cord-000354-05lnj3w0 2011 The sensitivity of the dynamin-independent entry pathway to inhibitors or dominant-negative mutants affecting actomyosin dynamics as well as to a number of specific inhibitors of growth factor receptor tyrosine kinases and downstream effectors thereof all point to the involvement of macropinocytosis in IAV entry. As factors present in serum are known for their potential to induce specific endocytic pathways, we further explored the conditions required for the novel DYNA-IND IAV entry pathway (using the Gluc-entry assay) by inoculating cells in PBS in the presence of increasing concentrations of fetal calf serum (FCS). Growth factor inducible activation of the tyrosine kinase src has also been linked to the induction of macropinocytosis [49] [50] [51] ; consistent with this observation the src inhibitor PP2 [52] specifically inhibited DYNA-IND entry of IAV (Fig. 9A-B) . cord-001017-4qfhltg4 2013 Previous studies demonstrated that microglia can be persistently infected by neurotropic mouse hepatitis virus (MHV) which causes meningoencephalitis, myelitis with subsequent axonal loss, and demyelination and serve as a virus-induced model of human neurological disease multiple sclerosis (MS). In our current studies, we have used RSA59 infection in vivo, in vitro, and ex vivo as a model to understand whether MHV can directly infect CNS resident microglia and the mechanism of microglial activation in the induction of chronic demyelination. To confirm the RSA59-induced CNS inflammation, brain and spinal cord sections from day 7 (peak of inflammation) and day 30 (peak of demyelination) postinfected mice were stained with H&E or LFB and examined. While Iba1 immunofluorescence was observed in both gray and white matter, double fluorescence/immunofluorescence demonstrated dual labelling of EGFP (viral antigen) positive Iba1 positive microglia/macrophages were present only in the white matter of RSA59 infected mice (Figure 1 ). cord-001065-j4hvyyoi 2013 title: In Vitro Infection of Pupae with Israeli Acute Paralysis Virus Suggests Disturbance of Transcriptional Homeostasis in Honey Bees (Apis mellifera) An experimental protocol to test these systems was developed, using injections of Israeli Acute Paralysis Virus (IAPV) into honey bee pupae reared ex-situ under laboratory conditions. Gene expression analyses of three separate experiments suggest IAPV disruption of transcriptional homeostasis of several fundamental cellular functions, including an up-regulation of the ribosomal biogenesis pathway. Little is known about the specific biology of the viruses in these families that infect honey bees, although they contain important bee pathogens, such as Deformed Wing Virus (DWV) and Israeli Acute Paralysis Virus (IAPV). Post-hoc tests of main treatment effects showed significantly higher gene expression in the IAPV-inoculated bees compared to the two control groups for Actin, 28S rRNA, and mGST1. The observed gene expression patterns could be due to viral manipulation of the cells to increase virus replication or present cell compensatory responses to IAPV infection. cord-001732-4eyn7pjq 2015 Evaluation of single-dose and repeated-dose toxicity of the vaccine candidate after i.d. administration to naive, non-infected mice did not reveal any safety concerns. To assess toxicity of LEISHDNAVAX in naive mice, sterile phosphate-buffered saline (PBS, placebo) and ascending doses of the vaccine were injected either once or five times in weekly intervals (Table 1) . Twenty-four hour after single or repeated injection, MIDGE-Th1 vector DNA was detected in almost all organs and tissues examined, suggesting that it was distributed systemically, most likely via the lymphatic system and the blood stream. Groups of 10 BALB/c mice (5 male, 5 female) were immunized i.d. at the tail base five times in weekly intervals with either PBS or LEISHDNAVAX (10, 50 or 100 μg per dose). In summary, we have shown here that LEISHDNAVAX, a novel DNA vaccine candidate against leishmaniasis is safe and well tolerated in both naive and Leishmania-infected mice. cord-002013-rb9xdzro 2016 To investigate whether EBOV infections can be controlled by virus neutralization alone, and to prevent the possible induction of serum sickness in humans that would be administered antisera, the post-exposure efficacy of F(ab′ ) 2 (immunoglobulin treated with pepsin to remove the Fc regions of the antibody) were also investigated side-by-side with equine antisera in all experiments as a potential alternate treatment. Three horses were immunized intramuscularly (IM) with 7 injections of eVLP over 11 weeks and the hyperimmune sera were collected from each animal at specified timepoints ( Fig. 1A) to determine the serum titers by neutralization assay against a recombinant HIV-1 virus pseudotyped with EBOV GP. The observation that administering F(ab′ ) 2 at 1 dpi is more efficacious than when the same treatment was given at 30 minutes post-exposure (Fig. 5A ) was also observed in past studies with therapeutic EBOV GP-specific monoclonal antibodies 25, 26 , and suggests that virus neutralization may play a bigger role in protection at a later timepoint after EBOV challenge. cord-002583-cgcf7mgj 2017 The results showed that the survival time of mice in the 500 mg Chinese herbs group and sulfadiazine group was significantly longer than that of the PBS control group. Also the parasite load in blood and tissues of 500 mg Chinese herbs and sulfadiazine groups was significantly lower than that of PBS group at 7 days post infection (dpi), which was in accordance with the result of histological detection. Results of spleen, lung, and liver tissues presented a similar pattern in that parasite loads were all largely increased from 3 to 7 dpi, and mice of the PBS control group had statistically significantly higher parasite load compared with sulfadiazine and 500 mg Chinese herb groups (P<0.05). The lungs of sulfadiazine and Chinese herbs-treated mice possessed a significantly lower parasite load than that of the PBS