpi. the specificities of a were also tested in nc sirna transfected cells and p -knockdown cells infected with tgev. pk- cells were transfected with p sirna or nc sirna for h and, then treated with . moi tgev and a for h. cells treated with tgev and dmso after transfection with sirna were used as controls. the virus yield in supernatants at hpi was detected by plaque assay. interestingly, no difference in the inhibitory activity of a against tgev was observed between p -knockdown cells and nc sirna-transfected cells (fig. c) . collectively, the results indicate that p is a target through which a inhibits tgev replication. lopac is a collection of high quality, innovative drug-like molecules that have been used for a broad range of studies including cell signaling and neuroscience, and they reflect the most commonly screened targets in the drug discovery community. in this study, lopac was used in the screening of anti-cov drugs using tgev as a surrogate model for cov. through successful hts, a new antiviral drug candidate, a , was identified and confirmed as the most prominent novel inhibitor of tgev. in addition, a inhibited infectious pedv, mhv and fipv replication with similar potencies (fig. ) . kumar et al. have shown that a and ag , two small-molecule rtkis each potently block influenza virus replication at multiple steps of the viral life cycle and that the anti-influenza activity of ag may be due to its suppression of trka signaling, while the precise inhibitory mechanism of a remains unknown (kumar et al., a; kumar et al., b) . the effects of a on tgev replication and their underlying mechanisms in vitro were further explored in the present study. the effective replication of the virus depends on the host cell machinery. similar to other viruses, cov modulates several cellular pathways to enhance its replication by regulating the phosphorylation and dephosphorylation of host proteins, which is also the case for pedv, sars-cov, mhv and mers-cov (banerjee et al., ; kim and lee, tgev activates the p mapk and jnk / signaling pathways in cultured cells. lysates from untreated control or tgev-infected pk- cells at the indicated times were subjected to sds-page and immunoblotting using antibodies against phosphorylated p , total p , phosphorylated jnk, total jnk and the tgev n protein. (c), (d) and (e) activation of p and jnk is abolished by a treatment. pk- cells were treated with dmso or a , followed by tgev infection or not at an moi of . or . untreated pk- cells were used as a mock control. lysates of cells collected at hpi were analyzed by immunoblotting using the indicated antibodies. gapdh was detected to verify equal loading. ; kindrachuk et al., ; lee et al., ; mizutani et al., ) . nevertheless, no studies to date have focused on detecting global phosphorylation events in host cells during cov infection. a is an rtki and we hypothesized that its antiviral effect is most likely its influence on the phosphorylation of molecules downstream of rtks in cells, which is required for viral replication. in this study, we applied quantitative phosphoproteome analysis to assess changes in cellular phosphoproteins involved in the host response to tgev infection; phosphoproteins from pk- cells and three viral proteins (matrix protein, spike glycoprotein and nucleoprotein) were identified (table s ). motif analysis suggested that the most significantly phosphorylated amino acid during tgev infection is tyrosine (fig. s b) , which is consistent with a acting as an inhibitor of rtk to inhibit the replication of tgev. several cov n proteins have been shown to be phosphorylated and the phosphorylated sites for tgev, avian infectious bronchitis virus (ibv) and mhv n proteins have been identified (calvo et al., ; chen et al., ; white et al., ) . chen and colleagues reported that phosphorylated ibv n protein presented a higher affinity for viral rna than for nonviral rna (chen et al., ) . phosphorylation also plays an important role in the immunoreactivity and specificity of the sars n protein (shin et al., ) . the phosphorylation status of the ibv n protein has the greatest impact on initiating the rescue of infectious virus from cdna (spencer et al., ) . phosphorylation modification is a unique strategy for cov n to facilitate the synthesis of longer viral rnas and subsequent viral progeny production, through the recruitment of cellular ddx (wu et al., ) . however, the function of the phosphorylated cov n protein in viral pathogenesis is unknown, and further investigation is needed to determine whether the spike glycoprotein and matrix protein could be phosphorylated during cov infection as well as the mechanism(s) by which coronaviral protein phosphorylation functions in viral replication. this study is the first to reveal global phosphorylation events in pk- cells induced by tgev infection using quantitative phosphoproteomics and may therefore provide a basis for better understanding the molecular mechanisms of tgev pathogenesis in addition to assisting in the development of antiviral drugs targeting tgev. several studies have indicated that many downstream targets of rtks are involved in cov replication. yang et al. found that pedv infection stimulated the activation of egfr and its downstream stat cascade, which increased viral infection by negatively regulating type i interferon (ifn-i) signaling (yang et al., ) . the erk/mapk and pi k/akt/mtor signaling responses play important roles in mers-cov infection (kindrachuk et al., ) , and jnk and phosphatidylinositol ′-kinase (pi k)/akt are required for establishing persistent sars-cov infection in vero e cells (mizutani et al., ) . our phosphoproteomic analysis found that tgev infection enhanced inhibitor-treated or dmso-treated pk- cells were infected with tgev (moi of . ) for h and then cultivated in the presence of each inhibitor or dmso. the viral rna level was quantified at the indicated times by real-time rt-pcr and normalized to internal control pig gapdh mrna. (d) chemical inhibition of p and jnk activation impairs viral protein synthesis. western blot analysis of viral n protein at the indicated times in cells infected with tgev (moi of . ) and treated with dmso or inhibitors. gapdh detection was used to confirm equal loading. each value is presented as the means of three independent experiments. error bars indicate sd. statistical analysis was conducted with student's t-test (*p < . , **p < . , ***, p < . ). phosphorylation of p and jnk by more than -fold and -fold, respectively, in tgev-infected cells compared to that in untreated control cells ( fig. a and b, fig. s b and table s ). in addition, p and jnk were not activated in pk- cells exposed to uv-inactivated tgev (data not shown). these results indicate that activation of p and jnk mapk is due to the actual replication of the virus in cells. however, tyrphostin a treatment can block tgev-induced p and jnk phosphorylation (fig. c and d and e and table s ), indicating that p and jnk map kinase function downstream of rtks. we further explored the effects of the downstream mediators of rtks, p and jnk , on tgev replication using the specific inhibitors birb and db , respectively. our results showed that pretreatment with both birb and db significantly reduced tgev n mrna and protein levels ( fig. c and d) , with a lower tgev titer ( fig. a and b) , compared to dmso-treated control cells. we speculate that the inhibitory activity of a against tgev replication might target multiple host kinases downstream of rtks. rtks are the primary mediators of many signals and control many downstream cascades, including phosphoinositide -kinase (pi k), mapk and ca + pathways (schlessinger, ) . several studies have shown that phosphoinositide -kinase (pi k) signaling and stat cascades, which are downstream of rtks, play important roles in the life cycle of cov (hu et al., ; kindrachuk et al., ; mizutani et al., ; yang et al., ) . further studies are required to explore whether these downstream cascades are involved in the inhibitory activity of a against tgev replication. to the extent that a might target multiple host components, drug-resistant viral variants are less likely to occur (kumar et al., a) . our results showed that the inhibitory effect of db on virus propagation was relatively weaker than that of birb (fig. ) . such a correlation suggests that a mainly inhibited the replication of tgev through the p pathway. we also used another p inhibitor to validate its essential regulatory role in tgev infection, and the results were consistent with those of birb , which can significantly reduce tgev replication (fig. e and f) . we also performed rna interference to further validate that p plays an important regulatory role in tgev replication ( fig. a and b) . as the level of p expression was reduced to only % (fig. a) , residual p would still be phosphorylated during tgev infection and promote virus replication. moreover, other pathways also play a role in tgev replication during a treatment, such as the jnk signaling pathway (figs. and ) . therefore, the inhibitory effect of p sirna was not as strong as the effect of the p inhibitor on viral replication. furthermore, the specificities of a were tested in wild type and p -knockdown cells with tgev infection. interestingly, the inhibitory activity of a against tgev in p -knockdown cells was similar to that of nc sirna-transfected cells (fig. c ). all our results suggest that the inhibition of a on tgev replication is mainly via targeting p . mapks are important cellular signaling molecules that regulate cell growth, apoptosis, metabolism and differentiation under both normal and pathological conditions. the well characterized mapks are composed of three major groups: extracellular signal-regulated kinases and (erk / , also known as p / mapk), p map kinases and jnks (schaeffer and weber, ) . the p mapk pathway can be activated by a variety of covs and plays a crucial role in cov infection. it has been reported that pedv infection activates p mapk and jnk / and can exploit these molecules for optimal replication (lee et al., ) . additionally, mhv infection activates p mapk and jnk, which is necessary for its replication (banerjee et al., ) . activation of p mapk by fipv regulates pro-inflammatory cytokine production which is a key contributor to the pathological changes observed in cats with fip (regan et al., ) . p mapk activation is required for human cov e (hcov- e) replication and chloroquine inhibits hcov- e replication may by suppressing p activation (kono et al., ) . our findings showed that a mainly inhibits tgev replication through the p pathway. moreover, a inhibited infectious pedv, mhv and fipv replication with similar potencies. hence, it is reasonable to speculate that a may inhibit the replication of other covs by the same mechanism. similar to other findings, tyrphostin a treatment inhibited gp -induced p phosphorylation (anand et al., ) . although the effect of p mapk on viral replication has been studied in a variety of viruses, little is known about the mechanisms by which p activation facilitates virus replication. the author of one study discussed that stimulation of p mapk may enhance the transcription of specific viral gene promoters, leading to the promotion of herpes simplex virus type proliferation (zachos et al., ) . in mhv, p mapk activation results in the phosphorylation of eif e, which facilitates virus protein synthesis and the subsequent production of infectious virus (banerjee et al., ) . cencic et al. reported that targeting the eif f complex is a strategy for blocking cov infection (cencic et al., ) . eif e is a downstream molecule of p mapk and fig. . p is one of the targets for the inhibitory activity of a against tgev replication. (a) the efficiency of p sirna was evaluated by western blotting. pk- cells in -well plates were transfected with the indicated sirna at pmol using transfection reagent. at h post transfection, the cells were lysed and subjected to western blotting with an antibody against p . protein levels were quantified with imagej software and normalized to the amount of gapdh. (b) knock down of p expression with sirna inhibited tgev replication at hpi. pk- cells in -well plates were transfected with p sirna and nc sirna at pmol, respectively. at h post transfection, the cells were infected with . moi tgev. virus yield in supernatants of the cultures were measured at different time points using the plaque assay. (c) the effects of downregulating p mapk on the inhibitory activity of a against tgev replication. pk- cells were transfected with p sirna or nc sirna for h, and then treated with . moi tgev and a for h. cells treated with tgev and dmso at h post transfection were used as controls. virus yield in the supernatants of the cultures was detected by plaque assay. the data shown are mean of three independent experiments (b and c). statistical analyses used student's t-test (*p < . , **p < . , ***p < . ). increased eif e phosphorylation usually leads to enhanced translation rates (gingras et al., ) . our results showed that tgev-specific protein synthesis was decreased when infected cells were treated with birb (fig. d) . therefore, whether viral replication is enhanced by p mapk by modulating the downstream molecule eif e to increase viral protein synthesis in tgev-infected cells remains to be determined. tgev infection induces high levels of p map kinase and jnk phosphorylation, both of which are required for tgev replication. a , an inhibitor of rtks, initiates a signaling cascade that involves phosphorylation of the tyrosine kinase, in turn inhibiting the phosphorylation of p and jnk , which are activated by tgev infection; the result is a robust decrease in tgev replication. in conclusion, although the signaling intermediates between tgev and rtk remain to be elucidated, we identified and validated that p mapk is a main downstream signaling node for the inhibitory activity of a against tgev replication. the findings reported here provide novel insight into the molecular mechanisms underlying tgev replication and offer new and promising therapeutic possibilities for combating infections caused by cov. in summary, our findings show that tgev infection can stimulate p and jnk phosphorylation and that a treatment can attenuate this phosphorylation status, which results in decreased tgev replication. there has been significant progress in both clinical and preclinical 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effect of phosphorylation on immunoreactivity and specificity role of phosphorylation clusters in the biology of the coronavirus infectious bronchitis virus nucleocapsid protein the tyrosine kinase inhibitor genistein blocks hiv- infection in primary human macrophages drug targets in severe acute respiratory syndrome (sars) virus and other coronavirus infections genistein treatment of cells inhibits arenavirus infection identification of mouse hepatitis coronavirus a nucleocapsid protein phosphorylation sites nucleocapsid phosphorylation and rna helicase ddx recruitment enables coronavirus transition from discontinuous to continuous transcription porcine epidemic diarrhea virus-induced epidermal growth factor receptor activation impairs the antiviral activity of type i interferon inhibition of herpes simplex virus replication by genistein, an inhibitor of protein-tyrosine kinase herpes simplex virus type blocks the apoptotic host cell defense mechanisms that target bcl- and manipulates activation of p mitogen-activated protein kinase to improve viral replication development and optimization of a direct plaque assay for human and avian metapneumoviruses inhibitors of sars-cov entryidentification using an internally-controlled dual envelope pseudovirion assay this work was supported by the national natural science supplementary data to this article can be found online at https:// doi.org/ . /j.antiviral. . . key: cord- - iwljdoi authors: chen, qin; li, jian; fang, xue-en; xiong, wei title: detection of swine transmissible gastroenteritis coronavirus using loop-mediated isothermal amplification date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: iwljdoi a conserved nucleic acid fragment of the nucleocapsid gene of swine transmissible gastroenteritis coronavirus (tgev) was chosen as the target, six special primers were designed successfully. loop-mediated isothermal amplification (lamp) was developed to detect the tgev by incubation at °c for h and the product specificity was confirmed by hphi digestion. standard curves with high accuracy for tgev quantization was constructed by adding × sybr greeni in the lamp reaction. the assay established in this study was found to detect only the tgev and no cross-reaction with other viruses, demonstrating its high specificity. by using serial sample dilutions as templates, the detection limit of lamp was about pg rna, times more sensitive than that of pcr and could be comparable to the nest-pcr. swine transmissible gastroenteritis coronavirus (tgev), as a member of the coronaviridae, is a kind of single-stranded rna virus, which produces villous atrophy and enteritis, leading to the serious financial loss to the whole pig industry. the traditional detection methods, including virus isolation, virus immunodiagnostic assays and pcr tests have the shortcomings, such as precise instruments requirement, elaborate result analysis demand, high cost, long detection time and so forth, which prevent these methods from being widely used [ ] [ ] [ ] [ ] . loop-mediated isothermal amplification (lamp) is a novel nucleic acid amplification method, which amplifies dna/rna with high specificity, sensitivity and rapidity under isothermal condition [ ] . it has already found wide application in rna virus detection, such as foot-and-mouth disease virus [ ] , swine vesicular disease virus [ ] , taura syndrome virus [ ] , severe acute respiratory syndrome coronavirus and h n avian influenza virus [ , ] . in this study, lamp method was applied in developing qualitative and quantitative detection system of tgev, while its specificity and sensitivity were assessed. swine transmissible gastroenteritis coronavirus (tgev, strain h), porcine reproductive and respiratory syndrome virus (prrsv), pesudorabies (prv), porcine parvovirus (ppv) derived from their passages in cell culture were provided by shanghai entry-exit inspection and quarantine bureau (shciq); nucleic acids of footand-mouth disease virus (fmdv) and classical swine fever virus (csfv) were obtained from chinese academy of inspection and quarantine (caiq). total genomic rna was extracted using trizol kit (invitrogen, usa). dna was extracted by dna blood mini kit (qiagen, germany). after elution in μl nuclease-free water, rna/dna samples were stored at - °c before use. the original concentration of rna/ dna sample was about ng/μl. complete genome sequences of fifteen different tgev strains/isolates and nine other similar viruses were obtained from genbank, and the homology was analyzed using the vector nti. the conserved fragment with high homology was chosen as the target region which and used to design the tgev lamp primers by the primer explorer v software http://primerexplorer. jp/e/. the target rna of tgev was first reverse transcripted using superscript™ ii (invitrogen, usa) and then amplified by pfu dna polymerase using forward primer: ggaagagaactgcaggtaa and reverse primer: ccatcttcctttgaagtcca. the amplified product was purified from agarose gels and then cloned into e. coli jm using the pmd -t vector. the target plasmid with the original concentration of . × copies/μl was extracted by the plasmid mini preparation kit and identified by the nm absorption spectroscopy, which was then used as the standard for the quantitative analysis. the amplified products were digested with hphi to confirm its specificity. the result of tgev-lamp was analyzed by agarose gel electrophoresis and fluorescence by adding × sybr greeni in the lamp reaction. the specificity of tgev-lamp was examined by the use of rna (or dna) extracted from five other pig disease viruses. the sensitivity of tgev-lamp was evaluated by comparing with pcr [ ] and nest-pcr[ ], using -serial tgev rna dilutions ( - to - ) as templates. lamp primers were designed using the primer explorer v software based on a conserved fragment of the nucleocapsid gene (fig. ) . the primers including outer primers (f and b ), inner primers (fip and bip) and loop primer (lf and lb) were shown in table . tgev deprived from the cell culture was first qualitatively analyzed by lamp. amplification products were analyzed by agarose gel electrophoresis. as shown in fig. (lane ), amplification could be carried out at °c and showed a ladder-like pattern on the gel while the negative control gave no bands (lane ). the specificity of the lmap product was confirmed by hphi digestion. predictable product of the -bp motif was resolved on the gel as theoretical expected (fig. , lane ) . standard control was used to develop real-time fluorescence lamp for quantitatively analyzing tgev. dynamic curves for tgev quantification was generated by serially diluting the standard control from . × to . × copies/μl (fig. ) . the log linear regression plot (standard curves) was obtained by plotting the time-to-positive (ttp) values against genome copies. the correlation coefficients were . (fig. ) five other pig viruses were used to confirm the specificity of the lamp for tgev detection. the results showed that only tgev detected gave amplification products while no amplification available to other viruses (fig. ) . the sensitivity of lamp was demonstrated by comparing with pcr tests using serial dilutions ( - to - ) of tgev rna samples as template. as shown in fig. , lamp and nest-pcr were able to detect - dilution (about pg rna), whereas pcr could only amplify the - dilution. therefore, the sensitivity of tgev-lamp could be comparable to nest-pcr, -fold higher than pcr. it is very important to find out a conserved nucleic acid fragment for designing specific lamp primers and developing efficient, accurate lamp assay [ , ] . in this study, the nucleic acid sequence homology of tgev strains/isolates and other similar viruses available from genbank were analyzed by vector nti software. the most conserved fragment of bp was found in the nucleocapsid protein gene which showed highly homology among different tgev strains/isolates (more than %) and low homology among other similar viruses (less than . %). the tgev lamp primers targeting the conserved sequence were designed successfully by the primer designer v software. the target region was amplified successfully by the lamp with a characteristic ladder-like pattern of bands from bp on the gel. this is because the final products of lamp are a mixture of stem-loop dnas with various stem lengths and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target sequence in the same strand [ , ] . after digestion with hphi, -bp motif was resolved on the gel as expected, demonstrating the specific structure of amplification products, which could also be validated by nucleic acid sequencing. as a kind of nucleic acid amplification method, lamp could not only qualitatively detect the tgev, but also quantitatively analyze the virus. in this study, real time fluorescence lamp for quantitatively detection of tgev was established by adding × sybr greeniin the lamp reaction. three standard curves established by tgev standards displayed the good correlation between the ttp and virus copies, implicating the great potential in quanlitatively detecting tgev. five other viruses were used in this study to confirm the specificity of lamp. the results showed no amplification in all viruses tested, which makes the lamp more accurate and reliable for tgev detection. the high specificity of lamp is most probably attributable to recognition of the target sequence by six independent sequences in the initial stage and by four independent sequences during the second reaction stage. the sensitivity of lamp was evaluated using various tgev-rna dilutions as templates. the assay exhibited almost equivalent sensitivity to the nest-pcr and -fold higher than the pcr, indicating that the lamp is a more powerful diagnosis tool to detect tgev in lower copy conditions [ ] . in addition, the tgev-lamp developed has advantages in its rapid detection and simple operation. the only equipment required for the reaction is a water bath or heat block. the assay developed is a faster detection method for the tgev detection, only taking about h, which means the whole diagnosis including rna extraction, amplification and product detection could be completed within one and a half hour after receiving of the samples. it is anticipated that with the advantages of specificity, sensitivity, reliability, rapidity and easy manipulation, lamp will turn out be a powerful molecular tool for the tgev detection in practice. in conclusion, this study demonstrates that the lamp method established could detect only the tgev and no cross-reaction with other viruses, the detection limit was about pg rna, which was times more sensitive than that of pcr and could be comparable to the nest-pcr. virus isolation and serum antibody-responses after infection of cats with transmissible gastroenteritis virus -brief report use of monoclonal antibodies in blocking elisa detection of transmissible gastroenteritis virus in faeces of piglets an indirect elisa for the detection of antibodies against porcine reproductive and respiratory syndrome virus using recombinant nucleocapsid protein as antigen detection of transmissible gastroenteritis virus by rt-pcr and differentiation from porcine respiratory coronavirus loop-mediated isothermal amplification of dna novel reverse transcription loopmediated isothermal amplification for rapid detection of foot-andmouth disease virus a: one-step reverse transcriptase loop-mediated isothermal amplification assay for simple and rapid detection of swine vesicular disease virus rapid and sensitive detection of taura syndrome virus by reverse transcription loop-mediated isothermal amplification development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus rapid diagnosis of h n avian influenza virus infection by newly developed influenza h hemagglutinin gene-specific figure standard curves of real-time fluorescence tgev-lamp negative control. figure sensitivity of lamp for tgev detection. a, lamp; b, pcr; c, nest-pcr the authors declare that they have no competing interests. key: cord- -k mtr j authors: ma, xuelian; zhao, xiaomin; wang, kaili; tang, xiaoyi; guo, jianxiong; mi, mi; qi, yanping; chang, lingling; huang, yong; tong, dewen title: identification and analysis of long non-coding rnas that are involved in inflammatory process in response to transmissible gastroenteritis virus infection date: - - journal: bmc genomics doi: . /s - - - sha: doc_id: cord_uid: k mtr j background: transmissible gastroenteritis virus (tgev) infection can cause acute inflammation. long noncoding rnas (lncrnas) play important roles in a number of biological process including inflammation response. however, whether lncrnas participate in tgev-induced inflammation in porcine intestinal epithelial cells (ipecs) is largely unknown. results: in this study, the next-generation sequencing (ngs) technology was used to analyze the profiles of lncrnas in mock and tgev-infected porcine intestinal epithelial cell-jejunum (ipec-j ) cell line. a total of lncrnas were differentially expressed. many differentially expressed lncrnas act as elements to competitively attach micrornas (mirnas) which target to messenger rna (mrnas) to mediate expression of genes that related to toll-like receptors (tlrs), nod-like receptors (nlrs), tumor necrosis factor (tnf), and rig-i-like receptors (rlrs) pathways. functional analysis of the binding proteins and the up/down-stream genes of the differentially expressed lncrnas revealed that lncrnas were principally related to inflammatory response. meanwhile, we found that the differentially expressed lncrna tcons_ might lead to a reduction of phosphorylation of transcription factor p (p-p ) in tgev-infected ipec-j cells by negatively regulating its antisense gene promyelocytic leukemia (pml). conclusions: the data showed that differentially expressed lncrnas might be involved in inflammatory response induced by tgev through acting as mirna sponges, regulating their up/down-stream genes, or directly binding proteins. virus can activate the inflammatory response by multiple means, including nuclear factor-kappa b (nf-κb), jak-stat, tlrs, t cell receptors (tcrs), nlrs, tnf, rlrs signaling pathway [ ] [ ] [ ] [ ] [ ] [ ] [ ] . previous studies have described that tgev can impair ipecs and trigger inflammatory response [ ] . ipecs are the targets for tgev, and play an important role in the nutrition absorption and inflammatory response against pathogens. the pathogenesis of tgev is strongly associated with the powerful induction of inflammatory response in host cells. a new study confirmed that the rlrs, tlrs and nf-κb signaling pathways are involved in tgevinduced inflammatory responses [ ] . non-coding rnas (ncrnas), including mirnas, circular rnas (circrnas), as well as lncrnas, typically do not encode proteins and functionally regulate many biological process [ ] . it has been demonstrated that many ncrnas are involved in inflammatory response in cells [ , , [ ] [ ] [ ] [ ] [ ] . in previous study, we determined that the profiles of mrnas, mirnas and circrnas were significantly changed in the ipec-j after tgev infection. the potential functions of differentially expressed mrnas, mirnas and circrnas were anlyzed and were closely related to inflammatory response [ ] . recently, increasing studies have indicated that lncrnas play important roles in inflammatory response [ ] [ ] [ ] [ ] . therefore, we proposed that lncrnas also might participate in regulating inflammatory response during tgev infection. the lncrnas play roles in regulating transcription, translation, and protein translocation [ ] [ ] [ ] [ ] [ ] . lncrnas can regulate translation by interacting with mirna or act as precursors of mirna [ ] [ ] [ ] . for example, lncrna sbf -as acts as a competing endogenous rna (cerna) to modulate cell proliferation via binding with mir- - p in acute myeloid leukemia [ ] . lncrna hotair functions as a cerna to upregulate sirtuin (sirt ) by sponging mir- a in diabetic cardiomyopathy [ ] . lncrnas can serve as scaffold to bind to different types of proteins or transcription factors at specific domains to activate or inhibit gene transcription. lncrna h decreases the transcriptional activity of p [ ] . lncrna snhg facilitates hepatocarcinogenesis and metastasis by modulating its homolog small cajal body-specific rna (scarna ) [ ] . lncrnas can also achieve the regulation of the expression of the target genes by recruiting some rna-binding proteins [ ] . this is the first study to demonstrate the expression profiles and regulatory mechanisms of lncrnas during tgev infection by ngs methods. the data showed that differentially expressed lncrnas might be involved in inflammatory response induced by tgev through acting as mirna sponges, regulating their up/down-stream genes, or directly binding proteins. this information will enable further research on the tgev infection and facilitate the development of novel tge therapeutics targeting lncrnas. to investigate the lncrna expression profiles of tgev infected ipec-j , ipec-j were infected with tgev strain (tgev-infected group, indicated by t and t ) and the normal ipec-j line (mock-infected group, indicated by m and m ) was used as a control. the rna-seq was performed with the total rna extracted from ipec-j infected with moi tgev at hpi. among all mapped transcripts , ( . %) were classified as known mrnas, , ( . %) were classified as new mrnas, ( . %) were classified as other rnas (including pseudogenes), and ( . %) were classified as lncrnas (including known lncrnas and new lncrnas) ( fig. a and additional file : table s ). among them, were antisense lncrnas, long intervening/intergenic non-coding rnas (lincrnas), other lncrnas, promoter-associated lncrnas, sense overlapping lncrnas, and utr lncrnas ( fig. b and additional file : table s ). the expression levels of transcripts were changed remarkably (fold change > . , and p < . ). among all remarkably changed transcripts, ( . %) were classified as known mrnas, ( . %) were classified as new mrnas, and ( . %) were classified as lncrna (fig. c) . among lncrnas, were antisense lncrnas, lincrnas, other lncrnas, promoter-associated lncrnas, sense overlapping lncrnas, and utr lncrnas (fig. d) . in the current study, lncrnas and , mrnas transcripts were identified. the lncrnas and mrnas transcripts were compared for their total length, exon number, exon length, and expression level. we found that known lncrnas and novel lncrnas, compared with mrnas, had significantly shorter transcript length (fig. a) , and longer exons (fig. b) . these properties were consistent with the lower estimated number of exons for known lncrnas and novel lncrnas compared with mrnas (fig. c) . the expression profiles of lncrnas and mrnas biotypes were presented as logarithmic distributions. the average mrna expression level was higher than that of the known lncrnas and novel lncrnas (fig. d) . the differential expression of multiple lncrnas in tgev-infected group compared with mock-infected group was observed in fig. . the expression levels of lncrnas were changed remarkably (fold change ≥ and ≤ . , fdr < . ). among them lncrnas were up-regulated and lncrnas were down-regulated. (additional file : table s ). lncrnas can be spliced into multiple small rnas which function as post-transcriptional regulators. to find potential mirna precursors, lncrnas were aligned to mirbase (version ). our result showed that there were lncrnas producing precursors of mirnas possibly (additional file : table s ). the secondary structures of these lncrnas and mirna precursors were predicted via the rnafold web server (http://rna.tbi.univie.ac.at/cgibin/rnawebsuite/rnafold.cgi). figure illustrates the secondary structure of tcons_ , which might release the precursor sequence of mir- by an endonuclease cleaving, and form mature mir- - p and mir- - p finally. the same to their precursors, these mirnas have no differences between tgev-infected group and mock-infected group. lncrnas can rescue the translation levels of mrna via pairing to mirnas to prevent the binding of mirnas and mrna untranslated regions (utr). in our study, we constructed a lncrna-mirna-mrna expression interaction network combinated with the mirna sequencing data [ ] . a total of differentially expressed lncrnas and differentially expressed mrnas targeted differentially expressed mirnas in the network respectively ( fig. a and additional file : table s ). to find the potential function of these significantly differentially expressed lncrnas acting as mirna sponges, kyoto encyclopedia of genes and genomes (kegg) analysis of the differentially expressed mrnas was performed and presented. the result showed that these mrnas were participated in the tlrs signaling pathway, herpes simplex infection, nlrs signaling pathway, tnf signaling pathway, and nf-κb signaling pathway primarily (fig. b) . we determined lncrna-protein interactions using the catrapid omics algorithm [ ] . the star rating system of catrapid helped us rank the results. the score was the sum of three individual values: ) catrapid normalized propensity, ) presence of rna/dna binding domains and disordered regions, and ) presence of known rna-binding motifs. three hundred seventytwo lncrna-protein interactions were predicted for differentially expressed lncrnas ( fig. a and additional file : table s ); the gene ontology (go) annotation of proteins with a ranking score > were next explored using go enrichment analysis. the result showed that lncrnas interacted with proteins, including complement c (c ), inhibitor of dna binding (id ), myc proto-oncogene (myc), interferon regulatory factor (irf ), which involve in immune system process (fig. b) . we predicted the up-and down-stream genes of differentially expressed lncrnas ( k). four hundred forty-three genes were obtained, some of which are shown in fig. a and additional file : table s . go analysis was conducted to enrich up-and downstream targets of differentially expressed lncrnas (http://www.geneontology.org/). the results exhibited that the up-and down-stream targets of differentially expressed lncrnas were primarily enriched in immune system process (fig. b) . to validate the rna-seq results of differentially expressed lncrnas, we tested the expression levels of them using qrt-pcr. the fold changes of lncrnas in tgev-infected cells were referred to that in mockinfected cells. the results indicated that our sequencing results were accurate. see fig. and additional file : table s . the software rnaplex [ ] (http://www.tbi.univie.ac.at/ rna/rnaplex. .html) was used to predict the complementary correlation of antisense lncrna and mrna. the prediction of best base pairing was based on the calculation of minimum free energy (mfe) through thermodynamics structure. the result showed that lncrna tcons_ was located in physical contiguity pml (mfe = − . ) (fig. a ). pml is a nuclear protein that forms sub-nuclear structures termed nuclear bodies associated with transcriptionally active genomic regions. previous studies have confirmed that pml promotes tnfα-induced transcriptional responses by promoting nf-κb activity. nf-κb signaling pathway plays an important role during tgev-induced inflammatory response. the antisense lncrna tcons_ was downregulated in tgev-infected group, and pml was upregulated in tgev-infected group. to further understand fig. genomic features of lncrnas. a transcript sizes of lncrnas, novel lncrnas, and mrnas. b exon sizes of lncrnas, novel lncrnas, and mrnas. c numbers of exons per lncrnas, novel lncrnas, and mrnas. d expression levels (fpkm values) of known lncrnas, novel lncrnas, and mrnas. a, b, d are standard boxplots, which display the distribution of data by presenting the inner fence (the whisker, taken to . × the inter quartile range, or iqr, from the quartile), first quartile, median, third quartile and outliers. the means are marked as tan diamonds the regulatory relationship between tcons_ and pml, ipec-j cells were transfected with shrna of tcons_ (sh-tcons_ ) (or negative control). the tcons_ level was downregulated by sh-tcons_ , while the pml level was up-regulated by sh-tcons_ (fig. b) . the string database (version . ) was used to further understand the regulatory relationship between pml and other differentially expressed mrnas related to inflammation process (fig. c and additional file : table s ). p is a subunit of nuclear factor nf-κb. the phosphorylation of p is a very significant symbol of nf-κb signaling pathway activity. to explore the function of pml in the process of tgev induced nf-κb activation, the sirna of pml (or negative control) were transfected into ipec-j cells respectively, then infected with tgev at moi for h. the pml level was down-regulated by si-pml- significantly (fig. d) . p-p was decreased by si-pml- ( fig. e and f) . the sirna sequences were shown in additional file : table s . lncrnas have been reported to be involved in the coronavirus infections [ , ] , but the roles of lncrnas during tgev induced inflammation response have not yet been elucidated. in our study, ngs techniques were used to investigate the lncrna expression profiles of tgev infected ipec-j . among the transcripts of ipec-j obtained in our study, a total of lncrnas across the entire genome were among them lncrnas were up-regulated and lncrnas were down-regulated (fold change > . , and p < . ) screened after sequencing and bioinformatics analysis. these lncrnas were characterized by shorter transcript length, longer exons, lower estimated number of exons and lower expression levels. these properties were also observed in other reported lncrnas within the genome [ , [ ] [ ] [ ] . in a previous study, tgev induced inflammatory response via nf-κb signaling pathway, tlrs signaling pathway, nlrs signaling pathway, jak-stat signaling pathway, tnf signaling pathway and rlrs signaling pathway [ ] . in our study, we identified lncrnas differential expression between tgev-infected group and mock-infected group, reminding us that lncrnas may be involved in the regulatory process of tgev infection. lncrnas can rescue the translation levels of mrna via pairing to mirnas to prevent the binding of mirnas and mrna utr. in this study, we found mir- , which we mentioned earlier, had three target genes, dexd/h-box helicase (ddx ), interferon regulatory factor (irf ) and signal transducer and activator of transcription (stat ) that might be involved in inflammatory response. additionally, ten lncrnas tcons_ , tcons_ , tcons_ , tcons_ , tcons_ , tcons_ , tcons_ , tcons_ , tcons_ and tcons_ , which were differentially expressed in tgev-infected group, were predicted to be targeted by this mirna, indicating that the lncrnas may compete with ddx , irf and stat to affect their expression levels and influence tgev-induced inflammatory response. some lncrnas can directly bind to proteins to regulate the functions of proteins [ , ] . we determined lncrna-protein interactions using the catrapid omics algorithm, the result showed that lncrnas interacted with proteins, including c , id , myc, and irf , which involve in immune system process. one of the important functions of lncrna is to act as antisense transcripts of mrnas or located adjacent to protein coding genes. in our data, many neighbouring genes correspond to compartments of the inflammatory response, such as pml (ensssct ), interferon beta (ifnb ) (ensssct ), radical s-adenosyl methionine domain containing (rsad ) (ensssct ), and interferon induced protein with tetratricopeptide repeats (ifit ) (ensssct ). previous studies have shown that nf-κb signaling pathway, one of the most important pathways, plays an important role during tgevinduced inflammatory response [ , , , ] . therefore, changes in the expression levels of genes, which related in nf-κb signaling pathway, might influence the tgev-induced inflammatory response. the differentially expressed lncrnas may affect tgev-induced inflammatory response by affecting nf-κb signaling pathway. it has been proved that pml promotes tnfα-induced transcriptional responses by promoting nf-κb activity [ ] . we further confirm that silencing pml gene expression rescued the tgev-induced nf-κb activity. in our study, lncrna tcons_ was identified as a potential antisense transcript of pml, which suppress transcription of pml. our work uncovered that lncrnas might act as regulatory elements of the host inflammatory response when tgev-infected. while, further efforts should be paied to confirm the present findings. the lncrna expression profile of ipec-j was compared between the ipec-j infected with tgev (n = ) and mock group (n = ). to identify lncrnas expressed in tgev infected ipec-j , cdna libraries were ipec-j cells were infected with tgev at moi for h (indicated by t and t ). meanwhile, the mock infection (indicated by m and m ) was carried out. total rna was extracted with trizol reagent (invitrogen, carlsbad, ca, us). after total rna was extracted, ribosomal rnas (rrnas) were removed to retain mrnas and ncrnas. following the purification, the enriched mrnas and ncrnas were iron-fragmented at °c. then, reverse transcriptase and random primers were used to generate the first strand cdna from the cleaved rna fragments. the second strand dna was amplified by pcr, qiaquick pcr extraction kit was used to purify the cdna fragments, then these fragments were end repaired, poly(a) added, and ligated to illumina sequencing adapters. the second-strand cdna was digested by uracil-n-glycosylase (ung), the products were size selected by pcr amplified, agarose gel reads containing adapters, low quality reads, and rrna reads were removed. the remaining reads of each sample were then mapped to sus scrofa reference genome (sus scrofa . ) by tophat (version . . . ), respectively. cufflinks (v . . ), which preferring to the program reference annotation-based transcripts (rabt), was used to reconstruct the transcripts. the influence of low coverage sequencing was fixed through cufflinks constructing faux reads based on reference. during the end of assembly, similar fragments were removed from all of the reassembled fragments by aligning with reference genes. then we used cuffmerge to merge transcripts from different replicates of a group into a to identify the novel transcripts, all of the reconstructed transcripts were aligned with reference genome and divided into twelve categories using cuffcompare (v . . ). we used the following parameters to identify reliable novel transcripts: the length of transcript was longer than bp and the exon number was more than . two softwares coding-non-coding index (cnci) (https://github.com/www-bioinfo-org/cnci) [ ] and coding potential calculator (cpc) (http://cpc.cbi.pku. edu.cn/) [ ] were used to assess the protein-coding potential of new transcripts by default parameters. the intersection of both results were chosen as long noncoding rnas. lncrna abundance was quantified by rsem (v . . ) and normalized to fragments per kilobase of transcript per million mapped reads (fpkm). the formula is shown as follow: c, the number of fragments that are mapped to transcripts; n, the total number of fragments that are mapped to reference genes; l, the number of base pairs of transcript. the edger package (http://www.r-project.org/) was used to identify differentially expressed lncrnas. a fold change ≥ and ≤ . , plus a false discovery rate (fdr) < . , were identified as significant differentially expressed lncrnas. lncrnas can be spliced into multiple small rnas which function as post-transcriptional regulators. to find potential mirna precursors, lncrnas were aligned to mirbase (version ). those with identity more than % were selected. based on the sequences of lncrnas, three softwares rnahybrid (v . . ) + svm_light (v . ), miranda (v . a) and targetscan (version: . ) were used to the candidate target genes. the interaction networks among lncrna and mirna were built and visualized using cytoscape (v . . ) (http://www.cytoscape.org/). one of the functions of lncrnas is cis-regulation of their neighboring genes on the same allele. the upstream lncrnas which have intersection of promoter or other cis-elements may regulate gene expression in transcriptional or post-transcriptional level. the downstream or 'utr region lncrnas may have other regulatory functions. lncrnas, which are classified as located in an "unknown region" in cuffcompare (v . . ) were annotated as up-or downstream of a gene. lncrnas in up/ down stream of a gene were likely to be cis-regulators. the interaction networks among lncrna and up-or downstream genes were built and visualized using cytoscape (v . . ) (http://www.cytoscape.org/). in order to reveal the interaction between antisense lncrna and mrna, the software rnaplex [ ] (http://www.tbi.univie.ac.at/rna/rnaplex. .html) was used to predict the complementary correlation of antisense lncrna and mrna. go and kegg analysis of differentially expressed lncrnas go database (http://www.geneontology.org/) and kegg database (http://www.genome.jp/kegg/) were used to annotate the pathways. the calculating formula is the same as the previous study [ ] .the interaction networks among lncrnas, mirnas, mrnas or proteins were built and visualized using cytoscape (v . . ) (http://www.cytoscape.org/). according to the manufacturer ′ s instructions, trizol reagent was used to extract the total rna of ipec- fig. function analysis of the antisense lncrna tcons_ . a the pattern diagrams showed that lncrna tcons_ was located in physical contiguity pml (mfe = − . ). b knockdown effect of si-tcons_ on tcons_ . the relative levels of tcons_ were measured by qrt-pcr (normalized toβ-actin and in reference to the control). c the regulatory relationship between pml and other differentially expressed mrnas related to immune system process (red). d knockdown effect of si-pml- and si-pml- on pml. the relative levels of pml were measured by qrt-pcr (normalized toβ-actin and in reference to the control). e and (f) the effects of si-pml- on p-p . data represent mean ± s.d. of three independent experiments. **p < . in comparison with the control j cells, then reverse transcription was carried out using m-mlv reverse transcriptase (invitrogen, us). qrt-pcr was performed on iq qrt-pcr system (bio-rad, us). the primers are shown in additional file : table s . ripa lysis buffer containing phenylmethylsulfonyl fluoride (pmsf) was used to treat samples to extract the protein, then using bca protein assay reagent (pierce, us) to measure the protein concentration. proteins were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and transferred onto polyvinylidene difluoride (pvdf) membranes (millipore, us) subsequently. block the pvdf membrane with % non-fat milk for h at room temperature and then incubate the pvdf membrane with phospho-nf-κb p (p-p ) rabbit monoclonal antibody (cst, us) overnight at °c and horseradish peroxidase (hrp)-conjugated secondary antibody (pierce, us) at room temperature for h subsequently. in the last step, the membrane was developed with enhanced chemiluminescence (ecl) (promega, us). statistical analysis spss . was used for statistical analysis. the data are presented as the means ± sem. statistical significance was analyzed by unpaired student ′ s t-test. p < . was defined as statistical significance. vaccinia virus inhibits nf-kappab-dependent gene expression downstream of p translocation toll-like receptor (tlr)-mediated innate immune responses in the control of hepatitis b virus (hbv) infection antagonizing cytokine-mediated jak-stat signaling by porcine reproductive and respiratory syndrome virus concanavalin a-mediated t cell proliferation is regulated by herpes virus entry mediator costimulatory molecule beyond the inflammasome: regulatory nod-like receptor modulation of the host immune response following virus exposure hepatitis c virus (hcv)-induced suppressor of cytokine signaling (socs) regulates proinflammatory tnf-alpha responses rig-i-like receptor regulation in virus infection and immunity. current opinion in virology mitophagy in tgev infection counteracts oxidative stress and apoptosis transmissible gastroenteritis virus infection induces nf-kappab activation through rlr-mediated signaling non-coding rna control of the rescue and replication of semliki forest virus recombinants by the insertion of mirna target sequences the mitochondrial lncrna asncmtrna- is induced in aging and replicative senescence in endothelial cells mirnas in mtdna-less cell mitochondria mitochondria-related mir- - p contributes to mitochondrial dysfunction in hfd-induced obesity by inhibiting mitochondria-related mir- a- p reduces cellular atp production by targeting cytb in asthenozoospermia differentially expressed non-coding rnas induced by transmissible gastroenteritis virus potentially regulate inflammation and nf-kappab pathway in porcine intestinal epithelial cell line the long noncoding rna lnczc h a promotes a trim -mediated rig-i antiviral innate immune response long noncoding rnas: novel regulators of virus-host interactions identification of lncrna- encoded by mir hg as a novel regulator of innate immunity against influenza a virus infection porcine endemic diarrhea virus infection regulates long noncoding rna expression rpsap lncrna is overexpressed in pituitary tumors and promotes cell proliferation by acting as mirna sponge for hmga proteins circrna_ /mir- a/csf axis modulates osteoclast differentiation to affect ovx-induced bone absorption in mice host relieves lnc-irak - -sequestered mir- b to attenuate apoptosis in enterovirus infection lncrna afap -as promotes malignant phenotype through binding with lsd and repressing hbp in non-small cell lung cancer long noncoding rna cps -it suppresses melanoma cell metastasis through inhibiting cyr via competitively binding to brg lncrnas with mirnas in regulation of gastric, liver, and colorectal cancers: updates in recent years long noncoding rna sbf -as act as a cerna to modulate cell proliferation via binding with mir- - p in acute myeloid leukemia. artificial cells, nanomedicine, and biotechnology long non-coding rna mirt relieves lipopolysaccharide-induced injury in pc cells by suppressing mir- rna sequencing and prediction tools for circular rnas analysis extracellular vesicles long rna sequencing reveals abundant mrna, circrna, and lncrna in human blood as potential biomarkers for cancer diagnosis lncrna snhg facilitates hepatocarcinogenesis and metastasis by modulating its homolog scarna via a positive feedback loop recirc: prediction of circrna expression and function through probe reannotation of non-circrna microarrays catrapid omics: a web server for large-scale prediction of protein-rna interactions effects of qi teng xiao zhuo granules on circrna expression profiles in rats with chronic glomerulonephritis. drug design, development and therapy the conservation and signatures of lincrnas in marek's disease of chicken the rnabinding protein rbm promotes cell proliferation in hepatocellular carcinoma by regulating circular rna scd-circrna production preliminary rna-seq analysis of long non-coding rnas expressed in human term placenta changing expression profiles of mrna, lncrna, circrna, and mirna in lung tissue reveal the pathophysiological of bronchopulmonary dysplasia (bpd) in mouse model tgev infection up-regulates fcrn expression via activation of nf-kappab signaling porcine transmissible gastroenteritis virus nonstructural protein contributes to inflammation via nf-kappab activation regulation of nf-kappab by pml and pml-raralpha utilizing sequence intrinsic composition to classify protein-coding and long noncoding transcripts cpc: assess the protein-coding potential of transcripts using sequence features and support vector machine rnaplex: a fast tool for rna-rna interaction search publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank zhanyong wei of college of veterinary medicine, henan agricultural university, for gifting ipec-j cell line. supplementary information accompanies this paper at https://doi.org/ . /s - - - .additional file : table s . classification of the assembled transcripts of ipec-j .additional file : table s . classification of the lncrnas of ipec-j .additional file : table s . the detailed information of differentially expressed lncrnas.additional file : function analysis of the antisense lncrna tcons_ . prediction of mirna precursor of lncrna.additional file : table s . the lncrna-mirna-mrna regulation network.additional file : table s . the lncrna-proteins regulation network. additional file : table s . the lncrna-up and down genes network.additional file : table s . the interactions of differential expression mrnas.additional file : table s . the sequences of tcons_ shrna and pml sirna.additional file : table s . primers designed for qrt-pcr. the raw data were submitted to the national center for biotechnology information (ncbi) sequence read archive (sra). (https://trace.ncbi.nlm.nih.gov/traces/sra/sra.cgi?view=run_browser). the accession numbers of the tgev-infected group (t , t ) and the mockinfected group (m , m ) are no.srr and no.srr .ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. key: cord- -v wlu eq authors: tô, long-thành; bernard, serge title: effect of fixation on the detection of transmissible gastroenteritis coronavirus antigens by the fixed-cell immunoperoxidase technique date: - - journal: journal of immunological methods doi: . / - ( ) -v sha: doc_id: cord_uid: v wlu eq abstract the effect of various fixatives and detergents on the in vitro detection of the viral determinants which are expressed in swine testis cells infected with the transmissible gastroenteritis coronavirus (tgev) was studied using a microwell immunoperoxidase technique. when compared with glutaraldehyde and formaldehyde, . % paraformaldehyde was found to be the fixative of choice for the detection of these determinants on the membranes of infected cells. among dehydrating fixatives, % acetone or a mixture of acetone and ethanol or of acetone, methanol and ethanol were found to be the best fixatives for the detection of these viral determinants which are expressed in infected cells. in the case of acetone, the temperature of fixation and its concentration in the fixative preparation were found to be important. the treatment of . % glutaraldehyde-fixed, infected cells with . % saponin or . % paraformaldehyde-fixed, infected cells with %np- led to satisfactory detection of viral determinants. using triton x- to render cells permeable, the quantities of n and m antigen detected in tgev-infected cells prefixed with either . % glutaraldehyde or . % paraformaldehyde were equal to those of % acetone-fixed, tgev-infected cells while the quantity of s antigen detected was diminished. the effect of other detergents such as zwittergent, embigen bb, chaps and n-lauroylsarcosine on the detection of viral determinants was also studied. transmissible gastroenteritis (tge) is a highly contagious enteric infection of swine caused by transmissible gastroenteritis coronavirus (tgev) (woode, major structural proteins: the nucleocapsid (n), which is associated with the rna genome; the peplomer glycoprotein (s) and the transmembrane glycoprotein (m) (laude et al., ) . in recent years there has been increasing interest in the development and application of the fixed-cell elisa system for virological and immunological investigations. for tge infections, this technique using infected cells as a source of antigen has been used to detect total anti-tgev antibodies (kodama et al., a) or iga antibodies (kodama et al., b) in the serum of infected swine. for other viruses, it has been used for screening hybridomas (russell et al., ) , for diagnosis (pauli et al., ; groin and bernard, ; van tiel et al., ) and for identification of epitopes on enveloped or naked viruses (anderson et al., ; boere et al., ; oosterlaken et ai., ; simkins et al., ). recently, we described a quantitative fixed-cell immunoperoxidase technique for the study of surface viral antigens induced by tgev (i" et al., ) . as the name of the technique implies, many fixatives have been used by different authors to fix infected cells to the plastic plates. however, there have not been any published reports concerning the effect of fixatives on the detection of viral antigens in infected cells by this technique. in the present study, we have established the effect of various fixatives and detergents on the detection of tgev determinants in infected cells, by means of mabs or pabs in a quantitative fixed-cell immunoperoxidase technique (ipt), the high-passaged purdue-ll strain of tgev (bohl et al., ) and the swine testis (st) cell line (mcclurkin and norman, ) , supplied by dr. e.h. bohl (wooster, oh, usa) were used in this study. eagle's minimal essential medium (emem) supplemented with % heat-inactivated fetal calf serum (fcs), penicillin ( iu/ml) and streptomycin ( /tg/ml) was used for cell growth. confluent monolayers of . × ceus/cm in -well, flat-bottomed plastic plates (falcon , becton dickinson) were inoculated with a volume of . ml/well of virus suspension at a multiplicity of infection (m.o.i) of . after rain of incubation at °c under . % co , the inoculum in each well was removed, washed twice with phosphate buffered saline (pbs) and replaced with . ml/well of emem containing % heatinactivated fcs. the infected cells were incubated at °c under . % co for an additional h, the unfixed, tgev-infecmd cells served as controls. the mabs were directed against m ( - ), s ( - ) and n ( - ) determinants of tgev and had been prepared and used as ascitic fluids following injection of balb/c mice with the antibody-producing hybridomas (laude et al., ) . the anti-tgev pabs ( - ) obtained from the serum of a sow orally immunized with vaccine containing the nouzilly strain of tgev (aynaud et al., ) , and having a neutralizing titer of : , , were used for comparative purposes. (pauli et al., ) , % acetone, % ethanol and % methanol (pauli et al., ) , % acetone and % ethanol (laude et al., ) . fixation procedure the tgev-infected cells were gently washed twice with pbs and the cells were fixed at - °c for min with the appropriate fixative ( . ml per well) which was previously frozen at - °c unless otherwise stated. the fixed cells were washed twice with pbs before addition of . ml/well of blocking solution and incubation for min at rt. an iit previously developed for the detection of surface viral antigens induced by the purdue- strain in infected st cells (t et al., ) was used. briefly, the fixed cells were washed gently with pbs and were incubated for min at °c with . ml/well of a predetermined dilution of anti-m, anti-s, anti-n mab or anti-tgev pabs. the reagents were removed from the plates by two rinses with tap-water and two washes with pbs containing . % tween (serva) and then replaced by . ml/well of a working dilution of peroxidase-labelled goat anti-mouse fc serum (icn immunobiologicals, israel) or peroxidaseu labelled rabbit anti-swine (heavy and light chain specific) serum (cappei, organon teknica, france) . after incubation at °c for min, the plates were washed as before and the enzymatic reaction was developed by incubation at 'c for h with , '-azino-bis ( -ethyl benzthiazoline- sulphonic acid) (abts (boehringer, mannheim, germany))/h chromogen/substrate solution. the supernatants were transferred to another plate containing . ml of % sodium dodecyl sulphate (sds) (serva) to stop the enzymic reaction and to permit the reading of the plate. the optical density (od) was measured at nm by the elisa reader (titertek multiskan) coupled to a microcomputer for data storage and statistical processing. the od signals obtained from the lit are composed of three components: (a) a specific od signal generated by the second-step conjugate specifically bound to the antigen-bound mona clonal antibody; (b) non-specific od generated by non-specifically bound second-step conjugate; and (c) nonspecific spontaneous od due to other reasons, e.g., spontaneous decay of the substrate or the intrinsic od of the cells. the first component is the one of interest. the other two contribute non-specifically to the od signal and therefore should be corrected in order to determine the specific signal. the quantity of each antigen, tested in quadruplicate, was expressed as the difference between the od at nm of virus-infected and uninfected cells using the formula: for comparative purposes the degree of relative antibody binding to a specified determinant was expressed as a percentage of antibody binding to unfixed infected cells. the effects of cell fixation with fa, ga and pfa at various concentrations on antibody bind-ing to viral determinants in the tgev-infected cells are shown in fig. . antibody binding to the n determinant was close to zero when cells were fixed with all these three aldehyde fixatives. fixation with fa at different concentrations led to irregular patterns for antibody binding to m and s determinants (fig. la) . fa at high concentrations caused a decrease of antibody binding to s determinant but an increase of the m determinant up to % of controls at a fa concentration of . %. the levels of antibody binding to m and s determinants were very low using fixation with ga at different concentrations (fig. lb) . and $ ( - ) determinants were expressed as percentages compared with the value obtained with . % paraformaldehyde-fixed, tgev-infected cells. the degree of antibody binding to the n ( - ) determinant was expressed as a percentage compared with that obtained with % acetone-fixed, tgev-infected cells. infected cells which were fixed with . % pfa and antibody binding profiles for these two determinants agreed well (fig. lc) . it is clear that, among the aldehydes tested, the m and s determinants were readily detected in tgev-infected cells which were fixed with . % pfa. the effects of cell fixation on antibody binding to viral determinants in the tgev-infected cells and at different temperatures are shown in fig. . antibody binding to m determinant was decreased whereas binding to s and n determinants increased in cells fixed with increasing acetone concentrations at - °c (fig. a) . when cell fixation occurred at °c, an increase in acetone concentration led to poor detection of m and s determinants although the latter could be detected in cells which were fixed with % or % acetone (fig. b) . in contrast, the antibody binding to the n ( - ) determinant reached a plateau at %. the antibody binding profiles to the m, s and n determinants in cells fixed with acetone at rt showed similarities with the profiles obtained in cell fixation at °c although their od values were lower (fig. c ). in conclusion, m and s determinants were not well detected when cell fixation occurred at °c and at rt while the n determinant was well detected in cells fixed with high acetone concentrations regardless of the fixation temperature. fig. . the effect of treating cells with . % glutaraldehyde or . % paraformaldehyde and then with various concentrations of saponin on antibody binding to viral determinants. the degree of antibody binding to the n ( - ) determinant was expressed as the optical density value obtained with tgev-infected ceils prefixed with the above-mentioned fixative preparation, treated with saponin to permeate the cell membrane and then incubated with anti-n ( - ) mab (block ), relative to the optical density obtained with % acetone-fixed, tgev-infected cells. the degree of antibody binding to the m ( - ) and s ( - ) determinants was expressed as the optical density value obtained with tgev-infected cells prefixed with above-mentioned fixative preparation, treated with saponin and then incubated with mabs having specificity for the m ( - ) (block ) and s ( - ) determinants (block ) relative to the optical densities of . % paraformaldehyde-fixed, tgev-infected cells. determinant was expressed as the optical density value obtained with tgev-infectad cells preflxed with the above-mentioned fixative preparation, treated with triton x- to permeate the cell membrane and then incubated with a mab having specificity for the n ( - ) determinant (block ), relative to the optical density obtained with % acetone-fixed, tgev-infected cells. the degree of antibody binding to the m ( - ) and s ( - ) determinants was expressed as the optical density value obtained with tgev-infected cells prefixed with above-mentioned fixative preparation, treated with triton x- and then incubated with mabs having specificity for the m ( - ) determinant (block ) and s ( - ) determinants (block ) relative to the optical densities of . % paraformaldehyde-fixed, tgev-infected cells. as shown in fig. , the degree of antibody binding to m, s and n determinants in methanol/acetone/ethanol-and methanol/acetone-flxed cells showed similarities with the results obtained in % acetone-fixed cells. the extent of antibody binding to these determinants in methanol/acetone/fa-, absolute methanol-, ethanol/acetic acid-, ethanol/ether-, absolute ethanol-fixed cells was lower than that detected in % acetone-fixed cells. it can be noted that antibody binding to the m and s determinants in . % pfa-fixed, tgev-infected cells was greater than those of cells fixed with any of these dehydrating fixatives. fig. . the comparative effect of treating cells with . % paraformaldehyde and then with different detergents on antibody binding to the viral determinants. the degree of antibody binding to the n ( - ) determinant was expressed as the optical density value obtained with tgev-infected cells prefixed with above-mentioned fixative preparation, treated with indicated detergent to perorate the cell membrane and then incubated with a mab having specificity for the n ( - ) determinant (block ), relative to the optical density obtained with % acetone-fixed, tgev-infected cells. the degree of antibody binding to the m ( - ) and s ( - ) determinants was expressed as the optical density value obtained with tgev-infected cells prefixed with above-mentioned fixative preparation, treated with detergetit and then incubated with mabs having specificity for the m ( - ) (block ) and s ( - ) determinants (block ) relative to the optical densities of . % paraformaldehyde-fixed, tgev-infected cells. the degrees of antibody binding to viral determinants in cells which were fixed with the aidehyde fixative, e.g., . % ga or . % pfa and then permeated with different concentrations of saponin are shown in fig. . the degree of antibody binding to the m, s and n determinants in . % ga-fixed cells which were permeated with . % saponin was similar to that ob/ained in % acetone-fixed cells. with the exception of the . % pfa control the degree of antibody binding to all other samples was lower than that detected in % acetone-fixed cells. when using triton x- (fig. ) , antibody binding to the m and n determinants in . % ga-or . % pfa-fixed cells which were then permeated with triton x- at various concentrations showed similarities with the results obtained in % acetone-fixed cells. the degree of antibody binding to the s determinant in . % ga-fixed cells which were then permeated with triton x- at . , . and . % was lower than that obtained with % acetone-fixed, infected cells. the degree of antibody binding to the s determinant in . % pfa-fixed cells which were permeated with different concentrations of triton x- was similar to that detected in % acetone-fixed cells. the od of cells incubated with anti-m mab was equal to that obtained from . % pfa-fixed, infected cells while the od of cells incubated with anti-s was significantly lower than that of . % pfa-f'lxed, infected cells. for some other permeabilizing agents (fig. ) , antibody binding to the n determinant in . % pfa-fixed cells which were permeated with either % zwittergent, % empigen bb, % np- , mm and mm chaps showed similarities with the results obtained in % acetone-fixed cells while antibody binding to this determinant in cells permeated with mm n-lauroyl and mm chaps was lower than that of % acetonefixed, infected cells. the degree of antibody binding to the s and m determinants in cells which were permeated with % zwittergent - or mm n-lauroyl was lower than that of % acetone-luted, infected cells. the degree of antibody binding to these determinants in cells permeated with % zwittergent - , or % empigen bb, % np- or chaps was similar to that obtained in % acetone-fixed, infected cells. the following experiments were carried out to identify differences in localisation of antigens detected in infected cells which were fixed with either . % pfa at °c (fig. a ) or with % acetone at - °c (fig. b) using two different fixation procedures. the degree of antibody binding to viral determinants in unfixed, infected control cells was compared with that measured in infected cells which were fixed (i) before their incubation with antibody or (ii) after their incubation with antibody. the antibodies used in this experiment were mabs and pabs as mentioned in the materials and methods section. whichever pfa fixation procedure was used, the degree of antibody binding to the m, s, n determinants and to virions in toev-infected cells was not significantly different from that of controls. however, antibody binding to viral determinants varied between cells fixed with % acetone at - °c and the control infected cells. the degree of antibody binding to the m determinant in cells fred according to the two different fixation procedures was similar to that obtained in the controls. the degree of antibody binding to the s determinant was lower than that with the controls suggesting that after acetone fixation the accessibility of this determinant to the mab was affected. the degree of antibody binding to the n determinant in cells fixed with % acetone after their incubation with mab was equal to that of controls but considerably lower than that of infected cells which were fixed with % acetone prior to their incubation with mab. the anti-tgev polyclonal binding profiles to viral antigens were similar to those obtained with the m and s determinants. the present study has shown that ga fixation alters the accessibility of tgev determinants to anti-m, anti-s and anti-n mabs and that fa fixation has the opposite effect on the detection of m and s viral determinants compared with pfa fixation. in another study smith et al. ( ) has shown that formalin fixation blocks, alters or destroys a specific antigen expressed in bovine viral disease virus (bvdv)-infected cells. similar results were also obtained by saunders ( ) for the detection of hog cholera virus antigen after ga f~ation at different concentrations. to investigate the influence of fixation temperature on viral antigen detection, the fixation step was carried out at - °c, °c or at rt. the detectable quantity of m antigen decreased with an increase in acetone concentration at all three test temperatures. the increase in acetone concentration parallelled the increase of detectable n antigen. our results suggest that the acetone concentration in fixative preparation and the fixa-tion temperature are two important criteria for a fixation procedure. similar results were also obtained (groen et al., ) by comparing the use of ethanol (- °c) or acetone (+ °c) fixation for the detection of hantaan virus. in his study on the development and evaluation of an enzyme labelled antibody test for the rapid detection of hog cholera antibodies, saunders ( ) showed that this test gave a good result only when the fixative solution contained less than % acetone. it should be noted that % acetone is a routinely used fixative which gives excellent results in immunohistochemistry. in our experiments, cell fixation with a high concentration of acetone ( %) resulted in adequate detection of tgev antigens. however, we could not use higher acetone concentrations to fix the cells because the microtiter plates would be distorted with acetone concentration higher than % or would melt with - % acetone (personal observation). therefore, we do not know if acetone concentrations higher than % would give the same results in fixed-cell immunoperoxidase. a comparison of cell fixation with dehydrating fixatives and mixtures of these fixatives (fig. ) suggested that the highest quantity of antigen detected was in tgev-infected cells which were fixed with either % acetone or mixtures of act/meth or act/meth/eth. it is possible that adequate detection of tgev antigens in act/ meth-and act/meth/eth-fixed cells is due to the acetone in the fixative preparations since viral antigens are not so readily detected in infected cells which are fixed with methanol or ethanol alone. similarly, comparisons of cell treatment with permeabilizing agents (figs. , and ) lead to the conclusion that the tgev antigens were readily detected in pfa-fixed cells which were then permeated with % np- and in ga-fixed cells which were then permeated with . % saponin. several authors have compared the effects of aldehyde fixative and dehydrating fixative in the fixed-cell elisa. cannon ( ) found that both methanol containing . % h and % fa were suitable fixatives for hep- cells infected with respiratory syncytial virus (rsv), although methanol/h was considered to be the fixative of choice. the author also found that where a mab specific for virus np was used in lit, staining was only seen following fa fixation but this was not the case for mab specific for rsv envelope glycoproteins. the latter generally gave good results with either fixative. similarly, fa fixation seemed to preserve borna virus-specific antigens better than acetone/methanol or acetone fixation and the labelling was pronounced when using mab (pauli et al., ) . smith et al. ( ) also noted that immunoperoxidase staining of bvdv-infected cells fixed with formaldehyde was less intense in cells fixed in acetone. our results (fig. ) showed that only viral antigens which are present on the membrane of infected cells may be detected after aldehyde fixation and that the accessibility of these antigenic determinants to the appropriate antibodies was not changed after pfa fixation. however, both surface and cytoplasmic antigens were detected after acetone fixation. this is due to the fact that after pfa fixation me cell membrane was still intact but became permeable after acetone fixation. thus mabs also bind to cytoplasmic viral antigens (t et al, ) . it seems that acetone fixation at - °(: had no effect on the accessibility of the n determinant to the mab. the same results (data not shown) were also obtained in tgev-infected cells which were fixed with either act/meth or act/meth/eth using two different fixation procedures. it would seem that the stronger the permeabilizing capacity of the fixative, the more n antigen but less m and s antigens were detected. consequently, the interpretation of results comparing aldehyde and dehydrating fixatives should take this observation into consideration. in conclusion . % pfa appears to be a suitable fixative for the detection of tgev antigens on the membrane of infected cells. for cellular viral antigens; % acetone, a mixture of act/meth or act/meth/eth are the fixatives of choice. identification of epitopes on respiratory syncytial virus proteins by competitive linking immunoassay transmissible gastrnenteritis (tge) of swine: survivor selection of tge virus mutant in stomach juice of adult pigs antigenic differences between virulent and avirulent strains of semliki forest viruses detected with mab antibody response in serum, colostrum and milk of swine after infection or vaccination with transmissible gastroenteritis virus microplate immunoperoxidase detection of infectious respirato~ ~ncytial virus in the lung of infected mice comparison of immunofluorescence and enzymelinked immunosorbent assays for the serology of hantaan virus infections virus enzyme-linked cell immunoassay (velcia): detection and titration of rotavirus antigen and demonstration of rotavirus neutralizing and total antibodies detection of antibody against transmissible gastroenteritis virus of pigs by indirect immunoperoxidase antibody test characterization of immunogiobusn a antibody in serum of swine inoculated with transmissible gastroenteritis virus ~ ) molecular biology of transmissible gastroenteritis viru!,~ antigenic ~ structure of transmissible gastroenteritis virus: . properties of mab directed against virion proteins studies on transmissible gastroenteritis of swine. ii. selected characteristics of cytopathogenie virus common to five isolates from transmissible gastrocnteritis plaque/focus immunoassays: a simple method for detecting antiviral monoclonal and other antibodies and viral antigens in cells a mierov~ll immunoperoxidase test for screening hybridomas and for diagnosing newcastle disease virus and sendal virus c ( ) development and evaluation of an enzyme labelled antibody test for the rapid detection of hog cholera antibodies epitope mapping and detection of transmissible gastroenteritis viral proteins in cell culture using biotinylated mab in a fixed-cen elisa detection of cytopathic and noncythopathic bovine viral diarrhea virus in cell culture with an immunoperoxidase test techniques immunoenzymatiques. soci~t~ fran~aise d'lmmunologie. les editions inserm fixed-ceu immunoperoxidase technique for the study of surface antigens induced by the coronavirus of transmissible gastruenteritis (tgev) enzyme immunoassay of mumps virus in cell culture with peroxidaselabelled virus specific monoclonal antibodies and its application for determination of antibodies transmissible gastroenteritis we are grateful to dr. j.m. aynaud for useful advice, to e. bottreau and i. lantier for help with the cell cultures and elisa. we also wish to acknowledge the fondation marcei-m rieux for providing a scholarship to the leading author. key: cord- -os kyvvf authors: wang, li; dai, xianjin; song, han; yuan, peng; yang, zhou; dong, wei; song, zhenhui title: inhibition of porcine transmissible gastroenteritis virus infection in porcine kidney cells using short hairpin rnas targeting the membrane gene date: - - journal: virus genes doi: . /s - - - sha: doc_id: cord_uid: os kyvvf the membrane (m) protein is the most abundant component of the porcine transmissible gastroenteritis virus (tgev) particle. to exploit the possibility of using rna interference (rnai) as a strategy against tgev infection, three plasmids (prnat- , prnat- , and prnat- ) expressing short hairpin rnas were designed to target three different coding regions of the m gene of tgev. the plasmids were constructed and transiently transfected into a porcine kidney cells, pk- , to determine whether these constructs inhibited tgev production. the analysis of cytopathic effects demonstrated that prnat- and prnat- could protect pk- cells against pathological changes specifically and efficiently. additionally, indirect immunofluorescence and % tissue culture infectious dose (tcid( )) assays showed that prnat- and prnat- inhibited the multiplication of the virus at the protein level effectively. quantitative real-time pcr further confirmed that the amounts of viral rnas in cell cultures pre-transfected with the three plasmids were reduced by , , and %, respectively. this is the first report showing that rnai targeting of the m gene. our results could promote studies of the specific function of viral genes associated with tgev infection and might provide a theoretical basis for potential therapeutic applications. transmissible gastroenteritis coronavirus (tgev) is a positive rna virus, which is a member of a large family of enveloped viruses [ ] . pigs of any age and breed can be infected. in particular, sucking piglets at about weeks old are the most susceptible, showing mortality rates up to %, which results in large economic loss in swine-producing areas worldwide [ , ] . however, the pathogenic mechanism of tgev remains unclear [ ] . at present, several vaccines to prevent tge are available; however, their efficacies are variable. attenuated tgev vaccines have the risk of returning to the virulent form and might even induce an adverse reaction and inactivated viruses are not sufficiently protective in pigs [ , ] . moreover, newborn piglets can suffer from gastroenteritis within h post-infection, and death can occur in - days [ ] , whereas current vaccines cannot provide complete protection in the first days after inoculation. thus, it is necessary to develop novel, highly effective, and rapid-acting antivirals to resist tgev infection [ ] . rna interference (rnai) is a precise gene silencing method that uses double-stranded rna (dsrna) molecules comprising - nucleotides (nt) . rnai in the form of small interfering rnas (sirnas) or short hairpin rnas (shrnas) has been studied for their interference with virus replication [ , ] . recent research suggests that the replication of various viruses, including many coronaviruses, could be inhibited effectively in vitro and in vivo [ ] [ ] [ ] [ ] [ ] [ ] . therefore, it might be possible to disrupt the replication of tgev in cell culture using shrnas targeting the m gene of tgev. tgev is a positive-sense, ssrna virus with a . kb genome that contains a leader sequence at the end and a poly (a) tail at the end, which encodes four structural proteins [spike (s), membrane (m), nucleocapsid (n), and envelope (e)] and five non-structural proteins [ ] [ ] [ ] . the s protein is a major membrane glycoprotein that plays important roles in inducing a protective immune response, and in virus attachment, membrane fusion, and viral pathogenicity [ ] [ ] [ ] . the n protein, together with the genomic rna, forms the viral nucleocapsid [ ] . the e protein regulates virion assembly and release [ ] . the m protein is the most abundant component of the coronavirus particle [ ] and differs from other viral proteins in terms of its structure, processing, and intracellular transport [ ] . the expressions of the m and e proteins might be sufficient to trigger the formation of virus-like particles (vlps). in addition, m is highly conserved among different strains, and our previous studies proved that the expression the m protein alone using a baculovirus expression system could lead to the formation of vlps, as observed under a transmission electron microscope, which further confirmed that the m protein of tgev is a decisive protein for the proliferation of viral proteins [ ] . as one of the important structural proteins of tgev particles, the m protein is exposed on the viral internal core [ ] , and associates with the golgi complex in the cell, which suggests that the m protein plays a mechanistic role at the site of virus assembly and budding [ ] , and suggest that m is an indispensable component for the replication of virus particles in host cells. in this study, we constructed three shrnas in a plasmid expression system that targeted the m gene and investigated whether shrna-mediated rna interference could inhibit tgev infection of pk- cells. virus and cells tgev strain cq was isolated from sick piglets with symptoms of diarrhea [ ] and stored in our laboratory. pk- cells were grown in high glucose dulbecco minimum essential medium (dmem) supplemented with % fetal bovine serum (gibco, usa), iu of penicillin, and streptomycin per ml, at °c in a % co atmosphere incubator. according to the general principles and guidelines for the design of rna interference, sequences from the m gene of tgev cq were designed based on the ambion's online sirna target design tool to choose the three best target sequences to target the m gene (http://www.ambion.com/ techlib/misc/sirna_finder.html). three theoretically effective sequences at nucleotide positions - (rnat- ), - (rnat- ), and - (rnat- ) were selected. the sequences were analyzed by blast to ensure that they did not have any similar sequences in the swine genome, but share % similarity with the published sequences of different tgev strains. these three sequences are listed in table . all the sequences were arranged in the following alignment: bamhi ? sense ? loop ? antisense ? termination ? hindiii. we designed the double-stranded oligo dna hairpin structures to target the m after annealing. all the shrnaexpressing plasmids were diluted with tris-edta buffer to a final concentration of lg/ll. the annealing reaction system ( ll) comprised ll of shrna sense template, ll of antisense template, and ll of ddh o. the mixture was heated to °c for min, cooled to °c for s, and then incubated at °c. the annealed shrna dna sequences (rnat- , rnat- , and rnat- ) and shrna expression vector, prnat-u . /neo (ribobio, china), were then double digested with bamhi and hin-diii, and inserted into bamhi-hindiii digested prnat-u . /neo to yield prnat- , prnat- , and prnat- , respectively. after transformation of escherichia coli dh a competent cells to obtain the recombinant plasmids, the positive clones were identified by pcr and sequencing analysis. the enhanced green fluorescence protein fusion gene in the plasmids was used as a reporter during the transfection efficiency analysis. one day before transfection, pk- cells were seeded into six-well plates and incubated for h at °c in a % co atmosphere without antibiotics. when the cells reached - % confluence, they were washed with . m pbs (ph . ) three times and overlaid with transfection complexes containing . lg of prnat- , prnat- , prnat- , or prnat-nc in ll of dmem medium mixed with lipofectamine tm (invitrogen, usa), according to the manufacturer's instructions. the transfection complexes were completely removed after incubating for h, and the medium was replaced with % fbs containing lg/ml g . after maintenance for d in selection media, resistant cell clones were selected, cultured, and infected with . moi of tgev per well in six-well plates. non-transfected cells were used as a virus genes ( ) : - control. cell transfection efficiency and cpe images were captured under an inverted fluorescence/phase-contrast microscopy (nikon, japan). shrna-transfected cells were collected h after viral infection, subjected to three freeze-thaw cycles, serially diluted tenfold from - to - , and added to -well plates. each dilution was added to eight wells. the tcid was calculated using the reed and muench method. to quantify the effect of shrna on viral replication at h post viral infection, total rna was extracted from pk- cells using the rnaiso plus (invitrogen, usa) reagent, according to the manufacturer's instructions, and reverse transcribed into cdna using the goscript tm reverse transcription system (promega, usa), also according to the manufacturer's instructions. quantitative real-time pcr (qpcr) analysis was performed to amplify m gene using the cdna as the template and the b-actin gene as the internal standard. western blotting pk- cells were transfected with prnat-nc, prnat- , prnat- , or prnat- and infected with tgev. cells as well as virus particle were lysed in phosphate buffered saline (pbs), and the total proteins were separated using % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and transferred onto a polyvinylidene difluoride membrane. the membranes were incubated with rabbit anti-tgev polyclonal primary antibodies ( : dilution, °c, overnight), washed, and then incubated with hrp-goat-anti-rabbit secondary antibody ( : dilution, room temperature, h). effects of shrna transfection pk- cells ( cells per well) were plated in six-well plates and transfected with shrna recombinant plasmids (prnat- , prnat- , or prnat- ) and empty plasmid (prnat-nc), separately, for h, before being examined by fluorescence and phase-contrast microscopy. the gfp gene expressed the green fluorescent protein from the cmv promoter, and more green fluorescent excitation by the blue wavelengths was observed in cells containing the empty plasmid (prnat-nc) compared with cells transfected with the recombinant plasmids (prnat- , prnat- , prnat- ). the normal pk- cells showed no fluorescence (fig. ) . the results showed that shrna recombinants were transfected into pk- cells successfully and that stably transfected cell lines were created. the transfection efficiencies were similar among the three recombinant plasmids, while that of the empty plasmid was higher. to study the tgev-induced cpe, pk- cells were infected with tgev at . moi. the virus infected cells (mock control) and empty plasmid (prnat-nc) exhibited obvious morphological changes at h post-infection, including cells shrinkage, turn round, and detachment, in contrast to the non-infected cells (normal) that remained tightly stuck to the plate and maintained their shape. as shown in fig. , the normal group grew well; however, the cells harboring the shrna-expressing plasmids prnat- and prnat- showed small patches of cpe, such as rounding, shrinking, and morphological changes of the cells, as well as shedding from the brim of the wells. interestingly, the cells harboring recombinant prnat- and prnat- were mostly capable of resisting the cpe as shown by the observation that the cells attached well and had reduced areas of cpe, which contrasted with the large area of severe cpe in the cells harboring prnat- . these results indicated that shrna-expressing plasmids prnat- and prnat- inhibited tgev-induced cpe to a certain degree and could relieve the specific cytopathic effect compared with the controls. to investigate the inhibition of tgev replication by the shrnas, virus titers in pk- cells were calculated by the reed-muench method. figure shows that the titers of tgev reached . , . , and . tcid /ml at h post-infection in cells harboring prnat- , prnat- , and prnat- , respectively. the titers at h postinfection corresponded to . -, . -, and . -fold reductions, respectively, compared with that of prnat-nc. the tgev titer was . tcid /ml in cells receiving no plasmid (mock) transfection, which was higher than the titer of . tcid /ml in cells pretransfected with prnat-nc. there was a significant difference between prnat- and prnat-nc (p \ . ), as well as between prnat- and prnat-nc (p \ . ). contrastingly, there was no significant difference between prnat- and prnat-nc. these data indicated that prnat- and prnat- resisted tgev infection by reducing the levels of progeny virus production significantly in pk- cells. in addition, prnat- and prnat- showed partial virus infection inhibition, with prnat- being the least effective shrna. the expression levels of the m gene in pk- cells treated with different interfering plasmids were examined using qpcr. figure shows the cellular expression of the m gene. when the cells were transfected with prnat- , the expression of m gene decreased by % compared with the cells transfected with prnat-nc. when the cells were transfected with prnat- or prnat- , the expression of the m gene decreased by and %, respectively, compared with cells transfected with prnat-nc. the results showed that prnat- and prnat- have a certain inhibitory effect on the proliferation of tgev in pk- cells, which is caused by degradation of the viral rna. to further investigate the levels of viral proteins in cells transfected with shrna plasmids and infected with tgev, the levels of viral proteins were assessed using western blotting. equal amounts of cell lysates from tgev-infected and mock-infected pk- cells at h were examined using positive anti-tgev serum. figure shows that the amount of viral protein recovered from cells transfected with prnat- or prnat- was reduced, while the amount of viral protein recovered from cells transfected with prnat- was similar to that recovered from cells without an interfering plasmid, which was consistent with the qpcr analysis. rnai has been used widely to silence target genes in mammalian and human cells [ ] [ ] [ ] . rnai can regulate specific gene expression and is closely related to anti-virus replication. rnai has an excellent prospect to improve the shortage of traditional anti-virus vaccines or related inhibitors. rnai has emerged as a potentially important therapeutic antiviral strategy [ , [ ] [ ] [ ] . recently, several kinds of animal viruses, such as porcine reproductive and respiratory syndrome virus [ , ] , newcastle disease virus [ ] , classical swine fever virus [ ] , porcine circovirus [ ] , infectious bursal disease virus [ ] , and porcine hemagglutinating encephalomyelitis virus [ ] have been silenced effectively, and most of these viruses are rna viruses. tgev is a porcine coronavirus with an rna genome; therefore, it should also be sensitive to rnai [ ] . several studies have reported the application of rnai against tgev replication. effective suppression of tgev infection in swine testicular (st) cells was achieved using dna-based vectors expressing sirnas or shrnas targeting the rna-dependent rna polymerase gene of tgev [ , ] . lei he, et al. reported the effective inhibition of tgev infection in st cells or pk- cells using dna-based vectors expressing an shrna targeting the transcription of tgev gene (a non-structural gene) [ , ] . however, there is no report showing that sirna/ shrna targeting the m gene of a coronavirus could efficiently inhibit viral infection. in this study, we constructed three shrnas plasmid expression systems to target the m gene and investigated whether shrna-mediated rna interference could inhibit tgev infection in pk- cells. our results demonstrated that the infection of tgev in cell culture could be disrupted by shrnas targeting the m gene of tgev: two of the three shrnas generated from the m gene of tgev blocked viral infection efficiently. the cpe and tcid assays revealed that cells transfected with prnat- , prnat- , and prnat- , harboring three sequencespecific shrnas, could trigger inhibition of tgev infection at h post-infection; prnat- in particular showed markedly suppression. western blotting and qpcr analyses further confirmed that the efficient inhibition of viral infection was caused by viral degradation. however, the qpcr analysis showed that transfection with prnat- and prnat- inhibited viral infection by the equivalent of %. the qpcr analysis and western blotting assays also demonstrated that, compared with the mock control, the amount of viral rnas in the prnat- group decreased a little, which suggested an inefficient inhibitory effect, which possibly indicated that the prnat- sequence results in non-specific inhibition or in 'off -target' effects. overall, the variability of viral suppression could be related to the following two aspects. one is that the regulation of rna transcription and protein expression is a very complex process, and represents the combined effect of various factors. the other possible explanation is the difference in the sensitivity and accuracy between tcid and qpcr. qpcr is highly sensitive to detect the suppression effect of rna interference. in addition to the potent inhibition shown by two sequence-specific shrnas, the tcid and qpcr analyses also demonstrated that, compared with the mock control, the amount of viral rnas in the negative control prnat-nc cells also decreased a little, which suggested a non-specific effect on tgev replication in pk- cells. similarly, other researchers have discussed an ''off-target'' effect induced by sirna or shrna in their reports. lu et al. [ ] found that the non-specific effect was positively related to the concentration of the shrnas. overall, compared with the low-efficiency inhibition and 'off-target' effects of prnat- , the other two sequencespecific shrnas exhibited the potential to silence tgev rnas. in conclusion, our results indicated that shrnas targeting the m gene in tgev genome could effectively block infection of tgev in pk- cells. this finding showed that shrnas could represent a potential novel tool against tgev infection. these results also provided an insight into the inhibition of tgev infection by targeting the m gene. taken together, the present data and the known advantages of shrna technology suggest that shrna represents a candidate agent for tgev therapeutic applications. the coronaviridae proc. natl. acad. sci. usa key: cord- -nvrl vyi authors: song, zhenhui; dai, xianjin; ye, cuifang; li, yuntian; wang, li; hu, yang title: morphogenesis and proliferative rule of porcine transmissible gastroenteritis virus in porcine intestinal epithelial cells date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: nvrl vyi to gain a better understanding of the replication, proliferation and infection characteristics of porcine transmissible gastroenteritis virus (tgev) in porcine intestinal epithelial cells (iecs), this study established a cell model of iecs infected with the chongqing (cq) strain of tgev. the morphogenesis and proliferative rule of tgev in porcine iecs were investigated using transmission electron microscopy, indirect immunofluorescence assays and real-time fluorescence quantitative pcr. observations under the tem indicated that the enveloped viral particles were roughly spherical, with diameters of between and nm. the virions entered porcine iecs by membrane fusion and the mature viruses in the vacuoles were transported to the cell membrane before release. the results also showed that from to h after tgev infection of porcine iecs, the intracellular viral rna content did not change significantly. logarithmic growth occurred from to h, after which it gradually decreased. moreover, the extracellular rna content began to rise at h after inoculation and then reduced gradually at approximately h. this study provided a theoretical foundation for further study on the infection characteristics of tgev in target cells. transmissible gastroenteritis virus (tgev), a member of the coronaviridae, is the etiological agent of transmissible gastroenteritis (tge), which is characterized by severe diarrhea, vomiting and dehydration in approximately -week-old suckling piglets (goodwin and jennings, ; mullan et al., ) . tge results in serious economic losses in swine-producing areas worldwide. tgev has a large, . kb single-stranded positive sense rna genome, which comprises four structural proteins encoded by the spike (s), membrane (m), envelope (e), and nucleoprotein (n) genes (lai and cavanagh, ; eleouet et al., ) . many cell types are susceptible to tgev, including pig thyroid cells, swine testis cells (st), pig kidney cells (pk- , ibrs- ) and salivary gland cells (nguyen et al., ; haelterman and hutchings, ) . ibrs- , pk- and st cell lines are used commonly to culture tgev in the laboratory (chen et al., ) . in vivo, tgev replicates in intestinal epithelial cells of susceptible animals, causing cellu-lar degeneration, villus shortening, malabsorption and diarrhea (haelterman, ; schwegmaan-wessels and herrler, ) . most studies concerning tgev pathogenesis in swine have been performed in animal infection models. only rarely have in vitro studies been used with cell lines originating from porcine intestines. cell lines ipec- , ipec-j and ipi- i have been used in in vitro model systems (fangning et al., ; valentina et al., ; peter et al., ) . however, data concerning these cell lines, and the morphogenesis and proliferation of the virus in the target cells, are scarce. thus, we developed an in vitro model based on porcine intestinal epithelial cells (iec) infected with tgev, and used transmission electron microscopy (tem), indirect immunofluorescence assay (ifa) and real-time fluorescence quantitative pcr (fq-pcr) to investigate the infection mechanism of tgev. tgev n genes are highly homologous and are essential for replication. the n gene encodes the nucleocapsid protein, which is a highly basic protein with a molecular mass ranging from to kda, depending on the species and strain (escors et al., ) . the n protein's function involves cessation of cell division and extension of the cell cycle to provide more suitable conditions for virus assembly (liu et al., motokawa et al., kapke and brian, ) . this is especially significant when the viral life cycle is longer than that of the host cell cycle (wurm et al., ) . therefore, to anahttp://dx.doi.org/ . /j.jviromet. . . - /© elsevier b.v. all rights reserved. lyze the tgev proliferation in target cells, a one-step growth curve was constructed according to the fq-pcr data for the transcription dynamics of the n gene. porcine intestinal epithelial cells (iecs) were developed in our institute, and were isolated from the jejunum of a newborn piglet (unpublished data). tgev chongqing (cq) strain was isolated from sick piglets with symptoms of diarrhea (song et al., ) . an anti-tgev-np mouse monoclonal antibody was produced in our laboratory. anti-rabbit igg/cy , anti-mouse igg/fitc and , -diamidino- -phenylindole (dapi) were purchased from sigma (usa). porcine iecs were grown in dulbecco's modified eagle's medium (dmem)/f- medium (gibco) with % fetal bovine serum (fbs, gibco brl) and maintained in maintenance medium (dmem/f- supplemented with % fbs) in a % co incubator. porcine iecs were seeded on cover slips and grown to % confluence. tgev ( × tcid ) was then inoculated into the cells. after culture for , , , and h, respectively, cells were fixed with % paraformaldehyde for min, the cover slips were washed three times with phosphate buffered saline (pbs), and then permeabilized using . % triton x- for min at room temperature (rt). the primary antibodies, diluted in % bovine serum albumin, were incubated with the coverslips for h at rt in a humid chamber, followed by washing three times with pbs. the secondary anti- bodies were then incubated with coverslips for min at rt. after the last incubation step, the cover slips were washed three times with pbs and twice with distilled water to remove precipitates. the coverslips were embedded in % polyvinyl-alcohol (mowiol) and stored at • c until examination under a fluorescence microscope. to detect whether tgev propagated in porcine iecs cells could infect st cells, st cells were infected by the supernatant of iecs infected with tgev for h. a monoclonal antibody against the tgev nucleoprotein (np), at a : dilution, was used to detect infected cells. the secondary antibody comprised fitc-labeled antimouse igg at a : dilution. an ifa was then performed using the method mentioned above. to demonstrate apoptosis, iecs were infected with tgev for , , , and h, and an apoptosis was detected using an anticaspase- antibody. the rabbit anti-caspase- antibody was used at a : dilution, followed by incubation with a cy -labeled anti rabbit igg as the secondary antibody at a dilution of : . cell nuclei were stained by incubation with dapi for min • c. an ifa was then performed using the method mentioned above. at , , and h post-infection, infected and mock-infected cells were harvested and washed with pbs ( . mol l − , ph . ) twice, and fixed with g l − glutaraldehyde at • c for h. the cells were scraped from the glass surface and centrifuged at × g for min. the supernatant was then discarded, and the cell pellet was resuspended in g l − low melting agarose at • c and cen-trifuged at × g for min. samples were post-fixed in g l − aqueous oso . after stepwise dehydration in acetone, the samples were embedded in epoxy resin and polymerized at • c for days. the embedded cells were cut into -nm slices and stained with uranyl acetate and lead citrate for subsequent examination using tem (hitachi h ; hitachi, tokyo, japan). a pair of primers to amplify the n gene of tgev was designed according to the published sequences obtained from genbank at the national center for biotechnology information (ncbi) website. the primer sequences were: sense primer: -ttcaaccccataaccctccaacaa- , and antisense primer: -ggcccttcaccatgcgatagc- . sangon biotech (shanghai, china) co., ltd synthesized the primers. total rna was isolated from infected porcine iecs using the tri-zol regent (takara biotechnology (dalian) co., ltd.), according to the manufacturer's protocol. the following reaction mixture was used to generate cdnas: mgcl l, × rt buffer l, rnase free dh o . l, dntp mixture l, rnase inhibitor . l, amv reverse transcriptase . l, random -mer . l, antisense primer . l and rna l. the reaction conditions were: • c for min, • c for min, • c for min, • c for min. the pcr amplification reaction comprised forward primer . l, reverse primer . l, ex taq hs . l, × pcr buffer l, cdna template . l and l of distilled water for a total volume of l. the pcr conditions were: • c for min, followed by cycles of dna amplification ( s at • c, s at • c, and min at • c) and a min incubation at • c. the pcr products were cloned into the pmd -t vector (takara biotechnology) and sequenced using the classical sanger dideoxy sequencing method (takara biotechnology). the concentration of the recombinant plasmid was determined using a micro-spectrophotometer. the copy number of the plasmid was calculated and the plasmid was then subjected to a -fold serial dilution (from − to − copies/l) for use as the standard template in real-time pcr method. the standard samples were amplified using real-time pcr using the following reaction mixture: ddh o l, sybr premix ex taq ii l, specific primers . l and dna l. the reaction conditions were: cycles of • c for s, • c for s and • c s. one high-concentration and one low-concentration plasmid template were selected for three repeats. the average value was then calculated, which permitted the construction of the kinetic curve and the standard curve. porcine iecs were infected with tgev cq strain, and the supernatant and cells were collected separately at different time points ( , , , , , , , , and h) . the total rna in the different samples was extracted and used to produce cdna as a template for quantitative pcr. the pcr reaction comprised: ddh o l, sybr premix ex taq ii l, specific upstream primer . l, specific downstream primer . l and cdna template l. the reaction conditions were: cycles of • c for s, • c for s and • c for s. the reaction for each sample was repeated three times and a negative control was included. the copy numbers of the gene were obtained from the test sample and used to construct a one-step growth curve for tgev. an indirect immunofluorescence technique was used to observe the distribution of tgev in porcine iecs at different time points. cell nuclei stained by dapi appeared blue, and the cytoplasm of the control group did not generate specific fluorescence compared with the treated cells (fig. a) . after h, h and h of infection, some green fluorescence was observed (fig. b, c, d) . at h after treatment, a large amount of green fluorescence was noted close to the dapi-stained cell nuclei (fig. e) . at h, the green fluorescence had decreased, although the nuclear fluorescence remained clearly visible (fig. f) . to determine whether st cells could be infected by tgev propagated in iecs, the growth of tgev in st cells infected by the culture supernatant of iecs was determined. the ifa results showed that st cells were susceptible to infection by tgev propagated in iecs (fig. ) . in addition, the apoptotic effect of tgev from iecs was assessed using an anti-caspase- antibody. as time increased, more caspase- -positive cells were observed (fig. ) , indicating that tgev induced apoptosis in iecs. to confirm tgev infection of the target cells, the morphogenesis of the tgev virions was observed under tem. at h after infection, virions with high electron density were observed attached to the membrane surface (fig. b) ; however, very few viruses appeared in the cytoplasm or vacuoles (fig. c) . at h after inoculation, the cytoplasm was filled with vacuoles containing tgev particles (fig. d, e) . fig. f shows that the viral envelope was obtained by budding into vacuoles. virions in vacuoles were transported to the cell membrane (fig. g) , with which they fused and then released into the extracellular space via exocytosis (fig. h) . at the later stage ( h post-infection), the golgi complex became disaggregated (fig. i) . a tgev cq strain was used to infect porcine iecs, and the supernatant and cells were collected at different time points for fq-pcr. the one-step growth curve of tgev was plotted based on amount of rna for the tgev n gene in different samples. the resulting growth curve (fig. ) showed that the amount of intracellular tgev rna did not change substantially during the first h post-infection. from h- h after infection, intracellular viral rna increased logarithmically, after which its proliferation slowed down. from h- h post-infection, the extracellular tgev rna content did not change significantly. thereafter, the viral rna level exhibited logarithmic growth, reaching a maximum at h. tgev can proliferate in many kinds cultured cells, especially in st cells and pk- cells (ren et al., ) . after infection by tgev for h, st cells showed a typical cytopathic effect, characterized by shrinkage and rounding, and a gradual weakening of their adherence ability: ultimately, the majority of the cells were floating in the medium (yin et al., ) . sirinarumitr et al. ( ) found that tgev could induce apoptosis during infection of st cells. nuclear chromatin condensation and fragmentation, as well as cell shrinkage and cytoplasmic vacuolation, were observed in pk- cells infected by tgev. this study showed that tgev's infection of porcine iecs has a partial cytopathic phase, characterized by cells changing to a fusiform shape, pyknosis, refraction and activation of caspase . however, the cytopathic effect in iecs was not as obvious as that in st or pk cells. tgev could induce porcine iecs to undergo apoptosis, which is consistent with the results of tong et al. ( ) . tgev-induced apoptosis of porcine iecs might function via blocking cell-cycle transfer from g to s phase; however, its specific mechanism requires further exploration. indirect ifa extends the application range of an antibody and further improves the detection sensitivity by labeling the specific secondary antibodies with fluorescein for signal amplification. in this study, both infected and uninfected cell cultures were detected by ifa using tgev antiserum. the results revealed the distribution of tgev in porcine iecs clearly. at h post-infection, many tgev particles bound to goat anti-rabbit igg-fitc showed green fluorescence when exposed to laser light and were located in the cytoplasm. at h, the fluorescence transferred to the perinuclear area, before decreasing gradually at h. porcine aminopeptidase n (papn), a cellular receptor for tgev, is abundantly expressed in the membrane of porcine iecs (data not shown), which suggested that papn plays a role in mediating tgev infection. in addition, we demonstrated that st cells could be infected with the culture supernatant of iecs infected tgev at h. taken together, these data indicated that iecs, which are derived from a target tissue for porcine coronaviruses, could be infected by tgev successfully, and that tgev propagated in iecs remains infectious for st cells. the tgev s protein is involved in the viral invasion process (trincone and schwegmaan-wessels, ) . combined with papn, the s protein mediates the fusion of tgev and the cell membrane, such that nuclear capsid is released into the cell (gelhaus et al., ) . a previous report also suggested that viral genome and structural proteins were assembled into virus particles in the area between the endoplasmic reticulum and the golgi, and that the particles were transported to the extracellular space through budding of vesicles (corse and machamer, ; lili and masters, ) . until now, the morphology of tgev in target cells had not explored in detail. in this study, tgev particles in porcine epithelial cells were observed to have a diameter ranging from to nm, which was consistent with previous reports. the infectious virions entered the target cells by membrane fusion and mature viruses budded into vacuoles, which were gradually transported to the cell membrane before being released. fq-pcr was used to quantify tgev inside porcine iecs in comparison with the sc-tgev virus inside pk- cells (dai et al., ) . the one-step growth curve of tgev in porcine iecs showed that the amount of viral rna did not change significantly from to h post-infection, whereas dai et al. showed that in pk- cells, the rna increased rapidly from to h. the viral rna displayed a logarithmic increase in porcine iecs from to h, while dai et al. showed that the viral rna was maintained at high level and entered into stationary phase gradually. extracellular tgev rna levels were unchanged at to h post-infection of porcine iecs, and increased logarithmically until reaching maximum at h, before decreasing gradually. in pk- cells, dai et al. demonstrated that extracellular tgev rna levels increased logarithmically at - h post-infection, and maintained relatively stable and high level at - h. the differences between the curves indicated that differences in virulence and cell types might lead to changes in virus replication. the onestep growth curve of tgev revealed the rule of virus proliferation, and thus will provide further guidance for clarifying the infection mechanism of tgev. in conclusion, this is the first study to report the proliferative rule and morphogenesis of tgev in porcine iecs. these results lay the foundation for further study of the infection characteristics between tgev and its target cells. genomic organisation of a virulent taiwanese strain of transmissible gastroenteritis virus infectious bronchitis virus e protein is targeted to the golgi complex and directs release of virus-like particles isolation and identification of 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partially replace the envelope protein in murine coronavirus assembly dna mediated immunization with encoding the nucleoprotein gene of porcine transmissible gastroenteritis virus comparison of the amino acid sequence and phylogenetic analysis of the peplomer, internal membrane and nucleocapsid protein of feline, canine and porcine coronavirus simulation of the economic impact of transmissible gastroenteritis on commercial pig production in australia transmissible gastroenteritis (tge) of swine: in vitro virus attachment and effects of polyanions and polycations characterization of a porcine intestinal epithelial cell line for in vitro studies of microbial pathogenesis in swine importance of cholesterol for infection of cells by transmissible gastroenteritis virus sialic acids as receptor determinants for coronaviruses transmissible gastroenteritis virus induced apoptosis in swine testes cell cultures isolation and identification of porcine transmissible gastroenteritis virus from chongqing, southwestern china influence of the transmissible gastroenteritis virus on activities of swine intestinal epithelial cells looking for a needle in a haystack: cellular proteins that may interact with the tyrosine-based sorting signal of the tgev s protein gene expression study of two widely used pig intestinal epithelial cell lines: ipec-j and ipi- i localization to the nucleolus is a common feature of coronavirus nucleoproteins and the protein may disrupt host cell division molecular cloning and phylogenetic analysis of orf region of chinese isolate th- from transmissible gastroenteritis virus this work was supported by the graduate scientific research in chongqing (cys , cys ), and the chongqing basic research program (cstc jcyja , cstc jcya ), and the fundamental research funds for the central universities (xdjk b ). key: cord- -wrztpeb authors: zhang, xin; shi, hongyan; chen, jianfei; shi, da; dong, hui; feng, li title: identification of the interaction between vimentin and nucleocapsid protein of transmissible gastroenteritis virus date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: wrztpeb nucleocapsid (n) protein of transmissible gastroenteritis virus (tgev) packages viral rna genome to form a ribonucleoprotein complex. in addition to its function as a structural protein, n protein is involved in cell apoptosis or cell-cycle regulation. n protein possibly interacts with host factors to modulate cellular functions. to identify cellular proteins that interacted with n protein of tgev, methods of gst pull-down and co-ip were utilized to precipitate cellular proteins of swine testicular (st). bound cellular proteins were resolved by sds-page. analysis of interacting proteins by mass spectrometry allowed identification of cellular protein bands representative of cellular proteins including vimentin that bound to n protein. furthermore, the function of vimentin cytoskeleton in st cells during tgev infection was examined. vimentin cytoskeleton was required for virus replication. the present study thus provides protein-related information about interaction of tgev n protein with host cell that should be useful for understanding host cell response to coronavirus pathogenesis infection and the underlying mechanism of coronavirus replication. coronaviruses (covs), a genus in the coronaviridae family, are pleomorphic, enveloped viruses (perlman and netland, ) . covs have been clustered in the cornavirinae subfamily, which includes three approved genera, alpha-, beta-and gammacoronavirus, as well as a tentative new genus, the deltacoronavirus (de groot et al., ; reguera et al., ) . transmissible gastroenteritis virus (tgev) is a representative cov in the alphacoronavirus genus; severe acute respiratory syndrome-related coronavirus (sars-related cov) is a representative of the betacoronavirus genus; infectious bronchitis virus (ibv) is a representative of the gammacoronavirus genus; and bulbul-cov is a representative of the deltacoronavirus genus (de groot et al., ) . the coronaviridae are involved in respiratory, enteric, hepatic and neuronal infectious disease in animals and humans (perlman and netland, ) . tgev infection causes severe diarrhea in suckling piglets (about weeks old), which results in enormous economic loss in swine-producing areas in the world (kim and chae, ; sestak et al., ) . about two-thirds of the tgev genome ( . kb) encodes the replicase gene (rep) at the end, and one-third of the genome encodes other viral genes at the end in the order -s- a- b-e-m-n- - (penzes et al., ) . the genome of the tgev encodes four structural proteins: spike (s), membrane (m), envelope (e), and nucleocapsid (n) . n proteins of covs are highly basic with a molecular mass ranging from to kda, depending on the species and strains. n protein binds to the rna genome, forming a helical nucleocapsid (escors et al., ; sturman et al., ) . recently, some reports showed that n protein of tgev play an important role in host cell for virus replication. n protein of tgev facilitates template switching and is required for efficient transcription (zuniga et al., ) . n protein underwent proteolysis in parallel with the activation of caspases within host cell and n protein of tgev is a substrate for caspases (eleouet et al., ) . tgev n protein nucleolus localization was found in transfection experiments and might induced a cell cycle delay or arrest to facilitate virus replication (wurm et al., ) . in contrast, tgev n protein was not accumulated in the nucleus in the infection context (calvo et al., ) . in addition, the role of tgev n protein in cell cycle arrest has been recently reported (ding et al., ) . n protein of tgev may function through direct or indirect interaction with cellular proteins. for better understanding of the mechanisms associated with pleiotropic functions of n protein, cellular proteins of swine testicular (st) cells were pulled down associated with n protein using glutathione (gst)-tagged full-length n proteins immobilized on gst agarose. and cellular proteins of st cells were precipitated using n protein mab. by sds-page coupled with mass spectrometry (ms), a total of cellular proteins interacting with n protein were successfully identified. information on the expanded repertoire of cellular proteins interacting with n protein will provide a framework for future biochemical analyses of these protein functions in tgev infection. st cells were grown in rpmi- medium supplemented with % fetal calf serum under standard culture conditions ( % co , • c). tgev infectious strain h (accession no. fj ) (wang et al., ) was propagated on an st cell monolayer. mouse mab to glyceraldehyde- -phosphate dehydrogenase (gapdh) (ab ) and rabbit poloclonal antibody vimentin (ab ) were purchased from abcam. fitc-labeled goat antimouse igg was purchased from kirkegaard and perry laboratories (kpl). tritc-labeled goat anti-rabbit igg was purchased from sigma. the mab to n protein of tgev were donated from mr. wang shao (institute of animal husbandry and veterinary science, fujian academy of agricultural science, china). st cells were plated in six-well plates day prior to infection with tgev infectious strain h at a multiplicity of infection (moi) of . tgev not-infected samples were exposed to culture medium alone. after adsorption for h, cells were washed twice and incubated in fresh rpmi- . the prokaryotic expression plasmid pgex-tgev-n was constructed previously . escherichia coli bl (de ) strain containing pgex-tgev-n plasmid was expressed under induction of mm isopropyl-␤-d-thiogalactopyranoside. gst pulldown assay was performed as previously described . expressed gst protein was used as a control. the lysate of tgev-infected st cells was prepared with ripa lysis buffer ( mm tris-hcl, ph . , mm nacl, % np- , . % deoxycholate) containing a protease inhibitor phenylmethanesulfonyl fluoride (pmsf) ( mm). after centrifugation at , × g for min, lysate supernatant was pretreated with l mouse igg control (beyotime) and protein a/g plus-agarose (santa cruz biotechnology) for min at • c to eliminate non-specific binding to agarose gel. the lysate supernatant ( g) was incubated with g of mab to n protein of tgev for overnight at • c. then, l resuspended protein a/g plus-agarose was added to this mixture and incubated at • c on a rocker platform for h. after washing four times with lysis buffer, isolated immunoprecipitated proteins (boiling min with page sample loading buffer) were then analyzed by % page analysis. the lysate of tgev not-infected st cells was used as a control. . . protein identification by matrix-assisted laser desorption/ionization time of flight (maldi-tof/tof) ms the gels described above stained with phastgel blue r (ge healthcare). protein bands of interest were manually excised from gels. maldi-tof/tof was performed as previously described (zhang et al., ). data were searched by gps explorer (ver. . ) with the search engine mascot (ver. . ). the search parameters were as follows: national center for biotechnology information non-redundant (ncbinr) database (release date, july ), and the database sus ( , sequences; , , residues); a trypsin digest was performed with one missing cleavage, ms tolerance was set at ppm and ms/ms tolerance at . da. known contaminant ions (tryptic autodigest peptides) were excluded. mascot protein scores (based on combined ms and ms/ms spectra) > were considered statistically significant (p ≤ . ). individual ms/ms spectrum, with a statistically significant (confidence interval ≥ %) ion score (based on ms/ms spectra), was accepted. to eliminate redundancy of proteins that appeared in database under different names and accession numbers, single protein member belonging to species sus or with the highest protein score (top rank) was separated from multi-protein family. equivalent amounts of cell lysates were subjected to % page and then transferred to . m nitrocellulose membranes (hybond-c extra, amersham biosciences). after blotting, membranes were incubated with mouse pab to vimentin or with mouse mab to n protein ( : ) at • c for h. after washing three times with pbst, membranes were inoculated with dylight tm -labeled antibody to mouse igg (h+l) ( : , , kpl, usa) or dylight tm -labeled antibody to rabbit igg (h+l) ( : , kpl, usa) at • c for min. images were visualized by odyssey infrared imaging system (li-cor). st cells inoculated with tgev were cultured for , , , , , and h. cells were washed twice with pbs and fixed with paraformaldehyde ( %) for min at • c, and then allowed to air dry. after blotting with % skimmed milk powder, the fixed cells were incubated with mab to tgev n protein ( : ) and rabbit pab to vimentin ( : , abcam) for h at • c in a humidified chamber. after washing three times with pbst, the fixed cells were incubated with fitc-labeled goat anti-mouse igg ( : , kpl) and tritc-labeled goat anti-rabbit igg ( : , sigma). additional nuclear staining with , -diamidino- -phenylindole (dapi, sigma) was performed as described previously (jungmann et al., ) . the triple-stained cells were washed three times with pbst and subsequently examined under a leica tcs sp laser confocal microscopy. . . transfection of sirna against vimentin sirna against vimentin (genepharma) was used for transfection. sequence of the sirna strands (two rounds of silencing) were as follows: -gcuaacuaccaagacacuatt- (sense) and -uagugucuugguaguuagctt- (antisense); -ccucugguu-gacacccauutt- (sense) and -aaugggugucaaccagaggtt- (antisense). negative control sirna strands were as follows: -uucuccgaacgugucacgutt- (sense), -acguga-cacguucggagaatt- (antisense). transfection with sirna was performed with lipofectamine reagent (invitrogen) by following the manufacturer's instructions. st cells were cultured overnight in six-well tissue culture plates. the sirna ( nm) was complexed with lipofectamine reagent by incubating together at room temperature for min. after removing the cell culture supernatant, the complex was added. after incubation for h, the cells were infected with tgev. total rna was extracted from the st cells transfected with sirna against vimentin and negative control sirna, using the rneasy mini kit (qiagen) according to the manufacturer's protocol. cdna synthesis was performed with total cellular rna using a reverse transcriptase m-mlv kit (takara), according to the manufacturer's protocol. real time rt-pcr was performed using a lightcycler ii (roche) in a total volume of l containing ng of cdna template, × sybr ® premix ex taq tm ii (perfect real time, takara), and a . m concentration of each primer. after initial denaturation at • c for min, the amplification was performed for cycles, each consisting of denaturation at • c for s and primer annealing at • c for s. melting curves were obtained, and quantitative analysis of the data was performed in a relative quantification ( − ct ) study model. parallel tgev notinfected st cells were used as control (relative expression = ) and gapdh as an internal reference gene. st cells were re-plated day before infection in well plates for the % infectious dose (tcid ) assays. treated samples and their paired controls were thawed as described and immediately serially diluted. cell cultures were then infected for h. after h of incubation, cpe was observed. tcid is calculated using the method of reed and munch. virus titer assay were performed three times for each condition and were performed using the student's t-test. the expressed gst-n protein immobilized on gst-agarose beads was used as a bait to pull down cellular proteins of tgevinfected st cells that form a complex with n protein. gst protein was used as control to eliminate non-specifically binding proteins. after extensive washing the resins with ripa buffer, cellular proteins bound to n protein were analyzed by sds-page and stained with phastgel blue r (ge healthcare). as shown in fig. , cellular protein bands that were not seen in gst control gel. the protein bands of interest were manually excised from gels and plated into -well microplates for ms identification. the mab to n protein was used as a bait to precipitate cellular proteins of tgev-infected st cells that form a complex with n protein. after extensive washing resins with ripa buffer, cellular proteins bound to n protein were analyzed by sds-page and stained with phastgel blue r (ge healthcare). as shown in fig. , cellular protein bands that were not seen in tgev not-infected st cells. the protein bands of interest were manually excised from the gels and plated into -well microplates for ms identification. tgev n protein-associated cellular proteins were identified using in-gel tryptic digestion, and maldi-tof/tof identification. protein bands binding to n protein were subjected to in-gel trypsin digestion, and peptides were analyzed by ms. database searches with the peptide masses resulted in positive identification for different cellular proteins (table , table s , and fig. s ). two atp-dependent rna helicase proteins were identified as interacting with n protein. protein band was identified as atpdependent rna helicase a (rha) and protein band was identified as atp-dependent rna helicase ddx (dbp-rb). four cytoskeleton proteins were identified as interacting with n protein. protein band and band were identified as ␣-actinin- (actn ). protein band and band were identified as vimentin (vim). protein band and band were identified as myosin. four ribosome-associated proteins were identified as interacting with n protein: heterogeneous nuclear ribonucleoprotein u (hnrnp u) (band and band ); s ribosomal protein s (band ); s ribosomal protein l (band ); and s ribosomal protein s (band ). one proteinbiosynthesis-associated protein (elongation factor -␣, band ) and one cell-cycle-arrest protein (vasohibin- , band ) were identified as interacting with n protein. maldi-tof ms spectra and tof/tof spectra of vimentin are shown in fig. . three cellular proteins, hnrnp u, actn , and vimentin, were identified both by gst-n pull down and co-ip in tgev-infected cells, which should have more biological importance in the context of infection. to verify the proteins identified by ms, western blotting was performed. many cytoskeleton associated proteins play important roles in virus infection (dohner and sodeik, ) . vimentin protein was identified not only in gst pull-down assay but also in co-ip assay. therefore, the identified protein vimentin was selected for western blot analysis. cellular proteins immobilized on gst-agarose beads in gst pull-down assay were examined with specific antibodies to vimentin (fig. ) . results validated the maldi-tof/tof identification of cellular proteins. to confirm the interaction between n protein of tgev and cellular vimentin, immunoprecipitation assay was utilized to identify whether tgev n protein interacted with cellular vimentin in tgevinfected st cells. from the immunoprecipitation results (fig. ) , we can see that the n protein of tgev was precipitated by the pab to cellular vimentin in tgev-infected st cells but not in tgev not-infected st cells. results demonstrate that cellular vimentin interacted with n protein of tgev. subcellular localization of vimentin was investigated in tgevinfected st cells using indirect immunofluorescence confocal microscopy. results indicated that subcellular localization of vimentin was distributed in cytoplasm with enrichment in the perinuclear region after tgev infection (fig. ) . cellular vimentin labeled with tritc was overlaid with n protein of tgev labeled with fitc, indicating that cellular vimentin was co-localized with n protein of tgev within the st cells during infection. to further investigate the role of vimentin in virus infection, vimentin protein of st cells was inhibited using sirna. the decrease level of vimentin mrna was validated by rt-pcr (fig. a) . st cells transfected with vimentin-specific sirna expressed lower level of vimentin by western blot analysis, compared to that transfected with the control sirna (fig. b ). after transfection with sirna, st cells were infected with tgev for another h after transfection with sirna at an moi of . virus titer assay were performed three times for each condition and were performed using the student's t-test. tcid of virus in control sirna group was . /ml and the vimentin sirna group was . /ml. fig. c shows that knock-down of vimentin resulted in significant reduction of cellassociated virus, which reflected viral replication. identifying host cell protein interacting with viral protein is a critical step for understanding protein function and viral replication. recently, affinity purification followed by ms has been widely used to find protein-protein interactions (dunham et al., ) . in the present study, using gst pull-down and co-ip coupled with ms identification, cellular protein bands representative of cellular proteins interacting with tgev n protein were identified. several cellular proteins found in this study were identical to those previously described interacting with cov n protein. ef a, ddx and hnrnp u were also observed in infectious bronchitis virus (ibv) n protein using quantitative proteomic approaches (emmott et al., ) . interaction between ef a and n protein of tgev and sars-cov were founded (zhou et al., ) . the hnrnp u protein was identified as binding preferentially tgev -end (galan et al., ) . a very recent work describes the key role of ibv n protein interaction with ddx in viral transcription (wu et al., ) . four cytoskeleton-associated proteins were identified that associated with n protein in this study. the cytoskeleton consists of a filament scaffold within cell. filaments are dynamic and divided into three types: microfilaments (actin filaments), microtubules, and intermediate filament (naghavi and goff, ) . during infection, viruses interact with components of the cytoskeleton to reach their appropriate intracellular sites of replication (radtke et al., ) . in previous study, differentially expressed cytoskeletal proteins were found in tgev infected st cells using two-dimensional difference gel electrophoresis (zhang et al., ) . cytoskeletal proteins found in this study provides new insights for understanding the mechanisms of tgev replication. cov replication uses complex mechanisms that involve viral and cellular proteins. similar to other positive-strand rna viruses, cov genome replication takes place in the cytoplasm in a membraneprotected microenvironment that contains all the protein required for viral rna synthesis (enjuanes et al., ) . electron microscopy studies of mouse-hepatitis-virus-infected cells have shown that replication complexes of virus consist of double-membrane vesicles (gosert et al., ) . cov replication complex formation possibly utilizes components of cellular autophagy (prentice et al., ) . although cov replication essentially takes place within the cytoplasm, the virus may control cell machinery by locating some of its proteins in host cell nucleus (enjuanes et al., ) . in the present study, vimentin was identified in pull-down assay and co-ip assay, suggesting that it plays an important role in tgev infection. costaining of cellular vimentin and n protein on immunofluorescence microscopy also supported an association between proteins. based on these findings, we speculate that vimentin plays a role in tgev replication. some studies demonstrate that vimentin plays important role in virus infection cycle. vimentin binding is critical for virus entry, such as human cytomegalovirus (cmv) (miller and hertel, ), japanese encephalitis virus (jev) (liang et al., ) , and cowpea mosaic virus (cpmv) (koudelka et al., ) . vimentin is required for virus replication, such as bluetongue virus (btv) (bhattacharya et al., ) , and dengue virus (denv) (kanlaya et al., ) . in this study, vimentin is required for tgev replication. therefore, these may potentially serve as novel therapeutic targets for controlling tgev infection. the efficiency of tgev infection depends on the presence and integrity of vimentin cytoskeleton, which may facilitate virus infection at different steps. vimentin may promote viral replication by interaction with n protein, which may help virions to transport through a functional golgi complex for viral maturation (risco et al., ) . additional work will be needed to pinpoint the exact steps during viral infection. overall, this study was the first to identified cellular proteins interaction with tgev n protein using proteomics method. a total of cellular proteins were identified, which provided new information to understand the mechanism of coronavirus replication. the interaction between the cellular vimentin and n protein of tgev was confirmed in tgev-infected st cells. knockdown of vimentin impairs tgev replication in st cells. the present study thus provides insights into interaction of tgev n protein with host cellular proteins that would be useful for further studying viral replication and pathogenesis. interaction between bluetongue virus outer capsid protein vp and vimentin is necessary for virus egress phosphorylation and subcellular localization of transmissible gastroenteritis virus nucleocapsid protein in infected cells coronaviridae tgev nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p signaling the role of the cytoskeleton during viral infection affinity-purification coupled to mass spectrometry: basic principles and strategies the viral nucleocapsid protein of transmissible gastroenteritis coronavirus (tgev) is cleaved by caspase- and - during tgev-induced apoptosis the cellular interactome of the coronavirus infectious bronchitis virus nucleocapsid protein and functional implications for virus biology biochemical aspects of coronavirus replication and virus-host 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virus: mutations that may contribute to attenuation nucleocapsid phosphorylation and rna helicase ddx recruitment enables coronavirus transition from discontinuous to continuous transcription localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division ef a interacting with nucleocapsid protein of transmissible gastroenteritis coronavirus and plays a role in virus replication identification of cellular proteome using two-dimensional difference gel electrophoresis in st cells infected with transmissible gastroenteritis coronavirus differential proteome analysis of host cells infected with porcine circovirus type the nucleocapsid protein of severe acute respiratory syndrome coronavirus inhibits cell cytokinesis and proliferation by interacting with translation elongation factor alpha coronavirus nucleocapsid protein facilitates template switching and is required for efficient transcription this work was supported by the national natural science foundation of china (grant nos. , ), heilongjiang provincial natural science foundation (grant no. jc ) and heilongjiang provincial department of education ( td ). we thank mr. zhou xin-wen (fudan university, china) for help with maldi-tof/tof mass spectrometry and mr. wang shao (institute of animal husbandry and veterinary science, fujian academy of agricultural science, china) for the donation of mab to n protein of tgev. