key: cord- -d borky authors: blaising, julie; polyak, stephen j.; pécheur, eve-isabelle title: arbidol as a broad-spectrum antiviral: an update date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: d borky arbidol (arb) is a russian-made small indole-derivative molecule, licensed in russia and china for prophylaxis and treatment of influenza and other respiratory viral infections. it also demonstrates inhibitory activity against other viruses, enveloped or not, responsible for emerging or globally prevalent infectious diseases such as hepatitis b and c, gastroenteritis, hemorrhagic fevers or encephalitis. in this review, we will explore the possibility and pertinence of arb as a broad-spectrum antiviral, after a careful examination of its physico-chemical properties, pharmacokinetics, toxicity, and molecular mechanisms of action. recent studies suggest that arb’s dual interactions with membranes and aromatic amino acids in proteins may be central to its broad-spectrum antiviral activity. this could impact on the virus itself, and/or on cellular functions or critical steps in virus-cell interactions, thereby positioning arb as both a direct-acting antiviral (daa) and a host-targeting agent (hta). in the context of recent studies in animals and humans, we will discuss the prospective clinical use of arb in various viral infections. arbidol (arb) has been administered for decades in russia and china against influenza, with no major adverse effects reported. its vast potential as a broad-spectrum antiviral agent, defined through in vitro and in vivo studies, lends hope for its clinical use against various infectious diseases that are at present not therapeutically controlled. however, evidence for beneficial effects in humans, especially in the perspective of long-term administration in chronic diseases, remains equivocal. this could be attributable to a relative lack of standardized animal studies and controlled clinical trials in healthy and infected subjects. in addition to influenza and pathogenic human respiratory viruses, arb shows mainly in vitro inhibitory activity against the hepatitis b virus (hbv), hepatitis c virus (hcv), chikungunya virus (chikv), reovirus, hantaan virus and coxsackie virus b . in this paper, we update current knowledge about arb, linking its physico-chemical properties to its molecular mode of action, toxicity and possible pharmaceutical forms. we will outline recent studies on the molecular and cellular mechanisms by which arb may inhibit several steps of viral life cycles, and discuss how arb is emerging as both a direct-acting agent (daa) and a host-targeting agent (hta). china, toxicity arb, or ethyl- -bromo- -[(dimethylamino)methyl]- -hydroxy- -methyl- [(phenylthio)methyl]-indole- -carboxylate hydrochloride monohydrate, is a small indole derivative (fig. a) . it is also called umifenovir. its invention is attributed to a joint consortium of russian scientists from the chemical-pharmaceutical scientific research institute of russia, the scientific research institute of medical radiology in obninsk and the leningrad-pasteur scientific research institute for epidemiology and microbiology, some years ago, as described in: arbidol.net/robert-nikolaevich-glushkov.html arbidol.org/ - -arbidol-invented-way-to-the discovery.pdf arbidol.org/article .html. one of the first descriptions of its chemical synthesis was published in (trofimov et al., ) , and modified later on miller and bergeron ( ) . the drug is manufactured by moscow-based masterlek™, a subsidiary of pharmstandard group (see below), and by shijiazhuang no. pharmaceutical™ in china (http://www.sjzsiyao.com/products_detail/&productid= .html). arb has been marketed for years in russia and has been used since in china for the prophylaxis and treatment of human pulmonary diseases caused by influenza a and b viruses and other human pathogenic respiratory viruses, as reviewed in boriskin et al. ( ) , brooks et al. ( ) . it is also used to prevent flu epidemics in poultry in china (berendsen et al., ) , and is available from chinese companies specialized in animal health products, such as: http://depond.b bage.com/product-chemical-auxiliary-agent/ /arbidol-hydrochloride-pharmaceutical-rawmaterial.html the first reports on the clinical efficacy of arb were published in russian in the s, in groups of students and industrial workers during epidemics of influenza a and outbreaks of acute respiratory diseases (gagarinova et al., ; obrosova-serova et al., ) . later studies performed in servicemen reported the efficacy and cost-effectiveness of prophylactic or curative treatments of arb against acute respiratory viral infections, where arb was shown to decrease the febrile period (shumilov et al., ; shuster et al., ) . when the information is available, the duration of arb treatment varies from to days. chinese clinical studies with similar design (patient inclusion criteria, arb doses and duration of administration) point to a comparable efficacy and tolerability of arb (wang et al., ) . arb efficacy compared well or even better with that of other commonly used antiviral molecules such as rimantadine (roflual Ò ), oseltamivir (tamiflu Ò ), ribavirin or interferon-alpha kolobukhina et al., kolobukhina et al., , leneva and shuster, ) . it potentiated the in vitro effect of rimantadine against influenza a and b viruses , enhanced the immunomodulatory properties of the anti-flu vaccine vaxigrip Ò , administered in a cohort of elderly patients (semenenko et al., ) , and had a beneficial effects on flu in patients with another infectious immunodeficiency (glushkov et al., ) . most of these studies point to a dual pharmacological action of arb: a specific effect on respiratory viruses and an immune-stimulating effect, with induction of serum interferon and activation of phagocytes. studies have also been conducted in children suffering from flu and other acute viral respiratory diseases (beliaev et al., ; drinevskii et al., ) . the latter study -and most documented one -was conducted on children of - years old, infected with influenza a or b or both or with other respiratory viruses. over a -day treatment, arb was efficient at reducing the duration of infection and the occurrence of complications, and its immunomodulating action was again suggested. in , masterlek™, the company currently marketing arb, sponsored a vast clinical trial conducted on children from to over years old. arb was given (i) either in prophylaxis twice a week for weeks or once daily for days; (ii) or in treatment thrice daily for days. in all cases, arb treatment led to a significant reduction of the duration of clinical signs, with no observed adverse effect or complications; see: http://arbidol.org/arbidol-childrens-study.pdf. interestingly, in studies comparing antivirals, most viral strains were sensitive to arb, whereas several resistant variants were found with rimantadine (cf also the recent iatsyshina et al., ; l'vov et al., ) (see also section .). since , arb is also patented by masterlek™ for its medicinal use as an antiviral agent against atypical pneumonia induced by the severe acute respiratory syndrome coronavirus (sars-cov); see: http://www.arbidol.org/arbidol-patent- -sars-russian.pdf most recently, a double-blind, randomized, placebo-controlled phase iv clinical trial has been launched by pharmstandard/mas-terlek™, to assess whether arb is effective in the treatment and prophylaxis of flu and common cold: http://clinicaltrials.gov/ct /show/ nct ?term=arbidol&rank= . two dosages will be evaluated: mg/day for days, or mg/day for days. completion of this study is expected in . apart from this recent trial and in spite of an abundance of studies in the s, the overall language barrier renders difficult the precise evaluation of the number of subjects enrolled per study, the way clinical trials were designed, and subsequent statistical analyses performed. moreover, an official russian site exists for arbidol (arbidol.ru), where more information could be collected; but no english translation is available. as to studies specifically addressing arb toxicity issues, initial literature is mostly in russian, when available. acute toxicity data report oral ld s of - mg/kg in mice, and > mg/kg in rats and guinea pigs: http://img .guidechem.com/msdspdf/ - - .pdf; glushkov, . these values are also reported elsewhere: http://arbidol.org/pre- -animal-human-test-results.pdf; http://arbidol.org/rat.html; (loginova et al., ; shi et al., ) . administered intravenously, arb exhibited ld s of mg/kg in mice and mg/kg in rats (eropkin et al., ). on long-term per os administration of arb in rats, guinea pigs, rabbits or dogs from to over months (with doses ranging from to mg/ kg), no pathological changes were observed in animals. these doses would roughly correspond to -to -fold the therapeutic doses in humans. arb is also reported not to induce embryo toxicity in pregnant female rats, nor alter the reproductive function of animals, over a day-administration period of mg/kg doses (http://arbidol.org/rat.html). recent data from a chinese group showed a good tolerability of arb administered to rats per os, at daily doses ranging from to mg/kg over a -week period (wang et al., ) . but in fact this study assessed the toxicity of a : . combination of arb with acetaminophen, which renders difficult to precisely evaluate the toxicity of each molecule individually. in healthy male volunteers receiving a single mg-dose of arb, an excellent tolerability was reported . from these data, it appears that arb is a well-tolerated molecule with a high therapeutic index, when administered on periods ranging from a few days to a month. to date, however, no studies have addressed the long-term administration of arb, for example in the context of chronic infections. as an indole derivative, arb is expected to be poorly soluble in aqueous media. this is of major repercussion on its bioavailability, forms of administration and pharmacokinetics. efforts to improve arb water solubility were undertaken, through the chemical grafting of acrylamide polymers to the arb molecule (eropkin et al., ) . antiviral properties of these complexes were maintained compared to the parent molecule. they also displayed a better in vitro pharmacological index than arb, defined as ic /vic (vic, virus-inhibiting concentration). however these polymers were not further developed. arb is soluble in hot glycerol, where it remains soluble down to °c. it can then be diluted into aqueous media and administered in vitro or in vivo (brooks et al., ) . however no pharmacokinetic nor metabolite studies were performed from this mode of administration. a very selective, sensitive and accurate method of detection of arb from human plasma by high-pressure liquid chromatography was designed, and allowed to conclude that no interference existed between arb and its expected metabolites (metz et al., ) . studies based upon this hplc method then evaluated the pharmacokinetic parameters of arb after oral or intravenous administration in rats. comparable plasma elimination half-lives (t / ) and maximum concentrations (c max ) were obtained for oral doses six times higher than those injected; however this was only performed on a small number of animals (liu et al., a) . the drug is manufactured in russia and china as tablets or capsules, each containing arb as its active ingredient. initial russian studies in humans revealed that the main site of arb metabolism is the liver. arb was rapidly distributed in tissues and organs with maximal accumulation in liver ( . % w/w), pituitary gland, kidneys, lymphatic nodes and thyroid, adrenal gland, bone marrow, lungs, plasma, thymus and spleen (less than % each) (glushkov, ) ; see: http://www.chemeurope.com/en/encyclopedia/arbidol.html. plasma c max was reached within h or . h after a mgor mg-dose, respectively, and t / was between and h. about % of the total intake dose of arb was excreted unchanged within h via feces. more recent studies reported much shorter plasma c max and t / in chinese healthy volunteers, concluding that russian and chinese populations differed in arb elimination rates (liu et al., b ). however doses administered differed, and technological improvements, especially in detection sensitivity, might also explain such discrepancies. single doses of mg of arb administered to healthy volunteers from dispersible tablets or capsules were found bioequivalent and well tolerated . pharmacokinetics of oral single vs multiple doses of arb were compared in healthy subjects, from plasma samples analyzed by hplc (metz et al., ; sun et al., ) . c max increased linearly with the intake dose for single dose administrations, peaking at . lg/ml for a mg-dose (sun et al., ) . arb exhibited little accumulation with repeated doses, and the pharmacokinetic profile differed from that observed after single dosage. based on arb's chemical structure, several metabolites can be predicted ( fig. a) : oxidation at the sulfur atom, loss of the -(dimethylamino)methyle group and n-demethylation, conjugation at the -hydroxyl moiety. in a pioneering study in rat urine, the formation of sulfone and sulfoxide forms was confirmed after hplc, from an oral administration of an arb/starch suspension (anisimova et al., ) . glucuronide or sulfate conjugations were also observed at position , and after demethylation of the (dimethylamino)methyle group (see also liu et al., ) . in human urine, after administration of a single mg-dose of arb to healthy subjects, metabolites could be identified, of which the major ones were arb glucuronide and sulfoxide-arb (or sulfinyl-arb; fig. b ) glucuronide (wang et al., ) . similar metabolites as identified in rats were observed. arb could also be glucuronidated in vitro, using purified human liver microsomes; this study also revealed that the microsomal (recombinant) udp-glucuronosyl-transferase (ugt) a was the major ugt isoform involved in arb glucuronidation (song et al., ) . conversely, arb was found to inhibit ugt a (ibid.; liu et al., ) . since ugt a is involved in the metabolism of several drugs (e.g. acetominophen, diclofenac), potential drug-drug interactions that could lead to adverse effects should be carefully examined if arb is administered with other molecules. a more complete picture could be obtained from a study in healthy volunteers receiving a single oral dose of mg arb, where urine, feces and plasma metabolites were analyzed (deng et al., ) . most of the metabolites were sulfoxidated, dimethylamine n-demethylated, glucuronide-or sulfate-conjugated. about . % of the total intake dose of arb was excreted unchanged via feces, as previously reported (boriskin et al., ) . sulfinyl-arb ( fig. b) was the major circulating component detected in plasma, followed by unmetabolized arb, n-demethyl-sulfinyl-arb and sulfonyl-arb ( fig. c ). urine samples contained mainly glucuronide and sulfate conjugates. in vitro experiments using human liver, intestine and kidney microsomes, and recombinant enzymes, showed that arb was metabolized by human microsomes from liver and intestines but not from kidney. in these organs, cyp a was identified as a key metabolic enzyme of arb. still, questions remain about the pharmacokinetic properties of arb metabolites and their potential antiviral activity. the following parameters were reported (deng et al., ) : tmax for arb and dimethylamine n-demethylated arb were comparable ( . and . h, respectively), while it was much longer for sulfinyland sulfonyl-arb ( and h). plasma elimination half-lives (t / ) of all metabolites were longer than that of arb ( . , , . and . h for n-demethylated, sulfinyl-, sulfonyl-arb and arb, respectively). exposure to metabolites is therefore greater than that to arb, and the main metabolite, sulfinyl-arb, is expected to accumulate on repeated arb doses. along these lines, sulfinyl-arb was reported to contribute for some of the pharmacological activities associated with arb, and sulfonyl-arb could inhibit protein kinase c ([ic ] = . lm) (demin et al., ) . therefore the potency and duration effect for the agent may be underestimated by measuring only arb concentrations. it is also predictable, based upon in vitro data with microsomes, that some of the in vivo metabolites could occur in cell cultures, especially in studies addressing the antiviral effect of arb on hepatotropic viruses using liver-derived cell lines. this could explain why arb demonstrated greater efficacy under pre-incubation conditions, where metabolites could already be produced and exert specific effects (see below section .). however, a recent study directly addressing the in vitro antiviral properties of sulfinyland sulfonyl-arb against the chikungunya alphavirus showed only moderate to weak activity as compared to that of the parent molecule (delogu et al., ) . this was reinforced by the observation that pre-incubation of cells with arb prior to infection did not improve antiviral effect, pointing to a minor role (if any) of arb metabolites against chikungunya infection, at least in vitro. in any case, further investigations will be necessary to: (i) understand the importance of metabolites in the efficacy and safety of arb, due to their high plasma exposure and long elimination half-lives; (ii) assess their stability in circulation, tissue binding and storage properties. arb has been shown to display antiviral in vitro and/or in vivo activity against a number of enveloped or non-enveloped rna or dna viruses, including influenza viruses a, b and c , respiratory syncytial virus, sars-cov, adenovirus, parainfluenza type , poliovirus , rhinovirus , coxsackievirus b , hantaan virus, chikungunya virus, hbv and hcv [reviewed in boriskin et al. ( ) , brooks et al. ( brooks et al. ( , , liu et al. ( a) ] (see also table ). numerous reports in russian describe the antiviral potency of arb against human or avian influenza a viruses, and notably the highly pathogenic h n leneva and shuster, ) and the pandemic h n subtype (fediakina et al., ) . in vitro studies report ic s in the . - lm range, and state an effect of arb comparable to that of ribavirin, but superior to that of rimantadine, with rimantadine-resistant strains sensitive to arb romanovskaia et al., ; leneva et al., ; fediakina et al., ) . a few studies state a potentiating effect of arb and rimantadine (or amantadine) on influenza a and b viruses burtseva et al., ) . however adamantane antivirals are scarcely used against influenza viruses, due to low barrier to resistance. in these studies, accessible information does not allow to evaluate the stage of the viral life cycle targeted by arb nor its mode of action. shi and coworkers showed a greater inhibitory effect on influenza a h n when arb was added before infection or when it was pre-incubated with the virus (shi et al., ) , suggesting that membrane impregnation and/or metabolites could underlie arb antiviral activity (see section .). arb demonstrated similar in vitro antiviral activity as the reference drug ribavirin (virazole Ò ) against the respiratory syncytial virus (rsv), an enveloped virus of the paramyxoviridae family (leneva et al., ) . arb was most efficient when added before infection (shi et al., ) , with an ic of lm. recently, tannock and coworkers reported a potent antiviral activity of arb on several virus families responsible of respiratory infections in animals and humans, in particular on influenza a h n (ic lm), and the non-enveloped picornaviridae poliovirus and rhinovirus (brooks et al., ; see also brooks et al., ) . concerning rsv, only a reduction in plaque size and not in number could be observed, hampering the estimation of an ic . in this study, arb was added to cells as an aqueous glycerol solution, instead of the classical dilution from dmso in other studies (leneva et al., ; shi et al., ) . this might explain the discrepancy of antiviral effect on rsv. arb also displayed an inhibitory effect on the coxsackievirus b , another member of the picornaviridae family responsible for a variety of pathologies including respiratory infections, myocarditis or encephalitis . arb was most active on the virus itself (virucidal test) or when added after infection, through the inhibition of late stages of viral replication. indeed it was shown to prevent the viral rna synthesis in a dose-dependent manner, with maximal effect obtained at lm. one study in russian describes in vitro antiviral activity of arb against the sars-cov, when added after viral infection and at high concentration ( lm) (khamitov et al., ) . depending on the cell type, arb cc was reported to vary between and lm (e.g. boriskin et al., ; brancato et al., ; brooks et al., ; shi et al., ) . the dose exhibiting anti-sars-cov activity may likely be cytotoxic. studies conducted on mouse-adapted flu models showed that arb was effective when administered orally at doses from to mg/kg leneva et al., ) , or up to mg/kg (shi et al., ) , especially when given in prophylaxis before infection. extrapolated to humans, these doses would correspond to - g per day, not evaluated clinically in terms of safety. recently, arb was found to be effective in vivo against two influenza a h n strains, responsible for seasonal or pandemic flu (liu et al., b) . reductions in lung viral titers and lesions were observed for oral doses of - mg/kg/day, and secretion of lung and macrophage cytokines was modulated, indicating an inhibitory effect of arb on virus-induced inflammation. however, no effect on interferon-alpha was observed, in line with (brooks et al., ) but contrary to initial reports (glushkov, ) . brooks et al. ( ) reported a minor effect of arb on flu a-infected mice at doses from to mg/kg/day. discrepancies between results from different groups might come from: (i) bioavailability issues, due to differences in solvents used to solubilize arb (dmso vs glycerol); (ii) animal models of flu, using viruses and viral strains adapted or not adapted to mice; (iii) doses administered to animals. however, an overall anti-flu effect of arb in vivo seems apparent. mice with rsv-induced pneumonia were responsive to arb at - mg/kg/day doses, with an observable but not significant reduction in lung infectious titers as compared to untreated animals (brooks et al., ) . while this study points to a potential promising effect of arb against rsv in vivo, the limited of global studies addressing the effect of arb against rsv should invite moderation and a call for additional studies. one study addressed the antiviral effect of arb in mice infected with the coxsackievirus b . mice developed interstitial pneumonia and myocarditis, and some received arb orally for days. at a dose of mg/kg, the drug prolonged survival and reduced viral propagation in lungs and heart. although this result completes the picture of the broad-spectrum antiviral activity of arb, it must again be taken with caution, since it is the sole study on this virus, conducted on a small number of animals and with a high dose of arb. recently, chinese studies demonstrated antiviral activity of arb against the hantaan virus, an enveloped virus from the bunyaviridae family wei et al., ) , causing an often lethal hemorrhagic fever with renal syndrome (hfrs). in vitro, arb was more efficient when added before infection, with an ic in the lm range. a direct virucidal effect was noted only for arb concentrations over lm. in vivo, it was able to increase the survival rate, reduce histopathological changes and viral loads in the lethal model of intracranially-infected suckling mice. also, serum levels of tnf-alpha were modulated. since these studies were performed by only one research group, with a limited number of animals, they should be reproduced by others before concluding to a beneficial effect of arb against hantavirus infection. however arb efficacy compared well in vivo with that of ribavirin, the reference treatment for such a disease (wei et al., ) . viruses from the rhabdoviridae family are known to induce neurological disorders, encephalitis or, more recently reported, hemorrhagic fever (grard et al., ) . the only study addressing the effect of arb against a virus of this family was conducted in our laboratory on the vesicular stomatitis virus (vsv) (blaising et al., ) . this enveloped rna virus mainly infects cattle and pigs, causing oral lesions, anorexia and lethargy. arb was shown to inhibit in vitro vsv infection in a very similar concentration range as that already shown to affect influenza a or rsv infection [ic of (blaising et al., ) , (brooks et al., ) or lm (shi et al., ) , respectively]. again, arb displayed optimal antiviral activity when incubated with cells before infection. this family of double-stranded rna viruses comprises animal and human pathogens, such as the rotavirus, a major agent of ( ), teissier et al. ( ) gastroenteritis in children. we recently addressed the potential of arb against the mammalian reovirus t l strain (blaising et al., ) . this virus is a prototypic member of the orthoreovirus genus, which infects a wide variety of host species without causing a significant pathology in humans. in spite of this, reovirus has proven to be a useful model for studying viral pathogenesis. in vitro, arb inhibited reovirus infection in the lm range, but interestingly, did not exert any effect on infectious subvirion particles (isvps), intermediates of reovirus infection (chandran et al., ) that could also directly infect cells via a different entry mechanism from that of reovirus (martinez et al., ) . this points to the molecular mechanisms of action of arb (detailed in section .). this alphavirus is an enveloped single-stranded rna virus from the togaviridae family, loosely related to flaviviridae (see below hcv). it is responsible for recent outbreaks of a rheumatological disease. some neurological complications were described, together with meningo-encephalitis. arb demonstrated potent in vitro activity against chikv infection (delogu et al., ) . arb did not show virucidal activity, contrary to data on respiratory viruses (shi et al., ; zhong et al., ) , and displayed the highest efficiency when preincubated with cells h before infection (ic . lm). in this study, the main metabolites sulfinyl-and sulfonyl-arb were assayed and exhibited only weak antiviral activity, with ic s > lm. arb activity was not improved when a h-preincubation with cells was performed, suggesting that metabolites or degradation products are not responsible for arb antiviral action. taken together, these results suggest an interference of the parent molecule arb with early steps of the viral life cycle, such as cell binding and entry. arb and derivatives demonstrated in vitro efficiency against the hepatitis b virus (hbv), an enveloped dna virus from the hepadnaviridae family zhao et al., ) . arb prevented hbv dna replication with an ic of lm, and reduced the production of the virion surface antigen hbsag at lm; however the % cytotoxic concentration was lm, suggesting that inhibitory concentrations are most likely cytotoxic. this work will be further discussed below in the section structure/activity relationship (sar; section .). we showed that arb exerts in vitro antiviral activity against the hepatitis c virus (hcv) (reviewed in boriskin et al. ( ) ), a member of the flaviviridae family of enveloped viruses. more specifically, arb was most efficient when incubated with cells before infection and left during infection (pécheur et al., ) . as already shown with other viruses, arb also displayed virucidal activity (haid et al., ; pécheur et al., ) . in the lm range, arb inhibited hcv entry, fusion in in vitro and in cellulo studies (blaising et al., ; haid et al., ; teissier et al., ) , and replication on longer times of cell treatment (boriskin et al., ; sellitto et al., ) . however, as previously reported in the case of influenza a infection in vivo (brooks et al., ) , arb was not found to induce interferon antiviral responses in vitro against hcv (boriskin et al., ) . from these studies, arb molecular mechanisms of action were proposed (see section below). several studies aimed at gaining a better understanding of the structural features of arb important for its broad antiviral activity, improving arb therapeutic index, or identifying novel lead compounds active against emergent viruses. compounds derived from the chemical structure of arb were synthesized and assayed against various influenza a and b viruses (brancato et al., ) . the amine in position and the hydroxyl moiety in position were found important for arb antiviral action, whereas the presence or absence of br in position had little effect (see fig. a ). insertion of a methyl group between the indole ring and -hydroxyl considerably increased antiviral potency of the resulting compound. this molecule was shown to directly bind ha , with a greater affinity than arb. the presence or absence of the -bromo group had also no influence on hbv or hcv infections (sellitto et al., ; zhao et al., ) . more specifically, the introduction of particular azote-based heterocyclic groups at position improved anti-hbv activity , while it had little effect against hcv (sellitto et al., ) . replacement of the s-phenyl group at position by a phenyl-sulfonyl decreased the cytotoxicity and increased the anti-hbv activity of the compound zhao et al., ) , while removal of this group was without any influence against hcv (sellitto et al., ) . the -hydroxy group was found dispensable against hcv. thus, it appears that different substituents of the arb molecule play a role in the antiviral activity, depending on the virus considered, the cellular model used and the test conditions. the combination of in vitro, in cellulo and in silico analyses will help refine the sar of arb. in particular, in silico molecular docking studies allowed the precise identification of amino-acid(s) involved in arb (or derivative) interaction with ha (nasser et al., ) . also, three-dimensional quantitative sar ( d-qsar) helped design novel anti-hbv compounds based upon a -hydroxy- h-indole- -carboxylate skeleton, and predict their antiviral potency (chai et al., ) . this type of approach is also now conceivable to study the potential interactions of arb with hcv envelope glycoproteins and clarify structural requirements for antiviral activity, since the d-structure of hcv e has recently been released (kong et al., ) . arb's broad-spectrum antiviral activity suggests that the molecule acts on common critical step(s) of virus-cell interactions. evidence indicates that arb directly exerts a virucidal effect, and can then be considered as a direct-acting antiviral (daa). most studies also report an effect of arb on one or several stages of the viral life cycle, such as cell entry (attachment, internalization) and replication. arb could therefore also act as a host-targeting agent (hta). in the following section, we will examine the mechanisms by which arb could exert such dual antiviral activity (recapitulated in table ). arb is an indole-based hydrophobic molecule susceptible to formation of supramolecular arrangements through aromatic stacking interactions with selective amino-acid residues of proteins (phenylalanine, tyrosine, tryptophan). by liquid-state nmr analysis, we showed that arb displays interfacial properties and intercalates in the shallow layer above the glycerol backbone of phospholipids (teissier et al., ) . it is even conceivable that arb could locally become more concentrated in viral or cellular membranes. it was also shown that arb interacts with aromatic residues within the viral glycoprotein involved in membrane interactions and destabilization necessary for fusion, aka the fusion protein (leneva et al., for influenza hemagglutinin; teissier et al., for hcv e ) . this could therefore underlie the virucidal (daa) effect of arb, interacting with the viral lipid envelope and/ or with key residues within structural proteins of virions (required for cellular receptor/captor recognition and/or membrane fusion). this effect has been described for enveloped [influenza a h n virus (shi et al., ) ; hantaan virus ; hcv (haid et al., ; pécheur et al., ) ] and non-enveloped viruses [coxsackie virus b , consistent with arb's dual physico-chemical properties. arb could also locally impair viral attachment to cell plasma membrane by stabilizing the membrane, and/or by masking key residues in a viral protein involved in receptor recognition, in a sort of daa + hta effect. this would have consequences on viral entry. as shown by fluorescence spectroscopy and surface plasmon resonance analyses, arb affinity for lipid membranes is even more pronounced at acidic ph, the optimal ph for the fusion step of several enveloped viruses, influenza viruses and hcv in particular (fig. ) (haid et al., ; pécheur et al., ; teissier et al., teissier et al., , . this interaction with phospholipids may perturb membrane fluidity, thereby rendering the lipid bilayer less prone to fusion. inhibition of viral entry and membrane fusion occurred in the lm range, in agreement with arb affinity for membranes and the concentration range achieved in healthy volunteers (sun et al., ) . mechanistically, the dual binding capacity of arb to lipids and proteins might also underlie alterations of protein/protein and/or protein/lipid interactions at other stages of the viral life cycles, such as replication, assembly and budding. for a number of viruses, in particular in the flaviviridae family, replication occurs in a subcellular compartment called the membranous web (heaton and randall, ; moradpour et al., ) . the membranous web is an emanation of the endoplasmic reticulum induced by viral proteins such as hcv ns b (gouttenoire et al., ; romero-brey et al., ) . since the web is created and maintained through interactions between viral and cellular proteins and lipids, it is plausible that arb could impair viral replication through its ability to bind proteins and lipids. concerning viral assembly and budding, intracellular membranes are obligate partners of nucleocapsids during packaging of enveloped viruses, and for the secretion of newly assembled viral particles. in the case of hcv, viral assembly is concomitant to the assembly of lipoproteins, giving rise to lipo-viro particles (bartenschlager et al., ) . arb could therefore interfere with these processes through its physico-chemical dual interactions with lipids and proteins. recently, we provided molecular details of how arb inhibits virus entry into target cells, in a study based on live-cell confocal imaging, using hcv as a model of an enveloped virus (blaising et al., ) (fig. ) . first, arb was found to drastically impede virion attachment to cell plasma membrane. arb subsequently impaired the release of clathrin-coated pits (ccps) from the plasma membrane, resulting in a slowing of clathrin-coated vesicle (ccv) intracellular trafficking. the net result was an intracellular accumulation of ccvs containing trapped virions. arb was also shown to affect clathrin-mediated endocytosis (cme) by impeding dynamin- -induced membrane scission, and thereby ccp formation. lastly, arb inhibited fusion between endocytic vesicles and endosomes and hindered viral intracellular trafficking. virions were not properly delivered to rab -positive endosomal compartments where fusion occurs and/or rab was not recruited to virioncontaining vesicles. as a result, fusion was greatly impaired and virions trafficked to endo-lysosomal lamp- -positive compartments for degradation. overall, the data suggest that arb's dual interactions with lipids and proteins may alter several aspects of intracellular trafficking with and maturation in endosomal compartments. arb may impede the recruitment and/or disassembly of machineries required for proper endosomal trafficking and viral entry. thus, arb inhibition of key actors of intracellular trafficking may be a likely explanation of its broad-spectrum antiviral activity (table , fig. ) , and suggest that arb acts as an hta. in most studies, arb exerted a maximal antiviral effect when used before infection, indicating an activity on early stages of viral infection and/or the requirement for arb to impregnate cells. in the current state of the literature, arb was shown to be active against viruses that enter cells by routes requiring at least one of these features: acidification, rab , dynamin- , actin. reovirus, vsv and hcv hijack cme (respectively: boulant et al., ; johannsdottir et al., ; meertens et al., ) , hbv also most likely enters via this pathway (yan et al., ) . chikv entry is mainly achieved via cme (leung et al., ) , but alternative clathrin-independent pathways have been described, dependent upon ph, dynamin- , rab and actin cytoskeleton integrity (bernard et al., ) . viruses such as influenza or hantaan can enter through clathrindependent and -independent endocytotic pathways that have acidification in common (reviewed in mercer and helenius ( ), lozach et al. ( ) ). rsv entry is achieved by macropinocytosis and, as shown for hcv, the intracellular trafficking of virions is rab -dependent (krzyzaniak et al., ) . in the picornaviridae family, group b coxsackieviruses entry in epithelial cells occurs via a complex process, combining caveolin-dependent endocytosis with features of macropinocytosis such as dependence upon rab (coyne et al., ) . also in this family, poliovirus relies on an actin-and tyrosine kinase-dependent endocytic pathway to invade its target cells (brandenburg et al., ) , and rhinovirus entry is ph-dependent and likely achieved by macropinocytosis (khan et al., ) . concerning sars-cov entry, the only consensus feature is its dependence on acidification in internal cell compartments (inoue et al., ; wang et al., ) . apart from lipid membranes, it is therefore conceivable that arb acts on several cellular targets common to the life cycle of various viruses. studies directly addressing arb as an hta and its interactions with proteins of intracellular trafficking are not available at present, but from our work with hcv, rab , dynamin- and elements of the clathrin coat could be potential targets (blaising et al., ) . it is also conceivable that elements of the cytoskeleton could be targeted; indeed, molecules based on an aryl-thio-indole skeleton (fig. d) , closely related to arb chemically, are inhibitors of tubulin polymerization, and thereby potent anticancer agents (la regina et al., ) . arb was reported to inhibit influenza-and hcv-mediated membrane fusion (leneva et al., ; teissier et al., ) . in vitro studies showed that arb increases the stability of the influenza virus hemagglutinin (ha) and hinders low ph structural reorganizations necessary for ha to adopt its fusiogenic conformation, thus blocking infection at the viral fusion step (leneva et al., ; see also below section .). concerning hcv, fusion inhibition is dosedependent but does not depend on the hcv genotype or on the lipid composition of target membranes (liposomes); it predominantly prevails at low ph (boriskin et al., ; haid et al., ; pécheur et al., ; teissier et al., ) . arb was found to directly interact with peptides from the hcv e glycoprotein (teissier et al., ) and within a pocket of the influenza ha subunit of hemagglutinin (nasser et al., ) , thereby exerting its effect as a daa. interestingly, these peptides and pocket contain aromatic residues such as tyrosines and tryptophans, which could engage in aromatic stacking interactions with arb molecules, as described above. arb may therefore inhibit fusion by impairing conformational changes in viral fusion proteins during initiation of fusion (daa activity) and by increasing membrane rigidity, rendering membranes refractory to the destabilization that is required for fusion (hta activity). arb was shown to inhibit hcv replication in replicon systems, i.e. a cell culture context where virus replicates without any production of infectious viral particles (boriskin et al., ; sellitto et al., ) . a progressive decline in both viral protein and rna expression was observed in arb-treated cells, and cells could be cured of replicating viral rna after weeks of arb treatment (boriskin et al., ) . since hcv modulates lipid metabolism (bassendine et al., ) and creates a lipid-rich internal membrane environment favorable for virus replication (i.e. the membranous web), arb could therefore impregnate these membranes to impede the formation and maintenance of the membranous web and in turn viral replication. chikv replication takes place in the host cell cytoplasm and is associated with cytoplasmic membrane alterations (solignat et al., ) . replication complexes are attached to the membrane of modified endosomes and lysosomes to form organelles characteristic of alphavirus replication called type cytopathic vacuoles. these vacuoles consist in vesicles of . - . lm in diameter harboring numerous spherules (grimley et al., ) , which are positive for lysosomal markers (kujala et al., ) . the vacuoles produce viral rna until cell death. as already described for hcv, lipid bilayers are therefore essential for chikv replication. it is thus plausible that arb may also impede the formation and stability of these vacuoles, thereby perturbing chikv replication. in the absence of studies aimed at addressing the potential interactions between arb and cellular proteins involved in viral replication, one cannot exclude that such interactions might occur, as already suggested at the viral entry/maturation stage. to date, no report has been made concerning an effect of arb on viral assembly; however, further investigations are still needed to address this question directly. concerning viral budding, a recent study supports the notion that arb could inhibit influenza virus egress because viral rnas accumulate in cells at later stages of infection (brooks et al., ) . arb impregnation of cellular membranes and/or the targeting of proteins involved in intracellular trafficking that relate to viral morphogenesis/budding could again underlie this observation. in spite of its usage in russia and china for several years in flu, arb does not seem to generate a high degree of viral resistance. epidemic strains of influenza a/h n and a/h n isolated in russia in - revealed resistance to oseltamivir and/or rimantadine, but were all sensitive to arb ). the pandemic swine influenza a/h n was found largely resistant to rimantadine, but had retained its sensitivity to oseltamivir (tamiflu Ò ) and arb (iatsyshina et al., ) . in - , influenza a/h n and b viruses were found to be the cause of a vast epidemic in russia; all tested strains were sensitive to oseltamivir, zanamivir (relenza Ò ) and arbidol, but resistant to rimantadine (l'vov et al., ) . however resistance to arb of various strains of influenza viruses has been reported, in particular in a population of influenza b . in a study aimed at understanding the anti-influenza mechanism of action of arb, leneva and colleagues isolated seven viral mutants from the influenza a/h n ''weybridge'' strain, that were refractory to arb doses above lm (leneva et al., ) . all mutants exhibited a single mutation in the ha subunit of the influenza hemagglutinin, the subunit involved in membrane fusion. this translated functionally into a . -unit increase in the ph required to induce ha conformational changes. arb was found to directly interact with ha , thereby increasing its stability to ph and impeding fusion in endosomes during virus infection. using an elegant proteomic approach, this interaction was further investigated by nasser and coworkers, and found confined to one peptide encompassing ha residues - . this region contains the arb already identified mutation resistance k r (leneva et al., ; nasser et al., ) . taken together, these data reveal that resistance of influenza viruses to arb mainly arises from mutations in the ha fusion protein, consistent with arb antiviral activity related to membrane fusion. addressing arb antiviral mechanism of action against chikv, delogu and coworkers isolated a mutant virus adapted to arb at lm (delogu et al., ) . this virus was characterized by a single mutation in the e viral envelope glycoprotein, in a region most likely involved in cell-surface receptor recognition, and maybe indirectly to membrane fusion. clearly, additional studies on arb resistance in the context of other viral infections are warranted. in conclusion, the broad-spectrum activity of arb may arise through duality of function: a capacity to interact with both membranes and with viral and/or cellular proteins. arb therefore has features of both a daa and a hta. these interactions would impede cellular processes and pathways that are hijacked by several viruses to infect their host cells. regarding hcv, we have shown that arb inhibits most steps of hcv entry, from attachment to internalization, until the final step of membrane fusion. arb also inhibits hcv replication, which may arise via alteration of intracellular membrane-protein structures essential for intracellular trafficking (e.g. clathrin coat components, elements of the cytoskeleton) and virus replication (e.g. membranous web), and could hinder membrane rearrangements necessary for the viral budding step. the broad-spectrum activity and the cellular mechanisms affected by arb are summarized in fig. . through these effects, arb could display an antiviral activity on viruses that hijack similar cellular pathways or have overlapping life cycles. in particular, endocytosis is used by several viruses and viral families including human immunodeficiency virus (von kleist et al., ) , adenoviridae, arenaviridae, coronaviridae, togaviridae to achieve productive infection (table ) . moreover, all positive-strand rna viruses of eukaryotes are known to reorganize intracellular membranes to create specific virus replication organelles. for these reasons, efforts should be pursued in order to determine the potential inhibitory effect of arb on a large class of viruses. a better understanding of its molecular mechanisms of action would also contribute to refine the conditions at which it could be given in long-term regimens against chronic infections (e.g. hepatitis b or c). indeed current data on toxicity issues are insufficient to evaluate the safety of arb in chronic administration. nevertheless, most studies point to a good tolerability of this molecule. in the present state of our knowledge, arb could therefore constitute a cost-effective pharmacological approach, affordable for emerging countries in urgent need for effective antiviral therapies. study of metabolism of the antiviral drug arbidol by mass spectrometry, thin-layer and high-performance liquid chromatography assembly of infectious hepatitis c virus particles arbidole -a new drug for prevention of influenza and acute viral respiratory infections in children quantitative trace analysis of a 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structures associated with hepatitis c virus replication synthesis and anti-hepatitis c virus activity of novel ethyl h-indole- -carboxylates in vitro characteristics of the immune status in specific and nonspecific prophylaxis of influenza in elderly persons antiviral activity of arbidol against influenza a virus, respiratory syncytial virus, rhinovirus, coxsackie virus and adenovirus in vitro and in vivo efficacy of arbidol in prophylaxis and treatment of acute respiratory viral infections in servicemen arbidol used in the prophylaxis of acute respiratory viral infections and their complications in servicemen replication cycle of chikungunya: a re-emerging arbovirus glucuronidation of the broad-spectrum antiviral drug arbidol by ugt isoforms pharmacokinetics of single and multiple oral doses of arbidol in healthy chinese volunteers targeting cell entry of enveloped viruses as an antiviral strategy mechanism of inhibition of enveloped virus membrane fusion by the antiviral drug arbidol synthesis of a new antiviral agent, arbidole role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition efficacy and safety of arbidol in treatment of naturally acquired influenza a -week oral toxicity study of an antiviral drug combination consisting of arbidol and acetaminophen in rats metabolite identification of arbidol in human urine by the study of cid fragmentation pathways using hplc coupled with ion trap mass spectrometry establishment of sybr green-based qpcr assay for rapid evaluation and quantification for anti-hantaan virus compounds in vitro and in suckling mice sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus synthesis and in vitro anti-hepatitis b virus activities of some ethyl -hydroxy- h-indole- -carboxylates antiviral activity of arbidol against coxsackie virus b in vitro and in vivo we thank steeve boulant for his invaluable contribution to live-cell imaging of hcv infection in blaising et al., . j.b. is the recipient of a doctoral grant from the rhône-alpes region (arc santé), and e-i. p. is supported by anrs (agence nationale pour la recherche sur le sida et les hépatites virales). key: cord- -x z o gs authors: artusi, sara; nadai, matteo; perrone, rosalba; biasolo, maria angela; palù, giorgio; flamand, louis; calistri, arianna; richter, sara n. title: the herpes simplex virus- genome contains multiple clusters of repeated g-quadruplex: implications for the antiviral activity of a g-quadruplex ligand date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: x z o gs guanine-rich nucleic acids can fold into g-quadruplexes, secondary structures implicated in important regulatory functions at the genomic level in humans, prokaryotes and viruses. the remarkably high guanine content of the herpes simplex virus- (hsv- ) genome prompted us to investigate both the presence of g-quadruplex forming sequences in the viral genome and the possibility to target them with g-quadruplex ligands to obtain anti-hsv- effects with a novel mechanism of action. using biophysical, molecular biology and antiviral assays, we showed that the hsv- genome displays multiple clusters of repeated sequences that form very stable g-quadruplexes. these sequences are mainly located in the inverted repeats of the hsv- genome. treatment of hsv- infected cells with the g-quadruplex ligand braco- induced inhibition of virus production. braco- was able to inhibit taq polymerase processing at g-quadruplex forming sequences in the hsv- genome, and decreased intracellular viral dna in infected cells. the last step targeted by braco- was viral dna replication, while no effect on virus entry in the cells was observed. this work, presents the first evidence of extended g-quadruplex sites in key regions of the hsv- genome, indicates the possibility to block viral dna replication by g-quadruplex-ligand and therefore provides a proof of concept for the use of g-quadruplex ligands as new anti-herpetic therapeutic options. g-quadruplexes are nucleic acids secondary structure that may form in g-rich sequences. they are based on the formation of g-quartets, which are stabilized by hoogsteen hydrogen bonds between guanines (sen and gilbert, ) . g-quartets stack on top of each other to give rise to g-quadruplexes that are further stabilized by the interaction with monovalent cations. dna strands in g-quadruplex may be oriented in anti-parallel, parallel, or hybrid configuration, and the nucleotide linkers between g-quartet stacks can adopt a multitude of loop structures (patel et al., ; phan, ; phan et al., ; simonsson, ) . g-quadruplexes occur in functionally important regions of the genome (huppert, ; neidle, ) : they have been identified in the promoters of a wide range of genes that are important in cell signaling (balasubramanian et al., ; duquette et al., ) , suggesting the possibility that g-quadruplexes behave as structural switches of cellular processes, therefore providing a basis for therapeutic intervention (neidle and parkinson, ; zhang et al., ) . one of the most potent g-quadruplex ligands is braco- (fig. a) , a , , -trisubstituted acridine derivative designed to stabilize the quadruplex dna structures formed in human telomeres (read et al., ) . has been shown to inhibit telomerase activity (harrison et al., ) and to display in vitro and in vivo antitumor activity (burger et al., ; gowan et al., ) . besides humans, other mammals (verma et al., ) , yeast (hershman et al., ) and prokaryotic cells (beaume et al., ; rawal et al., ; wieland and hartig, ) showed enrichment of putative g-quadruplex forming sequences. in viruses, evidence of significant g-quadruplex regions is increasingly being recognized. in the epstein-barr herpes virus (ebv), g-quadruplexes modulate ebv nuclear antigen (ebna ) activity and translation (murat et al., ) ; in particular, braco- inhibited ebna -dependent stimulation of viral dna replication http://dx.doi.org/ . /j.antiviral. . . - /Ó elsevier b.v. all rights reserved. (norseen et al., ) . in hiv- , we have demonstrated a g-quadruplex-mediated transcriptional silencing activity in the sp binding region of the long terminal repeat promoter (perrone et al., a) and in the nef coding gene (perrone et al., b) . in both cases, g-quadruplex ligands were able to stabilize the g-quadruplex conformations and to exert antiviral activity (perrone et al., ) . in the sars coronavirus, g-quadruplexes targeting by a key viral protein was proposed to control host's cell response to viral infection (tan et al., ) . the herpes simplex virus type (hsv- ) kbp genome has % guanosines (gs) and cytosines (cs) composition, which reaches . % in simple sequence repeats (ssrs), ubiquitously distributed in both coding and non-coding regions with similar relative frequency (ouyang et al., ) ; in contrast, gc content in the human genome is %. the hsv- genome is composed of two covalently linked regions of unique sequences, the unique long (u l ) and unique short (u s ) sequences, flanked by large inverted repeated sequences, tr l -ir l and ir s -tr s (fig. b) (hayward et al., ) . hsv replication is temporally regulated in rounds of transcription that yield immediate-early, early and late proteins. hsv- is a neurotropic virus: after the first lytic infection within mucosal epithelial cells, the virus enters sensory neurons where latency is established. the virus can later reactivate resulting in the generation of new virions that cause recurrent disease (roizman et al., ) . hsv- most commonly causes vesicular lesions affecting the mucous membranes; however, it can also cause infrequent but serious diseases, such as encephalitis, disseminated neonatal infections and visceral infections in immunocompromised patients (frangoul et al., ; genereau et al., ) . the presence of hsv increases sexual transmission of hiv- (freeman et al., ) . hsv- symptoms are not cured but treated with nucleoside analogues, such as acyclovir (acv) and its derivatives (vere hodge and field, ). however, the virus remains in the body for life, since no cure that eradicates herpes has yet been developed. in addition, emergence of resistance to current anti-herpetic drugs has long created an obstacle for the treatment of hsv- (bacon et al., ) . therefore new antiviral approaches able to suppress both lytic and possibly also latent infections are highly required. earlier work by roizman indicated the presence of an alternative conformation of a viral g-rich dna and rna sequence around the origin of dna replication in the hsv- genome (mccormick et al., ; roller et al., ) ; however no systematic analysis on the presence of g-quadruplex structures in the hsv- nucleic acid has ever been provided. here we present the first comprehensive genome analysis and evidence that extended g-rich sequences mainly located in repeats of the hsv- genome can fold in stable g-quadruplex structures. we demonstrate that treatment with the g-quadruplex ligand braco- greatly stabilizes these sequences resulting in decrease of infectious viral particles, reduction of late viral transcripts, inhibition of taq polymerase processing at the hsv- genome, specifically affecting viral dna replication at g-quadruplex regions. a detailed description of this section is provided in supporting information. . . highly conserved putative g-quadruplex forming sequences are present in repetitive regions of the hsv- genome we identified nine regions in the hsv- genome with highly repeated putative qgrs (quadruplex forming g-rich sequences) ( table ) . six of them (named gp a-f) were found in the leading strand of the gp gene ( fig. b) which encodes ul , the largest viral protein and essential viral tegument component (mcnabb and courtney, ) (table ). the gp series sequences displayed the g xtg xtg xtg xt common motif, where x was either a t or a c base, and covered about bps. four more putative qgrs were clustered at the terminal and internal repeats (both long and short) (fig. b, table ): they covered about bps in the leading strand and bps in the lagging strand. the exact role of these regions is as yet unknown and thus these positions were named ''un''. all identified sequences were highly conserved among different hsv- strains ( table ). the only exception was the unb sequence, which was identical to gp b, and was not conserved in the kos strain. to note that all identified sequences, apart from un , were characterized by four gggg-tracts, therefore possessing the ability to fold into -tetrad stacked g-quadruplex. this kind of tetraplex is in principle very stable, as denoted by the high g-scores, which indicate the propensity of a sequence to fold into unimolecular g-quadruplex, obtained by these oligonucleotides (table ). the un sequence displayed four ggg-tracts therefore gaining a lower g-score. folding of the hsv- putative qgrs was determined by circular dichroism (cd) spectroscopy. in physiological concentrations of k + , all tested oligonucleotides displayed cd g-quadruplex signatures that included three different conformations (vorlickova et al., ) : hybrid (gp ), antiparallel (un ) and parallel (un , un , fig. a) . usually, g-quadruplex folding occurs in the presence of k + , while in the absence of the monovalent cation oligonucleotides are mostly unfolded. the hsv- oligonucleotides, however, displayed peaks characteristic of the g-quadruplex conformation even in the absence of k + (fig. s ). stability of hsv- g-quadruplexes in the presence of k + was assessed by thermal denaturation experiments monitored by cd and uv spectroscopy. un and un were the most stable g-quadruplexes with t m above °c, table sequences of putative g-quadruplex forming oligonucleotides in the genome of hsv- . sequence repeats within hsv- main strains and g-scores are shown. followed by gp oligonucleotides. (fig. b , table ). because of the very low solubility of un in k + , t m values for this sequence were not obtained. in the absence of k + , un was the least stable sequence, followed by un . un and gp oligonucleotides showed comparable stability, with the exception of gp d, which was extremely stable even in the absence of k + ( table ). the ability of hsv- qgrs to fold into g-quadruplex was further assessed by emsa analysis. even in the absence of k + , all gp sequences run markedly faster than their bp-length control marker, indicating folding in these conditions (fig. c) . in particular, gp d displayed the fastest mobility, confirming its inherent property to fold into g-quadruplex in the absence of k + . un ( -bp) and un ( -bp) also migrated faster than the -long control marker (see * symbol in fig. c ), indicating effective folding. however, a band corresponding to the unfolded un was also observable (see § symbol in fig. c ). in contrast, the bp-long un migrated as a totally unfolded oligonucleotide (compare lane with lane m, fig. c ), confirming its lower tendency to fold in the absence of k + (see lower g-score, table , and lower stability, table ). in the presence of k + (fig. d) , all sequences displayed increased migration indicating effective folding. for the gp sequences, slower migrating species, ascribable to intermolecular dimeric g-quadruplex forms, were also observed (lanes a-f, fig. d) . the g-quadruplex ligand braco- was next tested by cd analysis for its ability to bind the hsv- qgrs in the presence of k + . braco- converted the gp hybrid conformation to an antiparallel-like g-quadruplex (fig. s ) and further stabilized all hsv- oligonucleotides, increasing t m to above °c (table ) . additional evidence of the intrinsic stability of the identified hsv- g-quadruplexes within a longer dna context and on the increased stabilization imparted by braco- was provided by the taq polymerase stop assay. all g-quadruplex forming sequences except un stopped the polymerase at the first g-rich region encountered by the polymerase even in the absence of k + (lanes , un , gp a, gp d, fig. ). when k + was added, strong stops were observed in all g-quadruplex templates (lanes , fig. ). upon addition of braco- the intensity of the initial stop sites dramatically increased (lanes , fig. ) ; moreover, an additional intense stop site corresponding to a second g-rich region was observed in the un template, possibly indicating the presence of multiple g-quadruplex structures. to note that braco- stopped the processing of the polymerase to such an extent that the full-length product did not form in all templates, with the exception of un , which resulted the sequence less susceptible to the activity of the compound (lanes , fig. ). in contrast, tmpyp , a porphyrin derivative which shares chemical features with braco- , i.e. a large aromatic surface and cationic moieties, but displays a different g-quadruplex binding mode and markedly lower g-quadruplex binding affinity (han et al., ; le et al., ) , used here for comparison, did not induce any stop at g-rich regions and it did not modify the amount of the fulllength amplicons (lanes , fig. ). moreover, a control sequence devoid of g-tracts did not show any stop site in the presence of k + and braco- (lanes - , non-g template, fig. ) , indicating that the observed polymerase inhibition was g-quadruplex dependent and specific. given the ability of braco- to recognize and stabilize hsv- g-quadruplexes, we tested the possibility that it displayed antiviral activity. tmpyp was used for comparison. cytotoxicity of both compounds was assessed on vero cells, a monkey kidney epithelial cell line which sustains hsv- infection. both compounds were non-cytotoxic up to lm, the highest tested dose (cc > lm). in hsv- infected vero cells, braco- demonstrated a statistical significant antiviral effect at lm, which increased up to % at lm (ec = lm) (fig. a) . in contrast, tmpyp displayed ec = lm. since both braco- and tmpyp , as most g-quadruplex binding molecules, are polycationic agents, we first excluded that the observed antiviral activity was due to the interaction of these molecules with the negatively charged heparan sulfate (campadelli-fiume et al., ; herold et al., ) , thus preventing virus attachment to the cells. braco- or tmpyp were added at different times relative to infection until the virus had completed its entry into cells (i.e. h.p.i.) (sodeik et al., ) . in this time range we did not observe any difference to the activity obtained by treating the cells h pre-infection (fig. s ) , indicating that neither braco- nor tmpyp act by inhibiting viral entry. one cluster of hsv- qgrs was embedded in the gp gene, which encodes the essential tegument protein ul ; we thus and tmpyp (t). extended un , un , gp a and gp d dna templates (table s ) in the absence and presence of k + mm (lanes and , respectively) and in the presence of tmpyp (t) and braco- (b) ( . lm) (lanes and , respectively) were used as templates for the taq polymerase. a non-g-quadruplex template (non-g lanes) was also used as negative control. p indicates primer. or h.p.i. total rna was isolated, retrotranscribed into cdna and expression of specific genes determined by rt-pcr (table s ). the mrnas of the immediate-early us , icp and ul , early ul dna polymerase and late proteins ul and ul were analyzed. rq are relative quantities. in all data sets: n p , mean ± s.d., student's t-test, ⁄⁄ p < . . investigated if treatment with braco- impaired ul transcription. transcript levels of a second tegument protein, ul , were also assessed as control for a protein whose coding sequence lacks important qgrs clusters. both viral transcripts were decreased to - % at h.p.i. (fig. b) and this effect was specific for the hsv- transcripts (fig. s a) . the mrna levels of representative immediate-early and early proteins, whose coding sequences lacked important g-quadruplex forming regions, were also analyzed: braco- did not affect transcription of these earlier proteins (fig. b) . moreover, tmpyp reduced hsv- transcript levels to a low extent ( - %) and it did not differentiate among the different kinetic groups of proteins. the fact that both analyzed late transcripts were affected by braco- to a similar degree indicates that the observed effect is independent of the presence of qgrs in the ul coding sequence; the absence of effect on earlier genes indicates that the compound likely exerts it activity against g-quadruplex-controlled viral mechanisms that mostly influence transcription of late proteins. since the identified clusters of qgrs were mostly present in non-coding repetitive-element regions, we tested the possibility that qgrs folding induced by braco- inhibited polymerase processing at the viral dna level. hsv- dna was extracted from a viral stock and treated with increasing concentrations of braco- and tmpyp . genome regions containing g-rich tracts were amplified by standard pcr using taq polymerase, which was used as a model dna polymerase enzyme. a non-g-rich/non-g-quadruplex forming sequence was also amplified as negative control sequence. amplified dna corresponding to g-quadruplex regions decreased in a concentration dependent manner in the presence of braco- (fig. a, un and gp , braco- ). in contrast, braco- was not able to disrupt polymerase processing in the non-g-quadruplex region (fig. a , non-g , and tmpyp had no effect in both g-rich and non-g-rich regions (fig. a, un, gp , non-g , tmpyp ). real-time pcr was employed for quantification purposes. amplification of the g-quadruplex region was inhibited up to % (with respect to the non-treated control, %) in the presence of braco- ( lm), while no effect was detected in the non-g-quadruplex region (fig. b) , confirming lack of activity of the compound against the polymerase enzyme. no activity of tmpyp was observed. this data demonstrate that braco- specifically interacts with g-quadruplex regions in the hsv- genome in vitro and inhibits polymerase processing likely due to the steric hindrance caused by multiple tetraplex structures stabilized by the ligand. we next tested the effect of braco- on viral dna amounts in infected cells. hsv- infected cells were treated with increasing concentrations of braco- or tmpyp and intracellular viral dna was extracted and quantified by real-time pcr. dna extraction was performed at different time points, before ( h.p.i.) and after ( h.p.i.) the viral dna was replicated in cells. braco- reduced intracellular viral dna levels to % of the untreated control ( %) at the highest concentration at h.p.i. (fig. c ). in contrast, at h.p.i. braco- and tmpyp did not affect viral dna amounts. moreover, addition of braco- did not modify cellular dna amounts (fig. s b) . to define the viral step targeted by braco- in infected cells, a time-of-addition experiment was set up (daelemans et al., ; pauwels et al., ) . this assay indicates the last viral step targeted by a compound. acv was used as reference compound with established mode of action in the time frame of replication events (james and prichard, ) . braco- was added to infected cells every h up to h.p.i. (fig. d) . a sharp increase in virus amounts (pfu/ml) was observed between and h.p.i., indicating that braco- was active at events that occur in this time range. acv showed a similar increase between and h.p.i. since acv acts by inhibiting the viral dna polymerase during dna replication, these data confirm the activity of braco- during viral dna replication. in this work we have found that the gc-rich hsv- genome presents multiple extended repeated clusters of g-quadruplex forming sequences, covering bp on the leading strand and bp on the lagging strand (fig. b, table ) . a first intriguing aspect is that these sequences display a remarkably high stability: in physiological conditions all but one sequence (un ) displayed t m around °c and were capable of folding into tetraplex even in the absence of k + . in particular, among the gp oligonucleotides, gp d standed out for its improved stability. interestingly, this is the only sequence that presents one c base in the loop and displays identical ct loops. it has been reported that c can also be involved in g-quartet formation (lim et al., ), therefore c bases may augment the folding stability of this sequence. to our knowledge the hsv- g-quadruplex structures reported in this work are the most stable ever reported in an organism. hsv- g-quadruplexes will likely form in the viral dna when the duplex is stimulated to unwind, i.e. during replication and transcription events. in eukaryotes g-quadruplex formation at the dna level has been shown to slow down replication and increase the likelihood of chromosomal breakage, genetic instability (ribeyre et al., ) and genomic rearrangements (cahoon and seifert, ); moreover, g-quadruplexes accumulation during the s-phase of the cell cycle, the phase at which replication occurs, has been reported (biffi et al., ) . dna processing is allowed by the activity of helicases (anand et al., ) , which disentangle the tetraplex structures. hsv- probably exploits similar cellular or viral enzymes to unwind its g-quadruplex sequences and allow replication/transcription events to proceed. moreover, g-quadruplex motifs have been associated with over % of dna replication origins (besnard et al., ) . here we have used braco- , a tri-substituted acridine characterized by excellent g-quadruplex binding properties, to investigate the effect of hsv- g-quadruplex stabilization. we have shown that braco- stabilized all tested sequences and that it was able to arrest viral dna processing in vitro. in infected cells, this activity resulted in less viral dna being synthesized in treated cells, shut down of late, but not immediate-early and early protein mrnas and overall, in inhibition of viral production. the activity of braco- was compared to that of tmpyp , a molecule that shares the chemical features of a g-quadruplex ligand, but has a much lower binding affinity toward g-quadruplexes (han et al., ) . at the highest concentrations, tmpyp displayed antiviral activity, which, based on the observation that tmpyp was essentially inactive in the polymerase assays and it did not modify the amount of intracellular dna, likely exploits a non-g-quadruplex related mechanism. the time-of-addition assay indicated an early time range of activity ( - h.p.i.) of braco- , similarly to acv, which targets viral dna polymerase. altogether these data indicate that braco- inhibits viral dna replication by stabilizing the extended clusters of g-quadruplex mainly present in non-coding regions of the viral genome. it has to be noted that ( ) the time-of-addition indicates only the last step affected by the antiviral molecule, therefore earlier steps may also be affected by ( ) only large clusters of g-quadruplex sequences have been considered in the present work. because of the abundance of g nucleotides in the hsv- genome, additional single tetraplexes in key regions of the hsv- genome may form to promote multiple yet non-identified functions required for the viral biology. since its first introduction in the s, acv has been the antiviral drug of choice for the treatment of hsv- infections (vere hodge and field, ) . however, the emergence of resistance to acv has created an obstacle for the treatment of hsv- (bacon et al., ) . because of the inherent different mechanism of action, g-quadruplex ligands could be envisaged as therapeutic options against hsv- strains resistant to current anti-herpetic drugs. in conclusion, this work provides a proof of concept for the development of selective anti-hsv- agents with an innovative mechanism of action. none to declare. (b) real-time pcr on a gp g-quadruplex sequence and a control non-g-quadruplex region was performed in the presence of braco- or tmpyp ( -fold dilutions from lm to . lm). quantification of amplified products was obtained by sybr Ò green detection. hsv- dna was extracted from hsv- -infected vero cells. (c) quantification of intracellular dna amounts obtained from infected cells at h and h.p.i., treated with increasing concentration of braco- (b ) and tmpyp (p ), with n p , mean ± s.d., student's t-test, ⁄⁄ p < . . rq are relative quantities. (d) time-of-addition assay of braco- . vero cells were infected with hsv- strain f and braco- was added at the indicated different time points after infection (x axis). virus was collected h.p.i. and quantified by plaque assay. the activity of braco- (b , lm) was compared with that of the negative control (cnt) and of acv as reference drugs. the left y axis refers to braco- and control data, whereas the right y axis refers to acv data. these results are representative of two independent experiments. overcoming natural replication barriers: differential helicase requirements herpes simplex virus resistance to acyclovir and penciclovir after two decades of antiviral therapy targeting g-quadruplexes in gene promoters: a novel anticancer strategy? genome-wide study predicts promoter-g dna motifs regulate selective functions in bacteria: radioresistance of d. radiodurans involves g dnamediated regulation unraveling cell type-specific and reprogrammable human replication origin signatures 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implications for meiosis g-quadruplex dna structures-variations on a theme microtubule-mediated transport of incoming herpes simplex virus capsids to the nucleus the sars-unique domain (sud) of sars coronavirus contains two macrodomains that bind g-quadruplexes antiviral agents for herpes simplex virus genome-wide computational and expression analyses reveal g-quadruplex dna motifs as conserved cis-regulatory elements in human and related species circular dichroism and guanine quadruplexes investigation of mrna quadruplex formation in escherichia coli g-quadruplex structures and their interaction diversity with ligands supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.antiviral. . . . key: cord- -b x fn authors: billiau, a. title: the interferon system as a basis for antiviral therapy or prophylaxis date: - - journal: antiviral res doi: . /s - ( ) - sha: doc_id: cord_uid: b x fn nan the interferon system is part of the so-called aspecific defense mechanism of vertebrates against virus infections. the term "aspecific" mainly refers to the fact that the system is operative against all taxonomic classes of viruses. it consists of a set of evolutionary related proteins, interferons, which possess the ability to tune the synthetic biochemistry of the cell so that various processes which are essential for virus replication are impaired. under "normal" conditions the organism synthesizes or releases little if any of the interferons. only under attack from viruses or other forms of biological stress is the interferon system triggered. hence, the two sets of fundamental questions which have governed interferon research : (i) the physiology and biochemistry of induction ( , ) and (ii) the physiology and biochemistry of action ( ) . possessing satisfactory answers to these questions is essential, not only to understand the natural role of the interferon system in virus disease but also to assess possible applications in medicine, in particular the use of interferons or interferoninducing substances as antiviral drugs. a striking feature brought to light by virtually each sector of interferon research is the pleomorphism of the system. thus, each animal species possesses a family of genes for interferon, and in some instances a single gene can give rise to multiple molecular forms. also there are several ways in which production of these interferons can be induced. finally, the antiviral effect is not due to a single cellular modification, blocking the virus at one particular junction in its replicative cycle. rather, virus replication is counteracted at different steps. another feature worth mentioning is that the interferon system is leaky blocking of virus replication or of the effects of viruses on the host is neither complete nor permanent. therefore, if the virus host is placed under the protective influence of interferon, some virus replication still occurs, and as the interferon effect wanes, residual virus can again multiply at full strength unless this would be blocked by some other mechanism (e.g. specific immunity) which has been generated in the. meantime. currently, interferons are defined as endogenous proteins which exert virusnonspecific, antiviral activity at least in homologous cells through cellular metabolic processes involving synthesis of both rna and protein ( ) . one of the main points in the definition is that it excludes all natural proteinaceous factors which inhibit virus infection at extracellular steps, e.g. adsorption. a limitation of this definition is that it would also call "interferon" any protein produced by cells which induces production of a known interferon. for instance, a monokine related to interleukin-i, has been shown to induce production of interferon-b by cells and thereby to exert an antiviral effect. in mammalians three (sero)types of interferon have been described : ifn-a, ifn-b and ifn-y. subtypes, reflecting the presence of multiple, slightly different genes, may occur. thus, in man there are several subtypes of ifn-a (huifn-al, -a , -a , •.. ), but only one ifn-b and -yo types of interferon (a, band y) are distinguished on the basis of reactivity with polyclonal antisera. antiserum against a given type neutralizes biological activity of all its subtypes but does not react with the other types. antigenic differences between the types reflect differences in primary structures. however, some homology in sequence exists between ifn-a and ifn-b. on the other hand, the eequence of ifn-y is completely different from that of either ifn-a or -b. the structural similarities and differences between the ifn types are reflected by the fact that ifn-a and -b, in acting on cells, share a common membrane receptor, while ifn-y uses a different receptor ( ) . interferons for animal experiments or for clinical trials are prepared in various ways. at present it is common and useful to make a distinction between preparations of so-called natural interferons and preparations of recombinant dna-derived interferon (rdd-interferons). among the natural interferon preparations, those most commonly used are as follows. interferon prepared from mouse cell lines infected with newcastle disease virus is a mixture of a and b ( ) . the two types can be separated but this has seldomly if ever been done for preparations destined to perform in vivo experiments. thus, literature data are representative for a mixture of ifn-a and -b. interferon prepared from suspensions of mouse splenocytes incubated in the presence of lymphocyte mitogens (concanavalin a, phytohemagglutinine, staphylococcus enterotoxin, ..• ) contain ifn-y. it should be mentioned that, depending on the degree of purity, these preparations also contain variable numbers and quantities of monokines and lymphokines such as interleukins, colony stimulating factors, etc. hence, all data obtained so far with these preparations represent effects of total lymphokine rather than of ifn-y preparations. the following interferons are currently available for clinical studies in immune interferon is produced from suspensions of blood leukocytes induced with mitogens. the antiviral activity present in these preparations is mainly due to ifn-y. small quantities of ifn-~ and -s may be present. however, the main contaminants are several lymphokines, some of which may indirectly affect viral disease, for instance by modulating the immune system. the prototype of rod-interferon is human ifn-a prepared from e.coli or yeasts containing an adequate expression plasmid into which the interferon genes have been inserted ( ) . in their crude form these preparations do not contain any products of human origin other than the interferons . bacterial products are completely removed by the purification process. the main difference between these products and their natural-source-derived counterparts is that they contain only a single subtype, namely a ' while it is known that different subtypes of huifn-~ may differ in their antiviral potency depending on the cells on which they are tested, it is not yet known whether this has any repercussion for the clinical effects. another currently available rod-interferon is huifn-~ produced in e.coli. this interferon differs from the natural counterpart by the absence of carbohydrate side-chains. although these side-chains do not playa significant role in the effects of the ifn-s on cells, the pharmacokinetic behavior of this rdd-ifn-s was found to be rather different from that of natural fibroblast-derived interferon ( ) . rdd-huifn-s possessing carbohydrate side-chains can be obtained by insertion and expression of the gene in yeasts or mammalian cells. finally, a rdd-huifn-y is available, which is produced from e.coli or mammlian expression systems. rdd-interferons for experiments in animals are becoming available at a much slower pace and in lesser quantities than those necessary for experiments in man. the genes for several animal interferons have been isolated and brought to expression in e.coli, in yeasts or in mammalian cells. this should allow to produce large quantities without great technical difficulty. especially in the case of muifn-y, this will be a great advantage over the cumbersome "natural" production method which depends on the availability of fresh mouse splenocytes. pharmacokinetic studies have been done with human interferons-a and -so in as far as they were done in man, they are of course quite relevant to the design of therapeutic trials. studies with these interferons in animals are of lesser value, especially as it has become clear that comparative studies between interferon-a and -s may yield quite different conclusions depending on the animal species used ( ). one of the key questions is to know whether interferon given by any route is concentrated in some organ. this question has so far not been studied in man although, with current techniques of production and radioactive scanning, it seems quite approachable. that the question is not trivial may be apparent from the observation that human interferon-a and -s, when injected by the intramuscular route, cause comparable increases in nk-cell activity, although blood levels with interferon-a are -to -fold higher ( ) . similarly, studies in mice have revealed that human interferon-a and -s given intraperitoneally, yield different blood levels but quite similar tissue levels (ii). there is evidence that some organs, in particular the brain, are rather difficult to penetrate by interferons. thus, during the initial phases of experimental mengo virus infection in mice, interferon levels in serum being quite high, no interferon was detectable in the brain ( ) . brain interferon became detectable only in later stages of the infection when full-blown virus replication had started in the brain itself. huifn-s on the other hand undergoes rapid uptake, suggesting that desialylation, wherever it may take place, is a first step toward degradation of huifn-s. the kidney was shown to playa key role in that interferons seem to pass easily through the glomerular sieve and to be taken up and degraded by tubular cells. of particular importance are pharmacokinetic studies with rdd-interferons. since these interferons often differ from the natural ones . by , the absence of carbohydrate side-chains it may be expected that their pharmacokinetics will be quantitatively different from those of their natural counterparts. it is not unthinkable that some of the artificial interferons . (e.g. those obtained by sitespecific mutagenesis) may be degraded slowly by the organism, allowing to decrease the doses. however, there is nothing that would lead one to think that site-specific mutagenesis . may enable one to target an interferon to a specific , the main lesson to be learned from these experiments is that interferon therapy is unlikely to achieve protective effects unless started before the major viral replication burst in the organism. in general one can say that this means that therapy has to be started before the first symptoms. as already mentioned, mouse model systems for chronic virus infections are scarce. one example is experimental viral leukemia. it has been known for a long time that continuous interferon administration started before or shortly after infection can significantly delay the development of the disease and can also prolong survival time ( ) . this finding may be expected to receive renewed in- ( , , ) . the question whether interferon therapy may be able to control chronic or recurrent disease at the level of the whole body, is confounded by the fact that much of the symptoms of these chronic virus diseases are not directly due to viral replication or cytopathogenicity but result from immunological and inflammatory reactions. therefore, it is not clear whether alleviation of symptoms by interferon is due to antiviral activity or to interference with secondary effects. thus, in the aforementioned example of friend leukemia in mice, it is not clear whether delay in splenomegaly development is due to reduction in viral load or to antimitotic or immunoregulatory effects of interferon. recurrent herpetic keratitis ( ) can benefit from topical interferon therapy if it is given in conjunction with debridement and/or with some of the older or newer antiherpetics ( ) . acute rhinopharyngitis due to rhinovirus or coronavirus infections can favorably be influenced if rather high doses of huifn-a (but not -s) ( ) are given and if treatment is started early, preferentially before infection. it has been envisaged to use daily instillations of interferon during autumn and winter periods as a means of prophylaxis against common cold. however, it is now being suggested that such long-term instillations cause damage to the nasal mucosa. condyloma accuminatum is amenable to treatment with ointments or gel lies containing interferon ( ) . a problem in interpreting these results is the possible confounding, especially in non-placebo-controlled trials, of the mere effects of hygiene. daily care of the lesions may by itself contribute to regression of the condylomas. however, the interpretation that the effects are largely due, indeed, to the action of the interferon is corroborated by the observation that other manifestations of hpv infections are also amenable to treatment with interferon. thus, skin warts can be made to disappear by repeated injections of fibroblast interferon in the perilesional skin ( ) . beginning or established acute (primary or recurrent) infections. systemic administration of interferons (a-or -type) has been found to favorably affect the course of a number of acute (prima ry or recurrent) virus infections : varicella in imrnunocompromised patients ( ) , zoster in cancer patients ( ) . the practical importance of these observations is rather minor, in that beginning or established hsv and vzv infections can now effectively be treated with nucleoside analogs. prevention of acute infections. systemic treatment with interferon can prevent herpes recurrence after surgical interventions on the trigeminal nerve ganglion (operation for "tic douloureux" ) ( ) . interferon therapy has a l so been considered as a possible means to cope with the increased incidence and severity of viral infections in renal transplant patients ( ) ; it appears that interferon therapy reduces virus-shedding as well as the severity of symptoms of cmv infections. chronic active virus infections . the following chronic active virus infections are currently investigated as pos s ible targets for systemic interferon therapy : persistent infections with hb virus, chronic infections with hpv (warts and wartlike diseases), chronic infection with leukemia viruses, e.g. htlv. the effects of interferon therapy on persistence of hb virus has rather extensively been investigated. in some patients transient and sometimes definitive disappearance of viral infectivity markers, accompanied by clinical improvements, were seen. it is difficult, however, to certify tha t these were not spontaneous cures or placeboeffects ( ) . the need for properly controlled trials, expressed early on by several investigators, has at this time still not been satisfied. therefore it remains impossible to even approximately assess the therapeutic potential of interferon in chronic hepatitis. the situation with chronic hpv infections is quite different. thus it is now generally recognized that juvenile laryngeal papilloma, a rather rare but serious complication of infection with certain hpv types, is quite amenable to control by therapy with various preparations of huifn-a ( ) . the therapy has to be given continuously for months before recurrence of the laryngea l warts is brought under control. arrest of treatment entails recurrence, but it is hoped that suitable low-dose maintenance treatments can be designed, by which protections can be prolonged to the age where the papillomas tend to regress spontaneously. retroviruses have only recently been implicated as etiologic fa c tors in human diseases, namely human t-cell leukemia (htlv-i and -ii) and aids (lav or htlv-iii). interferon therapy has already been investigated as a means to control aids and aids-associated malignancies (kaposi sarcoma), and some encouraging results have been reported in that the symptoms of the disease can in some patients be alleviated ( ) . a cure of aids however, has not been seen. mechanisms of production and action mechanisms of production and action proceedings of symposium on clinical use of interferon key: cord- -z b sd authors: zhong, yu; yoshinaka, yoshiyuki; takeda, tadahiro; shimizu, noriko; yoshizaki, sayaka; inagaki, yoshio; matsuda, shinobu; honda, gisho; fujii, nobutaka; yamamoto, naoki title: highly potent anti-hiv- activity isolated from fermented polygonum tinctorium aiton date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: z b sd a water-soluble extract of fermented polygonum tinctorium aiton (polygonaceae) called sukumo, exhibited a potent inhibitory activity against hiv type in vitro. the extract potently suppressed acute hiv- (iii(b)) infection in mt- cells with ec( ) values of . μg/ml but exhibited low cytotoxicity to mt- cells even at a high concentration (cc( ) > μg/ml). it also inhibited giant cell formation in co-cultures of hiv-infected cells and uninfected molt- cells. sukumo extract was found to interact with both the viral envelope glycoprotein and cellular receptors, thus blocking virus-cell binding and virus-induced syncytium formation. there was a good correlation between the extract's anti-hiv- activity and its inhibitory effects on hiv- binding. it also suppressed replication of herpes simplex virus type in vero cells with an ec( ) of . μg/ml. on the other hand, there was no appreciable activity against influenza a virus, poliovirus or sars corona virus when tested at concentrations ranging from . – μg/ml as shown by microscopic image analysis for cytopathic effect (cpe). physico-chemical studies revealed that the anti-hiv activity in the extract was essentially maintained after boiling at °c in n hcl or n naoh, and after treatment with mm naio( ). the inhibitory activity of the extract was also not reduced after pronase digestion. the active factor in the extract is likely to be a novel compound(s) having a polyanionic substructure and a molecular weight of , – , . one of the logical targets of the viral life cycle at which to inhibit hiv- replication is the step in the process where the infectious virion enters its host cell (moore and stevenson, ; lin et al., ) . therefore, the identification of hiv entry inhibitors, which can serve as novel anti-hiv drugs, is urgently needed. retroviral infection is initiated by the attachment of the virion to the cell surface, which even occurs before glycoproteins on the viral envelope interact with specific receptors on the host cell to trigger fusion. a great variety of polyanionic compounds have been described which act as virus adsorption inhibitors. this class of compounds also comprises the cosalane analogues, containing the polycarboxylate pharmacophore, as well as the sulfated polysaccharides extracted from sea algae (nakashima et al., a (nakashima et al., , santhosh et al., ; witvrouw and de clercq, ) . all of these compounds are assumed to exert their anti-hiv activity by shielding the positively charged sites in the v loop region of the viral gp envelope glycoprotein, and interrupting virus attachment to the negatively charged heparan sulfate proteoglycans on cell surface, and inhibiting the specific binding to the cd receptor of cd + cells. some of these compounds can also interfere with later events in receptor-mediated fusion by virtue of attachment to gp . these compounds probably do not penetrate into cells because of their mass and highly anionic charge, but rather, act as antiviral agents by impeding the attachment and subsequent entry of virus particles into the cell. a number of sulfated polysaccharides, including dextran sulfate and heparin, have been reported to have potential as antiviral drugs, since they inhibit the replication of a variety of viruses in vitro (baba et al., ; bartolini et al., ; nakashima et al., ; ylisastigui et al., ) . the extent of inhibition appeared to be dependent on both the viral strain and host cell type. dextran sulfate interferes with the association of gp with cxcr while having no detectable effect on gp -cd . the interaction between polyanions and x or x r gp was readily detectable, whereas weak or undetectable binding was observed with r gp (moulard et al., ) . cosalanes inhibited the binding of gp to cd as well as the fusion of the viral envelope with the cell membrane and is more potent against r hiv- rf in cem-ss cells than against vs x hiv- iiib in mt- cells (santhosh et al., ) . polygonum tinctorium has been used extensively in chinese and japanese folk medicine for the treatment of many infectious diseases and is believed to have effects such as detoxification, anti-pyrexia and anti-nociception. extracted constituents of this medicinal plant, such as tryptanthrin, has been shown to possess anti-fungal, cancer chemopreventive and anti-bacterial activities (honda and tabata, ; koya-miyata et al., ; kataoka et al., ; miyake et al., ) , while pigment (ptp) has an anti-anaphylactic activity (kim et al., ) . in this study, we report for the first time the potent anti-hiv- and hsv- activity of an aqueous extract from the fermented leaves of polygonum tinctorium (sukumo). this extract was found to be highly selective against hiv- and hsv- in vitro. sukumo extract suppresses production of hiv- by inhibiting the viral entry process through binding to the virus envelope and thus preventing hiv-induced syncytium formation with an exceedingly broad therapeutic window. based on the results of physico-chemical analysis of the anti-viral active factor, it is putatively a novel polyanionic high-molecular-weight compound containing a phenolic substructure in aqueous extract of sukumo. sukumo was collected from the leaves of polygonum tinctorium (tokushima, japan) and fermented for months, which was provided and identified by dr. matsuda. voucher specimens were deposited at the institute of hemorheological function of food co. ltd., hyogo, japan. sukumo powder ( g) was refluxed three times with . % ethanol, then with water ( l). the aqueous solution was clarified by filtering through a . m filter. the high-molecular compounds were precipitated from the aqueous extracts of sukumo by . % ethanol, which were collected by centrifugation ( , rpm, min) in a yield of . % ( . g). anti-hiv activity of the sukumo extract was tested and stored at • c before use. mt- , molt- cells and molt- cells chronically infected with the hiv- (iii b ) strain (molt- /iiib) were cultured in rpmi- medium supplemented with % fetal bovine serum (fbs) (cansera international inc., canada) and antibiotics ( g/ml penicillin/ g/ml streptomycin). t, vero and stably expressing cd -ccr of hos cells were maintained in dulbecco's modified eagle's medium (dmem) containing the same supplements. x /hiv- (iii b ) was prepared by propagation in molt- /iiib cells. hiv- molecular clones of the x hiv- strain nl - and the r hiv- strain jrcsf were prepared by transfection of t cells with nl - or jrcsf plasmids carrying full-length proviral dna. the culture supernatants were clarified by . m filters and frozen at − • c. herpes simplex virus type was propagated in vero cells (kindly provided by dr. shaku). cell-free virus stock was prepared by sonication of hsv-infected vero cells in % skim milk and stored at − • c until use. to determine the anti-hiv- activity and cytotoxicity of the sukumo extract, mt- cells were either infected with hiv- (iii b ) strains at a multiplicity of infection (moi) of . or un-infected (mock infection). cell viability was quantified with mtt (dojindo, kumamoto, japan) assay for mt- cells. ec values were calculated in infected cells for the anti-hiv- effect and cc values were calculated in un-infected cells for drug cytotoxicity (ichiyama et al., ) . peripheral blood mononuclear cells (pbmcs) from hiv- -seronegative donors were isolated by ficoll-hypaque density gradient centrifugation. pha ( g/ml, sigma-aldrich)/il- ( u/ml, shionogi, osaka, japan) activated pbmcs were infected for h with ng of hiv- p gag (x /nl - or r /jrcsf) in the presence or absence of the sukumo extract ( . - g/ml), washed three times with pbs and cultured for days in rpmi- medium/ % fbs plus u/ml il- with or without the sukumo extract. hiv- p gag of culture supernatant was determined by automated enzyme-linked immunosorbent assay (eilsa) (fuji rebio inc., tokyo, japan). in this assay the p antigen from zeptometrix (buffalo, new york) was used as the standard. chronically hiv- (iii b )-infected molt- cells were co-cultured with hiv non-infected molt- cells (ratio = : ) at • c for day in the presence of a test compound at graded concentrations. cell-cell fusion was analyzed by confocal microscopic assessment of syncytium formation. sukumo extract were also tested as an inhibitor of hsv- replication in vero cells using a standard plaque assay. two hour after treatment with sukumo extract, vero cells were subjected to a -h infection with about pfu of hsv- in the absence or presence of serially diluted sukumo extract and then cultured with medium supplemented with % fbs, human ␥-globulins ( g/ml, sigma-aldrich) and antibiotics, with or without compounds cultured for days. the cells were stained with giemsa solution. the numbers of viral plaques were calculated as a percentage of the tested control in order to determine the percent inhibition. the ec value is the concentration of compound that inhibits viral replication by % relative to control. human mt- cells ( × ) were suspended in fresh medium in the presence or absence of various concentrations of sukumo extract at • c for h. after washing, the cells were incubated with hiv- nl - ( ng of p gag) for h on ice or at • c in the absence or presence of extract. the cells were washed with pbs/ % fbs and the pellet resuspended with l of lysis buffer (pbs containing % tritonx- and % bsa). levels of p gag were quantified by an automated elisa system. mt- or hos/ccr cells ( × ) were pretreated with normal human igg (zymed, south san francisco) at . mg/ml in pbs containing % fbs buffer for min on ice to block the fc receptors and then were treated by anti-cxcr antibodies ( g/ml, g . r&d systems inc.) or anti-ccr antibodies ( . g/ml, d . biosciences-pharmingen, san diego) in the presence or absence of sukumo extract at • c for h. cells were washed with pbs/ % fbs and strained with fitc-conjugated anti-mouse igg ( . mg/ml, american qualex) for min on ice. pretreated mt- cells also were stained with fitc-conjugated anti-human cd or monoclonal mouse antibodies of isotype igg (negative control for flow cytometry) ( : dilution, dako). the cells were fixed in % paraformaldehyde-pbs solution and analyzed on facs calibur (becton dickinson), a flow cytometer with cellquest software (becton dickinson). pseudotyping vesicular stomatitis virus protein g (vsv-g) onto hiv cores from an env-defective reporter virus was carried out as follows. plasmid dna ( g) encoding envelope from vsv-g was co-transfected with pnl-e env(−) nef(−) ( g) into t cells using the calcium phosphate method. the virus titer was determined based on the level of p gag. time course assays were conducted to determine which steps in viral infection (entry and post-entry) were inhibited by sukumo extract. three treatment schedules were applied for hiv- infection with t cells, which were infected with pseudotyped hiv- /vsv-g viruses ( ng/ml of p gag). the amount of p gag in culture supernatant was determined to assess hiv- replication. sukumo extract was used at serial concentrations in a range of . - g/ml and added at different times. the levels of p gag were determined after days of incubation by an auto-elisa system. the binding assay was used to determine the affinity of sukumo extract for virions by viral replication assay. separation of sukumo extract and virus was carried out on a chromatography of gel filtration system with a column of sephacryl s- ( by cm) (amersham pharmacia biotech, sweden). the column was equilibrated and the compounds were eluted from column with pbs. the samples were separated into the following three samples; sukumo extract control: up to l of sukumo extract ( mg/ml), with l of rpmi- medium containing % fbs added; virus control: up to l of pbs, with l of cultured supernatant containing hiv- nl - added; sukumo extract-virus mixture: up to l of sukumo extract, with l of cultured supernatant containing hiv- nl - added. a volume of l of each sample was injected onto the analytical column after incubation at • c for h and one fraction of eluant was collected ( ml) on ice. the elution peak of the sukumo extract control fractions at a wavelength of nm and anti-hiv- iiib activity by mtt assay were monitored to determine the elution position of sukumo extract (fig. a ). the levels of p gag in the eluted fractions were measured with auto-elisa to determine the elution position of virus (fig. b ). viral infectivity was analyzed for the eluted fractions of the virus control and sukumo extract-virus mixture; the selected fractions and were clarified by . m filter. mt- cells ( × /ml) were infected by a mixture of the eluted fractions. two hours after infection, the cells were washed and added to fresh rpmi- / % fbs medium, cultured for days and the p gag of supernatant was measured. anion exchange chromatography was carried out on a deae-sephacel column, which had been equilibrated with phosphate buffer (ph . ). the bound sample was eluted by stepwise increases of the nacl concentrations in phosphate buffer. the eluted fractions were analyzed for anti-hiv activity using the mtt assay method. the sukumo extract was also separated with % sds-page. the gel was stained by silver reagent and cut as described in fig. b . sukumo was re-extracted with rpmi- medium from the sds-gel fractions and collected supernatants for anti-hiv- activity. the anti-hiv- activity of sukumo extract was first investigated by conventional mtt assay using mt- cells. sukumo extract completely inhibited hiv- (iii b strain) replication in mt- cells at a concentration as low as . g/ml. its and % effective concentrations (ec and ec ) were . and . g/ml, respectively. the % cytotoxic concentration (cc ) was found to be > g/ml (fig. a) , and the selectivity index (ratio of cc to ec ) of sukumo extract was > , indicating that this compounds is very potent and selective. sukumo extract was also evaluated for the inhibition of wild-type herpes simplex virus- replication in infected vero cells, using a standard viral plaque assay (fig. b) . sukumo extract, and exhibited anti-viral activity with an ec value of . g/ml. however, no inhibitory activity was observed against influenza a virus, poliovirus and sars virus when sukumo extract was tested at concentrations ranging from . to g/ml (data not shown). sukumo extract inhibited a variety of hiv- isolates, including a laboratory adapted isolate iiib strain, laboratory molecular clones x type nl - and r type jrcsf in a variety of cells, including molt- , jurkat, pm , and cd -ccr expressing hos cells (data not shown). the inhibitory activity of sukumo extract against x hiv- (nl - ) and r hiv- (jrcsf) replication in pbmcs was also demonstrated pha-stimulated pbmcs were infected for h at • c in the absence or presence of . - g/ml sukumo extract followed by washing. × /ml infected cells per well were seeded in -well plate and were incubated for days in the absence or presence of appropriate concentrations of compound. quantity of hiv- p gag was measured by auto-elisa system. by p assay of culture supernatants of the cells infected with the viruses exhibiting ec values of . and . g/ml, respectively (fig. ) . the p gag levels of untreated samples of hiv- nl - and jrcsf were . and . ng/ml, respectively. hiv- replication in mt- cells appeared to be more sensitive to sukumo extract than in pbmcs. in the various steps of the hiv- life cycle, we next investigated at which step sukumo extract exerts its effect as an hiv- antagonist. to determine whether the viral binding to cells is a target of sukumo extract, a binding assay was carried out to measure the effect of sukumo extract on virions/cell surface interactions. mt- cells were mixed with x virus nl - on ice for h, and then the cells washed to remove the unbound viruses. the results demonstrate that sukumo extract blocked virus-cell binding with an ec of . g/ml (fig. a) . the inhibitory effect of sukumo extract on hiv- entry to cells was also studied in mt- cells. cells were incubated with the same virus at • c for h, treated with trypsin to remove bound virions, and then the intracellular p gag of hiv- was measured. sukumo extract inhibited viral entry with an ec of . g/ml (fig. a) . the binding and entry of hiv- nl - in mt- cells was efficiently inhibited by sukumo extract in a dose-dependent manner. a similar result was also observed with r type hiv- jrcsf on hos/cd -ccr cells (data not shown). these experiments show that there is a good correlation between the anti-hiv activity and the inhibitory activity against virus binding/entry induced by the sukumo extract. sukumo extract also completely prevented syncytium formation through co-culture of molt- and hiv- -converted molt- cells at a concentration of g/ml and efficiently prevented it even at . g/ml (fig. b) . these data indicate that sukumo extract exerted its effect at an initial step of hiv- infection, such as viral entry and membrane fusion in the target cells. we then analyzed changes in cd and cxcr expression on mt- cells and ccr expression on hos/cd -ccr cells upon treatment with different concentrations of sukumo extract. only a high concentration of sukumo extract ( g/ml) caused down-expression of cd ( . % of control). when sukumo extract was used at the concentrations of , and . g/ml, the levels of cxcr expressed were only . , . and . % of control, respectively. in contrast, the levels of ccr expression on the surface of hos cells were . , . and . % of control at the same concentration range (fig. c) . to determine the sukumo extract and hiv- interaction, we applied a chromatographical analysis using a sephacryl s- for separation of the virus particles and the sukumo extract based on differential molecular size. when sukumo extract was fractionated, the main anti-hiv- activity was eluted in fractions - , as revealed by the mtt assay the eluted fractions and were selected and infected into mt- cells for h at • c. after washing, the cells were incubated for days and p gag of culture supernatant was measured by auto-elisa. (fig. a) . on the other hand, hiv- was eluted in fractions and as shown by p assay (fig. b left) . when an excess amount of sukumo extract was mixed with hiv- and separated with sephacryl, the viral peak was detected in fractions and once again (fig. b right) , while anti-hiv activity was still observed in fraction - (data not shown). to see whether these fractions contained an infective capacity of hiv- , the amount of p gag was assessed in the supernatant of mt- cells after infection. we used the same volume ( l) of eluted fractions and from viral control, which contained . and . ng of p antigen, or those from the sukumo extract-virus mixture which contained . and . ng of p antigen to infect × mt- cells, respectively. as shown in fig. c , fractions and obtained from the viral control exhibited high hiv- activity ( . and . ng/ml in p level) days after infection while fractions and from the sukumo extract-virus mixture had p levels as low as . and . ng/ml, respectively. these results strongly suggests that the sukumo extract specifically bound to viral particles and was efficiently trapped by viral particles so that viral infectivity was significantly abrogated due to the blockage of entry into the cells. although all the data provided evidence that hiv- entry could be a primary anti-viral target of sukumo extract, there still remained the possibility that sukumo extract exerts its effect on a late step of viral replication. to address this, a time course assay was performed using a single cycle infection with vsv-g pseudotyped hiv- and t cells. p gag in the supernatant was measured days post-infection. the result showed that a dose-dependent anti-viral activity of sukumo extract was observed when it was added at the time b (entry) step. in contrast, the inhibition was not seen when sukumo extract was added at the time a (pretreatment) or time c step (post-entry) at any concentrations studied (range . - g/ml) (fig. ) . a similar result was also observed when hos/cd -ccr cells were used (data not shown). these results suggest that sukumo extract does affect an early step, not a post-entry step, of the viral life cycle. based on these studies, we conclude that there is persuasive evidene that sukumo extract is a binding inhibitor that interferes with virion/cells interactions and that this inhibition is likely mediated through binding to the hiv- viral envelope. the anti-viral factor was extracted from sukumo using organic solvent and water. inhibitory activity was found in the aqueous extract. crude sukumo extract was fractionated by deae-sephacel column chromatography. the main fractions which had anti-viral activity was eluted from the column by . - . m nacl (fig. a) . the sukumo extract was also separated by using sds-page and anti-hiv- activity was detected in fractions - of the sds-gel extracts corresponding to a molecular weight of , - , (fig. b) . gas chromatography analysis of the acid hydrolysates of the sukumo extract revealed the carbohydrate contents of ara:xyl:man:gal:glc were . : : . : . : . ( table ) and sds-page/pas staining yielded a bright red band (zacharius et al., ) (data not shown). elemental analysis revealed that the sulfer content is . % in the fig. . effect of sukumo extract on vsv-g pseudotyped hiv- replication. t cells were infected with the hiv- nl-e strain lacking env and nef with vsv-g envelope of pesudotyped virus. . - g/ml sukumo extract was used and anti-hiv- activity was determined days later by measuring p gag. treatment a (a pre-entry step): the cells were incubated with sukumo extract for h at • c and washed before exposure to virus, and then the cells were infected and incubated in the absence of sukumo extract. treatment b (an entry step): the cells were exposed to virus in the presence of sukumo extract for h, then both sukumo extract and unabsorbed viruses were removed by washing. the cells were further incubated in the absence of sukumo extract. treatment c (a post-entry step): the cells were infected with virus for h, unabsorbed virus were removed and further incubated in presence of sukumo extract. sukumo extract ( table ). the anti-viral factor in sukumo extract was stable under a wide range of ph conditions. as shown in table , the anti-viral activity of sukumo extract was not reduced after treatment with n h so , n hcl or naio , but the value of ec ( . g/ml) was somewhat decreased when it was treated with n naoh. the activity was also not lost after being heated at • c for min and was not inactivated by protease (trypsin, proteinase k and pronase) digestion. finally, we addressed whether the anti-hiv- activities of sukumo extract, heparin and dextran sulfate were abrogated after they were treated with acid. when sukumo extract was boiled at • c in n h so and n hcl for h the % effective concentration (ec ) against hiv- was essentially unaffected. in sharp contrast, when heparin was boiled at • c in n h so for h or dextran sulfate in n hcl for h, the % effective concentrations against hiv- were > and > g/ml by mtt assay, while those of the untreated samples were . and . g/ml, respectively (table ) . sukumo extract potently and selectively inhibited hiv- replication in vitro. the compound was also evaluated for activity against various virus species with or without an envelope including vesicular stomatitis virus g protein enveloped hiv- pseudotyped type virus. whereas sukumo extract was active against herpes simplex virus, it was devoid of any activity against influenza a virus, sars virus and a non-enveloped poliovirus. based on the current knowledge of hiv, several stages of the viral life cycle are potentially vulnerable to inhibitors. these can be divided into the entry steps and post-entry steps. in this study, we have demonstrated by several different techniques that sukumo extract inhibits the hiv- infectious process at the cell entry step. the data presented in fig. indicate that sukumo extract is able to block viral binding to target cells and inhibits virus-induced cell-cell fusion. furthermore, a time-course experiment showed that the full protective activity of sukumo extract was achieved when the compound was present during the -h virus adsorption period, but none of the effect was seen when the compound was incubated with the cells prior to viral infection. also, the extract did not suppress the viral replication after the virus had entered the cells. thus, sukumo extract interferes with an early event of the virus replication cycle, most presumably the viral adsorption step. two classes of cell surface molecules, cd and chemokine receptors, as well as ccr or cxcr , are often viewed as hiv coreceptors which mediate hiv- entry. we found that the down-modulation of hiv- receptor cd or co-receptor ccr in target cells was induced by the sukumo extract. however, the inhibitory activity was rather weak. in addition, this activity of sukumo extract was lost if the cells were washed prior to addition of antibody, indicating that the compounds can only weakly associate with the cell surface. therefore, the results cannot perfectly explain why the sukumo extract is able to block virus entry of hiv so efficiently, especially the r hiv- virus. the effect of sukumo extract on the viral binding process was assessed directly, using a chromatography method (fig. ) . the results show that sukumo extract was bound to hiv- and was separated along with the larger virus particle fraction from a gel filtration column. from this study we hypothesized that sukumo extract exerts its anti-hiv activity by binding to the viral envelope glycoprotein. this results in prevention of virus attachment to the cell surface receptor or co-receptor, whereby interference with early adsorption and entry into the hiv replicative cycle. these findings are consistent with the hypothesis that sukumo extract interferes with virions rather than cell function. it might also explain why sukumo extract is less toxic to target cells in vitro. the biochemical features of water extract of sukumo prepared from polygonum tinctorium that selectively inhibited the replication of hiv- were studied. the anti-viral activity was extracted from sukumo in a variety of ways, using water and organic solvents (hexane, chloroform, acetone and ethanol). inhibitory activity was found in the aqueous extracts, whereas the extracts by organic solvents did not show any anti-hiv activity. indigo, a staining ingredient and tryptanthrin, a low molecular weight component from polygonum tinctorium, also did not exihibit any anti-hiv activity (data not shown). the main fraction of anti-hiv activity was eluted from the deae-sephacel column, a negative ion-exchange column at higher molar ( . - . ) nacl. this result indicate that the active factor(s) is highly anionic. it was also confirmed that the anti-hiv compound(s) consist of phenolic substructure by fecl -k fe(cn) staining (barton et al., ) (data not shown) and a polysaccharide containing sulfur atom by sugar analysis and elemental analysis, respectively ( table ) . the factor was estimated to be a high molecular weight compound of , - , by sephadex g- gel-filtration analysis (data not shown) and an sds-gel of sukumo extract (fig. b) . no protein was detected in the water extract of sukumo with sds-page/silver staining. the data confirm our observation that the inhibitory activity of sukumo extract was not inactivated by protease digestion or heating at • c for min. furthermore, boiling of the sukumo extract in the presence of n hcl, n h so and n naoh for h did not result in any loss of this activity. similarly, it was not inactivated by naio treatment (nakashima et al., b) , which breaks down carbohydrates (table ). this suggests that the sugar backbone is not essential for the anti-hiv activity of the sukumo extract. the pharmaceutical value of the sukumo extract is likely to be further enhanced by its stability over a wide range of ph values, as shown by the heating at • c min and treatment with acid and alkaline conditions. since the anti-hiv- activity of sukumo was higher than that of fresh leaves (data not shown), the possibility that the active substances were derived from bacteria could not be excluded. we compared difference between representative sulfated polysaccharides and sukumo extract for their susceptibility to acid treatment. the anti-hiv- effect was clearly abrogated by this treatment in the case of dextran sulfate and heparin, but not sukumo extract ( table ). the anti-hiv- activity of heparin was completely destroyed by n h so treatment as in the case of n hcl treatment of dextran sulfate. unlike epigallocatechin gallate, a polyphenolic substance from green tea (suzutani et al., ; yamaguchi et al., ) , sukumo extract did not exert any anti-hiv- activity on the post-virus entry process. further work on the characterization of the sukumo extract and its potency as an anti-viral candidate drug is in progress. mechanism of inhibitory effect of dextran sulfate and heparin on replication of human immunodeficiency virus in vitro susceptibility to highly sulphated glycosaminoglycans of human immunodeficiency virus type replication in peripheral blood lymphocytes and monocyte-derived macrophages cell cultures paper chromatography of phenolic substances isolation of antifungal principle tryptathrin, from strobilanthes cusia o. kuntze a duodenally absorbable cxc chemokine receptor antagonist, krh- , exhibits a potent and selective anti-hiv- activity antibacterial action of tryptanthrin and kaempferol, isolated from the indigo plant (polygonum tinctorium lour.), against helicobacter pylori-infected mongolian gerbils inhibition of mast celldependent anaphylactic reactions by the pigment of polygonum tinctorium (chung-dae) in rats prevention of azoxymethane-induced intestinal tumors by a crude ethyl acetateextract and tryptanthrin extracted from polygonum tinctorium lour cell surface ccr density determines the postentry efficiency of r hiv- infection promoting effect of kaempferol on the differentiation and mineralization of murine pre-osteoblastic cell line mc t -e new targets for inhibitors of hiv- replication selective interactions of polyanions with basic surfaces on human immunodeficiency virus type gp purification and characterization of an avian myeloblastosis and human immunodeficiency virus reverse transcriptase inhibitor, sulfated polysaccharides extracted from sea algae. antimicrob antiretroviral activity in a marine red alga: reverse transcriptase inhibition by an aqueous extract of schizymenia pacifica anti-hiv activity of dextran sulphate as determined under different experimental conditions inhibition of human immunodeficiency viral replication by tannins and related compounds correlation of anti-hiv activity with anion spacing in a series of cosalane analogues with extended polycarboxylate pharmacophores anti-herpesvirus activity of an extract of ribes nigrum l sulfated polysaccharides extracted from sea algae as potential antiviral drugs inhibitory effects of (−)-epigallocatechin gallate on the life cycle of human immunodeficiency virus type (hiv- ) soluble glycosaminoglycans do not potentiate rantes antiviral activity on the infection of primary macrophages by human immunodeficiency virus type glycoprotein staining following electrophoresis on acrylamide gels the authors thank dr. fumio shaku for performing the infection of hsv- experiments and gift of wild-type herpes simplex virus and vero cells. this work was supported by grants from the ministry of education, science, and culture and the ministry of health, labor, and welfare of japan. the manuscript was reviewed prior to submission by pacific edit. key: cord- -hqijd authors: bray, mike; di mascio, michele; de kok-mercado, fabian; mollura, daniel j.; jagoda, elaine title: radiolabeled antiviral drugs and antibodies as virus-specific imaging probes date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: hqijd a number of small-molecule drugs inhibit viral replication by binding directly to virion structural proteins or to the active site of a viral enzyme, or are chemically modified by a viral enzyme before inhibiting a downstream process. similarly, antibodies used to prevent or treat viral infections attach to epitopes on virions or on viral proteins expressed on the surface of infected cells. such drugs and antibodies can therefore be thought of as probes for the detection of viral infections, suggesting that they might be used as radiolabeled tracers to visualize sites of viral replication by single-photon emission computed tomography (spect) or positron emission tomography (pet) imaging. a current example of this approach is the pet imaging of herpes simplex virus infections, in which the viral thymidine kinase phosphorylates radiolabeled thymidine analogues, trapping them within infected cells. one of many possible future applications might be the use of a radiolabeled hepatitis c protease inhibitor to image infection in animals or humans and provide a quantitative measure of viral burden. this article reviews the basic features of radionuclide imaging and the characteristics of ideal tracer molecules, and discusses how antiviral drugs and antibodies could be evaluated for their suitability as virus-specific imaging probes. the use of labeled drugs as low-dose tracers would provide an alternative application for compounds that have failed to advance to clinical use because of insufficient in vivo potency, an unsuitable pharmacokinetic profile or hepato- or nephrotoxicity. the radionuclide imaging techniques of single-photon emission computed tomography (spect) and positron emission tomography (pet) employ short-lived radiolabeled tracers to visualize biochemical processes in animals and humans. the selective retention of an imaging probe at a site of interest is based either on its high-affinity binding to a specific target, such as a hormone receptor, or on the occurrence of a specific chemical modification, such as phosphorylation, that traps it within a cell (fig. ). many different tracer molecules are now employed to study a variety of pathophysiologic processes. when applied to virology research, spect and pet have been used almost exclusively to image host responses to infection, rather than to visualize viral replication. the only instance in which a pathogen-specific tracer has been employed to detect and track a virus has been the use of a radiolabeled thymidine analogue to image herpes simplex virus (hsv) infections, based on phosphorylation of the probe by the viral thymidine kinase (tk), which traps it within infected cells (fig. ) (brader et al., ; kuruppu et al., ) . this article examines the question of whether viruses other than hsv could be imaged by spect or pet, using radiolabeled antiviral drugs or antibodies as probes. in the case of hepatitis c, for example, such tracers might include molecules that block viral replication by binding to specific virus-encoded molecules, such as the ns protease (fig. ) . in contrast, an antiviral compound such as ribavirin, which is phosphorylated and retained within both infected and uninfected cells, would not be a suitable probe for virus-specific imaging. we begin by providing an overview of the principles of radionuclide imaging, noting the similarities and differences between the mechanisms of action of antiviral drugs and imaging probes. we then describe how spect and pet have been used to detect host responses to viral infection and examine how pet has been employed to track the distribution of radiolabeled antiviral drugs in uninfected subjects. next, we show how the recent development of recombinant oncolytic viruses encoding host reporter molecules provides a proof of concept for virus-specific imaging. we then examine how researchers have taken advantage of the selective phosphorylation of thymidine analogues by the hsv tk to image hsv infections, and how the tk has also been used as a recombinant marker to image tumors, gene therapy and other conditions. next, we identify a number of processes unique to viral replication that might serve as targets for radiolabeled pathogen-specific tracers. we then review nine different dna and rna virus families, identifying approved and experimental antiviral drugs that target virus-encoded molecules, and might have potential as radiolabeled probes. we conclude by explaining how researchers can determine the potential of an antiviral drug or antibody as a radiolabeled imag-ing probe by using the h-or c-labeled compound in simple in vitro and ex vivo experiments. antiviral therapy and nuclear medicine share a common origin in the efforts of paul ehrlich to develop antimicrobial medications in the early s. his concept of the "magic bullet" was based on the observation that certain dyes stained microbes, but not surrounding cells, in tissue sections, suggesting that the selective binding of other small molecules to pathogens could be exploited for therapeutic purposes. to find a substance that specifically inhibited treponema pallidum, ehrlich tested more than compounds with varied side chains until he found one that was toxic for the spirochete, but not its host. two decades later, georges de hevesy followed the same line of reasoning when he used radioactive compounds as "tracers" to explore mechanisms of drug action. at that time, bismuth was a component of a number of compounds employed for the treatment of syphilis. to determine their distribution within the body, de hevesy introduced radioactive bismuth into the medications, inoculated them into the bloodstream of rabbits and followed their distribution and clearance from blood and tissues (hevesy and paneth, ; lomholt, ) . the concept of using a low-dose radioactive tracer to study a biochemical process in which it directly participates is still followed today in developing probes that target cell surface and intracellular receptors, enzymes and other molecules (eckelman et al., ; phelps, ; rudin et al., ) . in some respects, the basic mechanisms that underlie radionuclide imaging are similar to those of antiviral therapy, in which a drug produces a beneficial effect within a region of interest (a locus of viral infection), while having a minimal impact on other tissues. the optimal pharmacokinetic properties of an antiviral agent differ, however, from those of an ideal imaging probe: continuous high drug levels in the blood and tissues are beneficial for therapy, but for spect or pet imaging, such persistence of the tracer would result in a high background signal and poor image contrast. as discussed below, an ideal imaging probe differs from a drug, in that it should be retained at sites of interest, but cleared rapidly from non-target tissues. spect and pet imaging are based on the use of radiolabeled probes that are selectively retained at the site of a specific physiological process. photons emitted from the probe are registered by a detector and the signals are translated by a computer into an antibody or peptide that is too large or too highly charged to diffuse through the cell membrane can be used to target an antigen or receptor on the cell surface. a small molecule that is transported or diffuses across the cell membrane may be retained in the cytoplasm or nucleus if it binds to a receptor or other target. alternatively, the probe may undergo phosphorylation by a cellular (e.g. hexokinase) or viral (hsv-tk) enzyme, so that it cannot diffuse out of the cell. (figure by fabian de kok-mercado) a visible image. in spect imaging, photons in the energy range of - kev impact on sodium iodide crystal detectors within camera heads that revolve around the subject, enabling the assembly of a three-dimensional image (fig. a ). photons from outside the region of interest and nonspecific scatter arising within the sub-ject are blocked by collimators (perforated metal blocks) placed in front of the detectors, increasing the signal-to-noise ratio (snr). because the spect cameras can determine the direction of a gamma ray source more accurately than its depth within the body, the method's spatial resolution is in the order of . - mm. in contrast to the registration of single photons over a range of energies by spect, pet detects the pair of kev photons that are released in opposite directions when a positron emitted by the probe within body tissues collides with an electron (fig. b ). pet instruments are therefore configured as a ring of detectors surrounding the subject. by registering a signal only when two photons of the correct energy strike opposite sides of the ring simultaneously, the snr is markedly increased. this intrinsic mechanism allows pet to provide better spatial resolution than spect (approximately mm for clinical imaging and - mm for animal micropet systems), and facilitates the accurate quantitation of radioactive decay within a region of interest. such quantitative data lend themselves to statistical analysis of pathologic abnormalities and assessment of response to treatment, and are especially useful for monitoring cancer therapy. . . considerations in the design of spect and pet probes . . . location of the imaging target: on the cell surface or within the cell? the first question to consider in designing an imaging probe is the location of its intended target: on the cell surface, where it can be accessed directly from the bloodstream, or within the cell, where it can be reached only by a tracer capable of traversing a lipid bilayer. cell-surface targets can be imaged using antibodies, antibody fragments and peptides, because these large, charged molecules can reach such targets from the plasma. for example, radiolabeled anti-cd antibodies are being used to detect and quantitate cd + t cells in lymphoid tissues of retrovirus-infected macaques (fig. ) (di mascio et al., a) . in contrast, when the target is in the cytoplasm or the nucleus, size and charge become critical considerations, and effective tracers are generally small, nonpolar molecules. target location and the chemical properties of the tracer, also affect the timing required to obtain suitable high-contrast images. because high molecular weight probes are cleared more slowly from non-target tissues than smaller molecules, it may be necessary to label them with a radionuclide with a longer half-life. as discussed in greater detail below, such pharmacokinetic considerations affect image contrast and the snr, by determining how long it takes for the tracer to be cleared from areas other than the site of interest. in the case of licensed medications that are being considered for use as radiolabeled probes, such clearance data may already have been obtained in pharmacokinetic studies. . . . nature of the target: saturable or non-saturable? the interactions of drugs and probes with their targets can be divided into two different types: those that reach a maximum level of activity ("become saturated") as the concentration of drug or probe increases, and those that continue to display greater activity as the concentration increases (non-saturable targets). saturable targets in antiviral therapy are generally those in which the therapeutic molecule occupies a target site, and only a limited number of sites are available for binding. examples include the binding of pleconaril within a cleft in the capsid of some picornaviruses, the blocking of the influenza m ion channel by amantadine, and the binding of a non-nucleoside reverse transcriptase (rt) inhibitor to a locus on the hiv rt, distinct from its active site. for radionuclide imaging, examples of saturable targets include beta-adrenergic, muscarinic and estrogen receptors, which bind labeled analogues (eckelman et al., ; katzenellenbogen et al., ) . several factors determine whether a radiolabeled probe that binds to a saturable target will generate a useful image. first, the probe must have the needed pharmacokinetic properties to reach the target (see below). second, it should have a high affinity for its binding site, as measured by the k d (the concentration at which % of binding sites are occupied at equilibrium). third, there must be a sufficient number of targets within the region of interest so that the accumulation of the bound tracer will result in a detectable signal. in practice, this means that it is possible to image lowdensity targets, as long as the k d of the tracer-target interaction is in the nanomolar range (or less) and the fraction of labeled probe molecules (the specific activity) is high. when these conditions are met, a minute quantity of tracer, much too small to cause any pharmacologic effect, can produce an image (jagoda et al., ; eckelman, ; eckelman et al., eckelman et al., , . compounds that are too toxic for therapeutic use might therefore still find application as imaging probes. in contrast, the target of a non-saturable probe is generally an enzyme that alters the chemical structure of the probe, in a manner that favors its retention at the target site. for antiviral therapy, the classic non-saturable target is the hsv tk, which phosphorylates acyclovir, causing it to accumulate within hsv-infected cells. as described below, this property of the hsv tk has also permitted the extensive use of the recombinant enzyme as an imaging reporter for spect and pet. the principal application of a non-saturable target in radionuclide imaging makes use of -deoxyglucose (dg), a glucose molecule lacking a -hydroxyl group that is readily phosphorylated by the host cell hexokinase, but cannot be further metabolized, and remains trapped within the cell (fig. ). dg can be labeled with fluorine- at the position, to form f-dg (fdg), without changing its metabolic activity. because malignant cells tend to have a high rate of glucose metabolism, dg was originally tested as an anti-cancer drug, but it proved too toxic for clinical use (woodward and hudson, ; pauwels et al., ) . at low dose, however, fdg is not toxic, and it can be used as a probe to label cells with high glycolytic activity. fdg is now widely used to identify foci of increased glucose metabolism, such as tumors or sites of inflammation, by pet imaging (fig. ) (de winter et al., ; kumar et al., ) . in contrast to imaging a saturable target, in which the signal strength is determined by the number of radiolabeled probe molecules that bind to the desired site, the success of imaging a nonsaturable target is not limited by the number of targets within the region of interest. for example, when f-fdg is used to visualize a site of high glucose metabolism, such as the influx of neutrophils that occurs in response to endotoxin, each affected cell accumulates a large amount of phosphorylated label, amplifying the signal (chen et al., ; chen and schuster, ) . by bringing about the selective retention of phosphorylated acyclovir and other thymidine analogues within infected cells, the hsv tk provides a similar advantage, both for antiviral therapy and for imaging. because of the signal amplification inherent in the use of a nonsaturable target, the specific activity of the probe and the number of target sites are less critical considerations in the acquisition of a useful image. in practical terms, this makes it possible for a tracer such as f-fdg to be stored longer between synthesis and administration to subjects because one can compensate for the diminished radioactivity of the probe by giving a larger dose. for a saturable target, in contrast, such a delay could impair image acquisition, because tracer molecules that have undergone radioactive decay will compete with the labeled probe for available binding sites. the radioisotopes with which virologists and medicinal chemists are most familiar, h and c, are weak beta-emitters with half-lives measured in years. because hydrogen and carbon are constituent elements of most, if not all-antiviral medications, these isotopes can be introduced into a drug during its synthesis, and the product will not differ in structure or biological properties from the unlabeled compound. the long half-lives also mean that h-and c-labeled drugs can be stored and used for years without loss of activity. when employed for in vivo pharmacokinetic studies, the compound can be detected in blood, urine or tissue samples by scintillation counting, in which beta decay triggers the emission of a photon of light. there are some key differences between such isotopes and the short-lived radionuclides used for pet and spect imaging. pet is based on positron-electron annihalations that yield pairs of kev photons, which because of their high energy are minimally attenuated or scattered during their passage through soft tissues. spect isotopes, on the other hand, emit photons in the lower energy range of - kev, which are more likely to be scattered or absorbed. in addition, degradation of image quality is worsened, the further the target is from the detector. an advantage of spect, however, is that because the various tracers emit photons with a range of energy peaks, it is possible to image more than one target in the same session, by setting the instrument's computer algorithms to separate signals of different energies. pet isotopes, in contrast, all produce photons of the same energy, making it necessary to perform separate imaging sessions if two different probes are used to study the same subject. as shown in tables and , many different radioisotopes can be used to label spect and pet probes. the choice of which one to use for a particular study will be based on a number of considerations: • the availability of an imaging instrument; • access to a cyclotron; • expertise of the radiochemistry laboratory; and • the technical challenges of incorporating a given isotope into the probe. the latter point includes questions such as the chemical stability of the resulting molecule and the retention of its biological activity. the half-life of the selected radionuclide should also be consistent with the expected clearance time of the tracer, to ensure that sufficient radioactivity will be detected in the region of interest to provide a useful image. the need for chemical stability and a short half-life can both potentially be met by performing pet imaging with a c-labeled probe, because the replacement of one carbon atom by another during synthesis leaves the molecule's physiologic activity unchanged, while the -min half-life makes it possible to image the same subject repeatedly, at intervals of a few hours. however, the isotope's short half-life means that its use is only practical if one possesses an on-site cyclotron and adequate radiochemistry support. those lacking such resources would be better off using a f-labeled probe, because its -min half-life makes it possible for synthesis to take place off-site, but still allows a subject to be imaged twice daily. the limitation of this approach is that the introduction of a fluorine atom into the probe may require a difficult chemical synthesis, and it may also alter the molecule's biochemical characteristics (in this case, its antiviral efficacy). it will therefore be essential to test the activity of the probe in vitro to ensure that its biological activity has been retained. table the radionuclides most commonly employed in pet imaging, with some of their advantages and disadvantages. the methods by which a radionuclide can be incorporated into an imaging probe fall into two broad categories: those in which the tracer molecule's original chemical structure remains unchanged, and those in which the structure is modified to permit addition of the radioactive moiety. the first approach includes the introduction of the radionuclide into the primary synthetic reaction and its direct exchange for its stable counterpart. the second includes three different techniques: chemical modification of the original molecule to permit the addition of a radiolabel; addition to the original molecule of a structure that already contains the radiolabel; and addition to the original molecule of a chemical structure that can noncovalently bind (chelate) the isotope. no matter how labeling is performed, the criteria for success are the same. most importantly, addition of the radionuclide should not interfere with the high-affinity binding of the tracer to its target. labeling also should not reduce the lipophilicity of a probe that must pass through cell membranes or cross the blood-brain barrier to reach its target, and should not prevent its interaction with a specific transporter system. finally, attachment of the radionuclide to the probe should be sufficiently stable to ensure that it remains in place while it is carried through the bloodstream to the target. successful in vivo imaging depends upon the proper systemic distribution, cellular uptake and elimination of a tracer following its administration. to ensure rapid and uniform delivery throughout the body, most imaging probes are administered by intravenous injection. as noted above, the pharmacokinetics of a virus-specific imaging probe should resemble those of an ideal antiviral drug, in terms of its uptake into infected cells, but should differ from a therapeutic agent in being cleared quickly from uninfected tissues. (such a pharmacokinetic pattern would provide optimal contrast between sites of viral replication and normal tissues.) because clearance takes time, the radioisotope must have a long enough half-life to ensure that adequate activity remains in the region of interest once the background signal has reached a low level, so as to maximize the target-to-background ratio. in contrast to antiviral therapy, in which the desired drug concentration in tissues tends to be in the micromolar range, radiotracer concentrations are typically in the pico-to nanomolar range. radionuclide imaging has been used to study viral diseases for more than three decades, by employing indirect methods to identify sites of infection. beginning in the s, it was found that cytomegalovirus pneumonitis and other infections in immunodeficient patients could be detected by administering a small intravenous dose of ga citrate and imaging them with a gamma camera (hamed et al., ; reinders folmer et al., ) . as a result of increased perfusion and enhanced vascular permeability, the radionuclide accumulated at sites of infection. other approaches include the intravenous infusion of in-labeled polyclonal human immunoglobulin or a patient's own in-tagged leukocytes (buscombe et al., ; kumar, ) . although these techniques can detect the presence of an infection, they cannot identify its causative agent, so a microbiologic diagnosis must be obtained by collecting appropriate samples and performing laboratory tests. fdg-pet imaging is increasingly used to study host responses to infection, especially in research settings. as described above, the technique relies on the preferential retention of phosphorylated f-fdg in cells with high levels of glucose metabolism, which occur in infectious and inflammatory processes (love et al., ; table the radionuclides most commonly employed in spect imaging, with some of their advantages and disadvantages. t mackie, ) . early in the hiv pandemic, for example, fdg-pet was used to demonstrate that zidovudine therapy had a beneficial effect in patients with hiv-related dementia, as shown by a measurable reduction in cerebral f-fdg retention (brunetti et al., ; yarchoan et al., ) . the same technique has shown that peripheral lymph nodes have a greater glucose uptake in untreated hiv-infected individuals than in patients on antiretroviral therapy, indicating that lymph nodes are sites of viral replication (brust et al., ) . fdg-pet was used recently to characterize a patient with severe swine-origin h n influenza, showing that an intense inflammatory response was present both in areas of dense pulmonary consolidation as seen by ct scan and in regions of aerated lung ( fig. ) (bellani et al., (bellani et al., , ). the value of such studies would obviously be enhanced if the same patient could also be imaged with an influenza-virus-specific probe, to determine the relative chronology and physical distribution of viral replication and host inflammatory responses. whole-body pet imaging has been used to supplement traditional methods of measuring the uptake, distribution and excretion of antiviral drugs in laboratory animals. for example, pet was used to study the pharmacokinetics of the tenofovir analogue f-pmpa in rats, revealing increased concentrations in the renal cortex that appear to correspond to the drug's occasional nephrotoxicity for humans ( fig. ) (di mascio et al., b) . similarly, in an effort to understand the cause of the neurologic side effects of the antiinfluenza drug oseltamivir, investigators employed pet to examine the uptake and retention of the c-labeled drug in the brains of mice (hatori et al., ; konno et al., ) . although a study of the pharmacokinetics of c-labeled stavudine in rats was based on measuring radioactivity in excised tissues, the authors noted that the same questions could have been answered by in vivo imaging (livni et al., ) . pet has also been used to follow the deposition and retention of an inhaled dose of radiolabeled zanamivir, another inhibitor of the influenza viral neuraminidase, in human subjects (bergstrom et al., ; cass et al., ) . these studies only involved uninfected individuals, but it is evident that the same procedure could be used to track the distribution of zanamavir in persons infected with seasonal influenza viruses, either before or after treatment. such experiments would help to determine the effect of therapy on the course of illness, and would reveal whether the radiolabeled drug could be used to detect and track the spread of virus in the respiratory tract. the nascent field of cancer "virotherapy" exploits the ability of viruses to replicate selectively in malignant cells as a novel approach to killing tumors. to monitor the replication of the oncolytic virus, recombinant agents have been constructed that encode a reporter for radionuclide imaging. examples include: • a recombinant vaccinia virus encoding the human norepinephrine transporter, which enables pet imaging by causing i-meta-iodobenzylguanidine (mibg) to accumulate within infected cells (fig. ) goel et al., ) . herpesviruses are also being used as experimental oncolytic agents; in this case, the virus' own tk provides a reporter for pet or spect imaging (see below). the strategy of introducing a gene encoding an imaging reporter into a viral genome provides proof of concept for the notion of employing a naturally occurring virus-encoded molecule as a target for a radiolabeled imaging probe. the approach described above, however, has a significant limitation that would not be seen using an unmodified virus: the introduction of an additional gene may interfere significantly with viral replication. this problem was observed, for example, when a gene encoding green fluorescent protein was introduced into ebola zaire virus; although the modified agent replicated normally in cell culture, it was attenuated for mice (ebihara et al., ) . the ideal imaging approach for the study of viral infections in laboratory animals should therefore make use of unmodified pathogens. the possibility of using a naturally occurring virus-encoded molecule as an imaging reporter was first explored in the early s, when acyclovir was approved as an antiviral drug for the treatment of hsv infections. the ability of this and other nucleoside analogues to selectively inhibit hsv replication is based on their ability to undergo phosphorylation by the viral thymidine kinase (tk), but not by corresponding host enzymes. tk supports dna synthesis in dividing cells or during pathogen replication by phosphorylating the hydroxyl group of deoxythymidine (dt) to produce dtmp, which is then converted to the triphosphate form by other cellular kinases (eriksson et al., ) . all multicellular organisms and bacteria possess a tk, and the enzyme is also encoded by large dna viruses (gentry, ) . two families of tk enzymes are recognized, based their chemical structure and substrate specificity (deville-bonne et al., ). the first group includes the tk enzyme found in the cytoplasm of human cells, a tetrameric molecule which phosphorylates only dt and deoxyuridine, using atp as the phosphate donor (welin et al., ) . tk is expressed during the s-phase of the cell cycle, and it is therefore present in significant quantity only in dividing cells. labeled deoxythymidine analogues such as f- -deoxy- fluorothymidine (flt) can therefore be used as probes to selectively image tumors by pet (langen et al., ; shields, ) . other members of this enzyme family include the poxviral tk, which has a similarly narrow range of substrate specificity (gentry, ) . the second class of tks consists of homodimeric enzymes that are able to phosphorylate a range of substrates, including a variety of nucleoside analogues (eriksson et al., ) . this group includes a second human enzyme, tk , that is found at low concentrations in mitochondria. it also includes the tk encoded by the alpha-herpesviruses, of which hsv is the best-known member. the broad substrate specificity of the hsv tk is the basis of antiviral therapy with acyclovir and other nucleoside analogues. acyclovir enters cells from the bloodstream by diffusion or active transport across the cell membrane. in uninfected cells, the drug is not modified by tk , and only a minimal amount is phosphorylated by the mitochondrial tk , so that almost all of it diffuses back into the bloodstream and is eliminated by the kidneys. in hsv-infected cells, however, the viral tk converts acyclovir to its monophosphate, which is then converted by cellular kinases to the triphosphate. because acycylovir triphosphate has a -to -fold greater affinity for the hsv dna polymerase than for the host enzyme, it is preferentially incorporated into replicating viral genomes, causing chain termination (elion, ) . the selective retention of acyclovir and related antivirals in hsv-infected cells and their rapid clearance from uninfected tissues enhances their potential utility as imaging probes, by increasing the target-to-background ratio. until recently, efforts to image hsv infections have focused on detecting and monitoring viral encephalitis in laboratory animals. beginning in the early s, investigators succeeded in showing that, even though the blood-brain barrier normally restricts the entry of deoxythymidine analogues into the central nervous system, the increased permeability associated with viral infection permitted the entry of a sufficient amount of probe to label infected cells. in a series of studies, rats were infected intraocularly with hsv- , and after an appropriate interval were injected intravenously with a small dose of c-labeled -fluoro- methyl- -␤-d-arabinosyluracil (fmau) or with i-or i-labeled e- -( -iodovinyl)- -deoxyuridine (ivdu) (gill et al., ; klapper et al., ; price et al., ; saito et al., ; cleator et al., ) . foci of encephalitis were identified either by killing animals and performing autoradiography on brain slices, or by imaging live animals using a gamma camera. however, only weak signals were obtained. similar studies have been performed more recently using c-labeled fmau, but the results of in vivo imaging were not shown (de vries et al., ) . in general, this experience has shown that the limited transport of probe across the blood-brain barrier significantly restricts the utility of radionuclide imaging for hsv encephalitis. in contrast to those discouraging results, success in pet imaging of hsv infections has recently been reported in a different area of research: the use of viruses for experimental cancer therapy. herpesviruses are one of a number of agents that are being explored for their capacity to infect and kill tumor cells. the fact that they naturally encode a tk means that their replication in laboratory animals can readily be monitored by radionuclide imaging. viral replication in tumors has been virualized using several different radiolabeled deoxythymidine analogues: [ f]- -fluoro- -deoxy- -␤-d-␤-arabinofuranosyl- -ethyluracil (feau) (fig. ) (brader et al., ); i- -iodo- -fluoro- -␤-d-arabinofuranosyl-uracil (fiau) (jacobs et al., ) ; or -( -[ f]-fluoro- -[hydroxymethyl]butyl)guanine (fhbg) (kuruppu et al., ) . this work thus represents the first success in imaging viral replication in a living animal by targeting a naturally occurring virus-encoded reporter with a radiolabeled probe. the beta herpesvirus cytomegalovirus (cmv) does not encode a tk, but employs its ul protein kinase to phosphorylate nucleosides. because ul recognizes acyclovir to only a limited extent, the drug cannot be used to treat cmv infections. in , however, a new compound with greater anti-cmv activity, ganciclovir, was introduced into medical practice. an f-labeled analogue of ganciclovir, -[( -fluoro- -hydroxy- propoxy)-methyl]guanine (fhpg), has been evaluated as a probe to image cmv infections (alauddin et al., ; de vries et al., ) . pet scanning using f-fhpg was shown to correctly identify cmv encephalitis in rats, as confirmed by autoradiography, but the utility of the approach was once again limited by the restricted entry of the probe across the blood-brain barrier (buursma et al., ) . because the hsv- tk gene is comparatively small, it should be possible to insert it into the genome of other viruses, to provide a reporter for radionuclide imaging. for example, recombinant hiv and simian immunodeficiency (siv) viruses encoding the hsv- tk has been constructed by using parental viruses lacking the nef gene, which both attenuates them and creates space for additional genomic material (chakrabarti et al., ) . although originally envisioned as live vaccines that would be safe for use, because of their sensitivity to ganciclovir, these recombinant agents could also be employed to study retroviral infections in animals by spect or pet. in addition to these uses, the hsv- tk molecule has been extensively exploited as a reporter for spect or pet imaging of malignancies and the assessment of gene therapy miyagawa et al., ; serganova et al., ) . in a novel therapeutic approach, hsv itself is now being harnessed to kill malignant cells, and its distribution and replication are being monitored by pet imaging, using i-fiau as the tracer (bennett et al., ; kuruppu et al., ) . it has recently been shown that treatment of herpesvirus-induced cancers, such as burkitt's lymphoma, with the anti-cancer drug bortezomib triggers expression of the viral tk, making it possible not only for the tumors to be visualized by pet, but potentially treated with ganciclovir (fu et al., ) . as noted above, poxviruses also encode their own tk. because it is a type ii enzyme, however, with a substrate specificity similar to that of human cells, poxviruses are not susceptible to acyclovir. treatment has instead relied on nucleotide analogues such as cidofovir, which enter cells by macropinocytosis, and are then converted to their bi-and triphosphates by cellular kinases. such drugs therefore inhibit both herpesviruses and poxviruses. because their initial phosphorylation is performed by a host enzyme, they accumulate in normal cells, making them unsuitable for use as virus-specific imaging agents. interestingly, recent research has identified some thymidine analogues that inhibit the replication of wild-type vaccinia virus significantly better than a virus lacking a tk gene, suggesting that their mechanism of action resembles that of acyclovir (prichard et al., (prichard et al., , . however, because these compounds can also be processed to some extent by the host tk, they probably have little value for imaging. in an interesting parallel to viral infections, many genera of bacteria encode a type i tk, making them susceptible to antiher- fig. . pet/ct image of septic arthritis in the right knee of a patient, using the bacterial tk as the reporter and i-fiau as the probe. from diaz et al. ( ) , with permission. pesviral drugs. imaging researchers have taken advantage of this property to visualize musculoskeletal infections, both in mice and in human patients. for example, a recent study employed i-fiau as a pet probe to detect staphylococcal infections following joint surgery ( fig. ) (bettegowda et al., ; diaz et al., ) . the same approach has been used to image mycobacterium tuberculosis in animals, even though the organism lacks its own tk. by introducing the gene for the escherichia coli tk into a mycobacterial strain, researchers were able to use i-fiau to visualize sites of infection by spect (davis et al., ). the only naturally occurring virus-encoded molecule that has been employed for radionuclide imaging is the hsv tk, but it seems unlikely that this enzyme should be the only possible target for virus-specific imaging. in the following sections, we review basic steps in viral replication that could be exploited for pet or spect. a number of these steps are identified in the hepatitis c virus replication cycle (fig. ) . to infect a cell, a virus must first bind to its surface (usually to a specific receptor), then undergo a process of fusion with the cell membrane that leads to the disassembly of the capsid and the release of its contents into the cytoplasm. during the fusion process, virion surface proteins undergo specific conformational changes that expose hydrophobic peptide sequences that interact with the lipid bilayer (fig. ) . while some agents, such as retroviruses, enter directly from the cell surface, for others membrane fusion takes place within endosomes, and is triggered by acidification. in both cases, peptides and other inhibitors have been identified for a wide range of viruses that specifically block the entry process. once the viral genome has been delivered into the cytoplasm, gene transcription and genome production depend upon a ready supply of nucleotides and deoxynucleotides for the synthesis of new rna, for all types of viruses, and dna (for dna viruses). a major antiviral strategy is therefore the design of analogues that are accepted as substrates by the viral rna or dna polymerase, resulting either in chain termination or their incorporation into polynucleotides, impairing their subsequent function. because most drugs that inhibit the viral polymerase are converted to their monophosphate by a cellular kinase, and are therefore trapped within both infected and uninfected cells, they would not be suitable as virus-specific imaging probes. an alternative approach, which has been successful for hiv therapy, and is now being used to target hepatitis c and other viruses, is to identify non-nucleoside molecules that bind to a locus on the surface of the viral polymerase, rather than to its active site, inducing a conformational change that blocks its enzymatic activity (fig. ) . because such molecules do not undergo initial processing by a host enzyme, they have the potential to serve as virus-specific probes. another potential target for pathogen-specific tracers is the virus-encoded helicase, which separates the strands of doublestranded dna or rna molecules (or, in the case of retroviruses, dna/rna molecules) during the course of transcription and genome replication. specific inhibitors of the helicase of a number of viruses are being developed as candidate drugs, and they may also have potential as imaging probes. while transcription is taking place, viral mrna must be modified to ensure its efficient translation, through the formation of a methylated cap. the methylation process is proving to be a fruitful target for antiviral therapy. however, although compounds that inhibit methylation by blocking the host cell enzyme, s-adenosylhomocyteine hydrolase, have potent broad-spectrum antiviral activity, they would not be suitable as virus-specific probes, because they do not bind to a viral target. in contrast, compounds that specifically block a virus-encoded methyltransferase, such as the flavivirus ns protein, might make good tracers for radionuclide imaging (dong et al., ) . translation of viral mrna by the host cell protein-synthetic machinery is often followed by cleavage of the protein products to their mature forms. in the case of the positive-sense, singlestranded rna viruses, for example, the entire genome is first translated into one large polyprotein, which is then cleaved into individual components by cellular enzymes and its own endogenous protease (fig. ) . because they differ from those of the host cell, the active sites of a number of viral proteases have proven to be rewarding targets for antiviral drugs. a number of compounds have been identified that bind irreversibly and with high specificity to the active sites of the proteases, and could potentially be used as radiolabeled probes. once nucleocapsids and other virion structural components have been generated, they are carried by endogenous transport pathways to the inner surface of the cell membrane, where assembly and release of viral particles takes place. efforts to develop specific inhibitors of this process are under way, but no drugs have yet been shown to have protective activity in vivo (harty, ). because the same transport pathways are shared by uninfected cells, it is unlikely that drugs that block such mechanisms could be used as pathogen-specific tracer molecules. viral envelope proteins accumulate on the cell surface during the course of replication, anchored by lipophilic peptide sequences (fig. ) . such surface markers, which make infected cells vulnerable to the immune system through the binding of specific antibodies and subsequent cell-mediated destruction, could also be targets for probes such as radiolabeled antibodies. interestingly, certain tumors induced by viral infection, such as ebv-associated lymphomas, hepatocellular carcinoma in patients with hepatitis b and c and cervical carcinomas induced by papillomaviruses, express viral antigens, making them vulnerable to destruction by antibodies tagged with high-energy alpha emitters (dadachova et al., ) . it has been suggested that the same strategy could be used to eradicate hiv in individuals with low levels of residual infection (casadevall et al., ) . a similar strategy, using antibodies labeled with gamma-ray or positron-emitting isotopes, might be used for spect or pet imaging. could drugs other than anti-hsv medications be used to visualize viral infections by spect or pet? most licensed antivirals are nucleoside or nucleotide analogues that undergo initial phosphorylation by cellular kinases, and therefore could not serve as pathogen-specific probes. however, as noted above, the replication cycles of various dna and rna viruses offer a variety of targets for drugs and probes that interact specifically with virus-encoded molecules. the following sections identify examples of candidate tracers for nine different virus families. • the small-molecule drug st- inhibits the replication of junin and other new world arenaviruses, while the benzimidazole derivative st- targets the old world agent, lassa virus (bolken et al., ; larson et al., ) . based on mutagenesis studies of the virion surface gp molecules, both compounds interfere with viral entry, but the specific mechanism is not known. • the extensive requirement for protein cleavage during the coronavirus replication cycle has made the viral papain-like (plpro) and chymotrypsin-like ( clpro) proteases major targets for drug development. highly specific small-molecule inhibitors of both enzymes have been identified that inhibit the sars coronavirus, and might also be used to image infections ratia et al., ) . fig. for identification of potential targets) • inhibitors of flaviviral proteases, such as the dengue virus ns , block replication without causing cellular toxicity, suggesting that they are specific for the viral target (lescar et al., ) . the small-molecule biln , which binds to the active site of the hepatitis c virus ns , was effective in reducing viral load in patients (lamarre et al., ) . although its development was halted by evidence of cardiotoxicity, this would not necessarily prevent the compound from being used at low dosage as an imaging probe. • drugs that directly inhibit the virus-encoded methyltransferase are currently under development for dengue, west nile and other flaviviruses (dong et al., ) . • non-nucleoside molecules have been identified that inhibit the rna-dependent rna polymerase of bovine pestivirus (paeshuyse et al., ) . others are now under evaluation for the treatment of hepatitis c. • the c-terminal portion of the flavivirus ns molecules is a helicase, which is essential for replication. inhibitors have been identified for hepatitis c and other flaviviruses (leyssen et al., ; lescar et al., ) . • the hepatitis c virion surface glycoproteins e and e are expressed on the surface of infected cells, making them potential targets for antibody-dependent cellular cytotoxicity and for imaging by a radiolabeled antibody fragment or a labeled dna aptamer (drummer et al., ; nattermann et al., ; chen et al., a) . • the ul protein kinase of cmv carries out multiple functions, including nucleoside phosphorylation (prichard, ). the recently discovered antiviral mirabavir inhibits this enzyme, suggesting that it could be used as a radiolabeled tracer to image cmv infections. • the prototypes of drugs that bind directly to virions to inhibit their replication are the adamantanes, amantadine and rimantidine, which block the m ion channel, preventing core acidification that is required for virion disassembly in the endosome (beigel and bray, ) . the specific nature of this interaction suggests that small doses of radiolabeled amantadine or rimantadine could be used to localize an influenza virus infection and track it's spread, both in laboratory animals and in humans. • two licensed drugs, oseltamivir and zanamivir, inhibit the cellto-cell spread of influenza a and b viruses by binding to the neuraminidase active site (beigel and bray, ) . this specific interaction might make them effective imaging probes. as noted, the distribution of inhaled c-zanamavir has been visualized by pet in healthy human volunteers bergstrom et al., ) . the same study, performed in influenza-infected patients, could provide useful information on drug distribution and the extent of viral infection. • two small-molecule compounds, vp- and jnj- , block the entry of respiratory syncytial virus (rsv) into cells at low nanomolar concentrations by binding to a small hydrophobic pocket in the center of the f protein (douglas et al., ) . • the rsv f protein is also the target of the human mab palivizumab, used to prevent infection in high-risk infants (georgescu and chemaly, ) . a new antibody, motavizumab, with a greater virus-specific binding affinity and a lower degree of nonspecific binding to host tissues, has recently been developed (wu et al., ) . • conserved heptad repeat regions of the fusion proteins of the highly virulent paramyxoviruses, hendra and nipah, are the targets of peptide mimics, which block infection in vitro at low nanomolar concentrations (porotto et al., ) . • the recently discovered compound rsv blocks the formation of rsv nucleocapsids by binding to a specific site on the n protein at submicromolar concentrations (chapman et al., ) . it is now in phase ii clinical trials. • the entry of picornaviruses into cells requires the disruption of interactions among the four capsid proteins, so as to release the viral genome into the cytoplasm. a number of compounds have been identified that insert into "pockets" in the virion surface, stabilizing the capsid and preventing its disassembly (de palma et al., ) . such compounds have protected mice against lethal poliovirus infection, and pleconaril, which acts by this mechanism, has been extensively tested for antirhinovirus activity in humans (de palma et al., ; mckinlay and steinberg, ). the highly specific interaction of such compounds with the viral capsid suggests that they could be used to image sites of picornaviral infection. • the c protease of human rhinovirus is inhibited by a number of compounds, including the small-molecule rupintrivir, which binds irreversibly to the enzyme's active site (de palma et al., ) . because the viral enzyme has no known human homologue, it should be a good target for virus-specific imaging. • the benzimidazole analogue enviroxime inhibits the replication of rhinoviruses and enteroviruses by blocking viral rna replication, apparently by directly inhibiting the action of the a protein. the compound protected mice against lethal coxsackie myocarditis and has shown activity in protecting humans against rhinoviruses (victor et al., ; de palma et al., ) . • the complex process by which poxviruses exit cells has provided targets for antiviral therapy. the small-molecule st- interacts specifically with the vaccinia virus f l protein, which is involved in the addition of a membrane layer needed to form extracellular enveloped virions (yang et al., ; duraffour et al., ) . st- is nontoxic for mammalian cells, animals and humans, suggesting that it uniquely targets the viral enzyme, and could therefore be used as a radiolabeled probe to image poxvirus infections. • the hiv fusion inhibitor, t- , is a polypeptide that binds to the hydrophobic fusion region of the viral gp , preventing it from undergoing the conformational change required for fusion with the cell membrane. a labeled version of this peptide might therefore be an effective tracer to identify sites of hiv replication. • non-nucleoside inhibitors of the hiv reverse transcriptase (rt) interfere directly with its activity by binding to loci other than the active site. a number of such compounds are now licensed for the treatment of hiv infection (sluis-cremer and tachedjian, ) . because no equivalent of the hiv rt is encoded by the human genome, radiolabeled derivatives of these drugs might prove useful in targeting sites of hiv replication. • the potential of protease inhibitors as effective antiviral drugs was first proven for the treatment of hiv infection, and a number of different compounds are now approved for clinical use. the most recently licensed, darunavir, has a strong binding affinity for the hiv protease (k d = . × − m), suggesting that it would make an effective probe (lefebvre and schiffer, ) . to assess whether a drug or antibody has promise as an imaging probe, it is not necessary to label it with one of the isotopes listed in tables and and attempt to image an infected animal by pet or spect. instead, a preliminary evaluation can be performed using the c-or h-labeled compound, which is often synthesized for pharmacokinetic studies and is therefore available for other types of research. three types of experiments can be performed. the first is an in vitro assay, in which the labeled drug is added to the liquid medium of a number of flasks or wells of virus-infected and uninfected cells. at various time points, the supernatant is removed, the cells are rinsed and harvested, scintillation counting is performed, and the amount of radioactivity retained by infected and uninfected cells is compared. the accumulation of a significantly larger quantity of radiolabeled drug in virus-infected than uninfected cells indicates that it may also be preferentially retained at sites of infec-tion in vivo. additional studies would include testing the specificity of binding, by adding a large excess of unlabeled drug; determination of the k d of the labeled drug, the concentration of binding sites (b max ) and the kinetics of binding; and competition studies with other unlabeled analogues (cheng and prusoff, ; deblasi et al., ; jeffries et al., ) . investigators could then move forward to an ex vivo experiment, in which virus-infected and uninfected animals are killed at various time points, necropsies are performed and relevant organs (for example, the lungs in influenza or the brain in encephalitis) are collected and frozen. thin sections are cut and placed onto microscope slides, which are then immersed in a solution of labeled cor h-labeled drug, rinsed to remove unbound tracer and placed against film or phosphor imaging plates for autoradiography (saito et al., ; frey and albin, ; cagnin et al., ; patel and gibson, ; stumpf, ) . once a sufficient exposure time has elapsed, the films are examined to determine if radiolabeled drug has preferentially been retained at sites of viral infection. to check for specificity, various amounts of unlabeled ("cold") drug can be added to the solution of labeled compound, to determine if it will compete for binding to sites of infection, reducing the detected signal. additional thin sections would be used for immunohistologic studies, to confirm the presence of virus-infected cells. at the same time that ex vivo studies are being performed, investigators can carry out in vivo experiments, in which c-or h-labeled drug is administered to infected and uninfected animals (preferably small rodents). after an appropriate delay, the animals are killed, organs of interest are removed, samples are collected for determination of viral titer by plaque titration and of probe concentration by scintillation counting or autoradiography, and sections are prepared for immunohistologic study. if sites of maximum viral replication also show the highest levels of radioactivity, and much lower signals are detected in uninfected tissues, this would suggest that the drug has potential as a virus-specific probe. as in ex vivo studies, the specificity of binding can be further evaluated by administering various amounts of unlabeled drug along with the labeled probe, to see if competition takes place. if initial testing of the c-or h-labeled drug confirms its potential as an imaging probe, the next step will be to work with a radiochemist to determine the necessary precursors and methods for synthesis of a spect or pet tracer. once that synthesis has been carried out, evaluation of the new radiolabeled probe should include in vitro and/or ex vivo studies to ensure that it has retained its biological activity. at the same time, imaging specialists should be consulted to design initial studies in an appropriate animal model of viral infection. in addition to offering a second application for certain licensed antivirals that directly target viral replication, radionuclide imaging may also provide a use for some compounds that are active in vitro and in animal models, but for various reasons have not moved forward to licensure. as in the case of deoxyglucose, which failed in trials as a cancer drug, but is now the most widely used tracer for pet imaging, this might include candidate antivirals that are nephro-or hepatotoxic at therapeutic doses. rather than discarding 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cord- - sqkh m authors: schmidt, alexander c; couch, robert b; galasso, george j; hayden, frederick g; mills, john; murphy, brian r; chanock, robert m title: current research on respiratory viral infections: third international symposium date: - - journal: antiviral res doi: . /s - ( ) -x sha: doc_id: cord_uid: sqkh m nan the third international symposium on respiratory viral infections was convened by the macrae group (new york, ny) in st. lucia, windward islands, on - december . for the third time, this symposium provided a forum for virologists, vaccinologists, clinicians, pharmacologists and public health specialists to discuss recent advances in respiratory virus research in an interdisciplinary fashion (kaiser et al., ; munoz et al., ) . the spectrum of discussion ranged from basic virology and pathogenesis to vaccinology, immunology, and management strategies for respiratory viral infections. epidemiology of respiratory viral disease and possible preparations for the next influenza pandemic were also an important part of the agenda. until , influenza viruses were the only known filterable human respiratory tract pathogens. in , shope recovered an influenza a h n virus from swine, which probably was the first human influenza virus isolated. two years later smith, andrews and laidlaw recovered the first influenza isolate from humans, and soon after, efforts to develop an inactivated influenza a vaccine began. influenza b and c were isolated in and by francis and taylor, respectively. in in the laboratory of infectious diseases (lid) at the national institutes of health, and hilleman, then working at the walter reed army medical center, recovered the first human adenoviruses and established their importance in acute febrile respiratory tract disease (rowe et al., ; hilleman, ) . in the following years, robert chanock discovered most of the remaining respiratory viruses that are considered important lower respiratory tract pathogens today. the first of them, a croup-associated myxovirus, was discovered during an outbreak of croup in cincinatti in , and it was later designated human parainfluenza virus type (piv ). in , morris and colleagues recovered the chimpanzee coryza agent (cca) during an outbreak of a cold-like illness in a chimpanzee colony, and a year later chanock and colleagues recovered two similar isolates from an infant with bronchopneumonia and from another infant with laryngotracheobronchitis, and characterized the human virus now known as respiratory syncytial virus (rsv) (chanock et al., ) . the discovery in of piv and piv , the single most common cause of croup and the second most common cause of serious viral pediatric lower respiratory tract disease, respectively, broadened our understanding of the etiology of acute lower respiratory tract disease (chanock et al., ) . the discovery of piv in followed in short order. in , a double-blind prospective study evaluating the use of tetracycline in the treatment of cold agglutinin-positive atypical pneumonia led to the identification of the etiologic agent of this disease. the agent, originally recovered by eaton from patients with this form of pneumonia, was known to be filterable and thought to be a virus but its role as an etiologic agent was heavily disputed. a large double-blind study by chanock, kingston and mufson, in which antibodies to the eaton 'virus' were used to define a subset of patients with pneumonia, showed that tetracycline therapy decreased duration of the disease, thereby excluding a virus etiology. subsequently, the eaton agent was shown by chanock, hayflick and barile to be a mycoplasma that grew in cell-free medium; later it was named mycoplasma pneumoniae (chanock et al., ) . serologic analyses and studies in adult volunteers confirmed the etiologic role of this organism in cold agglutinin-positive atypical pneumonia. renewed efforts in vaccine development against respiratory viruses began in the s with the observation that infants and young children, after having recovered from respiratory tract infection with adenoviruses, shed virus from their gastrointestinal tract for an extended period of time without experiencing gastrointestinal symptoms. this led to the hypothesis that one could potentially use the gastrointestinal tract to vaccinate against respiratory tract disease caused by these viruses. wild-type adenovirus type and administered orally in enteric-coated capsules was found to protect military recruits against respiratory tract disease caused by these viruses. gastrointestinal symptoms were not observed, and although virus was shed from the intestine, it did not infect close contacts (couch et al., ) . the development of vaccines against respiratory viruses suffered a major setback in when formalin-inactivated rsv vaccine not only failed to protect infants against rsv infection but instead potentiated rsv disease upon subsequent rsv infection (kim et al., ) . the inactivated vaccine did not induce a potent neutralizing antibody response but it stimulated an exaggerated cd + t cell response without stimulating cytotoxic cd + t cells. this unanticipated failure of a non-living vaccine reoriented the research agenda of the laboratory of infectious diseases towards the development of live-attenuated virus vaccines. a cold-passaged rsv strain (cp ) was selected in as the first candidate live-attenuated rsv vaccine strain (friedewald et al., ) . this candidate vaccine was safe and immunogenic in adults and older children but was insufficiently attenuated in seronegative infants (kim et al., ) . since then, the search for a live rsv vaccine strain has been a central focus of the lid. developing a live rsv vaccine candidate that is attenuated yet immunogenic in seronegative infants has proved to be a formidable task. since incidence and morbidity of rsv are highest in the second and third month of life, a vaccine candidate has to be safe for administration to neonates, able to stimulate an immature immune system, and able to overcome the immunosuppressive and antiviral effects of passively acquired maternal rsv antibodies. initial vaccine candidates were derived in the late s and early s by passage of virus at low temperature (cold passage) or by chemical mutagenesis. several different lineages of mutants, such as temperature sensitive mutants generated by -fluorouracil ( fu) mutagenesis, were evaluated in infants and young children but were insufficiently attenuated or genetically unstable. the cold-passaged (cp) mutant that was subsequently further attenuated by the acquisition of two missense mutations that conferred temperature sensitivity (ts) to yield cpts rsv / , has provided the most promising vaccine candidate tested thus far. this candidate vaccine virus was infectious, safe and immunogenic in -month-old seronegative infants, conferred protection against challenge with a second dose of vaccine virus weeks later, and caused only mild upper respiratory tract symptoms (wright et al., ) . recent development of a method for rescue of infectious rsv from cdna by collins enhanced our ability to develop rsv vaccine candidates rapidly (collins et al., ) . site-directed mutagenesis can now be used for the first time to construct viruses with one or more additional attenuating mutations. using recombinant cdna technology, viable rsv mutants with deletion of the ns , ns , sh or m - gene have been constructed as vaccine candidates that bear genetically stable attenuating mutations. for these reasons, it is likely that a live-attenuated vaccine that exhibits an acceptable balance between attenuation and immunogenicity can be developed within the next several years. this vaccine virus might possess one or more gene deletion mutations together with or without the earlier characterized cold-passaged (cp) and temperaturesensitive (ts) mutations. the first piv vaccine candidates were also prepared by formalin-inactivation. similar to the experience with formalin-inactivated rsv, these vaccines did not protect against piv disease. in the early s, belshe attenuated a piv isolate by passages at °c (cp ) (belshe and hissom, ) . in clinical studies, the piv cp vaccine candidate has proved to be safe, genetically stable and immunogenic in seronegative in-fants (karron et al., ) . this vaccine candidate is currently being tested in phase ii clinical trials. a recombinant version of piv cp has been rescued from cdna, and the genetic basis of its attenuation (att), temperature-sensitivity (ts) and cold-adaption (ca) phenotypes has been determined (skiadopoulos et al., ) . influenza a and b virus vaccine development followed this same path of serial passage at °c (cold-adaptation) to generate mutants with att, ts and ca phenotypes (maassab, ; maassab and bryant, ) . in contrast to piv and rsv, live attenuated influenza a vaccine strains were virus reassortants that were generated by mating the attenuated donor virus with an epidemic wild type virus so that the reassortant virus vaccine was a chimera that contained the attenuating genes of the donor virus, while the ha and na genes were derived from the current epidemic virus (murphy et al., ) . this strategy, developed by john maassab, of using the cold-adapted mutant virus a/aa/ / as the donor of the six attenuating internal and non-structural genes for construction of reassortant vaccine strains was validated by the large series of consecutive reassortants that have proven to be attenuated and immunogenic. analysis of the genetic basis of attenuation showed that the influenza a pb and pb genes each consistently specified the ts phenotype, and pa specified the ca phenotype. however, all three of these genes of the viral polymerase complex contribute to the attenuation of the trivalent influenza a (h n and h n ) and b vaccine viruses (murphy, ; maassab and bryant, ) . the safety, protective efficacy and phenotypic stability were confirmed in large phase iii trials, and licensure is expected in the near future (belshe et al., ) . evidence for the prophylactic effect of serum rsv neutralizing antibodies was demonstrated in the s (prince et al., b) . passive transfer of homologous rsv convalescent serum to cotton rats protected them against rsv replication in the lungs following subsequent intranasal challenge with wild type virus. a serum rsv neutralizing titer of : in recipient cotton rats conferred almost complete protection. this amount of neutralizing antibody necessary for protection was later confirmed in clinical trials and today forms the basis for passive rsv prophylaxis in high-risk infants (groothuis et al., ) . an increased incidence and severity of rsv disease is seen in preterm infants with or without chronic lung disease (cld), in children with congenital heart disease (chd), and in immunosuppressed children and adults. respiratory disease in general is a common cause for re-hospitalization of preterm infants. cunningham and colleagues compared a cohort of preterm infants (mean gestational age at birth weeks) to a cohort of term infants and found a -fold increase in readmission for respiratory disease in preterm infants without cld, and a -fold increase for preterm infants with cld ( . , and %, respectively) (cunningham et al., ) . cld patients in a home oxygen program were at even higher risk ( % hospitalization, % icu admission) (groothuis et al., ) . children with pulmonary disorders such as cystic fibrosis, lung malformation or recurrent aspiration pneumonitis, when admitted for rsv disease, are as likely to require icu treatment ( - %) and mechanical ventilation (up to %) as children with cld ( and %, respectively) (arnold et al., ) . earlier studies of high-risk infants admitted for rsv disease yielded similar results, with icu treatment necessary in - % and mechanical ventilation necessary in - % (meert et al., ; navas et al., ) . apart from preterm infants with or without cld, children with congenital heart disease (chd) are a second group of high-risk patients, particularly when they suffer from pulmonary hypertension. in a prospective study of children hospitalized in five consecutive rsv seasons ( ) ( ) ( ) ( ) ( ) , % of patients with rsv disease and chd (compared with % of patients without chd) required icu treatment and % died (macdonald et al., ) . in a prospective study of children with chd, the incidence of rsvrelated hospitalization during one rsv season was % for children younger than years and % for infants younger than months of age (simoes et al., ) . immunocompromised children are a very diverse population and not all of them are at equal risk for severe rsv infection. children receiving corticosteroid therapy have a much lower risk for rsv-related hospitalization and death than children receiving chemotherapy for malignancies or children with primary immunodeficiencies. rsvrelated mortality was % for chemotherapy-recipients and % for children with primary immunodeficiencies compared with % for steroid-treated children (hall et al., ) . rsv grew to very high titer in children receiving chemotherapy, and more than half the patients shed rsv for weeks or longer. for immunocompromised adults, the picture is not much different from that described for children. infections are often acquired nosocomially, virus shedding is prolonged, and the incidence of pneumonia and death is high. rsv is the most important viral respiratory pathogen in these patients, followed by picornaviruses, influenza and parainfluenza viruses: % of confirmed rsv infections in leukemia and bone marrow transplant patients resulted in pneumonia, and the fatality rate was greater than % . whimbey and colleagues reported rsv case fatality rates for bone marrow transplant recipients as high as % when therapy was administered early and adequately (ribavirin and ivig), and %, when therapy was initiated late or inadequately . hiv-infected children in an urban setting in south africa also have an increased burden of viral lower respiratory tract illness (lri) although respiratory viruses are less frequently isolated from nasopharyngeal aspirates of hiv-infected children than from children without hiv infection. the relative risk for severe lri caused by rsv was twice as high in hiv-infected than in uninfected children two years of age or younger (madhi et al., ) . little information is available regarding genetic and environmental factors in susceptibility to rsv infections. most rsv epidemiologic studies are conducted in affluent countries and temperate climates although rsv is thought to be the leading cause of severe viral acute respiratory infections (ari) in infants around the globe (weber et al., ) . a -year prospective surveillance study was conducted in the yukon-kuskokwim (yk) delta of south-western alaska to determine the rate and severity of rsv infections requiring hospitalization for infants in this yupik eskimo population (karron et al., ) . the annual rate of rsv hospitalization for yk delta infants less than year of age was unusually high, i.e. - / . one in children born in the yk delta, compared with between one in to one in infants in affluent countries (sims et al., ; martin et al., ; glezen et al., ) , required ventilatory support for rsv disease. rsv infection was the single most frequent cause of hospitalization of yk delta infants. as in temperate climates, rsv epidemics in the yk delta occur annually from november through june, with peak hospitalizations for rsv disease occurring between november and february. within sub-regions of the yk delta, epidemics were as brief as month, probably because there is very limited traffic between villages in the winter months. most of the infants admitted to hospital were less than year of age and had no medical risk factors for rsv disease. surprisingly, % of the admitted infants in the yk delta and % of infants in a comparison group admitted to johns hopkins hospital (jhh) were less than one month old. of children with severe disease, % in the yk delta and % at jhh were less than months old. disease severity in non-high risk children did not differ between children admitted to jhh or yk delta regional hospital, suggesting that differences in hospitalization practices could not account for the high rates of hospitalization for rsv in yk delta infants. in the yk delta, % of those admitted for rsv disease were readmitted within a single rsv season. severity of rsv disease, age at first illness and receipt of ribavirin were all associated with readmission. in infants less than months of age, a low neutralizing antibody titer in cord blood samples was strongly associated with severe disease. a questionnaire-based case control study that was matched for age and sub-region in the yk delta detected three risk factors influencing rsv hospital admission. medical risk factors (prematurity, chronic lung disease, congenital heart disease) increased the risk of admission . -fold. more than eight people living in one household doubled the risk of hospitalization while breastfeeding had a protective effect. smoking, food-pre chewing and economic status were not significantly associated with rsv hospitalization. this study may be useful in the continued analysis of the impact of rsv in developing countries. as in the yk delta, rsv is the leading cause of viral lower respiratory tract disease in most developing countries, but lack of access to diagnostic reagents and hospital facilities has made it difficult to quantify the impact of rsv. also, the rate of severe rsv disease in term neonates was much higher than in earlier studies, both in the yk delta population and the comparison group in baltimore. these findings should be confirmed in other populations because they have important implications for rsv vaccine development. . . respiratory syncytial irus (rsv) and human rhino irus (hrv) infections in children with aids and lower respiratory tract illness (lrti) acute respiratory infections (ari) cause a great burden of disease in developing countries (de arruda et al., ) . although a growing number of children from developing nations are hiv infected, there is little knowledge about the frequency and severity of viral ari in hiv infected children. earlier studies of viral pathogens in immunocompromised adults indicated that cmv, herpes simplex, influenza, parainfluenza, rhinovirus, adenovirus, enterovirus, and rsv cause lower respiratory infection (connolly et al., ) . a recent study assessed the frequency of rsv and rhinoviruses (hrv) in hospitalized children with or without aids, who presented with lower respiratory tract infection (lrti). about episodes of lrti in children with aids and in children without hiv infection, matched by age and sample collection month, were studied in a rural area of southern brazil. the frequency of rsv infection was highest in the fall and winter, between february and july, whereas hrv was detected throughout the year. rsv was found in / ( %) and / ( %) of lrti episodes in aids and non-hiv infected children, respectively. hrv was found in / ( %) and / ( %) of the episodes in children with aids and hiv-uninfected children, respectively. no difference was detected in frequencies of hrv and rsv infections between the two groups. hrv infections, however, tended to be more frequently associated with pneumonia in children with aids ( / , %) than in the control group ( / , %) (p= . ). other clinical presentations of lrti were observed with equal frequency. these findings did not confirm hrv as a causative agent of pneumonia in children with aids, but suggest that further studies of lrti are desirable and that interventions for hrv could be considered for immunocompromised children with lrti. the human adenoviruses (ads) are a large family of over serotypes, as well as numerous variants and intermediates. they are divided into six subgroups (a-f) that exhibit different tissue tropism. the clinical manifestations of adenoviral disease are protean. the most common are respiratory syndromes in both children and adults. it is estimated that ads cause - % of all respiratory disease in children, including pharyngitis, tonsillitis and pertussis-like syndrome. in children and adults, ads are also associated with lower respiratory tract infections such as bronchitis and pneumonia. subgroup d ads are associated with both epidemic and sporadic ocular infections including conjunctivitis and keratoconjunctivitis. due to their stability in the environment, these viruses are highly transmissible, particularly in nosocomial settings. in adults, ads cause largescale epidemics of acute respiratory disease (ard) in closed populations of military recruits, dormitory residents and long-term care facility occupants, which are primarily associated with ad serotypes and a, and to a lesser extent with serotypes , , , and . while replication in the gastrointestinal tract is a feature of most ad infections, only ad and ad (subgroup f) are associated with gastroenteritis in infants and young children. ad infections in immunocompromised subjects are increasing as their numbers increase, with severe consequences. clinical manifestations of ad infection in immunocompromised patients include pneumonia, hepatitis, encephalitis, and systemic and disseminated disease, with case fatality rates from to %, depending on the nature of the immunodeficiency. nearly all ad serotypes have been associated with these infections, but the higher numbered subgroup d serotypes that are usually not associated with clinical disease in the immunocompetent host have been especially common. a number of 'new' disease associations with ad infection have recently been reported, probably due to improved highly sensitive molecular diagnostic techniques that also increase the probability of laboratory contamination. detection of viral genome in the absence of positive viral culture has been described in cases of myocarditis and pericarditis in children and adults (martin et al., ; bowles et al., ; pauschinger et al., ) , sudden infant death (shimizu et al., ) , toxic shock-like syndrome (price, ) and 'unexplained death' (perkins et al., ) . isolation of ads from patients with central nervous system manifestations of fatal acute flaccid paralysis (cardosa et al., ) and encephalitis with cerebral edema (chatterjee et al., ) has also been reported. it is, perhaps, not surprising that the clinical manifestations of ad infection are evolving because the viruses themselves are in the process of continuous evolution. the ad mutational repertoire includes homologous recombination, illegitimate recombination, and single base mutation (sbm). homologous recombination occurs in conserved regions of the genome between closely related viruses within the same subgroup, and it requires regions of homology in the two parent strands. it is the primary mechanism responsible for intermediate ads, which are mosaic viruses with shared hexon characteristics, or with the hexon characteristics of one type and fiber of another. illegitimate recombination requires only short regions of homology of one to three nucleotides (short direct repeats), and is thought to be the result of polymerase stuttering or slippage. it causes deletions, insertions and duplications of short regions of dna. in ads it occurs in noncoding regions and hypervariable regions (hvrs) of hexon capsid proteins that tolerate structural variation. the hvrs of the hexon contain the viral neutralization epitopes, so that mutations in these regions result in deletion, formation or alteration of these epitopes, leading to antigenic shift. it is the primary mechanism by which new serotypes arise, particularly among the fastest growing group, the subgroup d ads. single base mutations accumulate gradually across the viral genome but can occur at a -fold higher rate in the hvrs, where they cause incremental antigenic drift and the creation of variant strains. ad evolution is compounded by all three mechanisms and perhaps others that have yet to be defined. as a result, serological identification of subgroup b and d ads has become extremely difficult. it seems reasonable that the time has come to consider a sequence-based ad classification system, similar to that in use for papillomaviruses and enteroviruses. adenovirus has re-emerged as a leading cause of febrile respiratory disease among military recruits. large and frequent epidemics were common at trainee camps before but were eliminated with the introduction of the live enteric type and vaccines. in , the sole vaccine manufacturer discontinued production of these live enteric coated vaccines because of contractual issues. while limited vaccine stores were still available in and , vaccine stores were completely depleted by early . to monitor the effect of discontinued vaccination, weekly surveillance for febrile respiratory infections (fri), defined as oral temperature \ . °f with respiratory disease symptoms, was conducted from october -june at four military training camps. during this interval, ( %) of throat cultures yielded adenovirus. during the winter of - , adenovirus infections caused more than % of fri at each of the four camps. ad , , , and accounted for , , , and % of the isolates, respectively. three training camps experienced a high prevalence of adenovirus type and the fourth camp experienced a type outbreak. among symptomatic trainees, those who did not receive vaccine were times more likely to be infected by ad or than vaccinated subjects (gray et al., ) . surveillance was extended to eight sites in june and virus isolation was attempted for adenovirus, influenza a and b, rsv, and parainfluenza - . large ad epidemics were observed in six training camps throughout the us, while rsv and influenza a and b viruses were isolated less frequently. the impact of adenovirus epidemics on basic training can hardly be overestimated. recruit camps were forced to convert barracks into special infirmaries to care for the ill, and hospitals were forced to halt elective surgeries. at one camp, the number of trainees that had to repeat their basic training because of extended illness increased -fold. this 'recycling' has an extremely negative effect on the morale of trainees and it impacts on the military's readiness. as many as preventable adenoviral trainee medical encounters occurred during the winter months of and in ; two military trainees died with molecular evidence of acute adenoviral infection, one with encephalomyelitis and another with acute respiratory distress syndrome (ards). vaccination against ad and has proven to be extremely safe and effective, and to prevent an enormous burden of disease in military trainees (howell et al., ) . it is urgent that a new manufacturer for adenoviral vaccines be identified, and vaccine production must resume as soon as possible. in recent years, much progress has been made in understanding virus-induced modulations of the host immune response to viral infections. for ad, more than viral gene products are known to participate in the modulation of immune responses, and many of these gene products are expressed from genes clustered in the early region (e ) (wold et al., ; horwitz, ) . the overall effect of ad e gene products on immune responses in vivo can be appreciated from the results of three studies in mice that investigated transplant rejection and development of autoimmune disease. in the first study, the expression of the complete ad e cassette in pancreatic islet cells as transgenes under the control of the rat insulin promoter (rip) enabled allogeneic islet donor cells containing the h- bxd class i mhc to be accepted long-term by h- d recipient mice (efrat et al., ) , indicating that ad e gene products could potentially be used as a powerful tool in the control of transplant rejection. the second study used the lymphocytic choriomeningitis virus (lcmv) model of autoimmune diabetes mellitus, in which the lcmv proteins np or gp are expressed on the surface of islet cells, and diabetes is induced by infection with lcmv that induces cd + (gp) or cd + and cd + (np) t-cell mediated immune responses. in this model, the co-expression of rip-e with lcmv-np or gp completely prevented the onset of diabetes after lcmv infection (von herrath et al., ) . similar protective effects of ad e transgenes were seen in a third study that used the non-obese diabetic mice (nod) model of diabetes mellitus, and the underlying mechanisms are currently being investigated (efrat et al., ) . thus, as the understanding of the mechanism of action of the ad e immunoregulatory genes are being pursued in various systems, they are being utilized to control selected immune reactions that might be involved in the genesis of autoimmune diabetes. some of the better-characterized gene products of the e region are (in order of increasing distance from the e promoter) gp , . , . , . and . k. only the functions of gp and . k shall be discussed here; the other ad e gene products have been reviewed elsewhere (horwitz, ) . in vitro, gp k reduces the expression of class i major histocompatibility complex (mhc) molecules by retaining the mhc heavy chain in the endoplasmic reticulum or retrieving it back from the golgi, and also by inhibiting peptide processing (bennett et al., ) . in the cotton rat model of adenovirus pneumonia, gp k deletion mutants replicate like wild-type virus but they induce a much stronger inflammatory response (ginsberg et al., ) , whereas in c bl mice an increase in pulmonary pathology is not seen (sparer et al., ) . the ad e . k protein inhibits tnfa-induced cell death by a process that does not involve down-regulation of the tnfa-receptor. in cotton rats (ginsberg et al., ) and c bl mice (sparer et al., ) deletions of ad e . k modify the pulmonary inflammatory response, i.e. an increase of polymorphonuclear leukocytes in cotton rats and more pronounced alveolar infiltration in mice. in order to determine how the ad . k protein prevents cell death, the cell proteins that interact with this viral protein were determined. using a yeast two-hybrid system, four . k-interacting proteins (fips) were identified. three of them have been characterized and have been shown to participate in quite diverse cellular pathways (li et al., (li et al., , (li et al., , b . fip- (also known as rag-a, a ras-related small gtpase) can bind to tctel, a component of the microtubule motor protein dynein, forming . k-fip- -tctel complexes (lukashok et al., ) , and . k has been postulated to affect microtubule dependent macromolecular transport or even modulate the transport of virus. however, because . k is not a structural protein of adenoviruses and must be made de novo from early viral transcripts, it is unlikely to play a role during viral entry, even though the process is known to be microtubule dependent. the role of . k during viral exit from cells has not been studied. fip- also binds to a second gtpase (gip- ) that localizes in the centrosome and in addition to potential effects during mitosis may be involved in transporting macromolecules between the nucleus and the cytoplasm. fip- binds to abnormal huntingtin, and more specifically to the expanded polyglutamine tract that appears to be associated with cell death of neurons in huntington's disease (faber et al., ) . whether or not ad e - . k and/or fip- can prevent huntingtin-induced cell death is currently being investigated. over-expression of fip- , which is also called nf-kb essential modulator (nemo) or inhibitor of kappa kinase gamma (ikkg) causes morphologic changes and eventually apoptosis in a variety of cell lines. the amount of apoptosis induced by fip- can be reduced by % when ad e - . k is present. apart from . k, fip- seems to interact with a number of key molecules in the tnf receptor and nf-kb signaling pathways such as the receptor interacting protein (rip), the inhibitor of kappa b kinase beta (ikkb) and the nf-kb inducing kinase (nik) (li et al., b) . these few examples of the effects ad e gene products have on the pathobiology of diseases as different as autoimmune diabetes, transplant rejection and huntington's disease indicate how much remains to be learned from studying adenovirus-host interactions. the rsv (strain a ) genome is a single stranded negative-sense rna of nucleotides that is transcribed into major subgenomic mrnas. three of the eleven encoded proteins are transmembrane proteins. the g protein mediates attachment to cell surface receptors, the f protein mediates virus-cell and cellcell fusion, and the function of the sh protein is unknown. other structural proteins are the m protein, which plays a role in virion assembly, the n, p and l proteins that make up the viral polymerase, and the m orf protein that functions as a transcription anti-termination factor. the other proteins include two non-structural species, ns and ns , and the m - protein encoded by the second orf of the m mrna. using a reverse genetics system to rescue infectious rsv from cdna, five of the genes of rsv can be ablated individually and in some cases in combination without rendering the virus non-viable jin et al., b) . these five non-essential genes are ns , ns , sh, g, and m - . since all of these genes confer a selective advantage to rsv in vitro and/or in vivo, they can be described as virulence factors -the deletion of which will lead to attenuation of the virus. deletion of the small hydrophobic (sh) transmembrane protein yields a recombinant rsv called rsva dsh, which replicates in vitro as well as wild type (wt) rsv and induces plaques in hep- cells that are larger than wt rsv plaques. there is no reduction in synthesis of rna or protein associated with the deletion of sh. in chimpanzees, however, the virus is slightly attenuated (whitehead et al., a) . deletion of the ns or ns gene results in a substantial reduction in replicative efficacy in vitro, and this reduction is more pronounced in hep- cells than in vero cells (which lack interferon a and b genes), suggesting that these two genes act as antagonists to type interferon effects. deletion of ns and ns from bovine rsv provided direct evidence that ns and ns cooperatively antagonize a/b interferon-induced antiviral responses (schlender et al. ) . in chimpanzees, the level of replication of both rsva dns and rsva dns is reduced greater than -fold in the lower respiratory tract. in the upper respiratory tract, the dns virus is more attenuated than the dns virus (teng et al., ) . deletion of the m orf not only identifies a markedly attenuated rsv mutant but reveals an important role for this orf in the replicative cycle of rsv (bermingham and collins, ) . during infection with wildtype rsv, transcription appears to shut off at approximately - h post infection while rna replication increases concurrently. in contrast, this apparent switch from transcription to rna replication was not observed for the rsvdm - virus, implying that m - is a regulatory protein involved in the shift. instead, transcription continued to increase while rna replication remained low compared to wild-type rsv. overall, gene expression was increased - fold. the synthesis of the g and f proteins also was increased and resulted in increased syncytium formation. replication of the rsvdm - virus in vitro was attenuated, probably due to reduced rna replication (bermingham and collins, ; jin et al., a) . in chimpanzees, comparison of the four rsv gene deletion mutants mentioned above with wt rsv and the incompletely attenuated rsv cpts- / vaccine candidate results in a hierarchy of increasingly more attenuated viruseswt rsv b dsh b dns b / b dns b dm - . the final rsv gene deletion mutant, rsva dg, was not evaluated in chimpanzees because the absence of this major protective antigen would not be desirable in a vaccine virus. rsva dg replicates as efficiently as wt rsv in vero cells, showing that g is not essential for efficient virus replication. however, the rsvdg virus is highly attenuated in balb/c mice, indicating the importance of the rsv g protein in vivo. it is evident from the above ranking that rsv reverse genetics is able to generate mutants exhibiting gradations in their level in attenuation ). this menu of viruses with different levels of attenuation is crucial in identifying an rsv vaccine that exhibits the desired balance between attenuation and immunogenicity in seronegative infants. since clinical data indicate that rsv / is just slightly under-attenuated in the -month-old target population, the dns mutant could be exactly what is needed. in order for rsv assembly to be an efficient process, viral structural proteins must be brought together in a coordinated fashion (peeples, ; lenard, ) . compared with other paramyxoviruses rsv exhibits several unique features. the g, m - and m - proteins are found only in the pneumo irus genus of the paramyxo iridae, and the role these proteins play in rsv assembly is much less well understood than the role of the f, hn and m proteins of other paramyxo iridae (collins et al., ) . comparison of multiple human, bovine and ovine rsv strains shows that the cytoplasmic domains of the f and g proteins are well conserved amongst human rsv strains and subtypes, and conserved to some degree between the three species. in analogy to other paramyxoviruses, it is likely that the cytoplasmic domain of f and g interact in the process of virion assembly with cellular proteins involved in protein trafficking and in the polarized budding process, as well as with other viral proteins. the rsv g and m proteins co-localize in the golgi apparatus, not only in rsv-infected cells but also in cells transfected with only the g and m proteins, indicating that other viral proteins are not needed for this interaction (peroulis et al., ) . the g-m interaction is seen with fulllength g protein but not with a secreted form of g that lacks the conserved cytoplasmic domain and transmembrane domains. systematic deletion and substitution mutagenesis of the cytoplasmic domain of g has identified a sequence-specific, six amino acid motif that directly interacts with m (peroulis et al., ) . during rsv infection m protein can initially be detected in the nucleus, but later in the infectious cycle it is found in inclusions within the cytoplasm (ghildyal et al., unpublished) . the n and p proteins of rsv were earlier shown to be necessary and sufficient for these inclusions to form, and the rsv m - and l proteins were also shown to be present in these inclusions (garcia et al., ) . these same investigators were unable to identify m protein in the inclusions (garcia et al., ) . using confocal immunofluorescent microscopy of infected and cotransfected cells the m protein was shown to be present in these inclusions . m protein does not localize to the inclusions unless m - is also present . the m and m - proteins not only co-localize by confocal microscopy but also interact in a protein overlay assay (ghildyal et al., unpublished) . taken together, these data suggest that, as with other single stranded negative-sense viruses (peeples, ; lenard, ) , the rsv m protein seems to play a crucial role in rsv assembly; bringing the nucleocapsid together with the envelope proteins by binding to the cytoplasmic domains of g and f and to the nucleocapsid proteins n and p together with or via m - . the interaction between m, m - and the nucleocapsid proteins might be more complex than outlined here, and might involve additional cellular or viral proteins. to better understand rsv assembly, the domains in the m protein that interact with g, f and m - will have to be defined. while influenza viruses readily develop resistance to older antivirals such as amantadine or rimantadine, resistance to neuraminidase inhibitors occurs much less frequently. nonetheless, resistant influenza a virus mutants can be isolated from patients treated with oseltamivir (treanor et al., ) . one of the resistant mutants carries an arginine to lysine mutation at position (r k) of the neuraminidase protein, causing a reduction in substrate binding and enzymatic activity, as well as resistance to oseltamivir (mckimm-breschkin, ) . the infectivity of influenza a viruses carrying the r k mutation was earlier found to be markedly reduced in mice and ferrets. transmission of an influenza a h n clinical isolate with a r k mutation was studied in ferrets in comparison to transmission of the parent wt h n virus that was isolated from the same patient. donor ferrets (four per group) were inoculated intranasally with the r k mutant or wt virus and housed with three naïve contact ferrets per donor ferret. the four donors inoculated with wt virus were infected and transmitted virus to each of the contacts. wild type virus replicated to between and pfu/ml nasal aspirate. only two of four donor ferrets inoculated with mutant virus became infected, and the level of replication of mutant virus was reduced - -fold compared with that of wt virus. however, both infected ferrets transmitted virus to contacts. one of them transmitted the r k virus to one of three contacts only, with virus detected at very low titer on day only. the other donor transmitted virus to all three contacts, and the transmitted virus replicated to titers greater than pfu/ml in the contact ferrets. sequence analysis showed that the donor virus was a mixed population of mutant and wt virus and that only wt virus was recovered from contacts. these data confirm the reduced infectivity of oseltamivir resistant r k mutants. effective transmission of the r k mutant virus in ferrets was not observed, suggesting that the transmission of oseltamivir-resistant virus from human to human will be unlikely, even during widespread use of na inhibitors in the treatment of influenza. respiratory virus infections in immunocompromised patients are characterized by persistence of viral infection, prolonged shedding of virus, a high rate of nosocomial acquisition, and a high frequency of pneumonia and death. similarly, respiratory virus infections in the elderly are responsible for a substantial amount of morbidity and mortality. detection of respiratory virus infections in these high-risk patients is important for several reasons. it enables the initiation of specific isolation procedures, initiation of specific antiviral therapy, cessation of unnecessary therapy, tests and procedures, and it can aid in identifying and preventing potential outbreaks. respiratory virus infections can be diagnosed using serology and culture techniques, as well as newer methods such as antigen-detection by enzyme-linked immunoassay (eia) or immunofluorescence (ifa), enzymatic detection by chemical reactions, or amplification of parts of the viral genome (pcr). serology is not useful in the acute phase of most illnesses and is of limited usefulness in immunocompromised patients, the elderly or those receiving blood products. recovery of virus in cell culture is still seen as the gold standard by many but the importance of obtaining a good specimen to ensure that the culture is not falsely negative is often overlooked. a good clinical specimen is the most critical factor in ensuring a correct diagnosis regardless of the diagnostic method used, although it is perhaps most important for culture. use of nasal wash to obtain a specimen for virus isolation is well-tolerated in cooperative adults and, compared to nasal swabs or throat swabs, increases the sensitivity of cell culture for virus detection. other factors critical to laboratory success are the use of appropriate transport media, temperature of transport/incubation and time until processing (atmar and englund, ) . this is particularly important in the case of rsv infections in immunocompromised or elderly patients, who often have a relatively low viral titer such as - pfu/ml, whereas children often have a titer greater than pfu/ml of nasal wash or bronchoalveolar lavage fluid (englund et al., ) . for rsv, parainfluenza and influenza virus there are a number of commercially available rapid detection kits. multiple simultaneous rt-pcr for detection of rsv, influenza a and b, and piv , and (hexaplex ® ) was tested in pediatric samples and yielded % sensitivity and % specificity as compared with culture (fan et al., ) , with only h processing time. in a separate study, pcr for respiratory viruses in adult patients with hematologic malignancies was found to be as sensitive as culture (van kraaj and van elden, ) . for influenza, several antigen detection kits ((directigen, fluoia and quick-vue) and one neuraminidase (zstatflu) assay are available to detect virus, with sensitivities ranging from to % and specificities ranging from to %. rapid diagnosis of influenza in pediatric patients leads to a decrease in the frequency and duration of antibiotic use and an increase in the frequency of antiviral therapy (noyola and demmler, ) . rapid diagnosis kits for the detection of rsv in children range in sensitivity from to % and in specificity from to %, with some test kits performing better than others (dominguez et al., ) . in immunocompromised adults, however, the sensitivity of antigen detection kits from nasal wash or throat swab sample was only %, while endotracheal or bronchoalveolar sampling increased sensitivity to or %, respectively, (englund et al., ) . several studies in recent years have highlighted the importance of upper respiratory tract infections in the exacerbation of asthma in children. freymuth and colleagues reported human rhinovirus (hrv) ( . %) and rsv ( . %) as the most frequent pathogens detected in patients with exacerbation of asthma, and noted that pcr increased detection rates . -and . -fold in hrv and rsv infections, respectively, over that of conventional assays (freymuth et al., ) . in a study of wheezing children between months and years of age, respiratory viruses were detected in %, with rsv in infants (detected in % of subjects) and hrv in older children ( %) as the predominant pathogens. both were strongly associated with wheezing (rakes et al., ) . in a community-based longitudinal study, respiratory viruses were detected in % of episodes of acute illness with reduced peak expiratory flow, % of episodes of wheezing, and in % of episodes of upper respiratory symptoms, cough, wheezing, and a fall in peak expiratory flow (johnston et al., ) . in these settings, rt-pcr provides a fast and sensitive method to detect rna viruses. real time quantitative pcr assays can be similar to conventional pcr in specificity and speed. however, real-time pcr can also quantify viral load (quantity of virus in respiratory secretions) during asthma exacerbations, and it is sometimes more sensitive than conventional pcr. real time taq-man quantitative pcr allows estimation of the input viral genome copy number by including a fluorescence reporter on one end and a quenching molecule on the other. the reporter does not fluoresce until the quencher has been cleaved off by the exonuclease activity of taq polymerase, permitting an estimate of the quantity of pcr product. fluorescence is being measured continuously every seven seconds, and quantification of the target is based on the number of pcr cycles it takes to produce detectable fluorescence. a retrospective study was conducted in asthmatic children - years old to compare viral loads in quiescent and exacerbation periods of asthma using the taqman technology. nasal aspirates had been collected earlier and records of peak flow measurement and clinical scoring were available. a quiescent period was defined as absence of clinical symptoms for two weeks or longer. real time quantitative pcr detected respiratory viruses (mostly rhinoviruses) in % of the children with an asthma exacerbation and in % of children in quiescence. viral load was higher in children with exacerbation of asthma than in those with quiescent asthma and higher viral loads also correlated with more severe clinical disease. although the study showed that real time quantitative pcr is more sensitive than nonnested conventional pcr and also allowed esti-mation of viral load, there is the caveat that the study had a retrospective design. modalities of immunity to acute viral respiratory infections are both specific and nonspecific, humoral and cell mediated. fever, interferon (ifn), tumor necrosis factor (tnf), natural killer cells and activated macrophages are non-specific modalities stimulated by infection that are capable of mediating antiviral effects. lung collectins such as sp-a, have also been implicated in the control of viral respiratory infections (ghildyal et al., ) . specific modalities are antibody in serum and secretions, lymphocyte proliferation responses with cytokine release, and cytotoxic lymphocytes (ctls). all modalities participate, to some degree, in containing an infection and promoting recovery either via inactivation of free virus or elimination of infected cells. thus, there is considerable redundancy in the mechanisms controlling the virus infection and promoting recovery. on the other hand, protection against infection is primarily conveyed by specific antibody. sufficient data is available to conclude that serum igg neutralizing antibody is the primary mediator of resistance to influenza virus infection, presumably because infection is initiated in the lower respiratory tract and igg antibody derived from serum is the dominant antibody isotype at that site (couch and kasel, ) . in contrast, iga antibody is the primary mediator of resistance to rhinovirus and coronavirus infection because evidence indicates these infections are initiated in the nasopharynx where iga is the dominant antibody isotype (cate et al., ) . since primary infections with influenza virus, rsv, or piv induce disease in both the upper and lower respiratory tract, both igg and iga antibodies are correlates of immunity to infection and disease (crowe, ) . although adenoviruses also cause upper and lower respiratory disease, only serum antibody has been shown to correlate with immunity. reinfection with homologous rsv, piv, rhinovirus and coronavirus can occur but is generally confined to the upper respiratory tract, presumably because igg antibody in protective quantities is more durable in serum (and lower respiratory secretions) than is iga antibody in nasopharyngeal secretions. for rsv, the risk of infection declined from % for naïve subjects to or % after one or two infections, respectively; the risk of lower respiratory disease in these groups declined from to to %, respectively, (glezen et al., ) . for a rhinovirus, resistance to reinfection of the nasopharynx correlated with the titer of iga antibody in secretions (cate et al., ) . rodent models of viral respiratory disease provided much of the early data on immune modalities conferring protection (crowe, ) . both cd + and cd + t cells can effect clearance of influenza a virus, rsv and piv in mice in the absence of the other cell type (lightman et al., ) . while cd + cytotoxic lymphocytes (ctl) mediate immunity through lysis of infected cells and expression of antiviral cytokines, cd + t cells exhibit limited direct antiviral activity but play a role in activating b cells and in inducing antiviral cytokine expression (epstein et al., ) . there is a general consensus that ctls contribute significantly to the resolution of primary infections with influenza, rsv and piv in rodents. while the correlation between antibody response and protection in humans was established decades ago, cellular immune responses were studied much later, and understanding of the development of these responses in infants and immunosenescence of them in the elderly is still incomplete. whether or not ctls in humans convey immunity to respiratory virus infection in the lower respiratory tract and whether they promote clearance of virus in the upper respiratory tract of humans remains to be elucidated. however, for rsv disease in humans, a correlation has been observed between the presence of rsvspecific ctls in year and absence of severe rsv disease in year (mbawuike, in press). morever, the level of influenza-specific ctls correlated inversely with the quantity of virus in nasal secretions after challenge of humans, and the ability of ctls to function at this site was demonstrated by a reduction in influenza virus titer in nasal turbinates of mice which were adoptively immunized with purified cd + ctls before intranasal challenge (mcmichael et al., mbawuike, personal communication). knowledge regarding viral-bacterial interactions in the respiratory tract goes back at least to the influenza pandemic, and interactions have been described for later influenza pandemics as well. epidemics and pandemics of influenza have been followed regularly by an increase in the incidence of bacterial pneumonia (schwarzmann et al., ; cartwright et al., ) . associations between rsv and haemophilus influenzae, bordetella pertussis, neisseria meningitidis and staphylococcus aureus infections have also been described (patel et al., ; jiang et al., ) . interactions between viral and bacterial diseases are fairly complex and the underlying mechanisms are only beginning to be elucidated. it is known that up-regulation of tnfa and il- increases adherence and uptake of pneumococci (cundell et al., ) , and that several cellular receptors for bacterial adherence are up-regulated by viral infections. s. pneumoniae and h. influenzae interact with paf receptors (swords et al., ) , and n. meningitidis interacts with cd and cd (raza et al., ) . not all interactions are regulated at the level of cell surface receptors. in the mouse influenza model, for instance, severe damage and desquamation of the respiratory epithelium enables access of s. pneumonia to the basal membrane and thereby increases the risk for invasive disease (plotkowski et al., ) . otitis media was traditionally thought to be a purely bacterial infection, with s. pneumoniae, h. influenzae and moraxella catarrhalis as the main pathogens. more recent studies, however, indicate that the majority of acute otitis media (aom) cases are a result of mixed bacterial and viral infection. heikkinen and colleagues reported an increase of rsv, parainfluenza virus, influenza or adenovirus infection in children with otitis media (heikkinen et al., ) . all respiratory viral infections of the nasopharynx are thought to predispose to aom but some viruses, e.g. rsv, are frequently found in the middle ear during aom. the mechanism underlying this respiratory tract infection-aom sequence involves eustachian tube obstruction, leading to negative middle ear pressure and inspissation of bacteria into the middle ear (giebink et al., ) . children with rsv, adenovirus or influenza virus infections have a % risk of developing aom within weeks of the onset of the respiratory tract infection (henderson et al., ) , and coinfection with bacteria and viruses also adversely influences the outcome of aom. if aom does not respond to antibiotic therapy within h, it is more likely to involve a viral infection (arola et al., ) . the effects of viral co-infection complicate the evaluation of clinical efficacy of anti-bacterial drugs in the treatment of aom (marchant et al., ) . with bacterial-viral co-infection in aom, there is also delayed clearance of bacteria from the middle ear during anti-bacterial therapy compared with disease attributable to bacterial infection alone (chonmaitree et al., ) . it is unclear whether the decrease in efficacy of antibiotics is due to impaired host responses (poor neutrophil function) or due to poor penetration of antibiotics into the middle ear. in a recent clinical trial heptavalent pneumococcal vaccine had % efficacy for prevention of bacteremia and % efficacy for prevention of aom caused by serotypes included in the vaccine. however, the frequency of aom caused by pneumococci not included in the vaccine increased (replacement phenomenon), so that the overall effect of the vaccine was reduced (eskola et al., ) . in contrast to the pneumococcal vaccine, viral vaccines seem very effective in preventing aom. belshe and colleagues reported considerable efficacy for an attenuated live influenza vaccine in preventing aom, and rsv prophylaxis with rsv antibodies was also associated with a marked decrease in otitis media (belshe et al., ; group, a ). the human adenoviruses consists of over known serotypes which have been divided into six subgroups (a-f) with distinctly different organ tropism. although other factors may influence infectivity and replication of these viruses, high affinity attachment of virions to host cell receptors represents a key determinant of tissue tropism. examples of this distinctly different organ tropism are a predominance of subgroup a adenoviruses (ad) such as ad in pneumonia in patients with primary immunodeficiencies; the preference of subgroup b viruses such as ad , and for the urinary tract in patients with kidney transplants; and the predominance of subgroup c viruses in hepatitis in liver transplant patients. receptor binding is mediated by the ad fiber protein, a homotrimeric molecule composed of an amino terminal region that anchors the fiber to the penton base capsid protein, an elongated central shaft domain (van raaij et al., ) , and the carboxy-terminal receptor binding knob. a high resolution structure of the ad fiber knob bound to its receptor, the coxsackie-adenovirus receptor (car), has recently been obtained by x-ray diffraction (bewley et al., ) , and amino acid residues directly involved in receptor binding were defined for several adenoviruses through mutagenesis studies (roelvink et al., ) . adenoviruses differ remarkably in the isoelectric point of the fiber protein knob domain, e.g. ph . for ad versus ph . for ad , suggesting that these fiber proteins cannot use the same receptor. besides car, heparin sulfate proteoglycans (dechecchi et al., ) and sialic acid (arnberg et al., ) mediate adenovirus attachment. since car is only expressed on the basolateral but not the luminal surface of epithelium, car can probably not be used to target adenoviral vectors in the therapy of cystic fibrosis. the important role that charge plays in virus-cell interactions can be deduced from the differential effect that removal of sialic acid from the cell surface by neuraminidase treatment has on adenovirus attachment. while ad p attachment is not affected by neuraminidase treatment, ad attachment is increased and ad attachment is decreased. the difference in ph optima may well be a determinant of tissue tropism. the major pathogens in adenoviral eye infections, ad , ad and ad , belonging to subgroup d, are very similar in their knob charge. chimerization of fiber proteins, whether evolved in nature or generated by mutagenesis, can cause a significant change in adenovirus pathogenesis. in , a new ad genotype (ad h) appeared in argentina, uruguay and chile, where it caused severe respiratory tract infections in children. analysis of the ad h fiber protein revealed that it was a chimera containing ad and ad sequences. in this new genotype and ad d also emerged in japan, where ad had been absent for years, and caused outbreaks of respiratory disease. the fiber protein, however, is not the only factor that determines adenovirus tropism. virus uptake is thought to also be mediated by an interaction between the penton base protein with integrin avß or avß . adenoviruses also differ in their ability to induce inflammatory responses. while ad is a potent inducer of interleukin (il- ), a hallmark cytokine of viral pneumonia, ad is not; this might explain why ad but not ad causes significant respiratory disease. in summary, it can be concluded that neither subgroup classification alone nor fiber protein knob charge alone determine adenovirus tissue tropism. key amino acids in the knob, as well as certain motifs of the penton base protein and possibly other adenovirus proteins interact in virus attachment and internalization. avian influenza viruses of the h n subtype were found to be transmitted directly from poultry to humans in in hong kong. these viruses were highly pathogenic in chickens and also caused severe clinical symptoms in humans, leading to the death of six of infected individuals. in order to obtain a better understanding of the pathogenesis, tropism and kinetics of replication of these viruses in primates, cynomolgus monkeys (macaca fascicularis) were infected with the highly pathogenic h n a/hong kong/ / isolate that was obtained from the index case. four monkeys were inoculated with . × tcid in a ml inoculum that was administered intratracheally, orally (tonsils) and onto the conjunctiva. two of the monkeys were euthanized on day and the remaining two on day post infection. the two monkeys that were euthanized on day , post infection developed a respiratory distress syndrome with high respiratory rate and fever on day . by day , one of the monkeys was lethargic and severely ill, with central cyanosis. the other monkey was also ill and developed fever. the virus replicated to a titer greater than tcid per g lung tissue on day but could not be isolated on day . macroscopic lung pathology was dominated by peribronchial consolidation and necrotic lesions. histopathologic examination of the tissues collected and days post infection revealed extensive pathologic changes in the respiratory tract characteristic of a viral necrotizing interstitial pneumonia. although rt-pcr for h n influenza virus was positive not only in the respiratory tract but in spleen, heart and also the cerebrum and cerebellum of one monkey, virus could only be demonstrated by immunoperoxidase staining in, and isolation from, the respiratory tract. the respiratory tract seems to be the major and probably the only target for the h n influenza virus. although cynomologous monkeys have been used earlier as a model for h n influenza disease (rimmelzwaan et al., ) , this is the first time a primate model for h n viruses has been described. influenza h n clinical symptoms observed in this study correlate well with what was seen in human disease caused by these highly virulent viruses, and was more severe than what is seen with h n viruses. therefore, cynomologous monkeys may provide a useful model for studying influenza h n pathogenesis and for developing h n vaccines. . . sb- , an orally acti e, selecti e p mitogen acti ated protein (map) kinase inhibitor impro es pulmonary functions in a murine influenza pneumonia model influenza infections are responsible for significant morbidity, especially in high-risk groups with underlying cardiopulmonary disease and in the elderly. the pathology results from a vigorous inflammatory response in the respiratory tract and damage to respiratory epithelial cells. in cells exposed to inflammatory cytokines, p mitogen activated kinase (map) activation leads to the upregulation of cytokines such as il- , il- and tnfa (ono and han, ) . a recent study examined the effect of sb- , a highly selective orally bioavailable inhibitor of p map kinase, on pulmonary function in mice infected with influenza a virus. mice were infected intranasally with influenza a/pr / and treated with sb- at different time points post infection. initiation of treatment on day , or post-infection resulted in a % (p b . ), % (pb . ) or % improvement in pulmonary capacity, respectively, compared with placebotreated control animals. no effect on virus clearance, survival, or antiviral immunity was observed. the efficacy of sb- in reducing pulmonary resistance and increasing blood oxygenation was similar to that of the neuraminidase inhibitor oseltamivir, the steroid dexamethasone, and the cox- inhibitor nimesulide. sb- was superior to non-specific nsaids indomethacin, naproxen, and ibuprofen. in mice and ferrets, sb- reduced airway neutrophilia, and treatment was well-tolerated without any adverse effects. these data suggest that p map kinase is involved in influenza-induced cell signaling and that inhibition of this enzyme might reduce the severity of pulmonary disease. fortunately, the number of viruses that cause respiratory disease severe enough to require hospitalization is limited. in children younger than five years, rsv (subgroup a and b viruses), the parainfluenza viruses (piv , - and - ), influenza a and b, and adenovirus types , , and are the major respiratory pathogens. influenza a and b remain, due to antigenic drift and shift, important agents in all age groups with very severe disease most commonly occurring in the elderly. rsv is the single most important cause of severe respiratory disease in infancy and childhood but it also causes significant morbidity in the elderly and in immunocompromised individuals. kim, chanock, brandt and parrott were the first to quantify the contribution of these viruses to the severe respiratory tract disease leading to hospitalization of infants and young children. rsv, piv , piv , piv , adenoviruses and influenza b caused , , , , , and %, respectively, of respiratory disease leading to hospitalization, while influenza a was responsible for % between and (h n era) and % between and (h n era) (brandt et al., ; parrott et al., ; kim et al., ; murphy et al., ) . what are the general principles underlying vaccine development for these viruses? first, the protective antigens of the virus and mediators of immunity to reinfection are largely known. it is generally accepted that neutralizing antibodies to surface glycoproteins (g and f of rsv, hn and f of piv, and ha and na of influenza) or to capsid proteins (hexon and fiber proteins) of adenoviruses are the major mediators of resistance to reinfection. second, serum and mucosal antibodies make independent contributions to immunity against reinfection. whereas serum antibody is needed to mediate immunity in the lower respiratory tract, mucosal antibody is needed to protect the upper respiratory tract (with the exception of adenovirus, where serum antibody alone can prevent uri). third, live virus vaccines are generally more immunogenic than nonliving virus vaccines in immunologically naïve subjects, but identifying a live vaccine virus that is both safe and sufficiently immunogenic can prove to be a difficult task. fourth, different age groups might need different vaccines. infants younger than four months of age make less antibody than older children due to immunosuppression by maternal antibodies and immaturity of the immune system, and only potent immunogens, e.g. live virus vaccines, will be successful in this population (murphy et al., ) . on the other hand, a live virus vaccine that is sufficiently attenuated for use in infants might be over-attenuated in the elderly (gonzalez et al., ) . fifth, viruses differ in their ability to cause disease in the presence of maternal serum antibody. while mucosally delivered rsv, piv and influenza are infectious in the presence of maternal antibody, piv , piv and adenovirus seem to be restricted in their replication in young infants by maternal antibodies. these five principles need to be considered when determining the optimal vaccination strategy (systemic versus mucosal, vectored antigen versus live attenuated virus) and the target age for vaccination. which vaccines are available? against influenza, the vaccine currently in use in the us is mostly a non-living, subvirion vaccine made from egg-grown purified virus disrupted with detergents. for practical purposes, the only relevant protein in the vaccine is the influenza hemagglutinin. the strains to be included in the vaccine for annual vaccination are selected by the public health service based on the antigenic profile of the currently circulating strains. subivirion or subunit vaccines plus adjuvant are also being developed (minutello et al., ; gluck et al., ) . live attenuated virus vaccines based on cold-adaptation of influenza a strain a/aa/ / -h n and influenza b strain b/aa/ / have been evaluated in phase iii clinical trials and should soon become available as a mucosal vaccine administered by nasal spray (maassab and bryant, ) . to date, an rsv vaccine has not been licensed. two non-living virus vaccines against rsv are being developed. one is a purified f protein subunit vaccine and the other consists of a g protein-specific peptide conjugated to the albumenbinding site of the streptococcus g protein (cano et al., ) . the purified f subunit vaccine is being evaluated for use in seropositive populations such as the elderly or high-risk older children (gonzalez et al., ) . used by itself, the f subunit vaccine has the disadvantage of not inducing a potent mucosal antibody response. also, this antigen induces a serum antibody response that is dominated by non-neutralizing antibodies (high ratio of titer of f protein-specific binding antibody to titer of neutralizing antibody). subgroup a and b live rsv vaccine candidates are being developed . one candidate vaccine bearing multiple attenuating mutations has been evaluated in - months old infants and was found to be attenuated and immunogenic but retained mild reactogenicity for the upper respiratory tract (wright et al., ) . it is likely that recombinant cdna technology will be used to develop or improve live attenuated rsv vaccines in the near future (whitehead et al., a) . for example, a subgroup b rsv vaccine candidate was developed by substituting the g and f glycoprotein genes of rsv b for the corresponding genes in an attenuated recombinant subgroup a virus (whitehead et al., b) , thereby rapidly generating a live attenuated subgroup b rsv vaccine candidate. to date, a piv vaccine has not been licensed. subunit and vectored vaccine candidates against piv have been developed and tested in animal models, but there are no reports of ongoing clinical trials. two live-attenuated piv vaccine candidates, a bovine piv (bpiv ) and a cold-adapted piv (cp ), have been evaluated in clinical trials, and both viruses were found to be safe and immunogenic in seronegative infants (karron et al., (karron et al., , . recombinant versions of these two viruses are also being evaluated as vaccines against piv and as vectors for the expression of foreign viral glycoproteins such as rsv g or measles ha (karron et al., ; durbin et al., ; schmidt et al., ) . the recombinant piv cp vaccine candidate is currently being tested in phase ii trials in seronegative infants. for adenoviruses, a live virus vaccine against serotypes and has been used successfully in military recruits from to . the vaccine made use of site-specific attenuation, i.e. a wt virus capable of inducing respiratory disease if administered to the respiratory tract was administered orally in an enteric coated capsule. the vaccine virus replicated only in the gastrointestinal tract and did not spread to the respiratory tract. the oral vaccine was found to be com-pletely attenuated and highly efficacious in inducing serum antibodies and protecting the upper and lower respiratory tract (couch et al., ; howell et al., ) . a similar tetravalent vaccine against serotypes , , and should be evaluated for use in young pediatric patients since these four viruses are responsible for over % of the adenovirus-related respiratory tract disease in this population. in virology, reverse genetics refers to the ability to rescue infectious virus from cdna. reverse genetic systems have been developed for all major viral respiratory pathogens, i.e. influenza a virus, paramyxoviruses, adenoviruses and most recently coronaviruses. this short review will only address the impact of reverse genetics on vaccine development for selected members of paramyxoviridae. for rsv, the generation of attenuated vaccine viruses started with conventional biological methods. first, wild-type rsv was passaged extensively at low temperature, and the resulting cold-passaged (cp)rsv was demonstrated to be attenuated in chimpanzees, as well as in seropositive adults and young children (friedewald et al., ). the cprsv virus was further modified by two rounds of chemical mutagenesis and biological selection for temperature-sensitivity (crowe et al., a) . a panel of resulting mutant viruses was evaluated in mice and chimpanzees and ranked according to increasing attenuation. several promising candidate vaccines then entered clinical trials. rsv cpts / , a cold-passaged virus with two additional attenuating point mutations, is the most promising vaccine candidate tested so far, but it still causes transient nasal stuffiness in infants (wright et al., ) . the development of a reverse genetics system for rsv provided a method for expediting development of further attenuated viruses. as a first step, the biologically derived mutant viruses were sequenced completely and their mutations were identified and evaluated by introduction, individually or in combination, into wt recombinant (r)rsv. the direct identification of attenuating mutations made it possible, for example, to improve the above mentioned cpts / mutation by incorporating one or more additional attenuating mutations from other vaccine candidates. furthermore, in many cases, the amino acid substitutions can be engineered to involve two nucleotide substitutions relative to wt, which should confer increased genetic stability. similarly, a reverse genetics system for piv was established and used to analyze all mutations in the biologically derived cold passaged piv cp (skiadopoulos et al., ) . however, the capabilities of reverse genetics go far beyond the analysis of existing biological mutants. a whole new set of methods for generating attenuated paramyxoviruses has been developed as described above. gene knock-out mutations, i.e. the deletion of non-essential genes, such as the rsv sh, ns , ns , m - or g gene, generated a number of vaccine candidates with different levels of attenuation. as another example, in some cases an attenuating point mutation was found to involve a residue conserved, for example, between the l proteins of rsv and piv or between the c proteins of sendai virus and piv . transfer of the attenuating mutation between the heterologous paramyxoviruses resulted in transfer of the attenuation phenotype. host range restriction could be employed as a basis of attenuation by creating antigenic chimeric viruses that use an animal virus as a platform for the expression of human rsv or piv glycoprotein protective antigen genes schmidt et al., ) . in this manner, an hpiv vaccine candidate was generated by replacing the bpiv f and hn glycoprotein genes in rbpiv with their hpiv counterparts (schmidt et al., ) . thus, the menu of available attenuating mutations for rsv and hpiv include numerous attenuating point mutations (which in some cases specify temperature sensitivity and in other cases do not), gene deletions, and host range restriction elements. the attenuating mutations or elements from each menu can be combined as desired to develop appropriately attenuated vaccine candidates. reverse genetics also expedites vaccine development because attenuated platforms can be used to make vaccines against additional viruses. for ex-ample, attenuated derivatives of rsv a (subgroup a) can be used to generate rsv subgroup b vaccine candidates by replacing the a f and g glycoprotein genes with their counterparts from the b strain of subgroup b (whitehead et al., b) . as another example, a live-attenuated vaccine candidate was developed for hpiv by replacing the f and hn coding regions of hpiv with their hpiv counterparts. the resulting virus thus bears the antigenic determinants of hpiv in a wt hpiv backbone, which can then be attenuated by the introduction of mutations from the hpiv attenuation menu. reverse genetics can potentially help us to make vaccines that are satisfactorily attenuated but more immunogenic than wt virus. protective antigen genes can be moved closer to the genomic promoter and codon usage can be optimized for translation in mammalian hosts. cytokines and/or chemokines can be co-expressed from additional genes (bukreyev et al., ) , and reactogenicity can potentially be reduced, for example by ablating the secreted form of rsv g, which might be a decoy for antibody and might also perturb the t helper lymphocyte response. vaccine specificity can be broadened by adding additional genes to existing vaccine viruses, e.g. the measles hemagglutinin (ha) gene can be expressed from an additional orf in rhpiv , generating a vaccine candidate that protects against both hpiv and measles (durbin et al., ) . this use of recombinant piv as vaccine and as a vector has several advantages over existing vector systems. it reduces the number of viruses to be administrated to infants and enables intranasal administration and thereby mitigates neutralization and immune suppression by maternal antibodies. the eradication of measles could be facilitated by a vaccine that does not include an infectious measles virus, which might cause prolonged infection in immunocompromised hosts. in summary, vaccines can be optimized through reverse genetics in a number of ways, such as the creation of novel combinations of mutations, the fine-tuning of the attenuation phenotype, the provision of a common attenuated platforms, and the generation of multivalent vaccines. . . predicti e alue of animal models in rsv accine de elopment rsv was initially isolated from chimpanzees that developed a common cold-like disease in . since then chimpanzees and other animal models for rsv disease have been studied extensively. principally, these higher primates have been used for three different purposes: the development of live virus vaccines, the recovery of rsv antibodies, and lastly to evaluate formalininactivated and subunit vaccines, and to study enhanced disease following vaccination with formalin-inactivated vaccine. a number of animal models are used to study rsv disease. among the apes, chimpanzees have been used most extensively for several reasons. they are permissive for rsv, their core body temperature is similar to that of humans, and symptomatic scoring of uri is possible (rhinorrhea score). the permissiveness of chimpanzees makes it possible to perform quantitative virologic studies in the upper and lower respiratory tract over the time course of an acute rsv infection. also, the restriction of rsv replication in the respiratory tract of chimpanzees correlates well with attenuation in seronegative human infants. in addition, human immunologic reagents can be used in chimpanzee studies. however, only young, carefully raised chimpanzees are rsv seronegative. other primates used include old world monkeys such as african greens, rhesus, cynomologous or bonnet monkeys and new world monkeys such as marmosets, tamarins and owl monkeys. although all these monkeys are semi-permissive for rsv, their core body temperature is higher than that of humans so that the level of attenuation of temperature-sensitive rsv mutants might be overestimated. although primate studies provide essential data for vaccine development, their use can be difficult. transport and care of primates, as well as sample collection are difficult and potentially dangerous, and they require well-trained personnel. primate research centers must provide an environment similar to that of a child-care facility and, therefore, maintenance costs are very high. lambs and calves are also used to study rsv infection, mostly because ovine and bovine rsv causes significant disease in their natural host. also, these viruses are important economically. cotton rats, mice and other rodents are used as small animal models. mice have the advantage of having a body temperature similar to that of humans and immunological reagents are readily available. also, gene knockout mice, transgenic mice and various inbred strains are available to study pathogenesis. the mouse model is not without difficulties, however. mice acquire a large portion of their maternal antibodies by suckling, and, therefore, they are not an optimal model for study of immunosuppression by maternal antibodies. peak rsv titers vary -fold among different mouse strains, and subgroup b rsv is poorly infectious and immunogenic in mice. immune mechanisms may also vary among different mice strains. rodent models can be used to study quantitative virology, immunology and airway pathophysiology (a model of wheezing), and weight loss can be used as a surrogate marker for rsv disease. whichever animal model one chooses to study respiratory syncytial virus, it is essential to clearly define whether the ultimate goal of the study is to prepare for clinical vaccine trials or to understand basic mechanisms of disease. several cold-passaged (cp) live attenuated rsv candidate vaccines have been evaluated in clinical trials. most of these candidate vaccines were derived by further attenuating the original cprsv through chemical mutagenesis and selection of ts mutants. the first two vaccines evaluated in the cpts lineage -cpts- / and cpts- / , were either insufficiently attenuated or were transmitted amongst seronegative children (karron et al., ) . a study of a more attenuated vaccine candidate, cpts- / , has recently been completed (wright et al., ) . this virus initially was evaluated in chimpanzees where it replicated to a peak titer of only . pfu/ml in the upper respiratory tract. due to this restriction in the respiratory tract, it was subsequently evaluated in clinical studies. cpts- / did not replicate in adults or older children. in seronegative children - months of age, however, cpts- / replicated to a peak titer of pfu/ml in the upper respiratory tract and induced an antibody response against rsv f and g glycoproteins. the virus was well tolerated in this age group, and the frequency of upper or lower respiratory tract disease, otitis media or fever was not different from that of the placebo group. based on these data cpts- / was selected to be the first rsv vaccine to be administered to - -month-old infants, which represent the primary target group for vaccination. in this youngest age group, of vaccine recipients developed a clinical syndrome characterized by nasal congestion that occurred most typically between days and and lasted for approximately h. since young infants are obligate nose breathers, this interfered with feeding and caused fussiness and difficulty in falling asleep. most vaccine recipients shed approximately pfu of rsv per ml nasal wash. age of the infant or level of maternal antibodies against rsv did nor affect virus shedding, indicating that virus replication in the nasopharynx was independent of these variables. in these young infants, neutralizing antibody responses and igg elisa responses to rsv f or g were rarely detected, most likely because these responses were masked by the presence of maternally derived antibody. these young infants did, however, develop serum and mucosal iga responses preferentially to the rsv g glycoprotein and detection of serum iga correlated with protection from re-infection with a second dose of vaccine. although cpts- / is not an acceptable vaccine in one to two month-old infants, several lessons were learned from the study of this vaccine candidate. first, seronegative infants are a much more susceptible host to rsv than chimpanzees. this means that for further attenuated vaccine candidates, the chimpanzee model of rsv infection will not be very useful. second, only viruses that do not replicate in adults and older children are attenuated enough for vaccination of infants. and third, reverse genetics is needed to further attenuate the existing biological rsv vaccine candidates. two of these recombinant rsv vaccine candidates are currently being evaluated in clinical trials. cpts- / dsh was generated by deleting the sh gene from the recombinant cpts- / virus, and cpts- / / dsh was engineered to contain an additional ( ) mutation in the polymerase protein. preliminary data suggest that both recombinant viruses are well tolerated in - -month-old children and are not associated with lower respiratory tract illness. whereas cpts- / dsh replicates as well as its parent biological virus without the sh gene deletion, the cpts- / / dsh mutant appears to be much more restricted in its replication in the nasopharynx. the more restricted cpts- / dsh induced both neutralizing and elisa igg antibodies in this age group. however, since this virus replicates, as well as cpts- / , it is likely not to be suitable for vaccination of one to two month old infants. cpts- / / ?sh is currently being evaluated in one to two month old infants. in summary, although cpts- / is close to an ideal vaccine candidate, further modifications of the virus through reverse genetics, such as addition of the mutation, are likely to be needed to generate an rsv vaccine that is immunogenic yet attenuated enough to be given to young infants. antibody preparations that neutralize free virus have been used as passive immunoprophylaxis to prevent a number of viral diseases, including hepatitis a and b, varicella and respiratory syncytial virus (rsv) disease. the efficacy of antibody preparations in preventing rsv disease was established first in cotton rat and chimpanzee models, and later in extended clinical trials. therapy of rsv infections with rsv antibodies, however, have so far failed to prove efficacious (malley et al., ; van woensel and kimpen, ) . the most important mechanism of action of antibody preparations is probably neutralization of free virus. this can occur via aggregation of free virus by bivalent or multivalent antibody, via receptor blockade, via antibody-complement lysis, or via fusion inhibition. receptor blockade is thought to result from steric inhibition of receptor binding rather than binding of antibody directly to the receptor-binding site itself. even if receptor binding does occur, virus infectivity can be neutralized through fusion inhibition. most rsv neutralizing antibodies are thought to use this mechanism of action. although complete antibody is most effective in neutralizing free virus, antibody binding fragments (fabs) alone can also neutralize. the minimal unit necessary to ablate infectivity is probably a peptide corresponding to one loop of the complementarity-determining region (cdr ). although neutralizing and non-neutralizing antibodies against infectious virus are often distinguished, it is not clear whether there really are antibodies that bind glycoproteins in virions without neutralizing the virion (sakurai et al., ) . many so-called non-neutralizing antibodies bind purified (conformationally relaxed) rsv f glycoprotein but not the (conformationally correct) f protein on infected cells. different immunoglobulin subclasses neutralize virus with varying efficacy, and mouse and human igg subclasses also bind complement with varying efficacy. the replication of sendai virus (murine piv ) and influenza a virus can be inhibited intracellularly by iga and similar effects of iga on rsv replication may occur. a last but essential determinant of neutralizing activity is defined by the amount of antibody available to neutralize virus. to prevent rsv disease in the lower respiratory tract, a serum neutralizing antibody titer of approximately : is required (groothuis et al., ; top et al., ) . upper respiratory tract infections, however, can only be prevented with serum antibody titers as high as : - : . such titers can only be achieved in experimental settings in small rodent models. there is no single monoclonal antibody directed against rsv g protein that neutralizes completely on its own but cooperative neutralization occurs when multiple mabs are used. effective treatment for rsv illness is very limited. corticosteroids, bronchodilators, ribavirin, and, more recently, rsv mabs have been used but none of these therapeutic interventions are accepted as reproducibly efficacious. it seems that neither antivirals nor anti-inflammatory agents alone improve the outcome of rsv disease. a single dose of topically administered human fabs has been shown to clear free virus from the respiratory tract of rodents, but their effect is shortlived since infected cells release newly synthesized infectious virus within a day or two (crowe et al. b ). whereas peak virus titers in humans usually occur around day , most patients with clinical rsv disease likely present no earlier than day . at this point, virus load is already in decline and it may be too late for antivirals, and antiinflammatory drugs such as map kinase inhibitors may be more effective in reducing cell injury. are there immunological consequences of passive immunoprophylaxis? in chimpanzees therapy with rsv antibodies suppresses the primary antibody response to rsv. this effect is mostly antigen-specific but may also have a non-specific aspect mediated by the fc portion of the antibody. the secondary antibody response to rsv in chimpanzees is enhanced by prior antibody therapy. however, this effect was not observed in mice or in clinical studies. t cell responses do not seem to be as affected by antibody therapy as humoral responses, and t cells might fill in for absent primary antibody responses. whether this is a desirable effect or not, remains uncertain. without a safe and effective rsv vaccine available, monthly infusion of rsv-igiv (respigam ® ) was the first effective measure to prevent rsv-induced lower respiratory tract infection (lri) and hospitalization in infants. the evaluation of rsv-ivig in cotton rats correctly predicted the serum concentrations necessary to protect against rsv-induced lri (prince et al., a) . protective concentrations in children were achieved by monthly infusions of mg/kg of rsv-ivig. in order to increase the potency of a rsv antibody preparation, and in order to be able to replace intravenous with intramuscular administration, a humanized monoclonal anti-rsv fusion protein antibody was developed. mab , a mouse monoclonal antibody developed in the laboratory of infectious diseases at nih against the rsv f protein antigenic site a, one of two antigenic sites that are conserved amongst different rsv strains, was selected for humanization based on in vitro and in vivo studies, and the complementarity determining regions (cdr) were transferred from the mouse antibody to a human igg. this humanized igg should have pharmacokinetics similar to human igg and permit repeated administration at monthly intervals. the chosen mab, medi- , had no crossreactivity with adult or neonatal tissue, broadly neutralized rsv subgroup a and subgroup b isolates at concentrations of ng/ml, and proved to be - times more active on a weight basis than rsv-ivig in the cotton rat model (johnson et al., ) . adult volunteers tolerated doses of the humanized antibody medi- from to mg/kg well, and only some volunteers developed a low-titer, transient anti-idiotypic antibody response. medi- had a half-life of days, as is expected for igg, and serum concentration after iv and im administration were comparable except for the initial bioavailability. medi- was safe and well tolerated in phase i/ii studies in high-risk children, and had no specific immunogenicity in and of itself. monthly dosage of mg/kg maintained serum concentrations greater than mg/ml, and a single intravenous dose of mg/kg reduced rsv titers in tracheal secretions of intubated children with rsv infection (malley et al., ) . the impact-rsv study that led to fda approval of medi- , also called palivizumab, was a : randomized, double-blinded, placebo-controlled phase iii multicenter study conducted at sites in the us, canada and the uk. study subjects received five doses of medi- or placebo at intervals of days and were followed for a total of days. the primary endpoint of this study, overall rsv-related hospitalization, was significantly reduced (pb . ) from . % for placebo recipients (n= ) to . % for medi- recipients (n= ). for premature infants with chronic lung disease, the incidence of rsv hospitalization was reduced from . to . % (p= . ), and for premature infants without chronic lung disease, it was reduced from . to . % (pb . ). total rsv hospitalization days, total days with increased oxygen requirement, total days with severe lri, rate of icu admission and total days in icu were secondary endpoints that occurred less frequently in the medi- treated group (p b . ). thus, administration of mg/kg medi- by im injection was found to be safe, well-tolerated, and to lead to a % reduction of rsv hospitalization in high-risk children (group, b) . this conclusion was confirmed in an outcome survey conducted in nine centers in and , in which high-risk children received mg/kg palivizumab. although direct comparison to the impact-rsv study is not possible, rsv hospitalization rates were low and similar to those observed in the impact-rsv study: . % (vs. . % in the impact-rsv study) of premature infants without cld and . % (vs. . %) of children with cld were hospitalized in the - rsv season (sorrentino and powers, ). an alternative strategy to protect neonates and young infants against severe rsv disease is vaccination of pregnant women. transfer of high concentrations of maternal rsv-specific igg to the fetus is expected to protect the infant against severe disease (glezen et al., ) . the safety and immunogenicity of respiratory syncytial virus purified fusion protein- (pfp- wyeth lederle vaccine and pediatrics, pearl river, ny) were recently evaluated for use in pregnancy. thirtyfive pregnant women were randomized at a ratio : to receive rsv pfp- or saline placebo at - weeks of gestation and were followed until the time of delivery. infants were followed during their first year of life and their first rsv season. rsv pfp- was safe and well tolerated by pregnant women, and there were no systemic reactions or serious adverse events associated with vaccine administration. all infants were born healthy, and there were no differences in the frequencies and outcomes of neonatal events between the groups. during the first rsv season, there was no increase in the frequency or severity of respiratory tract illnesses in infants of vaccine recipients. there is ample material from which to draw lessons relevant to needed preparations for pandemic influenza. but, to date, the response, and specifically preparations for dealing with a serious pandemic of influenza remain more in the realm of academic reflection than meaningful action. although much time has been devoted to the development of a national response plan, we do not seem to be much better equipped to deal with a new pandemic of influenza than we were in the spring of , when the h n strain of influenza a virus emerged. at that time, we faced a burgeoning epidemic, whose virulence and propensity for spread were as yet unknown. a program of surveillance and field epidemiology to better define the epidemic had to be developed and, at the same time, preparations were needed for distribution and use of a new influenza vaccine, if it arrived in time. it did not. it appears today that not much progress has been made over the past years, and still no real sense of urgency in dealing with the essentials of the problem can be felt. although much has been written about the epidemic, it is still perceived by most as an interesting but questionably relevant tale of death and disease during an earlier, pre-antibiotic era of medicine. many doubt that an epidemic of this severity would be possible today. but, here are a few of the facts. first, it is important to recognize that, for the us, it dwarfed all other outbreaks of the th century. at that time, more than million died worldwide and in the united states, there were more than registered deaths. from various studies, it is thought that, overall, perhaps % of the population became ill, of whom about % died. medical services were overwhelmed but, other than supportive care, there was little that curative medicine could offer. the deaths were so numerous that burials were greatly delayed because of the lack of morticians and grave diggers. pictures from the time provide at least a pale illumination of that catastrophic period. the effect of the disease was anything but uniform. surveys revealed morbidity rates ranging from % to more than % in different parts of the country. some remote and rural areas escaped the disease entirely but in some areas, it was remarkably lethal. western samoa, then a new zealand protectorate, registered deaths in a population of . this represented % of the entire population. curiously, american samoa, only miles away but with a quarantine in effect, was one of the few political entities to escape the epidemic entirely. what was surprising and unique about the epidemic was that more than half of all deaths were in persons between and years of age -an age bracket in which death is a relatively uncommon phenomenon. pregnant women and those with cardiac problems were at highest risk but most, who died, were otherwise in good health. a substantial number in this age group died of a fulminant, rapidly progressive pneumonia marked by severe cyanosis with death occurring within a matter of - days, in brief, what seemed to be almost certainly, a primary influenza pneumonia that would have benefited little from antibiotics, had they been available. today, a better outcome might be foreseen with better ventilatory support in intensive care units; with the administration of antiviral agents; and with the administration of antibiotics for the treatment of secondary pulmonary infections. but under epidemic circumstances, the bulk of cases in a new pandemic would occur over a period of only - weeks and only a fraction of patients could be accommodated in suitable hospital settings, let alone appropriate intensive care units. what quantity of antiviral drugs is available for emergency use? where do we obtain the added quantities of the standard antibiotics that today, are produced on a just-in-time basis -when, during a period when clinical facilities are not stressed, we are regularly experiencing antibiotic shortages on a regular, rotating basis. a cogent question is whether under epidemic circumstances, our modern health care system could provide a standard of clinical care that would be better than it was in . but there is still another dimension to the problem. do we appreciate that a 'pandemic', so characteristic of the emergence of a new influenza strain, means exactly that -a worldwide epidemic. many countries -developed and developing, have made no preparations and will inevitably turn to those with resources to help them in dealing with a catastrophe -a catastrophe that poses an international security threat. the rapid production and administration of large volumes of vaccine effective against the emergent new strain has been a basic building block of the strategy for dealing with pandemic influenza and, understandably so, given the limitations of curative medicine and the capacity of clinical services. the first real test of this strategy came during the epidemic. the first notice of a major outbreak occurred in mid april; specimens were received in the us on may; and field testing of new lots of the vaccine began in july. not a bad record. it was foreseen that million doses would be required. with good fortune in adapting the virus to grow at reasonably high titer on egg membrane and provided that sufficient fertilized eggs could be obtained, it was expected that a production target of february could be met. even this was a problematical date given that the seasonal peak of epidemic influenza usually occurs between the end of december and the end of february. still, it was believed that substantial numbers would be able to be protected. however, was not a typical year, and such, one must note, was the case when the epidemic strain first appeared. widespread epidemics began occurring in mid to late september, two months before they were anticipated. more than half of all counties reported epidemics by mid-october and by the end of october, the peak incidence had past, long before any substantial quantity of vaccine was available. given these experiences, could we expect another new strain to behave differently today? over the past years, extensive studies have been conducted in the search for a satisfactory live attenuated vaccine and for various approaches in production which would permit new antigenic variants to be produced rapidly and in quantity in tissue cell culture. however, today we are still producing influenza vaccine in the allantoic cavity of hens' eggs, as we were in ; procuring adequate supplies of fertile eggs in a timely manner remains a serious problem; and difficulties in adapting new strains to eggs remain. were we able to solve the vaccine production problem for our own country, this would not be the end to the practical quandary of dealing with the pandemic. few countries have influenza vaccine production capability and the international implications of the us having all or much of the vaccine supply are profound. following the swine influenza outbreak at fort dix, new jersey, the us had embarked on a program to rapidly produce an appropriate vaccine. vaccine production capability in europe and other countries was so marginal that a world health organization committee could only recommend that the fort dix situation be carefully monitored -a strategy of wait and hope. but as those at the who meeting commented in corridor conversations, they would not wish to face the ethical and political dilemma the united states would face were there a pandemic and the us was the only nation with a vaccine. in recent meetings with national and local hospital authorities, current capabilities of the medical system in us to deal with sudden surges in demand such as might follow release of a biological weapon were explored. such a release would result in an epidemic that would stress the system not unlike the way it would be stressed by a pandemic of severe influenza. from these meetings, it was evident that the elasticity of the nation's bed supply has been significantly reduced as drives for financial efficiency and the increasing use of out-patient procedures have sharply reduced the numbers of beds in all hospitals. meanwhile, managed care-driven market pressures and federal government reimbursement reductions have driven large numbers of hospitals into operating deficits. many of the municipal hospitals, once a primary source of care for the less prosperous and uninsured have been privatized. meanwhile, the hospitals are experiencing severe labor shortages, especially for nursing and technical personnel. few have either the resources or motivation to prepare to respond to the challenges posed by mass casualties due to any cause. reserves of antibiotics, as noted earlier, are marginal to nil; the public health infrastructure needed to deal with epidemic disease is grossly understaffed, underpaid and under trained; mechanisms for the development and implementation of community-wide plans are largely unexplored. it is not unreasonable to suggest that we are today less well-prepared to deal with an epidemic of influenza than we were years ago in most parameters that one can identify. what might a new strain of influenza mean in terms of numbers of cases and deaths. estimates of past pandemics suggest that perhaps % of the population were affected in a first wave. with rising proportions of the population in urban areas, the greater and more rapid mixing of populations through travel, a figure of not less than % would seem more reasonable. thus, in a city of . million, one might expect . million cases of influenza. it is doubtful that either antibiotics or antiviral agents would be of much help given the number of cases and the dearth of reserve supplies. the number of deaths would vary greatly depending on the strain. in hong kong, a recent new strain, h n , resulted in death among six of persons infected. it did not spread readily, however. it is estimated that the strain killed % of those who became ill, while the case-fatality rate was about one-tenth as large or . %. thus, in a city of million persons, one might anticipate between and deaths over a period of - weeks. medical care would consist largely of supportive therapy given the numbers of those ill. public health measures, likewise, would consist of little more than reassurance given the likelihood that vaccine supplies would not be available and that stocks of antiviral drugs would be too small to be of significance. many would argue for the closing of schools, churches and other places of public gathering but experiences suggests that such actions produce little benefit. the wearing of masks was once a favored intervention but this, too, has been discredited. in brief, without vaccine, there would be little that could be done of practical benefit except to reassure the community that the epidemic would someday pass and to accept the criticism of the public and political leadership for failure to make reasonable preparations. at least four lessons can be derived from past experience. first, the threat of pandemic influenza caused by an especially virulent strain is a continuing threat that has to be taken seriously. second, adequate supplies of an effective vaccine, available in a timely manner, are absolutely critical to a preventive effort. solving this problem should command top priority for research and development funding. third, special plans, programs and funding are needed within the health care system to permit development of an adequate community-wide response to the occurrence of mass casualties whatever the cause. lastly, additional research in influenza is needed to better understand its pathogenesis and epidemiology with the expectation that better preventative measures might eventuate. pandemics are the most dramatic presentation of influenza a virus and they cause considerable excess mortality as a result of pneumonia and exacerbation of cardiopulmonary or other chronic diseases. the epidemiologic success of influenza a is in large part due to antigenic variation that takes place in the two surface glycoproteins of the virus, i.e. its hemagglutinin (ha) and neuraminidase (na) proteins. to date, ha and na subtypes have been defined and are used to classify influenza a viruses. antigenic variation occurs either gradually through accumulation of point mutations (antigenic drift) or more abruptly though introduction of a new ha gene into virus circulating in the human population, be it through reassortment of animal and human viruses or through a change in host-specificity of an animal virus (antigenic shift). a 'pandemic virus' can be defined as a virus with a new ha with or without a novel na gene, acquired through antigenic shift, that spreads readily from person-to-person in a population that is highly susceptible to infection. the th century saw three pandemics caused by new ha subtypes. influenza a h n caused the 'spanish flu' in , subtype h n ('asian flu') was the causative agent of the pandemic and the h n subtype ('hong kong flu') caused a pandemic in . the excess mortality for the , and pandemics in the us alone can be estimated to have been , , and deaths, respectively. if one applies mathematical modeling to estimate the effects of a future pandemic, the us alone can expect between and excess deaths in the next pandemic (meltzer et al., ) . what are the viruses with pandemic potential? in , individuals in hong kong were infected with h n influenza, an avian subtype earlier not known to infect humans (subbarao et al., ) . of the patients - years of age, six died. molecular analysis established that all eight genes of the h n virus were of avian origin and that reassortment with human viruses had not occurred (subbarao et al., ) . the reported infections were most likely acquired from poultry; human-to-human spread, however, was a rare event. sequence analysis of the hong kong h n virus suggests that it is a reassortant made up from two or three different avian parent viruses: ha from a goose h n virus (xu et al., ) , na from a teal h n virus (hoffmann et al., ) , and internal genes from a quail h n (guan et al., ) or a teal h n virus (hoffmann et al., ) . although this virus did not spread readily from person to person, deep concern was caused by the fact that this was the first known avian influenza a virus that caused disease in humans. until this outbreak it was thought that the receptor specificity of avian ha proteins limited their infectivity to avian species. this notion, however, needs to be revised. in march , h n influenza a viruses were isolated from two children with febrile upper respiratory tract illness (peiris et al., ) . this virus, again, was an avian virus that was able to infect humans without passage through an intermediate host (lin et al., ) . what are our options for vaccination against potentially pandemic viruses? in the case of the h n viruses, there are four options for generating an effective influenza vaccine. a conventional inactivated vaccine can theoretically be generated by reassortment of internal genes from influenza a/pr/ / with h n glycoprotein genes. there are, however, problems with incompatibility between certain gene segments. secondly, a cold adapted live attenuated vaccine approach could be taken. the influenza a/ann arbor/ / coldadapted strain has been used to generate an h n vaccine in which the ha cleavage site was modified (li et al., a) . this virus could also be used to produce an inactivated vaccine. thirdly, a surrogate virus that is nonpathogenic but antigenically related (takada et al., ) could be sought. one of the closest antigenically related viruses found to date was of the h n subtype, but this virus was not highly immunogenic in humans. fourthly, purified protein could be used but again limited immunogenicity may be a problem. the development of vaccines against potential pandemic viruses poses a number of challenges. to begin with, most of the work has to be conducted in biosafety level + laboratories. there is a large array of avian viruses that could potentially become pandemic viruses and those with a genotype that confers transmissibility have to be identified. h n , h n and h n viruses that were the genetic precursors of the h n and h n viruses that caused human infections in hong kong (guan et al., ; xu et al., ; hoffmann et al., ) and that continue to circulate among birds should certainly be given priority in vaccine development. an adequate strategy for vaccine development has to be selected, and safety testing must be expedited in mice, ferrets, chickens, and eventually humans. use of recently described plasmid based reverse genetics systems may lessen the technical challenges faced in generating vaccine candidates. assays to evaluate immunity have to be optimized (hemagglutination inhibition assays do not perform well with avian ha proteins, yet neutralization assays are time consuming and technically more difficult), and serologic and molecular diagnostic reagents must be made available. collaboration between veterinary and public health authorities must be maintained and new technologies such as plasmid based reverse genetics systems must be used. last but not least, alternative substrates for vaccine production and new adjuvants are urgently needed. although influenza epidemics occur every winter, we have only had three pandemics this past century - , and . while annual influenza epidemics generally cause an excess mortality of - people in the us, it is only the pandemics that remain in memory. of the three pandemics that occurred in the twentieth century, the pandemic was the most devastating and claimed the most victims. although there is wide spread public interest in the pandemic, the event is mostly remembered as an interesting event in the distant past. whether due to denial or not, few feel that an influenza pandemic is a current threat. most journalists see their role not only as educators and advocators but also as entertainers. the interest of the readership has to be captured, and it can only be maintained if their curiosity is satisfied. while the aids pandemic and its effect on people's lives found a wide audience through most of the early s, the late s were characterized by fatigue and a reduced interest in the pandemic (although it was still on the increase). as a result, editors were much less willing to include related stories. reportage on influenza pandemics suffers a similar fate: the disease itself is old, and the last pandemic occurred too long ago for most to remember. the mere warning of a coming pandemic is almost perceived as an unfulfilled promise if the event does not occur shortly after the article is published. breast cancer, in contrast, is one of a few examples, where interest in a disease process can be maintained over an extended period of time. the risk of suffering from breast cancer is felt much more acutely, it seems, and there is a sense that proactive behavior will lead to an improved outcome. whether the mass media will take on a role in changing the public's attitude toward influenza as a threat is very much in doubt as long as journalists themselves perceive the next pandemic as an event as anonymous and inevitable as an earthquake. rsv infection is initiated by g glycoprotein attachment to cell surface receptors and followed by virus-cell fusion that is mediated by the f protein. the cleavage-activated rsv f protein is thought to interact with the target cell membrane through its n-terminal fusion peptide, which is released from a shielded position within the f homotrimer through a major conformational change. insertion of the f n-terminus into the cell membrane destabilizes the cell membrane and induces lipid mixing that is followed by mixing of contents, thereby enabling the viral nucleocapsid to enter the cytoplasm. the rsv f protein was recently found to interact with a small gtpase called rhoa through a domain that lies just carboxyterminal to the f fusion peptide (pastey et al., ) . rhoa is a member of the ras superfamily that is expressed intracellularly in all cell types. it induces bundling of actin filaments into stress fibers, focal adhesion plaque formation, cell-to-cell adhesion and organization of integral membrane proteins; it has additional roles in cell morphology and motility as well as cell cycle transition from g to s. a series of overlapping rhoa peptides from the interaction domain were evaluated in rsv plaque reduction neutralization assays. rhoa peptide - has an ic of about mg/ml against an inoculum of pfu. neutralizing activity is also seen against parainfluenza virus type (piv ), but not against a variety of other paramyxo, orthmyxo, corona and filoviruses (pastey et al., ) . intranasal administration of mg of peptide - prior to intranasal infection of mice with rsv reduces rsv replication more than -fold and also diminishes weight-loss. studies with recombinant rsv expressing green fluorescent protein indicated that rhoa peptide - inhibited rsv replication at a very early stage of infection. subsequently, it was shown that fusion of cells transfected with rsv f, g and sh was inhibited by this peptide, suggesting that it exerted an effect on rsv replication at the level of membrane fusion or entry. since rhoa is an intracellular molecule, one would have to hypothesize that the interaction of the n-terminal heptad repeat and fusion peptide with the target membrane causes enough membrane disruption to allow an interaction between rhoa and rsv f. alternatively, rsv f may not interact with rhoa during the entry process, and the rhoa-derived peptides may simply disrupt the f structure or function to render the virus noninfectious. the question of whether rhoa and rsv f interact during a natural infection is not yet resolved. rhoa can be found in the cytoplasm bound to gdp and a guanine nucleotide dissociation inhibitor, and, upon gtp exchange and isoprenylation, rhoa associates with the cell membrane. if the proposed model of rhoa-rsv f interaction at the membrane is correct, then inhibition of isoprenylation should inhibit rsv infection. indeed, inhibition of isoprenylation through hmg coa reductase inhibitors, such as lovastatin, inhibits rsv infection of hep- cells at an ic of mm and reduces peak rsv titers in lungs of mice greater than -fold with oral gavage of mg per day (gower, t.l., graham, b.s., submitted). the antiviral effect of lovastatin is also seen with piv in vitro. although it has not been formally proven that rsv f interacts with rhoa in the infected cell membrane, rhoa signaling activity is triggered in rsv-infected cells (gower, t.l. et al., submitted) . while rhoa signaling is not required for rsv replication, syncytium formation is diminished when rhoa signaling activity is inhibited. in addition, rhoa signaling may play a role in other aspects of rsv pathogenesis. rhoa kinase inhibitors reduce the transcription of il- and il- mrna in rsv infected cells (cytokines abundant in the nasal secretions of rsv-infected patients), and obviate airway hyperresponsiveness, one of the cardinal symptoms of rsv disease (hashimoto, k. et al., submitted). the balb/c mouse model was used to explore whether mycophenolic acid (mpa), an inhibitor of de novo purine synthesis in t and b lymphocytes, could improve the outcome of rsv disease, which was monitored by clinical symptoms such as ruffling of fur, increased respiratory rate and weight loss. oral administration of mg/kg mmf (the prodrug of mpa) daily from day to post-infection reduced weight loss on day post-infection from % in untreated animals to % in mpa treated mice (p b . ). a reduction of weight loss ( . %) was also observed when initiation of treatment was delayed until day post-infection. virus titers in lungs of mmf treated mice were similar to those of untreated controls but histological changes were reduced. on day post-infection, ifng levels were elevated . -fold in the treatment group while il , il and il levels were unchanged, indicating a shift toward a t helper cell type response that is thought to correlate with improved rsv disease outcome. these data suggest that inflammatory responses contribute to rsv disease in mice and that an immunomodulatory approach to the treatment of human rs virus disease is worth further consideration. using a cell-based assay to identify compounds which are able to inhibit fusion of hela cells infected with rsv, more than analogues of a lead compound were synthesized and one compound (termed r ) was selected for further evaluation. the in vitro % inhibitory concentration (ic ) of this benzimidazole derivative (mw ) was pm, and thus its potency exceeds that of ribavirin almost -fold (ic = mm). r exhibits in vitro antiviral activity against human rsv (subgroup a and b) and bovine rsv but not against pneumovirus of mice or other paramyxoviruses. rsv-induced cytopathic effect was reduced by r at . nm, and concentration of nm reduced rsv titers -fold in multi-cycle growth curves. time of addition studies indicated that both virus-cell fusion and cell-cell fusion were inhibited by this compound. selection of resistant viruses in vitro yielded two mutants with single point mutations in the f-protein: one upstream of the second heptad repeat motif and another within it (s l and d n). rsv titers, determined by quantitative rt-pcr, were reduced -fold in bronchoalveolar lavage fluid and in lung tissue of cotton rats treated once by inhalation with r prior to rsv infection. rfi- , a compound that was derived by chemical optimization of the earlier described antiviral cl- , is a small molecule antiviral drug that selectively inhibits rsv (wyde et al., ) . the molecule is water-soluble and not orally bioavailable, but it proves to be efficacious when administered intranasally or by inhalation. the in vitro ic varies between and ng/ml for laboratory strains or clinical isolates of rsv subgroup a and b. viral specificity and the large therapeutic window of rfi- (\ fold) indicate that the antiviral activity of the compound is not due to adverse effects on normal cells. addition of rfi- to cell culture prior to adsorption reduces rsv yield -fold at h post infection (wyde et al., ) . temperature shift experiments suggest that the rsv f protein is the target for rfi- , and this observation is confirmed by the inhibitory effect that rfi- has on rsv b cp- / b , a viable rsv mutant in which both the g and sh open reading frames are deleted. if rfi- is added to cell culture h post infection, it inhibits syncytium formation indicating that fusion inhibition occurs both in the early and late phase of the infectious cycle. rfi- -resistant viruses can be selected, albeit much less easily than amantadine resistant variants. resistance to rfi- is acquired by point mutations solely in the f protein, mostly upstream of the second heptad repeat motif, but not by mutations in the g or sh protein. when administered prophylactically by the intranasal route, rfi- inhibits rsv replication in vivo, with mice and cotton rats exhibiting a - -fold reduction in rsv replication on day post infection. in african green monkeys, rfi- reduces peak rsv titers not only when administered prophylactically but also when therapy is initiated at h post infection and continued for a total of days. a nebulized form of rfi- has been shown to be active in monkeys. the preclinical profile of this drug supports its development for treatment and prophylaxis of rsv disease in pediatric, adult and geriatric populations. phase clinical trials confirmed that rfi- is a potent antiviral, and that the safety profile of this drug is encouraging. . . de elopment of picona irus c protease inhibitors ag is a potent, irreversible inhibitor of human rhinovirus (hrv) c protease, the enzyme that is responsible for the cleavage of the viral polyprotein into its functional protein subunits (matthews et al., ) . ag was discovered by protein structure based rational drug design, and the compound exhibited activity against a large set of different hrvs. the % effective concentration (ec ) ranges between and mm. other piconaviruses such as coxsackievirus a or b , enterovirus , and echovirus are also sensitive to the compound. in a placebo controlled challenge study in which adult volunteers were infected with hrv and treated with ag ( mg per dose intranasally, times per day), ag reduced mean viral titers, as well as mucus weight, respiratory symptom score and total symptom score significantly. in this study viral titers were determined by culture and also by quantitative pcr (taqman) to exclude ex-vivo effects of ag on the reduction of virus titers. a phase ii clinical trial with subjects was conducted to determine the efficacy of ag in naturally acquired piconavirus infections. patients selected for the study had to present within h of onset of symptoms and had to suffer from at least two mild or one moderate cold symptom. a five step symptom score was used to record severity of rhinorrhea, cough, sneezing, sore throat, chills, headache, and malaise. the treatment group was stratified into two or four daily doses of mg ag per dose intranasally, and the mean respiratory symptom score for days - was chosen as the primary endpoint. only % of all enrolled patients were infected by picor-naviruses. no significant difference in respiratory or total mean symptom score for days - was detected. however, the drug was well tolerated and safe. the lack of efficacy in this particular study might have been due to a lower than expected frequency of picornavirus infections. in a retrospective analysis, stratification for start of therapy within h detected a trend to fewer respiratory symptoms and fewer total symptoms. there was a trend to earlier onset of relief, but the difference between treatment and placebo group again did not reach significance. . . pleconaril (p) treatment reduces the incidence of acute otitis media (om) in children with iral respiratory illness: a pilot study pleconaril, an orally bioavailable picornavirus c protease inhibitor, has in vitro antiviral activity against % of all rhinoviruses. to evaluate the efficacy of pleconaril in preventing acute otitis media (aom) in an outpatient population, a double-blind, placebo-controlled pilot study was conducted in children with viral respiratory illness. eighty-seven children with a median age of years and a history of - episodes of aom were randomized to pleconaril treatment ( mg/kg or . mg/kg) or placebo thrice daily for days following presentation with upper respiratory symptoms. picornavirus rna was detected by rt-pcr in nasal samples in % of patients at baseline. the overall incidence of physician-diagnosed aom during the day follow-up were . % ( / ), . % ( / ), and % ( / ) in the placebo, low dose and high dose groups, respectively. of the children developing aom, tested positive for picornavirus rna. pleconaril treatment reduced the frequency of nasal symptoms by % in the high dose ( . mg/kg, p= . ) and % in the low dose ( . mg/kg, p= . ) group. it reduced frequency of systemic symptoms by % in the . mg/kg group (p= . ) and % in the . mg/kg group (p= . ). pleconaril was well tolerated, and adverse events did not differ from the control group. these results encourage further expanded trials of pleconaril in children with viral respiratory infections. . . oral oseltami ir reduces influenza-related complications in all age groups influenza illness is associated with the development of secondary complications, often requiring antibiotic treatment or even hospitalization. insurance data indicate that up to % of influenza related hospital admissions occur in - year old individuals, often without recognized underlying disorders. influenza complications affect all age groups but the type of complication differs among them. while acute otitis media has been reported in over % of children with influenza, lower respiratory tract infections (lrti) such as bronchitis and less often pneumonia, are the most common complication in adults. in recent years six phase iii clinical trials were completed; two of them in pediatric populations, three in healthy adults and one in the elderly. the neuraminidase inhibitor oseltamivir (o) is already approved for the treatment of influenza in adults, and it was recently approved for treatment in children aged year and older by the fda. in six phase iii trials, most of the patients were enrolled within h of onset of symptoms, and oseltamivir was administered at mg/kg twice daily for children and mg twice daily for adults. subjects with febrile ( \ . °c) influenza were randomized to a day regimen of oseltamivir or placebo, and an important endpoint of all studies was the effect of oral oseltamivir (o) on the incidence of influenza-related complications requiring antibiotics. one thousand and twenty nine children between the age of and (mean age . years) were enrolled in the pediatric trials. in the adult and elderly populations, patients with a mean age of years and elderly patients with a mean age of years were enrolled. in the pediatric, adult and elderly trials , and %, respectively, tested positive for influenza a or b by culture or fourfold increase in hai titers. physician-diagnosed secondary complications (bronchitis, pneumonia, lrti, sinusitis or otitis media) requiring antibiotics were assessed in patients with confirmed influenza infection. oseltamivir reduced the rate of complications compared with placebo by % in children (p= / , o = / ; p = . ), by % in adults (p = / , o = / ; p = . ), and by % in the elderly (p= / , o= / ; p= . ). in the pediatric population, aom was the most common complication, followed by bronchitis, pneumonia and sinusitis. the frequency of aom was reduced from % in the placebo group to % in the oseltamivir group, a significant % reduction. in adults and the elderly, bronchitis and sinusitis were the most common complications of influenza. the frequency of bronchitis in the elderly was reduced from to %. thus, oral oseltamivir reduced the incidence of secondary complications and associated antibiotic use in all age groups. when all age groups were combined, antibiotic use for any indication was also significantly reduced. twelve patients in the placebo group versus four patients in the oseltamivir group required hospitalization for probable or possible influenza-related complications, suggesting a possible effect on hospitalization. . . neuraminidase inhibitors: clinical update . . . oseltami ir oseltamivir was recently approved in the us for prophylaxis of influenza in adults and adolescents over years of age after three successful phase three trials. one was conducted in healthy adults, one in a family setting with an infectious index case, and another evaluated seasonal prophylaxis in the elderly. the efficacy of oseltamivir in all three trials ranged between and %. an application for the approval of oseltamivir for treatment of influenza in children over one year of age was recently approved by the fda. in otherwise healthy children of - years, treatment with oseltamivir reduced the median time to freedom from illness by . days (p= b . ) and also reduced the relative risk of otitis media (aom) by %. in - year old asthmatic children, oseltamivir treatment, started within h of first symptoms, decreased the median illness duration by . days (p = . ), and airway function was significantly improved ( s forced expiratory volume on day improved by . % compared with baseline values among oseltamivir recipients and . % among placebo recipients (p= . )). the incidence of development of drug resistant virus under therapy was evaluated in more than subjects. none of subjects sampled on day of treatment harbored resistant virus. on day , however, / subjects ( %) shed virus with a resistant phenotype. in children, the frequency of viral resistance was % ( / ). all of these subjects recovered normally despite this incidental finding. so far, resistant mutants have been less infectious than wild type virus; no evidence of transmission has been detected in an experimental infection model in ferrets. the impact study evaluated the benefit of early intervention in reducing the total time with symptoms starting from the time of first onset of fever. this reflects the total burden of illness from a patient's point of view. a total of patients, - years of age were enrolled and treated within h of the first onset of symptoms. treatment was initiated within h in % of those recruited. using an accelerated time to failure model, earlier intervention was shown to strongly correlate with shorter duration of illness. the total duration of illness could be halved if treatment was started within h of the onset of symptoms compared with intervention at h. in phase iii trials, zanamivir reduced the duration of illness in adults over years of age and in children between and years of age by . days ( % ci= - . days). in high risk patients ( subjects, % with asthma and % with chronic obstructive pulmonary disease) duration of illness amongst all influenza positive subjects was reduced by . days. duration of illness was reduced in both vaccinated ( . day) and nonvaccinated ( day) subjects. during therapy, pulmonary function improved slightly between days and . zanamivir was well tolerated, and there were no differences in adverse effects between treatment and placebo groups. with subjects studied in clinical trials, resistant mutants were not isolated by sampling on days and . efficacy for treatment of influenza b was assessed in subjects and found to be similar to that of influenza a. a prophylaxis study in a family setting showed % protective efficacy. finally, in a nursing home study, zanamivir exhibited a % in-creased efficacy in preventing influenza disease compared with rimantadine. rwj- , an orally available once daily neuraminidase inhibitor that is active against influenza a and b in vitro and in animals, was evaluated in phase ii trials of experimentally infected healthy adults. therapy was started approximately h post infection with influenza a/texas h n or influenza b/yamagata. viral load over time was the primary endpoint, and nasal wash titers were determined every h on day - and daily thereafter. in the influenza a study, subjects were enrolled into four treatment ( to mg per day) and one placebo arm. high dose treatment ( mg once daily or mg twice daily) reduced viral load by - %, whereas or mg per day led to a reduction by %. shedding was reduced by - days in the high dose groups, and the incidence of fever declined from % in placebo controls to less than % in all treatment groups combined. to evaluate efficacy against influenza b, subjects were enrolled into two treatments ( or mg per day) and one placebo group. a % reduction of viral load was shown for the high dose treatment ( mg per day). no evidence for the emergence of resistant virus was detected, and oral rwj was well tolerated without excess gastrointestinal side effects. world-wide, acute respiratory infections (ari) are responsible for more than million deaths per year in children under five years of age. viral respiratory infections contribute significantly to this burden of disease, not only in children but also in adults. although there is a myriad of viral respiratory pathogens, the number of virus families that cause significant burden of disease is limited. in children, the paramyxoviridae are the most important family of viruses, with respiratory syncytial virus (rsv) and the parainfluenza viruses (piv) causing most disease, followed by adeno-and influenza viruses. in adults, influenza a virus with its constantly changing antigenic composition surpasses other viral pathogens, but the impact of rsv and rhinoviruses are being increasingly appreciated. the epidemiology of respiratory virus infections very much depends on host and environmental factors. while adenovirus infections are a major concern in military training camps and in immunocompromised patients, they are not as important in the general adult population. the immunocompromised are not only at increased risk for viral respiratory disease but diagnosis and management are much more difficult in this group. newer diagnostic methods based on antigen detection or viral genome amplification make rapid diagnosis possible and thereby affect clinical management of viral respiratory disease. vaccines are still the most powerful tool in preventing viral respiratory disease but for many important pathogens there is still no vaccine available. the inactivated influenza vaccine, with its antigenic composition changing annually, is still the mainstay of influenza prevention. coldadapted live attenuated influenza virus vaccines are on the horizon but are not yet approved by the fda. adenovirus vaccines have been used successfully in the military for many years, and the recent resurgence of large outbreaks of respiratory disease after discontinuation of vaccination emphasizes the importance of maintaining a preventive strategy. live virus vaccine candidates against rsv and piv, derived biologically or by reverse genetics, are currently in clinical trials, and there is hope that safe and efficient vaccines will become available within this decade. new insight into virus-host and virus-bacteria interactions in viral respiratory illness may help us understand the complexity of these disease entities and allows a re-evaluation of management options. diseases earlier thought of as purely bacterial, such as otitis media, might be more effectively prevented by viral vaccines than by bacterial vaccines. populations at high risk for complications resulting from respiratory viral infections are now better defined and a more targeted prophylaxis is possible, be it passive prophylaxis against rsv disease with monoclonal antibody preparations or active prophylaxis with influenza-or adenovirus vaccines. in the management of influenza virus disease, much has changed for the better. neuraminidase inhibitors (ni) are now established as an effective intervention to decrease the severity and to shorten the duration of illness when therapy is initiated within days of the onset of symptoms. zanamivir and oseltamivir are now licensed in the us and elsewhere for adults and adolescents twelve years of age and older, and a license for use in children over one year of age has recently been obtained for oseltamivir. both drugs are safe and well tolerated, and development of resistance occurs much less frequently than with older antivirals such as amantadine or rimantadine. rhinovirus infections, the cause of most colds, may now be amenable to treatment with the experimental c protease inhibitors pleconaril (given orally) and ag (given intranasally), and initial studies indicate that pleconaril treatment of colds in children might reduce their risk of developing otitis media. antivirals effective against rsv and piv are still in an early phase of development, but inhibitors of the viral fusion protein look promising. despite all progress, severe deficiencies remain in the preventative efforts. our level of preparedness for an influenza pandemic does not seem much different from that years ago. streamlining of hospital infrastructure and just-in-time production of antibiotics and antivirals limit our ability to respond effectively should a pandemic strike. although potential pandemic influenza viruses have been defined and novel techniques such as reverse genetics are being employed in vaccine development, the implementation of pandemic preparedness is still a daunting task. adenovirus type uses sialic acid as a cellular receptor variable morbidity of respiratory syncytial virus infection in patients with underlying lung disease: a review of the picnic rsv database. pediatric investigators collaborative network on infections in canada respiratory virus infection as a cause of prolonged symptoms in acute otitis media viral infections of humans cold adaptation of 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in tropical and developing countries community respiratory virus infections in immunocompromised patients with cancer recombinant respiratory syncytial virus (rsv) bearing a deletion of either the ns or sh gene is attenuated in chimpanzees replacement of the f and g proteins of respiratory syncytial virus (rsv) subgroup a with those of subgroup b generates chimeric live attenuated rsv subgroup b vaccine candidates immune responses to adenoviruses: viral evasion mechanisms and their implications for the clinic evaluation of a live, cold-passaged, temperature-sensitive, respiratory syncytial virus vaccine candidate in infancy cl exhibits marked and unusual antiviral activity against respiratory syncytial virus in tissue culture and in cotton rats genetic characterization of the pathogenic influenza a/goose/guangdong/ / (h n ) virus: similarity of its hemagglutinin gene to those of h n viruses from the outbreaks in hong kong key: cord- -cug fnf authors: hollingshead, melinda g.; westbrook, louise; ross, martha j.; bailey, jean; qualls, k.jeanine; allen, lois b. title: an elisa system for evaluating antiretroviral activity against rauscher murine leukemia virus date: - - journal: antiviral res doi: . / - ( ) -i sha: doc_id: cord_uid: cug fnf a system for evaluating the activity of antiviral agents against rauscher murine leukemia virus (r-mulv) has been developed using an enzyme linked immunosorbent assay technique. the activity of various antiviral compounds demonstrated in this assay system has been compared to their activity in the uv-xc plaque reduction assay, which has been used historically for evaluating anti-r-mulv compounds. the assay is based upon detection of r-mulv encoded p protein production in virus infected murine cells. the assay reagents are readily available and the assay system is amenable to automated data collection systems. cytotoxicity evaluations are conducted in parallel to the rauscher mulv elisa assay in order to assess drug-induced reductions in cell viability. cytotoxicity evaluations are important to interpretation of the elisa results since reductions in cell viability reduce viral protein production which would indicate an antiviral drug effect. this system is less sensitive than the classical uv-xc plaque reduction assay; however, it does offer an alternative to the time-consuming and labor-intensive plaque assay. rauscher murine leukemia virus (r-mulv) (rauscher, ) infection of mice has been used as a model for evaluating anticancer (chirigos et al., a; chirigos et al., b) and, more recently, anti-hiv compounds (ruprecht et al., ; ruprecht et al., ) . generally, in vitro assays are conducted prior to in vivo evaluations to assess the potential of the drugs to inhibit virusinduced disease. historically, these evaluations have been performed using the uv-xc plaque reduction assay (shannon et al., ) . this assay system is labor-intensive and requires experienced personnel to quantitate plaque formation. several workers have reported alternate assay systems for measuring mulv. generally these assays have relied upon immunofluorescence (decl ve et al., ) , in situ hybridization (rein et al., ) , purothionin a cytotoxicity (tahara et al., ) or radioimmunoassay (scolnick et al., ) for virus quantitations. more recently, focus forming assays have been described which use immunofluorescent (sitbon et al., ) or immunoperoxidase (nexo, ) labelling to aid in detection of mulvinduced foci. while these systems improve the visual counting efficiency of the assays, they are not amenable to automated data collection and analysis like the newer elisa methodologies have proven to be. thus, we have established an assay system which allows automated data collection. this system is similar in concept to assay systems described for quantitating antiviral activity or antiviral antibody responses to herpesviruses (rabalais et al., ) , dengue (fiqueiredo and shope, ) , bovine viral diarrhea (justewicz et al., ) and coronaviruses (smith and winograd, ) . for these assays, virus-infected monolayer cultures serve as the antigen source in an indirect elisa assay. multiwell ( ) culture plates were seeded with sc- cells (hartley and rowe, ) in eagle's minimum essential medium (emem) supplemented with % heat-inactivated fetal bovine serum (fbs), u penicillin/ml and #g streptomycin/ml [tissue culture medium]. following overnight incubation ( % co , °c) for cell adherence, the medium was removed and deae-dextran solution ( #g/ml in pbs) was added to each well. following incubation ( min) and deae-dextran removal, the wells were rinsed with phosphatebuffered saline (pbs). serial dilutions of the test compounds, prepared in tissue culture medium at . x the desired final concentration, were added to each of replicate wells ( #l/well). six virus control wells received # of tissue culture medium and cell control wells received # of tissue culture medium. each of the virus-challenged wells (test and virus controls)then received # of viral inoculum prepared at x the desired final concentration ( pfu/well). after days of incubation ( °c, % co ), the medium was aspirated from the plates and the wells were rinsed with pbs. the cells were fixed with cold ethanol:acetone ( : ) at °c for min. the fixative was decanted and the plates were allowed to air-dry overnight at room temperature (rt). for the elisa assay, the wells were blocked ( h, rt) with % fbs prepared in pbs. the primary antibody, consisting of a : dilution of goat anti-rauscher p antibody (nci/bcb repository), was added to each well and incubated for h at °c. the wells were rinsed times with wash solution ( . % tween- ® in pbs) and the second antibody, affinity-purified horseradish peroxidase-labelled anti-goat igg (tago immunologicals, burlingame, ca) was added. following incubation for h at °c, the wells were rinsed times with wash solution and , '-azino-di-[ -ethylbenzthiazoline sulfonate ( )] (abts/h ) substrate (kirkegaard and perry laboratories, inc., gaithersburg, md) was added. the optical density at nm (od o ) was determined spectrophotometrically on a microplate reader following incubation at °c for h. the average od was calculated for each set of replicates and for the virus and cell controls. the percent reduction in p protein was calculated as: the percent reduction was plotted and the % inhibitory concentration (ic ) was determined. the uv-xc plaque reduction assay has been described previously (shannon et al., ) . briefly, -well tissue culture plates were seeded with sc- cells in tissue culture medium. after overnight incubation ( °c, % co ), the medium was aspirated from the wells and ml of the drug solution ( . x ) and . ml of the virus inoculum were added to replicate ( ) wells. controls included cell controls (tissue culture medium only, no drug or virus) and virus controls (virus in medium, no drug). on day post-inoculation, the medium was decanted and the cultures were irradiated with ultraviolet light. each well then received xc cells (svoboda, ) . on day post-irradiation, the cultures were rinsed with pbs, fixed with °/ buffered formalin and stained with . o/ crystal violet. after air-drying, the plaques were enumerated with the aid of a dissection microscope. antiviral activity was expressed as the reduction in the mean number of plaques counted in the drug-treated, virus-infected cultures compared with the mean number of plaques in the untreated, virus-infected control cultures. the drug concentration which reduced the mean number of plaques by % (ic ) was calculated using regression analysis for semi-log curve fitting. cytotoxicity was determined in parallel by the mtt assay performed in well plates. cytotoxicity determinations were performed by a -( , -dimethylthiazol- yl)- , -diphenyltetrazolium bromide (mtt) dye reduction assay (mosmann, ) . for this, sc- cell monolayers were prepared in multiwell ( ) tissue culture plates in parallel to the elisa or uv-xc plates. for cytotoxicity assays performed in parallel to the elisa assay, the cells were pretreated with deae-dextran. the test compounds were added to each well at the desired final concentration in a volume of # . controls included cells incubated in tissue culture medium alone (cell controls) and color controls which contain no cells but received all the remaining reagents. the plates were incubated in parallel with the elisa ( days) or uv-xc ( days) plates. following incubation, each well received # of a mg/ml solution of mtt prepared in tissue culture medium. after a -h incubation at °c, /~ of a % sodium dodecyl sulfate (sds): . n hc solution were added to each well and the plates were incubated overnight at °c. the optical density at nm was determined spectrophotometrically on a microplate reader. the average od was calculated for each set of replicates and the percent of cell control was calculated for each drug dilution as: (mean drug treated od --color control od ) (mean cell control od -medium control od ) x these data were plotted in conjunction with the percent reduction in virus production. the minimum toxic concentration was defined as that concentration which reduced the cell viability to less than % of the cell control. a series of compounds have been assayed in the elisa and uv-xc systems. the icso and mtc results of compounds are presented in table . the results obtained with the two systems correlate well although the drug concentrations required to suppress p protein production, as measured in the elisa system, are generally higher than those required to suppress uv-xc plaque formation. one compound, diarylsulfone , demonstrated antiviral activity in the uv-xc plaque reduction assay; however, no activity was detected by the p protein reduction assay (elisa). this compound was inactive in a virus yield reduction assay (data not shown) thus, the elisa assay may be more indicative of the activity for some compounds. in the case of this compound, the activity detected by the uv-xc plaque reduction assay was believed to result from cytotoxicity to the xc cells used as an overlay. graphic presentation of the uv-xc and elisa data for ' ' dideoxythymidine (azt) is shown in fig. . reductions in virus production, either as percent plaque reduction or as percent reduction in p protein, are graphed on the left y-axis as the virus-infected cells. cytotoxicity, presented as the percent of cell control, is graphed on the right y-axis as the toxicity control cells. data presented in this form is readily interpreted visually without the need for extensive analysis of the numerical values. we have assayed the positive control drug, azt, multiple times. the average ids for azt in these assays ( ) was . #m with a median ics of . ~m and a standard deviation of . . we feel that this indicates that the results are reproducible from assay to assay. the primary difference between the-elisa assay and the uv-xc plaque reduction assay is in the drug concentrations required to suppress the virus production endpoint. the uv-xc plaque reduction assay measures the capacity of an antiviral compound to reduce production of infectious virus. when rat xc cells are placed into contact with mulv-infected cells, syncytia are formed. when these xc cells are overlaid onto mulv-infected sc- cell monolayers, they fail to infiltrate the sc- cell sheet in the mulv-infected areas resulting in lightly stained areas (plaques). mulv infected sc- cells that are killed by exposure to uv light are still capable of inducing syncytia. the xc cell overlay grows on the dying sc- cells as a uniform monolayer except where mulv infection is present. in these areas, there are 'holes' in the xc cell monolayer which contain multiple giant cells or focal masses of giant cells. production of these syncytia is presumably a function of the gp glycoprotein as typified by other retroviruses (i.e. gp in hiv). in contrast, the elisa system measures the capacity of a compound to reduce production of the virus protein, p , which forms part of the virion core similar to the p protein in hiv. this protein is encoded by the viral nucleic acid. reductions in p protein production will occur if a compound interferes with translation of the viral nucleic acid. thus, compounds which interfere with virus attachment, penetration, integration, transcription and translation will reduce p protein production. a compound which interferes with late stages in the virus production cycle (i.e. virus budding) would not necessarily reduce p protein levels. the only cases in which the elisa assay does not give results compatible with those found in the uv-xc assay are: ( ) assays of compounds which have toxic effects on the xc cells and ( ) assays of compounds which are active late in the virus replication cycle or which act by interfering with the infectivity of the progeny virus so that p protein is produced but the progeny virus is unable to infect new target cells• while the system described here is not as sensitive to antiviral drugs as the uv-xc plaque reduction assay, it does offer an alternative method for antiviral drug evaluations. we have found compounds that produce toxicity to the xc cell overlay which could be evaluated in this test system. also, smaller quantities of compound are required for the elisa since it is performed in well plates rather than the -well plates used in the uv-xc plaque reduction assay. we have used this system to evaluate analogs of known positive compounds for selecting the optimal drug for in vivo testing. further, the system can be modified at various steps including ( ) increasing the virus replication phase (i.e., -day incubation), ( ) altering the virus inoculum dose, ( ) altering the elisa reagent concentrations and incubation times and ( ) collecting supernatants for yield reduction assays. thus, this system can be adjusted to meet the desired goals. studies with the murine leukemogenic rauscher virus. i. chemotherapy studies with in vivo and in vitro assay systems studies with the murine leukemogenic rauscher virus. ii. chemotherapy of virus-induced lymphoid leukemia an improved murine leukemia virus immunofluorescence assay an enzyme immunoassay for dengue antibody using infected cultured mosquito cells as antigen clonal cell lines from a feral mouse embryo which lack hostrange restrictions for murine leukemia viruses bovine viral diarrhea virusinfected mdbk monolayer as antigen in enzyme-linked immunosorbent assay (elisa) for the measurement of antibodies in bovine sera rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays a plaque assay for murine leukemia virus using enzyme-coupled antibodies a virus-induced disease of mice characterized by erythrocytopoiesis and lymphoid leukemia in situ hybridization: general infectivity assay for retroviruses rapid herpes simplex virus susceptibility testing using an enzyme-linked immunosorbent assay performed in situ on fixed virus-infected monolayers suppression of retroviral propagation and disease by suramin in murine systems suppression of mouse viraemia and retroviral disease by '-azido- '-deoxythymidine radioimmunoassay of mammalian type c viral proteins i. species-specific reactions ofmurine and feline viruses inhibition of gross leukemia virus-induced plaque formation in xc cells by -deazauridine use of a focal immunofluorescence assay on live cells for quantitation of retroviruses: distinction of host range classes in virus mixtures and biological cloning of dual-tropic murine leukemia viruses two enzyme immunoassays for the detection of antibody to rodent coronaviruses a new method for titration of murine leukemia virus using purothionin a this work was supported by contract n -cm - from the national cancer institute. key: cord- -qdmunb l authors: zhao, yongkun; wang, chong; qiu, boning; li, chufang; wang, hualei; jin, hongli; gai, weiwei; zheng, xuexing; wang, tiecheng; sun, weiyang; yan, feihu; gao, yuwei; wang, qian; yan, jinghua; chen, ling; perlman, stanley; zhong, nanshan; zhao, jincun; yang, songtao; xia, xianzhu title: passive immunotherapy for middle east respiratory syndrome coronavirus infection with equine immunoglobulin or immunoglobulin fragments in a mouse model date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: qdmunb l middle east respiratory syndrome (mers) is a highly lethal pulmonary infection caused by a coronavirus (cov), mers-cov. with the continuing spread of mers-cov, prophylactic and therapeutic treatments are urgently needed. in this study, we prepared purified equine f(ab’)( ) from horses immunized with mers-cov virus-like particles (vlps) expressing mers-cov s, m and e proteins. both igg and f(ab’)( ) efficiently neutralized mers-cov replication in tissue culture. passive transfer of equine immune antibodies significantly reduced virus titers and accelerated virus clearance from the lungs of mers-cov infected mice. our data show that horses immunized with mers-cov vlps can serve as a primary source of protective f(ab’)( ) for potential use in the prophylactic or therapeutic treatment of exposed or infected patients. middle east respiratory syndrome (mers)-cov is an emerging pathogen that causes severe pneumonia in humans in the arabian peninsula and in travelers from this region (assiri et al., a; zaki et al., b; zumla et al., ) . human-to-human spread has been documented (assiri et al., b) . while infections of immunocompetent patients generally present with only mild symptoms, the elderly and patients with pre-existing illnesses such as diabetes or renal failure are likely to develop more severe disease (assiri et al., a) . as of september , , cases with deaths ( . % mortality) had been reported to the world health organization, although the actual number of infections could be much larger since mild, asymptomatic or undiagnosed cases are likely to be common (drosten et al., ) . as yet there are neither licensed vaccines nor any prophylactic or therapeutic treatments effective against mers-cov. given the ability of coronaviruses to rapidly adapt to new hosts, a major public health concern is that mers-cov will further adapt to replication in humans, triggering a global severe acute respiratory syndrome (sars)-like pandemic (peiris et al., ; zaki et al., a) . as of now, the most promising treatment is the passive administration of anti-mers-cov neutralizing antibodies. several research groups have developed and produced anti-mers patientderived or humanized monoclonal neutralizing antibodies in vitro that were able to protect mers-cov infected mice (corti et al., ; li et al., ; zhao et al., ) . however, since these antibodies react with a single epitope on the mers-cov spike (s) protein and since coronaviruses are prone to mutate, this approach has raised concerns about possible antibody escape (corti et al., ; sabir et al., ) . recently, we showed that sera from middle east dromedary camels contained high levels of anti-mers-cov neutralizing antibodies. passive immunotherapy with sera from these animals significantly reduced virus loads and accelerated virus clearance from the lungs of mers-cov infected mice . this provides proof of concept that immune animal sera are potentially useful in the treatment of patients with mers (hayden et al., ) . passive immunotherapy with animal sera or antibodies has been successfully used to prevent rabies and to neutralize snake venom (both et al., ; gutierrez et al., ) . convalescent plasma used to treat patients with sars has been found safe and has demonstrated some efficacy in a study with a small number of patients (mair-jenkins et al., ) . however, neutralizing antibody titers in mers patients are generally low and the limited number of mers survivors makes this approach impractical (drosten et al., ) . here, we show that immunization of healthy horses with mers-cov virus-like particles (vlps) expressing mers-cov s, m and e proteins induces strong polyclonal neutralizing antibodies against mers-cov. since administration of whole antibodies can induce allergic responses in some humans, we further tested f(ab') fragments prepared by digestion of antibody with pepsin. prophylactic or therapeutic treatment of mers-cov infected mice with either igg or f(ab') significantly decreased the virus load in their lungs. mers-cov vlps were produced and purified as previously described . in brief, army worm sf cells were infected with a single recombinant baculoviruses co-expressing mers-cov structural protein genes s, m, and e, at a multiplicity of infection (moi) of . . culture supernatants were harvested at h post-infection and centrifuged at g for min to remove cell debris. following centrifugation of the clarified supernatants at , g for h at c the resulting vlp pellets were resuspended in pbs and loaded onto a e e % discontinuous sucrose gradient. after an additional centrifugation at , g for . h at c, bands between and % sucrose containing mers-cov vlp were collected. four -year-old healthy horses received multi-point intramuscular injections of . , . , , , and mg mers-cov vlps in ml pbs at weeks , , , , and , respectively. freund's complete adjuvant (sigma) was included in the first dose, and incomplete adjuvant in the remaining ones. sera were collected from the jugular vein weeks after each injection, and stored at À c before further analysis. mers-cov specific antibodies in the sera were measured by an indirect enzyme-linked immunosorbent assay (elisa) using purified mers-cov receptor-binding domain (rbd) protein (i.e., s protein residues e cloned into the pet- a expression vector and purified by ni-nta affinity chromatograph column). briefly, -well microtitration plates (corning costar, usa) were pre-coated with ml purified rbd antigen diluted in . mol/l carbonate sodium buffer (ph . ) to a final concentration of mg/ ml and incubated at c overnight. after blocking with skimmed milk for h at c, ml twofold serially diluted serum samples were added to the wells, and incubated at c for h. the plates were washed three times with pbs containing . % tween- (pbst), before addition of ml hrp-labeled rabbit antibody against horse igg (bioss, china; : , ) and incubation at c for h. after washing with pbst, ml , , , '-tetramethylbenzidine (tmb) (sigma, usa) as substrate was added to each well and incubated for min. the reaction was stopped with ml m h so . optical densities at nm were measured in an elisa plate reader (bio-rad, usa). horse antiserum was diluted with vol of normal saline ( . % nacl) and a half volume of saturated ammonium sulfate was then added and mixed gently at room temperature for min before centrifugation at g for min. the resulting sediment was redissolved in saline and mixed with a one-third volume of saturated ammonium sulfate. after incubation at ambient temperature for min and centrifugation at g for min, the second sediments were dissolved in normal saline and dialyzed against normal saline to remove any remaining ammonium salt. immunoaffinity resins were prepared by coupling mg rbd protein to . m sodium periodate-activated sepharose b ( g), and then incubating with ml sodium borohydride for min. after reaction with m tris (ph . ) for min, a purified igg sample was diluted -fold with pbs and incubated with the rbd resin overnight at c with constant rotation. the flowthroughs (anti-rbd depleted) were collected, and then the flowthroughs were tested against the rbd protein by elisa to ensure rbdspecific igg all bound with the rbd sepharose b. after washing with pbs, the bound antibodies (anti-rbd) were eluted in . m glycine-hcl buffer (ph . ). the eluates were neutralized with m tris buffer (ph . ), and then dialyzed against pbs. all samples were adjusted to the same protein concentration and sterilized by passage through microspin filters ( . mm pore size; millipore). neutralizing activity of the igg, rbd-specific igg, and flowthroughs were tested. the ph of the horse antiserum was adjusted to . with mol/l hcl. following incubation with pepsin ( iu/ml) at c for . h, the reaction was stopped by adjusting the ph to . with mol/l naoh. the solution was then applied to protein-a and protein-g columns sequentially to remove whole immunoglobulins. the purity of the resulting f(ab') protein was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) followed by coomassie blue staining and the target fraction in the gel was analyzed in a thin layer chromatography scanner (transmission, zigzag scan, dual wavelength, swing width: mm, delta y: . mm) (cs- , shimadzu). specific pathogen-free week old balb/c mice were purchased from charles river laboratories international and maintained in the animal care facility, university of iowa. briefly, all mice were housed in thoren individually ventilated cages. caging and bedding were autoclaved. irradiated diet was fed. filtered water ( . mm filter) was provided with edstrom automatic watering system. hepa-filtered cage changing stations were used. all persons entering animal rooms worn autoclaved gowns, gloves, hair bonnets, face masks, and shoe covers. serum samples, purified igg or f(ab') were serially diluted in dmem and mixed with an equal volume of mers-cov containing pfu. following incubation at c for h, aliquots were added to cultures of vero cells in well plates and incubated at c in % co for h with gentle rocking every min. plates were then overlaid with . % agarose/dmem/ % calf serum. after further incubation for days, agarose plugs were removed using a small spatula, and the remaining plaques were visualized by staining with . % crystal violet. six-week-old female balb/c mice were lightly anesthetized with isoflurane and transduced intranasally with .  