TCID50 Assay Research Articles

Effectiveness of Cold Smoking on Inactivating Murine Norovirus in Salami-Like Pork Sausages (Mettwurst), and Hepatitis E Virus and Murine Norovirus in Solution

Hepatitis E virus (HEV) is a zoonotic virus that infects humans when virus-containing pork products are consumed. This study aimed to explore MNV (murine norovirus) and HEV inactivation during cold smoking and ripening/fermentation treatments used for salami-like sausages (mettwurst). MNV inactivation was monitored in culture medium solution and in sausage while being subjected to a salami-like sausage manufacturing process. The inactivation of MNV in the solution was also monitored at room temperature (RT) for four weeks. HEV inactivation was monitored in solution during the cold smoking process and at RT. A TCID50 assay was used to calculate the infectious MNV and HEV titres. MNV survival was modelled using Bayesian inference. MNV load in solution decreased by as much as 4.7 (SD 0.9) log10 TCID50/ml when it was subjected to the cold smoking process. Modelling revealed that 99.999% MNV might be inactivated during the treatment when observed at a level of 95% CI (Bayesian Confidence Interval). On the contrary, MNV load decreased by only 1.8 (0.2) log10 when stored at RT. The low-titre HEV in solution was inactivated (> 1.1 (0.2) log10) when treated, and at RT. However, MNV was resistant in the sausage matrix during the cold smoking process (log10 reduction of 1.9 (0.5) TCID50/ml). Based on modelling, a substantial amount of virus would remain in the product, even when the uncertainty was considered. Hence, viruses, here exemplified by MNV, may not be inactivated from salami-like pork sausages during manufacturing, which poses a risk for consumers in real-life situations.

Exploring potential of graphene oxide as an alternative antiviral approach for influenza A H1N1.

Aim: Graphene oxide (GO), known for its distinctive physicochemical properties, shows promise as a nanomaterial capable of combating infectious agents. This study investigates the efficacy of GO nanoparticles in restricting influenza A H1N1 replication in MDCK cells.Methods: GO nanoparticles were synthesized. After evaluating the toxicity of GO nanoparticles, the antiviral activity of the highest nontoxic concentration of GO against influenza A H1N1 in MDCK cells was studied.Results: GO treatments resulted in substantial decreases in virus titers, as shown via hemagglutination assay, TCID50 assay and real-time PCR analysis.Conclusion: This study emphasizes that GO nanoparticles have a high level of effectiveness against influenza A H1N1 viruses, making them an intriguing option for various antiviral uses.

Identification of porcine PARP11 as a restricted factor for pseudorabies virus.

PRV infection in swine can cause devastating disease and pose a potential threat to humans. Advancing the interplay between PRV and host is essential to elucidate the pathogenic mechanism of PRV and identify novel anti-PRV targets. PARP11-KO PK-15 cells were firstly constructed by CRISPR/Cas9 technology. Next, the effect of PARP11-KO on PRV infection was determined by RT-qPCR, TCID50 assay, RNA-seq, and western blot. In this study, we identified PARP11 as a host factor that can significantly affect PRV infection. Inhibition of PARP11 and knockout of PARP11 can significantly promoted PRV infection. Subsequently, we further found that PARP11 knockout upregulated the transcription of NXF1 and CRM1, resulting in enhanced transcription of viral genes. Furthermore, we also found that PARP11 knockout could activate the autophagy pathway and suppress the mTOR pathway during PRV infection. These findings could provide insight into the mechanism in which PARP11 participated during PRV infection and offer a potential target to develop anti-PRV therapies.