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.virusres. . . . key: cord- - s f wje authors: fu, yuguang; li, baoyu; liu, guangliang title: rapid and efficient detection methods of pathogenic swine enteric coronaviruses date: - - journal: appl microbiol biotechnol doi: . /s - - - sha: doc_id: cord_uid: s f wje abstract: porcine enteric coronaviruses (covs) cause highly contagious enteric diarrhea in suckling piglets. these cov infections are characterized by clinical signs of vomiting, watery diarrhea, dehydration, and high morbidity and mortality, resulting in significant economic losses and tremendous threats to the pig farming industry worldwide. because the clinical manifestations of pigs infected by different covs are similar, it is difficult to differentiate between the specific pathogens. effective high-throughput detection methods are powerful tools used in the prevention and control of diseases. the immune system of piglets is not well developed, so serological methods to detect antibodies against these viruses are not suitable for rapid and early detection. this paper reviews various pcr-based methods used for the rapid and efficient detection of these pathogenic covs in swine intestines. key points: . swine enteric coronaviruses (covs) emerged and reemerged in past years. . enteric covs infect pigs at all ages with high mortality rate in suckling pigs. . rapid and efficient detection methods are needed and critical for diagnosis. the coronaviruses (covs) critically threaten human and animal health because infection with them results in respiratory or enteric tract diseases (woo et al. ) . for instance, pneumonia occurs in humans infected with the new cov that emerged in china in the middle of dec. ; the virus has subsequently spread to many countries worldwide where it threatens the health of humans, resulting in tremendous economic losses. covs are enveloped, positive-sense, singlestranded rna viruses that possess a genome ranging from . to . kb; they belong to the order nidovirales, family coronaviridae, and subfamily coronavirinae (woo et al. ) . based on antigenic relationships, the classification of coronaviruses was traditionally divided into genera, but they were replaced by the following four genera: alphacoronavirus ( a l p h a -c o v ) , b e t a c o r o n a v i r u s ( b e t a -c o v ) , gammacoronavirus (gamma-cov), and deltacoronavirus (delta-cov), which are based on genetic and antigenic characteristics (woo et al. ). epidemiological surveys have indicated that bats and birds seem to be natural reservoirs for alpha-and beta-covs and gamma-and delta-covs, respectively (bolles et al. ; woo et al. ) . covs in four genera have been verified in a variety of species, e.g., canines, felines, and birds (chan et al. ) . six covs have been identified in swine (table ) : porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev), swine acute diarrhea syndrome coronavirus (sads-cov), and porcine respiratory cov (prcov) in the alpha-cov genus, porcine hemagglutinating encephalomyelitis virus (phev) in the beta-cov genus, and porcine deltacoronavirus (pdcov) in the delta-cov genus (jung et al. ; pan et al. ; pensaert and de bouck ; woo et al. ; zhang ; zhou et al. ). among the six swine covs, tgev, pedv, pdcov, and sads-cov are enteric viruses that cause diarrhea in the pig population, resulting in significant economic losses and tremendous threats to the pig industry worldwide. the four swine enteric covs causing highly contagious enteric diarrhea in neonatal and suckling piglets are clinically characterized by vomiting, watery diarrhea, dehydration, and high morbidity and mortality (gong et al. ; hsu et al. ) . because the clinical signs of pigs infected by these covs are very similar (table ) , it is difficult to differentiate the specific pathogens based on clinical symptoms. effective high-throughput detection methods are needed for their differential determination and would represent powerful tools to prevent and control diseases. as far as we know, many standard detection methods can be used to distinguish between causative agents, including virus isolation, electron microscopy, virus neutralization, and indirect immunofluorescence assays. however, these methods are time-consuming, laborious, and not suitable for the early and rapid detection of the four swine enteric covs (carman et al. ; dulac et al. ; van nieuwstadt et al. ). the enzyme-linked immunosorbent assay is a powerful and highthroughput method for detecting specific antibodies, but the immune system of piglets is not well developed, so serological methods for detecting antibodies against these viruses are also not suitable for rapid and early detection. polymerase chain reaction (pcr) methods have been widely used to detect pathogens since the pcr was invented; pcr has proven to be powerful and convenient tools for precise detection of diarrheal pathogens in pig populations (ben salem et al. ; collins et al. ; kim et al. ). this paper reviews various pcr-based methods for the rapid and efficient detection of these pathogenic covs in swine intestines. pan-cov rt-pcr assay for the detection of covs figure summarizes the basic workflow for the detection of the swine enteric coronaviruses from clinical samples. generally, porcine fecal or intestinal samples need to be suspended and homogenized in sterile pbs and centrifuged to remove debris. the supernatant should be collected and filtered through a . -μm filter to remove the debris and some potential bacteria. the yield supernatant can be used to extract the total rnas by trizol reagent or rna extraction kit. the total rnas are used to reverse transcription by random primers to generate cdna. this cdna is employed as a template to do pcr amplification either by specific individual coronavirus or by pan-cov primers first then followed by specific primers targeting individual swine enteric coronavirus when necessary. the pcr products are eventually subjected to dna electrophoresis and analyzed under uv light to identify the desired bands. the pan-cov pcr method is a powerful tool for detecting all known and unknown covs; it is based on the conserved gene sequences among them (moes et al. ). this assay is widely used to detect covs. pan-cov rt-pcr was employed to detect all known covs in the human respiratory tract (vijgen et al. ) and to detect distinct alpha-covs in five different bat species (escutenaire et al. ; lazov et al. ; vijgen et al. ) . for swine enteric covs, pan-cov pcr also played an important role in identification. during the early stage of investigating cases of diarrhea in piglets in the usa caused by the pedv variant and pdcov, pan-cov rt-pcr was applied to identify the causative agent together with electron microscopy and sequencing stevenson et al. ). in addition, during the identification of piglet diarrhea disease caused by sads-cov in china, pan-cov rt-pcr was employed (pan et al. ) . in , hu et al. reported an improved one-step pan-cov rt-pcr (hu et al. ) . though pan-cov pcr could detect all known covs in humans and animals, it could not make a differential detection, so it is not suitable for routine swine enteric cov detection. porcine epidemic diarrhea (ped) is an enteric disease caused by pedv that can infect pigs of all ages with different levels of clinical signs of vomiting, diarrhea, dehydration, and weight loss, but the disease is much more severe in suckling piglets (have et al. ; shibata et al. ; song and park ; sueyoshi et al. ) . ped was first reported in england in , but the pedv was isolated for the first time in belgium in (pensaert and de bouck ; song and park ) ; however, the epidemic was not controlled in europe before . in china, pedv was first identified in the s, after which it was reported in some asian countries, e.g., japan and korea (kusanagi et al. ; song and park ; takahashi et al. ). in october , a severe ped outbreak caused by a highly virulent pedv variant emerged in southern china with high mortality ranging from to %; the result was devastating damage to the pig farm industry and tremendous economic losses, and later, the pedv variant spreads to other countries, e.g., usa, canada, and mexico ( for the early and rapid detection of pedv, different types of pcr methods have been developed. a real-time reverse transcription recombinase polymerase amplification assay (rt-rpa) based on the nucleocapsid gene of pedv was reported in ; the assay was able to detect copies per reaction and was performed for min at °c . based on the advantage of increased thermal conductivity of solid gold nanometal particles that could reduce nonspecific amplification and increase specific amplification, yuan et al. developed a nanoparticle-assisted pcr assay for the detection of pedv on the basis of the n gene in , and the assay could detect . × − ng/μl of pedv rna (yuan et al. ) . ren and li designed six primers according to the sequence of the n gene and established a reverse transcription loop-mediated isothermal amplification (rt-lamp) for the rapid detection of pedv (within min) at °c. the detection limit of the method was × − μg pedv rna per reaction (ren and li ) . wang et al. designed five primers on the basis of the n gene sequence of pedv and established a reverse transcription cross-priming amplification-nucleic acid test strip (cpa-nast) for the detection of pedv. the method had high specificity for the detection of pedv and had the same sensitivity as traditional rt-pcr; the detection limit was a − dilution of plasmid containing the target gene ). xing et al. developed an rna extraction and transcription-free, nanoparticle-based pcr (nbp-pcr) method to specifically detect pedv, and the sensitivity of the schematic diagram of the workflow for swine enteric coronavirus detection by rt-pcr. porcine fecal or tissue samples are homogenized in sterile pbs and centrifuged to remove debris. the supernatant is filtered through a . -μm filter and used to total rna extraction. the total rnas are subjected to reverse transcription using random primer to generate cdna. with this cdna as template, the pcr amplification is carried out either by specific individual coronavirus or by pan-cov primers first then followed by specific primers targeting individual swine enteric coronavirus when necessary. the pcr products are subjected to dna electrophoresis and observed under uv light to identify the desired bands the method was -fold higher than that of conventional rt-pcr. in fecal samples, the positive detection rate of nbp-pcr specific was much higher than that of conventional rt-pcr ( . %) and sybr green real-time rt-pcr (xing et al. ). zhou et al. developed a conventional rt-pcr method, an sybr green i real-time rt-pcr, and taqman real-time rt-pcr reagents to detect the highly conserved m gene of pedv; the detection limit of the taqman real-time rt-pcr was copies/μl of the target gene, and the sensitivity of the taqman real-time rt-pcr was -fold and , -fold higher than that of sybr green i real-time rt-pcr and conventional rt-pcr, respectively ). in addition, there were variant pedv strains circulating in the field and the pcr methods for differentiating them had been established. song et al. analyzed pathogenicity and immunogenicity of pedv strain designated dr in piglets, which was a highly vero cell-adapted virus and could be employed as a vaccine candidate, and applied a restriction fragment length polymorphism (rflp) assay to differentiate dr from wild-type virus based on the difference of open reading frame (orf) sequence (song et al. ) . in , lee et al. reported an rt-pcr-based rflp assay targeting the n gene of pedv to distinguish field strains of pedv in korea from an attenuated-live vaccine j-vac, which was used in pig population to prevent pedv . for differentiation between attenuated-type pedvs including attenuated dr , kped- , and p- v that were used as live virus vaccine and wild-type pedvs including cv , brl/ , lzc, parent dr , and field samples, park et al. established an rt-pcr assay based on the difference of orf gene sequence of attenuated-and wild-type pedv, in which nucleotide deletions were found in all live pedv vaccine . in may , the virulent strain of pedv was verified in the usa resulting in significant economic losses in the swine industry. and a variant strain (oh ) of pedv emerged in the usa in december , which differs from the virulent strains of pedv in the nucleotides of the ′end of spike gene. to differentiate these two genotypes of pedv circulating in the usa, wang et al. reported a duplex probe-based real-time rt-pcr targeting the difference of spike gene among virulent and variant strains (wang et al. b ). sequence analysis of pedv genome indicated that pedv attenuated vaccine strains (e.g., the cv and zj in china, the p- v in japan, and kped- and dr in south korea in asia) have base pair deletion in the open reading frame (orf ); for differentiation of these cell-adapted vaccine strains from field strains, zhu et al. developed a nanoparticle-assisted rt-pcr assay targeting the orf (zhu et al. b) . because three major pedv types have been identified in the usa after including the original us pedv strains, the spike gene insertion-deletion pedv strains, and the pc strain that possess a amino acid-deletion in the s region of spike protein, liu and wang developed a reverse transcription-pcr method to differentiate these variants on the basis of differences in the s gene (liu and wang ) . since , pedv variants with base deletions and insertions in the s gene emerged in china and caused significant losses in piglets; zhao et al. developed a taqman probe-based real-time pcr method for the detection of different pedv variants and classical pedv strains based on the sequence difference of the pedv s gene, and the detection limit of the method was × target gene copies (zhao et al. ). su et al. established a duplex taqman probe real-time rt-qpcr method for detecting and differentiating classical and variant pedvs targeting the difference in the s gene; the detection limit of the method was . × genome copies/reaction for both the classical and variant pedv. the results of clinical sample detection showed that the assay was more sensitive than conventional pcr, and variant pedv was prevalent in china (su et al. ) . due to the wide use of a live attenuated pedv vaccine, classical and wild variant strains circulating in pig farms are common; therefore, he et al. established a multiplex rt-pcr to differentiate these strains based on the difference in s gene sequences, and the detection limit of the method was × . tcid / μl for pedv (he et al. ). due to variant pedv strains that emerged in china since and attenuated pedv vaccines (e.g., cv strain) being widely used in china, liu et al. reported a taqman probe-based realtime pcr to differentiate these virulent strains and attenuated vaccine strains based on the orf deletion region to detect virulent pedv strains in vaccinated pig population (liu et al. b ). transmissible gastroenteritis (tge) caused by tgev is an acute enteric diarrhea disease in pigs (garwes ) . tgev was isolated for the first time in , and then outbreaks of the virus occurred in many countries in america, asia, and europe (doyle and hutchings ; kim et al. ; stevenson et al. ) . tgev causes severe enteritis in piglets before weaning, and the clinical signs include diarrhea, vomiting, dehydration, and high mortality, resulting in significant economic losses (ding et al. ; penzes et al. ; saif ) . for the early and rapid detection of tgev, different types of pcr-based methods have been established. chen et al. designed six specific primers targeting the nucleocapsid gene of tgev and developed an rt-lamp assay for the detection of tgev, which involved incubation at °c for h. the sensitivity of rt-lamp was comparable to that of nested pcr described by rodríguez et al. in , and it was times more sensitive than the pcr reported by paton et al. in , which could detect pg of rna per reaction paton et al. ; rodríguez et al. ) . rt-lamp for the detection of the tgev targeting the n gene sequence by incubation at °c for min was reported by li et al., and the sensitivity of rt-lamp was much higher than that of a gel-based rt-pcr kit purchased from haigene company, china (li et al. ). vemulapalli et al. described a taqman probe-based real-time rt-pcr that specifically amplified conserved s gene sequences. the detection limit of the method was tcid /ml of tgev rna. the sensitivity of the assay was higher than that of the nested rt-pcr assay reported by kim et al. (kim et al. ; vemulapalli et al. ). wang et al. developed a rapid detection of tgev using real-time reverse transcription recombinase polymerase amplification (rt-rpa) based on the spike gene, and the method could detect copies of tgev rna after min at °c conditions . a real-time rt-pcr assay described by vemulapalli et al. was compared with the rt-rpa method in the analysis of clinical samples, and the two methods generated the same results (vemulapalli et al. ; wang et al. ). in the mid- s, prcov, which has a deletion of nucleotides of the s gene that is present in tgev (laude et al. ) , was reported in europe, north america, and asia (pensaert et al. ; wesley et al. ). paton et al. developed an rt-pcr assay to differentiate tgev and prcv based on the difference in the s gene of the two viruses (paton et al. ) . pdcov is a novel swine enteric diarrhea virus that causes severe diarrhea, vomiting, and dehydration in piglets (janetanakit et al. ; song et al. ) . pdcov was first discovered in samples from the healthy pig that were collected in in hong kong when a molecular surveillance study was performed (woo et al. ) . in february , the virus was detected in piglets with severe diarrhea in the usa (marthaler et al. ; wang et al. a ). subsequently, pdcov was reported in south korea, mainland china, and thailand (chen et al. a; janetanakit et al. ; lee and lee ; song et al. ) . to detect the virus with precision, pcr-based methods have been created. in , pigs on farms in ohio, usa, had clinical diarrheal disease. wang et al. developed a onestep rt-pcr targeting membrane and nucleocapsid gene of delta-cov and determined the causative agent as porcine delta-covs, but they did not evaluate the detection limit of the assay (wang et al. a ). in , song et al. established a nested rt-pcr method on the basis of the nucleocapsid gene sequence of the pdcov hku strain to identify the causative agent causing acute diarrhea in a pig farm in jiangxi, china, and the sensitivity of the assay was also not determined . to analyze the characteristics of porcine delta-covs in the usa, ma et al. established a real-time rt-pcr method for specific detection of the n gene, but the sensitivity of the assay was also not determined (ma et al. ) . for the analysis of pathogenicity and pathogenesis of a porcine delta-cov cell culture isolate, chen et al. developed an m gene-based real-time pcr method for detecting viral titers in different organs, but the sensitivity of the assay was not described in the publication (chen et al. b ). marthaler et al. developed a one-step probe-based real-time rt-pcr method targeting the m gene sequence of pdcov, which was applied by the university of minnesota veterinary diagnostic laboratory. the detection limit of the assay was viral rna copies per reaction, but the specificity of the assay was not evaluated (marthaler et al. ). the commercial reverse transcription-insulated isothermal pcr (rt-iipcr) pockit™ methods (pockit™ pedv reagent set and pockit™ pdcov reagent set, genereach usa, lexington, ma, usa) was used to analyze pedv and pdcov coinfection that occurred in diarrhea disease; zhang et al. described two singleplex rt-iipcr tests and a duplex real-time rt-pcr test for the detection of pedv and pdcov that were based on targeting the conserved m gene sequence. the detection limits of singleplex rt-iipcr were rna copies per reaction for pedv and rna copies per reaction for pdcov, and those of the duplex rt-iipcr were rna copies and rna copies per reaction for pedv and pdcov, respectively ). sads-cov, also designated seacov or peav, is a novel porcine enteric diarrhea virus that can cause severe and acute diarrhea and rapid weight loss in piglets (pan et al. ; zhou et al. ; zhou et al. ) . sads-cov was identified for the first time in southern china in late , and it caused more than , piglet deaths, resulting in significant economic losses (gong et al. ) . the clinical signs of infection with sads-cov are similar to those of other known swine enteric covs: tgev, pedv, and pdcov (dong et al. ; sun et al. ) . for specific detection of sads-cov, zhou et al. established an sybr premix ex taqii-based real-time pcr on the basis of the rna-dependent rna polymerase (rdrp) gene for the detection of sads-cov; the sensitivity of which was not evaluated ). zhou et al. developed a taqman-based real-time pcr assay for sads-cov detection based on the conserved sequence within the n gene; the detection limit of the assay was . × copies/μl, and the sensitivity of the method was -fold higher than that of conventional pcr, which also targeted the n gene (zhou et al. ). because the clinical signs caused by the four enteric covs in piglets are similar to each other, it is very difficult or timeconsuming to make a clear diagnosis of mixed infection in pigs using a single pcr method. therefore, it is critical to developing a multiplex polymerase chain reaction (mpcr) in which two or more loci can be simultaneously detected in the same reaction (chamberlain et al. ). tgev and pedv are traditional swine enteric diarrheal viruses, so several mpcr methods that can differentially detect the two viruses have been previously established. in , kim et al. reported a duplex rt-pcr assay to detect tgev and pedv in one pcr tube targeting the s gene of the two viruses, and the detection limit of the assay was tcid / μl (kim et al. ) . in , kim et al. established a multiplex realtime rt-pcr method based on the nucleocapsid (n) gene for the simultaneous detection and quantification of tgev and pedv, and the detection limits of this method were copies and copies for tegv and pedv, respectively (kim et al. ). zhu et al. designed two pairs of primers on the basis of the n gene sequences of tgev and pedv and established a nanoparticle-associated pcr assay; the sensitivity of the assay was -fold higher than that of conventional pcr (zhu et al. ) . apart from the four enteric covs, several enteric diarrheal viruses have been discovered, including porcine sapovirus (psav), porcine norovirus (pnov), porcine teschen virus (ptv), porcine kobuvirus (pkv), seneca valley virus (svv), porcine rotavirus (prv), porcine reovirus (reov), porcine bocavirus (pbov), and porcine astrovirus (pastv). therefore, some mpcr methods have been created to detect swine enteric diarrheal viruses, but they are not limited to enteric covs (ding et al. ) . ben salem et al. developed a nested rt-pcr method for the detection of pedv, tgev, and prv based on the sequence of the tgev purdue strain (accession no. nc_ ), the pedv strain cv (accession no. nc_ ), and the nsp gene of the prv-a osu strain (accession no. x ), respectively. the detection limits of multiplex nested rt-pcr for tgev and pedv were tcid /ml and . μg/μl of rna, respectively (ben salem et al. ) . in , zhao et al. developed a multiplex rt-pcr assay to identify pedv, tgev, prv-a, and porcine circovirus (pcv ) in one reaction, and the sensitivity of the assay for the detection of tgev and pedv was -fold lower than that of a single rt-pcr method ). zhao et al. established a multiplex rt-pcr assay for rapid and differential diagnosis of pedv, tgev, prv-a, and pcv targeting the s gene, segment region, and orf sequence, and the detection limits of the assay for tgev and pedv were . × and . × copies, respectively ). liu et al. developed a multiplex pcr assay to detect five diarrhea-related pig viruses: pedv (nucleoprotein), tgev (spike glycoprotein), prv-a, porcine group c rotaviruses (prv-c), and pcv . the detection limits of the assay for pedv and tgev were copies per reaction (liu et al. a ). wen et al. developed a multiplex real-time pcr method based on evagreen fluorescent dye to simultaneously detect and distinguish pedv-nucleoprotein (n), tgev-spike glycoprotein (s), prv-a, prv-c, and pcv , and the limits of detection ranged from to copies/μl (wen et al. ). there was no multiplex pcr method exclusively for differential detection of the four enteric covs until huang et al. developed a taqman-probe-based real-time rt-pcr method in targeting the m gene of pedv, the n gene of tgev, the m gene of pdcov, and the n gene of sads-cov. their multiplex real-time rt-qpcr assay could detect - copies of each target gene per pathogen (huang et al. ). as mentioned above, nested rt-pcr, rt-rpa, nanoparticleassisted pcr, rt-lamp, cpa-nast, sybr green-based real-time pcr, evagreen-based real-time pcr, and taqman probe-based real-time pcr represent single or multiplex pcr methods that have been developed to detect one, two, or four enteric pathogenic covs. in the field, the causative agents of swine enteric diarrhea are mixed; single rt-pcr methods are not suitable for rapid and efficient detection of covs even though they have higher sensitivity than multiplex rt-pcr. in addition, the pcr fragments of some single rt-pcr methods have to be subjected to agarose gel analysis to determine results, which is time-consuming. in particular, nested rt-pcr requires two pcr steps, resulting in an increased likelihood of contamination, so this method has not been widely used for pathogen detection. for rapid and efficient detection of pathogenic swine enteric covs, multiplex pcr methods, including conventional multiplex rt-pcr and multiplex real-time rt-pcr, are ideal options. although conventional multiplex rt-pcr can simultaneously differentially detect several different pathogens in one reaction, the method also possesses the disadvantages of single rt-pcr, e.g., risk of product contamination, and the inability to monitor developments in real time. the sensitivity of rt-pcr is - times lower than that of real-time rt-pcr, and the viral loads cannot be measured (keyaerts et al. ) . recently, real-time taqman probe-based rt-pcr methods have become increasingly used to detect targets because they own many advantageous characteristics: the ability to perform differential detection, high specificity, high sensitivity, high-throughput ability, high repeatability, quantification ability, and the ability to assess results in real time (slavov et al. ; teng et al. ; zhu et al. a ). therefore, huang et al. in our lab developed a taqman-probe-based real-time rt-pcr for the differential detection of pedv, tgev, pdcov, and peav (huang et al. ) . although real-time taqman probe-based rt-pcr possesses many merits and can be used to rapidly and efficiently detect pathogenic swine enteric covs, the method requires a high-precision and sophisticated instrument, practical technicians, and a good laboratory; therefore, it cannot be used for detection in under-equipped laboratories or on-site. rapid, accurate, and more practical detection methods are of great significance for the surveillance, prevention, and control of enteric diseases in pigs, so novel assays are still deserved further development. for instance, test strip detection methods, which can be used by under-equipped laboratories or on-site and can be easily operated to quickly generate results, are urgently needed. as mentioned earlier, apart from the four enteric covs, many other pathogens causing diarrhea in pigs have been identified. and the causative agents of swine enteric diarrhea are mixed in the field. to rapidly determine whether the pathogens are enteric covs, pan-cov pcr is the best option to initially detect from clinical samples and followed by specific primers targeting individual swine enteric coronavirus for further identification when the result from pan-cov pcr is positive. however, when the pan-cov pcr results are negative, specific primers targeting other enteric diarrheal viruses are required to determine the causative agents. authors' contributions yf and gl conceived the review. fy and bl collected all references and wrote the manuscript draft. gl edited the manuscript. conflict of interest the authors declare that they have no competing interests. ethical statement no ethical approval was required 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transmissible gastroenteritis virus from clinical specimens establishment of a nanoparticle-assisted rt-pcr assay to distinguish field strains and attenuated strains of porcine epidemic diarrhea virus publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- - jgqofbr authors: kocherhans, rolf; bridgen, anne; ackermann, mathias; tobler, kurt title: completion of the porcine epidemic diarrhoea coronavirus (pedv) genome sequence date: journal: virus genes doi: . /a: sha: doc_id: cord_uid: jgqofbr the sequence of the replicase gene of porcine epidemic diarrhoea virus (pedv) has been determined. this completes the sequence of the entire genome of strain cv , which was found to be , nucleotides (nt) in length (excluding the poly a-tail). a cloning strategy, which involves primers based on conserved regions in the predicted orf products from other coronaviruses whose genome sequence has been determined, was used to amplify the equivalent, but as yet unknown, sequence of pedv. primary sequences derived from these products were used to design additional primers resulting in the amplification and sequencing of the entire orf of pedv. analysis of the nucleotide sequences revealed a small open reading frame (orf) located near the ′ end (no – ), and two large, slightly overlapping orfs, orf a (nt – ) and orf b (nt – ). the orf a and orf b sequences overlapped at a potential ribosomal frame shift site. the amino acid sequence analysis suggested the presence of several functional motifs within the putative orf protein. by analogy to other coronavirus replicase gene products, three protease and one growth factor-like motif were seen in orf a, and one polymerase domain, one metal ion-binding domain, and one helicase motif could be assigned within orf b. comparative amino acid sequence alignments revealed that pedv is most closely related to human coronavirus (hcov)- e and transmissible gastroenteritis virus (tgev) and less related to murine hepatitis virus (mhv) and infectious bronchitis virus (ibv). these results thus confirm and extend the findings from sequence analysis of the structural genes of pedv. porcine epidemic diarrhoea virus (pedv) is a causative agent for diarrhoea in pigs, particularly in neonates. the disease has been recognised for approximately thirty years, but the causative virus was only first described in [ ] , while another ten years elapsed before a method was developed for propagation of the virus in cell culture [ ] . during this time, outbreaks of the disease were reported from numerous european countries as well as korea, china and japan. the epidemiology and pathogenesis of the disease have been well described by pensaert [ ] . the biological behaviour, electron microscopic appearance and polypeptide structure of pedv resulted in its provisional classification as a coronavirus [ , , ] . coronaviruses belong to the taxonomic order of nidovirales and contain a single stranded rna genome of positive polarity, which is approximately thirty kilobases in length. the genes encoding the structural proteins are located at the end of the genome. an astonishing two-thirds of the genome consist of the replicase gene, which is located at the end of the genome. the replicase proteins are encoded by orf a and orf b. these two long, slightly overlapping orfs are connected by a ribosomal frame shift site in all coronaviruses sequenced to date. this regulates the ratio of the two polypeptides encoded by orf a and the readthrough product orf ab. about ± % of the translation products are terminated at the end of orf a, and ± % continue to the end of orf b. the polypeptides are post-translationally processed by viral encoded proteases [reviewed by ]. these proteases are encoded within orf a; the polymerase-and the helicase-function are encoded by orf b. we have previously completed the sequencing of the nucleocapsid-(n), membrane-(m), small membrane-(e), orf and spike-(s) genes of the pedv strain cv [ ± ]. the alignment of the deduced amino acid sequences indicated that pedv occupies an interesting intermediate position between the two well-characterized members of the group i coronaviruses, transmissible gastroenteritis virus (tgev) and human coronavirus (hcov)- e. in this study, we have continued to determine and analyse nucleotide sequences of pedv. to our knowledge, only two group i coronaviruses have been sequenced completely, hcov- e and tgev [ , ] . in addition, two strains of mouse hepatitis virus (mhv), jhm and a belonging to the group ii coronaviruses, and infectious bronchitis virus (ibv) have been completely sequenced [ ± ] . therefore, the sequence presented in this paper is the sixth sequence of a coronavirus covering the entire genome. growth of cell adapted pedv strain cv was performed essentially as has been described elsewhere [ , ] , except that virus-infected cells were harvested at approximately h post infection. cells were freeze-thawed three times and cell debris removed by low speed centrifugation. virus was pelleted by centrifugation for h at , rpm and c in a sw rotor of a beckman centrifuge. virus pellets prepared from two cm flasks were pooled and resuspended in ml trizol tm (gibco-brl), and rna was prepared as recommended by the manufacturer. in order to obtain the first partial pedv specific sequences, the predicted amino acid sequences of the hcov- e and tgev polymerase orfs were aligned and homologous regions identified. the homologous regions were used to design degenerate primers [ ] that were used for rt-pcr amplifications. these initial amplicons were cloned and sequenced [ ] . later, a mixture of up to six antigenome sense primers based on pedv specific sequences or the degenerate primers and random hexamer primer (purchased from schmidheini ag; balgach, switzerland) was used for first strand cdna synthesis. rna prepared from two cm flasks of virus-infected cells was denatured for min at c and first strand cdna was performed in a ml total reaction volume using superscriptii tm (gibcobrl; basel, switzerland) according to the manufacture's protocol. this was modified to create the longer reverse transcription products by including a denaturation step at c for min following the first h incubation at c, followed by the addition of ml superscriptii tm and a second prolongation step of h at c. template rna was digested by adding ml rnaseh (gibcobrl; basel, switzerland) to the reaction mix and incubating at c for min. pcr amplification was performed as described elsewhere. in brief, pfu dna polymerase (stratagene; basel, switzerland) was used for the amplifications, which were performed on a dna engine (mj research) machine. pcr fragments were subsequently cloned into pbluescript ii ks or puc vectors using standard procedures. the nucleotide sequence was determined on these cdna clones. direct sequencing was performed on a rt-pcr product (see fig. b ), which was cleaned through an agarose gel. the contigs of the sequence determinations were constructed using seqman (dna*, lasergene, madison wi, usa). we previously reported the determination of the pedv leader sequence on the mrna encoding the n-gene [ ] . this sequence was used for the primer design in order to amplify the end of the genome. the leader sequence was used for the in silico construction of the genomic rna sequence, which is available on genbank database (accession number af ). virus sequences covering replicase genes were obtained from the genembl sequence database. the files with the accession numbers x , z , af , and m for hcov- e, tgev (purdue ), mhv-a , and ibv (beaudette) respectively were used. the deduced amino acid sequences were compared as indicated in the text using pileup and gap (gcg package version . ; madison, wi, usa). the files generated by pileup were used in distances (gcg package version . ; madison, wi, usa) to determine the kimura protein sequence distances, which were subsequently used for the construction of unrooted dendrogram using treegen on the cbrg server (http://cbrg.inf.ethz.ch/) the cloning approach we used previously to clone the pedv m and n genes involved designing primers based on conserved regions of the coronavirus m and n genes to amplify the equivalent to the unknown pedv sequence. in this study, we employed this technique to clone parts of the orf of pedv. such a method is useful for viruses which do not grow to high titre, avoids lengthy screening of clones and could potentially be applied to the cloning of any group i coronavirus. however, the large size of orf and the paucity of sequence data from other coronaviruses made this an ambitious objective. a number of conserved functional domains were identified in the predicted orf products, but these domains are mainly located in the orf b region and leave large regions of the orf a product with no known function and only a low level of sequence conservation between different coronavirus genomes. in order to clone and determine the sequences for the pedv orf , the predicted amino acid sequences of the hcov- e and tgev orf were aligned and homologous regions identified. the hcov- e and tgev orfs were sufficiently closely related to allow complete alignment of the predicted expression products. in contrast, the mhv and ibv sequences were much more divergent, and could only be aligned with the group i sequences in some of the conserved regions. degenerate primers were designed from regions conserved between the hcov- e and tgev and, where possible, mhv and ibv orf . these primers were used both to prime reverse transcription and for the pcr amplifications. sequence data derived from these pcr products allowed us to design sequence-specific primers which were then used to amplify the entire orf (see fig. b ). numerous small cdna clones, five large cdna clones and one rt-pcr product covering the twothirds of the pedv genome were used to determine the nucleotide sequence of the pedv orf (fig. ). this analysis completes the nucleotide sequence of pedv, and thereby the sixth entire sequence determined from a coronavirus genome [ ± , ] . the genome of pedv (cv ) excluding the poly a-tail is nt in length. analysis of the newly determined nucleotide sequence revealed a pattern of orfs typical of coronaviruses. a small orf with the potential to code for a -amino acid peptide was found at the end of the genome from nucleotide position ± . such small orfs (uorfs) are present in all coronaviruses sequenced so far. the uorfs of hcov- e [ ] and ibv [ ] are found to be eleven codons in length, while that of mhv is eight codons long [ , ] . that of tgev can only encode a three-amino acid peptide [ ] . two long orfs of and nt, which overlap by nt, covered most of the newly determined sequence. by analogy to published coronavirus sequences [ , , ] , the orfs were designated orf a and orf b. the predicted orf a of fedv extended from nucleotide to . this resulted in a -codon orf. the overlapping orf b starting at nucleotide and ending at nucleotide had the capacity to code for amino acids. it has been proposed for coronaviruses and other members of the order nidovirales [ ] that the nucleotide sequences in the overlapping regions of orf a and orf b are able to fold into a pseudoknot tertiary structure [ , ] . this region allows the ribosome shifting of the reading frame during translation of the orf a and subsequently continues the translation in orf b. the function of these rna structures as ribosomal frame shift sites was demonstrated for the analogous sequences of ibv [ ] and hcov- e [ ] . it seems likely that the translation of the pedv orf b is mediated by such a ribosomal frame shifting. the nucleotide sequences of pedv, hcov- e, and tgev covering the ribosomal frame shift site are more conserved to each other than to mhv-a or ibv. in order to identify the sequence which could be involved in the formation of the tertiary structure, the nucleotide sequences covering the end of orf a and the beginning of orf b from hcov- e [ ] and tgev [ ] were aligned with the corresponding sequence of pedv. fig. a shows the predicted frame shift region of pedv based on this comparison. the so-called slippery site (uuuaaac) at which frame shifting occurs is identical in all coronaviruses sequenced so far. the stems and loops required to provide the tertiary structure of the frame shift regions of tgev and hcov- e were compared and fig. b shows the predicted tertiary structure required for the frame shift of pedv based on this comparison. pairwise comparison of the deduced amino acid sequences (using gap) revealed that orf b of pedv is more conserved than orf a to corresponding sequences of other coronaviruses. the percentage of similarities and identities is shown in table . the putative protein sequence of orf a was most similar to the sequence of orf a of hcov- e ( . %) and less similar to the corresponding orf a of tgev ( . %), mhv-a ( . %) and ibv ( . %). the same relationship, but at a higher level of similarity, was true for the deduced amino acid sequence of the predicted pedv orf b. it was most similar to the amino acid sequence of hcov- e orf b and tgev orf b ( . % and . %, respectively). the similarity to the orf b from mhv-a and ibv was around %. the deduced amino acid sequences of orf a and orf b from pedv were aligned with the corresponding sequences of hcov- e, tgev, mhv-a , and ibv using pileup. the degrees of amino acid homologies are graphically presented as dendrograms (fig. a,b) . the multiple sequence alignments revealed several putative functional domains common to coronavirus sequences [ , ] located on the deduced amino acid sequence of orf ab of pedv. some of these had been used to design the primers for the rt-pcr amplification. in the orf a region the following motifs were observed. two motifs indicative of papain-like proteases (plp) were present at amino acid positions ± and ± . the plp motif is found twice in the replicase genes of hcov- e, tgev and mhv, but only once in that of ibv. in this respect, pedv resembles hcov- e, tgev and mhv rather than ibv. a highly conserved region (x-domain) was found between the two plp motifs. despite this motif being present in all coronavirus sequences, its function is not yet known. a picornavirus c-like ( c ) protease domain is located between amino acids and of the pedv orf a. all corona-and arteriviruses encode this motif, which is the main protease for the coronavirus mediated processing of the polyproteins. three markedly hydrophobic domains conserved among coronaviruses are found in orf a. the first is located after the second plp motif and the others flank the cl motif. finally, a growth factorlike (gfl) domain was located close to the end of orf a (amino acid position ± ). in the orf b region, three structural protein motifs could be recognized, which all play a role in viral replication. a sub-sequence at amino acid position ± containing the characteristic tripeptide orf of pedv sdd (or gdd in most rna viruses) [ ] is probably the active site for the rna dependent rna polymerase. a metal ion-binding domain covering amino acids ± and a helicase motif at amino acid positions ± were also observed in the pedv orf b product. alignments of the deduced amino acid sequences of the cl protease and the polymerase motif from five different coronaviruses are shown in fig. a and b, respectively. the findings concerning conserved domains are summarised in fig. a . a deletion of about amino acids located between the x-domain and the second plp motif in the putative orf a sequence of tgev compared to that of hcov- e was reported by eleouet et al. [ ] . this additional sequence was present in the pedv orf a product. the alignment (using gap) of the hcov- e and pedv amino acid sequences revealed . % similarity and . % identity in this region. earlier sequence analysis of pedv based on the structural protein sequences has shown that pedv is most closely related to hcov- e and tgev [ ± , ] , less related to mhv-a , and least related to ibv. however, it was not possible to determine the relative similarities of hcov- e, tgev and pedv. in this study, the similarities and identities of the amino acid sequence alignments based on orf a and orf b show clearly that pedv is most closely related to hcov- e and, moreover, that hcov- e is more similar in sequence to pedv than it is to tgev. in addition to the sequence analysis, the presented work offers various possibilities for future research on coronaviruses. functional analysis and processing of the as yet uncharacterised pedv orf is now possible. recently, almazan et al. and yount et al. achieved the generation of infectious tgev from cdna [ , ] and thiel et al. suceeded in generating full length cdna clones of hcov- e and ibv in a recombinant vaccinia virus system [ ] . the sequence and the cdna clones covering the entire genome of pedv would allow the development of a mini-genome system to study viral replication or the generation of an assembled, infectious cdna clone. bearing in mind the close relationship of pedv and hcov- e, the latter approach could be used to exchange functional parts of these viruses to gain new insights into the biology of these viruses. furthermore, the porcine epidemic diarrhea virus virus infections of porcines a reverse genetic system for coronaviruses the authors thank christa meyer for excellent technical assistance. these studies were supported by the swiss national science foundation, grant # - . . key: cord- -h fqijk authors: hu, weiwei; zhu, liqi; yang, xing; lin, jian; yang, qian title: the epidermal growth factor receptor regulates cofilin activity and promotes transmissible gastroenteritis virus entry into intestinal epithelial cells date: - - journal: oncotarget doi: . /oncotarget. sha: doc_id: cord_uid: h fqijk transmissible gastroenteritis virus (tgev), a coronavirus, causes severe diarrhea and high mortality in newborn piglets. the porcine intestinal epithelium is the target of tgev infection, but the mechanisms that tgev disrupts the actin cytoskeleton and invades the host epithelium remain largely unknown. we not only found that tgev infection stimulates f-actin to gather at the cell membrane but the disruption of f-actin inhibits tgev entry as well. cofilin is involved in f-actin reorganization and tgev entry. the tgev spike protein is capable of binding with egfr, activating the downstream phosphoinositide- kinase (pi k), then causing the phosphorylation of cofilin and f-actin polymerization via rac /cdc gtpases. inhibition of egfr and pi k decreases the entry of tgev. egfr is also the upstream activator of mitogen-activated protein kinase (mapk) signaling pathways that is involved in f-actin reorganization. additionally, lipid rafts act as signal platforms for the egfr-associated signaling cascade and correlate with the adhesion of tgev. in conlusion, these results provide valuable data of the mechanisms which are responsible for the tgev pathogenesis and may lead to the development of new methods about controlling tgev. is an enveloped enteropathogenic coronavirus (cov) with a large positive-sense single-stranded rna genome about . kb in length. similar to other covs,tgev has a diameter ranging from to nm, including the surface projections. tgev infects newborn piglets with mortality rates reaching %, resulting in significant losses in the swine industry. the cov spike protein binds to a cellular receptor and then mediates membrane fusion at the plasma membrane or by endosomal uptake. tgev, human coronavirus (hcov- e), serotype feline coronavirus (fcov), and canine coronavirus (ccov) all use the aminopeptidase n (apn) protein as the receptor [ ] . tgev infects epithelial cells through the small intestine and the respiratory tract. the virus enters epithelial cells from the apical or basolateral side, and is released from the apical plasma membrane into the gut lumen, where it propagates efficiently by cell-to-cell spreading [ ] . porcine intestinal columnar epithelial cells (ipec-j ) offer a practical model for studying porcine enteric pathogens [ ] . however, the precise molecular mechanisms responsible for tgev entry are largely unknown, and limited informations are available on the cell signaling pathways involved in coronavirus entry via the actin cytoskeleton. located beneath the plasma membrane, cortical actin is composed of a loosely organized network of actin cytoskeleton that is highly dynamic and is involved in many cellular processes. many pathogens facilitate cell entry and/or trafficking by stimulating actin remodeling [ , ] . cofilin plays an important role in actin polymerization and depolymerization [ ] . lim-kinases (limks) inhibit the activity of cofilin by phosphorylating the serine residue at position (ser- ). limks are activated by rho-associated kinase (rock), p -activated protein kinases (paks), which are downstream kinases of the rho family gtpases, rhoa, rac , and cdc [ ] . rho gtpases regulate actin polymerization, induce plasma membrane protrusion and control vesicle trafficking [ ] . the phosphoinositide- kinase (pi k) pathway is activated by a variety of extracellular stimuli and regulates a wide range of cellular processes, including cell cycle progression, cell growth, cell motility, cell adhesion and vesicular trafficking [ , ] . the serine/threonine kinase (akt) is a central node in cell signaling downstream of growth factors, cytokines, and other cellular stimuli [ ] . receptor tyrosine kinases (rtks) play an important role in transforming extracellular intracellular signals and activate pi k as well as extracellular signal regulated kinase (erk) / [ ] . the epidermal growth factor receptor (egfr) belongs to the rtk family, and is activated by a family of growth factors including epidermal growth factor (egf), transforming growth factor-α (tgf-α), and the neuregulins. it also interacts with three homologous transmembrane proteins erbb , erbb and erbb [ , ] . the binding of egf to its cell surface receptor activates the receptor's intrinsic tyrosine kinase and phosphorylates the tyrosine at its c-terminus. phosphorylated egfr is essential for the activation of ras gtpase and erk [ ] . egfr can be activated by many viruses, including influenza a, hepatitis c (hcv), herpes simplex type (hsv- ), and human cytomegalovirus (hcmv) [ ] [ ] [ ] [ ] . in this study, we found that tgev caused f-actin rearrangement and membrane ruffling early in infection. the phosphorylation of the egfr was also detected early in infection. we found that tgev acted via the egfr-pi k-rac /cdc -pak-limk signaling pathway to regulate the activity of cofilin and f-actin arrangement early in infection, and also demonstrated that egfr was a promoter for tgev entry. actin cytoskeleton assembly/disassembly dynamics are critical for many endocytic pathways [ ] . in order to explore potential interactions between tgev and f-actin, we stained cells shortly after infection with phalloidin-tritc and examined them using confocal microscopy ( figure a ). at min post-infection (mpi), f-actin filaments were observed close to the cell plasma membrane, and accumulated in this region as the experiment progressed. at mpi, actin stress fibers had became noticeably less abundant in the cytoplasm. at mpi, almost all f-actin was at the cell membrane. transmission electron microscopy (tem) confirmed that f-actin gathered underneath the plasma membrane, the podosome and lamellipodium were also observed in the cell membrane (indicated by the white arrows) ( figure b ). the relationship between tgev particles and f-actin early in infection were further examined by confocal fluorescence microscopy, with infected cells stained using phalloidin-tritc, tgev particles were labeled with fluorescent probe dylight ( figure c ). at mpi, f-actin was organized to produce a range of cell surface protrusions (red arrows), tgev particles had internalized into the cell membrane and begun to enter the cytoplasm. at mpi, tgev had entered the cytoplasm, we found that f-actin surrounded the tgev particles in spatial, part of the virus co-localized with the f-actin (yellow) (white arrows). these phenomena were also shown in d rendered images. together, these results suggest that the process of tgev infection causes f-actin accumulation around the cell membrane, possibly promoting viral binding, penetration and intracellular trafficking. to test these hypothesis, ipec-j cells were pretreated with three cytoskeleton inhibitors: latrunculin a (lat a), cytochalasin d (cyto d), and cucurbitacin e (cu e) at °c for h. the effect of these inhibitors on cell viability was shown in figure s . all three inhibitors reduced tgev entry in a dose dependent manner compared with mock control cells ( figure d, e, f) . these results provide evidence that f-actin is involved in the tgev entry process. to investigate the relationship between cofilin and tgev infection, we measured the levels of p-cofilin and cofilin in infected and uninfected cells by western blot ( figure g , h, i, j). in tgev-infected cells, the level of p-cofilin increased from mpi to mpi and decreased at mpi, but the level of cofilin was low from mpi to mpi and increased at mpi. in uninfected cells, the level of p-cofilin was low from mpi to mpi and increased at mpi, but the level of cofilin maintained a high expression level from mpi to mpi. we hypothesize that the process by which tgev causes f-actin filaments to gather around the cell membrane and the formation of cell surface protrusions require the participation of p-cofilin. to examine the role of cofilin more closely, we conducted series of experiments in which cofilin distribution and function were assessed in uninfected and tgev-infected cells. most p-cofilin was found in the nucleus of uninfected cells (figure a ). in contrast, early in tgev infection, p-cofilin redistributed to the cytoplasm and was less abundant in the nucleus. however, at mpi, p-cofilin appeared to shift from the cytoplasm back to the nucleus (figure a ). when the distribution of p-cofilin and f-actin were observed early in infection, we found most p-cofilin did not colocalize with f-actin, and multiple protrusions were noted around the cell membrance ( figure b ). we knocked cofilin down in normal ipec-j cells, and found that the actin stress fibers were also in the cytoplasm. usually underneath the cell membrane are the loosely organized network of f-actin filaments that are termed cortical actin [ ] , but the cortical actin underneath the cell membrane gathered together, the cell surface was smooth and couldn't form protrusions. in cofilin knockdown cells, early in tgev-infection, some actin stress fibers were also found in the cytoplasm, the protrusions in the cell membrance reduced significantly. the transient increased in p-cofilin level in the cytoplasm from mpi to mpi correlated with the f-actin filaments around the cell membrane and the formation of multiple tgev particles were labeled with fluorescent probe dylight , ipec-j cells were incubated with dylight labeled tgev at °c for h, then shifted to °c, fixed at mpi and mpi, f-actin stained with phalloidin (green). images were captured with a zeiss lsm confocal laser-scanning microscopy system and rendered three-dimensional ( d) images. scale bar = μm. d. to f. concentrationdependent inhibition of tgev (moi = ) entry by cytoskeleton inhibitors. g. and h. cells were incubated with tgev (moi = ) at °c for h, unbound virus removed, and cells were then incubated at °c. levels of p-cofilin, cofilin and p-limk were measured by western blotting using either mab specific for p-cofilin, or pab for p-limk, cofilin. i. and j. the amount of p-cofilin and cofilin were quantified. statistical significance was assessed by student's t-test. differences were considered significant at (*) . < p < . , (**) p < . . all experiments were performed separately three times. infection. cells were fixed at the indicated time points, stained with anti-p-cofilin antibody, and observed by confocal microscopy. scale bar = μm. b. the distribution of p-cofilin and f-actin early in tgev infection. at mpi, cells were fixed and stained with anti-p-cofilin antibody, followed by dylight -conjugated goat anti-rabbit igg. f-actin was stained with phalloidin-tritc (red). scale bar = μm. c. cofilin shrna block the destruction of actin cytoskeleton caused by tgev infection. cells were incubated with cofilin shrna for h, cells were infected with tgev for h, f-actin was stained with phalloidin-tritc (green), scale bar = μm. d. cofilin shrna verification. cells were transfected with three cofilin shrnas. after h incubation, the expression of cofilin was verified by western blotting. e. cofilin shrnas block tgev entry. cells were transfected with lentiviral vectors expressing cofilin shrnas. after h incubation, cells were infected with tgev for h and entry of tgev was evaluated by real-time pcr. f. verification of expression of wt cofilin and mutant cofilins s a and s e. cells were treated with lentiviral particles expressing wt cofilin or mutant cofilins s a and s e. after h incubation, expression of p-cofilin and cofilin were verified by western blotting. g. the wt cofilin and mutant cofilins s a and s e block tgev entry. cells were treated with lentiviral particles expressing wt cofilin or mutant cofilins s a and s e. after h incubation, cells were infected with tgev for h and entry of tgev was evaluated by real-time pcr. h. arp / inhibitor ck promotes tgev entry. ipec-j cells were pretreated with ck at different concentrations at °c for h, for details concerning binding and entry assays, see materials and methods. statistical significance was assessed by student's t-test. differences were considered significant at (*) . < p < . , (**) p < . . scale bar = μm. tgev at a multiplicity of infection of (moi = ). protrusions. all of these results demonstrate that cofilin involve in the regulation of f-actin early in tgev infection. to confirm the role of cofilin in tgev infection, we used molecular methods to modify the levels of cofilin and p-cofilin prior to infection by tgev. cells were transfected with cofilin targeting shrnas, the cofilin expression level was decreased significantly ( figure c) , and tgev entry level was decreased ( figure d ). cells were also transfected with vectors expressing wt cofilin, constitutively non-phosphorylated mutant cofilin (activated, s a), or constitutively-phosphorylated mutant cofilin (inactivated, s e) [ , ] . the expression of wt or constitutively inactivated cofilin s e significantly increased the expression of p-cofilin ( figure e ). all three constructs inhibited tgev entry ( figure f ). examination using confocal fluorescence microscopy showed that in uninfected cells, the f-actin partially depolymerized in wt cofilin-expressing cells and in constitutively activated cofilins a-expressing cells, while the f-actin in constitutively inactivated cofilins e-expressing cells, polymerized around the cell membrane ( figure s ). we also explored the role of actin-related protein / (arp / ) in tgev entry using its specific inhibitor ck . cells were treated with different concentrations of ck at °c for h prior to tgev infection. ck did not inhibit virus binding, but promoted tgev entry ( figure g ). we pretreated cells with several rho-family-gtpases inhibitors prior to tgev infection to determine whether some were the upstream regulators of cofilin. rock inhibitor y did not inhibit tgev binding as well as tgev entry ( figure a ). paks inhibitor ipa- also had no effect on tgev binding, but decreased tgev entry in a dose dependent manner ( figure b ). ipa- inhibited limk activation and cofilin phosphorylation, while rock inhibitor y had no inhibitory effect ( figure c ). using confocal fluorescence microscopy, we also observed that ipa- appeared to inhibit the destruction of f-actin by tgev infection, but ly couldn't inhibit the destruction of f-actin by tgev infection ( figure j ). the effect of these inhibitors on cell viability was shown in figure s . in order to study the role of rho-family-gtpases early in the entry process of tgev, cells were transfected with lentivirus constructs that expressed wild type, constitutively-activated, or constitutively-inactivated rho-family-gtpases. the expression of constitutively activated l rhoa, l rac , and l cdc , as well as constitutively inactivated n rac and n cdc , decreased tgev entry ( figure d , e, f). additionally, some rho-family-gtpases affected p-cofilin level early in tgev infection, the level of p-cofilin was significantly inhibited in cells expressing n rac , n cdc , l rac , or l cdc , but was not affected in cells expressing wt rhoa, n rhoa, or l rhoa ( figure g , h, i), these data was reinforced by results obtained using inhibitor drugs (y , ipa- ) ( figure c ). all of these results indicate that rho-family-gtpases play an important role in tgev entry. specifically, rac and cdc are involved in the regulation of cofilin early in tgev infection, while rhoa is not involved. neither pi k modulation of the rho-gtpase family, nor the involvement of pi k/akt pathway early in coronavirus infection, have been demonstrated conclusively. to investigate the role of pi k/akt pathway in tgev entry, we monitored the level of p-akt in cells shortly after infection. from mpi to mpi, the level of p-akt increased in tgev infected cells ( figure a ). in cells pretreated with ly , a highly specific inhibitor of pi k, virus binding was unaffected, but tgev entry was reduced % compared with untreated cells ( figure b ). examination by confocal fluorescence microscopy demonstrated that ly inhibited the destruction of f-actin early in tgev infection ( figure c ). we previously have showed that attenuated tgev (stc strain) and pedv (cv strain) could regulate the f-actin via the mapk pathways [ ] . in this study, we found that tgev infection induced the activity of erk / at mpi and increased it thereafter ( figure d ). u , a specific inhibitor of mek / , inhibited tgev entry in a dose dependent manner but did not affect the ability of the virus to bind to cells ( figure e ). u also inhibited the destruction of f-actin by tgev infection, as observed when cells were examined by confocal fluorescence microscopy ( figure c ). the phosphorylation of akt, limk and cofilin were inhibited when cells were treated with pi k inhibitor ly ( figure f) , suggesting that the pi k/akt pathway is involved in the regulation of actin cytoskeleton by cofilin. erk / , limk and cofilin phosphorylation were inhibited when cells were treated with mek / inhibitor u , but the activation of erk / was not inhibited by ly . these results indicate that the mek/erk pathway also involve in the regulation of the actin cytoskeleton by cofilin, but it is not activated by pi k/akt pathway. figure : egfr is involved in tgev entry. a. tgev is co-localized with egfr. after mpi, cells were fixed and stained with mouse anti-tgev mab, followed by dylight -conjugated goat anti-mouse igg and anti-p-egfr pab, followed by dylight -conjugated goat anti-rabbit igg. cells were examined by if microscopy. b. the rtk specific inhibitor, genistein (gen), inhibits tgev entry in a dose dependent manner. cells were pretreated with different concentrations of genistein. tgev binding and entry assays are described in materials and methods. c. and d. the egfr specific inhibitor, ag , inhibits tgev entry. ipec-j cells were pretreated with ag for h at °c. alternatively, ipec-j cells were incubated with tgev at °c for h, unbound viruses were removed, cells were shifted to °c, and ag was added at - mpi, - mpi, and - mpi. e. the activation of egfr promotes tgev entry. serum-starved ipec-j cells were pretreated with different low concentrations of egf at °c for h, cells were infected with tgev for h and entry of tgev was evaluated by real-time pcr. f. the internalized of egfr inhibits tgev entry. serum-starved ipec-j cells were pretreated with different high concentrations of egf at °c for h, cells were infected with tgev for h and entry of tgev was evaluated by real-time pcr. g.egfr shrnas block tgev entry. cells were transfected with lentiviral particles expressing egfr shrnas. h post-infection, cells were infected with tgev for h, and entry of tgev was evaluated by real-time pcr. h. tgev s interacts with egfr. cells were co-transfected with plasmids expressing tgev s -ha and egfr. immunoprecipitation and immunoblotting were performed to examine interactions between tgev s and egfr. statistical significance was assessed by student's t-test. differences were considered significant at (*) . < p < . , (**) p < . . all experiments were performed independently three times. scale bar = μm. tgev at a multiplicity of infection of (moi = ). www.impactjournals.com/oncotarget we have concluded that the pi k/akt pathway involve in tgev infection, but the involvement of egfr have not been demonstrated. to investigate the role of egfr more closely, we stained cells with an anti-p-egfr antibody after various treatments and then observed them using fluorescence microscopy. at mpi, both tgev and egf increased the phosphorylation level of egfr, but egfr specific inhibitor ag significantly impaired egfr phosphorylation ( figure a ). western blotting results confirmed that the phosphorylation level of egfr increased from mpi to mpi and reaching a peak at mpi ( figure b ). to study the relationship between egfr and downstream signaling pathways, we conducted experiments using different inhibitors and egfr shrnas. as expected, egfr-targeting shrnas significantly inhibited egfr expression ( figure c ). the activation of akt, limk, and cofilin phosphorylation were inhibited by egfr shrnas (figure d ), as well as with rtk inhibitor gen, and egfr inhibitor ag ( figure e ). the activity of erk / was also inhibited by egfr shrnas, gen, and ag . tgev infection caused the dissolution of stress fibers and stimulated the formation of membrane protrusions, but these phenomena were blocked by ag treatment ( figure f ). taken together, these results indicate that egfr is involved in the entry of tgev and the activation of downstream pathways early in the infection process. using fluorescence microscopy, we found that the spike protein of tgev co-localized with p-egfr ( figure a ). the rtk specific inhibitor genistein (gen) had no inhibition effect on the adhesion of tgev, but inhibited tgev entry in a dose dependent manner ( figure b ). although egfr specific inhibitor ag had no inhibition effect on the adhesion of tgev ( figure c ), when ipec-j cells were pretreated with ag for h at °c, tgev entry was significantly decreased ( figure d ). ag appeared to act only at early stage of tgev entry, shortly after the virus bound to the cell ( figure d ). when serum-starved cells were pretreated with low concentration of egf at °c for h, egfr was activated and tgev entry was increased significantly ( figure e ). when serum-starved cells were pretreated with high concentration of egf at °c for h, most of egfr internalized into the cytoplasm and tgev entry was decreased significantly ( figure f ). egfr targeting shrnas significantly decreased tgev entry ( figure g ). the interaction between tgev s and egfr were confirmed in hek t cells that were co-transfected with plasmids expressing tgev s -ha and egfr ( figure h ), providing further support that tgev s directly combined with egfr. most of egfr occured in the cell membrane in normal cells, part of egfr internalized into the cytoplasm upon low concentration egf ( ng/ml) stimulation, most of egfr internalized into the cytoplasm upon high concentration egf( ug/ml) stimulation, both tgev particles and egfr internalized into the cytoplasm upon tgev infection ( figure s ). taken together, these results indicate that egfr acts as a membrane promoter for tgev entry. when egfr in the lipid rafts is stimulated, the endocytosis of membrane microdomains via clathrindependent and clathrin-independent mechanisms occurs [ , ] . lipid rafts are mainly composed of sphingolipid and cholesterol [ ] . ganglioside gm serves as lipid rafts marker and can be labeled with fluorescein isothiocyanate (fitc)-conjugated cholera toxin beta subunit (ctxb) [ , ] . using this reagent, we found that lipid rafts distributed evenly in the cell membrane of untreated cells. in contrast, the lipid rafts clustered in egf treated or tgev infected cells ( figure a ). these results show that tgev infection causes lipid rafts function as a platform to concentrate apn and egfr. to analyze whether tgev or egfr is present in lipid rafts upon tgev infection, co-localization experiments were performed. lipid rafts were labeled with fitc-conjugated ctxb, tgev was stained with mouse anti-tgev mab, and egfr was stained with rabbit anti-egfr pab. both tgev and egfr co-localized with gm at the cell surface ( figure b ). therefore, we conclude that egfr and the attaching tgev particles are located together in the lipid rafts during infection. for studying the role of lipid rafts in the entry process of tgev, both methyl-β-cyclodextrin (mβcd) and nystatin were used to remove the cholesterol from the cell membrane. the effect of these inhibitors on cell viability were shown in s fig. cells were pretreated with different concentrations of mβcd or nystatin at °c for h prior to infection, both tgev binding and entry levels were inhibited in a dose dependent manner compared with mock control cells ( figure c, d) . these results suggest that lipid rafts play an important role in tgev binding and entry. during the early phase of tgev infection, the destruction of lipid rafts inhibited the activation of egfr, akt, and limk, and also inhibited the phosphorylation of cofilin ( figure e ). these results indicate that lipid rafts function as a platform to induce the activation of egfr the role of actin cytoskeleton in tgev entry process is unclear. we had shown that tgev induced f-actin rearrangement and caused membrane ruffles. tgev entry required f-actin gathered at the cell membrane. we identified cofilin as a critical factor for regulating cytoskeleton dynamics and tgev infection. our results also indicate that tgev binding induces the phosphorylation of cofilin through the egfr-pi k-rac / cdc -pak-limk signaling pathways, resulting in actin polymerization around the cell membrane and foming multiple poutrusions. the highly dynamic nature of actin cytoskeleton affects every stage of the viral life cycle, from entry through assembly to release. virion attachment to cells can stimulate the extension of cell surface protrusions [ ] . human immunodeficiency virus (hiv) and herpes simplex virus (hsv) virions cause cell surface extensions and viral-entry receptor clustering [ , ] . the entry of vesicular stomatitis virus (vsv) virions is enhanced by the formation of actin filaments, and actin filaments are recruited to increase the size of the endocytic structure [ ] . early in the infection of tgev, we observed the rearrangement of cortical actin and the formation of ruffles and protrusions in the cell membrane, as well as egfr and lipid rafts clustering in the cell membrane, the dissolution of actin stress fibers and their relocalization around the cell plasma membrane. these changes appear to mediate tgev entry. cofilin has a central role in controlling actin dynamics by regulating actin polymerization and depolymerization through its severing activity, as well as by inducing dendritic nucleation [ ] . the regulation of cofilin activity is a component of pathogen-mediated actin remodeling [ , ] . we conclude that phosphorylation of cofilin is critical for tgev entry. cofilin can be found in multiple cellular compartments, including the cytoplasm and nucleoplasm [ , ] . a nuclear localization signal (amino acid residues - ) with high homology to the consensus nuclear localization signal sequence of sv large t-antigen is found in the cofilin sequence [ ] . the nuclear translocation of cofilin is regulated by reversible phosphorylation of ser- [ ] . the phosphorylation of cofilin inhibits its f-actin severing activity. we found that p-cofilin was distributed mainly in the nucleus of normal ipec-j cells, however, early after tgev infection, most p-cofilin was found in the plasma membrane, derived both from the phosphorylation of cofilin and the migration of p-cofilin from the nucleus. late in infection, a portion of the p-cofilin was dephosphorylated, while the remaining p-cofilin returned to the nucleus. cofilin supports arp / complex-mediated actin branching by creating new actin filaments through its f-actin severing activity, making the actin filaments more stable at the cell membrane [ ] . cofilin is required for the early polymerization response to growth factor stimulation and the formation of protrusions, although the arp / complex is not involved in this process [ , ] . the inhibition of arp / caused the actin filaments to bind together more loosely, resulting in increased entry of tgev. we conclude that the structure of the cortical actin filaments, which underline the cell membrane, plays an important role in tgev early infection. the regulation of cofilin activity acts as a molecular switch to provide a functional link between actin cytoskeleton rearrangement and the tgev entry process. the actin cytoskeleton is mainly regulated by rhofamily-gtpases, which control the signaling pathway that links membrane receptors to the cytoskeleton [ ] . the rho family of gtpases contain more than twenty members. rac , a primary example, induces membrane ruffles or lamellipodia [ ] . pi k/akt is the main activation pathway for rac in many cell types [ ] . cofilin is inactivated by rac and cdc through pak and limk. the entry of ebola virions and vsv pseudovirions are decreased when rhoa is overexpressed [ ] . rhoa activation by pi k leads to the formation of stress fibers [ ] . constitutively activated mutant l rhoa caused the formation of stress fibers, and prevented the destruction of stress fibers caused by tgev and reduced the amount of f-actin gathered around the cell membrane. the inhibition of rock had no effect on the entry of tgev or the phosphorylation of cofilin. both constitutively inactivated mutant n rac and constitutively activated mutant l rac impeded the formation of lamellipodia and membrane ruffles, and both constitutively inactivated mutant n cdc and constitutively activated mutant l cdc impeded the formation of protrusive filopodia. these effects inhibited tgev entry. the activation of the pi k signaling pathway is involved in the early steps of virus infection, such as receptor/co-receptor engagement and virus internalization [ ] . pi k is a key mediator of the signaling pathway that regulates actin cytoskeleton reorganization and polarized cell migration [ ] . this study shows that the pi k/akt pathway is involved in the cellular entry of tgev, and is activated by egfr. our previous studies indicate that mapk pathways are activated early in tgev infection (for example, the ras-mediated activation of the raf/ mek/erk cascade). mapk pathways can also be activated by the egfr-pi k-rhogtpases signaling pathway [ , ] . these data demonstrate that tgev also activates mapk pathways to regulate the actin cytoskeleton through egfr early in infection. egfr, a member of the erbb receptor family, is expressed in many cell types. the primary signaling pathways activated by rtks include pi k/akt, ras/ www.impactjournals.com/oncotarget raf/erk / , and signal transduction and activator of transcription (stat) pathways. our data provides evidence that egfr acts as a signaling-promoter that is involved in the regulation of actin and tgev internalization. human papillomavirus (hpv) infection induces filopodia formation and the dissolution of stress fibers by mpi, and hpv entry is also blocked by rtk and pi k inhibitors [ ] . we find that the egfr specific inhibitor ag and pi k specific inhibitor y block stress fiber dissolution, suggesting that the egfr and pi k are involved in the signaling pathway that regulates the assembly of f-actin. our results indicate that egfr is activated by tgev binding and promotes tgev uptake into host cells. it has been reported that a -kda protein in st cells and in villous enterocytes in newborn pigs may be a second receptor for tgev and contribute to the age sensitivity of these animals to the virus [ ] , but the identity of this -kda receptor has not been determined. although egfr is a -kda transmembrane protein, we have found that it interacted with tgev s protein, and therefore should be considered as a factor in determining the high level of susceptibility of newborn pigs to tgev. the clustering of large lipid rafts is caused by protein modifications such as phosphorylation, which increase the number of protein-protein interactions [ ] . we found that tgev stimulated the clustering of large lipid rafts, and that activated egfr, and tgev virions were associated with the rafts. the clustering of the lipid rafts functions as a signaling platform, but the activation of downstream signaling pathways were inhibited by agents that remove the rafts. the integrity of lipid rafts is essential for the activation of src kinase and the pi k/ akt pathway [ ] . based on these data and our results, we hypothesize that tgev induces the clustering of lipid rafts by the binding of spike protein to apn and egfr. the activated egfr then transfers the signal to effector proteins downstream. apn is the specific receptor for tgev. our experiments identified egfr as another promoter for tgev entry. it is not known whether apn and egfr are related in any way, or whether other tgev receptors exist. actin plays an important role in the endocytosis and vesicle transport [ ] , and the cytoskeleton is involved in different stages of both clathrin-and caveola-mediated endocytosis. understanding the main endocytic pathway of tgev, and the molecular mechanisms regulating this process, will require further investigation. these findings are conducive to enhancing our understanding of the entry mechanism of tgev and providing a potential target for the development of new anti-tgev therapies. ipec-j cells are porcine intestinal columnar epithelial cells that were isolated from the middle jejunum of neonatal piglets. ipec-j cells were purchased from dsmz (germany). hek t cells were purchased from atcc (united states). both ipec-j and hek t cells were maintained in dulbecco's modified eagle's medium (dmem) with high glucose, hepes supplemented with % fetal bovine serum (fbs, gibco), % penicillinstreptomycin (invitrogen) at °c in a % co incubator (thermo scientific). transmissible gastroenteritis virus (strain shxb) was isolated in shanghai, china. the complete genome sequence for tgev shxb is available in genbank (kp . ) [ ] . for the binding assays, cells were incubated with tgev at a multiplicity of infection of (moi = ) for h at °c, and washed with pbs (ph . at °c) three times to remove unbound virus, then we added the trizol to collect the samples. for the entry assays, cells were incubated with tgev at moi = for h at °c and washed with pbs (ph . at °c) three times to remove unbound virus, then maintained in maintenance medium (dmem supplemented with % fbs and % penicillinstreptomycin) at °c in a % co incubator, after the indicated time, cells were washed with acidic pbs (ph . at °c) to remove the virus bound to the cell membrane (not enter the cell), then we added the trizol to collect the samples. total rna from ipec-j cells infected with tgev was extracted using trizol reagent (invitrogen) according to the manufacturer's instructions. the cdna was generated by reverse transcription using hiscript tm qrt supermix for qpcr (vazyme) according to the manufacturer's instructions. tgev entry and binding were assessed by detecting the viral nucleoprotein (n) gene using quantitative rt-pcr with the takara sybr green qpcr kit (takara). primer sequences were as follows: n-f (sense), '-caattcccgtggtcggaaga- '; n-r (antisense), '-tttacgttggcccttcacca- '; gapdh-f, '-tcatcatctctgccccttct- '; gapdh-r, '-gtcatgagtccctccacgat- '. pcr products were purified using a gel extraction kit (omega), and cloned into the pjet . vector (thermo). plasmids were diluted serially and used as standards for quantitative analysis. the initial copy number of tgev n gene and gapdh in each group was calculated using the following formula: x = -k log ct + b, where x is the initial copy number, k, ct, and b refer to the slope rate, cycle threshold, and constant, respectively. quantitative real-time pcr was performed with the abi prism detection system (applied biosystems, foster, usa). at indicated times post infection, cells were washed with pbs and lysed in radioimmunoprecipitation assay (ripa) buffer (thermo scientific). phosphatase inhibitor and protease inhibitor (thermo scientific) were added in the ripa according to the manufacturer's instructions. the concentration of the lysates were determined by a pierce bca protein assay kit based on the bicinchoninic acid spectrophotometric method (thermo scientific). after centrifugation at ×g for min, the supernatant ( - ug of protein) was fractionated by sds-page ( %- % gradient), the separated proteins were transferred to pvdf (merck millipore), the membranes were blocked for h in tris-buffered saline (tbs) containing % nonfat dry milk, and reacted with the indicated primary antibodies at °c overnight. membranes were exposed to species specific horseradish peroxidase (hrp)-conjugated secondary antibodies (dilution : ) followed by enhanced chemiluminescence (ecl, thermo scientific) detection by autoradiography. western blotting was quantified by quantity one (quantity one -d analysis software - , bio-rad). the intensity of the bands in terms of density was measured and normalized against gapdh expression. all data were expressed as means ± sd of three independent experiments. to construct the pcdna . vectors, rhoa, rac and cdc sequences were inserted into the nhe i/ hindiii site. plvx-dsred-monomer-n is an hiv- based, lentiviral expression vector that express the gene of interest fused to dsred-monomer (clontech). rhoa, rac and cdc sequences were inserted into the ecori/ bamhi site. to construct vectors expressing rhoa, rac , and cdc mutants (constitutively-activated mutants plvx-l rhoa, plvx-l rac , and plvx-l cdc ; constitutively-inactivated mutants plvx-n rhoa, plvx-n rac , and plvx-n cdc ), sequences were amplified from pcdna . -rhoa, rac , and cdc respectively. the constitutively-phosphorylated mutant (inactivated) plvx-cofilins e, and the constitutively non-phosphorylated mutant (activated) plvx-cofilins a, were amplified from pcdna . -cofilin. all mutants were generated using the mut express ii fast mutagenesis kit (vazyme) according to the manufacturer's instructions. all constructs were verified by dna sequencing. the primers used in pcr and for the site-directed mutagenesis are described in supplemental tables s and s . plvx-shrna is an hiv- -based, lentiviral expression vector designed to express a small hairpin rna (shrna) for rna interference (rnai) studies (clontech). the best silencing efficiency was observed with clone nm_ (porcine cofilin) and nm_ (porcine egfr). targeting sequences are described in supplemental table s . hek t cells were transfected with μg of specific expression plasmid per cells using the x-tremegene hp dna transfection reagent (roche) according to the manufacturer's instructions, with opti-mem (invitrogen) included in t- cell culture flask. lentivial particles (moi = ) were added to the ipec-j cells, and gently mixed. ipec-j cells were cultivated on t- cell culture flask and incubated with tgev at moi = for h at °c, then shifted to ℃ for min. samples were fixed in a . % glutaraldehyde solution in pbs for h. after fixation, the monolayer was gently removed from the flask with a rubber policeman and the cell suspension was pelleted by low speed centrifugation ( ×g for min). the cells were then washed twice by centrifugation in . m pbs. the pellet of cells was then fixed overnight at ℃ and then prepared for transmission electron microscope (tem) as described [ ] . samples were visualized using a hitachi- transmission electron microscope (tem, japan) at kv. ipec-j cells were grown on coverslips in -well tissue culture plates and incubated with tgev at moi= for h at °c, then shifted to °c. at the indicated time points, cells were fixed in % paraformaldehyde for min then permeabilised with . % triton x- for min. samples were blocked in % bovine serum albumin (bsa) for min and incubated with primary antibodies ( : for the cell signaling technology antibody and santa cruz antibody) at °c overnight then incubated with fluorochrome-conjugated secondary antibodies ( : ) at room temperature for h. to stain f-actin, phalloidin-tritc (green, red) (life) was added for min at room temperature. nuclei were stained using ug/ml dapi ( ', 'diamidino- -phenylindole dihydrochloride)-pbs for min at room temperature. images were captured with a zeiss lsm confocal laser-scanning microscopy system and a x objective lens. top view images were prepared as compacted z-stack images of non-permeabilised cells, using zen (zeiss) software. the co-localization of two channels was detected using the co-localization finder plug-in. x-y plane and z-axis views of confocal images were prepared using zen le software from zeiss, germany. for viral labeling, viruses were filtered with . μm filter, and then clarified by centrifugation at , ×g for . h, followed by ultra-centrifugation using %, %, % sucrose gradient at , ×g for . h, viruses were labeled with the fluorescent probe dylight nhs ester (thermo scientific) according to the manufacturer's instruction. unincorporated dye was removed by using commercial fluorescent dye removal columns (thermo scientific). to observe the clustering of lipid rafts in the cell membrane, ipec-j cells were incubated with tgev (moi = ) or egf ( ng ml - ), or left untreated at °c for h. subsequently, cells were incubated with fitcconjugated cholera toxin beta subunit (ctxb) ( ug ml - ) at °c for h and then incubated at °c for min. cells were fixed and observed by if microscopy. to observe the co-patching of lipid rafts with proteins of interest and for co-localization experiments, ipec-j cells were incubated with tgev (moi = ) at °c for h, and then incubated with fitc-ctxb at °c for h and at °c for min. egfr and tgev were visualized by the common immunofluorescence staining protocol. the cells were fixed and stained with mouse anti-tgev mab followed by dylight -conjugated goat anti-mouse igg or rabbit anti-p-egfr mab followed by dylight -conjugated goat anti-rabbit igg. cells were examined by if microscopy. cells were lysed in np- lysis buffer (beyotime). protease inhibitor (thermo scientific) was added according to the manufacturer's instructions. after centrifugation at ×g for min, supernatant was pretreated with protein g plus-agarose (santa cruz) and normal igg (from the same species as that of the immunoprecipitating antibody) at °c for h to eliminate nonspecific binding to the agarose beads or igg. the supernatant was incubated with immunoprecipitation antibody (ip) at °c for h and incubated with fresh agarose beads at °c for another h. the agarose beads were washed five times with pbs, and the immunoprecipitated proteins were analyzed by western blotting with specific primary and secondary antibodies. all results are presented as means ± standard deviation from three independent experiments. significant differences between control and experimental groups were analyzed using student's t test. differences were considered significant at * . < p < . , ** p < . . mechanisms of coronavirus cell entry mediated by the viral spike protein entry and release of transmissible gastroenteritis coronavirus are restricted to apical surfaces of polarized epithelial cells porcine ipec-j intestinal epithelial cells in microbiological investigations subversion of the actin cytoskeleton during viral infection the actin cytoskeleton as a barrier to virus infection of polarized epithelial cells functions of cofilin in cell locomotion and invasion signaling mechanisms and functional roles of cofilin phosphorylation and dephosphorylation rho gtpases and actin dynamics in membrane protrusions and vesicle trafficking signalling through class i pi ks in mammalian cells akt/pkb signaling: navigating downstream the epidermal growth factor receptor family as a central element for cellular signal transduction and diversification untangling the erbb signalling network epidermal growth factor receptor: mechanisms of activation and signalling internalization of the epidermal growth factor receptor: role in signalling egfr and epha are host factors for hepatitis c virus entry and possible targets for antiviral therapy activation of egfr on monocytes is required for human cytomegalovirus entry and mediates cellular motility the epidermal growth factor receptor (egfr) promotes uptake of influenza a viruses (iav) into host cells epidermal growth factor receptor-pi k signaling controls cofilin activity to facilitate herpes simplex virus entry into neuronal cells actin regulation in endocytosis reactivation of phosphorylated actin depolymerizing factor and identification of the regulatory site adf/cofilin: a functional node in cell biology transmissible gastroenteritis virus and porcine epidemic diarrhoea virus infection induces dramatic changes in the tight junctions and microfilaments of polarized ipec-j cells localization of epidermal growth factor-stimulated ras/ raf- interaction to caveolae membrane relationships between egfr signaling-competent and endocytosiscompetent membrane microdomains lipid rafts as a membraneorganizing principle a novel role for phagocytosis-like uptake in herpes simplex virus entry actindependent receptor colocalization required for human immunodeficiency virus entry into host cells vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization expression of rotavirus nsp alters the actin network organization through the actin remodeling protein cofilin hiv- nef interferes with host cell motility by deregulation of cofilin initiation of cofilin activity in response to egf is uncoupled from cofilin phosphorylation and dephosphorylation in carcinoma cells cytoplasmic localization and nuclear transport of cofilin in cultured myotubes cloning and characterization of porcine brain cofilin cdna. cofilin contains the nuclear transport signal sequence dephosphorylation of serine regulates nuclear translocation of cofilin cofilin produces newly polymerized actin filaments that are preferred for dendritic nucleation by the arp / complex. current biology the activity status of cofilin is directly related to invasion, www.impactjournals.com/oncotarget intravasation, and metastasis of mammary tumors egf-induced pip hydrolysis releases and activates cofilin locally in carcinoma cells mammalian rho gtpases: new insights into their functions from in vivo studies her /neu (erbb ) signaling to rac -pak is temporally and spatially modulated by transforming growth factor β rho gtpases modulate entry of ebola virus and vesicular stomatitis virus pseudotyped vectors phosphoinositide- kinase-akt pathway controls cellular entry of ebola virus kaposi's sarcoma-associated herpesvirus induces the phosphatidylinositol -kinase-pkc-ζ-mek-erk signaling pathway in target cells early during infection: implications for infectivity influenza virus propagation is impaired by inhibition of the raf/mek/erk signalling cascade virus activated filopodia promote human papillomavirus type uptake from the extracellular matrix evidence for a putative second receptor for porcine transmissible gastroenteritis virus on the villous enterocytes of newborn pigs cold spring harbor perspectives in biology critical role for lipid raft-associated src kinases in activation of pi k-akt signalling actin in the endocytic pathway: from yeast to mammals complete genomic sequence of the coronavirus transmissible gastroenteritis virus shxb isolated in china general morphology and capsid fine structure of african swine fever virus particles there is no conflict of interest key: cord- -ba dus j authors: he, lei; zhang, yan-ming; dong, ling-juan; cheng, min; wang, jing; tang, qing-hai; wang, gang title: in vitro inhibition of transmissible gastroenteritis coronavirus replication in swine testicular cells by short hairpin rnas targeting the orf gene date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: ba dus j background: transmissible gastroenteritis (tge) is a highly contagious viral disease of swine, characterized by severe vomiting, diarrhea, and high mortality. currently, the vaccines for it are only partially effective and no specific drug is available for treatment of tge virus (tgev) infection. rna interference has been confirmed as a new approach for controlling viral infections. in this study, the inhibitory effect of short hairpin rnas (shrnas) targeting the orf gene of tgev on virus replication was examined. results: four theoretically effective sequences of tgev orf gene were designed and selected for construction of shrna expression plasmids. in the reporter assays, three of four shrna expression plasmids were able to inhibit significantly the expression of orf gene and replication of tgev, as shown by real-time quantitative rt-pcr analysis of viral orf and n genes and detection of virus titers (tcid( )/ml). stable swine testicular (st) cells expressing the shrnas were established. observation of the cytopathic effect and apoptosis, as well as a cell proliferation assay demonstrated that the three shrnas were capable of protecting st cells against tgev destruction, with high specificity and efficiency. conclusions: our results indicated that plasmid-transcribed shrnas targeting the orf gene in the tgev genome effectively inhibited expression of the viral target gene and viral replication in vitro. these findings provide evidence that the shrnas have potential therapeutic application for treatment of tge. transmissible gastroenteritis (tge) is a highly contagious viral disease of swine, characterized by severe vomiting, diarrhea, and high mortality. these syndromes are especially serious in neonatal piglets aged < weeks old, frequently leading to a mortality as high as %; even surviving older pigs generally show growth retardation and low reward to feeding [ ] [ ] [ ] . tge causes enormous economic loss annually worldwide. currently, several vaccines for prevention of tge are available, but their efficacy is variable. both the attenuated and inactivated vaccines are partially effective; attenuated tge virus (tgev) vaccines have the risk of reverting to a virulent form and may even induce an adverse reaction, and the inactivated ones are poorly protective in swine [ , ] . moreover, newborn piglets infected with tgev may die within - days, whereas the vaccines for tgev do not provide effective protection at days after administration [ ] . however, no cure for tge is presently available apart from symptomatic treatment. rna interference (rnai) has been confirmed as a posttranscriptional gene silencing mechanism, which is widely considered to be the major antiviral system in plants and insects [ , ] . the effective inhibition of replication of several rna and dna viruses in animal cells also has been reported by means of rnai [ ] [ ] [ ] [ ] [ ] . therefore, rnai may be developed as a potential therapy for tge. tgev is a member of the genus alphacoronavirus in the family coronaviridae. it is a positive-sense, ssrna virus with a . -kb genome that contains a leader sequence at the end and a poly(a) tail at the end, and encodes four structural proteins [spike (s), envelope (e), membrane (m) and nucleoprotein (n)] and five nonstructural proteins (replicase a, b, a, b and protein ) [ ] [ ] [ ] . the genome itself, together with six sub-genomic mrnas transcribed discontinuously, forms a nested set of rnas of different lengths with co-terminal ends [ ] . orf is located at the end of the genome. it encodes a -amino-acid, . -kda hydrophobic protein, and has been reported to play a role in the process of membrane-associated replication complexes and/or virion assembly [ , ] . recently, more studies have shown that the deletion of tgev orf by reverse genetics may be related to the viral virulent and promotes an intensified dsrna-activated host antiviral response [ ] . compared to other viral proteins' gene (such as proteins s, e, m or n), the orf region is relatively conservative and rnai targeting to orf gene will result in the degradation of both the sub-genomic mrna for orf and the other sub-genomic mrnas [ , ] . besides, data showed that the ' end of tgev genome (orf gene and the downstream gene) could interact with host cell proteins and played a positive role in the replication of tgev [ , ] . according to the characteristics of rnai, the downstream gene will be degraded with the orf gene. then the interaction of ' end of tgev genome and host cell proteins will be interrupted and the viral replication will be reduced. thus, it is helpful to use rnai against it as a new therapeutic option. here, we demonstrate that rnai targeting of the orf gene of tgev, introduced by short hairpin rnas (shrnas), is capable of inhibiting virus replication and protecting swine testicular (st) cells from the destruction induced by tgev, which may be not only a new anti-tgev strategy, but also a new approach to the study of its pathogenesis. relative quantifications of both the orf and n genes were performed by real-time quantitative rt-pcr. comparative threshold (ct) cycle values in three independent experiments were calculated by the ct method, and the average relative amount of orf gene in each sample is represented in figure . the relative amount of orf gene in mock control cells was regarded as . , where the relative amounts of orf gene in cells infected with tgev after being transfected with pgpu -gfp/ , pgpu -gfp/ , pgpu -gfp/ , pgpu -gfp/ and pgpu -gfp/nc were . , . , . , . and . , respectively, and in cells infected with tgev before transfection was . , . , . , . and . , respectively. the sequence-specific shrna pgpu -gfp/ reduced the amount of orf gene by approximately % and %, which was better than with the other three plasmids. real-time quantitative rt-pcr of the n gene ( figure ) for cells infected with tgev after transfection with those shrnas was . , . , . , . and . , respectively. this indicated that these sequence-specific shrnas had approximately %, %, %, % and % reductions in tgev genome (included sub-genomic mrnas, except the shortest one). although the levels of viral genome in the cells infected with tgev before being transfected with shrnas were . , . , . , . and . , corresponding to a %, %, %, % and % reductions in tgev rna. taken together, rnai against orf gene showed a dramatic reduction in the tgev viral. the % tissue culture infective dose (tcid ) assay was performed to examine the effect of sirna on production of viable virus. figure shows that the titers of tgev were . , . , . , . and . tcid /ml in cells infected with tgev after being transfected with pgpu -gfp/ , pgpu -gfp/ , pgpu -gfp/ , pgpu -gfp/ and pgpu -gfp/nc at h post-infection, respectively, and the titers of tgev in cells infected with tgev before being transfected with shrnas were . , . , . , . and . tcid /ml. these data indicated that rnai against orf gene reduced the progeny virus production significantly; pgpu -gfp/ showed a maximum inhibition, whereas pgpu -gfp/ and pgpu -gfp/ showed partial virus replication inhibition, with pgpu -gfp/ being the least effective shrna. based on the results obtained by real-time quantitative rt-pcr and determination of tcid , cells stably expressing pgpu -gfp/ , pgpu -gfp/ , pgpu -gfp/ and pgpu -gfp/nc were selected by using g (pgpu -gfp/ abandoned). green fluorescence protein (gfp) was used as a reporter and cells were screened until % of them showed green fluorescence. as shown in figures and , a similar virus replication inhibition trend was observed in the cell lines in comparison with the transiently transfected cells. the three shrna-expressing cells exhibited potent ability in silencing tgev rnas, whereas cells expressing pgpu -gfp/nc showed a slightly nonspecific effect. to study the protective effect of shrnas against tgev destruction, the mts cell proliferation assay was performed on st cells stably expressing shrna plasmids. as the absorbance is proportional to the number of living cells in culture, it was measured at nm by using an elisa reader at h post-infection (including h incubation with mts). mean od of solutions in st cells stably expressing pgpu -gfp/nc and in control st cells was . and . , respectively, whereas in st cells expressing pgpu -gfp/ , pgpu -gfp/ or pgpu -gfp/ were . , . and . , respectively ( figure ). this showed that st cells expressing shrnas, especially pgpu -gfp/ , were protected from tgev destruction, leading to a significant increase in living cells compared with cells stably expressing pgpu -gfp/nc, or st control cells. to further investigate the effect of shrnas on protecting st cells against tgev-induced destruction, the cytopathic effect (cpe) and apoptosis of cells stably expressing shrnas were examined by fluorescence microscopy. the apoptotic cells were characterized by condensed nuclei, and were colored blue by fluorescent dye hoechst , whereas the cpe in cells that changed from cell fusion to lysis, and thereby formed large bodies, were stained red by propidium iodide (pi). as shown in figure , the negative control pgpu -gfp/nc had no apparent inhibitory effect on tgev-induced cpe and apoptosis, because many cells formed large bodies and showed condensed nuclei. in contrast, cells expressing pgpu -gfp/ had few condensed nuclei, and almost all cells expressing pgpu -gfp/ were capable of maintaining cpe resistance, because there were no apparent pi-stained cells. however, a small area of mild cpe was seen in the cells expressing pgpu -gfp/ and pgpu -gfp/ , indicating that these plasmids were less effective than pgpu -gfp/ at protecting st cells against tgev-induced destruction. although several veterinary coronavirus vaccines are currently available, their efficacy is variable. among them, the infectious bronchitis virus vaccine is very effective for chickens [ ] , whereas the canine and porcine vaccines are only partially effective. furthermore, there is currently no effective antiviral treatment against these virus infections [ ] ; therefore, rapid and potent anti-tgev therapeutic agents are urgently needed. rnai technology provides an important methodology for rational drug design and gene therapy for many viral diseases, which has proven to be a potent tool for host protection against viral infection, suppression of viral genome transcription, and blocking viral replication [ , ] . rnai can be introduced into the cells using two different approaches. the first is chemically synthesized sirnas. cells transfected with chemically synthesized sirnas can achieve rapid and effective silencing of a target gene, but the effects are transient. second, shrnas, which can be cleaved by dicer to produce sirnas in the host cell, can circumvent the disadvantages of chemically synthesized sirnas by using stably transfected plasmids or virus vectors [ ] [ ] [ ] . the present study demonstrated the use of rnai against tgev via shrna-expressing plasmid vector pgpu -gfp, which significantly reduced viral genomic rna replication and protected st cells from tgev destruction, by targeting orf gene. rnai is highly sequence-specific and requires % identity between the target and targeting sequences to achieve virus clearance from cell culture [ ] [ ] [ ] . the application of shrnas targeting the conserved region of the gene is one way to overcome the lack of knowledge of the target sequence. to ensure that shrnas can be used for a wide range of virus strains, we evaluated the cross-inhibitory capabilities of these shrnas by conducting multiple alignments of tgev orf gene sequences, based on the available sequences in genbank. our results showed that pgpu -gfp/ , pgpu -gfp/ and and pgpu -gfp/ could cover % ( / ), % ( / ) and % ( / ) of tgev strains, respectively (data not shown). interestingly, we have also conducted multiple alignments of the sequences among tgev, feline coronavirus virus and canine coronavirus, and have shown that they are also conservative. this conservative characteristic of the sequences between the three viruses means that they have greater potential application for the treatment of the diseases caused by these viruses, which also indicates that further studies are necessary to search for the cross-inhibitory effects in a range of tgev strains and other coronaviruses. in our study, two strategies were used to detect the inhibitory effect of shrnas on both the orf gene and tgev genome. the first approach was that cells were infected with tgev after being transfected with shrnas, in which the shrnas were used as preventive substances. as shown in figures and , there was a marked decrease in both the orf and n genes, which could be amplified simultaneously from all the same regions within the tgev genome rna and sub-genomic mrnas (except the shortest one). the reduction showed that the viral genome rna and its transcripts were decreased significantly. a similar suppression pattern was observed for virus titers. in the second strategy, shrnas of orf gene were used as therapeutic agents and transfected after the cells were infected with tgev. almost all the cells maintained their normal morphology before they were collected for real-time rt-pcr. the orf and n genes were reduced significantly when the shrnas were transfected at h after tgev infection (figures and ) . more experiments were carried out at and h after tgev infection, but the results were not as remarkable as those after h (data not shown), which indicates that shrnas affect the initial stage of tgev infection. in the study by ortego et al., recombinant tgev with orf gene deletion replicated in cell culture with similar efficiency to the wild-type virus, and stably maintained the modifications introduced into the genome [ ] . this observation seems to contrast with the results of the present study, which provides evidence that rnai of the orf gene could lead to decreased virus replication. there are three possible reasons for this. first, the genome of parental tgev could be degraded directly because it consists of a positive-sense ssrna. second, all the other sub-genomic mrnas could be degraded as the orf gene is included in all the sub-genomic mrnas. as shown in figures and , n gene was significant decreased in the shrna expressing cells. besides, the end of the tgev genome gene, the downstream gene of orf gene, could be degraded with the orf gene according to the characteristics of rnai, and its degradation could delay the viral replication, because it has been reported that it could interact with host cell proteins and have a great influence on the replication of tgev [ ] . in the study by cruz et al., it was reported that tgev without orf enhanced virulence in infected piglets [ ] . we speculate that this phenomenon won't happen on the rnai treated pigs, because the rnai method is different from the reverse genetics technique. by the rnai used in this study, the other viral sub-genomic mrnas were degraded (figure and ) . while the reverse genetics deletion will only affect the orf gene itself. nevertheless, whether the technology could be used in vivo still needs more explored. to elucidate the protection of shrnas against tgev in more detail, st cells stably expressing the shrnas were established. tgev replication in these cells was observed through detection of virus titers and real-time quantitative rt-pcr. cells expressing pgpu -gfp/ showed % inhibition in the viral genome, whereas pgpu -gfp/ showed up to % and pgpu -gfp/ % inhibition (figure ) . tgev destroys st cells in two ways, by apoptosis and necrosis [ , ] . therefore, cpe and apoptosis were analyzed. examination of tgev-induced cpe indicated that st cells were very sensitive to tgev infection. cells expressing pgpu -gfp/nc formed large bodies and were stained red by pi (figure ) , whereas cells expressing pgpu -gfp/ were not stained red, indicating that the cell membrane was intact. compared with the condensed, irregular nuclei of pgpu -gfp/nc-expressing cells, cells expressing pgpu -gfp/ were completely protected from apoptosis and the nuclei showed normal morphology. the sequence-specific shrnas, especially pgpu -gfp/ , exhibited potent ability to protect st cells from tgev-induced destruction. our present results indicate a close relationship between inhibition of tgev replication and cell viability by shrnas. our data also suggest that sirna targeting orf gene can elicit viral rna from infected cells and potentially offers an efficient therapeutic option for tgev infection. taken together, our results indicate that plasmidtranscribed shrnas targeting the orf gene in the tgev genome can inhibit expression of viral target genes and viral replication in st cells. this method merits further investigation in animal studies to define its therapeutic potential, and whether the technology could be used in vivo for anti-tgev therapy is still under investigation. st cells were cultured in high-glucose dulbecco's modified eagle's medium (dmem; gibco, uk) containing % heat-inactivated fetal calf serum (hyclone, china) and antibiotics ( μg/ml streptomycin and u/ml penicillin); the culture medium was replaced every days. tgev h strain was obtained from the national control institute of veterinary bioproducts and pharmaceuticals (beijing, china) and propagated in st cells. sequences from the orf gene of tgev h strain (genbank accession no: fj ) were designed based on the website sirna designing tools (http://www. ambion.com/techlib/misc/sirna_finder.html and https:// rnaidesigner.invitrogen.com/rnaiexpress/). the sequences were analyzed by blast to ensure that they did not have significant nucleotide sequence homology with the swine genome, but shared % identity with the other published sequences of tgev strains, from which four theoretically effective sequences at nucleotide positions - (orf - ), - (orf - ), - (orf - ) and - (orf - ) were selected. a nonspecific sequence (nc) was also scanned by blast analysis and served as a negative control. these five sequences are listed in table . all the sequences were arranged in the following alignment: bbsІ + sense + loop + antisense + termination signal + bamhІ and cloned into the pgpu -gfp vector to make shrna expressing plasmids: pgpu -gfp/ , pgpu -gfp/ , pgpu -gfp/ , pgpu -gfp/ and pgpu -gfp/nc. twenty-four hours before being transfected, st cells were seeded into six-well dishes in high-glucose dmem + % fetal bovine serum (fbs) without antibiotics. when they reached - % confluence, cells were transfected with μg/well pgpu -gfp/ , pgpu -gfp/ , pgpu -gfp/ , pgpu -gfp/ and pgpu -gfp/nc by lipofectamine (invitrogen, carlsbad, ca, usa) according to the manufacturer's recommendations. after being incubated at °c for h, the transfection complex was removed and the medium was replaced by high-glucose dmem with % fbs. the cells were inoculated with tgev at tcid h later. the plasmids contained the gfp gene sequence. therefore, gfp was expressed allowing a good discrimination between transfected and non-transfected cells. the transfection efficiency was determined by monitoring the percentage of gfp-expressing cells within the live cell population, and it showed that nearly % of the transfected cells were positive. cpe was evaluated and the cell images were captured under an inverted fluorescence/phase-contrast microscopy (nikon, japan) at different time points post-infection. the cell cultures were collected for a real-time quantitative rt-pcr analysis at h post-infection. to assess the influence of shrnas on tgev replication, the n gene of tgev was used as a standard for the tgev genome. another pair of primers was synthesized for quantification of the tgev genome in real-time rt-pcr: forward, -ggaagatggcgaccagatag- and reverse, -ccacttctgatggacgagca- . total rna of culture supernatants was isolated as described above, and a real-time quantitative rt-pcr was conducted using a sybr exscript™ rt-pcr kit (takara bio), according to the manufacturer's instructions. real-time rt-pcr was performed and the procedure was similar to that described above, expect that the reaction temperature for annealing was °c. data were analyzed according to the ct method, and the tests were performed in triplicate. tgev cultures in sirna-transfected cells were collected h after viral infection. after three freeze-thaw cycles, the cultures were serially diluted -fold from - to - , and added to st cells at - % confluence in -well plates. each dilution was added to four wells. after days of infection, the tcid was calculated by the reed-muench method. the pgpu -gfp vector carries the neomycin resistance gene; therefore, st cells stably expressing shrnas were selected using g . gfp in the plasmids was used as a reporter during the selection efficiency analysis. the st cells were seeded into six-well plates h before being transfected (up to - % confluence). cells were transfected with pgpu -gfp/ , pgpu -gfp/ , pgpu -gfp/ and pgpu -gfp/nc by lipofectamine as described before, and propagated in selection medium containing μg/ml g until % of the surviving cells stably expressed gfp. st cells stably expressing pgpu -gfp/ , pgpu -gfp/ , pgpu -gfp/ and pgpu -gfp/nc were seeded in -well plates at - % confluence and challenged with tgev at tcid . cell viability was assessed by adding μl/well of mts (promega, usa) to cell cultures, according to the manufacturer's instructions, at h after viral infection. after incubation with mts for h, light absorbance of each well was measured at nm. each measurement was performed six times, and the experiment was repeated three times. st cells stably expressing shrnas were seeded in sixwell tissue culture plates at - % confluence and challenged with tgev at tcid . thirty hours after infection, the cells were washed with hank's balanced salt solution (hbss) and incubated with hoechst ( ng/ml) at °c for min, and then washed three times with hbss. cell were then incubated with pi ( ng/ml) at °c for min and washed with dmem without serum. images were viewed by fluorescence microscopy (nikon, japan). transmissible gastroenteritis of pigs genetic basis for the pathogenesis of transmissible gastroenteritis virus binding of transmissible gastroenteritis coronavirus to brush border membrane sialoglycoproteins mucosal immunisation and adjuvants: a brief overview of recent advances and challenges increased litter survival rates, reduced clinical illness and better lactogenic immunity against tgev in gilts that were primed as 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vaccination against challenge with nephropathogenic infectious bronchitis virus pa/wolgemuth/ safety and efficacy of a modified-live canine coronavirus vaccine in dogs simultaneously inhibition of hiv and hbv replication through a dual small interfering rna expression system in vitro inhibition of csfv replication by retroviral vector-mediated rna interference a transgenic approach for rna interferencebased genetic screening in mice a system for stable expression of short interfering rnas in mammalian cells a large-scale rnai screen in human cells identifies new components of the p pathway short interfering rna confers intracellular antiviral immunity in human cells targeting marek's disease virus by rna interference delivered from a herpesvirus vaccine clearance of replicating hepatitis c virus replicon rnas in cell culture by small interfering rnas transmissible gastroenteritis virus induces apoptosis in swine testicular cell lines but not in intestinal enterocytes the viral nucleocapsid protein of transmissible gastroenteritis coronavirus (tgev) is cleaved by caspase- and − during tgev-induced apoptosis the authors declare that they have no competing interests. lei he took part in all the experiments, and wrote the manuscript. yan-ming zhang designed all the experiments. ling-juan dong and min cheng participated in plasmid construction, cell transfection and confocal microscopy. jing wang made a major contribution to the mts assay. gang wang and qing-hai tang carried out the cell culture and real-time rt-pcr. all authors read and approved the final manuscript. key: cord- -qxkxjxsz authors: pensaert, maurice b.; martelli, paolo title: porcine epidemic diarrhea: a retrospect from europe and matters of debate date: - - journal: virus research doi: . /j.virusres. . . sha: doc_id: cord_uid: qxkxjxsz abstract a retrospect is given on the emergence of porcine epidemic diarrhea (ped) during the early seventies in europe. while, at first, it appeared as a disease affecting feeder pigs, fattening- and adult swine, it later also became pathogenic for neonatal and suckling pigs hereby drastically increasing its economic impact. isolation of the causative virus revealed a new porcine coronavirus, the origin of which has never been clarified. pathogenesis studies with the prototype strain cv showed severe villous atrophy in neonatal pigs and the virus-animal interactions showed many similarities with transmissible gastro-enteritis virus (tgev), another porcine coronavirus. disease patterns in field outbreaks showed muchvariation but, while farm related factors played a role, possible genetic variations of virus strains in europe have not been examined and are thus unknown. cv in experimental pigs caused diarrheal disease and mortality rates similar to those later encountered in asia and more recently with the “original” us strains even though genomic typing of the prototype european strain have shown that it belongs to the s-indel strains. in europe, ped has become endemic during the eighties and nineties and subsequently regressed so that, after , swine populations in many countries have largely become seronegative. sporadic outbreaks have recently reappeared showing a large variety of clinical outcomes. one" or also "tge " and both these denominations referred to its clinical similarity to tge, a common cause of viral diarrhea in pigs in europe at that time. an important difference with tge was, however, that neonatal pigs were not affected. since these denominations were considered unsatisfactory from a scientific point of view, the name of the new syndrome was quickly changed to "epidemic diarrhea-ed". the first ed outbreak occurred on a farm in the spring of and the second months later, at a distance of miles from the http://dx.doi.org/ . /j.virusres. . . - /© elsevier b.v. all rights reserved. former. suckling piglets were not affected but pigs of weeks and older and also adults showed an acute diarrhea lasting one week. the outbreak lasted - weeks on the farm. during the autumn of and also the following winter, several new outbreaks were reported. a clinical diagnosis and possibility for differentiation from tge was thus based on the high morbidity in fatteners and adult animals in the absence of disease in neonatal and freshly weaned pigs. in most of these diarrheal cases, tgev was excluded by laboratory examination. the fact that ed was observed on farms with a recent history of a tge outbreak, and thus in tgev immune animals, increased the conviction that tgev was not involved. during , ed spread rapidly between pig farms, particulary fattening herds. mortality was rare and the effect of an outbreak was estimated at about weeks feed cost. a similar disease pattern was observed during the early seventies in belgium and a rapid spread occurred to neighbouring countries in western europe. here also, suckling pigs were not affected and remained free of diarrhea even when their mothers suffered from a watery diarrhea during several days. some neonatal pig mortality could occur by starvation because the sick mothers often suffered from agalactia. an additional sign observed in belgium and later also reported in gemany, but not mentioned in england, was that some fatteners were found dead, particularly towards the end of the fattening period, and this occurred repeatedly in some farms but not in others. a mortality rate as high as % could be encountered. this was not due to dehydration accompanying the diarrhea but animals suddenly died from acute back muscle necrosis. while the clinical link with an ed infection was clear, the pathogenetic background has never been revealed. belgian pigs were highly stress positive at that time and a severe belly ache often observed in adult animals during an outbreak of ed may have been a trigger. in general, as no baby pig mortality occurred due to the ed agent, not very much attention was given to this new diarrheal syndrome and the etiology was not intensively investigated. however, it was assumed that a viral agent was involved since bacteriological examination of faecal material did not reveal a specific bacterial cause. none of the known porcine viruses could be associated and a new virus was, therefore, suspected. much changed in when wood ( ) from the veterinary investigation centre in norwich (england) described a new diarrheic syndrome. it differed from ed in that it now affected pigs of all ages, including neonatal and suckling pigs. mortality was variable, restricted to young piglets and averaged around %. this new disease now resembled tge more closely than ed did, but tgev was again excluded using direct immunofluorescence on intestines and applying established serological techniques available for detection of anti-tgev antibodies. now, a differentiation with tge on a clinical basis only became difficult, often impossible. this new syndrome was called ed to differentiate it from the ed where no baby pigs were involved. ed was economically much more important than ed . ed also quickly spread to the european continent and was recognised in belgium in . reports from other countries including the netherlands, germany, france, bulgaria, hungary and switzerland, followed soon. in belgium, mortality rates in neonates on breeding farms varied considerably. they could be as high as % (large variation from to %) and the average was %. variation in mortality in neonatal pigs was litter bound, not explained at that time, but also farm bound where it appeared to be associated with farm size (still many small family farms at that time), farm struc-ture (one or several farrowing units), number of neonatal pig litters present at the start of the outbreak, number of pregnant sows due to farrow within one week of the appearance of disease signs and possibly other factors. differences in virulence of virus isolates was not given any attention. this new evolution to ed with the involvement of baby pigs and its larger economic impact yielded better opportunities for collecting material for etiological studies, for experimental reproduction of the disease and for the development of virological and serological techniques. in , chasey and cartwright ( ) reported the detection of virus like particles, and pensaert and debouck ( ) described the isolation of a new coronavirus-like agent (cvla) from diarrheic pigs, with both research groups succeeding in reproducing diarrhea in experimental pigs. soon after the isolation of this new coronavirus, extensive pathogenesis studies were performed in colostrum deprived pigs with one of the belgian isolates, designated coronavirus cv , (isolated in month of ) which became the prototype strain for pedv in europe (debouck and pensaert, ; debouck et al., ) . ed was soon named "porcine epidemic diarrhea" (ped) caused by ped virus (pedv) a denomination which still stands at present. from the early studies with pedv in neonates (debouck and pensaert, ; debouck et al., ) it was soon clear that the pathogenesis resembled very much that of tgev. experience gathered from research with tgev helped much in the approach to study this new enteric disease. lack of success to cultivate the virus in cell cultures forced to produce clean virus stocks by oral inoculation of colostrum deprived pigs, performing surgery h later and rinsing the in vivo produced virus from the lumen of the infected small intestines during h while keeping the pig in the incubator (debouck and pensaert, ) . such "clean" pig adapted virus stocks served for experimental pig inoculation experiments and to produce an hyperimmune serum for the preparation of a conjugate for an immunofluorescence (if) conjugate to detect the virus in tissues. serological tests were developed to detect antibodies by elisa and to study possible relationship with other coronaviruses by immuno-electron microscopy . genome analysis of the ped isolate(s) was not available at that time. by immuno-electron microscopy and if, pedv was not related to any of the known porcine coronaviruses (tgev, haemagglutinating encephalo-myelitis virus) . some discrete relationship with members of the genus alpha-coronavirus was later demonstrated using other and more sensitive tests. the origin of pedv was thus unknown and no potential parent coronavirus could be indicated. an elisa test was soon used for routine serology . a crucial question was whether or not the ed agent and ed /pedv were related or whether ed /pedv was totally new. infectious material containing the ed agent from earlier outbreaks was not available. a retrospective serological survey was carried out on sow sera that had been collected in slaughterhouses in belgium starting in and thus prior to the emergence of ed in on the european continent. antibodies to pedv were not found in sera collected in but were present in % of the sows collected in , in % of the sows in and in % of the sows in . these results indicated that the coronavirus ped had been responsible for first ed outbreaks, for the ed outbreaks and thus it can be accepted that pedv emerged in but later widened its host tropism from growing and adult swine towards neonatal pigs. this finding was interesting from an evolutionary point of view. thus, pedv that presumably started as a cause of diarrhea in in feeders, fatteners and adult swine, had suddenly acquired tropism for neonatal pigs and now became a rather devastating disease. but even after the emergence of ed /pedv, some outbreaks on breeding-finishing farms still did not involve neonatal pigs. while it was assumed that both ed and pedv were co-circulating in the swine population, it is also possible that some farms had experienced an earlier ed infection and that immune sows protected their offspring against pedv by lactogenic immunity while groups of fattening pigs had become susceptible. it must, however, be mentioned that cross-protection between ed and pedv has never been studied. also, after a first epidemic phase of the new pedv, the virus often persisted on breeding-finishing farms in weaned and feeder pigs (endemic ped). the sow population was immune, protecting its offspring, while feeder pigs became susceptible after loosing their maternal protection. the highly variable and mixed clinical picture was, at that time, ascribed to the possible co-circulation of the original ed agent and its presumed variant pedv in the population. genome analysis was not available and ed infectious material is also now no longer available to retrospectively examine this issue. ed /pedv is likely a variant of ed . that variants of pedv relatively easy emerge is not unusual as animal coronaviruses are known to easily undergo genetic alterations. recombination and insertions and deletions have repeatedly been demonstrated in pedv by genome analyses of isolates during more recent outbreaks in asia and in the usa (fan et al., ; li et al., ; jarvis et al., ; vlasova et al., ; oka et al., ) . even now ( ) in recent cases of ped in europe, varying types of clinical manifestations, either with or without affection of neonatal pigs, are observed (see later). the question on the origin of pedv (and thus of its presumed ancestor ed agent) in is unanswered. there are no indications for a possible evolution from another known so called "parental" coronavirus, even after comparative studies with the known coronaviruses using detailed genome analyses of different genes including the s gene. so far, only discrete antigenic relationship involving the n protein but without any cross protection was detected with some of the other animal members of the genus alphacoronavirus such as feline infectious peritonitis virus (fipv), tgev, porcine respiratory coronavirus (prcv), canine coronavirus (ccov) and mink coronavirus (mcv). by the use of monoclonal antibodies to the n proteins of the human alphacoronaviruses nl and e, no cross reactivity was detected with pedv (sastre et al., ) . the only alphacoronavirus in which also the m proteins cross reacts with pedv is mcv (have et al., ) . the nucleotide sequence of the pedv nucleocapsid gene and of typical coronavirus motifs show that pedv, within the region of the genome sequenced, shows indeed greatest homology to the human e, tgev, prcv, fipv, ccov and feline enteric coronavirus (fecv) (bridgen et al., ) . it is interesting to mention that, similar to pedv, several of the other alphacoronaviruses, including the human e, tgev, prcv, ccov, fecv and fipv use the cellular receptor aminopeptidase n (apn) for virus entry into cells in their host (weiss and navas-martin., ) and this seems to be a common evolutionary characteristic. still, the genetic and antigenic diversity between pedv and the other alphacoronaviruses is very largealso, no cross-reactivity has been reported between pedv and the coronaviruses belonging to the beta, gamma or delta genera.the genomic data presented above and the use of the same cellular receptor suggest a common origin of some of some of these alphacoronaviruses. a carrier-wild animal species as source of the virus, as often described with other coronaviruses, cannot be excluded. soon after its detection, experimental studies in neonatal pigs revealed that target cells of pedv were limited to the epithelium covering the intestinal villi and the pathogenesis was thus highly similar to that of tgev (debouck and pensaert, ; debouck et al., ) . cv virus infection in the villous enterocytes in neonatal pigs caused rapid cell desquamation throughout the small intestine within - h after inoculation which was somewhat slower than observed with tgev. still, the villous atrophy induced by pedv was so abrupt and extensive that rapid and severe dehydration occurred leading to death in neonatal pigs. due to this similarity in pathogenesis with tgev, much of the scientific knowledge acquired on tge diagnosis, −immunity, −prevention could be almost invariably applied to ped. an apparent difference with tgev, probably of minor importance from a clinical point of view, was that epithelial cells on colonic villi were also infected but desquamation was not observed. still now, it is a question if this colonic site of replication contributes to disease severity. ped diarrhea in fatteners and sows is often accompanied by an apparent belly ache, a clinical sign not seen with tge, and the question arises if the colon infection may contribute to this clinical manifestation. results of pathogenesis studies obtained in caesarean derived, colostrum deprived neonatal pigs upon inoculation with the european prototype strain cv of the seventies, were practically identical to those observed more recently in asia and in the usa epidemics with the so-called "original us pedv strains" stevenson et al., ; kim and chae, ) . a point of debate in the pedv evolution, particularly since its occurrence in asia and its emergence the usa, is the arising of pedv genetic variants influencing virulence. the history in europe, here presented, allows to assume that ed /pedv was a variant of ed which had acquired tropism for intestinal enterocytes in neonatal pigs. this new tropism expanded and increased the virus virulence since a vulnerable age became affected and piglets mortality became an important economic aspect of the disease. currently, two major pedv variants are described in the usa upon routine genome analyses of usa isolates. the first, also called "original us pedv ", appears to be "highly virulent" while the second, the so called s-indel strains, standing for insertions and deletions in the s gene of the virus, are associated with mild(er) clinical outbreaks. similar genotypic variants have been detected in asia, the s-indels already before and the highly virulent since . when adopting this genomic identification, it appears that cv is classified as a s-indel isolate apparently belonging to a different cluster compared to the us indels (carvajal et al., ) . considering the pathogenesis and virulence of the european prototype strain cv of the seventies as evaluated by the sites and degree of replication and the degree of villous atrophy, no real difference exists with the more recent highly virulent (original us pedv) isolates from the usa. for example, the pig adapted c when experimentally inoculated in neonatal piglets, caused villous atrophy with villous length reduction from the normal value of - m to as low as - m throughout the small intestine and within - h after the start of the diarrhea coussement et al., ) . much depends on how virulence of pedv is determined. if virulence of a pedv isolate is considered merely from the point of view of virus-neonatal pig interaction with parameters such as duration of incubation period, rapidity and severity of enterocyte desquamation, degree and extent of atrophy of villi, production of virulent virus quantities and severity of diarrhea, then cv can be classified as highly virulent despite its identification with s-indel isolates. that s-indels isolates do not systematically mean low virulence was recently shown in a us study (chun-ming et al., ) in which litters of - days old suckling pigs were inoculated with the s-indel iowa strain in the presence of their ped negative mothers. the severity of clinical signs and the mortality of the pigs varied between the liters (from % to %). severe clinical signs were observed in of the litters. two of the sows developed diarrhea. it was observed that, despite similar background of sows and environment in this experiment, the severity of disease was rather variable. it appeared that the pigs' body weight at birth and the sows health conditions and lactation were influential factors. in the same experiment mentioned above (chun-ming et al., ) , one litter was also inoculated with an original us pedv strain of high virulence. it was concluded that virulence of the s-indel isolate was generally lower based on the longer incubation period, the shorter duration of diarrhea, more limited regions of virus infection, overall lower pig mortality ( % vs % for "original") and some other additional parameters. the sites and extent of the deletions or insertions and the seqence differences in the s gene may play an important role. in a recent publication (chen et al., ) the pathogenicity differences between u.s.pedv prototype strains and a s-indel-variant stain were compared in conventional neonatal pigs under experimental infections and enteric disease, as evidenced by clinical signs,fecal virus shedding, gross and histopathological intestinal lesions, were significantly lower for the s-indel strain.however,the molecular basis for the virulence differences were not elucidated. since the early beginning in europe, it was clear that ped disease can show much variability even in different litters of pigs particularly when suckling their mother. such differences and the high variation in pig mortality in different litters (from to %) was an observation also made in the seventies in europe when the first epidemic occurred and the reason was never unravelled. even more variability is experienced when virulence and severity of ped disease is related to the interaction virus-farm population and thus in field outbreaks. the result of a ped outbreak will be much more difficult to predict, to evaluate and to define since, next to possible virulence differences of the isolates and next to variation among litters in suckling pigs, many additional factors play a role in determining the clinical outcome of the infection. they include immune status of sows, dose of virus exposure on the farm, herd size and pig farming management and others, all of which may be interacting in a different way. moreover, the procedures applied for intentional infection (feed back) of the sow population to speed up the induction of immunity to be passively transfered to the litter could be considered as a potential cause of worsening of the clinical status of the suckling pigs. in fact, that practice can also be a source of other pathogens for gilts/sows and/or for newborn piglets. it is thus possible that, particularly in a fully susceptible pig population and even with pedv strains of similar virulence, the mortality rates and losses are much higher in some continents or regions or farms with extensive and highly industrialised pig farming. the overall health status of the population apparently also plays an important role. while genomic changes surely will occur in ped virus isolates, it is advised to be careful when associating them with