pfu of ad -hdpp in ml dmem as described elsewhere (zhao et al., ) . five days post transduction, mice were infected intranasally with mers-cov (  pfu) in a total volume of ml dmem. mice were monitored daily for morbidity (weight loss) and mortality. all work with mers-cov was conducted in the university of iowa biosafety level (bsl- ) laboratory. separate groups were injected with ml horse antiserum or mg igg or f(ab') intraperitoneally (ip) day before or after intranasal infection with  pfu mers-cov. control mice were given an equal volume of normal horse serum (sigma). to obtain virus titers, lungs were harvested from subgroups of animals at the indicated time points (see results) and homogenized into ml of phosphate buffered saline (pbs), using a manual homogenizer. lung homogenates were aliquoted into micro tubes and kept in À c. virus was titered on vero cells. cells were fixed with % formaldehyde and stained with crystal violet three days post-infection (p.i.). viral titers are expressed as pfu/g tissue for mers-cov (zhao et al., ) . due to the biosafety risk, mers-cov must be handled in a bsl- laboratory, whereas vlps can be rapidly generated under bsl- conditions as an immunogen inducing high antibody titers. in addition, the horse provides little risk to humans and produces high antibody yields, making these animals an effective source for production of hyperimmune sera (zheng et al., ) . rbd-specific igg titers in the sera were all above : , after five immunizations (fig. ) as assessed by elisa. rbd contains the major neutralizing epitopes of the s protein, as shown by the observation that absorption of sars patient convalescent sera with sars-cov rbd removes the majority of neutralizing antibodies (he et al., ) . independent research groups have also shown more directly that the mers-cov rbd sequence contains the major antigenic determinants for inducing neutralizing antibodies, and that neutralizing epitopes within mers-cov s are also localized primarily in the rbd region (du et al., ; mou et al., ) . here, we have demonstrated that anti-rbd antibodies function as major components of neutralizing antibodies. we found that rbd-specific igg neutralized mers-cov infection with half maximal inhibitory concentration of . mg/ml, and .  mg/ml for flowthroughs (fig. ) , suggesting that the rbd of s protein act as an important neutralization determinant of mers-cov. our results demonstrate that equine antibodies are polyclonal and recognize more antigen determinants in mers-cov s protein than single mabs, which could potentially prevent antibody escape. the integrity of igg and f(ab') fragments was evaluated using an sds-page gel (fig. a) . the purity of the f(ab') fragments after protein-a/g chromatography was > % after gel electrophoresis (fig. b ). passive transfer of blood products from other humans poses a safety concern, with possible contamination with agents of blood-borne diseases (e.g., hiv, hepatitis). heterologous antibody carries a potential risk of allergic reaction, but generation of f(ab') fragments, results in antibodies being less immunoreactive and safer for use in humans. while we successfully generated equine antibodies against mers-cov vlps, their protective effect against authentic mers- fig. . robust mers-cov rbd-specific antibody in immunized horse sera. horses (n ¼ ) were injected intramuscularly with mers-cov vlps and boosted every two weeks an additional times. sera were collected weeks after each immunization. rbd-specific antibodies in immunized horse sera were detected using elisa. cov infection remained untested. using a plaque reduction neutralizing assay, we confirmed that immune sera significantly neutralized mers-cov infection in vitro, with a half effective maximal dilution of : , (fig. a, b) . further, we found that equine igg and f(ab') also neutralized mers-cov infection with half effective maximal concentrations (ec ) of . mg/ml and . mg/ml for igg and f(ab') , respectively (fig. c, d) . collectively, these results show that equine antibody products exhibit highly potent neutralizing activity against mers-cov. next we asked if adoptive transfer of equine antibodies could protect mice from mers-cov infection prophylactically and therapeutically. by using a mouse model we previously generated (zhao et al., ) , we injected animals with immune serum (fig. a, b) , purified igg (fig. c, d) or f(ab') (fig. e, f) i.p. day before (fig. a , c, e) or after (fig. b, d, f) mers-cov challenge. in both prophylactic and therapeutic settings, passive transfer of equine immune antibodies resulted in a e log reduction of virus titers in the lungs of mers-cov infected mice, and accelerated virus clearance in the serum treated group (fig. a, b) . we did not observe any difference in body weight loss and pathologic changes on the exterior surface of the lungs in treated and untreated mice after fig. . neutralizing activity of the rbd-specific antibodies in igg. in vitro neutralization tests of total igg, rbd-specific igg, and flowthroughs, were determined in a series of -fold dilutions and % neutralization was calculated using graphpad prism. infeciton, since in this model, mice only develope mild lung disease. rapid virus replication and inflammatory cell infiltration in the infected lungs are the major parameters to measure (zhao et al., ) . since the half-life of f(ab') in vivo is relatively short and mers-cov is cleared within days in this model (zhao et al., ) , we did not inject f(ab') antibodies before day À or after day p.i. of note, the purified igg seemed to have lower protective potency than that of the immune serum in vivo (fig. ) . the concentration of igg in serum is > mg/ml. we used ml of immune serum (equal to mg igg) per mouse which is much higher than the immune igg we used ( mg/mice). the other reason could be we purified immune igg using saturated ammonium sulfate precipitation method, which needed to be performed under room temperature. we speculated that some iggs were degraded or misfolded, and unable to bind to mers-cov spike protein under this circumstance. while, immune sera were properly stored at À c and contained high concentration of bsa and other proteins, which made the antiserum more stable. to date, there are several anti-mers-cov antibodies developed from different origins. each antibody contains its own advantages and disadvantages. for monoclonal antibodies, mouse-derived monoclonal antibody needs to be humanized before human use (li et al., ) ; a human neutralizing antibody derived from a convalescent mers patient can be produced in large amount from cho cells (corti et al., ) . however, the single clone antibody raises the concern of viral escape mutant when applied to human. administration of transchromosomic bovine human immunoglobulins (luke et al., ) or dromedary immune serum resulted in rapidly viral clearance in infected mouse lungs. the disadvantage of these antibodies is that these animals are not readily available. compared to the antibodies described above, the administration of equine igg-derived f(ab') fragment proved to be a versatile and feasible method (lu et al., ; zhou et al., ) . it provides a useful platform to produce therapeutics against emerging infectious diseases. in summary, by immunizing healthy horses with mers-cov vlps, we have successfully developed the first equine igg-derived f(ab') fragment that neutralizes mers-cov in vitro and in vivo. both prophylactic and therapeutic treatments decreased virus loads and accelerated virus clearance in the lungs of mers-cov-infected mice. therefore, horses immunized with mers-cov vlps can serve as a useful initial source for developing protective f(ab') fragments, for the purpose of preparedness and to serve as a strategic reserve for a potential mers epidemic and other emergent pathogens. the authors declare no competing interests. epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a 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targeting the receptor-binding domain of the spike protein passive immunotherapy for influenza a h n virus infection with equine hyperimmune globulin f(ab') in mice human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis the receptor binding domain of the new middle east respiratory syndrome coronavirus maps to a -residue region in the spike protein that efficiently elicits neutralizing antibodies severe acute respiratory syndrome co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia mers-cov virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques isolation of a novel coronavirus from a man with pneumonia in saudi arabia isolation of a novel coronavirus from a man with pneumonia in saudi arabia rapid generation of a mouse model for middle east respiratory syndrome passive immunotherapy with dromedary immune serum in an experimental animal model for middle east respiratory syndrome coronavirus infection treatment with hyperimmune equine immunoglobulin or immunoglobulin fragments completely protects rodents from ebola virus infection inhibition of infection caused by severe acute respiratory syndrome-associated coronavirus by equine neutralizing antibody in aged mice middle east respiratory syndrome ad -hdpp transduced balb/c mice ( wks, female) were injected intraperitoneally with ml horse serum key: cord- -q x xb authors: nan title: th icar abstracts: date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: q x xb nan the society was organized in as a non-profit scientific organization for the purpose of advancing and disseminating knowledge in all areas of antiviral research. to achieve this objective, the society organizes an annual meeting. the society is now in its th year of existence, and has about members representing countries. for membership application forms or further information, please contact dr. amy patick, secretary, isar; pfizer global r&d, department of virology, science center drive, san diego, ca ; phone + ; fax + ; e-mail amy.patick@pfizer.com. membership application forms will also be available at the conference registration desk, or from our website www.isar-icar.com. enzymes of the pol gene of hiv have been identified as important viral targets for the discovery anti-hiv therapeutic agents. while the viral targets, hiv reverse transcriptase and hiv protease, have been successfully investigated for the development of clinically useful therapeutic agents, research efforts on drug discovery on the third enzyme of the pol gene, hiv integrase, have not resulted in a single fda-approved drug. nevertheless, as integrase is essential for hiv replication, it remains an attractive target for the discovery of new anti-hiv agents. in this presentation, we report the discovery of a conceptually new beta-diketo acid, constructed on a nucleobase scaffold, that is a potent inhibitor of both the -processing and strand transfer steps of recombinant hiv integrase. this inhibitor and the positive control compound, azt, were tested in a pbmc cellbased, microtiter anti-hiv assay against the clinical isolate, hiv- teki (nsi phenotype) and hiv- nl - (si phenotype), and in a magi-x assay against hiv- nl - with hela-cd -ltr-beta-gal cells. our integrase inhibitor was found to have highly potent in vitro anti-hiv activity and efficacy. the discovery of this remarkably active molecule, representative of a unique set of diketo acids bearing nucleobase scaffolds, has uncovered a new chapter in the chemistry and biology of integrase inhibitors and their potential therapeutic applications. berta bosch , imma clotet-codina , julia blanco , eduard pauls , gemma coma , samandhy cedeño , francesc mitjans , anuska llano , margarita bofill , bonaventura clotet , jaume piulats , jose este retrovirology laboratory, fundacio irsicaixa, badalona, spain; laboratorio de bioinvestigación, merck farma y química, barcelona, spain macrophages are key cells for hiv infection and spreading inside the organism. macrophages cultured in vitro can be successfully infected after differentiation with cytokines such as macrophage colony stimulating factor (m-csf). in the monocyte to macrophage differentiation process with m-csf, av-integrins are upregulated concomitantly to the capacity of hiv to generate a productive virus infection. in the present study we show that an anti-av antibody, e , inhibited hiv- infection of primary macrophages. the effect of e on r or x hiv- replication in acutely infected macrophages was dose-dependent, with a % effective concentration (ec ) of ± g/ml in the absence of cytotoxicity. similarly, a monoclonal antibody targeting the avb integrin ( d .f ) also inhibited hiv- infection in this cell type. e reduced the detection of hiv- bal proviral dna in acutely infected macrophages but was completely ineffective against hiv- bal production in chronically infected macrophages, suggesting that e inhibited hiv infection at an early stage of the virus cycle. finally, a small molecular weight antagonist of the avb integrin reduced hiv replication at subtoxic concentrations. therefore, our results suggest that av-containing integrins could play a role in hiv replication in macrophages and indicate that small molecular weight compounds may be developed to interfere with hiv replication in macrophages through the interaction with av integrins. andrew vaillant , hong lu , shuwen liu , carol lackman-smith , roger ptak , jean-marc juteau , shibo jiang replicor inc., laval, que., canada; f. lindsay kimball research institute, new york blood center, new york, ny, usa; southern research institute, frederick, md, usa the sequence independent antiviral activity of phosphorothioate oligonucleotides in inhibiting hiv- by blocking interactions between the v loop and cd has been previously described. this activity was attributed to their polyanionic activity. here we show that ps-ons (and their fully -o-methylated derivatives) are also potent inhibitors of hiv- -mediated membrane fusion and hiv- replication in a sequence-independent, sizedependent (optimal size ∼ - bases) and phosphorothioation dependent manner (independent of stabilization). ps-ons interact with the heptad repeat regions of gp and the hiv- fusion inhibitory activity of ps-ons is closely correlated with their ability to bind to these heptad repeats and block gp six-helix bundle formation, a critical step during the process of hiv- fusion with the target cell. the requirement for ps-on interaction was also found to be dependent on phosphorothioation, suggesting that the v loop/ps-on interaction may also have a hydrophobic component. the increased hydrophobicity of longer (≥ base) ps-ons may contribute to their inhibitory activity against hiv- fusion and entry because these longer ps-ons have a greater hydrophobicity and are more potent in blocking the hydrophobic interactions involved in the gp sixhelix bundle formation than shorter ps-ons (< bases). this novel antiviral mechanism of action of long ps-ons has important implications for therapy against infection by hiv- and other enveloped viruses with type i fusion proteins. chris meier , soenke jessel , bastian reichardt , olaf ludek , jan balzarini university of hamburg, institute of organic chemistry, hamburg, germany; rega institute for medical research, katholieke universiteit leuven, leuven, belgium carbocyclic nucleoside analogues like abacavir showed very interesting antiviral properties. therefore, we are interested in a convenient stereoselective access to this class of compounds as potential antiviral agents. by using a new convergent synthetic strategy, starting from a chiral cyclopentenol, enantiomerically pure carbocyclic thymidine (carba-dt) was obtained as a key intermediate for further variations at the -position. this pathway allows an entry to d-and l-configurated nucleoside analogues. however, using this approach a mixture of side products avoids the formation of the product in very high yields. however, we will present that the side products can be recovered by a stereoselective hydroboration leading to one intermediate only that can be use as well for the synthesis of carbocyclic nucleosides. the condensation of the carbocyclic moiety and different pyrimidine and purine nucleobase was achieved by a mitsunobu reaction. various analogues have been prepared via this strategy, e.g. d-and l-carba-bvdu, nucleoside analogues known to be antivirally active against hsv- . additionally, carbocyclic ␣nucleosides and carbocyclic iso-nucleosides are accessible by this reaction sequence. all new nucleoside analogues were tested for their antiviral activity. particularly carba-dt was found to be a potent anti-hiv active derivative showing no toxicity. however, it can not be excluded that a non-activity of a compound is related to a missing phosphorylation to the monophosphate. in order to prove that, all nucleosides were converted into their cyclosal-phosphate trimesters and transferred into the nucleotides. detailed chemistry, enzymatic and antiviral activity data will be presented. in some cases the nucleotide releasing system showed improved antiviral activity as compared to the parent nucleoside. michela pollicita , candace pert , maria-teresa polianova , alessandro ranazzi , michael ruff , carlo-federico perno , stefano aquaro university of rome tor vergata, italy; georgetown university, washington, dc, usa monocytes/macrophages (m/m) are a strategic reservoir of hiv- commonly infected by ccr -using (r ) strains of hiv- . ccr is an attractive target for inhibition of ccr mediated hiv- entry. thus, ccr antagonists are expected to be a power-ful new class of receptor-based therapeutic agents against hiv- infection. d-ala-peptide t-amide (dapta) is an octapeptide derived from the gp v region of hiv- , able to bind ccr . dapta acts as selective viral entry inhibitor, displacing the binding of gp with ccr . dapta anti-hiv- activity was evaluated in m/m infected with two different hiv- r strains, bal and a, in presence of several doses of the compound. dapta inhibited hiv- replication in m/m (> % compared to control), measured by the p gag ag released in the cell culture supernatants, at concentration of - nm. pcr analysis of integrated hiv- proviral dna on cultured m/m proved that dapta is able to block hiv entry and so, to prevent hiv infection in m/m. moreover, the capability of different hiv- r strains produced and released by infected m/m in affecting neuronal homeostasis was assessed in a neuroblastoma cell line, sk-n-sh, expressing ccr . in sk-n-sh were evaluated cell morphology, propidium iodide binding and fluorescenceactivated cell sorting (facs) analysis. dapta, at concentration of - and - nm, strongly inhibited apoptosis in sk-n-sh of and %, respectively, compared to control. unexpectedly, tak- (a nonpeptidic ccr antagonist with potent anti-hiv- activity) inhibited apoptosis only of % compared to control. our results suggest that the development of new anti-hiv- compounds, such as dapta, could be important in synergistic combination with other antiretroviral treatments in prevent both central nervous system hiv-infection and the consequent neural damage. the mechanisms of dapta inhibition may include both suppression of hiv- r strains in the brain as direct inhibition of hiv- replication in m/m and gp related damage by ccr binding. pradimicin a (prm-a) is an antifungal non-peptidic benzonaphtacenequinone antibiotic that specifically inhibits human immunodeficiency virus (hiv) in cell culture. it markedly suppresses a variety of different hiv- clades in pbmcs, in primary macrophages and several hiv- and siv strains in laboratory cell lines (range of % effective concentrations: . - g/ml; % cytostatic concentration: > g/ml). prm-a also inhibits syncytium formation between persistently hiv- -infected hut- cells and uninfected sup t cells. prm-a behaves as an artificial lectin that selectively binds mannose-containing glycans. consequently, biacore experiments revealed that it binds to gp of hiv- /mn in the presence of ca + . prm-a is endowed with a high genetic barrier with regard to drug resistance development against hiv- . a variety of multiple mutations at n-glycosylation sites in hiv- gp are required before the virus looses marked sensitivity to the drug. there is no clustered pattern of hiv- gp glycan deletions that occur under prm-a drug pressure. the resistance spectrum and mode of action is unique among any of the existing anti-hiv drugs and warrant further (pre)clinical investigations. acknowledgement: this research was supported by the flemish "fonds voor wetenschappelijk onderzoek," the centers of excellence of the k.u. leuven (no. ef/ / ), and the european commission (empro). jan muench , ludger ständker , knut adermann , axel schulz , michael schindler , raghavan chinnadurai , wolf-georg forssmann , frank kirchhoff department of virology university of ulm, albert-einstein allee , ulm, germany; ipf pharmaceuticals gmbh, feodor-lynen-strasse , hannover, germany a variety of components in human blood might influence hiv- replication in infected individuals. peptide libraries derived from hemofiltrate (hf), an aqueous blood solution, contain essentially all circulating blood peptides with a molecular mass below kda, including chemokines, defensins, and cytokines. to identify the most potent natural occurring factors inhibiting hiv- replication, we screened a hf-derived peptide library for antiviral activities. the most active fraction contained a -residue peptide corresponding to a c-terminal fragment of ␣ -antitrypsin (␣ -at), a highly abundant serine proteinase inhibitor. further analysis of the corresponding chemically synthesized peptide, termed virus inhibitory peptide (virip), demonstrated that it inhibits infection by all hiv- variants tested, independently of their subtype and coreceptor usage. notably, virip also blocked multi-resistant hiv- variants and primary isolates. virip specifically inhibited hiv- env function, and did not affect infection by virions containing hiv- , siv, mlv, hcv, ebola or vsv env proteins. the antiviral activity proved to be highly specific for the -residue virip sequence since structurally closely related peptides were inactive. we found that virip inhibits hemolysis of erythrocytes induced by the hiv- gp fusion-peptide (fp). nmr spectroscopy confirmed that virip interacts directly with synthetic gp fp. our observations are evidence that a naturally occurring human substance inhibits hiv- infection by a new mode of action, i.e. binding of the highly conserved fp. furthermore, we performed a structure-activity-relation study with more than virip analogs and found that specific amino acid changes enhanced the antiviral potency of virip by up to two orders of magnitude. experiments in cell culture and animal models further demonstrated that virip exerted no cytotoxic effects. thus, virip derivatives might become a new class of hiv- entry inhibitors. stefano aquaro , valentina svicher , roberta d'arrigo , ubaldo visco-comandini , andrea antinori , mario santoro , giovanni di perri , sergio lo caputo , pasquale narciso , carlo-federico perno , university of rome tor vergata, italy; inmi l. spallanzani, italy; university of turin, italy; sm annunziata hospital, florence, italy to investigate gp -variability and correlation with viroimmunological parameters in hiv-infected patients (pts) receiving t added as a single active drug to a failing regimen. two hundred and ten hiv-gp sequences and clinical follow-up from t -treated patients were analyzed from baseline up to weeks (weeks) of treatment. the association of mutations with viremia (vl)/cd count (c/ul) was assessed by mann-whitney test. the addition of t to the failing antiretroviral regimen induced at weeks a significant vl decrease from . log (stable in the last weeks prior t ) to . log (p = . ) and a significant cd increase from c/ul (decreasing in the last weeks prior t ) to c/ul (p = . ). while vl rebounded to . - log at - weeks, respectively, cd increased to c/ul at weeks. t resistance mutations, absent at bl, occurred shortly after treatment and usually alone. v a was the most common sign of t failure ( . % of pts). the viroimmunological outcome of t -treated pts varied according to gp -mutations. v a/e ( . % of pts) was associated with a cd increase from bl ( c/ul) of . -fold ( c/ul) at weeks and . -fold ( c/ul) at weeks (p = . and . compared without v a/e, respectively). no significant correlation with vl was observed (from . log at bl to . - . at - weeks). by contrast, q h + l m ( . % of pts) was associated with cd loss from c/ul at bl to c/ul at weeks (p = . ), without significant changes in vl (from log at bl to log at weeks). mutation n k (observed in pts, but never found at bl) abrogates the th gp -glycosylation site and correlated with . -fold cd increase at weeks. conformational changes induced by v a/e in the highly conserved giv motif of gp -hr , are tightly related with a loss of hiv-induced damage of immune system. this facilitates cd -recovery through mechanisms that can be virus-(loss of fusion efficiency) and immune-mediated (exposure of new epitopes) not applicable to protease/rt-inhibitors, and thus important for innovative therapeutic strategies. the spread of highly pathogenic h n influenza viruses in humans in asia, with high mortality rates among infected individuals is a major public health concern. in the absence of a vaccine antigenically matching the pandemic virus, antiviral drugs can play an important role. in the present study we reported the antiviral activity of neuraminidase inhibitor oseltamivir against lethal h n influenza virus infection in ferrets, an appropriate animal model that closely resembles clinical signs of human influenza. inoculation of young adult ferrets with a viral dose as low as eid of a/vietnam/ / (h n ) influenza virus caused high fever ( . - . • c), weight loss ( . % of initial), anorexia, extreme lethargy and death of animals on days - post-virus inoculation (p.i.). oral administration of oseltamivir at a dose of mg/kg/day for days twice daily initiated h p.i. inhibited the febrile response, reduced weight changes ( . % of initial) and, most importantly, completely protected ferrets from lethal h n infection. in the treatment groups, virus replication in the upper respiratory tract of ferrets was prevented, whereas untreated animals shed virus at titers of . - . log eid /ml on days , and p.i. systemic spread of the h n virus was observed in untreated ferrets: virus was detected in multiple internal organs, including the brain. treatment with oseltamivir resulted in complete inhibition of virus replication in the lungs and small intestine on day p.i. in the brains of treated animals virus was detected in one of the two animals tested with > -fold reduction of titer. sequence analysis showed no amino acid substitutions at conserved residues in na or ha subunit in viruses isolated from ferret's internal organs after treatment. these results suggest that oseltamivir earlier treatment can prevent h n mortality in ferrets, however, further studies investigating optimal doses and treatment durations required to achieve protection against infection with highly pathogenic influenza viruses are much needed. natalia ilyushina, erich hoffmann, rachelle salomon, robert webster, elena govorkova st. jude children's research hospital, memphis, tn , usa in the present study we tested in the mouse model the hypothesis that combination chemotherapy with drugs targeting dif-ferent virus proteins may lead to more potent and beneficial effects. we applied plasmid-based reverse genetics technique to generate two recombinant a/vietnam/ / -like (h n ) viruses. one virus possessed asparagine at position of the m protein that was found in the naturally circulating virus (rgvn- ) and confers resistance to amantadine. the other recombinant virus possessed serine at that position and was sensitive to amantadine (rgvn- sens) . balb/c mice were administered oseltamivir ( or mg/kg/day) and amantadine ( . or mg/kg/day) twice daily for days by oral gavage; the first doses were given h before inoculation with mld of h n virus. combination treatment with mg/kg/day oseltamivir and mg/kg/day amantadine was given on the same schedule. single-drug oseltamivir produced a dose-dependent antiviral effect against both recombinant h n viruses (p < . ). treatment with oseltamivir at dosage of mg/kg/day significantly inhibited virus replication in the lungs, brain, spleen, and blood of mice at days , , and after inoculation (p < . ), but resulted in low survival rate ( %). single-drug amantadine showed dose-dependent effect only against rgvn- sens strain. notably, risk of death for mice that received mg/kg/day of amantadine or mg/kg/day of oseltamivir was similar (p < . ). in contrast, prophylactic treatment of mice with combinations of oseltamivir and amantadine completely inhibited virus replication in the animals infected with rgvn- sens (p < . ) compared to singledrug usage and protected % of animals. importantly combination chemotherapy completely protected h n virus spread to the brain of the mice: virus was not detected in brain of treated animals on days , , and after inoculation and neurological symptoms were not observed. our results suggest that combination chemotherapy provides an advantage over single-agent treatment. this strategy could be an option for the control of influenza virus infection, and combinations with other novel drugs should be explored. françois jean , reid asbury , meera raj , david lawrence , martin petric the university of british columbia, vancouver, bc, canada v t z ; ge healthcare bio-sciences, piscataway, nj , usa; british columbia center for disease control, vancouver, bc, canada v z r in late , severe acute respiratory syndrome (sars) became the first new severe and easily transmissible human disease to emerge in the st century. although it abated after six months, sars serves as a modern paradigm for human emerging infections with deaths reported from countries. the causative agent was found to be a new sars-associated coronavirus (sars-cov) . while the sequence of sars-cov genome was first reported by the bc genome sciences center, the full set of viral and cellular proteins that compose the sars-cov virion remains unknown. to approach this problem, we have utilized two-dimensional gel electrophoresis and liquid chromatography-tandem ms (lc-ms/ms) to identify the viral and cellular proteins in purified sars-cov virions obtained from human infected cells [huh : human liver] and primate (veroe : monkey kidney) infected cells. interestingly, analysis of the proteins from purified sars-cov preparations has revealed that the enveloped virions contain not only the predicted viral structural proteins (e.g. spike glycoprotein, nucleocapsid protein, and membrane glycoprotein) but also an important number of differentially incorporated host cellular proteins into or onto the newly formed viruses. we have unambiguously identified over host cellular proteins in sars-cov virions by lc-ms/ms. these proteins include members of the annexin superfamily, cytoskeletal proteins, chaperones, vesicular transport proteins, uracyl-dna glycosylases, and aldehyde oxidoreductases. this study provides the first comprehensive and comparative analysis of the viral and cellular proteins that compose infectious particles of sars-cov obtained from human and primate infected cells. the functions of these newly identified hostspecific proteins are currently being investigated using rna interfering systems; their contributions to structure, viral productive replication, and pathogenicity will be discussed. acknowledgement: supported by an early career ubc operating grant (f. jean) and cihr (m. petric) . dale barnard , craig day , robert montgomery , kevin bailey , matt heiner , larry lauridsen , robert sidwell , kurt berg institute for antiviral research, utah state university, logan, ut, usa; panum inst., immi, the ifn-lab, copenhagen, denmark severe acute respiratory syndrome (sars) is a life-threatening respiratory illness caused by sars-cov. there are no approved therapies for sars. some drugs inhibit sars-cov replication in vitro including human interferons and selected antiinflammatory agents (chihrin and loufty, . , - ) . interferons are very promising because of their potent in vitro inhibition of sars-cov. although anti-inflammatory agents are not very active in vitro, it is thought that they might be efficacious in reducing any deleterious inflammatory response associated with virus infections such as sars infections in humans. for example, troxerutin, a flavenoid with anti-inflammatory properties, is in clinical trials for treating rhinovirus (rv) infections, ameliorating rv-induced inflammation (turner et al., . apmis , - ) . therefore, troxerutin was tested for inhibition of sars-cov replication in the lungs of infected mice using a mix of four hydroxyethylrutosides that included troxuretin. in addition, mouse interferon-alpha, used as a model compound for human interferon-alpha, was evaluated for inhi-bition of virus lung titers. both mouse interferon-alpha administered i.p. daily beginning h pre virus exposure at doses of , and , iu and the hydroxyethylrutoside mix ( and mg/kg) administered i.p using the same schedule reduced virus replication in the lungs of mice to below detectable limits. when a hydroxyethylrutoside mix was given to mice in the drinking water at . mg/ml (likely equivalent to an i.p. dose of mg/kg, assuming that the mice drank freely), virus lung replication was also completely inhibited. all treatments appeared to be well tolerated, since all groups of mice gained weight. we also report on the efficacy of various combinations of two doses of these drugs administered i.p., using the same dosing regimen as described. these data support the supposition that interferon might be a useful therapy for treating human sars infections and that hydroxyethylrutosides should be investigated further as a potential therapy. acknowledgement: supported by contract no. n -ai- from the virology branch, niaid, nih. treatment options for human respiratory syncytial virus (rsv) are limited. an effective vaccine is not yet available. neutralizing polyclonal antibody (respigam tm , medimmune) and a humanized monoclonal antibody (synagis tm , medimmune), are licensed for prophylactic use. ribavirin is the only approved antiviral against rsv, but its efficacy is controversial and its use is limited to treatment of high-risk patients. there is a clear need for new anti-rsv therapeutics with improved efficacy and ease-of-use. many early efforts to identify anti-rsv compounds focused on blocking the process of fusion. we have developed a cell-based screening platform to identify antivirals that inhibit rsv transcription and replication. the assay does not require infection with wild-type virus. it is based on an rsv subgenomic replication system in baby hamster kidney (bhk- ) cells that express the essential viral replication proteins (n, p, l and m - ). the readout is expression of the reporter gene lacz from a subgenomic rna. screening of the apath small molecule library yielded compounds (hit rate = . %) with ec values ≤ m and with selectivity index (si) values ≥ . seventy-two compounds demonstrated antiviral activity against wild-type rsv (strain a ) in a cytopathic effects inhibition assay (ec < m; si > ). these anti-rsv compounds represent nine different chemical classes. two compounds, a -aminoquinoline (ec = . m) and a thienopyrimidine (ec = . m), were shown to have desirable pharmacokinetic profiles and were chosen for efficacy testing in the cotton-tail rat model of infection. sar studies to identify the pharmacophore of the compounds have been initiated. preliminary studies to characterize the mechanism-ofaction in virological assays will be discussed. acknowledgement: supported by nih r ai - . we have previously reported bicyclic furano pyrimidine nucleoside analogues (bcnas) as exquisitely potent and selective inhibitors of varicella zoster virus (vzv) , with subnanomolar activity for p-alkylphenyl substituted analogues such as lead compound cf (cf- ) ( ) . these compounds have entered preclinical development with fermavir pharmaceuticals. we now report the first chromatography-free synthesis of these agents, their scale up to multi-gramme amounts, and their pre-clinical characterisation. in addition, we were keen to address potential solubility and bioavailability issues of these highly lipophilic agents by the synthesis of more polar analogues in two categories; side-chain ethers ( ) as new analogues in their own right, and -phosphates ( ) as potential more soluble prodrugs. we report data on both of these new families at this meeting. finally, we note the application of our phosphoramidate pro-tide approach to this family, with a series of bcna protides ( ) designed as intracellular phosphate delivery forms to bypass the essential vzv thymidine kinase-mediated first phosphorylation step. graciela andrei , joos van den oord , pierre fiten , ghislain opdenakker , erik de clercq , robert snoeck rega institute for medical research, katholieke universiteit leuven, leuven, belgium; pathology department, u.z. leuven, leuven, belgium varicella (chickenpox) , the primary infection caused by vzv, is characterized by viremia and skin lesions. reactivation of the latent virus results in skin lesions characteristic of herpes zoster (shingles). as keratinocytes are one of the main target cells for productive infection in vivo for vzv, human epithelial cells represent a relevant model for the study of vzv pathogenesis and evaluation of antiviral compounds. organotypic epithelial raft cultures permit full differentiation of keratinocytes via culturing of the cells on collagen matrix at the air-liquid interface. we have previously shown that the susceptibility of cultures to infection with vzv depends on the stage of differentiation of the rafts. we have now quantified the activity of reference anti-vzv compounds by measuring viral dna load by realtime pcr. quantitative pcr for vzv dna was performed by using specific primers and a mgb-probe for the orf gene (single-stranded dna binding protein) by the taqman method. two series of raft cultures were infected with the wild-type oka strain after days of differentiation and treated with serial dilutions of the test compounds. at days post-differentiation one series of the cultures was processed for histology and the other one for viral dna quantification. acyclovir (acv), penciclovir (pcv) and brivudin (bvdu) at and . g/ml, foscarnet (pfa) at g/ml and cidofovir (cdv) at , . and . g/ml inhibited viral dna content by more than %. these results were in agreement with histological examination of the rafts, no cytopathic effect being observed at these concentrations. as expected, only cdv and pfa inhibited the replication of the thymidine-kinase deficient (tk-) - strain. a correlation between the degree of protection as determined by histological examination and viral quantification could also be demonstrated for cdv and pfa against the tk- - strain. since no animal model is available for the in vivo evaluation of antiviral agents against vzv, the organotypic cultures may be considered as a valuable ex vivo model to evaluate the efficacy of new anti-vzv antivirals. jae-seon hwang , oliver kregler , john c. drach , , leroy b. townsend , elke bogner institut für klinische und molekulare virologie, erlangen, germany; department of biologic and materials sciences, school of dentistry; interdepartmental graduate program in medicinal chemistry, college of pharmacy, university of michigan, ann arbor, mi, usa dna packaging is the key step in viral maturation and involves binding and cleavage of viral dna containing specific dnapackaging motifs. this process is mediated by a group of specific enzymes called terminases. we have previously demonstrated that the hcmv terminase is composed of two subunits, the large one encoding pul and the small pul , where each protein has a different function. while the large subunit mediates sequence specific dna binding and atp hydrolysis, pul is only required for duplex nicking. inhibitors targeting pul and/or pul are attractive alternatives as hcmv antivirals since mammalian cell dna replication does not involve cleavage of concatameric dna. we now have screened several members of the benzimidazole ribonucleoside class of replication inhibitors in order to determine if a compound has the capacity to block the atpase activity of the large terminase subunit pul . analysis by bioluminometric atpase activity assays identified bdcrb and one more compound [ -bromo- , , -trichloro- -( , , -tri-o-acetyl-␤-dribofuranosyl)benzimidazole (btcrb)] with inhibitory effects. although only btcrb and bdcrb were inhibitors of the atpase activity, two other compounds, dbdcrb and cl rb, inhibited virus replication in a plaque-reduction assay, thus indicating that those have a different mode of action. in addition, by electron microscopy of thin sections we observed that in the presence of btcrb only b-capsids and dense bodies were formed. furthermore, spherical capsids accumulated in the perinuclear cisternae indicating a block in nuclear egress thereby providing additional evidence that closely-related benzimidazole d-ribonucleosides may have differences in their antiviral modes of action. human cytomegalovirus (hcmv) is the cause of significant morbidity and mortality in a variety of immunocompromised patients. currently available anti-hcmv drugs interfere with dna replication; however, these drugs are highly toxic, pre-cluding their long-term use in humans. interrupting hcmv viral entry is largely unexplored as an antiviral drug development strategy and is potentially an ideal and tractable goal. hcmv is believed to rely upon formation of ␣-helical coiled coils in the viral glycoproteins gb and gh to promote virus-host membrane fusion; peptides encompassing heptad repeat sequences in these two proteins inhibit viral infection. we have explored nonnatural oligomeric molecules ("foldamers") that are designed to mimic elements of the putative ␣-helical segment of gb. this effort has led to the discovery of oligomers of ␤-amino acids ("␤-peptides") that block hcmv infection. the ␤-peptide scaffold offers several advantages for the design of protein-protein interaction inhibitors, as ␤-peptides are amenable to modular synthesis, resist proteolytic degradation, and can display large and tailored molecular surfaces. the most potent ␤-peptide inhibitor blocks hcmv infection with a micromolar ic in a cell-based assay. these compounds show specificity for hcmv relative to closely related viruses. mechanistic studies suggest that these inhibitors interfere with membrane fusion between hcmv particles and host cells. current efforts are focused on understanding in greater detail the origin of the observed biological activity, exploring other foldamer scaffolds as bases for inhibitor design, and developing specific fusion inhibitors for other herpesviruses. previous reports have indicated that herpes simplex virus (hsv) activates nuclear factor-kappab (nf-kb) during productive infections. nonsteroidal anti-inflammatory drugs (nsaids) have significant inhibitory effects on nf-kb. therefore, two nsaids, indomethacin and aspirin, were assayed for antiherpetic effects and utilized as tools to further study the role of nf-kb in hsv- infection. we report that indomethacin and aspirin inhibited hsv- replication at non-cytotoxic doses. in vero cells, um indomethacin and mm aspirin reduced hsv- titers . and . %, respectively. electromobility shift assays revealed that hsv- activation of nf-kb is inhibited by the nsaids at doses that coincide with reduction of hsv- titers. to investigate a pathway for nf-kb inactivation, protein levels of ikb-alpha, a cytoplasmic nf-kb inhibitor, were examined. ikb-alpha protein was present in uninfected samples, but decreased over time in all hsv samples, regardless of chemical treatment, suggesting localization of nf-kb to the nucleus. immunohistochemistry studies verified that p , a component of the dimeric nf-kb complex, translocated to the nucleus of hsv- infected cells in the presence or absence of the nsaids. finally, direct effects on viral gene activity were assayed by real-time rt-pcr analysis. indomethacin and aspirin reduced mrna for icp , an essential hsv immediate-early gene, . and . -fold, respectively, resulting in significant decreases of icp protein. but transcriptional analysis revealed that synthesis of mrna for thymidine kinase, an hsv early gene, was unaffected by chemical treatment. however, mrna for glycoprotein c, an hsv late gene was undetectable in indomethacin and aspirin treated samples. cumulatively, these data indicate that: (i) indomethacin and aspirin block hsv- replication and (ii) the in vitro anti-herpetic effects of nsaids may reside in their ability to block nf-kb activity within the nucleus, impairing activation of essentials hsv genes. increasing species-specificity constraints preclude study of human cytomegalovirus (hcmv) in animals, necessitating the use of rodent cmvs to model human disease. however, the susceptibility of animal cmvs to clinically useful antivirals is unpredictable. for example, the guinea pig cmv (gpcmv), a uniquely valuable virus for modeling congenital cmv infection, is highly resistant to ganciclovir (gcv) at medically relevant doses. we used a molecular virological approach to test the hypothesis that gcv susceptibility could be conferred on gpcmv by insertion of the human ul phosphotransferase gene into the gpcmv genome. the gpcmv genome, cloned as a bacterial artificial chromosome in e. coli, was modified by site-specific recombination, using a shuttle plasmid targeting the gp locus, and carrying the ul gene from hcmv strain towne. the resultant chimeric virus was replication competent, and was found to contain the hcmv ul by southern-blot and sequence analyses. northern-blot revealed that a hcmv ul -specific transcript was expressed with late gene kinetics. western-blot, using a hcmv ul -specific polyclonal antibody, detected protein in virus-infected cells. the chimeric virus was gcv-susceptible, compared to wild-type gpcmv, with an ic of m. chimeric virus also exhibited increased sensitivity to maribavir (mbv), exhibiting a -log reduction (compared to wild-type virus) in the presence of m mbv, and an ic of m. to study the in vivo pathogenesis of chimeric virus, cyclophosphamide-immunocompromised strain two guinea pigs were challenged intraperitoneally, resulting in evidence of disseminated infection, and mortality. ganciclovir treatment ( mg/kg/day) resulted in reduced weight loss, and mortality, compared to placebo. these studies confirm the key role of ul in cmv antiviral therapy, and demonstrate that a 'humanized' gpcmv can be generated with altered antiviral susceptibilities. genital herpes infections are a global health problem and impact hiv/aids epidemic. strategies to prevent transmission include treatment of infected subjects to suppress shedding and prophylaxis with vaginally-applied microbicides. we examined the in vitro and vivo activity of rep , a fully degenerate mer phosphorothioated oligonucleotide against hsv- infection of human cervical cells and in a vaginal murine model. rep has broad-spectrum anti-herpetic activity with potent in vitro activity against hsv- , hsv- , hcmv, vzv, ebv, and hsv- (vaillant et al., submitted for publication). at a concentration of m, rep inhibited -logs of hsv- infection if present during the entire experiment. synchronized infectivity assays demonstrate that, unlike sulfonated polyanions in clinical trials, which primarily block hsv attachment, rep acts at multiple steps and inhibits binding, entry and post-entry gene expression. in our in vivo studies, mice were treated once intravaginally with rep or pbs control at various times prior to vaginal challenge with a lethal dose of hsv- strain ( log pfu). rep prophylaxis provided protection to mice from hsv- infection and disease. protection was significant when challenged min after treatment (p < . ). additionally, treatment with an analog of rep , which cannot activate tlr- mediated immune stimulation, was at least as active as rep , suggesting that direct antiviral activity and not stimulation of innate immunity is the mechanism of action in vivo. utilizing this analog, protection was significant when challenged min after treatment (p < . ) with a trend toward protection when administered min prior to challenge (p = . ). in summary, treatment with the rep analog which has superior resistance to low ph and nuclease degradation was more effective than rep , in some experiments protecting % of mice from viral infection and disease. the testing of this ph resistant rep analog in a gel formulation is currently underway. acknowledgement: supported by contract no -ai- from the virology branch, niaid, nih. a phosphorodiamidate morpholino oligomer (pmo) designed to hybridize to a highly conserved region including the aug translation start site of hcv, called avi- , has been evaluated for efficacy, toxicity, and pharmacokinetic properties. avi- inhibits translation initiated at the aug start site with ec of nm ( . ug/ml) and shows positive cooperativity. this pmo retains most of the activity in the presence of point mutations in the hcv genome. huh- cells were incubated with normal human serum (nhs) or hcv infected human serum (is) and hcv replication observed by rt-pcr. avi- produced robust inhibition of hcv in a dose and sequence-specific manner. studies conducted in vivo with avi- in the hcv infected trimera mouse (xtl) show reduction in viral titer which is dose dependent with approximately % of mice with undetectable viral titer and the remaining mice show log reduction in viral titer with . mg/mouse/day for consecutive days. the fractional bioavailability of avi- from a sq dose is approximately . the apparent elimination half life in rat, nonhuman primate and humans was . , . and . h, respectively. the volume of distribution ranged from . to . l/kg and the cmax is linearly related to the dose in mg/m . a phase i study in healthy volunteers in which daily sq doses of and mg has been completed. no serious adverse events have been observed. treatment of infected patients is currently planned. inhibition of hcv polyprotein synthesis is anticipated to contain therapeutic benefis of both protease inhibitors and polymerase inhibition. hcv infection can progress to fibrosis, reduced liver function, hepatocellular carcinoma, and death. currently, the standard treatment for hcv infection involves treatment with pegylated interferon in combination with the nucleoside analogue ribavirin. this treatment regimen effects a cure in approximately - % of the genotype- (gt- ) population; therefore a significant unmet clinical need exists in hcv therapy. virus-encoded polymerases have proven to be excellent molecular targets for chemotherapeutic intervention in numerous viral mediated diseases. in the case of hiv, hbv and herpes virus infections, deoxy-nucleoside analogues, which act as chain terminating agents, have been shown to have invaluable clinical utility. by analogy, appropriate ribonucleoside analogues might be expected to inhibit the essential rna polymerase (ns b) encoded by hcv. here we describe the preparation of nucleoside analogues as inhibitors of the hcv polymerase. in our design of nucleoside analogs as potential anti-hcv agents, we chose to investigate the effect of -substituted ribonucleoside derivatives. we reasoned that after incorporation of a ribonucleoside containing a -substituent, a disruption in elongation of the growing rna could be effected through either steric hindrance or via a conformational change of the carbohydrate moiety. our investigations on several such analogues will be presented. of particular interest is -azido-cytidine, which shows good activity in the genotype b sub-genomic replicon (ic = . m) with no measurable cytotoxic or cytostatic behavior. in addition, we have shown that the triphosphate of -azido-cytidine is a potent and highly selective inhibitor of ns b (ic = . m). joanna e. boerner, sue ma, choilai tiongyip, michael p. cooreman, teresa compton, kai lin novartis institutes for biomedical research, technology square, cambridge, ma , usa current drug discovery efforts for hepatitis c virus (hcv) focus on developing specific inhibitors of two viral enzymes, ns b polymerase and ns - a protease. however, resistant viral mutants are likely to emerge during therapy, compromising the effectiveness of these inhibitors. an alternative and complementary strategy is to target host factors that are also essential for viral replication. cyclophilins, a family of peptidyl-prolyl isomerases and the cellular targets of cyclosporin a (csa), present such an opportunity. it was reported recently that cyclophilin b bound to hcv ns b polymerase and stimulated its rnabinding activity, and that these functions were blocked in the presence of csa (watashi k. et al., molecular cell ) . nim , a csa derivative, is a more suitable candidate for hcv therapy because it binds to cyclophilins with higher affinity than csa while lacking the immunosuppressive activity associated with csa. using the hcv replicon system we demonstrated that nim exhibited potent anti-hcv activities in vitro. moreover, the combination of nim with a specific non-nucleoside inhibitor of hcv polymerase led to synergistic antiviral effects with no significant increase of cytotoxicity. resistant clones against both inhibitors were obtained in vitro, however, it was much more difficult to generate resistance against nim than the polymerase inhibitor. also, there was no cross-resistance between the two inhibitors. finally, addition of nim to the hcv polymerase inhibitor drastically reduced the emergence of resistance compared to polymerase inhibitor alone. taken together, nim , with a novel mechanism of action and a favorable pharmacokinetics and safety profile, represents a promising clinical candidate for treating hepatitis c and provides a rationale for specific combination therapy. the nucleoside analog r was identified as a specific inhibitor of hcv replication in subgenomic hcv replicon cells. r -tp is a competitive inhibitor of cmp incorporation by hcv polymerase ns b. in a transient replicon system r inhibited hcv rna replication driven by genotype b polymerase with similar potency as compared to that driven by genotype a polymerase. r -tp inhibited native hcv replicase and recombinant ns b from genotype a and b with similar potency. in contrast, r -tp did not inhibit human dna polymerases alpha, beta or gamma, including reverse transcriptase activities of dna polymerases beta and gamma, which were highly sensitive to inhibition by azt-tp and tc-tp. no significant inhibition was observed with human rna polymerases i, ii and iii derived from hela cells. in addition, the functionally related native influenza virus rna dependent rna polymerase (rdrp) activity in vitro was not inhibited by r -tp at concentrations up to mm, suggesting high selectivity for the hcv rdrp. thus, r was identified as a potent and highly selective inhibitor of hcv polymerase mediated rna synthesis. guangxiang luo, zhaohui cai, chen zhang, kyung-soo chang, jieyun jiang microbiology, immunology, and molecular genetics, university of kentucky college of medicine, lexington, ky, usa the study of hepatitis c virus (hcv) replication and the search for specific antiviral agents against hcv infection have been hampered by the lack of an efficient stable cell culture system of hcv infection and propagation. we have successfully constructed stable human hepatoma cell lines that contain a chromosomally integrated-genotype a hcv cdna and constitutively produce and secrete high titers of infectious virus into the culture media. transcriptional expression of the full-length hcv rna genome is under the control of a cellular pol ii polymerase promoter at the end and a hepatitis delta virus ribozyme at the end. the resulting hcv rna was expressed and replicated efficiently, as shown by the presence of high levels of hcv proteins as well as hcv rna in the stable huh cell lines. hcv secreted from the stable cell lines was infectious, as determined by antibody neutralization, blockage of putative hcv receptors, and inhibition of hcv replication by interferon. our findings demonstrate the establishment of a stable cell culture system of infectious hcv production and propagation, which allows the study of the entire hcv infectious cycle. the stable hcv-secreting cell lines are now being pursued to develop high throughput screens for effective hcv inhibitors. additionally, we established a novel and powerful hcv replication system in the mouse hepatocyte and mouse embryo fibroblasts (mef). hcv rna was found to replicate efficiently in both pkr +/+ and pkr −/− mef cells, demonstrating that hcv rna replication in mef cells is a powerful system to study host-virus interaction by using diverse gene-knockout animals. interestingly, hcv rna replicates more efficiently in the pkr −/− cell than in the pkr +/+ cell, suggesting a role of pkr in the control of hcv rna replication. however, ifn inhibited hcv rna replication in the pkr −/− cell with an efficacy similar to that in the pkr +/+ cell, suggesting a pkr-independent antiviral mechanism. clearly both pkr-dependent and pkrindependent antiviral mechanisms are important for the control of hcv replication and the mediation of the ifn-induced anti-hcv response. our studies set a stage for the development of transgenic mouse models of hcv replication and open up new avenues to study hcv and host interactions in mefs derived from diverse gene-knockout animals. andrea cuconati , haitao guo , gael westby , anand mehta , timothy block , institute for hepatitis and virus research; drexel institute for biotechnology and virology research, doylestown, pa, usa the high levels of hepatitis b surface antigen (hbsag)-bearing non-infectious particles in the serum of infected individuals is thought to play a role in suppressing hepatitis b virus (hbv)specific immune response by titering out hbv-specific antibodies and lymphocytes. current hbv therapeutics do not directly reduce this viral antigenemia. our group has focused on the enhancement of the immune response through the inhibition of viral antigen secretion in the infected hepatocytes, with the therapeutic goal being the use of hbv vaccination for the treatment of acute and chronic infection. the high-throughput screening of a small molecule library of , drug-like compounds was undertaken to discover novel inhibitors of hbsag secretion. using the stably hbv-transfected, human hepatoma cell line hepg . . , we developed an hts-compatible elisa protocol for the detection of hbsag secreted in the culture media. the screen resulted in initially positive hits, a hit rate of . %. subsequent retesting for activity and toxicity by mtt assay has narrowed the number of confirmed, non-toxic hits to , currently categorized in twelve chemical series. we have previously reported on a trio of related pyrazolo-pyridines with ec measurements below . m and cc measurements > . m. nascent structure-activity relationship (sar) suggests that a central moiety of the molecules is essential to activity, with an aromatic side group contributing to potency. among recently confirmed inhibitors, two currently under investigation include: ( ) an isobutyl-acetamide with an ec of . nanomolar, and a cc of > m, and ( ) a carbothiamide with an ec of . micromolar and a cc of > m. measurement of secreted hbv l and m antigens and cellular markers indicated that the pyrazolo-pyridines are not specific inhibitors of viral antigens, while the isobutyl-acetamide and the carbothiamide are indeed specific. measurement of intracellular viral dna indicated that none of these molecules are inhibitors of replication. we will be reporting on our studies of the potency, specificity, and potential mechanisms of action of these novel anti-hbv compounds. background: entecavir (etv) is a potent competitive inhibitor of hepatitis b virus (hbv) polymerase with activity versus all three enzymatic functions including priming, minus, and plus strand dna synthesis. virologic rebound due to etv resistance (etvr) has only been observed in lamivudine resistant (lvdr) hbv (m v/i ± l m), and requires at least one additional change in the reverse transcriptase domain (rt) at residues t , s , or m . these substitutions surround the dntp binding site or primer grip of rt. the objectives of this work were to further characterize etvr and its mechanism(s) using cell culture, in vitro enzyme, and molecular modeling studies. methods: hbv cell culture assays used transfected hepg cells and quantitation of released, immunocaptured hbv nucleocapsids. gradient-purified intracellular nucleocapsids were used for in vitro rt assays. a d homology model based on the hiv- rt structure was used to model resistance changes in hbv. results: reduced etv susceptibility of etvr hbv was observed both in culture and enzymatically in vitro. kinetic studies showed various etvr substitutions in lvdr hbv selectively reduced etv-triphosphate (etv-tp) binding (k i ) to rt without markedly changing the affinity for dgtp (k m ) or inhibition by ddgtp. etvr rts also displayed reduced enzymatic activity (k cat ) relative to wildtype and etvr hbv appeared growth impaired. modeling studies suggested a novel etv-tp binding pocket in hbv rt that became constrained with etvr changes. m changes in the primer grip region of rt were unique in that resistance was primarily seen during synthesis of minus strand dna. etvr changes in the absence of lvdr substitutions had greatly reduced impacts on etv susceptibility, confirming models suggesting etvr is imparted through lvdr changes. summary: etv provides a high genetic barrier to resistance, requiring additional changes at residues t , s or m along with pre-existing lvdr substitutions m v/i ± l m. kinetic parameters and molecular modeling indicated that etvr substitutions selectively affected etv-tp binding and reduced the replication capacity of hbv. a nonhuman primate (nhp) model of classical, lesional smallpox has been used to test the efficacy of intravenous (iv) cidofovir treatment. cynomolgus macaques were infected with a high dose ( pfu iv) of variola to produce an artificial primary viremia, and then treated with cidofovir at , , or h postinfection (pi). later treatment times were not evaluated. treatment at or h pi halted increases in peak blood viral genome titers measured by quantitative taqman-mgb real-time pcr, which were more than -fold less in cdv-treated animals compared to placebo. historically, the number of pox lesions provided the best correlation with human smallpox clinical severity, and cdv treatment in our model significantly reduced maximum pox lesion counts by > %; the number and size of skin lesions, and in untreated animals contributed significantly to the total viral burden with lesions containing - genomes/g. to better understand the role of viral burden and disease progression in major organ systems, a serial sample study was undertaken. in untreated animals at h pi, viral replication in spleen exceeded genomes/g while liver and bone marrow yielded genomes/ml. in comparison, titers in other tissues ranged between and genomes/g and blood yielded genomes/ml at h, suggesting that the liver, spleen, and marrow may be initial sites of replication. levels of virus in the bone marrow reached a peak of approximately genomes/g at day , then decreased to quantities consistent with those in blood. viral load in the blood increased with time, peaking around days - at genomes/ml. virus was also detected in intestine, skeletal muscle, and late in infection, testes. the ability to successfully treat with cdv h pi despite early extensive organ infection in the accelerated nhp variola model suggests that this treatment could be effective in reducing viremia and mortality after onset of symptoms in human smallpox, which demonstrates a more protracted disease course. work involving variola virus conducted in who-sanctioned cdc, atlanta bsl- laboratory. earl kern , kathy keith , robert jordan , dennis hruby , debra quenelle department of pediatrics, university of alabama school of medicine, birmingham, al, usa; siga technologies, inc., corvallis, or, usa although cidofovir (cdv) has been approved as an investigational new drug for emergency treatment of smallpox, its lack of oral activity and dose limiting toxicity dictates a need for continued development of better therapeutic agents for this potential bioterror disease. it has been reported previously that st- , a low-molecular weight compound, inhibits replication of all the orthopoxviruses in vitro and protects mice infected with vaccinia or ectomelia virus. in the present study, we have utilized cowpox virus (cv) and vaccinia virus (vv) infections in vitro and in vivo to evaluate the efficacy of st- for treatment of orthopoxvirus infections. in plaque reduction assays in human foreskin fibroblast cells, both cv and vv were inhibited by about . - . um of st- . for in vivo studies, st- was administered once daily by oral gavage to mice using mg/kg for , , , or days beginning or h after intranasal inoculation with vv or cv. st- was highly effective (p < . ) in preventing mortality due to vv or cv even when treatment was delayed up to h post-infection. a dosing duration of days was adequate for vv infected mice, but duration of days or longer was required for efficacy in cv infected mice. when st- was given once daily for days at , , or mg/kg daily at , , or h post-cv inoculation, mortality was significantly altered at all dosage levels and time points. to determine the effect of treatment on virus replication in target tissues, mice were inoculated with cv or vv and treated once daily with mg/kg of st- . on various days post-infection tissues were harvested and assayed for virus. in cv or vv-infected mice, st- treatment successfully reduced virus titers from to logs in liver, spleen, and kidney. little effect was noted in lung tissue. these results indicate that st- has significant activity against vv and cv infections in vitro and in vivo and may be a potential chemotherapeutic agent for treatment of human orthopoxvirus infections. cidofovir (hpmpc) is a broad-spectrum anti-viral agent that is used (vistide ® ) to treat aids-related cmv retinitis. currently, cidofovir is of particular interest as a potential therapy for orthopox virus infections, including smallpox. an important limitation of cidofovir and analogous nucleotide drugs in a therapeutic role is their low oral bioavailability and poor transport into cells. in principle, bioavailability of a drug can be improved by structural modification targeting transporters expressed in human intestine. to be effective, the transported prodrug must be cleaved by endogenous enzymes to its parent compound. we will present synthetic studies of novel cidofovir and cyclic cidofovir (chpmpc) prodrugs incorporating amino acids or small peptides, comparing different drug-amino acid linkage strategies. the compounds were evaluated for transporter-mediated uptake and cellular and plasma hydrolysis. the results will be compared with similar studies carried out on a series of peptidomimetic conjugates of foscarnet, the trisodium salt of phosphonoformic acid (pfa), an anti-viral agent that also has very low oral bioavailability and poor cell penetration. the question addressed in this study is if wnv-reactive antibody can improve disease signs in a hamster model after the virus is demonstrated to be in the brain. the hypothesis is based on the high activity of a humanized monoclonal antibody, he , in a mouse model when administered later in infection (oliphant et al., . nat. med. , ) . in this study, virus was demonstrated to be in the brains of hamsters at days post-viral injection (dpi) by cell culture assay, quantitative rt-pcr, and immunohistochemical staining of wnv in neurons. eighty percent of hamsters treated i.p. dpi with mg/kg of humanized monoclonal antibody, he , survived wnv disease, whereas, % of placebo-treated hamsters survived ( *** p < . ). if administered at dpi, % survived. we tested the hypothesis that he is effective if delivered directly into the brain instead of by peripheral administration. the antibody was delivered into the brain dpi using convectionenhanced delivery through a cannula implanted into the brain. the he was detected in the cns, but none was detected in the kidney. the survival of he -treated hamsters was % as compared to % of placebo-treated animals ( *** p < . ). for additional proof, the majority of hamsters having wnv in their cerebrospinal fluid, a marker for cns infection, were protected with he administered i.p. at dpi. this humanized monoclonal antibody, therefore, is a possible treatment for the post-exposure, wnv-infected humans that develop signs of neuroinvasive disease. acknowledgement: supported by contract no -ai- from the virology branch, niaid, nih, and grant -u ai - from the rocky mountain regional centers of excellence, nih. hemorrhagic fever viruses are of serious worldwide health concern as well as potential biological weapons. lassa fever virus in particular annually infects several hundred thousand individuals in west africa, and the export of this pathogen outside of this region, either intentionally or unintentionally, presents a serious risk to the developed countries of the world. the cdc and niaid have identified lassa fever virus as a category a priority pathogen, indicating the highest degree of threat to public health. no arenavirus-specific antiviral drugs are currently approved for use in humans. the purpose of siga's biodefense program is to develop safe and effective drugs for preventing and treating diseases caused by category a viruses. to that end, a large and diverse library of small molecule compounds was screened using a viral pseudotype assay to identify inhibitors that target the essential lassa surface glycoprotein (gp) and thus block viral entry into the host cell. twenty-six compounds were identified as quality hits, as defined by potency, selectivity, and chemical tractability. antiviral activity against authentic lassa fever virus was assessed in cell culture through a collaboration with colleagues at usamriid. a number of these potent antiviral compounds and their related analogs have exhibited informative chemical structure-biological activity relationships (sar). two potential lead compound series have emerged from these studies, each with % effective concentrations (ec s) of less than nm against lassa fever virus and with ec s of less than nm against lassa gp-pseudotyped virus. characterization of the in vivo properties of these compounds is underway. the in vitro antiviral potency and selectivity, animal pharmacokinetics, and the development process will be presented. these inhibitors represent an important step toward the development of a small molecule antiviral drug for lassa fever virus. sven enterlein , pramila walpita , allison groseth , heinz feldmann , ramon flick university of texas medical branch, department of pathology, galveston, tx, usa; national microbiology laboratory, public health agency of canada, winnipeg, man., canada nipah (niv) virus (family paramyxoviridae) is a recently emerged human and animal pathogen that can cause severe encephalitis with fatality rates of up to %. since no treatment or vaccination is available, and cross-species spread was observed, the virus has been classified as biosafety level (bsl- ) agent. to avoid bsl- containment for the study of cis-acting signals as target for antiviral strategies, we used an optimized plasmid-driven t minigenome rescue system (without the need for recombinant vaccinia virus mva-t ) as well as an newly established rna polymerase i-based approach. minigenome rescue is based on transfection of the minigenome niv-cat and the plasmids encoding for the three nucleocapsid proteins n (nucleoprotein), l (polymerase), and p (phosphoprotein) and measured by enzymatic cat assays. we used the established plasmid-based minigenome rescue systems to screen for potential antiviral compounds. in a first step we tried to determine the optimal strategy for the delivery of small hairpin (sh) interfering rna molecules. for this we compared three shrna delivery systems against another bsl agent-reston ebolavirus (family filoviridae); (i) plasmid-mediated pol i and (ii) pol iii-driven shrnas, and (iii) exogenously (t ) produced shrna, for their ability to induce gene silencing. interestingly, beside the in vitro-generated or pol iii-driven shrnas, pol i transcripts showed very efficient inhibition of minigenome rescue. however, the most efficient delivery method was transfection of in vitro transcribed shrnas. we will present the results of this comparison and, based on the most efficient approach, also first results of shrnas targeted either to niv n, p, and l genes or to the leader/trailer noncoding regions to interfere with minigenome replication. conformative data with live virus experiments under bsl conditions will be included. filoviruses, which include ebola virus and marburg virus, are among the most notorious human pathogens because they cause sporadic outbreaks of severe hemorrhagic fever. unfortunately, very few therapeutic agents are available to treat infections with these viruses. antiviral screening methods that determine the effect of compounds on viral replication involve working with infectious virus, which is obviously not practical for these biosafety level (bsl- ) agents. we developed an antiviral screening method based on a cell-based, infection-independent, ebola subgenomic replication system in which the expression of an easily measurable enzyme is dependent on the rna replication and transcription factors of ebola virus. using this system we screened a synthetic compound library for antiviral activity against ebola virus and have identified a number of inhibitors. we also used it to identify a peptide inhibitor directed against vp . anti-ebola virus activity for many of the inhibitors was confirmed in a viral replication assay using a gfp-expressing zaire ' strain of ebola virus. fifty-two small molecule inhibitors from at least six classes of compounds had ec values in the low micromolar range and good selectivity. several of these compounds have promising chemical, biological, and pharmacological profiles to pursue as potential anti-filovirus drugs. we are currently preparing to test these compounds in a mouse model of ebola virus. we have also begun a lead optimization program to improve antiviral potency and selectivity of aryl sulfonamide and -aminoquinoline compounds. acknowledgement: supported by nih r ai - and r ai - . human papillomavirus (hpv) has been a difficult virus to target by traditional antiviral methods due to its small size, its small number of obvious therapeutic targets, and its resistance to propagation in vitro. nevertheless, antiviral compounds that reduce hpv dna load have the potential to prevent carcino-genic progression in infected patients. to that end, we developed an approach that dramatically reduces the hpv episomal dna load of keratinocytes in vitro by targeting viral dna sequences. pyrrole-imidazole polyamides, with some containing fluorescent probes to aid in cell localization studies, were designed to recognize the hpv ori. all fluorescent compounds rapidly localized to the nucleus of cultured keratinocytes following addition to the culture media. the compounds were then tested for their ability to alter keratinocyte hpv episomal dna content. two of the compounds caused a dose-dependent reduction in hpv episomes as measured by taqman tm realtime pcr. while control and vehicle-treated cells maintained ∼ copies of hpv per cell, compounds -ta and -ta both reduced hpv dna levels to below copies per cell after h incubation with m compound. an alternative taqman tm amplicon within the hpv e gene produced identical results. a multiplexed taqman tm real-time pcr reaction that followed the ratio of hpv dna to the human apoe gene also demonstrated dramatic loss of hpv dna copies, further confirming our initial observations. finally, cells were treated with polyamides for h, polyamide-containing media was removed, and episome levels were followed for days. at day , days after removal of polyamide and days after sub-culturing of the cells, viral episome levels remained approximately % lower than control samples. by day , days after removal of polyamide, viral dna levels were beginning to recover but still remained significantly lower than control samples. together our results demonstrate that targeting the hpv origin of replication with dna-binding compounds dramatically reduces episomal dna levels. small interfering rnas (sirnas) are potent tools for gene down-regulation but are minimally stable in cells. to improve the efficacy of sirna, we replaced non-bridging oxygens in the phosphodiester linkages of natural rnas with bh groups. the resulting boranophosphates have unique properties, including enhanced nuclease resistance, altered hydrogen bonding of the phosphate, different interactions with metal ions, and increased thermal stability of rna:rna and rna:dna duplexes. anti-egfp sirnas containing boranophosphate modifications were prepared by in vitro transcription with t rna polymerase from ribonucleoside -(alpha-p-borano)triphosphates, as well as normal and phosphorothioate sirnas. after confirming the presence of the borane modifications with maldi-ms, several properties of borano-modified sirnas were investigated: ( ) the double stranded rna with borane modifications maintained the a-form conformation characteristics according to the circular dichroism (cd) spectra; ( ) the borane groups in the sirnas increased the thermal stability, with an enhancement of t m by . - . • c per modification; and ( ) sirnas with borano-modifications were shown to be at least -fold more resistant to rnase a digestion than normal ones. when these modified sirnas were used to down-regulate egfp expression in hela cell cultures, it was found that: ( ) borano-modified sirnas were consistently more effective than sirnas containing the corresponding phosphorothioate modifications; ( ) borano-sirnas were more effective than normal sirnas provided that the center of the antisense strand was not heavily modified; ( ) borano-sirnas were more potent than normal or phosphorothioate sirnas at lower concentrations; and ( ) finally, the silencing activity of boranophosphate singlestranded sirna (ss-rna) was comparable to that of unmodified ds-sirna. the borano ss-rna had excellent maximum silencing activity and was highly effective at low concentrations, and silencing activity was durable up to one week after transfection. results with anti-hpv sirnas will be discussed. boranophosphate modification is a potential new class of anti-viral therapeutic agents. this report describes the antiviral structure activity relationships that led to the discovery of phosphonomethoxy- -fluoro- , dideoxydidehydroadenosine (fd ap, gs ), a novel ntrti, with an excellent resistance profile toward hiv- variants containing major n(t)rti resistance mutations. methods: phosphonomethoxy analogs on purine and pyrimidine dideoxydidehydro (d ) and dideoxy (dd) ribose scaffolds were prepared. antiviral activity was measured against wildtype and n(t)rti-resistant recombinant viruses using cytopathic assay in mt- cells. mitochondrial toxicity was assessed in hepg cells by measuring mitochondrial dna content. results: the d scaffolds displayed superior antiviral activity compared to the dd scaffold and adenine was superior to other nucleobases. phosphonomethoxy- , dideoxydidehydroadenosine (d ap) inhibited hiv- replication with a mean ec of . m and an . -, . -, and . -fold change in potency against viruses containing m v, k r, and thymidine analog mutations (tams), respectively. further exploration of d ap was limited by its mitochondrial toxicity, which was then addressed in ways: (i) preparation of l-d ap or (ii) fluorine substitution. l-d ap exhibited an ec of . m but had substantially reduced potency ( -fold) toward m v mutant viruses. fd ap exhibited an ec of . m, with . -, . -, and . -fold change in potency against viruses containing m v, k r, and tams, respectively. no cytotoxic effects were measured up to mm in mt- cells and no effects on mitochondrial dna were detected up to m in hepg cells for both fd ap and l-d ap. conclusion: fd ap is a novel phosphonate ntrti with antiretroviral activity toward wild-type and resistant mutant hiv- strains. compared to d ap, the -fluorine atom significantly improved the in vitro toxicity profile while retaining the favorable resistance profile. in subsequent studies, the monoamidate prodrug strategy was applied to fd ap to achieve optimal in vivo pharmacokinetic properties. entry inhibitors, and ccr- antagonists in particular, have become one of the most actively pursued treatments for hiv within the pharmaceutical industry. recently, multiple groups have disclosed piperidine-based ccr- antagonists that -to the medicinal chemist's eye -might appear to share a common three-point pharmacophore comprised of a tertiary amine, a phenyl ring, and a carboxamide or sulfonamide group. in several of these cases, these pharmacophoric elements are tethered together by a flexible, aliphatic chain. we sought to improve the potency of and introduce structural novelty into this class of compounds by rigidifying this tether. herein, we describe stereoselective syntheses and sar of a series of ccr- antagonists wherein the tether has been replaced with four stereochemical isomers of a rigidified cyclopropyl scaffold. the regulation of hiv transcription is a complex, multistage process that requires the concerted action of viral and cellular proteins. we discovered the n-aminoimidazoles (naims) as a unique class of hiv inhibitors targeted at the viral transcription level. a prototype naim, nr- , prevents the reactivation of dormant virus by inhibiting both the hiv- p and viral mrna production from latently hiv- -infected cell lines upon stimulation with tnf-␣, pma, or tsa. extensive research revealed that nr- was unable to inhibit the nf-b activation pathway or chromatin remodeling at the viral promoter, both known to be crucial for viral transcriptional activation. focusing on the viral transcription process, chromatin immunoprecipitation (chip) experiments revealed that nr- was able to inhibit the ser phosphorylation of the c-terminal domain (ctd) of rna polymerase ii. this step is mediated by the cdk subunit of p-tefb, which is recruited to the viral promoter by the hiv- tat protein. since we did not find an inhibition at the level of cdk activity or tat-mediated transcription in tat-expressing cell lines transiently transfected with a ltr-gfp construct, we infer that nr- must interfere with the transcription process by a unique mode of action. evidence points towards a kinase, not belonging to the cdk family, to be the target of the naims, resulting in an antiviral action at the level of retroviral transcription. clara e. cases-gonzález , sandra franco , miguel a. martínez , luis menéndez-arias centro de biología molecular "severo ochoa", csic-uam, madrid, spain; fundació irsicaixa, hosp. university germans trias i pujol, badalona, spain a ser-ser insertion at codons - together with substitutions t s and t y in the reverse-transcriptase (rt)-coding region of hiv- are known to confer resistance to zidovudine (azt) and stavudine (d t). phenotypic resistance correlates with increased atp-dependent phosphorolytic activity on inhibitor-terminated primers. we have previously shown that an rt derived from a clinical isolate (ss rt) that contained the insertion and additional mutations related to drug resistance (including t y) showed > -fold increased unblocking activity on azt-and d t-terminated primers, when compared with an rt containing the insertion together with mutations t s and t y, in an otherwise wild-type bh sequence. these results suggested that other mutations associated with the complex t sss/t y in clinically relevant rts contributed to increase atp-mediated excision activity and conferred high-level resistance to azt and d t in phenotypic assays. to identify residues increasing the excision activity, we obtained recombinant enzymes bearing ss rt residues - and wild-type bh rt residues - (l rt), or residues - of the bh rt and - of the ss rt (l rt), as well as an l rt variant with the substitution t y (l rt) and an l rt derivative with t sss (l rt). additional rts containing mutations m l, a v, or k r together with the combination t sss/t y in the bh background were also obtained. atp-mediated excision activities on azt-and d tterminated primers were determined and the effects of mutations were tested in phenotypic assays using recombinant hiv- . the l rt containing mutations t sss/t y and additional changes in the n-terminal region showed the highest atp-dependent phosphorolytic activity on blocked primers, giving values similar to those reported for the ss rt. results were consistent with phenotypic data. in contrast, l , l , and l rts displayed low-level activity. further experiments revealed that three amino acid changes at the n-terminal region of the polymerase (m l, a v and k r) were responsible for the increased excision activity shown by rts bearing mutations t sss and t y. from a series of phenyl-substituted thiazolobenzimidazoles, several compounds were identified as selective inhibitors of coxsackie b virus replication in vero cells. a structure-activity relationship was established, from which the -trifluoromethyl substituted analogs emerged as the most potent congeners. the compounds were active against all six coxsackie b strains tested. the in vitro antiviral activity of one of the most selective compounds, i.e. chi- , was assessed by (i) mts-based cytopathic effect assays, (ii) virus yield reduction assays, (iii) real-time quantitative pcr (rt-qpcr) and (iv) by monitoring viral antigen expression. in all assays a clear concentration-response effect was obtained. the % effective concentration (ec ) was . ± . g/ml, while the cc ( % cytotoxic concentration) of chi- for vero cells was more than g/ml, thus resulting in a selectivity index of > . detailed single cycle time-of-drug-addition studies (in which viral replication was monitored by means of rt-qpcr) revealed that the compound interacts with viral replication at a time that coincides with the onset of intracellular viral rna synthesis. chi- resistant virus is being generated by culturing the virus in the presence of increasing drug concentrations. drug-resistant virus will be genotyped, which should allow us to identify the (putatively viral) molecular target of this class of compounds. retroviruses hiromichi tanaka , kazuhiro haraguchi , hiroki kumamoto , takao nitanda , masanori baba , ginger e. dutschman , yung-chi cheng school of pharmaceutical sciences, showa university, tokyo, japan; center for chronic viral diseases, kagoshima university, kagoshima, japan; school of medicine, yale university, new haven, ct, usa our recent research program on the development of synthetic methods for -carbon-substituted nucleosides has led to a new strategy, ring opening of , -epoxy-nucleosides with organoaluminum and organosilicon reagents. this enabled us to introduce alkyl, alkenyl, and alkynyl groups to the -position. as a result of this study, -ethynylstavudine ( -ed t) was found to be more anti-hiv active than the parent compound stavudine (d t). this compound ( -ed t) has several additional appeals as a promising anti-hiv agent: much less toxic to various cells and also to mitochondrial dna synthesis, better substrate for human thymidine kinase than d t, very much resistant to catabolism by thymidine phosphorylase, its activity enhances in the presence of a major mutation k n known for nnrti-resistant hiv. in this conference, we present the synthesis and sar studies of -ed t analogues modified mainly in the sugar portion. negatively charged polymers (np) possess a broad immunoadjuvant and antiviral activity topically useful for vaccine, drug, and microbicide development. but their efficiency is limited over a reversibility of electrostatic kind of interference with virusspecific nano-objects. to overcome this limitation the purposemade intra-molecular modifications of np were studied among non-toxic maleic acid co-polymers (npsa), dextran and chitin derivatives (npps) within varied alicyclic modifiers application. the configurationally flexible alkyls (i), as non-alicyclic control, are ineffective synergist for np antiviral potency. monocycles (ii) are moderate active too. on the contrary the hardconformation frame-structured spheroids (iii-vi) exhibit ability (at optimal macromolecular parameters) to be super-effective synergists for strength and diapason of np antiviral action. unlike small molecular iii/iv-containing prototypes (amantadin, rimantadin, deitiforin, etc.), narrowly-effective inhibitors mainly of influenza a viruses, the np-coupled modifications become effective also against many other viruses, including the drugs resistant strains [antivir. res. ( ), ]. in focus of the anti-hiv potency the ivs provide a - -fold elevation of np activity. the more available and less toxic iii species are similarly active, but iii* (with spatial-optimally contactable double bond due to the exo-configuration) turns out the best synergist - -fold amplifying the anti-hiv- selectivity up to is∼ . augmentation of the frame cycles from iii-iv toward v-vi results in no essential enhancement of antiviral activity, but stimulates toxicity. the recently involved in the investigation vii, cholesterol-like systems, as tools for novel raft-targeted strategy, demonstrate capacity for at least -fold amplification of anti-hiv- potency our earlier studies showed that esterification of cidofovir (hpmpc) with alkoxyalkanols increased antiviral activity by more that two logs and promoted oral bioavailability. to evaluate this approach with purine based nucleoside phosphonates, we synthesized several alkoxyalkyl esters of acyclic purine phosphonates such as , ,-diamino-( -[ -phosphonomethoxyethyl]purine (pme-dap) and -amino- -cyclopropylamino-( -[ phosphonomethoxyethyl]-purine (pme-cpr-dap) these purine phosphonates have been reported to be active against a wide range of viruses such as human immunodeficiency virus (hiv- ), other retroviruses, herpesviruses, poxviruses and hepatitis b virus. for this study several alkoxyalkyl analogs of acyclic , diaminopurine nucleoside phosphonates were synthesized and evaluated against hiv- . the alkoxyalkyl esters were more inhibitory than the unmodified compounds in p reduction assays in mt- cells infected with hiv- . for example, hexadecyloxypropyl (hdp) and oleyloxyethyl (ole) esters of pme-cpr-dap were > logs more active than unmodified pme-cpr-dap. in spite of increased cytotoxicity in mt- cells, the selectivity indexes are more than -fold higher then for unmodified compound. in conclusion, esterification of pme-dap and pme-cpr-dap with hexadecyloxypropyl-or oleyloxyethyl-residues greatly increased their antiviral activity and selectivity against hiv- in vitro. victor kuz'min , eugene muratov , anatoly artemenko , ludmila koroleva , vladimir silnikov , v. lozitsky , a. fedchuk a.v. bogatsky physical-chemical institute, odessa, ukraine; institute of chemical biology and fundamental medicine, novosibirsk, russian federation; ukrainian mechnikov research anti-plague institute, odessa, ukraine "chemical" ribonucleases hold promise as tools for studying the structures of rnas and rna-protein complexes, as reactive groups in conjugates intended for cleavage of particular rnas, as therapeuticals inactivating virus genome rnas or certain mrnas, and as a promising antiviral agents. drug design and development of new medicines directed against hiv are permanently actual tasks. the usage of modern quantitative structure-activity relationship (qsar) methods could allow us to solve these problems more effectively. the objective of the present work is qsar analysis of antiviral activity of various tetrapeptides-artifical ribonucleases and consequent molecular design of new antiviral agents. qsar approach based on simplex representation of molecular structure (sirms) has been used for the solution of the formulated problem. usage of sirms allows us to develop the molecular design of the new effective antiviral agents. thorough researches of relationship between antiviral activity (hiv- , % of rna p-o bond cleavage) and a structure of artifical ribonucleases have been carried out. statistic characteristics for pls (partial least squares model) are quite satisfactory (r = . , q = . ). on the base of these models the molecular fragments with positive or negative influence on the explored property have been determined. thus, for example, guanidine and triethylenediamine fragments promote antiviral action. it gives a possibility to realize based on elucidated rules molecular design of compounds with the high level of antiviral activity. the results of prognosis are verifying by the experimental investigations. thus, quite adequate simplex qsar model "anti-hiv activity-artifical ribonucleases structure" was obtained and used for drug design. the cyclotriazadisulfonamide (cada) compound specifically down-modulates the cd receptor expression on the surface of lymphocytes and monocytes/macrophages, the primary receptors utilized by hiv for infection of its target cells. cada thus inhibits the entry of hiv and hhv- (vermeire et al., . virology , - ) . cada chemotherapy may not be susceptible to the production of drug resistant strains of viruses, as its mechanism of action is completely different from those of any other anti-hiv drugs currently in clinical use. the cd down-modulating and antiviral potencies of more than cada analogs have been described (vermeire et al., . mol. pharmacol. , - ) . structural modifications of cada were made to increase potency, reduce cytotoxicity, and improve physical properties. several head group analogs were synthesized with polar groups and good leaving groups ( fig. ) . the anti-hiv and cd down modulation activities of these compounds are being studied. some of these head groups may regenerate the double bond of cada by elimination reactions, potentially producing water-soluble pro-drugs. isocada (sa ), an isomer of cada, was synthesized by cyclization of , , -triazabicyclo-[ . . ]dec- -ene (tbd) (fig. ). this structural modification may reveal a relationship between the symmetry of the molecule and its biological activity. two new fluorine-containing analogs were also synthesized by modifying the toluenesulfonamide side arms (fig. ) . the anti-hiv and cd down modulation activities of these new cada analogs are summarized. the center for drug discovery, university of georgia, athens, ga , usa drug discovery targeted at the elusive viral enzyme, hiv integrase, has not resulted in a single fda-approved drug. in this presentation we describe our molecular modeling studies with conceptually novel inhibitors of hiv integrase that also possess potent in vitro anti-hiv activity. docking was performed on the catalytic core of integrase represented by chain c of pdb structure code bl . building of molecules and primary modeling was done with sybyl . on a silicon graphics onyx (r ) workstation. the program gold . (genetic optimization for ligand docking) was used extensively in evaluating the docking poses of these compounds with the active site of hiv integrase and to give information on key residues involved in the recognition and binding of these ligands. the gold function consists of three basic components: protein-ligand h-bonding energy, protein-ligand van der waals energy, and ligand internal energy. post-processing gold output was done with the program silver . , a utility program supplied with gold for evaluating hydrogen-bonding interactions, metal coordination and van der waals factors. for comparison purposes, additional docking was performed using other docking protocols, notably the sybyl module flexx. data obtained from these and related studies including binding poses, binding affinities, functional and conformational considerations, and gold function scores will be presented and explained. the center for drug discovery, university of georgia, athens, ga , usa hiv integrase is essential for hiv replication and is an attractive target for drug discovery against aids. however, research efforts on drug discovery pertaining to hiv integrase have not resulted in a single fda-approved drug for which mechanism of action is inhibition of hiv integrase. recently, we have been exploring a novel class of diketo acids that are constructed on nucleobase scaffolds and that have a specific arrangement of the functional and hydrophobic group on the scaffold. these compounds are inhibitors both key steps of hiv integrase. one lead compound from this group has also been found to have remarkable in vitro anti-hiv activity. however, the syntheses of the inhibitors are quite challenging. this presentation will describe the synthetic methodologies specifically developed in our laboratory for the preparation of some representative examples of these integrase inhibitors. purification approaches to produce highly purified compounds for biological studies will be explained. structural, functional and conformational data obtained from extensive spectroscopic studies will be discussed. representative anti-hiv integrase data and in vitro anti-hiv screening results will be presented. we have recently reported the synthesis and antiviral activities of a ring-expanded ("fat") nucleoside analogue, called nz- , that inhibits both hcv and hiv in vitro with ec values ranging in micromolar concentrations or less, with little or low toxicity to the host cells. in this part i of the presentation on this subject, we report our preliminary findings on the mechanism of anti-hiv activity of this compound, along with the synthesis and antiviral activity of a few additional analogues. in view of the fact that a number of hiv patients also suffer from hcv as a major coinfection, and that a number of them ultimately die of end-stage hcv-related complications including liver cirrhosis and hepatocellular carcinoma, a drug with dual inhibitory characteristics against both viruses is highly desirable and timely. marina burshtein , alexander serbin , alissa bukrinskaya d.i. ivanovsky institute of virology, moscow, russia; health research and development found, moscow, russia introduction: amantadine is a well-known effective antiinfluenza drug. it was modified to enhance its antiviral activity by chemically linkage with the water-soluble polyanionic matrix via different spacer groups. the other group of used compounds was norbornene derivatives, as norbornene is an adamantane analogue on anti-influenza activity. methods: the absence of cytotoxic effect was shown by mtt test for estimating cytotoxic dose (ctd ). the antiviral effect of the compounds was analyzed in lymphoblastoid mt- cells and in hela cd +/b-galactosidase cells ("magi" cells). the effect of the compounds was registered by immunoblotting of cell lysates and by measuring of b-galactosidase activity. results: the strong inhibition of hiv- replication was observed when the compounds were added with the virus and was expressed even when the compounds added with the virus were removed h after infection. the anti hiv- effect of the compounds was gradually decreased if they were added and h after infection, no inhibition was observed when the compounds were added h after infection. the compounds did not impair the virion structure. adamantane and norbornene derivatives were shown also to inhibit azt resistant viral strains. conclusion: adamantane and norbornene were shown to be active hiv inhibitors with the high selectivity index. the compounds are promising candidates for further investigation including preclinical studies. less is known about the effect of their intracellular half-lives on the maintenance of antiviral activity. to investigate this question, we developed a novel in vitro antiviral persistence assay. measurement of the antiviral persistence of tenofovir (tfv) and abacavir (cbv) was coupled to measurement of the half-lives of their tfv-dp and cbv-tp anabolites. methods: mt- cells or stimulated primary cd + t-cells were incubated with graded concentrations of tfv or cbv for h (h); then extracellular drug was removed by washing. cells were further incubated without drug for - h and then infected with hiv- (iiib or bal). p was quantified on day ; inhibition of hiv- replication due to intracellular drug persistence (pc ) was determined relative to a standard ec . decay of intracellular dp/tps in cd + t-cells was measured using lc/ms/ms. results: in mt- cells, the pc value for tfv h after drug removal remained unchanged relative to the ec (< -fold shift) whereas the pc for cbv shifted > -fold, indicating less persistence of cbv. in cd + t-cells, the pc value for tfv also showed a minimal shift relative to the ec ( . -fold) h after drug removal. cbv showed a much larger relative shift (> -fold). quantification by lc/ms/ms of intracellular tfv-dp and cbv-tp in cd + t-cells in vitro demonstrated that tfv-dp had the longest intracellular half-life of the two drugs (tfv-dp, h versus cbv-tp, h). conclusions: a novel antiviral persistence assay was developed to study the relationship between intracellular nrti halflives and antiviral activity. in both mt- cells and primary activated cd + t-cells, tfv had the longest persistence of antiviral activity. in cd + t-cells, tfv-dp also had the longest half-life of the two nrtis. cbv-tp had a much shorter half-life than tfv-dp and showed less antiviral persistence. although both drugs are approved for qd dosing, the half-life of intracellular tfv-dp maintains antiviral suppression in vitro over a timeframe most consistent with qd dosing. karen m. watson, tracy l. hartman, lu yang, robert w. buckheit jr. imquest biosciences, inc., frederick, md, usa isis is a phosphorothioate oligonucleotide with a molecular structure of t g t . the g-quartet possessing molecule has been shown to be a potent inhibitor of hiv attachment and cell-cell fusion and acts by specifically interacting at the v loop of gp . mapping studies with monoclonal antibodies targeting epitopes in and around the v loop have been used to define the binding site of isis . in vitro, isis inhibits all laboratory and clinical strains of hiv- and hiv- tested, including representative subtype viruses, drug resistant viruses (including mdr viruses) and viruses that utilize the cxcr and ccr chemokine receptors. serial passage of virus in the presence of increasing concentrations of the oligonucleotide did not result in the selection of drug resistant virus strains and combination assays resulted in additive to synergistic interactions with other approved hiv inhibitors. the antiviral and toxicity profiles of isis resulted in the performance of human clinical trials for the therapeutic use of the oligonucleotide to treat hiv infection. the antiviral properties and mechanism of action of isis suggest that it may be an excellent anti-hiv topical microbicide. isis was found to be highly active in a cervical explant model of hiv infection with highly significant inhibition of ccr -tropic strains of virus. activity was also observed in cell-free and cell-associated virus transmission assays, as well as in cd -dependent and cd -independent acute infection inhibition assays. in microbicidal specific combination assays, significant efficacy has been observed with isis used in combination with other microbicidal compounds. the results of these studies suggest that isis may represent a new and novel anti-hiv topical microbicide. karen m. watson, tracy l. hartman, lu yang, robert w. buckheit jr. imquest biosciences, inc., frederick, md, usa though a variety of compounds are being developed as anti-hiv topical microbicides, such as polyanionic molecules, surfactants, natural products, peptides, proteins, heterocycles, and virucidal agents, clinical efficacy studies that demonstrate the ability of these agents to impact virus transmission are still in progress. it has been estimated that a microbicide that is only % effective would have the capacity to prevent millions of new infections each year. thus, one of the challenges in hiv drug development is the discovery of compounds that will inhibit the sexual transmission of infectious organisms between sexual partners. the rapid mutability of hiv and the known presence of drug resistant viruses in wild type virus populations suggests that microbicide development will suffer from the same problems that exist for all hiv therapies, namely the selection of resistant virus strains that will bypass the microbicide barrier and infect target cells in the vaginal or rectal environment even in the presence of the microbicide. thus, it is likely that haart-like combination drug therapies will become the most effective means of inhibiting the sexual transmission of hiv. we have evaluated a wide variety of anti-hiv and anti-sti compounds in vitro alone and in combination with one another and have demonstrated that certain patterns of inhibition (additivity, synergy, antagonism) occur between the various classes of compounds. recently, we have compared the combination anti-hiv activity of microbicide compounds in fresh human pbmcs infected with clinical isolates of hiv to the combination activity of the same test agents in cem-ss-based cultures. in general, these two assay systems yield similar combination assay results. to provide a rationale for the combination use of the compounds in a microbicide setting, the same combination of compounds was evaluated in a microbicide-like virus transmission assay. these combination results suggest that higher levels of synergy between virus attachment and reverse transcriptase inhibitors might be expected in the microbicide environment compared to levels predicted for the systemic therapeutic environment. the results of the combination assays with various microbicides will be presented. during the onset of the hiv disease, hiv rna is continually produced in the face of treatment with haart in circulating reservoirs and rt inhibitors are almost ineffective in the postintegration events. among the classes of anti hiv- drugs, protease inhibitors (pi) are the unique to inhibit the hiv- production in chronically infected macrophages. in the progression of hiv infection, the role of the monocytes-derived macrophages (m/m) is further confirmed as they represent chronologically the first cytotype where the viral replication restarts as a consequence of failure or interruption of antiviral therapy. aim of the work was to evaluate the rebound of hiv- production when pi have been removed in hiv- chronically infected m/m and, moreover, to verify the effect of this removal on virus maturation, infectivity and ability to trigger apoptosis in uninfected peripheral blood lymphocytes (pbl). a rebound of p gag protein was measured starting from h after drug removal yet virus infectivity remained log lower than control up to week. inhibition of hiv- replication was still % and % upon amprenavir and m, respectively. these data were confirmed by western blotting and electronic microscopy showing production and release of immature viral particles. moreover, pi (amprenavir and indinavir) treatment dramatically reduced apoptosis of pbl co-cultured with chronically infected m/m and kept cd /cd ratio above the levels of untreated controls until the th day of co-culture. taken altogether, these findings suggest a wide clinical importance for amprenavir and indinavir for their relevant long-lasting antiviral effect in persistently-infected reservoirs of hiv even in case of drug interruption and/or when hiv infection can restart in districts where drugs find not sufficient concentration. moreover, these results strengthen the evidence for an unique positive utilize of pi against ongoing and productive hiv infection. weili jin, salvatore santino, michael wang gilead sciences, foster city, ca, usa background: effective inhibition of hiv reverse transcriptase (rt) currently represents a crucial objective of antiretroviral therapy. capravirine is a second-generation non-nucleoside rt inhibitor (nnrti) that is capable of blocking the replication of certain nnrti-resistant strains of hiv and was recently in clinical development. in this study, we report on the in vitro selection and characterization of viral resistance to capravirine. methods: viral resistance selection experiments were performed in mt- cells with the hiv iiib isolate and increasing concentrations of capravirine. viruses were analyzed genotypically by population sequencing and by single genome sequencing (sgs). recombinant viruses with nnrti mutations were generated from proviral dna clones. phenotypic analyses were performed in mt- cells. results: capravirine resistance selections were initiated at nm (ec of . nm for capravirine). following nine passages in the presence of increasing concentrations of capravirine, the l i mutation emerged in rt and additional passaging led to v d and f c mutations at higher concentrations ( - nm). further increases in capravirine concentrations led to the emergence of a l i + v d + f c triple mutant, which confers > -fold resistance to capravirine. sgs of mixed viral populations from different passages showed that l i, v d and f c were present on the same genome, with l i as the primary mutation, and f c and v d were acquired sequentially at later passages. through sgs analysis, a l i + k r + v d + f c quadruple mutation on the same genome was also observed at higher capravirine concentrations (> nm). recombinant viruses carrying these mutations were produced to assess their susceptibilities to capravirine. conclusions: after extensive in vitro passaging of hivinfected cells in the presence of capravirine, neither k n nor y c mutations in rt were observed. instead, the l i mutation was initially acquired, followed by mutations f c and v d. addition of the k r mutation to the triple mutant genome, l i + v d + f c, appears to further enhance hiv resistance to capravirine. oluwafemi olawuyi , adeyemi falegan medical microbiology, university college hospital, ibadan, nigeria; dentistry, university college hospital, ibadan, nigeria issue: the percentage of aids/hiv is increasing every year in the third worlds, and this is reinforced by the factor that majority of youth in third worlds do not know his/her hiv status. description: a self developed validated and reliable questionnaire [r = . ] was used to collect the data and percentage was used to analyze the data. the population of the study was made up of youth [female and male] in higher institutions, working places, market places and community streets in nigeria, , -sample size, selected through simple random sampling technique. the mean age is . years old. relative risk [rr] calculated is . , i.e. rr > , indicating that the factor is the risk factor, and the confidential interval [ci] for rr at % significant level is . < . < . from the formula, ci lower limit < rr < ci upper limit. lessons learned: seventy percent of the sample population did know his/her hiv status and had had sexual intercourse in the past before, out which % had the unprotected intercourse once or more, % had protected sex while % were not sure of using protection means. while, % have knowledge about own hiv status and had had sexual intercourse before. ten percent have no knowledge about own hiv status and had no sexual intercourse before. conclusion: aids/hiv still remains a killer disease in the third world. however, the lack of knowledge of individual's hiv status remains the only highest risk factor for the spread of the disease in the third worlds. yuichiro habu , , jacob barnor , , norio yamamoto , kahoko hashimoto , , naoko miyano-kurosaki , , koichi ishikawa , naoki yamamoto , david ofori-adjei , hiroshi takaku , , department of life and environmental sciences, chiba institute of technology, chiba, japan; high technology research center, chiba institute of technology, chiba, japan; japan foundation of aids prevention; department of molecular virology, bio-response, tokyo medical and dental university, tokyo, japan; aids research center, national institute of infectious disease, tokyo, japan; department of virology, noguchi memorial institute for medical research accra-ghana, accra, ghana; bach tech corp. rna interference (rnai) is a potentially strong gene interference tool, which had been successfully used to silence many pathogenic viruses including hiv. however, many recent reports have shown that, in long-term assay cultures involving rna viruses such as hiv, escape mutants breakthrough the silencing effect. in the light of this conundrum, it had been proposed that, vector designed to target multiple genes in a synergistic manner, may address the problem. hence, we designed a chimeric rna expression vector which express vif shrna and decoy tar rna by combining vif shrna and decoy tar rna with linker to which dicer was able to recognize for cleavage, as a second generation rnai expression vector system. the synergistic effect of these molecules enhanced the inhibition of hiv- replication in a long-term transduced pbmcs, h , and jurkat cell culture assays ( weeks) and prevented virus breakthrough associated with sirna-mediated escape variants. notably, hiv- replication was similarly suppressed in the control cells expressing only vif shrna for about weeks, but an increase in virus replication was observed afterwards. hiv viral rna extracted and sequenced at this point indicated escape mutants in the cells expressing the vif target in hiv. we confirmed substitution of bases in the vif shrna target sequence. on the other hand, the incidence of mutation was not observed in a sequence of viral rna from the culture expressing the vif shrna-decoy tar rna at the fourth week. interestingly, virus production was inhibited for a long-term by an effect of decoy tar rna, through the rna-protein interaction. combining shrna with decoy tar rna as second-generation anti-hiv shrna may provide practical basis for applying sirna-based gene therapy to the treatment of hiv/aids. introduction: this is a designed efficient gene therapy against aids/hiv. the novelty of this aids vaccine design/concept is seen in the fact that the 'pol' gene encoding for nonstructural proteins (polyproteins that generate three enzymes: reverse transcriptase, integrase and protease) is cloned in a suitable retroviral vector and adult stem cells are transfected by this and reinfused into the circulation to effectively counter hiv replication and antigenic variation. method: the mrna are isolated from adult stem cells and transcribed into cdna with reverse transcriptase. the cdna are then cloned in a suitable retroviral vector (vacinia) carrying 'pol' gene that confers resistance to a strong reverse transcriptase inhibitor drug. the adult stem cells are transfected by the recombinant mixture, and reinfused into the circulation of hiv infected person. result: the transfected stem cells are reinfused to provide renewable source of more and better empowered normal blood cell types that would disrupt and half hiv replication in the circulation. there would be efficient induction of both humeral and cellular mediated immunity with prolonged expression of antigens and protective immunologic memory generation against hiv antigenic variation. conclusions: this aids vaccine design would lead to both efficient prophylactic and therapeutic therapy against aids in that it would effectively take care of the problematic factor of hiv antigenic variation which has long been the main obstacle to potent aids vaccine development. because of the real risk of interspecies transmission and/or reassortment between avian, swine and human influenza a strains, drug susceptibility monitoring of circulating avian and porzine virus strains appears to be warranted for effective application of antiviral drugs like amantadine. this study was designed to gain insight into amantadine susceptibility of avian and porcine influenza a viruses isolated in germany between and . virus strains were isolated in embryonated chicken eggs and passaged one time in mdck cells. plaque reduction assays were applied to examine virus susceptibility to amantadine. genotyping was used to confirm drug resistance. in the result of these antiviral studies, only of the porzine isolates but all avian isolates were shown to be amantadine-susceptible. interestingly, the three amantadinesensitive porzine strains were isolated between and . all porzine influenza a viruses isolated later on were drugresistant and contained the aa substitutions g e, s n, and r q in the matrix protein (m ). additionally, l a was detected in two h n strains. s n and/or l a are well known amino acid substitutions in m that confer amantadine resistance. the role of the pig as an intermediate host of avian and human influenza a viruses, the possible involvement of genetic reassortment, and the high incidence of naturally amantadineresistant porcine influenza a viruses suggest a real risk of emergence of amantadine resistant human viruses. therefore, further studies are ongoing now to evaluate the circulation of the resistant phenotype in pigs, birds and human. recently much attention has been devoted to searching for effective chemotherapeutic agents and vaccines for eradication of this notorious disease. at present only chemotherapy is available to combat avian flu, for instance, tamiflu, approved for the treatment by the us-fda. development of a simple, novel molecule with potential antiviral activity against is essential to treat avian flu viral infection. isatin ( , -dioxoindole), is a versatile lead molecule for designing of potential antiviral agents and its derivatives were reported to possess broad spectrum antiviral activity. methisazone (nmethylisatin- -thiosemicarbazone) was first clinically approved for treatment of pox viral infections, and its derivatives were documented to have anti-influenza activity. based upon this evidence, the present work was initiated to determine the antiviral activity of novel isatin derivatives against avian flu (h n ) in mdck cells. antiviral activity was studied by virus yield assay (ec ), and cytotoxicity by neutral red uptake assay by uninfected mdck cells. all five compounds of a series inhibited the replication of avian flu (h n ) virus replication in mdck cells and compounds spiii- h and spiii- cl were most active (ec . g/ml, cc > g/ml and si > ). details of these studies and results of treatment of influenza-infected mice are discussed. acknowledgement: supported in part by contract noi-ai- and noi-ai- from virology branch, niaid, nih]. arginine-rich peptide conjugated phophorodiamidate morpholino oligomers (arp-pmo) are nuclease resistant antisense compounds that hybridize to target rna in a sequence-specific manner resulting in disrupted rna function. eight arp-pmo were designed to base-pair with various regions of a/pr/ / (h n ) rna and were then evaluated by hemagglutination and plaque assays for their ability to inhibit fluav production in vero cell culture. arp-pmo targeting the aug translation start site of the np or pb segment mrnas, or the -terminus of their respective vrnas, were highly effective, reducing influenza virus titer by - orders of magnitude in a dose-dependent and sequence-specific manner over a period of days. two of the p-pmo, targeting the pb translation start site region (pb -aug) and the terminus of np vrna (np v ), were evaluated by endpoint dilution (tcid ) or elisa assays against another h n strain (a/wsn/ ), as well as a/memphis/ / (h n ) and a/thailand/ (kan- )/ (h n ). the pb -aug arp-pmo generated over % specific reduction of virus level, regardless of viral subtype or methodology, at concentrations in the range of - m. the np v p-pmo yielded similar results, with the exception of considerably lower efficacy against the h n strain, with which it has two base mispairings. studies are planned to further evaluate of at least two arp-pmos in animal models for h n and h n fluva subtypes. macroheterocyclic compounds containing crown fragments and nitrogen atoms show large-scale biological activity. we synthesized series of aza-crown ethers and their derivatives. we also studied anti-influenza and antiherpetic action of some of them. anti-hsv action of studied compounds was tested using cyto-morphological method. hep- cells were infected with hsv- strain us in dose ifu/cell. the cells were incubated in eagle's medium that contained compounds in a dose of − m in experimental samples, or without them in control samples. then cells were fixed with % ethanol and stained with . % acridine orange solution. the amount of infected cells with dna-containing virus inclusion bodies was counted by fluorescent microscopy. anti-hsv activity of compounds was calculated as the difference between of the percentage of infected cells in treated cell cultures to the percentage of infected cells in untreated cell cultures. anti-influenza activity was studied on the model of replication of a/hong kong/ / (h n ) strain in tissue culture of chorio-allantoic membranes of chicken embryos. compounds were used in a dose of − m during the study of their anti-influenza action. diaza- crown- and two of its derivatives have showmen neither anti-hsv nor anti-influenza activity. diaza- crown- derivatives that contain -oxyethyl-or ethoxycarbonyl-fragments decreased amount of cells infected by hsv- by and %, respectively. both of these compounds inhibited replication of influenza virus on . log tid aza- crown- did not show antiviral activity, but both its derivatives proved to be active inhibitors of hsv and influenza virus reproduction. aza- crown- derivatives that contain -amino- -phenyl-propanoyl-or -benzyloxy- -oxapentyl-fragments decreased amount of cells infected by hsv- with virus-specific intranuclear inclusions by and %, respectively. first compound inhibited replication of influenza virus on . log tid and the second one decreased virus amount on . log tid . the results of this study show that aza-crown ethers are the perspective class of compounds for search of new antiviral agents. acknowledgement: this work was partially supported by stcu (grant # ) . robert w. sidwell , kevin w. bailey , min-hui wong , donald f. smee , dale l. barnard , shanta bantia institute for antiviral research, utah state university, logan, ut, usa; biocryst pharmaceuticals, inc., birmingham, al, usa the cyclopentane neuraminidase inhibitor, peramivir (bcx- , rwj- ) has striking inhibitory effects on a spectrum of influenza viruses in vitro, and has also demonstrated significant effects against influenza a (h n , h n ) and b virus infections when administered orally to mice and ferrets. unfortunately, clinical trials with the drug administered orally were not successful, probably due to low blood levels obtained after oral administration. significant plasma drug levels of peramivir persist up to h after intramuscular (i.m.) injection; more importantly, however, is the observation that peramivir remains tightly bound to influenza virus n neuraminidase for over h, suggesting single i.m. or intravenous (i.v.) therapy with the drug may be highly effective against an influenza infection. experiments now in press have indicated that single i.m. peramivir therapy administered up to h after virus exposure was protective to mice infected with influenza a (h n ) virus. in the present study, peramivir was administered i.m. or i.v. in a single injection h pre-virus exposure in separate experiments to mice infected with an influenza a (h n ) virus; efficacy was compared to similar dosages of oseltamivir and oseltamivir carboxylate run in parallel. dosages of and mg/kg of peramivir administered by either route significantly prevented deaths, lessened arterial oxygen (sao ) decline, inhibited development of lung consolidation, and inhibited lung virus titers. the lung assays were performed at varying times after virus exposure. oseltamivir and oseltamivir carboxylate, which do not have the same neuraminidase binding abilities seen with peramivir, were less efficacious in these experiments. delaying the single i.v. therapy up to h after virus exposure also significantly inhibited the virus infection. peramivir appeared to be well tolerated in toxicity control animals run concomitantly with these studies. these data indicate parenterally administered peramivir may hold promise as a therapy for clinical influenza a (h n ) virus infections. acknowledgement: supported by contract no -ai- from the virology branch, niaid, nih. hiroshi saitoh , naoko miyano-kurosaki , , hiroshi takaku , department of life and environmental sciences, faculty of engineering, chiba institute of technology, chiba, japan; department of life and environmental sciences, faculty of engineering and high technology research center, chiba institute of technology, chiba, japan background: influenza virus causes widespread infection in the human respiratory tract, but existing vaccines and drug therapy are of limited value. recently, small interfering rnas (sirnas) are a powerful tool for sequence-specific, post-transcriptional gene silencing and have a potential therapeutic and prophylactic application against cancer, as well as infectious diseases. here we show that short interfering rnas (sirnas) specific for conserved regions of the viral genome can potently inhibit influenza virus production in cell lines. the influenza virus np gene is a potential target for rnai technology. on the other hand, the baculovirus (acmnpv) can infect a variety of mammalian cells, facilitating its use as a virus vector for gene delivery in viral entry into cells. in this study, we describe the inhibition of influenza virus production by baculovirus-mediated shrna expression vectors. methods: the psv neo-u plasmid vectors and pvl based baculovirus vectors were used in this study. the influenza virus a and b np genes were made into the target and the shrna expression plasmid vectors were constructed under the control of the human u pol iii promoter. the shrna expression plasmids or shrna expression baculovirus vectors introduced into mdck cells, and h later the cells were infected with either a/pr or b/ibaraki virus at a moi of . . at h postinfection, culture supernatants were harvested and assayed to determine the virus titer by plaque assay. conclusion: the findings reveal that newly synthesized np proteins are required for influenza virus replication and provide a basis for the development of shrnas expression plasmids as prophylaxis and therapy for influenza infection in humans. julia serkedjieva, ekaterina krumova, tsvetanka stefanova, nadja nikolova, maria angelova institute of microbiology, bulgarian academy of sciences, sofia, bulgaria a semi-standardized polyphenol-rich extract (pre), obtained from geranium sanguineum l., inhibited the reproduction of influenza viruses types a and b in vitro and in ovo and protected mice from mortality in the experimental influenza virus infection (serkedjieva and manolova, ) . the selective in vitro virus-inhibitory activity of pre was fairly modest and this was in contrast with the significant protection in vivo. thus, the therapeutic effect of pre needed explanation. it was presumed that it might be attributed to a combination of more than one biological activities known for natural polyphenols. we have demonstrated previously that pre manifested strong antioxidant and radical-scavenging activities in model systems (sokmen et al., ) . the current study was undertaken to investigate the effect of the plant extract on the levels of the antioxidant enzymes superoxide dismutase (sod), catalase (kt) and peroxidase (po) in mice lungs during influenza virus infection as well as the effect of pre on the production of reactive oxygen species (ros) and reactive nitrogen intermediates (rni) by alveolar macrophages in influenza virus infected mice. mice were challenged intranasally (i.n.) with - ld of a/aichi/ / (h n ) influenza virus. pre was administered by i.n. instillation h before infection in the dose of mg/kg. it was established that influenza infection induced an increase in sod, kt and po production and on days and after infection their levels reached - % of placebo control. the application of pre brought enzymes values to control levels. influenza infection caused also a significant increase of h o , o •− and no production by alveolar macrophages; the generation of ros and rni peaked on day . pre-treatment before viral challenge reduced this excessive production. in conclusion, the obtained results outlined the antioxidant and radical scavenging properties of the plant extract; pre beneficially modulated the oxidative stress response in influenza virus-induced pneumonia. this alternative mechanism of action might contribute to the overall protective effect in the lethal murine experimental influenza infection. the antiviral activity of s , a natural herb extract, ji-sun kwon , hyun-jeong lee , chi-ung moon , jong-hwan kwak , youn-jeong lee , chang-seon song avian disease laboratory, college of veterinary medicine, konkuk university, seoul, korea; hanyang university, seoul, korea; sungkyunkwan university, seoul, korea; national veterinary research and quarantine services, seoul, korea the antiviral activity of s , one of the traditional korean medical herb extract, against influenza virus was investigated. the % effective concentration (ec ) using plaque reduction assay was . ug/ml and the mean % cytotoxic concentration (cc ) using wst- assay in the mdck cells was ug/ml. oral gavage treatment of the s to balb/c mice infected with a/pr/ / (h n ) influenza virus showed the therapeutic effects as delaying clinical signs, significant inhibition of death and reduction of lung virus titers. to identify the lead molecules, the s was subjected to further fractionation, purification, and isolation of active compounds. the antiviral activity of these natural herb compounds will be discussed. these results suggest that the s is a possible candidate for the development of new antiviral medicine for influenza therapy. hiroshi takaku , , , takayuki abe , hitoshi takahashi , naoko miyano-kurosaki , department life environ. sci., chiba inst. tech., chiba, japan; high tech. res. center, chiba inst. tech., chiba, japan; res. inst. microbial dis., osaka university, osaka, japan; bach tech corp background: the baculovirus autographa californica nuclear polyhedrosis virus (acnpv) has long been used as a biopesticide and as a tool for an efficient recombinant protein production in insect cells. in this study, we examined the immunization of a recombinant baculovirus expressing the influenza virus hemagglutinin (ha) against lethal influenza infection in mice. protection was observed in mice immunized intranasally with not only the recombinant baculovirus but also a wild-type baculovirus. baculovirus was also shown to induce secretion of inflammatory cytokines, such as tnf-␣ and il- , in murine raw . macrophage cell line. results: a varied route of immunization with a recombinant baculovirus expressing the influenza virus hemagglutinin protein of a/pr/ / (h n ) virus against lethal influenza infection was examined in mice. the recombinant baculovirus encoding the hemagglutinin gene under the control of chicken ␤ actin promoter was inoculated twice, weeks apart, at a dose of . × pfu per mouse by intramuscular, intradermal, intraperitoneal, and intranasal routes. mice intramuscularly and intraperitoneally immunized with the recombinant exhibited higher level of production of serum anti ha antibody than those immunized via the other routes, but protection was only achieved by the intranasal immunization. surprisingly, mice immunized with a wild-type baculovirus with intranasal route were also protected from the lethal influenza virus challenge. sufficient protection in mice was achieved by the intranasal immunizations with pfu of either the recombinant or wild-type baculovirus, as evaluated by the reduction of virus titer, production of inflammatory cytokines, and pulmonary consolidations in the lung. these results indicate that infection with a baculovirus induces a strong innate immune response and protection of mice from lethal influenza virus infection. conclusion: baculovirus (cpg motifs) induces a strong innate immune response and protection of mice from lethal influenza virus a and b infection. andrew vaillant , annie lebel , nathalie goyette , guy boivin , jean-marc juteau , phil wyde replicor inc., laval, que., canada; chuq-chul and laval university, st. foy, que., canada; baylor college of medicine, university of texas, houston, tx, usa potent antiviral activity of phosphorothioate oligonucleotides (ps-ons) was observed against influenza viral infections. antiviral activity was sequence-independent, size dependent (optimally active ps-ons were ≥ bases in length) and dependent on the presence of the phosphorothioate modification (hydrophobicity). binding studies showed that rep (a mer degenerate ps-on) interacts with both neuraminidase and hemagglutinin although the sialidase activity of neuraminidase was not affected, suggesting that the structural interactions of these proteins required for influenza activity are the target for this compound. the requirement for hydrophobicity further suggests that the alpha helical regions of hemagglutinin are one of the regions of interaction. the antiviral activity of rep was conserved in many influenza a and b strains suggesting potential therapeutic activity against avian flu and other newly emerging influenza strains. rep aerosol has excellent characteristics for lung deposition and aerosol treatment with rep was well tolerated and highly effective against infections with influenza a both in prophylaxis and h after infection. these results demonstrate the therapeutic potential of aerosolized ps-ons against influenza infection. acknowledgement: supported by nih contract no -ai- . irina v. alymova , y. sudhakara babu , allen portner virology division, department of infectious diseases, st. jude children's research hospital, memphis, tn , usa; biocryst pharmaceutical, inc., birmingham, al , usa bcx is a novel selective inhibitor of human parainfluenza virus infections, which design was based on the threedimensional structure of the hemagglutinin-neuraminidase (hn) protein of newcastle disease virus. compound exhibited striking activity against parainfluenza viruses in vitro and in vivo, and was efficacious in prophylaxis of lethal synergism between parainfluenza virus and streptococcus pneumoniae in a mouse model. present study was conducted to determine if bcx 's resistant variants of the recombinant sendai virus whose hn gene was replaced with that of human parainfluenza virus type (rsev(hpiv- hn) could be selected in tissue culture and animals. for this purpose virus was serially passaged in llc-mk cells at moi . in the presence of increasing (from to m) concentrations of compound; infected × /svj mice were treated with mg/kg/day of bcx twice for five days. treatment started h before infection. individual clones of viruses were analyzed for the presence of mutations. one mutation, e k, on the globular head region of the hn protein was selected in tissue culture after the fifth and eleventh passages of rsev (hpiv- hn) . several mutations in hn gene of rsev (hpiv- hn) were selected in an animal model after the second passage of virus from mice treated with bcx . two mutations, n s and p q, were located in the cytoplasmic domain of hn protein; mutations n s and t a were found on the globular head region of the glycoprotein. only nonconserved amino-acid residues of hn protein were involved in substitutions. all isolated mutant viruses were stable after the five passages in llc-mk cells without drug; did not develop other substitutions in the presence of drug and displayed no resistance to bcx both in vitro and in vivo. infectivity of all mutants was not altered to compare with the wild type of rsev (hpiv- hn) virus. taking together our results indicate that prophylaxis/treatment of human parainfluenza virus infections with bcx may not lead to appearance of clinically significant variant of viruses. kie-hoon jung , michelle mendenhall , lawrence m. blatt , robert w. sidwell , brian b. gowen institute for antiviral research, utah state university, logan, ut, usa; intermune, brisbane, ca, usa hantavirus pulmonary syndrome (hps) is an acute human respiratory disease with remarkably high case fatality rates ( - %) for which the etiological agents are members of the bunyaviridae family, genus hantavirus. maporal (map) virus is a recently identified hantavirus isolated in western venezuela, which is most similar phylogenetically to hantaviruses known to cause hps in southern regions of south america. despite the lack of evidence that map can productively infect humans and cause hps, infection of hamsters closely resembles disease manifestations associated with human hps. hantaviruses, in general, are known to produce little to no cytopathic effect (cpe) in cultured cell lines. unexpectedly, we found that map produces remarkable cpe in several vero cell lines facilitating the evaluation of known antiviral agents, ribavirin and interferon alfacon- . both drugs were highly effective at reducing cpe, as determined by visual examination and neutral red dye uptake, associated with map infection. since much of the observed cpe may be due to apoptosis of uninfected bystander cells, we also developed a quantitative (q)rt-pcr assay to detect copies of map genomic sequence to more directly assess the inhibition of viral replication. data obtained using the qrt-pcr-based assay were consistent with the visual cpe reduction and neutral red-uptake cytotoxicity findings. the development of in vitro antiviral testing methods for map are essential to the evolution of the in vivo hamster disease model of hps. the latter is of utmost importance considering the current need for effective antivirals for the treatment of hps and the lack of a suitable model that does not require biosafety level containment facilities. acknowledgement: supported by contract no -ai- from the virology branch, national institute of allergy and infectious diseases, national institutes of health. nucleoside analogues are widely used in antiviral and anticancer chemotherapy. for this class of drugs, intracellular conversion of the nucleoside analogue into the corresponding mono-, -di-, and -triphosphate after target cell penetration is a prerequisite for biological activity. because of the structural differences from natural nucleosides, this conversion is often inefficient and, as a consequence, therapeutic efficacy is sometimes limited. the free phosphates, or nucleotides, have limited utility in therapy on account of their poor membrane permeability and chemical stability. one approach to improve the therapeutic potential of nucleoside analogues is the delivery of the corresponding nucleotide entities via neutral, lipophilic prodrugs, or protides. the nucleoside aryl phosphoramidate approach, developed by mcguigan and co-workers ( ) has been successfully applied to a number of different nucleosides (azt, d t, dda, d a). the general structure of aryl phosphoramidates encompasses two masking groups, an amino acid ester and an aryl moiety bonded to the phosphate group. in order to apply this protide technology to nucleosides with the potential for anti-hepatitis c virus (hcv) activity, we have undertaken studies designed to probe the effect of varying the natural and unnatural amino acid esters and the aryl groups used as masking groups in the target phosphoramidates. these compounds have been synthesised and evaluated using genotipe b sub-genomic hcv replicon. we have prepared a variety of arylphosphoramidate derivatives from a range of -substituted nucleosides, including azido-cytidine, -azido-uridine, and , -protected variants. with certain nucleoside phophoramidates, we have observed dramatic enhancement (> -fold) of replicon activity relative to the parent nucleoside. the synthesis, biological activity and sar of these compounds will be presented. reference mcguigan, d. cahard, balzarini, j., . mini-review. med. chem. , - . we have identified a series of novel anthranilic acid derivatives that are potent, reversible inhibitors of hepatitis c virus (hcv) ns b polymerase, an essential enzyme for viral replication. the micromolar ns b polymerase inhibitors belong to the n-phenoxyacetylanthranilic acid chemotype. x-ray crystallography determined that the inhibitors bound to ns b between the thumb and palm regions adjacent to the active site. guided by crystallography, subsequent modifications to the hydrogen bonding and lipophilic regions of the inhibitors resulted in greatly improved activity against ns b. further sar studies revealed a second, more potent sub-series where the phenoxy group was replaced by an anilino group. analogs in both subseries showed antiviral activity in a cell-based replicon model of hcv. andrea brancale , dimitrios vlachakis , maria chiara barbera , romano silvestri , colin berry , johan neyts cardiff university, the welsh school of pharmacy, cardiff cf xf, uk; universita' degli studi "la sapienza", dipartimento di studi farmaceutici, roma, italy; cardiff university, cardiff school of biosciences, cardiff cf us, uk; rega institute for medical research, k.u. leuven, b leuven, belgium hepatitis c is a viral infection that affects million people worldwide, including million in the united states and million in europe. the virus establishes a chronic infection in - % of cases and % of affected individuals develop cirrhosis. at the moment there is neither a vaccine nor an effective antiviral therapy available and efforts to identify a specific anti-hcv inhibitor have dramatically intensified in the last few years. many research groups have focused their interest on the enzymes involved in the viral replication and, among these enzyme, the viral helicase/ntpase has proven to be a suitable target for developing novel anti-hcv compounds. compound is a potent inhibitor of the hcv helicase and, although its mode of action is still uncertain, it has been proposed that it acts as competitive inhibitor of rna binding. starting from this hypothesis, we have prepared a series of novel compounds based on the structure of where the benzimidazole moiety has been replaced by different chemical groups, including the negatively charged carboxylate moiety, which should mimic the phosphate backbone of the nucleic acid. the synthesis, the enzyme inhibition and the biological evaluation in replicon of these novel compounds will be presented and analyzed. dale r. cameron migenix inc., wesbrook mall, vancouver, bc, canada v s l hepatitis c virus (hcv), a leading cause of liver disease, continues to be an attractive target for new drug development. among the more favourable approaches to developing new hcv drugs is to target the rna-dependant rna (rdr) polymerase (ns b), which has been shown to be an essential enzyme for replication. there are several published non-nucleoside inhibitors of this polymerase (some in clinical development) and several published allosteric binding pockets on the protein they target. to be successful, traditional lead identification can be timeintensive, costly and have large infrastructure requirements. increasingly, a push towards effective computational-based screening has led to the development of virtual screening tools. such tools allow investigation of large quantities of compounds in silico for particular properties without the need for compound synthesis or high throughput screening. moreover, these techniques require only a modest infrastructure investment and are very efficient. we employed the openeye set of screening tools (omega, rocs and eon) in concert with publicly available hcv inhibitor information, and commercial databases to identify novel leads. the inhibitor coordinates from a protein-inhibitor complex crystal structure were utilized as the target. available compound databases (asinex and chembridge) were utilized as the testset of compounds. filtered compound conformers were generated using omega and compared with the template using rocs with post-analysis by eon. visual analysis to maximize particular desirable binding features while minimizing protein-inhibitor steric clash allowed the list of potential hits to be further narrowed. multiple classes of compound were identified from the above procedure and after sourcing a subset of the actual compounds or close analogs, they were tested for enzymatic inhibition activity and further characterized. iteration of the process resulted in the identification of a lead compound class containing multiple active compounds, one with reasonable replicon activity. in conclusion, readily available structural and database information and virtual screening tools can be successfully utilized to identify novel inhibitors of hcv rdr polymerase which, in turn, can serve as novel leads for developing new therapies for treating hcv. synthesis, antiviral activity, and cytotoxicity of some novel quinazolin- ( h)-one derivatives a series of novel -bromo/ , -dibromo- -( -oxo- -phenyl- h-quinazolin- y-l)-benzenesulphonamides were synthesized by condensation of -substituted benzo[ , ]oxazine- -ones and sulphonamide. their chemical structures were assigned by means of spectral analysis (ft-ir, pmr, ms). synthesized compounds were screened for in vitro antiviral activity against human pathogenic viruses (hiv, hcv, hsv, vv). -bromo- -( -oxo- -phenyl- h-quinazolin- y-l)benzenesulphonamide (sps-ii) and -( -oxo- -phenyl- hquinazolin- y-l)-benzenesulphonamide (sps-i) inhibits the replication of hiv- in acutely infected mt- cells at a concentration of approximately g/ml, while not being toxic to the host cell at a concentration of or > g/ml (selectivity index: and > ), respectively. in huh - cells sps-i inhibited hcv rna synthesis at ec of g/ml, while at cc for cell growth g/ml. sps-ii inhibited the virus-induced cytopathicity in human embryonic lung (hel) cell infection with hsv- , hsv- or vaccinia (vv) at a concentration of g/ml, while not being toxic to the cells up to a concentration of g/ml. further molecular modification in this series of compounds may help in optimising their antiviral activity. wengang yang, yongnian sun, avinash phadke, milind deshpande, mingjun huang achillion pharmaceuticals, new haven, ct , usa hcv nonstructural protein ns b is the catalytic subunit of the replication complexes, possessing a motif characteristic of rna-dependent rna polymerases. biochemical assays using recombinant ns b have been used to investigate ns b nonnucleoside inhibitors. however, the inhibitory effect of compounds often varies with the forms of recombinant ns b and the concentrations of the template and/or primer used in the assays. in addition, it does not always correlate to that obtained with replicon-containing cells. these observations have cast concerns about the validity of these cell-free assays. in the report, we explored replication complexes, isolated as crude membrane fractions from replicon-containing cells, for their competency to synthesize viral rna in vitro as well as their responsiveness to ns b inhibitors. after optimizing the experimental conditions, two species of nascent viral rna, one double-stranded and the other single-stranded, were readily detected. the addition of ns b nucleotide inhibitor blocked synthesis of both species. the presence of nonnucleoside inhibitors, however, inhibited mostly single-stranded rna (ssrna) synthesis. in addition, the replication complexes isolated from the cells containing a replicon that carried a resistant mutation in ns b to the nonnucleoside inhibitor were able to synthesize the same amount of ssrna in vitro regardless of the presence or absence of the inhibitor, demonstrating that the phenomenon is due to the specific inhibitory effect of the compound on ns b. combining with kinetic studies that ssrna synthesis was inhibited only when the nonnucleoside inhibitor was present during the pulse period, we conclude that ssrna synthesis catalyzed by the replication complexes in vitro is likely derived from the de novo initiation. we have recently reported the synthesis and antiviral activities of a ring-expanded ("fat") nucleoside analogue, called nz- , that inhibits both hcv and hiv in vitro with ec values ranging in micromolar concentrations or less, and little or low toxicity to the host cells. in this part ii of the presentation on this subject, we report our preliminary results on mechanistic studies of anti-hcv activity of this compound, along with the synthesis and antiviral activity of a few additional analogues in the series. in light of the fact that hcv is a major co-infection in patients infected with hiv, and that a number of them ultimately die of end-stage hcv-related complications including liver cirrhosis and hepatocellular carcinoma, a drug with dual inhibitory characteristics against both viruses is highly desirable and timely. nigel bourne, ronald veselenak, richard pyles, minkyung yi, stanley lemon the university of texas medical branch, galveston, tx, usa more than million people worldwide are estimated to be infected with hepatitis c virus (hcv). in the majority of these people a chronic infection is established which can result in serious long-term liver damage including progressive fibrosis, cirrhosis and hepatocellular carcinoma. in fact, hcv is believed to cause more than , cases of liver cancer annually worldwide and accounts for at least % of liver transplants in the us. current treatment options are limited and there is a high treatment failure rate. thus, there is a real need for new treatment options. amantadine has been evaluated as a treatment for chronic hcv infection in a number of clinical studies both as a monotherapy and in combination with other therapeutics. however, the results of these trials have been contradictory and at this time the clinical potential of amantadine as a therapy for chronic hcv infection remains unclear. recent studies have shown that the small hydrophobic hcv p protein forms an amantadine sensitive ion channel providing a possible basis for antiviral activity. we examined the ability of amantadine to reduce hcv replication in both subgenomic and full-length hcv replicons of genotypes a strain h c and genotype b strain n. in these studies amantadine failed to reduce viral rna replication in any of the replicons tested. further, in infectious virus assays using hcv genotype a strain jhf- um amantadine failed to reduce viral rna levels under any of the conditions tested. however, in these infectious virus studies, when the viral inoculum was treated with amantadine prior to infection of cell monolayers, or when the amantadine was added to cells h after virus adsorption there was a significant reduction in the number of infectious viral foci observed after h incubation (p < . and < . , respectively). these results suggest that even in the absence of a direct impact on rna replication amantadine has antiviral activity. we are currently evaluating amantadine for activity in infectious hcv genotype a assays to further define its antiviral spectrum of activity. studies to more fully define its mechanism of action in the virus life cycle are also underway. dominique dugourd, raymond siu, jeremy fenn migenix inc., vancouver, bc, canada celgosivir is an alpha glucosidase inhibitor that is being developed for the treatment of hepatitis c virus (hcv) infections in humans. the purpose of this study was to evaluate the in vitro antiviral activity of celgosivir and its primary active metabolite, castanospermine, when combined with current approved therapies (ribavirin, interferon ␣- b, or both) in a surrogate model of hcv (bovine viral diarrhea virus (bvdv)). compounds alone or in combination were tested against bvdv in infected madin-darby bovine kidney (mdbk) cells. synergies were analyzed using isobolograms and volume of synergy measurements (macsynergy ii tm software). the celgosivirinterferon ␣ b combination was significantly more synergistic than the celgosivir-ribavirin combination (∼ -fold), or the ribavirin-interferon ␣ b combination (∼ -fold). similarly, the castanospermine-interferon ␣ b double combination was more synergistic than the castanospermine-ribavirin combination (∼ -fold), or the ribavirin-interferon ␣ b combination (∼ . -fold). the combinations of celgosivir-interferon ␣ b or castanospermine-interferon ␣ b led to significant decreases in the ec s of celgosivir (up to > -fold) and castanospermine (up to > -fold). the effective ec s of celgosivir or castanospermine were further reduced by the addition of ribavirin. the cytotoxicity of the double and triple combinations was additive or less than additive, indicating that combinations of celgosivir or castanospermine with ribavirin and/or interferon ␣ b were generally less toxic than expected. these results indicate that the combination of celgosivir with interferon ␣ b or with interferon ␣ b and ribavirin may be effective in the treatment of hcv. pegylated interferon ␣ plus ribavirin is the current standard of care for the treatment of chronic hepatitis c virus (hcv) infections. this regimen results in sustained virologic response in only about % of patients and is associated with significant treatment-associated toxicities. a number of approaches are being used to identify novel therapeutic combinations with better tolerability and/or efficacy. inhibitors of endoplasmic reticulum (er) ␣-glucosidase have been shown to inhibit viral replication and secretion and may have utility as part of new multi-drug treatment cocktails. the ␣-glucosidase inhibitor celgosivir is currently being evaluated in combination with pegylated interferon ␣ and ribavirin in humans. the purpose of this study was to evaluate the antiviral effects of combinations of celgosivir and castanospermine, the primary active metabolite of celgosivir, with other antiviral agents having diverse mechanisms of action. the effect of the combination of celgosivir or castanospermine with the nucleoside analogue nm- , amantadine, and another iminosugar, n-butyl-deoxynojirimycin (nb-dnj) was determined in a cytopathic assay using the hcv surrogate virus bovine viral diarrhea virus in madin darby bovine kidney cells. synergies were analyzed using isobolograms and volume of synergy measurements (macsynergy ii tm software). volumes of synergy indicated that the castanospermine and nb-dnj combination was additive, while the celgosivir and nb-dnj combination was synergistic at high nb-dnj concentrations (> m). celgosivir and castanospermine were synergistic with both amantadine and nm- , with volumes of synergy between and m%. isobologram analysis confirmed these synergistic interactions. these results indicate that celgosivir could be considered in combination regimens containing drugs that directly target viral replication like nm- . mechanism(s) of synergy are under investigation. department of biotechnology, yonsei university, seoul - , korea hepatitis c virus (hcv) is an enveloped virus with positivestranded rna genome of approximately . kilobases and a major cause of non-a and non-b hepatitis, leading to liver cirrhosis and hepatocellular carcinoma. combination of interferon-␣ (ifn-␣) and ribavirin is the current standard therapy for the treatment of hcv infection, but there is no specific antiviral therapy available. the hcv viral genome encodes a single polyprotein of approximately amino acids, which is proteolytically processed by a combination of host and viral proteases into at least distinct structural and nonstructural proteins. the structural proteins include c, e , e , and p and the nonstructural (ns) proteins include ns , ns , ns a, ns b, ns a, and ns b. as new hcv specific therapies, small-molecule inhibitors against hcv enzymes including ns b protein, the viral rna-dependent rna polymerase (rdrp), and ns protease are in clinical tests. however, rapid emerging of drugresistant mutants has been hampering their practical clinical applications. recently, we have shown that phosphorylation of hcv rna polymerase by protein kinase c-like (prk ) regulates virus rna replication. hcv rna replication was inhibited when prk expression level was down-regulated by using a prk -specific sirna. in this study, we investigated the anti-hcv effect of prk inhibitors in an hcv subgenomic replicon system. treatment of the replicon cells with prk inhibitors suppressing the endogenous prk activity inhibited the phosphorylation of hcv rna polymerase and resulted in suppression of hcv rna replication in a dose-dependent manner. furthermore, the prk inhibitor in combination with ifn-␣ more effectively inhibited hcv rna replication than ifn-␣ alone. because the prk inhibitor did not show cytotoxicity in the cell-based drug inhibition studies and cellular proteins rarely get mutated, prk can serve as a cellular target for therapeutic intervention of hcv replication. specific inactivation of prk activity will provide an opportunity to interfere with hcv rna replication. haitao guo , tianlun zhou , ju-tao guo , andrea cuconati , anand mehta , timothy block drexel university college of medicine, doylestown, pa, usa; nucleonic inc., irvine, ca, usa; hepatitis b foundation, doylestown, pa, usa more than million people worldwide are chronically infected with hepatitis b virus (hbv). the major complication of chronic hepatitis b is the development of primary hepatocellular carcinoma (hcc), which causes an estimated , deaths annually. currently clinical treatments (␣-interferon and nucleoside analogs) of chronic hepatitis b rarely cure the virus infection. this is due, at least in part, to their failure to eliminate viral covalently closed circular (ccc) dna from the nuclei of infected hepatocytes. hbv cccdna is essential to the virus life cycle by serving as the template for the transcription of the pregenomic rna and of the subviral rna species. its elimination during chronic infection is considered critical to long-term therapy. however, cccdna has not previously been targeted in high throughput screens of small molecule libraries. to screen compound libraries for antiviral drugs targeting cccdna, we set out to develop a cell-based assay suitable for high throughput screening. since cccdna is time-consuming to assay, it was desirable to use a viral gene product that could serve as a reporter for intracellular cccdna level. we predicted that the secretion of hbv e antigen (hbeag) by hepad cells, a hepg -derived tetracycline inducible hbv expression cell line, would be cccdna-dependent. this is because a large portion of pre-core mrna leader sequence in the terminus of integrated viral genome was deleted, preventing hbeag expression from transgene, but could be restored from the terminal redundancy of pre-genomic rna during viral dna replication and subsequent cccdna formation. our experimental results showed that following induction, hepad produced and accumulated cccdna, which became detectable between and days. hbeag synthesis and secretion into culture fluid were dependent upon and proportional to the level of cccdna detected. therefore, the secretion of hbeag by hepad cells could potentially serve as a convenient reporter for the high throughput screening of novel antiviral drugs targeting hbv cccdna. kathy aldern, james beadle, karl hostetler university of california, san diego and the veterans medical research foundation, san diego, ca, usa (s)-hpmpa is a broad spectrum antiviral active against orthopoxviruses, hbv, cmv, hsv, and other herpes group viruses. we have shown that hdp-(s)-hpmpa has greatly enhanced antiviral activity against these viruses. in addition, while hpmpa itself is nearly inactive against hiv, we showed that hdp-(s)-hpmpa exhibited an ec > logs less than unmodified hpmpa in mt- cells by p reduction assay. to evaluate the metabolic basis for the increased antiviral activity, we studied and compared the cellular uptake of radiolabeled cdv, (s)-hpmpa and their hdp-esters and conversion to hpmpa-diphosphate (hpmpapp) and cdv-diphosphate (cdvpp) in mrc- human lung fibroblasts using hplc partisil sax ion exchange chromatography. cellular uptake of hdp-cdv and hdp-(s)-hpmpa was similar. however, when cells were exposed to the respective drugs for , and h, hpmpapp appeared much earlier than cdvpp and reached levels several fold greater than observed with hdp-cdv. drug wash out experiments were carried out in mrc- cells exposed to radiolabeled hdp-cdv and hdp-(s)-hpmpa. after h, the culture medium was removed and replaced with complete medium without drug and the levels of hpmpapp and cdvpp were measured by hplc every days for - days. levels of the diphosphates declined slowly with a t / of - days. in conclusion, hdp-(s)-hpmpa is converted to its diphosphate more rapidly than hdp-cdv and reaches higher intracellular levels. this may explain, in part, its greater antiviral activity. the antiviral activity and oral bioavailability of cidofovir (cdv) is enhanced when the phosphonate is esterified with various straight chain alkoxyalkyl groups. the length of this chain is an important determinant of antiviral activity and selectivity. however, in some cases, rapid metabolism to an inactive short chain metabolite was observed. to enhance the metabolic stability of these esters, we synthesized cidofovir alkoxyalkyl esters bearing methyl groups on the penultimate carbon of the alkyl chain. enzymatic stability of -me-hdp-cdv ( ) was tested in liver s fractions from various species. in mouse and human liver s fractions, compound was completely stable for min while - % of the straight chain hdp-cdv was metabolized. the branched alkoxyalkyl esters were then evaluated in cells infected with vaccinia, cowpox and ectromelia viruses. the branched methyl analogs were substantially more active than cdv and equal to or slightly more active than the straight chain analogs. compound retained full activity compared to hdp-cdv and compound showed greater activity against orthopoxviruses compared to its unbranched analog. we believe that the structural modification of the alkyl chain slows the formation of inactive metabolites, possibly by interfering with oxidation and may result in better pharmacokinetics and more potent antiviral activity against orthopoxvirus infection in vivo. cidofovir ([ -(s)- -hydroxy- -(phosphonomethoxy)propyl]cytosine, hpmpc) is a broad spectrum antiviral agent clinically used for treatment of aids-related cmv retinitis. cidofovir has limited oral bioavailability (< %), attributed to ionization of its phosphonic diacid moiety under physiological conditions. we have shown that masking of this group by conjugation of the cyclic form of the drug (chpmpc) via a ser side chain p-o ester linkage with x-ser dipeptides, where x = a hydrophobic amino acid, can result in prodrugs that afford significantly improved biological availability of parent drug in an animal model. here we describe the total synthesis of novel cyclic cidofovir prodrugs ab of l-val and l-phe using an alternative conjugation strategy, viz. via an ethylene glycol link utilizing p-o and c-o ester bonds. the preparation of the hpmpc synthon from r-glycidol used our modification of the literature procedure (brodfuehrer et al., ) , involving reaction of tritylated (r)-glycidol directly with unprotected cytosine to achieve regiospecific opening of the epoxide ring, followed by reaction with benzoic acid anhydride to obtain the desired n-benzoyl intermediate needed to continue the synthesis. pybop was used as condensing agent in a convenient, one-pot conversion of hpmpc to chpmpc and subsequent esterfication of the latter by the ethylene glycol-modified amino acids. the prodrugs were converted to drug by cellular (caco- , hff) and tissue (liver and intestinal) homogenates, but did not show enhanced oral bioavailability when evaluated in a rat model, suggesting that such compounds may be useful for understanding the effectiveness of in drug delivery. cidofovir (cdv) is a broad-spectrum anti-viral agent that is used to treat aids-related cytomegalovirus (cmv) retinitis and other cmv infections. cdv has good in vitro activity against orthopox viruses, including smallpox; however, its use is limited because of the drug's low oral bioavailability and poor transport into cells. in order to improve its oral bioavailability, our group has synthesized a series of dipeptide and amino acid prodrugs of the cyclic analog of cidofovir (ccdv). in the current project, we examined the cytotoxicity and antiviral activity of the prodrugs, showing that the compounds are not cytotoxic and have diverse activity against hcmv and orthopox viruses (vaccinia and cow pox) with % inhibitory concentrations ranging from . to . and m and greater, respectively for the two virus types. in vitro and in situ perfusion studies established that the permeability of the prodrugs is enhanced more than -fold and that the transport is mediated, at least in part, by the intestinal dipeptide transporter. we also have found that the bioavailability of the prodrugs is dependent upon the prodrug structure and that we can achieve up to an eight-fold increase in bioavailability over the parent compound in vivo. drug stability experiments showed that in gastrointestinal and liver homogenates, the ccdv prodrugs are enzymatically hydrolyzed to the parent compound. it is clear from this work that the biologically benign dipeptide moiety, strategically linked to the drug to mask its anionic properties, significantly enhances intestinal transport of ccdv, creating the possibility of an orally bioavailable form of ccdv with low toxicity. acknowledgement: supported by funds from tsrl inc, the university of michigan, and nih grants r ai and u ai . lawrence trost , bernhard lampert , lloyd frick , merrick almond , george painter chimerix, inc., durham, nc, usa; dmpk advisor, chimerix, inc., durham, nc, usa foscarnet, a pyrophosphate analog approved for the treatment of cmv retinitis and acyclovir-resistant herpes infections in immunocompromised patients, is active against highly drug resistant strains of hiv- . however, the clinical utility of foscarnet is limited because it requires controlled intravenous infusion and is associated with high risks of renal impairment and seizure caused by alterations in plasma minerals and electrolytes. lipid conjugation has been shown to increase the in vitro activity, improve oral bioavailability, and reduce the toxicity of several antiviral drugs requiring intravenous administration because of poor bioavailability. in the case of foscarnet, conjugation to methylbatyl alcohol (cmx ) decreases the apparent ec value against hiv- by up to -fold. cmx was esterified to produce cmx in order to increase solubility and to protect against decarboxylation of the foscarnet moiety during passage through the stomach. here we present the results of a preliminary toxicology and toxicokinetic study of the methylbatyl alcohol conjugate of foscarnet methyl ester (cmx ). rats were given oral doses of , and mg/kg cmx daily for days. there were no clinical signs of toxicity. body weight and food consumption were comparable to controls and serum biochemistry, hematology, coagulation parameters and urinalysis were normal. there were no gross findings at necropsy, no effects on organ weights and no findings by histopathological examination of a wide range of tissues. importantly, there were no changes in serum biochemistry parameters or histopathological examination that were indicative of the renal impairment or serum electrolyte changes that are associated with foscarnet. oral dosing resulted in significant plasma exposure to cmx (c max > g/ml), the biologically active deesterified form of cmx . in conclusion, cmx is absorbed after oral administration, converted to cmx , and has a good preliminary toxicity profile. these results support the development of cmx for the treatment of drug resistant hiv infection. zhiqian wu , julie breitenbach , ulrika erickson , john hilfinger , john drach , gordon amidon department of pharmaceutical sciences, college of pharmacy, the university of michigan, ann arbor, mi, usa; school of dentistry, the university of michigan, ann arbor, mi, usa; tsrl, inc., ann arbor, mi, usa vaccinia virus is a surrogate model system for study of pox virology and development of antiviral therapeutics. the potent anti vaccinia virus activity and various shortcomings of vidarabine make it a good candidate for improvement by utilizing prodrug strategy. vidarabine is a polar nucleoside drug with low membrane permeability and rapid degradation by adonesine deaminase. -monoester prodrugs of vidarabine with various amino acids promoieties (l-valine, l-isoleucine, l-phenylalanine. laspartic acid, l-proline) are synthesized and evaluated for their stability, permeability and activity against vaccinia virus. prodrugs exhibit different hydrolysis rate in caco- cell homogenate (t / : - min). -l-isoleucyl and -l-valyl monoester prodrugs exhibit comparable bio-conversion rate and hpept mediated uptake as well as caco- permeability with valacyclovir, a commercially marketed oral amino acid ester prodrug. both prodrugs have potent activity against vaccinia virus and are resistant to ada . preliminary animal study shows -lisoleucyl vidarabine results in > -fold increase in circulating vidarabine level. the results suggest that it may be possible to use amino acids prodrug strategy to improve vidarabine as anti vaccinia virus agent. vidarabine [ -␤-d-arabinofuranosyl)adenine or ara-a) was originally investigated as an anti-tumor agent and was later found to be active against herpes simplex virus (hsv) types and . it was the first fda-approved drug for treatment of systemic hsv infections. although replaced by acyclovir and analogs for most applications, vidarabine remains an alternative therapy for acyclovir-resistant hsv and varicella-zoster virus infections. despite its proven efficacy, vidarabine suffers some limitations including: (i) metabolism by adenosine deaminase (ada) to its inactive hypoxanthine homolog (ara-h); (ii) low lipophilicity and membrane permeability and (iii) poor aqueous solubility, thus limiting options for parenteral and peroral delivery. our recent interest in vidarabine was triggered by our discovery that it was ∼ -fold more active against vaccinia (vv) and cow pox (cpv) viruses than was cidofovir in plaque reduction assays. its activity was enhanced about -fold by combination with m -deoxycoformycin (pentostatin, a potent inhibitor of ada) thereby providing significant superiority to cidofovir. from these results and our earlier studies on -substituted vidarabine analogs (lipper et al., . mol. pharmacol. , - ), we determined that minimizing metabolism of vidarabine by synthesizing -amino acid substituted prodrugs gave a significantly more potent anti-pox virus agent. we found that amino acid ester prodrugs of vidarabine are active against vv at non-cytotoxic concentrations. further, using cell homogenates, purified enzyme and intact cell systems, we showed that the prodrugs are resistant to inactivation by ada. the prodrugs also had enhanced transport potential, most likely targeting the intestinal dipeptide transporter. finally, oral delivery of the prodrug to the small intestine resulted in a -fold increase in vidarabine plasma levels when compared to unsubstituted vidarabine. these properties make the prodrugs of vidarabine good candidates as orally bioavailable anti-pox virus agents that are stable in the presence of ada. acknowledgement: supported by funds from tsrl inc. and the university of michigan. ulf goerbig , anne baum , jan balzarini , chris meier university of hamburg, institute of organic chemistry, hamburg, germany; rega institute for medical research, katolieke universiteit leuven, leuven, belgium the cyclosal pronucleotide system has been designed for an intracellular delivery of therapeutically active nucleoside monophosphates. as part of recent work on the cyclosal approach, the interaction of cyclosal nucleotides with cholinesterases has been investigated. it is known that organo-phosphates may act as irreversible inhibitors of cholinesterases (suicide mechanism). in the case of cyclosal nucleotides, cholinesterase inhibition could lead to unwanted side effects in a possible therapeutic application. there are two types of cholinesterase found in humans, the highly specific, physiologically important acetylcholinesterase (ache) and the much more unspecific butyrylcholinesterase (bche) of unknown physiological importance. fortunately, no inhibition of ache was observed for a variety of different cyclosal nucleotides. in contrast, bche inhibition was found in some cases. the anti-hiv-active , -bis-tertbutyl- -fluoro-cyclosal-d t monophosphate is the first cyclosal derivative combining three desired properties: successful intracellular delivery of nucleotides, sufficient hydrolytic stability and strongly reduced inhibitor activity towards bche. because of the promising properties of this compound, we combined this mask developed for d t with the antiviral active nucleoside analogues like d a, dda, azt and acyclovir. in this contribution we present the synthesis, hydrolysis stability, inhibition behaviour towards bche and anti-hiv data of these new compounds. henning jessen , wolfgang fendrich , tilmann schulz , jan balzarini , chris meier university of hamburg, institute of organic chemistry, hamburg, germany, rega institute for medicinal research, katholieke universiteit leuven, leuven, belgium cyclosal-pronucleotides are used for the delivery of antivirally active nucleotides into cells via a ph triggered selective hydrolysis. to distinguish between intra-and extra-cellular environment enzyme-cleavable side chains were introduced in the aromatic moiety of the pronucleotide to enrich the compound inside cells. this behavior will further be described as "lock-in"effect. many other different cyclosal pronucleotides have been designed, all showing different hydrolysis properties and antiviral data, both originating from the nature of the cyclosal-moiety as well as of the nucleoside. to examine these differences, analytical tools of high accuracy and sensitivity were needed, being structurally as close as possible to the lead compounds. these requirements are met by intrinsically fluorescent nucleosides coupled to different cyclosal masking groups. for analysis of the purine-type nucleosides iso-da with high intrinsic fluorescence properties was chosen and converted into iso-a, iso-dda and iso-d a. for the pyrimidine-type series the fluorescent nucleoside m k was synthesized as well as the dideoxy-compound dm k. these nucleosides were transformed into different cyclosalpronucleotides and tested for their suitability for fluorescence analysis. in fact, an improvement of sensitivity by a factor of compared to uv-detection was found for some of the compounds (pmol detection). for all compounds fluorescence and absorbance spectra were recorded to determine the absorption and emission maxima. the new compounds lacked activity against hiv- and hiv- strains. however, the compounds showed low cytotoxicity, which is important for their usability as fluorescent probes in cells. due to the analytical sensitivity, a simple model uptake study could be carried out, employing an u-tube with two aqueous phases, which were separated by an unpolar organic solvent simulating a diffusion barrier. the properties of the aqueous phases were varied and an enzyme-driven enrichment of a "lock-in"-modified intrinsically fluorescent cyclosal-pronucleotide passing the diffusion barrier could be simulated. nicolas gisch , jan balzarini , chris meier university of hamburg, institute of organic chemistry, hamburg, germany; rega institute for medical research, katholieke universiteit leuven, leuven, belgium cyclosal-pronucleotides efficiently deliver therapeutically active nucleoside monophosphates in human cells. "lock-in"-cyclosal-pronucleotides -the so-called second generation of cyclosal-compounds -have been designed to trap the compound by intracellular cleavage of esterase-cleavable moiety. one disadvantage of the "lock-in"-compounds is their high chemical stability, which leads to a delayed drug delivery. therefore, conceptually different, enzymatically activated cyclosalpronucleotides have been developed. in this concept lipophilic donor substituents attached to the aromatic ring are converted into a polar acceptor substituent by intracellular enzymatic cleavage. as a consequence the liberated acceptor group leads to a strong decrease in hydrolysis stability and a rapid formation of a charged intermediate is the result. from the phosphodiester intermediate the nucleotide is released subsequently. the concept, synthesis, characterization and in vitro antiviral evaluation of the third generation of cyclosal-pronucleotides will be presented. tomas cihlar , richard mackman , adrian ray , dean boojamra , lijun zhang , deborah grant , hon hui , jennifer vela , neil parkin , yolanda lie , kirsten white , michael miller , gerry rhodes , manoj desai gilead sciences, foster city, ca, usa; monogram biosciences, so. san francisco, ca, usa background: n(t)rtis are currently used as a backbone of antiretroviral combination therapy. however, their long-term benefit can be limited by adverse effects, resistance development, drug-drug interactions, and sub-optimal efficacy in treatment-experienced patients. therefore, we searched for novel nucleotide analogs with improved pharmacological profiles. methods: phosphonomethoxy- -fluoro- , -dideoxydidehydroadenosine (gs ) was selected from a broad range of nucleoside phosphonate analogs. phosphoramidate prodrug technology previously explored with tenofovir was applied to gs , resulting in the identification of gs (ethylalaninyl phenyl ester of gs ). results: gs exhibits potent anti-hiv- activity in primary lymphocytes and t-cell lines (ec < nm). low cytotoxicity (cc > m) was observed in multiple cell types including renal cells. diphosphate metabolite of gs was shown to act as an obligatory dna chain terminator and a competitive inhibitor of hiv- reverse transcriptase (rt) (k i = . m). unlike ddi, d t, or d fc, neither gs nor its prodrugs inhibited mitochondrial dna replication in hepg cells. in a phenosense assay, gs retained its full activity against hiv- variants with k r, m v or l v mutations in rt. viruses with ≥ thymidine analog mutations showed ≤ fold reduced susceptibility to gs , a shift that was smaller than that of any other tested nrti. following an oral dose of mg/kg gs in dogs, the bioavailability of prodrug exceeded %, resulting in high intracellular levels ( . ± . m) and prolonged retention (t / > h) of gs diphosphate in blood lymphocytes. conclusions: both gs and its prodrug gs exhibit favorable in vitro pharmacological profiles including less resistance due to rt mutations than approved nrtis. gs possesses good in vivo pharmacokinetic properties and thus represents an attractive development candidate with potential for clinical efficacy in both treatment-naive and nrti-experienced patients. martin mcdermott, gabriel birkus, ruth wang, holly macarthur, xiaohong liu, nilima kutty, tomas cihlar, craig gibbs, swami swaminathan, arnold fridland, william lee gilead sciences, inc., foster city, ca, usa gs- and gs- are alkylalaninyl phenyl ester prodrugs of tenofovir (tfv; -[( -phosphonomethoxy)propyl]adenine) and a novel nucleotide analog fd ap (phosphonomethoxy- -fluoro- , -dideoxydidehydroadenosine), respectively. both gs- and gs- exhibit potent in vitro anti-hiv- activity, favorable resistance profile, and low cytotoxicity. compared to tenofovir disoproxil (viread), both prodrugs are significantly more stable in plasma and deliver > -fold greater levels of active diphosphate metabolites into pbmcs in vitro and in vivo. the initial step in the intracellular activation of gs- and gs- is the hydrolysis of the alanine carboxyester by an unknown hydrolytic enzyme. the isolation and identification of this enzyme from human pbmcs is reported here. results: a major enzyme capable of cleaving gs- and gs- was purified from human pbmcs and was separable from esterases able to cleave alpha napthyl acetate (ana). the increase in specific activity of prodrug hydrolase achieved was -fold. sds-page analysis showed the presence of a prominent protein band at kda, which was identified by ingel tryptic digestion and ms/ms sequencing of the resultant peptides as lysosomal carboxypeptidase a (cathepsin a, ec . . . , cata). the biochemical properties of purified prodrug hydrolase matched those of cata. recombinant cata and the isolated prodrug hydrolase displayed nearly identical susceptibility to hydrolase inhibitors and substrate preference against a panel of tenofovir phosphoramidate prodrugs. incubation of both enzymes with [ c]gs- and [ h]difluorophosphonate resulted in the labeling of an identical kda protein (catalytic subunit). both labeled bands reacted with polyclonal antibodies specific for human cathepsin a. finally, following incubation with gs- , approximately - -fold lower intracellular concentrations of tfv metabolites were detected in fibroblasts from patients expressing non-functional cat a (cat a-cells) compared to normal control fibroblasts (cat a+ cells). center for drug delivery and nanomedicine, university of nebraska medical center, omaha, ne, usa nucleoside -triphosphate (ntp) is the biologically active form of many antiviral nucleoside analogs capable of efficiently blocking the production of viral nucleic acids in infected cells. we describe novel microparticulate formulations for encapsulation of ntp, drug delivery and antiviral therapy of respiratory infections. polymer networks (poloxagels) consisted of crosslinked poloxamers and cationic polymer molecules were designed, synthesized and characterized by loading with ntp and interaction with cells. poloxagel-ntp formulations could be obtained by simple mixing of the aqueous solution of ntp with the aqueous dispersion of poloxagel and subsequent lyophilization. drug loading was equal up to % by weight. in this form, phosphates groups of ntp are complexed with amino groups of polycationic backbone of poloxagels, and the formulations could be stored at room temperature for many months without degradation of ntp. the particle size of aqueous poloxagel-ntp dispersions was low, with a hydrodynamic diameter of . - . m. the rate of passive drug release in physiological solution was from to % of loaded drug during the -h period. these formulations were effectively consumed by many types of cells. significant amounts of drug and poloxagels were detected in the cellular interior after only - h of incubation. in the presence of cellular membranes drug release from poloxagel-ntp formulations was dramatically increased. we attribute this effect to the triggered release of the bound ntp as a result of competitive interaction of polycationic backbone of poloxagels with phospholipids of cellular membranes. mucoadhesive properties of poloxamers may additionally enhance binding of poloxagels with airways/lung epithelium. -triphosphate of -␤-d-ribofuranosyl- h- , , -triazole -carboxamide (ribavirin) was synthesized using phosphorylation with a tris(imidazolyl) phosphate in a convenient one-pot approach. formulations of different poloxagels with the ribavirin -triphosphate were prepared and characterized as prospective antiviral formulations for prophylactic and therapeutic treatments of respiratory infections including influenza a virus. aerosolic route of application of these antiviral formulations and associated problems are discussed. edwin gong , ebrima gibbs , joel oger department of pharmacology and therapeutics, the university of british columbia, vancouver, bc, canada; neuroimmunology lab, ubc hospital, department of medicine, the university of british columbia, vancouver, bc, canada ifn-alpha and ifn-beta are currently employed in the treatment of many viral diseases, especially chronic hepatitis. ifn-beta is also employed for the treatment of multiple sclerosis (ms), a chronic and often debilitating disease of the central nervous system. however, as with other protein therapeutics, long-term ifn therapy can lead to the development of binding and neutralizing antibodies to ifns and thus lead to deceased clinical effect of ifns. in order to measure the bioavailability of ifns and the level of neutralizing antibody, we have developed a realtime rt-pcr (taqman) assay by quantitating the expression of mxa (an ifn-induced protein) mrna. the nucleotide sequences of mxa deposited in the genebank were aligned, and a pair of primers and the hybridization probe were designed based on the conserved regions. a house keeping gene, gapdh, was used as a calibrator for relative quantitation. the rna standards were generated by in vitro transcription from cloned mxa gene in a plasmid vector. the reaction parameters were optimized. the assay was validated using pbmcs of ms patients that were treated with ifn-beta. for evaluation of the ifn bioavailability, the total rna was extracted from pbmcs and quantitatively detected by one-step rt-pcr for both mxa and gapdh. the results calculated by the (− ct) method showed that the difference (signal-to-noise ratio) between samples with neutralizing antibodies and samples from untreated ms patients or healthy donors were approximately - -folds. this indicates that our assay is a reliable method for determination of ifn bioavailability. v. lozitsky , i. kravchenko , v. larionov research anti-plague institute, odessa, ukraine; national university, odessa, ukraine transdermal delivery (td) of drugs is a novel method for treatment of diseases. td is carried out by the help of transdermal therapeutic systems (tts), which are multilayer plasters that contain active ingredients. td have a number of advantages, such as: ( ) prolongation of the drug's action; ( ) drug's concentration is maintained in therapeutic range; ( ) there is no trauma to patient's skin while using td; ( ) removing tts from the skin immediately stops drug's entering to the organism; ( ) first-pass effect in the liver is reduced; and ( ) many highly active drugs are irritating the gastro-intestinal tract if administered orally, others have a short half-life time-these drugs do not have downsides mentioned above if used as tts. in our previous research we elaborated tts containing rimantadine (ttscr) and studied its efficacy during experimental influenza in mice. we had established that transdermal delivery of rimantadine is more effective than oral administration. the aim of this work was to increase the efficacy of ttscr. to solve this task we studied the influence of some permeability enhancers on anti-influenza efficacy of ttscr during experimental infection. applied tts had adhesion hydrogel matrix (polyvinyl alcohol and . -propylenglycol). they consisted of a base and a plastificator, which improves the administration of active substances through the skin and does not induce irritation. ttscr ( mg/mouse) were applied on shaven backs of experimental animals. tts for other groups additionally contained one of such permeability enhancers as: mg/mouse of dmso or mg/mouse of octanol or mg/mouse of papain. tts were applied on shaven backs of mice and stayed there from day before infection to th day after challenge. mice of all groups were infected intranasally with influenza virus a/pr/ / (h n ), which is highly pathogenic for them. challenge was carried out using four animals for each virus dilution within the range of − to − . deaths of animals were recorded for days. the results showed that proteolytic enzyme papain increased the anti-influenza efficacy of ttscr on log tid . dmso and octanol did not demonstrate such activity. mikhail dobrikov , serguei vinogradov , barbara ramsay shaw department of chemistry, duke university, durham, nc, usa; center of drug delivery and nanomedicine, and college of pharmacy, university of nebraska, omaha, ne, usa nucleoside reverse transcriptase inhibitors (nrti) are widely used in the antiviral chemotherapy. most nrtis require stepwise phosphorylation to the respective nucleoside triphosphates, which inhibit the viral dna synthesis. however, the emergence of hiv- reverse transcriptase-dependent drug resistance limits the effectiveness of treatment by nrtis. the ␣-p-borano-nucleotide analogues show several unique physico-chemical and biological properties: (i) enzymatic studies indicate that the rp-isomer of ␣-p-borano- , -ddndps is a better substrate for cellular ndp kinase than the parent ddndp; (ii) neither isomer of the ␣-p-borano-ddndps is a substrate for mammalian pyruvate kinase and shows very poor inhibitory properties to this enzyme; (iii) the rp-(␣-p-borano)-ddntp isomers are better inhibitors of drug-and multidrug-resistant viral reverse transcriptases and are poor substrates for dnadependent dna polymerises; and (iv) after incorporation into viral dna the borano-ddnmp residues are more resistant to atp-dependent removal from viral dna than parent ddntps. to by-pass inefficient phosphorylation of the nrtis, several prodrugs of ␣-p-borano-nucleotide analogues have been previously synthesized. a more efficient delivery system for ␣-p-boranonucleotide analogues based on nanosized cationic polymeric gel (nanogel) is proposed. selective inhibition of drug-and multi-drug resistant viral rts, poorer inhibition of intracellular kinases and dna polymerases by the ␣-p-borano-nucleotide analogues, and their specific delivery into infected cells in the complex with nanogel particles suggest a new approach to the design of more powerful antiviral drugs. acknowledgement: this work was supported by the nih grant r al to b.r.s. we have developed a high-throughput, cell-based assay to address the critical need for antiviral drugs for the treatment of influenza. in consideration of the demand to screen high volumes of compounds, we targeted the development of a microtiter plate format for the assay. in this assay, the inhibition of the influenza-induced cytopathic effect (cpe) in mdck cells was assessed using the celltiter-glo luminescent cell viability assay by promega. this reagent measures the amount of atp present in cells, which is directly proportional to the number of metabolically active cells. validation studies were executed to establish optimal cell density, viral concentration, dmso tolerance for compound dilution, incubation time for virus-induced cpe and effective control drug concentration. additional parameters, such as assay variability, reagent and read stability, edge effects, and ic stability were also investigated during validation. we are currently initiating use of the assay to screen chemical libraries and will report our findings from library screens in addition to the aforementioned validation. we believe the approach will also provide a mechanism for discovery of new antiviral leads for influenza as well as avian flu. fundacio irsicaixa, hospital universitari germans trias i pujol, badalona, spain we have developed bacteriophage lambda based genetic screen that can be used to isolate and characterize site-specific proteases. this genetic screen system is based on the bacteriophage lambda ci-cro regulatory circuit, in which the encoded repressor ci is specifically cleaved to initiate the lysogenic-to-lytic switch. we have adapted this simple, safe and rapid genetic screening system to predict the activities and phenotypes of human immunodeficiency virus type (hiv- ) proteases in the course of viral infection and antiretroviral therapy. a specific target for the hiv- protease, p -p , was inserted into the lambda phage ci repressor. the target specificity of the ci-hiv repressor was evaluated by coexpression of this repressor with an hiv- protease construct. upon infection of escherichia coli cells expressing the two constructs encoding the ci-hiv- repressor and hiv- protease, lambda phage replicated up to -fold more efficiently than in cells that did not express the hiv- protease. this assay responds appropriately to well-known hiv- proteases inhibitors and can be used to search for new proteases inhibitors. the high level of specificity of this system, in which modest differences in catalytic efficiency can be quantified, should be also useful for the characterization of different mutant viral proteases. we further demonstrated the broad applicability of this protease assay using other viral proteases and their cognate cleavage sites, including hepatitis c virus (hcv) ns protease and severe acute respiratory syndrome (sars) coronavirus (cov) (scov) c-like protease. compared with other protease assay methods, this assay has the following advantages: safe, highly sensitive, highly specific, easy quantification, and rapid generation of different protease cleavage substrates using molecular cloning and expression. this system may be useful for the development of a screening method to identify viral protease inhibitors and should be also useful to characterize cellular, viral, or other infectious agent proteases with different activities and specificities. karen m. watson, todd b. parsley, robert w. buckheit jr. imquest biosciences, inc., frederick, md, usa a virus transmission and rapid resistance selection assay has been developed in order to quickly evaluate the biological properties of anti-infective test compounds and rationally prioritize them for further development. the transmission assay specifically evaluates the ability of test agents to suppress and clear virus replication from cultures during the serial passage of virus in the presence of fixed concentrations of the test compounds alone or in combination. the growth and expansion of virus in the infected cultures has been shown to occur through the replication of originally infected cells in the absence of virus spread, through direct virus to cell infection, and through cell to cell transmission. in order to sterilize a culture a test compound must possess the ability to specifically and potently interfere with virus replication by each of these three methods and must be able to inhibit the replication of resistant viruses which pre-exist in the viral inoculum and which rapidly grow in the presence of the fixed concentrations of the test agent. twelve pyrimidinediones being evaluated for potential use as both anti-hiv therapeutic agents and topical microbicides were evaluated for their ability to inhibit virus transmission and to define their ability to rapidly select for resistant virus strains. these compounds were evaluated in parallel with known anti-hiv agents that inhibit virus entry (t ) and reverse transcription (sustiva, uc and azt). the results of the transmission assays suggest that significant biological differences exist between antiviral compounds and even between highly related congeners of the same class of pyrimidinediones, suggesting that the transmission and rapid resistant selection assay measures important antiviral properties of anti-hiv agents. biological studies that evaluate the mechanisms of virus growth in the presence of high concentrations of test compounds will be described. laure deflubé , kerstin angner , anna overby , david stein , patrick iversen , ramon flick utmb, department of pathology, galveston, tx, usa; avi biopharma, inc., corvallis, or, usa in the family bunyaviridae, several members on the genus phlebovirus have been reported to cause disease in humans or livestock. among these, rift valley fever (rvf) virus is an important human/animal pathogen. its widespread geographic distribution and its ability to produce severe human disease makes this virus a worldwide public health concern. the phosphorodiamidate morpholino-oligomers (pmo) are a class of dna-like antisense agents typically synthesized to a length of about subunits and contain purine or pyrimidine bases attached to a backbone composed of six member morpholine rings joined by phosphorodiamidate intersubunit linkages. they have been shown to be effective antiviral compounds for different virus families, e.g. coronaviridae and flaviviridae. pmo bind to rna preventing translation of the viral rnas. we used our recently developed plasmid-based minigenome rescue systems for uukuniemi (phlebovirus model virus) and rvf viruses to screen antiviral compounds based on the morpholino antisense oligonucleotide approach. for this the antiviral compounds were appraised on the basis of reporter gene activity (fig. b) . the inhibitory effects of the same compounds were also tested by measuring reduction in virus titer (fig. a) , by monitoring changes in viral antigen production using an indirect immunofluorescence procedure and facs analysis, and analysis of genome transcription/replication by rt-pcr. indeed several pmos could be identified with interfering effect at a low ic on bunyaviral minigenome rescue as well as virus proliferation. based on these results, we plan to confirm antiviral activity of the most promising compounds in suitable animal models. we have shown that the fractal approach to the problem of viruscell interaction gives the unique possibility to process the data through the sequence of the direct and inverse fourier transforms. the studies were carried on the herpes simplex virus us- interacting with the hep- sensitive cell culture. the object was imaged as system of bright peaks formed as a result of laser diffraction on the structural elements of the virus-cell system. the whole virus-cell interaction information is inserted into computer in a fastest parallel way. the laser intensity peaks, which form the speckle image of the system under consideration, could be transformed into the hierarchical system of the circles (or squares) according to the choice of the researcher, but conserving the same d value, which depends only on the true intermolecular interaction potential. this potential, being characteristic for every stage of virus-cell interaction, is responsible for the given structure of the dynamic virus-cell system and the unique, but the typical form of the fractal cluster corresponding both to the system itself and its image as well, was processed by computer techniques. the hierarchical fractal design of the virus-cell system, proposed here for the first time, gives the universality, needed for the quantitative description of any possible combination of the virus and corresponding sensitive cell. it should be noted, as well, that the fractal microscope use for viruscell dynamic system imaging have all the properties, required from all other experimental tools of monitoring, including the reliability, reproducibility and preciseness. this device could be used in drug design biological test stages with the scope of time and efforts economy during the compounds libraries screening. the fractal microscope combined with the qsar drug design technique makes the antiherpetic drug design more competitive as compared to the regular approaches. acknowledgement: the authors are indebted to the partial support of the stcu grant # . we have investigated experimentally the fractal properties of diffraction images obtained by laser irradiation of virus-cell system. it was shown that the diffraction process is mathematically equivalent to the direct fourier transform of the said system's components modeled with simple geometrical figures (e.g. circles). each viral family could be coded and described quantitatively with the average size of the free viral particle and the type of its symmetry. we propose here to use the inverse fourier transform of the virus-cell system in order to get the real enlarged image of the viruses attacking the sensitive cell as well as the cell's structural transformation caused by the sequent stages of virus reproduction process. the set of bright and dark spots, which forms the virus-cell system's diffraction image, could be coded into set of numbers (matrix form of correlation vector-function) using the quantification procedure. the correlation function was used as presented in polar coordinates because the system has the axial symmetry (laser beam taken as main physical axis). the full information included into the image peaks' diameters and color index is transformed using inverse fourier technique into set of intersecting bright and dark circles. the full in vitro dynamics of the structural changes of the virus-cell system are described by the changes of circles' diameters and the area of their intersection. it was shown, also, that the magnification of the fractal microscope could achieve , × to , ×, depending on the laser power used. proposed fractal microscope could be applied as well in vivo experiments until the required magnification will not make us to use projection laser with the output exceeding mw. we have shown that the fractal microscope based on the inverse fourier transform could be applied successfully in pharmaceutical antiviral drug design, laboratory and clinical trials of new antiviral preparations, especially effectively in hierarchic qsar research. acknowledgement: authors are grateful to the support under the stcu grant # . we have previously reported bicyclic furano pyrimidines as potent and selective inhibitors of varicella zoster virus (vzv) , with subnanomolar activity for palkylphenyl substituted analogues . these compounds however are highly lipophilic and poorly soluble in water. we then reported a series of p-alkyloxyphenyl compounds containing a phenolic ether aiming to enhance water solubility whilst retaining antiviral activity (mcguigan et al., ) . we will now report the synthesis, characterisation and antiviral evaluation of a novel series of p-alkyloxyphenyls where there is at least one methylene spacer between the phenyl and ether group to potentially boost the pharmacokinetic profile. the alkyl chain length was fixed to retain a high clogp value, a parameter that has previously been shown to correlate with high antiviral potency . the target structures were prepared by the pd-catalysed coupling of a series of para-substituted alkoxyphenyl acetylenes with -iodo- -deoxyuridine, to give intermediate -alkynyl nucleosides, which were subsequently cyclised in the presence of cui to give the desired bicyclic systems. the antiviral activity, cytotoxicity, and solubility of these compounds are to be reported. we have previously reported on some novel nucleoside analogues containing and unusual furano bicyclic pyrimidine base and long side chain , which were discovered to be both potent exquisitely and selective towards the varicella zoster virus. following this discovery, three main sites for modification were identified and explored: ( ) the side chain; ( ) the bicyclic base; and ( ) the sugar moiety. modification to the side chain by insertion of a phenyl ring, led to the most potent anti-vzv nucleoside to date (ec nm) . the investigation into modifications at the three sites stated above has continued and we herein report further adjustments to these analogues. replacement of the furo oxygen with sulfur on the parent nucleosides bearing an alkyl side chain has been reported to retain antiviral activity. however, those bearing a phenyl alkyl side chain are here shown to give a slight reduction in anti-vzv activity. modifications to the phenyl ring of the side chain have included halogen substitutions, and the fluorine in particular has produced some intriguing results in that, while the ortho and meta substitutions show some anti-vzv activity, the para analogue is completely devoid of antiviral activity. we now report further studies which include the di and tri substituted phenyl analogues. finally, we have also investigated sugar modification that has included substitutions of the hydroxyl group. previous modifications which have replacements of the hydroxyl groups, resulted in loss of activity against vzv . we now present some new substituted analogues which have provided interesting biological results. we have previously reported bicyclic furano pyrimidines as potent and selective inhibitors of varicella zoster virus (vzv) with subnanomolar activity for palkylphenyl substituted analogues srinivasan et al., ) . the sar is now further explored via the substitution of phenyl derivatives with electron withdrawing and electron donating groups. we now report the synthesis, characterisation, and biological evaluation of a novel series of mono substituted phenyl derivatives in order to probe the structure activity relationships in this region. the target compounds were synthesised under sonogashira conditions where a series of substituted phenyl acetylenes were coupled with -iodo- -deoxyuridine, to give intermediate alkynyl nucleosides that were subsequently cyclised in the presence of cui to give the desired bicyclic systems. diseases caused by herpes simplex virus (hsv) are widely distributed. prophylaxis and treatment of these infections are important health care tasks that require also the search, design and development of new antiherpetic drugs overcome drug resistance and toxic side effects of existing drugs. drug selection simply based on results of empirical screening is not very effective. computer-based technologies may help to optimize the structure of antiviral compounds as well as to design and develop new drugs. the objective of the present work is the quantitative structureactivity relationship (qsar) analysis of antiviral activity of various n,n -(bis- -nitropyrimidyl)dispirotripiperazines in connection with consequent drug design. the well-established simplex representation of molecular structure (sirms) qsar approach has been used to fulfill this objective. it allows the molecular design of new effective antiviral drugs. thorough investigation of the relationship between: (a) cytotoxic (hela cells and gmk cells, cc , g/ml); (b) antiherpetic activity (hsv- strain kupka, ic , g/ml); and (c) selectivity index (ratio of cc to ic ) and the structure of n,n -bis- -nitropyrimidyl derivatives of dispirotripiperazine have been conducted. statistic characteristics for pls (partial least squares) models are quite satisfactory (r = . - . , q = . - . ). the results are confirmed by experimental data. based on the obtained models, molecular fragments that promote and interfere with antiviral activity were defined. additionally, these models provide the possibility to predict molecular fragments that will enhance antiherpetic activity and to design new well tolerated highly virus-specific drugs. in summary, the developed simplex approach is an effective instrument for prediction and design of novel effective antiherpetic agents. several representatives of a series of -arylethynyl- deoxyuridines ( a) bearing bulky aryl groups were recently shown to possess unexpected activity towards hsv- . unlike common anti-hsv drugs, these compounds retain activity towards kinase-deficient acyclovir-resistant strains. therefore, an unusual mechanism of antiviral action is assumed. in order to investigate the mechanism and to discover more potent analogues we synthesized several novel -deoxy ( a) and -arabino ( b) uridine derivatives possessing different -arylethynyl substituents. dinucleosides containing two uridine moieties coupled to a single polycyclic aromatic hydrocarbon (e.g. pyrene) represent another type of structural variation of nucleosides a. these compounds as well as some of a and b possess bright fluorescence that can be used in biological evaluations. cytomegalovirus (cmv) is a wide spread opportunistic pathogen which belongs to the beta subfamily of the herpesviridae. primary infection is generally asymptomatic resulting in life long latency. however, morbidity and mortality rates post-transplantation are greatly increased following reactivation or recrudescence of cmv. ganciclovir (gcv) and cidofovir (cdv) have both been successful in suppressing cmv viral replication in immunocompromised patients. although sustained use of these drugs has resulted in the emergence of multi-drug resistant strains of virus. in this study we used plaque reduction assays to determine the antiviral efficacy of two ribonucleotide reductase inhibitors, didox (dx; , dihydroxybenzohydroxamic acid) and trimidox (tx; , , trihydroxybenzamidoxime) in inhibiting both wild type and drug-resistant strains of murine cmv (smith strain). the results presented here demonstrate that both dx and tx inhibit viral plaque formation in a dose dependent manner in both wild type and the resistant strain. a -and -fold increase in drug dose was required for cdv and gcv respectfully to inhibit plaque formation by % in the resistant strain (cdv wt: . m, r: . m/gcv wt: . m, r: . m). this compared to only a moderate increase in drug dose required for dx and tx to achieve % inhibition in the resistant strain (dx wt: . m, r: . m/tx wt: . m, r: . m), corresponding to a . -and . -fold increase respectfully. further work is currently underway to determine the possible mechanism of antiviral actions and toxicity profiles of these novel virostatics. in patients with human immunodeficiency virus (hiv) infection, coinfection with herpesviruses continues to be a problem for patients receiving antiviral hiv therapy. since treatment can be affected by the large number of drugs required for multiple infections, it would be useful to have antivirals that are active against both hiv and the herpesviruses. we reported previously that alkoxyalkyl ester prodrugs of cidofovir (cdv) are several logs more active against herpesvirus replication than unmodified cdv. to determine if this strategy would be effective for other acyclic nucleoside phosphonates which are active against hiv infections, hexadecyloxypropyl (hdp) esters were synthesized from -(phosphonomethoxyethyl)-cytosine (pme-c), -(phosphonomethoxyethyl)- -bromo-cytosine (pme- brc), -(phosphonomethoxyethyl)- -fluoro-cytosine (pme- fc), -(phosphonomethoxyethyl)- , -diaminopurine (pme-dap) and -(phosphonomethoxyethyl)- -amino- cyclopropylaminopurine (pme-cprdap) and assayed for activity against herpesvirus replication. overall, the hdp esters were more active than the unmodified acyclic nucleoside phosphonates, indicating that this is a useful strategy for increasing the antiviral activity of acyclic nucleoside phosphonates. one of the most active compounds was hdp-pme-cprdap which had ec values of . , . , and . m in hff cells infected with hsv- , hsv- or hcmv, representing a - -fold increase in efficacy over the parent pme-cprdap. another promising compound was hdp-pme-dap, which had ec values of . , . , and . um in hff cells infected with hsv- , hsv- , and hcmv, representing a - -fold increase over the parent pme-dap. the results presented here indicate that modified acyclic nucleotides with antiviral activity against hiv also inhibit the replication of some of the herpesviruses. further evaluation of their activity against other herpesviruses that are a problem in hiv-infected patients, such as human herpesviruses type and , is warranted and may provide new therapeutic options for patients with coinfections. julie m. breitenbach , katherine z. borysko , jiri zemlicka , john c. drach biologic & materials sciences, school of dentistry, university of michigan, ann arbor, mi, usa; karmanos cancer institute, wayne state university school of medicine, detroit, mi, usa we previously described first (qiu et al., . j. med. chem.) and second generation (zhou et al., . j. med. chem.) methylenecyclopropane purines that have potent and selective activity against hcmv. strains selected separately for resistance to first-generation analogs (synadenol, synguanol) were - -fold resistant to several first-generation purine analogs. similar resistance was observed to the second-generation guanine analog cyclopropavir [ic 's in plaque assays = . and m, respectively for wild-type (wt) and synguanol-resistant ( r) virus]. likewise a ul deletion mutant (prichard et al., . j. virol.) was resistant to both first and second-generation compounds (ic 's = . and . m in wt; and m in ul del , respectively for synguanol and cyclopropavir). ul from the hcmv strain selected for resistance to the synadenol was sequenced and two mutations were identified: m i and c y. because hcmv with either m i or the related c y mutation alone was sensitive to synadenol and synguanol (baldanti et al., . antiviral res.), we hypothesize that two mutations are required for resistance to first-and second-generation analogs. this hypothesis was tested by construction of three strains of hcmv from hcmv ad bac with one, the other, or both mutations in ul . as expected, the two strains with the single mutations were -to -fold resistant to ganciclovir but had little resistance to the first generation compounds synadenol and synguanol ( . -to -fold). both strains were somewhat more resistant to the second-generation compound cyclopropavir ( to -fold) but less so than observed in the r virus with two mutations. study of the resistance of the constructed virus with two mutations is underway. we conclude that a functional ul is required for activity against hcmv and that is likely that two mutations in ul are required for significant resistance. acknowledgement: supported by grants p -ai and r -ai from nih and funds from the university of michigan. svitlana zagorodnya , nadiya nesterova , inna alexeeva , larisa palchikovskaya , galina baranova , alexander kobko , anna golovan zabolotny institute of microbiology and virology of nas of ukraine, kiev, ukraine; institute of molecular biology and genetics of nas of ukraine, kiev, ukraine search of new effective preparations capable to inhibit herpesviruses reproduction is stipulated by their certain resistance to different groups of chemical preparations. new triazine bearing tricyclic bases and their n-glycosidic derivatives structures are widely used as potential antiviral agents. the objective of the present investigation was to study the activity triazine bearing tricyclic bases nos. and , as well as n-glycosidic derivatives no. against epstein-barr virus-lymphotropic and oncogenic virus from herpesviridae family. as a model of ebv-infection in vitro we used the line of lymphoblastoid b-cells raji, which infected by ebv. an inhibition of reproduction of ebv in a cell culture by no , no , and no was determined by reduction of a number of genome-equivalents of ebv dna on a cell, which were revealed by quantitative pcr with use of primers and reagents "amply-senc- r" (russia). the first stage of investigation of substances was the analysis of their cytotoxicity for cell line raji. they have been studied in concentrations of , , , , , , , , , . and . g/ml. the concentrations that inhibited the quantity of live cells on % (id ) were equal to substances no. - g/ml, no. - g/ml and no. - . the minimal inhibiting concentration (mic) of nos. , , and was equal to g/ml, because the amount of genome-equivalents of dna ebv on a cell was reduced with . up to . hence, the index of selectivity (is) was equal to and for triazine bearing tricyclic bases nos. and , for n-glycosidic derivatives- . in addition these compounds were also tested in transcription and replication model systems in vitro. our results indicate that bases and their n-glycoside derivatives effect rna and dna synthesis in different manner. r. sgarbanti , l. nencioni , g. macrì , c. nucci , u. benatti , m. magnani , e. garaci , a.t. palamara department public health sciences, university rome "la sapienza," rome, italy; department biopathology, physiopathological optics, university rome "tor vergata," rome, italy; department exp. medicine biochemistry section, university genova, genoa, italy; inst. biochemistry, university urbino, urbino, italy; department exp. med. biochem. sciences, university rome "tor vergata," rome, italy several studies have demonstrated that different viruses induce an imbalance in the intracellular redox state through a depletion of glutathione (gsh), the main intracellular antioxidant. the imbalance in the intracellular redox state represents a key event in the development of viral infection. indeed, our previous data showed that treatment with gsh prevents a decrease in intracellular gsh and inhibits replication of different rna and dna viruses in vitro and in vivo. our recent data demonstrated that a butanoyl derivative of gsh (gsh-c ), with increased hydrophobic properties, inhibited in vitro parainfluenza- and hsv- replication more efficiently than gsh. for this reason we evaluated the effectiveness of topical gsh-c administration in hsv- -induced keratitis in rabbits. for infection, the corneal epithelium, previously scratched, was inoculated with × pfu/ml of hsv- . gsh-c , dissolved in a saline solution ( mm, ul/eye), was administered as eyedrops four times daily for ten days. a saline solution was used for the control group. the clinical evaluation of conjunctival and corneal involvement, performed by using . % fluorescein sodium eyedrops and a slit lamp fitted with a cobalt blue filter, demonstrated that gsh-c treatment was effective in reducing the severity and progression of keratitis and conjunctivitis. moreover, in gsh-c treated animals, conjunctival hsv- titre, assayed by tcid on day post-infection, was significantly reduced as compared to that of control animals (mean = . × units/ml versus . × units/ml, n = for group). accordingly, similar results were obtained by measuring virus titre from the corneas of gsh-c -treated animals versus placebo animals (mean = . × units/ml versus . × units/ml, n = per group). such results highlight the antiviral activity of gsh-c in vivo and suggest that topical gsh-c treatment could be considered as complementary therapy of hsv- -induced keratitis. debra quenelle , deborah collins , latisha pettway , caroll hartline , james beadle , w. wan , karl hostetler , earl kern university of alabama school of medicine, birmingham, al, usa; department of medicine, university of california, san diego and veterans medical research foundation, san diego, ca, usa cytomegalovirus (cmv) can cause a wide variety of clinical manifestations in immunocompromised hosts or transplant recipients. we have utilized severe combined immunodeficient (scid) mice implanted with human fetal tissue and subsequently infected with hcmv or balb/c mice infected with mcmv to evaluate new antiviral therapies against cmv infection. in the current studies we used these two models to determine the efficacy of (s)- -[ -hydroxy- -(phosphonomethoxy)propyl]adenine ((s)-hpmpa), hexadecyloxypropyl-(s)-hpmpa (hdp-(s)-hpmpa), or octadecyloxyethyl-(s)-hpmpa (ode-(s)-hpmpa). in the hcmv model, human fetal thymus and liver (thy/liv) tissues were implanted under the kidney capsule of mice and inoculated - weeks later with pfu of hcmv. tissue samples were obtained at various time points for quantitation of hcmv titers by plaque assay. in general, replication of the toledo strain of hcmv in the implant tissue increased through - days and then gradually decreased to undetectable levels by weeks post-infection. to determine efficacy of these compounds, oral treatment with vehicle or mg/kg of (s)-hpmpa, hdp-(s)-hpmpa or ode-(s)-hpmpa was initiated h after infection and continued for days. cidofovir (cdv) at mg/kg was administered i.p. daily as a positive control. results indicated that (s)-hpmpa, hdp-(s)-hpmpa and ode-(s)-hpmpa were highly effective in significantly reducing replication when compared to the vehicle control. in mcmv infected mice, hdp-(s)-hpmpa was highly effective in preventing mortality when administered orally at or mg/kg beginning h post-viral inoculation and mg/kg when treatment was delayed until h postviral inoculation. these data indicate that these compounds were highly efficacious in two animal models of cmv infection and should be evaluated for use in hcmv infections in humans. cytomegalovirus (cmv) is a ubiquitous ␤-herpesvirus that asymptomatically infects immunocompetent individuals but leads to serious illness in immunocompromised individuals, such as transplant recipients, neonates and aids patients. thus, the need for well-tolerated and potent antiviral compounds with activity against cmv is well recognized. in our current studies, we have evaluated the in vivo activity of rep , a fully degenerate mer phosphorothioated oligonucleotide against murine cytomegalovirus infection (mcmv) in mice. rep has potent in vitro activity against hsv- , hsv- , hcmv, vzv, ebv, and hsv- (vaillant et al., submitted for publication). in our initial studies, infected mice were treated with rep and compared to saline-treated infected control mice. compound was administered intraperitoneally for consecutive days at mg/kg, starting at days prior to infection. mice were infected with × pfu mcmv on day , at h post-treatment. sera were collected at − h, at hpi, and at dpi for elisa analysis of ifn␥ production. spleens and livers were collected at dpi for determination of virus titers. at dpi, virus titers in the spleens and livers were significantly reduced by rep treatment as compared to control mice. splenomegaly was observed in infected mice treated with rep but not in saline treated, infected mice or in rep treated, uninfected mice. ifn␥ levels in mice treated with rep peaked at hpi compared to hpi for saline-treated control mice. these data suggests that immune stimulation might contribute to the antiviral activity of ps-ons, perhaps through ifn␥ levels. a second study comparing the in vivo activity of rep with two oligonucleotide analogs that do not activate tlr- mediated immune stimulation suggests that direct antiviral activity of rep and the analogs was the predominant therapeutic mechanism in vivo. moreover, one rep analog exhibited even greater antiviral activity than rep while causing no splenomegaly. additional experiments are underway to provide insights into the mechanism of action against mcmv infection. acknowledgement: supported by contract no -ai- from the virology branch, niaid, nih. kathy keith , joseph maddry , namita bansal , kochurani jacob , secrist john , earl kern department of pediatrics, university of alabama school of medicine, birmingham, al, usa; southern research institute, birmingham, al, usa a series of novel antiviral agents was prepared based on lead compounds related to acyclic nucleoside phosphonates. these agents consist of a purine nucleus bearing a pendant phosphonic acid group. the design strategy was two-fold: ( ) following the approach of the hostetler group, to mask or partially mask the anionic phosphonate as a lipophilic ester and/or as an amino acid phosphonamidate prodrug that could enhance cellular uptake and be cleaved intracellularly; and ( ) to investigate new substituents at the purine -and -positions. for proof of concept, a phosphonomethoxyethyl adenine (pmea) scaffold was employed. over analogs of pmea substituted at the purine -or at the adenine n- site have been synthesized and evaluated for activity against orthopoxvirus infections. many n- substituents other than the previously recognized n-cyclopropyl have shown antiviral activity, and these structure-activity relationships are being investigated. an exciting finding has been that introduction of several novel moieties at the purine -position particularly the hydrazino, hydroxylamino, or the cyclopropylamino groups resulted in several compounds with excellent in vitro activity. for example, octadecyloxyethyl (ode) -amino-n( )-cyclopropyl pmea had ec values of . - . m and ode -hydroxylamino-n( )-cyclopropyl pmea had ec values of . - . m against cowpox and vaccinia viruses, respectively, using a plaque reduction assay in hff cells. under these conditions the parent molecule, pmea, was completely inactive. these two compounds had cc values of - m giving selective indices of - . these studies indicate that several modifications in the pmea scaffold can result in good antiviral activity against orthopoxvirus infections in vitro and the most active compounds are currently being scaled up for evaluation in animal models. isatin ( , -dioxoindole), a versatile lead molecule for designing of potential bioactive agents, and its derivatives have been reported to possess inhibitory activity against a variety of pathogenic viruses. methisazone(n-methylisatin- thiosemicarbazone) was one of the first synthetic antiviral agents used clinically for the treatment of orthopox virus infections. the presence of the thiosemicarbazone, however, can result in immunosuppresion and we have attempted to replace the thiosemicarbazone with a sulphonamide in order to modify the antiviral activity. the present work was performed to evaluate the antiviral activity and cytotoxicity of some novel -[( , dihydro- -oxo- h-indol- -ylidene)amino]-n-( , -dimethyl- pyrimidiny)-benzene sulphonamides against pox viruses such as vaccinia and cowpox virus in human fibroblast cells and the activity was compared with cidofovir(cdv). among the compounds tested, -[( -methyl- , -dihydro- -oxo- h-indol- -ylidene)amino]-n-( , -dimethyl- -pyrimidiny)-benzene sulphonamide(spiii- me), was the most active compound with an ec value of mol, compared with cdv, which had an ec of mol against vaccinia virus. all the compounds were non-toxic (> lm)using a neutral red uptake assay. substitution of a halogen atom in th position of isatin was found to abolish the antiviral activity. this compound should be evaluated in orthopox infections in animal to determine its potential for development as an effective agents for treatment of these infections. acknowledgement: supported in part by contract no -ai- from virology branch, niaid, nih, usa. evgeny belanov , svetlana kotovskaya , nikolay bormotov , sergey balakhnin , olga serova , nataliya perova , zina baskakova , galina dzhumbaeva , valerii charushin , oleg chupakhin state research center of virology and biotechnology "vector", koltsovo, novosibirsk reg., russia; ural state technical university, yekaterinburg, russia; institute of organic syntheses, yekaterinburg, russia during this study, we synthesized a series of , , -benzotriazine ( fig. ) derivatives in order to evaluate the structural features required for anti-orthopoxviruses activity. these derivatives were tested for cytotoxicity and activity against the vaccinia, cowpox, mousepox, monkeypox, and in some experiments with variola viruses in vero and mk- cells. the results from studies of structure-activity relationship revealed that only compounds containing phenyl group at c- and the alkoxy and fluoro substitutes in the benzene ring of benzotriazines showed anti-orthopoxviruses activity. the antiviral activity was reduced or lost after substitution with other substitutes. thus, we find a new class of heterocyclic compounds with antiviral activity. acknowledgment: this research was funded by istc project # . yali chen, guang yang, kady honeychurch, dennis hruby, robert jordan siga technologies, inc., sw research way-suite , corvallis, or , usa we have recently discovered a highly specific and potent antiorthopoxvirus compound (st- ) via high throughput screening (yang et al., . j. virol. , - ) . marker rescue of st- resistant variants localized compound resistance to the f l gene that encodes a major orthopoxvirus envelope protein (p ), which is required for extracellular viral particle formation. p participates in wrapping of intracellular mature virus (imv) in membranes derived from the trans golgi or late endosomal compartment to produce intracellular enveloped virus (iev) that are transported to the cell surface to form extracellular virus particles. to gain insight into the mechanism of action of st- , we examined the effects of st- on the production of the extracellular viral particles in bsc cells infected with recombinant vaccinia virus containing a gfp-tagged p protein. in the presence of st- , iev particle formation was dramatically reduced, plaque formation was almost completely inhibited, and imv particles appeared to be retained in intracellular vesicles as revealed by electron microscopy. furthermore, st- prevented the intracellular localization of p to the late endosome compartment as measured by confocal microscopy. in contrast, st- did not affect localization of p expressed from a st- resistant virus variant. more intriguingly, the compound did not affect the intracellular localization of p in transfected cells. these results suggest that st- inhibits an unknown virusspecific activity that requires f l. this work underscores the exquisite specificity of st- and supports continued development of st- as a potential anti-orthopoxvirus drug. guang yang, chris harver, dennis hruby, robert jordan siga technologies, inc. sw research way, suite corvallis, or , usa st- is a potent, orally bioavailable anti-orthopoxvirus compound that is active in vitro and in vivo. the frequency of naturally occurring st- resistant variants was measured by fluctuation analysis and found to be . × − . marker rescue of drug resistant variants localized changes associated with reduced compound susceptibility to the vaccinia virus f l gene. the spectrum of mutations that confer st- resistance was determined using an error-prone pcr procedure to increase the frequency of compound resistance by -fold relative to the frequency of naturally occurring resistance. using this procedure, random point mutations were introduced into the f l coding sequence by error-prone pcr and the mutated f l alleles were transferred into wild-type virus genome by marker rescue. sequence analysis of the input error-prone pcr products prior to marker rescue identified numerous nucleotide changes in the f l coding sequence, some of which created nonsense mutations. virus recombinants were selected that formed plaques in the presence of drug selection. this powerful selection procedure enriched for viruses that produced functional, st- resistant, f l proteins. sequence analysis of the compound resistant f l alleles identified numerous silent mutations scattered throughout the f l coding sequence and point mutations leading to amino acid changes that clustered around amino acid positions - within the f l gene. seven of these mutations resulted in single amino acid changes and could be correlated with reduced compound susceptibility. taken together, these results suggest that: ( ) mutations in at least positions within f l can confer resistance to st- and ( ) st- resistant mutations cluster to a amino acid domain in a region of the protein of unknown function. several -substituted pyrimidine analogs were identified as having antiviral activity against cowpox virus (cv) and vaccinia virus (vv) in primary human foreskin fibroblast cells. molecules containing benzopyran, cyanovinyl, and pyrazolone moieties at this position exhibited significant antiviral activity against both these viruses. three compounds in this series had ec values below m in a plaque reduction assay against both cv and vv. the antiviral activity of these compounds was also determined against herpes simplex virus (hsv) in a plaque assay. two compounds with cyanovinyl derivatives at the position had ec values below m against both hsv- and hsv- , whereas other substituents at this position exhibited weaker activity against one or both of these viruses. analogs containing the benzopyran substituents were the most effective against varicella zoster virus (vzv) and yielded ec values below m in a plaque reduction assay. none of the compounds were active against human cytomegalovirus. interestingly, all of the compounds were much less effective in a thymidine kinase (tk) negative strain of cv suggesting that the activation by this enzyme was important in their mechanism of action. tk deficient strains of hsv were also comparatively resistant to some of the compounds. the tk dependence of these compounds in cv and hsv taken together with the lack of activity against cytomegalovirus replication suggests that activation by a viral tk is important in their mechanism of action. these results indicate that pyrimidine analogs with large substituents at the position are substrates for the distinct tk homologs encoded by the herpesviruses and orthopoxviruses and suggest that they may be effective against infections with these viruses. synthesis and testing of additional analogs is warranted and should help identify the most potent analogs for in vivo testing. department of pediatrics, university of alabama school of medicine, birmingham, al , usa n-methanocarbathymidine ((n)-mct) is a conformationally locked nucleoside analog that is active against some herpesviruses and orthopoxviruses in vitro. this compound inhibits the replication of herpes simplex virus (hsv) with ec values below g/ml, and vaccinia virus (vv) and cowpox virus (cv) with ec values of . and . g/ml, respectively. assays using a thymidine kinase (tk) negative strain of cv yielded ec values -fold greater than a tk positive isolate. similarly, a tk negative strain of hsv- was -fold less sensitive to the drug than wild-type strains. thus, the antiviral activity of this molecule is dependent on the type i tk in hsv and the type ii tk expressed by vv and cv viruses, suggesting that it is a substrate for these divergent forms of the enzyme. the drug is also a good inhibitor of viral dna synthesis in both viruses and is consistent with inhibition of the viral dna polymerase once it is activated by the viral tk homologs. it is also possible that the phosphorylated forms of the drug may inhibit other enzymes such as thymidylate synthetase and inhibit viral dna synthesis indirectly. the interesting tk dependence of this molecule explains the rather unusual spectrum of activity that includes orthopoxviruses, alphaherpesviruses, epstein-barr virus (ebv), and human herpesvirus (hhv- ), since these viruses all express molecules with tk activity that could phosphorylate and thus activate the drug. conversely, n-mct is ineffective against the betaherpesviruses because they do not encode tk homologs. the compound is also highly effective in reducing the mortality of mice infected with cv, vv, and hsv when treatment is initiated h after infection and at doses as low as mg/kg. these results indicate that (n)-mct is active in vitro and in vivo and its mechanism of action suggests that the molecule may be an effective and selective therapeutic for orthopoxvirus and certain herpesvirus infections and that it warrants further development. we have previously reported the isolation and characterization of drug-resistant mutants obtained following repeated passages of the vaccinia virus (vv, lederle strain) in the presence of increasing concentrations of cidofovir (cdv). cdvr mutants encoded two mutations (a t and a v) not related to genetic polymorphism. we have now introduced these mutations in the pathogenic strain w western reserve and characterized the drug-susceptibility profile of the recombinant viruses and their pathogenicity in mice. both the a t and the a v recombinant viruses proved to be resistant to cdv and related compounds, such as cyclic cdv and -propoxy]pyrimidine}. the virus bearing both substitutions proved to be more resistant to cdv than the single mutants. interestingly, the a t and the a v mutants differed in their sensitivity to phosphonoacetic acid (paa); the a t and the a v mutants being, respectively, hypersensitive and resistant to paa. in contrast, the double mutant showed no change in sensitivity to paa as compared to the wild-type strain. unlike the a v mutant that showed only a two to three-fold decrease in susceptibility towards the -hydroxy- -phosphonomethoxypropyl (hpmp) purine derivatives, the a t mutant showed cross-resistance to the hpmp purine derivatives. it should be noted that in the process of selection of cdv-resistant mutants in the presence of increasing concentrations of the compound, the a t mutation appeared before the a v substitution, and the latter mutation only occurred in conjunction with a t. when tested for virulence in a lethal intranasal infection model in mice, all cdvr recombinant viruses proved to be attenuated, suggesting that cdvr mutations are associated with reduced pathogenicity. furthermore, we found that cdv at a dose of mg/kg/day for days was still able to protect mice (in terms of body weight loss) against an intranasal challenge with the a t + a v recombinant virus. evaluating the use of cpg dna as an antiviral therapy amanda phelps, linda eastaugh, amanda gates, david ulaeto, arthur krieg defence science and technology laboratories (dstl), salisbury, wiltshire, uk; coley pharmaceuticals ltd., ottawa, ont., canada at present there are no licensed antivirals against orthopoxvirus infections such as variola or vaccinia virus (vacv). although a stockpile of smallpox vaccine exists and has utility as a post-exposure treatment to infection, it is a live viral vaccine and as such cannot be administered to those with contraindications. bacterial dna contains unmethylated motifs that, together with their flanking regions, can stimulate an innate immune response. synthetic cpg dna mimics the immunostimulatory activity of bacterial dna and is recognised by intracellular toll-like receptor . there are four classes of cpg dna all of which have different properties, eliciting distinct initial immune responses. previous studies using an established lethal respiratory model of poxvirus infection demonstrated that a class b cpg dna ( ) could provide protection from lethality against vacv in balb/c mice when administered up to days prior to challenge. in order to evaluate efficacy balb/c mice were challenged intra-nasally with vacv and treated with doses of ranging between and ug/mouse. treatment was administered intra-nasally under light anaesthesia either on the day of challenge, , , , or days post-challenge. efficacy was determined by percentage body weight loss post-challenge. the optimum survival rate observed was % when treated with ug day post-challenge ( mld challenge). a survival rate of % was observed when treated with ug days post-challenge ( mld challenge). the delay of treatment to either or days post-challenge was ineffective, indicating that the window of opportunity for delivery of is within days. multiple doses of were used to attempt to extend this window of opportunity, delivering twice within a -day period. interestingly, this had a considerable detrimental effect, actually accelerating the onset of disease and ultimately death. further work is required to optimise the use of cpg dna as a potential antiviral therapy, and there is evidence to suggest that they may have immense utility as part of a co-administration therapy with other antiviral compounds, an area of work currently under investigation. © crown copyright dstl . department of virology, hebrew university, hadassah medical school, jerusalem, israel the pathogenicity and immunogenicity of the lister (elstree) strain of vaccinia virus, used for vaccination against smallpox, was studied in the mouse model. the virus did not reach the brain when inoculated intranasally, but when injected intracranially at a dose of × plaque forming units (pfu), was lethal for % of the mice. lower doses of virus caused the mice to initially loose some weight but they completely recovered thereafter. a significant level of protection against a lethal dose of the wr strain was achieved in mice following immunization with the lister strain, while higher doses and repeated vaccination procedure, were required with modified vaccinia virus ankara (mva). we found that the lister vaccine strain applied in israel is comprised of heterogeneous virus population. we isolated and plaque-purified three virus variants differing in their plaque size in bs-c- cell cultures. they were named: l-large plaque, m-medium plaque and s-small plaque variants. these isolates could be neutralized by rabbit antibodies prepared against the western reserve strain of vaccinia virus and their one-step growth curves in bs-c- cells were quite similar. however, they differ in their pathogenicity to mice following intranasal inoculation of pfu, or an intracranial injection of × pfu; the s variant being more virulent than the other two variants and resembles the pathogenicity of the lister strain. activity was also determined against a thymidine kinase (tk) deficient vaccinia virus in mouse and monkey cells. the potency of (n)-mct was similar to that seen with wild-type virus, suggesting that a cellular enzyme may be more important than viral tk to phosphorylate the compound. mice were intranasally infected with cowpox and vaccinia viruses followed h later by intraperitoneal treatment with (n)-mct ( x/day for days) or cidofovir ( x/day for days). (n)-mct treatment at and mg/kg/day resulted in and % survival from cowpox virus infection, respectively, compared to % survival (placebo). statistically significant reductions in lung virus titers on day occurred in , , and mg/kg/day treated mice. these doses did not spare mice from lethal vaccinia virus challenge, however, but the and mg/kg/day treatments significantly reduced day virus titers and lung weights, and the mg/kg/day treatment reduced lung consolidation. cidofovir ( mg/kg/day) protected all animals from death in both models. the evaluation of (n)-mct may be limited to mice based upon its greatly reduced efficacy in the cells of higher animals. acknowledgement: supported in part by contract no- -ai- from the virology branch, niaid, nih. chelsea byrd , elena sbrana , shu-yuan xiao , marina siirin , robert tesh , dennis hruby , robert jordan siga technologies, inc., corvallis, or, usa; university of texas medical branch, galveston, tx, usa st- is a potent small molecule inhibitor of orthopoxvirus replication that has been shown to protect mice from lethal challenge with vaccinia and ectromelia viruses. here we report the results of preliminary trials that show efficacy of st- against severe monkeypox virus infection in the ground squirrel model. ground squirrels infected with less than pfu of monkeypox virus develop a fulminant disease resembling human hemorrhagic smallpox: the most severe and lethal form of the disease. oral administration of st- at mg/kg once per day for days protected ground squirrels from a lethal challenge with and pfu of monkeypox virus. compound treated animals showed no weight loss or evidence of disease, and blood chemistry values were similar to uninfected animals. in contrast, placebo-treated animals showed elevated liver enzyme (alt and ast) levels and all animals died by day post-infection. when treatment with , , and h, % protection was observed in the , , and h groups, and % protection in the h group. severe pathologic changes were observed in the organs of the animals receiving placebo, especially in the lungs, liver, and spleen. in contrast, the organs of the animals receiving st- at , , , and h postinfection appeared grossly and microscopically normal. thus, st- appears to be a promising candidate for continued development as a therapeutic agent for severe orthopoxvirus infection. inge vliegen , guang yang , dennis hruby , erik de clercq , robert jordan , johan neyts rega institute for medical research, k.u. leuven, belgium; siga technologies, inc. corvallis, or, usa st- is a potent inhibitor of the replication of various orthopoxviruses. resistance of cowpoxvirus to st- maps to a mutation in v , which is homologous to vaccinia virus f l (yang et al., . j. virol.) . the latter encodes the envelope protein p required for production of extracellular virus. deleting f l resulted in a virus ( f l-vac) that is replicationcompetent in cell culture but that produces smaller plaques than the wild-type wr-vac. whereas intravenous (i.v.) inoculation of nmri mice with × pfu of wr-vac resulted in ± pox tail lesions per mouse, the same inoculum of f l-vac caused no lesions (p < . ). athymic nude (nu/nu) or scid mice inoculated iv with × pfu f l-vac did not develop tail lesions. the mean day of death in nu/nu mice inoculated with f l-vac was ± days as compared to ± days for wr-vac-infected mice (p < . ); scid mice survived the infection. we next studied whether f l-vac is able to protect mice against a subsequent infection with wr-vac. to mimic the human vaccination protocol, nmri mice were infected intracutaneously (i.c.) by means of scarification at the lumbosacral area with × pfu f l-vac or placebo. none of the infected mice developed lesions at the inoculation site. one week later, animals were infected ic with × pfu of wr-vac. all placebo animals, but none of the f l-vac animals developed poxvirus lesions. in a second set of experiments, mice were again inoculated ic with placebo or f l-vac and were infected one week later with × pfu of wr-vac by the iv route. placebo animals developed an average of ± pox tail lesions; no lesions developed in the f l-vac animals (p < . ). in a third set of experiments, nmri mice were inoculated iv with either × pfu of f l-vac or placebo, and none of the mice developed lesions. one week later, animals were inoculated iv with × pfu wr-vac. the placebo group developed an average of ± lesions as compared to . ± . lesions in the f l-vac mice (p < . ). f l-vac may thus be considered as a severely attenuated virus that may have potential for use as a smallpox vaccine. ji yuan , travis lim , shuan coughlin , dexin qiu , zhen liu , dave stein , decheng yang the james hogg icapture centre for cardiovascular and pulmonary research, university of british columbia, vancouver, bc, canada; avi biopharma, inc., corvallis, or, usa background: coxsackievirus b (cvb ) is the most common cause of viral myocarditis, but existing drug therapy is of limited value. antisense oligonucleotides (asons) designed to pair with viral rna could inhibit viral replication. however, the effectiveness of traditional asons is limited due to poor cellular uptake and degradation by nucleases. phosphorodiamidate morpholino oligomers (pmos) contain backbone modifications, which make pmos more resistant to nucleases. in addition, an arginine rich peptide (p ) is conjugated to the end of the oligomer to improve its delivery into cells. these features make p -conjugated pmos (p-pmos) promising candidates for the inhibition of cvb infection. methods: total p-pmos targeting distinct regions of viral genome and one scrambled sequence were designed and chemically synthesized. fitc labeled p-pmos were used to observe their distribution of by confocal microscopy. viral protein vp , viral titre, and cell viability were measured by western-blot, plaque assay, and mts assay, respectively. results: p-pmos showed increased cellular uptake compared to non-conjugated pmos. among the p-pmos, p-pmo- , targeting the internal ribosomal entry site in the utr, showed the most potent anti-cvb ability in a dose-dependent manner. both infected hela and cardiomyocytes hl- cells treated with p-pmo- showed drastically reduced vp production and . log decreases in viral titres as compared to the controls. cell viability assay revealed that and % of treated hela and hl- cells were still alive as compared to and % of control-treated cells and % antiviral activity still existed after days treatment. in addition, cells treated post-infection showed similar inhibition of viral replication. furthermore, the specificity of the p-pmos was demonstrated by their inability to inhibit rsv infection in hela cells. we have showed that p-pmos can effectively inhibit viral replication in vitro, providing a new possibility for antiviral intervention. picornaviruses are responsible for various human viral diseases including common cold, encephalitis, meningitis, myocarditis, etc. up to now, there is no specific antiviral therapy to treat or prevent such viral disease. the usage of modern computer technologies may help to solve this problem more effectively. the objective of the present study was the quantitative structure-activity relationship (qsar) analysis of antiviral activity of a set of [(biphenyloxy)propyl]isoxazole derivatives that inhibit cvb replication in hela cells. based on results from qsar, the structure of new potential antiviral agents should be predicted by using consequent molecular design. the qsar approach applied is based on simplex representation of molecular structure (sirms). the relationship between: (a) antiviral activity against the pleconaril-sensitive clinical cvb isolate - (ic , g/ml); (b) cytotoxicity in hela cells (cc , g/ml); and (c) selectivity index (si = ratio of cc to ic ), and structure of [(biphenyloxy)propyl]isoxazole derivatives has been studied systematically. quite adequate qsar models (r = . - . , q = . - . ) have been obtained using pls (partial least squares) method for all parameters studied. the models are in close correlation with experimental data. structural fragments with positive or negative influence on cytotoxicity as well as antiviral activity have been determined on the base of these models. for example, qsar analysis of antiviral activity of [(biphenyloxy)propyl]isoxazole derivatives revealed that the presence of m-nitrophenyl or p-trifluorophenyl fragment has distinctly negative influence on antiviral action. compounds with strong antiviral activity have to contain an oxadiazole fragment. moreover, our data allow the virtual screening and molecular design of new well-tolerated compounds with strong anti-cvb activity. ivanka nikolova , roumena petkova , boris atanassov , stoyan chakarov , angel s. galabov institute of microbiology, bulgarian academy of sciences, sofia, bulgaria; scientific technological service, ltd., sofia, bulgaria; institute of organic chemistry, bulgarian academy of sciences, sofia, bulgaria analysis of the rna sequence of the disoxaril-resistant mutants of the coxsackievirus b was carried out. the wild-type disoxaril-sensitive strain (connecticut ) and two disoxarilresistant mutants (one of them produced in fl cells and the other one isolated from brains of newborn mice) infected with coxsackievirus b and treated with disoxaril and a disoxarildependent mutant strain obtained from the resistant strain by passages in cell culture were included in the present study. a rt-pcr assay with primer sets selected from a region of the coxsackievirus b genome coding for the capsid protein vp was carried out. a parallel comparative analysis of the sequences of resulting fragments from the disoxaril mutants studied and the genbank sequence of origin of the vp gene of coxsackievirus b was performed with the blast alignment tool. distinct alterations in the vp locus of the disoxaril-resistant and the disoxaril-dependent mutants compared to the sequence of origin from the genbank (namely, a deletion of uug at ntt. - and an insertion of uuu at nt. ) were observed. high-degree similarity ( %) between the resistant mutant produced in cell cultures and the dependent strain was observed, while the similarity to the wild strain was only %. the resistant mutant obtained in mice was found to be very similar to the strain, developed in cell cultures. a putative -d model of the spatial folding of the target protein in disoxaril mutants is proposed. ralitsa vassileva-pencheva, angel s. galabov in previous study of ours we presented a new approach to combined application of antivirals-consecutive administration of the partners. this schedule could be considered especially suitable for treatment of enteroviral infections, in which the development of resistance is very rapid due to the extremely high viral mutation rate. this approach aims to restrict the resistance development in experiments in vivo, using antivirals with proved high efficiency in experiments in cell cultures. the screening of various double, triple, and quadruple combinations that we carried out showed that two of the triple combinations, namely disoxaril (win compound)/oxoglaucin (a new antiviral drug, developed in our laboratory)/ptu- (a classic enteroviral inhibitor) and disoxaril/oxoglaucin/guanidinehydrochloride (a classic enteroviral inhibitor) manifest significant effect of protection in newborn mice with neurotropic coxsackievirus b infection. in the current study the role of the chronology of arrangement of the antivirals included in the combinations was investigated. in the experiments carried out with the triple combination disoxaril/oxoglaucin/guanidine-hydrochloride, it was found that the optimal treatment course should start with disoxaril. the treatment course is quite successful when disoxaril is followed by guanidine-hydrochloride. the effect of the triple combination starting with oxoglaucine, followed by guanidine-hydrochloride was moderate. the course starting with guanidine-hydrochloride proved to be ineffective. furthermore, we studied the virus sensitivity to the inhibitorspartners (ic values) and some other phenotypic characteristics of the brain isolates, e.g. the size of the plaques and the pathogenicity for mice. recently our contribution to the development of new antipicornavirus agents has led to the discovery of methylthio- -aryl-isoxazole- -carbonitrile derivatives whose in vitro anti-coxsackievirus b activity were dependent on the nature of the substituents on the para position of the phenyl ring. particularly, compounds -methylthio- -[ -( -phenyl- -propoxy)phenyl]isoxazol- -carbonitrile (on- ) and -methylthio- -[ -( -phenoxy- -buthoxy)phenyl]isoxazol- -carbonitrile (on- ) exhibited an interesting antiviral activity with high selectivity indexes. in the present study, we investigated on the mechanism of action of these compounds. studies on time of addition experiments suggested that these compounds exert a different interference with an early step of the viral replicative cycle. in fact, compound on- was effective when added within h after the end of the adsorption period and no reduction was observed if it was added during the adsorption period. whereas a reduction of virus titer was observed for on- when was added during the adsorption period, while no reduction was observed if the compound was added after this period (time ). the influence of the compounds on virus adsorption step, studied by the infective center assays, indicated that on- primarily interferes with coxsackie b cellular attachment. at a concentration times the id , inhibition of adsorption of coxsackievirus by on- was complete, while similar concentration of on- had no effect. our experiments on neutralization of viral infectivity and on thermal stabilization demonstrated that the compounds were able to directly inactivate coxsackievirus, and the infectious titer was restored to the original value after extraction of the compound with chloroform. however, the compounds did not protect the viral infectivity against heat inactivation at the different concentrations used. the blood-brain barrier (bbb) fulfills a vital protective function by limiting entry of potential pathogens, toxins, and inflammatory cells into the central nervous system (cns). disruption of the bbb is a common component of many cns diseases, including viral diseases such as that caused by west nile virus (wnv). transforming growth factor-␤ (tgf-␤ ) has previously been shown to improve the function of an in vitro model of the bbb. we evaluated the role of the bbb in wnv infection in mice by determining the ability of intraperitoneally (i.p.) administered sodium fluorescein to move from the circulating blood to the central nervous system. to demonstrate bbb permeability a mean and normal range of permeability values was determined in non-infected c /bl mice. in subsequent experiments, any animal expressing a permeability value greater than sd above the mean was considered abnormally high. we determined that elevations in bbb permeability can be detected in mice days after subcutaneous inoculation with wnv. wnv inoculated animals were treated with doses of , , or ng/kg/day of tgf-␤ or with drug vehicle once daily via the i.p. route on and days post-virus inoculation (dpi), and then assayed for bbb permeability on dpi. sixty-two percent ( / ) of placebo-treated animals had abnormally high permeability values, while animals treated with and ng/kg/day of tgf-␤ had % ( / ) and % ( / ) of animals with abnormally high permeability values, respectively. in contrast, none of the animals treated with ng/kg of tgf-␤ ( / ) expressed abnormally high permeability values, which was significantly lower (p < . ) than placebo-treated animals. these results suggest that tgf-␤ may improve the function of the blood-brain barrier in wnv infected mice. acknowledgement: supported by grant -u ai - from the rocky mountain regional centers of excellence, nih. and contract no -ai- from the virology branch, niaid, nih. people infected with west nile virus (wnv) usually see their physicians after showing symptoms suggestive of neurological infection. wnv infects the central nervous system (cns) of rodents - days after s.c. viral challenge. yet, most published animal studies begin therapeutic treatments before or soon after viral challenge. the question addressed in this study is if neuroprotective agents can be efficacious when administered early before brain infection or later after the virus is demonstrated to be in the brain. the drugs evaluated in wnvinfected rodents were nmda and ampa receptor antagonists, modulators of nitric oxide synthase (nos) and nitric oxide production, and riluzole for reducing glutamate excitotoxicity. serial doses of