Immunogenicity of various variants of Ebola and Marburg virus glycoprotein genes in recombinant adenoviral vectors

INTRODUCTION. Marburg and Ebola viruses cause severe haemorrhagic fever in humans and primates. Currently, there are no licensed prophylactic vaccines that can simultaneously prevent the spread or reduce the severity of both diseases caused by these filoviruses. The development of effective prophylactic vaccines requires studies aimed at selecting the most immunogenic forms of protective antigens.AIM. This study aimed to evaluate humoral immune induction in animals after administration of recombinant adenoviral vectors expressing various forms of Ebola and Marburg virus glycoproteins (GPs).MATERIALS AND METHODS. Samples of recombinant human adenovirus type 5 (rAd5) were obtained using homologous recombination in Escherichia coli, growth in HEK293 cells, and purification by CsCl gradient ultracentrifugation. The resulting rAd5 samples were characterised in terms of their identity (PCR and whole-genome sequencing), the concentration of viral particles (fluorescence spectroscopy), and the concentration of infectious viral particles (TCID50 assay). Enzyme-linked immunosorbent assay (ELISA) was used to evaluate the GP-specific IgG titres in the sera of immunised mice.RESULTS. The authors constructed rAd5 samples, and each construct contained an expression cassette with a GP gene form encoding a full-length GP, a GP without the mucin-like domain, or a GP without both the glycan cap and the mucin-like domain. Each of these forms was studied using the GPs of four filoviruses, including Zaire Ebola virus, Sudan Ebola virus, Bundibugyo Ebola virus, and Marburg virus. Neither of the forms had a critical effect on the rAd5 replicative capacity. Three weeks after immunisation, the highest GP-specific IgG production was induced by the rAd5 samples encoding either the full-length GP or the GP without the mucin-like domain. The GP without both the glycan cap and the mucin-like domain was the least immunogenic antigen regardless of the filovirus species.CONCLUSIONS. The most promising constructs for the development of filovirus vaccines based on recombinant adenoviral vectors are the constructs that include the genes encoding the fulllength GP or the GP without the mucin-like domain.

Protein characterization of an Indonesian isolate of foot and mouth disease virus inactivated with formaldehyde and binary ethylenimine.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-footed animals. It is a major threat to livestock production worldwide, causing significant economic losses. Inactivation of FMD virus (FMDV) is crucial for vaccine development and control of outbreaks. However, traditional inactivation methods can sometimes damage the viral protein, affecting vaccine efficacy. Therefore, finding new inactivating agents that effectively inactivate the virus while preserving the integrity of its proteins is an important research area. This study investigated the optimal materials (0.04% formaldehyde, 0.001 M binary ethylenimine [BEI], or a combination) for inactivating and preserving the specific molecular weight of Serotype O FMDV protein. This study used serotype O FMDV isolated from several areas of East Java. The virus was inoculated into baby hamster kidney-21 cells, and the titer was calculated using the TCID50 Assay. The virus was inactivated using 0.04% formaldehyde, 0.001 M BEI, or a combination of 0.04% formaldehyde and 0.001 M BEI. Inactive viral proteins were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting. Serotype O FMDV can be inactivated using 0.04% formaldehyde while preserving specific FMDV proteins, specifically VP0 and VP3 with a molecular weight (MW) of 36 kDa and VP3 with a MW of 24 kDa. Serotype O FMDV can be inactivated by 0.001 M BEI while preserving specific FMDV proteins, specifically VP0 with a MW of 35 kDa, VP3 with a MW of 28 kDa, and VP1 with a MW of 23 kDa. FMDV serotype O can be inactivated using a combination of 0.04% formaldehyde and 0.001 M BEI while preserving specific FMDV proteins, specifically VP0 and VP3 with a MW of 36 kDa and VP3 with a MW of 24 kDa. This study found that 0.04% formaldehyde, alone or in combination with 0.001 M BEI, was effective for inactivating and preserving the specific molecular weight of Serotype O FMDV protein. The limitation of this study was the inactivations of the virus have not yet been tested for their potency on experimental animals. Further research is warranted to investigate the inactivation kinetics of these materials, including their potency on experimental animals. Additionally, a comparison of the inactivation rates between 0.04% formaldehyde alone and the combination with BEI would help to determine the optimal inactivation agent for future applications.

Immunogenicity and Protective Efficacy of a Single Intranasal Dose Vectored Vaccine Based on Sendai Virus (Moscow Strain) against SARS-CoV-2 Variant of Concern.