control group (P<0.05) and the histological result verified this from the morphological perspective. cord-003915-kje8lvgl 2019 As orally transmitted viruses, densoviruses, are also challenged by the complexity of the insect gut barriers, more specifically by the chitinous peritrophic matrix, that lines and protects the midgut epithelium; how capsids stick to and cross these barriers to reach their final cell destination where replication goes has been poorly studied in insects. In addition, we showed that JcDV early infection results in (i) an arrest of N-Acetylglucosamine (GlcNAc) secretion by epithelial cells associated with a disorganization of the PM structure mimicking the effect of chitin-binding plant lectin; (ii) substantial changes in the expression of gut genes, which may also contribute to an early gut dysfunction and participate to viral pathogenesis. Results presented here show that JcDV capsids display carbohydrate-binding properties that insure recognition of the peritrophic matrix and determines caterpillars oral infection. cord-009800-tajkpgkj 2003 The cross‐reactivity of an immunological probe to the key photosynthetic enzyme Rubisco (ribulose‐1,5‐bisphosphate carboxylase/oxygenase) was characterized as part of a larger effort to determine maximal photosynthetic rates of individual phytoplankton cells. In order to use an immunoprobe to quantitatively measure Rubisco concentration within individual cells in a mixed-species assemblage, it is imperative to determine: first, whether the probe is truly specific to only the large subunit of Rubisco; second, whether the probe binds to the Rubisco large subunit of all phytoplankton species with equal affinity; and third, whether immunologically determined concentrations of Rubisco are well correlated with maximal photosynthetic rates. The results indicate that an affinity-purified anti-Rubisco probe can be used to quantify Rubisco concentrations and maximal photosynthetic potential of individual phytoplankton cells in mixed-species assemblages. When the more quantitative dot blot assays were used with purified Rubisco from different species, a high degree of variability in the binding affinity with the polyclonal antiserum was evident (Fig. 1) . cord-011106-h20vbmbo 2019 Here, we analyzed the antiviral activity of hibiscus (Hibiscus sabdariffa L.) tea extract against human IAV and evaluated its potential as a novel anti-IAV drug and a safe inactivating agent for whole inactivated vaccine. In addition, we assessed hibiscus tea extract''s potential as a candidate for novel anti-IAV drug and as an inactivating agent for whole-virus vaccines. PR8 virus propagated in allantoic fluid was mixed with an equal amount of neutral and acidic pH PBS, Hib[crude], frHibis, or PCA. 50 μl PBS, formalin-, β-PL-, or acidic Hib[crude]-inactivated PR8 virus vaccine was intranasally administered in mice (first vaccination) under light anesthesia with isolflurane (Intervet K.K., Tokyo, Japan). The neutralized Hib[crude] in the blood loses potent anti-IAV activity due to acid, and the low-pH-independent antiviral activity is inadequate to inactivate virus in vivo. cord-013265-qrfi6e5c 2020 To evaluate the efficacy of repeated hinokitiol administration in a pneumonia mouse model, all mice were intratracheally infected with S. Here, compared with PBS injection, intratracheal administration of 500 μg/mL hinokitiol in a pneumococcal pneumonia mouse model decreased inflammatory cell migration and prevented destruction of alveolar tissue (Fig 2) . However, in clinical settings, antimicrobial agents are administrated several times for the treatment of pneumococcal pneumonia, and thus, we next sought to investigate whether repeated hinokitiol injection decreased the bacterial load in (Fig 7; P < 0.05) . The present study showed that intratracheal administration of hinokitiol decreased the bacterial load in a mouse pneumonia model regardless of macrolide resistance of S. In this study, intratracheal hinokitiol administration reduced neutrophil infiltration and elastase release in the lungs of pneumonia mice, consequently diminishing lung injury and pneumococcal DNA detection in serum. cord-013526-6fip93l2 2020 Here we used Bluetongue virus (BTV) as a model of a non-enveloped arthropod-borne virus and discovered that the majority of viruses are released in EVs. Based on the cellular proteins detected in these EVs, and use of inhibitors targeting the cellular degradation process, we demonstrated that these extracellular vesicles are derived from secretory lysosomes, in which the acidic pH is neutralized upon the infection. Virus released in secretory lysosomes infectious EVs. However, inhibition of autophagosome-lysosome fusion with chloroquine (CQ), led to a significant reduction of infectious EVs released as compared to the control ( Fig 2B) , indicating that the late steps of autophagy are necessary for infectious EVs. In addition, inhibition of MVBs regulator protein HSP90 using geldanamycin in BTV-infected cells (MOI = 10) also led to a significant reduction of infectivity measured in the EVs fraction, as compared to the control, indicating a possible role for MVBs in the release of infectious EVs. In contrast, GW4869, a drug that inhibits the release of exosomes (small vesicles~200nm) derived from MVBs, did not affect the secretion levels of EVs containing BTV ( Fig 2B) . cord-026866-0hlre9i6 2020 In this study, we describe the prophylactic activity of orally delivered recombinant SIgA generated from two human monoclonal antibodies (CAA1 and CCG4) isolated for their reactivity against the flagellar-capping protein FliD, which is essential for bacteria motility and highly conserved across Campylobacter species associated with severe enteritis. In this study, we describe the prophylactic activity of orally delivered recombinant SIgA generated from two human monoclonal antibodies (CAA1 and CCG4) isolated for their reactivity against the flagellar-capping protein FliD, which is essential for bacteria motility and highly conserved