diethyldithiocarbamate (ddtc) and n(g)monomethyl-l-arginine (l-nmma), an inducer or inhibitor of nos, respectively, administered i.p. daily for days beginning h before viral challenge slightly improved survival of mice, but the difference was not statistically significant. tolerated doses of two nmda-receptor antagonists, flupertine ( mg/kg) and mk- ( mg/kg), and one ampa-receptor antagonist, gyki ( mg/kg), were administered twice daily (b.i.d.) on though days post-virus inoculation (dpi). gyki slightly improved mouse survival and weight gain, but the difference was not statistically significant. talampanel, an ampa-receptor antagonist and a derivative of gyki , slightly improved hamster survival (p ≤ . ) when treatment began on dpi, but repeated experiments using different doses and slightly different protocols gave mixed results. riluzole, the only drug shown to improved survival of amyotrophic lateral sclerosis (als), presumably by reducing glutamate excitotoxicity, was not effective against wnv disease when administered b.i.d. beginning dpi. overall, neuroprotective agents did not consistently improve wnv disease, although slight improvements in animal survival might be relevant to improvement of neurological sequelae in wnv-patients. acknowledgement: supported by contract no -ai- from the virology branch, niaid, nih. hamster and mouse models for west nile virus (wnv) disease were used in this study to identify infected cells of the central nervous system (cns) early in the course of infection. this information may be relevant to therapeutic strategies since most wnv-infected people visit their physicians after showing symptoms suggestive of neurological infection. we subcutaneously infected adult mice and hamsters using . tissue culture infectious doses of wnv. tissues of infected and control animals from to days post-viral injection (dpi) were fixed by cardiac perfusion using % paraformaldehyde. we localized wnv, neuronal and astroglial markers in the paraffin embedded tissue sections by immunofluorescence. the images were captured using the confocal microscope (bio-rad, mrc ). we observed the presence of wnv antigen in cns tissues of mice and hamsters as early as and dpi, respectively. a strong wnv-specific immunofluorescence staining was observed in the cytoplasm of neurons from the spinal cord, cerebellum, cerebral cortex, and midbrain of these rodents. the wnv-specific staining co-localized with neuron-specific markers; however, astroglial markers were not co-localized with wnv antigen in brain sections. the lack of tropism by wnv for astrocytes was also confirmed in primary murine astrocyte cultures. interestingly, infected neurons in the midbrain of -day infected hamsters co-localized with calbindin, which is a calcium-binding protein and mostly expressed in the interneurons of the cns. therapies were evaluated in hamsters or mice at a time-point when wnv-stained neurons were identified in the cns. acknowledgement: supported by grant -u ai - from the rocky mountain regional centers of excellence, nih. laboratory of virology, department of biological chemistry, school of sciences, university of buenos aires, argentina dengue virus (denv) is an arthropod-borne flavivirus that has re-emerged in recent years as an increasingly important public health threat with nearly million infections occurring each year. at present neither specific antiviral therapy nor vaccine exists for the treatment and prevention of denv infections. carrageenans are sulfated galactans that can be extracted from red seaweeds and comprise diverse structures with a wide range of biological properties useful in biomedicine. in a previous study we have demonstrated the antiviral activity of commercialand -carrageenans against denv type and in vero (monkey kidney cells) and hepg (human hepatoma cells), showing inhibitory concentration % (ic ) values in the range . - . g/ml and selectivity indexes (cc /ic ) in the range - . in the present work we studied the mode of action of these polysulfates against denv- in vero and hepg cells, first analyzing the influence of time of addition of compounds on anti-denv activity by an infectious centre assay. the highest inhibitory effect was observed when the compounds were added during adsorption or at h p.i., being ineffective at later times. then, the effect of compounds on virus adsorption and internalization was studied separately by a virus yield inhibition assay. significant antiviral efficacy was attained if compounds were present either only during denv- adsorption or internalization. the possible effect of carrageenans on viral protein synthesis, the subsequent stage of the virus cycle occurring during the first hour of infection, was analyzed by pulse-labeling with ( s)-methionine. no alterations in denv protein synthesis in carrageenan-treated cells were observed. when cells were transfected with purified denv- rna in the presence of -carrageenan no inhibition in fluorescent cell focus formation and virus production was detected. besides, no significant direct virucidal effect on denv- was shown by the compounds. these results indicate that both denv adsorption and internalization seem to be the main target for these compounds, lacking effect on the steps that occur once the viral genome is inside the cell during in vitro infection of human and monkey cells. multiple members of the flavivirus genus of the family flaviviridae cause lethal hemorrhagic fever or encephalitis. the public health significance of the hemorrhagic fever and encephalitis caused by such flaviviruses is enormous and global and there is a tremendous need for antivirals. imino sugar glucosidase inhibitors have been shown to have selective antiviral activity against viruses such as bovine viral diarrhea virus (bvdv) and west nile virus (wnv) that have common requirements for their glycoprotein processing during virus production. we are developing imino sugar deoxynojirycin (dnj) derivatives through chemical synthesis of compounds with various alkyl side chains and antiviral testing against bvdv and wnv as well as in wnv subgenomic replicon assays. briefly, using a single step growth (virus yield reduction) assay for bvdv and wnv, a series of dnj derivatives containing various conformational locking side chains were shown to have antiviral activity. pre-liminary structure-activity relationships (sar) were obtained for further modification of the alkyl side chain and improvement of these dnj derivatives. the activity and mechanisms of action of these compounds will be presented. several flaviviruses cause life-threatening diseases in man. currently, there is no therapy available for these infections. in recent years, several highly selective inhibitors of the replication of hepatitis c virus (hcv) were designed. most small molecule inhibitors of hcv that are in preclinical or clinical development are either protease or polymerase inhibitors. most of these compounds are highly selective for hcv and are unlikely to exhibit activity against flaviviruses. nucleoside polymerase inhibitors, however, may have the potential to inhibit the replication of flaviviruses as well. we evaluated in vitro whether the active component of the anti-hcv compound valopicitabine, i.e. -c-methylcytidine inhibits the replication of flaviviruses in cell culture. the compound was found to exhibit specific antiviral activity against yellow fever virus d (ec = . g/ml in cpe reduction assays and > % reduction at g/ml as assessed by qpcr) and dengue fever virus type (ec = . g/ml in cpe reduction assays). the compound also efficiently inhibited west nile virus replication (> % at g/ml as assessed by qpcr and > % by plaque reduction neutralization test at g/ml). in the absence of any drugs for the treatment of flavivirus infections, it may be envisaged that nucleoside polymerase inhibitors, when marketed for the treatment of hcv infections, could be used off-label for the treatment of lifethreatening flavivirus infections. even if such drug would not be able to completely inhibit flavivirus replication, a partial reduction of the viremia during the acute phase of the infection may be sufficient to prevent the development of a fulminate disease and thus protect against virus-induced mortality. yuri klimochkin , andrew shiryaev , igor moiseev , v sabynin , larisa rustamova , alexandr petkevich state technical university, samara, russia; institute of epidemiology and microbiology, minsk, belaruss arenaviruses are one of the most dangerous tools of bioterrorism in the view of pathogenicity and epidemiological threat. for the purpose of searching new remedies for treatment are-naviruses infections the synthesis of new derivatives of cage compounds has been carried out. the prepared compounds are bridgehead derivatives of cage compounds bearing different functional groups such as hydroxy, acylamino, alkoxycarbonylamino, alkylthiocarbonylamino groups as well as iminoalkyl adamantane derivatives, some adamantylated heterocycles and compounds containing two adamantane moieties in a molecule. the antiviral activity of the cage compounds has been studied in respect to arenaviruses lassa (sierra-leone strain) and pichinde (an- strain) on the vero cells culture. different level of antiviral activity was shown by compounds. the most active compounds are monosubstituted adamantane derivatives having sufur and nitrogen-containing substituent in the bridgehead position. the data on the activity confirm the availability of searching inhibitors of arenaviruses reproduction in the cage compounds series. brian gowen , donald smee , min-hui wong , anne pace , kie-hoon jung , kevin bailey , lawrence blatt , robert sidwell institute for antiviral research, utah state university, logan, ut, usa; intermune, brisbane, ca, usa several arenaviruses endemic to the south american (junin, machupo, and guanarito) and african (lassa) continents are known to cause frequently fatal hemorrhagic fever. with the exception of ribavirin, which has demonstrated efficacy in cases of lassa fever, there are no other effective therapeutics for the treatment of arenaviral hemorrhagic fever. the outcome of treatment is ultimately dependent upon early diagnosis and the tolerability of ribavirin by patients at the high doses required for effective antiviral activity. we have recently demonstrated that consensus interferon-alpha (ifn-alfacon ) can protect hamsters from lethal pichinde virus (pcv) infection (gowen et al., . antimicrob. agents chemother.), which serves as a model for acute arenaviral disease in humans. here we demonstrate highly effective therapy through the combined use of ribavirin and ifn alfacon- for the treatment of pcv infection in hamsters. ribavirin was given orally, twice per day for days, and ifn alfacon- was administered intraperitoneally, once per day for days. treatments were initiated - days post-infection with various dose combinations, many which were less than optimal when the drugs were given independently. combining suboptimal doses of ribavirin ( - mg/kg/day) with ifn alfacon- ( - mg/kg/day), we were able to show increased protection from mortality, reduced viral burden and liver disease, and greatly extended survival times as compared to treatments where drugs were administered alone. our data indicate that synergistic activity resulted from combination therapy and that this activity may slow down the progression of disease and decrease fatality rates seen with severe arenaviral infections. further, combination therapy reduces the effective dosage of ribavirin, which would serve to limit its toxicity. acknowledgement: supported by contract no -ai- from the virology branch, national institute of allergy and infectious diseases, national institutes of health. slobodan paessler , laure deflubé , andrew vaillant , jean-marc juteau , ramon flick department of pathology, university of texas medical branch, galveston, texas, usa; replicor inc., laval, que., canada rift valley fever virus (rvfv; genus phlebovirus, family bunyaviridae) is an arbovirus transmitted by many species of mosquitoes. this virus is a major public health concern in sub-saharan africa and egypt, which spread to yemen and saudi arabia. in this area, rvfv is responsible for dramatic epidemics/epizootics underlining the need for efficient antiviral/prophylactic measures. rep is a mer phosphorothioate oligonucleotide, which has previously been shown to have broad-spectrum activity in several viruses (vaillant et al., submitted for publication) . we used a vaccine strain (mp ) as well as the wild-type rvfv (zh ), to test the ability of rep to inhibit bunyavirus proliferation. in vitro data showed reduction of virus titer for both strains using rep at nanomolar concentrations. moreover, the absence of the phosphorothioate modification in a stabilized rep analog resulted in a loss of antiviral activity, suggesting that as in other viruses, the increased hydrophobicity of rep is essential for its antiviral activity. based on the inhibitory activity observed in vitro, we started with in vivo efficacy studies by utilizing a validated mouse model used in our laboratory. more animal experiments are ongoing to confirm the in vitro results and to evaluate the antiviral effect of the rep . adriana garozzo , rossella timpanaro , aldo stivala , gianna tempera , christian c.c. cutrì , angelo castro department of microbiological sciences, university of catania, via androne , catania, italy; department of pharmaceutical sciences, university of catania, viale a. doria , catania, italy our previous studies described the synthesis and the antiviral activity of , , -trisubstituted isothiazole derivatives that were found to be particularly effective against picornaviruses. compound -methylthio- -phenyl- -isothiazolecarbonitrile (is- ) exhibited an interesting anti-poliovirus activity with high selectivity index. in the present study, we investigated on the mechanism of action of this compound. studies on the time of is- addition to poliovirus type infected cells suggested that the compound may inhibit some early processes of viral replication. in order to determine its mechanism of action, we evaluated the rate of attachment and internalization of purified [ h]uridine-labeled poliovirus to hep- cells in the presence or absence of is- . no effect on poliovirus adsorption and internalization to host cells was detected. we also investigated the influence of the compound on virus uncoating using labeled poliovirus and measuring the radioactivity of oligoribonucleotides formed from viral rna susceptible to ribonuclease. these experiments demonstrated that poliovirus uncoating is influenced by is- action. justin julander , aaron olsen , john morrey , lawrence blatt , kristiina shafer , robert sidwell institute for antiviral research, utah state university, logan, ut, usa; intermune, brisbane, ca, usa alpha togaviruses are medically important arboviruses, with clinical cases occurring each year in north, south, and central america. the recent increase in the threat of the use of these viruses as bio-terrorism agents has led to increased efforts to develop therapeutic agents for treatment of these viruses. venezuelan (vee) and western equine encephalitis (wee) viruses have been listed as category b priority pathogens by the national institute of allergy and infectious disease (niaid). the goal of these studies was to characterize animal models for vee and wee for use in evaluation of antiviral therapies. c h/hen mice were infected through the intranasal (i.n.) route with a vaccine strain of vee, tc- . virus was detected in the brain days post-viral injection (dpi). brain titers increased to a peak titer of . % cell culture infectious doses per gram tissue (ccid /g) on dpi, maintained a titer of ccid /g through dpi, and dropped slightly to . ccid /g by dpi. virus was also detected in spleen, liver, and kidney. treatment of vee-infected mice with interferon alpha b/d, a human consensus interferon, resulted in % survival, whereas all placebotreated animals died by dpi. syrian golden hamsters were infected with ccid wee through intraperitoneal (i.p.) injection. morbidity, including hind limb paralysis, tremors, nasal bleeding, and hunching, and some mortality were seen as soon as dpi. the majority of deaths occurred on dpi. virus was detected in all organs assayed (brain, liver, and spleen) with peak titers occurring dpi. interferon alfacon (ifn alfacon), a human consensus interferon, active in hamsters, was effective in significantly reducing mortality (p < . as compared to placebo). there was a trend for reduction of brain titers in ifn alfacon-treated animals (mean titer . ccid /g) as compared with placebo (mean titer . ccid /g), although this difference was not statistically significant. these models will be useful in screening potential antiviral agents for efficacy against vee and wee. acknowledgement: supported by contract no -ai- from the virology branch, niaid, nih. justin julander , kristiina shafer , john morrey , lawrence blatt , robert sidwell institute for antiviral research, utah state university, logan, ut, usa; intermune, brisbane, ca, usa yellow fever virus (yfv) has caused significant morbidity and mortality for centuries. primates were the only animal models for visceral yfv. recently, hamsters were found to have morbidity and mortality when injected with a hamster-adapted jimenez strain of yfv (tesh et al., j. infect. dis. , - . the objective of this study was to characterize this model of yfv viscerotropic disease for the study of effects of antiviral compounds and to test compounds with known efficacy for use as a positive control. animals were challenged with a − dilution (a dilution previously shown to cause high mortality) of a liver homogenate made from livers taken days post-viral injection (dpi) from hamsters challenged with the jimenez strain. there was % mortality in animals challenged with the virus up to dpi. virus titers in the liver peaked dpi as determined by qrt-pcr. a significant increase in serum levels of alt ( dpi), alkaline phosphotase ( dpi) and bilirubin ( dpi), and a significant decrease in amylase ( dpi), albumin ( dpi), and glucose ( dpi) were observed. hepatic icterus was observed in hamsters that exhibited disease signs at the time of necropsy. hamsters were treated with ribavirin or interferon (ifn) alfacon , a consensus interferon. ribavirin and ifn alfacon both significantly (p < . ) reduced mortality as compared with placebo-treatment. there was also significant reduction in weight loss with ribavirin (p < . ) and ifn alfacon (p < . ) treatment as compared with placebo. disease signs, such as lethargy and lying prostrate, were also reduced with treatment of ribavirin and ifn alfacon . viral liver titers from treated animals were not significantly different from titers in placebotreated animals. the hamster model of yfv disease will serve as a suitable model for the evaluation of antiviral compounds for efficacy against the virus. acknowledgement: supported by contract no -ai- from the virology branch, niaid, nih. polyomaviruses are small dna tumor viruses that depend on the host cellular dna polymerase for their replication. three polyomaviruses have been associated with tumor formation in humans: jc virus (jcv), bk virus (bkv) and simian vacuolating virus (sv ). in addition, some of them have been associated with viral diseases. jcv can cause progressive multifocal leukoencephalopathy in immunosuppressed patients, while bkv is considered to be the causative agent of polyomavirusassociated nephropathy, which leads to kidney transplant failure. sv has not been associated with a well-defined disease, but viral dna sequences and protein expression have been detected mostly in central nervous system (cns) tumors which strengthens the evidence for the association of this virus with human cancer. the activity of various acyclic nucleoside phosphonates (anps) such as cidofovir and adefovir against murine polyomavirus and primate sv in vitro has already been demonstrated (andrei et al., . antimicrob. agents chemother. , - ) . here, the activity of a new class of anp's, namely -[ -(phosphonomethoxy)alkoxy]- , diaminopyrimidines, against polyomaviruses was assessed. confluent uc -b cells were infected with either of the four murine polyomavirus strains mn/rde toronto, pta, pta or lid- , while bsc- cells were infected with either the primate sv strain a , the sv pml- strain ek or the sv pml- strain dar. after removal of the residual virus, serial dilutions of the test compounds were added. the viral cytopathic effect was recorded microscopically after - days (murine polyoma virus) or - days (sv ). hpmpo-dapy ( , -diamino- -(r)-[ -hydroxy- -(phosphonomethoxy)propoxy]pyrimidine) and pmeo-dapy ( , -diamino- -[ -(phosphonomethoxy)ethoxy]pyrimidine) were less active/selective than cidofovir and adefovir against the three sv strains tested. hpmpo-dapy and pmeo-dapy proved to be equally active as cidofovir and adefovir against the murine polyomaviruses. naresh sunkara , sylvester mosley , brian bakke , joshua sadler , katherine seley(radtke) , sunny zhou university of maryland-baltimore county, baltimore, md, usa; washington state university, pullman, wa, usa inhibition of biologically significant enzymes critical to nucleotide metabolism and viral replication is a well-established chemotherapeutic approach to the treatment of many diseases. transcriptional -capping of viral mrna has been implicated as an "elongation checkpoint" critical to the replication cycle of many viruses. this capping process is accomplished by various methyltransferases, therefore disruption of methylation becomes an attractive target for therapy. this can be accomplished in several ways; in particular, by direct inhibition of methyltransferases (metase) and/or indirect inhibition of s-adenosyl-l-homocysteine hydrolase (sahase), both established cellular targets for antiviral, antiparasitic and anticancer agents. modified nucleosides, in particular carbocyclic nucleosides, have exhibited potent inhibitory activity against both sahase and metase. inspection of the recent literature has revealed a close correlation between sahase inhibition and potent biological activity against negative stranded (−)-rna viruses (i.e. arenaviridae, paramyxoviridae, rhabdoviridae), double stranded (ą)-rna viruses (reoviridae), poxviridae, as well as hiv- , thus supporting the importance of sahase as a viable chemotherapeutic target. herein we report the design, synthesis, and preliminary biological activity of a new class of structurally novel carbocyclic nucleosides. phosphorylation of ␣-p-borano substituted nucleoside diphosphates charlotta wennefors, mikhail dobrikov, barbara ramsay shaw chemistry department, duke university, durham, nc - , usa most nucleoside antiviral agents require stepwise phosphorylation to their respective triphosphates in order to be activated in the cell. ␣-p-borano substituted nucleoside triphosphates are of interest because they have proven to be good substrates for hiv- reverse transcriptase (rt) and may therefore be useful antiviral agents. studies in our laboratory have indicated that the ␣-p-borano substitution of -dideoxycytidine triphosphate (ddctp) resulted in a -fold increase in efficiency of incorporation by mmlv rt compared to non-substituted ddctp. however, the potency of these ␣-p-borano substituted nucleoside analogs as anti-viral drugs highly depends on their ability to be activated to nucleoside triphosphate (ntp). the phosphorylation of nucleoside analog diphosphates to their respective triphosphates has remained largely unexplored. here, the roles of several phosphorylating enzymes are examined. in our laboratory, nucleoside diphosphate kinase, creatine kinase, and pyruvate kinase are being evaluated for their specificity towards nucleoside analog diphosphates. the effects of nucleobase, ribose, ␣-phosphate substitution and stereochemistry of the boranophosphate group are of interest. the binding affinities of the substrates for creatine kinase (ck) and pyruvate kinase (pk) were determined using a fluorescence-quenching assay, which allowed us to investigate the substrate affinity in the pre-steady state. rabbit muscle ck and pk were titrated with a wide range of ndps and ntps by monitoring a decrease in enzyme intrinsic fluorescence. the affinities of these substrates were determined to establish a structure-activity relationship for ck and pk and to evaluate the effect of a substrate ␣-p-borano modification. ck showed stereospecificity towards the sp isomer of adp␣b whereas pk showed stereospecificity towards the rp isomer of adp␣b. negative cooperativity was observed for all studied substrates. steady-state experiments are also being performed directly following the product formation using uv-visible spectroscopy and high performance liquid chromatography (hplc). these kinases were investigated because they may serve as a means for activation of antiviral ␣-p-borano substituted ndps. traditional antiviral targets encoded by the small human papillomavirus (hpv) genome are lacking. for this reason, we chose to target dna sequences within the hpv genome in an effort to identify compounds that would block viral dna replication in cells. we chose compounds known as polyamides, which are related to distamycin and other natural products, as our dna binding agents. unlike many literature studies where polyamides were designed to block formation of the transcription complex for a particular gene, we chose to target sequences within the origin of replication (ori). thus, pyrrole-imidazole polyamides, with some containing fluorescent probes to aid in cell localization studies, were designed to recognize the hpv ori. the principles used to design these compounds will be described. we used "traditional" hairpin polyamides and some more unusual structures related to very recent literature reports. from the focused library that we prepared, two highly active molecules were identified. the rest of the molecules had minimal or zero activity. no cellular toxicity was observed, either in this project or in a related program where polyamides were used to affect cox- transcription (and subsequent expression) in rheumatoid synovial fibroblasts. of particular interest is the difference between the active molecules and two closely related compounds that were inactive: the active species bind and recognize two more hpv dna base pairs than do the related but inactive structures. this presentation will provide detailed chemistry background and structural information to complement our cell work that is also being presented at the meeting. discovery of the chemokine receptor ccr as a co-receptor for hiv- infections revealed a novel approach to hiv- treatments and preventions. ccr , a member from the family of tm g-protein coupled receptors, thus became an attractive target pursued in the pharmaceutical industry. with the recent successful developments of several small molecules in clinic, these ccr antagonists hold great promise to be the next generation of anti-hiv medicines. this poster will describe our efforts at the n-terminal piperidine ring of template a to improve pharmacological properties of derived molecules. according to current models, proteolytic processing of hiv- gag precursor occurs within the virions which detach from infected cells. meanwhile, the viral protease is activated much earlier, and gag p cleavage initiates in infected cells. we followed the fate of matrix protein cleaved in infected cells (cma) in comparison with ma cleaved in the virions (vma) and showed that both forms differ in their localization in the infected cells and in the virions, both forms are involved into virus pathogenesis and represent the targets for antiviral compounds. mt- cells were labeled with [ h]-leucine or myristic acid, and h after labeling protease inhibitor was added to separate the cleavage of cma from vma. cma was found in the nuclear and membrane fractions of infected cells while cca resided in cytoplasm. - h after labeling cma was found in the virions localizing in the cores. vma was located under lipoprotein envelope of the virions. new membranotropic antiviral compounds based on adamantane-and norbornene-related derivatives not toxic for the host cells were added to mt- cells before infection or - h later and at concentration - ug/ml blocked reverse transcription, the transport of cma into the nuclei, and the production of infectious virus. the compounds inhibiting very early step of virus life cycle are optimal candidates for microbicides. to enhance their antiviral activity, we plan to associate polyanionic matrix with ma imitating peptides and cholesterol-like fragments. kurt vermeire , thomas bell , sreenivasa anugu , noah duffy , roger le grand , erik de clercq , dominique schols rega institute for medical research, katholieke universiteit leuven, leuven, belgium; department of chemistry, university of nevada, reno, usa; service de neurovirologie, fontenay-aux-roses, france the cyclotriazadisulfonamide (cada) compounds were shown to be potent inhibitors of hiv replication in human t-cell lines, pha-stimulated pbmcs, and monocytes/macrophages (ec : . - . m). the prototype compound, cada, had consistent activity against laboratory adapted and primary clinical isolates of hiv- , irrespective of chemokine receptor preference (r , x , r /x ). cada acted synergistically when evaluated in combination with various other hiv drugs, such as reverse transcriptase (rt), protease, and virus entry inhibitors. flow cytometric analysis revealed a significant decrease in the cell surface and intracellular expression of the cd receptor in human cells after cada-treatment. moreover, the anti-hiv activity of cada correlated with its ability to down-modulate the cd receptor in human t-cells. here, we report the consistent antiviral activity of cada against: (i) drug-resistant viruses (i.e. viruses resistant to rt inhibitors, protease inhibitors, and enfuvirtide); (ii) different hiv- subtypes (a, b, c, d, a/e, f, h, o); and (iii) various hiv- strains examined. in addition, cada potently inhibited sivmac infection of pbmcs isolated from macaques (ec : . m). comparable results were obtained in human cells infected with sivmac . flow cytometric analysis also demonstrated a significant and dose-dependent down-regulation of the cd receptor expression at the cell surface of simian pbmcs after treatment with cada. the combination of cada with cellulose acetate , benzenedicarboxylate (cap), an enteric coating polymer for capsules and tablets, resulted in a synergistic antiviral activity. in summary, our data indicate that cada may qualify as a potential anti-hiv microbicide drug candidate for the prevention of the sexual transmission of hiv. the preparation of gel formulations of cada (as single drug and in combination with cap) for vaginal administration in non-human primates is currently under investigation. department of micro & immuno, suny upstate medical university, syracuse, ny, usa varicella zoster virus (vzv, human herpesvirus ) infection causes chicken pox, latency is established in neurons, and reactivation leads to shingles. acyclovir and its derivatives are the treatment of choice for both manifestations of vzv. new therapeutics are needed because acyclovir-resistant strains exist, and treatment must begin within h. we have studied the anti-vzv properties of roscovitine, a cyclin dependent kinase (cdk) inhibitor. here, we tested more compounds that block the cell cycle and determined that vzv is acutely sensitive to them. their effects on vzv replication were tested in human foreskin fibroblasts (hffs) because these primary cultures should have a normal cell cycle (unlike tumor cell lines). the cytotoxicity of the drugs was determined by neutral red dye uptake assays. hffs were inoculated with a low moi ( . ) of vzvinfected cells, which remains entirely cell-associated, and then treated with drugs or diluent for h. vzv spread and replication were measured by infectious focus assay and quantitative real time pcr. all of the drugs tested (acyclovir [acv], phosphonoacetic acid [paa], aphidicolin, aloisine a, purvalanol a, roscovitine, r-roscovitine, s-roscovitine, indole- -carbanol [i c], l-mimosine, dichloro-␤-d-ribofurano-sylbenzimidazole [drb]) had some anti-vzv activity. the selective indices of aphidicolin ( ), purvalanol a ( ), and i c ( ) were greater than the positive controls acv ( ) and paa ( ). aphidicolin inhibits mammalian dna polymerase and is in clinical use for cancer; purvalanol a, a , , -trisubstituted purine, primarily inhibits cdk ; and i c is derived from cruciferous vegetables and inhibits cell proliferation. the concentrations of these compounds that inhibited vzv replication were much less than those needed to cause cell cycle arrest, suggesting that vzv depends on the enzyme activities targeted by these compounds and not on cell proliferation per se. these three drugs will be studied next in skin organ culture and in the scid-hu mouse model of vzv pathogenesis. the results presented here demonstrate that targeting cell functions can inhibit vzv replication and help us better understand virus-host interactions. the viruses could be identified as supra-biopolymeric nanoscale complexes, parasitic intervention in cells of which occurs on an inter-polymeric reactions level. so the antiviral safety can not be fully provided without adequate nano-responsible antivirals (nav). here we discuss a strategy and methodology for the multi-functional nav development by rational macromolecular sar-cooperation of: ( ) polyelectrolyte-specific interferon induction and immunomodulation; ( ) electrostatic-selective prevention of viruses absorption on plasma membranes; ( ) membrane-targeted blocking of post-absorption steps (fusion); ( ) macromolecular prevention of structure-specific interactions of viral and cellular receptors; as well as ( ) polymericassociated disruption of the latest stage of viral replication (virions assembly and maturation). a cooperation of the ( ) and ( ) functions was explored by synthesis and sar-optimization of succinate and carbohydrate polymeric derivatives modified with controllable combinations of anionic (a /a ) groups. the immune-mediated protectors against tick-born, rabies, and other viral infections in vivo, and hiv- absorption inhibitors in vitro, were developed. this pre-nav generation was used as a macromolecular platform to step-by-step targeted design and synthesis toward high effective multi-functional nav where virusresponsible nano-selectivity was achieved by rational intra-or inter-molecular cooperation of virus-specific membranotropic vectors (bi), raft-targeted anchors (ci), and peptide-kind mimickers of virus usable receptors of human cells (pi), particularly ccr /cxcr . as a result, the novel nano-sensitive systems possessed unique wide multi-synergistic antiviral potency on a high level selectivity up to si = , (against hiv- strains) were created and purposed for advancement of antiviral vaccines, drugs, and microbicides. marina kukhanova , alexander ivanov , , georgii galegov , valeria andronova , maxim jasko engelhardt institute of molecular biology, russian academy of sciences, moscow, russia; centre for medical studies, university of oslo, moscow, russia; ivanovsky institute of virology, russian academy of medical sciences, moscow, russia novel acyclic purine phosphonate derivatives bearing a double bond conjugated with the nucleic base, namely, (z)-and (e)- -[ -(phosphonomethoxy)prop- -en- -yl]purines, were synthesized, and their efficacies against hiv- and hsv- were evaluated in cell cultures. the activity of (z)isomer was higher against hiv than that of the reference -[ -(phosphonomethoxy)ethyl]adenine (adefovir) and comparable in respect to the activity of adefovir against hsv. the (e)-isomer showed low antiviral activity against both viruses. the compounds were less toxic towards cell cultures if compared to adefovir. the diphosphates (z)-and (e)- -[ -(phosphonomethoxy)prop- -en- -yl]purines were evaluated as substrates towards hiv- reverse transcriptase and hsv dna polymerase. (z)-isomer was shown to be a more efficient substrate for both enzymes than the (e)-isomer. human dna polymerase alpha could incorporate neither of the diphosphates into the -end of the growing dna chain. available to this virus. jev genome is an approximately -kb single-stranded positive-sense rna that has a cap structure at its terminus but lacks a poly(a) tail at its -terminus. the coding region of the genome is flanked by -and -untranslated region (utr). the -utrs on both plus-and minus-strand jev genome contain important cis-acting elements required for the replication of the viral rna genome. peptide nucleic acid (pna) is a synthetic oligonucleotide, in which the phosphodiester backbone of dna/rna is replaced with a polyamine-( -aminoethyl) glycine skeleton. pna offers a potentially powerful approach for recognition of rna and silencing of gene expression. in this study, we investigated the antiviral effect of the pnas targeted to the -utr region of jev genome. to evaluate the pnamediated inhibitory effect on rna synthesis in vitro, the rnadependent rna polymerase (rdrp) of jev, ns protein, which plays a major role in replication of the viral genomic rna, was expressed in escherichia coli and purified to near homogeneity by sequential column chromatographies. the recombinant jev ns protein exhibited a primer-dependent rdrp activity in vitro on a homopolymeric template, poly(a). in addition, it was able to accept both plus-and minus-strand -utrs as templates for rna synthesis in the absence of an exogenous primer. it could utilize the -end -nt of jev genome as a minimal template. in vitro rdrp assays using this functional recombinant jev rdrp in the presence of the pnas targeted to the jev -utr -nt showed a dose-dependent rna synthesis inhibition. delivery of the inhibitory pnas to the jev-infected cells suppressed jev replication, as determined by western-blot analyses and plaque assays. our results showed a sequence specific inhibition of jev replication by antisense pnas, suggesting the possible application of pna as a novel anti-jev agent. julia serkedjieva , reneta toshkova , milena nikolova , reneta tsvetkova , stefka antonova , ivana roeva , munnever sokmen , bektas tepe , medine gulluce , fikrettin sahin , atalay sokmen institute of microbiology; institute of experimental pathology and parasitology; institute of botany, bulgarian academy of sciences; faculty of biology, department of microbiology, sofia university, sofia, bulgaria; faculty of art and science, department of biology, cumhuriyet university, sivas, turkey; faculty of art and science, department of biology, atatürk university, erzurum, turkey natural products can be an important source of new pharmaceuticals. research on antivirals of natural origin is mainly focused on plants, since, among other reasons, they can be selected on the basis of their ethnobotanical use. plant extracts and natural plant products exhibit also a variety of biological activities with pharmacophoric utility. the population of the balkan peninsula, like people from all continents, has long applied poultices and imbibed infusions of hundreds of indigenous plants. the present report summarizes the antiviral screening study of plant products, obtained from bulgarian and turkish medicinal plants. they were tested for inhibitory effect on the reproduction of selected influenza virus (flu) strains in mdck cells and herpes simplex virus (hsv) strains in mdbk cells. the reduction of virus-induced cpe and infectious virus yields were used as measures of viral inhibition. fifteen samples ( . %) inhibited flu reproduction, and twelve samples ( . %) were active against hsv. the anti-flu activity was confirmed in vivo for all tested samples. the most effective products were tested further for their antiproteolytic, antioxidant and immunogenic capacities and for potential antibacterial and antifungal effects. the following correlations among the variety of biological and pharmacological activities of the plant products were observed: the anti-flu effect was associated with anti-hsv effect and vice-versa in . %; the antiviral effect was connected with antioxidant activity in %; the anti-flu effect was associated with immunogenic properties in %; there was found no correlation between the antiviral effect and the antiproteolytic capacity, the anti-viral properties and bacterial or fungal inhibition, the anti-viral activity and the polyphenol contents. our previous investigations have revealed antiviral activity of some proteolysis inhibitors such as e-aminocaproic acid (e-aca) and para-aminomethylbenzoic acid (pamba) in vitro, in vivo and in clinic. construction of qsar computer-assisted hierarchical system for the effective anti-herpetic (anti-hsv) and anti-influenza (anti-flu) agents' selection as well as the elaboration of new methods of their synthesis are permanently the object of keen interest of our team. the objective of this study was to investigate the efficacy , -di-substituted pyridines and their analogs combined with the fragments of proteolysis inhibitors in the framework of the qsar approach. molecules of new compounds consisted of "nucleus" (py or ar) and two symmetrical fragments: e-aca-carbonyl or pambacarbonyl taken from the inhibitors' molecules. anti-flu activity in dose - m was studied in vitro on the model of a/hong kong/ / (h n ) reproduction in tissue cultures of chorioallantoic membranes of days old chick embryos. anti-hsv activity in doses - m was studied on models of reproduction of hsv- on cell culture hep- . compounds with py-nucleus, contained pamba-carbonyl or e-aca-carbonyl fragments, demonstrated sufficient anti-hsv activity ( . and % of reduction of intra-nucleus virus-specific inclusions on infected cells account accordingly). , -dihydrazine-carbonyl- , -dimethylpyridine showed high anti-hsv ( %) activity. the efficacy of the designed antiherpetic compounds obtained with the combined efforts of qsar computer-assisted design, properties prediction, synthesis, and biological testing as well as the correction introduced after the iteration circle passsage have proven to be the efficient modern way of drug design. acknowledgement: this research was supported in part by stcu grant # and all the authors are indebted to stcu foundation courtesy. lubomira nikolaeva-glomb , angelina trifonova , stephan filipov , angel s. galabov * institute of microbiology, bulgarian academy of sciences, sofia, bulgaria; institute of organic chemistry, bulgarian academy of sciences, sofia, bulgaria a series of aporphinoid alkaloids isolated from glaucinum flavum l. or obtained synthetically, were tested in vitro for antiviral activity against viruses belonging to picorna-, orthomyxo-, paramyxo-and herpesviruses. one of them, oxoglaucine, manifested a well-pronounced inhibitory effect on poliovirus replication in fl cells measured by the semi-quantitative agardiffusion plaque-inhibition test. in virucidal activity testing the compound did not show direct virucidal effect on the extracellular virus. oxoglaucine's % inhibitory concentration for poliovirus (mahoney) was found to be . g/ml in the cpeinhibition test and . g/ml in the classical plaque-inhibition test. similar values were obtained for the vaccinal poliovirus type strain, lsc- ab. the antiviral effect of oxoglaucine on the replication of viruses belonging to another enterovirus species was tested, i.e. coxsackie and echoviruses (hev-b). cva- , the six coxsackie b viruses and echoviruses were tested for their sensitivity against the antiviral effect of oxoglaucine by the endpoint dilution method in the multi-cycle cpe inhibition set-up in fl cells. oxoglaucine revealed a marked inhibitory effect on all tested enteroviruses. the concentrations that reduced virus titer by lg ranged from . to . g/ml. selectivity index was greater than and even greater than for some of the viruses tested. time-of-addition study by the one-step virus growth cycle set-up showed strong virus inhibition during the early periods of virus replication. milka mileva , angel s. galabov department of medical physics and biophysics, medical university, sofia, bulgaria; institute of microbiology, bulgarian academy of sciences, sofia, bulgaria in this study an investigation and comparison of the effects of plant flavonoid polyphenols quercetin and its sugar-containing homologue (rutinoside) rutin on the "oxidative stress" in liver, isolated from influenza virus a/aichi/ / (h n ) ( . of ld ) inoculated mice, is carried out. it was found that experimental influenza virus infection is accompanied with graduated oxidative disturbances in the liver of mice, despite the absence of virus and inflammation in this tissue. it was found that experimental influenza virus infection is accompanied with a significant increase of lipid peroxidation products, a decrease of natural antioxidants (vitamin e, glutathione) and cyp, an inhibition of cytochrome c-reductase and liver monooxygenases (analgin-ndemethylase and amidopyrine-n-demethylase) as compared to control (non-infected) animals. the preliminary ( days) supplementation of mice with rutin, quercetin or its combination, and their subsequent virus inoculation influence significantly all analyzed parameters as compared to controls. the protective effect of rutin against influenza virus-induced lipid peroxidation and activities of cyp and liver monooxygenases in liver was better expressed than the effect of quercetin may be due to containing of rutinoside part or difference of its metabolism. hyun-jeong lee , ji-sun kwon , chi-ung moon , jong-hwan kwak , youn-jeong lee , chang-seon song avian disease laboratory, college of veterinary medicine, konkuk university, seoul, korea; hanyang university, seoul, korea; sungkyunkwan university, seoul, korea; national veterinary research and quarantine services, seoul, korea one of the traditional korean medical herb extract named s was investigated to determine the anti-influenza virus activity in vitro and in vivo. the s showed potent antiviral activities against a/pr/ / (h n ) influenza virus with the % effective concentration (ec ) values of . g/ml and the % cytotoxic concentration (cc ) values of . g/ml in mdck cells. treatment with the s appeared capable of significantly ameliorating the influenza virus infection in mice by oral gavage treatment. the s treated mice showed significantly higher survival rate and lower pathogenic index as well as lung virus titers than untreated control mice. further, the s was extracted with chcl , etoac and n-buoh for isolation of active compounds. the anti-influenza effects of these active compounds will be discussed. the antiviral effect of aqeous and ethanol extracts of ocimum gratissimum (og), terminalia catappa (tc), gynostemma pentaphyllum (gp), newbouldia laevis (nl), aspilia africana (aa) and phyllantus amarus (pa) leaves was examined by cultivation of virus and extracts in embryonated chicken eggs. extracts were inoculated immediately after virus (zero time) or h after virus inoculation. virus replication was compared with those of controls by haemagglutination assay. at zero time, aqeous extracts of og, tc, pa, and gp inhibited virus growth by , , , and %, respectively whereas those of nl and aa did not. ethanol extracts of og, tc, pa and gp at same time inhibited by , , , and %, respectively whereas nl and aa did not. at two h after virus inoculation aqeous extracts of og, tc, pa and gp inhibited virus growth by , , , and %, respectively whereas nl and aa had no effect. ethanol extracts of tc, pa and gp inhibited the virus by , , and %, respectively whereas those of og, nl, and aa did not. the herbs were studied because they were being used by some herbalists in the treatment of human infectious diseases. the th international conference on antiviral research will be held in the palm springs, california area. the conference will begin on sunday, april , and will end on thursday afternoon, may , . all scientific sessions will be held at the westin mission hills resort, rancho mirage, ca. the purpose of the international conference on antiviral research is to provide an interdisciplinary forum at which investigators involved in basic, applied, and clinical research worldwide can meet to review recent developments in all areas of antiviral research. specific topics to be covered in the program include synthesis and chemistry, biochemistry and mechanism of action, molecular biology and drug targeting, in vitro evaluation, animal models, pharmacokinetics, toxicology, and clinical trials. within these areas of interest, there will be invited overview speakers, oral presentations, and poster presentations. the famous el paseo shopping district of palm desert and downtown palm springs offer not only a large variety of galleries, boutiques and shops too numerous to mention, but there are restaurants for virtually every palate. whether your tastes run to burgers, sushi, pizza, escargot, steak, mexican or continental you will find it here with a california flourish in every price range. we hope you will take advantage of this opportunity to combine an important learning experience with a magnificent travel experience and join us in palm springs, california for the th international conference on antiviral research. isar conference committee future conferences acknowledgement: the study was supported by rfbs - - and russian ministry of sciences (lot ). key: cord- -w g v g authors: nan title: isar news date: - - journal: antiviral res doi: . /s - ( ) - sha: doc_id: cord_uid: w g v g nan it is with great pleasure that i salute all isar members and friends in this new issue of the isar news. our th icar in atlanta was a success in many ways. we received excellent comments and congratulations for the excellence and quality of the invited speakers, and many oral and poster presentations. icar retains its flavor and personality, providing an interdisciplinary forum at which investigators involved in basic, translational, and clinical research worldwide meet to review recent developments in all areas of antiviral research, drug and vaccine development. as mentioned below, a new format of the icar report has been published in antiviral research with highlights and reviews of most oral presentations. additionally, satellite activities such as the women in science roundtable, the career development panel and the new member and first time attendee luncheon (the happy hour) provided an opportunity to discuss other issues of relevance. i take the opportunity to thank again the co-chairs of the program committee, justin julander and mark prichard, and all committee chairs and members for their unselfish commitment to organize the conference. the th icar meant a challenge to the society. with so many different competing conferences and meetings to attend and a long economic crisis of which scientific research did not escape, isar has gone through great financial efforts to continue supporting the participation of students, postdocs and young investigators. we are happy to learn that the th icar breaks a trend. the meeting becomes financially sound once again. we are indebted to all corporate sponsors, friends of the society and isar members for their contribution. we are encouraged by their financial support to continue organizing the best antiviral meeting possible. excellence in science and support to young investigators will continue driving our efforts. we are beginning to organize the st icar that will be held in porto, portugal. the conference will begin on monday, june and will conclude on porto has been selected for its easy access by flight from most european cities and major u.s. hubs, but also because we were dazzled with its warmth and beauty. porto is a city full of tradition and culture but modern and exiting. after many years organizing the meeting in a hotel venue, the st icar will be held at the alfândega congress centre (http://www.ccalfandegaporto.com/en). we hope to free icar participants from the constraints of staying at a venue hotel, allowing everyone to choose the accommodation that suits their needs. being a popular tourist location, porto has many types of accommodations to offer, from luxury hotels to small but comfortable hostels. isar will block a limited number of rooms at different hotels so i encourage everyone to make timely reservations for their stay in porto. in , we will continue supporting the participation of students, postdocs and young investigators. our travel support will be solely based on merit and excellence of the abstracts submitted for presentation. i encourage those wishing to apply to submit their best science. quality will be the prevailing criteria to receive financial support. keep an eye on further instructions on how to submit your abstracts at the isar-icar website http://www.isar-icar.com. i kindly remind all our members that the call for nominations for the isar awards is now open and encourage you to nominate a deserving colleague. the isar chu family foundation scholarship for women scientists is also now open for applications. awards will be given to advance the careers of women with potential for significant contribution in the field of antiviral research. information on how to submit nominations is posted in our website or by requesting information through the society's email address shown below. as we are in an early stage of organizing the st icar, i call on all those interested in participating to let us know your ideas, comments or suggestions. if you have a particular interest or a topic that should be highlighted, please feel free to contact us and let us know at info@isaricar.com. the isar held its annual business meeting at the atlanta hilton during the th icar on wednesday, may . brian gowen (treasurer) presented a brief summary of the society's finances. net assets at the end of the fiscal year was $ , . from several bank accounts, two investment accounts, and a cd, which matured in august of (table ). the net assets total is down from $ , . at the end of the fiscal year and is largely due to the $ , deficit from the th icar held in la jolla, ca (see isar news vol. . ). although there has been a downward trend in the society's holdings (figure ), projections from the review of revenue and expenses for the icar are favorable. we will not know the final numbers until late july or august when all revenue support has been received and meeting expenses paid. this information will be provided in an upcoming issue of isar news. also presented during the business meeting was the year-end financial statement reflecting isar's revenue sources and expenses for ( table ). the largest sources of revenue are the support we receive from our generous sponsors and the conference registration fees, with the major annual expense being the annual icar. graciela andrei (secretary) provided a report on the isar membership and on attendance at the th icar in atlanta ( figure ). twenty-one countries are represented in the society, with a total of members through may ( from usa). a total of attendees as of may , from different countries were registered for the th icar. this year, the society received applications for a travel grant award. because of the restricted budget available, some of the grant applications could not be e funded. based on the scientific review of the submitted abstracts, a ranking was established based on the scores provided by four independent reviewers. the society awarded travel merit grants ( from north america, from europe and from asia and australia who received an award of $ , $ , and $ , respectively) to help these members defray the costs of attending the conference. the society also awarded travel merit assistance awards to participants coming from low/middle-income countries to support their attendance to the meeting. for recipients of this award, besides a $ stipend, the registration fee was waived. as shown in figure , the society invested a total of $ , in travel awards this year, which highlights the considerable funds made available by the society during the past years to increase the attendance of young investigators at the meetings. this year's icar poster competition in atlanta was, as always, fierce and highly competitive! the international team of judges included graciela andrei, john bigger, andrea brancale, jinhong chang, cyril dousson, joana rocha-pereira, brian gentry, zlatko janeba, brent korba, chris meier, jennifer moffat, roger ptak, luis schang, enzo tramontano and the chair of the poster award committee, kathie seley-radtke. fifty-four posters registered to be judged in the three categories: undergraduate/graduate student, postdoctoral researcher and young investigator. each category had many excellent posters in the running for both poster prizes and the chance to give a shotgun talk at wednesday's opening session, which was co-chaired by kathie seley-radtke and her graduate student therese ku. the the poster committee would like to thank all the presenters for the excellent research presented and we look forward to judging next year's entries in porto! (rhonda cardin) the th annual women in science roundtable, the opening event of the th icar in atlanta, georgia, featured a panel discussion where participants engaged in interactive discussions with four leading women scientists in their respective fields that included priscilla yang (harvard medical school), inger damon (cdc), julie dyall (niaid, nih and tunnell consulting company) who were introduced by rhonda cardin (louisiana state university). each of the panel members shared personal and professional experiences as well as opportunities and challenges that they have encountered during their scientific careers. they brought forth many and varied viewpoints that provided food for thought for the participants provoking insightful questions leading to an enthusiastic discussion. we appreciate the panel members and participants for attending the event and look forward to seeing everyone at the th annual women in science roundtable in porto. in atlanta, the wis committee was pleased to present priscilla yang (harvard university) as the first wis sponsored speaker in recognition of her major contributions to antiviral research and leadership in mentoring young women. yang presented her research on "small molecule inhibitors of viral entry: pharmacological mimicry of the humoral immune response to viral infection" in the antiviral immunity symposium. we hope that this will be the first of many invited speakers to highlight the research accomplishments of women in science. thanks to a generous donation from the chu family, we are pleased to announce the chu family foundation (tcff) awards for women scientists. the tcff awards support the professional development of women with potential to make significant contributions to the field of antiviral research by providing funds to attend a conference, visit another laboratory, take a course, or acquire specialized training. these awards are given annually at the icar meeting. each award dengue fever disease is caused by the four serotypes of dengue virus (denv - ) and is mostly transmitted by the urban dwelling aedes aegypti mosquito. close to % of the global human population live in dengue endemic areas and are at risk of contracting the disease. poorly controlled urban development and vector spreading, which is thought to be associated with global warming, are possible contributors for the expansion of dengue (gubler, ) . it was estimated that around million symptomatic dengue cases occurred in out of a total of nearly million infections (bhatt et al., ) . disease manifestations range from asymptomatic to a mild undifferentiated dengue fever (df) and in up to % of symptomatic cases progresses to serious life-threatening complications as a result of vascular leakage, hemorrhage and organ dysfunction (simmons et al., , low et al . secondary dengue infections are often, but not consistently associated with severe dengue disease in part because of antibody dependent enhancement (ade) phenomenon (halstead, ) . vaccination, as a measure to counter the spread of dengue, has been on the agenda of national health authorities for many decades and still remains a high priority for the world health organization (who) (russell, ; recker et al., ) . at the who conference on dengue held in singapore in it was predicted that it would take years to develop a tetravalent dengue vaccine (russell, ) . however, the unexpected challenges of developing such a vaccine took nearly four decades. the first dengue vaccine, dengvaxia®, developed by sanofi pasteur has been licensed in several countries since december . the attenuated live recombinant vaccine was generated by splicing in the prm and e genes from denv - into the yellow fever virus (yfv) vaccine strain genome, replacing the yfv prm and e genes to yield the chimeric yellow fever dtetravalent dengue vaccine (cyd-tdv). dengvaxia is administered as doses on a / / month schedule and clinical studies found the overall efficacy to be . %, with serotype-specific efficacy for denv - to be . %, %, . % and . %, respectively. the vaccine is recommended for use in the individuals - years of age living in dengue endemic regions where the disease burden is high (who ). this achievement by sanofi pasteur should be commended as it marks an important milestone for dengue control through exemplary partnership with a very large number of public and private institutions around the world. valuable lessons have been learned and other dengue vaccine candidates are currently being explored to improve efficacy, extend the age coverage and also simplify the vaccination schedule in order to achieve wider vaccine acceptance and uptake. apart from vaccination, the vector control strategy using wolbachia bacteria to reduce the capacity of aedes aegypti mosquito to transmit dengue is being actively explored in many countries largely through the support of the bill & melinda gates foundation http://www.eliminatedengue.com dengue antiviral research and development as a potential intervention approach received a boost in when novartis pharma formed a public-private partnership with singapore's economic development board to establish the novartis institute for tropical diseases (nitd). the expectation was that a safe orally available drug for use in those > years old and with e activity against all four denv serotypes and capable of reducing dengue symptoms and the incidence of severe dengue could be achieved in - year period (keller et al., ) . although such a drug has not eventuated so far, the efforts of nitd have led to many promising leads targeting the ns and ns proteins, which contain the enzymatic activities necessary for viral rna replication. these leads have been reviewed by lim and colleagues (lim et al ) . in the meantime, major strides in the understanding of the structure and function of viral targets as well as dengue pathogenesis in general have been made through state-of-the-art research using d structural studies, cryoelectron microscopy, reverse genetics, rnai screens, crispr screens, whole genome deep sequencing and animal model studies. the contributions of data arising from these new approaches hold promise for the development of directly-acting antivirals (daa) or host factors that can target viral processes such as i) viral entry/fusion ii) translation/polyprotein processing, iii) rna replication and iv) packaging/virus maturation. however, in order to make an impact as one of the viable intervention approaches for dengue control, alongside with vaccines and vector control measures, it is important that an efficacious dengue antiviral drug reaches the clinic by the first half of the next decade. this is a significant challenge considering the bottlenecks in drug development during the late preclinical to clinical development phase. one way to mitigate this is through the formation of research consortia such as the partnership involving wellcome trust/ku leuven/janssen for dengue drug development http://www.janssen.com/partnerships/dengue to develop a pipeline of candidate drugs, which can be rapidly evaluated and developed as potent dengue antivirals. similar partnerships through the national institutes of health (usa), european union and other national agencies in other parts of the world can also help to link the large body of work from academic laboratories and small biotechnology companies to ensure potential candidate drugs can successfully enter the clinics for testing. if the task of managing the intellectual property expectations of partners within consortia can be streamlined, then work from these types of partnerships should result in more fruitful outcomes for diseases such as dengue or other emerging viral diseases. new opportunities for development of pan-flaviviral drugs have arisen especially since the closely related zika virus (zikv) outbreak (fauci and morens ). infection with zikv, which can result in congenital deformities in newborn babies when the mothers are infected during pregnancy as well as guillain-barré syndrome in otherwise healthy adults, has brought intense international focus on this emerging threat with calls for urgent development of antiviral drugs. flaviviruses carry a single-stranded positive sense rna genome of ~ nucleotides that encodes a ~ amino acid residues polyprotein precursor which is cleaved into three structural proteins (capsid, premembrane/membrane and envelope) and seven nonstructural (ns) proteins (ns , ns a, ns b, ns , ns a, ns b and ns ). the crystal structures of the enzyme targets, ns and ns from zikv, reveal extensive structural similarities with denv and other flaviviruses, which can form excellent starting point for the development of novel daas. in the case of zikv ns protease, it was shown that a binary expression construct where the ns b is not constrained by artificial linkers or modified native linker has resulted in high resolution crystal structure where the ns b wraps around the ns molecule in the closed conformation leaving the substrate binding site empty for binding small molecules. these crystals are amenable to fragment based screening and structuredirected antiviral development (zhang et al., ) . in the case of ns , the structures also show high similarity and the nucleoside inhibitor nitd is widely used as a control for drug screening efforts with different compound libraries (barrows et al., ; xu et al., ) . opportunities for non-nucleoside inhibitors also exist since the structural similarities reveal conserved pockets near regions that are critical for de-novo initiation for viral rna replication that can be targeted. important lessons have been learned about conducting dengue antiviral clinical trials using repurposed drugs, which has been reviewed recently (low et al., ) . this strategy is being more widely and systematically applied to discover novel individual or combination compounds as potential pan-flaviviral drugs. given the acute nature of dengue and the narrow window of opportunity for antiviral treatment, it is likely that the number of patients who can be recruited to evaluate the efficacy of these drugs may limit early phase clinical trials. additionally, the high cost of large trials with long study duration may limit the number of compounds entering phase ii/iii trials with detrimental effect on the whole process of drug discovery and development. in recent years, the limitations of large randomized clinical trials have been widely acknowledged as severely impairing medical advances. adaptive trial in part to further address clinical endpoints that require large sample size as used in dengue trials, an alternative robust biomarker, fluorodeoxyglucose (fdg), was recently examined in preclinical animal models. the goal was that drug efficacy could be directly correlated with reduction in fdg signals that were reflective of virus-induced inflammation by using positron emission tomography (pet) imaging (chacko et al ) . if the observations from the preclinical study translates to human cases, then it is conceivable that smaller phase ii trials can be rapidly conducted to triage a pipeline of drugs from various consortia. it would be valuable for other robust biomarker to be evaluated with a similar goal. in summary, the successful development of antivirals against the hepatitis c virus serve as an example that it is possible to develop potent dengue/pan-flaviviral antivirals. the bar may be viewed as being a little higher in the case of dengue because the available funding is probably a fraction of that available for hcv antiviral development. however, the task is not insurmountable if partnerships and consortia that led to the successful development of dengvaxia can be recapitulated for the dengue antiviral development to deliver a drug to be deployed for clinical use by . rega institute for medical research, ku leuven -university of leuven, b- leuven, belgium lieve.naesens@kuleuven.be this year, antiviral researchers celebrate the th anniversary of g. elion's first publication on acyclovir [ ] , as well as the th anniversary of zidovudine as the first drug to become approved for hiv therapy. both events represent landmarks in the clinical management of virus infections. the class of nucleos(t)ide inhibitors of hiv rt ripened to become the cornerstone in hiv treatment; some of these drugs proved to be equally valuable for hepatitis b. since the approval of sofosbuvir, hepatitis c is another viral disease for which nucleos(t)ide inhibitors appear hard to compete with. sofosbuvir is the first nucleotide drug acting as a chain terminator on a viral rna-dependent rna polymerase. newer analogues, such as the rsv inhibitor al- , and gs- that is active against ebola plus some other rna viruses, are in the clinical pipeline to, hopefully, address some important and unmet medical needs. when we look at which molecules have gained successful clinical approval, the structural variations on the nucleos(t)ide theme are still quite limited. this contrasts with the large chemical variety among experimental nucleos(t)ides that have been synthesized and proven to have antiviral activity in preclinical or early clinical studies. a few examples of rather 'exotic' sugar replacements are dioxolane [ ] ; six-membered rings [ ] ; or conformational locked rings [ ] . likewise, quite drastic variations in the base part are accepted, as exemplified by the two carboxamide compounds ribavirin and favipiravir, or the herpesvirus inhibitors brivudin and valnivudine (fv- ). it is evident that viral polymerases are highly promiscuous and able of recognizing nucleosidetriphosphate analogues with a very unusual and unnatural structure. for hiv rt and viral rnadependent rna polymerases that lack proofreading activity, this is not surprising. however, even herpesvirus polymerases, which do possess exonuclease activity, seem to be easily fooled. in fact, the defined antiviral spectrum that is often assigned to antiviral nucleo(s)tides is too simplistic. for instance, tenofovir inhibits hsv; cidofovir-diphosphate inhibits hiv rt; some dideoxynucleoside inhibitors of hiv are also active against adenovirus; penciclovir inhibits hepatitis b virus; and gs- is a recent example of a broad anti-rna virus agent. why is then the structural diversity of antiviral nucleos(t)ide drugs still quite limited? an important restriction lies within the host cell, which possesses a variety of purine and pyrimidine-converting kinases acting in tightly controlled pathways that are connected to nuclear or mitochondrial dna or rna synthesis. except for nucleoside diphosphatase (ndp) kinase, these kinases generally display narrow substrate specificity. for antivirals, this kinase dependency represents a daunting barrier since the unnatural nucleos(t)ide should not merely bind to the kinase, but, more importantly, act as an efficient substrate to be smoothly phosphorylated. to bypass the first and often rate-limiting first phosphorylation step, monophosphate formats like tenofovir and sofosbuvir have been designed. only recently, successful di-and triphosphate prodrug concepts have been developed by meier and colleagues [ , ] , creating the possibility to design antiviral nucleotides with less obvious chemical structures in the base or ribose part. given the lack of proofreading activity in the vast majority of rna viruses (coronaviruses being the only exception), such di-and triphosphate prodrugs could be one way to achieve broad anti-rna virus agents, which are desperately needed to tackle some emerging and potentially pandemic viruses. another issue worthy of thought is whether obligate/non-obligate chain termination, relying on incorporation of the antiviral nucleotide, is really an absolute requirement. whereas obligate chain terminators lack a -hydroxyl function, the term nonobligate chain termination is used for nucleotides with modifications in the sugar part (e.g. at the , or position) which hinder binding of the next nucleotide or cause termination during next-round synthesis of the complementary strand [ ] . chain termination was also reported for bcx , an immucillin-adenosine analogue that is active against ebola virus. although most antiviral nucleos(t)ides indeed possess unnatural (deoxy)ribose parts leading to nonobligate chain termination, exceptions with a natural ribose do exist, such as brivudin and favipiravir. for the latter molecule, the mechanism behind its inhibitory effect on viral rna synthesis remains uncertain. the central dogma is that chain termination ensures that viral dna/rna synthesis is fully shut off, leading to a powerful antiviral effect. if recognized by a cellular polymerase, such a nucleotide might disrupt cellular dna/rna synthesis but the chain termination event would exclude mutagenic effects upon the host cell. a valid alternative could be to design nucleotide mimetics that are not covalently incorporated into viral dna or rna but, instead, act as strong non-covalent blockers of the polymerase active site. one way is to introduce a triphosphate-like moiety (such as an αcarboxyphosphonate) that mimics the interactions of the α, β and phosphates [ ] . a related approach is to design mimetics of some transition state of the polymerase enzyme. this drug concept has yielded superior enzyme inhibitors in the field of hiv protease, influenza neuraminidase or some n-ribosyltransferase enzymes [ ] . given that viruses rely on very fast replication of their genomes to generate millions to billions of particles per day, a nucleotide showing tight binding to the viral polymerase could produce strong antiviral effects. even if such an active site blocking nucleotide would be recognized by a cellular polymerase, it is fair to assume that this would be free of mutagenicity. the new generation of antiviral nucleos(t)ides should, of course, be selected to have a high barrier to drug-resistance. in hiv rt, one common resistance mechanism is related to higher fidelity mutants that better discriminate between the natural and unnatural dntps, which is linked to the mutant having a more rigid active site structure [ ] . this suggests that unnatural analogues with higher structural flexibility can still adopt to the mutated site and, hence, remain active against the mutant virus. the downside is that ligand flexibility may reduce antiviral potency when it affects the binding parameters towards the protein target. so perhaps it is time to consider some less explored scaffolds in the search for new nucleos(t)ide antivirals? regardless the mechanism, one important issue is related to discrimination between viral and cellular polymerases. as mentioned above, the lack of proofreading activity in many viruses (i.e. in virtually all rna viruses), combined with their reliance on fast genome copying kinetics, are intrinsic factors that contribute to selectivity. in addition, for acute virus infections requiring only short-term treatment, a weak inhibitory effect on some cellular polymerase is expected to be harmless. another topic in the design of antiviral nucleos(t)ides is that of prodrug moieties to modulate drug lipophilicity, tissue distribution or organ tropism. brincidofovir is a lipid conjugate showing increased oral bioavailability and cns penetration than its cidofovir parent. a main advantage of the phosphoramidate drug sofosbuvir in the therapy of hepatitis c is its activation by liver-expressed enzymes. to expand the prodrug toolbox, some creativity is needed. perhaps we can exploit enzyme pathways that are upregulated in, for instance, phagocytes or granulocytes (which are attracted towards virus-infected tissues), lung alveolar cells or the blood-brain barrier. or perhaps the (immuno)pathology of virus infections may boost some specific enzyme reactions in the infected organs? the availability of genome-wide proteomic and metabolomic profiles can provide clues on which enzymes could be considered. one drawback is that these enzymes may be confined to primary cells or more complex tissues, which are harder to isolate, manipulate or infect with viruses for in vitro studies. to conclude, antiviral nucleos(t)ides are alive and kicking. these versatile and ubiquitous dna/rna building blocks are a relentless source of imagination for medicinal chemists, biochemists and virologists. new structural analogues, protides and prodrugs need to be further designed to consolidate the leading position of nucleos(t)ide analogues in the field of antiviral therapy. hard times came to me in when my grants ran out and other funding sources were drying up. it was then that i took a job as a contractor at usamriid, where i worked for nearly three years. then i returned to usu and have been there ever since, working currently as a research professor, an appointment that is still a soft money position. at the onset of my career at usu, i was uncomfortable being on soft money, since the money well can go dry as it did for me in . after usamriid, i had a much greater determination to make the soft money position work out. at usamriid, i conducted antiviral studies on orthopox viruses. i was able to obtain funding for poxvirus work at usu, which gave me a jump-start back in academia. moreover, i still interact with one of my colleagues from the usamriid era, mike bray, who is the current editorin-chief for antiviral research. one interesting thing occurred a few years after i left usamriid. i was interviewed by the u.s. postal service that was working with the fbi to investigate people who may have been involved in the anthrax terror attacks of . i was investigated because, while at usamriid, i was a colleague with steve hatfill, who was their main terrorist suspect at the time. later, steve was exonerated in the case. my career has taken many turns over the years, ebbing and flowing with the medical needs of the time. the most significant work i accomplished in my career was in the early 's when i participated in the preclinical development of ganciclovir. there were five companies vying for the rights to the compound, and syntex emerged victorious. it was an exciting time because ganciclovir was a very hot topic and there were many people interested in it. another significant part of my career has been the development of new animal models of viral infections. this has been diverse, and included developing different types of herpesvirus infections in immunocompromised mice, new orthopoxvirus models, and new models with arenaviruses, bunyaviruses, and orthomyxoviruses. in the latter categories, it has been exciting to work with a broad array of different rna viruses. isar news vol. ϭϰϲ e my passion over the years has been performing animal studies. animal experimentation is where the rubber meets the road in terms of an antiviral compound's potential to become a drug. i have read too many articles in the scientific literature where in vitro activity was touted, but with no evidence of efficacy in animals reported. later, most of the compounds fell by the wayside due to lack of in vivo efficacy. the approach we have taken at the institute for antiviral research is to publish the results of cell culture and animal testing data in the same article. animal work will forever play a key role in drug development. continuing research into the discovery of new animal models is essential. along these lines, one recent project from our group is the development of an enterovirus d (ev-d ) mouse model that we reported at the icar meeting. my colleague bart tarbet is the lead researcher on that project. since ev-d is related to rhinoviruses, the mouse model may serve as a surrogate for anti-rhinovirus compound evaluation if the compounds also inhibit ev-d . using this model, we demonstrated that guanidine is active at inhibiting virus replication but rupintrivir is not. rupintrivir is highly potent in vitro against picornaviruses, but failed in treating rhinovirus infections in the clinic. had this new animal model of ev-d infection existed in the early stages of rupintrivir development, the company would have been able to find out the ineffectiveness of the compound long before they invested in expensive clinical trials. mode-of-action studies are also highly interesting to me. i was able to do a lot of work in this area under the ganciclovir project, but not so much currently. what is satisfying is to take a compound for which nothing is known and actually figure out how the compound works. at the icar meeting each year, we often hear very interesting mode of action presentations. in my career, i have rubbed shoulders with some of the great men in science. my mentor in graduate school was robert sidwell, whom eight years later i worked with at utah state university and then until he retired in . we attended many icar meetings together year after year. his passion on those trips was to go to nice restaurants in the evenings and insisted that i go along with him. i would take him out into the woods and swamps looking for reptiles, which is what we did at the new orleans icar in . i worked closely with john martin (currently ceo of gilead sciences) when he was a chemist at syntex. he was a key player in the team that developed ganciclovir. many of the individuals that i collaborate with or do projects for are members of isar. the icar meeting brings diverse disciplines together and it is a chance to interact face-to-face with colleagues that i otherwise only visit through email or with occasionally on the telephone. i am involved with many viruses and research interests, and the icar is the most important meeting for me to get it all in just one venue. i have also enjoyed serving on various isar committees. emerging and re-emerging viruses arlington, va, usa (october - , ) this meeting, organized by elsevier conferences, will aim to ask how basic host-cell biology can contribute to understanding emerging and re-emerging viruses to help with surveillance as well as vaccine and drug development. the meeting will bring infectiousdisease specialists together with researchers interested in the host response to these viruses from immunological, cell-biological, and pharmacological viewpoints. the conference will focus on a range of october ϤϬϭϩ isar news vol. ϭϰϲ e human pathogenic viruses, which are constantly emerging and re-emerging, as highlighted by the recent ebola outbreak in west africa and the ongoing zika virus outbreak in the americas. the meeting will be held in arlington, va, usa (october - , ) . the hotel is conveniently located just minutes from washington dc, a city of variety and contrast, with a central area beautifully designed with broad avenues lined with magnificent buildings and monuments set in spacious green parks. for more information, go to the website http://cellsymposia.com/emerging-viruses- . (august - , ) the virology- conference will be centered on innovations and therapeutic approaches in virology. this meeting aims to be a perfect platform for the researchers, scientists, professors, and students to exchange and discuss on their ideas in the field of virology. virology- anticipates around participants around the globe and the three-day conference will include plenary sessions, keynote speeches, poster and oral presentations. more information regarding this conference can be found on the website http://www.virologycongress.com. the th annual meeting of the escv will be held in stresa located at the lago maggiore, italy, september - , . the scientific program, comprising presentations, symposia, discussions, open sessions and workshops, will be centered on the state of the art as well as the most recent discoveries and innovations in clinical virology. the main topics of the meeting will include respiratory viruses, gastrointestinal viruses, virus-associated neurologic syndromes, virus-associated tumors, hepatitis viruses, hiv and other retroviruses, viruses in immunocompromised patients, viral infections in pregnancy, emerging viruses, immune response to viral infections, advancements in diagnosis and monitoring advancements in prevention and treatment. the famous town of stresa ( inhabitants, m above sea level) enjoys a splendid location on lake maggiore in the gulf of borromeo. stresa is situated in northern italy, in an ideal position between mountains and the shores of borromean gulf. its beautiful countryside, mild climate, luxury villas and magnificent art nouveau hotels made the town of stresa a perfect combination of relaxation and culture. from stresa it is easy to reach the three borromean islands that are steeped in artistic, historical and botanical appeal. details of the meeting can be found on the escv meeting's website www.escv .com. dengue/dengue haemorrhagic fever: history and current status the global distribution and burden of dengue current status of dengue therapeutics research and development assessing dengue vaccination impact: model challenges and future directions who position paper -july finding new medicines for flaviviral targets ten years of dengue drug discovery: progress and prospects zika virus in the americas--yet another arbovirus threat a screen of fda-approved drugs for inhibitors of zika virus infection identification of small-molecule inhibitors of zika virus infection and induced neural cell death via a drugrepurposing screen adaptive design for clinical trials f-fdg as an inflammation biomarker for imaging dengue virus infection and treatment response selectivity of action of an antiherpetic agent synthesis and antiviral evaluation of -amino- -carbamoylpurine dioxolane nucleoside derivatives and their phosphoramidates prodrugs synthesis and antiherpes virus activity of , -anhydrohexitol nucleosides the history of n-methanocarbathymidine: the investigation of a conformational concept leads to the discovery of a potent and selective nucleoside antiviral agent lipophilic prodrugs of nucleoside triphosphates as biochemical probes and potential antivirals nucleoside diphosphate prodrugs: nonsymmetric dippro-nucleotides inhibitors of the hepatitis c virus rna-dependent rna polymerase ns b alpha-carboxy nucleoside phosphonates as universal nucleoside triphosphate mimics transition states, analogues, and drug development the high cost of fidelity we thank ooi eng eong for helpful discussion and gratefully acknowledge the support of national medical research council of singapore for support for dengue translational research. (nmrc/ctgcod/ / (jgl) and nmrc/cbrg/ / (sgv). key: cord- - tqfoecc authors: li, huixin; wang, yulong; han, zongxi; wang, yu; liang, shulin; jiang, lu; hu, yonghao; kong, xiangang; liu, shengwang title: recombinant duck enteritis viruses expressing major structural proteins of the infectious bronchitis virus provide protection against infectious bronchitis in chickens date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: tqfoecc to design an alternative vaccine for control of infectious bronchitis in chickens, three recombinant duck enteritis viruses (rdevs) expressing the n, s, or s protein of infectious bronchitis virus (ibv) were constructed using conventional homologous recombination methods, and were designated as rdev-n, rdev-s, and rdev-s , respectively. chickens were divided into five vaccinated groups, which were each immunized with one of the rdevs, covalent vaccination with rdev-n & rdev-s, or covalent vaccination with rdev-n & rdev-s , and a control group. an antibody response against ibv was detectable and the ratio of cd (+)/cd (+) t-lymphocytes decreased at days post-vaccination in each vaccinated group, suggesting that humoral and cellular responses were elicited in each group as early as days post-immunization. after challenge with a homologous virulent ibv strain at days post-immunization, vaccinated groups showed significant differences in the percentage of birds with clinical signs, as compared to the control group (p < . ), as the two covalent-vaccination groups and the rdev-s group provided better protection than the rdev-n- or rdev-s -vaccinated group. there was less viral shedding in the rdev-n & rdev-s- ( / ) and rdev-n & rdev-s - ( / ) vaccinated groups than the other three vaccinated groups. based on the clinical signs, viral shedding, and mortality rates, rdev-n & rdev-s covalent vaccination conferred better protection than use of any of the single rdevs. infectious bronchitis (ib) is a highly contagious viral disease of the upper respiratory and urogenital tracts of chickens that is caused by the infectious bronchitis virus (ibv). the disease is prevalent in nearly all countries with an intensive poultry industry, causing respiratory and renal diseases in chickens of all ages. it also reduces the quality and quantity of eggs produced by mature hens, causing heavy economic losses to the poultry industry. in addition, high mortality often occurs in young chickens infected with nephropathogenic strains as a result of renal pathology (cavanagh and gelb, ) . the ibv genome consists of a linear, single-stranded, positivesense rna, which encodes four major structural proteins, which include the spike (s) glycoprotein, the membrane (m) glycoprotein, the nucleocapsid (n) phosphoprotein, and the envelope or small membrane (e) protein. the n phosphoprotein is conserved among different ibv serotypes and can induce high titers of cross-reactive antibodies and cell-mediated immunity that protects chickens from acute infection, thus it is used as a target protein in designing vaccines against ib (williams et al., ; collisson et al., ; seo et al., ) . the s glycoprotein is responsible for receptor binding and membrane fusion (hofmann et al., ) , and consists of the nterminal s and c-terminal s subunits (bosch et al., ) . most of the conformation-dependent, neutralizing antigenic, and serotypespecific determinants in ibv have been mapped to s , while other immunodominant regions are located in the n-terminal regions of s (koch et al., ; kusters et al., ; lenstra et al., ) . in addition, interactions between the s and s subunits might affect the conformation of the s subunit, thereby accounting for differences in serologic protection (callison et al., ) . the m glycoprotein of coronaviruses gives the virion envelope its shape. it has been reported that the s glycoprotein interacts with the transmembrane region of the m glycoprotein and the cytoplasmic tail of the ibv e protein is responsible for its interaction with the ibv m glycoprotein (cavanagh, ) . measures to control ib in poultry rely primarily on vaccination. multiple live attenuated ibv vaccines are most often required because of poor cross protection between vaccines produced from different ibv serotypes (liu et al., (liu et al., , . live attenuated ibv vaccines do not provide adequate protection throughout the lifetime of layers or breeders, whereas inactivated vaccines convey certain advantages, such as slow antigen release and long-lasting immunity throughout the laying period. unfortunately, inactivated ibv vaccines are not effective when used alone, as birds require one or a series of vaccinations with live-attenuated ibv vaccines (live priming) prior to administration of an inactivated vaccine (cook et al., ) . conventional live ibv vaccines are attenuated by multiple serial passages in embryonated eggs (gelb and cloud, ; jackwood et al., ; bijlenga et al., ; huang and wang, ) , although this is a time-consuming process. genetically engineered vaccines present an alternative to inactivated and attenuated vaccines. in previous studies, a multivalent dna vaccine expressing s , n, and m conferred % protection , while a multivalent dna vaccine combined with an inactivated vaccine booster conferred complete protection (yan et al., ) . in addition, a recombinant newcastle disease virus expressing the s protein of ibv was shown to provide broad protection against ibv challenge (toro et al., ) . duck enteritis virus (dev) causes duck plague, an acute, contagious, and lethal disease that affects birds of all ages of the order anseriformes (davison et al., ) . dev is a member of the family herpesviridae with a genome approximately kb in size . because certain dev genes are not essential for viral replication in vitro (wang and osterrieder, ; liu et al., ) , dev has been used as a replicating vaccine vector in chickens to provide rapid protection against the h n influenza virus (liu et al., a,b) . in our current study, we used dev as a viral vector to construct three recombinant viruses expressing the n, s, and s proteins of ibv, and evaluated their protective efficacy in chickens against virulent ibv challenge. the nephropathogenic ibv strain ck/ch/ldl/ is an lx type (qx-like) strain that was first isolated in china in (sun et al., ) . the dev clone- was isolated from a commercial vaccine by plaque assay (li et al., ; liu et al., ) . primary chicken embryo fibroblasts (cefs) were used for dev propagation (li et al., ) . specific pathogen-free (spf) white leghorn chickens, chicken embryo eggs and duck embryo eggs were obtained from harbin veterinary research institute (hvri; harbin, china). the birds were maintained in isolators under negative pressure and provided with food and water ad libitum. all experiments were performed in strict accordance with the recommendations of the guide for the care and use of laboratory animals of the ministry of science and technology of the people's republic of china, and the study protocols were approved by the committee of the ethics of animal experiments of the hvri. dev genomic dna was extracted as previously described (prigge et al., ) . the left and right homologous arms of the transfer vector were amplified by polymerase chain reaction (pcr) using primers vl , vl , vr , and vr . the enhanced green fluorescent protein (egfp) cassette was amplified by primers r and r (table ) from the pegfp-n plasmid and cloned into the pmd tsimple plasmid to produce pt-egfp. the left and right arm pcr products were inserted through the clai and blni restriction sites and the mlui and avaiii restriction sites, respectively, of the pt-egfp plasmid to produce pus -egfp. complementary dna (cdna) of the n, s, and s genes of the virulent ibv strain ck/ch/ldl/ was synthesized from the viral genomic rna by reverse transcription (rt)-pcr (sun et al., ) . the pus -n, pus -s, and pus -s plasmids were produced by inserting the n, s, and s pcr products, respectively, between the xhoi and noti restriction sites flanking the egfp open reading frame (orf) in the pus -egfp plasmid. the strategy for the construction of the recombinant devs (rdevs) is depicted in fig. a . briefly, the genomic dna of dev and the pus -egfp transfer vector were cotransfected into cefs using turbofect transfection reagent (thermo fisher scientific, inc., waltham, ma, usa). rdev containing egfp (rdev-egfp) with deletion of the complete us gene was selected by plaque assay and used as the parental virus for constructing rdevs expressing the n, s, and s proteins of ibv. the selection of rdev-n, rdev-s, and rdev-s was conducted using fluorescence microscopy, and plaques without green fluorescence were purified by plaque assay. n f, n r, sf, sr, s f, and s r gene-specific primers (table ) were used to confirm the identity of rdev-n, rdev-s, and rdev-s by pcr. in addition, the primer pair df and dr, corresponding to the flanking sequence of the dev us gene (table ) , were used to differentiate wild-type (wt) dev from the rdevs. western blotting was performed to detect the expression of ibv proteins from the rdevs (han et al., ) . rabbit anti-gfp igg (sigmaealdrich corporation, st. louis, mo, usa), mouse anti-ibv n protein monoclonal antibody ( f ) (han et al., ) , and chicken anti-ibv serum were used as primary antibodies for detection of egfp, n, s, and s expressed from rdev-egfp, rdev-n, rdev-s, and rdev-s , respectively. in addition, an anti-chicken b-actin monoclonal antibody of mouse origin (sigmaealdrich corporation) and mouse anti-dev gl serum (prepared in our laboratory) were used to detect reference proteins and the efficacious replication of dev or rdevs in cefs. horseradish peroxidase-conjugated anti-rabbit igg, anti-mouse igg, or anti-chicken igg (sigmaealdrich corporation) were used as secondary antibodies. an indirect immunofluorescence assay was performed to detect protein expression in infected cefs. briefly, cefs were infected with rdevs at multiplicity of infection (moi) of . and then fixed with % paraformaldehyde at h postinfection. the antibodies described in section . were used as primary antibodies, while fluorescein isothiocyanate (fitc)-conjugated anti-mouse or anti-chicken igg (sigmaealdrich corporation) was used as the secondary antibody. expression of foreign proteins in recombinant viruses was observed by fluorescent microscopy. table primers used in this study. primer sequence template egfp, enhanced green fluorescence protein; ibv, infectious bronchitis virus. schematic of the insertion of the n, s, and s genes of ibv into the dev genome. rdev-egfp was used as the parental virus for construction of rdev-n, rdev-s, and rdev-s . (b) pcr identification by specific primers and differentiated primers. primer pairs nf and nr, sf and sr, and s f and s r were used to amplify specific genes from corresponding rdevs. the differentiated primer pair df and dr, corresponding to flanking regions of the us orf of dev, was used to analyze the purity and genetic stability of rdevs. there were differences in the fragment lengths amplified from the wt dev and each rdevs (dev clone- , bp; rdev-egfp, bp; rdev-n, bp; rdev-s, bp; and rdev-s , bp). (c) western blot analysis of the expression of the n, s, and s proteins of ibv from the rdevs. the expression of chicken b-actin and the gl protein of dev were used as internal references of cefs infected with the rdevs. (d) immunofluorescence detection of the n, s, and s proteins of ibv in rdev-infected cefs. for detection of the n, s, and s proteins, rdev-infected cefs were fixed using % paraformaldehyde at h postinfection. dapi was used to stain the nuclei and uninfected cefs were used as a control. to examine the growth kinetics of cefs infected with rdev-egfp, rdev-n, rdev-s, rdev-s , or dev clone- at an moi of . , the infected cefs and supernatants were harvested at , , , and h postinfection, and then titrated according to the method of reed and muench ( ) . to evaluate genetic stability, rdevs were passaged times in cefs. after passages , , and , the identity of each rdev was confirmed by pcr as described in section . . a total of four-week-old spf chickens were divided into six groups of birds each. five groups of chickens were inoculated intramuscularly with pfu of rdev-n, rdev-s, rdev-s , or received covalent vaccinations with rdev-n & rdev-s or rdev-n & rdev-s . the remaining chickens were immunized with dulbecco's modified eagle's medium, as a negative control. oropharyngeal and cloacal swabs were collected on post-vaccination days and for detection of the replication of the rdevs in chickens. peripheral blood samples from five vaccinated chickens were collected in sodium heparin-coated tubes to analyze the cellular immune responses to the rdevs on post-vaccination days , , , and , and on post-challenge day . on post-vaccination day , chickens, including five that were stable for cellular immune analysis, were selected from each group and challenged with % egg infectious dose (eid ) of the virulent ibv strain ck/ch/ldl/ by the oculonasal route. on post-challenge day , oropharyngeal swabs were collected from all chickens in each group to evaluate shedding of ibv. serum samples were collected from the remaining chickens in each group to monitor levels of anti-ibv antibodies at and weeks post-vaccination. chickens were monitored daily for clinical signs of infection. snicks (abnormal respiratory sounds) made by each of the birds were counted by three individuals over a -min period. birds were checked individually for tracheal rales, nasal discharge, watery eyes, and wheezing. the percentage of birds that died or exhibited clinical signs was recorded daily for days following ibv challenge. to determine whether the rdevs had the ability to replicate in chickens, cefs and -day-old spf embryonated chicken and duck eggs were used to re-isolate the viruses from the oropharyngeal and cloacal swabs of the vaccinated chickens. swabs maintained in phosphate-buffered saline (pbs) were centrifuged instantaneously at  g and the supernatants were filtered using a . -mm syringe filter (millipore corporation, billerica, ma, usa) and inoculated in cefs and chicken or duck embryos through the chorioallantoic membrane, respectively. three blind passages were conducted for each swab sample. samples cultured for days were detected by pcr using primers p and p (li et al., ) . serum samples were analyzed for the presence of anti-ibv antibodies using an enzyme-linked immunosorbent assay (elisa). briefly, -well plates were coated with homologous ibv strain ck/ ch/ldl/ , which was concentrated and purified by differential centrifugation and sucrose gradient ultracentrifugation. standard positive and negative sera, secondary antibodies, substrates, and stop solution were provided with the ibv antibody elisa kit (idexx corporation, westbrook, me, usa). titers were automatically calculated using a microplate reader at an optical density of nm. each serum titer was detected in triplicate, calculated based on the mean serum-to-positive (s/p) ratios (de wit et al., ; liu et al., ) , the cp value was determined as . and an s/p ratio ! . was considered positive. . . analysis of cd þ , cd þ , and cd þ t-lymphocytes lymphocytes were isolated from peripheral blood by ficollehypaque density gradient centrifugation using the chicken lymphocyte separation kit (beijing solarbio science & technology co., ltd., beijing, china), according to the manufacturer's instructions. peripheral blood mononuclear cells were isolated from each blood sample, adjusted to a concentration of  cells/ ml, and co-stained with mouse anti-chicken cd -sprd, mouse anti-chicken cd -pe, and mouse anti-chicken cd -fitc (south-ernbiotech, birmingham, al, usa) antibodies for h at room temperature. flow cytometry was performed using the bd facsaria cell sorter (bd biosciences, franklin lake, nj, usa) to calculate the percentages of cd þ , cd þ , and cd þ t-lymphocytes. viral shedding was quantified based on ibv rna levels in the oropharyngeal secretions of chickens challenged with strain ck/ch/ ldl/ . the oropharyngeal swabs contained in pbs were treated and detected as described previously (jones et al., ; cao et al., cao et al., , ). all samples were tested in triplicate and the data were analyzed using the lightcycler software, version . (roche diagnostics, basel, switzerland). the antibody titers of vaccinated chickens, the ratio of cd þ / cd þ t-lymphocytes, the growth kinetics of recombinant viruses, percentages of birds showing clinical signs, and mortality rates were statistical analyzed using multiple t-tests (graphpad prism ; graphpad software, inc., la jolla, ca, usa). all data are presented as means ± standard deviations. plaques formed by rdev-egfp exhibited green fluorescence, whereas those formed by rdev-n, rdev-s, and rdev-s did not (data not shown). the pcr screening and dna sequencing data confirmed that the n, s, and s cdnas from ibv were properly inserted into the dev genome with the deletion of the entire us gene. the primer pair df and dr, corresponding to the flanking regions of the us gene orf, were used to differentiate the wt from the rdevs. single fragments of varying lengths were amplified from the wt dev clone- , rdev-egfp, rdev-n, rdev-s, or rdev-s , indicating that the rdevs had been purified (fig. b) . western blotting analysis of the rdev-egfp-infected cells revealed a band of approximately kda, which corresponded to the size of egfp. specific bands corresponding to the n ( kda), s ( kda), and s ( kda) proteins of ibv were detected in the cells infected with rdev-n, rdev-s, and rdev-s , respectively. the gl protein of dev was detected in cells infected with wt dev clone- or rdevs, indicating efficient dev replication as a viral vector in cefs (fig. c) . the immunofluorescence analysis showed that the n, s, and s proteins were expressed in cefs infected with rdev-n, rdev-s, and rdev-s , respectively, whereas the mock-infected cefs exhibited no response to the anti-ibv or anti-ibv-n monoclonal antibodies (fig. d) . generally, the growth trends of rdev-egfp, rdev-n, rdev-s, and rdev-s were consistent with that of wt dev clone- , which showed that the viruses reached the highest titer at h and decreased at h postinfection ( fig. a) . however, statistical analysis demonstrated that the titers of the rdevs at each time point significantly differed from that of the wt dev clone- (*p < . , **p < . ). the primer pair df and dr was used to confirm the identity of three rdevs at passages , , , and . however, no -bp or -bp fragment (existed in wt dev or rdev-egfp, respectively) was detected, demonstrating that three rdevs expressing ibv proteins were genetically stable without reversion (fig. b) . oropharyngeal and cloacal swabs were collected postvaccination for the detection of rdevs in chickens. pcr detection showed that viral isolation was negative from the cefs, as well as the chicken and duck embryo cultures, suggesting that the vaccinated chickens do not shed rdevs from the respiratory and gastrointestinal tracts. chickens were challenged with eid of the virulent ibv strain ck/ch/ldl/ at days post-vaccination. the percentage of chickens that showed clinical signs in the rdev-n, rdev-s, rdev-s , rdev-n & rdev-s, rdev-n & rdev-s , and control groups was %, %, %, %, %, and % (p < . , very significant difference, each group compared with the control group only), respectively (table ) . birds began to exhibit ruffled feathers and snicks at days after challenge with virulent ibv, and developed depression and huddle, dark combs, and death as time went on. mortality rates in the rdev-n, rdev-s, rdev-s , rdev-n & rdev-s, rdev-n & rdev-s , and control groups were %, %, %, %, % and %, respectively (p > . , no significant difference) ( table ) . gross lesions were confined primarily to the kidneys. the renal parenchyma of the affected birds was pale, swollen, and mottled, while the renal tubules and urethras were distended due to accumulation of uric acid crystals. we evaluated viral shedding using qrt-pcr to quantify the ibv rna in oropharyngeal swabs. in the control group, % of the chickens shed viruses, while challenged chickens in the rdev-n, rdev-s, rdev-s , rdev-n & rdev-s, and rdev-n & rdev-s groups showed relatively lower viral shedding rates of only %, %, %, %, and %, respectively (p < . , each group compared to the control group only). the antibody titers of anti-n, -s, and -s of vaccinated chickens were measured weekly using an elisa with plates coated with the homologous virus ck/ch/ldl/ . in general, all groups showed relatively high antibody titers and seroconversion rates at week post-vaccination, which then decreased gradually. at week postvaccination, % ( / ) of the chickens vaccinated with rdev-n were seropositive, although seropositivity gradually decreased from week ( %) to week ( . %). in the rdev-s vaccinated group, % ( / ) of the chickens exhibited seroconversion at week , but this percentage decreased rapidly afterward to % at week and % at week post-vaccination. in the rdev-s group, the rdevs with the us deletion exhibited statistically significant lower viral titers (**p < . , *p < . ). viral titers are shown in tcid / ml. (b) the genetic stability of the rdevs expressing the major structural proteins of infectious bronchitis virus (ibv). the identity of the rdevs was confirmed by pcr using the differentiated primer pair df and dr following the th, th, th, and th passages. no reversion occurred during the passage from the rdevs to the wt dev clone- (dev clone- , bp; rdev-n, bp; rdev-s, bp; rdev-s , bp). % ( / ) of the chickens were seropositive, but this rate decreased sharply to % at week and to % at week . the antibody levels and seroconversion rates of the rdev-n & rdev-s covalent-vaccination group were similar to those of the rdev-n and rdev-s group. the antibody titers of the rdev-n & rdev-s covalent-vaccination group were higher than all other groups and all of the chickens in this group were ibv antibody-positive at weeks ( %) and ( %), although these rates then slowly decreased from week post-vaccination ( %). none of the chickens in the control group exhibited an antibody response (table ) . there were significant differences (**p < . , *p < . ) in antibody titers of each single vaccinated rdev group, as compared to the corresponding covalent-vaccination group (fig. ) , suggesting that the covalent rdev-n & rdev-s vaccine enhanced the humoral response. cd þ and cd þ t-lymphocytes are important parameters of cell-mediated immune responses in vaccinated chickens. to evaluate the cellular response induced by rdevs in vaccinated chickens, the percentages of cd þ cd þ and cd þ cd þ t-lymphocytes, respectively, in peripheral blood were analyzed by flow cytometry, which showed that the ratio of cd þ and cd þ t-lymphocytes decreased at day post-vaccination in each vaccinated group, and slightly increased from day post-vaccination, as compared with that of birds in the control group (table ) . there was a significant difference (p < . ) in the cd þ /cd þ ratio of the rdev-n group at day post-vaccination, as compared to the control group. there was no significant difference among the three single-vaccination groups and the two covalent-vaccination groups (p > . ) ( table ) . the ibv genome codes for four structural proteins, which play different roles in immune protection of vaccinated chickens. the n protein is an immunodominant antigen that induces high titers of cross-reactive antibodies. the m glycoprotein elicits low titers of antibodies with limited cross-reactivity, whereas the s glycoprotein induces production of serotype-specific and cross-reactive antibodies (ignjatovic and galli, ) . the s glycoprotein induces virus neutralizing and cross-reactive antibodies and cellmediated immune responses galli, , ) . moreover, ignjatovic and galli ( ) reported that immunization of chickens with purified m glycoproteins did not induce protection against virulent ibv challenge, whereas immunization with the s glycoprotein prevented replication of nephropathogenic ibv in the kidneys, but not the tracheas, of immunized chickens. hence, the n, s, and s proteins of ibv were chosen as the target proteins to construct rdevs in order to evaluate the immune protection of these proteins against the virulent ibv strain ck/ch/ldl/ . in the current study, the us gene of dev was replaced with the n, s, or s gene of ibv, respectively, to produce rdevs that expressed the n, s, and s proteins of ibv, respectively. virus titers of rdevs decreased -fold at h postinfection compared to that of the wt dev clone- . however, it was reported that virus titers of the ul gene deletion rdev decreased by e -fold, as compared to the parental virus (wang and osterrieder, ) , suggesting that gene deletion of dev does affect viral replication, while the influence depends on the deletion site in the viral genome. the antibody levels of the chickens in the rdev-n & rdev-s covalent-vaccination groups were higher than those of all other vaccinated groups. at week post-vaccination, the antibody levels in the rdev-s and rdev-s groups decreased sharply, whereas the covalent-vaccination groups had higher antibody levels. single rdev-n and single rdev-s vaccination invoked weak antibody responses, while covalent vaccination with these two rdevs induced strong antibody responses. the high antibody titers were related to the lower mortality in the rdev-n & rdev-s covalentvaccination group, indicating that covalent vaccination with the ibv structural proteins n and s provided better protection against challenge with virulent ibv strains. the ibv s glycoprotein consists ( ) a number of chickens shedding ibv on day post-challenge. b clinical signs (%) are presented as the percentage of birds showed clinical signs out of the total number of vaccinated chickens in the group after virulent ibv challenge. c p < . , very significant difference, each group was only compared to the control group. fig. . antibody responses of chickens vaccinated with recombinant duck enteritis viruses expressing the n, s or s gene of infectious bronchitis virus (ibv). sera from chickens immunized with rdev-n, rdev-s, rdev-s , rdev-n þ rdev-s, rdev-n þ rdev-s , or the control were tested for the presence of antibodies against ibv at week (n ¼ ), weeks (n ¼ ), weeks (n ¼ ), weeks (n ¼ ), and weeks (n ¼ ) post-vaccination. there were very significant differences in the antibody titers of the vaccinated groups as compared to that of the control group (p < . ). all single vaccinated groups were compared to the corresponding covalent vaccinated group. significant differences in antibody titers are indicated with dashes. of the s and s subunits. the s subunit induces efficient cellular immunity , while the n protein mainly induces cross-reactive antibodies (ignjatovic and galli, ) , which may be one of the reasons for the difference in antibody levels among the rdev-n, rdev-s and rdev-s groups. to evaluate the cellular immune responses induced by the rdevs, we detected the percentages of cd þ cd þ and cd þ cd þ t-lymphocytes in peripheral blood samples collected from vaccinated chickens on postvaccination days , , , and . the results showed that cd þ tlymphocytes increased and the causative ratio of cd þ /cd þ tlymphocytes decreased at day post-vaccination in each vaccinated group, suggesting the induction of cytotoxic t lymphocytes (ctls). reportedly, cd þ t-lymphocytes induce and enhance the immune response by secreting cytokines, while most ctls were shown to be cd þ cells, which play a major role in the control of viral infection. collisson et al. ( ) reported that ibv-specific ctl activity was dependent on the s and n proteins of ibv, which was consistent with the results of the present study. moreover, chickens infected with ibv gray strain showed ctl responses as early as days and peaked at days postinfection (collisson et al., ) , consistent with our results on the detection of cd þ t-lymphocytes. in this study, the antibody responses and increases in cd þ tlymphocytes levels were detected as early as days postvaccination, suggesting efficacious delivery of the foreign protein as a vaccine vector to the host, which elicited both humoral and cellular immune responses. chen et al. ( ) reported partial protection of chickens against a recombinant fowlpox virus expressing the ibv s protein with only % of birds showing clinical signs. in this study, clinical signs were detected in a significantly lower percentage of birds that were vaccinated with rdev-s, rdev-s , or rdev-n after challenge with virulent strain ck/ch/ldl/ when compared with the control group, suggesting vaccination with these three viruses conferred better protection. in addition, a lower percentage of birds vaccinated with rdev-s showed clinical signs after challenge when compared with birds vaccinated with the other two viruses, demonstrating that better protection was conferred by vaccination with rdev-s. furthermore, although covalent vaccination with rdev-n & rdev-s or rdev-n & rdev-s provided the same clinical protection ( % clinical signs) as chickens in the rdev-s group, vaccination with the covalent vaccines offered better protection in regard to viral shedding of virulent ibv. we did not test a live ibv vaccine in the present study because no commercial lx -type (qxlike) vaccine is currently available. in our previous study, we found that the h commercial vaccine did not provide efficacious protection against the ck/ch/ldl/ strain of ibv, resulting in a morbidity rate of % (sun et al., ) , which was higher than that observed in chickens vaccinated with rdev-s ( %), rdev-n ( %), dev-n & rdev-s ( %), or rdev-n & rdev-s ( %). the mortality induced by ibv ck/ch/ldl/ infection in the control group in this study was higher than that reported by sun et al. ( ) , which may be due to the higher dosages of the challenged viruses used in that experiment as compared to the present study ( vs. . eid per chicken, respectively). in a previous study, immunization with a recombinant fowlpox that coexpressed the s protein of ibv and chicken interleukin provided complete protection against ibv infection in chickens (chen et al., ) . therefore, the coexpression of immunomodulatory factors, such as cytokines, may improve the efficacy of rdevs to induce protection against ibv infection. hence, future studies are warranted to determine whether the use of combinations of our rdev vaccines or boosting with a live or inactivated vaccine may induce better efficacious protection against ibv infection in poultry. in addition, the protection provided by our rdev vaccine against other ibv serotypes, such as the massachusetts serotype, should also be evaluated. development and use of the h strain of avian infectious bronchitis virus from the netherlands as a vaccine: a review the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex infectious bronchitis virus s gene sequence variability may affect s subunit specific antibody binding proteomic analysis of chicken embryonic trachea and kidney tissues after infection in ovo by avian infectious bronchitis coronavirus proteomics analysis of differentially expressed proteins in chicken trachea and kidney after infection with the highly virulent and attenuated coronavirus infectious bronchitis virus in vivo coronavirus avian infectious bronchitis virus infectious bronchitis construction and immunogenicity of a recombinant fowlpox vaccine coexpressing s glycoprotein of infectious bronchitis virus and chicken il- cytotoxic t lymphocytes are critical in the control of infectious bronchitis virus in poultry the long view: years of infectious bronchitis research transmission of table ratio of cd þ /cd þ t-lymphocytes after vaccination and challenge. infectious bronchitis virus within vaccinated and unvaccinated groups of chickens effect of serial embryo passage of an arkansas-type avian infectious bronchitis virus isolate on clinical response, virus recovery, and immunity fine level epitope mapping and conservation analysis of two novel linear b-cell epitopes of the avian infectious bronchitis coronavirus nucleocapsid protein s protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients development of attenuated vaccines from taiwanese infectious bronchitis virus strains structural proteins of avian infectious bronchitis virus: role in immunity and protection the s glycoprotein but not n or m proteins of avian infectiousbronchitis virus induces protection in vaccinated chickens immune responses to structural proteins of avian infectious bronchitis virus attenuation, safety, and efficacy of an infectious bronchitis virus ga serotype vaccine development and validation of rt-pcr tests for the detection and s genotyping of infectious bronchitis virus and other closely related gammacoronaviruses within clinical samples antigenic domains on thepeplomer protein of avian infectious bronchitis virus: correlation with biologicalfunctions phylogeny of antigenic variants of avian coronavirus ibv antigenicity of thepeplomer protein of infectious bronchitis virus characterization of the genes encoding ul , tk and gh proteins from duck enteritis virus (dev): a proof for the classification of dev molecular characterization of the genome of duck enteritis virus recombinant duck enteritis virus works as a single-dose vaccine in broilers providing rapid protection against h n influenza infection a duck enteritis virus-vectored bivalent live vaccine provides fast and complete protection against h n avian influenza virus infection in ducks infectious bronchitis virus: s gene characteristics of vaccines used in china and efficacy of vaccination against heterologous strains from china molecular characterization of the herpes simplex virus (hsv- ) homologues, ul to ul , in duck enteritis virus (dev) origin and characteristics of the recombinant novel avian infectious bronchitis coronavirus isolate ck/ch/ljl/ altered pathogenicity, immunogenicity, tissue tropism and - kb region sequence of an avian infectious bronchitis coronavirus strain after serial passage in embryos recombinant duck enteritis virus expressing the ha gene from goose h subtype avian influenza virus construction and characterization of marek's disease viruses having green fluorescent protein expression tied directly or indirectly to phosphoprotein expression a simple method of estimating fifty percent endpoints the carboxyl-terminal -residue polypeptide of infectious bronchitis virus nucleocapsid induces cytotoxic t lymphocytes and protects chickens from acute infection phylogenetic analysis of infectious bronchitis coronaviruses newly isolated in china, and pathogenicity and evaluation of protection induced by massachusetts serotype h vaccine against qx-like strains infectious bronchitis virus s expressed from recombinant virus confers broad protection against challenge generation of an infectious clone of duck enteritis virus (dev) and of a vectored dev expressing hemagglutinin of h n avian influenza virus comparative analyses of the nucleocapsid genes of several strains of infectious bronchitis virus and other coronaviruses protection of chickens against infectious bronchitis virus with a multivalent dna vaccine and boosting with an inactivated vaccine multivalent dna vaccine enhanced protection efficacy against infectious bronchitis virus in chickens bacmam virus-based surface display of the infectious bronchitis virus (ibv) s glycoprotein confers strong protection against virulent ibv challenge in chickens. vaccine , e . glossary ibv: infectious bronchitis virus dev: duck enteritis virus rdev: recombinant duck enteritis virus cef: chicken embryo fibroblast rt-qpcr: rt and real-time pcr elisa: enzyme ratio of cd þ and cd þ t-lymphocytes dpv a dpv dpv dpv dpc rdev-n . ± . b . ± . a . ± . . ± . . ± . rdev-s . ± . . ± . . ± . . ± . . ± . rdev-s . ± . . ± . . ± . . ± . . ± . rdev-n & rdev-s . ± . . ± . . . ± . . ± . . ± . rdev-n & rdev-s . ± . . ± . . ± . . ± . . ± . control . ± . . ± . . ± . . ± . . ± . key: cord- - e zlt t authors: choy, ka-tim; wong, alvina yin-lam; kaewpreedee, prathanporn; sia, sin fun; chen, dongdong; hui, kenrie pui yan; chu, daniel ka wing; chan, michael chi wai; cheung, peter pak-hang; huang, xuhui; peiris, malik; yen, hui-ling title: remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov- replication in vitro date: - - journal: antiviral res doi: . /j.antiviral. . sha: doc_id: cord_uid: e zlt t an escalating pandemic by the novel sars-cov- virus is impacting global health and effective therapeutic options are urgently needed. we evaluated the in vitro antiviral effect of compounds that were previously reported to inhibit coronavirus replication and compounds that are currently under evaluation in clinical trials for sars-cov- patients. we report the antiviral effect of remdesivir, lopinavir, homorringtonine, and emetine against sars-cov- virus in vero e cells with the estimated % effective concentration at . μm, . μm, . μm and . μm, respectively. ribavirin or favipiravir that are currently evaluated under clinical trials showed no inhibition at μm. synergy between remdesivir and emetine was observed, and remdesivir at . μm in combination with emetine at . μm may achieve . % inhibition in viral yield. combinational therapy may help to reduce the effective concentration of compounds below the therapeutic plasma concentrations and provide better clinical benefits. within three months of the first identification of sars-cov- virus in wuhan, hubei province, china, the world is facing an escalating pandemic that will have significant impacts on global health systems and economy (who, ) . infection with the novel sars-cov- virus may lead to a wide range of clinical presentations from asymptomatic infection in % of laboratory confirmed cases to mild, severe, and critical infections in %, %, and % of symptomatic cases, respectively (wu and mcgoogan, ) . the estimated symptomatic case-fatality risk (scfr) among cases in wuhan was . %, and those aged above were . times more likely to die from infection than those aged - years . with an estimated basic reproductive number of . ( % ci, . - . ) (li et al., ) , the virus will continue to spread and infect % of the global population over time if no effective vaccine is developed (fine et al., ) . there is currently no effective antiviral compound licensed for the treatment against human coronaviruses or sars-cov- . the sars-cov- virus shared . % genetic homology to the sars-cov and both are descendants of bat coronaviruses within the betacoronavirus genus . antiviral compounds previously reported to show effect against sars-cov or other coronaviruses may be effective against sars-cov- (chu et al., ; de wilde et al., ; dyall et al., ; shen et al., ; cao et al., ) . in addition, remdesivir (gs- ), a prodrug of adenosine analog with a broad-spectrum antiviral activity against filoviruses, paramyxoviruses, and coronaviruses (brown et al., ; sheahan et al., ; de wit et al., ) , was recently confirmed to inhibit -ncov in vitro . according to the th edition of the novel coronavirus diagnosis and treatment plan issued by the national health commission of the people's republic of china, options for antiviral therapy include aerosolized α-interferon, lopinavir/ritonavir, ribavirin in combination with lopinavir/ritonavir, chloroquine phosphate, or arbidol (china national health commission, ). ongoing clinical trials are evaluating the efficacy of remdesivir, and various hivprotease inhibitors (lopinavir/ritonavir, asc /ritonavir, darunavir), reverse transcriptase inhibitor (azvudine), anti-influenza compounds, interferon alfa- b, or monoclonal antibody targeting pd- (camrelizumab) or il- (tocilizumab) (chinese clinical trial re). we evaluated the anti-sars-cov- effect of compounds that have been under development or already approved for other clinical applications; some compounds were previously reported to inhibit coronavirus replication in vitro, and some are evaluated in clinical trials in patients with coronavirus disease t sars-cov- virus, betacov/hong kong/vm / , was isolated from the nasopharynx aspirate and throat swab of a confirmed covid- patient in hong kong using vero e cells (atcc crl- ). stock virus ( . tcid /ml) was prepared after three serial passages in vero e cells in infection media (dmem supplemented with . g/l d-glucose, mg/l sodium pyruvate, % fbs, , u/l penicillin-streptomycin, and mm hepes). compounds were sourced from medchemexpress and sigma-aldrich and the stocks were prepared with dmso ( mm remdesivir, mm favipiravir, mm r- , mm tenofovir, mm fludarabine phosphate, mm baloxavir, mm chlorpromazine hydrochloride, mm dalbavancin hydrochloride, mm homoharringtonine, mm lopinavir, mm ritonavir) or with water ( mm emetine dihydrochloride, mm galidesivir hydrochloride, mm ribavirin, . mm oritavancin diphosphate). oseltamivir carboxylate ( mm in water) was provided by roche. to evaluate the effect of compounds in vitro, vero e cells were pretreated with compounds diluted in infection media for h prior to infection by sars-cov- virus at moi = . . antiviral compounds were maintained with the virus inoculum during the -h incubation period. the inoculum was removed after incubation, and the cells were overlaid with infection media containing diluted compounds. after h incubation at °c, supernatants were collected to quantify viral loads by tcid assay or quantitative real-time rt-pcr (taqman™ fast virus -step master mix) following the methods described (chu et al., ) . four-parameter logistic regression (graphpad prism) was used to fit the dose-response curves and determined the % effective concentrations (ec ) of the compounds that inhibit viral replication. cytotoxicty of selected compounds was evaluated in vero e cells using the celltiter-glo® luminescent cell viability assay (promega). among the compounds we tested, remdesivir, lopinavir, homoharringtonine, and emetine dihydrochloride were found to inhibit sars-cov- replication in vero e cells with ec under μm (table ) . importantly, we observed that some of the compounds currently undergoing clinical trials such as ribavirin, favipiravir, oseltamivir, or baloxavir showed no apparent antiviral effect against the sars-cov- virus in vitro at concentrations under μm (table ) . remdesivir is a ′-cyano-substituted adenosine analogue that has been shown to inhibit human coronaviruses (hcov-oc and hcov- e) sars-cov, mers-cov, and sars-cov- (brown et al., ; sheahan et al., ; de wit et al., ) . it is currently evaluated in phase clinical trials for sars-cov- . a recent study fitted viral load in linear scale (eg. the percentage of inhibition) under increasing concentrations of remdesivir reported ec against sars-cov- virus at . μm . we fitted viral load in logarithm scale (log tcid /ml and log viral rna copies/ml) under increasing concentration of remdesivir and determined ec at . μm and . μm, respectively ( fig. a and table ). two mutations (f l and v l) in the rna-dependent rna polymerase nsp of a murine hepatitis virus have been previously reported to confer resistance to remdesivir (agostini et al., ) . due to insertions and deletions in nsp , these two conserved residues are mapped at f and v in the sars-cov- isolate (gisaid# epi_isl_ ) used for the experiments, which should remain sensitive for remdesivir. other adenosine analogues (galidesivir, tenofovor, or fludarabine phosphate) or nucleoside analogues (favipiravir, ribavirin, r- ) did not inhibit viral replication under μm (table ) . however, nucleoside analogues require metabolic activation into their triphosphate forms by host cellular nucleoside kinases, which may differ among cell types. further evaluation of the effect of nucleoside analogues in primary human airway epithelial cells would facilitate the interpretation of the results. lopinavir in combination with ritonavir are fda approved hiv- protease inhibitors. lopinavir was more potent in inhibiting hiv- than ritonavir in vitro but showed poor bioavailability in vivo. ritonavir inhibits not only hiv- protease but also the host's cytochrome p a enzyme that metabolizes lopinavir (kempf et al., ) . lopinavir/ ritonavir in combination prolongs bioavailability of lopinavir in vivo (sham et al., ) . lopinavir but not ritonavir showed antiviral effect against sars-cov, mers-cov, and hcov- e in vitro, with mean ec ranged from . to . μm (de wilde et al., ) . lopinavir/ritonavir in combination with ribavirin were used previously to treat sars-cov patients under a non-randomized clinical trial. less sars patients developed into ards or death after receiving the combination of lopinavir/ritonavir with ribavirin than historical controls who received ribavirin and corticosteroids (chu et al., ) . efficacy of lopinavir/ ritonavir with or without ribavirin is currently evaluated in sars-cov- patients under randomized control trials. in agreement with previous reports, we observed antiviral effect of lopinavir (ec at . μm) but not ritonavir against sars-cov- in vitro ( fig. b and table ). hiv- patients treated with mg of lopinavir and mg of ritonavir twice daily may reach the minimal lopinavir serum concentration at . μm (iqr . - . μm), which is below the ec against sars-cov- virus in vitro (lopez-cortes et al., ) . currently, lopinavir/ritonavir at mg/ mg twice daily with or without ribavirin are part of the recommended treatment for managing covid- patients in china (china national health commission, ). a recent randomized control trial reported no significant benefit of lopinavir-ritonavir in hospitalized sars-cov- patients than standard care, as the time to clinical improvement, mortality at days, and viral loads at various time points were comparable between the two groups . combinational therapy of lopinavir with the other effective compounds against sars-cov- virus may increase synergy and reduce the inhibitory concentration of lopinavir. homoharringtonine is a plant alkaloid derived from cephalotoxus fortunei. it exhibits anti-tumor activity by binding to the ribosomal a site to inhibit protein translation, leading to rapid loss of short-lived proteins including mcl- and c-myc that promote the survival of leukemia cells (dong et al., ; lu and wang, ) . omacetaxine, a semi-synthetic form of homoharringtonine, is approved by fda for treatment of chronic myeloid leukemia. homoharringtonine has also been reported to exhibit potent anti-viral activity against herpesviruses (varicella-zoster virus, herpes simplex virus- , pseudorabies virus), coronaviruses (porcine epidemic diarrhea virus and murine hepatitis virus), rhabdoviruses (vsv and rabies virus), and other viruses (hepatitis b virus, newcastle disease virus, and echovirus ) (dong et al., ; andersen et al., ) . here, we observed homoharringtonine inhibits sars-cov- with ec at . μm ( fig. c and table ). previous pharmacokinetic study showed that patients treated with . mg/m omacetaxine every h by subcutaneous injection may reach the maximal plasma concentration at . ng/ml ( . μm) and . ng/ml ( . μm) on days and , respectively (nemunaitis et al., ) , which were below the ec against sars-cov- virus in vitro. emetine is a protein synthesis inhibitor that was used as anti-protozoan approved for treatment of ameobiasis; it also inhibits malaria by binding to the ribosomal e site of plasmodium falciparum (grollman, ; wong et al., ) . however, its potential cardiotoxicity has restricted its clinical use in the recent years. it was found to process antiviral activity against a broad range of rna and dna viruses, including zika virus, ebolavirus, cytomegalovirus, rabies virus, hiv- , echovirus , buffalo poxvirus, bovine herpesvirus , peste des petits ruminants virus, newcastle disease virus, herpes simplex virus- , metapneumovirus, rift valley fever virus, and influenza (andersen et al., ; chaves valadao et al., ; khandelwal et al., ; macgibeny et al., ; mukhopadhyay et al., ; yang et al., ) . emetine was also identified to inhibit hcov-oc , hcov-nl , sars-cov, mers-cov, and mhv-a in vitro with ec reported at low micromolar range (dyall et al., ; shen et al., ) . we observed emetine at around . μm may effectively inhibit sars-cov- virus replication ( fig. d and table ). the therapeutic plasma concentration of emetine may reach . μg/ml ( . μm) (regenthal et al., ) , which is below the ec against sars-cov- virus in vitro. the toxic plasma concentration is . μg/ml ( . μm) (regenthal et al., ) . table antiviral activity of compounds against sars-cov- in vero e cells. to reduce the effective concentration of individual compound below the maximal therapeutic plasma concentration, we explored the combinational effect of remdesivir and emetine in vitro. drug interaction was evaluated using the checkerboard assay with serially -fold diluted remdesivir ( - μm) and emetine ( - . μm) in combination. remdesivir at . μm in combination with emetine at . μm may achieve . % inhibition of viral yield, which can be further tested in vivo (fig. a) . the loewe additive model and the bliss independent model (malyutina et al., ) were used to analyse the interaction of the two compounds using synergyfinder (ianevski et al., ) . remdesivir and emetine in combination yielded a loewe synergy score of . (fig. b) and a bliss synergy score of . (fig. c) . we confirm the antiviral activity of four compounds that have been reported to inhibit other coronavirus or sars-cov- replication in vitro. our results suggest that combinational therapy may help to reduce the effective concentration against sars-cov- under the maximal therapeutic plasma concentration. there is an urgent research need to identify optimal dose combination of effective compounds against the sars-cov- virus for better clinical benefit. coronavirus susceptibility to the antiviral remdesivir (gs- ) is mediated by the viral polymerase and the proofreading exoribonuclease novel antiviral activities of obatoclax, emetine, niclosamide, brequinar, and homoharringtonine broad spectrum antiviral remdesivir inhibits human endemic and zoonotic deltacoronaviruses with a highly divergent rna dependent rna polymerase a screen of the nih clinical collection small molecule library identifies potential anti-coronavirus drugs a trial of lopinavirritonavir in adults hospitalized with severe covid- natural plant alkaloid (emetine) inhibits hiv- replication by interfering with reverse transcriptase activity novel coronavirus diagnosis and treatment plan role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings molecular diagnosis of a novel coronavirus ( -ncov) causing an outbreak of pneumonia screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture prophylactic and therapeutic remdesivir (gs- ) treatment in the rhesus macaque model of mers-cov infection the natural compound homoharringtonine presents broad antiviral activity in vitro and in vivo repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection herd immunity": a rough guide structural basis for inhibition of protein synthesis by emetine and cycloheximide based on an analogy between ipecac alkaloids and glutarimide antibiotics synergyfinder: a web application for analyzing drug combination dose-response matrix data pharmacokinetic enhancement of inhibitors of the human immunodeficiency virus protease by coadministration with ritonavir emetine inhibits replication of rna and dna viruses without generating drugresistant virus variants early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia lopinavir plasma concentrations and virological outcome with lopinavir-ritonavir monotherapy in hiv- -infected patients homoharringtonine and omacetaxine for myeloid hematological malignancies retrograde axonal transport of rabies virus is unaffected by interferon treatment but blocked by emetine locally in axons drug combination sensitivity scoring facilitates the discovery of synergistic and efficacious drug combinations in cancer efficacy and mechanism of action of low dose emetine against human cytomegalovirus pharmacokinetic study of omacetaxine mepesuccinate administered subcutaneously to patients with advanced solid and hematologic tumors drug levels: therapeutic and toxic serum/plasma concentrations of common drugs abt- , a highly potent inhibitor of the human immunodeficiency virus protease broad-spectrum antiviral gs- inhibits both epidemic and zoonotic coronaviruses high-throughput screening and identification of potent broad-spectrum inhibitors of coronaviruses remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro who coronavirus disease (covid- ) situation reports cryo-em structure of the plasmodium falciparum s ribosome bound to the antiprotozoan drug emetine characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention estimating clinical severity of covid- from the transmission dynamics in wuhan, china emetine inhibits zika and ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry a pneumonia outbreak associated with a new coronavirus of probable bat origin this study was supported by contract hhsn c from the national institute of allergy and infectious diseases of the national institutes of health, usa, and the theme-based research scheme (t - / n) from the research grants council, hong kong sar, china. key: cord- -uxyb vih authors: jockusch, steffen; tao, chuanjuan; li, xiaoxu; anderson, thomas k.; chien, minchen; kumar, shiv; russo, james j.; kirchdoerfer, robert n.; ju, jingyue title: a library of nucleotide analogues terminate rna synthesis catalyzed by polymerases of coronaviruses that cause sars and covid- date: - - journal: antiviral res doi: . /j.antiviral. . sha: doc_id: cord_uid: uxyb vih sars-cov- , a member of the coronavirus family, is responsible for the current covid- worldwide pandemic. we previously demonstrated that five nucleotide analogues inhibit the sars-cov- rna-dependent rna polymerase (rdrp), including the active triphosphate forms of sofosbuvir, alovudine, zidovudine, tenofovir alafenamide and emtricitabine. we report here the evaluation of a library of nucleoside triphosphate analogues with a variety of structural and chemical features as inhibitors of the rdrps of sars-cov and sars-cov- . these features include modifications on the sugar ( ’ or ’ modifications, carbocyclic, acyclic, or dideoxynucleotides) or on the base. the goal is to identify nucleotide analogues that not only terminate rna synthesis catalyzed by these coronavirus rdrps, but also have the potential to resist the viruses’ exonuclease activity. we examined these nucleotide analogues for their ability to be incorporated by the rdrps in the polymerase reaction and to prevent further incorporation. while all molecules tested displayed incorporation, exhibited immediate termination of the polymerase reaction (triphosphates of carbovir, ganciclovir, stavudine and entecavir; ’-ome-utp and biotin- -dutp), showed delayed termination (cidofovir diphosphate and ’-ome-utp), and did not terminate the polymerase reaction ( ’-f-dutp, ’-nh( )-dutp and desthiobiotin- -utp). the coronaviruses possess an exonuclease that apparently requires a ’-oh at the ’-terminus of the growing rna strand for proofreading. in this study, all nucleoside triphosphate analogues evaluated form watson-crick-like base pairs. the nucleotide analogues demonstrating termination either lack a ’-oh, have a blocked ’-oh, or show delayed termination. thus, these nucleotide analogues are of interest for further investigation to evaluate whether they can evade the viral exonuclease activity. prodrugs of five of these nucleotide analogues (cidofovir, abacavir, valganciclovir/ganciclovir, stavudine and entecavir) are fda-approved medications for treatment of other viral infections, and their safety profiles are well established. after demonstrating potency in inhibiting viral replication in cell culture, candidate molecules can be rapidly evaluated as potential therapies for covid- . the covid- pandemic, caused by sars-cov- , continues to have a devastating global impact. sars-cov- is a member of the orthocoronavirinae subfamily . coronaviruses, hcv and the flaviviruses are all positive-sense single-strand rna viruses that replicate their genomes using an rna-dependent rna polymerase (rdrp) . currently, there are no fda-approved antiviral drugs for the treatment of human coronavirus infections, including covid- . the rdrp of coronaviruses is a well-established drug target; the active site of the rdrp is highly conserved among positive-sense rna viruses (te velthuis ). these rdrps have low fidelity , allowing them to recognize a variety of modified nucleotide analogues as substrates. such nucleotide analogues may inhibit further rna-polymerase catalyzed rna replication making them important candidate anti-viral agents . rdrps in sars-cov and sars-cov- have nearly identical sequences . recently, the sars-cov- rdrp was cloned [not peer-reviewed] and the rna polymerase complex structure was determined ), which will help guide the design and study of rdrp inhibitors. remdesivir, a phosphoramidate prodrug containing a '-cyano modification on the sugar, is converted in cells into an adenosine triphosphate analogue, which was shown to be an inhibitor of the rdrps of sars-cov and sars-cov- (gordon et al. a; b) . it is currently in clinical trials in several countries as a therapeutic for covid- infections and was recently approved for emergency use by the fda. remdesivir triphosphate was shown to be incorporated with higher efficiency than atp by coronavirus rdrps, leading to delayed termination of rna synthesis, thereby overcoming excision by the viral exonuclease (gordon et al. a; b) . β-d-n -hydroxycytidine is another prodrug targeting the coronavirus polymerase and was shown to have broad spectrum activity against coronaviruses, even in the presence of intact proofreading functions ). we previously demonstrated that five nucleotide analogues inhibit the sars-cov- rdrp, including the active triphosphates of sofosbuvir, alovudine, zidovudine, tenofovir alafenamide and emtricitabine [not peer-reviewed]. emtricitabine and tenofovir alafenamide are used in fda-approved combination regimens for treatment of hiv/aids infections and as pre-exposure prophylaxis (prep) to prevent hiv infections . the fact that each of the previous five nucleotide analogues exhibited inhibition of the coronavirus polymerases indicates that the sars-cov- rdrp can accept a variety of nucleotide analogues as substrates. here we evaluate additional nucleotide analogues with a larger variety of modifications to identify those with more efficient termination; we also consider the chemical or structural properties of these molecules that may help overcome the virus' proofreading function. these nucleotide analogues were selected based on one or more of the following criteria. first, they have structural and chemical properties such as (a) similarity in size and structure to natural nucleotides, including the ability to fit within the active site of the polymerase, (b) presence of a small '-oh capping group or absence of a '-oh group resulting in obligate termination of the polymerase reaction; or (c) modifications at the ' or other positions on the sugar or base that can potentially lead to delayed termination. we previously showed that nucleotides with substantial modifications on the base can be incorporated by dna polymerases ). the criteria above will provide structural and chemical features that we can explore to evade viral exonuclease activity . second, if they have previously been shown to inhibit the polymerases of other viruses, even those with different polymerase types, they may have the potential to inhibit the sars-cov- rdrp, as we have previously shown for hiv reverse transcriptase (rt) inhibitors . third, ideally, the inhibitors should display high selectivity for viral polymerases relative to cellular dna or rna polymerases. fourth, there is an advantage in considering nucleotide analogues that are the active triphosphate forms of fda-approved drugs, as these drugs are known to have acceptable levels of toxicity and are more likely to be tolerated by patients with coronavirus infections, including covid- . using the criteria above, our study examines nucleotide analogues with sugar or base modifications (structures shown in fig. ) for their ability to inhibit the sars-cov- or sars-cov rdrps: ganciclovir '-triphosphate, carbovir '-triphosphate, cidofovir diphosphate, stavudine '-triphosphate, entecavir '-triphosphate, '-o-methyluridine- '-triphosphate ( '-ome-utp), '-o-methyluridine- 'triphosphate ( '-ome-utp), '-fluoro- '-deoxyuridine- '-triphosphate ( '-f-dutp), desthiobiotin- aminoallyl-uridine- '-triphosphate (desthiobiotin- -utp), biotin- -aminoallyl- '-deoxyuridine- 'triphosphate (biotin- -dutp) and '-amino- '-deoxyuridine- '-triphosphate ( '-nh -dutp). the nucleoside and prodrug forms for the fda-approved drugs are shown in fig. ; nucleoside and potential prodrug forms for three other nucleotide analogues are shown in fig. s . some of the uridine analogues listed above have been previously shown to be substrates of viral polymerases (arup et al. ; ). the '-o-methyluridine triphosphate is of particular interest since '-o-methyl nucleotides can resist removal by the '-exonuclease found in coronaviruses . we describe the properties of nucleotide analogues whose prodrug forms are fda-approved for other virus infections as follows. ganciclovir triphosphate (gan-tp) is an acyclic guanosine nucleotide (fig. ) . the parent nucleoside ganciclovir (cytovene, fig. ) is used to treat aids-related cytomegalovirus (cmv) infections. the drug can inhibit herpesviruses and varicella zoster virus. the valyl ester prodrug valganciclovir (fig. ) can be given orally. after cleavage of the valyl ester, ganciclovir is converted to ganciclovir triphosphate by viral and cellular enzymes to inhibit the viral polymerase . carbovir triphosphate (car-tp) is a carbocyclic guanosine didehydro-dideoxynucleotide ( fig. ) . the parent prodrug, abacavir (ziagen, fig. ), is an fda-approved nucleoside rt inhibitor for hiv/aids treatment . it is taken orally and is well tolerated. cidofovir diphosphate (cid-dp) is an acyclic cytidine nucleotide ( fig. ). its prodrug form cidofovir (vistide, fig. ) is an fda-approved intravenous drug for the treatment of aids-related cmv retinitis and has been used off-label for a variety of dna virus infections . a second prodrug form of cidofovir diphosphate, brincidofovir (fig. ) , is an oral antiviral drug with a lipid moiety masking the phosphate group and a candidate for treating smallpox infections. it is active against a wide range of dna viruses in animals, including poxviruses, adenoviruses, herpesviruses and cmv . both brincidofovir and a protide-based prodrug (fig. ) are expected to enter cells rapidly. interestingly, although cidofovir is incorporated into dna in the polymerase reaction by vaccinia virus dna polymerase, the termination of synthesis occurs after extension by an additional nucleotide, a delayed termination similar to that shown for remdesivir for coronavirus rdrp; cidofovir incorporated in the penultimate position of the dna extension strand by the vaccinia virus polymerase is not removed by the viral '-exonuclease . stavudine triphosphate (fig. ) , a thymidine analogue, is the active triphosphate form of stavudine (d t, zerit, fig. ), an antiviral used for the prevention and treatment of hiv/aids via inhibition of the hiv rt . the lack of a '-oh group makes it an obligate inhibitor. entecavir triphosphate (ent-tp, fig. ), the active triphosphate form of the oral drug entecavir (baraclude, fig. ) , is a guanosine nucleotide inhibitor of the hepatitis b virus polymerase (matthews , rivkina and rybalow ) . it shows little if any inhibition of nuclear and mitochondrial dna polymerases and has generally been shown to have low toxicity. entecavir triphosphate is a delayed chain terminator of the hiv- reverse transcriptase, making it resistant to phosphorolytic excision . we reasoned that once these nucleotide analogues are incorporated into a rna primer in the polymerase reaction, the fact that they lack either a normal sugar ring configuration or the '-and/or '-oh groups would make them unlikely candidates for removal by the '-exonuclease involved in sars-cov- proofreading. in contrast to many other rna viruses, sars-cov and sars-cov- have very large genomes that encode a '- ' exonuclease (nsp ) involved in proofreading , the activity of which is enhanced by the cofactor nsp ). this proofreading function increases replication fidelity by removing mismatched nucleotides . mutations in nsp led to reduced replication fidelity of the viral genome . interestingly, while the nsp /nsp complex efficiently excises single mismatched nucleotides at the ' end of the rna chain, it is not able to remove longer stretches of unpaired nucleotides or ' modified rna . for the nucleotide analogues to be successful inhibitors of the rdrps of these viruses, they need to overcome this proofreading function. the coronavirus exonuclease activity typically requires a '-oh group at the ' end of the growing rna strand . however, in instances of delayed termination in which the offending nucleotide analogue is no longer at the ' end, they will also not be removed by the exonuclease gordon et al. a; b) . nearly all the nucleotide analogues we selected lack the '-oh group, have modifications that block the '-oh group on the sugar, or are acyclic nucleotide derivatives. such nucleotides will not likely be substrates for viral exonucleases. nucleoside triphosphates and nucleoside triphosphate analogues were purchased from trilink biotechnologies (biotin- -dutp, desthiobiotin- -utp, '-ome-utp, '-ome-utp, '-f-dutp, '-nh -dutp, cidofovir-dp, ganciclovir-tp, dutp, ctp, atp and utp), santa cruz biotechnology (stavudine-tp, carbovir-tp), or moravek, inc. (entecavir-tp). oligonucleotides were purchased from integrated dna technologies, inc. the primer and template (sequences shown in figs. - , s -s ) were annealed by heating to °c for min and cooling to room temperature in x reaction buffer. the rna polymerase mixture consisting of µm nsp and µm each of cofactors nsp and nsp ) was incubated for min at room temperature in a : : ratio in x reaction buffer. then µl of the annealed template primer solution containing µm template and . µm primer in x reaction buffer was added to µl of the rna polymerase mixture and incubated for an additional min at room temperature. finally µl of a solution containing mm '-ome-utp (fig. a) , mm sta-tp (fig. b) , mm biotin-dutp (fig. c) , mm cid-dp + mm utp + mm atp (fig. ) , mm car-tp + mm utp + mm atp + mm ctp (fig. a) , mm ent-tp + mm utp + mm atp + mm ctp (fig. b) , mm gan-tp + mm utp + mm atp + mm ctp (fig. c ), . mm sta-tp (fig. s a) , . mm cid-dp + . mm utp + . mm atp (fig. s b) , mm desthiobiotin- -utp + mm atp (fig. s ) , mm '-ome-utp + mm dutp (fig. s ), mm utp, mm biotin-dutp and mm dutp (fig. s a) , mm '-f-dutp, mm '-ome-utp and mm dutp (fig. s b) , or mm '-nh -dutp, mm '-ome-utp and mm dutp ( fig. s c ) in x reaction buffer was added and incubation was carried out for hrs at °c. the final concentrations of reagents in the µl extension reactions were µm nsp , µm nsp , µm nsp , nm rna primer, nm rna template, µm '-ome-utp (fig. a) , µm sta-tp (fig. b) , µm biotin-dutp (fig. c) , µm cid-dp, µm utp and µm atp (fig. ) , µm car-tp, µm utp, µm atp and µm ctp (fig. a) , µm ent-tp + µm utp + µm atp + µm ctp (fig. b) , µm gan-tp, µm utp, µm atp and µm ctp (fig. c) , µm sta-tp (fig. s a) , µm cid-dp + µm utp + µm atp (fig. s b) , µm desthiobiotin- -utp + µm atp (fig. s ) , µm '-ome-utp + µm dutp (fig. s ) , µm utp, µm biotin-dutp and µm dutp (fig. a) , µm '-f-dutp, µm '-ome-utp and µm dutp (fig. s b) , and µm '-nh -dutp, µm '-ome-utp and µm dutp (fig. s c) . the x reaction buffer contains the following reagents: mm tris-hcl ph , mm kcl, mm mgcl and mm β-mercaptoethanol. following desalting using an oligo clean & concentrator (zymo research), the samples were subjected to maldi-tof-ms (bruker ultraflextreme) analysis. the primer and template above were annealed by heating to °c for min and cooling to room temperature in x reaction buffer (described above). the rna polymerase mixture consisting of µm nsp and µm each of cofactors nsp and nsp (kirchdoerfer and ward ) was incubated for min at room temperature in a : : ratio in x reaction buffer. then µl of the annealed template primer solution containing µm template and . µm primer in x reaction buffer was added to µl of the rna polymerase mixture and incubated for an additional min at room temperature. finally µl of a solution containing mm cid-dp + . mm utp + . mm atp (fig. s ) , mm car-tp + . mm utp + . mm atp + . mm ctp (fig. s a) , or mm gan-tp + . mm utp + . mm atp + . mm ctp (fig. s b) , mm '-ome-utp (fig. s a) , mm '-ome-utp (fig. s b) , or mm '-f-dutp (fig. s ) in x reaction buffer was added and incubation was carried out for hrs at °c. the final concentrations of reagents in the µl extension reactions were µm nsp , µm nsp , µm nsp , nm rna primer, nm rna template, µm cid-dp, µm utp and µm atp (fig. s ) , µm car-tp, µm utp, µm atp and µm ctp (fig. s a) , µm gan-tp, µm utp, µm atp and µm ctp (fig. s b) , µm '-ome-utp (fig. s a) , µm '-ome-utp (fig. s b ), and µm '-f-dutp (fig. s ) . following desalting using an oligo clean & concentrator, the samples were subjected to maldi-tof-ms analysis. we tested the ability of the active triphosphate forms of the nucleotide analogues (structures shown in fig. ) to be incorporated by the rdrp of sars-cov- or sars-cov. the rdrp of these coronaviruses, referred to as nsp , and its two protein cofactors, nsp and nsp , which were shown to be required for the processive polymerase activity of nsp in sars-cov , were cloned and purified as described previously (kirchdoerfer and ward ) . we then performed polymerase extension assays with the library of nucleoside triphosphate analogues (fig. ) either alone or in combination with natural nucleotides: '-ome-utp, '-ome-utp, '-f-dutp, '-nh -dutp, biotin-utp, desthiobiotin- -utp, sta-tp, cid-dp + utp + atp, car-tp + utp + atp + ctp, gan-tp + utp + atp + ctp, or ent-tp + utp + atp + ctp, following the addition of a pre-annealed rna template and primer to a pre-assembled mixture of the sars-cov and/or sars-cov- rdrp (nsp ) and the two cofactor proteins (nsp and nsp ). we also used combinations of nucleotide analogues in some cases to perform the polymerase reaction to compare their relative incorporation efficiencies. the polymerase reaction products were analyzed by maldi-tof mass spectrometry. the sequences of the rna template and primer used for the polymerase extension assay, which correspond to the ' end of the sars-cov- genome, are indicated at the top of figs. - and s -s . in the case of the utp and ttp analogues, because there are two a's in a row in the next available positions of the template for rna polymerase extension downstream of the priming site, if they are indeed terminators of the polymerase reaction at the relatively high concentration used, the extension is expected to stop after incorporating one nucleotide analogue. if they do not serve as terminators, two base extension by the utp or ttp analogue will be observed. in the case of cid-dp which is a ctp analogue, utp and atp must be provided to allow extension to the point where there is a g in the template strand. if the cid-dp is then incorporated and acts as a terminator, extension will stop; otherwise, additional incorporation events may be observed. similarly, for carbovir-tp, ganciclovir-tp and entecavir-tp, all of which are gtp analogues, utp, atp and ctp must be provided to allow extension to the point where there is a c in the template strand. if car-tp, gan-tp or ent-tp is incorporated and acts as a terminator, extension will stop; otherwise additional incorporation events occur. guided by polymerase extension results we obtained previously for the active triphosphate forms of sofosbuvir, alovudine, azt, tenofovir-dp and emtricitabine-tp [not peerreviewed], various ratios of the nucleotides were chosen in the current study. the results of the maldi-tof ms analysis of the primer extension reactions are shown in figs. - and s -s . the observed peaks generally fit the nucleotide incorporation patterns described above, however, additional peaks assigned to intermediate stages of the extension reaction (and in some cases extension beyond the incorporation of the nucleotide analogue) was also observed. we describe the results for the sars-cov- polymerase catalyzed reaction in detail; we obtained similar results for the subset of nucleotide analogues tested with the sars-cov rdrp, as shown in the supplementary material. the results for '-ome-utp, sta-tp (a t analog) and biotin-dutp are presented in fig. . in the case of extension with '-ome-utp (fig. a) , ms peaks representing incorporation by one '-ome-utp ( da observed, da expected) and to a lesser extent two '-ome-utps ( da observed, da expected) were observed. thus, '-ome-utp shows significant termination upon incorporation, indicating it can be a potential drug lead. '-o-methyl modification of rna occurs naturally and therefore should have relatively low toxicity. in addition, ribose- '-o-methylated rna resists viral exonuclease activity . for sta-tp (fig. b) , a single incorporation peak ( da observed, da expected) was seen, indicating that sta-tp is very efficiently incorporated and achieves complete termination of the polymerase reaction. a -fold lower concentration of sta-tp also resulted in termination of the polymerase reaction (fig. s a) . since sta-tp is a dideoxynucleotide without any hydroxyl groups on the sugar moiety, it may resist exonuclease activity. in the case of biotin-dutp (fig. c) , a single incorporation peak was evident ( da observed, da expected), suggesting that biotin-dutp is also a terminator of the polymerase reaction under these conditions. this indicates that the presence of a modification on the base along with the absence of a '-oh group in this nucleotide analogue leads to termination of the polymerase reaction catalyzed by the sars-cov- rdrp. the result for the ctp analogue cid-dp, which has an oh group, is presented in fig. . major peaks were observed indicating incorporation of a cid-dp at the th position from the initial priming site ( da observed, da expected) and a further base extension by one atp followed by one cid-dp at the th position ( da observed, da expected). there is no further extension beyond this position, indicative of delayed termination by cid-dp. a small intermediate peak was also observed indicating extension by an atp at the th position from the initial priming site following the first cid-dp incorporation ( da observed, da expected). a small partial utp extension peak ( da observed, da expected) was also observed. a -fold lower concentration of cid-dp also resulted in termination of the polymerase reaction (fig. s b ). an essentially identical result was obtained with the sars-cov polymerase (fig. s ) . delayed termination for cid-dp has been described for a vaccinia virus dna polymerase . the investigational drug remdesivir, which is currently being used for the treatment of covid- under emergency authorization, also displays delayed termination (gordon et al. a; b) ; this is a major factor in its ability to resist the nsp '- ' exonuclease activity. thus, cidofovir and its oral prodrugs are of interest for further investigation to evaluate whether they can evade the viral exonuclease activity. based on these results, and if potency for viral inhibition in cell culture is demonstrated with limited toxicity, cidofovir and its related prodrugs may be potential leads for covid- treatment. the results for the gtp analogues, car-tp, ent-tp and gan-tp are presented in fig. . in each case, extension to the first c position on the template occurs and further extension is blocked in the presence of atp, utp and ctp. in more detail, for car-tp (fig. a) , the major peak observed indicates extension by utp, atp and ctp followed by complete termination with a car-tp ( da observed, da expected). in addition, partial extension peaks were seen indicating a single utp incorporation ( da observed, da expected) and extension up to but not including the car-tp ( da observed, da expected). for ent-tp (fig. b) , a peak was observed indicating extension by utp, atp and ctp followed by complete termination by a single ent-tp ( da observed, da expected). additional peaks represent a single utp extension ( da observed, da expected) and a major peak indicating extension up to but not including the ent-tp ( da observed, da expected), suggesting that ent-tp is less efficiently incorporated than car-tp. and for gan-tp (fig. c) , a major peak observed indicated extension by utp, atp and ctp followed by complete termination with gan-tp ( da observed, da expected). a small peak representing extension up to but not including gan-tp ( da observed, da expected) was also seen. similar results were obtained for car-tp and gan-tp using the sars-cov polymerase (fig. s ). both car-tp and ent-tp are carbocyclic nucleotides. car-tp lacks the '-and '-oh groups, while ent-tp lacks the '-oh group. gan-tp is an acyclic nucleotide having an oh group but lacking a ribose ring. all three thus may resist the viral exonuclease activity. these results also indicate that car-tp and gan-tp are better terminators than ent-tp. fig. s shows a side-by-side comparison of the results with '-ome-utp and '-ome-utp using the sars-cov polymerase. the results for '-ome-utp are practically identical to those with sars-cov- in fig. a , indicating that '-ome-utp exhibits significant polymerase reaction termination. the '-ome-utp results are consistent with its being an obligate terminator, but with lower incorporation efficiency, represented by a small single-incorporation peak ( da observed, da expected). in fig. s , the results are shown for incorporation of '-f-dutp by sars-cov rdrp. '-f-dutp was incorporated very efficiently, but also was incorporated opposite the us in the template strand. this apparent mismatch incorporation may result from the high concentration of nucleotide analogues used and the relatively low fidelity of sars-cov rdrp. the results for desthiobiotin- -utp are presented in fig. s . desthiobiotin- -utp incorporation complementary to each a in the template was observed, just like a utp. thus, this nucleotide is incorporated and does not terminate the polymerase reaction. these results indicate that modification on the base of the utp does not affect its incorporation by sars-cov- rdrp. fig. s presents the results of an experiment where both '-ome-utp and dutp were added together at the same concentration. the major peak occurred at da ( da expected) representing incorporation by both dutp and '-ome-utp in adjacent positions. partial extension peaks of a single '-ome-utp ( da observed, da expected) and two dutps ( da observed, da expected) were found. the incorporation of a dutp, a '-ome-utp, both of which lack a '-oh group, or their combination would enable them to potentially resist the nsp '- ' exonuclease activity. fig. s shows three mass spectra of the polymerase reaction products using equimolar combinations of nucleotide analogues, (a) biotin-dutp, dutp and utp, (b) '-f-dutp, '-ome-utp and dutp and (c) '-nh -dutp, '-ome-utp and dutp, to determine their relative incorporation efficiencies. based on the results shown in fig. s a , biotin-dutp and dutp have lower incorporation efficiency than the natural utp for sars-cov- rdrp, since peaks are only observed for utp extension, either one utp ( da observed, da expected) or two utps ( da observed, da expected). in fig. s b , it is seen that '-f-dutp is incorporated far better than '-ome-utp and dutp, with the only evident peaks in the spectrum at da ( da expected) for extension by one '-f-dutp and at da ( da expected) for extension by two '-f-dutps. finally, as shown in fig. s c , '-nh -dutp is more efficiently incorporated than '-ome-utp and dutp as revealed by the presence of peaks only at da ( da expected) for extension by one '-nh -dutp and at da ( da expected) for extension by two '-nh -dutps. thus, '-f-dutp and '-nh -dutp behave like utp and do not terminate the polymerase reaction. neither '-f-dutp nor '-nh -dutp have a free '-oh group. it remains to be seen whether the rnas produced by these two nucleotide analogues will resist exonuclease activity. in summary, these results demonstrate that the library of nucleotide analogues we tested could be incorporated by the rdrps of sars-cov- and sars-cov. of the tested, exhibited complete termination of the polymerase reaction ( '-ome-utp, car-tp, gan-tp, sta-tp, ent-tp, biotin-dutp), showed incomplete or delayed termination (cid-dp, '-ome-utp), and did not terminate the polymerase reaction ( '-f-dutp, '-nh -dutp and desthiobiotin- -utp) using the rdrp of sars-cov and/or sars-cov- . their prodrug versions (figs. and s ) are available or can be readily synthesized using the protide approach ). the protide approach was used very successfully to develop sofosbuvir and remdesevir for treatment of hcv and covid- , respectively. it may be advantageous to use protide prodrug forms containing a phosphate masked by a hydrophobic phosphoramidate group for the five drugs whose structures are shown in fig. , because such prodrugs can be delivered into cells and converted to the triphosphate more rapidly, and potentially improve the bioavailability and potency of these molecules. the five drugs (ganciclovir/valganciclovir, cidofovir, abacavir, stavudine and entecavir (fig. ) ) are fda-approved medications for treatment of other viral infections and their toxicity profile is well established, while brincidofovir is an experimental oral antiviral drug. thus, our results provide a molecular basis for further evaluation of these prodrugs in sars-cov- virus inhibition and animal models to test their efficacy for the development of potential covid- therapeutics. this research is supported by columbia university, a grant from the jack ma foundation, a generous gift from the columbia engineering member of the board of visitors dr. bing zhao, and fast grants to j.j. and a national institute of allergy and infectious disease grant ai to r.n.k. structures of viral nucleoside/nucleotide inhibitors, example prodrugs and active triphosphate forms. the compounds ganciclovir, abacavir, cidofovir, stavudine and entecavir (left), example prodrug forms (middle) and their active triphosphate forms (right). fig. . incorporation of '-ome-utp, sta-tp and biotin-dutp by sars-cov- rdrp to terminate the polymerase reaction. the sequences of the primer and template used for this extension reaction, which are at the ' end of the sars-cov- genome, are shown at the top of the figure. polymerase extension reactions were performed by incubating '-ome-utp (a), sta-tp (b) and biotin-dutp (c) with preassembled sars-cov- polymerase (nsp , nsp and nsp ), the indicated rna template and primer and the appropriate reaction buffer, followed by detection of reaction products by maldi-tof ms. the detailed procedure is shown in the materials and methods section. the accuracy for m/z determination is ± da. fig. . incorporation of cid-dp by sars-cov- rdrp to achieve delayed termination of the polymerase reaction. the sequences of the primer and template used for this extension reaction are shown at the top of the figure. the polymerase extension reaction was performed by incubating cid-dp, utp and atp with pre-assembled sars-cov- polymerase (nsp , nsp and nsp ), the indicated rna template and primer and the appropriate reaction buffer, followed by detection of reaction products by maldi-tof ms. the accuracy for m/z determination is ± da. , and gan-tp, utp, atp and ctp (c) with pre-assembled sars-cov- polymerase (nsp , nsp and nsp ), the indicated rna template and primer and the appropriate reaction buffer, followed by detection of reaction products by maldi-tof ms. the accuracy for m/z determination is ± da. small-molecule antiviral β-d-n -hydroxycytidine inhibits a proofreadingintact coronavirus with a high genetic barrier to resistance coexpression of guanylate kinase with thymidine kinase enhances prodrug cell killing in vitro and suppresses vascular smooth muscle cell proliferation in vivo the protide prodrug technology: where next pharmacological considerations for tenofovir and emtricitabine to prevent hiv infection . '-fluoro and ,-amino- '-deoxynucleoside '-triphosphates as substrates for t rna polymerase rna '-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp /nsp exoribonuclease complex nucleotide analogues as inhibitors of sars-cov- polymerase clinical pharmacokinetics of the antiviral nucleotide analogues cidofovir and adefovir hepatitis c virus: life cycle in cells, infection and host response, and analysis of molecular markers influencing the outcome of infection and response to therapy cidofovir in the treatment of poxvirus infections approved antiviral drugs over the past years infidelity of sars-cov nsp -exonuclease mutant virus replication is revealed by complete genome sequencing ribavirin, remdesivir, sofosbuvir, galidesivir, and tenofovir against sars-cov- rna dependent rna polymerase (rdrp): a molecular docking study inhibitors of the hepatitis c virus polymerase; mode of action and resistance unique intracellular activation of the potent anti-human immunodeficiency virus agent u structural and molecular basis of mismatch correction and ribavirin excision from coronavirus rna structure of the rna-dependent rna polymerase from covid- virus the antiviral compound remdesivir potently inhibits rna-dependent rna polymerase from middle east respiratory syndrome coronavirus remdesivir is a direct-acting antiviral that inhibits rna-dependent rna polymerase from severe acute respiratory syndrome coronavirus with high potency cellular pharmacology of ', '-dideoxy- ', '-didehydrothymidine, a nucleoside analog active against human immunodeficiency virus selective action of ', '-didehydro- ', '-dideoxythymidine triphosphate on human immunodeficiency virus reverse transcriptase and human dna polymerases triphosphates of the two components in descovy and truvada are inhibitors of the sars-cov- polymerase four-color dna sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators nucleotide analogues as inhibitors of sars-cov polymerase structure of the sars-cov nsp polymerase bound to nsp and nsp co-factors development of cmx for the treatment of poxvirus infections enzymatic recognition of '-modified ribonucleoside '-triphosphates: towards the evolution of versatile aptamers structural basis and functional analysis of the sars coronavirus nsp -nsp complex mechanism of inhibition of vaccinia virus dna polymerase by cidofovir diphosphate entecavir for the treatment of chronic hepatitis b virus infection antiviral activity and mechanism of action of ganciclovir entecavir for treatment of hepatitis b virus displays no in vitro mitochondrial toxicity or dna polymerase gamma inhibition inhibitors of viral nucleic acid polymerases. pyrophosphate analogues discovery of an rna virus '--> ' exoribonuclease that is critically involved in coronavirus rna synthesis rational design of polymerase inhibitors as antiviral drugs mechanistic studies to understand the progressive development of resistance in human immunodeficiency virus type reverse transcriptase to abacavir chronic hepatitis b: current and future treatment options structural and functional basis of the fidelity of nucleotide selection by flavivirus rna-dependent rna polymerases remdesivir and sars-cov- : structural requirements at both nsp rdrp and nsp exonuclease active-sites an orally bioavailable broad-spectrum antiviral inhibits sars-cov- in human airway epithelial cell cultures and multiple coronaviruses in mice one severe acute respiratory syndrome coronavirus protein complex integrates processive rna polymerase and exonuclease activities delayed chain termination protects the anti-hepatitis b virus drug entecavir from excision by hiv- reverse transcriptase common and unique features of viral rna-dependent polymerases the efficacy and pharmacokinetics of brincidofovir for the treatment of lethal rabbitpox virus infection: a model of smallpox disease a novel coronavirus from patients with pneumonia in china coronaviruses -drug discovery and therapeutic options small-molecule antiviral β-d-n -hydroxycytidine inhibits a proofreadingintact coronavirus with a high genetic barrier to resistance coexpression of guanylate kinase with thymidine kinase enhances prodrug cell killing in vitro and suppresses vascular smooth muscle cell proliferation in vivo the protide prodrug technology: where next pharmacological considerations for tenofovir and emtricitabine to prevent hiv infection . '-fluoro and '-amino- '-deoxynucleoside '-triphosphates as substrates for t rna polymerase rna '-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp /nsp exoribonuclease complex nucleotide analogues as inhibitors of sars-cov- polymerase clinical pharmacokinetics of the antiviral nucleotide analogues cidofovir and adefovir hepatitis c virus: life cycle in cells, infection and host response, and analysis of molecular markers influencing the outcome of infection and response to therapy cidofovir in the treatment of poxvirus infections approved antiviral drugs over the past years infidelity of sars-cov nsp -exonuclease mutant virus replication is revealed by complete genome sequencing ribavirin, remdesivir, sofosbuvir, galidesivir, and tenofovir against sars-cov- rna dependent rna polymerase (rdrp): a molecular docking study inhibitors of the hepatitis c virus polymerase; mode of action and resistance unique intracellular activation of the potent anti-human immunodeficiency virus agent u structural and molecular basis of mismatch correction and ribavirin excision from coronavirus rna structure of the rna-dependent rna polymerase from covid- virus the antiviral compound remdesivir potently inhibits rna-dependent rna polymerase from middle east respiratory syndrome coronavirus remdesivir is a direct-acting antiviral that inhibits rna-dependent rna polymerase from severe acute respiratory syndrome coronavirus with high potency cellular pharmacology of ', '-dideoxy- ', '-didehydrothymidine, a nucleoside analog active against human immunodeficiency virus selective action of ', '-didehydro- ', '-dideoxythymidine triphosphate on human immunodeficiency virus reverse transcriptase and human dna polymerases triphosphates of the two components in descovy and truvada are inhibitors of the sars-cov- polymerase four-color dna sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators nucleotide analogues as inhibitors of sars-cov polymerase structure of the sars-cov nsp polymerase bound to nsp and nsp co-factors development of cmx for the treatment of poxvirus infections enzymatic recognition of '-modified ribonucleoside '-triphosphates: towards the evolution of versatile aptamers structural basis and functional analysis of the sars coronavirus nsp -nsp complex mechanism of inhibition of vaccinia virus dna polymerase by cidofovir diphosphate entecavir for the treatment of chronic hepatitis b virus infection antiviral activity and mechanism of action of ganciclovir entecavir for treatment of hepatitis b virus displays no in vitro mitochondrial toxicity or dna polymerase gamma inhibition inhibitors of viral nucleic acid polymerases. pyrophosphate analogues discovery of an rna virus '→ ' exoribonuclease that is critically involved in coronavirus rna synthesis rational design of polymerase inhibitors as antiviral drugs mechanistic studies to understand the progressive development of resistance in human immunodeficiency virus type reverse transcriptase to abacavir chronic hepatitis b: current and future treatment options structural and functional basis of the fidelity of nucleotide selection by flavivirus rna-dependent rna polymerases remdesivir and sars-cov- : structural requirements at both nsp rdrp and nsp exonuclease active-sites an orally bioavailable broad-spectrum antiviral inhibits sars-cov- in human airway epithelial cell cultures and multiple coronaviruses in mice one severe acute respiratory syndrome coronavirus protein complex integrates processive rna polymerase and exonuclease activities delayed chain termination protects the anti-hepatitis b virus drug entecavir from excision by hiv- reverse transcriptase common and unique features of viral rna-dependent polymerases the efficacy and pharmacokinetics of brincidofovir for the treatment of lethal rabbitpox virus infection: a model of smallpox disease a novel coronavirus from patients with pneumonia in china coronaviruses -drug discovery and therapeutic options j.j. and r.n.k. conceived and directed the project; the approaches and assays were designed and conducted by j.j., x.l., s.k., s.j., j.j.r., m.c. and c.t. and sars-cov/sars-cov- polymerases (nsp ) and associated proteins (nsp and ) were cloned and purified by t.k.a. and r.n.k. data were analyzed by all authors. all authors wrote and reviewed the manuscript. the authors declare no competing interests. key: cord- -fxlbx w authors: shephard, adrian; zybeshari, stela title: virucidal action of sore throat lozenges against respiratory viruses parainfluenza type and cytomegalovirus date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: fxlbx w most respiratory tract infections are self-limiting and caused by viruses, and do not warrant antibiotic treatment. despite this, patients with respiratory tract infections often receive antibiotics, fuelling the rise of antibiotic resistance. therefore, there is a need to encourage patients to try alternative non-antibiotic therapies, which ideally treat the symptoms and the cause. lozenges containing amylmetacresol and , -dichlorobenzyl alcohol (amc/dcba lozenges) as well as lozenges containing hexylresorcinol have been shown to provide effective symptomatic relief for sore throat. in this study, we investigated whether these lozenges also have virucidal effects in vitro against two viruses associated with respiratory tract infections, parainfluenza virus type and cytomegalovirus. both viruses were incubated with amc/dcba lozenge, placebo lozenge or the active ingredients (amc/dcba) as free substances, and parainfluenza virus type was incubated with hexylresorcinol lozenge, placebo lozenge or hexylresorcinol as a free substance. virucidal effects were observed with the active lozenges and the active ingredients as free substances against both parainfluenza virus type and cytomegalovirus. mean reductions in viral titre were significantly greater compared with placebo lozenge and peak effects were observed for the shortest incubation time, min. these findings suggest that amc/dcba lozenge and hexylresorcinol lozenge have the potential to have local antiviral effects in patients with sore throat due to viral respiratory tract infections. use of such over-the-counter treatments for self-limiting respiratory tract infections may satisfy patients’ desire for an anti-infective medication and reduce the demand for antibiotics. respiratory tract infections (rtis) are the most common illnesses to affect humans (denny, ) . typical symptoms include sore throat, rhinitis, cough and fever (dasaraju and liu, ; eccles, ) . most rtis are caused by a viral infection (denny, ) , for example, the symptom of sore throat is reported to be caused by viruses in - % of cases in adults, % in children under years and % in children aged - years (worrall, ) . viruses associ-ated with rtis include orthomyxoviruses (influenza), paramyxoviruses (parainfluenza viruses [piv] , respiratory syncytial virus [rsv] ), coronaviruses, picornaviruses, adenoviruses and herpes viruses (cytomegalovirus [cmv] , epstein-barr virus [ebv] ) (collier and oxford, ) . although rtis are generally self-limiting and have a predominantly viral cause, patients visiting healthcare professionals often expect antibiotics (or are perceived to expect antibiotics) and receive inappropriate antibiotic treatment (van der velden et al., ) . in europe, one-quarter of those who took antibiotics in the last year did so to provide symptom relief, and there remains much consumer confusion about the effects of antibiotics with % of europeans falsely believing that antibiotics kill viruses and % believing they were effective against colds and flu (european commission, ) . physicians respond to sore throat patients' demands (actual and perceived) by prescribing an antibiotic in approximately % of cases (barnett and linder, ; gulliford et al., ) but only about - % of sore throats in adults are caused by a bacterial infection (shulman et al., this unnecessary use of antibiotics drives the development of antibiotic resistance (goossens et al., ) , which is an increasingly serious threat to global public health (who, ) . therefore, there is a need to encourage patients with rtis to try alternative treatments, while antibiotics should be reserved for patients with a serious illness or those at increased risk of complications ). an ideal treatment would provide the symptomatic relief that patients seek as well as treat the cause. locally delivered formats such as lozenges and sprays are useful as they enable active ingredients to reach the site of infection directly; the localized delivery means that side effects are lower compared with systemically acting treatments (farrer, ) . lozenges containing the antiseptics and local anaesthetics amylmetacresol (amc) and , dichlorobenzyl alcohol (dcba) or hexylresorcinol have been developed to treat the symptoms of sore throat (buchholz et al., ; foadi et al., ; mcnally et al., mcnally et al., , wade et al., ) . these lozenges have demonstrated statistically significant reductions in sore throat symptoms in placebo-controlled clinical trials (mcnally et al., (mcnally et al., , wade et al., ) . amc/dcba lozenges have demonstrated antibacterial effects in vivo (richards et al., ) and in vitro (richards and xing, ) . amc/dcba lozenges have also been shown to have some virucidal effects in vitro on three enveloped viruses -rsv, influenza a virus and severe acute respiratory syndrome coronavirus (sars-cov) (oxford et al., ) . this study investigated the in vitro virucidal activity of amc/ dcba alone and in a lozenge on two other enveloped viruses that can cause sore throat and respiratory illness -piv type (piv ) and cmv (bisno, ) . it also assessed the in vitro virucidal activity of hexylresorcinol alone and in a lozenge on piv . to our knowledge, this is the first study to examine the virucidal effects of throat lozenges on these viruses. the american society for testing and materials (astm) international standard method e - for ''standard test method to assess the activity of microbicides against viruses in suspension" was followed. the challenge viruses, piv (strain c atcc vr- ) and cmv (strain atcc- - actt vr- ) were obtained from the american type culture collection (atcc). piv was propagated in vero cells (atcc ccl- ) and cmv in mrc- cells (atcc ccl- ). the test substances were honey and lemon amc/dcba lozenge, cherry menthol hexylresorcinol lozenge, placebo lozenge, amc/dcba as free active substances and hexylresorcinol as a free active substance (table ) . a positive control (sodium hypochlorite) and negative control (dilution medium) were also tested. a . ml aliquot of each test substance (table ) was transferred into a ml conical tube. then . ml of the virus stock (titre of - tcid /ml for piv and . - . tcid /ml for cmv) was added and mixed immediately (by vortexing) at ambient room temperature ( °c). different contact times ( , or min) were used for each test (table ) . upon completion of the contact time, the reaction mixture was immediately mixed (by vortexing) with an equal volume of neutralizer (rpmi medium + % newborn calf serum + % hepes + % nahco for the amc/dcba and piv experiments, mem for the hexylresorcinol and piv experiments and mem + % fetal bovine serum + % hepes + % nahco for the amc/dcba and cmv experiments). the quenched sample was serially diluted -fold with dilution medium. serial dilutions were inoculated onto host cells in a -well plate ( ml inoculum per well, n = per dilution). the inoculated plates were incubated at ± °c in ± % co for days for the amc/dcba and piv experiments, - days for the hexylresorcinol and piv experiments and days for the amc/ dcba and cmv experiments. after the incubation period, the titre of infectious virus was determined by tcid assay using the spearman-karber formula. the results from the negative control was used as the input viral load and compared with the test substances to evaluate the viral reduction by each test substance. each experiment (for each test substance, each control and each contact time) was run in triplicate. the mean reductions in viral titre were compared between the test substances and the placebo lozenge using the two-sided student t-test. additional control tests were also conducted to assess the effectiveness of the neutralizer and cytotoxicity of the test substances. a . ml aliquot of the test substance (each lozenge and each combination of free active substances) was mixed with . ml of dilution medium, incubated at ambient room temperature ( °c) for each contact time, and then an equal volume of neutralizer was added. following serial dilution of the reaction mixture in dilution medium, ll of a low titred virus stock (containing no more than approximately units of virus) was added to . ml of each dilution and incubated at ambient room temperature ( °c) for at least min. these were then inoculated onto the host cells which were assessed for the presence of infectious virus at the end of the incubation period. to evaluate cytotoxicity, the amc, amylmetacresol; cmv, cytomegalovirus; dcba, , -dichlorobenzyl alcohol; mem, minimum essential medium; piv , parainfluenza virus type . * . g sodium bicarbonate, . g sodium chloride, and . g potassium carbonate was added to $ ml of sterile deionized water. g of bovine serum albumin was added to the solution and the ph adjusted to . ± . . sterile deionized water was added to reach a total volume of ml. condition of the host cells was recorded at the end of the incubation period. the viability of the host cells and sterility of the cell culture medium were tested by inoculating control cells with medium during the incubation period of the study. the titre of the virus stock was confirmed by inoculating virus stock onto the host cells. amc/dcba lozenge exhibited a . - . log (mean . log ) reduction in viral titre at min, . - . log (mean . log ) reduction at min, and . - . log (mean . log ) reduction at min (fig. ) . placebo lozenge exhibited - . log (mean . log ) reduction at min and . - . log (mean . log ) reduction at min (fig. ) . amc/dcba as free active substances exhibited . - . log (mean . log ) reduction at min and . - . log (mean . log ) reduction at min (fig. ) . the positive control exhibited a complete inactivation of the virus within min (fig. ) . the negative control did not exhibit any negative effect on the titre of virus or cell viability (data not shown). neither the amc/ dcba lozenge nor amc/dcba as free active substances exhibited any direct cell cytotoxicity within the assay (data not shown). hexylresorcinol lozenge exhibited a . - . log (mean . log ) reduction in viral titre at min, p . log reduction at and min (fig. ) . placebo lozenge exhibited . - . log (mean . log ) reduction at min and . - . log (mean . log ) reduction at min (fig. ) . hexylresorcinol as a free active substance exhibited p . log reduction at and min (fig. ) . the effects of the positive control were similar to that of hexylresorcinol lozenge and hexylresorcinol as a free active substance (fig. ) . the negative control did not exhibit any negative effect on the titre of virus or cell viability (data not shown). neither the hexylresorcinol lozenge nor hexylresorcinol as a free active substance exhibited any direct cell cytotoxicity within the assay (data not shown). both amc/dcba lozenge and amc/dcba as free active substances, as well as the positive control, exhibited a p . log reduction in viral titre at all time-points assessed (fig. ) . placebo lozenge exhibited . - . log (mean . log ) reduction at min and . - . log (mean . log ) reduction at min (fig. ) . the negative control did not exhibit any negative effect on the titre of virus or cell viability (data not shown). neither the amc/ dcba lozenge nor amc/dcba as free active substances exhibited any direct cell cytotoxicity within the assay (data not shown). this study helps to further elucidate the action of long-standing active ingredients used for the relief of sore throat by showing that amc/dcba and hexylresorcinol exhibit virucidal activity, hexylresorcinol against piv and amc/dcba against piv and cmv in vitro. there are four subtypes of piv (piv , piv , piv and piv ), all of which can cause rtis, although less is known about piv (henrickson, ) . the prevalence and seasonality of piv is reported to vary. in a study in china from to , . % of children and adults with rti were positive for piv ( . % were positive for piv ), with seasonal peaks of piv and piv occurring from april to july and september to november (liu et al., ) . in a study in brazil from to , . % of children and adults with influenza-like illness were positive for piv , with the prevalence peaking in the second half of each year (freitas, ) . in contrast, in the usa from to , seasonal peaks of piv occurred in the spring, sometimes with a second smaller peak during the autumn (fry et al., ) . piv can cause the symptom of sore throat (bisno, ) ; in the study in china, % of patients with parainfluenza virus infection had the symptom of pharyngeal discomfort (liu et al., ) . cmv, a member of the herpes virus family, infects and establishes latency in approximately % of the global population (emery, ) . although primary cmv infection in healthy adults is usually asymptomatic or associated with a mild mononucleosislike syndrome, cmv can cause severe, life-threatening disease and mortality in neonates and in immunocompromised adults (lancini et al., ) . cmv can also cause sore throat (bisno, ) . in a study of hospitalized non-immunocompromised adults with confirmed or presumed cmv, % had pharyngitis (bonnet et al., ) . in this study, both the active lozenges and the free active substances (amc/dcba and hexylresorcinol) exhibited virucidal activity within min, with minimal further virucidal effects beyond min. a greater virucidal effect against piv was observed with amc/dcba as free active substances than with amc/dcba lozenge, whereas hexylresorcinol as a free active substance had a similar effect to that of hexylresorcinol lozenge. possible reasons include the binding of amc and/or dcba by an excipient in the amc/dcba lozenge, and/or interference or possible protective effect from an excipient in the lozenge. in sore throat treatments, hexylresorcinol is routinely used at a higher dose than either amc or dcba, and this may also be a factor. this difference in virucidal effect between amc/dcba as free active substances and amc/dcba lozenge was seen with piv but not with cmv, suggesting that the mechanism of virucidal action of amc/dcba lozenge is different for the two viruses. also, the placebo lozenges for both the amc/dcba and hexylresorcinol experiments appeared to exert some virucidal activity, potentially due to the presence of tartaric acid in the placebo lozenge (for the amc/dcba experiments), osmotic pressure changes or stickiness induced by the sugar in the placebo lozenge (for both the amc/ dcba and hexylresorcinol experiments) (oxford et al., ) or due to the action of other excipients (e.g., menthol in the placebo lozenge for the hexylresorcinol experiments). this study adds to the previous observations of in vitro virucidal activity of amc/dcba lozenge against rsv, influenza a virus and sars-cov by oxford and colleagues ( ) . these data suggest that amc/dcba and hexylresorcinol lozenges have the potential to inactivate free virus during the infection cycle, although the limitation of both studies is that the observed in vitro effects may not occur in vivo. phenols (amc and hexylresorcinol) and alcohols (dcba) are thought to disrupt lipid membranes, and alcohols can also cause rapid denaturation of proteins (mcdonnell and russell, ) . the virucidal activity of these substances is likely to be due to their effects on viral lipid membranes or protein-lipid interaction (oxford et al., ) . consistent with this theory, amc/dcba lozenge has thus far been shown to exert virucidal activity against enveloped viruses but not as yet against non-enveloped viruses (oxford et al., ) . the findings of this study as well as previous research (oxford et al., ) suggest that amc/dcba lozenge and hexylresorcinol lozenge have the potential to have local antiviral effects in patients with sore throat due to viral rtis. in addition, both lozenges have demonstrated efficacy against sore throat symptoms in clinical trials (mcnally et al., (mcnally et al., , wade et al., ) . taken together, these data support the use of these lozenges as a first-line treatment for sore throat caused by viral rtis. further investigation into the mechanisms of action and the clinical effects of amc/dcba and hexylresorcinol for the control of rtis is warranted. use of products containing such active ingredients, which not only relieve symptoms but also help treat the infectious cause of rtis, should help to curb patient demand for antibiotics by satisfying their need for treatment of infection and hence reduce the burden of antibiotic resistance. this study was funded by reckitt benckiser healthcare ltd. the authors, who are employees of reckitt benckiser healthcare ltd, were involved in the study design, analysis and interpretation of data, and took the decision to submit this article for publication. medical writing assistance for this article was funded by reckitt benckiser healthcare ltd. adrian shephard and stela zybeshari are employees of reckitt benckiser healthcare ltd. antibiotic prescribing to adults with sore throat in the united states acute pharyngitis clinical and laboratory findings of cytomegalovirus infection in hospitalized nonimmunocompromised adults topical antiseptics for the treatment of sore throat block voltage-gated neuronal sodium channels in a local anaesthetic-like manner human virology infections of the respiratory system the clinical impact of human respiratory virus infections mechanisms of symptoms of the common cold and influenza cytomegalovirus: recent progress in understanding pathogenesis and control a framework for the non-antibiotic management of upper respiratory tract infections: towards a global change in antibiotic resistance sprays and lozenges for sore throats a combination of topical antiseptics for the treatment of sore throat blocks voltage-gated neuronal sodium channels sentinel surveillance of influenza and other respiratory viruses, brazil seasonal trends of human parainfluenza viral infections: united states outpatient antibiotic use in europe and association with resistance: a crossnational database study continued high rates of antibiotic prescribing to adults with respiratory tract infection: survey of uk general practices parainfluenza viruses cytomegalovirus disease in immunocompetent adults epidemiology and clinical presentation of the four human parainfluenza virus types antiseptics and disinfectants: activity, action, and resistance rapid relief of acute sore throat with amc/dcba throat lozenges: randomised controlled trial randomised, double-blind, placebocontrolled study of a single dose of an amylmetacresol/ , -dichlorobenzyl alcohol plus lidocaine lozenge or a hexylresorcinol lozenge for the treatment of acute sore throat due to upper respiratory tract infection a throat lozenge containing amyl meta cresol and dichlorobenzyl alcohol has a direct virucidal effect on respiratory syncytial virus, influenza a and sars-cov in vivo investigations of the antibacterial activity of lozenges and mouthwashes on the aerobic bacterial flora of the mouth and throat in vitro evaluation of the antimicrobial activities of selected lozenges clinical practice guideline for the diagnosis and management of group a streptococcal pharyngitis: update by the infectious diseases society of america prescriber and patient responsibilities in treatment of acute respiratory tract infections -essential for conservation of antibiotics a multicentre, randomised, double-blind, single-dose study assessing the efficacy of amc/dcba warm lozenge or amc/dcba cool lozenge in the relief of acute sore throat antimicrobial resistance global report on surveillance acute sore throat this study was conducted by microbiotest division of microbac laboratories, inc. (sterling, va, usa). statistical analyses were conducted by bartosz jenner of reckitt benckiser (hull, east yorkshire, uk). medical writing assistance was provided by papia das of elements communications ltd (westerham, kent, uk). enveloped viruses: viruses that have an outer lipid membrane that contains viral proteins; non-enveloped viruses lack this membrane serial dilution: the repeated dilution of a solution where the dilution factor is usually constant, for example, a -fold serial dilution involves diluting a solution : so it is one-tenth the concentration of the original solution, then diluting : again so that it is one-hundredth of the original solution, and so on spearman-karber formula: a formula used to determine viral titre: m = x k + (d/ ) À d p p i , where m = log of the titre relative to the test volume; x k = log of the smallest dosage which induces infection in all cultures; d = log of the dilution factor; p i = the proportion of positive results at each dilution tcid : the amount of virus required to kill % of inoculated tissue culture cells key: cord- -jzmwy yi authors: lin, jung-chung; cherng, jaw-ming; hung, man-shan; baltina, lidia a.; baltina, lia; kondratenko, rimma title: inhibitory effects of some derivatives of glycyrrhizic acid against epstein-barr virus infection: structure–activity relationships date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: jzmwy yi glycyrrhizic acid ( β-gl or gl) is a herbal drug with a broad spectrum of antiviral activities and pharmacological effects and multiple sites of action. previously we showed that gl inhibits epstein-barr virus (ebv) infection in vitro by interfering with an early step of the ebv replication cycle (possibly attachment/penetration). here we tested the effects of gl derivatives against ebv infection by scoring the numbers of cell expressing viral antigens and quantifying ebv dna copy numbers in superinfected raji cells. the derivatives were made either by transformation of gl on carboxyl and hydroxyl groups or by conjugation of amino acid residues into the carbohydrate part. we identified seven compounds active against ebv and all showed dose-dependent inhibition as determined by both assays. among these active compounds, the introduction of amino acid residues into the gl carbohydrate part enhanced the antiviral activity in three of the seven active compounds. however, when glu(oh)-ome was substituted by glu(ome)-ome, its antiviral activity was completely abolished. introduction of potassium or ammonium salt to gl reduced the antiviral activity with no significant effect on cytotoxicity. the α-isomer ( α-gl) of β-gl was as potent as the β-form, but its sodium salt lost antiviral activity. the metabolic product of gl, β-glycyrrhetinic acid ( β-ga or ga), was . -fold more active against ebv than its parental compound gl but, concomitantly, exhibited increased cytotoxicity resulting in a decreased therapeutic index. glycyrrhizic acid ( ␤-gl or gl) is one of the bioactive compounds of licorice roots (glycyrrhiza radix) and is composed of one molecule of glycyrrhetinic acid ( ␤-ga or ga), which has a steroid-like structure, and two molecules of glucuronic acid. after oral administration or intravenous injection, gl is hydrolyzed by glucuronidase in intestinal bacteria to its active principle aglycone, ␤-ga, which is then absorbed into the blood (takeda et al., ) . gl and ga have been shown to possess several beneficial pharmacological activities (dirnagl et al., ; shibata, ; abe et al., ; armanini et al., ; cherng et al., ; cinatl et al., ; fiore et al., ) , which include an anti-ulcerative effect, anti-inflammatory activity (fujisawa et al., ) , interferon (ifn)-␥ induction, anti-hepatotoxic effect (chan et al., ; gumpricht et al., ; zheng and lou, ) , anti-tumor activity (hibasami et al., ) , and it is active against a range of viruses (lin, ; * corresponding author. tel.: + x ; fax: + . e-mail address: jclin@mail.tcu.edu.tw (j.-c. lin). cinatl et al., ; crance et al., ; briolant et al., ; cherng et al., ; hoever et al., ) . clinically, gl has been used to treat patients with chronic active hepatitis (van rossum et al., ; bean, ; coon and ernst, ) . in previous years, we have shown that many nucleoside analogs selectively inhibit replication of epstein-barr virus (ebv) in vitro (lin et al., (lin et al., , (lin et al., , (lin et al., , (lin et al., , (lin et al., , lin and machida, ; mar et al., ; for a review see gershburg and pagano, ; lin, ) . the molecular target of all nucleoside analogs is the virus-encoded dna polymerase. recently, we have reported that gl is active in a dose-dependent fashion against ebv replication in superinfected raji cells (lin, ) . our results also indicated that gl interferes with an early step of the ebv replication cycle (possibly adsorption and/or penetration) (lin, ) . thus, gl represents a new class of anti-ebv compounds with a mode of action different from that of the nucleoside analogs that inhibit the viral dna polymerase. in the present studies we extend our previous findings after a search for more potent compounds against ebv. we tested the antiviral activity of gl derivatives synthesized in the laboratory. here we report the identification of seven active compounds against ebv infection in vitro and elucidate their structure-activity relationships. glycyrrhizic acid (glycyrrhizin, ␤-gl or gl) and ␤glycyrrhetinic acid ( ␤-ga or ga) were purchased from sigma-aldrich chemie gmbh, germany. the synthesis of gl derivatives was reported previously (baltina, ; baltina et al., ; kondratenko et al., ) . l-aminoacids were used for the synthesis of gl glycopeptides. unless otherwise specified, all drugs were dissolved in phosphate-buffered saline, ph . . the chemical structures are shown in chart . a latently ebv-infected non-virus producing cell line (raji) was maintained at between × and × cells/ml in rpmi medium containing % fetal calf serum (fcs) supplemented with iu penicillin/ml and g streptomycin/ml, as described previously (lin et al., ) . a highly productive virus-producer cell line, p hr- (ls), derived by low-serum cloning (lin, ) , was maintained in the same culture medium as for raji cells, except that the serum concentration was reduced to %. viral stocks were prepared from cultures of p hr- (ls) that had been treated with -o-tetradecanoyl-phorbol- -acetate (tpa) at a concentration of ng/ml (lin, ) . briefly, the p hr- (ls) cells were induced with tpa for days without additional medium. the cells were removed by centrifugation at × g for min, virus was then pelleted from the cell-free supernatant fluids by centrifugation at , × g for min in a gs rotor (ivan sorvall, inc.). the centrifuged bottles were swabbed to remove residual medium chart . and the virus pellets were suspended in rpmi medium. the viral suspension was clarified to remove cellular debris by filtering through . -, . -, and . -m-pore-size filters and stored at − • c. as an assay system for drug effects we used raji cells superinfected with p hr- (ls) virus, which results in reactivation of the latent ebv infection and replication of virus. raji cells were first propagated in medium containing a sub-effective dose of compound ( m). for superinfection (lin, ) , raji cells growing exponentially were pelleted and resuspended in . ml of rpmi medium containing % fcs and appropriate concentrations of drug as specified. then units of ebv early antigen (ea)-inducing virus were added to the cells. one unit of virus is defined as the amount of virus capable of inducing % of infected raji cells to express ea. after h adsorption at • c in a co incubator, the cells were pelleted and suspended in ml of the medium and incubation continued for h. the levels of ebv early antigen (ea) and viral capsid antigen (vca) were monitored by indirect immunofluorescent assay (ifa) at h after infection with ebv-positive sera from npc patients. the specificity of sera for ebv ea/vca was confirmed by western blots as reported previously (lin and pagano, ) . the detailed protocol for ifa was described previously (lin, ) . briefly, superinfected raji cells were smeared on slides and fixed in methanol for min at room temperature. the fixed cells were first reacted with npc patients' serum, followed by fluorescein-conjugated goat anti-human igg (h + l) (santa cruz biotechnology, inc.). after counterstaining with , -diamidino- -phenylindole (dapi) ( . g/ml in pbs) for min in the dark, the slides were analyzed under the fluorescent microscope. the drug effects on ebv dna in superinfected raji cells were determined using a real-time quantitative pcr system toward the bamhi-w fragment region of the ebv genome as described (lo et al., ) . real-time quantitative pcr is based on the continuous optical monitoring of the progress of a fluorogenic pcr reaction. in this system, the amplification primers used in conventional pcr toward the bamhi-w were: w- f ( -cccaacactccaccacacc- ) and w- r ( -tcttaggagctgtccgaggg- ). the duallabeled fluorogenic hybridization probe was w- t[ -(fam)ca-cacactacacacacccacccgtctc(tamra)- ]. the fluorescent probes contained a -blocking phosphate group to prevent probe extension during pcr. the effect of gl derivatives on raji cell cytotoxicity was carried out by mtt ( -[ , -dimethylthiazol- -yl]- , -diphenyltetrazolium bromide; thiazolyl blue) cell-proliferative kit i (roche, mannheim, germany). raji cells were seeded in wells of a -well plates and treated with various concentrations of drug for days. then l of sterile mtt dye were added, and the cells were incubated for h at • c, followed by adding l of acidic isopropanol ( . m hcl in isopropanol). spectrometric absorbance at nm (for formazan dye) was measured with the absorbance at nm for reference. to determine the dose-dependent effect of gl derivatives, raji cells were superinfected with p hr- (ls) virus in the presence of various concentrations of compounds. the expression of ea and vca were monitored by ifa at h after superinfection. a representative result from compound (xv) is shown in fig. . without drug treatment, approximately % of infected cells became positive for viral antigens (fig. , panel a) . in the presence of drugs, a dose-dependent inhibition of the expression of viral antigens was observed (fig. , panels b, c, d, and e) . the concentration of drug required to inhibit ebv antigen expression by % (ec ) was determined from the plot of drug concentrations against % of viral antigen-positive cells, assuming no-drug control as . fig. a shows graphical quantitation for compound (xv). the ec determined from the graph was approximately m. as shown in table , seven gl derivatives were active against ebv infection with ec ranging from to m. all these active compounds exhibited a dose-dependent inhibition manner. the parental compound gl was used as a positive drug control. in addition, ga, the metabolite of gl was also tested in parallel with the derivatives. it is interesting to note that ga was the most effective compound to block ebv infection with an ec of m, which was . -fold lower than for gl ( m). however, ga was approximately -fold more toxic (cc = m) than gl (cc = m). the cytotoxicity of the gl derivatives was determined in -well microtiter plates by the mtt assay and the results are expressed as the concentration of the compound that decreased cell viability to % (cc ) of the no drug control (table ) . these compounds exhibited variable cytotoxicity, the cc ranging from to m. to more precisely determine the drug effects, ebv dna copy numbers were measured using a real-time quantitative pcr system that amplified a dna segment in the bamhi-w fragment region of the ebv genome (lo et al., ) . a dose-dependent inhibition of viral genome copy numbers by compound (xv) was observed (fig. b) . the ec obtained by this method is approximately m, in contrast to m determined by ifa (table ) . data obtained by pcr and ifa are shown side by side in table . it should be noted that the ec values obtained by real-time quantitative pcr is approximately - % lower than that obtained by ifa. however, the inhibitory profile of these compounds remains unchanged, indicating that ifa is as reliable as the quantitative pcr method for initial drug screening. evaluation of antiviral agents effective against ebv has been hampered by the lack of a permissive cell system for the replication of this virus. superinfection of raji cells with p hr- virus results in shut-down of host cell dna synthesis and stimulation of viral dna replication and lytic antigen synthesis and production of virus. we have previously shown that the percentage of ea-and vca-positive cells is directly proportional to the amount of ebv dna in the cells (lin, ) . furthermore, we have also demonstrated that gl blocks ebv replication at an early step of the viral replication cycle. however, gl has no effects on ebv dna in the spontaneously virus-producing cell line p hr- and the latently infected nonvirus-producing raji cells (lin, ) . the lack of effect of gl in p hr- and raji cells, which are already infected, is consistent with our proposed mechanism of gl action, i.e., inhibition of virus attachment/penetration. thus, superinfection of raji cells represents a useful system for evaluation of compounds that block early steps in the ebv replication cycle. our results indicate that gl and several of its derivatives are highly selective in their antiviral action in that they inhibit ebv infection at concentrations far below the cytotoxic concentration. as demonstrated previously (lin, ) , pretreatment of cells with sub-effective dose of gl markedly reduced cytotoxic effects while decreasing the amount of drug (approximately -fold reduction) needed to reach the same effective level. the marked reduction of ec obtained by pretreatment of cells with sub-effective doses of the compounds has clinical implication with regard to prophylactic and therapeutic effects. among seven active compounds (table ) , three of them contain amino acid residues in the gl carbohydrate part. the ala-ome containing glycopeptide (xii), the glu(oh)-ome containing glycopeptide (xiii), and leu containing glycopeptides (xv) were active against ebv. however, compounds (xii) (ec = m) and (xv) (ec = m) were approximately -fold more active against ebv than compound (xiii) (ec = m). the cc values of glycopeptides (xii), (xiii), and (xv) were, respectively, , , and m, resulting in a therapeutic index of , , and . in contrast, two other glycopeptide containing compounds such as (i) and (ix) did not exhibit activity against ebv at concentrations up to mm. interestingly, when glu(oh)-ome was substituted by glu(ome)-ome as in compound (xiv), its antiviral activity was completely abolished. introduction of a hydroxyl or methoxyl group at the r position of the aglycon moiety of gl, such as in compounds (i), (ii), and (iii) did not show any antiviral activity. compounds (ix) and (xii) both contain a hydroxyl group at the r position of ga molecule, but with different substitution at the r position of the carbohydrate part of gl. compound (xii) with an ala-ome group at the r position showed good antiviral activity, whereas substitution of compound (ix) with an ala-obu group at the same position abolished the antiviral activity. interestingly, other substitutions at the r position of ga, such as lys(z)-oh in compound (v) and l-glu(ome)-ome in compound (xiv), did not confer any antiviral activity. thus, it appears that the modification of the carbohydrate moiety of gl rather than the r position of aglycon may affect the antiviral activity. these findings may provide a lead for further development of more active antiviral compounds. the two commercially available compounds gl and ga were tested in parallel with semisynthetic gl derivatives. unexpectedly, ga, the metabolic product of gl, was . -fold more active (ec = m) against ebv than its parental compound (ec = m), but was -fold more toxic (cc = m) than gl (cc = m), resulting in a therapeutic index of and , respectively. introduction of potassium [compound (vi)] or ammonium salt [compound (vii)] to gl reduced the antiviral activity with a significant effect on cytotoxicity. the ␣-isomer [ ␣-gl, compound (viii)] derived from ␤-gl was as potent as the ␤-form, with ec of and cc of , resulting in a therapeutic index of . in contrast, the sodium salt of ␣-isomer of ␤-gl [compound (xi)] lost antiviral activity as compared to its ␤-form [compound (x)]. previous study indicated that the sugar moiety of gl is essential for anti-sars-cov activity . the present study clearly demonstrated that compounds (vi), (vii), (viii), (x), (xii), (xiii), and (xv) are active inhibitors of ebv infection and all contain a sugar moiety. in summary, among the newly identified analogs four showed better or equal activity than the parent gl. recently we have demonstrated that gl interferes with an early step of ebv replication cycle (possibly attachment/penetration) (lin, ) . however, other possible mechanism of gl may also involve inhibition of ebv replication. both gl and ga have been shown to elicit a dose-dependent increase in no production and the level of inos mrna in macrophages (jeong and kim, ) . as a host defense molecule, no has an antimicrobial activity against a variety of pathogens including viruses. previous studies indicated that no is involved in maintaining ebv latency through inhibition of ebv reactivation (mannick et al., ; gao et al., ) . the molecular mechanism underlying the gl inhibition of viral attachment is not well understood. a complement protein called c d is a breakdown product of complement c . receptors for c d, called type complement receptors (cr or cd ), are expressed on mature b lymphocytes. when b lymphocytes recognize an antigen by their antigen receptors and simultaneously recognize c d bound to the antigen by the complement receptor, the b cells are activated. the major ebv outer envelope glycoprotein gp / is the cd ligand (nemerow et al., ; tanner et al., ) . cd is the only b-lymphocyte surface protein that binds to gp / (nemerow et al., ) , which mediates virus attachment to the ebv/c dg receptor (cr ) of human b lymphocytes. a recent study indicated that complement c is a gl-binding protein (kawakami et al., ) . synthetic peptides corresponding to two regions in gp / , which have a similar amino acid sequence with the complement c dg protein, were used to identify a receptor binding epitope. a peptide corresponding to the n terminus of gp / , edpgffnvei, bound to purified cr and to cr positive but not cr negative b and t lymphoblastoid cell lines (nemerow et al., ) . this peptide sequence edpgffnvei is similar to the peptide sequence edpgkqlynvea, through which the c d component of complement binds to cd (nemerow et al., ) . synthetic peptides containing the lynvea c d sequence block c d binding to cd (lambris et al., ) , whereas peptides containing the edpgffnvea sequence block ebv infection (nemerow et al., (nemerow et al., , . taken together these studies open the possibility that the inhibitory effect of gl could be in part due to the blockade of ebv receptor. the anti-ebv activity of glycopeptides (xii), (xiii), and (xv) could be attributed to the similarity of their amino acid residues with the peptide sequence edpgkqlynva of c d component of complement that binds to cd . ascertaining this possibility would require further study and is currently under investigation. from the target perspective, the drugs that might be candidates for treatment of ebv infection fall into two groups (for reviews see refs. gershburg and pagano, ; lin, ) . the first group targeting viral dna polymerase includes acyclic nucleoside analogues (acyclovir, ganciclovir, penciclovir, valaciclovir, valganciclovir and famciclovir); acyclic nucleotide analogues (cidofovir and adefovir); pyrophosphate analogues (phosphonoformic acid and phosphonoacetic acid); possibly -oxo-dihydroquinolines (pnu- and pnu- ). the second group contains compounds of mixed nature that have a distinct structure such as maribavir, ␤-l- -iododioxolane uracil and indolocarbazole nigc-i. mechanisms of action of these drugs are still under study, but they do not involve inhibition of the viral dna polymerase. the molecular target in herpesviruses of all nucleoside analogs are the virus-encoded dna polymerases from which the mode of action of gl and its derivatives differs. the therapeutic and prophylactic effects of gl on chronic active viral hepatitis (bean, ; coon and ernst, ; shibata, ; van rossum et al., ) and the inhibitory effect on ebv replication observed in the present study together with its relative lack of toxicity at the cellular level all suggest that gl and selected derivatives may be worth further evaluating for their efficacy and safety in the treatment of active ebv infection. glycyrrhizin enhances interleukin- production by liver dendritic cells in mice with hepatitis history of the endocrine effects of licorice chemical modification of glycyrrhizic acid as a route to new bioactive compounds for medicine synthesis of glycyrrhizic acid conjugates containing l-lysine containing the spread of hiv infection among high-risk groups in vitro inhibition of chikungunya and semliki forest viruses replication by antiviral compounds: synergistic effect of interferon-alpha and ribavirin combination inhibition of glycyrrhizic acid on aflatoxin b -induced cytotoxicity in hepatoma cells a quantitive bioassay for hiv- gene expression based on uv-activation: effect of glycyrrhizic acid inhibition of nuclear factor b is associated with neuroprotective effects of glycyrrhizic acid on glutamate-induced excitotoxicity in primary neurons glycyrrhizin, an active component of liquorice roots, and replication of sarsassociated coronavirus development of antiviral therapy 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derivatives against sars-coronavirus induction of inducible nitric oxide synthase expression by ␤-glycyrrhetinic acid in macrophages characterization of complement c as a glycyrrhizin (gl)-binding protein and the phosphorylation of c ␣ by ck- , which is potently inhibited by gl and glycyrrhetinic acid in vitro synthesis and immunomodulating activity of new glycopeptides of glycyrrhizic acid containing residues of l-glutamic acid mapping of the c d receptor (cr ) binding site and a neoantigenic site in the c d domain of the third component of complement strategies for evaluation of antiviral agents against epstein-barr virus in culture mechanism of action of glycyrrhizic acid in inhibition of epstein-barr virus replication in vitro pathogenesis and therapy of epstein-barr virus infection: novel therapeutic approaches comparison of two bromovinyl nucleoside analogs, -␤-d-arabinofuranosyl-e- -( -bromovinyl)uracil and e- -( -bromovinyl)- -deoxyuridine, with acyclovir in inhibition of 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epstein-barr virus dna in plasma of patients with nasopharyngeal carcinoma nitric oxide produced by human b lymphocytes inhibits apoptosis and epstein-barr virus reactivation some nucleoside analogs with anti-human immunodeficiency virus activity inhibit replication of epstein-barr virus identification of gp as the viral glycoprotein mediating attachment of epstein-barr virus (ebv) to the ebv/c d receptor of b cells: sequence homology of gp and c complement fragment c d identification of an epitope in the major envelope protein of epstein-barr virus that mediates viral binding to the b lymphocytes epstein-barr virus receptor (cr ) soluble recombinant cr (cd ) inhibits epstein-barr virus infection a drug over the millennia: pharmacognosy, chemistry, and pharmacology of licorice bioavailability study of glycyrrhetic acid after oral administration of glycyrrhizin in rats; relevance to the intestinal bacterial hydrolysis epstein-barr virus gp / binding to the b lymphocyte c d receptor mediates adsorption, capping, and endocytosis intravenous glycyrrhizin for the treatment of chronic hepatitis c: a doubleblind, randomized, placebo-controlled phase i/ii trial pathologic characteristics of immunologic injury in primary cultured rat hepatocytes and protective effect of glycyrrhizin in vitro we thank joseph pagano for his critical reading and comments on this manuscript. this work was supported in part by grants from the national science council of the republic of china (nsc - -b- - ; nsc - -b- - ) and by an institutional grant (tcirp - y ) of tzu chi university. russian authors thank the federal agency on science and innovation (russia) for financial support (contract . . . ). key: cord- -y xuxj authors: liu, rui; an, liwei; liu, ge; li, xiaoyu; tang, wei; chen, xulin title: mouse lung slices: an ex vivo model for the evaluation of antiviral and anti-inflammatory agents against influenza viruses date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: y xuxj the influenza a virus is notoriously known for its ability to cause recurrent epidemics and global pandemics. antiviral therapy is effective when treatment is initiated within h of symptom onset, and delaying treatment beyond this time frame is associated with decreased efficacy. research on anti-inflammatory therapy to ameliorate influenza-induced inflammation is currently underway and seems important to the impact on the clinical outcome. both antiviral and anti-inflammatory drugs with novel mechanisms of action are urgently needed. current methods for evaluating the efficacy of anti-influenza drugs rely mostly on transformed cells and animals. transformed cell models are distantly related to physiological and pathological conditions. although animals are the best choices for preclinical drug testing, they are not time- or cost-efficient. in this study, we established an ex vivo model using mouse lung slices to evaluate both antiviral and anti-inflammatory agents against influenza virus infection. both influenza virus pr (h n ) and a/human/hubei/ / (h n ) can replicate efficiently in mouse lung slices and trigger significant cytokine and chemokine responses. the induction of selected cytokines and chemokines were found to have a positive correlation between ex vivo and in vivo experiments, suggesting that the ex vivo cultured lung slices may closely resemble the lung functionally in an in vivo configuration when challenged by influenza virus. furthermore, a set of agents with known antiviral and/or anti-inflammatory activities were tested to validate the ex vivo model. our results suggested that mouse lung slices provide a robust, convenient and cost-efficient model for the assessment of both antiviral and anti-inflammatory agents against influenza virus infection in one assay. this ex vivo model may predict the efficacy of drug candidates’ antiviral and anti-inflammatory activities in vivo. influenza a virus (iav) is still a threat to human health and poses a global concern due to its unpredictable, pandemic potential and pathogenesis. as high evolutionary rates of the influenza virus makes vaccination strategies difficult, anti-influenza drugs are crucial for the control of influenza pandemics. antiviral therapy is generally licensed for use within h of influenza illness onset, and delaying treatment is associated with decreased drug efficacy and increased morbidity and mortality (kandun et al., ) . although influenza-induced pathology is still unclear, the uncontrolled immune response may be the major contributor to influenza virus-induced mortality (de jong et al., ; iwasaki and medzhitov, ; kobasa et al., ) . thus, studies on the modulation of the host immune response are currently underway. several animal studies have shown that anti-inflammatory agents http://dx.doi.org/ . /j.antiviral. . . - /Ó elsevier b.v. all rights reserved. abbreviations: tnf-a, tumour necrosis factor alpha; il- , interleukin ; rantes, regulated on activation, normal t cell expressed and secreted; mip- a, macrophage inflammatory protein alpha; ip- , interferon-gamma induced protein ; il- , interleukin ; il- b, interleukin beta; ifn-c, interferon gamma; ppar-c, peroxisome proliferator-activated receptor gamma; egcg, epigallocatechin gallate; cxcr , chemokine (c-x-c motif) receptor ; tnfr , tumour necrosis factor alpha receptor ; il- r, interleukin receptor; mip- , macrophage inflammatory protein ; ccr , c-c chemokine receptor type ; gm-csf, granulocyte-macrophage colony-stimulating factor; i.p., intraperitoneal; balf, bronchoalveolar lavage fluid; rig-i, retinoic acid-inducible gene i; tlr / , toll-like receptor / ; tlr , toll-like receptor ; munana, -( -methylumbelliferyl)-a-d-acetylneuraminic acid. can protect mice from death against influenza infection. -deoxy-d , -prostaglandin j ( d-pgj ) has been shown to protect % of mice from death against lethal influenza infection through manipulation of the ppar-c pathway, whereas it does not inhibit virus replication (cloutier et al., ) . the p inhibitor significantly protects mice from lethal influenza infection without affecting virus replication (borgeling et al., ) . statins not only reduce the levels of ldl-cholesterol, but they also counteract the inflammatory changes associated with acute coronary syndrome and improve survival in patients with influenza. similarly, in patients hospitalised with laboratory-confirmed seasonal influenza, statin treatment is associated with a % reduction in -day mortality (fedson, ) . therefore, strategies targeting aberrant host immune responses may be good complements for existing antiviral drugs. the routine strategies for anti-influenza drug development rely primarily on cell-based assays in primary screening followed by animal studies. however, the cost of in vivo studies is very high due to the use of animals and a large quantity of investigational compounds. lung slices, which provide a bridge between single cells and whole animals, are broadly used in physiology and toxicity studies (morin et al., ; sanderson, ) . different from single cell lines, lung slices possess multiple cell types and preserve the physiological and functional cellular relationships within the body. cell-cell and cell-matrix interactions result in lung slices closely resembling the morphology and functionality of the lung. whereas the evaluation of the efficacy of a drug based on animal studies is expensive and time-consuming, the lung slice model may serve as a valuable tool for efficacy tests of compounds for the treatment of influenza. with the development of tissue slicers, which produce slices rapidly and reproducibly, an increasing number of studies have employed lung slices to explore the interaction of hosts and pathogens (chakrabarty et al., ; londt et al., ; punyadarsaniya et al., ; seehase et al., ; van poucke et al., ; wu et al., ) . recently, several studies have shown that pig lung slices can support influenza virus replication (londt et al., ; van poucke et al., ) . another study demonstrated that influenza virus infection can induce robust cytokine and chemokine responses in human lung slices (wu et al., ) . theoretically, the three-dimensional lung slice culture system can be used to evaluate the potency of both antiviral and anti-inflammatory agents against influenza virus infection. in the current study, we showed that influenza viruses can efficiently replicate in mouse lung slices and induce significantly elevated levels of cytokines and chemokines. a panel of antiviral and anti-inflammatory agents were tested for their antiviral activities and/or anti-inflammatory effects in the mouse lung slices. the results from the lung slice model are consistent with those from mouse studies. our results showed that the lung slice model provides a robust, convenient and cost-economical method for the screening and evaluation of both antiviral and anti-inflammatory agents against influenza virus infection in one assay. - -week old balb/c mice were purchased from changsha laboratory animal center (hunan province, china) and were housed under specific-pathogen-free condition. all experiments were conducted according to the protocol approved by the animal care and use committee of wuhan institute of virology, chinese academy of sciences (wiva ). mouse adapted a/puertorico/ / (h n ) and mouse adapted a/human/hubei/ / (h n ) were propagated in the allantoic cavity of -day-old specific-pathogen-free embryonated chicken eggs for h. the allantoic fluids were collected and filtered with . lm filter and stored at À °c. the virus strains were provided by the virus collection at wuhan institute of virology, chinese academy of sciences, china. -deoxy-d , -prostaglandin j ( d-pgj ), ribavirin and the neuraminidase substrate -( -methylumbelliferyl)-a-d-acetylneuraminic acid (munana) were purchased from sigma-aldrich. egcg was purchased from sichuang weikeqi biological technology co. ltd. (china). glycyrrhizin was purchased from shanghai hanxiang biological technology co. ltd. (china). the p pathway inhibitor sb , the erk pathway inhibitor u and the sapk/jnk pathway inhibitor sp were purchased from beyotime institute of biotechnology (china). oseltamivir carboxylate (gs ) was obtained from toronto research chemicals (canada). all compounds were initially dissolved in dimethyl sulfoxide (dmso, sigma-aldrich). mouse lung slices were prepared using a modification of a protocol that has been previously reported (bauer et al., ) . after anesthetisation by intraperitoneal injection of sodium pentobarbital ( mg/kg), the mouse was bled through the abdominal aorta. then, the trachea was exposed, dissected from surrounding tissues and was cannulated with an -gauge needle. through the cannula, the lung was inflated with . ml of % low-melting agarose (bio-rad) dissolved in hank's buffered saline solution (hbss) solution. the whole animal was cooled with ice for min to solidify the agarose and, thereby, the lung. then, the lung was taken out en bloc from the thoracic cavity and placed in the slice culture medium (dulbecco's modified eagle medium: nutrient mixture f- , dmem/f- , gibco) at °c for an additional min to completely solidify the agarose. the culture medium was supplemented with units/ml of penicillin, lg/ml streptomycin and ng/ml of amphotericin to avoid contamination. the lung lobe was afterwards dissected and cut to create a flat surface at the end of the primary bronchus. another flat surface was cut approximately . cm from the first surface. the cube was maintained in the pre-chilled slice culture medium prior to or during the slicing. the cube was cut into slices of desired thickness using a vibratome slicer (leica, vt s). each mouse lung cube generated at least -lm slices. the slices were then transferred into a -well cell culture plate and covered with ll of slice culture medium in each well. the medium was changed every hour at least three times before virus infection to remove cell debris. the lung slice viability was assessed by bronchoconstriction and live/dead staining. the bronchoconstriction was monitored under a microscope when adding or removing À m acetyl-ß-methylcholine chloride (sigma-aldrich). the photos were taken using a nikon inverted research microscope ti eclipse. for live/dead staining experiments, the slices were incubated with calcein am ( lm) and propidium iodide (pi, lg/ml) for min at room temperature. a nikon multiphoton confocal microscope a mp + was used to record the images. the lung slices were infected with ll of pfu/ml influenza viruses for h. virus diluent was used as a negative control. after the incubation, the viruses were discarded, and the slices were washed twice with phosphate buffered saline (pbs); then, fresh medium was added. at the indicated time points, the supernatants were harvested, and the virus titres and expression levels of cytokines and chemokines were detected. the slices were stored at À °c until rna extraction was performed. for the drug addition assay, -lm-thick lung slices were prepared as described in section . . after the last wash, the slices were infected with ll of pfu/ml pr virus. after incubation for h, the virus was discarded, and the slice was washed with pbs. for the measurement of the z factor, each -well plate (one lung slice per well) was processed as follows: one column was uninfected, one column was infected and mock treated, the rest of columns were infected and treated with ll of six concentrations of -fold dilutions of ribavirin starting from lm. in total, four plates were processed. for the lung slice model validation assay, each -well plate contained two agents, and each data point had three lung slices. na activity and ip- level were determined h post infection. the virus titres were determined by a tcid assay. briefly, madin-darby canine kidney (mdck) cells were maintained in dmem supplemented with % foetal bovine serum and units/ml of penicillin/streptomycin. mdck cells were seeded into -well cell culture plates. twenty-four hours later, -fold serial dilutions of the supernatant were inoculated on an mdck monolayer at °c for h, and the cytopathic effects were examined. the virus titres were calculated by the reed-muench method (reed and muench, ) . -( -methylumbelliferyl)-a-d-acetylneuraminic acid (munana) is a fluorometric substrate of the neuraminidase of the influenza virus. the virus containing sample was mixed with lm munana, which is dissolved in a mes solution ( mm -[n-morpholino] ethanesulfonic acid and mm cacl , ph = . ), and incubated at °c for h in a -well black optiplate (perkinelmer). the reactions were stopped by adding . m naoh in % ethanol, and the fluorescence signal was recorded at nm (excitation) and nm (emission) using the wallac envision multilabel reader (perkinelmer). the protein levels of tnf-a, il- , il- , il- b, ifn-c, mip- a, ip- , and rantes were measured using commercial elisa kits (boster biotechnology company, wuhan, china). for the anti-inflammatory assay using ip- as readout, the protein levels of ip- were measured using duoset elisa development kits (r&d). drug toxicity was assessed by mtt assay. the slices were incubated with ll serially diluted concentrations of drugs for h. then the drugs were removed and the slices were washed once with pbs and incubated with mtt ( . mg/ml) for min at °c. after the formazan was completely dissolved in ll of dmso, ll of dmso solution was transferred to a -well plate. the optical density (od) of the dissolved formazane was measured at nm. the lm thick lung slices were cut and exposed to ll of pfu/ml pr (h n ) or hubei (h n ) viruses. after incubation for h, the viruses were removed and fresh medium were added. h later, the slices were fixed with % paraformaldehyde for h at room temperature. the slices were then incubated in blocking buffer (pbs containing % bsa, % triton x- and % normal goat serum) for another h. afterwards, the slices were immunoprobed with antibody against nucleoprotein of influenza a virus ( : , santa cruz sc- ) and in binding buffer (pbs containing % bsa and % triton x- ) overnight at °c. after washing three times with pbs, the slices were incubated with dapi ( lg/ml, sigma-aldrich) and tritc-conjugated goat anti-mouse igg ( : ) for h. fluorescent images were obtained using a nikon multiphoton confocal microscope a mp + (nikon). total rna was extracted from lung slices with the e.z.n.a™ microelute total rna kit according to the animal tissue protocol (omega). reverse transcription was conducted using a random hexamer primer. real-time quantitative pcr was performed using the itaq™ universal sybr Ò green supermix (bio-rad). the pcr conditions were °c for s and cycles of °c for s and °c s. the primers for cytokines and chemokines were synthesised according to previously published data (giulietti et al., ) . mice were anesthetized by intraperitoneal injection of sodium pentobarbital ( mg/kg). twenty microlitres of pfu/ml pr virus was inoculated nasally. the mock group was inoculated with virus diluent. on day post infection, the mock mice and infected mice were sacrificed the tracheas and lungs were removed and washed three times with an injection of ml of pbs containing . % bsa. after centrifugation at rpm, the bronchoalveolar lavage fluids (balf) were stored at À °c. statistical analysis was performed by the unpaired t-test. correlation of cytokines/chemokines between ex vivo and in vivo was evaluated using a linear regression analysis using graphpad prism . software. cc and ec values were determined using non-linear regression using graphpad prism . . statistical significance was determined as follows: *, . < p < . ; **, . < p < . . the data in the figures are represented as the means ± sem. the z factor was calculated according to the method described previously (zhang et al., ) . to establish the ex vivo lung slice model for influenza virus infection, the viability of lung slices must be maintained. we first tested how long the mouse lung slices can remain viable under the ex vivo culture condition. mouse lung slice viability was assessed based on the bronchoconstriction assay and live/dead viability assay. as shown in fig. , a strong bronchoconstriction (compare a with b) could be induced on days , (data not shown) and after treatment with . lm acetyl-ß-methylcholine chloride. the removal of the drug resulted in relaxation of the airway (fig. c) . to further assess the viability of lung slices, the live/dead staining method was used to evaluate whether the alveolar architecture remained intact and alive after preparation and ex vivo culture. as shown in fig. , the slices were almost % green (viable cells) directly after preparation (fig. d ), whereas treatment of the slices with % triton- resulted in only red nuclei staining (dead cells, fig. e ). in addition, the mouse lung slices stayed alive and the alveolar architecture in lung slices remained intact up to days after preparation ( fig. f-h) . collectively, these results suggest that the mouse lung slices can be kept alive for at least days after preparation. because the lung slices can survive under ex vivo conditions, we assessed whether influenza viruses can infect and replicate in the mouse lung slices. first, lung slices with different thicknesses were generated to determine the optimal thickness of lung slice suitable for the infection of influenza viruses. as expected, -lm thick slices produced more infectious virions h post-infection than -lm thick slices. however, the virus yields of and -lm thick slices were similar to those of -lm thick slices ( fig. a) . these results suggest that -lm thick lung slices are the best for infection with influenza virus, balancing cost and efficiency. next, the growth curves of influenza viruses in mouse lung slices were obtained. as shown in fig. d and e, both pr (h n ) and hubei (h n ) viruses demonstrated time-dependent replication and reached their maximum titre h post infection, though the virus titres ex vivo are much lower than those in cell culture. neuraminidase (na) activity results ( fig. b and c) showed that both virus strains have similar kinetics in virus replication and infectivity ( fig. d and e) , suggesting that na activity can precisely represent the virus replication and infectivity in lung slices. to further correlate na activity and virus titre, ribavirin and u were tested for dose dependent inhibition. as shown in fig. s , both compounds exhibit dose dependent inhibition of na activity or virus infectivity, indicating that na activity can be used to the -lm thick lung slices were prepared, and the bronchoconstriction was observed days after preparation. the untreated slices (a) were incubated with . lm acetyl-ß-methylcholine chloride at °c for min (b) in a -well cell culture plate. then, the drug was removed, lung slices were washed twice with pbs and incubated with fresh culture medium at °c for min (c). the images were captured using a nikon's eclipse ti inverted microscope. red, blue and black arrows show the lung bronchus, pulmonary artery and pulmonary vein, respectively. scale bar = lm. (d-h) live/dead staining assay. lung slices were stained with calcein-am ( lm) and propidium iodide (pi, lg/ml) for min at room temperature on days , and after preparation (lower panels, f-h). the middle panel shows a viable slice directly after preparation (d), and a slice with complete loss of activity (treated by % triton- ) (e) for comparison. calcein-am and pi were used to simultaneously determine the live and dead cells. after being washed twice with pbs, images were taken using a nikon multiphoton confocal microscope a mp + with excitation at nm, and an emission filter of / nm for calcein-am and / nm for pi. scale bar = lm. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) represent virus replication in the primary screening. to further confirm the infection of the lung slices by influenza virus, an immunofluorescence assay (ifa) was used to stain the nucleoprotein (np) of influenza virus on the lung slices. as shown in fig. f , the lung slice was strongly infected by influenza virus. overall, these results demonstrated that lung slices support influenza virus replication. one aim of our study was to evaluate the anti-inflammatory effects of potential agents in lung slices. many cytokines and chemokines are elevated in influenza-infected mice and seem important in influenza-induced inflammation. despite complications in the cytokine storm induced by influenza virus infection, we chose eight important cytokines and chemokines (belisle et al., ; brandes et al., ; lauder et al., ; szretter et al., ; wang et al., ; weiss et al., ) to study the inflammatory response following influenza virus infection. first, we examined the mrna levels of cytokines and chemokines in influenza virus-infected lung slices by real time rt-pcr. as shown in fig. a and b, exposure to pfu/ml doses of pr and hubei viruses resulted in elevated mrna levels of most cytokine and chemokine genes tested. at h post infection, the gene transcriptions were elevated and were highly activated at h post infection. the cytokines and chemokines with the highest levels of induction in transcription by both viruses were rantes and ip- . because the cytokine and chemokine responses mediated by the two viruses at transcription level are similar, the pr influenza virus was used for the steady protein level analysis. we then further determined the protein levels of selected cytokines and chemokines in response to influenza virus infection at h and h post infection. as shown in fig. c , after exposure to pr virus for h, the protein levels of all tested cytokines and chemokines were significantly elevated except for il- b and il- . ifn-c was undetectable in the supernatant of the lung slices. the fold increase over mock for tnf-a, mip- a, il- , rantes and ip- were . , . , . , . and . , respectively. at h post infection, higher levels of cytokines and chemokines were induced (fig. d) , and the fold increases were , . , , and , respectively. ip- was the most highly stimulated chemokine at both h and h post infection, which is consistent with the results of its mrna response to virus infection. taken together, our results suggest that a strong the supernatants were collected at indicated time points to perform the na activity assay (b and c) and tcid assay (d and e). (f) infection of lung slices was assessed by the ifa assay. slices with a thickness of lm were exposed to ll of pfu/ml pr (h n ) or hubei (h n ) viruses for h. the slices were fixed with % paraformaldehyde and analysed by ifa. the images were captured using a nikon multiphoton confocal microscope a mp + . the virus diluent was used as a negative control (nc) in the infection. scale bar = lm. the right panels show enlarged regions of interest from the left panels. inflammatory response represented by the highly elevated mrna and protein levels of cytokines and chemokines are triggered by the infection of influenza viruses in the ex vivo mouse lung slice. our results demonstrated that the ex vivo model supports influenza virus infection (section . ) and exhibits an inflammatory response following influenza virus infection (section . ). to meet the goal of this study in the establishment of an ex vivo mouse slice model for the screening and evaluation of both antiviral and anti-inflammatory drugs against influenza infection in one assay, ensuring that the ex vivo model has similar patterns in influenza-induced cytokine and chemokine responses is critical. as shown in fig. s , the inflammatory responses in vivo on day post-infection appeared to be most robust. the cytokine/chemokines levels in vivo on day were chosen to compare with that ex vivo. the results from mouse studies showed that ifn-c, il- b, tnf-a, and il- exhibited lower levels of activation ( pg/ml, pg/ml, pg/ml and pg/ml, respectively), whereas mip- a, il- , rantes and ip- exhibited higher levels of activation in bronchoalveolar lavage fluid (balf, pg/ml, pg/ml, pg/ml and pg/ml, respectively). the elevated expression of most of the tested cytokines and chemokines in the supernatant of lung slices demonstrated the same expression pattern compared with that in the mouse model (figs. c, d and a) . to evaluate the correlation between ex vivo and in vivo levels of cytokines and chemokines, a linear regression analysis model was employed. the levels of cytokines and chemokines in the supernatants of the lung slices h post infection (fig. c ) were compared to that in the balf of mice day post infection. the results show that there is a positive correlation between ex vivo and in vivo expression of selected cytokines and chemokines with a coefficient of correlation of . (fig. b ). more specifically, ip- , rantes and mip- a are perfectly correlated. the coefficient of correlation between cytokines and chemokines in the supernatant of lung slices h post infection (fig. d) and that in the balf of mice day post infection was . (fig. s ). our results here demonstrated that the ex vivo mouse lung slice model resembles the in vivo mouse model in response to influenza virus infection. in addition, ip- , rantes and mip- a may serve as readouts in the screening and evaluation of anti-inflammatory agents against influenza infection. to assess the quality of the mouse lung slice model, we used ribavirin, an antiviral drug, as a reference in a pilot experiment to test the z factors for the measurements of antiviral and anti-inflammatory activities. the z factor, a screening window coefficient, can be used to evaluate the quality of assays (zhang et al., ) . neuraminidase was chosen as a readout for antiviral activity, which was confirmed to correlate well with the production of infectious virions (fig. b-e) . ip- was chosen as a readout f nc pr hubei fig. (continued) to represent the influenza virus-induced inflammation for three reasons: (a) it exhibits good correlation between in vivo and ex vivo models at the protein level, (b) it is the most highly induced chemokine in the mouse model and in the lung slice model, and (c) ip- À/À mice or mice treated with anti-ip- antibody demonstrate global down-regulated cytokines and chemokines and can survive after challenged by a lethal dose of influenza virus (wang et al., ) . therefore, ribavirin was used to determine its na and ip- inhibition effects to test the performance of the lung slice model. four replicate plates were processed to determine the z factor. as shown in table , a z factor ranging from . to . for na inhibition and . to . for ip- inhibition demonstrated that the quality of the lung slice model for the evaluation of both antiviral and anti-inflammatory effects is satisfactory. to further validate our mouse lung slice model, a panel of agents with known antiviral or anti-inflammatory activities in vivo was tested for antiviral and anti-inflammatory effects in the lung slice model. among these agents, ribavirin, oseltamivir and gemacrone are anti-influenza drugs. egcg and u have both antiviral and anti-inflammatory effects in vivo. d-pgj and sb have anti-inflammatory activities in vivo. all of these to show the induced changes, cytokines and chemokines were placed in separate panels according to scales of the protein levels. each data point was from three lung slices, and virus diluent was used as a negative control. comparisons between the infected group and the negative control were performed by the unpaired ttest (*, . < p < . ; **, . < p < . ). the data are expressed as the means ± sem. nd, not detected (for real-time pcr, no amplification was detected during the cycles. for elisa, the signal was below the detection limit of the commercial kit). agents have been reported to protect mice from death against lethal influenza infection. as shown in table , all of the antiviral agents (ribavirin, oseltamivir, and gemacrone) efficiently inhibited virus replication in the lung slices, and the ip- levels were decreased as a result of the inhibition of viral replication (fig. s ) . unexpectedly, oseltamivir inhibited only virus replication in the lung slices without interfering with ip- expression (fig. s ) . in addition to their antiviral effects, u and egcg possess anti-inflammatory effects (droebner et al., ; ling et al., ; pinto et al., ; ranjith-kumar et al., ) . we found that both u and egcg can dose dependently decrease na levels and ip- levels in the lung slice model. d-pgj and sb were chosen because they protect mice from influenza infection by anti-inflammatory effects only (borgeling et al., ; cloutier et al., ) . as expected, both d-pgj and sb dose dependently decreased ip- levels, whereas neither inhibited virus replication in lung slices, suggesting they improved mouse survival through their anti-inflammatory mechanisms (fig. s ) . to further validate the model, we also tested six agents (dicyclomine, clotrimazole, fenofibrate, proadifen, benzydamine and nafronyl oxalate) with known antiviral activity in mdck cells ) that failed to protect mice from lethal doses of influenza virus infection (fig. s ) . results showed that dicyclomine, clotrimazole, fenofibrate, proadifen and benzydamine inhibited neither viral replication represented by na activity nor inflammatory response represented by ip- levels in mouse lung slices (fig. s ) . however, nafronyl oxalate, although not showing any antiviral activity, inhibited the inflammatory response (with ip- -ec of . lm, fig. s ), although it did not improve mouse survival rate (fig. s ) . taken together, our results suggest that with the benefits of avoiding the overuse of animals and expensive compounds, the mouse lung slice model is robust and cost-efficient for screening and evaluating both antiviral and anti-inflammatory drugs against the influenza infection in one assay. overall, the mouse lung slice can serve as a predictive model of the efficacy of drug candidates on the mouse model. current anti-influenza drug screening is based primarily on transformed cells in the form of cell-based assays. though the transformed cells are convenient for high throughput screening and the in vitro study of potency and mechanisms, they are distantly related to physiological and pathological conditions. as a consequence, results based on transformed cells may suffer from simplification. current treatments for influenza rely on the use of antiviral drugs; however, the window for antiviral therapy is narrow. several reports have shown that anti-inflammatory agents can be effective in reducing the death rate against the infection of lethal influenza (borgeling et al., ; cloutier et al., ) . comparisons between infected mice and mock mice were performed by the unpaired t-test (*, . < p < . ; **, . < p < . ). the data are expressed as the means ± sem. nd, not detected (the signal was below the detection limit of the commercial kit). (b) comparison of the cytokine and chemokine levels in response to influenza infection between the ex vivo and in vivo models. after subtraction of the corresponding levels of the mock group, the correlation of the virus-induced il- b, ifn-c, tnf-a, il- , mip- a, il- , rantes and ip- in balf of day post infection ( fig. a ) and lung slice supernatants (fig. c) were analysed by a linear regression analysis model. table the pilot experiment of lung slices model using ribavirin. lm thick lung slices were prepared as described in section . . the lung slices were infected with ll of pfu/ ml pr virus. after h incubation on °c, the viruses were discarded and the lung slices were washed twice with pbs. then six concentrations of ribavirin starting from lm and with -fold serial dilution were added. na activities and ip- levels in culture supernatants were determined h post infection as described in sections . and . . s/b-na a s/b-ip- b z -na c z -ip- d na-ec (lm) e ip- -ec (lm) f moreover, current antiviral drug screening is performed primarily using cell-based assays. the cell-based assays available for the development of anti-inflammatory agents against influenza virus infection are largely targeting single host factors or pathways that play a role in the influenza-induced inflammatory response. such strategies could be very risky for the development of anti-inflammatory drugs and lead to failure in animal tests or in clinical trials. whereas animal models, e.g., mice and ferrets, are good for efficacy studies in of both antiviral and anti-inflammatory drugs, they are not time-or cost-efficient. to overcome this problem, at least in part, we have established the mouse lung slice model to test drugs for antiviral and anti-inflammatory activities in one ex vivo assay with the convenience of the in vitro cell-based assay. the goals for this model are as follows: (i) the mouse slices can remain alive long enough for efficient influenza virus infection and inflammatory response; (ii) the inflammatory response is similar to that in the lung of mouse; (iii) to choose the best readouts for the antiviral and anti-inflammatory activities in the model; and (iv) to validate the model using antiviral and anti-inflammatory agents with confirmed efficacy in vitro and in vivo. the tissue viability of the lung slices was the first concern in establishing the lung slice model. mouse lung slices can survive and maintain their native structure for at least three days after preparation (sanderson, ; siminski et al., ) . the methods to monitor the viability of lung slices include the measuring of the ldh release, ciliary activity, bronchoconstriction induced by stimuli, and live/dead staining (sanderson, ) . in our research, results from bronchoconstriction experiments and live/dead staining experiments demonstrated that lung slices remained alive for at least days. moreover, the infection of lung slices by influenza viruses and the kinetics of virus growth are better indications that the mouse lung slices stay viable for at least the first days after preparation. our second goal was to establish an ex vivo model for the infection of mouse lung slices by influenza viruses and the induction of inflammatory responses similar to that in the mouse model. our results demonstrated that influenza viruses replicate efficiently in the mouse lung slices. these results are consistent with previous reports showing that the influenza virus can replicate in pig lung slices (van poucke et al., ). by ifa, mouse lung slices were clearly shown to be infected by influenza virus. to analyse the inflammatory response of lung slices following the infection of the influenza virus, we measured the levels of cytokine and chemokine and compared them with those in the balf of mice. our results showed that a positive correlation exists between the ex vivo and in vivo models in the expression of selected cytokines and chemokines with a coefficient of correlation of . . ifn-c and il- b were significantly induced in vivo but not ex vivo. this may be due to an influx of multiple types of immune cells in the lung in vivo but not in the lung slice. the lower levels of il- b in lung slices may be due to the observation that the epithelial cells and macrophages secreted less il- b than neutrophils in responds to influenza infection (brandes et al., ) . the observation that ifn-c secretion was not detected in lung slices can be explained by the fact that the primary source of ifn-c are nk cells, cd + th cells and cd + t cells (schroder et al., ) , which are absent in the lung slice. despite the discrepancy in some of the cytokine and chemokine responses, ip- , rantes and mip- a are perfectly correlated between the ex vivo and in vivo models. therefore, ip- , rantes and mip- a may represent the inflammatory response following influenza virus infection in the ex vivo model. the choice of readout for antiviral effects is straight forward and was determined previously in our lab. the fluorometric substrate of the neuraminidase of the influenza virus, munana, is used for detection of virus neuraminidase activity, which represents the levels of virus replication . the virus replication level determined by na activity is proportional to that of the tcid as shown in fig. (compare b and d, c and e). however, the choice of readout for anti-inflammatory activity is much more complicated because no known cytokine or chemokine is widely accepted to represent the inflammatory response to influenza infection. based on the comparison between ex vivo and in vivo responses of the selected cytokines and chemokines following influenza virus infection, ip- , rantes and mip- a may serve as readouts for influenza-induced inflammation. in the recent literature, to elucidate which cytokine and chemokine is more important in the influenza-induced innate immune response, cytokines or their receptor gene were knocked out in mice. tnfr À/À , il- À/À , mcp- À/À , il- r À/À , mip- À/À , ccr À/À , and gm-csf À/À mice did not survive influenza infection; however, ip- À/À mice did survive influenza infection, suggesting that ip- may play an table the evaluation of a panel of antiviral and anti-inflammatory agents in mouse lung slice model. the lm thick lung slices were prepared as described in section . . the slices were infected with ll of pfu/ml pr virus. after h incubation on °c, the viruses were removed and the lung slices were washed twice with pbs. then drugs with serially diluted concentrations were added and maintained for h, and the na activities and ip- levels were determined as described in sections . and . . important role in the aberrant cytokine responses during influenza infection (darwish et al., ; wang et al., ) . ip- has also been proposed to be a poor prognostic indicator for patients with severe acute respiratory syndrome (sars), which is clinically similar to severe influenza infection (nelli et al., ) . although we did not compare the proteome changes induced by influenza, the rationale to choose a single factor to prove the concept has been previously reported (cameron et al., ) . taking these results into consideration, we chose ip- as a readout for the evaluation of anti-inflammatory drugs, and ip- appeared to be valid for representing the inflammatory response in the validation test of a panel of antiviral and anti-inflammatory agents. of course, if a critical study of the efficacy of a candidate compound is required, multiple readouts determined by the levels of multiple cytokines and chemokines can be used to assess the inhibitory effects against the inflammatory response following influenza virus infection. very often, compounds showing antiviral or anti-inflammatory activity in the single cell system fail in the animal assay. to validate this precision cut mouse lung slice model in screening and evaluation of both antiviral and anti-inflammatory drugs against influenza virus infection in one assay using na and ip- as readouts, four categories of known antiviral and anti-inflammatory drugs were tested for their antiviral and anti-inflammatory activities. the results were compared with that from mouse studies conducted by others and us. the four categories of agents are: (i) agents with only antiviral effects in vitro and in vivo; (ii) agents with both antiviral and anti-inflammatory effects in vitro and in vivo; (iii) agents with only anti-inflammatory effects in vitro and in vivo; (iv) agents with antiviral effects in vitro which failed in the mouse model. the agents of the first three categories showed antiviral or anti-inflammatory effects in the lung slice model. among the agents from the category iv, all failed to show any antiviral or anti-inflammatory activity on the mouse lung slice model, except that nafronyl oxalate was shown to inhibit ip- release. overall, this validation testing of a group of agents with known antiviral or anti-inflammatory activities demonstrated that the mouse lung slice model is valid, convenient and cost-efficient for screening and evaluation of both antiviral and anti-inflammatory drugs in one ex vivo assay. it appears that only drugs showing either antiviral or anti-inflammatory activity in the mouse lung slice model may protect mice from infection of lethal influenza, though exceptions may occur. in summary, we established and validated a mouse lung slice model to evaluate compounds for antiviral and anti-inflammatory effects in one assay. importantly, employing the lung slice model is economical in term of experiment costs, in compound synthesis and animal use. for the development of antiviral drugs, the mouse lung slice model is more similar to the mouse model. for the development of anti-inflammatory drugs, the mouse lung slice model is suitable for drugs targeting multiple targets and pathways involved in the inflammatory response following influenza infection and more similar to the mouse model. this model can be useful in the secondary screening for selection of drug candidates with potent antiviral or (and) anti-inflammatory activities before testing in an animal model. notably, the mouse lung slice model should be used with caution in testing anti-inflammatory drugs against influenza viruses other than h n in two aspects. firstly, to make sure that the virus being tested can replicate efficiently on mouse lung slices and induce cytokine/chemokine responses similar to that in vivo. secondly, the readout(s) for the inflammatory response can be different and should be determined experimentally ex vivo and in vivo. considering that the infection of many respiratory viruses causes acute inflammation in the human lungs and induce similar inflammatory responses, it is likely that the anti-inflammatory drugs developed using the influenza virus infected mouse lung slice model may also be effective in the treatment of infection of other respiratory viruses. taken together, our research demonstrates that it is beneficial to use the mouse lung slice model to develop new drugs with either antiviral or anti-inflammatory activities in the treatment of influenza virus infection. screening and identification of inhibitors against influenza a virus from a us drug collection of drugs treating viral 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article can be found, in the online version, at http://dx.doi.org/ . /j.antiviral. . . . key: cord- - c r os authors: spisakova, martina; cizek, zdenek; melkova, zora title: ethacrynic and α-lipoic acids inhibit vaccinia virus late gene expression date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: c r os smallpox was declared eradicated in . however recently, the need of agents effective against poxvirus infection has emerged again. in this paper, we report an original finding that two redox-modulating agents, the ethacrynic and α-lipoic acids (ea, la), inhibit growth of vaccinia virus (vacv) in vitro. the effect of ea and la was compared with those of β-mercaptoethanol, dtt and ascorbic acid, but these agents increased vacv growth in hela g cells. the inhibitory effects of ea and la on the growth of vacv were further confirmed in several cell lines of different embryonic origin, in epithelial cells, fibroblasts, macrophages and t-lymphocytes. finally, we have analyzed the mechanism of action of the two agents. they both decreased expression of vacv late genes, as demonstrated by western blot analysis and activity of luciferase expressed under control of different vacv promoters. in contrast, they did not inhibit virus entry into the cell, expression of vacv early genes or vacv dna synthesis. the results suggest new directions in development of drugs effective against poxvirus infection. vaccinia viruses (vacvs) of various strains have been used in the past for a wide-spread vaccination against smallpox, leading to eradication of this life-threatening disease. in , the world health organization smallpox eradication program declared the disease as eradicated (who, ) and vaccination of the general population has been stopped. along with the rising concerns about the smallpox misuse in a bioterrorist attack, certain countries have been re-introducing vaccination programs among selected groups of population and re-considering a vaccination of the general population. however, the old vaccines that are available (new york city board of health strain of vaccinia virus -dryvax; wyeth laboratories), and probably also the new vaccines based on the same virus strain but prepared in tissue culture (e.g. acam ; acambis) reveal a substantial risk of post-vaccination complications (artenstein, ) . the most severe complications described during the eradication campaign include progressive vaccinia (vaccinia necrosum), post-vaccination encephalitis, and eczema vaccinatum (aragon et al., ) . a new complication, myopericarditis, was described based on the recent vaccination of the military personnel with the old dryvax vaccine by the us department of defense (arness et al., ) , but it has also occurred during a trial with the new, tissue culture-based vaccine acam (artenstein, ) . * corresponding author. tel.: + ; fax: + . e-mail address: zmelk@lf .cuni.cz (z. melkova). these authors contributed equally to this work. in addition to the need of an efficient therapy of vaccinia virusrelated post-vaccination complications or variola infection, there is a risk of the infection of humans with monkeypox virus. this virus is endemic in africa, but cases were also reported in the united states in (ligon, ; reed et al., ) . current therapeutic possibilities of poxvirus infections are rather limited. vaccinia immune globulin or vig, has been used for a passive immunization, providing immediate, - weeks lasting protection against infection, as well as for treatment of postvaccination complications. however, its effectiveness has never been assessed in properly controlled trials (bray and wright, ) . the only drug currently allowed for use by the us food and drug administration (fda) for treatment of certain reactions to the smallpox vaccine is cidofovir (cdv), a nucleoside analogue originally approved for treatment of cmv infections in immunocompromised individuals (de clercq, ; de clercq and holý, ) . a disadvantage of this compound is its intravenous administration and nephrotoxicity (safrin et al., ). an ether-lipid analogue of cdv, cmx , reveals a good oral bioavailability and a low toxicity with similar antipoxvirus efficiency as cdv (parker et al., ) . this compound is currently evaluated in clinical trials (http://www.chimerix-inc.com/pdfs news/ chimerix% initiates% cmx % % multi-dose.pdf). another drug, st- , inhibits a late step in vacv morphogenesis and its egress from the cell (yang et al., ) . it has been also evaluated in clinical trials (http://www.siga.com/?id= ). this experimental drug was permitted for use in a single case of eczema vaccinatum together with vig and cidofovir (kaiser, ) . additionally, thiosemicarbazone derivatives had been used in the past for anti-poxvirus prophylaxis as well as treatment, but the effectiveness of individual derivatives was never properly assessed in placebocontrolled human trials (pirrung et al., ; smee and sidwell, ) . a recent study in mice, however, suggested that neither isatin-␤-thiocarbazone (ibt) nor its derivative marboran may be useful in therapy of poxvirus infections. they effectively reduced the mortality of mice infected systemically only with vacv but not with cowpox virus, while they did not reduce replication of either virus or clinical illness in mice infected cutaneously (quenelle et al., ) . in this paper, we report a new finding that ethacrynic and ␣lipoic acids inhibit growth of vaccinia virus in vitro. ethacrynic acid (ea) is a well known clinically used thiol-reactive diuretic agent and a na + /k + /cl − co-transport system inhibitor (available under various trade names, e.g., edecrin, edecril, reomax; (griffiths and simmons, ; koechel and rankin, ) . it has been also used to decrease the intraocular pressure in glaucoma (melamed et al., ) . finally, it is known to inhibit glutathione-s-transferase (piclass; gst), and thus to deplete both cytosolic and mitochondrial stores of gsh, serving as an experimental pro-oxidant (meredith and reed, ) . the inhibition of gst by ea has been clinically employed to potentiate the effects of anti-cancer drugs by decreasing their conjugation and elimination from the organism (gate and tew, ) . ␣-lipoic acid (la) is a thiol-containing, redoxcycling agent. as the lipoamide, it serves as a cofactor of pyruvate dehydrogenase and other multienzyme complexes that catalyze the oxidative decarboxylation of ␣-keto acids. additionally, it acts as an antioxidant involved in free radical quenching, antioxidant recycling, and metal chelation. it has been shown to be effective in various experimental and clinical studies focused on therapy of alzheimer's and other neurodegenerative diseases, diabetes, myocardial and cerebral ischemia-reperfusion injury holmquist et al., ; packer et al., ) . la has been used as a food additive (available under various trade names, e.g. alpha lipoic acid, heparlipon, thioctsan) and it has been also proposed to ameliorate the hiv-induced redox-stress in the organism (jariwalla et al., ) . here we describe that both ea and la inhibit growth of vaccinia virus in vitro in several cell types in a dose-dependent manner. in contrast, other redox-modulating agents such as ␤mercaptoethanol, dtt or ascorbic acid did not reveal any inhibitory effects at similar concentrations; on the contrary, they promoted vacv growth. the effective concentrations of ea were found in the low micromolar range, while those of la in the high micromolar range. the mechanism of action seems to be, however, similar for both compounds-they appear to inhibit the expression of vacv late genes, resulting in decreased levels of infectious virus progeny. the two compounds do not seem to affect either vacv entry into the cell or viral dna synthesis. all the media and growth supplements were purchased from invitrogen corporation (carlsbad, ca) or paa laboratories gmbh (pasching, austria). l-ascorbic acid, dithiothreitol (dtt), ␤-mercaptoethanol, ethacrynic acid, ␣-lipoic acid, cytosine arabinoside (arac), methylthiazolyldiphenyl-tetrazolium bromide (mtt) and atp were purchased from sigma co. (st. louis, mo), mouse recombinant ifn␥ from r&d systems (minneapolis, mn). luciferin was purchased from promega (madison, wi). all the enzymes, primers, probes and chemicals for real-time pcr were purchased from applied biosystems (foster city, ca). other chemicals and supplements were purchased from sigma co., unless otherwise specified. stock solutions of the redox-modulating and antiviral agents were prepared as follows: m ␤-mercaptoethanol in water, m dtt in water, mm ascorbic acid in water, . mm ea in pbs, mm la in ethanol, and . mg/ml arac in water. the aliquots of stock solutions were kept in − • c and diluted shortly before experiments. human cervical carcinoma-derived hela g (melková et al., ; scherer et al., ) and african green monkey kidney-derived bsc- (hruby et al., ; melková and esteban, ) cell lines were grown in dulbecco's modified eagle's medium (dmem; glucose . g/l) supplemented with % heat-inactivated neonatal calf serum (ncs; % ncs-dmem) and antibiotics (penicilin × u/l, streptomycin mg/l). mouse fibroblast l (lai et al., ; sanford et al., ) and mouse monocyte/macrophage j .g (melková and esteban, ; unkeless et al., ) cell lines were grown in dmem (glucose . g/l), supplemented with % heatinactivated fetal bovine serum ( % fbs-dmem) and antibiotics. macrophages were also pre-treated with u/ml of ifn␥ (r&d systems) for h before the infection. the human t-cell derived jurkat cell line (clone e - ; atcc tib- ) was grown in rpmi supplemented with % heat-inactivated fetal bovine serum ( % fbs-rpmi), antibiotics, . mm hepes, ph . , and mm glutamine. the cells were maintained at • c, in % co atmosphere and % humidity. viruses used included wild-type (wt) vacv, strain western reserve (wr; atcc vr- ; (humlová et al., ) , and several vacv recombinants (rvacv) expressing reporter genes. all the recombinants were prepared by homologous recombination into the wt vacv (strain wr) tk gene and reveal similar growth properties in vitro. rvacv expressing luciferase either under control of a vacv early/late promoter p . (luc e/l) or under control of a vacv late promoter p b (luc l) were described previously (rodriguez et al., ; rodriguez and smith, ) . rvacv expressing chloramphenicol acetyltransferase or bcl- genes under control of an iptg-inducible vacv late promoter p b were generated and kindly provided by dr. s.b. lee (lee et al., . the viruses were propagated in bsc- cell line in dmem supplemented with % ncs and antibiotics ( % ncs-dmem) and virus titers were determined by serial dilutions and plaque assays in bsc- cells. for virus yields determination, the infected cells were collected directly in the culture medium, lysed by two cycles of freezing-thawing and sonication, and virus yields were determined by -fold dilutions in dmem and plaque assays in bsc- cells. briefly, bsc- cells were plated in -well plates at a density of . × cells per well in % ncs-dmem. and h later, l of each dilution was allowed to adsorb to bsc- cells for h with shaking every - min. after removal of virus inocula, cells were supplemented with % ncs-dmem and incubated for about h. then, the cells were fixed with % paraformaldehyde in pbs and stained with % crystal violet in pbs with addition of % ethanol. virus plaques were counted and virus yields expressed as pfu/ml. plaque reduction assays were performed using a similar protocol with minor modifications. briefly, the cells were infected with approximately pfu of a crude stock of wt vacv or rvacv lucl and fixed and stained at h.p.i. for the experiments, adherent cells were plated in fresh culture medium h before each experiment in -or -well plates at a density of . × or . × cells per well, respectively. suspension cells were diluted with a fresh culture medium h before the experiments. before infection, cells were washed once with serumfree medium. virus inocula were added in a serum-free medium at a multiplicity of infection (m.o.i.) = and allowed to adsorb for h with shaking every - min. crude stocks of the viruses were used for infection of hela g, bsc- and l cell lines. for infection of macrophage and t-cell lines, viruses were purified through a sucrose gradient (joklik, ) . after removal of virus inocula, cells were supplemented with % ncs-dmem (hela g and bsc- ), % fbs-dmem (l and j .g ) or % fbs-rpmi (jurkat), and with appropriate concentrations of the tested compound or vehiculum. at h after infection (h.p.i.) or at the time indicated in each experiment, the cells were collected, processed and analyzed by serial dilutions and plaque assays, by measuring luciferase activity, western blot analysis or real-time pcr. the inhibitory concentration of each compound was then expressed as ic and ic , the concentrations of the tested compound that reduced virus growth or luciferase activity to or % of mock-treated controls, respectively. these values were calculated from the linear or exponential regressions characterizing the dependence of virus yields or luciferase activity on the concentration of the tested compounds. hela g cells were plated in -well plates at a density of . × cells per well in % ncs-dmem. h later, the cells were infected with a crude stock of rvacv luc l (rodriguez et al., ; rodriguez and smith, ) as described above. m ea, m la, or vehicula were included during inoculum and during the whole incubation period or added at various h.p.i. at h.p.i., the cells were collected and virus growth characterized by luciferase activity. . × of the collected cells were washed twice with pbs and processed according to a published protocol with minor modifications (brasier et al., ; rodriguez et al., ) . briefly, the cells were lysed in l of luciferase extraction buffer containing mm glycylglycine, ph . , % triton x- , mm mgso , mm egta, mm dtt and protease inhibitors (complete, roche, basel, switzerland). luciferase activity was then determined with mm atp, ph . , and . mm luciferin in mm hepes, ph . , mm nacl, mm edta, mm mgcl , and . % polyethylene glycol, using a luminometer microlite tlx (dynatech laboratories). . × of cells were collected and lysed in a laemmli reducing sample buffer, boiled and analyzed by sds-page and western blotting as previously described (ausubel et al., ) . vacv polypeptides were detected with a rabbit antiserum against vacv (dilution : ; antiserum raised by immunization with a live vacv, generated and kindly provided by drs. d. and j.r. rodriguez ( fig. a ; (rodriguez et al., ) , or with an inactivated purified vacv, prepared by seva-imuno praha, czech republic ( fig. b) ), peroxidase-conjugated goat anti-rabbit igg (dilutions : ; mp biomedicals -cappel, solon, oh) and a chromogenic substrate ␣chloronaphtol. luciferase was detected with a rabbit polyclonal antibody (dilution : ; santa cruz biotechnology, santa cruz, ca), peroxidase-conjugated goat anti-rabbit igg (dilution : , ; mp biomedicals -cappel, solon, oh) and chemiluminescence (west femto, thermo fisher scientific-pierce, rockford, il). the cells were collected by pipetting, washed twice with pbs and total dna was isolated using a treatment with proteinase k and a phenol-chloroform extraction (ea samples; (ausubel et al., ) or using a pcr lysis buffer containing proteinase k (la samples; (schmidtmayerova et al., ) . vacv dna was then quantified by real-time pcr, using the applied biosystems real-time pcr system. primers and probes were designed by applied biosystems as custom taqman ® gene expression assay. the set of primers and the probe were designed to the end-terminal part of vaccinia virus genome, bps - (forward primer, bps - , cgtatca-cactattgagacagaaaaagaaga; reverse primer, bps - , gacactatattccggtttgcaaaaca; fam probe, bps - , tcgcgagaggtaactttttgtga; strain western reserve, genbank accession no. ay ). for the absolute quantification of the vacv growth, a standard containing the amplification site cloned into pcr ® blunt-topo was generated by generi-biotech (hradec kralove, czech republic). to generate the calibration curve, the standard was diluted to . × to . × copies/reaction. ng of the total dna were subjected to real-time pcr in a reaction mixture consisting of the taqman ® universal pcr master mix (no amperase ® ung, applied biosystems, foster city, ca) and the assay mix containing the set of primers and the labeled probe (custom taqman ® gene expression assays, applied biosystems, foster city, ca). the reaction was carried out in a final volume of l, and the universal thermal cycling conditions were used: initial denaturation • c/ min and cycles consisting of denaturation • c/ s and annealing/extension • c/ min. all samples, including standards and the non-template control, were run in duplicates. the data were analyzed by the sequence detection software version . (applied biosystems, foster city, ca). cytotoxicity of the tested compounds was characterized by the inhibition of cell growth using a protocol adapted according to tox- kit (sigma co., st. louis, mo). briefly, adherent cells were plated in -well plates at a density of . × ml − /well in culture medium. and h later, the cells were supplied with a fresh medium containing increasing concentrations of the individual compounds. suspension cells were diluted with a fresh culture medium and h later, they were plated in -well plates at a density of . × ml − /well in culture medium containing increasing concentrations of the individual compounds. after days of incubation, the cell growth and viability were characterized by activity of mitochondrial dehydrogenases using the mtt assay. the conversion of mtt to formazan was determined photometrically at nm after dissolving the product in an acidified isopropanol. the cytotoxic concentration was expressed as cc , the concentration of the tested compound that reduced cell growth to % as compared to vehiculum-treated controls. results are presented as means ± s.d. (standard deviation). statistical differences within each group were evaluated using anova. statistical differences between the mock-treated control and different concentrations of each compound or between two distinct groups were determined using a two-sample two-tailed student's t-test. table antiviral and cytotoxic effects of the ethacrynic and ␣-lipoic acids against vaccinia virus in different cell lines. . × of cells of different origins were infected with the recombinant vacvs expressing luciferase; m.o.i. = . after removal of virus inoculum, the cells were treated with the indicated concentration of the ethacrynic or ␣-lipoic acids. macrophages were also pre-treated with u/ml of ifn␥. at h.p.i., the cells were collected and virus yields or luciferase activity were determined. the inhibitory concentration of each compound was expressed as ic and ic , the concentration required to inhibit virus growth characterized by luciferase activity or virus titers by or %, respectively. for cytotoxicity experiments, . × of cells were treated with increasing concentrations of the tested compounds. after days of incubation, cytotoxicity was characterized using the mtt assay. cc , the concentration of the tested compound that reduced activity of mitochondrial dehydrogenases to % compared to vehiculum-treated controls. the results represent means of - experiments performed in duplicates ± s.d. lipoic acid luc titer luc titer we have observed that two redox-modulating agents, ea and la, inhibit growth of vacv in vitro. therefore, we have first compared the effect of different redox-modulating agents on the growth of vacv, characterized by activity of luciferase expressed by a rvacv. hela g cells were infected with a rvacv expressing luciferase under control of a vacv late promoter p b (luc l; the results indicated that all tested concentrations of ␤mercapthoethanol, dithiothreitol and l-ascorbic acid significantly increased luciferase activity expressed by rvacv ( m concentration of these agents increased luciferase activity to , , and % of the mock-treated controls; p < . ). in contrast, la and ea revealed statistically significant inhibitory effects (p < . ). luciferase activity was decreased to % of the mock-treated controls in the presence of or m la and to % in the presence of mm la. the inhibitory effect of ea was stronger-luciferase activity was decreased to % and % in the presence of or m ea, respectively, while it was almost completely abolished in the presence of m ea. in conclusion, ea and la revealed inhibitory effects on vacv growth, while the other agents, ␤mercapthoethanol, dithiothreitol or l-ascorbic acid, promoted it. based on the findings described above, we decided to further characterize the effects of ea and la on vacv growth in cell lines of different origin-in human and monkey epithelial cell lines hela g and bsc- , respectively, mouse fibroblasts l , mouse macrophages j .g untreated and pre-treated with interferon ␥ table . in general, values of ic and ic were lower for rvacv than for wt vacv, and values of ic were lower when calculated from virus titers than from luciferase activity. for both rvacv and wt vacv, values of ic ( . and . m, respectively) and ic ( . and . m, respectively) were the lowest in hela g cells treated with ea. for the rest of the cells, values of ic of ea were in the range of - m for both viruses. in the case of la, values of ic were in the range of - m for rvacv and - m for wt vacv. consequently, we have tested cytotoxicity and growth inhibitory properties of ea and la in actively dividing cells by determining activity of mitochondrial dehydrogenases using the mtt assay. as indicated in table , ea affected growth and viability of epithelial cells hela g and bsc- , and of fibroblasts l relatively modestly. in contrast, macrophages j .g and especially jurkat t-cells were more inhibited by this compound, resulting in low selectivity indices (si). analogously, la was not found to inhibit growth of hela g, bsc- and l cells up to mm (on the contrary, it promoted the cell growth), while it inhibited growth of macrophages j .g at relatively low concentrations (cc = m) and started to inhibit jurkat cells at mm, the highest concentration tested. on the other hand, neither ea nor la induced any obvious cytotoxicity in conditions of the antiviral assays (i.e. higher cell density, % serum, h incubation) up to m and mm, respectively (data not shown; also see fig. ). to further evaluate the effect of ea and la on vacv growth, plaque reduction assays were performed. since vacv does not generate virus plaques in hela g cells, bsc- cells were used. they were plated in -well plates and infected with about pfu of wt vacv or rvacv in the presence of increasing doses of ea and la for h. as shown in fig. , m ea reduced the number of plaques formed by wt vacv in these cells, but their size remained almost unchanged. in contrast, m la reduced the size of plaques without affecting their number. similar results were obtained also with rvacv luc l (not shown). in conclusion, both ea and la revealed inhibitory effects on vacv growth and luciferase activity in all cell lines tested. based on the results of the mtt assays, both compounds were found relatively non-toxic for the epithelial and fibroblast cell lines (si indices - for ea; promotion of growth by la), while they revealed inhibitory effects on macrophage and t-cell lines. . at h.p.i., the cells were collected and lysed, and luciferase activity was determined in cell lysates. (a) ethacrynic acid. graph represents means of independent experiments performed in duplicates ± s.d. *significant difference between luc e/l and luc l groups (p < . ); two-sample t-test. (b) ethacrynic acid and arac. graph represents means of independent experiments performed in duplicates ± s.d. *significant difference between luc e/l and luc l groups (p < . ); two-sample t-test. (c) ␣-lipoic acid. graph represents means of independent experiments performed in duplicates ± s.d.