virulence changes. when a different clinical picture is observed on farms, it is often too hastily concluded that variants with varying virulence have arisen based on genome analysis only and without testing for virulence factors in experimental pigs. while genome analysis is certainly useful and may be directional, repeated comparative animal inoculation experiments with so called new isolates, clinically denominated as candidate "virulence-variants", need to be carried out in a standardized way before solid conclusions about virulence are made. this is indicated by the large variations very often observed with one and the same isolate. the neonatal, non suckling pig, preferably colostrum deprived, is reliable and even essential for this purpose. parameters as duration of incubation period, a timewise follow up of site and degree of villous affection in the small intestine are needed and must be repeated before calling a pedv isolate a variant with impact on virulence. it should be stressed that pig adapted virus strains should be used as it is known that major genomic modifications can arise when pedv is cell culture passaged, as well in porcine as in non-porcine cells, such as in vero cells. the epidemiology of ped in europe has been and still is quite puzzling. pedv outbreaks in the late seventies and early eighties occurred both on breeding and fattening swine farms. acute outbreaks with neonatal pig mortality were encountered in the breeding-fattening farms which became infected for the first time. pedv often became endemic. in farrowing-finishing farms, successive groups of pigs became infected upon weaning and after losing their lactogenic protection from their immune mothers, so that the virus could persist. whether the virus persisted or not after the original outbreak was somewhat unpredictable, as it could also disappear from the farm. the farm size (number of sows) and its structure (number of units) played a role. also ped persistence regularly occurred in fattening farms using the system of continuous introduction of feeder pigs originating from numerous and different breeders. a typical case of persistent diarrhea caused by pedv lasting months on a breeding-finishing farm was described in the netherlands (pijpers et al., ) and this was a feature regularly observed in europe in the eighties. recent experience in the usa ( ) ( ) ( ) , has shown that management practices adopted in the epidemic phase of the infection can turn ped to a endemic/enzootic and long lasting form (jung and saif, ) . pedv infections were a regular cause associated with viral diarrheal picture in weaned and feeder pigs. in a serological study in belgium in , pedv was associated with diarrhea in out of groups of feeder pigs after arrival in fattening farms (callebaut et al., ) but, the virus remained prevalent in the swine populations of western europe during the eighties. a serosurvey in belgian sows using sera collected in slaughterhouses, and thus mostly originating from different farms, showed pedv antibodies % in and % in . similar percentages of sows were positive in germany (on regional locations), france, spain, the netherlands and bulgaria while no antibodies were found in scandinavia, northern ireland, usa or australia . in , antibodies were detected in sera received from taiwan (the first evidence of the presence of pedv in asia). as the eighties advanced, fewer outbreaks on breeding farms were seen even though the virus was still detected but the general economic impact of ped had become lower. in belgium in , groups of feeder pigs from commercial finishing herds, using the all in-all out production system, were examined for serocoversion to pedv and tgev. none of the groups seroconverted to tgev while seroconverted to pedv with diarrhea observed in all (van reeth and pensaert, ) . in an hungarian study published in , . % of faecal samples from weaned pigs with diarrhea tested positively for pedv (nagy et al., ) . during the nineties, an acute ped outbreak which was described in spain involving a fattening unit of pigs with diarrhea starting in - weeks old pigs in one barn affecting pigs from to kg and subsequently spreading to the other barns (carvajal et al., ) . an isolated outbreak was described, in in england, in a large finishing herd where weaners were brought in over a month period and positive sows were found in the breeding herds supply-ing the weaners (pritchard et al., ) . but, no further epidemic of ped occurred despite a very low pedv seroprevalence as only . % of fatteners from different finishing units were positive for antibodies to pedv(may -january . interest from a disease and economic point of view became very low in europe and no further research was performed on ped. practically no serosurveys were carried out. a serological survey in sows from farrow to finish herds carried out in belgium in revealed that gilts were positive for pedv antibodies in only of the considered farms, and in , fattening farms were examined for pev antibodies and none were positive (pensaert, unpublished) . it appeared that pedv, except for a focal case, was disappearing from the european swine population towards the turn of the century. for this reason, no attention or follow up was given anymore to this viral infection while its field of interest had fully moved to asia. however, a somewhat atypical ped outbreak occurred unexpectedly in the po valley in northern italy in , (martelli et al., ) . it occurred between may and june in an area densely populated with pigs. the outbreak started with four cases occurring in fattening farms from may to july. no clinical cases were detected during august and september. in october, two new cases appeared: the first in a fattening unit and the second in the nursery of a three-site production unit. the disease spread during the winter of - , affecting more than farms including fattening units as well as farrow-to-finish or farrow-to-weaner farms. some pedv positive farms were still detected between mid- and the end of , but the disease progressively disappeared (sozzi et al., ) . from - , only sporadic outbreaks were observed in grower and finisher herds (efsa, ) . this epidemic in italy in - inclined us to forecast a new episode of ped epidemics in europe but it did not occur. recently and due to an increased attention following the epidemic in the usa, single or limited ped outbreaks have sporadically been diagnosed in europe. one case in ukraine, (dastjerdi et al., ) occurred in a -sow farm ( farrowings a week) and mortality in pigs less than days old approached %. the virus was closely related to "original us" strains reported form north america (sequence identity of . %). isolates from other cases reported from belgium (theuns et al., ) , holland (van der wolf et al., ) , france (grasland et al., ) , germany hanke et al., ) and portugal (mesquita et al., ) and italy were, on the basis of genetic sequence, closely related to each other. when sequenced, they were classified as s-indel strains, and the german isolate showed . % identity to the oh strain isolated in the usa in january . the size and clinical disease in these outbreaks were very variable. the outbreak involved in belgium: fattening farm (no mortality), in france: farrow to finish farm (mortality % in pigs at one week and % at weaning), in germany: farms ( fattening with . % and % mortality, breeding with % and no mortality, respectively) and in portugal where it started in one farm (with pig mortality, but not further defined) and where the virus spread to other pig farms during a period of months. from these data, it can be seen that, again, there was much and unexplained variation in ped clinical disease and outcome. except for the possibility of the outbreak in ukraine, it is very doubtful that the other european isolates have anything to do with those involved in the us epidemic. similar focal cases must have occurred in europe before the us outbreak but were, most likely, neither recognized nor diagnosed nor reported as ped. s-indel strains have been present in europe as cv appears to be the earliest known representative (carvajal et al., ) . it is remarkable that, in many european countries, no large epidemic occurred despite several indications that the breeding population in their densely populated swine raising regions is negative for antibodies to pedv and thus presumably fully susceptible. it is difficult to understand why a virus such as pedv has gradually regressed in the swine population in europe in the absence of any special control measures. vaccination has not been applied and no control programmes have been installed. the puzzling aspect is that, during the last decennia, pedv was and still is focally present in europe and did not cause an epidemic despite the high numbers of susceptible-seronegative farms and despite the very dense swine populations in some regions. one would expect that a virus such as pedv, which replicates to very high titers in swine and which can easily and rapidly spread from one swine farm to another, would be able to maintain itself in the swine population. as previously explained, once an outbreak has occurred on a breeding farm, pedv virus could persist easily when successive litters of pigs, after losing their lactogenic protection at weaning, become a susceptible target for infection. in fact, persistence for a virus such as pedv would be almost as a "natural" feature similar to the endemic character observed with other porcine enzootic enteric viruses such as swine rotaviruses, swine enteroviruses and others. tgev cannot serve as an example here because, in europe, it has largely been eliminated from the swine population due to the emergence, in the early eighties, of the closely related porcine respiratory coronavirus (prcv). prcv is a tgev deletion mutant which has acquired respiratory tropism and shows an epidemiological advantage of rapid aerogenic spreading while causing a protective immunity to tgev. endemic prcv has thus "replaced" tgev in europe. it would be interesting to study the mechanisms behind the regression/waning of pedv in europe. could it be that the virus has a non swine ancestor which has temporarily become adapted to swine but which is not really swine-borne? such evolution would not be unique for animal coronaviruses. could it be that the virus can maintain itself in the population only when present at a sufficient high dose allowing it to continue the infection chain but once reaching a low level quantity,e.g on a farm basis, is not longer able to do so? the waning of pedv has apparently not occurred in parts of asia within its - decennia of presence on that continent to the same degree as it did in europe, and it will be intriguing to closely follow the epidemiological course and evolution of pedv in the usa, once the epidemic phase has passed. surveillance and control of ped coronavirus in pig in italy seqence determination of the nucleocapsid protein gene of the porceni epidemic diarhoea viris confirms that this virusis a coronavirus relatyed to human coronavirus e and porcine transmissible gastroenteritis virus enzyme-linked immunosorbent assay for the detection of the coronaviruslike agent and its antibodies in pigs with porcine epidemic diarrhea 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coronavirus-like agent cv experimental infection of pigs with a new porcine enteric coronavirus cv scientific opinion on porcine epidemic diarrhea and emerging deltacoronavirus complete genome sequence of a novel porcine epidemic diarrhea virus in south china complete genome sequence of a porcine epidemic diarrhea s gene indel strain isolated in france comparison of porcine epidemic diarrhoea viruses from germany and the united states serological evidence of infection with a coronavirus related to transmissible gastroenteritis virus and porcine epidemic diarrhea virus genomic and evolutionary inferences between american and global strains of porcine epidemic diarrhea virus pathology of us porcine epidemic diarrhea virus strain pc a in gnotobiotic pigs porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis experimental infection of piglets with a korean strain of porcine epidemic diarrhoea virus genome sequencing and analysis of a 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differentiation betyween human coronaviruses nl and e using a novel double-antibody sandwich enzyme-linked immunosorbent assay based on specific monoclonal antibodies emergence of porcine epidemic diarrhea virus in southern germany emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences complete genome sequence of a porcine epidemic diarrhea virus from a novel outbreak in belgium first case of porcine epidemic diarrhea (ped) caused by a new variant of ped virus in the netherlands prevalence of infections with enzootic respiratory and enteric viruses in feeder pigs entering fattening units distinct characteristics and complex evolution of pedv strains, north america coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. microbiol an apparent new syndrome of porcine epidemic diarrhea key: cord- - o upns authors: pascual-iglesias, alejandro; sanchez, carlos m.; penzes, zoltan; sola, isabel; enjuanes, luis; zuñiga, sonia title: recombinant chimeric transmissible gastroenteritis virus (tgev)—porcine epidemic diarrhea virus (pedv) virus provides protection against virulent pedv date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: o upns porcine epidemic diarrhea virus (pedv) is an enteric coronavirus causing high morbidity and mortality in porcine herds worldwide. although both inactivated and live attenuated vaccines have been extensively used, the emergence of highly virulent strains and the recurrent outbreaks even in vaccinated farms highlight the need of effective vaccines. engineering of genetically defined live attenuated vaccines is a rational approach for novel vaccine development. in this line, we engineered an attenuated virus based on the transmissible gastroenteritis virus (tgev) genome, expressing a chimeric spike protein from a virulent united states (us) pedv strain. this virus (rtgev-rs-spedv) was attenuated in highly-sensitive five-day-old piglets, as infected animals did not lose weight and none of them died. in addition, the virus caused very minor tissue damage compared with a virulent virus. the rtgev-rs-spedv vaccine candidate was also attenuated in three-week-old animals that were used to evaluate the protection conferred by this virus, compared with the protection induced by infection with a virulent pedv us strain (pedv-nvsl). the rtgev-rs-spedv virus protected against challenge with a virulent pedv strain, reducing challenge virus titers in jejunum and leading to undetectable challenge virus rna levels in feces. the rtgev-rs-spedv virus induced a humoral immune response specific for pedv, including neutralizing antibodies. altogether, the data indicated that rtgev-rs-spedv is a promising vaccine candidate against virulent pedv infection. acute infectious diarrhea is a major cause of high morbidity and mortality in piglets worldwide. enteric infections in animals are frequently associated with viruses, including rotaviruses and coronaviruses (covs) [ ] . a metagenomics analysis of diarrheic and healthy samples from china in found porcine covs in % of the diarrheic samples, and only in around % of the healthy samples, highlighting the potential relevance of covs as enteric porcine pathogens [ ] . porcine epidemic diarrhea virus (pedv) was found in more than % of the diarrheic samples [ ] , in agreement with the importance that this virus has for the porcine industry worldwide. pedv was first described in europe in the s and the virus has, since then, remained endemic in the european porcine herds [ ] . in asian countries, pedv was detected in the s, with highly virulent outbreaks observed since viruses , , of in piglets. subsequently, an attenuated chimeric vaccine candidate was developed by introducing duplication of transcription regulating sequences (trss), as previously described by our group [ ] . this attenuated vaccine candidate conferred partial protection to pigs from a challenge with a virulent pedv. baby hamster kidney cells stably transformed with the gene coding for porcine aminopeptidase n (bhk-papn) [ ] were grown in in dulbecco's modified eagle's medium (dmem) supplemented with % fetal calf serum (fcs) and g ( . mg/ml) as selection agent. vero cells were grown in dmem supplemented with heat-inactivated % fcs. a us virulent pedv strain (pedv-nvsl, genbank accession number kf ) was kindly provided by ceva animal health. a recombinant virulent us pedv virus (rpedv), recovered from an infectious cdna engineered by our group (a. pascual-iglesias, l. enjuanes and s. zuñiga, unpublished data), was used as challenge virus in the animal experiments. pedv infectious cdna was maintained as a bac, including pedv usa/iowa/ / sequence (genbank accession number kf ). pedv viruses and recombinant tgev viruses obtained in this work were grown in vero cells supplemented with infection medium (dmem supplemented with . % tryptose phosphate broth, . % yeast extract, and µg/ml trypsin). the culture medium was supplied daily with % of the initial trypsin amount. a dna fragment containing the s gene sequence from virulent pedv usa/iowa/ / (genbank accession number kf ) [ ] was chemically synthesized and purchased from geneart (regensburg, germany). a pgem-t plasmid containing nucleotides , to , of tgev-sc genome (genbank accession aj ) and engineered paci and mlui restriction sites [ ] were used as an intermediate plasmid. overlapping polymerase chain reaction (pcr) was used to fuse pedv and tgev s gene sequences. the pedv s sequence encoding the ectodomain was amplified using purchased s sequence as template and oligonucleotides pac-s-vs ( -ggattaattaagaagggtaagttgctcattagaaataatggtaagttactaaactttggtaa ccacttcgttaacacaccatgaagtctttaacctac- , paci restriction site in italics) and pedv-s- -rs ( -ccacacataccaaggccacttgatgtatgtctc- ). tgev sequence encoding s protein transmembrane domain and endodomain was amplified using pgem-t-tgev-sc intermediate plasmid as a template and oligonucleotides tgev-s- -vs ( -gagacatacatcaag tggccttggtatgtgtgg- ) and tgev-s- -rs ( -tccaacgcgtaagtttag- , mlui restriction site in italics). in all cases, pedv sequences are indicated underlined. the obtained bp and bp pcr products were used as templates and amplified with oligonucleotides pac-s-vs and tgev-s- -rs. the bp pcr product was digested with paci and mlui and cloned into the same sites of pgem-t-tgev-sc plasmid, generating pgem-tgev-spedv plasmid. this plasmid was digested with paci and mlui restriction enzymes and the fragment containing chimeric s gene was cloned into the same sites of pbac-tgev-sc -p-m or pbac-tgev-sc -rs [ ] . all cloning steps were checked by sequencing of the pcr fragments and cloning junctions. for each mutant sequence, two independent cdnas were constructed. bhk-papn cells grown to % confluence in mm plates were transfected using µg of the corresponding pbac and µl of lipofectamine (invitrogen, carlsbad, ca, usa), according to manufacturer's specifications. at h post-transfection (hpt), bhk-papn transfected cells were trypsinized, washed twice with dmem, and plated over confluent vero monolayers grown in -mm-diameter plates. one hour later, trypsin was added to a final concentration of µg/ml. after a -day incubation period, the cell supernatants were harvested (passage ) [ ] . the rtgevs were cloned by limiting dilution method, grown, and titrated as previously described [ ] . twenty-one five-day-old suckling piglets, born from tgev and pedv seronegative sows, were randomly divided in two groups of nine piglets and one group of three animals. the nine-piglet groups were orally inoculated with tcid /animal of the different rtgevs in phosphate buffered saline (pbs). the three piglets in the other group were mock inoculated. infected animals were monitored daily to detect signs of enteric disease, and body weights were determined each day. three animals per group were euthanized and necropsied at days , , and after inoculation. intestinal macroscopic lesions were evaluated. front, mid, and end sections of jejunum were collected in all cases. samples were kept frozen for subsequent virus titration, stabilized with rnalater stabilization reagent (life technologies, carlsbad, ca, usa) for rna extraction, or fixed in zinc formaline fixative (sigma-aldrich, st. louis, mo, usa) for histopathology. fixed jejunum sections were washed twice with pbs and stored in % ethanol at • c. paraffin embedding, sectioning, and hematoxylin-eosin staining were performed by the histological laboratory (autopsy path kft., budapest, hungary). samples were examined with a zeiss axiophot fluorescence microscope. determination of the jejunum damage was obtained from unbiased measurement of three full-length perceived villi and crypt per location. at least two different locations per section per animal were measured. the villous height to crypt depth (vh/cd) ratio was calculated from the measurements. twenty-one -day-old piglets, born from tgev and pedv seronegative sows, were randomly divided in four groups ( table ) . piglets of groups and were orally inoculated with tcid /animal of rtgev-rs-spedv or pedv-nvsl, respectively. piglets of groups and were mock inoculated. three weeks after the immunization, piglets of groups , , and were challenged with tcid of rpedv per animal by oral route. infected animals were monitored daily to detect signs of enteric disease, and body weights were determined every days. fecal swabs were collected at days , , , , , and post-first inoculation. serum samples were taken at days , , , and post-immunization. saliva samples were collected, using salivette tubes (sarstedt, nümbrecht, germany), at days , , and post-first inoculation. three animals per group were euthanized and necropsied at days and post-challenge ( and post-immunization, respectively). intestinal macroscopic lesions were evaluated. front, mid, and end sections of jejunum were collected in all cases. samples were kept frozen for subsequent virus titration, stabilized with rnalater stabilization reagent (life technologies) for rna extraction, or fixed in zinc formaline fixative (sigma-aldrich) for histopathology. antibodies induced against tgev and pedv viruses were detected by elisa as described before [ ] . briefly, elisas were performed using . µg per well of partially purified tgev, pedv-nvsl, or rpedv viruses. antigens were bound to -well microplates, saturated with % bovine serum albumin (bsa) in pbs for h at • c, and incubated with serial dilutions of the serum sample in wash buffer ( . % bsa, . % tween in pbs) for min at • c. microplates were washed three times with wash buffer. bound antibodies were detected by incubation with peroxidase-conjugated protein a, goat anti pig igg (fc domain), or goat anti pig iga (biorad, hercules, ca, usa), diluted : , in pbs with . % bsa. elisa was developed with k-blue tmb substrate (neogen, lexington, ky, usa) for min at room temperature. reactions were stopped with . m h so , and the absorbance was read at nm. the elisa values of the sera were expressed as sample to positive ratio [sp-ratio = (od of sample − od of negative control)/(od of positive control − od of negative control)]. heat-inactivated sera were incubated for h at • c in the presence of pfus of pedv virus in dmem containing % fcs. serial dilutions of the mixtures were added to confluent vero cells in -well plates. after one hour of incubation at • c, medium was removed and infection medium was added. after h, cells were fixed with % formaldehyde in pbs, stained with a crystal violet solution, and virus titer was determined. the neutralization index was defined as the logarithm of the ratio of tcid in the presence of medium or in the presence of serum sample. total intracellular rna was extracted from vero cells or tissue samples. total rna was purified with rneasy mini kit (qiagen, hilden, germany), according to the manufacturer's specifications. to isolate viral rna from fecal swabs, the swab was resuspended in µl of pbs with antibiotics ( u/ml penicillin-streptomycin, µg/ml gentamicin, µg/ml amphotericin b). rna was isolated from µl of that solution using qiaamp viral mini kit (qiagen) following the manufacturers' instructions. in all cases, total cdna was synthesized using ng of total rna as a template, random hexamers, and the high-capacity cdna transcription kit (life technologies), following the manufacturers' recommendations. the viral genome region from b gene to utr (nt. - ) was sequenced in all cases. to that end, overlapping pcrs were performed using oligo pairs described in table . the pcr fragments were then sequenced using specific oligonucleotides. pedv genomic rna (grna) levels were measured by rt-qpcr analysis using a custom taqman assay detecting a conserved orf a region (taqman probe -fam-tgtact ggcttactggtgtt-mgb; forward primer -tgttgctatgtttgtgcattgg- ; reverse primer -tctgaatcactaggctgacctttg- ). tgev grna was evaluated using a custom taqman assay set up in our laboratory [ ] . the porcine β-glucuronidase (gusb) gene (taqman code ss _u ) was used as a reference housekeeping gene, since its expression remains constant in infected cells compared to that in non-infected cells [ [ ] and data not shown]. data were acquired with a viruses , , of real-time pcr system (applied biosystems, foster city, ca, usa) and analyzed with software v . . . the relative quantifications were performed using the −∆∆ct method [ ] . all experiments and data analysis were miqe (minimum information for publication of quantitative real-time pcr experiments guidelines) compliant [ ] . reverse size (bp) two-tailed, unpaired student t tests were used to analyze the difference in mean values between groups. all results were expressed as means ± the standard deviations of the means. p values < . were considered significant. a chimeric s protein (spedv-tgev) was designed. the n terminus of the protein contained the exposed domain of pedv s protein from a virulent us strain ( figure a ), including the epitopes recognized by neutralizing antibodies, and the heptad-repeats domains (aa to ). the c-terminus of the protein was that from the enteropathogenic tgev sc virus [ ] , including the transmembrane domain and c-terminus (aa to ). the chimeric protein was cloned in the tgev infectious cdna [ ] , replacing tgev s protein (rtgev-spedv, figure b ). in addition, chimeric spedv-tgev protein was cloned in an infectious cdna from an attenuated tgev, which contained duplications of transcription regulating sequences (trss) and engineered unique restrictions sites (rs) to avoid the overlapping of consecutive genes [ ] (rtgev-rs-spedv, figure c ). recombinant viruses rtgev-spedv and rtgev-rs-spedv were recovered after transfection of vero cells with the wild-type and attenuated cdnas, respectively. these viruses required trypsin to efficiently infect vero cells, and form syncytia, similarly to pedv virus. growth kinetics in cell culture was analyzed by infecting vero cells at two multiplicities of infection (moi)moi of . and . . both viruses reached peak titers at or hpi, depending on the moi ( figure a ). nevertheless, rtgev-rs-spedv virus peak titers were five-to -fold lower than those for parental rtgev-spedv virus ( figure a ). vero cells with the wild-type and attenuated cdnas, respectively. these viruses required trypsin to efficiently infect vero cells, and form syncytia, similarly to pedv virus. growth kinetics in cell culture was analyzed by infecting vero cells at two multiplicities of infection (moi)moi of . and . . both viruses reached peak titers at or hpi, depending on the moi (figure a ). nevertheless, rtgev-rs-spedv virus peak titers were five-to -fold lower than those for parental rtgev-spedv virus ( figure a ). to analyze the virulence of control rtgev-spedv and attenuated rtgev-rs-spedv viruses, fiveday-old piglets were orally inoculated with tcid /animal. interestingly, despite the difference in viral titers observed in cell cultures, viral titers in the jejunum of infected piglets were similar for both viruses at and dpi ( figure b ). at dpi, rtgev-spedv titers were slightly lower than those for rtgev-rs-spedv virus, suggesting a decreased number of gut epithelial cells available for reinfection. piglets inoculated with parental rtgev-spedv virus lost weight after infection (figure to analyze the virulence of control rtgev-spedv and attenuated rtgev-rs-spedv viruses, five-day-old piglets were orally inoculated with tcid /animal. interestingly, despite the difference in viral titers observed in cell cultures, viral titers in the jejunum of infected piglets were similar for both viruses at and dpi ( figure b ). at dpi, rtgev-spedv titers were slightly lower than those for rtgev-rs-spedv virus, suggesting a decreased number of gut epithelial cells available for reinfection. piglets inoculated with parental rtgev-spedv virus lost weight after infection ( figure a ) and % died ( figure b ). this data indicates that the chimeric rtgev-spedv control virus was virulent. in contrast, animals infected with rtgev-rs-spedv virus maintained weight ( figure a ) and all of them survived ( figure b ), indicating that the virus was attenuated. interestingly, virus shedding in the feces of piglets infected with attenuated rtgev-rs-spedv virus was up to -fold lower than that observed in the animals infected with the virulent parental rtgev-spedv virus ( figure c ). a) and % died ( figure b ). this data indicates that the chimeric rtgev-spedv control virus was virulent. in contrast, animals infected with rtgev-rs-spedv virus maintained weight ( figure a ) and all of them survived ( figure b ), indicating that the virus was attenuated. interestingly, virus shedding in the feces of piglets infected with attenuated rtgev-rs-spedv virus was up to -fold lower than that observed in the animals infected with the virulent parental rtgev-spedv virus ( figure c ). in agreement with the observed clinical signs, epithelial degeneration and exfoliation in the different parts of the jejunum was observed in animals infected with the virulent rtgev-spedv virus, accompanied by inflammatory infiltration and shortening of the villi ( figure a ). interestingly, the jejunum damage was significantly lower in animals infected with the attenuated rtgev-rs-spedv virus ( figure b ). altogether, the data indicate that rtgev-rs-spedv virus was partially attenuated. in agreement with the observed clinical signs, epithelial degeneration and exfoliation in the different parts of the jejunum was observed in animals infected with the virulent rtgev-spedv virus, accompanied by inflammatory infiltration and shortening of the villi ( figure a ). interestingly, the jejunum damage was significantly lower in animals infected with the attenuated rtgev-rs-spedv virus ( figure b ). altogether, the data indicate that rtgev-rs-spedv virus was partially attenuated. the main concern for the use of modified live vaccines based on attenuated viruses is their biosafety. reversion to virulence or recombination with circulating strains is in the basis for the emergence of novel virulent pedv strains, especially in asia [ , ] . the rtgev-rs-spedv virus genetic stability, which may influence its safety as vaccine candidate, was evaluated in cell culture. after passages in cell culture, viral genome region comprising from b gene to utr (nucleotides to ) was sequenced. no changes appeared in the passed virus compared with the parental one, strongly suggesting that the engineered virus was genetically stable in cell culture. to address virus stability in vivo, the -ends (nt to ) of rtgev-spedv and rtgev-rs-spedv viruses were sequenced, using rna isolated from the jejunum of five-day-old infected piglets at six days post-infection. no modifications were found in the parental rtgev-spedv virus. in the rtgev-rs-spedv virus, modifications were only detected in the engineered trs duplicated sequences ( figure ). small deletions and point mutations were found between e and m genes, eliminating the engineered fsei restriction site, but maintaining duplicated sequence ( figure ). extensive deletion was observed between m and n genes, reverting the sequence to that of the wild-type virus ( figure ). no changes were observed in the engineered mutations between n and genes ( figure ). interestingly, viruses , , of when viral rna was isolated from feces of -day-old piglets at seven days post-vaccination (see below) and rtgev-rs-spedv virus was sequenced, the same modifications were observed. this data strongly suggests that the rtgev-rs-spedv genome recovered from piglets may represent the in vivo evolution of the engineered virus. it is worth noting that tgev attenuation was similar with the three trss duplications and with just the duplication between n and genes [ ] , suggesting that in vivo evolved rtgev-rs-spedv virus may still be attenuated. the main concern for the use of modified live vaccines based on attenuated viruses is their biosafety. reversion to virulence or recombination with circulating strains is in the basis for the emergence of novel virulent pedv strains, especially in asia [ , ] . the rtgev-rs-spedv virus genetic stability, which may influence its safety as vaccine candidate, was evaluated in cell culture. after passages in cell culture, viral genome region comprising from b gene to ′utr (nucleotides to ) was sequenced. no changes appeared in the passed virus compared with the parental one, strongly suggesting that the engineered virus was genetically stable in cell culture. to address virus stability in vivo, the ′-ends (nt to ) of rtgev-spedv and rtgev-rs-spedv viruses were sequenced, using rna isolated from the jejunum of five-day-old infected piglets at six days no changes were observed in the engineered mutations between n and genes ( figure ) . interestingly, when viral rna was isolated from feces of -day-old piglets at seven days postvaccination (see below) and rtgev-rs-spedv virus was sequenced, the same modifications were observed. this data strongly suggests that the rtgev-rs-spedv genome recovered from piglets may represent the in vivo evolution of the engineered virus. it is worth noting that tgev attenuation was similar with the three trss duplications and with just the duplication between n and genes [ ] , suggesting that in vivo evolved rtgev-rs-spedv virus may still be attenuated. pedv infects animals of all ages, although the severity of the clinical signs is age-dependent [ ] . pregnant sows are the most suitable models to evaluate the effect of pedv vaccine candidates, as the target animals are neonatal piglets, and lactogenic immunity has an important role in protection [ ] . nevertheless, availability, cost, and housing resources limited its use in preliminary vaccine candidate trials [ , ] , and a young pig model has been proposed for preliminary vaccine efficacy trials [ , ] . therefore, to evaluate the protection conferred by rtgev-rs-spedv virus, -day-old pigs were used. group (g ) was vaccinated with rtgev-rs-spedv virus, group (g ) was vaccinated with a virulent us pedv strain (pedv-nvsl), and two groups of animals (g and g ) were not vaccinated ( table ) . twenty-one days after vaccination, animals from g , g , and g were challenged with a virulent recombinant pedv strain (rpedv) ( table ) . as expected, due to the animal age, no clinical sings of enteric disease such as diarrhea, vomiting, or dehydration were observed after vaccination or challenge (data not shown). nevertheless, animals vaccinated with the virulent pedv-nvsl strain showed a delay in weight gain the first week after vaccination, compared with non-infected animals or those vaccinated with rtgev-rs-spedv virus ( figure ). similar observations were reported for other virulent us isolates [ ] . these data indicate that rtgev-rs-spedv virus was also attenuated in -day-old piglets. virus titers were evaluated in the jejunum of challenged animals. the challenge virus replicated efficiently in the gut of non-vaccinated animals ( figure a ). in contrast, the titers were reduced more than -fold or -fold in animals vaccinated with rtgev-rs-spedv and pedv-nvsl viruses, respectively ( figure a ). similar results were obtained when viral rna presence was evaluated, with up to -fold reduction in the accumulation of pedv rna ( figure b ). interestingly, after challenge, pedv virus was only shed by non-vaccinated animals ( figure c ). altogether, these data indicate that the challenge virus did not efficiently infect the vaccinated animals. furthermore, these results indicate that rtgev-rs-spedv protected the animals against virulent pedv challenge. pigs were used. group (g ) was vaccinated with rtgev-rs-spedv virus, group (g ) was vaccinated with a virulent us pedv strain (pedv-nvsl), and two groups of animals (g and g ) were not vaccinated (table ) . twenty-one days after vaccination, animals from g , g , and g were challenged with a virulent recombinant pedv strain (rpedv) ( table ) . as expected, due to the animal age, no clinical sings of enteric disease such as diarrhea, vomiting, or dehydration were observed after vaccination or challenge (data not shown). nevertheless, animals vaccinated with the virulent pedv-nvsl strain showed a delay in weight gain the first week after vaccination, compared with non-infected animals or those vaccinated with rtgev-rs-spedv virus ( figure ). similar observations were reported for other virulent us isolates [ ] . these data indicate that rtgev-rs-spedv virus was also attenuated in -day-old piglets. figure . weight gain in vaccinated animals. three-week-old pigs were divided into four groups. animals from two groups were vaccinated with tcid /animal of rtgev-rs-spedv virus (red) or a virulent us pedv strain (pedv-nvsl, green). three weeks after vaccination ( dpv indicated in red), non-vaccinated animals were either mock infected (mock, grey) or challenged with tcid /animal of a virulent pedv strain (non vaccinated, black). vaccinated animals were also challenged with the same dose as non-vaccinated animals. clinical signs and weight were observed daily after vaccination and challenge, and once per week during the course of the experiment. average daily gain represents the mean from six different animals. error bars represent the standard deviation for each value; p value, ** < . . virus titers were evaluated in the jejunum of challenged animals. the challenge virus replicated efficiently in the gut of non-vaccinated animals ( figure a ). in contrast, the titers were reduced more than -fold or -fold in animals vaccinated with rtgev-rs-spedv and pedv-nvsl viruses, respectively ( figure a ). similar results were obtained when viral rna presence was evaluated, with up to -fold reduction in the accumulation of pedv rna ( figure b ). interestingly, after challenge, pedv virus was only shed by non-vaccinated animals ( figure c ). altogether, these data indicate that the challenge virus did not efficiently infect the vaccinated animals. furthermore, these results indicate that rtgev-rs-spedv protected the animals against virulent pedv challenge. figure . weight gain in vaccinated animals. three-week-old pigs were divided into four groups. animals from two groups were vaccinated with tcid /animal of rtgev-rs-spedv virus (red) or a virulent us pedv strain (pedv-nvsl, green). three weeks after vaccination ( dpv indicated in red), non-vaccinated animals were either mock infected (mock, grey) or challenged with tcid /animal of a virulent pedv strain (non vaccinated, black). vaccinated animals were also challenged with the same dose as non-vaccinated animals. clinical signs and weight were observed daily after vaccination and challenge, and once per week during the course of the experiment. average daily gain represents the mean from six different animals. error bars represent the standard deviation for each value; p value, ** < . . the antibody response specific for pedv was evaluated in the sera from vaccinated and nonvaccinated animals. before challenge ( dpv), vaccination elicited pedv-specific antibodies to a higher level in pedv-nvsl vaccinated animals than in rtgev-rs-spedv vaccinated ones ( figure a ). this result was expected, as the elisa test was developed against the whole pedv virus, and the chimeric rtgev-rs-spedv virus only expressed pedv s protein. after challenge at and dpv, pedv total antibodies increased in all challenged groups, as expected. interestingly, the increase was slower in the vaccinated pigs, in agreement with a decreased challenge virus replication in these animals. total anti-tgev antibodies were also evaluated by elisa. all animals were seronegative at the time of vaccination. when challenge was performed, the level of tgev antibodies was not significant in any group, and the all the animals remained seronegative after pedv challenge. this result was expected, as s protein is the main inducer of antibodies against tgev and it was not present in any of the viruses used for vaccination or challenge. in addition, it has been shown that the antibody response specific for pedv was evaluated in the sera from vaccinated and non-vaccinated animals. before challenge ( dpv), vaccination elicited pedv-specific antibodies to a higher level in pedv-nvsl vaccinated animals than in rtgev-rs-spedv vaccinated ones ( figure a ). this result was expected, as the elisa test was developed against the whole pedv virus, and the chimeric rtgev-rs-spedv virus only expressed pedv s protein. after challenge at and dpv, pedv total antibodies increased in all challenged groups, as expected. interestingly, the increase was slower in the vaccinated pigs, in agreement with a decreased challenge virus replication in these animals. total anti-tgev antibodies were also evaluated by elisa. all animals were seronegative at the time of vaccination. when challenge was performed, the level of tgev antibodies was not significant in any group, and the all the animals remained seronegative after pedv challenge. this result was expected, as s protein is the main inducer of antibodies against tgev and it was not present in any of the viruses used for vaccination or challenge. in addition, it has been shown that there is no cross-reactivity between tgev and pedv [ ] . to further analyze the humoral response specific for pedv, the levels of pedv-specific iggs ( figure b ) and igas ( figure c ) were evaluated. the data were similar to those obtained for total anti-pedv antibodies. it has been demonstrated that protection against pedv infection correlates with iga levels [ , , , ] . it is worth noting that iga levels in the sera of vaccinated animals, after challenge, were significantly reduced compared with non-vaccinated animals ( figure c ), strongly suggesting that challenge virus did not elicit a potent iga response in the gut of vaccinated animals, most likely due to reduced replication in the enteric tract, correlating with protection. the neutralization capability of the antibodies elicited was evaluated. as expected, non-vaccinated animals only induced the production of neutralizing antibodies after challenge (figure ). in contrast, pigs vaccinated with both pedv-nvsl and rtgev-rs-spedv virus induced a significant level of neutralizing antibodies before challenge, which were slightly increased after challenge (figure ). the neutralization capability of the antibodies elicited was evaluated. as expected, nonvaccinated animals only induced the production of neutralizing antibodies after challenge (figure ). in contrast, pigs vaccinated with both pedv-nvsl and rtgev-rs-spedv virus induced a significant level of neutralizing antibodies before challenge, which were slightly increased after challenge (figure ). . neutralizing antibodies produced in vaccinated animals. virus neutralization assay was performed using serum samples, collected before ( dpv) or after ( dpv) challenge, from rtgev-rs-spedv virus (red) or pedv-nvsl (green) vaccinated animals, and from non-vaccinated and challenged animals (black). the values represent neutralization index from three different animals. error bars represent the standard deviation for each value. altogether, the data indicate that rtgev-rs-spedv virus conferred protection against pedv infection in the young pig model and could be the basis for an effective vaccine candidate. an attenuated chimeric rtgev virus expressing the ectodomain of a virulent us pedv s protein (rtgev-rs-spedv) was engineered as vaccine candidate for pedv and evaluated in a young piglet model system. the rtgev-rs-spedv virus elicited a neutralizing humoral response specific for pedv. in fact, vaccinated animals were protected against challenge with a virulent pedv virus. the attenuated live vaccine candidates are the best option for pedv vaccination because nonreplicative antigens, as in inactivated vaccines, do not induce mucosal immunity and protection [ , ] . traditionally, attenuated live vaccines were developed for classical pedv strains, consisting of serial passages of the virulent virus in cell culture, as tissue culture adaptation led to attenuation in vivo. these vaccines were not effective to control the infection, as they were extensively used in countries were novel pedv strains emerged [ , ] . as a consequence, many groups have developed traditionally attenuated live vaccines based on novel epidemic pedv strains. in general, it has been shown that more than passages of the virulent virus in cell culture are required to attenuate pedv in vivo [ ] [ ] [ ] [ ] [ ] [ ] . it is worth noting that in our rtgev-rs-spedv vaccine candidate, the attenuating mutations were specifically designed and introduced in several locations of the viral genome. in contrast, traditionally attenuated vaccines contain non-controlled mutations throughout the genome figure . neutralizing antibodies produced in vaccinated animals. virus neutralization assay was performed using serum samples, collected before ( dpv) or after ( dpv) challenge, from rtgev-rs-spedv virus (red) or pedv-nvsl (green) vaccinated animals, and from non-vaccinated and challenged animals (black). the values represent neutralization index from three different animals. error bars represent the standard deviation for each value. altogether, the data indicate that rtgev-rs-spedv virus conferred protection against pedv infection in the young pig model and could be the basis for an effective vaccine candidate. an attenuated chimeric rtgev virus expressing the ectodomain of a virulent us pedv s protein (rtgev-rs-spedv) was engineered as vaccine candidate for pedv and evaluated in a young piglet model system. the rtgev-rs-spedv virus elicited a neutralizing humoral response specific for pedv. in fact, vaccinated animals were protected against challenge with a virulent pedv virus. the attenuated live vaccine candidates are the best option for pedv vaccination because non-replicative antigens, as in inactivated vaccines, do not induce mucosal immunity and protection [ , ] . traditionally, attenuated live vaccines were developed for classical pedv strains, consisting of serial passages of the virulent virus in cell culture, as tissue culture adaptation led to attenuation in vivo. these vaccines were not effective to control the infection, as they were extensively used in countries were novel pedv strains emerged [ , ] . as a consequence, many groups have developed traditionally attenuated live vaccines based on novel epidemic pedv strains. in general, it has been shown that more than passages of the virulent virus in cell culture are required to attenuate pedv in vivo [ ] [ ] [ ] [ ] [ ] [ ] . it is worth noting that in our rtgev-rs-spedv vaccine candidate, the attenuating mutations were specifically designed and introduced in several locations of the viral genome. in contrast, traditionally attenuated vaccines contain non-controlled mutations throughout the genome introduced during passages in cell cultures that, after passages in vivo, may lead to vaccine virus reversion to virulence. the engineered rtgev-rs-spedv vaccine candidate has the advantage of replicating in the jejunum of infected piglets as efficiently as a virulent control virus, without causing significant pathology, and most likely leading to a protective iga response. indeed, vaccinated animals were protected against virulent pedv challenge. in contrast, traditionally attenuated vaccines, as a consequence of adaptation to cell culture, have a dramatically reduced replication in the enteric tract [ ] and, as a consequence, these vaccines do not actively replicate to induce good iga and lactogenic immune responses [ , , ] . recently, a swine enteric coronavirus (secov) that is a recombinant between tgev and pedv has been described in europe [ ] [ ] [ ] . the secov contains a genome background identical to tgev, but expresses the s protein from pedv. in addition, recombination led to extensive deletions in the coding sequence and in trs of tgev gene a, and, as a consequence, protein a may not be expressed by secov. it is worth noting that secov was isolated from diarrheic samples in all cases, suggesting that it is virulent. the engineered parental rtgev-spedv virus resembles the recombinant secov and, as shown by our data, is virulent in suckling piglets. in contrast, the engineered virus (rtgev-rs-spedv) used as a vaccine candidate was attenuated. there are some genetic differences that may eventually allow the discrimination between secov and the rtgev-rs-spedv vaccine candidate as (i) rtgev-rs-spedv virus did not contain the full pedv s gene sequence, (ii) it expressed tgev protein a, and (iii) because of the tgev strain used to engineer the infectious cdna, it did not express tgev protein b [ ] . 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impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking '-o-methyltransferase activity mutagenesis of coronavirus nsp reveals its potential role in modulation of the innate immune response we thank ceva animal health for providing vero cells and pedv-nvsl virus. we are grateful to dora katona and máté halas (prophyl ltd., hungary) for in vivo experiments. we thank marga gonzalez (cnb-csic) for her technical assistance.conflicts of interest: z.p. is the global director of bio r&d at ceva animal health. the authors declare no other conflict of interest. key: cord- - x bidq authors: ding, li; li, jiawei; li, weihao; fang, zhenhua; li, na; wu, shannan; li, jiangyue; hong, meiling title: p - and ros-mediated aif pathway involved in tgev-induced apoptosis date: - - journal: j vet med sci doi: . /jvms. - sha: doc_id: cord_uid: x bidq we previously demonstrated that transmissible gastroenteritis virus (tgev) could induce apoptosis through caspase signaling. however, apoptosis was not completely prevented by caspases inhibitors, suggesting that there may be a caspase-independent pathway involved in tgev-induced cell apoptosis. in this study, we investigated the regulation of apoptosis-inducing factor (aif) on tgev-induced apoptotic pathway. results indicated that aif translocated from the mitochondria to nucleus during tgev infection, and the aif inhibitor, n-phenylmaleimide (np), significantly attenuated the apoptosis. in addition, the translocation of aif was inhibited by veliparib (abt- ), an inhibitor of poly (adp-ribose) polymerase (parp). and the reactive oxygen species (ros) scavenger, pyrrolidinedithiocarbamic (pdtc), redistributed aif in the mitochondria and nucleus in tgev-infected cells. moreover, the protein levels in nucleus and the mrna levels of aif were inhibited in the presence of the p inhibitor, pifithrin-α (pft-α) or in tgev-infected p −/−cells. furthermore, tgev-induced apoptosis was blocked by combination of three or more inhibitors, such as pan caspase inhibitor z-vad-fmk, np, abt- , pdtc, pft-α, to treat pk- cells. taken together, these results suggest that the p - and ros-mediated aif pathway and caspase-dependent pathway were involved in tgev-induced apoptosis. apoptosis-inducing factor (aif), normally locates in the inner mitochondrial membrane and exert a cytoprotective role [ , , ] . recently, in contrast to its cytoprotective role in the mitochondria, aif was found to translocate from the mitochondria to the cytosol, and subsequently to the nucleus, serving as a powerful proapoptotic trigger [ , ] . upon induction of various apoptotic stimuli, aif partly bound to dna, stimulated dnase activity and triggered condensation and large-scale fragmentation of chromatin, to induce apoptosis [ , , ] . however, the proapoptotic effects were not prevented by caspase inhibitors, suggesting that aif mediated a caspase-independent manner to trigger nuclear apoptosis [ , , ] . numerous lines of research have showed that a caspase-independent manner is involved in various stimuli-induced apoptotic programs [ , ] , including viruses [ , ] . transmissible gastroenteritis virus (tgev) belongs to the alphacoronavirus genus in the coronaviridae family according to viral taxonomy of international committee of taxonomy of viruses [ , ] . after infecting piglets, tgev then replicates in enterocytes, subsequently results in lethal watery diarrhea and dehydration [ ] . when infects several cell lines in vitro, tgev could induce morphological and biochemical changes in these cells [ , ] , and these findings were consistent with in vivo pathologic changes. we previously proved that tgev infection induced apoptosis by caspase signaling [ ] . p and reactive oxygen species (ros) play pivotal roles in tgev-induced cell apoptosis [ , ] . however, tgev-induced apoptosis is not completely inhibited by caspases inhibitors, suggesting that a caspase-independent pathway may be involved in tgev-induced cell apoptosis. thus, the study presented here stemmed from our previous findings and investigated the role of aif in tgev-induced apoptosis. our results demonstrated that ros and p levels mediated aif released to the nucleus from the mitochondria during tgev infection, thereby leading to apoptosis. doi: . /jvms. - pk- cells were cultured in dulbecco minimal essential medium (dmem) with % heat-inactivated newborn bovine serum (gibco brl, now york, md, u.s.a.), at °c in a humidified % co chamber. the tgev was used as previously described by ding et al. [ ] . primary antibodies against aif (sc- ), β-actin (sc- ), cox (sc- ), p (sc- ), parp- (sc- ), and histone h (sc- ) were purchased from santa cruz biotechnology (santa cruz, delaware, ca, u.s.a.). the antibody against tgev n protein was prepared by our laboratory. fluorescein isothiocyanate (fitc), rhodamine and horseradish peroxidase-conjugated secondary antibodies were purchased from the thermo scientific pierce (pierce, rockford, il, u.s.a.). n-phenylmaleimide (np) and pifithrin-α (pft-α) were purchased from sigma (sigma-aldrich, burlington, ma, u.s.a.). z-vad-fmk and veliparib (abt- ) were purchased from medchemexpress (medchemexpress, monmouth junction, nj, u.s.a.). pyrrolidinedithiocarbamic (pdtc) was purchased from merck (merck kgaa, darmstadt, germany). cells were fixed with % paraformaldehyde for min, permeabilized with . % triton x- for min. then the cells were incubated with a primary antibody against aif ( : ) or tgev n at °c overnight after blocked with % serum albumin for min. samples were washed three times with phosphate-buffered saline (pbs). subsequently, secondary antibody (rhodamineconjugated igg) was incubated for hr in the dark. then, ', -diamidino- -phenylindole (dapi) were used to counterstain cell nuclei for min. images were taken under a fluorescence microscope (amg evos, hatfield, pa, u.s.a.). rna extraction and qrt-pcr were performed as our previously described [ ] . specific primers that were used are as follows: aif sense, tgtggaacgtcttcaaccga; aif antisense, agaggaaggctgcgcaataa. actin sense ggacttcgagcaggagatgg; actin antisense: aggaaggagggctggaagag). the mitochondrial and nuclear proteins were isolated as the description of the mitochondria fractionation kit and the cellytic™ nuclear™ extraction kit (sigma-aldrich, burlington, ma, u.s.a.), respectively. protein concentrations were measured using the bca protein assay reagent (pierce, rockford, il, u.s.a.). the western blot assay was conducted as we described previously [ ] . the detection of apoptosis was performed as described by ding et al [ ] . summarily, harvested cells were washed with pbs for three times and then resuspended in µl binding buffer, followed by the addition of µl of annexin v-fitc and µl of pi. after incubation for min at room temperature, a total of , cells were examined. the positive cells was analyzed by flow cytometry using beckman cytoflex fcm (beckman coulter, brea, ca, u.s.a.). the crispr/cas system (genome engineering. broad institute cambridge, ma, u.s.a.) was used to mutate p gene, according to the description by xu et al [ ] . briefly, annealed fragments of grna (f: agtcacgaactggctggatg, r: tcagtggcttgaccgacctac) were cloned into lenticrisprv plasmid (addgene, cambridge, ma, u.s.a.), then with the pspax packaging plasmid co-transfected into hek t cells to generate the lentivirus. lentiviral titers were measured by qpcr. lentiviral-infected pk cell lines grown in puromycin dmem medium were used for selecting p knockout cells. puromycinresistant pk cells were selected and amplified and were subsequently examined by western blot. our previous study demonstrated that caspase inhibitors did not completely prevent apoptosis in tgev-infected cells [ ] . thus, to investigate whether aif pathway was activated in tgev-induced apoptosis in pk- cells, the protein levels of aif in mitochondrial and nuclear fractions were measured by western blot. results showed that the mitochondrial fraction levels of aif decreased significantly after hr of infection with tgev, while the nuclear fraction of aif increased after hr post infection, and keep rising with infection time (fig. a) . to further confirm aif translocation to the nucleus during tgev infection, immunofluorescence was performed with an antibody against aif (red fluorescence). as predicted, aif translocated to the nucleus after tgev infection (fig. b) , suggesting that aif was activated in tgev-infected pk- cells. in addition, to confirm the aif activation was induced by tgev infection, we measured the tgev n protein levels by immunofluorescence and western blot assays. results showed that tgev n protein were observed in tgev-infected pk cells ( fig. c and d) . to verify whether aif activation was involved in tgev-induced apoptosis, the aif inhibitor, np, was used to co-treat pk- cells with tgev, and the apoptotic rate was measured. our results showed that np partly abrogated tgev-induced apoptosis (fig. c) . these results suggest that aif pathway might be involved in tgev-induced apoptosis. previous studies demonstrated that poly (adp-ribose) polymerase (parp- ) contributed to the release of aif from the mitochondria to nucleus, activating the aif apoptotic pathway [ ] . our previous research showed that tgev infection induced parp- activation and subsequent cleavage [ ] . to determine whether the translocation of aif was mediated by parp- during tgev-induced apoptosis, the parp- inhibitor, abt- , was used to co-treat pk- cells with tgev. western blot assay was used to measure the aif protein levels in the nucleus. as shown in fig. a , abt- markedly inhibited the total protein and cleaved protein levels of parp- , compared with tgev infection alone, however the total protein levels of parp- were no significant difference in abt- -and tgev-co-treated pk- cells, compared with abt- treatment alone. moreover, abt- significantly inhibited the activation of parp- and the aif translocation was prevented by abt- (fig. b) . these results indicated that parp- may be crucial in the aif translocation during tgev infection. virus infection could induce ros accumulation [ , ] , and excessive ros could change mitochondrial permeability transition pore (mptp) and activate parp- , thereby leading to aif translocation into the nucleus [ , ] . our previous work revealed that ros is an important early mediator in tgev-induced apoptosis [ ] . to further explore the roles of ros accumulation in the aif-mediated apoptotic pathway during tgev infection, we investigated the effects of pdtc, an ros scavenger, on the cleavage of parp- and aif translation. as predicted, pdtc significantly attenuated the cleavage of parp- (fig. a) , as evidenced by the redistribution of aif in the mitochondria and nucleus in pk- cells infected by tgev (fig. b) . the result indicated that ros may play a vital role in tgev-induced aif activation. our previous research showed that tgev infection can activate ros/p -mediated apoptotic pathways [ ] . in addition, several lines of research showed that aif could be regulated by p [ , ] . we therefore sought to whether p regulated the aif expression. to that end, we used the p inhibitor, pft-α, to co-treat the cells with tgev and analyzed the aif mrna levels. result showed that the mrna levels of aif decreased in the presence of pft-α compared to those without pft-α (fig. a) . to further explore the effect of p on aif translocation, the aif protein levels in the mitochondria and the nucleus were detected by western blot. our results showed that pft-α treatment significantly reduced aif protein levels in the nucleus (fig. b) , suggesting that p may be involved in aif activation upon tgev infection. to rule out the contribution of the pft-α inhibitor on the virus, we used the crispr/cas system to mutate the p gene. result showed that the p protein was completely eliminated by the grna-generated clone (fig. c) . next, the wild-type pk cells (wt) and the pk p knockout cells (pk p −/− ) were infected with tgev, and the activation of aif was detected by qpcr and western blot. as shown in fig. d and e, aif mrna expression and the translocation of aif were reduced markedly in the absence of p . the findings were consistent with the changes associated with pft-α treatment. to further confirm that tgev infection activated both caspase-dependent pathway and aif pathway, we combined these inhibitors to treat the cells, and then measured the apoptotic rate upon tgev infection. result showed that tgev-induced apoptosis was almost completely inhibited by combination of pan caspase inhibitor z-vad-fmk and aif inhibitor np treatment, compared to tgev infection alone. the same blocking were also observed when the cells were treated by combination of three or more inhibitors, such as z-vad-fmk, np, abt- , ros scavenger pdtc, p inhibitor pft-α (fig. ) . the result is further evidence that caspase-dependent and caspase-independent pathway were included in tgev-induced apoptosis, and, p and ros played vital roles during apoptosis process. aif can participate in apoptosis upon translocation to the nucleus [ , , ] . it binds to dna in the nucleus, and induces chromatin condensation and dna fragmentation by recruiting downstream nucleases, resulting in apoptosis [ ] . actually, aif is considered to play a centric role in regulating caspase-independent pathways in cells [ , ] . our present study showed that tgev infection activated aif, which was released to the nucleus, thereby resulting in apoptosis. combination the inhibitors of pan caspase and aif blocked the apoptosis. in agreement with our previous research [ ] , we demonstrated that tgev-induced apoptosis involved a caspase-independent pathway as well as caspase-dependent pathways in infected cells. similar findings were seen with the rabies virus and the vesicular stomatitis virus [ , ] . several researches indicated that parp- can induce poly (adp-ribosyl) ation translocation to the mitochondria, then triggering aif translocation to the nucleus, resulting in dna fragmentation, to activate the aif apoptotic pathway [ , ] , consistent with these results, the current study showed that parp- inhibition could reduce the redistribution of aif in the mitochondria and nucleus, suggesting that parp- may be a prerequisite for tgev-induced translocation of aif. however, the total protein levels of parp- were no significant difference in abt- and tgev co-treated pk- cells, compared with abt- treatment alone, but remarkably decreased when compared to that tgev infection alone. and also the cleaved parp- were not absolutely inhibited in tgev-infected cells. these results suggest that tgev might activate parp- to induce aif translocation, while the cleavage of parp- was partly promoted by caspases. in multiple signaling pathways, ros is believed to be a second messenger [ ] . many studies have shown that high levels of ros can change the mitochondrial permeability transition pores (mptps) and activate parp- , leading to aif translocation to the nucleus [ , ] . we previously demonstrated that tgev-induced ros accumulation reduced mitochondrial membrane potential, leading to apoptosis [ ] . in the present study, we show that the inhibition of ros by pdtc attenuated the translocation of aif, indicating that ros may be an important factor in a caspase-independent cell apoptosis pathway during tgev infection. the accumulation of ros may initiate cellular safeguard machinery p [ ] . the signal would be returned to the mitochondria, leading to the increase of mitochondrial outer membrane permeabilization, subsequently the release of pro-apoptotic factors, thus triggering the apoptosis cascade [ ] . p not only takes effects on caspase-dependent apoptosis [ , ] , but also drives the efficient activation of aif through regulating aif gene expression, to mediate a caspase-independent death pathway [ ] . in the current study, the p inhibitor, pft-α, significantly reduced the mrna levels of aif and decreased the protein levels in the nucleus, and the similar results were also found in tgev-infected p −/− cells. these results showed that p might mediate the transcription of aif, and induce aif translocation to the nucleus. in the present study, we elaborated that tgev infection induced aif translocation to the nucleus, which may be mediated by p activation and ros accumulation. these results provided further insight into the mechanism of tgev-induced cell death. ratification vote on taxonomic proposals to the international committee on taxonomy of viruses the role of p in apoptosis apoptosis-inducing factor (aif): key to the conserved caspase-independent pathways of cell death? apoptosis-inducing factor (aif): a novel caspase-independent death effector released from mitochondria apoptosis-inducing factor (aif): caspase-independent after all role of aif in caspase-dependent and caspase-independent cell death transmissible gastroenteritis virus infection induces apoptosis through fasl-and mitochondria-mediated pathways regulation of ros in transmissible gastroenteritis virus-activated apoptotic signaling transmissible gastroenteritis coronavirus induces programmed cell death in infected cells through a caspase-dependent pathway early activation of the mitochondrial apoptotic pathway in vesicular stomatitis virusinfected cells nuclear and mitochondrial conversations in cell death: parp- and aif signaling transmissible gastroenteritis virus infection induces cell apoptosis via activation of p signalling essential role of the mitochondrial apoptosis-inducing factor in programmed cell death cadmium-induced apoptosis is mediated by the translocation of aif to the nucleus in rat testes ros and p : a versatile partnership. free radic apoptosis inducing factor (aif): a phylogenetically old, caspase-independent effector of cell death nadh oxidase activity of mitochondrial apoptosis-inducing factor possible contribution of apoptosis-inducing factor (aif) and reactive oxygen species (ros) to uvb-induced caspase-independent cell death in the t cell line jurkat the evolutionary processes of canine coronaviruses rabies virus-induced apoptosis involves caspase-dependent and caspase-independent pathways ros-mediated parp activity undermines mitochondrial function after permeability transition pore opening during myocardial ischemia-reperfusion p induces apoptosis by caspase activation through mitochondrial cytochrome c release role of reactive oxygen species (ros) in apoptosis induction intracellular reactive oxygen species production by polymorphonuclear leukocytes in bovine leukemia virus-infected dairy cows regulation of aif expression by p molecular characterization of mitochondrial apoptosisinducing factor mechanisms of aif-mediated apoptotic dna degradation in caenorhabditis elegans wnt/β-catenin signaling reduces bacillus calmette-guerin-induced macrophage necrosis through a ros -mediated parp/aif-dependent pathway p signaling modulation of cell cycle arrest and viral replication in porcine circovirus type infection cells dna binding is required for the apoptogenic action of apoptosis inducing factor mycotoxin zearalenone induces aif-and ros-mediated cell death through p -and mapk-dependent signaling pathways in raw . macrophages key: cord- -admlzeu authors: wang, gang; liang, rui; liu, ziwei; shen, zhou; shi, jiale; shi, yuejun; deng, feng; xiao, shaobo; fu, zhen f.; peng, guiqing title: the n-terminal domain of spike protein is not the enteric tropism determinant for transmissible gastroenteritis virus in piglets date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: admlzeu transmissible gastroenteritis virus (tgev) is the etiologic agent of transmissible gastroenteritis in pigs, and the n-terminal domain of tgev spike protein is generally recognized as both the virulence determinant and enteric tropism determinant. here, we assembled a full-length infectious cdna clone of tgev in a bacterial artificial chromosome. using a novel approach, the clustered regularly interspaced short palindromic repeat (crispr)/crispr-associated protein (cas ) systems efficiently and rapidly rescued another recombinant virus with a -amino-acid deletion in the n-terminal domain of the tgev spike gene (s_ntd ), which is analogous to the n-terminal domain of porcine respiratory coronavirus. s_ntd notably affected the tgev growth kinetics in pk- cells but was not essential for recombinant virus survival. in animal experiments with two-day-old piglets, the tgev recombinant viruses with/without s_ntd deletion induced obvious clinical signs and mortality. together, our results directly demonstrated that s_ntd of tgev mildly influenced tgev virulence but was not the enteric tropism determinant and provide new insights for the development of a new attenuated vaccine against tgev. importantly, the optimized reverse genetics platform used in this study will simplify the construction of mutant infectious clones and help accelerate progress in coronavirus research. coronaviruses (covs) are single-stranded, positive-sense rna viruses closely related to animal and human health [ ] [ ] [ ] . covs belong to the coronaviridae family, which consists of the alpha-, beta-, gamma-and deltacoronavirus genera [ ] . since , covs, including severe acute respiratory syndrome cov (sars-cov), middle east respiratory syndrome cov (mers-cov), and porcine epidemic diarrhea virus (pedv), have swept across the world and caused considerable global economic losses [ ] [ ] [ ] [ ] [ ] . as the largest rna genome viruses, covs have at least six typical overlapping open reading frames pk- cells were cultured in dulbecco's modified eagle's medium (gibco, waltham, ma, usa) supplemented with % fetal bovine serum at • c with % co . tgev strain wh- (genbank accession number hq ) was propagated at • c in a % co incubator in dulbecco's modified eagle's medium (gibco, waltham, ma, usa) supplemented with % fetal bovine serum (gibco, waltham, ma, usa). all the experiments using live viruses were performed under biosafety level (bsl) conditions. total rna was extracted from virus-infected cultures using trizol reagent (invitrogen, carlsbad, ca, usa), and cdna was reverse transcribed with reverse transcriptase (takara, amv, kusatsu, japan) using random primers (takara, kusatsu, japan, mer). all the fragments were amplified by polymerase chain reaction (pcr) with phanta super fidelity dna polymerase (vazyme, nanjing, jiangsu, china). the natural complete genome of tgev wh- was determined by sequencing (genscript, nanjing, china) the overlapping pcr products cloned into the corresponding vectors in triplicate. compared with the parental tgev wh- from ncbi (bethesda, md, usa), several site mutations, including t c, g t, g c, c t, and c g, were observed, and these mutations were maintained during the cloning of the tgev full-length genome. a point mutation, a t, was introduced by overlap extension pcr to remove the natural van i site and maintained as the rescue marker. as described in previous research, the egfp gene was inserted into the tgev genome [ , ] to replace the original sequence positioned from , to , . the virus genome was divided into six continuous fragments (a to f), and each fragment was amplified from the total cdna using specific primers (available upon request). fragments a, b, d, and e were cloned into the pmd -t vector (takara, kusatsu, japan). fragment a was cloned with the saci and van i sites, and fragment e was cloned with the van i and kasi sites. notably, fragment a was cloned to contain a saci site (gagctcgtttagtgaaccgt) [ ] located in the terminal of the tgev genome sequence. fragments b and d were cloned with van i sites. fragment c was cloned into a bac plasmid (kindly provided by prof. cao gang) that was modified from pbelobac to include a van site [ ] . fragment f was also cloned into the bac plasmid with saci and kasi sites introduced at the and termini of the tgev genome, respectively. as the final recipient bac vector, subclone f also contained the synthesized essential element sequences, such as the cmv promoter, the poly(a) tail sequence ( a), the hdv rz sequence (hepatitis delta virus self-cleaving ribozyme sequence, rz), and the bovine growth hormone (bgh) transcription terminal signal (genscript, nanjing, china) [ ] . after the six subclones were sequenced in their corresponding vectors, subclones a and e were first digested with saci and kasi, respectively, and then treated with calf intestinal alkaline phosphatase (ciap, scientific). all the subclones were then digested with van i except subclone f, which was digested with saci and kasi. subsequently, all the digested products were purified with a gel extraction kit (omega, norcross, ga, usa), and fragments a to f were ligated for more than h at • c and transformed into chemically competent dh b cells (biomed, beijing, china). after determination of all the fragments by bacterial pcr, the positive clones were further determined by restriction fragment length polymorphism with kpni, and the correct clone was designated ptgev-gfp bac after sequencing (genscript, nanjing, china). pk- cells were seeded in a six-well plate and incubated for h, and the recovery of tgev-gfp or tgev-gfp-∆s_ntd was then performed by transfecting µg of the corresponding bac into pk- cells with µl of lipofectamine (invitrogen, carlsbad, ca, usa). at h post-transfection, the collected virus progenies were purified once by fluorescent plaques. subsequently, the purified virus clone was amplified and stored until use at − • c. for the design of a sgrna to mediate cleavage of the targeted site, a constant reverse primer (ssdna-r) and two forward primers (ssdnaa-f and ssdnab-f) specific for sites a and b were synthesized as shown in table , similar to the protocol described in a previous report [ ] . to anneal the primers ssdnaa-f and ssdnab-f with the reverse primer ssdna-r, pcr was conducted for cycles at • c for s, • c for s, and • c for s using × utaq mastermix (zoman). the pcr products were then purified with cp buffer (omega, norcross, ga, usa) and transcribed using a t transcription kit (neb, ipswich, ma, usa) according to the manufacturer's instructions to produce the targeted sgrna a and sgrna b. the purity of the sgrna products was analyzed by electrophoresis on agarose gels using . µg of each sgrna product. to modify the sequence of tgev s_ntd , ptgev-gfp bac was digested using the targeted sgrnas. specifically, ptgev-gfp bac was digested in a µl reaction mixture with µg of ptgev-gfp bac, µl of cas (neb, ipswich, ma, usa), µg of sgrna, and µl of nuclease reaction buffer incubated at • c for more than h or, preferably, overnight. for purification of the digested ptgev-gfp bac, an equivalent volume of solution i (plus rnase; omega, norcross, ga, usa) was first added to digest the sgrna at room temperature for min, and the cp buffer (omega, norcross, ga, usa) was then applied to recycle the digested bac according to the manufacturer's instructions. the pcr products with the -bp deletion were constructed by two-cycle pcr. first, the primers rec- sf and δs-ntdr or the primers rec- sr and δs-ntdf (table ) were used to amplify the primary pcr products from the template of the ptgev-gfp bac. second, the two primary pcr products were annealed to produce pcr products of the -bp deletion using the primers rec- sf and rec- sr (table ). homologous recombination was then performed using the clonexpress ii one step cloning kit (vazyme, nanjing, jiangsu, china) according to the manufacturer's instructions using ng of recycled linearized ptgev-gfp bac, ng of pcr products of s_ntd and two pairs of primers (rec- sf/δs-ntdr and rec- sr/δs-ntdf) ( table ) . subsequently, a pair of primers (primerf/primerr) ( table ) was designed to amplify the sequence of the modified s_ntd area for sequencing (genscript, nanjing, china). the recombinant virus corresponding to the correct ptgev-gfp-∆s_ntd bac was then recovered as described above. pk- cells in six-well plates were inoculated with -fold serially diluted recombinant virus. after virus adsorption for min, monolayer cells were washed three times with pbs and overlaid with a mixture of % low-melt agarose and times the concentration of dmem (invitrogen, carlsbad, ca, usa) supplemented with % fetal bovine serum (gibco). the overlay was then solidified at • c for min. subsequently, the plates were cultured at • c in a % co incubator, and days post-infection, the fluorescent plaques were visualized by fluorescence microscopy. thirteen -day-old piglets from a tgev-free sow were randomly divided into three groups and fed fresh liquid milk diluted in warm water every h. all piglets were confirmed to be free of tgev, pedv, porcine delta coronavirus (pdcov), and rotavirus (rv) through a rt-pcr assay of piglet feces before viral challenge. the piglet weights were measured and recorded at the beginning of the challenge. the piglet challenge group was intranasally and orally inoculated with µl ( × tcid ) of chimeric virus, and the mock-infected control group was intranasally and orally inoculated with µl of dmem. the piglets were monitored for their clinical status every h. any piglet exhibiting moribund signs were euthanized. at days post-inoculation, all surviving piglets were euthanized consecutively to reduce the stress of the other piglets. before necropsy, the weight of each piglet was recorded. at necropsy, five sections of the duodenum, jejunum, ileum, colon and stomach were collected, fixed in % formalin for histopathological examination and stained with hematoxylin and eosin (he). after necropsy, samples of jejunal contents and lung tissue were collected for virus detection by nested rt-pcr using the specific primers f /r and f /r (table ) [ ]. the animal experiments were performed according to the protocols approved by the scientific ethics committee of huazhong agricultural university (permit number: hzausw- - ). the animal care and maintenance protocols complied with the recommendations detailed in the regulations for the administration of affairs concerning experimental animals made by the ministry of science and technology of china. to construct an infectious clone of tgev, six overlapping cdna fragments designated a to f were generated by reverse transcriptase pcr (rt-pcr) using total rna extracted from pk- cells infected with tgev wh- ( figure a ,b). fragments a, b, d, and e were cloned into the pmd -t vector, and fragments c and f were cloned into the bacterial artificial chromosome (bac) to produce the corresponding subclones. subclone f was also constructed as the final recipient bac vector by inserting the cytomegalovirus (cmv) promoter at the terminus of fragment f and a -bp poly(a) tail ( a) followed by the hepatitis delta virus ribozyme (rz) and bovine growth hormone (bgh) transcription terminal signal sequences at the terminus of fragment f ( figure b ). to more conveniently observe the chimeric virus and exclude the influence of the accessory gene orf on tgev enteric tropism and virulence, the gene encoding orf at the genome position from , to , was replaced by the egfp gene ( figure a ,b). through the one-step assembly of fragments a to f, we successfully obtained a full-length cdna infectious clone of tgev, designated ptgev-gfp bac ( figure b ,c). the full-length ptgev-gfp bac was verified by sequencing. after propagation for more than generations in e. coli dh b cells, ptgev-gfp bac digestion with the kpni enzyme, which produced six different fragment products, also confirmed the correct ptgev-gfp bac clone ( figure c ,d). in other words, the cloning of fragment c into the bac yielded no toxic sequences in any of the experiments. produce the corresponding subclones. subclone f was also constructed as the final recipient bac vector by inserting the cytomegalovirus (cmv) promoter at the ′ terminus of fragment f and a -bp poly(a) tail ( a) followed by the hepatitis delta virus ribozyme (rz) and bovine growth hormone (bgh) transcription terminal signal sequences at the ′ terminus of fragment f ( figure b) . to more conveniently observe the chimeric virus and exclude the influence of the accessory gene orf on tgev enteric tropism and virulence, the gene encoding orf at the genome position from , to , was replaced by the egfp gene ( figure a ,b). through the one-step assembly of fragments a to f, we successfully obtained a full-length cdna infectious clone of tgev, designated ptgev-gfp bac ( figure b ,c). the full-length ptgev-gfp bac was verified by sequencing. after propagation for more than generations in e. coli dh b cells, ptgev-gfp bac digestion with the kpni enzyme, which produced six different fragment products, also confirmed the correct ptgev-gfp bac clone ( figure c,d) . in other words, the cloning of fragment c into the bac yielded no toxic sequences in any of the experiments. to rescue the recombinant cov of tgev-gfp (corresponding to ptgev-gfp bac), the ptgev-gfp bac was transfected into pk- cells using lipofectamine . sporadic green fluorescence was observed h post-transfection, as depicted in figure a , but the infected pk- cells grew and showed a normal morphology. however, compared with the mock-infected group, obvious green fluorescence and the cytopathic effect (cpe) could be observed h post-transfection ( figure b ). western blot and rt-pcr assays were performed to further confirm the recombinant virus tgev-gfp. the . -kilodalton band of tgev n protein ( figure c ) and the marker mutation at position in the tgev genome ( figures b and d ) confirmed the recovery of the tgev-gfp recombinant virus. to rescue the recombinant cov of tgev-gfp (corresponding to ptgev-gfp bac), the ptgev-gfp bac was transfected into pk- cells using lipofectamine . sporadic green fluorescence was observed h post-transfection, as depicted in figure a , but the infected pk- cells grew and showed a normal morphology. however, compared with the mock-infected group, obvious green fluorescence and the cytopathic effect (cpe) could be observed h post-transfection ( figure b ). western blot and rt-pcr assays were performed to further confirm the recombinant virus tgev-gfp. the . -kilodalton band of tgev n protein ( figure c ) and the marker mutation at position in the tgev genome ( figures b and d ) confirmed the recovery of the tgev-gfp recombinant virus. spike with n-terminal aa deletion in tgev wh is analogous to the spike of a reported natural prcv strain ( figure a ). to verify whether s_ntd is the enteric tropism determinant for tgev, we used the crispr-cas system to efficiently manipulate the tgev gene. briefly, two specific enzyme sites encompassing the sequence of s_ntd were selected. we then synthesized two types of single-stranded dna forward primers (ssdnaa-f or ssdnab-f) and a constant reverse primer (ssdna-r) ( table ) corresponding to the two enzyme cutting sites, designated sites a and b ( figure c ). after annealing pcr using the forward and reverse primers, the purified pcr products of short dna fragments were transcribed by t rna polymerase ( figure b ). the transcribed products corresponding to sites a and b (designated sgrna a and sgrna b) ( figure c ) were incubated with the nuclease cas to digest the ptgev-gfp bac in vitro, and the digestion yielded a linearized bac and a~ . -kb dna fragment that included the sequence of s_ntd ( figure a,d) . tgev, we used the crispr-cas system to efficiently manipulate the tgev gene. briefly, two specific enzyme sites encompassing the sequence of s_ntd were selected. we then synthesized two types of single-stranded dna forward primers (ssdnaa-f or ssdnab-f) and a constant reverse primer (ssdna-r) ( table ) corresponding to the two enzyme cutting sites, designated sites a and b ( figure c ). after annealing pcr using the forward and reverse primers, the purified pcr products of short dna fragments were transcribed by t rna polymerase ( figure b ). the transcribed products corresponding to sites a and b (designated sgrna a and sgrna b) ( figure c ) were incubated with the nuclease cas to digest the ptgev-gfp bac in vitro, and the digestion yielded a linearized bac and a ~ . -kb dna fragment that included the sequence of s_ntd ( figure a,d) . to construct the mutant infectious clone of the s_ntd deletion (designated ptgev-gfp-Δs_ntd bac) from the ptgev-gfp bac, we produced a -nucleotide deletion-specific mutation pcr product using two pairs of primers (rec- sf/δs-ntdr and rec- sr/δs-ntdf) ( table ). the mutation pcr products were then recombined into the linearized bac vector cleaved from the full-length ptgev-gfp bac ( figure d ). after the recombination products were transformed into dh b competent cells, all monoclonal colonies were identified as positive clones by pcr using the primer pair rec- sf/rec- sr ( figure e ). the sequencing of three randomly selected monoclonal colonies also confirmed the positive ptgev-gfp-Δs_ntd bac, and we then constructed an infectious clone with the corresponding -aa deletion in the n-terminus of the tgev-gfp s protein by sequencing one entire genome of the three positive clone ( figure e ). to construct the mutant infectious clone of the s_ntd deletion (designated ptgev-gfp-∆s_ntd bac) from the ptgev-gfp bac, we produced a -nucleotide deletion-specific mutation pcr product using two pairs of primers (rec- sf/δs-ntdr and rec- sr/δs-ntdf) ( table ). the mutation pcr products were then recombined into the linearized bac vector cleaved from the full-length ptgev-gfp bac ( figure d ). after the recombination products were transformed into dh b competent cells, all monoclonal colonies were identified as positive clones by pcr using the primer pair rec- sf/rec- sr ( figure e ). the sequencing of three randomly selected monoclonal colonies also confirmed the positive ptgev-gfp-∆s_ntd bac, and we then constructed an infectious clone with the corresponding -aa deletion in the n-terminus of the tgev-gfp s protein by sequencing one entire genome of the three positive clone ( figure e ). we rescued the recombinant virus tgev-gfp-∆s_ntd from pk- cells as previously described, and sporadic and more obvious green fluorescence was observed at and h post-transfection, respectively ( figure a ). we then verified the modified virus tgev-gfp-∆s_ntd by rt-pcr using the primers primerf and primerr (table ). an obvious deletion of approximately bp was observed in tgev-gfp-∆s_ntd in comparison with tgev-gfp ( figure b ). subsequently, the rt-pcr product was sequenced using the primers primerf and primerr ( figure c) . comparison with the ptgev-gfp bac showed that all the modified nucleotides were correct, as depicted by the model shown in figure d . to validate whether s_ntd determines the enteric tropism of tgev, two-day-old piglets were randomly divided into three groups, with five piglets in each virus-infected group and three piglets in the mock-infected control group. the piglets in the two virus-infected groups were to further evaluate the role of s_ntd in tgev, we measured the growth kinetics of the wild-type virus, tgev-gfp and tgev-gfp-∆s_ntd. the replication kinetics of the tgev-gfp and wild-type viruses were comparable to each other and considerably different from those of tgev-gfp-∆s_ntd ( figure e ). twelve hours after inoculation, the titer of tgev-gfp was more than -fold greater than that of tgev-gfp-∆s_ntd ( figure e ). to further identify the effect of s_ntd on tgev, we also analyzed the fluorescent viral plaques. the plaque size of tgev-gfp-∆s_ntd was notably different from that of tgev-gfp ( figure f ), which also indicated that tgev-gfp infected cells more effectively than tgev-gfp-∆s_ntd. to validate whether s_ntd determines the enteric tropism of tgev, two-day-old piglets were randomly divided into three groups, with five piglets in each virus-infected group and three piglets in the mock-infected control group. the piglets in the two virus-infected groups were inoculated intranasally and orally at a dose of × % tissue culture infective dose (tcid ) with the respective chimeric virus, and the mock-infected control piglets were inoculated with dulbecco's modified eagle's medium (dmem). all the piglets in the tgev-gfp group exhibited severe clinical symptoms and weight loss, and those in the tgev-gfp-∆s_ntd group showed ameliorated but still obvious clinical symptoms ( figure a,b) . in addition, the piglets in the tgev-gfp and tgev-gfp-∆s_ntd groups appeared moribund within days postinoculation, whereas the piglets in the mock-infected control group remained healthy ( figure c ). in addition, the piglets in the tgev-gfp group showed a higher mortality rate (as high as %) and presented earlier symptoms compared with those in the tgev-gfp-∆s_ntd group, which showed % mortality at days postinoculation ( figure a,c) . to better detect the presence of the inoculated virus in the euthanized piglet intestine, the presence of both tgev-gfp and tgev-gfp-∆s_ntd in intestinal tissue was detected by nested pcr using the primer pairs f /r and f /r (table ) . tgev-gfp and tgev-gfp-∆s_ntd were detected in intestinal tissue from the moribund piglets ( figure d ), but no chimeric virus was detected in the two piglets in the tgev-gfp-∆s_ntd group that were euthanized at days post-inoculation ( figure d ). tgev-gfp-Δs_ntd groups appeared moribund within days postinoculation, whereas the piglets in the mock-infected control group remained healthy ( figure c ). in addition, the piglets in the tgev-gfp group showed a higher mortality rate (as high as %) and presented earlier symptoms compared with those in the tgev-gfp-Δs_ntd group, which showed % mortality at days postinoculation ( figure a ,c). to better detect the presence of the inoculated virus in the euthanized piglet intestine, the presence of both tgev-gfp and tgev-gfp-Δs_ntd in intestinal tissue was detected by nested pcr using the primer pairs f /r and f /r (table ) . tgev-gfp and tgev-gfp-Δs_ntd were detected in intestinal tissue from the moribund piglets ( figure d ), but no chimeric virus was detected in the two piglets in the tgev-gfp-Δs_ntd group that were euthanized at days post-inoculation ( figure d ). the postmortem of the moribund piglets in the tgev-gfp and tgev-gfp-Δs_ntd groups revealed that the small intestines were filled with watery contents. in particular, the intestinal walls in the jejunal section of the intestines of these piglets were clearly thinner and more transparent compared with those of the mock group ( figure a) . he staining compared with the normal mock group also revealed that the tgev-gfp chimeric viruses caused more severe intestinal tissue the postmortem of the moribund piglets in the tgev-gfp and tgev-gfp-∆s_ntd groups revealed that the small intestines were filled with watery contents. in particular, the intestinal walls in the jejunal section of the intestines of these piglets were clearly thinner and more transparent compared with those of the mock group ( figure a ). he staining compared with the normal mock group also revealed that the tgev-gfp chimeric viruses caused more severe intestinal tissue damage than tgev-gfp-∆s_ntd ( figure b ). more severe villous atrophy was observed in the small intestine, particularly the jejunum and ileum, of the piglets in the tgev-gfp and tgev-gfp-∆s_ntd groups compared with those of the mock group ( figure b ). collectively, these results suggested that s_ntd has not altered the enteric tropism for tgev but exerts a mild influence on tgev virulence. the n-terminal domain of spike protein is recognized as the tgev enteric tropism determinant in piglets, as demonstrated through comparisons of the sequences of natural tgev isolates or those obtained after continuous passage in cell culture [ , , ] , but more direct evidence is still needed. in this study, based on a dna-launched infectious clone, we used a novel cov gene editing method to efficiently perform cov targeted gene editing. using reverse genetics, we found that s_ntd was not the enteric tropism determinant for tgev. the relevant insights regarding the novel cov targeted gene editing method and tgev s_ntd are discussed below. because covs are the viruses with the largest rna genomes, the construction of a cov infectious clone is hampered by two main challenges: large full-length cdna and toxic sequences in the bacterial clone [ , ] . although the problem of cov cdna sequence instability has been overcome by several methods, the manipulation of the large full-length cov genome remains a considerable challenge. until now, the direct editing of the full-length cdna of covs has not been reported. in this study, we constructed a tgev-gfp infectious clone by ligating six fragments in one the n-terminal domain of spike protein is recognized as the tgev enteric tropism determinant in piglets, as demonstrated through comparisons of the sequences of natural tgev isolates or those obtained after continuous passage in cell culture [ , , ] , but more direct evidence is still needed. in this study, based on a dna-launched infectious clone, we used a novel cov gene editing method to efficiently perform cov targeted gene editing. using reverse genetics, we found that s_ntd was not the enteric tropism determinant for tgev. the relevant insights regarding the novel cov targeted gene editing method and tgev s_ntd are discussed below. because covs are the viruses with the largest rna genomes, the construction of a cov infectious clone is hampered by two main challenges: large full-length cdna and toxic sequences in the bacterial clone [ , ] . although the problem of cov cdna sequence instability has been overcome by several methods, the manipulation of the large full-length cov genome remains a considerable challenge. until now, the direct editing of the full-length cdna of covs has not been reported. in this study, we constructed a tgev-gfp infectious clone by ligating six fragments in one step [ ] . the crispr/cas system was then used to finish the construction of ptgev-gfp-∆s_ntd. to our knowledge, this study provides the first demonstration of the direct in vitro manipulation of full-length coronavirus cdna. to edit specific cov genes, targeted cleavage of the bac was completed by cas protein through a reaction mediated by two types of sgrna transcribed together or separately ( figure b,d) . sgrna can be easily obtained by annealing pcr and transcription using an available kit. moreover, regardless of the exonuclease trimming activities of cas [ ] , in the experiment, we were able to insert the mutated fragments in the linearized bac with -bp overlapping sequences through homologous recombination. notably, numerous mutation fragments can be inserted efficiently into the linearized bac at the same time ( figure d ), which is perfect for the construction of a viral mutant library [ ] [ ] [ ] . similar to traditional plasmid manipulation, we edited the targeted gene by recombination in vitro with overlapping pcr products (e.g., mutations, deletions, or insertions). moreover, as little as ng of linearized, digested bac was sufficient to complete the recombination reaction. to determine the mutation of the targeted bac, we only needed to amplify the fragments by bacterial pcr using a pair of primers in duplicate, and this assay can be used to sequence the site of recombination and the modified fragment area. furthermore, almost any area of the targeted bac can be simply cleaved by the crispr/cas system with two types of specific sgrna. throughout the process, we accomplished recombinant virus recovery using only one plasmid of bac in a single week ( figure ) . namely, once an infectious clone was constructed, the recombinant coronaviruses was more efficiently and conveniently rescued in this study than in previous research, and the proposed approach thus greatly accelerates the gene editing speed of large rna virus rescue. moreover, the simple manipulation of a bac vector and modification of the specific small region throughout the procedure would theoretically lower the mutation probability of the full-length cov cdna. thus, this method is not only cost-effective but also reduces the probability of introducing additional mutations during the bac modification procedure. the spike gene of tgev has been shown to alter tgev virulence or enteric tropism. however, recombinant tgev with s protein n-terminal amino-acid deletion was constructed through targeted recombination and passaged several times, which might cause other locus mutations in addition to the s protein deletion [ ] . in particular, an early study reported that a -residue deletion in prcv corresponding to the n-terminal domain of the tgev s protein, as depicted in figure a , is likely responsible for the loss of replication observed in the enteric tract [ ] . no other studies have provided direct evidence demonstrating that only the n-terminal region of the spike gene changes the tgev virulence or enteric tropism [ ] . here, we emphasize the importance of the n-terminus of the tgev s protein for the enteric tropism of the virus. to that end, we constructed a recombinant virus with an s protein analogous to that of prcv ( figure a) , tgev-gfp-∆s_ntd, and this recombinant virus showed titers and fluorescent plaque sizes that greatly differed from those of tgev-gfp ( figure e,f) . these results indicated that the amino acids of the n-terminal of the tgev spike protein are not essential for viral survival but important for viral replication or infection, which was analogous to the findings obtained for pedv and other covs [ ] [ ] [ ] [ ] . our animal experiments revealed that tgev-gfp-∆s_ntd caused % mortality in piglets and obvious intestinal tissue damage, which indicates that s_ntd has a mild influence on virulence but does not alter the enteric tropism of tgev [ ] . using reverse genetics, we confirmed that changes in s_ntd alone altered, albeit not completely, the virulence of tgev. one reason explaining no detection of tgev-gfp-∆s_ntd in the two piglets euthanized at days post-inoculation might be related to immunity of recovering piglets. the role of s_ntd might be analogous to that of the -amino-acid region in the n-terminus of the pedv s gene when used as a viral virulence marker. consistent with previous reports, our experiments also detected tgev-gfp and tgev-gfp-∆s_ntd in the jejunal contents tissue by nested rt-pcr [ , ] , which indicates that changes in s_ntd alone do not alter tgev enteric tropism in vivo. and it is also possible that other genes in addition to the amino acids of the n-terminal of the tgev spike protein might regulate changes in tgev tissue tropism [ , ] . additional research is needed to determine the detailed mechanism of tgev enteric tropism in vivo. in summary, using the reverse genetics method, we have provided direct evidence showing that the n-terminal domain of spike protein is not the determinant of tgev enteric tropism in piglets, although s_ntd exerts a mild influence on tgev virulence. these results provide new insights into the development of a new attenuated vaccine against tgev. furthermore, the method developed in this study allows the efficient and rapid editing of the full-length cov genome in vitro and can theoretically be applied to all viruses with 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s protein facilitates infection by porcine transmissible gastroenteritis coronavirus deletion of a -amino-acid region in the n-terminal domain of spike protein attenuates porcine epidemic diarrhea virus in piglets identification and comparison of receptor binding characteristics of the spike protein of two porcine epidemic diarrhea virus strains crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor crystal structure of bovine coronavirus spike protein lectin domain increased litter survival rates, reduced clinical illness and better lactogenic immunity against tgev in gilts that were primed as neonates with porcine respiratory coronavirus (prcv) recovery of transmissible gastroenteritis virus from chronically infected experimental pigs isotype-specific antibody-secreting cells to transmissible gastroenteritis virus and porcine respiratory coronavirus in gut-and bronchus-associated lymphoid tissues of suckling pigs the authors declare no conflict of interest. key: cord- - vqe authors: ding, li; huang, yong; dai, meiling; zhao, xiaomin; du, qian; dong, feng; wang, lili; huo, ruichao; zhang, wenlong; xu, xingang; tong, dewen title: transmissible gastroenteritis virus infection induces cell cycle arrest at s and g /m phases via p -dependent pathway date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: vqe p signaling pathway plays an important role in the regulation of cell cycle. our previous studies have demonstrated that tgev infection induces the activation of p signaling pathway. in this study we investigated the effects of tgev infection on the cell cycle of host cells and the roles of p activation in this process. the results showed that tgev infection induced cell cycle arrest at s and g /m phases in both asynchronous and synchronized pk- and st cells, while uv-inactivated tgev lost the ability of induction of cell cycle arrest. tgev infection promoted p accumulation, down-regulated cell cycle-regulatory proteins cyclins b , cdc , cdk and pcna. further studies showed that inhibition of p signaling could attenuate the tgev-induced s- and g /m-phase arrest by reversing the expression of p and corresponding cyclin/cdk. in addition, tgev infection of the cells synchronized in various stages of cell cycle showed that viral genomic rna and subgenomic rna, and virus titer were higher in the cells released from s-phase- or g /m phase-synchronized cells than that in the cells released from the g /g phase-synchronized or asynchronous cells after h p.i. taken together, our data suggested that tgev infection induced s and g /m phase arrest in host cells, which might provide a favorable condition for viral replication. viral infection could activate a variety of signal transduction pathways to induce subversion of the host cell cycle, which plays important roles in the viral life cycle by facilitating the replication of progeny virus after viral infection (davy and doorbar, ) . a variety of rna viruses have been shown to induce g /g , s or g /m arrest in infected cells. for example, human immunodeficiency virus (hiv)-infected t lymphocytes isolated from patients are arrested in g /m (zimmerman et al., ) ; influenza a virus a/wsn/ (h n ) infection results in g /g -phase accumulation of infected cells, which increase viral protein expression and progeny virus production (he et al., ) ; hepatitis c virus (hcv) ns protein induces cell cycle arrest in the s-phase in mammalian cells to facilitate hcv viral replication (yang et al., ) . in term of coronaviruses, murine coronavirus mouse hepatitis virus (mhv) has been shown to induce g /g arrest (chen and makino, ) ; sars-cov nucleocapsid (n) protein can disrupt cytokinesis and block s-phase progression in mammalian cells (surjit et al., ) ; bronchitis virus (ibv) infection can induce g /m phase arrest to facilitate viral replication (dove et al., ) . transmissible gastroenteritis virus (tgev) infection has been shown to alter some cell signaling pathways implicated in cell cycle regulation in our previous study (huang et al., ) . however, the effects of tgev infection on the cell cycle of host cells and the significance of cell cycle regulation in tgev replication need to be further investigated. the cell-cycle progression is tightly regulated through a complex network of cell-cycle regulatory molecules. cyclind-cdk complex and cycline-cdk complex regulate cell cycle progression in the g /g phase. cyclina-cdk complex regulates cell cycle progression in the s phase. cyclinb-cdc complex is a crucial regulator for progression through late g and early m (malumbres and barbacid, ). these cell-cycle regulatory molecules have been shown to be regulated by some upstream pathways like p signaling. p signaling can control the expression of p , which directly bind to some cdk-cyclin complexes to inhibit their kinases activity (he et al., ) . our previous studies have demonstrated that tgev infection induced the activation of p signaling pathway to regulate cell growth and apoptosis (huang et al., ) . in this study, we further investigated the effects of tgev infection on the cell cycle of host cells, the roles of p signaling activation in regulation of cell cycle progression in tgev-infected cells, and the significance of cell cycle regulation in tgev replication. results showed that that tgev infection could perturb the progression of cell cycle and facilitate virus gene replication. pk- cells (atcc, ccl- ) and st cells (atcc, crl- ) were grown in dulbecco minimal essential medium (d-mem) (gibco brl, md, us) supplemented with % heat-inactivated fetal bovine serum (gibco), iu of penicillin and g of streptomycin per ml, at • c in a % co atmosphere incubator. the tgev shaanxi strain was isolated from intestinal tract contents of tgev-infected piglets in shaanxi province of china and propagated in pk- cells and st cells (ding et al., ) . virus titers determined by % tissue culture infective doses (tcid ) as described previously (reed and muench, ) . cell cycle analysis was measured by propidium iodide staining. briefly, cells were fixed in % ethanol for min at • c. after several washes with phosphate-buffered saline (pbs), the cell pellets were resuspended in . ml pbs containing . % triton x- (sigma-aldrich, us), g /ml rnase a (sigma) and g/ml propidium iodide (sigma) for min, prior to facs analysis (beckman the results are shown as mean ± sem of three independent experiments. *p < . , **p < . versus mock infection. (b) serum-starved pk- cells were mock infected or infected with . moi of tgev. cells were co-stained with brdu and pi and analyzed using flow cytometry. the histograms were analyzed to determine the percentage of cells in s phase of the cell cycle. s phase, upper gate; g , lower left; g , lower right. the results are shown as mean ± sem of three independent experiments. **p < . versus mock infection. coulter, inc. fullerton, ca, us). at least nuclei were counted for each sample. the thymidine analog bromodeoxyuridine (brdu) (sigma) is incorporated into actively replicating dna and thus accurately determines the proportion of cells in s phase. briefly, m brdu was added to cell medium and incubated at • c for min to allow brdu incorporation. cells were collected and then fixed in % ethanol. then cells were pelleted and incubated in m hcl in pbs at • c for min. after incubating in wash buffer and pelleting, the cell pellet was resuspended in . m sodium borate. samples were then pelleted before addition of l of anti-brdu antibody (cell signalling technology) and incubated for min at room temperature. samples were then incubated by fluorescein isothiocyanate (fitc)-labeled antibody for min in the dark. samples were stained by pi before analyzed using a facs calibur analyzer. pk- and st cells were synchronized at g /g phase using serum deprivation by maintenance of cells in dmem containing no fbs supplementation for h. synchronized cells were mock infected or infected with . moi of tgev. after h of virus adsorption, cells were treated with medium containing % fbs and harvested at various times post infection (p.i.) for cell cycle analysis. pk- and st cells were synchronized at the g /s phase border using double-thymidine treatment by incubation for h in maintenance media supplemented with mm thymidine (sigma). cells were then washed three times with pbs and incubated for h in maintenance media, followed by additional h incubation in maintenance media supplemented with mm thymidine. then, synchronized cells were mock infected or infected with . moi of tgev. pk- and st cells were synchronized at g /m phase using nocodazole (sigma) treatment by incubation of cells in . at the indicated times, cells were lysed and equal amounts of proteins from the samples were tested by western blot analysis. the same membranes were also probed with ␤-actin as a loading control. the numbers below the proteins indicated the relative folds of mock infection after normalized to ␤-actin. maintenance media supplemented with ng/ml nocodazole for h. cells were washed three times with pbs, and then infected with . moi of tgev. at indicated times p.i., cells were processed for rt-pcr and flow cytometric analysis. cell extracts were prepared as described previously (ding et al., ) . protein concentrations were measured using bca protein assay reagent (pierce, rockford, il, us). equivalent amounts of proteins were loaded and electrophoresed on % sodium dodecyl sulfate-polyacrylamide gel (sds-page). subsequently, proteins were transferred to polyvinylidene difluoride (pvdf) membranes (millipore corp, atlanta, ga, us). the membranes were blocked with % nonfat dry milk at room temperature for h, and then incubated with indicated primary antibodies over night at • c, followed by hrp-conjugated secondary antibodies at room temperature for h. the signal was detected using ecl reagent (pierce, rockford, il, us). total rna extraction and reverse transcription were performed as described (ding et al., ) . quantitative analysis of genomic rna (grna) and subgenomic mrnas (sgrnas) from tgev-derived replicons was performed by real-time rt-pcr using bio-rad iq real time pcr system. the primers for qrt-pcr in this study were described in previous study (dufour et al., ) . reactions were carried out in l volume containing × sybr premix ex taqtm ii (takara, dalian, china), sense and anti-sense primers and target cdna. the relative quantification of gene expression was analyzed by the two-ddct method. pifithrin-␣ (ptf-␣) was purchased from sigma and stored as a mm stock solution in dmso. to reduce the activation of p , ptf-␣ ( m) was diluted in cell culture medium without serum and added to cultures h prior to infection. inhibitor was not included in the virus inoculum. after h of tgev adsorption, the virus inoculum was removed and fresh basal medium containing fresh inhibitor was added to the culture. at indicated times, cells were harvested and correlative indicators were detected. data are mean ± sem of three independent experiments. results were analyzed by one-way analysis of variance (anova). for each assay, student's t-test was used for statistical comparison. a value of p < . was considered significant. to investigate the influence of tgev infection on cell cycle progression, mock infected or tgev-infected asynchronously growing pk- and st cells were harvested at different times p.i., and cell cycle profiles were detected by flow cytometry. representative cell cycle histograms and profiles in pk- and st cells were presented in fig. a and b, respectively. in tgev-infected pk- cells, there was a significant increase in the proportion of cells in the s phases and g /m phases of the cell cycle from h p.i. and h p.i., respectively, and continued to increase with infection time, when compared to mock-infected cells (fig. a) . in tgev-infected st cells, the proportion of cells in the g /m phases and s phases significantly increased from h p.i. and h p.i., respectively, when compared to mock-infected cells (fig. b) . these results suggest that tgev infection induced the arrest of the cell cycle in the s and g /m phase. in tgev-infected cells, tgev infectious levels were evaluated by detection of the grna and sgrnas (n sgrna, m sgrna and orf sgrna) of tgev using qrt-pcr. results showed that the levels of tgev grna increased significantly from h p.i. in pk- ( fig. a , left panel) and st cells (fig. b, left panel) . the sgrna levels of n, m, orf could not been detected at h p.i., detected at h p.i., significantly increased at h p.i., and continued to increase with following infectious time in tgev-infected cells ( fig. a and b, right panel) . moreover, the titers of tgev were determined at various times postinoculation. results showed that little infectious virus was detected at and h p.i., thereafter the virus titer showed a rapid increase between and h p.i. and then reached a plateau (fig. c) . to further determine whether the tgev-induced s and g /m arrest requires virus replication, uv-inactivated tgev was inoculated in pk- and st cells, and cell cycle profiles were measured at h p.i. by flow cytometry. results showed that cell cycle arrest were not observed in cells infected with uv-inactivated tgev, while there was no significant difference between mock-infected cells and cells treated with uv-inactivated tgev (fig. d) , suggesting that viral replication was required for induction of cell cycle arrest in tgev-infected cells. to further confirm that tgev replication caused s and g /m cell cycle arrest, we infected serum-starved quiescent cells with tgev and examined cell cycle progression after serum stimulation. as shown in fig. a , prior to infection, approximately % of serum-starved cells were arrested at the g /g phase in pk- cells. at , and h p.i., the cells population at g /g -phase significantly decreased, while the cells population at s-phase dramatically increased in tgev-infected cells compared to that in mock-infected cells, and apparent increase of g /m-phase cells was observed at h p.i. (fig. a) . similar feature were also seen in quiescent st cells after infection (fig. b) . these results suggest that tgev-infected cells exhibited s-phase delay and g /m-phase arrest. to determine whether tgev replication may induce the release of some soluble factors to affect the cell cycle progression of uninfected cells, we used high mois of tgev to infect serumstarved quiescent pk- cells and detect cell cycle profiles. the results showed that s-phase delay induced by and moi of tgev occurred at h p.i., and the number of s-phase arrested cells induced by moi and moi of tgev were approximately two and four folds as many as that by . moi tgev, respectively (fig. a) . however, the s-phase and g /m-phase arrested cells number induced by moi and moi of tgev were not significantly higher than that by . moi tgev at h p.i. (fig. a) . these results suggested that tgev replication just affected the cell cycle progression of infected cells, and that over moi of tgev infection would interfere the progression of cell cycle due to the involvement of cell apoptosis. to further confirm the changes of tgev-infected cells in the s phase of cell cycle, we used brdu incorporation assay to accurately determine the cell number in s phase. results showed that the proportion of cells in s phases significantly increased at h p.i., and further increased with infection time, when compared to mockinfected cells (fig. b) , which were in consistent with the results of pi staining. to determine the molecular mechanism underlying the tgevinduced cell cycle arrest, we detected the levels of associated cell cycle regulatory factors at , , , , and h p.i. in tgev-infected cells. results showed that cdc , acting as a main coordinator complexed with cyclin a and cyclin b to advance the cell cycle into m phase, began to decrease at h p.i., and continued to decrease with infection time in infected cells compared to that in mock-infected cells (fig. a) , suggesting that the cell cycle might be arrested at g /m phase in tgev-infected cells. in addition, the levels of cdk significantly decreased from h p.i. in tgev-infected cells compared to mock-infected cells, but cdk and cdk did not show significant difference between in mock-infected and tgevinfected pk- cells (fig. a, left panel) . the levels of cyclin b began to significantly decrease at h p.i. in tgev-infected pk- cells compared to mock-infected cells, but cyclin a, cyclin d and cyclin e did not show significant difference between mock-infected and tgev-infected pk- cells (fig. b, left panel) . in parallel experiments, western blot analysis showed similar or analogous results in the expression of these cdks and cyclins in tgev-infected st cells ( fig. a and b, right panel) . among them, the reduction of cyclin b was more significant in tgev-infected st cells than in tgevinfected pk- cells when compared with mock infection. taken together, these results suggest that tgev infection lead to an accumulation of cells in the s and g /m phases of the cell cycle through decreasing certain cell cycle factors that regulate the proceeding of s and g /m phase. our previous study showed that tgev infection induced the accumulation and activation of p in cells. here, we examined the levels of p and p in . moi tgev-infected cells. western blot analysis showed that . moi of tgev infection could also increase the protein level of p and induce phosphorylation of p at serine and in pk- and st cells (fig. a) . consequently, the level of p increased in tgev infected pk- and st cells from h p.i., and significantly increased at h p.i., while proliferating-cell nuclear antigen (pcna), a mediator involved in p -regulated dna replication within s phase, decreased accordingly in tgev-infected pk- and st cells (fig. a) . these results suggest that tgev infection could up-regulate p expression to regulate cell cycle through activation of p signaling. to further determine the roles of p in tgev-induced cell cycle arrest, we investigated the effects of pft-␣, a specific inhibitor of p that does not affect the mrna levels of tgev genes (huang et al., ) , on the cell cycle profiles and the expression of p , cdks and cyclins in tgev-infected pk- and st cells. as shown in fig. b , pre-incubation of pk- and st cells with pft-␣ attenuated cell cycle arrest at s and g /m phase induced by tgev infection. in addition, cdc , cdk and cyclin b expression increased in tgevinfected pk- and st cells with pft-␣ compared to that with dmso (fig. c) . as expected, pre-incubation of pk- and st cells with pft-␣ also attenuated p up-regulation and pcna reduction induced by tgev infection (fig. c) . these results suggest that p and p might play key roles in mediation of tgev-induced cell cycle arrest. since the proportion of cells in the s and g /m phases was greater in tgev infected cells compared to mock-infected cells, to investigate the effects of cell accumulation at s and g /m phases on tgev replication, synchronized cells in g /m phase, g /g phase, g /s phase or asynchronous cells were simultaneously released from their different phase of cell cycle, and then either mock infected or infected with . moi of tgev, cell cycle profiles were determined by flow cytometry and tgev grna and sgrnas levels were determined by qrt-pcr at h p.i. results showed that using serum deprivation treatments, over % of pk- and % of st cells were synchronized at the g phase (fig. a ). using nocodazole treatments, over % of pk- cells and % of st cells were synchronized at the g /m phase (fig. b ). using double-thymidine treatment, approximately % of cells were synchronized at the g /s phase border in pk- and st cells (fig. c ). in the cells released from the g -phase-synchronized cells, the proportion of s phase cells was greater in tgev infected cells compared to mockinfected cells at h p.i. (fig. a) , which was in consistent with the results above. in the cells released from the g /s and g /m-phasesynchronized cells, the cells population at g /m phase significantly increased in tgev-infected cells compared to that in mock-infected cells at h p.i. (fig. b and c) . real-time rt-pcr analysis of the grna and sgrnas of tgev showed that the replication levels of grna and the synthesis of sgrna were higher in the cells released from g /m or g /ssynchronized cells than that in the cells released from g /g phase-synchronized or asynchronous cells (fig. a ). in addition, the titers of the virus also were higher in the cells released from g /m or g /s-synchronized cells than that in the cells released from g /g phase-synchronized or asynchronous cells (fig. b) . these results suggest that tgev induction of host cell staying at s and g /m phase might be beneficial for virus replication. cell cycle manipulation is crucial event occurred in virusesinfected cells (davy and doorbar, ; kannan et al., ) . in this study we demonstrated that tgev-infected cells accumulated in the s and g /m phase of the cell cycle, and the effect was not cell type specific and could be reproduced in synchronously replicating cells. the cell arrest at the s and g /m phases was dependent upon tgev replication and was controlled by viral modulation of certain cyclins/cdks. many viruses employ a variety of mechanisms to utilize or manipulate the cell cycle pathways of infected cells (chowdhury et al., ; de bolle et al., ; li et al., ) . throughout the cell cycle, progression is regulated by a series of cyclins and cdks (king and cidlowski, ) . the g /m transition in the cell cycle is positively controlled by complex of cdc (cdk ) and cyclin b. members of the cdki family of proteins (cdk inhibitors) bind to the cdk/cyclin complexes and inhibit the kinase activity, to regulate the g /m-phase transition (king and cidlowski, ) . cyclin a-cdk complex is the main cyclin-cdk complex in the s phase, and the activity of the complex is required for s phase transition and control of dna replication (elledge, ) . it is possible that the regulation of s and/or g /m phase is one of the crucial mechanisms that viruses employed to manipulate the cell cycle (li et al., ; martin-lluesma et al., ) . in tgev-infected cells, we observed that a significant decrease of cdk , cyclin b and cdc , involved in s phase and g /m phase transition, which regulated the cell cycle to arrest at the s and g /m phases. p is a key element in the induction of cell cycle arrest in viruses infected cells (casavant et al., ) . as a dna damage response protein, p transcriptionally activates numerous genes involved in dna repair and cell cycle arrest (vogelstein et al., ) , and p dependent arrest of cells at g /s or g /m phase is an important component of the cellular response to genotoxic stress including virus infection (casavant et al., ; de bolle et al., ) . the first transcriptional target of p was p , a cki of the cip/kip family, which bridges the function of p with the cell cycle and plays important roles in regulation of cell cycle progression or arrest (he et al., ) . the p is recognized to be an important mediator to delay transit from g to s phase and/or from g to m phase, thus preventing the effects of dna damage on gene functions (abbas and dutta, ). p has also been shown to regulate dna replication within the s phase in vitro, by binding to proliferating-cell nuclear antigen (pcna)/cdk complexes and by dissociating cyclin a/cdk /p /e f transitional complexes (abbas and dutta, ) . in this study, we showed that tgev infection induced p and p accumulation and pcna decrease. furthermore, pft-␣, a specific inhibitor of p , prevented the arrest of the cell cycle in tgevinfected cells. the observation that tgev infection induced cell cycle arrest at s and g /m phases in both pk- and st cells virtually required the possible involvement of p in these processes. this observation is not consistent with the findings in other coronavirus-infectious bronchitis virus, which induced cell cycle arrest at both s and g /m phases independent of p (li et al., ) , suggesting that p might play different roles in viruses-infected cells. some viruses can regulate the cell cycle progression through interaction between virus and host cells in order to induce the cell cycle arrest and provide an advantageous environment for viral replication (dove et al., ; li et al., ) . infectious bronchitis virus (ibv) could increase protein accumulation and progeny virus production in cells enriched in the g /m phase of the cell cycle (li et al., ) . human immunodeficiency virus (hiv) infection also increased the transduction of hiv in the g /m phase compared to other stages of the cell cycle (groschel and bushman, ) . many substrates (i.e., nucleotides) in s phase are abundantly available for viral replication (yang et al., ) . furthermore, there is evidence that one consequence of g /m arrest is to establish a pseudo-s phase state for viruses to replicate of their genomes (belyavskyi et al., ) . to specifically investigate whether a particular stage of the cell cycle favored tgev replication, we synchronized cells in various stages of the cell cycle and compared efficiencies of virus infection. results showed that tgev replication levels, sgrna synthesis were greater in the cells released from g /s phase or g /m phase of the cell cycle after h p.i., when compared to either cells released from the g /g phase or asynchronous cells. our results also provided evidences that tgev titer in the cells released from g /s phase or g /m phase synchronized cells was higher than that in cells released from g /g phase synchronized cells. this might be that cells synchronized at g /g phase need longer time to reenter cell cycle progression than synchronized cells at the g /s phase, which support the hypothesis that s phase state was beneficial for virus replication. therefore, we favor the hypothesis that tgev induces s and g /m phase cell cycle arrest in order to provide a more favorable condition for virus production. in conclusion, our study revealed that tgev infection induced the host cells to arrest at s and g /m phases of the cell cycle, which were responsible by the certain key players in the s and g /m transition. moreover, the cell cycle arrest induced by tgev infection might provide a more favorable condition for viral replication. li ding and yong huang designed the experiments and interpreted the data and wrote the article. li ding performed the experiments with assistance and advice from meiling dai, xiaomin zhao, qian du, feng dong, lili wang, ruichao huo, wenlong zhang and xingang xu. yong huang and dewen tong revised the manuscript. all authors have read the manuscript and approved to submit it to your journal. there is no conflict of interest of any authors in relation to the submission. p in cancer: intricate networks and multiple activities the structural protein odv-ec of autographa californica nucleopolyhedrovirus is a multifunctional viral cyclin potential role for p in the permissive life cycle of human cytomegalovirus murine coronavirus replication induces cell cycle arrest in g /g phase hiv- vpr activates cell cycle inhibitor p /waf /cip : a potential mechanism of g /m cell cycle arrest g /m cell cycle arrest in the life cycle of viruses human herpesvirus infection arrests cord blood mononuclear cells in g phase of the cell cycle isolation and identification of porcine transmissible gastroenteritis virus shaanxi strain and clone and sequence analysis of its gene transmissible gastroenteritis virus infection induces apoptosis through fasl-and mitochondria-mediated pathways cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus replication structure and functional relevance of a transcription-regulating sequence involved in coronavirus discontinuous rna synthesis cell cycle checkpoints: preventing an identity crisis cell cycle arrest in g /m promotes early steps of infection by human immunodeficiency virus induction of p by p following dna damage inhibits both cdk and cdk activities influenza a virus replication induces cell cycle arrest in g /g phase transmissible gastroenteritis virus infection induces cell apoptosis via activation of p signaling hepatitis c virus infection causes cell cycle arrest at the level of initiation of mitosis cell cycle regulation and apoptosis cell cycle arrest and apoptosis induced by the coronavirus infectious bronchitis virus in the absence of p human herpesvirus suppresses t cell proliferation through induction of cell cycle arrest in infected cells in the g /m phase cell cycle, cdks and cancer: a changing paradigm hepatitis b virus x protein affects s phase progression leading to chromosome segregation defects by binding to damaged dna binding protein a simple method of estimating fifty per cent endpoints the nucleocapsid protein of severe acute respiratory syndrome-coronavirus inhibits the activity of cyclin-cyclindependent kinase complex and blocks s phase progression in mammalian cells surfing the p network hcv ns protein inhibits cell proliferation and induces cell cycle arrest in the s-phase in mammalian cells through down-regulation of cyclin a expression human immunodeficiency virus type vpr induces dna replication stress in vitro and in vivo this work was supported by grants from the national natural science foundation of china (grant no. /c ), the doctoral program of higher education of china (grant no. ), and the scientific research program of northwest a&f university (no. z ). key: cord- - x s g n authors: stoian, ana; rowland, raymond r.r.; petrovan, vlad; sheahan, maureen; samuel, melissa s.; whitworth, kristin m.; wells, kevin d.; zhang, jianqiang; beaton, benjamin; cigan, mark; prather, randall s. title: the use of cells from anpep knockout pigs to evaluate the role of aminopeptidase n (apn) as a receptor for porcine deltacoronavirus (pdcov) date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: x s g n the coronaviruses, porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev), and porcine deltacoronavirus (pdcov) represent important sources of neonatal diarrhea on pig farms. the requirement for aminopeptidase n (apn) as a receptor for tgev, but not for pedv, is well established. in this study, the biological relevance of apn as a receptor for pdcov was tested by using crispr/cas to knockout the apn gene, anpep, in pigs. porcine alveolar macrophages (pams) from anpep knockout (ko) pigs showed resistance to pdcov infection. however, lung fibroblast-like cells, derived from the anpep ko pam cultures, supported pdcov infection to high levels. the results suggest that apn is a receptor for pdcov in pams but is not necessary for infection of lung-derived fibroblast cells. the infection of the anpep ko pigs with pdcov further confirmed that apn is dispensable as a receptor for pdcov. coronaviruses belong to the family coronaviridae, order nidovirales, and are important pathogens of humans and animals. coronaviruses are divided into four genera, alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. infection of neonatal pigs with the alphacoronaviruses, transmissible gastroenteritis virus (tgev) or porcine epidemic diarrhea virus (pedv), results in mal-absorptive diarrhea, which can lead to dehydration and death (madson et al., ; saif et al., ) . the first outbreak of pedv on u.s. farms in resulted in the death of nearly million pigs or about % of u.s. pig production for that year (stevenson et al., ) . in , a diarrhea-causing porcine deltacoronavirus (pdcov) was isolated from five pig farms in ohio (wang et al., ) . the m and n gene sequences were % identical to another pdcov, hku , from china. since , pdcov has rapidly spread throughout pig-producing regions in the u.s. to date, four host proteins, aminopeptidase n (apn), angiotensinconverting enzyme (ace ), dipeptidyl peptidase (dpp ), and carcinoembryonic antigen-related cell adhesion. molecule (ceacam ), have been described to function as coronavirus receptors (delmas et al., ; li et al., ; raj et al., ; dveksler et al., ) . porcine apn (papn), a amino acid, type ii membrane metallopeptidase, participates in the removal of nterminal amino acids from protein substrates during digestion. delmas et al. ( ) were the first to characterize an apn peptide sequence, located between amino acids and , as required for infection of cells with tgev. the corresponding receptor region on tgev is located on the s subunit c-terminal domain (s -ctd) of the tgev spike (s) protein (godet et al., ) . recent studies have focused on the potential role of apn as a receptor for pdcov. the data have yielded three different observations and conclusions. in , wang et al. showed that cell lines, such as vero and bhk- , were resistant to infection with pdcov and tgev. permissiveness was acquired after transfection of both cell lines with a plasmid expressing a papn cdna. pdcov and tgev growth curves on the transfected cells showed a yield greater than log tcid /ml. the authors concluded that papn is a functional receptor for pdcov. also, in , zhu et al., reported similar results. however, hela cells, which were papn-negative, and resistant to tgev, supported pdcov infection. furthermore, a knockout of papn expression in the porcine ipihttps://doi.org/ . /j.virol. . . received october ; received in revised form december ; accepted december cell line was prepared by using crispr to inactivate anpep, the gene which codes for apn. the anpep ko ipi- cells were completely resistant to tgev, but retained permissiveness for pdcov. the authors concluded that "papn is likely not a critical functional receptor for pdcov, although it is involved in pdcov infection". the third outcome related to papn and pdcov is found in li et al. ( ) , who evaluated the permissiveness of anpep knockout (ko) st cells to infection with tgev and pdcov. st cells lacking papn were completely resistant to tgev but retained a small capacity to support pdcov infection. the authors concluded that pdcov utilizes papn as a primary receptor for virus attachment, but the presence of a second co-receptor contributes to the permissiveness of cells for infection. furthermore, the pdcov coreceptor can retain function independent of papn. in this study, we investigated the role of papn as a receptor for pdcov by evaluating the permissiveness of different cell populations derived from the lungs of anpep ko and wild-type (wt) pigs. porcine alveolar macrophages from anpep ko pigs were resistant to pdcov and tgev. however, lung fibroblast-like cells, which appeared following the outgrowth of anpep ko pam cultures, were susceptible to pdcov but remained resistance to tgev. furthermore, anpep ko pigs supported pdcov infection. the results support a role for papn as a receptor for pdcov, but the presence of a second, unknown receptor or factor can substitute for papn function. porcine alveolar macrophages (pams) from anpep ko and wt pigs were used to evaluate the permissiveness of cells for infection with pdcov and tgev. as shown in figure panels a and b, the anpep wt pams were permissive for infection with pdcov and tgev, while no infected tgev or pdcov cells were detected in pams from the anpep ko pigs. the results showed that pams from pigs lacking a functional anpep gene are resistant to tgev and pdcov infection. the long-term culture of pam cultures typically results in the outgrowth of a minor population of lung mesenchymal stem cells (mscs), which exhibit a fibroblast-like morphology (khatri et al., ) . by two weeks, the pam cultures were completely overgrown with fibroblastlike cells along with the disappearance of macrophages. the fibroblastlike cells from the wt and ko pigs were passaged at least two times and then infected with pdcov or tgev. the fibroblast-like cells derived from the anpep wt pigs were permissive for both tgev and pdcov ( fig. c and d) . however, the anpep ko fibroblast-like cell cultures showed no evidence of tgev infection, but showed several pdcovinfected cells, all possessing a fibroblast-like morphology. these data confirmed the requirement of apn for the permissiveness of the fibroblast cells to tgev; however, the absence of apn had no effect on infection of fibroblast-like cells with pdcov. the permissiveness of anpep wt and ko pams and fibroblast-like cells for tgev and pdcov infection was also evaluated by determining percent virus antigen-positive cells after infection with different mois of virus. the results for wt pams are shown in fig. a . an moi = produced % and % antigen-positive cells for pdcov and tgev, respectively. the corresponding virus dilution endpoints were . and . moi. increasing the moi to increased the percent pdcov the results for virus yields on anpep wt and ko fibroblast-like cells are shown in fig. . at an moi = , pdcov grew to near equal levels on fibroblast-like cells derived from the anpep wt and ko pigs; whereas, tgev could only be propagated on fibroblast-like cells derived from the anpep wt pigs. previous work from our lab showed that anpep ko pigs are completely resistant to infection with tgev (whitworth et al., ) . we tested the ability of pdcov to infect anpep ko pigs. the results for virus infection are summarized in table . all pigs were positive for pdcov nucleic acid at one day after infection. on the second day, two anpep wt and three ko pigs remained rt-pcr-positive. by three days after infection, only one pig, an anpep ko pig, was positive for pdcov nucleic acid. by four days after infection, all pigs were negative for virus nucleic acid in feces. serum samples obtained at three days after infection were negative for virus nucleic acid (data not shown). at four days after infection, one wt and one ko pigs were removed from the study and necropsied. rt-pcr amplification of pdcov nucleic acid in intestines and mesenteric lymph nodes showed that both pigs were negative for pdcov. the presence of pdcov infection was also assessed by virus-specific neutralizing activity measured in serum at the termination of the study, days after infection. the results, summarized in table , showed that two of the remaining six pigs seroconverted; one wt and one ko pig. clinical signs, such as diarrhea and fever, were not detected in any of the infected wt or ko pigs. the absence of clinical signs is likely a result of the older-age pigs used in the infection study. together, the rt-pcr and serological results showed that anpep ko pigs are not resistant to infection with pdcov. the results from this study showed no pdcov infection of pams obtained from anpep ko pigs (see fig. a ), which supports the observations of wang et al. ( ) , who concluded