across Campylobacter species associated with severe enteritis. In this study, an immunocompetent mouse model of Campylobacter infection was used to evaluated the prophylactic activity of orally delivered recombinant SIgA generated from human monoclonal antibodies isolated and selected for their reactivity against the flagellar-capping protein FliD, which is pivotal for bacteria motility (25), and highly conserved across Campylobacter species frequently associated with severe neonatal enteritis (26) . cord-259094-5qzbctan 2013 title: The effect of dietary intake of the acidic protein fraction of bovine colostrum on influenza A (H1N1) virus infection In this study, we isolated the acidic protein fraction of bovine colostrum (AFC, isoelectric point <5) by anion-exchange chromatography, and investigated the effect of its dietary intake on influenza A (H1N1) virus infection. In this study we isolated the acidic protein fraction of bovine colostrum (AFC) and investigated how dietary intake of AFC modulates the symptoms of influenza infection. The three mouse groups were given oseltamivir, PBS and AFC orally for 14 days prior to infection with influenza A virus. Therefore, the effect of total bovine colostrums on influenza virus infection in mice was significantly different from our expectation. In this study, we provide the first evidence that orally supplemented AFC is effective in reducing the symptoms associated with influenza A virus infection. cord-259364-8zoav7b0 2019 title: Production of anti-Trichophyton rubrum egg yolk immunoglobulin and its therapeutic potential for treating dermatophytosis The aim of this study was to estimate the therapeutic potential of specific egg yolk immunoglobulin (IgY) on dermatophytosis caused by Trichophyton rubrum. rubrum cell wall proteins IgY (anti-trCWP IgY) presented a certain degree of cross-reactivity with different fungi. In the in vitro and in vivo activity researches, Anti-trCWP IgY showed a significant dose-dependent growth inhibitory effect on T. To estimate the cross-reactivity (CR) of anti-trCWP IgY against other fungi, indirect ELISAs were performed by the use of antigens from different fungi according to the method described previously. rubrum and the protective effect of anti-trCWP IgY against dermatophytosis of experimentally infected mice. In our study, the cell wall proteins showed good immunogenicity, highest titer of anti-trCWP IgY reaching 1:16000. rubrum in a dose-dependent manner compared with IgY from unimmunized hens,and significantly reduced lesion scores of mice experimentally infected with T. cord-260834-v254de8k 2019 The phage ELISA was conducted to measure the performances and effects of different blocking agents on the binding of αUbi phages against crude rUbi. Out of the four samples, the combination of PTM buffer and lysate showed the highest signal (1.226) detected with anti-c-myc-HRP as shown in Fig. 4c . Lysate preblocking and phage preincubation effects towards αUbi and M13KO7 phages against rUbi. To further optimize the biopanning conditions against crude antigens, the ''Yin-Yang'' (capture and eliminate) approach was designed to reduce non-specific binding by background phages. coli proteins in the crude fraction of rUbi with the actual concentration of rUbi being expected to be lower than the purified rUbi, the binding capability of αUbi is still unaffected as shown in phage ELISA assay upon affinity selection against crude rUbi. The blocking effect of PTM and E. cord-264267-weat0qs6 2020 Four polymorphisms (K267E, K267N, A291P and Δ346-348) strongly reduced binding of MERS-CoV S to DPP4 and S protein-driven host cell entry, as determined using soluble S protein and S protein bearing rhabdoviral vectors, respectively. For host cell entry, the surface unit, S1, of MERS-CoV S binds to the cellular type-II transmembrane protein dipeptidyl peptidase 4 (DPP4, CD26) [15] . For the binding studies with solMERS-S1-Fc, a similar protocol was followed as described for the analysis of DPP4 surface expression with the exceptions that sol-MERS-S1-Fc was used instead of the primary antibody (1:10 dilution in PBS/BSA) and that an AlexaFluor488conjugated anti-human antibody (goat, 1:500 dilution in PBS/BSA, ThermoFisher Scientific) was employed as the secondary antibody. Reduced MERS-CoV S-driven host cell entry is caused by inefficient S protein binding to DPP4 harboring polymorphic amino acid residues. cord-265740-wjdeps3h 2020 Given that SARS-CoV-2 viral RNA has demonstrated stability, we posited that phosphate buffered saline (PBS) may be a viable transport medium, as an alternative to VTM), for clinical qPCR testing. We assessed the intraand inter-individual reliability of SARS-CoV-2 qPCR in clinical endotracheal secretion samples transported in VTM or PBS, evaluating the stability of the RT-qPCR signal for three viral targets (N gene, ORF1ab, and S gene) when samples were stored in these media at room temperature for up to 18 hours. We report that using PBS as a transport medium has high intra-and inter-individual reliability, maintains viral stability, and is comparable to VTM in the detection of the three SARS-CoV-2 genes through 18 hours of storage. SARS-CoV-2 detection using standard testing of upper airway secretions requires a nasopharyngeal (NP) or oropharyngeal (OP) swab that is transported to a clinical laboratory using viral transport media (VTM) (https://www.fda.gov/medical-devices/emergency-situations-medical-devices/faqs-diagnostic-testingsars-cov-2#offeringtests, last accessed April 29 2020). cord-267736-rya9w6sh 2012 The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus. When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. cord-273035-sewfb3q8 2005 Background: Definitive early-stage diagnosis of severe acute respiratory syndrome (SARS) is important despite the number of laboratory tests that have been developed to complement clinical features and epidemiologic data in case definition. Results: The discriminatory classifier with a panel of four biomarkers determined in