*, significant difference between luc e/l and luc l groups (p < . ); two-sample t-test. (d) ␣-lipoic acid and ara c. graph represents means of independent experiments performed in duplicates ± s.d. *significant difference between luc e/l and luc l groups (p < . ); two-sample t-test. a possible effect of the ea and la on vacv entry into the cells was tested in hela g and bsc- cells. the cells were pretreated or mock-treated for h before infection with the individual agents in appropriate concentrations and the agents or vehicula were included also in the virus inoculum. the agents were then added also to the culture medium after removal of the inoculum, i.e. h after infection (h.p.i.), and they were present during the whole incubation period. then, activity of luciferase and virus titers were determined. however, the pre-treatment of the cells with ea or la was not found to cause any differences in comparison with the samples treated only at h.p.i. (data not shown). consequently, we wanted to characterize the step in vacv growth cycle that was inhibited by ea and la. vacv growth cycle is a sequential process in which a successful accomplishment of one step is pre-requisite for the next one (schramm and locker, ) . thus, expression of vacv late genes governed by vacv late promoters can occur only after replication of vacv dna. consequently, recombinant vacvs expressing luciferase under control of two different promoters, the early/late p . (luc e/l) and the late p b (luc l), in combination with the inhibition of vacv dna synthesis by cytosine arabinoside (arac), can be used to distinguish if the inhibitory effect occurs before or after dna synthesis. hela g cells were infected with luc e/l or luc l in the absence or presence of arac, an inhibitor of dna polymerase, and treated with indicated concentrations of ea or la. concentration of arac used, . g/ml, was experimentally determined to inhibit vacv growth and not to induce death of the host cells. at h.p.i., the cells were collected and activity of luciferase was determined. in agreement with the previous experiments, both ea and la revealed a dosedependent inhibitory effect on activity of luc e/l and luc l in the absence of arac ( fig. a and c) . in the presence of arac, the activity of luciferase expressed by luc l was decreased to approximately . % or less of the mock-treated controls grown in the absence of arac, i.e. to the levels very close to the background; neither ea nor la affected this result ( fig. b and d, respectively) . in contrast, the activity of luciferase expressed by luc e/l in the presence of arac was decreased less, only to about or % of the controls, due to the persisting expression of luciferase governed by the early portion of the promoter p . . different concentrations of neither ea nor la further decreased this measurable activity of luciferase ( fig. b and d, respectively), suggesting that neither ea nor la affect vacv early gene expression. additionally, we have analyzed the protein levels of luciferase in the samples used to determine luciferase activity by western blot analysis. as shown in fig. , both ea and la caused a gradual decrease in the levels of luciferase expressed by either luc e/l or luc l in the absence of arac, while no signal could be detected in the presence of arac. these results reflect a different sensitivity of the assays determining activity of luciferase and of western blot analysis. next, we analyzed the overall levels of vacv proteins in hela g cells infected with different recombinant vacvs by western blotting. as shown in fig. a , m ea did not seem to decrease protein levels of two different recombinant vacvs, m ea induced a little decrease, while m ea almost completely inhibited vacv protein expression. similarly, la did not induce any major changes in protein levels of two distinct recombinant vacvs in lower concentrations, while the highest concentration used, mm, decreased the protein expression slightly (fig. b) . the overall pattern of vacv proteins expressed seemed to be qualitatively comparable in all samples, resembling the pattern of vacv late proteins expressed in the mock-treated controls. it should be also noted, that the concentrations of ea or la that were found to inhibit vacv growth in hela g cells in the previous experiments, did not seem to considerably affect vacv protein levels. thus, the inhibitory effects of ea or la seemed to affect vacv late gene expression. since levels of the reporter gene luciferase expressed under control of a vacv late promoter p b as well as the overall levels of vacv late proteins were found to be decreased in the presence of ea or la, the inhibition of vacv growth mediated by ea or la was likely to occur at the level of dna replication or expression of vacv late proteins. to analyze the effect of ea and la on vacv dna synthesis, we have quantified number of copies of the vacv genomic dna during a -h time course using real-time pcr. however, the absolute quantification using an external standard and a calibration curve indicated that levels of vacv genomic dna were not changed in fig. s ). thus, these two agents seem to inhibit vacv late gene expression. to further analyze the effects of ea and la at distinct stages of vaccinia virus growth cycle, a time of addition assay was performed. hela g cells were infected with rvacv luc l in which expression of luciferase occurs only after dna synthesis, and the two compounds were added at indicated times post-infection. at h.p.i., the cells were collected, lysed and activity of luciferase determined. as shown in fig. , addition of m ea at any time tested significantly decreased activity of luciferase, with the inhibitory effect being smaller when ea was added at h.p.i. addition of m la significantly decreased activity of luciferase when added up to h.p.i., i.e. the time of beginning of the vacv dna synthesis. a smaller but significant inhibition could be observed also when la was added at h.p.i., the time when vacv dna synthesis was essentially completed as indicated by real-time pcr (supplementary data fig. . effect of the time of addition of ethacrynic and ␣-lipoic acids on vacv growth characterized by activity of luciferase. a total of . × of hela g cells were infected with recombinant vacv luc l at m.o.i. = . m ethacrynic acid, m ␣-lipoic acid, or vehicula were added at indicated h.p.i. at h.p.i., the cells were collected and lysed, and luciferase activity was determined. the graph represents means of two independent experiments performed in duplicates ± s.d.; *significant difference between the indicated concentration of ea or la and the mock-treated controls (p < . ); two-sample t-test. fig. s ). no significant inhibition was observed when la was added at h.p.i. these results are compatible with the previous conclusion that ea and la seemed to inhibit vacv late gene expression. in this paper, we report a new finding that ethacrynic and ␣lipoic acids reveal inhibitory effects on the growth of vaccinia virus in several cell lines. both agents seem to inhibit vacv at the same stage of its growth cycle, i.e. at the level of expression of late genes. ea has been found to be effective in low micromolar range, while la was effective in high micromolar range. first, we have compared the inhibitory properties of ea and la with other redox-modulating agents, ␤-mercaptoethanol, dtt and ascorbic acid. in contrast to ea and la, none of these agents inhibited vacv growth in hela g cells characterized by activity of the reporter luciferase expressed by a rvacv. since ␤-mercaptoethanol and dtt are prototypical reducing agents, ascorbic and ␣-lipoic acids act predominantly as anti-oxidants, while ea acts as a prooxidant, it seems difficult to find a common denominator for ea and la that might help to suggest their mode of action. nevertheless, we assume that both ea and la affect some specific thiol-dependent function in the vacv growth cycle. it is possible to imagine, that the two agents might interfere with disulfide bond formation and/or exchange or with redox cycling of a thioredoxin, glutaredoxin, or similar compounds. the difference in the effective concentrations of ea and la may reflect their mode of modification of the thiol moieties. ea is an alkylating agent (bowes and gupta, ; han et al., ) and it could irreversibly bind to the essential cysteine residues. in contrast, action of la may be reversible due to its redox cycling (packer et al., ) . alternatively, it is possible to suggest that the net effect of both ea and la would be pro-oxidant. ea inhibits gst, and thus inhibits regeneration of the gsh pool. in contrast, la promotes regeneration of gsh. however, dihydrolipoic acid (dhla) that is supposed to form quickly from la inside the cell has been suggested to release iron from its storage protein ferritin and reduce fe + to fe + ; fe + can initiate lipid peroxidation, and consequently decrease gsh levels (packer et al., ) . in any case, the effects of ea and la on vacv late gene expression might be also indirect, possibly through the action in the same cascade of events but at distinct steps. evidently, the same tasks cannot be fulfilled by ␤mercaptoethanol, dtt or ascorbic acid, the compounds that favor reducing conditions. on the contrary, these compounds increased vacv growth, suggesting that different redox conditions differently influence vacv growth. these effects might be related to a cytoplasmic redox pathway that is encoded by vacv, as discussed below. both ea and la were shown to negatively regulate nf-b, possibly through different mechanisms. ea was suggested to inhibit activation of the nf-b pathway at multiple points, including covalent modification of nf-b itself and inhibition of its binding to dna (han et al., ) , while la is considered to ameliorate redox stress and to decrease nf-b activation in this way (kiemer et al., ; petersen shay et al., ) . along these lines, la can decrease hiv provirus reactivation (merin et al., ; pande and ramos, ) . in contrast, vacv itself inhibits nf-b pathway through several gene products (graham et al., ) . it is, therefore, unclear if inhibition of nf-b by ea or la could further inhibit nf-b activation, and prevent vacv growth in this way. we have tested the inhibitory effects of ea and la on vacv growth using several recombinant vacvs and wt vacv. the effects were characterized by virus titers and activity of luciferase in cells of different embryonic origin. it should be emphasized that all the recombinant viruses used in this study reveal similar growth properties in vitro. the inhibitory concentrations were found lower for rvacv than for wt vacv, and values of ic were lower when calculated from virus titers than from luciferase activity. based on ic , ea was most effective in hela g cells, while la was so in macrophages. for the rest of the cells, values of ic for ea were in the range of - m for both viruses, while for la, values of ic were in the range of - m for rvacv and - m for wt vacv. under conditions of the antiviral assays, the two compounds did not induce any obvious cytotoxic effects in any concentration used, except for jurkat cells in which m ea seemed to be toxic, as assessed by optical microscopy. however, based on the results of the mtt assays in actively dividing cells, ea and la were found relatively non-toxic only for the epithelial and fibroblast cell lines, while they revealed inhibitory effects on macrophage and t-cell lines. possibly, different redox milieu of the distinct cell types might be responsible for different effects of the two agents. alternatively, different pattern of expression of gst isoenzymes and their interaction with ea or the elimination of ea from the cell may play a role (gate and tew, ) . ea and la inhibited vacv growth even in macrophages stimulated with ifn␥, in which vacv growth was already potently inhibited. this inhibition has been known to be at least partially mediated by ifn␥-induced expression of inos and production of nitric oxide (karupiah et al., ; melková and esteban, , ) . in this respect, la has been shown to inhibit lps-induced nf-b-and ap- -mediated inos and tnf-␣ expression in kupffer cells and raw . macrophages (kiemer et al., ) . similarly, we have observed that addition of ␤-mercaptoethanol decreased accumulation of nitrite, a stable oxidation product of nitric oxide, and increased vacv growth in ifn␥-stimulated j .g macrophages (melková, ) . in the assays performed in this study, ic values for ea were found in the range of - m in most cells using wt vacv (strain wr), while for la in the range - m. based on the published results, ic values for cidofovir and a prodrug of hpmp- -azac (hexadecyloxyethylester of hpmp- -azac) were found . and . g/ml in human embryonic lung fibroblasts hel using vacv (strain lederle; krecmerová et al., ) . in another study, ic value for cidofovir was found and m in vero cells, and m in human foreskin fibroblasts hff, and < m in bsc- cells using two different vacvs (strain wr; becker et al., ). yet in another study, ic values for st- , cmx and cidofovir were found . , . , and m, respectively, in hff cells using vacv (strain wr; quenelle et al., ) . it is, however, difficult to compare the ic values determined in different assays under different conditions. our assays were performed using m.o.i. = and h incubation period, while most other assays published employed much lower m.o.i. and usually day incubation. finally, we have tried to identify a step in vacv growth cycle inhibited by ea and la in hela g cells. neither ea nor la were found to affect virus entry into the cell. in contrast, several lines of evidence suggest, that the affected step is at the level of virus late gene expression, or possibly later. first, we used recombinant viruses expressing luciferase under control of two different promoters, the early/late p . (luc e/l) and the late p b (luc l). in combination with the inhibition of vacv dna synthesis by arac, these viruses can be used to distinguish if the inhibited process occurs before or after dna synthesis. the overall activity, as well as protein levels of the reporter luciferase expressed by both viruses, were found decreased in hela g cells in the presence of increasing concentrations of ea or la. it should be emphasized that uninhibited, absolute activities of luciferase expressed by luc l are several times higher than those expressed by luc e/l. upon inhibition of vacv dna synthesis by arac, levels of luciferase expressed by either rvacv were undetectable by western blot analysis. in the presence of arac, the activity of luciferase expressed by luc l was strongly inhibited, while the activity of luciferase expressed by luc e/l was decreased less, with absolute levels several times higher than those in luc linfected samples; these results confirm that luciferase expression by luc l really occurs after dna synthesis, while luciferase expression by luc e/l occurs also earlier, before dna synthesis takes place. however, neither ea nor la decreased luciferase activity expressed under control of the early promoter, suggesting that these agents did not affect expression of early genes. second evidence supporting the hypothesis that ea and la inhibit vacv late gene expression is western blot analysis revealing that ea and la somewhat affected the levels of vacv proteins, while they did not change the overall pattern of vacv late proteins. third, quantification of vacv dna by real-time pcr yielded no differences in dna levels due to ea or la treatments, the result demonstrating that the inhibitory action of ea and la takes place at the level of late gene expression or later. this conclusion is further supported by results of the time of addition assays. they indicated that addition of ea or la up to h.p.i., i.e. the time when dna synthesis was accomplished according to real-time pcr, significantly decreased virus growth characterized by luciferase activity. it should be noted that concentrations of ea that inhibited vacv growth by or logs in hela g cells ( or m ea, respectively), did not seem to considerably affect the overall vacv protein levels as characterized by western blot analysis. analogous results could be observed with la. similarly, ic values were found lower when calculated from virus titers than from luciferase activity. thus, these findings might suggest that ea and la could additionally act in a step after expression of vacv late proteins, i.e. during virus morphogenesis. vacv morphogenesis involves a cytoplasmic redox pathway constituted by several viral enzymes that catalyze formation of intramolecular disulfide bonds within the cytoplasmic domains of certain virion membrane proteins . these virion membrane proteins contain multiple cysteines that participate in virus particle formation, maturation or in virus entry and cell-cell fusion (ojeda et al., ; townsley et al., ) . it can be therefore expected that both ea and la would interfere with these processes as well. ea derivatives, namely amids, were suggested as new lead structures for non-peptidic active-site-directed inhibitors of the coronavirus main protease, m pro (kaeppler et al., ) . this protease belongs to the cystein protease family, the members of which play important roles in host cell physiology and pathology, as well as at various stages of life cycle of the viruses and other parasites (johnston et al., ; vicik et al., ) . cystein proteases participate in proteolysis of virus polypeptides as well as in core maturation of several dna viruses, including vacv (alejo et al., ; byrd et al., ; byrd and hruby, ; greber, ) . it is therefore possible that ea could also inhibit vacv growth at this stage. these and other effects of ea and la would need to be explored in a different set of experiments. additionally, the antiviral effects of these compounds should be evaluated in experiments in mice. in conclusion, we demonstrate inhibitory properties of ea and la on the growth of vacv. this inhibition was found to occur at the level of vacv late gene expression. the results suggest new directions in development of drugs effective against poxvirus infection and bring new information about the scope of the effects of ea and la. african swine fever virus proteinase is essential for core maturation and infectivity risks of serious complications and death from smallpox vaccination: a systematic review of the united states experience myopericarditis following smallpox vaccination new generation smallpox vaccines: a review of preclinical and clinical data isolation and characterization of cidofovir resistant vaccinia viruses induction of mitochondrial fusion by cysteinealkylators ethacrynic acid and n-ethylmaleimide optimized use of the firefly luciferase assay as a reporter gene in mammalian cell lines progressive vaccinia vaccinia virus 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of nitric oxide synthase bcl- prevents nitric oxidemediated apoptosis and poly(adp-ribose) polymerase cleavage status of the mitochondrial pool of glutathione in the isolated hepatocyte alpha-lipoic acid blocks hiv- ltr-dependent expression of hygromycin resistance in thp- stable transformants entry of vaccinia virus and cell-cell fusion require a highly conserved cysteine-rich membrane protein encoded by the a l gene alpha-lipoic acid as a biological antioxidant. free radic nuclear factor kappa b: a potential target for anti-hiv chemotherapy efficacy of therapeutic intervention with an oral ether-lipid analogue of cidofovir (cmx ) in a lethal mousepox model is alpha-lipoic acid a scavenger of reactive oxygen species in vivo? evidence for its initiation of stress signaling pathways that promote endogenous antioxidant capacity combinatorial optimization of isatin-beta-thiosemicarbazones as anti-poxvirus agents in vitro and in vivo evaluation of isatin-[beta]-thiosemicarbazone and marboran against vaccinia and cowpox virus infections synergistic efficacy of the combination of st- with cmx against orthopoxviruses the detection of monkeypox in humans in the western hemisphere inducible gene expression from vaccinia virus vectors expression of the firefly luciferase gene in vaccinia virus: a highly sensitive gene marker to follow virus dissemination in tissues of infected animals insertional inactivation of the vaccinia virus -kilodalton gene is associated with attenuation in mice and reduction of viral gene expression in polarized epithelial cells clinical uses of cidofovir the growth in vitro of single isolated tissue cells studies on the propagation in vitro of poliomyelitis viruses. iv. viral multiplication in a stable strain of human malignant epithelial cells (strain hela) derived from an epidermoid carcinoma of the cervix human immunodeficiency virus type t-lymphotropic strains enter macrophages via a cd -and cxcr -mediated pathway: replication is restricted at a postentry level cytoplasmic organization of poxvirus dna replication complete pathway for protein disulfide bond formation encoded by poxviruses a review of compounds exhibiting antiorthopoxvirus activity in animal models vaccinia virus a virion membrane protein is required for cell entry and fusion fc-receptor variants of a mouse macrophage cell line inhibitors of cysteine proteases vaccinia virus g l glutaredoxin is an essential intermediate of a cytoplasmic disulfide bond pathway required for virion assembly an orally bioavailable antipoxvirus compound (st- ) inhibits extracellular virus formation and protects mice from lethal orthopoxvirus challenge we are grateful to dr. sarka nemeckova for stimulating discussions and suggestions, and we thank dr. barbora lubyova with dr. karel holada for careful reading, helpful comments and corrections of the manuscript. the work of m.s. and z.c. was performed in partial fulfillment of the requirements for phd degree at the st medical faculty, charles university. the work was supported by the grant agency of the czech republic -projects nos. / / , and / /h , and by the ministry of education of the czech republic -project no. msm . supplementary data associated with this article can be found, in the online version, at doi: . /j.antiviral. . . . key: cord- -p b mtbl authors: theerawatanasirikul, sirin; kuo, chih jung; phecharat, nanthawan; chootip, jullada; lekcharoensuk, chalermpol; lekcharoensuk, porntippa title: structural-based virtual screening and in vitro assays for small molecules inhibiting the feline coronavirus cl protease as a surrogate platform for coronaviruses date: - - journal: antiviral res doi: . /j.antiviral. . sha: doc_id: cord_uid: p b mtbl feline infectious peritonitis (fip) which is caused by feline infectious peritonitis virus (fipv), a variant of feline coronavirus (fcov), is a member of family coronaviridae, together with severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), and sars-cov- . so far, neither effective vaccines nor approved antiviral therapeutics are currently available for the treatment of fipv infection. both human and animal covs shares similar functional proteins, particularly the cl protease ( cl(pro)), which plays the pivotal role on viral replication. we investigated the potential drug-liked compounds and their inhibitory interaction on the cl(pro) active sites of covs by the structural-bases virtual screening. fluorescence resonance energy transfer (fret) assay revealed that three out of twenty-eight compounds could hamper fipv cl(pro) activities with ic( ) of . ± . μm to . ± . μm, and ki values of . ± . to . ± . μm, respectively. evaluation of antiviral activity using cell-based assay showed that nsc and nsc could strongly inhibit the cytopathic effect and also reduced replication of fipv in crfk cells in all examined conditions with the low range of ec( ) ( . ± . to . ± . μm and . ± . to . ± . μm, respectively), less than those of ribavirin and lopinavir. analysis of fipv cl(pro)-ligand interaction demonstrated that the selected compounds reacted to the crucial residues (his and cys ) of catalytic dyad. our investigations provide a fundamental knowledge for the further development of antiviral agents and increase the number of anti-cov agent pools for feline coronavirus and other related covs. feline infectious peritonitis (fip), which is caused by feline infectious peritonitis virus (fipv), is a life threatening, immunopathogenic disease affecting multiple organs in cats (pedersen, a) . and % antibiotics-antimycotics (invitrogen tm , carlsbad, usa) at °c with %co for h. the cytopathic effect (cpe) was observed daily under an inverted microscope (olympus ckx , tokyo, japan). after the fifth passages of viral propagation, the virus stock was quantitated as decribed previously (lekcharoensuk, et al., ) presented as means and standard deviation, and qpcr data were analyzed and reported as the percentage of viral reduction for each compound. selectivity index (si) was determined as the ratio of cc to ec for each compound. in this study, the structure-based virtual screening was performed in which the compounds were placed within the binding pocket of the cl pro due to high similarity of amino acid residues among various covs. then, the most favorable binding energies between the compounds and the catalytic dyads within the binding pocket of covs- cl pro were analized. the binding affinity scores of the top-ranked compounds and covs- cl pro structure were ranged from - . to - . annotate the active sites of all covs, which their d structures were highly conserved especially at his and cys numbering based on the positions on the fipv cl pro analysis of other covs- cl pro in respect to the fipv cl pro sequence exhibits highly conserved amino acid residues at leu , pro , val , phe , cys , his , glu , leu and asp ) in the catalytic dyad which strongly interact with the candidate compounds (supplementary figure d) . these results indicates that fipv cl pro has a potential use as a surrogate platform to screen the candidate compounds that could inhibit the covs- cl pro activities. according to the fipv cl pro structurral based study, we determined if the candidate compounds that could bind to the active site and inhibited the protease activity still actively impeded the viral growth in cell culture. the candidate compounds possessing the inhibitory effect on protease activity had ic values of . ± . μm (nsc ), . ± . μm (nsc ), and . ± . μm (nsc ) as shown in table . the ki values of these three compounds were . ± . , . ± . , and . ± . μm, respectively ( figure ) . in addition, the protease inhibition assay of pedv cl pro and sars-cov cl pro was also performed and the inhibition results are corresponding that of fipv cl pro (figure c ). after screening using in silico and in vitro protease assay, the three selected compounds were further examined for their cytotoxicity. the results showed that compound nsc had no effect on the crfk cells even though at the concentration as high as . μm, whereas the cc values of nsc and nsc were . ± . μm and . ± . μm, respectively (table ) . prophylaxis experiment was designed to allow the entry of small compounds into the host cells, and also determined that the compounds remaining in the supernatant still have a sufficient viral inhibition potency. we found that compounds nsc and nsc had inhibitory effect on fipv with ec = . ± . μm and . ± . μm, respectively. although, the protease inhibitory assay showed that nsc could strongly inhibit fipv cl pro activity with very low ki value (figure (figure ) . in this study, % dmso was used instead of the compound in the mock-treated fipv infected crfk cells as a non- inhibitor control, which was not influent on fipv infectivity (data not shown). we also studied the interaction of nsc and nsc with fipv cl pro binding pocket using molecular docking simulation and protein ligand interaction analysis ( figure ) . the results showed that the molecule of nsc folded and fit well within the active site of the protease (figures a) . two nitrogen atoms of a benzonitrile group formed hydrogen bonds with thr and his residues. the compound buried perfectly in the binding pocket and formed hydrophobic interaction with hydrophobic residues (amino acid residue: leu , val , and leu ), and asn , thr , his , and cys , respectively. in addition, cys residue can form non-covalent bonds, such as π-alkyl and π-donor interactions with benzofuran and nitrile functional groups, respectively. for nsc , the compound favorably located into the active site of fipv cl pro (figure b) . particularly, his residue in the catalytic dyad links with oxygen atom by hydrogen bond, and also form π-π stack bond to the -chlorophenol functional group of this compound. most hydrophobic bonds were well defined which involve amino acid residues: leu , thr , cys , leu , asp , gln and pro , respectively. in addition, pro can form amide-π stack with -chlorophenol functional group and form alkyl bond to the chloride atom of this functional group. cys and his , the active residues of fipv cl pro , formed the π-alkyl interaction and hydrogen bond to the compounds, respectively. the absorption, distribution, metabolism, excretion and toxicity (adme/t) properties of the three compounds are shown in supplementary table . all three compounds -nsc , nsc , and nsc - did not violate the druglikeness of lipinski's rule of five when analyzed using swissadme tract. moreover, compounds nsc and nsc might be able to permeate blood- brain barrier as shown in supplementary table clinical features of patients infected with novel coronavirus in wuhan triple combination of interferon beta- b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid- : an open-label, randomised, phase trial mechanism of the maturation process potent inhibition of feline coronaviruses with peptidyl compounds targeting coronavirus c-like protease broad-spectrum antivirals against c or c-like proteases of picornaviruses, noroviruses, and coronaviruses characterization of sars main protease and inhibitor assay using a fluorogenic substrate ligplot+: multiple ligand-protein interaction diagrams for drug discovery cloned cdna of a/swine/iowa/ / internal genes as a candidate backbone for reverse genetics vaccine against influenza a viruses chloroquine for covid- infection coronavirus emerging in china -key questions for impact assessment the nucleoside analog gs- strongly inhibits feline infectious peritonitis (fip) virus in tissue culture and experimental cat infection studies open babel: an open chemical toolbox the nucleoside analog gs- strongly inhibits feline infectious peritonitis (fip) virus in tissue culture and experimental cat infection studies remdesivir in adults with severe covid- : a randomised, double-blind, placebo-controlled, multicentre trial inhibitory effects of ribavirin alone or combined with human alpha interferon on feline infectious peritonitis virus replication in vitro update on antiviral therapies. august's consult the interactions are presented as dashed lines for alkyl/π-alkyl bonds (pink), π-donor (dark blue), hydrogen bonds (green) and hydrophobic interaction (red circles and ellipses) in the right panel. the visualizations are generated using ucsf chimera version . . and ligplot software version . . of tgev and pedv and also shared superclustering with the betacoronaviruses (sars-cov, sars-cov- and mers-cov) (a). the analysis of covs- cl pro alignment by t-coffee and visualized by jalview analysis of complexes between covs- cl pro with the two best compounds revealed that both compounds are completely buried within the binding pockets. the nsc and nsc are presented in cyan and dark purple key: cord- -n dwcx authors: levanova, alesia a.; kalke, kiira m.; lund, liisa m.; sipari, nina; sadeghi, mohammadreza; nyman, marie c.; paavilainen, henrik; hukkanen, veijo; poranen, minna m. title: enzymatically synthesized '-fluoro-modified dicer-substrate sirna swarms against herpes simplex virus demonstrate enhanced antiviral efficacy and low cytotoxicity date: - - journal: antiviral res doi: . /j.antiviral. . sha: doc_id: cord_uid: n dwcx chemical modifications of small interfering (si)rnas are used to enhance their stability and potency, and to reduce possible off-target effects, including immunogenicity. we have earlier introduced highly effective antiviral sirna swarms against herpes simplex virus (hsv), targeting bp of the essential ul viral gene. here, we report a method for enzymatic production and antiviral use of ’-fluoro-modified sirna swarms. utilizing the rna-dependent rna polymerase from bacteriophage phi , we produced ’-f-sirna swarms containing either all or a fraction of modified adenosine, cytidine or uridine residues in the antisense strand of the ul target. the sirna containing modified pyrimidines demonstrated high resistance to rnase a and the antiviral potency of all the ul -specific ’-f-sirna swarms was -fold in comparison with the unmodified counterpart, without additional cytotoxicity. modest stimulation of innate immunity signaling, including induced expression of both type i and type iii interferons, as well as interferon-stimulated gene , by ’-f-cytidine and ’-f-uridine modified sirna swarms occurred at early time points after transfection while the ’-f-adenosine-containing sirna was similar to the unmodified antiviral sirna swarm in this respect. the antiviral efficacy of the ’-f-sirna swarms and the elicited cellular innate responses did not correlate suggesting that innate immunity pathways do not significantly contribute to the observed enhanced antiviral activity of the modified sirnas. the results support further applications of enzymatically produced sirna molecules with incorporated adenosine nucleotides, carrying fluoro-modification on ribose c ’ position, for further antiviral studies in vitro and in vivo. rna interference (rnai) is a natural antiviral defense mechanism in plants, fungi, invertebrates, and, under specific conditions, in mammals schuster et al., ) . antiviral rnai comes about via a sequence-specific binding of small interfering rnas (sirnas) to viral mrnas or genomic rnas, resulting in their degradation or translational repression. in general, sirna is prone to fast degradation with rnases, and the delivery to the target cells might be difficult. a number of chemical modifications introduced to different positions in the sirna (sense, antisense, or both strands) have been tested to improve sirna stability and reduce offtarget activities (braasch et al., ; choung et al., ; czauderna et al., ; fedorov et al., ; jackson et al., ; watts et al., ; hassler et al., ) . instead of chemical synthesis and post-synthesis hybridization of rna oligonucleotides, we produce high-quality molecules for rnai using viral rna polymerases and recombinant giardia dicer enzyme (levanova and poranen, ) . this results in a swarm of -bp-long dicer-substrate sirnas (dsirnas) (macrae et al., ; romanovskaya et al., ) , which can cover several kbp sequence of the target (nygardas et al., ; niehl et al., ; jiang et al., ) . when introduced into a cell, such sirnas are processed by the endogenous dicer, which enhances the rnai potency and efficacy (kim et al., ) . in the enzymatically synthesized sirna swarms, each individual sirna species is present only in low concentrations, diluting out the potential off-target effects (levanova and poranen, ) . the sirna swarm contains predominantly '-monophosphates as only the single sirnas originating from the very ends of the produced dsrna molecules contain the '-triphosphate moiety, which could induce innate immunity responses. the swarm, comprising of numerous different sirnas against the j o u r n a l p r e -p r o o f target, also widens the antiviral spectrum (paavilainen et al., ; jiang et al., ) and minimizes the potential of emergence of viral variants resistant to a single sirna. we have previously generated three antiviral sirna swarms against herpes simplex virus type (hsv- ) mrnas encoding essential viral proteins, including glycoprotein b (ul ), the infected cell protein (icp ; ul ), and icp (ul ) (romanovskaya et al., ; paavilainen et al., ) . the sirna swarm targeting mrna of hsv single-stranded dna binding protein icp , encoded by the ul gene, harbors a most pronounced antiviral effect (paavilainen et al., ) and induces only minimal non-specific cellular responses (romanovskaya et al., ) . the hsv ul -derived sirna swarm elicits modest ifn-λ (il- ), ifn-λ / , isg , and tlr expression . however, the observed slight activation of innate immune responses did not cause a decrease in cell viability (romanovskaya et al., ; paavilainen et al., ) . the anti-hsv-ul sirna swarm also controlled hsv infection in mice and inhibited local virus replication in corneal epithelia in vivo (paavilainen et al., ) . here, we set up an enzymatic production method for modified dsirnas using t dnadependent rna polymerase (ddrp), phi rna-dependent rna polymerase (rdrp) and giardia dicer. using this method, we produced ul -specific sirna swarms harboring 'fluoro-modifications in the ribose moieties of uridine, adenosine or cytidine residues. the incorporated fluoro-modifications generally improved sirna stability. the '-f-sirna swarms were well tolerated by cells and had enhanced hsv-specific antiviral activity compared to unmodified sirna swarms. j o u r n a l p r e -p r o o f the bp sequence of the ul gene from hsv- prototype strain ( +) (genbank jn . , nucleotides , to , ) was pcr amplified to produce a dna template for ssrna production (romanovskaya et al., ) dicer (romanovskaya et al., ; paavilainen et al., ) . the sirna molecules were purified by anion-exchange chromatography on a cimac qa column (bia separations, slovenia, . - . ) using an Äktapurifier fplc system (ge healthcare) (romanovskaya et al., ) at the instruct-hilife biocomplex unit (university of helsinki). the same protocol was used to produce non-specific unmodified control sirna swarms from a bp long sequence of escherichia coli laci gene in pet b vector using primers: fwd_pet b_ : '-taatacgactcactatagggctgcctgcactaatgttcc- ' and rev_pet b_ : '-ggaaaaaaatcgtcgtatcccactacc- '. as confirmed with blastn, the selected j o u r n a l p r e -p r o o f control sequence has no significant similarities with human, herpesviral or mouse transcriptomes. the bp dsrna was produced as described previously (jiang et al., ) . chemidoc touch imaging system (bio-rad) and the band intensities measured using fiji (schindelin et al., ) . the relative amount of undegraded sirna was calculated according to the formula: a%=(s rnasea × )/s untreated , where s rnasea is a density of rnasea-treated sirna and s untreated is a density of the equivalent amount of untreated sample. an astrocytoma-glioma cell line (u mg) obtained originally from atcc (manassas, va) was used. the line has been subsequently reclassified as u human glioblastoma, but referred here as u mg to ensure continuity with our previous studies (paavilainen et al., , romanovskaya et al., ) . u mg cells were maintained in dmem with % heat j o u r n a l p r e -p r o o f inactivated fetal bovine serum (fbs) and mm l-glutamine at ˚c, % co . vero cells (atcc), used for plaque titration, were maintained in m medium with % fbs. green fluorescent protein (gfp)-expressing hsv- strain, hsv- ( +)lox-p mcmv gfp (abbreviated here as hsv- -gfp), originally received from prof. beate sodeik (mhh hannover medical school, germany), was used for antiviral studies (snijder et al., ; mattila et al., ) and propagated as previously described (romanovskaya et al., ) . the u mg cells were transfected on -well plates with either modified or unmodified sirna swarms, bp dsrna or water (mock transfection) using lipofectamine rnaimax (# - ; invitrogen, carlsbad, ca) according to the manufacturer's forward transfection protocol (romanovskaya et al., ) . depending on the experiment, the cells were transfected with - pmols of rna per well, which is equivalent to a concentration of - nm. all experiments were repeated at least twice with three or more biological replicates each. cellular viabilities in sirna treatments were assessed hours post transfection (hpt) with celltiter-glo (#g ; promega, madison, wi) luminescent assay (romanovskaya et al., ; turunen et al., ) . the luminescence was quantified with victor nivo multimode plate reader (perkin elmer, waltham, ma). viability of more than % compared to mock-and untreated cells was considered acceptable. j o u r n a l p r e -p r o o f the total cellular rna was extracted from sirna-treated u mg cells at , , and hpt with tri reagent (invitrogen, carlsbad, ca) according to the manufacturer's protocol. the rna was processed to complementary dna using revertaid h reverse transcriptase (thermo fisher scientific, waltham, ma) with random hexamer primers (thermo fisher scientific) after treatment with dnase (thermo fisher scientific) (romanovskaya et al., ; paavilainen et al., , . quantitative pcr (qpcr) was performed with rotor-gene q, as previously described (nygardas et al., ) . the gapdh housekeeping mrna values of the corresponding sample were used for normalization of mrna expression levels. the mrna expression of interferon beta (ifn-β) (peri et al., ) , lambda (il- ; ifn-λ ) (paavilainen et al., ) , interferon stimulated gene (isg ) (romanovskaya et al., ) , and gapdh (nygardas et al., ) was quantified using previously published primer sequences. the rna-or control-treated cells were challenged with a clinically relevant infectious dose of hsv- -gfp at plaque forming units ( we tested the capability of phi rdrp to utilize '-f-dntps and '-ome-ntps in the dsrna synthesis. hsv ul gene-specific ssrna, produced using t ddrp, was used as a template for the phi rdrp-catalyzed dsrna synthesis. initially, the reactions were done by replacing one of the canonical ntps in the reaction mixture with the corresponding modified ntp. although dsrna synthesis was not detected in the presence of '-ome-modified ntps (fig. s ) and the quality of the dsrna product was somewhat compromized in the presence of '-f-dgtp ( fig. s a) , phi rdrp was able to produce full-length dsrna products using '-f-datp, '-f-dutp, and '-f-dctp substrates (fig. a) , suggesting that the adenosines, uridines, and cytidines in the antisense strand of the hsv ul target sequence can potentially be substituted with their fluoro-modified counterparts. wild-type phi rdrp and a mutant rdrp j o u r n a l p r e -p r o o f containing substitution k→a at position (k a) were initially tested for dsrna production. the k a rdrp consistently provided higher dsrna yields (fig. s b) and was therefore used for the subsequent sirna production. complete replacement of a canonical rntp with the corresponding '-f-dntp resulted in slightly decreased yields, which was especially pronounced when all three ntps (atp, utp and ctp) carried the modification (fig. a) . therefore, to produce sirna swarms for the cellular experiments we used '-f-datp, '-f-dctp, or '-f-dutp, but not their mixture. furthermore, we set up a dsrna synthesis reaction in which only a fraction ( %) of a given ntp was fluoro-modified (fig. s ). the mass spectrometry analysis confirmed that the " % modified" dsrna products contained '-fluoromodified nucleotides (fig. s ) . furthermore, the giardia dicer could process the produced ul - '-f-dsrna into a swarm of dicer-substrate '-f-sirnas (fig. b) . rnase a treatment in low salinity conditions was performed to assess sensitivity of the '-f-sirna swarms to rnase. under the conditions used, about % of the unmodified sirna was degraded ( fig. c) , whereas fluoro-modifications in the uridine and cytidine residues significantly enhanced the stability of the sirnas. cellular toxicity of the '-f-sirna swarms was assessed using u mg cells in -well plates. cell were transfected with each of the hsv-specific ul -gene derived- '-f-sirna swarms, an unmodified ul -sirna swarm, a non-specific sirna swarm derived from e. coli laci gene, a j o u r n a l p r e -p r o o f cytotoxic -bp-long control dsrna, or water. the viability of the cells was assessed hpt with luminescent assay (fig. ) . transfections with the '-f-sirna swarms resulted in similar levels of cellular viability to that with unmodified ul -sirna swarms, with the exception of the fully '-f-dctp modified swarm, which was significantly less tolerated than the unmodified sirna swarm at and nm. nevertheless, all the '-f-sirna swarms were well tolerated, with relative cellular viability consistently higher than % at doses relevant for antiviral applications. hence, '-f-modifications had no effect on tolerability of the sirna swarms in the studied concentration range. the ul -targeting sirna swarms did not display increased cytotoxicity in comparison to the non-specific sirna swarm, targeting a non-relevant bacterial sequence. the bp dsrna used as a toxic control resulted in a clear decrease in cell viability at nm concentration (fig. ) . the cytotoxicity profile of the modified sirna swarms was confirmed with non-cancerous human corneal epithelial cells, in which only the fully '-f-dctp modified swarm was significantly less tolerated than the unmodified sirna swarm (fig. s ) . the antiviral efficacy of the ul - '-f-sirna swarms was quantified at hpt by plaque titration from culture supernatants of hsv- -gfp-infected u mg cells. before infection, cells were treated with hsv-targeted modified or unmodified ul -sirna swarms, a nonspecific sirna swarm, water (mock-treated), or left untreated (fig. ) . after treatment with the unmodified ul sirna swarm, the viral titer was reduced at least three orders of magnitude compared to the untreated samples (fig. a) . the antiviral effect of the ul - '-f-sirna swarms was even more pronounced, resulting in a five orders of magnitude reduction in hsv j o u r n a l p r e -p r o o f titer (fig. b ). compared to treatment with the non-specific sirna swarm, treatment with the modified and unmodified ul sirna swarms resulted in a significant decrease of titer (p = . and p = . , respectively), confirming the sequence specificity of the antiviral effects (fig. a) . furthermore, treatment with the ul - '-f-sirna swarms in comparison with the unmodified counterparts resulted in significantly lower titers (p = . ) ( fig. a) and an extensive decrease of viral shedding (p = . ) (fig. c) . the difference in antiviral efficacy between the % and % modified swarms was non-significant (fig. a) . nonetheless, the '-f-sirna swarms harboring modifications in cytidine or adenosine residues, i.e. '-f-dcmp-sirna and '-f-damp-sirna swarms respectively, yielded significantly higher antiviral potency compared to those having uridine modifications ( '-f-dump-sirna), when using the unmodified ul -sirna swarm as a reference (fig. b ). the % modified '-f-dump-sirna swarm was the only ul - '-f-sirna swarm that did not show improved antiviral efficacy in comparison with the unmodified ul -swarm (fig. b ). the efficacy of the hsv-targeted sirna swarms against hsv- -gfp infection on u mg cells was visualized by living cell fluorescent imaging at hpt (at hpi; fig. d ). the virusderived gfp signal was prominent in infected cells, untreated or control-treated, whereas in cells pretreated with unmodified ul -sirnas or ul - '-f-sirnas, the gfp signal could not be detected using standard imaging parameters. '-f-dump-and '-f-dcmp-sirnas induced higher ifn-β, il- and isg expression compared to unmodified ul -sirna swarm, particularly at the earliest time point (fig. ) . innate immunity signaling induced by '-f-damp-sirna swarms and that by the unmodified ul sirna were at equal levels, or even lower. overall, the induction of interferon signaling by modified or unmodified sirna swarms was not extensive (only ca. -fold compared to mock-and untreated cells). we have previously reported a novel enzymatic approach to synthetize large pools of sirna molecules, which we refer to as sirna swarms. the antiviral sirna swarms targeting the current target of choice, the ul gene of hsv- , have previously demonstrated their efficacy against circulating pathogenic hsv strains in vitro and corneal infection in vivo, unveiling their potential as antiviral drugs. in the current study, we pursued the modification of the rnas using nucleotides with '-modified ribose, in order to further enhance the antiviral potency and stability of the sirna preparations. eventually, we are able to report successful enzymatic incorporation of '-f-dntp nucleotides to biologically active dsrna molecules. all '-f-sirna swarms were well tolerated and highly effective against hsv- in vitro. the observed j o u r n a l p r e -p r o o f enhanced antiviral efficacy of the '-f-sirna swarms was unrelated to the elicited cellular innate immunity responses, proving the sequence specificity of the modified sirnas. we produced sirna swarms, in which either all or a fraction of adenosines, cytidines or uridines were replaced by '-f-dnmps. replacement of a '-oh group of ribose with a fluorine has been shown to stabilize rna molecules against nucleases (monia et al., ) . sirnas are primarily degraded by rnase a (turner et al., ) , which recognizes the '-oh of pyrimidine nucleosides for cleavage (cuchillo et al., ) . therefore, a common strategy is to modify all pyrimidines (hassler et al., ) to increase the sirna stability. accordingly, substitution of the canonical cmps or umps by the corresponding '-f-dnmps resulted in a significant increase in sirna stability in our experiments (fig. c) . conventionally, modifications are introduced into rna molecules using chemical solid phase synthesis. some wild-type or mutant ddrps of autographiviruses (t , syn ) can be used for enzymatic synthesis of single-stranded '-f-rnas (sousa and padilla, ; zhu et al., ) which may be further hybridized to produce dsrna. we demonstrate here that bacteriophage phi rdrp efficiently utilizes '-f-dntps allowing enzymatic production of perfectly duplexed dsrnas without the often error-prone post-synthesis hybridization. similarly, to other wild-type bacteriophage polymerases (sousa and padilla, ; zhu et al., ) , phi rdrp did not tolerate '-ome modifications (fig. s ) . all of the modified sirna swarms were non-toxic for u mg cells at the relevant antiviral concentrations. the findings were reproducible in human corneal epithelial cells (fig. s ) , which represent another potential target tissue for antiviral therapy of diseases caused by hsv- . furthermore, modified swarms had higher antiviral activity than the unmodified swarm, as demonstrated by the significant reduction of viral titer in plaque assays. notably, there was no difference between sirna swarms containing only a fraction of modified dnmps and those containing all modified dcmps, dumps, or damps in the antisense strand (fig. ) . the antiviral effect of sirnas containing only a fraction of modified dumps was not statistically different from the unmodified sirna swarm. interestingly, a higher stability to nucleases of sirna swarms containing '-f-dumps or '-f-dcmps did not translate into higher antiviral efficacy than those with '-f-damps. the immunostimulatory potential of '-f-damp-sirnas resembled that of the unmodified sirna swarm and was thus minimal. at the early time point ( hpt the authors declare no conflict of interest. zhu, b., hernandez, a., tan, m., wollenhaupt, j., tabor, s., richardson, c.c., . synthesis of '-fluoro rna by syn rna polymerase. nucleic acids res. , e . http://doi.org/ . /nar/gkv since the number of cmps in the sequence is significantly higher than that for amps or umps rna interference in mammalian cells by chemically-modified rna chemical modification of sirnas to improve serum stability without loss of efficacy bovine pancreatic ribonuclease: fifty years of the first enzymatic reaction mechanism structural variations and stabilising modifications of synthetic sirnas in mammalian cells off-target effects by sirna can induce toxic phenotype small rna-based antimicrobial immunity comparison of partially and fully chemically-modified sirna in conjugate-mediated delivery in vivo positionspecific chemical modification of sirnas reduces "off-target" transcript silencing innate immune responses in human monocyte-derived dendritic cells are highly dependent on the size and the ' phosphorylation of rna molecules efficient inhibition of avian and seasonal influenza a viruses by a virus-specific dicer-substrate small interfering rna swarm in human monocyte-derived macrophages and dendritic cells synthetic dsrna dicer substrates enhance rnai potency and efficacy rna interference as a prospective tool for the control of human viral infections structural basis for double-stranded rna processing by dicer an investigation of herpes simplex virus type latency in a novel mouse dorsal root ganglion model suggests a role for icp . in reactivation evaluation of '-modified oligonucleotides containing '-deoxy gaps as antisense inhibitors of gene expression synthetic biology approach for plant protection using dsrna treatment of experimental autoimmune encephalomyelitis in sjl/j mice with a replicative hsv- vector expressing interleukin- inhibition of coxsackievirus b and related enteroviruses by antiviral short interfering rna pools produced using phi rna-dependent rna polymerase inhibition of clinical pathogenic herpes simplex virus strains with enzymatically created sirna pools innate responses to small interfering rna pools inhibiting herpes simplex virus infection in astrocytoid and epithelial cells topical treatment of herpes simplex virus infection with enzymatically created sirna swarm herpes simplex virus type us gene deletion influences toll-like receptor responses in cultured monocytic cells enzymatically produced pools of canonical and dicer-substrate sirna molecules display comparable gene silencing and antiviral activities against herpes simplex virus high-throughput purification of double-stranded rna molecules using convective interaction media monolithic anion exchange columns fiji: an open-source platform for biological-image analysis antiviral rnai in insects and mammals: parallels and differences. viruses single-cell analysis of population context advances rnai screening at multiple levels a mutant t rna polymerase as a dna polymerase maldi-tof mass spectral analysis of sirna degradation in serum confirms an rnase a-like activity hsv- infection modulates the radioresponse of a hpv -positive head and neck cancer cell line chemically modified sirna: tools and applications ## ### ### *** key: cord- -mv sk aa authors: kumaki, yohichi; salazar, andres m.; wandersee, miles k.; barnard, dale l. title: prophylactic and therapeutic intranasal administration with an immunomodulator, hiltonol(®) (poly ic:lc), in a lethal sars-cov-infected balb/c mouse model date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: mv sk aa hiltonol(®), (poly ic:lc), a potent immunomodulator, is a synthetic, double-stranded polyriboinosinic-polyribocytidylic acid (poly ic) stabilized with poly-l-lysine and carboxymethyl cellulose (lc). hiltonol(®) was tested for efficacy in a lethal sars-cov-infected balb/c mouse model. hiltonol(®) at , , . or . mg/kg/day by intranasal (i.n.) route resulted in significant survival benefit when administered at selected times h prior to challenge with a lethal dose of mouse-adapted severe acute respiratory syndrome coronavirus (sars-cov). the infected balb/c mice receiving the hiltonol(®) treatments were also significantly effective in protecting mice against weight loss due to infection (p < . ). groups of mice were dosed with hiltonol(®) at . or . mg/kg by intranasal instillation , , and days before virus exposure and a second dose was given h later, prophylactic hiltonol(®) treatments ( . mg/kg/day) were completely protective in preventing death, and in causing significant reduction in lung hemorrhage scores, lung weights and lung virus titers. hiltonol(®) was also effective as a therapeutic when give up to h post virus exposure; % of the-infected mice were protected against death when hiltonol(®) was administered at mg/kg/day h after infection. our data suggest that hiltonol(®) treatment of sars-cov infection in mice leads to substantial prophylactic and therapeutic effects and could be used for treatment of other virus disease such as those caused by mers-cov a related coronavirus. these properties might be therapeutically advantageous if hiltonol(®) is considered for possible clinical use. severe acute respiratory syndrome coronavirus (sars-cov) is the causative agent of an emerging human infectious disease, severe acute respiratory syndrome (sars) ksiazek et al., ; peiris et al., b; rota et al., ) and is related to the middle east respiratory syndrome (mers-cov). infections from either virus are associated with high morbidity and mortality. due to its high morbidity and mortality, sars has evolved as an important global respiratory disease. even in a situation of no new infections, sars remains a potential major health hazard, as re-emerging epidemics may arise. the serious consequences posed by virulent emerging pathogens such as sars-cov and mers-cov to which our populations have little or no immunity was highlighted by sars outbreak in e . the principal containment strategy to date has emphasized rapid diagnosis and isolation of infected patients and to some extent contacts with those patients. however, in spite of enormous effects and funds expended, no agents have been approved for treating sars-cov or mers-cov. sars-cov has posed a serious threat to the human population and still represents a challenge for antiviral drug development and administration (groneberg et al., (groneberg et al., , . thus, numerous types of agents have been tested against sars-cov both in vitro and in vivo . notably, it has been shown that antibodies to the sars-cov spike protein block entry of the virus into cells (sui et al., ) and small peptides derived from the heptad repeat (hr) regions of sars-cov s protein have been shown to inhibit sars-cov infection by interfering with sars-cov fusion to target cells (bosch et al., ; ho et al., ) . additionally, the main protease of sars-cov, which is essential for the replication cycle of sars-cov, has been a key target for developing anti-sars-cov drugs barnard and kumaki, ; yang et al., ) . another approach that was explored for treating sars-cov infections was to evaluate rna species as therapies. thus, antisense ribonucleic acid (rna) and rna interference (rnai) technologies have shown potential in treating some severe diseases including sars-cov infection (ahlquist, ; gibson, ; johnson-saliba and jans, ; leonard and schaffer, ) . using sirnas to inhibit sars-cov infection in rhesus macaques, it was demonstrated that sirnas were effective both prophylactically and therapeutically (chang et al., ) . another approach has been the use of drug combination. the combination of ribavirin and corticosteroids was the most frequently administered antiviral therapy used during the sars outbreak (booth et al., ; ho et al., ; peiris et al., a; tsang et al., ; tsui et al., ) . however, ribavirin alone at nontoxic concentrations was found to have little in vitro activity against sars-cov , although an improved clinical outcome was reported among sars patients receiving early administration of kaletra plus ribavirin and corticosteroids (tsang and seto, ) . in several mouse models and in vitro, ribavirin was even found to enhance the sars-cov infection (barnard et al., a (barnard et al., , b day et al., ). more recent data demonstrated that urtica dioica agglutinin (uda) treatment of sars-cov-infected mice lead to a substantial therapeutic effect that protected mice against death and weight loss that result from the infection (kumaki et al., b) . a class of "natural" molecules, interferons are considered as a first line of defense against viral infections in humans (isaacs and lindenmann, ; isaacs et al., ) . in earlier studies, we evaluated a few compounds approved for therapeutic use in humans and some in vitro inhibitors of sars-cov for inhibition in the mouse sars-cov replication model (barnard et al., a) . a hybrid interferon, interferon alpha (ifn-a) b/d, and a mismatched doublestranded (ds) rna interferon inducer, ampligen (poly i:poly c ), were shown to potently inhibit virus titers in the lungs of infected mice (barnard et al., a) . to evaluate the prophylactic potential of antivirals directed against sars-cov infection, new lethal animal models for sars are needed to facilitate antiviral research. we adapted and characterized a new strain of sars-cov (strain v ) that was highly lethal in -to -week-old balb/c mice (day et al., ) . a number of compounds were tested for the efficacy in sars-cov-infected balb/c mice (day et al., ) . the use of adenovirus vectored mouse interferon-alpha (mdef ) as a prophylactic treatment and a therapeutic countermeasure for treating lethal sars-cov infection in balb/c mice was very efficacious in protecting mice against death (kumaki et al., a) . in other studies, treatment with an interferon inducer, polyriboinosinicpolyribocytidylic acid stabilized with poly-l-lysine and carboxymethyl cellulose (poly ic:lc), given by the intranasal route, was effective in protecting mice against a lethal infection with mouseadapted sars-cov and reduced viral lung titers (kumaki et al., ) . the active immunomodulator, poly ic:lc (hiltonol ® ), was effective when therapy was initiated h before infection or as late as h after virus inoculation, at a time when clinical signs of sars were being manifested in the balb/c mice. in this report, we further evaluated the use of hiltonol ® as an extended prophylactic or therapeutic countermeasure for treating lethal sars-cov infection in balb/c mice caused by a mouse-adapted sars-cov. in addition, its value as a vaccine adjuvant was assessed. vero cells were obtained from american type culture collection (atcc, manassas, va), and were routinely grown in minimal essential medium (mem) supplemented with % heatinactivated fetal bovine serum (fbs, thermo fisher scientific co., logan, ut) . for in vitro antiviral assays, the serum was reduced to % fbs and gentamicin was added to the medium up to a final concentration of mg/ml. -cov, strain urbani ( ) , was obtained from centers for disease control and prevention (cdc, atlanta, ga, usa). this strain was propagated and titrated in vero cells. the mouse-adapted sars-cov strain has previously been described (day et al., ) . briefly, the mice were infected with the urbani strain. three or five days after infection, the lungs were removed and homogenized and then used to re-infect a subsequent group of mice. this infection step was continued times through balb/c mice lungs. the virus was then plaque-purified times and yielded a virus causing severe lung disease and mortality in infected mice. the virus was verified as sars-cov by enzyme-linked immunosorbent assay (elisa) and polymerase chain reaction (pcr). all experiments involving infectious viruses were conducted in an approved biosafety level þ (bsl- þ) laboratory. the interferon inducer, polyriboinosinic-polyribocytidylic acid stabilized with poly-l-lysine and carboxymethyl cellulose hiltonol ® (poly ic:lc) was obtained from andres m. salazar (oncovir, inc., washington, dc ) . hiltonol ® was diluted in physiologically sterile saline (pss) for in vivo experiments just before use. ampligen ® was provided by hemispherx biopharma (philadelphia, pa ). specific pathogen-free female e g balb/c mice were obtained from charles river laboratories (wilmington, ma) for this study mainly. female e g b . s -il tm kopf/j and the parent strain c bl/ j were obtained from jackson laboratories (bar harbor, maine). female e g c bl/ j/ scnj and the parental strain mice c bl/ j were obtained from charles river laboratories (wilmington, ma). they were maintained on wayne lab blox and fed with standard mouse chow and tap water ad libitum. all the mice were quarantined for h prior to use. the animal studies were done in an approved bio-safety level þ animal facility. personnel entering the facility wore powered air-purifying respirators ( m hepa air-mate; m, saint paul, mn) and tyvek body protection suits. animal studies had been approved by utah state university animal care committee. the general experimental design is described below. the balb/c mice were anesthetized with a . ml intraperitoneal injection of mg/kg of ketamine ® and the mouse-adapted sars-cov was administered intranasally (i.n.) in a volume of . ml. groups of mice were administered hiltonol ® or vehicle placebo i.n. at selected times prior to challenge with .  pfu of mouse-adapted sars-cov. ampligen was administered i.n. h before virus exposure and h after exposure to virus and served as a positive control for controlling the virus infection. fifteen mice were treated i.n. with pss at various times prior to virus exposure. mice in this group represented the placebo controls. sars-cov-infected and mock-infected mice were weighed every day and clinical signs of disease were also observed and recorded daily. animal deaths were recorded for up to days post virus exposure. following intranasal administration of sars-cov, five mice from each group were sacrificed on day and for lung score, lung weight and lung titer determinations. animals that lost greater than % of their initial body weight were humanely euthanized by co asphyxiation, and the day of euthanization was designated as the day of death due to infection. for hiltonol ® , a dose range finding experiment was carried out to determine the maximum tolerated concentration. three mice were used per treatment group. toxicity was evaluated in terms of weight change and adverse events. mice were weighed every day from h prior to virus infection to day post virus exposure. adverse events for which observations were made included ruffling of fur, lethargy, paralysis, incontinence, repetitive circular motion, and aggression. samples from each mouse lung lobe were weighed and placed in a petri dish. lungs were scored based on surface appearance of lungs. lungs were then assigned a score ranging from to , with indicating that the lungs looked normal and denoting that the entire surface area of the lungs was inflamed and exhibited plum colored lung discoloration (sidwell et al., ) . significant differences in lung scores were determined by kruskal-wallis test followed by dunn's pairwise comparison post tests. one-way analysis of variance (anova) was used to probe for significant differences in lung weights. pairwise comparisons were made by sidak's multiple comparison tests. lung virus titers were analyzed from mice sacrificed on day and post virus exposure. a lobe from each mouse lung was homogenized in mem supplemented with % fbs and the tissue fragments were allowed to settle. the varying dilutions of the supernatant fluids were assayed in triplicate for infectious virus in vero cells by cytopathic effect (cpe) assay. the titers ( % tissue culture infectious dose, ccid values) were calculated using the reed-muench method (reed and muench, ) . significant differences were detected by anova. pairwise comparisons were made by sidak's multiple comparison tests. sera were harvested by submandibular bleeding from surviving mice at day and day after virus challenge. ml aliquot of each serum sample was added to approximately ml of mem, mixed, then serially diluted by / to achieve / to / dilutions in -well plates. virus stock was diluted in mem to approximately ccid per ml. next ml of virus was added to each well, the plates vibrated for approximately min, and then incubated for h at c for neutralization. ml of the liquid from each well was then transferred to -well plates containing sub-confluent monolayers of vero cells, and ml of mem þ % fbs was added to each well. plates were sealed with tape, incubated for days at c with % co , and scored for the presence or absence of virus cytopathic effect. uninfected wells served as a negative cell control, and a serum sample with known anti-sars antibody served as a positive control. results were reported as the inverse of the greatest dilution where virus cpe was not detected. group of mice infected as described previously were sacrificed on day and either on or post virus exposure. the lungs from these mock-infected or infected mice were collected and formalinfixed. the lungs from each group were sectioned and stained with h&e stain and evaluated by a board certified veterinary pathologist for histopathological changes. mice were weighed in groups prior to treatment and then every day thereafter to determine the average weight change for all animals in each treatment group. weights were expressed as group averages for each day and evaluated by the two-way analysis of variance for significant differences among treatment groups followed by pairwise comparisons using dunnett's multiple comparison tests. survival analysis was done using the kaplan-meier graphical method and a logrank test. the analysis revealed significant differences among the treatment groups. therefore, pairwise comparisons of survivor curves (pss versus any treatment) were analyzed by the gehan-breslow-wilcoxon pairwise comparisons test, and the relative significance was adjusted to a bonferronicorrected significance threshold for the number of treatment comparisons done. mean day of death was calculated and analyzed by the kruskal-wallis test, followed by dunn's post tests for evaluating the significant pairwise comparisons. live numbers per total mice in a group differences were evaluated by contingency table analysis. fisher's exact tests were used to make pairwise comparisons to placebo-treated mice. most mice randomly assigned to the toxicity control groups gained weight at rates nearly equal to mice receiving pss (data not shown). the mice treated with hiltonol ® at . or . or . mg/kg/ day were the groups to lose noticeable amounts of weight, which occurred at day . however, they regained the lost body weight after the nadir of weight loss at day . the mice in these groups managed to rapidly regain all lost weight by the end of the experiment. no other adverse events were observed for any of the toxicity control mice used in the experiment. in a previous study, balb/c mice inoculated intranasally with mouse-adapted sars-cov, the infected mice died between and days, with e % mortality rate achieved by day . the lungs were severely inflamed and exhibited extreme lung consolidation. the efficient viral replication was observed from day e in the lungs (day et al., ) . virus titers often exceeded /ml at peak replication during day e . weight loss was excessive. in the current study, group of mice were administered hiltonol ® or vehicle placebo i.n. at selected times prior to or after virus challenge with mouse-adapted sars-cov. some mice received additional doses of hiltonol ® after virus challenge. all doses and treatment regimens of hiltonol ® significantly protected mice against a lethal infection of balb/c mice exposed to sars-cov (fig. a , p < . ; table ). only three mice died using hiltonol ® , one mouse at day post virus exposure when dosed with mg/kg/day once h before infection, and one mouse at day when receiving hiltonol ® at mg/kg/day dose given once h after infection, and one mouse at day when treated three times with a lower dose of hiltonol ® ( mg/kg/day before, and at and h after infection). these treated, sars-covinfected mice receiving the various hiltonol ® dosing regimens were also significantly protected against weight loss due to virus infection (table , p < . -p< . ) from days e post virus exposure when the greatest weight loss occurred in this mouse model. we also evaluated how far before the initial virus challenge time prophylactic dosing regimens would be efficacious in protecting mice from the lethal challenge with sars-cov. groups of mice were dosed with hiltonol ® at . or . mg/kg by intranasal (i.n.) instillation on days , , or before virus exposure. a second dose was given h later. hiltonol ® when administered at . mg/kg/day beginning at day or before virus exposure protected e % of infected mice against death due to the virus infection ( fig. , p < . ). the only hiltonol ® dosing regimen that was not very protective was when it was administered at . mg/kg, one time, days prior to exposing mice to virus. ampligen ® used at mg/ kg, significantly (p < . ), yet only protected % of the mice from death using the designated therapeutic treatment regimen. at the critical time of day post virus exposure, when many mice began to die in the placebo treated groups, mice treated with hiltonol ® at . mg/kg or days before infection seemed to be least susceptible to weight loss due to virus infection compared to the corresponding the placebo controls (data not shown). the placebo-treated mice usually died at day e (table ) , whereas mice treated with the higher dose of hiltonol ® survived in significant numbers (p < . ). it was also obvious that mice treated with lower dose of hiltonol ® survived in significant numbers provided that hiltonol ® was administered or days before virus infection. when administered at . mg/kg/day days before virus infection, almost all treated mice in the hiltonol ® group died with a mean day of death equivalent to the corresponding placebo control group (table ) . ampligen ® was given i.p. twice a day beginning h before infection at mg/kg. the mice receiving ampligen ® were slightly yet significantly protected against death ( % survivors table , p < . ). . . effects of hiltonol ® on lung scores, lung weights, and virus lung titers of female balb/c mice infected with a lethal dose of mouseadapted sars-cov at day post virus exposure other parameters measured to determine the extent of the efficacy of the two prophylactic doses of hiltonol ® included effects on gross lung pathology, lung weights, and virus lung titers. at day after mice were exposed to virus, the . mg/kg dose of hiltonol ® significantly ameliorated the extent of damage induced by the virus infection was observed on the surface of lungs of infected mice (p < . , table ). there was little or no surface hemorrhaging observed. in addition, almost all mice treated with this dose of hiltonol ® , regardless of the time of administration, were protected to the same extent (table ). the treatment with the lower dose of hiltonol ® led to similar results of less observable surface lung pathology, especially for mice treated with hiltonol ® beginning at day before virus exposure (p < . ). another indicator of the extent of an inflammatory response in the lungs is edema. edema can be indirectly measured by evaluating the weight of an infected lung. at day of the virus infection, dosing with the . mg/kg of hiltonol ® using any treatment regimen significantly prevented an increase in lung weights of treated mice, a manifestation of edema (table , p < . -p< . ). however, the efficacy of repression edema was much less pronounced in mice receiving the . mg/kg dose of hiltonol ® . ampligen ® treatment resulted in a similar efficacy profile in preventing edema as was seen with mice dosed with hiltonol ® at . mg/kg. did this apparent moderation of the inflammatory response both in the lungs and on the surface of the lungs due correlate with a reduction of virus replication in the lungs of infected mice, the presence of virus protein the likely culprit for inducing edema? in general, virus lung titers at day after inoculation with virus were almost . log lower or more in mice treated with . mg/kg hiltonol ® compared to the placebo-treated control (table ) . however, only the virus lungs titers of mice a b fig. . a. effects of hiltonol ® on survival of balb/c mice with a lethal sars-cov infection. ***p < . versus pss. the sars-cov-infected balb/c mice were treated with pss (c: À h, þ h, þ h); hiltonol ® at mg/kg/day (a: þ h), (b: À h); hiltonol ® at . mg/kg/day (,: À h, þ h, þ h), (△: À h, þ h, þ h), (▽: À h, þ h, þ h), (>: À h). b. effects of various long-term dosing regimens of hiltonol ® on survival of balb/c mice with a lethal sars-cov infection. ****p < . versus pss. the sars-cov-infected balb/c mice were treated with pss (c: day À ), (b: day À ), (>: day À ); hiltonol ® (-: . mg/kg/ day À ), (,: . mg/kg/day À ), (;: . mg/kg/day À ); (:: . mg/kg/ day À ), (▽: . mg/kg/day À ), (△: . mg/kg/day À ). a second dose was given h later. (a) ampligen ® mg/kg/day (bid x beg, h). treated with . mg/kg hiltonol ® days before infection were significantly reduced compared to the virus titers of mice receiving pss. they were reduced by almost one log compared to the placebo treated mice (table ) . none of the other treatments, including ampligen ® , achieved such a reduction in virus lung titer. . . effects of various long-term dosing regimens of hiltonol ® on neutralizing antibody titers of balb/c mice at day and post challenge with a lethal dose of sars-cov it is conceivable that an immune modulator such as an interferon inducer could suppress normal adaptive immune responses such as the formation of neutralizing antibody. the effects of hiltonol ® treatment on immune responsiveness were also investigated by analyzing the neutralizing antibody levels to the virus and days after virus challenge. sera from all groups of mice used in the long term prophylaxis experiment were analyzed for neutralizing antibody ( fig. a and b) . the data suggest that the mice treated with the high dose of hiltonol ® could still mount a robust virus neutralization response, even though the drug did significantly reduce virus lung titers in only one case. in fact, mice receiving pretreatment with hiltonol ® beginning at day and day before virus challenge had significant higher neutralizing antibody titers than did the corresponding placebo-treated mice ( fig. a ; p < . , p < . ; respectively). the neutralizing antibody titers remained equivalently high or higher at day post table effects of various dosing regimens of hiltonol ® on a lethal sars-cov infection in a balb/c mouse model. live/total mean day of death ± sd weight loss from day to day (g) average weight through day (g) ± sd pss (À , þ , þ h) / . ± . *** . . ± . hiltonol ® mg/kg/day (À , þ , þ h) / > . . ± . *** hiltonol ® mg/kg/day (À , þ , þ h) / > . . ± . *** hiltonol ® mg/kg/day (À , þ , þ h) / > . . ± . *** hiltonol ® mg/kg/day (À h) / . . ± . * hiltonol ® mg/kg/day (þ h) / . . ± . *** hiltonol ® mg/kg/day (À , þ , þ h) / . . ± . * hiltonol ® mg/kg/day (À , þ , þ h) / > . . ± . * hiltonol ® mg/kg/day (À , þ , þ h) / > . . ± . * hiltonol ® mg/kg/day (À h) / > . . ± . *** hiltonol ® . mg/kg/day (À , þ , þ h) / > . . ± . * hiltonol ® . mg/kg/day (À , þ , þ h) / > . . ± . * hiltonol ® . mg/kg/day (À , þ , þ h) / > . . ± . * hiltonol ® . mg/kg/day (À , þ , þ h) / > . . ± . ** hiltonol ® . mg/kg/day (À , þ , þ h) / > . . ± . ** hiltonol ® . mg/kg/day (À , þ , þ h) / > . . ± . *** *p < . , **p < . , ***p < . . effects of various long-term dosing regimens of hiltonol ® on survival of balb/c mice with a lethal sars-cov infection. dosing regimen (day prior to virus exposure) survivors/total mean day of death ± sd pss / . ± . hiltonol ® ( . mg/kg/day) / *** > *** hiltonol ® ( . mg/kg/day) / . ± . pss / . ± . hiltonol ® ( . mg/kg/day) / *** . ± . hiltonol ® ( . mg/kg/day) / *** . ± . pss / . ± . hiltonol ® ( . mg/kg/day) / *** > *** hiltonol ® ( . mg/kg/day) / *** > *** ampligen ® ( mg/kg/day) bid x , beg À h / * . ± . *p < . , ***p < . compared to pss control. virus challenge (fig. b) . interestingly, the group of mice receiving the lowest dose of hiltonol ® appeared to have the higher neutralizing antibody titers at day than at day . one could speculate that this observation was driven by the fact that virus titers were higher in these mice. treatment of mice with low dose hiltonol ® and ampligen ® also showed similar results. this would suggest that this dose of hiltonol ® did not adversely affect the adaptive immune response. in general, lungs infected with sars-cov should show acute to subacute alveolitis with some perivascular edema in some sections. lungs of mice inoculated with sars-cov showed no significant changes, and mock-infected controls showed no marked changes except moderate rims of lymphocytes surrounding scattered vessels (fig. ) . also, the pathological changes were not observed in the sars-cov-infected, hiltonol ® -treated lungs (fig. ) . however, significant pathological differences in the distribution of inflammatory cells between the sars-cov-infected and the mock-infected lungs were not observed. three sars-cov-infected, hiltonol ® -treated lung samples ( . mg/kg days before virus exposure) were evaluated for pathological change on day after inoculation. in ampligen ® -treated mice, the positive control-treated group, the infection was very limited with little or no evidence of an inflammatory response in the lungs from the mouse that was observed, although there was a tremendous amount of erythrocyte infiltration in the air spaces (fig. f) . the efficacy of hiltonol ® ( mg/kg/day) as a treatment for sars-cov infection was evaluated with hiltonol ® at , , , , or h post virus challenge. survival and weights were monitored daily for at least days. hiltonol ® given at mg/kg/day resulted in % survival of treated, infected mice when administered h post virus challenge, at a time when clinical signs of sars are starting to be manifested in the untreated balb/c mice (p < . , table ). in addition, treatment with hiltonol ® at mg/kg/day by intranasal route resulted in % survival when it was given twice, and then h post virus exposure (table , p < . ). these data suggest that hiltonol ® treatment of sars-cov infection in mice leads to substantial therapeutic effect that protects mice against death only when administered within h after virus exposure or when using a therapeutic multiple dosing regimen. hiltonol ® given one time at mg/kg/day was not protective against death when treatment was at , or h after sars-cov infection (table ). when hiltonol ® was administered intranasally or h post virus challenge, the data showed partial survival (table ). in another subsequent therapeutic experiment, hiltonol ® was administered intranasally and h post virus exposure. at day , % of mice receiving hiltonol ® and h after exposure to virus were still alive compared with the other therapeutic regimens in which all were dead or only one mouse had survived (data not shown). this data suggest that treatment beginning h after virus exposure might temporarily prolong survival, but that the pathogenesis caused by the virus finally overwhelms the mice. interleukin (il- ) is a multifunctional cytokine that regulates the immune response, hematopoiesis, the acute phase response, and inflammation (hirano, ) . we have previously reported that il- levels were increased in sars-cov-infected mice, and high il- expression was associated with mortality (day et al., ). therefore, we evaluated the effects of virus infection in mice deficient in il- (b . s -il tmlkopf/j). since these il- mutant mice show defects in responses to various viruses and in inflammatory responses to infection, we hypothesized that mortality and disease parameters might be reduced in il- -/-mice infected with sars-cov because increased il- levels contributed greatly to the lethality of the infection in normal mice (day et al., ). this a b fig. . effects of various long-term dosing regimens of hiltonol ® on neutralizing antibody titers of balb/c mice at day (a) and (b) post virus challenge with a lethal dose of sars-cov. **p < . versus pss (day À ), ***p < . versus hiltonol ® . mg/kg (day À ) or hiltonol ® . mg/kg (day À ). the sars-cov-infected balb/c mice were treated with pss (c: day À ), (b: day À ), (>: day À ); hiltonol ® (-: . mg/kg/day À ), (,: . mg/kg/day À ), ( ; : . mg/kg/day À ); (:: . mg/ kg/day À ), (▽: . mg/kg/day À ), (△: . mg/kg/day À ), (a) ampligen ® mg/kg/day (bid x beg, h). hypothesis assumed that il- production was a major source of lung disease in sars-cov-infected mice. when both sars-covinfected mice (c bl/ j, il- þ/þ and b . s -il tm kopf/j, il- -/-) were treated with hiltonol ® at . mg/kg/day, the typical depression of weight gain by hiltonol ® treatment was observed at days e , although it was significantly more severe in the b . s -il tm kopf/j mice (il- -/-) (data not shown). however, the surviving mice gained back the lost weight by the end of the experiment. hiltonol ® did not significantly protect mice from death in either strain of mouse compared to the placebo-treated mice of both strains (table ) and il- -/-mice still supported virus lung replication. in addition, the mean day of death was the same for all groups. however, the virus lung titers in these mice were significantly reduced at day post virus exposure (table , p < . ). all effects of various long-term dosing regimens of hiltonol ® on death of balb/c mice infected with a lethal dose of sars-cov post virus exposure. dosing regimen (hour post virus exposure) live/total mean day of death pss À , þ , þ , h / . hiltonol ® ( mg/kg/day) þ h / *** undefined hiltonol ® ( mg/kg/day) þ h / . hiltonol ® ( mg/kg/day) þ h / . hiltonol ® ( mg/kg/day) þ h / . hiltonol ® ( mg/kg/day) þ h / . hiltonol ® ( mg/kg/day) þ h / . hiltonol ® ( mg/kg/day) þ , þ h / *** undefined hiltonol ® ( mg/kg/day) À , þ , þ h / *** undefined ***p < . versus pss control. il- -/-mice had significantly highly virus titers than did the normal c bl/ j mice (table , p < . ), suggesting that il- was necessary to control virus replication in the lungs. interestingly, at day , hiltonol ® -treated il- -/-mice had significantly increased virus lung titers compared to untreated, infected il- þ/þ mice (table , p < . ). in addition, the virus infection of the lungs likely induced a significant yet slight inflammatory response in untreated, infected il- -/-mice (p < . ), as measured by the increase in lung score but there was no induction of edema in these mice as can be seen by the normal lung weights ( table ). the inability to significantly control virus replication may be one of reasons why il- -/-mice were not protected against death. thus, virus cytopathic effects may have sufficiently destroyed enough lung cells to contribute to poor lung function that may have lead to death for mice that succumbed because of the virus infection. our data suggest that these mice ( bl/ j mice, b . s -il tm kopf/j mice, il- -/-) lack a pathway with which hiltonol ® interacts to prevent lethal sars-cov infection in mice. khanolkar et al. reported that c h/hej mice harboring a natural mutation in the gene that encodes toll-like receptor (tlr ) that disrupts its normal function exhibited the enhanced morbidity and mortality following i.n. mouse hepatitis virus strain (mhv- ) infection, indicating that tlr- plays an important role in respiratory cov pathogenesis (khanolkar et al., ). in the current study, we hypothesized that intranasal infection of tlr -/-mice with mouse-adapted sars-cov would result in an acute respiratory disease with a higher lethality. when both strains (c bl/ j and c bl/ j/ scnj tlr- -/-mice) were infected with sars-cov and treated with hiltonol ® , infected, untreated c bl/ j mice lost weight, but onset of weight loss was delayed relative to hiltonol ®treated, infected mice (data not shown). an exception was the one c bl/ j/ scnj tlr- -/-mouse that died at day post virus exposure. despite not inducing death of these strains of mice, the virus was able to replicate quite rigorously in the lungs of c bl/ j mice (table ) as previously seen in other mice (table ). yet the virus induced no significant inflammatory response, as manifested by lack of increased lung scores and lack of induction of edema manifested as normal lung weights (table ) . nevertheless, virus lung titers were dramatically reduced in both strains of mice treated with hiltonol ® (p < . ), but more so in the c bl/ j/ scnj tlr- -/-mice, especially at day . in addition, the c bl/ j/ scnj mice were less suitable hosts for prolonged lung virus replication than the c bl/ j mice, because virus lung titers at day in c bl/ j/ scnj tlr- -/-mice were significantly lower than in c bl/ j mice (p < . ). these data indicated that hiltonol ® action appeared to be independent of the tlr- locus. the host innate immune response against viral insult includes the production of interferon type i (ifn-a/b), which is initiated to limit viral replication. the virus-infected cells usually cause the activation of several transcription factors, such as interferon regulatory factor (irf- ), which play a central role in downstream gene activation (lin et al., ) . once interferon is synthesized and secreted from the cells, it binds to cell surface receptors and induces transcription, which results in an anti-viral state in the target cells. epithelial cells secrete interferon-b as an initial response to viral infection (marie et al., ) . dendritic cells are able to produce infa subtypes (diebold et al., ) . however, the production of interferon type i by sars-cov-infected cells is limited (chen and subbarao, ; spiegel et al., ) and neither endogenous interferon transcripts nor interferon promoter activity are detected (spiegel et al., ) . this lack of interferon activity has been attributed to proteins, which antagonize interferon and block transcription factors necessary for the expression of interferon (kopecky-bromberg et al., ) . although sars-cov antagonizes the production and effect of endogenous interferon, it remains susceptible to exogenous interferons (chen and subbarao, ; kumaki et al., a) . interferon induction occurs mainly by an intracellular pathway: double-stranded (ds) rna and '-triphosphorylated singlestranded (ss) rna trigger a signaling chain, which activates interferon-b gene expression (hornung et al., ; pichlmair et al., ; weber et al., ) . retinoic-acid-inducible gene i (rig-i) table effects of hiltonol ® on the death of c bl/ j and b . s -il m kopf/j mice infected with a lethal dose of mouse-adapted sars-cov. dosing regimen (hour post virus exposure) live/total mean day of death pss c bl/ j À , þ , þ h / . ± . hiltonol ® c bl/ j À , þ , þ h / . ± . pss b . s -il tm kopf/j À , þ , þ h / . ± . hiltonol ® b . s -il tm kopf/j À , þ , þ h / . ± . ***p < . versus pss control. effects of hiltonol ® on various lung parameters measured in il- knockout mice (il- -/-) mice infected with mouse adapted sars-cov. virus titer (log ccid /g) ± sd visual lung score ± sd lung weight (g) ± sd day pss c bl/ j . ± . . ± . . ± . * hiltonol ® ( mg/kg/) c bl/ j . ± . *** . ± . . ± . pss b . s -il tm kopf/j . ± . . ± . * . ± . hiltonol ® ( mg/kg/) b . s -il tm kopf/j . ± . *** . ± . . ± . day pss c bl/ j . ± . . ± . . ± . hiltonol ® ( mg/kg/) c bl/ j . ± . *** . ± . . ± . pss b . s -il tm kopf/j . ± . . ± . . ± . hiltonol ® ( mg/kg/) b . s -il tm kopf/j . ± . *** . ± . . ± . ***p < . versus pss control. and melanoma-differentiation-associated gene (mda- ) are the main intracellular receptors of viral rna (andrejeva et al., ; kato et al., ; yoneyama et al., ) . the binding of a viral rna to rig-i and mda- induces a signaling chain which results in the phosphorylation of the transcription factor irf- (fitzgerald et al., ; sharma et al., ) . , '-oligoadenylate synthetases ( , -oas) (silverman, ) and the protein kinase r (pkr) (williams, ) have also been characterized in detail. , -oas catalyses the synthesis of short - oligoadenylates that activate the latent endoribonuclease (silverman, ) . pkr is a serinethreonine kinase that phosphorylates the alpha subunit of the eukaryotic translation initiation factor eif (williams, ) . in addition, interferon induction also does occur by an endosomal pathway in selected cells. myeloid dendritic cells (mdcs) (diebold et al., ) and plasmacytoid dendritic cells (pdcs) (colonna et al., ) are the main interferon producers of the lymphatic system. myeloid dendritic cells (mdcs) can sence dsrna by the classic intracellular pathway (diebold et al., ) and, the endosomal toll-like receptor (tlr) (alexopoulou et al., ) . tlr- / and tlr- recognize viral ssrna and dsrna, respectively, and activate interferon-a/b transcription via the transcription factors irf- , irf- , and nf-kb. the pathogenesis of severe acute respiratory syndrome has not yet been fully characterized. one hypothesis is that the pathogenesis of sars-cov is caused by a disproportionate immune response, illustrated by elevated levels of inflammatory cytokines and chemokines, such as interferon gamma inducible protein (ip- ), monocyte chemoattractant protein- (mcp- ), interleukin (il- ) and interleukin (il- ) (jiang et al., ; wong et al., ) . chemokines are involved in the recruitment of leukocytes into sites of tissue inflammation (sauty et al., ) . sars-cov has been shown in vitro to induce changes of cytokines and chemokines in various human and animal cells (spiegel and weber, ; yen et al., ) . il- is a monomor of amino acids secreted by tcells, macrophages and endothelial cells to stimulate immune response during the infection (ray et al., ; sehgal et al., sehgal et al., , . xiao et al. speculated that dampening of the proinflammatory cytokine response in sars-cov infection, in particular the production of il- , could have a clinically beneficial effect (xiao et al., ) . we noticed that sars-cov infection increased il- in mice, and high il- expression corrected with mortality (day et al., ) . levels of il- dropped when balb/c mice were treated with ribavirin, uda ( mg/kg/day), ampligen ® or hiltonol ® ( mg/kg/ day) (day et al., ; kumaki et al., b) . ip- and other cytokines are released from both the apical and basolateral sides, while il- is secreted through the apical surface (yoshikawa et al., ) . it can be speculated that ip- in sars patients might directly be produced by virus-infected cells, whereas upregulation of il- is likely a secondary response due to the activation of the immune system. in addition, the trif pathway leads to the production of type i interferons via interferon regulatory factor (irf- ) and also cause delayed nf-kb activation via activation of tnf receptorassociated factor (traf ) (hoebe et al., ; sato et al., ) . imai et al. also showed that trif-dependent lung injury is likely to be mediated by production of interleukin (il- ), as il- -deficient mice were protected from injury (imai et al., ) . toll-like receptors (tlrs), a family of evolutionarily conserved pathogen recognition receptors, are a class of proteins that play a key role in the innate immune system. the tlr family consists of mammalian members. viral protein binds to tlr- and tlr- . the single stranded rna binds to tlr- and tlr- . the double stranded rna binds to tlr- while viral dna binds to tlr- . tlr- , tlr- , tlr- , and tlr- recognize viral nucleic acids on endosomal membrane. the binding of ligands to tlrs might trigger downstream signaling pathways that are involved in both the cytokine release during the primary induction of inflammation and secondary activation of anti-inflammatory mechanism (netea et al., ) . each tlr has its own intrinsic signaling pathway and induces specific biological responses against microorganisms, such as dendritic cell maturation, cytokine production, and the development of adaptive immunity murawski et al., ) . tlr- recognizes the viral proteins, such as the fusion (f) protein from respiratory syncytial virus and the envelope protein of mouse mammary tumor virus (uematsu and akira, ) . tlr- mutated c h/hej mice are sensitive to respiratory syncytial virus infection (murawski et al., ) . tlr- triggers the induction of proinflammatory cytokines by myeloid differentiation primary response gene (myd ) pathway, and tlr- also interacts with tir-domain-containing adapter-inducing interferon-b (trif)mediated signaling pathways that are involved in interferon induction (uematsu and akira, ) . the mechanisms of action behind these encouraging results remain to be further elucidated. hiltonol ® (poly ic:lc, or polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose) is a chemically stabilized active doublestranded rna (dsrna) therapeutic that activates host innate and adaptive immunity by various pathways/mechanisms leading to induction of the immediate antiviral state. the immediate antiviral state is characterized by induction of a 'natural mix' of interferons, cytokines, and chemokines; activation of dsrna-dependent host defense systems such as rig-i, mda- , , -oas, pkr, stimulation of mdcs, of natural killer (nk) cells and others via toll-like receptors. toll-like receptors are also implicated in lung airway injury as well as leading to beneficial responses to antigen insult. imai et al. demonstrated that acute lung injury (ali) is triggered by the signaling of oxidative stress through toll-like receptor (imai et al., ) . khanolkar et al. reported that c h/hej mice harboring a natural mutation in the gene that encodes toll-like receptor (tlr ) that disrupts its normal function exhibited the enhanced morbidity and mortality following i.n. mouse hepatitis virus strain table effects of hiltonol ® on various lung parameters of c bl/ j mice and tlr- -/-mice infected with a lethal dose of mouse-adapted sars-cov. virus titers (log ccid /g) ± sd visual lung scores ± sd lung weights (g) ± sd day pss in c bl/ j mice . ± . . ± . . ± . hiltonol ® ( mg/kg/) in c bl/ j mice . ± . *** . ± . . ± . pss in c bl/ j/ scnj tlr- -/-mice . ± . . ± . . ± . hiltonol ® ( mg/kg/) in c bl/ j/ scnj tlr- -/-mice . ± . *** . ± . . ± . day pss in c bl/ j mice . ± . . ± . . ± . hiltonol ® ( mg/kg/) in c bl/ j mice . ± . *** . ± . . ± . pss in c bl/ j/ scnj tlr- -/-mice . ± . . ± . . ± . hiltonol ® ( mg/kg/) in c bl/ j/ scnj tlr- -/-mice . ± . *** . ± . . ± . ***p < . versus pss control. (mhv- ) infection, indicating that tlr- plays an important role in respiratory cov pathogenesis (khanolkar et al., ) . in this study, we hypothesized whether intranasal infection of tlr- -/mice with mouse-adapted sars-cov would result in an acute respiratory disease with a higher lethality. however, the data presented is not consistent with this hypothesis. toll-like receptor (tlr- ) is a member of the toll-like receptor family of pattern recognition receptors of the innate immune system (rock et al., ) . tlr- is expressed on respiratory epithelium as well as on mdcs, especially in response to respiratory infections such as influenza. using tlr- -deficient mice, gowen et al. indicate that tlr- plays an important role in punta toro virus (ptv) infection (gowen et al., ) . ichinohe et al. have also demonstrated an even earlier activation of lung tlr- in response to nasal hiltonol ® or poly-ic in mice (ichinohe et al., ) . while these host defense mechanisms are typically activated by presentation of 'foreign' dsrna species generated by viral replication, many of them are also frequently the target of inhibition by many neoplasms and viruses, including influenza virus and sars-cov. treatment with mucosally applied hiltonol ® may preempt or reverse some of those viral evasive mechanisms. the apparent modulation by hiltonol ® of the usual relationship between host survival and replication of sars-cov, as manifested by clinical protection of animals in spite of some evidence of viral replication, is also of much interest. for example, complete inhibition of viral replication might be counterproductive to the generation of longer term adaptive immunity. thus, while some of the dsrna dependent antiviral/antiproliferative pathways mentioned above may help control replication, the potential activation of mdcs via tlr- hiltonol ® 's can be seen as improving the efficiency of antigen presentation and may be particularly suited to generation of a th cellular antiviral immunity. in fact, the tlrs have been proposed as a 'bridge' between innate and adaptive immunity. it remains to be further determined whether tlr- is involved in sars-cov infection in mice. hiltonol ® has been in extensive preclinical and experimental clinical therapeutic use, with evidence of clinical safety and potent antiviral, immune adjuvant, and antineoplastic actions, alone or combined with other agents (levy et al., ) . initial high-dose studies were quasi-empirical and driven by its induction of interferon, yet only recently have its effectiveness at lower doses and broad mechanisms of action begun to be more fully elucidated. there is an extensive early, empirical experience on the broad in vivo antiviral actions of hiltonol ® , yet these actions have not been fully exploited in the context of prophylaxis of emerging infections, pandemic containment and biodefense. mucosal (intranasal) hiltonol ® provides rapid and complete protection from respiratory viruses such as influenza virus, sars-cov, and rsv (guerrero-plata et al., ; kumaki et al., ; wong et al., wong et al., , . wong et al. has demonstrated e % protection lasting up to weeks provided by intranasal aqueous or liposomal hiltonol ® in otherwise lethal murine challenge models of influenza a and avian (h n ) influenza viruses. hiltonol ® has been shown to have efficacy against yellow fever , simian hemorrhagic fever (levy et al., ) , japanese encephalitis (harrington et al., ) , rabies (baer et al., ) and rift valley fever (kende et al., ) . the antiviral activity of hiltonol ® is believed to be mediated by its ability to augment the production of interferon-a, -b and -g (levy et al., ) . hiltonol ® also activates natural killer cells and macrophages. the antiviral efficacy of hiltonol ® was previously evaluated against avian influenza virus and hiltonol ® appeared to provide effective and broad-spectrum prophylaxis against avian influenza virus (wong et al., ) . for our in vivo studies, we recommend to administer the safe dose of hiltonol ® at . mg/kg/ day. in a pilot trial, salazar et al. reported that low dose hiltonol ® (about e mcg/kg) was given intramuscularly two to three times weekly for up to months to malignant brain tumor patients (salazar et al., ) . patients tolerated the regimen well, with little or no toxicity and a preserved quality of life. we conclude that the concept of long-term, broad spectrum stimulation of host defenses with non-toxic, inexpensive double-stranded ribonucleic acids, such as hiltonol ® at mcg/kg, might be applicable to the treatment of virus infections. in the current study, the data suggest that nasal hiltonol ® treatment of sars-cov infections in mice leads to a substantial long-term prophylactic and somewhat less robust therapeutic effect that protects mice against death and weight loss resulting from the infection. host-targeted therapeutics such as hiltonol ® that activate innate immunity and provide immediate broad spectrum resistance could fill the early gap in protection by allowing time for more specific vaccination strategies to take effect, and could thus become an important element of the rapid response to a bioterror attack or pandemic outbreak. hiltonol ® represents a relatively new host-directed paradigm in therapeutics that seeks to activate nonspecific and specific host defense systems that have been highly evolved over millions of years. hiltonol ® also has the potential to be used prophylactically in those who have been potentially exposed to the sars-cov, but not yet showing symptoms, thus enabling clinicians to "ring" and isolate the focus case with individuals prophylactically treated and unlikely get severe disease or unlikely not get disease at all. the demonstrated clinical safety of hiltonol ® 's, its immediate induction of an innate immune persistent broad spectrum antiviral state, its relatively low cost, its stability in storage and relative ease of use make it a potentially very 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macrophages and dendritic cells we thank aaron j. smith, kevin w. bailey, michael a. morrey for kindly providing technical assistance; craig w. day for scientific discussion and ramona t. skirpstunas for professional consultation. we are grateful to other colleagues for their support. this work was supported by a contract n -ai- and hhsn i/ hhsn /a from the virology branch, national institute of allergic and infectious diseases, national institutes of health. key: cord- - t ld authors: park, il-hyun; kwon, young-chan; ryu, wang-shick; ahn, byung-yoon title: inhibition of hepatitis b virus replication by ligand-mediated activation of rnase l date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: t ld rnase l is a cellular endoribonuclease that is activated by ′, ′-linked oligoadenylates ( – a), which are unique and specific ligands synthesized by a family of interferon-inducible, dsrna-activated enzymes named oligoadenylate synthetases. in the typical antiviral pathway, activated rnase l degrades viral and cellular rnas, thus limiting viral replication and spread. although the antiviral activity of rnase l has been demonstrated for several rna viruses, there is little evidence regarding its role against dna viruses. in the present study, the potential antiviral activity of rnase l against hepatitis b virus (hbv) was explored utilizing the recently reported infection protocol based on human hepatoma hepg cells stably complemented with the virus entry factor ntcp. viral replication and expression in this cell type was markedly inhibited by poly(i:c)- or – a-mediated activation of rnase l; however, the inhibition was significantly reversed by rnase l knockdown. further analysis in hbv . -transfected huh- hepatoma cells indicated that the antiviral activity of rnase l depends on its ribonuclease function. we also provide evidence for the specific roles of oas family members in this process. these results suggest that hbv replication can be regulated through interferon-mediated rna decay pathways and that activation of these host antiviral factors may represent a novel therapeutic strategy for hbv infection. rnase l is a latent endoribonuclease that is constitutively expressed in almost all mammalian cells. this enzyme is activated upon the binding of a specific ligand, , -linked oligoadenylate ( - a), which is synthesized by a family of enzymes named oligoadenylate synthetases (oass). oas expression is induced by type i interferons (ifn-a/b), but the enzymes require dsrna for the activation of their catalytic activity (kristiansen et al., ) . in the typical antiviral pathway, viral dsrna binds to an oas, which results in the activation of the oas and the synthesis of - a. rnase l, activated upon binding to - a, degrades viral and cellular rnas, thus limiting viral replication and spread (chakrabarti et al., ) . in addition to the direct antiviral effect, rnase l can activate intracellular signaling pathways through the small rna cleavage products that it generates, leading to the induction of type i ifn production (malathi et al., ) . the antiviral role of rnase l has been established for a number of rna viruses. rnase l-deficient mice exhibited increased susceptibility to infection by these viruses (e.g., the picornavirus emcv and the flavivirus wnv) (silverman, ) . recent findings of viral functions that antagonize oas or rnase l, most notably the murine mhv coronavirus ns -encoded phosphodiesterase that can degrade - a, underscore the importance of rnase l pathway in antiviral immunity (zhao et al., ) . compared to its roles against rna viruses, evidence regarding the antiviral role of rnase l against dna viruses (e.g., herpesviruses and vaccinia virus) is limited (rasmussen et al., ; xiang et al., ; zheng et al., ) , although dna viruses can produce dsrna (weber et al., ) . hepatitis b virus (hbv) is a hepatotropic dna virus that can cause acute and chronic hepatitis in humans. more than million people worldwide are chronic carriers of the virus and are at risk of progression of the infection to liver cirrhosis and hepatocarcinoma (lok and mcmahon, ) . ifn-a is currently the approved treatment for chronic hepatitis b (perrillo, ) . however, the mechanism by which ifn-a inhibits hbv replication is not completely understood. a recent etiological study that aimed to identify a potential link between naturally occurring rnase l gene variations (e.g., r q) and prostate cancer suggested that this gene is correlated with chronic hepatitis b and hiv infections (arredondo et al., ) . however, there are few experimental data on the association between hbv and rnase l. in an early study, hbv replication was notably inhibited following poly(i:c) http://dx.doi.org/ . /j.antiviral. . . - /Ó elsevier b.v. all rights reserved. administration to hbv transgenic mice, but the extent of inhibition was not significantly different between groups of mice that were rnase l +/+ or rnase l À/À (guidotti et al., ) . while these data indicated that rnase l is not likely to mediate the antiviral activity of ifn against hbv, it was not determined whether the catalytic activity of rnase l was activated in these mice. in the present study, we adopted human hepatoma cells that permit hbv infection to address whether the oas/rnase l system plays a role in the inhibition of hbv, although hbv is not known to carry or produce dsrna. our data indicate that ligand-mediated activation of these enzymes results in a marked inhibition of hbv replication in both infected and transfected cells. we also provided evidence for differential roles of oas family members in this process. these results suggest that hbv replication can be regulated through the activation of rnase l-mediated rna decay pathways. hbv stock was prepared from the culture supernatant of hepg . . cells (sells et al., ) . the cells were maintained in dmem supplemented with % fbs (hyclone), lg/ml g (a.g. scientific) and lg/ml gentamicin (invitrogen). the cells were split : every days, and the culture supernatant was concentrated as described (vincent et al., ) . to prepare viral dna the viral stock was treated with lg/ml proteinase k for h at °c in mm tris (ph . ), mm edta and % sds, followed by phenol/chloroform/isoamyl alcohol (pci) treatment. viral dna was measured by real-time qpcr with a sybr qpcr kit (kapa biosystems) and a myiq system (bio-rad laboratories) using hbv-specific primers (suppl. table ). pcr was conducted by denaturation at °c for s, followed by cycles of denaturation at °c for s and annealing/elongation at °c for s. the viral genome copy number was calculated based on a standard curve generated with pgem-hbv . and was indicated as genome equivalents (geq). for the infection assay, hepg cells (atcc hb- ) were stably transfected with the ntcp gene (a generous gift from wenhui li of nibs, china) and maintained in dmem with lg/ml blasticidin (invivogen). the ntcp-supplemented cells were incubated for days before infection in primary hepatocyte maintenance media (pmm). pmm consists of williams e medium supplemented with % fbs, ng/ml egf (invitrogen), lg/ml transferrin, lg/ml insulin, mm l-glutamine, lg/ml hydrocortisone, ng/ml dexamethasone, ng/ml sodium selenite, % dmso (all from sigma) and lg/ml gentamicin. the cells were seeded in -well plates ($ cells/well) and inoculated the following day with hbv at $ geq/cell in pmm (without dmso) containing % peg . the viral inoculum was removed h later, and the cells were further incubated (up to days in most experiments) in pmm; the media was changed every days. for the transfection assay, huh- cells grown in dmem with % fbs were transfected with lg/well of pgem-hbv . , a replicon containing the . -mer hbv genome. viral polymerase gene expression was nullified in the hbv . (p-) construct due to a t to c mutation in the first atg codon and a deletion of t from the second atg codon; these changes did not alter the expression of the core gene that overlaps in the same region of the genome (ryu et al., ) . in hbv . (x-), the expression of the viral x gene was blocked with two stop codons introduced adjacent to the first and second atg codons of the x gene by substitution of t for c at nt positions and (cha et al., ). hbv . (c- ) expresses an assembly-defective core protein due to deletion of the nd codon (leu) of the capsid gene (koschel et al., ) . capsid-associated viral dna was extracted from infected cells that were lysed for min on ice in buffer [ mm tris-hcl (ph . ), mm edta, . % np- and mm nacl]. the lysate was clarified by centrifugation at , g, and the supernatant was transferred to a new tube and gently mixed for h with an anti-hbc antibody (dako) and protein a/g plus-agarose (santa cruz biotechnology). the beads were collected by centrifugation at g and washed times in the lysis buffer. viral dna was hbcag merge extracted with lg/ml of proteinase k for h at °c in mm tris (ph . ), mm edta and % sds; it was then extracted by pci and measured by southern blot and real-time pcr as described above. viral cccdna was extracted by pci from infected cells that were lysed for h at °c in lysis buffer [ mm tris-hcl (ph . ), mm edta, mm nacl and % sds] supplemented with proteinase k ( lg/ml). the extracted dna ($ ng) was treated with u of plasmid-safe dnase (epicentre technologies) for h at °c followed by dnase inactivation at °c for min. an aliquot ($ ng) of extracted dna was denatured at °c for min and amplified by qpcr using cccdna-specific primers (suppl. table ). the amplification protocol included cycles of denaturation at °c for s, annealing at °c for s and elongation at °c for s. the hbv cccdna copy number was calculated based on a standard curve generated with pgem-hbv . . viral pgrna was measured by real-time qpcr. total rna was extracted from infected cells with rnaiso plus (takara), treated with rq dnase and reverse transcribed with the improm-ii rt system (promega). cdna derived from ng of total rna was amplified by qpcr as described for the capsid dna assay. transcripts of rnase l, p , oas , oas and oas were measured by real-time pcr from lg of total cellular rna under the same conditions described above. gapdh mrna was used as a normalization control. viral rna and capsid dna were measured by northern and southern blotting as previously described (park et al., ) . infected cells were fixed with . % paraformaldehyde, permeabilized with . % triton x- and immunostained with anti-hbcag (dako) followed by alexa -conjugated goat anti-rabbit igg (invitrogen). rnase l was immunoblotted and detected with anti-rnase l (invitrogen) or anti-flag antibody (sigma). tubulin was probed with an antibody obtained from applied biological materials. the rnase l expression plasmid p xflag-rnase l and a nuclease-defective construct, r a, have been previously described (kwon et al., ) . poly(i:c) was obtained from sigma. trimeric , adenylates, either in -monophosphorylated (p - a) or . intracellular pgrna at days post-infection was measured by rt-qpcr ( ⁄ p < . , ⁄⁄ p < . ). (b) aliquots of huh- or hepg cells either grown in dmem or further incubated in pmm for days were immunoblotted for rnase l ($ kda). human lung carcinoma (a ) cells grown in dmem were also probed. tubulin served as a loading control. (c) rnase l mrna levels in hepg cells incubated in pmm for the indicated periods were measured by rt-qpcr and are shown as fold-induction relative to the initial level. for comparative purposes, p mrna is also shown. unphosphorylated ( - a) forms were provided by chemgenes corp. pcr primers and sirnas were purchased from integrated dna technologies and genepharma, respectively, and their nucleotide sequences are shown in suppl. table . jetpei (polyplus) was used for dna transfection. lipofectamine (invitrogen) was used for transfection of poly(i:c), - as and sirnas. ifn-a was obtained from r&d systems, inc. studies on hbv have been hampered by the lack of a robust cell culture system that permits the full life cycle of viral growth. a recent study identified a bile acid transporter protein named sodium taurocholate cotransporting polypeptide (ntcp) that binds the nterminal pre-s domain of hbv envelope protein l and mediates the entry of hbv and its surrogate virus hdv (yan et al., ) . it was shown that supplementation of human hepatoma cells such as huh- and hepg with ntcp supports hbv infection. to address the role of rnase l in this process, we prepared a pool of hepg cells stably transfected with the ntcp gene. while the ntcpexpressing cells were routinely maintained in dmem, for experiments involving hbv infection, the cells were incubated in pmm for several days before infection, which appeared to render the cells more susceptible to viral infection. infected cells were further incubated (up to days in most experiments) in pmm, and the media was changed every days. immunostaining indicated that the number of cells positive for hbcag, the viral capsid protein, increased with time, although it was less than % of the total cells at days post-infection (fig. a) . to follow viral replication, we measured the viral pregenomic rna (pgrna) by rt-qpcr. at days post-infection, pgrna reached up to $  copies per lg of total cellular rna (fig. b) . in contrast to these results, expression of hbcag and pgrna was markedly reduced when infected cells were transfected with poly(i:c). viral dna in the capsids, measured by real-time pcr of intracellular viral capsids immunoprecipitated with anti-hbc antibody, was similarly decreased. intracellular viral cccdna was also significantly reduced at days post-infection, while no inhibition was observed at days post-infection (suppl. fig. s ). these results demonstrate the profound antiviral effect of poly(i:c) on hbv expression and replication in this cell-based infection system. the antiviral effect of poly(i:c) observed above was likely due to the type i ifn-inducing response mediated by dsrna-binding cellular receptor proteins such as tlr and rig-i. indeed, we observed strong induction of ifn-b and oas mrnas in hepg -ntcp cells approximately h after poly(i:c) transfection (suppl. fig. s ). among the ifn-related cellular factors, we focused on rnase l because while the induction of oas depends on the ifn response, the activation of its endonuclease function requires dsrna. to more specifically address the antiviral activity, if any, of rnase l against hbv, we transfected the infected cells with synthetic -monophosphorylated trimeric , -adenylates (p - a), a specific activator of rnase l. viral pgrna expression was inhibited by up to $ % in a p - a dose-dependent manner, whereas no inhibition was observed in the cells either mock-transfected or transfected with unphosphorylated - a ( fig. a) . because p - a is a unique and specific activator for rnase l, these results strongly indicated that the observed inhibition of hbv was the result of rnase l activation. however, unlike the situation in liver tissue, where abundant expression of rnase l was observed, huh- and hepg cells express only low levels of rnase l (kwon et al., ; malathi et al., ; zhou et al., ) . interestingly, we identified rnase l proteins in an amount that was detectable by immunoblotting of hepatoma cells that were incubated for days in pmm, whereas it was not detected in the same type of cells grown in dmem (fig. b) . our analysis indicated a steady increase of rnase l mrna in the two hepatoma cell lines during incubation in pmm (up to $ -fold in days), whereas the level of p mrna remained stable (fig. c) . although we could not explain this phenomenon, the elevated expression of rnase l provided an opportunity to address the role of rnase l through sirna-mediated suppression. inhibition of pgrna by p - a was almost fully reversed in the rnase l knockdown cells, demonstrating a specific role for rnase l in the inhibition of hbv (fig. ) . compared with this result, poly(i:c)-mediated inhibition was rescued by $ %, suggesting that other effectors in addition to rnase l also contributed to the inhibition. the antiviral activity of rnase l was further characterized in another hepatoma cell line, huh- , following transient transfection of the viral genome construct hbv . . including the . -kb pgrna, all viral mrnas ( . , . and . kb in length) were markedly reduced when the replicon cells were further transfected with rnase l (fig. a) . as in the infection experiments, poly(i:c) was required for the antiviral activity of rnase l, although a mild inhibitory effect was observed even without poly(i:c) (fig. b ). this mild inhibitory effect of rnase l (shown in the lane vs. lane of fig. a) suggested that some oas activation occurred under these conditions, most likely due to non-specific dsrnas (e.g., read-through transcripts) produced off the plasmid dna templates. in contrast, no inhibition was observed with r a, which harbors a missense mutation in the ribonuclease domain of rnase l (dong et al., ) . the strong antiviral effect and concomitant occurrence of rrna cleavage (shown in the lane ) confirmed the poly(i:c)-mediated activation of ectopically expressed rnase l. poly(i:c) alone (without ectopic rnase l expression) had no effect (lanes vs. ; lanes vs. ). this result is consistent with the low level of endogenous rnase l expression, as we reported previously (kwon et al., ) , and no induction of ifn-b or isg expression was observed in this type of hepatoma cell upon poly(i:c) transfection (described below) as reported (li et al., ) . therefore, the observed antiviral activity of transfected poly(i:c) was primarily contributed by the activated ribonuclease function of rnase l. the activation of rnase l was also confirmed with p - a transfected huh- cells (suppl. fig. s ). because the first step in the hbv replication cycle is the synthesis of viral mrna and pgrna off the viral genome, downregulation of viral transcripts by rnase l would subsequently affect viral dna replication as well. as expected, the capsid-associated viral dna was barely detectable in rnase l-activated cells, indicating that the downregulation of viral transcripts resulted in the severe inhibition of viral dna synthesis, including the new synthesis of viral relaxed-circular (rc) and duplex-linear (dl) dnas (fig. c, nd lane of the southern blot). to further substantiate this result, we examined hbv with mutations that have critical effects on viral dna replication. hbv . (p-) is incapable of dna replication because viral pol gene expression is nullified due to a t to c mutation in the first atg codon and a t deletion in the second atg codon; however, these mutations do not alter the expression of the core gene that overlaps the same region (ryu et al., ) . hbv . (c- ) is also replication-defective because the core gene lacks the nd codon (leu) and thus cannot form assembled capsids (koschel et al., ) . our analyses indicated that the transcripts of these mutants, which were not affected despite their replication defects, were degraded by poly(i:c) and rnase l similar to those of the wild-type genome. a similar result was obtained for the third mutant, hbv . (x-), in which the viral x gene was nullified due to stop codons introduced adjacent to the first and second atg codons (cha et al., ) . despite nullification of this critical protein, viral transcription and dna replication were not affected as reported previously for this type of hepatoma cell (melegari et al., ) . likewise, all viral transcripts of this mutant were degraded by poly(i:c) and rnase l similar to those of the wild-type genome. thus, rnase l-dependent downregulation of viral rnas (and dna) as observed for wild-type hbv was observed for all three mutants regardless of their replication capability. rnase l activation requires - a, which is synthesized by oas. in humans, three closely related and linked genes, oas , oas and oas , code for oas proteins. oas expression is induced by type i ifns, but dsrna is required for the activation of their catalytic activity. our rt-pcr analysis showed that oas and oas , but not oas , were constitutively expressed in huh- and hepg cells before all three genes were further induced by ifn-a treatment (fig. ) . as mentioned above, poly(i:c) had no effect on the expression pattern of the three oas genes in huh- cells, yet it induced all three oas genes in hepg cells. therefore, it is likely that in huh- cells, the constitutively expressed endogenous oas and/or oas proteins were catalytically activated by poly(i:c) and contributed to rnase l activation as observed above. to address this hypothesis, we attempted to knock down oas expression using a specific sirna. despite the notable suppression of oas (by up to %), inhibition mediated by rnase l and poly(i:c) was not affected (fig. ) , suggesting that oas does not play a major role in this process. interestingly, a moderate rescue of viral rna was observed in the oas knockdown cells even prior to the transfection of rnase l and poly(i:c). most likely, oas was activated by non-specific dsr-nas produced off the plasmid dna templates, resulting in the activation of endogenous rnase l that was present at a low level. compared with oas , viral transcripts were partially but significantly rescued by oas sirnas, although smearing of the signals might be still indicative of rna degradation, consistent with incomplete suppression of oas (fig. ) . this result indicated that oas plays an important role in activating rnase l in the presence of poly(i:c). however, unlike oas , oas knockdown had no effect without poly(i:c). because both oas and oas are constitutively expressed in these cells prior to poly(i:c) treatment, as shown in fig. , these results suggest that the two enzymes differ in their requirement for dsrna; specifically, oas might be activated more efficiently with poly(i:c). in vitro data indicated that oas was more sensitive to dsrna ($ times less dsrna is required for activation) (marie et al., ) . to examine the role of oas , we stimulated its expression by ifn-a treatment following transfection with oas sirna. viral rna was partially rescued with one of the two sirnas, and this rescue was correlated with the extent of oas mrna suppression (fig. ) . ifn-a treatment alone did not show apparent inhibition of hbv transcripts, as we previously reported for this type of hepatoma cell (park et al., ) . our results indicate that oas and oas , but not oas , are dependent on poly(i:c) under these conditions. thus, oas family members differ in their requirement for dsrna and contribute differentially to rnase l activation under different conditions. among ifn-related cellular factors, rnase l has been shown to inhibit a number of rna viruses and few dna viruses. in the typical antiviral pathway, viral dsrna activates oas, which in turn activates rnase l through the synthesis of - a. it is thought that rnase l-mediated viral rna decay leads to the inhibition of these viruses. in this study, we attempted to address whether the oas/ rnase l system plays a role in cellular restriction or ifn-mediated inhibition of hbv, although hbv is not known to carry or produce dsrna. with the newly established hepg -ntcp cell culture system, which permits hbv infection, we showed that viral gene expression and replication can be profoundly reduced by - aor poly(i:c)-mediated activation of rnase l. in hbv . -transfected huh- cells, it was further confirmed that this type of inhibition occurred mostly through viral rna degradation via the ribonuclease function of rnase l. in contrast to the effect of the specific ligand - a, the poly(i:c)-mediated antiviral effect was not fully rescued by rnase l knock down. while the latter result implies that other effectors also contribute to the inhibition observed, it might be worth considering the ways by which poly(i:c) might have functioned in our study and in previous studies performed with hbv-transgenic animals. intravenous administration of poly(i:c) resulted in a dramatic reduction of viral replication; however, no significant difference was observed between groups of rnase l +/+ or rnase l À/À animals with respect to the extent of inhibition (guidotti et al., ) . a subsequent study showed that poly(i:c) inhibited hbv through induction of type i ifn responses, as evidenced by intrahepatic isg expression and requirement for the cytokine receptors in this process (isogawa et al., ) . while these data indicate that rnase l is not likely to mediate the antiviral activity of ifn against hbv, it was not determined whether the catalytic activity of rnase l was activated in these mice. most likely, poly(i:c) that was injected intravenously into the animals triggered ifn induction via tlr signaling. however, the lack of apparent difference in viral replication between rnase l +/+ or rnase l À/À animals (despite intrahepatic induction of oas) suggested that rnase l was most likely not activated. consistent with this notion, the steady state level of viral rna was not affected in these animals, whereas hbv dna was profoundly inhibited. in contrast, all forms of hbv rnas were profoundly reduced in our cell-based assays, indicating the efficient activation of rnase l in poly(i:c)transfected cells. this direct rnase l-activating effect of poly(i:c) was more evident in the huh- cell, in which tlr -mediated signaling is known to be inefficient (li et al., ) . our rnase l knockdown experiment performed in hepg cells, showed $ % rescue of poly(i:c)-mediated viral rna reduction, suggesting that other factors in addition to rnase l contributed to the inhibition. indeed, we observed strong induction of ifn-a and oas mrnas in hepg cells within h of poly(i:c) transfection. studies exploring how the ifn-mediated antiviral responses (those triggered by the secreted ifns or by the direct activation of prr with pamp) inhibit hbv indicated that various steps of viral replication are affected (reviewed in chang et al., ) . although many of these studies highlight posttranscriptional inhibition mechanisms, cellular effectors responsible for this type of regulation have not been identified. recently, it was reported that zinc finger antiviral protein (zap), a cellular protein known initially as a retroviral restriction factor, inhibits hbv through the viral rna decay process (mao et al., ) . while zap itself retains no ribonuclease activity, it is known to bind viral (and cellular) rnas through its n-terminal domain, which contains four zinc finger motifs and stimulates rna degradation by recruiting various host rna processing machineries, including exosomes. according to these authors, expression of at least one isoform of zap was induced by ifn signaling, and ectopic expression of zap markedly reduced viral rna. while rnase l differs from zap in that it requires specific ligands to activate its ribonuclease function, it is also possible that rnase l acts in combination with other cellular factors or as part of the cellular machinery during viral rna decay. regarding the suggested relationship between naturally occurring rnase l gene variation (e.g., r q) and chronic hepatitis b (arredondo et al., ) , we did not find any defect in hbv inhibition with this variant in our transfection assay (data not shown). this result was consistent with the reduced - a-mediated oligomerization activity but intact ribonuclease function of this allele (xiang et al., ) . we showed that oas and oas were constitutively expressed in huh- cells before their expression was further induced, along with that of oas , by ifn-a treatment. elevated oas activity has been found in the sera of hepatitis c patients following ifn therapy, and the level of oas activity was found to be correlated with their virological responses to this therapy (kim et al., ) . among chronic hepatitis b patients, the frequency of a nonsense mutation in the r codon of oas was reported to be higher among non-responders to ifn therapy compared with responders (ren et al., ) . although we did not test this allele of oas , our knockdown data support the role of oas , along with that of oas , in the activation of rnase l. in summary, our data indicated that hbv replication can be regulated through the activation of rnase l-mediated rna decay pathways with exogenously provided ligands. specifically, small molecules that mimic natural ligands (such as - a) would represent a novel therapeutic strategy. short communication: rnasel alleles and susceptibility to infection by human retroviruses and hepatitis viruses stimulation of hepatitis b virus genome replication by hbx is linked to both nuclear and cytoplasmic hbx expression new insights into the role of rnase l in innate immunity the innate immune response to hepatitis b virus infection: implication for pathogenesis and therapy basis for regulated rna cleavage by functional analysis of rnase l and ire p interferon-regulated pathways that control hepatitis b virus replication in transgenic mice toll-like receptor signaling inhibits hepatitis b virus replication in vivo -, -oligoadenylate synthetase response ratio predicting virological response to peg-interferon-alpha b plus ribavirin therapy in patients with chronic hepatitis c hepatitis b virus core gene mutations which block nucleocapsid envelopment the oligoadenylate synthetase family: an ancient protein family with multiple antiviral activities the ribonuclease l-dependent antiviral roles of human , -oligoadenylate synthetase family members against hepatitis c virus distinct poly(i-c) and virusactivated signaling pathways leading to interferon-beta production in hepatocytes chronic hepatitis b small self-rna generated by rnase l amplifies antiviral innate immunity inhibition of hepatitis b virus replication by the host zinc finger antiviral protein -kda and -kda isoforms of interferon-induced ( - )oligoadenylate synthetase exhibit differential catalytic parameters cloning and characterization of a novel hepatitis b virus x binding protein that inhibits viral replication pkr-dependent mechanisms of interferon-alpha for inhibiting hepatitis b virus replication benefits and risks of interferon therapy for hepatitis b herpes simplex virus infection is sensed by both toll-like receptors and retinoic acid-inducible gene-like receptors, which synergize to induce type i interferon production polymorphisms of interferon-inducible genes oas associated with interferonalpha treatment response in chronic hbv infection hepatitis b virus polymerase suppresses translation of pregenomic rna via a mechanism involving its interaction with stem-loop structure production of hepatitis b virus particles in hep g cells transfected with cloned hepatitis b virus dna viral encounters with , -oligoadenylate synthetase and rnase l during the interferon antiviral response hepatitis b virus impairs tlr expression and function in plasmacytoid dendritic cells doublestranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses blockade of interferon induction and action by the e l double-stranded rna binding proteins of vaccinia virus effects of rnase l mutations associated with prostate cancer on apoptosis induced by , -oligoadenylates sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns protein is required for virus replication and liver pathology increased severity of hsv- keratitis and mortality in mice lacking the - a-dependent rnase l gene mapping of the human rnasel promoter and expression in cancer and normal cells we are grateful to wenhui li for providing us with the ntcpexpressing plasmid. this work was supported by the basic science research program of the national research foundation (# - ) of the republic of korea. some preliminary studies were supported by a grant from korea university in - . supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.antiviral. . . . key: cord- -cgky ar authors: otaki, momoko; sada, kiyonao; kadoya, hiroyasu; nagano-fujii, motoko; hotta, hak title: inhibition of measles virus and subacute sclerosing panencephalitis virus by rna interference date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: cgky ar subacute sclerosing panencephalitis (sspe) is a rare, but fatal outcome of measles virus (mev) infection. sspe develops after prolonged persistence of mutated mev called sspe virus. although a combination therapy using interferon and inosiplex or ribavirin appears to prolong survival time to some extent, there is currently no effective treatment to completely cure sspe and a new treatment strategy is greatly needed. in this study, we adopted rna interference (rnai) strategy and examined whether small interfering rnas (sirnas) can be used to inhibit replication of mev and sspe virus. we report here that sirnas targeted against l mrna of mev, either synthetic sirnas or those generated by pcpur + u i-based expression plasmids, effectively and specifically inhibited replication of both mev and sspe virus without exhibiting any cytotoxic effect. the l protein of mev is a major component of rna-dependent rna polymerase that is essential for viral rna replication, and yet it is least abundant among all the mev proteins expressed. therefore, mrna encoding the l protein would be a good target for rnai strategy. the present results imply the possibility that our sirnas against mev l mrna are among the potential candidates to be used to treat patients with sspe. measles virus (mev), a member of the genus morbillivirus, the family paramyxoviridae, is nonsegmented, negativestranded rna virus. the mev genome contains six genes, such as n, p/c/v, m, f, h and l (lamb and kolakofsky, ) . the p/c/v gene is transcribed to the overlapping p/c/v mrnas while the other genes each to a single mrna. the mev genes are flanked by the extragenic -terminal leader and the terminal trailer regions, and are connected by short stretch of conserved sequences. these gene boundary sequences consist of three regions, called the transcription end, intercistronic and transcriptional start sequences, which function as the transcription regulatory sequences. the l mrna encodes the l protein that contains the catalytic motif common to the rna-dependent rna polymerase of negative-strand rna viruses. subacute sclerosing panencephalitis (sspe) is a rare, but fatal outcome of mev infection. it has been estimated that approximately - out of a million mev-infected individuals develop sspe, which is caused by mutated mev, or sspe virus, after a prolonged incubation period of an average of years (okuno et al., ) . although a combination therapy using interferon (ifn) and inosiplex (anlar et al., ; gascon, ) or ribavirin (hosoya et al., ) appears to improve clinical conditions and survival time to some extent, there is currently no specific treatment available to cure patients with sspe and a new treatment strategy for sspe is greatly needed. rna interference (rnai), which involves small interfering rna (sirna) of - -bp, is known to play an important role in host defense mechanisms in plants and insects (bernstein et al., ; elbashir et al., b; hamilton and baulcombe, ; zamore et al., ) . sirnas function in mammalian cells as well (elbashir et al., a; gitlin et al., ; mccaffrey et al., ; paddison et al., ; paul et al., ; rubinson et al., ; xia et al., ) and are shown to suppress replication of a wide variety of viruses, including human immunodeficiency virus (coburn and cullen, ; huelsmann et al., lee et al., ; takaku, ) , hepatitis b and c viruses (radhakrishnan et al., ; randall and rice, ) , severe acute respiratory syndrome (sars)-coronavirus (wang et al., ; wu et al., ) , human rhinovirus (phipps et al., ) , human respiratory syncytial virus and parainfluenza virus (barik, ; bitko et al., ; zhang et al., ) , the latter two of which belong to the family paramyxoviridae. however, little information is available so far on rnai against mev and sspe virus infection. we report here that sirnas targeted against l mrna of mev, either synthetic ones or those generated by pcpur + u i-based expression plasmids, effectively inhibit replication of both mev and sspe virus. to our knowledge, this is the first report describing effective inhibition of mev and sspe virus by means of sirna. chemically synthesized rna oligonucleotides were purchased (japan bio service, saitama, japan). in order to increase the stability of the sirna, overhang of each strand was made of thymidine residues instead of uridine (elbashir et al., a (elbashir et al., , b . sirna duplex was prepared as described previously (takigawa et al., ) . in brief, m each of sense and antisense rna oligonucleotides were heated at • c for min and incubated at • c for min in an annealing buffer ( mm potassium acetate, mm hepes-koh (ph . ), mm magnesium acetate). the sirna thus prepared was transfected to cultured cells using lipofectamine (invitrogen) according to the manufacturer's instructions. plasmid vectors expressing sirnas were constructed as reported previously (takigawa et al., ) with some modifications. briefly, chemically synthesized dna oligonucleotides were purchased (espec oligo service corp., tsukuba, ibaraki, japan). sense and antisense dna oligonucleotides were annealed and cloned into the unique bspmi site of pcpur + u i downstream of the u promoter (miyagishi and taira, ) . pcpur + u i was kindly provided by dr. k. taira, graduate school of engineering, the university of tokyo, tokyo, japan. the sirna expression plasmids or an empty control vector were transfected into cultured cells by using fugene reagent (roche) according to the manufacturer's instructions. a marmoset b-lymphoblastoid cell line b a was described previously (itoh et al., ; katayama et al., ; kobune et al., ; shibahara et al., ) . vero/slam cells (ono et al., ) were a kind gift from dr. y. yanagi, department of virology, kyusyu university graduate school of medicine, fukuoka, japan, and were maintained in dulbecco's modification of eagle's minimum essential medium supplemented with % fetal calf serum and g ( g/ml). wild-type mev strains, clinical isolates k and t , were described previously (katayama et al., ) . k and t strains belong to mev genotypes d and d , respectively. a laboratoryadapted standard strain, edmonston, which belongs to genotype a, was also used as described previously (shibahara et al., ) . sspe-kobe- , an sspe virus strain, was recently isolated from a patient with sspe, and belongs to genotype d (manuscript in preparation). sspe-kobe- has characteristics similar to other strains of sspe virus, such as the lack of cell-free virus production and accumulation of mutations in the viral genome (data not shown). encephalomyocarditis virus (emcv; dk- strain), a member of the family picornaviridae, was prepared as described previously (song et al., ; taguchi et al., ) and used as an irrelevant control virus in this study. serially diluted culture supernatants of cells infected with wild-type mev were inoculated onto vero/slam cells and virus adsorption was done at • c for h. after being cultured overnight, the number of syncytium formed on the monolayer cells were counted. to assess the effect of sirna on sspe virus replication, sspe virus-infected cells were transfected with either synthetic sirna or a pcpur + u i expression plasmid, and then cocultured with fresh vero/slam cells. after overnight culture, the number of newly formed syncytia was counted. emcv titers were determined by plaque assay on vero/slam cells, as described previously with minor modifications (song et al., ; taguchi et al., ) . we first assessed the efficacy of synthetic sirnas, which were designed to target l mrna of mev. the sequences and positions of the sirnas are summarized in table . sirna targeted against ns of hepatitis c virus, named ns - (takigawa et al., ) , served as a negative control. in the initial experiments, vero/slam cells were first transfected with sirna for h and then inoculated with the k strain of mev. after h, virus titers in the culture supernatants were determined. as shown in fig. a , mev replication was markedly inhibited by sirnas mv-l , -l and -l , and moderately by mv-l , -l and -l . on the other hand, mv-l only slightly inhibited mev replication. when cells were first inoculated with mev and then transfected with sirna, marked inhibition of mev replication by sirnas mv-l , -l and -l was also observed. treatment with mv-l sirna after , or h postinfection efficiently inhibited mev replication (fig. b) . however, the inhibitory effect was no longer observed when the sirna was added to the cells h postinfection. similar results were obtained with mv-l and -l sirnas (data not shown). the inhibitory effect was dose-dependent and the %-inhibiting dose was nm for mv-l (fig. c) . we constructed pcpur + u i-based plasmids expressing mv-l to -l and mv-l , and tested their possible inhibitory effects on mev replication. the ns - sirna-expressing plasmid served as a negative control. vero/slam cells were transfected with the expression plasmid and then inoculated with the virus. after days, virus replication was monitored. the results clearly demonstrated that pcpur + u i-mediated sirna, mv-l , -l , -l and -l , markedly inhibited replication of the k strain of mev ( fig. a) . the inhibitory effects of mv-l and -l appeared to be weaker than those of mv-l to -l in this series of experiments. the plasmid-based sirna, mv-l , -l and -l , also inhibited replication of mev t strain, which belongs to genotype d and shares the target sequences in common with genotype d strains (data not shown). on the other hand, only mv-l sirna, but not mv-l or -l , inhibited replication of the edmonston strain, which belongs to genotype a (fig. b) . it should be noted that there are one and two nucleotide substitutions in the target sequences for mv-l and -l , respectively, between genotypes d and a. on the other hand, the target sequence for mv-l is completely conserved between the two genotypes. as had been expected, replication of an unrelated virus, emcv, was not affected by mv-l sirna (fig. c) . these results suggested mev sequence-specific inhibition by the sirnas. we then tested to see whether or not mv-l sirna, either synthetic or pcpur + u i-mediated, inhibits sspe virus infection. vero/slam cells persistently infected with sspe-kobe- and uninfected cells were transfected with either synthetic sirna or pcpur + u i-mv-l overnight. ns - sirna, either synthetic or plasmid-mediated, served as a control. after transfection, the sspe virus-infected cells were cocultured with uninfected cells and the number of syncytium that emerged on the monolayer cells was counted , and h thereafter. this experimental procedure was used to examine the effect of sir-nas on both virus multiplication in the cell and cell-to-cell viral spread. the results obtained clearly demonstrated that both syn-thetic and plasmid-mediated mv-l sirna markedly inhibited sspe virus replication and/or cell-to-cell spread ( fig. a and b) . in the next series of experiments with sspe virus, vero/slam cell cultures persistently infected with sspe-kobe- were transfected with synthetic mv-l sirna h after cell seeding (coculture). after overnight incubation with sirna, the cells were refed with fresh medium without containing sirna and maintained in the culture to observe syncytium formation. under this experimental condition too, sspe virus replication in the infected cells was markedly inhibited by synthetic mv-l sirna (fig. c) . similarly, pcpur + u i-mediated mv-l sirna brought about efficient inhibition of sspe-kobe- replication when the plasmid was transfected h after cell seeding (fig. d) . the l protein of mev is an rna-dependent rna polymerase that is essential for viral rna replication, and yet it is least abundant among all the mev proteins expressed (barik, ; lamb and kolakofsky, ) . therefore, viral mrna encoding the l protein would be a good target for the rnai strategy to inhibit replication of mev and sspe virus. indeed, we demonstrated in the present study that sirnas targeted against selected portions of l mrna of mev, especially mv-l -l and -l , either synthetic sirnas or those expressed by pcpur + u i-based plasmids, effectively inhibited replication of both mev and sspe virus (figs. - ) . the inhibitory capacity of these otherwise effective sirnas became barely detectable, if any, when the sirnas were transfected to the cells h after mev infection (fig. b) . this result suggests that, once virus replication reaches its maximal level, viral mrna molecules have accumulated abundantly in the cells to a level beyond the inhibitory capacity of sirna. this idea might discourage an attempt to use sirna for therapeutic purposes. however, it should also be stated that sirna renders neighboring, uninfected cells resistant to virus infection, preventing the viral spread to the neighboring cells. in fact, the spread of sspe virus from persistently infected cells was efficiently blocked by mv-l sirna (fig. a and b) . it is also noteworthy that, even in the transient transfection system where sirna expression plasmids were introduced to at most % of the cells, mev replication was inhibited by > % vero/slam cells were transfected with each of the pcpur + u i-based expression plasmids ( g/well) overnight and then inoculated with the k strain of mev. after h, virus titers in the culture supernatants were determined. the ns - sirna-expressing plasmid served as a control (cont). mock indicates cultures treated with the transfection reagent without sirna. data represent the mean ± s.e.m. obtained from two independent experiments, which are expressed as the percentages of the number of syncytium seen in the wells treated with the ns - sirna-expressing plasmid. * p < . , compared with the control. (b) vero/slam cells were transfected with each of the pcpur + u ibased expression plasmids ( g/well) overnight and then inoculated with the edmonston strains of mev (genotype a). after h, virus titers in the culture supernatants were determined. data represent the mean ± s.e.m. obtained from two independent experiments. * p < . , compared with the control. (c) vero/slam cells were transfected with each of the pcpur + u i-based expression plasmids ( g/well) overnight and then inoculated with emcv. after h, virus titers in the culture supernatants were determined. data represent the mean ± s.e.m. obtained from two independent experiments. by the sirnas (fig. a and b) . the most likely explanation for this remarkably efficient inhibition is that virus-infected cells form syncytia through fusion with their neighboring cells, at least some of which are expected to harbor the sirna-expressing plasmid, and that de novo-produced sirna is introduced to the virus-infected cells by virtue of syncytium formation. although some sirnas may activate the ifn system (kim et al., ; sledz et al., ) , the sirnas used in our study were unlikely to induce ifn activation since they did not inhibit replication of emcv (fig. c) . emcv is known to be highly sensitive to ifn (song et al., ; taguchi et al., ) . this result also indicates that the sirnas used in this study do not bring about any cytotoxic effect. currently, there is no satisfactory specific treatment for sspe and, therefore, the rnai strategy might be considered as a potential therapeutic measure against this fatal disease. the sequence targeted by mv-l sirna is well conserved across different genotypes of wild-type mev and sspe virus (data not shown). considering its efficient silencing effect and the lack of cytotoxicity together, mv-l sirna would be one of the good candidates for a clinical trial. however, a number of important issues should be addressed. it was recently reported that some viruses could escape rnai by mutating the targeted viral sequences (boden et al., ; das et al., ; gitlin et al., ; leonard and schaffer, ; wilson and richardson, ) . characteristic to an rna virus, whose rna-dependent rna polymerase is error-prone, sspe virus frequently undergo genetic mutations during its long-term persistence in a patient (lamb and kolakofsky, ) . therefore, a combined usage of multiple sirnas that target different sequences would be recommended. at the same time, the possible off-target effect(s) of each sirna should be examined thoroughly. we also need to establish a good system to efficiently introduce sirnas into the brain cells. we are currently generating recombinant adenovirus vectors expressing sirnas against different portions of mev/sspe virus l mrna. the usage of nonviral vectors, such as organically modified silica nanoparticles (bharali et al., ) , would be another promising option. in any case, the safety of the vectors when applied in vivo should be completely warranted. in conclusion, we have developed sirnas that can effectively inhibit replication of mev and sspe virus. although a number of important issues should be addressed before its possible clinical application, these sirnas may be a potential candidate for therapeutic measures against the currently incurable sspe. graduate school of medicine, fukuoka, japan, for providing pcpur + u i and vero/slam cells, respectively. this work was supported in part by research programs for slow virus infection from the ministry of health, labour and welfare, japan, and was also carried out as part of the coe program at kobe university graduate school of medicine. retrospective evaluation of interferon-beta treatment in subacute sclerosing panencephalitis control of nonsegmented negative-strand rna virus replication by sirna role for a bidentate ribonuclease in the initiation step of rna interference organically modified silica nanoparticles: a nonviral vector for in vivo gene delivery and expression in the brain inhibition of respiratory viruses by nasally administered sirna human immunodeficiency virus type escape from rna interference potent and specific inhibition of human immunodeficiency virus type replication by rna interference human immunodeficiency virus type escapes from rna interference-mediated inhibition duplexes of -nucleotide rnas mediate rna 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interference molecular epidemiology and changing distribution of genotypes of measles virus field strains in japan interferon induction by sirnas and ssrnas synthesized by phage polymerase marmoset lymphoblastoid cells as a sensitive host for isolation of measles virus paramyxoviridae: the viruses and their replication expression of small interfering rnas targeted against hiv- rev transcripts in human cells computational design of antiviral rna interference strategies that resist human immunodeficiency virus escape rna interference in adult mice u promoter-driven sirnas with four uridine overhangs efficiently suppress targeted gene expression in mammalian cells incidence of subacute sclerosing panencephalitis following measles and measles vaccination in japan measles viruses on throat swabs from measles patients use signaling lymphocytic activation molecule (cdw ) but not cd as a cellular receptor short hairpin rnas (shrnas) induce sequence-specific silencing in mammalian cells effective expression of small interfering rna in human cells small interfering rna molecules as potential anti-human rhinovirus agents: in vitro potency, specificity, and mechanism rna interference as a new strategy against viral hepatitis interfering with hepatitis c virus rna replication a lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by rna interference increased binding activity of measles virus to monkey red blood cells after long-term passage in vero cell cultures activation of the interferon system by short-interfering rnas the ns a protein of hepatitis c virus partially inhibits the antiviral activity of interferon hepatitis c virus ns a protein interacts with , -oligoadenylate synthetase and inhibits antiviral activity of ifn in an ifn sensitivity-determining region-independent manner gene silencing of hiv- by rna interference suppression of hepatitis c virus replicon by rna interference directed against the ns and ns b regions of the viral genome inhibition of severe acute respiratory syndrome virus replication by small interfering rnas in mammalian cells hepatitis c virus replicons escape rna interference induced by a short interfering rna directed against the ns b coding region inhibition of sars-cov replication by sirna sirna-mediated gene silencing in vitro and in vivo rnai: doublestranded rna directs the atp-dependent cleavage of mrna at to nucleotide intervals inhibition of respiratory syncytial virus infection with intranasal sirna nanoparticles targeting the viral ns gene the authors are grateful to dr. k. taira, graduate school of engineering, the university of tokyo, tokyo, japan, and dr. y. yanagi, department of virology, kyusyu university key: cord- -ihj hi authors: takano, tomomi; endoh, misaki; fukatsu, hiroaki; sakurada, haruko; doki, tomoyoshi; hohdatsu, tsutomu title: the cholesterol transport inhibitor u a inhibits type i feline coronavirus infection date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: ihj hi feline infectious peritonitis (fip) is a feline coronavirus (fcov)-induced fatal disease in wild and domestic cats. fcov exists in two serotypes. type i fcov is the dominant serotype worldwide. therefore, it is necessary to develop antiviral drugs against type i fcov infection. we previously reported that type i fcov is closely associated with cholesterol throughout the viral life cycle. in this study, we investigated whether u a, the cholesterol synthesis and transport inhibitor, shows antiviral effects against type i fcov. u a induced cholesterol accumulation in cells and inhibited type i fcov replication. surprisingly, the antiviral activity of u a was suppressed by the histone deacetylase inhibitor (hdaci), vorinostat. hdaci has been reported to revert u a-induced dysfunction of niemann-pick c (npc ). in conclusion, these findings demonstrate that npc plays an important role in type i fcov infection. u a or other cholesterol transport inhibitor may be considered as the antiviral drug for the treatment of cats with fip. coronaviruses are single-stranded positive-sense rna viruses in the subfamily coronavirinae of the family coronaviridae, and cause respiratory and gastrointestinal diseases in mammals and birds (su et al., ) . coronaviruses are important pathogens causing emerging and re-emerging infectious diseases. for example, outbreaks of severe acute respiratory syndrome coronavirus (sars-cov) and middle-east respiratory syndrome coronavirus (mers-cov) occurred mainly in asia in and , respectively (de wit et al., ) . feline infectious peritonitis (fip) is known as a highly fatal disease caused by coronaviruses, similar to sars and mers (dandekar and perlman, ; pedersen, ) . feline coronavirus (fcov) belongs to alphacoronavirus of the family coronaviridae (de groot et al., ) . the fcov virion is mainly composed of nucleocapsid (n), envelope, membrane, and peplomer spike (s) proteins (motokawa et al., ) . fcov has been classified into types i and ii according to the amino acid sequence of its s protein (motokawa et al., ) . type ii fcov was previously suggested to be from recombination between type i fcov and type ii canine coronavirus (ccov) (herrewegh et al., ; terada et al., ) . separate from these genotypes/serotypes, fcov consists of two biotypes: low pathogenic feline enteric coronavirus (fecv: lowvirulent fcov) and high pathogenic fip virus (fipv: virulent fcov) (pedersen, ) . fipv can cause immune-mediated inflammatory disease with high mortality in domestic and wild felidae. although antiviral drugs and vaccines against fip have been investigated, no method has yet been established for practical use. serological and genetic surveys revealed that type i fcov is dominant worldwide (hohdatsu et al., ; kummrow et al., ; wang et al., ) ; therefore, antiviral drugs and vaccines need to be developed against type i fcov infection. however, a few studies on type i fcov have been performed because of its low replication ability in feline cell lines. we previously reported that type i fcov is closely associated with cholesterol throughout the viral life cycle (takano et al., ) . we also confirmed that an increase in plasma membrane cholesterol enhances type i fcov infection. these findings suggest that cell membrane cholesterol plays an important role in type i fcov infection. cellular cholesterol is derived from de novo cholesterol biosynthesis and low density lipoprotein uptake (simons and ikonen, ) . biosynthesized or entrapped cholesterol is transported in cells and heterogeneously distributed in organelles. association of the cholesterol biosynthesis and transport systems in cells with virus replication has been reported (aizaki et al., ; carette et al., ; mackenzie et al., ; zheng et al., ) . recent studies have shown that intracellular cholesterol synthesis and transport inhibitors potently reduced viral replication. for example, human hepatitis virus c (hcv) rna replication is disrupted by hmg-coa reductase inhibitors (honda and matsuzaki, ) , and cholesterol transporter inhibitors inhibit replication of ebola virus (carette et al., ) . however, it remains unclear whether cholesterol biosynthesis and intracellular transport inhibitor can suppress type i fcov replication. u a is a cationic amphiphilic drug (cad) impairing cholesterol biosynthesis and intracellular transport (cenedella, ). u a inhibits intracellular cholesterol biosynthesis by suppressing oxidosqualene cyclase (cenedella et al., ) . u a also inhibits cholesterol release from lysosomes through impairing the function of a cholesterol transporter, niemann-pick type c (npc ) (ko et al., ) . it has been reported that u a suppresses replication of ebola virus, dengue virus, and human hepatitis c virus (elgner et al., ; lu et al., ; poh et al., ) . however, no study on the influence of u a on fcov infection has been reported. in this study, we investigated whether u a inhibits fcov infection. felis catus whole fetus (fcwf)- cells (kindly supplied by dr. m. c. horzinek of universiteit utrecht) were grown in eagles' mem containing % l- medium, % fetal calf serum (fcs), u/ml penicillin, and mg/ml streptomycin. the type i fcov ku- strain (fcov-i ku- ) was isolated in our laboratory, and the type i fcov black strain (fcov-i black), the type i fcov ucd- strain (fcov-i ucd- ), and the type i fcov ucd- strain (fcov-i ucd- ) were kindly supplied by dr. j. k. yamamoto from the university of florida. the type ii fcov wsu - strain (fcov-ii - ) was kindly provided by dr. m. c. horzinek of universiteit utrecht. the type ii fcov wsu - (fcov-ii - ) was kindly provided by dr. a. j. mckeirnan from washington state university. the type ii ccov - strain (ccov-ii - ) were supplied by dr. e. takahashi of the university of tokyo. these viruses were grown in fcwf- cells at c. u a, methyl-b-cyclodextrin (mbcd), ro - , ay- , amorolfine, simvastatin, clomiphene, and alendronate were purchased from sigma aldrich (usa). lovastatin, fluvastatin, and itraconazole were purchased from wako pure chemical (japan). vorinostat was purchased from cayman chemical (usa). u a and mbcd were dissolved in water as mm (u a) and m (mbcd) stock, respectively. other compounds were dissolved in dimethyl sulfoxide (dmso) as mm stock. on the day of the experiments, these compounds were diluted to the desired concentrations in maintenance medium. the fcwf- cells were seeded on -well plates. the compounds were added in triplicate to the wells. after incubation for defined periods, the culture supernatants were removed, wst- solution (kishida chemical, japan) was added, and the cells were returned to the incubator for h. the absorbance of formazan produced was measured at nm with a -well spectrophotometric plate reader, as described by the manufacturer. percentage viability was calculated using the following formula: cytotoxicity (%) ¼ [(od of compound-untreated cells -compound-treated cells)/(od of compound-untreated cells)]  . the cellular cholesterol content of fcwf- cells was evaluated by the cholesterol cell based detection assay kit (cayman chemical, usa) according to the manufacturer's instructions. briefly, fcwf- cells were grown on an -well lab-tek chamber slide (thermo fisher scientific, usa). semi-confluent fcwf- cell monolayers were cultured in medium containing mm u a at c for , , , or h. after fixing and staining, filipin iii-binding cells were analyzed using a leica dm b microscope and las x integrated imaging system (leica microsystems, germany). the amount of intracellular cholesterol was determined using the amplex red cholesterol assay kit (molecular probes, usa) following the manufacturer's instructions. confluent fcwf- cell monolayers were cultured in medium containing compounds at the indicated concentrations in -well multi-plates at c for h. cells were washed and the virus (moi . ) was adsorbed into the cells at c for h. after washing, cells were cultured in carboxymethyl cellulose (cmc)-mem or mem. in the case of cells cultured in cmc-mem, the cell monolayers were incubated at c for h, fixed and stained with % crystal violet solution containing % buffered formalin, and the resulting plaques were then counted. the percentage of the plaque decrease or increase was determined using the following formula: percentage of the plaque reduction (%) ¼ [(plaque number of compoundÀtreated cells)/(plaque number of compoundÀuntreated cells)]  . in the case of cells cultured in mem, the culture supernatants were collected h post-infection, and virus titers were determined by the tcid assay. fcwf- cells were grown on an -well lab-tek chamber slide (thermo fisher scientific, usa). semi-confluent fcwf- cell monolayers were cultured in medium containing mm u a at c for h. cells were washed and the virus (moi . ) was adsorbed into the cells at c for h. after washing, cells were cultured in mem. the cell monolayers were incubated at c. after h, n protein levels were determined by an immunofluorescence assay (ifa), as described previously (hohdatsu et al., ) . for recognizing fcov n protein, mab e - (mouse igg ) prepared by our laboratory was used (hohdatsu et al., ) . the confluent fcwf- cells were cultured in medium containing mm u a for , , , , , or h. after washing, the virus (moi . ) was adsorbed into cells at c for h. cells were washed and cultured in cmc-mem. the cell monolayers were incubated at c for h, fixed and stained, and the resulting plaques were then counted. the percentage of the plaque reduction was measured with reference to the method described above. confluent fcwf- cell monolayers were cultured in medium containing mm u a in -well multi-plates at c for h. after washing, cells were cultured in medium containing vorinostat at the indicated concentrations for h. cells were washed and the virus (moi . ) was adsorbed into the cells at c for h. cells were washed and cultured in cmc-mem. the cell monolayers were incubated at c for h post-inoculation, fixed and stained, and the resulting plaques were then counted. the percentage of the plaque reduction was measured with reference to the method described above. the influence of vorinostat on the intracellular distribution of cholesterol was investigated as described above. the influence on virus infection in vorinostat-treated cells was confirmed by ifa as described above. data from only two groups were analyzed with the student's ttest (welch's t-test or bartlett's test), and multiple groups were analyzed with a one-way anova followed by tukey's test. cytotoxicity assay was performed to determine the non-toxic concentration of u a against fcwf- cells (fig. a) . the % cytotoxic concentration (cc ) and the % cytotoxic concentration (cc ) value of u a were . ± . (mean ± se) mm and . ± . (mean ± se) mm, respectively. we investigated changes in the cellular cholesterol content of u a-treated fcwf- cells. u a caused accumulation of cholesterol as measured by filipin iii staining (fig. b) . to determine whether u a affected the cellular cholesterol content, the level of cholesterol in fcwf- cells was measured. although the level of cellular cholesterol significantly decreased in mm mbcd-treated cells, neither mm nor mm u a decreased cellular cholesterol levels (fig. c) . in order to confirm the effects of the accumulation of cholesterol on fcov infection, fcwf- cells were pretreated with u a, followed by virus inoculation. pretreatment with u a demonstrated reduction of fcov-i ku plaque formation ( fig. d and e) . in contrast, the percentage of plaque reduction of fcov-ii - was not affected by pretreatment with u a. we investigated the expression of viral proteins in order to further determine the effects of u a on fcov infection. the n protein levels of fcov-i ku- specifically decreased in fcwf- cells pretreated with u a (fig. f) . in contrast to fcov-i ku , pretreatment with u a did not affect the n protein levels of fcov-ii - in fcwf- cells. the relationship between the fcov serotypes and antiviral effects of u a were investigated. the influence of u a on replication of strains of type i fcov (fcov-i ku , fcov-i black, fcov-i ucd , and fcov-i ucd ), strains of type ii fcov (fcov-ii - and fcov-ii - ), and strain of type ii ccov (ccov-ii - ) in fcwf- cells was investigated. pretreatment with u a reduced type i fcovs plaque formation in a dose-dependent manner ( fig. a) . in contrast, the percentage of plaque reduction in type ii fcovs and type ii ccov was slightly affected by pretreatment with u a (fig. b) . the virus titers of type i fcovs significantly decreased in the culture supernatant of cells pretreated with mm or higher u a (fig. c) , and production of fcov-i ku , fcov-i ucd , and fcov-i ucd into the culture supernatant was completely inhibited in cells pretreated with mm u a. in contrast to type i fcovs, pretreatment with u a did not affect the titer of type ii fcovs and type ii ccov in the supernatant of fcwf- cells (fig. d ). pretreatment experiments were performed to determine the period at which u a exerted its antiviral effects. pretreatment with u a significantly increased the degree of the reduction of fcov-i ku plaque formation at h prior to infection (fig. a) . cholesterol accumulation was observed in u a-pretreat cells by the reaction time (fig. b ). cholesterol accumulation was noted in cells pretreated with u a for h. cholesterol accumulation were mostly lost in cells pretreated for h, and completely disappeared in cells pretreated for h. inhibition of fcov infection by compounds with biochemical properties similar to those of u a was investigated. pretreatment with cads, except amorolfine, significantly increased the degree of reduction of fcov-i ku plaque formation (fig. ) . when cells were pretreated with cholesterol biosynthesis inhibitors, only itraconazole significantly inhibited fcov-i ku plaque formation. no other cholesterol biosynthesis inhibitors showed significant inhibitory effects on fcov-i ku plaque formation. no drug exhibited significant inhibitory effects on type ii fcov - plaque formation. the selectivity index (si) was calculated from the cc and half maximal effective concentrations (ec ) of each drug. for type i fcov, the si values of u a, ay- , clomiphene, ro - , and itraconazole were high (table ). in contrast, for type ii fcov, the si values of all drugs were low. ay- , clomiphene, ro - , and itraconazole, which significantly inhibited fcov-i ku plaque formation, inhibit the function of cholesterol transporter npc , unlike the other drugs (amorolfine, fluvastatin, lovastatin, simvastatin, and alendronate). based on this, we assumed that inhibition of type i fcov replication by u a is associated with reduction of npc function, and investigated whether hdaci, which improves reduced npc function, resolves u a-induced cholesterol accumulation and inhibition of type i fcov replication. when an hdaci, vorinostat, was added to u a-pretreated fcwf- cells, cholesterol accumulation were reduced (fig. a) . the influence of vorinostat on fcov-i ku replication was investigated using plaque reduction assay. when vorinostat was added to u a-pretreated fcwf- cells, the percentage of plaque reduction significantly decreased ( fig. b and c). the influence of vorinostat on the n protein expression level in fcov-infected cells was investigated using the fluorescent antibody method. the n protein levels of fcov-i ku- specifically decreased in fcwf- cells pretreated with u a. on the other hand, the addition of vorinostat to u a-pretreated fcwf- cells increased the level of fcov-i ku- n protein (fig. d ). several studies have been made on the role of cholesterol in coronavirus infection. (thorp and gallagher, ; nomura et al., ; ren et al., ) . however, the influence of the intracellular synthesis and transport systems of cholesterol has not been investigated. u a suppresses cholesterol biosynthesis and the function of cholesterol transporter npc . in this study, we investigated anti-fcov activity of u a. u a induced intracellular cholesterol accumulation and strongly inhibited type i fcov replication. in addition, treatment of cells with u a did not influence the cellular cholesterol level, suggesting that u a inhibits type i fcov replication at a concentration suppressing cholesterol transporter but not influencing the synthesis system. in contrast to type i fcov, no inhibitory effects of u a on type ii fcov replication were noted. we previously reported that type i fcov infection is dependent on plasma membrane cholesterol, but type ii fcov does not (takano et al., ) . our findings of the present and previous studies suggest that type i fcov requires cholesterol to infect cells, but type ii fcov does not require cholesterol for its infection. it was suggested that type ii fcov was arisen from a double recombination between type i fcov and type ii ccov (herrewegh et al., ; terada et al., ) . in fcov-ii - , the genomic region from orf b, orf (encoding s protein), and orf c is derived from type ii ccov and the other regions are derived from type i fcov (herrewegh et al., ) , suggesting that these genes are factors leading to differences in the virological properties between the fcov serotypes. of these genes, the homology of the orf gene sequence between the serotypes is low similarity ( %) (motokawa et al., ) , suggesting that orf is involved in the difference effects of u a between type i and ii fcov. s protein (encoded by orf ) is an important protein for binding to the virus receptor and cell entry (gallagher and buchmeier, ) . the physical properties of s protein are markedly different between types i and ii fcov. for example, type ii fcov s protein binds to the virus receptor, aminopeptidase n (apn), but type i fcov s protein does not bind to apn (hohdatsu et al., ) . in addition, type i fcov s protein binds to heparin, but type ii fcov s protein does not (de haan et al., ) . the differences in these s protein properties between the serotypes may also influence the differences in the effects of u a. to demonstrate this assumption, it is necessary to prepare a recombinant fcov containing the type i fcov orf gene replacing the type ii fcov orf gene. confirmation of the influence of u a on replication of the recombinant fcov is expected. in this study, u a did not inhibit type ii fcov and type ii ccov production. on the contrary, it was previously reported that mbcd, a cholesterol extracting agents, inhibited type ii ccov but not type ii fcov (pratelli and colao, ; takano et al., ) . mbcd disrupts cholesterol-and caveolin-rich membrane domain (caveolae) (d'alessio et al., ) . van hamme et al. ( ) reported that type ii fcov was shown to enter cells through caveolaeindependent endocytosis. on the basis of this report and above, it is suggested that type ii ccov enters cells via caveolae. the addition of u a h before viral infection significantly inhibited type i fcov replication. the duration of exposure to u a required for inhibiting type i fcov replication was mostly consistent with that required for cholesterol accumulation in cells, strongly suggesting that when the cholesterol transporter is inhibited, type i fcov replication is also inhibited. hcv replication is inhibited by u a. it has been reported that replicated virions were not released from endosomes when hcv-replicating cells were treated with u a for h, i.e., hcv virions were accumulated with cholesterol in late endosomes (elgner et al., ) . probably, type i fcov replication is also inhibited by accumulation in late endosomes. u a is an inhibitor of the cholesterol transporter npc (ko et al., ) . u a binds npc and block its function (lu et al., ) . in this study, ay- , clomiphene, ro - , and itraconazole, which block npc function similarly with u a (shoemaker et al., ; trinh et al., ) , also exhibited type i fcov replication-inhibitory effects, suggesting that type i fcov replication was inhibited by blocking the npc function. to verify this hypothesis, we investigated the influence of hdaci on the type i fcov replication-inhibitory effects of u a. in contrast to u a, hdaci reverts npc dysfunction (pipalia et al., ) . when the vorinostat was added to u a-treated cells, intracellular cholesterol accumulation disappeared and type i fcov replicability resumed. to our knowledge, reversion of u ainduced inhibition of virus replication by hdaci has not previously been reported. it is necessary to determine whether the same phenomenon is observed in ebolavirus, dengue virus, and hcv. it is suggested that breeds of cats can influence the susceptibility to fip. pesteanu-somogyi et al. ( ) reported that the incidence of fip among breeds of cats. they showed that the incidence of fip was the lowest in siamese cats. interestingly, the siamese cat breed seems to have a genetic predisposition to npc disease (wenger et al., ) . npc dysfunction is a cause of npc disease. these facts and the finding of our study suggest that the impaired npc function is the cause of the low incidence of fip in siamese cats. that is to say, npc -deficient cats may be less sensitive to type i fcov, which is dominant serotype in the field (hohdatsu et al., ; kummrow et al., ; wang et al., ) . further investigation of type i fcov sensitivity of siamese cats is necessary. u a strongly inhibited type i fcov replication, but it did not inhibit type ii fcov replication. it was suggested that u a inhibits type i fcov replication by suppressing the intracellular cholesterol transporter. the experiment using hdaci suggested that dysfunction of npc is involved in the antiviral effects of u a. as type i fcov is widely present in the field, u a may be applied as a therapeutic drug against fip. it is necessary to investigate whether administration of u a exhibits therapeutic effects in cats with fip and determine the cause of reversion of u a-induced inhibition of virus replication by hdaci. critical role of virion-associated cholesterol and sphingolipid in hepatitis c virus infection ebola virus entry requires the cholesterol transporter niemann-pick c cholesterol synthesis inhibitor u a and the role of sterol metabolism and trafficking in numerous pathophysiological processes direct perturbation of lens membrane structure may contribute to cataracts caused by u a, an oxidosqualene cyclase inhibitor caveolae participate in tumor necrosis factor receptor signaling and internalization in a human endothelial cell line immunopathogenesis of coronavirus infections: implications for sars the intracellular cholesterol transport inhibitor u a inhibits the exosomedependent release of mature 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seroprevalence and association with disease in switzerland identification of npc as the target of u a, an inhibitor of lysosomal cholesterol export and ebola infection cholesterol manipulation by west nile virus perturbs the cellular immune response molecular cloning and sequence determination of the peplomer protein gene of feline infectious peritonitis virus type i comparison of the amino acid sequence and phylogenetic analysis of the peplomer, integral membrane and nucleocapsid proteins of feline, canine and porcine coronaviruses human coronavirus e binds to cd in rafts and enters the cell through caveolae an update on feline infectious peritonitis: virology and immunopathogenesis prevalence of feline infectious peritonitis in specific cat breeds histone deacetylase inhibitors correct the cholesterol storage defect in most niemann-pick c mutant cells u a, an intracellular cholesterol transport inhibitor, inhibits dengue virus entry and replication role of the lipid rafts in the life cycle of canine coronavirus importance of cholesterol for infection of cells by transmissible gastroenteritis virus multiple cationic amphiphiles induce a niemann-pick c phenotype and inhibit ebola virus entry and infection how cells handle cholesterol epidemiology, genetic recombination, and pathogenesis of coronaviruses differential effect of cholesterol on type i and ii feline coronavirus infection emergence of pathogenic coronaviruses in cats by homologous recombination between feline and canine coronaviruses requirements for ceacams and cholesterol during murine coronavirus cell entry triazoles inhibit cholesterol export from lysosomes by binding to npc an eight-year epidemiologic study based on baculovirus-expressed type-specific spike proteins for the differentiation of type i and ii feline coronavirus infections sars and mers: recent insights into emerging coronaviruses nef increases the synthesis of and transports cholesterol to lipid rafts and hiv- progeny virions this work was supported by jsps kakenhi (grant-in-aid for scientific research (b)) grant number jp h . key: cord- -vzyrcmu authors: pizzorno, andrés; padey, blandine; dubois, julia; julien, thomas; traversier, aurélien; dulière, victoria; brun, pauline; lina, bruno; rosa-calatrava, manuel; terrier, olivier title: in vitro evaluation of antiviral activity of single and combined repurposable drugs against sars-cov- date: - - journal: antiviral res doi: . /j.antiviral. . sha: doc_id: cord_uid: vzyrcmu in response to the current pandemic caused by the novel sars-cov- , identifying and validating effective therapeutic strategies is more than ever necessary. we evaluated the in vitro antiviral activities of a shortlist of compounds, known for their cellular broad-spectrum activities, together with drugs that are currently under evaluation in clinical trials for covid- patients. we report the antiviral effect of remdesivir, lopinavir, chloroquine, umifenovir, berberine and cyclosporine a in vero e cells model of sars-cov- infection, with estimated % inhibitory concentrations of . , . , . , . , . and μm, respectively. virus-directed plus host-directed drug combinations were also investigated. we report a strong antagonism between remdesivir and berberine, in contrast with remdesivir/diltiazem, for which we describe high levels of synergy, with mean loewe synergy scores of and peak values above . combination of host-directed drugs with direct acting antivirals underscore further validation in more physiological models, yet they open up interesting avenues for the treatment of covid- . in vitro evaluation of antiviral activity of single and combined repurposable drugs against sars-cov- authors: andrés pizzorno a , blandine padey a,b , julia dubois a , thomas julien a,c , aurélien traversier a , victoria dulière a,c , pauline brun a,c , bruno lina a,d , manuel rosa-calatrava a,c* † , olivier terrier a* † author affiliations: abstract: in response to the current pandemic caused by the novel sars-cov- , identifying and validating effective therapeutic strategies is more than ever necessary. we evaluated the in vitro antiviral activities of a shortlist of compounds, known for their cellular broad-spectrum activities, together with drugs that are currently under evaluation in clinical trials for covid- patients. we report the antiviral effect of remdesivir, lopinavir, chloroquine, umifenovir, berberine and cyclosporine a in vero e cells model of sars-cov- infection, with estimated % inhibitory concentrations of . , . , . , . , . and µm, respectively. virus-directed plus host-directed drug combinations were also investigated. we report a strong antagonism between remdesivir and berberine, in contrast with remdesivir/diltiazem, for which we describe high levels of synergy, with mean loewe synergy scores of and table ) . a similar trend was observed when we analyzed the berberine dose-response profile in the presence of . µm remdesivir but not at higher remdesivir concentrations (figure a , right panel and table ). to further explore this combination, we used synergyfinder targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases arbidol as a broad-spectrum antiviral: an update a trial of lopinavir-ritonavir in adults hospitalized with severe covid- favipiravir versus arbidol for covid- : a randomized clinical trial zhang, zhan, . efficacy of hydroxychloroquine in patients with covid- : results of a randomized clinical trial remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov- replication in vitro role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings the broad-spectrum antiviral ribonucleoside ribavirin is an rna virus mutagen cyclosporin a inhibits the replication of diverse coronaviruses antiviral combinations for severe influenza favipiravir (t- ), a novel viral rna polymerase inhibitor vitro and in vivo activities of anti-influenza virus compound t- arbidol (umifenovir): a broad-spectrum antiviral drug that inhibits medically important arthropod-borne flaviviruses clinical features of patients infected with novel coronavirus in wuhan synergyfinder: a web application for analyzing drug combination dose-response matrix data the effect of arbidol hydrochloride on reducing mortality of covid- patients: a retrospective study of real world date from three hospitals in wuhan viral dynamics in mild and severe cases of covid- outcomes of hydroxychloroquine usage in united states veterans hospitalized with covid- . medrxiv . . covid- : consider cytokine storm syndromes and immunosuppression. the lancet s lusakibanza a randomized, controlled trial of ebola virus disease therapeutics characterization and treatment of sars-cov- in nasal and bronchial human airway epithelia repurposing of drugs as novel influenza new world hantaviruses activate ifnλ production in type i ifn-deficient vero e cells broad-spectrum antiviral activity of virazole: -f -d-ribofuranosyl- , , -triazole- -carboxamide discovery of berberine, abamectin and ivermectin as antivirals against chikungunya and other alphaviruses the antiviral alkaloid berberine reduces chikungunya virus-induced mitogen-activated protein kinase signaling remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( - ncov) in vitro anti-influenza activity of berberine improves prognosis by reducing viral replication in mice viral load dynamics and disease severity in patients infected with sars-cov- in zhejiang province, china a novel coronavirus from patients with pneumonia in china in response to the current pandemic caused by the novel sars-cov- , identifying and validating effective therapeutic strategies is more than ever necessary we evaluated the in vitro antiviral activities of a shortlist of compounds, known for their cellular broad-spectrum activities, together with drugs that are currently under clinical evaluation we report the antiviral effect of remdesivir, lopinavir, chloroquine, umifenovir, berberine and cyclosporine a in vero e cells we report a strong antagonism between remdesivir and berberine, in contrast with remdesivir/diltiazem, for which we describe high levels of synergy combinations of host-directed drugs with direct acting antivirals open interesting avenues for the treatment of covid- key: cord- - a u authors: tan, chee wah; chan, yoke fun; quah, yi wan; poh, chit laa title: inhibition of enterovirus infection by antisense octaguanidinium dendrimer-conjugated morpholino oligomers date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: a u enterovirus (ev- ) infections are generally manifested as mild hand, foot and mouth disease, but have been reported to cause severe neurological complications with high mortality rates. treatment options remain limited due to the lack of antivirals. octaguanidinium-conjugated morpholino oligomers (vivo-mos) are single-stranded dna-like antisense agents that can readily penetrate cells and reduce gene expression by steric blocking of complementary rna sequences. in this study, inhibitory effects of three vivo-mos that are complementary to the ev- internal ribosome entry site (ires) and the rna-dependent rna polymerase (rdrp) were tested in rd cells. vivo-mo- and vivo-mo- targeting the ev- ires showed significant viral plaque reductions of . and . log( )pfu/ml, respectively. both vivo-mos reduced viral rna copies and viral capsid expression in rd cells in a dose-dependent manner. in contrast, vivo-mo- targeting the ev- rdrp exhibited less antiviral activity. both vivo-mo- and remained active when administered either h before or within h after ev- infection. vivo-mo- exhibited antiviral activities against poliovirus (pv) and coxsackievirus a but vivo-mo- showed no antiviral activities against pv. both the ires-targeting vivo-mo- and vivo-mo- inhibit ev- rna translation. resistant mutants arose after serial passages in the presence of vivo-mo- , but none were isolated against vivo-mo- . a single t to c substitution at nucleotide position was sufficient to confer resistance to vivo-mo- . our findings suggest that ires-targeting vivo-mos are good antiviral candidates for treating early ev- infection, and vivo-mo- is a more favorable candidate with broader antiviral spectrum against enteroviruses and are refractory to antiviral resistance. enterovirus (ev- ) is a single-strand, positive-sense rna virus. ev- usually cause mild hand, foot and mouth disease (hfmd) characterized by fever with papulovesicular rash on the palms and soles . in recent years, ev- infections were also associated with neurological complications with high mortalities among infants and young children < years old . to date, no effective antiviral agent is available for clinical use (shang et al., b; tan et al., ) . thus, there is an urgent need to develop effective antiviral agents to treat ev- infection. considering the morbidity caused by ev- , new approaches to the development of therapeutics are needed. a number of promising rna-based therapeutics designed to inhibit ev- infections have shown promising results, including sirna and shrna (deng et al., ; sim et al., ; tan et al., a,b; wu et al., ). however, the limitations of rna-based therapeutics are short half-life and it required a delivery agent which might be toxic to the host. therefore, nucleic acid-based therapeutics should be designed to possess favorable pharmacological properties such as in vivo stability and low toxicity. a phosphorodiamidate morpholino oligomer (pmo) is a singlestranded dna-like compound that has the ability to bind to the mrna and inhibit gene expression by steric blockage of complementary rna. pmos are highly nuclease-resistant and do not require the rnase h or other catalytic proteins for their activity (kole et al., ; summerton, ) . pmos have been conjugated with various cell-penetrating compounds such as cell-penetrating peptides and octaguanidinium dendrimers which are able to enhance their uptake by cells (moulton and jiang, ). peptide conjugated-pmos (ppmo) have been demonstrated to inhibit various viral infections, including ebola virus (warfield et al., ) , west nile virus (deas et al., ) , dengue virus (kinney et al., ) , sindbis virus (paessler et al., ) , coronavirus (neuman http (opriessnig et al., ; patel et al., ) , foot-and-mouth disease virus (vagnozzi et al., ) , poliovirus, rhinovirus (stone et al., ) , and coxsackievirus b (yuan et al., ) . in this study, three octaguanidinium dendrimer conjugatedmorpholino oligomers (vivo-mos) targeting the ev- internal ribosome entry site (ires) core sequence and the rna-dependent rna polymerase (rdrp) were tested for their inhibitory effects against ev- . we demonstrated that the two vivo-mos targeting the ires core sequence showed significant inhibition of ev- infection. rhabdomyosarcoma (rd, atcc) cells were grown in dulbecco's modified eagle's medium (dmem, hyclone) supplemented with % fetal bovine serum (fbs). ev- strains (genbank accession number: af ), brcr (genbank accession number: ab ) and uh / (genbank accession number: am ); coxsackievirus a (cv-a ), pv and chikungunya virus (chikv) strain my/ / (genbank accession number: fn ) were propagated in rd cells. all vivo-mos were synthesized by gene tools llc (usa). the -mer vivo-mos were designed to be complementary to the ev- (strain ) ires stem-loop v-vi and the rdrp gene (table , fig. ). all the vivo-mos were dissolved in phosphate buffer saline (pbs) at concentration of . mm. the cytotoxicity of the vivo-mos were evaluated using cell titer aqueous cell proliferation reagent (promega) according to the manufacturer's instructions. rd cells were seeded at .  cells or .  cells within each well of a -well plate or -well plate, respectively and incubated overnight at °c in % co . after overnight incubation, the growth medium was removed and replaced with ev- inoculum with a multiplicity of infection (moi) of . (pfu per cell) and incubated at °c for h. after incubation, the inoculum was removed and replenished with maintenance medium (dmem with % fbs) with or without vivo-mos. the inhibitory effects of the vivo-mos were evaluated by plaque assay, taqman real-time rt-pcr and western blot analysis h post-infection (hpi) as previously described (tan et al., . the western blot signal was enhanced using supersignal Ò western blot enhancer (pierce biotechnology). vivo-mos were added to rd cells at various time points relative to viral inoculation. rd cells were pre-incubated with vivo-mos at final concentration of lm for h before ev- inoculation at a moi of . . in concurrent studies, both vivo-mos and ev- were added into rd cells for h followed by replacement of medium without vivo-mo. for post-infection studies, rd cells were infected with ev- for , , and h before vivo-mos were applied. the viral titers for each experiment were quantitated hpi by plaque assays. to evaluate the efficacy of vivo-mos against different enteroviruses including cv-a and pv, rd cells were pre-incubated with vivo-mos at the final concentration of lm for h before viral table sequence of the -mer vivo-mos and target locations in ev- rna. sequence ( inoculation at a moi of . for h at °c. the viral titers were determined hpi by plaque assay. chikv was used as a negative control virus in this experiment. ev- strain infectious clone was constructed using the fulllength genome pcr approach according to yeh et al. ( ) with modifications. the primers involved in the infectious clone constructions were listed in table s . full-length genome rt-pcr was performed with superscript iii reverse transcriptase (invitrogen) and iproof high-fidelity polymerase (bio-rad) using pt / eagi-r and psp /ev -f primers. ev- expressing enhanced green fluorescence protein (egfp) was constructed by overlapping extension pcr strategy using q high-fidelity polymerase (neb). the egfp gene was fused into the ev- genome between the ' utr and vp gene followed by the a cleavage site (-aittl-) as previously described (shang et al., a) . the full-length pcr product was then cloned into pcr-xl-topo (invitrogen). in vitro transcription was performed with linearized dna using ribo-max-sp large scale rna production system (promega) and the rna was transfected into rd cells using lipofectamine (invitrogen) according to the manufacturer's instructions. cell free translation assay was performed with lg of in vitro transcribed rna using -step human coupled ivt kit (pierce biotechnology) either in the presence or absence of vivo-mos according to the manufacturer's instructions. an aliquot of ll of in vitro translated sample was subjected to sds-page and western blot analysis as described previously. the immunoblot was developed with clarity™ western ecl substrate (bio-rad) and detected by chemiluminescence. rd cells in a -well plate were infected with ev- -egfp for an hour at °c. after incubation, the inoculum was removed and replaced with maintenance medium containing . lm of vivo-mos. the egfp expression was observed at hpi using fluorescence microscopy. ev- was passaged in rd cells with increasing concentrations of either vivo-mo- or vivo-mo- . for the first passage, rd cells were infected with ev- at a moi of . for h at °c and the inoculum was removed and replaced with maintenance medium containing lm of vivo-mo- or vivo-mo- . each selection was passaged by adding ll of the supernatant into new rd cells. viruses were passaged once at each of the concentrations ranging from lm to lm, followed by passaging four times at lm. to identify the mutation(s) which conferred resistance to vivo-mos, viral rna of an individual plaque population was amplified and subjected to dna sequencing. point mutations were incorporated into the ev- infectious clone by using quickchange lightning site-directed mutagenesis kit (agilent technologies) according to the manufacturer's instructions. four mutants were constructed with different nucleotide substitutions at the vivo-mo- targeted region ( table ). the degree of resistance was evaluated using inhibitory assay as described in section . . the data presented are the means ± standard deviations (sd) obtained from at least two independent biological replicates. error bars represent the sd. statistical significance was calculated using the mann-whitney test. a p value of < . was considered statistically significant. to evaluate the effects of vivo-mos on ev- infectivity in rd cells, rd cells were treated with vivo-mos an hour after infection. as shown in fig. , both vivo-mos targeting the ev- ires stemloop region exhibited significant antiviral activity against ev- infection with reduction of virus-induced cpe ( fig. a) , plaque formation (fig. b) , rna (fig. c ) and capsid expression (fig. d ) in a dose-dependent manner. the vivo-mo- and vivo-mo- significantly reduced ev- plaque formation by up to . and . log -pfu/ml at lm, respectively. significant inhibition was observed at concentrations higher than lm. the ic values of vivo-mo- and vivo-mo- were . lm and . lm, respectively. however, vivo-mo- exhibited less inhibitory effect against ev- infection in rd cells with a plaque reduction of only . log pfu/ml (fig. b) . the vivo-mo-c which has no homologous sequence to the ev- genome has no inhibitory effects at all against all ev- strains tested. none of the vivo-mos caused more than % reduction of cell viability at concentrations less than lm as measured by the mts assay (fig. ) . to further characterize the efficacy of vivo-mos at multiple time points relative to ev- infection, vivo-mos were applied for h before, or , , , or h after ev- infection. both vivo-mo- and vivo-mo- remained effective when administered before or after ev- infection. however, the efficacies were reduced when treatments were delayed. when vivo-mos and ev- were added together into the rd cells for h, the inhibitory effect was reduced, which could have resulted from the incomplete uptake of the vivo-mos by the cells. nonetheless, the antiviral effects were retained for both ires-targeting vivo-mos even when administered hpi, with . % plaque inhibition. vivo-mo- had no observable inhibitory effects on ev- infection when administered h before infection and , , or hpi (fig. a) . next, we investigate whether any of the vivo-mos could inhibit different ev- strains as well as other picornaviruses. we evaluated each of the vivo-mos against two other ev- strains (brcr table sequence of vivo-mo- and the in vitro transcribed infectious rna with target sequences. sequences mismatch(es) and uh / ), pv, cv-a and chikv as the control virus. the vivo-mo- which targets the highly conserved region of the ires stemloop structure exhibited significant inhibitory activity against ev- strains brcr and uh / , pv and cv-a with viral plaque reduction ranging from . to . log pfu/ml (fig. b) . however, vivo-mo- only exhibited antiviral activities against ev- strains brcr and uh / , cv-a , but not against pv (fig. b) . ev- strain brcr which has a single nucleotide mismatch in the middle of the vivo-mo- targeted site (fig. ) remained sensitive to the vivo-mo- treatment. the efficacy of vivo-mo- against cv-a ( . log pfu/ml reduction) was significantly lower when compared to ev- ( . log pfu/ml reduction). this could be due to three nucleotide mismatches with the vivo-mo- . pv with five nucleotide mismatches was completely resistant to the inhibitory effect of vivo-mo- . all the vivo-mos did not show any inhibition of chikv infection. to investigate the mechanism of action of the antiviral vivo-mos, cell-free translation analysis was used. as depicted in fig. a , the presence of either vivo-mo- or vivo-mo- significantly blocked the ires-dependent translation when compared to the control. vivo-mo- exhibits reduced efficacies as compared to ires-targeting vivo-mos. in the ev- -egfp inhibition assay, the presence of the vivo-mo- or vivo-mo- greatly reduced egfp expression in rd cells hpi with ev- -egfp (fig. b) . we explored whether ev- could become resistant to vivo-mo treatments. ev- was serially passaged in the presence of increasing concentrations of either vivo-mo- or vivo-mo- . interestingly, only ev- mutants resistant to vivo-mo- were isolated after eight passages. we failed to isolate ev- mutants that were resistant to vivo-mo- . to investigate the determinant(s) of resistance, viral rna was isolated from the resistant population and was sequenced. a single point mutation from t to c at position was sufficient to confer resistance to vivo-mo- (fig. a) . to characterize the loss of inhibitory activity by vivo-mo- , we constructed ev- mutants carrying different mismatches at the vivo-mo- target site (table ) , and the inhibitory effects of vivo-mo- against each of the mutants (  objective) were observed hpi. the total infectious particles or total viral proteins were harvested hpi and evaluated by (b) plaque assay, (c) quantitative taqman real-time pcr, and (d) western blot analysis, respectively. ev- viral capsid protein was detected by mouse anti-ev- monoclonal antibody (millipore) and cellular b-actin was detected using mouse anti-b-actin monoclonal antibody (sigma). the data presented were obtained from at least two independent biological replicates. fig. . cell viability analysis. various concentrations of vivo-mos were incubated with rd cells ( .  cells) for h in maintenance medium (dmem supplemented with % fbs) followed by mts assay using cell titer aqueous one solution cell proliferation (promega). the absorbance reading at nm was obtained using a microtiter plate reader after h of incubation at °c. the percentage of cell viability (%) was determined by multiplying the ratio of the absorbance readings obtained from cells treated with vivo-mos over the nontreated cells with %. the data presented were obtained from at least two independent biological replicates. were evaluated. the mismatched rna target sequences were designed to reflect the most likely natural variations that would arise in the ev- sequence. as shown in fig. b , the ev- mo- -mutant- , which carried a single point mutation at position (t to c substitution), required higher vivo-mo- concentrations to achieve a similar inhibitory effect when compared to the wild type. the ev- mo- -mutant- with a single point mutation in the middle of the targeted sequence (a t to c substitution at position ) remained sensitive to vivo-mo- , but vivo-mo- had reduced inhibitory efficacy when compared with the wild type. increasing the number of mutations on the targeted sequence significantly reduced the inhibitory efficacy of vivo-mo- . the viral plaque inhibition at lm of vivo-mo- against ev- mo- -wt was . % ± . , and reduced to . % ± . for the mo- mutant- that carried a point mutation at the ' end of the target sequence. the viral plaque inhibition by vivo-mo- reduced to . % when the number of nucleotide mismatches increased to three. to date, there is no fda approved vaccine available to prevent infection and treatment options remain limited due to a lack of effective antivirals (chong et al., ; shang et al., b; . highly negatively-charged compounds like suramin or its analog, nf that disrupt the virus-host receptor interactions are ineffective when ev- mutants acquire glu gln and lys arg substitutions in the vp capsid protein (arita et al., ; . a single point mutation, val met in egfp expression and nuclei are shown in green and blue, respectively. the data presented were obtained from at least two independent biological replicates. (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) the vp , is sufficient to confer resistance to the capsid binder bproz- (shih et al., ) . therefore, there is an urgent need to develop effective antiviral agents to treat patients with severe ev- infection. the use of antisense mechanisms to inhibit pathogen replication has been under investigation and has shown promising results. fomivirsen, a -mer phosphorothioate oligonucleotide antisense agent used for intravitreal treatment of cytomegalovirus retinitis, is the only antisense agent that has been approved by the fda to date (de smet et al., ) . there are advantages of vivo-mos over rna-based antisense oligomers as they are, nucleaseresistant and readily penetrate the cells (kole et al., ; moulton and jiang, ) . these have an advantage over peptidebased cell penetrating molecules, which are susceptible to protease degradation that can impact the effectiveness of ppmos (youngblood et al., ) . amongst the three vivo-mos being examined, the two vivo-mos targeting the ev- ires stem-loop region exhibited significant antiviral activity. the vivo-mos blocked ev- viral rna translation in a cell-free inhibition assay, as well as in a cell-based egfp reporter inhibition assay. previous studies have also reported that only pmos targeting the positive strand ires regions or the aug start codon sites were effective when compared to pmos targeting other regions of the viral rna (stone et al., ; vagnozzi et al., ; yuan et al., ) . the pmo may bind with the complementary viral rna and disrupt the integrity of the ires stem-loop secondary or tertiary structures, hence arresting ires-dependent translation (kole et al., ; warren et al., ) . the pmos are likely to block s ribosomal subunit binding the viral rna in the vicinity of the stem-loop v in the picornaviral type i ires (belsham, ) . furthermore, it has been suggested that the ires stem-loop v-vi of ev- interacts with ires-specific trans-acting factors such as fuse-binding protein and heterogeneous nuclear ribonucleoprotein al (lin et al., a,b; shih et al., ) . unlike synthetic double-stranded sirna which involve cleavage of mrna, pmos are translation-suppressing oligonucleotides which do not lead to rna degradation (kole et al., ; warren et al., ) . this could explain why sirna targeting the similar viral sequences as vivo-mo- was effective, but not acting as a translation suppression oligonucleotide which down-regulates gene expression via steric blockage. ires-targeting vivo-mo- exhibited broad-spectrum activities against multiple enteroviruses, but with different antiviral efficacies. this could have resulted from the ires stemloop structure differences between enteroviruses which affect the accessibility of the vivo-mos to the targeted sites (dias and stein, ) . in this study, an ev- mutant that was resistant to vivo-mo- carried a single substitution of t to c at position . we failed to isolate any ev- resistant to vivo-mo- even after eight passages in vitro, implying that the vivo-mo- targeted region is intolerant to mutation in nature. our mutagenesis studies further confirm that the position of mutation and the number of mutations affect the antiviral efficacies in rd cells. a single mismatch present in the middle of the targeted sequence (t to c substitution at position ) was more tolerable than a mutation appearing at the end of the targeted site (t to c substitution at position ). these findings correlated well with previous findings that pv, fmdv and wnv that showed resistance to ppmo also carry a single point mutation at the end of the ppmo-targeted sequence (stone et al., ; vagnozzi et al., ; zhang et al., ) . a similar observation has been made for influenza virus as the degree of inhibition was associated with the number of mismatches (ge et al., ) . in summary, we have established two critical locations that are involved in viral translation initiation in the ev- genome as targets for vivo-mo antiviral intervention. the vivo-mos worked well with low micromolar concentrations to inhibit ev- infection and showed little cytotoxicity in rd cells. the potent inhibition of several enteroviruses by vivo-mo- raises the possibility that it could be developed as a broad-spectrum antiviral agent. furthermore, the degree of tolerance of mismatches by vivo-mos suggests a favorable characteristic for their use as a potential antiviral agent. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.antiviral. . . . the effect of sequence mismatches between vivo-mo- and target rna on inhibition assay. several in vitro transcribed rna, each having a different number of nucleotide mismatches in the target region as described in section were analyzed. viral titers were quantitated hpi using standard plaque assay. the data presented were obtained from at least two independent biological replicates. characterization of pharmacologically active compounds that inhibit poliovirus and enterovirus infectivity divergent picornavirus ires elements production of ev vaccine candidates fomivirsen -a phosphorothioate oligonucleotide for the treatment of cmv retinitis inhibition of flavivirus infections by antisense oligomers specifically suppressing viral translation and rna replication the highly conserved ' untranslated region as an effective target towards the inhibition of enterovirus replication by unmodified and appropriate '-modified sirnas antisense oligonucleotides: basic concepts and mechanisms inhibition of multiple subtypes of influenza a virus in cell cultures with morpholino oligomers inhibition of dengue virus serotypes to in vero cell cultures with morpholino oligomers rna therapeutics: beyond rna interference and antisense oligonucleotides far upstream element binding protein interacts with enterovirus internal ribosomal entry site and negatively regulates viral translation hnrnp a interacts with the ' untranslated regions of enterovirus and sindbis virus rna and is required for viral replication inhibition of herpes simplex virus ocular infection with morpholino oligomers targeting icp and icp gene knockdowns in adult animals: ppmos and vivomorpholinos antisense morpholino-oligomers directed against the ' end of the genome inhibit coronavirus proliferation and growth clinical features, diagnosis, and management of enterovirus inhibition of porcine reproductive and respiratory syndrome virus infection in piglets by a peptide-conjugated morpholino oligomer inhibition of alphavirus infection in cell culture and in mice with antisense morpholino oligomers peptide-conjugated morpholino oligomers inhibit porcine reproductive and respiratory syndrome virus replication development and characterization of a stable egfp enterovirus for antiviral screening antiviral drug discovery for the treatment of enterovirus infections host factors in enterovirus replication mutation in enterovirus capsid protein vp confers resistance to the inhibitory effects of pyridyl imidazolidinone rna interference against enterovirus infection virology, epidemiology, pathogenesis, and control of enterovirus a morpholino oligomer targeting highly conserved internal ribosome entry site sequence is able to inhibit multiple species of picornavirus morpholino antisense oligomers: the case for an rnase hindependent structural type enterovirus receptors: promising drug targets? inhibition of enterovirus (ev- ) infections by a novel antiviral peptide derived from ev- capsid protein vp recent developments in antiviral agents against enterovirus infection enterovirus uses cell surface heparan sulfate glycosaminoglycan as an attachment receptor enhanced potency and efficacy of -mer shrnas in inhibition of enterovirus inhibition of enterovirus in virus-infected mice by rna interference inhibition of foot-and-mouth disease virus infections in cell cultures with antisense morpholino oligomers gene-specific countermeasures against ebola virus based on antisense phosphorodiamidate morpholino oligomers advanced morpholino oligomers: a novel approach to antiviral therapy identification of small interfering rnas which inhibit the replication of several enterovirus strains in china a single nucleotide in stem loop ii of '-untranslated region contributes to virulence of enterovirus in mice stability of cell-penetrating peptide-morpholino oligomer conjugates in human serum and in cells inhibition of coxsackievirus b in cell cultures and in mice by peptide-conjugated morpholino oligomers targeting the internal ribosome entry site co-selection of west nile virus nucleotides that confer resistance to an antisense oligomer while maintaining long-distance rna/rna base pairings key: cord- -m nyt authors: hong, wei; li, tian; song, yu; zhang, runhong; zeng, zhengyang; han, shisong; zhang, xianzheng; wu, yingliang; li, wenxin; cao, zhijian title: inhibitory activity and mechanism of two scorpion venom peptides against herpes simplex virus type date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: m nyt herpes simplex virus type (hsv- ) is a widespread human pathogen that causes severe diseases, but there are not effective and safe drugs in clinical therapy besides acyclovir (acv) and related nucleoside analogs. in this study, two new venom peptides from the scorpion heterometrus petersii were identified with effective inhibitory effect on hsv- infection in vitro. both hp and hp peptides exhibited potent virucidal activities against hsv- (ec( ) = . ± . and . ± . μm, respectively) and effective inhibitory effects when added at the viral attachment (ec( ) = . ± . and . ± . μm, respectively), entry (ec( ) = . ± . and . ± . μm, respectively) and postentry (ec( ) = . ± . and . ± . μm, respectively) steps. both hp and hp peptides adopted α-helix structure in approximate membrane environment and resulted in the destruction of the viral morphology. moreover, hp and hp peptides entered vero cells and reduced the intracellular viral infectivity. taken together, hp and hp peptides are two anti-viral peptides with effective inhibitory effect on multiple steps of hsv- life cycle and therefore are good candidate for development as virucides. herpes simplex virus type (hsv- ) is a widespread human pathogen that infects primarily epithelial tissues and causes severe diseases including mucocutaneous lesions in the oral mucosa (cold sores), encephalitis, meningitis, and blinding keratitis (gopinath et al., ) . after an initial infection, hsv- spreads to the nervous system and establishes latent infection of neurons in sensory ganglia of the host (hill et al., ) . approximately % of the world populations are carriers of hsv- and about % suffer from recurrent infection (gold and corey, ; palem et al., ) . current therapeutic drugs against hsv infection are nucleotides, nucleosides or pyrophosphate analogues, such as acyclovir, valacyclovir, penciclovir and famciclovir (hsiang and ho, ) . after uptake by virus-infected cells, these drugs are activated by the viral thymidine kinase and inhibit the viral dna polymerase. however, hsv infection remains a serious challenge because of the viral resistance and side effect (crute et al., ; field, ) . therefore, the development of new safe and effective anti-hsv molecules is urgently needed. many antimicrobial peptides (amps) have been shown to have inhibitory activities against hsv infection. according to the structure characteristics, these peptides are classified into five types: a-helix, b-sheet, cyclic b-sheet, b-turn and extended (jenssen et al., ) . proposed anti-viral mechanism of a-helix mainly includes cellular target and viral inactivation, including magainin, cecropin, mellitin, ll- and brevinin- (aboudy et al., ; albiol matanic and castilla, ; yasin et al., ) . two kinds of b-sheet peptides, human and rabbit defensins, were shown to interact with hsv membrane/glycoprotein and cellular targets but not heparan sulfate (sinha et al., ; yasin et al., ) . another b-sheet peptide from frog, dermaseptin, was shown to have potent inhibitory effect when applied to the virus before or during virus adsorption to the target cells (belaid et al., ) . two b-sheet peptides tachyplesin and protegrin were proved to have viral inactivating effect (yasin et al., ) . cyclic b-sheet peptides such as h-defensin was shown to bound to gb protein and blocked hsv- attachment (yasin et al., ) . the b-turn peptides lactoferrin (lf) and lactoferricin (lfcin) are antimicrobial peptides that were found to block hsv entry into vero cells. lf had no effect against hsv after the virus had entered the cells, while lfcin exerted anti-viral activity also after the initial binding of the virus to the host cells jenssen et al., ). an extended peptide from bovine, indolicidin, showed a direct inactivation effect on cell-free hsv- virons by targeting viral membrane/ glycoprotein (albiol matanic and castilla, natural amps from scorpion venoms have attracted much attention due to their anti-viral bioactivities. some of them have been identified as anti-enveloped virus agents in our previous works. the peptide mucroporin-m was shown to be virucidal against the measles, sars-cov and influenza h n viruses, and it inhibited hbv replication in vitro and in vivo zhao et al., ) . a natural a-helical peptide, hp , was proven to have the property of killing hcv . two histidine rich peptides were designed on the molecular template of a short virucidal peptide ctry and were confirmed to have enhanced bioavailability, which resulted in potent inhibitory effect on hcv proliferation (hong et al., ) . another mutational peptide, kn - , was also effective in inhibiting hiv- infection (chen et al., ) . these studies indicated that scorpion venom is a rich source of anti-viral peptides. in the present study, we screened and identified two peptides from the venom of scorpion heterometrus petersii, which exhibited effective inhibitory effect on hsv- infection. the hp , hp , hp , hp , hp , hp peptides were from the non-amplified cdna library of h. petersii venom gland that was constructed in our previous work (ma et al., ) . peptides were chemically synthesized using the solid-phase synthesis method and amidated at the c-terminus (gl biochem ltd., china). the synthetic peptides were confirmed by rp-hplc and maldi-tof-ms (purity > %). african green monkey kidney cells (vero) were grown in minimum essential medium (mem) supplemented with % (vol/vol) fetal bovine serum (fbs), u/ml penicillin, and lg/ml streptomycin sulfate at °c in a % co incubator. cells infected with virus were grown in mem supplemented with % serum. to prepare high-titer stocks of hsv- (f strain) virus, vero cells were infected at a low multiplicity of infection (moi) of . in a t flask (nest biotechnology co. ltd. china). when the cytopathic effect (cpe) was - %, the infected monolayers were harvested and subjected to three freeze-thaw cycles using a dry ice-ethanol bath, centrifuged at , g for min to remove debris, and stored at À °c. viral titers were determined by plaque forming assay on vero cells. cells were seeded in a -well plate ( - , cells per well) and cultured at °c for h. a series of concentrations of peptides were added into the medium, and the plate was incubated for at °c for h, at which time ll of mtt solution ( mg/ml in pbs buffer; invitrogen) was added to each well, and the plate was incubated at °c for h. the medium was removed, ll dmso was added, and then the plate was shaken for min at room temperature to completely dissolve the crystal purple formazan. the absorbance was measured at nm. freshly obtained human red blood cells were washed three times with hepes buffer (ph . ) by centrifugation for min at g. the cells were then resuspended in . % saline and seeded in a -well plate with - cells per well. a series of concentrations of peptides were added and incubated at °c for h. a . % saline solution was used as a negative control, and . % triton x- was used as a positive control. the plate was centrifuged for min at g, and the absorbance of hemoglobin released in the supernatant was measured at nm. six-well plates containing vero cell monolayers with about % confluence were infected with virus. after h of absorption at °c, the inocula were removed. cells were washed three times with phosphate-buffered saline (pbs) and replenished with a maintenance cover layer (mem with % fbs and . % carboxymethylcellulose). after an incubation period of days, the cells were stained with % crystal violet containing % methanal and the plaques were counted. the viral titer was calculated according to the plaque number and dilution. six-well plates containing vero cell monolayers with about % confluence were infected with virus to yield about plaques per well. the anti-viral effects of peptides were determined in the following assays. peptides at appointed concentrations were incubated with virus at indicated conditions. at the indicated time, the virus-peptide mixtures were diluted and added to vero cell monolayers. after h of absorption at °c, the inocula were removed. cells were rinsed and replenished with cover layer as described above. after days, the inhibitory effects were determined by plaque reduction assay. vero cells were incubated with peptides for h at °c, at which time the peptides were removed and the cells were washed with pbs for three times. the peptide-treated cells were then infected with virus at °c for h. cells were rinsed and replenished with cover layer as described above. after days, the inhibitory effects were determined by plaque reduction assay. vero cells were cooled at °c for min and peptides were added to vero cells together with virus at °c for h. at the indicated time the cells were rinsed with pbs for three times and shifted to °c for viral entry. after h incubation, cells were rinsed and replenished with cover layer as described above. after days, the inhibitory effects were determined by plaque reduction assay. vero cells were infected with virus at °c for h, at which time cells were rinsed with pbs for three times and replenished with complete mem containing peptides. cells were then shifted to °c for viral entry. after h incubation, cells were rinsed and replenished with cover layer as described above. after days, the inhibitory effects were determined by plaque reduction assay. vero cells were infected with virus at °c for h, at which time the cells were rinsed with pbs for three times and replenished with cover layer containing peptides. after days, the inhibitory effects were determined by plaque reduction assay. the secondary structure of peptides was measured by circular dichroism (cd) spectroscopy. measurements were performed in the uv range of - nm at °c in water and % tfe using a jasco- spectropolarimeter, at a concentration of . mg/ml. spectra were collected from three separate recordings and averaged after subtracting the blank spectrum of pure water. a large quantity of hsv- supernatant was prepared and cleared by differential centrifugation method or passed through a . -lm filter unit. the viral particles in the media were concentrated by ultracentrifugation at , rpm at °c for h in a beckman sw rotor. the obtained hsv- pellets were dissolved in pbs for subsequent studies. virus was treated with peptides and incubated at °c. samples were placed on mesh form var/carbon-coated copper grids by dipping the grids into the virus sample solutions. each sample was then negatively stained by placing a drop of % phosphotungstic acid (ph , adjusted with naoh) on the grid. the sample grid was then dried in air for approximately min. finally, various areas of the grid were examined and photographs were taken from hit-achi h- transmission electron microscope operating at kv. n-terminus fitc-labeled peptide was added to the cells at a final concentration of lm, and then was incubated at °c. after incubating for the indicated time, cells were washed with pbs, fixed with % paraformaldehyde, and washed twice. cell nuclei were stained with dapi (diluted : in pbs). cells were rinsed with pbs for three times. the cellular localization of the peptide was analyzed by confocal microscopy. vero cells were infected with hsv- (moi . ) at °c for h. at which time the cells were rinsed with pbs for three times and replenished with medium containing peptide. infected cells were harvested at and h postinfection and the intracellular viruses were by freeze-thaw method. the intracellular infectivity was determined by plaque forming assay. data are expressed as the mean ± standard deviation from at least three separate experiments. sequence alignments of these peptides were performed using genedoc software (fig. a) and showed the typical characters of cationic host defense peptides. the anti-hsv- activities of these scorpion venom peptides were determined by plaque reduction assay (pra) (fig. b) . the result showed that among the six peptides, hp and hp exhibited the most effective anti-viral activities. they could significantly inhibit the plaque forming unit (pfu) of hsv- , with an inhibition rate > % at a concentration of lm (fig. c) . these results indicated that hp and hp were the most potential anti-hsv- agents, and then they were chosen for further studies. the cytotoxicities of hp and hp peptides on vero cells were determined using mtt assay. the concentrations of hp and hp that inhibited % of cell growth (cc ) were . ± . and . ± . lm, respectively. at the concentration of lm, the viability of the peptide-treated vero cells was greater than %, indicating that lm or less peptides was were used to infect cells for h at °c. the virus-peptide mixtures were removed, and the cells were rinsed and replenished with cover layer without peptide. (ii) cell inactivation assay. peptide was added to cells for h at °c and the peptide-treated cells were rinsed before virus infection; (iii) viral attachment assay. cells were precooled at °c for min and peptides were added together with virus for h at °c. at the indicated time the cells were rinsed and shifted to °c for viral entry. (iv) viral entry assay. virus was attached to cells for h at °c and the cells were rinsed before peptides were added. the cultures were then shifted to °c for viral entry. (v) postentry assay. cells were infected with virus for h at °c, at which time the virus was removed. the cells were rinsed and replenished with cover layer containing peptides throughout the experiment. (b, c) antiviral activities of hp and hp peptides in time of addition assay. after h postinfection, all cells were replenished with cover layer and cultured for h, at which time, the inhibitory effects of peptides were determined by plaque reduction assay. minimally cytotoxic to vero cells ( fig. a) . thus, this peptide concentration was chosen for further anti-viral studies. hemolytic assay indicated that the hc of hp and hp peptides were . ± . and . ± . lm, respectively (fig. b) . since having shown that hp and hp inhibited the plaque forming unit of hsv- (fig. ) in vitro, we sought to determine the exact step that was blocked by the two peptides. to determine whether hp and hp could inhibit hsv- infection or replication in vero cells, lm of each peptide was added to the virus or cells at different times relative to incubation (fig. a) . as shown in fig. b and c, both hp and hp exhibit potent inhibitory activities when added to the virus before infection or added to the cells together with viral attachment and entry step, suggesting an initial inhibitory effect of viral infection. in contrast, they displayed less inhibitory activities when added to the cells h after viral infection and much less inhibitory activities when added to the cells for h and removed before the virus was added. these results suggest that the peptides hp and hp could potently inhibit the initial infection of hsv- , but with less inhibitory activities of the viral replication. the above results suggest that hp and hp peptides have potent inhibitory activities against the initial viral infection. we further determined the detailed viral inactivation activities of them. the inhibitory activities against hsv- infection were time-dependent at the concentration lm (fig. a and b ). when treated for min with hp or hp , the pfu of hsv- decreased to about %, suggesting a rapid viral inactivating effect. when treated for h, the inhibitory activity against hsv- was the most effective. under this condition, the inhibitory effect on hsv- infection was dose-and temperature-dependent ( fig. c and d). the most effective temperature is °c and when treated at °c for h, the % effective concentrations (ec ) of hp and hp were . ± . and . ± . lm, approximate -fold and -fold lower than their cc against vero cells, respectively (table ) . various concentrations of peptides were added to vero cells h before hsv- infection and the inhibitory effects were determined by plaque assay. as shown in fig. a, hp and hp slightly inhibited hsv- infection when preincubated with vero cells. in addition, hp and hp peptides inhibited hsv- attachment and entry in a dose-dependent manner, although weaker than their virucidal activities. in the viral attachment assay, the % effective (ec ) concentrations of hp and hp were . ± . and . ± . lm, respectively (fig. b) , approximate -fold and -fold lower than their cc against vero cells. in the viral entry assay, the % effective (ec ) concentrations of hp and hp were . ± . and . ± . lm, respectively (fig. c ). finally, when added h postinfection, hp and hp inhibited hsv- proliferation with ec of . ± . and . ± . lm, respectively (fig. d ). the pharmacological profiles of hp and hp peptides were listed in table . circular dichroism (cd) analysis was used to analyze the secondary structures of both hp and hp peptides. the result showed that the synthetic peptides hp and hp both exhibited a-helix structure in tfe ( fig. a and c) . when plotted as a helical wheel projection ( fig. b and d) , hp and hp were divided into two parts: one part was the hydrophobic face, and the other was the hydrophilic face. these data indicated that hp and hp are amphipathic peptide molecules. to confirm whether the viral structural integrity was destabilized by the treatment of hp or hp , we performed transmission electron microscopy (tem) experiments to monitor the morphological changes of hsv- . as shown in fig. , the morphology of untreated hsv- virions were mostly integrated, which contains compact envelop ($ nm). treatment by either hp or hp significantly changed the structure of hsv- , including rupture of viral envelop and dissociation of proteins from the virons, generally displayed incompact morphology. these results strongly supported the underlying mechanism of action of hp and hp that destabilized the integrity of hsv- and inhibited its infectivity. to explore the intracellular anti-viral mechanism of hp and hp against hsv- , confocal microscopy was used to investigate whether hp and hp peptides could penetrate the cell membrane and enter the vero cells. as shown in fig. a and c, fitc-labeled hp and hp peptides slightly entered the vero cells and accumulated in granular structures in the cytoplasm after incubated for h. however, they exhibited a higher amount of cellular uptake after incubated for h, with a dispersed distribution in the cytoplasm. moreover, when infected cells were incubated with hp and hp for and h, the infectivity of intracellular virus significantly decreased ( fig. b and d) . these results suggest that hp and hp peptides enter the vero cells and inactivated intracellular viral particles. currently, various virucidal agents have been identified and they provided a rich source of developing effective anti-viral drugs. but the challenge is how to solve the lack of inhibitory effect on viral postentry step. inhibition of cell-to-cell spread is important for topical microbicide development because a source of sexual acquisition also may be cell-associated virus (sinha et al., ) . the derived virucidal peptide, c a, disturbs the integrity of the viral membrane and exhibits potent virucidal activities against hcv, hiv- and hsv- . it is worth mentioning that c a has a property of suppressing viral entry and established infection, which differs from common virucidal agents (bobardt et al., ; cheng et al., ; de witte et al., ) . in this study, we screened and identified two new anti-viral peptides from the peptide library of scorpion venom. the peptides, hp and hp , with potent inhibitory effect on the proliferation of hsv- (fig. ) , were selected for further analysis. we proved that both hp and hp inhibited multiple steps of hsv- life cycle (fig. ) . at noncytotoxic concentration, hp and hp peptides effectively inactivated hsv- virons in time-, dose-and temperature-dependent manners in vitro (fig. ) . hp and hp also blocked hsv- attachment and entry to vero cells. it was worthy to be attention that hp and hp could prevent viral proliferation in the postentry assay (fig. ) . in the comprehensive anti-viral assay, we found that hp and hp induced poor cellular resistance to infection, suggesting that hp and hp might not effectively target to cellular components. however, hp and hp displayed effective inhibitory activity in the viral attachment and entry stages. according to the time-dependent virucidal experiment in fig. a and b, the peptides hp and hp inactivated more than % virus in min and % in min, indicating a rapid action mode of hp and hp . for this property, we can speculate that hp and hp may inactivate hsv- when they attach to the cell surface and also effectively inactivate the virus that has already bound to the cells but not entry yet. in our previous studies, a serious of anti-viral peptides from scorpion venom have been identified, such as ctry (hong et al., ) , hp (yan et al., ) and kn - [ ] . these peptides shared the same a-helix structure and similar virucidal activities against enveloped virus. however, they lacked the inhibitory activities when added to the cells after viral infection. this disadvantage badly reduced the medical value of this type of antiviral peptide. in this study, we found that both hp and hp peptides exhibited potent inhibitory effects against hsv- proliferation in the postentry assay. obviously, they are different from the above virucidal peptides, but are similar to the mutant virucidal peptide ctry -h (hong et al., ) . the virucidal activities of hp and hp mostly depend on the sequence and structure characters. recent studies had shown that sialic acid on hsv- envelope glycoprotein was required for efficient infection and a b-peptide possibly binded to sialic acid, which resulted in inhibitory effect on the infection of hsv- . (akkarawongsa et al., ; teuton and brandt, ) in this study, cd analysis displayed that both hp and hp peptides adopted amphipathic a-helix structure in approximate membrane environment, with positive charged amino acids exposed outside the helix wheel (fig. ) . therefore, we can speculate that the peptides hp and hp interact directly with viral membranes via binding to the negatively charged sialic acid units on the virus and inactivate the viral particles. nevertheless, future studies will be needed to identify the target on the virus for hp and hp . transmission electron microscopy has been used to monitor the morphological changes of adenovirus type , showing that the conformational changes of the viral proteins result in the destruction of the viral morphology (yoon et al., ) . in the present study, we also observed the significant structure change of hsv- , especially the rupture of viral envelop and the incompact morphology of virons (fig. ) . this is the first time to show the morphology change of enveloped virus by anti-viral peptides, providing a direction of revealing the mechanism of virucidal drugs. to find the intracellular mechanism of the inhibitory effect on viral proliferation, we firstly determined the cellular localization of hp and hp . as shown in fig. a and c, fitc-labeled hp and hp peptides slightly entered vero cells at h postincubation, but displayed a big amount of uptake and equally dispersed distribution in the cytoplasm after h. moreover, hp and hp had inhibitory activities against intracellular viral infectivity ( fig. b and d) . the intracellular mature hsv- virions rapidly accumulated to high levels at and h postinfection, at which time the intracellular virucidal activities of hp and hp were significantly reflected. thus, we can conclude that hp hp enter the vero cells, target to the mature viral particles and inactivated them. previously, we designed two histidine rich peptides with enhanced cellular uptake and advanced intracellular distribution, which resulted in potent anti-viral activities against hcv (hong et al., ) . while in the present study, we found two natural peptides with the same properties and anti-viral functions, indicating the diversity of biomolecules from scorpion venom. many anti-viral drugs readily lead to the generation of drugresistant virus variants. for more than two decades, anti-viral drugs targeting to the viral dna polymerase such as acyclovir (acv) have been widely used for the treatment of hsv infection. unfortunately, the extensive clinical use of these drugs has led to the emergence of drug-resistant viral strains. (greco et al., ; krawczyk et al., ) using the virucidal peptides such as hp and hp for the treatment of hsv infection, the generation of drug-resistant virus variants is likely to be reduced. amphipathic a-helix peptides, such as hp and hp , have potent virucidal activity and inhibitory effect on viral proliferation, exhibiting potential medical value for the treatment of viral infection. furthermore, because of the advantage of therapeutic alliance, anti-viral strategy became diversification and already achieved remarkable successes. in this way, the virucidal molecules may act as a potential member to participate the therapeutic alliance, with its unique anti-viral pattern. in brief, this kind of membrane-targeting peptides could be combined with drugs that target intracellular components of the viral life cycle. thus, a virucidal peptide used in combination with protease and/or polymerase inhibitors targeting multiple steps of viral infection cycle would have advantages over single drug therapy in reducing viral titers as well as in suppressing the emergence of viral resistance (liu et al., ) . in summary, the present study revealed for the first time that scorpion venom cationic peptides are indeed effective against hsv- infection in vitro. the screened peptides hp and hp not only exhibited extracellular virucidal activities against hsv- , but also had potent inhibitory effect when added at the viral attachment and entry steps. the virucidal activities depended on the morphological changes of hsv- virons by the treatment of hp and hp . their properties of amphipathic a-helix conformation under appropriate membrane environment possibly contributed to their membrane penetrating and virucidal activities, resulting in the intracellular anti-viral effect. hp and hp easily entered vero cells. naturally, both peptides also inactivated intracellular viral particles and inhibited viral proliferation at the postinfection step. our work provided a new direction of developing potential biofunctional molecules from animal venoms and opened new avenues for discovering anti-viral agents or sources. activity of two synthetic amphiphilic peptides and magainin- against herpes simplex virus types and inhibition of herpes simplex virus type infection by cationic beta-peptides antiviral activity of antimicrobial cationic peptides against junin virus and herpes simplex virus anti-hsv activity of lactoferrin and lactoferricin is dependent on the presence of heparan sulphate at the cell surface in vitro antiviral activity of dermaseptins against herpes simplex virus type hepatitis c virus ns a anchor peptide disrupts human immunodeficiency virus anti-hiv- activity of a new scorpion venom peptide derivative kn - cellular localization of hp and hp peptides in vero cells. the cellular localization of hp and hp peptides were determined by confocal microscopy after incubation for and h. (b) intracellular viral inactivation effects of hp and hp peptides. vero cells were infected with hsv- (moi . ) and hp and hp ( lm) were added at indicated time postinfection. infected cells were treated for h and h, the intracellular viruses were harvested by freeze-thaw method a virocidal amphipathic {alpha}-helical peptide that inhibits hepatitis c virus infection in vitro herpes simplex virus helicase-primase inhibitors are active in animal models of human disease herpes simplex virus antiviral drug resistance-current trends and future prospects acyclovir prophylaxis for herpes simplex virus infection aptamer that binds to the gd protein of herpes simplex virus and efficiently inhibits viral entry novel targets for the development of anti-herpes compounds quantitation of herpes simplex virus type dna and latency-associated transcripts in rabbit trigeminal ganglia demonstrates a stable reservoir of viral nucleic acids during latency design of histidine-rich peptides with enhanced bioavailability and inhibitory activity against hepatitis c virus emodin is a novel alkaline nuclease inhibitor that suppresses herpes simplex virus type yields in cell cultures anti-hsv activity of lactoferricin analogues is only partly related to their affinity for heparan sulfate peptide antimicrobial agents overcoming drug-resistant herpes simplex virus (hsv) infection by a humanized antibody virucidal activity of a scorpion venom peptide variant mucroporin-m against measles, sars-cov and influenza h n viruses a peptide derived from hepatitis c virus e envelope protein inhibits a post-binding step in hcv entry molecular diversity of toxic components from the scorpion heterometrus petersii venom revealed by proteomic and transcriptome analysis manzamine a as a novel inhibitor of herpes simplex virus type- replication in cultured corneal cells np- , a rabbit alphadefensin, prevents the entry and intercellular spread of herpes simplex virus type sialic acid on herpes simplex virus type envelope glycoproteins is required for efficient infection of cells a new natural alpha-helical peptide from the venom of the scorpion heterometrus petersii kills hcv evaluation of the inactivation of infectious herpes simplex virus by host-defense peptides theta defensins protect cells from infection by herpes simplex virus by inhibiting viral adhesion and entry virucidal mechanism of action of nvc- , a novel antimicrobial drug for the treatment of adenoviral conjunctivitis mucroporin-m inhibits hepatitis b virus replication by activating the mitogen-activated protein kinase (mapk) pathway and down-regulating hnf alpha in vitro and in vivo this work was supported by grants from the national key basic research program in china (nos. cb and cb ), the china specific project for developing new drugs (nos. zx - and zx - - ), and the fundamental research funds for the central universities in china. key: cord- -hh h t authors: tseng, chin-kai; hsu, sung-po; lin, chun-kuang; wu, yu-hsuan; lee, jin-ching; young, kung-chia title: celastrol inhibits hepatitis c virus replication by upregulating heme oxygenase- via the jnk mapk/nrf pathway in human hepatoma cells date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: hh h t background and purpose: celastrol, a quinone methide triterpene isolated from the root extracts of tripterygium wilfordii, can greatly induce the gene expression activity of heme oxygenase- (ho- ) to achieve disease prevention and control. ho- induction was recently shown to result in anti-hcv activity by inducing type i interferon and inhibiting hepatitis c virus (hcv) ns / a protease activity. the aim of the present study is to evaluate the anti-hcv activity of celastrol and characterize its mechanism of inhibition. methods: the anti-hcv activity of celastrol was evaluated using the hcv subgenomic replicon and hcvcc infection systems. the anti-hcv mechanism of celastrol targeting ho- expression was clarified using specific inhibitors against several signaling pathways. the transcriptional regulation of celastrol on target gene expression was determined using promoter-based reporter activity assay. the synergistic effect of celastrol and a numbers of clinically used anti-hcv drugs was determined via a drug combination assay. results: celastrol inhibited hcv replication in both the hcv subgenomic and hcvcc infection systems with ec values of . ± . and . ± . μm, respectively. celastrol-induced heme oxygenase (ho- ) expression promoted antiviral interferon responses and inhibition of ns / a protease activity, thereby blocking hcv replication. these antiviral effects were abrogated by treatment with the ho- -specific inhibitor snmp or silencing of ho- expression by transfection of shrna, which indicates that ho- induction contributes to the anti-hcv activity of celastrol. jnk mitogen-activated protein kinase and nuclear factor erythroid -related factor (nrf ) were confirmed to be involved in the inductive effect of celastrol on ho- expression. celastrol exhibited synergistic effects in combination with interferon-alpha, the ns a inhibitor daclatasvir, and the ns b inhibitor sofosbuvir. conclusion: celastrol can serve as a potential supplement for blocking hcv replication. targeting the jnk/nrf /ho- axis presents a promising strategy against hcv infection. hepatitis c virus (hcv) is an enveloped single-stranded positive-sense rna virus belonging to the hepacivirus genus of the flaviviridae family (brass et al., ) . the genome of the virus includes base pairs encoding structural proteins (c, e , e and p ) and nonstructural proteins (ns , ns , ns a, ns b, ns a and ns b) (brass et al., ) . hcv infection is a risk factor of chronic liver diseases, including cirrhosis, fibrosis and hepatocellular carcinoma. hcv-infected patients have been estimated to number up to million worldwide (alter, ) . in the past, the standard of care (soc) for hcv infection is treatment with a combination of pegylated interferon (peg-ifn-a) and ribavirin (rbv); however, the efficacy of this treatment is only %e % in patients infected with genotype hcv (ghany et al., ) . as well, this soc therapy is associated with several adverse effects, such as anemia, headache, fatigue and depression (schoggins et al., ) . in , telaprevir and boceprevir, the first daas were approved by the us food and drug administration (fda) and found to exhibited higher rates of sustained virologic responses (svrs) in patients infected with genotype hcv when combined with peg-ifn-a, which provided a new soc for the treatment of chronic hcv infection (kiser et al., ) . despite the increasing efficacy of the treatment modes developed for hcv infection, however, undesirable side effects continue to be observed during therapy (kiser et al., ) . the fda recently approved the two-agent combo harvoni and the fouragent combo viekira pak for distribution; these treatments could achieve svrs up to % in patients infected with genotype hcv (koretz, ) . unfortunately, while these antiviral agents achieve higher rates of svr while avoiding the adverse effects often induced by ifn (koretz, ) , they also exist the risk to reactivate the hepatitis b virus (hbv) in treating patients with hcv and hbv co-infection (yeh et al., ) . moreover, these two drugs relatively more expensive than other drugs. thus, development of a more safety and cost-effective substitute for treatment is an important endeavor. celastrol is a quinone methide triterpene isolated from tripterygium wilfordii, a medicinal plant used to treat a range of illnesses including inflammation, swelling, fever, sores, and pain in india, japan, china, korea, and other asian countries (ju et al., ; lee, j.y. et al., ; youn et al., ) . celastrol is a meal supplement that is widely used in herbal medicine because of its diverse biological activities, which include anti-inflammation, anti-cancer, hepatoprotection, and anti-microbial properties (ju et al., ; lee et al., ) . several reports have demonstrated that celastrol exhibits inhibitory effects on human immunodeficiency virus (youn et al., ) , dengue virus (yu et al., a) , and other severe acute respiratory syndrome-associated coronaviruses (ryu et al., ) . celastrol can induce heme oxygenase- (ho- ) gene expression via nuclear factor erythroid -related factor (nrf ) activation to prevent circulatory failure prevention and protect against myocardial ischemia (der sarkissian et al., ) . ho- induction was recently shown to result in anti-hcv activity by inducing type i interferon and inhibiting hcv ns / a protease activity (lehmann et al., ; zhu et al., ) . ho- is an inducible and rate-limiting enzyme that degrades heme to produce biliverdin, carbon monoxide (co) and ferrous iron (fe þ) via the heme catabolic pathway (maines, ) . the enzyme and its metabolites exhibit protective effects against oxidative stress-induced tissue damage upon exposure to multiple stimuli such as viral and bacterial products including lipopolysaccharides, cytokines, oncogenes, mitogens, and various growth factors (farombi and surh, ; paine et al., ) . upon hcv infection, down-regulation of ho- expression has been observed in hcv-infected patients and hcv core protein expression although the biological role of ho- suppression and the detailed mechanism of hcv against ho- expression were unclear (abdalla et al., ) . ho- is a detoxifying enzyme that is mainly regulated by nrf (shan et al., ) . activation of ho- expression occurs when nrf binds to the antioxidant response element (are) of the ho- promoter region. by contrast, bach suppresses ho- expression by competing with the are binding site (shan et al., ) . under normal conditions, nrf is sequestered in the cytoplasm by kelchlike ech-associated protein (keap )-mediated proteasome degradation (shan et al., ) . upregulating nrf /are-dependent ho- expression to achieve anti-inflammatory and anti-oxidative stress functions is mediated by several host factors, including the mitogene-activated protein kinase (mapk) molecules p mapk, extracellular signal-regulated kinase and (erk / ), and c-jun nterminal kinase (jnk) (paine et al., ) . in this study, our data revealed that celastrol significantly inhibits hcv replication, and that the anti-hcv effect of celastrol was attenuated by the ho- specific inhibitor tin mesoporphyrin (snmp) or ho- gene expression silencing. celastrol-mediated ho- induction contributed to the anti-hcv action through inducing antiviral ifn response and inhibiting hcv ns protease activity. moreover, the jnk/nrf / are axis was the critical pathway involved in celastrol-mediated ho- induction against hcv replication. combinations of celastrol and ifn-a, sofosbuvir or daclatasvir were tested to determine their ability to enhance of anti-hcv activity. human hepatoma (huh- ), ava (huh- cells containing hcv genotype b subgenomic rna replicon cells) (blight et al., ) , and huh . /j /jfhemcviresrlucneo (huh- cells harboring hcv genotype a subgenomic rna and renilla luciferase reporter gene) obtained from apath llc (st. louis, mo) were maintained in dulbecco's modified eagle's medium containing % fetal bovine serum, % non-essential amino acids, and % antibioticeantimycotic in a % co atmosphere at c. huh- . cells stably expressing the pef/jfh -rz/n plasmid were grown to produce cell culture-derived infectious hcv particles (hcvcc), and the conditional medium was collected to harvest viral particles according to a protocol described previously (kato et al., ) . celastrol (pubchemcid: ) was purchased from fusol-material co., ltd (tainan, taiwan). ifn-a- a (roferon © -a) was purchased from roche ltd. an ho- specific inhibitor [tin mesoporphyrin (snmp)], and mapk-specific inhibitors (sp , sb , and pd ) were purchased from sigma (st. louis, mo). daclatasvir and sofosbuvir was purchased from shanghai haoyuan chemexpress co., ltd. the final concentration of dmso in all reactions was maintained at . %. ava cells were seeded in -well plates at a density of  cells per well and treated with celastrol at the indicated concentrations. after days incubation, the cell viability was determined by the celltiter aq ueous one solution cell proliferation assay (promega, madison, wi, usa) according to the manufacturer's instructions. pho- -luc (hill-kapturczak et al., ) and p xare-luc (liao et al., ) vectors were used to measure the transcriptional activity of ho- and nrf , respectively. pisre-luc, a reporter vector containing firefly luciferase under the control of an ifn-stimulated response element (isre), was used to measure ifn response-dependent transcriptional activity (stratagene, agilent technologies, ca, usa). all cloned dna fragments were verified by dna sequencing. western blotting was performed as described previously (lee et al., ) . in brief, mg of cell lysates was analyzed by sds-page, and then transferred to a pvdf membrane. the membranes were probed with anti-hcv ns b ( : ; abcam, cambridge, ma, usa), anti-ho- ( : ; abcam), anti-nrf ( : ; genetex, ca, usa), anti-phospho-erk / ( : ; cell signaling technology, inc. danvers, ma, usa), anti-phospho-p ( : ; cell signaling), anti-phospho-jnk ( : ; cell signaling), anti-erk / ( : ; cell signaling), anti-p ( : ; cell signaling), or anti-jnk ( : ; cell signaling)antibody. a loading control was determined using anti-gapdh antibody ( : ; genetex). rna isolation and quantitative real-time rt-pcr (qrt-pcr) were performed as described previously (lee et al., ) . relative mrna levels were determined by normalization against the cellular endogenous glyceraldehyde- -phosphate dehydrogenase (gapdh) gene. the primers used in the study are listed in table . to evaluate the transcriptional regulation of ho- , nrf , or ifn response by celastrol, the ava cells were seed on the -wells plates at a density of  cells per well. after h incubation, the . mg of promoter-driven firefly luciferase plasmids, including pho- -luc, p xare-luc, or pisre-luc, were respectively transfected into pre-seeded ava cells using t-pro™ transfection reagent (ji-feng biotechnology co., ltd. taiwan), according to the manufacturer's instructions. the transfected cells were then treated with celastrol at various concentrations for days. cell lysates were prepared for the luciferase activity assay and western blotting using the bright-glo luciferase assay system (promega, madison, wi, usa). the cell-based ns / a activity assay was performed as described previously (lee et al., ) . in brief, the huh- cells were seed on the -wells plates at a density of  cells per well. after h incubation, the cells were co-transfected with the ns / a protease reporter vector peg(ded ab)seap and ns / a expression vector pns / a, followed by celastrol treatment with or without snmp or days. the . mm telaprevir treatment server as the positive control. supernatants were harvested for the seap activity assay using the phospha-light assay kit. each transfection mixture contained . mg of the firefly luciferase expression vector (pfluc) as a transfection control for normalization against seap activity. cells were seeded in -well plates at a density of  cells/ well and treated with various concentrations of celastrol for days. the cell culture medium was harvested to measure ifn-a concentration using a human ifn-a elisa kit (uscnk life science inc., usa) according to the manufacturer's protocol. absorbance was detected at nm using an epoch microplate spectrophotometer. ava cells were seeded in a -cm dish at a density of .  cells per -cm dish, and treated with increasing concentrations of celastrol for days. the cells were then collected for preparation of a nuclear fraction as described previously (lee et al., ) . ava cells were treated with serially diluted celastrol ( , . , . , or . mm) in combination with serially diluted ifn-a ( . , , , or u/ml), telaprevir ( . , . , . , and . mm) sofosbuvir ( , , , or nm), or daclatasvir ( . , , and pm). after days of incubation, hcv rna levels were determined by qrt-pcr. multiple drug combination data were analyzed using calcusyn ™ software (biosoft, cambridge, uk) which compares single and multiple drug dose effects and determines the combination index (ci) value. the effect of a multiple drug combination is presented as antagonism (ci > ), additivity (ci ¼ ), or synergism (ci < ). data were presented as the mean ± standard deviation of at least three independent experiments. statistical significance was analyzed using student's t-test. a significant difference was considered as *p < . . celastrol is a quinone methide triterpene (fig. a) possessing anti-denv activity (lee et al., ) . to discover whether celastrol exhibits anti-hcv activity, we first treated hcv subgenomic replicon ava cells with celastrol at increasing concentrations for days. western blotting assay and qrt-pcr analysis were performed to determine hcv protein and rna levels under celastrol treatment, respectively. the cytotoxic effect of celastrol on ava cells was also tested using the mts assay. the results indicated that celastrol dose-dependently reduced hcv protein synthesis (fig. b) and rna replication with a % effective concentration (ec ) of . ± . mm (fig. c ) without cytotoxicity at effective antiviral concentrations ( % cytotoxicity concentration: cc ¼ . ± . mm) (fig. d ). based on the results collected from hcv replicon, the selectivity index of celastrol against hcv replication approximates . the jfh replicon and hcvcc infectious assay was performed to confirm the anti-hcv activity of celastrol. here, huh . /j /jfhemcviresrlucneo replicon cells and jfh infected huh- cells were treated with increasing concentrations of celastrol for days. as shown in fig. e and f, qrt-pcr analysis revealed that celastrol dose-dependently reduced hcv rna levels and fully inhibited hcv replication at a concentration of . mm. celastrol was recently shown to induce on ho- gene expression for antiviral activity (youn et al., ) . we first examined whether celastrol could induce ho- expression by determining ho- promoter activity, as well as ho- rna and protein levels, in ava cells in the presence of celastrol. ava cells were transfected pho- -luc containing a firefly luciferase gene driven by the ho- promoter. then, the transfected-cells were treated with celastrol at increasing concentrations for days and subjected to luciferase activity assay. the results indicated that celastrol significantly induced the ho- promoter activity in a concentrationdependent manner ( fig. a) . as expected, the ho- rna and protein levels were also dose-dependently induced by celastrol ( fig. b and c). to investigate whether celastrol-induced ho- expression is involved in the anti-hcv activity of celastrol, ava cells were cotreated with a fixed concentration of celastrol and increasing concentrations ho- specific inhibitor snmp for days. western blotting assay was preformed to determine the restorative effect of snmp on hcv protein synthesis upon celastrol treatment. as shown in fig. d , celastrol inhibited hcv protein synthesis compared with non-celastrol treated cells (lanes and ) by contrast, snmp treatment dose-dependently attenuated the suppressive effect of celastrol on hcv protein synthesis (lanes e ). as expect, the ho- specific shrna mediated ho- gene silencing also can attenuate the suppressive effect of celastrol on hcv protein synthesis (fig. e) . these results reveal that celastrol inhibited hcv replication is correlated with ho- induction. the results of qrt-pcr analysis indicated that ifn-a- and ifn-a- rna levels were gradually induced by celastrol (fig. a) . by contrast, the inductive effect of celastrol on ifn-a- and ifn-a- rna levels was significantly attenuated by ho- inhibitor snmp in a concentration-dependent manner (fig. b) . we next measured ifn-a protein secretion levels in the culture medium after celastrol treatment. as expected, elisa results indicated that ifn-a protein secretion levels were gradually induced by celastrol after days of treatment (fig. c ). an antiviral effect on cells may be attributed to the interaction of ifn-a and cell surface ifn-a receptors, leading to the activation of isre and upregulation of downstream antiviral genes (yu et al., b) . to evaluate whether celastrol-induced ifna could activate downstream antiviral genes, isre activity and the expression of three critical ifn-mediated antiviral genes, including - -oligoadenylate synthetase e (oas e ), were measured. ava cells were transfected with isre-driven firefly luciferase reporter plasmid followed by treatment with celastrol for days. luciferase assay results indicated that the isre promoter activity was increased by approximately . e . -fold by celastrol (fig. a ). oas e gene expression levels were significantly upregulated approximately . ± . , . ± . , and . ± . -fold by celastrol, respectively (fig. b) . another proposed anti-hcv action of ho- induction is inhibition of ns / a protease activity (zhu et al., ) . therefore, we performed a cell-based hcv protease assay to examine the ability of celastrol to target hcv ns / a protease activity. huh- cells were co-transfected with peg(de ab)seap reporter and hcv pns / protease expression plasmids, followed by celastrol treatment for days. as shown in fig. a , hcv ns / a protease activity decreased by approximately . e -fold in comparison with that of the control by celastrol. by contrast, the inhibitory effect of celastrol on hcv ns / a protease activity was dose-dependently attenuated by ho- inhibitor snmp (fig. b) . these data indicate that targeting hcv ns / a protease is an alternative anti-hcv action of celastrol. nrf functions as an important upstream regulator in the mediation of ho- expression by binding to the are response element (farombi and surh, ) . to investigate whether celastrol-induced ho- induction is mediated by nrf activation, we first investigated nrf nuclear translocation. ava cells were treated with increasing concentrations of celastrol for days, after which the total cells lysate and nuclear fraction were harvested and subjected to western blotting assay. as shown in fig. a , total nrf protein levels were not affected by celastrol (upper panel). by contrast, nuclear nrf protein levels significantly accumulated upon celastrol treatment at a concentration of . mm, which is the effective dosage against hcv replication. we then examined the nrf -mediated are activation caused by celastrol. here, ava cells were transfected with are-driven firefly luciferase reporter plasmid. the transfected-cells were treated with increasing concentrations of celastrol for days. as expected, are-driven firefly luciferase activity was elevated by celastrol in a concentrationdependent manner (fig. b ). considering these results, the nrf -are-ho- axis can be concluded to be strongly associated with the anti-hcv activity of celastrol. activation of the mapk signaling cascade, which includes p , erk / , and jnk, has been reported to be involved in ho- induction resulting in anti-hcv activity (huang et al., whether mapks are involved in anti-hcv effect of celastrol, ava cells were treated with . mm celastrol for e min. the phosphorylation levels of p , erk / , and jnk were then measured by western blotting with phospho-specific antibodies. ava cells were incubated with celastrol for days, and the total cell lysate and cellular nuclear fraction were harvested. total nrf (t nrf ) and nuclear nrf (ne nrf ) levels were analyzed by western blotting using anti-nrf antibody. lamin b served as the internal control for the nuclear fraction, and gapdh served as internal control for the total cell lysate. (b) celastrol increases nrf -mediated are transactivation. ava cells were transfected with . mg of p xare-luc followed by celastrol treatment for days, and then harvest to analyze their luciferase activity. here, luciferase activity was used to represent isre activity. error bars denote the mean ± sd of three independent experiments. *p < . . as shown in fig. a , the jnk phosphorylation levels were elevated by celastrol in a time-dependent manner compared with that at the time point of min. by contrast, celastrol showed no significant effect on erk/ / or p phosphorylation at any time point. to clarify the role of jnk in the ho- induction of celastrol, we used specific inhibitors against erk / (pd ), jnk (sp ), and p (sb ), to measure ho- rna expression. as shown in fig. b , ho- rna expression levels were induced by celastrol compared with non-celastrol treated cells and the jnk inhibitor sp significantly reduced the ho- inductive effect of celastrol. by contrast, the erk / inhibitor pd and p inhibitor sb showed no significant effect on celastrol-induced ho- induction. these results suggest that the anti-hcv effect of celastrol is associated with jnk-mediated ho- induction in ava cells. . . celastrol synergistically or additively inhibits hcv replication when combined with ifn-a, sofosbuvir, daclatasvir or telaprevir to determine whether celastrol can enhance the anti-hcv activity of several of clinically used anti-hcv drugs, such as ifn-a, the ns / a inhibitor telaprevir, the ns b inhibitor sofosbuvir (lam et al., ) and the ns a inhibitor daclatasvir (lee et al., a) . ava cells were co-treated with each drug and celastrol at various concentration ratios for days. the synergistic effect of each combination was evaluated by calcusyn . as described by chou and talalay ( ) . the ci values for the ec , ec , and ec of ifn-a ranged from . to . , those of telaprevir ranged from . to . , sofosbuvir ranged from . to . and daclatasvir is ranging from . to . (table ) . no significant cytotoxicity was observed in any combination treatment, as assessed by a colorimetric mts assay (data not shown). these findings reveal that celastrol may serve as a dietary supplement for enhancing the therapeutic effect of clinically used anti-hcv drugs. the type i ifn system presents important innate immunity to effectively block virus replication (jones et al., ; morrison et al., ) . hcv infection has been reported to inhibit antiviral ifn responses that facilitate virus replication by promoting the hcv ns / a protease-mediated cleavage of mitochondrial antiviralsignaling protein (mavs)/tir-domain-containing adapterinducing interferon-b (trif) (gokhale et al., ) . in the present study, we found that a natural product celastrol could effectively inhibit hcv ns / a protease activity and enhance ifn-mediated antiviral gene expression through ho- induction (figs. e ) . the ho- metabolite biliverdin has been proven to be a blocker of hcv ns / a protease activity. therefore, the regulatory effect of celastrol on the mitochondria-mediated inf signaling pathway against virus replication presents promising prospects for future investigations. our data revealed that nrf- -mediated ho- induction contributed to the anti-hcv activity of celastrol based on the accumulation of nuclear nrf- and enhancement of nrf- binding activity on the are response element (fig. ) . given that the activation of nrf nuclear translocation is regulated by keap dependent ubiquitination and bach , a competitor of nrf for binding to are in the ho- promoter region (maines, ) , future studies should be performed to determine whether celastrol alters the expression levels of keap- or bach for regulating ho- induction. knowledge in this area will help provide alternative targets for screening anti-hcv agents. we further found that the ho- inductive effect of the celastrol was also associated with jnk activity (fig. ) . however, several kinases are involved in jnk activation, including mitogen-activated protein kinase kinase (mkk ), mkk and mixed-lineage kinases (mlks) (wasserman et al., ) . future studies should examine the effect of celastrol on the kinases involved in jnk activation comprehensively describe the relationship between celastrol and ho- induction. several studies have indicated that celastrol exhibits anti-inflammatory activity by inhibiting of nuclear factor-kb (nf-kb) and downstream cycloxygenase- (cox- ) (ding et al., ; joshi et al., ) . on the basis of earlier findings on a promising tactic against hcv infection via down-regulation of nf-kb-mediated cox- expression lee et al., b) , we propose that the inhibitory effect of celastrol on nf-kb-mediated cox- expression may, at least in part contribute to its anti-hcv activity. hence, more work is necessary to elucidate additional signaling pathways involved in the anti-hcv activity of celastrol. drug combination therapy is considered to be a promising approach to increase therapeutic efficacy and decrease drug resistance in comparison with mono-drug therapy (hajj et al., ) . the two-agent combo harvoni and the four-agent combo viekira pak, which could achieve the svrs of up to % in patients infected with genotype hcv. however, the low fidelity of hcv polymerase during viral replication may lead to the emergence of drug resistance, which poses a major challenge in treating hcv infection (lee et al., ) . targeting host factors could be an alternative strategy to eliminate drug resistance in hcv therapeutic regimens because the mutation rate of the host genome is lower than that of the rna virus genome (liao et al., ) . in this study, we showed that celastrol can be considered as a suitable candidate for hcv therapy to minimize the risk of drug resistance by targeting host ho- signaling pathway and synergistically inhibiting hcv replication in combination with clinically used drugs against different viral targets (table ) . further studies are warranted to clarify the potential clinical relevance of our findings. in summary, the data indicated that celastrol efficiently inhibited hcv replication via the induction of the jnk/nrf /ho- axis, which may represent a therapeutic target for the future development and discovery of anti-hcv drugs. celastrol exhibited synergetic effects on anti-hcv activity in combination with ifn, sofosbuvir or daclatasvir. these results reveal that celastrol may serve as a dietary supplement for enhancing therapeutic effects of the anti-hcv drugs currently available. down-regulation of heme oxygenase- by hepatitis c virus infection in vivo and by the in vitro expression of hepatitis c core protein epidemiology of hepatitis c virus infection efficient initiation of hcv rna replication in cell culture aqueous extract of the edible gracilaria tenuistipitata inhibits hepatitis c viral replication via cyclooxygenase- suppression and reduces virus-induced inflammation quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors celastrol protects ischaemic myocardium through a heat shock response with up-regulation of haeme oxygenase- celastrol, an inhibitor of heat shock protein beta potently suppresses the expression of matrix metalloproteinases, inducible nitric oxide synthase and cyclooxygenase- in primary human osteoarthritic chondrocytes american association for the study of liver diseases, diagnosis, management, and treatment of hepatitis c: an update hepatitis c virus. strategies to evade antiviral responses combination of acamprosate and baclofen as a promising therapeutic approach for parkinson's disease an internal enhancer regulates heme-and cadmium-mediated induction of human heme oxygenase- mechanistic link between the anti-hcv effect of interferon gamma and control of viral replication by a ras-mapk signaling cascade dengue virus inhibits alpha interferon signaling by reducing stat expression celastrol modulates inflammation through inhibition of the catalytic activity of mediators of arachidonic acid pathway: secretory phospholipase a group iia, -lipoxygenase and cyclooxygenase- celastrol ameliorates cytokine toxicity and pro-inflammatory immune responses by suppressing nf-kb activation in rinm f beta cells production of infectious hepatitis c virus of various genotypes in cell cultures review and management of drug interactions with boceprevir and telaprevir acp journal club: review: telaprevir, boceprevir, simeprevir, or sofosbuvir improves response in hcv type genotype and subtype profiling of psi- as a nucleotide inhibitor of hepatitis c virus. antimicrob the hepatitis c virus ns a inhibitor (bms- ) alters the subcellular localization of the ns a non-structural viral protein anti-hepatitis c virus activity of acacia confusa extract via suppressing cyclooxygenase- andrographolide exerts anti-hepatitis c virus activity by up-regulating haeme oxygenase- via the p mapk/nrf pathway in human hepatoma cells celastrol blocks binding of lipopolysaccharides to a toll-like receptor /myeloid differentiation factor complex in a thiol-dependent manner cinnamaldehyde inhibits the tumor necrosis factor-alpha-induced expression of cell adhesion molecules in endothelial cells by suppressing nf-kappab activation: effects upon ikappab and nrf the heme oxygenase system: update dengue virus co-opts ubr to degrade stat and antagonize type i interferon signaling signaling to heme oxygenase- and its anti-inflammatory therapeutic potential regulation of hepatitis c virus replication and gene expression by the mapk-erk pathway sars-cov clpro inhibitory effects of quinone-methide triterpenes from tripterygium regelii a diverse range of gene products are effectors of the type i interferon antiviral response role of bach and nrf in up-regulation of the heme oxygenase- gene by cobalt protoporphyrin a novel c-jun n-terminal kinase (jnk)-binding protein wdr is recruited to stress granules and mediates a nonclassical jnk activation oxidative stress induces anti-hepatitis c virus status via the activation of extracellular signal-regulated kinase reactivation of hepatitis b in patients of chronic hepatitis c with hepatitis b virus infection treated with direct acting antivirals celastrol ameliorates hiv- tat-induced inflammatory responses via nf-kappab and ap- inhibition and heme oxygenase- induction in astrocytes celastrol inhibits dengue virus replication via up-regulating type i interferon and downstream interferon-stimulated responses schisandrin a inhibits dengue viral replication via upregulating antiviral interferon responses through stat signaling pathway biliverdin inhibits hepatitis c virus nonstructural / a protease activity: mechanism for the antiviral effects of heme oxygenase we are grateful to dr. charles rice (rockefeller university and aapth, lcc, usa) for kindly supporting con b replicon plasmid, human hepatoma cell; huh- and hcv subgenomic replicon containing cell line; ava , huh . /j /jfhemcviresrlucneo hcv replicon cells and dr. t. wakita (national institute of infectious diseases, japan) for providing the jfh plasmid. 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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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- -lsfmcj c authors: geller, c.; fontanay, s.; mourer, m.; dibama, h. massimba; regnouf-de-vains, j.-b.; finance, c.; duval, r.e. title: antiseptic properties of two calix[ ]arenes derivatives on the human coronavirus e() date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: lsfmcj c facing the lack in specific antiviral treatment, it is necessary to develop new means of prevention. in the case of the coronaviridae this family is now recognized as including potent human pathogens causing upper and lower respiratory tract infections as well as nosocomial ones. within the purpose of developing new antiseptics molecules, the antiseptic virucidal activity of two calix[ ]arene derivatives, the tetra-para-sulfonato-calix[ ]arene (c[ ]s) and the , -bis(bithiazolyl)-tetra-para-sulfonato-calix[ ]arene (c[ ]s-btz) were evaluated toward the human coronavirus e (hcov e). comparing these results with some obtained previously with chlorhexidine and hexamidine, (i) these two calixarenes did not show any cytotoxicity contrary to chlorhexidine and hexamidine, (ii) c[ ]s showed as did hexamidine, a very weak activity against hcov e, and (iii) the c[ ]s-btz showed a stronger activity than chlorhexidine, i.e. . and . log( ) reduction in viral titer after min of contact with (− ) mol l(− ) solutions of c[ ]s-btz and chlorhexidine, respectively. thus, the c[ ]s-btz appeared as a promising virucidal (antiseptic) molecule. facing the lack in specific antiviral treatment, it is necessary to develop new means of prevention. in the case of the coronaviridae this family is now recognized as including potent human pathogens causing upper and lower respiratory tract infections as well as nosocomial ones. within the purpose of developing new antiseptics molecules, the antiseptic virucidal activity of two calix [ ] arene derivatives, the tetra-para-sulfonato-calix[ ]arene (c[ ]s) and the , -bis(bithiazolyl)tetra-para-sulfonato-calix[ ]arene (c[ ]s-btz) were evaluated toward the human coronavirus e (hcov e). comparing these results with some obtained previously with chlorhexidine and hexamidine, (i) these two calixarenes did not show any cytotoxicity contrary to chlorhexidine and hexamidine, (ii) c[ ]s showed as did hexamidine, a very weak activity against hcov e, and (iii) the c[ ]s-btz showed a stronger activity than chlorhexidine, i.e. . and . log reduction in viral titer after min of contact with − mol l − solutions of c[ ]s-btz and chlorhexidine, respectively. thus, the c[ ]s-btz appeared as a promising virucidal (antiseptic) molecule. © elsevier b.v. all rights reserved. the lack in specific antiviral treatments is still persisting, considering the large variety of viruses already circulating among human population and the potential emerging ones. the coronaviridae family illustrates this problem. indeed, no specific treatment is available to fight coronaviruses infections, while they are known to be responsible for upper and lower tract infections as well as nosocomial ones. thus, efficient means of prevention, as an adapted antisepsis-disinfection (ats-d), should be developed to prevent the environmental spread of such infections. human coronaviruses (hcov) were historically known to be responsible for about % of common colds and other upper respiratory tract infections (larson et al., hcov were known, the e strain and the oc strain. this serious outbreak, due to a newly discovered hcov, the sars-cov (ksiazek et al., ; peiris et al., ) , reinforced the interest into the coronaviridae family. indeed, coronaviruses were since involved in more serious respiratory diseases, i.e. bronchitis, bronchiolitis or pneumonia, especially in young children and neonates (gagneur et al., ; gerna et al., ) , elderly people (falsey et al., ) and immunosuppressed patients (gerna et al., ; pene et al., ) . furthermore, they have been shown to survive for at least several hours under different environmental conditions (ijaz et al., ; lai et al., ; rabenau et al., a; sizun et al., ) . finally, their adaptive properties and their ability of species barrier crossing, involve a significant possibility of new coronaviruses emergence (laude et al., ; li et al., ; vijgen et al., ) . thus, these specificities (i.e. pathogenicity, potential environmental resistance and evolutionary ability) make the coronaviridae family a pertinent model for studying ats-d activity. new antiviral molecules are urgently needed. within this purpose, some macrocyclic compounds belonging to the calixarene family (de fátima et al., ; rodik et al., ) , have already been shown to be interesting as anti-hiv and anti-hsv agents (coveney and costello, ; harris, harris, , hwang et al., ; kral et al., ; motornaya et al., ) . in this field, our team described antiviral properties of various derivatives, such as , -bis(bithiazolyl)-tetra-para-sulfonato-calix to evaluate these properties, a protocol described elsewhere (geller et al., ) has been implemented. it responded to the general imperatives of the only european standard existing (nf en + a ) to evaluate ats-d antiviral activity in human medicine (afnor, ) . according to this standard, a product should induce a log reduction in viral titers to quality as an ats-d antiviral activity. for comparison, american standards recommend a reduction of log as efficiency criterion (astm, (astm, , . the general principle of our protocol is: (i) to incubate viruses with the test product, at room temperature, for a defined contact time, (ii) to neutralize product activity and (iii) to estimate the loss in viral titers. the neutralization process allows: (i) to stop the potential antiviral activity of the product, (ii) to remove its eventual cytotoxicity and (iii) to prevent interference, due to the test itself, in viral infectivity. it was achieved thanks to a gel filtration method, using sephadex tm g- columns, developed and validated previously (geller et al., ). these assays required appropriate controls especially to check the non retention of viruses by sephadex tm columns, the absence of interference with viral infectivity, the efficiency of neutralization and the absence of cytotoxicity (supp. data ). as recommended by the european standard nf en + a , controls are validated if the difference between viral titers, with and without treatment, is less than . log (afnor, ). molecular masses of c[ ]s and c[ ]s-btz were . g mol − and . g mol − respectively. thus, they were susceptible to be retained by the sephadex tm g- columns. to assess the non cytotoxicity of the filtrates, cytotoxic assays and spectrophotometric measurements were conducted. two concentrations for both molecules were tested, i.e. − and − mol l − . mtt (methylthiazole tetrazolium) and nr (neutral red) assays were first performed to evaluate cytotoxicity of c[ ]s and c[ ]s-btz on l- cells. for both molecules, ic (inhibitory concentration %) and cc (cytotoxic concentration %) were higher than − mol l − , even after h, the time required for obtaining the hcov e cytopathogenic effect. the same assays were then performed with the filtrates obtained after filtration of both molecules on sephadex tm g- columns and cytotoxicity was also higher than − mol l − even after h of incubation. spectrophotometric analyses, coupled with regression analyses, allowed to determine the specific parameters of each molecule (supp. data ). retention rates by sephadex tm g- columns were then estimated after evaluation of residual concentration in the filtrates. retention rates of c[ ]s solutions by sephadex tm g- columns were . % and . % for solutions at − mol l − and − mol l − , respectively. the lower retention rate of − mol l − solution was due to calculation limitations. retention rate of the solution at − mol l − was considered as significant. in the case of c[ ]s-btz, retention rates were . % and . % for solutions of − and − mol l − , respectively (supp. data ). ats-d antiviral assays were then conducted. each experiment was performed in triplicate. to validate these tests, controls, mentioned above, were done the same time, and results are shown for both molecules in table . the c[ ]s was tested at a concentration of − mol l − and contact times of min and min. because of the very weak activity against hcov e, i.e. . and . log reduction for contact times of min and min, respectively, no further experiments were performed with c[ ]s (fig. ) . results obtained with the c[ ]s-btz were markedly better. indeed, at a concentration of − mol l − , it induced log reductions of . , . , . and . after contact times of , , and min, respectively. at − mol l − , it induced reductions of . , . , . and . log in viral titers for the same contact times (fig. ) . previous experiments have been conducted with two largely used antiseptics in human medicine, hexamidine (hxm) and chlorhexidine (chx) (geller et al., ) . first of all, both of these molecules showed cytotoxicity towards l- cells. indeed, chx showed ic and cc values of . × − and . × − mol l − , respectively, after h. in the same way, ic and cc of hxm were . × − and . × − mol l − after h of contact time. thus, the first interest of these two calix [ ] arenes was the absence of cytotoxicity (> − mol l − after h), contrary to hxm and chx. the hxm, in the manner of the c[ ]s, showed a very weak activity on the hcov e, i.e. . and . log reduction after contact times of min and min, respectively. the chx showed a better activity, since it induced . , . , . and . log reduction at − mol l − for contact times of , , and min, respectively, and . , . , . and log reduction at − mol l − and for the same contact times (fig. ) . when comparing c[ ]s-btz and chx activities, the first important point is that, even if they showed a certain anti-hcov e activity, they did not reach the threshold fixed by european and american standards, except for chx at − mol l − and min of contact time. however, this contact time could not be considered as really representative time for ats-d in field conditions. thus, a really attractive characteristic of the c[ ]s-btz was its fast action at − mol l − , as soon as min, compared to chx, which appeared concentration-and time-dependent. furthermore, this activity persisted until min of contact time. several items should be yet taken into consideration when analyzing these results. first, the different molecules were tested alone, i.e. without any additive as alcohol and without any interfering substances. in this way, their own anti-coronavirus activity could be estimated, but this was not really representative of field conditions, since viruses are normally found embedded in organic materials, preventing them from the action of ats-d. these results are consistent with previous studies, which showed that chx did not have ats-d anticoronavirus activity unless it was associated with cetrimide and % (v/v) ethanol (sattar et al., ) . it would be of interest to associate the fast and persistent action of c[ ]s-btz with that of alcoholic solutions. indeed, even if ethanol showed a good ats-d activity, in particular against coronaviruses (rabenau et al., b; sattar et al., ) , its volatile nature involves a transient action, which could potentially be improved by c[ ]s-btz activities. furthermore, the absence of cytotoxicity made the c[ ]s-btz even more promising, considering toxicity risks involved with the currently used ats-d (skin reactions, allergy or occupational diseases). chemical disinfectants and 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bithiazolyl podands coronavirus as a possible cause of severe acute respiratory syndrome coronavirus e-related pneumonia in immunocompromised patients stability and inactivation of sars coronavirus efficacy of various disinfectants against sars coronavirus calixarenes in bio-medical researches chemical disinfection of non-porous inanimate surfaces experimentally contaminated with four human pathogenic viruses survival of human coronaviruses e and oc in suspension and after drying on surfaces: a possible source of hospitalacquired infections complete genomic sequence of human coronavirus oc : molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event financial support was provided by the french ministry of further education and research and french national scientific research center. the authors declare no conflict of interest. supplementary data associated with this article can be found, in the online version, at doi: . /j.antiviral. . . . key: cord- -ni qfjk authors: choi, hwa-jung; kim, jin-hee; lee, choong-hwan; ahn, young-joon; song, jae-hyoung; baek, seung-hwa; kwon, dur-han title: antiviral activity of quercetin -rhamnoside against porcine epidemic diarrhea virus date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: ni qfjk porcine epidemic diarrhea virus (pedv) is the predominant cause of severe entero-pathogenic diarrhea in swine. the lack of effective therapeutical treatment underlines the importance of research for new antivirals. in this study, we identified q r, which actively inhibited pedv replication with a % inhibitory concentration (ic( )) of . μg/ml. the % cytotoxicity concentration (cc( )) of q r was over μg/ml and the derived therapeutic index was . several structural analogues of q r, quercetin, apigenin, luteolin and catechin, also showed moderate anti-pedv activity. antiviral drugs and natural compounds revealed ribavirin, interferon-α, coumarin and tannic acid have relative weaker efficacy compared to q r. q r did not directly interact with or inactivate pedv particles and affect the initial stage of pedv infection by interfering of pedv replication. also, the effectiveness of q r against the other two viruses (tgev, prcv) was lower compared to pedv. q r could be considered as a lead compound for development of anti-pedv drugs to may be used to during the early stage of pedv replication and the structure-activity data of q r may usefully guideline to design other related antiviral agents. porcine epidemic diarrhea virus (pedv), family coronaviridae, causes porcine epidemic diarrhea, an enteric disease characterized by acute watery diarrhea, dehydration, vomiting, and high mortality in nursery piglets (debouck and pensaert, ) . infection with this virus has become a serious issue in the swine industry and outbreaks have lead to serious economic losses in many countries (hofmann and wyler, ) . unfortunately, now there are no effective commercial vaccines or specific treatments available and the only measures to control the disease are those directed to preventing the entrance of the virus on the farm. medicinal plants are increasingly being pursued as suitable alternative sources for discovery of antiviral agents (briskin, ; cowan, ; jassim and naji, ; vlietinck and vanden berghe, ; williams, ) . recent studies have demonstrated that houttuynia cordata thunb. (saururaceae) is effective in treating anaphylaxis and cancer (kwon et al., ; li et al., ; lu et al., ) . it has also been shown that the essential oil prepared * corresponding author. tel.: + ; fax: + . e-mail address: dhkwon@kribb.re.kr (d.