that apn is a receptor for pdcov. in contrast, the pdcov-positive infection results for fibroblast-like cells derived from the anpep ko cultures support the observations of zhu et al. ( ) , who concluded that apn is not a functional receptor for pdcov (see fig. c ). when taken together, our data support the observations of li et al. ( ) who concluded that there are apn-dependent and apn-independent receptors for pdcov. we hypothesize that pams possess only the apn receptor, while the lung-derived fibroblast-like cells possess both receptors. however, since the apn-independent receptors for pdcov have not been identified, we currently cannot easily prove this hypothesis and it remains to be elucidated in future studies. ultimately, the importance of papn as a receptor for pdcov is determined by the permissiveness of anpep ko pigs for infection. the results in table , showing positive results for the presence of pdcov nucleic acid in the feces of infected anpep ko pigs confirms the results obtained following the infection of ko fibroblast-like cells. these data support the notion that apn is not required for pdcov infection of pigs. however, the conclusion that anpep ko pigs retain permissiveness for infection with pdcov comes with a few caveats. for example, all fecal samples on day after infection were positive for pdcov nucleic acid and by days, the virus was only detected in one ko pig. intestinal tissues from the two pigs tested, one wt and one ko, were negative for pdcov. the positive results obtained for feces can be explained by the presence of environmental contamination caused by the residual inoculum used for infection. however, support for a productive infection of the ko pigs comes from the seroconversion of one ko pig at days after infection (see table ). a second caveat comes from the transient nature of pdcov infection in the wt and ko pigs used in this study, which made it difficult to determine if apn plays a role in the disease process. previous studies show that neonatal piglets are more susceptible than weaned pigs to enteric virus infection jung et al., ) . the relationship of pig age and pdcov infection outcome has not been studied in detail; however, it is speculated that pdcov infection may be age-dependent, similar to pedv, with more severe outcomes in younger piglets. in this study, - day-old anpep ko and wt pigs were used for pdcov inoculation. in future studies, a more sensitive neonatal pig infection model can be used which may yield a more accurate determination regarding the role of apn in pdcov infection of its natural host. the anpep-edited pigs used in this study were derived by breeding founder animals created using direct zygote injection of crispr/cas along with two crispr guides directed at exon of anpep . along with sequencing of the anpep gene, the apn phenotype for each knockout allele was confirmed by the absence of cd expression in intestines and by the resistance of anpep ko pigs to infection with tgev (whitworth et al., ) . a. stoian, et al. virology ( ) - the pdcov isolate, south dakota, was used for the infection of cells and pigs (vitosh-sillman et al., ) . pdcov stocks were prepared on st cells maintained in mem supplemented with % fbs, pen-strep ( units/ml and μg/ml, respectively), μg/ml fungizone, mm hepes mem, and . μg/ml l- -tosylamide- -phenylethyl chloromethyl ketone (tpck) trypsin, as described in chen et al. ( ) . after h, the media was replaced with mem-fbs with antibiotics. tgev, purdue strain, was obtained from iowa state university. tgev was maintained in the same media without tpck. cells were maintained at °c and % co . after h, the virus was harvested and titrated on st cells. serial : dilutions of virus in triplicate were performed on a well plate containing st cells. twenty-four h later, the cells were fixed for min in % acetone and air-dried. for detection of pdcov, cells were stained with anti-n protein mab sd - (kindly provided by steve lawson at south dakota state university), which was diluted : in pbs with % goat serum (pbs-gs). detection of tgev was performed using anti-fipv - mab (custom monoclonals international, usa), diluted : in pbs-gs. after h incubation with antibody at o c, the cells were washed with pbs and bound antibody detected with alexafluor -labeled goat anti-mouse igg (cat. no. a- , invitrogen) diluted : in pbs-gs. after h incubation at °c, the plates were washed with pbs, nuclei counterstained with propidium iodide (pi), and cells viewed under a fluorescence microscope. the % tissue culture infectious dose (tcid /ml) was calculated according to the method of reed and muench ( ) . the recovery of porcine alveolar macrophages is described in wells et al. ( ) . lungs were removed from euthanized pigs and lavaged by pouring ml of cold pbs into the trachea. pams were sedimented by centrifugation at ×g for min at °c and cells re-suspended and washed once in cold sterile pbs. the final cell pellet was re-suspended in freezing medium containing % rpmi with antibiotics, % fbs, % dimethylsulfoxide (dmso) and stored in liquid nitrogen until use. for the infection, approximately pams were added to each well of a well plate and incubated overnight at o c in % co . the cells were gently washed to remove non-adherent cells. serial : dilutions of virus in media were added to wells in triplicate. after an overnight incubation, the cells were washed with pbs and fixed for min in % acetone. presence of either pdcov or tgev in the infected pams was confirmed as described above. for enrichment of lung mesenchymal stem cells, pams were cultured in rpmi- supplemented with % fbs, l-glutamine, and antibiotics. the continuous maintenance of pams for two weeks resulted in the overgrowth of cultures by fibroblast-like cells, which comprise only about % of the lung lavage material. the confluent cells were removed by trypsinization and passaged at least times. the infection of fibroblast-like cells was carried out as described for the pam cultures. all experiments involving animals and viruses were performed in accordance with the federation of animal science societies guide for the care and use of agricultural animals in research and teaching, the usda animal welfare act and animal welfare regulations; and after approval by the kansas state university and university of missouri institutional animal care and institutional biosafety committees. three ko piglets were obtained from an anpep −/− sow mated with an anpep −/− boar. the anpep ko genotype of each piglet was confirmed by pcr and dna sequencing. to avoid unnecessary stress, the weaned piglets were transported from the university of missouri rearing facility to kansas state university at three weeks of age. three anpep wt piglets, obtained from a separate mating, were included as positive infection controls. at the time of virus infection, the ko and wt pigs were and days old, respectively. pigs were infected with . log tcid of pdcov administered as a single oral dose in ml of culture medium. a cm tube was attached to the end of the inoculating syringe to allow the virus to flow down the back of the throat. throughout the study, the infected wt and ko pigs were housed in the same pen to allow for continuous pig-to-pig contact. caretakers and researchers were blind as to the genotype of each pig. fecal swabs were collected daily from each pig beginning one day prior to infection until the end of the study. each swab was placed in a ml conical tube containing ml of mem with pen-strep and fungizone. the tube was vortexed briefly to mix the swab contents, aliquoted into . ml cryovial storage tubes, and stored at − °c. blood for serum was collected into serum separator tubes at , , , and days after infection. total rna was extracted from fecal and serum samples using a magmax tm - total rna isolation kit (invitrogen tm ) according to the manufacturer's instructions. rt-pcr was performed using a commercial kit, ez-ped/tgenatur/pdcov mpx . (tetracore) on a cfx- realtime pcr system (bio-rad). results were reported as ct. serum samples were serially diluted : , starting at a dilution of : in μl of mem supplemented with % fbs, antibiotics and . μg/ml tpck trypsin. one hundred microliters of diluted serum were mixed with μl of pdcov south dakota an tcid virus in μl media tcid / μl on a well plate and incubated h at °c. samples were transferred to a -well plate of confluent st cells and incubated for h at °c, % co . infected cells were detected by ifa using an anti-pdcov mab. the neutralizing antibody titer was reported as the highest serum dilution at which % of virus infection was inhibited. pathogenicity and pathogenesis of a united states porcine deltacoronavirus cell culture isolate in -day-old neonatal piglets aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev determinants essential for the transmissible gastroenteritis virus-receptor interaction reside within a domain of aminopeptidase-n that is distinct from the enzymatic site cloning of the mouse hepatitis virus (mhv) receptor: expression in human and hamster cell lines confers susceptibility to mhv major receptor-binding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein comparative pathogenesis of us porcine epidemic diarrhea virus (pedv) strain pc a in conventional -day-old nursing piglets vs. -day-old weaned pigs porcine lung mesenchymal stromal cells possess differentiation and immunoregulatory properties broad receptor engagement of an emerging global coronavirus may potentiate its diverse cross-species transmissibility angiotensin-converting enzyme is a functional receptor for the sars coronavirus characterization of porcine epidemic diarrhea virus isolate us/ iowa/ / , infection in -day-old cesarean-derived colostrum-deprived piglets dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc a simple method of estimating fifty percent endpoints coronaviruses emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences effect of porcine epidemic diarrhea virus infectious doses on infection outcomes in naive conventional neonatal and weaned pigs experimental infection of conventional nursing pigs and their dams with porcine deltacoronavirus porcine deltacoronavirus engages the transmissible gastroenteritis virus functional receptor porcine aminopeptidase n for infectious cellular entry detection and genetic characterization of deltacoronavirus in pigs replacement of porcine cd scavenger receptor cysteine-rich domain with a cd -like homolog confers resistance of pigs to genotype but not genotype porcine reproductive and respiratory syndrome virus zygote injection of crispr/ cas rna successfully modifies the target gene without delaying blastocyst development or altering the sex ratio in pigs resistance to coronavirus infection in amino peptidase n-deficient pigs contribution of porcine aminopeptidase n to porcine deltacoronavirus infection the work was supported by genus plc and food for the st century at the university of missouri. the authors acknowledge many people, including undergraduate students who made many of the experiments possible. key: cord- -sp tai authors: jiang, xinpeng; hou, xingyu; tang, lijie; jiang, yanping; ma, guangpeng; li, yijing title: a phase trial of the oral lactobacillus casei vaccine polarizes th cell immunity against transmissible gastroenteritis coronavirus infection date: - - journal: appl microbiol biotechnol doi: . /s - - - sha: doc_id: cord_uid: sp tai transmissible gastroenteritis coronavirus (tgev) is a member of the genus coronavirus, family coronaviridae, order nidovirales. tgev is an enteropathogenic coronavirus that causes highly fatal acute diarrhoea in newborn pigs. an oral lactobacillus casei (l. casei) vaccine against anti-transmissible gastroenteritis virus developed in our laboratory was used to study mucosal immune responses. in this l. casei vaccine, repetitive peptides expressed by l. casei (specifically the mdp and tuftsin fusion protein (mt)) were repeated times and the d antigenic site of the tgev spike (s) protein was repeated times. immunization with recombinant lactobacillus is crucial for investigations of the effect of immunization, such as the first immunization time and dose. the first immunization is more important than the last immunization in the series. the recombinant lactobacillus elicited specific systemic and mucosal immune responses. recombinant l. casei had a strong potentiating effect on the cellular immunity induced by the oral l. casei vaccine. however, during tgev infection, the systemic and local immune responses switched from th to th -based immune responses. the systemic humoral immune response was stronger than the cellular immune response after tgev infection. we found that the recombinant lactobacillus stimulated il- expression in both the systemic and mucosal immune responses against tgev infection. furthermore, the lactobacillus vaccine stimulated an anti-tgev infection th pathway. the histopathological examination showed tremendous potential for recombinant lactobacillus to enable rapid and effective treatment for tgev with an intestinal tropism in piglets. the tgev immune protection was primarily dependent on mucosal immunity. lactic acid bacteria (lab) are a group of gram-positive bacteria that includes species of lactobacillus, leuconostoc, pediococcus and streptococcus. consumed for centuries, lab has enjoyed a long and safe association with humans and animals for healthy food. over the past decade, there has been increasing interest in the use of lab as mucosal delivery vehicles. the application of lab stems from research into effective strategies to deliver vaccine antigens, which may come into contact with the mucosal tissues for the first time, such as the intranasal, oral and genital mucosal surfaces (lavelle and o'hagan ; malik et al. ). mucosal delivery of therapeutics or vaccines for chronic diseases and infections of mucosal origin could increase their potency and specificity. because the mucosal immune system builds an effective iga barrier in no more than days, contact with the mucosal tissues will neutralize the pathogenic microorganism outside of the host. there are abundant studies in the field of lab vaccines. one major advantage of the use of lab as a delivery vehicle for vaccines is that the bacteria can elicit both antigen-specific secretory immunoglobulin (ig) a and an effective systemic immune response. the specific igas have the same function as the neutralizing iggs. some candidate lab vaccines elicited antigen-specific iga responses in faeces, saliva or bronchoalveolar and intestinal lavage fluids (wells and mercenier ) . additionally, lactobacilli are probiotics that may confer health benefits to the host (bengmark and gil ; corthesy et al. ; isolauri et al. ; saarela et al. ) , and there is accumulating evidence that lactobacilli are effective at preventing intestinal disease in both humans and animals due to their ability to inhibit pathogen adhesion to the intestinal wall and prevent inflammatory processes (blum and schiffrin ; de vrese and marteau ; ouwehand ; sartor ; sheil et al. ). because the porcine digestive tract is similar to the digestive tract of human infants with respect to anatomical and histological features and digestive physiology (kararli ; oswald et al. ; tadros et al. ) , the pig has been used as an animal model to study gastrointestinal diseases of human infants (gunzer et al. ) and vaccination studies against these diseases. coronaviruses (covs) comprise a large family of enveloped, positive-stranded rna viruses that infect a broad range of animal hosts as well as humans. well-known representatives are porcine transmissible gastroenteritis virus, porcine respiratory cov, porcine epidemic diarrhoea virus, canine cov, feline cov, bovine cov, human covs, severe acute respiratory syndrome-associated cov, murine hepatitis virus (mhv), avian cov infectious bronchitis virus (ibv) and turkey cov (tcov). the most famous and critical coronavirus is the middle east respiratory syndrome virus found in africa and asia (siddell et al. ). this study investigates whether mucosal immunization is effective in stimulating a protective immune response against cov infection. transmissible gastroenteritis coronavirus (tgev), which is a member of the genus coronavirus, family coronaviridae, and order nidovirales, is an enteropathogenic coronavirus that causes highly fatal acute diarrhoea in newborn pigs (cavanagh ) . the viral rna consists of a single strand comprised of three major structural proteins: a phosphorylated nucleoprotein (n protein) and two glycoproteins (the membrane (m) and the spike (s) proteins) (schwegmann-wessels and herrler ). the s protein has four sites (a, b, c and d). both the a and d sites were demonstrated to induce tgev-neutralizing antibodies (di-qiu et al. ) ; however, the a site is highly glycosylated and thus is not suitable for expression in the lab prokaryotic expression vector. additionally, the different tgev sites induce different immune responses. following infection with virulent transmissible gastroenteritis coronavirus, isolated mesenteric lymph node cd + t cells mounted a specific proliferative response against infectious or inactivated purified virus upon secondary in vitro stimulation (anton et al. ) . the peptide n defines a functional t helper epitope that elicits t cells capable of collaborating with b cells specific for different tgev proteins (anton et al. ) . the most important finding is that oral immunization with a recombinant lactobacillus vaccine and infection with tgev elicit various immune responses, such as humoral immunity and cellular immunity. here, an oral lactobacillus casei vaccine against anti-transmissible gastroenteritis virus developed in our laboratory was used to study the mucosal immune response (jiang et al. ) . in this l. casei vaccine, repetitive peptides expressed by l. casei (specifically the mdp and tuftsin fusion protein (mt)) are repeated times and the d antigenic site of the tgev spike (s) protein is repeated six times. the pig model was developed to study intestinal mucosal immune responses (ruan and zhang ) . probiotic feed supplementation may benefit the animal host directly by preventing infection and combating the causative agent of the intestinal disorder by balancing the disrupted equilibrium of the enteric flora and augmenting the host's immune responses. however, lab vaccine has not received national law or certification for human or animal use against coronaviruses. the first clinical trial to use recombinant lab demonstrated that the containment strategy for l. lactis expressing recombinant il- was effective against crohn's disease (braat et al. ) . our laboratory has researched the lab vaccine for more than one decade, and we have developed many lab vaccines in the field of piglet diarrhoea (di-qiu et al. ; liu et al. ; qiao et al. ; yigang and yijing ) . this study is the last step to obtain new drug certification in china. this lactobacillus vaccine has been demonstrated to increase the treg population in the mouse model (jiang et al. ) . tregs effectively depressed t and b cell proliferation, and some studies demonstrated that this regulation also depressed proliferation in inflammatory bowel disease. our study investigated whether the recombinant lactobacillus vaccine gradually increased the breg and treg populations during the immunization process at the first step of immunization (unpublished data). the intestinal immune system of the pig maintains its ability to mount an active immune response against pathogens and exhibits tolerance to at least food antigens and probably commensal flora through an extremely complex network of cellular and humoral immune interactions. consequently, it is important to elucidate the immunological inductive sites of the protective mucosal immune response following oral immunization in pigs. this vaccine could induce tgev antibody immune responses in both the humoral and mucosal immune systems. mdp and tuftsin possess substantial immunopotentiating properties and can induce cellular-mediated immune responses upon oral administration in mice. however, their use in oral vaccines against tgev challenge in the pig host may have different results. furthermore, the only host (and target of the vaccine) of the transmissible gastroenteritis coronavirus is the weaning piglet. there have been no clear reports concerning whether tgev infection stimulates th or th type immune response. the relationship between humoral and cellular immune responses is not clear; moreover, whether the systemic or mucosal lymphoid response will be the primary immune response following oral immunization is unknown. at present, we are not certain which pathway the lactobacillus vaccine will provoke against tgev infection. our study is the first to analyse immunity in response to oral immunization and tgev infection. virus, bacterium and cell line the l. casei atcc strain used in this study was deposited in atcc and is a plasmid-free strain grown in man-rogosa-sharpe (mrs) medium (sigma) at °c without shaking. the recombinant l. casei (pg: - mt d) was generated in previous study (jiang et al. ) . chloramphenicol (cm) and kanamycin (sigma) were each used at a final concentration of μg/ml. tgev was previously isolated and purified in our laboratory. swine testicle (st) cells were cultured in dulbecco's modified eagle's medium (dmem, gibco) supplemented with % foetal bovine serum (fbs, gibco) at °c with % co . the virus stocks were clarified by centrifugation at ×g for min to remove cell debris, titrated using the cytopathic effect assay and then stored in aliquots at − °c until needed. tgev-seronegative crossbreed (large white) piglets were obtained from a local breeding farm after birth. the piglets were housed separately in specialized cages that were maintained in sterile stainless steel isolators (five piglets/isolator) and fed commercial sterile milk and water. four groups (n = each) of piglets were orally dosed with cfu of pg: - mt d in ml of pbs or pbs alone (jiang et al. ) ; this formulation was used to immunize piglets via an intragastric route in a different immunization protocol. the first group was immunized with pg: - mt d in ml for priming. the second group was immunized with pg: - mt d in ml for priming. the third group was immunized with pg: - mt d in ml for consecutive hours. the forth group was immunized with pg: - mt d in ml for consecutive hours. the control group was immunized with pbs. the piglets were handled and maintained under strict ethical conditions according to international recommendations for animal welfare. seven days after immunization, serum samples were prepared from collected blood samples. the intestinal mucus was collected by rectal swab and subsequently homogenized for min in μl of sterile pbs (ph . ) containing . mol/ l edta-na and then incubated for h at °c. clear extracts of all samples were collected by centrifugation at ×g for min and stored at − °c with protease inhibitors for subsequent analysis. enzyme-linked immunosorbent assay (elisa) plates were coated for and h at °c with full tgev and the vp protein, respectively, which were previously isolated and purified in our laboratory. cultured st cells were used as a negative antigen control. after the wells were blocked for h at °c with pbs containing % skim milk, the plates were washed three times with pbs + tween ( . %) (pbst). mucus and serum (diluted : ) samples were added to the wells in triplicate and incubated for h at °c. afterwards, the plates were washed three times with pbst, and a horseradish peroxidase-conjugated goat anti-pig igg or iga antibody (invitrogen) was added to each well ( : ) and incubated for an additional h at °c. after another round of washing, colour development was accomplished using ophenylenediamine dihydrochloride as a substrate, and the absorbance was measured at nm. naive purified spleen t cells ( × cells/ml) were cultured in -well tissue culture plates and stimulated with . μg ml − of plate-bound anti-cd (pe) antibody (abcam) in complete rpmi medium. single-cell suspensions were stimulated in culture with ionomycin ( μg ml − ) in the presence of monensin ( μm) for - h of culture. the cells were surface labelled and then fixed, permeabilized and intracellularly labelled with ifn-γ and il- antibodies as previously described (moore-connors et al. ; zhou et al. ). for th and th differentiation, the cells were stimulated in the presence of ng ml − anti-ifn (fitc) and ng ml − anti-il- (fitc) (abcam) antibodies. the cells were labelled with carboxyfluorescein succinimidylester (cfse) according to a previous protocol (jiang et al. ) . the data were acquired by gating on the cd + cell population with a facscalibur cytometer. the sequential loss of cfse fluorescence was used to measure cell division and proliferation. groups were housed in the same facility but separated by room and ventilation system. pigs in each room were confined by pens on a solid floor that was rinsed daily, fed a balanced diet ad libitum based on weight and provided free access to water. tgev-challenged pigs received a ml dose of × plaque-forming units (pfu)/ml via oral-gastric gavage on days post-inoculation (dpi). pigs in the control group were administered volume-matched virus-free cell culture media. the control, immunized and no challenge group pigs were randomly selected for necropsy on the fourth day. to assess histological changes in the intestinal tissues, both the intestine and other major organs were examined at necropsy. after h of fixation in % neutral buffered formalin, tissue sections were trimmed, processed, and embedded in paraffin, sectioned, stained with haematoxylin and eosin (h&e) and then examined for pathological changes by light microscopy using a model microscope (olympus, tokyo, japan). real-time rt-pcr (qrt-pcr) was employed to determine the amount of tgev and cytokine gene products (isgs) in rectal swab samples and the intestinal tissues using a cfx tm real-time pcr detection system (bio-rad). total rna was extracted from faecal samples and splenic and intestinal tissues using viral rna extraction and total rna extraction kits (intron) according to the manufacturer's instructions. the extracted rna was subjected to real-time qrt-pcr using a one-step sybr® qrt-pcr reagent kit (takara, shiga, japan). following reversetranscription of the viral rna at °c for min, the resulting cdnas were used for real-time pcr amplification. a standard curve was generated by plotting threshold values against serially diluted plasmid dna encoding the fragment of the tgev spike protein (lee et al. ). all determinations were performed using data from wells evaluated in duplicate to ensure reproducibility. the copy number of the experimental samples was determined by interpolating the threshold cycle values using the standard curve. real-time quantitative pcr was utilized to quantify the products of interest (trl- , − , − , il- , il- , ifn-γ and tgf-β) relative to the quantity of messenger rna (mrna) in the total rna isolated from the splenic and intestinal tissues (dirisala et al. ; kiros et al. ) ; the specific primers are listed in table . the livak method (ΔΔct method) was used to calculate the fold change compared to the β-actin gene control. gene expression data were expressed relative to unimmunized and uninfected piglets. to phenotype immune cells in the spleen and mesenteric lymph nodes, single-cell suspensions were isolated and labelled with fluorochrome-conjugated antibodies. to determine the cell type and the frequency of ifn-γ and il- -producing th and th cells, single-cell suspensions were stimulated in culture with ionomycin ( μg ml − ) in the presence of monensin ( μm) for - h. the cells were surface labelled to detect cd and then fixed, permeabilized and intracellularly labelled with ifn-γ and il- antibodies as previously described (moore-connors et al. ; zhou et al. ). comparison of the piglets' iga and igg titres was conducted by a paired t test. the th cell, cytokine expression and faecal pedv rna shedding titres among litters were compared using one way analysis of variance (anova) followed by duncan's multiple range test. the mucosal immune response of the piglets was evaluated by measuring the iga response in diluted intestinal lavage fluid post-intragastric immunization. as shown in fig. a , the newborn piglets that received ml of recombinant lactobacillus pg: - mt d had the highest mucosal iga levels after immunization. the iga levels at h in the piglets that received ml were slightly lower than the newborn piglets that received ml throughout the process. the vaccine doses also provoked systemic immunity based on the serum analysis and elicited specific igg responses from the immunized piglets (fig. b) . newborn piglets that received ml of the vaccine also had the highest specific igg level. the specific igg titre reached as high as : . finally, the antibody kinetics in the serum and intestinal lavage samples from the animals showed that the specific antibody igg and iga levels were increased on the seventh and eighth days and the titre was decreased during the last week without immunization. to analyse the effect of recombinant lactobacillus pg: - mt d on t helper cells, we evaluated t helper cell polarization. throughout the process, we utilized the model of immunization described here. as shown in fig. , the immune response balance mediated by th and th was broken in favour of th -mediated immunity. the pg: - mt d/l. casei group exhibited % protection within days of challenge with tgev (pg: - mt d/l. casei) (fig. ) . in contrast, the control group piglets immunized with pbs all died after tgev challenge. all piglets that died/were killed were emaciated and had yellow faeces coating the skin and hair. in some piglets, the intestinal lumens were filled with large amounts (approximately - ml) of yellowish foamy fluid. in other piglets, the walls of the small intestine were transparent and thin and the intestinal lumens were empty. no significant gross lesions were observed in other major organs (lung, kidney, liver and heart). formalin-fixed intestinal tissue sections from piglets treated with different treatment modalities were blindly analysed for histopathological changes associated with tgev infection. according to the histopathological analysis, the small intestine samples from the three groups (positive, negative and immunized groups) showed obvious differences. as indicated in fig. , different degrees of pathological changes were detected after infection, especially in the positive group where serious damage was observed. the representative pathological changes included intestinal villi hyperaemia, atrophy and destruction. in the pbs infection group, the jejunum villi were severe atrophied and destroyed, and the ilea exhibited severe lymphocyte proliferation in the lamina propria. the recombinant lactobacillus group showed jejuna with intact villi but low-grade hyperaemia and lymphocyte proliferation, and the ilea exhibited lymphocyte proliferation in the lamina propria. both the pbs and vaccine groups had severe inflammatory responses. the negative control piglets that were not infected showed normal histology. the sequences of the two primers were checked using the ncbi blast software, and no significant alignment with any other animal virus gene was found piglets inoculated with virulent tgev shed the virus for h, followed by profuse diarrhoea that led the piglets to the verge of death - days after inoculation. the tgev load shed in the diarrhoea was . × copies at the th hour, peaked at . × copies at the th hour and then decreased until death in the pbs group (fig. ) . the recombinant lactobacillus vaccine group (pg: - mt d) exhibited the same trend. the tgev copy number was at the th hour, and the copies reached a peak at the th hour. the copy numbers were similar and followed the same trend after reaching the peak. however, the copy numbers in the pg: - mt d group were significantly lower than in the pbs group. next, we investigated the generation of lactobacillus vaccineinduced regulatory cells after infection in piglets. as shown in immunized with pg: - mt d and pbs piglets were orally challenged with tgev. tgev-challenged pigs received a -ml dose of × plaqueforming unit (pfu)/ml via oralgastric gavage on days postinoculation. pigs (control group) were administered volumematched virus-free st cell culture media. all piglets were euthanized at days for necropsy examination fig. , there was a marked increase in the production of il- in cd + t cells. the th immune response induced by the vaccine was seriously broken in favour of th -mediated systemic and mucosal immunity post-infection. the systemic th immune response was higher than the mucosal th mediated immune response. after tgev infection, the body activated more th to protect itself in response to the infection. as shown in fig. , toll-like receptor (tlr) expression was detected in the splenic lymphocytes (sl) and mesenteric lymph node cells (ml) in the piglets in the pbs and vaccine groups. tlr- was higher in the vaccine group than in the pbs group. in contrast, there were no significant differences between tlr- and tlr- . however, the three tlrs exhibited the same trends in the mesenteric lymph node cells compared with the pbs and lactobacillus vaccine. tgev infection induced tlr expression and especially enhanced tlr- and tlr- expression, but the expression levels in the lactobacillus vaccine group were significantly lower than the levels in the pbs group. cytokine expression in the splenic lymphocytes and mesenteric lymph node cells was also analysed and compared in our study. the ifn-γ, il- and il- levels in the splenic lymphocytes from the lactobacillus vaccine group showed marked changes, whereas no significant difference was observed in the level of tgf-β. tgev infection stimulated cytokine expression in the pbs group, including ifn-γ and il- . the vaccine group did not express a notably higher level of ifn-γ and il- compared with the pbs group. the tgf-β expression level in the mesenteric lymph node cells was lower in the vaccine group than in the pbs group; the same trend was observed with ifn-γ and il- . the vaccine group provoked higher il- expression than the pbs group following tgev infection. the il- expression levels in both the splenic lymphocytes probiotics are well known to have additive effects on human health in terms of improving the gut microflora and modulating immune responses (villena et al. ) . studies have also reported that probiotic feeding results in an increased spleen mass, followed by higher levels of total serum proteins, increased globulin levels and enhanced production of secretory iga (dock et al. ) . in humans, lactobacilli colonize the distal small bowel and the large intestine. different probiotic bacteria possess various mechanisms, including adhesins and/ or coaggregation factors, which aid in adhesion and colonization (friedrich ) . during this period, immunization with recombinant lactobacillus is crucially important on the effects of immunization, such as the timing of the first immunization in the protocol and the dose. from these results, we show that the immune response in response to the first priming immunization dose is better than the response to the second immunization because the priming dose is better at initiating adhesion and colonization in the piglet. interestingly, the mucins are large glycoproteins that are the major organic components of mucus. the mucin protein content of the mucus is %, whereas the carbohydrates comprise to % by weight. intestinal mucin has been shown to inhibit the replication of rotavirus in vitro (chen et al. ; superti et al. ). additionally, a high dose of lactobacillus adversely affects the immunization. there is some evidence of diarrhoea after a double dose of the lactobacillus vaccine, but from this result, we find that the two-dose immunization is still the best immunization plan. the reason for the diarrhoea after immunization is the overdose of lactobacillus, which is a type of microbe that causes a disturbance in the intestinal microbial flora for short time. many enteric pathogens must first adhere to the intestinal epithelial cells to initiate intestinal disease. limiting access of the pathogens to intestinal epithelial cells is one strategy to prevent disease that has been investigated previously. for example, the competitive inhibition of bacterial adherence by mimicry of receptors on the apical surface of enterocytes using oral administration of sialylated glycoproteins has been shown to protect newborn calves from the enterotoxigenic e. coli strain k (mouricout et al. ). lactobacillus must colonize the gut soon after birth; therefore, the vaccine could play a role in non-specific immunity. probiotic strains with a high adherence capacity have been demonstrated to enhance the immunoglobulin a response to rotavirus (kaila et al. ) . in this study, we observed a significant increase in the anti-tgev iga titre in the intestinal tract of piglets administered recombinant l. casei. furthermore, we showed that the diarrhoea state of piglets administered l. casei was significantly lower than that of piglets administered saline. in the murine model of ifv infection, the virus moves from the upper respiratory tract to the lower respiratory tract (hori et al. ) . hrv-vaccinated and lactobacillus acidophilus-fed pigs had a significantly higher magnitude of hrv-specific iga and igg antibody-secreting cell responses in the ileum and serum igg antibody and virus neutralizing antibody titres compared to hrv-vaccinated pigs without l. acidophilus colonization (zhang et al. ) . our immunization stimulated the same systemic specific igg titres. the specific antibody response neutralized the challenged tgev, and the load of tgev in the diarrhoea was significantly decreased. the mucosal immune response formed the first barrier function to neutralize tgev. in large scale swine farm surveillance, lower piglet birth weight and higher within-litter variability in birth weight were factors associated with higher losses from birth to weaning (yuan et al. ) . during tgev infection, it is likely that the stronger piglets obtained more iga than their non-immunized counterparts and thus were more likely to survive until the intestinal villi regenerated and immunity developed. during the histopathological analysis, some damage was observed in the small intestine. for instance, the villus wall of the control groups was thin and almost transparent, probably due to atrophy. lymphocyte proliferation in the intestinal lamina propria was also found in some piglets administered an oral dose of recombinant lactobacillus, indicating that recombinant lactobacillus induced a mucosal immune responses in the piglets. taken together, our data show the tremendous potential for recombinant lactobacillus to enable rapid and effective treatment for tgev infection with intestinal tropism in piglets. we evaluated the specific t cell immune responses induced by the recombinant l. casei vaccine compared with the pbs control group in the piglets. we demonstrated that l. casei significantly enhanced the immunogenicity of the tgev vaccine as indicated by the significantly higher magnitude of specific ifn-γ-producing cd + t cell responses. there has reported that mice fed recombinant l. casei with the adjuvant mdp and tuftsin have significantly higher th and th production than control group mice (jiang et al. ) . these results were the same and indicated that l. casei had a strong potentiating effect on both the cellular and humoral immunity induced by the oral l. casei vaccine. similarly, a previous study showed that oral intake of l. fermentum cect fig. cytokines and tlr expression. the rna of splenic lymphocyte (sl) and mesenteric lymphocyte (ml) in immunized piglets, pg: - mt d and pbs groups, were used to analysed the cytokines and tlr expression. ifn-γ, il- , il- , tgf-β, tlrs expressing were detected in splenic lymphocyte (sl) and mesenteric lymph cells (ml) piglets, such as pbs and vaccine groups. *significant difference by student t test (p < . ). data shown were compared using one-way analysis of variance (anova) followed by duncan's multiple range test. representative for three independent experiments significantly enhanced serum th type cytokine production and influenza-specific iga antibody responses to an intramuscular influenza vaccine in adults (olivares et al. ) . the mesenteric lymph nodes were primarily used to analyse tgev infection and the immunoprotection provided by the lactobacillus vaccine in terms of mucosal immunization and infection. ifn-γ induction by tgev results from interactions between an outer membrane domain of el and the pbmc membrane (charley and laude ) ; however, these authors did not study the expression of il- by pbmcs. the expression of ifn-γ was higher than il- in the immunized group, and the th /th balance was broken in our study. after immunization with recombinant lactobacillus, ifn-γ played a major role in the mucosal immune response. however, after tgev infection, the systemic and local immune responses shifted from th to th . the systemic humoral immune response was stronger than the cellular immune response after tgev infection. this is the first study to demonstrate that tgev infection polarized the immune response to th immunity and that recombinant lactobacillus could weaken tgev infection in the form of th immunity. from these results, we found that the immunization did not polarize th immunity more seriously compared to the pbs control group. the proteinbased sars coronavirus vaccine boost induced similar levels of th and neutralizing antibody responses that protected vaccinated mice from subsequent sars-cov challenges but induced stronger th and ctl responses (zheng et al. ) . the uv-inactivated sars coronavirus vaccine retained its immunogenicity and promoted th type immune responses (tsunetsugu-yokota et al. ). the activation of th responses such as il- stimulate b cell proliferation, which can produce specific and nonspecific anti-infection antibodies (grodeland et al. ) ; similarly, both t and b cells have functions following lactobacillus vaccination. the production of il- by th cells results in the proliferation of mast cell growth, and il- stimulates epithelial cell growth (tukler henriksson et al. ) . the proliferation of epithelial cells is crucial for tgev infection. the th response could also stimulate the production of mucus by epithelial cells (zhang et al. ) . il- cells play an essential role in mhv-induced immunopathology, and ifn-γ is important for maintaining the immune balance between th and th responses during acute viral infection (yang et al. ). however, the th /th balance in the negative control group was different than the balance in the immunized group. tgev affects both systemic and local cellular immunity without immunization. il has been reported to have both pro-and anti-inflammatory effects (lafdil et al. ; nagata et al. ) . our results showed that the immunized piglets provoked il- expression form both the systematic and mucosal immune responses after tgev infection. compared with the mucosal immune response, il- expression in the mesenteric lymph nodes was markedly lower than the expression in the spleen cells. however, il- expression in the pbs group was lower than the expression level in the immunized groups, indicating that the lactobacillus vaccine group activated il- expression during tgev infection. swine-origin influenza a virus-infected patients exhibited rapid lymphopenia, t cell activation and a preferential loss of the th subset during the early stage of acute infection (jiang et al. ) , which was consistent with the first reports that swine-origin virus inhibited th proliferation after infection. our study also found the same phenomenon after coronavirus infection in swine compared with the immunized group. the most important finding was that the oral recombinant lactobacillus vaccine stimulated th cell proliferation. the proliferation was involved in cytokine and chemokine production, neutrophil recruitment, promotion of t cell priming and antibody production (dirisala et al. ) . th responses are protective against lethal influenza virus infection in il- deficient mice (mckinstry et al. ). in contrast, a deleterious role of il- has been proposed to contribute to the acute lung injury associated with il- -mediated neutrophil recruitment during influenza virus infection (crowe et al. ). there were significant changes in the il- and ifn-γ expression levels in the mesenteric lymph node cells compared with the pbs group. moreover, il- expression was higher than ifn-γ expression in the spleen cells. the expression of il- and ifn-γ indicated that the recombinant lactobacillus effectively inhibited inflammation after tgev infection. in this study, we also evaluated tlr- , tlr- and tlr- expression in piglets immunized with recombinant l. casei and then challenged with tgev. previously, tlr expression in pigs has been studied only at the mrna level in lymphocytes using real-time pcr because antibodies against porcine tlrs are not currently available. tgev infection did not induce tlr- , tlr- and tlr- expression in the spleen cells. however, tlr expression was significantly different in the mesenteric lymph node cells compared with the pbs and lactobacillus vaccine groups. tlr expression was extremely high in the pbs group compared with the other groups. the cytokine and tlr expression levels in the splenic lymphocytes are indicators of systemic immunity, whereas the expression in the mesenteric lymph node cells was associated with the local and mucosal immune responses. the expression levels of all tlrs in mesenteric lymph node cells were higher in the pbs group than the vaccine group, suggesting that the lactobacillus vaccine effectively inhibited tlr expression. the recombinant lactobacillus groups exhibited jejuna and ilea with lymphocyte proliferation in the lamina propria. transmissible gastroenteritis (tge) coronavirus infection resulted in antibody production from primed mesenteric lymph node cells following an in vitro boost with viral antigen (berthon et al. ); as an intestinal infectious disease, tgev would attack the intestinal tissue and local immune system. exposure of pigs to tgev or prcv results in distinct disease patterns related to differences in tissue tropism (saif ) . however, the increased frequencies of tlr- and tlr- expression in pigs may simply be due to the significantly higher counts of l. casei or mdp and tuftsin, which may translate to an increased magnitude of tlr agonists available to stimulate the host mucosal immune system. it is likely that the higher lab count in the lab plus hrv group played a more pertinent role in the significant increases in tlr and tlr expression (wen et al. ). taken together, tgev immune protection was primarily dependent on the mucosal immune response. systemic immunity did not play a key role after tgev infection. interestingly, il- expression in the vaccine group was significant higher than il- expression in the pbs group challenged with tgev, and th played an anti-inflammatory role in mucosal immunity. il- also stimulated intestinal epithelial cell differentiation and growth. in conclusion, our study suggests that the recombinant 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epithelial cells from chronic rhinosinusitis patients studies of sars virus vaccines critical role of the interleukin- /interleukin- receptor axis in regulating host susceptibility to respiratory infection with chlamydia species acknowledgments this work was supported by the national natural science foundation of china ( ). conflict of interest the authors declare that they have no competing interests.ethical statement the piglets were handled and maintained under strict ethical conditions according to international recommendations for animal welfare. this article does not contain any studies with human participants performed by any of the authors. key: cord- - hrjzapj authors: chen, fangzhou; knutson, todd p.; rossow, stephanie; saif, linda j.; marthaler, douglas g. title: decline of transmissible gastroenteritis virus and its complex evolutionary relationship with porcine respiratory coronavirus in the united states date: - - journal: sci rep doi: . /s - - -z sha: doc_id: cord_uid: hrjzapj the epidemiology and genetic diversity of transmissible gastroenteritis virus (tgev) in the united states (us) was investigated by testing clinical cases for tgev by real time rt-pcr between january and november . prevalence of tgev ranged between . – . % and peaked during cold months until march , in which prevalence decreased to < . %. nineteen complete tgev genomes and a single strain of porcine respiratory coronavirus (prcv) from the us were generated and compared to historical strains to investigate the evolution of these endemic coronaviruses. sixteen of our tgev strains share unique deletions and distinct amino acid changes, which might greatly affect the biological characteristics of the variant tgev, and resulted in a “variant” genotype of tgev. the “variant” genotype shared similar unique deletions and amino acid changes with the recent prcv strain identified in this study, suggesting a recombination event occurred between the ‘‘variant’’ tgev and prcv. moreover, the results indicate the “variant” genotype is the dominant genotype circulating in the us. therefore, this study provides insight into the occurrence, origin, genetic characteristics, and evolution of tgev and prcv circulating in the us. is also associated with protease and adp-ribose "-monophosphatase activities . the functions of other nsps of tgev are unknown. the s glycoprotein attaches to the host cellular receptor porcine aminopeptidase n (papn) or sialic acid, induces cellular fusion, stimulates neutralizing antibodies, and has hemagglutination activity [ ] [ ] [ ] [ ] . the papn-binding domain of the s protein has two major antigenic sites, a and b . in addition, deletions in the orf gene led to attenuation and reduced pathogenesis of tgev and in vivo . in , porcine respiratory coronavirus (prcv), which is believed to have evolved from tgev since prcv and tgev shared a high nucleotide percent identity, was first identified in belgium. compared to the s gene of tgev strains, the s gene of prcv has - nt deletions at n-terminal, including in strains from asia . thus, the s gene is used to differentiate prcv from tgev. recently, a novel strain of prcv (oh ) was identified in the us, but the origin and evolutionary relationship to current tgev strains in the us is unknown. while the decline of tgev is believed to occur in response to partial immunity from prcv infections [ ] [ ] [ ] [ ] , the united states swine industry also made significant changes (increased biosecurity, site production model, etc.) to raise healthier pigs, which may have contributed to the reduction of tgev infections as well. the porcine enteric coronaviruses (including tgev, porcine epidemic diarrhea virus [pedv] , and porcine deltacoronavirus [pdcov]) cause similar clinical presentation, and co-infection of these enteric coronaviruses can occur . in , a highly virulent pedv emerged in china and later spread to the us in april . within less than a year, pdcov was identified in the us , . pedv quickly spread throughout the us , and by march , approximately % of the sow herds were infected with pedv . while the identification of the pedv lead to an increased biosecurity measure within the us swine industry, prevalence of pedv in the sow herds did not significantly decrease until july . within the past couple of years, a chimeric tgev and pedv virus (consisting of a tgev backbone and the spike of pedv) was identified in multiple countries in europe [ ] [ ] [ ] , illustrating the potential emergence of a chimeric tgev and pedv virus in the us. the occurrence and genetic diversity of tgev was investigated prior to and after the identification of pedv and pdcov in the us from the diagnostic cases submitted to the university of minnesota veterinary diagnostic laboratory since pedv recently emerged in the us, and a chimeric tgev and pedv virus was identified in europe. nineteen tgev strains (us, n = and mexico, n = ) and a single prcv strain from the us were sequenced, analyzed, and compared with other global tgev and prcv strains to characterize historical and currents strains. this research will further our understanding of the occurrence, genetic variability, and evolution of an endemic coronavirus in the us and will provide guidance for future efforts to prevent, monitor, and control endemic coronaviruses. decline of tgev positive cases from - . between january and november , , porcine enteric cases, distributed across states in the us and mexico, were tested for tgev by real time rt-pcr, and . % of the cases (n = ) were positive for tgev ( table ). the percentage of tgev positive cases was . % in , increased to . % in , and decreased to . % in (table ) . after the introduction of pedv in the us, the prevalence of tgev decreased further to less than . %. positive tgev cases were detected in the main pig raising regions (midwest, south-central, and southeast) of the us (fig. a) between january and november . most of the cases (n = , ) were from minnesota where . % (n = ) were positive for tgev. the percent of positive tgev cases per state ranged between . - . %, with the highest percentage found in tennessee. a seasonality trend occurred with the positive cases between winter and spring (november to april) compared to summer and fall (may to october) (fig. b) . genomic characteristic, entropy and recombination analyses. the genomic nucleotide sequence alignments of the tgev and pcrv strains revealed two main genotypes (traditional and variant genotypes) ( fig. a) there were major regions of insertion or deletion (indels) between the traditional and variant tgev strains. in the variant group, deletion regions occurred within nsp , deletion regions occurred between the s and orf a genes, deletion regions occurred in orf a genes, deletion region occurred between the orf a and orf b genes and a single deletion occurred in the m genes ( table and fig. b) . interestingly, the same orf a and orf b deletions were present in some of the historical tgev strains. however, these deletions were not present in the tgev strain purdue, which was used to create the attenuated tgev vaccine in the us. the variant tgev strain illinois / had a -nucleotide deletion in orf a, which resulted in the truncated protein compared with our variant tgev strains. the traditional tgev strains z/ , hb/ , and mex/ / shared the same nucleotide deletions in the s gene that were present in the tgev strain, purdue. surprisingly, the pcrv strains contained an assortment of these deletions in their genomes, and compared to the historical tgev strains, the tgev strains from our study (excluding z/ , hb/ and mex/ / ) contained deletions and amino acid changes similar to the recently reported prcv strain oh and the single prcv strain minnesota / from our study. www.nature.com/scientificreports www.nature.com/scientificreports/ to determine the level of nucleotide or amino acid variation across the tgev genomes, entropy analysis was conducted with an alignment of the complete genomes and concatenated amino acid sequences (fig. ) . based on the level of diversity in the dataset and previous work , entropy values greater than . were considered highly variable for the nucleotide and amino acid sequence alignments. the orf and s genes had the highest number of nucleotides with entropy levels above . (n = and n = , respectively) while the orf b, e and, orf genes lacked diversity in nucleotide positions (fig. a ). within orf , the nsp and nsp had the highest number of nucleotide positions with entropy values greater than . (n = and n = , respectively) while a single position was identified in nsp , nsp , nsp , nsp , nsp , and nsp . within the amino acid alignment, the m, n and orf genes lacked positions with entropy levels above . while orf and s proteins (n = and n = , respectively) had residues with entropy values higher than . (fig. ). there were high-entropy positions at orf b www.nature.com/scientificreports www.nature.com/scientificreports/ gene in the nucleotide sequence entropy value analysis while entropy value higher than . were lacking in the amino acid sequence entropy value analysis. recombination analysis was performed with the tgev and prcv strains. a single recombination event was detected by chimera, bootscan, maxchi, and siscan and rdp within the dataset between the recently identified variant tgev strains and the novel prcv strain minnesota / (fig. ) . the tgev variant oklahoma / shares a high nucleotide identity of the first , nucleotides (breakpoint) with prcv strain minnesota / while the remainder of the genome shares a high nucleotide identity with tgev strain minnesota / , indicating a complex nucleotide relationship between prcv and tgev. phylogenetic analyses of tgev and prcv strains. phylogenetic trees of complete genome sequence (n = ) and complete s gene (n = ) revealed two distinct genotypes, representing the traditional and variant tgev strains from the us (fig. a,b, respectively) . in the complete genome and s gene phylogenetic trees, the traditional genotype contained historical strains from the us, the recent strain from mexico, and historical and recent strains from china. from our study, the recent tgev strains from the us formed the variant tgev group, which share a common ancestor with the prcv strain ohio-oh / . also, the variant tgev strains were identified in the three main swine production regions of the us (midwest, south-central, and southeast) indicating substantial geographical distribution. interestingly, whole genome phylogenetic analysis of prcv strain minnesota / clustered within the variant genotype and not with historical prcv strains, indicating significant genetic diversity within prcv strains circulating in the us. since additional partial s gene sequences (first nt) were available from geographically different locations, a partial s gene phylogenetic tree was constructed to further investigate the global evolutionary relationship between tgev and prcv strains (fig. c) . the traditional tgev group consisted of tgev strains isolated from the us, china, japan, south korea, england and mexico. the variant tgev group consisted our us tgev (fig. a) . the different amino acid between traditional and variant tgev strains were highlighted in the predicted crystal structure of rbd ( fig. b-d) . four of eight amino acid substitutions within the rbd (l f, f l, v a, and d e) were exposed on the viral protein while two of the four amino acid changes in papn (d e and t a) were exposed on the viral protein ( fig. b-d) . porcine enteric coronaviruses (pedv, pdcov, and tgev) are significant, emerging pathogens causing severe enteric diseases in the global swine industry. while global historical strains of tgev caused severe enteritis and prcv is endemic in europe, asia, and the us, the mortality rate due to tgev infections has declined in these countries , , . however, a virulent tgev or prcv strain could emerge since the epidemiology of tgev and prcv is different in china. in , prcv was first reported in china and was not identified or reported in china afterwards while tgev is constantly reported as a significant viral pathogen for the chinese pig industry , , . the different clinical status of tgev in china and the western countries could be due to the stringent biosecurity measures in western countries compared to china. tgev was consistently detected in the us between and , but after the introduction of pedv into the us, the swine industry significantly increased their biosecurity to prevent and control pedv infections and the prevalence of pedv were reduced after march in us , , , which indirectly may have reduced the prevalence of tgev to less than . %. our study illustrates a seasonality pattern with tgev in the us with infection peaking during cold months, which has been identified in our swine pathogens as well , , . while tgev has been identified throughout europe , , , , asia , , , , and north america , , only the variant tgev genotype was detected in us pig farms since , suggesting that variant tgev is the dominant genotype currently circulating in the main pig raising midwest, south-central, and southeast regions. in addition, the first tgev genome from mexico was characterized, which helps us to understand the evolutionary relationship of tgev strains from different countries. surprisingly, the mex/ strain is phylogenetically related to the traditional tgev, and not the variant tgev strain. given the close geographical proximity of mexico and us and that the mexican and us pedv strains are phylogenetically related , the variant tgev strains could be circulating in mexico, but were not represented in our study since our study had only a single tgev strain from mexico. the circulation of tgev and pedv in the us indicates a chimeric virus could emerge in the us similar www.nature.com/scientificreports www.nature.com/scientificreports/ as the chimeric virus emerged in europe. characterizing of the current tgev strains will aid in understanding the emergence of virulent or chimeric tgev strains in the us, if such an event would occur in the future. prcv was first identified in belgium in and has been identified multiple countries including belgium , china , japan , uganda and the us . the evolutionary relationship between the recently identified prcv strain (ohio-oh / ) and our prcv strain (minnesota / ) was unclear given the limited number of globally available prcv strains. ideally, additional prcv strains would have been sequenced, but routine screen of prcv does not occur since prcv is not considered as a significant pathogen in swine. the lab accidently isolated the prcv strain, which was added to the study since the genetic and evolutionary relationship of prcv is unknown in the us. the variant tgev strains shared the same nucleotide mutations and amino acid deletions with the novel prcv strain ohio-oh / , and a recombination event between prcv and tgev was identified, illustrating the complex evolutionary relationship between tgev and prcv strains in the us. www.nature.com/scientificreports www.nature.com/scientificreports/ hopefully, future us studies will assess the genetic and evolutionary of prcv strains to fully elicit the complex relationship with tgev. historically, a partial gene fragment or a single gene of tgev was sequenced to differentiate viral strains due to limitations in technology and economic constraints. in this study, ngs technology generated the whole tgev genome, revealing a total of unique indels located in nsp , s, orf a, orf b and m protein, and these regions, excluding the m protein, had high entropy levels in our study (fig. a) . the nsp , s, orf a and orf b proteins of tgev are associated with enteric tropism, immunogenicity, neutralization, sialic acid and other receptor binding activity ability, virulence, protease and adp-ribose ″-monophosphatase activities [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and changes in these proteins might reduce indels efficacy to cause clinical disease. mutations in the spike gene in other coronaviruses has impact on initiation of membrane fusion , sialic acid binding activity , confer resistance to virus neutralization , and render trypsin independent for cell entry and fusion , which could be contribution to a reduction of the overall fitness of the indels. the attenuation of murine coronavirus in the natural host occurred due to deletion of the he, a/he, and / a genes , epidemics of feline infectious peritonitis were contributed to deletions in the orf a and orf b of feline coronavirus , , and disruption of functional expression of accessory protein occurred due to deletion of orf in human severe acute respiratory syndrome coronaviruses . given the results from the previous studies, we hypothesis the indels are significantly attenuated compared to the traditional strains of tgev. further studies are needed to confirm this hypothesis and investigate the effect of these deletions and mutations on the biological characteristics and fitness of the new tgev genotype. in conclusion, a variant genotype of tgev is the predominant in the us and evolutionary relationship between tgev and prcv is complex. the decline of tgev positive diarrhea cases after may in the us may be indirectly associated with the outbreak of ped in the us via increased biosecurity. compared with the www.nature.com/scientificreports www.nature.com/scientificreports/ traditional tgev strains, the unique amino acid indels might affect the biological characteristics of the variant tgev, which could lead to changes in pathogenesis or chimeric virus in the future. veterinarians routinely send samples to the university of minnesota veterinary diagnostic laboratory (mnvdl) to determine potential pathogenic agents contributing to disease and to promote the health of swine herds. the samples may represent clinical outbreaks of diarrhea or are for routine monitoring of enteric pathogens in swine herds. upon arrival, ownership of the samples belongs to the mnvdl and client(s) confidentiality is retained by removing identifiers associated with client(s) information. between january and november , a total of , samples, including fresh intestines and fecal samples from of clinical cases were submitted from us states and mexico and tested for tgev by real time rt-pcr under standard operation procedures (available upon request) and various bacterial and viral enteric pathogens dependent on the veterinarian's request. request may include beta-hemolytic escherichia coli, non-beta-hemolytic escherichia coli, rotavirus a, b, and c, pedv, and pdcov. randomly selected historical positive tgev samples from the mnvdl (n = ) were saved from previous enteric studies, two tgev samples from ohio (z and hb) were supplied from dr. linda saif. routine testing for prcv does not occur since it is not considered a major swine pathogen. however, a prcv isolate from mn was obtained from a nasal swab in during an attempted to isolate other viruses. all samples (n = ) were selected for whole genome sequencing using next generation sequencing as previously described . tgev prevalence information was exported from the mnvdl database and analyzed at the case-level with r software using the ggplot and maps . to investigate the differences in tgev strains and phylogenetic relationship with prcv, our sequences and available sequences from genbank (table s ) were aligned using clustalw in geneious v . . . nucleotide and amino acid entropy analyses of the concatenation orfs (orf a/b, s, orf a/ b, envelope, membrane, nucleocapsid and orf ) was performed using the matlab to determine regions of diversity within the alignment. entropy values higher than . in the nucleotide and amino acid alignments were identified as high variation positions . recombination analysis was performed using recombination detection program (rdp) v with rdp, bootscan, geenecov, siscan and maxchi algorithms (window size is bp). recombination event was represented using similarity plot, with oklahoma as the query strain. the similarity plot was implemented in the simplot, v. . . package , using the two-parameter (kimura) distance model with a sliding window of bp and step size of bp. whole genome (n = ), the whole s gene (n = ), and the partial s gene (first nt) (n = ) (table s ) phylogenetic trees were constructed using the maximum likelihood algorithm, with a gtr nucleotide substitution model (bootstrap analysis with , replicates) by mega v . . the s protein receptor binding domains (rbds) of tgev and prcv were modeled using the open-source modeling server swiss-model provided by the swiss institute of bioinformatics. predicted tertiary structure of the rbds of tgev and prcv were modeled using prcv rbd (pdb accession no. szs) reported in the previous study. 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and porcine respiratory coronavirus pedv orf encodes an ion channel protein and regulates virus production porcine epidemic diarrhea virus orf gene prolongs s-phase, facilitates formation of vesicles and promotes the proliferation of attenuated pedv accessory proteins of sars-cov and other coronaviruses the group-specific murine coronavirus genes are not essential, but their deletion, by reverse genetics, is attenuating in the natural host deletions in the a orf of feline coronavirus associated with an epidemic of feline infectious peritonitis field strain feline coronaviruses with small deletions in orf b associated with both enteric infection and feline infectious peritonitis the -nucleotide deletion present in human but not in animal severe acute respiratory syndrome coronaviruses disrupts the functional expression of open reading frame r: a language and environment for statistical computing. r foundation for statistical computing maps: draw geographical maps. r package version geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data rdp : detection and analysis of recombination patterns in virus genomes full-length human immunodeficiency virus type genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination molecular evolutionary genetics analysis version . swiss-model: modelling protein tertiary and quaternary structure using evolutionary information glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy the pymol molecular graphics system, version . schrödinger, llc the study was funded by the mnvdl. the authors thank the faculty and personal at the university of minnesota veterinary diagnostic laboratory (mnvdl) for their technical services. supplementary information accompanies this paper at https://doi.org/ . /s - - -z. publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -fudeixy authors: xu, kui; zhou, yanrong; mu, yulian; liu, zhiguo; hou, shaohua; xiong, yujian; fang, liurong; ge, changli; wei, yinghui; zhang, xiuling; xu, changjiang; che, jingjing; fan, ziyao; xiang, guangming; guo, jiankang; shang, haitao; li, hua; xiao, shaobo; li, julang; li, kui title: cd and papn double-knockout pigs are resistant to prrsv and tgev and exhibit decreased susceptibility to pdcov while maintaining normal production performance date: - - journal: elife doi: . /elife. sha: doc_id: cord_uid: fudeixy porcine reproductive and respiratory syndrome virus (prrsv) and transmissible gastroenteritis virus (tgev) are two highly infectious and lethal viruses causing major economic losses to pig production. here, we report generation of double-gene-knockout (dko) pigs harboring edited knockout alleles for known receptor proteins cd and papn and show that dko pigs are completely resistant to genotype prrsv and tgev. we found no differences in meat-production or reproductive-performance traits between wild-type and dko pigs, but detected increased iron in dko muscle. additional infection challenge experiments showed that dko pigs exhibited decreased susceptibility to porcine deltacoronavirus (pdcov), thus offering unprecedented in vivo evidence of papn as one of pdcov receptors. beyond showing that multiple gene edits can be combined in a livestock animal to achieve simultaneous resistance to two major viruses, our study introduces a valuable model for investigating infection mechanisms of porcine pathogenic viruses that exploit papn or cd for entry. porcine reproductive and respiratory syndrome (prrs) is a highly infectious viral disease characterized by reproductive disorders including premature birth, late abortion, stillbirth, weak and mummy fetuses, and respiratory dysfunction in piglets and in growing pigs (wensvoort et al., ) . since its discovery in the united states in , prrs has rapidly spread worldwide, with frequent outbreaks causing large economic losses (holtkamp et al., ) . three surface receptors on porcine alveolar macrophages (pams) have been shown to function in prrsv invasion in vivo: heparin sulphate (hs), sialoadhesin (sn), and cd (calvert et al., ; crocker and gordon, ; jusa et al., ) . multiple studies have reported that cd is an essential receptor for prrsv infection, with scavenger receptor cysteine-rich domain (srcr ) serving as the core domain for virus recognition (calvert et al., ; van gorp et al., ; patton et al., ) . gene editing technology has been emerging as an important approach of livestock animal and plant germplasm improvement. the technology makes possible for precise modification of more than one gene simultaneously, which is particularly desirable for obtaining important economic traits that are controlled by multiple genes. in , prather's group was the first to use crispr/cas technology to generate srcr domain-targeted cd knockout pigs. they demonstrated that a cd knockout line was completely resistant to genotype prrsv infection (whitworth et al., ) . subsequently, several laboratories have generated anti-prrsv pigs targeting cd . for example, the cd srcr domain was replaced with human cd -like srcr domain to generate prrsv genotype resistance (wells et al., ) . wei et al., reported homozygous geneedited large white pigs with a bp deletion in exon of the cd gene (wei et al., ) that are fully resistant to genotype prrsv. there are also examples of deletion of the srcr domain seeking resistance to both prrsv genotypes (burkard et al., ) , or introducing a premature termination in the cd srcr domain to generate hp-prrsv (highly pathogenic prrsv)-resistant duroc pigs . deleting the srcr lbp region has also been reported to generate a prrsv genotype resistant pigs . all these studies demonstrate that prrsvresistant pig breeds can be generated by editing the cd gene, enabling alleviation of the severity of prrsv. in addition to prrsv, transmissible gastroenteritis virus (tgev), an acute high-contact infectious virus, is known to frequently occur to co-infect with other porcine diarrhea-associated viruses such as porcine epidemic diarrhea virus (pedv), porcine rotavirus (porv) (zhang et al., ) . tgev is globally distributed and causes tremendous economic losses in pork production (gerdts and zakhartchouk, ) . characterized by vomiting, severe diarrhea, and dehydration, the mortality rate of tgev-infected piglets under the age of days approaches %. tgev is a single-stranded, positive-sense rna coronavirus which targets pig intestinal epithelium for infection (brierley et al., ; wesley and lager, ) . studies have shown that the papn protein acts as a receptor in mediating tgev infection. the viral glycoproteins bind to papn receptors on the surface of small intestinal epithelial cells and mediate membrane fusion, thus resulting in the virus entering into elife digest pig epidemics are the biggest threat to the pork industry. in alone, hundreds of billions of dollars worldwide were lost due to various pig diseases, many of them caused by viruses. the porcine reproductive and respiratory virus (prrs virus for short), for instance, leads to reproductive disorders such as stillbirths and premature labor. two coronaviruses -the transmissible gastroenteritis virus (or tgev) and the porcine delta coronavirus -cause deadly diarrhea and could potentially cross over into humans. unfortunately, there are still no safe and effective methods to prevent or control these pig illnesses, but growing disease-resistant pigs could reduce both financial and animal losses. traditionally, breeding pigs to have a particular trait is a slow process that can take many years. but with gene editing technology, it is possible to change or remove specific genes in a single generation of animals. when viruses infect a host, they use certain proteins on the surface of the host's cells to find their inside: the prrs virus relies a protein called cd , and tgev uses papn. xu, zhou, mu et al. used gene editing technology to delete the genes that encode the cd and papn proteins in pigs. when the animals were infected with prrs virus or tgev, the nonedited pigs got sick but the gene-edited animals remained healthy. unexpectedly, pigs without cd and papn also coped better with porcine delta coronavirus infections, suggesting that cd and papn may also help this coronavirus infect cells. finally, the gene-edited pigs reproduced and produced meat as well as the control pigs. these experiments show that gene editing can be a powerful technology for producing animals with desirable traits. the gene-edited pigs also provide new knowledge about how porcine viruses infect pigs, and may offer a starting point to breed disease-resistant animals on a larger scale. epithelial cells (delmas et al., ; hansen et al., ) . inhibition or direct knockout of papn in small intestinal epithelial cells can mitigate tgev infection zhu et al., ) . papn knockout pigs are resistant to tgev (luo et al., ; whitworth et al., ) . pdcov is a highly virulent porcine coronavirus discovered in that causes watery diarrhea and vomiting in sows and piglets, with piglet mortality rates of % to % woo et al., ) . there is controversy about whether or not papn is a functional receptor for pdcov. wang et al., showed that papn functions as a receptor to promote pdcov entry into cells , while zhu et al., confirmed its involvement but showed that papn was an unnecessary important functional receptor for pdcov infection (zhu et al., ) . li et al., suggested that pdcov infection may require a co-receptor, in addition to papn . using cells isolated from papn knockout pigs, however, stoian et al., showed that these pig cells were still susceptible to pdcov infection in vitro. it was suggested that papn may be one of the receptors for pdcov, and an unknown receptor or factor may compensate for papn function in the absence of papn (stoian et al., ) . however, whether papn knockout pigs may be resistant to pdcov infection in vivo remains unknown. although gene-edited cd knockout (prrv resistant) pigs and papn knockout (tgev resistant) pigs have been previously generated, respectively, pigs that are resistant to the infection of both viruses are lacking. our objectives in the present study were ( ) to knockout cd and papn simultaneously using a gene editing approach; ( ) to verify if the resultant dko pigs are simultaneously resistant to infection by prrsv and tgev; ( ) to use the dko pigs as an in vivo experimental model to test for potential papn-mediated resistance to pdcov infection. we report successfully generated gene-edited large white pigs with both cd and papn gene knockouts using crispr/cas and somatic cell nuclear transfer (scnt). through viral challenge experiments, we found that these dko pigs exhibit complete resistance to genotype prrsv and tgev, and exhibit decreased susceptibility to pdcov infection. in addition, with the exception of meat color score and iron content, no differences in the production performance, reproductive performance, or pork nutrient content were observed between dko pigs and wt pigs. thus, in addition to demonstrating that our dko pigs are robustly resistant to both prrsv and tgev without suffering deleterious effects for production performance, our study also provides insights into ongoing controversy about the papn protein as a potential receptor for pdcov infection of pigs. in order to generate cd and papn dko cloned pigs, we constructed sgrna delivery plasmids targeting these genes, and selected successful dko pig fetal fibroblasts (pefs) as nuclear transfer donors ( figure a ). for cd , the srcr domain-binding site for prrsv in exon (van gorp et al., ; ma et al., ) was selected as the sgrna recognition site. to inactivate the papn protein, a sgrna target site in exon two immediately downstream of the atg start codon was selected ( figure b) . successful dko colonies were cultured as donor cells for scnt (supplementary file ). the cloned pigs generated in this experiment were obtained via both primary and secondary clonings. for primary cloning, the selected dko cells are used as donors for nuclear transplantation. for secondary cloning, the ear-derived fibroblasts of the primary cloned pigs are re-cloned, which rapidly provided a large number of high-quality dko donor cells, thus improving cloning efficiency and resulting in many genotypically identical pigs. in our primary cloning, a total of reconstructed embryos were transplanted into surrogate sows, of which two were pregnant and gave birth to eight live piglets. of these piglets, four survived after weaning ( figure c and supplementary file ). we determined the cd and papn genotypes of the four surviving piglets using pcr and sanger sequencing. the genotypes of the three piglets (# , # , and # ) matched that of cell colony # , which had an bp deletion on both copies of cd near the target site, and a copy of papn carrying a bp deletion on one copy and a bp deletion on the other, both resulting in frameshift mutations or premature termination after the target site ( figure d ). papn protospacer pam in order to generate more dko pigs for viral challenge experiments, we collected ear tissue samples from three piglets (# , # , and # ) and isolated ear-derived fibroblasts. a total of reconstructed embryos generated from ear-derived fibroblasts of # were transplanted into nine surrogates. four sows successful gave birth to a total of live piglets, among which survived post-weaning (supplementary file ). the genotypes of these piglets matched that of # , and the three dko primary clones used for subsequent experiments. we used flow cytometry and western blotting for cd , immunohistochemistry (ihc) and western blotting for papn, and confirmed that expression of both proteins was undetectable in dko pigs but detectable in wt pigs of the same age and breed ( figure e -g). we designed multiple pairs of amplification primers for the px vector backbone to confirm that no random integration of px vector fragments were in cloned pigs (figure -figure supplement ). we also tested for off-target modifications in dko pigs using potential off-target sites for each of the two sgrnas and found no alteration in any of these predicted sites in the cloned pigs (supplementary file ). this data demonstrates that clones of sus scrofa line with multiple gene-edited can be generated through primary and secondary cloning with high efficiency and no off-target detected. for testing of prrsv resistance in pams derived from dko pigs, we selected the highly pathogenic genotype prrsv strain wuh to challenge dko and wt pams at a multiplicity of infection (moi) of . . qrt-pcr and western blot analyses were used to assess prrsv proliferation in pams. at hr post-infection (hpi), dko pams carried a significantly lower prrsv load compared with wt pams, and no viral rna or prrsv-n protein was detected thereafter in dko pams (figure a and b). the low level of prrsv rna that was initially detectable in the dko line at hpi is likely attributable to the adsorbed prrsv independent of the existence of cd , as cd is thought to be primarily responsible for the uncoating and viral rna release processes of prrsv infection van gorp et al., ) . we next sought to examine if dko pigs are resistant to prrsv in vivo. four -day-old dko pigs and six wt control pigs of the same age were challenged with the prrsv strain wuh . nasal intubation drip ( ml: tcid /ml) and intramuscular injection ( ml: tcid /ml) were used to infect both experimental groups. the phenotypic data of body temperature, feed intake, respiration, defecation, and mental condition were recorded daily after infection. as shown in figure c , while fever (over ˚c) began at day post-infection (dpi) and persisted throughout the remainder of the experimental period in the wt group, the body temperature of the dko pigs stayed normal throughout the days of the post-viral challenge observation period. scoring for other clinical symptoms of prrsv at dpi showed that wt pigs exhibited decreased appetite, shortness of breath, cough, malaise, drowsiness, and difficulty walking, whereas the dko group displayed no abnormalities except for a brief cough and diarrhea in two pigs at dpi and dpi, respectively ( figure d ). the body weight of the dko pigs increased, throughout the day post viral challenge observation period: the detected body weights of the wt pigs were all lower than dko pigs after dpi ( figure e ). of the six challenged wt pigs, one was slaughtered at dpi to harvest pams, and the five remaining wt pigs died within dpi. in sharp contrast, all four pigs in the dko group remained healthy, and survived for the entire duration of the -day experiment ( figure f ). among the dead and slaughtered wt pigs, the lungs were swollen, with severe bleeding, and obvious lesions, while the lung tissues of dissected dko pigs did not exhibit lesions or any other distinct symptoms associated with prrsv ( figure g ). hematoxylin and eosin (h and e) staining showed thickening of the alveolar walls and infiltration of a large number of inflammatory examination of prrsv antigens in lung tissue via ihc, it was revealed that the viral antigens were present in the lungs of the wt group, but not that of the dko pigs ( figure h , lower panel). moreover, we measured the prrsv viral load in the serum of both groups at , , , , and dpi and found that in the wt group, the prrsv load increased rapidly and significantly by dpi, reaching its maximum at dpi. in agreement with other experiments showing viral resistance, the prrsv viremia in the dko group remained negative throughout the challenge ( figure i ). we also tested the prrsv viral load in pams, lung tissues, and tonsil tissues of the two groups of pigs after viral challenge. whereas a high titer of prrsv was detected in all tissues examined in the wt group, prrsv was almost undetectable in dko pigs ( figure j ). from dpi, the amount of prrsv-specific elisa antibodies in the serum of wt pigs increased significantly, and antibody levels were positive (s/ p! . ) at and dpi, while such antibodies in dko pigs remained consistently negative (s/p< . ) ( figure k ). taken together, these results provide compelling in vitro and in vivo evidence that the dko pigs are resistant to prrsv infection. following characterization of prrsv resistance, we next sought to determine if double knockout of cd and papn also conferred resistance against tgev. four -day-old dko pigs and six wt control pigs of the same age and breed were fed under the same conditions and infected with tgev. a total of ml of tgev (  tcid /ml) were orally administered to each pig in two doses (day and day , ml/day). at dpi, one dko pig and one wt pig were slaughtered to collect intestinal tissues for pathological examination, and the remaining pigs were housed under regular husbandry conditions until slaughter, and tissues were sampled at dpi. body temperature was recorded daily beginning at day , prior to inoculation, and piglet weighing and blood sampling for serum separation were conducted at , , and dpi. during the viral challenge period, no abnormalities were observed among the pigs, with the exception of two wt pigs that had diarrhea. there was no significant difference in weight gain between the two groups (data not shown). detection of tgev-specific neutralizing antibodies in serum showed no neutralizing antibodies in the dko pigs throughout the experiment, while two of the wt pigs were positive for neutralizing antibodies at dpi, and all wt pigs were positive by dpi ( figure a) . all slaughtered pigs from both wt and dko groups (sampled at dpi and dpi) were dissected to examine potential lesions in small intestine tissues. for the dko group, no lesions were found in the small intestine samples collected at either dpi or dpi ( figure b ). in marked contrast, wt group tissues collected at dpi demonstrated a thin and yellowing small intestine wall, with hemorrhages typical of tgev clinical symptoms, and by dpi there were notable duodenum, jejunum, and ileum hemorrhages, accompanied by intestinal wall thinning and enlarged mesenteric lymph nodes ( figure b ). pathological examination of small intestine tissue sections revealed pathological changes, including necrosis and shedding of intestinal mucosal epithelial cells, intestinal villi fusion, plasma cells accumulating in the lamina propria, and infiltration of eosinophils in the duodenum, figure continued in serum. wt: to dpi, n = ; dpi, n = ; dpi, n = . dko: to dpi, n = . data are expressed as means ± sd. statistical significance was determined by student's t test; ns, p> . ; *p< . ; **p< . ; ***p< . . the online version of this article includes the following source data for figure : source data . the qrt-pcr detection of prrsv load in pams. source data . rectal temperatures of pigs. source data . clinical symptoms scores of pigs. source data . body weights of pigs. source data . the survival rate of pigs. source data . the qrt-pcr detection of prrsv load in serum. source data . the qrt-pcr detection of prrsv load in pams, lung tissues and tonsil tissues. source data . prrsv-specific antibodies in serum (s/p ration). jejunum, and ileum of wt pigs at dpi and dpi, while the same small intestine tissues in dko pigs appeared healthy ( figure c ). we also analyzed the ratio of intestinal villus height (vh) to the crypt depth (cd). the smaller the ratio, the more severe the intestinal villi atrophy. we found that compared with the mock group, the three intestinal segments of the wt group had significant intestinal villous atrophy, and the intestinal villi of these intestinal segments in the dko group did not show atrophy; that is, the degree of intestinal villous atrophy in the three intestine segments in the wt group was significantly higher than that in the dko group ( figure d) . these results consistently demonstrate that our cd /papn dko pigs exhibit strong resistance to tgev infection. pdcov is a highly pathogenic virus that has recently been shown to cause diarrhea in newborn piglets, although the functional receptors for pdcov have not yet been confirmed stoian et al., ; zhu et al., ) . whether papn functions as a receptor or co-receptor in pdcov infection of pigs remains controversial. to test the hypothesis that papn may functionally mediate pdcov infection, we tested the susceptibility of our dko pigs to this virus. two -day-old dko pigs and four wt pigs of the same age and breed were challenged with pdcov. a total of ml of pdcov ( .  tcid /ml) was orally administered to each pig in two doses (day and day , ml/day). during the days of pdcov challenge study, both the dko and wt pigs appeared normal, with no distinct differences in body temperature or weight (data not shown). blood was collected at , , and dpi to assay for levels of virus-specific antibodies. at and dpi, wt pigs were all antibody-positive, while the dko pigs were all antibody-negative at dpi, but carried antibody levels comparable to that of the wt group by dpi ( figure a ). this suggests that the double-gene knockout led to a delayed onset of humoral immunity in pigs, possibly due to delayed-immune system exposure to the virus. all pigs were slaughtered at dpi, and the small intestine tissues were collected to evaluate disease severity. it was found that the intestinal wall of the wt had become thinner, with watery fluid in the small intestine, and mesenteric hyperemia, none of which was observed in the small intestine of the dko pigs ( figure b ). pathological examination of small intestine tissue sections revealed significant lesions in the small intestine tissues of both of the wt and dko groups, which included intestinal villi fusion, infiltration of lymphocytes in the intestinal mucosa, with many lesions in the intrinsic membrane in the duodenum and jejunum tissues. in the ileum, there were signs of necrosis and shedding of intestinal mucosal intraepithelial cells and naked lamina propria. the extent of lesions in the wt pigs was more severe than that of the dko pigs ( figure c ). we also detected the ratio of intestinal villus height to the crypt depth, and found that compared with the mock group, the three intestinal segments of both of the wt group and the dko group had intestinal villous atrophy, but the degree of villous atrophy in the ileal tissue in the dko group was lower than that of the wt group ( figure d) . in addition, we tested the resistance of pams derived from dko pigs to pdcov. dko and wt pams were infected with pdcov, and indirect immunofluorescence assays (ifa), tissue culture infectious dose (tcid ) assays, qrt-pcr, and western blot analyses to assess pdcov proliferation in pams all indicated that dko pams exhibit significantly decreased susceptibility of pdcov infection compared to wt pams (figure -figure supplement ) . these data suggest that although the dko line is still susceptible to pdcov infection, the viral invasion and damage to the small intestines was partially inhibited compared to that of the wt line. we next evaluated the growth and performance indices of dko pigs. three -month-old dko large white boars and three wt large white boars of the same age were selected for slaughter testing. the live weight at slaughter, carcass weight and length, dressing percentage, ham percentage, lean rate, loin eye area, average backfat thickness, muscle ph, marbling, and drip loss were determined. as shown in table , with the exception of meat coloring score, dko pigs showed no difference in comparison with wt pigs for these indices. in addition, there was no significant difference in birth weight or in the average daily gain between wt and dko pigs (supplementary file ). most notably, the meat color score in the dko pigs ( . ± . n= ) was significantly higher than that of wt pigs ( . ± . n= ), although both were within the normal range of to according to the guideline of 'rules for performance testing of breeding pigs' document published by the ministry of agriculture and rural affairs of pr china (ny/t - ) ( table and figure a -b). since the cd protein is known to play a role in the degradation of haemoglobinhaptoglobin (hb-hp), and considering that fe is an important component of haemoglobin, we reasoned that the increased meat color score (redness) may be due to the decreased hb metabolism as a consequence of cd knockout, and subsequently mild accumulation of fe containing hb in the meat. to test this hypothesis, the meat fe level was analyzed, it was found that the concentration of fe was significantly higher in dko pigs compared to wt pigs ( figure c ). we also tested the serum haptoglobin (hp) content and found that the hp content in dko pigs was significantly higher than that of wt control pigs ( figure d ). evaluation of the nutritional components of pork such as total protein, total fat, ash, moisture, specific minerals, and amino acid content was also performed. as shown in table and supplementary file , no differences in these indices were observed between the two groups. in order to test the reproductive performance of the dko boars, semen from dko male pigs (n = ) and that of wt pigs (n = ) of the same age and breed were analyzed. it was revealed that the concentration, motility, and velocity distribution of the sperm from dko boars did not differ from wt boars (table ) . furthermore, there was no difference in the litter size between the two genotypes: dko litters were . ± . (n = , litter size from to ) and the wt litters were . ± . (n = , litter size from to ). in addition, these three dko pigs did not show any growth abnormalities or disease phenomena during the -month rearing process, and no abnormalities were observed in the main tissues and organs after slaughter (data not shown). taken together, with the exception of slight meat coloring score increase, these results show that the simultaneous, editing-based disruption of the cd and papn loci, does not affect the normal growth and reproductive performance of the resultant dko pigs. conventional breeding for complex traits using molecular marker-assisted selection is a lengthy process, requiring multiple rounds of crosses and backcrosses to introgress each individual gene. crispr/cas gene editing not only allows bypassing of this long process, but also provides a possible means to obtain multiple beneficial genotypes in a single generation while also avoiding gene penetration from donor species, thus maintaining the desirable qualities of the original species. zhou et al., first used crispr/cas in combination with scnt to generate knockout of park and pink genes, whose dysfunction are known to contribute to the early onset of parkinson's disease in humans . huang et al., got the pig model with metabolic disorder successfully by editing apolipoprotein e and low density lipoprotein receptor genes simultaneously . our study is the first report on how multiple gene edits can be combined in livestock animal to offer simultaneous resistance to two major viral infection. similar to the previous reports above, double knockout efficiency using crispr/cas -mediated dual gene editing method without any drug or flow cytometry screening was high in our study, reaching . % ( dko cell colonies out of cell colonies). in this experiment, we quickly generated a large number of dko pigs by re-cloning. we found that the re-cloning efficiency ( . %, / ) was much higher than the primary cloning efficiency ( . %, / ). a possible reason for this elevated efficiency could be that the monoclonal cells used for the primary cloning must be cultured in vitro for a long time, which has been reported to inhibit cloning efficiency (li et al., ; magnani et al., ; mastromonaco et al., ) . the donor cells used in re-cloning were ear-derived fibroblasts isolated directly from dko pigs, eliminating the requirement for a long-term, in vitro screening process. our findings support the notion that the efficiency of this approach is not gene specific, and may be applicable to the knockout of other genes that allow improving disease resistance or animal production. in , calvert et al. first discovered that cd functions as a prrsv receptor protein during pams infection, which has since been confirmed by several studies (calvert et al., ; van gorp et al., ; guo et al., ; patton et al., ) . structural studies of cd revealed that the srcr domain corresponding to cd exon seven is necessary to mediate prrsv infection (van gorp et al., ) . in recent years, several groups have successfully generated prrsv-resistant gene-edited pigs by targeting exon of the pig cd gene (burkard et al., ; the online version of this article includes the following source data for the online version of this article includes the following source data for table : source data . comparison of the concentration, motility, and velocity distribution of the sperm between dko and wt large white pigs. ; whitworth et al., ; yang et al., ) . in the present study, we used a single sgrna targeting exon of cd , generated an bp double-stranded deletion that terminated protein translation near the target site. our finding on the complete resistance to prrsv genotype in our knockout line is consistent with those previous reports. cd is known to play a role in promoting the clearance of plasma free haemoglobin (kristiansen et al., ) . our finding that the dko pigs have higher meat fe content and have elevated serum hp levels is consistent with this idea, and may explain the observed darker red color in our dko meat. interestingly, and consistent to our finding, wells et al., also reported that the serum hp levels are elevated in cd knockout pigs (wells et al., ) . despite the slight color score increase, no abnormal growth or reproductive performance was observed in our dko pigs, and the meat color of both dko pigs and wt pigs were within the normal range. production performance evaluations and identification of pork nutritional components showed that our dko pigs were indistinguishable from that of the wt pigs in growth rate and reproductive performances, except for the meat color score and iron content. however, the number of dko pigs tested by us is still small, and the production performance of dko pigs still needs to be verified in large populations in the future. apn is known to be a receptor for many coronaviruses, and studies have shown that separate domains function in virus recognition vs. hydrolase catalytic activity (reguera et al., ) . two research groups have recently demonstrated that papn knockout pigs block tgev but not pedv infection (luo et al., ; whitworth et al., ) . our data showing that papn knockout can completely prevent tgev virus infection are consistent with these recently published findings. in addition to tgev and prrsv, we also determined if papn deletion conferred protection against pdcov. apn is a receptor for multiple coronaviruses and is abundantly expressed on small intestinal epithelial cells, which has led to the speculation that papn may also be a receptor for pdcov. wang et al., and li et al., proposed that papn functions as a receptor in mediating pdcov infection wang et al., ) . however, another study found that knockout of papn in ipi- i cells inhibited but did not completely block pdcov infection, suggesting that papn was not essential for viral recognition (zhu et al., ) . taken together, these studies suggest that papn may be involved in pdcov infection, but pdcov may also be able to enter cell through other pathway(s). our results on the delayed pdcov-specific neutralizing antibodies production, and a reduced extent of gross and histopathological lesions on small intestine in dko pigs compared to wt pigs are consistent with this previous suggestion that papn may play a role but is not the only path for pdcov cell entry. interestingly, a recent study showed that pams, but not lung fibroblast-like cells, from papn knockout pigs showed resistance to pdcov infection (stoian et al., ) , a finding consistent with our in vitro experiments showing that dko pams exhibit decreased susceptibility to pdcov infection. in addition, papn knockout pigs are susceptible to pdcov when virus levels were detected using qrt-pcr, and virus neutralization activity was measured, although the extent of tissue lesions between the ko and wt groups was not compared (stoian et al., ) . our findings are in line with this study reporting that papn knockout pigs are still susceptible to pdcov. however, as reflected by the delay in neutralizing antibody response, and much lighter intestine damage in the dko pigs, the susceptibility of the papn knockout group to the virus is reduced compared that of the wt pigs, indicating the potential role of papn in mediating pdcov infection. additionally, the effect of cd knockout in the delayed adaptive immune response cannot be ignored. despite the important role of cd in innate immunity, an inhibiting effect of soluble cd on the adaptive immune system has also been reported (frings et al., ; o'connell et al., ) . it is thus possible that the delayed adaptive immune response we observed in pdcov-infected dko pigs may be associated with cd knockout-induced immunosuppression. in summary, the dko pigs generated in this study are simultaneously resistant to prrsv and tegv, and exhibit decreased susceptibility to pdcov, while maintaining the same growth and reproductive production traits when compared to wt animals. these pigs may offer breeding starting points for disease-resistant pig colony generation and will be a valuable model to help deepen our understanding of the role and mechanisms of these receptor proteins in the infection mechanisms of multiple viruses. the cd and papn genotypes of colonies and piglets born after nuclear transfer were detected by pcr and sanger sequencing. one third of the cells in the -well plate and the ear tissue were used to extract the genomic dna. the primer pairs cd -f/cd -r and papn-f/papn-r were used to amplify the sequences near the sgrna target sites in the cd and papn genes, respectively. the primer sequences are shown in supplementary file . the pcr products were genotyped by sanger sequencing. potential off-target sites were predicted using an online software: crispor (http://crispor.tefor.net/ ). we identified the potential off-target sites for each of the two sgrnas. twenty pairs of primers were designed to amplify the potential off-target sites from the genomic dna isolated from the dko pigs ( #, #, #). sanger sequencing was performed to determine whether any mutations occurred. the primer sequences are shown in supplementary file . the total protein extracted from lung tissue, liver tissue, and spleen tissue of non-challenged wt pigs and dko pigs was used to detect cd , and protein extracted from duodenal, jejunal, and ileal tissues were used to detect papn expression. whole cell lysates of prrsv-infected pams and pdcov-infected pams were used to quantify the expression levels of prrsv nucleocapsid (n) protein and pdcov nucleocapsid (n) protein, respectively. the protein samples were separated by % or % sds-page and transferred to a polyvinylidene fluoride membrane (millipore). the membrane was blocked with % skim milk for hr, and then incubated with primary antibody at ˚c overnight and secondary antibody at room temperature for hr. chemiluminescent signals were developed with supersignal west pico plus chemiluninescent substrate (thermos scientific) and captured with a tanon- (tanon). cd rabbit polyclonal antibody ( - -ap; proteintech) was used to detect porcine cd . apn polyclonal antibody (a ; abclonal) was used to detect papn, anti-prrsv-n antibody (made in our laboratory) was used to detect prrsv-n protein, anti-pdcov-n antibody (made in our laboratory) was used to detect pdcov-n protein, gapdh rabbit antibody ( ; cell signaling) or b-actin rabbit antibody (ac ; abclonal technology) was used to stain gapdh or b-actin as a loading control. hrp-conjugated affinipure goat anti-rabbit igg(h+l) (sa - ; proteintech) and hrp-conjugated affinipure goat anti-mouse igg(h+l) (sa - ; proteintech) were used as the secondary antibody. pams were isolated from dko piglets and wt piglets. the lungs were obtained from the euthanized piglets. the lung surfaces were rinsed with pbs, and pams were subsequently obtained by bronchoalveolar lavage with prmi- medium (gibco, usa). the collected lavage solution was dispensed into a ml centrifuge tube, centrifuged at g for min, and the supernatant was discarded. pams were washed again with prmi- medium and then frozen in cryopreservation solution containing % fbs and % dmso. for further in vitro infection experiments, pams were cultured in rpmi- medium with % fbs and  antibiotic antimycotic ( ; invitrogen) at ˚c/ % co , and then infected with a highly pathogenic prrsv (hp-rrsv) strain wuh (gen-bank accession number hm ) (li et al., ) at a dose of moi = . and pdcov strain chn-hn- (genbank accession number kt ) at a dose of moi = . the production of progeny prrsv was evaluated through western blot, and qrt-pcr assays, and the production of progeny pdcov was evaluted through ifa, tcid , qrt-pcr and western blot assays. pams were fixed in % formaldehyde for min at room temperature. the cells were then blocked with % bsa overnight at ˚c and incubated with mouse anti-pig cd mabs (mca pe; bio-rad) at ˚c for hr in the dark. after washing with pbs three times, pams were resuspended in pbs and immediately analyzed using a bd facsverse flow cytometer (bd biosciences, ca) and flowjo software (treestar, ca). all wt pigs used in the infection experiment were born from natural breeding, and they were matched by age and breed with the dko pigs. the four dko and six wt pigs used for prrsv wuh viral challenge were both about days old. viral inoculation was conducted by nasal intubation drip ( ml: tcid /ml) and intramuscular injection ( ml: tcid /ml). during the days of prrsv challenge, piglet rectal temperature and clinical symptoms data (feeding, breathing, defecation, mental state) were collected every morning. at the same time, piglet survival rate was recorded, blood was collected, and the piglets were weighed regularly. if any pigs died during the course of the prrsv challenge, pictures were immediately taken and samples were collected. all surviving pigs were slaughtered at days post-infection (dpi) and lung tissue was examined for disease symptoms. four dko pigs and six wt pigs were used for tgev challenge. pigs were inoculated with a total of ml of tgev strain wh- (genbank accession number hq ) (  tcid /ml) that were orally administered to each pig in two doses (day and day , ml/day). for the pdcov challenge, two dko pigs and four wt pigs were orally administered a total of ml of pdcov strain chn-hn- ( .  tcid /ml) divided into two doses delivered on day and day ( ml/day). for both the tgev and pdcov groups, the rectal temperature of the pigs was measured daily for the full day experiment and the diarrhea of the piglets was observed. blood was collected and the piglets were weighed regularly. in the tgev group, a dko pig and a wt pig were slaughtered on day , and the remaining pigs in the tgev group and all pigs in the pdcov group were slaughtered at dpi. after slaughter, the pigs were dissected to observe the gross lesions in small intestine tissue, to collect small intestine tissue samples, and to detect any pathological changes by h and e staining. meanwhile, during these days, wt pigs and dko pigs were reared under the same conditions without any virus infection, and these pigs were used as the mock group. lung tissues of pigs in the prrsv challenge group, and duodenum, jejunum, and ileum tissues in the tgev and pdcov groups were collected. the tissues were fixed in % paraformaldehyde fixative, dehydrated, embedded, and cut into ~ mm-thick sections. for histopathology, the sections were stained by h and e. for ihc, tissue sections were stained with antibodies specific to the corresponding protein antigens. tissue sections were then observed and photographed with a fluorescence microscope. the antibodies used to detect papn protein were purchased from abcam (ab ); the antibody used to detect prrsv-n protein was made by our laboratory. the blood tissues of three experimental groups of pigs were collected at different times after viral challenge and the sera were separated. for the prrsv group, the sera from all samples were subjected to prrs antibody detection by commercially available enzyme-linked immunosorbent assay (elisa) kit (idexx, me). the antibody level was determined to be negative or positive according to the s/p value. if s/p< . , the antibody is negative, and if s/p! . , the antibody is positive. in order to detect tgev-specific and pdcov-specific antibody levels in serum, we used a serum neutralization test (snt). briefly, sera were heat inactivated by min of incubation in a ˚c water bath. then serial -fold dilutions of serum samples in four replicates were mixed with tcid of tgev strain wh- in a : ration. after incubation, ml of the mixture was added into st cells (a swine testicular cell line permissive of tgev infection; atcc crl- ) at a confluence of~ %, seeded in well cell culture plates. appropriate serum, virus ( tcid , tcid , tcid , and . tcid ), and cell controls were included in this test. for about hr after incubation, the cells were monitored for tgev-specific cytopathic effects. neutralization titers were calculated as the reciprocal of the highest dilution resulting in complete neutralization. similarly, sera were diluted mixed with tcid of pdcov strain chn-hn- . in contrast, pdcov titers were assessed using llc-pk cells (a porcine kidney cell line permissive of pdcov infection; atcc cl- ) that were washed twice with dulbecco's modified eagle's medium (dmem) (invitrogen, ca), and supplemented with . mg/ ml trypsin (gibco, usa) prior to and after hr incubation with these mixtures. cells were then cultured in dmem supplemented with . mg/ml trypsin for approximately hr, and the neutralization titers of sera from pdcov group were calculated. to quantify the copies of prrsv and pdcov in the infected experimental group, we extracted prrsv rna from pams, serum, lung tissue, and tonsil tissue from both the challenge and the mockinoculated group, and extracted pdcov rna from dko pams and wt pams after infected with pdcov. rna extraction was performed using trizol reagent (omega bio-tek). the rna was reverse transcribed into cdna according to the instructions for a transcriptor first strand cdna synthesis kit (roche). the cdna was then amplified with sybr green real-time pcr master mix (applied biosystems) in an abi real-time pcr system (applied biosystems). rna copy numbers were calculated from a standard curve drawn from positive standards at different dilutions. the primers used for qrt-pcr are listed as follows: '-gcaattgtgtctgtcgtc- ' and '-cttatcctccctgaatc tgac- ' for prrsv; '-gccctcggtggttctatctt- ' and '-tccttagcttgccccaaata- ' for pdcov. dko pams and wt pams in -well cell culture plates were infected or mock-infected with pdcov at a multiplicity of infection (moi) of . at hpi, cells were fixed with % paraformaldehyde for min and permeabilized with methanol for min at room temperature. the cells were then blocked with bovine serum albumin ( %) diluted in phosphate-buffered saline (pbs) for hr, and incubated with a pdcov-n-protein-specific monoclonal antibody for hr and an alexa fluor -conjugated donkey anti-mouse igg for hr. the cell nuclei were counterstained with ', -diamidino- -phenylindole (dapi) for min at room temperature. after three washes with pbs, the stained cells were observed with an inverted fluorescence microscope (olympus ix , japan). pdcov-infected pams were frozen and thawed repeatedly to completely release viruses. next, llc-pk cells (a pig kidney cell line known to be highly permissive to pdcov infection) were seeded in -well plates and were infected with -fold serial dilutions of virus samples in eight replicates. at hpi, pdcov titers were calculated based on cytopathic effects and expressed as the tcid value per milliliter, using the reed-muench method. the amount of hp in serum was measured using an enzyme-linked immunosorbent assay (elisa) kit ( - , alpha diagnostic) specific to pig hp, as previously described . assays were performed in triplicate for each sample. the quality and performance of pigs related to slaughter were determined by a third-party testing center (the national breeding swine quality supervision and testing center (chongqing), ministry of agriculture and rural affairs of china). all testing followed the guidelines stipulated in the 'rules for performance testing of breeding pigs' document published by the ministry of agriculture and rural affairs of pr china (ny/t - ) . briefly, dko pigs and control wt pigs were weighed before slaughter, euthanized after fasting for hr, and hairs, heads, hoofs, and internal organs were removed after carcass dissection. the weight of carcass, length of carcass, loin eye areas, thickness of skin, and backfat thickness of carcass were all measured. ham, skin, bone, lean, and fat were dissected from the left side of the carcass and their individual weights were determined. to evaluate meat quality, we measured muscle ph, meat color score, intramuscular fat, marbling, and drip loss of longissimus dorsi. for analysis of pork nutrition, total protein, total fat, ash, moisture, amino acid, and individual minerals, amino acids were analyzed for the longissimus dorsi. the nutritional content of the pork was tested by the beijing institute of nutritional sources. semen from dko pigs and wt control pigs were collected and returned to the laboratory in a ˚c incubator for testing their quality. the detection system was hamilton-thorne research ivos ii computer-assisted sperm analyzer to measure the concentration, motility, and velocity distribution of the sperm. all data are presented as the mean ± standard error of mean (sem). data from each of the two groups of pigs were compared with an unpaired t-test when a normal distribution was not obtained. the significance levels were set at . , . , and . , as indicated by *, **, ***, respectively. the data was analyzed with graphpad prism . . for windows (graphpad software, la jolla, california). animal experimentation: all experimental protocols related to animal work described in this study were reviewed and approved by the institutional animal care and use committee (iacuc) at the institute of animal sciences, chinese academy of agricultural sciences. all experiments were performed in accordance with the approved guidelines for animal care and management of research projects. (ias - ). decision letter and author response decision letter https://doi.org/ . /elife. .sa author response https://doi.org/ . /elife. .sa supplementary files . source data . amino acid content of dko lean meat and wt lean meat. . source data . birth weights and average daily gains of wt pigs and dko pigs from birth weight to slaughtering weight. table , table and table . quantitative proteomic analysis reveals that transmissible gastroenteritis virus activates the jak-stat signaling pathway characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot precision engineering for prrsv resistance in pigs: macrophages from genome edited pigs lacking cd srcr domain are fully resistant to both prrsv genotypes while maintaining biological function cd expression confers susceptibility to porcine reproductive and respiratory syndrome viruses generation of pigs resistant to highly pathogenic-porcine reproductive and respiratory syndrome virus through gene editing of cd properties and distribution of a lectin-like hemagglutinin differentially expressed by murine stromal tissue macrophages aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev isolation, genomic characterization, and pathogenicity of a chinese porcine deltacoronavirus strain chn-hn- only the soluble form of the scavenger receptor cd acts inhibitory on phorbol ester-activated t-lymphocytes, whereas membrane-bound protein has no effect vaccines for porcine epidemic diarrhea virus and other swine coronaviruses modulation of cd expression by metalloprotease adam regulates porcine reproductive and respiratory syndrome virus entry highly efficient generation of pigs harboring a partial deletion of the cd srcr domain, which are fully resistant to porcine reproductive and respiratory syndrome virus infection the coronavirus transmissible gastroenteritis virus causes infection after receptor-mediated endocytosis and acid-dependent fusion with an intracellular compartment assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers crispr/cas -mediated apoe-/-and ldlr-/-double gene knockout in pigs elevates serum ldl-c and tc levels aminopeptidase-n-independent entry of porcine epidemic diarrhea virus into vero or porcine small intestine epithelial cells n-acetylpenicillamine inhibits the replication of porcine reproductive and respiratory syndrome virus in vitro effect of heparin on infection of cells by porcine reproductive and respiratory syndrome virus identification of the haemoglobin scavenger receptor effect of the number of passages of fetal and adult fibroblasts on nuclear remodelling and first embryonic division in reconstructed horse oocytes after nuclear transfer recombination in vaccine and circulating strains of porcine reproductive and respiratory syndrome viruses broad receptor engagement of an emerging global coronavirus may potentiate its diverse crossspecies transmissibility porcine deltacoronavirus (pdcov) infection suppresses rig-i-mediated interferon-b production aminopeptidase n-null neonatal piglets are protected from transmissible gastroenteritis virus but not porcine epidemic diarrhea virus the crystal structure of the fifth scavenger receptor cysteine-rich domain of porcine cd reveals an important residue involved in porcine reproductive and respiratory syndrome virus infection developmental capacity of porcine nuclear transfer embryos correlate with levels of chromatin-remodeling transcripts in donor cells role of chromosome stability and telomere length in the production of viable cell lines for somatic cell nuclear transfer monocytelymphocyte cross-communication via soluble cd directly links innate immune system activation and adaptive immune system suppression following ischemic stroke modulation of cd receptor expression and replication of porcine reproductive and respiratory syndrome virus in porcine macrophages structural bases of coronavirus attachment to host aminopeptidase n and its inhibition by neutralizing antibodies highly efficient crispr/cas -mediated transgene knockin at the h locus in pigs the use of cells from anpep knockout pigs to evaluate the role of aminopeptidase n (apn) as a receptor for porcine deltacoronavirus (pdcov) sialoadhesin and cd join forces during entry of the porcine reproductive and respiratory syndrome virus identification of the cd protein domains involved in infection of the porcine reproductive and respiratory syndrome virus porcine coronavirus hku detected in us states porcine deltacoronavirus engages the transmissible gastroenteritis virus functional receptor porcine aminopeptidase n for infectious cellular entry generation and propagation of cluster of differentiation biallelic gene edit ing pigs replacement of porcine cd scavenger receptor cysteine-rich domain with a cd -like homolog confers resistance of pigs to genotype but not genotype porcine reproductive and respiratory syndrome virus mystery swine disease in the netherlands: the isolation of lelystad virus increased litter survival rates, reduced clinical illness and better lactogenic immunity against tgev in gilts that were primed as neonates with porcine respiratory coronavirus (prcv) gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus resistance to coronavirus infection in amino peptidase n-deficient pigs discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus cd knockout pigs are fully resistant to highly pathogenic porcine reproductive and respiratory syndrome virus occurrence and investigation of enteric viral infections in pigs with diarrhea in china generation of crispr/cas -mediated gene-targeted pigs via somatic cell nuclear transfer contribution of porcine aminopeptidase n to porcine deltacoronavirus infection the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. for the cd gene, the sgrna was designed to target exon , and for the papn gene, the sgrna was designed to target exon . the sequences of the two sgrnas are as follows: ggaaacc-caggctggttggaggg (cd -sgrna) and gcatcctcctcggcgtggcgg (papn-sgrna). the pam is indicated in bold font. the two sgrna sequences were cloned into the px vector (addgene plasmid # ) and named px -cd and px -papn, respectively. two plasmids were extracted (tiangen, dp ) in large quantities and used to transfect the fetal fibroblasts of large white pigs. the fetuses of large white pigs at -day-old were used to isolate pefs, which were then cultured in dmem medium containing % fbs. when the cells grew to % confluence, approximately cells were transfected with px -cd ( . ug) and px -papn ( . ug) plasmids. a lonza b nuclear transfection system was used for transfection with nucleofector program t- . the entire transfection process was performed according to the kit instructions (lonza, vpi- ). cells were cultured for hr after transfection and then seeded into cm dishes at a density of cells/dish. the culture medium was changed every days, and cells were cultured for days to form singlecell colonies. single-cell colonies were transferred to -well plates for expansion culture. when cells in the -well plates reached confluence, / of the cells were taken for genotype identification, and the remaining cells continued to expand. cells with genotypes identified as double-gene mutations were cultured and frozen for scnt. the oocytes for scnt were derived from a nearby slaughterhouse, and the nuclear donor cells were the dko fibroblasts. the nuclear transfer donor cells were transferred into enucleated oocytes, and the reconstructed embryos were activated and cultured to develop into blastocysts. we then selected well-developed recombinant embryo clones to be surgically transferred into the oviduct of recipient gilts on the day after estrus was observed. after the embryo transfer, the technicians observed the estrus of the sow, and regularly checked the pregnancy by b-ultrasound. changli ge: is affiliated with shandong landsee genetics co., ltd. the author has no financial interests to declare. key: cord- -dpgexphf authors: hu, weiwei; zhang, shuai; shen, yumeng; yang, qian title: epidermal growth factor receptor is a co-factor for transmissible gastroenteritis virus entry date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: dpgexphf transmissible gastroenteritis virus (tgev) causes severe diarrhea and high mortality in newborn piglets. it is well established that porcine intestinal epithelium is the target of the tgev infection, however the mechanism that tgev invades the host epithelium remains largely unknown. aminopeptidase n (apn) is a known receptor of tgev. this study discovered that the extracellular receptor binding domain pertaining to epidermal growth receptor (egfr) interact with tgev spike protein. apn and egfr synergistically promote tgev invasion. tgev promotes apn and egfr clustering early in infection. furthermore apn and egfr synergistically stimulate pi k/akt as well as mek/erk / endocytosis signaling pathways. tgev entry is via clathrin and caveolin mediated endocytosis in ipec-j cells. tgev binds with egfr, and subsequently promotes egfr internalization by a clathrin-mediated endocytosis pathway. these results show that egfr is a co-factor of tgev, and that it plays a synergistic role with apn early in tgev infection. procine transmissible gastroenteritis virus (tgev) is a member of the enteropathogenic alpha-coronavirus (αcov) family. tgev infects intestinal epithelial cells resulting in severe and frequently fatal diarrhea in newborn pigs, with mortality rates reaching % (doyle and hutchings, ) . tgev is an enveloped cov, with a large positivesense single-stranded rna genome, about . kb in length. it has a diameter ranging from to nm, including surface projections. porcine intestinal columnar epithelial cells (ipec-j ) offer a practical model for studying porcine enteric pathogens (brosnahan and brown, ) . we will use this model to study the entrance mechanism of tgev. aminopeptidase n (apn), also known as cd , is a typeⅡtransmembrane glycoprotein, about kda, belonging to a membrane-bound metalloprotease family (delmas et al., ) . most alpha coronavirus use apn as cellular receptors for virus entry, such as human coronavirus e (hcov- e), feline infectious peritonitis virus (fipv), canine coronavirus (ccov), porcine epidemic diarrhea virus (pedv), and transmissible gastroenteritis virus (tgev) (delmas et al., (delmas et al., , kolb et al., ; li et al., ; tresnan et al., ) . the high degree of tropism of tgev for the villous enterocytes of newborn pigs is well established and has been suggested as being a factor in age sensitivity of newborn pigs to the virus (schwegmann-wessels and herrler, ) . there has been some confusion around the question that if apn is the only receptor for tgev entry, why older piglets are not susceptible to tgev, especially since apn was found to be highly expressed in villous enterocytes of both newborn and older piglets. research suggest a known protein, approximately -kda in size, only expressed in the upper villi of newborn piglets, it has high affinity for tgev (weingartl and derbyshire, ) . it has also been demonstrated that apn is not essential for pedv cell entry (ji et al., ; li et al., ) . it is most likely that tgev do have more than one receptor. many cell surface components have been identified as virus receptors, including: chemokine receptors (feng et al., ) , fibroblast growth factor receptors (qing et al., ) , the tumor necrosis factor receptor family (terry-allison et al., ) , and integrin (wang et al., ) . pidermal growth factor receptor (egfr), a member of the receptor tyrosine kinases (rtk) family, is widely expressed on many cells including epithelial and mesenchymal cells (wells, b) . it has been demonstrated that many viruses interact with egfr to facilitate viral entrance, including: influenza a virus (iav), hepatitis c virus (hcv), herpes simplex virus (hsv), and human cytomegalovirus (hcmv) (chan et al., ; eierhoff et al., a; lupberger et al., ; zheng et al., b) . ligand binding to egfr induce receptor dimerization and cross phosphorylation, which in turn actives the intracellular signaling cascades critical for cellular protein synthesis, cytoskeleton reorganisation, apoptotic inhibition, transcriptional activation, and cell motility. the ligand binding to egfr results in rearrangement of the cytoskeleton network through egfr-mediated signaling, and subsequently ligand-egfr complexes are internalize through clathrin-coated pits (zheng et al., a) . many researchers have shown that numerous viruses utilize egfr endocytosis to mediate virus internalization (mercer et al., a) . the interactions between viruses and their receptors are specific, but the affinity is low. many multiple receptor binding sites exist on virus particles which are likely to cluster receptor proteins. it is known that multiple viruses use more than one type receptor to aid uptake into host cells (marsh and helenius, ) . egfr also has been identified as a co-receptor for many viruses, such as human cytomegalovirus (hcmv), hepatitis c virus (hcv), and adenoassociated virus serotype (aav ) (lupberger et al., ; wang et al., ; weller et al., ) . previous studies that we have conducted demonstrated that tgev spike protein interacts with egfr, and stimulates phosphorylation of cofilin as well as stimulating polymerization of f-actin through the pi k-rac /cdc -pak-limk signaling pathway. this is required for efficient tgev entry (hu et al., ) . we further demonstrate whether egfr is another co-factor for tgev. egfr is a transmembrane protein with two dimer forms, it can be divided into extracellular, transmembranal, and intracellular regions. its extracellular region contains two receptor-binding domains receptor ( - aa) and receptor ( - aa). its intracellular protein associated with tyrosine kinase pi k/akt and ras/raf/erk / pathways are activated by phosphorylated tyrosine located in egfr cytoplasmic tails (fig. a) . the objective of our present study was also to study the relationship between apn and egfr in the early stage of tgev infection. ipec-j cells are porcine intestinal columnar epithelial cells that are isolated from the middle jejunum of neonatal piglets. ipec-j cells were purchased from dsmz (germany). hek t cells and swine testis (st) cells were purchased from atcc (united states). ipec-j , st, and hek t cells were cultured in dulbecco's modified eagle's medium (dmem) with high glucose, hepes containing with % fetal bovine serum (fbs, gibco), % penicillin-streptomycin (invitrogen) at °c in a % co incubator (thermofisher scientific). transmissible gastroenteritis virus (strain shxb) was isolated in shanghai, china. the complete genome sequence for tgev shxb is available in genbank (kp . ) (weiwei et al., ) . to analyze viral entry, cells were incubated with tgev at a multiplicity of infection of (moi = ) for h at °c. subsequently, the cells were washed with phosphate-buffered saline (pbs), and maintained in a maintenance medium (dmem supplemented with % fbs and % penicillin-streptomycin) for h at °c in a % co incubator. for viral labeling, viruses were filtered with . µm filter, and then clarified by centrifugation at , g for . h, followed by ultra-centrifugation using %, %, and % sucrose gradient at , g for . h. viruses were labeled with the fluorescent probe dylight , , and nhs ester (thermofisher scientific, waltham, usa), according to the manufacturer's instruction. unincorporated dye was removed by using commercial fluorescent dye removal columns (thermofisher scientific). flow cytometry analysis for the entry of tgev was performed as follows: fluorescent probe labeled "tgev particles" were incubated with ipec-j cells for h at °c. subsequently, the cells were washed with pbs and maintained in dmem for h at °c in a % co incubator, the cells were harvested by . % trypsin, and then washed with pbs three times. cells acquisition was performed by facs (becton dickinson), and the date was analyzed using flowjo software. antibodies used in the present study were obtained from commercial sources. these antibodies included: rabbit anti-human egfr, rabbit anti-human phospho-egfr (tyr ), rabbit anti-human akt, rabbit anti-human phospho-akt, rabbit anti-human phospho-erk / , and rabbit anti-human erk / (cell signaling technology, danvers, usa). mouse anti-procine apn antibody was donated by prof. zhu guoqiang in yangzhou university. mouse monoclonal antibodies to ha and gfp (cmctag, milwaukee, usa). goat anti-rabbit igg (h + l) secondary antibody, dylight conjugate, goat anti-mouse igg (h + l) secondary antibody, dylight conjugate (thermofisher scientific). anti-gapdh monoclonal antibody, hrp-conjugated goat anti-rabbit igg (h + l), and hrp-conjugated goat anti-mouse igg (h + l) (vazyme, nanjing, china). ipec-j cells were washed with pbs and lysed in an ice-cold cell lysis buffer, phosphatase inhibitor and protease inhibitor (thermofisher scientific) were added in the cell lysis buffer according to the manufacturer's instructions. the supernatant of lysates were obtained by centrifugation at , g for min at °c, and subsequently equal protein levels of the prepared lysates were fractionated by sds-page ( - % gradient). the separated proteins were transferred to pvdf (merck millipore), and the membranes were blocked for h in tris-buffered saline (tbs), containing % nonfat dry milk. after which they were reacted with indicated primary antibodies at °c overnight. membranes were exposed to species-specific horseradish peroxidase (hrp)-conjugated secondary antibodies, (dilution : ) followed by enhanced chemiluminescence (ecl, thermofisher scientific) detection by use of autoradiography. western blotting was quantified by quantity one (quantity one -d analysis software - , bio-rad). the intensity of the bands in terms of density was measured and normalized against gapdh expression. all data were expressed as means ± sd of three independent experiments. the plvx-dsred-monomer-n is an hiv- -based, lentiviral expression vector that expresses the gene of interest fused to the dsred-monomer (clontech, palo aito, ca). egfr and apn sequences were inserted into the ecori/bamhi site. egfr receptor and egfr re-ceptor sequences were cloned into a pacgfp -c vector (sali/bamhi) (clontech), and tgev spike sequence was cloned into pcmv-c-ha (bamhi/xbai) (d , beyotime, china). table showed the primers used for cloning. all constructs were verified by dna sequencing. hek t cells were optimized for lentivirus production, we transfected apn or egfr lentiviral overexpression vector and lenti-x htx packaging mix (vsv-g, plp , plp ) into hek t cells using the x-treme-gene hp dna transfection reagent (roche, switzerland), according to the manufacturer's instructions. ipec-j cells were treated with apn, egfr overexpressing lentiviral particles (moi = ), and after h of incubation, infected cells were maintained with fresh dmem, and continued for extra - h to allow the overexpressing lentiviral particles to achieve their maximum effect. his-egfr receptor and his-egfr receptor were cloned into and expressed in escherichia coli bl- , then purified using ni-nta resin. the purified proteins were eluted with elution buffer containing m urea, which was removed using a dialysis bag against buffer ( mm nah po , mm nacl) with gradual reduction in the concentration of urea ( m, m, buffer alone). the concentrations of the purified proteins were measured with the bca assay. the two purified proteins were verified by sds-page and western blot, respectively. plvx-shrna is an hiv- -based, lentiviral expression vector designed to express a small hairpin rna (shrna) used for rna interference (rnai) studies (clontech) ( table ). the best silencing efficiency was observed with clone kf (porcine apn), nm_ (porcine egfr), nm_ . (procine clathrin), nm_ (procine caveolin). these shrna oligonucleotides were inserted into the bamhi/ecori site. all constructs were verified by dna sequencing. his-tagged egfr extracellular receptor binding domain or expressed in e.coli bl and purified in ni-nta columns, the purified products were separated using sds-page and stained with coomassie brilliant blue. (c) the purified egfr extracellular receptor-binding domain or were verified by western-blot. (d) tgev (moi = ) was incubated in dmem containing his- a, his-egfr receptor or his-egfr receptor at °c for h, then incubated with ipec-j cells and cultured for h. the invasion of tgev was detected by rt-pcr. (e) tgev (moi = ) was incubated in dmem containing his- a, his-egfr receptor and his-egfr receptor at °c for h, then incubated with ipec-j cells, and cultured for h, the viral titers of intracellular tgev were analyzed by tissue culture infectivity dose tcid . (f) ipec-j cells were pretreated with his-egfr receptor at different concentrations at °c for h, then incubated with ipec-j cells, and cultured for h. the invasion of tgev was detected by rt-pcr. (g) intracellular tgev were analyzed by viral plaque morphology in st cells. (h) the lysates of tgev-infected ipec-j cells were immunoprecipitated with rabbit anti-egfr or normal rabbit igg. immunoblotting was then performed to determine the presence of egfr and tgev in the egfr immunoprecipitate. (i and j) t cells were co-transfected with a ha-tagged tgev s expression plasmid together with gfp-tagged egfr receptor or gfp-tagged egfr receptor expression plasmid, cell lysates were immunoprecipitated with an anti-ha antibody or an anti-gfp antibody, the resulting precipitates were examined by immunoblotting using an anti-ha or an anti-gfp antibody to examine the interaction between ha-tgev s and gfp-tagged egfr. (** p < . ). ipec-j cells were treated with apn, egfr interference lentiviral particles (moi = ), after h of incubation, infected cells were maintained with fresh dmem and continued for - h to allow the interference lentiviral particles to achieve their maximum effect. in regards to detecting the interaction between tgev s and egfr, cells were lysed in a lysis buffer after infection of tgev (moi = ). cell lysates were cleared by centrifugation at , g for min, and the supernatant was immune-precipitated with rabbit anti-human egfr monoclonal antibody ( s, cell signaling technology, danvers, usa) or normal rabbit igg (a , beyotime, china) at °c for h, and fresh protein a/g magnetic beads (b , bimake, usa) were added and incubated with cells at °c for another h. magnetic beads containing protein complexes were washed five times with pbs and incubated with concentrated tgev at °c for h. magnetic beads containing egfr proteins and tgev complexes were washed five times with pbs. complexes were subsequently boiled for min in × protein loading buffer (takara, janpan), then analyzed by western blotting with specific primary and secondary antibodies. the primary antibodies used to probe membranes were rabbit anti-egfr mab and rabbit anti-tgev n pab. table primers used for cloning. primer sequence ( ′- ′) vector