the training set could precisely detect 36 of 37 (sensitivity, 97.3%) acute SARS and 987 of 993 (specificity, 99.4%) non-SARS samples. We established a decision tree algorithm consisting of four unique biomarkers for acute SARS in the training set and subsequently validated the accuracy of this classifier by use of a completely blinded test set. To identify the serum biomarkers that could distinguish SARS from non-SARS samples, we used a training set of specimens (37 SARS acute and 74 controls; Tables 1 and 2) and constructed the decision tree classification algorithm using 10 989 peaks [99 peaks ϫ (37 ϩ 74) spectra] of statistical significance identified in the low energy readings (see Materials and Methods). cord-274396-l611eisi 2020 While the lopinavir-ritonavir-, hydroxychloroquine sulfate-, or emtricitabine-tenofovir-treated group exhibited lower overall clinical scores than the phosphate-buffered saline (PBS)-treated control group, the virus titers in nasal washes, stool specimens, and respiratory tissues were similar between all three antiviral-candidate-treated groups and the PBS-treated control group. Compared to the PBS-treated control group, azathioprine-immunosuppressed ferrets exhibited a longer period of clinical illness, higher virus titers in nasal turbinate, delayed virus clearance, and significantly lower serum neutralization (SN) antibody titers. In order to determine the antiviral efficacies of lopinavir-ritonavir, hydroxychloroquine (HCQ) sulfate, or emtricitabine-tenofovir for treatment of SARS-CoV-2 infection, SARS-CoV-2 antibody-free ferrets (10/group) were inoculated with 10 5.8 50% tissue culture infective doses (TCID 50 )/ml of an NMC-nCoV02 strain through the intranasal (i.n.) route ( Fig. 1 ). Therefore, although clinical symptoms were attenuated in ferret groups treated with antiviral candidates, we also evaluated virus titers in respiratory and gastrointestinal tracts using nasal washes and stool samples, respectively, from SARS-CoV-2-infected ferrets. cord-275635-d50bxe7c 2015 To explore the possibility of developing a vaccine against transmissible gastroenteritis virus (TGEV) infection, a recombinant swinepox virus (rSPV-SA) expressing a TGEV protective antigen has been constructed. Results from the passive immunity protection test of new born piglets demonstrated that the recombinant live-vector vaccine, rSPV-SA, could 100% protect piglets from the SPV infection, and there was no significant clinical symptom in the rSPV-SA treatment group during this experiment. Eight one-month-old swine (Large White) were randomly divided into four groups (2 pigs per group) and were immunized twice at 0 and 28 days with infectious rSPV-SA (1 × 10 8 PFU/ml in 2 ml of PBS), inactivated-TGEV (1 × 10 8 PFU/ml in 2 ml of PBS), wtSPV (1 × 10 8 PFU/ml in 2 ml of PBS) or PBS, each time via three routes: oral, nasal, and intraperitoneal. To explore whether mice or swine generated TGEV neutralizing antibodies, serum from the PBS, wtSPV, inactivated-TGEV and rSPV-SA treated mice and pig were collected at 0, 14, 21, 35, 42 days post-primary immunization (1:100-1:12,800 dilution in a 100 l volume). cord-275779-ocbygkyb 2017 title: Antibody-Dependent Cell-Mediated Cytotoxicity Epitopes on the Hemagglutinin Head Region of Pandemic H1N1 Influenza Virus Play Detrimental Roles in H1N1-Infected Mice Engaging the antibody-dependent cell-mediated cytotoxicity (ADCC) for killing of virus-infected cells and secretion of antiviral cytokines and chemokines was incorporated as one of the important features in the design of universal influenza vaccines. In this study, we determined the ADCC and antiviral activities of two putative ADCC epitopes, designated E1 and E2, on the HA head of a pandemic H1N1 influenza virus in vitro and in a lethal mouse model. The phenotype was potentially due to an exaggerated inflammatory cell infiltration triggered by ADCC, as an upregulated release of cytotoxic granules (perforin) was observed in the lung tissue of E1-vaccinated mice after H1N1 influenza virus challenge. While the antiviral efficacy provided by the stalk-specific ADCC antibodies has been confirmed (12) , our data raised concerns on the side effect of certain HA head epitopes in devising a universal influenza vaccine. cord-276732-u2d1z4ip 2017 -Arterial blood gas analysis for gas exchange -Wet-to-dry ratio as index of edema -Micro-CT scan to measure change over time in non-aerated lung tissue expressed as percentage of the whole lung tissue, with more negative values representing larger decrease of alveolar collapse; -Histopathology examination performed according to previous study [12] evaluating alveolar serofibrinous exudate and alveolar hemorrhage -Bronchoalveolar lavage for differential cell count, total protein content (with bicinchoninic acid method) and keratinocyte chemoattractant (CXCL1, previously named KC), and tumor necrosis factor-α (TNF-α) were assayed by ELISA -Blood withdrawal for PTX3 levels measurement in plasma (ELISA assay) (b)In 1 week from lung injury D-dimer (marker of fibrinolysis) [20] and matrix metalloproteinase 13 (MMP13), an enzyme that participates in collagen degradation [21] , were detected by ELISA and by western blot in lungs lysate, respectively. cord-277054-eq4obbte 2009 The potency of modified DNA vaccines assessed by total antibody response, antibody isotypes, cytokine profile, neutralizing antibody titer and protection conferred against in vivo challenge; was enhanced in comparison to native unmodified vaccine, but the response elicited did not pertain to the type of target sequence and the directed arm of immunity. The ability of these DNA vaccines to elicit protective responses in immunized mice was assessed by intracerebral challenge with 20 LD 50 of virulent rabies virus CVS strain. In an effort to develop an optimal DNA vaccine against rabies virus, this study was aimed at evaluating the immune enhancement potential of different antigen targeting strategies to selectively improve responses mediated by CD8 + and CD4 + T