-h. kwon) . from fresh plants of h. cordata possessed direct inhibitory activity against herpes simplex virus type (hsv- ), influenza virus and human immunodeficiency virus type (hiv- ), without showing cytotoxicity (lu et al., ) . in particular, quercetin, a flavonoid present in h. cordata, has been reported to have inhibitory effects on several viruses (mucsi and pragai, ) . although a variety of pharmacological activities associated with chemicals in h. cordata has been demonstrated, antiviral activities against pedv have not been reported. in this study we examine the positional effects of the hydroxyl group within basic flavonoid structure, and the type and position of attached sugars against pedv replication. furthermore, to elucidate the action of q r on pedv multiplication in more detail, we investigated the effect of q r on the infection cycle of pedv through time-of-addition study, rt-pcr analysis, effects of q r on the infectivity of pedv particles. tamiflu (f. hofmann-la roche ltd., switzerland), relenza (laboratoire glaxosmithkline, france) and lamivudine (glaxosmithkline australia pty ltd., australia) were purchased from a pharmacy in korea as prescribed by a medical doctor. acriflavine, acycloguanosine, azidovudine caffeine, cumarin, glycyrrhizin, interferon-␣, riboflavin, tannic acid, quercetin, apigenin, luteolin, catechin, quercetrin, genistin, hesperidin, rutin and sulforhodamine b (srb) were purchased from sigma-aldrich (st. louis, mo, usa). all chemicals were a reagent grade. vero (african green monkey kidney cell line; atcc ccr- ), st (pig testis cell line; atcc crl- ), tgev (transmissible gastroenteritis virus; atcc vr- ) and prcv (porcine respiratory coronavirus; atcc vr- ) were kindly provided by atcc (american type culture collection, manassas, va, usa). pedv cv (porcine epidemic diarrhea virus) was obtained from national veterinary research & quarantine service in korea. vero and st cell lines were maintained in minimal essential medium (mem) supplemented with % fetal bovine serum (fbs) and . % antibiotic-antimycotic. antibiotic-antimycotic, trypsin-edta, fbs and mem were supplied by gibco brl (grand island, ny, usa). the tissue culture plates were purchased from falcon (bd biosciences, nj, usa). the aerial parts of h. cordata were collected at gyeongsangnamdo agricultural research & extension services, korea, in august . the dried and ground materials ( kg) were extracted three times with meoh ( × l) at room temperatures for days, and this extract was performed to srb-based cytotoxicity and antiviral activity assay ( table ). the meoh extract ( g; ic , . g/ml; cc , . g/ml) was separated into two fractions using etoac as the non-aqueous phase. the concentrated etoac fraction ( . g; ic , . g/ml; cc , . g/ml) was loaded on a silica gel (merck, - mesh, g) column, and eluted with a step-gradient of chcl :meoh ( : , . : . , : , . : . , . : . , : , : and : ; each l) to afford eight fractions. the active fraction (chcl :meoh, : fraction; . g; ic , < g/ml; cc , > g/ml) was separated using sephadex lh- (amersham pharmacia biotech ab, sweden, - mesh, g), eluting successively with % methanol/water (v/v = / ; each . l), and fractions were obtained. for further purification, the active fraction (combination of fractions from to , . g; ic , < g/ml; cc , > g/ml) was subjected to analytical hplc (ymc-pack ods-ma; i.d. mm × mm; eluent, % aq. acetonitrile; flow rate, . ml/min; uv, nm), and gave the pure compounds ( mg; ic , . g/ml; cc , > g/ml). based on electron spray ionization-mass spectrometry, h nmr and c nmr spectral data, the purified compound was verified as quercetin -rhamnoside (awaad et al., ) . one day before infection, vero cells were seeded onto a well culture plate at a concentration of × cells per well. next day, medium was removed and then washed with × phosphate buffered saline (pbs). infectivity of virus stock was determined by the srb method using cytopathic effect (cpe) reduction and was determined as infectivity of the virus by srb id ( % infective dose). following this, . ml of diluted virus suspension of pedv containing ccid ( % cell culture infective dose) of the virus stock to produce a appropriate cytopathic effects within days after infection and . ml of medium supplemented with typsin-edta containing an appropriate concentration of the compounds were added. the antiviral activity of each test material was determined with a -fold diluted concentration ranging from . to g/ml. three wells were used as virus controls (virus-infected non-drug-treated cells) while three wells were used as cell controls (non-infected non-drug-treated cells). the culture plates were incubated at • c in % co for days. after washing times with × pbs, l of cold (− • c) % acetone were added to each well and left for min at − • c. % acetone was removed and -well plates were left at dry oven for min. l of . % (w/v) srb in % acetic acid solution were added to each well and left at room temperature for min. unbound srb was removed and the plates were washed times with % acetic acid before oven drying and were then left in a dry oven for day. bound srb was solubilized with l of mm unbuffered tris-base solution and plates were left on a table for min. the absorbance was read at nm by using a versamax microplate reader (molecular devices, palo alto, ca, usa) with a reference absorbance at nm. to calculate the ic values, the results were transformed to percentage of controls and the ic values were graphically obtained from the dose-response curves. the percent protection achieved by the test compound in pedv-infected cells was calculated by the following formula: where (od t ) pedv is the optical density measured with a given concentration of the test compound in pedv-infected cells; (od c ) pedv is the optical density measured for the control untreated pedvinfected cells; and (od c ) mock is the optical density measured for control untreated mock-infected cells. the concentration achieving % protection according to the formula above was defined as the % inhibitory concentration (ic ). the therapeutic index was defined as cc /ic . to test the effect of q r on the infectivity of pedv particles, pedv was pre-incubated with q r of g/ml for h at • c and vero cells were infected with pretreated or untreated pedv for h at • c. unbound virus was removed and washed times with pbs, ant then cells were incubated in infection medium supplemented with or without q r of g/ml at • c. antiviral activity was determined by srb assay after days. the time-of-addition effect of q r was examined according to previously described procedures with minor modifications (chiang et al., a,b) . vero cells were seeded onto -well culture plates at density of × cells per well and incubated for day. after washing with × pbs, each g/ml of the q r and ribavirin was then added onto the cells at either before (− h), during ( h) or after ( , , , , , and h) the period of pedv infection. after days, antiviral activity was carried out as above described. ribavirin was used as positive control. the cytotoxicity was evaluated by modified the srb assay previously described (lin et al., ) . vero cells were seeded onto a -well culture plate at a concentration of × cells per well. next day, medium was removed and the -well plates were replaced with media containing the serially diluted compounds and the cells were further incubated for h. the culture medium was removed and washed with × pbs. the next step was conducted by antiviral activity assay obove described. to calculate the cc values, the results were transformed to percentage of controls and the cc values were graphically obtained from the dose-response curves. vero cells were seeded onto a -well culture plate at a concentration of × cells per well. the next day, medium was removed and the cells were washed with × pbs. then, . ml of diluted virus suspension and . ml of medium supplemented with typsin-edta containing q r or ribavirin of g/ml were added. after incubation at • c in % co for or h, the next step was performed. total rna was extracted from the cultured cells by the acid guanidinium thiocyanatephenol-chloroform extraction method (chomczynski and mackey, ) . total rna was dissolved in . % diethylpyrocarbonate (sigma)treated water. the amount of rna was determined by measuring spectrometric absorbance at nm. to check purity of the s ribosomal rna, rna of g/ml were loaded on a . % rna gel in umops buffer, and the s ribosomal rna bands were compared. first-strand cdna was synthesized from total rna in an rnasefree condition. the reaction was performed with g of total rna using a prostar first-strand rt-pcr kit (stratagene, la jolla, ca, usa), according to the manufacturer's instructions. pcr was performed in a geneamp pcr system (perkinelmer/cetus, norwalk, ct, usa) using the first-strand cdna and taq polymerase (takara shuzo, kyoto, japan) as follows for each primer: membrane (m) gene of pedv, -cggttctattcccgttgatg- and -ccacaaccgaatgctattga- ; ␤-actin, -gccatgtacg-ttgctatccaggctg- and -agccgtggccatctcttgctcgaag- . pcr-amplified products were separated on . % agarose gels containing . g/ml ethidium bromide and visualized under uv light. current antiviral drugs, natural compounds and flavonoids were further studied for their inhibitory effects on replication of the pedv and cytotoxicity in vero cells, among which ribavirin, tannic acid, coumarin and interferon-␣ exhibited the activities with ic of . , . , g/ml and . unit, respectively. their cc were . , . , . g/ml and > unit, and the therapeutic indices were . , . , . and > . , respectively. quercetin, apigenin, luteolin and catechin showed anti-pedv activity with ic of less than g/ml and cc of . , > , . and > g/ml, respectively. furthermore, q r showed the highest ti (> . ) for pedv tested with ic of . g/ml and cc of > g/ml (table ) . on the basis of the above results, the relationship between flavonoid structure and antiviral activity against pedv was further evaluated. among the flavonoids tested, flavones (i.e. apigenin and luteolin), flavonol (i.e. q r) and flavan (i.e. catechin), which all have o-dihydroxy functional groups at c- and c- , showed significant anti-pedv activity. in addition, flavonols q r possessed one rhamnoside at position with a , -dihydroxyl group was also found to have an antiviral activity than higher that of the , dihydroxylated flavonoids. but, the anti-pedv activity of quercetin with five hydroxyl groups decreased (ic value of less than %). this indicates that sugars groups at c- of a-ring are an important feature for the anti-pedv activity of flavonoids. to observe the effects of q r on the infectivity of pedv particles, this exam was performed. antiviral activity of pre-incubation with q r and ribavirin resulted in . %, . %, respectively. in contrast, continuous presence of q r during infection led to a significant increase in antiviral activity. a similar result was obtained in control infections with treated ribavirin (fig. ), but antiviral activity was shown to be lower than that of q r. this indicates that q r does not interact with the particles of pedv, as pre-exposure of the virus to q r did not alter the infectivity of pedv particles. the q r was added at different periods (before, during, and after) of pedv infection. results showed that the q r suppressed pedv infection, when added just after the virus inoculation ( h) results are presented as the mean ic values obtained from three independent experiments carried out in triplicate ± s.d. a concentration required to inhibit virus-induced cpe by % (g/ml). b concentration required to reduce cell growth by % (g/ml). c therapeutic index = cc /ic . fig. . the effects of q r on the infectivity of pedv particles. pedv particles were incubated with q r of g/ml for h at • c. afterwards, vero cells were incubated with q r-treated or untreated virus for h at • c. unbound virus was removed by extensive washing and infection was continued by cultivating cells in infection medium with or without q r of g/ml at • c. antiviral activity was determined by titration using srb assays days post-infection. preinc, pre-incubation was expressed incubation without q r of g/ml after washing; inc., incubation was expressed incubation with q r of g/ml after washing. and also early after the virus inoculation ( , and h). ribavirin also showed a weak effect on pedv infection. the inhibitory either amount of level of q r was higher than % ( fig. ) but that of ribavirin was lower than %. however, the inhibitory rate of two compounds declined to % or less when added at either prior (− h) or post ( , , and h) infection. this observation indicated that q r affects the initial stage of pedv infection. further evidence of the inhibitory effects of q r on infection pedv and viral replication in vero cells was provided by pcr analysis. the rna extraction was performed at and h after pedv infection. q r ( g/ml) decreased the intensity of the product fig. . rt-pcr analysis. replication of pedv from vero cells before and at and h after infection by pedv in the presence of q r ( g/ml) or ribavirin ( g/ml) or vehicle alone (control, . % dmso), as detected by rt-pcr. band of pedv at and h after infection, but ribavirin ( g/ml) did not decrease the intensity of the product band of pedv at h after infection (fig. ) . the two viruses selected for these experiments included tgev and prcv in st cells. when the antiviral activity and cytotoxicity of q r or ribavirin was examined by srb assay, the two compounds did not exhibit any cytotoxicity at the highest concentrations tested ( table ). the ic value of q r and ribavirin in tgev was . and . g/ml, respectively, while that in prcv was . and . g/ml, respectively. furthermore, ti value was no great different between the two viruses. several veterinary coronavirus vaccines are currently available, but their efficacy is variable. the vaccine for prevention of infectious bronchitis virus (ibv), which infects chickens, is effective (ladman et al., ) , while the canine and porcine vaccines are only partially effective (pratelli et al., ) . ribavirin has been used as treat against various dna and rna virus infections, although virusacquired resistance to it was isolated from various virus populations and observed in some patients (jason and craig, ) . antiviral studies have shown that essential oil prepared from fresh plants of h. cordata has an inhibitory effect on herpes simplex virus type (hsv- ), influenza virus and human immunodeficiency virus type (hiv- ), without showing cytotoxicity (lu et al., ) . quercetin has been reported to have inhibitory effects on several viruses (mucsi and pragai, ) . these studies indicate that h. cordata possesses compound exhibited antiviral effects. in this study, q r is an effective antiviral compound against pedv. q r did not have a marked inhibitory effect on the growth of tgev or prcvinfected cells. the antiviral activity of this compound appeared to be strongly influenced by the strain of the coronaviruse tested. ribavirin, which is a broad-spectrum nucleoside analogue, exhibited results are presented as the mean ic values obtained from three independent experiments carried out in triplicate ± s.d. a concentration required to inhibit virus-induced cpe by % (g/ml). b concentration required to reduce cell growth by % (g/ml). c therapeutic index = cc / ic . expected antiviral activity. this broad-spectrum antiviral drug was previously shown to be inhibitory to dna and rna viruses in cell cultures (sidwell et al., ) . trials of ribavirin in this study showed that the drug had favorable effects on antiviral activity in vero cells infected with pedv. in tgev or prcv, ribavirin had a weaker influence on survival of in vero cells after infection with tgev or prcv. we were however able to ascertain that ribivirin does possess some antiviral activities. to elucidate the action of q r on pedv multiplication in more detail, we investigated the effect of q r on single steps during the infection cycle of pedv. as a result, it can be concluded that q r does not interact with the particles of pedv, as pre-exposure of the virus to q r did not alter the infectivity of pedv particles (fig. ) . based on the results of the time-course study, pre-incubation of the vero cells with q r did not protect the cells from pedv infection (fig. ) . furthermore, q r can inhibit pedv infection only when it was added on, during, and early stages after the virus inoculation ( , , h), but not after h or later (fig. ) . this suggests that the mode of action is not deriven from inhibiting the absorption of virus but results from inhibition at an early stage of viral replication after infection ( figs. and ). among the flavonoids tested, only q r possessed significant activity against pedv. in a previous report, rutin (quercetin- rutinoside) did not express antiviral activity whereas quercitrin (quercetin -rhamnoside) possessed similar activity to quercetin against human herpesviruses and adenoviruses (chiang et al., a,b) . they found that the antiviral activity among the flavonoid glycosides containing the quercetin moiety might be correlated with the species of sugar group at the position. our results suggest that the antiviral activity of flavonoids against pedv is correlated with the species of sugar group at the position. in conclusion, the present study described that q r possesses strong anti-pedv activity among the flavonoids. it will be interesting to further investigate the antiviral activity of the q r in preventing various pedv-mediated injuries in in vivo pathological situations. further studies will be required to explore the detailed antiviral mechanism of q r. hepatoprotective activity of schouwia thebica web medicinal plants and phytomedicines linking plant biochemistry and physiology to human health in vitro antiviral activities of caesalpinia pulcherrima and its related flavonoids in vitro cytotoxic, antiviral and immunomodulatory effects of plantago major and plantago asiatica substitution of chloroform by bomochloropropane in the single-step method of rna isolation plant products as antimicrobial agents experimental infection of pigs with a new porcine enteric coronavirus cv study of the occurrence of epizootic viral diarrhea in swine in switzerland mechanisms of action of ribavirin against distinct viruses novel antiviral agents: a medicinal plant perspective herba houttuyniae extract induces apoptotic death of human promyelocytic leukemia cells via caspase activation accompanied by dissipation of mitochondrial membrane potential and cytochrome c release protection of chickens after live and inactivated virus vaccination against challenge with nephropathologenic infectious bronchitis virus pa/wolgemuth/ inhibitory effects of houttuynia cordata water extracts on anaphylactic reaction and mast cell activation sulforhodamine b assay for measuring proliferation of a pigmented melanocyte cell line and its application to the evolution of crude drugs used in the treatment of vitiligo anti-inflammatory effect of houttuynia cordata injection inhibition of virus multiplication and alteration of cyclic amp level in cell cultures by flavonoids safety and efficacy of a modified-live canine coronavirus vaccine in dogs broad-spectrum antiviral activity of virazole: -beta-d-ribofuranosyl- , , -triazole- -carboxamide can ethnopharmacology contribute to the development of antiviral drugs review of antiviral and immunomodulating properties of plants of the peruvian rainforest with a particular emphasis on una de gato and sangre de grado this work was supported by a grant from research institutes of bioscience & biotechnology (kribb), republic of korea. this research was also partially supported by a grant from biogreen program rural development administration, by the ministry for food, agriculture, foresty and fisheries, republic of korea. key: cord- -nkv h uh authors: matyushenko, victoria; kotomina, tatiana; kudryavtsev, igor; mezhenskaya, daria; prokopenko, polina; matushkina, anastasia; sivak, konstantin; muzhikyan, arman; rudenko, larisa; isakova-sivak, irina title: conserved t-cell epitopes of respiratory syncytial virus (rsv) delivered by recombinant live attenuated influenza vaccine viruses efficiently induce rsv-specific lung-localized memory t cells and augment influenza-specific resident memory t-cell responses date: - - journal: antiviral res doi: . /j.antiviral. . sha: doc_id: cord_uid: nkv h uh respiratory syncytial virus (rsv) can cause recurrent infection in people because it does not stimulate a long-lived immunological memory. there is an urgent need to develop a safe and efficacious vaccine against rsv that would induce immunological memory without causing immunopathology following natural rsv infection. we have previously generated two recombinant live attenuated influenza vaccine (laiv) viruses that encode immunodominant t-cell epitopes of rsv m protein in the neuraminidase or ns genes. these chimeric vaccines afforded protection against influenza and rsv infection in mice, without causing pulmonary eosinophilia or inflammatory rsv disease. the current study assessed the formation of influenza-specific and rsv-specific cd and cd t-cell responses in the lungs of mice, with special attention to the lung tissue-resident memory t cell subsets (t(rm)). the rsv epitopes did not affect influenza-specific cd effector memory t cell (tem) levels in the lungs. the majority of these cells formed by laiv or laiv-rsv viruses had cd (+)cd (-) phenotype. both laiv+na/rsv and laiv+ns/rsv recombinant viruses induced significant levels of rsv m ( ) epitope-specific lung-localized cd tem cells expressing both cd and cd t(rm) markers. surprisingly, the cd (+)cd (+) influenza-specific cd tem responses were augmented by the addition of rsv epitopes, possibly as a result of the local microenvironment formed by the rsv-specific memory t cells differentiating to t(rm) in the lungs of mice immunized with laiv-rsv chimeric viruses. this study provides evidence that laiv vector-based vaccination can induce robust lung-localized t-cell immunity to the inserted t-cell epitope of a foreign pathogen, without altering the immunogenicity of the viral vector itself. while there are currently a wide variety of vaccines against influenza a and b viruses, no vaccine against other respiratory infections has yet been licensed, despite attempts over many years to develop such vaccines (tannock et al., ) . in particular, there is an urgent need to develop a safe and efficacious vaccine against respiratory syncytial virus (rsv). rsv infection causes lower respiratory disease in infants and young children, with an estimated annual global burden of . million episodes of acute lower respiratory tract infection and over , deaths (shi et al., ) . the development of an rsv vaccine suffered a major setback in the s, when an alum-adjuvanted formalin-inactivated rsv (fi-rsv) vaccine resulted in enhanced respiratory disease (erd) in infants and toddlers after subsequent exposure to natural rsv infection (kim et al., ) . development of an rsv vaccine is more problematic than an influenza vaccine because of striking differences in the virology and host response to these two pathogens. influenza infection induces mucosal iga and systemic igg antibodies, as well as broadly reactive t-cell responses, which help to protect against re-infection by neutralizing the virus, or ameliorate the disease by killing infected cells. rsv, in contrast, can re-infect an individual even in the presence of high levels of virus-specific neutralizing antibodies (hall et al., ) . this is because rsv can modulate the host immune system, impairing b-cell memory and reducing t-cell generation and functionality (reviewed in (ascough et al., ) ). fi-rsv vaccination has been shown to induce t helper type (th )-biased cd t cells with low levels of cytotoxic cd t cells and less protective antibodies, causing erd in animal models after rsv challenge, accompanied by high pulmonary eosinophilic infiltrates graham et al., ; knudson et al., ) . the ability of rsv m -specific cd t cells to protect against a secondary infection and inhibit severe pulmonary eosinophilia by reducing the number of th cd t cells in the lungs after rsv challenge (graham et al., ; olson and varga, ; srikiatkhachorn and braciale, ) provided the rationale for designing a new class of cd t-cell-based rsv vaccines. however, excessive generation of rsv m epitopespecific systemic memory cd t cells can result in exacerbated morbidity and mortality following rsv infection, indicating that caution is needed in designing t-cell-based rsv vaccines (schmidt et al., ) . importantly, schmidt et al. demonstrated that a booster immunization with m rsv epitope, delivered directly to the lungs by a recombinant influenza virus expressing this epitope, generated rsv-specific lung-localized memory cd t cells, which were associated with reduced immunopathology following rsv challenge (schmidt et al., ) . these data suggest that the site of administration of t-cell-based rsv vaccines is crucial. for example, these epitopes can be delivered to the respiratory tract by live attenuated influenza vaccine (laiv) viruses (isakova-sivak et al., a) , which are known to be effective inducers of cross-reactive t-cell responses (isakova-sivak et al., ; korenkov et al., ; mohn et al., ) . in addition, such bivalent laiv-rsv vaccines could be used for simultaneous prophylaxis of the two respiratory pathogens. we have previously generated two prototype laiv-rsv viral vectored vaccines carrying several conserved t-cell epitopes of rsv inserted into the neuraminidase (na) or ns gene of h n laiv virus. these vaccines induced functional cytotoxic t-cell immune responses, targeted at both influenza virus and the rsv m epitope (isakova-sivak et al., b; kotomina et al., ) . subsequently, we demonstrated that these recombinant viruses were able to protect vaccinated mice against virulent influenza virus and, most importantly, rsv, without causing inflammatory disease . in the latter experiment, rsv m epitope-specific t-cell recall responses were measured in lungs and bronchoalveolar lavage (bal) five days after rsv challenge; levels in the laiv-rsv groups were significantly higher than those in groups given the laiv vector or fi-rsv . the precise mechanisms of immune protection afforded by such laiv-vectored rsv vaccines still need to be established. of particular interest is whether laiv-delivered rsv epitopes can induce memory t-cell responses that persist at the site of infection of the target pathogen. for the majority of respiratory pathogens, the establishment of lung tissue-resident memory cd t cells (t rm ) can provide a first line of adaptive cellular defense on exposure to the same or similar pathogen, because cd t cells are targeted primarily to the relatively conserved viral proteins (reviewed in (takamura, ; topham and reilly, ) ). a recent study found that laiv, but not inactivated influenza vaccine (iiv), can efficiently promote robust long-lived influenzaspecific t rm responses that provide protection against heterologous influenza virus challenge, independent of circulating t cells and neutralizing antibodies (zens et al., ) . the current study was undertaken to obtain a deeper understanding of how influenza-specific and rsvspecific memory t-cell immunity is established in the lungs after intranasal (i.n.) immunization with the recombinant laiv-rsv viruses, and in particular whether the insertion of an immunodominant foreign cd t-cell epitope can affect anti-influenza t-cell immunity in the lungs. hep- cells (atcc ccl- ) were maintained in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs) (capricorn, germany) and antibiotic-antimycotic solution (aa) (thermo fisher scientific, usa). recombinant h n laiv-rsv viruses were previously generated by means of reverse genetics, on a/leningrad/ / / backbone (kotomina et al., (saint petersburg, russia) and propagated in hep- , as described previously . to prepare the fi-rsv vaccine, formalin was added to the rsv-containing supernatant to a concentration of : , incubated for h at °c and centrifuged at g and °c for one hour. the pellet was suspended in dulbecco's phosphate-buffered saline (pbs), and stored in aliquots at - °c. the rsv titer was determined by plaque assay in -well plates seeded with hep- cells. serially diluted rsv was inoculated onto the cell monolayer, and incubated for hours at °c. the cells were then covered with an overlay containing dmem and . % agarose (thermo, usa). after days' incubation, the cells were fixed in % formaldehyde and the immune plaques were developed using primary anti-rsv f monoclonal antibody (mab , emd millipore corp., usa), secondary horseradish peroxidase (hrp)-conjugated goat anti-mouse igg antibody (southern biotech, usa) and , 'diaminobenzidine (dab) substrate (thermo scientific, usa). the rsv titer was expressed in plaque-forming units (pfu) per ml. rsv strain a matrix protein peptide m - (syigsinni) was chemically synthesized by almabion ltd (russia) with a purity of more than %, as measured by high-performance liquid chromatography. the peptide was reconstituted in dimethyl sulfoxide at a concentration of mm and stored at - °c in single-use aliquots. immunization procedures, as well as influenza virus and rsv challenge were performed as previously described . briefly, groups of mice were given i.n. immunization with either h n laiv or one of the laiv-rsv vaccines [laiv+na/rsv and laiv+ns/rsv], at a dose of eid in a volume of µl, twice at a three-week interval. a control group received two i.n. doses of pbs. there was an additional vaccine group (fi-rsv, n= ), in which mice were given two -μl intramuscular injections of µg of formalininactivated purified rsv with alumvax hydroxide adjuvant formulation ( µg) (ozbiosciences, france) at a two-week interval. three weeks after the second immunization five mice from each group were infected intranasally with × pfu of rsv a . they were euthanized on day after rsv infection and lungs were collected for virological and histopathological studies. lung rsv titers were determined as described by and expressed as pfu per gram of lung tissue. on day after the second immunization, spleens were collected from five mice and single splenocytes were isolated in conditioned media (rpmi- , capricorn scientific, germany) with aa solution (thermo fisher scientific, usa), mm hepes (gibco, usa) and μm -mercaptoethanol (sigma, usa), using -µm cell strainers (bd biosciences, usa). red blood cells were then lysed with ammonium-chloride-potassium lysing buffer. for intracellular cytokine staining (ics), × cells were plated into u-bottom well microplates in µl of conditioned media; µl of sucrose-purified influenza virus was added for laivstimulation to a final multiplicity of infection (moi) of . . samples for non-peptide and peptide stimulation received µl of conditioned media and were placed in a co -incubator for one hour, after which µl of conditioned media was added with % fbs, to give a final fbs concentration of %. after - hours incubation in a co -incubator, µl of conditioned media with golgiplug™ solution (bd biosciences, usa) was added to a final dilution of : ; μm of rsv m peptide was added to the peptide stimulation group, and incubated for an additional hours. samples were then stained for minutes at °c in the dark with live/dead fixable stain (zombieaqua, invitrogen) and surface antibody-conjugates anti-cd (rm - ) and anti-cd l (mel- ) (from biolegend, usa), and anti-cd ( - . ) and anti-cd (im ) (from thermo). a cytofix/cytoperm kit (bd biosciences, usa) was used for fixation/permeabilization, then samples were stained with intracellular cytokine antibody anti-ifnγ (xmg . ) and anti-tnfα (mp -xt ) (from biolegend) for minutes at °c in the dark. samples were fixed with % paraformaldehyde and analyzed by navios flow cytometer (beckman coulter). positive controls with phorbol myristate acetate (pma) stimulation (sigma), non-stimulated controls and isotype controls were also prepared. the number of ifnγ-and tnfα-positive cells in stimulated groups were counted, and the spontaneous cytokine secretion level of non-stimulated controls subtracted. gating strategy for measuring ifnγ-and/or tnfαsecreting cd tem cells in mouse spleens is shown in fig.s . on day after the second immunization, the lungs of five mice from each group were perfused with ml of pbs through the right ventricle. the perfused lungs were cut into small pieces and homogenized using -mm beads in tissuelyser lt (qiagen, germany), then digested with collagenase (sigma, usa) and dnase i (sigma, usa) for min at °c. the cells were isolated using -µm cell strainers (bd biosciences, usa), and red blood cells were lysed by ammonium-chloride-potassium lysing buffer. to determine the numbers of live and dead cells, staining with zombieaqua (biolegend, usa) was used for minutes at room temperature in the dark. lung lymphocytes were phenotyped by staining with surface fluorescent antibodies anti-cd (rm - ), anti-cd l (mel- ) and anti-cd ( e ) (from biolegend, usa), anti-cd (h . f ), anti-cd ( - . ) and anti-cd (im ) (from thermo, usa) for minutes at room temperature in the dark. samples were fixed by % paraformaldehyde and analyzed with a navios flow cytometer (beckman coulter). gating strategy for phenotyping ifnγ-secreting cd and cd t cells in the lungs is shown in fig.s . for ics, × lungderived cells were used with the protocol described above, with additional staining with surface antibody-conjugates anti-cd ( e ) (biolegend) and anti-cd (h . f ) (thermo). for lung ics, only intracellular cytokine antibody anti-ifnγ (xmg . ) was used. histopathological changes in the lungs were analyzed in five mice from each test group on day after rsv challenge. we used the method described by (albert et al., ) to detect eosinophils in tissues. briefly, -μm paraffin-embedded lung sections were rehydrated, stained for one hour in a % ethanolic solution of congo red (sigma), counterstained in karazzi's haematoxylin for min, and incubated for min with alcian blue gx stain at ph . to detect mucus in the bronchial lumen. the degree of rsv-induced pathology was evaluated as described by (matsuse et al., ) . briefly, the lung sections were reviewed and evaluated for epithelial damage, peribronchovascular cell infiltrate, and interstitial-alveolar cell infiltrate. epithelial damage was scored as: (no damage); (increased cytoplasm of epithelial cells without desquamation); (epithelial desquamation without bronchial exudate composed of inflammatory cells); or (bronchial exudate composed of desquamate epithelial cells and inflammatory cells). peribronchovascular cell infiltrate was scored as: (no infiltrate); (infiltrate up to cells); (infiltrate - cells); or (infiltrate > cells). interstitial-alveolar cell infiltrate was scored as: (no infiltrate); (mild, generalized increase in cell mass of the alveolar septa without thickening of the septa or significant air space consolidation); (dense septal infiltrate with thickening of septa); or (significant alveolar consolidation in addition to interstitial inflammation). eosinophils were counted in - perivascular areas of each section. data were analysed with the graphpad prism . software (graphpad software inc). the statistical significance of virological and immunological outcomes was determined by oneway or two-way anova followed by holm-sidak's multiple comparisons test. p values less than . were considered statistically significant. consistent with our previous report , the vaccine viruses the two laiv-rsv vaccines induced high levels of m -specific ifnγ-secreting cd tem cells (fig. ) , reflecting our earlier findings that these vaccines induce systemic cd t-cell responses (kotomina et al., ) . it is noteworthy that the majority of the ifnγ-secreting cd tem cells were also positive for tnfα cytokine (fig. ) . these data are in accordance with the tcell recall responses seen in bal and lung tissues of laiv-rsv-immunized mice after rsv challenge . interestingly, the laiv+na/rsv recombinant virus induced significantly higher levels of cytokine-secreting cd t cells specific to the rsv epitope than the laiv+ns/rsv candidate, suggesting that the replication capacity of the recombinant virus has an impact on the vaccine's immunogenicity. the fi-rsv vaccine was unable to induce systemic cd t-cell responses to the rsv m epitope, which is in line with other studies (krause et al., ; olson and varga, ) (fig. ) . since the laiv-rsv vaccine candidates are delivered intranasally, i.e. at the site of entry of influenza virus and rsv, it was important to ascertain the magnitude of the induced t-cell responses directly in the lungs. no significant differences were observed in the numbers of cd and cd cells in the lungs of immunized and control mice on day after the second vaccine dose ( fig. a) . however there were significant differences in the relative proportions of these t cells. the cd to cd ratio was significantly higher in the laiv-rsv vaccine groups than in the control group, suggesting that robust cd lung-localized t-cell responses were induced by both recombinant viruses (fig. b) . the cd response in the laiv+na/rsv group was even more pronounced, showing a significant difference with the cd /cd ratio in both the laiv and fi-rsv groups. cd , a mucosal lymphocyte surface marker integrin αeβ , and cd have been widely accepted as the common markers of tissue-resident cells in mucosal and some nonmucosal tissues (liu et al., ) . seven days after the second immunization, the levels of effector memory cd t cells expressing cd early activation marker were not significantly higher in the lungs of immunized mice than in those of the control group (fig. b) . however, significant levels of cd tem cells, expressing both cd and cd markers associated with tissue residence, were identified in the lungs of mice immunized with either of the laiv-rsv vaccine candidates (fig. b ). this is somewhat surprising because cd is usually expressed only by some subsets of cd t rm (mueller and mackay, ) . nevertheless, several studies have identified distinct populations of cd t cells that carry typical t rm markers in both mouse and human lungs, with the cd t rm cells usually carrying less cd or expressing cd a instead of cd (reviewed in (liu et al., ) ). in this study, the cd + cd + cd tem cells were induced only by the recombinant viruses carrying rsv epitopes, and not by laiv alone, which suggests that the rsv cassette is involved in the formation of cd t rm cells after prime-boost immunization. similarly, the proportion of cd tem cells possessing either cd or both cd and cd surface markers in the lungs was significantly increased by immunization with a recombinant laiv-rsv virus (fig. ) . although laiv provoked the development of cd t cells with dual t rm phenotype in the lungs, the proportion of these cells among all cd +cd l-effector memory cd t cells was relatively low and substantially lower than in the laiv-rsv groups. our data suggest that the inserted cd rsv epitope is immunodominant, and capable of generating resident memory cd t cells in the lungs after local delivery to the mucosal sites. this is in agreement with other studies involving intranasal delivery of rsv m epitope using different viral vectors (morabito et al., ; schmidt et al., ) . as expected, the laiv and both laiv-rsv vaccines induced robust influenza virusspecific cd and cd t-cell responses in the lungs, with similar levels of ifnγ-positive cd t cells (fig. ) . influenza-specific cd t-cell responses in the lungs were more pronounced in mice immunized with laiv than in the two laiv-rsv vaccine groups. this suggests that the insertion of the rsv epitopes in the laiv virus can change the distribution of cd t-cell specificity in the lungs after intranasal immunization (fig. ) . indeed, high levels of rsv m specific ifnγ-secreting cd t cells were observed in the lungs of mice immunized with laiv-rsv, confirming that the rsv peptide is highly immunogenic and efficiently establishes rsvspecific effector memory in the lungs (fig. ) . the rsv-specific cd t-cell responses were more robust in the laiv+na/rsv group than in the laiv+ns/rsv group, which is in line with the systemic cd responses measured in spleens (fig. ) . since influenza-specific cd tcell responses are identical in these vaccine groups (fig. ) , the differences in the expression of the rsv cassette by these two vaccine viruses could be the reason for diverse rsv-specific responses. the vast majority of influenza virus-specific cd t cells had cd + cd phenotype, which is in line with previous findings that the cd t rm cells usually express cd a instead of rsv-vaccinated mice expressed cd marker, the second canonical t rm marker cd was present at much higher frequencies in the laiv-rsv groups than in the laiv group (fig. a) . given that the proportion of the ifnγ-secreting cd tem cells in the lungs of immunized mice was significantly higher in the laiv group than the laiv-rsv groups, it can be speculated that the rsv insert had a significant impact on influenza-specific cd t rm differentiation or maintenance. this suggestion is supported by the evidence that there were more influenzaspecific cd t rm in the laiv+na/rsv group than in the laiv+ns/rsv group, and the namodified recombinant virus had higher rsv-specific cd responses than the ns-modified laiv-rsv candidate, both in spleen (fig. ) and in the lungs (fig. ) . nevertheless, the rsv m epitope-specific cd t rm levels were similar in the two laiv-rsv groups (fig. b) . importantly, only - % of lung-localized rsv-specific cd tem cells possessed the cd marker, whereas almost all influenza-specific cd tem were cd -positive (fig. a) . after challenge with live rsv a strain, the infectious virus was detected at significantly lower levels in both laiv-rsv groups, compared with the pbs and laiv only groups lived immunological memory. in particular, it has been shown that rsv infection leads to suboptimal activation of dendritic cells, which subsequently leads to impaired immunological synapse formation, causing insufficient activation of naïve lymphocytes, failure of differentiation, and absence of long-lived t-cell memory in the tissue (gonzalez et al., ) . in kotomina et al., ) . in accordance with other studies, truncation of ns protein in the laiv+ns/rsv candidate led to the inability to cause a productive infection in the respiratory tract, without affecting the immunogenicity against influenza (ferko et al., ; talon et al., ; zhou et al., ) . for viruses like rsv, it is important to establish the precise mechanisms of the development and maintenance of virus-specific immunity, especially in light of the severe immunopathology caused by the excessive generation of rsv m epitope-specific systemic memory cd t cells after natural rsv infection (schmidt et al., ) . our previous studies demonstrated that our laiv-rsv vaccine candidates generate systemic rsv m -specific cd t cells and a robust recall response in the bal and lungs of immunized mice following rsv challenge, which accelerated viral clearance without exacerbation of disease kotomina et al., ) . however, we have not previously measured the magnitude of different memory t-cell subsets, such as effector memory and tissue-resident memory, which have rsv-or laiv-specificity. since the vaccines being developed are assumed to be effective against both influenza and rsv, it is important to understand the balance between t-cell responses to both pathogens after vaccination. the current study found that both vectored laiv-rsv vaccine candidates generated significant levels of both ifnγ-and tnfα-secreted rsv m -specific cd tem cells, most of which were double cytokine producers. this is in accordance with the recall response data from the previous study . importantly, in contrast to the study by (schmidt et al., ) , these functional systemic rsv-specific double cytokine-producing cd memory t cells did not cause immunopathology following rsv infection. a number of recent experimental studies reported the establishment of a specialized subset of memory t cells that remain in the tissue and do not re-enter the circulation following the resolution of infection (reviewed by (nolz, ; takamura, ) ). these resident memory t cells can quickly respond to the invading pathogen, either by secreting cytokines and chemokines to recruit immune cells to the infected tissue (mcmaster et al., ) or by rapid proliferation and killing of the infected cells (schenkel and masopust, ) , thus providing local protection immediately after infection. such t rm in the lungs are commonly identified by the expression of cd , an early activation marker, and the αe integrin (cd ) chain of αeβ which binds e-cadherin and helps to retain t rm specifically in tissues (cepek et al., ) . a recent study found that immunization with laiv could efficiently induce lung-localized t rm (zens et al., ) . we therefore assessed the levels of tem cells (cd + cd l -) with surface markers t rm cd and cd in the lungs of mice immunized with laiv, laiv-rsv candidates and fi-rsv. phenotyping of lung lymphocytes showed that both the vector itself and the vectored vaccines induced cd t rm with the double phenotype cd + cd + ; however, the vectored vaccines generated a significantly higher level of t rm than laiv. unexpectedly, we found that the vectored vaccines also stimulated the formation of cd tem cells with cd + cd + phenotype. this can be explained by the inclusion in the rsv construct of the cd epitope of the m - rsv protein amino acids to , which is specific for th and stimulates the production of interferon in balb/c mice (mcdermott et al., ) . the response to this peptide was not assessed in the current study, since our early experiments showed an absence of detectable systemic cd t-cell responses to this epitope after vaccination with the laiv-rsv vaccine candidates (unpublished data). however, in the light of the current study, it is worthwhile to estimate the level of rsv М - -specific cd t-cell responses in the lungs. the induction of such cd t rm could play a significant role in keeping a balance of rsvspecific cd /cd t-cell responses after immunization with laiv-rsv vaccine, thus preventing lung immunopathology. in addition, cd -expressing cd t cells are not usually detected in the lungs (mueller and mackay, ) ; the cd t rm are rather localized in the interstitium of the lung and express cd b as a typical marker (richter et al., ) . therefore, in future experiments, both cd and cd b markers should be used to assess rsv-specific cd t-cell responses in the lungs of mice immunized with laiv-rsv vaccine candidates. the laiv vector itself induced rather weak t rm responses, which is in contrast to a previous study that found high levels of cd -positive cd t rm in mice immunized with commercial laiv (zens et al., ) . in that study, a quadrivalent vaccine formulation was used, including two influenza b viruses (flumist) at a dose of . - . ffu per strain, whereas in our study all vaccines were administered as a single strain at a dose of . eid . the antigen load could have played a role in the formation of memory t-cell responses in the lungs of immunized mice. in addition, zens et al. used c bl /j mice, which differ significantly from balb/c mice in the development of t-cell immune responses to influenza (califano et al., ; sellers et al., ) . we further estimated the levels of influenza virus-specific cd and cd t rm cells in the lungs of immunized mice, as well as rsv m epitope-specific cd t rm . specificity was determined by ifnγ secretion upon in vitro stimulation of isolated lung cells with the corresponding antigen. for maximal influenza epitope coverage we used whole virus for lymphocyte stimulation. as expected, the laiv and both laiv-rsv groups induced strong virus-specific cd and cd tem responses after vaccination. while cd t-cell responses to influenza were similar in the laiv and laiv+rsv groups, the influenza-specific cd tem cell levels were significantly higher in the laiv group than in the laiv-rsv groups. interestingly, the vast majority of influenza-specific cd t cells in the lungs possessed the cd + cd phenotype, with no differences between the laiv and laiv-rsv groups, further indicating that the cd + cd + cd memory t cells found in the laiv-rsv groups are not specific for influenza. surprisingly, - % of influenza-specific cd tem cells induced by laiv+rsv chimeric viruses were positive for both t rm markers cd and cd , whereas the corresponding proportion in the laiv group was significantly lower. these data suggest that the robust t rm response to the inserted rsv immunodominant epitope could have boosted the influenza-specific t rm responses as well. indeed, the recombinant vaccines induced high levels of rsv-specific cd tem, about % of which carried cd , while at last half had the "dual" t rm cd + cd + phenotype. there was a clear dependence on the magnitude of induced rsv-specific and influenza-specific cd t rm responses, with higher frequencies in the laiv+na/rsv group, compared with laiv+ns/rsv group. a possible explanation for the co-stimulatory effect of the rsv epitope on t rm formation to influenza virus epitope(s) is that differentiation of effector cd + t cells into tissue-resident memory cd + t cell populations occurs in response to cytokines, such as transforming growth factor (tgf-β) and, to a lesser extent, il- and tumor necrosis factor alpha (tnfα) (mackay et al., ; nolz, ; wakim et al., ; zhang and bevan, ) . it is possible that the local microenvironment formed by the rsv-specific memory t cells in the lungs can increase influenza-specific cd t rm formation, which is relevant since all these epitopes are expressed together within an infected cell and can be simultaneously processed through the mhc-i pathway. about % of the rsv-specific lung-localized tem did not have t rm markers and were probably migratory tem cells. the major limitation of our study is that we did not use an intravascular antibody-labeling procedure to differentiate the population of t cells that reside permanently in the lungs and those that transit the tissue and re-enter the bloodstream (anderson et al., ) . however, the identification of virus-specific cd t cell subsets expressing both cd and cd markers suggest that they remain in lung tissue (liu et al., ) . it should be noted that the localization of t rm s in peripheral tissue is important and depends directly on their phenotype. thus, two "lines of defense" can be distinguished: the first is t rm cells located directly in the respiratory tract, which are the first to encounter infection. they possess cd marker (receptor for e-cadherin), which determines their location within the epithelial cells of the respiratory tract, and this is probably why the proportion of cd + cd + cd t cells is relatively low. the second line of defense against reinfection is t rm s located in the interstitium of the lungs and basal membranes of the epithelium of the respiratory tract. these possess cd a marker, the vla- receptor that binds to type i and iv collagen (takamura, ). in the current study, we observed a decrease in rsv titer at only one time point ( days post challenge), while it would be important to assess the impact of the cd t rm levels on the kinetics of rs virus clearance from the lungs of immunized mice. overall, the results of our study indicate that the designed laiv-rsv recombinant viruses carrying immunodominant t-cell epitopes of rsv are capable of inducing balanced cellmediated immune responses in the lungs that afford strong protection against both influenza and rsv without causing pathological changes in the lung tissues. the laiv vector not only delivered the rsv epitopes to the site of viral entry, but also correctly presented these epitopes to the immune system. this is the first evidence that laiv vector-based vaccination can induce robust lung-localized t-cell immunity to the inserted t-cell epitope of a foreign pathogen, without altering the immunogenicity of the viral vector itself. since the laiv-rsv vaccine is also designed for the elderly as a risk group for rsv infection, it is important to evaluate the immunogenicity of the recombinant viruses in aged mice as well. h n pandemic laiv was used in this study for modelling purposes, as this strain is known to replicate well in the upper (but not lower) respiratory tract, inducing high virusspecific neutralizing antibody titers, as well as cell-mediated immunity (stepanova et al., ) , but any other laiv strain could be used for this purpose. it is of note that the insertion of foreign epitopes in the na other than n might yield slightly different immunogenicity to the target pathogen, whereas insertion in the ns gene of laiv virus is universal and can be combined with any other ha and na genes of laiv viral vector. our results provide a rationale for generating a universal multivalent vaccine against various pathogens that naturally target the human respiratory tract, such as rsv, parainfluenza viruses, adenoviruses, coronaviruses, metapneumoviruses, and rhinoviruses. to conclude, the laiv-rsv constructs evaluated in this study were generated as a proofof-concept for detailed assessment of the establishment of cd and cd t-cell subsets in mice following intranasal immunization. the promising data obtained allow the further development of t cell-based rsv vaccines by incorporating human rsv epitopes into the laiv genome. rsv human challenge models will help to accelerate this vaccine development (habibi and chiu, ) . in particular, new data are emerging regarding rsv epitopes for cd airwayresident t cells (guvenel et al., ) and cd resident memory t cells (jozwik et al., ) with a defined immunodominance hierarchy. in addition, a combination of b-cell and t-cell rsv epitopes can be considered for rsv vaccine design (retamal-diaz et al., ) . it is desirable that the safe and immunogenic rsv vaccine should contain a b-cell epitope with neutralizing activity, a cd epitope (most likely th , so that there is no possible bias towards th and th , which are associated with asthma) and cd epitopes, both highly immunogenic and less dominant (ruckwardt et al., ) . all authors have no conflicts to disclose. the current research was supported by russian science foundation grant № - - . cd t cells in the lungs on day after the second immunization. data were analyzed with two-way anova. b. ratio of cd to cd t cells in the lungs. data were analyzed with one-way anova (* p < . , ** p < . , *** p < . ). cd and cd tem cells. data were analyzed with one-way anova (* p < . , ** p < . , *** p < . , **** p < . ). expression of cd and cd markers by influenza-specific cd and cd tem cells induced by immunization with indicated vaccine. b. expression of cd and cd markers by rsv-specific cd tem cells induced by immunization with indicated vaccine. data were analyzed by -way anova with holm-sidak's multiple comparison test (* p < . , **** p < . ). comparisons test (* p < . , ** p < . , *** p < . , **** p < . ). figure s . gating strategy for measuring ifnγ-and/or tnfα-secreting cd tem cells in mouse spleens using an ics assay. figure s . 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determining the breadth of the respiratory syncytial virus-specific t cell response airway-resident memory cd t cells provide antigen-specific protection against respiratory virus challenge through rapid ifn-gamma production boosting of cross-reactive and protection-associated t cells in children after live attenuated influenza vaccination intranasal administration of rsv antigen-expressing mcmv elicits robust tissue-resident effector and effector memory cd + t cells in the lung tissue-resident memory t cells: local specialists in immune defence molecular mechanisms of cd (+) t cell trafficking and localization. cellular and molecular life sciences : cmls cd t cells inhibit respiratory syncytial virus (rsv) vaccineenhanced disease contribution of resident memory cd (+) t cells to protective immunity against respiratory syncytial virus and their impact on vaccine design collagen distribution and expression of collagen-binding alpha beta (vla- ) and alpha beta (vla- ) integrins on cd and cd t cells during influenza infection responses against a subdominant cd + t cell epitope protect against immunopathology caused by a dominant epitope tissue-resident memory t cells memory cd t cells mediate severe immunopathology following respiratory syncytial virus infection immunological variation between inbred laboratory mouse strains: points to consider in phenotyping genetically immunomodified mice virus-specific cd + t lymphocytes downregulate t helper cell type cytokine secretion and pulmonary eosinophilia during experimental murine respiratory syncytial virus infection amino acid substitutions n d and n d in hemagglutinin molecule enhance immunigenicity of live attenuated influenza h n vaccine strain in experiment persistence in temporary lung niches: a survival strategy of lung-resident memory cd (+) t cells influenza a and b viruses expressing altered ns proteins: a vaccine approach why are vaccines against many human viral diseases still unavailable; an historic perspective tissue-resident memory cd (+) t cells: from phenotype to function antibodytargeted vaccination to lung dendritic cells generates tissue-resident memory cd t cells that are highly protective against influenza virus infection vaccine-generated lung tissue-resident memory t cells provide heterosubtypic protection to influenza infection transforming growth factor-beta signaling controls the formation and maintenance of gut-resident memory t cells by regulating migration and retention ns-based live attenuated h n pandemic vaccines protect mice and ferrets key: cord- -oxohdfpu authors: noble, christian g.; li, shi-hua; dong, hongping; chew, sock hui; shi, pei-yong title: crystal structure of dengue virus methyltransferase without s-adenosyl-l-methionine date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: oxohdfpu flavivirus methyltransferase is a genetically-validated antiviral target. crystal structures of almost all available flavivirus methyltransferases contain s-adenosyl-l-methionine (sam), the methyl donor molecule that co-purifies with the enzymes. this raises a possibility that sam is an integral structural component required for the folding of dengue virus (denv) methyltransferase. here we exclude this possibility by solving the crystal structure of denv methyltransferase without sam. the sam ligand was removed from the enzyme through a urea-mediated denaturation-and-renaturation protocol. the crystal structure of the sam-depleted enzyme exhibits a vacant sam-binding pocket, with a conformation identical to that of the sam-enzyme co-crystal structure. functionally, equivalent enzymatic activities (n- methylation, ′-o methylation, and gmp-enzyme complex formation) were detected for the sam-depleted and sam-containing recombinant proteins. these results clearly indicate that the sam molecule is not an essential component for the correct folding of denv methyltransferase. furthermore, the results imply a potential antiviral approach to search for inhibitors that can bind to the sam-binding pocket and compete against sam binding. to demonstrate this potential, we have soaked crystals of denv methyltransferase without a bound sam with the natural product sinefungin and show that preformed crystals are capable of binding ligands in this pocket. cap formation: (i) an rna triphosphatase removes the c-phosphate from the triphosphate of the nascent rna (pppn-rna ? ppn-rna); (ii) an rna guanylyltransferase (gtase) transfers the gmp moiety from gtp to ppn-rna to generate gpppn-rna (gtp + ppn-rna ? gpppn-rna); (iii) an n- methyltransferase (mtase) methylates the guanine n- position to produce m gpppn-rna (gpppn-rna ? m gpppn); (iv) a -o mtase methylates the ribose -o position of the rna to complete the type- cap formation (m gpppn-rna ? m gpppnm). both n- and -o methylations use s-adenosyl-l-methionine (sam) as a methyl donor and generate s-adenosyl-l-homocysteine (sah) as a by-product (decroly et al., ) . the sam molecule always co-purifies with dengue virus (denv) mtase. this is also true for almost all known flavivirus mtases (assenberg et al., ; bollati et al., ; egloff et al., ; geiss et al., ; jansson et al., ; mastrangelo et al., ; zhou et al., ) . the only known exception is the modoc virus mtase (jansson et al., ) . for the denv mtase, we have performed extensive dialysis to remove the bound sam, but the ligand always remained bound to the mtase (unpublished data). this observation raises the question of whether the sam molecule is an integral structural component required for the folding of denv and closely related flavivirus mtases (noble and shi, ) . to address this question, we investigated the structural and enzymatic integrity of denv mtase in the absence of sam. denv belongs to the flavivirus genus within the flaviviridae family. it contains a single-strand, plus-sense rna with a type- cap. the n-terminal third of the flavivirus nonstructural protein (ns ) encodes n- mtase (ray et al., ) , -o mtase (egloff et al., ) , internal rna mtase (dong et al., ) , and gtase (bollati et al., ; egloff et al., ; issur et al., ). here we report that structurally and functionally integrated recombinant denv- mtase without a sam molecule can be obtained using a urea-mediated denaturation-and-renaturation process. the crystal structure of the sam-depleted mtase is identical to that of the sam-mtase complex. the sam-depleted and sam-containing mtases exhibited comparable enzymatic activities (n- mtase, -o mtase, and covalent gmp-mtase complex formation the natural product sinefungin, showing that this is a viable approach to identify novel small molecules that bind to the same pocket. preparation of sam-depleted recombinant mtase: we used a urea-mediated denaturation-and-renaturation procedure to remove the sam molecule that co-purified with the denv- mtase. the expression and purification protocol was reported previously (lim et al., ) . briefly, the mtase domain representing the amino acids of denv- ns was fused with an n-terminal glutathione-s-transferase and purified by glutathione affinity. the tag was removed by cleavage with prescission protease. to remove the co-purified sam, we denatured the purified protein in mm hepes, ph . , mm nacl, and mm dtt by addition of m urea and exhaustively dialyzed the protein against a buffer containing m urea, mm hepes, ph . , mm nacl, and mm dtt. the mtase was refolded by concentrating the protein, followed by -times rapid dilution into the same buffer lacking urea, to give a final concentration of m urea. the refolded mtase was further purified by size-exclusion chromatography. as shown in fig. a , the sam-containing mtase and sam-depleted mtase migrated at equivalent positions on a % sds-page gel. the absence of sam was demonstrated by precipitating the protein with perchloric acid and removing the precipitant by centrifugation as described previously (noble et al., ) . the supernatant containing the solubilized nucleotide was then analyzed by measuring the absorbance at nm. the native recombinant mtase protein treated this way showed a typical adenosine absorbance spectrum, whilst the refolded mtase, showed no absorbance (data not shown). the lack of a bound sam was confirmed by determining the crystal structure of the sam-depleted protein (see below). enzymatic activities of sam-depleted mtase: to examine the effect of sam depletion on the function of mtase, we compared three enzymatic activities between the wild-type (wt) mtase with co-purified sam and the refolded mtase depleted of sam. we first analyzed cap-methylation activities in the presence of additional lm sam. equivalent n- methylation (fig. b) and -o methylation ( fig. c) were detected for both the sam-containing and sam-depleted mtases. the n- and -o methylation assays were performed as previously described (dong et al., ) . briefly, the p-labeled g ⁄ pppa-rna and m g ⁄ pppa-rna (the asterisk indicates that the following phosphate is labeled with p) were used for n- and -o methylations, respectively. the rna substrate consisted of the first nucleotides of the denv- genome. the n- methylation was performed in a -ll reaction containing mm bis-tris [ph . ], mm nacl, mm dithiothreitol [dtt], $  cpm g ⁄ pppa-rna, lm sam, and lg mtase; the reaction was incubated at °c for min. the -o methylation was performed in a -ll volume containing mm tris-hcl [ph . ], mm mgcl , mm dtt, $  cpm m g ⁄ pppa-rna, lm sam, and lg mtase; the reaction was incubated at °c for h. both n- and -o reactions were stopped by phenol-chloroform extraction followed by ethanol precipitation. the methylated rna products were re-suspended in rnase-free h o, digested with nuclease p (sigma-aldrich) overnight, and analyzed on polyethyleneimine cellulose thin-layer chromatography (tlc) plates (jt baker) using an aqueous solution containing . m licl. the radioactive cap variants (g ⁄ pppa, m g ⁄ pppa, and m g ⁄ pppam) on tlc plates were quantified by a phosphorimager (typhoon; ge healthcare). next, we analyzed the formation of a covalent gmp-mtase complex. a gtase capping is mediated by a two-step ping-pong reaction: the gmp moiety from gtp is first covalently linked to the gtase to form a gmp-gtase complex; the gmp-gtase complex then transfers the gmp to the -end of ppn-rna to produce gpppn-rna (shuman and hurwitz, ) . for flavivirus, the gmp-mtase (functioning as a gtase) complex can be readily formed via a phosphoamide bond to an attacking lys of the enzyme, whereas the conversion to the product gpppn-rna is not efficient (bollati et al., ; issur et al., ) . as shown in after incubation at °c for . h, the reaction was terminated by heating at °c for min and analyzed on a % sds-page gel. after drying the gel, the a- p-gmp-mtase complex was visualized and quantified with a phosphorimager. taken together, the above results showed that depletion of sam from the recombinant mtase does not significantly affect its n- mtase, -o mtase, or gmp-mtase complex-formation activities. crystal structure of sam-depleted mtase: to compare the conformation of the sam-depleted mtase with the structure of the sambound mtase, we determined the high resolution crystal structure of the refolded denv- mtase to . Å resolution using the crystallization conditions described previously (lim et al., ) . briefly, ll of the protein at mg/ml was mixed with ll of a reservoir solution of . % peg , . m nacl, . m tris-hcl, ph . , and mm tri-sodium citrate and crystallized by hanging-drop vapor diffusion. crystals were transferred to the same solution supplemented with % glycerol for shock-cooling. the data were processed using the global phasing software suite (www.globalphasing.com) and the structure solved using pdb id p as an initial model (lim et al., ) . the overall structure of the sam-depleted mtase is very similar to the sam-bound structure with a root mean square deviation (rmsd) of . Å for all ca atoms (table and fig. ) . a surface representation of the ligand-free mtase ( fig. a) shows that the denv mtase in the absence of a ligand retains the large cavity for binding sam. to determine whether this cavity of the enzyme is still capable of binding ligands, we soaked pre-formed crystals of the samdepleted mtase with the natural product sinefungin and determined the structure. crystals were soaked by adding ll of the reservoir solution supplemented with mm sinefungin directly to the crystallization drop and equilibrating the crystals overnight. the structure of the complex shows that this enzyme is capable of binding the sam analog sinefungin in this pocket, without significant structural rearrangement; the rmsd for the free mtase and sinefungin-bound structures is . Å for all ca atoms, whilst between the sam-and sinefungin-bound structures it is . Å for all ca atoms. a comparison of the three structures (bound to sam, bound to sinefungin, and ligand free) shows that there is no conformational change to the ca backbone upon ligand binding ( fig. b) and only modest movements of amino acid sidechains. the most obvious of these is a flip of glu , which is pointing towards the pocket in the free structure, but oriented away in the sam-and sinefungin-bound structures. phe moves slightly towards the pocket in the free structure ($ . Å) and lys moves slightly away ($ . Å). the structures of the complex show that the sam-depleted mtase can be used for screening and structure-based design of novel inhibitors that bind to the sam pocket. taken together with the biochemical data, they strongly indicate that the presence of the ligand sam is not required for the correct folding of denv mtase. targeting the sam-binding pocket as a potential antiviral approach: denv represents the most prevalent mosquito-borne viral pathogen in humans. there is currently no clinically-approved vaccine or antiviral for denv. although great advances have been made toward to the development of denv vaccines and drugs lim et al., ) , new approaches are urgently needed to combat this global human pathogen. recent studies showed that mutant viruses defective in -o mtase are potential vaccine candidates for denv (zust et al., ) , japanese encephalitis virus , and severe-acute respiratory-syndrome coronavirus (menachery et al., ) . mutagenesis analysis revealed that defective n- mtase was lethal for flavivirus replication, suggesting that mtase is a potential antiviral target (dong et al., ) . the current study demonstrates that sam is not an essential structural element for the folding of denv mtase. this conclusion was supported by two complementary structural and functional approaches: (i) the crystal structure unequivocally shows that absence of sam does not affect the overall conformation of denv mtase, including the sam-binding pocket; (ii) depletion of sam from the recombinant mtase did not change the activities of cap methylations and gmp-mtase complex formation. these results, together with the established protocol for preparation of samdepleted mtase, have made it possible to search for compounds that can bind to the sam-binding pocket and compete against sam binding. a number of biophysical assays, such as surface-plasmon resonance or differential-scanning fluorimetry, can be envisioned. one potential challenge of this approach is the requirement of inhibitors with high affinity for the sam-binding pocket in order to efficiently compete against the authentic ligand sam. in line with this idea, sah analogs that interact with a flavivirus mtase-specific pocket (located adjacent to the sam-binding pocket) were reported to selectively inhibit denv mtase without suppression of cellular mtases (lim et al., ) . since flavivirus mtase is a genetically-validated antiviral target (zhou et al., ) , the proposed approach warrants further studies for flavivirus antiviral development. crystal structure of the murray valley encephalitis virus ns methyltransferase domain in complex with cap analogues recognition of rna cap in the wesselsbron virus ns methyltransferase domain: implications for rna-capping mechanisms in flavivirus conventional and unconventional mechanisms for capping viral mrna -o methylation of internal adenosine by flavivirus ns methyltransferase biochemical and genetic characterization of dengue virus methyltransferase flavivirus rna methylation an rna cap (nucleoside- -o-)-methyltransferase in the flavivirus rna polymerase ns : crystal structure and functional characterization structural and functional analysis of methylation and -rna sequence requirements of short capped rnas by the methyltransferase domain of dengue virus ns live attenuated vaccine: the first clinically approved dengue vaccine? analysis of flavivirus ns methyltransferase cap binding enzymology of rna cap synthesis the . a structure of vaccinia protein vp : a bifunctional enzyme that participates in the modification of both mrna ends the flavivirus ns protein is a true rna guanylyltransferase that catalyzes a two-step reaction to form the rna cap structure structure of the methyltransferase domain from the modoc virus, a flavivirus with no known vector rational design of a flavivirus vaccine by abolishing viral rna -o methylation small molecule inhibitors that selectively block dengue virus methyltransferase ten years of dengue drug discovery: progress and prospects structural bases for substrate recognition and activity in meaban virus nucleoside- -o-methyltransferase attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking -o-methyltransferase activity structure of a nucleotide-bound clp -pcf polyadenylation factor structural biology of dengue virus enzymes: towards rational design of therapeutics west nile virus -cap structure is formed by sequential guanine n- and ribose -o methylations by nonstructural protein mechanism of mrna capping by vaccinia virus guanylyltransferase: characterization of an enzyme-guanylate intermediate structure and function of flavivirus ns methyltransferase rational design of a live attenuated dengue vaccine: -omethyltransferase mutants are highly attenuated and immunogenic in mice and macaques we thank colleagues at novartis institute for tropical diseases for helpful discussions during the course of this study and the beamline scientists at the swiss light source for assistance with data collection. key: cord- - norumv authors: vere hodge, r. anthony title: meeting report: th international conference on antiviral research, in raleigh, nc, usa date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: norumv the th international conference on antiviral research (icar) was held in raleigh, north carolina, usa from may to , . this article summarizes the principal invited lectures. john drach (elion award) described the early days of antiviral drugs and their novel modes of action. piet herdewijn (holý award) used evolutionary pressure to select dna polymerases that accept nucleoside analogs. replacing thymine by -chlorouracil led to the generation of a new form of escherichia coli. adrian ray (prusoff award) demonstrated how prodrugs can markedly improve both the efficacy and safety of potential drugs. the keynote addresses, by david margolis and myron cohen, tackled two emerging areas of hiv research, to find an hiv “cure” and to prevent hiv transmission, respectively. these topics were discussed further in other presentations – a cure seems to be a distant prospect but there are exciting developments for reducing hiv transmission. tdf-containing vaginal rings and gsk- , as a long-lasting injection, offer great hope. there were three mini-symposia. although therapy with tdf/ftc gives excellent control of hbv replication, there are only a few patients who achieve a functional cure. myrcludex, an entry inhibitor, is active against both hbv and hdv. the recent progress with hbv replication in cell cultures has transformed the search for new antiviral compounds. the hbv capsid protein has been recognized as key player in hbv dna synthesis. unexpectedly, compounds which enhance capsid formation, markedly reduce hbv dna synthesis. the development of bcx , which is active against marburg and ebola viruses, is of great current interest. this article provides an overview of the invited lectures at the th international conference on antiviral research, sponsored by the international society for antiviral research (isar), which was held in raleigh, north carolina, usa from may to , . it begins with reports of lectures by the recipients of isar's three major awards, held in memory of gertrude elion, antonín holý and william prusoff. these are followed by brief summaries of the keynote addresses and the three mini-symposia on ''hepatitis b virus'', ''research triangle park'' and ''challenges in hiv infection, treatment and prevention''. because this review article simply provides short accounts of oral presentations, it is not generally accompanied by references to the scientific literature. any descriptions of favorable treatment outcomes should not be taken as recommendations for clinical use. . gertrude elion memorial award lecture: collaborative antiviral studies for the discovery of drugs to treat cytomegalovirus infections john c. drach, ph.d., university of michigan, ann arbor, michigan, usa (fig. ) . gertrude b. (trudy) elion was born in new york city and was pleased to work for the burroughs wellcome co. when based in new york but was concerned when it transferred to research triangle park, north carolina, not many miles from this year's meeting site. however, within just a few months she declared that she was ''at home'' in north carolina. she was awarded the nobel prize in physiology or medicine in for her pioneering work in purine biosynthesis which paved the way for the discovery of drugs to treat organ rejection, cancer and viral diseases. the focus of john's presentation was on the research conducted in his own and his collaborators' laboratories that ultimately led to the invention of three compounds which were discovered to have antiviral activity against human cytomegalovirus (hcmv) and which later entered clinical trials: bdcrb pyranoside (gw x) (phase i), maribavir (phases i, ii and iii) and cyclopropavir (phase i). his major collaborators included karen biron, charles shipman, leroy townsend, and jiri zemlicka. to date, there are only five fdaapproved drugs for treatment of hcmv infections: cidofovir, fomivirsen, foscarnet, ganciclovir and valganciclovir. being inspired by the presence of a naturally-occurring , dimethylbenzimidazole nucleotide in vitamin b , research on benzimidazole nucleosides was initiated by medicinal chemists in the s and ' s. this led to the synthesis of a trichloro analog in townsend's laboratory at the university of utah and later the discovery of its activity against hcmv in john's laboratory. much work, in both their laboratories at the university of michigan, established that it and its -bromo analog (bdcrb) have excellent activity against hcmv with very low cytotoxicity. surprisingly, it was found to be inactive against other herpes viruses and it did not need conversion to a triphosphate to be active against hcmv. collaborative studies with karen biron at burroughs wellcome established that, unlike many other anti-virals that inhibit viral dna synthesis such as ganciclovir (gcv), these compounds acted by a novel mechanism, inhibition of viral dna processing. it was the viral resistance studies which revealed the viral targets, pul and pul . these two proteins, with pul , form a complex known as the terminase which cuts newly synthesised hcmv dna into unit lengths for packaging into virions. although bdcrb had many desirable properties in vitro, it had poor pharmacokinetics in mice and monkeys due to hydrolysis of its glycosidic bond; therefore it was not developed for human use. much additional work in drach's and townsend's laboratories at michigan and by biron's group at burroughs wellcome ultimately led to two potential drug candidates, bdcrb pyranoside and maribavir (fig. ) . both compounds have excellent activity against hcmv, low toxicity, and excellent pharmacokinetics. clearly, their modes of action differed markedly from that of gcv. quite unexpectedly, they have different mechanisms of action. bdcrb pyranoside has a mechanism of action very similar to its parent compound bdcrb, inhibition of dna processing. in contrast, maribavir inhibits dna synthesis, albeit indirectly. it is a isopropylamine derivative of bdcrb except that it has the unnatural l-sugar configuration. its mechanism of action involves inhibition of the viral kinase (pul ), which phosphorylates another viral protein, pul . phosphorylated pul is necessary for viral dna synthesis. thus inhibition of pul by maribavir inhibits viral dna synthesis. interestingly, pul is also the kinase that activates (phosphorylates) gcv. resistance studies confirmed that a single mutation in ul , resulting in a mutation in the kinase (leu arg), was necessary and sufficient for resistance to maribavir. in a further study of resistance, virus already resistant to bdcrb was passaged in increasing concentrations of maribavir and resistant virus was isolated. this strain grew at the same rate as the wild-type virus and was resistant to both bdcrb and maribavir. as expected, resistance to bdcrb was due to known mutations in ul and ul . however, no mutations were found in ul . further investigation showed that a single base change in ul (t c) was necessary and sufficient for resistance to maribavir. the role of the encoded protein was then unknown but the amino acid mutation (leu pro) is in the middle of the protein. similarly, biron's group detected resistance due to mutations in the ul gene. further research studies on maribavir have been summarized in previous icar scientific reports. cyclopropavir (cpv, fig. ) was synthesized in the laboratory of jiri zemlicka, karmanos cancer institute, detroit, michigan. it is a guanosine nucleoside analog which is very active against hcmv. unlike the benzimidazole nucleosides, it also inhibits epstein-barr virus (ebv) and human herpesvirus (hhv- ). like gcv, it is phosphorylated by the kinase encoded by ul . it is more potent in vitro and in vivo than ganciclovir but has a somewhat different pattern of resistance. in one resistant strain, the key mutation formed a stop codon resulting in a truncated pul kinase protein. the phosphorylation of cpv by pul is more efficient than that of gcv, with a considerably lower k m and higher v max . interestingly, the phosphorylation of cpv to its monophosphate (cpv-mp) by pul is stereoselective; only the (+) isomer of cpv-mp is formed. a single enzyme, gmp kinase, phosphorylates cpm-mp to both its di-and triphosphates. in contrast, acyclovir and gcv require additional cellular enzymes to convert their diphosphates to active triphosphates. cyclopropavir is currently in phase i clinical trials for the treatment of hcmv infections. . the antonín holý memorial award lecture: from modified nucleoside to a chemically modified genome piet herdewijn, rega institute for medical research, ku leuven, belgium (fig. ) . the icar began with a symposium, on the legacy of the late antonín (tony) holý , at which the establishment of a new isar award in medicinal chemistry was announced. the awardee is to be a senior scientist of international stature in medicinal chemistry and who has made innovative contributions impacting antiviral drug discovery or development. piet is, therefore, the first to receive this award. in the late s, the potent activities of bvdu and bvarau against herpes simplex virus type (hsv- ) and varicella zoster virus (vzv) were discovered; this work motivated piet to start antiviral research with the synthesis of carbocyclic bvdu. through to the early s, he synthesized several other nucleoside analogs with bicyclic bases having good activity against hsv- and vzv. during the s, emphasis switched to investigating the effect of modifying the sugar ring, in particular the synthesis of six-membered rings containing an oxygen or a double bond. piet showed examples of compounds with activity against hsv- , hsv- , vzv and hcmv. back in , erik de clercq showed piet a paper on aids, one of the authors being phil furman. this publication stimulated the search for anti-hiv compounds. many compounds were discovered with potent activities (and good selectivity indices) against hiv. piet worked out the first structure-activity relationships of anti-hiv dideoxy nucleosides. starting in the late s, tony holý synthesised a series of phosphonates. at the icar, erik de clercq recalled how this work led, ultimately, to tenofovir, which was to become a major success for treating hiv-infected patients. from its first introduction in , its market share has increased to well over %. in , having a single-pill regimen was agreed as a way forward to simplify, and thereby enhance, hiv therapy. this led to atripla being approved in , complera in and stribild in . tenofovir, in its various prodrug forms, is now available in over countries and is distributed widely to the known hivinfected population. in line with this research, piet synthesized phosphonate nucleosides, with a threose sugar moiety, which showed anti-hiv activity in the same range as -( -phosphonylmethoxyethyl) adenine (pmea). piet's work had taken a different pathway. it is possible to link several nucleotides together to form aptamers. for example (fig. ) , the above antiviral nucleosides, which have a -membered ring in place of the natural furanose, could be incorporated into hexitol nucleic acid (hna) aptamers. x-ray studies revealed the structures of hna-rna duplexes and hna-hna duplexes, the latter having a similar overall form to that of an rna-rna duplex with the same base sequence. hna-containing aptamers were shown to be potent and specific inhibitors of trans-activating region (tar)mediated transcription. normally, an hiv encoded protein, transactivator of transcription (tat), binds to cellular factors and to the viral tar rna regulatory element, resulting in a vastly increased rate of transcription of all hiv genes. hna-containing aptamers prevents this interaction and so inhibit hiv replication. it took four years to engineer a polymerase that would utilise hnas to assemble a strand complementary to a dna template. in line with this research, hexitol-modified sirna has shown good activity in an in vivo anti-hbv model. this success stimulated the concept that it may be possible to generate new forms of biologically active dna. in order to pursue this idea, a culture system with twin growth chambers was devised. alternative nutrient media could be fed into the chambers and the culture from one chamber could be used to seed the second chamber, the former culture being removed. in this example, the aim was to replace thymine with -chlorouracil ( fig. ) using escherichia coli. initially, the nutrient contained % -chlorouracil and % thymine. with each cycle, seeding one chamber from the previous one, the proportion of -chlorouracil was increased. after days, in which there had been about generations of e. coli, thymine had been replaced totally by -chlorouracil. an interesting outcome was that the alternative base led to a change not only in the genotype but also in the phenotype; the ''new'' e. coli cells were much longer than the original. this is the first example of a dna polymerase being adapted through evolutionary pressure to accept a nucleotide analog, resulting in the generation of a new living organism. prusoff young investigator award lecture: use of nucleotide prodrugs to enhance selectivity of anti-hiv and -hcv agents adrian s. ray, gilead sciences inc., foster city, ca, usa (fig. ) . adrian started his lecture with photos of william (bill) prusoff and reminisced of his days with bill, raymond schinazi and yung-chi (tommy) cheng. adrian presented examples to illustrate two models of how a prodrug strategy can transform a potential drug into a much improved clinical candidate. in the first, the prodrug alters the distribution of the pharmacologically active nucleotide analog to tissues where viral infection is taking place (on-target) and away from tissues resulting in adverse events (off-target). in the second, the prodrug enables one to select a drug candidate based more directly on the intrinsic properties of the active nucleotide-triphosphate analog via by-passing an inefficient activation (phosphorylation) of the corresponding nucleoside analog. sofosbuvir (sovaldi Ò ), a prodrug of -f- -c-meump, was approved in the usa on th december, for treatment of patients with hepatitis c. this is a fine example of a prodrug enhancing the activity of the parent compound. the nucleoside analogue, -f- -c-meu, is poorly active due to restricted phosphorylation to the monophosphate. sofosbuvir, a nucleotide analogue prodrug of -f- -c-meu, delivers the monophosphate into the cell and this is then further phosphorylated efficiently to give high levels of the triphosphate which inhibits hcv rna polymerase. adrian recalled being much impressed by a result reported at the meeting in of the american association for the study of liver diseases (aasld). in a phase ii monotherapy trial in patients with hcv, at day , the viral loads were reduced by log . and log . for vx- ( mg bid, n= ) and rg- ( mg bid, n= ), respectively. however, from day to , the polymerase inhibitor (rg- ) had continued to reduce the viral load, reaching a reduction of log . . on the other hand, the protease inhibitor (vx- ) did not give a sustained reduction, with the viral load starting to increase from day . at day , the viral load was only log . less than baseline. nucleotide analogues have two advantages over other classes of inhibitors. there is a high genetic barrier to resistance selection, due to the hcv rna polymerase being highly specific for its natural substrates and template. this specificity can be altered but only under extreme evolutionary pressure (see section ). also, nucleotide analogs often have pan-genotype activity because the active site of the hcv ns b polymerase is so highly conserved. as an example of how prodrugs can impact a discovery program, allowing for more targeted delivery and for the optimization of the intrinsic properties of the triphosphate, adrian presented the history of the gs- program. the c-adenine analogue ( -c-me- -aza- , -dideazaa, c-nuc ) was compared to the corresponding n-nucleoside, mk . in a genotype b replicon assay, the ec values were . lm and . lm respectively. however, their triphosphates were equally effective against hcv ns b polymerase (ic values both . lm). in the replicon system, the triphosphate of the n-nuc (mk ) was formed more efficiently than that of the c-nuc , thus explaining the lower activity of the c-nuc . however, in primary human hepatocytes, c-nuc was phosphorylated to the triphosphate more efficiently than the n-nuc (mk ). this illustrates the importance of using primary human cells. c-nuc seemed to have a benign in vitro toxicity profile, including not inhibiting the mitochondrial dna polymerase-gamma, but it had very significant toxicity in animals. in a collaboration between gilead and craig cameron at pennsylvania state university, the researchers sought to identify the toxicity target(s) for ribonucleotide analogues, including c-nuc and others that had been stopped in phase ii trials. these studies showed a correlation between c-nuc and the phase ii candidates, r , nm and bms /idx . all the latter were efficiently incorporated into rna by the mitochondrial rna polymerase (> % of the corresponding natural nucleotide). the triphosphate of c-nuc was also an efficient substrate ( % the rate of atp). in contrast, the active nucleotide analogs, formed by drugs approved for the treatment of hcv, were poor substrates. ribavirin was poorly incorporated (about %) and sofosbuvir was below the limit of detection (= . %). more extensive in vitro and cell culture evaluation of the compounds could have saved the expense of taking them into clinical trials. understanding that the mitochondrial rna polymerase is an important target for ribonucleotide toxicity, the gilead team sought analogs that were not incorporated by this polymerase. adding a cn group to the position of c-nuc did not change its activity as an hcv ns b polymerase inhibitor (ic . lm) but it did reduce incorporation in the mitochondrial rna assay (< . %). however, in the absence of a nucleotide prodrug to bypass the first phosphorylation step, the resulting di-substituted nucleoside analog would not be a drug candidate because it was not efficiently activated in cells. application of a nucleotide prodrug strategy allowed this nucleotide to be pursued further. oral absorption, delivery of the monophosphate into hepatocytes and high hepatic extraction were criteria used as part of the prodrug optimization process. a nucleotide prodrug, gs- (a mixture of diastereoisomers at phosphorous) was well absorbed in dogs (> %). comparing the pre-hepatic and post-hepatic plasma drug levels, about % of the absorbed drug was taken up by the liver. inside cells, gs- was converted to the corresponding monophosphate which was efficiently converted to the triphosphate. at h, the triphosphate levels remained about -fold above the ic value. a pure stereoisomer was selected and later named gs- . in a phase ii trial ( mg, bid days), the mean reduction in hcv load was about log . . two subjects achieved hcv rna < iu/ ml. however, the pharmacokinetics and antiviral responses were highly variable. whereas the activity results were disappointing, clinical proof of concept was observed in terms of safety. gs- did have a markedly improved safety profile relative to c-nuc , progressing through chronic toxicology studies in rats and dogs at relatively high doses. the story of gs- illustrates both how nucleotide prodrugs enable further progression of candidates and also the complexity of predicting the behavior of nucleotide prodrugs across species. one wonders what cell culture test or animal model may have predicted such variability. when selecting famciclovir as the prodrug for penciclovir, one potential prodrug was rejected because the pharmacokinetics in rats varied widely between individual animals (vere hodge et al., ) . a recent publication by adrian and his team highlights the metabolism of gs- by carboxylesterase , an enzyme highly expressed in the human small intestine but not uniformly expressed in different animal species, as a possible reason for the highly variable and suboptimal intestinal absorption of gs- in humans (murakami et al., ) . the focus of adrian's talk then switched to hiv. over the last or years in north america, the hiv-infected population has been changing, becoming older (now % over years old vs < % in ) and more likely to be obese (in every usa state, > % adults with bmip ). this has led to a shift in the focus of antiretroviral therapy (art), from solely control of hiv replication to now include tolerability in older, possibly obese, patients. the first example given for hiv was how application of a different prodrug strategy can markedly change the distribution even when delivering the same pharmacologically active nucleotide analog. the first approved prodrug of tenofovir (tfv) was tfv disoproxil fumarate (tdf). more recently, tfv alafenamide (taf) has been progressed into clinical development. a key difference in the properties of the two prodrugs is their stability in plasma, with half-lives of . and min, respectively. even with a short halflife, tdf gave better delivery of tfv into cells, as indicated by the hiv ec values in cell culture assays but there clearly was room for improvement; the ec values for tfv, tdf and taf are . , . and . lm respectively. whereas the gain in cell culture ec value may be modest, this is not the only gain. the increased stability of taf allows it to load on-target cells and tissues (e.g., lymph nodes) for a longer period of time resulting in increased lymphoid cell and tissue levels at greatly reduced circulating tfv levels, leading to less exposure to off-target tissues (e.g., kidney). in monotherapy studies after oral dosing with tdf ( mg) and taf ( mg), the plasma tfv auc is reduced from to ng.h/ml respectively whereas the reduction in hiv load from baseline is improved, from log . to log . copies/ml, respectively, reflecting the more efficient delivery of taf to target cells and tissues. clearly the lower dose of taf ( mg) relative to tdf ( mg) will give taf a marked advantage when considering combination pill therapy. understanding how marked a difference a prodrug can make from the taf example, adrian went on to describe how a prodrug approach transformed a new nucleotide project in which intrinsic properties of the pharmacologically-active nucleotide analog were optimized. their starting point was gs- (d api), which had good activity against both wild-type and resistant hiv strains but was an active inhibitor of mitochondrial polymerase-gamma. on comparing the known structures of hiv rt and mitochondrial polymerase-gamma, differences in the -binding pocket were noted. this led to gs- in which -f was added to gs- (fig. ) . compared to tfv, gs- was about -fold less active against wild-type hiv but maintained better activity against resistant strains (k r and multiple thymidine analog resistance mutations). most importantly, it was inactive (ic > lm) against mitochondrial polymerase gamma. more than prodrugs were synthesized and evaluated in metabolism studies and in dogs (intravenous and oral administration). then the enantiomers were tested separately in dogs. this led to the selection of gs- . whereas tfv is efficiently utilised by renal uptake transporters, gs- was poorly taken into the kidney. no adverse renal findings were observed with the prodrug (gs- ) in -day studies in rats, dogs and monkeys at the highest doses tested ( mg, mg and mg/kg daily, respectively). in summary, this work has given examples of the prodrug approach being used successfully both to increase selectivity (by loading on-target tissues vs off-target tissues) and to increase activity (via by-passing metabolic constraints). adrian presented cases in which a prodrug strategy was able to fulfil the full potential of a selective, active triphosphate analog and enable its further progression as a clinical candidate. the keynote speakers were david margolis and myron cohen (fig. ) . david margolis, university of north carolina, nc, usa in hiv-infected patients, there is a long-lasting reservoir of hiv in the form of integrated viral dna in resting cd + memory cells of the host immune system. therefore, even if it were possible to eliminate % of viral replication, a reservoir of hiv would remain. there may be reservoirs in other long-lived cells. to date, there is only one known hiv patient who has been cured of his infection, the ''berlin patient''. he was treated for cancer by chemotherapy followed by a bone-marrow transplant. being ccr +/À, the chemotherapy had a greater chance to remove all the ccr +ve cells. the bone marrow donor was ccr Àve. although this patient continues to have no sign of hiv infection, this is hardly a viable treatment option for most hiv-infected patients. even in subjects with hiv replication well controlled by therapy, % have detectable plasma viremia which does not appear to decay over time (at least two years). to improve the sensitivity of the assay for hiv, billion lymphocytes are mixed with antibody attached to magnetic beads. this selects for the cd + t cells, about . - billion cells. the limit of detection is copy of hiv rna/million cells, limit of quantitation is copies/million cells. to reduce the reservoir of hiv, it was suggested that activation of integrated hiv in resting cd + t cells would give renewed hiv rna synthesis and possibly result in cell death either due to viral cytopathic effects or resulting from hiv-specific immune responses. a small clinical trial was set up to test this hypothesis. vorinostat (vor), a clinically approved drug for treating certain cancers, has been shown to bind to the active site of histone deacetylases. after a single dose, there was an increase in hiv rna ( . to -fold, mean . -fold). of these subjects, elected to continue with multiple doses. from the th to nd vor dose, acetylation of histones and activation of hiv rna synthesis became refractory to therapy. also, it is not known what proportion of cells, with latent hiv, can be activated. whereas a single vor dose did increase the expression of hiv rna, this is not an effective therapy for removing the hiv reservoir. myron cohen, university of north carolina, nc, usa myron noted that there are . million new hiv infections each year. in this context, anal sex may be an important factor because just one or a few virions of hiv can be infective; within weeks, there is rapid virus replication throughout the body and latent hiv reservoirs of ''founder virus'' are already formed. although anal sex has been associated with homosexual couples, myron pointed out that it is not uncommon amongst heterosexual couples. although behavioral education should be encouraged, it can never be the whole answer. various approaches to the prevention of hiv transmission are being evaluated. monoclonal antibodies, broad neutralising antibody (bnab) and vaccines may have potential for prevention of transmission, but most progress is being made with dapivirine rings containing tdf. these are designed to stay in the vagina for a month. phase iii trials are ongoing. a long-acting hiv integrase inhibitor, gsk (generally known as gsk ), is administered i.m. once every months; a two-year safety trial will be required. phase i trial has been completed and phase ii trial is being planned. by analogy with tuberculosis therapy, in which the infectious state is disabled prior to a complete cure, one wonders if hiv transmission rates may decrease with effective art use. in , the hiv prevention trials network (hptn) initiated a study (hptn ) which enrolled , hiv sero-discordant couples (couples that have one member who is hiv-infected and the other who is hiv-uninfected), mostly ( %) heterosexual couples. the infected partner had to be well enough not to require immediate art. the couples were randomised to have either immediate or delayed art. both groups received the same care including counselling on safe sex practices, free condoms, treatment for sexually transmitted infections and regular hiv testing. in may , it had been announced that there had been hiv transmissions in the delayed art group ( couples) compared to only in the immediate art group ( couples), a % reduction. in these cases, the hiv strain was linked to the partner. this is the first randomised clinical trial to show that treating an hivinfected individual with art can reduce the risk of hiv transmission to an uninfected partner. even with ''safer-sex'' counselling, there were pregnancies in the delayed art group, despite that group having more incentive for safer-sex. following the announcement of this result, all infected participants were offered art. myron reported the th annual review of this study. in the delayed art group, there had been a total of cases of hiv transmission with the hiv strain linked to the partner and cases of unlinked transmission. in the one case of hiv transmission in the immediate art group, infection had been detected at day of the study and further investigation suggested that the infection event was on day . clearly, early art is highly beneficial. cdc guidelines now recommend that all hiv infected patients should have art. . mini-symposium: hepatitis b virus anna lok, university of michigan, mi, usa the number of people infected with hbv world-wide, as estimated by the who and cdc in , was between and million, but was declining due to vaccination. in the usa, vaccine use has led to a steady decline in the rate of new infections, decreasing from about / , residents in the s to about / , today. in contrast, the prevalence of chronic hepatitis b among immigrants remains high, with no decreasing trend. when infection is acquired early in life, chronic infection is the norm. high viral load is associated with progression to liver cancer. there are fda-approved drugs to treat chronic hbv infection, including entecavir (etv), emtricitabine (ftc) and tdf. with several years of continuous therapy, hbeag loss is achieved in about % of patients but hbsag loss (the ultimate goal, seen as a ''cure'') is still a distant prospect for most patients. however, cirrhosis can be reduced by long-term antiviral treatment. in one tdf trial at years, / patients had a liver biopsy which showed that % of patients had improved fibrosis scores (p units) and that most other patients had no worsening. tdf has now been used for years without detecting hbv resistance, making it one of the first line drugs. tdf is generally well tolerated but its rare side effects include nephrotoxicity (see above for a possible switch to taf when it is approved), reduction in bone mineral density and very rarely lactic acidosis. despite the major progress made in hbv therapy, there remain various challenges. one is cost, about $ , -$ , for -year tdf therapy. pharmacy claims show that adherence is a problem; doses used are less than doses prescribed. there is a lack of accurate prediction of how hbv disease will progress in individuals. hbv dna can be integrated into the human genome at an early stage of infection. fortunately, the integrated viral dna is usually not the complete viral genome and patients, who achieve hbsag loss, rarely relapse. stefan mehrle, university of heidelberg, germany (stephan urban, head of hepatitis b research group, university of heidelberg, was originally scheduled to give this presentation). some chronic hbv-infected subjects are co-infected with hepatitis delta virus (hdv). this is a defective virus that replicates only in the presence of hbv. current antiviral drugs do not inhibit hdv. recently, heparan sulphate proteoglycan (hspg) has been shown to be essential for binding both hbv and hdv to primary hepatocytes. in , human sodium taurocholate co-transporting polypeptide (hntcp) was identified as a functional receptor for hbv and hdv. hntcp is also designated as a solute carrier protein a (slc a ). hntcp was shown to be a binding factor for the pres domain of the hbv l envelope protein. this interaction was found to be essential for hbv and hdv infection. whereas hbv replication is poor in cell lines derived from hepatocytes (e.g. hepg and huh- ) in which hntcp is usually weakly expressed, hbv replication is possible in primary human hepatocytes. the critical discovery was that over-expression of hntcp in hepg or huh- cells conferred susceptibility to hbv and hdv infection. myrcludex-b is a lipopeptide derived from amino acid residues - of the pres region of the hbv l protein. because it quickly (within min) targets the liver, it is being developed for liver imaging and for drug targeting. it also acts as an entry inhibitor for hbv and hdv by interrupting binding between the hbv l protein and hntcp. it specifically inhibits hntcp-mediated taurocholate transport but the effect on hbv replication is much greater. myrcludex-b activity has been investigated in vivo using scid mice reconstituted with human hepatocytes. with prophylactic treatment, not one infected hepatocyte was seen. following therapeutic treatment, at week post-infection, there were a few isolated infected cells. after the end of therapy, the infection seems to spread but only to neighboring cells. myrcludex-b has been synthesised on a g scale. toxicology evaluation in chimpanzees has been completed and clinical trials have been initiated. in a phase i trial using a mg dose, myrcludex-b was well tolerated. results of a further phase i trial are due to be reported later this year ( ). a dose-ranging phase ii trial has been started. kyong-mi chang, university of pennsylvania, pa, usa anti-hbs antibodies clearly play a critical role in controlling hbv disease. their presence has been accepted as an indication of an ''effective cure''. however, these antibodies appear late in the disease course and so they must have a limited role in the early stages of the disease. what is the role of t-cell responses? in contrast to other viruses, there is a delayed onset, about - weeks rather than days. cd + t cells regulate the adaptive response, cd + t cells attack hbv-infected cells. the national institute of diabetes and digestive and kidney diseases (niddk), part of the usa national institutes of health (nih), is supporting a prospective clinical trial to investigate hbv-specific t cell responses during the course of hbv disease. there are no clear t cell differences relative to hbv genotype. the t cell responses are highest during acute hbv infection. during the chronic phase of hbv disease, t cell responses remain suppressed. in conclusion, there are a lot of players in the immune control of hbv infection but their relative contributions and how they adapt to control hbv replication are still largely unknown. . . diversifying the hepatitis b pipeline: current efforts to explore novel mechanisms andrea (andy) cuconati, institute for hepatitis & virus research, pennsylvania commonwealth institute, pa, usa current hbv therapy using nucleotide anti-virals has been highly effective in controlling the infection but a ''cure'', as defined by hbsag seroconversion, has remained elusive. at best after years, the rate is about %. other approaches are needed. myrcludex-b (see section . ) is the lead entry inhibitor. nvr- , an encapsidation inhibitor, is entering clinical trials. in addition. studies with novel nucleotide analogues are ongoing. the hbv field has been transformed recently by the introduction of cell-based antiviral assays. stefan mehrle (see section . ) has been leading the way. the assay read-out will need to be optimized for high-throughput screening (hts) but, already, the assay has shown some ''hits''. a few compounds inhibited encapsidation of viral rna. (the hbv virion contains partly double stranded (ds) dna but the reverse-transcription from rna to dna occurs within the capsid.) within the cell, hbv dna is transported into the nucleus where the viral dna forms covalently closed circles (cccdna). two specific inhibitors of cccdna formation have been found. current nucleotide anti-hbv compounds do give large reductions of hbv dna in plasma but only a minimal reduction in levels of the hbs antigen (about log . ). in contrast, one ''hit'', hbf- inhibited surface antigen production but not genomic replication. structure-activity-relationship (sar) studies have given the current lead compound, hbv- . in conclusion, the cell-based assay, with complete replication of hbv, has markedly improved the screening for anti-hbv compounds although further optimization is still needed to give hts capability. adam zlotnick, university of indiana, in, usa. over the last few years, there has been much progress towards understanding the critical role of the hbv core protein -it is much more than just a protective coat for the genome because it plays a major role in the hbv life cycle. the core protein, being amino acids long, is known as cp . the first amino acids are involved in core assembly whereas the last residues, rich in serines and arginines, bind to rna. phosphorylation of the serines, particularly s , s and s , is required for specific packaging of full length hbv rna complexed to the polymerase (reverse transcriptase -pregenomic rna; rt-pgrna). this rt-pgrna complex initiates encapsidation. the core consists mainly of cp but also includes other proteins (about . %). adam showed us a computer model of the core, using different colours to highlight the various critical components. inside the core, the area of highest density (highlighted in red) represented the polymerase which was attached to the inner surface of the core. the ''other proteins'' in the core were shown in blue. the current thinking is that the polymerase, initially acting as a reverse transcriptase, is attached to, and guided by, an ''inside railway track''. this enables the polymerase to jump to the other end of the rna to start the reverse transcription into dna and then jump again to the other end to start, but never complete, the replication of the complementary dna strand. the self-assembly of the core is an energetically ''downhill'' process. somewhat surprisingly, it is possible to get mutations in which the core is even more stable but the rt activity is reduced. the phenylpropenamide derivative, at- , fills a pocket in the core and so stabilizes it, similar to the change in amino acids in the mutants. in the presence of at- , core assembly occurs faster; hence it is known as a core assembly enhancer (as adam mentioned, not a term much loved by industry, their preference is for core assembly inhibitors). regardless, the whole capsid structure changes. the binding of only a few drug molecules is required to make the core non-functional. it seems that it is easier to find compounds to enhance core assembly than inhibitors. . . targeting cccdna to cure chronic hepatitis b massimo levrero, sapeinza universita' di roma, italy. the current hbv therapies of choice are tdf alone or with etv. these drugs have an extensive safety record with use up to years. however, as for other nucleoside/nucleotide analogs, there is only a limited (about log ) reduction in the levels of hbv cccdna. the half-life of hbv cccdna seems to be long, but is still unknown. hbv replication parallels host gene expression, in that they involve the acetylation of histones, for example h and h . both host transcription factors and viral proteins bind to the cccdna. massimo summarized various assays to study different stages of cccdna during the replication cycle. potentially, these assays would allow the study of various approaches: to reduce or clear cccdna, to silence cccdna or to prevent the formation of new cccdna so that it would eventually be removed by dilution and cell death. for proof-of-concept, known ''epigenetic'' compounds, which act as transcription inhibitors, have shown that cccdna can be silenced. by reducing histone acetylation, the cccdna becomes too compact to allow transcription. this approach mimics, partly, therapy with interferon. this research is still at an early stage. due to time constraints, the next two speakers were asked to present brief summaries. john morrey (utah state university, ut, usa) described four mouse models but all stages of the life cycle of hbv can be studied only in the chimeric mouse model, in which human hepatocytes are used. however, this model lacks the potential to study the immune system and it is very expensive. stephan menne (georgetown university, dc, usa) described the woodchuck model. woodchuck hepatitis virus (whv) resembles the human virus and the disease in animals has many similarities to that in humans. neonatal infection becomes chronic in about - % of cases. these chronic cases have virtually a % life-time risk of developing cancer, the time scale being about year of chronic infection, followed by cancer at years to . the use of microbicides is an active area of research for the prevention of transmission of hiv. david katz (duke university, nc, usa) described how mathematical models may aid drug product design. for example, if it is assumed that the microbicide gel is microns thick, the epithelium is microns and the stroma (connective tissue) is microns and if the partition coefficient between gel and epithelium in known, then it is possible to model drug transfer and suggest how various other parameters, for example the size of the subject, may modify drug delivery. it is important that different disciplines work together, for example biophysicists with behavioral scientists. biophysics can help an understanding of complex physical phenomena but human behavior can be both complex and highly variable. ralph baric (university of north carolina, nc, usa) noted that a particular infective agent, for example norovirus (nov), may cause subclinical or serious disease in different individuals. in general, animal models are designed to give consistent outcomes rather than aiming to mimic the genetic diversity found in human subjects. in a collaborative effort, mice from ''founder'' strains, including wild-derived strains, were selected. the founder laboratory strains were all derived ultimately from a single female mouse ca . the susceptibility of the founder strains to severe acute respiratory syndrome coronavirus (sars-cov) differed widely (ld p - ). the founder strains were cross-bred. although ca % of the genes was equally distributed among the new mouse lines, there were gene combinations not seen previously. after infecting mice from the different founder strains with a constant sars-cov inoculum and measuring virus load at a set time after infection, there was a correlation between virus load and disease (as measured by vascular cuffing). it was possible to relate the effect to chromosomes ( %) and ( %). hopefully, identification of the important genes may be achieved. by keeping the virus inoculum constant, this system better represents the clinical spectrum of disease. when using this system to evaluate a potential vaccine, it was found that mice, under the age of one year, could be protected. however, there was a range of effectiveness, from good protection to inactive. these variations may give a representation of human diversity. angela kashuba, university of north carolina at chapel hill, nc, usa in four clinical studies, truvada [a combination pill containing tdf and emtricitabine (ftc)] was taken once daily to prevent hiv transmission, known as pre-exposure prophylaxis (prep). the adherence rates were unexpectedly poor in all four studies, particularly low in the study including at risk women. for example in one study, ''high adherence'' was defined as subjects taking at least % of drug doses and was achieved by only % of subjects. possible reasons may have been the apparent risk of side-effects (the long consent form included pages of side-effects) and the perception that the subjects, as individuals, were not particularly at risk of infection by hiv. importantly, the trial did confirm the concept that prep could be effective. there was > % protection in those subjects generally taking doses/week and there was some protection, albeit much less, in subjects taking doses/week. adherence rates, reported by subjects, were appreciably higher than the rates evidenced by drug blood level measurements taken just before the next dose (i.e. h after previous dose). in an attempt to better understand and model these data, the drug concentrations (tdf/tfv and ftc) in various tissues were measured. the ratio between drug concentrations in blood and tissue samples differed greatly for tdf/tfv, with less variations for ftc. concentration ratios of tdf/tfv were about in rectal tissue but only . in vaginal tissue. for ftc, the ratios were . and . , respectively. when considering the possible consequences of missed doses, the time scale for hiv infection is an important factor. it is thought that hiv takes about - h to reach the epithelial cells. clearly, adherence is a critical factor for efficacy and so a real-time objective method for measuring adherence is urgently needed before further clinical studies are initiated. . . the novel nucleoside analog bcx exhibits broad-spectrum antiviral activity and confers post-exposure protection against ebola and marburg viruses travis k. warren, usamriid, fort detrick, md, usa ebola and marburg viruses are members of the filovirus family. even in recent outbreaks of these diseases, including the current ebola epidemic in west africa, care workers are becoming infected and dying. drugs, which are being investigated for treating these diseases, are progressed under the fda ''animal rule''. bcx is a c-nucleoside adenine analog (fig. ) which is being progressed by biocryst pharmaceuticals inc. (warren et al., ) . in cell culture assays, bcx is active against ebola and marburg viruses, (ec ca lm). with bcx at lm, there was no detectable incorporation into host dna or rna. in rats, bcx is efficiently activated (phosphorylated) to the triphosphate. in a primer-extension assay, there is some read-through beyond a single residue of bcx , but there is effective chain termination after the first bcx residue where the template has two consecutive uridine residues. bcx has been tested in rodent and nonhuman primate models of marburg hemorrhagic fever. in mice, there was a dose response ( , , . and . mg/dose, bid) with full protection at the two higher doses (survivors, %, %, % and % respectively). in an experiment with dosing starting at different times ( h pre-infection, , , , and h post-infection vs placebo), the placebo-treated mice died on days , and with one survivor ( %). in the treated groups, the percent survival was , , , , and , respectively. in guinea pigs, bcx (bid) with treatment starting at different times ( h pre-infection, , and h post-infection) there was full protection ( % survival) for the pre-infection and h groups, with reduced efficacy at the later start times. in cynomolgus monkeys, bcx treatment was started at , and h post-infection. in the placebo group, all animals died within days to . in all the treated groups, virus loads were reduced by more than log . there was one late death in the h group but the other monkeys survived. various markers of potential organ damage were reduced in all treated groups. encouraged by these results, -day toxicology trials have recently been completed without any serious concerns. biocryst is developing bcx under the fda animal rule and indenabling work is ongoing. when asked about viral resistance, travis explained that it is not ethically permissible to create resistant strains of marburg virus, but samples collected from the monkeys are being sequenced to look for mutations indicative of drug resistance. as yet, mitochondrial toxicity has not been examined. had similar bone marrow transplants, initially seemed to have been ''cured'' but hiv was detected after and days, respectively. latent hiv can survive in various long-lived cells for decades, especially in memory t cells. when these cells proliferate, the integrated hiv genome is duplicated as the cell divides and the cells survive so long as hiv remains silent. compounds known to activate all t-cells are too toxic to become a clinical therapy. however, latency-reversing agents (lra) have greater specificity, ideally activating only the integrated hiv, leading to the death of hiv-containing t-cells. there remains another possibility (perhaps a less popular view) that there is continued low rate of hiv replication. two clinical studies have been initiated in subjects with undetectable plasma hiv levels. raltegravir, an hiv integrase inhibitor, was added to the background therapy. latent hiv is mostly integrated into host dna but hiv may also form episomal circular dna. the proportion of the circular form increases with raltegravir treatment. in the two clinical studies, / and / subjects, respectively, had detectable hiv circles which then decayed. this implies that some de novo infection of cells is ongoing. on the other hand, art works well, with no evidence of sequence evolution in the hiv circles at weeks. is it possible that raltegravir is inducing a single round of hiv replication, to give an increase in hiv circles? derek sloan, gilead sciences, foster city, ca, usa like vorinostat, (vor), romidepsin (rmd) is a histone deacetylase inhibitor which is used clinically to treat cancer. memory cd + t cells were taken from hiv subjects on suppressive art; ex-vivo treatment with rmd ( nm) induced a -fold increase in intracellular hiv rna which persisted for h. in contrast, a much higher concentration of vor ( lm) gave a to -fold lower response which was only transient. rmd also increased levels of extracellular hiv rna and virions. encouragingly, this ex-vivo induction of latent virus was seen at rmd concentrations that are below the levels of drug achieved in humans by clinical doses of rmd. accordingly, in a phase i/ii trial in hiv-infected subjects on art, rmd gave a better and more sustained response than vor. about . % of cells containing hiv provirus were activated. although this is far too low a percentage to eliminate the latent hiv reservoir, it is hoped that combination of such lra, which give improved results in ex-vivo cell assays, may give better clinical efficacy. gilead scientists have started screening for novel lras. ''gs- '' has been identified as a hit by hts. research on this lead is at a very early stage. gilead workers are also investigating other approaches. for example, gs- is a toll-like receptor (tlr ) agonist and it acts as an immune stimulator. although it is being evaluated in phase ii studies for the treatment of chronic hbv infections, the potential effect on hiv reservoirs is being investigated. in siv-infected monkeys, oral dosing of tlr agonist induced the activation of immune effector cells such as cd + t cells and nk cells. based on these data, tlr agonists are being further investigated for their effect on latent siv reservoirs in monkeys which have good virological suppression. another approach is to use anti-envelope antibodies. broadly neutralising antibodies (bnabs) are very effective in preventing siv infection when the viral load is low but less effective against a high-load virus challenge. in addition, a prophylactic cmv-vector-based siv vaccine was effective in preventing siv infection in rhesus monkeys. this and similar vaccines are being tested in vivo for their effects on the latent siv reservoirs. in summary, lras are able to activate hiv provirus in memory cd + t cells and thereby may enhance the recruitment of immune effector cells to destroy provirus-containing cells. however, a ''cure'' for hiv infection is still a distant prospect. furthermore, latent hiv reservoirs are heterogeneous and so a combination of approaches will likely be required. gerardo garcia-lerma, centers for disease control and prevention, atlanta, ga, usa proof-of-concept studies for prep, are mostly conducted in nonhuman primates. these can be used either to model a single highdose infective challenge or repeated low inoculations, about - tissue culture infective doses (tcid ). since , rhesus macaque models have been used in a long series of investigations. in a study, in which the monkeys were treated daily with either oral tdf or tdf/ftc and given a weekly siv inoculum rectally, tdf/ftc gave a longer delay in infection than did tdf alone. when using the vaginal infection route, tdf/ ftc gave % protection. in contrast, there was far less protection in clinical trials -why? one possible reason may have been that women were having the contraceptive injection, depot medroxyprogesterone acetate (dmpa). a study, in macaque monkeys given dmpa, confirmed that dosing with tdv/ftc gave good drug levels in plasma and in vaginal secretions. therefore, this did not explain the poor protection in the clinical trial. the macaque model has been used successfully to investigate various situations that are presented in the clinic. when macaques were co-infected with siv and a bacteria and treated with tdf/ftc for weeks, there was good, but not complete, protection ( %). with ftc-resistant virus, tdf/ftc remained protective. in this case, ftc-resistant virus has increased susceptibility to tdf. with the k r mutant hiv, there was protection against a low inoculum but only partial protection (ca %) against a high inoculum. whereas daily dosing seems to be acceptable for patients living with hiv, another option for prep is desirable. gsk- (generally known as gsk- ) is an hiv integrase inhibitor. it can be formulated with nano-particles to provide an injectable drug depot. in the macaque model, gsk- , injected once monthly, gave full protection against repeated rectal and vaginal exposures. because metabolism of gsk- is much slower in humans than macaques, it was expected to remain effective in humans for up to three months. a phase i study confirmed that drug levels remained above the predicted effective level with a -week dosing interval. a phase ii trial is planned. another approach is to use vaginal rings, which have been in clinical use as contraceptive devices for years. in the macaque model, tdf-containing rings, replaced every weeks, gave full protection. a phase iii trial has just been initiated. another option, elvitegravir (evg) and taf are being evaluated in a biodegradable polymer. although daily dosing with tdf/ftc has not proved sufficiently successful as prep in clinical use, it has proved that prep is an achievable aim and this has encouraged the progression of other options. courtney fletcher, university of nebraska, omaha, ne, usa atripla was the first triple combination pill taken once daily for hiv therapy. it contained tdf, ftc and efavirenz (efv). the macaque model has been used to investigate the differing tissue distributions of these drugs and how viral replication may be continuing wherever the drug concentrations are lowest. there are two approaches: tissue homogenates and tissue cells. tissue homogenates give both the intracellular and extracellular drug amounts. from tissues, mononuclear cells (mncs) are collected and the intracellular drug concentration measured. this approach is preferred by courtney but this option may be constrained by sample size and the drug concentration may be underestimated. for exam-ple, with raltegravir, after the mncs have been washed times, the drug concentration is very low. much higher raltegravir concentrations are found when the mncs are cleaned by a rapid spin through oil. comparing an oil spin and repeated washes, the oil process gives higher drug levels, typically about % higher. following initial studies in macaques, a clinical study, in subjects, investigated distribution of the drugs from atripla in peripheral blood mononuclear cells (pbmc) and various tissues (see above). in / subjects, there are data on the time to reduce hiv load to < copies/ml. in plasma, the time was - months. in lymphoid tissues, there was a much slower rate of hiv decline. also, patient variability was noted, with the faster responders having the higher drug levels. a drug may be absorbed from the gastrointestinal tract either going via the portal vein to the liver and then into blood circulation or via the lymphoid system. blood flow is about times faster than lymphoid flow. when the water/ -octanol partition-coefficient (logp) of a drug is < , absorption tends to be via the blood route. the prodrug approach can be used to alter absorption or, as for tfv, stability of the prodrugs (tdf and taf) can influence the relative concentration in lymphoid tissues (see above). this year, the three major award lectures exemplified the strength of icar, covering very different areas of research. john drach (elion award) described his journey through the early days of antiviral research, which led to the identification of novel modes of antiviral action that had not been envisaged previously. piet herdewijn (holý award) used evolutionary pressure to select dna polymerases that accept novel nucleoside analogs. the replacement of thymine by -chlorouracil led to the generation of a new form of e. coli. i suggest that this work has important implications in conventional antiviral research. with hiv and hcv protease inhibitors, the genetic barrier is limited by the ability of the viral protease and its substrate (the viral polyprotein cleavage sites) to co-mutate so that the virus can become resistant to the antiviral drug. so far, polymerase inhibitors have not suffered the same fate but this work shows that a poor choice of nucleotide analog could result in a resistant virus with a new type of rna in which the drug replaces a natural nucleoside. adrian ray (prusoff award), describing work at gilead, demonstrated how the prodrug concept can markedly improve both the efficacy and safety of potential drugs. their progress with hiv and hcv therapies has been remarkable. the keynote addresses tackled two emerging areas of hiv research. david margolis summarized work aiming to eradicate hiv from infected subjects and myron cohen described current progress with approaches to prevent hiv transmission. i found both these presentations to be informative and stimulating. hiv ''cure'' still seems to be a distant prospect. in contrast, prior to exposure prophylaxis (prep) has been shown to be an achievable aim although the need for daily dosing is a barrier to success. gerardo garcia-lerma described recent progress which is likely to radically change the prospects for therapeutic convenience and success. tdf-containing vaginal rings, which need replacing only once a month, are being evaluated. another exciting prospect is gsk- which has been formulated as a long-lasting injection. a phase i trial confirmed that the drug may be administered at month intervals. in the absence of a proven hiv vaccine, prep with drugs has become the most promising strategy to reduce hiv infection rates among high-risk populations. this conference also included three interesting mini-symposia: ''hepatitis b virus'', ''research triangle park'' and ''challenges in hiv infection, treatment and prevention''. an innovation this year was a session devoted to the european training network, euvirna and introduced by frank van kuppeveld. all the euvirna fellows, who attended icar, gave short presentations at this session. for further information, please see the isar news ( . ) in the september issue of antiviral research for an account by frank van kuppeveld. for many years, the clinical symposium was, for me, a major highlight of icar. in my report for the icar, i expressed a hope regarding hcv therapy: ''there is the prospect that the first nucleotide analogue will be licensed by the time of our next icar meeting. the combination of a nucleotide analogue and a ns a inhibitor looks set to transform hcv therapy across all genotypes. as for hiv, single-pill, once-daily regimens are following on quickly''. on th december , sofosbuvir (sovaldi Ò ) was the first nucleotide analog to be approved in the usa by the food and drug administration (fda) for treatment of patients with hcv. approval by the european union followed soon afterwards, in january . a ns a inhibitor, ledipasvir, formulated as a single fixed-dose combination pill with sofosbuvir, is progressing quickly through clinical trials. with such remarkable progress being achieved since the icar, i was disappointed to discover that there was no presentation on this topic at this year's icar. a paper (sofia, ) , which was part of a symposium in antiviral research on ''hepatitis c: next steps toward global eradication'', emphasizes recent successes. after completing therapy, a sustained virological response for weeks (svr ) is regarded as a cure for hcv-infected patients. the combination of sofosbuvir/ledipasvir has shown remarkable results in clinical trials, with svr in the range - % across genotypes. this combination was well tolerated. a nda for the sofosbuvir/ledipasvir combination pill was submitted recently. i do not recall any previous antiviral trials in which the ''intention-to-treat'' analyses showed % success rates. perhaps similar to the hcv symposium in antiviral research, i hope that the icar, which will be held in rome, will have a mini-symposium which will include an account of this remarkable progress. it would be interesting to have an update on the clinical impact of this combination therapy for hcv and to have an assessment on the prospects for global eradication of hcv. beside this one disappointment, there were many excellent presentations and i would like to add my thanks to the isar officers and conference committee for organizing another interesting and successful icar. metabolism and pharmacokinetics of anti-hepatitis c virus nucleotide prodrug gs- beyond sofosbuvir: what opportunity exists for a better nucleoside/nucleotide to treat hepatitis c? selection of an oral prodrug (brl ; famciclovir) for the antiherpesvirus agent brl protection against filovirus diseases by a novel broad-spectrum nucleoside analogue bcx acknowledgements i wish to thank all those authors who have kindly provided me with copies of their presentations and for giving me valuable comments. also, i thank the president of isar for asking me to prepare this meeting report. key: cord- - wsyl vk authors: li, wenmiao; xu, cuijing; hao, cui; zhang, yang; wang, zhaoqi; wang, shuyao; wang, wei title: inhibition of herpes simplex virus by myricetin through targeting viral gd protein and cellular egfr/pi k/akt pathway date: - - journal: antiviral res doi: . /j.antiviral. . sha: doc_id: cord_uid: wsyl vk myricetin, a common dietary flavonoid, was reported to possess many different biological activities such as anti-oxidant, anti-inflammatory, and antiviral effects. in this study, we explored the anti-hsv effects and mechanisms of myricetin both in vitro and in vivo. the results showed that myricetin possessed anti-hsv- and hsv- activities with very low toxicity, superior to the effects of acyclovir. myricetin may block hsv infection through direct interaction with virus gd protein to interfere with virus adsorption and membrane fusion, which was different from the nucleoside analogues such as acyclovir. myricetin also down-regulate the cellular egfr/pi k/akt signaling pathway to further inhibit hsv infection and its subsequent replication. most importantly, intraperitoneal therapy of myricetin markedly improved mice survival and reduced virus titers in both lungs and spinal cord. therefore, the natural dietary flavonoid myricetin has potential to be developed into a novel anti-hsv agent targeting both virus gd protein and cellular egfr/pi k/akt pathway. herpes simplex virus type (hsv- ) and type (hsv- ) are enveloped double stranded dna viruses belonging to herpesviridae (fatahzadeh and schwartz, ) . hsv infection causes skin lesions that are generally localized at the oral, nasal, and ocular level with hsv- infection, whereas with hsv- , infections most commonly occur at genital-skin and mucosa sites (liu and cohen, ; smith and robinson, ) . approximately %- % of the adult human population worldwide is sera-positive for hsv- (carr et al., ) . the current fda approved antiviral agents for herpesviruses involve mainly nucleoside analogues, such as acyclovir (acv) and pencyclovir, which mainly inhibit viral genome replication. despite these successes, drug resistance, and side effects remain unresolved issues in the fight against hsv infections (morfin and thouvenot, ) . hence, the development of novel anti-hsv agents with active mechanisms different from nucleoside analogues is of high importance. myricetin ( , , -trihydroxy- -( , , -trihydroxyphenyl)- -benzopyrone) is a common dietary flavonoid from plant sources such as vegetables, fruits, and tea, with antioxidant properties (ong and khoo, ; ross and kasum, ) . like other flavonoids, myricetin is reported to possess many different biological activities such as antimicrobial, anti-thrombotic, neuroprotective, and anti-inflammatory effects (cushnie and lamb, ; dajas et al., ; gupta et al., ; ong and khoo, ; santhakumar et al., ; semwal et al., ; tian et al., ) . pasetto et al. reported that myricetin possessed anti-hiv- activities with low toxicity, and it mainly inhibited the activity of hiv reverse transcriptase (pasetto et al., ) . lyu et al. found that some flavonoids such as myricetin and quercetinin showed moderate inhibitory effects against hsv- in vero cells (lyu et al., ) . yu and co-workers found that myricetin and scutellarein potently inhibited the sars-cov helicase protein in vitro by affecting the atpase activity, suggesting that myricetin and scultellarein might serve as sars-cov chemical inhibitors (yu et al., ) . thus, myricetin has the potential to be developed into a novel anti-viral agent. to further correlate the anti-viral applications of myricetin with its underlying molecular mechanisms, the anti-hsv effects and mechanisms of myricetin were investigated both in vitro and in vivo in this study. the results showed that myricetin possessed anti-hsv- and hsv- activities with very low toxicity. myricetin may block hsv https://doi.org/ . /j.antiviral. . received october ; received in revised form december ; accepted january infection through direct interaction with virus gd protein to interfere with virus adsorption and membrane fusion. moreover, intraperitoneal therapy of myricetin markedly improved mice survival and reduced virus titers in both lungs and spinal cord. thus, myricetin merits further investigation as a novel anti-hsv agent in the future. myricetin (with purity > %) was purchased from topscience co., ltd. (shanghai, china) . acyclovir was purchased from sigma aldrich (st. louis, mo, usa). vero, hela and hep- cells were routinely cultured in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (excell bio, china), penicillin ( u/ml), and streptomycin ( μg/ml) at °c in % co . hsv- strain f was purchased from atcc (vr- ). hsv- strain was obtained from wuhan institute of virology, chinese academy of sciences. the antiviral activity was evaluated by the cpe inhibition assay (dai et al., ) . briefly, vero cells in -well plates were infected with hsv- or hsv- at a multiplicity of infection (moi) of . , and then treated with indicated concentrations of myricetin in triplicate after removal of virus inoculum. after h incubation, the cells were fixed with % formaldehyde for min at room temperature (rt). after removal of the formaldehyde, the cells were stained with . % (w/v) crystal violet for min at °c. the plates were washed and dried, and the intensity of crystal violet staining for each well was measured at nm. the concentration required for a test compound to reduce the cpe of hsv by % (ic ) was determined. hsv- or hsv- ( - pfu/well) was pre-incubated with or without myricetin for min at °c before infection. then the virusmyricetin mixture was transferred to vero cell monolayers in -well plates, and incubated at °c for h with gentle shaking every min. after that, the inoculum was removed and each well was overlaid with ml of agar overlay media containing . % agarose, u/ml penicillin, and μg/ml streptomycin. the cells were then incubated at °c until plaque sizes were adequate. then the cells were fixed with % paraformaldehyde (pfa) and stained with % crystal violet for plaque counting. vero cells were infected with hsv- or hsv- (moi = . ) under four different treatment conditions: i) pretreatment of virus: myricetin ( μm) pretreated hsv was added to vero cells and incubated at °c for h. then after adsorption, the virus innuculum containing myricetin was removed and the cells were overlaid with compound-free media. ii) pretreatment of cells: vero cells were pretreated with μm of myricetin before hsv infection. iii) adsorption: vero cells were infected in media containing myricetin ( μm) at °c for h. after that, the virus innuculum was removed and the compound-free media were added into cells. iv) post-adsorption: after removal of unabsorbed virus, myricetin ( μm) was added to the cells. at h p.i., virus yields were determined by plaque assay. the indirect immunofluorescence assay was performed as previously described . briefly, for hsv binding assay, hsv- (moi = . ) was adsorbed to hela cells for h at °c in the absence or presence of μm myricetin before washing with pbs. for entry assay, hsv- (moi = . ) was firstly adsorbed to hela cells at °c for h before washing with pbs. then myricetin ( μm) was added to cells and incubated at °c for h. after that, cells were treated with proteinase k to remove adsorbed but not internalized virus. then the cells were washed with pbs and fixed with % paraformaldehyde for min. then cells were permeabilized using . % (v/ v) triton x- in pbs for min before incubated with % bsa for h at °c. after washing, cells were incubated consecutively with anti-icp antibodies ( : dilutions) and dylight ®-conjugated secondary antibodies. then the cell nucleus was stained with dapi for min before confocal imaging. images were recorded using a nikon confocal microscope, and analysed by imagej (nih) version . u (usa). the total rna was extracted from hsv infected vero cells using an rnaiso™ plus kit (takara, japan), and analysed by using the one step sybr primescript rt-pcr kit (takara, japan). the primer pairs for hsv gd and cellular β-actin mrna were listed as follows: gd mrna, ′-agcatcccgatcactgtgtacta- ′ and ′-gcgatggtcaggttgt acgt- '; monkey β-actin mrna, ′-ctccatcctggcctcgctgt- ′ and ′-gctgtcaccttcaccgttcc- '. the real-time rt-pcr was performed at °c min, °c s, cycles of °c s, °c s, followed by melting curve analysis, according to the instrument documentation (abi prism , applied biosystems, usa). all reactions were performed in triplicate and the results were normalized to β-actin. the relative amounts of hsv gd mrna molecules were determined using the comparative ( -ΔΔct ) method, as previously described (livak and schmittgen, ) . darts experiments for identifying the targets of myricetin were performed as previously reported (lomenick et al., ). briefly, hsv- (moi = . ) infected vero cells were lysed with np- cell lysis buffer (beyotime, nantong, china) and treated with or without myricetin ( μm) for h at room temperature (rt) followed by digested with pronase ( μg/ml) in reaction buffer ( mm tris-hcl (ph . ), mm nacl, mm cacl ) for min at rt. the digestion was stopped by directly add × sds-page loading buffer and inactivation by boiling. protein samples were separated with % sds-polyacrylamide gels and analysed by western blot with antibodies against hsv- gd, gb, gc, gh/gl proteins or cellular hvem, nectin- , actin, tubulin proteins as controls. spr assays were conducted on a spr biosensor instrument ge biacoret (ge, usa). hsv- gd proteins (biodragon, beijing, china) were firstly immobilized onto the surface of a carboxymethylated dextran sensor chip (cm ) via amino group coupling as described previously (geng et al., ) . to assess real-time binding of myricetin to the gd proteins on cm chips, myricetin with different concentrations ( , , . , . , . , . μm) dissolved in dmso, was injected over the sensor chip surface with gd immobilized within min, followed by a -min wash with × pbst buffer. the sensor chip surface was then regenerated by washing with naoh ( mm) for s. all binding experiments were carried out at °c with a constant flow rate of μl/s pbs buffer. to correct for non-specific binding and bulk refractive index change, a blank channel without gd was used and run simultaneously for each experiment. then, the biacoret spr evaluation software was used to calculate the kinetic parameters, and the changes in mass due to the binding response were recorded as w. li, et al. antiviral research ( ) resonance units (ru). after drug treatment, the cell lysate was separated by sds-page and transferred to nitrocellulose membrane. after being blocked in trisbuffered saline (tbs) containing . % tween (v/v) and % bsa (w/ v) at rt for h, the membranes were rinsed and incubated at °c overnight with primary antibodies against phosphorylated or nonphosphorylated egfr, pi k, akt antibodies, or anti-α-tubulin and gapdh antibodies (cell signaling technology, danvers, usa) as control. the membranes were washed and incubated with ap-labeled secondary antibody ( : dilutions) at rt for h. the protein bands were then visualized by incubating with the developing solution (pnitro blue tetrazolium chloride (nbt) and -bromo- -chloro- -indolyl phosphate toluidine (bcip)) at rt for min. the relative densities of proteins were all determined by using imagej (nih) v. . u (usa). the cell fusion inhibition assay was performed as described with modifications (du et al., ) . monolayers of vero cells grown in well plates were infected with hsv- (moi = . ) at °c for h. after removal of virus inoculum, compound free media were added to cells and incubated at °c for h. then myricetin ( or μm) was added to cells, and incubated at °c for another h. after that, cells were fixed and detected with hematoxylin-eosin (he) staining. the inhibition of syncytium formation was examined by light microscopy. the influence of myricetin on hsv glycoprotein expression during - h post infection was also evaluated by western blotting. molecular docking was conducted in moe v . (moe, ) . the d structures of myricetin was drawn in chembiodraw and converted to d structure in moe through energy minimization. the d structure of the protein gd was downloaded from rcsb protein data bank (pdb id: myv). prior to docking, the force field of amber : eht and the implicit solvation model of reaction field (r-field) were selected. moe-dock was used for molecular docking simulations of myricetin with gd. the docking workflow followed the "induced fit" protocol, in which the side chains of the receptor pocket were allowed to move according to ligand conformations, with a constraint on their positions. the weight used for tethering side chain atoms to their original positions was . for each ligand, all docked poses of which were ranked by london dg scoring first, then a force field refinement was carried out on the top poses followed by a rescoring of gbvi/wsa dg. the conformations with the lowest free energies of binding were selected as the best (probable) binding modes. molecular graphics were generated by pymol. all animal experiments were performed in accordance with the national institutes of health guide for the care and use of laboratory animals and approved by the institutional animal care and use committee at ocean university of china. three-week-old female balb/ c mice (average weight, . ± . g) were purchased from beijing vital river laboratory animal technology co., ltd. (beijing, china) and raised in a pathogen-free environment ( ± °c and % ± % humidity). mice were randomly divided into five experimental groups ( mice each). mice were anaesthetized and infected via the intranasal route with pfu of hsv- (f strain) in μl of pbs. four hours after inoculation, mice received intraperitoneally treatment of acyclovir ( mg·kg- ), myricetin ( . or mg·kg- ), or placebo, and the treatments were repeated once daily for five days. each day mice were weighed and monitored for signs of illness for days, and those suffering a severe infection or having lost > % of their original body weight were euthanized. to determine virus titers in organs, mice were euthanized, and the lungs, brains and spinal cords were removed, homogenized and clarified by centrifugation on day after inoculation. samples were assayed for virus titers by plaque assay and quantitative rt-pcr assay. histopathological analysis was also performed using h&e staining on lung and brain samples collected on day . all data are representative of at least three independent experiments. data are presented as means ± standard deviations (s.d.). statistical significance was analysed using graphpad prism software using oneway anova with turkey's test. p values < . were considered significant. myricetin is a member of the flavonoid class of polyphenolic compounds, with antioxidant properties, and its structure is shown in fig. a . herein, the anti-hsv effects and mechanisms of myricetin were investigated in vitro. the cytotoxicity of myricetin in vero, hep- and hela cells was firstly assessed by mtt assay (livak and schmittgen, ) . the results showed that myricetin exhibited no significant cytotoxicity at the concentrations from to μm (fig. b) . the cc ( % cytotoxicity concentration) value for myricetin in vero, hep- and hela cells was about . ± . μm, . ± . μm, and . ± . μm, respectively (table ) . myricetin was then assayed for its ability to inhibit hsv multiplication in vitro using cpe inhibition assay and plaque assay . briefly, hsv- (f strain) or hsv- ( strain) (moi = . ) was pretreated with myricetin ( . , . , , μm) for h at °c before infection, then after adsorption, the media containing myricetin at indicated concentrations were added to cells. at h p.i., the viral titers in the culture media were determined by plaque assay, and cell viability was measured by cpe inhibition assay. as shown in fig. c , myricetin significantly reduced the virus titers of both hsv- and hsv- in vero cells when used at the concentration > . μm (p < . ). cpe inhibition assay showed that the ic values of myricetin was about . ± . μm and . ± . μm for hsv- and hsv- , respectively, and the selectivity index (cc /ic ) for myricetin was about . and . , respectively, superior to that of acyclovir (acv) (si = . and . ) ( table ) . myricetin also inhibited hsv replication in hep- and hela cells with si > (table ). in addition, the plaque reduction assay showed that pretreatment of hsv with myricetin at the concentrations of . - μm significantly reduced the number of plaques in both hsv- and hsv- infected cells (fig. d-f) , suggesting that myricetin may be able to inactivate viral particles directly. the time-of-addition assay was performed to determine the stage(s) at which myricetin exerted its inhibition actions in vitro . in brief, myricetin was added to vero cells under four different conditions: pre-treatment of viruses, pre-treatment of cells, during virus adsorption, or after adsorption. subsequently, the antiviral activity was determined by plaque assay. as shown in fig. a , pretreatment of hsv- or hsv- with μm myricetin for h before infection significantly reduced the virus titers of hsv, compared to the non-treated virus control group (p < . ), suggesting that myricetin may have direct w. li, et al. antiviral research ( ) interaction with hsv particles. treatment of myricetin during adsorption or after adsorption also possessed obvious inhibition on virus multiplication (p < . ) ( fig. a) . however, pretreatment of cells did not significantly reduce virus titers in vitro ( fig. a) , suggesting that myricetin may not interact with vero cells directly. moreover, the indirect immunofluorescence assay under these four conditions (pachota et al., ) indicated that pretreatment of virus with myricetin ( μm) before infection also significantly reduced the expression of icp in hsv- infected cells (fig. b) , which was in concern with the results in plaque assay ( fig. a) . thus, myricetin may interact with virus particles to block the adsorption of hsv or inhibit some steps after virus adsorption. since myricetin may be able to block adsorption and post-adsorption processes of hsv, we further explored the potential inhibition of myricetin on hsv binding and entry process by using immunofluorescence assay. briefly, for hsv binding assay, hsv- (moi = . ) was adsorbed to hela cells for h at °c in the absence or presence of μm myricetin before washing with pbs. for entry assay, hsv- (moi = . ) was firstly adsorbed to hela cells at °c for h before washing with pbs. then myricetin ( μm) was added to cells and incubated at °c for h. after that, cells were treated with proteinase k to remove adsorbed but not internalized virus. then the immunofluorescence assay was performed to determine the amount of hsv virions binding to or entering into hela cells. as shown in fig. c and e, myricetin treatment ( μm) during adsorption significantly decreased the fluorescence of icp protein on cell surface, compared to that in non-treated virus control cells, suggesting that myricetin may block virus adsorption process of hsv. moreover, at h p.i., many fluorescence spot of icp could be found at the nucleus area (white arrow indicated), suggesting that hsv- nucleocapsid had been egressed from late endosomes and delivered to the nuclear periphery in hela cells (fig. d) . however, myricetin treatment during entry process dramatically reduced the fluorescence of icp in cytoplasm, and only very little fluorescence could be observed in cell nucleus, suggesting that myricetin may also be able to block hsv entry process in hela cells (fig. d, f and g ). thus, myricetin may possibly block hsv infection mainly through interfering with hsv binding and entry process. since myricetin may block hsv binding and entry process, we then asked whether myricetin could inhibit the virus-induced membrane fusion process. as shown in fig. a , in hsv- (moi = . ) infected vero cells, obvious syncytia with multinuclear cells were observed in non-treated virus control group (hsv- ) at h p.i. however, treatment with myricetin ( , μm) during - h p.i. markedly blocked syncytium formation only with a limited number of small syncytia, suggesting that myricetin may inhibit hsv-induced cell fusion. in addition, myricetin treatment during - h p.i. did not obviously influence the expression of virus surface glycoproteins such as gd protein (fig. b) . thus, myricetin may directly block hsv-induced membrane fusion through inhibiting the function of virus surface glycoproteins rather than reducing their expression. since myricetin can inhibit both hsv adsorption and membrane fusion, we then questioned whether myricetin directly interacts with virus surface gd protein which is mainly required for hsv entry process. we employed a drug affinity responsive target stability assay (darts) (lomenick et al., ) , which relies on the reduction of protease susceptibility of the target protein upon drug binding, to detect the potential interaction between myricetin and gd protein. in brief, hsv- infected vero cell lysates were digested with pronase in the presence or absence of myricetin ( μm), then the amount of gd protein in cell lysates was detected by western blotting. as shown in fig. c , hsv- gd protein was obviously protected by protease digestion in the extracts of myricetin ( μm) treated cells especially under pronase treatment at μg/ml, suggesting that myricetin may interact with hsv gd protein in hsv- infected vero cells. however, myricetin ( μm) could not protect other virus surface glycoproteins such as gb, gc, and gh/gl proteins from protease digestion in hsv- infected cells (fig. d) . similarly, the cellular surface receptors of hsv- such as hvem and nectin- could also not be protected from protease digestion by myricetin ( μm) (fig. d) . thus, myricetin may interfere with hsv binding and entry process through targeting virus gd protein rather than cellular receptors of hsv. moreover, the direct interaction between myricetin and gd protein was further evaluated by using spr assay (geng et al., ) . briefly, with hsv- gd proteins being immobilized on the chip, myricetin at the concentrations of . - μm was flowed over the biosensor chip surface, respectively. data revealed a marked binding of myricetin to virus gd protein in a concentration-dependent manner with a kd equivalent to about . e- m ( . nm), implicating a high affinity of myricetin for hsv- gd protein (fig. e) . thus, pretreatment of hsv were infected with hsv- or hsv- (moi = . ) using four different treatment conditions. i) pre +virus: hsv was pretreated with myricetin ( μm) at °c for h before infection. ii) pre+cell: vero cells were pretreated with μm of myricetin before infection. iii) adsorption: vero cells were infected in media containing myricetin ( μm) at °c for h. iv) post-adsorption: after removal of unabsorbed virus, myricetin ( μm) was added to the cells. at h p.i., virus yields were determined by plaque assay. the results were presented as mean ± s.d. from five independent experiments. *p < . vs. virus control group. (b) vero cells were infected with hsv- (f strain) under four treatment conditions of myricetin, and the expression of virus icp protein was detected by immunofluorescence assay. scale bar represents μm. (c and d) hsv- (moi = . ) infected hela cells were treated with or without myricetin ( μm) during adsorption process (c) or entry process (d), then after washing three times with pbs, the immunofluorescence assay was performed by using anti-icp antibody to evaluate the amount of virions binding to or entering into cells. scale bar represents μm. (e-g) the average fluorescence intensity of icp proteins during adsorption process (e) or entry process (f) was measured by imagej (nih) version . u (usa) to calculate the average intensity per cell of different images (n = ). the average intensity of icp in cell nucleus during entry process (g) was also measured by imagej (usa) to calculate the average intensity per unit area of cell nucleus of different images (n = ). the average intensity for nontreated virus control cells (hsv) was assigned values of and the data presented as mean ± s.d. (n = ). significance: **p < . vs. virus control group (hsv). w. li, et al. antiviral research ( ) with myricetin before infection may allow myricetin to fully bind gd protein and form a stable myricetin-gd complex. furthermore, the impact of myricetin on hsv- gd binding to vero cells was further explored using a cell enzyme-linked immunosorbent assay, as previously described (tiwari et al., ) . briefly, vero cells were first treated with or without myricetin ( , , , μm) for h, then the hsv- gd protein or myricetin ( , , , μm) pretreated gd protein ( μg/ml) were added to cells and incubated for another h, the hsv- gd proteins were firstly immobilized onto the surface of a carboxymethylated dextran sensor chip (cm ). to assess realtime binding of myricetin to the gd proteins on cm chips, myricetin ( , , . , . , . , . μm) was flowed over the biosensor chip surface. the sensorgram for all binding interactions were recorded in real time and were analysed after subtracting the sensorgram from the blank channel. then, the changes in mass due to the binding response were recorded as resonance units (ru). (f) hsv- gd binding assay. vero cells were first treated with or without myricetin ( , , , μm) for h, then the hsv- gd protein or myricetin ( , , , μm) pretreated gd protein ( μg/ml) were added to cells and incubated for another h, respectively. then the amount of gd protein binding to vero cell surface was detected by elisa assay using anti-gd antibody and hrp labeled secondary antibody sequentially. values are means ± sd (n = ). significance: **p < . vs. virus control group. (g and h) the d (g) and d (h) binding mode of myricetin with hsv gd protein (pdb id: myv) was shown. myricetin is colored in purple, the surrounding residues in the binding pockets are colored in cyan. the backbone of the receptor is depicted as lightblue cartoon. (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) w. li, et al. antiviral research ( ) respectively. then the amount of gd protein binding to vero cell surface was detected by sequentially incubated with anti-gd antibody and hrp labeled secondary antibody. as shown in fig. f , pre-incubation of gd protein with myricetin ( , , , μm) significantly reduced the binding of gd to cell surface in a dose-dependent manner, however, pretreatment of cells did not significantly decrease the amount of gd protein on cell surface. this finding strongly supports that myricetin may interact with hsv gd protein rather than cellular receptors to block virus binding and entry. to further investigate the binding mode of myricetin with gd, docking simulation studies were carried out using the crystal structure of hsv- gd protein (pdb code: myv). the results indicated that the oxygen atom of carbanyl group of myricetin, regarded as hydrogen bond acceptor, forms two hydrogen bonds with the nitrogen atom of amide group of gln as well as asn in gd (fig. g) . the oxygen atom of phenolic hydroxyl group of myricetin, regarded as hydrogen bond donor, forms a hydrogen bond with the oxygen atom of backbone of leu in gd. the oxygen atom of hydroxyl group of myricetin, regarded as hydrogen bond donor, forms a hydrogen bond with the oxygen atom of carboxyl group of glu in gd ( fig. g and h) . thus, myricetin may interact with gln , leu , glu and asn of gd through hydrogen bond interactions. since myricetin may inhibit hsv entry process in hela cells, we further explored whether myricetin could influence the hsv infectionrelated signaling pathways by using western blot assay. the egfr/ pi k/akt signaling pathway was reported to be required for virus endocytosis and replication, and the inhibitors of pi k signaling could inhibit both entry and fusion of hsv (eierhoff et al., ; tiwari and shukla, ) . herein, treatment with myricetin ( , μm) for h significantly decreased the levels of phosphorylated egfr from about . to about . and . -fold of normal control group, respectively (p < . ) (fig. a and d) . moreover, the activation of downstream signal pi k was also evaluated, and the results showed that treatment with myricetin ( , , μm) for h significantly decreased the levels of phosphorylated pi k proteins from about . to about . , . , and . -fold of normal control group, respectively (p < . ) (fig. b and e). in addition, treatment with myricetin ( , , μm) for h significantly reduced the activation of akt from about . to about . , . , and . -fold of normal control group, respectively (p < . ) ( fig. c and f) . however, myricetin ( . , , , μm) treatment did not significantly influence the total expression levels of egfr, pi k and akt proteins in hsv- infected vero cells (fig. g and h) . thus, cellular egfr/pi k/akt signaling pathway may be involved in the anti-hsv fig. . involvement of egfr/pi k/akt signaling pathway in the anti-hsv actions of myricetin. (a-c) hsv- (moi = . ) infected cells were treated with or without myricetin ( . , , , μm) for h, and then the phosphorylation of egfr (a), pi k (b), and akt (c) was evaluated by western blot. blots were also probed for gapdh and α-tubulin proteins as loading controls. the result shown is a representative of three separate experiments. (d-f) quantification of immunoblot for the ratio of p-egfr (d), p-pi k (e), p-akt (f) protein to gapdh or tubulin, respectively. the ratio for non-infected cells (m) was assigned values of . and the data presented as mean ± s.d. (n = ). significance: ##p < . vs. normal control group (mock); *p < . , **p < . vs. virus control group (hsv). (g) hsv- (moi = . ) infected cells were treated with or without myricetin ( . , , , μm) for h, and then the total expression levels of egfr, pi k, and akt were evaluated by western blot. the result shown is a representative of three separate experiments. (h) quantification of immunoblot for the ratio of egfr, pi k, akt protein to α-tubulin, respectively. the ratio for non-infected cells (mock) was assigned values of . and the data presented as mean ± s.d. (n = ). w. li, et al. antiviral research ( ) actions of myricetin in vitro. furthermore, myricetin ( . , , , μm) treatment could not significantly influence the activation of egfr, pi k and akt proteins in the non-infected vero cells ( fig. a and b) , suggesting that the inhibition of egfr/pi k/akt pathway by myricetin may be related to its inhibition of hsv infection. moreover, treatment with hsv- gd protein ( μg/ml) could significantly enhance the phosphorylation of egfr, pi k, and akt proteins in the non-infected vero cells (p < . ), suggesting that the activation of egfr/pi k/akt pathway in hsv infected cells may be related to the binding of gd to cellular receptors ( fig. c and d) . however, after myricetin treatment ( , , μm), the levels of phosphorylated egfr, pi k, and akt proteins were significantly reduced as compared to the gd treated control group (p < . ) ( fig. c and d) . in addition, myricetin treatment ( , , μm) also significantly reduced the expression level of gd mrna in hsv- and hsv- infected vero cells (p < . ) ( fig. e and f) . considered that egfr/pi k/akt pathway is required for efficient hsv replication, we suppose that myricetin may be able interact with virus gd protein to interfere with the activation of egfr/pi k/akt pathway to further inhibit subsequent replication of hsv. the anti-hsv effects of myricetin were further tested in a murine intranasal model of hsv pneumonia and encephalitis (de clercq and luczak, ) . in brief, hsv- (f strain)-infected mice received intraperitoneal treatment of myricetin ( . or mg·kg- ), acyclovir ( mg·kg- ), or placebo (pbs) once daily for five days. as shown in fig. a , intraperitoneal treatment of myricetin ( . or mg·kg- ) significantly increased survival rates as compared to the placebotreated control group (p < . ). by day post infection, only % of the individuals in the placebo group survived whereas % of animals in the myricetin-treated group survived, superior to that in acyclovir ( mg·kg- )-treated group ( %). moreover, the body weights of mice in virus control group (placebo) began to decrease at days p.i., losing up to % of initial weight, before gradually recovering. however, myricetin ( . or mg·kg- )-treated mice gradually increased their body weights only with a limited weight loss of less than % during the infection, superior to those in the acyclovir ( mg·kg- ) treated group (fig. b) . furthermore, four days post-infection, four mice of each treatment group were sacrificed and the tissue samples were removed for further analysis. subsequently, the viral titers in the lung and spinal cord were determined by plaque assay and quantitative rt-pcr. the results showed that after treatment of myricetin for four days, the pulmonary virus titers significantly decreased compared to that of the virus control group (p < . ) (fig. c ). the relative virus gd mrna levels in spinal cord also significantly reduced compared to that of the placebo group, suggesting that intraperitoneal therapy with myricetin could inhibit hsv multiplication in mice (fig. d) . acyclovir ( mg·kg- ) treatment also showed significant reduction of virus titers in mice lungs and spinal cord ( fig. c and d) . to further evaluate the effects of myricetin on viral pneumonia and encephalitis in mice, histopathology analysis was also performed. the results showed that the lung tissues in virus-control group showed marked infiltration of inflammatory cells and the presence of massive hyperaemia in the lumen (fig. e) . however, mice treated with myricetin ( . or mg·kg- ) following infection had intact columnar epithelium even in the presence of tiny serocellular exudates in the lumen fig. . the inhibition of egfr/pi k/akt pathway by myricetin may be related to its inhibition of gd protein. (a) non-infected vero cells were treated with or without myricetin ( . , , , μm) for h, and then the phosphorylation of egfr, pi k, and akt was evaluated by western blot. blots were also probed for α-tubulin proteins as loading controls. the result shown is a representative of three separate experiments. (b) plots quantifying the immunoblots (as ratios to tubulin) for p-egfr, p-pi k and p-akt proteins. the ratios for non-treated cells (mock) were assigned values of . and the data presented as mean ± s.d. (n = ). (c) vero cells were treated with or without gd proteins in the presene or absence of myricetin ( , , μm) for h, and then the phosphorylation of egfr, pi k, and akt was evaluated by western blot. blots were also probed for gapdh proteins as loading controls. the result shown is a representative of three separate experiments. (d) quantification of immunoblot for the ratio of p-egfr, p-pi k, p-akt protein to gapdh, respectively. the ratio for non-treated control cells (mock) was assigned values of . and the data presented as mean ± s.d. (n = ). significance: ##p < . vs. normal control group (mock); *p < . , **p < . vs. gd treated control group (gd). (e and f) hsv (moi = . ) infected vero cells were treated with myricetin ( . , , , μm), and incubated at °c for h. after that, total rna was extracted for real-time rt-pcr assay of hsv- (e) and hsv- (f) gd mrnas and cellular β-actin mrna. the relative amounts of hsv gd mrnas were determined using the comparative ( -ΔΔct ) method. rna levels for non-treated virus control cells (hsv) were assigned values of . . values are means ± sd (n = ). significance: *p < . , **p < . vs. virus control group. w. li, et al. antiviral research ( ) fig. . the anti-hsv actions of myricetin in vivo. (a) survival rate. hsv- infected mice were received intraperitoneally treatment of myricetin ( . or mg·kg- ), acyclovir ( mg·kg- ), or placebo (pbs) once daily for five days. results are expressed as percentage of survival, evaluated daily for days. significance: *p < . , **p < . vs. virus control group (placebo). (b) hsv infected mice were received intraperitoneally treatment with indicated compounds for five days. the average body weights in each group were monitored daily for days and are expressed as a percentage of the initial value. (c and d) viral titers in lungs and spinal cord. after intraperitoneal therapy with indicated compounds for five days, the viral titers in lungs (c) and spinal cords (d) were evaluated by plaque assay and quantitative rt-pcr assay on day p.i., respectively. values are means ± s.d. (n = ). significance: *p < . , **p < . vs. virus control group. (e and f) histopathology analyses of lung and brain tissues on day p.i. by he staining ( × and × ). the representative micrographs from each group were shown. mock: non-infected mice tissues; hsv- : hsv- infected tissues without drugs; hsv- +acyclovir- : hsv- infected tissues with acyclovir ( mg·kg- ) treatment; hsv- +myricetin- : hsv- infected tissues with myricetin ( mg·kg- ) treatment; hsv- +myricetin- . : hsv- infected tissues with myricetin ( . mg·kg- ) treatment; the black arrows indicate the presence of hyperaemia and infiltration of inflammatory cells in the lumen. ( fig. e ). in addition, treatment of myricetin ( mg·kg- ) nearly had no hyperaemia and infiltration of inflammatory cells in brain tissues (fig. f) . myricetin ( . mg·kg- ) treated mice only had tiny hyperaemia in the brain tissues, comparable to the effects of acyclovir ( mg·kg- ) (fig. f) . thus, myricetin may be able to attenuate pneumonia and encephalitis symptoms in hsv infected mice. myricetin is a member of the flavonoid class of polyphenolic compounds, which was reported to possess antiviral activities against lots of viruses including hiv and sars-cov (ong and khoo, ) . in this study, we discovered that myricetin possessed anti-hsv- and hsv- activities in different cells with very low toxicity (si > ). myricetin may block hsv infection through direct interaction with virus gd protein to interfere with virus adsorption and membrane fusion, which was different from the nucleoside analogues such as acyclovir. most importantly, intraperitoneal therapy of hsv- -infected mice with myricetin markedly improved their survival and reduced virus titers in both lungs and spinal cord. pretreatment of hsv by myricetin before infection significantly reduced the virus titers in hsv infected cells, suggesting that myricetin may have direct interaction with hsv particles. in addition, myricetin can block hsv induced membrane fusion and interfere with virus binding and entry process, suggesting that myricetin may interact with some surface glycoproteins of hsv. the results of darts assay and gd binding assay indicated that myricetin may interact with virus gd protein rather than cellular receptors of hsv to block virus binding and entry (fig. ) . the data from spr assay verified that myricetin can truly bind to hsv- gd protein with high affinity (kd ≈ . nm) (fig. e) . the molecular docking analysis indicated that myricetin may interact with gln , leu , glu and asn of gd protein through hydrogen bond interactions ( fig. g and h) . furthermore, myricetin treatment inhibited the activation of egfr, pi k and akt proteins in hsv infected or gd treated non-infected cells, and reduced the mrna levels of virus proteins in hsv-infected cells (fig. ) . considered that egfr/pi k/akt pathway is required for efficient hsv replication (tiwari and shukla, ) , we suppose that myricetin may interact with virus gd protein to interfere with the activation of egfr/pi k/akt pathway so as to further inhibit subsequent replication of hsv. the mouse model of hsv intranasal infection has been used for studying hsv induced pneumonia and encephalitis (gamba et al., ) . in this study, intraperitoneal therapy of hsv- -infected mice with myricetin markedly improved their survival and inhibited hsv multiplication in both lungs and spinal cord (fig. ) . moreover, the histopathological analysis indicated that myricetin treatment also significantly attenuated the pneumonia and encephalitis symptoms in hsv-infected mice, comparable to the effects of acyclovir. in contrast to acyclovir, myricetin can block hsv binding and entry process with low toxicity, suggesting that it may be used for therapy and prophylaxis of hsv infection. however, oral administration of myricetin as an antiviral may be unlikely to be successful because of its rapid metabolism. thus, myricetin may be used for prevention and treatment of herpes simplex pneumonia and encephalitis by intravenous administration in the future. in summary, myricetin possessed anti-hsv activities both in vitro and in vivo with low toxicity. myricetin may block hsv infection through direct interaction with virus gd protein to interfering with virus adsorption and membrane fusion. in addition, cellular egfr/ pi k/akt pathway was also involved in the anti-hsv actions of myricetin. thus, myricetin merits further investigation as a novel anti-hsv agent targeting both virus gd protein and host egfr/pi k/akt pathway. further studies of the anti-hsv effects of myricetin against clinical strains especially the acyclovir-resistant strains will be required to advance it for drug development. nevertheless, myricetin has the potential to be developed into a novel antiviral injection for therapy of herpes simplex encephalitis and pneumonia in the future. none declared. the immune response to ocular herpes simplex virus type infection antimicrobial activity of flavonoids antiviral effects of abma against herpes simplex virus type in vitro and in vivo neuroprotection by flavonoids. braz intranasal challenge of mice with herpes simplex virus: an experimental model for evaluation of the efficacy of antiviral drugs a novel glycoprotein d-specific monoclonal antibody neutralizes herpes simplex virus the epidermal growth factor receptor (egfr) promotes uptake of influenza a viruses (iav) into host cells human herpes simplex virus infections: epidemiology, pathogenesis, symptomatology, diagnosis, and management early inhibition of nitric oxide production increases hsv- intranasal infection the potential 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national natural science foundation of china ( , , ), nsfc-shandong joint fund (u , u ), shandong provincial natural science foundation (zr mh ), and qingdao marine biomedical science and technology innovation center ( -cxzx - - ). supplementary data to this article can be found online at https:// doi.org/ . /j.antiviral. . . key: cord- - san m authors: martin, baptiste; hoenen, thomas; canard, bruno; decroly, etienne title: filovirus proteins for antiviral drug discovery: a structure/function analysis of surface glycoproteins and virus entry date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: san m this review focuses on the recent progress in our understanding of filovirus protein structure/function and its impact on antiviral research. here we focus on the surface glycoprotein gp( , ) and its different roles in filovirus entry. we first describe the latest advances on the characterization of gp gene-overlapping proteins sgp, ssgp and Δ-peptide. then, we compare filovirus surface gp( , ) proteins in terms of structure, synthesis and function. as they bear potential in drug-design, the discovery of small organic compounds inhibiting filovirus entry is a currently very active field. although it is at an early stage, the development of antiviral drugs against ebola and marburg virus entry might prove essential to reduce outbreak-associated fatality rates through post-exposure treatment of both suspected and confirmed cases. entry into the host cell via a complex mechanism that is, to date, only partially understood. drawing on the present knowledge, numerous research groups have recently developed antiviral strategies aiming to inhibit viral entry. this review presents current knowledge on filovirus glycoproteins, and compares their structures and associated functions. after briefly placing filoviruses in viral classification and nomenclature, we describe the soluble gp derivatives sgp, ssgp and d-peptide, and review all structural features of the surface gp , . we detail step by step the mechanism by which gp , triggers virus entry. in this part, the aim is to highlight the relationship between structural features and their role in the various phases of the filovirus entry process, with attention to idiosyncrasies within the family filoviridae. lastly, we recapitulate existing and ongoing antiviral strategies, in order to connect mechanisms of action to structure/function analysis aiming at potent anti-filovirus therapies. the family filoviridae belongs to the negative strand, nonsegmented (nns) rna viruses of the mononegavirales order. this family groups highly pathogenic viruses such as those found in the marburgvirus and ebolavirus genera (ascenzi et al., ) , responsible for severe hemorrhagic fevers, as well as the genus cuevavirus (negredo et al., ) , the latter being found so far only in form of rna sequenced from bats (fig. ) . the marburgvirus genus is represented by viruses within a single species, marburg marburgvirus (marburg virus -marv). it was the first filovirus genus and species discovered in during related outbreaks in frankfurt (germany) and belgrade (yugoslavia) upon importation of infected monkeys from uganda to marburg (germany) (siegert et al., ) . the ebolavirus genus consists of five virus species. they are known as zaire ebolavirus (ebola virus -ebov), which is the first ebolavirus species identified in in the democratic republic of the congo (formerly northern zaire) near the ebola river, sudan ebolavirus (sudan virus -sudv), taï forest ebolavirus (taï forest virus -tafv), bundibugyo ebolavirus (bundibugyo virus -bdbv) and reston ebolavirus (reston virus -restv) according to the new nomenclature (kuhn et al., ) . while restv has not been described to cause human disease yet, the other species, including marv, are highly pathogenic with fatality rates ranging from % up to % (feldmann and geisbert, ) . the cuevavirus genus was established after the discovery of sequences in most likely belonging to a new filovirus, lloviu cuevavirus (lloviu virus -llov), presumably infecting bats in asturias (spain) (negredo et al., ) . since it is a novel entry in the filovirus phylogeny, only little is known about its biology and putative infectivity in humans. with their high infectivity and their ability to impair the immune system (feldmann and geisbert, ; ramanan et al., ) , filoviruses trigger an abrupt onset of symptoms including fever, headache, myalgia and gastrointestinal disorders. next, hemorrhagic manifestations can arise during the peak of illness. shock, convulsions, coagulopathy and multi-organ failure appear later and are fatal in many cases (feldmann and geisbert, ; nina, ) . unfortunately, there are no approved antivirals or vaccines available yet, although significant progress has been made lately in this respect (mendoza et al., ) , but supportive treatments such as rehydration and control of fever and pain might help patients to overcome infection. lately, a lot of efforts have been put together to identify key viral targets in order to inhibit the viral cycle and help to cure the infection (choi and croyle, ) . filoviruses share a common genomic organization. their nns rna genome of around kb carries seven main genes leading to the synthesis of the different viral proteins (figs. and ) (ascenzi et al., ) . all these proteins are essential to establish an infection leading to efficient virus replication (fig. ) . the sole surface protein gp , triggers the first steps of cell infection, which requires attachment to factors present at the surface of target dendritic cells (dcs) and monocytes/macrophages, and on endothelial cells of liver sinusoids and lymph node sinuses. once attached, the virions are internalized, and endosomal events induce fusion (feldmann et al., ) allowing the release of the viral particle content into the cytoplasm. the nucleocapsid is composed of the genomic rna in complex with the nucleoprotein np, the two cofactors vp and vp , and the large protein l, which form a large macromolecular complex protecting the rna genome and facilitating genome replication/transcription (reviewed by mühlberger, ) . the l protein harbors the rna-dependent rna polymerase (rdrp) activity, which is essential for both genome replication and transcription. in addition, this protein carries yet uncharacterized enzymatic activities involved in rna transcriptional modifications such as rna capping and polyadenylation, protecting viral mrna from both degradation and detection by the host cell innate immunity guardians (mühlberger, ; liang et al., ) . the nucleoprotein np enwraps and protects the nns rna from host nucleases. the vp protein acts as a transcription cofactor, while vp is the polymerase cofactor (mühlberger, ) . after replication of the viral genome and rna transcription, nascent viral particles are assembled in a process mediated by the matrix protein vp , and virus budding occurs at the cell surface membrane in a process that involves hijacking the host escrt machinery (hartlieb and weissenhorn, ; noda et al., ) . early stages of cell infection have been shown to be mediated by the class i viral glycoprotein gp , exposed at the virus membrane surface (reviewed by lee and saphire, ). this protein is synthesized as a precursor gp after translation of an edited gp open reading frame (fig. ) , which is cleaved to yield an ectodomain gp fig. . filovirus genome organization. filoviruses are a family of non-segmented negative single stranded rna viruses, including the genera ebolavirus, marburgvirus, and cuevavirus, with the respective prototype viruses ebola virus (ebov), marburg virus (marv) and lloviu virus (llov) sharing a common genome organization. their genome of about kb codes for at least well defined monocistronic mrnas with the exception of one bicistronic mrna in the llov genome. for ebov and marv the first and last nucleotides in the mrnas are indicated, whereas for llov exact mrna ends are still unclear, but lengths are roughly estimated (*). and a trans-membrane fusion domain gp . viral cell entry being a critical point for infection, this step has been targeted for the design of antiviral molecules. it is also noteworthy that the gp gene codes for additional proteins, whose functions are not completely understood. the mechanism driving the expression of these proteins is described below (fig. ) . the gp gene is the fourth gene along the genome of every filovirus (sanchez et al., ; negredo et al., ) . all filovirus gp genes encode a trans-membrane protein gp , localized at the virus surface. in ebolaviruses and presumably in cuevaviruses, unedited and edited transcripts produce several forms of gp, which, together with host furin-dependent proteolysis, lead to the expression of additional proteins: the soluble gp (sgp) described in ebolaviruses and cuevaviruses, the dÀpeptide, and the small soluble gp (ssgp) (fig. ) . it has been proposed that this edition mechanism limits the surface gp-associated cytotoxicity (see .) (volchkov et al., ; mohan et al., ) . the rna editing of ebolaviruses involves a slippage region composed of seven consecutive template uridines where viral polymerase stuttering results in a frameshift in the middle of the gp sequence (volchkov et al., ; sanchez et al., ; mehedi et al., ) . indeed, this editing mechanism has been recently shown to be regulated by neighboring sequences of the uridine template, probably in synergy with vp as a transacting factor (mehedi et al., ) . thus, the ebolavirus gp gene is able to generate three different mrnas coding for protein precursors pre-sgp, pre-gp and ssgp in a ratio of approximately : : , respectively, albeit the exact ratio is cell type dependent (fig. ) (mehedi et al., ) . in ebolaviruses, the most abundant product is the unedited transcript pre-sgp mrna, which leads to the synthesis of the protein precursor pre-sgp. remarkably, it is not incorporated into the virion structure per se. this precursor of about kda is cleaved by cellular proteases of the furin family at the c-terminus of the r-x-r-ry conserved motif at position . this cleavage forms the final sgp and the d-peptide (fig. ) . the amino-acid carboxy-terminal d-fragment is subsequently highly modified at the post-translational level (o-glycosylation) before being secreted. although the d-peptide function has not completely been understood yet, it has been suggested that this peptide regulates filovirus entry since its expression limits infection on filovirus-permissive cells . moreover, based on in silico analysis this peptide was also proposed to act as a virulence factor forming a lytic viroporin, although experimental evidence for such a function is lacking (gallaher and garry, ) . efforts have been devoted to characterize sgp because it shares its first n-terminal residues with gp , and ssgp (volchkov et al., ) . after cleavage, sgp monomers bind to each other in a parallel orientation by means of two disulfide bonds involving residues cys and cys (falzarano et al., ) . due to the lack of a transmembrane domain, sgp forms a soluble dimer, mainly n-glycosylated, of kda. its roles have been recently investigated in vitro and in vivo. indeed, sgp has been suggested as a virulence factor , although currently it is not entirely clear whether or not this is the case, with one study indicating that abolishment of sgp production did not lead to a noticeable attenuation of the virus . however, the fast emergence of revertants both in vivo and in vitro (in certain cell lines) when sgp production is reduced by introducing an a genotype (i.e. uridine residues in the virus genome, leading to production of predominantly a mrnas) in the editing site suggests an important role of sgp in the biology of ebolaviruses (volchkova et al., ; kugelman et al., ; hoenen et al., ; tsuda et al., ) . in vitro, sgp exerts vascular effects, notably the restoration of the barrier function suggesting an anti-inflammatory role (wahl-jensen et al., ) ; however, the relevance of this finding is currently not clear. also, there is increasing evidence that sgp might reduce viral cytotoxicity by limiting the amount of expressed gp , (see .) (iwasa et al., ; mohan et al., ) . finally, it has been shown that secreted sgp might also lead to immune subversion, and act as a decoy for antibodies directed against gp , (ito et al., ; mohan et al., ) . however, for all these roles of sgp (reviewed by de la vega et al., ) further investigations are required to ascertain what relevance they really have for ebolavirus biology, and whether this protein represents a potential antiviral target. another protein product of the ebolavirus and cuevavirus gp gene is ssgp, a small protein of kda that is synthesized from a transcript in which either one adenosine is deleted or two are added during transcriptional editing (fig. ) . it has been shown that ssgp is secreted as a kda dimer carrying a disulfide linkage between cys of each monomer, the latter being largely n-glycosylated (mehedi et al., ) . although ssgp shares similar structural properties with sgp (and gp , ), it does not seem to exert the same anti-inflammatory function on endothelial cells (mehedi et al., ) , and its role in viral pathogenicity, as well as its potential as antiviral target, remains unclear. . schematic ultrastructure of a filoviral particle. the viral protein assembly leads to the formation of filamentous viral particle able to infect host target cell and carrying the required material to complete a viral replication cycle. the surface glycoprotein gp , triggers viral attachment and entry. then, the nucleocapsid components, the rna-protecting nucleoprotein np, the viral proteins (vp) and and the "large" (l) polymerase, are released into the cytoplasm for replication and transcription, resulting in synthesis of new viral genomes and proteins. vp aids in nucleocapsids assembly, while the matrix protein vp orchestrates the formation of new virions. the gp , rna transcript codes for the gp precursor. this transcript results from a polymerase slippage on its template resulting in an additional adenosine on ebolavirus (and presumably cuevavirus) gp , mrnas, unlike in the case of the unedited marburgvirus mrnas ( fig. ) (volchkov et al., ; sanchez et al., ) . mrnas are then translated into the gp precursor, which transits through the endoplasmic reticulum and the golgi apparatus, where it is cleaved by furin-like protease(s) into two proteins, gp and gp . the position of the conserved cleavage site is variable inside the family filoviridae, but gene structure and functional organization are homologous (volchkov et al., ; manicassamy et al., ; maruyama et al., ) . indeed, these two proteins together form a trimeric chalice structure made of three gp and three gp subunits (figs. and ) assembled by gp /gp and gp / gp interactions (simmons, ) . the bowl of the chalice is shaped by the gp subunits, while gp organizes and anchors the complex to the membrane. in the trimer, gp , s are bound to each other by disulfide bonds -between the cys of gp and the cys of gp as described for ebolaviruses (lee et al., ) -leading to a complex and metastable intricacy. the ectodomain gp is constituted of a core protein and a mucin-like domain (mld), which is largely glycosylated (simmons, ) . this mld has not been structurally defined yet, but there are slight differences in terms of sequence, length, and position relative to the cleavage site (volchkov et al., ; sanchez et al., ) between different filovirus genera. indeed, ebolaviruses hold their mld as a single unit on gp whereas marburgviruses and cuevaviruses carry it in two blocks, one in gp and the other in gp . despite these differences, it has been proposed that this feature plays a common role both in the attachment of the virus via lectins and in immune escape by hiding potential conserved epitopes (simmons, ) . actually, known epitopes targeted by neutralizing anti-ebolavirus antibodies such as kz and f have been shown to lie in an uncovered domain present at the interface between gp and gp (lee et al., ; dias et al., ) . similarly, the antibody mr , which was identified in a marburgvirus disease survivor, targets key residues for receptor binding, which are masked in case of ebolaviruses by the mld, but more accessible in case of marburgviruses (hashiguchi et al., ) . the fact that such a cross-reactive antibody targeting the receptor-binding site (rbs) was found in a marburgvirus disease survivor, but not in ebolavirus disease survivors, is used as a further argument to strengthen the case for a function of the mld in immune escape. the core of gp is subdivided into three domains: the glycan cap, the head, and the base (lee et al., ) . the glycan cap is the outer part of gp forming the chalice. the head supposedly helps structuring the metastable pre-fusion conformation. this part is exposed to the host membrane surface carrying the putative rbs. the base subdomain supports the linkage with gp and stabilizes the metastable pre-fusion conformation. thus, gp has the required structural features to mediate viral attachment to cell receptors (see . ). the trans-membrane gp protein anchors the complex to the viral membrane, but also manages virus entry and fusion (figs. and ) (simmons, ) . its structure/function complexity has been well described for ebolaviruses (lee et al., ) . briefly, its structure incorporates a transmembrane domain, a short cytoplasmic tail, an internal fusion loop defined by a disulfide bound between gp cys and cys , and two heptad repeat regions (hrr and hrr ) surrounding the fusion peptide. this domain constitutes the unstable pre-fusion conformation of gp , which rearranges itself at low ph to trigger fusion. to maintain the structure in the pre-fusion state, the gp head packs the gp hydrophobic fusion peptide and stabilizes gp . such features have not ( ), the viral particle is processed in the endosome by proteases ( ) leading to receptor recognition ( ) that triggers fusion and release of nucleocapsids into the host cytoplasm ( ) . negative strand rna is transcribed into messenger rnas ( ), allowing translation and protein synthesis to occur, which facilitates further secondary transcription, as well as replication through a complimentary positive sense rna ( ). gp , transits through the rough endoplasmic reticulum/golgi apparatus pathway ( ). then, budding occurs by diverting host trafficking machinery ( ), leading to the formation of new virions ( ). been clearly described yet for marburgviruses and cuevaviruses, but sequence alignments and in vitro data suggest similar conformations and entry/fusion mechanisms (manicassamy et al., ; maruyama et al., ; liu et al., ) . . gp, a fusion protein mediating filovirus cell entry gp , orchestrates viral entry, which can be seen as a three step mechanism: attachment, uptake, and fusion ( fig. ) (olejnik et al., ) . the attachment between a filovirus and its target cell is mediated by gp , but the identity of the main cellular attachment factor remains unclear (reviewed by takada, ) , and in fact it appears that there are numerous proteins at the cell surface that can fulfill this function. first, c-type lectin family members such as hmgl on immature dendritic cells and macrophages (takada et al., ; matsuno et al., a) or asialo-glycoprotein, dc-sign, l-sign, l-sectin ebolavirus gp gene products. gp genes are roughly similar for ebolaviruses, marburgviruses, and cuevoviruses, with the notable exception that the marburgvirus gp gene does not undergo transcriptional editing, but only encodes gp . the ebolavirus gp gene, like the cuevovirus gp gene, contains a poly-u repeat (stuttering region, sr), facilitating an editing mechanism that results in the synthesis of three different mrnas, leading to the synthesis of sgp (shown on the left), gp (shown in the center), and ssgp (shown on the right). all these mrnas contain a signal sequence (ss) and the coding sequence for the different proteins. they share a common sequence (grey) leading to an identical aminoterminus for all gp-proteins. mrnas are translated into pre-proteins transiting through the endoplasmic reticulum and the golgi apparatus. during this intracellular trafficking, the signal peptide (sp) is removed; the protein is glycosylated (n-and o-glycosylations), and gp matures by cleavage by furin-like proteases in gp (red) and gp (green). the mucinlike domain is part of gp (red) for ebolaviruses, whereas for marburgviruses and cuevoviruses it is part of both gp and gp . together, gp and gp form gp , , which assembles further into trimers. the surface gp , can shed as a soluble trimer upon cleavage by the host tnfa-converting enzyme (tace). three other proteins, sgp (blue), the d-peptide (purple), and ssgp (yellow) are synthetized by ebolaviruses, and presumably also by cuevoviruses. and dc-sign(r) on liver endothelial cell and lymphocytes alvarez et al., ; lin et al., ; simmons et al., ; marzi et al., ; gramberg et al., gramberg et al., , dominguez-soto et al., ; powlesland et al., ; maruyama et al., ) are expected to interact with a set of n-and o-linked glycans on the mld and the glycan cap of gp , since these lectins increase filovirus attachment. however, the binding to such molecules does not seem to be sufficient to trigger virus internalization marzi et al., ; matsuno et al., b) . additionally, b -integrins have been proposed to serve as attachment factors for ebolaviruses, since infection decreases in the presence of antibodies targeting these proteins in cell lines and primary cell types (takada et al., ; simmons et al., ) . however, no direct interaction between both molecules has been demonstrated yet, and recent studies suggest that a b -integrin is not required for gp-mediated binding of internalization, but rather is a positive regulator of cathepsins, which play an important role in processing gp into its fusion-competent form within the endosomes of infected cells (schornberg et al., ) . axl, member of the tyro /axl/mer (tam) receptor family, has also been proposed as a co-receptor for ebov attachment but, similarly to b -integrins, it may promote viral entry indirectly (shimojima et al., ; schornberg et al., ; brindley et al., ; hunt et al., ) . other cell-surface molecules, the t-cell immunoglobulin mucin domain- and (tim- and tim- ), have been described to interact with ebolavirus gp , , leading to virus internalization moller-tank et al., ; yuan et al., ; rhein et al., ) . nevertheless, despite this clearly demonstrated role, only epithelial cells and some antigenpresenting cells subsets significantly express tim- and tim- respectively, suggesting that there are other attachment factors involved in other filovirus-susceptible cell types. tam, tim- and tim- could target phosphatidyl-serine (ptdser), which is exposed on the outer leaflet of the filovirus membrane, strengthening an interplay promoting efficient attachment (reviewed in moller-tank and maury, ). finally, a last attachment mechanism has been described, reminiscent to an antibody-dependent enhancement (ade) process, by which filoviruses divert virus-specific antibodies to get attached to immune system cells through cellular fc receptors or via the complement component c q and its ligands identified in most mammalian cells takada et al., takada et al., , nakayama et al., ) . interestingly, viral pathogenicity seems to correlate with filovirus ade nakayama et al., ) . in all cases, it appears that attachment requires a set of proteins that interact in a complex manner to promote entry. the uptake is a key step in filovirus entry, as it serves to transform the pre-fusion gp , conformation into a primed gp , that triggers fusion events (fig. ) . first, the internalization has been proposed to involve different endocytic pathways. the precise mechanisms were controversial in the past, since clathrin-dependent and caveolin-dependent uptakes have been shown to occur (bavari et al., ; empig and goldsmith, ; sanchez, ; bhattacharyya et al., bhattacharyya et al., , . however, latest data support that the filovirus uptake mechanism is mainly mediated by macropinocytosis and depends among other factors on the host cell and virus particle size (nanbo et al., ; saeed et al., ; aleksandrowicz et al., ) . after internalization, macropinocytosis vesicles are routed to endosomal vesicles of the host cell, where proteolytic events occur to prime gp , (fig. ) . once in the endosome, ebov gp , is sequentially processed by the cysteine proteases cathepsin b (catb) and/or cathepsin l (catl) under acidic ph and reducing conditions (chandran et al., ; schornberg et al., ; brecher et al., ) . concisely, it appears that, for ebov, bdbv, and tafv, catb removes the major part of gp (glycan cap and mld). the proteolytic events are slightly different for sudv, restv, marv and llov, as catb has been shown to be dispensable (chan et al., ; gnirss et al., ; xia et al., ; maruyama et al., ; ng et al., ) . it is thought that catl partially removes a part of the gp cap and subsequent proteolysis e by catb, catl and/or other proteases e results in a smaller gp form of less than kda. with latest structural inputs, further information regarding cathepsin cleavage have become available. indeed, amongst the minor differences between ebov and marv gp , , a catb cleavage site identified on ebov has been shown to be disordered, and thus potentially easily accessible, fig. . ebolavirus surface gp , and its conservation among the family filoviridae. surface gp , (pdb: csy) is a trimer composed of three dimers of gp (red) and gp (green), forming a chalice at the viral envelope. the left panel presents a surface representation of the three-dimensional structure of ebolavirus gp , , and gp and gp are shown in red and green, respectively. the right panel highlights the conserved residues (from dark red to light red or dark green to light green according to their conservation) derived from a sequence alignment of every filovirus species using hierarchical clustering (multialin server). the figure shows that conserved residues are localized at the center of the trimeric complex (indicated with a grey dotted circle), which contains all features for priming and fusion, as well as in the external domain targeted by the cross-genus neutralizing antibody mr . while in marv the homologous region is an a-helix (hashiguchi et al., ) . however, there remain open questions regarding filovirus gp proteolysis processing, since other proteases were also proposed to participate to this process. further, while much of the cathepsin work was done using gp-pseudotyped vsv or retrovirus particles, work with infectious ebolaviruses challenges the importance of cathepsins for the viral life cycle. particularly, virus replication in vitro as well as in vivo was shown to be independent of catb and catl, and cathepsin knockout mice succumbed to ebolavirus challenge, albeit not to vsv infection (marzi et al., ) . in any case, these cleavages trigger an increase in binding and infectivity for ebola virus by unmasking the potential rbs, which was firstly described as a residue peptide on gp (k , k , k , g , p , k ) (lee et al., ) . recently, the niemann-pick c protein (npc ), which is an anchored late endosomal/ lysosomal protein physiologically implicated in cholesterol absorption and homeostasis, has been shown to interact with this region of gp (fig. ) white and schornberg, ) . indeed, the latest structural data define the rbs as a negatively charged crest and a hydrophobic trough at the apex of gp trimer that interact mostly with the domain c (loops and ) and partially with the domain ntd of npc (bornholdt et al., ; gong et al., ; zhao et al., ) . this structural characterization of the npc / gp interaction provides key information to design promising antiviral strategies. more importantly, further analyses described npc as a crucial receptor that potentially determines the species susceptibility to filoviruses (ng et al., ; hoffmann et al., ; ndungo et al., ) , paving the way towards filovirus-specific antiviral molecules (see . .). even if deeper investigation is needed into the mechanism of npc during filovirus entry, its interaction with cleaved gp and the endosomal environment triggers a conformational change of gp initiating membrane fusion (fig. ) (lee et al., ; kuroda et al., ) . fusion necessitates large conformation changes representing a high-energy barrier. with gp cleavages and a low-ph environment, it is hypothesized that both gp constraint removal from the metastable gp and histidine protonation generate the energy triggering the fusion events (kampmann et al., ; lee et al., fig. . surface gp endosomal processing. after attachment mediated by interaction between the filovirus surface protein gp , (pdb: csy) and various attachment factors, the complex is internalized and routed to the endosome, where gp , is processed to trigger fusion of viral and host membranes. first, in the endosomal low ph environment, cathepsin proteases l&b (catl & catb) and others remove the mucin-like region (mld) and the glycan cap (gc) domain of gp (red). a receptor binding domain (rdb), also carried by gp , is unmasked, leading to the interaction with a mainly hydrophobic pocket in the n-terminal domain (blue) of the endosomal protein niemann-pick c (npc ) (pdb: f b). this interaction together with other only partially understood molecular events remove gp constraints on gp (green), forming the primed-gp , capable to induce fusion. the gp heptad repeat regions and (hrr and hrr ) then rearrange themselves, pushing out the fusion peptide (pink) to anchor it in the host membrane. this intermediate pre-hairpin conformation destabilizes membrane bilayers, and a folding-back into a six-bundle helices conformation (pdb: ebo) merges membranes, opening a fusion pore for the release of viral nucleocapsids. , markosyan et al., ) . at this point, the disordered hrrs rearrange themselves into a-helices. these helices push through the host membrane, and then anchor the hydrophobic fusion peptide in target membrane. within the membrane, key residues, amongst them pro , trigger bilayer destabilization with an extended pre-hairpin intermediate conformation (fig. ) . therefore, elongated gp spans and thus branches both host and viral membranes. the fusion finally occurs by the collapse of this intermediate into a folding-back conformation that distorts both viral and host membranes with their simultaneous rapprochement. gp acts as a clamp to reach its low energy state characterized as a six helices bundle, which is the gp hairpin post-fusion structure (fig. ) . this novel conformation leads to a merge into a hemifusion stalk and then to the opening of a fusion pore allowing the release of the nucleocapsid into the host cytoplasm (fig. ) . while the principle function of gp , is cell infection, several lines of evidence suggest that it might also be involved in pathogenesis. cellular gp , -related cytotoxic events occur in infected cells. this cytotoxicity is reflected in rounding of cells in vitro, due to masking of various host cell surface molecules such as b -integrins (takada et al., ; francica et al., ). this masking has been shown to be a "glycan umbrella" mediated steric shielding of these adhesion proteins mediated by surface-expressed gp , , leading to a loss of accessibility and function of these host surface proteins (reynard et al., ; francica et al., ) . the masking of such adhesion molecules leads to cell detachment, and may contribute to the disruption of blood vessel integrity and hemorrhages developed during a filovirus infection. indeed, in primary endothelial cell cultures overexpression of gp , after transduction with an adenovirus vector resulted in a loss of adherence resulting in apoptosis (ray et al., ) . additionally, steric shielding by gp , also affects mhc-i surface expression (francica et al., ) , a phenomenon also known to occur in infected primary endothelial cells (harcourt et al., ) . this might alter immune cells recruitment, and thus may participate in the immune suppression and inflammatory dysfunction linked to a filovirus infection (harcourt et al., ; reynard et al., ) . as mentioned in . ., sgp has been shown to regulate gp , expression thanks to transcriptional editing in the gp gene (volchkov et al., ; mohan et al., ) . interestingly, when ebov is serially passaged in certain cell lines like vero cells, the gp gene gets modified to predominantly generate gp , by addition of an th u in the editing site (n.b., in contrast to transcriptional editing this occurs on the genome level) (volchkova et al., ) . however, when this ebov is further passaged in animals or in other cell lines like huh cells, the genotype reverts back so that sgp is again produced as the main product of transcription (volchkova et al., ; hoenen et al., ; tsuda et al., ) . this observation suggests that either the importance of the function of sgp or the importance of gp gene modulation is dependent on the host cell environment. additionally, if gp , is over-expressed, the cellular glycosylation machinery is overwhelmed, which competitively inhibits physiological processes (volchkov et al., ; mohan et al., ) . nevertheless, at later stages of viral infection, gp , gets highly expressed, which is associated with a massive release of viral particles by infected cells. thus, cytotoxicity appears even in these situations to be under control by transcriptional editing. however, viral particles produced at this point have been shown to be less pathogenic, suggesting that a quantitative balance in gp , expression might also be required to regulate infectivity (mohan et al., ) . finally, another form of gp , , known as shed gp or gp , dtm, has been shown to be released during ebolavirus infection (dolnik et al., . indeed, gp , dtm is released from surfaceexpressed gp , by the host tnfa-converting enzyme (tace), a member of the disintegrin and metalloproteinase (adam) family, leaving only the transmembrane and cytoplasmic parts of gp on the cell surface (fig. ) . this secreted protein may play a role in viral pathogenesis, blocking neutralizing antibodies or stimulating ade. it might also contribute to an impaired inflammatory response (escudero-p erez et al., ) . a last potential role of shed gp might be the modulation of endothelium homeostasis, thus triggering increase of vascular permeability, and disseminated intravascular coagulation, ultimately causing multi-organ failure and death (escudero-p erez et al., ). since filovirus gp , plays a key role in virus entry, strategies targeting different key points in this process are supposed to block replication at an early stage, thus reducing the chance of virus spread and its potential evolution towards drug resistance. therefore, numerous methods targeting filovirus entry have been investigated. (i) immune-based therapies have been rapidly developed (reviewed by choi and croyle, ) leading to efficient monoclonal antibody cocktails (olinger et al., ; qiu et al., qiu et al., , a qiu et al., , b such as the promising antibody-based drug against ebolaviruses zmapp ® (qiu et al., ) . (ii) alternatively, peptidebased antiviral molecules like tat-ebo and analogs were designed in order to block cell membrane fusion (miller et al., ; higgins et al., ) . the inhibition is based on peptides limiting hrr and interactions to block gp extension. such compounds have still to be optimized in terms of treatment window and dosage. (iii) other strategies based on a broad range of small molecules have been built up to disrupt the entry step. these potential antiviral compounds have been heterogeneously characterized from in vitro high throughput screening hits to in vivo studies (reviewed by nyakatura et al., ; rhein and maury, ) . here are reported the latest advances related to small entry inhibitors, which can be sorted as broad-spectrum molecules, filovirus-specific compounds, and fdaapproved therapeutics (summarized in table ). with regards to antibody-based therapies the interested reader is referred to (zeitlin et al., ) . as broad-spectrum molecules we define inhibitors targeting key points of the entry process that are effective against multiple virus multiple rna viruses indeed, filoviruses share entry steps with other viruses using class i envelope glycoproteins such as hiv or influenza. the first target that has been considered is attachment. various soluble mannose-specific lectins from plants (e.g., concanavalin a and cyanovirin n) have been proposed because of their potential antiviral effect on hiv- (balzarini et al., ) . another example is griffithsin, a lectin of terminal mannose residues of asparagine (n)linked man - glcnac structures purified from red-algae, which might also have antiviral potential since these carbohydrate residues are found on hiv- and , hcv, sars-cov and, relevant to our discussion, on ebolaviruses (barton et al., ) . similarly, recombinant human mannose binding lectin has been shown in a mouse model to be protective against ebolaviruses (michelow et al., ) ; however, the mouse model, while a necessary and important early step in drug evaluation for ebolaviruses, has only limited predictive value regarding the effectivity of a treatment in other, more stringent animal models of evd, or even human patients. the inhibition of catl and catb, involved in ebov gp , endosomal processing, could also have a broad-spectrum antiviral effect. various more or less specific inhibitors have been described: the unselective cys and ser protease inhibitor leupeptin, unselective cys protease inhibitors e- and e- d and its recent derivatives, the mixed catl and b inhibitor fy-dmk, the specific catb inhibitors ca- , ca- me and nafamostat mesilate (which has a dual action also targeting factor viia for an anticoagulation action), and catl-specific inhibitors oxobarzate, zy(t-bu)-dmk (also known as cid ), triazine derivatives and , and k (chandran et al., ; schornberg et al., ; barrientos and rollin, ; shah et al., ; gnirss et al., ; elshabrawy et al., ; nishimura and yamaya, ; zhou et al., ; van der linden et al., ) . all these compounds have been characterized in ebolavirus entry modelling systems, such as pseudotypes, and/or studies with infectious virus. they are mechanismbased suicide inhibitors, since they carry an epoxide or diazomethane functional group. they are not only active on filoviruses, but also on sars-cov as well as hendra and nipah viruses (elshabrawy et al., ) . as far as both anti-filovirus attachment and anti-uptake are concerned, these inhibitors need further optimization, especially concerning their toxicity. molecules targeting fusion constitute another class of inhibitors. viruses being unable to repair membrane damage, the use of membrane intercalating agents such as aryl methyldiene rhodamine derivative lj (wolf et al., ), duy (st vincent et al., , a rigid amphipathic fusion inhibitor (rafi), the indole based hydrophobic molecule arbidol (p echeur et al., ), presumably synthetic hc oxysterols (liu et al., ; schoggins and randall, ) and teicoplanin (y. may impair virus/host cell membrane fusion. to date, development is at a proof-of-concept stage, but the strategy showed a broad efficiency against ebolaviruses, influenza, hiv, pox-, arena-, bunya-, herpes-, paramyxo-and flaviviruses (wolf et al., ) . other broad-spectrum compounds can be mentioned such as genistein, a broad tyrosine kinase inhibitor, or tyrphostin ag , an epidermal growth factor receptor tyrosine kinase blocker. both trigger the disruption of endocytosis and endosome formation used by numerous viruses (chandran et al., ) . the aminoquinoline, long known as the antimalarial agent chloroquine (savarino et al., ) , showed beneficial effects, from endocytosis to exocytosis with increase of endosomal ph, diminution of inf-g and tnf-a production, on hiv- , sars-cov and ebolaviruses in vitro (savarino et al., ; keyaerts et al., keyaerts et al., , madrid et al., ) . unfortunately, for some of these compounds animal studies have shown that they are not effective against ebolaviruses in vivo (dowall et al., ; falzarano et al., ; akpovwa, ) , whereas other compounds still need to be tested in such models, before any conclusions regarding their effectiveness can be made. importantly, different in vitro and in vivo models have different predictive values regarding the effectiveness of drugs, with in vitro experiments providing the weakest evidence (although they remain an essential starting point), followed by the various mouse models, whereas guinea pigs provide a more stringent model, and non-human primates constitute the most stringent model for (balzarini et al., ; barton et al., ) endosomal processing protease inhibitors cys&ser protease inhibitors (leupeptin) (chandran et al., ; schornberg et al., ; barrientos et al., ; shah et al., ; gnirss et al., ; elshabrawy et al., ; nishimura et al., ; zhou et al., ; van der linden et al., ) cys protease inhibitors (e- and e- d) catl&catb inhibitors (fy-dmk) catb inhibitors (ca- , ca- me, nafamostat mesilate) catl inhibitors (oxobarzate, zy(t-bu)-dmk, triazine derivatives and , and k ) endosome disruption genistein, tyrphostin ag , chloroquine (savarino et al., ; keyaerts et al., ; keyaerts et al., ; madrid et al., ) fusion intercaling agents lj , duy , arbidol (st vincent et al., ; wolf et al., ; p echeur et al., ) unclear hc oxysterols, teicoplanin (liu et al., ; schoggins et al., ; attachment lectin competitors tridecafullerenes (muñoz et al., ) fusion npc inhibitors u a, imipramine, ro - , compounds . and . , and mbx and mbx (cenedella, ; côt e et al., ; kolokoltsov et al., ; lee et al., ; shoemaker et al., ; basu et al., ) gp inhibitors compound (basu et al., ) approved drugs against other targets attachment glycosaminoglycan competitor heparin (salvador et al., ; cheng et al., ; o'hearn et al., ) uptake cytoskeleton inhibitors vinblastine, vincristine, colchicine, nocodazole, cytochalasin b and d, latrunculin a, chondramides (kouznetsova et al., ; beck et al., ) endosomal processing g protein-coupled receptor antagonsits benztropine mesylate endosomal ph increase omeprazol, esomeprazol (long et al., ) unclear estrogen receptor modulators clomiphene, toremiphene, raloxifene, taxomifene shoemaker et al., ; gehring et al., ; kouznetsova et al., ; sakurai et al., ; zhao et al., ; beck et al., ) ion channel inhibitors amiodarone, dronedarone, verapamil, tetrandine, nimodipine, diltiazem, digoxin, rottlerin, noricumazole a) in addition to serological and peptide-based approaches, numerous studies report potential small anti-filovirus molecules. these compounds can be sorted in three categories (broad-spectrum molecules, filovirus-specific compounds and repurposed fda-approved therapeutics). in each category, molecules have been arranged according to their targeted entry process and then following their specific activity when described. ebolavirus and marburgvirus disease. these characteristics have to be kept in mind when evaluating these and other antiviral drugs against filoviruses, or when interpreting study results. further, all these compounds target a broad spectrum of pathogenic viruses; however, since they often target the cellular machinery, selectivity and toxicity will inevitably remain an important issue. an alternate strategy would be to select molecules targeting filoviruses specifically. the gp /npc interaction that triggers fusion events in filoviruses has been highly considered and recently characterized (bornholdt et al., ; gong et al., ; zhao et al., ) . such a strategy is challenging, since the interface between npc and gp is large, dynamic and mainly hydrophobic, but also very promising for the development of filovirus specific antiviral molecules. development of protein-protein interaction inhibitors towards both gp and npc has been reported. several compounds have been described targeting npc , such as u a, imipramine, cathionic amphiphiles ro - and the terconazole adamantane class of compounds like benzylpiperazine adamantane diamides . and . , or the lately discovered sulfonamide derivative mbx and the triazole thioether derivative mbx (rodriguez-lafrasse et al., ; cenedella, ; côt e et al., ; lee et al., ; shoemaker et al., ; basu et al., ) . most of these inhibitors helped to identify npc as a critical factor for filovirus entry. interestingly, the tertiary amine imipramine inhibits both npc and sphingomyelinase, resulting in ebolavirus entry inhibition (m.e. . as npc is involved in other cellular functions, its inhibition disrupts its primary role in cholesterol homeostasis and triggers cholesterol accumulation in endosomes, as is the case in niemann-pick disease type c shoemaker et al., ) . therefore, further development might be required to uncouple the npc function in cholesterol traffic from its implication in filovirus entry. this unbundling should be tricky, since both cholesterol management and ebolavirus binding are facilitated by the same domains of npc . however, as ebolavirus disease constitutes an acute infection, it should be possible to keep the treatment time relatively short, so that this side effect might have limited detrimental effects in practice. an alternative strategy avoiding unwanted host-directed effects might be to target gp , since it is a viral protein with no cellular homolog. indeed, a benzodiazepine derivative (referred to as compound ) has been shown -by computer calculation and mutagenesis -to mask the hydrophobic conserved s pocket defined at the gp /gp interface, inhibiting ebolavirus replication (basu et al., ) . these results also show that this s pocket does not correspond to the trough of the rbs, suggesting that it might be another binding site or a steric regulation region of the ncp binding site. although the mechanism of inhibition has yet to be clarified, s pocket and rbs pharmacophores might be key targets to design filovirus-specific antiviral molecules. lately, another filovirus-specific strategy has been described using tridecafullerene derivative compounds ( a and c) (muñoz et al., ) . these compounds are giant globular multivalent molecules carrying sugar motifs able to compete with lectins. dependent on the sugar motifs linked on such "superballs", it is possible to target specific lectins. targeting dc-sign for example resulted in inhibition of ebolavirus in vitro at subnanomolar concentrations with no cytotoxicity notable, and perfect solubility in water. as a very new technology, these tridecafullerene derivatives require deeper investigations to depict their mechanism of action in vitro and their effectiveness in vivo, as well as their specificity and their delivery to target organs. given that the development of new therapeutics is a long and expensive process, one approach to rapidly develop drugs against filovirus infection is to reposition compounds that have been already approved by national and international health agencies (kouznetsova et al., ) . using this strategy, cationic amphiphiles such as the estrogen receptor modulators clomiphene (infertility treatment to induce ovulation), toremiphene (breast cancer chemotherapeutic) raloxifene (osteoporosis prevention) and taxomifene (breast cancer chemotherapeutic), and ion channel inhibitors (cardiac arrhythmias and vascular modulation) such as amiodarone, dronedarone, verapamil, tetrandine, nimodipine, diltiazem, digoxin, and rottlerin have been shown to inhibit filovirus entry shoemaker et al., ; gehring et al., ; kouznetsova et al., ; sakurai et al., ; nelson et al., ) . other channel blocking compounds such as noricumazole a from myxobacteria have been also described as potent inhibitors (beck et al., ) . their inhibition mechanisms are not yet understood, but are distinct from gp /npc interaction-based inhibitors. another group of therapeutics targeting the cytoskeleton has been described to have anti-ebov potential (vinblastine, vincristine, colchicine, nocodazole, cytochalasin b and d, latrunculin a and others) (kouznetsova et al., ; beck et al., ) . however, these molecules are supposed to be quite toxic, since they are mostly used for anti-cancer chemotherapies. additionally, as a large class of approved therapeutics, g protein-coupled receptor (gpcr) antagonists have been screened. benzotropine mesylate, initially used against parkinson's disease, exhibits in vitro anti-filovirus activity targeting a post-attachment step . such molecules deserve a thorough mechanistic investigation, since there is no other literature mentioning the involvement of gpcr in filovirus entry. other drugs such as omeprazole and esomeprazole (acid reflux disease) have off-target inhibitory activity triggering increase of endosomal ph, which inhibits late entry events during a filovirus infection (long et al., ) . also, several studies described heparin as a potent inhibitor of early attachment and co-reception via glycosaminoglycans (salvador et al., ; cheng et al., ; o'hearn et al., ) . however, further studies are needed to unravel the antiviral mechanism of those fda-approved compounds. although these compounds have already passed safety tests, it is important to underline that dosing and treatment window will likely be a critical issue before use. antibodies such as zmapp ® have demonstrated that inhibitors targeting filovirus entry might provide a potent antiviral strategy. however the development of small molecules inhibiting this crucial viral step is still hampered by both the insufficient molecular characterization of the attachment/uptake mechanism, and the multiple molecular events occurring in endosomal compartments. to date, the identification of the main characters is clear, but the fine-tuning of each step has to be clarified. the inhibitors presented herein appear to function at several entry steps, but the most susceptible one has yet to be identified. targeting pre-fusion gp , or the gp /npc interface might represent the best strategy to develop filovirus-specific treatments. with a more integrative vision in mind, antiviral therapies might advantageously complement vaccines and serological therapies during filovirus outbreaks with their simple logistics especially in west and central africa. chloroquine could be used for the treatment of filoviral infections and other viral infections that emerge or emerged from viruses requiring an acidic ph for infectivity ebola virus enters host cells by macropinocytosis and clathrin-mediated endocytosis c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans ebolavirus and marburgvirus: insight the filoviridae family mannose-specific plant lectins from the amaryllidaceae family qualify as efficient microbicides for prevention of human immunodeficiency virus infection release of cellular proteases into the acidic extracellular milieu exacerbates ebola virus-induced cell damage activity of and effect of subcutaneous treatment with the broad-spectrum antiviral lectin griffithsin in two laboratory rodent models identification of a small-molecule entry inhibitor for filoviruses novel small molecule entry inhibitors of ebola virus lipid raft microdomains: a gateway for compartmentalized trafficking of ebola and marburg viruses identification of entry inhibitors of ebola virus pseudotyped vectors from a myxobacterial compound library the asialoglycoprotein receptor is a potential liver-specific receptor for marburg virus differential requirements for clathrin endocytic pathway components in cellular entry by ebola and marburg glycoprotein pseudovirions ebola virus uses clathrin-mediated endocytosis as an entry pathway host-primed ebola virus gp exposes a hydrophobic npc receptor-binding pocket, revealing a target for broadly neutralizing antibodies cathepsin cleavage potentiates the ebola virus glycoprotein to undergo a subsequent fusion-relevant conformational change tyrosine kinase receptor axl enhances entry of zaire ebolavirus without direct interactions with the viral glycoprotein ebola virus entry requires the cholesterol transporter niemann-pick c cholesterol synthesis inhibitor u a and the role of sterol metabolism and trafficking in numerous pathophysiological processes endosomal proteolysis of the ebola virus glycoprotein is necessary for infection distinct mechanisms of entry by envelope glycoproteins of marburg and ebola (zaire) viruses inhibition of ebola and marburg virus entry by g protein-coupled receptor antagonists emerging targets and novel approaches to ebola virus prophylaxis and treatment small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection the multiple roles of sgp in ebola pathogenesis a shared structural solution for neutralizing ebolaviruses shedding of ebola virus surface glycoprotein is a mechanism of self-regulation of cellular cytotoxicity and has a direct effect on virus infectivity ectodomain shedding of the glycoprotein gp of ebola virus the dc-sign-related lectin lsectin mediates antigen capture and pathogen binding by human myeloid cells chloroquine inhibited ebola virus replication in vitro but failed to protect against infection and disease in the in vivo guinea pig model identification of a broad-spectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and ebola, hendra, and nipah viruses by using a novel high-throughput screening assay association of the caveola vesicular system with cellular entry by filoviruses shed gp of ebola virus triggers immune activation and increased vascular permeability structure-function analysis of the soluble glycoprotein, sgp, of ebola virus. chembiochem eur lack of protection against ebola virus from chloroquine in mice and hamsters ebola haemorrhagic fever the glycoproteins of marburg and ebola virus and their potential roles in pathogenesis requirements for cell rounding and surface protein down-regulation by ebola virus glycoprotein steric shielding of surface epitopes and impaired immune recognition induced by the ebola virus glycoprotein modeling of the ebola virus delta peptide reveals a potential lytic sequence motif the clinically approved drugs amiodarone, dronedarone and verapamil inhibit filovirus cell entry cathepsins b and l activate ebola but not marburg virus glycoproteins for efficient entry into cell lines and macrophages independent of tmprss expression structural insights into the niemann-pick c (npc )-mediated cholesterol transfer and ebola infection lsectin interacts with filovirus glycoproteins and the spike protein of sars coronavirus interactions of lsectin and dc-sign/dc-signr with viral ligands: differential ph dependence, internalization and virion binding ebola virus selectively inhibits responses to interferons, but not to interleukin- beta, in endothelial cells filovirus assembly and budding structural basis for marburg virus neutralization by a cross-reactive human antibody c-peptide inhibitors of ebola virus glycoprotein-mediated cell entry: effects of conjugation to cholesterol and side chain-side chain crosslinking soluble glycoprotein is not required for ebola virus virulence in guinea pigs the glycoproteins of all filovirus species use the same host factors for entry into bat and human cells but entry efficiency is species dependent the tyro receptor kinase axl enhances macropinocytosis of zaire ebolavirus ebola virus glycoprotein: proteolytic processing, acylation, cell tropism, and detection of neutralizing antibodies sgp serves as a structural protein in ebola virus infection fda-approved selective estrogen receptor modulators inhibit ebola virus infection the role of histidine residues in low-ph-mediated viral membrane fusion antiviral activity of chloroquine against human coronavirus oc infection in newborn mice in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine t-cell immunoglobulin and mucin domain (tim- ) is a receptor for zaire ebolavirus and lake victoria marburgvirus identification of compounds that block ebola virus-like particle entry via a repurposing screen of approved drugs ebola virus genome plasticity as a marker of its assaging history: a comparison of in vitro passaging to non-human primate infection proposal for a revised taxonomy of the family filoviridae: classification, names of taxa and viruses, and virus abbreviations interaction between tim- and npc is important for cellular entry of ebola virus structure of the ebola virus glycoprotein bound to an antibody from a human survivor ebolavirus glycoprotein structure and mechanism of entry inhibition of ebola virus infection: identification of niemann-pick c as the target by optimization of a chemical probe the roles of histidines and charged residues as potential triggers of a conformational change in the fusion loop of ebola virus glycoprotein structure of the l protein of vesicular stomatitis virus from electron cryomicroscopy differential n-linked glycosylation of human immunodeficiency virus and ebola virus envelope glycoproteins modulates interactions with dc-sign and dc-signr structural and functional studies on the marburg virus gp fusion loop interferoninducible cholesterol- -hydroxylase broadly inhibits viral entry by production of -hydroxycholesterol antiviral therapies against ebola and other emerging viral diseases using existing medicines that block virus entry. f research , a systematic screen of fda-approved drugs for inhibitors of biological threat agents characterization of marburg virus glycoprotein in viral entry induction of cell-cell fusion by ebola virus glycoprotein: low ph is not a trigger characterization of the envelope glycoprotein of a novel filovirus, lloviu virus dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus analysis of the interaction of ebola virus glycoprotein with dc-sign (dendritic cell-specific intercellular adhesion molecule -grabbing nonintegrin) and its homologue dc-signr cathepsin b & l are not required for ebola virus replication different potential of c-type lectin-mediated entry between marburg virus strains c-type lectins do not act as functional receptors for filovirus entry into cells a new ebola virus nonstructural glycoprotein expressed through rna editing ebola virus rna editing depends on the primary editing site sequence and an upstream secondary structure progression of ebola therapeutics during the - outbreak high-dose mannose-binding lectin therapy for ebola virus infection inhibition of ebola virus entry by a cpeptide targeted to endosomes ebolavirus requires acid sphingomyelinase activity and plasma membrane sphingomyelin for infection antigenic subversion: a novel mechanism of host immune evasion by ebola virus less is more: ebola virus surface glycoprotein expression levels regulate virus production and infectivity role of the phosphatidylserine receptor tim- in enveloped-virus entry phosphatidylserine receptors: enhancers of enveloped virus entry and infection filovirus replication and transcription synthesis of giant globular multivalent glycofullerenes as potent inhibitors in a model of ebola virus infection antibody-dependent enhancement of marburg virus infection enzyme-linked immunosorbent assay for detection of filovirus species-specific antibodies ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner a single residue in ebola virus receptor npc influences cellular host range in reptiles. msphere , e discovery of an ebolavirus-like filovirus in europe clomiphene and its isomers block ebolavirus particle entry and infection with similar potency: potential therapeutic implications cell entry by a novel european filovirus requires host endosomal cysteine proteases and niemann-pick c filovirus receptor npc contributes to species-specific patterns of ebolavirus susceptibility in bats ebolavirosis: a review for clinicians. acta m edica port a synthetic serine protease inhibitor, nafamostat mesilate, is a drug potentially applicable to the treatment of ebola virus disease assembly and budding of ebolavirus chemical and structural aspects of ebola virus entry inhibitors role of ext and glycosaminoglycans in the early stage of filovirus entry intracellular events and cell fate in filovirus infection delayed treatment of ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques the synthetic antiviral drug arbidol inhibits globally prevalent pathogenic viruses a novel mechanism for lsectin binding to ebola virus surface glycoprotein through truncated glycans sustained protection against ebola virus infection following treatment of infected nonhuman primates with successful treatment of ebola virus-infected cynomolgus macaques with monoclonal antibodies reversion of advanced ebola virus disease in nonhuman primates with zmapp mabs and ad-vectored ifn-a therapy rescue ebolainfected nonhuman primates when administered after the detection of viremia and symptoms ebolavirus delta-peptide immunoadhesins inhibit marburgvirus and ebolavirus cell entry filoviral immune evasion mechanisms ebola virus glycoprotein-mediated anoikis of primary human cardiac microvascular endothelial cells ebolavirus glycoprotein gp masks both its own epitopes and the presence of cellular surface proteins ebola virus entry into host cells: identifying therapeutic strategies characterization of human and murine t-cell igv domain residues critical for ebola virus entry abnormal cholesterol metabolism in imipramine-treated fibroblast cultures. similarities with niemann-pick type c disease cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes ebola virus. two-pore channels control ebola virus host cell entry and are drug targets for disease treatment filoviruses utilize glycosaminoglycans for their attachment to target cells analysis of filovirus entry into vero e cells, using inhibitors of endocytosis, endosomal acidification, structural integrity, and cathepsin (b and l) activity sequence analysis of the ebola virus genome: organization, genetic elements, and comparison with the genome of marburg virus the virion glycoproteins of ebola viruses are encoded in two reading frames and are expressed through transcriptional editing effects of chloroquine on viral infections: an old drug against today's diseases? the anti-hiv- activity of chloroquine lipids in innate antiviral defense alpha beta -integrin controls ebolavirus entry by regulating endosomal cathepsins role of endosomal cathepsins in entry mediated by the ebola virus glycoprotein a small-molecule oxocarbazate inhibitor of human cathepsin l blocks severe acute respiratory syndrome and ebola pseudotype virus infection into human embryonic kidney t cells tyro family-mediated cell entry of ebola and marburg viruses multiple cationic amphiphiles induce a niemann-pick c phenotype and inhibit ebola virus entry and infection on the etiology of an unknown human infection originating from monkeys filovirus entry dc-sign and dc-signr bind ebola glycoproteins and enhance infection of macrophages and endothelial cells ebola virus glycoproteins induce global surface protein down-modulation and loss of cell adherence rigid amphipathic fusion inhibitors, small molecule antiviral compounds against enveloped viruses filovirus tropism: cellular molecules for viral entry epitopes required for antibody-dependent enhancement of ebola virus infection antibody-dependent enhancement of ebola virus infection human macrophage c-type lectin specific for galactose and n-acetylgalactosamine promotes filovirus entry the pathogenesis of ebola hemorrhagic fever downregulation of beta integrins by ebola virus glycoprotein: implication for virus entry an improved reverse genetics system to overcome cell-typedependent ebola virus genome plasticity genomic rna editing and its impact on ebola virus adaptation during serial passages in cell culture and infection of guinea pigs delta-peptide is the carboxyterminal cleavage fragment of the nonstructural small glycoprotein sgp of ebola virus rna editing of the gp gene of ebola virus is an important pathogenicity factor s es gp mrna of ebola virus is edited by the ebola virus polymerase and by t and vaccinia virus polymerases proteolytic processing of marburg virus glycoprotein recovery of infectious ebola virus from complementary dna: rna editing of the gp gene and viral cytotoxicity effects of ebola virus glycoproteins on endothelial cell activation and barrier function ebola viral glycoprotein bound to its endosomal receptor niemann-pick c teicoplanin inhibits ebola pseudovirus infection in cell culture a new player in the puzzle of filovirus entry a broad-spectrum antiviral targeting entry of enveloped viruses bidentate and tridentate metalion coordination states within ternary complexes of rb dna polymerase tim- acts a dual-attachment receptor for ebolavirus by interacting directly with viral gp and the ps on the viral envelope antibody therapeutics for ebola virus disease structure of glycosylated npc luminal domain c reveals insights into npc and ebola virus interactions protease inhibitors targeting coronavirus and filovirus entry this work was supported by a dga-mris and aix-marseille universit e scholarship and by the anr grant anr-st -astr- for the project vmtasein. key: cord- -rlpzejjt authors: coutard, b.; valle, c.; de lamballerie, x.; canard, b.; seidah, n.g.; decroly, e. title: the spike glycoprotein of the new coronavirus -ncov contains a furin-like cleavage site absent in cov of the same clade date: - - journal: antiviral res doi: . /j.antiviral. . sha: doc_id: cord_uid: rlpzejjt in , a new coronavirus ( -ncov) infecting humans has emerged in wuhan, china. its genome has been sequenced and the genomic information promptly released. despite a high similarity with the genome sequence of sars-cov and sars-like covs, we identified a peculiar furin-like cleavage site in the spike protein of the -ncov, lacking in the other sars-like covs. in this article, we discuss the possible functional consequences of this cleavage site in the viral cycle, pathogenicity and its potential implication in the development of antivirals. in , a new coronavirus ( -ncov) infecting humans has emerged in wuhan, china. its genome has been sequenced and the genomic information promptly released. despite a high similarity with the genome sequence of sars-cov and sars-like covs, we identified a peculiar furin-like cleavage site in the spike protein of the -ncov, lacking in the other sars-like covs. in this article, we discuss the possible functional consequences of this cleavage site in the viral cycle, pathogenicity and its potential implication in the development of antivirals. human coronaviruses (cov) are enveloped positive-stranded rna viruses belonging to the order nidovirales, and are mostly responsible for upper respiratory and digestive tract infections. among them sars-cov and mers-cov that spread in and respectively, have been associated with severe human illnesses, such as severe pneumonia and bronchiolitis, and even meningitis in more vulnerable populations (de wit et al., ) . in december , a new cov ( -ncov) has been detected in the city of wuhan, and this emerging viral infection was associated with severe human respiratory disease with a~ - % fatality rate . the virus that was presumed to have initially been transmitted from an animal reservoir to humans possibly via an amplifying host. however human-to-human transmission has been reported, leading to a sustained epidemic spread with > , confirmed human infections, including > deaths, reported by the who in early february . the estimated effective reproductive number (r) value of~ . ( %: . - . ) at the beginning of the outbreak raises the possibility of a pandemics . this prompted who to declare it as a public health emergency of international concern. this is especially relevant because so far there are no specific antiviral treatments available or vaccine. based on its genome sequence, -ncov belongs to lineage b of betacoronavirus (fig. a) , which also includes the sars-cov and bat cov zxc , the latter and cov zc being the closest to -ncov. -ncov shares~ % amino acid sequence identity in the spike (s)-protein sequence with sars-cov and % with cov zxc (chan et al., ) . in this article, we focus on a specific furin-like protease recognition pattern present in the vicinity of one of the maturation sites of the s protein ( fig. b ) that may have significant functional implications for virus entry. the proprotein convertases (pcs; genes pcsks) constitute a family of nine serine secretory proteases that regulate various biological processes in both healthy and disease states (seidah and prat, ) . by proteolysis, pcs are responsible for the activation of a wide variety of precursor proteins, such as growth factors, hormones, receptors and adhesion molecules, as well as cell surface glycoproteins of infectious viruses (seidah and chretien, ) (table ) . seven pcs cleave precursor proteins at specific single or paired basic amino acids (aa) within the motif (r/k)-( x)n-(r/k)↓, where n = , , , or spacer aa (seidah and chretien, ) . because of their role in the processing of many critical cell surface proteins pcs, especially furin, have been implicated in viral infections. they have the potential to cleave specifically viral envelope glycoproteins, thereby enhancing viral fusion with host cell membranes (izaguirre, ; moulard and decroly, ) . in the case of human-infecting coronaviruses such as hcov-oc (le coupanec et al., ) , mers-cov (millet and whittaker, ) , and hku (chan et al., ) the spike protein has been demonstrated to be cleaved at an s /s cleavage site (fig. ) generating the s and s subunits. the above three viruses display the canonical (r/k)-( x)n-(r/k)↓ motif (table ) . additionally, it has been demonstrated that variation around the viral envelope glycoprotein cleavage site plays a role in cellular tropism and pathogenesis. for instance, the pathogenesis of some cov https://doi.org/ . /j.antiviral. . received february ; received in revised form february ; accepted february has been previously related to the presence of a furin-like cleavage site in the s-protein sequence. for example, the insertion of a similar cleavage site in the infectious bronchitis virus (ibv) s-protein results in higher pathogenicity, pronounced neural symptoms and neurotropism in infected chickens (cheng et al., ) . similarly, in the case of influenza virus, low-pathogenicity forms of influenza virus contain a single basic residue at the cleavage site, which is cleaved by trypsin-like proteases and the tissue distribution of the activating protease(s) typically restricts infections to the respiratory and/or intestinal organs (sun et al., ) . conversely, the highly pathogenic forms of influenza have a furin-like cleavage site cleaved by different cellular proteases, including furin, which are expressed in a wide variety of cell types allowing a widening of the cell tropism of the virus (kido et al., ) . furthermore the insertion of a multibasic motif rerrrkkr↓gl at the h n hemagglutinin ha cleavage site was likely associated with the hyper-virulence of the virus during the hong kong outbreak (claas et al., ) . this motif exhibits the critical arg at p and basic residues at p and p , as well as p and p and an aliphatic leu at p ' positions (table ) (schechter and berger nomenclature (schechter and berger, ) ), typical of a furin-like cleavage specificity (braun and sauter, ; izaguirre, ; seidah and prat, ) . the coronavirus s-protein is the structural protein responsible for the crown-like shape of the cov viral particles, from which the original name "coronavirus" was coined. the~ aa long s-protein belongs to class-i viral fusion proteins and contributes to the cell receptor binding, tissue tropism and pathogenesis (lu et al., ; millet and whittaker, ) . it contains several conserved domains and motifs table comparative sequences of envelope protein cleavage site(s) in coronaviruses (above) and in other rna viruses (below). empty boxes: no consensus motif detected.. (fig. ) . the trimetric s-protein is processed at the s /s cleavage site by host cell proteases, during infection. following cleavage, also known as priming, the protein is divided into an n-terminal s -ectodomain that recognises a cognate cell surface receptor and a c-terminal s membrane-anchored protein involved in viral entry. the sars-cov s protein contains a conserved receptor binding domain (rbd), which recognises the angiotensin-converting enzyme (ace ) (li et al., ) . the sars-cov binds to both bat and human cells, and the virus can infect both organisms (ge et al., ; kuhn et al., ) . the rbd surface of s /ace implicates aa in the s of sars-cov (li et al., ) . among them, residues are strictly conserved in -ncov, supporting the hypothesis that ace is also the receptor of the newly emerged ncov (wan et al., ) . the s -protein contains the fusion peptide (fp), a second proteolytic site (s ′), followed by an internal fusion peptide (ifp) and two heptad-repeat domains preceding the transmembrane domain (tm) (fig. ) . notably, the ifps of the -ncov and sars-cov are identical, displaying characteristics of viral fusion peptides (fig. ) . while the molecular mechanism involved in cell entry is not yet fully understood, it is likely that both fp and ifp participate in the viral entry process (lu et al., ) and thus the sprotein must likely be cleaved at both s /s and s ′ cleavage sites for virus entry. the furin-like s ′ cleavage site at kr↓sf with p and p basic residues and a p ′ hydrophobic phe (seidah and prat, ) , downstream of the ifp is identical between the -ncov and sars-cov (fig. ) . in the mers-cov and hcov-oc the s /s site is replaced by rxxr↓sa, with p and p basic residues, and an ala (not aliphatic) at p ′, suggesting a somewhat less favourable cleavage by furin. however, in the other less pathogenic circulating human cov, the s ′ cleavage site only exhibits a monobasic r↓s sequence (fig. ) with no basic residues at either p and/or p needed to allow furin cleavage, suggesting a less efficient cleavage or higher restriction at the entry step depending on the cognate proteases expressed by target cells. even though processing at s ′ in -ncov is expected to be a key event for the final activation of the s-protein, the protease(s) involved in this process have not yet been conclusively identified. based on the -ncov s ′ sequence and the above arguments, we propose that one or more furin-like enzymes would cleave the s ′ site at kr↓sf. in contrast to the s ′, the first cleavage between the rbd and the fp (s /s cleavage site, fig. ) has been extensively studied for many covs (lu et al., ) . interestingly the s /s processing site exhibits different motifs among coronaviruses (fig. , site & site ), with many of them displaying cleavage after a basic residue. it is thus likely that the priming process is ensured by different host cell proteases depending on the sequence of the s /s cleavage site. accordingly the mers-cov sprotein, which contains a rsvr↓sv motif is cleaved during virus egress, probably by furin (mille and whittaker, ) . conversely the sprotein of sars-cov remains largely uncleaved after biosynthesis, possibly due to the lack of a favourable furin-like cleavage site (sllr-st). in this case, it was reported that following receptor binding the sprotein is cleaved at a conserved sequence ayt↓m (located aa downstream of sllr-st) by target cells' proteases such as elastase, cathepsin l or tmprss (bosch et al., ; matsuyama et al., matsuyama et al., , millet and whittaker, ) . as the priming event is essential for virus entry, the efficacy and extent of this activation step by the proteases of the target cells should regulate cellular tropism and viral pathogenesis. in the case of the -ncov s-protein, the conserved site sequence ayt↓m may still be cleaved, possibly after the preferred furincleavage at the site (fig. ) . since furin is highly expressed in lungs, an enveloped virus that infects the respiratory tract may successfully exploit this convertase to activate its surface glycoprotein (bassi et al., ; mbikay et al., ) . before the emergence of the -ncov, this important feature was not observed in the lineage b of betacoronaviruses. however, it is shared by other cov (hcov-oc , mers-cov, mhv-a ) harbouring furin-like cleavage sites in their s-protein ( fig. ; table ), which were shown to be processed by furin experimentally (le coupanec et al., . the sp, s ↓s and s ′ cleavage sites are indicated by arrows. the sequence of different cov s /s and s ′ cleavage sites were aligned using multalin webserver (http://multalin.toulouse.inra.fr/multalin/) with manual adjustments and the figure prepared using espript (http://espript.ibcp.fr/espript/espript/) presenting the secondary structure of sars-cov s-protein at the bottom of the alignment (pdb x ) (yuan et al., ) . insertion of furin like cleavage site is surrounded by a black frame. red asterisks indicate the presence of a canonical furin-like cleavage motif at the s /s site. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) ; mille and whittaker, ) . strikingly, the -ncov s-protein sequence contains additional nucleotides upstream of the single arg↓ cleavage site (figs. b and ) leading to a predictively solventexposed prrar↓sv sequence, which corresponds to a canonical furinlike cleavage site (braun and sauter, ; izaguirre, ; seidah and prat, ) . this furin-like cleavage site, is supposed to be cleaved during virus egress (mille and whittaker, ) for s-protein "priming" and may provide a gain-of-function to the -ncov for efficient spreading in the human population compared to other lineage b betacoronaviruses. this possibly illustrates a convergent evolution pathway between unrelated covs. interestingly, if this site is not processed, the s-protein is expected to be cleaved at site during virus endocytosis, as observed for the sars-cov. obviously much more work is needed to demonstrate experimentally our assertion, but the inhibition of such processing enzyme(s) may represent a potential antiviral strategy. indeed, it was recently shown that in an effort to limit viral infections, host cells that are infected by a number of viruses provoke an interferon response to inhibit the enzymatic activity of furin-like enzymes. it was also demonstrated that hiv infection induces the expression of either the protease activated receptor (par ) (kim et al., ) or guanylate binding proteins and (gbp , ) (braun and sauter, ) that restrict the trafficking of furin to the trans golgi network (par ) or to early golgi compartments (gbp , ) where the proprotein convertase remains inactive. altogether, these observations suggest that inhibitors of furin-like enzymes may contribute to inhibiting virus propagation. a variety of approaches have been proposed to inhibit furin activity to limit tumour growth, viral and bacterial infection. thus, a variant of the naturally occurring serine protease inhibitor α- antitrypsin harbouring a consensus furin cleavage, called α- antitrypsin portland (α -pdx), inhibits furin and prevents the processing of hiv- env (anderson et al., ) . the addition of a chloromethylketone (cmk) moiety to the c-terminus of a polybasic cleavage motif and a decanoyl group at the n-terminus to favour cell penetration (dec-rvkr-cmk) irreversibly blocked the enzymatic activity of furin, pc , pc , pace and pc (decroly et al., ; garten et al., ) . finally, the elucidation of the crystal structure of furin resulted in the design of a , dideoxystreptamine-derived inhibitor, where two molecules of the inhibitor form a complex with furin (dahms et al., ) . as furin-like enzymes are involved in a multitude of cellular processes, one important issue would be to avoid systemic inhibition that may result in some toxicity. accordingly, it is likely that such small molecule inhibitors, or other more potent orally active ones, possibly delivered by inhalation and exhibiting a slow dissociation rate from furin to allow for sustained inhibition, deserve to be rapidly tested to assess their antiviral effect against -ncov. inhibition of hiv- gp -dependent membrane fusion by a furin-directed α -antitrypsin variant targeting proprotein convertases in furin-rich lung cancer cells results in decreased in vitro and in vivo growth cathepsin l functionally cleaves the severe acute respiratory syndrome coronavirus class i fusion protein upstream of rather than adjacent to the fusion peptide furin-mediated protein processing in infectious diseases and cancer spike protein, s, of human coronavirus hku : role in viral life cycle and application in antibody detection genomic characterization of the novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan the s subunit of qx-type infectious bronchitis coronavirus spike protein is an essential determinant of neurotropism human influenza a h n virus related to a highly pathogenic avian influenza virus structural studies revealed active site distortions of human furin by a small molecule inhibitor sars and mers: recent insights into emerging coronaviruses identification of the paired basic convertases implicated in hiv gp processing based on in vitro assays and expression in cd + cell lines processing of viral glycoproteins by the subtilisin-like endoprotease furin and its inhibition by specific peptidylchloroalkylketones isolation and characterization of a bat sars-like coronavirus that uses the ace receptor the proteolytic regulation of virus cell entry by furin and other proprotein convertases role of host cellular proteases in the pathogenesis of influenza and influenza-induced multiple organ failure neuroinflammation-induced interactions between protease-activated receptor and proprotein convertases in hiv-associated neurocognitive disorder angiotensin-converting enzyme : a functional receptor for sars coronavirus cleavage of a neuroinvasive human respiratory virus spike glycoprotein by proprotein convertases modulates neurovirulence and virus spread within the central nervous system structure of sars coronavirus spike receptor-binding domain complexed with receptor early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia angiotensin-converting enzyme is a functional receptor for the sars coronavirus bat-to-human: spike features determining "host jump" of coronaviruses sars-cov, mers-cov, and beyond efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease tmprss mediated enhancement of severe acute respiratory syndrome coronavirus infection comparative analysis of expression of the proprotein convertases furin, pace , pc and pc in human lung tumours host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein host cell proteases: critical determinants of coronavirus tropism and pathogenesis host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein maturation of hiv envelope glycoprotein precursors by cellular endoproteases on the active site of proteases. . mapping the active site of papain; specific peptide inhibitors of papain proprotein and prohormone convertases: a family of subtilases generating diverse bioactive polypeptides the biology and therapeutic targeting of the proprotein convertases modifications to the hemagglutinin cleavage site control the virulence of a neurotropic h n influenza virus receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains preliminary estimation of the basic reproduction number of novel coronavirus ( -ncov) in china, from to : a data-driven analysis in the early phase of the outbreak supplementary data to this article can be found online at https:// doi.org/ . /j.antiviral. . . key: cord- - oltss authors: patel, deendayal; opriessnig, tanja; stein, david a.; halbur, patrick g.; meng, xiang-jin; iversen, patrick l.; zhang, yan-jin title: peptide-conjugated morpholino oligomers inhibit porcine reproductive and respiratory syndrome virus replication date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: oltss porcine reproductive and respiratory syndrome (prrs) has been devastating the global swine industry for more than a decade, and current strategies to control prrs are inadequate. in this study we characterized the inhibition of prrs virus (prrsv) replication by antisense phosphorodiamidate morpholino oligomers (pmo). of peptide-conjugated pmo (ppmo), four were found to be highly effective at inhibiting prrsv replication in cell culture in a dose-dependant and sequence-specific manner. ppmo up and hp are complementary to sequence in the ′ end of the prrsv genome, and p and p to sequence in the translation initiation regions of orf and orf , respectively. treatment of cells with up or hp caused a . log( ) reduction in prrsv yield, compared to a control ppmo. combination of p and p led to higher level reduction than p or p alone. up , hp, and a combination of p and p inhibited prrsv replication in porcine alveolar macrophages and protected the cells from prrsv-induced cytopathic effect. northern blot and real-time rt-pcr results demonstrated that the effective ppmo led to a reduction of prrsv rna level. up and hp inhibited virus replication of other strains of prrsv. results from this study suggest potential applications of ppmo for prrs control. porcine reproductive and respiratory syndrome (prrs) causes an estimated us$ million in losses per year to the swine industry in the usa (neumann et al., ) . the clinical manifestations of prrs includes severe reproductive failure, post-weaning pneumonia, growth reduction, and increased mortality (keffaber, ; loula, ) . the causative agent of this disease is prrsv, an enveloped, single-stranded and positivesense rna virus (j.j. . prrsv is a member of the family arteriviridae, which includes equine arteritis virus (eav), lactate dehydrogenase-elevating virus for years; however, there is evidence of reversion to virulence of at least one of the current vaccine strains following use in pigs (opriessnig et al., ) . therefore, alternative strategies are needed for effective prrs control. this study explored the inhibition of prrsv propagation in cell cultures by peptideconjugated phosphorodiamidate morpholino oligomers (ppmo) specific for prrsv sequences. pmo are structurally similar to single-stranded dna in that each subunit includes a purine or pyramidine base. in pmo each base is joined to a novel backbone consisting of one morpholine ring and phosphorodiamidate linkage per subunit (summerton, ; summerton and weller, ) . pmo are uncharged, water-soluble, and highly resistant to nuclease degradation (hudziak et al., ) . pmo bind to target mrna by watson-crick base pairing and exert an antisense effect by preventing access to critical segments of rna sequence, such as a translation initiation site, through steric blockade. this is a distinctly different process from the rnase h-dependent mechanism induced by the often-used antisense structural type phosphorothioate dna (summerton, ) . a number of studies have shown that pmo can effectively and specifically block translation of target mrna in vitro and in vivo via intravenous, intraperitoneal, subcutaneous, transdermal or oral administration (arora et al., a,b; brent and drapeau, ; hudziak et al., ; qin et al., ; stein et al., ; taylor et al., ) . it has been shown that pmo conjugated to short arginine-rich cell penetrating peptides have a significantly higher efficiency of delivery into cultured cells than do non-conjugated pmo (moulton et al., ; deas et al., ) . ppmo were found to be fairly stable in cells and human serum for at least h (youngblood et al., ) . sequence-specific antiviral efficacy of ppmo in cell cultures has been documented against sars coronavirus (neumann et al., ) , eav (van den born et al., ) , flaviviruses (deas et al., ; kinney et al., ) , influenza a virus (ge et al., ) , and kaposi's sarcomaassociated herpesvirus (zhang et al., ) . recently ppmo were also applied in vivo and protected animals from challenge with ebola virus (enterlein et al., ) , coxsackievirus b (yuan et al., ) , and murine coronaviruses (burrer et al., ) . prrsv has an rna genome approximately -kb in size, which consists of untranslated region (utr), nine open reading frames (orf a, orf b, orf a, orf b, and orfs through ) and utr (meng et al., ; j.j. meulenberg et al., ) . two large orfs, la and lb, together occupy over kb and encode non-structural proteins, which are likely involved in genome replication and transcription (j.j. . the remaining orfs, a through encode six membraneassociated proteins (gp , e, gp , gp , gp , and m) and nucleocapsid (n) protein (meulenberg et al., ) . in prrsvinfected cells, a set of six or seven nested viral subgenomic rna is formed (conzelmann et al., ; j.j.m. . all of the subgenomic rna have an identical -leader sequence derived from the end of genomic rna, as well as an identical terminal sequence preceding poly-(a) tails of variable length. the generation of co-terminal mrna species is believed to be accomplished through a process known as dis-continuous subgenomic mrna synthesis (sawicki and sawicki, ; van marle et al., ) . the end of each subgenomic mrna of north american strains contains a common leader sequence of bases (nelsen et al., ) , whereas in lelystad virus, the prototype strain of the european prrsv genotype, the leader is bases (j.j. . the utr of north american and european prrsv strains are and bases, respectively, excluding the poly-(a) tail, and is common to all of their subgenomic mrna. prrsv subgenomic rnas are polycistronic in structure, but it is believed that only the first open reading frame (orf) of each subgenomic rna is translated into a viral protein (meng et al., b) . in a previous study (zhang et al., ) , we tested six ppmo and found one of them ( up ), designed to target the terminal region of the prrsv genome, to be a highly effective inhibitor of prrsv replication in a sequence-specific and dose-dependent manner. considering the lengthy genome and sequence heterogeneity of prrsv strains, we sought to evaluate ppmo that target other sites of the genome and that have broad reactivity against different strains of prrsv, and also to further refine our evaluation of efficacy and ppmo mechanism of action, in the hopes of defining a potential strategy for addressing prrsv with ppmo technology. in this study, we evaluated a dozen ppmo targeting a variety of different sites in prrsv genomic, subgenomic and negativesense rna. the peptide component of the ppmo used in this study was recently developed, and possesses improved characteristics compared to the peptide used in our previous ppmo study. we found that it was possible to obtain a greater-thanadditive effect by using a combination of two ppmo. the effects of ppmo on prrsv rna synthesis were also assessed in an attempt to gain insight into the mechanism of ppmo-mediated inhibition of prrsv replication. ppmo are also shown in this study to inhibit prrsv replication in pam cultures, the primary target cells for prrsv infection in pigs. cell line atcc crl (meng et al., a) was maintained in dmem medium supplemented with % fetal bovine serum (fbs). prrsv strains atcc vr (meng et al., a) , lelystad (j.j. , and nvsl - (nvsl, ames, ia) were used to inoculate crl cells at . multiplicity of infection (moi) for ppmo testing. other prrsv strains used in this study include fl- , , , b, , , , and (kindly provided by dr. fernando osorio, university of nebraska-lincoln), and ingelvac mlv (kindly provided by dr. kay s. faaberg, university of minnesota). for virus titration, a series of fold dilutions of virus were added to monolayer crl cells in a -well plate. the degree of cytopathic effect (cpe), characterized by cell rounding, clumping and detachment, was assessed microscopically h after prrsv inoculation, in comparison with mock-infected cells. tissue culture infectious dose (tcid ) per milliliter was calculated based on cpe develop-ment according to the method of reed and muench, as described previously (zhang et al., ) . for pam culture, broncho-alveolar lavage was collected from to weeks old prrsv-negative piglets, as described previously (shibata et al., ) . broncho-alveolar lavage fluid was centrifuged at × g for min, and cell pellets resuspended in rpmi- medium supplemented with % fbs, mm lglutamine, u/ml penicillin, g/ml streptomysin sulfate and g/ml gentamycin. the cells were plated at a density of × cells/well in -well cell culture plates. pam were incubated for h at • c in a humidified % co incubator and washed once with plain rpmi- media before virus inoculation. ifa was carried out as reported previously (zhang et al., ) with an n-specific monoclonal antibody sdow (nvsl, ames, ia) (nelson et al., ) . specific reactions between sdow and n protein were detected with goat anti-mouse igg-fluorescein isothiocyanate (fitc) conjugate (sigma, st louis, mo) and observed with fluorescence microscopy. for pam cells, ifa was performed by fixation of pam cells with % paraformaldehyde and permeabilization with . % triton x- , followed by incubation with antibody sdow . pmo were synthesized at avi biopharma inc. (corvallis, or) by methods previously described (summerton and weller, ) . the peptide nh -(rxr) xb-cooh (where r = arginine, x = -aminohexanoic acid, and b = ␤-alanine) was covalently conjugated at the end of each pmo (fig. a) . the conjugation, purification, and analysis of ppmo were similar to the methods described elsewhere (abes et al., ; moulton et al., ) . a random sequence ppmo (named 'cp ') having little agreement with prrsv or primate mrna sequences was used as control for non-sequence-specific activity of the ppmo chemistry. ppmo were dissolved in water at a concentration of mm and stored at • c. for experiments, ppmo were diluted to desired concentrations in dmem. crl cells were seeded in -well plates at × cells per well and grown overnight to near confluency. treatment of crl cells was conducted as previously described (zhang et al., ) . briefly, cells were inoculated with virus at . moi for h at • c. after inoculum removal, ppmo diluted in dmem was added to the cells and incubated for h at • c. dmem medium without ppmo was included as a mock treatment control and ppmo cp as a negative control. after removal of ppmo, maintenance medium (dmem supplemented with % fbs) was added. the cells were then incubated for h at • c, after which both supernatant and cells were harvested for further analysis. for experiments using ppmo combination treatments, each of the two constituent ppmo were present at equal molar concentration. for the cross strain inhibitory assay, similar procedures as described above were used, except that different prrsv strains were used. macrophages were incubated in -well cell culture plates for h prior to treatment. cells were inoculated with prrsv strain vr at . moi and incubated for h. the cells were washed once with plain rpmi medium before addition of ppmo. the ppmo were diluted in plain rpmi medium, added to the cells and incubated for h at • c. the ppmo solution was removed and culture medium (rpmi supplemented with % fbs) was added. the cells were cultured and observed for cpe development. total rna was isolated from prrsv-infected crl cells by trizol ® reagent (invitrogen, carlsbad, ca). ppmotreated crl cells (same procedure as above) were grown in -well cell culture plates and harvested at h p.i. by direct lysis in trizol ® reagent after removal of culture supernatant. cells treated with cp or mock-treated were included as controls. rna was quantified by quant tm universal microplate spectrophotometer (biotek instruments, winooski, vermont) and stored at − • c for further analysis. rna samples ( . g) were denatured at • c for min and immediately placed on ice for min. the denatured rna was separated at v for h in % agarose gel containing formaldehyde. the separated rna were transferred onto nylon membrane and hybridized with a dna probe derived from prrsv orf sequence with the pcr primers sb f , -gcgga tccca aataa caccg gcaag c- and sb r , -cgtct agatg ccagc ccatc atgct gag- . the probe was labeled with dig- -dutp by pcr dig probe synthesis kit (roche diagnostics, indianapolis, in). the blotted membrane was incubated with anti-digoxigenin-alkaline phosphates for min, cspd chemiluminescent substrate (roche) for min, and then exposed on x-ray film. for quantitative rt-pcr analysis, rna was first treated with rnase-free dnase (promega, madison, wi) to remove carryover dna from the rna isolation procedure. reverse transcription was carried out using superscript tm iii first-strand synthesis system and random hexamers (invitrogen). real-time pcr primers were the same as reported previously (zhang et al., ) . a fragment of bases from the end of the prrsv genome was cloned into pcdna vector, and used as template to generate standard curves for the real-time pcr, which was conducted in chromo tm four-color real-time system (bio-rad laboratories, hercules, ca) with iq sybr green supermix (bio-rad). transcripts of ␤-actin were also amplified from the same samples in order to assure normalized quantitative rt-pcr detection of prrsv rna from the cells. pairs of dna oligonucleotides corresponding to ppmo target sequence in prrsv were duplexed and subcloned upstream of luciferase coding sequence in a t promoter-containing reporter plasmid, pcineoluc, as described previously (zhang et al., ) . dna sequencing was conducted to confirm the presence of desired sequences in the resulting plasmids. cp was used as a negative control in the reporter assay. each plasmid dna was linearized downstream of the luciferase sequence. in vitro transcription was conducted with the t ribomax tm express large scale rna production system (promega). in vitro translation was carried out with rabbit reticulocyte lysate translation system (promega). bright-glo tm luciferase assay system (promega) was used to detect luciferase yield. luminescence signals were measured with victor tm multilabel counter (perkin-elmer life and analytical sciences, wellesley, ma). the viability of crl and pam cells after ppmo treatment was determined with celltiter-glo ® luminescent cell viability assay (promega). briefly, cells were treated with ppmo under conditions similar to those described above in "ppmo treatment of crl cells". mock-treated cells were included for comparison. celltiter-glo reagent was added and incubated for min at room temperature. the luminescence signal was measured with victor tm multilabel counter. relative percentages of luminescence intensity were calculated by comparison to mock-treated controls. the student's t-test was used to assess the significance of differences of viral yield or rna level between the groups of ppmo-treated cells. a two-tailed p-value of less than . was considered significant. prrsv genomic sequences were retrieved from the gen-bank database. parallel sequence alignments of prrsv strains from each of the two major genotypes show that the and utrs, and the translation initiation regions of all orfs, within each genotype, are highly conserved. these regions likely participate in essential events of the viral life cycle (tan et al., ; verheije et al., ) , making them rational sites for antisense ppmo targeting. for this study, ppmo were designed against prrsv vr , a virulent strain of the north american genotype. ppmo sequences and target site locations in the prrsv genome are specified in table and depicted schematically in fig. b . the terminal region has proven to be a productive ppmo target region in a number of positive-strand rna viruses (burrer et al., ; deas et al., ; van den born et al., ; zhang et al., ) . ppmo up and hp were designed to basepair to the end region of prrsv rna genome, in an attempt to interfere with translation of viral rna replicases. hp is complementary to the hairpin loop region, which was predicted from secondary structure analysis ( ). ppmo complementary to negative-sense eav rna inhibited eav replication in cell culture (van den born et al., ) . nsp is complementary to the end of negative-sense prrsv rna, and was designed to interfere with the production of positivesense viral rna. nsp is complementary to negative-sense prrsv rna in the transcription regulatory sequence (trs) region, in an attempt to interfere with synthesis and/or transla- tion of the viral subgenomic rna. up is complementary to the end of prrsv rna genome, in an attempt to interfere with negative-strand rna synthesis. ppmo bp , p , p , p , p , p , and p are complementary to the translation initiation regions of orfs b, , , , , , and , respectively, to attempt blocking the translation of these orfs. ppmo up , which is complementary to nucleotide - of prrsv ' terminal sequence and was previously shown to be highly effective (zhang et al., ) and cp were included as positive and negative controls, respectively. a virulent prrsv strain, vr , was used to evaluate the ability of the ppmo to inhibit prrsv replication in crl cells. at . moi, vr reached peak replication in these cells at approximately h post-inoculation. in initial experiments, ppmo were applied to monolayer cells at a final concentration of m in dmem and incubated for h immediately after the virus inoculation period. the cells were then observed daily for cpe development and supernatants were collected at days p.i. for virus titration in crl cells to determine prrsv yield. of the ppmo tested, up and hp were found to be highly effective at inhibiting prrsv cpe development compared to cp -or mock-treatment. the cells treated with p and p also showed less severe cpe than the controls. the other ppmo had no effect on preventing cpe development. titration of prrsv in cell culture supernatants showed that treatment of crl cells with ppmo up or hp caused a . log tcid reduction in prrsv yield in comparison to cp ( fig. a) . treatment of the cells with p and p led to a reduction in prrsv yield of and log tcid , respectively, compared to cp . none of the other ppmo reduced prrsv yield significantly. ppmo up , hp, p , and p were selected for further characterization in this study. confirmation of the cpe observations and prrsv yield titration was obtained by ifa on crl cells after virus inoculation and ppmo treatment (fig. b ). ifa using a monoclonal antibody against the prrsv n-protein showed that treatment of cells with up led to an absence of prrsv-positive cells. in cells treated with hp, a few fluorescent-positive cells were observed. in cells treated with p and p , some prrsv-positive cells were observed, but far fewer than those treated with cp and up . these results indicate that the presence of up , hp, p , and p inhibited prrsv replication in vr -inoculated cells. ppmo up , hp, p , p , and cp were further tested at doses between and m for inhibition of vr replication in crl cells (fig. ) . cp had little effect on virus replication in comparison with the mock treatment control. each of the other four antisense ppmo reduced virus yield in a dose dependent manner (fig. ) . treatment of cells with up at m or hp at m concentration led to . log tcid reduction in prrsv yield compared to cp . ppmo p or p at m concentration led to reduction in prrsv yield to below the detection level of this assay. to assess the cytotoxicity of the ppmo used in this study, cell viability assays were conducted on crl and pam cells treated with ppmo at m. the experiment was repeated twice, each with three replicates. in comparison with mock treatment control, the average relative percentages of cell viability for crl cells after treatment was % ( up ), % ( hp), % ( p ), % ( p ), and % (cp ). the average percentages of viable pam cells after ppmo treatment were % (cp ), % ( up ), % ( hp), and % ( p + p ). these results virus titers are shown as tcid (log /ml). "mock" samples is from cells receiving virus inoculation but no ppmo treatment. statistical significance of difference in viral yields between ppmo and mock treatments: * p < . ; ** p < . . cells that were treated with m up or hp had virus yields not detectable (nd) in this assay, and a bar is arbitrarily drawn to show the presence of the samples in the graph. the experiment was repeated three times and error bars are shown. (b) immunofluorescence assay with sdow monoclonal antibody. specific fluorescence is clearly visible at h post prrsv (vr ) infection, while no fluorescence is observed in uninfected (−) control. treatment with ppmo up , hp, p , and p resulted in reduction of virus replication, while up and cp did not appear to have any effect under identical conditions. the images below the green fluorescence images were taken with a dapi filter to show the total number of living cells. demonstrated that ppmo generated little cytotoxicity in both crl and pam cells, further indicating that the suppression of virus replication observed in the antiviral experiments above was due to sequence-specific effects. in an attempt to derive a "selectivity index" (si) (the ratio of the concentration of drug causing % cytotoxicity [cc ] divided by the concentration of drug causing a % inhibition of viral production) relevant to the various conditions of this study, we sought to determine the cc for ppmo up , hp, p , and p . crl cells were treated for h with concentrations of each ppmo from to m. cell viability was determined at h after the treatment period. the cc for up , hp, p , and p are , , , and m, respectively. based on a % inhibition of viral production of approximately , . , fig. . dose-dependent inhibition of prrsv replication. virus yield in cell culture is reduced concomitantly with increasing concentrations of up , hp, p , and p . cells receiving some of the treatments had virus yields not detectable (nd) in this assay, and a bar is arbitrarily drawn to show the sample presence in the graph. the dose-dependent inhibition of prrsv replication by up , hp, p , and p is significantly different from cp (p < . ). the experiment was repeated three times and error bars are shown. . , and m, respectively, for these four ppmo (fig. ) , we conclude that the si for these antisense ppmo is , , , and under these conditions. to investigate the effect of treatment with ppmo combinations on prrsv replication, ppmo were paired and tested at equal molar concentrations, for example, a combination at m final concentration consisted of each ppmo being present at m. the ppmo up was not included in this test since it was already established as effective at m. the ppmo that target regions in prrsv thought to be involved in transcription events were tested in pairs: hp + p , hp + p , up + nsp , up + nsp . other pairs of ppmo targeting the translation initiation regions of orfs to were also tested: p + p , p + p , p + p , and p + p . the results of virus titration showed that only the combination of p fig. . combinatory inhibition assay of ppmo. two ppmo were added at equal molar concentration to cells after prrsv inoculation. the combination treatment of p and p led to significant lower virus yield (p = . ) than did either ppmo alone. cells that were treated with or m p + p had virus yields not detectable (nd) in this assay, and a bar is arbitrarily drawn to show the presence of the samples in the graph. and p ( p + p ) caused significant reduction (p = . ) in prrsv yield in comparison with a constituent individual ppmo (fig. ) . none of the other ppmo combinations showed significantly higher inhibitory activity than that of a constituent individual ppmo. to determine ppmo effect on prrsv rna synthesis, crl cells were inoculated with vr , treated with ppmo and harvested at h p.i. for rna isolation. ppmo up , hp, nsp , nsp , and up were included in this experiment as they were designed to block prrsv rna synthesis. the combination of ppmo p + p was also used to see if it had any effect on prrsv rna synthesis. ppmo p , p , and cp were included, for comparison. in northern blot analysis, at least seven distinct rna species were detected in the mocktreated sample, consistent with what would be expected from using a probe that detects orf , and indicating that all prrsv genomic and subgenomic rna containing orf sequence was no detectable rna is seen after treatment with up or hp. rna molecular weight markers are on the left. prrsv rna transcript designations are indicated on the right. as a loading control, the transcript of ␤-actin was detected and is shown in the lower panel. (b) quantitation of prrsv rna by real-time rt-pcr with primers designed to amplify a region of orf a. treatment of the cells with ppmo up , hp, p + p , up , nsp and nsp at m led to significant reduction of prrsv genomic rna level (p < . ). the cells were harvested at h p.i. and total rna was isolated for real-time rt-pcr. cells treated with up had no prrsv rna detectable (nd) in this assay and a bar is arbitrarily drawn to show the sample in the graph. the prrsv rna copy numbers in each sample was calculated in comparison with a standard curve, after normalization with ␤-actin transcript levels (see section . ). detected. prrsv rna in samples treated with up or hp was below the detection level (fig. a) . ppmo p or p had no effect on viral rna level, but the combination of p and p led to significant reduction of viral rna level (fig. a) . ppmo up , nsp , and nsp also apparently led to a modest reduction of viral transcription in comparison with cp . the northern blot shows that the, control ppmo, cp , produced a small yet noticeable effect on prrsv rna level compared to mock-treatment. however, the slight non-specific inhibition caused by cp was far less than the high level of inhibition produced by several of the antisense ppmo. real-time rt-pcr with primers specific for prrsv orf a was also used to evaluate rna levels. only prrsv genomic rna could be detected with the primers. in cells treated with up , prrsv rna was not detectable (fig. b) . in cells treated with hp or p + p , prrsv genomic rna was reduced by -and -fold, respectively, compared to cp -treated cells. the presence of up , nsp , and nsp also led to -, -, and -fold reduction of prrsv genomic rna in comparison to cp -treated cells. treatment with either p or p did not reduce prrsv rna. these results are consistent with the northern blot analysis above. to further characterize the sequence-specificity of the pmomediated inhibition of prrsv replication, we conducted a cell-free luciferase reporter assay. the target sequences of ppmo up , hp, p , and p were subcloned upstream of luciferase in a reporter expression plasmid. in this assay, ppmo binding to its rna target typically results in inhibition of translation of the downstream luciferase coding sequence. effects of ppmo treatment on target rna translation were assessed by measurement of luciferase production. incremental concentrations of ppmo were added to in vitro translation reactions to determine ppmo inhibitory effect on translation. as shown in fig. , nm or higher concentration of up , p , hp, and p reduced luciferase yield by more than %, while cp had no or little effect on translation of corresponding rna. ppmo p was slightly less effective than the other three ppmo in this test. the results of this assay indicate that the antisense ppmo specifically bound complementary rna and thereby inhibited the translation of luciferase. the ppmo in this study were designed against the vr strain of prrsv. to determine the efficacy of the ppmo against eleven other prrsv strains, nvsl - , fl- , b, , , , , , , mlv, and lelystad, cross strain inhibition assays were conducted. the lelystad strain is a prototype of the european genotype. the rest are strains of the north american prrsv genotype. ppmo up , hp, and the combination of p + p were tested against fig. . inhibition of target rna translation by ppmo in a cell-free luciferase reporter assay. relative percentages of luciferase level were calculated in comparison with signal of control ppmo sample at nm. the level of luciferase production decreased in response to increasing concentrations of ppmo up , hp, p , and p , while cp has little effect on luciferase rna translation. each of the prrsv strains. virus inoculation and ppmo treatment were conducted in the same manner as described above for vr . virus titration results showed that up and hp effectively suppressed replication in all strains except lelystad (fig. a) . the p + p combination led to a noticeable reduction in virus titer of a majority of the north american isolates, including nvsl, b, , , , and mlv. replication of lelystad was not inhibited by any of the ppmo tested. sequence alignment of the ppmo target sites in the prrsv strains showed that lelystad had low sequence identity with the other strains (fig. b ). prrsv nvsl, , , and b have one nucleotide mismatch with vr in the end of the up target site. in the hp target site, the only mismatch found was in nvsl, which has a single nucleotide difference in the middle of the ppmo target region. in the p target site, nvsl and have two mismatches, lelystad has five mismatches and two deletions, and all others have one mismatch. in the p target site, one mismatch occurs in all strains except lelystad, which has five mismatches. the sequence alignment results indicate that nucleotide mismatches in the prrsv target sites of p and p are likely responsible for the low level of inhibition produced by these two ppmo. alveolar macrophages are the primary target cells for prrsv infection in pigs (yoon et al., ) . evaluation of ppmo effect on virus replication in pam has relevance to the biology of a natural infection. to facilitate adaptation to laboratory culturing, pam cells were pre-incubated for h before their inoculation with prrsv. after virus inoculation, pam were treated with ppmo up , hp, p + p , or cp , and then observed at h p.i. for cpe development. the pam that were treated with fig. . cross strain inhibition assay. (a) virus yield titration shows inhibition of ten north american prrsv strains by ppmo up and hp. lelystad is a prototype of european prrsv genotype. all other strains are within the north american prrsv genotype. "mock" denotes virus infected cells receiving no ppmo. treatment of cells with ppmo up or hp led to suppression of prrsv replication of all north american strains, producing virus yields not detectable (nd) in this assay. bars are arbitrarily drawn for those samples with no detectable virus ("nd") to show the presence of samples in the graph. (b) sequence analysis identifies nucleotide mismatches between ppmo and their complementary target sites in prrsv rna. prrsv strain names are listed in the first column. the sequence of strain vr is used as the reference sequence, as the ppmo were designed against it. ppmo names are listed above the blocks of prrsv sequences. "lely" stands for lelystad strain, which has little similarity in the utr to other strains, as indicated by symbol "-" in the alignment of up and hp target sequences. for all other sequences, only nucleotides differing from reference sequence are shown, and identical nucleotides are indicated with ".". missing nucleotides are indicated with "-". the initiation codons atg of orfs and are underlined. genbank accession numbers for prrsv strains in the alignment are listed in parenthesis: ppmo up , hp, or combination of p + p showed no or little cpe development, while the cp -or mock-treated control cells suffered severe cpe (fig. a ). this result indicates that ppmo treatment protected the cells from prrsv-induced cpe development, and also that prrsv replicates rapidly in pam. to investigate the ppmo-mediated protection of pam from cpe development, we treated of pam with up twice, h apart. no cpe was observed in the cells at days p.i. (data not shown), indicating that ppmo treatment protected pam from cpe for at least week after prrsv inoculation. ifa was conducted with pam cells after prrsv inoculation and ppmo treatment. the cells were fixed at h p.i. to mini-mize cell death in mock-treated control. results showed that the approximate percentages of prrsv-positive cells were % with up treatment, % with hp, % with p + p , and % in mock-treated control (fig. b) . the results demonstrated that treatment with antisense ppmo reduced prrsv infection in pam cells. prrsv yield in pam culture was titrated in crl cells. treatment of pam with ppmo up , hp, and p + p led to -, -, and -fold reduction in virus yield, respectively, compared to mock-treated control (fig. c) . the reduction in virus titer in pam was considerably less than had been observed in crl cells after similar ppmo treatment. employing morpholino oligomers to inhibit rna virus replication has become an appealing prospect after reports of the successful knockdown of cellular proteins via inhibition of pre-mrna splicing and/or translation of mrnas (heasman, ) . in rna virus genomes, there are a variety of regions for pmo targeting. in our previous study, six ppmo, designed to target the and utrs, the trs-region of genomic rna, and the end of negative-sense prrsv rna, were evaluated (zhang et al., ) . only one of those ppmo, up , targeting the terminal region of the genome, was found to be effective at inhibiting prrsv replication. in the present study, ppmo were designed to target a wider variety of sites within the prrsv genome, including the and utrs, translation initiation regions of orfs through , and negative-sense prrsv rna. of the ppmo tested here, up was found to be the most highly effective ppmo, followed by hp, p , and p . in this study, ppmo up appeared more effective than up did in our previous study (zhang et al., ) . the target sites for up and up are located in the terminal region of the prrsv rna genome and overlap by nucleotides. we note, however, that comparing results between the two studies is complicated by the fact that the first study employed ppmo in which the peptide component was r f r (p ), while in the present study we utilized the more recently developed (rxr) (p ) peptide. ppmo hp complements a hairpin sequence in the utr, and was also quite effective, reducing prrsv yield by . log ( fig. a) . a ppmo targeting the corresponding region in eav was likewise found to be highly effective (van den born et al., ) . it has previously been reported that a combination of pmo compounds can outperform a single agent. this was shown in vivo with non-conjugated pmo against ebola virus , and the authors of that study suggested that slowing virus replication may allow sufficient time for development of antiviral immune responses and viral clearance. likewise ge et al. showed that a combination of ppmo produced greater efficacy than either ppmo alone against influenza a virus in cell culture (ge et al., ) . our results showed that treatment with a combination of p and p led to significantly higher prrsv inhibition than did either of the two ppmo alone. however, many other ppmo combinations in this study failed to outperform their constituent single agents. to elucidate the mechanism of pmo-mediated inhibition, we conducted northern blot and rt-pcr analyses (fig. ) . treatment of cells with up or hp reduced prrsv rna to the level of below detection, indicating inhibition of prrsv rna synthesis, likely accomplished through blocking translation of the replicase encoded by orf a/b. treatment with a combination of p and p also produced a reduction in prrsv rna level, probably due to the blockage of the translation of the transcripts for orfs and . based on their target locations in the end of prrsv genome, we speculate that the combination of p and p may have reduced prrsv genome replication by interfering with assembly or progression of the replicase complex. our results clearly show a reduction of prrsv rna level in cells treated with up or hp. a ppmo-associated reduction in prrsv rna production was also observed by rt-pcr in our previous study (zhang et al., ) . conversely, similar experiments against eav showed no significant change in the level of any viral rna transcripts, as assessed by gel hybridization analysis (van den born et al., ) . this seeming discrepancy may have resulted from differing experimental conditions. for rna isolation, cells were harvested at h p.i. in the prrsv, but h in the eav experiments (van den born et al., ) . there likely are some differences in the replication mechanics between prrsv and eav, since ppmo designed to target the respective trs-regions were reported to be effective against eav (van den born et al., ) , yet ineffective against prrsv (zhang et al., ) . among the ppmo tested in this study, only were found to be significantly efficacious. the reasons for the ineffectiveness of the other ppmo are unclear, but could include inaccessibility of prrsv target sequence, or, that successful ppmo/target rna duplexing did not affect prrsv replication. ppmo up , hp, and combination of p + p effectively inhibited prrsv-induced cpe development in primary pam, the major target cell type of prrsv infection in host animals. ifa confirmed the ppmo inhibition of virus replication. these results indicate that ppmo may indeed have potential to control, or reduce the severity of, prrsv infection in pigs. it was unexpected that ppmo-mediated reduction of virus yield in pam was less than that observed in crl cells. this difference but may be because prrsv has a higher replication rate in pam than it does in crl cells, and the ppmo were unable to significantly interfere with the more rapid viral replication. alternatively, differential uptake of ppmo by crl and pam cultures may have had an effect. however, virus replication in pam after treatment with up did not result in cytopathic effect, and the cells were healthy for at least week after virus inoculation. further study is being undertaken to further investigate ppmo-mediated protection of primary cells. ppmo up and hp inhibited replication of ten north american prrsv strains in our cross-strain inhibition assay. sequence alignment (fig. b) showed that the region targeted by these ppmo contains highly conserved sequences. in addition, analysis of prrsv sequences from the genbank indicates that the complementary sequence of up and hp are highly conserved across north american prrsv strains. thus, these two antisense ppmo may be suitable for application against most north american prrsv strains. the broad inhibition by up and hp also further confirms that the utr of prrsv is a highly productive target region for ppmo, and that this region likely plays an important role in arterivirus replication. a one base mismatch between p or p ppmo and their respective target sites in ten north american prrsv strains may explain the low activity observed. (fig. a and b) . it is noteworthy that all strains had identical sequences in both the p and p target sites. thus, a minor sequence modification of ppmo targeting these regions may lead to improved efficacy. a cell viability assay of ppmo-treated crl cells and pam detected no cytotoxicity, indicating that the suppression of prrsv replication observed in the antiviral assays was due to ppmo-specific inhibition of prrsv molecular events. the absence of ppmo-induced cytotoxicity at effective antiviral concentrations is an important attribute of these compounds, when considering potential in vivo applications. the prevalence of prrsv and financial losses associated with prrsv infection in swine herds is high, and current strategies to control prrs, including the use of commercial and autogenous vaccines, are inconsistent and generally less than adequate (meng, ; opriessnig et al., ) . specific anti-prrsv drugs would be useful to complement other strategies of prrsv prevention and control. under the conditions of this study, four ppmo showed potent and specific anti-prrsv activity in cell culture, and can be considered potential drug candidates for use in prrsv control. further investigation into the pharmacokinetic, toxicological and antiviral properties of these ppmo in vivo is warranted. vectorization of morpholino oligomers by the (r-ahx-r) peptide allows efficient splicing correction in the absence of endosomolytic agents phosphorodiamidate morpholino antisense oligomers inhibit expression of human cytochrome p a and alter selected drug metabolism bioavailability and efficacy of antisense morpholino oligomers targeted to c-myc and cytochrome p- a following oral administration in rats comparison of porcine alveolar macrophages and cl for the detection of porcine reproductive and respiratory syndrome (prrs) virus and anti-prrs antibody characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) targeted 'knockdown' of channel expression in vivo with an antisense morpholino oligonucleotide antiviral effects of antisense morpholino oligomers in murine coronavirus infection models molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group inhibition of flavivirus infections by antisense oligomers specifically suppressing viral translation and rna replication vp knockdown inhibits ebola virus amplification and protects against lethal infection in mice inhibition of multiple subtypes of influenza a virus in cell cultures with morpholino oligomers morpholino oligos: making sense of antisense? resistance of morpholino phosphorodiamidate oligomers to enzymatic degradation antiproliferative effects of steric blocking phosphorodiamidate morpholino antisense agents directed against c-myc reproductive failure of unknown etiology enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line inhibition of dengue virus serotypes to in vero cell cultures with morpholino oligomers mystery pig disease: an update for the practitioner heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development molecular cloning and nucleotide sequencing of the terminal genomic rna of porcine reproductive and respiratory syndrome virus phylogenetic analyses of the putative m (orf ) and n (orf ) genes of porcine reproductive and respiratory syndrome virus (prrsv): implication for the existence of two genotypes of prrsv in the usa and europe characterization of a high-virulence us isolate of porcine reproductive and respiratory syndrome virus in a continuous cell line, atcc crl a nested set of six or seven subgenomic mrnas is formed in cells infected with different isolates of porcine reproductive and respiratory syndrome virus lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav characterization of proteins encoded by orfs to of lelystad virus subgenomic rnas of lelystad virus contain a conserved leader-body junction sequence cellular uptake of antisense morpholino oligomers conjugated to argininerich peptides porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents differentiation of u.s. and european isolates of porcine reproductive and respiratory syndrome virus by monoclonal antibodies assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states comparison of molecular and biological characteristics of a modified live porcine reproductive and respiratory syndrome virus (prrsv) vaccine (ingelvac prrs mlv), the parent strain of the vaccine (atcc vr ), atcc vr , and two recent field isolates of prrsv in vivo evaluation of a morpholino antisense oligomer directed against tumor necrosis factoralpha coronaviruses use discontinuous extension for synthesis of subgenome-length negative strands in vivo replication of porcine reproductive and respiratory syndrome virus in swine alveolar macrophages and change in the cell population in bronchoalveolar lavage fluid after infection inhibition of vesivirus infections in mammalian tissue culture with antisense morpholino oligomers morpholino antisense oligomers: the case for an rnase h-independent structural type morpholino antisense oligomers: design, preparation, and properties comparison of the leader sequences of north american isolates of reference and field strains of porcine reproductive and respiratory syndrome virus (prrsv) antiviral activity of morpholino oligomers designed to block various aspects of equine arteritis virus amplification in cell culture arterivirus discontinuous mrna transcription is guided by base pairing between sense and antisense transcription-regulating sequences kissing interaction between noncoding and coding sequences is essential for porcine arterivirus rna replication gene-specific countermeasures against ebola virus based on antisense phosphorodiamidate morpholino oligomers mystery swine disease in the netherlands: the isolation of lelystad virus isolation of a cytopathic virus from weak pigs on farms with a history of swine infertility and respiratory syndrome stability of cell-penetrating peptide-morpholino oligomer conjugates in human serum and in cells inhibition of coxsackievirus b in cell-cultures and in mice by peptide-conjugated morpholino oligomers targeting the ires monoclonal antibodies against conformationally dependent epitopes on porcine reproductive and respiratory syndrome virus suppression of porcine reproductive and respiratory syndrome virus replication by morpholino antisense oligomers inhibition of replication and transcription activator and latency-associated nuclear antigen of kaposi's sarcoma-associated herpesvirus by morpholino oligomers we are grateful to the chemistry department of avi bio-pharma inc. for the synthesis and quality control of all ppmo used in this study, to dr. fernando osorio at university of nebraska-lincoln for his gifts of prrsv isolates fl- , , , b, , , , and , and to dr. kay s. faaberg at university of minnesota for her gift of prrsv ingelvac mlv used in cross strain inhibition assay in this study. this project was supported by a grant from the national pork board. key: cord- -il g w authors: akkina, ramesh; ellerbrok, heinz; hall, william; hasegawa, hideki; kawaguchi, yasushi; kleanthous, harold; mcsweegan, edward; mercer, natalia; romanowski, victor; sawa, hirofumi; vahlne, anders title: international meeting of the global virus network date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: il g w the global virus network (gvn) was established in in order to strengthen research and responses to current viral causes of human disease and to prepare against new viral pandemic threats. there are now gvn centers of excellence and affiliate laboratories in countries. gvn scientists meet annually to learn about each other's current research, address collaborative priorities and plan future programs. the meeting was held from october – in hokkaido, japan, in partnership with the japanese society for virology, the national institute of infectious diseases of japan and the research center for zoonosis control of hokkaido university. this report highlights the accomplishments of gvn researchers in many priority areas of medical virology, including the current zika epidemic, infections by human papillomavirus, influenza, ebola, lassa, dengue, hiv, hepatitis c, and chikungunya viruses, and the development of improved diagnostics and new vaccines. the global virus network (gvn) was established in in order to strengthen research in response to viral causes of human disease and to prepare for new viral pandemic threats (mann, ) . the gvn now has centers of excellence and affiliates in countries. network scientists meet annually to address research and collaborative priorities, learn about each member's current work and plan future programs. these international conferences have become critical platforms for the exchange of ideas (for further information, go to http://www.gvn.org). the international gvn meeting was held from october e in hokkaido, japan, in partnership with the japanese society for virology (jsv), the national institute of infectious diseases of japan and the research center for zoonosis control of hokkaido university. it brought together directors of gvn centers and their colleagues for two days of scientific presentations and discussions of emerging and re-emerging viral threats. this meeting report highlights accomplishments of gvn researchers in many priority areas of human virology, including the current zika epidemic, infections by human papillomavirus, influenza, ebola, lassa, dengue, hiv, hepatitis c and chikungunya viruses, and the development of improved diagnostics and new vaccines. the sapporo meeting was the first gvn event held jointly with a local national virology society. more than jsv members had the opportunity to attend scientific sessions, thereby expanding opportunities for collaborative dialogue, particularly with young japanese virologists. the main objectives of the meeting were to present and discuss current findings in medical virology, including advances in research on hiv vaccines and other important retroviruses; provide a framework to encourage collaborations among world experts; and address the gvn's annual strategy for continued development. the gvn was founded because there was no research organization independent of national governments which had the depth of expertise needed to respond promptly to new pandemic viral outbreaks and to prepare for such outbreaks. the gvn was not intended to replace organizations such as the centers for disease control and prevention (cdc) or the world health organization (who), but to complement their activities with independent opinions and expertise. in parallel, a second major goal of the gvn was to advocate for the strengthening of virology as a core discipline in infectious diseases and to help develop the next generation of virologists (dimaio, ) . in march , of the world's leading virologists gathered in washington, d.c. to pledge their support for an international coalition of virology institutions, ready to act in times of outbreaks and committed to advancing knowledge of pathogenic viruses. today, the gvn is comprised of centers of excellence and affiliates in countries (fig. ). centers are led by world-class virologists who have expertise in two or three areas of virology, strong publication records, a commitment to building technical and scientific capacities in resource-poor countries, and the readiness to support the gvn infrastructure. albert osterhaus, phd, dvm, director of the research center for emerging infections and zoonoses (riz) at the university of veterinary medicine hannover, germany, a gvn center of excellence, was presented with the robert c. gallo award for scientific excellence and leadership for his pioneering contributions to influenza and coronavirus research as well as his contributions to advancing the gvn mission. osterhaus was cited for his decades-long career, which has included the discovery of more than new human and animal viruses and helping the who to combat outbreaks of mers (middle east respiratory syndrome), sars (severe acute respiratory syndrome), and pandemic influenza. he was also praised as a leader within the gvn for his active participation in international conferences and virology training programs, for making important basic science contributions to medical virology and for translating those contributions into broad public health initiatives. the award is named for robert c. gallo, md, the director of the institute of human virology at the university of maryland at baltimore, and the co-founder and scientific director of the gvn. dr. gallo and colleagues discovered the first human retroviruses, human t-cell leukemia virus (htlv)- and - ; and human herpesvirus- , which was subsequently demonstrated by others to cause roseola (gallo, ) . gallo co-discovered the human immunodeficiency virus (hiv) as the cause of aids and together with colleagues discovered one of the first cytokines (il- ). il- later became critical to growing continuous in vitro cell-lines, and is now important in many immune therapy protocols. dr. gallo and colleagues also developed the first blood tests for htlv- and hiv. robert gallo reviewed recent hiv vaccine trials that used four different methods. the thai-u.s. army trial (rv ) appeared to have given very modest protection in the early months after vaccination, while the others failed to prevent infection or in one case was associated with greater infection. gallo explained that for the last several years, the emphasis has been on induction of antibodies to the hiv envelope component, gp , rather than to a primary induction of cellular immunity to prevent infection. this approach was based on the logic of trying to prevent integration and establishment of a permanent infection. this effort has mainly evolved around two programs. the first was a detailed analysis of the rv results, particularly correlates of protection, which have mainly highlighted antibodies interacting with viv gp epitopes and epitopes induced by gp interactions with cd , referred to as cd i abs, which target conserved envelope regions needed for gp interaction with ccr . functional correlations were mainly with fc effector functions rather than neutralizing antibodies (nab). the second major approach is one that is geared toward inducing broad nab, which is a reasonable goal, but one that is not being met, as it is proving far more difficult than supposed. moreover, in studies in nonhuman primates (nhp), as well as the results with rv , correlation of protection is rare with nab, even when they are generated. gallo also described a candidate immunogen expressed as a chimeric protein of gp and cd , which has shown some efficacy against heterologous challenges in primates, and correlates with cd i antibodies that have fc effector activity (fouts et al., ) . this vaccine is in phase i trials (https://clinicaltrials.gov/show/ nct ). like all anti-gp antibodies correlating with protection, these antibodies do not persist, and attempts to increase persistence have resulted in hyper-activation of t-cells, which then form more targets for hiv infection. persistent boosting to maintain the antibodies leads to changes in the antibody isotype, which can reduce their efficacy. based on the rv trial and recent nhp studies, gallo concluded that two critical basic science problems remain to be solved for the development of a successful antienvelope hiv vaccine: durability of the antibodies and a correct immune balance. yasushi kawaguchi, at the university of tokyo, described cell factors that are involved in a unique nuclear-pore-independent export system for macromolecular complexes in the nucleus, and which may be potential targets for novel anti-herpetic drugs. vesicle-mediated nucleocytoplasmic transport is a unique mechanism for nuclear export of macromolecular complexes in which a complex in the nucleus buds through the inner nuclear membrane to form a vesicle in the perinuclear space (primary envelopment). the vesicle then fuses with the outer nuclear membrane to release the complex into the cytoplasm (de-envelopment) . this type of transport is observed in herpesvirus-infected mammalian cells for export of viral nucleocapsids, but is not common in other types of cells, indicating that herpesviruses may expropriate this mechanism (oda et al., ) . however, the cellular mechanism for vesicle-mediated nucleocytoplasmic transport remains largely unknown. he went on to describe recently identified host cell proteins involved in this unique nuclear export system, and how these proteins could be targets for novel anti-herpetic drugs, since this transport system is essential for the life cycle of herpesviruses and is unique in biology. hideki hasegawa, a gvn center director at the national institute of infectious diseases in tokyo, explained that secretory iga antibodies on mucosal surfaces play an important role in protection against influenza virus infection. secretory, polymeric