tgev infection causes the co-localization of apn and egfr. ipec-j cells were infected with tgev (moi = ) and cultured for min, then stained for fluorescence microscopy using mouse anti-apn pab, followed by dylight -conjugated goat anti-mouse igg and rabbit anti-p-egfr mab, followed by dylight -conjugated goat anti-rabbit igg. mock-infected cells served as controls. the data shown are from two independent experiments. for detecting the binding domain of tgev s , hek t cells lysates were prepared by ice-cold np- lysis buffer (p f, beyotime, china), and protease inhibitor (thermofisher scientific) was thereafter added according to the manufacturer's instructions. cell lysates were cleared by centrifugation at , g for min, and supernatant was incubated with protein a/g magnetic beads (b , bimake, usa) and normal igg (from the same species as that of the immunoprecipitating antibody) at °c for h, to eliminate nonspecific binding to the magnetic beads or igg. after centrifugation at g for min, the supernatant was incubated with ha (at , cmctag) or gfp (at , cmctag) antibody at °c for h. fresh protein g plus-magnetic beads were added and incubated with cells at °c for another h. magnetic beads, containing protein complexes, were washed five times with pbs, and complexes were boiled for min in × protein loading buffer (takara, janpan). at this point they were analyzed by western blotting with specific primary and secondary antibodies. tgev-infected and mock-infected cells were seeded onto -well plates ( × ) and cultured for h. tgev was collected by freezing and thawing the plates three multiple times, and were determined by the tissue culture infectious dose (tcid ) in st cells. tgev plaque assays were performed on monolayers of st cells seeded in -well plates. cells were inoculated with ten-fold dilutions of stock virus, and incubated for h at °c. these cells were overlaid with dmem containing % fbs and % low melting point agarose (sigma-aldrich). after which they were incubated for about h until plaques were visible. plaques were stained with % crystal violet in methanol. ipec-j cells were grown on coverslips in -well tissue culture plates, after tgev infection, at room temperature, cells were fixed in % paraformaldehyde in pbs for min. they were washed three times with pbs, and incubated with . % triton x- in pbs for min, washed three times with pbs. at this point, cells were blocked in % bovine serum albumin (bsa) for min. for studies analyzing the co- localization of apn and p-egfr, cells were incubated with apn and p-egfr primary antibodies ( : ) at °c, overnight. for studies analyzing the internalization of egfr, cells were incubated with egfr primary antibody ( : ) at °c, overnight, and were followed by three washes with pbs, then subsequently incubated with fluorochrome-conjugated secondary antibodies ( : ) at room temperature for h. cells were washed three times with pbs and the nucleus were stained using μg/ml dapi ( ′, ′diamidino- -phenylindole dihydrochloride)-pbs for min at room temperature. images were captured with a zeiss fluorescence microscopy system and a × objective lens. the co-localization of the two channels was detected using zen (zeiss) software. cells were incubated with tgev at moi = for h at °c and washed with pbs (ph . at °c) three times to remove unbound virus, then maintained in maintenance medium (dmem supplemented with % fbs and % penicillinstreptomycin) at °c in a % co incubator, after the indicated time ( min, min, min, min), cells were washed with acidic pbs (ph . at °c) to remove the virus bound to the cell membrane (not enter the cell), then cell membrance protein was performed according to the manufactures'instructions. ligand egf was used as a positive control, egf ( ng/ml) was added to the cell culture medium for h at °c, and washed with pbs (ph . at °c) three times to remove unbound egf, then maintained in maintenance medium (dmem supplemented with % fbs and % fig. . apn and egfr synergistically activate pi k/akt and mek/erk / signaling pathways. (a) ipec-j cells were transfected with overexpression vector plvx-dsred, plvx-apn-dsred, plvx-egfr-dsred, or plvx-apn-dsred + plvx-egfr-dsred through lentiviral supernatant. cells were infected with tgev at an moi of and cultured for min. normal cells and tgev infected-normal cells served as controls. the activation of downstream signaling pathways analyzed by westernblot with anti-p-egfr, anti-egfr, anti-p-akt, anti-akt, anti-p-erk / , anti-erk / , and anti-gapdh antibodies. (b) ipec-j cells were transfected with interference vector plvx-shrna-apn, plvx-shrna-apnctrl, plvx-shrna-egfr, plvx-shrna-egfrctrl, or plvx-shrna-apn + plvx-shrna-egfr through lentiviral supernatant. cells were infected with tgev at an moi of , and cultured for min normal cells and tgev infected-normal cells served as controls. the activation of downstream signaling pathways analyzed by western-blot with anti-p-egfr, anti-egfr, anti-p-akt, anti-akt, anti-p-erk / , anti-erk / , and anti-gapdh antibodies. (c-h) the ratio of p-egfr, egfr, p-akt, akt, p-erk / or erk / to gapdh was normalized to control conditions. data shown are the mean results ± sd from three independent experiments. (* . < p < . , ** p < . ). penicillinstreptomycin) at °c in a % co incubator, after the indicated time ( min, min, min, min), then cell membrance protein was performed according to the manufactures'instructions (mem-per™ plus membrane protein extraction kit, catalog number: ,thermo). human transferrin-fitc labele was obtained from nanocs (usa). cholera toxin beta subunit (ct-b)-fitc labele was purchased from sigma-aldrich (usa). ipec-j cells were prepared on coverslips in well tissue culture plates, and incubated with dylight -tgev with μg/ml of fitc-transferrin or μg/ml of fitc-ctxb for min at °c to synchronize entry. at this point they were then shifted to °c for min. cells were fixed in % paraformaldehyde for min, and nucleus were stained using μg/ml dapi for min. images were captured with a zeiss lsm confocal laser-scanning microscopy system and a × objective lens. top view images were prepared as compacted z-stack images of non-permeabilised cells. x-y plane and z-axis views of confocal images were prepared using zen le software from zeiss, germany. all results were presented as means ± standard deviation (sd) from three independent experiments and performed using graphpad prism software (sandiego, ca), p values for all data were determined using student's t-test and one-way analysis of variance (anova) (* . < p < . , ** p < . ). purified escherichia coli-expressed fusion protein his-tagged receptor binding protein and were verified by sds-page and western blot, respectively ( fig. b and c) . tgev was incubated with with interference vector plvx-shrna-clathrin, plvx-shrna-clathrinctrl, plvx-shrna-caveolin, or plvx-shrna-caveolinctrl through lentiviral supernatant. normal cells served as controls. cells were infected with tgev at an moi of and cultured for h. the invasion of tgev was detected by rt-pcr. (e-g) ipec-j cells were transfected with interference vector plvx-shrna-clathrin, plvx-shrna-clathrinctrl, plvx-shrna-caveolin, or plvx-shrna-caveolinctrl through lentiviral supernatant. cells were infected with tgev at an moi of , and cultured for h. the invasion of tgev was detected by flow cytometry (e and f). the viral titers of intracellular tgev were analyzed by tcid (f). (h and i) the co-localization of transferrin and cholera toxin with tgev, (scale bar = µm), the immunofluorescence experiment was repeated two times, every time there are three groups of parallel samples. the data shown are the mean results ± sd from three independent experiments. (* . < p < . , ** p < . ). receptor ( ng/ml) and receptor proteins ( ng/ml) at °c incubator for two hours. receptor protein was able to bind with tgev and inhibit tgev invasion ( fig. d and e). receptor protein was able to inhibit tgev invasion in a dose dependent manner (fig. f ). receptor protein inhibition tgev invasion was proven by plaque assays (fig. g) . to investigate the direct interaction between tgev s and egfr, co-immunoprecipitation was performed on lysates from tgevinfected cells precipitated with antibody against egfr. egfr and tgev n protein were detected in the precipitates of tgev-infected cells, indicating that tgev interacts simultaneously with egfr in infected cells (fig. h) . the interaction between tgev s and egfr receptor / receptor were validated in precipitates from t cells co-transfected with plasmids expressing tgev s -ha and egfr receptor / receptor -gfp ( fig. i and j) . this further confirmed that tgev s directly interacts with egfr receptor but not egfr receptor . apn is a receptor of tgev, we wanted to determine whether egfr had any interaction with apn. hence, the localization of apn and egfr the protein of the cell membrane was extracted. cell membrane egfr was analyzed by westernblot using rabbit anti-egfr pab. (b) the ratio of egfr to the mean of e-cadherin and gapdh was normalized to control conditions. the data shown are the mean results ± sd from three independent experiments. was assessed by fluorescent microscopy. in mock-infected cells, it was noticed that apn and p-egfr distributed evenly on the surface of the cell membrane. in tgev-infected cells, egfr was activated. the level of p-egfr increased, apn and egfr were co-localization significantly (fig. ) . to determine the role of egfr and apn in mediating tgev infection, lentivirus interference methods were used to reduce the expression of egfr and apn. ipec-j cells were transfected with lentivirus constructs that expressed apn or egfr targeting shrnas. apn or egfr expression level reduced significantly ( fig. a and b) . the results of real-time reverse transcription pcr (rt-pcr) for detection of tgev invasion revealed that apn-targeting shrna, egfr-targeting shrna, and apn + egfr-targeting shrnas, significantly inhibited tgev entry. the inhibition of apn + egfr-targeting shrnas was seen to be more significant (fig. e) . these results were further verified by flow cytometry, tgev particles were labeled with fluorescent probe dylight , apn-targeting shrna, and egfr-targeting shrna inhibited the invasion of tgev with the similar results. apn+egfr-targeting shrnas showed a more significant inhibitory effect ( fig. d and e) . tgev infection was measured in the supernatant of infected ipec-j cells by tcid (fig. f) . plaque formation in st cells by the intracellular of infected ipec-j cells showed that apn + egfr-targeting shrnas inhibited tgev entry more significantly (fig. g) . all of these results indicated to us that egfr and apn synergistically promote tgev invasion. the activation of phosphatidylinositol- kinase (pi k) and the extracellular signal-regulated kinase / (erk / ) has been identified in many viruses as part of their entry mechanism. egfr is an upstream mediator of pi k/akt signaling pathway and erk / . to determine whether egfr can induce the endocytosis signaling pathway to support tgev entry, the activation of egfr, pi k/akt and erk / were investigated. cells were incubated with tgev at °c for h, and unbound viruses were removed. then cells were incubated at °c for min, and the phosphorylation level of egfr was increased in the early infection stage of tgev. in apn overexpressed, egfr overexpressed, and apn+egfr overexpressed cells, overexpression of egfr and apn were confirmed in figs. a and s , tgev infection caused an a comparably increased phosphorylation level of egfr, akt, as well as erk / than that of the tgev infection control group ( fig. a and c) . in apn-targeting shrna, egfr-targeting shrna, and apn+egfr-targeting shrnas cells, tgev infection resulted in the decreased phosphorylation level of egfr, akt, and erk / significantly in comparison to the tgev infection control group (fig. f-h) . apn-targeting shrna inhibited tgev-induced egfr activation more significantly than egfr-targeting shrna (fig. b and f) . these data demonstrated that apn is required for tgev-induced egfr activation. hence, tgev binding to apn induces egfr activation and is required for viral entry. to explore whether the endocytosis pathway supports tgev entry into ipec-j cells, ipec-j cells were transfected with lentivirus that expressed clathrin or caveolin targeting shrnas. the clathrin or caveolin expression level was reduced significantly (fig. a and b) . rt-pcr for detection tgev invasion results showed that both clathrin and caveolin targeting shrnas significantly inhibited tgev entry (fig. c and d) . this data was reinforced by flow cytometry, tgev particles were labeled with fluorescent probe dylight , clathrin-targeting shrna, and caveolin-targeting shrna which inhibited the invasion of tgev ( fig. e and f) . the viral titers of intracellular tgev were also analyzed by tcid (fig. g ). our previous studies have found that nystatin, an cholesterol removing agent which also functions by inhibiting the lipid/caveolin pathway can in fact inhibit tgev binding and entry. this result also show us that caveolin-mediated endocytosis is a method that can be utilized in tgev internalization. to verify and confirm our results, clathrin and caveolin mediated endocytosis specific markers were used to provide direct evidence in an attempt to prove the tgev endocytosis pathway. we found that both fitc-transferrin and fitc-ct-b were co-localized with dylight -tgev ( fig. h and i) . taken together, we confirmed that both clathrin and caveolin mediated endocytosis are important for tgev entry. to explore the role of egfr in tgev internalization, the internalization of egfr was investigated by confocal laser-scanning microscopy. in normal ipec-j cells, most egfr was located at the cell surface membrane, and egfr was internalized upon egf stimulation. tgev infection also promotes egfr internalization ( fig. a and b) . we also detected cell surface membrane egfr expression levels ( fig. c and d). egfr ligands (egf and tgf-α) induced receptor dimerization, activation, and internalization (wells, a) . ipec-j cells were incubated with tgev for different times at °c for h, and ligand egf was used as a positive control. when egf-stimulated cells were transferred at °c, egfr internalized rapidly. at min post-infection (mpi), most cell membrane egfr was internalized into the cytoplasm. at mpi, egfr was recruited to cell membrane. tgev infection caused egfr internalization in a time dependent manner. egfr was internalized into the cytoplasm from mpi to mpi. in normal cells, most egfr resided on the cell membrane. this data suggests that tgev particles cause the internalization of egfr early in tgev infection. to explore the role of caveolin and clathrin in egfr internalization early in tgev infection, we reduced clathrin or caveolin down in normal ipec-j cells through targeting shrnas, and investigated cell membrane egfr expression levels during tgev invasion. clathrin targeting shrna significantly inhibited egfr internalization. in caveolin targeting shrna cells, egfr was internalized from mpi to mpi, and recruited to the cell membrane at mpi. egfr circulation was faster than that of normal cell groups ( fig. a and b) . these results indicate that tgev causes egfr internalization through the clathrin- fig. . apn and egfr synergistically promote tgev invasion. egfr is a cofactor for tgev invasion. tgev s protein interacts with egfr extracellular receptor binding domain . tgev infection induces egfr internalization and causes apn and egfr clustering. apn and egfr synergistically promote tgev invasion. apn and egfr synergistically activate pi k/akt and mek/erk / signaling pathways. clathrin and caveolin mediate the endocytosis of tgev and egfr internalization through clathrin endocytosis pathway. mediated endocytosis pathway. in this study, we demonstrated that egfr is an another co-factor of tgev, and the tgev spike protein can interact with egfr extracellular receptor binding domain . tgev infection causes the co-localization of apn and egfr. furthermore apn is required for tgev-induced egfr activation. pi k/akt and erk / signaling pathways are involved in tgev internalization. in addition to this, tgev particles and egfr internalize through clathrin-mediated endocytosis. egfr has also been demonstrated to participate in the invasion of other viruses, including: adeno-associated virus serotype , influenza a, hepatitis c virus, and human cytomegalovirus (chan et al., ; eierhoff et al., a; lupberger et al., ; weller et al., ) , suggesting that egfr activation and internalization may be common mechanisms utilized in virus invasion. the activation of pi k and erk / has been found in many virus entry mechanisms, such as: human cytomegalovirus, influenza a, and herpes simplex virus (eierhoff et al., b; johnson et al., a johnson et al., , b zheng et al., b) . pi k activation has been demonstrated to regulate vesicular uptake, and trafficking of ebola virus (saeed et al., ) . ligand binding to egfr on the cell surface induces receptor dimerization and cross phosphorylation, which leads to the activation of downstream signaling cascades of which include: mapk, pi k, jak/ stat, and plcγ signaling pathways (zheng et al., a) . in this study, we found that early in the tgev infection process, apn and egfr synergistically stimulate pi k/akt and mek/erk / signaling pathways, and promoted tgev entry. many pathogens enter the host cell by endocytosis which results in cell surface receptor, ligand, and membrane component internalization (mosesson et al., ) . enveloped viruses are capable of entering directly through the cell membrane surface receptors, or are able to be internalizing via endocytosis via fusion taking place in the endosomal compartment (belouzard et al., ) . our previous published research found that tgev can be internalized into ipec-j cells, however the mechanism of tgev entry is still not known. it has been confirmed that pedv invades via clathrin-mediated endocytosis independent of caveolin-mediated endocytosis (park et al., ) . our results suggest that tgev infection reduces in clathrin or caveolin targeting shrnas cells. furthermore, co-localization between endocytosed fitc-transferrin/ fitc-ctxb and fluorochrome-labeled tgev support the conclusion that both clathrin and caveolin mediated endocytic uptake are the pathways of tgev entry. typically for particle sizes internalized through caveolin ranging from to nm and through clathrin less than nm (aleksandrowicz et al., ) . tgev particles sizes are between and nm, some are smaller tgev particles that are internalized through caveolin-mediated endocytosis. the actin cytoskeleton has been thought of as participating in the formation of the clathrin-mediated endocytosis structure, as well as providing mechanical force to enable complete endocytosis (kaksonen et al., ; smythe and ayscough, ) . our previous research has also found that tgev infection induces actin cytoskeleton rearrangement through cofilin, and actin surround tgev particles in the spatial part of the virus co-localize with the actin, which indicates that clathrin and actin are involved in the early invasion of tgev. receptor clustering is an actin-dependent process that uses rho-family gtpase signaling, and the actin regulatory proteins filamin and cofilin (jimenez- baranda et al., ; yoder et al., ) . the co-localization of apn and egfr is also found in the early infection of tgev, suggesting that egfr phosphorylation and pi k-rac /cdc -limk-cofilin signaling pathways participate in the regulation of actin cytoskeleton. tgev particles movement towards entry sites, the co-localization of apn and egfr are all mediated by actin cytoskeleton re-modeling. in the resting state, most egfr reside on the cell membrane surface. upon ligand binding, egfr is activated and internalized via clathrin-coated pits (wells, a; zheng et al., b) . numerous viruses utilize egfr endocytosis to mediate virus internalization and trafficking to the site of replication during the infection of the host cell (mercer et al., b) . some previous studies have suggested clathrin-coated pits can form naturally at °c in the presence of egf, but are not internalized into the cytoplasm (brown and petersen, ; jiang et al., ) . clathrin-coated pits connected to the cell surface and clathrin-coated vesicles are intracellular membranal structures. lipid rafts function as platforms to active intracellular egfr effector signals and egfr internalization (puri et al., ) . in this study we found that when egf was incubated with cells at °c for h, egf formed clathrin-coated pits connected to the cell surface and egfr became localized in the coated pits. when cells were transferred at °c, egfr was rapidly internalized into the cytoplasm through clathrin-coated vesicles. for tgev infected cells, tgev particles bound with cells at °c. as soon as cells were transferred at °c, tgev stimulated clustering of lipid rafts and activated egfr and effector signals. this process took comparatively more time, hence at mpi, egfr was found to begin internalization of tgev particles. in this study, we found that tgev infection caused egfr internalization, and clathrin-targeting shrna inhibited egfr internalization. egfr also was identified as an another receptor for tgev. we can get to the conclusion that in the early infection stage of tgev, tgev particles bound with apn and egfr, the virus-receptors complex are subsequently internalized by clathrin. caveolin targeting shrna has no effect on the internalization of egfr. however further research is needed to specify the mechanism of tgev particles internalized by caveolin. all our data confirms that egfr is a co-factor for tgev invasion, and that tgev s protein interacts with egfr extracellular receptor binding domain . tgev infection induces egfr internalization and causes apn and egfr clustering. plus, apn and egfr not only synergistically promote tgev invasion, but they also active pi k/akt and mek/erk / signaling pathways. clathrin and caveolin mediate endocytosis of tgev and egfr internalization through the clathrin endocytosis pathway (fig. ) . these findings are conducive to enhancing our understanding of the entry mechanism of tgev, and for providing a potential target for the development of new anti-tgev therapies. ebola virus enters host cells by macropinocytosis and clathrin-mediated endocytosis mechanisms of coronavirus cell entry mediated by the viral spike protein porcine ipec-j intestinal epithelial cells in microbiological investigations an image correlation analysis of the distribution of clathrin associated adaptor protein (ap- ) at the plasma membrane activation of egfr on monocytes is required for human cytomegalovirus entry and mediates cellular motility determinants essential for the transmissible gastroenteritis virus-receptor interaction reside within a domain of 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role of mkk / kinase activity in human cytomegalovirus infection human cytomegalovirus up-regulates the phosphatidylinositol -kinase (pi -k) pathway: inhibition of pi -k activity inhibits viral replication and virus-induced signaling harnessing actin dynamics for clathrinmediated endocytosis characterization of functional domains in the human coronavirus hcv e receptor porcine aminopeptidase n is a functional receptor for the pedv coronavirus aminopeptidase n is not required for porcine epidemic diarrhea virus cell entry egfr and epha are host factors for hepatitis c virus entry and possible targets for antiviral therapy virus entry: open sesame virus entry by endocytosis virus entry by endocytosis derailed endocytosis: an emerging feature of cancer clathrin-and serine proteases-dependent uptake of porcine epidemic diarrhea virus into vero cells relationships between egfr signaling-competent and endocytosis-competent membrane microdomains human fibroblast growth factor receptor is a co-receptor for infection by adeno-associated virus phosphoinositide- kinase-akt pathway controls cellular entry of ebola virus sialic acids as receptor determinants for coronaviruses actin regulation in endocytosis hvea (herpesvirus entry mediator a), a coreceptor for herpes simplex virus entry, also participates in virus-induced cell fusion feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i integrin αvβ is a coreceptor for human cytomegalovirus epidermal growth factor receptor is a cellular receptor for human cytomegalovirus evidence for a putative second receptor for porcine transmissible gastroenteritis virus on the villous enterocytes of newborn pigs complete genomic sequence of the coronavirus transmissible gastroenteritis virus shxb isolated in china epidermal growth factor receptor is a co-receptor for adeno-associated virus serotype egf receptor egf receptor hiv envelope-cxcr signaling activates cofilin to overcome cortical actin restriction in resting cd t cells viruses exploit the function of epidermal growth factor receptor epidermal growth factor receptor-pi k signaling controls cofilin activity to facilitate herpes simplex virus entry into neuronal cells this work was supported by from the national science grant of china and a project funded by the priority academic program development of jiangsu higher education institutions (papd). supplementary data associated with this article can be found in the online version at http://dx.doi.org/ . /j.virol. . . . key: cord- -zpstb h authors: cui, tingting; theuns, sebastiaan; desmarets, lowiese m. b.; xie, jiexiong; de gryse, gaëtan m. a.; yang, bo; van den broeck, wim; nauwynck, hans j. title: establishment of porcine enterocyte/myofibroblast co-cultures for the growth of porcine rota- and coronaviruses date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: zpstb h a stable culture of primary porcine enterocytes is necessary to study porcine enteric virus replication characteristics. because the direct cultivation of primary porcine enterocytes is difficult, alternatives have to be considered. as subepithelial myofibroblasts secrete extracellular matrix and growth factors contributing to the attachment, proliferation and differentiation of epithelial cells, co-cultures of primary porcine enterocytes (ileocytes and colonocytes) with myofibroblasts were developed and evaluated for their susceptibility to enteric viruses. first, it was demonstrated that the co-cultured ileocytes and colonocytes were susceptible to an archival rotavirus strain rva/pig-tc/bel/rv / /g p[ ] and different other rotavirus genotypes (fecal samples containing g p[ ], g p[ ], g p[ ], g p[ ]). next, the tgev purdue strain infected both ileocytes and colonocytes whereas the miller strain only infected ileocytes. last, the pedv cv vero adapted and non-adapted (fecal suspension) strains could infect co-cultured ileocytes but not colonocytes. the infectivity of the cv vero adapted strain was higher when the cells were cultured without fetal bovine serum and the cv fecal suspension only infected the ileocytes cultured without fetal bovine serum. in conclusion, a novel co-culture of porcine enterocytes with myofibroblasts was established, which can be used for the investigation of the replication of enteric viruses. diarrhea and dehydration in piglets and destroy villous enterocytes in the small intestine. the severity and lethal outcome are strain dependent . different non-intestinal cell lines have been used in the past for virus cultivation in vitro , , but at low efficiency. tgev can be propagated in swine testicle (st) and porcine kidney (pk- ) cells. upon adaptation, pedv may infect african green monkey kidney (vero) cells. like tgev and pedv, rotavirus is mainly transmitted by fecal-oral route and virus infection causes the destruction of mature small intestinal enterocytes. similarly, most rotavirus research was conducted on non-polarized ma cells (african green monkey kidney epithelial cells) which are easy to culture and permissive for certain rotavirus strains of different genotypes. but there are still a lot of genotypes of rotavirus strains that do not grow in ma cells . data concerning the replication cycles of these enteric viruses in their target cell (mature intestinal enterocytes) are scarce. to this end, it is essential to obtain cultures of porcine intestinal enterocytes. the gastrointestinal tract is lined with a rapidly proliferating simple columnar epithelium. the epithelial cells migrate from the crypts where mitosis takes place towards the top of the villi (small intestine) or towards the top of the intercrypt (large intestine) as they mature. mature epithelial cells are replaced by a steady supply of crypt cells. in suckling piglets, intestinal epithelial cells renew every - days. because of the highly dynamic and rapid renewal properties of intestinal epithelial cells, it is difficult to successfully culture them in vitro. in , a porcine mid-jejunum epithelial cell line was established from neonatal piglets by immortalization upon transfection/ transduction with human telomerase reverse transcriptase (htert) gene . because the immortalization alters the biology of the original cells, field viruses replicate more efficiently in primary cells than in continuous cell lines. therefore, it is advisable to use primary cells for virus research. intestinal enteroids have been developed by culturing intestinal crypts onto matrigel which is enriched with laminin α and α . enteroids enhanced the viability of the cells and were already used for the study of rotaviruses , noroviruses and enteroviruses . the successful cultivation of enteroids is dependent on many growth factors, critically including wnt a, r-spondin, and noggin, which is an expensive method. intestinal myofibroblasts, one of the intestinal mesenchymal cells, are directly subjacent to the basement membrane of epithelial cells and they have been reported to support the proliferation and differentiation of epithelial cells . myofibroblasts are identified by the expression of intracellular cytoskeletal microfilament α-smooth muscle actin (α-sma). myofibroblasts contribute to the growth and differentiation of intestinal enterocytes by secreting several growth factors (hepatocyte growth factor, transforming growth factor beta (tgf-β ) , , insulin-like growth factors (icfs) ), extracellular matrix proteins (collagen type iv, laminin-β and γ , and fibronectin), cytokines and chemokines. to date, a mouse colonic myofibroblast cell line established by hirokawa has been reported to stimulate colonoid formation and human myofibroblasts isolated from small intestine were able to support human intestinal epithelial cell growth in vitro . all the information suggested the potential role of myofibroblasts in intestinal enterocytes cultivation in vitro. in this study, a porcine co-culture system of primary intestinal enterocytes with intestinal myofibroblasts was established which mimics the enterocytes growth in vivo. the morphological and functional features of co-cultured enterocytes were characterized. to determine the usability of this co-culture system, enteric rotaand coronaviruses were used to infect the co-cultured enterocytes. after euthanasia of a three-day old piglet, ileum and colon tissues were embedded immediately and cryosections were made to visualize the distribution of epithelial cells and myofibroblasts in vivo. immunofluorescence stainings were performed against the epithelial cell marker cytokeratin and myofibroblast marker α-smooth muscle actin. in the porcine ileum, a lot of myofibroblasts were located in the lamina propria directly underneath the epithelial cell layer of the villi, representing the largest cell population in the lamina propria. fewer myofibroblasts (< %) were observed in the lamina propria underneath the epithelial cell layer in colon. in contrast, the myofibroblasts formed an integral line underneath the epithelial cell layer in the colon crypts (fig. ) . these results show that porcine small and large intestinal epithelial cells grow in close contact to myofibroblasts in vivo. the contact communication between these two cell types is supposed to be an important element for the support of myofibroblasts in the attachment, proliferation and migration of epithelial cells. myofibroblasts were isolated from the subepithelial layer of porcine ileum. cell clusters were observed days post isolation and had a cobblestone-like morphology with stellate edges. the clusters continued to grow into larger structures which contain more than cells. meanwhile, the fibroblasts started to expand. the biggest cluster was marked by making a circle with a pen on the bottom of the plate and other cells were scraped away using a sterile tip. afterwards, this circled cluster was split using trypsin. cells maintained their morphology and could be continuously passaged ( fig. a) . the obtained cells were characterized by immunofluorescence staining. cells were stained positive for α-smooth muscle actin (α-sma), vimentin and fibronectin, which are all markers of myofibroblasts. the cells were negative for desmin, cytokeratin and sucrase-isomaltase (fig. b) . these results confirmed that the cells isolated from the ileum subepithelial layer were myofibroblasts. primary ileum epithelial cells were used as control, which were positive for cytokeratin and sucrase-isomaltase (fig. c ). myofibroblasts serve as supporting cells for porcine enterocytes. twenty-four hours post seeding, most of the ileum epithelial cells were attached and became confluent (> %) when seeded on porcine type i and iii collagen coated wells. however, days post seeding, the epithelial cells started to detach, and days post seeding, most of the epithelial cells were dead. only a few big epithelial cell clusters could be visualized. six days post seeding, fibroblasts took over the wells and a few tiny epithelial clusters were still present (fig. a) . the use of % conditioned medium did not give an improvement: most of the epithelial cells were dead at day (fig. b ). when monolayers of myofibroblasts were used as supporting layers for ileum and colon epithelial cell cultivation, epithelial clusters attached to the myofibroblasts and became visible at hours post seeding. the epithelial cells continued to expand and grew into monolayers days post isolation. they maintained their polygonal morphology and confluent layers for more than week (fig. c,d) . characterization of enterocytes co-cultured with myofibroblasts. cells isolated from ileum and colon co-cultured with myofibroblasts were characterized by immunofluorescence staining days post co-cultivation. antibodies against cytokeratin and vimentin were used to determine the epithelial nature. in the co-culture system, most of the cells were found to be cytokeratin positive ( ± . % of all cells in the ileum epithelial cell co-cultures and ± . % of all cells in the colon epithelial cell co-cultures), confirming the epithelial nature of the polygonal cells (fig. ) . interestingly, the vimentin positive cells (myofibroblasts) clustered into aggregates in both co-cultures, which suggests that the expansion of epithelial cell growth squeezed the myofibroblasts into aggregates. and after co-culture with myofibroblasts (in vitro). the differentiation status of porcine intestinal epithelial cells of days old piglets and the co-cultured enterocytes were analyzed by scanning electron microscopy. as shown in fig. , the epithelial cells of the ileum and colon had a different appearance in vivo. a few epithelial cells on the tip of a villus of the ileum were fully covered with microvilli, while most of the cells were immature without microvilli on the surface. in contrast, almost all epithelial cells of the colon had short microvilli. in the co-cultured ileum and colon epithelial cells, a lot of microvilli were present on the cell's surface at three days post co-cultivation and showed a different appearance (some microvilli were longer). these results demonstrate that myofibroblasts could support the differentiation of both ileum and colon epithelial cells in vitro. replication kinetics of rotavirus rva/pig-tc/bel/rv / /g p [ ] strain in co-cultured enterocytes. to determine the percentage of rotavirus infected cells in primary porcine enterocytes, cells were fixed at different time points ( , , , , , h) post inoculation with a low-passage archival rva strain. viral antigens were stained by immunofluorescence (fig. a ). the first antigen-positive ileum epithelial cells appeared at h p.i and increased over time. the percentage of rotavirus infection in ileum epithelial cells increased to . ± . % at h p.i. in colon epithelial cells, the first antigen-positive cells appeared at h p.i and the percentage of infection increased to . ± . % at h p.i (fig. b) . to determine the kinetics of virus production in ileum and colon epithelial cells, viral titers and rna copies of supernatant (extracellular virus) and cells (intracellular virus) were assessed (fig. c,d) . the intracellular and extracellular virus titers were determined from to h post inoculation. the intracellular virus titer of ileum epithelial cells increased from . ± . ccid /ml to . ± . ccid /ml. the extracellular virus titer of ileum epithelial cells increased from . ± . ccid /ml to . ± . ccid /ml. for colon epithelial cells, the intracellular and extracellular virus titers increased from . ± . ccid /ml to . ± . ccid /ml and from . ± . ccid /ml to . ± . ccid /ml, respectively. the rt-qpcr showed that viral rna started to be synthesized in ileum and colon epithelial cells from h p.i. viral rna increased up to . ± . log /ml in ileum epithelial cells and . ± . log /ml in colon epithelial cells. the viral rna started to be released into the supernatant between and h p.i; at h p.i, . ± . log /ml rna copies were detected in both ileum and colon epithelial cell cultures. susceptibility of primary porcine enterocytes to different rotavirus genotypes present in fecal suspensions of diarrheic pigs. a major restriction of rotavirus research is the lack of cell cultures supporting the growth of different genotypes of field strains. therefore, the susceptibility of primary porcine enterocytes co-cultured with myofibroblasts to four different genotypes of rotavirus a contained in fecal suspensions was tested by rt-qpcr (fig. ) . the results showed that these four genotypes of rotavirus could infect both ileum and colon epithelial cells. rotavirus r (g p [ ] ) strain demonstrated the highest infectivity to primary enterocytes, followed by the r (g p [ ] ) strain. for rotavirus r (g p [ ] ) strain, an increase of more than fold rna copies/ml was detected at h post inoculation of ileum and colon epithelial cells. ileum and figure . evaluation of different methods to support the growth of primary porcine ileum and colon epithelial cells. (a) porcine ileum epithelial cells were cultured without myofibroblasts on a porcine collagen type i/iii coated plate. they could live approximately days before detaching and dying, fibroblasts took over days after seeding. (b) porcine ileum epithelial cells were cultured without myofibroblasts on a porcine collagen type i/iii coated plate with medium supplemented with % conditioned medium collected from ileum myofibroblasts. epithelial cells could be maintained for less than days and a few tiny epithelial clusters were present at days post seeding. porcine ileum (c) and colon (d) epithelial cells were cultured in the presence of ileum myofibroblasts. epithelial cells grew into a monolayer days post co-cultivation and maintained their morphology for more than days. colon epithelial cells were less susceptible to rotavirus r (g p [ ] ) strain. these results demonstrated that the primary enterocytes could be infected by rotavirus field strains containing different genotypes but that the susceptibility of enterocytes to rotavirus differed among the different genotypes. susceptibility of co-cultured enterocytes to transmissible gastroenteritis virus. next, the susceptibility of primary enterocytes in co-cultures with myofibroblasts was tested for the tgev purdue and miller strains. tgev antigen expression kinetics were assessed in primary enterocytes (fig. ). tgev purdue infected both ileum and colon epithelial cells. at h p.i, the purdue strain had infected . ± . % and . ± . % of the ileum epithelial cells and colon epithelial cells, respectively, and the virus titer in both ileum and colon epithelial cells increased to . ± . ccid /ml. tgev miller only infected ileum epithelial cells. the highest infection ( . ± . %) appeared at h p.i. no tgev antigens of miller strain were found in colon epithelial cells on h post inoculation. pedv cv vero adapted strain and pedv cv positive fecal suspensions were used to infect co-cultured primary ileum epithelial cells to confirm the usability of primary enterocytes. twenty-four hours post inoculation, viral antigens of vero adapted cv were observed in a low number of ileum epithelial cells cultured with/without fbs. two times more infection was found in epithelial cells cultured without fbs. for cv fecal suspension, virus infection was only observed in ileum epithelial cells which were cultured without fbs (fig. ). in this study, a co-culture system of porcine ileum subepithelial myofibroblasts with porcine small (ileum) and large (colon) intestinal epithelial cells was established and the use of this co-culture system for enteric virus research was assessed. the cross talk between epithelium and subepithelial myofibroblasts has been reported to promote epithelial cell proliferation and differentiation in both mice and humans. in this study, a myofibroblast cell line was established from porcine ileum which was identified by the presence of α-smooth muscle actin, vimentin and fibronectin. the in vivo distribution of epithelial cells and myofibroblasts shows that a lot of myofibroblasts directly grow underneath the epithelium in porcine ileum and that myofibroblasts form an integral line along colon crypts. this initial contact may be an important factor for the support of myofibroblasts towards epithelial cells. at present, many mechanical and enzymatic seperation methods have been used for the isolation of intestinal epithelial cells from human, mice, rat, bovine, porcine and feline intestines. however, the successful cultivation of intestinal epithelial cells still poses a big challenge because of the rapid death/apoptosis of isolated epithelial cells which in vivo renew every - days. this apoptosis may be triggered by the disruption of the epithelial cell contact with extracellular matrix. a dispase and collagenase combination was used for epithelial cell isolation in the present study, which preserves more cell-to-cell interactions and reduce the damage of cell-matrix adhesions . the contamination with stromal cells is a huge problem for epithelial cell cultivation. in order to decrease the contamination with mesenchymal cells, we removed these cells by d-sorbitol density centrifugation and plastic adhesion for hours. according to the specific property that stromal cells attach to plates faster than epithelial cells, most stromal cells were separated from epithelial cells after hours incubation. in the presence of ileum myofibroblasts, both ileum and colon epithelial cells are growing longer than one week and maintain their polygonal, cobblestone-like morphology. in the absence of myofibroblasts, epithelial cells died after - days, even when supplemented with % conditioned medium collected from myofibroblast cultures. our data indicate that the supporting effect of myofibroblasts for epithelial cell growth is very dependent on the direct contact between these two cell types. we also demonstrated that myofibroblasts not only support the growth of intestinal epithelial cells from newborn piglets, but also the epithelial cells of weeks old pigs (data not shown), which confirms the important role of myofibroblasts on epithelial cell proliferation independently of the age of the donor. the epithelial cells in co-cultures were identified by the presence of cytokeratin which is regarded as an important marker of epithelial cells. most of the cells (> %) preserved their epithelial nature with a positive staining of cytokeratin after days of co-cultivation. remarkably, the myofibroblasts clustered into aggregates in this co-culture system. it seems that myofibroblasts retracted into aggregates during the expansion of epithelial cells growth. in earlier reports, it was shown that myofibroblasts can migrate to wound tissue and demonstrate high contractile activities to generate tissue contractures, which help wound healing and organ remodeling by secretion of extracellular matrix proteins and exerting strong contraction force [ ] [ ] [ ] . in addition, human and porcine myofibroblasts express s a proteins which have been demonstrated to be implicated in cancer cell migration , . taken together all this information, we hypothesize that myofibroblasts first secrete extracellular matrix proteins, such as collagen and laminin, coordinating the attachment and proliferation of epithelial cells and migration of myofibroblasts in clusters. epithelial cells co-cultured with myofibroblasts showed microvilli after days of co-cultivation which is in accordance with the reported data that myofibroblasts not only support the growth of epithelial cells, but also stimulate the differentiation of epithelial cells . rotavirus research is hampered by the lack of susceptible enterocyte cell lines. although some cell lines, including ma , marc, ipec-j and caco- cells [ ] [ ] [ ] are susceptible to some rotavirus strains, a lot of genotypes of rotavirus, such as p [ ] , p [ ] , p [ ] , p [ ] do not grow efficiently in these cell lines. therefore, enterocyte cultures are an essential tool to investigate rotavirus-cell interaction. in this study, the susceptibility of primary enterocytes to different rotavirus genotypes were explored. rotavirus rva/pig-tc/bel/rv / /g p [ ] strain, which was first isolated in belgium in from a pool of watery diarrhea of pigs , is a typical g porcine rotavirus. g is the most common vp genotype of human group a rotavirus, but is rarely found in porcine rotavirus strains . it is suggested that all human g vp genes originate from porcine rotavirus transmission to humans and that this interspecies transmission was followed by human-to-human transmissions . although rotavirus was reported to have an exclusive tropism for small intestinal enterocytes , rotavirus rva/pig-tc/bel/rv / /g p [ ] strain could infect both primary ileum and colon epithelial cells with a trypsin treatment. the antigen expression kinetics did not show significant differences in cell tropism of this rotavirus strain. studies showed that some animal group a rotaviruses, especially porcine rotaviruses recognize sialic acid as host receptor for virus attachment. on porcine small and large intestinal epithelium, sialic acid receptors were clearly detected and colon crypts showed even a greater abundance of sialic acid than small intestines . rotavirus genotype p [ ] has been classified as a sialic acid-dependent genotype , which may explain why rotavirus g p [ ] strain could also infect colon epithelial cells in vitro. both ileum and colon epithelial cells were also susceptible to four fecal suspensions containing different rotavirus genotypes (g p [ ] , g p [ ] , g p [ ] and g p [ ] ). g p [ ] and g p [ ] rotaviruses, which are the predominant g/p genotype combination of circulating rotaviruses in belgium, were more infectious than the other two strains, which was also observed on ma cells . the higher infectivity in enterocytes in vitro may explain the wide prevalence of these genotypes in the field. rotavirus p [ ] genotype is one of the major human rotavirus genotypes. it is associated with symptomatic infection in children and is frequently detected in africa. in pigs, p [ ] rotaviruses are also regularly detected and most of these strains display a high genetic similarity to human p [ ] rotavirus strains , . animal rotaviruses are regarded as a potential reservoir for the genetic and antigenic diversity of human rotaviruses and interspecies transmission from swine to humans has been reported increasingly for p [ ] genotype. eight hungarian human g p [ ] rotavirus strains were supposed to originate from pigs by independent events of zoonotic transmission . therefore, studying porcine rotavirus is a key step to deeply understand the evolution of human rotavirus. recent studies demonstrated that p [ ] rotavirus strains recognize human histo-blood group antigens (hbgas), which explains why most human rotavirus strains cannot be cultured in ma cells. in our study, rotavirus g p [ ] could infect both ileum and colon epithelial cells which demonstrates that the primary enterocytes contain the cellular receptor for rotavirus p [ ] , likely containing the right hbgas. this receptor is still not identified. rotavirus almost exclusively infects mature enterocytes of the small intestinal villi in vivo , while in vitro, the large intestinal epithelial cells could also be infected, which leads us to think that other factors in the large intestines block the rotavirus infection of colon epithelial cells in vivo. in the enterohepatic circulation, bile salts are synthesized in the liver, travel to the gall bladder and are secreted into the descending part of the duodenum helping in the digestion of fats and other substances. up to % of secreted bile salts are collected in the ileum and return back to the liver via the portal system. we hypothesize that the lack of bile salts in the lumen of the large intestines may be the reason why rotavirus cannot infect the epithelial cells of large intestines in vivo. in order to test this hypothesis, we collected bile from the porcine gall bladder and used it for the infection of rotavirus to primary ileum epithelial cells. the results show that the infectivity of rotavirus in ileum epithelial cells increased after treatment with bile ( supplementary fig. s ), which is in accordance with the results that bile/bile acids are essential for porcine enteric calicivirus, hepatitis c virus and human norovirus infection in cells , , . porcine epidemic diarrhea virus and transmissible gastroenteritis virus primarily replicate in the villous enterocytes of the small intestine. porcine aminopeptidase n (papn), one of the type ii cell surface metalloproteases, was reported to be a cellular receptor for both pedv and tgev [ ] [ ] [ ] . although these two viruses bear similarities in structure and disease, they are clearly distinct. in vitro, pedv does not grow in continuous cell cultures which are permissive to tgev. therefore, it is of interest to investigate the susceptibility of the target cell, primary porcine enterocytes, to both pedv and tgev. interestingly, we found that tgev purdue can infect both ileum and colon epithelial cells, whereas the virulent miller strain only infects ileum epithelial cells and that the infectivity of the purdue strain is much higher than the miller strain in ileum epithelial cells. the purdue and miller strains are two virulent american tgev strains, which originally caused % mortality of less than -weeks-old piglets due to the lytic infection of enterocytes. the purdue strain used in this study has been passaged times in primary porcine kidney cells and then adapted to grow in st cells by one more passage . a nucleotide mutation (t to g at nucleotide position ) of the purdue s protein, which causes a serine (s) to alanine (a) mutation at aa was reported . this mutation is present in the domain of the s protein encoded by nucleotide - which is used for the cell receptor papn recognition . this mutation may make the virus more suitable to grow in papn negative cultures/primary porcine kidney cells due to the recognition of other cell receptors. it may also be responsible for the growth of the purdue strain in primary porcine colon epithelial cells and porcine myofibroblasts which are both negative for papn ( supplementary fig. s ). due to the absence of papn, the miller strain cannot infect the primary colon epithelial cells. pedv cv strain has been adapted to grow in the apn negative vero cells by several blind passages and has been widely used for pedv research. we found that primary ileum epithelial cells were more susceptible to the cv vero adapted strain when the cells were grown in absence of fbs and the cv fecal suspension only infected the cells which were cultured without fbs. fbs is usually added in culture medium for cell growth because it contains various biological factors. but its compositional complexity also causes a lot of side-effects, such as the inhibition of cell differentiation and virus replication. fbs may inhibit viral replication by blocking cellular proteins, as shown for another nidovirus porcine reproductive and respiratory syndrome virus (prrsv) . cv strain shows a lower infectivity in ileum epithelial cells cultured with fbs suggesting that fbs contains factors that interfere with pedv infection or that without fbs the epithelial cells could differentiate better which makes them more susceptible to pedv infection. the susceptibility of primary ileum epithelial cells to a pedv fecal suspension represents a big step for further investigating pedv field isolates. in this study, the primary epithelial cells were susceptible to rota-and coronaviruses, however the infection efficiency was relatively low. this might be caused by the antiviral response of interferons, especially type iii ifns (ifn-λ). interferons belong to the class of cytokines, which are made and released by host cells to trigger protective defenses to several pathogens, such as viruses, bacteria and parasites. intestinal epithelial cells abundantly produce type iii ifns and elicit antiviral defenses in response to viral infections. zhang and colleagues demonstrated that ifn-λ possesses a strong antiviral effect on pedv replication in the porcine intestinal epithelial cell line ipec-dq, a subclone obtained from the nontransformed ipec-j cells by limited serial dilutions . a similar strong ifn-λ might have been rapidly induced in the currently described co-cultures. furthermore, rotavirus, pedv and tgev infect mature villous epithelial cells. in our co-culture system, not only the mature villous epithelial cells were cultured but likely also the less differentiated intestinal crypt epithelial cells. these undifferentiated crypt epithelial cells might cause the low infection efficiency. in future work, efforts will be made to stimulate the differentiation of primary epithelial cells and control the ifn antiviral response in order to increase the infection efficiency. in conclusion, porcine enterocyte/myofibroblast co-cultures were successfully established in the present study, demonstrating that myofibroblasts are necessary for small and large intestinal epithelial cell attachment, proliferation and differentiation in vitro. primary enterocytes were susceptible to both cell line adapted and non-adapted rota-and coronaviruses. this co-cultivation system will be a great assett in future research on rota-, coronavirus and other enteric viruses. ethical statement. euthanizing piglets was done in agreement with the european legislation on animal experiments. all experimental procedures were approved by the local ethical committee of the faculty of veterinary medicine, ghent university, and all methods were carried out in accordance with the approved guidelines. piglets and virus samples. healthy conventional -day-old suckling piglets were purchased from a conventional pig farm. using tissues of euthanized animals was in accordance of the requirements of the local ethical committee of the faculty of veterinary medicine. the piglets were euthanized by intravenous injection of % sodium pentobarbital ( ml/ . kg, kela laboratories, hoogstraten, belgium). rotavirus rva/pig-tc/bel/ rv / /g p [ ] strain grown on ma cells, tgev purdue and miller strain grown on st cells and pedv cv strain grown on vero cells were used in this study. in addition, four diarrheic fecal suspensions containing rotavirus of suckling pigs less than weeks old (table ) and pedv cv fecal suspension from an experimentally inoculated -day-old sucking piglet were also included. twenty percent fecal suspensions were prepared in phosphate buffered saline (pbs) containing u/ml penicillin (continental pharma, puurs, belgium), mg/ml streptomycin (certa, braine l' alleud, belgium), mg/ml gentamicin (gibco brl, merelbeke, belgium) and . % v/v fungizone (bristol-myers squibb, braine l' alleud, belgium) . establishment of porcine intestinal subepithelial myofibroblasts. the ileum was collected from a euthanized -day-old piglet and brought in ice-cold dulbecco's modified eagle medium (dmem; gibco brl, merelbeke, belgium), containing u/ml penicillin, . mg/ml streptomycin, . mg/ml gentamycin (flushing medium) and % fetal bovine serum (fbs; gibco brl). subsequently, the intestine was cut into - cm long segments and turned inside-out, mucosal side facing outwards. intestinal contents were removed by one washing with ice-cold flushing medium containing % fbs and two washings with flushing medium without fbs. the intestinal segments were incubated in pbs containing mm edta and shaken at rpm/min for min at °c. the edta suspension was removed and this edta incubation step was repeated times. next, intestinal segments were incubated in dmem containing dispase ii ( . mg/ml, sigma, st. louis, mo, usa) and collagenase i ( . mg/ml, invitrogen, paisley, uk) for min at °c. subsequently, the digested mucosa was gently scraped with a sterile scalpel blade, scrapings were collected and incubated in dmem containing dispase ii ( . mg/ ml) for min whilst pipetting. after centrifugation for min at × g and °c, the pellet was resuspended in dmem containing % d-sorbitol (sigma) and . % fbs, and centrifuged at × g for min. the pellet was resuspended in dmem/f- (gibco brl) culture medium supplemented with u/ml penicillin, . mg/ml streptomycin, % fbs, and % non-essential amino acids (gibco brl) and incubated at °c and % co . culture medium was refreshed on day , afterwards medium was changed every days. morphology of the cells was evaluated daily by light microscopy (olympus). once myofibroblasts (cobblestone-like clusters) grew into big clusters (≈ cells), they were marked and other cells (e.g. epithelial cells, fibroblasts) were removed by scraping. then the cobblestone-like clusters were detached by trypsinization with μg/ml trypsin- . % edta, and sub-cultured in a -well plate (split ratio : ) with culture medium containing % fbs and evaluated daily for cobblestone-like features by light microscopy. subsequently, the cobblestone-like cells were digested by trypsinization and further expanded in flasks to generate a long-term semi-continuous culture. to characterize the obtained primary cells, immunofluorescence stainings were performed to visualize cytokeratin, vimentin, α-smooth muscle actin, fibronectin, desmin and sucrase-isomaltase. a third passage of cells was fixed with % paraformaldehyde for min at room temperature (rt) followed by permeabilization with . % triton x- for min at rt. the cells were respectively incubated with mouse monoclonal anti-human cytokeratin antibodies (dako, denmark a/s), mouse monoclonal anti-human vimentin antibodies (bio-rad), mouse monoclonal anti-human α-smooth muscle actin antibodies (dako), mouse monoclonal anti-human desmin antibodies (dako) sheep polyclonal anti-human fibronectin antibodies (bio-rad) or mouse monoclonal anti-human sucrase-isomaltase (santa cruz) for h at °c. afterwards, cells were washed and incubated with goat anti-mouse-igg fitc labeled antibodies (molecular probes) or rabbit anti-sheep-igg fitc labeled antibodies (molecular probes) for h at °c. nuclei were stained with hoechst (molecular probes) for min at rt. the slides were mounted using glycerin solution with . % , -diazabicyclo[ . . ]octane (janssen chimica, beerse, belgium) and analyzed using fluorescence microscopy (dm b fluorescence microscope, leica microsystems gmbh, heidelberg, germany). of supporting cells. two days before enterocyte isolation, myofibroblasts were seeded in -well plates at a density of cells/ml in dmem/f- culture medium containing u/ml penicillin, . mg/ml streptomycin, and % fbs, and % non-essential amino acids and cultured at °c and % co . monolayers of myofibroblasts were used as support layer for enterocytes growth and differentiation. preparation of conditioned medium collected from myofibroblasts. when myofibroblasts grew to % confluency, their medium was refreshed by dmem/f- culture medium. next, the refreshed medium was collected after h, centrifuged at × g for min and the supernatant was collected and used as conditioned medium. the conditioned medium was stored at − °c until later use. isolation of porcine enterocytes. after euthanasia, around cm long segments of ileum and colon were removed from a piglet. the intestinal segments were turned inside-out, mucosal side facing outwards. the intestinal contents were removed by one washing with ice-cold flushing medium with % fbs and two washings with ice-cold flushing medium without fbs. in order to fully expand the contact area of intestinal mucosa layer to digestion enzymes, one side of the intestinal piece was closed with a surgical clamp and the lumen was filled with warm ca + -and mg + -enriched pbs containing penicillin and streptomycin. then, the other side was also closed with a surgical clamp. the intestinal mucosa was digested in dmem containing dispase ii ( . mg/ml) and collagenase i ( . mg/ml) for min at °c. then, the pbs was released from the lumen by removing one clamp. the mucosa was gently scraped with a sterile scalpel blade. afterwards, the intestinal lumen was filled again with pbs and the mucosa was digested in dmem containing dispase ii ( . mg/ml) and collagenase i ( . mg/ml) again for another min (ileum) or min (colon) at °c. subsequently, the digested mucosa was deeply scraped with a sterile scalpel blade. the scrapings were incubated in dmem containing dispase ii ( . mg/ml) for min whilst pipetting. after centrifugation ( × g, min at °c) the pellet was resuspended in dmem containing % d-sorbitol and . % fbs and centrifuged at × g for min at °c to separate single stromal cells. this sorbitol process was repeated at least times. afterwards, the pellet was resuspended in dmem supplemented with antibiotics and filtered using a μm cell strainer (falcon). after centrifugation ( × g, min), the pellet was finally resuspended in dmem/f supplemented with u/ml penicillin, . mg/ml streptomycin, . mg/ml gentamycin, % fbs, . % fungizone, ng/ml epidermal growth factor (sigma), % insulin-transferrin-selenium-ethanolamine (gibco brl) and % non-essential amino acids and seeded in -well plates in order to let the mesenchymal cells attach and separate enterocytes from mesenchymal cells. after h incubation at °c and % co , the non-adherent cell clusters in the -well plates were collected and reseeded on top of the monolayer of myofibroblasts. to confirm the support effect of myofibroblasts, cell clusters were also seeded on porcine collagen type i/iii (gentaur, kampenhout) coated plates and cultured with/without % conditioned medium. the cells were further incubated at °c and % co and the medium was refreshed every days. morphological features of primary enterocytes were evaluated by light microscopy (olympus). to characterize the origin of enterocytes which were co-cultured with myofibroblasts, immunofluorescence staining was performed against cytokeratin and vimentin. three days post isolation, co-cultured enterocytes grown on coverslips were fixed with % paraformaldehyde for min at rt followed by permeabilization with . % triton x- for min at rt. the cells were incubated with mouse monoclonal anti-human cytokeratin or mouse monoclonal anti-human vimentin antibodies containing % goat serum for h at °c, followed by goat anti-mouse-igg fitc labeled antibodies for h at °c. nuclei were stained with hoechst for min at rt. the percentage of cytokeratin positive cells were analyzed by fluorescence microscopy (leica microsystems gmbh). immediately after euthanasia of a -day-old piglet, mm square pieces of the ileum and colon were collected. tissues were embedded in methocel (fluka, sigma) and μm thick cryosections were made. immunofluorescence staining with markers for epithelial cells and myofibroblasts were performed. in brief, cryosections of ileum and colon tissues were fixed with methanol for min at − °c and then incubated with mouse monoclonal anti-human α-smooth muscle actin antibodies for h at °c, followed by goat anti-mouse-igg fitc labeled antibodies for h at °c. afterwards, the sections were incubated with rabbit polyclonal anti-bovine cytokeratin antibodies, followed by goat anti-rabbit-igg texas red labelled antibodies for h at °c. after washing, nuclei were stained with hoechst for min at rt. the slides were analyzed by fluorescence microscopy. scanning electron microscopy. in order to determine the differentiation status of intestinal epithelial cells, the presence of microvilli was assessed by scanning electron microscopy (sem). the protocol for sem was performed as described by glorieux and colleagues . tissue samples (ileum and colon from -day-old piglet) and cell samples (porcine enterocytes days post co-cultivation) were fixed in hepes-buffered glutaric aldehyde for hours. then, the samples were treated with % osmiumtetroxide for hours at rt, followed by ascending grades of alcohol dehydration. in order to avoid the water vaporization obstructing the electron beam and interfering with image clarity, the dehydrated samples were transferred to a critical point drier (cpd, bal-tec, balzers, liechtenstein) for complete drying. finally, the dried samples were mounted on a metal stub and sputter-coated with platinum. the microvilli of all the samples were acquired with a jeol jsm lv scanning electron microscope (jeol ltd., tokyo, japan). replication of ma grown rotavirus in co-cultured enterocytes. three days post seeding, primary enterocytes co-cultured with myofibroblasts were inoculated with μl rotavirus rva/pig-tc/bel/ rv / /g p [ ] at an m.o.i. of . after h of inoculation ( °c and % co ), cells were washed times with warm dmem and further incubated in serum-free dmem/f culture medium containing μg/ml trypsin. at different time points ( , , , , and h) post inoculation, cells were fixed with % paraformaldehyde for min and permeabilized with . % triton x- for min at rt. double-immunostainings were performed as described above and the percentage of infected enterocytes was counted by fluorescence microscopy. in addition, the supernatant and cells were collected at different time points ( , , , , and h) post inoculation for virus titration and qpcr quantification. cell culture supernatant was collected and centrifuged for min at × g. supernatant was collected (extracellular virus) and the pellet was collected together with cells that were scraped with serum free dmem/f culture medium (intracellular virus). the cells were lysed by freeze-thaw cycles to release virus particles. the virus titer was determined using virus titration and rt-qpcr. titration of intraand extracellular virus was performed. monolayers of ma cells growing in -well plates were inoculated with -fold dilution of supernatant and cells ( − - − ). five days after inoculation, the cytopathogenic effect (cpe) was visualized using a light microscope and the cell culture infective dose (ccid susceptibility of co-cultured enterocytes to different rotavirus genotypes present in fecal suspension of diarrheic pigs. four fecal suspensions containing different genotypes of rotavirus collected from less than -week-old diarrheic suckling pigs were used to determine the susceptibility of porcine primary enterocytes to rotavirus field strains. these samples were collected at a private diagnostic laboratory for etiology diagnosis as described before . three days after co-cultivation, enterocytes were inoculated with these four fecal suspensions at viral rna copies/ml using the method described above. the supernatant was harvested at h and h post inoculation. rt-qpcr was performed to determine the increased number of viral rna copies. susceptibility of co-cultured enterocytes to transmissible gastroenteritis virus. three days post seeding, monolayers of primary enterocytes co-cultured with myofibroblasts were inoculated with μl tgev strains (miller and purdue) at an m.o.i. of . after h inoculation at °c and % co , cells were washed times with warm dmem. serum free dmem/f supplemented with u/ml penicillin, . mg/ml streptomycin, . mg/ml gentamycin, ng/ml epidermal growth factor, % insulin-transferrin-selenium-ethanolamine and % non-essential amino acids was added for further incubation. at different time points ( , , , , and h) post inoculation, cells were fixed with % paraformaldehyde for min and the supernatant was collected. double-immunostainings against both tgev antigens and cytokeratin were performed to specifically visualize the infected enterocytes. the cells were permeabilized with . % triton x- for min at rt and incubated with swine polyclonal anti tgev antibodies containing % negative goat serum for h at °c, followed by goat anti-swine-igg fitc labelled antibodies for h at °c. afterwards, cells were incubated h at °c with mouse monoclonal anti-human cytokeratin antibodies, followed by h at °c with goat anti-mouse-igg texas red labelled antibodies. nuclei were stained with hoechst for min at rt. the percentage of infected enterocytes was determined by fluorescence microscopy. virus titration of supernatant was performed using st cells. monolayers of st cells were inoculated with -fold dilutions ( - − ) of supernatant. five days after inoculation, cytopathogenic effect (cpe) was visualized and cell culture infective dose (ccid ) was calculated using the formula of reed and muench. hours post seeding, ileum epithelial cells were refreshed with culture medium with/without % fbs. three days post cultivation, monolayers of primary ileum epithelial cells cultured with/without fcs were inoculated with μl pedv cv vero adapted strain at . ccid /ml or viral rna copies/ml of fecal suspension with µg/ml trypsin. after h inoculation at °c and % co , cells were washed times with warm dmem and serum free dmem/f supplemented with u/ml penicillin, . mg/ml streptomycin, . mg/ml gentamycin, ng/ml epidermal growth factor, % insulin-transferrin-selenium-ethanolamine and % non-essential amino acids was added for further incubation. twenty-four hours post inoculation, cells were fixed with % paraformaldehyde for min and permeabilized with . % triton x- for min at rt. the cells were incubated with mouse monoclonal anti-pig pedv antibodies containing % normal goat serum, followed by h at °c with goat anti-mouse-igg fitc labelled antibodies. nuclei were stained with hoechst for min at rt and the infection was analyzed by fluorescence microscopy. the datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. uniformity of rotavirus strain nomenclature proposed by the rotavirus classification working group (rcwg) candidate new rotavirus species in sheltered dogs vp -sequence-based cutoff values as a criterion for rotavirus species demarcation species h rotavirus detected in piglets with diarrhea, brazil complete genome analyses of the first porcine rotavirus group h identified from a south african pig does not provide evidence for recent interspecies transmission 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interaction of porcine viruses with the respiratory tract serotypic similarity and diversity of rotaviruses of mammalian and avian origin as studied by plaque-reduction neutralization a simple method of estimating fifty per cent endpoints we are grateful to prof j. t. patton for supplying the antibody against rotavirus. special thanks go to ytse noppe and marthe pauwels for the excellent technical assistance. supplementary information accompanies this paper at https://doi.org/ . /s - - - . the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -beb k authors: zhang, shuai; hu, weiwei; yuan, lvfeng; yang, qian title: transferrin receptor is a supplementary receptor that assists transmissible gastroenteritis virus entry into porcine intestinal epithelium date: - - journal: cell commun signal doi: . /s - - - sha: doc_id: cord_uid: beb k background: transmissible gastroenteritis virus (tgev), the etiologic agent of transmissible gastroenteritis, infects swine of all ages causing vomiting and diarrhea, in newborn piglets the mortality rate is near %. intestinal epithelial cells are the primary target cells of tgev. transferrin receptor (tfr ), which is highly expressed in piglets with anemia, may play a role in tgev infection. however, the underlying mechanism of tgev invasion remains largely unknown. results: our study investigated the possibility that tfr can serve as a receptor for tgev infection and enables the invasion and replication of tgev. we observed that tgev infection promoted tfr internalization, clustering, and co-localization with tfr early in infection, while tfr expression was significantly down-regulated as tgev infection proceeded. tgev infection and replication were inhibited by occluding tfr with antibodies or by decreasing tfr expression. tgev infection increased in tgev-susceptible st or ipec-j cell lines and tgev-resistant caco- cells when porcine tfr was over-expressed. finally, we found that the tgev s protein interacts with the extracellular region of tfr , and that pre-incubating tgev with a protein fragment containing the extracellular region of tfr blocked viral infection. conclusions: our results support the hypothesis that tfr is an additional receptor for tgev and assists tgev invasion and replication. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. transmissible gastroenteritis virus (tgev), a porcine enteropathogenic coronavirus, can cause watery diarrhea, vomiting, and rapid dehydration. infection with tgev results in nearly % mortality in neonatal piglets [ , ] , and is responsible for enormous economic losses in the swine industry [ ] . tgev is an enveloped virus with a large positive-sense single-stranded rna genome that encodes structural proteins: spike protein (s), envelope protein (e), membrane protein (m), and nucleoprotein protein (n) [ , ] . the structure of the s glycoprotein changes radically as it binds to specific epithelial cell receptors and mediates the fusion of cellular and viral membranes [ ] [ ] [ ] [ ] . significant effort has been made to characterize the cellular receptors for tgev infection. porcine aminopeptidase n (papn), also known as cd , has been identified as a primary cell-surface receptor for tgev [ ] [ ] [ ] [ ] [ ] . our laboratory recently reported that epidermal growth factor receptor (egfr) also promotes clathrin-mediated endocytosis of tgev [ ] . tgev may use a third protein ( kda), currently uncharacterized, for cell entry [ , ] . identification of this receptor would provide additional insight into tgev-mediated disease and define a novel therapeutic target for protecting swine from tgev infection. tfr is a homo-dimeric type ii transmembrane glycoprotein with two apparent kda subunits (total kda). tfr contains a large extracellular c-terminal domain ( amino acids, includes a binding site for its ligand transferrin), a single transmembrane domain ( amino acids), and a short intracellular n-terminal domain ( amino acids) [ ] [ ] [ ] . in normal intestinal epithelium, transferrin receptor (tfr /cd ) is predominantly expressed in the crypt enterocytes (observed on the basolateral surface and in the cytoplasm of crypt epithelial cells) [ ] [ ] [ ] , with little expression in the epithelial cells at the apical of the villus. in contrast, under conditions of iron-deficiency anemia and low iron stores, the iron-responsive element/iron regulatory protein (ire/ irp) network prevents tfr mrna degradation [ ] [ ] [ ] [ ] [ ] and up-regulates tfr substantially, resulting in expression all across the intestinal epithelium [ , , ] . in addition to its functions in iron uptake and homeostatic maintenance of the intestinal epithelium [ , , ] , tfr had also been identified as a cellular entry receptor for several viruses, including the new world arenaviruses, new world hemorrhagic fever arenaviruses, machupo virus (macv) [ , ] , mouse mammary tumor virus (mmtv) [ , ] , the canine and feline parvovirus, feline panleukopenia virus [ , ] , and hepatitis c virus (hcv) [ ] . additionally, junín virus (junv) uses tfr as a cellular receptor for entry into susceptible cells by clathrin-mediated endocytosis [ ] . because tfr is distributed widely along the surface epithelium of newborns with anemia, and intestinal epithelial cells of newborn piglets are targets of tgev, it is possible that tfr is the as yet uncharacterized kda protein that mediates tgev infection. to test this hypothesis, we used cultured porcine intestinal columnar epithelial cells (ipec-j ), derived from the neonatal piglet mid-jejunum [ , ] , as a model to characterize the interaction between tfr and tgev in vitro. our results indicate that tfr serves as a receptor for tgev, contributes specifically to virion binding and endocytosis, and determines tgev tropism. rabbit anti-human tfr (abcam). mouse monoclonal antibodies to his ha, and gfp (cmctag, milwaukee, usa). rabbit monoclonal anti-tgev n protein was prepared in our laboratory. anti-gapdh monoclonal antibody, hrp-conjugated goat anti-rabbit and anti-mouse igg (h + l) (vazyme, nanjing, china). fitc-conjugated anti-tgev polyclonal antiserum (vmrd). anti-rabbit igg antibody (beyotime). ferristatin ii and lmp agarose (sigma-aldrich). protease inhibitor cocktail, enhanced chemiluminescence (ecl), and pierce bca protein assay kit (thermo scientific). protein a/g magnetic beads (b , bimake, usa). lenti-x htx packaging mix (clonetech). x-tremegene hp dna transfection reagent (roche, switzerland). pvdf membrane (millipore). np- lysis buffer (beyotime). trizol reagent (takara, dalian, china). ripa lysis buffer and sds-page sample loading buffer ( x) (fcmacs, nanjing, china). caco- and ipec-j cell lines (guangzhou jennio biotech co, ltd., china), st cell line (atcc,usa), and hek t cell line (atcc,usa) were cultured in dulbecco's modified eagle's medium (dmem from life technologies) supplemented with % fetal bovine serum (fbs, gibco), mm hepes (life technologies), and μg/ml penicillin/streptomycin (invitrogen) in a humidified atmosphere containing % co at °c. cells were routinely seeded at a density of × /ml in cm plastic tissue culture flasks (corning) and passaged every - days for a maximum of passages. tgev (strain shxb) was provided by the jiangsu academy of agricultural sciences and propagated in st cells. the complete tgev shxb genome sequence is available in genbank (kp . ) [ ] . viruses were labeled with the fluorescent probe dylight nhs ester (thermo scientific) according the manufacturer's recommended protocol. a dna fragment encoding tfr was amplified by pcr and then inserted into the plvx-dsred-monomer-n expression vector (ecori/bamhi) (clontech). similarly, tfr -out (encoding the extracellular region of tfr ) was cloned into pacgfp -c (using sali/bamhi) (clontech) and pet- a-c(+) (using ecori/sali). tgev spike (s ) was cloned previously into pcmv-c-ha (bamhi/xbai) in our laboratory [ ] . shrna sequences targeted against tfr were designed using tools available at http://rnaidesigner.lifetechnologies.com/ rnaiexpress/insert.do and block-it rnai designer. the best silencing efficiency was exhibited by clone nm_ . (sus scrofa transferrin receptor ). shrnas were cloned into the plvx-shrna vector (ecori/bamhi) (takara, dalian, china). all primers used in pcr are described in table . cells were seeded with or without treatment for the indicated times, inoculated with tgev at a moi of at °c for h, and then washed three times with cold phosphate-buffered saline (pbs) to remove unattached virus. the cells were shifted to °c in a % co incubator and maintained in dmem supplemented with % fbs and μg/ml penicillin/streptomycin. infected cells and supernatants were harvested at the conclusion of the incubation period. to determine whether tfr and tgev co-localize, ipec-j cells were seeded on cover slips in -well tissue culture plates, infected with tgev or -conjugated tgev at °c for h, and then incubated at °c for various times. cells were fixed in % paraformaldehyde for min and incubated with . % triton x- in pbs for min. after the cells were blocked with % bovine serum albumin (bsa), they were incubated with primary antibodies ( : ) overnight at °c. cells were then rinsed and incubated with fluorochrome-conjugated secondary antibodies ( : ) for min at room temperature, washed again three times with pbs and incubated with μg/ml dapi for min. images were captured using a zeiss lsm confocal microscope (carl zeiss, germany) and analyzed using zen (blue edition) (carl zeiss). at the indicated times post infection, cells were washed with pbs and lysed in ice-cold cell lysis buffer with protease inhibitor cocktail. total protein concentration was determined with a pierce bca protein assay kit, using the bicinchoninic acid spectrophotometric method. samples containing equal amounts of protein were separated sds-page and transferred to a pvdf membrane. the membrane was blocked with % nonfat milk in tris-buffered saline (tbs) containing . % tween , incubated overnight at °c with primary antibodies ( : ), and then incubated with the corresponding hrp-conjugated secondary antibodies ( : ) for min at °c. antibody binding was detected by autoradiography using ecl. ipec-j cells were incubated in ripa lysis buffer containing protease inhibitor cocktail and then centrifuged at , ×g for min. the cleared lysate was pretreated with μl anti-rabbit igg control and fresh protein a/g magnetic beads for h at °c to eliminate nonspecific binding to the beads. the beads were removed by magnetic separator and the lysate was incubated with μg of anti-tfr ab overnight at °c on a rocker platform. μl of fresh protein a/g magnetic beads were then added to the mixture, and incubated for h at °c on a rocker platform. the beads were washed four times with pbs, combined with page loading buffer, and incubated for min at °c to release the immunoprecipitated proteins. the proteins were analyzed by western blotting using anti-tfr ab. in parallel, magnetic beads loaded with tfr were incubated with purified tgev for h at °c on a rocker platform. beads were washed four times with pbs, and prepared for western blotting using the same methods. the blot was probed with anti-tgev-n ab and anti-tfr ab. to identify the tfr binding domain, we co-transfected plasmids expressing tfr and tgev-s (or tfr -out and tgev-s ) into hek t cells that had reached - % confluence. transfection was conducted using the x-tremegene hp dna transfection reagent following the manufacturer's instructions. after h, cells were lysed in np- lysis buffer containing a protease inhibitor cocktail then centrifuged at ×g for min. the supernatant was pretreated with protein a/g magnetic beads and igg (from the same species as the immunoprecipitating antibody) for h at °c to eliminate nonspecific binding to the beads. the beads were removed by centrifugation and the supernatant was then incubated on a rotary mixer overnight at °c with the immunoprecipitating antibody. fresh protein a/g magnetic beads were added to the mixture and incubation continued for additional hours at °c. the complexes were analyzed by western blotting using antibodies against ha, gfp, and tfr . to examine virus entry, ipec-j cells were infected with tgev at a moi of for h at °c. trizol reagent was used to extract total rna, following the manufacturer's protocol. rna was analyzed by rt-qpcr, as previously described [ ] . gene expression was calculated using the comparative ct method and results from three independent experiments. rna levels were normalized using endogenous gapdh rna as a reference. primer sequences used for qrt-pcr are listed in table . flow cytometry was used to detect tgev entry into cells as follows: fluorescently tagged tgev was incubated with cells for h at °c. the cells were washed with pbs and maintained in dmem for h at °c in % co , then harvested using . % trypsin. acquisition of the fluorescent cells done was by facs (becton dickinson) and the data were analyzed using flowjo software (version . . ). to produce lentivirus, we transfected the tfr expression construct or shtfr into hek t cells using the lenti-x htx packaging mix (plasmids plp , plp , and vsv-g) and the x-tremegene hp dna transfection reagent, following the manufacturer's instructions. h post transfection, the culture medium was renewed and incubation continued for and days. virus-containing supernatants were collected, filtered (pore size . μm), and stored at − °c. ipec-j , st, and caco- cells were infected with lentiviral particles (moi = ) containing the tfr expression construct. twenty-four hour post infection the culture medium was refreshed, and incubation continued for - h to allow for maximum protein expression. ipec-j and st cells were transfected with the tfr shrna (shtfr ) and the negative control (scrambled) vectors (shctrl). to generate cells that were stably transformed with ipec-j -shtfr and st-shtfr , lentiviral particles (moi of ) were added to the cells in the presence of μg/ml polybrene. after incubation for h, the cell cultures were expanded and maintained for weeks in dmem with μg/ml puromycin. surviving cells were maintained in medium supplemented with μg/ ml puromycin. lysates from transduced cells were analyzed by western blotting. expression and purification of his-tagged tfr -out recombinant protein tfr -out recombinant protein was expressed in escherichia coli bl- and purified using ni-nta resin, following the manufacturer's protocol as previously described [ ] . expressed a protein was used as a control. confluent monolayers of st cells in -well plates were inoculated with serial ten-fold dilutions of virus suspension and incubated for h at °c. the cells were then overlaid with . % low melting point agarose in dmem containing % fbs and incubated about h at °c. to visualize plaques, cells were stained with % crystal violet in methanol. data are presented as means ± standard deviation (sd) from three independent experiments. statistical analysis was performed using statistical program for social sciences (spss) . . differences between control and experimental groups were analyzed using student's t-test and one-way analysis of variance (anova). differences were considered statistically significant at * . < p < . , ** p < . . to investigate whether tfr is involved in tgev infection, we used confocal microscopy to visualize the subcellular locations of tgev and tfr during tgev infection. in the first experiment, tgev was directly labeled with dylight -labeled tgev, while tfr was labeled indirectly using a dylight -conjugated antibody (fig. a) . the results indicated that tfr co-localized with tgev. the experiment was repeated using a dual-label immunofluorescence strategy (fig. b) . the results were consistent with our initial observation, and also showed that tfr appeared to re-localize, internalize, and cluster early in tgev infection (fig. b and c) . based on fluorescence, tfr protein levels were significantly reduced compared to levels in uninfected cultures at h post infection (h p.i.) (fig. c) . western blotting analysis confirmed that tfr expression decreased significantly until h p.i. (fig. d) . the co-localization of tfr and tgev, and the decrease in tfr expression during tgev infection, suggest that tfr and tgev interact reciprocally. to test this hypothesis, endogenous tfr was enriched from ipec-j cell lysates using magnetic beads bound to anti-tfr ab. empty magnetic beads (i.e., without antibody) and magnetic beads bound to allogeneic anti-rabbit igg were used as negative controls (fig. a) . the beads were then tested for their ability to bind tgev. only beads that had previously captured endogenous tfr were able to bind tgev (fig. b) . importantly, pre-incubating tgev (moi ) with the precipitated endogenous tfr blocked viral replication at h p.i. (fig. c and d) . these data indicate that endogenous tfr interacts with tgev. to verify that tfr is involved in tgev infection, we assessed the ability of tgev (moi or ) to infect cells after blocking cellular tfr with anti-tfr ab. the cells were fixed, stained for tgev-n protein using a fluorescence-tagged antibody, and then examined by microscopy. fluorescence was significantly decreased at h p.i. in blocked cells (fig. a) . at h p.i., western blotting showed that expression of tgev-n protein was significantly lower in ipec-j cells that had been pre-incubated with anti-tfr ab (fig. b and c) . in contrast, tgev invasion was not affected by treatment with the tfr ligands holo-tf or apo-tf (additional file : figure s ). ferristatin ii, a tfr inhibitor that causes degradation of tfr ( ) , was used to confirm the role of tfr in tgev invasion. preliminary experiments showed that a μm dose of ferristatin ii did not result in detectable cytotoxicity (additional file : figure s ). tgev replication was significantly inhibited in ipec-j cells pre-incubated with this concentration of ferristatin ii at h p.i. (fig. d and e) . given the evidence that tfr functions in tgev infection, we performed analogous experiments using shrna to target tfr in ipec-j and st cell lines. both total tfr protein (fig. f ) and membrane tfr protein (fig. g ) decreased in the knockdown cells compared to normal cells. tgev-n mrna levels were also significantly lower in tfr knockdown cells at h p.i. (fig. h) . to determine whether tfr knockdown affects tgev replication in ipec-j cells, we used immunofluorescence staining to detect changes in tgev-n protein levels. tgev-n protein was obviously reduced in cells treated with the tfr knockdown shrna at h p.i., relative to cells treated with the scrambled shrna control (fig. i) . likewise, we observed a significant decrease in tgev replication in western blotting assays ( fig. j and k) (see figure on previous page.) fig. tgev co-localizes with tfr and decreases its expression. a ipec-j cells were infected with dylight -tgev (green), and cultured for h. the cells were then stained for confocal microscopy using a rabbit anti-tfr pab, followed by dylight -conjugated goat anti-rabbit igg (red). the panel shows a three-dimensional rendering of a representative field obtained using imaris . , and the arrows indicate co-localized signals (scale bar = μm). b ipec-j cells were infected with tgev (moi ) and fixed at h p.i. cells were then stained for confocal microscopy using a rabbit anti-tfr ab, followed by dylight -conjugated goat anti-rabbit igg (red) and fitc-conjugated anti-tgev polyclonal antiserum (green). the arrows indicate co-localized signals (scale bar = μm). c indirect immunofluorescence images of tfr in mock-and tgev-infected cells at h p.i. and h p.i. (moi ). fixed cells were stained for tfr (red) and the nucleus was stained with dapi (blue). scale bar = μm. d ipec-j cells were uninfected or infected with tgev (moi ) and harvested at different time intervals ( - h p.i.) . the cell lysates were analyzed by western blotting using anti-tfr , anti-tgev-n, and anti-gapdh antibodies as probes fig. tgev interacts with endogenous tfr . a endogenous tfr was enriched from ipec-j cell lysates using magnetic beads coated with anti-tfr ab. tfr immobilized on the beads was detected by western blotting. b the enriched endogenous tfr described in panel a was used as bait to bind tgev. empty magnetic beads and magnetic beads with allogeneic anti-rabbit igg served as negative controls. the n protein of tgev was precipitated using immobilized beads coated with anti-tfr ab but not anti-rabbit igg. c and d tgev (moi ) was pre-incubated with the precipitated tfr protein for h at °c before cells were infected. at h p.i., tgev replication was analyzed by western blotting. pre-incubation with tfr protein inhibited viral replication. the tgev-n to gapdh ratio was normalized to control conditions. data shown are means ± sd from three independent experiments. (* . < p < . , ** p < . ) and plaque assays (fig. l and m) at h p.i. similar results were also obtained in st cells (fig. n and o) . because the experiments described above indicate that tfr is involved in tgev infection, we investigated whether overexpression of tfr would enhance tgev infectivity. ipec-j and st cells were infected with lentiviruses (moi ) containing the tfr expression construct or the empty control vector. as expected, cells infected with the tfr-expressing lentivirus particles showed significantly increased tfr expression over cells infected with lentivirus containing the empty vector (fig. a) . to determine whether overexpression of tfr impacts the tgev infection process, we used flow cytometry to test the ability of dylight -labeled tgev to associate with ipec-j cells (fig. b and c) . tgev mrna levels were also were measured in ipec-j cells at h p.i. using rt-qpcr (fig. d) . both experiments demonstrated that overexpression of tfr protein significantly increases viral infectivity. tgev replication was also significantly higher in tfr -overexpressing st cells at h p.i. (fig. e and f ) . finally, when tfr was overexpressed i. for western blotting. tgev replication was inhibited by tfr knockdown. the tgev-n to gapdh ratio was normalized to control conditions. the data shown are means ± sd from three independent experiments. (* . < p < . , ** p < . ) in tgev-resistant caco- cells, they became susceptible to tgev infection (fig. g, h and i) . extracellular tfr interacts with tgev s protein in vitro tgev s protein binds to specific receptors on the membrane of susceptible cells [ ] . to examine the interaction between tgev s and tfr , we obtained lysates from t cells that had been co-transfected with plasmids expressing tgev s -ha and tfr , and then conducted a co-immunoprecipitation assay. the results confirmed that tgev s directly interacts with tfr (fig. a) . we then constructed a plasmid encoding the extracellular region of tfr (designated tfr -out) and transfected it into cells along with the tgev s plasmid described earlier. the co-precipitation assay confirmed that the extracellular region of tfr interacts with tgev s protein in vitro (fig. b) . finally, his-tagged tfr -out fusion protein was prepared using an e. coli expression system. protein quality was verified by sds-page (fig. c ) and western blotting (fig. d) . when cells were pre-incubated for h with tfr -out ( ng/ml) prior to infection by tgev, viral replication as reflected by tgev-n levels, was inhibited ( fig. e and f ) . this result was consistent with a plaque assay for virus particles present in the cell culture medium (fig. g and h ). tgev invades the epithelial cells of the intestine via a receptor-mediated fusion mechanism [ , ] . the species-specific virus tropism or host-range is usually determined by entry receptors [ ] . the intestinal epithelium of neonatal piglets is particularly susceptible to tgev [ ] . identifying the proteins that mediate the association between the host cell and the virus is therefore a crucial step for understanding virus-host interactions. in this study, we conducted experiments to determine if tfr can function as a receptor for tgev invasion. the results show that tgev induces the internalization, clustering, and down-regulation of cellular tfr . overexpression of tfr enhances tgev invasion, and infection by tgev can be inhibited if access to tfr is blocked, or if tfr levels are reduced. finally, we determined that tgev-s protein interacts with the recently, li et al. observed that the ability of tgev to bind mdck cells is enhanced when the cells express porcine aminopeptidase n (papn) [ ] . we found that overexpression of tfr in the refractory caco- cell line is sufficient to allow tgev entry, synthesis of viral rna and protein, and release of infectious tgev. papn has been shown to function as a receptor for tgev infection [ ] [ ] [ ] [ ] . however, this protein seems to be widely distributed on enterocytes and probably on other tissues, irrespective of age [ ] . an entry receptor or co-receptor usually mediates virus entry, as is the case for human immunodeficiency virus (hiv) and poliovirus [ ] [ ] [ ] [ ] . a kda protein that is restricted to the villous enterocytes of newborn pigs has been identified as a possible additional receptor for tgev, and may account for the age sensitivity of these animals to the virus [ ] . newborn piglets with iron-deficiency anemia usually present elevated levels of tfr on the entire surface of the intestinal epithelium [ , , ] . tfr may therefore be a extracellular tfr interacts with tgev s protein in vitro. a and b t cells were co-transfected with a ha-tagged tgev s expression plasmid together with plasmids expressing tfr or gfp-tagged tfr -out. cell lysates were immunoprecipitated with anti-ha antibody and anti-tfr antibody, or anti-ha antibody and anti-gfp antibody, respectively. the precipitates were examined by western blotting using anti-ha antibody and anti-tfr antibody (a) or anti-ha antibody and anti-gfp antibody (b) to examine the interaction between ha-tgev-s and tfr , or ha-tgev-s and gfp-tagged tfr -out, respectively. c his-tagged tfr -out was expressed in e.coli bl and purified using a ni-nta column. purified products were separated using sds-page and stained with coomassie brilliant blue. d purified tfr -out was verified by western blotting. e and f ipec-j cells infected with tgev (moi ) were pre-incubated with tfr -out ( ng/ml) for h at °c, and cell lysates were harvested for western blotting. the ratio of tgev-n to gapdh was normalized to control conditions, and culture supernatants were collected for viral plaque assays in st cells. plaques developed days after infection. normal cells and cells treated with pet- a-c(+) served as controls. data shown are means ± sd from three independent experiments. (* . < p < . , ** p < . ) major contributing factor to the high level of tgev susceptibility exhibited by anemic newborn pigs. tfr plays a role in hcv entry via the clathrin endocytic pathway, and the expression of tfr is down-regulated in hcv-infected huh cells [ ] . junv also uses tfr as a cellular receptor for entry into vero cells and is rapidly internalized by clathrin-mediated endocytosis [ ] , and canine parvovirus (cpv) utilizes limited tfr clustering on the surface to enter host cells through the clathrin endocytic pathway [ , ] . studies in our laboratories have demonstrated that egfr is another promoter for tgev entry via the clathrin-dependent endocytic pathway [ ] . upon binding to its ligand holo-tf (iron-bound transferrin), tfr that is associated with clathrin-coated pits of the plasma membrane is internalized by dynamin-mediated endocytosis. the small gtpase rab , and its upstream activator dennd , regulate membrane trafficking of tfr from recycling endosomes to lysosomes [ ] . we observed that a decrease in tfr expression follows tgev invasion. we hypothesize that the decrease occurs because tfr is degraded by lysosomes after binding to tgev, and that tfr plays a critical role in the clathrin-mediated endocytosis of tgev. one of the four structural proteins encoded by the tgev genome is the large transmembrane s glycoprotein. trimers of s molecules form the surface spikes on coronaviruses which mediate virus entry and is a primary determinant tissue tropism cells [ , ] . the ectodomain of the s protein consists of an n-terminal variable domain called s that is responsible for receptor binding, and a c-terminal conserved domain called s that is responsible for fusion. [ , , ] . the tgev-s protein interacts with papn and egfr [ , ] . in our study, we demonstrated that tgev interacts with endogenous tfr and that tgev-s protein interacts with tfr and the extracellular region of tfr . the extracellular region of tfr is located between amino acid residues - . our experiments confirmed that pre-incubating tgev with endogenous tfr or soluble tfr -out blocked viral invasion, supporting the hypothesis that these extracellular amino acids residues are important for the recognition of tgev s . in addition, the anti-tfr antibody used in our blocking experiments recognizes amino acid residues - of tfr . this reagent also blocks tgev invasion, presumably by occluding the site at which tgev binds to tfr . in contrast, neither holo-tf nor apo-tf interfered with the ability of tgev to infect ipec-j cells, indicating the use of non-overlapping binding sites. based on these results, we hypothesize that tfr amino acid residues - encode the binding site for s protein. our experimental results strongly support the conclusion that tfr functions as a receptor for tgev invasion. the results are also consistent with the hypothesis that tfr may be responsible for the high level of tgev susceptibility exhibited by newborn pigs. additional work will be required to verify this hypothesis. finally, this study will facilitate the development of vaccines to combat tgev infection. additional file : figure s . tgev invasion didn't affect with the treatment of tfr ligands, holo-tf and apo-tf. figure s . the molecular biology of coronaviruses development of protection against coronavirus induced diseases simulation of the economic impact of transmissible gastroenteritis on commercial pig production in australia complete sequence ( kilobases) of the polyprotein-encoding gene of transmissible gastroenteritis virus mechanisms of coronavirus cell entry mediated by the viral spike protein morphogenesis and proliferative rule of porcine transmissible gastroenteritis 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transferrin receptor as a hepatitis c virus entry factor cell entry by human pathogenic arenaviruses the ipec-j cell line porcine ipec-j intestinal epithelial cells in microbiological investigations complete genomic sequence of the coronavirus transmissible gastroenteritis virus shxb isolated in china how viruses enter animal cells entry and release of transmissible gastroenteritis coronavirus are restricted to apical surfaces of polarized epithelial cells aminopeptidase n is not required for porcine epidemic diarrhea virus cell entry identification of a major co-receptor for primary isolates of hiv- hiv- entry into cd + cells is mediated by the chemokine receptor cc-ckr- hiv- entry cofactor: functional cdna cloning of a seven-transmembrane, g protein-coupled receptor a monoclonal antibody that blocks poliovirus attachment recognizes the lymphocyte homing receptor cd characterization of junin arenavirus cell entry limited transferrin receptor clustering allows rapid diffusion of canine parvovirus into clathrin endocytic structures small gtpase rab regulates constitutive degradation of transferrin receptor targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence major receptor-binding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein epidermal growth factor receptor is a co-factor for transmissible gastroenteritis virus entry we thank dr. qinghua yu and dr. jian lin for helpful suggestions and comments on the manuscript, and dr. jie peng for her technical assistance. this work was supported by an award from the national natural science foundation of china ( ) and by the priority academic program development (papd) of jiangsu higher education institutions. we declare that materials described in the manuscript, including all relevant raw data, will be freely available to any scientist wishing to use them for non-commercial purposes, without breaching participant confidentiality. ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -lkd mj authors: sungsuwan, suttipun; jongkaewwattana, anan; jaru-ampornpan, peera title: nucleocapsid proteins from other swine enteric coronaviruses differentially modulate pedv replication date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: lkd mj porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev) and porcine deltacoronavirus (pdcov) share tropism for swine intestinal epithelial cells. whether mixing of viral components during co-infection alters pathogenic outcomes or viral replication is not known. in this study, we investigated how different coronavirus nucleocapsid (cov n) proteins interact and affect pedv replication. we found that pdcov n and tgev n can competitively interact with pedv n. however, the presence of pdcov or tgev n led to very different outcomes on pedv replication. while pdcov n significantly suppresses pedv replication, overexpression of tgev n, like that of pedv n, increases production of pedv rna and virions. despite partial interchangeability in nucleocapsid oligomerization and viral rna synthesis, endogenous pedv n cannot be replaced in the production of infectious pedv particles. results from this study give insights into functional compatibilities and evolutionary relationship between cov viral proteins during viral co-infection and co-evolution. porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev) and porcine deltacoronavirus (pdcov) belong to the family coronaviridae (enjuanes, ) . pedv and tgev have been classified into the alphacoronavirus genus, whereas pdcov belongs to the deltacoronavirus genus (jung et al., a; jung and saif, a) . they share similar genome architectures, with a - kb positivesense, single-stranded rna genome. the ′ two-thirds of the viral genome encodes non-structural proteins from open reading frames (orf) a and b necessary for viral genome replication. the rest of the genome encodes a number of unique accessory proteins such as pedv orf , tgev a/ b/ , pdcov ns /ns , and four common structural proteins, namely the spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins (kocherhans et al., ; lee and lee, ; penzes et al., ) . these enteric swine coronaviruses (covs) infect epithelial cells lining the small intestine and cause villous atrophy, resulting in malabsorption and severe diarrhea (jung et al., a ). an outbreak of these viruses, especially pedv, can lead to up-to- % mortality in neonatal piglets, prompting huge economic losses in the swine production industry worldwide. unless they are examined by laboratory-level diagnosis, these covs produce almost indistinguishable pathogenesis. co-infection of enteric pathogens are common. tgev and pdcov have been found to co-circulate with pedv in the field (song et al., ; wang et al., ) . in pdcov-positive samples, the rate of pedv co-infection as detected by rt-pcr varies from % to % (jung et al., a; jung and saif, a) . although tgev infection has become rarer nowadays, it has still been detectable in samples in china, and often together with pedv and/or pdcov (dong et al., ; wang et al., ) . despite substantial epidemiological evidence of co-infection, the effects of these events on disease outcomes have not yet been formally described. since these enteric swine covs share cell tropism, co-infection of these viruses can theoretically cause mixing of viral components in the same cellular compartments, possibly leading to direct or indirect effects on viral replication kinetics or pathogenic outcomes. to the best of our knowledge, there are currently no reports on studies at molecular or cellular levels on how viral components from different cov species interact with or affect other viruses. investigation of possible molecular interactions between components of pedv, pdcov and tgev and their influence on replication of each virus would provide a crucial insight into comprehensive understanding of these covs. of all viral proteins, we have chosen to start with the n protein, as it is among the most abundant and ubiquitous structural proteins in infected cells. the cov n protein is functionally conserved across the family coronaviridae (chang et al., ; cong et al., ) , with its primary function being to form a scaffold for packaging viral genomic https://doi.org/ . /j.virol. . . received september ; received in revised form november ; accepted november rna (grna) into the internal core of virions (de haan and rottier, ) . besides scaffolding, other functions of the cov n protein (primarily based on studies of common representatives of the family like severe acute respiratory syndrome-cov (sars-cov) or mouse hepatitis virus (mhv)) include acting as rna chaperones (zuniga et al., (zuniga et al., , , promoting viral genome transcription or replication (hurst et al., (hurst et al., , masters et al., ; zuniga et al., zuniga et al., , , facilitating viral assembly (de haan and rottier, ; kuo et al., ) , suppressing antiviral rna-interference activity from their hosts (cui et al., ) , and suppressing host immunity (ding et al., (ding et al., , xu et al., ; zhang et al., ) . based on sequence alignment and limited structural data from some representative covs, all cov n proteins are predicted to consist of three structural domains: the n-terminal domain (ntd), linker region (lkr) and c-terminal domain (ctd) (chang et al., ; mcbride et al., ) . ntd binds rna through electrostatic interaction with its charged amino acids as well as interaction between conserved aromatic residues in the proteins and nucleotide bases in the rna (chang et al., ; huang et al., ; tan et al., ) . lkr is a disordered domain between ntd and ctd. studies of sars-cov n indicate roles for lkr in rna binding, virion assembly and self-association (chang et al., (chang et al., , he et al., a) . although it is also reported to have rna binding capacity, ctd is a more hydrophobic domain mainly responsible for self-association to form stable dimers and subsequent oligomers of cov n (chen et al., ; yu et al., ) . studies of n proteins from sars-cov and mhv revealed that n dimerization followed by multimerization is a common process in the formation of viral ribonucleoprotein that initiates viral genome packaging in the virion assembly process among covs (cong et al., ; fan et al., ; ma et al., ; yu et al., ) . examples from other viruses suggest that cross-association between viral nucleocapsid proteins could determine different outcomes during co-infection. for instance, mixed infection between two types of plant tospoviruses results in interspecies interaction between their n proteins and more severe symptoms compared to single infections, suggesting synergy between these viruses (bag et al., ; tripathi et al., ) . on the other hand, intertypic interference between types a and b influenza viruses could be partially explained by the inhibitory effect of type b influenza virus nucleoprotein exerted on its type a counterpart (aoki et al., ; jaru-ampornpan et al., ) . given structural similarities, we expect that cov n proteins would likely cross-interact with each other during a co-infection event. whether this cross-interaction has beneficial or detrimental effects on viral replication has never been explored. investigation of the interaction between cov n proteins could lead to insights into virus evolution, anti-cov drug development and vaccine design (chang et al., ; lo et al., ) . human embryonic kidney (hek) t cells, wild-type veroe cells and veroe -based cell lines stably expressing cov n were maintained in optimem supplemented with % fetal bovine serum (fbs) and antibiotics at °c with % co . the virus pedv-avct -mcherry (pedv-mcherry), its infectious clone (psmart-bac-mcherry-pedv avct [ppedv-mcherry]) and pcaggs-pedv n-myc, a plasmid for c-terminally myc-tagged pedv n expression, have been described previously (jaru-ampornpan et al., ; jengarn et al., ) . tgev and pdcov n encoding genes were codon-optimized for high expression in mammalian cells and to avoid expression of pdcov ns embedded in the pdcov n gene ) (genbank: aag . , afd . , respectively). pcr products of the corresponding n genes with indicated tags at the c-termini were ligated into pcaggs vectors to give plasmids for expression of tagged cov n (pcaggs-tgev n-flag and pcaggs-pdcov n-ha). pcaggs-ha-pedv n for n-terminally ha-tagged pedv n expression was similarly constructed based on the same pedv n dna sequence in pcaggs-pedv n-myc. pgex- t -pedv n was constructed by in-frame insertion of the pedv n gene sequence, codon-optimized for bacterial expression based on the sequence from avct strain (accession number lc ), into the pgex- t vector (amersham) following the coding sequence of n-terminal gst. all plasmids were verified by dna sequencing. cells were lysed with lysis buffer ( mm tris-hcl ph . , mm nacl, mm edta, % np- and % glycerol, supplemented with a protease inhibitor cocktail (halt™ protease inhibitor cocktail, thermo scientific)). proteins in the cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page), and then transferred onto nitrocellulose membranes (bio-rad laboratories). the membrane was blocked in % skim milk prior to incubation with indicated primary antibodies. horseradish peroxidase (hrp)-conjugated goat anti-mouse igg (biolegend) or (hrp)-conjugated donkey antirabbit igg (biolegend) was used as secondary antibodies. primary antibodies used in this study include mouse-anti-myc (thermo scientific), rabbit-anti-flag (cell science technology), rabbit-anti-ha (cell science technology), mouse-anti-s (a kind gift from dr. qigai he, huazhong agricultural university), and mouse-anti-pedv n (sd - , medgene labs). hek t cells were co-transfected with pcaggs-pedv n-myc (or the empty pcaggs vector for negative controls) and either pcaggs-ha-pedv n, pcaggs-tgev n-flag or pcaggs-pdcov n-ha using fugene hd (promega) according to the manufacturer's instructions. at h post-transfection (hpt), cell lysates were prepared as described previously and pre-adsorbed to control agarose beads (thermo scientific) for h at °c to reduce non-specific binding. then, the treated lysates were incubated with anti-myc-conjugated agarose beads (thermo scientific) at °c overnight. the mixtures were washed three times with lysis buffer supplemented with mm nacl. ip complexes were eluted by boiling the bead mixtures in × non-reducing sample buffer (thermo scientific) for min and supplemented with mm dtt before sds-page and western blot analysis. gst and gst-pedv n were expressed in bl (de *) cells. after h protein induction by . mm isopropyl ß-d- -thiogalactopyranoside, bacteria were lysed by sonication in × phosphate-buffered saline (pbs). the lysates were clarified and stored in -μl aliquots at - °c until use. hek t cells were transfected with plasmids as indicated and were lysed at hpt with lysis buffer. the bacterial lysate containing the bait protein (gst/gst-pedv n) and the hek t cell lysate containing the prey protein (ha-pedv n, tgev n-flag, pdcov n-ha, or combinations thereof) were incubated with pre-equilibrated gsh ff resin (amersham) overnight and washed twice in high-salt gst buffer and four times in low-salt gst buffer ( mm tris-hcl ph . , % glycerol, mm edta, . % np- and m/ mm nacl) (nguyen and goodrich, ) . the bound proteins were released by boiling the resin in × sds-page loading buffer and analyzed by coomassie staining (for bait) and western blotting with indicated antibodies (for prey). figures were representatives of three independent experiments. for fig. , quantification of eluted protein bands in each gst pulldown reaction was performed using the biorad image lab software . . and expressed as the ratios of pulled-down pedv n (western blot with anti-ha) to eluted gst-pedv n (coomassie stain). because of variability between independent experiments, these ratios were normalized to the conditions without extra cov n protein performed side-by-side in each experiment before statistical comparison. for the rnasea experiment, the combined lysates were treated with μg of rnasea (thermo scientific) or pbs buffer for min at °c to avoid protein precipitation at high temperature. to verify the absence of rna after the treatment, total rnas were purified from parts of treated lysates by rna purification kit (thermo scientific) and were used at equal volumes as templates for rt-pcr. the rt-pcr products were visualized by ethidium bromide staining. veroe cell lines expressing pedv n-myc, tgev n-flag and pdcov n-ha were constructed using the same protocol as veroe -pedv n cells as described previously (liwnaree et al., ) . briefly, lentiviruses carrying each cov n gene were generated by co-transfection of psin-csgw-ubem carrying the inserted cov n gene with a packaging plasmid encoding gag, pol, rev and tat (pcmv-Δr . ) and a plasmid expressing lentiviral vsv envelope glycoprotein (pmd .g) into hek t cells. at hpt, supernatants containing the lentiviruses were harvested and filtered through a . -μm filter. the filtered supernatants were then used to transduce veroe cells. a single clone of the transduced cells expressing the corresponding n protein was selected based on similar levels of the transgene protein expression. uniform expression of exogenous n proteins in the engineered veroe cell lines was verified by immunofluorescence analysis. briefly, cells were grown in an eight-well slide. at h, cells were fixed with % cold acetone for min. after washing twice with pbs, cells were blocked with % fbs, % bovine serum albumin (bsa) in pbs for h at room temperature. cells were then incubated with mouse anti-pedv n antibodies (sd - ) or anti-pdcov n (sd - , medgene labs) or anti-tgev n ( . , median diagnostics, republic of korea). after washing twice with pbs, cells were incubated with goat antimouse igg alkaline phosphatase antibodies (abcam). fluorescence was developed by adding alkaline phosphatase substrate (immpact™ vector® red). fluorescence images were taken under a fluorescence microscope (olympus). the n-deficient pedv infectious clone (ppedv-mcherry-Δn) was constructed using a strategy described previously (jengarn et al., ; wanitchang et al., ) . a frameshift mutation was used to silence n expression by mutating the original start codon in the n orf, atggct, to atatgt in an intermediate plasmid, ptz-gh, containing a pedv genome fragment (designated 'gh'; fig. a ) with the ′ end of the n gene as described previously (jengarn et al., ) . the site-directed mutagenesis was performed in the ptz-gh cloning vector. the mutated fragment was amplified by primers that facilitated subsequent in-fusion ligation (in-fusion hd, clontech) into a pre-digested psmart-bac plasmid containing the rest of the pedv genome (jengarn et al., ) to yield psmart-bac-pedv-mcherry-Δn (designated ppedv-mcherry-Δn). hek t cells were transfected with μg of the indicated infectious clone (or co-transfected with the indicated amount of a cov n expressing plasmid) using fugene hd (promega) according to the manufacturer's instructions. at hpt, cell lysates were prepared for western blot analysis as described previously, and supernatants were transferred for adsorption on veroe cells for h at °c. for viral rescue of pedv-mcherry-Δn, veroe cells stably expressing pedv n was further transfected with μg of pcaggs-pedv n for h before virus adsorption to ensure sufficient expression of pedv n in the infected cells. the inocula were then removed, and the cells were washed once with pbs and supplied with ml fresh optimem with . % tryple (thermo scientific). at indicated time points, infected cells were imaged under a fluorescence microscope (olympus), and the supernatants were collected for subsequent experiments or viral titer analysis by tcid assay. veroe -based cell lines ( × cells/ml) were plated in a six-well plate. at h, the cells were washed once with pbs and treated with ml of virus at the indicated multiplicity of infection (moi). at h post-adsorption, the inoculum was removed, and the cells were washed once with pbs and supplied with ml of fresh optimem containing . % tryple. extent of pedv infection was monitored by mcherry fluorescence under a fluorescence microscope. cell lysates or supernatants were harvested at the indicated time points for further analysis. for viral infection in the transient cov n expression experiment, veroe cells were transfected with or μg of pcaggs-pedv n-myc, pcaggs-pdcov n-ha, pcaggs-tgev n-flag, or the empty pcaggs vector and were incubated for h to allow for protein expression. cells were then infected with pedv-mcherry (moi = . ) as described above. monolayers of veroe cells in -well plates were washed once with pbs. one hundred microliters of -fold serially diluted virus in optimem with . % tryple was added to the cells ( wells per each dilution). at h post-infection (hpi), the infected cells were scored by mcherry expression under a fluorescence microscope. tcid titers were calculated using the reed-muench method (reed and muench, ) . for analysis of viral rna synthesis, total rna was extracted from veroe cell lines expressing various n infected with pedv-mcherry (moi = . ) at indicated time points using the rna extraction kit (thermo scientific). dnasei (fermentas) was used to treat the rna ( min at °c) before inactivation with edta ( min at °c). onestep rt-qpcr was performed with the luna universal one-step rt-qpcr mix (new england biolabs) as described previously (liwnaree et al., ) . relative quantities of rna accumulation were evaluated using the -ΔΔct method normalized against viral rna levels from infected veroe cells at hpi. values are reported as averages ± sem from three independent experiments. during co-infection, viruses can influence the course of infection of other viruses via direct physical interactions between viral components. to explore the possibility that swine cov n proteins might affect replication of other co-infecting covs, we first investigated cross-species interaction between these n proteins. specifically, we asked if n proteins from tgev or pdcov can interact with pedv n and possibly affect pedv replication. we first assessed interspecies protein-protein interaction by co-immunoprecipitation. hek t cells were co-transfected with pcaggs-pedv n-myc and pcaggs expressing ha-pedv n, other cov n proteins bind competitively to pedv n. gst pulldown by gst-pedv n was performed with hek t cell lysates containing ha-pedv n and varying amounts ( - μg transfected plasmids) of pdcov n-ha or tgev n-flag. the input and eluates were separated by sds-page. coomassie staining was used to detect gst-pedv n, and western blotting was used to detect haand flag-tagged proteins. the figure is representative of three independent experiments. quantification was performed using the biorad image lab software . . as described in materials and methods. the pulldown ratios from the conditions without pdcov or tgev n proteins in each independent experiment were set to one. *p < . , **p < . compared to the conditions without pdcov or tgev n proteins. fig. . effect of rna on homo-and hetero-oligomerization of pedv n. gst pulldown by gst-pedv n was performed with hek t cell lysates containing ha-pedv n, pdcov n-ha or tgev n-flag. the lysate mixtures were treated with μg rnasea (+) or buffer (-) for min at °c prior to incubation with gsh ff resin. the input and eluates were analyzed with sds-page (coomassie staining for gst-pedv n) and western blotting for the other n proteins. rna was extracted from a portion of the lysate mixtures, treated with dnasei, and subjected to rt-pcr with primers specific to the nucleocapsid genes studied in each experiment. expected rt-pcr products were about bpand were analyzed by agarose gel electrophoresis. pdcov n-ha or tgev n-flag. at hpt, lysates were prepared and incubated with agarose beads coupled to anti-myc antibodies prior to elution and analysis by western blotting. as expected, ha-tagged pedv n could be precipitated with myc-tagged pedv n but not with empty beads, indicating specificity of the homo-oligomeric interaction (fig. a) . interestingly, both n proteins from related covs also displayed specific interaction with pedv n, both co-eluting with myctagged pedv n (fig. a) . nevertheless, we observed slight non-specific binding in the case of pdcov n-ha despite stringent washing conditions. therefore, we utilized a gst pulldown assay as an alternative and independent verification of these results. for gst pulldown, the bait proteins, gst and gst-pedv n, were expressed in bacterial bl (de *) cells, and the prey proteins, ha-pedv n, pdcov n-ha and tgev n-flag, were expressed in hek t cells. bacterial and hek t cell lysates were incubated with gsh ff resin overnight to allow for bait-prey interaction. after removing nonspecific binding by multiple rounds of washing, the bound proteins were released and analyzed by sds-page. bait and prey proteins were detected by coomassie staining and western blotting with indicated antibodies, respectively. ha-pedv n could only be observed when pulled down with gst-pedv n, while no ha-pedv n was observed when pulled down with gst, demonstrating the specificity of the assay (fig. b) . next, we performed the gst pulldown experiment with pdcov n and tgev n as prey proteins. similarly, we found that pdcov n-ha and tgev n-flag only co-eluted with gst-pedv n, indicating that n proteins from both tgev and pdcov bound to pedv n specifically (fig. b) . both co-immunoprecipitation and gst pulldown results indicated that interspecies hetero-oligomers of cov n proteins are possible, suggesting conservation of the oligomerization motifs and function of nucleocapsid proteins among swine covs. these results encouraged us to further probe the factors affecting the nature of these interactions. the cross-association capability of pdcov n or tgev n could possibly interrupt homo-oligomerization between pedv n and lead to competitive binding between pedv n and other cov n proteins. using the gst pulldown assay, we further probed the interplay between these cov n proteins to see how pedv n homo-oligomers would be affected in the presence of other cov n proteins. hek t cells were cotransfected with pcaggs-ha-pedv n and varying amounts of pcaggs-pdcov n-ha or pcaggs-tgev n-flag. at hpt, lysates were prepared and incubated with gst-pedv n-containing bacterial lysate and gsh ff resin overnight. gst pulldown revealed that, upon increasing amounts of co-eluting pdcov or tgev n, co-eluting hatagged pedv n decreased reciprocally (fig. ) . this indicates, to a certain level, a competition between homo-oligomerization of pedv n and hetero-oligomerization with nucleocapsid proteins from other covs, and suggests that these cov n proteins utilize the same motifs or binding interfaces in oligomeric complex formation or harness common factors such as rna that can mediate oligomeric interactions. one of the primary functions of cov n proteins is to bind and organize viral rna genomes for viral assembly (mcbride et al., ) . however, there have been conflicting reports about the necessity of viral rna in promoting or assisting cov n self-oligomerization. some reports observed rnasea susceptibility of the oligomeric complex (narayanan et al., ; verheije et al., ) , while others detected intact higher cov n oligomers despite the absence of rna (cong et al., ; jayaram et al., ; ma et al., ) . to investigate whether the presence of rna influences interspecies association between these swine enteric cov n proteins, gst pulldown experiments were performed with or without rnasea treatment. first, however, we tested whether or not rna affected pedv n homo-oligomerization in this assay setup. hek t cell lysate containing ha-tagged pedv n was mixed with gst-pedv n bacterial lysate and split into two equal parts. the lysate mixtures were either treated with rnasea or pbs buffer for min at °c. the absence of rna after the treatment was assured by performing rt-pcr reactions with primers specific to the pedv n gene (fig. ) . the lysate mixtures were then incubated with gsh ff resin overnight to compare the amount of ha-pedv n pulled down by gst-pedv n in the presence or absence of rna. our results across three independent experiments consistently showed a slight but noticeable increase in gst-pedv n in the elution fraction after rnasea treatment (fig. ) . moreover, the amount of ha-pedv n pulled down in the absence of rna seemed to be greater than that observed in the presence of rna, suggesting that rna interferes with the ability of pedv n to bind to each other. we also consistently observed a slight decrease in the level of tagged pedv n in the input fraction after the lysates were treated with rnasea, implying decreased overall stability of pedv n in the absence of rna. similar decreases in protein levels were not observed with tgev or pdcov n proteins (see below), suggesting specificity of the phenomenon. interestingly, when the experiments were performed on lysates containing pdcov n-ha or tgev n-flag, a completely opposite trend was observed for hetero-oligomerization. while increased gst-pedv n was still similarly observed in the eluted fraction in the absence of rna, both pdcov n and tgev n were pulled down by gst-pedv n much less efficiently in the absence of rna (fig. ) . it should be noted that the total amount of the tagged cov n proteins did not decrease, arguing against the possibility that tgev or pdcov n proteins were more prone to degradation without the bound rna (fig. , 'input' lanes) . these results suggest that the presence of rna immensely helps strengthen hetero-oligomerization between pedv n and other cov n proteins but renders homo-oligomerization between pedv n weakened. although not an absolute requirement for interspecies complex formation, rna could act as a bridge for n proteins from different species to form the complex, implying the heterologous protein-protein interactions are significantly weaker or less specific than the homologous interaction. even though the levels of rna purified from rnasea-treated lysates were below the detection limit of spectrophotometer (data not shown) and could not produce visible rt-pcr products, we could not rule out the possibility of leftover small rna fragments mediating heterologous cov n protein binding, giving rise to much fainter tgev n or pdcov n bands that co-eluted with gst-pedv n. in summary, the results in this section demonstrate conserved association capability among different cov n proteins but suggest some differentiating features, especially the influence of rna on each complex, between homo-and hetero-oligomers involving pedv n. hetero-oligomer formation as demonstrated in the previous section formed a basis for our investigation into n-mediated virus-virus interaction. as it has been shown for other viruses, it is not unreasonable to assume that the specific and competitive binding between n proteins of pedv and other covs could interrupt regular functions of pedv n during viral growth. to investigate how this protein-protein interaction might affect pedv replication, we transiently transfected veroe cells with varying amounts of the pcaggs plasmid expressing n proteins from either pdcov or tgev for h before infection with pedv-mcherry (moi = . ) and followed the course of viral replication for each condition. the levels of cov n expression were verified by western blotting against tags attached to each n protein (fig. a) . cell culture supernatants were collected at , and hpi to determine virus titers by tcid assay (fig. b-d) . the results showed that veroe cells transiently transfected with either pedv n or tgev n showed a dose-dependent increase, up to an order of magnitude, in pedv-mcherry titers compared to veroe cells transfected with a blank vector ( fig. b and d) . interestingly, veroe cells transiently transfected with pdcov n showed suppression on pedv-mcherry growth, especially at high levels of pdcov n expression (fig. c ). due to relatively low transfection efficiencies in veroe cells and the uncontrollable proportion of exogenous n expression and pedv infection occurring in the same cells, the actual effects of these cov n proteins on pedv growth might be even more pronounced than observed from the transfection experiments. to test this, we constructed veroe -based cell lines stably expressing n from pedv, pdcov and tgev by lentivirus transduction. veroe clones expressing relatively equal amounts of n proteins from either pedv, pdcov and tgev were selected based on western blot analysis (fig. a) . uniform expression of exogenous n proteins in these engineered veroe cells was also assessed by immunofluorescence to eliminate the concern from the transient transfection experiment (fig. a) . equal numbers of cells were plated to confluence in -well plates and infected with pedv-mcherry (moi = . ). spread of pedv infection and syncytium formation was monitored daily by fluorescence microscopy. supernatants were collected at , and hpi to monitor pedv growth by tcid assay. veroe cells expressing n from pedv and tgev clearly accelerated pedv syncytia formation and spread. by hpi, most cells displayed signs of profuse infection; by hpi, infected cells were dead and detached compared to infection in wild-type veroe cells (fig. b) . interestingly, pdcov n stably expressed in veroe cells significantly suppressed pedv spread; even at hpi, no extensive syncytia had formed (fig. b) . quantification of supernatant pedv-mcherry titers by tcid assay also reflected the extent of syncytium formation. at hpi, veroe cells expressing tgev n or pedv n yielded about a hundred times more infectious pedv virus particles than veroe cells, while those expressing pdcov n resulted in an infectious titer about ten times lower. by hpi, pedv replication in wild-type veroe cells had started to catch up and closed the gap to about one order of magnitude. however, veroe -pdcov n cells still showed significantly slower pedv replication kinetics (fig. c ). as expected, we noted that the suppressive effect from veroe -pdcov n cells was stronger than that observed in transiently transfected veroe cells. this could be because the stable cell line homogeneously expresses pdcov n which can directly exert an effect on the infecting virus, while only a fraction of infected cells was transfected (resulting in the effect being diluted out by cells that were infected but not transfected). previously, we observed a significant enhancement of pedv rna synthesis and increased viral titers in veroe -pedv n cells (liwnaree et al., ) . to explore if similar mechanisms were employed by tgev n and pdcov n on pedv replication, we then probed viral rna production inside these veroe -based cell lines using rt-qpcr. total rna was extracted from cells infected with pedv-mcherry at , and hpi. specific primers were used to quantify viral genomic rna (orf a gene; grna) and subgenomic rna ( ′utr-s gene; sgrna) production. the levels of each rna species were normalized to the levels of gapdh mrna from the same conditions and were expressed as relative to rna levels at hpi from infected veroe cells. as previously observed, veroe -pedv n cells drastically increased sgrna and grna production at and hpi (fig. d ). on the other hand, veroe -pdcov n cells displayed stunted pedv rna transcription and replication; even at hpi, the levels of sgrna and grna were still lower than those observed in veroe cells at hpi (fig. d ). veroe -tgev n cells moderately enhanced pedv rna synthesis compared to wild-type veroe cells but did not reach the same level as veroe -pedv n cells (fig. b ). according to the results, the presence of extraneous n proteins posed greater effects on grna replication than sgrna transcription. these data corroborated with visual observation and quantitative measurement of infectious viral particle production. together, they strengthened the findings in the transient expression experiments and suggest opposing directions of influence on pedv replication mediated by n proteins derived from related covs. based on their ability to cross-associate and affect pedv rna synthesis and replication, we speculated that n proteins from other covs, especially tgev, may have interchangeable functions with pedv n in pedv replication. in an attempt to answer which roles and functions of pedv n could be replaced by other cov n proteins, we constructed an n-deficient pedv infectious clone, ppedv-mcherry-Δn, and determined whether supplying cov n proteins in trans could replace the missing pedv n protein from the pedv's viral genome. since the nucleotide fragment of the n gene in the viral genome may play other important roles in viral genome packaging or transcription, we employed a frameshift mutation strategy to silence n expression from the infectious clone while minimally disturbing the nucleotide sequence of the n gene. the original start codon in the n orf, atggct, was mutated to atatgt, thereby shifting the start codon by two nucleotides, resulting in a + frameshift during translation (fig. a) . the mutated sequence theoretically produced a short unrelated peptide of amino acids with early termination. we intentionally kept the mcherry gene in the construct as a visual indicator for sub-genomic mrna transcription and protein expression in the virus rescue process. to verify the lack of n expression during pedv reverse genetics rescue, we transfected the n-deficient infectious clone into hek t cells. western blot analysis confirmed that n expression was completely diminished in cells transfected with the n-deficient infectious clone compared to the wild-type ppedv-mcherry (fig. b) . with the n-deficient infectious clone in hand, we next investigated pedv reverse genetics rescue in the presence of other cov n proteins. s. sungsuwan, et al. virology ( ) - hek t cells were co-transfected with ppedv-mcherry-Δn and a pcaggs plasmid expressing cov n protein. expression of mcherry from the infectious clone was used as a visual proxy for production of viral rna and proteins in hek t cells during the reverse genetics step. no detectable fluorescence was observed from co-transfection with the empty pcaggs vector, confirming the role of pedv n protein during at least the first round of viral rna transcription and viral protein translation (fig. a ). co-transfection with pcaggs-pedv n-myc yielded a substantial fraction of red cells, while slightly lower amount of mcherry protein expression was observed from cells cotransfected with pcaggs-tgev n-flag. co-transfection with pcaggs-pdcov n-ha resulted in very low, yet detectable, numbers of mcherrypositive cells (fig. a ). cell lysates were prepared at hpt and probed for s expression using anti-s antibody to corroborate visual inspection. as a negative control, cells transfected with ppedv-mcherry-Δn and pcaggs vector displayed no detectable trace of s (fig. b) . cells co-transfected with pcaggs expressing cov n proteins showed varying levels of s expression, with pedv n-myc giving the highest s expression and pdcov n-ha giving the lowest (fig. b) . tgev n-flag expression resulted in noticeably lower s expression compared to pedv n-myc but still higher than that of pdcov n-ha (fig. b) . the levels of spike protein expression from the n-deficient clone in each condition correlated with the level of mcherry expression as observed by fluorescence microscopy (fig. a and b) . these results suggested that cov n proteins from alphacoronaviruses can aid viral rna and protein production from infectious pedv clones more efficiently than that from deltacoronavirus. to determine if pedv rescue successfully produced viable infectious particles with help from various cov n proteins in trans, hek t cell supernatants were collected at hpt to