lymphocytes and by antibodies, induced after intramuscular immunization with DNA plasmids. also reported that DNA vaccine encoding rabies virus glycoprotein lacking transmembrane domain though enhances antibody response but does not confer protection [35] . cord-278802-bverdk5w 2010 On 0, 7, 14, 21, 28 and 35 post first vaccination, the dynamic changes of peripheral lymphocyte proliferation, cytokine assays and serum antibody titers were assayed respectively by MTT method, ELISA and hemagglutination inhibition assay (HI). The results showed that sodium new houttuyfonate significantly raised IB antibody titer in the chickens and also markedly promoted lymphocyte proliferation. Five chickens were sampled randomly from each group for assay of lymphocyte proliferation by MTT method and 10 chickens from each group for assessment of serum HI antibody titer by the micro-method and cytokines by ELISA kits at 0, 7, 14, 21 and 35 days post the primary immunization (dpi). In this study, with chicken IBV H120 strain live attenuated vaccine in chickens, by measuring peripheral blood lymphocyte proliferation and serum cytokines and antibody titers, the humoral and cellular immunity induced with SNH was researched. cord-292862-ezrkg0dc 2020 We show that polystyrene nanoparticles and five liposome formulations do not accumulate in injured lungs, indicating that nanostructures that are not based on protein are not intrinsically drawn to marginated neutrophils in acute inflammation. 6, 10, 14, 18 Single cell suspensions prepared from mouse lungs were probed by flow cytometry to further characterize pulmonary neutrophils in naïve mice and in mice following LPS-induced inflammation. The protein component of each particle was labeled with 125 I for tracing in biodistributions, and assessed 30 minutes after IV administration of NPs. Both absolute LDNG lung uptake and ratio of lung uptake to liver uptake registered a ~25-fold increase between naïve control and LPS-injured animals (Figure 2A , Supplementary Table 1) . As with LDNGs and albumin NPs in Figure 2C -H, single cell suspensions were prepared from LPS-inflamed and naïve control lungs after circulation of fluorescent DBCO-IgG liposomes. cord-295033-5fd9bu60 2011 The data suggest that immunization of hens with Stx2B could be a strategy to obtain at low cost a relatively high concentration of anti-Stx2 egg yolk IgY, able to neutralize Stx2 lethal activity. The data suggest that immunization of hens with Stx2B could be a strategy to obtain at low cost a relatively high concentration of anti-Stx2 egg yolk IgY, able to neutralize Stx2 lethal activity. Specific anti-Stx2B polyclonal antibodies obtained from chicken egg yolk and rabbit sera recognized not only the denatured and native form of B subunit, used for immunization but the antibodies were also able to recognize the denatured and native wild type Stx2 holotoxin in western blot (Fig. 3) , indirect ELISA (Fig. 4) and sandwich ELISA (Fig. 5) . In the present report, specific egg yolk IgY antibodies with binding and neutralizing capabilities against the wild type Stx2 toxin were obtained after immunization of laying hens. cord-295416-y3lvcjqd 2020 These data suggest that in the absence of preimmunity, stabilized PreF antigens may still be associated with aberrant Th2 responses that induce lung pathology in response to RSV infection, and can be prevented by formulation with more Th1/Th2-balanced adjuvants that enhance CD4+ and CD8+ IFNγ+ T cell responses. The objective of this study was to determine the capacity of RSV pre-fusion (PreF) vaccines formulated with different adjuvants to generate neutralizing antibody, prevent virus replication, and protect from pulmonary pathology following RSV challenge. Despite lower neutralizing antibody titers in mice immunized with PreF/Advax-SM, as compared to PreF/Alum, both immunization groups had undetectable RSV in their lungs at 4 dpi ( Figure 1F ). Taken together, these data indicate that RSV PreF immunization formulated with Th1-skewing adjuvants, like Advax-SM, provide protection against RSV infection in naïve BALB/c mice as compared to alum by inhibiting viral replication without eliciting enhanced pulmonary pathology. cord-298224-flyx85lr 2020 Following optimization with antiglyceraldehyde 3-phosphate dehydrogenase (GAPDH) siRNA, PEI and PEI-LPEG anti-IL8 siRNA nanoparticles were assessed for efficacy using polarised Calu-3 human airway epithelial cells and a twin stage impinger (TSI) in vitro lung model. This work demonstrates the potential of nebulised PEI-PEG siRNA nanoparticles in modulating pulmonary inflammation and highlights the need to move towards more relevant in vitro and in vivo models for respiratory drug development. In contrast, the nebulised PEI-LPEG siRNA nanoparticles demonstrated significantly greater levels of GAPDH knockdown versus the PBS-treated controls at higher doses. Using the differential cell staining of BAL samples with Eosin Y and azur/methylene blue, it was In the case of the PEI-LPEG siRNA nanoparticle-treated groups, both the non-targeting (NT) and anti-CXCL-1 siRNA-treated groups demonstrated 10-fold decreases in the CXCL-1 gene expression compared to the PBS-LPS samples (4-vs. cord-303381-xvzhb7ix 2015 The effect of environmental acidification on Ibaraki virus (IBAV) infection was tested using endosomal inhibitory chemicals and low pH treatment. Treatment of target cells with endosomal inhibitors significantly decreased the progeny virus production. HmLu-1 (hamster lung) cells were infected with IBAV in the presence of three different endosome inhibitors, bafilomycin A1 (Baf A1, Sigma, St. Louis, MO, U.S.A.), chlorpromazine (CPZ, Abcam, Cambridge, U.K.) and dynasore (Wako, Osaka, Japan). These results indicated that IBAV utilizes clathrin-dependent endosomal pathway for infection and coincided with the previous research on bluetongue virus entry [8] . To confirm the effect of low pH on virus infectivity, purified IBAV was incubated in PBS (−) with several pH (pH=4, 7 or 9) for five min and infected