iga antibodies induced by intranasal, inactivated influenza vaccine have higher neutralizing and cross-neutralizing ability against homologous and heterologous influenza viruses compare to monomeric iga antibodies in humans (suzuki et al., ) . a method of producing secretory multimeric iga antibodies in vitro was established recently, and hasegawa and colleagues examined the improvement of neutralizing effects by multimerization using monoclonal iga antibodies. the method of in vitro production of multimeric iga antibodies was established by introducing cdna constructs of h, l, and j chains of iga with secretory component (sc) into expi f cells. this method allowed production of large amounts of recombinant monoclonal polymeric iga antibody. the antibody-coding cdna was isolated and cloned from single plasma cell from the peripheral blood mononuclear cells of a subject previously vaccinated intranasally with inactivated influenza a (h n ) vaccine. monomeric and multimeric iga antibody specific for influenza virus was prepared, and functional analysisdsuch as elisa and neutralization testsdwas performed. similar to secretory iga antibodies in human nasal washes, the recombinant iga showed that polymerization of the antibody molecule enhanced the neutralizing activity. mass spectrometry analysis found that the antibodies had a molecular weight of kda, suggesting they consisted mostly of the tetramer form. these tetrameric iga antibodies may be clinically useful as mucosal agents for the prevention and treatment of influenza. erica ollmann saphire, a gvn center co-director at the scripps research institute (usa), reported on recent outbreaks of ebola virus disease and other viral diseases and the urgent need for novel therapeutics. monoclonal antibodies offer a promising solution to the lack of currently available therapies, but it is often unclear how to select the most effective antibodies or combinations for optimal therapy (saphire and aman, ) . integrated analysis of a large array of antibodies provides a rational basis for the selection of optimized therapeutics, and can lead to rapid understanding of the mechanisms underlying protective efficacy. she described the results of a field-wide analysis of antibodies against ebola virus, a particularly potent antibody against marburg virus, and a novel panel of antibodies against lassa virus. a newly formed international group, the viral hemorrhagic fever immunotherapeutic consortium, aims to galvanize and focus efforts against ebola and similar viruses, using multidisciplinary approaches (saphire et al., ) . sharon lewin, a gvn center director at the doherty institute for infection and immunity (australia), reported on hiv cure research (lederman et al., ) . hiv persists predominantly in long-lived resting cd þ t cells that are found in blood and at greater frequency in tissue. hiv can also persist in specific t-cell subsets enriched in certain tissue sites, including t follicular helper cells in lymph nodes and th (ccr þ) cells in the gastrointestinal tract. long-lived infected macrophages, specifically microglia in the brain or kupffer cells in the liver, may also play a role, though this remains controversial. on art, hiv-specific t cells express multiple exhaustion markers, and these cells are often unable to recognize and kill infected cells due to multiple immune escape mutations that are archived in long lived latently infected cells. furthermore, in some tissue sites such as b-cell follicles in lymph nodes, there is limited penetration of hiv-specific t-cells. lewin described several approaches that are being tested to achieve hiv remission, including reducing latently infected cells through early art or latency reversal, boosting immune clearance, reducing immune activation and homeostatic proliferation, eliminating tissue reservoirs, and gene therapy to make cells resistant to hiv. recently completed clinical trials of latency-reversing agents (lra) in hiv-infected patients on art have included histone deacetylase inhibitors (hdaci) and high-dose disulfiram (delagr everie et al., ) . other interventions, including activation of protein kinase c (pkc) with bryostatin and ingenols, are under evaluation. studies of hdaci and disulfiram have demonstrated that hiv transcription can be activated in vivo with varying efficiency, but to date there is no evidence that these interventions alone can clear latently infected cells. in addition, recent in vitro studies have shown that activating transcription with hdaci, but not other lras, may lead to profound changes in hiv rna splicing, therefore limiting potency. other concerns regarding hdaci include short-and long-term toxicity, changes in host gene expression and adverse effects on immune function. more potent and specific lras are necessary and in development for use alone and in combination. additional interventions to promote cell death may be needed. targeting bcl or inhibitors of anti-apoptotic proteins (iap) is being evaluated in vitro, and immune-mediated clearance with bispecific antibodies, broadly neutralizing antibodies and t-cell vaccines are in clinical development. however, even if the number of latently infected cells is reduced, long-term immune control will be required through therapeutic vaccination and/or immunomodulation. immune checkpoint blockers, including antibodies to ctla- and pd- that have recently been licensed for the treatment of melanoma, are potentially attractive tools to enhance hiv-specific t-cells and reverse latency. clinical trials of these agents are now starting in hiv-infected individuals with malignancies. shyamasundran kottilil, at the institute of human virology (usa), described the challenges of current treatments of chronic hepatitis c. eighty-five percent of hcv infections become chronic; e % of those cases will develop cirrhosis, and e % of these patients will progress to hepatocellular carcinoma and/or decompensation of the liver, leading either to death or liver transplantation. in contrast to chronic hepatitis b or hiv infection, chronic hcv infection can be cured with direct-acting antiviral (daa) treatment. "cure" is defined in terms of sustained virologic response (svr), which is the absence of detectable levels of hcv rna in the plasma after or weeks of daa treatment (jazwinski and muir, ) . current hcv inhibitors act against the ns / protease (e.g., asunaprivir and grazoprevir), the ns a protein (e.g., daclatasvir and velpatasvir) and the ns b polymerase (e.g., sofosbuvir and beclabuvir). treatment with a combination of the two inhibitors, sofosbuvir and velpatasvir, for weeks, will result in a cure of more than % of treated patients. still, clinical challenges remain, including treatment of patients with end-stage liver and renal disease owing to metabolism and/or toxicity of the drugs. other challenges include the treatment of hiv/hcv co-infections, injection drug users, pediatric patients and maternal-fetal transmission and pregnancy. estaban domingo, at the centro de biología molecular severo ochoa in spain, presented studies on the population dynamics of hcv after antiviral interventions in infected human hepatoma huh- . cells. unexpectedly, serial passage of hcv in these cells in the absence of antiviral drugs resulted in increased resistance to inhibitors that target viral or cellular proteins, and was associated with a fitness gain of the virus. if daa-resistance mutations become widespread in human populations, alternative drug treatments will be needed. one such drug is favipiravir (furuta et al., ) . this is a broad-spectrum antiviral agent active against numerous rna viruses, including influenza, norovirus, rabies, and rift valley fever virus. it acts as a lethal mutagen (de avila et al., ) . efficacy and safety trials of favipiravir for treating influenza, as well as ebola virus disease, already have been done. in cell cultures], the efficacy of favipiravir against hcv was found to be comparable to its efficacy against other rna viruses. in hcv-infected huh- . cells, favipiravir demonstrated features typical of lethal mutagenesis. it was concluded that favipiravir offers an alternative for patients chronically infected with hcv. research on biomarkers of progression in human papillomasvirus (hpv)-related cancers was highlighted in this session by franco buonaguro (national cancer institute, italy). buonaguro described persistent infections with high-risk hpvs that are often associated with progression to mucosal cancers in anogenital as well as oropharyngeal areas of the body. he also noted that for cervical cancers, for which screening programs are available, it is difficult to identify the lesions likely to progress to invasive cancers. given that only in hpv -positive cervical dysplasias will eventually progress further, biomarkers clearly are needed. tumor progression is characterized by increased expression of the viral e gene and e -dependent degradation of p , and increased expression of e , which is known to bind and inactivate prb; and integration of viral dna into the host genome with the consequent disruption of the e viral gene (annunziata et al., ) . molecular markers able to identify viral infections associated with progressing cervical neoplasia are strongly needed for screening and triage. in particular, predictive biomarkers are needed to detect lesions at high risk of recurrence or progression, in order to implement appropriate treatment and to avoid overtreatment of patients with a high probability of regression. to achieve such goals, expression profile analysis of p -related genes in hpv- -positive genital carcinomas, along with autologous nontumor tissue, was performed, and significant differences in the expression levels of genes involved in regulation of apoptosis, cell cycle, proliferation and dna repair pathways were identified. moreover, oncogenic driver mutations in critical genes (such as pik ca and tp ), recurrent chromosomal gain/loss, and oncogene activation by hpv integration have all been recognized to cause deregulation of cell signaling pathways and cancer progression (tornesello et al., ) . more recently, analysis of the regulatory regions of human genes encoding for cell proliferation proteins has shown a high frequency of mutations in lesions associated with less oncogenic hpv genotypes, and even in hpv-negative lesions. validation of these candidate biomarkers is currently under way on a larger number of cases, including different grades of hpv-related neoplastic lesions (cin - and invasive cervical cancer). such studies will contribute to the development of new tools for the identification of premalignant lesions at high risk of progression to invasive cervical carcinoma. jonathan gershoni, a gvn center director at tel aviv university (israel), described an analysis of the vast repertoire of antibodies circulating in polyclonal serum (the "igome.") he highlighted how the paratope region of an antibody mirrors the corresponding region of an antigen (e.g., a virus protein) and therefore favors an indirect approach of igome profiling by describing all peptides interacting with the entity of antibodies. the spectrum of antibody specificities is dynamic and varies with age, physiology, and exposure to pathological insults. the igome is an extraordinarily rich source of informationea molecular record of all previous encounters, as well as a status report of current immune activity (weiss-ottolenghi and gershoni, ) . the ability to profile antibody specificities of polyclonal serum at very high resolution has been an important and serious challenge, which can now be met by probing, recording, and qualifying the archive of antibodies present in the serum of an individual with a vast library of peptides. gershoni also described "deep panning", a methodology that merges the flexibility of combinatorial phage-display peptide libraries with the power of next-generation sequencing to enable high-resolution/high-throughput interrogation of the igome (ryvkin et al., ) . this approach produces heat-maps that characterize the interaction between the immune system and antigens. this method was used to characterize and differentiate sera from hiv-infected individuals versus hcv-infected individuals. by analyzing the immune response to influenza antigens in birds, jonathan showed that this technique could be used to monitor and characterize the efficiency of vaccines, suggesting that igome analysis may become a very useful tool for vaccine development. since the appearance of chikungunya virus (chikv) in the western hemisphere in , the gvn has had a special focus on arboviruses. in , the network sponsored a joint symposium with the american society of tropical medicine and hygiene on "the global spread of chikungunya: epidemiology, evolution, pathogenesis and global needs" and published a special report describing research goals and public health priorities for the outbreak (mcsweegan et al., ) . a task force of international experts also was formed to provide a source of expert information and a platform for collaboration. marc lecuit at the gvn center at the institut pasteur in paris described chikv, its geographical spread, the clinical presentation of infection and the development of a mouse model that has allowed pathophysiology studies and the evaluation of novel therapeutics. murine fibroblasts of skeletal muscles, joint capsules and dermis were found to be primary viral targets, and type i ifn-sensing, non-hematopoietic cells were critical for innate immune control. a genome-wide sirna screen identified proviral and antiviral host factors affecting viral replication, which allowed identification of potential drug targets. twenty-one small molecule inhibitors that affect six host pathways have been identified; three showed prophylactic effects in the mouse model. a calmodulin inhibitor (pimozide) and a fatty acid synthesis inhibitor (tofa) showed antiviral effects in mice when used together (karlas et al., ) . other experimental data showed that chikv infection does not directly target the placental barrier, therefore vertical transmission from viremic mothers to neonates likely occurs via laborinduced placental breaches. chikv also was found to be a significant cause of cns disease in larger human outbreaks, with the virus detected in affected tissues (g erardin et al., ) . scott weaver, chair of a gvn task force on zika virus (zikv), reviewed the sudden emergence of zikv from relative obscurity to global menace. serosurveys have shown its historical incidence in parts of asia and africa (weaver et al., ) . a widespread outbreak in the micronesian island of yap in later expanded to neighboring islands in south pacific. coincident with these outbreaks, a -fold higher incidence of guillain-barre syndrome (gbs) was noted. the explosive outbreak in brazil that began in and has involved millions of people has since spread to other countries and territories in the western hemisphere. the peridomestic mosquito, aedes aegypti, and the invasive aedes albopictus mosquito have been identified as the major insect vectors. surprisingly, sexual transmission of zikv also was confirmed in travelers returning to non-endemic regions. a most disturbing aspect of the recent outbreaks is the high incidence of fetal microcephaly and other birth defects directly related to infection in pregnant women. weaver also discussed important knowledge gaps. when and how should infants be tested for possible congenital zika infection? is microcephaly among some neonates just the tip of the neurological iceberg? why does the overall risk of microcephaly and other birth defects seem to differ in other parts of latin america and the caribbean, compared to brazil? it appears that these new outbreaks in the western hemisphere have not been caused by novel viral strains. instead, the introduction of zikv into naïve populations via rapid global travel, the growth of tropical cities, and changing vector populations seem to have allowed virus amplification and subsequent epidemic spread. prospects for control are dependent on mosquito control, new therapeutics and development of an effective vaccine. vaccine prospects appear good, with about candidates now in development and animal models available for preclinical testing. two dna-based vaccines have entered phase i trials, and an inactivated vaccine is nearing phase i. deploying a vaccine, however, remains dependent on developing an accurate test for zikv. current tests are hampered by antigenic cross-reactivity between zikv and other closely related flaviviruses such as dengue, yellow fever, and st. louis encephalitis, some of which are endemic in the same geographic areas. richard scheuermann, gvn center co-director at the j. craig venter institute (usa), discussed using the influenza research database (ird, fludb.org) and the virus pathogen resource (vipr, viprbrc.org) to identify diagnostic peptide regions in zika virus (zikv) and other flaviviruses. these databases identified and diagnostic amino acid sites from the zikv ns and e proteins, respectively, that distinguish it from other flaviviruses, with sensitivity/specificity above % (sun et al., ) . similar numbers of such diagnostic sites were also identified for other flaviviral species. sliding window analysis revealed several contiguous peptide regions that contain multiple diagnostic sites specific for each of the different flaviviruses. these regions are now being used to develop peptide arrays for the detection of antibodies specific for each of the viruses including zikv. these peptide arrays could have a significant impact on diagnosis and epidemiological analysis of future disease outbreaks. diane griffin at the gvn center at johns hopkins university (usa) noted that insect-borne viruses causing encephalitis have become a global health concern due to their recent expansion into new regions. while the fatality rates vary, many patients recovering from serious episodes of viral encephalitis are likely to have lifelong physical and mental disabilities. viral persistence and immunemediated clearance are important factors in these sequelae. griffin described experiments with sindbis virusda prototype alphavirusdusing a variety of mouse models (griffin, ) . infection of neurons in adult mice resulted in non-fatal encephalomyelitis. recovery was mediated through a coordinated effort between antibody to the e viral surface protein and ifn-g. while ifn-g signaling leads to clinical disease via pro-inflammatory cytokines, it synergistically helps clear the virus by increasing b-cellattracting chemokine production for recruitment of antibodyproducing cells to the central nervous system (cns). however, the non-cytolytic immune phenomenon does not lead to elimination of intracellular viral rna from long-lived neurons, so that continued suppression of virus and the prevention of reactivation and reemergence in the cns require immune control after recovery. evidence was presented that b and t cells that infiltrated during the acute phase of infection remain there after recovery and continue to exert their protective effect locally. long-term residence of these immune cells in the cns is likely facilitated by the continued presence of viral proteins. arthropods make up the largest phylum of the animal kingdom and are known to harbor and transmit viruses of agricultural, veterinary and medical importance. the true extent of the 'virosphere' of arthropods, however, is largely unknown and needs to be more completely explored. yong-zhen zhang, an investigator at the national institute for communicable disease control and prevention in beijing, china, presented work on newly identified rna viruses from arthropods collected in different regions of china. among recent examples are jingmen tick virus (jmtv), and viruses belonging to a new family, chuviridae (qin et al., ) . jtmv has four genomic segments, and, sequence analysis has revealed an unexpected connection between segmented and non-segmented viruses. chuviruses exhibit various genomic organizations, including non-segmented, bi-segmented, and circular genomes. these viruses provided evidence of an evolutionary link between segmented and non-segmented negative strand viruses. overall, many novel viruses representing five new families, have been discovered in china and await further characterization. peter palese, a gvn center director at the icahn school of medicine at mount sinai (usa), described ongoing efforts to design a universal influenza vaccine and to understand the immune responses to such constructs. he said that, despite the availability of fda-approved vaccines and antivirals, seasonal and pandemic influenza remain a serious threat associated with substantial morbidity and mortality. while annual seasonal influenza virus vaccination is effective e albeit underutilized in most countries e a safe, universal vaccine providing broad and long-lasting immunity would represent a major breakthrough. today's licensed vaccines focus on eliciting humoral immunity to the globular head (ha ) of the hemagglutinin (ha) glycoprotein, an antigen subject to antigenic drift, which requires continued surveillance of circulating influenza viruses. since the h n pandemic, research efforts on universal vaccines have refocused, looking to shift immunity to the more conserved domains of ha, which is expected to cover many more circulating influenza viruses. palese also has developed vaccine constructs that express chimeric hemagglutinins (cha) that result in the redirection of the immune response away from the immunodominant (variant) ha head domain towards the much more conserved stalk (ha ) region, and which also express the highly conserved neuraminidase (na) (tran et al., ) . this type of immunogen has been demonstrated to be efficacious following vaccination in mice, conferring heterosubtypic protection against virus challenge. the mechanism by which this approach confers antiviral activity suggests that redirecting immunity to the stalk region preferentially supports induction of antibody-dependent cell-mediated cytotoxicity (adcc). data presented showed that broadly neutralizing ha stalk-specific antibodies require fcgr interactions for protection. palese proposed a two-contact model for optimal induction of adcc by influenza virus-specific mabs targeting the stalk regiondand not the globular headdand he noted the importance of the ha binding to effector cells via its sialic acid receptor was required for optimal adcc induction. passive transfer studies in mice further supported the role of h -specific stalk antibodies and protection against weight loss following virus challenge. this novel technology is being supported through government and industry support, and gmp-quality batches of two cha immunogens (ch / n & ch / n ) that display heterogenous globular heads have been produced for evaluation in the clinic. such studies will make it possible to assess the role of additional effector mechanism in protecting against influenza virus infection and to evaluate the importance of reducing the immunodominance of the variable ha head domain. this type of translational study will bring us a step closer to a universal influenza vaccine. hiroshi kida at hokkaido university in japan reported on the establishment of a library of avian influenza virus strains isolated from ducks, and reassortant viruses generated in the laboratory, with combinations of the nine na and sixteen ha subtypes (virusdb.czc.hokudai.ac.jp). because influenza a viruses of all known subtypes can perpetuate among migratory ducks and contribute genes to the generation of reassortant viruses in pigs, none of the combinations can be ruled out as possible future pandemic strains. this virus library is kept as a source for vaccine candidates to assure effective preparedness for future pandemics (haredy et al., ) . vaccine constructs prepared from h n , h n , h n , h n , h n and h n viruses in the library elicited sufficient immune responses to protect chickens, mice, and macaques from challenge with isolates from poultry and humans, suggesting their utility against pandemic influenza. in addition, he noted that current seasonal vaccines prepared by ether or detergent disruption are not sufficiently immunogenic, especially in children and the elderly, and need to be significantly improved. furthermore, methods for controlling pandemic influenza should be based on measures that are used for the control of seasonal influenza. hiroshi noted that inactivated whole virusbased vaccine candidates might provide solutions to the limitations of current vaccines. to accomplish the goal of a new and effective influenza vaccine, a collaborative research group, the all japan collaborating study group for the development of influenza vaccines, was established in april . five japanese manufacturers of influenza vaccines participate in the program. the goals of the study group are: to compare the immunogenicity and safety of whole virus particle vaccine and "split" vaccines; use the results of pre-clinical and clinical trials to revise the standard of influenza vaccines for human use; and determine the optimum route of inoculation and evaluate proprietary adjuvants. massimo palmarini, a gvn center director at the mrc-university of glasgow center for virus research (scotland), reported on studies of bluetongue, a major infectious disease of ruminants, caused by the double-stranded rna virus known as bluetongue virus (btv). studies of btv in sheep offer unique perspectives for understanding the pathogenesis of arboviral diseases, as observations made in the naturally occurring disease can be effectively reproduced in a convenient experimental setting, using the same animal species (caporale et al., ) . the clinical outcome of btv infection in sheep is extremely variable, but similar to other arbovirus infections, in that a rapid onset of the antibody response correlates with a more favorable clinical outcome. btv infection of its natural sheep host was used to examine a previously uncharacterized mechanism adopted by an arbovirus to manipulate host immunity at the early stages of infection. btv is transported rapidly via the lymph to the peripheral lymph nodes, where it infects and disrupts follicular dendritic cells (fdc). these cells of stromal origin promote the formation and maintenance of the germinal centers in which b cells differentiate into memory cells and plasma cells, and are also responsible for supporting antibody class-switching and affinity maturation. this work showed that btv hindered b-cell division in germinal centers, resulting in the delayed production of high-affinity and virus-neutralizing antibodies. importantly, the humoral immune response to a second antigen is also hampered in infected sheep. thus, an arbovirus can evade the host antiviral response by inducing an acute immunosuppression. although transient, this immunosuppression occurs at the critical, early stage of infection, when a delayed host humoral immune response likely affects the systemic dissemination of the virus and the clinical outcome of disease (melzi et al., ) . ab osterhaus, the gvn center director at the research center for emergin, reviewed the complex relationships among human and animal species that have promoted cross-species transmission, emergence and the eventual evolution of a plethora of human pathogens. changes affecting modern human populations worldwide and their dramatic impact on the global environment have taken domestication, agriculture, urbanization, and industrialization to unprecedented levels. this has created new and global multi-faceted human-animal interfaces, associated with major epidemiological transitions, accompanied by an unexpected surge of emerging and re-emerging human infectious diseases that have their origin in animal reservoirs. until the beginning of the last century, infectious diseases were the major cause of human mortality. around , infections caused about fifty percent of deaths in the western world, but in the following decades, this percentage decreased to less than a few percent. this was largely due to the implementation of public health measures such as the installation of sewers and the development of clean drinking water systems, but also to development of vaccines and antimicrobials. major successes in this regard were the eradication of smallpox and rinderpest through wellorchestrated vaccination campaigns in humans and cattle, respectively. these successes prompted policymakers and scientists to predict that infectious diseases of humankind and of their domestic animals would eventually be brought under control in the industrialized world. paradoxically the following decades confronted the world with an ever-increasing number of emerging or re-emerging infectious diseases, some causing true human or animal pandemics. pathogens spilling over from wildlife reservoirs, either directly or via intermediate hosts, were the basis of most of the outbreaks. striking examples in humans were the emergence of aids from chimpanzees, avian flu from migratory birds, and sars, mers, and ebola virus disease from bat reservoirs. a complex mix of predisposing factors in our globalizing world, linked to major changes in the societal environment and global ecology, collectively created opportunities for viruses and other pathogens to infect and adapt to new animal and human hosts. this paved the way for the unprecedented spread of infections, with dramatic consequences for public and animal health, animal welfare, food supplies, economies, and biodiversity. it is important to realize that, because of the complex and largely interactive nature of the predisposing factors, it is virtually impossible to predict the next pathogen threat, where it will come from and when it will strike. however, a better understanding of the underlying processes may eventually lead to enhanced predictive capacity, improving preparedness for outbreaks in humans and animals. importantly, the increased emergence of viral infections has been largely paralleled by medical, veterinary, technological, and scientific progress. investments to better understanding human-animal interfaces should offer a future head start in the struggle against infectious diseases of humans. ayato takada at the global institution for collaborative research and education at hokkaido university in sapporo, japan, described an ebola virus glycoprotein-specific monoclonal antibody (mab d ) that was broadly cross-reactive with all known ebola virus species. this mab recognized the putative epitope in the highly conserved internal fusion loop (ifl) and neutralized infectivity by inhibiting membrane fusion. mab d may, therefore, have potential as a therapeutic agent in animal models. he also described a novel antibody-dependent enhancement (ade) mechanism in which fc receptor-mediated intracellular signaling increased uptake of ebola virus into cells in vitro (furuyama et al., ) . activation of the fc-receptor-mediated signaling pathway was essential for ade of ebola virus infection; this finding provides new insights into mechanisms of ade and the development of treatments for ade-associated diseases. finally, he noted that neutralizing and ade antibodies show balanced effects depending on total antibody concentration, raising theoretical concerns about the use of convalescent human sera or plasma with low antibody titers. therapeutic treatment with convalescent sera with in vitro neutralizing activity was not sufficient to protect against ebola virus infection in nonhuman primates (mire et al., ) . it may be that ade antibodies counterbalanced the neutralizing capacity of other antibodies. these laboratory phenomena warrant further evaluation. luc willems, a gvn investigator at gembloux agro-bio tech in belgium, described how bovine delta-retroviral ribonucleic acids induce cell transformation and oncogenesis. among the strategies developed by viruses to escape the host immune response, mechanisms involving viral non-coding rnas have recently been discovered. viruses can express micrornas that directly target cellular transcripts, while others produce long non-coding rnas acting as microrna sponges or epigenetic modulators. these strategies, based on ribonucleic acids, control the cell's fate without eliciting immune responses due to the absence of viral proteins. virally encoded mirnas were first identified in dna viruses. most of them are generated from endonucleolytic cleavage of long viral transcripts. however, retroviruses were initially assumed not to encode mirnas because of the potential self-cleavage of their rna genome (gillet et al., ) . to date, four retrovirusesbovine leukemia virus (blv), bovine foamy virus (bfv), avian leucosis virus (alv-j) and simian foamy virus (sfv) e have been shown to express viral mirnas in infected cells. blv naturally infects bovine species to cause a benign lymphocytosis, but approximately % of infections lead to b-cell leukemia/lymphoma. remarkably, blv persists and replicates in vivo in the absence of significant levels of viral mrna transcription. in contrast, blv mirnas are highly expressed in infected cells, representing up to % of all cellular mirnas. consequently, because ribonucleic acids likely are less immunogenic than viral protein antigens, blv mirnas could alter the cell's fate by escaping immune recognition (gillet et al., ) . as with the appearance of chikv in the western hemisphere, the sudden emergence and dissemination of zikv have driven similar cooperative responses at the gvn, including the formation of a second task force of experts to exchange information and share resources; the organization of a webinar for business leaders to inform them about the virus and prospects for therapies and vaccines (http://www.btsmeetings.com/zika); providing expert information to the news media to ensure accurate reporting; and acquiring a grant from the allergan foundation to establish a bank of acute and convalescent sera from confirmed zikv patients to aid in developing better diagnostics. among other accomplishments, shyamasundran kottilil and colleagues at the institute of human virology in baltimore began a project with the gvn, and funded by the gilead foundation, to develop a training model for physicians using daa treatment for hcv patients in india. members of the htlv- task force published a commentary on the need for htlv- screening prior to organ transplant, and an agenda for future research (gallo et al., ; willems et al., ) . other gvn investigators published new research on west nile virus and rabies virus (kobayashi et al., ; phongphaew et al., ; anindita et al., ) . during the past year, the network expanded the number of cooperating research centers by adding the university of miami, emory university and tulane university school of medicine in the usa and the international vaccine institute in south korea (http:// gvn.org/coe/). these centers are expected to strengthen cooperative research on vaccines, antiviral drugs, and emerging viruses such as zikv and ebola virus. among other projects, the gvn staff in baltimore expects to assist with a pilot study of hepatitis b in arunachal pradesh, india. funded by the john martin foundation, the project will screen , people, provide vaccinations for those who are not infected, and develop a longitudinal cohort to treat patients with chronic hepatitis b infections. members of the gvn task force on htlv- expect to publish additional commentaries and reviews during . similarly, a gvn task force report on zikv emergence and research priorities is in preparation. gvn staff also will continue to seek additional patient serum samples for the zikv serum bank based at the university of texas medical branch in galveston, texas. additionally, a project to sequence and conduct phylogenetic analyses of collected zika virus isolates has begun in cooperation with the world reference center for emerging viruses and arboviruses (wrceva), which is funded by the national institute of allergy and infectious diseases (usa). the gvn will host its fourth annual short course on medical virology for young investigators. the short course attracts an international class of e postdoctoral students and fellows for five days of meetings and lectures with experts in medical virology research and clinical practice. information about the course and registration is available online at http://www.gvn.org. the international gvn meeting will be in melbourne, australia during september e . it will be co-hosted by the gvn centers of excellence, the institut pasteur (paris) and the peter doherty institute for infection and immunity (melbourne generation of recombinant rabies viruses encoding nanoluc luciferase for antiviral activity assays characterization of the human papillomavirus (hpv) integration sites into genital cancers lethal mutagenesis of hepatitis c virus induced by favipiravir virus and host factors affecting the clinical outcome of bluetongue virus infection ongoing clinical trials of human immunodeficiency virus latency-reversing and immunomodulatory agents is virology dead? mbio balance of cellular and humoral immunity determines the level of protection by hiv vaccines in rhesus macaque models of hiv infection favipiravir (t- ), a novel viral rna polymerase inhibitor fcg-receptor iia-mediated src signaling pathway is essential for the antibody-dependent enhancement of ebola virus infection history of the discoveries of the first human retroviruses: htlv- and htlv- global virus network's task force on htlv- chikungunya virus-associated encephalitis: a cohort study on la r eunion island bovine leukemia virus small noncoding rnas are functional elements that regulate replication and contribute to oncogenesis in vivo alphavirus encephalomyelitis: mechanisms and approaches to prevention of neuronal damage an mdck cell culture-derived formalin-inactivated influenza virus whole-virion vaccine from an influenza virus library confers cross-protective immunity by intranasal administration in mice direct-acting antiviral medications for chronic hepatitis c virus infection a human genome-wide loss-of-function screen identifies effective chikungunya antiviral drugs rab b regulates transport of west nile virus particles from recycling endosomes a cure for hiv infection new organization pledges scientific expertise for viral outbreaks the global virus network: challenging chikungunya follicular dendritic cell disruption as a novel mechanism of virus-induced immunosuppression passive immunotherapy: assessment of convalescent serum against ebola virus makona infection in nonhuman primates the interaction between herpes simplex virus tegument proteins ul and ul and its role in virion morphogenesis valosin-containing protein (vcp/p ) plays a role in the replication of west nile virus a tick-borne segmented rna virus contains genome segments derived from unsegmented viral ancestors deep panning: steps towards probing the igome feverish quest for ebola immunotherapy: straight or cocktail? how to turn competitors into collaborators comprehensive annotation of mature peptides and genotypes for zika virus relationship of the quaternary structure of human secretory iga to neutralization of influenza virus tp and pik ca gene mutations in adenocarcinoma, squamous cell carcinoma and high-grade intraepithelial neoplasia of the cervix cryo-electron microscopy structures of chimeric hemagglutinin displayed on a universal influenza vaccine candidate zika virus: history, emergence, biology, and prospects for control profiling the igome: meeting the challenge reducing the global burden of htlv- infection: an agenda for research and action we thank the participants who presented their data at the international gvn meeting. we thank the japanese society of virology and hokkaido university organizers for their outstanding management of the meeting and related events, and the japanese hosts: hokkaido university's research center for zoonosis control, the global institution for collaborative research and education (gi-core) global station for zoonosis control (gsz) and the national institute of infectious diseases (niid). support also was provided by numerous sponsors who are noted at http://gvn.org/sapporo_ . key: cord- -cco aq n authors: okamoto, mika; toyama, masaaki; baba, masanori title: the chemokine receptor antagonist cenicriviroc inhibits the replication of sars-cov- in vitro date: - - journal: antiviral res doi: . /j.antiviral. . sha: doc_id: cord_uid: cco aq n cenicriviroc (cvc) is a small-molecule chemokine receptor antagonist with highly potent and selective anti-human immunodeficiency virus type (hiv- ) activity through antagonizing c-c chemokine receptor type (ccr ) as a coreceptor of hiv- . cvc also strongly antagonizes c-c chemokine receptor type b (ccr b), thereby it has potent anti-inflammatory and immunomodulatory effects. cvc is currently under clinical trials in the patients for treatment of nonalcoholic steatohepatitis, in which immune cell activation and dysregulation of proinflammatory cytokines play an important role in its pathogenesis. in this study, cvc was examined for its inhibitory effect on the replication of sars-cov- , the causative agent of covid- , in cell cultures and found to be a selective inhibitor of the virus. the % effective concentrations of cvc were . and . μm in the assays based on the inhibition of virus-induced cell destruction and viral rna levels in culture supernatants of the infected cells, respectively. interestingly, the ccr -specific antagonist maraviroc did not show any anti-sars-cov- activity. although the mechanism of sars-cov- inhibition by cvc remains to be elucidated, ccr b does not seem to be its target molecule. considering the fact that the regulation of excessive immune activation is required to treat covid- patients at the late stage of the disease, cvc should be further pursued for its potential in the treatment of sars-cov- infection. the pandemic of severe pneumonia caused by the transmission of the new coronavirus sars-cov- is a serious threat to humanity (di gennaro et al., ; harapan et al., ; helmy, et al., ) . at present, no vaccines exist, and the nucleoside analog remdesivir (rdv) has recently been approved for treatment of sars-cov- infection (grein et al., ) . rdv has broad-spectrum antiviral activity against several viruses, including ebola virus and coronaviruses. in addition to rdv, there is an attempt to treat covid- with existing drugs developed for other purposes. these include hydroxychloroquine and chloroquine (pastick et al., ) , the anti-influenza virus agent favipiravir (pilkington et al., ) , and the human immunodeficiency virus type (hiv- ) protease inhibitor lopinavir/ritonavir . more recently, the anti-parasitic agent ivermectin has been shown to inhibit sars-cov- replication in cell cultures (caly et al., ) . however, these drugs are not optimized for sars-cov- , so that they may have inevitable adverse effects due to the requirement of higher dosages. furthermore, these antiviral agents must be used at an early stage of infection, since severe deterioration of pneumonia in some patients is considered to be closely associated with not viral replication but "cytokine storm" caused by dysregulated and excessive cytokine release from activated immune cells (ye et al., ) . therefore, the use of anti-inflammatory and immunomodulatory agents is mandatory in the late stage of this disease (alijotas-reig et al., ) . we have examined several compounds for their inhibitory effect on sars-cov- replication in cell cultures and found that the chemokine receptor antagonist cenicriviroc (cvc) inhibits the replication of sars-cov- . cvc is a c-c chemokine receptor type (ccr ) antagonist with potent and selective anti-hiv- activity (baba et al., ) . in addition, cvc antagonizes not only ccr but also c-c chemokine receptor type b (ccr b). since ccr b is the receptor of monocyte chemoattractant protein (mcp- ), cvc exerts anti-inflammatory and immunomodulatory effects in vivo (dawson et al., ; xia and sui, ) . in fact, cvc is currently under clinical trials for the treatment of nonalcoholic steatohepatitis (nash), in which immune cell activation and dysregulation of proinflammatory cytokines play an important role in its pathogenesis (pedrosa et al., ) . veroe cell line expressing transmembrane protease serine (veroe /tmprss ) highly susceptible to sars-cov- infection was obtained from japanese collection of research bioresources (jcrb) cell bank in japan (jcrb no. jcrb ) and used for virus propagation and antiviral assays after removal of mycoplasma contamination. vero cells were also used for experiments. the cells were cultured in dulbecco's modified eagle medium (nacalai tesque, kyoto, japan) supplemented with % heat-inactivated fetal bovine serum (fbs), u/ml penicillin g, µg/ml streptomycin, and mg/ml g (nacalai tesque). for antiviral assays, the cells were cultured in the absence of g . sars-cov- (wk- strain, gisaid database id epi_isl_ ), a clinical isolate from a covid- patient, was provided by national institute of infectious diseases, tokyo, japan . the infectious virus titer was determined in veroe /tmprss cells and expressed as % cell culture infectious dose (ccid ) per ml. cvc and maraviroc (mrv) were purchased from medchemexpress (monmouth junction, nj). the nucleotide/nucleoside analogs rdv and favipiravir (fpv) were obtained from chemscence (monmouth junction, nj) and selleck chemicals (houston, tx), respectively. mcp- was purchased from petpotech (rocky hill, nj). mrv is the ccr antagonist clinically approved for treatment of hiv- infection (woollard and kanmogne, ) . except for mcp- , these compounds were dissolved in dimethyl sulfoxide (dmso) at a concentration of mm or higher to exclude the cytotoxicity of dmso and stored at - °c until use. mcp- was dissolved in distilled water. veroe /tmprss cells ( × cells/well) were cultured in a -well microtiter plate and incubated at °c. after h, the cells were mock-infected or infected with sars-cov- at a multiplicity of infection (moi) of . and cultured in the absence or presence of various concentrations of test compounds. after days, the number of viable cells was determined by a tetrazolium dye method. briefly, μl of culture medium was removed from each well, and μl of water-soluble tetrazolium dye solution (dojindo, kumamoto, japan) was added. after incubating at °c for h, μl of isopropanol acidified with hydrochloric acid was added, and the absorbance was read at two wavelengths ( and nm) with a microplate reader (pauwels et al., ) . the % effective concentration (ec ) of each compound was calculated from a dose-dependent curve based on the viability of infected and uninfected cells. all experiments using sars-cov- were conducted in biosafety level (bsl ) facilities of kagoshima university. for immunofluorescence microscopy, vero cells ( × cells/well) were cultured in a microtiter plate and incubated. after h, the cells were infected with sars-cov- at a moi of . in the absence of cvc and incubated. after h, the cells were washed with phosphate buffered saline (pbs) to remove unadsorbed virus particles and further incubated in the absence or presence of μm cvc. after days, the cells were fixed with % paraformaldehyde in pbs for min. then, the solution was removed, and the cells were washed with pbs and permeabilized with methanol. after washing with pbs, the cells were treated with pbs containing % bovine serum albumin (sigma-aldrich, st. louis, mo) and . % tween (fujifilm wako, osaka, japan) and incubated overnight at °c with an anti-sars-cov- nucleocapsid rabbit antibody (genetex, irvine, ca). after incubation, the cells were washed with pbs and stained with the secondary antibody goat anti-rabbit igg h&l (alexa fluoro ® ; abcam, cambridge, uk). the cells were washed with pbs, stained with ', -diamidino- -phenylindole (dapi; bio-rad, hercules, ca), and observed under a fluorescent microscope (bz-x ; keyence, osaka, japan). the inhibitory effect of compounds on sars-cov- replication was also evaluated by the viral rna levels in culture supernatants of the infected cells. veroe /tmprss cells were infected with the virus and incubated for days in the absence or presence of test compounds, as described above. ten μl of each culture supernatant was mixed with μl of sidestep lysis and stabilization buffer (agilent technologies, santa clara, ca) and diluted with μl of distilled water. the amount of viral rna was measured by real-time reverse transcription polymerase chain reaction (rt-pcr) using taqman gene expression cells-to-ct tm kit (thermo fisher scientific, waltham, ma), according to the manufacturer's protocol except for its cell lysis step. the primer pair '-aaattttggggaccaggaac- ' and '-tggcagctgtgtaggtcaac- ' and the probe '-fam-atgtcgcgcattggcatgga-tamra- ' were used for real-time pcr reat-time rt-pcr to determine the amount of viral rna, as described above. when veroe /tmprss cells were infected with sars-cov- and incubated in the absence of compounds for days, the cells were completely destroyed by the virus-induced cytopathic effect (fig. b) . such cell destruction was not observed for the infected cells in the presence of μm cvc, although some morphological changes were identified (fig. d ). in contrast, mrv did not inhibit the virus-induced cell destruction even at μm (fig. f) . the ec s of cvc and mrv were ± . and > μm, respectively, based on the inhibition of virus-induced cell destruction (table ) . both compounds did not show apparent cytotoxicity at concentrations up to μm. dose-dependent protection of the infected cells from virus-induced cell destruction was observed for cvc but not for mrv (fig. ) . these results indicate that cvc is a selective inhibitor of sars-cov- replication. as previously reported , rdv proved to be a potent and selective inhibitor of sars-cov- replication in our assay, whereas fpv did not show any selective inhibition even at a concentration of μm (table and fig. s ). the anti-sars-cov- activity was also examined by the inhibition of viral antigen expression in vero cells. vero cells is less susceptible to sars-cov- replication than veroe /tmprss cells. in fact, vero cells were not destroyed by the virus-induced cytopathic effect on day after infection (fig. a) . however, viral antigen expression was observed for many cells in the absence of cvc (fig. b ). in contrast, the antigen expression was completely inhibited in the presence of μm cvc (fig. d) . when the anti-sars-cov- activity of cvc was evaluated by the viral rna levels in culture supernatants of the infected cells, cvc significantly and dose-dependently reduced the amount of viral rna in culture supernatants (fig. a ). its ec in this assay was . μm, and more than % inhibition was achieved by cvc at a concentration of μm. in contrast, mrv did not reduce the viral rna levels at concentrations up to μm in , we have reported that the small molecule and orally bioavailable ccr antagonist cvc inhibits hiv- replication at subnanomolar concentrations in vitro (baba et al., ) . the anti-hiv- activity of cvc is attributed to the inhibition of viral entry using ccr as a coreceptor. however, different from other anti-hiv- ccr antagonists such as mrv, cvc also suppresses the binding of mcp- to ccr b-expressing cells. this unique feature of cvc encouraged its manufacturer to develop this compound as an agent for treatment of nash. a recent randomized, placebo-controlled trial of cvc for treatment of nash demonstrated that twice as many subjects achieved improvement in fibrosis and no worsening of steatohepatitis compared with placebo (friedman et al., ; tacke, ) . safety and tolerability of cvc were found to be comparable to placebo, suggesting that it can be administered safely to covid- patients for preventing severe deterioration of pneumonia due to the cytokine storm. in fact, alveolar macrophage-borne mcp- was reported to be a key agent in the initiation of the systemic inflammation of alveolar hypoxia in rats, and a ccr b receptor antagonist prevented the mesenteric inflammation of alveolar hypoxia (chao et al. ) . furthermore, a recent study on transcriptome sequencing of the rnas isolated from the bronchoalveolar lavage fluid and peripheral blood mononuclear cells specimens of covid- patients revealed the association between its pathogenesis and excessive cytokine release including mcp- (xiong et al. ). it will be claimed that the anti-sars-cov- activity of cvc in vitro is insufficient for the treatment of covid- patients. however, our antiviral assay system using (table and data not shown). the broad-spectrum anti-rna virus agent fpv was not inhibitory to sars-cov- replication even at μm (table and fig. s ), which is consistent with a previous report (choy et al. ) . the anti-sars-cov- activity of cvc was more obvious, when determined by the inhibition of viral rna levels in culture supernatants. its ec was . μm (fig. ) , which was similar to those of rdv ( . μm) and ivermectin ( . - . μm) (data not shown and caly et al., , respectively) . thus, although the anti-sars-cov- activity of cvc in vitro is modest, we cannot exclude the possibility that cvc exhibits some antiviral efficacy in covid- patients. the target molecule of cvc remains to be elucidated. our preliminary studies on its mechanism of action revealed that cvc did not interfere with the entry of sars-cov- (data not shown). unlike hiv- , ccr is not the target of cvc for inhibition of sars-cov- replication, since another potent ccr antagonist mrv was totally inactive (fig. b and fig. b ). furthermore, mcp- could not block sars-cov- infection (data not shown), suggesting that ccr b is not the target molecule either. further studies, such as a time-of-addition experiment and biochemical approaches, are required to elucidate the mechanism of action, and they are currently in progress. in conclusion, in addition to the potent anti-inflammatory activity of cvc in vivo, the present study has identified its anti-sars-cov- activity in vitro. since not only the inhibition of viral replication but also the control of excessive immune activation is mandatory to save covid- patients at the late stage of the disease, cvc should be further pursued for its potential in the treatment of sars-cov- infection. the data of cvc and rdv represent mean ± range for two separate experiments and mean ± standard deviation for three separate experiments, respectively. immunomodulatory therapy for the management of severe covid- . beyond the anti-viral therapy: a comprehensive review tak- inhibits ccr -mediated human immunodeficiency virustype infection in vitro and has favorable pharmacokinetics in humans the fda-approved drug ivermectin inhibits the replication of sars-cov- in vitro a trial of lopinavir-ritonavir in adults hospitalized with severe covid- monocyte chemoattractant protein- released from alveolar macrophages mediates the systemic inflammation of acute alveolar hypoxia remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov- replication in vitro targeting monocyte chemoattractant protein- signaling in disease coronavirus diseases (covid- ) current status and future perspectives: a narrative review a randomized, placebo-controlled trial of cenicriviroc for treatment of nonalcoholic steatohepatitis with fibrosis compassionate use of remdesivir for patients with severe covid- coronavirus disease (covid- ): a literature review the covid- pandemic: a comprehensive review of taxonomy, genetics, epidemiology, diagnosis, treatment, and control enhanced isolation of sars-cov- by tmprss -expressing cells rapid and automated tetrazolium-based colorimetric assay for the detection of anti-hiv compounds a randomized, double-blind, multicenter, phase b study to evaluate the safety and efficacy of a combination of tropifexor and cenicriviroc in patients with nonalcoholic steatohepatitis and liver fibrosis: study design of the tandem trial a review of the safety of favipiravir -a potential treatment in the covid- pandemic? development of genetic diagnostic methods for novel voronavirus cenicriviroc for the treatment of non-alcoholic steatohepatitis and liver fibrosis remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro maraviroc: a review of its use in hiv infection and beyond recent developments in ccr antagonists transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid- patients the pathogenesis and treatment of the `cytokine storm' in covid- we thank national institutes of biomedical innovation, health and nutrition and national institute of infectious diseases for kindly providing veroe /tmprss cells and sars-cov- (wk- strain), respectively. kagoshima university is applying for a patent of cvc as a sars-cov- inhibitor, and m.o., m.t. and m.b. are its inventors. key: cord- -bwn bmf authors: mohan, ketha v.k.; rao, shilpakala sainath; atreya, chintamani d. title: antiviral activity of selected antimicrobial peptides against vaccinia virus() date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: bwn bmf antimicrobial peptides (amps) are gaining importance as effective therapeutic alternatives to conventional antibiotics. recently we have shown that a set of nine synthetic antimicrobial peptides, four originating from thrombin-induced human platelet-derived antimicrobial proteins named pd –pd and five synthetic repeats of arginine-tryptophan (rw) repeats (rw - ) demonstrate antibacterial activity in plasma and platelets. using wr strain of vaccinia virus (vv) as a model virus for enveloped virus in the present study, we tested the same nine synthetic peptides for their antiviral activity. a cell culture-based standard plaque reduction assay was utilized to estimate antiviral effectiveness of the peptides. our analysis revealed that peptides pd , pd , and rw were virucidal against vv with pd demonstrating the highest antiviral activity of -fold reduction in viral titers, whereas, pd and rw peptide treatments resulted in – -fold reduction. the ec( ) values of pd , pd and rw were found to be μg/ml, μg/ml and . μm, respectively. in vv-spiked plasma samples, the virucidal activity of pd , pd and rw was close to % ( – -fold reduction). overall, the present study constitutes a new proof-of-concept in developing peptide therapeutics for vaccinia virus infections in biothreat scenarios and as in vitro viral reduction agents. ever since their discovery, antimicrobial peptides (amps) have been gaining attention as an important therapeutic intervention alternative in the field of disease prevention and care against a number of microbes (brogden, ; hancock and sahl, ; oyston et al., ; zaiou, ) . this can be mainly attributed to the rising microbial drug resistance, associated toxicity and higher production costs involved with conventional antimicrobial drugs. as a result of intense research in this field over the last decade, approximately , amps have so far been listed in the amps database (brogden, ; hancock and sahl, ; oyston et al., ; zaiou, ) . these peptides are either derived from nature (from microbes, vertebrates including humans) or are synthetic forms (brogden, ; hancock and sahl, ; zaiou, ) . each amp could demonstrate their antimicrobial property either on a single class of organisms i.e., anti-bacterial, anti-viral, anti-parasitic, anti-fungal or on multiple classes of pathogens. the mechanism of action of many amps include either direct microbicidal activity or indirect action by blocking or inhibiting an important pathway in the microbial life cycle (brogden, ; chan et al., ; hancock and sahl, ; oyston et al., ; zaiou, ) . though most of the currently reported amps are known to be either anti-bacterial or anti-fungal peptides, the number of antiviral peptides is slowly going up. some of the amps that have been shown to be effective antivirals have been against viruses such as influenza a virus, severe-acute respiratory syndrome coronavirus (sars-cov), west nile virus (wnv), and other viruses (bai et al., ; basu et al., ; chu et al., ; daher et al., ; guo et al., ; oyston et al., ; zaiou, ) . interestingly these antiviral peptides were originally either elicited due to an immune response by the host to the viral infection or virus-encoded (bai et al., ; basu et al., ; chu et al., ; daher et al., ; guo et al., ; oyston et al., ; zaiou, ) . besides ␣defensins two other classes of amps are produced by mammalian cells: ␤-defensins and cathelicidins (gallo et al., ; harder et al., ) . between the two, cathelicidin represented by ll- is the most famous antimicrobial peptide that has demonstrated potent anti-bacterial, anti-viral and anti-fungal properties (dorschner et al., ; howell et al., ) . the ␣-defensins are known to inhibit replication of herpes simplex virus (hsv), cytomegalovirus (cmv), vesicular stomatitis virus (vsv) and influenza a virus (daher et al., ) . the mechanism of action of these peptides has been suggested to involve disruption of the microbial membrane and/or penetration of the microbial membranes to interfere with intracellular functions (brogden, ; harrison and ramshaw, ; howell et al., ; liu et al., ; tang et al., ) . so far, the ␤-defensins, cathelicidins (ll- ), caragenins (synthetic amps), peptide mimetics of ␥-interferon, the broad spectrum antiviral eb and peptide aptamers have been shown to possess antiviral activity against vaccinia virus (vv) (ahmed et al., ; altmann et al., ; harrison and ramshaw, ; howell et al., howell et al., , howell et al., , saccucci et al., ) . we have recently demonstrated the antibacterial activity of two types of amps, the thrombin-induced human platelet-derived antimicrobial peptides (pd) and the arginine-tryptophan (rw) repeat peptides, against aerobic bacterial contaminants encountered in blood products (mohan et al., a) . the structure and mechanism of action of pd and rw peptides has been elucidated previously in detail and these peptides are also known to be noncytotoxic and non-hemolytic (chan et al., ; liu et al., ; yeaman et al., ; yeaman and bayer, ) . because vaccinia virus has been used as a model to test methods of decontaminating blood products, and it has been found to be relatively resistant to solvent/detergent inactivation methods, we selected this virus to evaluate the antiviral activity of pd and rw peptides (lin et al., ; remington et al., ; roberts, ; wu and snyder, ) . in the present study we evaluated the same pd and rw peptides that were previously evaluated against bacteria (mohan et al., a) for their antiviral activity against vv as a model virus for enveloped viruses and observed that pd and rw peptides do demonstrate antiviral activity on vv in cell culture as well as in spiked plasma. simian kidney epithelial cell lines vero and b-sc- cells (atcc, manassas, va) were cultured in dulbecco's modified eagle's medium (dmem) (mediatech, herndon, va) containing % fetal bovine serum (fbs), mm l-glutamine (mediatech, herndon, va) and penicillin-streptomycin (invitrogen, gaithersberg, md). vaccinia virus (wr strain, kind gift from b. moss) was propagated in vero cells as described previously (jones-trower et al., ; kotwal et al., ; mohan et al., b) . vv seed stocks were prepared by infecting three t flasks of vero cells (jones-trower et al., ; kotwal et al., ) . infected cells were freeze-thawed three times at h post-infection (h.p.i.), followed by low speed centrifugation and the supernatant was collected, estimated for viral titers by plaque assay, aliquoted and stored at − • c, until used (jones-trower et al., ; kotwal et al., ; mohan et al., b) . vv infection of b-sc- (simian kidney epithelial) cells for antiviral analysis was performed in -well plates. a total of nine antimicrobial peptides were synthesized at cber core facility. four were tpmp- consensus sequence derived (pd) peptides of amino acids in length and other microbicidal peptides were - repeats of arg-trp (rw - ) amino acids (table ) (liu et al., ; mohan et al., a; tang et al., ; yeaman and bayer, ) . reconstitution of the peptides was essentially as per a published protocol except for the solvent used (mohan et al., a) . both the pd and rw peptides were reconstituted in phosphate buffered saline (pbs), ph . . since pd peptides were of uniform length ( -mers), the stock solutions were made at g/l by reconstituting the lyophilized powder in pbs and stored at - • c until used. since the rw peptides were of variable length ( -, -, -, -, and -mer), the stocks were made at mm concentration for these peptides by dissolving in pbs, ph . . all the nine amps were evaluated for their antiviral potential. amps were tested at different stages of virus infection: pre-, during-and post-infection. vv-infection without the peptides was taken as control and all experiments were performed in triplicate for statistical analyses. to analyze whether the amps act on the virus and/or cells both the vv and b-sc- cells were treated individually with the nine amps prior to infection and assessed for viral titers. effect of amps on vv was performed by taking l of pfu/ml concentration of wr virus, mixed individually with pd -pd or rw -rw peptides made up to a final volume of ml with dmem and incubated at room temperature for h. final concentration of the pd peptides was at g/ml whereas rw peptides were diluted to a m final concentration. the inoculum was then added to b-sc- cells maintained in -well plates. following virus binding to the cells at • c for h, virus inoculum was aspirated and agar overlay containing dmem was added to the cells and incubated for h (jones-trower et al., ; kotwal et al., ; mohan et al., b) . at the end of incubation period the agar overlay was removed and cell monolayer was stained with crystal violet for enumeration of virus plaques. results were expressed in plaqueforming units (pfu)/ml. similarly, effect of pd and rw peptides on b-sc- cells was analyzed by individually treating cells with each of these peptides for h at the same concentrations as above. following incubation the peptide mixture was aspirated and cells washed once with fresh dmem. virus infectivity was assessed by inoculating a ml mixture of vv ( pfu/ml) onto these treated-cells and incubated for h at • c. viral titers were measured by plaque assay as described above. l of pfu/ml concentration of wr virus was mixed with individual pd and rw peptides (final concentration g/ml and m, respectively) and made up to ml with fresh dmem and the mixture was added directly to b-sc- cells. cells were incubated at • c for h. following virus binding, the inoculum was aspirated and an agar overlay containing dmem was added to the cell culture plates and incubated at • c for h (jones-trower et al., ; kotwal et al., ) . cell monolayer was then stained with crystal violet for viral plaques as described above. b-sc- cells maintained in -well culture plates were infected with ml of vv inoculum ( pfu/ml) and incubated at • c for h. following virus binding, the inoculum was aspirated and fresh dmem containing individual pd peptides at a final concentration of g/ml and rw peptides at m were added to each well and cell culture plates were incubated at • c. following h of virus infection supernatant was aspirated and cell monolayer was freeze-thawed thrice to extract virus particles as described previously (jones-trower et al., ; kotwal et al., ) . virus particles extracted from each well was then quantified by the plaque assay as described above. to evaluate whether the pd and rw peptides were microbicidal at concentrations lower than g/ml and m, respectively, a serial doubling dilution analysis was performed with pd , pd and rw peptides. since pd peptides were of uniform length ( aa) they were constituted in g/ml concentration. rw peptides though were of varying length ( , , , and aa) and hence expressed in m concentrations. we maintained these two different concentration expression units to be consistent with our previously published work (mohan et al., a) . the final concentration of the pd peptides for the assay were , , and . g/ml whereas the rw peptides concentration was , , . and . m. pfu/ml of wr virus was mixed individually with each of these peptides and the antiviral assay was performed as per the pre-infection protocol described above. to assess whether these three amps would maintain their antiviral activity in a biological material, human plasma samples were spiked with vv and the peptides were tested for their activity. human plasma samples were acquired from the nih blood bank, bethesda, md and were spiked with pfu of vv-wr virus. pd , pd and rw peptides were each incubated with the virus inoculum for h at room temperature and tested for antiviral activity by performing the plaque assay as described in the pre-infection experiment. all assays described here were performed a minimum of three times and mean values ± sd (standard deviation) were calculated using microsoft excel ® . statistical analyses were performed using a two-tailed student's t-test and values were considered significant when p < . . dose-response curves (ec values) for pd , pd and rw peptides were estimated using the graphpad tm prism . software. in order to evaluate at which stage of the virus infection are the pd and rw peptides demonstrate antiviral activity, we tested all nine amps in a pre-infection incubation step. results from this experiment revealed that preincubation of amps with vv were able to inhibit viral infection (fig. ) . platelet-derived peptides pd and pd were able to bring down the vv titer by - -fold. the rw peptide was able to elicit a -fold reduction in vv titers (fig. ) . remaining six peptides (pd , pd , rw , rw , rw and rw ) did fig. . antiviral activity of pd and rw peptides on vv during the pre-infection stage. wr strain of vv was incubated with pd and rw peptides at g/ml and m concentration, respectively, for h at rt and titers were measured by counting plaques on b-sc- cells. since pd peptides were of uniform length ( aa) they were constituted in g/ml whereas rw peptides though were of varying length ( , , , and aa) and hence expressed in m concentrations. these two different concentration expression units were maintained to be consistent with our previously published work (mohan et al., a) . vv infection without the peptides was included as control. pd , pd and rw peptides demonstrate significant (p < . , indicated by *) reduction in viral titers. not demonstrate any antiviral activity above what was observed with the control group. once we observed that the amps were able to inhibit vv, the next step was to address whether the amps would still inhibit vv post a virus-binding step or a post-infection stage. analysis of the post virus-binding experiment revealed that none of the pd or rw peptides were able to inhibit virus infection as there was no significant difference in the viral titers between the test and control groups (fig. ) . similarly, the pre-and post-infection treatment of b-sc- cells with pd and rw peptides did not have any significant effect on virus titers between control and the peptide-treated groups (fig. ) . and resulting viral titers were compared to that of vv-infection lacking the peptides. in the pre-infection experiment b-sc- cells were incubated with pd and rw peptides for h at • c, followed by washing of the cells and infection with vv. for the virus-binding stage experiment peptides were mixed individually with vv and the mixture was added to cells. following incubation for h inoculum was aspirated and agar overlay with dmem was added. the post-infection assay was performed by first infecting b-sc- cells with vv for h and then adding fresh medium containing pd and rw peptides. note that both pd and rw peptides do not exhibit significant antiviral activity (p > . ) during these three stages of treatment. fig. . dose-response curves or ec estimation of pd and rw peptides. serial doubling dilution analysis was performed with pd , pd and rw peptides. the final concentration of the pd peptides for the assay were , , and . g/ml whereas the rw peptides concentration was , , . and . m. ml of wr virus ( pfu/ml) was mixed individually with each of these peptides at rt for h, followed by infection of b-sc- cells for h. following virus binding agar overlay with dmem was added to cells and incubated at • c for h. viral titers were estimated by counting plaques at the four different concentrations of the peptides tested and ec values were deduced by using the graphpad prism . software. peptide concentrations are represented on the x-axis (log scale) with pd and pd expressed in g/ml and rw concentration expressed in m. analysis reveals that the ec values for pd (a), pd (b) and rw (c) were g/ml, g/ml and . m, respectively. in order to evaluate the minimal inhibitory concentration of the pd , pd and rw peptides further analysis by serial doubling dilution of these peptides and their effect on virus inhibition was performed. dose-response curves for pd , pd and rw were generated using the graphpad prism . software and their ec values calculated. analysis revealed that pd (fig. a) and pd (fig. b ) exhibited an ec value of g/ml and concentration, respectively. rw on the other hand revealed an ec dose at . m concentration (fig. c) . since these peptides have already been known to act as potent antibacterial agents (mohan et al., a) in blood products, we analyzed whether these amps could inhibit vv in a vv-spiked plasma sample as well. analysis of the effect of pd , pd and rw peptides on vv in spiked plasma samples revealed that pd was the most potent peptide with % virus inhibition at g/ml concentration. pd peptide on the other hand demonstrated a -fold reduction in virus titer at g/ml concentration (fig. ) . rw was able to inhibit vv by ∼ % at m concentration (fig. ) . we had previously reported the proof-of-concept of usage of pd and rw peptides as bactericidal agents in experimentally contaminated blood products (mohan et al., a) . ideally, to be the most useful, any antimicrobial agent has to exhibit a broad-spectrum antimicrobial activity viz. anti-bacterial, anti-viral, anti-fungal and anti-parasitic. hence, in the present study we evaluated the efficacy of the same amps against vaccinia virus reduction in experimentally infected plasma. though current virus inactivation procedures are considered highly efficient and the transmission risks due to viruses is said to be minimal, the possibility still does exist. in order to address this hypothetical but possible occurrence we selected vaccinia virus as a model virus as it poses considerable challenge in being the most resistant virus to solvent/detergent inactivation methods (lin et al., ; remington et al., ; roberts, ; srinivasan et al., ; wu and snyder, ) . of the nine amps tested, three peptides (pd , pd and rw ) exhibited virucidal activity against vaccinia virus. the same three peptides also exhibited potent bactericidal activity in plasma and platelets stored in plasma (mohan et al., a) . the mechanism of action of these fig. . antiviral activity of pd and rw peptides against vv in spiked-plasma samples. human plasma samples spiked with pfu of vv-wr virus were incubated with pd , pd and rw peptides individually for h and tested for antiviral activity by infecting b-sc- cells and measuring virus titer by performing the plaque assay as described in the pre-infection experiment. note that pd , pd and rw treatment results in - -fold reduction of viral titers (p < . , indicated by *). amps on bacteria is well known but their mechanism of action on viruses is not yet clearly understood (liu et al., ; yeaman and bayer, ) . antimicrobial peptides have been reported against a variety of viruses that include influenza a virus, sars co-v, wnv, hsv and many others (bai et al., ; basu et al., ; chu et al., ; guo et al., ). the precise mechanism of action of the various amps is similar in some and different between other viruses. these amps could even have a stage-specific action on different viruses such as some may act on the extracellular virus, some may do so on the intracellular form, and some at the time of virus release (bai et al., ; basu et al., ; chu et al., ; guo et al., ). our analysis of the pd and rw peptides indicate that all three peptides exhibit their antiviral activity on the virus during in vitro conditions. these peptides do not have any effect once the vv is bound to the cell membrane or during post-infection. more interestingly, pre-treatment of b-sc- cells with pd and rw peptides did not adversely affect virus titers suggesting that these peptides do not affect virus entry and may not be able to penetrate the cell as reported with some of the recently tested amps (snyder and dowdy, ) . our analysis of the vv-spiked plasma samples further confirm that the pd , pd and rw peptides are able to inhibit the virus and are able to retain their antiviral activity in biological fluids as well. while one would argue that a peptide that provides a fold reduction in vv titers is not an effective therapeutic agent, it could make a difference in blood transfusion settings where blood donor deferral takes care of the high titer symptomatic individuals and inadvertently collected blood from an asymptomatic individual (i.e. with very low titers). in this scenario a reduction of ∼ vv particles to less than or, particles to would certainly make a difference, if the peptide can achieve a -fold reduction of the virus. in the present study, the highest concentration of the pd and rw peptides tested was only g/ml and m, respectively, based on our previous experience with bacteria (mohan et al., a) . regardless of whether there is substantial risk of acquiring vv by blood products or not, the demonstration of anti-vaccinia properties of these amps in plasma samples is very promising. the antiviral potency of these amps may have potential applications, as observed with caragenins, for topical therapy of poxviral infections (howell et al., ) . interestingly, the caragenins are synthetic antimicrobial compounds designed to mimic the structure and function of endogenous amps (howell et al., ) . future application of these amps in other potentially challenging scenarios include treatment of various microbial infections, especially the drug-resistant ones, through systemic administration (groneberg et al., ) . more pertinently, small animal testing of these amps would provide further evidence of such an application for these peptides and additionally reveal peptide clearance or safety aspects of these amps in vivo. the distinct advantage of the pd and rw peptides is that these are non-immunogenic, non-cytotoxic and non-hemolytic and thus have very minimal safety concerns. additionally, since these amps are synthetic they are easy to produce in large quantities with a good product consistency. a more comprehensive analysis using these amps on some of the more recently emerging viruses such as h n , west nile virus (wnv), sars co-v, cmv could bring about the value of these amps in virus inactivation/intervention strategies in general. peptide mimetics of gamma interferon possess antiviral properties against vaccinia virus and other viruses in the presence of poxvirus b r protein inhibition of vaccinia virus entry by a broad spectrum antiviral peptide antiviral peptides targeting the west nile virus envelope protein novel influenza virus ns antagonists block replication and restore innate immune function antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? tryptophan-and arginine-rich antimicrobial peptides: structures and mechanisms of action fusion core structure of the severe acute respiratory syndrome coronavirus (sars-cov): in search of potent sars-cov entry inhibitors direct inactivation of viruses by human granulocyte defensins cutaneous injury induces the release of cathelicidin anti-microbial peptides active against group a streptococcus biology and clinical relevance of naturally occurring antimicrobial peptides molecular mechanisms of pulmonary peptidomimetic drug and peptide transport identification of a new region of sars-cov s protein critical for viral entry antimicrobial and host-defense peptides as new antiinfective therapeutic strategies a peptide antibiotic from human skin cytokines, skin, and smallpox-a new link to an antimicrobial peptide selective killing of vaccinia virus by ll- : implications for eczema vaccinatum ceragenins: a class of antiviral compounds to treat orthopox infections antiviral activity of human beta-defensin against vaccinia virus identification and preliminary characterization of vaccinia virus (dryvax) antigens recognized by vaccinia immune globulin mapping and insertional mutagenesis of a vaccinia virus gene encoding a , -da secreted protein inactivation of viruses in platelet concentrates by photochemical treatment with amotosalen and long-wavelength ultraviolet light length effects in antimicrobial peptides of the (rw)n series evaluation of antimicrobial peptides as novel bactericidal agents for room temperature-stored platelets the proteoglycan bamacan is a host cellular ligand of vaccinia virus neurovirulence factor n l novel peptide therapeutics for treatment of infections inactivation of west nile virus, vaccinia virus and viral surrogates for relevant and emergent viral pathogens in plasmaderived products resistance of vaccinia virus to inactivation by solvent/detergent treatment of blood products inhibition of vaccinia virus replication by peptide aptamers cell penetrating peptides in drug delivery absence of detectable viremia in plasma and peripheral blood mononuclear cells from smallpox vaccinees: implications for blood safety antimicrobial peptides from human platelets safety of the blood supply: role of pathogen reduction antimicrobial peptides from platelets modular determinants of antimicrobial activity in platelet factor- family kinocidins multifunctional antimicrobial peptides: therapeutic targets in several human diseases the authors wish to acknowledge dr. bernard moss, nih, bethesda, for providing the wr virus strain. ssr is a recipient of a postdoctoral fellowship at the center for biologics evaluation and research (cber) administered by the oak ridge institute for science and education (orise) through an intra-agency agreement between the u.s. department of energy and the u.s. food and drug administration. review of the manuscript by drs. tahir malik and zhiping ye, cber is gratefully acknowledged. 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 transmembrane glycoprotein e genome sequence of chinese porcine parvovirus strain ppv a novel, stable, helical scaffold as an alternative binder -construction of phage display libraries identification of model peptides as affinity ligands for the purification of humanized monoclonal antibodies by means of 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of the purdue virus cluster importance of cholesterol for infection of cells by transmissible gastroenteritis virus phages harboring specific peptides to n protein of prrsv distinguish the virus from other viruses heterologous expression of fused genes encoding the glycoprotein from prrsv: a way for producing functional protein in prokaryotic microorganism phage displayed peptides recognizing porcine aminopeptidase n inhibit transmissible gastroenteritis coronavirus infection in vitro action mechanisms of lithium chloride on cell infection by transmissible gastroenteritis coronavirus cholesterol dependence of pseudorabies herpesvirus entry membrane protein molecules of transmissible gastroenteritis coronavirus also expose the carboxy-terminal region on the external surface of the virion the coronavirusmembrane glycoprotein transmissible gastroenteritis and porcine respiratory coronavirus phage display allows identification of zona pellucida-binding peptides with species-specific properties: novel approach for development of contraceptive vaccines for wildlife antigenic homology among coronaviruses related to transmissible gastroenteritis virus searching for peptide ligands with an epitope library coronaviruses: structure and genome expression antiviral effect of diammonium glycyrrhizinate and lithium chloride on cell infection by pseudorabies herpesvirus mechanisms of transmissible gastroenteritis coronavirus neutralization phage displayed peptides to avian h n virus distinguished the virus from other viruses a phage-displayed peptide can inhibit infection by white spot syndrome virus of shrimp cholesterol is important for a post-adsorption step in 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- - sw wt i authors: naesens, lieve; vanderlinden, evelien; rőth, erzsébet; jekő, józsef; andrei, graciela; snoeck, robert; pannecouque, christophe; illyés, eszter; batta, gyula; herczegh, pál; sztaricskai, ferenc title: anti-influenza virus activity and structure–activity relationship of aglycoristocetin derivatives with cyclobutenedione carrying hydrophobic chains date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: sw wt i previous studies have demonstrated that glycopeptide compounds carrying hydrophobic substituents can have favorable pharmacological (i.e. antibacterial and antiviral) properties. we here report on the in vitro anti-influenza virus activity of aglycoristocetin derivatives containing hydrophobic side chain-substituted cyclobutenedione. the lead compound e displayed an antivirally effective concentration of . μm, which was consistent amongst influenza a/h n , a/h n and b viruses, and a selectivity index ≥ . structural analogues derived from aglycovancomycin were found to be inactive. the hydrophobic side chain was shown to be an important determinant of activity. the narrow structure–activity relationship and broad activity against several human influenza viruses suggest a highly conserved interaction site, which is presumably related to the influenza virus entry process. compound e proved to be inactive against several unrelated rna and dna viruses, except for varicella-zoster virus, against which a favorable activity was noted. previous studies have demonstrated that glycopeptide compounds carrying hydrophobic substituents can have favorable pharmacological (i.e. antibacterial and antiviral) properties. we here report on the in vitro anti-influenza virus activity of aglycoristocetin derivatives containing hydrophobic side chainsubstituted cyclobutenedione. the lead compound e displayed an antivirally effective concentration of . m, which was consistent amongst influenza a/h n , a/h n and b viruses, and a selectivity index ≥ . structural analogues derived from aglycovancomycin were found to be inactive. the hydrophobic side chain was shown to be an important determinant of activity. the narrow structure-activity relationship and broad activity against several human influenza viruses suggest a highly conserved interaction site, which is presumably related to the influenza virus entry process. compound e proved to be inactive against several unrelated rna and dna viruses, except for varicella-zoster virus, against which a favorable activity was noted. © elsevier b.v. all rights reserved. currently available drugs for the treatment of influenza virus infections comprise the m ion channel blockers amantadine and rimantadine, and the neuraminidase inhibitors oseltamivir and zanamivir (de clercq, ; moscona, ) . stockpiling of oseltamivir and, to a lesser extent, zanamivir has been advocated in the context of pandemic preparedness (schünemann et al., ) , yet the recent isolation of oseltamivir-resistant seasonal influenza virus mutants, even from untreated patients, warrants for continued caution (lackenby et al., ; van der vries et al., ) . additional anti-influenza virus compounds should be urgently developed, having a novel antiviral target that is highly conserved amongst influenza virus (sub)types and, hence, less prone to genetic variation and resistance selection. one of the attractive therapeutic strategies would be a blockade of the viral entry into the host cell. the cellular entry process of influenza viruses has been unraveled since many years (reviewed in skehel and wiley, ) . a key role is being played by the viral envelope glycoprotein hemagglutinin (ha), which contains the receptor-binding site for initial attachment to the sialylated cellular receptors, and governs the receptor specificity of human versus avian influenza virus subtypes (chandrasekaran et al., ; nicholls et al., ) . in addition, after cellular uptake of the virus by endocytosis, the ha mediates the low ph-induced fusion of the viral envelope with the endosomal membrane, leading to release of the viral ribonucleoprotein in the cytosol. although the ha has been extensively studied from a biochemical and epidemiological perspective, specific antiviral drugs blocking the ha-receptor interaction remain to be clinically developed. several reports are available on the in vitro activity of small-molecule inhibitors of influenza virus fusion, which act by preventing the conformational change of the ha at low ph deshpande et al., ; plotch et al., ) . unfortunately, their development has been slow due to their inferior activity against some human influenza virus (sub)types, rapid selection for resistance and/or unsatisfactory outcome in animal models (yagi et al., ) . glycopeptide compounds represent a large series of natural, semisynthetic or fully synthetic compounds, which are widely recognized for their potent activity against gram-positive bacteria (nicolaou et al., ) . several studies have demon- strated that hydrophobic derivatives of glycopeptide antibiotics (e.g. vancomycin and eremomycin), and/or their aglycones exert antibacterial activity against glycopeptide-resistant enterococci. (cooper et al., ; printsevskaya et al., ; pace and yang, ) . remarkably, some of these lipophilic glycopeptide compounds were found to have inhibitory activity against coronaviruses and hiv, the latter being ascribed to inhibition of the hiv entry process (balzarini et al., (balzarini et al., , preobrazhenskaya and olsufyeva, ) . we here report on the chemical synthesis, anti-influenza virus activity and structure-activity relationship of novel glycopeptide compounds carrying a hydrophobic side chain on an aglycoristocetin backbone ( fig. ) . high-yield synthesis of aglycovancomycin ( ) and aglycoristocetin ( ) (fig. ) was performed as originally described by wanner et al. ( ) and consisted of deglycosidation of the parent antibiotics with hydrogen fluoride in anisole at neutral ph ( fig. ) (sztaricskai et al., ) . the aglycones were converted into the squaric acid amide esters ( and ) by coupling with dimethyl squarate ( ). this was followed by reaction with primary amines ( a-j) to yield the corresponding asymmetric squaric diamides ( a-g, a-j), using a regioselective procedure without the requirement for a protecting group strategy (tietze et al., ; sztaricskai et al., ) . the primary amines were: aminohexanol ( a); -aminohexanecarboxylic acid ( b); triglycine ( b ); dopamine ( c); n-( -aminophenyl)piperidine ( d) and phenyl-benzylamine ( e). based on the observation that compound e displayed favorable anti-influenza virus activity (see below), subsequent modifications were performed to study the impact of an increasing steric bulk in the rigid aromatic side chain of f-g and f-h, which were prepared with -naphthylamine ( f), -aminoterphenyl ( g) or -aminoanthracene ( h). it has been shown that introduction of hydrophobic substituents into glycopeptide antibiotics enhances their activity against glycopeptide-resistant bacteria (cooper et al., ; printsevskaya et al., ; pace and yang, ) , but, unfortunately, these products have lower water-solubility. to improve solubility, the squaric acid amide ester was reacted (sztaricskai et al., b) with d-glucosamine ( i) or d-galactosamine ( j), resulting in the asymmetric squaric diamides i and j, respectively. in these products, the carbohydrate moiety is linked to the aglycone in an unusual manner, i.e. through a cyclobutandione moiety, and not directly through one of the hydroxyl groups, as in the parent glycopeptide antibiotics. the structure, reaction yield and physico-chemical data for the aglycones, their squaric acid amide esters and corresponding asymmetric diamides, are summarized in table . the homogeneity of all compounds was checked by tlc and hplc, and the structures were confirmed by mass spectrometry. ( ) were first converted into their squaric acid amide esters ( and , respectively), followed by conversion to the asymmetric squaric diamides ( a-g and a-j, respectively). see table for the structures of the r group, as present in the primary amines a-j and the final products a-g and a-j. ( ) into their squaric acid amide esters ( and , respectively), these were converted to the asymmetric squaric diamides ( a-g, derived from aglycovancomycin, and a-j, derived from aglycoristocetin). b hplc conditions: instrument: waters with uv nm detection; column: lichrospher rp- ( mm × mm; m); injection volume: l (corresponding to g compound); solvents: table , several asymmetric squaric diamides derived from aglycoristocetin exerted marked activity against influenza virus, the most potent compounds being the phenylbenzyl derivative e [average antiviral ec : . m; selectivity index (si), defined as the ratio of mcc to ec : ]; the hexanol deriva-tive a (ec : m; si: ) and the naphthyl derivative f (ec : . m; si: ). their activity was -to -fold higher than that of the squaric acid amide ester of aglycoristocetin (ec : . m; si: ). an intermediate activity (ec : m) was observed for the triglycyl derivative b which, surprisingly, was comparably active as unsubstituted aglycoristocetin . the , -dihydroxybenzyl derivative c and the d-galactosamine derivative j were active against two of the three influenza virus strains tested, and the derivatives containing carboxypentyl ( b) and d-glucosamine ( i) substituents had activity against only one virus strain. the compounds carrying -aminophenylpiperidine ( d), terphenyl ( g) and anthracene ( h) substituents were completely inactive. thus, the intrinsic antiinfluenza virus activity of aglycoristocetin is markedly increased by squaric acid amide coupling and addition of a hydrophobic side chain, with the phenylbenzyl group being the optimal substituent. the favorable effect of this side chain appears to depend on different factors, namely: neutral charge (the alcoholic aliphatic derivative a is clearly more active than the corresponding carboxypentyl compound b) and steric bulkiness ( e and f are active while the more bulky compounds g and h are not). the aglycoristocetin backbone structure was shown to be critical for inhibition of influenza virus, since no activity was observed for compound e, which represents the aglycovancomycin analogue of e. aglycoristocetin and aglycovancomycin both contain a central heptapeptide core and nonproteinogenic phenolic amino acids, i.e. ␤-hydroxytyrosine (c and e units), -hydroxyphenylglycine (b and d units) and , -dihydroxyphenylglycine (a unit) (fig. ) . the main structural differences between both glycopeptide aglycones are as follows (fig. ) : (i) whereas aglycovancomycin has five aromatic rings (a-e) and two known amino acids (l-aspartic acid and n-methyl-d-leucine), aglycoristocetin has seven aromatic moi- table cytotoxicity of selected aglycoristocetin derivatives in human and animal cell lines. a . cytotoxic concentration ≥ > > a human embryonic lung (hel) fibroblasts; human cervix epithelial (hela); african green monkey kidney (vero); and crandell feline kidney (crfk) cells; nd: not done. b the cytotoxic concentration was defined as the minimum cytotoxic concentration (mcc) or % cytotoxic concentration (cc ); cf. legend to table . eties (a-g); (ii) the additional f and g rings of aglycoristocetin are interconnected via a diphenylether linkage (constituting the ristomycinic acid moiety); (iii) aglycoristocetin lacks the chloro substituents on the c and e rings, present in aglycovancomycin; (iv) the c-terminal carboxyl function is free in aglycovancomycin, but contains a methyl ester in aglycoristocetin and (v) aglycoristocetin has a primary amine function, whereas aglycovancomycin contains a secondary amine group (crowley et al., ) . at present, we cannot speculate on which of these structural components explain the antiviral specificity of the aglycoristocetin derivatives. our basic test panel of influenza viruses contained one chimeric virus (a/x- ; h n subtype), one a/h n virus and one b virus strain (table ) . when the lead compound e was further evaluated for activity against a/h n (strain a/puerto rico/ / ), its antiviral ec value was . ± . m, which is in the same range as that for the a/h n and b strains. determination of its cytostatic activity in mdck cells, using a cell counting assay, revealed an ic value of ± m. thus, e emerged as a potent inhibitor of influenza virus replication, with broad activity against different (sub)types and favorable selectivity. the glycopeptide compounds were evaluated for activity against other viruses besides influenza virus. none of the following rna viruses was found to be significantly inhibited by any of these glycopeptides, as evaluated by cpe or plaque reduction assay: feline coronavirus [examined in crandell-rees feline kidney cells]; vesicular stomatitis virus, coxsackie b virus and respiratory syncytial virus [performed in human epithelial hela cells]; parainfluenza- virus, reovirus- , sindbis virus and punta toro virus [tested in african green monkey vero cells]. in human embryonic lung fibroblast cells infected with various dna viruses, the only glycopeptide displaying some activity was compound e, with antiviral ec values of m (herpes simplex virus type ; wild-type or thymidine kinase-deficient); m (herpes simplex virus type ); > m (cytomegalovirus) and m (vaccinia virus), and a selectivity index of - . of note, e was found to be highly active against varicella-zoster virus (vzv; wild-type or thymidine kinasedeficient), with an antiviral ec value of . m and a selectivity index of . the diverse antiviral assays performed in human or animal cell lines permitted to obtain a more detailed insight into the cytotoxicity of the aglycoristocetin derivatives selected from the influenza virus experiments (table ). all compounds were either not or minimally cytotoxic at a concentration of - m. the cytotoxicity values for the lead compound e were in the same range as previously obtained in mdck cells. some glycopeptides in this study were previously evaluated for antibacterial activity (sztaricskai et al., ) , with e emerging as a highly active compound. our present anti-influenza virus data thus agree with the view that hydrophobic substitution has a positive impact on the pharmacological (i.e. antibacterial and antiviral) activities of glycopeptide compounds (printsevskaya et al., ; balzarini et al., balzarini et al., , . with regard to the antiviral mode of action, time-of-addition studies suggested that e blocks the viral entry process, since optimal anti-influenza virus activity was obtained when the compound was added to mdck cells min prior to or simultaneously with virus infection. a detailed analysis is currently ongoing to determine the effect of e on the virus-receptor interaction (i.e. binding of the viral ha to the sialic acid terminus of cell surface glycans), endocytosis or membrane fusion (i.e. fusion of the viral envelope with the endosomal membrane). whatever the precise mode of action, the subtype-independent activity of e provides strong support that the interaction site of e is highly conserved amongst human influenza virus strains. this is consistent with our observation that influenza virus fully retained its sensitivity to e after eleven sequential virus passages in mdck cells in the presence of e (at concentrations up to m). within human influenza virus ha sequences, only few residues are fully conserved, in particular in the receptor-binding site and fusion peptide (skehel and wiley, ) . the aglycoristocetin compounds described here and represented by the lead compound e are not the first glycopeptides reported to have activity against influenza virus. in , naruse et al. reported on the isolation, characterization and anti-influenza virus activity of two kistamicin antibiotics (naruse et al., ) . similarly to our compounds, both kistamicins showed strong activity against influenza virus and low activity against herpes simplex virus type . the kistamicins were reported to be inactive in hiv syncytium assays (naruse et al., ) . the observation that the anti-influenza virus activity of the kistamicins was higher when a lipophilic substituent was present at the terminal amine function, is reminiscent of our findings with the aglycoristocetin derivatives. in conclusion, the broad and robust anti-influenza virus activity of the aglycoristocetin derivatives described in this study creates a new avenue for the development of anti-influenza virus agents with a novel mode of action. the relatively narrow structure-activity relationship points to a highly specific interaction with the antiviral target, which is probably related to interaction of the influenza virus hemagglutinin with its cellular receptor. further studies to unravel the structure-activity relationship and precise mode of action are underway in our laboratories. antiretroviral activity of semisynthetic derivatives of glycopeptide antibiotics inhibition of feline (fipv) and human (sars) coronavirus by semisynthetic derivatives of glycopeptide antibiotics glycan topology determines human adaptation 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exemplified with vancomycin, ristocetin, and ramoplanin development of anti-influenza virus drugs. i. improvement of oral absorption and in vivo anti-influenza activity of stachyflin and its derivatives this study was supported by grants from the fonds voor wetenschappelijk onderzoek vlaanderen (fwo no. . . ), the international consortium for anti-virals (icav) and the hungarian national scientific research foundation (no. otka t ). we thank leentje persoons, frieda de meyer and vicky broeckx for dedicated assistance, and dr. sándor kéki (department of applied chemistry, university of debrecen) for recording the mass spectra. we thank dr. t. cihlar (gilead sciences, usa) for the generous gift of oseltamivir carboxylate. key: cord- - evk g authors: röcker, annika e.; müller, janis a.; dietzel, erik; harms, mirja; krüger, franziska; heid, christian; sowislok, andrea; riber, camilla frich; kupke, alexandra; lippold, sina; von einem, jens; beer, judith; knöll, bernd; becker, stephan; schmidt-chanasit, jonas; otto, markus; vapalahti, olli; zelikin, alexander n.; bitan, gal; schrader, thomas; münch, jan title: the molecular tweezer clr inhibits ebola and zika virus infection date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: evk g ebola (ebov) and zika viruses (zikv) are responsible for recent global health threats. as no preventive vaccines or antiviral drugs against these two re-emerging pathogens are available, we evaluated whether the molecular tweezer clr may inhibit ebov and zikv infection. this small molecule has previously been shown to inactivate hiv- and herpes viruses through a selective interaction with lipid-raft-rich regions in the viral envelope, which results in membrane disruption and loss of infectivity. we found that clr indeed blocked infection of ebov and zikv in a dose-dependent manner. the tweezer inhibited infection of epidemic zikv strains in cells derived from the anogenital tract and the central nervous system, and remained antivirally active in the presence of semen, saliva, urine and cerebrospinal fluid. our findings show that clr is a broad-spectrum inhibitor of enveloped viruses with prospects as a preventative microbicide or antiviral agent. zika virus (zikv) was first isolated in from a febrile rhesus macaque in the zika forest of uganda (dick et al., ) . since then, sporadic zikv infections occurred in africa and asia (haddow et al., ; hayes, ) . until , zikv infection usually has been associated with mild symptoms and thus, the virus had not been considered a threatening pathogen. however, since the recent outbreaks it is evident that zikv causes drastic birth defects, most prominently microcephaly (mlakar et al., ; rasmussen et al., ) , and is associated with neurological disorders, such as guillain-barré syndrome (cao-lormeau et al., ; krauer et al., ) . since , countries and territories reported ongoing zikv transmission (http://www. who.int/emergencies/zika-virus/en/). in brazil alone, . million persons had been infected (shuaib et al., ) , prompting the who to declare zikv a public health emergency of international concern. currently, there are no specific treatments or vaccines against zikv making development of effective preventive measures an urgent public health need (barrows et al., ; paixão et al., ) . zikv is transmitted mainly via mosquito bites but cases of sexual transmission also https have been reported (d'ortenzio et al., ; moreira et al., ; petersen et al., ) . like other members of the flaviviridae family (e.g. dengue virus), zika virions are surrounded by a host-membranederived lipid bilayer containing envelope glycoproteins responsible for cell entry (sirohi et al., ) . ebola virus (ebov) was discovered in near the ebola river in the former zaire. since then, outbreaks sporadically occurred in africa, with the most severe in , reaching epidemic proportions and a death toll of more than , people (centers for disease control and prevention (cdc), ) . ebov belongs to the family of filoviridae, forms enveloped filamentous infectious particles, and causes hemorrhagic fever, a rare and deadly disease with a high fatality rate. in addition to being a global health concern, the virus also is considered a potential biological threat agent. ebov likely is transmitted from infected bats to humans where it may spread through personal contact, contaminated objects, or sexual intercourse (mate et al., ; pandey et al., ) . noteworthy, even after recovery, ebov is found in some body fluids, including semen, up to several months (deen et al., ) . as for now, no licensed vaccine or medicine is available to prevent or manage future ebov outbreaks (sharma and ketki jangid, ) . molecular tweezers are novel drug candidates for the treatment of amyloidosis and related conditions. a lead compound, clr , has been used in many in vitro and in vivo studies to date schrader et al., ) . binding of clr to lysine residues in polypeptides disrupts noncovalent molecular interactions that are important for the abnormal self-assembly that leads to the formation of toxic oligomers and amyloid aggregates (schrader et al., ; sinha et al., ) . in fact, clr prevents assembly and promotes disaggregation of amyloid fibrils that are associated with neurodegenerative diseases ferreira et al., ; prabhudesai et al., ; richter et al., ) . therapeutic effects of clr have been demonstrated in animal models of parkinson's disease (lulla, ; prabhudesai et al., ; richter et al., ) , alzheimer's disease malik et al., ) , familial amyloidotic polyneuropathy (fap) (ferreira et al., ) , and desmin-related cardiomyopathy and was found to be safe in mice at doses -times higher than those showing therapeutic effects . we have recently established that clr also disrupted the formation of amyloid fibrils in semen (lump et al., ) , which enhance hiv- infection (münch et al., ; usmani et al., ) . surprisingly, clr also inhibited hiv- infection through a direct virusinactivating mechanism: the tweezer preferentially bound to raft-rich regions in the viral membrane resulting in the disruption of the lipid bilayer and loss of infectivity. in line with the membrane targeting activity, clr is inactive against non-enveloped viruses such as human adenovirus but destroyed other enveloped viruses, including hepatitis-c virus, human cytomegalovirus, and herpes simplex virus, demonstrating that clr represents a broadly active antiviral compound with prospects for microbicide or drug development (lump et al., ) . thus, we evaluated here whether clr might also block infection of re-emerging enveloped viruses. we show that clr indeed inhibits infection of replication-competent ebov and zikv as well as marburg, rabies, and sars corona virus (sars-cov) pseudoparticles. clr lost antiviral activity in the presence of serum, but remained active in the presence of semen, urine, saliva or cerebrospinal fluid. thus, this broadly active compound represents a promising lead for further development as a microbicide to protect from the sexual acquisition of zikv or ebov, or as a topically applied drug for the therapy of viral infections of the skin, mucosa or the respiratory tract. clr and clr were synthesized as described previously (fokkens et al., ; talbiersky et al., ) and . mm stock solutions were prepared in pbs. zikv envelope protein had been ordered from fitzgerald industries international, acton, usa. vero e cells were used for propagation and infection with zikv as described . tcid values (tissue culture infectious dose ; tcid /ml) were determined according to reed and muench, multiplied with a factor of . to obtain pfu/ml (plaque forming units) and, by normalizing to the number of cells, used to calculate the respective moi (multiplicity of infection). sw (human epithelial colon carcinoma) cells were kindly provided by ninel azoitei (center for internal medicine i, university of ulm, ulm, germany). hff (human foreskin fibroblasts), human glioblastoma (a ) or neuroglioma (h ) cells were kindly provided by jens von einem and karin danzer (ulm university medical center, ulm, germany). hepatic huh- cells were kindly provided by s. pöhlmann (göttingen, germany). the reporter cell line tzm-bl was obtained through the nih arrrp. virus stocks of r -tropic hiv- nl - th were generated by transient transfection of t cells as described (münch et al., ) . methods describing the effect of clr on pseudotyped lentiviral particles ( . .), ebola virus infection ( . .), the detection of zikv infection by a colorimetric mtt assay ( . .) or by cell-based zikv immunodetection assay ( . .), flow cytometry ( . .) and confocal microscopy ( . .) as well as the rna release assay ( . .) and the antiviral activity of clr in body fluids ( . ) can be found in the supplement. we have shown previously that clr inhibits hiv- infection by disrupting the viral membrane (lump et al., ) , suggesting that the antiviral activity of the tweezer is independent of the presence of the viral glycoproteins. to test this hypothesis, we generated luciferaseencoding retroviral particles harboring glycoproteins derived from ebov, the closely related filovirus marburg virus, rabies virus (rhabdoviridae) or sars-cov (coronaviridae). these pseudoparticles were exposed to clr and then added to huh- cells. clr , which lacks hydrophobic side arms and has no anti-hiv- activity (lump et al., ) , served as a negative control. infection rates were determined three days later by quantifying luciferase activity and showed that clr , in contrast to clr , efficiently blocked infection by all tested pseudoparticles (fig. a) . the half-maximal inhibitory concentrations (ic ) of clr against the four pseudoparticles were similar and ranged between . μm for rabies and . μm for marburg virus (fig. a) . these data corroborate our previous findings that clr targets the viral membrane rather than a viral glycoprotein (lump et al., ) . we tested next whether clr blocked infection by the replication-competent bsl pathogen ebov. vero e cells were inoculated with gfp-encoding ebov (ebihara et al., ; hoenen et al., ) that had been exposed to clr or clr . after days, the number of virus-induced plaques revealed that clr , but not clr , reduced the infectious titer in a dose-dependent manner with an ic of . μm (fig. b) . next, we determined the effect of clr on zikv infection. vero e cells were inoculated with the african zikv mr strain that was isolated in from a sentinel rhesus macaque (dick et al., ) in the absence or presence of clr or clr and viral replication was monitored by light microscopy. in the absence of clr , zikv caused a profound cytopathic effect (cpe) as evidenced by large plaques caused by detachment of cells. clr had no effect on this phenotype ( fig. a) . however, exposure to μm clr prevented formation of the virus-induced cpe entirely. to quantitatively assess the antiviral activity, we used a colorimetric mtt assay measuring the virally induced cpe (müller et al., ) . in the absence of compounds or in the presence of clr , zikv resulted in more than % dead (detached) cells. in contrast, clr suppressed cpe formation with an ic of . μm (fig. b ). the mtt assay allows an indirect measurement of zikv infection as the tetrazolium dye is metabolized only in living, non-infected cells. to evaluate the effect of clr on zikv more directly, infected cells were stained for the viral e protein and analyzed by confocal microscopy (schandock et al., ) . upon treatment with or μm of clr , e protein-specific fluorescence could not be detected, whereas clr had no effect, as expected ( fig. c ). lack of e protein expression in cells infected with clr -treated virus was confirmed by flow cytometry (fig. d, supplementary fig. s a ). of note, clr was not cytotoxic at concentrations active against zikv ( supplementary fig. s b ), and did not cause alterations in cell morphology (compare confocal images and dot plots of uninfected cells vs. μm clr exposed cells in fig. c and d). thus, clr prevents zikv infection of vero e cells. we hypothesized that if clr inhibited zikv infection by a similar mechanism to other enveloped viruses, its activity would be directed against the viral membrane (lump et al., ) . indeed, when clr was added to cells, rather than to the virus, and washed out prior to infection, no antiviral effect was observed in a cell-based zikv immunodetection assay (fig. a) . we performed next a time-course analysis in which zikv was exposed to μm clr for to s prior to infection. already after exposure for only s, clr reduced infection by ∼ %. infection declined further with increasing incubation time and was nearly at the level of uninfected cells after s incubation (fig. b) . in agreement with a direct activity against the , rhabdoviridae (rabies virus) or the coronaviridae family (sars-cov), were exposed to indicated concentrations of clr or clr and then used to infect huh- cells. after three days, infection rates were determined by quantifying firefly luciferase activity and subtracting background activity derived from uninfected cells. values represent % reporter gene activities ± sd derived from triplicate infections, normalized to values obtained for cells infected in the absence of tweezers. (b) analysis of replication competent ebov was performed using confluent vero e cells in -well plates. rgebov-egfp was preincubated with clr or clr and mixtures were added to the cells. after days, samples were analyzed by counting the number of plaques per well and calculating the corresponding plaque forming units per milliliter (pfu/ml) for each inhibitor and dilution. displayed values represent the mean of three independent experiments ± sd. ic values were calculated by graphpad prism. one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare the different clr /clr concentrations to cells infected in the absence of compound (* denotes p < . ; ** denotes p < . ; *** denotes p < . ). light microscopy images of vero e cells infected with zikv mr in the absence or presence of μm clr or clr . images were taken days post infection (dpi). (b) zikv mr was incubated with . - μm clr or clr before these mixtures were used to infect vero e cells. after days, when significant cytopathic effects were visible, the number of adherent, viable cells were determined using the mtt assay (müller et al., ) . values represent mean ± sd of percentages derived from triplicate infections. ic was determined using graphpad prism. one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare the different clr /clr concentrations to cells infected in the absence of compound (*** denotes p < . ) (c) confocal microscopy images of vero e cells infected with clr -or clr -treated zikv at day post infection. cells were stained for zikv e protein (green) and nuclei (hoechst, blue) and imaged using confocal microscopy. scale bar: μm. (d) flow cytometry of infected vero e cells. virus was pretreated with pbs, clr or clr and added to cells. h later, cells were fixed, permeabilized, and stained with an anti e protein antibody, and quantified using an alexa coupled secondary antibody. percentages indicate the fraction of protein e positive cells. ic, isotype control. a.e. röcker et al. antiviral research ( ) - virion, the ic of clr increased from . μm to . μm when a fold higher multiplicity of infection was applied (supplementary fig. s ). clr was shown to destroy hiv- membrane integrity through interaction with raft-rich regions in the retroviral envelope (lump et al., ) . in contrast to hiv- , the zikv particle is relatively densely packed with glycoproteins (sirohi et al., ) , which might hamper the interaction of the tweezer with the zikv membrane. to determine whether clr might inhibit zikv infection through direct interaction with glycoproteins, increasing amounts of recombinant viral e protein were titrated to clr , and then these solutions were assayed for anti-zikv activity. as shown in fig. c , even e-antigen concentrations of μg/ml did not affect the antiviral activity of clr , suggesting that the tweezer did not reduce zikv infectivity through binding to the viral e protein. interestingly, we also observed that elevated e-antigen concentrations of and μg/ml reduced infection in the absence of clr (fig. c) , which is likely due to the competition between the virion-associated e protein and the recombinant e protein for cellular receptors. to test whether clr interaction with the viral particle results in loss of membrane integrity, as was shown in the case of hiv- , (lump et al., ) , we measured viral rna genome release. sucrose cushion purified zikv was incubated with clr , clr , pbs or triton x- , as a positive control, for min at °c and the amount of total rna in the solution was determined fluorometrically. as expected, no viral for virus treatment, zikv was incubated with clr for min at room temperature before the mixtures were added to vero e cells. for cell treatment, clr was added directly to the cells; after h, the medium was replaced and the cells were infected with zikv mr . dpi, cell-based zikv immunodetection assay was performed. values represent mean ± sd (n = ). one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare the different clr /clr concentrations to cells infected in the absence of compound (* denotes p < . ; *** denotes p < . ) (b) zikv was incubated for the indicated times with pbs or μm clr before the mixture was added to vero e cells. dpi, cell-based zikv immunodetection assay was performed. values represent mean ± sd (n = ). one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare cells infected with clr -treated zikv to cells infected with zikv that had been incubated with pbs for the same time period (*** denotes p < . ). (c) indicated concentrations of zikv e protein were titrated to μm clr or pbs before zikv was added. mixtures were used to inoculate vero e cells. dpi, the number of adherent, viable cells were determined using the mtt assay (müller et al., ) . the values shown are mean ± sd from triplicate infections. one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare cells treated with different concentrations of zikv e protein and clr to cells that had been treated with the same concentrations of e protein and pbs (*** denotes p < . ). (d) zikv mr was incubated with pbs, μm triton x- , . - μm clr or μm clr for min at °c. samples were inactivated by uv light of a laminar flow for min. then, μl of the samples were used to determine rna concentrations using the quantifluor ® rna system and a quantus fluorometer (promega). background values obtained from control samples using cell supernatant of uninfected cells were subtracted from the respective signals. rna levels of virus stock incubated with pbs were subtracted from these values. data points represent mean ± sd (n = ). one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare samples treated with different clr concentrations, clr or triton x- to pbs-treated virus (*** denotes p < . ). a.e. röcker et al. antiviral research ( ) - rna was detectable when zikv was incubated with buffer or clr (fig. d ). in contrast, incubation with triton x- resulted in readily detectable viral rna suggesting that the detergent lysed the zikv particle. similarly, elevated amounts of rna were detected upon treatment of zikv with clr , demonstrating the physical destruction of the viral particle by the tweezer. of note, clr itself did not act as detergent but targets the viral membrane (lump et al., ) . next, we analyzed whether clr was active against epidemic zikv strains. the fb-gwuh- isolate was derived in from the fetus of a pregnant finnish tourist returning from south america (driggers et al., ) , and the prvabc- isolate represents the current american epidemic strain, isolated in from a puerto rican patient (lanciotti et al., ) . both zikv strains were exposed to clr upon infection of vero e cells. cell-based immunodetection assay and flow cytometry experiments demonstrated that the tweezer suppressed infection of both strains with ic values of . μm for fb-gwuh- ( fig. a and supplementary fig. s ) , and . μm against prvabc- (fig. b) , respectively. as zikv can be transmitted via sexual intercourse, we studied whether zikv infects cells derived from the anogenital region and if so, whether infection can be blocked by clr . zikv effectively infected cell lines derived from cervix (fig. a, supplementary fig. s ) , colon (fig. b, supplementary fig. s ) , and primary foreskin cells (fig. c, supplementary fig. s ). viral infectivity was suppressed by clr but not clr , with ic values in the μm range ( fig. and supplementary fig. s ). because zikv is neurotropic (cao-lormeau et al., ; mlakar et al., ) and clr has been shown previously to penetrate through the blood-brain barrier (bbb) when administered systemically richter et al., ) , we also tested whether clr blocked zikv infection of brain-derived cells. both a glioblastoma and h neuroglioma cells were susceptible to zikv infection and clr entirely abrogated infection at μm (fig. a) . finally, we confirmed these findings obtained in human cell lines using primary murine cerebellar neurons. zikv infected neuronal bystander cells, and this was blocked by clr (fig. b) . there was no apparent toxicity of clr in the primary neurons at this concentration. . . the antiviral effect of clr is abrogated by serum but not urine, saliva, semen, or csf its broad antiviral activity makes clr an interesting lead compound for antiviral prevention or treatment. for a systemic application, clr must retain antiviral activity in blood. to test whether this was the case, clr was diluted in human serum, and then exposed to zikv. as shown in fig. a , serum concentrations of . % and higher abrogated the anti-zikv activity of the tweezer. similarly, serum also abrogated the anti-hiv- activity of clr ( supplementary fig. s ), precluding its use as a systemically applied antiviral drug. in contrast, clr remained active and inhibited zikv infection in the presence of up to % of urine (fig. b) , saliva (fig. c ), cerebrospinal fluid (fig. e ) and % human semen (fig. d) . thus, clr inactivates zikv in the presence of body fluids associated with virus transmission, and could be administered locally to halt virus replication, or applied topically as a microbicide to prevent e.g. sexual virus transmission. thereafter, vero e cells were infected and days later, infection rates were determined via a cell-based zikv immunodetection assay. data points represent mean ± sd (n = ). ic values were determined with graphpad prism. one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare the different clr concentrations to cells infected in the absence of compound (** denotes p < . ; *** denotes p < . ). zikv was incubated for min at °c with . - μm clr or clr . next, mixtures were used to inoculate hela (a), sw (b), or hff (c) cells. days later, infection rates were determined via quantification of the viral e protein using a cell-based zikv immunodetection assay. data points represent mean ± sem (n = ). one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare the different clr concentrations to cells infected in the absence of compound (* denotes p < . ; ** denotes p < . ; *** denotes p < . ). a.e. röcker et al. antiviral research ( ) - . discussion our results establish clr as a broad-spectrum antiviral agent, which not only inhibits infection of established viral pathogens (lump et al., ) , but also of emerging viruses, such as ebov and zikv. we characterized here mainly the effect of clr on zikv, as there is currently no specific antiviral therapy nor a preventive vaccine available. clr blocked zikv infection of primary brain-derived murine cells (fig. b) as well as human cell lines derived from the anogenital region (fig. , supplementary fig. s ) and the brain (fig. a) with ic values between and μm. these values are in the same range as ic values obtained against ebov ( μm, fig. b ) and hiv- ( - μm) (lump et al., ) . several lines of evidence demonstrate that the antiviral activity of clr is directed against the zikv particle itself. first, treatment of cells with clr prior to infection had no antiviral effect (fig. a) . second, the ic of clr increased with increasing viral concentrations ( supplementary fig. s ) . third, the integrity of the zikv particle was lost upon clr treatment, as shown by the release of viral rna from clr -treated virions (fig. d) . these findings were in agreement with our previous observations that clr selectively disrupted viral membranes (lump et al., ) . interestingly, the selectivity stems from clr 's preferential interaction with membranes containing high levels of sphingomyelin and cholesterol, two lipids that are enriched in membranes of enveloped viruses, such as hiv- or ebov (bavari et al., ; brügger et al., ; chazal and gerlier, ; lorizate et al., ) . the selective interaction of clr with lipids that are enriched in the viral but not the cellular membrane may also explain its minimal effects on cell viability ferreira et al., ; lopes et al., ; prabhudesai et al., ; sinha et al., ) (fig. c , d, supplementary fig. s b ). clr is slightly cytotoxic at concentrations of ∼ mm, corresponding to selectivity indices of - , which is in a reasonable range for drug development (buschmann and mannhold, ; food and drug administration, ) . . clr inhibits infection of human and murine brain cells. (a) human glioblastoma (a ) or neuroglioma (h ) cells were infected with zikv in the presence or absence of . - μm clr . two days later, cells were stained for zikv e protein (green) and nuclei (with hoechst; blue) and imaged by confocal microscopy. scale bar: μm. (b) primary murine cerebellum cultures were infected with zikv mr that had been incubated with . - μm clr for min at °c. dpi, cultures were fixed, permeabilized and stained for the neuronal protein βiii tubulin (red), zikv e protein (green) and nuclei (with hoechst; blue). scale bar: μm. clr lost antiviral activity in the presence of serum, precluding application as a systemic antiviral drug. this finding was unexpected because clr has been found previously to be stable in mouse and human plasma, with a half-life of ∼ . h in mice after sc or iv injection . moreover, clr showed therapeutic effects in animal models of alzheimer's and parkinson's diseases malik et al., ; prabhudesai et al., ; richter et al., ) . these data suggest that the loss of antiviral activity in the presence of serum likely was due to binding of clr to serum proteins. the concentrations of clr required to prevent amyloid formation in vitro and in vivo are in the sub-micromolar range, which is - orders of magnitude lower than those needed to block viral infection. presumably, the vast majority of clr was bound to serum proteins and hence unavailable to act on the virus, whereas the minority of the tweezer remains free at concentrations allowing to exert anti-amyloid activity. this interpretation also is supported by the observation that clr retained anti-zikv activity in semen, urine, saliva, and csf. the total protein concentrations in these body fluids are ∼ - orders of magnitude lower than in serum (hu et al., ; vibhakar et al., ) . thus, a larger percentage of clr molecules likely are free in these biofluids and hence antivirally active. clr has been described previously as a novel bi-functional microbicide counteracting sexual hiv- transmission both through directly targeting virus infectivity and by inhibiting the infection-promoting activity of amyloids in semen (lump et al., ) . we show here that the tweezer also inhibits pathogenic zikv infection of cells derived from the anogenital region in the presence of semen. interestingly, both zikv and ebov are present in semen of infected individuals (atkinson et al., ; deen et al., ; moreira et al., ) and can be transmitted by sexual intercourse, similar to hiv- (d'ortenzio et al., ; mate et al., ; moreira et al., ) . thus, application of clr as a microbicide in the vaginal or rectal tracts might protect from acquiring different viral pathogens. interestingly, clr has been shown to penetrate the bbb richter et al., ) , where it may also block replication of neurotropic viruses such as zika or rabies (cao-lormeau et al., ; ludlow et al., ; mlakar et al., ) . however, it needs to be considered that clr may only block cell-free virus infection but not cell-to-cell viral spread, which may limit its utility as therapeutic agent. therefore, the effect of clr on cell-tocell viral transmission needs to be explored. another means of application of clr is topical, e.g., by directly administering clr on virus-infected body surfaces, such as the skin or mucous membranes, to treat, e.g., hsv-induced herpes labialis or genitalis. topical medications may also be inhalational, such as medications against flu or respiratory syncytial virus (rsv), or applied to the surface of tissues other than the . after min of incubation, the mixtures were used to infect vero e cells. days later, infection rates were determined via a cell-based zikv immunodetection assay. data points represent mean ± sem (n = ), except for csf and serum: mean ± sd (n = ). one-way anova (non-parametric, grouped), followed by bonferroni's multiple comparison tests were applied to compare the different clr concentrations to cells infected in the absence of clr but in presence of the same respective body fluid concentration (* denotes p < . ; *** denotes p < . ). a.e. röcker et al. antiviral research ( ) - skin, such as eye drops applied to the conjunctiva, for example, in cmvinduced retinitis. to address the challenge of emerging and re-emerging viral pathogens, it is imperative to develop broad-spectrum classes of antiviral agents as the conventional one-bug-one-drug paradigm is insufficient (zhu et al., ) . current direct-acting antiviral drugs are highly successful but have a narrow spectrum of coverage and are only available against a very limited number of viral pathogens. moreover, drug development is slow and expensive, and it typically takes more than a decade to get market approval. currently, no specific antiviral treatment exists against most, if not all, emerging and re-emerging viruses. broad-spectrum antivirals may offer certain advantages as they reduce time and cost of drug development, allow off-label use, and could be applied even before a viral threat is diagnosed (bekerman and einav, ; zhu et al., ) . clr is broadly active against enveloped viruses as it disrupts lipid bilayers containing elevated levels of sphingomyelin and cholesterol (lump et al., ) , which are typically enriched in viral membranes (bavari et al., ; brügger et al., ; chan et al., ; chazal and gerlier, ; lorizate et al., ) . we have shown that clr inactivates major viral pathogens such as hiv- , hsv- , cmv, and hcv, as well as zikv and ebov. it is highly likely that clr also blocks other enveloped viruses, such as severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) corona viruses, influenza or rsv. most notably, resistance development against clr is very unlikely to occur, as this would require changes in the lipid composition of the cellular and the viral membrane, which is difficult to envisage. in conclusion, clr represents a promising prototype of a broad-spectrum antiviral agent. all data were analyzed using graphpad prism version . for windows, graphpad software, la jolla california usa (www.graphpad. com). the significance level was calculated using one-way one-way analysis of variance (anova) (non-parametric, grouped), followed by bonferroni's multiple comparison test. p values of < . were considered significant (*, p < . **, p < . ***, p < . ). detection of zika virus in semen safety and pharmacological characterization of the molecular tweezer clr -a broad-spectrum inhibitor of amyloid proteins' toxicity protection of primary neurons and mouse brain from alzheimer's pathology by molecular tweezers a screen of fda-approved drugs for inhibitors of zika virus infection lipid raft microdomains combating emerging viral threats the hiv lipidome: a raft with an unusual composition drug selectivity: an evolving concept in medicinal chemistry guillain-barré syndrome outbreak associated with zika virus infection in french polynesia: a case-control study ebola outbreak in west africa -case counts retroviruses human immunodeficiency virus and murine leukemia virus are enriched in phosphoinositides virus entry, assembly, budding, and membrane rafts. microbiol ebola rna persistence in semen of ebola virus disease survivors -final report zika virus (i). isolations and serological specificity evidence of sexual transmission of zika virus zika virus infection with prolonged maternal viremia and fetal brain abnormalities in vitro and in vivo characterization of recombinant ebola viruses expressing enhanced green fluorescent protein molecular tweezers targeting transthyretin amyloidosis a molecular tweezer for lysine and arginine guidance for industry; antiviral product development-conducting and submitting virology studies to the agency. department of health and human services genetic characterization of zika virus strains: geographic expansion of the asian lineage zika virus outside africa a novel ebola virus expressing luciferase allows for rapid and quantitative testing of antivirals human body fluid proteome analysis zika virus infection as a cause of congenital brain abnormalities and guillain-barré syndrome: systematic review phylogeny of zika virus in western hemisphere molecular tweezers inhibit islet amyloid polypeptide assembly and toxicity by a new mechanism comparative lipidomics analysis of hiv- particles and their producer cell membrane in different cell lines neurotropic virus infections as the cause of immediate and delayed neuropathology neurotoxicity of the parkinson's disease-associated pesticide ziram is synuclein dependent a molecular tweezer antagonizes seminal amyloids and hiv infection using molecular tweezers to remodel abnormal protein self-assembly and inhibit the toxicity of amyloidogenic proteins molecular evidence of sexual transmission of ebola virus zika virus associated with microcephaly sexually acquired zika virus: a systematic review inactivation and environmental stability of zika virus development of a highthroughput colorimetric zika virus infection assay semen-derived amyloid fibrils drastically enhance hiv infection history, epidemiology, and clinical manifestations of zika: a systematic review strategies for containing ebola in west africa zika virus a novel "molecular tweezer" inhibitor of α-synuclein neurotoxicity in vitro and in vivo zika virus and birth defects-reviewing the evidence for causality a molecular tweezer ameliorates motor deficits in mice overexpressing α-synuclein macromolecular antiviral agents against zika, ebola, sars, and other pathogenic viruses molecular tweezers for lysine and arginine-powerful inhibitors of pathologic protein aggregation ebola vaccine: how far are we? re-emergence of zika virus: a review on pathogenesis, clinical manifestations, diagnosis, treatment, and prevention lysine-specific molecular tweezers are broad-spectrum inhibitors of assembly and toxicity of amyloid proteins the . a resolution cryo-em structure of zika virus molecular clip and tweezer introduce new mechanisms of enzyme inhibition direct visualization of hiv-enhancing endogenous amyloid fibrils in human semen salivary total protein levels and their correlation to dental caries inhibition of mutant αb crystallin-induced protein aggregation by a molecular tweezer broad-spectrum antiviral agents a.e.r. is funded by a fellowship of the landesgraduiertenförderung baden-württemberg and is part of the international graduate school in molecular medicine ulm. j.a.m. is indebted to the baden-württemberg stiftung for the financial support of this research project by the eliteprogramme for postdocs. authors declare to have no conflict of interest. supplementary data related to this article can be found at http://dx. doi.org/ . /j.antiviral. . . . key: cord- -f lmstyn authors: struck, anna-winona; axmann, marco; pfefferle, susanne; drosten, christian; meyer, bernd title: a hexapeptide of the receptor-binding domain of sars corona virus spike protein blocks viral entry into host cells via the human receptor ace date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: f lmstyn in vitro infection of vero e cells by sars coronavirus (sars-cov) is blocked by hexapeptide tyr-lys-tyr-arg-tyr-leu. the peptide also inhibits proliferation of coronavirus nl . on human cells both viruses utilize angiotensin-converting enzyme (ace ) as entry receptor. blocking the viral entry is specific as alpha virus sindbis shows no reduction in infectivity. peptide ( )ykyryl( ) is part of the receptor-binding domain (rbd) of the spike protein of sars-cov. peptide libraries were screened by surface plasmon resonance (spr) to identify rbd binding epitopes. ( )ykyryl( ) carries the dominant binding epitope and binds to ace with k(d) = μm. the binding mode was further characterized by saturation transfer difference (std) nmr spectroscopy and molecular dynamic simulations. based on this information the peptide can be used as lead structure to design potential entry inhibitors against sars-cov and related viruses. the sars-associated corona virus (sars-cov) has been identified as the causative agent of severe acute respiratory syndrome (sars) which emerged as an alerting epidemic in winter of - resulting in over infected cases with approximately % deaths ksiazek et al., ; marra et al., ; peiris et al., ; rota et al., ; who, ) . sars-cov infects human host cells by an initial interaction of its spike glycoprotein (s) and the receptor on human cells, angiotensinconverting enzyme (ace ) (dimitrov, ; holmes, ; li et al., ) . functional characterization of the s protein suggests that the receptor-binding domain (rbd) is located between amino acid residues and (xiao et al., ) . flow cytometry indicated that amino acids - are the minimal receptor binding region of the s glycoprotein (babcock et al., ) . further studies located the rbd from residues to . the rbd fused to the fc region of human igg (rbd-fc) binds ace with higher affinity (k d $ nm) than does the full length s -ig chimera (li et al., b; wong et al., ) . the crystal structure of residues - of the s in complex with the receptor ace reveals that a loop within the rbd (residues - ) makes all the contacts to ace and is referred to as the receptor-binding motif (rbm). six tyrosine residues are involved in direct binding to the receptor (li et al., a) . studying the virus adaptation to humans the spike protein also seems to play a major role in the species specificity of coronavirus infection. especially, the introduction of a threonine residue at position and an asparagine instead of a charged lysine residue at position of the spike protein seem to be responsible for its high affinity to human ace (holmes, ; li et al., b; qu et al., ) . yi et al. demonstrated that a single amino acid substitution (r a) in a full-length spike protein dna vaccine failed to induce neutralizing antibodies (nabs) and that the same mutation yielded pseudoviruses that were unable to enter the human cells (yi et al., ) . furthermore, the rbd-fc bearing the same r a mutation shows no affinity to ace and is not capable of blocking s protein-mediated pseudovirus entry (he et al., ) . the interaction of sars-cov with its receptor ace is an attractive drug target as epitopes of the rbd on the spike protein may serve as leads for the design of effective entry inhibitors (du et al., ) . another drug target is the fusion process of the spike protein with the host cell membrane that is characterized by the presence of two heptad repeat (hr) regions, hr and hr , which are postulated to form a fusion-active conformation similar to those of other typical viral fusion proteins (sainz et al., ; van der hoek et al., ; yuan et al., ) . peptides were synthesized on solid phase using a fmoc-protecting group strategy on a fmoc-pal-peg-ps resin (applied biosystems) with o-(benzotriazol- -yl)-n,n,n ,n -tetramethyluronium tetrafluoroborate (tbtu, iris biotech) as activator. a mos x synthesizer (advanced chemtech) and a liberty microwave synthesizer (cem) were used for peptide syntheses starting with lmol amino groups each. after each coupling step the growing peptide was capped with an acetyl residue by % acetic anhydride in dmf. cysteine residues were substituted by serines to avoid dimerization. using trifluoroacetic acid (tfa), triisopropylsilan and h o ( : : , v/v), peptides were cleaved off the resin leaving an amide at the c-terminus. the cocktail was applied twice for and min, respectively. preparative rp-hplc was carried out on a biocad e instrument (perseptive biosystems) using a h o/acetonitrile gradient ( . % tfa) on a vp / nucleodur c pyramid l column (macherey & nagel). peptides were characterized by maldi-tof mass spectrometry on a biflex iii instrument (bruker daltonics) in reflector mode using , dihydroxybenzoic acid (dhb) or a-cyano- -hydroxycinnamic acid (cca) as a matrix. peptide rbd- b (ykyryl, y -l ) and related peptides were further characterized by d-and d-nmr spectroscopy (not shown). spr studies were carried out using a biacore or biacore t instrument. for all experiments a temperature of °c, flow rate of ll/min (biacore ) or ll/min (t ), and a tbs running buffer ( mm tris, . m nacl, lm zncl at ph ) were used. the carboxymethylated sensorchip surface of a cm chip (biacore) was activated by nhs/edc followed by immobilization of rhace (r&d systems) in acetate-buffer (ph . , biacore). rhace was obtained in tbs that had to be changed for the immobilization of the enzyme to pbs containing additional lm zncl (ph . ) using a slide-a-lyzer mini unit (pierce biotechnology) with a molecular weight cut-off of at °c for at least h. sensorgrams of rbd- and rbd- were recorded with a chip that had fmol of ace immobilized, that of rbd- on a chip with fmol, those of rbd- b and of the rbd- b related peptide library on a chip with fmol, respectively. carboxyl groups of the activated chip surface that had not reacted with the protein were capped with ethanolamine (biacore). . . saturation transfer difference (std) nmr spectroscopy . . . sample preparation nmr samples were prepared in deuterated tris-buffered saline (d-tbs) containing mm perdeuterotris(hydroxymethyl)aminomethane (tris-d ), . m nacl and lm zncl (ph . ) in deuterium oxide (d o, . %). tbs of the commercial rhace (r&d systems) was changed to d-tbs in slide-a-lyzer mini units (pierce biotechnology) with a molecular weight cut-off of twice for at least h at °c. ykyryl was added from mm stock solution in d-tbs with sample volume adding up to ll in a mm shigemi nmr micro tube with c(ace ) = . lm and peptide concentration between . and lm ( - fold excess over ace ). all std spectra were recorded at a temperature of k with a spectral width of ppm on a bruker avance drx mhz spectrometer equipped with a mm inverse triple resonance cryoprobe. selective saturation of the protein was achieved by a train of gauss-shaped pulses of ms length each, truncated at %, and separated by a ms delay leading to a total length of saturation time of s. the on-resonance irradiation of the protein was performed at a chemical shift of À . ppm. off-resonance irradiation was set at ppm. total scan number in the std experiments was . nmr spectra were multiplied by an exponential linebroadening function of . hz prior to fourier transformation. water suppression was achieved by an excitation sculpting pulse sequence. spectra processing was performed using topspin . software (bruker). the sars-cov inhibition assay was performed as described previously (vassilatis et al., ) . in brief, vero cells in -well plates were infected in the biosafety level laboratory (bni hamburg) with sars-cov (frankfurt isolate) at a multiplicity of infection (moi) of . . the inoculum was removed after h and replaced with fresh medium complemented with different concentrations of compound. the virus rna concentration in the supernatant was measured by real-time pcr after days. rna was prepared from ll supernatant using diatomaceous silica (pfaff et al., ) . quantitative real-time reverse transcription-pcr (rt-pcr) was performed with the purified rna according to a published protocol . in vitro transcripts of the target region were used in the pcr to generate standard curves for quantification of the virus rna. the md simulations were carried out with the software maestro/desmond on an hp z workstation (one quadcore cpu), using the opls_aa/ force field. the starting structure was placed in a water box with orthorhombic boundary conditions and salt concentration of mmol/l (spc solvent model, , water molecules,   Å). md simulations over , ps were performed to equilibrate the system at k. the simulation in equilibrium was performed over ps at k, with the nose-hoover thermostat method and a relaxation time of . ps. the recording interval was . ps. before starting md simulations the system was minimized three times over steps, respectively. the period of the md after equilibration with constant potential energy was used for the analysis. table synthetic peptide library of sixteen mer peptides comprising rbd-residues n -t of sars-cov spike protein. the peptides rbd- , rbd- and rbd- show binding to ace . residues of the spike protein (s) amino acid sequence a library of linear peptides was synthesized via solid phase synthesis using fmoc strategy containing sixteen mer rbd peptides (rbd- to rbd- ) which together comprised residues n -t (cf. table ). cysteine residues were substituted by serine amino acids to avoid dimerization of the peptides. the compounds were used to identify binding motifs in interaction with the human receptor ace by surface plasmon resonance (spr) binding studies. this method allows the determination of the binding specificity, as table synthetic peptide library of fourteen mer peptides comprising rbd-residues n -e and a -s of sars-cov spike protein. the k d of rbd- b (ykyryl) is lm. the on-and off-rates are .  m À s À and . s À , respectively. peptide rbd- c (rylrhg) shows k d = lm, k on = .  m À s À and k off = . s À . residues of the spike protein (s) amino acid sequence well as the association and dissociation rates of ligands interacting with protein receptors. the receptor protein ace was immobilized on the sensor chip and the peptides were passed over the sensor surface. the affinity of the interaction is determined from the level of binding at equilibrium as a function of sample concentration and can also be determined from analysis of the binding kinetics. spr screening of the sixteen mer rbd peptides resulted in positive responses of three peptides, i.e. rbd- (y -r -ykyrylrhgklr), rbd- (s -t -sywplndygfyt) and rbd- (t -v -ttgigyqpyrvv), respectively. all other rbd peptides showed no or insignificantly small response signals and were thus not interacting with ace . to analyze whether the interaction of the peptides rbd- , rbd- and rbd- is based on a specific binding event, the spr sensorgrams of each compound were recorded at several different concentrations. the signal at equilibrium measured in response units [ru] obtained from the sensorgrams was plotted against the concentration of ligand passed over the sensor surface (cf. fig ) . assuming a one site binding model the specific saturable binding affinity of the ligand peptides is calculated to k d = ± lm for rbd- , k d = ± lm for rbd- and k d = ± lm for rbd- . the spr sensorgrams were also used to determine kinetic parameters of binding, the association rate constant k on [s À m À ] and the dissociation rate constant k off [s À ]. rbd- shows a fivefold higher association rate constant k on = .  s À m À compared to rbd- and rbd- with k on = .  s À m À and .  s À m À , respectively. the dissociation rate of all three peptides is comparable with k off = . s À (rbd- ), . s À (rbd- ) and . s À (rbd- ), respectively. the dissociation constant k d = k off /k on resulting from analysis of the kinetic data of the peptides are k d = lm (rbd- ), lm (rbd- ) and lm (rbd- ) in excellent agreement with the dissociation constants obtained from thermodynamic data analysis. the on-rates suggest that the bioactive conformation of peptide rbd- is more similar to its solution conformation than for rbd- and rbd- . peptides rbd- , rbd- and rbd- contain % of the residues, namely y , y , y , n , y , t , t , g and y that were identified by x-ray crystal structure analysis to be in contact with ace (li et al., a) . several mutations in the viral protein were necessary to change the host organisms from wild animals, like civet cats, to humans, i.e. s t and k n (holmes, ; li et al., b; qu et al., ) . these amino acids are included in the peptides rbd- and rbd- . in a screening of a library hu in all spectra water was suppressed by an excitation sculpting pulse sequence and spectra were acquired in d-tbs at k in a ll (hwang and shaka, ) . (b) determination of binding affinity from std nmr titration data. the titration curve of the aromatic protons hd,d of tyr is shown. the k d value was determined from the std nmr titration by using the one site binding model. the resulting k d value is ± lm. (c) std nmr epitope mapping of rbd- b. the spots indicate the range of relative std% for the protons that were saturated according to their proximity to the human receptor protein ace . a strong binding is detected for the aromatic protons of the three tyrosines. the highest degree of saturation of . std% (absolute) obtained for he,e of tyr was set to % relative std. could inhibit the binding of rbd to ace and the plaque formation of sars-cov in vero cells with an ec value of . lm . ho et al. showed biological activities of small peptides derived from s protein to inhibit the spike protein and ace interaction. among others peptide sp- (residues - , fytttgi-gyqpy), which overlaps in sequence with the identified peptides rbd- and rbd- , shows inhibitory activity at a million fold excess relative to ace (ho et al., ) . however, the mer peptide p comprising residues - (palncywplndygfyttsgi) of zheng et al. overlapping with peptide rbd- shows no inhibition in a cytopathic effect (cpe)-based assay . in contrast, as mentioned above, the partially overlapping peptides a -l and sp- (ho et al., ) showed virus inhibition in their respective assays. to further characterize the binding epitope we synthesized a second peptide library of fourteen hexamer peptides comprising residues n -e (rbd- a to rbd- e) and a -s (rbd- a to rbd- d and rbd- a to rbd- e) with an overlap of three amino acids in each sequence (cf. table ). these peptides were used to further locate strongly interacting amino acids by spr and saturation transfer difference (std) nmr spectroscopy. spr screening was performed with ligand concentrations of up to lm in tbs and fmol of immobilized ace . rbd- b (y -l ; ykyryl) showed the highest spr response signal of ru at a concentration of lm. also the flanking peptides rbd- a (n -y ; nynyky) and rbd- c (r -g ; rylrhg) showed significant binding of ru (c = lm) and ru (c = lm), respectively. the spr response of peptides rbd- a (a -w ; alnsyw) and rbd- b (s -n ; sywpln) was comparable to that of rbd- a ( and ru, respectively) (cf. fig. ) . the other hexapeptides gave no significant spr signal. therefore, only peptide rbd- b (y -l ; ykyryl) was further investigated by spr and std nmr experiments. a concentration dependent spr affinity plot was performed with fmol of receptor protein immobilized. fig. shows the concentration dependent binding of rbd- b (ykyryl) by spr resulting in a dissociation constant k d = ± lm which is two-fold higher affinity than that of peptide rbd- . the binding kinetics are k on = .  s À m À and k off = . s À and also result in a dissociation constant of k d = lm. the two-fold increase of the binding affinity compared to rbd- results from a two-fold higher on-rate which is probably due to a better defined conformation in solution such that binding can occur more rapidly. the off-rate is the same as found for the dodecapeptide rbd- . std nmr spectroscopy is a well-established method to characterize ligand-protein interactions meyer, , ) . here it was used to determine dissociation constant and binding epitope of the interaction of rbd- b with a soluble construct of the receptor protein ace in deuterated tris-buffered d o. fig. shows the h std nmr spectrum of rbd- b (ykyryl) and receptor ace at a fold excess of the ligand over the protein. dependence of the std amplification factor on the concentration of rbd- b yields the k d value. a one site binding model fits the experimental data well and gives k d values in the low micromolar range: hd,d of the tyr results in k d = ± lm. the binding epitope was determined from std spectra at a fold excess of the ligand. it shows that all tyrosine residues have a close contact to ace . furthermore, the ha atom of arg also has a close proximity to the receptor surface. the ha atoms of tyr and leu interact also strongly with the cellular receptor. the positively charged groups of lysine and arginine side chains also show moderately strong interactions with the surface. the c-terminal leucine seems to be involved in a hydrophobic contact via its side chain. the highest level of saturation of . % std (absolute) is found for the he,e of tyr . the x-ray crystal structure analysis of the complex of rbd (residues - ) with ace shows only contacts of tyr and tyr of the rbd with ace (li et al., a) . however, in the isolated hexapeptide ykyryl, tyr is also binding to ace . according to mutation studies arg of sars-cov spike protein is important for binding affinity (he et al., ) . this fact is confirmed by std nmr analysis. the side chain protons hd,d of arg exhibit . % absolute std, which suggests an ionic interaction of the guanidinium group with the receptor molecule. the x-ray structure of the complex revealed no direct contact between arg of rbd and ace . thus in the free form the hexapeptide adopts a different binding mode and conformation compared to the case when integrated into the rbd. lys also makes a contact via its positively charged group evidenced by the std effect on the he,e protons that receive a saturation of . %. protons of leu methyl groups show a std effect of . % and . % std, respectively, indicating a contact of the methyl groups to the receptor. the biological activity of the lead structure rbd- b (y -l ; ykyryl) was assayed with respect to its ability to inhibit virus replication in cell culture (drosten et al., ) . veroe cells were infected with the sars-cov isolate frankfurt. as described previously, growth of the virus in vero cells is not associated with a cytopathic effect (vassilatis et al., ) . cells were infected with a virus titer of . moi. the inoculum was removed after h and replaced with fresh medium complemented with different concentrations of peptide rbd- b in a one-time dose. two days post infection virus rna concentration in the supernatant was measured by real-time pcr (cf. fig. ). there was no evidence for toxicity of the compound in the concentration range tested with mtt cell proliferation assay on subconfluent cells. after two days a onetime dose of the peptide rbd- b (ykyryl, . mm) reduced the virus rna level compared to the untreated control by a factor of . further, the inhibition of virus proliferation by the peptide is concentration dependent. after two days, relative to an untreated control virus rna is reduced by a factor of at a mm concentration of the peptide. the final amount of virus rna is in fact lower than the initially added amount. these data suggest that the hexapeptide blocks indeed the binding site necessary for the initial viral attachment to the human receptor ace and effectively inhibits viral entry into vero cells. thus, the virus is not capable to replicate and viral particles are exposed to the degradation process. these results are in good agreement with the occupation of the receptor's binding site given the peptide's dissociation constant of k d = lm. at peptide concentrations of mm the occupation of binding sites is . %, at . mm it is . % and at mm . %, respectively. the peptide inhibits the infection of the corona virus specifically. this is proven by the fact that rbd- b does not inhibit infection of vero cells with the alpha virus sindbis, which cause high fever in humans. (tesh, ) furthermore, the peptide was also tested in an inhibition assay with another corona virus nl in llc-mk and caco cells. nl corona virus causes severe colds in human and uses ace also as a functional receptor. (van der hoek et al., ; wu et al., ) inhibitory effects were observed for both cell lines at a concentration of mm. for caco cells inhibition is also observed at a peptide concentration of mm. we have synthesized various hexapeptides closely related to peptide rbd- b (ykyryl) including an alanine scan library and analyzed by spr the importance of individual amino acids for binding to ace . the alanine scan reveals that the tripeptide motif kyr is essential for the binding (cf. fig. ) . the other binding curves show that the positively charged side chains of the amino acids lys and arg may be important for the receptor binding. no binding was observed for the peptides ydyryl and ykydyl with the negatively charged aspartate replacing the positively charged lys and arg. changing positively charged residues to uncharged residues (k s, r s) reduces the binding affinity but does not abolish it. these results indicate a clear role of lys and arg in the binding process in agreement with the std nmr data shown above. the binding mode of peptide rbd- b to the human receptor ace was also analyzed by docking of the peptide to the binding site followed by a molecular dynamics (md) simulation that was recorded for ps in equilibrium using the opls_aa/ force field as realized in the program desmond (schrödinger) (jorgensen amino acid sequence , ) . the conformation of peptide rbd- b in the crystal structure of the rbd of the viral spike protein in complex with the receptor ace was used as starting structure (li et al., a) . the protein peptide complex was placed in a water box with , water molecules and a sodium chloride concentration of . mol/l. the peptide shows high dynamics during the md but stays always in contact with the receptor surface. fig. shows the starting and the final conformation of the complex. the side chains of amino acids tyr and tyr interact with ace in the crystal structure. at the end of the md simulation lys , tyr and arg show interactions with the receptor. the binding mode of hexapeptide rbd- b to ace differs from the binding mode of the hexapeptide as part of the full length s protein. the positively charged residue of lys forms ionic interactions with the carboxyl group of glu of ace . the average distance from the lysine e-nitrogen atom and an oxygen atom of the carboxyl group over the course of the md simulation is . ± . Å. the guanidinium group (ng) of arg interacts with the carboxyl group of asp of the receptor with an average distance of . ± . Å. the crystal structure shows the first three carbohydrate units of a high mannose n-glycan on the receptor surface resolved. in the md simulation tyr shows contact to the glycan molecule by interaction of its aromatic residue with the second n-acetylglucosamine and the mannose residue. the average distance of the interaction of the aromatic ring with the n-acetylglucosamine is . ± . Å and with the mannose . ± . Å, respectively. during the md simulations the peptide shifted . Å at the c-terminus and . Å at the n-terminus from its initial position to its final pose (fig. ) . the results of the md studies support the affinity data obtained from the alanine scan, which indicates that the tripeptide motif kyr is important for the binding to ace . sars belongs to the major new emerging virus diseases. in it caused a major outbreak with about deaths. it is a respiratory disease with about % mortality that is caused by a variant of the common coronaviruses (sars-cov). rigid political measures helped to contain the epidemic within a short period of time. so far no specific treatment against sars is available. in search for molecules that can specifically block the attachment of the virus to the human cell, we analyzed binding modes of viral peptides to the human receptor. we identified and characterized the focal point of the viral protein that is used by the virus for its attachment to the human cell. we found a hexapeptide in the receptor-binding domain (rbd) of the s protein of sars-cov that carries a significant portion of the binding affinity of the virus to the human cell. the s protein mediates the attachment of the virus to its functional receptor ace . the attachment of the virus to ace does not interfere with the natural function of the receptor. therefore, it is easy to block the attachment site of the virus in the upper respiratory tract as a preventive measure against sars. we could clearly demonstrate that hexapeptide tyr-lys-tyr-arg-tyr-leu reduces viral infection of epithelial cells, as found in the upper respiratory tract, by a factor of . this peptide was shown to be specific against coronaviruses that attach to the ace receptor. its mode of action is specific as it does not interfere with other infections by viruses that utilize different receptors, like alpha virus sindbis. combination of several biophysical methods, e.g. spr, std nmr and molecular dynamics simulations, were used to characterize the specific binding mode of the inhibitory peptide. although there is currently no sars outbreak the need of an antiviral drug, e.g. based on the hexapeptide, is still present. a viral reservoir is present in wild animals like bats and civet cats and a new epidemic is likely someday. amino acids - of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor the secret life of ace as a receptor for the sars virus evaluation of advanced reverse transcription-pcr assays and an alternative pcr target region for detection of severe acute respiratory syndrome-associated coronavirus identification of a novel coronavirus in patients with severe acute respiratory syndrome the spike protein of sars-cov -a target for vaccine and therapeutic development a single amino acid substitution (r a) in the receptorbinding domain of sars coronavirus spike protein disrupts the antigenic structure and binding activity design and biological activities of novel inhibitory peptides for sars-cov spike protein and 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structure of nl respiratory coronavirus receptor-binding domain complexed with its human receptor the sars-cov s glycoprotein: expression and functional characterization single amino acid substitutions in the severe acute respiratory syndrome coronavirus spike glycoprotein determine viral entry and immunogenicity of a major neutralizing domain suppression of sars-cov entry by peptides corresponding to heptad regions on spike glycoprotein synthetic peptides outside the spike protein heptad repeat regions as potent inhibitors of sars-associated coronavirus key: cord- -zemns wd authors: calvo-pinilla, eva; de la poza, francisco; gubbins, simon; mertens, peter paul clement; ortego, javier; castillo-olivares, javier title: antiserum from mice vaccinated with modified vaccinia ankara virus expressing african horse sickness virus (ahsv) vp provides protection when it is administered h before, or h after challenge date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: zemns wd previous studies show that a recombinant modified vaccinia ankara (mva) virus expressing vp of ahsv serotype (mva-vp ) induced virus neutralising antibodies in horses and protected interferon alpha receptor gene knock-out mice (ifnar −/−) against challenge. follow up experiments indicated that passive transfer of antiserum, from mva-vp immune donors to recipient mice h before challenge, conferred complete clinical protection and significantly reduced viraemia. these studies have been extended to determine the protective effect of mva-vp vaccine-induced antiserum, when administered h before, or h after challenge. in addition, passive transfer of splenocytes was undertaken to assess if they confer any degree of immunity to immunologically naïve recipient mice. thus, antisera and splenocytes were collected from groups of mice that had been vaccinated with mva-vp , or wild type mva (mva-wt), for passive immunisation of recipient mice. the latter were subsequently challenged with ahsv- (together with appropriate vaccinated or unvaccinated control animals) and protection was assessed by comparing clinical signs, lethality and viraemia between treated and control groups. all antiserum recipients showed high protection against disease ( % survival rates even in mice that were immunised h after challenge) and statistically significant reduction or viraemia in comparison with the control groups. the mouse group receiving splenocytes from mva-vp vaccinates, showed only a % survival rate, with a small reduction in viraemia, compared to those mice that had received splenocytes from mva-wt vaccinates. these results confirm the primarily humoral nature of protective immunity conferred by mva-vp vaccination and show the potential of administering mva-vp specific antiserum as an emergency treatment for ahsv. african horse sickness (ahs) is an arthropod-borne viral disease of solipeds transmitted by haematophagous insects of the genus culicoides, the horse being the most severely affected species. mortality rates of ahsv outbreaks in immunologically naïve populations may exceed % (mellor and hamblin, ) . all of the different isolates, strains and serotypes of african horse sickness virus (ahsv) are classified within the species african horse sickness virus, genus orbivirus, family reoviridae. ahsv is closely related to bluetongue virus, which causes bluetongue disease in ruminants. the orbiviruses are characterised by a genome composed of ten linear segments of dsrna, with one copy of each of the ten segments packaged within each virus particle. the spherical, non-enveloped capsid of ahsv is approximately nm in diameter, is composed of three concentric protein layers (roy et al., ) . the outer-capsid layer is formed by two major structural proteins, vp and vp (encoded by genome-segments and respectively), and is primarily involved in cell attachment and cell entry. vp is the most variable antigen of ahsv and is responsible for serotype definition (burrage et al., ) . ahsv infection in horses most often results in severe clinical disease and death, although animals that do survive, exhibit a solid, life-long but serotype-specific immunity. the humoral nature of ahs immunity has been associated with virus neutralising antibodies (vnab) in both horses and mice using colostrum and (blackburn and swanepoel, ; burrage et al., ; crafford et al., ) . the main target of vnab is ahsv vp (burrage et al., ) and several studies have mapped neutralising epitopes to the amino terminal half of the vp protein (bentley et al., ; martinez-torrecuadrada and casal, ; martinez-torrecuadrada et al., ) . although less important in this respect than vp , ahsv-vp appears to be both directly involved in the formation of virus neutralising epitopes and indirectly involved by influencing the conformation of vp (martinez-torrecuadrada and casal, ; martinez-torrecuadrada et al., ) . unsurprisingly, experimental vaccines based on the outer-capsid proteins vp and/or vp , were successful (castillo-olivares et al., ; chiam et al., ; romito et al., ; roy and sutton, ; scanlen et al., ; stone-marschat et al., ) . recently, cell-mediated immune responses have been detected in horses following inoculation with live attenuated ahsv vaccines (pretorius et al., ) , or recombinant canarypox virus expressing ahsv vp and vp (el garch et al., ) . more recently, cell-mediated immune responses have also been observed in interferon alpha receptor gene knock-out mice (ifnar À/À) after vaccination with single mva recombinant viruses expressing ahsv vp or ns (de la poza et al., ) . since cellular immunity has been associated with protection against btv, it is generally assumed that the same applies to ahsv. after showing that mva expressing ahsv vp (mva-vp ) was protective against ahsv infection in mice (castillo-olivares et al., ) and horses (alberca et al., ) , the mechanisms of immunity underlying the protective effect of mva-vp vaccination were investigated. passive transfer of ahsv neutralising antiserum, from mva-vp vaccinated mice to immunologically naïve recipients, was shown to be highly protective against a lethal ahsv challenge . in this paper, follow-up studies investigating the relative contribution of humoral and cell-mediated immunity to the protection conferred by mva-vp vaccination, using passive immunisation of naïve recipient ifnar À/À mice with splenocytes or antiserum from mva-vp vaccinated mice, are described. chicken embryo fibroblast (df- ) (atcc, cat. no. crl- ) and vero cells (atcc, were grown in dulbecco's modified eagle's medium (dmem) supplemented with mm glutamine, penicillin ( units/ml), streptomycin ( lg/ml) and % foetal calf serum (fcs). ahsv- (madrid/ ) (ahsv- ) was grown in vero cells and mva viruses grown in df- cells. virus stocks were generated by infection of confluent cells using a multiplicity of infection (moi) of . . at h post-infection, or when a total cytopathic effect (cpe) was visible, the cells and supernatants were harvested and centrifuged. the virus was released from the cells by three freeze and thaw cycles and then titrated by plaque assay. the mva-vp virus used in this study has been previously described (chiam et al., ). ifn a/b ro/o ifnar À/À mice on an a background were purchased from b&k universal ltd (uk). eight-week old mice were used throughout. mice were maintained under pathogen-free conditions and allowed to acclimatise to the biosafety level (bsl ) animal facilities at the centro de investigacion en sanidad animal, inia, madrid (inia-cisa), for week before use. all experiments with live animals were performed under the guidelines of the european community ( / ) and were approved by the ethical review committee of inia-cisa (ceea - ). . . murine immunisations with mva-vp and mva-wt and preparation of donor antisera and splenocytes groups of mice were vaccinated ( pfu/mouse) with either mva-vp (group a and group b) or mva-wt (group ) and used as donors of serum and splenocytes as shown in table . a third dose was given on day to mice from group a (mouse . mouse . ). mice from group b and group were euthanised on day . mice from group a were euthanized on day . splenocytes and blood samples were collected at termination and subsequently used for passive immunisation. the blood was used to prepare donor antisera. pools of spleen cells, obtained from mice from group b (mva-vp ) and group (mva-wt), were transferred via intravenous injection (  cells/mouse) to immunologically naïve recipients groups and (table a) . transfers were performed h before infection with ahsv- ( pfu/mouse). donor antisera s a, s b and s were prepared by pooling individual serum samples from groups a, b and respectively (table b ). the donor antisera were inactivated ( min at °c) and used for intra-peritoneal injection of groups of -weekold recipient ifnar À/À mice: (a) group , received ll of undiluted s a antiserum administered h before challenge; (b) group received ll of diluted s b antiserum, administered h before challenge; (c) group received ll of s b h before challenge and (d) group received ll of s b antiserum after challenge. passively immunised mice were challenged with pfu of ahsv- administered subcutaneously. mva-wt (group ) and mva-vp (group ) vaccinated control mice were infected at the same time as the recipients. clinical signs and viraemia were evaluated in all the animals following challenge. following challenge, animals were monitored twice daily and more regularly (at least three times per day) at the onset of any signs of morbidity, including: changes in behaviour and activity, table vaccination and preparation of splenocytes and antiserum donors. day day day day a ( mice) mva-vp pfu/mouse mva-vp pfu/mouse mva-vp pfu/mouse collection of serum (s a) b ( mice) mva-vp pfu/mouse mva-vp pfu/mouse collection of serum (s b) and spleen (l ) ( mice) mva-wt pfu/mouse mva-wt pfu/mouse collection of serum (s ) and spleen (l ) changes in water or food intake, alterations in the hair coat appearance, body weight loss, presence of ocular signs (conjunctivitis, ocular discharge, swelling), changes in hydration and presence of neurological signs (i.e. paresis, paralysis, ataxia). the humane end-points for euthanasia included: persistent hunching, severe conjunctivitis, signs of dehydration, loss of more than % of body weight, presence of any neurological signs, or any other condition that prevented food or water intake. animals that displayed any of these clinical signs were humanely euthanised by cervical dislocation following anaesthesia with % isoflurane. a clinical scoring system was used (table ) whereby each animal received a clinical score value per day. the value used for analysis was the individual mean clinical score for the study period (sum of daily scores of each animal/number of days). determination of vnab titres of serum samples was carried out as described previously . briefly, twofold serial dilutions of serum samples were incubated with tcid of ahsv- for h and then overnight at °c. the following day,  vero cells were added to each well and incubated for days at °c, % co . the end-point of the assay was the highest dilution that prevented ahsv cytopathic effect in % of the wells. antibody titres were then calculated according to karber ( ) . antibody levels of vp -specific antibodies in serum were determined as previously described (calvo-pinilla et al., ). whole blood from animals was collected in edta at regular intervals after challenge. volumes of ll of blood were washed in pbs, mixed with ll of water to lyse the cells and then ll of  pbs was added to the sample. infectious virus was measured by standard plaque assay on vero cells. spleens were homogenised and then filtered through lm cell strainers. erythrocytes were lysed with nh cl and the remaining cells were re-suspended in rpmi media. ahsv-specific ifn-c producing cells were measured by elispot assay as described previously (calvo-pinilla et al., ) . splenocytes were stimulated for h with either ahsv- cell extract that had been inactivated by min exposure to ultraviolet light ( j/cm ) or with affinity purified ahsv- vp expressed from recombinant baculovirus (chiam et al., ) . pha (phytohemagglutinin, lg/ml) stimulated control cells used as positive control and uninfected cell extract stimulated cells were used as the negative control. the spot count of antigen-specific cells was calculated as the spot count difference between ahsv -stimulated cells and non-stimulated cells. unless otherwise stated, the methods used were the same for the splenocyte and serum transfer experiments. virus neutralising titre (vnt) in donor mice: titres in mva vp vaccinated and mva-wt vaccinated mice (splenocyte transfer) and in mice vaccinated twice or three times with mva-vp (serum transfer) were compared using wilcoxon rank-sum tests. elispot data for donor mice: the numbers of cells excreting ifnc were compared using a kruskal-wallis test, with mice grouped according to vaccination status (mva-vp or mva-wt) and stimulus (ahsv or vp ) (i.e. there are four groups in total). differences between groups were then explored using post hoc wilcoxon rank-sum tests. clinical scores in recipient mice: clinical scores in different groups of mice were compared using a wilcoxon rank-sum test (splenocyte transfer) or a kruskal-wallis test (serum transfer). in the latter case, differences between groups were then explored using post hoc wilcoxon rank-sum tests. viraemia in recipient mice: differences in viraemia amongst groups of mice were assessed using linear mixed models with log pfu as the response variable, days post infection (as a factor) and group as fixed effects and mouse as a random effect. model selection proceeded by stepwise deletion of non-significant terms (as judged by the akaike information criterion) starting from a model including days post infection and group as main effects. survival in recipient mice: survival in different groups of mice were compared using log-rank tests. vnt in recipient mice: titres at experiment end point (either death or days post infection) were analysed using a one-way analysis of variance, followed by post hoc tukey tests to identify differences between groups. a significance level of p = . was used in all analyses. all methods were implemented in r version . . (r core team ). it has recently been demonstrated that immunologically naïve mice can be protected against challenge with a virulent strain of ahsv- , by administering serum from mice previously vaccinated with mva-vp ). this suggests that the protection afforded by mva-vp vaccination is mediated mainly by antibodies (calvo-pinilla et al., ). however, it was shown in that study that mva-vp vaccination also induced a cd + t-cell response. these studies have been extended to determine: (a) whether adoptive transfer of splenocytes from mva-vp vaccinates could protect against ahsv- infection; and (b) whether protection by passive immunisation with antibodies from mva-vp vaccinated mice could be observed when administered h before, or h after the challenge. serum and splenocytes were obtained from mice that had been vaccinated with mva-vp or mva-wt, as indicated in section . consistent with previous findings castillo-olivares et al., ) , the mice did not show any signs of infection following mva-vp vaccination and developed an ahsv-vp -specific vnab response. titres at day (following two vaccinations) ranged between . and . (average . ) and these were subsequently boosted on day (average titre . ). ahsv-vp -specific antibodies in mva-vp vaccinates were also detected by elisa on days (mean od value . ; sd . ) and (mean od value . ; sd . ). antisera collected on days and were used for passive immunisation. adoptive transfer of splenocytes was carried out using spleens harvested from mva-vp and mva-wt vaccinates collected on day . results of ifn-c elispot assays (fig. ) confirmed that the splenocytes from mva-vp vaccinated donors (mice . - . ) were functional and secreted ifn-c, with the frequency of responder cells being significantly higher than in the mva-wt vaccinates (mice . - . ) regardless of whether they were stimulated with ahsv or vp . samples from mice . and . could not be used in this assay. once donor splenocytes were confirmed as viable and functional in the ifn-c elispot assay, they were transferred to groups of recipient mice, which were subsequently challenged with ahsv- to assess the protective capacity of the cell-mediated immunity. all mice from group (receiving splenocytes from mva-wt vaccinates) and three from group (receiving mva-vp splenocytes) succumbed to the infection (table ) , and ahsv was detected in the blood of all mice in these groups (fig. ) . the survival rates ( %) and viraemia levels of group did not differ significantly (p > . ) from those of group . however, a reduction of clinical score values in group relative to group was statistically significant (p = . ). altogether, these data indicate that cell mediated immunity does not play a critical role in the high level of protection that was afforded against the homologous serotype by mva-vp vaccination (see table ). in this study, the influence of the time of administration and dose of mva-vp -specific antiserum in protection against ahsv was evaluated. thus, two groups of mice were passively immunised, as indicated in section , and were subsequently infected with ahsv- , together with two vaccinated groups. as expected, the mice that were vaccinated with mva-wt (group ) developed clinical signs typical of ahs in the ifnar À/À mouse model (table ). these started on day post-infection and reached the humane end-point between days and post-challenge. all mice from this group were euthanised. in contrast, mice from all other groups survived the challenge and developed, at most, very mild clinical signs. survival was significantly (p < . ) lower for mice in group compared with all other groups. the clinical scores were also significantly higher (p < . ) in group compared with all of the other groups, and in group compared with groups , and . differences in viraemia levels were also significant (p < . ), being highest in group relative to all other groups, but also evident in group relative to groups and (fig. ) . thus, the levels of protection from the serum transfer experiment can be ranked in the following order: group > group > group = group > group > group . these data collectively indicate that transfer of antiserum was protective when given in a time window of plus or minus h around the time of challenge. this protection is defined by a complete prevention of lethality of ahsv infection, a significant reduction of clinical signs and a significant reduction of viraemia for every sampling point. protection was maximum when the dose was increased (group , ll/mouse) and when the administration was performed prior to virus challenge (group , serum transferred h before challenge). as expected the administration of the antiserum h after challenge (group ) was less protective than in groups , and , but even in these conditions the affected mice recovered and clinical signs had completely disappeared by day post-infection. despite the inability of mva to replicate in most mammalian cells in vitro and in vivo, even in immunocompromised individuals (gilbert, ; gomez et al., ) recombinant mva vaccines can express foreign antigens in host cells upon inoculation and induce immune responses specific for the foreign antigen. mva vector vaccines for viral infections have proven to be effective against a wide range of viral diseases (gilbert, ) , such as chikungunya (garcia-arriaza et al., ) or severe acute respiratory syndrome (chen et al., ) . humoral and cellular immune responses elicited against a foreign mva-encoded antigen are higher than those triggered with the vaccinia western reverse strain, in part due to the higher levels of expression of the foreign antigen by mva (ramirez et al., ) . moreover, mva is able to elicit strong, specific responses under conditions of pre-existing immunity to the vector (ramirez et al., ) , as seen in previous studies using mva in both prime and boost immunisations. the use of mva based vaccines, in heterologous prime-boost vaccination regimes, is most often pursued for diseases where cell-mediated immunity definitely plays an important role in protection, as in the case of plasmodium falciparum (ogwang et al., ) , hiv (gomez et al. ; cosma et al., ) , or influenza (berthoud et al., ) . however, mva based vaccines are also able to elicit strong antibody responses (draper et al., ) . mva has been used to develop a promising antibody-inducing vaccine against viral surface proteins, such as the haemagglutinin (ha) of influenza virus (kreijtz et al., ) , or the spike glycoprotein of sars (chen et al., ) . in vaccination studies for btv, mva has been very effective as a vaccine, inducing a robust vnab response against the expressed vp protein (calvo-pinilla et al., ; jabbar et al., ) . similarly, mva expressing ahsv-vp has been shown to elicit high levels of neutralizing antibodies (castillo-olivares et al., ; chiam et al., ) . a previous passive immunisation study showed that mva-vp vaccination induces a highly protective humoral immune response against ahsv . in that study, ahsv-specific interferon-c secreting cd + t cells were also induced by vaccination with mva-vp , but their role in protection could not be evaluated at that time. previously, pretorius et al. ( ) demonstrated an increase of virus specific cd + t-cells in horses, in response to vaccination with live attenuated ahsv. vaccination studies in mice using recombinant mva expressing ahsv- ns , administered in combination with mva-vp , also showed specific t-cell responses and a reduction in viraemia following heterologous challenge (de la poza et al., ) . in addition, a recombinant canarypox expressing ahsv-vp and vp was able to induce vp specific cd + t-cell responses, alongside vnab. this suggests cellmediated immunity could play a significant role in the overall level of protection against ahsv (el garch et al., ) . however, a clear demonstration of the protective effect of this effector mechanism of immunity, through adoptive transfer of cd + t-cells has not yet been reported. in the present study, adoptive transfer of splenocytes from mva-vp vaccinated mice, significantly reduced the clinical score of ahsv challenged recipient mice, and resulted in a % survival, although changes in the survival rate and the reduction in viraemia were not considered to be mathematically significant. this suggests that the role of cell-mediated immune responses is less important than the antibody responses in the protection generated by mva-vp vaccination. in addition, it can be argued that the marginal protection against ahsv shown by the recipients of splenocytes could be due to ahsv-vp -specific b cells contained in the transferred cells. additional adoptive transfer experiments using cell-sorted cd + lymphocytes from donors before challenge could help to elucidate the precise contribution of cell-mediated immunity following mva-vp vaccination. in studies with the related orbivirus btv, adoptive transfer of cd + cytotoxic cells from a btv infected sheep, partially protected recipient sheep against infection with either homologous or heterologous btv serotypes (jeggo et al., a) . in contrast, when passive transfer of antibodies from infected animals was performed, the protection was complete against virulent virus (jeggo et al., b) . in the case of ahsv, the humoral nature of protection against the virus has been demonstrated by the passive transfer of a monoclonal neutralising antibody against vp protein in the neonatal mouse model (burrage et al., ) . passive transfer of neutralising antibodies has also been described from ahsv vaccinated mares to foals via colostrum (blackburn and swanepoel, ; crafford et al., ) . in the present study, consistent with previous observations, the highly protective effect of antiserum derived from mva-vp vaccinated mice was also demonstrated even when transferred h post-infection. when the antiserum dose was increased (group ), the protective effect was very close to that shown by the actively immunized mva-vp vaccinated mice. the limited clinical signs that were still evident in mice infected h before transfer, eventually disappeared and the animals recovered completely (group ). this suggests that passive immunisation with mva-vp antiserum could be used for therapeutic applications, in particular for emergency treatment of ahs in horses during the initial stages of an outbreak, before containment measures or widespread vaccination campaigns are initiated. in this and previous studies it has been shown that mva-vp vaccination induces high vnab responses both in mice and horses and is highly protective against ahsv in both experimental systems. it can be concluded that protection is mainly associated with antibodies, since passive transfer of immune serum from mva-vp vaccinated mice is sufficient to provide clinical protection. vaccination of horses with a recombinant modified vaccinia ankara virus (mva) expressing african horse sickness (ahs) virus major capsid protein vp provides complete clinical protection against challenge identification of antigenic regions on vp of african horse sickness virus serotype by using phage-displayed epitope libraries potent cd + t-cell immunogenicity in humans of a novel heterosubtypic influenza a vaccine, mva-np+m observations on antibody levels associated with active and passive immunity to african horse sickness neutralizing epitopes of african horse sickness virus serotype are located on vp vaccination of mice with a modified vaccinia ankara (mva) virus expressing the african horse sickness virus (ahsv) capsid protein vp induces virus neutralising antibodies that confer protection against ahsv upon passive immunisation heterologous prime boost vaccination with dna and recombinant modified vaccinia virus ankara protects ifnar(À/À) mice against lethal bluetongue infection a modified vaccinia ankara virus (mva) vaccine expressing african horse sickness virus (ahsv) vp protects against ahsv challenge in an ifnar À/À mouse model therapeutic vaccination with mva-hiv- nef elicits nef-specific t-helper cell responses in chronically hiv- infected individuals passive transfer and rate of decay of maternal antibody against african horse sickness virus in south african thoroughbred foals response of memory cd + t cells to severe acute respiratory syndrome (sars) coronavirus in recovered sars patients and healthy individuals induction of antibody responses to african horse sickness virus (ahsv) in ponies after vaccination with recombinant modified vaccinia ankara (mva) ns is a key protein in the vaccine composition to protect ifnar(-/-) mice against infection with multiple serotypes of african horse sickness virus utilizing poxviral vectored vaccines for antibody induction-progress and prospects an african horse sickness virus serotype recombinant canarypox virus vaccine elicits specific cellmediated immune responses in horses a novel poxvirus-based vaccine, mva-chikv, is highly immunogenic and protects mice against chikungunya infection clinical development of modified vaccinia virus ankara vaccines the poxvirus vectors mva and nyvac as gene delivery systems for vaccination against infectious diseases and cancer protection of ifnar (À/À) mice against bluetongue virus serotype , by heterologous (dna/rmva) and homologous (rmva/rmva) vaccination, expressing outer-capsid protein vp a study of the role of cell-mediated immunity in bluetongue virus infection in sheep, using cellular adoptive transfer techniques role of neutralising antibody in passive immunity to bluetongue infection % end-point calculation mva-based h n vaccine affords cross-clade protection in mice against influenza a/h n viruses at low doses and after single immunization identification of a linear neutralization domain in the protein vp of african horse sickness virus full protection against african horse sickness (ahs) in horses induced by baculovirus-derived ahs virus serotype vp , vp and vp definition of neutralizing sites on african horse sickness virus serotype vp at the level of peptides african horse sickness safety and immunogenicity of heterologous prime-boost immunisation with plasmodium falciparum malaria candidate vaccines, chad me-trap and mva me-trap virus-specific cd (+) t-cells detected in pbmc from horses vaccinated against african horse sickness virus attenuated modified vaccinia virus ankara can be used as an immunizing agent under conditions of preexisting immunity to the vector immune responses in a horse inoculated with the vp gene of african horse sickness virus african horse sickness virus structure new generation of african horse sickness virus vaccines based on structural and molecular studies of the virus particles the protective efficacy of a recombinant vp -based african horse sickness subunit vaccine candidate is determined by adjuvant immunization with vp is sufficient for protection against lethal challenge with african horse sickness virus type this work was funded by department for food, environment and rural affairs, uk (defra project number: se ). javier ortego is recipient supported by grant agl- - from the spanish ministerio de economia y competitividad. we would like to thank prof malcolm mccrae for critically reading the manuscript. key: cord- - hcb xc authors: qi, yan-fei; zhang, hong; wang, juan; jiang, yanfang; li, jinhua; yuan, ye; zhang, shiyao; xu, kun; li, yangguang; li, juan; niu, junqi; wang, enbo title: in vitro anti-hepatitis b and sars virus activities of a titanium-substituted-heteropolytungstate date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: hcb xc a structural determined heteropolytungstate, [k( )(h( )o)( )cl][k( )(h( )o)( )pti( )w( )o( )]·nh( )oh , has been synthesized and evaluated for in vitro antiviral activities against hepatitis b (hbv) and sars virus. the identity and high purity of compound were confirmed by elemental analysis, nmr, ir analysis and single-crystal x-ray diffraction. the compound , evaluated in hepg . . cells expressing permanently hbv, significantly reduced the levels of hbv antigens and hbv dna in a dose-dependent and time-dependent manner. ec( ) values were determined to be μm for hbeag, μm for hbsag and . μm for supernatant hbv dna, as compared to , , μm, respectively, for the commercially-available hepatitis b drug adefovir dipivoxil (adv). intracellular cccdna, pgrna and hbcag were also found to be decreased by compound in a concentration-dependent manner. cytotoxicity results showed that compound has low toxicity in hepg cells with cc( ) value of . μm. the results indicate that compound can efficiently inhibit hbv replication in hepg . . cells line in vitro. additionally, compound also shows high anti-sars activity at an ec( ) of . μm and toxicity with a cc( ) of . μm against mdck cells. hepatitis b virus (hbv) infections continue to be a major public health problem worldwide (barraud et al., ) . more than million people worldwide are currently infected with hepatitis b virus. approximately % of hbv patients will develop chronic hepatitis, and are at significant risk of developing cirrhosis or liver hepatocarcinoma. hbv is the prototype of hepadnaviridae, a family of small enveloped hepatotropic dna viruses that can infect the liver of human (marion and robinson, ) . chronic hepatitis b patients are commonly treated with either interferon alpha (inf-a), or the nucleoside analog lamivudine ( tc), adefovir, entecavir or telbivudine which are the synthetic reverse transcrip-tase inhibitors (de clercq, ; delmas et al., ; buster and janssen, ) . however, none of these therapies are completely safe and effective. although direct antiviral therapy with amivudine or adefovir could efficiently control chronic active hepatitis b, drug resistance or renal toxicity could develop progressively several months after the initiation of therapy. it is thus still urgently required to identify effective anti-hbv agents. polyoxometalates (poms) are inorganic cluster-like complexes and constituted from oxide anion and transition metal cations. these complexes have shown potential applications in multitudinal fields such as catalysis, medicine and functional materials müller, , ) . especially, the medicinal properties of poms have been a subject of interest (witvrouw et al., ; judd et al., ; shigeta et al., ; yamase, ) . these compounds have low toxicity for cultured cells, and relatively less expensive than the ''chemical'' antiviral drugs. recently, poms have been reported to inhibit the replication of rna viruses and dna viruses in vitro and in vivo, such as the human immunodeficiency virus, severe acute respiratory syndrome (sars) virus, influenza virus and herpes simplex virus (rhule et al., ; dan et al., ) . the activity of poms against hepatitis b virus was also suggested by zoulim ( ) . the mechanism of action of poms remains to be fully elucidated, but may occur at any of the life cycle stages, including viral adsorption, penetration, or reverse-transcription (dan et al., ; shigeta et al., (domaille and knoth, ; ozeki and yamase, ) shows high antiviral activity. the interesting biological results of [pw ti o ] À prompted us to explore the antiviral activity of compounds containing [pw ti o ] À anions as potential anti-hbv agents. it was also reported that the heteropolyoxotungstates [pw ti o ] À have broad-spectrum anti-rna virus activity (dan and yamase, ) . therefore, it is necessary to further explore the antiviral activity of compounds containing [pw ti o ] À . severe acute respiratory syndrome (sars), a disease seriously threatening human health caused by the single-stranded rna coronavirus, spread in countries in early , presenting a worldwide public health concern drosten et al., ; ksiazek et al., ) . the research attention in exploitation of anti-sars treatments has been mainly focused on vaccines, antiviral drugs, and their integration of traditional chinese medicine and western therapy. however, no effective therapeutic drug is available to date (stadler et al., ) . as a part of our ongoing antiviral drug discovery program, a number of pom analogs have been synthesized and evaluated for their potential antiviral activity (li et al., a,b the results indicate that compound exhibits strong antiviral activities against the hbv and sars viruses with low cytotoxicity, indicating that it is a potential medicinal candidate against hbv and sars viruses. all the chemicals were of analytic grade and used without further purification. compound , was freshly prepared and characterized. w and ti in were determined by a leaman inductively coupled plasma (icp) spectrometer. infrared spectrum was recorded in the range - cm À on an alpha centaurt ft/ir spectrophotometer using kbr pellets. the w nmr spectrum was obtained on a bruker am- spectrometer operated at mhz with d o as the solvent. quantitative rt-pcr was performed with abi sequence detection system (roche, germany). (domaille et al., ) was added into ml distilled water with stirring. to this solution, . g ( . mmol) nh ohÁhcl and . g ( . mmol) lacl Á h o was added in sequence. the solution was heated to °c for more than h in a water bath, then filtered and kept slow evaporation in an undisturbed place at room temperature. colorless crystals of compound were isolated in week with the yield of % based on k pti w the measurement for compound was collected on a rigaku r-axis rapid ip diffractometer with mo-ka monochromated radiation (k = . Å a ) at k. empirical absorption correction was applied. the structure was solved by the direct method and refined by the full-matrix least-squares on f using the shelxl- software (sheldrick, ) . all of the non-hydrogen atoms except the disordered atoms o( ), ow( ), ow( ) and cl( ) were refined anisotropically. all the crystallographic parameters are tabulated in table . images were created with the diamond program. hepg . . cells (provided by the department of infectious diseases of the st hospital, jilin university, pr china) were cultured in dulbecco's modified eagle's medium (dmem; gibco) containing a % fetal bovine serum (fbs; gibco), u/ml penicillin, u/ml streptomycin, and lg/ml g (growth medium). during the experiments, the cells were grown in the media as described above without g . the cell lines were incubated at °c in % carbon dioxide atmosphere. prior to exposures to drugs, the cell viability was verified to be > % according to the standard trypan blue exclusion test. hepg (provided by the department of infectious diseases of the st hospital, jilin university, pr china) were cultured in dulbecco's modified eagle's medium (dmem; gibco) containing a % fetal bovine serum (fbs; gibco), u/ml penicillin, and u/ml streptomycin. the cell lines were incubated at °c in % carbon dioxide atmosphere. sars virus (provided by academy of military medical sciences, beijing, pr china) was propagated in african green monkey kidney cells (vero-e ). vero cells were propagated in dmem supplemented with % fbs, mm l-glutamine, u/ml penicillin, lg/ml streptomycin and bicabouate. the cell lines were incubated at °c in % carbon dioxide atmosphere. drugs were sterilized by filtration prior to use. each agent was dissolved in dmem to generate the appropriate doses for experimentation. non-treated cells (dmem alone) were used as negative controls. . . anti-hbv activity of compound the cytotoxicity of compound was determined using -( , -dimethylthiazol- -yl)- , -diphenyl tetrazolium bromide table crystal data and structure refinements for . formula h clk no pti w fw . (mtt) assay as previously described (kodama et al., ) . the optical absorbance was read on a plate reader (bio-rad co.) at a wavelength of nm. briefly, hepg cells were plated on -well plates at a density of .  cells/ml. after a h in period of incubation, the dilutions of , and adv at different doses were added. hepg cells in the negative control group were treated with the same volume of medium. after a period of incubation, mtt solution ( . ml, mg/ml in . m pbs) was added to each well. the cells were incubated for another h at °c, and then dmso ( . ml) was added. the cytotoxicity was measured by the reduction of mtt observed in mitochondria at , , and h after the initial treatment. the cc was defined as the concentration of drug that achieved % cytotoxicity against cultured cells, as calculated by the bliss method (han et al., ) . supernatants of cells treated with compound or adv and nontreated cells were collected. the semi-quantitative detection of hbsag and hbeag were estimated using elisa assay kits (shanghai kehua co., ltd., china). the supernatants of treated and non-treated cells were collected. the hbv dna from the medium was extracted using hbv real quant pcr kit (qiagen kits, china). the abi sequence detection system (applied biosystems) was used to quantify the purified dna. pcr was performed under the following conditions: °c for min and °c for min, followed by amplification cycles at °c for s and °c for s. hepg . . cells were harvested by trypsin digestion and washed three times with phosphate buffered saline (pbs, ph . ). cells were counted and used to evaluate the presence of hbv replicative intermediates:  cells for cccdna,  cells for pgrna. total rna was isolated from cells using the trizol reagent (invitrogen). for rt-pcr analysis, rna was reverse-transcribed at °c for min using a commercially available cdna synthesis kit (invitrogen). pcr was performed with gene specific primers for hbv and b-actin: hbv nt - (forward), -ctcaatctcgggaatctcaatgt- ; hbv nt - (reverse), -tggataaaacctagcaggcataat- ; b-actin nt - (forward), -acggccaggtcatcaccat- , b-actin nt - (reverse), -aggctggaagagtgcctcag- . qpcr was performed on the abi sequence detection system using the sybr green kit (invitrogen). a standard curve was constructed by the simultaneous amplification of serial dilutions of the expression plasmid encoding hbv. target cdnas were normalized to the endogenous rna levels of bactin. gene expression was determined using the relative quantifi- where ct was the fractional cycle number that reached a fixed threshold, ctest was the test of hbv in compound and adv exposed groups, and ccontrol was the reference control (rna from non-treated cells). the fold increase was calculated using Àddct (livak and schmittgen, ) . since hbv cccdna is structurally similar to plasmid, intracellular cccdna was extracted from cells by an alkali lysis procedure with a qiagen plasmid mini kit (ca) in order to remove most of cellular chromosomal dna and non-supercoiled relaxed circular (rcdna). purified cccdna was dissolved in ll te buffer ( mm, ph . ), and ll of the product was further treated with plasmid-safe™ atp-dependent dnase (psad; epicentre technologies, wi) to remove any remaining single-stranded virus dna, rcdna or cellular chromosomal dna. intracellular cccdna was quantified by selective fluorescent pcr with taqman Ò mgb probe capable of amplifying and checking cccdna more efficiently than rcdna. the hbv primers were: nt - (forward), -ttctcatctgccggaccg- ; nt - (reverse), -cacagcttggaggcttgaac- . the taqman mg b probes were: nt - (reverse), fam-cctaat catctc ttgttcat-mgb . qpcr was performed to detect intracellular cccdna by using the realquant pcr kit (invitrogen). cell lysates were prepared by using ripa (radio immunoprecipitation assay) lysis buffer supplemented with % (v/v) phenylmethanesulfonylfluoride (pmsf; biyuntian co., ltd., china). proteins were separated by electrophoresis on an % sodium dodecyl sulfate-polyacrylamide gel, transferred onto immobilon-p membranes (millipore), and analyzed by standard western blot technique using anti-hbcag antibodies (santa cruz biotechnologies). b-actin (santa cruz biotechnologies) was used as the normalization control. the anti-sars virus activity was estimated by mtt assay (sigma). the optical absorbance was read on a plate reader (bio-rad co.) at a wavelength of nm for mtt. vero-e cell cultures (  cells/ml) were prepared in a -well plate. after a h period of incubation, sars virus stock ( . ml per well) and different doses of compound ( . ml per well) were added. in the control group, . ml medium was added. the plates were incubated at °c in a humidified % co atmosphere for days until maximum cytopathic effects were observed in the untreated, negative control cultures. the cytopathic effects were quantitated by the mtt assay. briefly, ll of mtt solution prepared in dmem was added to each well and plates were incubated at °c for h. the mtt solution was removed without disturbing the cells and ll of dmso was added to each well to dissolve formazan crystals. after gently shaking the plates for min, the absorbance from each well was measured at nm. the percentages of protection were calculated as [(a À b)/(c À b)  ], where a, b, and c stand for the absorbances of wells containing compound and virus (a), virus (b) and cell (c) only, respectively. data were expressed as mean ± sd (standard deviation). all experiments were performed in triplicate. statistical significance was evaluated by the one-way analysis of variance (anova) and student's t-test. multiple comparisons were statistically analyzed using sas software version . (significance was established at p < . ). x-ray crystallography shows that compound consists of a -d polyoxoanion framework [k (h o) pti w o ] À , one-dimensional ( -d) chainlike [k (h o) cl] + cations and the isolated hydroxylamine nh oh. in compound , the polyoxoanion [pti w o ] À exhibits a well-known keggin structure (fig. a) . the central p atom is surrounded by a cube of eight oxygen atoms with each oxygen site half-occupied. the p-o distance is . ( ) Å. in the two crystallographically unique surrounding metal sites of the polyoxoanion, there exists a site occupancy disorder in the w center, that is, both ti and w atom share the same site with % occupancy, respectively, forming the polyoxoanion [pti w o ] À . (fig. a) . in asymmetric unit of compound , the k center (see fig. b ) is octa-coordinated with four l -o atoms of one polyoxoanion (o and o ), two terminal oxygen atoms of other two polyoxoanions (o ), and two coordination water molecules (ow and ow a). the k-o distances range from . ( ) to . ( ) Å. thus, the k centers can be regarded as a joint to connect all the polyoxoanions together. the k center (fig. c ) also exhibits the eight coordination environment with two terminal o atoms (o ) of two polyoxoanions, four coordination water molecules (ow and ow ) and two cl anions. the k-o distances are in the range of . ( )- . ( ) Å, while the k-cl distance is . ( ) Å. it is interesting that all coordination atoms are shared by two k centers. based on this connection mode, the k centers form a -d chain via these double-bridging atoms ( fig. a) . in the asymmetric unit of , ow , ow and cl possess the %, % and . % occupancies, respectively, thus, the -d chainlike cations can be described as [k the growth of the hepg cells in the presence of various concentrations of compound was examined. the results of the cytotoxicity experiments are given in fig. . the cytotoxicity of fig. . (a) the structure of polyoxoanion of compound showing the coordination sites with k centers. coordination environments of (b) k center and (c) k center. the levels of hbsag, hbeag and extracellular hbv dna in the medium were measured at different time points in the control group, adv group, and compound group, respectively, as shown in fig. . after treatment, the levels of hbeag, hbsag and extracellular hbv dna in the drug-treated groups were decreased significantly compared to that in the control group in a concentrationdependent manner (p < . ). the levels of hbeag, hbsag and extracellular hbv dna decreased with time in hepg . . cells, indicating that the anti-hbv activity of compound is time-dependent (p < . ). as shown in fig. a , the inhibition ratio of hbeag in compound -treated cultures reached the peak value . % at . lm at day and still kept about . % at day . at the same time, the inhibition ratio of hbsag (shown in fig. b ) reached the peak value of . % at . lm at day and was still about . % at day . the inhibition ratios of hbeag and hbsag in the adv group were . % and . % at lm, respectively. the inhibitory values of hbeag and hbsag in adv group decreased at day . interestingly, the inhibition rates of compound on extracellular hbv dna at day ( days after the end of exposure to the drug) were lower than those observed on day . however, they were higher than those on day . at the same time, the inhibition values of adv for extracellular hbv dna declined and they were lower than the values at day , as shown in fig. c . these result indicate that compound may have a persistent effect on suppressing hbv. the % effective concentration (ec ) values of hbeag, hbsag and extracellular hbv dna for compound and adv are shown in table . the therapeutic index (ti) of compound was higher than that of adv. to characterize the anti-hbv mechanism of compound , the amounts of intracellular viral pgrna and dna were measured in the control group, adv group, and compound group at different concentrations, respectively, at day , as shown in fig. . the results revealed that the levels of intracellular hbv pgrna and cccdna were decreased with elevation of the compound concentration, as compared to those detected from the control group (p < . ). the maximum inhibition ratios of pgrna and dna were . % and . % at lm in the compound group, which are higher than the ratios in the positive control group (avd group, p < . ). these results suggested that compound apparently impacting viral cccdna replication and viral pgrna transcription in hepg . . cells, and that the anti-hbv mechanism of compound seems to be similar to that of adv. the inhibitory effects of adv and compound on hbs(c)ag protein levels were determined with western blot. hepg . . cells were treated with compound and adv, respectively. the cell extracts were prepared and analyzed at day . as shown in fig. , the levels of intracellular protein levels of hbcag were reduced with elevation of the compound concentration in comparison with that in the control group (p < . ), indicating that the anti-hbv hbcag activity of compound is concentration-dependent. in addition, the inhibitory effect of compound was higher than that of adv at the same dose. aiming at evaluating the antiviral activity against sars virus of compound , the cytotoxicity of compound in vero-e cells was first measured. the results showed that the maximal noncytotoxic concentration (cc ) and the % cytotoxic concentration (cc ) of compound were . lm and . lm, respectively. according fig. . cytotoxicity of compound and adv in hepg cells. a p < . for the drug groups vs. control group. for compound : b p < . for the drug group vs. drug at lm group. c p < . for the drug groups vs. drug at lm group. d p < . for the drug group vs. drug at lm group. e p < . vs. drug at lm group. for adv: b p < . for the drug group vs. drug at lm group. c p < . for the drug groups vs. drug at lm group. d p < . for the drug group vs. drug at lm group. e p < . vs. drug at lm group. to the cytotoxic results, compound was diluted at five non-toxic concentrations ( . , . , . , . , and . lm) and the anti-sars virus activity was checked with mtt assay. as shown in fig. , the inhibition ratio of compound increased with concentration, indicating that the anti-sars virus activity of compound is concentration-dependent. it is noteworthy that the sars virus was completely inhibited at . lm. the % effective concentration (ec ) and the % effective concentration (ec ) were . and . lm, respectively. ti of compound was . . currently, nucleoside analogs play an important role in the therapy of hbv, however, some disadvantages of these agents include side effects, drug resistance and costs which limit their clinical use in hbv-infected patients. polyoxometalates, as nonnucleoside analogs, have been proven to exhibit broad inhibitory activity against human immunodeficiency viruses (hiv- and hiv- ), herpes simplex virus, and influenza virus. especially, the titanium-containing polyoxotungstates have shown antiviral activity against a variety of enveloped rna or dna viruses. herein, we synthesized a titanium-substituted-heteropolytungstate [k (h o) cl][k (h o) pti w o ]Ánh oh, and examined the anti-hbv activities in vitro. hepg . . cells as a useful ''in vitro'' model are widely used for the evaluation of novel anti-hbv drugs and chosen in our experiments. the cell line contains multiple copies of hbv genome that can stably integrate into the host cell genome. the results indicate that compound inhibited hbv dna, hbsag and hbeag antigens in the culture medium in a concentrationand-time dependent manner. a lower concentration of the compound was required to effectively inhibit secreted hbv dna than to inhibit secreted antigens. it is possible that the compound acts on the exported virions outer protein coats. although the mechanisms mediating the antiviral effects by compound remain un- fig. . the inhibitor effect of control group, adv groups, and compound groups on secretion of hbeag (a), hbsag (b) and extracellular viral dna (c) from the medium of hepg . . cells. a p < . vs. the corresponding negative control. b p < . vs. the corresponding days outcome of the same dose. c p < . vs. the corresponding days outcome of the same dose. table anti-hbv activity, cytotoxicity and the selective index (ti) of compound and adv in vitro. hbsag hbv dna clear, we have deduced that compound might block the secretion of hbv containing nucleocapsids or destabilize hbv dna-containing nucleocapsids. interestingly, we observed that the in vitro anti-hbv properties of compound against rebound of serum hbv dna, hbsag and hbeag were more robust than those of positive group, adv, as indicated by evaluation of treated cells days after termination of treatment. this result shows that compound has a sustained anti-hbv activity. the levels of intracellular hbv dna, rna and hbcag protein were also reduced by compound in a concentration-dependent manner. the adv drug competitively inhibits hbv polymerase, which is structurally similar to datp. it is known to reduce the levels of hbv dna and hbsag both in vitro and in vivo by a phosphorylation event that facilitates its physical incorporation into nascent fig. . the inhibition effect on the levels of intracellular pgrna (a) and cccdna (b) in control group, adv groups, and compound groups at different concentrations. a p < . vs. the corresponding negative control. b p < . vs. the corresponding adv groups at the same concentration. viral dna by the activity of hbv polymerase during replication. in our study, we observed an apparent reduction in the levels of intracellular hbv-specific rna and dna following compound treatment. these data also reveal that the anti-hbv mechanism of compound may be similar to that of the nucleoside analog. since the hbv pgrnas were transcribed from cccdna, we presume that hbv-specific transcripts may be affected by compound . it is also important to consider that the hbv pgrna was inhibited in drugtreated cells (he et al., ) . hbcag, hbsag and hbv polymerase are translated from pregenome mrna, and the minus strand hbv dna are transcribed from the pregenome mrna template. in this study, it was found that compound could inhibit the levels of hbcag, hbsag and hbv dna protein expressed in a concentration-dependent manner in vitro. these results suggested that compound apparently interfered with viral pgrna transcription in hepg . . cells. moreover, compound shows broad-spectrum antiviral activity. it can efficiently inhibit sars virus in vero-e cells in vitro with low toxicity against mdck cells. in summary, a heteropolytungstate has been prepared and characterized. the in vitro experimental results show that compound has potential anti-hbv and anti-sars virus activities. further experiments are underway now, which include evaluation of in vivo activities and determination of the mechanism of anti-hbv activity of compound . enhanced duck hepatitis b virus gene expression following aflatoxin b exposure antiviral treatment for chronic hepatitis b virus infection-immune modulation or viral suppression? the memory effect of heteropolyoxotungstate (pm- ) pretreatment on infection by herpes simplex virus at the penetration stage prevention of the interaction between hvem, herpes virus entry mediator, and gd, hsv envelope protein, by a keggin polyoxotungstate perspectives for the treatment of hepatitis b virus infections inhibitory effect of adefovir on viral dna synthesis and covalently closed circular dna formation in duck hepatitis b virus-infected hepatocytes in vivo and in vitro ti w po À and preparation, properties, and structure determination by tungsten- nmr identification of a novel coronavirus in patients with severe acute respiratory syndrome aetiology: koch's postulates fulfilled for sars virus in vivo and in vitro antihepatitis b virus activity of total phenolics from oenanthe javanica 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polyoxotungstates sars -beginning to understand a new virus potent anti-hiv (type and type ) activity of polyoxometalates: structure-activity relationship and mechanism of action anti-tumor, -viral, and -bacterial activities of polyoxometalates for realizing an inorganic drug therapy of chronic hepatitis b virus infection: inhibition of the viral polymerase and other antiviral strategies the inhibition of to sars virus in different concentrations supplementary data associated with this article can be found, in the online version, at doi: . /j.antiviral. . . . key: cord- -o c x authors: tai, wanbo; zhang, xiujuan; he, yuxian; jiang, shibo; du, lanying title: identification of sars-cov rbd-targeting monoclonal antibodies with cross-reactive or neutralizing activity against sars-cov- date: - - journal: antiviral res doi: . /j.antiviral. . sha: doc_id: cord_uid: o c x sars-cov- -caused covid- cases are growing globally, calling for developing effective therapeutics to control the current pandemic. sars-cov- and sars-cov recognize angiotensin-converting enzyme (ace ) receptor via the receptor-binding domain (rbd). here, we identified six sars-cov rbd-specific neutralizing monoclonal antibodies (nabs) that cross-reacted with sars-cov- rbd, two of which, f and b , neutralized sars-cov- infection. f recognized conserved epitopes on sars-cov and sars-cov- rbds, whereas b recognized epitopes on sars-cov rbd not fully conserved in sars-cov- rbd. the f -recognizing epitopes on rbd did not overlap with the ace -binding sites, whereas those recognized by b were close to the ace -binding sites, explaining why b could, but f could not, block sars-cov or sars-cov- rbd binding to ace receptor. our study provides an alternative approach to prevent sars-cov- infection using anti-sars-cov nabs. which, f and b , neutralized sars-cov- infection. f recognized conserved epitopes on sars-cov and sars-cov- rbds, whereas b recognized epitopes on sars-cov rbd not fully conserved in sars-cov- rbd. the f -recognizing epitopes on rbd did not overlap with the ace -binding sites, whereas those recognized by b were close to the ace -binding sites, explaining why b could, but f could not, block sars-cov or sars-cov- rbd binding to ace receptor. our study provides an alternative approach to prevent sars-cov- infection using anti-sars-cov nabs. controls. briefly, elisa plates were precoated with respective rbd proteins ( μg/ml) overnight at ℃, which were blocked with % fat-free milk in pbst for h at ℃. sars-cov rbd-specific mouse mabs ( and μg/ml) were added to the plates and incubated for h at ℃. after washes, the plates were further incubated with horseradish peroxidase (hrp)-conjugated anti-mouse igg antibody (fab specific, : , , thermo fisher scientific) for h at ℃. substrate , ', , '-tetramethylbenzidine (tmb) (sigma, st. louis, mo) was the added to the plates, and the reactions were stopped by addition of h so ( n). the absorbance at nm (a ) was measured using an elisa plate reader (tecan, san jose, ca). mapping of epitopes of the selected sars-cov rbd-specific mabs on sars-cov rbd was performed using a protocol similar to that described above, except for coating of the elisa plates with respective sars-cov rbd wt and mutant proteins ( μg/ml), followed by the addition of mabs at serial dilutions for detection. binding affinity at μg/ml ( fig. a) . in contrast, all these mabs could bind sars-cov rbd protein, and most had strong binding affinity at and μg/ml (fig. b) . these data suggest that sars-cov rbd-specific mabs could cross-react with sars-cov- rbd. pseudovirus infection with about % neutralization at μg/ml (fig. c) . however, all these mabs neutralized sars-cov pseudovirus infection, most of which had > % neutralizing ability at μg/ml, and a few reached > % neutralization at μg/ml (fig. d) . notably, several mabs, such as s , g , and f , had potent neutralizing activity against sars-cov pseudovirus, but failed to neutralize sars-cov- infection, even at μg/ml (fig. c-d) , suggesting that these mabs may recognize epitopes on the rbd of sars-cov different from those of sars-cov- rbd. therefore, we identified two sars-cov rbd-targeting nabs with proven cross-neutralizing ability against sars-cov- s protein-mediated viral entry. we further detected the epitopes on sars-cov rbd potentially recognized by the two cross-neutralizing mabs, b and f , and included two non-cross-neutralizing mabs, b and g , as controls. we have previously shown that b may recognize epitopes at residues r and d of sars-cov rbd (he et al., a) . here, we constructed a series of sars-cov rbd mutant proteins based on the interaction between the rbd and viral receptor (li et al., ) , and performed an elisa to test binding ability of the above mabs to these mutant proteins. compared with the binding to sars-cov rbd wild-type (wt) protein, neither b nor f bound to the rbd containing d and v mutations. moreover, b did not bind to the rbd containing d and f mutations, and b did not bind to the rbd containing i and a mutations or the rbd containing k mutation (fig. ) . in addition, b showed reduced binding to the rbds containing v /a , a /k , or y mutations (fig. ) . this line of evidence suggests that the above residues were the epitopes recognized by respective mabs, among which residues d and v in sars-cov rbd were conserved neutralizing epitopes corresponding to residues d and v in sars-cov- rbd, and residues i , a , and k in sars-cov rbd were neutralizing epitopes not fully conserved in sars-cov- rbd (fig. a) . receptor, the results revealed that b and f could not block such binding (fig. b, c). this might be due to that most or all epitopes recognized by b or f did not overlap with the ace binding sites on sars-cov or sars-cov- rbd (li et al., ; yan et al., ), while most epitopes recognized by b were very close to the ace binding sites (fig. a ). in addition, control mab g only blocked sars-cov rbd, but not sars-cov- rbd, binding to the ace receptor (fig. b, c) , partially explaining why this mab did not neutralize sars-cov- infection. moreover, the two cross-neutralizing mabs, f and b , respectively recognized two conserved, as well as several non-conserved, neutralizing epitopes on the rbds of sars-cov and sars-cov- . while f could not block the binding between rbd and ace receptor, b did block this binding, indicating that they recognized epitopes different from, or close to, the receptor binding sites on the rbds. notably, b had a relatively higher neutralizing activity against sars-cov- infection than that against sars-cov infection, whereas its ability to inhibit the sars-cov- rbd-ace binding was relatively lower than that to inhibit the sars-cov rbd-ace binding, partially because that the neutralizing activity of nabs could not always be positively correlated with the inhibition of their binding to the receptor. other reasons might be due to that the binding between sars-cov- rbd and ace receptor was much stronger than that between sars-cov rbd and ace (tai et al., ) , potentially resulting in the reduced inhibition. sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor don't rush to deploy covid- vaccines and drugs without sufficient safety guarantees an emerging coronavirus causing pneumonia outbreak in wuhan, china: calling for developing therapeutic and prophylactic strategies neutralizing antibodies against sars-cov- and other human coronaviruses a distinct name is needed for the new coronavirus structure of sars coronavirus spike receptor-binding domain complexed with receptor interaction between heptad repeat and regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors characterization of the receptor-binding domain (rbd) of novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine recombinant receptor-binding domains of multiple middle east respiratory syndrome coronaviruses (mers-covs) induce cross-neutralizing antibodies against divergent human and camel mers-covs and antibody escape mutants potent binding of novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody. emerg microbes infect coronavirus disease (covid- ) situation report- structural basis for the recognition of sars-cov- by full-length human ace epidemiology and cause of severe acute respiratory syndrome (sars people's republic of china a pneumonia outbreak associated with a new coronavirus of probable bat origin. nature tan, w., . a novel coronavirus from patients with pneumonia in china ). (b) inhibition of sars-cov rbd-specific mabs for the binding of sars-cov or sars-cov- rbd to ace receptor by flow cytometry analysis. percent (%) inhibition was calculated based on the relative fluorescence intensity with or without respective mabs sars-cov or sars-cov- rbd ( μg/ml) was used for the binding to hace / t the experiments were repeated twice and obtained similar results. (c) representative flow cytometry images of sars-cov rbd-specific mabs ( μg/ml) in inhibition of the binding between sars-cov or sars-cov- rbd and ace receptor μg/ml) to hace / t cells is shown in red line, and the blockage of this binding by mabs ( g , b , f , and b ) is shown in blue line. higg-fc protein the authors declare no competing interests. du, l., he, y., zhou, y., liu key: cord- -et sli authors: du, ruikun; wang, lili; xu, hao; wang, zhiying; zhang, tao; wang, manli; ning, yunjia; deng, fei; hu, zhihong; wang, hualin; li, yi title: a novel glycoprotein d-specific monoclonal antibody neutralizes herpes simplex virus date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: et sli the worldwide prevalence of herpes simplex virus (hsv) and the shortage of efficient vaccines and novel therapeutic strategies against hsv are widely global concerns. the abundance on the virion and the major stimulus for the virus-neutralizing antibodies makes gd a predominant candidate for cure of hsv infection. in this study, we generated a monoclonal antibody (mab), termed m f, targeting to glycoprotein d (gd) of hsv- , which also has cross-reactivity against hsv- gd. it has a high level of neutralizing activity against both hsv- and hsv- , and binds to a highly conserved region (residues – ) within the pro-fusion domain of gd. it can effectively block hsv cell-to-cell spread in vitro. the pre- or post-attachment neutralization assay and syncytium formation inhibition assay revealed that m f neutralizes hsv at the post-binding stage. moreover, therapeutic administration of m f completely prevented infection-related mortality of mice challenged with a lethal dose of hsv- . our newly identified epitope for the neutralizing antibody would facilitate studies of gd-based hsv entry or vaccine design, and m f itself demonstrated a high potential for adaptation as a protective or therapeutic drug against hsv. herpes simplex virus (hsv) is a prevalent worldwide human pathogen that infects epithelial cells before it establishes latency in trigeminal or sacral nerve root ganglia (steiner and benninger, ) , causing mucocutaneous lesions, keratitis, and encephalitis (dropulic and cohen, ) . there are two serotypes of hsv, hsv- and hsv- , also known as human herpesvirus and (hhv- and hhv- ). the most common clinical sign of hsv- is herpes labialis, as well as ocular diseases, including conjunctivitis and keratitis (everett, ) . hsv- is the main cause of recurrent genital herpes, which is one of the most predominant sexually transmitted diseases, and hsv- infection of newborns during delivery is usually accompanied by a high mortality rate (cleary et al., ; domeika et al., ; overall, ) . moreover, epidemiological and biological studies have revealed that hsv- dramatically increases the risk of hiv infection (barnabas and celum, ; freeman et al., ; sartori et al., ) and may also act in conjunction with human papillomavirus (hpv) infection to increase the risk of invasive cervical carcinoma (smith et al., ; zhao et al., ) . according to world health organization (who) statistics, in , there were million people aged - years living with hsv- infection worldwide (looker et al., ) , and the newly infected population is estimated to be million per year (looker et al., ) . therefore, the development of vaccines and novel therapeutic strategies against hsv has become very urgent. both initial entry of the virion into cells and subsequent lateral spread of hsv require the interaction of four viral glycoproteins, gb, gd, gh and gl, with at least one of the cell receptors, either herpesvirus entry mediator (hvem) or nectin- . among these viral glycoproteins, gd acts as the receptor-binding glycoprotein, and gh, gl and gb are required for membrane fusion execution. the gd ectodomain is organized into two structurally and functionally differentiated regions. the n terminus (residues - ) carries the receptor binding sites, and the c terminus functions as the pro-fusion domain (residues - ), which is required for triggering virus-cell fusion. in nature, the c terminus of the gd ectodomain binds to its n-terminal region and masks the receptor-binding site, resulting in a closed conformation (cocchi et al., ; fusco et al., ) . after binding to a cell surface receptor, either hvem or nectin- , gd undergoes conformational changes to adopt an open conformation, which is a key step in activating the core fusion machinery of gh/gl and gb. gd is the most abundantly expressed glycoprotein on the virion, and it induces both humoral and cellular immunity and is the major stimulus of virus-neutralizing antibodies together with gb (bender et al., ; eisenberg et al., ; fotouhi et al., ; nicola et al., ; whitbeck et al., ) . therefore, gd has been the predominant candidate for hsv vaccine or novel therapeutic strategies (awasthi and friedman, ; shin and iwasaki, ) . monoclonal antibodies (mabs) represent a useful tool for the study of the structure and function of target proteins, and virus-host interactions, as well as for the development of promising therapeutic agents. anti-gd mabs have been arranged into groups that recognize distinct discontinuous native and continuous denatured epitopes. most mabs that have virus-neutralizing activity recognize discontinuous epitopes. residues to , to , and to of the extracellular domain are major continuous antigenic determinants on gd (isola et al., ) . mabs recognizing epitopes located in these continuous determinant regions have no or low virus-neutralizing ability, except for the mab d , which can neutralize viral infectivity by blocking gdreceptor interaction (cairns et al., ) . our current investigation found a mab, m f that recognizes a new continuous epitope (residues to ) within the pro-fusion domain of hsv and possesses a high level of virus-neutralizing activity. it showed a high degree of neutralizing activity against both hsv- and hsv- , completely abrogated viral cell-to-cell spread, and inhibited syncytium formation in vitro. in addition, it also exhibited highly therapeutic effects in a hsv- infected mouse model, implying its high potential for adaptation as a protective or therapeutic interventions. cell lines used in the study are african green monkey kidney (vero) cell line (atcc, and baby hamster kidney (bhk) cell line (atcc, . cells were cultured in dulbecco's modified eagle medium (dmem) (thermo fisher scientific) with % fetal bovine serum (fbs) (thermo fisher scientific). hsv- f strain and hsv- strain were kindly provided by microorganisms and viruses culture collection center, wuhan institute of virology, cas (serial number: ivcas . , ivcas . ). viruses were propagated in vero cells and cell lysate stocks were prepared as previously described (morrison and knipe, ) . virus titers were determined in vero cells (navarro et al., ) . female balb/c mice, six-weeks of age, were purchased from the hubei provincial center for disease control and prevention (wuhan, china) and maintained under specific pathogen-free conditions at the the structure of full-length gd protein is shown at the top; the truncated gd proteins used for the sequential mapping experiment are shown below. numbers indicate amino acid positions. the domain (pro-fusion) was defined according to fusco et al. ( ) . (b) recognition region mapping of mabs m f and c . the truncated gd proteins expressed by pmal-c x vector were presented as antigens. m f and c were used as the primary antibodies respectively for western blot analysis. central animal laboratory of wuhan institute of virology, chinese academy of sciences (wiv, cas. license number: syxk - ). all animal experiments were approved by the institutional animal ethical committee of wiv, cas (approval: wiva ). all procedures were carried out under pentobarbital sodium (sigma) anesthesia and all efforts were made to minimize any suffering and the number of animals used in the study. mabs against hsv- gd protein were prepared as described previously (evan et al., ) . in brief, the nucleotide sequence encoding the ectodomain (residues to ) plus the natural signal sequence of hsv- gd protein, gd - t, was cloned into the pet- a vector (novagen) and expressed in escherichia coli bl . the protein was purified under denaturing conditions in the presence of m urea by using a ninitrilotriacetic acid (nta) column and then dialyzed (pirestani et al., ) . the quantity of purified protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page). balb/c mice (n = ) were immunized with μg of purified gd - t in complete freund's adjuvant (sigma). after , , and weeks, the immunizations was repeated with gd - t conjugate in incomplete freund's adjuvant (sigma). fusion with sp / myeloma cells was carried out by using standard methodology (de stgroth and scheidegger, ) . hybridomas culture supernatant was screened by enzyme-linked immunosorbent assay (elisa) and western blot analysis. totally strains of positive hybridoma cells were obtained. the mabs were purified by protein a-sepharose™ (sigma) according to the manufacturer's instructions. for epitope mapping, the ectodomain of hsv- gd protein was dissected into seven truncated fragments, gd - t, gd - t, gd - t, gd - t, gd - t, gd - t and gd - t, with any two adjacent fragments sharing an overlap of - amino acids (fig. a) . amino acids to of the gd protein were further truncated into eight fragments, gd - t, gd - t, gd - t, gd - t, gd - t, gd - t, gd - t and gd - t (fig. a ). the nucleotides encoding the above fragments were pcramplified using the primer pairs listed in table s , cloned into pmal-c x (neb) and then expressed in e. coli dh β. for characterization of recognizing speciality, vero cells infected with hsv- or hsv- at an multiplicity of infection (moi) of pfu per cell were harvested at h post infection (p.i.) and rinsed with pbs. the protein samples were separated by % sds-page, and transferred onto a polyvinylidene fluoride (pvdf) membrane (merck millipore, massachusetts, usa) by a trans-blot apparatus (bio-rad). the western blot analyses were performed with primary antibodies generated from above-mentioned screening positive hybridoma cells which against hsv- gd protein: m f and c . pre-immune mouse serum and β-actin (sigma) antibodies were used as controls. after washing, the blots were incubated with horseradish peroxidase-conjugated goat anti-mouse antibody. the immunoreactive signals were visualized with supersignal west pico chemiluminescent substrate (fermentas) and the microchemi bioimaging system (dnr, usa). vero monolayers were infected with hsv- or hsv- at an moi of pfu per cell. at h p.i., the cells were fixed with % paraformaldehyde for min, permeabilized with . % triton x- , and washed three times with phosphate-buffered saline (pbs). to characterize the recognizing speciality of m f and c , we immunostained cells with them respectively for h at °c. pre-immune mouse serum and mouse immunoglobulin g (beyotime) were used as controls. all the antibodies were diluted : in blocking solution ( % bsa in pbs). after being washed for three times with pbs, the cells were incubated with the secondary antibody, fluorescein isothiocyanate (fitc)-conjugated goat anti-mouse igg (abcam, hangzhou, china). the cells were observed by fluorescence microscopy. the hsv neutralization assay was based on a previously described protocol (bag et al., ) . in brief, vero cell were seeded into well plates (costar) and incubated overnight to achieve confluent monolayers. m f and c were serially diluted in dmem supplemented with % fbs and then combined with hsv- or hsv- ( pfu/well) respectively (final concentration of m f or c for hsv- ranged from μg/ml to , and final concentration of m f or c for hsv- ranged from μg/ml to ). pre-immune mouse serum was used as negative control. the virus-antibody mixtures were incubated for h at °c with gentle rocking. after h incubation, the mixtures were inoculated over vero cell monolayers in plates at °c for h. after h adsorption the cells were covered with overlay media with % methylcellulose to form plaques. the plaques developed after h of incubation were fixed and visualized by staining with crystal violet. plaques were counted and the percentage inhibition was determined relative to the negative control. the effective concentration of mabs that inhibited the number of viral plaques was interpolated from the dose-response curves. virus neutralization assays were repeated at least twice and reported as number of plaques relative to control ± sem. the pre-or post-attachment neutralization assays were performed as described with modifications (krawczyk et al., ) . briefly, virusantibody mixture was incubated for h at °c before inoculating over the vero cells (pre-attachment), while prechilled ( °c for min) vero cells were infected with hsv- or hsv- ( pfu/well) at °c for h to allow virus adsorption before serial dilutions of antibodies were added (post-attachment). inoculated vero cells were then incubated for h at °c. neutralization efficacy was determined after h as described above for the standard neutralization assay. cell-to-cell spread assay was based on a previously described protocol (krawczyk et al., ) . bhk cells were grown on glass coverslips and incubated overnight to achieve confluent monolayers. cells were infected with hsv- or hsv- at an moi of . pfu per cell. after h of adsorption at °c, the virus inoculum was removed and cells were washed twice with pbs and then incubated in dmem containing % fbs in the presence of antibodies, m f ( μg/ml) and c ( μg/ml). mouse igg ( μg/ml), or medium alone was used as control. to visualize the viral spread by immunofluorescence, after h p.i., cells were fixed with % paraformaldehyde, permeabilized with . % triton x- , and washed three times with pbs. cells were incubated with mouse polyclonal antibody against gd as primary antibody and fitc-conjugated goat anti-mouse igg as secondary antibody. immunofluorescence images were observed by fluorescence microscopy. to quantify the cell-to-cell spread, the number of infected cells with gfp fluorescence per total cells was calculated in nine randomly chosen areas, representing ∼ cells for each group. the percentage infection was determined relative to control. the experiment was repeated twice. syncytium formation inhibition assay was performed as described with modifications (navarro et al., ) . in brief, monolayers of vero cells grown in -well plates were infected with hsv- or hsv- at an moi of pfu per cell. after h of adsorption at °c, the virus inoculum was removed and cells were washed with pbs twice then incubated in dmem containing % fbs in the presence of antibodies, m f ( μg/ml) and c ( μg/ml), mouse igg ( μg/ml), or medium alone as a control. after h p.i., cells were fixed and then stained with hematoxylin-eosin (he) staining. the inhibition of syncytium formation was examined by light microscopy. to quantify the inhibition of syntytium formation, the numbers of nuclei in syncytia were counted in randomly chosen area, and the percentage inhibition was determined relative to no antibody treatment group. only syncytia containing or more nuclei were analyzed. three independent experiments were calculated. mouse intravaginal hsv- challenge was established as previously described (cena-diez et al., ) . six-weeks-old female balb/c mice were rendered susceptible to infection by subcutaneous injection with . mg of medoxyprogesterone acetate (sigma) days prior to challenge (parr et al., ) . mice were randomly assigned to five groups of or each. mice were anesthetized with pentobarbital sodium and intravaginally challenged with × pfu/ μl of hsv- . the infected mice were passively immunized by intravenous injection of , , and mg/kg of m f at , , and h after intravaginal hsv- challenge. pbs group was set as control. mice in each group were examined daily for symptoms of genital disease, body weight over days, as well as mortality for days. disease score was graded from to : no apparent infection scored ; genital erythema scored ; moderate genital infection scored ; purulent genital ulceration, hair loss and generally poor condition scored ; severe ulceration of genital and surrounding tissue, and hind limb paralysis scored . mice that developing % loss in body weight, debilitating symptoms of disease or paralysis were sacrificed and considered to have died the following day in all survival analyses. surviving mice were sacrificed at the indicated times after infection. data are presented as means ± standard error of the mean (sem). sequence alignment was performed with dnaman v . statistical analysis was performed using graphpad prism . significant differences were analyzed by log-rank (mantel-cox) test and gehan-breslow-wilcoxon test. p-values were calculated, and statistical significance is reported as highly significant using *(p < . ), ** (p < . ), or *** (p < . ). (original magnification, × ) (c) and (d) virus neutralization assay. hsv- or hsv- ( pfu/well) was incubated with indicated serially diluted antibodies m f, c or preimmune serum. at h p.i., cells were stained and the number of plaques counted. the percentage inhibition was determined relative to the negative control. representative data (mean ± sem) from three independent experiments are shown. to develop functional monoclonal antibody against hsv- gd, we immunized balb/c mice with purified extracellular domain of gd protein. after the primary screening of hybridomas by elisa, western blot analysis and immunofluorescence assay (ifa), a total of strains of positive hybridoma cells were obtained (table s ). epitope mapping of these mabs was determined using the expressed truncated gd proteins, including gd - t, gd - t, gd - t, gd - t, gd - t, gd - t and gd - t, with an overlap of - amino acids between each pair of adjacent fragments (fig. a) , by western blot analysis (data not shown). among these mabs, m f and another mabs recognized truncated gd - t, which is located in the c-terminus (residues - ) of the gd ectodomain (fig. b) . in contrast, c (fig. b ) and the remaining mabs recognized both nterminal fragments, gd - t and gd - t, suggesting that they recognized the overlap region (residues - ) of the two fragments. therefore, two kinds of mab, as represented by m f and c , recognizing different epitopes of hsv- gd, were chosen for further characterization. to characterize the specific recognition ability of m f and c , we first tested their ability to recognize denatured and native forms of hsv- gd and hsv- gd by western blot assay and ifa. western blot analysis revealed that m f reacted with the expected gd band in the sample of both hsv- and hsv- infected vero cells, while c only recognized gd in the hsv- infected vero cells sample, but not the hsv- infected sample ( fig. a) . the ifa results demonstrated that m f detected native forms of gd in the cells infected with either hsv- or hsv- , while c only detected native gd in the cells infected with hsv- (fig. b) . these results suggested that m f could recognize the denatured and native forms of gd of both hsv- and hsv- , while c could only recognize the denatured and native forms of gd of hsv- . to further confirm whether m f and c could neutralize hsv infection, virus neutralization assays were performed. either hsv- or hsv- ( pfu/well) were pre-incubated with serially diluted mabs, m f and c , before infection into vero monolayer cells. pre-immune mouse serum was used as negative control. neutralization assay demonstrated that m f neutralized both hsv- and hsv- in a dosedependent manner, with about ∼ % hsv- infection and about ∼ % hsv- infection being inhibited at an antibody concentration of μg/ml. in contrast, c demonstrated no neutralizing activity of hsv- and low neutralizing activity of hsv- infection groups (only about % virus infection being inhibited at a concentration of μg/ ml) (fig. c and d) . these results suggested that m f but not c , is a neutralizing mab against hsv- and hsv- infection. as shown above, m f had a high level of neutralization activity against both hsv- and hsv- , and m f recognized a linear epitope, gd - t, which is located in the c-terminus of the gd ectodomain. to identity accurately the epitope recognized by m f, we constructed a series of further truncations of the c-terminal region (residues to ) of hsv- gd protein (fig. a) . by western blot analysis, gd - t but not gd - t that could be recognized by m f, indicating that p is the last c-terminal amino acid critical for m f binding (fig. b) . similarly, p is the first n-terminal amino acid critical for m f binding. on this basis, pnwhip was considered to be the precise epitope for m f binding. sequence alignment showed that these residues are highly conserved among hsv- and hsv- (fig. c) . these results explain why m f recognized the gd protein of both hsv- and hsv- . direct transmission between adjacent cells (cell-to-cell spread) is an efficient route for hsv to bypass the host immune system. not all neutralizing mabs can prevent cell-to-cell spread of herpesviruses (co et al., ; hooks et al., ) . therefore, blocking of cell-to-cell spread is considered to be an important aspect in evaluating the in vivo protective efficiency of neutralizing antibodies. we herein analyzed the ability of m f to inhibit cell-to-cell spread of hsv- and hsv- in vitro. confluent bhk cell monolayers were initially incubated with either hsv- or hsv- at an moi of . pfu per cell. to prevent viral spread through viral particles moving across the cell culture supernatant (cellfree spread), bhk cell monolayers were treated with an excess of m f or c , and treatment with mouse igg or culture medium alone was performed as controls. after h p.i., the transmission of virus was detected by immunostaining with mouse polyclonal antibody against gd. as shown in fig. a , m f completely inhibited cell-to-cell spread of both hsv- and hsv- , as evidenced by observing the limited fluorescence caused by virus infection. in contrast, neither c nor mouse igg could sufficiently inhibit transmission of hsv- or hsv- , and extensive virus spread on bhk cell monolayer was observed by immunostaining. when the infected cells were quantified, the results clearly showed that m f is able to inhibit cell-to-cell transmission in hsv-infected cells (fig. b) . to further characterize the mode of action of m f, we analyzed whether this mab could inhibit receptor binding. we compared the neutralization efficacy of m f when the antibody was added before (pre-attachment) or after (post-attachment) hsv virions interacted with vero cells. c was chosen as a negative control. by pre-or postattachment assays, when m f was added before virus-host interaction (pre-attachment), about % hsv- infection was inhibited at a concentration of μg/ml (fig. a) , about % hsv- infection was inhibited at a concentration of μg/ml when m f was added after hsv- virions interacted with vero cells (post-attachment) (fig. b) . almost equal neutralization efficacy of m f was observed, irrespective fig. . m f blocks cell-to-cell spread of hsv- and hsv- . (a) monolayers of bhk cells were infected hsv- or hsv- at an moi of . pfu per cell. cell monolayers were treated with an excess of antibodies, m f and c ( μg/ml each). mouse igg ( μg/ml) or culture medium alone was performed as controls. at h p.i., cells were fixed and observed by fluorescence microscopy. bar represents μm. the experiments were repeated at least twice. (b) the number of infected cell with gfp fluorescence per total cells was calculated in randomly chosen areas. the percentage infection was determined relative to control. the experiment was repeated twice. * p < . , ** p < . , *** p < . . of whether the antibody was added before or after hsv- virions interacted with vero cells. in contrast, the negative control, c , had no neutralizing ability either before or after virions were incubated with cells. the hsv- infection group showed a similar result. about % hsv- infection was inhibited in the pre-attachment treatment (fig. c ) and about % hsv- infection was inhibited in the postattachment treatment with an antibody concentration of μg/ml of m f (fig. d ). in contrast, c had low ( % hsv- infection being inhibited) or no neutralizing ability in the pre-attachment or post-attachment treatments, respectively ( fig. c and d) . the existing data indicated that m f does not interfere with the virus binding process, m f therefore neutralizes hsv entry at the post-binding stage. after binding of gd to a cell surface receptor, it undergoes a conformational change, which is also a key event for triggering later steps leading to fusion. since m f might neutralize hsv entry at the post-binding stage, we asked whether m f could inhibit the virus-induced membrane fusion process. monolayers of vero cells were infected with hsv- or hsv- at an moi of pfu per cell. after h of adsorption at °c, the virus inoculum was removed and cells were washed twice with pbs, the cells were then incubated with an excess of m f, c , mouse igg, or medium alone as a control. after h, syncytium formation was observed by staining with he staining. as shown in fig. a , in both hsv- and hsv- infection groups, obvious syncytia with multinuclear cells were observed when infected cells were treated with preimmune serum, mouse igg or c . however, when cells were incubated with m f, syncytium formation was almost completely inhibited. the syncytium formation inhibition of vero cells with different treatment was quantified in fig. b . this result suggested that m f indeed inhibited the hsv-induced membrane fusion. to investigate the protective efficacy of m f in vivo, we exploited a fig. . m f neutralizes hsv- and hsv- at the post-binding stage. comparison of neutralization efficacy of m f when serial dilutions of antibodies were added before (a and c, preattachment) or after (b and d, post-attachment) hsv virions interacted with vero cells. c was used as a control. neutralization efficacies were determined after h as described above for the standard neutralization assay. the percent inhibition was determined relative to the negative control. representative data (mean ± sem) from at least independent experiments performed are shown. r. du et al. antiviral research ( ) - lethal female balb/c mice model for hsv infection. to assess the therapeutic efficiency of m f, mice were randomly assigned to five groups of or each. in pbs-treated group, intravaginal infection of hsv- ( × pfu/ μl) of six-weeks-old female balb/c mice resulted in rapid progressive disease (fig. a ) and steady decrease of body weight from day post infection (fig. b) , with a median survival time of days (fig. c) . the experimental groups of balb/c mice were treated intravenously with , , , or mg/kg of antibody at , and h after intravaginal hsv- challenge. as shown in fig. , mice receiving mg/kg of m f were slightly protected against lethal infection by hsv- , the body weight of this group decreased as fast as the pbs-treated group, with a median survival time of days longer than those of control mice treated with pbs. however, mice treated with mg/kg or higher concentrations of m f were completely protected from lethal hsv- infection (fig. c) . no symptom of hsv- infection (fig. a ) or loss of body weight was observed among these groups (fig. b) . glycoprotein d (gd) is the most abundant glycoprotein on the virion and the major stimulus for virus-neutralizing antibodies of hsv. for both vaccine design and novel therapeutic strategies, it is important to study epitopes on gd that stimulate virus-neutralizing antibodies. according to the properties of a panel of mabs to gd and gd mutants, which were used to define the relationship between gd structure and function, anti-gd mabs have been previously arranged into groups that recognize distinct discontinuous native and continuous denatured epitopes. most mabs that recognize discontinuous native epitopes have virus-neutralizing activity. for example, mc (which recognizes residues and ), dl (which recognizes residues and ), mc (which recognizes residues - ) and mc (which recognizes residue ) have been reported previously to be neutralizing mabs that recognize discontinuous conformational epitopes. lazear et al., ; muggeridge et al., ; nicola et al., fig. . m f inhibits syncytium formation of hsv- and hsv- . (a) vero cells were infected with hsv- or hsv- at an moi of pfu per cell and then incubated in dmem containing % fbs in the presence of antibodies, m f ( μg/ml) and c ( μg/ml). mouse igg ( μg/ml), or medium alone were used as control. after h, cells were fixed and then stained with he staining. the inhibition of syncytium formation was observed by light microscopy. bar represents μm. the experiments were repeated at least twice. (b) syncytium formation of vero cells with different treatment as shown in panel a was quantified by counting the number of nuclei in syncytia of randomly chosen area. the percentage inhibition was determined relative to no antibody treatment group. only syncytia containing or more nuclei were analyzed. data were representative of three independent experiments. * p < . , ** p < . , *** p < . . ; whitbeck et al., ) . there are also a number of reported mabs that recognize continuous denatured epitopes of hsv gd. among these mabs, most have no or low virus-neutralizing ability, except for the mab d (which recognizes residues to ), which can neutralize viral infectivity by blocking gd-receptor interaction (cairns et al., ) . in this study, we found a novel mab, m f, to be another virusneutralizing mab whose epitope is located within a continuous antigenic determinant. epitope mapping assays indicated that m f recognized conserved residues to which located in the c-terminal pro-fusion domain (residues - ) of gd ( figs. and ) . sequences alignment analysis showed that this epitope is highly conserved among a broad range of type- and type- hsv strains (data not shown). this may also explain why the recognition specificity of m f was type-common. it is likely that m f can be efficacious among divergent isolates yet the breadth of m f neutralizing activity against a panel of hsv- and - isolates in vitro need to be performed. in addition, the resistance evaluation of m f and alanine scanning of - should also be carried out in the future. the recognition specificity of m f was type-common, and m f had a high level of hsv virion neutralizing activity (fig. ) . the epitopes of three reported type-common mabs, bd (which recognizes residues to ), dl (which recognizes residues to ), and bd (which recognizes residues to ), were continuous and also located in the c-terminal pro-fusion domain of gd. however, they had no neutralization activity against both hsv- strain kos and hsv- strain (isola et al., ; lazear et al., ) . previously, hooks and his colleagues found hsv- , human and murine cmv and vaccinia could spread in the presence of neutralizing antibodies generated in rabbits (hooks et al., ) . co et al. humanized two murine monoclonal antibodies against hsv gb and gd, which had been shown to neutralizing hsv- in vitro, however, neither humanized and murine anti-gd mab was able to protect against viral spread. while humanized and murine anti-gb mab protected cells from viral spread (co et al., ) . our research showed that m f is able to inhibit cell-to-cell spread of hsv- and hsv- in vitro (fig. ) , which is an efficient way for hsv to bypass the host immune system through the direct transmission adjacent cells. to our knowledge, m f is the first epitope-mapped neutralizing mab against hsv gd reported to be capable of inhibiting cell-to-cell spread of hsv- and hsv- . antibodies against entry glycoproteins have the ability to neutralize virions via interfering with receptor binding or subsequent fusion events. for example, antibodies targeting both stalk (s ) and globular (s ) subunits of the spike protein of severe acute respiratory syndrome (sars) coronavirus were able to abolish the binding between s protein and its cellular receptors. mabs neutralize hiv by inhibiting the binding of gp to the cd receptor (wibmer et al., ; zeng et al., ) . the above-mentioned neutralizing mabs mc and dl neutralize virus by blocking binding of gd to both receptors, hvem and nectin- . in addition, there are many reports of mabs blocking virus entry at the post-receptor-binding step. for instance, neutralizing mabs specific for the c-terminal domain of the murine leukemia virus (mulv) surface (su) envelope protein subunit interfere with a post-attachment event necessary for membrane fusion (burkhart et al., ) . mab d against the f subunit of the helicoverpa armigera nucleopolyhedrovirus f protein neutralizes virus entry by inhibiting membrane fusion (zou et al., ) . our data from pre-attachment and post-attachment neutralization assays indicated that m f does not interfere with the virus binding process; instead, it might neutralize hsv entry at the post-binding stage (fig. ) . by observing hsv-induced membrane fusion following treatment with m f, we determined that m f could almost completely inhibit syncytium formation (fig. ) , which further confirms that m f could inhibit the membrane fusion event rather than the virus binding process. consistent with current findings, earlier studies (cocchi et al., ; fusco et al., ) established that the profusion domain (residues - ) is required for viral infectivity and fusion but not for receptor binding and the substitutions of some pro residues in pfd (amino acids , , , and ) affected steps in entry and fusion. however, the detailed mechanism of how m f inhibits the hsv fusion process needs further study. based on the high level of hsv virion neutralizing activity, we also investigated whether application of m f could confer protection in vivo by exploiting a lethal hsv- intravaginally infected female balb/c mouse model to evaluate the potency of m f as therapeutic alternative to counteract hsv infection. our data demonstrated that mab m f conferred protection in a dose-dependent manner and that mg/kg or more of m f provided full protection (fig. ) . consistently, intracutaneously injection human recombinant neutralizing mab hsv specific for gd (type-common and its epitope located in residues to ) can protect mice effectively when administered systemically zeitlin et al., ) . passive immunization of immunocompetent animals with gd specific mab hd (a type-common fig. . m f protects mice from lethal hsv- infection. (a) symptoms of genital disease in each group was scored daily as described in the text for days after intravaginally hsv- challenge. data was shown as the mean values of mice in each group. (b) mice were weighed daily and body weight are expressed as a percentage of the value prior to treatment. data represent mean ± standard error. (n = animals per group for pbs; n = for all other groups) (c) survival of mice in each group monitored for indicated days. (n = animals per group for pbs; n = for all other groups). data were analyzed by log-rank (mantel-cox) test and gehan-breslow-wilcoxon test. * p < . , *** p < . . neutralizing mab, and its discontinuous epitope located in residues to ) postexposure at appropriate times demonstrated protection against hsv- strains (htz, mp, and a mutant strain mp) and hsv- strain g -induced neurological disease (dix et al., ; pereira et al., ) . furthermore, intravaginally administered murine anti-gb mab post hsv- infection conferred full protection in an immunodeficient non-obese diabetic (nod)/severe combined immunodeficiency (scid) mouse model (krawczyk et al., ) . these results encourage further potency of neutralizing mabs for clinical therapy against severe hsv diseases. in conclusion, our results have demonstrated for the first time that mab m f targeting a new continuous epitope (residues to ) within the pro-fusion domain has a high level of virus-neutralizing activity. these finding will enrich the hsv glycoprotein d-specific neutralizing antibodies and will facilitate the development of vaccine design or novel therapeutic strategies. status of prophylactic and therapeutic genital herpes vaccines an indole alkaloid from a tribal folklore inhibits immediate early event in hsv- infected cells with therapeutic efficacy in vaginally infected mice infectious co-factors in hiv- transmission herpes simplex virus type- and hiv- : new insights and interventions antigenic and mutational analyses of herpes simplex virus glycoprotein b reveal four functional regions distinct mechanisms of neutralization by monoclonal antibodies specific for sites in the n-terminal or c-terminal domain of murine leukemia virus su dissection of the antibody response against herpes simplex virus glycoproteins in naturally infected humans prevention of vaginal and rectal herpes simplex virus type transmission in mice: mechanism of antiviral action type-specific screening for asymptomatic herpes infection in pregnancy: a decision analysis humanized antibodies for antiviral therapy the soluble ectodomain of herpes simplex virus gd contains a membrane-proximal pro-fusion domain and suffices to mediate virus entry production of monoclonal antibodies: strategy and tactics use of monoclonal antibody directed against herpes simplex virus glycoproteins to protect mice against acute virus-induced neurological disease guidelines for the laboratory diagnosis of genital herpes in eastern european countries the challenge of developing a herpes simplex virus vaccine herpes virus fusion and entry: a story with many characters isolation of monoclonal antibodies specific for human c-myc proto-oncogene product hsv- biology and life cycle expression of the herpes simplex virus type glycoprotein d in baculovirus expression system and evaluation of its immunogenicity in guinea pigs herpes simplex virus infection increases hiv acquisition in men and women: systematic review and meta-analysis of longitudinal studies the pro-fusion domain of herpes simplex virus glycoprotein d (gd) interacts with the gd n terminus and is displaced by soluble forms of viral receptors viral spread in the presence of neutralizing antibody: mechanisms of persistence in foamy virus infection fine mapping of antigenic site ii of herpes simplex virus glycoprotein d impact of valency of a glycoprotein b-specific monoclonal antibody on neutralization of herpes simplex virus herpes simplex virus glycoprotein d can bind to poliovirus receptor-related protein or herpesvirus entry mediator, two structurally unrelated mediators of virus entry antibody-induced conformational changes in herpes simplex virus glycoprotein gd reveal new targets for virus neutralization an estimate of the global prevalence and incidence of herpes simplex virus type infection global estimates of prevalent and incident herpes simplex virus type infections in mechanisms of immunization with a replication-defective mutant of herpes simplex virus identification of a site on herpes simplex virus type glycoprotein d that is essential for infectivity domains of herpes simplex virus i glycoprotein b that function in virus penetration, cell-to-cell spread, and cell fusion monoclonal antibodies to distinct sites on herpes simplex virus (hsv) glycoprotein d block hsv binding to hvem herpes simplex virus infection of the fetus and newborn a mouse model for studies of mucosal immunity to vaginal infection by herpes simplex virus type type-common and type-specific monoclonal antibody to herpes simplex virus type evaluation of immunogenicity of novel isoform of eg (eg - g ) from echinococcus granulosus in balb/c mice. iran protection of nude mice by passive immunization with a type-common human recombinant monoclonal antibody against hsv herpes simplex virus type infection increases human immunodeficiency virus type entry into human primary macrophages generating protective immunity against genital herpes herpes simplex virus- as a human papillomavirus cofactor in the etiology of invasive cervical cancer update on herpes virus infections of the nervous system the major neutralizing antigenic site on herpes simplex virus glycoprotein d overlaps a receptor-binding domain hiv broadly neutralizing antibody targets topically applied human recombinant monoclonal igg antibody and its fab and f(ab') fragments protect mice from vaginal transmission of hsv- quantitative comparison of the efficiency of antibodies against s and s subunit of sars coronavirus spike protein in virus neutralization and blocking of receptor binding: implications for the functional roles of s subunit a novel multiplex real-time pcr assay for the detection and quantification of hpv / and hsv / in cervical cancer screening characterization of two monoclonal antibodies, f and d , against the major envelope fusion protein of helicoverpa armigera nucleopolyhedrovirus we thank members of our laboratory for discussions and the core facility and supplementary data related to this article can be found at http://dx. doi.org/ . /j.antiviral. . . . key: cord- -sw xjpbr authors: guo, chao-tan; takahashi, tadanobu; bukawa, wakoto; takahashi, noriko; yagi, hirokazu; kato, koichi; hidari, kazuya i.-p. jwa; miyamoto, daisei; suzuki, takashi; suzuki, yasuo title: edible bird's nest extract inhibits influenza virus infection date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: sw xjpbr edible bird's nest (ebn) is the nest of the swift that is made from its saliva. although ebn has been widely used for enhancing immunocompetence, its antiviral efficacy has not been studied in detail. we found that ebn extract could strongly inhibit infection with influenza viruses in a host range-independent manner when it was hydrolyzed with pancreatin f. western blotting assay showed that the ebn extract bound to influenza virus. furthermore, ebn extract could neutralize the infection of mdck cells with influenza viruses and inhibit hemagglutination of influenza viruses to erythrocytes, but it could not inhibit the activity of influenza virus sialidase. fluorometric hplc indicated that the major molecular species of sialic acid in ebn is n-acetylneuraminic acid. the results suggest that ebn is a safe and valid natural source for the prevention of influenza viruses. edible bird's nest (ebn) is the nest of the swift that is made from its saliva, which contains sialylglycoconjugates. the composition of the swift's saliva resembles that of salivary mucin. many studies have been carried out on the tonic effects of ebn, and it has been shown that ebn stimulates mitosis hormones and the growth factor for epidermal growth, resulting in repair of cells and stimulation of the immune system (kong et al., ; ng et al., ) . recently, several carbohydrate molecules, including new sialic acid-containing compounds and glycoconjugates, have been detected in ebn (pozsgay et al., ; reuter et al., ; wieruszeski et al., ; kakehi et al., ; martin et al., ; yu-qin et al., ) , but the importance of sialic acid residues in ebn is not clear. sialic acid residues of many glycoconjugates are involved in biologically important ligand-receptor interactions such as specific cell-to-cell, pathogen-to-cell, or drug-to-cell interactions. we found that an ebn extract could neutralize infection with influenza viruses in mdck cells and inhibit the hemagglutination of influenza a viruses (such as human, avian, and porcine strains) to human erythrocytes. we used two kinds of samples from bird nests collected in indonesia. sample (s ) was obtained from a bird's nest collected in a natural cave and sample (s ) was obtained from a bird's nest that had been house-cultured. the bird's nests were dried for h at • c and then ground and sifted through a mesh ( m in pore size) for combing out plume and foreign substances. the grounded samples were kept in distilled water ( g sample/ ml water) at • c for h and then heated at • c for min. the suspension was treated with pancreatin f (final concentration of . mg/ml, amano enzyme inc., japan) at • c for h at ph . - . and then heated at • c for min for enzyme deactivation. the treated extracts were filtrated with a filter paper (advantec filter paper no. ), and the filtrate was freeze-dried. for neuraminidase treatment, the ebn extracts were incubated in pbs containing mu/ml of neuraminidase from c. perfringens (n , sigma) for h at • c and then heated at • c for min for enzyme deactivation. madin-darby canine kidney (mdck) cells were cultured in eagle's minimum essential medium (emem; nissui pharmaceutical co., ltd., japan) containing % (v/v) heat-inactivated fetal calf serum (fcs; biological industries). human lung carcinoma a cells were purchased from atcc and cultured in dulbecco's modified eagle medium (dmem; nissui pharmaceutical co., ltd.) supplemented with % (v/v) heat-inactivated fcs, mm glutamine, u/ml penicillin, and g/ml streptomycine (gibco) at • c in humidified air containing % co . the influenza viruses used in this study (except strains a/shizuoka/ / and a/shizuoka/ / , see table ) were propagated in the allantoic sacs of -day-old embryonated eggs and purified by sucrose density gradient centrifugation (suzuki et al., ) . a/shizuoka/ / (h n ) and a/shizuoka/ / (h n ) strains were grown with a cell monolayers in fcs-free dmem supplemented with g/ml acetyl-trypsin, mm glutamine, u/ml penicillin, and g/ml streptomycine at . • c in humidified air containing % co . viral ha units were determined in micrometer plates using . % human type o erythrocytes as described previously (suzuki et al., ) . rabbit anti-influenza virus antibodies were raised by immunization of a/memphis/ / (h n ) virus grown in eggs as described previously (suzuki et al., ) . hemagglutination inhibition (hai) assay was carried out using -well microtiter plates as described previously (suzuki et al., ) . phosphate-buffered saline (pbs, ph . ) containing . % gelatin was used as a dilution buffer. human erythrocytes were used as indicator cells. virus suspension ( ha units in . ml of pbs) was added to each well containing the ebn extract in two-fold serial dilutions with the dilution buffer. the plates were incubated for h at • c. after . ml of . % (v/v) human type o erythrocytes, in which the total sialic acid content for blood group o is -fold higher than that for blood group a or b (bulai et al., ) , in pbs had been added to the plates, the plates were kept for h at • c. the maximum dilution of the samples showing complete inhibition of hemagglutination was defined as the hai titer. a fluorometric assay was used to determine the inhibitory effect of the ebn extract on influenza virus sialidase activity (suzuki et al., ) . five microliters of influenza virus suspension ( ng protein) in mm sodium acetate buffer (ph . ) containing two-fold serial dilution of the ebn extracts was stored at • c for min and then incubated with l of mm -( -methylumbelliferyl)-␣-d-n-acetylneuraminic acid ( -mu-neu ac) (sigma-aldrich, st. louis, mo) at • c for min. the reaction was stopped by the addition of ml carbonate buffer (ph . ). the fluorescence of the released methylumbelliferone was measured with a fluorescence spectrophotometer (f- ; hitachi, tokyo) with excitation at nm and emission at nm. neutralization of influenza virus by the ebn extract was determined as previously described (guo et al., ) . briefly, mdck cell monolayers were maintained in emem containing % fcs in a -well plate. one hundred microliters of tcid ( % tissue culture infectious dose) of viruses in the presence of the ebn extract ( . - g/ml) was inoculated at . • c for h. after removal of the inoculum, the monolayers were washed three times with emem. the cells were examined using a light microscope for the progression of viral-induced cytopathic effects (cpe) after incubation at . • c for h. lactate dehydrogenase (ldh) that was released from mdck cells was examined for virus neutralization by a slightly modified colorimetric assay. ldh activities in the medium were determined according to the manufacturer's instructions. briefly, the medium ( . ml) was diluted to : (v/v) with pbs and mixed with . ml of ldh reagent (shinotest, japan). the mixture was incubated at • c for min, and the reaction was stopped by the addition of . ml of . n hcl. absorbance was measured at nm (reference at nm). the assays were performed in triplicate for three independent experiments. as cytopathic effects appear on all cells, the ldh activity was indicated % as a relative activity. western blotting was used to detect the binding activity of the viruses to the glycoprotein from the ebn extracts. total amounts ( g) of proteins from the ebn extracts were subjected to sds-page through a - % polyacrylamide running gel. proteins were electrophoretically transferred to immune-blot pvdf membranes (bio-rad lab., usa). the membranes were blocked for h in pbs containing % skin milk and . % tween- . binding of proteins to influenza virus (a/aichi/ / (h n ), hau/ml) was carried out at • c by incubation overnight (more than h) and then probing with anti-h n (anti-mem) rabbit serum at a dilution of : , followed by horseradish peroxidase (hrp)-conjugated protein a (icn pharmaceuticals inc.) at a dilution of : . finally, the immunoreactive proteins were visualized using a solution containing mm citrate buffer (ph . ), mm n,n-dimethyl-p-phenylenediamine dihydrochloride in acetonitrile, mm -chloro- -naphthol in acetonitrile, and % h o aqueous ( : : : . , by volume) for min at room temperature. fluorometric determination of -n-acetyl-neuraminic acid (neu ac) and -n-glycolylneuraminic acid (neu gc) was conducted by a high-performance liquid chromatography (hplc) method using , -diamino- , -methylenedioxy-benzene (dmb) as previously described (suzuki et al., ) . the ebn extract was hydrolyzed with l of mm sulfuric acid. the hydrolysate was reacted with dmb reagent and heated at • c for . h in the dark to develop fluorescence for determination of sialic acids. a -l aliquot of the resulting solution was used for determination of sialic acids. the fluorescence of dmb derivatives was detected at an excitation wavelength of nm and an emission wavelength of nm. for the establishment of calibration curves, standard mixtures of neu ac and neu gc (sigma, usa) were used. to identify sialyoligosaccharides reactive with sialic acid (sa) ␣ , galactose (gal)-or sa␣ , gal-specific lectins (glycan determination kit; boehringer mannheim biochemicals, mannheim, germany), each section was incubated with l of digoxigenin (dig)-labeled sambucus nigra agglutinin (sna) lectin [ g/ml; specific for sa␣ , gal/nacetylgalactosaminide (galnac)] or maackia amurensis agglutinin (maa) lectin ( g/ml; specific for sa␣ , gal) for h at room temperature. after three washes with cold pbs, the membrane was incubated with hrp-conjugated anti-dig antibody (boehringer mannheim biochemicals) for h at room temperature and then after three additional washes in cold pbs and buffered glycerol (ph . ) were visualized according to the protocol provided by the manufacturer (glycan determination kit; boehringer mannheim biochemicals). in order to check the reaction of ebn extract with influenza viruses, we used the human influenza virus strain a/aichi/ / (h n ), which bound to both sialyl ␣ - and ␣ - galactose linkages in salylglycoproteins and sialylglycolipids, and carried out an sds-page western blotting assay to analyze the glycoprotein molecules. as shown in fig. , strain a/aichi/ / could bind to the ebn extract both treated (molecular weight is lower than kda) and not treated (molecular weight is over than kda) with a pancreatic enzyme that hydrolyzes protein to polypeptide (fig. a) . the virus binding activity of ebn extract from s (from a bird's nest collected in a cave) was stronger than that of ebn extract from s (from a house-cultured nest). furthermore, the effect of ebn extract from s was markedly reduced after treatment with neuraminidase (fig. b) , indicating that the virus binding is associated with sialic acid of the ebn. pancreatin f-treated ebn extract from s was further treated with mu/ml of neuraminidase from c. perfringens (n , sigma) to cleave terminal sialic acid residues in the ebn extract. to measure the biological response of the ebn extract, we carried out an hai assay and a neutralization assay (suzuki et al., ) . as shown in fig. a , in case of no treatment with the pancreatic enzyme, neither the ebn extract from s nor that from s inhibited the hemagglutination of influenza a virus (strain a/aichi/ / ) to human type o erythrocytes. however, ebn extracts (s -pan and s -pan) treated with pancreatin f at • c for h had stronger hai activities. the hai activities of the extracts from s -pan and s -pan were or g/ml, respectively. the difference in their hai activities indicates that s -pan and s -pan contain different sialylconjugates, such as sialic acidic species and linkages of sialic acid to galactose. on the other hand, after treatment with both the pancreatic enzyme and neuraminidase, the ebn extracts from s (s -pan-na) and s (s -pan-na) had a reducing effect. we also investigated the neutralization of infection with influenza a virus (strain a/aichi/ / ) in mdck cells by the ebn extract. as shown in fig. b , the results were similar to those of the hai assay. the inhibitory effect of the ebn fig. . effects of the ebn extracts from s and s treated with pancreatin f (pan) and/or a neuraminidase (na) on the inhibitory effects of hemagglutinin of influenza virus (a/aichi/ / ) (a) and the neutralization (b) of influenza virus infection as described in section . s (the wild ebn) and s (the cultured ebn) are ebn extracts that were not treated with any enzyme. s -pan and s -pan are ebn extracts which were treated with pancreatin f (final concentration of . mg/ml) at • c for h at ph . - . . s -pan-na and s -pan-na are s -pan and s -pan that were further treated with mu/ml of neuraminidase from c. perfringens (n , sigma) at • c for h. the starting dilution concentration of the ebn extracts is mg/ml. extract from s treated with pancreatin f (s -pan) on strain a/aichi/ / was stronger (ic , ∼ g/ml) than that of the ebn extract from s (ic , ∼ g/ml). after treatment with neuraminidase, the inhibitory effects of the ebn extracts (s -pan-na and s -pan-na) were -fold and -fold less than those of s -pan and s -pan, respectively. taken together, the results showed that the ebn extract from s after treatment with a pancreatic enzyme has stronger inhibitory activity than that of the ebn extract without enzyme treatment. the results indicate that the lower molecular peptides ( - kda) compared with the higher molecular protein (over than kda) of the ebn extract are propitious to inhibit influenza a virus infection. on the other hand, the ebn extract ( mg/ml) did not inhibit the neuraminidase activity of influenza viruses as using mg/ml of their higher concentration (data not shown), indicating that the ebn extract acts on viral hemagglutinin but not viral neuramindase. in the hai and neutralization experiments, the ebn extract at mg/ml had no side effects, such as hemolysis and cytolysis, on human erythrocytes and mdck cells. we further tested the effects of the ebn extracts (s -pan and s -pan) against on strains isolated from humans, ducks, and pigs. as shown in table , the ebn extracts (s -pan and s -pan) had strong effects on the hai and neutralization of all influenza viruses used in this test, indicating that its effect is independent of virus strain. many studies have indicated that various bird nests have abundant sialic acid-containing sugar chains (pozsgay et al., ; reuter et al., ; wieruszeski et al., ; kakehi et al., ; martin et al., ) . in order to determine the mechanism underlying the antiviral effect of ebn extract, we carried out fluorometric hplc and a lectin binding assay for analyzing the molecular species of sialic acid and the linkage between sialic acid and galactose. neu ac was detected as the major molecular species of sialic acid in the ebn extract from s ( . % of total sialic acid) ( fig. a-a, b) . peak in panel c is much higher than that in panel b. after alkali hydrolysis with n naoh for h at • c, peak became lower and peak became higher (panel d), indicating that the ebn extract from s has an o-acetyl sialic acid species. we further examined the linkages between nonreducing terminal sialic acid to gal in the ebn extract using sialic acid-gal linkage-specific lectins by sds-page (fig. b ). since maa (specificity for sialic acid terminally linked - to gal in n-glycan and o-glycan) bound strongly to the ebn extract from s and weakly to the ebn extract from s , it is possible that maa can not bind to o-acetyl sialic acid to gal linkage. on the other hand, sna [specificity for sialic acid terminally linked - to gal (or galnac) in n-glycan] did not react with either of the ebn extracts. these findings suggest that the sialyl-glycoconjugates of the ebn extract from s have neu ac␣ - gal linkages and that the ebn extract from s also have an o-acetylated neu ac, while neu ac is the major sialic acid species. these sialyl glycoconjugates containing neu ac - gal linkages may act as the main inhibitor of influenza virus infection. in this study, we found that the ebn extract had a potent inhibitory activity against infection of host cells with human, avian, and porcine influenza viruses. the effect is mediated by the neu ac residues of sialyl-sugar chains in the ebn extract. we also found that the inhibitory activity of ebn extract against influenza virus was markedly enhanced by treatment with a pancreatic enzyme (pancreatin f) that contains protease to hydrolyze glycoproteins to glycopeptides. we noted the ebn extracts that were not treated with a pancreatic enzyme could be bound by influenza viruses but had no effect on hai or the neutralization of influenza virus infection in mdck cells, indi- cating small molecular of the ebn extract was more propitious for its anti-viral effect. in a general way, the hemagglutination of viruses propagated in chicken eggs preferentially binds to receptors with a silaic acid bond to the adjacent sugar via ␣ - linkage, whereas the ha of human mdck-grown viruses preferentially binds to receptors with a silaic acid bond to the adjacent sugar via ␣ - linkage. we determined the effects of the ebn extract on viruses propagated in chicken eggs and also on recent viruses [strains a/shizuoka/ / and a/shizuoka/ / (h n )] isolated from influenza patients in february and cultured in a cells, human lung carcinoma cells that express both sialyl ␣ - and ␣ - linkages. the inhibitory effects of the ebn extract on strains a/shizuoka/ / and a/shizuoka/ / (h n ) were similar to those on other strains used in this study. the data obtained from lectin blotting showed that neu ac␣ - gal linkage was the major molecular species of sialic acid in the ebn extract. why could the ebn extract inhibit infection of recently isolated influenza viruses? we have recently reported that sialic acid-free components from bacteria (guo et al., ; nakata et al., ) and sulphatide (suzuki et al., (suzuki et al., , also have some inhibitory effects on influenza a virus infection. the ebn extract may contain some neu ac␣ - gal linkages since the lectin sna can not detect the neu ac␣ - gal linkage in o-glycan and glycolipids (dalziel et al., ) . we do not know why there is a difference between ebns from the wild and cultured environments. one possible reason is that the saliva of the wild swift and that of the cultivated swift are different. another possibility is that the o-acetylated sialic acid residue in the ebn from the wild environment or in its harvest process is destroyed. further investigation of this issue is needed. chinese have been consuming bird nests for hundreds of years, and most of them believe that ebn consists of several proteins and minerals that promote the generation and growth of human cells, rejuvenate human skin, and strengthen the immune system. although ou et al. ( ) reported that bird's nest is the common cause of food-induced anaphylaxis in children, which could lead to potentially life-threatening allergenic reactions, and the presence of a major allergen of kda in ebn . in this study, the proteins or peptides which had the best anti-influenza effect were some - kda sialylglycoproteins of the ebn extract after with pancreatin f. although the proteins of over than kda (including a major allergen of kda) untreated with pancreatic enzyme could bind to influenza a virus, the neutralization against the virus infection not detected or was very low. furthermore, the ebn extract showed no side effects, such as hemolysis and cytolysis, on erythrocytes and mdck cells even at a higher concentration ( mg/ml). therefore, we think that the ebn extract (lower than kda) treated with pancreatin f will become an effective and secure agent for anti-virus. many studies have validated several different sialyl linkages to galactose or n-acetylgalactosamine (pozsgay et al., ; reuter et al., ; wieruszeski et al., ; kakehi et al., ; martin et al., ) in bird nests. not only influenza a viruses but also other microbes, such as influenza b and c viruses, parainfluenza virus, newcastle disease virus, rotavirus, coronavirus and bacteria (cholera), recognize sialic acid and its derivatives, o-acetylated sialic acid-containing sugar chains as its receptor (guo et al., ; schultze et al., ; suzuki et al., a,b; sillerud et al., ) . although the o-acetylated sialic acid-containing sugar chains in the ebn are not a good source of the inhibitory activity for influenza a viruses, they may have an effective roles against other viruses which recognize o-acetylated sialic acid, such as influenza c virus and coronavirus. we have searched the way of the harvest of the cultured house and wild cave bird nests and also their processing in the market, but could not obtain any more detailed information on them. therefore, we have no clear answer on the difference of the binding and inhibitory activities between ebn from the wild and from cultured environments. one reason of the low binding and inhibitory activity in s rather than s may due to in the presence of o-acetylated sialyl sugar chains in s which do not bind and inhibit the influenza a and b viruses. taken together, the results suggested that ebn is a safe and valid natural source for the prevention of influenza viruses in vitro, however, the detailed in vivo effect of the inhibition of the influenza viruses by ebn should be evaluated. diversity of the human erythrocyte membrane sialic acids in relation with blood groups lectin analysis of human immunoglobulin g n-glycan sialylation immunochemical characterization of edible bird's nest allergens a unique phosphatidylinositol bearing a novel branched-chain fatty acid from rhodococcus equi binds to influenza virus hemagglutinin and inhibits the infection of cells an o-glycoside of sialic acid derivative that inhibits both hemagglutinin and sialidase activities of influenza viruses synthetic sialylphosphatidylethanolamine derivatives bind to human influenza a viruses and inhibit viral infection highperformance capillary electrophoresis of o-glycosidically linked sialic acid-containing oligosaccharides in glycoproteins as their alditol derivatives with low-wavelength uv monitoring evidence that epidermal growth factor is present in swiftlet's (collocalia) nest a facile procedure for the isolation of n-acetylneuramic acid from edible bird's-nest influenza a virus-binding activity of glycoglycerolipids of aquatic bacteria potentiation of mitogenic response by extracts of the swiftlet's (collocalia) nest identification of a serine protease inhibitor homologue in bird's nest by an integrated proteomics approach -anhydro-nacetylneuraminic acid. isolation from edible bird's nest and structure determination a detailed study of the periodate oxidation of sialic acids in glycoproteins hemagglutinating encephalomyelitis virus attaches to n-acetyl- -oacetylneuraminic acid-containing receptors on erythrocytes: comparison with bovine coronavirus and influenza c virus observation by c nmr of interactions between cholera toxin and the oligosaccharide of ganglioside gm broad distribution of the jc virus receptor contrasts with a marked cellular restriction of virus replication swine influenza virus strains recognize sialylsugar chains containing the molecular species of sialic acid predominantly present in the swine tracheal epithelium receptor specificities of human respiroviruses sulphatide binds to human and animal influenza a viruses, and inhibits the viral infection inhibition of influenza a virus sialidase activity by sulfatide the hemagglutinins of the human influenza viruses a and b recognize different receptor microdomains structural determination of gangliosides that bind to influenza a, b, and c viruses by an improved binding assay: strain-specific receptor epitopes in sialo-sugar chains action of ortho-and paramyxovirus neuraminidase on gangliosides. hydrolysis of ganglioside gm by sendai virus neuraminidase isolation and characterization of receptor sialoglycoprotein for hemagglutinating virus of japan (sendai virus) from bovine erythrocyte membrane structure of the monosialyl oligosaccharides derived from salivary gland mucin glycoproteins of the chinese swiftlet (genus collocalia) determination of edible bird's nest and its products by gas chromatography support for this work was provided by a grant of the jsps postdoctoral fellowship for foreign researchers ( ) from the japan society for the promotion of science, and a grant-in-aid ( ) from the ministry of education, culture, sports, science and technology of japan. key: cord- - yecwdlo authors: lin, min-han; moses, david c.; hsieh, chih-hua; cheng, shu-chun; chen, yau-hung; sun, chiao-yin; chou, chi-yuan title: disulfiram can inhibit mers and sars coronavirus papain-like proteases via different modes date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: yecwdlo severe acute respiratory syndrome coronavirus (sars-cov) emerged in southern china in late and caused a global outbreak with a fatality rate around % in . ten years later, a second highly pathogenic human cov, mers-cov, emerged in the middle east and has spread to other countries in europe, north africa, north america and asia. as of november , mers-cov had infected at least people with a fatality rate of about % globally, and hence there is an urgent need to identify antiviral drugs that are active against mers-cov. here we show that a clinically available alcohol-aversive drug, disulfiram, can inhibit the papain-like proteases (pl(pro)s) of mers-cov and sars-cov. our findings suggest that disulfiram acts as an allosteric inhibitor of mers-cov pl(pro) but as a competitive (or mixed) inhibitor of sars-cov pl(pro). the phenomenon of slow-binding inhibition and the irrecoverability of enzyme activity after removing unbound disulfiram indicate covalent inactivation of sars-cov pl(pro) by disulfiram, while synergistic inhibition of mers-cov pl(pro) by disulfiram and -thioguanine or mycophenolic acid implies the potential for combination treatments using these three clinically available drugs. before , human coronaviruses (covs) had the reputation of occasionally emerging from zoonotic sources and causing mild respiratory tract infections. in late , however, without any warning, severe acute respiratory syndrome (sars) emerged and spread by coronaviral infection to become a pandemic, mainly in asia but also in other regions, with a fatality rate of % (hilgenfeld and peiris, ) . ten years later, when sars had almost been forgotten, a second highly pathogenic human cov, mers, caused the severe respiratory syndrome in the middle east and then spreading to other countries due to human activity (zaki et al., ) . mers-cov has infected at least people with a high mortality rate of % since (http://www.who.int/csr/ don/ -november- -mers-saudi-arabia/en/). because of international travel and climate change, we cannot rule out the possibility of the emergence of additional highly pathogenic covs in the near future (menachery et al., (menachery et al., , . thus, the development of antiviral drugs effective against covs is urgently needed. covs are positive-sense single-stranded rna viruses. after the virion has entered the host cell, two polyproteins, pp a and pp ab, are directly translated and then cleaved by two viral proteases, main protease (m pro ) and papain-like protease (pl pro ) (perlman and netland, ). pl pro is responsible for the cleavage of non-structural proteins (nsp) , and while m pro cleaves all junctions downstream of nsp (perlman and netland, ). in addition, pl pro can deubiquitinate or deisgylate host cell proteins, including interferon factor (irf ), and inactivate the pathway of nuclear factor κ-light-chain-enhancer of activated b cells (nf-κb), resulting in the immune suppression of host cells (clementz et al., ; frieman et al., ; yang et al., ; zheng et al., ) . due to its multiple roles in viral replication and host cell control, pl pro is considered a potential antiviral target. disulfiram is a drug which has been approved by the united states food and drug administration (fda) for use in alcohol aversion therapy since (bell and smith, ; krampe and ehrenreich, ; moore et al., ) . it is known to irreversibly inhibit hepatic aldehyde dehydrogenase (lipsky et al., ) . recent studies indicate t that disulfiram is able to inhibit other enzymes, such as methyltransferase, urease and kinase, all by reacting with important cysteine residues, suggesting broad-spectrum characteristics (diaz-sanchez et al., ; galkin et al., ; paranjpe et al., ) . in addition, there has been a clinical trial investigating the usage of disulfiram for reactivating latent hiv in order to make it accessible to highly active anti-retroviral therapy (elliott et al., ) , and the drug has also been shown to act as a "zinc ejector" with respect to hepatitis c virus ns a protein (lee et al., ) . however, the effect of disulfiram on viral cysteine proteases is still unknown. in the present study, we demonstrate that disulfiram is an inhibitor of mers-cov and sars-cov pl pro s, and furthermore that disulfiram acts on mers-cov and sars-cov pl pro via different inhibition mechanisms. moreover, we investigated the synergies between a number of known pl pro inhibitors and disulfiram, and our results point to the possibility of using combination treatments involving disulfiram and other clinically available drugs against covs. the sars-cov pl pro c a mutation was introduced using the quikchange mutagenesis kit (stratagene) and was verified by dna sequencing. the forward primer was ′-gtacactggtaactatcaggcgggtcatt acactcatata and the reverse primer was ′-tatatgagtgtaatgacccgcctgatagttaccagtgtac. the mers-cov and sars-cov pl pro s and the sars-cov pl pro c a mutant protein were produced and purified as previously described (chou et al., lin et al., ) . briefly, the cultures were grown at °c for h, then induced with . mm isopropyl β-d- -thiogalactopyranoside and grown at °c for h. the cell pellet was resuspended in lysis buffer ( mm tris, ph . , mm nacl, % glycerol, . % triton x- , mm β-mercaptoethanol (βme)), lysed by sonication and then centrifuged to remove the insoluble pellet. the target protein was purified from the fraction of soluble proteins via nickel affinity chromatography, then loaded onto an s- gel-filtration column (ge healthcare) equilibrated with running buffer ( mm tris, ph . , mm nacl, mm dithiothreitol). for the crystallization of sars-cov pl pro in complex with glycerol, the reductant was removed and μm disulfiram was added to each buffer during the purification process. the purity of the fractions collected was analyzed by sds-page and the protein was concentrated to mg/ml using an amicon ultra- -kda centrifugal filter (millipore). the dub assay was carried out as previously described (cheng et al., ; chou et al., ; lin et al., ) . the fluorogenic substrate ub- -amino- -trifluoro-methylcoumarin (ub-afc) (boston biochem) was added at a concentration of . μm along with various concentrations of inhibitors into mm phosphate (ph . ) and each mixture was incubated at °c for min. after adding . μm coronaviral pl pro , enzymatic activity was determined by continuously monitoring fluorescence intensity at excitation and emission wavelengths of and nm, respectively. the data was fitted to obtain ic according to eq. ( ): ( ) in which v is the initial velocity in the presence of inhibitor at concentration [i] and v is the initial velocity in the absence of inhibitor, while n is the hill constant. in addition, to test for the recoverability of activity, coronaviral pl pro was incubated with or without μm disulfiram for h and then desalted using a sephadex g- column. the dub activity of . μm treated enzyme was then determined in the presence or absence of mm βme. the peptidyl substrate dabcyl-frlkggapikgv-edans was used to measure the proteolytic activity of pl pro . fluorescence intensity was monitored at nm (excitation) and nm (emission) and converted to the amount of hydrolyzed substrate based on previous studies (cheng et al., ; chou et al., ) . for inhibition studies, the reaction mixture contained - μm peptide substrate with - μm disulfiram in mm phosphate (ph . ). mers-cov pl pro at . μm and wild-type sars-cov pl pro and c a mutant at . μm was used, respectively. after adding the enzyme to the reaction mixture, fluorescence intensity was continuously monitored at °c. the increase in fluorescence was linear for at least min, and thus the slope of the line represented the initial reaction velocity (v). the data obtained for the inhibition of mers-cov pl pro by disulfiram was found to best fit a noncompetitive inhibition pattern in accordance with eq. ( ): while the data obtained for the inhibition of sars-cov pl pro by disulfiram was found to best fit a competitive inhibition pattern in accordance with eq. ( ) or a mixed inhibition pattern in accordance with eq. ( ): in which k cat is the rate constant, [e], [s] and [i] denote the enzyme, substrate and inhibitor concentrations, and k m is the michaelis-menten constant for the interaction between the peptide substrate and the enzyme. k is is the slope inhibition constant for the enzyme-inhibitor complex and αk is is the slope inhibition constant for the enzyme-substrate-inhibitor complex. the program sigmaplot . (systat software inc., usa) was used for data analysis. to characterize the mutual effects of disulfiram and other known pl pro inhibitors, the activity of mers-cov pl pro was measured with and without either -thioguanine ( tg) ( and μm) or mycophenolic acid (mpa) ( and μm) in the presence of various concentrations of disulfiram ( - μm), and that of sars-cov pl pro was measured with and without either tg or n-ethylmaleimide (nem) in the presence of various concentrations of disulfiram ( - μm). the concentrations of the peptidyl substrate and mers-cov pl pro were and . μm, respectively, while those of the substrate and sars-cov pl pro were and . μm, respectively. data obtained from the reactions were fitted to eq. ( ): where v is the initial velocity in the presence of both inhibitors, [i] and [j] are the concentrations of the two inhibitors, v is the velocity in the absence of inhibitors, k i and k j are the apparent dissociation constants for the two inhibitors, and α is a measurement of the degree of interaction between the two inhibitors (copeland, ; yonetani and theorell, ) . release of zinc ions from coronaviral pl pro s was monitored as the increase in fluorescence emission from the zinc-specific fluorophore fluozin- (thermo fisher scientific) (lee et al., ) . briefly, the protein and fluozin- were mixed in mm phosphate buffer (ph . ) to concentrations of μm and μm, respectively, in the presence or absence of μm disulfiram. fluorescence emission was continuously measured at °c using emission and excitation wavelengths of nm and nm, respectively, in a perkinelmer ls b luminescence spectrometer. the change in secondary structure of coronaviral pl pro s in the absence and presence of μm disulfiram was continuously measured using ellipticity at nm as the temperature was ramped from to °c in a jasco j- spectropolarimeter. the protein at μm was dissolved into mm phosphate buffer, ph . . the width of the cuvette was mm. for the inactivation studies, sars-cov pl pro ( . μm in mm phosphate buffer, ph . ) was incubated with different concentrations of disulfiram and peptide substrate, and enzymatic activity was traced for min. all progress curves recorded showed an exponential course and were analyzed according to the following integrated rate equation (eq. ( )) (copeland, ) : in which v i is the initial velocity, v s is the steady-state velocity, and d is the displacement on the y-axis. the replot of k inact versus the concentration of disulfiram was fitted to a saturation curve according to eq. ( ) (copeland, ) : in which k inact is the dissociation constant of the enzyme-disulfiram complex and k max is the maximum inactivation rate constant. crystals of sars-cov pl pro in complex with βme or glycerol were obtained at °c by the sitting-drop vapor-diffusion method. for the pl pro -βme complex, the protein at mg/ml was incubated with . mm disulfiram for h and then crystallized. single crystals were grown in reservoir solution containing % (w/v) peg and . m bis-tris propane (ph . ). for the pl pro -glycerol complex, protein purified with the addition of μm disulfiram into each buffer during the purification process was crystallized at . mg/ml. single crystals were grown in reservoir solution containing % (w/v) peg and . m hepes (ph . ). all crystals were cryoprotected in reservoir solution supplemented with % and % (v/v) glycerol for pl pro -βme and pl pro -glycerol, respectively, and then flash-cooled in liquid nitrogen. . . data collection and structure determination x-ray diffraction data was collected at k on the spxf beamline a at the national synchrotron radiation research center, taiwan, roc using a rayonix mx he ccd detector at a wavelength of Å. the diffraction images were processed and then scaled with the hkl- package (otwinowski and minor, ) . the structure was solved by the molecular-replacement method with phaser (mccoy et al., ) using the structure of wild-type sars-cov pl pro (pdb entry fe ; (ratia et al., ) ) as the search model. manual rebuilding of the structure model was performed with coot (emsley and cowtan, ) . structure refinement was carried out with refmac (murshudov et al., ) . data-processing and refinement statistics are summarized in table . the crystal structures of the sars-cov pl pro -βme complex and sars-cov pl pro -glycerol complex have been deposited in the protein data bank (pdb entries y q and y e for pl pro -βme and pl pro -glycerol, respectively). . . the inhibition of mers-cov and sars-cov pl pro s by disulfiram pl pro s are cysteine proteases that use the thiol group of cysteine as a nucleophile to attack the carbonyl group of the scissile peptide bond han et al., ; verma et al., ) . inhibition can be expected if the catalytic cysteine of a pl pro is interfered with or modified (cheng et al., ; chou et al., ) . disulfiram is known to be a thiol-reactive compound that can covalently modify cysteine residues (diaz-sanchez et al., ; galkin et al., ; lipsky et al., ; paranjpe et al., ) . to determine whether disulfiram can inhibit coronaviral pl pro s, the dub activity of mers-cov and sars-cov pl pro was measured in the presence of various concentrations of disulfiram. interestingly, disulfiram showed a dose-dependent inhibitory effect on both proteases with ic values in the micromolar range ( fig. ) . next, to elucidate the kinetic mechanisms of the interactions between disulfiram and the two pl pro s, the proteolytic activity of each enzyme was measured in the presence of various concentrations of a peptidyl substrate and disulfiram. the results were then fitted to different kinetic models (competitive, noncompetitive, uncompetitive and mixed inhibition). surprisingly, disulfiram showed a noncompetitive inhibition pattern against mers-cov pl pro ( fig. a ) but a competitive inhibition pattern against sars-cov pl pro (fig. b ). this inconsistency is quite intriguing since the two enzymes share a similar overall structure and an identical catalytic triad (bailey-elkin et al., ; chou et al., ; lei et al., ; ratia et al., ) , albeit the inhibition constant (k is ) of disulfiram for mers-cov pl pro is . -fold higher than that for sars-cov pl pro (table ). perhaps this discovery should not be surprising given that disulfiram is also a noncompetitive inhibitor for citrullus vulgaris urease with a k is of . μm (diaz-sanchez et al., ), while its ic for giardia lamblia carbamate kinase is . - . μm (chen et al., ) . similarly, a previous study mentions that their compound also has different recognition specificity for the two pl pro s . our study once again suggests broad-spectrum potency for disulfiram, given the versatility it shows even against two coronaviral pl pro s. the inconsistent inhibitory effect of disulfiram against the two pl pro s suggests that the binding modes of disulfiram on the two enzymes may be different. to verify this, multiple inhibition assays using disulfiram and other known pl pro inhibitors, including tg, mpa and nem, were performed (fig. ) (chen et al., ; cheng et al., ; yonetani and theorell, ) . interestingly but not surprisingly, we found that disulfiram displays a synergistically inhibitory effect with either tg or mpa on mers-cov pl pro , with the lines in the yonetani-theorell plots intersecting above the x-axis and α values below in both cases (fig. a and b) (copeland, ) . in contrast, in the case of sars-cov pl pro , each of the plots displays two parallel lines and both α values are significantly higher than (fig. c and d) , suggesting that binding of disulfiram and of tg or nem are mutually exclusive on sars-cov pl pro (copeland, ) . since tg is a competitive inhibitor of both pl pro s (cheng et al., ; chou et al., ) , the contrasting synergy of disulfiram and tg on the two pl pro s confirms the inconsistent inhibitory pattern of disulfiram (figs. and ) . furthermore, mpa has previously been shown to be a noncompetitive inhibitor of mers-cov pl pro and to work synergistically with tg to inhibit mers-cov pl pro (cheng et al., ) . combining those results with our results regarding the binding synergy of disulfiram and tg or mpa (fig. a and b), we propose that disulfiram may occupy a third binding site on mers-cov pl pro , neither a site at the active center nor the mpa binding site. next, we evaluated pl pro inhibition in the presence of disulfiram combined with tg and/or mpa by proteolytic assays using a peptidyl substrate. we found that the ic of disulfiram against mers-cov pl pro showed a . -fold decrease in the presence of μm tg and a . -fold decrease at μm tg when it was tested in combination with μm mpa (table ) . for comparison, in the case of disulfiram against sars-cov pl pro , there is no enhanced inhibitory effect in the presence of tg or nem. our results suggest a potential for using the above three fdaapproved drugs in combination treatments against mers-cov. incidentally, previous studies have suggested that mpa may be used in combination treatments with interferon against mers-cov (chan et al., ) . the experiments were repeated to ensure reproducibility. kinetic parameters such as k m , k cat and k is from the best-fit results are shown in table . in the presence of disulfiram, the best-fitted kinetic parameters and k is were determined in accordance with competitive (eq. ( )) or mixed inhibition (eq. ( )) and noncompetitive (eq. ( )) inhibition models for sars-cov and mers-cov pl pro , respectively. c the value is αk is , the inhibition constant for the enzyme-substrate-inhibitor complex. d k inact and k max values are from the best fit to the saturation equation (eq. ( )). previous studies suggested that disulfiram can bind to the zincbound cysteines in hepatitis c virus ns a protein (lee et al., ) . as there are four cysteines bound to a zinc ion in pl pro s ( fig. s c and s d ) (bailey-elkin et al., ; chou et al., ) , we performed zinc ejection assays to test whether these zinc-bound cysteines may be a candidate for the aforementioned "third binding site" occupied by disulfiram on mers-cov pl pro . in the present study, the zinc-specific fluorophore, fluozin- , was used to identify the release of zinc ion due to the binding of disulfiram to the enzyme (fig. a) . unexpectedly, we observed significant zinc release in the presence of disulfiram not only from mers-cov pl pro but also from sars-cov pl pro . this result indicates that disulfiram may bind not only to the active site but also to the zinc-binding sites in sars-cov pl pro . following this finding, we tried to fit our inhibitory results to a mixed inhibition model (fig. s ). the two k is for the enzyme-substrate and enzyme-substrate-inhibitor complexes were . and . μm, respectively, showing a . -fold difference in the binding affinity for the two putative binding sites (table ). this significant difference may explain why the inhibitory pattern of disulfiram against sars-cov pl pro looks more like competitive inhibition. next, the thermostability of the two pl pro s in the absence and presence of disulfiram was evaluated (fig. b) . not surprisingly, the melting temperature of both pl pro s decreased - °c in the presence of disulfiram. these results conform to our earlier finding that the release of zinc ion can destabilize pl pro (chou et al., ) . )). (c) the observed inactivation rate constants (k inact ) from panel b were replotted against disulfiram concentration. the solid line is the best-fit result in accordance with the saturation equation (eq. ( )). kinetic parameters k inact and k max corresponding to the best-fit curve are shown in table . disulfiram is known to covalently modify cysteine residues and leave a diethyldithiolcarbamate (ddc) moiety to inactivate the carbamate kinase of giardia lamblia (galkin et al., ) . in the presence of mm βme, however, the inhibitory effect of disulfiram against pl pro s is minor and the ic is larger than μm (table ). this suggests that the reductant can protect the enzyme and, therefore, that disulfiram may inhibit the enzyme by modifying the cysteine in the catalytic triad (cys -his -asp ). to further investigate this possibility, the dub activity of the enzyme was measured after incubation with μm disulfiram for h followed by removal of the small molecules using a sephadex g- column. this treatment resulted in an % loss of activity, suggesting irreversible inhibition of sars-cov pl pro by disulfiram (fig. a, right panel) . similarly, in a previous in vivo study, disulfiram-treated aldehyde dehydrogenase showed % enzyme inhibition as compared to the activity of the control (lipsky et al., ) . next, the disulfiram-treated sars-cov pl pro was incubated with mm βme for min, after which activity was measured to test for re-activation. we found that % of the enzyme's activity was restored after treatment with βme (fig. a, right panel) . the rescuing effect of the reductant suggests that the modification was due to the disulfide bonding interaction between the enzyme and the inhibitor. however, in the case of mers-cov pl pro , we found that treatment with disulfiram resulted in an irreversible loss of activity which was not rescued by the addition of the reductant (fig. a, left panel) . previous studies have suggested that the release of zinc ion following treatment with edta will lead to a % loss of pl pro activity (chou et al., ) . this result is consistent with the effect of disulfiram on pl pro s. also, the inability of the reductant to rescue the dub activity of mers-cov pl pro , suggesting that disulfiram cannot influence its active site, is compatible with disulfiram's noncompetitive mode of inhibition of the enzyme. on the other hand, proteolytic assays of sars-cov pl pro at various concentrations of disulfiram showed dose-and time-dependent decay when enzyme activity was measured for min (fig. b) . by fitting the data to eq. ( ), different k inact values at various concentrations of disulfiram were determined and then plotted versus those disulfiram concentrations (fig. c ). the saturated curvature suggests a slowbinding phenomenon due to covalent inactivation (copeland, ) , a conclusion supported by the irrecoverability of enzyme activity after disulfiram removed (fig. a) . best-fit analysis determined a k inact of . μm and a k max of . s − (fig. c and table ). interestingly, the k inact value is close to k is , indicating that disulfiram may inactivate the enzyme very soon after binding. for comparison, previous studies have indicated that -mercaptopurine and tg are also slow-binding inhibitors against the same enzyme, albeit enzyme activity was recovered after removing the inhibitors (chou et al., ) . the structure of sars-cov pl pro in complex with disulfiram should allow us to understand the binding mechanism more clearly. accordingly, we attempted to crystallize sars-cov pl pro in the presence of disulfiram. unfortunately, although crystals of the protein were formed in the presence of . mm disulfiram, the crystal structure showed only βme-like electron density near the active-site cysteine with no omit electron density shown near the zinc-binding site ( fig. s a and s c ). βme is a reducing agent that is added into the purification buffer to stabilize the protein, and which is also known to reverse the effect of disulfiram (table , fig. a and kitson, ) . to avoid this effect, we eliminated all reducing agents from the purification process, added μm disulfiram into all purification buffers, and then attempted to crystallize the protein purified under these conditions. although we were able to grow crystals under different crystallization conditions, we again obtained an unexpected result, as the only omit electron density near the catalytic site was fitted as a glycerol molecule ( fig. s b and s d ). this result might be due to the crystals having been cryoprotected in reservoir solution supplemented with % (v/v) glycerol. nevertheless, the binding of βme and glycerol near the active site suggests that the active site may be accessible to disulfiram. next, using the aforementioned two complex structures, a disulfiram and a ddc molecule were docked into the glycerol and βme binding sites, respectively (fig. ) . ddc may be able to covalently bind to residue cys in a manner similar to that of βme (fig. a ), while disulfiram may be able to occupy the glycerol site (fig. b) . interestingly, in the docking structure of the pl pro -disulfiram complex, we can see that one sulfur atom of the disulfide bond of disulfiram is within Å of residue cys at blocking loop (bl ), which is very important for substrate and inhibitor binding ratia et al., ) . for comparison, there is a valine at the same site in mers-cov pl pro (bailey-elkin et al., ; chou et al., ) . to verify the possible inhibitory effect of disulfiram due to binding to residue cys , inhibition of the sars-cov pl pro c a mutant by disulfiram was measured (fig. s ) . interestingly, we can see a . -fold increase in ic ( table ) compared with that for inhibition of wild-type sars-cov pl pro by disulfiram. in addition, the decrease of the melting temperature of the c a mutant following treatment with disulfiram is °c, lower than that of wild-type sars-cov pl pro treatment with the same inhibitor ( fig. b and c) . these findings suggest that disulfiram may inhibit sars-cov pl pro partly via the residue cys and support the reliability of the docking of disulfiram on the glycerol binding site. based on our kinetic and structural results, we propose kinetic mechanism schemes for the inhibition of the two pl pro s by disulfiram (fig. ) . similar to the mechanism in the case of disulfiram-treated urease (diaz-sanchez et al., ) , disulfiram may form a covalent adduct with sars-cov pl pro and then leave a ddc on the active-site cys , preventing downstream acylation and thereby inactivating the fig. . binding of disulfiram to sars-cov pl pro . overlay of model structure of sars-cov pl pro in complex with ddc (magenta) (a) or disulfiram (orange) (b) with the crystal structure of sars-cov pl pro in complex with ubiquitin (gray, pdb code: m w). ddc and disulfiram are modeled based on the binding sites of βme and glycerol, respectively. the red dashed lines show putative polar interactions while the black dashed line shows the distance between residue cys and disulfiram as . Å. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) enzyme. in contrast, disulfiram shows a noncompetitive inhibitory effect against mers-cov pl pro and can synergistically inhibit that enzyme with tg and mpa. in this study, we found that disulfiram is, respectively, a noncompetitive and competitive (or mixed) inhibitor of mers-cov and sars-cov pl pro s. multiple inhibition assays also support a kinetic mechanism by which disulfiram together with tg and/or mpa can synergistically inhibit mers-cov pl pro , but not, due to its competitive mode of inhibition, sars-cov pl pro . on the other hand, the results of kinetic assays, continued inactivation after the removal of disulfiram, reactivation by reductant, and the phenomenon of slow-binding inhibition suggest that disulfiram may act at the active site of sars-cov pl pro , forming a covalent adduct with residue cys . crystal structures of the enzyme in complex with glycerol and βme imply that the active site is solvent-exposed and accessible for disulfiram or ddc binding. fig. . schemes of proposed kinetic mechanisms for the inhibition of sars-cov and mers-cov pl pro by disulfiram. the upper diagram denotes enzyme catalysis, mixed inhibition and inactivation of sars-cov pl pro by disulfiram. the lower diagram shows noncompetitive inhibition of mers-cov pl pro by disulfiram and triple inhibition with two other fda-approved drugs, tg and mpa. sh symbolizes the thiolate of catalytic triad residue cys. crystal structure of the middle east respiratory syndrome coronavirus (mers-cov) papainlike protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression preliminary report on clinical trials of antabuse broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus a homogenous luminescence assay reveals novel inhibitors for giardia lamblia 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features structural and functional characterization of mers coronavirus papainlike protease in vivo inhibition of aldehyde dehydrogenase by disulfiram phaser crystallographic software a sars-like cluster of circulating bat coronaviruses shows potential for human emergence sars-like wiv -cov poised for human emergence sheep liver cytosolic aldehyde dehydrogenase: the structure reveals the basis for the retinal specificity of class aldehyde dehydrogenases refmac for the refinement of macromolecular crystal structures processing of x-ray diffraction data collected in oscillation mode disulfiram is a direct and potent inhibitor of human o -methylguanine-dna methyltransferase (mgmt) in brain tumor cells and mouse brain and markedly increases the alkylating dna damage coronaviruses post-sars: update on replication and pathogenesis a noncovalent class of papain-like protease/deubiquitinase inhibitors blocks sars virus replication severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme cysteine proteases: modes of activation and future prospects as pharmacological targets proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease studies on liver alcohol hydrogenase complexes. . multiple inhibition kinetics in the presence of two competitive inhibitors isolation of a novel coronavirus from a man with pneumonia in saudi arabia plp , a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production we would like to thank ziad omran for helpful suggestions. this research was supported by grants from the ministry of science and technology, taiwan, roc ( - -b- - , - -b- - and - -b- - ) to cyc and a cgmh-nymu joint research grant (cmrpg f ) to cys and cyc. we are grateful for the experimental facilities and the technical services provided by the synchrotron radiation protein crystallography facility, which is supported by the national core facility program for biotechnology, ministry of science and technology, taiwan, roc, and the national synchrotron radiation research center, a national user facility supported by the ministry of science and technology, taiwan, roc. supplementary data related to this article can be found at http://dx. doi.org/ . /j.antiviral. . . . key: cord- -vgdjpjkj authors: chen, sunrui; wang, yining; li, pengfei; yin, yuebang; bijvelds, marcel; de jonge, hugo; peppelenbosch, maikel p.; kainov, denis; pan, qiuwei title: drug screening identifies gemcitabine inhibiting rotavirus through alteration of pyrimidine nucleotide synthesis pathway date: - - journal: antiviral res doi: . /j.antiviral. . sha: doc_id: cord_uid: vgdjpjkj although rotavirus infection is usually acute and self-limiting, it can cause chronic infection with severe diseases in immunocompromised patients, including organ transplantation recipients and cancer patients irrespective of pediatric or adult patients. since no approved medication against rotavirus infection is available, this study screened a library of safe-in-man broad-spectrum antivirals. we identified gemcitabine, a widely used anti-cancer drug, as a potent inhibitor of rotavirus infection. we confirmed this effect in d cell cultures and d cultured human intestinal organoids with both laboratory-adapted rotavirus strains and five clinical isolates. supplementation of utp or uridine largely abolished the anti-rotavirus activity of gemcitabine, suggesting its function through inhibition of pyrimidine biosynthesis pathway. our results support repositioning of gemcitabine for treating rotavirus infection, especially for infected cancer patients. rotavirus infection is the leading cause of severe dehydrating gastroenteritis among children under five-year-old (greenberg and estes, ) . although rotavirus infection is usually acute and self-limiting, it can cause chronic infection with severe diseases in immunocompromised patients, in particular organ transplantation recipients irrespective of pediatric or adult patients (yin et al., b) . in addition, cancer patients have compromised immune system especially when undergoing chemotherapy or radiotherapy treatment, which make them prone to infections with worse outcomes (hotchkiss and moldawer, ) . rotavirus infections have been widely reported in pediatric or adult cancer patients causing prolonged diarrhea (akhtar et al., ; ghosh et al., ) . therefore, specific and effective antiviral treatment is urgently needed for these special populations who are infected with rotavirus, but there are no fda-approved medications available against rotavirus infection. developing new drugs usually takes more than ten years with enormous investment and high risk of failure. given that only the specific population with rotavirus infection require antiviral treatment, the pharmaceutical industry will likely not develop new anti-rotavirus drugs. we propose that repurposing existing drugs represents a costeffective approach to identify antiviral treatment that can readily benefit patients (qu et al., ) . in this study, we screened a library of safe-in-man broad-spectrum antiviral agents (bsaas, https://drugvirus.info) (andersen et al., ; ianevski et al., ) on rotavirus infection in experimental models. these compounds are known to target viruses belonging to two or more viral families and have passed phase clinical trials. this greatly enhances the probability of identifying novel activities of some of these agents against rotavirus infection and facilitates their clinical translation. we first screened bsaas in human intestinal caco cell line infected with simian rotavirus sa strain (table s ) . to minimize non-specific effects on host cells, we used low concentration of µm and treated for hours. by qrt-pcr (primers listed in table s ) quantification of rotavirus genomic rna, we identified candidates exerting over % inhibitory effects, and with inhibition over % (fig. a) . among these, gemcitabine was one of the most effective candidates (fig. a) . it is a cytidine analog that has been widely used for cancer treatment (cerqueira et al., ; zhang et al., ) . it has been shown to inhibit a broad range of rna viruses including severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), zika virus and hepatitis c virus (hcv) in experimental models (beran et al., ; dyall et al., ; kuivanen et al., ) . nucleotide and nucleoside analogues are excellent examples of bsaas that have been widely used in the clinic for treating infections of rna and dna viruses (ianevski et al., ) . we have previously demonstrated that nucleotide, including purine and pyrimidine, biosynthesis pathways are essential in regulating rotavirus infection and can be pharmacologically targeted yin et al., a; yin et al., ) . as a cytidine analog, gemcitabine has been reported to inhibit pyrimidine biosynthesis, resulting in nucleotide depletion (lee et al., ) . therefore, we focused on the effects and mode-of-action of gemcitabine on rotavirus infection in this study. we next tested a series of gemcitabine concentrations ( . - µm) in the caco cell model to assess both antiviral and cytotoxic effects. we confirmed the potent antirotavirus effect and observed a large therapeutic window between cytotoxic and antiviral activities, as shown the cc value of . mm and ic value of . µm ( fig. b, c) . we performed same experiments in the monkey ma cell line that is widely used for propagating rotavirus in laboratory. similar trends were observed with cc value of . mm and ic value of . µm (supplementary fig. s ). by harvesting supernatant of caco cells at hour post-treatment, we performed tcid assay to determine the titers of secreted viruses. consistently, the titers of produced rotavirus with infectivity were significantly reduced by gemcitabine treatment (fig. d) . at protein level, we found potent inhibition of viral protein (vp ) expression determined by western blotting (fig. e) . furthermore, immunofluorescent staining of viral capsid protein (vp ) showed significant reduction of the number of infected caco cells by gemcitabine treatment (fig. f, g, supplementary fig. s ). the responsiveness to antiviral therapy can vary dramatically in patients. this mainly attributes to host and viral factors. the nucleoside analog ribavirin has been used for treating hcv infection for decades. reduced cellular uptake by the host and mutagenesis of the viral genome have been linked to treatment resistance in chronic hcv patients (ibarra et al., ) . as recently repositioned for treating chronic hepatitis e virus (hev) infection (kamar et al., ) , viral mutagenesis during ribavirin treatment or pre-deposition of resistance mutations are thought to contribute to treatment failure (ikram et al., ) . our previous studies have evaluated the effects of the nucleoside analog ribavirin and mycophenolic acid (mpa), and the antiviral cytokine interferon alpha (ifn-α) on rotavirus in cell culture models. we found the responsiveness to these agents dramatically vary among different clinical isolates from potent, moderate, minimal to even pro-viral effects (yin et al., a; yin et al., ) . this imposes major challenges for clinical application as how to personalize the selection of potential responders. thus, we extended our evaluation of gemcitabine to different rotavirus strains/isolates. we found that gemcitabine significantly inhibits the replication of rhesus rotavirus rrv strain ( fig. a) and five clinical isolates (fig. b- f) . based on these results, we postulate that the response to gemcitabine in rotavirus patients could be universally effective. we have previously established modeling of rotavirus infection in intestinal organoids, which allows the study of virus-host interactions and assessment of antiviral drugs (yin et al., a; yin et al., a; yin et al., b; yin et al., ) . intestinal organoids, also called mini-gut, are stem cells-derived epithelial cultured in d structure. these organoids are much better in recapitulating the architecture, composition, diversity, organization and functionality of cell types of the intestine. potently inhibited viral rna synthesis and secretion of rotaviruses with approximately % inhibitory effects for both at µm concentration treated for hours ( fig. a; supplementary fig. s ). this effect was further confirmed at vp protein level by western blot assay (fig. b) . importantly, rotavirus infection led to morphological and pathological changes in organoids. based on optical imaging, we observed that rotavirus infected organoids without gemcitabine treatment showed opaque, wizened and disorganized morphology. in contrast, gemcitabine treated groups, in particular with µm concentration, most of the organoids were hyaline and in a spheroidal shape (fig. c upper panel) . in confocal immunostaining, vp protein was detected in all groups, but the intensity and frequency of the viral protein were lower in treatment groups (fig. c middle panel) . fluorescence staining of cell viability by propidium iodide (pi) showed that cell death (determined as described in supplementary fig. s ) in un-treated group is more obvious, and dead cells were diffused in almost all organoids (fig. c lower panel) . this is consistent with the percentage of deteriorated organoids (fig. d) . thus, inhibition of rotavirus replication by gemcitabine can protect organoids from rotavirus induced cytopathogenesis. the antiviral activity of gemcitabine has been linked to the inhibition of pyrimidine biosynthesis pathway, especially in salvage pathway (lee et al., ) . we thus supplemented utp or uridine to rotavirus infected caco cell model when treated with gemcitabine. we found that exogenous supplementation of pyrimidine dosedependently abolished the anti-rotavirus effect of gemcitabine. the anti-rotavirus effect was almost completely abolished by addition of μm utp or uridine (fig. a, b) . hence, the anti-rotavirus activity of gemcitabine was largely dependent on the salvage pyrimidine biosynthesis pathway. combination approaches are often used in clinic to achieve optimal antiviral efficacy and avoid resistance development. we finally assessed the combinatory effects of gemcitabine with ribavirin, mpa or ifn-α, as we previously have demonstrated the anti-rotavirus effects of these three agents (yin et al., a; yin et al., ) . we found enhanced anti-rotavirus activity when combined with ribavirin or ifn-α, but not mpa (fig. c) . in summary, we have identified gemcitabine as a potent inhibitor against rotavirus infection through screening of a bsaa library. the antiviral activity was largely dependent on the pyrimidine biosynthesis pathway. because gemcitabine has been widely used as chemotherapy for cancer patients, and these patients are at risk of infections (hotchkiss and moldawer, ) . the use of gemcitabine in cancer patients probably mitigates the risk of viral infections in general. our results support repositioning of gemcitabine for treating rotavirus infection, especially in infected cancer patients. furthermore, the approach of discovering new antiviral therapy from bsaas bears essential implications in combating emerging viral pathogens, such as the ongoing coronavirus disease covid- pandemic (mahase, ). and secreted viruses calculated as genomic copy number were analyzed by qrt-pcr (n = ). standard curve for calculation of genomic copy number is included in supplementary fig. s . (b) the expression of viral structural protein vp was stained and quantified by western blot assay (n = ). (c) optical microscopy images, confocal images and indirect fluorescence microscope images of hios from mock group and gct treated groups. in confocal images, vp protein was stained in red, green signal represents epcam, and nuclei were visualized by dapi (blue). in fluorescence microscope images, hios were stained by pi (red) indicating dead cells, hoechst (blue) for nuclear, and calcein (green) as live cells. (d) rate of deteriorated hios was calculated as dead organoids/ total organoids (n = ). data represent means ± sem; *p < . ; **, p < . . cell model (n = ). (c) the combinatory effects of µm gct with u ifn-α, µg/ml ribavirin (rib) or . µg/ml mycophenolic acid (mpa), respectively (n = ). data represent means ± sem; *p < . ; **p < . . detection of rotavirus in paediatric oncology patients with diarrhoea: the impact of rotavirus vaccine discovery and development of safe-in-man broad-spectrum antiviral agents cellular growth kinetics distinguish a cyclophilin inhibitor from an hsp inhibitor as a selective inhibitor of hepatitis c virus understanding ribonucleotide reductase inactivation by gemcitabine suppression of pyrimidine biosynthesis by targeting dhodh enzyme robustly inhibits rotavirus replication repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection viral associated diarrhea in immunocompromised and cancer patients at a large comprehensive cancer center: a -year retrospective study rotaviruses: from pathogenesis to vaccination parallels between cancer and infectious disease novel activities of safe-inhuman broad-spectrum antiviral agents host-based ribavirin resistance influences hepatitis c virus replication and treatment response modeling rotavirus infection and antiviral therapy using primary intestinal organoids -thioguanine inhibits rotavirus replication through suppression of rac gdp/gtp cycling pi k-akt-mtor axis sustains rotavirus infection via the e-bp mediated autophagy pathway and represents an antiviral target rotavirus in organ transplantation: drug-virus-host interactions mycophenolic acid potently inhibits rotavirus infection with a high barrier to resistance development gemcitabine and cisplatin induction chemotherapy in nasopharyngeal carcinoma gemcitabine, a widely used anti-cancer drug, has potent antiviral activity against rotavirus infection the antiviral effect of gemcitabine has been confirmed with both laboratoryadapted strains and clinical isolates gemcitabine exerts its anti-rotavirus effect through inhibiting pyrimidine biosynthesis pathway the authors declare that they have no competing key: cord- - t yuq v authors: takano, tomomi; katoh, yasuichiroh; doki, tomoyoshi; hohdatsu, tsutomu title: effect of chloroquine on feline infectious peritonitis virus infection in vitro and in vivo date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: t yuq v feline infectious peritonitis (fip) is a feline coronavirus-induced fatal disease in domestic and wild cats. several studies have investigated potential treatments for fip. however, there have been no reports on agents that have exhibited a therapeutic effect. recently, chloroquine has been reported to antiviral effect. we investigated whether chloroquine can be used to treat fip in vitro and in vivo. it was demonstrated that chloroquine has inhibitory effect against the replication of fipv and anti-inflammatory effect in vitro. in vivo study using cats with experimentally induced fip, the clinical score of chloroquine-treatment groups were better than in chloroquine-untreated group. however, alanine aminotransferase levels increased in the chloroquine-treated groups. it will be necessary to further investigate the possibility of fip treatment with a combination of chloroquine and other agents. coronaviruses cause diseases of the respiratory, digestive, and nervous systems in mammals and birds. after severe acute respiratory syndrome coronavirus (sars-cov) was newly identified as a pathogen of fatal respiratory disease in (ksiazek et al., ) , new coronavirus species have been isolated from various animals including humans. recently, it has been suggested that coronavirus may be transmitted from animals to humans (müller et al., ) . feline infectious peritonitis virus (fip virus: fipv), a feline coronavirus (fcov) of the family coronaviridae, causes a fatal disease called fip in wild and domestic cat species. cats that developed fip were affected in several organs, including the liver, lungs, spleen, and central nervous system, forming lesions accompanied by necrosis and pyogenic granulomatous inflammation (pedersen, ) . in some cats, pleural effusion and ascitic fluid accumulated. monocytes and macrophages (monocytes/macrophages) play an important role in the pathogenesis of fip. it has been reported that virus replication in monocytes/macrophages induced interleukin (il)- b, il- , and tumor necrosis factor (tnf)-a production (regan et al., ; takano et al., b takano et al., , . fipv replication and cytokine production are enhanced when monocytes/macrophages are inoculated with fipv in the presence of anti-fipv s antibodies (antibody-dependent enhancement: ade) (hohdatsu et al., ; corapi et al., ; olsen et al., ) . the phenomenon of ade is involved in the aggravation of the pathology of fip (pedersen and boyle, ; takano et al., b) . over the past forty years, several studies have investigated potential treatments for fip (hartmann and ritz, ) . antiviral, immunostimulating, and immunosuppressive drugs have been experimentally used for the treatment of fip, but none of these have exhibited a sufficient therapeutic effect. several agents that significantly inhibit fcov replication in vitro have been identified (balzarini et al., ; barlough and shacklett, ; hsieh et al., ; kim et al., ; takano et al., ) ; however, whether or not these agents exhibit a therapeutic effect in cats with fip has not been investigated. the anti-malarial drug, chloroquine, has been reported to inhibit the replication of human immunodeficiency virus (hiv), influenza a/h n virus, sars-cov, and human coronavirus e (kono et al., ; murray et al., ; savarino et al., ; vincent et al., ; yan et al., ) . chloroquine also has been used to treat immune-mediated inflammatory disorders (karres et al., ; landewé et al., ; lesiak et al., ) . fip is a viral infection that causes an immune-mediated inflammatory disease. on the basis of these findings, chloroquine may be effective as a therapeutic drug for fip. we investigated whether or not chloroquine inhibited fipv replication in vitro and the fipv-induced ade activity of monocytes. furthermore, we investigated the effect of chloroquine for cats with experimentally induced fip. felis catus whole fetus- (fcwf- ) cells were grown in eagles' minimum essential medium containing % l- medium, % fetal calf serum (fcs), and antibiotics. feline monocytes were cultured in rpmi medium containing % fcs and antibiotics. strain fipv - was grown in fcwf- cells. strain fipv - was supplied by dr. m.c. horzinek (utrecht university). mab - - (igg a) used in the present study recognizes the spike (s) protein of type ii fipv, as demonstrated by immunoblotting. it has been reported that mab - - exhibits a neutralizing activity in fcwf- cells, and an enhancing activity in feline monocytes/macrophages depending on the reaction conditions (hohdatsu et al., ) . feline monocytes were isolated from specific pathogen-free (spf) cats as previously described by dewerchin et al. ( ) . confluent fcwf- cell monolayers were cultured in medium containing chloroquine (wako, japan) in -well multi-plates at °c for h. after washing, fipv strain - (  tcid ) was added to the culture and adsorbed to the cells at °c for h in the presence of chloroquine. after washing, cells were cultured in medium containing chloroquine, and culture supernatants were collected after day. the experiment using feline monocytes from spf cats was performed as follows: feline monocytes (  cells) were cultured in this medium in -well multiplates at °c for h. after washing, fipv strain - (  tcid ) reacted with or without mab - - was added to the culture and adsorbed to the cells at °c for h in the presence of chloroquine. after washing, cells were cultured in medium containing chloroquine, and culture supernatants and cells were collected after days. as the virus titer in the culture supernatant, tcid was calculated by the method of reed-muench. cells were used to measure il- b, tnf-a, and il- mrna, and fcov n genes. to evaluate the cytotoxic effects of chloroquine in feline cells, cell viability was measured by the wst- assay as described before (takano et al., b) . the percent viability was calculated using the following formula: cell viability (%) = (od of chloroquine-treated cells/od of chloroquine-untreated cells)  . fipv strain - (  tcid ) was added to the culture and adsorbed to the fcwf- cells at °c for h. after washing, cells were cultured in medium containing chloroquine, and culture supernatants were collected after day. the experiment using feline monocytes from spf cats was performed as follows: fipv strain - (  tcid ) reacted with or without mab - - was added to the culture and adsorbed to the feline monocytes at °c for h. after washing, cells were cultured in medium containing chloroquine, and culture supernatants and cells were collected after days. the experiment using feline monocytes from cats with fip (fip cats) was performed as follows: feline monocytes were cultured in medium containing chloroquine, and culture supernatants and cells were collected after days. as the virus titer in the culture supernatant, tcid was calculated. cells were used to measure cytokine mrna, and fcov n genes. rna isolation and cdna preparation were performed employing the method of takano et al. ( a) . . . determination of feline cytokine mrna and fcov n gene expression levels cdna was amplified by pcr using specific primers as shown in table . pcr was performed using the method of takano et al. ( a) . band density was quantified under appropriate uv exposure by video densitometry using image j software (nih, u.s.a.). cytokine mrna and fcov n genes were quantitatively analyzed in terms of the relative density value to the mrna of the housekeeping gene gapdh. nine spf cats were randomly assigned to three experimental groups. in accordance with the experimental schedule indicated in fig. . group a: chloroquine ( mg/kg) was subcutaneously administered every days from days before fipv inoculation. strain fipv - ( tcid /ml) was inoculated orally to cats. group b: hanks' balanced salt solution (hbss) was subcutaneously administered every days from days before fipv inoculation. strain fipv - ( tcid /ml) was inoculated orally to cats. hbss administration was completed days after fipv inoculation, and chloroquine ( mg/kg) was subcutaneously administered every days thereafter instead of hbss. group c: hbss was subcutaneously administered every days from days before fipv inoculation. strain fipv - ( tcid /ml) was inoculated orally to cats. one animal in the group c died unexpectedly before the challenge of fipv, and was excluded from this study. cats were checked daily for clinical signs, and we measured their body temperature and weight. fip diagnoses were confirmed upon postmortem examination, revealing peritoneal and pleural effusions, and pyogranuloma in the major organs. to evaluate the condition of cats, we monitored karnofsky's score. karnofsky's score was calculated in reference to the report by hartmann and kuffer ( ) , ritz et al. ( ) . the protocol for the experiments in the present table the cytotoxic effects of chloroquine in fcwf- cells and monocytes. cell viability was evaluated by wst- assay. chloroquine ( blood ( ml) collected from cats using a syringe coated with edta were used for the collection of samples for blood biochemistry and hematological analyses. a half milliliter of the blood sam-ple was centrifuged ( g) and the supernatant was used as a plasma sample for blood biochemistry. the remainder of blood samples was used for hematological analyses. heparinized blood ( ml) was -fold diluted with phosphate-buffered saline (pbs), and subjected to ficoll-hypaque , followed by inoculation with fipv at °c. after h, the supernatant was removed and cells were then treated with chloroquine at °c in the pre/post treatment and post treatment groups. after day, the virus titer in the fcwf- cell culture supernatant was measured. n = . (b) monocytes were cultured with chloroquine at °c for h in the pre/post treatment group, but not in the post treatment group, followed by inoculation with fipv at °c. after h, the supernatant was removed and cells were then treated with chloroquine at °c in the pre/post treatment and post treatment groups. after day, the virus titer in the feline primary monocyte culture supernatant was measured. n = . ⁄⁄ p < . vs. without chloroquine ( lm), ⁄ p < . vs. without chloroquine ( lm). monocytes were cultured with chloroquine for h in the pre/post treatment group (black bar), but not in the none group (white bar) or post treatment group (gray bar), followed by treatment with a reaction mixture of fipv and anti-fipv-s antibodies (fipv + mab - - ) for h. then, the supernatant was removed and cells were treated with chloroquine at °c in the pre/post treatment and post treatment groups, but not in the none group. after days, the virus titer in the cell culture supernatant (c) or the intracellular fcov n gene level (d) was measured. the fcov n gene was quantitatively analyzed in terms of the relative density value to the mrna of the housekeeping gene gapdh. n = . with chloroquine at °c in the pre/post treatment and post treatment groups, but not in the none group. after days, intracellular il- b, tnf-a, and il- mrna levels were measured. cytokine mrnas were quantitatively analyzed in terms of the relative density value to the mrna of the housekeeping gene gapdh. n = . density gradient centrifugation at g for min. the pbmc layer was collected, washed twice with pbs, and resuspended with growth medium at  cells/ml. . . statistical analysis data from two groups were analyzed by the student's t test, and multiple groups were analyzed by a one-way anova. the effect of chloroquine on fipv infection in fcwf- cells and spf cat-derived monocytes was investigated. the cytotoxic effect by chloroquine in feline cells was showed in table . in fcwf- cells, fipv replication was inhibited in a chloroquine concentration-dependent manner in both pre/post and post treatment groups (fig. a) . similarly, concentration-dependent inhibition of viral replication was noted in feline monocytes treated with chloroquine (fig. b) . however, the antiviral effect of chloroquine was slightly lower in the post treatment group than in the pre/post treatment group. the effect of chloroquine on the antibody-dependent enhancement of fipv infection in monocytes was investigated. the virus titer and intracellular expression level of the fipv n gene were significantly higher in the culture supernatant of monocytes inoculated with a mixture of fipv and mab - - than in monocytes cultured with fipv alone ( fig. a and b) . in monocytes inoculated with a mixture of fipv and mab - - , the virus titer was significantly lower in the post and pre/post treatment groups than in the none group (fig. c) . the intracellular expression level of the fipv n gene was significantly lower in the pre/post treatment group than in the none group (fig. d) . intracellular il- b, tnfa, and il- mrna expression levels were significantly higher in monocytes inoculated with a mixture of fipv and mab - - than in monocytes cultured with medium or fipv alone (fig. a) . in monocytes inoculated with a mixture of fipv and mab - - , il- b, tnf-a and il- mrna expression levels were significantly lower in the pre/post treatment group than in the none group (fig. b) . however, no significant differences in these mrna expression levels were noted between the post treatment and the none groups (fig. b) . the influence of chloroquine on cytokine mrna and fcov n gene expressions was investigated in monocytes from cats with fip. il- b, tnf-a, and il- mrna expression levels were signifi-cantly higher, with an increase in the level of fcov n gene expression, in monocytes from cats with fip than in monocytes from spf cats (fig. a) . these mrna expression levels were significantly lower in monocytes from cats with fip cultured in medium containing lm of chloroquine than in monocytes cultured without chloroquine (fig. b) . however, no significant difference was noted in the level of fcov n gene expression with or without chloroquine. based on the in vitro results, the therapeutic effect of chloroquine was investigated using cats with experimentally induced fip. the experiment was performed following the schedule shown in fig. . the fcov n gene and cytokine mrna were detected and clinical parameters were measured after fipv inoculation until the onset of fip. pbmc was collected and days after fipv inoculation, viral mrna expression level was measured. the viral mrna expression level was measured by semiquantitative rt-pcr. on day , viral mrna expression level was slightly lower in group a than in group b and c. on day , viral mrna expression level was slightly lower in group a and b (chloroquine-treated groups) than in group c. (fig. ) . inflammatory cytokine mrna expression levels in pbmc on day - after fipv inoculation were measured, and the ratios of mrna expression levels to those before inoculation were determined (fig. ) . inflammatory cytokine mrna expression ratios were slightly lower in group a and b (chloroquine-treated groups) than in group c. changes in body temperature after fipv inoculation were measured (table ) . no major change in body temperature was noted in any group. lymphocyte and neutrophil counts in peripheral blood decreased in all cats on day after fipv inoculation (table ), but the level of this reduction was smaller in chloroquine-treated groups than in group c. increases in total protein (tp), alkaline phosphatase (alp), gamma-glutamyl transferase (ggt), and direct bilirubin (d-bil) levels were smaller in group a than in the other groups (table ) . on the other hand, alanine aminotransferase (alt) levels increased in the chloroquine-treated groups. when the clinical condition of the cats in each group was evaluated using the karnofsky score, the score was high in the chloroquine-treated groups ( table ). the mean number of survival days were . , . , and . in groups a, b, and c, respectively. unfortunately, one animal in the group c died before the challenge of fipv, statistical significance was not shown for any of the differences between . . cytokine mrna levels (ratio to the control) il- il- tnf- fig. . il- b, tnf-a, and il- mrna expression levels of pbmc in fipv-infected cats treated with chloroquine. after inoculation with fipv - for - days, pbmcs were collected from cats. intracellular il- b, tnf-a, and il- mrna levels were measured. cytokine mrnas were quantitatively analyzed in terms of the relative density value to the mrna of the housekeeping gene gapdh. data are expressed as a ratio of the control value (at the day of inoculation with fipv). the chloroquine-treatment groups and the group c in the days of survival. it has been reported that chloroquine is useful for the treatment for viral infections (kono et al., ; murray et al., ; savarino et al., ; vincent et al., ; yan et al., ) . however, most of studies were obtained by in vitro experiments; only a few studies have investigated the antiviral effect of chloroquine using experimental animals (pallister et al., ; vigerust and mccullers, ) . we investigated the effect of chloroquine as a therapeutic drug for fip both in vitro and in vivo. fipv was enhanced proliferation in monocytes treated with a reaction mixture of fipv and anti-fipv-s antibodies. in contrast, virus proliferation was inhibited in chloroquine-treated monocytes, i.e., chloroquine inhibited the fipv-induced ade activity of monocytes. in addition, a significant inhibitory effect on fipv gene expression was noted in cells with pre/post treatment. in contrast, no significant inhibitory effect was noted in cells with post treatment, and the treatment of fipv-infected monocytes collected from fip cats with chloroquine did not change fipv n gene expression, suggesting that the treatment of cells already infected with fipv with chloroquine induces no antiviral effect. fipv-infected monocytes/macrophages are major component cells that produce the inflammatory cytokines involved in pathological deterioration (regan et al., ; takano et al., b takano et al., , . in this study, chloroquine significantly inhibited inflammatory cytokine mrna expression levels in fipv-infected monocytes. the mechanism of inhibiting inflammatory cytokine mrna expression by chloroquine is still unclear. weber and levitz ( ) suggested the presence of lysosomotropic and non-lysosomotropic mechanisms in the chloroquine-induced inhibition of tnf-a production. the former inhibits tnf-a release from cells and the latter inhibits tnf-a mrna expression and transcription, which suggests that the significant inhibition of inflammatory cytokine mrna expression levels by chloroquine in fipv-infected monocytes involves the non-lysosomotropic mechanism. however, in a more recent paper by jang et al. ( ) , it was shown that chloroquine does inhibit tnf-a release, but does not change the tnf-a mrna levels or the synthesis of the tnf-a precursor. the reason for this discrepancy is unclear. furthermore, in addition to the lowering of tnf-a mrna expression, the expression of il- b and il- mrna was decreased. jang et al. ( ) reported that the stability of il- b and il- mrna was decreased by chloroquine. in the present study, the same mechanisms might decrease the expression of cytokine mrna. chloroquine is administered orally or intravenously for the treatment of malaria. however, oral or intravenous administration of chloroquine is a burden on veterinarians and/or owners. thus, in the present study, we chose a subcutaneous route. unfortunately, there are no pharmacokinetic data on chloroquine after subcutaneous administration. therefore, we must rely on pharmacokinetic data on chloroquine after intravenous administration examined under similar conditions to our study. aderounmu et al. ( ) reported pharmacokinetic data after a single intravenous dose of mg/kg chloroquine. in our present study, the subcutaneous dose of chloroquine was . - . mg/kg. thus, pharmacokinetic data on chloroquine after subcutaneous administration to cats may be similar to those reported by aderounmu et al. ( ) . we administered chloroquine when there were some clinical symptoms in cats after inoculation with fipv strain - . the timing of administration was established based on the report of de groot-mijnes et al. ( ) and our previous experiments (takano and hohdatsu, unpublished data). therefore, the cq treatment timing in group b was set to days after inoculation with fipv strain - . the clinical score of chloroquine-treatment groups were better than in chloroquine-untreated group, which suggests that chloroquine has therapeutic effect against fip. if chloroquine has antiviral effect against fipv in vivo, this drug may be useful for the treatment of fip. to obtain the antiviral effect, it is necessary to increase the chloroquine dosage. however, as mentioned above, when the chloroquine concentration is increased, a severe side effect may be induced. combining chloroquine with other ''anti-fipv agents'' overcomes this problem. hsieh et al. ( ) succeeded in inhibiting fipv replication in fcwf- cells using the low-cytotoxic antiviral agents. it will be necessary to further investigate the possibility of fip treatment with a combination of chloroquine and other agents. in conclusion, it was demonstrated that chloroquine has inhibitory effect against the replication of fipv and anti-inflammatory effect. it was also noted experimentally that chloroquine may be an effective drug for the treatment of fip. the present experiments only examined whether cq is effective for treating fip. future studies are necessary to collect preclinical data using the pharmacokinetics data of cq reported by aderounmu et al. ( ) and others (aderounmu and fleckenstein, ; gustafsson et al., ; ducharme and farinotti, ) . table sequences of pcr primers for feline gapdh, il- b, tnf-alpha, il- , and fcov n. nucleotide sequence location length ( pharmacokinetics of chloroquine diphosphate in the dog comparison of the pharmacokinetics of chloroquine after single intravenous and intramuscular administration in healthy africans pyridine n-oxide derivatives are inhibitory to the human sars and feline infectious peritonitis coronavirus in cell culture antiviral studies of feline infectious peritonitis virus in vitro monoclonal antibody analysis of neutralization and antibody-dependent enhancement of feline infectious peritonitis virus replication of feline coronaviruses in peripheral blood monocytes treatment of cats with feline infectious peritonitis clinical pharmacokinetics and metabolism of chloroquine. focus on recent advancements natural history of a recurrent feline coronavirus infection and the role of cellular immunity in survival and disease disposition of chloroquine in man after single intravenous and oral doses karnofsky's score modified for cats a study on the mechanism of antibody-dependent enhancement of feline infectious peritonitis virus infection in feline macrophages by monoclonal antibodies enhancement and neutralization of feline infectious peritonitis virus infection in feline macrophages by neutralizing monoclonal antibodies recognizing different epitopes synergistic antiviral effect of galanthus nivalis agglutinin and nelfinavir against feline coronavirus chloroquine inhibits production of tnf-alpha, il- beta and il- from lipopolysaccharide-stimulated human monocytes/macrophages by different modes chloroquine inhibits proinflammatory cytokine release into human whole blood potent inhibition of feline coronaviruses with peptidyl compounds targeting coronavirus c-like protease inhibition of human coronavirus e infection in human epithelial lung cells (l ) by chloroquine: involvement of p mapk and erk a novel coronavirus associated with severe acute respiratory syndrome a randomized, double-blind, -week controlled study of low-dose cyclosporine versus chloroquine for early rheumatoid arthritis effect of chloroquine phosphate treatment on serum mmp- and timp- levels in patients with systemic lupus erythematosus human coronavirus emc does not require the sarscoronavirus receptor and maintains broad replicative capability in mammalian cell lines reduction of immune activation with chloroquine therapy during chronic hiv infection monoclonal antibodies to the spike protein of feline infectious peritonitis virus mediate antibody-dependent enhancement of infection of feline macrophages chloroquine administration does not prevent nipah virus infection and disease in ferrets a review of feline infectious peritonitis virus infection: - immunologic phenomena in the effusive form of feline infectious peritonitis activation of p mapk by feline infectious peritonitis virus regulates pro-inflammatory cytokine production in primary blood-derived feline mononuclear cells effect of feline interferon-omega on the survival time and quality of life of cats with feline infectious peritonitis effects of chloroquine on viral infections: an old drug against today's diseases? neutrophil survival factors (tnf-alpha, gm-csf, and g-csf) produced by macrophages in cats infected with feline infectious peritonitis virus contribute to the pathogenesis of granulomatous lesions a ''possible'' involvement of tnf-alpha in apoptosis induction in peripheral blood lymphocytes of cats with feline infectious peritonitis tnf-alpha, produced by feline infectious peritonitis virus (fipv)-induced macrophages, upregulates expression of type ii fipv receptor feline aminopeptidase n in feline macrophages analysis of the mechanism of antibody-dependent enhancement of feline infectious peritonitis virus infection: aminopeptidase n is not important and a process of acidification of the endosome is necessary chloroquine is a potent inhibitor of sars coronavirus infection and spread chloroquine is effective against influenza a virus in vitro but not in vivo chloroquine interferes with lipopolysaccharideinduced tnf-alpha gene expression by a nonlysosomotropic mechanism anti-malaria drug chloroquine is highly effective in treating avian influenza a h n virus infection in an animal model this work was in part supported by kakenhi (grants-in-aid for young scientists (b), no. ) from the ministry of education, culture, sports, science and technology, and kitasato university research grant for young researchers ( ). key: cord- - fue x authors: chang, chung-ke; hou, ming-hon; chang, chi-fon; hsiao, chwan-deng; huang, tai-huang title: the sars coronavirus nucleocapsid protein – forms and functions date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: fue x the nucleocapsid phosphoprotein of the severe acute respiratory syndrome coronavirus (sars-cov n protein) packages the viral genome into a helical ribonucleocapsid (rnp) and plays a fundamental role during viral self-assembly. it is a protein with multifarious activities. in this article we will review our current understanding of the n protein structure and its interaction with nucleic acid. highlights of the progresses include uncovering the modular organization, determining the structures of the structural domains, realizing the roles of protein disorder in protein–protein and protein–nucleic acid interactions, and visualizing the ribonucleoprotein (rnp) structure inside the virions. it was also demonstrated that n-protein binds to nucleic acid at multiple sites with a coupled-allostery manner. we propose a sars-cov rnp model that conforms to existing data and bears resemblance to the existing rnp structures of rna viruses. the model highlights the critical role of modular organization and intrinsic disorder of the n protein in the formation and functions of the dynamic rnp capsid in rna viruses. this paper forms part of a symposium in antiviral research on “from sars to mers: years of research on highly pathogenic human coronaviruses.” the severe acute respiratory syndrome coronavirus (sars-cov) nucleocapsid (n) protein is the most abundant protein in the virusinfected cells. its primary function is to package the kb single stranded, -capped positive strand viral genome rna molecule into a ribonucleoprotein (rnp) complex called the capsid. ribonucleocapsid packaging is a fundamental part of viral self-assembly and the rnp complex constitutes the essential template for replication by the rna-dependent rna polymerase complex. in addition, the n-protein of the sars-cov has been shown to modulate the host cellular machinery and may serve regulatory roles during its viral life cycle (ababou and ladbury, ; hsieh et al., ; surjit et al., ) . there have been several excellent reviews on the coronavirus n protein (laude and masters, ; masters, ) , including one on sars-cov n protein (surjit and lal, ) . here we will review the recent findings on the structure and function of sars-cov n and its interaction with nucleic acid from a more biophysical point of view. coronavirus assembly is localized at membranes of the endoplasmic reticulum-golgi intermediate compartment, likely mediated by species-specific interactions of the matrix (m) protein with spike (s), nucleocapsid (n), and envelope (e) proteins (de haan et al., ; krijnse-locker et al., ) . however, the detailed molecular packaging of n inside the virion and the interaction between n and other proteins are unknown. early em studies of coronaviruses have shown that coronavirus rnps are helical, consisting of coils of - nm in diameter and a hollow interior of approximately - nm (caul and egglestone, ; davies et al., ; macneughton and davies, ) . more recently, chang et al. ( a) . the ribbon representations of the structures of ntd (green) and ctd (blue and gold) are generated with pymol from coordinates in the protein data bank (pdb ids: ntd, ofx; ctd, cjr). the relative orientation of ntd and ctd, as well as the conformations of the disordered regions (n-arm, lkr and c-tail), are drawn randomly to reflect the dynamic nature of the n protein, as revealed by saxs data (chang et al., ). the ribbon structures were generated using pymol (the pymol molecular graphics system, version . . . schrödinger, llc). neuman et al. have employed single particle image analysis of d electron cryo-microscopy (cryo-em) to investigate the structural organization of sars-cov at nm resolution (neuman et al., ) (fig. a) . they observed overlapping lattices arranged near the viral membrane surrounded by a disordered core. the rnp particles displayed a coiled shape when released from the viral membrane. edge views revealed that most of the viral rnp was located within nm of the inner face of the membrane. fifteen-nanometer-wide strands of electron-dense material can be seen emerging from a spontaneously disrupted sars-cov particle. the rnp is maintained in a spherically packaged form at the inner face of the membrane with no indication of icosahedral symmetry. the sars-cov nucleocapsid is separated from the envelope by a gap, which contains thread-like densities that connect the m protein density on the inner face of the viral membrane to a two-dimensionally ordered ribonucleoprotein layer (fig. b) , a feature also seen in tgev (risco et al., ) . since the carboxyl tail of m protein has been shown to interact specifically with n (escors et al., ; kuo and masters, ; narayanan et al., ; sturman et al., ) , these results suggest that the m-n interactions constrain some n molecules in close apposition to the envelope. glycoprotein spikes were found to be aligned with the membrane-proximal layer of rnp densities, implying that protein location within the envelope is constrained by consistent s-m, m-m, and m-n interactions. the organization further implies that m is also organized in a two-dimensional lattice which was proposed to be a likely scaffold for viral assembly (de haan et al., ) . the stoichiometry of the unit cell at the virion surface was estimated to be approximately s : m: n to s : m: n proteins, where s is a spike trimer, with the remainder of the n protein distributed throughout the virion core. nucleoprotein molecules in the paracrystalline rnp shell appeared to be partially organized through interactions at points of contact in the rnp lattice. the distribution of density in the viral core was consistent with a membrane-proximal rnp lattice formed by local approaches of the coiled ribonucleoprotein. the cryo-em images did not reveal any internal features within the nmthick rnp zone proximal to the envelope. this suggests that inner core densities of mature coronaviruses are not consistently ordered with respect to the membrane. a model based on interpretation of the d cryo-em data is shown on fig. b. koster and associates, on the other hand, employed d cryoelectron tomography to study the structure of mouse hepatitis virus (mhv) particles (barcena et al., ) . they showed that the viral envelope has a thickness that is almost twice that of a typical biological membrane. the extra internal layer was attributed to the c-terminal domains of the m protein. in the interior of the particles coiled structures and tubular shapes are observed, consistent with a helical nucleocapsid formed by self-association of the n protein and the genomic rna. the rnp seems to be relatively densely packed and disorganized underneath the envelope. consistent with previous observations, they also observed quasi-circular density profiles approximately nm in diameter enclosing an empty space approximately nm in diameter inside the otherwise relatively disorganized interior. the observation of only short coiled fragments in the reconstructions strongly suggests that the helical nucleocapsid is a very flexible structure that extensively twists and folds upon itself, adopting orientations that are not easily recognizable as coils in tomographic sections. the general features and global architecture observed for mhv were also observed in tgev, suggesting a general model for the architecture of covs. the pleomorphic nature of the coronavirus particle has hampered the effort to obtain high-resolution virion image at atomic resolution. nonetheless, the cryo-em images have provided considerable insights regarding the organization of various structural proteins, especially the virion envelope and the rnp. it also revealed a structural plasticity that may play an essential role in the virus life cycle. the presence of partially organized and flexible n protein regions could facilitate packaging of the genomic rna by covs. it is perhaps surprising that prior to the outbreak of sars the structure of coronavirus n proteins were never studied in detail. the earliest structural model of coronavirus n protein was proposed by parker and masters (masters, ; parker and masters, ) in the s based on sequence comparison and evolutionary studies of mhv, a prototypical group ii coronavirus. in their model, the n protein comprised three domains separated by two spacers. the central domain acted as the rna-binding domain, whereas the remaining two acidic domains presumably played a role in protein-protein interactions. although the model provided a general overview of coronavirus n protein structure at the time, it lacked the necessary details to provide a clear picture of the structure-function relationship of the protein. the sars pandemic ushered a new era of structural studies on coronavirus protein structure. the sars-cov n protein is a kda phosphoprotein of amino acids, sharing - % sequence identity with the n proteins of other coronaviruses (marra et al., ; rota et al., ) (fig. ) . it forms a dimer, which constitutes the basic building block of the nucleocapsid, through its c-terminus surjit et al., ; yu et al., ) . huang et al. first solved the solution structure of the n-terminal domain ( fig. c ), which they coined as rbd (residues - ) and demonstrated that this domain is capable of binding to rna with micromolar affinity (huang et al., b) . the term rbd is misleading since rna binds to n at multiple sites other than rbd. to avoid confusion we will use the acronym, ntd, from now on instead. the structure of the dimerization domain (residues - ) was solved by x-ray crystallography and nmr (chen et al., ; takeda et al., ; yu et al., ) (fig. c ). since the dimerization domain is not just a dimerization domain and it also binds to nucleic acid we refer it as ctd instead. as shown by nmr, chromatography, and small-angle x-ray scattering (saxs), the ntd and ctd forms two independent domains that do not interact with each other (chang et al., ) . it was evident at this point that the original three-domain model would require extensive revision in light of these new developments. the modular organization of sars-cov n was further defined in more detail by a combination of bioinformatics and biophysical methods by chang et al. who showed that the two structural domains are interspersed by intrinsically disordered regions (idrs) that account for % of the amino-acid residues ( fig. c ) (chang et al., (chang et al., , . a relatively new concept in structural biology, intrinsically disordered proteins (idps) or idrs lack a defined tertiary structure in the native state, but play important roles in biological processes, particularly in macromolecular interactions (dunker et al., ; dyson, dyson, , dyson and wright, ; xie et al., ) . in the case of sars-cov n protein, all three idrs (residues - , - , and - ) are able to modulate the rna-binding activity of the ntd and ctd (chang et al., ) . the middle idr, which we coined lkr, and c-terminal idr have both been implicated in the oligomerization of the n protein (he et al., a; luo et al., ) . the lkr includes a ser/arg-rich region that contains a number of putative phosphorylation sites, which may regulate n protein function (peng et al., ; surjit et al., ; wu et al., ) and n-m interaction (he et al., b) . based on these new findings, chang et al. proposed a structure-based domain arrangement for sars-cov n protein where the ntd and ctd are sandwiched between three idrs. sequence alignments suggested that other coronavirus n proteins might share the same structural organization based on intrinsic disorder predictor profiles and secondary structure predictions (fig. ) . determination of the ntd and ctd structures of the n proteins from infectious bronchitis virus (ibv) (fan et al., ; jayaram et al., ) , mhv (grossoehme et al., ; ma et al., ) and human coronavirus oc were in general agreement with the structure-based model. the n protein sequence of the recently discovered middle-east respiratory syndrome coronavirus (mers-cov) also shares the same intrinsic disorder and secondary structure profile, which further supports the universality of the structure-based model (van boheemen et al., ) . although the original three-domain model has been partially superseded by the structure-based model, some features of the earlier model may be reconciled with the latter one. first, the second spacer and the c-terminal acidic region in the three-domain model can be mapped to the c-terminal idr in the structure-based model. similar to the sars-cov n protein, the c-terminal acidic region of mhv n protein has been shown to self-interact (hurst et al., ) , and it has also been reported that a c-terminal idr in the n protein of human coronavirus strain e is involved in oligomerization (lo et al., ) . second, the rna-binding domain in the three-domain model could be re-defined to span both the ntd and the ctd. in fact, hurst et al. noticed that effective binding to rna by mhv n protein in host cells required the presence of both the ntd and ctd (hurst et al., ) , suggesting that the ntd and ctd formed a single bipartite rna interaction site, a feature to be reiterated in the final sars-cov rnp model. in this regard, the structure-based model is an evolution of the original three-domain model that provides a more refined framework for linking the structure and function of coronavirus n proteins. modular structures are found in many rna-binding proteins, including other viral nucleocapsid proteins (draper, ; lunde et al., ) . for example, the nucleocapsid protein from bumyamwera virus is a single-stranded rna-binding protein with two modular domains . constructing a protein with a modular architecture confers many advantages which would not be possible with single-domain proteins. these include: (i) enhanced binding specificity and affinity through cooperative coupled allosteric binding of individual domains. the modular organization of a protein also allows it to present a large and flexible surface for binding to complex structural features, or multiple and extended regions of the target molecules such as rnas. (ii) facilitated regulation and functional expression. the relatively weak interactions of individual domains make it easier to regulate the formation and disassembly of rnp complexes when needed. assembly and disassembly can proceed via the (un)zipping action of one module at a time with moderate free energy cost. (iii) the multiple binding sites can evolve independently, and thus enhance environmental adaptation. the modular nature of sars-cov n protein and n proteins from coronaviridae in general, is clearly essential for packaging rnp and viral function. the structure of the ntd of sars-cov n was first determined by nmr by huang et al. ( b) . the protein adopts a unique fivestranded antiparallel b-sheet with the topology of b -b -b -b -b (fig. a) . the middle strands b and b are connected by a protruding b-hairpin (b -b ). the residues in the extended b-hairpin are predominantly basic with of the residues being arginines or lysines. the d folding created a positively charged pocket at the junction between the hairpin and the core structure which served as the rna binding site, as confirmed by nmr chemical shift perturbation upon addition of a -mer or -mer rna (fig. b) . nmr relaxation and heteronuclear noe data indicate that the b- hairpin is highly flexible, suggesting that this region may undergo conformational adaptation upon rna binding (clarkson et al., ) . the structural features of the ntd are reminiscent of the b-sheet rna recognition proteins found in many rnps (draper, ) . this class of proteins has a babbab fold in which the middle first and third b-strands contain characteristic aromatic residues. in the crystal structure of u a-rna hairpin complex three bases are stacked against conserved aromatic residues while a flexible long b-hairpin grasp the rna against the b-sheet (oubridge et al., ) . these aromatic residues are thought to orient bases on the protein surface, rather than select particular protein-rna sequences. in sars-cov ntd there are also many conserved aromatic residues in the same structural region. although not confirmed, it is probable that some of these aromatic residues in sars-cov n, in particular tyr , tyr , tyr , tyr , tyr , and trp , are on the same face of b-sheet and are conserved in coronaviruses and may play similar roles in rnp packaging (fig. c) .the ntd structure of sars-cov n was later on determined by x-ray crystallography in two crystal forms (saikatendu et al., ) . the overall folding of the crystal structure is similar to that observed in solution by nmr, with a root mean square deviation (rmsd) of . Å over superimposed ca atoms of the monoclinic form. significant inward shift of loops l and l and outward hinge motion of the b-hairpin were observed, resulting in the rna-binding cleft being significant narrower and shallower in the crystal structure. it is not clear whether the difference is due to the insufficient noe constraints in the solution structure or due to crystal packing or both. nonetheless, the difference observed in the two structures further supports the concept that the rna-binding cleft is deformable and is likely to adopt a different conformation upon rna binding. intriguingly, in the cubic form the individual monomers organized as trimeric units and the consecutive trimers stack in a right-handed twist, resulting in an overall packing of a helical tubule. at present the physiological relevance of the helical packing is unclear. the c-terminal structural domain (ctd) of sars cov n exists in dimeric form (chang et al., , yu et al., ) . the crystal structure of ctd was solved in two different constructs, ctd - and ctd - (chen et al., ) . alignment of corresponding ca atoms showed a rmsd of . Å, indicating that these two structures are practically identical. however, the absence of the n-terminal amino acid peptide between residues and in ctd - significantly diminished the proteinprotein interaction and crystal packing, as well as its interaction with nucleic acids, as described below. each ctd monomer is composed of eight a-helices and a b-hairpin in the following topology: a a a a a a b b a a (fig. e) . the dimer has the shape of a rectangular slab in which the four-stranded b-sheet forms one face and the a-helices form the opposite face. the two c termini are located at the diagonal apices on the b-sheet face and the two n termini are located at the center of two opposing edges of the slab. the dimerization interface of the ctd dimer is composed of four b-strands and six a-helices with each protomer contributing one b-hairpin and helices a , a and a . the long b strand of one protomer pairs with the b strand of the other protomer to form the four-stranded intermolecular antiparallel b-sheet that is stabilized through extensive hydrogen bonding . each hairpin also interacts extensively with the hairpin from the other protomer in a domain-swapped manner. the other part of the dimerization interface is composed of helices a and a , where strong hydrophobic interactions involving trp , ile , pro , phe and phe were observed. the dimer is further stabilized by hydrophobic interactions between the longest helix, a , and the intermolecular b-sheet. the combination of hydrogen bonds and hydrophobic interactions results in a very stable dimer with a buried surface area of Å , suggesting that the dimer is likely the native structure of coronavirus n protein. takeda et al. have solved the solution structure of ctd - and showed that the nmr structure is almost identical to the crystal structure. the backbone rmsd between the protomers of the mean nmr structure and the crystal structure of the ctd spanning residues - is . Å if residues - and - are superimposed. however, in the nmr structure the two n-termini (residues - ) protruding from the dimer core are disordered and lack a short helix formed by residues - , whereas, in the crystal structure, they are involved in a number of intra-monomer and intra-dimer contacts and are more rigid. interestingly, chang et al. also observed the formation of an octamer in the asymmetric unit of the ctd crystal. translational stacking of the octamer forms a hollow twin helix structure with an outer diameter of Å and an inner diameter of Å, with a pitch of Å. the groove of the twin helix, which is lined with several positively charged residues, has a depth of . Å. the nterminal amino acid residues from a.a. - play an important role in protein-protein interaction in the octamer, accounting for the absence of the octamer in the crystal structure of ctd - . studies of the nmr chemical shift perturbations caused by the binding of single-stranded dna and mutational analyses have identified this mostly disordered region at the n-termini as the prime site for nucleic acid binding (takeda et al., ) . in addition, residues in the b-sheet region also showed significant perturbations. mapping of the locations of these residues onto the helical model observed in the crystal structure of ctd - revealed that these two regions are parts of the interior lining of the positively charged helical groove (fig. f) . this observation led them to propose a helical packaging model of sars-cov rnp, as will be elaborated in more detail in the following sections. due to difficulties arising from protein stability and dynamic behavior, there are no structures available for any of the full-length n proteins from coronaviruses. fitting of the small angle x-ray scattering (saxs) data led chang et al. to propose a structural model for a di-domain (dd) construct spanning the ntd, lkr, and ctd of sars-cov n protein (a.a. - ) (chang et al., ) (fig. c) . the dd dimer adopts a clamp-like open conformation in the model with lkrs serve as the two arms connecting the two ntds to the ctd dimer. the model is consistent with the known structural features of coronavirus n proteins, namely the dimerization of the ctd and intrinsically disordered nature of the lkr, and currently remains the only structural model spanning multiple domains of coronavirus n proteins. comparison of sars-cov n protein structure with those of other viral n proteins provides valuable mechanistic and evolutionary insights. the ntd from avian infectious bronchitis virus (ibv) (fan et al., ) (jayaram et al., ) , mouse hepatitis virus (mhv) (grossoehme et al., ) , and human coronavirus oc (hcov-oc ) , as well as the ctd of ibv (jayaram et al., ) have been reported. the sequence identities of sars-cov ntd with those of ibv and hcov-oc are % and %, respectively, yet the d structures of these three proteins are highly homologous. the rmsd between sars-cov n-ntd (pdb id: ofz) and ibv-ntd (pdb id: gec) is . Å for aligned ca atoms and that between sars-cov ntd and hcov-ntd is . Å for aligned ca atoms of the two proteins . interestingly, the surface charge distribution of the ndt of ibv, mhv and hcov-oc are significantly different from that of the sars-cov ntd (fig. d) , suggesting that they may interact with rna differently. the structure of ibv-ctd (a.a. - , pdb id: ge ) is also highly homologous to that of sars-cov ctd (pdb id: cjr). the rmsd of aligned ca atoms in a dimer between the two structures is . Å and both exist as a domainswapped dimer. three types of interactions (s-, l-and f-types) were observed in three forms of ibv ctd crystals. intriguingly, type s interaction observed in crystal form and ( fig. a and b in jayaram et al. ( ) ) bears high resemblance to that observed in the helical packing of sars-cov ctd (fig. a) . furthermore, the surface charge distribution of ibv ctd dimer also contains a positively charged strip spanning the region observed for sars-cov ctd dimer ( fig. f and g) implying similar interaction between ctd and rna for the two coronaviruses. sequence alignment coupled with secondary structure prediction show that many coronavirus ctds share the bba topology observed in sars-cov . these results raise the possibility that all coronaviruses employ the same interface mechanism for dimerization and they belong to the same structural class. the structural arrangement of ctd is also reminiscent of the dimer-interface of the nucleocapsid protein from porcine reproductive and respiratory syndrome virus (prrsv), an arterivirus . thus, there are common principles that underlie the architecture of the nucleocapsid protein in both sars-cov and prrsv. the structural similarity between the n proteins of sars-cov and prrsv provides valuable information for understanding the evolutionary links between corona-and arteriviruses, suggesting a possible common origin of these two proteins . reports in the literature suggested that n can oligomerize through the sr-rich region or the c-terminal fragments in a concentration dependent manner (surjit and lal, ) . however, these early studies were carried out using fragments that often cut through the structured region that could have adverse effect on their structures and oligomerization behavior as well. crystal structures of coronavirus n protein led to several proposed n-polymers that could bind rna and mimic the rnp packaging (chen et al., ; fan et al., ; jayaram et al., ; saikatendu et al., ) . it is unclear whether the oligomer structure is biologically relevant, since there have been no reports of oligomer species being detected in solution. to test the possibility that the oligomer structure reflects the existence of transient interactions that have been trapped during the crystallization process, chang et al. applied an in vitro disulfide trapping technique in an attempt to capture these transient interactions in solution . specifically, using the crystal structures as guides they engineered single-site cysteine mutations at various locations and tested the ability of these mutants to spontaneously form disulfide linkages through size-exclusion chromatography. sars-cov n contains no cysteine and none of the mutants are located close enough to form intra-dimer disulfide linkages, thus any disulfide linkage must be due to inter-dimer protein disulfide bond formation. the results suggested that fragments containing the ctd of sars-cov n protein are capable of transient self-association through the oligomer interface identified in the crystal structure, even though the long-lived stable helical structure of ctd was not observed in solution. thus, the ctd dimer-dimer interaction observed in the crystal is also the preferred interaction in solution but the oligomer can form only transiently due to weak interaction as shown in the small interface area between ctd dimers ( Å ). presumably these weak interactions can be augmented by rna binding and binding of the other n protein domains linked to the ctd through lkr in a synergistic manner and the conformation of the ctd oligomer will be further modified by n-rna interactions. a similar strategy was applied to engineer ntd mutants. however, no significant oligomer formation was observed for the ntd fragments, suggesting that the ntd either does not form oligomers or forms oligomers through an unidentified intermolecular interface other than that identified in the ntd crystal structure. sars-cov n is a highly basic protein containing an excess of positive charges. these charges are considered important for rna binding, but they are also potentially deterring for the self-association of the protein through electrostatic repulsion (huang et al., b; takeda et al., ) . chang et al. tested whether salt concentration affects sars-cov n transient self-association by disulfide trapping experiment as described above using the q c mutant. gln is located at the interface between two dimers and the two gln in the dimer are far apart, formation of disulfide bonds in the q c mutant would require at least two dimers to draw close together in space, resulting in the formation of tetramers or higher oligomers. the relative amount of tetramer and larger oligomers in solution increases with increasing salt concentration, suggesting that reducing charge repulsion by increasing salt concentration enhances self-association of ctd. the n protein is heavily phosphorylated at the ser/arg-rich portion of the lkr region (peng et al., ; surjit et al., ; zakhartchouk et al., ) and phosphorylation may affect nucleocytoplasmic shuttling of the n protein (surjit et al., ; wu et al., ). peng et al. demonstrated that phosphorylation of the lkr by the sr protein kinase- (srpk ) partially impaired the self-association of the full-length protein (peng et al., ) . chang et al. examined whether changing the electrostatic properties of the protein itself could affect transient self-association . they chose the putative phosphorylation sites on the flexible linker as prime target, and assayed the effect of negative charges on n protein self-association by changing these sites from ser to glu in the q c mutant of di-domain constructs containing the ntd, lkr and ctd (dd q c , a.a. - ). they observed that gradual introduction of negative charges on the unstructured linker had a positive effect on the oligomerization of the dd when compared to the dd q c control, with maximum effect achieved when negative charges were introduced per each chain. further increases in negative charges were less effective in enhancing dd q c oligomerization. overall, the results suggest that hyperphosphorylation of the lkr, which reduces the total positive charge of the n protein, can enhance and regulate oligomerization of dd through electrostatic effects. the results suggest a biophysical mechanism where electrostatic repulsion may act as a switch to regulate n protein oligomerization. . protein-nucleic acid interaction the primary function of the coronavirus n protein is to package the viral genome into a ribonucleoprotein (rnp) particle to protect the genomic rna and for incorporation into a viable virion. thus, n must bind to rna tightly. during viral infection the n protein must also be readily dissociated to expose the genomic rna for efficient expression, transcription and replication (lai and cavanagh, ; tahara et al., tahara et al., , . this function demands a low energy barrier for n to dissociate from rna. viruses have evolved a clever tactic to achieve these two seemingly contradictory functions. the secret lies on the modular structural organization and the dynamic nature of the n protein rendered by the intrinsically disordered regions. much information about the interaction between the n protein and rna in coronaviruses have been gathered through studies on mhv model systems, including detection of general binding activity (robbins et al., ) and identification of rna sequences that bind with high affinity to the protein (nelson et al., ) . however, it was the discovery of sars-cov that spurred research on the mechanisms behind the interaction between coronavirus n protein and nucleic acids. studies on the nucleic acid-binding behavior of sars-cov n protein at the domain level have started to provide much needed insight into the binding mechanism of coronavirus n proteins. sars-cov n protein is a highly basic protein with excess positively charged residues mostly localized in three regions: the sr-rich region of the lkr (residues - ,+ charges), the n-terminal region of the ctd (residues - ,+ charges) and the c-terminal disordered region (residues - ,+ charges). the nucleic acid-binding activity of the ntd was tested and confirmed early on huang et al. ( a) due to the presence of the classic rna-binding motif first detected in the u -rnp (nagai et al., ) . the effect of the other structural domain, the ctd, on nucleic acid binding was not expected since initial structures of the domain did not include the residues that interacted with nucleic acids . structures of longer constructs of ctd later revealed a positively charged groove on the surface of the molecule that could act as a binding site for nucleic acids (chen et al., ) , and follow-up studies demonstrated that the ctd was capable of binding to both ssdna and ssrna with similar affinity as the ntd (k d lm) (chang et al., ; takeda et al., ) . the idrs, on the other hand, have not been studied individually due to stability issues (mark et al., ) . however, inclusion of the idrs to any of the structural domains resulted in significantly increased binding affinity and binding cooperativity towards a poly (u) ssrna under in vitro conditions (k d . lm), suggesting that the idrs are able to modulate the nucleic acid binding activity of sars-cov n protein (chang et al., ) . of particular interest is the role of the lkr in n protein-nucleic acid interaction since it contains a sr-rich motif where most of the putative phosphorylation sites are located. it has been reported that sars-cov n protein is hypophosphorylated within the virion , and deletion of the sr-rich motif within the lkr resulted in formation of larger than normal rnps that were sensitive to rnase treatment (peng et al., ) . these observations suggest that phosphorylation of the sars-cov n protein at the lkr not only affects n oligomerization, it may decrease the nucleic acid binding affinity as well. by itself, the sars-cov n protein is a non-specific nucleic acidbinding protein. it has been shown to bind to single-stranded rna (ssrna), single-stranded dna (ssdna) and double-stranded dna (dsdna) under in vitro conditions (takeda et al., ; tang et al., ) . the non-specific nature of this binding is also expected since encapsidation of the entire viral genome would require the n protein to bind to diverse sequences with reasonable affinity. although one could argue that the n protein may bind to a particular sequence with high affinity and package the rest of the rna by relying on protein-protein interaction alone, such a scenario is unlikely to happen because the interaction between n protein dimers is extremely weak in the absence of nucleic acids (chang et al., . moreover, the highly charged regions are exposed to the solvent and the electrostatic forces might be the main driving force behind protein-nucleic acid binding. indeed, nucleic acid-binding sites on the ntd and ctd identified from nmr studies were found to have strong positive surface charges (huang et al., b; takeda et al., ) . takeda et al. also found that mutating lys and lys to gln in the ctd resulted in decreased binding affinity towards ssdna, whereas mutating the same residues to arg had no effect on the binding strength (takeda et al., ) . these lines of evidence strongly indicate that sars-cov n protein binds to nucleic acids in a non-specific manner through electrostatic interactions. the discovery that multiple regions within the sars-cov n protein are capable of interacting with nucleic acids provides critical insights into the binding mechanism. although the binding strength of individual binding sites towards nucleic acids is only in the micromolar range, the concerted action of these sites confers higher nucleic acid-binding affinity to the n protein as a whole. the idrs play a special role, since their inclusion not only increases the binding affinity, but also enhances the binding allostery, enabling the n protein to bind rna with high cooperativity (chang et al., ) . a variety of functions were found to be associated with domains containing conserved disorder with dna/rna binding among the most common function (dunker et al., ; dyson, ) . the intrinsic disorder in protein confers several advantages in performing its biological functions, including promiscuous basal activity, enhanced specificity, higher capture radius for formation of complexes, facilitating regulation by post-translational modification (dyson, ) . two possible causes for the binding enhancement can be argued. first, the extended conformation of the n protein due to the presence of the idrs increases the collision radius with nucleic acids. if the binding were further coupled to changes in protein conformation, the rate of binding would be enhanced through the ''fly-casting mechanism'' proposed by shoemaker et al. ( ) . second, the flexibility of the idrs allows the optimal alignment of the multiple nucleic acid-binding sites to interact with the same nucleic acid molecule in an allosteric fashion, resulting in a ''coupled allostery'' effect that enhances the binding affinity of the protein towards the nucleic acid (hilser and thompson, ) . in addition to the idrs, the structural domains, ntd and ctd, also could act in conjunction to enhance the binding affinity. the ntd contains a number of aromatic residues conserved among coronavirus n proteins that may interact with nucleotide bases by forming stacking interactions, whereas the strong electropositive surface formed by the ctd dimer is perfectly suited for interacting with the phosphate backbone (chen et al., ) . consistent with this model, mutagenesis studies conducted by grossoehme and coworkers have found that tyr on the ntd of mhv n protein was important for binding to a transcriptional regulatory sequence rna (grossoehme et al., ) . accommodation of the exceptionally large ( kb) sars-cov genome into newly formed virion spherules < nm in size necessitates an extremely well-packed, largely helical, supercoiling of the nucleic acid within the rnp core. the inability to observe a well-structured rnp layer inside the sars-cov particle and only short coiled fragments of rnp in mhv in the cryo-em reconstructions strongly suggests that the helical nucleocapsid is a very flexible structure that extensively twists and folds upon itself (bárcena et al., ) . such a dynamic structure of coronavirus rnp is not totally unexpected since rna is known to be dynamic and exists in multiple folded and unfolded states (dyson, ) . thus, rna-protein recognition often involves an induced-fit process, in contrast to protein-b dna interaction which most often manifests itself as molding of the protein onto the b-form dna structure. furthermore, the modular organization with three long idrs of the n protein provides the n protein with considerable flexibility. no existing data supports the presence of a long-lived sars-cov n oligomer or intermediate in solution and the sars-cov genomic ssrna by itself is unlikely to exist as a helix of the length observed in cryo-em. thus, packaging of sars-cov rnp proceeds most likely through a rna binding-coupled packaging mechanism, as also proposed for mhv, which showed that the product rna of mouse hepatitis virus synthesized was mostly of genome length and was found to be encapsidated by n protein (compton et al., ) . this suggests that coronavirus rna synthesis is coupled to the encapsidation of nascent rna, analogous to the replication of viruses with helical negative-strand rna nucleocapsids. based on available detailed d structural information of the sars-cov n protein modules and our understanding of n-rna interaction we propose a probable model derived from the crystal structure of the ctd (chen et al., ) , which was shown to exist transiently in solution by disulfide trap experiment (fig. a and b). a putative scenario of the molecular events leading to the formation of rnp is as follows: ( ) initiation: in solution initial binding of rna at either ntd or ctd facilitates binding of other modules to rna in a coupled-allostery manner with rna molecule threads between the two structural domains. this initial n-rna binary complex (rnp ) is highly stable and each rna molecule may have several n protein bound at a particular time. ( ) growth: the rnp could grow by either recruiting more n to the adjacent rna sites, or it could slide or hop along the linear rna molecule and combine with other smaller n-rna oligomers to form a larger oligomer (rnp n ) of various sizes. the n proteins in rnp n would pack in a structure with ctd forming the helical core, similar to that observed in the ctd crystal structure, and rna wraps and twists around the helical groove through mostly electrostatic interaction between the positively charge residues in the groove and the phosphate backbone of the rna molecule ( fig. c and d) . ( ) packaging of ntd: the ntd module will cap on the outside of the helical ctd-rna complex with the charged surface in the junction between the b-sheet and the b-hairpin covering the free phosphate groups of the rna molecule. furthermore, rna bases sticking out of the groove could intercalate in between the aromatic rings on the ntd core at the bottom of the b-sheet (figs. c, and c) . the presence of the long disordered lkr permits the two structural domains considerable freedom to adapt a wide range of orientations and positions for optimal packing of the rnp complex. likewise, the rna molecule also possesses high freedom to adjust to local conformation by an induced-fit process. thus, the n protein binds to rna in a fashion resembling that of an octopus clinching onto its prey (rna) using all its tentacles (modules) (fig. e and f). ( ) thermodynamic basis: electrostatic interaction drives the formation of n-rna complex but the multitude of weak protein-protein interactions contributes towards the selfassembly of the helical rnp. this is consistent with the concept for virus assembly that capsid proteins associate through locally weak interactions to form globally stable structures (zlotnick, ) . the rnp structure proposed above would have an outer diameter of nm and an inner diameter of nm, consistent with that observed by cryo-em. each n dimer would bind to rna bases. the two termini would stick out of the helix and the lkr linker would be accessible to interact with the matrix protein m. the combination of a modular structure incorporating idrs, multiple sites of moderate rna binding affinity, and weak dimer-dimer interaction in the n protein not only allows the packaging of a stable rnp but also offers an energetically favorable condition for the expression of the viral genomic information. one can envision an unzipping mechanism for unwinding of the viral rna molecule and dissociation of the rna molecule from the n protein in a stepwise manner, one module at a time, without the need to overcome a high-energy barrier of dissociating a whole n protein at once. the weak interactions between n protein dimers also minimize formation of kinetic traps and allow a greater degree of regulation of rnp assembly. at present the structures of several helical viral rnp of rna viruses have been reported. these include rabies virus (rv) (albertini et al., ) , vesicular stomatitis virus (vsv) (green et al., ) , respiratory syncytial virus (rsv) (tawar et al., ) , lassa virus (hastie et al., ; hastie et al., ) , rift valley fever virus (rvfv) (raymond et al., ) , bunyamwera virus (bunv) (ariza et al., ; li et al., ) and leanyer orthobunyavirus (leav) (niu et al., ) . the n proteins of these rna viruses all possess the modular organization similar to that of the sars-cov n protein, namely they all consist of an n-terminal arm, two domains which are connected by a flexible hinge, and a flexible c-terminal tail. with the exception of lassa virus, the rna binds to the positively charged crevice between the n-and c-terminal domains that shield rna from the environment. thus, rna sequestering by nucleoproteins is likely a common mechanism used by rna viruses to protect their genomes from host defense mechanism. it also suggests that conformational change in the rna packing is required during expression and translation. the number of rna bases bound per n protein varies from in rvfv to in leav. over the past years considerable insights regarding the structure and function of the sars-cov n protein have been revealed. it is remarkable that the coronavirus n protein family shares a common modular structure organization incorporating functionally important idrs even when they share only moderate sequence identity. new biophysical information, together with recent studies employing classical genetics and biochemical meth-ods, have started to provide a clearer picture of how the n protein forms the rnp and what factors affect the process. however, the quest for understanding how the sars-cov n protein (and coronavirus n proteins in general) carries out its roles during the viral life cycle is still far from over. a critical piece of missing information lies in the atomic structure of the rnp complex, whose elucidation has been hampered by the low solubility of the complex and labile nature of the full-length n protein. it will be probably not enough to only obtain the structure of the sars-cov rnp, but the determination of a number of coronavirus rnps will be necessary to ascertain whether they share a common structural code. another topic is the role of the n protein in the viral replication-transcription complex (rtc), which is composed of various coronavirus nonstructural proteins (nsp's). in mhv, the n protein has been shown to dynamically associate with the rtc (verheije et al., ) . keane and giedroc recently found that mhv n bound to nsp with high affinity through the ntd and lkr (keane and giedroc, ) . co-localization of the n protein with the rtc has also been observed in cells infected with sars-cov (stertz et al., ) , although whether there is direct physical interaction between the two remains to be seen. one problem in this field is the lack of knowledge on the functions of the individual nsp's, making it extremely difficult to interpret the biological relevance of n-nsp interactions. the n protein might also associate with the rna-dependent rna polymerase (rdrp) in coronaviruses (van der meer et al., ) , but the interaction is poorly defined and more effort will be required to verify the association and clarify its role. the sars-cov n protein has been reported to interact with numerous host cell proteins, such as the b phosphoprotein (zeng et al., ) , smad , the chemokine cxcl (zhang et al., ) , translation elongation factor- alpha (zhou et al., ) , pyruvate kinase (wei et al., ) , and - - (surjit et al., ) . unfortunately, there have been few follow-up studies that independently verify these interactions, and the large variance in experimental conditions used to initially identify these interactions makes it extremely difficult to obtain a coherent picture of the sars-cov n protein interactome in the host cell. on the other hand, a recent ibv study employing high-throughput mass spectrometry yielded a list of cellular proteins that may potentially bind to the n protein (emmott et al., ) , and the same strategy could be applied to sars-cov and other coronaviruses (especially mers-cov) for interactome mapping. comparisons between different coronavirus n protein interactomes should provide valuable information on host specificity and evolution of the interactions between n and host cell proteins, and may offer insight into the development of antiviral agents against coronaviruses that target interactions between 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nucleoprotein-rna complex reveals unique architecture for rna encapsidation crystal structure at . a resolution of the rna-binding domain of the u a spliceosomal protein complexed with an rna hairpin sequence comparison of the n genes of five strains of the coronavirus mouse hepatitis virus suggests a three domain structure for the nucleocapsid protein phosphorylation of the arginine/serine dipeptide-rich motif of the severe acute respiratory syndrome coronavirus nucleocapsid protein modulates its multimerization, translation inhibitory activity and cellular localization phleboviruses encapsidate their genomes by sequestering rna bases the transmissible gastroenteritis coronavirus contains a spherical core shell consisting of m and n proteins rna-binding proteins of coronavirus mhv -detection of monomeric and multimeric-n protein with an rna overlay-protein blot assay ribonucleocapsid formation of sars-cov through molecular action of the n-terminal domain of n protein speeding 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dimerization domain of sars coronavirus nucleocapsid protein solved by the sail-nmr method accurate determination of leucine and valine side-chain conformations using u-[ n/ c/ h]/[ h-(methine/methyl)-leu/ val] isotope labeling, noe pattern recognition, and methine cgamma-hgamma/cbeta-hbeta residual dipolar couplings: application to the -kda enzyme iia(chitobiose) crystal structure of a nucleocapsid-like nucleoprotein-rna complex of respiratory syncytial virus localization of mouse hepatitis virus nonstructural proteins and rna synthesis indicates a role for late endosomes in viral replication the coronavirus nucleocapsid protein is dynamically associated with the replication-transcription complexes sars-cov nucleocapsid protein interacts with cellular pyruvate kinase protein and inhibits its activity glycogen synthase kinase- regulates the phosphorylation of sarscoronavirus nucleocapsid protein and viral replication functional anthology of intrinsic disorder. . biological processes and functions of proteins with long disordered regions recombinant severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein forms a dimer through its c-terminal domain crystal structure of the severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein dimerization domain reveals evolutionary linkage between corona-and arteriviridae augmentation of immune responses to sars coronavirus by a combination of dna and whole killed virus vaccines the nucleocapsid protein of sars-associated coronavirus inhibits b phosphorylation cxcl interact with sars-cov n protein in and out cell severe acute respiratory syndromeassociated coronavirus nucleocapsid protein interacts with smad and modulates transforming growth factor-{beta} signaling the nucleocapsid protein of severe acute respiratory syndrome coronavirus inhibits cell cytokinesis and proliferation by interacting with translation elongation factor {alpha} are weak protein-protein interactions the general rule in capsid assembly? acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . references . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . acknowledgement this work was supported by grants nsc - -b- - from the national science council and nhri-ex - b from the national health research institute of the republic of china. the nmr experiments were carried out with nmr spectrometers of the high-field nuclear magnetic resonance center (hfnmrc) supported by core facility for protein structural analysis, national core facility program for biotechnology, the national science council of the republic of china. crystal structure was determined at the taiwan beamline bl b (in spring- ) of the national synchrotron radiation center (nsrrc). key: cord- -mua qvst authors: cui, zhanding; li, dengliang; xie, yinli; wang, kai; zhang, ying; li, guohua; zhang, qian; chen, xiaoxueying; teng, yue; zhao, shihui; shao, jiang; xingmeng, fan; zhao, yanli; du, dongju; guo, yanbing; huang, hailong; dong, hao; hu, guixue; zhang, shuang; zhao, yongkun title: nitazoxanide protects cats from feline calicivirus infection and acts synergistically with mizoribine in vitro date: - - journal: antiviral res doi: . /j.antiviral. . sha: doc_id: cord_uid: mua qvst feline calicivirus (fcv) is a highly contagious pathogen that causes acute upper respiratory infections and oral disease in cats, thus seriously endangering feline health. recently, there have been outbreaks of particularly virulent variant strains of fcv, which can cause both acute symptoms and fatal systemic disease. the discovery of effective antiviral agents to treat fcv infection is, therefore, gradually assuming increased importance. in this study, we showed that both nitazoxanide and mizoribine had antiviral activity in f cells infected with different strains of fcv and also demonstrated a synergistic effect between the two drugs. experiments in cats challenged with fcv showed that nitazoxanide significantly reduced the clinical symptoms of fcv infection, reduced viral load in the trachea and lungs, and reduced viral shedding. our results showed that nitazoxanide and mizoribine could potentially be used as therapeutic agents to treat fcv infection. feline calicivirus (fcv), the major representative of the family caliciviridae, primarily causes upper respiratory tract disease in cats. highly virulent strains of fcv (vs-fcv) can cause systemic diseases, including subcutaneous edema, necrosis in multiple organs (liver, spleen, pancreas) and interstitial pneumonia (desselberger, ) . fcv vaccines have been commercially available for years but fcv remains highly prevalent among cats, and none of the vaccines currently on sale can protect cats from all strains of fcv (sato et al., ) . in the past decade, severe systemic diseases caused by vs-fcv have been reported in many countries (caringella et al., ; guo et al., ; radford and gaskell, ; schulz et al., ) . one reason for the incomplete protection afforded by vaccines may be that the broad spectrum of genetic and antigenic variability of fcv results in different strains with little cross-reactivity (bergmann et al., ) . some cats also occasionally spread the virus after being inoculated with live viruses (afonso et al., ) . the cat is a companion animals of humans and, in recent years, the development of companion animal culture has aroused interest in treating diseases caused by fcv. a variety of drugs have been shown to have antiviral effects against fcv, but most of these were tested only in in vitro studies (aboubakr et al., ; tian et al., ; wu et al., ) . whether these compounds can also be used in vivo to treat fcv infection is unknown. nitazoxanide (ntz, -[( -nitro- , -thiazol- -yl) carbamoyl] phenyl acetate) was initially identified as an anti-parasitic agent and is approved by the us food and drug administration (fda) as an orally active treatment for protozoal diarrhoea. ntz has since been shown to have antiviral effects against a variety of rna and dna viruses (dang et al., ; jasenosky et al., ; zhou et al., ) , including norovirus, a member of the caliciviridae family, which is a major cause of human gastroenteritis (dang et al., ) . mizoribine (mzr, -[( r, r, s, r)- , -dihydroxy- -(hydroxymethyl)oxolan- -yl]- -hydroxy- h-imidazole- -carb oxamide) is as an imidazole nucleoside that has anti-proliferative activity against some immune cells. mzr has been used in several countries or regions as an immunosuppressant to treat autoimmune diseases and steroid-resistant nephrotic syndrome after renal transplantation (ishikawa, ) . recently, mzr has been shown to have antiviral activity against cytomegalovirus, respiratory syncytial virus, severe acute respiratory syndrome-associated coronavirus, bovine viral diarrhea virus, and foot-and-mouth disease virus saijo et al., ; shigeta, ; shiraki et al., ; stuyver et al., ) . here, we showed first that ntz and mzr had low cytotoxicity in transformed feline kidney fibroblast (f ) cells. we then evaluated the antiviral effects of the two compounds against fcv. both compounds were found to be effective against several strains of fcv and the antiviral effects were found to be dose-dependent. there was also a synergistic effect between mzr and ntz in vitro. animal experiments showed that ntz significantly reduced viral load in the trachea and lungs, and also reduced viral shedding. in cats, both clinical score and mortality decreased on administration of ntz, and blood biochemistry and immunohistochemistry showed that ntz may be an effective clinical treatment for cats infected with fcv. feline kidney fibroblast-like monolayer cells (f ) and the ch-sh strain of fcv were provided by the institute of military veterinary medicine (changchun, china). ch-jl , ch-jl , ch-jl and ch-jl strains of fcv were isolated and stored in our laboratory zhao et al., ) . mzr (#m ) and ntz (#n ) were purchased from aladdin, china. fcv strain ch-jl was used for all experiments unless otherwise stated. f cells were seeded into a -well plate and grown in minimum essential medium (mem; gibco, usa) containing % fetal bovine serum (fbs). when the cells had formed monolayers, the medium was replaced by mem containing % fbs and different concentrations of mzr ( , , , , , and μm) or ntz ( , , , , , and μm) and % fbs. mem containing . % dmso was used as the blank control. the cells were incubated for or h at °c under an atmosphere containing % co and then washed twice with phosphate-buffered saline (pbs). fbs-free mem ( μl) and cck ( μl, biosharp, china) were then added and the cells were incubated at °c for - h. a cmax plus plate reader (molecular devices, usa) was used to read the optical density (od) at nm. cell viability was calculated using the following equation: fcv ( × half-tissue culture infectious dose (tcid ), μl) and different concentrations of mzr or ntz were added to -well plates containing monolayers of f cells and the cells were incubated at °c under an atmosphere containing % co for h. each drug concentration was tested in three replicate wells and . % dmso was used as the blank control. tcid values were determined and reverse transcription-quantitative polymerase chain reaction (rt-qpcr) of fcv was carried after three freeze-thaw cycles to evaluate the antiviral effects of mzr and ntz against fcv. half-maximum inhibitory concentrations (ic ) were determined, and the results were plotted using graphpad prism . an indirect immunofluorescence assay (ifa) was performed to observe the antiviral effects against fcv more directly. after immobilization with cold acetone and washing, anti-fcv antibodies ( : , vmrd, usa) were used as primary antibodies and fitc-labeled rabbit anti-cat igg antibodies ( : ; bioss antibodies, china) were used as secondary antibodies (cui et al., ) . fluorescence was observed using a leica dmi inverted fluorescence microscope (leica, germany). a solution of virus was diluted in a -fold gradient. aliquots ( μl per well) of solutions with different concentrations of virus, together with mem containing % fbs ( μl), were added to each plate column. the blank control wells contained no virus. the plates were cultured h at °c under an atmosphere containing % co and virus tcid values were then calculated using the reed and muench formula. relative rt-qpcr was used to evaluate fcv gene expression. briefly, viral rna was extracted using a simply p total rna extraction kit (bioflux, china), retranslated into cdna using revertaid first strand cdna synthesis kit (thermo fisher, usa), followed by rt-qpcr using tb green premix ex taq ii (tli rnaseh plus, takara, china). the upstream and downstream primers were: fcv ′-gcaggttgggataaacatgga- ′ and ′-cacgaggcgattgagttgag- ′; gapdh ′-tggaaagcccatcaccatc- ′, and ′-actccacaacatactcagcacca- ′. the antiviral effects of mzr and ntz against other stains of virus (ch-jl , ch-sh, ch-jl and ch-jl ) were determined as described in section . . briefly, solutions of virus were diluted to × tcid and added, together with mzr or ntz, to -well plates containing monolayers of f cells. tcid and rna expression levels were determined after incubation at °c under an atmosphere containing % co for h, followed by three freeze-thaw cycles. the checkerboard method, with serial dilutions and mixtures of the two compounds, was used to determine the effect of combinations of mzr and ntz. tcid values were determined and synergyfinder was used to evaluate the effects of the combinations (ianevski et al., ) . the zero interaction efficacy (zip) model was used to calculate the synergistic combination score of different concentrations of the drugs (yadav et al., ) . all animals conformed to the general requirements for animal experiments in china (gb/t - ). healthy female cats ( - weeks old, . - . kg) were randomly assigned to eight groups (four animals per group) once they had been confirmed to be virus-free by elsa and pcr detection of fcv rna and antibodies. the prophylactic effect of ntz was investigated first. on day - (- dpi, the day before viral infection) animals in the treatment groups received oral doses of ntz ( mg/kg, mg/kg or mg/kg) in pbs ( μl). these were designed groups a, b and c, respectively. the control group received the same volume of pbs. on day , the cats were infected intranasally with fcv ( . × tcid ) in mem ( μl). the therapeutic effect of ntz after onset of infection was examined next. on day , days post-infection (dpi), infected cats were treated orally with ntz ( mg/kg, mg/kg or mg/kg) in pbs ( μl). these were designated groups d, e and f, respectively. the control group received an equal volume of pbs. clinical signs and survival rates of the animals were monitored as described previously, and cat health was assessed on days and (see table for details) (cui et al., ) . virus titers were measured in oral swabs and different tissues by quantitative rt-qpcr. the sequences of the fcv primers were the same as those used for the relative rt-qpcr. blood samples were collected from the cats. a complete blood count was carried out using a hematology analyzer (pe- vet , prokan, china) and biochemical analysis was carried out using a vettest plasma chemistry analyzer (idexx, usa). immunohistochemistry tests were performed as described before (cui et al., ) . briefly, paraffin sections were prepared after incubation with anti-fcv antibody ( : , vmrd) for h followed by treatment with hrp-labeled rabbit anti-cat secondary antibody (gibco). unless otherwise stated, all experiments were simulated with . % dmso and × tcid fcv, and all were repeated three times. * p < . ; ** p < . ; *** p < . ; **** p < . . a cck kit was used to test the cytotoxicity of the two compounds in f cells. cc values of both compounds were > μm (results not shown). ifa was then used to measure the antiviral activity of the two compounds against fcv (supplementary material s ) and the results were analyzed using image j software (figure a and e) . at a concentration of μm, the optical densities of samples containing the two compounds were significantly different to the control group (p < . ). fcv tcid values and levels of gene expression at different compound concentrations were compared with values for the control group. the tcid values of fcv was significantly reduced (p < . ) after treatment (figure b and f) and gene levels of fcv were significantly downregulated (p < . ) (figure c and g) . the ec of ntz was . μm and the ec of mzr was . μm (figure d and h) . (a) once the antiviral activity of ntz and mzr against the fcv ch-jl strain had been established, ch-jl , ch-jl , ch-jl and ch-sh strains (diluted to × tcid ) were treated with ntz ( μm) or mzr ( μm). tcid values were measured after h. (b) relative rt-qpcr was used to assess changes in gene levels of different fcv strains. (c and d) virus tcid values were calculated to determine the combined effect of different concentrations of ntz ( - . μm) and mzr ( - μm) on fcv. the zip model in synergyfinder was used to analyze the combined effects of the drugs and plot the results. the synergy score for the zip model was expressed as the average of all δ scores in the dose-response landscape, and the red portion of the graph indicates synergy. all experiments were repeated three times. * p < . ; ** p < . . ntz and mzr not only had antiviral efficacy against the fcv ch-jl strain, but the tcid values of the ch-jl , ch-jl , ch-jl and ch-sh strains were also significantly different after drug treatment (p < . ) (figure a ). fcv gene expression was also significantly downregulated by treatment with the two compounds (p < . ) ( figure b ). the checkerboard method was used to determine the combined effects of ntz and mzr. drug solutions were diluted and the zip model was used to calculate the scores of different drug dose combinations. the average synergy score was . ( figure d ) and the most synergistic area score was . ( figure c ), indicating that ntz and mzr had synergistic effects over the concentration range tested. table. (d and h) survival rates for different groups. n = cats/group; ns p > . ; *** p < . ; **** p < . . once we had established that ntz and mzr both have antiviral activity against fcv, we chose the fda-approved oral medication ntz for subsequent animal experiments. we found that, at - dpi, rectal temperature was not significantly increased in group b. the maximum average temperature of groups a, b and c was significantly lower than the control group (p < . ). the rate of weight change was not significantly reduced in groups a and b and the difference was significant at day , compared with the control groups (p < . ). when different doses of ntz were administered orally at dpi (groups d, e and f), there was little difference in rectal temperature of any of the groups, compared with the control group (p < . ). the rate of weight change, however, was significantly lower in groups d and e than in the control group. we also calculated clinical scores for all groups on days and . on dpi, the average scores of the control groups (treatment at - dpi and treatment at dpi) were . and . , which were significantly different to the scores of the other groups (p < . ). the survival rate of group c was %, and that of the relevant control group was %. the survival rate of groups e and f was % and that of the relevant control group was %. figure . nitazoxanide reduces virus shedding and viral load (a and b) fcv viral shedding in cat's mouth and nose test paper on dpi, measured by rt-qpcr. (c and d) at dpi, one cat was euthanized in each group, the trachea, lung, spleen, liver and kidney were collected, and shedding of fcv from the different tissues was measured. (e) cats' tracheas and lungs were sectioned ( -fold) and the sections were treated with cat anti-fcv primary antibody and hrp-labeled rabbit anti-cat secondary antibody, followed by color development. brown areas indicated by black arrows are fcv-positive. (f) image-pro plus . was used to analysis optical density in two selected slices ( -fold); n = cats/group; - dpi, mg/kg ntz; dpi, mg/kg ntz; control, μl pbs (to simulate mg/kg ntz orally); ns p > . ; * p < . ; ** p < . ; *** p < . ; *** p < . . in the − dpi oral ntz cats, one cat in group c died on day ; viral shedding was copies/μl on the ninth day ( figure a ). in the dpi oral ntz cats, one cat in group d died on day and one cat in group f died on day ; viral shedding at day and day was copies/μl and copies/μl, respectively ( figure b ). as expected, ntz reduced viral load in both trachea and lungs ( figure e ). after oral ntz treatment, either on - dpi or dpi, levels of fcv in the trachea and lungs were significantly lower than in the control group (p < . ) ( figure f ). wbc counts and lym percentages fluctuated little in cats treated orally with ntz. mid counts fluctuated, with both mid and gran firstly increasing and then decreasing ( figures. a -f) . plt counts showed a slight downward trend but the plt counts of cats in the − dpi oral drug group were within the normal range ( figure f ). one cat in the control group did not recover from the infection and died on day . the biochemical results showed that there were no significant fluctuations in alt, amyl, tbil, urea or glu in cats that received ntz orally at − dpi or dpi, indicating no significant organ damage in these animals compared with the control group ( figure g -l). there is currently little choice of drugs for the treatment of fcv and, in the clinic, broad-spectrum antiviral drugs, such as famciclovir and ganciclovir, remain the first choice. recent research has shown, however, that famciclovir has limited efficacy against fcv (fumian et al., ) . nitd , fexaramine and cmc are also candidate drugs to treat fcv infection. ntz and cmc have been shown to have synergistic anti-fcv activity, and have been used to treat fcv-infected cats (enosi tuipulotu et al., ; fumian et al., ; kim and chang, ) . mzr has been shown to have antiviral activity against caprine alpha herpes virus (cphv- ), but it had not been determined whether it also has activity against fcv (camero et al., ) . in our study, we found that ntz and mzr have low cytotoxicity. the ec values of ntz and mzr against fcv ch-jl were . μm, and . μm, respectively, and both compounds showed activity against other fcv strains. we also showed that ntz and mzr act synergistically, with the most synergistic area scoring . . to our knowledge, this is the first report of the antiviral activity of mzr against fcv. we chose the fda-approved orally active medication ntz for animal experiments. previous studies found that ntz caused diarrhea and vomiting (siddiq et al., ) , but we did not observe these side effects in our study. when we treated cats with different doses of ntz, either at - dpi or dpi, viral shedding and mortality were both reduced. the cats did not show significant changes in body temperature after oral ntz at dpi, but weight loss was reduced compared with the control group. there was significant relief of the symptoms of oral ulcers, especially in cats who received ntz at dpi, (supplementary material s ) . the amount of fcv in the trachea and lungs, which are easily infected by fcv (pesavento et al., ) , was significantly reduced after treatment with different doses of ntz, and this result was confirmed by immunohistochemistry. by analyzing complete blood counts and biochemical results, we found that systemic symptoms were alleviated in cats that received ntz. in conclusion, we have confirmed the antiviral activity of ntz and mzr against fcv in vitro, and shown that the compounds acted synergistically. ntz was shown to reduce viral load in the trachea and lungs and still had a therapeutic effect on fcv-infected cats when administered at dpi. the synergy between ntz and and mzr in vivo should now be evaluated and drug resistance studies should be carried out. these studies will provide further evidence that ntz and mzr can be used as therapeutic agents for diseases caused by fcv. table table in vitro antiviral activity of clove and ginger aqueous extracts against feline calicivirus, a surrogate for human norovirus a multi-national european cross-sectional study of feline calicivirus epidemiology, diversity and vaccine cross-reactivity antibody response to feline calicivirus vaccination in healthy adult cats enhancement of the antiviral activity against caprine herpesvirus type of acyclovir in association with mizoribine feline calicivirus infection in cats with virulent systemic disease equine immunoglobulin f(ab') fragments protect cats against feline calicivirus infection opposing effects of nitazoxanide on murine and human norovirus caliciviridae other than noroviruses the adenosine analogue nitd has potent antiviral activity against human and animal caliciviruses potential therapeutic agents for feline calicivirus infection isolation and molecular characterization of a virulent systemic feline calicivirus isolated in china. infection synergyfinder: a web application for analyzing drug combination dose-response matrix data mizoribine and mycophenolate mofetil the fda-approved oral drug nitazoxanide amplifies host antiviral responses and inhibits ebola virus fexaramine as an entry blocker for feline caliciviruses in vitro and in vivo antiviral activity of mizoribine against foot-and-mouth disease virus distribution of the feline calicivirus receptor junctional adhesion molecule a in feline tissues dealing with a potential case of fcv-associated virulent systemic disease inhibitory effect of mizoribine and ribavirin on the replication of severe acute respiratory syndrome (sars)-associated coronavirus intranasal immunization with inactivated feline calicivirus particles confers robust protection against homologous virus and suppression against heterologous virus in cats two outbreaks of virulent systemic feline calicivirus infection in cats in germany recent progress in antiviral chemotherapy for respiratory syncytial virus infections effects of cyclosporine, azathioprine, mizoribine, and prednisolone on replication of human cytomegalovirus norovirus gastroenteritis successfully treated with nitazoxanide inhibitors of the impdh enzyme as potential anti-bovine viral diarrhoea virus agents identification of inonotus obliquus polysaccharide with broad-spectrum antiviral activity against multi-feline viruses isolation, genomic characterization and pathogenicity of a feline calicivirus strain ch-jl from chinese stray cats in vitro antiviral effect of germacrone on feline calicivirus searching for drug synergy in complex dose-response landscapes using an interaction potency model isolation and phylogenetic analysis of three feline calicivirus strains from domestic cats in jilin province inhibitory effects of antiviral drug candidates on canine parvovirus in f cells the authors declare no conflicts of interest. a cat in group e that started oral ntz at dpi developed a large area of oral ulcers at dpi but the ulcerated area gradually decreased with time. a cat in group b that started oral ntz at - dpi had no visible oral ulcers at dpi. a cat in the control group had a mouth ulcer at dpi. key: cord- -v p rze authors: livonesi, márcia cristina; moro de sousa, ricardo luiz; badra, soraya jabur; figueiredo, luiz tadeu moraes title: in vitro and in vivo studies of the interferon-alpha action on distinct orthobunyavirus date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: v p rze oropouche, caraparu, guama, guaroa and tacaiuma viruses (orthobunyavirus genus) cause human febrile illnesses and/or encephalitis. to achieve a therapeutical agent to prevent and/or treat these diseases we evaluated the antiviral action of interferon-alpha (ifn-α) on these orthobunyaviruses. in vitro results showed that all the studied orthobunyaviruses are susceptible to antiviral action of ifn-α, but this susceptibility is limited and dependent on both concentration of drug and treatment period. in vivo results demonstrated that ifn-α present antiviral action on oropouche and guaroa viruses when used as a prophylactic treatment. moreover, a treatment initiated h after infection prevented the death of guaroa virus infected-mice. additionally, mortality of mice was related to the migration and replication of viruses in their brains. our results suggest that ifn-α could be potentially useful in the prevention of diseases caused by oropouche virus and in the prevention and/or treatment of diseases caused by guaroa virus. the oropouche (orov), caraparu (carv), guama (gmav), guaroa (grov) and tacaiuma (tcmv) viruses belong to distinct antigenic serogroups of the genus orthobunyavirus in the bunyaviridae family. these viruses are enveloped with trisegmented single-stranded rna genome of negative or ambisense polarity, replicate in the cytoplasm and bud into the golgi apparatus (elliot et al., ) or upon the plasma membrane (goldsmith et al., ) . orov (simbu serogroup) is transmitted mainly by the biting midge (culicoides paraensis) and has been associated with dengue-like acute febrile illness. orov fever has emerged over the past years as a serious public health problem in tropical and subtropical areas of central and south america, having caused at least reported outbreaks involving more * corresponding author. tel.: + ; fax: + . e-mail addresses: pink@rpm.fmrp.usp.br, livonesi@zipmail.com.br (m.c. livonesi). than half a million people (watts et al., ; pinheiro et al., ) . clinical features of orov fever include abrupt onset of fever, chills, severe headache, dizziness, myalgia, arthralgia, nausea, and vomiting. about half of the patients have a recurrence of symptoms within - days after they become afebrile. aseptic meningitis by orov has been reported during outbreaks. all ages and both sexes appear to be equally susceptible to infection (leduc and pinheiro, ) . similarly to orov, carv (group c serogroup), gmav (guama serogroup), grov (california encephalitis and bunyamwera serogroups) and tcmv (anopheles a serogroup) have also been associated with febrile illness in humans. these viruses are transmitted by mosquitoes and cause disease mainly in the south america countries (brinton et al., ; tavares-neto et al., ; jonkers et al., ; march and hetrick, ; travassos da rosa et al., ; iversson et al., ; iversson, ) . an antiviral therapy, if available, would be a very helpful intervention, reducing symptoms and disease period of orov fever as well as of those febrile illnesses caused by other orthobunyaviruses. however, until the moment, there is not treatment or vaccine for these viral diseases, and a recent study demonstrated that these viruses are resistant to antiviral action of the ribavirin, a broad-spectrum antiviral drug (livonesi et al., ) . the interferon (ifn) system is the first line of defense against viral infection in mammals. this system is able to block the spread of virus infection in the body, sometimes at the expense of accelerating the death of the infected cells. interferons are divided into two types, type i and type ii, both of which have antiviral activity (sen, ; galligan et al., ) . the type i interferons include the ifn-␣ and -␤ and they are secreted by virus-infected cells, and exhibit multiple biologic properties including antiproliferative, antiviral, and immunomodulatory effects (platanias et al., ; stark et al., ; goodbourn et al., ) . the type ii interferon present only one member, the ifn-␥, which is not virus-inducible, but it is secreted by activated t lymphocytes and nk cells and shows antiviral activity directly, through the induction of effector molecules (e.g. nitric oxide), and indirectly, through enhanced antigen presentation and the induction of apoptosis (boehm et al., ) . due to the antiviral properties of ifn-␣, it has been used clinically to treat chronic infections caused by hepatitis b and c viruses (davis et al., ; mchutchison et al., ) and in the treatment of infections caused by hpv (sen, ) . moreover, in vitro and/or in vivo experiments have demonstrated that ifn-␣ is able to inhibit the replication of many viruses as: sandfly fever sicilian (crance et al., ) , dengue (diamond et al., ) , severe acute respiratory syndrome-related coronavirus (sars cov) (tan et al., ; ströher et al., ; galligan et al., ) , vaccinia (liu et al., ) , and ebola (mahanty et al., ) . thus, in an effort to characterize antiviral agents that could attenuate infections caused by orov, carv, gmav, grov and tcmv, we tested the in vitro and in vivo actions of ifn-␣ on these viruses. oro (bean ), gma (bean ), gro (beh ), and tcm (bean ) viruses were kindly supplied by dr. pedro vasconcelos and dr. amélia travassos da rosa (evandro chagas institute, brazilian ministry of health, belém, brazil and university of texas medical branch, galveston, tx, usa). carv (span ) was kindly supplied by dr. terezinha lisieux coimbra (adolpho lutz institute, são paulo, brazil). viral stocks were obtained from the brains of intracerebrally infected newborn mice. brains were mixed with pbs (dilution : , w/v), macerated, and centrifuged at × g for min at • c. the supernatants were harvested and stored at − • c until use. african green monkey kidney (vero e ) cells were grown in minimum essential medium (mem, cultilab, brazil) supplemented with % inactivated, mycoplasma-free, fetal bovine serum (fbs, cultilab, brazil), % l-glutamine and . % sodium bicarbonate. interferon-alpha- a (ifn-␣- a) or roferon ® -a (hoffmann-la roche, usa) was used in the in vitro experiments. recombinant murine interferon-alphaa (ifn-␣a) (sigma-aldrich; st. louis, mo, usa) was prepared following the instructions of manufacturer and was used in the in vivo experiments. swiss newborn mice were obtained from the laboratory animal facility of the university of são paulo (ribeirão preto, brazil). the mice were maintained in microisolator cages in the animal housing facility of the center for research in virology (university of são paulo, ribeirão preto, brazil). the experiments were approved by the ethical committee on vertebrate animal experiments of the university of são paulo (no. / ). in vitro antiviral evaluation was done with a plaque assay (livonesi et al., ) . vero e cells were seeded in -well plates in mem with % fbs, for h at • c and % co . medium was removed, serial -fold dilutions of viral stocks diluted in mem with % fbs were added ( . ml/well) in quadruplicates, and the cells were incubated for h at • c. subsequently, the viral inoculum was removed, and . ml of a combination (v/v) of % low-melting-point agarose plus × mem ( % fbs) was added to each well; the plates were incubated at • c for days for orov and gmav, days for carv and grov, and days for tcmv. the plaques were visualized after staining with a naphtol blue black solution ( min) (morens et al., ) , preceded of removal of the agarose plug. the plaques were counted under an inverted microscope, and the virus titer was determined as log pfu ml − . ifn-␣- a was diluted in the medium and added to cells h before, or , , , h after viral infection. comparisons between the virus titers obtained from ifn-␣- a-treated and non-treated cell cultures were done and the results were plotted as percentage of inhibition on plaque formation (table ) . the ifn-␣- a was added to the cell cultures at the concentrations ≤ , iu ml − , because this concentration is not toxic to vero e cells as previously described (tan et al., ) . the most frequent adverse reactions caused by administration of ifn-␣ in humans include fatigue, myalgia, arthralgia, headache, fever, chills, anorexia, nausea, vomiting, diarrhea and abdominal pain, and usually occur in patients treated with high doses of ifn-␣ (the italian cooperative study group on cml, ; schmutz et al., ; márquez-peiró et al., ) . thus, the parameter chosen to evaluate the ifn-␣a toxicity in mice was a significant weight loss of ifn-␣a-treated mice in comparation with placebo-treated mice. the ifn-␣a doses used were , , , and , iu ml − ( l per mouse/day). the mice were treated intraperitoneally (i.p.) daily for days. the animal weights were determined daily and the concentration chosen of ifn-␣a was , iu ml − , because it was well tolerated by mice, which presented an increase of weight similar to the placebo-treated mice (fig. ) . furthermore, a dose of , iu ml − approximates to the highest levels of the recombinant ifn-␣ given to humans ( million units daily) (freireich et al., ) . three-day-old swiss mice were infected i.p. with orov ( ld ), carv ( ld ), gmav ( ld ), grov ( ld ), or tcmv ( ld ) in a volume of l per mouse. the mice were treated i.p. with ifn-␣a ( , iu ml − ) or placebo in a volume of l per mouse. the treatment was initiated h before, or or h after infection and maintained every day. the animals were monitored daily for mortality. suckling mice infected i.p. with orov, carv, gmav, grov and tcmv usually die after development of encephalitis, showing paralysis of forefoot, tremor and difficulty in eating, and presenting high viral titers in the brain some days before death . considering the brain as the principal target organ to viral replication, the virus titers obtained in this organ were used as parameter to demonstrate the efficacy of drug. thus, the brains of mice (two mice per group) were taken aseptically on days , , , , , and after infection. brains were mixed with pbs (dilution : , w/v), macerated and centrifuged at × g for min at • c. supernatants were harvested and stored at − • c before plaque assay on vero e cells. virus titers in brain were expressed as log pfu ml − . analysis of variance followed by the parametric tukey-kramer test was used in the in vitro experiments, while fisher's exact test was used in the in vivo experiments (instat soft-ware, graphpad, san diego, ca). a p value less than . was considered to indicate statistical significance. firstly, vero e cells were treated with ifn-␣- a ( , iu ml − ) at different periods of the cell infection by orov, carv, gmav, grov and tcmv. table shows that ifn-␣- a was able to inhibit the replication of all the studied orthobunyaviruses when treatment occurred either h before or h after infection (p < . ). moreover, ifn-␣- a presented a significant inhibitory activity on replication of gmav (p < . ) and tcmv (p < . ) when treatment was initiated h after infection. additionally, ifn-␣- a significantly inhibited the tcmv replication when cell treatment was initiated h after infection (p < . ). these results show that ifn-␣ present inhibitory effect on the orov, carv, gmav, grov, and tcmv replication, but this effect is dependent on timing of the treatment. next, we verified whether doses lower than , iu ml − would have inhibitory effect on the replication of distinct orthobunyavirus. thus, cells were treated h after viral infection with ifn-␣- a doses ≤ , iu ml − or medium. fig. shows that the concentration of , iu ml − is able to significantly inhibit carv, gmav, grov, and tcmv replication, but not of orov. furthermore, the concentration of iu ml − produces a significant inhibitory effect on carv, grov and tcmv replication (fig. ) , suggesting that antiviral effect of ifn-␣ on these orthobunyaviruses is also dependent on the concentration used. we firstly examined whether ifn-␣a treatment beginning day before viral infection would be effective on preventing lethal encephalitis caused by orov, carv, gmav, grov, and tcmv. intraperitoneal administration of the maximum nontoxic dose of ifn-␣a ( , iu ml − ) (fig. ) prevented the death of all mice infected by orov or grov (table ). these high survival rates were associated with the inhibition of viral migration and replication in brains of the mice (fig. ) . however, treatment of carv-, gmav-or tcmv-infected mice with ifn-␣a did not result in an increase in survival or prolongation of the mean time to death and did not prevent virus replication in the brain of animals (table and fig. , respectively) . next, the effect of administration of ifn-␣a initiated h after infection of animals by orov or grov was analysed. table shows that treatment with ifn-␣a was either unable to increase the survival time or inhibit the viral replication in the brain tissue of the mice infected by orov (fig. ) . on the other hand, ifn-␣a-treated-grov-infected mice had a survival rate of % (table ) , which was associated with inhibition of viral replication in the brain tissue of mice (fig. ) . treatment with ifn-␣a initiated h after mice infection by grov was unable to inhibit either the death of animals or the viral replication in the brain (table and fig. , respectively). fig. . ifn-␣-treated orov-or grov-infected mice did not show virus replication in the brain when treatment was initiated h before infection. groups of sixteen -day-old swiss mice were infected i.p. with orov, carv, gmav, grov, or tcmv and they were treated i.p. with ifn-␣ ( , iu ml − ) or placebo. mouse brains (two mice per group) were taken on days , , , , , and after infection. the virus titer in the brain was measured by plaque assay. the scale bars represent the mean of pfu ml − . similar results were obtained in a second experiment. h orov / ( %) / ( %) . ± . . ± . grov / / ( %) b . ± . > h grov / / ( %) . ± . . ± . a mean time to death. b p < . ; fisher's exact test. fig. . ifn-␣-treated grov-infected mice did not show virus replication in the brain when treatment was initiated h after infection. groups of sixteen -dayold swiss mice were infected i.p. with orov or grov and they were treated with ifn-␣ ( , iu ml − ) or placebo. mouse brains (two mice per group) were taken on days , , , , and after infection. the virus titer in the brain was measured by plaque assay. the scale bars represent the mean of pfu ml − . similar results were obtained in a second experiment. fig. . ifn-␣ treatment initiated h after infection did not inhibit replication of grov in the brain tissue of mice. groups of nine -day-old swiss mice were infected with grov and treated with ifn-␣ ( , iu ml − ) or placebo. the treatment was initiated h after infection. mouse brains (two mice per group) were taken on days , , , and after infection. the virus titer in the brain was measured by plaque assay. the scale bars represent the mean of pfu ml − . similar results were obtained in a second experiment. these results also show that the in vivo antiviral activity of ifn-␣ on orov and grov is dependent on timing of the treatment. in this study, we evaluated in vitro and in vivo antiviral activity of ifn-␣ on orov, carv, gmav, grov and tcmv. ifn-␣ had a significant antiviral effect in vitro on all studied orthobunyaviruses. the antiviral action observed was dependent on both administration timing ( table ) and concentration of the drug (fig. ) . in vivo experiments confirmed the antiviral activity of ifn-␣ on orov and grov, but not on carv, gmav and tcmv, being that this antiviral activity was dependent on the administration period of the drug (tables and ). the antiviral effect of ifn-␣ observed in vivo was related to its ability to inhibit viral replication in the brain tissue of infected mice . the antiviral action of exogenous ifn-␣ observed in vitro was probably due to its ability to induce an antiviral state on cells through the secretion of proteins that ultimately inhibit viral replication. for instance, the dsrna-dependent protein kinase (pkr) leads to phosphorylation of eif ␣ with a consequent blockade of translation of most cellular and viral mrnas; the , -oligoadenylate synthetases (oas), which are also activated by viral dsrna, produces , -oligoadenylates that in turn activate the rnase l, resulting in the degradation of viral and host rnas; and the mx proteins, gtpases of the dynamin family, whose mechanisms of action and functions await elucidation (stetson and medzhitov, ) . similarly, the antiviral effect observed in vivo after administration of exogenous ifn-␣ might have been either due to the above-mentioned cell-intrinsic mechanisms of this cytokine or the actions of type i ifns on the immune system. in fact, type i ifns are able to enhance the expression of mhc class i proteins on cells and thereby promote cd + t cell response (goodbourn et al., ) . moreover, type i ifns play an essential role in the differentiation and function of effector cd + t cells (stetson and medzhitov, ) . type i ifns are also able to enhance the cytotoxicity of nk cells by up-regulating the levels of perforins, and can stimulate the proliferation of nk cells to a limited degree (stetson and medzhitov, ) . ordinarily, cd + t and nk cells can eliminate infected cells, defending the host against intracellular infections (stetson and medzhitov, ) . however, the mechanisms through which the exogenous ifn-␣ performed its functions were not analysed in the present study. in vitro and in vivo results showed that ifn-␣ was able to prevent viral replication in a limited manner, exerting antiviral effect only when administrated early and in high doses, suggesting that orov, carv, gmav, grov and tcmv present some escape mechanism from antiviral actions of the ifn-␣. resistance to the ifn system was previously described for three members of bunyaviridae family, carv (brinton et al., ) , rift valey fever virus (bouloy et al., ) , and bunyamwera virus (weber et al., ; léonard et al., ) . brinton et al. ( ) , corroborating the results obtained by us, observed that carv resisted antiviral action of immmunomodulators, such as ifn-␣ and ifn-␤. however, the escape mechanism of carv was not studied by the authors. rift valey fever and bunyamwera viruses presented resistance to the ifn system associated with the non-structural protein nss which is encoded during viral replication. this protein showed to be able to block the production of ifn␣/␤ through the inhibition of transcription factors (bouloy et al., ; weber et al., ) . additionally, a recent study has demonstrated that the nss protein is also able to inhibit the ifn response through the interaction with the med component of mediator, a protein complex necessary for mrna production (léonard et al., ) . in our study, both cells and mice received high doses of exogenous ifn-␣ and a possible inhibition of the production of endogenous ifn-␣ by orthobunyaviruses would not be sufficient to block the antiviral effects derived from exogenous ifn-␣. moreover, it is known that vero e cells have an ifn i gene deficiency and thus they are unable to express endogenous type i ifn (mosca and pitha, ) . however, the ifn-dependent pathways are functional and can be activated by exogenously provided ifn (mosca and pitha, ). thus, it is possible that the studied orthobunyaviruses present a escape mechanism similar to that recently described for bunyamwera virus (léonard et al., ) , being able to inhibit the responses produced by ifn after association with the cell surface receptor. it would be interesting to further elucidate the escape mechanisms used by different bunyaviruses, especially those etiologically related to human diseases. it is important to mention that the cytokines tested in this study are part of a large group of existing ifns-␣ (foster and finter, ; yeow et al., ) . therefore, we cannot affirm whether other ifns-␣ would present a similar antiviral activity to that observed here. for instance, tan et al. ( ) evaluated the in vitro antiviral activity of four types of ifn-␣, roferon ® -a (ifn-␣- a), intron a (ifn-␣- b), wellferon (ifn-␣-n ) and alferon (ifn-␣-n ), on sars coronavirus (sars-cov) and observed that only two out of them, wellferon and alferon, were able to inhibit the cytophatic effect caused by this virus in vero e cell cultures. mechanisms for explaining these differences in activity of ifns-␣ are unknown. we may suppose that it could be related to distinct ifn-␣ sources because some preparations are derived from human lymphoblastoid or leukocyte cells, while other preparations are recombinantly produced in escherichia coli or mammalian cell culture (foster and finter, ) . in particular, the ifns-␣ used in our experiments were the roferon ® -a and the ifn-␣a, both produced in e. coli. in conclusion, our results demonstrated that the ifn-␣ presents in vitro antiviral activity on carv, gmav and tcmv, but this activity is limited and was not confirmed by in vivo experiments. therefore, ifn-␣- a could not be considered a suitable therapeutic drug for illnesses caused by these viruses. however, ifn-␣ presented in vitro and in vivo prophylactic antiviral activities on orov and grov and also showed therapeutic antiviral action on grov. thus, ifn-␣- a could be potentially useful in the prevention of diseases caused by oropouche virus and in the prevention and/or treatment of diseases caused by guaroa virus, mainly during epidemics or after occurrence of a laboratory accident. cellular responses to interferon-gamma genetic evidence for an interferon-antagonistic function of rift valley fever virus nonstructural protein nss characterization of murine caraparu bunyavirus liver infection and immunomodulator-mediated antiviral protection inhibition of sandfly fever sicilian virus (phlebovirus) replication in vitro by antiviral compounds interferon alfa- b alone or in combination with ribavirin for the treatment of relapse of chronic hepatitis c modulation of dengue virus infection in human cells by alpha, beta, and gamma interferons bunyaviridae are all type i human interferons equivalent? quantitative comparison of toxicity of anticancer agents in mouse, rat, hamster, dog, monkey and man interferons and viruses: signaling for supremacy ultrastructural characteristics of sin nombre virus, causative agent of hantavirus pulmonary syndrome interferons: cell signalling, immune modulation, antiviral responses and virus countermeasures current status of the eco-epidemiological knowledge on arboviruses pathogenic to humans in the atlantic forest region of the state of são paulo circulation of eastern equine encephalitis, western equine encephalitis, ilhéus, maguari, and tacaiuma viruses in equines of the brazilian pantanal laboratory studies with rodents and native to trinidad oropouche fever interation of bunyamwera orthobunyavirus nss protein with mediator protein med : a mechanism for inhibiting the interferon response prevention of letal respiratory vaccinia infections in mice with interferon-␣ and interferon-␥ in vitro and in vivo studies of the ribavirin action on brazilian orthobunyavirus protection from lethal infection is determined by innate immune responses in a mouse model of ebola virus infection studies on guaroa virus identificación de oportunidades de mejora del tratamiento de la hepatitis c interferon alfa- b alone or in combination with ribavirin as initial treatment for chronic hepatitis c simplified plaque reduction neutralization assay for dengue viruses by semimicro methods in bhk- cells: comparison of the bhk suspension test with standard plaque reduction neutralization transcriptional and posttranscriptional regulation of exogenous human beta interferon gene in simian cells defective in interferon synthesis oropouche fever differences in interferon ␣ and ␤ signalling side effects of interferon alpha a and b viruses and interferons how cells respond to interferons type i interferons in host defense severe acute respiratory syndrome-related coronavirus is inhibit by interferon-␣ inhibition of sars coronavirus infection in vitro with clinically approved antiviral drugs serologic survey for yellow fever and other arboviruses among inhabitants of rio branco, brazil, before and three months after receiving the yellow fever d vaccine ifn-␣ a as compared with conventional chemotherapy for the treatment of chronic myeloid leukemia doenças infecciosas e parasitárias-enfoque amazônico oropouche virus transmission in amazon river basin of peru bunyamwera bunyavirus nonstructural protein nss counteracts the induction of alpha/beta interferon antiviral activities of individual murine ifn-alpha subtypes in vivo: intramuscular injection of ifn expression constructs reduces cytomegalovirus replication we thank dr. pedro vasconcelos, dr. amélia travassos da rosa, and dr. terezinha lisieux coimbra for kindly supply viruses used in this paper. this work was supported by a fapesp grant to luiz t.m. figueiredo (no. / - ) and by a fellowship from capes to márcia c. livonesi. key: cord- -xqxznv r authors: kohyama, shunsuke; ohno, satoshi; suda, tatsuya; taneichi, maiko; yokoyama, shoichi; mori, masahito; kobayashi, akiharu; hayashi, hidenori; uchida, tetsuya; matsui, masanori title: efficient induction of cytotoxic t lymphocytes specific for severe acute respiratory syndrome (sars)-associated coronavirus by immunization with surface-linked liposomal peptides derived from a non-structural polyprotein a date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: xqxznv r spike and nucleocapsid are structural proteins of severe acute respiratory syndrome (sars)-associated coronavirus (sars-cov) and major targets for cytotoxic t lymphocytes (ctls). in contrast, non-structural proteins encoded by two-thirds of viral genome are poorly characterized for cell-mediated immunity. we previously demonstrated that nucleocapsid-derived peptides chemically coupled to the surface of liposomes effectively elicited sars-cov-specific ctls in mice. here, we attempted to identify hla-a* -restricted ctl epitopes derived from a non-structural polyprotein a (pp a) of sars-cov, and investigated whether liposomal peptides derived from pp a were effective for ctl induction. out of peptides predicted on computational algorithms, nine peptides could significantly induce interferon gamma (ifn-γ)-producing cd (+) t cells in mice. these peptides were coupled to the surface of liposomes, and inoculated into mice. six liposomal peptides effectively induced ifn-γ-producing cd (+) t cells and seven liposomal peptides including the six peptides primed ctls showing in vivo killing activities. further, ctls induced by the seven liposomal peptides lysed an hla-a* positive cell line expressing naturally processed, pp a-derived peptides. of note, one of the liposomal peptides induced high numbers of long-lasting memory ctls. these data suggest that surface-linked liposomal peptides derived from pp a might offer an efficient ctl-based vaccine against sars. the outbreak of severe acute respiratory syndrome (sars) in early led to thousands of infected patients and hundreds of deaths (groneberg et al., ) . sars is caused by a novel coronavirus termed sars-associated coronavirus (sars-cov) (drosten et al., ; ksiazek et al., ; peiris et al., ) . this is a plusstranded rna virus with an approximately kb long genome encoding replicase gene products and the structural proteins containing spike, envelop, membrane and nucleocapsid (groneberg et al., ) . although the viral receptor has been identified (li et al., b) , the pathogenesis of sars remains poorly understood and the apparent latency of sars-cov in animal reservoirs continuously provides us a serious threat of reemergence. spike protein of sars-cov is a major target for neutralizing antibodies because this protein interacts with the cellular receptor (li et al., b) . high titers of neutralizing antibodies to sars-cov were detected in sera of the recovered patients (li et al., a) , and humoral immunity induced by a dna vaccine contributed to the protection against sars-cov challenge in mice (yang et al., ) . these data strongly suggest that neutralizing antibodies play a central role in the clearance of sars-cov. on the other hand, a rapid loss of both cd + and cd + t cells was observed in patients suffering from severe sars, and the cell counts gradually returned to normal ranges as the patients recovered (tang et al., ) . furthermore, certain hla class i alleles have been reported to correlate with sars susceptibility (lin et al., ; ng et al., ) , suggesting that cytotoxic t lymphocytes (ctls) play an important role in the elimination of sars-cov as well. several ctl epitopes have been identified from spike and nucleocapsid proteins of sars-cov (wang et al., a; wang et al., b; chen et al., ; tsao et al., ; zhou et al., ; ohno et al., ) . however, ctl epitopes have not been found in non-structural proteins of sars-cov. in general, non-structural proteins are more conserved and synthesized earlier than structural proteins, and therefore, they could be preferable as an antigenic target for ctls. a synthetic peptide vaccine is a potential candidate for a ctl-based vaccine against pathogenic viruses because short peptides rarely cause undesirable responses including general toxicity, immunosuppression and autoimmunity. however, the immunogenicity of this type of vaccine is very weak. liposomes have extensively been investigated as a delivery system for antigen (alving et al., ) . in most cases, it has been prepared by antigen entrapment within the aqueous lumen of liposomes. in contrast, we have previously demonstrated that an ovalbumin (ova)-derived peptide, ova - conjugated on the surface of liposomes induced ova - -specific ctls in mice more effectively than did liposomes containing ova - inside (taneichi et al., ; nagata et al., ) . furthermore, we have recently shown that surfacelinked liposomal peptides derived from nucleocapsid of sars-cov effectively induced sars-cov-specific ctls in mice (ohno et al., ) . these data suggest that liposomes would become an excellent adjuvant vehicle for a synthetic peptide vaccine when a peptide(s) is chemically coupled to the surface of liposomes. in the current study, we attempted to identify hla-a* restricted ctl epitopes derived from a largest non-structural polyprotein a (pp a) of sars-cov using computational algorithms and hla-a* transgenic mice. there are several reasons why we focused on this protein. first of all, pp a protein is a regulatory protein, and hence, more conserved and synthesized earlier than spike and nucleocapsid proteins (groneberg et al., ) . second, since pp a is a largest protein composed of amino acids among sars-cov-associated proteins (groneberg et al., ) , it is more likely to find highly immunogenic, dominant epitopes in pp a protein than in any other proteins of sars-cov. peptides identified were then chemically conjugated on the surface of liposomes and evaluated for their abilities to induce sars-cov-specific ctls. to define potential hla-a* -binding peptides derived from pp a of sars-cov (urbani strain) (genbank accession number: aap ), we used two computer-based programs, syfpeithi (http://www.syfpeithi.de/) (rammensee et al., ) and bimas (http://www-bimas.cit.nih.gov/molbio/hla bind/) (parker et al., ) . as shown in table , peptides with superior scores were selected for predicted ctl epitopes. these peptides were synthesized by operon biotechnologies (tokyo, japan). in addition, three known epitopes derived from the sars-cov spike protein (spike- : litgrlqsl; spike- : rlnevaknl; spike- : fiagliaiv) (wang et al., a; wang et al., b) were synthesized as well. t (salter et al., ) is a transporter associated with antigen processing (tap)-deficient, human lymphoblastoid cell line expressing natural hla-a* . c r-a is a human b cell line, hmy .c r that was transfected with an hla-a* gene (winter et al., ) . t and c r-a cells were maintained in rpmi- medium (sigma-aldrich, st. louis, mo) supplemented with % fetal calf serum (fcs) (jrh biosciences, lenexa, ks) (r- ) and r- containing g/ml g (sigma-aldrich), respectively. peptide binding assay was performed as described by ohno et al., . briefly, t cells were suspended in aim v serum-free medium (life technologies, rockville, md) containing nm human beta- microglobulin (␤ m) (sigma-aldrich) and were incubated with each synthetic peptide at various concentrations overnight at • c. cells were then stained with the anti-hla-a* monoclonal antibody (mab), bb . (parham and brodsky, ) , followed by fluorescein isothiocyanate (fitc)-labeled goat anti-mouse igg (sigma-aldrich). the mean fluorescence intensity (mfi) was measured by flow cytometry (facscan, bd biosciences, san jose, ca). the concentration of each peptide that yields the half-maximal mfi of t cells pulsed with a control peptide derived from hepatitis c virus, ns - (ohno et al., ) was calculated as the half-maximal binding level (bl ). experiments were performed three times, and data are given as mean values ± standard deviation (sd). mice express a transgenic hla-a* monochain, designated as hhd, in which human ␤ m is covalently linked to a chimeric heavy chain composed of hla-a* (␣ and ␣ domains) and h- d b (␣ , transmembrane, and cytoplasmic domains) (pascolo et al., ; matsui et al., ) . eight-to twelve-week-old mice were used for all experiments. mice were housed in appropriate animal care facilities at saitama medical university, saitama, japan, and handled according to international guidelines for experiments with animals. surface-coupled liposomal peptides were prepared as described previously by taneichi et al., via disuccinimidyl suberate (dss) . briefly, ml of anhydrous chloroform solution containing . mm dioleoyl phosphatidyl ethanolamine (dope) and l of triethylamine was mixed with . ml of anhydrous chloroform solution containing . mm dss and stirred for h at • c. the solvent was evaporated, and ml of a : mixture of ethyl acetate and tetrahydrofuran was added to dissolve the residue. thirty six milliliters of mm sodium phosphate (ph . ) and ml of saturated nacl aqueous solution were added to the solution, shaken for min, and allowed to separate. the upper layer was washed with the same buffer again. after evaporation of the solvent, ml of acetone was added to dissolve the residue. ice-cold acetone ( ml) was added in drops and kept on ice for min to precipitate. crystals were collected and dissolved in ml of chloroform. after evaporation, . mg of dope-dss was obtained. for preparation of unsaturated liposomes, dope-dss ( . mm), dioleoyl phosphatidylcholine ( . mm), cholesterol ( . mm), and dioleoyl phosphatidyl glycerol ( . mm) were dissolved in ml of chloroform/methanol. the solvent was then removed under reduced pressure and . ml of phosphate buffer (ph . ) was added to make a . % lipid suspension. the vesicle dispersion was extruded through a . m polycarbonate filter to adjust the liposome size. a ml suspension of dss-introduced liposomes and . ml of mg/ml peptide solution were mixed and stirred for days at • c. the liposome-coupled and -uncoupled peptides were separated using cl- b column chromatography. liposomes used in the experiments were prepared under the regulation of good manufacturing practice and involved no detectable endotoxin. for identification of ctl epitopes, mice were immunized intravenously (i.v.) with × syngeneic spleen cells pre-pulsed with m of each synthetic peptide. in the case of immunization with liposomal peptides, mice were subcutaneously (s.c.) immunized once or several times at one-week intervals with each of (rammensee et al., ) at http://www.syfpeithi.de/. b peptide binding scores to hla-a . were determined by the bimas database (parker et al., ) at http://www-bimas.cit.nih.gov/molbio/hla bind/. c data of peptide binding assays are shown as bl , indicating a concentration of each peptide that yields the half-maximal mfi of t cells pulsed with a control peptide, ns - . data are given as mean values ± sd of three independent experiments. surface-coupled liposomal peptides ( g/mouse) together with cpg-odn ( -tccatgacgttctgatgtt- , hokkaido system science, sapporo, japan) ( g/mouse) in l pbs in the footpad. ics was performed as described by matsui et al., . briefly, after one week following immunization, × spleen cells of immunized mice were incubated with m of a relevant peptide for h at • c in the presence of brefeldin a (golgiplug tm , bd biosciences). after blocking fc receptors with the rat antimouse cd /cd mab (fc block tm , bd biosciences), cells were stained with fitc-conjugated rat anti-mouse cd ␣ mab (bd biosciences) for min at • c. cells were then fixed, permeabilized, and stained with phycoerythrin (pe)-conjugated rat anti-mouse interferon-gamma (ifn-␥) mab (bd biosciences). after washing the cells, flow cytometric analyses were performed. in vivo ctl assay was carried out as described before by suvas et al., . in brief, spleen cells from naive hhd mice were equally split into two populations. one population was pulsed with m of a relevant peptide and labeled with a high concentration ( . m) of carboxyfluorescein diacetate succinimidyl ester (cfse) (molecular probes, eugene, or). the other population was unpulsed and labeled with a lower concentration ( . m) of cfse. an equal number ( × ) of cells from each population was mixed together and adoptively transferred i.v. into mice that had been immunized once with a liposomal peptide two weeks earlier. twelve hours later, spleen cells were prepared and analyzed by flow cytometry. to calculate specific lysis, the following formula was used: % specific a multiepitope minigene that encodes multiple predicted epitopes and natural flanking amino acid sequences proximal to the n and c termini of each epitope (fig. a) was synthesized by invitrogen japan k.k. (tokyo, japan). this minigene contains the kozak sequence and the xflag-epitope-tag sequence upstream of the multiepitope sequence (fig. a) . the minigene was then cloned into the nhe i and hind iii sites of a mammalian expression vector, pacgfp -hyg-n (clontech laboratories inc., mountain view, ca). since this vector encodes a green fluorescent protein (gfp) downstream of the multiple cloning site, the resultant pacgfp -pp a plasmid expresses a gfp fusion protein containing pp a-derived ctl epitopes under the control of the cmv promoter in mammalian cells. this plasmid was transfected into c r-a cells by electroporation (gene pulser, bio-rad laboratories inc., hercules, ca). after selection with hygromycin b (invitrogen, carlsbad, ca) at a final concentration of . mg/ml, the transfectant, c r-a -pp a was confirmed for their gfp expression by flow cytometry (fig. b) . . . cr-release assay cr-release assays were carried out as described before by matsui et al., . in brief, after two weeks following immunization, spleen cells of immunized mice were cultured with m of a relevant peptide for one week, and used as effector cells in standard cr-release assays. c r-a -pp a cells were labeled with ci of na cro , and used for target cells. c r-a cells transfected with pacgfp -hyg-n vector were used as a negative target control. after a h incubation, supernatant of each well was harvested and the radioactivity was counted. results were calculated as the mean of a triplicate assay. percent specific lysis was calculated according to the formula: % specific lysis = [(cpm sample − cpm spontaneous )/(cpm maximum − cpm spontaneous )] × . spontaneous release represents the radioactivity released by target cells in the absence of effectors, and maximum release represents the radioactivity released by target cells lysed with % triton x- . statistical comparisons between two groups were performed by the student's t test. one-way anova followed by post-hoc tests were used for statistical analyses between multiple groups in fig. . all statistic analyses were performed using graphpad prism software. a value of p < . was considered statistically significant. the amino acid sequence of pp a protein of sars-cov was searched for potential hla-a* -restricted ctl epitopes by two computer-based programs, syfpeithi (rammensee et al., ) and bimas (parker et al., ) . based on the scores calculated, nonameric peptides were selected and synthesized (table ) . these peptides were then evaluated for their binding affinities to hla-a* molecules as described before by ohno et al., (table ) . twenty-four out of the peptides were high binders displaying bl values less than m, and four of them were medium binders displaying bl values ranging from to m, suggesting that most epitopes should be properly predicted. in contrast, two peptides showed low affinity binding. to examine whether the predicted peptides could elicit peptidespecific ctls in vivo, hhd mice were immunized i.v. with syngeneic spleen cells pre-pulsed with each of pp a-derived peptides. one week after immunization, spleen cells of the immunized mice were prepared and stimulated in vitro with a relevant peptide for h. cd + t cells were then analyzed for their peptide-induced intracellular expression of ifn-␥. as shown in fig. , significant numbers of ifn-␥-producing cd + t cells (p < . ) were detected in mice immunized with syngeneic cells pulsed with each of nine pp aderived peptides including pp a- , - , - , - , - , - , - , - , and - , suggesting that these nine peptides may be hla-a* -restricted ctl epitopes derived from sars-cov pp a protein. since the nine pp a peptides were expected to be ctl epitopes (fig. ) , these peptides were conjugated on the surface of liposomes. the resultant surface-linked liposomal peptides were then evaluated for their capabilities of ctl induction in mice. after hhd mice were immunized once with one of the nine liposomal peptides, spleen cells of them were prepared, stimulated with a relevant synthetic peptide, and stained for their expression of surface cd and intracellular ifn-␥. as shown in fig. , six liposomal peptides including lip-pp a- (p < . ), - (p < . ), - (p < . ), - (p < . ), - (p < . ), and - (p < . ), were able to significantly induce ifn-␥-producing cd + t cells compared with each negative control stimulated without a rel- fig. . intracellular ifn-␥ staining of cd + t cells specific for pp a-derived peptides in spleen cells of mice immunized i.v. with peptide-pulsed spleen cells. hhd mice were immunized i.v. once with syngeneic spleen cells pre-pulsed with each of pp a-derived peptides. one week after immunization, spleen cells were prepared and stimulated with or without (no pep) a relevant peptide for h. cells were then stained for their surface expression of cd and their intracellular expression of ifn-␥ (y axis). the data are shown as percentages of intracellular ifn-␥ + cells within cd + t cells. three mice were used in each group and the data are shown as the mean ± sd of three mice. one-way anova was used for comparison of data between groups in each of the panels (a-f). *p < . compared to data of negative controls (no pep). evant peptide. however, numbers of ifn-␥ + cd + t cells varied among these liposomal peptides (fig. ) , suggesting a variety of immunogenicity. in particular, lip-pp a- was most effective for the induction of peptide-specific ifn-␥ + cd + t cells (fig. ) . in contrast, lip-pp a- and lip-pp a- marginally elicited ifn-␥-producing cd + t cells, and lip-pp a- failed to induce ifn-␥ + cd + t cells in mice. to further assess immunogenicity of the liposomal pp a peptides, three known ctl epitopes derived from sars-cov spike protein, spike- , - and - (wang et al., a; wang et al., b) were conjugated on the surface of liposomes (lip-spike- , - , and - ), and were compared to the pp a-derived peptides for their induction of ifn-␥-secreting cd + t cells. as shown in fig. , high percentages of ifn-␥ + cells within cd + t cells were detected in mice immunized with lip-spike- . however, this liposomal peptide was likely to be slightly less effective than lip-pp a- in the induction of ifn-␥ + cd + t cells (fig. ) . on the contrary, either lip-spike- or lip-spike- did not elicit ifn-␥-producing cd + t cells in mice (fig. ) . we next examined in vivo killing activities of peptide-specific ctls in mice immunized with liposomal peptides. two weeks after immunization, both peptide-pulsed cfse high and unpulsed cfse low target cells were delivered into the mice via i.v. injection, and then peptide-specific lysis was analyzed by flow cytometry (fig. ) . in agreement with the ics data in fig. , high killing activities were observed in mice immunized with lip-pp a- as well as lip-spike- (fig. ) . lip-pp a- also induced a high level of killing activity in mice (fig. ) . on the other hand, modest killing responses were elicited in mice immunized with each of five liposomal peptides involving lip-pp a- lip-pp a- , - lip-pp a- , - lip-pp a- , - , whereas any significant cell lysis was not observed in mice injected with either lip-pp a- , -pp a- , -spike- , or -spike- (fig. ) . these data also suggest that each of the liposomal peptides exhibits different immunogenicity. to address whether the pp a-derived peptides identified are naturally processed and presented, liposomal peptide-induced ctls were tested for their capacity to lyse c r-a -pp a cells. c r-a -pp a cells carry the multiepitope minigene that encodes multiple predicted epitopes with several natural flanking amino acid residues at the n and c termini of each epitope (fig. a) , and thereby express naturally processed epitopes. expression of the multiepitope minigene was confirmed by detection of gfp in c r-a -pp a cells (fig. b) . hhd mice were immunized twice with each of seven liposomal pp a-derived peptides including lip-pp a- lip-pp a- , - lip-pp a- , - lip-pp a- , - lip-pp a- , - , and - that significantly induced ifn-␥ + ctls (fig. ) and/or in vivo killing activities (fig. ) . ctl activities in spleen cells of the mice were then determined by cr-release assays using c r-a -pp a cells as a target. as shown in fig. , c r-a -pp a cells were significantly (p < . or p < . ) lysed by liposomal peptide-induced ctls, whereas c r-a cells were not recognized by them. these data indicate that the seven pp a-derived peptides are naturally processed and presented to ctls. we next examined whether long-lasting peptide-specific ctls could be elicited by immunization with liposomal peptides. hhd mice were immunized three times at one-week intervals with either lip-pp a- or lip-pp a- . spleen cells were then prepared at various days after the final immunization, and stimulated in vitro once with a relevant peptide for h at • c. cd + t cells were then analyzed for their peptide-induced expression of intracellular ifn-␥. in both of the cases, the total numbers of ifn-␥-producing cd + t cells per mouse spleen were slightly increased at day after immunization, rose to a peak at day , and gradually decreased as days went by (fig. a and b) . as shown in fig. , ifn-␥-producing cd + t cells were still detected in mice days after immunization with any of the two liposomal peptides. these results demonstrate that immunization with these liposomal peptides generated long-lasting memory ctls. especially, the frequency of ifn-␥ + cd + t cells in mice injected with lip-pp a- was quite high (fig. ) , indicating that lip-pp a- may be an excellent vaccine candidate. in the current study, high-performing computational algorithms have extensively been utilized for the prediction of ctl epitopes derived from pp a protein of sars-cov. as shown in table , peptides were selected as potential ctl epitopes using syfpei-thi (rammensee et al., ) and bimas (parker et al., ) . out of them, only nine peptides could significantly induce ifn-␥-producing cd + t cells in mice (fig. ) . furthermore, there was not always a good correlation between peptides identified by the algorithms and their activity in the biological assays. for instance, pp a- and pp a- showed relatively inferior scores in bimas (table ) , whereas both peptides stimulated good ctl responses (figs. - ) . thus, currently available algorithms have limited accuracy to find actual epitopes (chentoufi et al., ) , fig. . in vivo killing activities specific for peptides derived from pp a and spike of sars-cov in mice immunized with surface-linked liposomal peptides. hhd mice were immunized once with either each liposomal peptide (lip-peptide) or liposomes alone (lip) together with cpg. one week later, an equal number of a relevant peptide (pp a- , pp a- , pp a- , pp a- , pp a- , pp a- , pp a- , pp a- , pp a- , spike- , spike- spike- )-pulsed cfse high targets (m ) and unpulsed cfse low targets (m ) were transferred into the immunized mice by i.v. injection. after h, cfse-labeled cells were recovered from spleens of recipient mice and analyzed by flow cytometry. the numbers are the percentages of specific lysis shown as mean values ± sd of three independent experiments. however, they are still quite useful because we can easily choose a cluster of promising peptides within a huge protein such as pp a on the programs. multiple immunological screenings have been advanced to validate predicted ctl epitopes. when used individually, each screen is not sufficient for identifying actual epitopes. however, various combinations of these screens are usually successful (chentoufi et al., ; ohno et al., ). therefore, we performed multiple screenings, including cell surface stabilization of hla-a* molecules on t cells, detection of antigen-driven ifn-␥-producing cd + t cells, and functional in vivo and in vitro ctl assays. in these experiments, we took advantage of highly reactive hla-a* transgenic mice, termed hhd mice (pascolo et al., ) . in hhd mice, the innate h- d b and mouse ␤ m genes have been disrupted by homologous recombination, and therefore, the only mhc class i molecule on the cell surface, hla-a* , is efficiently utilized by hla-a* -restricted ctls. as a consequence, six peptides conjugated on the surface of liposomes significantly induced ifn-␥-producing cd + t cells (fig. ) , and seven liposomal peptides including the six peptides primed ctls showing peptidespecific killing activities in mice (fig. ) . however, it has to be taken account that there may be differences between the immunogenic variation observed in hla class i transgenic mice and that in humans primarily because the antigen processing, presentation and ultimately, immunodominance may differ between them. in fact, it was shown that several hla-a* -restricted ctl epitopes derived from human papillomavirus were not processed in hdd mice although these epitopes were naturally processed in hla-a* + humans (street et al., ) , indicating that cross-species incompatibility in antigen-processing and presentation machinery skews the presentation of some ctl epitopes. because pp a-specific ctls induced were generated by stimulation with synthetic peptides, it was necessary to test whether they would recognize naturally processed peptides. to this end, we generated an hla-a* positive c r-a -pp a cell line in substitution for sars-cov-infected target cells because it is quite fig. . recognition of naturally processed epitopes derived from pp a. hhd mice were immunized twice with either lip-pp a- , lip-pp a- , lip-pp a- , lip-pp a- , lip-pp a- , or liposomes alone. two weeks after immunization, spleen cells were prepared and stimulated in vitro with a relevant peptide. after one week, cr release assays were performed at an e:t ratio of with c r-a -pp a cells (gray bars) or c r-a (black bars) as targets. data are shown as the means ± sd of triplicate wells. the experiment was repeated twice with similar results. at least three mice per group were used in each experiment. *p < . ; **p < . ; ns, not significant. difficult to obtain live sars-cov in japan. c r-a -pp a cells carry the multiepitope minigene that encodes nine predicted epitopes with several natural flanking amino acid residues at the n and c termini of each epitope (fig. ) . the basic idea to utilize flanking amino acids (fig. ) comes from the observation that flanking amino acid sequences modulate antigen processing of ctl epitopes (le gall et al., ) . in fact, it was shown that several mutations at nterminal (draenert et al., ; milicic et al., ) and c-terminal (allen et al., ; milicic et al., ) flanking residues of ctl epitopes disrupt proteasomal processing of hiv gag and nef proteins. therefore, addition of flanking amino acid sequences allows natural antigen processing of ctl epitopes in c r-a -pp a cells. as shown in fig. , ctls induced by seven liposomal peptides recognized hla-a* positive c r-a -pp a cells. these data indicate that the seven peptides are naturally processed epitopes. as a matter of course, it will be necessary to examine whether pp a-derived peptides can induce protective ctls using sars-cov at the final screening. so far, several ctl epitopes have been identified from spike and nucleocapsid proteins of sars-cov (wang et al., a; wang et al., b; chen et al., ; tsao et al., ; zhou et al., ; fig. . kinetics of pp a-specific cd + t cell responses after immunization with surface-linked liposomal peptides. spleen cells were prepared from mice at various days after immunization with either lip-pp a- (open circles in a), lip-pp a- (open circles in b) or liposomes alone (closed circles in a and b). after stimulation with a relevant peptide for h, cells were stained for their surface expression of cd and their intracellular expression of ifn-␥. the data indicate the total numbers of intracellular ifn-␥ + cd + t cells per mouse spleen, and are shown as the means ± sd of three mice per group. significant (*p < . ) difference compared to mice immunized with liposomes alone on the same day post immunization. ohno et al., ) . to our knowledge, however, the current study is the first report to demonstrate ctl epitopes derived from a nonstructural protein of sars-cov such as pp a protein. in fact, several liposomal peptides derived from pp a induced high frequencies of ifn-␥-producing cd + t cells (fig. ) in comparison with liposomal peptides derived from nucleocapsid which we have recently published (ohno et al., ) . in particular, lip-pp a- turned out to be most effective in the induction of antigen-driven ifn-␥producing cd + t cells (fig. ) , indicating that pp a- is a highly immunogenic, dominant ctl epitope. it was demonstrated that the surface-linked liposomal peptide was effective for peptide-specific ctl induction in the current study as well as in the previous study (ohno et al., ) . it is noteworthy that long-lasting memory ctls were detected in mice days after immunization with liposomal peptides (fig. ) . these data suggest that the surface-linked liposomal peptide may be an effective tool for ctl-based immunotherapy against infectious diseases such as sars. the surface-linked liposomal peptide might be similar to the lipopeptide, a form of palmitoyl-lipidated peptide that is currently under intense investigation as human vaccines (zhu et al., ; zhang et al., ) . although both effectively induce peptide-specific ctls, there are several differences between them. the lipopeptide is self-adjuvanting to stimulate peptide-specific ctls via toll-like receptor (trl)- (zhu et al., ; zhang et al., ), but the surface-linked liposomal peptide requires external tlr ligands such as cpg (nagata et al., ) . however, cpg causes toxicity in humans (davila et al., ) , and hence, it is essential to find out a safe adjuvant for clinical use of liposomal peptides. on the other hand, the lipopeptide alone without a cd + t-cell epitope fig. . induction of long-lasting ifn-␥-producing cd + t cells in mice immunized with surface-linked liposomal peptides. hhd mice were immunized three times at one-week intervals with either lip-pp a- , lip-pp a- or liposomes alone (lip-no peptide). spleen cells were then prepared days after the final immunization, and stimulated in vitro once with (+) or without (−) a relevant peptide for hours at • c. cells were then stained for their surface expression of cd (x axis) and their intracellular expression of ifn-␥ (y axis). the numbers shown indicate the percentages of intracellular ifn-␥ + cells within cd + t cells. the experiment was repeated twice with similar results, and the data shown are representative of the two independent experiments. at least three mice per group were used in each experiment, and spleen cells of mice per group were pooled. failed to induce ctl-based protective immunity, whereas a helper peptide is not necessary for the surface-linked liposomal peptide to induce peptide-specific ctls. although we focused this study on ctl epitopes restricted by hla-a* which is the most common hla class i allele in the world, this is just a model system that could be applied to any haplotypes. the hla polymorphism should hinder the development of our system, but the supertypes of hla class i may solve this issue. sette and sidney, defined only nine hla class i supertypes that almost cover the entire repertoire of hla class i molecules. epitopes related to all of the nine supertypes should be identified and incorporated into the liposomal vaccine. in summary, we have identified seven hla-a* -restricted ctl epitopes derived from pp a protein of sars-cov using computational algorithms, hla-a* transgenic mice and the surface-linked liposomal peptide. it was shown that one of the liposomal pp a peptides was effective for peptide-specific ctl induction in mice, and efficiently elicited long-lasting memory ctls. these data suggest that surface-linked liposomal peptides derived from pp a protein may offer an effective and safe ctl-based vaccine against sars. selection, transmission, and reversion of an antigen-processing cytotoxic t-lymphocyte escape mutation in human immunodeficiency virus type infection liposomes as carriers of peptide antigens: induction of antibodies and cytotoxic t lymphocytes to conjugated and unconjugated peptides response of memory cd + t cells to severe acute respiratory syndrome (sars) coronavirus in recovered sars patients and healthy individuals hla-a* -restricted cd + cytotoxic t lymphocyte epitopes identified from herpes simplex virus glycoprotein d generation of antitumor immunity by cytotoxic t lymphocyte epitope peptide vaccination, cpg-oligodeoxynucleotide adjuvant, and ctla- blockade immune selection for altered antigen processing leads to cytotoxic t lymphocyte escape in chronic hiv- infection identification of a novel coronavirus in patients with severe acute respiratory syndrome molecular mechanisms of severe acute respiratory syndrome (sars) severe acute respiratory syndrome: global initiatives for disease diagnosis a novel coronavirus associated with severe acute respiratory syndrome portable flanking sequences modulate ctl epitope processing profile of specific antibodies to the sars-associated coronavirus angiotensin-converting enzyme is a functional receptor for the sars coronavirus association of hla class i with severe acute respiratory syndrome coronavirus infection adjuvant activities of novel cytokines, interleukine (il)- and il- for induction of hepatitis c virus-specific cytotoxic t lymphocytes in hla-a* transgenic mice t-bet is required for protection against vaccinia virus infection cd + t cell epitope-flanking mutations disrupt proteasomal processing of hiv- nef peptides coupled to the surface of a kind of liposome protect infection of influenza viruses association of human-leukocyte-antigen class i (b* ) and class ii (drb * ) genotypes with susceptibility and resistance to the development of severe acute respiratory syndrome synthetic peptides coupled to the surface of liposomes effectively induce sars coronavirus-specific cytotoxic t lymphocytes and viral clearance in hla-a* transgenic mice immunogenic variation between multiple hla-a* -restricted, hepatitis c virus-derived epitopes for cytotoxic t lymphocytes partial purification and some properties of bb . : a cytotoxic monoclonal antibody with specificity for hla-a and a variant of hla-a scheme for ranking potential hla-a binding peptides based on independent binding of individual peptide sidechains hla-a . -restricted education and cytolytic activity of cd + t lymphocytes from ␤ microglobulin (␤ m) hla-a . monochain transgenic h- d b ␤ m double knockout mice coronavirus as a possible cause of severe acute respiratory syndrome syfpeithi: database for mhc ligands and peptide motifs genes regulating hla class i antigen expression in t-b lymphoblast hybrids nine major hla class i supertypes account for the vast preponderance of hla-a and -b polymorphism limitations of hla-transgenic mice in presentation of hla-restricted cytotoxic t-cell epitopes from endogenously processed human papillomavirus type e protein cd + cd + t cells regulate virus-specific primary and memory cd + t cell responses antigen chemically coupled to the surface of liposomes are cross-presented to cd + t cells and induce potent antitumor immunity measurement of subgroups of peripheral blood t lymphocytes in patients with severe acute respiratory syndrome and its clinical significance hla-a* t-cell epitopes in severe acute respiratory syndrome (sars) coronavirus nucleocapsid and spike proteins identification of an hla-a* -restricted cd + t-cell epitope ssp- of sars-cov spike protein t-cell epitopes in severe acute respiratory syndrome (sars) coronavirus spike protein elicit a specific t-cell immune response in patients who recover from sars the pocket of hla-a . plays a role in presentation of influenza virus matrix peptide and alloantigens a dna vaccine induces sars coronavirus neutralization and protective immunity in mice a genital tract peptide epitope vaccine targeting tlr- efficiently induces local and systemic cd + t cells and protects against herpes simplex virus type challenge screening and identification of severe acute respiratory syndrome-associated coronavirus-specific ctl epitopes lipopeptide epitopes extended by an n ε -palmitoyllysine moiety increase uptake and maturation of dendritic cells through a toll-like receptor- pathway and trigger a th -dependent protective immunity this work was supported by a grant from the ministry of health, labor and welfare of japan. the authors are grateful to dr. f. a. lemonnier (pasteur institute, paris, france) for providing hhd mice. key: cord- -xemarhlv authors: goswami, biswendu b.; kulka, michael; ngo, diana; cebula, thomas a. title: apoptosis induced by a cytopathic hepatitis a virus is dependent on caspase activation following ribosomal rna degradation but occurs in the absence of ′– ′ oligoadenylate synthetase date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: xemarhlv we have presented previously evidence that the cytopathogenic f strain of hepatitis a virus (hav) induced degradation of ribosomal rna (rrna) in infected cells [arch. virol. ( ) – ]. in contrast, the non-cytopathogenic parent virus hm clone had no effect on rrna integrity. we present here data showing that rrna degradation is followed by apoptosis accompanied by characteristic dna laddering in the cytoplasm of f infected cells. the dna laddering coincided with the detection of caspase and parp- cleavage and was dependent upon activation of the caspase pathway, since treatment with z-vad-fmk, a pan-caspase inhibitor, inhibited both events. rnase l mrna was present in both virus-infected and uninfected cells. messenger rna for the interferon inducible enzyme ′– ′ oligoadenylate synthetase ( ′– ′ oas), which polymerizes atp into ′– ′ oligo adenylate ( – a, the activator of rnase l) in the presence of double-stranded rna, was not detected following virus infection. ′– ′ oas mrna was induced by treatment of the cells with interferon-β (ifn-β). ifn-β mrna was marginally induced following infection. however, phosphorylated stat , a key regulator of interferon-stimulated gene transcription was not detected in virus infected cells. stat phosphorylation in response to ifn treatment was lower in virus-infected cells, compared to uninfected cells treated with interferon, suggesting that f virus infection interferes with interferon signaling. the results suggest that f infection causes the induction of a – a independent rnase l like activity. hepatitis a virus is a picornavirus and is the only known member of the genus hepatovirus. it is the major causative agent for infectious hepatitis worldwide and is easily disseminated by person to person contact as well as via contaminated foods and water, due to its resistance to environmental factors such as heat, low ph, etc. (siegl et al., ; gust, ) . most of the known hav strains establish a slowly replicating and non-cytopathogenic persistent infection in permissive cells in vitro (provost and hillemen, ; flehmig, ; binn et al., ; vallbracht et al., ) . this is in contrast to in vivo infection, where persistence is restricted to a few weeks (lemon, ; vallbracht et al., ) , although sporadic cases of chronic infection have been reported (fagan et al., ; inoue et al., ) . several laboratories have isolated rapidly replicating cytopathogenic (cp) strains that produce visible morphological changes primarily in continuous cell lines of monkey kidney origin (divizia et al., ; venuti et al., ; anderson, ; cromeans et al., ; nasser and metcalf, ; zhang et al., ; brack et al., ) . the mechanism of the cytopathogenic effect (cpe) produced by the rapidly replicating hav strains has been intensely investigated during the past few years to understand why wild type (wt) and cell culture adapted (cc) strains replicate slowly and are unable to induce cpe in these cell lines. these studies have revealed that the genome of the cpe-inducing (cp) strains contain point mutations, compared to the wt strains from which they were derived, scattered throughout the genome, as well as a -bp repeat in the non-translated region (ntr), which harbors the internal ribosomal entry site or ires (lemon et al., ; brack et al., ) . the biological significance of these mutations for the rapid replication phenotype of the cp strains is not completely understood. experiments with chimeric viral genomes containing mutated regions of cp strains on a genetic background of cc strains suggested an effect of these mutations on the expression of the viral genome, possibly due to altered interaction of the mutated genomes with cellular proteins that either positively or negatively regulate viral gene expression whetter et al., ; zhang et al., ; funkhouser et al., ; yi et al., ) . however, the view that hav ires is inefficient compared to other picornaviruses is not universally accepted (jia et al., ; graff and ehrenfeld, ; gauss-müller and kusov, ) . we have recently reported that in the permissive monkey kidney cell line frhk infection with the cp strain hm f (hereafter referred to as f) resulted in degradation of ribosomal rnas (rrnas), whereas viral genomic rna was not degraded. in contrast, the parental cc strain did not cause such degradation either after an acute infection or extended persistent infection (kulka et al., ) . based on the pattern of rrna degradation in intact ribosomes, we suggested that the f virus activates the interferon (ifn) controlled - oligoadenylic acid-dependent rnase l pathway (for recent reviews, see stark et al., ; barber, ; sen, ) . since this pathway is essentially an antiviral mechanism mounted by cells in defense against viral infection, the usurpation of this pathway by the f virus could be a mechanism employed by this virus to reduce competition from cellular mrnas for the host protein synthetic machinery. this virus lacks an active a protease (schultheiss et al., ; harmon et al., ) capable of degrading either eif- g or other cellular translation factors required for translation of capped cellular mrnas, therefore, the importance of such an rna degradative mechanism cannot be overstated. the activation of the rnase l pathway is controlled by ifn (stark et al., ; barber, ; sen, ) through the induction of - oligoadenylate synthetase ( - oas), which synthesizes - oligoadenylic acid or - a, the proximal activator of the rnase l from atp, in the presence of double stranded rna (dsrna). overexpression of rnase l has been shown to inhibit the growth of diverse rna viruses (zhou et al., ) . whether hav infection can induce synthesis of type i ifn (ifn ␣/␤) in cultured cells or intact organisms and consequently activate the - oas/rnase l pathway remains unclear. cell cultures persistently infected with culture-adapted hav do not induce type ifn, and the replication of the virus in persistently infected cells remains sensitive to exogenously added ifn (vallbracht et al., ; brack et al., ) . acute infection with cc or cp strains of hav did not cause induction of ifn ␣/␤ mrna transcription and cc strains also blocked ifn mrna transcription and secretion of ifn in response to dsrna in frhk and mrc- cells (brack et al., ) . exogenous ifn inhibited virus replication when added either before or after exposure to virus in a dose dependent manner in plc/prf/ cells (crance et al., ) . inhibi-tion of virus replication in the above study also showed a strong dependence on the multiplicity of infection (moi). a significant increase in the activity of the ifn-induced - oas was observed following ifn treatment, suggesting that the rnase l pathway may be involved in the inhibition of virus replication (crance et al., ) . constitutive expression of - oas results in the inhibition of picornavirus replication (chebath et al., ) , and ectopic expression and activation of rnase l induces apoptosis in animal cells (diaz-guerra et al., ; castelli et al., ) . the goal of this investigation was to study the role of the ifn -regulated - a activated rnase l on rna degradation and apoptosis in f infected cells. frhk monkey kidney cell line was a kind gift of dr. g. kaplan (fda, center for biologics evaluation and research, bethesda, md). the cells were grown in eagles minimal essential medium (emem) containing % heat inactivated fetal bovine serum (fbs), mem non-essential amino acids and sodium pyruvate (all from invitrogen, carlsbad, ca), with routine weekly sub-culturing. under these conditions the cells increase about -fold in - days. the cell line is contact inhibited and can be kept in growth medium or maintenance medium ( % fbs) for several weeks without degeneration of the monolayer. hav strains hm / f (cytopathogenic in frhk cells) and hm /clone (non-cytopathogenic in frhk cells) were obtained from atcc. viruses were grown in frhk cells. virus stocks were prepared as described before (kulka et al., ) . f was titered by plaque assay, while clone virus was titered by eia in -well plates as described previously (goswami et al., ) using the havab eia diagnostic kit (abbott laboratories, abbott park, il). human ifn-␤ and the pancaspase inhibitor z-vad-fmk were obtained from sigma (st. louis, mo). confluent cultures of frhk cells in cm flasks ( × cells) were infected with a multiplicity of infection (moi) of pfu/cell of f or tcid /cell of clone in . ml of mem containing % heat inactivated fbs or were mock infected. after h of adsorption, . ml of the same medium was added to each flask and incubation continued until the desired time of harvest. cells were harvested by scraping, centrifuged to remove medium, and the pellets washed once with pbs (ca + and mg + free) and stored frozen at − • c or used immediately. rna isolation was carried out as previously described (kulka et al., ) . a persistent infection of frhk cells with clone virus was established as described (kulka et al., ) . the cells were maintained by routine subculturing in the same manner as for normal frhk cells. to monitor virus replication, cells were periodically seeded into -well plates along with uninfected cells, and viral antigen in methanol fixed cells measured by the havab eia procedure (goswami et al., ; kulka et al., ) . all experiments with this cell line (hereafter referred to as clone ) were carried out between months and year of routine subculture. cell pellets of uninfected or virus infected cells were resuspended in mm tris-hcl, mm edta, ph . and lysed by the addition of triton x- to . % (brack et al., ) . cell lysates were kept on ice for min with intermittent vortex mixing, followed by centrifugation at , × g for min. the supernatant was carefully removed and digested with proteinase k ( . mg/ml) in the presence of . % sds. the digest was extracted with an equal volume of phenol:chloroform:isoamyl alcohol (pcia), and the aqueous phase removed to a fresh tube. nucleic acids were precipitated by the addition of sodium acetate to . m and . volume of ethanol. the precipitates were collected by centrifugation, washed once with % ethanol, dried and dissolved in rnase/dnase free water. nucleic acids were quantitated by a measurement. equal amounts of sample were incubated at • c for min in l te with or without u of cloned rnase (ambion, austin, tx), and analyzed by % agarose gel electrophoresis in tbe. denaturing agarose gel electrophoresis was performed as described before (kulka et al., ; chomczynski and mackey, ) . the gel was photographed under uv light. the induction of apoptosis was investigated by the incorporation of biotin labeled deoxyribonucleotides and detection of biotin incorporation according to the manufacturer's instructions (deadend colorimetric tunel system, promega). cells were seeded in four well chamber slides, and infected at confluency with f at pfu/cell. cells were fixed for tunel assay at h pi. persistently infected cells were seeded in four well chamber slides, and processed when the monolayers reached confluence. viral antigen-positive cells were displayed by immunohistochemistry, using an antibody to vp (kulka et al., ) . briefly, cells were fixed in buffered formalin, permeabilized with . % tween in pbs, and reacted with : dilution of rabbit anti-vp antibody in pbs containing % nonfat dry milk (nadala and loh, ) . after several washes with pbs containing . % tween , the cells were incubated with hrp labeled anti-rabbit igg diluted in % non-fat dry milk. following several washes to remove unbound antibody, color was developed using cn-dab substrate (pierce chemical co.). total cytoplasmic rnas were digested with rnase free dnase (promega) in the presence of rnasin rnase inhibitor followed by pcia extraction and ethanol precipitation, as above, to remove any contaminating dna. rna samples ( g) each were reverse transcribed using amv reverse transcriptase in a total volume of l using g oligo(dt) as primer as described previously (goswami et al., ) . ifn-␤ mrna was amplified using the primer pairs accaacaagtgtctcctcca (sense) and gaggtaacctgtaagtctgt (antisense), with an annealing temperature of • c. this primer pair amplifies a -bp fragment (brack et al., ) . amplification of the p / and the p / forms of - oas were carried out with the primers p / sense ( gaagcctgtcaaagagagag ) and p / antisense ( tgtgttttcatgctccctcg ) which amplifies a -bp fragment (nucleotides - ), p / sense ( caatcagcgaggccagtaatc ), and p / antisense ( cttgacgattttgtgccgctc ), which amplifies a -bp fragment (nucleotides - ), with annealing at and • c, respectively (takahashi et al., ) . to amplify rnase l mrna, the following primers were synthesized based on the sequence of human rnase l (genbank accession number nm ): rnase l sense ( agatgaggaaactaaggacctc ), and rnase l antisense ( gaggtttgtttggactgtggg ), which amplifies a -bp fragment (nucleotides - ). the annealing temperature was • c. ␥-actin mrna levels were used to normalize the rt-pcr signal generated by the other mrnas. the primers for ␥-actin were aagtaccccattgagcatggc (sense) and cacagcttctccttgatgtcgc (antisense). the annealing temperature was • c. viral rna in total or cytoplasmic rna samples were amplified using primers ccgtttgcctaggctataggcta (sense) and cagctccatgctaatcatggagt (antisense). this primer pair is directed to the end of the hav genome and can differentiate between the genomes of f and clone strains of hav based on the presence of an additional bases in the ntr of the f genome (goswami et al., ; kulka et al., ) . all pcr amplifications were carried out in a total volume of l, and contained - l of the cdna pool, . mm mgcl , m of each deoxynucleoside triphosphate, pmol of each primer, and u of taq dna polymerase (promega) for - cycles. frhk cells were mock infected or infected with pfu/cell of f, tcid /cell of clone or pfu/cell of coxsackievirus b (cbv ) in mem containing % heat inactivated fbs. after h adsorption period, medium was added to each flask and incubation continued for the indicated time period. for infected cultures treated with z-vad-fmk, the virus adsorption media was removed prior to the addition of mem/ % fbs containing - m inhibitor and subsequently incubated for h. at the indicated times post-treatment, cell culture monolayers were scraped into their media and pelleted by centrifugation ( × g, • c). cell extracts were prepared and the protein content estimated as described previously (kulka et al., ) . equal concentrations of each protein extract were adjusted to equivalent volumes in denaturing sample buffer and heated at • c for - min and subjected to sds-page under reducing conditions, followed by electrophoretic transfer onto nitrocellulose membranes in transfer buffer containing methanol. the membranes were blocked with tbs-t (tris buffered saline/ . % tween ) containing % nonfat dried milk (nfdm) for min at room temperature (rt) and washed four times with tbs-t. membranes were incubated either overnight at • c or h at rt with primary antibody diluted in tbs-t containing either % bsa or % nfdm. primary antibodies were used that recognize (i) stat (phosphorylation state independent) or stat phosphorylated on tyrosine at position (cell signaling technologies), used at a : dilution ( % bsa) for incubation overnight at • c; (ii) full length/large (cleaved) fragment caspase- (cell signaling technologies), used at a : dilution ( % nfdm) for overnight incubation at • c; (iii) full length/large (cleaved) fragment parp (sigma), used at a : dilution ( % bsa) for h incubation at rt, and (iv) ␤-actin (sigma), used at a : dilution ( % nfdm) for h incubation at rt. secondary antibody-hrp conjugates (pierce-endogen) were used at a : , dilution. all other steps including detection were performed as previously described (kulka et al., ) . confluent cultures of frhk cells in -well plates were infected with f at . , , or pfu/cell, as described previously. replication of virus was assessed by the level of viral antigen in each well as described before using the havab eia kit (goswami et al., ; kulka et al., ) . previous investigations have demonstrated that a cp strain of hav, havcyt/hb . , derived from the cc strain hm , induced apoptosis in - % of the cells by h post-infection (pi), increasing to . % by days pi (brack et al., ) . similar slowly developing cpe and apoptosis were reported in cells infected with another cp strain, hm / a (gosert et al., ) . in contrast, cells infected with the f strain develop extensive cpe in approximately % of the cells by h, and an almost total cpe is observed by h (kulka et al., ) . using a colorimetric tunel assay at h pi we investigated whether cpe induced by the f strain is a result of apoptosis (fig. ) . tunel positive cells were present in the f infected monolayers, whereas positively stained cells were only occasionally observed in the absence of virus infection. persistently infected clone cells at days or days pi also showed an occasional positively stained cell. the culture remained viable, however, and f like cpe was not observed until more than a year of weekly passages. immunohistochemical staining was performed on the persistently infected clone cells to determine if the persistently infected status of these cells is due to the lack of actively replicating virus in a majority of the cell population (fig. ) . almost all of the cells showed positive staining for viral antigen. in addition, replication of the virus as measured by eia confirmed the presence of actively replicating virus (data not shown). taken together, these results show that the f strain causes apoptosis in infected cells much earlier than cells infected with other cp strains, and that the lack of apoptosis in persistently infected cells is not due to lack of active virus replication. we have previously shown that rrna degradation is dependent upon replication of f, since treatments that inhibited replication also resulted in inhibition of rrna degradation. however, certain cell lines are sensitive to perturbation of cellular macromolecule synthesis and can be driven to apoptotic cell death by treatment with metabolic inhibitors. in such instances, apoptosis causes s rrna fragmentation (houge et al., ) . therefore, to determine whether rrna degradation is a cause, or the effect of apoptosis in f infection, it was necessary to establish the temporal sequence of these two events. frhk cells were infected with f and total nucleic acids from the cytoplasm were prepared at different times as described in section . as control, cytoplasmic nucleic acids were also isolated from mock infected cells and persistently infected (clone ) cells. nucleic acids were analyzed by agarose gel electrophoresis before and after digestion with rnase (fig. ) . degradation of the s and s molecules into discrete products was fig. . rapid induction of apoptosis in f infected frhk cells. confluent cultures of frhk cells were mock infected or infected with the cytopathic f strain of hav. cells were processed for the detection of tunel positive cells at h pi as described in section . persistently infected cultures following infection by the non-cytopathic hm (clone ) strain of hav were established as described in section . cells were processed for tunel positive cells at days or days post infection. evident at h pi, and by h pi very little intact rrnas could be detected. the appearance of apoptotic dna laddering in the cytoplasm could be detected following elimination of rrnas from total nucleic acid preparations by rnase and clearly lagged behind rrna degradation as a result of f infection (fig. , panel a) . neither mock infected cells nor persistently infected (clone ) cells showed any evidence of rna or dna degradation (fig. , panel b) . no evidence of rna or dna degradation could be seen in nucleic acid preparations from cells infected with f virus, at or h pi (data not shown). the data indicate that rrna degradation in f infected cells occurs prior to apoptosis, and is not a consequence of apoptosis or dna fragmentation. previous studies have shown that rrna degradation in cells over-expressing the ifn regulated - a activated rnase l results in apoptosis due to activation of caspase (rusch et al., ) . caspase is the terminal enzyme in the caspase cascade, and activation of caspase by upstream caspases occurs due to proteolytic cleavage (for a review, see roulston et al., ) . to investigate if a similar activation of caspase takes place due to infection with the f virus, we investigated caspase activation by western blotting experiments (fig. ) . proteolytic cleavage of caspase could be detected in f infected cells at h pi, which coincided with the appearance of a dna ladder in the cytoplasm (compare fig. , panel a with fig. , panel a) . proteolysis of caspase was not detectable at earlier times, nor in persistently infected (clone ) cells (fig. , panel a) , confirming that caspase activation follows rrna degradation (fig. ) . activation of caspase was accompanied by the degradation of parp- (poly adp-ribose polymerase ), a target for caspase during the apoptotic response (kaufmann et al., ) . cleavage of parp- was evident at h pi and coincided with the appearance of activated caspase ; parp was no longer detectable in f infected cells at h pi (fig. , panel b) . commensurate with the lack of caspase activation or rna degradation, there was no cleavage of parp in persistently infected cells. having shown the proteolytic activation of caspase in f infected cells, it was of interest to determine if apoptotic dna degradation was a consequence of caspase activity. frhk cells were infected with f and then treated with different concentrations of the caspase inhibitor z-vad-fmk for h. cultures were processed for the analysis of caspase activation by western blotting as described in section . caspase activation was prevented in a dose dependent manner by treatment with z-vad-fmk (fig. ). significant inhibition of caspase activation was achieved with m z-vad-fmk, and complete inhibition was observed at m of the inhibitor. rna and dna analyses by agarose gel electrophoresis in the cytoplasm of f infected cells were carried out to establish a connection between caspase activation and apoptotic dna laddering (fig. ) . treatment of f infected cells with m z-vad-fmk did not prevent rrna degradation (panel a). the appearance of apoptotic dna laddering in the cytoplasm was completely prevented (panel b). these results show that rrna degradation is independent of caspase induced dna degradation, fig. . effect of the caspase inhibitor z-vad-fmk on proteolytic activation of caspase in response to f infection. cells were infected with f ( pfu/cell), and incubated in growth medium containing - m of the inhibitor for h, prior to protein extraction. electrophoresis, transfer, and immunodetection with antibody against caspase were performed as described in section . the lane marked m contains protein extracts from mock infected cells harvested and processed h following mock infection in the absence of inhibitor. fig. . effect of z-vad-fmk on rrna degradation and apoptotic dna laddering in the cytoplasm of infected cells. cells were infected with f ( or . pfu/cell) or mock infected, and further incubated in growth medium containing m z-vad-fmk for h, prior to isolation of total cytoplasmic nucleic acids. samples were separated in an % agarose gel before (a) or after (b) digestion with rnase . and is not a consequence of apoptosis due to perturbation of cellular macromolecule synthesis as a result of virus infection. we have previously shown that despite rrna degradation, cellular protein synthesis remains unaffected in f infected cells between and h pi (kulka et al., ) . in agreement with the lack of effect of z-vad-fmk on rrna degradation which requires replication of the virus (kulka et al., ) , virus replication was not inhibited by treatment with this compound (table ) . ribosomal rna degradation is one of the three antiviral mechanisms controlled by ifn. it is brought about by the activation of a latent ribonuclease rnase l by - a, and requires ifn treatment of cells prior to virus infection fig. . effect of ifn on rrna degradation and dna ladder formation in response to f infection. cells were either pretreated with u/ml ifn-␤ for h ( h pre) before infection or treated with ifn following virus infection ( h post). at h pi, cytoplasmic nucleic acids were isolated as described in section and analyzed by agarose gel electrophoresis before (a) or after (b) treatment with rnase to assess rna or dna degradation. the bracketed areas indicate the position of the degradation products of s and s rrna. (stark et al., ; barber, ; sen, ) . in contrast, rna degradation or apoptosis induced by f did not require ifn pretreatment of the cells (figs. and ) . to investigate whether ifn is involved in f induced rrna degradation and apoptosis, cells were pretreated with u/ml ifn-␤ (human) for h, prior to exposure to f virus at a moi of or . pfu/cell. dna and rna from the cytoplasm of infected cells were analyzed at h pi as described in section (fig. ) . pretreatment with ifn had a modest inhibitory effect on both rrna degradation and apoptotic dna degradation, particularly at low moi. when ifn was added to the cells following the h virus absorption period, neither rna degradation nor dna laddering was affected by ifn treatment (fig. ) . ifn treatment had a significant growth inhibitory effect on f only at low moi ( . pfu/cell) infection (table ) , and had no effect on virus replication in clone cells over a h treatment period (data not shown). thus, the involvement of the canonical ifn regulated - a system seemed unlikely. the relatively slow replication of even the f strain might allow the infected cells to synthesize and excrete ifn, however, which may stimulate the - a pathway in neighboring uninfected cells causing rna degradation. we addressed this question by two different approaches. first, the level of ifn, - oas and rnase l mrnas were investigated by rt-pcr to determine if any of these rnas were induced following virus infection (fig. ) . in agreement with previous reports that hav infection does not induce ifn, we did not observe significant induction of ifn message up to h pi with f virus (fig. , panel a) . fig. . rt-pcr amplification of selected cellular and viral mrnas. two microgram of rna isolated from virus infected, ifn-␤, or dsrna treated cells were reverse transcribed in a volume of l using oligo(dt) as primer as described in section . five microliter of each rt reaction was amplified for cycles with sense and antisense primers for ifn-␤ (a), - oas (b), ␥-actin (c), rnase l (d), or hav genomic rna (e) as described in section , and l pcr reactions were analyzed by % agarose gel electrophoresis. rna from uninfected cells (ui) and cells transfected in the absence of dsrna (trfn. control) were included in the rt-pcr analyses. takahashi et al. ( ) successfully demonstrated the application of rt-pcr for the detection of mrnas, which encode the - oas isoforms that synthesize the biologically active forms of - a. using these primers, we determined that the mrnas for either the p / form (fig. , panel b) or the p / form (data not shown) of - oas was not induced following virus infection. treatment of the cells with u/ml ifn-␤ induced the message for the / form (panel b), but not the / form, although the primer pair is capable of amplifying this mrna from both mouse and human cells (data not shown). thus, the frhk cells are competent to mount an ifn response by inducing the / form of - oas. rt-pcr experiments with primers for rnase l, on the other hand, revealed that this mrna was present in cells constitutively, with some increase in the level of this mrna following virus infection (panel d). to ascertain that both the f and clone cells were actively replicat- fig. . stat phosphorylation in response to virus infection and/or ifn-␤ treatment. (a) frhk cells were treated with ifn-␤ for or h prior to total protein extraction. (b) frhk cells were mock infected (m), infected with f for various times, or infected with coxsackie b (cbv ) prior to protein extraction. (c) frhk cells were infected with pfu/cell f for h followed by the addition of growth medium. after h, cells were treated with u/ml ifn-␤ in growth medium. nt means not treated with ifn-␤. proteins were extracted after or h of ifn treatment. clone cells were used at d pi. western blotting with an antibody to unphosphorylated stat (anti-stat), phosphorylated stat (anti-stat-p), or actin was performed as described in section . ing virus, viral genomic rna was amplified using primers directed to the ntr (panel e). the results clearly show the presence of undiminished amount of viral genome in clone cells after several months of cultivation, with no indication that the virus has acquired the base repeat present in the genome of f and other cp strains (brack et al., ; zhang et al., ) . second, we determined whether stat phosphorylation occurs following virus infection. phosphorylation of stat on y is indicative of the activation of the jak-stat pathway, and is a prerequisite for the transcriptional induction of isgs by ifn (for a review, see stark et al., ) . to investigate if this signaling pathway is activated following viral infection due to synthesis of low levels of ifn, phosphorylation of stat was investigated by immunoblot analysis (fig. ) . in ifn treated frhk cells, stat phosphorylation was observed as early as min post-treatment (data not shown), and the peak level of phosphorylation was detected at h post-treatment (panel a). these results confirm that frhk cells are capable of responding to ifn (panel a). however, no phosphorylation was detectable following infection with f, up to h pi (panel b), when rrna degradation is occurring. previously, we followed stat phosphorylation for up to h pi and found no evidence of activation of this pathway (kulka et al., ) . surprisingly, cells infected with the f virus for h showed diminished phosphorylation of stat when treated with ifn, compared to uninfected or clone cells treated with ifn (panel c). thus, the cp strains of hav not only interfere with the induction of ifn by dsrna (brack et al., ) , but can also interfere with ifn signaling. similarly, coxsackievirus b , a fast-replicating picornavirus that induces similar rna degradation in frhk cells, did not induce phosphorylation of stat (panel b). the data clearly suggest the existence of a virus inducible, - a independent rna degradative pathway that is activated by f infection in the apparent absence of ifn induction of - oas. in this study, we report a novel pathway for rrna degradation in cytopathogenic hepatitis a virus infected cells. the observed cleavage of both species of rrna suggests that this phenomenon is different from the cleavage of s rrna in mouse hepatitis a virus (a coronavirus) infected cells (banerjee et al., ) . we have also shown that the virus induced rrna cleavage is not a consequence of apoptosis, as has been reported to occur as a result of perturbation of cellular macromolecular synthesis (houge and døskeland, ) . in a previous study, we have shown that the cleavage of rrnas is also accompanied by a reduction in the levels of some cellular mrnas, while viral rna was not significantly affected (kulka et al., ) . the appearance of specific sizes of rrna degradation products is reminiscent of ifn regulated - a activated rnase l. the activation of this pathway, which is part of the cellular defense against viral infection, requires prior exposure of cells to ifn (stark et al., ; barber, ; sen, ) . rna degradation during f infection, however, occurs without prior treatment of the cells with ifn. it is conceivable that a slowly replicating virus such as hav could induce production of low levels of ifn to activate the - a pathway in surrounding uninfected cells. we showed that this is not the case, since the jak/stat pathway was not activated following virus infection, even though ifn-␤ mrna was marginally induced in f and clone infected cells. these results make it unlikely that the canonical - a dependent rna degradation pathway is activated during f infection. we also showed that rna is not degraded in persistently infected cells despite the presence of comparable levels of viral rna and antigens (proteins). our results complement the results of brack et al. ( ) and vallbracht et al. ( ) , that ifn-␤ does not play a major role in hav infection. the rnase l enzyme is one member of a multi-component system for rna degradation, consisting of ifn, - oas (the enzyme that synthesizes - a from atp in the presence of dsrna), and the latent endoribonuclease rnase l. rnase l is generally present in most cells and may function in situations other than virus infections, particularly during differentiation, apoptosis, hormonal regulation, chronic fatigue syndrome, and certain types of prostate cancer (for a review, see silverman, ) . rnase l levels are usually unaffected by virus infection or ifn treatment, but it is activated only in the presence of the proximal activator - a, a series of unique - phosphodiester linked oligoadenylic acid molecules. the synthesis of - a is dependent upon the induction of the enzyme - oas which is activated by dsrna or other activator molecules such as the hepatitis c virus core protein (naganuma et al., ) . we showed that - oas was not induced in f infected cells, despite the occurrence of massive rna degradation. we also showed that the failure to detect - oas mrna was not due to a failure of the rt-pcr protocol or the genetic background of the frhk cells, since treatment of this cell line with ifn-␤ resulted in an induction of the / form of - oas mrna. we also showed that the mrna for rnase l was present in both virus infected and uninfected cells, with some increase in the level of its mrna following virus infection. we conclude that either rnase l may be activated by factors other than - a, or that the enzyme responsible for rna degradation is a novel rnase specifically induced or activated in f and coxsackievirus infected cells. in this regard, ifn treatment has been shown to stimulate the expression of a second ribonuclease, identified as isg (nguyen et al., ) . however, this enzyme is an exonuclease, and the negative effect of ifn treatment on f-induced rna degradation (fig. ) argues against the role of this enzyme. unlike most picornaviruses, persistent infection is the norm rather than the exception for hav, at least in vitro in tissue culture cells. persistent infection by most cc strains of hav has been suggested to be the result of inefficient translation initiation from its ires (whetter et al., ; funkhouser et al., ; yi et al., ) . recent results suggest that hav ires is as efficient as the ires of other picornaviruses in initiating translation, and the switch from translation to negative strand synthesis is the rate-limiting step in hav replication (gauss-müller and kusov, ) . it has also been suggested that the slow replication (even the replication of cp strains are slow compared to other picornaviruses) of hav is a result of down regulation of its replication by the virus itself by unknown mechanisms (de chastonay and siegl, ) . in the discussion of slow replication of hav, the lack of a functional a protease has often been overlooked. picornaviral a protease is responsible for promoting cap-independent translation by inactivating eif g, and possibly other translation factors (ziegler et al., ; kerekatte et al., ) . a protease is also a strong inducer of apoptosis when expressed ectopically (goldstaub et al., ) . in the absence of a functional a protease, hav viral rna has to compete with cellular mrnas for the host cell translation machinery. we suggest that induction of an rnase activity may partially compensate for the absence of an active a protease, by reducing competition from cellular mrnas and contributing to the rapid replication phenotype of f and possibly other cp strains of hav. apoptosis is generally thought to be a host defense response to virus infection (for a review, see barber, ) . premature induction of apoptosis results in abortive virus replication. however, a late induction of apoptosis might favor the virus by allowing it to evade the host immune system. many viruses have thus evolved mechanisms to delay the onset of an apoptotic response (roulston et al., ) . similarly, induction of ifn mediated antiviral pathways is a defensive response of the host to virus infection, and many viruses have evolved mechanisms to block the induction of ifn mediated antiviral pathways (for reviews, see barber, ; sen, ) . it is therefore not surprising that the two major ifn mediated antiviral pathways, namely the - a/rnase l pathway and the dsrna activated pkr pathway, are also mediators of apoptosis. among the picornaviruses, hav is unique in its slow replication in permissive cells, and also in its ability to initiate and maintain a persistent infection. persistent infection is known for other picornaviruses, but usually requires restriction of virus replication and gene expression, through the use of inhibitors in cultured cells and presumably the host ifn regulated system or the immune system in the intact organism (agol et al., ) . moreover, cp strains of hav kill cells by apoptosis in cell culture, while in the whole organism, a role for apoptosis in the destruction of infected cells, or evading the host immune response is uncertain. the late depletion of parp- clearly indicates that hav is capable of avoiding the induction of necrosis in infected cells and evade a host immune response. in conclusion, we present evidence that unlike some other picornaviruses, notably encephalomyocarditis (emc) virus, hav is relatively insensitive to the antiviral effect of ifn. furthermore, the degradation of rrna in hav f infected cells occurs in the absence of - oas, the key enzyme that regulates the - a/rnase l antiviral pathway. the f virus is able to suppress the expression of isgs including - oas by interfering with jak/stat signaling by an unknown mechanism, and it utilizes the degradation of rna as a means to augment its replication and subsequently induce apoptosis in the host cell. competing death programs in poliovirus infected cells: commitment switch in the middle of the infectious cycle cytopathology, 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picornavirus a proteinase-mediated stimulation of internal initiation of translation is dependent on enzymatic activity and the cleavage products of cellular proteins we thank dr. joseph e. leclerc and dr. david reese for critical reading of the manuscript, ms. marcia meltzer for the graphics, and ms. mobolanle ayodeji for her excellent technical assistance. key: cord- -bqaqhp m authors: sui, xiuwen; yin, jiechao; ren, xiaofeng title: antiviral effect of diammonium glycyrrhizinate and lithium chloride on cell infection by pseudorabies herpesvirus date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: bqaqhp m diammonium glycyrrhizin (dg), a salt from glycyrrhizinate (gl) that is a major active component of licorice root extract with various pharmacological activities was investigated for its inhibitory effect on pseudorabies virus (prv) infection. in parallel, lithium chloride (licl), a chemical reagent with potential antiviral activity was compared with dg for their inhibitory ability against prv infection in vitro. virus plaque-reduction assays, pcr and rt-pcr analysis indicated that both drugs inhibited cell infection by prv. moreover, addition of the drugs resulted in fewer apoptotic cells during prv infection. glycyrrhizin (gl) is the most important bioactive compound of licorice root (glycyrrhiza radix), and it has been used as a traditional chinese medicinal herb for treating hepatitis due to its anti-inflammatory property (yuan et al., ) . gl is active against herpes simplex type (hsv- ), varicella-zoster virus (vzv), epstein-barr virus (ebv), human cytomegalovirus, hepatitis a, b, and c viruses, influenza virus, human immunodeficiency virus (hiv), and sars coronavirus (arase et al., ; baba and shigeta, ; crance et al., ; hoever et al., ; ito et al., ito et al., , lin, ; numazaki et al., ; pompei et al., ; sato et al., ; utsunomiya et al., ) . diammonium glycyrrhizinate (dg) is derived from the glycyrrhizin modified in the two carboxyl groups at the and position ( fig. ) . dg is more stable, soluble and has more significant bioactivities than gl. lithium salts have been used as therapeutic substances for the treatment of 'gout and rheumatic gout', 'bright's disease', epilepsy, syphilis, acute mania and depressive episodes (skinner et al., ) . the effect of lithium salts on the replication of herpes simplex virus and vaccinia virus was documented (skinner et al., ; ziaie and kefalides, ) . recently, it was reported that lithium chloride inhibits infectious bronchitis virus, an rna coronavirus in cell culture and such inhibition was due to a cellular rather than virucidal effect (harrison et al., ) . pseudorabies virus (prv) is a swine neurotropic herpesvirus that has common genome arrangements with those of hsv- . prv is a useful model organism for exploring herpesvirus biology. in swine and cattle, prv infection can cause aujeszky's disease (ad), which is one of the list b diseases of the office international des épizooties (oie). although pig is the natural reservoir for the virus, prv has a broad host range and can infect most mammals and some avian species (klupp et al., ) . in susceptible species, prv infection is often lethal and infected animals may die from central nervous system disorders (tirabassi and enquist, ) . prv infection poses a severe threat to animal husbandry and the development of effective antiviral agents is one important strategy to decrease the occurrence of prv infection in addition to vaccination. in this study, we demonstrated the inhibitory effect of dg and licl on prv infection. moreover, the effect of the drugs on the cell apoptosis induced by prv was investigated. the data is helpful for better understanding the antiviral mechanism of both compounds. vero cells (african green monkey kidney cells) were maintained in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (shanghai excell biology, inc.) at • c in an incubator containing % co . prv (strain bartha k- ) was propagated in vero cells. vero cell monolayers in -well culture plates at a density of cells/well were washed three times with phosphate-buffered saline (pbs), then dg (zhengda pharmacy, china) and licl (sigma, st. louis, mo) diluted serially in serum-free dmem were inoculated into the wells ( wells/each drug). mock-treated cells were served as control. all the cells were cultured at • c for h, and the cell morphology was analyzed by light microscope. cell monolayers in -well plates were incubated with drugs as above. after washing with pbs, the cells were incubated with l/well dmem culture and l/well . % mtt solution at • c for h. after washing with pbs, l dmso were added to each well. the tray was shaken gently for min to dissolve the precipitate of formazan and the optical density (od) value of the wells was determined using a plate reader at a test wavelength of nm. cell survival rate was calculated as drug average od value/control average od value and % above cell survival rate was regarded as a non-toxic concentration of the drug. trypan blue staining was used to confirm the maximum nontoxic concentration of drug. briefly, ml of the trypan blue mix ( . % (w/v) in pbs) was added into each dish of cells, and incubated for min at room temperature. the percentage of viable cells was determined by dividing the number of unstained cells by the total number of cells and multiplying by . the equation is as follows: (number of unstained cells/total number of cells) × = percent viable cells. virus titration or subsequent infectivity analysis was performed by plaque-reduction assays (burleson et al., ) . briefly, cell monolayers in -well plates were inoculated with serially diluted viruses. the inoculums were replaced with % methylcellulose in dmem, after h absorption of the virus. the cell monolayers were incubated at • c for h and the overlay medium was removed. then the cells were gently washed three times with pbs and fixed with % formalin in pbs for min at room temperature. then, % crystal violet (v/v) diluted in % etoh in pbs was used to stain the cells for min at room temperature. the clear plaque number was visualized post-washing and the virus titer in plaque-forming units (pfu) was calculated. the experiment was done in triplicate. to analyze the effect of both drugs on virus per se, × pfu/ml viruses were treated with serially diluted drugs and incubated at • c for h. then, vero cells were infected by the viruses and subjected to virus plaque assays as above. to analyze the inhibition of drugs to prv-infected cells, the cells in -well plates were infected with the × pfu/ml viruses at • c for h. after washing three times with pbs, the cells were treated with serially diluted drugs at • c for h post-infection (hpi). the infectivity of the viruses was analyzed as above. to analyze the sensitivity of the cells to the drugs, the cells in well plates were treated with serially diluted drugs at • c for h. the cells were infected with × pfu/ml viruses, after washing with pbs. then, the cells were subjected to plaque assays. to confirm the effect of the drugs on prv infection in vitro, polymerase chain reaction, pcr was used to assess the viral load using prv gd-specific primers. both cells infected with drugtreated viruses and virus-infected cells treated with increasing concentrations of licl were subjected to dna extraction with a commercial kit (qiagen, germany). according to the published sequence of prv gd gene (genbank no. aj ), the sense primer -cccgaattcatgctgctcgcagcgc- and anti-sense primer -gggctcgagacggaccgggctgcgc- were used to amplify the prv gd gene (about bp in length). the pcr system included l dna template, . l × gc pcr buffer, . l each primer, l dntp, . l rtaq polymerase, . l sterile h o. the pcr was performed at • c for min and cycles of • c s, • c s, • c min. a final extension at • c for min. the pcr product was analyzed in % agarose gel electrophoresis. at the same time, gapdh ( bp in length) used as an internal standard was amplified with the sense primer -atcaccatcttccaggagcgaga- and the anti-sense primer, -gcttcaccaccttcttgatgtca- . the temperature profile for pcr amplification of the gene was • c for min, • c for min and • c for min, followed by cycles at • c for min, • c for min and • c for min, followed by a final extension of min at • c. cells infected with drug-treated viruses or prv-infected cells treated with increasing concentrations of licl were subjected to rna extraction with a commercial kit (qiagen, germany). reverse transcription (rt)-pcr was performed using a rt-pcr kit (takara, japan). the anti-sense primers for amplifying either gd or gapdh gene were used for rt, and the subsequent pcr procedures were performed as above. cell apoptosis was analyzed with annexin v-fitc kit (nanjing keygen biotech. co., ltd.). briefly, fitc-conjugated annexin v ( l/well) and propidium iodide, pi ( l/well) were added the cells infected by drug-treated viruses, or the virus-infected cells treated with licl in -well plates and incubated at room temperature for min in the dark prior to fluorescence observation. the detection of cell early apoptosis was based on the observation that soon after initiating apoptosis, cells translocate the membrane phosphatidylserine (ps) from the inner face of the plasma membrane to the cell surface. thereafter, ps can be easily detected by staining with a fluorescent conjugate of annexin v, a protein that has a high affinity for ps. cells stained by pi were detected as cellular late apoptosis. the results were analyzed by fluorescence microscope (leica, germany) at hpi. in parallel, flow cytometry technique was used to further analyze cell apoptosis. above-mentioned cell samples were trypsinized and then centrifuged at rpm for min. the cells were re-suspended with l binding buffer at a concentration of cells/ml, after washing two times with pbs at rpm for min. then, l fitc-conjugated annexin v and l pi were added to the cells and incubated at room temperature for min in the dark. the samples were analyzed within h post-staining. the effects of dg and licl on vero cell viability and proliferation were determined as above and the maximum non-toxic concentrations of dg and licl diluted in dmem medium were g/ml and mm, respectively. under above-mentioned drug concentrations, there were no difference between the drug-treated cells and mocktreated cells in terms of cell morphology. the cell proliferation was not significantly affected under the maximum non-toxic concentrations of both drugs as indicated in mtt assay (fig. ) . trypan blue staining showed that the cells were completely viable under above-mentioned concentrations of both drugs (fig. ) . the viruses were used to infect the cells, after they were treated with the drugs. the plaque-reduction assays showed that both drugs inhibited the virus infection in a dose-dependent manner. at the maximum non-toxic concentration of the drugs, the inhibition rate of licl and dg for prv infection in cell culture reached % (fig. ) . the effect of both drugs on prv-infected cells was analyzed using plaque-reduction assays. no reduction of the virus infectivity was observed at any of the different samples in the dg treatment group (data not shown). when the effect of licl was analyzed, the formation of virus plaques was inhibited in a dose-dependent manner (fig. ) . after the cell monolayers were treated with licl or dg, the viruses were applied to the cells and the plaque-reduction assays fig. . trypan blue staining. the mock-treated cells and cells treated with maximum non-toxic concentrations of drugs were subjected to trypan blue staining and a representative comparison is provided. fig. . pcr targeting the amplification of prv gd gene. the cells infected by drugtreated viruses were subjected to pcr using prv gd-specific primers. the effect of licl and dg on the gene amplification is shown in panels a and b, respectively. prvinfected cells were treated with licl and then subjected to pcr for detecting the gd gene. the pcr result is shown in panel c. housekeeping gene gapdh is served as an internal reference. done h later showed that no inhibitory effect was observed for either of both drugs (data not shown). the viral genome load assessed by pcr analysis of the gd gene was inhibited partially at mm licl or g/ml dg and was inhibited completely at mm licl or g/ml dg. the gd gene amplification was also inhibited in prv-infected cells treated by licl. there was no positive pcr product in the mm licl treatment group. the amplification of housekeeping gene gapdh was positive in all pcr analyses (fig. ) . to confirm the effect of both compounds on gd gene, rt-pcr was performed to analyze the amount of amplified dna from the gd gene. as shown in fig. , the rt-pcr analysis gave rise to a similar result with that of pcr analysis. the cell apoptosis analyzed with annexin v-fitc and pi dual staining kit showed that prv-infected cells displayed both early and late apoptosis. the late apoptosis rate of cells infected by drug-treated viruses was decreased in a drug dose-dependent manner. the late apoptosis of virus-infected cells treated with licl was inhibited significantly. in contrast, dg did not prevent virus-infected cells from apoptosis. these results were agreement with virus infection analysis. however, as early as hpi, the early apoptosis of the cells was affected lightly. a representative staining picture regarding the effect of the drugs at maximum non-toxic concentration on the cell apoptosis is shown in fig. a . the inhibition rate of late apoptosis is shown in fig. b and c. to confirm the cell apoptosis induced by prv, flow cytometry technique was utilized. as shown in fig. , the maximum nontoxic concentration of drugs had an inhibitory activity against prv-induced cell apoptosis. many antiviral agents have been tested for their effect on human herpes simplex viruses, the counterparts of prv in the subfamily alphaherpesvirinae in herpesviridae family (de clercq, ; naesens and de clercq, ; sergerie and boivin, ; cheng et al., ) . due to the potential threat of prv infection to the animal husbandry, it is necessary to develop effective antiviral agents against prv infection. it was reported that licl was able to inhibit the replication of the dna virus herpes simplex and interfered with the herpes simplex virion-associated inhibition of host protein synthesis (skinner et al., ; ziaie and kefalides, ) . in this study, we further investigated the effect of this reagent on prv infection in vitro and its mode of action. the results showed that the prv infectivity was decreased in a dose-dependent manner after the treatment of licl either to the virus-infected cells or to the virus per se. licl has been known for its inhibitory effect on the rna coronavirus, infectious bronchitis virus (ibv) in cell culture, and such inhibition was due to a cellular, rather than virucidal, effect (harrison et al., ) . therefore, licl may be a perspective and broad-spectrum antiviral. our apoptosis analysis indicated that prv infection initiated cell apoptosis. both drugs used in this study inhibited the virus-induced cell apoptosis, although some cells with early apoptosis were not affected by the drug treatment. we also investigated the cell apoptosis at hpi, which gave the similar results with that at hpi. it has been reported that point mutations in the atp binding site of prv us protein kinase prevented bad phosphorylation and cell survival after apoptosis appearance and p mapk and jnk/sapk signaling were involved in prv apoptosis (deruelle et al., ; yeh et al., ) . the mechanisms regarding the effect of both drugs on apoptosis pathways are under investigation. our flow cytometry analysis also suggested that licl had a more significant effect than dg in terms of their inhibitory activity against cell apoptosis. the microscopic scoring of fluorescent samples and the flow cytometric data indicated a similar reduction trend in cell apop-tosis by the addition of the drugs, however, there was difference in the degree of reduction of apoptosis. this discrepancy might be attributed to the difference from the both methods per se, such as criterion, cell status, system error, etc. the pcr results showed that the amplified amount of prv gd gene was decreased with the increasing drug concentration, manifesting that both drugs appear to reduce the viral genome load via an unresolved mechanism. the rt-pcr results suggested that the compounds interfered with the viral gd gene. it is possible that the observed anti-apoptotic effect of dg and licl during prv infection is indirect and due to the reduced virus replication caused by the drugs. our future work will further try to elucidate whether the inhibition of prv infection by licl is attributed to its direct virucidal effect or to its inhibitory activity against the replication of prv. dg is a salt derived from gl, and it may have a similar antiviral activity with gl. in this study, we were especially interested in understanding its mode of action regarding its inhibitory activity against prv infection in vitro. it was reported that gl was active against herpes viruses such as hsv- , vzv, ebv (lin, ) . however, in above-mentioned experiment, ebv was incubated with . mm gl at • c for h, and then the ebv infectivity was analyzed. no significant reduction in viral infectivity was observed (lin, ) . the author assumed that the antiviral activity of gl was not attributed to a direct inactivation of the virus. in another report, it was found that mm gl could inactivate herpes simplex virus particles irreversibly in min (pompei et al., ) . our data showed that the pre-treatment of the viruses with dg inhibited the cell infection by prv in a dose-dependent manner. we suppose that the difference in the reported modes of action of the dg/gl may be attributed to either the different nature of individual virus in the herpes virus family or the discrepant concentrations of the drugs applied in each assay. compared with licl, dg did not inhibit the prv infection after virus adsorption, and it seems that this drug is only virucidal to prv. this finding would be interesting, because the effects of gl on cellular protein kinase c and p were reported (abe et al., ; ito fig. . cell apoptosis analysis by flow cytometry. the cells were stained with annexin v-fitc and pi, after either they were infected by maximum non-toxic concentration of drug-treated viruses or virus-infected cells were treated with licl at maximum non-toxic concentration. the cell apoptosis was analyzed by flow cytometry. data are presented as dual-parameter fl of annexin v versus fl of pi. the upper right areas are apoptotic cells. the apoptosis rates of cells are indicated. ohtsuki and iahida, ) and it was proposed that the anti-hiv effects of gl may be attributed to inhibitory effects on the cellular protein kinases. in our study, whilst dg has no observable effect on cell morphology or virus-infected cells, it has effects on cell signaling which could easily lead to changes in virus replication (hsiang et al., ) . therefore, we will further investigate the effects of the dg. our data showed that pre-treatment of the cells with the both drugs did not result in a decrease of prv infectivity, suggesting that cellular factors including cellular receptors for prv might be not sensitive to the drugs. like licl, dg showed a good inhibitory effect on cell apoptosis, after incubation of the drug with viruses. these data indicate that prv infection induces cell apoptosis and addition of drugs also results in fewer apoptotic cells during prv infection. glycyrrhizin prevents of lipopolysaccharide/d-galactosamine-induced liver injury through downregulation of matrix metalloproteinase- in mice the long term efficacy of glycyrrhizin in chronic hepatitis c 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reduces morbidity and mortality of mice infected with lethal doses of influenza virus tnf-alpha mediates pseudorabies virus-induced apoptosis via the activation of p mapk and jnk/sapk signaling anti-inflammatory effect of diammonium glycyrrhizinate in a rat model of ulcerative colitis lithium chloride restores host protein synthesis in herpes simplex virus-infected endothelial cells the authors thank dr. erik de clercq, the editor and three anonymous reviewers of the journal antiviral research for their useful comments. the funds from national natural science foundation of china ( ) key: cord- -cdn epy authors: artuso, maría c.; ellenberg, paula c.; scolaro, luis a.; damonte, elsa b.; garcía, cybele c. title: inhibition of junín virus replication by small interfering rnas date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: cdn epy junín virus (junv), the etiological agent of the argentine hemorrhagic fever, has a single-stranded rna genome with ambisense expression which encodes for five proteins. in previous works we have demonstrated that the z arenavirus matrix protein represents an attractive target for antiviral therapy. with the aim of studying a new alternative therapeutic mechanism, four z-specific sirnas (z - to z -sirnas) were tested showing variable efficacy. the most effective inhibitor was z -sirna targeted at the region encompassed by nt – of z gene. the efficacy of this z -sirna against junv was also demonstrated in virus-infected cells, by testing infectious virus plaque formation ( . % junv yield reduction), viral rna level or antigen expression, as well as in cells transfected with z-specific reporter plasmids ( % reduction in expression of z-egfp fusion protein). furthermore, the lack of effect of this z-sirna on the expression of other junv proteins, such as n and gpc, confirmed the specificity of action exerted by z -sirna on z transcript. thus, the present study represents the first report of virus inhibition mediated by rna interference for a new world arenavirus. viruses are among the most important causes of morbidity and mortality worldwide, but especially in the developing countries where the national health care system is deficient. particularly, arenaviruses are among the etiological agents that can be considered within the neglected viral infections in south american countries where the human hemorrhagic fevers are often fatal to the infected patients. junín virus (junv) is the agent of argentine hemorrhagic fever (ahf), with mortality rates ranging from to % in the absence of the administration of standardized doses of convalescent plasma that is today the best therapy against ahf (damonte and coto, ) . junv is an enveloped, single stranded, ambisense rna virus with a segmented genome consisting of two segments, designated large (l) and small (s). the s segment encodes the nucleocapsid protein (n) and a pre-envelope glycoprotein precursor (gpc) which is processed post-translationally into a signal peptide, the external glycoprotein g and the transmembrane g , whereas the l fragment encodes the rna polymerase l and the matrix protein called z. the z protein has been implicated in several aspects of arenavirus biology. it has been shown that z exerted an inhibitory effect on viral rna synthesis de la torre, , ; lópez et al., ) . in addition to this regulatory role, z was also implicated as a virion component with matrix functions, similar to other enveloped negative-strand viruses (neuman et al., ; perez et al., ; salvato et al., ; salvato, ; strecker et al., ) . consistent with its features, z presents a ring finger and a canonical late domain motif that enable z to interact with the host cell (borden et al., ; campbell-dwyer et al., ; djavani et al., ) and viral proteins. furthermore, z-mediated budding requires its myristoyl modification (perez et al., ) , which likely facilitates z association with membranes at budding sites. thus, z has an essential role in the particle formation. rna interference (rnai) is a post-transcriptional gene silencing process in which double-stranded rna (dsrna) initiates specific cleavage of cytoplasmic mrna. initially, dsrna is recognized and processed by dicer, an rnase iii enzyme (hutvágner et al., ) . the resulting processed dsrna consists of - nt fragments with - nt overhangs at the end of each rna strand (bernstein et al., ; zamore et al., ) . dicer-processed dsrna is recognized by the dsrna-induced silencing complex (risc) and used as a template to guide degradation of mrna that is homologous in sequence to the risc-bound dsrna fragment, resulting in greatly reduced protein production (hammond et al., ) . synthetically produced small interfering dsrna molecules (sir-nas) have been shown to induce the rnai effect in vitro and greatly decrease specifically targeted transcripts (elbashir et al., ) . such efforts have been expanded to include targeting of viral transcripts present in the cytoplasm for degradation by the risc complex. the list of the human viral pathogens that have (leung and whittaker, ) , poliovirus (gitlin et al., ) , adenovirus (chung et al., ) , human immunodeficiency virus (hannon and rossi, ; naito et al., ) , hepatitis b virus (li et al., ) , hepatitis c virus (liu et al., ; lupberger et al., ) , influenza a (ma et al., ) , marburg virus (fowler et al., ) , human papillomavirus (jiang and milner, ) , and coronavirus (zhang et al., ) , among others. to the present, only two reports were published describing the use of sir-nas with the old world arenaviruses lymphocytic choriomeningitis virus (lcmv) (sánchez et al., ) and lassa virus (lasv) (müller and günther, ) . since the matrix z protein is involved in processes that are fundamental to the success of viral infection, we selected z as candidate target gene in order to determine the efficacy of rnai for inhibition of junv gene expression. here we report the identification of sirna target regions on z that are able to silence its expression and impair the production of infectious viral progeny. vero and bhk- cells were grown as monolayers in eagle's minimum essential medium (mem) (gibco) containing % fetal bovine serum (gibco) and g/ml gentamycin (sigma). medium was supplemented with m hepes buffer (sigma) when incubated at • c under % co . maintenance medium (mm) consisted of mem with . % fetal bovine serum. the attenuated strains iv and xjcl of junv (candurra et al., ) , the trlv strain of tacaribe virus (tcrv), and the armstrong b strain of lcmv were used. arenavirus stocks were prepared in bhk- cells and titrated by plaque formation in vero cells. four double-stranded sirnas were designed targeting the z transcript of junv-iv (genbank accession no. dq ) by using the algorithm programs from different companies. blast analysis was performed for ensuring the lack of homology with cellular genes. the sirnas targeted to z transcript (z -to z -sirnas) and a sirna without homology to any known gene transcript used as a non-silencing control (x-sirna) were supplied as annealed duplexes by invitrogen (table ) . vero cells grown in six-well plates at approximately % confluence were transfected with sirna using lipofectamine (invitrogen) according to manufacturer's instructions with minimum modifications. briefly, cell supernatant was removed and l opti-mem (gibco) containing l lipofectamine and m sirna was added. after h of incubation at • c, supernatant was removed and fresh advanced-mem (gibco) containing % fetal bovine serum was added to the cells. after h incubation at • c, transfected monolayers were infected with junv at a moi of . . at h post-infection (p.i.), supernatants were collected to determine virus yield by plaque assay and cells were processed to measure viral rna by real time pcr (qrt-pcr). vero cells grown as monolayers were infected with serial dilutions of the supernatants of z-sirna-transfected and junv-infected vero cells. after h adsorption, cells were washed, covered with mm containing . % methylcellulose, and incubated at • c under % co . at day p.i. virus plaques were counted and % inhibition with respect to the viral control was calculated. each value was the mean of two independent experiments performed in duplicate ± standard deviation. rna was isolated from sirna transfected and junv-infected vero cells using trizol (invitrogen) according to the manufacturer's instructions. to monitor rna replication, cdna was generated by using murine reverse transcriptase m-mlv ( u/l, invitrogen) and random primers. this cdna was amplified by real time pcr using sybrgreen (roche) detection. the mix reaction volume was l including l of cdna, dna polymerase gotaq ( u/l, promega) and specific primers to amplify gene z: z forward -atgggcaactgcaacggggcatc- and z reverse -agccaacagcaccaccaccatag- . real time pcr was carried out with an initial incubation at • c during min followed by cycles of s at • c, s at • c, s at • c, s at • c and a final incubation of s at • c. amplification plots were expressed as c t values to be analyzed with opticon monitor . . software where c t values represent the reaction cycle at which pcr products reach a threshold level of detection. c t values were normalized by using actin as standard. vero cell monolayers grown on coverslips were transfected with m z-sirna and infected with junv at h post-transfection as described in section . . at h p.i., cells were fixed with methanol for min at − • c for cytoplasmic immunofluorescence. indirect staining was carried out by incubation with a rabbit anti-junv polyclonal serum for h at • c followed by fluorescein (fitc)conjugated goat anti-rabbit igg in the same conditions. after a final washing, cells were mounted in a glycerol solution containing . % , -diazabicyclo[ , , ]octane (dabco) and visualized by a fluorescence microscope. a truncated z lacking a stop codon was obtained by pcramplification using plasmid pgem containing a full-length cdna copy of the z gene as template dna together with oligonucleotides z-f ( -ggatccatgggcaactgcaacggggcatc- ) and z-trunc-r ( -aaaagcggccgcctggtggtggtgctgttggct- ) containing a bamhi and noti site, respectively (underlined). the insert dna was subsequently subcloned into the bamhi and noti sites of the mammalian expression vector pcdna . to generate pcdna . -z trunc. oligonucleotides egfp-f ( -aaaagcggccgcatggtgagcaagggcgaggag- ) and egfp-r ( -aaaatctagattacttgtacagctcgtccatgcc- ), which contain a noti and xbai site, respectively (underlined), were used with plasmid pegfp c as template dna to pcr amplify the sequence encoding the enhanced green fluorescent protein (egfp). the amplicon was cloned into the noti-xbai digested pcdna . -z trunc vector to generate the reporter plasmid pcdna . -z-egfp, which resulted in the expression of a fusion protein z containing egfp in its cterminus. the same strategy was used to generate the reporter plasmid pcdna . -z-flag (dykddddk). in order to generate the n and gpc proteins encoding plasmids, the pcdnahismax a vector was modified and agei and clai restriction sites respectively (underlined) were added into the hindiii and apai vector sites using the following designated primers: -ctggctaactagagaacccactgc- and -tatgggccccccggcgcccccatcgatcccaccggtccccatggtttc-ggaggccg- . the n and gpc genes were obtained by pcramplification from junv-infected vero cells using the following primers: n agei sense ( -aaaaccggtatggcacactccaaagagg- ), n clai antisense ( -aaaatcgatcagtgcataggctgcc- ), gpc agei sense ( -gggaccggtatggggcaattcatcagc) and gpc clai antisense ( -tttatcgatgtgtcctcttgcgcc- ). then, pcr amplification fragments were cloned into modified pcdnahismax a vector to obtain the pcdnahismax a-n and pcdnahismax a-gpc constructs, respectively. vero monolayers grown to % confluence on mm diameter glass coverslips were co-transfected with g plasmid pcdna . -egfp, pcdna . -z-egfp, pcdna . -z-flag, pcdnahismax a-n or pcdnahismax a-gpc and m z-sirnas using lipofectamine (invitrogen) as described previously. as control, cells were transfected with the respective plasmid without z-sirna. to record egfp expression, at h post-transfection cells were fixed with methanol for min at − • c, mounted in glycerol with dabco and observed in a fluorescence microscope. for z-flag detection, cells fixed at h post-transfection were incubated with rabbit anti-flag polyclonal serum (cell signaling technology) overnight at • c, followed by incubation with rhodamine-conjugated goat anti-rabbit igg (sigma) for h at room temperature. then, cells were mounted and visualized. for n or gpc detection, cells fixed as above were incubated first with monoclonal antibodies sa bg or qc -bf (sánchez et al., ) , respectively, then with fitc-labeled goat anti-mouse igg. vero cells co-transfected with g pcdna . -z-flag with or without m z-sirna as described above were harvested at h post-transfection in sample buffer ( % sds, % -mercaptoetanol, % glycerol and . % bromophenol blue in . m tris-hcl, ph . ) (promega). after boiling the lysates during min, proteins were separated by % sds-page and blotted onto a pdvf membrane (millipore). membranes were incubated with a rabbit anti-flag serum overnight at • c. after washing with tris-buffered saline (tbs, mm tris-hcl, mm nacl, ph . ) containing . % tween , a second incubation was performed with horseradish peroxidase-conjugated anti-rabbit igg during h at room temperature. as control, the presence of actin was revealed by incubation with mouse monoclonal antiactin antibody (jla , calbiochem) followed by incubation with peroxidase-conjugated anti-mouse igg. for protein detection blots were treated with the western lightning luminescence system (perkinelmer). to evaluate the effect of z-sirnas on infectious junv production, vero cells were transfected with the respective sirna (z -sirna, z -sirna, z -sirna, z -sirna or x-sirna), and h later cultures were infected with junv at a moi of . . as a control, nontransfected junv-infected cells were also included. supernatants were collected at h p.i. and titrated by plaque assay. cells transfected with the non-specific x-sirnas did not exhibit a reduction in virus yield ( . × pfu/ml), showing similar levels to nontransfected junv-infected vero cells ( . × pfu/ml), indicating that transfection of the cells with the control x-sirna did not induce a non-specific interference with virus replication during the h incubation period. by contrast, reduction in virus titer was observed in cells treated with the sirnas corresponding to the z transcript (fig. a) . whereas z -sirna reduced the virus titer by . % compared to the viral control, z -sirna induced a . % yield reduction. in vero cells treated with both z -sirna and z -sirna, the virus yield was reduced by . %, showing that the combination of different silencing agents did not present any advantage (data not shown). on the other hand, z -sirna and z -sirna were less effective, with only . and . % of virus yield inhibition, respectively. interestingly, the silencing effect of z -sirna, the most effective inhibitor, was observed also when the virus yield inhibition was determined at longer period of incubation ( h p.i.) with . % of virus yield inhibition (fig. b) , and even when junv infection was performed at higher mois of and , with . and . % of virus yield inhibition, respectively (fig. c) . since the gene regions targeted for z-sirna design were conserved among arenaviruses (fig. ) , we looked for a possible interference of the selected z-sirnas on xjcl , another attenuated strain of junv, and also on the close antigenically related tcrv and the non-antigenically related lcmv. when z-sirnas originally designated against junv-iv were evaluated against junv-xjcl , a moderate virus yield inhibition was observed with z -to z -sirnas, with . , . , . and . % of virus yield inhibition, respectively. however, when junv z-sirnas were evaluated against the other two arenaviruses species, no inhibition was observed ( fig. a for tcrv and not shown for lcmv), confirming the specificity of the rna interference mechanism. since sirnas function by identifying and degrading mrna with its complementary sequence, we next examined the effect of z -sirna and z -sirna on the abundance of viral rna in junv-infected cells by qrt-pcr using z-specific primers. in contrast to vero cells transfected with the control non-silencing x-sirna, viral rna amplification was reduced in vero cells transfected with both zspecific sirnas (fig. d) . again, z -sirna was the most effective inhibitor with a -fold reduction in viral rna whereas z -sirna induced approximately a -fold rna inhibition with respect to control x-sirna (fig. d) . vero cells treated with z -sirna and infected with junv were also examined for viral antigen expression at h p.i. by an indirect immunofluorescence assay with a rabbit anti-junv polyclonal serum. hyperimmune junv antiserum allows the detection of the main viral proteins, the nucleoprotein n and the precursor and mature glycoproteins gpc and g . in accordance with the reduction in virus yield produced by z -sirna shown in fig. a , a concomitant the xjcl (dark grey bars) strains of junv, or tcrv (black bars). supernatants collected at h p.i. were titrated by plaque assay. inhibition of virus yield was calculated comparatively to the titer obtained in cells transfected with x-sirna. each value represents the mean of two independent experiments performed in duplicate ± standard deviation (s.d.). (b) vero cells were transfected with z -sirna and infected with junv, moi = . . supernatants were collected at or h p.i. and titrated by plaque assay. inhibition of virus yield was calculated as above. (c) vero cells were transfected with z -sirna and infected with junv at different mois. supernatants were collected at h p.i., and titrated by plaque assay. inhibition of virus yield was calculated as above. (d) amount of viral rna in vero cells transfected with z -sirna or z -sirna and infected with junv was quantified by real time rt-pcr. values, standardized to those of actin mrnas, were expressed as relative rna levels comparatively to the amount obtained in cells transfected with x-sirna and represented as mean of two independent experiments performed in triplicate ± s.d. (e) expression of viral antigens in vero cells transfected with z -sirna or x-sirna and infected with junv was detected by immunofluorescence assay using a rabbit anti-junv polyclonal serum. magnification = ×. decrease in cytoplasmic junv antigen expression was observed (fig. e) . to confirm that the z-sirnas were effective as specific inhibitors of the expression of z protein, the effect of z-sirnas on the expression of recombinant z fusion proteins was analyzed. first, the inhibitory action of z-sirna on the expression of the fusion protein z-egfp was determined by co-transfection of vero cells with the plasmid pcdna . -z-egfp and either the z-sirna or the non-silencing x-sirna. a series of co-transfections of the reporter plasmid pcdna . -egfp and the sirnas were assayed in parallel. at h post-transfection, egfp fluorescence was analyzed. the egfp protein was highly expressed in cells transfected with the reporter plasmid in the presence of co-transfected z-sirna or x-sirna (fig. a, panels a and b) . the control non-silencing x-sirnas had no apparent effect on z-egfp expression (fig. a, panel c) , whereas the four specific z-sirnas produced a marked reduction in fluorescence (fig. a , panels d-g). as observed in virus yield assay, z -sirna appeared to silence z-egfp gene expression more efficiently than the other z-sirnas. in fact, the quantification of fluorescent cells showed that z -to z -sirna reduced expression of the z-egfp fusion protein in comparison to x-sirna by . , . , . and . %, respectively (fig. b) . the specificity of z -sirna to block expression of z protein was further assessed by testing the expression of other recombinant protein, the z-flag fusion protein expressed by the plasmid pcdna . -z-flag, by immunofluorescence and western blot assays performed with rabbit anti-flag antibodies. it was evident from the results shown in fig. c and d that the z-specific sirna reduced the amount of detectable z-flag fusion protein in both assays whereas the co-transfection of vero cells with the control non-silencing x-sirna failed to block the synthesis of z-flag fusion protein. it is also worth to note that the expression in the same cell samples of a non-targeted host protein, actin (ca. kda), remained almost unaffected (fig. d) . to gain further evidence for the specificity of z-sirnas, we investigated whether the presence of z -sirna could decrease other transiently expressed junv proteins. to this end, cells were co-transfected with the z -sirna or x-sirna and plasmids pcd-nahismax a-n or pcdnahismax a-gpc encoding n and gpc junv proteins, respectively. as shown in fig. , no effect on n and gpc expression could be detected by indirect immunofluorescence using specific monoclonal antibodies against these proteins, respectively, suggesting that off-target effects are not the cause of the junv yield reduction observed in fig. . . effect of z-sirna on junv nucleocapsid and glycoprotein expression. vero cells grown on glass coverslips were untransfected (panels a and d), co-transfected with pcdnahismax c-gpc or pcdnahismax c-n and x-sirna (panels b and e) or z -sirna (panels c and f). at h post-transfection, cells were fixed, and protein expression was detected using specific monoclonal antibodies against gpc/g and n, followed by fitc-conjugated goat anti-mouse igg (magnification = ×). altogether, these data showed that the sirna targeting the z transcript exerts its inhibitory action exclusively through a significant and specific reduction in the expression of the z protein. in conclusion, results presented here have shown the effective inhibitory action against the arenavirus junv by sirnas directed against the sequence of the z transcript. four z-specific sirnas were tested showing variable efficacy. the most effective inhibitor was z -sirna, targeted to the region encompassed by nt - of z gene. the efficacy of this agent against junv was also demonstrated in virus-infected cells by testing infectious virus plaque formation, viral rna or antigen expression, as well as in cells transfected with z-specific reporter plasmids. furthermore, the lack of effect of this sirna on the expression of other junv proteins, such as n and gpc, confirmed the specificity of action exerted by z -sirna on z transcript. interestingly, the inhibitory effect observed in the release of junv infectious progeny by z -sirna (> % inhibition at h p.i.) is similar to the effect described in transient transfection of sirnas for other viruses, but higher than the activity recently reported for sirnas targeted to the termini of n and l genes in the arenavirus lasv (müller and günther, ) . it remains to be investigated if the antiviral potential of rna interference for junv infections here shown after transient transfection of synthetic sirna targeted to z gene can be improved by using a viral vector of a short hairpin rna that resembles sirnas precursors. however, sirnas elicit potent effects at relatively low doses; so as to minimize the interference with naturally occurring processes, current therapeutic efforts seem to be focused towards the use of a pool of synthetic unmodified naked sirnas (davis, ) . likewise, our results provide a clear evidence of the central role of z protein in the junv life cycle and assess the good perspectives of this protein as antiviral target in arenaviridae, in accordance with previous studies reporting the efficacy against junv and lcmv infections of compounds reactive with the ring finger motif of the z protein (garcía et al., (garcía et al., , . the present study represents the first report of virus inhibition mediated by rna interference for a new world arenavirus, a group in the family including four agents of severe viral hemorrhagic fevers in south america (junv, machupo, sabiá and guanarito viruses). it would be an ideal situation to get an antiviral approach effective against all these pathogens. regarding infectious diseases, ongoing clinical trials in the field of sirna are developed by different companies and most of them are in the preclinical stage, with the exception of hbv and hiv studies that are in phase i, and the most advanced program concerning the treatment of infection by rsv using sirna developed by alnylam pharmaceuticals that has just finished phase ii (lópez-fraga et al., ) . given the diversity of arenaviruses, it will be difficult to design a sirna treatment able to cross-inhibit several species in the family, even using a highly conserved sequence as antiviral target. in contrast with the cross-reactivity among the old world arenaviruses lasv, lcmv and mopeia virus reported with sirnas targeted to lasv (müller and günther, ) , in our study we observed no efficacy when junv designed z-sirnas were used to inhibit the close antigenically related new world arenavirus tcrv. even, the effectiveness against other junv strain was reduced when there is more than one nucleotide change in the target sequence, as shown for z -and z -sirna against iv and xjcl junv strains (figs. a and ). thus, given the variability observed among natural and experimentally isolated junv strains (as shown in fig. a for the z gene of iv , rumero, xjcl , xj and candid strains), more detailed studies will be necessary in the design of rnai-based therapeutics for successful clinical intervention of human pathogenic arenaviruses. role for a bidentate ribonuclease in the initiation step of rna interference an arenavirus ring (zincbinding) protein binds the oncoprotein promyelocyte leukemia protein (pml) and relocates pml nuclear bodies to the cytoplasm the lymphocytic choriomeningitis virus ring protein z associates with eukaryotic initiation factor e and selectively represses translation in a ring-dependent manner antigenic relationships between attenuated and pathogenic strains of junin virus silencing e a mrna by rna interference inhibits adenovirus replication characterization of the arenavirus ring finger z protein regions required for z-mediated inhibition of viral rna synthesis ring finger z protein of lymphocytic choriomeningitis virus (lcmv) inhibits transcription and rna replication of an lcmv s-segment minigenome treatment of arenavirus infections: from basic studies to the challenge of antiviral therapy the first targeted delivery of sirna in humans via a selfassembling, cyclodextrin polymer-based nanoparticle: from concept to clinic the proline-rich homeodomain (prh/hex) protein is down-regulated in liver during infection with lymphocytic choriomeningitis virus functional anatomy of sirna for mediating efficient rnai in drosophila melanogaster embryo lysate inhibition of marburg virus protein expression and viral release by rna interference differential inhibitory action of two azoic compounds against arenaviruses arenavirus z protein as an antiviral target: virus inactivation and protein oligomerization by zinc finger-reactive compounds poliovirus escape from rna interference: short interfering rna-target recognition and implications for therapeutic approaches an rna-directed nuclease mediates post-transcriptional gene silencing in drosophila cells unlocking the potential of the human genome with rna interference a cellular function for the rna-interference enzyme dicer in the maturation of the let- small temporal rna selective silencing of viral gene expression in hpv-positive human cervical carcinoma cells treated with sirna, a primer of rna interference rna interference: from gene silencing to genespecific therapeutics combination of small interfering rna and lamivudine on inhibition of human b virus replication in hepg . . cells rna interference effectively inhibits mrna accumulation and protein expression of hepatitis c virus core and e genes in human cells transcription and rna replication of tacaribe virus genome and antigenome analogs require n and l proteins: z protein is an inhibitor oh these processes rna interference-based therapeutics: new strategies to fight infectious disease rnai-a powerful tool to unravel hepatitis c virus-host interactions within the infectious life cycle rna interference and antiviral therapy broad-spectrum antiviral activity of small interfering rna targeting the conserved rna termini of lassa virus optimal design and validation of antiviral sirna for targeting hiv- complementary in the supramolecular design of arenavirus and retroviruses revealed by electron cryomicroscopy and image analysis the small ring finger protein z drives arenavirus budding: implications for antiviral strategies myristoylation of the ring finger z protein is essential for arenavirus budding molecular biology of the prototype arenavirus, lymphocytic choriomeningitis virus biochemical and immunological evidence that the kda zinc-binding protein of lymphocytic choriomeningitis virus is a structural component of the virus rna interferencemediated virus clearance from cells both acutely and chronically infected with the prototypic arenavirus lymphocytic choriomeningitis virus junin virus monoclonal antibodies: characterization and cross-reactivity with other arenaviruses lassa virus z protein is a matrix protein sufficient for the release of virus-like particles rnai: double-stranded rna directs the atp-dependent cleavage of mrna at to nucleotide intervals silencing sars-cov spike protein expression in cultured cells by rna interference this work was supported by funding to the group from the consejo nacional de investigaciones científicas y técnicas (conicet), agencia nacional de promoción científica y tecnológica, universidad de buenos aires, and fundación bunge and born, argentina. ccg, las and ebd are members of the research career from con-icet. we are thankful to dr a. sanchez and dr t. ksiazek (cdc, atlanta, ga) for providing junv mabs. key: cord- -zy zwr authors: xu, jinfang; qian, ping; wu, qunfeng; liu, shasha; fan, wenchun; zhang, keshan; wang, rong; zhang, huawei; chen, huanchun; li, xiangmin title: swine interferon-induced transmembrane protein, sifitm , inhibits foot-and-mouth disease virus infection in vitro and in vivo date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: zy zwr the interferon-induced transmembrane protein (ifitm ) is a widely expressed potent antiviral effector of the host innate immune system. it restricts a diverse group of pathogenic, enveloped viruses, by interfering with endosomal fusion. in this report, the swine ifitm (sifitm ) gene was cloned. it shares the functionally conserved cd domain and multiple critical amino acid residues (y , f , f , r and y ) with its human ortholog, which are essential for antiviral activity. ectopic expression of sifitm significantly inhibited non-enveloped foot-and-mouth disease virus (fmdv) infection in bhk- cells. furthermore, sifitm blocked fmdv infection at early steps in the virus life cycle by disrupting viral attachment to the host cell surface. importantly, inoculation of -day-old suckling mice with a plasmid expressing sifitm conferred protection against lethal challenge with fmdv. these results suggest that sifitm is a promising antiviral agent and that can safeguard the host from infection with fmdv. the innate immune system is the first line of defense against pathogenic invasion (tarakhovsky and kroemer, ) . type i/ii interferons (ifns) are critical for generating cell intrinsic antiviral activity and hindering pathogens infection by inducing expression of a large number of ifn-stimulated genes (isgs) . the human ifn-induced transmembrane protein (ifitm ) was recently identified as a new antiviral effector of the isgs family. overexpression of ifitm blocks infection of a wide range of highly pathogenic enveloped viruses, including influenza a virus (iav), vesicular stomatitis virus (vsv), west nile virus, dengue virus, and severe acute respiratory syndrome coronavirus, as well as one non-enveloped reovirus (anafu et al., ; brass et al., ; huang et al., ; weidner et al., ) . ifitm is a small isg protein with an approximate molecular weight of kda, and it belongs to the cluster of differentiation (cd ) domain family (john et al., ) . amino acid (aa) sequence analysis of ifitm and its other four human paralogs (ifitm , ifitm , ifitm and ifitm ) reveals that they have two intramembrane domains (im and im ) and a conserved intracellular loop (cil), flanked by short variable n-terminal and c-terminal domains (john et al., ) . the intramembrane domains and cil constitute the cd domain that is shared by hundreds of cd domain family members (punta et al., ) . brass et al. ( ) first demonstrated the antiviral activity of ifitm proteins using a functional rnai genomic screen. ifitm expression accounted for - % of ifns' ability to block iav infection and exhibited early viral infection restriction (brass et al., ) . ifitm potently restricts the viral entry by altering the properties of cellular endolysosomal membranes, thus creating an unfavorable milieu for viral fusion with membranes (amini- bavil-olyaee et al., ; feeley et al., ) . ifitm further blocks cytosolic entry of viruses and the release of viral genomes, resulting in degradation of pathogens trapped within later endosomal or lysosomal compartments. foot-and-mouth disease virus (fmdv) is a non-enveloped virus with a positive single-stranded rna genome of approximately . kb, enclosed within an icosahedral capsid formed from copies of four structural proteins (vp , vp , vp and vp ) (grubman and baxt, ) . fmdv has seven serotypes, a, o, c, asia , sat , sat , and sat , with little cross-protection (bronsvoort et al., ; cao et al., ; rodriguez and gay, ) . these make it difficult to prevent and control fmdv by current vaccines (golde et al., ) to the action of a/b-ifns (dias et al., ; golde et al., ; kim et al., ; molinari et al., ) . inoculation of swine with adenovirus-delivered type i ifn confers % protection against fmdvcaused fever, vesicular lesions, and viremia and this protection can be maintained for - days (chinsangaram et al., ) . in addition, ifns interact with cognate receptors on the cell surface and activate isgs expression to safeguard the host through intracellular signaling cascades (siegrist et al., ) . fmdv enters host cells via receptor recognition and clathrindependent endocytosis. later, an acidic ph within the endosome is required to disassemble the capsid and translocate viral rna into the cytoplasm (johns et al., ) . ebola virus, iav and vsv that exhibit similar entry patterns are inhibited by human ifitm (bhattacharyya et al., ; huang et al., ; lakadamyali et al., ; sun et al., ; weidner et al., ) . the aim of this study was to clone swine ifitm (sifitm ) and examine if it could restrict fmdv infection. our results demonstrate that sifitm has a similarly functional cd domain and several critical conserved aa residues that correspond to its human ortholog. ectopic expression of sifitm inhibited infection of non-enveloped fmdv in baby hamster kidney (bhk- ) cells by disrupting viral attachment, and this differs from human ifitm restriction on enveloped viruses. inoculation of suckling mice with a plasmid expressing sifitm confers protection against lethal fmdv challenge. these data suggest that sifitm is a novel and potent antiviral agent that could be used to enhance animal resistance to fmdv infection. bhk- and ft cells were grown in dulbecco's modified eagle's medium (dmem; invitrogen, usa) containing % fetal bovine serum, u/ml penicillin, and lg/ml streptomycin sulfate at °c in a humidified % co incubator. type o fmdv strains o/es/ , o/gd/ , o/ah/ and type asia fmdv strain asia /js/ were amplified, and titrated by plaque assay and/or % tissue culture infective dose (tcid ) assay in bhk- cells (pengyan et al., ) . a blast search identified a swine expressed sequence tag (est) sequence with high homology to the human ifitm sequence (genbank: nm_ . ). a reverse primer (sifitm -r: -ctagta gcctctgtaatcctttatgagc- ) was designed using this est sequence, and a forward degenerate primer (sifitm -f: -accatg aactgcgcttcccag- ) was designed via multiple sequence alignment with human and predicted bovine ifitm (xm_ . ). the cdna of sifitm was amplified by reverse transcriptase-polymerase chain reaction (rt-pcr) using total rna extracted from a swine lymph node. the pcr product was cloned into the pmd -t vector (takara bio inc, japan) for sequence verification. the coding region of sifitm was subcloned into the plenti . / v -gw/lacz vector (invitrogen) at bamh i/xho i sites to replace the lacz gene, resulting in plenti . -sifitm . it was also subcloned into the previously described pca vector (niwa et al., ) , resulting in pca-sifitm . briefly, ft cells were seeded at a density of  cells per cm tissue culture plate and co-transfected with plenti . -sifitm or plenti . -egfp (expressing the emerald green fluorescence protein, egfp) and viralpower™ packaging mixture (invitrogen) using lipofectamine (invitrogen). fresh medium was added h later. the pseudotyped lentiviruses lv-sifitm expressing sifitm and lv-egfp expressing egfp were collected h and h post transfection and filtered through a . lm filter. bhk- cells were transduced by incubation with equal volumes of fresh dmem and lentivirus containing medium in the presence of lg/ml of polybrene. twenty-four hours post transduction, cell clones were developed by limiting dilution of the cell populations expressing sifitm or egfp. the bhk- stable cell lines expressing sifitm (bhk-sifitm ) or expressing egfp (bhk-egfp) were identified by fluorescence microscopy analysis and western blotting assay. bhk-sifitm or bhk-egfp were seeded in six-well plates at a density of  cells per well and cultured for h. next, cells were ice-chilled and incubated with fmdv strain o/es/ (henceforth referred to as o/es/ ) at a multiplicity of infection (moi) of for h on ice to allow viral attachment but impede viral entry. after three washing steps with ice-cold phosphate-buffered saline (pbs), o/es/ binding to the host cell surface was measured by real-time quantitative rt-pcr and flow cytometric analysis. to assay viral entry into cells, viral inocula were removed after h of binding on ice. cells were intensively washed with ice-chilled pbs, and pre-warmed medium was added, followed by incubation for an additional min at °c incubator. this was followed by one washing step with cold pbs, treatment with . % trypsin, and three more washing steps with cold pbs to remove any cell-associated virions that had not entered the cytoplasm. real-time rt-pcr and plaque assay were used to determine the amount of virus that had entered cells. bhk-sifitm or bhk-egfp cell lines were cultured for h, followed by infection with o/es/ . cells were harvested prior to being infected or at indicated time points post infection. total cellular rna or viral rna were extracted with trizol reagent (invitrogen), treated with dnase (promega, usa) and reverse transcribed with revertra ace Ò (toyobo; shanghai, china). real-time pcr was performed with specific primers and/or probe targeted at the b gene of o/es/ ( b: -acgaaacacggacccgactt- / -ccttgaccccagcggccaattcct- ; probe: were used as an endogenous reference control. thermal cycling conditions were min at °c, then cycles of s at °c, s at °c and s at °c. gene expression was analyzed as described previously wu et al., wu et al., , . bhk-sifitm and bhk-egfp cell lines were seeded into six-well plates h prior to infection with different doses of o/es/ . cells and supernatants containing virus were collected at different time points and stored at À °c. to determine quantities of intracellular virus, cell suspensions were subjected to three freeze/thaw cycles to release virions. for tcid assay, o/es/ titer was determined by endpoint dilution as described previously (pengyan et al., ) . briefly, -fold serial dilutions of fmdv samples were incubated in eight replicates in -well plates with - % confluent bhk- cells. two hours later, the supernatant was replaced with fresh culture medium. plates were incubated for an additional - h to determine cytopathic effect (cpe). the tcid was calculated with reed-muench formula (reed and muench, ) . for plaque assay, bhk- cells were seeded in six-well plates h prior to infection with -fold serially diluted o/es/ samples. a % methylcellulose overlay was added post h incubation. plaques were fixed with % formaldehyde and visualized with crystal violet staining h post infection. infected or non-infected bhk-sifitm and bhk-egfp cells were detached with trypsin, washed with pbs by centrifugation at g for min, and fixed with % paraformaldehyde. cells were washed and suspended with solution containing an anti-vp monoclonal antibody (mab) ( : dilution; prepared in our laboratory) for h at room temperature, followed by three round of washing steps. after centrifugation, the cells were stained with goat antimouse cy -conjugated secondary antibody (abcam, usa). finally, cells were washed, resuspended in pbs and analyzed by flow cytometry facscalibur (bd biosciences, usa). the percentage of egfp-and cy -positive cells in each sample was analyzed with the cellquest software (bd biosciences). the protein concentration of cell lysates was determined with bicinchoninic acid protein assay kit (pierce, usa). equal protein amounts were separated using % sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. the membrane was blocked with % non-fat milk and incubated with primary rabbit anti-sifitm polyclonal antibody ( : dilution; prepared in our laboratory), probed with a secondary peroxidase-conjugated anti-rabbit antibody (sigma, usa), followed by enhanced chemiluminescence staining (thermo fisher, usa). expression of b-actin was detected with an anti-b-actin mab (sigma) as internal reference. groups of -day-old suckling mice ( - mice/group) were obtained from the wuhan institute of biological products of china national biotec cooperation (wuhan, china). they were housed and handled according to the hubei provincial animal care and use committee guidelines. the mice were inoculated subcutaneously with ll ( lg/ll) pca-sifitm , pca or ll pbs as control. at h post immunization, the mice were subcutaneously infected with mouse lethal dose (mld ) of o/es/ at the immunization site. the mice were observed for clinical signs for weeks. all data were analyzed with one-way analysis of variance by the origin software (origin lab; northampton, ma, usa). p values < . were considered statistically significant. the survival curves were analyzed with the kaplan-meier method and compared with a log-rank test (v test) (wu et al., ) . the sifitm fragment was amplified by rt-pcr from the swine lymph node with primers sifitm -f and sifitm -r. the full-length cdna of sifitm is base pairs (bp) and it encodes aa residues (genbank: hq . ). multiple sequence alignments against ifitm sequences from other species show that sifitm nucleotide sequence shares % identity with bovine ifitm , % with human ifitm , % with mouse ifitm and % with chicken ifitm . corresponding aa alignments exhibit %, %, % and % identity, respectively (fig. a) . phylogenetic analysis indicates that sifitm belongs to the group containing bovine ifitm (fig. b) . to predict the functional characteristics of sifitm , the putative aa sequence was compared with human ifitm . sifitm protein sequence is aa longer than its human ortholog, and heterogeneity is mainly displayed at the c-terminal (fig. c) . comparison of the sifitm and human ifitm functional domains shows that aa residue identity is % at the n-terminal domain (ntd), % at the im domain, % at the cil domain, % at the im domain, and % at the cd domain overall, indicating that the functional domains are conserved between human and swine ( fig. c and d). in particular, sifitm aa residues y , f , f , r , and y (corresponding to human ifitm aa residues y , f , f , r , and y ) are completely retained ( fig. c ; highlighted with asterisk). these domains and residues are required for antiviral activity and cellular distribution of human ifitm (jia et al., ; john et al., ) . importantly, these data suggested that sifitm might possess the potential antiviral activity as its human ortholog. to investigate the antiviral activity of sifitm , we constructed the pseudotyped lentiviruses lv-sifitm and lv-egfp ( fig. a) . bhk- cells were transduced with these lentiviruses to generate the stable cell lines bhk-sifitm and bhk-egfp. western blotting assay confirmed that sifitm was efficiently expressed in bhk-sifitm cells (fig. b ). in addition, flow cytometric analysis showed that almost % of the cells were positive for sifitm or egfp expression (data not shown). these data demonstrated that these two cell lines could be used for the following antiviral study. fmdv has several serotypes that do not confer cross-protection against each other. as such, we firstly evaluated the antiviral activity of sifitm against different serotypes fmdv. low moi (moi = . ) were used, as fmdv exhibits high replication ability and pathogenesis in bhk- cells. two days post infection of the cell lines with viruses, plaque assay showed that bhk-sifitm was less susceptible to infection by type o fmdv strains o/es/ , o/gd/ , and o/ah/ than the control bhk-egfp, and reduced plaque formation numbers by over -to -fold (fig. c ). in addition, post infection with type asia fmdv asia /js/ , the plaque formation number was reduced to over fold in bhk-sifitm cells (fig. c) . next, fmdv viral rna was quantified using real-time rt-pcr. sifitm restricted the replication level of serotype o fmdv o/es/ , o/gd/ and o/ah/ about -fold, . -fold and -fold lower, respectively, when compared with bhk-egfp cells (p < . ) (fig. d) . furthermore, replication of fmdv serotype asia (asia / js/ ) was inhibited approximately -fold in bhk-sifitm cells when compared with bhk-egfp cells (p < . ). to characterize the dynamic curve of viral infection inhibited by sifitm , the appearance of cpe and viral titers were monitored in fig. . alignment, phylogenetic tree and architecture of sifitm . (a) aa sequence alignment of swine, human, mouse, bovine, and chicken ifitm . the numbers indicate the aa position. identical aa residues (black) and similar aa residues (red is identity from % to less than % and blue is identity from % to less than %) are marked. (fig. a and b) . to determine the infection step specifically disrupted by sifitm , we first examined whether o/es/ attachment and entry could be blocked by ectopic expression of sifitm in bhk cells, with regard to the successive steps of virus infection. as shown in fig. a , sifitm expression substantially blocked virus binding to cell surface, as viral rna copies extracted from bhk-sifitm cells were significantly fewer than those extracted from bhk-egfp cells. to analyze viral entry, cells were incubated an additional min at °c, and real-time pcr was performed on intracellular viral rna. the intracellular viral load was -fold lower in bhk-sifitm cells than in bhk-egfp cells (p < . ) (fig. a) . moreover, plaque assay demonstrated that viral entry into bhk-sifitm cells was significantly lower than that in bhk-egfp cells (p < . ) (fig. b) . to further confirm that sifitm inhibits o/es/ attachment to cell surface, the immunofluorescence and flow cytometric assays were performed to probe the virions binding on cell surface with mab against o/es/ vp . fluorescence intensity by microscope observation indicated that fewer virus particles were attached to cells expressing sifitm when compared with control cells (data no shown). moreover, flow cytometric analysis showed that over % of bhk-egfp cells exhibited an anti-vp mabpositive signal, compared with less than % of bhk-sifitm cells (p < . ) (fig. c ). mean fluorescence intensity analysis showed that each bhk-egfp cell captured more viral particles than bhk-sifitm cell (p < . ) (fig. d) . (fig. a ). all pbs-treated mice died within days, and . % of the pca-treated mice died within days post viral challenge (fig. b) . six of the mice inoculated with pca-sifitm survived the viral challenge, and this difference was statistically significant from the pbs and pca control groups (p < . ). these data indicated that sifitm conferred protection against lethal o/ es/ challenge in the suckling mice. one out of the nine mice in pca group survived. it is possibly due to the non-specific innate immune response induced by plasmid dna in vivo. ifns are part of the first line of defense against pathogen invasion and they confer antiviral activity via several diverse mechanisms. antiviral ifn signal amplification is primarily effected by isg, and many isg suppress virus infection at multiple steps in the virus life cycle (sadler and williams, ) . ifitm is a transmembrane protein that is mainly stimulated by type i ifn. it has been implicated in a broad range of cellular processes including adhesion, apoptosis, immune cell signaling, oncogenesis cell adhesion, heart development, germ cell homing, and repulsion (siegrist et al., ) . ifitm is also responsible for ifn-induced cell growth inhibition by transduction of antiproliferative signals (brem et al., ) . however, our data show that expression of sifitm in bhk- cells does not interfere with cell growth in comparison with the bhk-egfp, as previous studies (data not shown) (brass et al., ; weidner et al., ) . more importantly, ifitm is a potent antiviral effector against a number of viruses infection (anafu et al., ; brass et al., ; jiang et al., ) . to date, the characteristics and antiviral activity of ifitm have been mainly investigated in human species and human-associated pathogens. despite the moderate degree of inter-species similarity ( %) to the human ortholog, sifitm shares the similar molecular structure with human ifitm and retains the highly conserved ( % identity) ntd with aa from to . the conserved distal ntd is essential for complete antiviral activity of ifitm , especially the critical tyrosine y (y in sifitm ), whose dephosphorylation would result in mislocalization of ifitm from endosomes and lysosomes to the cell periphery and loss of the restriction of virus infection (jia et al., ; john et al., ) . sifitm shares the % identity to im and % identify to cil of the main functional cd domain of human ifitm and still keeps all the conserved residues f , f , r and y , which are indispensable for viral inhibition, intermolecular interaction, protein trafficking and phosphorylation (john et al., ) . in this study, it has been observed that sifitm acts at early steps in the fmdv life cycle, and these findings are consistent with ifitm proteins to inhibit the early replication of iav and flaviviruses (brass et al., ) . surprisingly, we found that sifitm disrupts fmdv attachment to the cell surface. this result differs from previous studies, in which human ifitm interferes with fusion of intraluminal virion-containing vesicles and endosomal membranes, thereby blocking genome release from iav and vsv into the cytoplasm (amini- bavil-olyaee et al., ; feeley et al., ) . one recent study suggests that ifitm exerts antiviral effect by eliciting a marked accumulation of cholesterol within endosomes, ultimately impairing the function of endosome membrane-virus fusion (amini-bavil-olyaee et al., ) . in addition, integrins recycling on endosomes membrane serve as main receptors recognizing fmdv particles (johns et al., ) . siljamaki et al. reported that cholesterol stability plays a vital role in integrin a uptake and in the formation of integrin-specific multi-vesicular bodies during virus infection (siljamaki et al., ) . cholesterol aggregation can halt integrin a uptake and prevent internalization. it has also been reported that ifitm , the paralog of ifitm , interacts with hepatitis c virus receptors cd and occludin to disrupt the process of viral entry (wilkins et al., ) . in future studies, it will be interesting to explore the relationship between the biological features, steady state of fmdv receptors and sifitm expression. it has further been demonstrated that ifitm alters endosomal structure and its acidic milieu (amini-bavil-olyaee et al., ; anafu et al., ; feeley et al., ) . therefore, we postulate that virus attachment is not the only step that is targeted by sifitm during fmdv infection. we cannot exclude the possibility that sifitm also alter endosomes in bhk- cells in order to prevent the disassembly of the viral capsids and viral rna release. we suppose that the -to -fold decrease in viral attachment associated with sifitm expression cannot fully account for over -fold decrease in viral production. control of animal infectious pathogens is a key component for the development of livestock farming. fmdv is a highly contagious disease among cloven-hoofed animals that can have a devastating economic impact on the international trade of animals and animal products (carpenter et al., ; yang et al., ) . our data indicate that sifitm is a potent swine-originated factor defense against fmdv infection in bhk- cells and suckling mice. therefore, we propose that this novel antiviral factor sifitm could potentially be employed to improve animal resistance to fmdv infection. all authors declare no competing interests. the antiviral effector ifitm disrupts intracellular cholesterol homeostasis to block viral entry interferoninducible transmembrane protein (ifitm ) restricts reovirus cell entry ebola virus uses clathrin-mediated endocytosis as an entry pathway the ifitm proteins mediate cellular resistance to influenza a h n virus, west nile virus, and dengue virus inhibition of proliferation by - u in interferon-alpha-responsive and non-responsive cell lines serological survey for foot-and-mouth disease virus in wildlife in eastern africa and estimation of test parameters of a nonstructural protein enzyme-linked immunosorbent assay for buffalo poly(i:c) combined with multi-epitope protein vaccine completely protects against virulent foot-and-mouth disease virus challenge in pigs epidemic and economic impacts of delayed detection of foot-and-mouth disease: a case study of a simulated outbreak in california novel viral disease control strategy: adenovirus expressing alpha interferon rapidly protects swine from foot-and-mouth disease novel antiviral therapeutics to control foot-and-mouth disease ifitm inhibits influenza a virus infection by preventing cytosolic entry vaccination against foot-and-mouth disease virus confers complete clinical protection in days and partial protection in days: use in emergency outbreak response foot-and-mouth disease fig. . sifitm protects suckling mice from lethal fmdv challenge. 