inoculate veroe -pedv n cells pre-transfected with pcaggs-pedv n. we found that additional transfection could ensure sufficient pedv n expression in infected cells and facilitated pedv replication better than untreated veroe -pedv n cells (data not shown). limited syncytium formation was observed only in veroe -pedv n cells inoculated with the supernatant derived from hek t cells co-transfected with pcaggs-pedv n-myc (fig. c) . however, the infecting viruses could not continuously propagate to produce extensive syncytia as routinely observed with the rescue of ppedv-mcherry. this is probably due to insufficient pedv n protein levels as supplied by the stable cell line compared to virally encoded pedv n. remarkably, supernatants from hek t cells co-transfected with a plasmid encoding tgev n or pdcov n did not contain infectious progeny from virus rescue, displaying no second-round pedv-mcherry replication in veroe -pedv n cells as determined by mcherry expression or syncytia formation (fig. c) . these results suggest that, despite some conserved function of n proteins among porcine enteric covs in self-oligomerization and assistance during viral rna synthesis or protein expression, n is not fully interchangeable among covs for generating pedv infectious virus progeny. these results imply that the suppressive or enhancing effects on pedv replication in cells expressing pdcov n or tgev n may reflect their effects on viral rna replication and/or viral protein expression, rather than their abilities to supply complementary n proteins during rnp core formation or virion assembly. although the swine industry has focused on pedv alone as one of its largest threats, few have attempted to understand the implications of swine enteric cov co-infection despite multiple epidemiological studies. as the true impact of co-infection on disease outcomes still awaits further investigation, these viruses could predictably exert influence on each other, considerably shaping the course of their infection or evolution. molecular interactions underlying these possible viral interferences also remain to be identified and characterized. in this work, we tried to understand how different enteric swine cov n proteins, one of the most abundant structural proteins, interact and affect functions of pedv n and replication kinetics of pedv. first, we provided evidence of cross-interaction between cov n proteins despite low sequence similarity among them. amino acid sequence analyses on pedv, tgev and pdcov n proteins revealed that the proteins can be divided roughly into two structural domains (ntd fig. . construction of the n-deficient pedv infectious clone, ppedv-mcherry-Δn. (a) schematic representation of a frameshift mutation strategy to silence n expression from the ppedv-mcherry-derived infectious clone to yield ppedv-mcherry-Δn. cmv, cytomegalovirus immediateearly promoter; hdv, hepatitis delta virus ribozyme self-cleavage site; bgh, bovine growth hormone termination; t, transcription terminator. (b) western blot analysis of lysates prepared from hek t cells transfected with ppedv-mcherry or ppedv-mcherry-Δn ( μg each) at hpt. and ctd) with relatively high conservation and three intrinsically disordered regions (idrs) with relatively low conservation located in the middle and at the two termini (fig. ) . this domain analysis is consistent with previous crystallographic studies on sars-cov and mhv n, which also suggested conserved general structural organization of cov n (chang et al., ; cong et al., ; ma et al., ; yu et al., ) . moreover, immunological data documenting cross-reactivity of antibodies against n proteins from tgev or pdcov with pedv n further implied shared topological similarity between these cov n proteins (gimenez-lirola et al., ; lin et al., ; ma et al., ) . nevertheless, precise molecular mechanisms mediating interspecies oligomerization of cov n proteins remain to be determined. structural and biochemical studies of sars-cov n strongly suggest a role of ctd as a major mediator for cov n dimerization. moreover, ctds of n proteins from sars-cov and porcine reproductive and respiratory syndrome virus (prrsv), despite diverse sequences, could adopt well-conserved structures, implying similar approaches to oligomerization, at least at a gross structure level (yu et al., ) . however, since cov n ctds formed intricate molecular interaction networks during dimerization (chang et al., ) , it is unlikely that cov n proteins from different species could replace all these specific molecular interactions. consistent with our results that hetero-oligomer formation is highly sensitive to the presence of rna, a more likely possibility is that rna-protein interactions play a more dominant role in interspecies cov n interactions than protein-protein interactions. next, we asked if n proteins from different covs can replace pedv n function during viral rna and protein synthesis. during the rescue of the n-deficient pedv infectious clone, we showed that supplying n protein from any cov in trans can initiate first-round sgrna transcription and protein expression. most likely, this function stems from the shared intrinsic ability of cov n to bind viral rna and act as an rna chaperone that initiates rna replication and/or transcription processes (zuniga et al., (zuniga et al., , . these results support previous notions that cov n proteins play important roles during cov rescue. for instance, rescue of tgev infectious rna required at least co-transfection of mrna encoding the n protein (yount et al., ) . schelle et al. demonstrated that an n-deficient hcov e rna vector replicon carrying the gfp gene can produce sgrna and express gfp from the vector when co-transfected with n from pedv, a closely related virus, but not from mhv, a more distantly-related one (schelle et al., ) . we noted fig. . other cov n proteins can partially function to replace pedv n. for pedv rescue experiments, hek t cells were co-transfected with ppedv-mcherry-Δn and μg of pcaggs plasmid expressing n from pedv, pdcov or tgev. at hpt, (a) cells were imaged under fluorescence microscopy to monitor mcherry expression from ppedv-mcherry-Δn, and (b) cell lysates were prepared for western blot analysis with an anti-s antibody. supernatants from (a) were adsorbed onto veroe -pedv n cells, which were additionally transfected with μg pcaggs-pedv n to ensure sufficient n expression. at hpi, (c) cells were imaged under fluorescence microscopy to monitor mcherry expression and syncytium formation as a sign for successful pedv infection into veroe -pedv n cells (scale bar, μm). a similar preference in our experiments. between tgev n and pdcov n, the former is more effective in replacing the indigenous pedv n than the latter. since both pedv and tgev belong to the alphacoronavirus genus, while pdcov is classified as a deltacoronavirus, tgev n would be expected to be functionally and structurally closer to pedv n, and hence able to form replication/transcription complexes and participate in pedv replication with higher compatibility than pdcov n. although both tgev n and pdcov n can oligomerize and perform some functions during g/sgrna transcription, they alone cannot totally replace all pedv n's functions. without native pedv n, infectious pedv virions could not be detected in the presence of tgev or pdcov n, implying that other functions are not interchangeable. these critical functions might involve viral core assembly as cov n participates in many steps during viral core or virion assembly (mcbride et al., ) . for instance, several reports showed that cov n is involved, directly or indirectly, with recognition of specific packaging signals for genome packaging (hsieh et al., ; hsin et al., ; kuo et al., ) . since rna packaging signals among covs are distinct in terms of length and sequence, even closely related cov n might not be able to recognize pedv packaging signals. similar compatibility issues have been raised in the case of viral interference between different types of influenza viruses (baker et al., ) . another critical step that could be disrupted by heterologous cov n is viral assembly through interactions between n and m proteins. it is widely accepted that cov n and m proteins interact to form the viral core, but different covs utilize different regions of n in binding to their own m proteins. for instance, while mhv utilizes the very c-terminal end of n to bind m, sars-cov n uses the middle disordered linker for the interaction (he et al., b; narayanan et al., ) . in addition, although both tgev and mhv n proteins used their c-termini to bind m, the n binding sites on tgev m and mhv m are very distinct (hurst et al., ; kuo and masters, ) , pointing to high specificity for each virus. it could be argued that our use of c-terminally tagged cov n proteins might have contributed to failure in rescuing infectious pedv from ppedv-mcherry-Δn, possibly due to interference with n-m protein interaction. nevertheless, our results showed that addition of a myc tag at the c-terminus of pedv n did not completely ruin protein-protein interaction required for viral assembly as pedv n-myc remained capable of producing infectious virions. moreover, using untagged pedv n and tgev n during reverse genetics rescue of ppedv-mcherry-Δn gave similar results to those presented in fig. (data not shown) , suggesting that the inability of tgev n or pdcov n to rescue infectious pedv lacking endogenous nucleocapsid protein stemmed largely from incompatibility of interspecies protein interaction rather than the interference from epitope tags. in all, it is conceivable that not all of n functions could be replaced by the protein originating from other viral species, even though n proteins from pedv, tgev and pdcov can cross-assemble, form highorder oligomers and aid in rna synthesis processes. notably, we observed completely opposite effects of tgev and pdcov n proteins on pedv replication. we noticed that, in the presence of rna, homologous binding between pedv n itself is weakened and cross binding between pedv n and the other n proteins is strengthened. during co-infection, where viral grna is ubiquitous, the rna could act as a tethering bridge to stabilize the formation of heterologous oligomers, which may not be a productive core structure for pedv virion assembly and may interrupt the optimal processes in viral fig. . analysis of cov n sequences reveals low sequence similarity but conserved structural organization. multiple amino acid sequence alignment was performed with t-coffee (notredame et al., ) . the color scheme denotes consistency between different alignment methods ranging from bad (low agreement; blue and green) to good (high agreement; pink). bold and normal lettering denote ordered and disordered regions as predicted by the prdos program (ishida and kinoshita, ) . (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) replication that prefer the homo-oligomer of n. such interruption could explain the suppressive effect of pdcov n. nevertheless, the existence of interspecies cov n oligomers might not be the only factor dictating the effect of extraneous cov n proteins on pedv replication. tgev n also forms rna-dependent interspecies oligomers with pedv n and could have resulted in similar suppression. instead, we observed a small enhancement effect on pedv replication in the presence of tgev n. this could be the net outcome of other oligomerization-independent roles of tgev n that indirectly enhance pedv replication. a study by zhao et al. showed that, when compared to single-virus infection, coinfection of tgev and pedv could synergistically enhance rupture to tight and adherens junctions of the intestinal epithelial cells ipec-j by suppressing expression of proteins that help form the junction (zhao et al., ) . such altered barrier integrity is possibly caused by cov n and facilitate viral replication. another possible role of pedv or tgev n that could lead to enhancement of pedv replication is to act as a viral suppressor of rna silencing (cui et al., ) . using mhv n as a model, they found that cov n can bind and suppress host antiviral rnai, leading to increased viral protein expression and subsequent viral replication. more remarkably, they found that n proteins of alphacoronaviruses, including tgev and pedv, also have this ability. indeed, among different cov n proteins, they found that tgev n was among the most active and mers-cov n was the least active (cui et al., ) . therefore, n from tgev may also employ this mechanism in promoting pedv replication, while some other cov n proteins might not be capable of this function. these are interesting hypotheses currently under investigation, which could reveal more complexity of co-infection between these viruses. in summary, this work shows how other swine enteric cov n proteins can substitute for endogenous pedv n in some functions but not in others, demonstrating both conservation and specificity among related viruses in the coronaviridae family. furthermore, co-expression experiments revealed interesting phenomena in which different cov n proteins exert vastly different effects on pedv replication. these results raise even more questions. for example, in the context of co-infection, in which all other viral components are present, what would be the net outcomes on replication kinetics of each virus? are there effects from other viral components? considering the opposing effects on pedv replication of n proteins from tgev, an older virus emerging prior to pedv, and pdcov, a newly emerged one, is it possible that nucleocapsid proteins could act as one of the factors in permitting or limiting emergence of enteric swine covs? if so, what is the effect of pedv n on newer covs such 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of coronavirus genomes that express nucleocapsid protein newly emerged porcine deltacoronavirus associated with diarrhoea in swine in china: identification, prevalence and full-length genome sequence analysis amino acid residues critical for rna-binding in the n-terminal domain of the nucleocapsid protein are essential determinants for the infectivity of coronavirus in cultured cells movement and nucleocapsid proteins coded by two tospovirus species interact through multiple binding regions in mixed infections the coronavirus nucleocapsid protein is dynamically associated with the replication-transcription complexes porcine epidemic diarrhea in china porcine epidemic diarrhea virus variants with high pathogenicity a single v f substitution in the spike protein of field-isolated pedv promotes cell(-)cell fusion and replication in veroe cells porcine epidemic diarrhea virus n protein prolongs s-phase cell cycle, induces endoplasmic reticulum stress, and up-regulates interleukin- expression strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model crystal structure of the severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein dimerization domain reveals evolutionary linkage between corona-and arteriviridae type iii interferon restriction by porcine epidemic diarrhea virus and the role of viral protein nsp in irf signaling transmissible gastroenteritis virus and porcine epidemic diarrhoea virus infection induces dramatic changes in the tight junctions and microfilaments of polarized ipec-j cells coronavirus nucleocapsid protein facilitates template switching and is required for efficient transcription coronavirus nucleocapsid protein is an rna chaperone the authors would like to thank dr. qigai he for anti-pedv s antibody, and benjamas liwnaree for technical assistance. we thank the members of virology and cell technology research team, especially dr. samaporn teeravechyan, for critical comments and language help on the manuscript. this work was supported by biotec's fellow grant [grant number p- - ]. key: cord- - t uxmj authors: lamphear, barry j.; jilka, joseph m.; kesl, lyle; welter, mark; howard, john a.; streatfield, stephen j. title: a corn-based delivery system for animal vaccines: an oral transmissible gastroenteritis virus vaccine boosts lactogenic immunity in swine date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: t uxmj recombinant plant expression systems offer a means to produce large quantities of selected antigens for subunit vaccines. cereals are particularly well-suited expression vehicles since the expressed proteins can be stored at relatively high concentrations for extended periods of time without degradation and dry seed can be formulated into oral vaccines suitable for commercial applications. a subunit vaccine candidate directed against porcine transmissible gastroenteritis virus and expressed in corn seed has been developed for oral delivery to swine. here, we show that this vaccine, when administered to previously sensitized gilts, can boost neutralizing antibody levels in the animals’ serum, colostrum and milk. thus, this vaccine candidate is effective at boosting lactogenic immunity and is appropriate to pursue through large-scale field trials preceding commercialization. oral administration of vaccines has the potential to greatly cut the cost and increase the safety of vaccine delivery. in the case of human vaccines, avoiding the use of needles reduces equipment costs, removes the requirement for trained medical personnel to supervise delivery and eliminates safety concerns associated with needle disposal. the economic benefits of oral over parenteral delivery are also apparent with animal vaccines, where equipment and labor costs can be substantially reduced. also, in the cases of farmed animals destined for meat markets, carcass quality may be compromised by repeated injections and oral vaccines overcome this concern. subunit vaccines are generally considered to have a low safety risk since they are well defined and do not contain attenuated or inactivated pathogens with the potential for adverse affects. thus, oral delivery of subunit vaccines is a particularly attractive option for safe, inexpensive vaccination programs. however, oral delivery generally requires very high levels of specified antigens to be administered in order to attain efficacy. this is presumably because of degradation of the selected proteins in the digestive tract and only a small proportion of the relatively intact molecules being presented to the immune system in a manner favoring an immunogenic response. several recombinant systems are being utilized to generate large amounts of subunit vaccines, including, for example, the use of yeast to produce the surface protein of hepatitis b. however, despite the development of such recombinant vaccines, the economic production of large quantities of desired antigens is severely limited. recently, certain recombinant plant expression systems have begun to offer a means to produce very large quantities of proteins in a sufficiently concentrated form to make oral delivery feasible for a wider array of antigens. levels of expression have been achieved with various antigens in plants that allow practical oral delivery of a sufficient dose to elicit desired immune responses in humans and target animals (reviewed in [ ] ). the approaches followed to achieve these high levels of expression include the use of tissue-specific promoters to direct expression to tissues well suited to the stable storage of proteins [ ] and the targeting of the expressed proteins to sub-cellular locations conducive to their accumulation [ ] . many plant-based oral vaccine candidates have been tested in animal studies and several responses have been noted, including the generation of serum and mucosal antibodies (reviewed in [ ] ) and raised cytokine levels [ ] . protective efficacy has also been recorded with some of these vaccine candidates in model species trials (reviewed in [ ] ). a few plant-based vaccine candidates have advanced into early phase human clinical trials or target animal trials. among human vaccines, these include those directed against travelers' diarrhea and norwalk virus delivered in potato tubers [ , ] , against hepatitis b virus delivered in lettuce leaves [ ] and against rabies virus delivered in spinach leaves, themselves infected with a recombinant plant virus [ ] . immune responses were observed during these trials, and although there were some reports of nausea, presumably resulting from the administration of up to g of unprocessed, unpalatable plant material, the vaccine candidates were generally well tolerated. in the case of farmed animals, a corn-based vaccine directed against transmissible gastroenteritis virus (tgev) can induce protective immunity in piglets [ , ] . a key issue in producing plant-based oral vaccines is the selection of plant material that both expresses high levels of a chosen subunit vaccine candidate and is also suitable for extensive storage and oral delivery. the chosen plant material must also be ready for direct administration or must be suitable for inexpensive processing into an appropriate form for oral delivery to the target species. much of the work to date on plant expression systems has been conducted using tobacco leaf tissue (discussed in [ ] ). however, tobacco leaves are inedible, and therefore protein extraction is required prior to delivery. several edible options have also been pursued, including the tubers and leaves of certain vegetable crops such as potato and lettuce, respectively [ ] [ ] [ ] . some fruits, such as bananas, are also being considered. however, perishable items are not practical for extended storage and expression levels can vary considerably between, for example, potato tubers taken from a single harvest [ , ] . cereal seeds are particularly well-suited systems for the oral delivery of subunit vaccines. they have low water contents and naturally store proteins over long periods of time without degradation. corn (zea mays) is an especially attractive option because of its intensively studied genetics and the availability of established transformation procedures. several vaccine candidate antigens have been expressed at high levels in corn seed and the proteins are stable when stored in this tissue for periods of at least a year and probably for much longer, obviating the requirement for a cold chain during distribution and storage [ ] . furthermore, the antigen concentration is uniform across a corn grain harvest, facilitating even dosing [ ] . a wide range of processing alternatives have been developed by the food and feed industries to convert corn grain into readily edible forms and pilot-scale processes have been developed that ensure antigens are not degraded during processing [ ] . in the case of farmed animals, such processing is unnecessary since the livestock can consume corn grain directly. here, we focus on the development of a corn seed-based subunit vaccine directed against swine tgev. this virus causes a contagious enteric disease that is particularly severe for piglets. it results in severe diarrhea and vomiting and is associated with high mortality rates among piglets under weeks of age [ ] . tgev is a coronavirus and has a large surface glycoprotein referred to as the spike (s) protein displayed on its surface [ ] . the tgev vaccine candidate assessed here comprises the s protein expressed in corn seed. feeding studies have been conducted with this vaccine candidate delivered orally to piglets. the animals showed a priming of their immune system and were protected against infection [ , ] . here, we extend these swine feeding studies to assess the potential for this oral tgev vaccine candidate to boost immunogenic responses in gilts (young sows) previously sensitized with a commercially available modified live viral vaccine. we focus particularly on the level of antibodies in the colostrum and milk as a guide to whether immunity could be acquired passively by piglets through suckling. the subunit vaccine candidate comprised milled yellow grain corn expressing the s protein of tgev. a single dose corresponded to kg of corn containing mg of the antigen. the placebo for the study comprised kg of non-transformed milled yellow grain corn. a commercially available modified live tgev vaccine (intervet inc., millsboro, de) with a titer of . tcid (tissue culture infectious doses)/ ml was used to prime all animals and to provide booster treatments to a positive control group. this vaccine was administered according to label directions. a total of specific pathogen free gilts of suitable age for breeding were included in the study. they were taken from a low disease incidence herd and were seronegative for tgev at the outset of the study. the gilts were randomized into six treatment groups with from five to eight animals in each group (table ) . duplicate ear tags were used for identification purposes. all gilts in all groups were orally administered the modified live tgev vaccine on the day of breeding ( days before farrowing) and also days before farrowing. they were then administered the tgev modified live vaccine by intramuscular injection days before farrowing. the subsequent immunization regimen for each group is outlined in table . all animals were fasted overnight prior to oral administrations of the corn-based vaccine to test groups. standard lactation gestation rations were administered to all gilts throughout the study. during the period comprising the three administrations of modified live virus to all gilts, the groups were housed together and allowed pen-to-pen contact. prior to days before farrowing gilts were separated into their separate groups and during subsequent vaccine administrations, animals were individually isolated. blood samples were collected from gilts on the day of breeding ( days prior to farrowing), and days prior to farrowing and on the day of farrowing. blood was allowed to clot and was sedimented by centrifugation, so allowing the serum to be collected. tgev neutralizing titers were determined by incubating a specific dilution of tgev with multiple serum dilutions for h at • c. these mixtures were then inoculated onto a swine testicular cell line and the capacity of the serum to interfere with the viral infection was assessed after days. sample titers were calculated using a spearman-karber % endpoint table. colostrum samples of at least ml were collected on the day of farrowing and at least ml milk samples were collected , , and days after farrowing. the samples were sedimented by centrifugation, and the central region was collected. tgev neutralizing titers were determined as with serum samples. for all tgev neutralization data, geometric mean titers were compared and differences in excess of four-fold were considered to be significant. the generation of transgenic corn containing the s protein of tgev has been previously described [ , ] . in brief, sequence encoding the s protein was synthesized with optimal codon usage for expression in z. mays. an n-terminal cell surface targeting signal was included to direct accumulation of the protein to the cell wall. dna encoding the s protein was introduced into immature zygotic z. mays embryos by agrobacterium tumefaciens mediated transformation and selection was imposed for transgenic callus. transgenic plants with sequence encoding the s protein integrated into the nucleus were regenerated, and those expressing the highest levels of the s protein were taken through a plant-breeding scheme to increase and stabilize expression levels. this culminated in a large-scale grain harvest in which the s protein was present at mg kg − , as determined using a sandwich enzyme linked immunosorbent assay [ ] . at this concentration a practical antigen dose of - mg can be delivered in an amount of corn material easily consumed at a single feeding. all animals were seronegative for tgev at the time of breeding. subsequent serum neutralization titers are summarized for each study group in fig. . the modified live virus vaccine, which was administered twice orally and then once intramuscularly resulted in gilts in all groups having similar tgev serum neutralizing titers days prior to farrowing. analysis of serum samples taken from gilts at days prior to farrowing showed that animals that had received the oral corn-based tgev vaccine (groups a-c) had notably higher serum neutralization titers than those that had received no material at this stage (groups d and f). the difference between the test and control groups was significant in all cases except for that of a single administration of corn-based vaccine (day − to group c) over group d. animals that had received the modified live virus vaccine as a single intramuscular boost (day − to group e) responded to an almost identical level to those that had received a single oral administration of the corn-based vaccine (group c). although more oral administrations of the corn-based vaccine appeared to increase the neutralization titer, differences between the treatment groups (a-c) were not significant and none of the treatments induced a significantly stronger response than the intramuscular boost of modified live vaccine delivered to group e. similarly, at the time of farrowing the tgev serum neutralization titers in gilts administered the corn-based tgev vaccine as a boost (groups a-c and f) were raised over those observed with animals that had received the corn placebo (group d). this difference was significant in all but the case of gilts that had received six administrations of the corn-based vaccine (group b) compared to those that had received the placebo (group d). animals given intramuscular administrations of the modified live virus vaccine as a boost (group e) responded similarly to those that received the oral corn-based vaccine, again with two administrations of either vaccine giving almost identical results (groups c and e). gilts administered a boost of the corn-based tgev vaccine only during the second week before farrowing (group f) showed the most marked increase in the serum neutralization titer at the time of farrowing, although differences between the groups that received the corn-based vaccine (groups a-c and f) were generally not significant. interestingly, for groups that received two blocks of booster administrations (a-c and e), in no case did the second set of treatments elevate the serum neutralization titers over those observed with the first set. indeed, neutralization titers appeared to decline with the second set of administrations, although in no case was the drop statistically significant. colostrum and milk neutralization titers are summarized for each study group in figs. and , respectively. each of the groups of gilts that were orally administered the corn-based tgev vaccine as a booster (groups a-c and f) showed a greater level of neutralizing antibodies than did gilts administered two intramuscular injections of the tgev modified live virus vaccine as a booster (group e). however, these differences were not sufficient to be considered significant, and therefore all of the booster treatments with either the corn-based oral vaccine or with the modified live intramuscular vaccine are considered similarly effective. all of the booster regimens with the corn-based vaccine (groups a-c and f) resulted in significantly greater neutralizing antibody levels than those observed among animals that were administered the control corn placebo material (group d). gilts in all groups that received an oral corn-based tgev vaccine boost (groups a-c and f) showed similar levels of neutralizing antibodies in their milk, with levels trailing off steeply between and days after farrowing and continuing to decline thereafter. these levels correspond closely to those observed with the modified live tgev vaccine delivered intramuscularly (group e). with all groups that received a corn-based oral booster treatment (groups a-c and f) the neutralizing antibody titer in milk days after farrowing is considerably higher than for the group that received the control corn placebo (group d). this difference is significant in all cases except that of the group that received two blocks, each of seven consecutive days, of the corn-based vaccine (group a). by days after farrowing differences in the neutralization titers between the placebo (group d) and other groups are not significant. the corn-based tgev vaccine candidate described here shows great potential for expediting the administration of an efficacious vaccine to large herds of swine. the current standard regimen for administrating a vaccine comprises both priming and boosting stages. during the priming phase of the regimen the modified live tgev vaccine is often administered along with other swine vaccines. thus, at this point no reduction in labor costs is achieved through delivering an oral corn-based tgev vaccine separately. however, replacing subsequent injections of the modified live tgev vaccine with oral corn vaccine boosters would clearly save considerable time and effort. the orally administered corn-based tgev vaccine is effective in boosting the serum neutralizing titer response in animals previously sensitized to tgev using the modified live virus vaccine. when administered as a booster to gilts the corn-based vaccine also results in increased levels of neutralizing antibodies in the colostrum and early milk. milk antibodies of the igg class have typically disappeared within h of farrowing, so the neutralizing antibody activities observed in milk samples collected days after farrowing most likely reflect iga levels. protection against tgev amongst nursing piglets has been linked to iga levels [ ] , indicating that the corn-based tgev vaccine is inducing an immune response with the potential to confer protection. in this regard, a potato-based vaccine candidate directed against rotavirus, and assessed in a mouse feeding study, has been shown to confer passive immunity to pups when administered orally to dams [ ] . protective efficacy has previously been demonstrated with a corn-based oral vaccine directed against tgev and administered to piglets [ , ] . the neutralizing antibody levels achieved here in the colostrum and early milk of gilts extends the scope for how this vaccine candidate can be deployed. future studies with this tgev oral vaccine candidate will focus on optimizing the administration reg-imen for maximum responses and on conducting larger scale trials. these will include an assessment of whether the lactogenic immunity observed here results in protection being conferred to piglets. the results presented here successfully demonstrate a commercial application for a corn-based vaccine and indicate that there is great promise for plant-based vaccines that can be easily administered to large farmed animals by oral delivery. plant-based vaccines expression of a synthetic e. coli heat-labile enterotoxin b sub-unit (lt-b) in maize corn as a production system for human and animal vaccines a plant-based multicomponent vaccine protects mice from enteric diseases immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes a plant-derived edible vaccine against hepatitis b virus expression in plants and immunogenicity of plant virus-based experimental rabies vaccine plant-based vaccines: unique advantages delivery of subunit vaccines in maize seed medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants congress symposium on molecular farming: development of an edible subunit vaccine in corn against enterotoxigenic strains of escherichia coli molecular biology of transmissible gastroenteritis virus immunity to transmissible gastroenteritis virus and porcine respiratory coronavirus infections in swine key: cord- -mn r x authors: hodgins, douglas c.; chattha, kuldeep; vlasova, anastasia; parreño, viviana; corbeil, lynette b.; renukaradhya, gourapura j.; saif, linda j. title: mucosal veterinary vaccines: comparative vaccinology date: - - journal: mucosal immunology doi: . /b - - - - . - sha: doc_id: cord_uid: mn r x infections of mucosal surfaces are major causes of morbidity, mortality, and economic loss in species of veterinary interest, and a concern for animal welfare. vaccines are used extensively in veterinary medicine, and innovative vaccine technologies such as recombinant dna-vectored and distinguishing infected from vaccinated animals (diva) vaccines and automated in ovo vaccination (of embryonated chicken eggs) have been rapidly adopted commercially. immunological research using outbred, nonrodent animal models has contributed to a broader understanding of mucosal defenses, and has provided the initial impetus for investigation of the common mucosal immune system. studies of the potential of novel adjuvants to improve vaccine efficacy against genetically unstable, immune-subverting rna viruses, such as porcine reproductive and respiratory syndrome virus in pigs, should assist in the control of pathogens with similar characteristics in other species. successful development of vaccines to prevent and treat ascending infections of the reproductive tract of cattle set a precedent for applications in other species including humans. studies of mucosal adjuvants and delivery systems continue at the interface between passive and active immunity, with the goal of inducing the earliest possible protection against enteric and respiratory pathogens of neonates. despite advances in nutrition, genetics, housing, and therapeutics, diseases of the respiratory, reproductive, and gastrointestinal tracts of domestic animals and poultry continue to be major causes of morbidity and mortality. although vaccines have been developed and licensed for prevention of many of these diseases, there is a need for improvement in vaccine efficacy and for new vaccines. reasons for low vaccine efficacy include inappropriate, unstable, or outdated antigens in vaccine preparations (e.g., in vitro expressed antigens instead of in vivo expressed antigens), inappropriate immune responses (e.g., systemic instead of mucosal, or th type instead of th type or vise versa), and inappropriate use of otherwise efficacious vaccines (e.g., inappropriate timing of vaccination) (tizard, ). an overview of some of the vaccines currently licensed for vaccination of domesticated animals and wildlife by mucosal routes is provided in table . many of these vaccines consist of pathogens attenuated by traditional methods, but engineered virus-vectored vaccines are now used extensively (by both mucosal and parenteral routes) in veterinary medicine. the majority of current poultry vaccines are attenuated agents delivered in ovo, orally, intranasally (in) or by other mucosal routes, for reasons of ease of administration, economy, and protective efficacy. in comparison, fewer vaccines for domestic mammals are delivered by mucosal routes. management practices for mammals differ from those for poultry; mass vaccination techniques for mucosal delivery have been developed for poultry, but have not been pursued as zealously for mammals. recently however, a number of attenuated live vaccines for in administration have been developed for respiratory tract infections, using traditional methodologies. improved protective efficacy and rapid onset of immunity compared to killed vaccine products have led to widespread acceptance. attenuated live oral vaccines for enteric diseases have also been marketed, but in many cases efficacy has been disappointing due to lack of potency in adults or interference by maternal antibodies in suckling animals. better strategies for induction of immunity in the gastrointestinal tract are needed, especially for neonates in the presence of maternal antibodies. in contrast, effective parenteral vaccines for the most common diseases of the reproductive tract in veterinary species have been available for years, and there has been little motivation to develop mucosal vaccines. many of the diseases of the respiratory and gastrointestinal tracts are most devastating in the neonatal period. for these diseases active immunization may not provide protection before natural exposure to the pathogen. maternal vaccination to enhance passive immunity has been widely used in veterinary medicine, especially for control of enteric diseases. practical difficulties arise, however, with diseases such as parvovirus enteritis in puppies in which a smooth transition must be made from protection by passive maternal antibodies to protection by active immunity, without permitting a window in between of disease susceptibility. this transition is difficult to achieve because induction of active immunity is commonly inhibited by maternal antibodies. various strategies are used to address this problem, but improved vaccines, adjuvants, and antigen delivery systems would improve the reliability of neonatal immunization. although progress is being made in disease prevention in veterinary species, ever changing management practices (e.g., earlier weaning of piglets, larger animal operations) generate new patterns of disease, requiring new control strategies. the emergence of new pathogens (e.g., porcine reproductive and respiratory syndrome virus (prrsv), porcine circovirus- ) provides new challenges for vaccine research before some of the older challenges have been met. in this chapter, we focus on mucosal veterinary vaccines and vaccine concepts related to selected pathogens of economic importance. our intent is to highlight progress, to review existing and future vaccination strategies, and to acknowledge the unique contributions of this research to our understanding of mucosal vaccines and immunology. respiratory tract infections are a major cause of morbidity and mortality in farm animals, poultry, and pets. disease conditions are intensified by current management practices such as mixing of recently weaned, stressed beef calves from multiple sources in auction barns. certain disease conditions, such as atrophic rhinitis of pigs, result from the interplay of several pathogens, and multiple agents must be represented in vaccines. some respiratory pathogens such as mycoplasma hyopneumoniae in young pigs are causing new patterns of disease as management practices change (e.g., weaning at an earlier age), requiring changes in vaccine strategies. other pathogens such as prrsv have only recently emerged and improved vaccines await advances in understanding of the agent and of disease pathogenesis. a discussion of respiratory vaccines for even the major pathogens of veterinary species is beyond the scope of the present review. this section will focus on vaccines for prrsv infections in pigs and influenza in horses to illustrate general principles. porcine reproductive and respiratory syndrome (prrs) is a chronic reproductive and respiratory disease of pigs caused by prrsv, a member of the family arteriviridae (benfield et al., ) . signs associated with prrs are anorexia, fever, lethargy, pneumonia, red/blue discoloration of the ears and vulva, delayed return of sows to estrus after weaning, abortion, fetal mummification, stillborn or weak born piglets, and high preweaning mortality (mengeling et al., ) . prrsv is prevalent in swine-producing countries worldwide. annual economic losses to the pork industry in the united states due to prrs have been estimated at $ million (holtkamp and kliebenstein, ) . according to the american animal and plant health inspection service (aphis) report of january , . % of unvaccinated pigs in the united states are seropositive to prrsv. infected pigs excrete prrsv in saliva, nasal secretions, urine, milk, colostrum, and feces at low levels or intermittently, and also in semen of infected boars (rossow et al., ) . in the absence of control measures, prrsv is spread by aerosols, fomites, and personnel. prrsv is divided into two distinct genotypes, type i (european) and type ii (north american). each genotype contains subtypes and strains, which are genetically diverse and vary in virulence and pathogenicity (kim et al., ) . immunity to one genotype of prrsv may provide partial or no protection against reinfection, reflecting the complexity of prrsv genetics and immunological variation among strains (botner et al., ) . swine are the only known species susceptible to prrsv. alveolar macrophages (mΦs) are the primary permissive cells to prrsv; mΦs present in pulmonary intravascular spaces, lymph nodes, thymus, heart, spleen, placenta, and umbilical cord may also be infected by the virus (halbur et al., ) . the most efficient and rapid host response against viruses consists of production of type i interferons (ifns) (ifnα and ifnβ). in prrsv-infected pigs, innate ifnα secretion is significantly suppressed (albina et al., ) and the virus dampens innate nk cell-mediated cytotoxicity as early as day postinfection (renukaradhya et al., ; dwivedi et al., ) . in pigs, a significant correlation has been observed between the prrsv infection and an increased expression of il- , associated with reduced expression of ifnα, ifnγ, il- , and tnfα (gómez-laguna et al., ) . induction of il- is mediated by interaction between prrsv proteins and mΦs/dendritic cells (dcs). the prrsv nucleocapsid protein induces il- production by peripheral blood mononuclear cells (pbmcs) and alveolar mΦs (wongyanin et al., ) , delaying the onset of protective cell-mediated immunity (cmi) to prrsv (dwivedi et al., a . dysregulated immune responses in infected pigs affect viral pathogenesis, disease severity, and susceptibility to secondary microbial infections (thanawongnuwech et al., ; renukaradhya et al., ) . immune responses against prrsv are inadequate to completely clear the virus. viremia disappears in - weeks, but the virus persists in the tonsils and lymphoid tissues for several months (moyron-quiroz et al., ) . prrsv infection in both germ-free and conventionally raised pigs is associated with polyclonal b cell activation, hypergammaglobulinemia, lymphoid adenopathy, renal lesions, and lymphoid hyperplasia (cooper et al., ) . polyclonal lymphoplasia also occurs in mice infected with lactate dehydrogenase-elevating virus, a related virus (grossmann et al., ) . primary antibody responses occur promptly after prrsv infection (loemba et al., ) , but the majority of secreted antibodies are autoantibodies or prrsv nonneutralizing antibodies (lemke et al., ) . the virusneutralizing antibody (na) response is weak, and delayed (mateu et al., ) . both killed and modified live virus (mlv) prrsv vaccines are available commercially for intramuscular or intradermal administration (charerntantanakul, ) . mlv vaccines confer protection against genetically homologous prrsv, but incomplete protection against heterologous viruses (mengeling et al., ) . in both prrsv-infected and mlv-prrsv-vaccinated pigs, virus-specific cell-mediated immune responses are either delayed or dampened . reversion to virulence is a major concern with modified live vaccines. there are numerous reports of reversion of prrsv vaccine virus to virulence and of the presence of reverted vaccine strain prrsv in unvaccinated sows and pigs . vaccine-derived virus has been isolated from fetuses, stillborn, and dead piglets, indicating the spread of disease from vaccinated pigs. identification of mutations in multiple vaccine-derived isolates at identical positions of the viral genome suggests a strong selective pressure on critical viral proteins in field situations. available killed prrsv vaccines fail to protect even against homologous virus, do not elicit na, and induce weak cell-mediated immune responses (bassaganya-riera et al., ) . pregnant sows and gilts and breeding boars are not protected from prrsv due to a lack of safe and protective vaccines. control of prrsv in breeding stock is critical in preventing vertical and horizontal transmission of the virus. early attempts to develop killed prrsv vaccines using recombinant prrsv proteins, plasmids expressing viral genes, and inactivated prrsv administered with or without adjuvants have been unsuccessful ( charerntantanakul, ) . however, recent studies show promise. killed prrsv vaccine coadministered in with cpg oligodeoxynucleotide adjuvant (a tlr- ligand) induced antibodies, virus-specific t cells, and secretion of ifnγ and il- (zhang et al., ) . in another study, prrsv inactivated by uv light or binary ethylenimine induced na and protected pigs against homologous viral challenge. the suppressive effect of live prrsv on ifnα production was lost when the virus was inactivated by uv irradiation (vanhee et al., ). an oral immunization strategy using prrsv nucleocapsid protein genetically fused to cholera toxin (ct) stimulated prrsv antibody responses in the intestines, but no detectable response in vaginal secretions (hyland et al., ) . lack of success in developing protective killed prrsv vaccines may reflect an inability to present killed prrsv antigens effectively to the pig immune system, or (like live prrsv) killed virus may induce immunosuppressive effects. in addition, the antigenic mass used in killed vaccines may be insufficient, suggesting the need for potent adjuvants or novel delivery systems. attempts have been made to improve mlv-prrsv vaccines using adjuvants. in one study, pigs were injected with recombinant porcine il- , il- , il- , or ct within week of intramuscular administration of mlv-prrsv. il- and ct enhanced ifnγ and gp antibody responses, respectively (foss et al., ) . however, use of these adjuvants did not reduce the severity of viremia. unfortunately, prrsv (na) responses were not assayed, and protection against heterologous challenge was not assessed in this study. prrsv gains entry through respiratory and reproductive mucosal surfaces and causes disease primarily at mucosal sites. therefore, a mucosal vaccine against prrsv may be an effective strategy for controlling the disease. until recently, attempts to elicit protective mucosal immunity against prrsv have been unsuccessful, probably due to a lack of appropriate adjuvants. because prrsv infection rapidly subverts the host immune responses, an effective adjuvant must overcome immunosuppression caused by vaccine virus and simultaneously potentiate virus-specific adaptive immunity. a panel of bacterial preparations derived from mycobacterium, vibrio, and streptococcus species, which were potent adjuvants in rodent models, were tested in with mlv-prrsv (bonavida et al., ; barral and brenner, ) . based on mucosal and systemic immune responses, three of the preparations, mycobacterium tuberculosis whole cell lysate (m. tb wcl), ct b subunit, and ok- (a product of streptococcus pyogenes), were selected for viral challenge trials. only m. tb wcl significantly dampened prrsv immunosuppressive effects, and enhanced virus-specific adaptive responses (renukaradhya et al., ; dwivedi et al., b) . historically, heat-killed mycobacteria are recognized to have as potent adjuvant effects as components of freund's complete adjuvant (fca) and have been used extensively in experimental animals. the use of fca in humans and food animals is unacceptable due to severe granulomatous inflammatory reactions to toxic cell wall components ( bekierkunst, ). however, a nontoxic water-soluble purified fraction of m. tb wcl (werner et al., ) has adjuvant properties in rodents, guinea pigs, and rabbits. pigs inoculated in with m. tb wcl have no detectable signs of toxicity locally or systemically (dwivedi et al., a,b) . a recombinant vaccine (prrsv gp and m expressed in bacillus calmette-guerin (bcg)) is reported to reduce clinical signs of prrs with decreased viremia and viral load in the tissues (bastos et al., ) . cross-protective immunity against prrsv has been evaluated in pigs receiving mlv-prrsv and m. tb wcl in, by challenging with virulent heterologous prrsv virus (kim et al., ) . reduced clinical disease, viremia, and virus-mediated immunosuppression were noted. in addition, secretion of ifnγ and il- by lung and blood lymphocytes in response to prrsv m and nucleocapsid proteins was enhanced (dwivedi et al., a) . guillonneau et al. ( ) reported similar findings in mice vaccinated in with adjuvanted influenza vaccines. enhanced virus-specific cytotoxic t cell and memory responses to internal viral proteins were noted, as well as cross-protective immunity. studies of passive protection of pigs by prrsv na have indicated that modest na titers (≤ / ) protect pregnant sows against reproductive failure, block placental transmission of infection, prevent viremia in piglets, and provide sterilizing immunity (osorio et al., ) . pigs immunized in with mlv-prrsv alone or with m. tb wcl developed na titers < / and / , respectively, postimmunization (dwivedi et al., b) . in pigs immunized (mlv-prrsv + m. tb wcl) and challenged with prrsv mn , an na titer > / persisted until pid (dwivedi et al., a) . relatively low numbers of circulating virus-specific ifnγ secreting cells have been reported for pigs vaccinated in with mlv-prrsv without adjuvant, ( - cells per million pbmcs) (dwivedi et al., a) . in contrast, meier et al. ( ) have reported - ifnγ secreting cells per million pbmcs in pigs vaccinated against aujeszky's disease virus. pigs immunized in with mlv-prrsv with m. tb wcl and challenged with prrsv had greater than ifnγ secreting cells per million pbmcs, and more than a twofold higher frequency of cd + cd + t cells (dwivedi et al., a) . the literature concerning mucosal immune responses in the pig respiratory tract is limited compared to studies of rodents and humans. recent research highlights the advantages of activating the mucosal immune system using vaccines delivered with potent adjuvants. induction of adequate immune responses in the respiratory and reproductive tract will be essential for control of prrs in swine. in-delivered, potent mucosal vaccines generate better cross-protective immunity against genetically variable prrsv field viruses. efforts to control prrs outbreaks using conventional parenterally delivered vaccines have had limited success; development of an alternative approach of generating immunity in the respiratory tract should be a priority. in particular, mucosal adjuvants based on components of mycobacterial species show promise. respiratory tract disease affects virtually every aspect of equine husbandry, including working, pleasure, and race horses. considerable efforts are expended to prevent epizootics of respiratory disease in stables, fairs, shows, and race tracks. equine influenza virus will be discussed in detail as an example of past, current, and future vaccine approaches. equine influenza virus causes epizootics of upper and lower respiratory tract disease almost worldwide. until equine influenza was excluded from the continent of australia through import restrictions and quarantine. in august of that year, importation of an infected horse led to an explosive epizootic of equine influenza with an estimated , horses infected over a -month period (callinan, ) , demonstrating the potential of the virus to spread in a naïve, unvaccinated population. infection can occur in horses of all ages, but epizootics often involve younger animals (van maanen and cullinane, ) . clinical signs include high fever, a persistent dry cough, nasal discharge, anorexia, and depression. secondary bacterial pneumonia may complicate the clinical picture. equine influenza viruses are classified as type a influenza. antigenic differences in the hemagglutinin (h) and neuraminidase (n) glycoproteins define the two recognized subtypes, a/equine/ (h n ) and a/equine/ (h n ) (wilson, ) . two lineages of a/equine/ , american and european, have been identified; multiple virus strains are included in vaccines since protection against heterologous strains is incomplete (yates and mumford, ) . antigenic drift is sufficient to require regular reappraisal of strains included in vaccines (mumford and wood, ) . natural infection induces iga antibodies in nasal secretions, igga and iggb antibodies in serum nelson et al., ) , and circulating cytotoxic t lymphocytes . protection against reinfection persists for at least a year (hannant et al., ) . vaccination with inactivated virus vaccines induces serum igg (t) antibodies without detectable iga in nasal secretions (nelson et al., ) and without cytotoxic t cell activity (van maanen and cullinane, ) . two or three doses of vaccine are typically administered in the primary series, with booster doses at least once a year thereafter. more frequent vaccination is advised for horses at high risk of infection (wilson, ) . protection is typically incomplete and of limited duration. improved adjuvants can enhance the level and duration of antibody responses to inactivated virus vaccines (mumford et al., c) . suppressive effects of maternal antibodies on responses to inactivated vaccines have led to recommendations not to vaccinate foals before months of age (van oirschot et al., ) . a subunit equine influenza vaccine based on the immune stimulating complex (iscom) adjuvant technology has been licensed and marketed in europe since (newmark, ) . antibody responses to iscom-based vaccines typically are of higher titer and are more persistent than for conventional inactivated vaccines (mumford et al., a) . protection has been demonstrated against experimental challenge, months after a three dose vaccination series (mumford et al., b) . protection may be due in part to the ability of iscom-adjuvanted vaccines to induce cytotoxic t lymphocytes (morein et al., ) . although iscom-based vaccines can induce iga antibody responses following in administration (hu et al., ) , the commercial influenza iscom vaccine is administered parenterally. in administration of inactivated equine influenza virus with ct b has been reported to induce local iga antibodies, and protection against experimental challenge (hannant et al., ) . a cold-adapted, temperature-sensitive live vaccine for in administration is available commercially . protection against experimental challenge has been demonstrated months after a single vaccination . this is a notable improvement in efficacy and practicality over conventional killed vaccines. experimental plasmid vaccines encoding the hemagglutinin gene of equine influenza have been examined in horses. three doses of plasmid administered to the skin and mucosal sites (tongue, conjunctiva, and third eyelid) induced protection against clinical disease and partial protection against viral shedding. protection against clinical disease was reduced if plasmid was administered only to the skin (lunn et al., ) . a canarypox-vectored equine influenza vaccine, expressing hemagglutinins from two strains of h n equine influenza, has been available commercially (intramuscular administration) since (toulemonde et al., ) . in the australian epizootic of , this vaccine was used extensively during the government program to eradicate equine influenza. the vectored vaccine was chosen for this program because of its diva (distinguishing infected from vaccinated animals) characteristics ( kirkland and delbridge, ) . infected horses mount antibody responses to the viral nucleoprotein in addition to the viral hemagglutinin, whereas vaccinated horses respond only to the hemagglutinins expressed by the vaccine vector. a recent report indicates that adequate antibody responses are generated with as little as days between primary and secondary vaccination (el-hage et al., ) . protection against experimental challenge has also been noted weeks after a single dose of vaccine in ponies ( toulemonde et al., ) . for some respiratory pathogens (e.g., m. hyopneumoniae in pigs) the critical antigens associated with protective immune responses have not been identified. for other pathogens (e.g., prrsv) there is also a need to identify the appropriate type of immune response (th or th ) needed for protection. for some complex disease conditions (e.g., bovine respiratory disease complex) there is continuing uncertainty whether all of the relevant contributing pathogens have been identified. although many parenteral vaccines are efficacious in reducing lower respiratory tract disease, there is a need to investigate whether induction of mucosal immunity in the upper airways, in combination with systemic immunity, can further reduce infection rates, transmission of pathogens, and economic losses. finally, there is a need to devise and implement changes in management procedures to reduce disease exposure (by nonimmunological methods), and to optimize immune interventions by improved timing of vaccinations. vaccines to prevent reproductive tract disease have received much emphasis in veterinary medicine. this is especially true of food-producing animals because reproductive failure is a major economic problem. although vaccines to prevent reproduction are of interest for abandoned pets or deer in areas of overpopulation, this section will deal only with vaccines designed to prevent infectious disease of the reproductive mucosa. infections causing adverse pregnancy outcome can be classified by route of infection: hematogenous or ascending. several hematogenous infections have a predilection for the gravid uterus resulting in early to late abortions . these include leptospirosis, chlamydial infection, and brucellosis in several animal species, histophilus somni infection in cattle and sheep and neospora caninum in cattle. although vaccines are available for several of these infections, the vaccine for brucellosis has been available since the s and is the most well known. several brucella species cause abortion or epididymitis/orchitis in the primary hosts ( brucella abortus in cattle, brucella suis in swine, brucella melitensis in goats, brucella ovis in sheep, brucella canis in dogs, and brucella marinum (or brucella delphini) in marine mammals). infection may be acquired via the gut mucosa or the conjunctiva/upper respiratory mucosa and localizes in the reticuloendothelial system and endometrium/placenta by systemic spread. thus, systemic vaccines are effective. a modified live b. abortus vaccine, along with a "test and slaughter" eradication program, has been successful in controlling bovine brucellosis in north america. the modified live vaccine (b. abortus strain ) is very effective in stimulating cmi that is critical for protection against this facultative intracellular pathogen. strain now has been largely replaced by a new attenuated strain, rb , which does not stimulate an antibody response known to interfere with diagnostic serologic assays. there is considerable information on mechanisms of systemic immunity to brucellosis. neospora caninum also causes abortion by a hematogenous route and a vaccine is available (weston et al., ) . histophilus somni can infect either by the hematogenous route or by an ascending genital route to cause abortion or infertility and vaccines are available. since the focus of this volume is mucosal immunity, no more will be said concerning protection against hematogenous infections of the genital tract. ascending local infections of the reproductive tract are usually transmitted sexually. the two best examples of vaccines for sexually transmitted infections of animals are campylobacter fetus subsp. venerealis (formerly vibrio fetus subsp. venerealis) and tritrichomonas foetus. both are host-specific bovine sexually transmitted diseases (stds) that only infect the reproductive mucosa. both are extracellular pathogens that do not invade the mucosa of the reproductive tract but may be found in the placenta and fetus. the localized nature of these infections and transmission limited to coitus suggest that mucosal immunity must be important. because a vaccine has been available for c. fetus subsp. venerealis for several decades and its use has controlled the disease in developed countries, that vaccine will be discussed first. vibriosis (or campylobacteriosis) is a chronic bacterial genital infection with no overt clinical signs other than reproductive failure (corbeil et al., ) . after months of infection, the uterus is cleared first, followed by the vagina. convalescent immunity is partially protective for a limited time. antibody is effective in protection against this extracellular pathogen as demonstrated by systemic passive immunization (berg et al., ) . the antibody response to infection is primarily iga in the vagina and igg in the uterus (corbeil et al., ) . systemic immunization with a whole cell vaccine results in both igg and igg antibody responses to surface antigen in serum, and uterine and vaginal secretions (corbeil et al., ) . this response prevents infection and can rapidly clear infected cows (corbeil and winter, ) . that is, the vaccine can be used prophylactically and even therapeutically. immunization is efficacious even though surface antigenic variation occurs in the face of a local immune response (corbeil and winter, ) . presumably, immune clearance occurs when the dynamic interaction between protection and evasion is shifted in the favor of the host. this appears to occur earlier when the response is primarily igg than when iga predominates (corbeil et al., ) . this may be related to the ability of the igg antibodies to mediate opsonization and intracellular killing of the bacterium, an ability which iga antibodies lack (corbeil and winter, ) . although this work was done many years ago, it sets a precedent for systemic immunization for prophylaxis and therapy of reproductive mucosal infections. trichomoniasis is a similar chronic genital mucosal infection of cattle. it is caused by the protozoan, t. foetus, and results in pregnancy loss. trichomonas vaginalis causes a human std also associated with adverse pregnancy outcome. thus, bovine trichomoniasis serves as a model for immune prevention of both bovine and human reproductive mucosal infections. because of the economic significance of bovine trichomoniasis and because no chemotherapy is approved, investigations have focused on immunoprophylaxis and immunotherapy. t. foetus colonizes the vaginal and uterine or preputial surfaces for months, like c. fetus subsp. venerealis infection. in fact, mature bulls are often infected for life whereas young bulls may clear the infection with time (cobo et al., ) . this is probably related to innate immunity. trichomonads are anaerobic parasites and are found deep in uterine glands and epithelial crypts of the penis and prepuce (rhyan et al., ) where the oxygen tension is probably lowest. older bulls have deeper epithelial crypts, which may have lower oxygen tension. in order to understand protective acquired immune responses, monoclonal antibodies (mabs) with putative protective functions were chosen for immunoaffinity purification of a highly glycosylated surface antigen (bondurant et al., ; corbeil et al., ) . analysis of many isolates of t. foetus indicated that two mabs recognized different epitopes of the same antigen, which was conserved in all isolates tested. this glycosylated surface antigen was later shown to be a lipophosphoglycan (lpg)/protein complex. systemic immunization with the immunoaffinity-purified lpg-containing surface antigen, followed by intravaginal challenge with t. foetus, resulted in significantly earlier clearance of the parasite from vaccinated animals than from controls (bondurant et al., ; corbeil et al., ) . more importantly, clearance of immunized animals most often occurred before weeks of infection. parsonson et al. ( ) showed that significant inflammation accompanied by reproductive failure did not occur until after weeks of infection, so the vaccine should protect against fetal loss. analysis of vaccine-induced antibody responses demonstrated predominantly igg responses in the serum and iga plus igg antibodies in secretions (corbeil et al., , . ige antibodies were also increased during infection. as ige antibodies increased, mast cells degranulated and clearance of t. foetus occurred. these studies raised several questions. first, why is the systemic response to the lpg/protein complex skewed toward igg (a th response in cattle) and not igg (a th response)? this question is under investigation. second, are igg or iga antibodies to the lpg-containing antigen most protective and how can that ig isotype be enriched to enhance protection? to address the latter questions, preliminary studies were done in mice to determine the best routes and adjuvants to enrich for igg or iga in genital secretions . subcutaneous priming with the immunoaffinity-purified lpg-containing surface antigen (called tf . antigen) in quil a adjuvant and subcutaneous boosting with whole cells enriched for igg anti-tf . antibodies in genital secretions whereas subcutaneous priming and intravaginal boosting greatly enriched for iga antibodies in genital secretions. when cattle immunized by these two methods were challenged intravaginally with t. foetus, those with predominantly iga or predominantly igg anti-tf . antibodies in genital secretions were equally protected. later studies with similar in immunizations showed that stimulation of the common mucosal immune system gave results similar to those of intravaginal immunization . this raised the question of inductive sites for local immune responses in the genital tract. others have suggested that the genital tract is not an inductive site because m cells and mucosa-associated lymphoid tissue (malt) are not present. this is true of cattle as well as mice and people. however, even though control cows did not have histologically demonstrable malt in the uterus and vagina, cows experimentally infected with t. foetus did . similar lymphoid nodules and follicles under a modified epithelium were detected in preputial and penial surfaces of bulls infected with t. foetus (rhyan et al., ) . immunostaining of parallel sections with mab to tf . antigen indicated uptake of antigen by epithelial cells and large mΦ or dc under the basement membrane near the lymphoid follicles (rhyan et al., ) . similar antigen uptake has been detected in the infected female uterine and vaginal mucosa (corbeil et al., ) . thus, even though the parasite is noninvasive, released tf . antigen appears to be taken up by epithelial cells. rat uterine epithelial cells can present antigen to t helper cells (wira and rossol, ) . also, mΦ/ dc positive for antigen should be antigen-presenting cells (apcs). detection of igg and iga antibodies in genital secretions of infected bulls (cobo et al., ; rhyan et al., ) and cows and the histologic demonstration of follicles and putative apcs suggests that inductive sites in the genital tract are formed in response to antigenic stimulation. like c. fetus subsp. venerealis, t. foetus has mechanisms for evasion of immune responses. these include coating of the surface with ig nonspecifically , epitope variation (ikeda et al., ) , and cleavage of igg , igg , and complement component by extracellular cysteine proteinase (talbot et al., ; kania et al., ) . studies with cysteine proteinase inhibitors demonstrated decreased cytotoxicity of t. foetus for bovine trophoblast cells and decreased infectivity in a mouse model, confirming the likely role of cysteine proteinases in pathogenesis (cobo et al., ) . even with these mechanisms for immune evasion, as with c. fetus, it is clear that the dynamic interaction between host and parasite can be made to favor the host by systemic or local immunization. the usefulness of whole cell vaccines in preventing t. foetus infection and reproductive failure in cows has been demonstrated in clinical trials (kvasnicka et al., ) . first-generation whole cell vaccines are now commercially available for prevention of trichomoniasis in cows. earlier, clark et al. ( ) demonstrated efficacious immunization of bulls with whole t. foetus cells or crude membrane glycoproteins that probably contained tf . antigen. this study indicated that vaccination of bulls could both prevent infection and clear already established infection. so vaccines for bovine trichomoniasis are both immunoprophylactic and immunotherapeutic. recent studies showed that vaccination of bulls with a commercially available whole cell killed t. foetus vaccine in oil adjuvant resulted in protection against challenge and high levels of igg antibodies in serum and preputial secretions (cobo et al., ) . igg antibodies to whole cell t. foetus antigens predominated but fairly high levels of igg antibodies were also detected. the above studies with c. fetus subsp. venerealis and t. foetus show that: . stds can be prevented or even cured by systemic vaccination of both males and females. . at least for one std, igg and iga of the same antigenic specificity are equally protective at the mucosal surface. either systemic immunization or mucosal (in or intravaginal) immunization with systemic boosting is protective. . inductive sites are formed in the mucosa of infected male and female genital tracts even with noninvasive pathogens. . strong and appropriate immune responses will clear microbial infection from the genital tract even when the microbe has multiple immune evasive mechanisms. . the fact that protection against two stds has been demonstrated in the natural outbred host (cattle) has advantages over murine models of std vaccines. in the latter, the human pathogen is usually inoculated into the unnatural murine host and the disease does not mimic the human infection. furthermore, although inbred mice provide a homogeneous experimental animal, they do not represent the variation in immune responses seen in the outbred human population. the work on bovine vibriosis and trichomoniasis demonstrates protection under field conditions for two stds that cause adverse pregnancy outcome in an outbred host. this is an encouraging precedent for control of human stds and related adverse pregnancy outcomes. future needs include identification of the protective antigens for most stds. for antibody-mediated protection of the genital mucosa, several questions have not yet been addressed. as far as we know, the role of ige in the genital tract is largely unstudied even though it does seem to be involved in clearance of trichomonads from the bovine genital tract. lastly, manipulating genital immune responses to enhance th or th type responses is an unexplored research area. enteric disease is a major cause of mortality and morbidity in animals. agents causing diarrhea in animals include viruses (e.g., adenoviruses, pestiviruses, caliciviruses, coronaviruses, parvoviruses, rotaviruses, toroviruses), bacteria (e.g., campylobacter spp., clostridium spp., diarrheagenic escherichia coli, salmonella spp., yersinia spp.), and parasites (e.g., coccidia spp., cryptosporidium parvum). these infections occur most commonly in suckling animals or in poultry under weeks of age, but may also be common postweaning and in susceptible seronegative or stressed adult animals (saif and jackwood, ) . induction of local secretory iga (s-iga) antibodies (prevent attachment, invasion, and neutralize toxins or infectious agent) and mucosal cellular immune responses (against intracellular bacteria and viruses) by vaccines are essential for protection from enteric pathogens. peyer's patches (pp) and mesenteric lymph nodes are two important organized gut-associated lymphoid tissues (galt) in domestic animals and serve as the induction site for gut immune responses. ileal pp in some domestic animals (sheep, cattle, pigs, dogs) differ from jejunal pp, serving as a primary lymphoid organ similar to the bursa of fabricius in chickens, unlike in humans and rodents where pp are secondary lymphoid organs (chu and liu, ; yasuda et al., ) . cryptopatches or clusters of lymphoid cells in the basal lamina propria occur in mice, but are absent in pigs (pabst et al., ) . mucosal lymphoid tissues and lymphoid cells in domestic animals and humans differ in toll-like receptor (tlr) expression and function (after binding their ligand) when compared to mice (iwasaki and medzhitov, ; tohno et al., ; guzylack-piriou et al., ) . these differences in immune components suggest that vaccine studies carried out in mice may fail to translate to domestic animals or humans. enteric pathogens have different characteristics related to their intestinal tropism and replication, requiring different vaccination strategies. enteric viruses have predilections for replication in distinct vertical and longitudinal regions of the small or large intestines. cytolytic infection of enterocytes leads to varying degrees of villous loss and fusion, reduced small intestinal absorptive capacity, and a malabsorptive, maldigestive diarrhea (saif, a) . secretory diarrhea is also induced by some viruses such as rotavirus (rv), which involves the viral enterotoxin, nonstructural protein (nsp ), and/or stimulation of the enteric nervous system (ball et al., ; lundgren et al., ) . thus, viral diarrheas can be of variable severity and act via multiple mechanisms that differ from those of enteric bacteria, most of which cause secretory diarrhea mediated by enterotoxins (fairbrother and gyles, ) . enteropathogenic viruses can be divided into three types (types i, ii, and iii) according to their preferred site of replication in the intestine (reviewed by saif ( a) ). type i (transmissible gastroenteritis virus (tgev) and rv) and ii virus infections can be prevented by inducing local intestinal immunity, whereas type iii viruses (parvovirus), which infect crypt enterocytes basolaterally, can be prevented by inducing systemic immunity. the enteropathogenicity of bacteria is determined by their virulence factors including adhesion factors (fimbriae or pili) and enterotoxins; therefore, bacterial vaccines generally need to prevent attachment and toxin action within the intestine (fairbrother and gyles, ) . in the following sections, we will review vaccine strategies for these different types of enteric infections. tgev and rv vaccines in pigs will be reviewed to illustrate findings using domestic outbred animals instead of inbred laboratory rodent models. neonates can be protected from enteric infections by providing passive lactogenic immunity, which can be achieved by immunizing mothers preparturition. pioneering work done by bohl and saif in the early s with live oral virulent tgev in pigs was the foundation for the gut-mammary gland-s-iga immunologic axis, which later became the basis for the concept of the common mucosal immune system. their studies showed that in tgev seronegative sows, only oral immunization with virulent tgev induced high rates of protection in suckling neonates, which was associated with high titers of s-iga antibodies in colostrum and milk, whereas systemic immunization induced mainly igg antibodies saif et al., ) . rotavirus is a major cause of dehydrating diarrhea in young livestock, infants, and poultry (saif and fernandez, ) . multiple rv serogroups, based on common inner capsid vp antigens (a-h), and multiple g (vp , glycoprotein) and p (vp , protease-sensitive) serotypes (based on neutralizing epitopes) or genotypes (based on sequence analysis) for group a rvs have been detected in humans, sheep, swine, cattle, horses, dogs, cats, poultry, and wildlife (estes and kapikian, ; martella et al., ) . among the distinct rv serogroups and g/p serotypes/genotypes, cross-protection is limited. the antigenic divergence among different sero/genotypes of rvs presents a challenge for the design of vaccines that are capable of inducing heterotypic protection. commercial modified live and killed rv vaccines for rv diarrhea in livestock and poultry are limited to group a rvs (saif and fernandez, ) . mebus et al. ( ) developed the first oral rv vaccine for calves in ( year prior to the discovery of human rv) using a cell cultureadapted neonatal calf diarrhea rv strain. although a significant reduction in morbidity and mortality was observed in a field trial among vaccinated calves, in the majority of herds (compared to previous years), subsequent field studies revealed variable efficacy. experimental studies suggested that maternal antibodies interfered with live oral vaccine replication and suppressed development of active immunity (saif and fernandez, ) . the neonatal gnotobiotic pig model has been used to investigate immune responses to rv vaccines and infection for nearly three decades (saif et al., , yuan and saif, ; gonzalez et al., ) . gnotobiotic pigs are free of maternal antibodies (placental transfer of igs does not occur in swine), but they are immunocompetent at birth. they are maintained aseptically and are free of exposure to extraneous wildtype rvs, assuring that exposure to a single pathogen can be analyzed. initial studies were conducted to mimic natural rv infection (bohl et al., ) and to study immune correlates of protection gonzalez et al., ; yuan et al., ) . understanding the sequence and kinetics of immune responses stimulated by virulent rvs allows for the determination of markers of protective immunity and pathogenicity, which can then be used to design vaccines that will stimulate protective immunity without inducing pathology. gnotobiotic pigs orally inoculated with virulent porcine or human rvs were completely protected from homotypic but not heterotypic (distinct g/p types) rv challenge (hoshino et al., ; saif et al., ) . significant correlations were observed between the protection to rv-induced diarrhea and shedding and the following immune parameters: the number of intestinal iga rv antibody-secreting cells (ascs), serum and intestinal iga rv antibody titers, and the frequency of intestinal rv-specific ifnγ producing cd + and cd + t cells yuan et al., ; to et al., ; yuan and saif, ; yuan et al., ) . these immune parameters were significantly higher in virulent rv inoculated pigs than in those inoculated with attenuated or inactivated rv (ward et al., b) . pigs inoculated with two or three doses of attenuated rv were moderately protected against diarrhea ( %- doses, %- doses) and virus shedding ( %- doses and %- doses) after homotypic challenge, suggesting a need for multiple doses and suitable mucosal adjuvants to enhance the efficacy of rv vaccines yuan et al., yuan et al., , yuan and saif, ) . gnotobiotic pigs inoculated with virulent rv developed an acute proinflammatory serum cytokine profile (il- , tnfα) coinciding with peak diarrhea and viremia, followed immediately by increased th (il- , ifnγ) cytokines and convalescent th (il ) and tr (il- ) cytokine responses. gnotobiotic pigs inoculated with one dose of attenuated rv showed lower early ifnγ and proinflammatory cytokine responses compared to virulent rv inoculated pigs. both attenuated and virulent rv inoculated pigs developed th /th /tr cytokine-secreting cell (csc) responses at - weeks postinoculation; however, attenuated rv inoculated pigs developed lower intestinal ifnγ and higher intestinal and splenic il- cscs compared to virulent hrv inoculated pigs (azevedo et al., ) . virulent rv inoculated pigs had significantly higher protection rates against rv challenge ( % and % against diarrhea and shedding, respectively) compared to one dose of attenuated rv inoculated pigs ( % and % against diarrhea and shedding, respectively) (azevedo et al., ) . these findings suggest that higher protection rates are associated with early proinflammatory and th cytokine responses, which promote cytotoxic t cell activity and viral clearance, and later th induced cytokines, which are important for protective s-iga antibody responses. thus, protection against rv in pigs requires balanced th /th /treg responses (azevedo et al., ; gonzalez et al., ) . infection of gnotobiotic pigs with virulent rv causes early recruitment of innate apcs (monocytes/mΦ and dcs) and γδ t cells into the ileum, and enhanced tlr , tlr , and tlr expression among apcs in spleen (zhang et al., c; wen et al., wen et al., , . in virulent rv-infected pigs, plasmacytoid dcs are major producers of serum ifnα and, along with other innate immune cells (γδ t cells and apcs) and cytokines (tnfα, ifnγ and il- ), are important for controlling early rv viral replication and subsequent development of protective adaptive immune responses . development of attenuated rv vaccines with mucosal adjuvants that mimic immune responses to virulent rv may improve existing vaccines. immunogenicity and protective efficacy of rv vaccine formulations (attenuated replicating virus, inactivated virus, and recombinant baculovirus-expressed virus-like particles (vlp)), administration routes, and adjuvants have also been evaluated using the gnotobiotic pig model (bohl et al., ; yuan and saif, ; gonzalez et al., ) . inactivated oral or intramuscular rv vaccines failed to protect against virulent rv challenge, despite high igg antibody responses induced in serum and systemic lymphoid tissues. serum igg antibodies did not correlate with protection. however, a recent study showed that an inactivated reassortant rv strain (cdc- strain) formulated with aluminum phosphate and administered systemically in gnotobiotic pigs resulted in induction of serum igg antibody titers, coinciding with partial protection against shedding and diarrhea, suggesting that adjuvant may have stimulated local specific (gut iga antibodies) or nonspecific immune responses, which were not assessed in this study (wang et al., ) . rotavirus subunit vaccines consisting of double-layered vlp composed of rv inner capsid proteins vp and vp ( / -vlps) administered in or orally with mutant heat-labile toxin of e. coli (mlt) or iscoms as adjuvants (yuan and saif, ) induced igg asc responses in systemic lymphoid tissues and low or no iga asc responses in intestinal lymphoid tissues and also failed to mediate protection, contrary to results in adult mouse studies (yuan and saif, ) . the failure of in or oral / -vlp vaccines suggests that protective immunity to rv diarrhea in neonatal pigs requires mainly the presence of systemic or intestinal iga antibodies to the outer capsid rv proteins, vp and vp , each of which induces na. however, when / -vlps adjuvanted with mlt or iscom were used as in or oral booster doses in pigs orally primed with attenuated rv, the protective efficacy increased significantly. an oral attenuated rv prime and in / -vlp-iscom boost regimen (attrv/ / -vlp) induced the highest numbers of intestinal iga ascs and serum and intestinal iga antibody titers, and protection rates were similar to or higher than those induced by three oral doses of attenuated rv (gonzalez et al., azevedo et al., ) . interestingly, priming with two doses of / -vlp (in or oral) followed by live attenuated rv was ineffective for inducing iga antibodies or protection. thus the use of a replicating vaccine to prime at one inductive site (galt) followed by boosting with a nonreplicating vaccine at a second mucosal inductive site (nasal-associated lymphoid tissue, nalt) is an effective strategy for stimulating protective mucosal immune responses, which can be applied to other enteric viral vaccines. furthermore, efficacy of the prime/boost strategy (replicating/nonreplicating vaccine, attrv/ / -vlp) was examined in the presence of high and low titers of passive antibodies to mimic neonatal pigs receiving maternal antibodies (nguyen et al., a,b) . similar to attrv/ / -vlp prime/boost vaccine studies, plasmid dna containing vp was used as a booster subsequent to oral attenuated rv vaccine priming. this regimen showed high protection against shedding, but poor efficacy against rv diarrhea (yuan et al., ) . collectively these findings suggest that mucosal boosters are effective in enhancing iga antibody titers to rvs in orally primed animals . studies have shown that immunogenicity and efficacy of mucosal vaccines can be improved by the use of appropriate strains of probiotics that modulate mucosal and systemic immune responses, by interaction with epithelial cells and the underlying intestinal immune cells (fukushima et al., ; sanz and de palma, ) . certain probiotic strains are reported to enhance immune responses to rv vaccines in children; others reduce rv diarrhea severity, but the mechanisms are not well defined (fang et al., ; holscher et al., ) . supplementation with lactobacillus acidophilus in attenuated rv vaccinated pigs is reported to enhance intestinal ifnγ-producing cd + t cells, intestinal iga and igg rv ascs, and serum iga and igg rv antibody titers (zhang et al., b) . these findings suggest that probiotics are an alternative, cheap, and safe adjuvant for enhancing efficacy of oral attenuated rv vaccines in animals and potentially in humans. colonization of pigs by two different strains of lactic acid bacteria (lab, l. acidophilus and lactobacillus reuteri) and subsequent virulent rv infection resulted in higher and balanced th /th /treg cytokine responses (il- , ifnγ, il- , il- , tgfβ), higher total intestinal iga-secreting cells, total serum igm, and intestinal igm and igg titers, although the numbers of iga rv ascs and serum and intestinal rv antibody titers did not differ compared to virulent rv-only infected pigs. no overall difference in protection rates was noted when compared to virulent rv-only inoculated pigs, which was likely because of the short interval (only days) between lab colonization and virulent rv challenge (zhang et al., a; azevedo et al., ) . dual colonization of the aforementioned lab strains also modulated innate immune components in virulent rv inoculated pigs as follows: ( ) enhanced frequency of γδ t cells in the intestine and their distribution; ( ) enhanced tlr and tlr -expressing cd + apc, and tlr -expressing cd − apcs in the blood, but reduced tlr -and tlr expressing cd − apcs in the spleen, and ( ) reduced frequency of mΦ and cdcs in the spleen. collectively, these findings suggest that probiotics may reduce systemic inflammatory responses induced by virulent rv. effects on tlr expression on apcs in the ileum were not determined (zhang et al., c; wen et al., wen et al., , . probiotics may not only modulate immune responses to rvs or other enteric vaccines, but may also directly ameliorate diarrhea/infection by enhancing gut barrier integrity and maintaining intestinal permeability, by stimulating nonspecific immune responses, by changing gut microbial populations, and by aiding in the regulation and prevention of apoptosis ( madsen et al., ; yan and polk, ; preidis et al., ) . using the tgev model for evaluation of active protection against diarrhea in pigs, researchers also delineated compartmentalization in the common mucosal immune system and its impact on mucosal vaccine strategies and protection (vancott et al., (vancott et al., , . the natural occurrence of a deletion mutant of tgev with exclusive respiratory tropism, referred to as porcine respiratory coronavirus (prcv), provided a unique opportunity to study asc responses and protective immunity to two antigenically related porcine coronaviruses with enteric (tgev) versus respiratory (prcv) tropism. oral immunization of pigs with tgev induced high numbers of iga asc in the intestine and provided complete protection against tgev challenge, whereas in immunization of pigs with prcv induced mainly systemic responses (igg asc) and provided only partial protection against tgev challenge. thus, the in prcv alone failed to elicit sufficient intestinal iga asc to provide full protection against the enteric pathogen, tgev. findings from this study and rv vaccine studies suggest that the use of multiple mucosal inductive sites in a prime/boost vaccination regimen may be an effective approach for overcoming compartmentalization in the common mucosal immune system. canine parvovirus (cpv) infects crypt enterocytes causing hemorrhagic gastroenteritis in pups (saif and jackwood, ) . because cpv is likely disseminated to the basolateral surface of crypts by the hematogenous route, serum na (derived maternally or actively produced) is protective against the disease. pollock and carmichael ( ) demonstrated that pups with hemagglutination inhibition (hi) serum antibody titers of > : were immune to oronasal cpv type challenge. cpv is highly stable in the environment and pups were susceptible to infection as soon as maternal antibodies declined to hi titers of : - : . a maternal hi antibody titer as low as : severely affected the efficacy of a live low titer cpv vaccine ( - th passage in culture) in generating active immune responses (carmichael et al., ) , which resulted in a window of susceptibility for pups. others have shown that an hi titer of less than : in cpv- (low passage, high titer) vaccinated pups did not severely affect active antibody responses (burtonboy et al., ; hoare et al., ) , suggesting that increasing the dose or reducing the attenuation of the virus may help to overcome inhibitory effects of maternal antibodies. studies with modified live variant cpv- b strain (low titer) have shown that these vaccines, when given either parenterally ( th passage) or in ( th tissue culture passage), elicited almost % protection in pups with maternal antibody titers of : to : and even % protection in pups with antibody titers of : . higher efficacy of these vaccines can be attributed to either strong inherent immunogenicity of these new vaccines or antigenic differences among cpv- and cpv- a and cpv- b (pratelli et al., ) . overall, during the past four decades of cpv vaccine development, modified live viruses have proven superior to inactivated intramuscular (im) vaccines (appel, ) . in brief, various strategies such as the use of less attenuated virus (low serial passage), high titer vaccines, multiple doses, or the use of the in route of immunization have been attempted to overcome inhibitory influences of maternal antibodies and to reduce the window of susceptibility in pups. recently new variant cpv- c has emerged, which is highly pathogenic and causes more severe diarrhea in dogs. currently used vaccines (cpv- or cpv- b strains) are effective in protecting dogs against challenge with cpv- c virus under experimental conditions (spibey et al., ) , although efficacy in the field is unknown (reviewed by decaro and buonavoglia ( ) ). antigenic differences between original cpv- and its variants may reduce the efficacy of current cpv vaccines, which is supported by in vitro virus-neutralization tests conducted on vaccinated animals that showed low cross-reactivity between heterologous cpv variants (cavalli et al., ) . however, this may not represent actual cross-protection in clinical cases. there is a need not only to make current vaccines effective in the presence of maternal antibodies, but also to update them based on continuous epidemiological surveillance studies. dna plasmids expressing vp (jiang et al., ) , replicon-based cpv dna vaccine expressing vp (dahiya et al., ) , b cell epitope ( l peptide of vp ) fused to ct b subunits expressed in transgenic tobacco chloroplasts (molina et al., ) , chimeric virus particles expressing cpv peptide (different prime/boost strategies) (nicholas et al., ) , and recombinant vlps formed by baculovirusexpressed vp (casal, ) have been evaluated in dogs or rodents without maternal antibodies and have demonstrated good immunogenicity and/or protective efficacy. further efficacy tests in pups with maternal antibodies are needed to assess their commercial potential. oral vaccines for the induction of active immunity against bacterial diarrhea are not commonly used in livestock, although e. coli diarrhea is an important problem in neonatal and postweaning calves and pigs. f (k ) and f are the major fimbrial adhesins present on swine enterotoxigenic e. coli (etec) and are major targets for e. coli vaccines (fairbrother et al., ) . whole bacteria vaccines are routinely administered parenterally to pregnant cattle, sheep, and swine to protect their suckling neonates against etec infections (moon and bunn, ) . such vaccines are practical and effective because: ( ) fimbriae are required for the adhesion-colonization of bacteria early in the pathogenesis of the disease; ( ) most fatal etec infections in farm animals occur in the neonatal period; ( ) more than % of the etec in farm animals belong to a small family of fimbrial antigen types, ( ) and mothers are seropositive to etec so booster responses are elicited. recent vaccine studies have focused on administration of purified bacterial subunits (transgenically expressed adhesin of f fimbriae in plants) and mucosal adjuvants (verdonck et al., ; joensuu et al., ) and have shown promising results. overall, the vaccine strategy used, parenteral vaccination of field-exposed seropositive mothers, to induce lactogenic immunity is the same as that shown to be effective for parenteral application of rv vaccines in rv seropositive mothers (bohl et al., ; saif and jackwood, ; saif and fernandez, ) . studies of live oral enteric vaccines in animals have clarified the mechanisms of induction of protective immunity against enteric disease and contributed to our understanding of the common mucosal immune system. however, commercial live oral vaccines often have shown inadequate or inconsistent efficacy under field conditions (saif and jackwood, ; saif and fernandez, ) . major obstacles to improved efficacy of oral vaccines include: ( ) maternal antibodies in the intestine of neonates (mainly colostrum and milk antibodies), which interfere with live vaccine replication; ( ) qualitative and quantitative limitations in the neonatal immune system, although neonates are immunocompetent; ( ) the inability of attenuated vaccine strains to adequately infect (too high attenuation) or stimulate s-iga antibodies in the intestine; ( ) the use of inappropriate (or unstable) antigens or route for subunit vaccines; ( ) the lack of oral delivery vehicles or mucosal adjuvants for subunit vaccines; and ( ) infection by pathogens prior to vaccination. studies of neonatal pigs indicate that protection rates against rv diarrhea upon challenge correlate with the magnitude of iga asc and memory b cell responses in intestinal lymphoid tissues gonzalez et al., ; yuan and saif, ) . studies conducted in immunodeficient or specific gene knockout adult mice have shown that neither cd + or cd + t cells nor antibodies were essential for induction of protective immunity to rv infection, but usually one of these effectors (t or b cells) was necessary for elimination of primary rv infection (mcneal et al., (mcneal et al., , . in pigs, recent studies have shown that cd + and cd + ifnγ producing t cells play a role in protection against rv diarrhea and infection (yuan et al., ) , but it is difficult to create genetically modified pigs similar to knockout mice, to assess the contribution of b cells and t cells. the redundant nature of immune responses to rv in mice, the multiple immunologic and possibly nonimmunologic pathways to resolve rv infections (franco and greenberg, ; ward, ) , the age factor and host differences in rv pathogenesis in mice and pigs (saif et al., , ward, ) , and the use of inbred mouse strains contribute to the discrepancies seen between the adult mouse and the neonatal pig models. the majority of pathogens enter and initiate infection at mucosal surfaces, making mucosal sites relevant targets for vaccines to prevent infection. to develop effective mucosal vaccines, it is important to determine correlates of protection against enteric pathogens. generally for localized gut infections, s-iga antibodies and intestinal t cells play an important role. improved vaccines that induce high levels of intestinal iga antibodies against the appropriate microbial antigens can be achieved by choosing the proper vaccine formulation and delivery method. vaccines should also induce: ( ) heterotypic protection; ( ) active immunity in the presence of maternal antibodies; and, ( ) long-lasting immunological memory. novel vaccines (e.g., vlps, transgenic plants), adjuvants (e.g., mlt, iscoms, tlr ligands (e.g., cpg), mycobacterial extracts, α -dihydroxyvitamin d , retinoic acid, probiotics, and cytokines), and vaccine delivery systems (e.g., recombinant plant or animal viruses or bacterial vectors, genetically engineered probiotics, iscoms, liposomes, and nanoparticles) should be explored and evaluated in relevant animal models to further enhance the efficacy of current or new vaccines. recent studies have shown that the targeting of antigens directly to apcs (via surface receptors) is an effective way to tailor immune responses to optimize protection and should be explored further in large animals (trumpfheller et al., ) . the passive transfer of maternal immunity provides essential protection in newborns. although most neonatal immune systems are competent to mount primary immune responses against pathogens, primary responses often do not develop quickly enough to prevent disease. maternal immunologic transfer provides a critical (though temporary) aid to survival for neonates. the enhancement of passive immunity through vaccination of the mother has been a successful disease prevention strategy in domesticated animals. vaccinated mothers develop higher levels of specific antibodies in colostrum and milk and increased levels of immunity in their offspring (glezen, ; saif and fernandez, ) . passive immunity can also be enhanced by oral administration of immune milk or heterologous antibody preparations (e.g., chicken egg yolk igy (ikemori et al., ; kuroki et al., ) or monoclonal antibodies) or by parenteral administration of hyperimmune plasma (becu et al., ) . recent studies using monoclonal, single-chain antibodies (variable heavy domain (vhh) nanoantibodies) of llama origin open new possibilities for providing passive immunity to humans and animals (garaicoechea et al., ) . immunoglobulins of the igg and igg isotypes of camelids consist of heavy chains without associated light chains (hamers-casterman et al., ) . the distinctive biochemical characteristics and binding qualities of vhh antibodies overcome some key limitations of conventional antibodies (see below). unfortunately, passive antibodies often interfere with active immunization of young animals and birds. various vaccination strategies have been developed to minimize the suppressive effects of maternal antibodies, but improved adjuvants and antigen-delivery systems are needed to facilitate efficient induction of active immunity in the presence of maternal antibodies. this section will address past, current, and future approaches for enhancing passive immunity in veterinary species. the transfer of systemic passive immunity from mother to offspring can occur prenatally, via the placenta or yolk sac, or postnasally via ingestion of colostrum and milk, depending on the species. the main ig isotype transferred in most mammalian species is igg. in avian species igy, the functional equivalent of mammalian igg, is transferred to the yolk to passively protect the developing chick (kovacs-nolan and mine, ) . in primates and rabbits, maternal igg is transferred across the placenta to the fetus. in rodents, transplacental transfer occurs in combination with prolonged ( - days) postnatal transfer by means of colostrum and milk (husband, ) . in dogs and cats transfer of igg occurs by a combination of prenatal and postnatal mechanisms, with % to % of total transfer occurring before birth (tizard, ) . in ruminants, horses, and pigs, offspring are born virtually agammaglobulinemic and transmission of ig occurs only via colostrum for a limited time after birth (tizard, ) . after the transition from production of colostrum to milk, ig are no longer absorbed from the intestines and only act locally in the gastrointestinal tract. immunoglobulin absorption in neonates of large domestic species is facilitated by the presence of protease inhibitors in colostrum and its efficiency declines rapidly after birth, with maximal absorption occurring in the first h. the cessation of absorption of intact macromolecules is termed "gut closure," and occurs at different ages in different species. in calves and pigs closure normally occurs by - h after birth. absorption of colostrum ig can be highly effective, supplying the newborn with serum ig at concentrations similar to those of the dam. failure of passive transfer (fpt) is a common problem, however, in newborn calves and foals (besser and gay, ; tyler-mcgowan et al., ) . fpt may occur because of the production of low quantities of colostrum, because of production of colostrum with low concentrations of maternal antibodies, because of ingestion of low quantities of colostrum, or because of inefficient absorption (quigley and drewry, ) . colostrum supplements, colostrum replacers, and plasma products have been developed commercially to address this problem, with variable success. vaccination of the dam in late pregnancy can enhance antibody titers in colostrum and after suckling in the serum of the offspring (saif and fernandez, ) . the benefits of vaccination of the dam for enhancing passive immunity can be lost if colostrum is of low quality or if absorption of colostral ig is inefficient. the half-life of ig varies considerably among species of domestic animals and with the ig isotype. neonatal receptor for fc of igg (fcrn) is involved in homeostasis of serum levels of igg in general, but distinct mechanisms may function in neonates. the main route of clearance of passively acquired maternal igg in calves is transfer from the serum to the intestine (besser et al., ) . approximately % of passively acquired igg is eliminated by this route. if titers of passive circulating antibodies are high enough, the transfer of antibodies from the circulation to the intestinal lumen can mediate short-term protection against rotavirus diarrhea (besser et al., ) . the same mechanism may be functional in piglets (saif and wesley, ; parreño et al., ; ward et al., a) . the persistence of titers of circulating maternal antibodies is generally considered in designing vaccination strategies for young animals because of suppressive effects of maternal antibodies on active immune responses. induction of immune memory can occur in the absence of a detectable serum antibody response (boersema et al., ; parreño et al., ) . the presence of passive antibodies in the intestines can interfere with mucosal immune responses to natural infection as well as to vaccination parreño et al., ; nguyen et al., a,b) . experiments in colostrum-deprived lambs (jones et al., ) and calves (mosier et al., ) have demonstrated the ability of parenterally administered immune antisera to mediate protection following experimental challenge with mannheimia haemolytica. parenteral administration of hyperimmune plasma raised against rhodococcus equi has been shown to protect against pneumonia in young foals in experimental (hooper-mcgrevy et al., ) and field studies (becu et al., ) . hyperimmune plasma is available commercially for use in foals. prepartum vaccination of beef (van donkersgoed et al., ) and dairy (hodgins and shewen, ) cows has been demonstrated to increase antibody titers to m. haemolytica in their colostrum and in the serum of their calves. vaccination of broiler breeder chickens can be used to provide passive protection against respiratory/septicemic disease caused by avian pathogenic e. coli (kariyawasam et al., ) . rodents have been a popular model for the study of passive protection by milk antibodies. however, rats and mice actively transport igg from the gut into the circulation during the first - weeks of life. thus, antibodies in ingested milk contribute to both local and systemic immunity in rodents, in contrast to the strictly local effects occurring in humans and most domestic animals. in pigs, horses, dogs, and cats, igg is the most abundant ig in colostrum but iga predominates in milk. parenteral vaccination, by enhancing serum igg antibody titers, contributes to igg antibodies in colostrum but has limited effects on iga antibodies in milk. milk antibodies provide passive protection to the neonatal intestinal tract by immune exclusion preventing the attachment of viruses, bacteria, and parasites and by neutralizing viruses or enterotoxins. s-iga antibodies, because of their resistance to cleavage by digestive enzymes, and higher levels in milk are more efficient in mediating protection in the gut of pigs and other monogastrics (saif and jackwood, ) , but high persisting levels of passive igg antibodies are also protective. in ruminants igg antibodies, relatively resistant to proteolytic enzymes (brock et al., ) and predominant in milk, have functions similar to those of s-iga. numerous vaccines are marketed for vaccination of cows and sows to provide lactogenic immunity to rotavirus, coronavirus, and e. coli in suckling offspring. vaccine efficacy has been variable. ideally, suckling animals become subclinically infected with enteric pathogens while receiving adequate passive antibodies to prevent disease, and develop active immunity to prevent subsequent diarrhea. this balance between passive immunity and disease can be disrupted in intensive animal production systems by exposing animals to pathogens in confined, contaminated environments. earlier weaning practices reduce intake of milk antibodies. maternal enteric vaccines are commonly used in two populations of pregnant animals. to control epidemic infections, they are used in naïve, seronegative animals to induce primary immune responses. to control endemic infections (such as rotavirus and e. coli), booster vaccines are used in seropositive, field-exposed animals to stimulate anamnestic memory responses. virulent tgev given to pregnant sows stimulates high levels of iga antibodies in milk and passive protection (saif and jackwood, ) . oral attenuated tgev vaccines, which replicate poorly in sows, induce lower iga milk antibody titers and low or variable efficacy in the field (moxley and olson, ) . parenteral killed tgev vaccines induce only low igg milk antibody titers and have the lowest protection rates. attempts to develop maternal tgev recombinant subunit vaccines based on the surface tgev spike (s) protein that induces na, or live vector vaccines expressing the s protein, have also been of limited success in tgev seronegative swine (saif and wesley, ) . however, prime/boost strategies such as im administration of tgev s protein following oral/in priming with attenuated tgev have shown promise as a means of enhancing iga milk antibody titers (park et al., ) . booster vaccination strategies are required to enhance lactogenic immunity to endemic enteric pathogens, such as rotavirus and e. coli, because antibody titers in milk decline dramatically during lactation. breast milk iga antibodies are increased in women endemically exposed to cholera following parenteral boosting with a cholera vaccine (svennerholm et al., ) . in pigs infected with rotavirus, iga memory b cells initially reside in the ileal pps but are subsequently present in substantial numbers in spleen (yuan et al., ) . systemic stimulation of such iga memory b cells by parenteral booster vaccines can yield dimeric iga antibodies in serum for transport to mucosal secretions via the polymeric ig receptor. under field conditions, antibodies to endemic intestinal pathogens are also common in bovine colostrum and milk, but without the boosting effect of highly immunogenic vaccines, antibody titers are often too low to protect calves (besser and gay, ; saif and fernandez, ) . thus vaccines are marketed for prepartum vaccination of cows against rotavirus, coronavirus, and e. coli to enhance passive immunity in their calves, but the field efficacy of these vaccines has been questioned (waltner-toews et al., ) . vaccination of pregnant dairy cows with modified live or binary ethyleneamine inactivated rotavirus or recombinant / / / vlps has been shown to increase igg and virus na titers to rotavirus in colostrum and milk and mediate passive protection in calves against oral rotavirus challenge (saif and fernandez, ; kim et al., ) . prepartum vaccination of cows and sows with bacterins prepared from enteropathogenic (epec) e. coli for prevention of diarrhea in their offspring is also commonly practiced. under modern farming practices, dairy and veal calves rarely are fed whole milk from their dams for more than or days. thus vaccine efficacy is based on antibodies absorbed from colostrum or retained temporarily in the gut, rather than on a continuing supply of immune milk. piglets, in contrast, continue to receive immune milk until weaning at - weeks of age. the importance of a continuous supply of passive antibodies for protection against tgev has been demonstrated (saif and wesley, ) . numerous commercial ig preparations with antibody activity against specific enteric pathogens have been marketed. products intended for prevention of e. coli enteritis in calves include dried bovine colostrum and whey, hyperimmune sera raised in horses, and mouse monoclonal antibodies to the k (f ) antigen of e. coli. these products are administered orally in the first h of life to prevent adhesion of epec e. coli. orally administered bovine colostral whey containing rotavirus antibodies also passively protects piglets against rotavirus (schaller et al., ) . immunization of chickens shows promise as an efficient method for producing polyclonal antibodies for passive protection. specific antibodies of the igy isotype are induced by vaccination and are concentrated in egg yolk. laying hens can produce about g of igy per year. yolk antibodies with virus na provide calves with partial protection against diarrhea caused by rotavirus (kuroki et al., ; vega et al., ) , and etec e. coli (ikemori et al., ) . in a recent study, supplementation of the milk ration of neonatal calves with egg yolk containing igy antibodies to rotavirus for days provided % protection against rotavirus diarrhea after challenge, and also enhanced mucosal asc numbers in the duodenum (vega et al., ) . egg yolk lacking rotavirus specific igy did not provide clinical protection, but did enhance asc responses, suggesting the presence of immune modulators in egg yolk. protective effects of yolk antibodies are dependent on antibody titers in oral preparations (marquardt, ) . development of better means to protect yolk antibodies from digestive processes will improve both the efficacy and the economic viability of yolk antibodies for clinical applications (kovacs-nolan and mine, ). for many diseases of newborns and neonates, passive immunity is the only practical means of providing timely protection. unfortunately, it is well documented that maternal antibodies can suppress active immune responses following vaccination. this effect has been observed with both live and nonreplicating vaccines, and for both systemic and mucosal responses (siegrist et al., ; hodgins et al., ; parreño et al., ; nguyen et al., a,b) . antibody responses especially are affected; t-lymphocyte responses may not be suppressed (siegrist et al., ) . titers of maternal antibodies are maximal for most species of interest in the first week of life and then decline gradually over the next few months, but variability of titers among individuals is high. with many vaccines, a "window of disease susceptibility" of variable duration occurs when titers of maternal antibodies are too low to mediate protection, but are too high to permit effective vaccination. a number of strategies are used to cope with this problem. some veterinary vaccines for cattle are sold with the disclaimer that "animals vaccinated before months of age should receive a booster dose of vaccine at months of age." this provides little solace for the many diseases of cattle occurring in the first weeks or months of life. a common strategy for vaccines of dogs and cats is to administer a series of doses of vaccine from an early age (at which time only a few individuals will be responsive) and to continue vaccinating until an age at which virtually all can respond to vaccination. this strategy has economic disadvantages for the pet owner. some manufacturers produce low passage, high virus titer vaccines especially for use in situations where high titers of maternal antibodies and high pathogen exposure are anticipated. this is similar to a strategy once (but no longer) approved by the world health organization for vaccination of children in developing countries against measles (gellin and katz, ) . preliminary evidence suggests that incorporation of vaccine antigens in highly structured iscoms or in application of vaccines can enhance immune responses in the presence of maternal antibodies (van binnendijk et al., ; brockmeier et al., ) . immunoglobulin g in most mammalian species is composed of two heavy chains, covalently linked by disulfide bonds, and two light chains. "conventional" heavy chains consist of one variable domain and three constant domains (ch , ch , and ch ); light chains are composed of a variable and constant domain. in contrast camelid species produce "heavy chain immunoglobulins" that lack light chains and the first constant heavy domain (hamers-casterman et al., ) . the antigen-binding site of these unusual heavy chain antibodies is formed by a single domain, designated vhh in camelids. vhh are easily produced as recombinant proteins, designated single domain nanoantibodies or nanobodies ® and represent the smallest molecule in nature capable of binding a specific antigen. the cdr region of these nanobodies has the capacity to form long loops that can extend into cavities on antigens, e.g., the active site crevice of enzymes. other advantageous features of nanobodies include their high solubility, thermal stability (resist pasteurization), refolding capacity, and optimal tissue penetration in vivo (reviewed by vanlandschoot et al. ( ) ). nanobodies have demonstrated efficacy as agents of passive immunity for infectious diseases. vhh specific for rotavirus inner capsid protein vp are able to broadly neutralize rotaviruses independently of serotype, and in mouse experiments provide passive protection against challenge with human rotavirus (pant et al., ; garaicoechea et al., ) . nanobodies against other viral diseases with veterinary impact have been developed (foot-and-mouth disease, influenza, rabies (vanlandschoot et al., ) ) and represent a promising next-generation biologic platform for passive immunity. maternal vaccination to enhance passive immunity is already widely used in veterinary medicine. some of these vaccines, especially vaccines against enteric viruses, have limited efficacy. new approaches to enhance immunogenicity are promising, but await commercial development. a clearer understanding of protective mechanisms and immune modulation mediated by passive antibodies would contribute to more effective interventions. there is an urgent need for adjuvants and delivery systems capable of reliably inducing active immunity in neonates despite the presence of maternal antibodies. the ability to provide continuity of immune protection from birth, by combining passive immunity with active immunization, would have a major impact on neonatal morbidity and mortality. the rapid expansion of commercial fish farming (aquaculture) in many countries in recent decades has been accompanied by an urgent need to develop vaccines to prevent (infectious) fish diseases that were previously unknown or obscure. added to the difficulties involved in identifying the pathogens responsible, there has been the challenge of delivering vaccine efficiently with minimal stress to very large numbers of fish. some commercial vaccines for fish are administered by intraperitoneal or im injection, but mucosal vaccines are also widely used (reviewed by brudeseth et al. ( ) ). fish are routinely vaccinated by immersion in tanks of diluted bacterin, modified live bacteria or viruses, with exposure times ranging from s up to min, depending on the vaccine and age of the fish. it is unclear whether the main route of antigen uptake is oral or via the mucosal surface of the gills. several commercial vaccines are now available that consist of recombinant viral proteins for mixing into the feed (evensen and leong, ) ; days of feeding is recommended by the manufacturer. research on mucosal veterinary vaccines has contributed new concepts to the field of mucosal immunity. investigations of pathogen-host interactions in outbred animals have illustrated the complexity of these interactions. early studies of an enteric coronavirus infection of swine (tgev) led to the concept of the gut-mammary gland s-iga immunologic axis and provided part of the basic tenet for a common mucosal immune system. later studies of tgev and a deletion mutant of tgev with respiratory tropism (prcv) revealed functional compartmentalization within the common mucosal immune system whereby in inoculation of pigs with prcv failed to elicit sufficient intestinal iga antibody to fully protect against the enteric pathogen tgev (vancott et al., (vancott et al., , . subsequent studies have explored new prime/boost mucosal immunization strategies to elicit intestinal immunity to rotavirus in naïve pigs (saif, b; yuan and saif, ; gonzalez et al., ) . in these studies only oral priming with attenuated virus led to successful in booster responses using nonreplicating (vlp) vaccines combined with mucosal adjuvants such as iscom or mlt. thus use of a replicating vaccine to prime lymphocytes at a major mucosal inductive site (galt) followed by boosting with a nonreplicating vaccine at a second inductive site (nalt) effectively stimulated intestinal iga antibodies and induced active protection against rotavirus diarrhea. although there is progress in developing safe and effective nonreplicating vaccines to boost mucosal immune responses (saif and fernandez, ) , the dilemma remains to develop effective vaccines to prime for mucosal immunity. mucosal adjuvants (mlt, iscom, cpg, cytokines) and new delivery systems (replicating vectors, microparticles) have shown promise in animal studies reviewed in this chapter. however, their economical production and final evaluation under field conditions, including in the presence of maternal antibodies as relevant, are needed. considerable research effort has been devoted to development of vaccines for respiratory diseases of domestic animals. in some instances attenuated organisms delivered by mucosal routes have demonstrated improved efficacy over nonreplicating antigens given by systemic routes. for many respiratory diseases, however, further progress in the development of mucosal vaccines will have to await advances in understanding of disease pathogenesis and identification of protective antigens. in contrast, studies of ascending infections of the reproductive tract in cattle have demonstrated the efficacy of systemic vaccination to clear established infections, and highlight the possibility of therapeutic vaccines. finally it is important to realize that there are considerable species differences in mucosal immunity. for example, the primary ig in mammary secretions of ruminants is igg which is actively transported to the mammary gland from serum and provides effective passive immunity to the nursing offspring against enteric pathogens. thus parenteral immunization of the dam stimulates passive immunity in ruminants against enteric pathogens. in contrast, in monogastrics, iga predominates in milk and iga lymphoblasts that traffic to the mammary gland originate in the intestine. therefore oral vaccines in monogastrics may provide a more effective vaccine strategy to induce iga antibodies in milk (saif and fernandez, ) . by applying the aforementioned vaccine concepts with new and effective mucosal 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acidophilus enhances the immunogenicity of an oral rotavirus vaccine in gnotobiotic pigs lactic acid bacterial colonization and human rotavirus infection influence distribution and frequencies of monocytes/macrophages and dendritic cells in neonatal gnotobiotic pigs key: cord- -a yoz w authors: zhao, xiaomin; song, xiangjun; bai, xiaoyuan; fei, naijiao; huang, yong; zhao, zhimin; du, qian; zhang, hongling; zhang, liang; tong, dewen title: mir- b attenuates apoptosis induced by transmissible gastroenteritis virus (tgev) infection via targeting runt-related transcription factor (runx ) date: - - journal: peerj doi: . /peerj. sha: doc_id: cord_uid: a yoz w transmissible gastroenteritis virus (tgev), belonging to the coronaviridae family, is the key cause of the fatal diarrhea of piglets and results in many pathological processes. micrornas (mirnas) play a key role in the regulation of virus-induced apoptosis. during the process of apoptosis induced by tgev infection in pk- cells, the mir- b is notably down-regulated. thus, we speculate that mir- b is involved in regulating the process of apoptosis in pk- cells. in this study we demonstrated that the over-expression of mir- b led to the inhibition of tgev-induced apoptosis, reduction of bax protein level, and decrease of caspase- and − activities. conversely, silencing of mir- b by mir- b inhibitors enhanced apoptosis via up-regulating bax expression and promoting the activities of caspase- and − in tgev-infected cells. subsequently, the runt-related transcription factor (runx ) is a candidate target of mir- b predicted by bioinformatics search. we further identified that the mir- b directly bound to the ′ utr of runx mrna and suppressed runx expression, which indicates runx is the direct target gene of mir- b. the over-expression of runx increased apoptosis and knockdown runx blocked apoptosis of viral-infected cells via regulating bax expression and the activities of caspase- and − . our data reveal that mir- b may repress the mitochondrial pathway of apoptosis by targeting runx , indicating that tgev may induce apoptosis via down-regulating mir- b and that mir- b may act as a target for therapeutic intervention. tgev, a member of coronaviridae family, is an enveloped virus with a positive-sense single-stranded rna genome (weiss & leibowitz, ) . tgev infection primarily causes transmissible gastroenteritis (tge) that is characterized by highly contagious and fatal gastroenteritis for pigs of all ages, especially for piglets under weeks old (chae et al., ; kim & chae, ) . apoptosis is a process of self-destruction in response to a variety of stimuli such as viral infection. infection of coronavirus such as porcine epidemic diarrhea virus (pedv) (kim & lee, ) , infectious bronchitis virus (ibv) (li, tam & liu, ) , severe acute respiratory syndrome coronavirus (sars-cov) (krahling et al., ) , may result in host cell apoptosis. we have reported that tgev infection induced apoptosis via mitochondria mediated apoptotic pathway in pk- cells (ding et al., ; ding et al., ) . mirnas are a class of small non-coding rna molecules and may regulate gene expression at post-transcription level (bartel, ) . numerous experimental studies have demonstrated that mirnas play important roles in cell apoptosis (wilson & doudna, ) . mir- b effectively inhibits apoptosis via directly targeting caspase- and nuclear apoptosis inducing factor (naif ) in madin-darby bovine kidney (mdbk) cells infected with bovine viral diarrhea virus (bvdv) (fu et al., ) . mir- b promotes doxorubicin-induced apoptosis in hepatoblastoma cell line (hepg ) (mu et al., ) . moreover, mir- b is an endogenous inhibitory factor of apoptotic peptidase activating factor (apaf- ) expression and decreases the apoptotic rate of neurons (chen et al., ) . mirnas are also involved in regulating the process of virus-induced apoptosis (smith et al., ; zhang et al., ) . hs_ promotes wnv-mediated apoptosis via inhibiting the expression of ccctc-binding factor (ctcf) and the epidermal growth factor receptor (egfr) (smith et al., ) . our previous studies showed that mir- b was significantly down-regulated during tgev-induced apoptosis (song et al., ) . thus, we supposed that mir- b may play a role in apoptosis induced by tgev infection. in the present study, we investigated the role of mir- b in tgev-induced apoptosis in pk- cells and demonstrated that the mir- b attenuated tgev-induced apoptosis by targeting runx , suggesting tgev may use mir- b to regulate apoptosis in pk- cells. monoclonal runx (mab ) was purchased from r& d systems (r& d systems, minneapolis, mn, usa). monoclonal antibodies against bax (sc- ) and β-actin (sc- ) were purchased from santa cruz biotechnology (santa cruz, inc., santa cruz, ca, usa). horseradish peroxidase (hrp)-conjugated secondary antibody was purchased from pierce (pierce, rockford, il, us). pk- cells were obtained from atcc (ccl- ) and cultured in dulbecco's minimal essential medium (dmem) supplemented with % fetal bovine serum (gibco brl, gaithersburg, md, usa), iu of penicillin, and mg of streptomycin per ml, at • c in a % co atmosphere incubator. the tgev shaanxi strain was isolated from tgev-infected piglets by ding et al. ( ) . total rna was extracted using trizol reagent (invitrogen, carlsbad, ca, usa) from pk- cells reverse transcription reactions and real-time pcr were performed as described previously (song et al., ) . the relative quantification of mirnas was normalized to u using the two-ddct method (livak & schmittgen, ) . annexin v-fitc apoptosis kit (invitrogen, carlsbad, ca, usa) was used to detect apoptosis according to the manufacturer's protocol. briefly, cells were washed twice with ice-cold pbs and resuspended in µl ×annexin v binding buffer followed by adding µl annexin v-fitc and µl pi. after incubation for min at room temperature, analysis was done by flow cytometry (beckman coulter, ca, usa). caspases activities were measured by colorimetric assay kits (keygen biotech, nanjing, china) following the manufacture's protocol. briefly, protein concentrations were measured by bca protein assay reagent (vazyme biotech nanjing, china). then mg protein of each sample was incubated with each caspase substrate at • c in a microplate for h. absorbance at the wavelength of nm was read in microplate spectrophotometer (infinite pro nanoquant; tecan, switzerland). the utr sequence of runx mrna containing the target sites of mir- b was amplified by pcr using the primers of table s . the utr of runx mrna was cloned into psicheck- (promega, madison, wi, usa) between xho i and not i cloning sites to obtain the wild type plasmid runx -wt. the binding sequences of mir- b seed region in utr of runx -wt were mutated following a mutagenesis protocol (heckman & pease, ) to generate runx -mut. mir- b mimics, mirna mimics control, mir- b inhibitors and mirna inhibitors control were synthesized by ribo biotech (ribobio, guangzhou, china). mir- b inhibitors were modified with -o-methyl. the sequences of mirna in this study were shown in table s . for the luciferase reporter assay, pk- cells were grown in -well plates and then co-transfected with plasmid runx -wt (or runx -mut) and mir- b mimics (or mir- b inhibitors) using lipofectamine (invitrogen, carlsbad, ca, usa). the luciferase activities were detected at h post transfection (hpt) using a dual-glo r luciferase assay system (promega, madison, wi, usa) following the manufacturer's manual. three sirnas (sirunx - , sirunx - , sirunx - ) of runx were synthesized by genepharma (genepharma, shanghai, china). the most effective sirna (si-runx - ) was applied for the further experiments. pk- cells were transfected with nm runx specific sirunx - (table s ) or irrelevant sirna using lipofectamine (invitrogen, carlsbad, ca, us). cell viability was detected using cell counting kit- (cck- ) (vazyme biotech nanjing, china). briefly, cells were seeded in -well culture plate and cultured at • c in a humidified atmosphere with % co . twelve hours later, cells were transfected with nm sirunx - or irrelevant sirnas. the cell viability was tested following the manufacturer's manual at hpt. to construct the runx expression plasmid, the full-length runx gene was amplified by pcr and was cloned into plasmid pci-neo (promega, madison, wi, usa) between ecor i and not i cloning sites to gain pci-neo-runx . the primer sequences of pcr are shown in table s . pk- cells were transfected with pci-neo (control) or pci-neo-runx . cells were infected with tgev at multiplicity of infection (moi) of at h post infection (hpi). the total rna was extracted using trizol reagent (invitrogen, carlsbad, ca, usa) according to manufacturer's instructions. a total of µg of rna was transcribed into cdna using the first-strand cdna synthesis kit (invitrogen, carlsbad, ca, us). real-time pcr was carried out using the accupower r × greenstar qpcr master mix (bioneer, daejeon, korea) on bio-rad iq real-time pcr system. the relative fold changes were calculated using the -ddct method. the real-time pcr primers are shown in table s . cells were treated with radioimmunoprecipitation assay (ripa) lysis buffer containing phenylmethyl sulfonylfluoride (pmsf). protein concentration was tested with bca protein assay reagent (pierce, rockford, il, usa). the proteins were separated on a % sodium dodecyl sulfate-polyacrylamide (sds-page) gel and subsequently transferred to a polyvinylidene difluoride (pvdf) membrane (millipore corp, atlanta, ga, usa). the membrane was blocked with % non-fat dry milk for h at room temperature and then incubated with the primary antibodies overnight at • c. the hrp-conjugated secondary antibodies were used to incubate for h at room temperature. the blotting was visualized using enhanced chemiluminescence (ecl) reagent. all data are representative of results from at least separate experiments. all data points are the average of triplicates, with error bars representing standard deviations. the difference between two groups was statistically analyzed by a two-tailed student's t test using spss software. p value < . was considered significant. to investigate whether mir- b is involved in the apoptosis induced by tgev, the cells were transfected with mir- b mimics or mir- b inhibitors and infected with tgev at a moi of at hpt. the apoptotic rate was analyzed by flow cytometry at and hpi. the results showed that the over-expression of mir- b led to a decrease of apoptotic rate and the down-expression of mir- b increased apoptotic rate at and hpi (fig. a) , indicating that mir- b attenuated apoptosis induced by tgev infection in pk- cells. we previously found that tgev infection could cause pk- cell apoptosis through mitochondria-mediated pathway and fasl-mediated apoptotic pathway ( ding et al., ) . therefore, we analyzed caspase- activity of mitochondria-mediated pathway and caspase- activity of fasl-mediated apoptotic pathway. however, mir- b did not affect the caspase- activity during tgev infection (data not shown). as expected, the caspase- activity was decreased by mir- b mimics and up-regulated by mir- b inhibitors in the tgev-infected pk- cells (fig. b) . moreover, we investigated the effect of mir- b on caspases- activity in tgev-infected pk- cells. the caspase- activity was decreased by mir- b mimics and increased by mir- b inhibitors in the tgev-infected cells compared with control (fig. c) . taken together, these results suggest that mir- b plays a negative role in the tgev-induced apoptosis in pk- cells via mitochondria-mediated pathway. to identify the targets that affect tgev-induced apoptosis, we predicted the targets of mir- b using targetscan and miranda and found that runx was a potential target of mir- b (song et al., ) . moreover, runx was reported to regulate apoptosis (wu et al., ) . thus, we investigated the direct interaction between mir- b and utr of runx mrna through dual-luciferase assay. the sequence of the utr of runx was obtained by pcr and the two binding sites of mir- b seed region in the utr of runx were mutated with a -bp substitution. the utr of runx and its mutational version were respectively sub-cloned into the utr of the renilla luciferase dual-luciferase plasmid psicheck- ( figs. a and b ). the constructs were co-transfected into pk- cells with mir- b mimics (or negative control) or mir- b inhibitors (or negative control). compared with the mirna mimics control, introduction of mir- b mimics decreased the runx -wt reporter activity (fig. c) . to determine whether mir- b inhibits runx expression, mir- b mimics or mirna mimics control were transfected into pk- cells. runx expression was assessed by western blot. the result showed that mir- b decreased expression of runx in pk- cells compared with the control (fig. d ). together, our data conclusively demonstrate that runx is a direct target of mir- b in pk- cells. runx shows transactivate effect on bax expression (wu et al., ) .we previously found that tgev infection could cause pk- cell apoptosis through mitochondria-mediated pathway (ding et al., ) . therefore, we proposed a hypothesis that mir- b could restrain tgev-induced apoptosis of pk- through mitochondria-mediated pathway by decreasing expression of bax. in order to confirm this hypothesis, we analyzed the effect of mir- b on bax. at hpt of mir- b mimics or mir- b inhibitors, the cells were infected with tgev at moi. the mrna and protein levels of bax were analyzed by real-time pcr and western blotting at and hpi. over-expression of mir- b resulted in a decrease of bax at both mrna and protein levels, down-expression of mir- b increased the mrna and protein expression of bax (figs. a and b ). taken together, these results indicate that mir- b attenuates tgev-induced apoptosis by suppressing bax. to investigate the effect of runx on apoptosis, we silenced and over-expressed runx respectively using sirunx and pci-neo-runx . the runx mrna and protein levels were remarkably reduced by sirunx - (figs. a and b); however, the cell viability was for each schematic, the upper sequence is the binding site of mir- b in utrs of swine runx , the middle sequence is the mature mir- b, and the lower sequence is the mutated sequence of utr. the seed sequence is underlined. (b) schematic drawing of the putative binding sites or mutations of mir- b binding-sites in utr of runx mrna. the locations of the potential binding sites or their mutations are presented by blank boxes. (c) the runx luciferase reporter construct was co-transfected with mir- b mimics (or negative control) or mir- b inhibitors (or negative control) into pk- cells (normalized to the firefly luciferase activity). data are expressed as relative luciferase activities to control. (d) western blot analysis of runx in cells transfected with mir- b mimics or mimics control. data represent means ± s.d. of three independent experiments. * p < . in comparison with the control. * * p < . in comparison with the control. not affected in comparison with irrelevant sirna (fig. c) , indicating that the runx gene was knocked down in pk- cells by the sirunx - . in addition, runx was notably over-expressed using pci-neo-runx (fig. d) . to determine the effect of runx on bax, the runx were silenced with sirunx - and over-expressed by pci-neo-runx followed by infection with tgev. the bax expression was decreased by knockdown of runx and up-regulated by over-expression of runx (fig. e) . the activities of caspase- and - were respectively reduced and increased by silence and over-expression of runx (figs. f and g) . therefore, runx promoted tgev-induced apoptosis of pk- cells. taken together, our findings suggest that mir- b shows a negative regulatory effect on tgev-induced apoptosis of pk- cells via targeting runx . accumulating evidence has shown that mirnas play an important role in apoptosis induced by virus infection (fu et al., ; guan et al., ; smith et al., ) . we previously found that tgev infection induced apoptosis in pk- cells via mitochondria-mediated apoptosis pathway (ding et al., ) . in addition, we reported that mir- b was significantly downregulated during apoptosis triggered by tgev infection in pk- cells (song et al., ) . however, the role of mir- b in the process of apoptosis induced by tgev infection is unclear. here we proved that mir- b suppressed the tgev-induced apoptosis via regulating mitochondrial pathway. during the viral infection, mirnas may regulate the interaction between virus and host cell via targeting viral or cellular genes. mir- c, which is down-regulated by hcv infection in hepatocytes, suppresses homeobox a expression (hoxa ) and its downstream molecules stat and stat to regulate cell growth by targeting e and ns a sequences of hcv (mukherjee et al., ) . mir- represses hiv infection by inhibiting transcription of hiv- viral protein r-binding protein (vprbp) in monocytes (ma et al., ) . we assessed the effects of mir- b on the replication and transcription of tgev genes and found that the replication and transcription of tgev structural and non-structural genes were not affected by mir- b (data not shown). we tested the effects of mir- b on apoptotic rate of tgev-infected pk- cells. the results showed that mir- b decreased tgev-induced apoptosis. we previously reported that mir- b was decreased by tgev infection (song et al., ) , so we propose that mir- b may present an antagonistic effect with tgev infection on apoptosis via regulating mitochondrial pathway. an analogous situation has been reported that mir- b alleviate hypoxia-induced neuronal apoptosis (chen et al., ) . it indicates that mir- b not only down-regulates cancer cell apoptosis, it also suppresses virus-induced apoptosis. apoptosis is primarily modulated by two pathways, extrinsic pathway and intrinsic pathway (mitochondrion-mediated), which regulate cell death involving the continuous activation of caspases (lavrik, ) . caspase- and caspase- are factors of extrinsic pathway and mitochondrial pathway respectively (sakamaki et al., ; thornberry & lazebnik, ) . bax, a member of the bcl- family, functions as an apoptotic activator to activate caspase- and - (chipuk et al., ; ding et al., ) . in this study, we showed that mir- b inhibited bax expression and reduced the activities of caspase- and - during tgev-induced apoptosis. we demonstrated that mir- b regulated apoptosis via down-regulation the expression of bax and activation of caspase- and caspase- , implying that mir- b are involved in regulating tgev-induced apoptosis via regulating mitochondrial pathway. the mirnas suppress gene expression by imperfectly binding to the utr of target mrna in mammalian cells. the imprecise binding make each mirna has the ability to target multiple mrnas. for example, mir- b could suppress adipogenesis in hmads cells by targeting peroxisome proliferator-activated receptor gamma (pparγ ) (karbiener et al., ) and promote differentiation of myeloblasts through targeting runx (feng et al., ). in the present study, we confirmed that runx is the target of mir- b during tgev infection, while whether mir- b regulates apoptosis via runx is unclear. runx is a transcription factor, and play important roles in regulating the growth, development, and/or differentiation of multiple lineages of haematopoietic cells (feng et al., ) . we found over-expression of runx reduced bax and decreased the activities of caspase- and - , and silencing runx up-regulated bax and increased the activities of caspase- and - . this suggests that mir- b regulates tgev-induced apoptosis via targeting runx , which is an important supplement of runx function. in summary, our finding suggests mir- b represses tgev-induced apoptosis by directly targeting runx , which may have potential for therapeutic strategies. micrornas: target recognition and regulatory functions prevalence of porcine epidemic diarrhoea virus and transmissible gastroenteritis virus infection in korean pigs microrna- a/b and microrna- a/b suppress apaf- protein and alleviate hypoxia-induced neuronal apoptosis the bcl- family reunion the research in this article did not generate any raw data. supplemental information for this article can be found online at http://dx.doi.org/ . / peerj. #supplemental-information. the authors declare there are no competing interests. • xiaomin zhao analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.