to HmLu-1 at MOI=0.01. The result obtained above implicated that low pH treat-ment removes IBAV outer capsid proteins from the particle and initiates its infection. cord-305648-majanu8l 2007 We developed a 1-hour field enzyme immunoassay (EIA) for detecting antibody to Sin Nombre virus in deer mice (Peromyscus maniculatus). Samples were retested under laboratory conditions with PAGEIA and standard Centers for Disease Control and Prevention (CDC) enzyme immunoassay (EIA) (5) . One sample (HA-2564) was scored negative by fi eld and laboratory PAGEIA, but (low) positive (optical density [OD] of 0.327) by conventional EIA (Table) . One sample (TS-0830-7) scored as 1+ in the fi eld was determined to be negative on subsequent laboratory testing by both PAGEIA and conventional EIA. The other 4 samples (HB-2628, HA-2609, HA-2616, HB-2710) were scored as positive by fi eld and laboratory PAGEIA but negative by conventional EIA. Similar laboratorybased PAGEIAs have also been used to detect antibody to antigens of agents causing other infectious diseases, including severe acute respiratory syndrome coronavirus-like viruses and Nipah virus in bats (13) (14) (15) . cord-308461-4lhh3du0 2020 Unlike ex vivo methods, which involve isolated or sliced lungs, in vivo imaging using two-photon excitation microscopy of live animals enables researchers to observe hemodynamics, migration and extravasation of immune cells, as well as interactions among immune cells during influenza virus infection. To detect multiple fluorescent signals excited simultaneously by a two-photon excitation laser, fluorochromes with different spectra and equal brightness must be selected; however, there is currently no comprehensive database of fluorescent reagents, fluorescent reporter viruses, and reporter mouse lines available for lung in vivo imaging. Our system uses suction-based lung stabilization 16, 28 to improve an existing in vivo two-photon imaging system for influenza virus-infected lung as a model of an acute inflammatory respiratory disease 5 . In vivo two-photon imaging is performed under conditions of single stimulation with a two-photon excitation laser; limitations exist regarding available fluorescent reagents/proteins for multiple labeling of target cells and lung architecture. cord-309742-fd1qmr87 2016 Therefore, we assayed A/PR/8/34 (H1N1), A/Japan/305/57 (H2N2), and X31 (H3N2) influenza virus strains for binding and infection on fully differentiated primary cultures of airway epithelia isolated from human bronchus, grown on semiporous filters at an air–liquid interface. These data show that influenza viruses with SAα2,3Gal binding specificity, like Japan, productively infect differentiated human airway epithelia from the apical surface. For measurement of the kinetics of influenza virus production from infected airway cells in one infectious cycle, 100 l of the virus preparation was diluted in Dulbecco''s PBS (Life Technologies, Inc., Grand Island, NY) and applied to the apical surface of the epithelia (m.o.i. of 0.1). To infect airway epithelia with different influenza virus strains from the basolateral side, the Millicell culture insert was turned over and virus preparations diluted in PBS were applied in a 100-l volume to the bottom of the membrane (m.o.i. ϭ 0.1). cord-327568-5vo4nmei 2019 title: Delivery of SA35 and SA40 peptides in mice enhances humoral and cellular immune responses and confers protection against Cryptosporidium parvum infection parvum proteins, were tested for their ability to induce immune responses in adult mice and for protection on neonate BALB/c mice born from females immunised by mucosal delivery of both peptides. The IP immunisation of adult BALB/c mice to a single antigen (SA35 or SA40) or to a mixture of the two antigens (SA35/40 mix) induced specific anti-Cryptosporidium IgG in serum after day 14 following initial administration. The mucosal delivery of SA35/40 mix in female BALB/c mice induced specific anti-Cryptosporidium IgG (mainly IgG1) in serum 21 days after initial immunisation. In humans, maternal immunisation with tetanus toxoid has Fig. 9 Quantification of COWP gene DNA copies by qPCR in the intestinal content of neonate mice infected with 5 × 10 3 Cryptosporidium parvum oocysts. cord-332221-6ea6gz9s 2019 The expression of inflammatory cytokines in the serum was detected by enzyme linked immunosorbent assay; lung injury was observed by hematoxylin-eosin staining; the viral proliferation in the lung was detected by real-time quantitative PCR; and the protein expression of the main molecules in the phosphatidylinositol-3-kinases/protein kinase B (PI3K/AKT) and mitogen-activated protein kinase (MAPK) signaling pathways was detected by Western blot. The expression of inflammatory cytokines in the serum was detected by enzyme linked immunosorbent assay; lung injury was observed by hematoxylin-eosin staining; the viral proliferation in the lung was detected by real-time quantitative PCR; and the protein expression of the main molecules in the phosphatidylinositol-3-kinases/protein kinase B (PI3K/AKT) and mitogen-activated protein kinase (MAPK) signaling pathways was detected by Western blot. To explore the mechanism by which IGF1 regulates acute inflammatory lung injury induced by IAV infection, a Western blot was used to detect the expression of molecules in key signaling pathways in the lung tissue of PR8-infected mice (50LD 50 PR8). cord-333639-usgpe1cz 2018 title: Macromolecular prodrugs of ribavirin: Polymer backbone defines blood safety, drug release, and efficacy of anti-inflammatory effects We focus on the choice of the macromolecular backbone as a carrier for the conjugated drug and analyze blood coagulation, binding to albumin, albumin aggregation, inhibitory activity on polymerases, and cytotoxicity for polymers differed by their anionic charge (carboxylates, phosphates and phosphonates, sulfonates). As a result, we identify polymers and macromolecular prodrugs that are devoid of blood anti-coagulation activity but are strong as