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protects mice against a lethal challenge of fmdv efficient selection for high-expression transfectants with a novel eukaryotic vector inhibition of foot-and-mouth disease virus replication in vitro and in vivo by small interfering rna a simple method of estimating fifty per cent endpoints development of vaccines toward the global control and eradication of foot-and-mouth disease interferon-inducible antiviral effectors the small interferon-induced transmembrane genes and proteins cholesterol dependence of collagen and echovirus trafficking along the novel alpha beta integrin internalization pathway innate immunity interferoninduced cell membrane proteins, ifitm and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms ifitm is a tight junction protein that inhibits hepatitis c virus entry a pseudotype baculovirus-mediated vaccine confers protective immunity against lethal challenge with h n avian influenza virus in mice and chickens protective immunity elicited by a pseudotyped baculovirusmediated bivalent h n influenza vaccine enhanced immunogenicity induced by an alphavirus replicon-based pseudotyped baculovirus vaccine against porcine reproductive and respiratory syndrome virus epidemiological characteristics and financial costs of the foot-and-mouth disease epidemic in taiwan key: cord- -v z tld authors: chu, hsu-feng; chen, chiao-che; moses, david c.; chen, yau-hung; lin, chao-hsiung; tsai, ying-chieh; chou, chi-yuan title: porcine epidemic diarrhea virus papain-like protease can be noncompetitively inhibited by -thioguanine date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: v z tld porcine epidemic diarrhea virus (pedv) is a coronavirus (cov) discovered in the s that infects the intestinal tract of pigs, resulting in diarrhea and vomiting. it can cause extreme dehydration and death in neonatal piglets. in asia, modified live attenuated vaccines have been used to control pedv infection in recent years. however, a new strain of pedv that belongs to genogroup a appeared in the usa in and then quickly spread to canada and mexico as well as asian and european countries. due to the less effective protective immunity provided by the vaccines against this new strain, it has caused considerable agricultural and economic loss worldwide. the emergence of this new strain increases the importance of understanding pedv as well as strategies for inhibiting it. coronaviral proteases, including main proteases and papain-like proteases, are ideal antiviral targets because of their essential roles in viral maturation. here we provide a first description of the expression, purification and structural characteristics of recombinant pedv papain-like protease , moreover present our finding that -thioguanine, a chemotherapeutic drug, in contrast to its competitive inhibition on sars- and mers-cov papain-like proteases, is a noncompetitive inhibitor of pedv papain-like protease . discovered in the s, porcine epidemic diarrhea virus (pedv) is a coronavirus that causes severe agricultural loss (chasey and cartwright, ; lee, ) . pedv infects the intestinal tract and causes diarrhea and vomiting in older pigs. the mortality of pedv-infected piglets can reach % due to extreme dehydration (stevenson et al., ) . recently, modified live attenuated vaccines for pedv genogroup has been an important way to control the spreading of pedv in asia (song and park, ) . in , a new vaccine-resistant pedv strain, appeared in the usa (stevenson et al., ) . since then, this new usa pedv strain, which was later assigned to genogroup a, has caused a continuous pandemic in north america, europe and asia (huang et al., ; song et al., ) . the resistance of the new pedv strain to the conventional vaccine highlights the issue that although vaccination is a powerful strategy, it can still fail due to the genetic diversity of epitopes between genogroups. as a result, it is necessary to develop another antiviral strategy for pedv. unlike two other highly pathogenic human covs, severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov), which belong to the beta group, pedv belongs to the alpha group (chan et al., ) . like other covs, pedv depends on its own proteases, including main protease (m pro ) and papain-like protease (pl pro ) to cleave the polyprotein, and alpha covs have both papain-like protease (pl pro ) and papain-like protease (pl pro ) (lee, ; wojdyla et al., ) . polyprotein cleavage is required for viral maturation and thus these proteases are ideal antiviral targets (bacha et al., ; cheng et al., ; chou t et al., ; kumar et al., ; lin et al., ; park et al., ; ratia et al., ; wu et al., ) . furthermore, in contrast to highly variable spike proteins targeted by antibodies (li et al., (li et al., , wang et al., ; wu et al., ) , proteases with more conserved structure and catalytic function may serve as a general target across different covs bailey-elkin et al., ; chou et al., ; ho et al., ; ratia et al., ; yang et al., ) . like other coronaviral pl pro s, pedv pl pro is not only a deubiquitinating (dub) protease but also a multifunctional protein which plays a role in regulating host antiviral immune response (chaudhuri et al., ; clementz et al., ; mielech et al., ; xing et al., ; zheng et al., ) . dub activity of pl pro is required for the suppression of host immunity by blocking type interferon signaling. however, up to now, the structural characteristics and the detailed catalytic mechanism of pedv pl pro are still unclear, as are strategies for its inhibition. according to sequence alignment (fig. s ) and homology modeling, pedv pl pro may be composed of four domains: ubiquitin-like (ubl), palm, thumb and fingers domains. the latter three domains form a catalytic core and may retain catalytic activity, like that of sars-and mers-cov pl pro s (chou et al., ; clasman et al., ; lei et al., ) . in the present study, we made a recombinant catalytic core pedv pl pro and demonstrated its secondary, tertiary and quaternary structural characteristics. further studies suggest that pedv pl pro exhibits much higher dub activity than that of sars-and mers-cov pl pro s and can be inhibited by the anti-leukemia drug -thioguanine ( tg). tg has been found to be able to inhibit multiple dub enzymes including human usp as well as sars-and mers-cov pl pro s (chen et al., ; cheng et al., ; chou et al., ; chuang et al., ; lin et al., ) . inhibition assays and docking simulations were used to clarify the detailed inhibition mechanism of tg against pedv pl pro . our findings increase understanding of the structure and catalytic activity of pedv pl pro and identify the first potent inhibitor against this enzyme. the coding sequence of the usa strain of pedv pl pro ubl and catalytic domain (genbank accession number ahc . ; polyprotein residues - ) was full-gene synthesized (genomics co., taiwan). cdna of pl pro digested by ndei and xhoi was then inserted into pet- a and pet- a expression vectors, respectively. for the catalytic core of pl pro (residues - ) , the primers ′-ggctcc gcgaattgggattccc- ′ and ′-aaaactcgagtcattcggacaccac cacattg- ′ were used for polymerase chain reaction. the product was digested with xhoi and then ligated into a phd vector with a × histag and a sumo (smt ) sequence at the n-terminus (lee et al., ) . the primer sequences for site-directed mutagenesis of the t w mutant were ′-gaatggccgtcgtgtgctgaaatggaccgataataattgc tgg- ′ and ′-ccagcaattattatcggtccatttcagcacacgacggc cattc- '. the reading frames of the above plasmids were verified by dna sequencing. the expression vector was transformed into e. coli bl (de ) cells (novagen). cultures were grown in lb medium at °c until the absorbance at nm reached . . the cells were then induced with mm isopropyl-β-d -thiogalactopyranoside and incubated for h at °c. after centrifugation, the cell pellet was suspended in lysis buffer ( mm tris-cl, ph . , mm nacl, % glycerol, . % triton x- and mm β-mercaptoethanol) and lysed by sonication. next, the crude lysate was centrifuged at , rpm for min. the soluble lysate was incubated with ml of ni-nta agarose slurry (qiagen co., usa) at °c for h. after the unbound lysate removed, the beads were washed with wash buffer ( mm tris-cl, ph . , mm nacl, mm imidazole and mm β-mercaptoethanol). the beads with × his-tag protein bound were then transferred into . ml cutting buffer ( mm tris-cl, ph . , mm nacl, and mm β-mercaptoethanol), . mg ulp was added, and the mixture was incubated at °c for h to remove the sumo fusion. finally, unbound pedv pl pro catalytic core was loaded onto an s- gel-filtration column (ge healthcare) equilibrated with running buffer ( mm tris-cl, ph . , mm nacl and mm dithiothreitol). each protein fraction was checked for purity using sds-page. fractions containing a protein band of the correct size were then pooled and concentrated using an amicon ultra- -kda filter (millipore) to mg/ml. the protein with % glycerol was flash-frozen with liquid nitrogen and stored at − °c. the typical yield of protein was - mg per liter of cell culture. a jasco j- spectropolarimeter was used to analyze the secondary structure. recombinant pedv pl pro at a concentration of mg/ml in mm phosphate buffer (ph . ) was used for cd scanning from nm to nm at °c. the cuvette width was . mm. the far-uv cd spectrum data was analyzed using the cdsstr program at the dichroweb server (whitmore and wallace, ) . the normalized root mean square deviation was calculated to evaluate goodness of fit. the fluorescence spectrum of . μm protein dissolved in mm phosphate buffer (ph . ) or . m guanidine hydrochloride was monitored at °c using a perkinelmer ls b luminescence spectrometer. the excitation wavelength was nm and the emission spectrum was scanned from nm to nm. measurements of maximal peak and intensity were used to identify conformational change. auc experiments were performed on an xl-a analytical ultracentrifuge (beckman coulter) using an an- ti rotor. sedimentation velocity (sv) experiments were performed using a double-sector epon charcoal-filled centerpiece at °c at a rotor speed of , rpm. a protein solution of . mg/ml pedv pl pro catalytic core and reference solutions ( mm tris-cl, ph . , mm nacl) were loaded into the centerpiece. absorbance at nm was monitored continuously with a time interval of s and a step size of . cm. multiple scans at different time intervals were fitted to a continuous c(s) and c(m) distribution model using the sedfit program (schuck, ) . for dub assays, the reaction mixtures contained . μm of fluorogenic substrate ubiquitin- -amino- -trifluoro-methyl-coumarin (ub-afc) (boston biochem) and . μm of sars-or mers-cov pl pro or . μm of pedv pl pro in mm phosphate buffer (ph . ) for a total volume of . ml. enzymatic activity at °c was determined by monitoring excitation and emission at and nm, respectively. to determine the inhibitory effect, velocity data at various concentrations of inhibitors was fitted to obtain ic according to eq. ( ): in which v is the initial velocity in the presence of inhibitor at concentration [i] and v is the initial velocity in the absence of inhibitor, while n is the hill constant. the program sigmaplot . (systat software) was used for data analysis. the peptidyl substrates dabcyl-frlkggapikgv-edans and dabcyl-fkkkgggdvke-edans (synthesized by genscript) were used to measure the proteolytic activity of the pedv pl pro catalytic core and the t w mutant. increases in fluorescence intensity were monitored at excitation and emission wavelengths of and nm, respectively, in a perkinelmer ls b luminescence spectrometer. fluorescence intensity was converted to the concentration of hydrolyzed substrate using a standard curve determined by fluorescence measurements of defined concentrations of products. for kinetic analysis, the reaction mixtures contained - μm peptide substrate in mm phosphate buffer (ph . ) for a total volume of ml. after the addition of enzyme to the reaction mixture to a concentration of . μm, the fluorescence intensity was monitored at °c. the increase in fluorescence intensity was linear for min and thus the slope of the line represented the initial velocity. steady-state kinetic parameters were determined by fitting the data to the michaelis-menten equation. enzyme kinetic assays were performed by a method similar to that described above at peptidyl substrate concentrations of - μm and inhibitor concentrations of - μm. the velocity data was found to best fit a noncompetitive inhibition model in accordance with eq. ( ): in which k cat is the rate constant, [e], [s] and [i] denote the enzyme, substrate and inhibitor concentrations, respectively, and k m is the michaelis constant for the interaction between the peptide substrate and the enzyme. k is is the inhibition constant. the model structure of pedv pl pro was generated by swissmodeling (arnold et al., ) and molecular docking was performed using autodock vina (trott and olson, ) . several grid boxes of Å ( Å × Å x Å) with different centering coordinates were set to cover the entire putative structure. the docking parameters were set as default and the best models in each coordinate set were listed for further inspection. the model that scored the best among these sets was selected as the final binding model. initially, attempts at expressing pedv pl pro including the ubl and catalytic domains (polyprotein residues - ) with either an nterminal or a c-terminal × his-tag were not successful. previous studies suggested that the ubl domain was not involved in the catalytic activity of sars-and mers-cov pl pro s (chou et al., ; clasman et al., ) . therefore we removed the ubl domain and applied a sumo fusion protein at the n-terminus of the catalytic core (residues - ) to enhance solubility. fortunately, it was possible to express the pedv pl pro catalytic core in e. coli and purify it following the removal of sumo. after further purification using size-exclusion chromatography, highly pure pl pro was obtained (fig. s , left panel, lane ). mass spectrometry was performed to identify the sequence of the recombinant protein. in total, peptides originating from pedv pl pro were identified (fig. s , right panel) . alignment of these peptides with the pedv pl pro sequence shows % coverage, and both the n-terminus and c-terminus of the protein were confirmed (fig. s , bottom panel) . as this is the first time that pure pedv pl pro has been obtained, its secondary, tertiary and quaternary structures were further analyzed. cd spectrometry and analysis of the spectrum by cdsstr (fig. s a) showed that pedv pl pro consists of % α-helix, % β-sheet and % random coil. these proportions are similar to those of the sars-cov pl pro core (residues - ), which consists of % α-helix, % βsheet and % random coil (data not shown). for comparison, previous studies suggested that mers-cov pl pro consists of % α-helix, % βsheet and % random coil, where the higher content of β-sheet is because of the inclusion of the ubl domain (lin et al., ) . our results suggest that the three coronaviral pl pro s show similar secondary structural content. protein emission was used to reveal tertiary conformational change in the absence and presence of denaturant (fig. s b) . the results demonstrated that the fluorescence emission of pedv pl pro in phosphate buffer (native form) shows a major broad peak at nm which splits to two peaks at nm and nm in . m guanidine hydrochloride. the two peaks match the maximal emission wavelengths of tyrosine and tryptophan, respectively. this result suggests that the addition of denaturant induces exposure of the hydrophobic core of the protein. similar folding/unfolding change at maximal emission wavelength was also observed in the case of sars-cov pl pro , albeit the fluorescence intensity of denatured pedv pl pro is higher than that of the native enzyme (chou et al., ) . previous studies on the stability of the p core domain suggested that the higher fluorescence intensity of denatured p is because of aggregation (bullock et al., ) . sv experiments were carried out to determine the quaternary structure of pedv pl pro (fig. s a ). using continuous c(s) and c(m) distribution analysis, we found one major peak with a sedimentation coefficient of . and molecular weight of kda (figs. s b and s c ). this value is close to the predicted monomeric mass ( . kda). previous studies using the same sv experiment suggested that both the sars-and mers-cov pl pro s are also monomers lin et al., ) . overall, the secondary, tertiary and quaternary structures of the pedv pl pro catalytic core are similar to those of sars-and mers-cov pl pro s, even though their sequence identity is only - % (fig. s ). next, in order to compare the dub activity of pedv pl pro with other coronaviral pl pro s, activity assays using ub-afc as the substrate were carried out. the activity was determined and then normalized to give the fold increase in dub activity of pedv pl pro (fig. ) . the results showed that sars-and mers-cov pl pro s have similar dub activity. surprisingly, at a given substrate concentration, pedv pl pro shows activity comparable to that of the other two pl pro s at only onefortieth of the protein concentration. this indicates that pedv pl pro has considerably greater dub activity. furthermore, sequence alignment indicates that pedv pl pro may also have a zinc fingers motif like other coronaviral pl pro s (fig. s , green ovals) (bailey-elkin et al., ; lei et al., ; ratia et al., ; wojdyla et al., ) . previous studies have shown that the dub activity of coronaviral pl pro s can be inhibited by the addition of a chelator like edta, which removes intrinsic zinc ions (chou et al., ; lin et al., ) . in line with our expectations, inhibition of pedv pl pro by edta displays a similar dose-dependent pattern, suggesting that the removal of endogenous metal ions can inhibit pedv pl pro (fig. ) . furthermore, like other coronaviral pl pro s, pedv pl pro can be inhibited by adding extra external zinc ions (chou et al., ; lin et al., ) . in addition, the proteolytic activity of pedv pl pro was investigated using fluorogenic peptidyl substrates (table ) . for comparison, we used a peptidyl substrate with a sequence matching the p to p residues of the cleavage site of sars-cov pl pro , frlkgg, and a peptidyl substrate optimized for pedv pl pro whose cleavage site p to p residues are fkkkgg, based on the non-structural protein (nsp) to cleavage site (from genbank accession number ahc . ). interestingly, less saturation was observed while using the optimized peptidyl substrate (fig. s a) . we failed to improve it due to the fact that the proteolytic activity at concentrations of substrate higher than μm cannot be appropriately measured because of the inner-filter effect. after fitting the data to the michaelis-menten equation, k m of . μm and k cat of . min − for the sars-cov-derived substrate and the k m of . μm and k cat of . min − for the optimized substrate were determined (table ). the optimized substrate shows a . -fold higher k cat /k m , as a result of a . -fold higher k m and . -fold higher k cat , compared with the sars-cov-derived substrate. the dissimilar kinetic parameters for the two peptidyl substrates suggest that pedv pl pro may recognize various p residues between beta covs (l/f/v/g) and alpha covs (k/r/a). by contrast, sars-cov pl pro has a hydrophobic s subsite, with the result that it cannot cleave the pedv-optimized substrate and substrates of hcov- e and ibv whose p residue is also a lysine han et al., ; lei et al., ; wojdyla et al., ) . furthermore, in contrast to its considerably greater dub activity, k cat of pedv pl pro for the optimized peptidyl substrate is fold lower than that of sars-cov pl pro (lin et al., ) . the inconsistent efficacy between dub and proteolytic activities indicates that pedv pl pro is more like a usp enzyme (avvakumov et al., ; renatus et al., ) . previous studies have suggested that pedv pl pro , but not pl pro , is an interferon antagonist via its dub activity (xing et al., ) . in addition, a recent review suggests that pl pro and pl pro of alpha covs may show different levels of efficiency for cleaving the nsp to , to and to sites (lei et al., ) . previous studies of sars-cov pl pro demonstrated that the oxyanion is within hydrogen-bonding distance of the side chain of trp during catalysis (ratia et al., ) . although it is not conserved, mutation of the equivalent residue leu of mers-cov pl pro to tryptophan showed a -fold increase in k cat (lin et al., ) . again, according to sequence alignment, the equivalent residue of pedv pl pro is thr (fig. s , purple oval) . to verify this, a t w mutant was produced and its kinetic parameters were characterized by using the two peptidyl substrates (fig. s b and table ). interestingly, using the sars-covderived substrate produced a . -fold increase in activity based on k cat / k m as a result of a . -fold increase in k m and a -fold increase in k cat . this result demonstrates that the mutation enhances hydrolysis of the sars-cov-derived peptidyl substrate. in contrast, there is no significant difference in hydrolysis of the optimized substrate between wild-type pedv pl pro and the t w mutant (table ). this result indicates that the existence of residue thr may be quite enough to support proteolytic ability. further structural information on pedv pl pro in complex with ub or the optimized peptidyl substrate is required to demonstrate the detailed catalytic mechanism, especially for the formation of the oxyanion hole. several coronaviral pl pro inhibitors have been identified in previous studies (chen et al., ; cheng et al., ; chou et al., ; lin et al., ; ratia et al., ) . in the present study, these compounds were screened to determine whether they can inhibit pedv pl pro (table ) . among the compounds, two thiopurine analogs, -mercaptopurine ( mp) and tg, were found to be able to inhibit the dub activity of pedv pl pro with ic of . and . μm, respectively ( fig. and table ). ic of tg is . -fold lower than that of mp, suggesting that the amino group of tg may play the role of an active pharmacophore in the inhibition. in addition, two mp/ tg analogs, hypoxanthine, and -amino- -methyl-mercaptopurine, were also used for structure-function relationship studies. we found that replacement of the thiocarbonyl group of mp/ tg with either a hydroxyl or a methylthio group resulted in compounds devoid of inhibitory activity, suggesting its importance in the inhibition (table ) . due to its higher inhibition capability, tg was chosen for further investigation. kinetic assays at various concentrations of peptidyl substrates and tg were carried out to further investigate the inhibition mechanism (fig. s ) . interestingly, the observed kinetic parameters showed a decrease in the apparent k cat at increasing tg concentrations, whereas the apparent k m was not affected significantly. this is clearly indicative of a noncompetitive pattern of inhibition. indeed, the data was found to best fit to a noncompetitive inhibition model with the k is of . μm ( fig. and table ). this result suggests that tg and the peptidyl substrate may bind to different sites. for comparison, tg shows a competitive inhibitory effect against sars-and mers-cov pl pro s chou et al., ) but shows a noncompetitive inhibitory effect against human usp (chuang et al., ) , albeit their k is are close. these results indicate that tg can be a broad spectrum inhibitor against human and viral dub enzymes via different mechanisms. as tg is a noncompetitive inhibitor of pedv pl pro , recognition of the binding site of tg will allow us to understand its inhibitory mechanism more clearly. as no detailed structural information was . inhibition of pedv pl pro by - mm edta or μm zn + were also measured. activity data of each set was normalized to that of pedv pl pro . available, we generated a structural model of pedv pl pro and tried to discover a putative binding site of tg using in silico docking. interestingly but not surprisingly, we found that the putative binding site of tg with the highest affinity score is near the active site and on the left side of the blocking loop (residues to ), while the ubiquitin c-terminal tail is located on the right side (fig. a ). in our model, tg has polar interactions with residues asp , gly (on the blocking loop), val and thr and shows hydrophobic contact with lys and pro . binding of tg at this site may render the blocking loop less flexible and therefore disfavor catalysis. a series of noncovalent inhibitors such as compound grl shows a similar blocking effect, although they are competitive inhibitors and bind to the s -s subsite (ratia et al., ) . for comparison, besides residue val , the alignment shows no sequence identity for the putative binding residues (fig. s , red ovals) . furthermore, the same region is occupied by residue pro of sars-cov pl pro and residue lys of mers-cov pl pro (fig. b) , indicating that this binding site may only exist in pedv pl pro . in contrast, previous studies suggested that the binding site of tg for sars-and mers-cov pl pro s may be near the catalytic triad's cysteine residue due to its competitive pattern of inhibition chou et al., ) . in this study, we provide a first description of the expression, purification and structural properties of pedv pl pro as well as its potent inhibition by thiopurine analogs. the broad spectrum inhibitor tg was found able to inhibit not only the dub activity but also the proteolytic activity of pedv pl pro . these results shed light on the possibility of inhibition of pedv infection by small molecules instead of antibodies. furthermore, based on its noncompetitive inhibitory effect, we proposed an allosteric tg binding site which can stabilize the blocking loop near the active site, resulting in inhibition. the present study suggests that tg may be suitable as a lead compound for further ( )). b proteolytic activity at various concentrations of sars-cov-derived peptidyl substrate and tg was measured ( fig. s and fig. ) and the apparent k is was determined from the best global fit of the data to a noncompetitive inhibition model (eq. ( )). r sqr was . . c nd: ic was not determined due to lack of inhibition at a compound concentration of μm. 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of a large multi-domain protein crystal structure of the papain-like protease of mers coronavirus reveals unusual, potentially druggable active-site features cell attachment domains of the porcine epidemic diarrhea virus spike protein are key targets of neutralizing antibodies structure of sars coronavirus spike receptor-binding domain complexed with receptor structural and functional characterization of mers coronavirus papainlike protease disulfiram can inhibit mers and sars coronavirus papain-like proteases via different modes mers-cov papain-like protease has deisgylating and deubiquitinating activities diarylheptanoids from alnus japonica inhibit papain-like protease of severe acute respiratory syndrome coronavirus a noncovalent class of papain-like protease/deubiquitinase inhibitors blocks sars virus replication severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme structural basis of ubiquitin recognition by the 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acute respiratory syndrome cl protease crystal structure of nl respiratory coronavirus receptor-binding domain complexed with its human receptor the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor plp , a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production we would like to thank prof. ralph kirby for helpful suggestions. we also appreciate mrs. yen-su lin's help in the synthesis of compound grl . this research was supported by grants from the ministry of science and technology, taiwan, roc ( - -b- - , - -b- - , - -b- - , and - -b- - ) to cyc. hfc was supported by the ministry of education, taiwan, r.o.c. and bened biomedical co. supplementary data related to this article can be found at https:// doi.org/ . /j.antiviral. . . . key: cord- -x q vyf authors: millet, jean k.; séron, karin; labitt, rachael n.; danneels, adeline; palmer, kenneth e.; whittaker, gary r.; dubuisson, jean; belouzard, sandrine title: middle east respiratory syndrome coronavirus infection is inhibited by griffithsin date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: x q vyf highly pathogenic human coronaviruses associated with a severe respiratory syndrome, including middle east respiratory syndrome coronavirus (mers-cov), have recently emerged. the mers-cov epidemic started in and is still ongoing, with a mortality rate of approximately %. no vaccine is available against mers-cov and therapeutic options for mers-cov infections are limited to palliative and supportive care. a search for specific antiviral treatments is urgently needed. coronaviruses are enveloped viruses, with the spike proteins present on their surface responsible for virus entry into the target cell. lectins are attractive anti-coronavirus candidates because of the highly glycosylated nature of the spike protein. we tested the antiviral effect of griffithsin (grft), a lectin isolated from the red marine alga griffithsia sp. against mers-cov infection. our results demonstrate that while displaying no significant cytotoxicity, griffithsin is a potent inhibitor of mers-cov infection. griffithsin also inhibits entry into host cells of particles pseudotyped with the mers-cov spike protein, suggesting that griffithsin inhibits spike protein function during entry. spike proteins have a dual function during entry, they mediate binding to the host cell surface and also the fusion of the viral envelope with host cell membrane. time course experiments show that griffithsin inhibits mers-cov infection at the binding step. in conclusion, we identify griffithsin as a potent inhibitor of mers-cov infection at the entry step. coronaviruses belong to the coronaviridae family. they are enveloped viruses with a large single stranded, positive-sense rna genome and infect both humans and animals. until , human coronaviruses were responsible for only mild respiratory diseases, mainly common colds. this changed with the emergence of a highly pathogenic coronavirus associated with a severe respiratory syndrome, namely the severe acute respiratory syndrome coronavirus (sars-cov). in , a new deadly human coronavirus emerged in the arabian peninsula, middle east respiratory syndrome coronavirus (mers-cov) responsible for severe pneumonia that can be associated with additional clinical symptoms such as vomiting, diarrhea or renal failure (alhogbani, ; zaki et al., ) . the mers-cov epidemic is still ongoing, and so far more than laboratory-confirmed cases have been diagnosed with a mortality rate of approximately %. there are neither clinically approved antivirals nor vaccines available against mers-cov and therapeutic options for mers-cov infections are limited to palliative and supportive care. the mers-cov spike protein is a main determinant of virus entry into host cells as it mediates both binding to the dpp (dipeptidyl peptidase ) receptor and fusion of the viral envelope with host cell membrane (millet and whittaker, ; raj et al., ) . it is a type i fusion protein and is highly glycosylated with predicted n-glycosylation sites. one option to block mers-cov infection is to take advantage of the highly glycosylated nature of its spike protein by using lectins in order to inhibit mers-cov entry and thus virus propagation. griffithsin (grft) is a amino acid long lectin that was isolated from the red marine alga griffithsia sp. griffithsin has been shown to have antiviral activity against hiv- within the picomolar range (mori et al., vero- cells were treated with increasing concentrations of griffithsin (a) or with mg/ml (À) or mg/ml (þ) of the lectin (b). for (a), after h of incubation, supernatants were replaced with medium without griffithsin and cells were incubated at c for h. in (b), cells were left treated with griffithsin for h or h. cell viability was measured using a luminescence-based atp quantitation assay. results are expressed as average of cell viability (% of mg/ml control), with error bars representing sd from the average of three independent experiments. data were statistically analyzed using one-way anova test (a) or using a two-tailed student t-test (b), with the following convention for p-value significance: not significant (n.s.), p > . . (c) dose-response analysis of griffithsin on mers-cov infection. immunofluorescence assay of mers-cov-infected huh- , mrc- , and vero- cells in presence of increasing concentrations of griffithsin. cells were infected with mers-cov strain emc/ at an m.o.i. of , in presence of increasing amounts of griffithsin for h at c. pbs was used for the non-treated control condition. cells were washed with pbs and the inoculum replaced with cell growth medium. the cells were incubated at c % co for an additional h. cells were then fixed, immunolabeled for mers-cov s, and stained for nuclei (dapi) . n.i.: non infected control. (d) quantification of mers-cov-positive cells in presence of increasing concentration of griffithsin. for each condition, five  objective fields were randomly acquired and analyzed for total number of ). characterization of griffithsin structure has demonstrated that it is a domain-swapped homodimer with three carbohydrate binding domains on each monomer that bind to terminal mannose residues linked on hiv envelope protein gp n-glycans (zi ołkowska et al., ) . griffithsin has also been identified as a potent inhibitor of hepatitis c virus (hcv) and sars-cov infection in vitro and in vivo with minimal toxicity (meuleman et al., ; o'keefe et al., ) . therefore, we evaluated the anti-mers-cov activity of griffithsin. hek- t (atcc), huh- (nakabayashi et al., ) , mrc- (atcc), and vero- (atcc) cells were grown at c % co in dmem (corning) supplemented with % fetal bovine serum (thermofisher), mm hepes (corning), iu/ml penicillin and mg/ml streptomycin (corning). baric (unc chapel hill) and bart haagmans (utrecht university). the virus was propagated in vero- cells, with viral titrations performed using tcid assays. all experiments involving authentic mers-cov were performed in a biosafety level facility (animal health diagnostic center, cornell university). mannonanose-di-(n-acetyl-d-glucosamine) (man- glycan), was purchased from sigma aldrich. celltiter-glo luminescent cell viability assay reagents and luciferase assay kit were purchased from promega. .  huh- , mrc- , and vero- cells were seeded in well plates and incubated for h at c % co incubator. the cells were then treated with increasing concentrations of griffithsin, from (pbs) up to mg/ml, a range used in subsequent experiments. the treated cells were incubated at c % co for h. the medium was replaced with fresh growth medium without griffithsin and cells were incubated for another h at c % co . to test the effect of griffithsin for longer incubation times, the cells were left treated with either or mg/ml griffithsin for h or h. the cells were then lysed and viability assayed using the celltiter-glo kit according to manufacturer's guidelines. luminescence (relative luminescence units -rlu) readings were performed using a glomax / luminometer (promega). the percent cell viability was calculated by the following equation: the assays were carried out using triplicate wells, and data corresponds to the average of three independent experiments. . e .  huh- , mrc- , and vero- cells were seeded in wells of -well chamber slides (ibidi) and incubated at c % co for h. increasing concentrations of griffithsin ( e mg/ml) were added to mers-cov strain emc/ viral inoculums that were used to infect cells at a multiplicity of infection (m.o.i.) of . noninfected cells and pbs-treated virus inoculum were used as negative controls. the infected cells were incubated at c % co for h, after which the inoculum was removed, followed by pbs washing to remove unbound virions and addition of cell culture medium. the infection was left to proceed at c % co for an additional h. the wells of chamber slides were then processed for immunofluorescence assay as described in . . immunofluorescence microscopy assays.  huh- and mrc- cells were seeded in -well plate wells and incubated at c % co for h. cells were infected with virus inoculum that was untreated or treated with mg/ml griffithsin. the infected cells were incubated at c % co for h, after which the inoculum was removed, followed by pbs washing to remove unbound virions and addition of cell culture medium containing or not mg/ml griffithsin. cells were left to incubate at c % co . at h and h post infection time points, cell culture supernatants were harvested and stored at À c. cell titrations were performed using a tcid assay on vero- cells. results are expressed as log tcid /ml and represent averages of two independent experiments. particles pseudotyped with either mers-cov-s envelope proteins (merspp), genotype a hcv envelope proteins (hcvpp), or the g envelope glycoprotein of vesicular stomatitis virus (vsv-gpp) were produced as previously described (belouzard et al., ; op de beeck et al., ) . pseudotyped virions were used to inoculate huh- cells in -well plates for h in the presence of griffithsin. the inoculum was removed and cells were further incubated with culture medium for h. cells were lysed and luciferase activity was detected by using a luciferase assay kit (promega) and light emission measured by using a tristar lb luminometer (berthold technologies). for competition experiments with man- , griffithsin was preincubated with man- at different concentrations for h at c before being added to merspp during the h inoculation step of huh- cells. the inoculum was removed and luciferase activity quantified h post inoculation as described above. .  huh- cells were seeded in wells of -well chamber slides (ibidi) and incubated at c % co for h. virus infection was divided into four steps: ) inoculation and attachment of virus for h at c, ) pbs wash to remove unbound virus, followed by further binding of bound virus for h at c, ) internalization for h at c, and ) infection for h at c. in all conditions, cells were infected with mers-cov strain emc/ at an m.o.i. of , with griffithsin ( mg/ml) added or not at different steps during virus entry. in experimental condition a, griffithsin was not present at any step. in condition b, griffithsin was only present during initial attachment of virus on cells. in condition c, griffithsin was added cells (dapi nuclei stain) and s-positive cells (infected cells). results are expressed as percentage infected cells, with error bars representing sd from the average of three independent experiments. data were statistically analyzed using one-way anova test with the following convention for p-value significance: very highly significant (***), p . . only during the further binding step. in condition d, griffithsin was present only during internalization. finally, in condition e, griffithsin was present during initial attachment, further binding and internalization steps ( e ), but not during infection. after mers-cov infection was left to proceed for h at c, the wells of the chamber slides were processed for immunofluorescence assay as described in . . immunofluorescence microscopy assays. immunofluorescence were performed as previously described (millet and whittaker, ) . for quantifications of each condition, fields were randomly acquired. for each field, the total number of cells (dapi stain) and number of infected cells (spike labeling) were counted using automatic cell counting software (imaris . . ). the results of quantification are expressed in percent-infected cells. the experiment was performed using duplicate wells, and data corresponds to the average of three independent experiments. purified v -tagged soluble mers-cov spike proteins were incubated in % triton x- in pbs in presence or absence of mg/ ml of griffithsin for h at c followed by addition of polyclonal anti-griffithsin antibodies and a h incubation. the proteinantibodies complexes were precipitated with protein a sepharose beads pre-incubated with % bsa to avoid nonspecific binding. proteins were separated by % sds-page and were transferred to nitrocellulose membrane (amersham). spike proteins were immunodetected with an anti-v antibody (thermofisher). analysis of experimental data, calculations of standard deviations (s.d.) and standard error of the mean (s.e.m.), and graph plotting were performed using graphpad prism . . statistical analyses were performed using one-way anova test (dose-responses for cell viability and viral infectivity) and two-tailed student's t-test (viral titer assays, competition with man , and binding assay). for p-value significance, the following convention was used: not significant (n.s.) p > . , significant (*) p . , highly significant (**) p . , very highly significant (***) p . . we assessed the effect of griffithsin on cell viability and tested its potential cytotoxicity by treating huh- , mrc- and vero- cells with a range of griffithsin concentrations that was used in subsequent experiments, e mg/ml (fig. a) , or with mg/ml for longer treatment exposure times (fig. b) . griffithsin does not decrease cell viability at the doses and times tested, results that are in agreement with a previous investigation (nixon et al., ) . to determine the inhibitory activity of griffithsin on mers-cov infectivity, we have carried out dose-response infection experiments in three different cell lines (fig. c and d) . huh- , mrc- , and vero- cells were infected with strain emc/ of mers-cov at an m.o.i. of in presence of increasing concentrations of griffithsin ( e mg/ml). the infected cells were fixed at an early infection time point ( h) and assayed by immunofluorescence microscopy analysis (fig. c ) followed by quantification of the percentage of infected cells for each concentration (fig. d) . griffithsin significantly decreased infectivity by mers-cov, with a clear, dosedependent inhibitory effect in all cell lines tested ( fig. c and d) . even at the most diluted concentration of . mg/ml tested, the inhibitory activity of griffithsin on mers-cov infection was significant, with a minimal drop of . % in infectivity for huh- cells and a maximal drop of . % for vero- cells compared to the condition without the lectin. at mg/ml, griffithsin inhibited viral infectivity by more than % in all cell lines tested. because we have performed our assay using high m.o.i. and a short infection time, these results show the strong inhibitory activity of griffithsin on early steps of the mers-cov viral cycle. we investigated the effect of griffithsin on mers-cov virus titers (fig. ) . huh- and mrc- cells were infected with mers-cov in presence or not of mg/ml griffithsin. at h and h post infection, cell supernatants were harvested and analyzed for virus titers by performing a tcid assay (fig. ) . for both cell lines, griffithsin significantly decreased virus titers at both and h fig. . effect of griffithsin on mers-cov virus titers. huh- and mrc- cells were seeded in wells of -well plates and infected with mers-cov in presence or not of mg/ml griffithsin. after h of incubation, the inoculum was removed, and cells were incubated in presence or absence of mg/ml of griffithsin. at h and h post infection, supernatants were harvested. tcid assays were performed on the collected supernatants using vero- cells. results are expressed as average of log tcid /ml, with error bars representing sd from the average of two independent experiments. data were statistically analyzed using two-tailed student t-test with the following convention for p-value significance: significant (*), p . . time points, confirming the inhibitory role of the lectin on mers-cov infection. griffithsin has been shown to inhibit cellular entry of numerous viruses including sars-cov. to confirm the results obtained with mers-cov and determine the activity of this molecule on entry mediated by mers-cov spike protein, mers-cov pseudotyped virions (merspp) were produced and used to infect huh- cells. a dose-dependent inhibition of merspp infection was observed in the presence of griffithsin (fig. a ) suggesting that this compound is able to inhibit mers-cov entry. as expected, griffithsin was also able to inhibit hcvpp infection and had no effect on vsv-gpp infection as previously described (meuleman et al., ) . griffithsin is known to interact with n-linked high mannose oligosaccharides. the antiviral effect of this compound was shown to be due to the interactions of griffithsin with the mannoses linked to the viral envelope protein (moulaei et al., ) . to confirm that the inhibition observed is specific of griffithsin activity, a competition assay using n-linked high mannose oligosaccharides mannonosedi-(n-acetyl-d-glucosamine) (man- glycan) was performed. as shown in fig. b , incubation of merspp with griffithsin preincubated first with man- glycan significantly restores the infectivity of pseudotyped particles which suggests that griffithsin interacts with mannoses of mers-cov s envelope and impairs its functions during entry. to further confirm that griffithsin interacts with mers-cov spike protein, we performed a coimmunoprecipitation assay. soluble mers-cov spike proteins were incubated in presence or absence of griffithsin. the presence of spike proteins complexed with griffithsin was analyzed by western blot after immunoprecipitation with a polyclonal antigriffithsin antibody (fig. c) . western blot analysis revealed that spike protein could be coimmunoprecipitated only in presence of griffithsin. to better define which stage in the virus life cycle griffithsin acts on, we performed an infection assay using authentic mers-cov with griffithsin present at different times during viral entry steps (fig. a) . in this experiment, viral entry steps were divided into attachment at c, further binding at c, internalization at c. in the control conditions, griffithsin was either not present during entry steps (a) or present during all entry steps (e). in the latter condition, griffithsin had a significant inhibitory effect on infectivity compared to control condition (a), a result which recapitulates well the dose-response results ( fig. b and c) . we do note however that the infectivity in condition (a) ( . %) is lower for h at c before being added to the merspp for infection of huh- . intracellular firefly luciferase signal was measured as described above. experiment was performed three times in duplicate or triplicate. error bars represent sem. data were statistically analyzed using two-tailed student's t-test comparing results with non-treated condition with the following convention for p-value significance: not significant (n.s.), significant (*) p > . ; highly significant (**) p . , very highly significant (***), p . . (c) soluble mers-cov spike protein was incubated in presence or absence of mg/ml griffithsin. then griffithsin was immunoprecipitated and mers-cov spike protein was detected in western blot. than observed in the non-treated control of the dose response experiments ( . %). we attribute this drop to differences in the infection assays used, such as shorter inoculum contact time and multiple pbs washes. in condition (b), where griffithsin is only present during the first attachment step, a significant decrease in infectivity was measured (fig. c) . such significant inhibition was not observed for conditions (c), and (d), where griffithsin was present only at later steps in entry (further binding without inoculum, and internalization, respectively). taken together, these data reveal that griffithsin acts on very early stages of virus entry, probably by directly interacting with the glycosylated part of mers-cov spike protein and inhibiting its binding function. mers-cov emerged in in saudi arabia causing severe pneumonia less than years after the sars-cov emergence in china. in the case of mers-cov, person-to-person transmission has during the second step, the viral inoculum was removed and cells were washed three times with cold pbs and kept at c for h, enabling further binding of virus to cell surfaces, in presence (c and e) or absence (a, b, and d) of mg/ml griffithsin. for the third step, the cells were placed at c for h allowing for internalization of virions to occur, in presence (d and e) or absence (a, b, and c) of mg/ml griffithsin. the infection was left to proceed in a last h step at c in absence griffithsin. (b) immunofluorescence assay of mers-covinfected huh- cells with griffithsin present at different stages of infection. cells were infected and treated as described in (a) then fixed, immunolabeled and analyzed as described previously in fig. . (c) quantification of mers-cov-positive huh- cells for each condition tested. cells were analyzed as described in fig. . results are expressed as percentage infected cells, with error bars representing sd from the average of three independent experiments. data were statistically analyzed using two-tailed student's t-test comparing results with non-treated condition (a) with the following convention for p-value significance: not significant (n.s.), p > . ; very highly significant (***), p . . occurred, however transmission is not sustained, with a basic reproduction number below . mers-cov is a zoonotic virus, and camels and bats are its most likely reservoir. sars-cov-like viruses have been isolated in bats and it is likely that bats are the reservoir of a sars-cov ancestor. considering the increasing number and diversity of coronaviruses identified/isolated in bats and the proven propensity of coronaviruses to cross the species barrier and infect new hosts, it is likely that new coronaviruses causing severe diseases in humans may emerge in the future. currently, there are no drugs with proven efficacy to treat mers-cov or any other coronavirus infection. treatments are limited to supportive care for respiratory and circulatory functions and preventive care to protect renal, hepatic and neurological functions and to prevent secondary infection. as a consequence, new therapies are urgently needed. virus features conserved among the coronavirus family are attractive targets for the development of antiviral therapy to fight current and future emerging coronavirus infections. coronavirus spike proteins are the main determinant of virus entry and tropism (belouzard et al., ) . these glycoproteins are variable in sequence and length but share the same organization with two domains s and s . the s domain is responsible for receptor binding and attachment of the virus to the host cell surface whereas s contains the fusion machinery. a common feature of coronavirus spike proteins is their high glycosylation with e potential glycosylation sites, coronavirus spike proteins being exclusively n-glycosylated. therefore, the carbohydrate-binding proteins lectins are attractive anti-coronavirus candidates. griffithsin is a lectin that was isolated from the red marine alga griffithsia sp. and has been shown to inhibit hiv, hcv, and sars-cov within the nanomolar range. we demonstrated that while griffithsin has minimal to no effect on cell viability, it is a potent inhibitor of mers-cov infectivity and production in vitro. by using particles pseudo-typed with the mers-cov spike protein, we have shown that griffithsin acts during mers-cov entry. among the viral proteins anchored in the mers-cov envelope, in addition to the spike protein, only the m protein harbors a potential n-glycosylation site in its short n-terminal domain. for mhv, it has been shown that the m protein may be an additional target for lectins and enhance its antiviral effects. griffithsin inhibits mers-cov pseudotyped-particles to the same extent than mers-cov infection, suggesting that the spike protein is the main target of griffithsin for its antiviral effect. indeed, our results indicate a direct interaction of griffithsin with mers-cov spike protein. for hiv, griffithsin binds to the terminal mannoses of the n-glycans of gp , this binding slightly interferes with cd binding and likely prevents conformational rearrangement required during entry (mori et al., ) . for sars-cov, it has been shown that griffithsin does not affect ace binding and it has been suggested that griffithsin acts at a post-binding step during entry . other plant lectins, galanthus nivalis agglutinin (gna), hippeasttrum hybrid lectin (hha) and urtica dioica agglutinin (uda) have also been shown to prevent mhv entry at a post-binding step probably by interfering with the conformational rearrangements of the spike protein that drive the fusion of the viral envelope with the host cell membrane (van der meer et al., ) . for mers-cov, our results show that griffithsin inhibits infection with a different mechanism. in fact, we found that griffithsin interferes with infection as early as during the attachment step, likely by preventing interaction between spike and dpp , but is not able to prevent mers-cov infection when added at a postbinding step. indeed, a similar mechanism of receptor binding interference has already been observed for hcv (meuleman et al., ) . crystal structures of mers-cov receptor binding domain (rbd, residues to ) have been resolved in combination with dpp or in its absence. the mers-cov rbd contains a core domain with an accessory subdomain lying on the edge of the core structure (chen et al., ; lu et al., ) . both subdomains contain nlinked glycans on n in the core domain and on n in the accessory domain. the contributions of these sugar moieties to the virus-receptor interaction are unknown, but one can imagine that any involvement of one of these n-glycans in receptor binding could be impaired by griffithsin. in conclusion, griffithsin has a low cytotoxicity, likely interacts with any coronavirus spike proteins because of their highly glycosylated nature and is able to hamper coronavirus spike protein functions. griffithsin should be considered as an interesting drug candidate to develop for the treatment and/or prevention of current but also future emerging coronavirus infections. acute myocarditis associated with novel middle east respiratory syndrome coronavirus activation of the sars coronavirus spike protein via sequential proteolytic cleavage at two distinct sites mechanisms of coronavirus cell entry mediated by the viral spike protein crystal structure of the receptor-binding domain from newly emerged middle east respiratory syndrome coronavirus molecular basis of binding between novel human coronavirus mers-cov and its receptor cd griffithsin has antiviral activity against hepatitis c virus host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein isolation and characterization of griffithsin, a novel hiv-inactivating protein, from the red alga griffithsia sp monomerization of viral entry inhibitor griffithsin elucidates the relationship between multivalent binding to carbohydrates and anti-hiv activity growth of human hepatoma cells lines with differentiated functions in chemically defined medium griffithsin protects mice from genital herpes by preventing cell-to-cell spread broad-spectrum in vitro activity and in vivo efficacy of the antiviral protein griffithsin against emerging viruses of the family coronaviridae characterization of functional hepatitis c virus envelope glycoproteins the carbohydratebinding plant lectins and the non-peptidic antibiotic pradimicin a target the glycans of the coronavirus envelope glycoproteins domain-swapped structure of the potent antiviral protein griffithsin and its mode of carbohydrate binding this work was supported by the french national agency for research (anr, anr- -ce - ) (ks, jd, and sb). work in the whittaker lab is supported by the national institutes of health grant r ai . we are grateful to sophana ung for his assistance in the illustrations. we also thank susan daniel and the whittaker lab for helpful discussions. key: cord- - jgmxtzd authors: zhu, qingyuan; hu, hui; liu, haixia; shen, hong; yan, zhipeng; gao, lu title: a synthetic sting agonist inhibits the replication of human parainfluenza virus and rhinovirus through distinct mechanisms date: - - journal: antiviral res doi: . /j.antiviral. . sha: doc_id: cord_uid: jgmxtzd stimulator of interferon genes (sting), as a signaling hub in innate immunity, plays a central role for the effective initiation of host defense mechanisms against microbial infections. upon binding of its ligand cyclic dinucleotides (cdns) produced by the cyclic gmp-amp synthase (cgas) or invading bacteria, sting is activated, leading to the induction of both type i interferon responses and autophagy, which are critical for the control of certain microbial infections. rna viruses, such as parainfluenza virus (piv) and rhinovirus (hrv), are among the leading causes of respiratory infections that affect human health without effective treatments. activation of sting pathway may provide a new therapeutic approach fighting against these viruses. however, the role of sting in the control of rna virus infection remains largely unexplored. in this study, using dimeric amidobenzimidazole (diabzi), a newly discovered synthetic small molecule sting receptor agonist with much higher potency than cdns, we found that activation of sting elicits potent antiviral effects against parainfluenza virus type (piv ) and human rhinovirus (hrv ), two representative respiratory viral pathogens. notably, while anti-piv activity was depend on the induction of type i interferon responses through tank-binding kinase (tbk ), anti-hrv activity required the induction of autophagy-related gene (atg )-dependent autophagy, indicating that two distinct antiviral mechanisms are engaged upon sting activation. antiviral activity and individual specific pathway was further confirmed in infected primary bronchial epithelial cells. our findings thus demonstrate the distinct antiviral mechanisms triggered by sting agonist and uncover the potential of therapeutic effect against different viruses. stimulator of interferon genes (sting) is a signaling molecule located in the endoplasmic reticulum (er) and is essential for the activation of host innate immune responses against microbial infections (barber, ) . upon binding of its cyclic dinucleotides (cdns) ligand produced by the cyclic gmp-amp synthase (cgas) or invading bacteria, sting is activated. the activation of sting leads to the recruitment and activation of tank-binding kinase (tbk ), which phosphorylates and activates the transcription factor interferon regulatory factor (irf ) to initiate the transcription of innate immune genes with antiviral functions, such as type i interferons (ifn) and interferon stimulated genes (isgs) (suresh and mosser, ) . recently, sting activation has been reported to induce autophagy-related gene (atg )-dependent autophagy, which mediates the clearance of dna and viruses in the cytosol and has a crucial role in antiviral defense (gui et al., ; liu et al., a) . the known natural sting agonists are nucleosides guanosine (g) and/or adenosine (a)-based cdns and several of modified cdns are currently being pursued as immunotherapy agents for cancers. however, cdns are susceptible to hydrolysis by phosphodiesterases and contain negatively charged phosphate groups impeding passive diffusion through the plasma membrane, which limits their the therapeutic application only to patients with accessible solid tumors via intratumoral delivery (yum et al., ) . recently, the amidobenzimidazole (abzi) family, a new class of synthetic small molecule sting agonists with physicochemical properties completely different from those of cdns and suitable for systemic administration, was j o u r n a l p r e -p r o o f reported (ramanjulu et al., ) . dimeric abzi (diabzi) demonstrated much higher binding affinity and cellular potency than cdns. the reported ec of sting activation is at nm, which is folds more potent than cgamp) (ramanjulu et al., ) . thus, abzi-based compounds have the potential to treat a much broader scope of indications, including cancers and infectious diseases. soon after the discovery of sting, it became evident that sting plays a critical role in restricting many dna viruses, including papilloma virus, herpes simplex virus (hsv ), adenovirus and vaccinia virus (dai et al., ; lam et al., ; reinert et al., ; sunthamala et al., ) . in contrast to its well-documented role in innate immune responses to dna viruses, its function in controlling rna virus infections remains largely unexplored. although neither synthetic rna nor the rna virus genome directly activates sting-related signaling pathway, an accumulating body of evidence suggests that sting is also required for optimal host protection against multiple rna viruses, including vesicular stomatitis virus (vsv), sendai virus (sev), dengue virus (denv) and influenza a virus (iav) (aguirre et al., ; holm et al., ; ishikawa and barber, ; ishikawa et al., ). in addition, some recent studies have reported that several rna viruses, such as denv, severe acute respiratory syndrome (sars) coronavirus and influenza a virus (iav), can antagonize sting functions (aguirre et al., ; holm et al., ; sun et al., ) . thus, whether sting agonists could exert antiviral effects against a broader range of rna viruses than currently accepted, as well as the nature of the mechanisms by which sting suppresses the replication of rna viruses needs further study. j o u r n a l p r e -p r o o f parainfluenza virus (piv) and rhinovirus (hrv) are among the leading causes of respiratory infections that affect human health, and there are currently no approved antiviral treatments or vaccines for these viral infections (bloom et al., ) . piv is a negative-strand rna virus belonging to the paramyxoviridae, whreas hrv is a positive-strand rna virus belonging to the picornaviridae. in this study, we aimed to evaluate whether sting agonists could inhibit piv and hrv infections, using abzi compounds as tools, and if so, their underlying downstream mechanisms upon sting activation against infections with selected subtypes of piv and hrv. our results suggest that abzi compound-mediated sting activation elicits potent antiviral effects against piv and hrv via two distinct antiviral mechanisms. abzi, diabzi derivative (patent application wo ) and diabzi (ramanjulu et al., ) were synthesized > % purity according to patents wo and wo , respectively. the tbk inhibitor bx , ', '-cgamp and digitonin were purchased from sigma (sml , sml and ) and autophagy inhibitor chloroquine was purchased from selleck chemical (s ). hep cells (atcc, ccl- ) were cultured in dmem/f medium supplied with % fbs (gibco) and % penicillin/streptomycin (sigma). h -hela cells from atcc j o u r n a l p r e -p r o o f (atcc, crl- ) were cultured in dmem supplied with % fbs (gibco) and % penicillin/streptomycin (sigma). thp- -dual (thpd-nfis), thp- -dual ko-sting (thpd-kostg), raw-lucia isg (rawl-isg), raw-lucia isg-ko-sting (rawl-kostg), and hek-blue isg-ko-sting (hkb-isgkostg) cells were purchased from invivogen and cultured according to manufacturer's instruction. anti-sting (cst, s), anti-atg (cst, s) and anti-lc (cst, s) antibodies were obtained from cell signaling technology (cst). the anti-p-irf antibody (abcam, ab ) and antiactin antibody (abcam, ab ) were purchased from abcam. the ifnr antibody ( - ) was purchased from pbl. (zhang et al., ) was purchased from viratree (viratree, p ) and propagated in llc-mk (atcc, ccl- . ) cells according to the manufacturer's protocol. enhanced gfp was inserted between the p/c/d/v and m coding regions of piv (zhang et al., ) . rhinovirus was obtained from atcc (atcc, vr- pq) and propagated in h -hela cells (lee et al., ) . the viruses were stored in - °c until use. h -hela cells were seeded at , cells per well in a -well plate. after incubation overnight, cells were infected with hrv virus at a multiplicity infection (moi) of . . immediately following the addition of virus, the indicated concentration of compound was added to the culture medium and returned to °c incubator for days. following incubation, cell viability was determined using a cell counting j o u r n a l p r e -p r o o f kit (dojindo, cat.ck ) and a envision reader (pe) according to the manufacturer's protocol. hep , h -hela or pbec were seeded at x e cells per well in a cell carrier ultra -well cell culture plate one day prior to infection. after overnight incubation, cells were infected with piv -gfp virus addition and treated with the indicated dose of the compound in infection medium (opti-mem for piv or opti-mem with µg/ml tpck treated trypsin for mpv) and returned to a °c incubator for days of incubation. the number and intensity of gfp puncta were determined with an enzyme-linked immune absorbent spot (elispot) reader (aid elispot vspot spectrum) and quantified with aid elispot reader version . software. human thp- or murine raw . cells containing an interferon regulatory factor (irf)-inducible luciferase reporter were seeded at x e cells per well in well plates. after overnight incubation, cells were stimulated with the indicated compound for hours. luciferase activity in the supernatant was measured using quanti-luc assay (invivogen) and an envision reader (pe) according to manufacturer's instructions. hep or h -hela cells were seeded at . x e cells per well in a -well plate. after overnight incubation, lipofectamine rnai max (life technologies, j o u r n a l p r e -p r o o f ) was used according to the manufacturer's protocol to transfect cells with indicated sirna. forty-eight hours after transfection, the protein level of the target of interest was determined by western blotting. anti-viral assays were performed at hours post transfection. the control, sting and atg stealth sirna sets were obtained from life technology (assay id s sisting and s siatg ). to determine the expression levels of cellular proteins, cells were lysed in ripa buffer (life technology) containing proteinase inhibitor cocktail (thermo fisher). lysates were mixed with loading buffer (life technology), heated at °c for minutes, and subjected to sds-page and western blotting using preset gels and a western blot system (life technology) according to the manufacturer's instructions. total rna was isolated from cells with a rneasy mini kit (qiagen), and firststrand cdna was generated from total rna using random primers and a reverse transcriptase system (invitrogen). real-time pcr was performed on a roche light cycler with taqman qpcr master mix universal (abi). the primer and probe sequences for detecting hrv are shown below. forward primer: '-cgctcagctgttaacccaaca - '. reverse primer: '-cagccacgcaggctagaac - '. probe: fam '-tagagattcccctccggcgacgg - ' bhq. (sachs et al., ) the sequences for detecting piv are shown below. reverse primer: '-atgatagctccaccagctgatttt- '. probe: fam '-cgtaggcaagaaaacataa- ' bhq. (hu et al., ) the cycling conditions were as described previously (hu et al., ; sachs et al., ) . the expression values were normalized to that of hgapdh (applied biosystems, f). hep or h -hela cells were seeded at . x e per well in -well plate. the cells were treated with indicated compound for hr. the sample were collected from cells by quantigene cell culture sample processing kit (life technology), then subjected to quantigene multiplex kits (life technology) for luminex reader according to manufacturer's instructions. pbec was obtained from atcc (pcs- - , lot ). cells were revived and cultured in atcc epithelial cell basal medium supplied with atcc epithelial cells growth kit (pcs- - ). a total of x e cells/ml in . ml/ . ml of growth medium were seeded in collagen coated or -well plate. cells were cultured for at least hours and were then used for different anti-viral assays. statistical analysis was performed using prism software (graphpad). a value of p < . was considered statistically significant. the chemical structures of two small molecule abzi-based sting receptor agonists are shown in fig. a . the abzi compound was derived from the original hits identified through high-throughput screening of small molecules competing with cgamp for binding to sting. the diabzi compound with enhanced binding affinity and cellular functions is a designed dimer of the key binding element of abzi linked through a four-carbon linker to synergize the effect of the two symmetry-related abzibased compounds (ramanjulu et al., ) . the ability of these compounds to activate the sting pathway was first evaluated using a thp- irf-inducible luciferase reporter cell line, which is a human monocytic cell line containing a secreted luciferase reporter gene under the control of an irf-inducible promoter. the natural sting ligand cgamp activated the reporter only in wild type but not in sting knockout (ko) cells, thus validating this assay ( fig. b and c ). we observed that both the abzi and diabzi compounds activated the irf reporter in a dose-dependent manner (fig. b) . compared to the µm half-maximal effective concentration (ec ) value of abzi, diabzi showed a much higher potency, with an ec value of . µm. in contrast, there was no induction of irf reporter in sting ko thp- cells, which confirmed that the irf pathway activation induced by the abzi compounds is j o u r n a l p r e -p r o o f sting-dependent (fig. b) . similar results were observed when a murine macrophage-derived cell line, raw . , was used, suggesting that unlike the murine sting selective agonist dmxaa (conlon et al., ) , the abzi-based sting receptor agonists can activate both human and murine sting with similar potency (fig. c) . with activities much improved over that of cgamp ( fig. b and c ), these non-nucleotide abzis represent a novel class of synthetic sting receptor agonists with the potential to be further developed for therapeutic applications. next, we sought to determine the antiviral activity of the abzi-based sting agonists against selected subtypes of piv and hrv. piv , which belongs to one of the four subtypes of piv, is endemic year-round and can cause serious viral respiratory tract disease in infants and children (bloom et al., ) . hrv is the most common cold-causing virus and it can be classified into major and minor groups based on the use of cellular receptors. hrv belongs to the major group, which binds human intercellular adhesion molecule (icam- ) (greve et al., ) . first, gfp-labelled piv virus (piv -gfp) was used to infect hep cells seeded in -well plates at an moi of . in the presence of sting agonists. two days after infection, the cells were fixed, and green fluorescence images of each well were acquired with a fluorescence elispot reader. the gfp signal intensity, which reflects the level of viral replication, was quantified. both abzi and diabzi dose-dependent inhibited the gfp signal, as demonstrated by the substantial, simultaneous reduction in both the number of gpf-positive spot puncta and the gfp signal intensity, suggesting that viral spread and replication were profoundly impaired ( fig. a and b) . the half-j o u r n a l p r e -p r o o f maximal inhibitory concentration (ic ) values of abzi and diabzi to gfp signal intensity were calculated as . µm and . µm, respectively, which correlated well with their irf inducing activity as shown in figure . in the meanwhile, no significant impact on cell viability was observed at up to µm in hep cells (supplementary fig. a) , therefore the selectivity index (si) values of abzi and diabzi were calculated as > and > ,respectively. in addition, the viral rna levels were also measured, and the results were well correlated with the gfp intensity quantification results. (fig. c ). to evaluate anti-hrv activity, h -hela cells infected with hrv at an moi of . were treated with abzi or diabzi. after hours, the cell viability was measured via a cck- assay to quantify cytopathic effects. although the anti-hrv activity was weaker than the anti-piv activity, both the abzi and diabzi compounds dose-dependently protected the cells from cpes, with ic values of . µm and . µm, respectively, indicating a significant anti-hrv activity (fig. d ). in addition, the half-maximal cytotoxic concentration (cc ) values of both compounds were greater than µm in h -hela cells, suggesting that the observed antiviral effects were not related to nonspecific cytotoxicity (supplementary fig. b) , therefore the selectivity index (si) values of abzi and diabzi were calculated as > . and > . , respectively. in addition, the viral rna levels were also measured and the results were correlated with the cpe assay results (fig. e ). in contrast to the strong antiviral activity of the diabzi sting agonist, the natural sting ligand cgamp, showed only modest anti-piv and anti-hrv activity at high concentrations in the presence of digitonin permeabilization (supplementary j o u r n a l p r e -p r o o f fig. c and d) . collectively, these results show that the diabzi sting agonist, but not cgamp (with digitonin permeabilization), has potent anti-piv and anti-hrv activity. to determine whether the antiviral activity of the diabzi compound depends on sting, we investigated the impact of reducing sting expression on the antiviral potency of the diabzi compound. to this end, we first used sirna to knockdown the expression of sting in hep and h -hela cells. three sting targeting sirnas were tested and sisting- , which demonstrated the highest knockdown efficiency in both cell lines as assessed by western blotting, was selected (fig. a) . sisting- was transfected into hep or h -hela cells for hours, the cells were then infected with piv -gfp or hrv , respectively, in the presence of diabzi. knockdown of sting dramatically reduced the anti-piv activity of diabzi in hep cells and completely abolished the anti-hrv activity of diabzi in h -hela cells ( fig. b and c ). in addition, the anti-piv and anti-hrv activity of diabzi were completely abolished in sting knockout hek cells, but were fully restored via exogenous expression of sting (supplementary fig. ) . moreover, similar to the results shown in fig. , diabzi exhibited much stronger activity against piv than against hrv activity (supplementary fig. b and c). to further characterize the antiviral effect of diabzi, time-of-addition experiments were performed to determine the effects of varying the treatment initiation time of the sting agonist diabzi on the replication of piv and hrv . hep cells and h -hela cells were infected with piv -gfp and hrv , respectively, at an moi of . , and diabzi was added at different time points during viral infection. the anti-piv activity of diabzi was minimally affected even when the treatment was initiated at hours after infection (fig. d) . in contrast, delaying treatment initiation time point substantially impaired the anti-hrv activity of diabzi, and no antiviral effect was observed when diabzi was added at hours after infection (fig. e ). in summary, although these results confirmed that the antiviral activity of sting agonist diabzi is dependent on sting expression, the antiviral mechanisms against piv and hrv infections engaged upon sting activation may differ. activation of sting leads to the induction of type i interferon (ifn) responses via tbk , which are believed to play critical roles in the control of certain viral infections. in addition, recent reports demonstrate that sting also directly activates autophagy, which is important for the clearance of dna and viruses in the cytosol (gui et al., ; liu et al., a) . consistent with previous reports, in hep and h -hela cells treated with diabzi, we observed clear activation of both the irf and autophagy pathways, as indicated by the increased levels of phosphorylated-irf (p-irf ) and lc -ii conversion (fig. a) . therefore, to determine the contribution of these two pathways to the antiviral activity of diabzi, the tbk inhibitor bx or the autophagy inhibitor chloroquine (cq) was applied during diabzi treatment. in both piv -infected hep and h -hela cells, bx completely abolished the anti-piv activity of diabzi, whereas no significant impairment was observed with cq treatment, j o u r n a l p r e -p r o o f suggesting that the activation of immune responses via tbk plays a dominant role in sting-mediated anti-piv activity (figs. b and c). interestingly, cq, but not bx , substantially reduced the anti-hrv activity of diabzi, suggesting that the induction of the autophagy pathway plays a major role in sting-mediated anti-hrv activity, in sharp contrast to the effect seen on sting-mediated anti-piv activity (fig. d ). in addition, neither cq nor bx at test concentration do not affected the gfp intensity of piv ,, as well as the cpe ability of hrv at test concentration (supplementary fig. ). consistent with these observations, we found that pegasys (pegylated interferon alpha) treatment greatly suppressed the replication of piv , but not hrv (supplementary figs. a and b) . furthermore, blocking the type i interferon receptor (ifnr) with a neutralizing antibody modestly affected the anti-piv activity of diabzi, but did not affect its anti-hrv activity (supplementary figs. c, d and e) . in addition, we observed similar inductions in the expression of several representative isg mrnas upon diabzi treatment in the presence or absence of viral infection, indicating that the sting downstream signaling pathway is not affected by viral infection (supplementary fig. ). these results support the hypothesis that ifn responses significantly contribute to the anti-piv activity, but not the anti-hrv activity of diabzi, mediated by sting activation. furthermore, knockdown atg expression, which is required for sting-induced autophagy, completely abolished anti-hrv , but not anti-piv activity of diabzi (supplementary fig. ). interestingly, we found a diabzi derivative could induce p-irf in both hep and h -hela cell, but with a much weaker ability to induce lc -ii conversion j o u r n a l p r e -p r o o f (supplementary fig. a and fig. a) . moreover, this compound demonstrated strong anti-piv activity (ic = . µm), but without any anti-hrv activity (ic > µm, supplementary b). further studies are warranted to better understand the mechanistic differences engaged by the diabzi and diabzi derivative compounds. taken together, these results suggest that sting activation inhibits piv and hrv via activation of the tbk -mediated ifn responses and induction of autophagy, respectively. immortalized cell lines, such as hep and hela, which can be conveniently cultured to support robust viral replication, have been used extensively to study virus-host interactions. however, these cell lines are poorly representative of the complexity of the respiratory epithelium. pbecs are the primary targets of many respiratory viruses in vivo, including piv and hrv, and are thus a more physiologically relevant model for studying these viruses (bossios et al., ; moskwa et al., ) . therefore, we sought to determine whether the sting agonist diabzi could inhibit piv and hrv replication in pbecs. to this end, pbecs were seeded in collagen-coated plates and were then infected with piv -gfp or hrv in the presence of diabzi. for piv -gfp-infected cells, at hours after infection, green fluorescence images of each wells were acquired with a fluorescence elispot reader. the gfp signal intensity was quantified, and the inhibition percentage relative j o u r n a l p r e -p r o o f to dmso treatment was plotted ( fig. a and b ). the ic value was calculated as . µm, which was similar to that observed in hep cells. hrv infection did not cause apparent cpes in pbecs (data not shown). therefore, we harvested the cells and measured the viral rna level with rt-pcr at hours and hours after infection. we observed a substantial reduction in the hrv rna level upon diabzi treatment, confirming its anti-hrv activity (fig. c ). in addition, the pathway analysis experiments were also conducted in pbecs. we observed that cq, but not anti-ifnr antibody, blocked hrv replication, while anti-ifnr mab, but not cq, inhibited piv replication in pbecs, which were consistent with the results from cell lines (fig d and e ). in summary, using virusinfected pbecs, we further confirmed the antiviral activity and distinct antiviral mechanisms induced by sting activation. the host innate immune system acts as the first line of defense against viral infections. as an essential innate signaling molecule, sting is required for protecting the host against a broad range of viral infections. thus, sting agonists has the potential to be developed as broad-spectrum antiviral agents. however, cdns type agonists derived from natural sting ligands have stability and permeability issues that limit their therapeutic applications. using the newly discovered diabzi sting agonist as a tool, we demonstrated potent antiviral activity against the representative respiratory rna viruses, piv and hrv , upon sting activation. compared to j o u r n a l p r e -p r o o f cgamp, diabzi showed a much stronger ability to trigger sting activation, which correlated well with its high antiviral potency. we speculate that this enhanced cellular performance is likely derived from its improved permeability (data not shown). in addition to the highly differentiated physicochemical properties and increased sting binding affinity of abzi-based agonists compared with cdns, these agonists efficiently activate sting in an open conformation without the need for lid closure, which may also contribute to their improved potency (ramanjulu et al., ) . similar to many other rna viruses, such as hcv, vsv and sev, piv is highly sensitive to ifn treatment (rabbani et al., ) . type i ifn treatment has been reported to efficiently block piv replication by inducing the expression of isgs with antiviral functions (atreya and kulkarni, ; rabbani et al., ) . it has been wellcharacterized that sting activation leads to type i ifn production and isgs induction (ishikawa and barber, ) . indeed, diabzi strongly suppressed piv replication with a similar potency across all cell culture systems tested, which correlated well with its irf induction activity. however, some rna viruses, such as respiratory syncytial virus (rsv), which belongs to the same paramyxoviridae as piv, as well as hrv , are resistant to ifn treatment (supplementary fig. e ) (atreya and kulkarni, ) . consistent with these results, we observed much reduced potency against hrv and rsv upon diabzi treatment, implying the resistance of these viruses to the conventional ifn response-driven antiviral mechanisms ( fig. c and data not shown). it is unlikely that the virus actively antagonizes ifn responses, as we did not observe a significant difference in the isg mrna levels between infected and noninfected cells upon diabzi treatment (supplementary fig. ). despite its resistance to host ifn responses, diabzi showed clear dosedependent anti-hrv activity, with an ic in the micro molar range, which was not affected by treatment with the tbk inhibitor bx or the ifnr blocking antibody, indicating that other antiviral mechanisms might be involved (fig. d and c ). indeed, using cq and sirna knockdown, we confirmed that atg -dependent autophagy triggered by sting activation is responsible for the anti-hrv activity of diabzi ( fig. d and supplementary fig. c ). autophagy is a major pathway for degradation of cellular components inside eukaryotic cells (fader and colombo, ) . viruses have coevolved strategies to manipulate this pathway to ensure their own replication and advantage. most previous studies focused on the proviral mechanisms of autophagy in infected cells. however, piv and hrv have been reported to induce and subvert the autophagic machinery to promote their own replication (ding et al., ; klein and jackson, ) . interleukin- receptor-associated kinase m (irak-m) promotes human rhinovirus infection of lung epithelial cells via the autophagic pathway (wu et al., ) . however, accumulating evidence suggests that autophagy also contributes to the host defense against microbial infections (rey-jurado et al., ) . induction of autophagy has been shown to inhibit the growth of vsv and hsv (orvedahl et al., ; shelly et al., ) . furthermore, sting induced autophagy restricts zikv infection in the adult fly brain (liu et al., b) . picornaviruses, such as poliovirus (pv) and hrv, permeabilize endosomes to deliver their genomes into the cytoplasm, where they can be detected and inhibited by galectin- via autophagic degradation (staring et al., ) . in addition, the results of the time-of-addition experiments result j o u r n a l p r e -p r o o f suggest that diabzi functions early during hrv infection and that delaying treatment initiation substantially impacts its antiviral efficacy. therefore, the anti-hrv activity observed upon sting activation is likely driven by autophagymediated virion degradation, thus viral genome release is prevented. interestingly, diabzi showed much lower potency against hrv than against piv , and this discrepancy cannot be explained by assay methodologies and endpoint differences. the autophagy-mediated antiviral response, which targets only early steps in the viral life cycle, is likely less efficient than the irf-mediated antiviral response which is believed to block multiple steps in the viral life cycle via induction of ifn and isgs. alternatively, induction of the autophagy-mediated antiviral response may have slower kinetics or require a higher sting agonist concentration than induction of the ifn response. further studies are thus needed to elucidate the details regarding the difference in the potency of diabzi against different type of viruses. in this study, the diabzi sting agonist demonstrated potent antiviral activity against multiple respiratory rna viruses, represented by piv and hrv , which broadly affect human health and have no effective treatment or vaccine. therefore, sting agonists hold promise further development as therapeutic or prophylactic agents in respiratory infections caused by these viruses. however, caution is required, because sting activation also induces the production of pro-inflammatory cytokines, such as il- β, il- , and tnf-α, which may exacerbate inflammation-related symptoms (chen et al., ) . thus, the timing, dose level and duration of the treatment with sting agonists need to be evaluated carefully to prevent exacerbated hours. the gfp signal intensity was quantified and the percent inhibition relative to dmso treatment was plotted. (e) pbecs were infected with hrv at an moi of and treated with µm of diabzi for hours in the presence or absence of cq ( µm) / ifnr antibody (anti-ifnr mab µg/ml) /isotype control antibody (control mab, µg/ml). total cellular rna was isolated and reverse transcribed to cdna. viral rna and gapdh mrna were then quantified via rt-pcr. the hrv rna level was normalized to the gapdh mrna level and the percent inhibition relative to dmso treatment was plotted. the data are presented as the means ± sd of triplicate j o u r n a l p r e -p r o o f samples from one experiment and are representative of at least three independent experiments. *p < . , **p < . . j o u r n a l p r e -p r o o f were infected with piv -gfp at moi of . and treated with indicated concentrations of diabzi in the presence of µg/ml ifnr antibody (anti-ifnr mab) or isotype control antibody (control mab) for hours. the gfp signal intensity was quantified and the percent inhibition relative to dmso treatment was plotted. the inhibition of gfp intensity was determined with an elispot reader and related software. (g) h -hela cells were infected with hrv at moi of . and treated with indicated concentrations of diabzi in the presence of µg/ml ifnr antibody or isotype control antibody for hours. the cpe was determined by cck- and the percent inhibition relative to dmso treatment was plotted. the data are the means ± sd of triplicate samples from one experiment and are representative of at least three independent experiments. p values were determined using student t test, **p < . ; *p < . . denv inhibits type i ifn production in infected cells by cleaving human sting respiratory syncytial virus strain a is resistant to the antiviral effects of type i interferons and human mxa sting: infection, inflammation and cancer summary health statistics for u.s. children: national health interview survey rhinovirus infection induces cytotoxicity and delays wound healing in bronchial epithelial cells regulation and function of the cgas-sting pathway of cytosolic dna sensing mouse, but not human sting, binds and signals in response to the vascular disrupting agent , -dimethylxanthenone- -acetic acid modified vaccinia virus ankara triggers type i ifn production in murine conventional dendritic cells via a cgas/sting-mediated cytosolic dna-sensing pathway phosphoprotein of human parainfluenza virus type blocks autophagosome-lysosome fusion to increase virus production autophagy and multivesicular bodies: two closely related partners the major human rhinovirus receptor is icam- autophagy induction via sting trafficking is a primordial function of the cgas pathway influenza a virus targets a cgas-independent sting pathway that controls enveloped rna viruses development of a realtime rt-pcr assay for detection and quantitation of parainfluenza virus sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling sting regulates intracellular dna-mediated, type i interferondependent innate immunity human rhinovirus induces the autophagic pathway and replicates more efficiently in autophagic cells adenovirus detection by the cgas/sting/tbk dna sensing cascade growth of human rhinovirus in h -hela cell suspension culture and purification of virions inflammation-induced, sting-dependent autophagy restricts zika virus infection in the drosophila brain innate immune response to viral infections in primary bronchial epithelial cells is modified by the atopic status of asthmatic patients hsv- icp . confers neurovirulence by targeting the beclin autophagy protein identification of interferon-stimulated gene proteins that inhibit human parainfluenza virus type sensing of hsv- by the cgas-sting pathway in microglia orchestrates antiviral defence in the cns contribution of autophagy to antiviral immunity quantitative realtime pcr for rhinovirus, and its use in determining the relationship between tcid and the number of viral particles autophagy is an essential component of drosophila immunity against vesicular stomatitis virus pla g represents a switch between entry and clearance of picornaviridae coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling interleukin- receptor-associated kinase m (irak-m) promotes human rhinovirus infection in lung epithelial cells via the autophagic pathway roles of the cgas-sting pathway in cancer immunosurveillance and immunotherapy infection of ciliated cells by human parainfluenza virus type in an in vitro model of human airway epithelium cells were transfected with pm siatg - or sicontrol for hours, then infected with hrv at moi of . and treated with diabzi at indicated concentrations for hours. to determine the anti-hrv activity, the cpe was determined by cck- . the percent inhibition relative to dmso treatment was plotted. the data are the means ± sd of triplicate samples from one experiment and are representative of at least three independent experiments. key: cord- -gcfx authors: ianevski, aleksandr; zusinaite, eva; kuivanen, suvi; strand, mårten; lysvand, hilde; teppor, mona; kakkola, laura; paavilainen, henrik; laajala, mira; kallio-kokko, hannimari; valkonen, miia; kantele, anu; telling, kaidi; lutsar, irja; letjuka, pille; metelitsa, natalja; oksenych, valentyn; bjørås, magnar; nordbø, svein arne; dumpis, uga; vitkauskiene, astra; Öhrmalm, christina; bondeson, kåre; bergqvist, anders; aittokallio, tero; cox, rebecca j.; evander, magnus; hukkanen, veijo; marjomaki, varpu; julkunen, ilkka; vapalahti, olli; tenson, tanel; merits, andres; kainov, denis title: novel activities of safe-in-human broad-spectrum antiviral agents date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: gcfx according to the who, there is an urgent need for better control of viral diseases. re-positioning existing safe-in-human antiviral agents from one viral disease to another could play a pivotal role in this process. here, we reviewed all approved, investigational and experimental antiviral agents, which are safe in man, and identified compounds that target at least three viral diseases. we tested of these compounds against eight different rna and dna viruses. we found novel activities for dalbavancin against echovirus , ezetimibe against human immunodeficiency virus and zika virus, as well as azacitidine, cyclosporine, minocycline, oritavancin and ritonavir against rift valley fever virus. thus, the spectrum of antiviral activities of existing antiviral agents could be expanded towards other viral diseases. dozens of viruses, such as fluav, hsv- , vzv, cmv and nov, constantly infect human population and represent substantial public health and economic burden (dalys and collaborators, ; disease et al., ) . emerging and re-emerging viruses, such as ebov, marv, lasv, chikv, zikv, denv, rvfv, mers-and sars-cov, surface from natural reservoirs approximately one each year and also represent global threats (howard and fletcher, ; who, ) . according to who, there is an urgent need for better control of these viruses, including drug-resistant and vaccine immunity escaping viral strains (bekerman and einav, ; de clercq and li, ) . antiviral drugs and vaccines are the most powerful tools to combat viral diseases. most drugs and vaccines, however, selectively target a single virus, thereby providing a "one drug-one bug" solution. by contrast, broad-spectrum antivirals (bsas) can cover multiple viruses and genotypes and reduce the likelihood of development of resistance. therefore, some bsas can be used for rapid management of new or drug-resistant viral strains, for treatment of viral co-infections reducing therapy complexity, as well as a first-line treatment or the prophylaxis of acute virus infections. thus, to overcome time and cost issues associated with the development of virus-specific drugs and vaccines, the development of bsas should be prioritized (bekerman and einav, ) . nucleotide and nucleoside analogues are excellent examples of bsas. they inhibit transcription and/or replication of different rna and dna viruses (de clercq, ) . in particular, valaciclovir inhibits replication of different herpesviruses and hbv (laube et al., ; vere hodge and field, ) . cidofovir and its lipid conjugate brincidofovir also inhibit replication of dsdna viruses, such as herpesviruses, adv, bkv, and hpv (andrei et al., ) . ribavirin blocks viral rna synthesis of fluav, hcv and rsv (hong and cameron, ) . favipiravir and bcx also inhibit replication of different rna viruses (mckimm-breschkin et al., ) . however, viruses are able to develop resistance to some of these nucleotide and nucleoside analogues. other examples of bsa agents include inhibitors of cellular pathways, which are exploited by different viruses for efficient viral replication (debing et al., ) . these agents overcome the problem of antiviral drug resistance. for example, lipid-lowering statins (atorvastatin, lovastatin, simvastatin, and fluvastatin) inhibit cellular hmg-coa reductase and attenuate replication of some enveloped viruses (bernal et al., ; enserink, ) . anti-malaria quinolones (chloroquine and hydroxychloroquine) inhibit acidification of endosomes, which is an essential process for uncoating of ssrna viruses (al-bari, ). anticancer kinase inhibitors dasatinib, imatinib, gefitinib, nilotinib, erlotinib and sunitinib impair intracellular viral trafficking and exert bsa effects (bekerman et al., ; schor and einav, ) . the anti-duchenne muscular dystrophy agent, alisporivir, targets cellular cyclophilin and inhibits the folding of hcv, hiv, mers-and sars-cov proteins, and, therefore, prevents formation of infectious virus particles (boldescu et al., ; de wilde et al., ; soriano et al., ) . thus, both host-directed antivirals and nucleotide/nucleoside analogues could possess bsa activity. here, we hypothesised that some of the identified safe-in-human bsas could possess novel antiviral activities and, therefore, could be used for treatment of many different viral infections. to prove this hypothesis, we reviewed safe-in-man approved, investigational and experimental antiviral agents. we identified compounds that target at least three viral diseases. we tested of the compounds against different viruses and found novel activities for of these agents. we conclude that the spectrum of antiviral activities for existing bsa agents could be expanded towards other viral diseases. information on the viruses and associated human diseases is summarized in table s . information on approved, investigational and experimental safe-in-human antivirals is summarized in tables s -s . this information was extracted from drugbank ( ), clinical trials websites ( ) and pubmed. information on approved, investigational, and experimental antivirals, which target ≥ viral diseases, is summarized in table s . eye diagrams and interaction network plots were created with javascript library d .js v ( ). a structural similarity plot for the drugs was constructed and visualized using a c-spade web application (ravikumar et al., ) . the compounds used in this study, their suppliers and catalogue numbers are summarized in table s . to obtain mm stock solutions compounds were dissolved in % dimethyl sulfoxide (dmso, sigma-aldrich) or milli-q water. the solutions were stored at − °c until use. bhk- cells (baby hamster kidney fibroblasts) were grown in glasgow's minimal essential medium (gmem) containing . % fetal bovine serum (fbs; gibco, paisley, uk), % tryptose phosphate broth (tpb), mm hepes, u/ml penicillin and . mg/ml streptomycin (penstrep, lonza basel, switzerland). ach- cells, a model for chronic hiv- infection, which possesses a single integrated copy of the provirus hiv- strain lai (nih aids reagent program), were grown in rpmi- medium supplemented with % fbs and penstrep. madin-darby canine kidney epithelial (mdck) cells, human embryonic kidney cells (hek t) and african green monkey kidney epithelial cells (vero) were grown in dulbecco modified eagle's medium (dmem; sigma-aldrich, st. louis, mo, usa) supplemented with mm l-glutamine (lonza; basel, switzerland), u/ml penstrep and % fbs. human telomerase reverse transcriptase-immortalized retinal pigment (rpe) cells were grown in dmem-f medium supplemented with u/ml penstrep, mm l-glutamine, % fbs, and . % sodium bicarbonate (sigma-aldrich). tzm-bl cells were grown in dmem supplemented with % heat-inactivated fbs and penstrep. human lung adenocarcinoma epithelial cells, a , were cultured in dmem containing . g/l nahco , mm hepes (euroclone, milan, italy), penstrep, and % fetal bovine serum (fbs, gibco) at °c. all cell lines were grown in humidified incubator at °c in the presence of % co . human influenza a/wsn/ (h n ) virus was generated using eight-plasmid reverse genetics system in hek and vero-e cells, as described previously (hoffmann et al., ) . ev strain was propagated in a monolayer of vero cells, as described earlier (myllynen et al., ) . hsv- was amplified in vero cells, as described previously (nygardas et al., ) . zikv fb-gwuh- strain was cultured in vero e cells, as described earlier (driggers et al., ) . for production of hiv- , × ach- cells were seeded in ml of full culture media, and hiv- production was induced by the addition of nm phorbol -myristate -acetate (viira et al., ) . the induced cells were incubated for h, and subsequently, the hiv- containing media was collected and filtered. the amount of hiv- in the stock was estimated by quantification of p protein in the media. quantity of p was measured using a reference recombinant purified p protein and anti-p -elisa, which was developed in-house. chikv- sg-nanoluc strain was generated from icdna clone of picres representing lr opy strain belonging to east/central/ south african genotype (utt et al., ) . rrv- sg-nanoluc strain derived from rrv-t strain (jupille et al., ) . sequence encoding nanoluc protein was placed between non-structural and structural regions of chikv or rrv genomes under the control of native subgenomic promoters of the viruses. to ensure expression of viral structural proteins, a copy of subgenomic promoter (residues − to + for chikv and residues − to + for rrv, positions given with respect to start site of subgenomic rna) was inserted immediately downstream of sequence encoding for nanoluc. recombinant rvfv expressing the far-red fluorescent protein katushka instead of the deleted nss protein (rrvfvΔnss::katushka) was used in this study (islam et al., ) . the virus stocks were stored at − °c. all the experiments with viruses were performed in compliance with the guidelines of the national authorities using appropriate biosafety laboratories under appropriate ethical and safety approvals. fluav virus was titrated in mdck cells using plaque assay as described previously (denisova et al., ) . ev titers were determined by plaque assay on a cells, as described earlier (marjomaki et al., ) . zikv titers were determined by plaque assay on vero-e cells, as described earlier (kuivanen et al., ) . hsv- titers were determined by plaque titration in vero cells in the presence of human immunoglobulin g ( μg/ml), as described earlier (paavilainen et al., ) . chikv- sg-nanoluc and rrv- sg-nanoluc were titrated in bhk- cells using plaque assay, as described previously (oo et al., ; taylor et al., ) . for testing the viability and death of compound-treated mock-, fluav-, ev -, chikv- sg-nanoluc-, rrv- sg-nanoluc-, zikv-and hsv -infected cells, approximately × rpe cells were seeded in each well of a -well plate. the cells were grown for h in cell growth medium. the media was replaced with virus growth medium (vgm) containing . % bsa, mm l-glutamine, . % nahco , and μg/ml l- -tosylamido- -phenylethyl chloromethyl ketone-trypsin (tpck)-trypsin (sigma-aldrich) in dmem-f . in the case of zikv, the media was replaced with dmem-f medium supplemented with u/ ml penstrep, mm l-glutamine, % fbs, and . % sodium bicarbonate. the compounds were added to the cells in three-fold dilutions at seven different concentrations starting from μm. no compounds were added to the control wells. the cells were mock-or virus-infected at a multiplicity of infection (moi) of one. when virus induced a cytopathic effect in cells (typically - hpi), celltox green express cytotoxicity reagent (ctxg, : dilution in the assay well, promega, madison, wi, usa) was added in vgm and the fluorescence was measured with a pherastar fs plate reader (bmg labtech, ortenberg, germany) or hidex sense microplate reader (hidex oy, turku, finland). the media was removed from the cells and stored at − °c. celltiter-glo viability reagent (ctg, promega, madison, wi, usa) containing firefly luciferase and luciferin was added ( μl per well). the luminescence was measured with a pherastar fs plate reader. for testing virus titers in compound-treated and non-treated fluav-, ev -, zikv-and hsv -infected cells the media was collected, serially diluted in pbs, and added to mdck (fluav), a (ev ), and vero-e (zikv and hsv- ) cells. the media was changed, and the cells were overlaid with plaque assay media. the cells were fixed, and viral titers were calculated. the titers were expressed as plaque-forming units per ml (pfu/ml). the presence of chikv- sg-nanoluc and rrv- sg-nanoluc in the media from non-or drug-treated rpe cells was evaluated by passaging the viruses in fresh rpe cells. the viruses expressed nano-luciferase from viral promoter, which was measured from lysed cells h post infection using renilla luciferase assay (promega, madison, wi, usa). for testing compound toxicity and efficacy against hiv- , approximately × tzm-bl cells were seeded in each well of a -well plate. tzm-bl cells express firefly luciferase under control of hiv- ltr promoter allowing quantitation of the viral infection (tat-protein expression by integrated hiv- provirus) using firefly luciferase assay. the cells were grown for h in cell growth medium. compounds were added to the cells in three-fold dilutions at seven different concentrations starting from μm. no compounds were added to the control wells. the cells were infected with hiv- (media that contained ng of hiv- p was used per well) or mock. at hpi, celltox green express cytotoxicity reagent (ctxg, : dilution in the assay well) was added and the fluorescence was measured with a pherastar fs plate reader. the media was removed from the cells, the cells were lysed, and firefly luciferase activity was measured using the luciferase assay system (promega, madison, wi, usa) and pherastar fs plate reader. to determine the efficacy of compounds against rvfv, the fluorescent intensity of individual infectious cell foci was quantified. briefly, a cells ( × /well) were grown in -well black plates with transparent bottoms (greiner bio-one international). compounds were serially diluted in dmso in two-fold steps from μm to . μm and mixed with pfu of rrvfvΔnss::katushka virus in a total volume of μl medium (moi, . ). the final concentration of dmso in the assay was %. the growth medium was removed from the wells and μl of compound and virus mixture was added to the cells for each compound concentration. at hpi the medium was removed, and cells were fixed with % paraformaldehyde (pfa) for h and washed with phosphate-buffered saline (pbs). μl pbs was added to each well and the plate was analyzed in the trophos plate runner hd (trophos, roche group) to count the number of virus infected cells per well, by identifying all individual cells expressing the far-red fluorescent protein katushka (islam et al., ) . the toxicity of compounds was analysed using ctg assay. the half-maximal cytotoxic concentration (cc ) and the halfmaximal effective concentration (ec ) for each compound were calculated after non-linear regression analysis with a variable slope using graphpad prism software version . a (graphpad software la jolla, ca, usa) or using hill curve fit with sigmastat . software (spss inc., chicago, il, usa). the relative effectiveness of drugs were quantified as the selectivity indexes (si = cc /ec ). there is limited information available on approved, investigational and experimental bsas. to identify all potential bsas, we reviewed approved, investigational and experimental safe-in-human antiviral agents in drug bank, clinical trials websites and pubmed, respectively. first, we searched for drugs, which have been approved for treatment of viral diseases in human. by excluding vaccines and discontinued drugs, we identified active antiviral substances. these compounds target viral, human and unknown factors and inhibit viruses (table s ). the approved antivirals include neutralizing antibodies, interferons, antisense oligonucleotide, and small molecules. only of the molecules target viruses or host factors associated with ≥ viral diseases. fig. shows bsas and other approved antiviral drugs linked to viral and host targets through viruses they inhibit. next, we reviewed all investigational antivirals and their safety profiles. by excluding vaccines from the search parameters, we found active compounds. these compounds target viral, human and unknown factors and inhibit viruses (table s ). the drug candidates include neutralizing antibodies, antisense oligonucleotides, interferons, enzyme and small molecules. only of the small molecules target ≥ viral diseases. fig. shows these and other investigational antiviral agents linked to cellular and host factors through targeted viruses. next, we searched for drugs, which have been approved for treatment of non-viral diseases, but for which antiviral activity has been reported. we found active compounds, which are all small-molecules. these compounds target viral, human and unknown factors, and inhibit human viruses (table s ) . thirty-nine of the antiviral agents target ≥ viral diseases. fig. shows these and other experimental antiviral agents connected to their host and viral targets through viruses they inhibit. altogether, we identified antiviral agents with available safety information in humans. these agents target host, viral and unknown factors and inhibit different viruses, belonging to viral families. fifty-nine agents have bsa activity (table s ). these agents target of the reported viruses (except andv, emcv and rabv) (fig. s ). structural analysis of bsa revealed that several drugs (such as, acyclovir, famciclovir and valacyclovir; imatinib and disatinib; minocyclin and doxycycline; ritonavir and lopinavir; hydroxychloroquine and chloroquine; esomeprazole and omeprazole) have similar scaffolds (fig. s ) . these drugs target a limited number of host and viral factors (fig. s ). in particular, statins (fluvastatin, lovastatin and simvastatin) target human hmg-coa; dalbavancin, oritavancin, teicoplanin and telavancin target human cyp a ; acyclovir, valacyclovir, famciclovir, azacitidine, brincidofovir, cidofovir, foscarnet, trifluridine, and vidarabine inhibit viral dna polymerases; bcx , favipiravir and ribavirin inhibit viral rna polymerases; and lamivudine and tenofovir disoproxil inhibit viral reverse transcriptases. we hypothesized that approved, investigational and experimental antiviral agents, which target ≥ viral diseases, could inhibit other viral infections. as a proof of concept, we tested of the identified bsas against fluav, ev , chikv, rrv and hsv- infections in rpe cells (alisporivir, cyt , sunitinib and thymalfasin were excluded; table s ). we also tested of the bsas against zikv infection in rpe cells (alisporivir, cyt , sunitinib, thymalfasin, dalbavancin, pentosan polysulfate and rapamycin were excluded). we evaluated viability and death of virus/mock-infected cells using ctg and ctxg assays, respectively. the ctg assay quantifies atp, an indicator of metabolically active living cells, whereas ctxg assay uses fluorescent cyanine dye that stains the dna of dead cells. after initial screening we found several hits, which kept infected cells alive or rescued infected cells from virus-mediated death. these hits are gemcitabine, gefitinib and vibarabine (fluav); gemcitabine, pirlindole dibucaine, fluoxetine and dalbavancin (ev ); gemcitabine, imatinib, ivermectin, lopinavir, lovastatin, ezetimibe, fluoxetine, bcx , chloroquine and hydroxychloroquine (zikv); chloroquine and mycophenolic acid (chikv); chloroquine, mycophenolic acid, dibucaine and itraconazole (rrv); as well as -azacitidine, gemcitabine, trifluridine and vidarabine (hsv- ). we repeated the ctg and ctxg assays with selected compounds and titrated fluav, ev , zikv and hsv- produced in drug-treated and non-treated cells. the presence of chikv and rrv viruses in the media collected from non-or drug-treated rpe cells was evaluated by infecting fresh rpe cells and measuring reporter protein expression from viral promoter (nanoluciferase activity). these experiments confirmed antiviral activity of gemcitabine against fluav (fig. s ) ; gemcitabine, pirlindole, dibucaine, fluoxetine, and dalbavancin against ev (fig. s ) ; gemcitabine, lopinavir, ezetimibe, bcx , chloroquine, and hydroxychloroquine against zikv (fig. s ) ; chloroquine and mycophenolic acid against chikv (fig. s ) ; mycophenolic acid against rrv (fig. s ) ; and gemcitabine against hsv- (fig. s ) . importantly, dalbavancin and ezetimibe demonstrated novel antiviral activities against ev and zikv, respectively. in particular, they rescued infected rpe cells from virus-mediated cell death, and lowered production of infectious virus particles without detectable cytotoxicity (table ) . we also examined toxicity and antiviral activity of of the bsa agents (excluding alisporivir, cyt , sunitinib and thymalfasin) against hiv- infection in tzm-bl cells. our primary screen identified ezetimibe, minocycline and rapamycin as anti-hiv- agents. validation experiment confirmed all three hits (fig. s , table ). interestingly, ezetimibe is a novel inhibitor, whereas minocycline and rapamycin are known anti-hiv- agents (heredia et al., ; singh et al., ) . in addition, we examined antiviral activity and toxicity of of the bsa agents (alisporivir, cyt , sunitinib, thymalfasin, dalbavancin, pentosan polysulfate and rapamycin were excluded) against rvfv expressing the far-red fluorescent protein katushka in a cells. our screen identified azacitidine, bortezamibe, cyclosporine, doxycycline, ezetimibe, fluoxetine, gefitinibe, minocycline, oritavancin, ritonavir and topotecan. azacitidine, cyclosporine, minocycline, oritavancin, ritonavirand and bortezamib remained after excluding compounds with si < ( fig. s ; table ). interestingly, azacitidine, cyclosporine, minocycline, oritavancin and ritonavirand are novel, whereas bortezamib is a known inhibitor of rvfv infection (keck et al., ) . thus, we tested several known bsa agents against (−)ssrna, (+) ssrna, ssrna-rt and dsdna viruses and identified novel activities for dalbavancin against ev , ezetimibe against zikv and hiv- , as well as azacitidine, cyclosporine, minocycline, oritavancin and ritonavir against rvfv. fig. shows known, validated and novel interactions between bsas and viruses. several approved and investigational agents, as well as safe in man chemical probes, were discovered for potential treatment of various viral diseases (bekerman and einav, ; de clercq, ; de clercq and li, ) . re-purposing such therapeutics from one viral disease to another could save resources and time needed for development of novel drugs. in this study, we tested approved, investigational and experimental bsa agents against fluav, rvfv, ev , zikv, chikv, rrv, hiv- and hsv- in vitro. we identified novel antiviral activities for dalbavancin (against ev ), ezetimibe (against hiv- and zikv), azacitidine, cyclosporine, minocycline, oritavancin and ritonavir (against rvfv) (fig. ) . dalbavancin is a lipoglycopeptide antibiotic, which is approved by the fda for the treatment of acute bacterial skin infections caused by staphylococcus aureus and streptococcus pyogenes. dalbavancin also inhibits mers-cov and sars-cov infections. it binds cathepsin l in the late endosomes/lysosomes and blocks the entry of ebov (zhou et al., ) . our study demonstrates that it also inhibits replication of ev , which also enters the cells via an endocytic route (krieger et al., ) . ezetimibe is an fda-approved medication that lowers plasma cholesterol by decreasing its absorption in the small intestine. ezetimibe also inhibits hbv and hdv infections by impairing viral entry mediated by pres -specific receptor hntcp (blanchet et al., ; lempp and urban, ; monrroy-bravo et al., ) . our study shows that ezetimibe also inhibited hiv- and zikv infections. however, the entry of these viruses into the cell is mediated by other receptors (hamel et al., ; wilen et al., ) . most probably, the anti-hiv- , ev , as well as hcv action of ezetimibe is associated with depletion of cholesterol which is required for entry of these viruses into the host cells (sainz et al., ) . importantly, this drug was successfully tested in combination with antiretroviral therapeutics to lower cholesterol levels in serum of hiv-infected patients (saeedi et al., ; wohl et al., ) . the question remains whether ezetimibe alone reduces hiv titers in these patients. azacitidine is a chemical analogue of cytidine, which is used in the treatment of myelodysplastic syndrome. it also inhibits fluav, adv, hiv- and hiv- replication by blocking viral rna or dna synthesis (beach et al., ; rawson et al., ) . cyclosporine is an immunosuppressive agent used for the treatment of rheumatoid arthritis, psoriasis, crohn's disease, nephrotic syndrome and keratoconjunctivitis sicca. in addition, cyclosporine is used to prevent graft rejection in organ transplant recipients. cyclosporine also inhibits hcv, fluav, wnv and zikv replication through blocking interaction of cellular cyclophilins with viral proteins and attenuating viral rna synthesis (barrows et al., ; firpi et al., ; qing et al., ) . minocycline is a broad-spectrum antibiotic and antiviral agent, which possesses activity against denv, hiv- and wnv (leela et al., ; quick et al., ; singh et al., ) . oritavancin is a semisynthetic glycopeptide antibiotic used for the treatment of gram-positive bacterial skin infections. it also inhibits ebov, mers-cov, and sars-cov infections (zhou et al., ) . ritonavir is an antiretroviral medication. it has also antiviral activity against mers-cov (chan et al., ) . we showed that azacitidine, cyclosporine, minocycline, oritavancin and ritonavir are active against rvfv. we also confirmed previously reported activities of chloroquine against chikv and zikv, mycophenolic acid against chikv and rrv, fluoxetine, pirlindole and dibucaine against ev , bcx , lopinavir and hydroxichloroquine against zikv, as well as gemcitabine against ev , fluav, zikv and hsv- (cao et al., ; delogu and de lamballerie, ; delvecchio et al., ; denisova et al., ; julander et al., ; kang et al., ; khan et al., ; kuivanen et al., ; lee et al., ; soderholm et al., ; ulferts et al., ; yuan et al., ) (fig. ) . the number of compounds with novel and confirmed antiviral properties may have been higher if we had used other cell lines, other viruses and viral strains, concentration ranges and purity of compounds, as well as endpoint measurements. also testing other compounds, which target < viral diseases, could reveal novel antiviral properties of these compounds and increase the number of potential bsas. for conducting this properly, a harmonized antiviral bioactivity data annotation, standardization, curation, and intra-resource integration is needed. excellent antiviral profiles from cell-line-based assays might not be reflected in vivo because systemic mechanisms may compensate the blocked target effect. therefore, the identification of bsa targets that are essential for viral replication but redundant for the cell is critical for reducing putative toxicities associated with blocking cellular pathways. thus, follow-up mechanistic studies and in vivo experiments are needed to validate our in vitro results. in conclusion, repositioning of safe-in-man agents from one viral disease to another could play a pivotal role in development of broadly acting antivirals. our study demonstrated the potential value of such approach with some examples, such as dalbavancin, ezetimibe, azacitidine, cyclosporine, minocycline, oritavancin and ritonavir, confirming a principle that "rich becomes richer". effective bsa treatment may shortly become available, pending the results of further pre-clinical studies and clinical trials. the most effective and tolerable compounds will expand the available therapeutics for the treatment of viral diseases. some of these compounds could be used as first-line therapeutics to combat emerging and re-emerging viral threats, having global impact by improving preparedness and the protection of the general population from viral epidemics and pandemics. the authors declare no financial and non-financial competing interests. half-maximal cytotoxic concentration (cc ), the half-maximal effective concentration (ec ) and minimal selectivity indexes (si) for selected broad-spectrum antivirals. ctxg -celltox green express cytotoxicity assay, ctg -celltiter-glo viability assay, otherplaque assay or reporter gene expression assay, n.a.not available. fig. . the interaction network between viruses and bsas, which are safe in man. drug-like shapes represent antiviral agents. blue spheres represent viruses. the diameter of spheres corresponds to the number of interactions between the viruses and the drugs. novel interactions between bsas and viruses are shown in red, validatedin blue, and known -in grey. 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vitro and in vivo glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) this study was supported by the european regional development fund, the mobilitas pluss project mobtt (to denis e. kainov), jane and aatos erkko foundation grant # (to the vejo hukkanen). we thank qiuwei abdullah pan from the department of gastroenterology and hepatology, erasmus mc, university medical center rotterdam for advice. we thank ritva kajander for the hsv- culture. supplementary data related to this article can be found at http://dx. doi.org/ . /j.antiviral. . . . key: cord- -ic q ke authors: sun, ying; wang, zidao; tao, jiali; wang, yi; wu, andong; yang, ziwen; wang, kaimei; shi, liqiao; chen, yu; guo, deyin title: yeast-based assays for the high-throughput screening of inhibitors of coronavirus rna cap guanine-n -methyltransferase date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: ic q ke the ′-cap structure is a distinct feature of eukaryotic mrnas and is important for rna stability and protein translation by providing a molecular signature for the distinction of self or non-self mrna. eukaryotic viruses generally modify the ′-end of their rnas to mimic the cellular mrna structure, thereby facilitating viral replication in host cells. however, the molecular organization and biochemical mechanisms of the viral capping apparatus typically differ from its cellular counterpart, which makes viral capping enzymes attractive targets for drug discovery. our previous work showed that sars coronavirus (sars-cov) non-structural protein represents a structurally novel and unique guanine-n -methyltransferase (n -mtase) that is able to functionally complement yeast cellular n -mtase. in the present study, we developed a yeast-based system for identifying and screening inhibitors against coronavirus n -mtase using both -well and -well microtiter plates. the mtase inhibitors previously identified by in vitro biochemical assays were tested, and some, such as sinefungin, effectively suppressed n -mtase in the yeast system. however, other compounds, such as ata and adohcy, did not exert an inhibitory effect within a cellular context. these results validated the yeast assay system for inhibitor screening yet also demonstrated the difference between cell-based and in vitro biochemical assays. the yeast system was applied to the screening of natural product extracts, and three were observed to more potently inhibit the activity of coronavirus than human n -mtase. the cellular messenger rnas (mrnas) of higher eukaryotes and many viral rnas are sequentially methylated at the n- and -o positions of the -guanosine cap. the cap structure has several important biological roles, such as protecting mrna from degradation by - exoribonucleases (schwer et al., ) and directing pre-mrna splicing and mrna export from the nucleus (darnell, ) . in addition, the cap structure confers stability to mrnas and ensures their efficient recognition by translation initiation factor f for translation (filipowicz et al., ; schibler and perry, ) . in contrast, host and virus rna molecules with unprotected -ends are degraded in cytoplasmic compartments (liu and kiledjian, ) . uncapped rnas, such as nascent viral transcripts, may also be detected as ''non-self'' by the rna sensors rig-i, mda- , and ifit in host cells (abbas et al., ; bowzard et al., ; hornung et al., ; zust et al., ) , triggering antiviral innate immune responses through the production of interferon or ifit (interferon-induced protein)-mediated antiviral activity (daffis et al., ; nallagatla et al., ; rehwinkel et al., ) . the following four cap-forming enzymes are involved in the formation of the cap- structure. (i) rna triphosphatase hydrolyzes the -triphosphate of nascent pre-mrna to a -diphosphate. (ii) rna guanylyltransferase caps the diphosphate rna with gmp. (iii) rna guanine-n -methyltransferase (n -mtase) methylates the gpppn cap at the n position of guanine, resulting in the cap- structure (m gpppn) (shuman, ) . (iv) ribose -o-mtase further methylates the first nucleotide of higher eukaryotic cellular and viral mrnas at the ribose -oh position to form cap- (m gpppnm) structures (furuichi and shatkin, ) . both n and -o-mtases utilize s-adenosyl-l-methionine (adomet) as a methyl http://dx.doi.org/ . /j.antiviral. . . - /Ó elsevier b.v. all rights reserved. abbreviations: sars, severe acute respiratory syndrome; sars-cov, sars coronavirus; nsp, nonstructural protein; n -mtase, guanine-n -methyltransferase; -o-mtase, -o-methyltransferase; adomet, s-adenosyl-l-methionine; adohcy, s-adenosyl-l-homocysteine; ata, aurintricarboxylic acid; ic , inhibitory concentration at % activity. donor and generate s-adenosyl-l-homocysteine (adohcy) as a byproduct. eukaryotic viruses that replicate in the cytoplasm encode their own capping apparatus, and the structure and mechanisms of the viral rna capping apparatus are different from those of host cells (furuichi and shatkin, ) , which could be useful for the development of antiviral drugs. indeed, a number of biochemical and functional studies have previously addressed methyltransferases as potential inhibitor targets (chrebet et al., ; schwer et al., ; woyciniuk et al., ) . moreover, the enzymes involved in the coronavirus capping pathway are increasingly considered to be promising targets for potential anti-coronavirus drugs . coronaviruses (covs), infect many species of animals, including humans, and cause acute or chronic respiratory diseases (e.g., severe acute respiratory syndrome coronavirus [sars-cov] , middle east respiratory syndrome coronavirus [mers-cov] (de groot et al., ; zaki et al., ) , and infectious bronchitis virus [ibv] , enteric diseases (e.g., transmissible gastroenteritis virus [tgev]), and central nervous system (cns) diseases (murine hepatitis virus [mhv] ) (weiss and navas-martin, ) . covs are the largest rna viruses, are enveloped, and contain a single-stranded, positive-sense rna genome ranging from to . kb in length. the genome of sars-cov contains open reading frames (orfs) (snijder et al., ) and generates nonstructural proteins (nsps) produced by the autocatalytic processing of the polyprotein by viral proteases (ziebuhr et al., ) . covs replicate in the host cytoplasm and encode their own capping enzymes. among the four capping enzymes involved in coronavirus m gpppam-cap formation, guanine-n -methyltransferase (n -mtase) was identified as nsp in our previous work using a yeast genetic system (chen et al., ) , and -o-methyltransferase ( -o-mtase) is formed by nsp with nsp as a cofactor (chang et al., ; chen et al., ; decroly et al., ; lugari et al., ) . sars-coronavirus nsp was previously characterized as a -to -exoribonuclease (exon) (chen et al., ; minskaia et al., ) , and the n -mtase domain was mapped to the carboxy-terminal part of the protein (chen et al., ) . interestingly, the exon active site is dispensable, though the exon domain is required for n -mtase activity. the combination of the two functional domains (chen et al., (chen et al., , , a unique feature among all n -mtases, indicates that sars-cov n -mtase is a novel form of rna-processing enzyme and thus suggests it as an attractive target for the development of antiviral drugs. it has been shown that the capping functions in yeast cells can be replaced by the cap-forming enzymes of mammals or dna viruses (ho et al., ; saha et al., saha et al., , , and we previously found that coronavirus nsp could replace yeast cap n -mtase in vivo (chen et al., (chen et al., , . in the present study, we further developed the yeast genetic system as a high-throughput enzymatic activity assay platform of various n -mtases to identify coronavirus n -mtase inhibitors. the system was validated using mtase inhibitors previously identified by in vitro biochemical assays and then used for the screening of natural product extracts. the coding sequences of nonstructural protein of sars-cov, mhv, tgev, and ibv were pcr amplified from cdnas of the sars-cov whu strain (genbank accession no. ay ) (hussain et al., ) , mhv-a (genbank accession no. ay . ), tgev (genbank accession no. fj . ), and ibv (genbank accession no. aj . ) and inserted into the bamhi and xhoi sites of the yeast vector pmcek a ( lm, trp ) (chen et al., ) . the cdna of mers-cov nsp was chemically synthesized according to the deposited sequence of mers-cov (genbank accession no. kf . ) and cloned into pmcek a. for expression in e. coli cells, the coding sequences of sars and mhv nsp were inserted into the ndei and sali sites of pet a, and tgev nsp (kindly provided by dr. luis. enjuanes) was cloned into the bamhi and sali sites of petduet- . the plasmid for ibv expression (pdest -ibv-nsp ) was a kind gift from dr. eric j. snijder. e. coli bl (de ) cells (novagen) were separately transformed with the pet a-sars-nsp , pet a-mhv-nsp , pet-duet -tgev-nsp , and pdest -ibv-nsp plasmids. the cells were cultured at °c in l of lb medium supplemented with kanamycin ( lg/ml) or ampicillin ( lg/ml) until the culture density (a ) reached . - . and then induced with . mm isopropylb-d- -thiogalactopyranoside (iptg) for h at °c. the cells were harvested, and the recombinant proteins were purified by affinity chromatography using previously described protocols (chen et al., ) . the protein was concentrated by ultra-filtration (millipore), and the buffer was changed to mm tris-hcl (ph . ), mm nacl, % glycerol, and . mm dtt. the protein was stored at - °c until use. n -mtase activity assays were carried out in a -ll reaction mixture ( mm tris-hcl [ph . ], mm mgcl , mm dtt, units rnase inhibitor, . mm sam) with lci of s-adenosyl [methyl- h] methionine ( . ci/mmol, . mci/ml). the purified coronavirus nsp proteins were added at a final concentration of nm using . mm gpppa cap analog as the substrate. after incubation at °c for . h, the reaction was stopped by adding an equal volume of stop solution ( . % sds and mm edta). the samples were purified using deae-sephadex columns, and the methylation of the rna substrates was quantitated in counts per minute (cpm) using a scintillation counter (beckman coulter ls ). to measure the inhibitory effects of coronaviral n -mtase activity, adohcy, aurintricarboxylic acid (ata), ribavirin, and sinefungin were used at a maximum concentration of lm in the assays. typically, the enzyme and gpppa substrate were pre-incubated with each compound at room temperature (rt) for min, and the reactions were initiated by the addition of s-adenosyl [methyl- h] methionine. a control reaction was performed in the presence of % dmso instead of each tested compound. the ic (concentration of the drug to repress % of the n -mtase activity) value of sinefungin against sars-cov nsp was determined using graphpad prism . the data were adjusted to a logistic doseresponse function. the yeast n -mtase gene abd has been shown to be essential for yeast cell growth (mao et al., ) . saccharomyces cerevisiae strain ybs (mata leu ade trp his ura can abd ::hisgp -abd ) (mao et al., ) was transformed with yeast expression plasmids carrying coronavirus nsp and human n -mtase (pyx -hcm ) as a positive control (chen et al., ; saha et al., ) . trp+ transformants were selected at °c on an agar medium lacking tryptophan. the cells were then streaked onto an agar medium lacking tryptophan and containing . mg/ml of -fluoroorotic acid ( -foa) (sikorski and boeke, ) at °c for up to days to counter-select the ura + plasmid carrying the yeast abd gene. the formation of foa-resistant colonies indicated that the transformation of the mutants could replace or complement the endogenous cap n -methyltransferase function residing in the ura plasmid (chen et al., (chen et al., , . a solution of sinefungin (adenosylornithine, calbiochem - ), ribavirin ( -b-d-ribofuranosyl- , , -triazole- -carboxamide, sigma r- ), and adohcy (adenosine-homocysteine, sigma a- ) was prepared in h o or dmso (decroly et al., ; luzhkov et al., ; milani et al., ) . ata (aurintricarboxylic acid, sigma a- ) was dissolved in . mm naoh (decroly et al., ) . concentrations of the stock solutions were set to mm, and the compounds were stored at À °c. microtiter plates ( -or -well plates) were used for the yeast growth suppression test and inhibitor screening. a single transformed colony of the ybs strain containing plasmids expressing human n -mtase (mt-human), sars-cov n -mtase (mt-sars), n -mtases of other coronaviruses (mt-mhv, mt-tgev, and mt-ibv), and the pmcek a vector as control (representing the yeast n -mtase [mt-yeast]), were inoculated separately into ml of a basic medium (min sd base) lacking tryptophan and incubated at °c for - h until reaching a similar final cell density in the stationary phase ( . - .  cells/ml) (chrebet et al., ) . the starting cell density of the yeast-based assay was a = . and contained different inhibitor candidates or % dmso as a negative control. for high-throughput screening, lg/ml or lg/ml natural product extract was added into the -well or -well microtiter plates. the starting volumes for the -well and -well microtiter plates were ll and ll, respectively, and the final cell density (a ) was measured using a multifunctional microplate reader (tecan genios) after incubation for h at °c in a shaker. after incubation, yeast cells were collected and then ground in liquid nitrogen. total rna was isolated from cells using trizol reagent (invitrogen) and subjected to real-time pcr analysis to measure mrna expression. real-time pcr was performed using faststart universal sybr green mastermix (roche) and analyzed on real time pcr system (applied biosystems). gene-specific primer sequences are as follows: n -mtase of the mt-sars, forward: -gatcattactggtcttcatcctaca- and reverse: -aatc cacgcacgaacgtgacgaata- ; n -mtase of the mt-human, forward: -agctcaacagtggctgcccattaca- and reverse: -atc ggcaatatcagtacaaactagc- ; n -mtase of the mt-yeast and ybs , forward: -tgtcacaagaagactatgaccgtca- and reverse: -atggatctatttgcagtcatctcta- ; beta-actin, forward: -aagacaccaaggtatcatggtcgg- and reverse: -cggaag agtacaaggacaaaacggc- . the microbial natural product extracts were obtained from the microbial natural product library at hubei biopesticide engineering research centre (hberc) and consisted of purified secondary metabolites, semi-purified fractions and extracts from actinomycetes and fungi isolated from soil, lichen, fresh leaves, organic samples, and mushrooms using classical isolation methods. for the soil or lichen, mg was quantified and placed into a tube containing ml of sterilized sodium lauryl sulfate solution and shaken for min. the other samples were seeded aseptically and chopped into small pieces using a blender. the samples were then plated on two different agar media and incubated at °c for - days. the actinomyces and fungi were isolated to pure culture and incubated at °c. the microbial isolates were morphologically characterized, stored and archived. the ratio of actinomycetes/fungi in the collection of microorganisms at hberc was around . : . the microbial identity will be further characterized by classical and molecular methodology when active compounds are identified from the corresponding microbial extracts. for preparation of the microbial extracts, each microbial isolate was fermented in ml fermentation medium and the resulting cultures were extracted with ethyl acetate. for making the working solution of the microbial extracts, the extracts were dissolved in ethanol to a concentration of mg/ml (w/v). in total, microbial culture extracts were used for mtase inhibitor screening in this study, which were prepared from actinomycetes and fungi. our previous study showed that sars-cov nsp possesses n -mtase activity that could substitute for cellular n -mtase in yeast (chen et al., (chen et al., , . in this system, the gene abd encoding yeast n -mtase is knocked out from the yeast genome and complemented by expression of abd from a plasmid with a ura selective marker, resulting in yeast strain ybs , whereas the exogenous n -mtase is expressed from another plasmid with a trp selective marker. the expression of a functional ura gene encoding orotine- -monophosphate decarboxylase results in the conversion of the nontoxic -foa compound to toxic -flurouracil in yeast. in a medium containing -foa, which counter-selects ura -expressing cells, the yeast cells can grow normally only when the exogenous protein can replace the function of the cellular n -mtase (fig. a) . in this study, we confirmed that sars-cov and human n -mtase could rescue the growth of yeast cells that were deficient in n -mtase. we further tested whether this n -mtase activity is universal for nsp from other coronaviruses. as shown in fig. b , nsp from other coronaviruses, including tgev (group ), mhv (group a), sars-cov (group b), mers-cov (group c), and ibv (group ), showed n -mtase activity that was functional in vivo by complementing the endogenous yeast enzyme. these results indicate that n -mtase is well conserved among different coronaviruses and that nsp appears to be a target for the development of a universal inhibitor against coronaviruses. we then generated three yeast strains that grew dependently on the n -mtase activity from sars-cov (mt-sars), humans (mt-human), and yeast (mt-yeast). to compare the expression levels of these n -mtases, we analyzed the mrna levels of the n -mtases using real-time pcr. as shown in fig. c , there are no significant differences between the mrna levels of mt-sars and mt-human, which are under the control of the same yeast tpi promoter. the mrna of mtyeast, which was transformed with pmcek a vector as control, is similar with yeast train ybs , because the mrnas of yeast n -mtases are transcribed from the complementary plasmids (ura ) bearing the yeast-mt. in the strains with mt-sars and mt-human, the expression of yeast n -mtase could not be detected (data not shown). as the expression of mt-sars and mt-human is at the similar level, these yeast strains could be used to identify the inhibitors that inhibit sars-cov mtase more potently than human mtase. however, the mrna levels of mt-sars and mt-human are four times higher than that of mt-yeast, the latter being expressed from a low-copy-number plasmid with centromeric element (cen), the inhibitory activity on sars-cov and yeast mtases cannot be directly compared. to establish a drug screening system using these yeast strains, we first tested whether the expression of viral or human n -mtase influenced yeast growth. individual colonies of mt-yeast, mt-human, and mt-sars were cultured in a medium lacking tryptophan, and the pre-cultures were diluted with fresh medium to an a = . . a -ll aliquot of the culture was incubated in each well of two -well plates, one of which was used for measuring yeast propagation by spectrophotometry, whereas the other plate was used for the direct counting of the cell number. measurements were performed at -h intervals over h. as shown in fig. d and e, all three yeast strains (mt-sars, mt-human, and mt-yeast) showed similar growth curves within h, indicating that both the sars-cov and human mtases could support the normal growth of yeast cells. at such a growth condition, starting with a liquid culture at a = . , the time period between and h corresponded to the logarithmic growth phase (fig. d and e) . during this growth phase, the optical density (a ) was well correlated with the cell number as demonstrated for the yeast strain mt-sars by linear regression analysis (fig. f ). in the linear regression equation, y = . x À . (r = . , r is regressive coefficient), it shows that there is a correlativity between the a of culture liquid and cell number in the logarithmic phase ( - h) of mt-sars when the least square method was used to evaluate the interrelationships between the two variables (x and y), where x represents the spectrophotometric measurement (a ) and y represents the corresponding cell number in the logarithmic phase ( - h) of mt-sars. the linear regression equation for the mt-sars strain, y = . x À . (r = . , r is regressive coefficient), was obtained through normative analysis in least square method with excel program. in this linear regression equation, there is a correlativity between the a of culture liquid and cell number in the logarithmic phase ( - h) of mt-sars when the r > . . the r > . . similarly, there was also a high correlativity between the two optical absorbance and the cell number for strains mt-yeast and mt-human, with linear regression equations y = . x À . (r = . ) and y = . x À . (r = . ), respectively. as spectrophotometric measurements can facilitate rapid and high-throughput evaluation of microtiter plates, the optical density of the cell culture was applied to measure yeast growth in the ensuing experiments. to validate the yeast-based system for identifying and screening inhibitors against viral n -mtases, we tested previously reported mtase inhibitors, including adohcy, sinefungin, aurintricarboxylic acid (ata), and ribavirin . we first detected the n -mtase activities of coronaviruses nsp in vitro when incubated with different compounds at lm using biochemical assays. as shown in fig. a , sinefungin effectively inhibits the activities of all four n -mtases and adohcy and ata weakly inhibit the n -mtase activities of sars, mhv and tgev with approximately - % inhibition at lm, except for ibv with - % inhibition. in contrast, ribavirin, which is a guanosine (ribonucleic) analog used to interfere with viral rna synthesis and viral mrna capping of many different viruses (kim and lee, ; scholtissek, ; zhao et al., ) , did not inhibited the coronavirus n -mtases. the doseresponse curve of sinefungin, which was the best inhibitor of the three effective compounds, showed an ic value of sinefungin against sars-cov nsp of . ± . nm (fig. b) . sinefungin could widely inhibit the activities of mtases from diverse sources including viruses and yeast. for example, the ic values of sinefungin for n -mtase and o-mtases of vaccinia virus in vitro are . and . nm, respectively (pugh et al., ) , and the ic values for o-mtase (ns mtase) activity of dengue virus in vitro are nm (li et al., ) and nm . the ic values of sinefungin for yeast n -mtase are nm in vivo (chrebet et al., ) and nm in vitro (zheng et al., ) , respectively. it was reported previously that the ic values of adohcy are and . mm for n -mtase and o-mtases activities of vaccinia virus in vitro, respectively (pugh and borchardt, ) . in this study, the inhibitory effects of sinefungin, adohcy and ata on mtases were consistent with that of previous reports. as sinefungin, adohcy and ata were effective inhibitors of coronaviruses n -mtases in biochemical assays, they were used to validate the yeast-based assay system established in this study (fig. ) . the yeast strains mt-yeast, mt-human, and mt-sars were incubated with lm of each compound, and the cell growth was measured at -h intervals over a -h period. remarkably, sinefungin significantly repressed the growth of all three yeast strains (fig. ) , confirming the previous report of sinefungin as a potent inhibitor (chrebet et al., ; li et al., ; pugh et al., ) . in contrast, ata, adohcy, and ribavirin did not exhibit inhibitory activities against the three yeast strains. because ado-hcy and ata could inhibit the activity of coronavirus n -mtase in biochemical assays, the results showed a clear difference between in vitro biochemical assays and in vivo cell-based assays, indicating that n -mtase inhibitors identified using in vitro biochemical assays may not necessarily be effective within a cellular context. such discrepancy may be attributed to the low membrane permeability to adohcy and ata or that the ic of adohcy and ata are far greater than lm. indeed, sinefungin has been reported to be actively transported across yeast membranes, whilst adohcy and ata are not (zheng et al., (zheng et al., , . sinefungin, which is produced by streptomyces, is a natural sulfur-modified analog of adohcy, a well-characterized, nonspecific mtase inhibitor. as shown in fig. , sinefungin exhibited a similar inhibitory effect on all three yeast strains at high concentration ( lm). however, stronger inhibitory effect on mt-yeast was observed than that on mt-human and the strains harboring the mtases of other coronaviruses at low concentration ( . lm) (fig. a and b). this could be explained by intrinsic difference of inhibitory effect to different mtases or the difference of the expression levels of the mtases as we showed that higher mrna levels of mt-human and mtases of coronaviruses were expressed than that of mt-yeast (fig. c) . to further characterize sinefungin as an mtase inhibitor in the yeast-based system, yeast strain ybs , expressing different n -mtases, was incubated with sinefungin at different concentration for h (fig. c and d) . the ic (n = , mean values ± sd) of mt-yeast, mt-human and mtases of coronaviruses were calculated and shown in table . these results indicate that sinefungin is a broad-spectrum inhibitor against various species of n -mtases, including those from yeasts, humans, and coronaviruses and demonstrate that the yeast-based system could be used for analysis of n -mtase inhibitors. however, sinefungin is not an ideal antiviral inhibitor due to its lack of specificity on coronavirus n -mtases compared to yeast and human mtases. therefore, it is still necessary to screen for specific viral n -mtases inhibitors, an effort that will benefit the functional study of viral mtases and clinical drug development. . . screening for effective microbial natural products using the yeastbased assay as the three yeast strains, mt-yeast, mt-human, and mt-sars, have the same genetic background and their growth depends on yeast, human, and sars-cov mtases, respectively, the yeast-based system described above can be adopted to screen specific inhibitors against coronavirus n -mtase. it is of note that the mrna levels of mt-sars and mt-human are similar but they are higher than that of mt-yeast (fig. c) . however, this would not influence the screening for inhibitors with more specific inhibition on sars-cov mtase over human mtase. for the screening of inhibitors to sars-cov n -mtase, over microbial natural product extracts were tested in the yeastbased system. the microbial extracts were prepared from liquid culture of actinomycetes and fungi as described in the materials and methods section. the overnight cultures of mt-yeast, mt-human, and mt-sars were diluted to a = . , and a -ll aliquot of each cell culture was seeded into -well microtiter plates in the presence of lg/ml of microbial natural product extract. twenty hours post-incubation, the cell density was monitored by measuring the optical density at a . in the first round, the extracts that did not repress mt-human were selected for further testing. the primary candidate extracts were tested twice for reproducibility and inhibitory effects using independent samples of each extract. in summary, we obtained three candidates extracts, pf (natural product extract from a fungus species, preliminarily characterized as penicillium spp.), pa and pa (natural product extracts from actinomycetes, preliminarily characterized as streptomyces spp.), which could potently inhibit the growth of mt-sars and mt-yeast but significantly less effectively inhibit mt-human (fig. ) . the inhibition ratios of the extracts on mt-sars to mt-human were . , , and , respectively. in contrast, sinefungin suppressed the growth of all yeast strains at a final concentration of lm ( . lg/ml). the fungal and actinomyces natural product extracts used in this study were composed of complex ingredients, and these three extracts could be used for the isolation of the active ingredients in future work. taken together, the yeast-based assay system could provide a platform for screening n -mtase inhibitors that are more effective against viral mtases and less effective against human mtases. efficient drug development often depends on high-throughput screening, which offers rapid and sensitive data acquisition coupled with high-content analyses. to explore the possibility of miniaturizing the yeast-based assay and to increase its throughput scale, yeast cells were seeded in -well microtiter plates, and the growth of the mt-sars yeast cells was monitored at -h intervals over h at °c in a shaker ( fig. a and b) . during the logarithmic growth phase ( - h), the optical density of the culture at a was well correlated with the cell number ( fig. a-c) . the following linear regression equation was established for the mt-sars strain: y = . x À . (r = . ), indicating that they are more correlative in -well microtiter plates than -well microtiter plates (fig. c) . to validate the yeast-based system for the highthroughput screening of inhibitors against n -mtases, we performed a growth suppression assay and measured the ic of sinefungin against the yeast strains that had been tested in the -well format ( fig. d and e) . the ic values (n = , mean values ± sd) for sinefungin against mt-yeast, mt-human, mt-sars, mt-mhv, mt-tgev, and mt-ibv were . ± . nm, ± . nm, ± . nm, ± . nm, ± . nm, and ± . nm, respectively (table ) , which were highly consistent with that of the -well microtiter plates (figs. and and table ). these results indicate that the yeast-based high-throughput screening system could be used for the discovery of specific antiviral inhibitors. in comparison to the -well microplate assays, the -well assay system was more reliable, and cost effective. many coronaviruses cause severe human and animal diseases. recently, a novel sars-like coronavirus, the middle east respiratory syndrome coronavirus (mers-cov) belonging to the genus betacoronavirus, was identified in saudi arabia and subsequently spread to the united kingdom (uk), germany, and france (al-tawfiq, ; lim et al., ; wiwanitkit, ) . as there are no clinically approved antiviral therapies, including effective antiviral drugs, available to date for the treatment of coronavirusassociated diseases, the establishment of a high-throughput antiviral inhibitor screening system is important for the development of novel anti-coronavirus drugs. a the numbers represent the mean ± standard deviations (sd) with n = . fig. . the inhibitory effects of microbial natural product extracts screened using the yeast-based system. sinefungin as an n -mtase inhibitor was used as a positive control, and yeast culture in the absence of extracted compounds was used as the mock control. pf , pa , and pa represent the three microbial natural product extracts. the growth suppression in percentage was calculated as [(x À y)/x]  % where x is the a value of the mock culture and y is the a value in the presence of sinefungin or microbial natural product extracts. the experiments were repeated three times. the results are shown as the percent suppression of cell growth of yeast strains mt-sars, mt-human, and mt-yeast. although adohcy, ata and sinefungin, were previously reported to be inhibitors of coronavirus rna mtases in vitro , only sinefungin significantly suppressed the growth of the mt-yeast, mt-human, and mt-sars yeast cells (fig. ). among these three strains, sinefungin was more effective against mt-yeast than mt-sars (fig. ) . thus, the observed differences between in vitro biochemical assays and yeast-based assays indicate that the inhibitors identified by an in vitro biochemical assay may not be effective in cells. as the yeast cell-based assay system is more similar to host cells with regard to the complexity of chemical composition than an in vitro biochemical assay, the former may have advantages over the latter for the identification of antiviral inhibitors that can function effectively in living cells. although the yeast-based n -mtase assay system was validated by testing previously identified inhibitors, no specific inhibitors against coronavirus n -mtases were identified. as the yeast-based system developed in this study included three yeast strains carrying yeast, human, or coronavirus n -mtase activity, this method could be used to screen and identify inhibitors that are specific against coronaviruses and not (or significantly less effective) to human n -mtase. in addition, the structural and mechanistic differences among coronavirus, yeast, and human mtases also provide the rationale for developing specific inhibitors. accordingly, we exploited these differences and screened over natural product extracts. interestingly, three extracts showed more potent inhibitory effects on sars-cov and yeast n -mtase than human n 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proteolytic processing in the nidovirales ribose -o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda we thank dr. s. shuman for kindly providing the yeast plasmids and strains and dr. eric j. snijder for the ibv-nsp e. coli expression plasmid. we are grateful to dr. luis. enjuanes for providing the tgev cdna. this study was supported by the china '' '' basic research program ( cb and cb ), china nsfc grants ( , , , and ) and china specialized research fund for the doctoral program of higher education ( ). d.g. is supported by hubei province' s outstanding medical academic leader program. key: cord- - yimrpaz authors: nicholls, john m.; moss, ronald b.; haslam, stuart m. title: the use of sialidase therapy for respiratory viral infections date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: yimrpaz das is an inhaled bacterial sialidase which functions by removing sialic acid (sia) from the surface of epithelial cells, preventing attachment and subsequent infection by respiratory viruses that utilize sia as a receptor. das is typical of bacterial sialidases in cleaving sia α - and sia α - linkages, and it also has a demonstrated effect against acetylated and hydroxylated forms of sia. the potency of the compound has been enhanced by coupling the active sialidase with an amphiregulin tag, allowing a longer duration of action and minimizing spread to the systemic circulation. das is now in phase ii development for the treatment of influenza, and it has also demonstrated activity in individual cases of parainfluenza in immunosuppressed patients. continued evaluation of the roles and activities of bacterial sialidases is required to expand the range of successful antiviral therapies targeting sia or its derivatives. -an interaction involving viral polypeptides or attachment proteins and cognate cell-surface receptors. das is the first antiviral compound in phase ii development that functions by blocking this pathogen-host interaction, by destroying the influenza host-cell receptor, sialic acid (sia), on the surface of respiratory epithelial cells. in this paper, we provide background information on sia and sialidases; discuss the potential role of bacterial sialidases as antiviral agents; review the in vitro and phase ii evaluation of das for the treatment of influenza; and note evidence that the drug would also be useful against parainfluenza virus infections. current antiviral strategies focus on prevention of viral replication within the cell or inhibition of release of newly formed virions. neuraminidase (na) inhibitors primarily act at this step, but they play a minor role in modulating viral entry (matrosovich et al., ) . the first step of viral binding often involves close contact between the viral attachment protein and host receptor. before the development of das , studies in the s demonstrated that natural compounds present in serum, in particular alpha -macroglobulin, inhibited virus adsorption to surfaces (hanaoka et al., ; . later studies identified sia as being the key determinant of binding for these compounds (pritchett et al., ; pritchett and paulson, ). lentz proposed a number of strategies to prevent cell entry by blocking the host receptor (lentz, (lentz, , , but these were mainly directed at developing an antibody-mediated approach, rather than receptor destruction. in , weis and colleague used x-ray crystallography to map the haemagglutinin-sia interaction (weis et al., ) . with this structure solved, it was envisioned that it might be possible to develop synthetic analogs of sia to block binding (pritchett et al., ; pritchett and paulson, ) , though it was acknowledged that such drug design would have a number of structural obstacles (weis et al., ) . this route of anti-influenza therapy was not pursued in as much detail as blocking the viral neuraminidase site, but there have been two recent reports on developing drug design by blocking the ha binding site, using databases of commercially available compounds (zinc) nandi, ) and other techniques (al-qattan and mordi, a,b; blessia et al., ) . in , springer and ansell found that influenza viruses and bacteria had a common enzyme property: the ability to remove recep-tors from red blood cells and prevent haemagglutination (springer and ansell, ) . to investigate this requires an understanding of the nature of sia, its presence in the respiratory tract and the structure and function of bacterial sialidase. sialic acid is the term used for a family of nine-carbon monoscaccharides found in animals and certain bacteria (chen and varki, ) . they are typically found at the terminal portions of oligosaccharide chains attached to proteins and lipids to form glycoproteins and glycolipids, respectively. there are over naturally occurring variants of these nine-carbon keto-sugars based on a neuraminic acid (neu) or a -keto- -deoxynononic acid (kdn) backbone (schauer, ; varki, ) . the discovery of neuraminic acid by klenk and colleagues has been reviewed by schaeur (schauer, ) with the term sialic acid proposed by blix, gottschalk and klenk based on the greek word sialion (salivea) as these carbohydrates were isolated from the mucin of submaxillary glands. . . sialic acid: structure and function . . . sialic acid structure the diversity of sia present in nature is due to the substitution at the - carbons by o-acetyl, sulfate, methyl and lactate groups and by glycosidic linkage by the anomeric c to the or hydroxyl group of galactose (gal) (referred to as the a - or a - ), n-acetyl galactosamine (galnac) or n-acetyl glucosamine (glcnac) (varki and varki, ) (fig. ) . eleven different types of sialic acid have been identified on the human erythrocyte membrane, using gas chromatography and mass spectrometry (bulai et al., ) . galnac is typically present on o-glycans, and glcnac is normally a component of n-glycans. the binding of sia to gal or galnac requires a family of enzymes called sialyltransferases (st), with each st producing a specific linkage (harduin-lepers et al., ; paulson and rademacher, ; xia et al., ) . in some instances, multiple sia may be linked together to form polysialic acid, most commonly in a a - configuration (hildebrandt et al., ) . the linkage of sia to the adjacent sugar is in an a configuration, which means the bond is acid-labile and is readily destroyed by enzymes known as sialidases. it should be noted that in solution, unbound sia is mainly in the ß-configuration, with the c hydroxyl positioned axial to the ring and in equilibrium with the minor a-form (vimr, ) . the acidic nature of sia is due to the carboxylate at c , which is ionized at physiological ph and has a pk of . - . , giving the sia a number of useful features, as highlighted by schauer (schauer, fig. . structures of different sialic acid derivatives. the numbering of carbons is listed in green; aco refers to acetlylation. two common linkages of sia with galactose to form sialyllactose (siaa - galß - glu) and sialyllactose (siaa - galß - glu) are shown. ) and vimr (vimr et al., ) . the negatively-charged glycoproteins will mutually repel each other, allowing trapping of water molecules to produce a highly viscous aqueous solution that comprises the glycocalyx of many epithelial surfaces. the importance of this shield was highlighted by varki and varki, who indicated that > % of the mammalian genome encodes proteins involved in this glycosylation pathway (lehmann et al., ; varki and varki, ) . the high sia content of mucin acts as a lubricant for epithelial surfaces in the cornea, nasal mucosa, respiratory and gastrointestinal tracts and other tissues in contact with the environment. this mucin protects the cells against mechanical and chemical damage, such as gastric acid and digestive enzymes. the sia content of mucin also functions as a decoy for pathogens that bind to sia, such as respiratory viruses (lehmann et al., ) . the mucin is produced by goblet cells and normally is a benefit to the host (nicholls et al., ) . however, in certain diseases such as cystic fibrosis, changes in mucin composition allow increased bacterial growth and pathogenesis (xia et al., ) . the second main function of sia is to enable host proteins avoid and evade recognition by the body's defense mechanisms. the capping of terminal galactose by sia masks the recognition of certain oligosaccharides by the asialoglycoprotein receptor of the liver (grewal et al., ) or similar receptors present on macrophages. the sialylation of compounds allows plasma macromolecules to have a longer period of time in the circulation; the number of glycosylation sites is related to biological functions (banks, ; sun et al., ) . conversely, the half-life of many serum glycoproteins such as thyrotropin, erythropoietin, and follicle stimulating hormone is decreased upon desialylation. glycosylation of proteins as a shield to prevent recognition is not limited to host factors, but is also employed by pathogens such as the influenza viruses, leading to the development of antigenic variation (sun et al., ) . finally, the presence of sia on glycans may also modulate the immune response, through recognition by sia-binding lectins, now known as siglecs. the first siglec to be identified, sialoadhesin, was found on macrophages (o'neill et al., ) , but more than different siglec molecules have since been discovered (reviewed in varki and gagneux ( ) ). as mentioned previously, sia can undergo modification, and the two most common changes are acetylation and hydroxylation to glycolylneuraminic acid (neu gc), a variant present in many animal species but not in humans. in the former situation, this occurs at c and c - (cao et al., ; schauer et al., ) with identification of the enzyme sialate-o-acetyltransferase as a candidate recently identified (arming et al., ) . modifications of sia are important to consider, as these changes, in particular o-acetylation, may have an effect on enzymatic cleavage by sialidases. schauer demonstrated that the position of acetylation has an effect on sialidase activity: an o-acetyl group on c reduces cleavage by % and at c by about % (schauer, ) . bacteria and some viruses (notably influenza c virus and coronaviruses) have circumvented this protective mechanism through the development of esterases located in the cytosol or in lysozymes. although influenza c virus appeared to lack sialidase activity, it was still able to agglutinate red blood cells from certain species, which led to the finding of an esterase combined with the haemagglutinin and fusion protein to form the hef that is present on influenza c and some coronaviruses (martin et al., ) . o-acetylation is one of the most common modification of sialic acids, and it was initially thought to be a species-specific determinant (klein and roussel, ) . the tissue localization of the -oacetylation variant has been investigated by the use of specific monoclonal antibodies (argueso and sumiyoshi, ) , or binding by influenza c virus or its recombinant soluble form, coupled with the fc portion of human immunoglobulin (che-fcd) (mandal et al., ) . these studies showed that o-acetylation of sialic acid in humans appears localized to the suprabasal plasma membranes of cells exposed to a ''wet'' surface, such as the cornea, conjunctiva, laryngeal and vaginal epithelium. there is a variation in the degree of o-acetylation between ethnic backgrounds, depending on whether individuals are slow or fast acetylators. apart from the sialidase from arthrobacter urefaciens, actinomyces viscosus and to a lesser extent the sialidase from newcastle disease virus, other sialidases are apparently not effective at cleaving the terminal sialic acid. the o-acetylation inhibits sialidase sterically, and is not competitive. thus, o-acetylation of mucins can protect against the action of bacterial sialidases. this o-acetlyation is conspicuous on the terminal aspect of muc , a glycoprotein with a long extracellular domain that prevents infection of the intact epithelium. the other common modification is the addition of an oxygen to the c to form n-glycolylneuraminic acid (neu gc), through the action of the enzyme cytidine monophosphate n-acetylneuraminic hydroxylase (cmah). many studies by ajit varki and colleagues have shown a high expression of this enzyme in many animal species. humans lack cmah activity and the only source of neu gc is from the diet. this additional oxygen is important from a sialidase point of view in that many bacterial and viral sialidases preferably cleave neu ac over neu gc in the a - and a - configuration (davies et al., ) . viral sialidases from newcastle disease virus, fowl plague and most influenza viruses also show poor cleavage of neu gc over neu ac. molecular dynamic studies suggest that there is a conformational change induced by the extra oxygen that may explain this decreased activity. if bacterial sialidases are to be used as potential therapeutic agents, these two changes of o-acetylation and neu gc will have practical implications, as the agent will have to be one that is able to break through the mucin barrier to cleave sialic acid on the surface of epithelial cells. in the case of enzymatic cleavage of neu gc, this enzymatic cleavage is unlikely to be a major concern, because of the lack of cmah enzyme in humans, and thus the mucin is exclusively neu ac. however, with o-acetylation, this enzymatic cleavage may be of concern if topical therapy is to be used in regions of the body where there is a high degree of o-acetylation of non-keratinized stratified squamous epithelium, as is present in the eye and larynx. in addition to recognition by siglecs, sia also functions as a receptor for a number of respiratory viral agents, some of which were discovered by their ability to cause clumping or agglutination of red blood cells in vitro (haemagglutination). as viruses replicate intracellularly, it follows logically that successful attachment to the cell requires a matching up of the receptor with the pathogen. this is often a two-step process: first, a low-affinity ''browsing'', followed by a higher-affinity interaction with the virus, and more specific receptor interactions (helander et al., ) . an extensive list of viruses that utilize sia and which may infect the respiratory tract has been reviewed by lehmann and colleagues (lehmann et al., ) . even though influenza virus has been the most well characterized of the pathogens studied, it must be noted that other viruses, including cytomegalovirus (taylor and cooper, ) , rhinovirus (blomqvist et al., ) , mumps urabe am (reyes- leyva et al., ) and the paramyxoviruses all utilize sia (suzuki et al., ) (paulson et al., ) , suggesting that sialidase treatment may potentially be useful for these infections. bacteria are able to produce sia both through de novo synthesis and by using sialyl precursors scavenged from animal hosts. the syn-thesis of sia in bacteria is limited to a few, mostly pathogenic species that incorporate sia into surface structures, such as the capsular polysialic acid found in neisseria meningitidis and escherichia coli k (plumbridge and vimr, ) . the sia that is incorporated or synthesized is used as a growth factor or a source of carbon (kim et al., ; stafford et al., ; vimr et al., ) . bacteria that utilize sia include neisseria, haemophilus, bacteroides, fusobacteria and streptococci. to achieve this scavenger effect, a bacterium first require a sialidase to remove the sia from the host environment, then enzymes for catabolism, followed by uptake into the bacterium for utilization. the latter two steps will not be elucidated further, but an excellent review on these processes is available (vimr et al., ) . another outcome of the sialylation of bacterial surfaces is similar to that mentioned above for viruses, in that they are able to avoid recognition by the host defense mechanisms, as sialylation results in recognition as ''self'' rather than ''foreign''. over microorganisms have sialidase activity, and several bacteria such as streptococcus pneumoniae produce more than one sialidase (nana, nanb, and nanc) (gut et al., ) . the sialidases of s. pneumoniae may expose host receptors and desialylate host glycoproteins such as lactoferrin and immunoglobulin that clear bacteria. they may also lead to the desialylation of the lipopolysaccharides of other bacteria such as n. meningitidis and haemophilus influenza, giving the pneumococcus a competitive growth advantage (pettigrew et al., ) . the nana is cell surface-anchored, and functions in adherence. it may also provide a carbon source for the bacterium. similar to many other bacterial sialidases, nanb is secreted as an extracellular enzyme and implicated in the severity of pneumococcal infection (manco et al., ) . desialylation may also involve the respiratory epithelium, and there have been previous concerns as to whether the desialylation of an epithelium by bacterial or viral sialidases may lead to increased bacterial adherence and superinfection. it has been well recognized that patients with influenza are susceptible to secondary bacterial infection (van der sluijs et al., ) , and the high mortality of the influenza pandemic has been attributed to secondary infections (brundage and shanks, ) . if sialidase is to be used as a prophylactic or therapeutic strategy, there have therefore been concerns that this may lead to increased bacterial adherence and secondary bacterial pneumonia (zhang, ) . in reality, increased adherence is due to epithelial necrosis and exposure of the basement membranes as a result of viral-induced cell death (plotkowski et al., ) , rather than surface-induced desialylation, which was not associated with epithelial cell death (nicholls et al., ) . although sequence homologies among the different bacterial sialidases are less than %, there are unique motifs and conserved regions in the sialidase domains. the catalytic domain of clostridium perfringens sialidase has been determined by nmr, and shares key features with other bacterial sialidases (newstead et al., ) . these include a cluster of three arginines that interact with the carboxylate of c , an acid/base catabolic asp and a hydrophobic pocket to accommodate the n-acetyl group. three water molecules are involved to bridge the side chains and ligands. despite many overall similarities in structure, each bacterial sialidase displays a preference for a different linkage, in a manner that is not clearly related to the bacterial species. the range and diversity of bacterial sialidase specificities was initially studied and characterized by schauer in the early s (corfield et al., ; corfield et al., ) . for instance, bacteroides fragilis stb prefers the a - to the a - / linkage, but b. fragilis appears quite flexible in its preference. it is even possible for the same bacteria to have different specificities: the nana of s. pneumonia can cleave a - , a - or a - linkages, but the extracellular nanb prefers a substrate with the a - linkage. the broad range of specificities has been attributed to a number of mechanisms, including the configuration of the active site and the role of water molecules as nucleophiles. the crystal structure of nanb has been used to demonstrate a - cleavage specificity, proposing that an additional rim of residues above the arginine triad may only allow an a - configuration to bind (gut et al., ) . because sia are the primary recognition sites for many viral respiratory pathogens, it would seem appropriate that a bacterial sialidase could be used as an inhibitory agent to cleave sia from the surface receptor, and thus block binding. achieving this would require a number of properties. the first is that an effective sialidase would need to have a broad catalytic activity, because with agents such as influenza virus, binding is dependent on the linkage between the sia and galactose. the first evidence of this in influenza was demonstrated by rogers and paulson, who desialylated red blood cells, then re-sialylated them using specific sialyltransferases that were either a - or a - linked . avian or equine viruses agglutinated the former, while human and swine viruses agglutinated the latter. from these studies the paradigm emerged that avian viruses were primarily a - binding and human and swine viruses were a - binding (wilks et al., ) . as ducks and waterfowl contain mainly a - terminated sia in their intestines, there would be little selection pressure for avian viruses to acquire a - binding, and cross-species transmission would require a number of mutations. in ferrets, for example, up to nine passages with a high inoculum has been required for adaptation of an h n virus to the avian configuration (sorrell et al., ) . recent studies in the usa (imai et al., ) and the netherlands (herfst et al., ) have shown that h n undergoes natural purifying or negative selection, rather than a diversifying positive selection, and that multiple introduced mutations are required for avian viruses to develop greater affinity for a - receptors and to replicate in the ferret upper respiratory tract, leading to transmission. lectin binding studies have shown that a - and a - n-linked sialic acids are distributed throughout the upper and lower respiratory tract in humans, with an increased distribution of a - o-linked sialic acids in the lower respiratory tract (nicholls et al., ) . if a sialidase was to be used to prevent influenza in humans, it would therefore have to be one that cleaves both types of linkages present in the upper and lower airways. to be used as therapy for respiratory tract infections, a sialidase also have to be effective as a topical or inhalational agent. it was mentioned previously that one of the properties of sia is to protect ''self'' glycoproteins from being recognized as ''foreign''. if a sialidase were administered systemically, this could lead to widespread desialylation of glycoproteins and suppression of the siglec pathways. for instance, since erythrocytes are removed from the circulation once they are desialylated, it has been proposed that systemic sialidase treatment might result in widespread erythrophagocytosis (biondi et al., ; bratosin et al., ) . furthermore, as many viral respiratory pathogens infect both the upper and lower respiratory tract, including the alveoli, the sialidase would need to be in an aerosolized or nebulized form, to allow even distribution throughout the airway. finally, a mechanism for retention in the extracellular environment, avoiding rapid catabolism, would be advantageous for effective pharmacokinetics. sialidases have been demonstrated to inhibit influenza virus infection in a number of in vitro experiments. even before sia were proven to be the receptors for influenza viruses, it was observed that when sia was enzymatically removed from cell surfaces, the cells were less susceptible to infection (gottschalk et al., ) . further work using bacterial sialidases and cell lines showed a reduction in influenza virus infection (bergelson et al., ; griffin et al., ) . the sialidase of micromonospora viridifaciens was used in to destroy influenza receptors (air and laver, ) but these findings were not translated into a practical approach until , when there were two reports of using sialidase to successfully block respiratory virus infection. the first approach used the sialidase from vibrio cholera to treat influenza and parainfluenza virus three infections (thompson et al., ) , while the second used a bacterial sialidase that was coupled to amphiregulin and tested for anti-influenza activity (malakhov et al., ) . this second compound, called das , used the sialidase from a. viscosus. the coupling of sialidase to amphiregulin allowed it to bind to the respiratory epithelium, thus prolonging the duration of drug effect. a. viscosus is one of the first bacteria to colonize the surface of teeth. it is abundantly present in patients with gingivitis (haffajee et al., ) , and has been implicated in the immunopathogenic role of periodontal disease (mahanonda et al., ) . it has been proposed that continuous swallowing of this bacterium leads to a low-grade immunological response and the induction of oral bacterial tolerance (sosroseno et al., ) . the sialidase from a. viscosus was chosen for das three reasons. like many bacterial sialidases, it was effective at cleaving many types of sia; it had a high specific activity; and as it was a normal commensal of the oral cavity, there would not be natural antibodies against it, as a result of oral tolerance. the sialidase from a. viscosus had been studied in by teufel and colleagues who found that it was not primarily secreted, but cell-bound (teufel et al., ) . though it had a higher hydrolysis rate against the a - linkage, it was also effective at cleaving a - linkages, and was also able to cleave the ganglioside gd a, an uncommon property among bacterial sialidases. acetylation at c , and of sia still allowed cleavage, but -o-acetylation, gm and neu gc gm were not attacked at all. amphiregulin is an -amino acid glycoprotein that was discovered in (shoyab et al., ) , as reviewed in (busser et al., ) . one of its features is an epidermal-growth factor (egf)-like domain, which is homologous with other members of the egf-like growth factor family, meaning that it can bind to the egf receptor present on epithelial cells. unlike many other members of the egf family, amphiregulin binding does not lead to egfr degradation, but to recycling of the receptor and continued accumulation at the surface (willmarth et al., ) . at the other end of the protein is a heparin-and glycosaminoglycan-binding domain, which is involved in the inhibition or transformation of this glycoprotein (hence the name amphi-regulin). in the case of das , the sialidase was combined with the gag-binding portion of human ar. by binding to the negatively-charged glycosaminoglycans on the surface of airway epithelial cells, the cationic c-terminal amphiregulin tag anchors das on the respiratory epithelium, improving its potency. this net effect of combining the sialidase with amphiregulin means that the sialidase will be targeted to epithelial cells lining the respiratory tract, but without activating the egfr pathway. das was tested in ferret and human airway epithelium models and was shown to have activity against a wide range of influenza a and b viruses (malakhov et al., ) . compared to other sialidases, das had higher activity and was stable over a wide range of ph values that would be present in the respiratory tract. after a single treatment, there was effective desialylation of mdck cells for days and recovery to % after days; however, later treatment regimens adopted a daily dosing schedule. the amphiregulin attachment increased the potency - -fold (malakhov et al., ) . there was no appreciable cell toxicity in a number of established cell lines, and no endogenous production of interferon or tnfa was seen, indicating that the amphiregulin tag was not detrimental to the host cells. inhalation toxicity was evaluated in sprague-dawley rats. repeated daily exposures of inhaled das for days at achieved doses of up to . mg/kg/day were well tolerated, and clinical signs were not affected by treatment with das . minimal but significant decreases in red blood cells, haematocrit and hemoglobin were identified, together with an increased reticulocyte count, in keeping with a mild anemia; systemic absorption from the respiratory tract was estimated at % (larson et al., ) . in this study there was also a -fold increase in alkaline phosphatase (alp) from the liver, which was attributed to diminished clearance, owing to competition for the asialoglycoprotein receptor. the rise in alp was not associated with any evidence of hepatotoxicity. it is thought that desialylation of the serum proteins may lead to saturation of the asialoglycoprotein receptors (asgr) in the liver and spleen, and reduced clearance of certain glycoproteins, including alp. further studies were done on human airway epithelial cells . in this study, a % desialylation effect was seen after min, and the presence of mucus did not interfere with desialylation (fig. ) . similar changes were also seen in freshly isolated human bronchial ex vivo cultures . in the hae model, re-sialylation was achieved by days, whereas without the amphiregulin tag re-sialylation was much faster. a h postexposure regimen also produced a reduction in viral titre in h n infection, thus demonstrating that das could have a therapeutic was well as a prophylactic role. this was also demonstrated in an animal model, in which treatment at h postinfection reduced mortality and viral load in mice infected with a/pr / (h n ) and a/victoria/ / (h n ) . additional studies were done on lung ex-vivo infections, using h n h n pdm viruses (triana-baltzer et al., ) , showing a similar reduction in viral infection without toxicity. das was formulated into dry-powder microparticles with a diameter ranging from to lm using a proprietary temperaturecontrolled organic solvent assisted precipitation (tosap) technology, and drug deposition was tested using a cast of the human airway. as expected, smaller particles ( lm) were associated with increased deposition in the deep lungs ( %) and higher systemic exposure, compared to . % for lm particles. using the same airway model, relenza Ò had a . % deposition in the distal bronchi and lungs (data on company file). in the phase a trial, the lm size was used, but current phase b clinical studies are comparing lm and lm particles. a single phase ii study of das has been completed, which comprised participants with pcr-confirmed influenza. the inclusion criteria were: male and female participants age - who gave informed consent, were in generally good health, were febrile with oral temperature > . °c or reported temperature > . °c, or had felt ''feverish'' in the past h, and who had one or more respiratory (cough, sore throat, nasal symptoms) or constitutional symptoms (headache, myalgia, sweat/chills, prostration). there were two treatment arms: one with a single dose of mg, and the second using mg/day over days. the mean age of the patients in the multiple-dose arm was . years, in the single-dose arm . years and in the placebo arm years, with a slight female predominance in the treatment arms. thirty-seven percent of the participants had confirmed infection with influenza b, % with seasonal h n and % with h n pdm. no h y mutations were identified that would confirm the oseltamivir-resistant phenotype in the h n subtype of influenza viruses. the trial was conducted over many geographical areas, thus allowing an evaluation of a broad range of commonly circulating influenza viruses. the study demonstrated a significant reduction in viral load over days, for the day dosing group, and a significantly shorter time to sustained decrease in viral shedding in this group, compared to the placebo group. the day dose schedule was more effective than the single-day dose regimen, but no change in flu-like symptoms was seen between the control or treatment groups. however, the study lacked statistical power and was not designed to assess clinical differences among the treatment groups. there was a low incidence of serious adverse events, and only a mild increase in serum alp, possibly a desialylation-mediated effect . there did not appear to be a reported increase in secondary bacterial pneumonia. there are a number of potential concerns with sialidase therapy, including systemic dissemination of the drug, development of antibodies and the role of secondary bacterial infection. it was mentioned earlier that a. viscosus was chosen because it is a natural commensal of the oral cavity, and there have been no reported changes of immunity with exposure to das . a mild increase in alp was seen in the phase ii study, which may be due to systemic desialylation, but no changes in hematological parameters were identified . it was mentioned previously that patients with influenza had increased bacterial infection with pneumococcus, and there were concerns that the same may be found with das treatment (zhang, ) . the interaction between influenza virus infection and secondary pneumonia is a complex one that has been investigated by mccullers and others (grijalva and griffin, ; iverson et al., ; karlstrom et al., ) . since the h n pandemic, there has been an association of increased bacterial infection following influenza infection. the recent h n pandemic was associated with an increase in invasive pneumococcal infection (nelson et al., ) , suggesting that pneumococcal vaccination should be included as a vaccine strategy in future pandemics. indeed, vaccination strategies using pneumococcal vaccination reduced the incidence of influenza-associated pneumonia. however, the link between influenza and pneumococcal infection has been complicated by the finding that not all pneumococcal serotypes are associated with secondary pneumonia, and that influenza virus infection may select out and favor the growth of particular serotypes (mccullers et al., ) . it has been proposed that the desialylation produced by the viral neuraminidase exposes receptors that allow increased binding by pneumococci, followed by invasion. as we pointed out previously, a clear distinction needs to be made between desialylation in viral infection and that induced by topical therapy (nicholls et al., ) . it is therefore important to study whether sialidase therapy will lead to an increased risk of secondary pneumonia. studies by hedlund and colleagues using das showed that, in contrast to expectations, desialylation appeared to protect mice against secondary bacterial colonization . also, in a study previously cited, there was no evidence of increased adherence of pneumococci to desialylated tracheal epithelial specimens (nicholls et al., ) . the contrast between changes induced by influenza virus infection and by topical desialylation can be explained by the different pathologies: influenza virus infection of an epithelial surface causes necrosis and desialylation, while the changes induced by bacterial sialidase consist only of desialylation. in a virus infection, there is acute epithelial denudation and exposure of the basement membrane, allowing adherence of s. pneumoniae. with sialidase treatment alone, because there is no cell cytotoxicity or epithelial loss, there is no increased adherence of pneumococi. as detailed earlier, the phase ii clinical study did not document any increase in pneumococcal infection. even though influenza viruses are one of the leading pathogens causing acute respiratory illness, infections with parainfluenza virus , and make up a significant proportion of the causative agents isolated during these illnesses, especially in the pediatric population (weigl et al., ) . although parainfluenza viruses typically cause bronchitis and bronchiolitis, they are increasingly recognized as agents of lower respiratory tract disease, where they have a similar clinical presentation to infections by other respiratory viruses. parainfluenza viruses are of increasing concern in pediatric and immunocompromised populations, in which they may cause severe pulmonary disease. piv - resemble influenza viruses in using sia as a receptor, but binding differs among strains (villar and barroso, ) . use of a sialylated microarray has recently demonstrated that hpiv has the narrowest specificity, binding to neu ac, but stronger binding to neu gc with a methylated derivative. in contrast, hpiv showed a broader range of binding to neu ac and neu gc derivatives, while hpiv showed a similar range of binding, but at a much lower magnitude than hpiv . all three strains preferentially bound glycans with an a - , rather than an a - linkage (song et al., ) . the mechanism of binding and release of piv differs from influenza viruses, as they have a different replication pathway, and thus may not be amenable to standard anti-influenza therapy. thus, although zanamivir inhibited piv neuraminidase activity, it did not prevent release of virus from the cell (moscona, ; porotto et al., ) . as the parainfluenza viruses use a - and a - bound sia for attachment, infection with these viruses could be amenable to sialidase treatment. even though most studies utilizing das have used influenza a as a target, it has also been used in patients with parainfluenza. in an experimental setting, das treatment of human parainfluenza virus-permissive cell lines and of infected cotton rats led to effective desialylation and reduced infection (moscona et al., ) . das has also been used on a compassionate, emergency basis in immunocompromised patients infected with parainfluenza virus, with a beneficial effect. the first case was a year old female stem cell transplant recipient who developed nasal congestion and cough, followed by clinical and radiological features of viral bronchiolitis and worsening respiratory function. she received three daily inhaled administrations of das using a dry powder inhaler, and showed improvement in clinical, radiological and pulmonary function parameters after treatment. piv infection was confirmed, and there was a reduction in viral load after treatment . in another report, two immunocompromised lung or stem cell transplant recipients who developed piv infection were successfully treated after developing lower respiratory tract disease. in these cases, there was a day treatment with improvement in clinical symptoms and respiratory function, though changes in quantitative viral load did not match the clinical findings (guzman-suarez et al., ) . however, a more recent case of das treatment of piv pneumonia in a lung transplant patient was associated with both clinical improvement and reduction in viral load (drozd et al., ) . a novel bacterial sialidase, das , has been developed over the past six years from in vitro testing to phase ii studies for influenza virus infection. this agent shows promise in the repertoire of antivirals for influenza, and it should also show potential benefit against other respiratory viruses that use sia as a receptor, such as severe parainfluenza virus infection. red cells bound to influenza virus n neuraminidase are not released by the n neuraminidase activity docking of sialic acid analogues against influenza a hemagglutinin: a correlational study between experimentally measured and computationally estimated affinities site-directed fragment-based generation of virtual sialic acid databases against influenza a hemagglutinin characterization of a carbohydrate epitope defined by the monoclonal 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intermediates sialic acid receptor detection in the human respiratory tract: evidence for widespread distribution of potential binding sites for human and avian influenza viruses comment on: concerns of using sialidase fusion protein as an experimental drug to combat seasonal and pandemic influenza sialoadhesin -a macrophagerestricted marker of immunoregulation and inflammation glycan terminator restoration of specific myxovirus receptors to asialoerythrocytes by incorporation of sialic acid with pure sialyltransferases variation in the presence of neuraminidase genes among streptococcus pneumoniae isolates with identical sequence types adherence of type i streptococcus pneumoniae to tracheal epithelium of mice infected with influenza a/pr virus convergent pathways for utilization of the amino sugars n-acetylglucosamine, n-acetylmannosamine, and n-acetylneuraminic acid by escherichia coli human parainfluenza virus type hn-receptor interaction: effect of -guanidino-neu ac en on a 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protein as an experimental drug to combat seasonal and pandemic influenza key: cord- -d l cgc authors: nan title: mers: progress on the global response, remaining challenges and the way forward date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: d l cgc this article summarizes progress in research on middle east respiratory syndrome (mers) since a fao-oie-who global technical meeting held at who headquarters in geneva on – september . the meeting reviewed the latest scientific findings and identified and prioritized the global activities necessary to prevent, manage and control the disease. critical needs for research and technical guidance identified during the meeting have been used to update the who r&d mers-cov roadmap for diagnostics, therapeutics and vaccines and a broader public health research agenda. since the meeting, progress has been made on several key actions in animal populations, at the animal/human interface and in human populations. this report also summarizes the latest scientific studies on mers since , including data from more than research studies examining the presence of mers-cov infection in dromedary camels. since its identification in the kingdom of saudi arabia (ksa) (zaki et al., ) and jordan (hijawi et al., ) in , middle east respiratory syndrome (mers) has become a global public health threat. typical of an emerging zoonosis, middle east respiratory syndrome coronavirus (mers-cov) has an animal reservoir, i.e. dromedary camels in which the virus causes little to no disease (mohd et al., ) . many details about the extent of circulation and the mechanisms of transmission within dromedary camel herds, or factors related to zoonotic transmission and differences in circulating mers-cov strains, remain unknown. the virus has repeatedly spilled over from dromedary camels to humans, principally in countries on the arabian peninsula, causing significant morbidity and mortality (world health organization, a; azhar et al., ) . clusters of cases in the community and among family members are rare (world health organization, a; . however, delays in diagnosis in hospitals has sometimes led to secondary cases among health care workers, patients sharing rooms or family members as a result of unprotected direct contact with a patient before isolation. this humanto-human transmission in health care facilities can sometimes be amplified, causing very large outbreaks, as has been seen in the middle east and in the republic of korea, with significant public health and economic impacts (hijawi et al., ; assiri et al., ; drosten et al., ; al hosani et al., ; ki, ; park et al., ) . as of august , more than human cases from countries have been reported to the world health organization (who) (world health organization, a) . the fao, oie and who tripartite have regularly brought together affected member states, public health and animal officials, and academics to discuss what is known and unknown about the zoonotic origin of mers-cov (world health organization, ; fao, fao, , ; who regional office for the eastern mediterranean, a). the purposes of these meetings and workshops have been to advocate for more surveillance and research on mers-cov in animals and humans, to share information about how mers-cov is transmitted between animals, from animals to humans and between humans, to describe the diseases it causes, and to develop policies and guidelines for detection, https://doi.org/ . /j.antiviral. . . received august ; accepted september reporting of animal and human infections, and prevention of human cases and clusters. in the two years since the last international technical consultation on mers-cov in , there have been notable improvements in surveillance and reporting of human cases, multidisciplinary research, cross-sectoral collaboration at country level, public awareness about the disease, and laboratory and surveillance capacity in affected countries. in addition, a number of countries in the arabian peninsula and in africa have engaged in research activities and surveillance of camel populations to shed light on the wider distribution of this virus or investigate transmission patterns and routes for viral shedding. as a follow-up to previous meetings (world health organization, ; fao, fao, , ; who regional office for the eastern mediterranean, ; who regional office for the eastern mediterranean, b, c), fao, oie and who tripartite held a global technical meeting on mers-cov with representatives from ministries of health and ministries of agriculture, subject matter experts, researchers, funders and industrial partners from to september in geneva, switzerland (see supplementary information) (world health organization et al., ) . the objectives were to review the latest scientific evidence on mers-cov, further enhance cross-sectoral collaboration and communication during preparedness and response activities, and identify research priorities given the advancements in our knowledge. with participants, this was the largest mers-cov technical meeting to date and the first meeting attended by representatives from both affected and at risk countries. that is, countries which have reported human infection, countries with evidence of mers-cov in dromedary camels but no reported human cases, and countries at risk for importation (countries without infected camels that have close ties to affected countries through expatriate workers, travel to affected countries for medical procedures and/or frequent international travel). there is strong consensus among all stakeholders that dromedary camels are the main source of transmission to humans. in , oie identified mers-cov as an emerging disease with zoonotic potential in camels and thereby creating expectations of reporting positive camels by countries (oie, a) and recently published a mers-cov case definition (oie, ) for the reporting of confirmed and suspected infection in camels. not all countries face the same risks. for example, countries that have the infected reservoir (dromedary camels) differ from those countries in which dromedary camels show no evidence of current or past infection (fig. ). there may also be differences in spillover potential in countries with documented zoonotic transmission, compared to those without, due to several factors including potential differences in husbandry practices, cultural, social, medicinal, occupational exposures, prevalence of underlying chronic medical conditions, or genetic factors in human populations, and mers-cov viral differences (wong et al., ) . as such, technical and risk mitigation guidance to protect human health and research priorities differ by region. the findings from the global technical meeting are summarized below: i. surveillance needs: surveillance in animals and humans to limit zoonotic transmission routine human surveillance for mers-cov in ksa (abdulaziz et al., ) and throughout the middle east has improved since the identification of the virus in humans in , but there is significant variation in the quality and extent of surveillance between countries. in other parts of the world, surveillance is limited. since it is known that mers-cov is enzootic in areas of africa and asia where dromedary camels are found, heighted awareness and surveillance for zoonotic mers is required. this is currently lacking and remains a knowledge gap. one exception is the notable effort to identify potential mers-cov infection among pilgrims travelling back from the middle east. since , event-based surveillance among pilgrims returning from hajj, umrah and other religious events in ksa has been conducted by ksa and countries sending pilgrims. while many return reporting respiratory symptoms, no mers-cov infections have been identified among returning pilgrims (muraduzzaman et al., ; barasheed et al., ; atabani et al., ; koul et al., ; annan et al., ; ma et al., ; memish et al., a memish et al., , b refaey et al., ; al-abdallat et al., ; matthew et al., ; alqahtani et al., ; win et al., ; yavarian et al., ; kapoor et al., ) . among animals, field surveys conducted to date have included several domestic and wildlife species including dromedary camels (camelus dromedarius) and bactrian camels (camelus bactrianus), goats, bats, cattle, sheep, chickens, swine, ducks, buffalo and equids. field studies in dromedary camels have been conducted in a number of countries (table ) . to date, mers-cov rna or mers-cov-specific antibodies have been identified in dromedary camels a number of countries (table ) except australia (hemida et al., ) , kazakhstan (miguel et al., ) , and the netherlands (reusken et al., a) . other livestock such as alpacas (vicugna pacos), llamas (llama pacos), young goats, rabbits and pigs have been shown to be susceptible to experimental infection (crameri et al., ; adney et al., ; vergara-alert et al., ) . despite improvements, routine surveillance in dromedary populations is limited. the lack of surveillance information about mers-cov circulation in dromedary camels restricts our understanding of the transmission dynamics and epidemiology in dromedary camel populations. meeting participants agreed that surveillance should be integrated into existing surveillance systems, particularly in at-risk countries, similar to one health approaches developed for avian influenza, and existing human respiratory disease surveillance systems set up for influenza-like illness (ili) or severe acute respiratory infections (sari). currently, a limitation in our ability to mitigate spillover from dromedary camels to humans is a lack of clarity on the mode(s) of transmission between dromedary camels and humans, the extent and epidemiology of mers-cov circulation in dromedary camels in large parts of africa and south asia, and on why zoonotic transmission is limited across africa, large parts of the middle east, and some parts of south asia despite high seroprevalence in dromedary camels (chu et al., ) (table ) . fao has outlined the meeting participants conclusions on priorities for mers-cov surveillance and management of pcr positive dromedary camels, coordinated outbreak investigation of community acquired cases with dromedary exposure, testing of animals at quarantine and entry points, food safety and environmental contamination, risk communication and awareness raising for mers-cov among animal owners and intersectoral collaboration and coordination in an updated doha declaration first published in (fao, ) (ref/hyperlink). in dromedary camels, longitudinal studies to evaluate the natural history, shedding profile and immunity were highlighted as key research priorities. meeting participants agreed that further understanding of differences in viral strains and transmission dynamics, including the role of immunity in acquiring infection and shedding virus, the geographic range of spillover events, and environmental, behavioral or host-related risk factors for zoonotic transmission should be prioritized. countries face significant challenges in the early identification and diagnosis of mers in humans due to the non-specificity of clinical symptoms ( the spectrum of illness ranges from no symptoms (or asymptomatic infection) to severe disease including pneumonia, acute respiratory disease syndrome, organ failure and death, with a case fatality ratio . % among reported cases (world health organization, ). the delay in identification and recognition of signs and symptoms compatible with mers and delay in early isolation of patients has reduced the ability to prevent transmission between people in health care settings, notably in emergency departments, cardiac care centers and renal dialysis units (hijawi et al., ; assiri et al., ; drosten et al., ; al hosani et al., ; ki, ; park et al., ; ahmed et al., ; amer et al., ) . our understanding of human-to-human transmission in health care settings has improved through experimental and observational studies conducted in countries during such outbreaks. for example, studies of respiratory pathogens (yu et al., ; tran et al., ; thompson et al., ) and mers-cov conducted in the middle east (assiri et al., ; oboho et al., ; hunter et al., ; balkhy et al., ) and the republic of korea (bin et al., ; kim et al., a kim et al., , b nam et al., ) illustrate that aerosol-generating procedures and non-invasive ventilation, combined with inappropriate infection prevention and control practices and lack of adherence to standard practices had an important role in facilitating human-to-human transmission in health care settings. the role of environmental contamination has been evaluated in a number of hospitals following the outbreak in the republic of korea and collaborative, experimental studies are being conducted to evaluate the viability and persistence of mers-cov on surfaces and in the air (bin et al., ; kim et al., a; van doremalen et al., ) . the role of mild or asymptomatic cases in transmission chains, however, remains unclear (omrani et al., ; memish et al., c; al-gethamy et al., ; moon and son, ; al-abdely et al., ) , warranting targeted epidemiological and clinical studies to be conducted among contacts during outbreaks, especially in health care facilities. countries which have reported health care-associated outbreaks have implemented a variety of strategies to improve infection prevention and control and reduce human-to-human transmission in hospitals, including the introduction of a visual triage system prior to entrance to the emergency departments, the restructure of emergency department layouts for better triage of patients with respiratory symptoms, the standardization and training and re-training of infection prevention and control practices at facilities with high hospital staff turnover, and the auditing of health care facilities for adherence to infection prevention and control measures. iii. product research and development needs: clinical management, diagnostics and medical interventions the who r&d blueprint, a global strategy and preparedness plan that allows the rapid activation of r&d of epidemic pathogens, aims to fast-track the development and use of effective point-of-care diagnostic tests, vaccines and medicines that can be used to save lives and avert large scale crises. since , mers-cov has been included in the annual who r&d blueprint list of prioritized pathogens for accelerated research and development on diagnostics, vaccines and therapeutics (world health organization). in addition, mers-cov has a specific roadmap for product research and development, outlined by who in ( the nonspecific, and sometimes unusual, clinical presentation of mers in humans, makes early diagnosis difficult in health care facilities. while several highly specific and sensitive molecular and serologic assays exist for diagnosis in animals and humans corman et al., a corman et al., , b lu et al., ; perera et al., a; reusken et al., b; müller et al., ; song et al., ) , there was a clear call from representatives from affected countries for the development of a rapid diagnostic test to improve identification and isolation of primary human cases in health care facilities. a full landscape analysis of mers-cov diagnostics will be published separately (van kerkhove, personal communication). at the global technical meeting, several therapeutics (including convalescent plasma, lopinavir/ritonavir, ribavirin, interferon and novel therapies including polyclonal antibodies and broad-spectrum antivirals) in development were presented. however, small case numbers make the evaluation of their impact on morbidity and mortality from mers-cov infection difficult (arabi et al., a; arabi et al., b; al-dorzi et al., ; sheahan et al., ; ko et al., ; arabi et al., c) . several pre-clinical and phase i-iii studies are under way or in the design phase (outlined by the who r&d blueprint: http://who-blueprint-mapping-tool.surge.sh/). who is currently evaluating all available evidence on therapeutics to update guidance on clinical management of patients and in the process to develop standardized clinical trial protocols that could be used in affected countries to evaluate promising therapeutic candidates. who has identified target product profiles for mers-cov vaccines which include a dromedary camel vaccine for the reduction of zoonotic transmission, a human vaccine for long term protection of high risk individuals, such as those working with infected dromedary camels or health care workers, and a human vaccine for reactive use in outbreak settings (world health organization, b) . currently, no mers-cov-specific or licensed human vaccines are available (modjarrad et al., ; world health organization, c) . several human vaccine candidates for coronaviruses, including mers-cov, are at various stages of development and five general vaccine technology platforms have been developed and target the mers-cov spike protein (modjarrad et al., ; okba et al., ) . who, the ministry of health in ksa and the international vaccine institute (ivi) have continued to further align efforts to develop coronavirus vaccines (excler et al., ) and the coalition for epidemic preparedness and innovation (cepi) has included mers-cov as one of three priority pathogens for financing of a human vaccine. understanding correlates for protection and having a reliable animal model remain essential for evaluating coronavirus vaccine candidates, including mers-cov. there was a clear call from global technical meeting participants to accelerate the development of a dromedary camel vaccine in order to evaluate the potential to reduce spillover transmission to humans. the acceptability, cost-effectiveness and feasibility of a dromedary camel vaccine will also need to be evaluated and compared to other intervention strategies, such as human vaccination of high risk groups (e.g., those with occupational exposure). because mers-cov is endemic in dromedary camel populations in the middle east and elsewhere, multiple intervention strategies, including personal protective measures and the strategic implementation of a camel vaccine, are likely needed to reduce transmission from dromedary camels to humans. (farag et al., ) antibodies to mers-cov s were found in of ( . %) dromedary camels tested by micro-array technology (farag et table list of prioritized research and progress on mers-cov research, as discussed at the september meeting. *based on an enhanced understanding of the virus, the doha declaration (fao, ) is undergoing revision with a focus on guiding surveillance techniques, management of dromedary camels shedding the virus, research, regional and inter-sectoral coordination, risk communication, food and environmental safety practices, and biosecurity measures. the update includes explicit guidance on import testing, quarantine procedures, and management of shedding animals. these recommendations and priority actions in the doha declaration will be delivered as a separate document after validation by stakeholders in affected and at risk countries. at least two promising camel vaccine candidates are currently in development and being evaluated in field trials (haagmans et al., ; alharbi et al., ) . stakeholders agreed that the current funding mechanisms need to include risk-mitigating options that target the animal-human interface to prevent zoonotic transmission. these funding pathways would enable the development of camel vaccine candidates. stakeholders also agreed that prior to camel vaccine implementation, consultation with camel owners and government agencies, feasibility studies including exploring opportunities for commercial manufacturing and incentives for camel vaccination and an assessment of potential trade implications, would all be critical. in june , who and the international vaccine institut (ivi) held a joint workshop update the status of human and animal mers-cov vaccine development, and identify and prioritize activities to accelerate vaccine research and development. the meeting was held in seoul, korea on - june and included experts and professionals from industry, academia, international organizations and government agencies around the world, including the coalition for epidemic preparedness innovations (cepi), the korean ministry of food and drug safety (kmfds) and the korea centers for disease control and prevention (kcdc). the global technical meeting served as an opportunity to review the available evidence and best practices for control of this epidemic threat six years after the virus was first detected in humans. this covered our understanding of the virus, our ability to detect and respond to cases in animal and human populations, how we communicate our findings and how our work impacts policy decisions to protect animals and prevent human infections. during the global technical meeting, the latest findings from scientific studies and knowledge gained from collaborative research and surveillance were shared across animal, environmental and human sectors. fao, oie and who strongly believe that to effectively address zoonoses, including mers-cov, a one health approach to prevent, detect, contain, eliminate and respond to animal and public health risks from zoonotic high threat respiratory pathogens such as mers-cov and should involve all relevant sectors, the public and animal health and academic research community, industry and affected communities. we acknowledged the progress that has been made, and importantly, discussed the challenges that need to be addressed so that we can minimize the future public health and economic impacts of this epidemic prone virus. our aim was to articulate a clear action plan to address these remaining unknowns and to foster better collaboration between sectors and with subject matter experts willing to support member states. given the marked expansion in research related to mers-cov conducted in the past two years, fao, oie and who agree that global surveillance and research activities must now be focused on achieving the following major public health goals: reducing zoonotic transmission, detecting and identifying suspected cases early, providing safe and effective treatment to reduce human morbidity and mortality, and significantly reducing human-to-human transmission in health care settings. the critical needs for research and technical guidance identified during the global technical meeting have been used to inform the who r&d mers-cov roadmap (modjarrad et al., ) and a broader research agenda for mers-cov and other high threat coronaviruses. this research agenda serves as a catalyst to focus, align and mobilize partners to address outstanding knowledge gaps in relation to mers-cov across five technical areas: i) virus origin and characteristics, ii) epidemiology and transmission, iii) clinical management and infection prevention and control, iv) product development and implementation (modjarrad et al., ) and v) impact of interventions and operational research. the meeting identified a large number of remaining priorities and the organizing committee has summarized the main research needs in table . now is the time to devote more 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camels, saudi arabia we gratefully acknowledge the input from all those who attended the fao-oie-who global technical meeting on mers-cov in september . the authors also thank malik peiris for his review of the final manuscript. the opinions expressed in this article are those of the authors and do not necessarily reflect those of the institutions or organizations with which they are affiliated. the authors have no competing financial interests. supplementary data to this article can be found online at https:// doi.org/mmcdoino. key: cord- -wtmmewiz authors: warren, travis k.; shurtleff, amy c.; bavari, sina title: advanced morpholino oligomers: a novel approach to antiviral therapy date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: wtmmewiz phosphorodiamidate morpholino oligomers (pmos) are synthetic antisense oligonucleotide analogs that are designed to interfere with translational processes by forming base-pair duplexes with specific rna sequences. positively charged pmos (pmoplus™) are effective for the postexposure protection of two fulminant viral diseases, ebola and marburg hemorrhagic fever in nonhuman primates, and this class of antisense agent may also have possibilities for treatment of other viral diseases. pmos are highly stable, are effective by a variety of routes of administration, can be readily formulated in common isotonic delivery vehicles, and can be rapidly designed and synthesized. these are properties which may make pmos good candidates for use during responses to emerging or reemerging viruses that may be insensitive to available therapies or for use during outbreaks, especially in regions that lack a modern medical infrastructure. while the efficacy of sequence-specific therapies can be limited by target-site sequence variations that occur between variants or by the emergence of resistant mutants during infections, various pmo design strategies can minimize these impacts. these strategies include the use of promiscuous bases such as inosine to compensate for predicted base-pair mismatches, the use of sequences that target conserved sites between viral strains, and the use of sequences that target host products that viruses utilize for infection. antisense oligonucleotides (asos) are designed to bind to complementary rna by watson-crick hybridization. asos are typically designed to arrest translational processes either by inducing cleavage mechanisms involving endogenous cellular nucleases such as - /$ -see front matter Ó elsevier b.v. all rights reserved. doi: . /j.antiviral. . . rnase h (giles et al., ) or by sterically blocking enzymes involved in target gene translation ( fig. ) (stein et al., ) . alternatively, asos can be designed to target viral genomic regions or rna secondary structures involved in transcription or genomic-replication processes (deas et al., ; stone et al., ) . aso-mediated interference with these virological processes inhibits production of infectious virus particles in infected cells, potentially reducing cytopathic effects. as illustrated in fig. , one unproven hypothesis about the mechanism of action of asos postulates that production of defective interfering particles following aso treatment may facilitate the generation of productive immunological responses that clear virus following infection. since the discovery of antisense molecules, there has been keen interest in optimizing the molecular scaffolds of antisense molecules to improve the pharmaceutical properties and in vivo activity for therapeutic applications. improvements to increase solubility, reduce toxicity and increase broad spectrum activity have been engineered into phosphorodiamidate morpholino oligomers (pmos) over the last few years of research. this article focuses on pmo-based antiviral therapeutic agents. overviews of efforts to develop other antisense-based therapeutics for treatment of both viral and bacterial pathogens have been presented in several recent reviews (haasnoot and berkhout, ; spurgers et al., ; stein, ; warfield et al., a) . in , zamecnik and stephensen provided the first report of the use of a synthetic oligonucleotide to inhibit pathogen replication through antisense mechanisms. in these experiments, a tridecamer oligodeoxyribonucleotide (fig. ) complementary to rous sarcoma virus rna introduced to chick embryo fibroblast cells inhibited production of virus . further investigations revealed that hybridization of this oligodeoxyribonucleotide likely inhibited replication by blocking viral protein translation (campbell et al., ; stephenson and zamecnik, ; wickstrom, ) . while the findings of zamecnik and stephensen suggested that administration of com-plementary antisense oligonucleotides (asos) might prove to be a useful therapeutic approach to achieve gene-specific knockdown, limitations to this approach were quickly realized. unmodified oligodeoxynucleotides are susceptible to degradation by endogenous nucleases, which cleave phosphodiester linkages, present in bodily fluids (wickstrom, ) . in one of the first oligonucleotide analog derivatives designed to resist nuclease degradation, linkages of natural oligonucleotides were replaced with phosphorothioate groups (fig. ) (campbell et al., ) . findings from wheat-germ and reticulocyte cell-free translation assays showed that these phosphorothioate oligonucleotides (ptos) inhibited translation through rnase h-dependent cleavage of mrna (minshull and hunt, ) . matsukura et al. first exploited the antisense properties of ptos, to inhibit replication of hiv (matsukura et al., ) . the benefits of resistance to nuclease degradation conferred by the pto chemical modifications are diminished by a number of off-target effects, including cleavage of non-target sequences, interaction with a variety of proteins, activation of b lymphocytes, and activation of complement (henry et al., ; krieg and stein, ; stein et al., ; summerton, ) . despite these limitations, the fda has approved a pto antisense drug -fomiversen, for intravitreal treatment of cytomegalovirus retinitis in patients with aids -which to date is the only antisense agent to receive approval for therapeutic application (geary et al., ; perry and balfour, ) . various other strategies have been adopted in attempts to improve the resistance of asos to degradation by nucleases, to limit off-target effects, and to enhance binding affinity with target rna. incorporating o-methyl or o-methoxyethyl groups at the position of the pto ribose ring increases the potency and nuclease resistance compared to unmodified ptos ( fig. ) (dean and bennett, ; turner et al., ) . these modifications do not support rnase h-activity, which has led to development of chimeric strategies, in which gaps of contiguous unmodified phosphorothioate nucleotide analogs are incorporated to maintain rnase h activity while retaining the favorable properties of modified ptos (turner et al., ; zheng, ) . the locked nucleic acid (lna) aso design incorporates a o -c -methylene linkage of the ribose fig. . comparison of translational arrest mechanisms mediated by antisense molecules. during typical translational processes, assembly of the ribosomal complex at the aug start codon facilitates de novo protein synthesis from template rna. complementary antisense molecules can arrest translation either by sterically blocking ribosomal assembly or by supporting rnase h cleavage activity. aso mediated translational arrest may interfere with critical processes required for virion production and assembly and could promote the formation of defective interfering viral particles. the presence of these altered proteins or defective particles may serve to stimulate productive immunological clearance mechanisms. rings, a design which supports rnase h recruitment ( fig. ) (kurreck et al., ; veedu and wengel, ) . peptide nucleic acids (pnas) possess no ribose rings and rely instead on a peptide-based structure for the molecular backbone (fig. ) . as a result, pnas do not support rnase activity but effect translational arrest through steric-block mechanisms. pnas investigated to date have exhibited poor cellular penetration and in vivo pharmacokinetic properties (larsen et al., ; mcmahon et al., ) . phosphorodiamidate morpholino oligomers are antisense molecules in which the ribose rings of dna are replaced with -membered morpholine rings and phosphodiester linkages are replaced with phosphorodiamidate linkages (fig. ) . pmos are nonionic, a charge status that contrasts with the negative charges associated with native nucleotides and many other aso design approaches. pmos do not support rnase h activity and effect translational arrest or mrna processing, depending on the target site, through steric block mechanisms. the untranslated region ( utr) and transcript sites at or near the aug translational start codons are most often targeted to achieve translation arrest. although it has been suggested that the nonionic characteristics of pmos hinder cellular uptake, pmos readily enter primary cells (arora et al., ; iversen, ) . the in vivo activity of pmo antisense agents against a variety of viral pathogens has been demonstrated and is discussed in greater detail herein. while a number of neutralcharge pmos have entered human clinical trials (iversen, ; iversen et al., ) , none has yet been licensed for treatment of viral infection or other indications. attempts to improve the in vivo efficacy of pmos have included conjugation with peptides designed to enhance transport across cellular membranes. peptide conjugated pmos (ppmos; fig. ), which include positively charged amino acid residues such as arginine, have been viewed as particularly promising transporters and have shown efficacy in multiple in vivo models of viral infection (enterlein et al., ; paessler et al., ; stein, ; swenson et al., ) . the enhanced efficacy of ppmos has been hypothesized to result from the enhanced affinity resulting from ionic interactions between the net positive charge of the peptides and the negative charge of the complementary rna and through enhanced pharmacokinetic properties and cellular uptake (iversen, ) . however, maldi-tof mass spectrometry analyses have shown that while the pmo portion of ppmo conjugates are stable in cells, serum or tissue homogenate, the peptide portion is susceptible to degradation (amantana et al., ; youngblood et al., ) . this degradation impacts the effectiveness of ppmos by preventing the efficient escape of the conjugated molecule from endosomal or lysosomal vesicles . while ppmos are efficacious in multiple in vivo models of viral infections, ppmos are more poorly tolerated in vivo compared to neutral-charge pmos. in mice, dose-dependent reductions in weight, behavioral alterations, and mild liver histopathology were observed following repeat administration of - lg doses of a pmo conjugated to an arginine-rich peptide (deas et al., ) . in rats, administration of or mg/kg of an anti-c-myc ppmo (containing an arginine-rich peptide) produced reduction in body weight and elevation of serum blood urea nitrogen and creatinine concentrations, changes not observed following dose-matched administration of an unconjugated version of this pmo (amantana et al., ) . various attempts to introduce non-natural amino acids or other chemical changes in the conjugated peptide to offset toxicity and improve cellular uptake and intracellular distribution have met with varying degrees of success (abes et al., ; wu et al., ; youngblood et al., ) . the recognition that positively-charged peptides can enhance the potency of neutrally charged pmos led to the design of a new class of positively charged pmos (pmoplus tm ), which contain positively-charged piperazine groups along the molecular backbone ( fig. ). two recent reports (swenson et al., ; warren et al., ) , showing that these therapeutics are well tolerated and provide improved efficacy in numerous in vivo viral infection models, have broad implications for therapeutic development against viruses and other highly pathogenic microbes. in , swenson et al. provided preliminary findings showing that intraperitoneal administration of pmoplus tm molecules targeting ebola virus (ebov) vp mrna protected mice against lethal infection (swenson et al., ). these results were expanded upon in a report by warren et al. that showed that treatment conferred postexposure protection against ebov and, separately, marburg virus (marv) in nonhuman primates (nhps) (warren et al., ) . to achieve postexposure efficacy against ebov in nhps, a combination therapeutic (avi- ) containing pmoplus tm molecules targeting ebov vp and vp mrna was administered either intravenously or at intraperitoneal and subcutaneous sites (warren et al., ) . reducing production of vp and vp may facilitate viral clearance by host innate and adaptive immunological mechanisms by limiting the interference of these viral products on type i interferon induction and signaling processes with which they are involved (hartman et al., ; kash et al., ; reid et al., ) . in studies showing protection against marv, avi- , a combination of pmoplus tm molecules targeting marv vp and nucleoprotein (np), was delivered either intravenously, subcutaneously, or both intraperitoneally and subcutaneously. inhibiting translation of marv np and vp may interfere with the release of virus from infected cells (bamberg et al., ) and with marv genome synthesis by affecting formation and activity of nucleoprotein complexes (dicarlo et al., ) . the inclusion of cross-over, negative control treatments in nhp trials confirmed that the efficacy of both avi- and avi- was sequence-specific and was not mediated by nonspecific mechanisms. in all in vivo studies, treatment was well tolerated in all species tested (rat, mouse, guinea pig, rhesus monkey, and cynomolgous macaque). open investigational new drug applications are on file with the us food and drug administration (fda) and placebo-controlled, double-blinded phase i clinical trials of patients for each of avi- and avi- are in progress to evaluate the safety of up to mg/kg dose levels. an advanced development plan for these candidates is being actively pursued and relies on licensure under the fda's animal efficacy rule, a regulatory mechanism designed to facilitate the development of new drug products for indications in which clinical efficacy studies are neither feasible nor ethical (shurtleff et al., ; snoy, ) . there currently are no licensed vaccine or therapeutic products for treatment of ebov or marv infection in humans, and reports of therapeutic agents besides pmoplus tm showing substantive postexposure protection against these highly virulent pathogens in nonhuman primate models are few (geisbert et al., a; geisbert et al., b) . findings showing these therapeutics protect against marv and ebov -viruses associated with high case fatality rates, up to % for certain outbreaks -provide encouragement that this class of therapeutics may be efficacious against other, more prevalent, human viruses. antisense therapeutics possess multiple drug properties that are particularly favorable for use as therapeutics during outbreak scenarios. because the timing of viral outbreaks can be highly unpredictable, an ideal therapeutic will need to be stable and amenable to large-scale manufacturing procedures to allow the drug to be prepared in advance of an outbreak. pmoplus tm agents have been amenable to the mid-scale, quality-controlled manufacturing needs required for phase i clinical trials and nonhuman primate efficacy trials conditions reported herein, and these molecules are highly stable in lyophilized form following purification or in solution. further, they are highly soluble in aqueous solutions or other isotonic solutions in which they may be administered in clinical settings. initial investigations in nonhuman primates demonstrate that the agents are well tolerated by intravenous administration (warren et al., ) , suggesting that their use in emergency-response personnel are not likely to impact the ability of these individuals to perform their critical duties during public health or medical crises. the pharmacokinetic properties of the agents comprising avi- and avi- have been examined in rats following administration of a single iv or ip dose (warren et al., ) . the plasma elimination half-life following iv administration of . - mg/ kg ranges from . to . h (warren et al., ) . these half-lives are comparable to that reported from a rat pharmacokinetic analysis involving a neutral-charge c-myc pmo but somewhat shorter than that of an arginine-rich peptide conjugated version of the cmyc pmo (amantana et al., ) . the plasma elimination half-life for neutral-charge pmos is sequence-dependent and can range from . to h (amantana et al., ; arora et al., ; iversen, ) . further studies are required to determine whether the pharmacokinetic behavior of these agents is sequence-specific. for the two pmoplus tm components of avi- , the primary route of excretion is renal with - percent recovery of the total iv dose from urine within h of administration. the agents of avi- exhibited reduced renal clearance relative to avi- , with . - . percent of the total dose excreted in urine within h. administered via the iv route, renal half-life for the components of avi- was . - . h and for avi- was . - . h. following ip administration, the agents are generally rapidly aborbed with a time to maximal plasma concentration of . - . h. plasma elimination half-life following ip administration ranges from . to . h. the plasma elimination half-life may underestimate tissue residence time, which may have important implications for efficacy. for neutral-charge pmos, tissue residence time in kidney and liver has been reported to range from to days (amantana and iversen, ; iversen, ) . studies in rats have shown that conjugation of a c-myc pmo with an arginine-rich peptide increases the tissue uptake and increases tissue retention, especially in liver, kidney, and spleen (amantana et al., ) . tissue distribution studies and interspecies pharmacokinetic comparisons involving avi- , avi- or other agents have not yet been reported. while investigations are ongoing to further explore the routes by which pmoplus tm agents can be administered to confer in vivo efficacy against viral pathogens, results from studies involving neutrally charged pmos or ppmos suggest that efficacy can be achieved using administration via any of a number of routes. recent mouse studies (eide et al., ; moerdyk-schauwecker et al., ) have shown that ppmos directed against immediate early genes icp( ) and icp of hsv- and hsv- , when administered topically to the eye or genital tract epithelium, for these viruses, respectively, significantly reduced the viral loads in tissues and reduced the number of deaths from these infections. ppmos can be successfully administered intranasally for treatment of respiratory diseases such as influenza in experimental models. lupfer et al. showed that fluorescently labeled ppmos given by intranasal instillation to mice could be detected in lung tissue by fluorescence visualization (lupfer et al., ) . grossly, they observed the presence of fluorescent pmos in a concentration gradient, which decreased towards the lower lobes of the lung (lupfer et al., ) . microscopically, ppmos were at highest concentrations in and around bronchioles (lupfer et al., ) . using this intranasal instillation method for ppmo delivery, mice were protected from challenge with influenza virus a subtype h n , demonstrating that ppmos were present in situ at concentrations that were sufficient for antiviral activity (lupfer et al., ) . mouse models of other systemic viral infections, such as alphaviruses, flaviviruses and filoviruses, have been used to test intraperitoneal, subcutaneous, intramuscular or intravenous routes, or even some combination thereof, of neutral-charge pmos, ppmos, and pmoplus therapeutic agents (paessler et al., ; warfield et al., b) . a limited number of published studies describe administration of ppmos, neutrally charged pmos, or pmoplus tm to larger animals such as guinea pigs, cats, or nonhuman primates for treatment of infectious diseases. cats were treated for calicivirus infection with mg doses of neutrally charged pmos delivered subcutaneously, resulting in up to % protection from disease (smith et al., ) . intraperitoneal treatment of guinea pigs with mg doses of neutrally charged pmos directed against ebov proteins greatly enhanced survival in this lethal infection model (warfield et al., b) . a prophylactic dosing regimen consisting of subcutaneous, intraperitoneal, and intramuscular doses of up to mg of ebov-specific pmos protected rhesus macaques against lethal ebov infection (warfield et al., b) . there exists a keen interest in developing antiviral therapeutics that can be administered rapidly to a large number of individuals with minimal involvement by medical professionals. accordingly, therapeutics that can be delivered by oral, intramuscular, or transdermal routes are particularly appealing for use in emergency response scenarios and for use in remote regions that lack a modern medical infrastructure. moreover, a host of promising new technologies are currently under development for drug administration, including gene-gun methods (tavernier et al., ) , aerosol and nasal deliveries (wong et al., ) , and transdermal dissolving microneedle technologies (escobar-chavez et al., ) . exploration of the efficacy of antisense therapeutics delivered by methods such as these may further expand the treatment options. through use of animal models for investigations of experimental efficacy, pmos and ppmos have shown activity against human pathogenic viruses from a number of families of dna and rna viruses. published efficacy data against these viruses range from early in vitro antiviral activity studies to in vivo advanced preclinical development studies in animal models of viral infection, with most of the published work concentrating on rna viruses (moerdyk-schauwecker et al., ; neuman et al., ; smith et al., ; smith et al., ; van den born et al., ; warren et al., ) . because viruses that belong to the same taxonomic class or order generally have similar genomic structure and gene functions, a gene target that can be inhibited effectively by asos for one virus, is likely to implicate promising targets in related viruses (neuman et al., ; van den born et al., ) . in fact, some viruses possess conserved sequence regions across different virus isolates or strains, and these areas sometimes offer targets against which pmos can be designed (gabriel et al., ; holden et al., ) . ppmos have inhibited replication of herpes simplex virus- (hsv- ) in cell culture and in vivo when directed against hsv- icp or icp (moerdyk-schauwecker et al., ) . gene expression for another large dna virus, the iridovirus, has been further characterized using morpholinos to knock down viral gene expression to assess the roles of viral regulatory genes and proteins (sample et al., ) . the replication of a calicivirus, an rna virus, has been reduced up to % in cell culture by neutrally charged pmos at a concentration of lm (smith et al., ) . pmos used in this study were designed to target the feline calicivirus (fcv) orf- . fcv can cause a severe and contagious febrile respiratory disease in cats and kittens, and human caliciviruses, categorized as category b priority pathogens by niaid (niaid, ) , cause serious infections. when given at the time of disease onset and visible signs, subcutaneous administration of . mg/kg of this fcv orf- pmo resulted in protection of up to % of cats tested in a veterinary field trial (smith et al., ) . prophylactic efficacy was not investigated in this veterinary clinical setting. viruses of the order nidovirales include viruses from the coronaviridae family, e.g. severe acute respiratory syndrome coronavirus (sars-cov), and human coronaviruses which cause respiratory and gastrointestinal diseases. coronaviruses and arteriviruses are rna viruses that share similar genomic structures. pmos and arginine-rich peptide-conjugated ppmos directed against the utr of equine arteritis virus (eav) and against the utr of porcine reproductive and respiratory syndrome virus (prrsv), respectively, have exhibited antiviral activity in in vitro translation assays and in cell culture (han et al., ; patel et al., ; van den born et al., ; zhang et al., ) . regions of the arterivirus genome which can be inhibited by pmos have also shown to be target regions for inhibiting cov. these viruses possess a region within the utr of the genome termed the transcription regulatory sequence (trs). arginine-rich peptide-conjugated ppmos directed against this sequence have been shown to reduce sars cov yield in vitro by fold (neuman et al., ) . prophylactic administration of a ppmo targeting the utr genomic region reduced liver viral load and diminished hepatic pathophysiology in mice infected with murine hepatitis virus (mhv), another betacoronavirus related to sars cov (burrer et al., ; neuman et al., ) . however, viral challenge with delayed ppmo treatment given as a therapeutic regimen did not protect the animals (burrer et al., ) . a variety of studies of other human viruses have been published demonstrating the antiviral efficacy of pmos or ppmos, either in cell culture or in mouse infection models. subtypes of influenza a viruses, such as h n and h n , represent common seasonal respiratory pathogens for humans, yet highly pathogenic h n viruses can emerge and cause natural outbreaks. a collection of in vitro and in vivo studies have shown that ppmos targeting the translation start site for pb and the terminal region of np rna have high efficacy against several subtypes of influenza a: h n , h n , h n , h n and h n (gabriel et al., ; ge et al., ; lupfer et al., ) . the same ppmos, which were tested in vitro against strains of h n and h n , protected up to % of mice from lethal influenza infection when administered intranasally before and after virus challenge (gabriel et al., ; lupfer et al., ) . venezuelan equine encephalitis virus (veev) is a fast-replicating category b alphavirus that causes a rapid onset of debilitating illness in humans which is usually nonfatal. arginine-rich peptideconjugated ppmos directed against the utr and the aug region of the genomic rna of veev were administered as a combination therapeutic to mice before and/or after challenge with a lethal dose of virulent veev strain zpc (paessler et al., ) . the combination ppmo therapy was administered intranasally and intraperitoneally, with lg and lg given at each location, respectively. the prechallenge doses given both at day and h before infection imparted superior protection, compared to simple daily administration of the combination therapeutic at both dose sites after virus challenge. mice receiving ppmos before and after challenge were % protected from mortality due to veev infection, while only % of mice survived challenge when given the post-infection dose regimen of ppmos within a few hours after infection, as a postexposure prophylactic (paessler et al., ) . flaviviruses, many of which are categorized as niaid category a or b priority pathogens, rely on conserved structures within the and utrs of their viral genomes to form secondary stem-loop structures to direct and regulate viral transcription and translation [reviewed in (shurtleff et al., ) ]. west nile virus, dengue virus types - , japanese encephalitis virus (jev) and st. louis encephalitis virus can be blocked by arginine-rich peptide-conjugated ppmos directed against areas in the utr and the conserved region number (deas et al., ; deas et al., ; holden et al., ; kinney et al., ) . one study described the requirement for the presence of the arginine-rich peptide conjugated to the pmo for protection of mice from wnv infection, since subsequent experimental trials using unconjugated pmos did not protect (deas et al., ) . increased survival was observed in dengue- -infected ag mice treated using a prophylactic regimen, but not a post-infection regimen, with arginine-rich ppmos directed towards viral cyclization sequences in the and utr, in comparison to treatment with unconjugated versions of these pmos . as with other flaviviruses, arginine-rich ppmos targeting the viral cyclization sequence were able to inhibit jev infection of different cell types in vitro and prophylactically in a mouse model of intracerebral infection (anantpadma et al., ) . arenaviruses are another virus family that causes significant human disease and for which there are limited treatment options. peptide-linked pmos, termed ppmo term-s and ppmo term-l, were designed against conserved sequences in the termini of the arenavirus short and long rna segments, and evaluated in vitro against related arenaviruses: lymphocytic choriomeningitis virus (lcmv), tacaribe virus, pichinde virus and junin virus vaccine strain candid # (neuman et al., ) . in vitro, ppmos directed against the l terminus were able to reduce viral np and gp expression and reduce lcmv growth by fold (neuman et al., ) . when the ppmos were administered prophylactically to mice at or mg/kg, just prior to infection with lcmv and daily for days thereafter, significant reduction of viral growth in spleen and liver tissues was observed upon sampling at day (neuman et al., ) . these ppmos were only administered using a prophylactic regimen, and were not evaluated as strictly therapeutics. collectively, these studies suggest that modifying pmos to include positive charges improves their in vivo efficacy. additional investigations are currently in progress to evaluate the in vivo efficacy of pmoplus tm agents against flaviviruses, influenza viruses, hemorrhagic fever viruses, and other clinically important viral pathogens. while the complementary design of antisense molecules for target rna confers a high degree of selectivity to these agents, this aspect of antisense therapeutics poses limitations on the use of antisense molecules against virus variants that possess mismatches at the target site. two design strategies were implemented in avi- to enhance the binding of pmoplus tm agents with sequence variations known to occur in marv variants (fig. ) . first, positive piperazine moieties were selectively positioned at sites where mismatches were predicted, in an effort to stablilize non-complementary bases by strengthening the ionic interactions of the pmoplus tm molecules with the targeted viral rna. second, inosine bases, which indiscriminately pair with adenine, uracil, or cytosine, were incorporated at two positions in the agent targeting marv vp mrna and at one position in the agent targeting marv np mrna. inosine residues were positioned to produce complementarity at locations where bases were known to vary between marv variants, based on previous sequence analyses. efficacy results obtained in mouse, guinea-pig, and nhp infection models suggest that the pmoplus tm design strategy used in avi- successfully accommodated the sequences predicted in the mouse-adapted ravn virus isolate and in the marv-musoke isolates used in guinea-pig and nhp models of marv infection (warren et al., ) . a similar strategy may prove useful in designing agents against other viruses, to accommodate limited mispairing that would be predicted among virus variants or closely related species. the ability to rapidly develop and produce therapeutics is integral to efficiently responding to emergency situations involving pathogens that may be resistant or otherwise insensitive to available drugs. highly specific pmos can be rapidly synthesized in response to such a need. in , anti-ebov morpholino-based oligomers were designed, synthesized, and shipped to a biocontainment medical facility as a contingency therapeutic within days of a suspected accidental laboratory exposure event (kortepeter et al., ) . pmoplus tm -based therapeutics have also been designed and prepared as part of formal rapid-response exercises in only days (patrick iversen, personal communication). for these events, the viral agent was identified and the complete viral genomic rna target sequence was available at the time pmos were requested for immediate synthesis. while some of these efforts have formed part of biodefense preparations, they will also serve to refine the rapid responses required for scenarios involving emerging viruses and other pathogens. a rapid response to a veterinary medical crisis was generated in when a lethal outbreak of wnv in humboldt penguins occurred at the milwaukee county zoo. pmos were designed, synthesized and delivered for treatment within days (iversen, ) . all pmo-treated penguins survived, while of untreated penguins died. the small number of animals involved in this event limits any conclusions that can be drawn regarding the efficacy of the pmo treatment, but the event illustrates that pmos can be quickly designed and delivered in response to a medical crisis. this situation required only a relatively small-scale synthesis of pmos; obstacles associated with large-scale production technologies will need to be overcome to produce the large quantities of therapeutics needed for emergency mass treatment during outbreaks involving large numbers of patients or potentially infected individuals. it is well established that viruses can escape the effects of antiviral therapeutics through genomic alterations. this is a concern for aso-based therapeutics, for which antiviral resistance could conceivably occur through the propagation of minority variants containing nucleotide polymorphisms that prevent the efficient binding of the antisense agents to the target nucleotides. several reports document the rapid selection of rna viruses containing compensatory mutations -specifically sars cov, wnv, poliovirus type (pv ), and foot-and-mouth disease virus (fmdv) -enabling these viruses to overcome pmo antiviral activity following serial passage in cultured cells (deas et al., ; neuman et al., ; stone et al., ; vagnozzi et al., ) . nucleotide variations that alter the complementarity of the target rna with the antiviral antisense agents were responsible for conferring resistance in sars cov, fmdv, and pv ; however, in wnv, resistance determinants were identified outside of a ppmo target site when virus was serially passaged in the presence of a ppmo targeting the untranslated conserved sequence i ( csi) (deas et al., ) . it has been hypothesized that these mutations compensate for csi-mediated disruptions of wnv genome cyclization (stein, ; stein and shi, ) . wnv isolated from brains of infected mice treated with csi ppmo showed no mutations compared to original virus challenge stock (deas et al., ) . likewise, sequence obtained from virus isolated from the muscle of pv ppmo-treated mice did not show mutations . whether escape mutants appear during the replication of ebov and marv in infected nonhuman primates treated with antiviral pmoplus tm agents is an active area of research in our laboratory. rigorous deep-sequence-based investigations are in progress to characterize the occurrence and prevalence of potentially resistant minority quasispecies in virus challenge stocks and in viruses isolated from infected nonhuman primates. pmos possess multiple chemical properties favorable for use as therapeutic agents: they are highly soluble, stable during storage and in biological fluids, and can be designed with either a fine degree of antiviral specificity or with broad-spectrum efficacy against multiple viruses. investigators striving to develop antisense-based therapeutics have been challenged to identify aso chemical mod-ifications that enhance the in vivo efficacy of candidate therapeutics while minimizing the potential for toxicity. recent studies have shown that pmoplus tm therapeutic agents confer a high degree of protection against filoviruses. earlier-generation morpholino oligomers, i.e. neutrally charged pmos and ppmos, have been shown to be efficacious by a variety of routes of administration and against a wide range of pathogens. the stability and multiple-route efficacy of pmoplus tm agents make them well suited for use in a variety of settings, including regions lacking a modern medical infrastructure. two therapeutic agents, avi- and avi- , have entered phase i clinical safety studies, and preclinical efficacy evaluations are in progress for additional therapeutics in efforts to enhance the antiviral activity already achieved by other morpholino asos against a number of viral pathogens. because the molecular backbone of all pmoplus tm agents is identical, establishment of clinical safety profiles for one therapeutic may serve to expedite the regulatory review and approval of additional agents. while the pmoplus tm design can accommodate limited sequence mispairing, development of broad-spectrum antisense-based therapeutic agents will likely benefit from a parallel approach targeting both pathogen-specific products and host-based processes that viruses exploit during infection. as new discoveries are made of host pathways that divergent viruses rely on for entry, replication, or exit from infected cells, new potentially targetable genes will be identified to assist in efforts to develop broad-spectrum antisense-based therapeutics. opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the us army. delivery of steric block morpholino oligomers by (r-x-r) peptides: structure-activity studies genomic sequences from representative marv variants/isolates are shown as cdna complementary to genomic rna. the marv isolates shown include those against which avi- has exhibited antiviral activity in in vivo models (mouse-adapted ravn virus, guinea-pig adapted marv-musoke, and non-adapted marv-musoke), or which were isolated during outbreaks pharmacokinetics and biodistribution of phosphorodiamidate morpholino antisense oligomers pharmacokinetics, biodistribution, stability and toxicity of a cell-penetrating peptide-morpholino oligomer conjugate inhibition of japanese encephalitis virus replication in cultured cells and mice by a peptide-conjugated morpholino oligomer neutrally charged phosphorodiamidate morpholino antisense oligomers: uptake, efficacy and pharmacokinetics vp of marburg virus influences formation of infectious particles antiviral effects of antisense morpholino oligomers in murine coronavirus infection models oligodeoxynucleoside phosphorothioate stability in subcellular extracts, culture media, sera and cerebrospinal fluid antisense oligonucleotide-based therapeutics for cancer in vitro resistance selection and in vivo efficacy of morpholino oligomers against west nile virus inhibition of flavivirus infections by antisense oligomers specifically suppressing viral translation and rna replication nucleocapsid formation and rna synthesis of marburg virus is dependent on two coiled coil motifs in the nucleoprotein reduction of herpes simplex virus type- replication in cell cultures and in rodent models with peptide-conjugated morpholino oligomers vp knockdown inhibits ebola virus amplification and protects against lethal infection in mice microneedles: a valuable physical enhancer to increase transdermal drug delivery morpholino oligomers targeting the pb and np genes enhance the survival of mice infected with highly pathogenic influenza a h n virus inhibition of multiple subtypes of influenza a virus in cell cultures with morpholino oligomers fomivirsen: clinical pharmacology and potential drug interactions postexposure treatment of marburg virus infection postexposure protection of non-human primates against a lethal ebola virus challenge with rna interference. a proof-of-concept study chimeric oligodeoxynucleotide analogues: enhanced cell uptake of structures which direct ribonuclease h with high specificity nucleic acids-based therapeutics in the battle against pathogenic viruses enhanced inhibition of porcine reproductive and respiratory syndrome virus replication by combination of morpholino oligomers inhibition of irf- activation by vp is critical for the high level of virulence of ebola virus activation of the alternative pathway of complement by a phosphorothioate oligonucleotide: potential mechanism of action inhibition of dengue virus translation and rna synthesis by a morpholino oligomer targeted to the top of the terminal stem-loop structure antisense drug technology morpholinos efficacy of antisense morpholino oligomer targeted to c-myc in prostate cancer xenograft murine model and a phase i safety study in humans global suppression of the host antiviral response by ebola-and marburgviruses: increased antagonism of the type i interferon response is associated with enhanced virulence inhibition of dengue virus serotypes - in vero cell cultures with morpholino oligomers managing potential laboratory exposure to ebola virus by using a patient biocontainment care unit phosphorothioate oligodeoxynucleotides: antisense or anti-protein? cpg motifs in bacterial dna trigger direct b-cell activation design of antisense oligonucleotides stabilized by locked nucleic acids antisense properties of peptide nucleic acid inhibition of influenza a h n virus infections in mice by morpholino oligomers phosphorothioate analogs of oligodeoxynucleotides: inhibitors of replication and cytopathic effects of human immunodeficiency virus pharmacokinetics and tissue distribution of a peptide nucleic acid after intravenous administration the use of single-stranded dna and rnase h to promote quantitative 'hybrid arrest of translation' of mrna/dna hybrids in reticulocyte lysate cell-free translations inhibition of hsv- ocular infection with morpholino oligomers targeting icp and icp development of peptide-conjugated morpholino oligomers as panarenavirus inhibitors inhibition, escape, and attenuated growth of severe acute respiratory syndrome coronavirus treated with antisense morpholino oligomers antisense morpholino-oligomers directed against the end of the genome inhibit coronavirus proliferation and growth niaid biodefense research agenda for category b and c agents inhibition of alphavirus infection in cell culture and in mice with antisense morpholino oligomers peptide-conjugated morpholino oligomers inhibit porcine reproductive and respiratory syndrome virus replication ebola virus vp binds karyopherin alpha and blocks stat nuclear accumulation inhibition of iridovirus protein synthesis and virus replication by antisense morpholino oligonucleotides targeted to the major capsid protein, the kda immediate-early protein, and a viral homolog of rna polymerase ii genetic variation in the non-coding region of dengue viruses nonhuman primates as models for the discovery and development of ebolavirus therapeutics virus-specific antiviral treatment for controlling severe and fatal outbreaks of feline calicivirus infection antisense treatment of caliciviridae: an emerging disease agent of animals and humans establishing efficacy of human products using animals: the us food and drug administration's ''animal rule oligonucleotide antiviral therapeutics: antisense and rna interference for highly pathogenic rna viruses a specificity comparison of four antisense types: morpholino, -o-methyl rna, dna, and phosphorothioate dna inhibition of rna virus infections with peptide-conjugated morpholino oligomers treatment of ag mice with antisense morpholino oligomers increases survival time following challenge with dengue virus nucleic acid-based inhibition of flavivirus infections inhibition of rous sarcoma viral rna translation by a specific oligodeoxyribonucleotide a morpholino oligomer targeting highly conserved internal ribosome entry site sequence is able to inhibit multiple species of picornavirus morpholino, sirna, and s-dna compared: impact of structure and mechanism of action on off-target effects and sequence specificity chemical modifications of antisense morpholino oligomers enhance their efficacy against ebola virus infection mrna as gene therapeutic: how to control protein expression targeting the hiv- rna leader sequence with synthetic oligonucleotides and sirna: chemistry and cell delivery antiviral activity of morpholino oligomers designed to block various aspects of equine arteritis virus amplification in cell culture locked nucleic acid as a novel class of therapeutic agents antisense treatments for biothreat agents gene-specific countermeasures against ebola virus based on antisense phosphorodiamidate morpholino oligomers advanced antisense therapies for postexposure protection against lethal filovirus infections oligodeoxynucleotide stability in subcellular extracts and culture media aerosol and nasal delivery of vaccines and antiviral drugs against seasonal and pandemic influenza cell-penetrating peptides as transporters for morpholino oligomers: effects of amino acid composition on intracellular delivery and cytotoxicity stability of cell-penetrating peptide-morpholino oligomer conjugates in human serum and in cells inhibition of rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide suppression of porcine reproductive and respiratory syndrome virus replication by morpholino antisense oligomers technology evaluation: gem- the authors thank dr. james c. burnett for providing the structural figures presented in fig. . support for this work has been provided by the us defense threat reduction agency and by transformational medical technologies (w m- -c- ). key: cord- -sn o ugb authors: xue, qiao; liu, huisheng; zhu, zixiang; yang, fan; xue, qinghong; cai, xuepeng; liu, xiangtao; zheng, haixue title: seneca valley virus c protease negatively regulates the type i interferon pathway by acting as a viral deubiquitinase date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: sn o ugb the mechanisms that enable seneca valley virus (svv) to escape the host innate immune response are not well known. previous studies demonstrated that svv c(pro) suppresses innate immune responses by cleavage of host proteins and degradation of irf and irf protein expression. here, we showed that svv c protease ( c(pro)) has deubiquitinating activity. overexpressed c(pro) inhibits the ubiquitination of cellular substrates, acting on both lysine- - and lysine- -linked polyubiquitin chains. svv infection also possessed deubiquitinating activity. the ubiquitin-proteasome system was significantly involved in svv replication. furthermore, c(pro) inhibited the ubiquitination of retinoic acid-inducible gene i (rig-i), tank-binding kinase (tbk ), and tnf receptor-associated factor (traf ), thereby blocking the expression of interferon (ifn)-β and ifn stimulated gene (isg ) mrnas. a detailed analysis revealed that mutations (h a, c a, or h a/c a) that ablate the cys and his residues of c(pro) abrogated its deubiquitinating activity and the ability of c(pro) to block ifn-β induction. together, our results demonstrate a novel mechanism developed by svv c(pro) to promote viral replication, and may also provide a novel strategy for improving ubiquitination-based therapy. seneca valley virus (svv), belonging to the picornaviridae, is a positive-sense, single-stranded rna virus that is most closely related to cardiovirus (hales et al., ) . svv was first isolated in the united states in as a contaminant in the cell culture of human fetal retinoblasts (segales et al., ) . afterward, a large number of svv infections, which were characterized by porcine idiopathic vesicular disease, were observed in the united states, canada, brazil, and china (xue et al., ) . in china, the first case of svv infection was identified in guangdong province in . subsequently, new svv isolates were identified in guangdong and hubei provinces (qian et al., ; zhao et al., ) . in , we also identified a novel svv strain in fujian province in china (zhu et al., ) . many viruses have evolved strategies to evade innate immune response by inhibiting the host ubiquitination to promote their survival. for instance, human immunodeficiency virus- inhibits the antiviral response by the ub-mediated degradation of irf (okumura et al., ) , porcine reproductive and respiratory syndrome (prrs) virus inhibits nuclear factor kappa-light-chain-enhancer of activated b cells (nf-ĸb) activation by inhibition of the polyubiquitination process of iĸbα . to date, svv c pro has evolved mechanism to cleave or degrade innate immune adaptors to escape the host antiviral innate immune response (qian et al., ; xue et al., ) . however, other mechanisms that enable svv to escape the host innate immune response remain unclear. to determine whether svv can evade innate immune response by inhibiting the host ubiquitination, hek t cells were transfected with flag-tagged vp , vp , ab, b, c, d, c plasmids along with ha-ub plasmid. at h post-transfection (hpt), the ubiquitinated cellular proteins was assessed by western blotting. the svv a, c, and c pro inhibited the level of ubiquitinated cellular proteins, and c pro most significantly inhibited this process ( supplementary fig. ). human dubs are classified into five subfamilies based on their catalytic domains structures. they have a high degree of homology in the two regions known as cys and his boxes that surround the catalytic cys and his residues (nijman et al., ) . similar to other picornaviruses, svv c pro also possesses a conserved catalytic box with cys and his residues (qian et al., ) . therefore, the svv c pro was selected for further studies. to determine whether c pro functions as a dub, hek t cells were transfected with increasing amounts of plasmid encoding c pro along with ha-ub or empty vector. at hpt, the effect of c pro on all https://doi.org/ . /j.antiviral. . . received july ; received in revised form september ; accepted october ubiquitinated cellular proteins was assessed by western blotting. as shown in fig. a , expression of c pro resulted in a dose-dependent reduction of the level of ubiquitinated cellular proteins compared with that in the empty vector-transfected cells. to further determine which ub linkage type is targeted by c pro , hek t cells were transfected with ha-k -ub or flag-k -ub in lieu of ha-ub. at hpt, the cells were collected for western blotting. both k -and k -linked ub chains were also processed by c pro in a dose-dependent manner ( fig. b and c) . we also analyzed dub activity during svv infection. hek t cells were mock infected or infected with svv at a multiplicities of infection (moi) of for h. as shown in fig. a, the levels of endogenous ub, k , and k ubiquitinated cellular proteins were reduced in svv-infected cells compared to those in uninfected cells. taken together, these results confirm that svv exerts dub activity during viral infection and c pro is a potent viral dub that inhibits ub conjugates formed through k or k linkages in cellular substrates. svv c pro contains cys and his residues. therefore, the three mutants, namely the single-site mutants h a or c a, and the doublesites mutants h a/c a ( cdm) (xue et al., ) , were used to confirm whether the cys and his residues are involved in the dub activity of svv c pro . hek t cells were co-transfected with ha-ub-, ha-k -ub-, and flag-k -ub-expressing plasmids along with flag- c-expressing plasmid or flag- c mutants expressing plasmids. at hpt, the cells were collected for western blotting. all the c pro proteins without cys and his residues lost the ability to inhibit ub conjugates compared with the overexpression of wild-type svv c pro ( cwt) (fig. a-c) . to further confirm the dub activity of svv c pro , hek t cells were transfected with flag- c-expressing plasmid, flag- cdm expressing plasmid, or empty vector. at hpt, the cells were collected for western blotting. it showed that the c pro proteins without cys and his residues could no longer reduce levels of endogenous ubiquitinated ub, k , and k (fig. b) . taken together, these results indicate that the catalytic cys and his residues of svv c pro are required for its dub activity. we also investigated whether c pro is associated with the deubiquitination of rig-i, tbk , and traf . to do so, hek t cells cultured in -cm dishes were transfected with various plasmids. at hpt, cell lysates were immunoprecipitated with anti-flag or anti-myc antibody and analyzed by western blotting. overexpression of cwt significantly inhibited the ubiquitination of rig-i ( overexpression of cdm, which lacks dub activity, had no such effects. in addition, our results also demonstrated an interaction between c pro and either tbk , or traf ( fig. b and c). to investigate the interaction between c pro and rig-i, hek t cells were transfected with flag- c-expressing plasmid or empty vector. at hpt, cell lysates were immunoprecipitated with anti-rig-i antibody and analyzed by western blotting. rig-i pulled down flag- c (fig. d ), which confirmed that c pro interacted with rig-i. the interaction of c pro and rig-i, tbk , or traf in context of viral infection was further confirmed. hek t cells were mock-infected or infected with svv (moi ) for h. the cell lysates were immunoprecipitated with anti- c antibody and subjected to immunoblotting analysis. c pro pulled down rig-i, tbk , and traf in svv-infected cells (fig. e) . a reverse immunoprecipitation experiment was subsequently performed using anti-rig-i, tbk , or traf antibody, which showed that rig-i, tbk , or traf also immunoprecipitated c pro (fig. f ). these results indicate that the interaction of c pro with rig-i, tbk , and traf in the context of viral infection is involved in the suppression of ubiquitination levels. endogenous ubiquitination levels of rig-i, tbk , and traf in svv-infected cells were further assessed. hek t cells were mock infected or infected with svv (moi ) for h. cell lysates were immunoprecipitated with anti-rig-i, tbk , or traf antibody and analyzed by western blotting. the results showed that the ubiquitination levels of endogenous k for tbk , and k for rig-i and traf were reduced during svv infection (fig. g ). taken together, these results indicate that svv and c pro inhibit the ubiquitination of rig-i, tbk , and traf in a dub-dependent manner. ubiquitination and deubiquitination are important mechanisms that are involved in regulating type i ifn signaling pathways (peisley et al., ) . to date, many cellular ub ligase enzymes can regulate these processes. for example, the e ubiquitin ligase rnf or nrdp directly enhance ubiquitination of tbk , which facilitates the activation of tbk (song et al., ; wang et al., ) . meanwhile, many cellular dubs negatively regulate type i ifn signaling pathways. for example, usp , usp , and usp target rig-i, tbk , and traf for deubiquitination, respectively, thereby blocking the activation of type i ifn signaling pathways (cui et al., ; gu et al., ; lin et al., ) . dub activity also has been demonstrated in many bacteria and viruses, such as salmonella enterica serovar typhimurium, fmdv, prrsv, herpesviruses, coronaviruses, and bunyaviruses, and dub fig. . svv c pro inhibits the ubiquitination of cellular proteins in a manner that is dependent on its dub activity. (a) hek- t cells were seeded in six-well plates, and the monolayer cells were transfected with μg ha-ub-expressing plasmid along with μg flag- cwt-expressing plasmid, μg flag- c h a-expressing plasmid, μg flag- c c a-expressing plasmid, or μg flag- cdm-expressing plasmid. the empty flag vector was used in the transfection process to ensure that the same number of cells received the same amount of total plasmids. at hpt, the cells were collected for western blotting. similar transfection and analysis were performed for k -ub (b) and k -ub (c) as described above. (caption on next page) q. xue et al. antiviral research ( ) - enzymes play multiple roles in regulating bacterial or viral infections (fiskin et al., ; sun et al., ; van wijk et al., ; wang et al., ) . here, svv c pro also has dub activity. to investigate whether svv c pro can block the type i ifn signaling pathway, quantitative polymerase chain reaction (qpcr) analysis was performed to determine the transcript levels of the ifn-β, isg , and isg genes during viral infection. relative expression of mrna was calculated based on the comparative cycle threshold (ct) ( −ΔΔct ) method (schmittgen and livak, ) . the qpcr primers used in this study are listed in table . it showed that only flag- cwt was able to inhibit sev-induced ifn-β, isg , and isg mrna expression (fig. a ). to determine whether c pro affected the replication of svv, hek t cells were seeded in -well plates, and the monolayer cells were transfected with flag- c-expressing plasmid or empty vector. at hpt, the cells were infected with equal amounts of svv (moi ). at h post-infection (hpi), viral rna and protein levels were examined. overexpression of c pro significantly enhanced replication of svv (fig. b) . the ubiquitin-proteasome system (ups) plays important roles in the degradation of proteins, the immune response, and signal transduction (casorla-perez et al., ) . the ups is a double-edged sword in viral pathogenesis: the ups is necessary for many viruses replication by maintaining the proper functions of viral proteins (barrado-gil et al., ; luo, ; wang et al., ) ; the ups can constitute host immune system to reduce viral replication (luo, ) . for example, the ups was essential for coronavirus replication (raaben et al., ) , whereas coronavirus papain-like proteases can act as dubs that block type i ifn production (clementz et al., ) . proteasome inhibitors mg can inhibit the ups. to determine the impact of the ups on the replication of svv, hek t cells were infected with an equal amount of svv (moi ). at hpi, the cells were incubated with or without mg for h. viral rna and protein levels were examined. mg significantly inhibited replication of svv (fig. c ), which indicated that the ups was also essential for svv replication. therefore, we speculated that the ups plays an important role in maintaining functions of svv proteins. the abundance of rig-i, tbk , and traf during svv infection remained unclear. hek t cells cultured in -well plates were mock infected or infected with svv. the abundance of rig-i, tbk , and traf were compared at , , and h after svv infection. the results showed that svv infection had no impact on the abundance of rig-i and traf , but did inhibit expression of tbk (fig. d) . it is well known that reduction of the k -linked polyubiquitin can stabilize tbk . however, our results indicated that svv or c pro reduced expression of tbk . to clarify this unexpected result, a broad-range deubiquitinase inhibitor, pr- (sigma-aldrich), was selected for further study (altun et al., ) . the ubiquitination levels of endogenous k for tbk was enhanced after pr- incubation ( supplementary fig. ). hek t cells were transfected with flag- cexpressing plasmid or infected with svv (moi ). then, the cells were treated with or without pr- . cell lysates were analyzed by western blotting. the expression of tbk in the c pro -transfected and svv-infected cells was enhanced comparing with that in the pr- -treated cells (fig. e ), which indicated that c-induced reduction of the k linked polyubiquitin of tbk enhanced the expression of tbk . however, as a whole, c pro and svv still reduced the expression of tbk . svv c pro possesses protease activity. therefore, we speculated that c pro reduced the expression of tbk by its protease activity, and c pro partly stabilized tbk by its dub activity. to further investigate whether svv c pro can block rig-i-and tbk -induced type i ifn signaling pathway, we performed a luciferase reporter assay. hek t cells grown in -well plates were co-transfected with . μg/well of the plasmid ifn-β-luciferase (ifn-β-luc) along with . μg/well of plasmid prl-tk and plasmids encoding flag-rig-i, myc-tbk , flag- cwt, flag- cdm, or empty vector. at hpt, the cells were lysed and analyzed with a dual-specific luciferase assay kit. as shown in fig. f , rig-i-, and tbk -mediated ifn-β promoter activity was reduced in the presence of flag- cwt but not flag- cdm. to further confirm this effect, we conducted a qpcr analysis to determine the levels of the rig-i-and tbk -mediated endogenous transcription of the ifn-β and isg genes. the results showed that flag- cwt also inhibits rig-i-and tbk -induced ifn-β and isg mrna expression (fig. g) , which is in accordance with previous findings showing that svv c pro significantly inhibited the endogenous transcription of the ifn-β and isg genes that is mediated by rig-i and tbk (qian et al., ) . taken together, these results indicate that svv c pro promotes replication of svv and suppresses rig-i-and tbk induced type i ifn production in a manner that is dependent on its dub activity. in the present study, we provide direct evidence that svv c pro is a novel viral deubiquitinating enzyme. however, more work will be required to determine if other picornavirus c pro , such as encephalomyocarditis virus, enterovirus , coxsackievirus a , or fmdv, have dub activity. our data also uncover a novel mechanism by which svv c pro antagonizes type i ifn induction, i.e., by deubiquitinating the critical signaling moleculars rig-i, tbk , and traf . our results comparing various c pro mutants suggest that the dub activity of c pro enables it to block induction of the ifn-β promoter and the fig. . svv c pro inhibits the ubiquitination of rig-i, tbk , and traf . hek- t cells were seeded in -mm dishes, and the monolayer cells were cotransfected with μg ha-ub-expressing plasmid, μg flag- cwt-expressing plasmid, μg flag- cdm-expressing plasmid, and the rig-i (a), tbk (b), or traf (c) expression plasmids ( μg). mg ( nm) was added at hpt. cell lysates were prepared at h after treatment and immunoprecipitated with anti-flag or anti-myc antibody, and the ub conjugation of the proteins was detected by western blotting with anti-ha antibody. the input tagged proteins were detected with the indicated antibodies. (d) hek- t cells were seeded in -mm dishes, and the monolayer cells were transfected with μg flag- c-expressing plasmid or empty vector. at hpt, cell lysates were immunoprecipitated with anti-rig-i antibody and analyzed by western blotting. the whole-cell lysates and ip antibody-antigen complexes were analyzed by ib using anti-flag and anti-rig-i antibodies. (e, f) hek- t cells were seeded in -mm dishes, and the monolayer cells were mockinfected or infected with svv (moi ) for h. cell lysates were immunoprecipitated with anti- c antibody and analyzed by western blotting (e). similar infection and ip experiments were carried out as described above. however, the lysates were immunoprecipitated with anti-rig-i, tbk , or traf antibody and subjected to western blotting (f). (g) hek t cells were cultured in -cm dishes, and the monolayer cells were mock infected or infected with svv (moi ) for h. cell lysates were immunoprecipitated with anti-tbk , rig-i, or traf antibody and analyzed by western blotting. the whole-cell lysates and ip antibody-antigen complexes were analyzed by ib using anti-k , k , tbk , rig-i, traf , or vp antibodies. hc represents heavy chain. lc represents light chain. the qpcr primers used in this study. sequences ( ′- ′) target gene xue et al. antiviral research ( ) - endogenous transcription of the ifn-β and isg genes. studies have indicated that svv c pro mutants that abrogate cys and his residues lost protease activity (qian et al., ; xue et al., ) . here, our results indicated that svv c pro mutants that abrogate cys and his residues also lost dub activity. therefore, we speculate that the cys and his residues of svv c pro may contribute to both protease and dub activities. c pro might suppress type i ifn response by different mechanisms in host cells. svv has been identified as a novel oncolytic virus against several human cancers (burke, ) . ubiquitination and deubiquitination are mechanisms that also play important roles in regulation of cancer (kaushal et al., ) . whether svv can inhibit cancer progression through c pro dub activity is still unclear and will need to be investigated in future studies. it may provide a novel method for cancer therapy. (caption on next page) q. xue et al. antiviral research ( ) - fig. . svv c pro inhibits rig-i-and tbk -induced type i ifn production. (a) hek t cells were seeded in -well plates, and the monolayer cells were transfected with . μg flag- cwt-expressing plasmid, flag- cdm-expressing plasmid, or empty vector. at hpt, the cells were mock infected or infected with sev ( hemagglutinating activity units) for h. the expression of ifn-β, isg , and isg mrnas was determined with qpcr assay. (b) hek t cells were seeded in -well plates, and the monolayer cells were transfected with μg flag- c-expressing plasmid or empty vector. at hpt, the cells were infected with an equal amount of svv (moi ) for h. expression of viral rna was determined by qpcr assay. expression of viral vp protein was detected by western blotting. (c) hek t cells were seeded in -well plates, and the monolayer cells were infected with svv (moi ). at hpi, the cells were incubated with or without μm mg for h. expression of viral rna was determined by qpcr assay. expression of viral vp protein was detected by western blotting. (d) hek t cells were seeded in -well plates, and the monolayer cells were mock infected or infected with svv (moi ) for , and h. expression of tbk , rig-i, traf , and viral vp proteins were detected by western blotting. (e) hek t cells were seeded in -well plates, and the monolayer cells were transfected with μg flag- c-expressing plasmid or infected with svv (moi ). then, the cells were treated with or without μm pr- for h. cell lysates were analyzed by western blotting. (f) hek t cells grown in -well plates were co-transfected with . μg/well of ifn-β-luc along with . μg/well of prl-tk plasmid and . μg/well of plasmids encoding flag-rig-i, myc-tbk , flag- cwt, flag- cdm, or empty vector. at hpt, the cells were lysed. the dual-specific luciferase assay kit was used to analyze the luciferase activities of firefly and renilla. the data represent the means and standard deviations from three independent experiments. (g) hek- t cells were seeded in six-well plates, and the monolayer cells were co-transfected with μg flag-rig-i-expressing plasmid, μg flag- cwt-expressing plasmid, μg flag- cdm-expressing plasmid, or μg empty vector. the empty vector was used in the transfection process to ensure that the same number of cells received the same amount of total plasmids. similar transfection were performed for myc-tbk as described above. at hpt, the expression of ifn-β and isg mrnas was determined with qpcr assay. activity-based chemical proteomics accelerates inhibitor development for deubiquitylating enzymes the ubiquitin-proteasome system is required for african swine fever replication oncolytic seneca valley virus: past perspectives and future directions the ubiquitin-proteasome system is necessary for the efficient replication of human astrovirus deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases usp inhibits type i interferon signaling by deubiquitinating rig-i-like receptors global analysis of host and bacterial ubiquitinome in response to salmonella typhimurium infection usp suppresses cellular type i interferon signaling by targeting traf for deubiquitination complete genome sequence analysis of seneca valley virus- , a novel oncolytic picornavirus deubiquitinating enzymes in cancer stem cells: functions and targeted inhibition for cancer therapy usp inhibits type i interferon signaling by editing tbk ubiquitination through nlrp signalosome interplay between the virus and the ubiquitin-proteasome system: molecular mechanism of viral pathogenesis a genomic and functional inventory of deubiquitinating enzymes hiv- accessory proteins vpr and vif modulate antiviral response by targeting irf- for degradation structural basis for ubiquitinmediated antiviral signal activation by rig-i seneca valley virus suppresses host type i interferon production by targeting adaptor proteins mavs, trif, and tank for cleavage the ubiquitin-proteasome system plays an important role during various stages of the coronavirus infection cycle analyzing real-time pcr data by the comparative c(t) method e ubiquitin ligase rnf promotes innate antiviral immunity through k -linked ubiquitination of tbk the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein possesses deubiquitinating and interferon antagonism functions linear ubiquitination of cytosolic salmonella typhimurium activates nf-kappab and restricts bacterial proliferation the e ubiquitin ligase nrdp 'preferentially' promotes tlr-mediated production of type i interferon the leader proteinase of foot-and-mouth disease virus negatively regulates the type i interferon pathway by acting as a viral deubiquitinase the ubiquitin-proteasome system is essential for the productive entry of japanese encephalitis virus complete genome sequence of seneca valley virus ch- - identified in china seneca valley virus c(pro) abrogates the irf -and irf -mediated innate immune response by degrading irf and irf emergence of novel seneca valley virus strains in china this work was supported by grants from the national natural sciences foundation of china (no. u and ), the national key research and development program of china ( yfd ), the gansu science foundation for distinguished young scholars (no. rjda ) and the gansu science foundation for young scholars ( rjya ). the authors declare no competing financial interest. supplementary data to this article can be found online at https:// doi.org/ . /j.antiviral. . . . 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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