• xiangjun song performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.• xiaoyuan bai, naijiao fei, yong huang, zhimin zhao, qian du, hongling zhang and liang zhang reviewed drafts of the paper.• dewen tong conceived and designed the experiments, contributed reagents/materials/ analysis tools, reviewed drafts of the paper. the following information was supplied regarding data availability: key: cord- - kbggzk authors: galán, carmen; enjuanes, luis; almazán, fernando title: differential role of n-terminal polyprotein processing in coronavirus genome replication and minigenome amplification date: journal: the nidoviruses doi: . / - - - - _ sha: doc_id: cord_uid: kbggzk nan providing active components of the replication-transcription complex. coronavirus orf a encodes one or two papain-like proteinases (plps) that are responsible for the cleavage at the n-proximal region of the replicase. although plp- cleavage products have been identified in hcov- e virus-infected cells, no specific roles or functional requirements have been studied so far for the hcov- e or tgev proteins p (nsp ) and p (nsp ). in the present study, we characterize by reverse genetics the effect of a point mutation at position , which mapped in the putative plp- cleavage site at the p /p junction, on tgev and minigenome replication. a correlation was found between predicted cleaving and noncleaving mutations and the different nucleotides selected for virus or minigenome replication, respectively. point mutations at genome position were engineered in pbac-tgev fl using an overlapping pcr strategy. bhk cells stably transformed with the porcine aminopeptidase n gene were transfected with the cdna clones using lipofectamine (invitrogen). after a -h incubation period at ºc, cells were trypsinized and plated over a confluent monolayer of swine testis (st) cells. cell supernatants were harvested for titration at , , and h post-transfection. the cdnas encoding tgev-derived rna minigenomes di-c ( . kb) and m ( , kb) were previously described. the m l minigenome was derived from the m minigenome including a -bp linker sequence. m l and di-c cdnas with point mutations at position were generated by restriction fragment exchange from plasmids with the corresponding mutations. for the trans-cleavage assay, the plp- domain (glu to ser ) from pp a, and the n-terminal amino acids with either gly ( g) or asp ( a) at position , were cloned into pcdna (invitrogen). t -driven in vitro transcripts of m l minigenome mutants were transfected in st cells previously infected with tgev pur -mad strain, and five serial passages were performed on fresh st cells. total rna from each passage was used as template for realtime rt-pcr reactions (q-rt-pcr) with minigenome-specific primers. tgev plp- proteinase and pp a substrates were expressed using the tnt t coupled reticulocyte lysate system (promega). different enzyme to [ s]met-labeledsubstrate [e/s] ratios were incubated overnight at ºc. cleavage reactions were resolved by sds- % page and processed for fluorography. during the construction of the tgev full-length cdna clone, a point mutation that was present in the defective minigenome di-c ( -t) was maintained as a genetic marker. interestingly, while other markers remained stable in the recombinant virus rescued (rtgev), nucleotide reproducibly reverted to the parental virus sequence g or c, indicating a strong selective pressure at this position. to study the role of nucleotide in virus replication, tgev cdna mutants at this position were generated. nucleotide substitution of the viral sequence g at position by c, t, or a produces an amino acid change at position of the pp a from gly to ala, val, or asp, respectively. virus recovery efficiency (fig. a ) and plaque morphology (fig. b) were analyzed after cdna transfection. no differences between the viruses carrying a g or c at position were detected. however, a reduction of three logarithmic units and small plaque morphology was observed for viruses with t or a at position . mutations that severely affected virus recovery from the cdna correlated with more drastic amino acid substitutions. no correlation was found between rna secondary structure predictions of the mutants and virus phenotypes (data not shown), indicating that the effect of mutations at position was most likely at the protein level. to study the sequence requirements at position for minigenome replication, m l minigenome mutants at this position were generated, and their rescue efficiency was determined by q-rt-pcr (fig. c) . controls of rna transfection (rna input) and background levels (mock; tgev; ntc) were included. only m l minigenomes with a or t at position were efficiently rescued, in contrast with the sequence requirements at the same position for efficient virus recovery from the full-length cdna clone. a late increase in the rna accumulation observed for m l- g and c was due to a genotypic reversion at position to a. no differences in virus titers that could explain minigenome phenotypes were observed (data not shown). the same mutational analysis with the rna di-c ( . kb) showed identical results to that of m l ( . kb) indicating that the same sequence requirements at nucleotide were necessary for both di-c and m l minigenome amplification regardless of their differences in size (data not shown). sequence alignments reported for the hcov- e and tgev replicase polyproteins, proposed two potential n-terminal cleavage sites in the tgev pp a, although no experimental data have been provided to date. mutations at nucleotide affects the nature of amino acid that occupies the p ' or p residues relative to the thr /gly or gly /ala putative cleavage sites, respectively, according with these predictions ( fig. a) . in both cases, a drastic amino acid change at position could affect plp -mediated processing. to support this hypothesis a trans-cleavage assay was performed with the tgev plp- domain and a n-terminal pp a substrate with either the wild-type sequence g at position or an a at the same position, leading to the least conservative amino acid change (gly asp) (fig. b) . in the absence of enzyme, only the -kda substrate and a minor protein, probably resulting from a premature termination event, were detected. when the plp- was included, a processed form of the substrate appeared with the substrate containing a gly residue at position , with the expected size of the cterminal cleavage product generated after the release of the n-terminal p protein, but not with the substrate presenting an asp residue at the same position. these results indicated that mutations at nucleotide affected the plp- -mediated n-terminal polyprotein cleavage in vitro. because predicted cleaving mutations were required for virus recovery, we propose a critical role of this processing event for viral replication. in contrast, the minigenomes selected predicted noncleaving mutations, suggesting that the processing of the minigenome-encoded fusion protein led to the generation of pp a products that interfere with minigenome amplification. further analysis will be required to assign specific functions to the n-terminal replicase proteins in tgev replication. virus-encoded proteinases and proteolytic processing in the nidovirales engineering the largest rna virus genome as an infectious bacterial artificial chromosome replication and packaging of transmissible gastroenteritis coronavirus-derived synthetic minigenomes proteolytic processing at the amino terminus of human coronavirus e gene -encoded polyproteins: identification of a papain-like proteinase and its substrate key: cord- -zp h k authors: zhao, xiaomin; bai, xiaoyuan; guan, lijuan; li, juejun; song, xiangjun; ma, xuelian; guo, jianxiong; zhang, zhichao; du, qian; huang, yong; tong, dewen title: microrna- promotes transmissible gastroenteritis virus (tgev)-induced mitochondrial damage via targeting rb , upregulating interleukin- receptor accessory protein (il rap), and activating p mapk pathway in vitro date: - - journal: mol cell proteomics doi: . /mcp.ra . sha: doc_id: cord_uid: zp h k transmissible gastroenteritis virus (tgev), a member of the coronaviridae family, could cause fatal diarrhea of piglets and result in numerous economic losses. previous studies demonstrated that tgev infection could lead to mitochondrial damage and upregulate mir- level. so mir- may play an important regulatory role in the control of mitochondrial function. to explore the potential role of mir- in mitochondrial damage, we adopted a strategy consisting of quantitative proteomic analysis of porcine kidney (pk- ) cells in response to mir- and tgev infection. eventually, differentially expressed proteins were gained. the target of mir- was identified. the effects of mir- and its target rb on mitochondrial ca( +) level, mitochondrial membrane potential (mmp), interleukin- receptor accessory protein (il rap), p mapk signaling pathway were investigated. the results showed that mir- elevated mitochondrial ca( +) level, reduced mmp, targets retinoblastoma (rb ), upregulated il rap, and induced activation of p mapk pathway during tgev infection. rb was identified as the direct targets of mir- and downregulated il rap, suppressed the activation of p mpak, and attenuated tgev-induced mitochondrial damage. in addition, il rap played a positive role in activating p mapk signaling and negative role in tgev-induced mitochondrial damage. the data indicate that mir- aggravates tgev-induced mitochondrial damage by repressing expression of rb , promoting il rap, and activating p mapk pathway. transmissible gastroenteritis virus (tgev) is an enveloped enteropathogenic coronavirus with positive-sense singlestranded rna genome ( ) . tgev infects pigs and especially causes piglets up to days of age high mortality, which can reach to % ( , ) and result in numerous economic losses. our previous work demonstrated that tgev infection can reduce mmp, leading to mitochondrial damage ( , ) . mitochondrion, an organelle of eukaryocyte, not only acts as the energy metabolism factory, but also is involved in many key biological processes, such as apoptosis, pathogenic infection ( , ) . mitochondrion is a very sensitive organelle to microenvironmental changes in cells and much easier becomes dysfunction than other organelles ( ) . mitochondria, the major source of energy, in the form of atp, are closely interconnected with cell death. ca ϩ is essential for maintain mitochondrial function. however, ca ϩ accumulation in mitochondria can impair mitochondrial function, resulting in a transient depolarization of mmp and reducing atp production ( - ) . both ca ϩ accumulation and mmp depolarization can cause mitochondrial damage, so the alterations of ca ϩ level and mmp are used to assess mitochondrial function ( ) . mirnas are small noncoding rna species containing about nucleotides and contribute to regulating many cellular processes including apoptosis, development, differentiation, and cell cycle ( ) . we previously reported that mir- level was upregulated during tgev-induced mitochondrial dysfunction ( ) . whereas, whether mir- participate in regulating tgev-induced mitochondrial damage in pk- cells is unclear. according to go enrichment and kegg pathway analysis of mir- targets, mir- targets can be enriched in mitochondria and mitochondria-related pathways. therefore, we postulate mir- can participate in regulating mitochondrial dysfunction in pk- cells. to investigate the regulatory effects of mir- on tgev-induced mitochondrial damage, mir- mimics or mimics control was transfected into pk- cells and the proteomic analysis was performed using lc-ms/ms labeled by tandem mass tags (tmt). then we identified the targets of mir- and detected the effects of mir- target on mitochondrial damage and p mapk pathway. we demonstrated that mir- increased mitochondrial ca ϩ level and decreased mmp via activating p mapk pathway and upregulating expression of il rap. moreover, mir- activated p mapk pathway via targeting rb and upregulating il rap. our data suggest that mir- could facilitate tgev-induced mitochondrial damage in pk- cells via targeting rb , promoting il rap, and activating p pathway. antibodies and plasmids-anti-rb was purchased from santa cruz biotechnology. horseradish peroxidase (hrp)-conjugated secondary antibody was purchased from pierce (us). anti-p-p and anti-␤-actin antibody were purchased from cell signaling technology (us). anti-il rap and anti-gapdh primary antibody was purchased from genetex (us). the psicheck- plasmid and dual-luciferase reporter assay system were purchased from promega (us). the primers and sirnas were synthesized by ribo biotech (ribobio, china). virus and cells-the tgev shaanxi strain was isolated from tgevinfected piglets ( ) . pk- cells were obtained from atcc (ccl- ) and grown in dulbecco's minimal essential medium (dmem) supplemented with % fetal bovine serum (hyclone, us), iu of penicillin, and mg/ml streptomycin, at °c in % co atmosphere incubator. experimental design and statistical rationale for proteomics-in order to investigating the effect of mir- on mitochondrial damage in response to tgev infection, pk- cells were transfected with nm mir- mimics or negative control using lipofectamine and infected with tgev at an moi of . at h post-transfection (hpt). the cells were collected for quantitative proteomic analysis at h post-infection (hpi). two biological replicates preparations labeled with tmt were analyzed. to evaluate the transfection efficiency, the mir- level was measured by real-time pcr. reverse transcription reactions and real-time pcr were performed as described previously ( ) . the relative quantification of mirnas was normalized to u using the two-ddct method ( ) . protein isolation, digestion, and labeling with tmt-the pk- cells were sonicated on ice in lysis buffer ( m urea, % triton- , mm dtt and . % protease inhibitor mixture) and centrifuged at , ϫ g for min at °c. the clarified supernatant was collected. finally, the protein was precipitated with cold % tca for h at Ϫ °c and centrifuged at °c for min. the precipitate was redissolved in buffer ( m urea, mm teab, ph . ). one hundred micrograms protein of each sample was digested with trypsin. the two control samples and two treatment samples were respectively labeled with tmt ( , , , and ). lc-ms/ms-the samples were fractionated by high ph reversephase high performance liquid chromatography (hplc). briefly, peptides were dried by vacuum centrifugation. then the peptides were dissolved in . % formic acid and directly loaded onto a reversed-phase precolumn (acclaim pepmap , thermo scientific). peptides were separated using a reversed-phase analytical column (acclaim pepmap rslc, thermo scientific) and analyzed by q exactive tm hybrid quadrupole-orbitrap mass spectrometer (thermo fisher scientific). the peptides were subjected to nsi source followed by tandem mass spectrometry (ms/ms) in q exactive tm (thermo fisher scientific) coupled online to the hplc. intact peptides were detected in the orbitrap at a resolution of , . peptides were selected for ms/ms using nce setting as . ion fragments were detected in the orbitrap at a resolution of , . a data-dependent procedure that alternated between one ms scan followed by ms/ms scans was applied for the top precursor ions above a threshold ion count of e in the ms survey scan with . s dynamic exclusion. the electrospray voltage was . kv. automatic gain control (agc) was used to prevent overfilling of the ion trap. e ions were accumulated for generation of ms/ms spectra. for ms scans, the m/z scan range was to . of tmt -plex reporter ions intensity, and calculation of tmt plex reporter ions intensity ratios. for each ms/ms spectra, top most intense peaks in every da window were extracted for database search. then mascot search was performed by searching tandem mass spectra against uniprot sus scrofa database (uniprot release _ , entries) (http://www.uniprot.org) concatenated with reversed decoy database and protein sequences of common contaminants. cleavage enzyme was specified as trypsin/p, maximum number of missing cleavages was set to . mass tolerance for precursor ion was set to ppm and mass tolerance for ms/ms ions was set to . da. carbamyiodomethylation on cys and tmt plex (nterm) and tmt plex (k) were specified as fixed modification and oxidation on met, acetylation on protein n-terminal were specified as variable modifications. false discovery rate (fdr) thresholds for protein, peptide and modification site were specified at %. mascot software assembled the peptide/protein groups, calculated false discovery rates, filter the identifications. protein quantification was set to use only unique peptides bearing any modification. the median ratio of tmt -plex reporter intensity of unique peptides was set as protein relative abundance changes. in this study, we prepared two duplicate samples. for each protein, we set the average ratio calculated from two duplicate samples as the final quantitation of protein. student's test was explored to calculate differential significance degree of protein relative abundance changes. t test p value less than . was considered significantly differentially. the mass spectrometry proteomics data have been deposited to the proteomexchange consortium via the pride partner repository with the data set identifier pxd (http://www.ebi.ac.uk/pride/archive/) ( ) . bioinformatic analysis of differentially expressed proteins-gene ontology (go) annotation of proteome was derived from the uniprot-goa database (http://www.ebi.ac.uk/goa). kyoto encyclopedia of genes and genomes (kegg) database was used to annotate proteins pathways. the wolf psort database (https://wolfpsort.hgc.jp/) was used to predict subcellular localization. the differentially expressed protein name identifiers (uniprot accession) were searched against the string database (version . ) for protein-protein interactions ( ) . vector construction-to overexpress rb and il rap, the fulllength sequences of rb and il rap were obtained by pcr from pk- cells and cloned into plasmid pci-neo (promega, usa). the constructions were respectively named pci-neo-rb and pci-neo-il rap. the primer sequences are shown in supplemental information s . western blot analysis-pk- cells were treated with ripa lysis buffer containing phenylmethyl sulfonylfluoride (pmsf). the proteins were separated using % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and transferred onto the polyvi- nylidene difluoride (pvdf) membrane. the membrane was blocked with % bsa for h at room temperature and then incubated with primary antibodies overnight at °c. the hrp-conjugated secondary antibodies were used to incubate the membrane for h at room temperature. reverse transcription pcr and real-time pcr-total rna was extracted with trizol (invitrogen, us). g of total rna was treated with dnase i (fermentas, germany) for min at °c and reversely transcribed using the first-strand cdna synthesis kit (invitrogen). the cdna was used as the template for real-time pcr. the real-time pcr was performed on bio-rad iq real-time pcr system (bio-rad). the reverse transcription primers and real-time pcr primers are shown in supplemental information s . dual-luciferase reporter assay- Ј utrs of candidate targets containing the mir- binding sites were respectively amplified by pcr and were cloned into the vector psicheck- (promega). the binding sites of mir- in rb -wt Ј utr were mutated following the mutagenesis protocol ( ) to generate rb -mut. the pcr primers are shown in supplemental information s . pk- cells were cotransfected with plasmid rb -wt (or rb -mut) and mir- mimics (or mir- inhibitors) using lipofectamine (invitrogen, us). the luciferase activities were detected at hpt using a dual-glo luciferase assay system (promega). rna interference-the sirnas of rb and il rap were synthesized by genepharma (genepharma, china) (supplemental information s ). pk- cells were transfected with nm sirna or irrelevant sirna using lipofectamine (invitrogen, us). evaluation of mmp-mmp of pk- cells was detected using jc- as an indicator ( ) . pk- cells were transfected with mir- mimics or mir- inhibitors and were subsequently infected with tgev at moi. pk- cells were treated with pbs containing jc- and incubated at °c for min. the absorbance was measured at ex/ em. measurement of mitochondrial ca ϩ level-the mitochondrial calcium level was detected using rhod- kit (genmed, china) following the manufacturer's protocol. the absorbance was measured at ex/ em. the relative fluorescence unit (rfu) was calculated. decreases mmp-pk- cells were infected with tgev at moi for h. the mitochondrial ca ϩ level and mmp of pk- cells were evaluated. the results showed that tgev infection a) and decreased mitochondrial mmp (fig. b) . gene ontology enrichment analysis of mir- targets-mir- targets were predicted using targetscan and mi-randa, by which targets were obtained (supplemental information s ). the targets of mir were searched against gene ontology (go) database to provide enrichment information on biological processes, molecular functions, and cellular components. among total predicted targets, targets were related to mitochondria, . % of total targets. go enrichment of the targets of mir- showed that . % of targets were enriched in metabolic process and . % of targets were linked to mitochondria in cellular component (fig. ) . we previously reported that tgev infection caused the reduction of mmp through inducing ros accumulation and increased mir- level ( , ) . to investigate the effects of mir- on mitochondria damage during tgev infection, the pk- cells were transfected with mir- mimics, or mirna mimics control, mir- inhibitors, inhibitors, and subsequently infected with tgev at moi for h. the mir- level remarkably increased by mir- mimics (fig. a) and suppressed by mir- inhibitors (fig. b) . mir- mimics led to a decrease of mitochondrial ca ϩ level (fig. c) and an increase of mmp (fig. e) . mir- inhibitors reduced mitochondrial ca ϩ level (fig. d ) and increased mmp (fig. f) . quantitative proteomic analysis-a total of proteins were identified by high-resolution lc-ms/ms analysis, among which proteins were quantified (supplemental information s ). when setting Ն . -fold as the upregulated threshold and Յ . -fold as the downregulated threshold, differentially expressed proteins were obtained, including upregu-lated proteins and downregulated proteins (table i) . raw data of ms are available via proteomexchange with identifier pxd . go enrichment analysis of the differentially expressed proteins-the differentially expressed proteins were searched against wolf psort database (https://wolfpsort. hgc.jp/) for prediction of subcellular localization. the subcellular localization analysis revealed that the distribution of differentially expressed proteins was distributed in cytosol ( . %), nuclear ( . %), mitochondria ( . %), extracellular ( . %), plasma membrane ( . %), cytosol and nuclear ( . %), endoplasmic reticulum ( . %), and peroxisome ( . %) (fig. a) . the differentially expressed proteins were annotated by uniprot-goa database or interproscan. the proteins were enriched by go annotation based on three categories: biological process, cellular component, and molecular function. the differentially expressed proteins are primarily enriched in cellular process ( . %), metabolic process ( . %), and single-organism process ( . %). the molecular functions of the differentially expressed proteins network nodes represent proteins. splice isoforms or post-translational modifications are collapsed, i.e. each node represents the protein produced by a single, protein-coding gene locus. colored nodes represent query proteins and the first shell of interactors. white nodes represent the second shell of interactions. small nodes are the proteins with unknown d structure. large nodes are the proteins with known or predicted d structure. lines represent protein-protein interactions. known interactions: represents from curated databases; represents experimentally determined. predicted interactions: represents gene neighborhood; represents gene fusions; gene co-occurrence. others: represents textmining; represents co-expression; represents protein homology. phosphorylation, ca ϩ signaling pathway, mapk signaling, and nf-b signaling pathway (fig. ) . the differentially expressed proteins were introduced into the web-tool string to generate protein-protein interaction networks (fig. ) . the transcription and expression levels of some differentially expressed proteins were verified using western blot and real-time pcr. the western blot analysis revealed that the protein levels of il rap and rela in pk- cells were dramatically increased by mir- overexpression (fig. a) . the mrna levels of mitochondriarelated differentially expressed proteins, including il rap, cox a, ndufb , marpl , marpl , rela, camka d, pck , and bcat , were confirmed by real-time pcr (fig. b) . the western blot and real-time pcr results were consistent with the proteomic data. il rap promotes tgev-induced mitochondrial damage and activates p mapk signaling-according proteomic analysis, overexpression of mir- may lead to an increase of il rap protein level. it is reported that il rap is a component of il- protein complex ( ) and il- is a activator of p mapk ( ) . we previously found that tgev infection reduced mmp and activated p mapk pathway ( ) . therefore, we presume that il rap might play a role in tgevinduced decrease of mmp and activation of p mapk. to explore the effects of il rap on mitochondria and p mapk pathway, we constructed pci-neo-il rap to overexpress il rap and synthesized sirna of il rap, siil rap, to silence il rap. the results showed that pci-neo-il rap led to an increase il rap at mrna level (fig. a ) and protein level (fig. b) and siil rap reduced mrna level (fig. a ) and protein level (fig. b) . mitochondrial ca ϩ level was upregulated by pci-neo-il rap and downregulated by siil rap (fig. c ). in addition, mmp level was suppressed by pci-neo-il rap and increased by siil rap (fig. d ). as expected, phosphorylation level of p was enhanced by pci-neo-il rap and attenuated by siil rap (fig. e) . activation of p mapk pathway facilitates tgev-induced mitochondrial damage-to investigate whether the activation of p mapk promotes tgev-induced mitochondrial damage, pk- cells were treated with sb , the specific inhibitor of p mapk, and infected with tgev. as predicted, tgev-induced phosphorylation of p was attenuated by sb (fig. a) . inhibition of p phosphorylation led to a decrease of mitochondrial ca ϩ level (fig. b) and an increase of mmp during tgev infection (fig. c) , indicating that p promotes tgev-induced mitochondrial damage. mir- promotes tgev-induced activation of p mapk pathway-we demonstrated that mir- could upregulated il rap expression via quantitative proteomic analysis and il rap contributed to activation of p mapk pathway via phosphorylating p . however, whether mir- can activate p pathway is unknown. to investigate the effect of mir- on p mapk, mir- mimics or inhibitors was introduced into pk- cells. the phosphorylation level of p was detected. the result showed that tgev-induced phosphorylation of p was enhanced by mir- mimics and attenuated by mir- inhibitors (fig. ) , suggesting mir- caused activation of p mapk pathway. rb is the direct target of mir- -to identify whether mir- directly binds to the Ј utrs of mir- targets, the mir- binding sites of target mrna were mutated with a -bp substitution (fig. a) . the wild type sequences of Ј utrs containing mir- binding sites and mutated Ј utrs sequences of mir- targets, in which mir- seed sequence was mutated, were cloned into the Ј utr of renilla luciferase in dual-luciferase reporter plasmid psi-check- to generate constructions, rb -wt and rb -mut (fig. b) . the constructs were co-transfected into pk- cells with either mir- mimics/mimics control or mir- inhibitors/inhibitors control. the renilla luciferase activities of rb -wt and rb -mut were respectively reduced and improved by mir- mimics (normalized to firefly luciferase activity) (fig. c) . in contrast, the renilla luciferase activities of rb -wt and rb -mut were respectively raised and decreased by mir- inhibitors (normalized to firefly luciferase activity) (fig. d) . the results revealed that mir- directly binds to Ј utr of rb mrna. to determine whether mir- inhibits rb expression, either mir- mimics or mirna inhibitors control were transfected into pk- cells. rb protein level was tested using western blot, showing that expression of rb was suppressed by mir- mimics and improved by mir- inhibitors (fig. e) . level and improves mmp during tgev infection-we demonstrated that mir- facilitated mitochondrial damage and directly targeted rb . however, whether mir- affects mitochondria through targeting rb is unclear. to investigate the effect of rb on tgev-induced mitochondrial damage, three sirnas of rb , sirb - , sirb - , and sirb - , were synthesized and respectively transfected into pk- cells to silence rb . the silencing efficiency of rb sirnas were evaluated, showing sirb - was the most effective sirna for rb silencing (fig. a and b) . moreover, rb gene was cloned into eukaryotic expression plasmid pci-neo, named pci-neo-rb , to overexpress rb . pci-neo-rb and pcineo were respectively transfected into pk- cells, followed by evaluation of mrna and protein level. expectedly, both the mrna and protein level were upregulated (fig. c and d) . then, pk- cells were transfected with either sirb - or pcineo-rb and subsequently infected with tgev for h. the mitochondrial ca ϩ level and mmp were measured. the results showed that mitochondrial ca ϩ level was decreased by pcineo-rb and increased by sirb - (fig. e ). in addition, mmp was upregulated by pci-neo-rb and decreased by sirb - (fig. f) . the results reveal that mir- promotes tgevinduced mitochondrial damage via targeting rb . rb suppresses il rap expression and p mapk signaling-we showed that mir- could aggravate tgevinduced mitochondrial damage via targeting rb and activating p mapk pathway. therefore, we presumed that mir- is likely to regulate il rap and p mapk signaling mir- promotes mitochondrial damage via p mapk pathway through its target rb . we silenced rb using sirb - and overexpressed rb using pci-neo-rb . il rap, p , and p-p were analyzed by western blotting. it was found that rb was suppressed by sirb - (fig ) . silencing rb caused an upregulation of il rap and p-p (fig. ). reversely, overexpression of rb led to a suppress of il rap and p-p (fig. ). in this study, we investigated the effects of mir- on mitochondrial damage induced by tgev infection via target-ing rb . the results showed that mir- promoted tgevinduced mitochondrial damage via targeting rb , upregulating il rap, and enhancing tgev-induced activation of p pathway. our findings reveal a novel regulatory effect of mir- on tgev-induced mitochondrial damage. it is reported that tgev infection caused the alterations of proteomes in pk- cells, including upregulated stat and many isgs (isg , ifit , ifit , ifit , ifit , oas , oas , and mx ) ( ) . in this study, mx was differentially decreased by mir- in contrast to upregulation in tgev-infected pk- cells. it indicates that mir- plays a negative role in tgev-induced mx expression. in addition, although stat and many isgs, including isg , ifit , ifit , ifit , oas , oas , mx , were detected using tmt approach, only mx was altered by mir- in tgev-infected pk- cells. the likely reason is that mir- downregulated tgev-induced stat and these isgs, including isg , ifit , ifit , ifit , oas , oas , to normal level. in contrast to upregulation in tgev-infected pk- cells, mx was differentially decreased by mir- in tgev-infected pk- cells. it indicates that mir- plays a negative role in tgev-induced mx expression. in this study, rb is identified as the target of mir- and affects mitochondrial function. rb is a suppressor of cell death via binding and repressing transcription factor e fs. the hypophosphorylated rb , the active form of rb , binds to and sequesters transcription factor e f to operate as an inhibitor of e fs ( ) . c-terminal region of rb has a e f -specific binding site that is sufficient to repress e f -induced apoptosis, so the c-terminal interaction site on rb acts as a potent inhibitor of e f ( ) . a pool of evidence shows rb localizes in mitochondria ( ) . therefore, a set of reports highlights a series of links between rb and mitochondrial function. rb promotes mitochondrial biogenesis for erythropoiesis ( ) . loss of rb caused a decrease of mitochondrial mass, downregulated mitochondrial function, oxidative phosphorylation, mmp, and accumulation of hypopolarized mitochondria ( ) , implying that suppression of rb leads to mitochondrial damage. these results further support our conclusion that rb weakens tgev-induced mitochondrial damage. here, rb was identified as the target of mir- and inhibited by mir- . whereas, rb was not identified as the differentially expressed protein using tmt, it may be because of low expression level of rb and the insufficient sensitivity of this technology. the epidermal growth factor receptor regulates cofilin activity and promotes transmissible gastroenteritis virus entry into intestinal epithelial cells bacillus subtilis and surfactin inhibit the transmissible gastroenteritis virus from entering the intestinal epithelial cells transmissible gastroenteritis virus infection induces apoptosis through fasl-and mitochondria-mediated pathways regulation of ros in transmissible gastroenteritis virus-activated apoptotic signaling mitochondrial diseases part i: mouse models of oxphos deficiencies caused by defects in respiratory complex subunits or assembly factors. mitochondrion mitochondria-associated apoptosis in human melanoma cells induced by cardanol monoene from cashew nut shell liquid mitochondria as sources and targets of damage in cellular aging mitochondria and calcium: from cell signalling to cell death mitochondrial calcium overload is a key determinant in heart failure oxidative stress caused by mitochondrial calcium overload development or disease: duality of the mitochondrial permeability transition pore molecular mechanisms of rna interference transmissible gastroenteritis virus (tgev) infection alters the expression of cellular microrna species that affect transcription of tgev gene isolation and identification of porcine transmissible gastroenteritis virus shaanxi strain and sequence analysis of its n gene analysis of relative gene expression data using real-time quantitative pcr and the -⌬⌬ct method a practical guide to the maxquant computational platform for silac-based quantitative proteomics update of the pride database and its related tools the string database in : functional interaction networks of proteins, globally integrated and scored gene splicing and mutagenesis by pcr-driven overlap extension effect of rb on il rap and activation of p mapk camkii determines mitochondrial stress responses in heart the interleukin- receptor family interleukin alpha (il- alpha) induced activation of p mitogen-activated protein kinase inhibits glucocorticoid receptor function quantitative proteomic analysis reveals that transmissible gastroenteritis virus activates the jak-stat signaling pathway conserved rb functions in development and tumor suppression prb contains an e f -specific binding domain that allows e f -induced apoptosis to be regulated separately from other e f activities the retinoblastoma protein induces apoptosis directly at the mitochondria rb intrinsically promotes erythropoiesis by coupling cell cycle exit with mitochondrial biogenesis proteomic analysis of prb loss highlights a signature of decreased mitochondrial oxidative phosphorylation targeting the interleukin- pathway in patients with hematological disorders the structural pathway of interleukin (il- ) initiated signaling reveals mechanisms of oncogenic mutations and snps in inflammation and cancer the e ubiquitin ligase march negatively regulates il- beta-induced nf-kappab activation by targeting the il rap coreceptor for ubiquitination and degradation transmissible gastroenteritis virus infection induces nf-kappab activation through rlr-mediated signaling tgev infection upregulates fcrn expression via activation of nf-kappab signaling promotes mitochondrial damage via p mapk pathway cytokines of the il- family are considered critical regulators of intestinal mucositis ( ) . il- is a key proinflammatory cytokine that can initiate a series of signaling resulting in activation of nf-b signaling pathway. il- interacts with l rap and il- receptor type i (il- ri) to form il- complex ( ) , which mediates the activation of p mapk signaling in presence of il- ␤ treatment ( ) . it is in consistent with our finding that il rap can lead to the activation of p mapk pathway. moreover, we found mir- caused an upregulation of rela, which is the p subunit of nf-b. nf-b is a key activator of inflammation and is activated during tgev infection ( , ) . therefore, we speculate mir- may function as a regulator of tgev-induced nf-b pathway through regulating il rap and rela. the mass spectrometry proteomics data have been deposited to the proteomexchange consortium via the pride partner repository with the data set identifier pxd (http:// www.ebi.ac.uk/pride/archive/) ( key: cord- -d f a ds authors: pensaert, m. b.; de bouck, p. title: a new coronavirus-like particle associated with diarrhea in swine date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: d f a ds coronavirus-like particles were detected by electron microscopy in the intestinal contents of pigs during a diarrheal outbreak on swine breeding farms. diarrhea was reproduced in experimental pigs with one of the isolates, designated cv , which was found to be distinct from the known porcine coronaviruses, transmissible gastroenteritis virus and hemagglutinating encephalomyelitis virus. coronavirus-like particles were detected by electron microscopy in the intestinal contents of pigs during a diarrheal outbreak on swine breeding farms. diarrhea was reproduced in experimental pigs with one of the isolates, designated cv , which was found to be distinct from the known porcine coronaviruses, transmissible gastroenteritis virus and hemagglutinating encephalomyelitis virus. in , do yr.e and hutc~i~gs ( ) described a viral diarrhea, in swine and called it transmissible gastroenteritis. until recently, transmissible gastroenteritis virus was the only virus known to be specifically associated with diarrhea in swine of all ages. in , following the discovery of rotaviruses in different, animal species, a porcine rotavirus was detected in the feces of pigs with diarrhea ( ) . diarrhea could be reproduced experimentally in piglets with this virus. in a search for rotaviruses on belgian swine breeding farms with diarrheal problems, a new coronavirus-like particle was detected b y electron microscopic examination of intestinal or fecal samples from sick pigs. the present report describes the morphology of this coronavirus-like particle, and shows that it is distinct from the known porcine coronaviruses and causes diarrhea. up to now, the only coronaviruses isolated from swine have been transmissible gastroenteritis virus {tgev) and hemagglutinating encephalomyelitis virus (hev). tgev has been described as a cause of diarrhea in swine in countries all over the world ( ) . numerous studies have been performed on the v i r u s --a n i m a l interactions of tgev, which is usually detected either by its isolation from fecal these studies were supported by the institute for the encouragement of l~esearch in industry and agriculture (iwonl), brussels, belgium. material in cell cultures or by immunoflu orescence in the smm intestinal epithelium of infected pigs ( , ) . tgev infections can also be diagnosed serologically. j:iev was first described in canada in as a cause of centrm nervous disorders in pigs ( ). the same virus was later associated with a disease syndrome called vomiting and wasting disease in several european countries ( , ) . the virus can easily be detected by cultivation in several porcine cell cultures ( ). both tgev and hev have been classified as coronaviruses mainly on the basis of their specific morphology ( ). in , a sudden outbreak of diarrhea was observed in swine of all ages on belgian swine breeding farms. the morbidity in sows was very variable and the animals recovered after a diarrhea which lasted to days. all the pigs showed a watery diarrhea. death occurred up to the age of days and the overali mortality rate in these piglets was approximately per cent ( ) . it decreased with increasing age. tgev was suspected as the cause of this diarrhea. however, the direct immunofluorescence test for the diagnosis of tgev, which is routinely applied on cryostat sections of the small intestine of sick pigs, was negative for these pigs. the absence of seroneutralizing antibodies to tgev in the blood of sows collected to weeks after the outbreak confirmed that tgev was not involved. in an attempt to arrive at an etiologie diagnosis, fecal material and intestinal contents from pigs of each farm were subsequently processed for examination in an electron microscope by negative staining. they were diluted t to (v/v) in phosphate-buffered saline, ph . and clarified at × g, at ° c, for minutes. the supernatant was layered on top of a per cent sucrose solution and centrifuged at , x g, at ° c, for minutes. the resulting pellet was resuspended in a few drops of distilled water, placed on mesh formvar coated grids, and stained with per cent k-phosphotungstate, ph . . grids were examined using a zeiss em s- electron microscope at an acceleration voltage of kv. micrographs used for particle size measurement were taken at an instrumental magnification of , x, which were then photographically-enlarged to , x or , ×. gotavirus particles were not detected. however, eoronavirus-like particles were observed in specimens of pigs from each of the breeding farms. one of the fecal samples containing these coronavirus-like particles was designated cv and was used for further studies. the etiologic relationship between the corona virus-like particles, cv , and the occurrence of diarrhea was established by oral inoculation of a per cent suspension of the fecal material contmning cv into a one day old colostrumdeprived pig. the experimental pig was killed hours later, at the height of diarrhea, and a virus stock was prepared from an homogenate of its small intestine and contents. a bacteria free filtrate of the supernatant of a per cent suspension of this material was used for inoculation of colostrum-deprived-hysterectomy-derived piglets, kept i n isolation. seven control pigs were used. the pigs were inoculated at the age of to days. all the inoculated pigs developed a watery diarrhea within to hours after inoculation whereas the control animals remained normal. coronavirus-like particles were detected by electron microscopic examination in the watery feces or intestinal contents of each of the experimentally inoculated pigs. such particles were not found in the feces of the same pigs prior to inoculation or in the fecal samples of the control animals. the particles, shown in figure , had typicm coronavirus morphology. they were pleomorphic with a range in diameter of to nm, including the projections, which were approximately nm in length. most particles were between t and t nm in diameter. the projections formed a single fringe radiating from the core. they appeared to be club-shaped. only the dilated distal ends of the projections were seen on the micrographs. the negative stain also appeared to settle on the surface of some particles and an electron opaque central area covered by surface projections was often seen (fig. a --a r r o w s and lc) . no internal structure was observed. it was impossible to distinguish these coronavirus-like particles morphologically from tgev or h e v particles from similar preparations. other particles, different from these coronavirus-like particles, were also observed in the majority of the fecal samples. as seen in figure , they were pleomorphic and very variable in size, ranging in diameter from to nm with an average diameter of to rim. they carried numerous short projections, of approximate length nm, on their surfaces. similar particles of unkno~m identity have been described in human and animal fecal samples ( , , ) . in the present studies such particles have also been found in the solid fecal samples of the control pigs. they appeared, therefore, not to be associated with diarrhea. rotaviruses and other recognizable virus particles were not seen in control or experimentally inoculated pigs. as already mentioned, tgev was eliminated as the cause of the diarrhea on the origina farms. additionmly out of the experimentally inoculated pigs, killed at, the heigth of diarrhea, were negative for tge viral antigens in their small intestinal epithelium by the direct immunofluorescenee test. furthermore, the remaining pigs, inoculated with cv at the age of days, were allowed to recover after a diarrhea which lasted -- days. a serum sample, collected from these pigs weeks later, did not contain neutralizing antibodies against the cell culture adapted purdue strain of tgev. fig. . one eoronavirus-like particle cv (arrow) together with pleomorphie particles of unknown identity. bar represents nm the possibility that the cv particles consisted of h e v was less likely since the latter virus does not cause diarrhea in pigs. cryostat sections of the sma~ intestine of experimentmly inoculated pigs were negative for fluorescence by the direct test using a conjugate directed against the vw isolate of h e v ( ) . furthermore, the pigs that had been allowed to recover did not possess hemagglutination-inhibiting or seroneutralizing antibodies against this t t e v isolate. preliminary attempts were made to cultivate the coronavirus-like particle, cv , in primary pig kidney cell cultures and in secondary porcine thyroid cells. four weekly blind passages were made. the cells were examined for cytopathic effect and hemadsorption with chicken red blood cells, and the cell culture fluids were examined for hemagglutination. no evidence of viral replication in the cell cultures was obtained. it is kno~n that the tiev can easily be isolated in primary pig kidney cell cultures using the same criteria ( ) . the present data suggest that, as well as tgev and hev, another previously unrecognized coronavirus-like virus is prevalent in swine. the results indicate that diarrhea can be reproduced in experimental pigs with this virus and that it is associated with certain outbreaks of epizootic diarrhea on belgian swine breeding farms. more details on the clinical disease in the field and on the results of the experim e n t a l infections will be reported later. vomiting and wasting disease of piglets a transmissible gastroenteritis in pigs the hunt for viruses in infections of the alimentary s y s t e m : a n immuno etectronmicroscopicat approach a hemagglutinating virus producing encephalomyelitis in baby pigs pleomorphic virus-like particles in human faeces virus-like particles in calves' faeces diagnosis of transmissible gastroenteritis in pigs by immunofluorescence characteristics of a coronavirus causing vomition and wasting in pigs a virus isolated from an apparently new epizootic diarrhea in swine. proe. th world int the size and morphology of transmissible gastroenteritis and vomiting and wasting disease viruses of pigs titration of two porcine respiratory viruses in mammalian cell cultures by direct fluorescent antibody staining isolation of the virus of transmissible gastroenteritis (tge) from naturally infected piglets in cell culture transmissible gastroenteritis of swine the isolation of reovirus-like agents (rotaviruses) from acute gastroenteritis of piglets key: cord- - rgcc h authors: pedersen, n. c.; ward, j.; mengeling, w. l. title: antigenic relationship of the feline infectious peritonitis virus to coronaviruses of other species date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: rgcc h utilizing the direct and indirect fluorescent antibody procedure, the antigenic relationship of the feline infectious peritonitis virus (fipv) to other human and animal coronaviruses was studied. fipv was found to be closely related to transmissible gastroenteritis virus (tgev) of swine. transmissible gastroenteritis virus and fipv were in turn antigenically related to human coronavirus e (hcv- e) and canine coronavirus (ccv). an interesting finding in the study was that the coronaviruses selected for this study fell into one of two antigenically distinct groups. viruses in each group were antigenically related to each other to varying degrees, but were antigenically unrelated to coronaviruses of the second group. the first antigenically related group was comprised of mouse hepatitis virus, type (mhv- ), hemeagglutinating encephalomyelitis virus n (hev- n) of swine, calf diarrhea coronavirus (cdcv), and human coronavirus oc (hcv-oc ). the second antigenically related group was comprised of fipv, tgev, hcv- e and ccv. utilizing the direct and indirect fluorescent antibody procedure, the antigenic relationship of the feline infectious peritonitis virus (fipv) to other human and animal coronaviruses was studied. fipv was found to be closely related to transmissible gastroenteritis virus (tgev) of swine. transmissible gastroenteritis virus and fipv were in turn antigenically related to human eoronavirus e (i-icv- e) and canine coronavirus (ccv). an interesting finding in the study was that the eoronaviruses selected for this study fell into one of two antigenically distinct groups. viruses in each group were antigenieally related to each other to varying degrees, but were antigenically unrelated to coronaviruses of the second group. the first antigenically related group was comprised of mouse hepatitis virus, type (mi-iv- ), hemeagglutinating encephalomyelitis virus n (hev- n) of swine, calf diarrhea coronavirus (cdcv), and human coronavirus oc (hcv-oc ). the second antigenically related group was comprised of fipv, tgev, hcv- e and ccv. the family coronaviridae is a recently characterized group of animal and human viruses ( ) . coronaviruses are to nm in diameter, have a buoyant density in sucrose of . to . g/cm , are sensitive to lipid solvents, contain a large single strand of ribonucleic acid, have regularly spaced surface projections n.c. pedersen, j. wai~d, and w. l. mesrg~ll~g: t h a t are to n m in length, and bud from profiles of endoplasmic reticulum into cytoplasmic vesicles in the infected cells ( ) . coronaviruses cause bronchitis in chickens ( ), humans ( , ) and rats ( ) , acute enteric infections in b a b y pigs ( ) , calves ( ) a n d puppies ( ) , hepatitis in mice ( ), and encephalomyelitis a n d chronic vomition and wasting in swine ( , ) . feline infectious peritonitis (fip) is a viral disease of cats t h a t is characterized b y peritonitis, pleuritis or disseminated granulomata ( ) . feline infectious peritonitis represents an uncommon secondary form of a common i n a p p a r e n t or mild p r i m a r y illness of cats ( ) . the f i p agent has strong morphologie and physical similarities to known coronaviruses ( , , , ) . a possible antigenic relationship between feline infectious peritonitis virus (fipv) and the transmissible gastroenteritis virus (tgev) of swine has been recently reported ( , , ) , which further supports the assumption t h a t f i p v is a coronavirus. the antigenic relationship of f i p v to h u m a n and animal coronaviruses other t h a n t g e v has not been studied, and confirmation of the antigenic relationship of f i p v and t g e v using monospeeific antiserum is needed. the purpose of this s t u d y is twofold: to confirm the antigenic relationship of f i p v to tgev, and to demonstrate the antigenic relationship of different animal and h u m a n coronaviruses to f i p v , and to each other. the viruses selected for this s t u d y were h u m a n coronavirus c (hcv- c ), h u m a n eoronavirus e (hcv- e), t g e v and hemagglutinating encephalomyelitis virus n (hev- n) of swine, mouse hepatitis virus t y p e (miiv- ), calf diarrheal coronavirus (cdcv), and canine coronavirus (ccv). antigenic comparisons were m a d e utilizing the direct and indirect fluorescent a n t i b o d y technique. this procedure has been utilized to s t u d y serologic differences between several h u m a n coronaviruses (/ ). monospecific antiserum to f i p v was prepared in specific pathogen free kittens (liberty laboratories, liberty corners, nit). the cats were inoculated intraperitoneally with . g equivalents of liver suspension containing approximately id of the ucd- strain of fipv. the origin of this strain and the preparation of the inoeula have been previously described ( ) . serum was harvested prior to the animms' death, from to days after inoculation. by the indirect fluorescent antibody technique ( ) this antiserum had a titer of : against fipv. mouse anti-mttv- serum was produced in specific pathogen free adult. swiss white mice. mice were inoculated intraperitoneally with a sublethal dose of a per cent mouse liver suspension containing the craig strain of mitv (mhv- ). this material was obtained from the american type culture collection, rockville, md. three weeks later the mice were challenged intraperitonemly with a second sublethal dose of mhv- , followed by challenge weeks later with a lethal dose of virus. serum was harvested weeks after the final challenge dose. this serum had a titer by the indirect fluorescent antibody technique of : against mi-iv infected nctc- cells. bovine anti-cdcv antiserum was obtained from calves that. had been experimentally infected with the virus. the globulin fraction of this serum was conjugated with fluoreseein isothiocyanate (fitc). conjugated antiserum was provided by dr. c. a. mebus, lincoln, nebraska.. the conjugated antiserum produced maximum fluorescence in cdcv infected bovine fetal lung cells at dilutions of : or less. swine anti-tgev serum was obtained from a specific pathogen free sow that was experimentally infected with the miller strain of tgev. this serum was kindly provided by dr. roger woods, ames, iowa. both swine anti-tgev and ttev serum produced maximum fluorescence by the antibody technique at di utions of t : or less. guinea pig anti,hcv- e serum was provided by dr. harold kaye, communicable diseases center, atlanta, georgia. before use, the serum was absorbed with swine testicle, human embryo flbroblasts, nctc-t , african green monkey (cv- ), bovine fetal lung cells and with cat liver homogenate. this serum produced maximum fluorescence by the indirect fluorescent antibody technique at dilutions of : or less. mouse anti-i-icv-oc ascitie fluid was kindly provided by dr. harold kaye, communicable diseases center, atlanta, georgia. ascitic fluid was collected from virus free mice that had been experimentally infected with hcv-oc . this serum produced maximum fluorescence by the indirect fluorescent antibody technique at dilutions of : or tess. canine anti-ccv globulin conjugated with fluorescein isothioeyanate was provided by dr. l. q. binn, walter reed army institute of research, washing*on, d.c. it was prepared from convalescent serum of puppies experimentally infected with ccv. the conjugated antiserum produced maximum fluorescence in ccv infected canine fetal thymus cells at dilutions of : or less. cryostat microtome sections of liver from fib¥ infected cal~s were used as the antigen source of fibv. the preparation of these slides has been previously described ( ) . prior to use, the fixed liver sections were immersed for minutes in . ~ glycine-i.ic buffer, pit . to remove immunoglobulin bound in rive. when this bound immunoglobulin was not removed, a false positive reaction was seen in the indirect fluorescent antibody test, especially when the second antibody was rabbit anti-cat igg. after treating in buffer, the slides were washed immediately in phosphate buffered saline (bbs), followed by a minute and minute wash in bbs. mhv- infected cell monolayers were prepared as follows. nctc- cells (microbiological associates, bethesda, md.), adapted to grow in eagle's minimum essential media (mem) and per cent fetal calf serum (fcs), were grown in well culture chamber slides (lab-tek, microbiological associates, bethesda, md.). when confluent, the cell monolayer was exposed ~o mhv- by placing .t nil of a : dilution of per cent infected mouse liver suspension in each well. the slides were fixed in absolute acetone when significant cytopathic effect was noticed. calf diarrhea coronavirus was obtained from dr. c. a. mebus, lincoln, nebraska.. one-tenth ml of infectious tissue culture media was placed in each well of a culture chamber slide containing a three-fourth confluent monolayer of low passage fetal bovine lung cells. the slides were fixed in absolute acetone after -- days. tgev and i.iev were cultivated in swine embryonic testicle cells {national animal diseases center, ames, iowa). tgev (miller strain) infected tissue culture fluid was provided by dr. roger woods, ames, iowa,. i.iev ( n strain) infected tissue culture fluid was obtained from the national animal disease center, ames, iowa. swine testicle cells were grown in eagle's mem in per cent fcs in well culture chamber slides. when the cultures were almost confluent they were exposed to the n strain of ttev or miller strain of tgev by placing .i ml of infected tissue culture fluid in each well. the slides were fixed in absolute acetone after to days. ttcv- e was obtained as infected tissue culture fluid from dr. harold kaye, communicable diseases center, atlanta, georgia. low passage human embryonic fibroblasts were grown in eagle's mem in per cent fcs in well culture chamber slides. when the cultures were ahnost confluent, . nil of infected tissue culture fluid was placed in each well. slide cultures were fixed after days. n.c. ped~ase~, j. ward, and w. l. mengeling: hcv-oc was obtained as a mouse brain suspension from dr. harold kaye, communicable diseases center, atlanta, georgia. high passaged african green monkey kidney cells (cv- ) were grown in eagle's mem with per cent fcs in well culture chamber slides. when nearly confluent, . ml of a : dilution of brain suspension was placed in each well. slides were fixed in absolute acetone after to days. canine eoronavirus (i- ) in tissue culture fluid was obtained from the american type culture collection, izockville, ,md. low passage dog thymus cells were cultivated in well culture chamber slides. when nearly confluent, . ml of infected tissue culture fluid was placed in each well. cultures were fixed in absolute acetone after eytopathie effect became noticeable. rabbit anti-mouse igg globulin-fitc, rabbit a~nti-cat igg globulin-fitc, rabbit anti-pig igg-fitc, and goat anti-guinea pig igg-fitc were obtained from antibodies incorporated, davis, ca. conjugated anti-igg globulins were free of anticoronavirus activity as determined by reacting antigen substrates with the diluted conjugates alone. goat anti-guinea pig igg-fitc was absorbed with eat liver homogenate and human embryo cells. indirect fluorescent antibody staining was carried out as follows. antigen substrate slides were overlaid with a : dilution in pbs of the appropriate antiserum and incubated at ° c for hour in a humidified chamber. the slides were immediately rinsed with pbs and then washed for minutes in pbs. the slides were blotted dry and then overlaid with the appropriate anti-igg conjugate diluted : in pbs. the slides were incubated for i hour at ° c in humidified chamber and then washed in pbs. this treatment was followed by a minute wash in pbs containing a : dilution of a per cent stock solution of aqueous evans blue, and a minute wash in pbs. slides were then blotted dry, and eoverslips mounted with per cent glycerol in pbs. direct fluorescent antibody staining was carried out essentially as above, except tha~ the first antibody reaction was omitted. slides were photographed with a zeiss reflected light fluorescent microscope, powered by a watt ac mereury~vapor bulb, using exciter and barrier filters specific for fluorescein isothioeyanate. photomicrographs were made using ektachrome mm daylight slide film, asa (kodak co., l%oehester, ny), with second exposures. all photomicrographs were prepared from black and white negatives made from the colored slides. the cross-reactivity by immunofluorescence of antisera to different coronaviruses is listed in table . antigenic cross-reactivity varied from nondetectable, barely detectable, weak, moderate, to very strong (equal to that produced by the homologous serum). cross-reactivity, varying from barely detectable to very strong, was seen between mhv- , cdcv, hev- n and hcv-oc . there was no detectable antigenic cross-reactivity between these viruses and fipv, tgev, hcv- e, or ccv. antiserum to fipv reacted strongly with tgev, and vice versa. antiserum to both of these viruses had barely detectable to weak antigenic cross-reactivity with hcv- e, and antiserum to iicv- e had weak to moderate cross-reactivity with fipv and tgev. antiserum to ccv did not react with any of the other coronaviruses, although antiserum to fipv and tgev reacted very strongly with ccv. photomicrographs of some of the strongly positive eross-reae~ions are shown in figures and . it was concluded from these studies that, miiv- , hcv- c , cdcv, and hev- n are all antigeniemly interrelated to varying degrees, but a~ not related antigenieally to any of the other [ eoronaviruses. similany, fipv, tgev, hcv- e and ccv share antigens to varying degrees with each other, but appear antigenieally unrelated to mhv- , hcv-oc , cdcv, and hev- n. in the ease of ccv, however, the reactivity was largely in. one direction only, in t, hat antiserum to ccv showed no reactivity with any of the other eoronaviruses, whereas antiserum to fipv and tgev reacted strongly with ccv. these studies demonstrate conclusively that the fip virus has antigenic similarities to known coronaviruses, namely tge virus of swine, e virus of humans, and canine eoronavirus. this finding, coupled with the morphologic and it was interesting that the coronaviruses selected for this study segregated into distinct groups on the basis of antigenic cross-reactivity by immunefluorescence. although viruses within each group were antigenieally related to each other, there appeared to be no antigenic relationship of viruses from one group with viruses of the other group. the first antigenically related group was comprised of mhv- , hev- n, cdcv and i-icv-oc , and the second group was comprised of tgev, fipv, hcv- e and ccv. these findings confirm a number of published reports on antigenic relationships among recognized coronaviruses. on the basis of serum neutralization or complement fixation tests, antigenie relationship has been previously reported between rat eoronavirus and mi-iv ( ) , hcv-oc and mhv ( ), hev- n and ci)cv ( ) , hcv-oc and tiev- n ( ), and ccv and tgev ( ) . it has also been previously reported that hcv- e appeared to be antigenically unrelated to mhv- and hcv-oc ( ). the lack of relationship by immunofluorescence of hcv- c and hcv- e has also been reported ( ) . although the results of our studies were in agreement with most of the published literature on antigenic relationships among various corona viruses, there were several reports that we could not confirm. we could find no antigenic relationship by immunofluoreseenee between mhv- and hcv- e, and i-icv-oc and hcv- e. a relationship between these viruses has been previously described ( ). we also found no antigenic relationship between hev- n and tgev, although a relationship using the immnnopreeipitation technique has been reported ( ) . it has been reported that antiserum to tgev does not react against fipv in the fluorescent antibody test ( ) . in contrast, we found that ~ntiserum to tgev reacted strongly with fipv. finally, we are at a loss to explain the failure of anti-ccv globulin to react with tgev and fipv with immunefluorescence, especially considering the strong reaction against ccv demonstrated by both anti-fipv and tgev serum. dog anti-ccv serum will apparently neutralize tgev ( ), and it is strange that this reaction was not detected with immnnofluoreseence. recovery and characterization of a eoronavirus from military dogs with diarrhea the detection of transmissible gastroenteritis viral antibodies by immunodiffusion antigenic relationships amongst eoronaviruses a virus related to that causing hepatitis in mice (mi-iv) studies on the infectious bronchitis virus of chickens isolated in finland a hemagglutinating virus producing encephalomyeiitis in baby pigs a new virus isolated from the human respiratory tract feline infectious peritonitis virus. zbl antigenic relationship between human corona virus strain c and hemagglutinating encephalomyelitis virus strain n of swine: antibody responses in human and animal sera i~ecovery in tracheal organ cultures of novel viruses from patients with respiratory disease antigenic relationship among the coronaviruses of man and between human and animal coronaviruses detection of eoronavirus infection of man by immunofluorescence seroepidemiology of feline infectious peritonitis virus infections using transmissible gastroenteritis virus as antigen rat corona virus (rcv) : a prevalent, naturally occurring pneumotropic virus of rats serologic studies of naturally occurring feline infectious peritonitis morphologic and physical characteristics of feline infectious peritonitis virus and its growth in autochthonous peritoneal ceil cultures feline infectious peritonitis: something old, something new characteristics of a coronavirus causing vomition and wasting in pigs detection of transmissible gastroenteritis virus neutralizing antibody in cats characterization of a calf diarrheal coronavirus characterization of a calf diarrheal coronavirus morphology-of transmissible gastroenteritis virus of pigs morphogenesis of a virus in cats with experimental feline infectious peritonitis untersuchungen fiber die antigenverwandtschaft der viren der felinen infekti ser peritonitis und der transmissiblen gastroenteritis des sehweines ultrastruetural evidence for the viral etiology of feline infectious peritonitis received october , key: cord- -t py oyw authors: yin, jiechao; glende, joerg; schwegmann-wessels, christel; enjuanes, luis; herrler, georg; ren, xiaofeng title: cholesterol is important for a post-adsorption step in the entry process of transmissible gastroenteritis virus date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: t py oyw cholesterol is a major constituent of detergent-resistant membrane microdomains (drms). we localized transmissible gastroenteritis virus (tgev) spike (s) protein in drms in the viral envelope. though s protein was not solubilized by cold non-ionic detergents, this behavior was unchanged when cholesterol was depleted from viral membrane by methyl-β-cyclodextrin (mβcd) and the protein did not comigrate with cellular drm marker proteins in flotation analyses. therefore, the s protein is not anchored in the viral membrane drms as they are known to occur in the plasma membrane. cholesterol depletion from viral membrane may not affect the adsorption process as neither the sialic acid binding activity nor the binding to aminopeptidase n was reduced post-mβcd treatment. reduced infectivity of cholesterol-depleted tgev was observed only when the adsorption process occurred at °c but not when the virus was applied at °c. cholesterol is important for a post-adsorption step, allowing membrane rearrangements that facilitate virus entry. transmissible gastroenteritis virus (tgev) belongs to the family coronaviridae and can cause severe gastroenteritis in young seronegative pigs (saif and wesley, ; schwegmann-wessels et al., ) . coronaviruses are enveloped viruses with nonsegmented, single-stranded, positive-sense rna (enjuanes et al., , . the spike (s) glycoprotein projects approximately - nm from the virion surface. other structural proteins are the integral membrane (m) glycoprotein, a minor envelope (e) protein, and the nucleocapsid (n) protein. functionally, the s glycoprotein is the major target of neutralizing antibodies (garwes et al., ; jiménez et al., ; laude et al., ; antón et al., ; sune et al., ) , and it is also related to cell tropism (jacobs et al., ; torres et al., ; sánchez et al., ; schwegmann-wessels et al., ) , interaction with its cellular receptor (collins et al., ; delmas et al., ; schwegmann-wessels et al., liu et al., ; shulla and gallagher, ) , pathogenicity (krempl et al., ; garwes et al., ; tuboly et al., ) , fusion (collins et al., ; spaan et al., ; sune et al., ) and hemagglutination activity (krempl and herrler, ; krempl et al., ) . detergent-resistant membranes (drms) are plasma membrane microdomains characterized by insolubility in cold nonionic detergents such as triton x- or brij- and by enrichment of cholesterol and sphingomyelin (glebov and nichols, ) . accumulating evidence suggests the involvement of drms in virus life cycles (simons and ehehalt, ) . cholesterol, a major constituent of drms is important in the entry of nonenveloped viruses such as simian virus (sv ), rotavirus, enterovirus and rhinovirus (suzuki and suzuki, ) . the binding of enveloped viruses to specific cellular receptors and fusion of the viral membrane with a cellular membrane are indispensable for virus entry. correspondingly, the initiation of virus infection may require cholesterol in either of the two membranes involved or in both. previous data show that the infectivity of influenza virus and canine distemper virus is sensitive to cholesterol depletion from the viral membrane (imhoff et al., ; sun and whittaker, ) . in contrast, murine leukemia virus, ebola virus, and marburg virus are sensitive to cholesterol depletion from the cellular membrane (bavari et al., ; lu et al., ) . cholesterol in both membranes is required for the infection by human immunodeficiency virus (hiv) and herpes simplex virus (smith and helenius, ; nayak and hui, as far as coronaviruses are concerned, the depletion of cellular cholesterol by the drug methyl-␤-cyclodextrin (m␤cd), a cholesterol depletion reagent, inhibits virus entry of several coronaviruses: mouse hepatitis virus (thorp and gallagher, ; choi et al., ) , severe acute respiratory syndrome (sars)-coronavirus (li et al., ; glende et al., ) , human coronavirus e (nomura et al., ) and avian infectious bronchitis virus (imhoff et al., ) . more recently, we showed the importance of cholesterol in both the cellular and viral membranes for tgev infection (ren et al., ) . this finding raises the question whether there are drms in the viral envelope. here we analyzed whether the s protein of tgev is located within drms in the viral envelope. though the viral protein was found to be insoluble in non-ionic detergents at low temperature, this solubilization behavior was not abolished by cholesterol depletion. furthermore, in a flotation analysis the s protein did not appear at the position of drm marker proteins. therefore, the s protein in viral membranes does not show the characteristic features of cellular drm proteins. a functional analysis suggests that cholesterol depletion affects a post-adsorption step in the virus entry process that requires membrane rearrangements. swine testicle (st) cells and baby hamster kidney cells (bhk ) were maintained in mem medium supplemented with % newborn bovine serum (excell bio) and passaged twice a week, respectively. tgev strain pur -mad and vesicular stomatitis virus (vsv, strain indiana) were propagated in st and bhk cells, respectively as previously described (ren et al., ) . all viruses used for the experiments were grown in serum-free medium. for cholesterol depletion, virus samples ( × pfu/ml or × pfu/ml) were treated with - mm methyl-␤-cyclodextrin (m␤cd) at • c for min followed by ultracentrifugation to remove residual m␤cd. the virus pellets were resuspended and their infectivity was determined with plaque assays in either -well or -well plates. for cholesterol replenishment, after extraction of viral membrane cholesterol using mm m␤cd, the virus suspensions were replenished with water-soluble cholesterol (sigma) by applying final concentrations ranging from to m at • c for min as previously described (ren et al., ) . the experiment was performed in triplicate. a clarified cell supernatant of ml tgev or vsv ( × pfu/ml) was pelleted at , × g at • c for h. the purified virions were homogenized and solubilized with ml % triton x- in the cold for h. the viral extract was centrifuged at , g at • c for . h to yield a supernatant (non-drms) and a pellet. the pellet was treated with ml np buffer overnight at • c followed further ultracentrifugation as above. the resulting supernatant was designated as drms (detergent-resistant microdomains). the non-drm and drm fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and western-blot analysis. a mouse monoclonal antibody against tgev s protein ( a.c ) and a polyclonal rabbit antiserum against vsv g protein (kindly provided by dr. gert zimmer) were used for detecting the respective proteins. the effect of cholesterol depletion was analyzed by treating virions with mm m␤cd at • c for min prior to detergent extraction. for a flotation analysis, a known lipid raft marker, flotillin- , and cholesterol-deprived virions (see above) of tgev and vsv were solubilized at • c for h and then mixed with the same volume of % sucrose in pbs (w/v). the mixtures were loaded at the bottom of a gradient tube and overlaid by sucrose-pbs cushions ( ml each) of and %. after centrifugation at , × g for h at • c, the fractions were collected and the protein in each fraction was precipitated with water-free ethanol ( %) at − • c overnight prior to % sds-page and semi-dry western blot analysis using mouse monoclonal antibody ( a.c ) against tgev s protein or rabbit polyclonal antibody against vsv g or flotillin- protein (sigma-aldrich), respectively, for the detection of the proteins. haemagglutination-positive virions of tgev were grown in desialylated cells treated with neuraminidase (na) at • c for h as described previously (krempl et al., ) . virus ( × pfu/ml) was treated with m␤cd ( , and mm) at • c for min. the treated viruses and % chicken erythrocytes in physiological salt solution were used for a standard hemagglutination (ha) analysis in micro-titer plates. the experiment was performed in triplicate. after treatment of purified tgev with mu na, virions were ultracentrifuged to remove the residual na. the pellet was resuspended ( × pfu/ml) and treated with m␤cd ( , , , and mm) at • c for min. the virons were ultracentrifuged and resuspended in pbs and the amount of viral protein was determined via photometrical measurement of the uv absorption at nm. st cell monolayers in micro-titer plates were washed three times with pbs, and each well was incubated for h at • c with mu na. after washing with pbs, the cells were incubated with the treated viruses ( g of viral protein per well) at • c for h. the cells were washed three times with pbs and fixed with % paraformaldehyde for min at room temperature, and incubated with . m glycine for min. the fixed cells were incubated with the monoclonal antibody ( a.c ) directed against the s protein and then with a peroxidase-conjugated rabbit anti-mouse antibody. for the detection of bound antibody, the abts [ , -azinobis( ethylbenzthiazolinesulfonic acid)] peroxidase substrate was used. the reaction was stopped with % sds. extinction was measured at nm (schwegmann-wessels et al., ) . the experiment was performed in triplicate. . . the effect of cholesterol depletion on infection by tgev at • c or • c tgev was harvested from na-treated st cells as described above. the harvested viruses ( × pfu/ml) were subjected to mock-treatment or mm m␤cd treatment. st cells were treated with mu na and the desialylated cells were incubated with the viruses at either at • c or • c for h. the cells were washed three times with pbs and overlaid with % (w/v) methyl-cellulose in mem and cultured at • c for h when the plaques were counted. the reduced infectivity of the virus was calculated according to the equation: reduction of virus titer = (the plaque number of cells infected with drug-treated viruses/plaque number of cells infected with non-drug treated viruses) × . at least three independent experiments were carried out. each data point was presented as mean ± sd. statistical significance was evaluated using the t-test. "*" means a value of p < . was considered statistically significant; "**" means a value of p < . was considered statistically highly significant; "***" means extremely significant in statistics. an increased content of cholesterol is a characteristic feature of drms derived from the plasma membrane. as cholesterol depletion from tge virions results in a reduction of viral infectivity (ren et al., ) , we were interested to find out whether the surface protein s that mediates virus entry is localized in drms. for this purpose, we analyzed whether the tgev s protein is resistant to solubilization by triton x- at • c. as shown in fig. a , the s protein was found in the pellet, i.e. it was associated with the triton x- -insoluble fraction. for comparison, we used vesicular stomatitis virus that is not affected in its infectivity when cholesterol is depleted from the viral membrane. the surface protein g of this virus was only detected in the soluble fraction. the same result was obtained when two other non-ionic detergents were used, tween or brij (data not shown). this solubilization behavior would be compatible with the presence of the s protein in detergent-resistant microdomains. to further characterize the membrane association of the s protein, tgev particles were treated with m␤cd to deplete cholesterol from the viral membrane. m␤cd is commonly used to destroy the integrity of drms. application of this drug at a concentration of mm reduced approximately % infectivity of tgev (p < . ), compared with the infectivity of nontreated viruses (fig. b) . to analyze the association of cholesterol with the infectivity of tgev, a cholesterol replenishment assay was performed. as shown in fig. c , the infectivity of tgev restored to % of the value determined prior to cholesterol depletion, after addition of m exogenous cholesterol to m␤cd-treated tgev (fig. c) . the data indicate that the viral infectivity is significantly reduced by m␤cd and can be restored by cholesterol. however, the s protein was still only detected in the detergent-insoluble fraction (fig. a) . this result indicates that the surface protein of tgev is arranged in the viral envelope in a detergent-resistant way that is not affected by cholesterol depletion. to get more information about the membrane association of the s protein, a flotation analysis was performed. overlaid by a - % sucrose gradient, drm proteins are floating to the top fraction upon ultracentrifugation. this approach was applied to the s protein and the vsv g protein after solubilization by % triton x- at • c. as shown in fig. , the vsv g protein was detected in fractions - , but not in fractions - where drm proteins are expected. the s protein was found to have an even higher buoyant density as it was present mainly in fraction . the distribution within the gradient was not affected by prior cholesterol depletion from the viral membrane. in contrast, after the treatment with mm m␤cd, the lipid raft marker protein, flotillin- , migrated from fractions - to a higher density fractions, most of which concentrated in fractions - . these data indicate that the s protein is not localized in typical solubility of viral proteins. the s protein of tgev treated with or without mm m␤cd and vsv g protein were extracted with % triton x- at • c and were detected by western blot by corresponding antibody. drms is the abbreviation of detergent-resistant microdomains (panel a); the infectivity of tgev ( × pfu/ml) treated with mm m␤cd was compared with that of mocktreated tgev (panel b). recovery of tgev infectivity after exogenous cholesterol addition to cells treated with mm m␤cd is shown (panel c). symbol "**" means highly significant difference in statistics, "***" means extremely significant difference in statistics. drms as they have been described to occur in the plasma membrane. nevertheless, the behavior of the s protein upon detergent solubilization is different from that of the vsv g protein. possible reasons are discussed below. the s protein of tgev has two binding activities, binding to n-glycolylneuraminic acid on sialoglycoconjugates and binding to porcine aminopeptidase n (papn). to analyze the effect of cholesterol depletion on the sialic acid binding activity of tgev, we applied a hemagglutination assay. maximum ha titers are obtained with virions that have been enzymatically desialylated to remove fig. . flotation density of viral proteins by sucrose density gradient centrifugation. the flotation density of either tgev s protein or vsv g protein (with or without mm m␤cd) was analyzed using a - % sucrose gradient after treatment of % triton x- at • c the drug concentrations, viral protein and gradient sequence are indicated. sialic acids from the viral surface (krempl et al., (krempl et al., , . therefore, purified virus was treated with neuraminidase prior to cholesterol depletion by m␤cd. as shown in fig. , a concentration of mm m␤cd significantly reduced the viral infectivity. the same concentration did not affect the ha activity of tgev. this result indicates that the effect of cholesterol depletion on infectivity is not related to the sialic acid binding activity. in order to analyze the importance of cholesterol in the viral membrane for binding to papn, the cellular receptor of tgev, viral cholesterol was depleted as described above. to exclude binding to cellular sialoglycoconjugates, st cells were enzymatically desialylated to allow only virus binding to papn (schwegmann-wessels et al., ) . the neuraminidase-treated cells were incubated with cholesterol-depleted virus or with control virus, respectively, for h at • c. unbound virions were removed by thorough washing and bound virus was measured by an indirect elisa. as shown in fig. b , m␤cd treatment lowered tgev infectivity significantly, but had no effect on the binding to desialylated cells. from this result we conclude that cholesterol depletion from the viral membrane does not affect the binding of tgev to papn (fig. a ). to characterize a potential postadsorption step affected in the infection by the cholesterol-depleted tgev we performed an infection fig. . effect of m␤cd-treatment of virions on infectivity and ha actvity of tgev. tgev was harvested from neuraminidase-treated st cells and the resulting viruses ( × pfu/ml) were treated with m␤cd ( , and mm) at • c for min. the treated viruses were used to determine the infectivity and the ha activity. assay where the inoculum was applied for h at either or • c, respectively. the former conditions should allow binding but not concurrent rearrangement of cellular or viral proteins. following the adsorption period, cells were overlaid by methylcellulose and at h.p.i., the number of plaques was determined. as shown in fig. , when the virus was applied at • c, the infectivity titer of cholesterol-depleted tgev was significantly lower compared to the control virus. on the other hand, when adsorption occurred at • c, the titer of m␤cd-treated virus was not reduced, it was cholesterol of neuraminidase treated tgev was depleted with m␤cd ( , , , and mm) at • c for min and then the viruses were used to bind neuraminidase treated st cells. the binding activity between them was analyzed by indirect elisa. the od value was measured at nm wavelength (panel a); the infectivity of tgev ( × pfu/ml) treated with various concentrations of m␤cd was reflected by virus plaque-reduction assays (panel b). the od is from three independent experiments and each experiment was performed in triplicate. binding of tgev virions. desialylated st cells were incubated with untreated or m␤cd-treated tgev ( × pfu/ml) harvested from na-treated st cells for h at • c or • c prior to further incubation at • c for subsequent infection assay. the infectivity of the treated viruses was compared with that of mock-treated viruses at • c (a) or • c (b), respectively. each sample was done in triplicate, and bars indicate standard deviation. symbol "**" means highly significant difference in statistics. even slightly increased when compared to the untreated virus. this result indicates that the effect of cholesterol depletion from the viral membrane on the infectivity of tgev is evident only when infection is initiated at • c but not when adsorption occurs at • c. lipid rafts have been reported to play important roles in different stages of the virus life cycle such as viral entry, protein transport and targeting, as well as assembly and budding (nayak and hui, ) . cholesterol, a characteristic structural component of lipid rafts, is thought to function as a dynamic glue that keeps the raft assembly together (simons and toomre, ) . as far as virus entry is concerned most of the information available is related to the role of drms in the plasma membrane of the target cell where they may act as platforms for concentration of virus receptors and/or for an efficient penetration process. however, the exact mechanism how membrane microdomains contribute to virus entry is not known. this is also true for coronaviruses which are reduced in their infectivity when the integrity of drms is abolished by cholesterol depletion of the target cell membrane (bavari et al., ; li et al., ; ren et al., ; thorp and gallagher, ) . despite this similarity, coronaviruses differ with respect to the location of the cellular receptor. aminopeptidase n, the cellular receptor of human coronavirus e, tgev and some other members of this virus family is constitutively present in drms. by contrast, the receptor for mouse hepatitis virus, ceacam, is usually detected in the detergent-soluble fraction but may be recruited to the drm fraction during the entry process (choi et al., ; thorp and gallagher, ) . in recent years, it has been reported for a few viruses that cholesterol depletion from the viral membrane also results in a reduction of infectivity: hiv- (ono and freed, ) , human herpesvirus (huang et al., ) , canine distemper virus (imhoff et al., ) , varicella-zoster virus (hambleton et al., ) , pseudorabies virus (ren et al., ) , duck hepatitis b virus (funk et al., ) as well as influenza virus (sun and whittaker, ) . as these viruses belong to quite diverse families, the importance of cholesterol in the viral membrane may be a more general feature of many enveloped viruses. the importance of cholesterol in the viral membrane may suggest that drms are present in the viral envelope and may be important for the function of the viral membrane proteins. the evidence, however, is circumstantial as drm association of viral proteins has been analyzed only in infected cells. it has not been demonstrated that the association of viral protein with membrane microdomains is maintained in the viral envelope. it gets even more complicated with viruses like herpesviruses, coronaviruses or hepatitis virus b which mature at intracellular membranes. the concentration of cholesterol and sphingolipids is increasing in the membranes along the secretory pathway, i.e. from the er to the plasma membrane. despite budding from intracellular organelles, surface glycoproteins may reach the cell surface which makes it difficult to distinguish the subsets of membrane glycoproteins that enter virus particles. membrane microdomains are characterized by light buoyant density, insolubility in cold non-ionic detergents, and relatively high levels of glycosphingolipids and cholesterol, which contribute to their detergent-resistant, liquid-ordered structure. drms are typically recovered as low-density material in equilibrium flotation gradients and measuring drm association provides valuable information regarding the preference for rafts by proteins or lipids of interest (ono et al., ) . sedimentation analysis indicated that the tgev s protein cannot be solubilized by cold non-ionic detergents. though this behavior is compatible with the location of a protein in drms, other pieces of evidence do not support this conclusion. cholesterol depletion did not render the s protein soluble by cold non-ionic detergent and in the flotation analysis it was not transferred to fractions where drm proteins are expected. therefore, factors other than cholesterol-rich detergent membranes are responsible for the insolubility in cold non-ionic detergents. interaction with other viral proteins such as the m and/or the e protein may contribute to this behavior. in this respect, it is interesting to note that the vsv g protein behaves different from the tgev s protein. the former protein may interact with other proteins in the viral envelope not as tightly making it easier to solubilize g by detergent treatment. though the effect of cholesterol depletion on virus infectivity cannot be explained by the lipid raft concept, it is evident that cholesterol is important for virus entry. our analysis did not provide any evidence that the two binding activities of the s protein, binding to sialic acids on sialoglycoconjugates and binding to aminopeptidase n, are reduced in cholesterol-depleted virions. therefore, a post-adsorption step appears to require cholesterol for optimal infectivity. stages in the infection cycle following the binding to cellular receptors have also been implicated in the importance of cholesterol for other viruses (funk et al., ; hambleton et al., ) . to understand the role of cholesterol, one should keep in mind that cholesterol depletion results in a reduction but not in the abolishment of virus infectivity. virus entry may occur also at lower cholesterol levels but increased cholesterol makes this process more efficient. in this context it is interesting that cholesterol depletion reduced infectivity only when virus binding occurred at elevated temperatures. when the adsorption step was performed at • c, the infectivity was somewhat lower but unaffected by cholesterol depletion. interestingly choi and coworkers reported a similar finding when they analyzed the infection by mouse hepatitis virus with cholesterol-depleted cells (choi et al., ) . based on these findings we conclude that virus entry can be optimized when binding of virions to the cell surface can be combined with membrane rearrangements that are possible at • c but not at • c. future work has to address the question whether cholesterol facilitates coronavirus entry by affecting the membrane fluidity or whether other molecular interactions depend on an increased content of cholesterol. ) and a visiting scholarship from deutscher akademischer austauschdienst (daad) to x.r. are acknowledged. g.h. was supported by deutsche forschungsgemeinschaft (sfb ) and by the german ministry of education and research (bmbf: fkz ki ) cooperation between transmissible gastroenteritis coronavirus (tgev) structural proteins in the in vitro induction of virus-specific antibodies lipid raft microdomains: a gateway for compartmentalized trafficking of ebola and marburg viruses murine coronavirus 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in the viral envelope characterization and translation of transmissible gastroenteritis virus mrnas critical epitopes in transmissible gastroenteritis virus neutralization characterization of the sialic acid binding activity of transmissible gastroenteritis coronavirus by analysis of haemagglutination-deficient mutants sialic acid binding activity of transmissible gastroenteritis coronavirus affects sedimentation behavior of virions and solubilized glycoproteins point mutations in the s protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus sequence and n-terminal processing of the transmembrane protein el of the coronavirus transmissible gastroenteritis virus lipid rafts play an important role in the early stage of severe acute respiratory syndrome-coronavirus life cycle expression and functional analysis of porcine aminopeptidase n produced in prokaryotic expression system asymmetric requirement for cholesterol in receptorbearing but not envelope-bearing membranes for fusion mediated by ecotropic murine leukemia virus the role of lipid microdomains in virus biology human coronavirus e binds to cd in rafts and enters the cell through caveolae plasma membrane rafts play a critical role in hiv- assembly and release association of human immunodeficiency virus type gag with membrane does not require highly basic sequences in the nucleocapsid: use of a novel gag multimerization assay importance of cholesterol for infection of cells by transmissible gastroenteritis virus cholesterol dependence of pseudorabies herpesvirus entry transmissible gastroenteritis and porcine respiratory coronavirus targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence binding of transmissible gastroenteritis coronavirus to cell surface sialoglycoproteins binding of transmissible gastroenteritis coronavirus to brush border membrane sialoglycoproteins role of spike protein endodomains in regulating coronavirus entry cholesterol, lipid rafts, and disease lipid rafts and signal transduction how viruses enter animal cells coronaviruses: structure and genome expression role for influenza virus envelope cholesterol in virus entry and infection mechanisms of transmissible gastroenteritis coronavirus neutralization virus infection and lipid rafts requirements for ceacams and cholesterol during murine coronavirus cell entry tropism of human adenovirus type -based vectors in swine and their ability to protect against transmissible gastroenteritis coronavirus immunogenicity of the s protein of transmissible gastroenteritis virus expressed in baculovirus national natural science foundation of china ( ) to j.y. national natural science foundation of china ( ; ), funding supported by program for new century excellent talents in heilongjiang provincial university ( -ncet- key: cord- - vj oasa authors: song, xiangjun; zhao, xiaomin; huang, yong; xiang, hailing; zhang, wenlong; tong, dewen title: transmissible gastroenteritis virus (tgev) infection alters the expression of cellular microrna species that affect transcription of tgev gene date: - - journal: int j biol sci doi: . /ijbs. sha: doc_id: cord_uid: vj oasa transmissible gastroenteritis virus (tgev) is a member of coronaviridae family. tgev infection has emerged as a major cause of severe gastroenteritis and leads to alterations of many cellular processes. meanwhile, the pathogenic mechanism of tgev is still unclear. micrornas (mirnas) are a novel class of small non-coding rnas which are involved in the regulation of numerous biological processes such as viral infection and cell apoptosis. accumulating data show that mirnas are involved in the process of coronavirus infection such as replication of severe acute respiratory syndrome coronavirus (sars-cov). however, the link between mirnas and tgev infection is unknown. in this study, we performed microrna microarray assay and predicted targets of altered mirnas. the results showed tgev infection caused the change of mirnas profile. then we selected mir- for further analysis and subsequently identified cell division cycle-associated protein (cdca ) as the target of mir- . moreover, mir- showed the ability to inhibit transcription of tgev gene (a non-structure gene) via directly targeting cdca . in conclusion, differentially expressed mir- that is caused by tgev infection can suppress transcription of tgev gene via targeting cellular cdca . our key finding is that tgev selectively manipulates the expression of some cellular mirnas to regulate its subgenomic transcription. the mirnas are small rna species (containing about nucleotides) which are found in animals, plants, and some viruses. it has been shown that mirnas play a variety of roles in many cellular processes including development, differentiation, apoptosis, cell cycle (reviewed in reference [ ] ). generally, mirnas are derived from introns or intergenes of eukaryotic organisms or viruses and serve as regulators of gene expression [ ] . the mirna-mediated rna interference includes two patterns. one is perfectly base-paring with ' utr of target mrna leading to degradation of mrna. the other is imperfectly binding to target ' utr of mrna resulting in the inhibition of mrna translation. in mammalians, mirnas perform rnai through translation inhibition mediated through imperfect complementarity [ ] . as a result, a mirna can directly interact with multiple targets and a gene can be targeted by many mirnas [ ] . during the interactions between virus and host, mirnas are also playing important regulatory roles [ , ] . increasing evidence indicates that viral infection results in the changes of cellular mirnas species. in turn, the altered mirnas also affect viral infection [ , ] . for example, sars-cov infection up-regulates mir- *, - - p, and - , and down-regulates mir- and mir- . of these mirnas, mir- *, - - p, and - suppress sars-cov replication and ivyspring international publisher contribute to immune evasion in bronchoalveolar stem cells (basc) [ ] . tgev is an enveloped virus with a positive-sense single-stranded rna genome and causes a highly contagious, fatal gastroenteritis primarily in less than -week age piglets around the world. the mortality rate of seronegative suckling piglets infected with tgev can reach to as high as % [ , ] . the tgev genome contains a leader sequence at the ' end and a poly (a) tail at the ' end. the tgev genes are arranged in the order '-rep-s- a- b-e-m-n- - ' [ ] . the tgev gene locates at the 'end of the genome. during tgev infection, protein attenuates host antiviral response by interacting with protein phosphatase catalytic subunit (pp c), a key regulator of host antiviral defense [ ] , and reduces apoptosis, immune response, interferon response, and inflammation [ ] . the mammalian cellular mirnas are profoundly affected by viral infection and, in turn the mirnas also regulate viral gene expression via targeting viral genes or cellular genes. the mir- promotes hepatitis c virus (hcv) rna translation through hybridizing with the ' end of viral transcript [ , ] . the cellular mir- , mir- b, mir- , mir- , and mir- play important roles in the regulation of human immunodeficiency virus (hiv) gene expression through targeting the viral nef gene in cd + t-cells [ , ] . the mir- a binds to cellular ring finger (rnf) mrna to increase hendra virus replication [ ] . to date, complex regulatory networks associated with mirnas have not been comprehensively assessed in tgev infection. overall, we observed that tgev infection caused the change of mirna profile and mir- suppressed transcription of tgev gene via directly targeting cdca . cdca polyclonal antibody ( - -ap) was obtained from proteintech group (proteintech group, chicago, il, us). monoclonal β-actin (sc- ) was purchased from santacruz biotechnology (santa cruz, inc., ca, us). horseradish peroxidase (hrp)-conjugated secondary antibody was purchased from pierce (pierce, rockford, il, us). pk- cells were obtained from american type culture collection (atcc) (ccl- ) and grown in dulbecco's minimal essential medium (dmem) supplemented with % fetal bovine serum (gibco brl, gaithersburg, md, us), iu of penicillin and mg of streptomycin per ml, at ℃ in a % co atmosphere incubator. the tgev shaanxi strain was isolated from tgev-infected piglets by ding l et al [ ] . confluent pk- cells in -mm cell culture dish were infected with tgev for h at an moi of . . meanwhile, the mock infection was carried out. at hpi, total rna was extracted with trizol reagent (invitrogen, carlsbad, ca, us). microarray assay was performed as described previously [ ] using an updated version of the chip (swine mirna version , http://www.mirbase.org/). targets of differentially expressed mirnas were predicted by tar-getscan and miranda. the total rna was obtained using trizol reagent (invitrogen, carlsbad, ca, us) from pk- cells infected with tgev at . moi for h for the microarray analysis. reverse transcription reactions were performed as described previously [ ] . briefly, μg of total rna initially treated with dnase i (fermentas, st. leon-rot, germany), nm stem-loop rt primers, ×first strand buffer, . u/μl rnase inhibitor, u/μl m-mlv, and mm dtt (invitrogen, carlsbad, ca, us), were incubated at ℃ for min, ℃ for min, and ℃ for min. real-time pcr was carried out using the accupower ×greenstar qpcr master mix (bioneer, daejeon, korea) in a μl reaction volume including . μl ×greenstar master mix, . μl ×rox dye, . μl rt product, mm forward primer, and mm reverse primer. reactions were incubated at ℃ for min, followed by cycles of ℃ for sec, and ℃ for min on bio-rad iq real-time pcr system (bio-rad, usa). the relative quantification of mirnas was normalized to u using the ∆∆ct method [ ] . total rna was obtained using trizol reagent (invitrogen, carlsbad, ca, us) according to manufacturer's instructions. the primers for genomic rna (grna) and subgenomic mrnas (sgmrna) of tgev were described previously [ ] . a total of μg of rna was treated with dnase i (fermentas, st. leon-rot, germany) for min at ℃. the treated total rna was reversely transcribed with the first-strand cdna synthesis kit (invitrogen, carlsbad, ca, us). real-time pcr was performed using the accupower ×greenstar qpcr master mix (bioneer, daejeon, korea) in a μl reaction volume on bio-rad iq real-time pcr system (bio-rad, usa). fold variations of the sgmrnas were calculated (normalized to grna). ' utrs of candidate target genes containing the binding site of mir- were respectively amplified by pcr using primers containing sequences of xho i and not i cloning sites and were cloned into the vector psicheck- (promega, madison, wi, usa). to obtain mutation of mir- complementary sites within the ' utr of cdca , seed region was mutated following a mutagenesis protocol [ ] . the mir- mimics (sense strand '-uguggcug ugguguaggccagc- '; antisense strand '-gcug gccuacaccacagccac a- '), a negative control for mimics (an unrelated mimic, sense strand '-ucacaaccuccuagaaagaguaga- '; antisense strand '-ucuacucuuucuaggagguu guga- '), an inhibitor for mir- ( '-gcuggc cuacaccacagccaca- '), and a control rna inhibitor (a random sequence, '-ucuacucuuu cuaggagguuguga- ') were designed and synthesized by ribo biotech (ribobio, guangzhou, china). the mirna inhibitors were modified with '-o-methyl. for the luciferase reporter assay, pk- cells were seeded in -well plates and then co-transfected with ng plasmid and nm of mir- mimics, mir- inhibitors, or negative control, using lipofectamine (invitrogen, carlsbad, ca, us). at h post transfection (hpt), the luciferase activities were measured using dual-glo luciferase assay system (promega, madison, wi, usa) according to the manufacturer's manual. three sirnas (sicdca - , sicdca - , sicdca - ) silencing cdca gene and irrelevant sirna were synthesized by ribo biotech (ribobio, guangzhou, china). the most effective sirna (si-cdca - ), identified by western blot, was applied for the experiments. the sequence of si-cdca - is: sense sequence '-gaaguugauuuccaugg aadtdt- ' and antisense sequence '-dtdt cuucaacuaaagguaccuu- '. the negative control sirna is an irrelevant sirna. pk- cells were transfected with nm cdca -specific sicdca - or irrelevant sirna using lipofectamine (invitrogen, carlsbad, ca, us) according to the manufacturer's guidelines. cells were grown at ℃ for h and then infected with the tgev at moi of . . the total rna was isolated at and hpi. cell viability assay was carried out using cell counting kit- (cck- ) reagent (vazyme, piscataway, nj, usa). pk- cells were plated in -well dishes at a density of × cells/well. cells were transfected with nm sicdca - or irrelevant sirna using lipofectamine (invitrogen, carlsbad, ca, us) according to the manufacturer's guidelines. after h of incubation, the cells were treated with cck- reagent for h. absorbance was recorded with the microplate reader at nm. to construct the cdca expression plasmid, full-length cdca clone was produced by pcr amplification (forward '-ctagctagctagctgg ccgtcagcatggatg- '; reverse '-ctagtcta gactagcaagtggaaggcaggtagtgg- '). pcr products were digested and ligated into pci-neo (promega, madison, wi, usa) at nhe i and xho i sites. the correct constructed plasmid was named pci-neo-cdca . pk- cells were transfected with pci-neo vector (control) or pci-neo-cdca . at hours post-infection (hpt), cells were infected with tgev at moi of . . total rnas were extracted by trizol reagent (invitrogen, carlsbad, ca, us) at and hpi according to the manufacturer's protocol. real-time pcr was performed to detect the sgmrnas level. cells were treated with ripa lysis buffer with phenylmethyl sulfonylfluoride (pmsf). protein concentration was determined with bca protein assay reagent (pierce, rockford, il, us) and separated on a % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) gel and subsequently transferred to a polyvinylidene difluoride (pvdf) membranes (millipore corp, atlanta, ga, us). the pvdf membrane was blocked with % non-fat dry milk for h at room temperature, and then incubated with primary antibodies overnight at ℃ and subsequently with hrp-conjugated secondary antibody at room temperature for h. the signal was detected with enhanced chemiluminescence (ecl). pk- cells were seeded in -well plate ( × cells/well) in growth medium and transfected using lipofectamine with nm mir- mimics, mir- inhibitors, or negative control respectively. at hpt, the cells were infected with tgev at moi of . . the transcription of virus was determined by real-time pcr analysis after and hpi. to gain the overview of the impact of tgev in-fection on host cellular mirnas expression, pk- cells were infected with tgev shaanxi strain at a multiplicity of infection (moi) of . for microarray. the microarray was performed with the total rna extracted from pk- cells infected with tgev at hpi. the differential expression of multiple mirnas in tgev-infected cells in comparison with mock-infected cells was observed in fig. (a) . the expression levels of mirnas were changed remarkably (fold change > . , and p < . ). among them mirnas were up-regulated and mirnas were down-regulated. to validate the microarray results of differentially expressed mirnas, we tested the expression levels of them using real-time pcr. the fold changes of mirnas in tgev-infected cells were referred to that in mock-infected cells. the mirnas levels were normalized to u . the results were correlated with microarray (fig. b) . a total of differentially expressed mirnas were chosen for target prediction. the potential targets of differentially expressed mirnas were predicted with tar-getscan and miranda and , potential target genes were gained (supplementary table s ). to investigate the impact of differentially expressed mirnas on tgev transcription, we selected mir- as a representative based on p value (p < . ), fold change of mirna expression (fold change > . ), and mean fluorescence intensities (mfi) (mfi > ). pk- cells were transfected with mir- mimics, mimics control, inhibitors, or inhibitors control. subsequently all the samples and controls were infected with tgev. the mir- level of the samples was compared with the controls and normalized to u . the mir- level increased after transfection with the mirna mimics and decreased after transfection of the inhibitors in comparison with the control ( fig. a) . the transcription of tgev gene was decreased by overexpression of mir- and increased by inhibition of mir- at and hpi (fig. b) . except for gene , the replication of tgev genome and transcription of other subgenomes were not affected by mir- (data are not shown). to identify the targets that affect the transcription of tgev gene , targets of mir- were predicted by targetscan and miranda (supplementary table s ), and tgev gene does not contain mir- binding site. therefore, we hypothesized that mir- should target cellular gene to inhibit transcription of gene . in order to screen mir- target gene, recombinant dual-luciferase reporter plasmids that contain the binding site of mir- target gene at ' utr of the reporter were constructed for the dual-luciferase assay. the luciferase activity of plasmid containing the ' utr of cdca was markedly lower than that of the control (fig. a) . to generate ' utr mutant of cdca mrna containing mutations of conserved mir- binding site, the site-directed mutagenesis was performed using the dna sequence of wild-type ' utr as the template. two binding sites of mir- seed sequence at cdca ' utr were respectively mutated with the -bp substitution (fig. b) . to determine the direct interaction between mir- and ' utr of cdca mrna, the porcine cdca ' utr or its mutant was respectively subcloned into the ' utr of a renilla luciferase gene in a luciferase reporter psicheck- (fig. c) . the constructs were co-transfected into pk- cells with mir- mimics, mir- inhibitors, or negative control. compared with the control, introduction of exogenous mir- decreased the cdca -wt reporter activity by %, while overexpression and knockdown of mir- using mir- mimics and inhibitors did not affect cdca -mut reporter activity (fig. d) . to assess the specificity of mir- binding, mir- mimics, inhibitor, and cdca -wt reporter are co-transfected. the results showed that the mir- mimics decreased the cdca -wt reporter activity and the mir- mimics and inhibitors had specific binding activity (fig. e) . to determine whether mir- inhibits cdca expression, mir- mimics or mimics control was transfected into pk- cells, and cdca expression was assessed by western blot. introduction of exogenous mir- decreased expression of cdca in pk- cells compared with the control (fig. f) . knockdown of endogenous mir- using mir- inhibitors failed to increase the cdca expression (fig. g) . this is likely due to the lower level of endogenous mir- in pk- cells. moreover, we observed that tgev infection decreased cdca protein levels in pk- cells (fig. h) , while downregulation of endogenous mir- using mir- inhibitors reversed the effect of tgev infection on cdca expression (fig. i) . together, our data conclusively demonstrate that cdca is a direct target of mir- in pk- cells. the mirnas regulate target gene expression via binding to the ' utr of target mrna, and one mirna may target multiple genes. we confirmed that mir- suppressed transcription of gene and targeted cdca directly, but whether mir- inhibits transcription of gene by targeting cdca is unclear. to test the effect of artificial sirna of cdca , three sirnas with different sequences were respectively transfected into pk- cells and the effects of silencing were assessed by western blot analysis. the data indicated that sicdca - was the most effective sirna (fig. a) . the mrna level of cdca was significantly silenced by sicdca - (fig. b) , however the viability of cells was not affected by sicdca - in comparison with irrelevant sirna (fig. c) . to detect the overexpression effect of pci-neo-cdca , we measured the protein level of cdca at hpt using western blotting. as expected cdca expression level in transfected group increased compared with the control (fig. d) . to investigate the effect of cdca on the transcription of tgev gene , we detected transcription level of gene using real-time pcr. the results showed that silence of cdca using sicdca - decreased transcription of gene at and hpi and that overexpression of cdca facilitated transcription of gene at and hpi (fig. e) . to understand the relevance of cdca expression in viral infection, we determined the effect of cdca silencing and overexpression on tgev grna, other sgmrnas, and viral titers. the results showed that cdca had no effect on tgev grna level, other sgmrnas level, and viral titers (data are not shown). taken together, the data presented here demonstrated that tgev infection induced mirnas profiling changes and, among these altered mirnas the up-regulated mir- induced by tgev infection inhibited transcription of gene via directly targeting cdca . the major findings in this study are that tgev infection leads to the change of cellular mirnas expression profile, and altered mirnas regulate transcription of tgev gene through targeting cellular cdca . increasing evidence shows that mirnas play important roles in virus-host interactions including viral transcription, cell apoptosis, and antivi-ral effects of host [ ] . in this study, we examined the connection between mirnas and tgev infection and assessed global expression patterns of cellular mir-nas. based on the findings of this study, we proposed that mir- expression altered by tgev infection is involved in regulating transcription of tgev gene . the infection of coronavirus, such as sars-cov and infectious bronchitis virus (ibv), affects many cellular processes such as viral replication, host innate immunity or signaling pathways [ ] . in some cases, the altered processes are directed by the virus for its own advantage and other altered processes are cellu-lar defense responses to viral infection. in this study, we observed that the expression levels of cellular mirnas are regulated positively or negatively by tgev infection. the mirnas regulate viral infection through interacting with targets. therefore, the mirnas and targets form a complicated regulatory network during viral infection which suggests that this greatly expands the range of possible virus-host interactions. this is in good agreement with the notion that mirnas have evolved as a necessary and critical component to regulate host-virus interactions [ ] . in this study, we observed that tgev altered the mirnas profile and mir- was involved in regulating the interplay between tgev and pk- cells by modulating transcription of tgev gene . it was reported that sars-cov infection resulted in differentially expressed mirnas profile and some of altered mirnas regulated the interaction between sars-cov and host [ ] . these indicate coronaviruses may affect cellular mirnas expression and the altered mirnas are able to regulate viral infection. a key observation in this study was that mir- suppresses transcription of tgev gene . tgev gene , a non-structure gene, contributes to viral invasion and inhibits apoptosis in host cells [ ] . previously we reported that tgev induces apoptosis by multiple pathways [ ] [ ] [ ] . these observations suggest that mir- might be involved in regulating apoptosis through gene . therefore it will be important to detect effects of mir- on apoptosis. mir- was detected in porcine intestinal and milk by deep sequencing [ , ] , but the current studies related to mir- are very few. to identify what gene mir- will target to regulate the transcription of tgev gene , the potential targets of mir- were predicted using targetscan and mi-randa. more than potential targets were obtained (supplementary table s ). since tgev genes were not the potential targets of mir- , mir- must target the cellular genes. we identified the potential targets and obtained a new finding that exogenous mir- directly interacts with the ' utr of cdca mrna and suppresses cdca protein level. cdca is a transcriptional regulator that is directly targeted and activated by transcription factor e f [ ] . the e f plays a crucial role in controlling cell cycle and action of tumor suppressor proteins [ ] . in addition, cdca is a target gene of c-myc and the phosphorylated cdca regulates c-myc-dependent apoptosis and transformation [ ] . therefore it will be very important to discover the regulatory mechanism of cdca during tgev infection. in conclusion, the results of the present study provide evidence that tgev infection resulted in altered profiles of mirnas in pk- cells and the differentially expressed mir- was involved in regulation of tgev transcription by targeting cellular cdca . these findings will give insight into the regulatory mechanism of mirna to transcription of tgev gene . in addition, this opens a new avenue to target host cellular factors using sirna technologies to combat tgev transcription. there are limitations in this study that need to be addressed and resolved in future studies. the mir-nas play regulatory role at post-transcriptional level [ ] . an mirna may target multiple genes, and one gene may be targeted by many mirnas [ , ] . for mir- , it is supposed to take part in regulating the tgev infection process via targeting other genes. for the whole study, numerous differentially expressed mirnas and a quantity of targets of these mirnas form a complex regulatory network to modulate viral-host interactions at any stage of tgev infection. therefore, more functions of altered mirnas on tgev-host interactions need to uncover and more targets need to identify in the future studies. molecular mechanisms of rna interference microrna: a master regulator of cellular processes for bioengineering systems mechanisms of mirna-mediated gene regulation from common downregulation to mrna-specific 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mirna-mediated mechanisms in mammalian cells role of mir- in the regulation of lymphocyte immune function and disease runx : a microrna hub in normal and malignant hematopoiesis xiangjun song and xiaomin zhao designed the experiments, interpreted the data and wrote the article. xiangjun song and xiaomin zhao performed the experiments with assistance and advice from yong huang, hailing xiang, wenlong zhang. dewen tong revised the manuscript. all authors have read the manuscript and approved to submit it to this journal. supplementary the authors have declared that no competing interest exists. key: cord- - gq e f authors: staroverov, sergey a.; volkov, alexei a.; mezhenny, pavel v.; domnitsky, ivan yu.; fomin, alexander s.; kozlov, sergey v.; dykman, lev a.; guliy, olga i. title: prospects for the use of spherical gold nanoparticles in immunization date: - - journal: appl microbiol biotechnol doi: . /s - - - sha: doc_id: cord_uid: gq e f recent years have seen extremely fast development of new viral nanovaccines and diagnostic agents using nanostructures prepared by biological and chemical synthesis. we used spherical gold nanoparticles (average diameter, nm) as a platform for the antigen for swine transmissible gastroenteritis virus (tgev). the literature data demonstrate that immunization of animals with the tgev antigen coupled to gold nanoparticles (gnps) not only activates antigen-presenting cells but also increases the proliferative activity of splenic lymphoid (antibody-forming) cells. the contents of γ-ifn, il- β, and il- in animals immunized with gnp-antigen conjugates were found to be higher than those in intact animals or in animals given the antigen alone. the increased concentration of il- β in the immunized animals directly correlated with the activity of macrophages and stimulated b cells, which produce this cytokine when activated. the increased concentration of il- indicates that the injected preparations are stimulatory to cellular immunity. immunization with the tgev antigen conjugated to gnps as a carrier activates the respiratory activity of lymphoid cells and peritoneal macrophages, which is directly related to their transforming activity and to the activation of antibody generation. furthermore, the use of this conjugate allows marked improvement of the structure of the animals’ immune organs and restores the morphological–functional state of these organs. the microanatomical changes (increased number of follicles) indicate the activation of the b-dependent zone of the spleen and, consequently, the development of a humoral-type immunological reaction. the degradative processes observed in the animals immunized with tgev antigen alone are evidence of weak resistance to pathogen attack. these results can be used to develop vaccines against this infection by employing tgev antigen coupled to gold nanoparticles as a carrier. swine transmissible gastroenteritis is an acute, rapidly spreading, highly contagious enteric disease of pigs of all ages. it is characterized by vomiting, watery and yellow diarrhea, weight loss, and rapid dehydration, leading to a high mortality rate, especially in piglets (mortality decreases with age). swine transmissible gastroenteritis is caused by transmissible gastroenteritis virus (tgev) of the genus coronavirus in the family coronaviridae. the major route of virus transmission is fecal-oral. infected animals cannot digest food properly and die from dehydration. the disease causes great economic losses in pig farms all over the world, and the primary means of animal protection against it is vaccination (cheng and niu ; straw et al. ) . therefore, the development of new vaccines and the implementation of large-scale vaccination programs are important in pig breeding (moxley and olson ( ) . in several countries, vaccination against swine transmissible gastroenteritis is conducted effectively but only with inactivated monovalent and combined vaccines not recognized internationally. traditional inactivated vaccines have high contents of contaminant substances (up to %), causing side effects, and/or may contain, as inactivating compounds, toxic agents such as phenol and formaldehyde, normally used to inactivate microbial cells. moreover, traditional vaccines are usually low immunogenic, because killed microorganisms cannot induce fully functioning cellular immunity. therefore, there is a need to develop new, more effective, and less costly vaccines against swine transmissible gastroenteritis (mout et al. ) . recent years have seen extremely fast development of new viral nanovaccines and diagnostic agents using nanostructures prepared by biological and chemical synthesis. in particular, this can be attributed to the development of biocompatible inorganic nanomaterials-gold nanoparticles. gold nanoparticles are stable metallic nanoparticles which have excellent surface functional chemistry, high biocompatibility, and nil toxicity. in addition, gold nanoparticles can be easily synthesized, giving them shapes such as spheres, rods, cubes, stars, and pyramids. several reports have described the development and use of nanostructures for the delivery of biologically active agents to target cells (mout et al. ; bhattacharya and mukherjee ; staroverov et al. ). to reach their target cells in vivo, nanoparticles must bypass many barriers that protect animals against antigens (dreaden et al. ) . gold nanoparticles (gnps) have been used widely as carriers of antigens and therapeutic agents in human medicine, veterinary practice, and biology . of particular interest is the ability of gnps to induce humoral immune response to weakly immunogenic antigens and haptens (dykman et al. a; dykman et al. b ). m o r e o v er, g n p s c on j u ga t ed t o t g e v h a v e a n immunostimulatory effect (dykman et al. b) . we studied the immunological variable and morphological changes in the immune organs of guinea pigs immunized with tgev antigen conjugated to gnps as a nanocarrier. antigen tgev strain vn- was taken from the museum of strains of the laboratory of virology (scientific research veterinary institute, russian academy of sciences, saratov) and deposited in the all-russian state collection of microbial strains used in veterinary and animal husbandry by the number bvn- ^(patent ru a ). tgev strain vn- with a titer of . - . lg tcd ml − was used. test cultures were inoculated with the same strain at . lg tcd ml − after h of cultivation. tgev was purified in a sucrose gradient according to horzinek et al. ( ) . the resultant virus suspension was used for protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) at a constant voltage of v for min and then at v for h (maniatis et al. ) . for molecular m a s s d e t e r m i n a t i o n s , p r e c i s i o n p l u s p r o t e i n ™ kaleidoscope™ prestained protein standards no. were used. the results of protein separation were processed with the gel-imager and gel explorer, version . , programs (dna-technology, moscow). genome identity was checked by polymerase chain reaction (pcr) using a specific primera conservative part of tgev's s genome, containing base pairs (bp) (herrington and mcgee ) . rna was isolated from tgev by using a nucleos+tm suspension silicate sorbent (biokom, moscow) according to the manufacturer's instructions. pcr results were recorded on a transilluminator with a dna-analyzer video system. pcr products obtained from a preparation of the tgev strain vn- were analyzed by electrophoresis in agarose gel ( . %) using dna ladder as a molecular size marker and dna of bacteriophage λ as a negative control. as viral nucleic acid is responsible for virulence, it was inactivated, after being isolated from tgev and purified, with ribonuclease (rnaase), a hydrolase enzyme that catalyzes rna hydrolysis. the inactivation was done by the procedure described by fedorova et al. ( ) . the antigen resulting from nucleic acid inactivation was used for animal immunization. spherical gold nanospheres (average diameter, nm) were made by the citrate method of frens ( ) . for reduction, . ml of . % aqueous tetrachloroauric acid (haucl ; sigma-aldrich) was heated under reflux to °c on a magnetic stirrer in an erlenmeyer flask. this was followed by the addition of . ml of % aqueous sodium citrate to the flask. the particle diameter was found as described earlier (khlebtsov and dykman ) , by using a libra transmission electron microscope (carl zeiss, germany), a specord s spectrophotometer (analytik jena, germany), and a zetasizer nano-zs particle size and zeta potential analyzer (malvern). to prepare antigen-gold nanoparticle conjugates, we found the bgold number^(minimal amount of antigen that protects the sol against salt aggregation) for the antigen solution. to this end, μl of an antigen solution in pbs (initial concentration, mg ml − ) was titrated twofold on a -well microtiter plate. each well received μl of gnps and μl of . m nacl. the minimal stabilizing concentration for the antigen was . μg ml − . conjugation was done by simple mixing (without the use of coupling agents) (dykman et al. ). thirty six male guinea pigs, weighing - g at the time the experiment started, were used. the animals were cared for and handled in compliance with the guide for the care and use of laboratory animals, the european convention for the protection of vertebrate animals used for experimental and other scientific purposes, and the legislation of the russian federation. for immunobiological studies, the guinea pigs were divided into four groups of nine each. the animals were immunized subcutaneously along the spinal column by two injections with an interval of days between. the guinea pigs in group were injected with . ml of tgev antigen-gnps (antigen concentration, . μg per animal). group received ml of tgev antigen solution ( . μg per animal). group served as the control, which received ml of physiological saline. group served as the control gnps, which received ml of gnps solution. gnp-antigen group was in comparison with group which received only physiological saline (control group). the animals were killed days after the last injection, and blood serum samples were taken for immunological studies. immediately after removal, all pieces of tissues and organs were placed in a fixing solution ( % aqueous neutral formalin), and then -μm-thick histological sections were prepared on a freezing microtome, model (reichert wien, austria). all histological samples were stained with hematoxylin-eosin. for isolating peritoneal macrophages, the animals were killed and then fixed on their backs. an incision was made along the midline of the anterior abdominal wall, and the skin flap was carefully separated, with care taken to keep the peritoneum intact. after a puncture had been made with a needle connected to a syringe, ml of pbs, ph . , was injected into the peritoneal cavity. the anterior abdominal wall was then gently massaged, and after - min, peritoneal fluid was collected with a pasteur pipet through a cut made in the peritoneum and was filtered into a test tube through a nylon filter. the cells were washed three times by centrifugation in pbs at g, after which they were resuspended in ml of pbs and counted in a goryaev chamber. peritoneal macrophages were cultured by standard procedures (leiter ) . for isolating splenic lymphocytes, an incision was made along the white line of the peritoneum after peritoneal macrophages had been isolated, and the spleen was removed. the spleen was minced with scissors, and the tissue pieces were mashed through a fine sieve into a petri plate containing sterile pbs. the resulting suspension was subjected to ficoll-urografin density-gradient centrifugation. the lymphocyte ring was collected in a new test tube. the lymphocytes were washed three times by centrifugation in pbs, ph . , at g for min, and the cell sediment was resuspended in ml of pbs. the lymphocytic cells were counted with a haemascreenvet hematology analyzer (hospitex diagnostics, italy). the titer of antibodies in the sera was estimated by enzyme-linked immunosorbent assay (elisa) with horseradish peroxidase-labeled antibodies against guinea pig igg (jackson immunoresearch, uk). the synthetic peptide was used as the immobilized antigen. the reaction results were recorded on a plate screen microplate spectrophotometer. the interleukin concentration in the sera was measured with a plate screen analyzer (hospitex diagnostics, italy) and using reagents of il- β, il- , and inf-γ (vector-best, russia). respiratory activity was measured conventionally (bernas and dobrucki ) by the ability of cells to reduce nitrotetrazolium blue [ -( , -dimethylthiazol- -yl)- , diphenyl tetrazolium bromide, mtt (sigma-aldrich)] to formazan. briefly, suspensions of known concentrations of isolated animal cells (macrophages and lymphocytes) were centrifuged at g for min, and the sediment was resuspended in ml of . % mtt and incubated at °c for h. after incubation, the cells were centrifuged at g for min and the sediment was resuspended in . ml of dimethyl sulfoxide (fluka, switzerland). the amount of reduced formazan was measured with a genesys s uv-vis spectrophotometer at nm. to construct the calibration curve, we used commercial formazan (sigma-aldrich) at . , . , . , and mg ml − . for studying possible morphological changes in the spleen as an important part of the macrophage system, the animals were immunized with tgev antigen-gnps. the control group comprised of nonimmunized animals. we used the spleens of guinea pigs of the same physiological age. longitudinal and transverse spleen sections ( -μm-thick) were prepared on a freezing microtome, model (reichert wien, austria), by following standard procedures. histological sections were differentially stained with hematoxylin-eosin. the results were statistically processed by the standard procedures using student's t test to evaluate the reliability of the differences between samplings in the experimental and control studies. after the arithmetic mean and the standard deviation for a given data sample were found, we determined the standard error of the arithmetic mean and its confidence limits by student's t coefficient (n, p) at a significance level of % (p = . ). the obtained results were statistically processed by the standard procedures integrated in excel software (microsoft corp., usa). after the arithmetic mean and the standard deviation for a given data sample had been found, we determined the standard error of the arithmetic mean and its confidence limits with account of student's t coefficient (n, p) [number of measurements n = , significance level = % (p = . )]. these results are presented as histograms. the significance of differences between individual samples was evaluated by a two-sample unpaired student's t test with unequal variances. differences were considered significant when the experimentally found p exp value was ≤ . . the reliability of the changes recorded in the results of each of the experiments described above for the entire set of antigen formulations examined was assessed by one-way analysis of variance (anova) by using fisher's ratio test. the dependences found were considered significant at f > f crit , where the critical value f crit at n = and m = - (number of data sets) corresponded to p = . (with the number of degrees of freedom (df) lying between and ) and the effective value p eff was < . . swine transmissible gastroenteritis is caused by an rna virus belonging to group а of the genus alphacoronavirus in the family coronaviridae. the virion has a spherical shape with a diameter of - nm (martins et al. ) . the viral nucleocapsid is a flexible spiral containing single-stranded rna and a large number of nucleocapsid protein molecules (delmas et al. ; sturman et al. ) . the virus replicates in the cytoplasm of mature epithelial cells located at the ends of the intestine villi. a general scheme of the experiment is depicted in fig. . after the antigen was isolated and its protein composition was analyzed by sds-page, we found that the molecular masses of the separated proteins were identical to those of the tgev proteins. the antigen contained the following proteins: p protein (large surface glycoprotein s; molecular mass, - kda), n protein (basic nucleocapsid protein, - kda), and m protein (matrix membrane protein, - kda) (fig. ) . a protein fraction of - kda was also present. according to wesley et al. ( ) , kda is the size of an unglycated structural protein of tgev. thus, all known tgev proteins were separated. the tgev nucleic acid was identified by pcr using a specific primer-a conservative part of tgev's s genome, containing bp. analysis of the pcr products from a purified preparation of the tgev strain vn- , which was done by agarose gel electrophoresis with dna ladder , demonstrated sufficiently high separation of cdna (fig. ) . the presence of only a characteristic cdna fraction (size, bp) indicated the presence of the tgev genome in the sample. after the virus's nucleic acid was inactivated with ribonuclease, the resultant antigen (a mixture of viral capsid proteins) was used for conjugation with gnps and for subsequent animal immunization. the gnp diameter was measured by tem, the average particle size was . nm. analysis of the antibody titer data showed that the titer produced by immunization with gnp-antigen conjugates ( : . ; p = . ) was higher than that produced by immunization with unconjugated tgev antigen ( : . ). in immunology, the mononuclear phagocyte system (mps) (also known previously as the reticuloendothelial or macrophage system) is part of the