inhibitors of polymerases and efficacious as delivery vehicles for ribavirinthus being attractive for the development of broad-spectrum antiviral agents. This observation echoes our recent findings on the apparent unique pairing of negative character and hydrophobicity of the polymer backbone that renders PEAA an efficacious inhibitor of e.g. hepatitis C virus intracellular replication [55] and a lead polymer with broad-spectrum antiviral activity [21] . Polyanionic macromolecular prodrugs of ribavirin: antiviral agents with a broad Spectrum of activity cord-334165-7gfk554m 2020 Basic Protocol 1: Mammalian cell transfection and protein purification Basic Protocol 2: A two‐stage ELISA for high‐throughput screening of human serum samples for antibodies binding to the spike protein of SARS‐CoV‐2 We reported in our earlier work that individuals not exposed to SARS-CoV-2 are completely naïve to the spike protein, and their serum samples show little or no reactivity in an ELISA (Amanat et al., 2020) . We developed this as a two-stage ELISA in which the first stage (''a'' steps below) includes relatively high-throughput screening of samples in a single serum dilution against the RBD (which expresses very well and therefore can be produced in greater quantities). This is followed by a second stage (''b'' steps below) in which positive samples from the first stage undergo a confirmatory ELISA against the full-length spike protein (which is harder to express; therefore there is usually less available). i. Thaw the required number of vials of antigen (SARS-CoV-2 RBD protein) to coat 96-well microtiter ELISA plates at a concentration of 2 μg/ml. cord-335121-ro3x3qa3 1988 Abstract Antibodies against Crithidia fasciculata choanomastigotes were detected in green toad (Bufo viridis) sera by direct agglutination, indirect haemagglutination (IHA), complement-fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA), Correlation coefficients (r) were calculated for comparisons between each of the techniques and regression formulae derived in order to convert antibody levels as determined by one immunological method to that of another. In this paper we present the results of acomparative assessment of four serological tests (direct agglutination, indirect haemagglutination, complement-fixation test and ELISA) used to detect and to determine the levels of antibodies in the sera of green toads (B. fasciculata and the number of control and immune sera containing detectable antibodies were the lowest for DA and CFT, intermediate for IHA and highest for the ELISA method (Table 1) . Nevertheless the method of antigen preparation ma> not be a salient criterion for antibody estimation in immune sera because correlation values of over 89% (f''< 0.001) were found when IHA titres were compared to those of ELISA. cord-335310-61wibso4 2016 Herein, a facile approach to formulate synthetic virus-like particles (sVLPs) is demonstrated by exploiting the phenomenon of protein corona formation induced by the high-energy surfaces of synthetic nanoparticles. As compared to inoculation with free proteins, vaccination with the sVLPs showed enhanced lymphatic antigen delivery, stronger antibody titers, increased splenic T-cell response, and reduced infection-associated symptoms in an avian model of coronavirus infection. Comparison to a commercial whole inactivated virus vaccine also showed evidence of superior antiviral protection by the sVLPs. The study demonstrates a simple yet robust method in bridging viral antigens with synthetic nanoparticles for improved vaccine application; it has practical implications in the management of human viral infections as well as in animal agriculture. In the present study, vaccination with the sVLPs resulted in enhanced humoral and cellular immune responses, improving protection against an avian model of coronavirus infection as compared to free protein antigens and a commercial WIV vaccine. cord-335591-r0x8yaqj 2007 The hybridomas produce monoclonal antibodies that recognize viral component molecules, including the spike protein (S) and the nucleocapsid protein (N), enabling the immunological detection of SARS-CoV by immunofluorescence staining, immunoblot, or an antigen-capture ELISA system. Based on clinical experience, several options have been considered in the quest to develop the capacity to accurately diagnose SARS-CoV infection, including molecular biology techniques and serological tests such as antigen-capture ELISA assay and immunofluorescence assay to detect virus-infected cells in respiratory swabs (3-7) . These mAbs enable the general immunological detection of SARS-CoV by methods such as immunofluorescent staining, immunoblotting, and immunohistology, in addition to the construction of a highly sensitive antigen-capture sandwich ELISA (6). The UV-inactivated purified SARS-CoV samples (see Note 1), which are serially diluted with 1% OVA/PBS-Tween, are added to the wells and incubated for 1 h at room temperature cord-338108-3rn6fwx3 2013 title: Immunomodulatory Activity and Protective Effects of Polysaccharide from Eupatorium adenophorum Leaf Extract on Highly Pathogenic H5N1 Influenza Infection In this study, we evaluated the immunomodulatory activities and protective effect of Eupatorium adenophorum polysaccharide (EAP) against the highly pathogenic H5N1 subtype influenza virus. EAP treatment significantly increased the production of IL-6, TNF-α, and IFN-γ both in vivo and in vitro as measured by qPCR and ELISA. In a mouse infection model, intranasal administration of EAP at a dose of 25 mg/kg body weight prior to H5N1 viral challenge efficiently inhibited viral replication, decreased lung lesions, and increased survival rate. To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model. cord-339694-sp212tai 2016 title: A phase trial of the oral Lactobacillus casei vaccine polarizes Th2 cell immunity against transmissible gastroenteritis coronavirus infection casei) vaccine against anti-transmissible gastroenteritis virus developed in our laboratory was