immune system that consists of phagocytic cells located in reticular connective tissue. the cells are primarily monocytes and macrophages, which accumulate in lymph nodes and in the spleen. the effectiveness of interaction of the gnp-antigen conjugate with cells of the reticuloendothelial system was evaluated using the mtt test. on day after immunization, an increase in the respiratory activity of macrophages was noted (fig. ) . after immunization with the gnp-antigen conjugate, the activity increased by % (p = . ) as compared with the control and by % (р = . ) as compared with the immunization with tgev antigen and % (р = . ) as compared with gnp. a study of the respiratory activity of splenic lymphoid cells (fig. ) showed that after immunization with the conjugate, the activity increased . -fold, as compared to the control, whereas after immunization with tgev antigen alone, it did not change much. the data demonstrate that immunization of animals with tgev antigen-gnps not only activates antigen-presenting cells but also increases the proliferative activity of splenic lymphoid (antibody-forming) cells. this indicates that gnps can present antigens to the organs of the endothelial system. the effect of the conjugate on humoral immunity was assessed by comparing changes in the cytokine concentration between the control and experimental animals. there was a statistically significant increase in the content of the γ-ifn in animals immunized with gnp-antigen conjugates (fig. ) . these animals had the highest content of γ-ifn ( . ± . pgml - ; p = . ) this group was comparison with group which received only physiological saline (control group). we determined the p value error probability when deviating from the null hypothesis (errors of the first kind). we determined that for our research, p was ≤ . .when the animals were given the native antigen and gnps, no appreciable changes in γ-ifn content were found. the content of il- β in animals immunized with gnpantigen conjugate was . ± . pgml - (p = . ), which is higher than it was in intact animals ( . ± . pgml - ) or in animals given the antigen alone ( . ± pgml - ; p = . ) and gnps ( ± . pgml - ; p = . ) (fig. ) (p value for gnp-antigen group was comparison with group which received only physiological saline (control group). gnp-antigen group was comparison with group which received only physiological saline (control group). the increased concentration of il- β in the immunized animals directly correlated with the activity of macrophages and stimulated b cells, which produce this cytokine when activated. this point is so important, since it is desirable to avoid an inflammatory process during immunization, which results in the appearance of granulomas. similar data were obtained with il- ( fig. ) . this fact indicates that the injected preparations are stimulatory to cellular immunity. it is known that interferons, which possess protective properties, are produced in the body only when the virus enters or tumor diseases. that is why the analysis of the effect of immunization with the help of gold nanoparticles on the change in the level of interferon was made. it was found that in the immunized group, the concentration of interferon increased by % (compared to the control) against % for the group immunized with the antigen. when immunizing animals with native antigen, no significant changes in the concentration of interferon in blood serum of the immunized animals were observed. thus, an increase in the concentration of interferon indicates the stimulation of the introduced drugs of the cell link of immunity. the results obtained correlate with research in this area by other scientists. for example, in work (zhang et al. ) showed that tgev n protein induced er stress and activated nf-κb, which was responsible for the upregulation of il- markers protein markers (kda) protein p protein and bcl- expression. tao et al. ( ) also revealed an increase in the concentration of cytokines including interferon gamma and interleukin in response to immunization aunp-m e + scpg. but he mainly considered the reaction of splenocytes to a given complex (their reaction, which was expressed in the production of cytokines, on the effect of aunp) was studied. in addition, aunp-m e was administered together with scpg which is a strong immunomodulator. it was interesting for us to look at the production of these cytokines in the body as a whole and to reveal the general clinical changes that can develop on the introduction of antigen and aunp. one can speculate that gnps facilitate antigen expression with the major histocompability complex on the surface of antigen-presenting cells. this subsequence ensures that the viral peptides are effectively presented to cytotoxic t lymphocytes and natural killers. our data for the increase in γ-ifn activity are in agreement with those for the increase in the respiratory activity of splenocytes on day after the last injection, another indication that colloidal gold can stimulate the release of γ-ifn by t cells, thereby enhancing the activity of lymphoid immunity. in guinea pigs, the spleen forms leucocytes and removes old red blood cells (erythrocytes). the spleen framework is formed by trabeculae, which consist of reticular connective tissue, smooth muscle cells, and blood vessels (at the same time, the spleen contains only efferent lymphatic vessels). the reticular connective tissue includes both red and white pulp. the white pulp is a lymphoid tissue that produces immune and blood cells. the red pulp functions to filter blood for antigens, microorganisms, and defective red blood cells; therefore, it contains various blood corpuscles, including large and small lymphocytes, immature leucocytes, leucocytes at the final stage of graininess, and disintegrating erythrocytes. the spleen is also important for the development of humoral immunity and is a b-system organ. in the animals immunized with gnp-antigen conjugates, the lymphoid follicles had distinct boundaries and contained large number of densely spaced lymphoid cells (fig. c . the lymphoid tissue was well structured and was located in a compact manner. there was a clear boundary between the red and white pulp regions, and there were full of cellular elements. for comparison, we analyzed morphological changes in the spleens of the animals immunized with tgev antigen alone. the observed changes were characterized by the existence of perivascular edema (fig. d) , sometimes accompanied by proliferation and desquamation of the vessels' endothelium. moreover, the lymphoid tissue of the follicles became sparse, and the number and density of lymphoid cells decreased. in spleen tissue sections from the animals immunized with tgev antigen alone, the boundary between red and white pulp was less clear, the size and total number of follicles in the microscopic field decreased, and no germinative centers could be detected. these results point to degradative processes in the immune organs of the animals. in the control group of guinea pigs and gnps, injected with physiological saline, no morphological changes in spleen tissue were observed (fig. a, b) . gold nanoparticles are stable metallic nanoparticles which have excellent surface functional chemistry, high biocompatibility, and nil toxicity (pissuwan et al. ( ) . in addition, gold nanoparticles can be easily synthesized, giving them shapes such as spheres, rods, cubes, stars, and pyramids. this quality allows the use of gold nanoparticles for a variety of purposes, including vaccine delivery (versiani et al. ). there are a series of works devoted to the use of gold nanoparticles as carriers of antigens for pathogens such as the virus grippe (tao et al. ) , virus foot-and-mouth (chen et al. ) , tetanus toxoid (barhate et al. ) , and yersinia pestis (gregory et al. ) . in our work, we used spherical gold nanoparticles (average diameter, nm) as a platform for the antigen for tgev. the data demonstrate that immunization of animals with tgev antigen-gnps not only activates antigen-presenting cells but also increases the proliferative activity of splenic lymphoid a b c d fig. a spleen tissue section from the animals immunized gnps. hematoxylin-eosin staining; magnification, × . b spleen tissue section from the animals control group. hematoxylineosin staining; magnification, × . c spleen tissue section from the animals immunized with tgev antigen-gnps. hematoxylin-eosin staining; magnification, × . lymphoid follicles have distinct boundaries. d spleen tissue section from the animals immunized with tgev antigen alone. hematoxylineosin staining; magnification, × . perivascular edema and sparse lymphoid tissue in the follicles can be observed fig. concentration of il- in animals immunized with gnpantigen conjugates (tgev antigen р = . ; gnp-antigen conjugate р = . ; gnps р = . ) (antibody-forming) cells. the contents of γ-ifn, il- β, and il- in animals immunized with gnp-antigen conjugates were higher than those in the intact animals or in animals given the antigen alone. the increased concentration of il- β in the immunized animals directly correlated with the activity of macrophages and stimulated b cells, which produce this cytokine when activated. the increased concentration of il- indicates that the injected preparations are stimulatory to cellular immunity. several reports have described the development and use of nanostructures for stimulating cellular immunity. for example, xu et al. ( ) showed that gold nanorods associated with the dna vaccine may cause increased formation of γ-ifn in hiv-infected mice. assis et al. ( ) showed that gold nanorods, functionalized with cysteamine and associated with the protein rsm , are stimulatory to il- . in a work, tao et al. ( ) showed that the use of nanostructured vaccine led to an increase in the concentration of igg a, iga, and il- a, il- , il- , tnf-и rantes. our research correlates with these studies but in the present work we used gold nanoparticles for the first time as a platform for antigen for tgev. also, we did nonspecific adsorption of the antigen and did not perform a chemical surface functionalization. this made it possible to simplify the conjugation process, as well as to remove additional procedures related to the purification of the drug from toxic components. additionally we analyzed the morphological changes in the spleens of the animals immunized with gnp-antigen conjugates and tgev antigen. the microanatomical changes (increased number of follicles) indicate the activation of the b-dependent zone of the spleen and, consequently, the development of a humoral-type immunological reaction (galaktionov, ) . the degradative processes observed in the animals immunized with tgev antigen alone are evidence of weak resistance to pathogen attack. in conclusion, immunization with tgev antigen conjugated to gnps as a carrier activates the respiratory activity of lymphoid cells and peritoneal macrophages, which is directly related to their transforming activity and to activation of antibody generation. furthermore, the use of this conjugate allows marked improvement of the structure of the animals' immune organs and restores the morphological-functional state of these organs. the results of this study can be used in the further development of nanovaccines against swine transmissible gastroenteritis. the microanatomical changes (increased number of follicles) indicate the activation of the b-dependent zone of the spleen and, consequently, the development of a humoral-type immunological reaction (galaktionov, ) . the degradative processes observed in the animals immunized with tgev antigen alone are evidence of weak resistance to pathogen attack. funding information this study was funded by the russian science foundation (project no. - - ). conflict of interest the authors declare that they have no conflict of interest. ethical statement all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. the use of gold nanorods as a new vaccine platform against schistosomiasis quillajasaponaria extract as mucosal adjuvant with chitosan functionalized gold nanoparticles for mucosal vaccine delivery: stability and immunoefficiency studies the role of plasma membrane in bioreduction of two tetrazolium salts, mtt, and ctc biological properties of bnakedm etal nanoparticles assessment of gold nanoparticles as a size-dependent vaccine carrier for enhancing the antibody response against synthetic foot-and-mouth disease virus peptide investigation on the porcine epidemic diarrhea prevalent on qinhai antigenic structure of transmissible gastroenteritis virus. ii. domains in the peplomer glycoprotein the golden 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dna vaccine adjuvant for hiv- treatment transmissible gastroenteritis virus n protein causes endoplasmic reticulum stress, upregulates interleukin- expression and its subcellular localization in the porcine intestinal epithelial cell key: cord- -k d a authors: yuan, peng; yang, zhou; song, han; wang, kai; yang, yang; xie, luyi; huang, shilei; liu, jia; ran, lin; song, zhenhui title: three main inducers of alphacoronavirus infection of enterocytes: sialic acid, proteases, and low ph date: - - journal: intervirology doi: . / sha: doc_id: cord_uid: k d a transmissible gastroenteritis virus (tgev) and porcine epidemic diarrhea virus (pedv) are similar coronaviruses, causing diseases characterized by vomiting, diarrhea, and death from severe dehydration in piglets. thus, they have caused huge losses to the swine-breeding industry worldwide. nowadays, they are easily transmitted among the continents via vehicles, equipment, and cargo. both viruses establish an infection in porcine enterocytes in the small intestine, and their spike (s) proteins play a key role in the virus-cell binding process under unfavorable conditions when the intestine with a low ph is filled with a thick layer of mucus and proteases. sialic acid, proteases, and low ph are three main inducers of coronavirus infection. however, the details of how sialic acid and low ph affect virus binding to the host cell are not determined, and the functions of the proteases are unknown. this review emphasizes the role of three factors in the invasion of tgev and pedv into porcine enterocytes and offers more insights into alphacoronavirus infection in the intestinal environment. two porcine coronaviruses, transmissible gastroenteritis virus (tgev) and porcine epidemic diarrhea virus (pedv), are clustered as different species into the alphacoronavirus genus. they both are important viral pathogens in piglets, causing similar pathological characteristics with acute diarrhea and dehydration [ , ] , leading to massive losses in the modern swine-breeding industry worldwide. although their transmission route is limited to the fecal-oral route, as economic globalization increases rapidly and transportation develops remarkably, vehicles, equipment, and cargo have been convenient for these viruses to spread to all continents. tgev and pedv replicate in enterocytes of the small intestine and are the causative agent of a fatal diarrhea in newborn piglets. doyle and hutchings [ ] described the hku , and munia coronavirus hku . both γ-and δ-covs have also been found in mammals [ ] . α-, β-, γ-, and δ-covs are genera of coronaviridae that are clustered together based on numerous studies and serological and genotypic criteria (table ) . each genus has its own representative cov species. tgev, pedv, and human coronaviruses (hcov- e and hcov-nl ) are typical viruses of the genus α-cov. the representative species of β-cov are mouse hepatitis virus and severe acute respiratory syndrome coronavirus (sars-cov). infectious bronchitis virus is currently the most studied virus in the γ-cov genus [ ] . little is known about δ-cov. this review emphasizes the role of three factors (sialic acid, proteases, and low ph) in the invasion of tgev and pedv into porcine small intestine epithelial cells and provides information with respect to α-cov infection that brings new insights into virus research. the spike (s) protein of covs is essential for the interaction with receptors and the fusion of the viral particles and for cellular membranes. it also plays a crucial role in the interspecies transmission of covs. the interactions of cov s glycoproteins with receptors on the cell surface determine the host range and tissue tropism of covs [ , ] . virus infection begins with the interplay between the fig. . phylogenetic tree of the coronavirinae subfamily. the phylogenetic tree was built on the basis of the nucleotide sequences of complete spike genes from coronaviruses. the nucleotide sequence alignment and the construction of the phylogenetic tree were completed using the mega . program with a proper substitution model: d = transitions + transversions, and all other settings were maintained as default. the final depiction of the phylo-genetic tree was completed using itol on the internet. as the map shows, the coronavirus family is divided into main groups, and coronaviruses are regarded as representatives, which are shown in various colors. interestingly, the spike gene of pedv isolate strain zj hz is separated into a single group in the phylogenetic tree. s protein and its specific receptors, followed by penetration into the cells by a fusion event [ , ] . the s protein, a class i fusion protein, is a membrane protein. it is the largest glycoprotein of covs [ ] , projecting out from the surface of cov particles and forming a homotrimer structure called a peplomer. the s protein is responsible for the corona-like appearance of the surface projections in the electron microscope. the peplomer includes a globular portion and a protein stalk. by adopting the helical structure that is characteristic of class i virus fusion proteins, the protein stalk connects the globular portion to the transmembrane domain [ ] . the nterminal s domain constitutes the globular region, and the stalk is made up of the membrane-proximal s domain. the n-terminal s domain and c-terminal s domain of the s protein play a similar role in all covs, the s region is related to receptor binding, and the s domain plays a role in the membrane fusion process. in addition, the s domain contains two subdomains, an nterminal domain (ntd) and a c-terminal domain (ctd) (fig. ) . the two subdomains, called rbds (receptor binding domains), bind with specific cell receptors, including a series of proteins and sugars. thus, determinants in the s domain are not only crucial for initiating virus entry into cells, they also determine the cell and host tropism of covs [ ] . tgev and pedv s proteins have many similarities in their secondary structure. the tgev s protein is produced as a , -amino acid precursor polypeptide with a -residue signal peptide. according to analysis of the s protein sequences of covs with different biological phenotypes, there are antigenic sites (c, b, d, and a in that order) in the n-terminal half of the s protein, among which the a site can induce neutralizing antibodies [ ] and is highly conserved in tgev and porcine respiratory coronavirus (prcov), a respiratory variant of tgev. the s protein encoded by prcov lacks about amino acids in the n-terminal region that contain determinants related to the enteropathogenicity of tgev [ ] . pedv has a -to -kda spike glycoprotein with a homotrimeric structure. the pedv s protein is also a type i transmembrane glycoprotein that contains regions, a signal peptide (residues - ), an s region (residues - ), an s region (residues - ), a transmembrane domain (residues - ), and a cytoplasmic tail (residues - ) [ ] . the s region is responsible for virus particle binding to the cellular receptors, whereas the s region participates in membrane fusion of the virus and host cells. like other covs, the s region possesses two subdomains comprising an ntd (residues - ) and a ctd (residues - ). the ctd of the s domain binds to a functional cellular receptor for pedv infection. sequence analyses of pedv prototype and variant strains reveal that the n terminus of the s protein changes more easily than the c terminus. in addition, a previous report suggested an interaction of ntd of the s domain with a coreceptor [ , ] . simplified graphic of the structural domains of the main coronaviruses' spike proteins. the spike protein structure can be divided into the s and s domains, and the structural domains in the spike protein are located in the order (from c to the n terminus) as: transmembrane (tm), heptad repeats (hrs) in the s domain, c-terminal domain (ctd), and n-terminal domain (ntd) in the s domain as well as the signal peptide (sp). in the graphic, compared with tgev, prcov lacks an ntd, which is associated with enteric tropism. the rbd in the sars spike protein is shown as discontinuous in the graphic. the α-cov rbd comprises about residues that adjoin the ctd in the s region ( fig. ) . previous studies on the ctd concluded that the structure had the characteristic of independent expression of the s protein and preservation of its native structure maintained the binding specificity [ ] . several structural studies indicate that the α-cov rbd adopts a β-barrel fold with highly twisted β-sheets, in which β-strands (β , β , and β ) run parallel and disulfide bonds exist in the β-structures [ ] . the rbd of tgev is located within the ctd of the s domain [ ] . in the tgev rbd crystal structure, the bent-strand β crosses both β-sheets. n-linked glycans are concentrated at one side of the β-barrel; the opposite side is not glycosylated and might be closer to other s protein domains. the n-and c-terminal ends of the rbd are located on the same side of the domain (terminal side); at the opposite side, β-turns form the tip of the barrel in the tgev rbd [ ] . like tgev, the crystal structure of a single domain unit in the prcov rbd adopts a β-barrel fold with highly twisted β-sheets located in the ctd of the s domain and engages in binding to the host cell surface receptor. compared with the tgev s domain, the related prcov lacks the ntd, which is related to enteric tropism. the tgev or prcov rbd tips consist of protruding β-turns (β -β and β -β ), each having a solvent-exposed aromatic residue (tyrosine or tryptophan) [ ] . in the tertiary structure of the prcov rbd, loops (β -β , β -β , and β -β ) at the tips of the β-barrel domains are responsible for receptor binding. some researchers found that single amino acid mutations in the loops completely or significantly reduced the ability of prcov rbd to bind to host receptors, and mutations outside the rbd had no effect on receptor recognition [ ] . to date, there have been few reports on the rbd structure of pedv. it is interesting that receptor binding mutant proteins, rbm - , rbm - , and rbm - proteins, did not significantly reduce pedv papn-binding activities in virus infection [ ] , suggesting the pedv s protein uses a receptor-binding mechanism different from tgev and prcov. pedv is further confirmed to have a broader receptor range than other α-covs [ ] . during the progress of evolution and adaptation to diverse hosts, covs have evolved to use various receptors to enter host cells. different hosts or virus strains produce evolutionary diversity in the same virus family, and the binding of covs to susceptible cells seems to show variation in the receptors used that correspond to viral groups and species. these covs recognize distinct cellular receptors and coreceptors, such as proteins and sugars, to facilitate their penetration into cells [ ] . currently, there are main protein receptors: apn, angiotensin-converting enzyme (ace ), carcinoembryonic antigen-related cell adhesion molecule (ceacam ), and dipeptidyl peptidase (dpp ). most members of α-cov use apn as the receptor for infecting host cells, such as tgev, pedv, and hcov- e. apn, also known as cd , is a -kda type ii transmembrane protein that belongs to a membrane-bound metalloprotease family [ ] . interestingly, hcov-nl , as an α-cov, shares the same cell entry receptor, identified as ace , with sars-cov, which is a β-cov. ace is a type i integral membrane glycoprotein with an n-terminal extracellular domain comprising α-helical lobes, between both of which there is a catalytic site with a coordinated zinc ion [ ] . by contrast, the β-cov mouse hepatitis virus utilizes ceacam as a cell surface receptor for the s protein. ceacam , the first identified cov receptor [ ] , is a type i transmembrane multifunctional protein and a member of the immunoglobulin superfamily termed igsf. middle east respiratory syndrome-related cov, belonging to β-cov, has been shown to use dpp as a cell entry receptor. dpp (also known as cd ), a type ii membrane protein, is a multifunctional membrane-bound serine protease that forms homodimers on the surface of host cells. the dpp ectodomain comprises about amino acids and has domains, an α/β-hydrolase domain and an -bladed propeller [ ] . among these cellular receptors and coreceptors, apn is a major cell entry receptor for covs. apn exists on the epithelial cell surface of different tissues. in particular, it is expressed abundantly in the brush border membrane of the small intestine, the kidney, and the respiratory tract [ ] . most α-covs use apn as cell entry receptor. for example, previous studies showed that tgev uses porcine (p) apn as the receptor in the entry process [ ] , whereas human (h) apn is a receptor for the infection by hcov- e [ ] . the reason why tgev uses apn as an entry receptor has not been clarified. it might be linked to its abundance on the surface of epithelial cells rather than its biological function, which seems to be dispensable for cov binding capacity [ , ] . in the small intestine mucosa, apn occupies about % of the total protein content of the differentiated enterocytes [ ] . for pedv, although there are many articles in which papn was proposed as the recep-doi: . / tor in pedv infection, this view has been questioned due to the lack of robust direct evidence. the characteristic structure of apn is a large glycosylated ectodomain with a zinc metal ion at the active site, which functions as a zinc-dependent protease responsible for cleavage of the n-terminal amino acids, mediated by the helah motif [ ] . the enzymatic function of the papn catalyzes the removal of amino acid residues from the n termini of oligopeptides, and apn has been termed the "moonlighting enzyme" because of its many cell functions. apn can be cleaved into n-terminal ( kda) and c-terminal ( kda) subunits by trypsin digestion and comprises domains (di-div) [ , ] . it is heavily glycosylated and forms dimers through extensive div-div interactions. sequence conservation in the rbd tip of α-covs exerts a crucial function in which the apn recognition mode is highly conserved [ ] . moreover, the specificity of apn with the recognition structure in the s protein is linked to the structure of the apn n-linked glycan and fusion with the rbd β -β turn. in addition, the covs tyrosine and tryptophan residues are critical in forming the tgev rbd-apn structure. hcov- e does not have a tyrosine in its rbd β -β turn, hence it recognizes the human apn that lacks this form of glycosylation [ , ] , meaning that hcov- e recognition of apn must be unique. there have been some studies that determined the structure of a protruding tip for binding to small apn cavities in this human α-cov. the s proteins of pedv and tgev share high homology, but they have different host preferences. in addition, pedv has been verified to use a different receptor recognition model compared with tgev, prcov, and hcov-nl . the n-terminal region in the pedv s domain binds to sugars, which are regarded as its coreceptor [ ] . in addition to binding to defined protein receptors, some covs show a sialic acid-binding activity. at present covs in α-, β-, and γ-cov have developed variant sialic acid binding activities [ ] . according to current research, types of sugars have been characterized as receptors or coreceptors for cov entry into host cells: -nacetylneuraminic acid (neu ac), -n-glycolylneuraminic acid (neu gc), and -n-acetyl- -o-acetyl neuraminic acid (neu , ac ) [ ] . recognition of sugars as co-receptors of tgev and pedv seems to be a strategy by which these viruses adapt to the living organism, meaning that tgev and pedv bind to sialic acid to survive under unfavorable intestinal tract conditions. tgev was first described to have a sialic acid binding activity in [ ] . the sialic acid binding activity resides in the n-terminal portion of the s subunit that has been linked to the enteropathogenicity of tgev and that is absent from the s protein of prcov. the sialic acid preferentially recognized by tgev is n-glycolylneuraminic acid (neu ac) [ ] . the spike protein has a trimeric structure and retains its sialic acid binding activity in soluble forms of the protein [ , ] . tgev recognizes and binds the sugar moieties of glycoconjugates that are highly o-glycosylated which promotes binding but is not sufficient for initiation of infection. it is believed that abundant sialic acid in mucins aids tgev to penetrate the mucus layer and then to get access to papn on the surface of the intestinal epithelial cells. thus, the efficiency of infection can be enhanced under unfavorable conditions. binding to papn and sialic acid are two independent processes. interestingly, binding to papn is more efficient in the absence of sialic acid [ ] . there are few studies on the binding of pedv to sialic acids. pedv has been proven to have the ability to bind sialic acids. neu ac presented the highest binding affinity with pedv s in experiments using a glycan array screening. moreover, the sialic acid binding region of pedv was confirmed as being in the ntd of the s protein (residues - ), similar to other covs [ ] . however, it is unknown how the binding of sialic acid to the s protein can affect pedv entry into cells. increasing numbers of proteases have been demonstrated to participate in pedv and tgev infection of host cells in mechanisms where they do not act as receptors. these proteases are reportedly involved not only in adaptation of virus to innate immune response, but also in proteolytic processing of the s protein. covs always produce two types of cysteine proteases, a chymotrypsin-like main protease and papain-like proteases (pl pro and pl pro). in general, they are essential for replicase polyprotein processing and viral replicationtranscription. pl pro, a nonstructural protein of tgev, resides in the nonstructural protein nsp subunits of tgev [ ] . the structure of tgev pl pro resembles a right hand with distinct regions, the palm, thumb, and fingers (fig. ) cysteine residues binding the zinc ion, and a catalytic triad formed by residues cys , his , and asp [ ] [ ] [ ] . the tgev pl pro protein, which contains a socalled usp-like binding site, exhibits deubiquitinating enzyme activity in vitro [ ] . deubiquitinating enzymes play essential roles in the innate immune response with interferon (ifn) secretion into the intestine mucosa. in the intestine, the tgev entry is restricted by ifns in epithelial mucosa. the pathogen-associated molecular patterns of tgev, virus rnas, are sensed by pattern recognition receptors in host cells [ ] , triggering the ifn expression. tgev arouses a early ifn-α production in intestinal secretions [ ] , ifn-α can improve b-cell response to provide protection against reinfection [ ] . ifn-λ is the main mucosal antiviral cytokine responsible for resisting virus invasion in the gut, such as tgev and pedv [ ] , together with interleukin- , which forms the tissue barrier in intestinal epithelial cells to reduce viral infection [ ] . tgev pl protease possesses deubiquitinating activity and hydrolyzes the peptide that binds both lys -and lys -linked polyubiquitin chains [ ] . regulation of signaling molecules by ubiquitin has a significant function in the activation of the ifn response. tgev pl binds and deubiquitinates retinoic acid-induced gene rig and stimulator of interferon gene sting, which are regulators in the ifn signaling pathway, and then the levels of phosphorylated ifn regulatory factor exhibit reduced activity [ ] , which counteracts ifn regulatory factor translocating into the nucleus to activate the transcript of ifns, such as ifn-α/β and ifn-λ. tgev adopts the strategy to interfere and inhibit ifn secretion into the intestine gut to improve the efficiency of virus invasion. previous reports confirm that proteolytic processing of the s protein contributes to the fusion of the viral membrane with cellular membranes and is necessary for virus entry. proteases in virus entry are capable of cleaving the s protein. these proteases in the pig small intestine potentially facilitate pedv infection of host cells [ ] . pedv successfully infects african green monkey kidney (vero) cells in vitro with extracellular trypsin [ ] . the use of trypsin improves the possibility of pedv entry, because trypsin facilitates s protein-mediated fusion with the plasma membrane to deliver viral genomes into host cells. it has been shown that trypsin is helpful for syncytium formation in pedv infection of mdck cells [ ] interestingly, pedv can also propagate without trypsin, suggesting that trypsin might be relevant for cellcell fusion rather than viral envelope-cell membrane fusion [ ] . for pedv entry into vero cells under the trypsin-free conditions, endogenous proteases in endosome may adopt trypsin-like function, prompting pedv s-mediated fusion. ttsp is a type of trypsin-like serine protease termed type ii transmembrane serine protease. ttsp is confirmed to cleave and activate proteins on the surface of influenza viruses and covs, allowing multicycle replication in the absence of trypsin. ttsps are reportedly involved in the release of pedv virions [ ] . tmprss and mspl are members of ttsps. tmprss and mspl exhibit trypsin-like features in the amplification of pedv in vitro in the absence of trypsin and play a vital role in cellcell fusion and virus-cell fusion. it is found that pedv s protein is colocalized with tmprss and mspl extensively and cleaved by coexpression with tmprss or mspl. tmprss and mspl could cleave pedv s protein into two fragments of the same size. the two ttsps interact with the pedv s protein to promote viral entry into cells by promoting cell-cell fusion and virus-cell fusion. moreover, mspl exhibited the strongest effect in the replication of pedv compared to tmprss . interestingly, the adaptive capacity and the growth of pedv in vero cells expressing tmprss and mspl are higher than those in cells treated with trypsin [ ] . it is comfirmed that pedv requires serine and serinelike proteases (fig. ) for its entry through endocytosis in the early stages of infection. for cell-cell fusion, serine proteases are involved in pedv entry in an acidic phindependent manner [ ] . cellular serine proteases pos- fig. . ribbon drawing of the tgev pl pro [ ] . the entire structure resembles a hand, with the palm, thumb, and fingers represented in yellow, green, and blue, respectively. the catalytic triad is presented as magenta spheres, and the zinc ion is a gray sphere. color version available online doi: . / sess a catalytic triad of amino acids comprising his, asp, and ser, which are located in similar positions at the -dimensional structure. the nucleophilic ser is responsible for cleavage and is often replaced by a functionally and spatially equivalent cys in viral trypsin-like proteases [ , ] . the asp of the active-site residues can be substituted by an equivalent glu. serine or serine-like proteases in the cytoplasm are required for the fusion between the pedv envelope and the host endosomal membrane. serine proteases activate pedv entry in a low ph-independent manner. new evidence has emerged that papn in the small intestine acts as a protease for pedv, which contrasts with the idea that papn plays role as a cell surface receptor. the structure of apn is shown in figure . with regard to the similarities between pedv and tgev and the accordance of the pathology of pedv infection with the tissue distribution of papn, papn used to be regarded as a receptor for pedv, which was supported by some indirect evidence [ , ] . however, pedv can also infect and propagate in papn-negative vero cells [ , ] , indicating that there is no direct evidence to support the view that papn is a receptor for pedv. in current research, by employing cpk cells to express porcine homologs of papn, ace , ceacam , and dpp , results were obtained showing that cpk cells were susceptible to tgev, but not to pedv. in addition, pedv infection was not affected by soluble papns, suggesting that pedv utilizes different receptors compared with other covs. by contrast, another study showed that when papn was overexpressed in porcine cpk cells, papn enabled pedv multiplication [ , ] . another study showed that nonpermissive st cells expressing a papn gene supported productive infection of pedv of the host cells, showing that constitutive overexpression of papn directly promotes pedv multiplication [ ] . in summary, papn more likely functions as a protease in pedv infection rather than as a receptor, which is supported by research evidence; however, the mechanism by which papn promotes pedv infection remains unknown. it is unquestionable that ph is important for the process of cov entry into cells. low ph is necessary for conformational changes of viral glycoproteins and proteolytic activation of viral glycoproteins by endosomal proteases [ ] . it is reported that ph-dependent conformational changes occur in the cov peplomer [ ] , and the s domain is released from peplomers on the surface of the virus. inside the endosome, endogenous proteases participating in membrane fusion are active in a low-ph environment. tgev and pedv tend to multiply at a low ph. the amount of tgev in the medium at ph . was -fold greater than that at ph . , and the yield was almost -fold higher than that at ph . [ ] . tgev binds to apn and enters the host cell via endocytosis. upon attaching to the porcine apn receptor, tgev is incorporated into the cell membrane and enters by way of caveola-dependent endocytosis [ ] . subsequent membrane fusion is promoted by active cellular proteases in the endosome in a low-ph environment [ , ] . using mdck cells overexpressing porcine apn, it has been demonstrated that acidification of the intracellular compartment promotes membrane fusion inside the endosome and cellular proteases are activated in a phdependent manner to facilitate membrane fusion [ , ] . hxe) . the x-ray diffraction method was used to test the structure. the structure comprises α-helixes (α -α ) and β-sheets (β -β ) presented in diverse colors in the light of different domains. there is an na + site close to β (yellow), shown in stick form. different from the caveola-mediated uptake of tgev, pedv enters cells using endocytosis via clathrin-coated pits. after the first step of interacting with receptors on the cell surface, pedv penetration is facilitated by viral envelope fusion with the host cell plasma membrane, which happens inside a ph-dependent endosomal compartment. the endosomal cellular elements are very important for the activation of low phdependent proteases, rather than the virus needing an acidic environment to trigger its entry [ , ] . the acidic conditions do not affect pedv itself, suggesting that acidic ph is not the only factor that affects viral infection of host cells. interaction between the s protein and the host cells is an indispensable step for viral entry into host cells. in the process of tgev and pedv entry into cells, the environmental conditions in the intestinal gut are complex and unfavorable. a thicker layer of mucus containing sialic acid covers the intestinal epithelium, and the small intestine generally has a low ph. in addition, the intestine is filled with proteases from the stomach and intestinal wall. these conditions make it hard for viruses to infect intestinal cells compared with infection of cultured cells. the selection of tgev and pedv rbds interacting with cell surface receptors is the result of pressure from immune surveillance. sialic acid usage by tgev and pedv promotes the efficient invasion into host cells. pedv and tgev invade enterocytes in the intestinal epithelium by exploiting proteases in the small intestine. tgev induces pl pro to hijack cellular canonical pathways to prevent viral protein degradation, facilitating later virus entry into targeted cells. tmprss and mspl interact with the pedv s protein to promote viral entry into cells by promoting cell-cell fusion and virus-cell fusion. recently, porcine apn has been observed to function as a protease for pedv, not as a receptor, to promote viral multiplication during pedv entry into the cell, the mechanism of which requires further study. low ph in the initial stage of pedv entry into intestinal epithelial cells allows structural changes 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mediated polarized infection by porcine epidemic diarrhea virus in target cells clathrin-and serine proteases-dependent uptake of porcine epidemic diarrhea virus into vero cells monoclonal antibodies to the peplomer glycoprotein of coronavirus mouse hepatitis virus identify two subunits and detect a conformational change in the subunit released under mild alkaline conditions the influence of ph on the growth and stability of transmissible gastroenteritis virus in vitro evidence for a putative second receptor for porcine transmissible gastroenteritis virus on the villous enterocytes of newborn pigs the coronavirus transmissible gastroenteritis virus causes infection after receptor-mediated endocytosis and acid-dependent fusion with an intracellular compartment a lysinemethionine exchange in a coronavirus surface protein transforms a retention motif into an endocytosis signal inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry we thank zhangcheng li (college of animal science, southwest university) for his guidance in constructing the evolutionary tree. this project has been supported by the fundamental research funds for the central universities (xdjk d ). no potential conflict of interest is reported by the authors. key: cord- -g dqiki authors: costantini, v.; lewis, p.; alsop, j.; templeton, c.; saif, l. j. title: respiratory and fecal shedding of porcine respiratory coronavirus (prcv) in sentinel weaned pigs and sequence of the partial s-gene of the prcv isolates date: - - journal: arch virol doi: . /s - - -z sha: doc_id: cord_uid: g dqiki porcine respiratory coronavirus (prcv), a spike (s) gene deletion mutant of transmissible gastroenteritis virus (tgev), causes mild or subclinical respiratory infections in pigs. the shedding of prcv/tgev was studied at different days post-arrival in fecal and nasal swabs from prcv/tgev seronegative sentinel pigs introduced into a prcv seropositive herd with questionable tgev serology and diarrhea. nasal shedding of prcv was detected in % and % of samples by nested-rt-pcr and cell culture immunofluorescence (ccif), respectively. however fecal shedding of prcv was detected in % of the samples by nested-rt-pcr and % by ccif. four respiratory and fecal prcv strains were isolated in swine testicle cells including nasal/fecal prcv pairs (isolated at the same time) from pigs. comparison of nasal/fecal prcv pairs from individual pigs revealed different deletions in the spike (s) gene ( or nt) in pairs and a consistent change in nt / (aa t to v) for all pairs. in preliminary studies, inoculation of gnotobiotic pigs with each plaque-purified pair of the nasal and fecal prcv isolates, revealed no clinical disease but different tropisms. the nasal isolate was shed both nasally and in feces, but the fecal isolate was shed only marginally in feces, and not nasally. our results show that nested-rt-pcr was as sensitive as ccif for prcv detection in nasal swabs, but was more sensitive than ccif for prcv detection in fecal samples; alternatively prcv shed in feces was more labile with loss of infectivity. the s-gene sequence differences found between the fecal and respiratory prcv isolates may influence their tissue tropism. these new prcv isolates should be useful to understand the molecular basis of coronavirus tropism and evolution in infected swine. porcine respiratory coronavirus (prcv) is a deletion mutant of transmissible gastroenteritis coronavirus (tgev) with altered respiratory tissue tropism [ , ] . transmissible gastroenteritis virus causes fatal diarrhea in neonatal piglets. it selectively infects and replicates in the villous enterocytes of the small intestine, causing subsequent malabsortion and dehydration characteristic of transmissible gastroenteritis (tge) [ ] . transmissible gastroenteritis virus has also been shown to replicate in the upper respiratory tract tissue of infected swine [ , ] . porcine respiratory coronavirus is genetically and antigenically related to tgev, but it has a selective tropism for respiratory tissue causing mild or subclinical respiratory infections with limited to no replication in the intestinal tissue of infected swine [ , , , ] . during routine serological surveillance of pig herds in great britain, belgium, holland and france in the s, an increase in the number of herds with antibodies to tgev was noted but without concomitant increases in clinical enteric disease. a coronavirus, prcv, was isolated in from respiratory tissues of affected pigs in belgium [ ] , great britain [ ] and later in other parts of europe. several years later another strain, prcv-ind was isolated from pigs in the u.s. [ ] . both tgev and prcv contain a single-stranded positive-sense rna genome of about kb and produce - subgenomic mrnas during viral replication. the major structural proteins, the spike (s), the integral membrane (m) glycoprotein and the nucleocapsid (n) protein are translated from mrnas , and , respectively. the mrnas , - or a, b encode two putative nonstructural proteins [ , ] . comparison of tgev and prcv strains revealed that prcv has a large deletion in the region of the s gene, and minor deletions in genes / a and - / b [ , ] . most european prcvs have an identical deletion of nt in the same position at the end region, suggesting that they were derived from the same precursor [ ] . in contrast u.s. prcv strains, have deletions of various sizes ( - nt) located in different positions, suggesting that they originated independently [ ] . because this deletion is present in all independently derived prcvs, it has been proposed that the size and position of the deletion is related to the differences observed in tissue tropism between prcv and tgev [ , ] . investigators have suggested that amino acid changes at the n-terminal region of the tgev s protein also affect the enteric tropism of the pur strain of tgev [ ] . the s protein has a glycosylated membrane anchoring domain and is thought to be the viral attachment protein that interacts with the cell receptor, porcine aminopeptidase n (apn) [ , ] . however a second region in the s protein (around amino acid ) also influences the enteric tropism of tgev [ ] . the s protein of tgev has four major antigenic sites, with site a being the major inducer of neutralizing antibodies and conserved in both tgev and prcv strains [ , ] . the s protein of prcv is smaller due to the deletion with loss of one or two antigenic sites (c and b or d depending on the nomenclature) in the deletion region [ , ] . because most virus neutralization (vn) antibodies are directed to site a, conventional antibody assays fail to differentiate between pigs infected with prcv or tgev. blocking elisa tests using monoclonal antibodies to antigenic sites in the prcv deletion region of the s protein (one to conserved site a and a second to deleted site d) are used to serologically differentiate between prcv and tgev-infected pigs [ , , ] . because site a is conserved on tgev and prcv, only sera from pigs infected with either virus will contain antibodies to this site and compete with site a mabs for binding to the viral protein in blocking elisa. in contrast, because site d is only present on tgev, but absent on prcv, sera from pigs infected with tgev compete with site d mabs [ , ] . however, sometimes these tests result in false tgev positives or borderline reactions that are difficult to interpret making it problematic to accurately define the tgev status or diagnose tgev in prcv infected swine herds [ ] . we investigated the shedding of prcv/tgev in prcv/tgev seronegative sentinel pigs introduced into a prcv seropositive herd with questionable tgev serology and diarrhea of uncertain etiology in weaning pigs. our objectives were to isolate and characterizate tgev or prcv strains from this field outbreak and to determine their genetic relationships to one another and to reference strains. because previous studies have suggested that the s gene deletion area may influence viral tissue tropism, we focused on analysis of this region [ , ] . these new prcv isolates derived from both nasal swabs and feces should serve as tools to gain a better understanding of the molecular basis and evolution of the pathogenesis of coronaviruses. we attempted to isolate and characterize tgev and prcv strains from a prcv seropositive herd in canada with questionable tgev serology (inconclusive results in blocking elisa, svanovir, uppsala, sweden) and diarrhea of uncertain etiology. thirty-one prcv/tgev seronegative weaned sentinel pigs were introduced into the herd. the herd was a sow farrow-to-finish unit. the average inventory was nursing piglets, nursery (weaned) pigs and grow-finish pigs. the sentinel pigs were all placed in one room, and then dispersed among pens with or sentinel pigs in each pen, in addition to - recently weaned resident pigs. the average weaning age in the herd was days and the sentinel pigs were - -weeks-old when introduced. we investigated the shedding of prcv/tgev in sentinel pigs introduced into the herd. although resident pigs consistently tested positive for serum antibodies to prcv in a commercial blocking elisa test, occasionally some pigs also tested tgev seropositive in this test, suggesting the presence of false positives or tgev cases in the weaning pigs with diarrhea. in an attempt to isolate and characterize tgev and prcv strains from this herd, fecal and nasal swabs were collected from of the sentinel pigs at , , , and days post-arrival (dpa). a total of nasal swabs and fecal samples were collected, with nasal swabs and fecal sample pairs collected concurrently and tested by nested-rt-pcr and cell culture immunofluorescence (ccif) to detect prcv or tgev. each swab was identified v. costantini et al. as follows: pig number-dpa-origin (fecal "f" or nasal "n" sample). for example - f: pig , dpa, fecal sample (fig. ) . four respiratory (nasal) and enteric (fecal) prcv strains, but no tgev strains, were isolated. the designation of prcv was based on the presence of the typical s-gene deletion in all the isolates (described in a subsequent section). the swine testicle cells were used for virus isolation, propagation and cell culture immunofluorescence tests (ccif) as previously described [ ] . each strain was isolated and plaque-purified once or twice in swine testicle (st) cells. fecal and nasal swabs from the field cases (sentinel pigs), cell culture isolates and fecal and nasal swabs from gnotobiotic pigs were tested by nested-rt-pcr to detect and to differentiate tgev and prcv viral rna as previously described by kim et al. [ ] . the rt-pcr primers f ( -gggtaagttgctcattagaaataatgg- ) and r ( -cttcttcaa agctagggactg- ), and the nested-pcr primers f ( -ttgtggtyttggtygtaa tkcc- ) and r ( -ggctgtttggtaactaatttrcca- ) associated with the open reading frame b and the s-gene deletion area for u.s. and european strains of prcv were used [ ] . the rna from the fecal or nasal swabs or cell culture isolates were extracted using a commercial rna extraction kit (trizol ls reagent, life technology, ny, u.s.a.) according to the manufacturer's recommendation. briefly, µl of the nasal or fecal swab fluids (diluted in mem-e) were mixed with µl of trizol and were incubated for min at room temperature. following incubation, µl of chloroform were added. the samples were incubated for min at room temperature and centrifuged at . g for min at • c. the rna was precipitated with isopropanol. purified rna was resuspended in µl of depcwater. the reference strains, isu- (prcv) and m milller (tgev) were used as positive controls and negative controls included mem-e or prcv/tgev negative fecal or nasal swabs from unexposed gnotobiotic pigs. the conditions for the nested-pcr were as follows: cycles of denaturation at • c for min, annealing at • c for . min and extension at • c for . min, followed by a final cycle of extension at • c for min. pcr products were analyzed on . % agarose gels stained with ethidium bromide [ , , ] . the predicted size of the amplified product was bp for tgev and - bp for prcv [ ] . fecal and nasal swab supernatant fluids from the sentinel field cases or the cell culture passaged prcv isolates and nasal and fecal swabs from gnotobiotic pigs were diluted in minimum essential medium (mem-e) and tested by ccif to detect infectious virus using previously described procedures [ ] . briefly, or -fold serial dilutions of the nasal and fecal swab supernatants or prcv cell culture isolate, respectively were inoculated onto st cell monolayers in -well plates and incubated for hours. the cells were fixed with % acetone, stained with hyperimmune porcine anti-tgev serum conjugated to fluorescein isothiocyanate, and analyzed by fluorescent microscopy [ ] . four respiratory ( - n, - n, - n, - n) and fecal ( - f, - f, - f, - f, - f) prcv strains (including the pairs of nasal/fecal samples from three pigs one day after diarrhea, designated - n/ - f, - n/ - f, - n/ - f) were isolated from the nasal and fecal swab fluids, respectively of the sentinel pigs in contact with the resident pigs. each isolate was first passaged once or twice in st cells and then plaque purified in st cells. sequence analysis of the partial s-gene of - of times passaged in cell culture (number of times plaque-purified)] and reference strain isu- was performed with primers f and r . the rt-pcr products were purified using a geneclean spin kit (bio , ca) and sequenced by dideoxynucleotide chain termination procedures using an automated sequencer [abi , perkin elmer, ca]. sequence data were aligned using the dnastar software program (dnastar, madison, wi) and compared with the published sequence using the clustal methods. rectal and nasal swab fluids collected from gnotobiotic pigs from to dpi were tested by das-elisa to detect virus antigen as described previously [ ] . briefly, elisa plates were coated with the monoclonal antibody (mab) c and c to the tgev s protein, and mab h to the n protein (positive coating) or with negative ascites sp / (negative coating). all samples were tested in duplicate wells, one with positive and one with negative coating. viral antigen captured on the plate was detected with the purified, biotinylated igg fraction of a tgev hyperimmune antiserum, followed by streptavidin-peroxidase and - azino-bis ( -ethylbenzthiazoline- -sulfonic acid) as substrate. two hysterectomy-derived, colostrum-deprived -day-old gnotobiotic pigs were oronasally inoculated with the cell culture adapted, plaque purified - n * ( ) prcv isolate [ × plaque forming units (pfu)/pig]. as a control a third gnotobiotic pig was oronasally inoculated with mem-e. clinical parameters including diarrhea and fecal scores ( = normal, = pasty, = semiliquid, = liquid) were recorded. fecal and nasal shedding of viral rna or virus were assayed by nested-rt-pcr, ccif and das-elisa [ , ] from days post inoculation (dpi) to . one infected and one control pig were euthanized at dpi and sections of duodenum, jejunum, ileum and lung were collected for immunofluorescence assay (ifa) and histopathology. for ifa impression smears were made on glass microscope slides, air dried, fixed in acetone and stained with fitc-conjugated anti-tgev serum. for histopathology studies, tissues were processed in prefer fixative solution, embedded in paraffin and stained with hematoxylin and eosin as previously described [ , ] . in a second experimental group, two hysterectomy-derived, colostrum-deprived -dayold gnotobiotic pigs were oronasally inoculated with either the cell culture adapted, plaquepurified - f * ( ) prcv isolate [ × plaque forming units (pfu)/pig] or with ml of a : dilution of the pooled rectal swab fluids recovered (dpi - ) from one of the initial gnotobiotic pigs inoculated with - n * ( ) prcv. in addition, a third gnotobiotic pig was oronasally inoculated with mem-e as a control. pigs were euthanized at dpi and sections of duodenum, jejunum, ileum and lung were collected for ifa and histopathology. the sentinel pigs remained prcv/tgev seronegative at and dpa, but developed diarrhea at dpa and / ( %) seroconverted to prcv but not tgev detected by blocking elisa at dpa. at dpa, one sentinel pig died but neither prcv nor tgev were detected in the tissues. nasal shedding of prcv was detected in % ( fig. a and b . the peak of prcv nasal (fig. a) shedding was detected at dpa ( day after diarrhea outbreak) by nested-rt-pcr ( %) and ccif ( %). both techniques showed similar sensitivity to detect prcv in nasal swabs, with the percentage of positive samples increasing until dpa, and decreasing at dpa ( days after diarrhea outbreak). the peak of prcv fecal shedding (fig. b) was also detected at dpa ( day after diarrhea outbreak) by nested-rt-pcr ( %) but at dpa by ccif ( %). nested-rt-pcr was more sensitive than ccif for detecting prcv in the fecal swabs. four respiratory and fecal prcv field strains were adapted to growth in st cells and plaque purified. the partial s-gene of these cell culture adapted, plaque-purified strains was sequenced (fig. ) . the strains were assigned to groups according to the size and position of the s-gene deletion (fig. ) . the group isolates [ - f * ( ) and - f * ( )] had a nt deletion in the sgene starting from nt to nt . the group isolates [ - f * ( ), - n * ( ), - f * ( ) and - f * ( )] had a nt deletion starting from nt to nt and the group isolates [ - n * ( ), - n * ( ) and - n * ( )] had a nt deletion starting from nt to nt . sequence analysis revealed that the selected region of the s-gene of the prcv field isolates had higher homology to u.s. prcv strains than to european prcv strains and the size and position of the deletion was similar to u.s. strains. analysis of the sequence of cell culture adapted, plaque-purified - f * ( ), - n * ( ), - f * ( ), - n * ( ), and - f * ( ), - n * ( ) (each fecal/nasal pair collected concurrently as nasal and fecal swabs of different pigs) showed nt differences (nt , , and region - ) between the fecal and respiratory strains isolated from the same pig on the same day. however only changes in nt , and region - resulted in amino acid changes (fig. , table ). when the nucleotide and amino acid sequences of these paired samples were compared with tgev and prcv reference strains, a fifth nt substitution (nt ) between the field and the reference strains was identified. this change resulted in another v. costantini et al. table . amino acid change, in this case between the reference and the field strains (fig. , table ). two gnotobiotic pigs were oronasally inoculated with the cell culture adapted, plaque purified - n prcv nasal isolate ( × pfu/pig) and an additional pig was mock-inoculated as a control (table ) . no clinical signs including diarrhea were evident in any of the pigs after inoculation. nasal shedding of prcv was detected until dpi by rt-pcr, ccif and elisa in both exposed pigs and until dpi in exposed pig - by nested-rt-pcr. using nested-rt-pcr, fecal shedding was detected from dpi until dpi, and by elisa from dpi until dpi in both exposed pigs, but ccif failed to detect virus shedding from rectal swab samples in either exposed pig. the control pig was negative at all times by all tests ( table ). the results of the ifa on the duodenum, jejunum, ileum, lung and nasal turbinate impression smears were negative for exposed pig - which was euthanized at dpi. the histopathology examination showed normal length villi in the duodenum, jejunum and ileum. mild multifocal subacute lymphohistiocytic bronchointerstitial pneumonia with lobular atelectasis was detected in lung. in a second experiment, one gnotobiotic pig (gp - ) was oronasally inoculated with the cell culture adapted, plaque-purified - f prcv isolate ( × pfu/pig) and a second pig (gp - ) was inoculated with ml of a : dilution of the pooled rectal swab fluids recovered from gp - described above at to dpi (table ) . neither of the inoculated pigs showed clinical signs up to dpi when they were euthanized. fecal shedding was detected on and dpi in gp - and on dpi in gp - by nested-rt-pcr and on dpi in gp - by elisa. no nasal shedding was detected by nested-rt-pcr or ccif in either pig ( table ). the control pig remained negative at all times in all tests. the ifa on impression smears from duodenum, jejunum, ileum and lung of gp - was negative. likewise for gp - , the ifa on the duodenum, jejunum, ileum and lung was negative. the histopathology results showed normal length villi in the duodenum and jejunum for both gp - and gp - , but in gp - , villi in the ileum were slightly shortened. the lung tissues did not differ from the control (data not shown). in this study we investigated prcv/tgev nasal and fecal shedding in sentinel pigs introduced into a prcv seropositive herd with questionable tgev serology and diarrhea of uncertain etiology in weaning pigs. kim et al. [ ] recently reported a similar field case in a u.s. swine herd where the presence of prcv antibodies in the herd may have complicated the diagnosis of tgev infection. in this latter case tgev infection was confirmed by isolation of tgev in st cells from the gut contents of diarrheic sentinel pigs. a prcv strain was also isolated in st cells from nasal swabs from clinically normal tgev-seronegative sentinel pigs in contact with the diarrheic pigs. in the present outbreak, prcv/tgev seronegative - -week-old sentinel pigs, were introduced into the prcv seropositive herd with diarrhea occurring in weaned pigs. the sentinel pigs remained prcv/tgev seronegative at and dpa. at dpa, both the sentinel and resident pigs developed diarrhea. one day later, one sentinel pig died and laboratory testing failed to confirm a diagnosis, but the tissues were prcv/tgev negative by the immunoperoxidase test. fiftysix percent ( / ) of sentinel pigs seroconverted to prcv at dpa without respiratory clinical signs. because investigators have reported that only tgev has an enteric tropism and causes diarrhea, but the serology suggested the presence of prcv and was questionable for tgev, our overall goal was to clarify if pigs were shedding tgev or prcv and to determine if the isolates were similar or identical to each other and to previously described tgev or prcv strains. fecal and nasal shedding of prcv/tgev was detected by ccif and a nested-rt-pcr assay was used to detect and differentiate prcv and tgev [ ] . no shedding of tgev was detected by nested-rt-pcr. nasal shedding of prcv/tgev was detected in % of the samples by ccif and % of the samples were prcv positive by nested-rt-pcr. the nested-rt-pcr was as sensitive as ccif to detect prcv and there was a good agreement between both techniques, with the peak of prcv nasal shedding at dpa ( day after diarrhea). however the detection of prcv in fecal swabs showed different results. the nested-rt-pcr was more sensitive than ccif for prcv detection in fecal samples ( % and %, respectively, fig. ). the peak of prcv fecal shedding was at dpa by nested-rt-pcr ( %) but only . % of the fecal samples were positive by ccif. previous reports showed that nested-rt-pcr is more sensitive than ccif for detection of rectal shedding of tgev. in a study by kim et al. [ ] , gnotobiotic pigs were inoculated with the cell culture adapted tgev strain bw b or the original field pig intestinal content sample. viral shedding was detected in rectal swabs from dpi until by nested-rt-pcr, but only at dpi using ccif [ ] . it is also possible that intestinal antibodies to prcv, present in the intestinal contents of the prcv seropositive pigs could interfere with the detection of fecal shedding of prcv by ccif, but not by the nested-rt-pcr assays. in addition, it is possible that prcv is more labile in feces or particles may lose their spike protein [ ] resulting in loss of infectivity detected by ccif assays, but not the rna in these viral particles, detected in nested-rt-pcr assays. the prcv has a different tissue tropism from that of tgev. the tgev replicates in both respiratory and intestinal epithelial cells and causes gastroenteritis, whereas prcv replicates to high titers in the upper respiratory tract and lung tissue of swine [ , ] . however, prcv strain ar was the first prcv strain isolated from the small intestine of a field pig from an arkansas, u.s. swine herd [ ] . cox et al., , reported the isolation of prcv strain tlm from various tissues including the intestine of hysterectomy-derived colostrumdeprived pigs which were inoculated by aerosol with tcid prcv at six days of age. virus was consistently isolated in highest titers from respiratory tract tissues, but also from stomach, small intestine and colon. however none of the pigs showed respiratory signs or diarrhea [ ] . their results showed that prcv infected only a few unidentified cells at villous or crypt sites in the small intestinal mucosa and spread from the ileum to the duodenum. although unclear, the authors suggested that the aerosol inoculation of pigs with tlm caused a respiratory infection followed by viremia and ingestion of prcv to infect the intestinal cells. in our studies the detection of prcv from nasal swabs of gnotobiotic pigs gp - and - inoculated with the respiratory - n prcv isolate confirms the respiratory tropism, but the presence of fecal shedding in both pigs in the absence of villous atrophy (gp - ) also suggests an intestinal infection like that reported for prcv strain tlm [ ] . however, when a gnotobiotic pig (gp was oronasally inoculated with the fecal - f prcv isolate (isolated from the fecal swab fluids of the same sentinel pig as - n) and a second gnotobiotic pig (gp - ) was inoculated with the fecal prcv strain recovered from gp - (originally inoculated with the respiratory strain - n), virus shedding was detected only in rectal swab fluids. because the virus was inoculated by both the oral and nasal routes and detected only from rectal swab fluids and not from nasal swab fluids, the positive signal was unlikely to be derived from the virus inoculum. the histopathology results showed normal length villi in the small intestine of gp - , but slightly shortened villi in the ileum of gp - . these collective results suggest that the detection of prcv in the rectal swab fluids from the gnotobiotic pigs inoculated with - n, is not just the consequence of a respiratory infection followed by ingestion of prcv with shedding of ingested virus in feces, but another factor may exists to account for the change in tropism of these prcv strains. failure to detect villous atrophy or prcv antigen in the small intestine of gp - given - n prcv may have been due to the later time that this pig was euthanized ( dpi) versus pigs gp - and gp - that were euthanized at dpi. however because no clinical signs were evident it was difficult to establish optimal times to euthanize these pigs for antigen or lesion detection. failure to detect prcv antigen by ifa in the intestinal tissue of any of these pigs may have been due to the timing, the presence of too few infected cells for detection or to use of impression smears which may not adequately reflect cells in the crypt regions or in the intestinal submucosal region. alternatively failure to detect fecal shedding of prcv in these pigs by ccif may reflect a lower sensitivity of this assay or the lability of prcv strains in feces. differences in the tropism between tgev and prcv strains have been attributed to deletions in the region of the s-gene [ , , ] . four respiratory [ - n * ( ) - n * ( ) - n * ( ), - n * ( )] and fecal [ - f * ( ), - f * ( ), - f * ( ), - f * ( ), - f * ( )] strains were isolated and plaque-purified in st cells and the s-gene was partially sequenced. all u.s. prcv strains reported in the literature have - nt deletions within the region of the s-gene resulting in loss of or antigenic sites [ , ] . in contrast tgev strains have an intact s-gene. sequence analysis allowed grouping of the isolates into groups according to the size and position of the s-gene deleted. the deletion size ranged between to nt, starting at nt , or to nt , and . the sequences were aligned by the clustal method and the analysis showed a high homology between the prcv isolates and the other u.s. strains of prcv. previous reports indicated that the s glycoprotein domain recognized by the cellular receptor papn is close to the antigenic sites a and d (nt - ) but this domain is present in both tgev and prcv strains [ ] indicating that its presence is not sufficient for infection of enterocytes. analysis of the tropism of tgev recombinant isolates have demonstrated that nucleotide changes of the s gene that result in amino acid changes at the n-terminal region of the tgev s protein also affect the enteric tropism of tgev. because of this observation, we focused on analysis of this region [ ] . to analyze if sequence differences between the prcv fecal and respiratory strains were involved in the enteric tropism, the sequence of the fecal and nasal pairs - , - and - were compared. ballesteros et al., described aa changes at residues (g → a) and (g → t) of the s-gene that were responsible for the loss of the enteric tropism of the ptv-ts-mad tgev strain [ ] . as shown in table , only differences were found between the fecal and nasal prcv isolates. three of them where at nt , , and the fourth difference was in the region of nt - . a fifth difference (nt ) was found when the prcv field strains were compared with the reference strains. when the amino acid (aa) sequences were analyzed, only changes in nt , and the region - resulted in amino acid changes. the nucleotides - encoded the same amino acid (aa or depending on the strain). the segment from nt - (aa - ) is inside the deletion area of the nasal prcv strains. however the possibility that the change in the tropism was a consequence of the differences found in nt - was unlikely because the same sequence was found in the respiratory prcv isu- strain which causes little or no respiratory disease or fecal shedding in infected gnotobiotic pigs [ ] . the fifth difference in sequence was detected in all prcv field strains compared to the reference prcv strains. as shown (table ) , an identical nt change (nt a → g) was found in each fecal and nasal prcv pair when they were compared with the prcv reference strains. however this change would not affect the tropism because it is within the leader peptide of the s protein and not present in the mature virus. surprisingly however, based on a limited pathogenesis study in a gnotobiotic pig, the fecal - f prcv isolate appears to have lost the respiratory tropism. the two nt changes between the respiratory and fecal isolates at nt - caused an aa change from threonine to valine, respectively ( table ) . the same aa (t) was found in this position in the tgev pur mad strain, which has both enteric and respiratory tropism and in the prcv isu- strain, which has only respiratory tropism. our results would suggest that the loss of the respiratory tropism of the - f prcv strain could be a consequence of the change in this aa. it is possible that the changes in the tropism of the present prcv isolates are a consequence of the changes in nt - . however, further genetic analysis of these strains and pathogenesis studies are needed. a better understanding of the molecular basis of virus tropism may help to clarify the mechanism of disease for tgev and prcv strains and understand their evolution in infected swine in the field. two amino acid changes at the n-terminal of the transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism porcine pararotavirus: detection, differentiation from rotavirus, and pathogenesis in gnotobiotic pigs new porcine coronavirus? sites of replication of a porcine respiratory coronavirus related to transmissible gastroenteritis virus four major antigenic sites of the coronavirus transmissible gastroenteritis virus are located on the amino-terminal half of the spike glycoprotein s molecular basis of transmissible gastroenteritis virus epidemiology experimental reproduction of pneumonia in gnotobiotic pigs with porcine respiratory coronavirus isolated ar field isolates of transmissible gastroenteritis virus differ at the molecular level from the miller and purdue virulent and attenuated strains and from porcine respiratory coronaviruses development of a reverse transcription-nested polymerase chain reaction assay for differential diagnosis of transmissible gastroenteritis virus and porcine respiratory coronavirus from feces and nasal swabs of infected pigs molecular characterization and pathogenesis of transmissible gastroenteritis coronavirus (tgev) and porcine respiratory coronavirus (prcv) field isolates co-circulating in a swine herd point mutations in the s protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus interferon induction in rotavirus and coronavirus infections: a review of recent results porcine respiratory coronavirus: molecular features and virus-host interactions immune response of sows vaccinated with attenuated transmissible gastroenteritis virus (tgev) and recombinant tgev spike v. costantini et al.: respiratory and fecal shedding of prcv protein vaccines and protection of their suckling pigs against virulent tgev challenge exposure detection of transmissible gastroenteritis virus by rt-pcr and differentiation of porcine respiratory coronavirus isolation of a porcine respiratory, non-enteric coronavirus related to transmissible gastroenteritis transmissible gastroenteritis and porcine respiratory coronavirus targeted recombination demostrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence genetic evolution and tropism of transmissible gastroenteritis coronavirus antigenic homology among coronavirus related to transmissible gastroenteritis virus transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has a sialic acid (n-glycolylneuraminic acid) binding activity evaluation of the baculovirus-expresssed s glycoprotein of transmissible gastroenteritis virus (tgev) as antigen in a competition elisa to differentiate porcine respiratory coronavirus from tgev antibodies in pigs competition elisa, using monoclonal antibodies to the transmissible gastroenteritis virus (tgev) s protein, for serologic differentiation of pigs infected with tgev or porcine respiratory coronavirus antigenic variation among transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus strain detected with monoclonal antibodies to the s protein of tgev sequence comparison of porcine respiratory coronavirus isolate reveals heterogenecity in the s, and - genes three new isolates of porcine respiratory coronavirus with various pathogenicities and spike (s) gene deletions antigenic and biological diversity among transmissible gastroenteritis virus isolated of swine evidence for a porcine respiratory coronavirus, antigenically similar to transmissible gastroenteritis virus, in the united states we thank drs jeff smiley, mustafa hasoksuk, armando hoet, sonia cheetham and mr arden agnes for technical advice and also the staff of the oardc/osu molecular and cellular imaging center for sequencing. salaries and research support were provided by state and federal funds appropriated to the ohio agricultural research and development (oardc), the ohio state university. this study was supported in part by the national pork producer's council on behalf of the u.s. national pork board. key: cord- -a mjj dt authors: abid, nabil ben salem; chupin, sergei a.; bjadovskaya, olga p.; andreeva, olga g.; aouni, mahjoub; buesa, javier; baybikov, taufik z.; prokhvatilova, larisa b. title: molecular study of porcine transmissible gastroenteritis virus after serial animal passages revealed point mutations in s protein date: - - journal: virus genes doi: . /s - - - sha: doc_id: cord_uid: a mjj dt porcine respiratory coronavirus is related genetically to porcine transmissible gastroenteritis virus with a large deletion in s protein. the respiratory virus is a mutated form that may be a consequence of the gastroenteritis virus’s evolution. intensive passages of the virus in its natural host may enhance the appearance of mutations and therefore may contribute to any attenuated form of the virus. the objective of this study was to characterize the porcine transmissible gastroenteritis virus tmk strain after passages in piglets from until . a typical experimental infection, molecular characterization, and serological analysis were also carried out to further characterize and to evaluate any significant difference between strains. the sequence analysis showed two amino acid deletions and loss of an n-glycosylation site in transmissible gastroenteritis virus s protein after passages in piglets. although these deletions were positioned at the beginning of the antigenic site b of s protein, no clinical differences were observed in piglets infected experimentally either with the native virus or the mutated one. serological tests did not show any antibody reactivity difference between the two strains. in this article, we report that the s protein deletion did not affect the virus’s pathogenicity. the variety of the virus’s evolutionary forms may be a result, not only of the multiple passages in natural hosts, but also of other factors, such as different pathogens co-infection, nutrition, immunity, and others. further studies need to be carried out to characterize the mutated strain. porcine transmissible gastroenteritis virus (tgev) is an enteropathogenic coronavirus [ , ] . it infects pigs of all ages; however, infection is most severe in newborn piglets, resulting in a fatal diarrhea [ , ] . the disease outbreaks were observed in many countries such as japan, canada, russia, and causing considerable economic damages in swine industries [ ] . the mortality of the newborn piglets can reach % [ , ] . tgev has three major structural proteins: the spike (s), the nucleoprotein (n), and the membrane (m) [ , ] . protein s is the major inducer of tgev neutralizing antibodies (abs). studies of tgev mutations were enhanced by the detection of porcine respiratory coronavirus (prcv) in the winter of - [ ] . this virus was closely related to tgev [ , , ] and the main difference, which was found between the two viruses, was the large deletion in the s gene ( - bp for the american strains and bp for the european strains) [ , ] . as a result, prcv had a deletion of amino acids (european strain) or amino acids (american strains) and the loss of the antigenic sites c and b in the spike s protein [ , , , , ] . furthermore, some prcv strains differ from tgev by two minor deletions [ , , ] . based on sequence and phylogenetic analysis, sanchez and collaborators proposed that the european prcv strains have been derived by a -nt deletion from an enteric tgev and the origin of prcv may be the consequence of tgev evolution by sequence deletion in the s protein [ ] . although the nature of the events responsible for the genomic diversity between prcv and tgev remains an open question, both homologous and heterologous rna recombinations, and an accumulation of point mutations were proposed to be a driving force for coronavirus evolution [ ] . unlike tgev, prcv replicated in the respiratory tract yet with low extent in the gut. this deletion changed the primary affinity of the virus from the digestive to the respiratory tract. in this respect, mutants of mouse hepatitis virus encoding a point-mutated or a truncated s protein have been shown to be neuron-attenuated for mice [ ] . prcv constitutes a good example of genotypic and phenotypic changes as a result of sequence deletion. the infection with prcv induced the production of abs which were able to neutralize both tgev and prcv. as a result, the incidence and the severity of tgev infection have been decreased dramatically since the widespread of prcv infection in swine herds. it was proposed that prcv behaved as a natural vaccine against tgev, which makes the study of its origin and evolution interesting [ ] . the mutation of tgev s gene could be enhanced by intensive passages of the virus in its natural host under the influence of many environmental factors. the objective of this study was to carry out a typical experiment to compare two tgev tmk strains, one of which was cell culture, adapted and maintained in the laboratory for about years, and the other virus was passed in animals during the same period of time. cell culture adapted tgev tmk strain (the collection of microorganism strains of the federal governmental institute, all-russian research institute for animal health, fgi arriah) was used in this study. it was detected in a -week-old piglet with diarrhea in a swine farm in the mid s. the strain was cell culture adapted and propagated in swine testis (st) cells as described previously [ ] . these cells were found to be permissive to porcine hemagglutinating encephalomyelitis virus (hev), swine influenza virus (siv), and porcine tgev. in addition, st cells were shown to be not permissive to porcine reproductive and respiratory syndrome virus (prrsv). st cells are almost used for the isolation of tgev. the continuous st cell culture was started from trypsinized testicular tissue from swine fetuses, as described previously [ ] . cells were seeded in earle's balanced salt solution with . % lactalbumin hydrolysate (lah) and % pig serum. after a period of adjustment, the cells were trypsinized and sub-cultured at weekly intervals. tgev was propagated in st cells with mem medium buffered with mm hepes and . % (w/v) sodium bicarbonate, supplemented with % fetal bovine serum, % (v/v) antibiotics, and mm l-glutamine. cells were examined daily for cytopathic effects (cpes). when cpe was shown in % of cell monolayer, cells and supernatant fluids were frozen and thawed three times to release intracellular virus into the medium. the fluid was clarified by low-speed centrifugation ( g for min). the virus was then titrated by growth of serial -fold dilutions in vero cells. the virus titer was estimated by reed and muench method [ ] , and the virus amount per ml volume was calculated. tgev tmk strain was passaged annually in -weekold piglets during the period from until . these piglets were sacrificed, and the intestinal contents were taken, clarified, and stored at - °c until use. this tgev strain was referred in this study to as ptmk . rna extraction and rt-pcr rna extraction was carried out according to the procedure of gribanov et al. [ ] with modifications using gf/f nitrocellulose glass filters (whatman, england), as described previously [ ] . the tgev genome was detected using the reverse-transcription polymerase chain reaction (rt-pcr) method, as described in the original article [ ] . rt-pcr and dna sequencing were carried out using primers s and s . the primers were located in prcv s gene region, which were able to differentiate prcv and tgev viruses. the nucleotide and the amino acid sequences of tgev tmk strains were compared with the corresponding sequences of tgev strains, available in genbank database. sequences were analyzed with the computer program mega version . [ ] . in this study, go-taq polymerase (promega, moscow, russia) was used and showed high fidelity by sequencing of more than pcr products for the detection of porcine epidemic diarrhea virus and porcine rotavirus. nevertheless, dna fragments of ptmk strain were sequenced three times to be sure of the given mutations and to exclude any spontaneous mutations introduced by the dna polymerase. to compare the pathogenicity of the two tgev strains, six -week-old piglets were used in this typical experiment, whereas four -week-old piglets were used to study the neutralizing abs production. before use, all animals were confirmed negative for coronavirus abs by a blocking enzyme linked immunosorbent assay (elisa) (ingezim corona differential, spain). for the first lot ( -week-old piglets): two piglets were inoculated with cell culture adapted tgev tmk strain; the next two piglets were inoculated with ptmk strain, and the remaining two piglets were used as a control group. the piglets were inoculated orally with ml of cell culture supernatant, containing tgev tmk strain or ptmk at a titer of tcid /ml. the control piglets were inoculated with uninfected cell culture supernatant. for the second lot ( -week-old piglets): one piglet was inoculated with cell culture adapted tgev tmk strain at a titer of tcid /ml, one piglet was inoculated with ptmk strain at a titer of tcid /ml, one piglet was inoculated with a mixture of the two strains, and the remaining one piglet was used as control. all piglets were housed individually and fed with a commercial sterile milk substitute. clinical signs and rectal temperature were recorded daily. the stool samples were collected after the manifestation of diarrhea. major organs, including liver, lung, kidney, spleen, and intestine were collected aseptically post mortem for virus genome detection using rt-pcr analysis. the detection of the tgev antigens in stool samples was carried out using a commercial kit (anigenrapid tge ag test kit, south korea) according to manufacturer's protocol. the elisa test is an useful diagnostic method for the differentiation between tgev and prcv viruses [ ] . there are several available tests using mabs based on the s protein epitopic differences that have been developed to differentiate tgev and prcv abs [ , , , , ] . sera were collected before infection and every day during the first days after inoculation, and then samples were taken every days. abs against tgev in serum was established using blocking elisa test (ingezim corona differential, spain). in brief, the coronavirus antigen was fixed to a solid support (polystyrene plate) [ , , ] . serum sample was added in two wells. after incubation, a specific peroxidaseconjugated mab against one common epitope of both coronaviruses was added to one of the wells. if the serum sample contains abs against any one of the viruses, they will not permit the binding of the labeled mab to the antigen, whereas if it does not contain specific abs, the mab will bind to the antigen on the plate. in the other well, a specific peroxidase-conjugated mab against one specific epitope of the tgev was added. the experiment was performed as described above, the different combinations of the results in both wells permitted us to know if the serum sample contains abs against tgev or against prcv, or do not contain abs against either of them. the presence of anti-tgev abs was considered confirmation that the tgev infection had succeeded. after elisa testing was complete, sera were subsequently heated at °c for min and evaluated for virus neutralizing ab against porcine coronaviruses using standard microtiter virus neutralizing assay (vn). serial -fold dilutions of sera were made in -well microtiter plates. tgev strain was added to each well (approximately tcid ). after h of incubation at °c, st cells were added, and the plates were incubated at °c in a % co atmosphere for - days. each well was then examined for cpe. the antibody titer was determined to be the dilution of sera where % of the wells were infected. positive and negative controls were included in each reaction. results were presented as the reciprocal of the highest dilution of the test sample capable of neutralizing the virus. sequence analysis of tgev ptmk strain revealed a deletion of six nucleotides in the s gene fragment. the deduced amino acid sequence of ptmk strain revealed two amino acid deletions in two positions and of the s protein and two amino acid substitutions in position (threonine by lysine) and (asparagine by threonine) resulting in a loss of the glycosylation site upstream the b antigenic site (fig. ) . although no amino acid deletion was detected in ptmk s protein (data not shown), virus may undergo other nucleotide changes elsewhere in the genome. the nucleotide sequence of the amplified s gene fragment of the mutated tgev was deposited in genbank under accession number gq . to further characterize the mutated strain, typical experimental infections using tmk and ptmk strains were carried out. clinical signs were reproduced successfully, and the virus was recovered from the fecal samples of the infected newborn piglets. overall, the clinical signs observed in piglets, infected with the cell culture adapted tmk and ptmk strains, were consistent with a typical tgev infection and did not show any clinical difference in these typical experiments. for the -week-old piglets: clinical signs showed loss of appetite, vomiting, a yellowish diarrhea with smell of foul steatorrhea due to maldigestion and depression h post infection (hpi). infected piglets developed hyperthermia (rectal temperature [ °c) hpi. severe dehydration and death were observed days post infection (dpi). post mortem, the main pathological findings were the gas-filled stomach and intestine, coagulated transparent milk in the intestine, intestinal swelling, and congestion. the control piglets did not exhibit clinical signs consistent with tgev infection (table ) . for the -week-old piglets: clinical signs were less severe. these piglets were less susceptible to death in comparison to the newborns. the clinical signs were depression, loss of appetite, and diarrhea dpi. rectal temperature increased for the first dpi and it returned to its normal levels. in contrast to -week-old piglets, all the -week-old piglets recovered dpi. the kinetic of these clinical signs were shown to be the same for all piglets infected either by tmk , ptmk , or tmk /ptmk strains. no clinical signs were shown for the non infected piglets (controls) ( table ). as shown in table , the genome of tgev was detected by rt-pcr in stool samples of the all -week-old infected piglets either by tmk or ptmk strains hpi. the isolation of tgev in cell culture failed and was not possible before hpi. the detection of tgev antigen using immunochromatographic test strip was detected in stool samples of the infected piglets before hpi. in the acute phase of infection, tgev neutralizing abs were not detected by virus neutralization assay. all the infected piglets were dead by the virus dpi. no antibody response was seen for the non infected piglets. post mortem, tgev genome was detected in intestine, lung, kidney, spleen, and liver samples of the infected piglets either by tgev tmk or ptmk strains. vn test was carried out using antisera taken from the -week-old infected piglets. blood samples were taken daily after infection to monitor the tgev abs level. table , the highest ab titer was detected during a period from to dpi, and then the titer slightly declined until dpi. although tgev abs were not detected dpi, it may present in serum at low level. in general, tgev ab titer in infected animals during the acute phase of infection was less than this titer in the infected animals during the convalescent phase [ ] . the infected piglets remain healthy after the typical experiment. the infection has produced adequate immunity due to the ability of viruses (tmk and ptmk ) to infect the intestinal tract and consequently stimulate b cells for the production of immunoglobulin class a (iga) [ ] . in addition, cell-mediated immunity plays a direct role in the protection and the recovery from infection, and the production of abs is regulated by various cytokines derived from activated mononuclear cells during the immune response [ ] . although the use of -week-old piglets is limited to study the neutralizing ab production, stool samples were tested for the presence of viral genome. the same results were obtained for the two virus strains (data not shown). genetic variability has been observed for all rna viruses examined, and their potential for rapid evolution is increasingly recognized as the basis of their ubiquity and adaptability [ , ] . the molecular mechanisms underlying rna virus variations are: mutation, homologous and non homologous recombinations, and genome reassortment in viruses with a segmented genome such as reoviruses. the genetic evolution of viruses is an important aspect of the epidemiology of viral diseases and sometimes causes problems in the development of successful vaccines. however, for some viruses, such evolutionary behavior may generate new variations in favor of their natural host as the case of tgev and prcv (in favor of pigs) where abs produced after primary infection with prcv may prevent pigs to die after infection with tgev. the incidence and the severity of tgev infection have decreased dramatically in the world since the widespread of prcv infection. however, tgev outbreaks still occur at several prcv antibody-positive farms [ ] . such report enhances investigators to use tgev as a model to study gastroenteritis infections. experimental studies provided further signs of the ability of extensive virus passages in animals to generate new mutants and new variants [ ] . however, the degree of tgev mutation in infected pigs over time is not known. to address this question, we examined the genetic and antigenic changes that occurred in tgev tmk strain using pcr and dna sequence analysis, for viral rna variability study, we can detect and characterize recombination events with extreme precision [ , , , , ] . in this study, we have focused on nucleotide deletion that occurred in tgev tmk strain after passages on animals for a long period of time resulting in two amino acid deletions in s protein. although the homologous rna recombination between virus genomes (tmk and ptmk ) in piglets infected experimentally was not proved, it is not excluded. it is possible that rna recombination among virus particles of the same strain occurs naturally and under experimental conditions. our results showed no difference in pathology caused by either virus (tmk and ptmk ) in either piglet population ( -week-old and -week-old piglets) in these typical experiments. although no clinical differences were observed in piglets infected experimentally with native and mutated strains, tgev s gene showed some degrees of change. furthermore, the mutated strain undergoes a modified n-glycosylation site upstream the antigenic site b. it has been shown in other studies that site b is fully dependent on glycosylation for proper folding [ ] . the loss of n-glycosylation at the beginning of the b antigenic site needs to be further investigated by the analysis of the in silica d protein modeling of the s protein. further investigation needs to be undertaken to analyze host-pathogen interaction by studying protein-protein and protein-rna interactions, and experimental co-infection of piglets with gastroenteritis pathogens to better characterize the tgev mutated strain and to explore any interference phenomena. according to results of this study, we cannot suggest that tgev s gene mutations is responsible for changing any biological function (host-pathogen interactions) of the virus unless we carry out further biochemical, structural, and proteomic analysis. nd not detected the coronaviridae pathogenesis of transmissible gastroenteritis of swine viral infections of the gastrointestinal tract viral diarrhea of man and animals diseases of swine diseases of swine we are grateful to dr. nikolay zinyakov from viral molecular diagnostics laboratory of avian diseases for sequencing the dna samples. key: cord- -s w yr authors: hohdatsu, t.; okada, s.; koyama, h. title: characterization of monoclonal antibodies against feline infectious peritonitis virus type ii and antigenic relationship between feline, porcine, and canine coronaviruses date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: s w yr seven monoclonal antibodies (mabs) with neutralizing activity against feline infectious peritonitis virus (fipv) strain - (type ii) were prepared. when the polypeptide specificity recognized by these monoclonal antibodies (mabs) was investigated by western immunoblotting, all of the mabs reacted with peplomer glycoprotein (s) of the virus. by competitive binding assay these mabs were found to recognize at least different epitopes. the reactivity of these mabs with viruses classified as fipv type i (ucd- , ucd- , ucd- , ucd- , nw- , and black), feline enteric coronavirus (fecv) type ii strain - , canine coronavirus (ccv) strain - , and transmissible gastroenteritis virus (tgev) strains to- and sh was examined by neutralization tests. all mabs neutralized fecv strain - , ccv strain - , and tgev strains to- and sh, while they did not neutralize the fipv type i viruses. moreover, the mab against tgev strain to- , which has strong neutralizing activity against tgev viruses, neutralized ccv strain - , fecv strain - , and fipv strain - , but did not neutralize the fipv type i viruses. these results demonstrated that there are at least epitopes involved in the neutralization of fipv type ii strain - , and that these epitopes are not present in fipv type i viruses but are present in fecv strain - which does not induce feline infectious peritonitis, tgev strains to- and sh, and ccv strain - . these results suggest the presence of serotypes of fipv which can be clearly distinguished by the neutralization test using mabs. the mammalian members of the family coronaviridae are divided into distinct antigenic groups on the basis of serologic tests [ , ] . one antigenic group t. hohdatsu et al. contains mouse hepatitis virus, neonatal calf diarrhea coronavirus, human coronavirus hcv-oc , hemagglutinating encephalomyelitis virus of swine, and rat coronavirus. the second antigenic group consists of human respiratory coronavirus hcv- e, transmissible gastroenteritis virus (tgev) of swine, canine coronavirus (ccv), and feline infectious peritonitis virus (fipv) [ , ] . in addition to these members, a feline enteric coronavirus (fecv) has been isolated, which is very closely related to fipv, tgev, and ccv [ , ] . feline coronaviruses are divided into fipv types i and ii, and fecv types i and ii, on the basis of the disease types, that is, whether it causes feline infectious peritonitis (fip) or not, the ability of the viruses to proliferate in cell cultures, and the antigenic relationship to tgev and ccv [ ] . among these types, fipv type ii and fecv type ii are reported to be antigenicatly closer to tgev and ccv. between tgev purdue strain and fipv - strain a high degree of homology in the primary structure of the peplomer protein (s, k) has been described ( % for amino acid residues to and % for residues to ) [ ] . the authors prepared mabs which react only with the feline coronaviruses fipv type ii strain - and fecv type ii strain - . some of these mabs reacted with either tgev or ccv in indirect fluorescent antibody assays. also, some differences in antigenically between fipv type i virus strains were noted in an analysis using mabs (unpubl. obs.). in this study, we prepared mabs with neutralizing activity against fipv strain - , and investigated the differences in the epitopes recognized by these mabs, using a competitive binding assay. using these mabs and mabs with neutralizing activity against tgev strain to- , we examined the neutralizing epitopes for differences between feline coronaviruses, and tgev and ccv viruses. feline whole fetus cells (fcwf- ), crandell feline kidney cells (crfk), and swine kidney cells (cpk) were grown in eagle's minimum essential medium (mem) containing % l- medium, % fetal calf serum, units/ml penicillin and gg/ml streptomycin. the maintenance medium was mem containing % l- and antibiotics as above. the cells were maintained in a humidified % co incubator at °c. the coronavirus isolates used in this study and their sources are shown in table . among the fipv strains used in the study, strains ucd-t, nw- , ucd- , ucd- , ucd- , and black show cell-associated growth, and are therefore regarded as type i virus strains in the classification of pedersen et al. [ ] . fipv and fecv, tgev, and ccv were passaged or times in fcwf- cells, cpk cells, and crfk cells, respectively, and were used for the study. the antigen was prepared with the fipv - strain grown in fcwf- cell cultures. infectious culture fluid concentrated about tenfold by ammonium sulfate precipitation was layered onto a discontinuous sucrose density gradient ( and %) in a rps rotor (hitachi koki co., ltd, japan) and centrifuged at , r.p.m, for h. the virus bands formed at the interface of % and % sucrose layers were collected, diluted in nte buffer ( . m nac , . m tris-hc , ph . , . m edta) and centrifuged at , x g for h. the ,arus-containing pellet was suspended in a / volume of nte buffer. for preparation of neutralizing mabs against fipv - strain, balb/c mice, about weeks of age, were inoculated intraperitoneally with a mixture of ~tg of the viral antigen prepared as above and t cells of pertussis adjuvant. four or weeks later the mice received an intravenous booster dose of gg of viral antigen, and spleen cells were obtained for fusion days later. the fusion was carried out by essentially the same method described by k hler and milstein [ . polyethyleneglycol- , (merck, federal republic of ger-many) was used as a fusing agent and the ratio of mouse spleen cells and mouse myeloma cells (p- /x- -ag - , , ) was : . the selective medium contained hypoxanthine ( - m), aminopterin ( x - m) and thymidine ( . x -sm). the fused cells, at a concentration of . x spleen cells per ml, were dispended in ~tl volumes into wells of -well, flat-bottomed microplates (coming glass works, corning, ny) and incubated at °c in a humid atmosphere containing % cq. after incubation for weeks, the wells were examined and those which contained hybridoma cultures were tested for feline coronavirus specific antibody by a neutralization (nt) test (see below). the colonies in antibody positive wells were passaged in -well multiplates (corning glass works, corning, ny) and incubated in medium containing hypoxanthine ( - m) and thymidine ( . x - m). the cells were then cloned by the soft agar method. the neutralizing mabs against tgev to- strain used were previously reported by hohdatsu et al. [ ] . the supernatant fluid of antibody-secreting hybridoma cultures were concentrated tenfold by % saturation of ammonium sulfate and used for determination of antibody class and subclass by double diffusion in % agar gel containing . % nan . rabbit antisera against mouse immunoglobulins, igg , igg a, igg b, igg , igm and iga, and • and )~ chains (miles laboratories, naperville, u.s.a.) were placed in center wells and test samples were added to peripheral wells. the plates were incubated overnight at room temperature in a humidified chamber. sds-page was performed in slab gets under a nonreducing condition. the separation gel contained % polyacrylamide and the stacking gel contained % polyacrylamide. the sample buffer contained final concentration of % (w/v) sds, % (v/v) glycerol, . % (w/v) bromphenol blue and mm tris-hc (ph . ). the electrode buffer (ph . ) contained . g/ tris, . g/ glycine and . % sds. samples were mixed with sample buffer and kept at room temperature for h without boiling. electrophoresis was performed at room temperature for h at a constant current of ma. viral antigen separated in polyacrylamide gel by sds-page were transferred to nitrocellulose sheets of . gm pore size. the transfer was carried out electrophoretically by the method adapted from towbin et al. [ ] in a transfer-blot cell apparatus (bio-rad laboratories, richmond, ca) at ma and v for h using transfer buffer consisting of g/ tris (ph . ), % methanol and . g/ glycine. the nitrocellulose sheets were then cut into strips and incubated at °c for h in pbs containing % fetal calf serum. the sur)ernatant fluid of antibody-secreting hybridoma cultures were added in ml volumes to individual strips and incubated at °c for h. the strips were then washed times with pbs containing . % tween- , and incubated at °c for h with horseradish peroxidase-conjugated rabbit antibody against mouse igg, iga, and igm (miles lab., u.s.a.) diluted : with pbs containing % fetal calf serum. the strips were then washed and treated with substrate solution containing . g diaminobenzidine, lal of % h in ml of . m tris-hc , ph . . when distinct bands appeared about min later, the reaction was stopped by pouring off the substrate solution and rinsing with distilled water. this was carried out by a modified enzyme-linked immunosorbent assay (elisa) [ ] . serial tenfold dilutions in gl volumes of each competing mab from ascitic fluid were added to wells of a flat-bottomed microelisa plate coated with virus antigen (see above), and incubated at °c for rain. after washings with washing solution ( . % nac solution containing . % tween- ), gl of biotin-labeled mab diluted so as to show an optical density (od) of . was added, and incubated at °c for min. after washings with washing solution, peroxidase conjugated avidin (cappel, cooper biomedical, inc. malvern, pa) were diluted to the optimal concentration with pbs containing % fetal calf serum and . % tween- and gl of the dilution was added to each well of the plates. after incubation at °c for min, each well received gl of substrate solution and incubated at °c for min in a dark room. after completion of the incubation, the reaction was stopped with n h so solution and the od at nm was determined. the amount of competitive binding was estimated from the od in the presence or absence of unlabeled competing antibodies. the percentage of competition was determined by the formula, (a -n)/(a -b), where a is the od in the absence of competing antibody, b is the od in the presence of homologous antibody ( elisa units), and n is the od in the presence of a competitor. serial twofold dilutions of the mab were mixed with an equal volume of a virus suspension diluted so as to contain approximately tcid / . ml. the mixtures were incubated at °c for rain. each mixture was then inoculated into cell cultures in flat-bottomed microplates (coming glass works, coming, ny), and incubated in an atmosphere of % co in air at °c for days. two wells were employed for each antibody dilution. the anitbody titer was expressed as the reciprocal of the highest dilution of mab that completely inhibited cytopathic effect in the test. using the spleen cells of mice immunized with fipv strain - , cell fusion was conducted times. as a result, mabs ( - - , - - , - - , - - , - - , - - , and - - ) with neutralizing activity against fipv strain - were obtained. the immunoglobulin isotype and polypeptide specificity of these mabs are shown in table . polypeptide specificity was determined by western immunoblotting. all mabs were found to recognize the s protein of the viruses. figure shows an example of the reaction. to determine the differences in the epitope specificity of the neutralizing mabs, competitive binding assay was performed, and the results are summarized in table . binding of the biotin-labeled mabs - - , - - , - - , and - - to virus antigen was completely blocked by all of these unlabeled mabs. - - , - - , - - , - - , and - - by .t'-~ . %. on the basis of these results, epitopes recognized by these mabs were named i, ii, and iii. reactivity of the mabs which recognize the neutralizing epitopes in fipv strain - , with feline, porcine, and canine coronaviruses was investigated with the neutralization (nt) test. as shown in table , these mabs did not neutralize the fipv type i strains, but neutralized fecv strain - , as well as the porcine and canine coronaviruses. in particular, mabs which recognize epitope iii ( - - and - - ) also showed a high neutralizing activity against these viruses, as against fipv strain - . the the results are shown in table . while mab - neutralized the fipv type ii strain - , fecv type ii strain - , and ccv strain - , it did not neutralize fipv type i viruses. mabs -a and -a did not neutralize any coronaviruses other than tgev. in this study, we prepared mabs with neutralizing activity against fipv strain - , and used them to investigate the serological relationships between feline, porcine, and canine coronaviruses. it is generally accepted that epitopes associated with neutralization are present on the s protein of viruses. fiscus and teramoto have reported this phenomenon in feline coronaviruses i- ]. all mabs with neutralizing activity were observed to recognize s protein. this finding confirms the presence of a neutralizing epitope on the s protein. however, there have been no reports on the characteristics of this epitope. the results of the competitive binding assay using the mabs revealed the existence of at least different neutralizing epitopes (i, ii, and iii) in fipv strain - (table ) . we believe that the topographical relationship between epitopes i, ii, and iii is as follows. since (fig. a) resulting in blocking by steric hindrance, or that epitope iii overlaps part of epitopes i and ii (fig. b) . differences in the epitopes recognized by the mabs were determined. the nt activity of these mabs against several feline, porcine, and canine coronaviruses was examined. these mabs did not neutralize the fipv type i viruses, but neutralized fecv strain - , tgev strains to- and sh, and ccv strain - , which do not cause feline infectious peritonitis (table ) . moreover, a mab against tgev strain to- , which neutralized the tgev strains, neutralized fipv strain - , fecv strain - , and ccv strain - (table ). these results support the classification by pedersen et al. [ ] . when the virus groups (fipv type i, fipv type ii, fecv type ii, tgev, and ccv) are serologicalty compared, fipv type ii, fecv type ii, tgev, and ccv are found to form a group. fipv type i virus seems to be a virus of a distinct serotype. in other words, fipv inducing feline infectious peritonitis seem to consist of viruses of at least serotypes. pedersen etal. [ ] have indicated that many cases of feline infectious peritonitis in cats are induced by type i virus infection. we also found that, out of cats with feline infectious peritonitis-like symptoms and antibody-positive by indirect fluorescent antibody assay, only were positive for neutralizing antibody against fipv strain - (fipv type ii) (unpubl. data). it is probable that fipv type i is more prevalent in a natural environment. in the future, examination of the effects of different virus types on the anitbody-mediated enhancement of fipv infection [ , ] , as seen in dengue virus infection [ , ] , would be valuable. at present, we are investigating the effects of the neutralizing mab prepared in this study against in vivo fipv infection. differences between tgev strain purdue and fipv type ii strain - at the gene level of s protein have been reported [ ] . analysis of fipv type i at the gene level is also desired. recovery and characterization of a coronavirus from military dogs with diarrhoea recovery and in-vitro cultivation of a coronavirus from laboratoryinduced cases of feline infectious peritonitis (fip) antigenic comparison feline coronavirus isolates: evidence for markedly different peplomer glycoproteins comparison between virulent and attenuated strains of transmissible gastroenteritis virus heterogeneity of infection enhancement of dengue strains by monoclonal antibodies studies on transmissible gastroenteritis in pigs. iii. isolation of cytopathogenic virus and its use for serological investigation antigenic variation of porcine transmissible gastroenteritis virus detected by monoctonal antibodies antigenic relationships among homologous structural polypeptides of porcine, feline and canine coronaviruses spaan wjm (t ) the nucleotide sequence of the peplomer gene of porcine transmissible gastroenteritis virus (tgev): comparison with the sequence of the peplomer protein of feline infectious peritonitis virus (fipv) topographical analysis of antigenic determinants on envelope glycoprotein v (e) of japanese encephalitis virus, using monoclonal antibodies continuous cultures of fused cells secreting antibody of predefined specificity isolation of feline coronaviruses from two cats with diverse disease manifestations dengue virus monoclonal antibodies identify epitopes that mediate immune infection enhancement of dengue viruses attempted immunization of cats against feline infectious peritonitis, using avirulent live virus or sublethal amounts of virulent virus immunologic phenomena in the effusive form of feline infectious peritonitis experimental studies with three new strains of feline infections peritonitis virus: fipv-ucd , fipv-ucd and fipv-ucd pathogenic differences between various feline coronavirus isolates infection studies in kittens utilizing feline infections peritonitis virus propagated in cell culture an enteric coronavirus infection of cats and its relationship to feline infectious peritonitis pathogenicity studies of feline coronavirus isolates - and - morphologic and physical characteristics of feline infectious peritonitis virus and its growth in autochthonous peritoneal cell cultures antigenic relationship of the feline infectious peritonitis virus to coronaviruses of other species electrophoretic transfer of proteins from polyacrytamide gets to nitrocellulose sheets: procedure and some applications antibody-mediated enhancement of disease in feline infectious peritonitis: comparison with dengue hemorrhagic fever this work has been funded by the kitasato research foundation under grant no. . key: cord- - zu e y authors: piñeyro, pablo enrique; lozada, maria inez; alarcón, laura valeria; sanguinetti, ramon; cappuccio, javier alejandro; pérez, estefanía marisol; vannucci, fabio; armocida, alberto; madson, darin michael; perfumo, carlos juan; quiroga, maria alejandra title: first retrospective studies with etiological confirmation of porcine transmissible gastroenteritis virus infection in argentina date: - - journal: bmc vet res doi: . /s - - - sha: doc_id: cord_uid: zu e y background: in , a notification of porcine transmissible gastroenteritis virus (tgev) was made by the national services of animal health of argentina (senasa) to the world organization of animal health (oie). the notification was based on a serological diagnosis in a small farm with a morbidity rate of . % without enteric clinical signs. in order to determine if tgev was circulating before the official report, a retrospective study on cases of neonatal diarrhea was performed. the selection criteria was a sudden increase in mortality in - to -day-old piglets with watery diarrhea that did not respond to antibiotics. based on these criteria, three clinical cases were identified during – . results: all animals that were evaluated presented histological lesions consistent with enteric viral infection. the feces and ultrathin sections of intestine that were evaluated by electron microscopy confirmed the presence of round particles of approximately nm in size and characterized by finely granular electrodense nucleoids consistent with complete particles of coronavirus. the presence of the tgev antigen was confirmed by monoclonal specific immunohistochemistry, and final confirmation of a metabolically-active virus was performed by in situ hybridization to detect a tge mrna encoding spike protein. all sections evaluated in this case were negative for pedv and rotavirus a. conclusions: this is the first case series describing neonatal mortality with etiological confirmation of tgev in argentina. the clinical diagnosis of tgev infections in endemic regions is challenging due to the epidemiological distribution and coinfection with other enteric pathogens that mask the clinical presentation. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. there are five coronaviruses (covs) known to infect swine, and the clinical disease is mainly associated with neonatal diarrhea, but respiratory and neurological signs have also been reported [ ] [ ] [ ] [ ] . porcine transmissible gastroenteritis virus (tgev) and porcine epidemic diarrhea virus (pedv) belong to the coronaviridae family, coronavirinae subfamily, and genus alphacoronavirus [ ] . a new coronavirus genetically distinct from tgev and pedv, porcine deltacoronavirus (pdcov) (genus deltacoronavirus), has recently been associated with enteric disease in pigs [ ] . enteric porcine coronaviruses including tgev, pedv and pdcov are characterized by acute diarrhea and anorexia with rapid dissemination in naïve populations. the severities of clinical diarrhea, vomiting, and anorexia can vary based on the age of the affected pigs [ , ] . without adequate passive lactogenic immunity, the mortality rate in neonatal piglets can reach up to % [ , [ ] [ ] [ ] . etiological diagnosis relies mainly on molecular tools like pcr and serology, as the clinical signs and enteric lesions associated with tgev and pedv are indistinguishable [ , , ] . the epidemiology of tgev is rather complex, and infection in neonates can arise from multiple sources. in addition to swine, it has be documented that cats, dogs, and foxes can host tgev [ ] . the virus can be shed in feces for approximately months, and milk shedding from infected sows can result in vertical transmission. historically, tgev infection has followed a seasonal pattern, becoming more prevalent during winter months perhaps due to increased viral survival in colder temperatures and with less exposure to sunlight. tgev is susceptible to most commercial disinfectants, but resistant to digestive bile and stable at ph [ ] . only one tgev genotype has been described, however differences in pathogenicity among strains has been reported in field outbreaks, although not confirmed by an experimental study [ ] . infection with tgev has two different clinical presentations: epidemic and endemic. in epidemics, tgev enters a naïve herd and all pig categories are affected, particularly piglets that are - weeks old. the duration of the clinical presentation is short, approximately weeks, and in small, farrow-to-finish herd, the infection can be self-limiting [ , ] . endemic disease scenarios, those occurring after the epidemic phase, are observed in farms with incomplete aiao management or in breeding farms that have a continuous flow of naïve gilts. in breeding herds, the varying levels of humoral and lactogenic immunity lowers piglet mortality, but may lengthen the course of the disease [ ] . since the first description provided in the united states [ ] , tgev infections have been reported all over the world. in south america, it has been reported in colombia [ ] , venezuela [ ] , bolivia, and is currently seen in brazil [ ] . in argentina, an episode of high pre-weaning mortality related to isospora suis infection alone or in association with an unknown enteric virus was reported in [ ] . further studies using negative stain electron microscopy demonstrate the presence of viral particles consistent with coronavirus in feces of pre-weaning diarrheic ( . %) and post-weaning ( %) piglets [ ] . a retrospective histopathological study performed on cases of neonatal diarrhea at our laboratory during showed that % of neonatal diarrhea cases had lesions consistent with viral enteritis [ ] . in , a notification of tgev infection was reported by the national services of animal health of argentina (senasa) to the world organization of animal health (oie). it was detected by serology in a small farm with an apparent morbidity rate of . % without clinical signs [ ] . this is an unusual presentation of tgev infection and might be related to passed infection or interspecies transmission [ ] . in order to clarify the situation prior to the first official report of tgev infection in argentina, a retrospective study was performed on cases suspected of tgev-like disease recorded at the laboratory of special veterinary pathology at the college of veterinary sciences, la plata university. benchmarking analyses of epidemiological behavior and clinical histories were the criteria for herd selection. etiological diagnosis was confirmed by electron microscopy (em), immunohistochemistry (ihc), and in situ hybridization (ish-rna) of archived paraffin blocks. clinical, pathological and etiological findings case thirteen -to -day-old piglets with body weights ranging from to . kg were submitted for pathological investigation. pigs with clinical diarrhea were dirty, wet, had stained perinea, and showed moderate dehydration characterized by sunken eyes and diffusely pale mucosa. their small intestinal walls were thin with unremarkable mesenteric lymphatic vessels, and were distended by gas or occasionally contained yellow watery digesta with a ph of - ( fig. a) . their stomachs were empty or contained floccules of undigested milk. no other macroscopic findings were observed. the histopathological evaluation showed a shortening of intestinal villi with a crypt-villous ratio of : ( fig. b) , that villi were fused and lined by vacuolated cuboidal or attenuated epithelium, and that the lamina propria was expanded by moderate edema. microscopic lesions were limited to the jejunum and ileum. immunohistochemistry and ish-rna against tgev showed strong staining in the epithelium of sections that presented minimal epithelial villous changes (fig. c, d) . conversely, sections with the most severe epithelial damages were ihc negative. all sections evaluated in this case were negative for pedv and rotavirus. a gross evaluation of five piglets between and days old showed marked dehydration, wet and stool-stained perinea, and poor body conditions ( . kg average body weight) (fig. a) . the small intestine contained abundant yellow-watery diarrhea with a ph of - in two pigs and alkaline ph in three pigs (fig. b) . a histopathology examination of multiple sections of jejunum and ileum showed mild to moderate villous shortening and fusion (fig. c) . the villous enterocytes showed a marked cytoplasmic vacuolization (fig. d) . the lamina propria was minimally infiltrated by lymphocytes and plasma cells, and was expanded by edema. the superficial villous enterocytes in the ileum showed strong hybridization signals characterized by active-replicating tgev (fig. e) . no microscopic lesions were seen in the sections of colon. all sections evaluated in this case were negative for pedv and rotavirus a. in the ultrathin sections, rounded particles measuring approximately nm in diameter were located in the cytoplasm of the intestinal epithelial cells. the particles were characterized by finely granular electrodense nucleoids with electron lucent centers compatible with complete particles of coronavirus (fig. f ) . three neonatal piglets that died naturally presented with fecal stained perinea and were markedly dehydrated. the stomachs were empty, the small intestinal wall was thin/translucent, and scant yellow watery contents were sometimes apparent. the histopathology examination of the jejunum and ileum showed moderate villous fusion and shortening, and the villi were lined by low-cuboidal to flattened/attenuated epithelium (fig. a) . the lamina propria was infiltrated by numerous lymphocytes and plasma cells, was expanded by edema, and exhibited lymphangiectasia (fig. b) . the tgev antigen was detected by ihc in multiple sections of the jejunum and ileum (fig. c) . no significant lesions were observed in the section of colon. all sections evaluated in this case were negative for pedv and rotavirus a. the epidemiological and clinical presentations of outbreaks of neonatal mortality associated with enteritis and the detection of tgev started in the gestation units. both gilts and sows showed anorexia, diarrhea, and vomiting before enteric signs were observed in neonatal piglets. however, the prevalence of clinical signs in the breeding stock was low and no mortality was reported. when tgev enters in a naïve herds, an epizootic form characterized by a % mortality of pre-weaning piglets due to diarrhea and dehydration is normally observed [ , ] . although in the present study, the farms had a high prevalence of diarrhea in suckling pigs, only farms a and c showed almost % neonatal mortality, while in farm b had approximately % neonatal mortality. the clinical presentation and epidemiological pattern observed in farm b resembled the tge endemic form. although no other etiological diagnosis was confirmed, the low mortality associated with tgev that was observed in farm b might be the result of previous exposure to prcv. prcv infection can confer cross-protection against tgev, reducing the enteric clinical signs and pre-weaning mortality [ ] . therefore, herds concomitantly infected with prcv and tgev develop less severe clinical signs, making the clinical differentiation from other enteric infections such as rotavirus or e. coli infections more challenging [ , ] . another potential reason for the low mortality rate due to tge infection that was observed in farm b could be the intermittent viral exposure of the breeding stock that provided partial immunity to the neonatal piglets [ ] . in all herds in this study, it was suspected that the virus entered the farms through the subclinically infected replacement animals although the pre-weaning mortality rate in farm b was lower than that in farm a and c, the pre-weaning mortality was higher in this farm than the mortality rate seen in similar production systems due to other enteric causes such us i suis [ , ] , c. perfringes type a [ ] , or c. difficile [ ] . according to a previous study, the pre-weaning mortality due to diarrhea should not exceed % [ ] . usually, in porcine covs infections, the course of the clinical disease is short and normally does not exceed - weeks [ ] due to the establishment of a rapid herd immunity, as early as one week post-infection. however, in farm b the clinical presentation persisted for approximately months. potential reinfection due to poor husbandry and incomplete aiao management are just few potential causes of viral persistence in the environment that can predispose reinfection of the farrowing units. in clinically affected litters, most of the pigs are dirty, wet, and dehydrated, with diminished body weights. diarrhea is watery, yellowish, and with an acidic smell from the presence of undigested milk [ ] . the mortality rate is inversely correlated with the age of the piglets, reaching % in - day-old piglets. this predisposition is due to the slow replacement rate of villus epithelial cells (around days) in neonatal piglets compared with the replacement rate of -week-old piglets (around - days) [ ] . in this study, mortality varied from to %. the jejunum and ileum are the target segments of the small intestine for virus multiplication. however, tgev infection is segmental, so multiple segments should be included for histopathology or etiological diagnoses in situ such as ihc or ish-rna. due to the retrospective nature of this study, fixed tissue was used to confirm the presence of tgev and the morphological changes consistent with viral infection in the intestinal mucosa. it is important to highlight that although tgev was detected by different means in each farm, due to the segmental nature of the lesions and viral distribution, viral arn or viral antigen was not detected in all of the intestinal segments that were evaluated. in piglets that are less than weeks old, the reduction of villus length and the fusion of jejunum and proximal ileum are the main histological changes [ ] . the normal villous/crypt length ratio is : , however, after - h post-infection, the villous/ crypt ratio can be reduced to : or : [ ] . since tgev replicates in mature absorptive epithelial cells [ ] , a false negative diagnosis can be observed by ihc or ish in specimens that display villous shortening. few absorptive vacuolated cells are seen normally in the intestine, however, during tgev infection, they are found in great numbers with a cuboidal shape [ ] . in endemic tgev infection, histopathological diagnosis is more difficult because only % of pigs display typical tge lesions. in addition, immunofluorescence tests and ihc often fail in endemic farms due to a low number of enterocytes in the tgev-infected because of partial protection conferred by colostral antibodies [ , ] . different diagnostic techniques have been used to detect tgev infection such as ihc, ish, electron microscopy, and immunoelectron microscopy and pcr [ ] ; however, histopathology remains the most useful tool for screening diagnosis [ ] . in this study, although all cases were selected using clinical features and epidemiological information, the histological evaluation consistently showed lesions compatible with viral infection. the application of ihc and ish-rna on archived paraffin blocks from cases of neonatal diarrhea with high morbidity and mortality allowed retrospective identification of tgev infection. diagnosis of tgev infection in endemic regions, such as in argentina, is complicated due to the epidemiological distribution and clinical signs that might be masked with other enteric infections. further studies are necessary to determine the true prevalence of this pathogen and the correlation with neonatal enteric cases observed in confined production systems. case selection was based on the clinical history reported by the farm managers and referring veterinarians. the selection criteria was a sudden increase in mortality that included, significant increment of more than sd from average pre-weaning mortality and last for a period of a week. in addition reported mortality should be associated with the presence of watery diarrhea in -to -day-old piglets that did not respond to antibiotics. first screening of cases was done by histopathological evaluation, and only cases presenting features of viral enteritis with no other detected pathogen were included. based on these criteria, three clinical cases were identified from to . a -sow farrow-to-finishing herd located in buenos aires served as the first case. the farm produced its own replacement breeding stock, however, two months before the outbreak, gilts were introduced from a breeding company. the parity of the breeding stock was distributed as % gilts while the rest of the reproductive stock parity varied from to . on september , approximately % of the pregnant sows presented acute vomiting while % of the pregnant sows presented acute diarrhea. during the period when the sows showed gastro-enteric clinical signs, -to -day-old piglets presented vomiting ( - %) and diarrhea ( %), and the mortality rate of suckling pigs reached %. the course of the disease in both breeder stock and piglets lasted for approximately three weeks. case involved a one-site herd of sows with its own replacement gilts and the following parity distribution: % gilts, % parity - , and . % distributed amongst parity - . boars were purchased from a breeding company and were incorporated into the reproductive herd without quarantine. the farm was located within a few miles of a swine slaughterhouse in buenos aires. in february , the pregnant sows showed anorexia ( - %) and diarrhea ( %) associated with heat returns and abortions ( . %). in the farrowing houses, approximately % of the lactating sows presented with anorexia. pre-weaning mortality associated with the presence of diarrhea varied from . % at the beginning of the outbreak to . % to weeks after the initial clinical signs. an anatomopathological evaluation showed that . % of the total pre-weaning mortality was due to diarrhea. a one-site herd of sows was the subject of case . in july , two boars were located close to the gestation unit. a week later, gestation sows showed anorexia ( . %) and diarrhea ( . %). thereafter, in the gilts, diarrhea was evident in the nursery ( - %) and fattener ( - %). two-day-old piglets showed watery diarrhea ( %) with a mortality rate of %. affected piglets died from severe dehydration within two days of the onset of clinical signs. the course of the disease lasted approximately two months with an overall pre-weaning mortality of % during that period. at the onset of the outbreak, clinically affected suckling pigs were submitted for postmortem examination. tissue samples from different organs, including multiple segments of small intestine (duodenum, jejunum, and ileum) and large intestine, were fixed in % buffered formalin, processed for routine histopathologic examination, and stained with hematoxylin and eosin. etiological diagnosis in tissues and feces by immunohistochemistry, in situ hybridization, and electron microscopy immunohistochemestry ihc was carried-out briefly to differentiate tgev [ ] from pedv [ ] . monoclonal antibodies against tgev (osu: #. e - c) and pedv (osu c ) at dilutions of : were used. antigen retrieval was performed with humid heat and revealed with peroxidase (novocastra a , leica biosystems, il, usa). to rule out other potential viral enteritis, rotavirus a was evaluated in all sections by ihc [ ] . rotavirus ihc was performed using a monoclonal antibody against rotavirus a (santa cruz: sc- ) at a : dilution. antigen retrieval was performed with epitope retrieval solution for min, as programmed on leica bond iii, and revealed with powervision poly-hrp anti-mouse (leica pv ). all samples were tested in duplicate and sections were controlled appropriately for tgev, pedv and rotavirus a with positive and negative controls (additional file : figure s ). in situ hybridization ish-rna was developed through the rnascope platform (advanced cell diagnostics, inc., ca), targeting the specific reverse complementary nucleotide sequence of the tge viral mrna ( - region of spike gene, genbank: kc . ). therefore, positive hybridization signals represent a metabolically-active virus characterized by the tge mrna encoding spike protein. unstained paraffin tissue sections were processed as previously described [ ] . briefly, tissues were deparaffinized and treated with hydrogen peroxide at room temperature for min. the slides were hybridized using a hybridization buffer, and sequence amplifiers were added. the red colorimetric staining detected the tge hybridization signal, and counterstaining occurred with hematoxylin. representative sections of small intestine were fixed in . % glutaraldehyde and % paraformaldehyde in . m, ph . phosphate buffer (pbs) and post-fixed in % osmium tetroxide in pbs. after dehydration in an alcohol series, the fragments were embedded in epoxy resin, quetol (nisshin em co., ltd., tokyo). ultrathin sections were cut, double-stained with uranyl acetate-lead citrate, and observed under a jem- ex (jeol co. ltd., tokyo). diseases of swine new variant of porcine epidemic diarrhea virus hemagglutinating encephalomyelitis coronavirus infection in pigs porcine respiratory coronavirus: molecular features and virus-host interactions a comparative sequence analysis to revise the current taxonomy of the family coronaviridae pathogenicity and pathogenesis of a united states porcine deltacoronavirus cell culture isolate in -day-old neonatal piglets retrospective testing and case series study of porcine delta coronavirus in u.s. swine herds porcine epidemic diarrhea: a review of current epidemiology and available vaccines emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis does circulating antibody play a role in the protection of piglets against porcine epidemic diarrhea virus? molecular characterization and pathogenesis of transmissible gastroenteritis coronavirus (tgev) and porcine respiratory coronavirus (prcv) field isolates co-circulating in a swine herd development of pcr-based techniques to identify porcine transmissible gastroenteritis coronavirus isolates porcine coronavirus. in: trends in emerging viral infections of swine a transmissible gastroenteritis in pigs coronavirus en porcinos: importancia y presentación del virus de la diarrea epidémica porcina (pedv) en colombia detección de focos de gastroenteritis transmisible en venezuela diagnosis to detect porcine transmissible gastroenteritis virus (tgev) by optical and transmission electron microscopy techniques infección por isospora suis sola o asociada a virus entéricos como causa de alta morbimortalidad en lechones lactantes identificación e índice de detección de partículas virales en materia fecal por microscopía electrónica análisis de los cuadros entéricos en cerdos remitidos al laboratorio de patología especial veterinaria transmissible gastroenteritis, argentina. in: oie, editor. servicio nacional de sanidad y calidad agroalimentaria (senasa), ministerio de agricultura, ganadería y pesca: world organization of animal health endemic transmissible gastroenteritis: difficulty in diagnosis and attempted confirmation using a transmission trial fibrinonecrotic enteritis of piglets in a commercial farm: a postmortem study of the prevalence and the role of lesion associated agents isospora suis and clostridium perfringens neonatal piglets mesocolon edema and colitis due to clostridium difficile infection: prevalence, clinical disease and pathological studies why should piglets dead at the pre-weaning period be postmortem examined and statistically analysed at weekly intervals? lesions of the gastrointestinal tract of pigs infected with transmissible gastroenteritis mechanisms of porcine diarrheal disease transmissible gastroenteritis in endemically infected breeding herds of pigs in east anglia immunohistochemistry of transmissible gastroenteritis virus antigens in fixed paraffin-embedded tissues monoclonal antibody-based immunohistochemical detection of porcine epidemic diarrhea virus antigen in formalin-fixed, paraffin-embedded intestinal tissues immunohistochemical detection of porcine rotavirus using immunogold silver staining (igss) rnascope: a novel in situ rna analysis platform for formalin-fixed, paraffin-embedded tissues additional file : figure s . panel of pathogens used as control for detection of tgev by immunohistochemistry. row one include immunostaining of tgev clinical cases against tgev, pedv, and rotavirus specific antibodies. a moderate to severe immunostaining is observe only reacting against tgev. in row two include positive controls for each pathogen detected by immunohistochemistry. row three present section tested negative by pcr for tgev, pedv, and rotavirus that were used as negative control of the immunohistochemistry techniques. ( the authors declare that all the data supporting the findings of this report is available in the article.authors' contributions pep, cjp, maq performed histological examination, data analysis and conclusion and were the major contributors in writing the manuscript; dmm, performed ihc; fv, performed ish; mil, rs, jac, emp, aa, lva field data collection and case identification. all authors read and approved the final manuscript.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.