used to study mucosal immune responses. We found that the recombinant Lactobacillus stimulated IL-17 expression in both the systemic and mucosal immune responses against TGEV infection. Following infection with virulent transmissible gastroenteritis coronavirus, isolated mesenteric lymph node CD4+ T cells mounted a specific proliferative response against infectious or inactivated purified virus upon secondary in vitro stimulation (Anton et al. Here, an oral Lactobacillus casei vaccine against anti-transmissible gastroenteritis virus developed in our laboratory was used to study the mucosal immune response (Jiang et al. In conclusion, our study suggests that the recombinant Lactobacillus vaccine provokes specific mucosal and systemic immune responses to protect piglets from infection. cord-340125-il35gs97 2006 title: Genome-wide gene expression profiling of human mast cells stimulated by IgE or FcεRI-aggregation reveals a complex network of genes involved in inflammatory responses A substantial number of genes were regulated by IgE sensitization alone; and following FcεRI aggregation, a wide range of genes were triggered, including genes for cytokines, chemokines, transcription factors, anti-apoptosis, and several genes involved in innate and acquired immunity. Other than these, several genes coding for other receptors involved in immune-responses; immunoregulatory genes; adhesion and/or cytoskeleton remodeling; regulators of apoptosis; signal transduction; transcription factors; were also upregulated by monomeric IgE (Table. Here we studied, whether monomeric-IgE alone, may activate FcεRI intracellular signaling pathways, leading to physiological responses of mast cells, by analyzing the overall tyrosine phosphorylation; fluctuations in cytosolic Ca 2+ concentration; and degranulation by measuring β-hexosaminidase release (Fig. 6A,B &6C) . cord-343409-oao75pzy 2013 As there were differences in the start position of this ORF in the various group VII bat βERVs (PvERV-βE -I), likely due to random mutation since integration, a nucleotide alignment of the region was generated (Additional file 1: Figure S2 ). To determine if the groupings we had assigned were congruent with known functional differences between retroviruses with respect to betaretroviral RNA nuclear export strategies, we analyzed the bat βERVs, alongside known exogenous and endogenous betaretroviruses, for evidence of motifs indicative of the major export strategies (Additional file 2: Table S4 ). We coupled our analysis of the genomic features of the bat βERVs with the phylogenetic patterns observed in the Gag, Pol, and Env trees (with primacy given to the phylogeny of the highly conserved polymerase sequences) to generate a hypothetical series of events that may have led to the current state of diversity in the genus Betaretrovirus ( Figure 5 ). cord-347661-q9lgliph 2007 The use as antigens of either synthetic peptides (coupled to a carrier protein) or proteins expressed in Escherichia coli is described, and detailed protocols for immunization and preparation of test bleeds are provided. Furthermore, in the context of specific research questions, polyclonal antisera may sometimes even be preferred over monoclonal antibodies since they contain a mixture of immunoglobulin molecules, derived from different B-cell lines in the immunized animal, often recognizing multiple epitopes of the target protein. The antiserum used here was raised against a domain in the C-terminal region of pp1a of the arterivirus EAV (8) and recognizes a large set of processing intermediates and end products, which are indicated by arrowheads: (A) Western blot analysis: EAV-and mock-infected cell lysates were prepared at 8 h postinfection, run on an SDS-polyacrylamide gel, blotted to PVDF membrane, and incubated with the postimmune serum at a 1:1000 dilution. cord-351498-bmq6zcb0 2018 In the present study, we developed and tested three indirect ELISAs using rFhpCL1, rFhpCL2, and rFhpCL5 and evaluated their recognition by sera from sheep and cattle naturally infected with F. In the present study, we developed and tested indirect ELISAs based on recombinant procathepsin L1 (rFhpCL1), rFhpCL2, or rFhpCL5, in order to investigate which of these targets are best recognized by sera from sheep and cattle naturally infected with F. Finally, the r values obtained on comparing the four ELISA methods for Fasciola-infected cattle and sheep sera are shown in Table 3 . These results suggest that the use of a single recombinant cathepsin/ procathepsin as target antigen in ELISAs for serodiagnosis of fascioliasis may limit the sensitivity of the assay when testing sera from some species, particularly cattle. (2001) who tested sera from sheep and cattle harboring other parasites, mainly nematodes, suggesting that the cross-reactivity may be due to common epitopes between recombinant Fasciola cathepsins and antigens present in other parasites. cord-356207-tpn5cg4n 2006 The emergence in 2003 of a new coronavirus (CoV) responsible for the atypical pneumonia termed severe acute respiratory syndrome (SARS) was a stark reminder that hitherto unknown viruses have the potential to cross species barriers to become new human pathogens. Here we describe the SARS-CoV ''spike'' structure determined by single-particle cryo-EM, along with the docked atomic structures of the receptor-binding domain and prefusion core. SUPPLEMENTARY INFORMATION: The online version of this article (doi:10.1038/nsmb1123) contains supplementary material, which is available to authorized users. SARS-CoV-enriched fractions were checked by SDS-Page and Western blotting, and rendered non-infectious by irradiation in a gamma cell on dry ice with a 2 Mrad exposure for 90 minutes. Formvar-carbon coated 400-mesh nickel grids were floated on drops of purified SARS-CoV (40μl) for 1 minute. Structure of SARS coronavirus spike receptor-binding domain complexed with receptor Solution structure of the severe acute respiratory syndrome-coronavirus heptad repeat 2 domain in the prefusion state