The mouse paramyxovirus Sendai, which is capable of limited replication in human bronchial epithelial cells without causing disease, is well suited for the development of vector-based intranasal vaccines against respiratory infections, including SARS-CoV-2. Using the Moscow strain of the Sendai virus, we developed a vaccine construct, Sen-Sdelta(M), which expresses the full-length spike (S) protein of the SARS-CoV-2 delta variant. A single intranasal delivery of Sen-Sdelta(M) to Syrian hamsters and BALB/c mice induced high titers of virus-neutralizing antibodies specific to the SARS-CoV-2 delta variant. A significant T-cell response, as determined by IFN-γ ELISpot and ICS methods, was also demonstrated in the mouse model. Mice and hamsters vaccinated with Sen-Sdelta(M) were well protected against SARS-CoV-2 challenge. The viral load in the lungs and nasal turbinates, measured by RT-qPCR and TCID50 assay, decreased dramatically in vaccinated groups. The most prominent effect was revealed in a highly sensitive hamster model, where no tissue samples contained detectable levels of infectious SARS-CoV-2. These results indicate that Sen-Sdelta(M) is a promising candidate as a single-dose intranasal vaccine against SARS-CoV-2, including variants of concern.

Rehmmannia glutinosa polysaccharide exerts antiviral activity against pseudorabies virus and antioxidant activity

Pseudorabies virus (PRV) is an important pathogen harming the global pig industry. Vaccines available for swine cannot protect against PRV completely. Furthermore, no antiviral drugs are available to treat PRV infections. Rehmmannia glutinosa polysaccharide (RGP) possesses several medicinal properties. However, its antiviral activity is not reported. In the present study, we found that RGP can inhibit PRV/XJ5 infection by western blotting, immunofluorescent assay (IFA), and TCID50 assay quantitative polymerase chain reaction (qPCR). We revealed RGP can inhibit virus adsorption and invasion into PK-15 cells in a dose-dependent manner via western blotting, IFA, TCID50 assay, and quantitative polymerase chain reaction (qPCR), and suppressed PRV/XJ5 replication through western blotting, and qPCR. Additionally, it also reduced PRV/XJ5-induced ROS, lipid oxidation, and improved SOD levels in PK-15 cells, which was observed by using corresponding test kits. To conclude, our findings suggest that RGP might be a novel therapeutic agent for preventing and controlling PRV infection and antioxidant agent.

Evaluation of Novel Nasal Mucoadhesive Nanoformulations Containing Lipid-Soluble EGCG for Long COVID Treatment.

Nanoparticle size and Zeta potential were measured using the ZetaView Nanoparticle Tracking Analysis system; mucociliary safety was determined using the MucilAir human nasal model; contact antiviral activity and post-infection inhibition against the OC43 viral strain were assessed by the TCID50 assay for cytopathic effect on MRC-5 cells. The saline-based EC16 mucoadhesive nanoformulations containing 0.005 to 0.02% w/v EC16m have no significant difference compared to saline (0.9% NaCl) with respect to tissue integrity, cytotoxicity, and cilia beat frequency. A 5 min contact resulted in 99.9% inactivation of β-coronavirus OC43. OC43 viral replication was inhibited by >90% after infected MRC-5 cells were treated with the formulations. The saline-based novel EC16m mucoadhesive nasal nanoformulations rapidly inactivated human coronavirus with mucociliary safety properties comparable to saline, a solution widely used for nasal applications.

Human Norovirus Surrogate Is Highly Stable in Berry Smoothies and under In Vitro Simulated Digestion.

Human noroviruses are major causes of foodborne outbreaks linked to berries. The overall goal of this study was to investigate the persistence of a human norovirus surrogate, Tulane virus (TV), in berry smoothies and under simulated digestion through the gastrointestinal track. Two types of smoothies were prepared from blueberries and strawberries. Tulane virus was spiked into each smoothie and incubated either at 37 or 4 °C for 2, 60, and 120 min. Furthermore, the virus-spiked smoothies were subjected to sequential oral (2 min), gastric (10 and 60 min), and intestinal (15 and 120 min) digestion according to the standardized INFOGEST model. Quantification of infectious TV was carried out using the TCID50 assay. At 4 °C, in both berry smoothies, TV infectivity did not show significant changes throughout the 120 min period. At 37 °C, TV infectivity showed significant reduction (~0.5 log TCID50/mL) only in blueberry smoothies starting at 60 min. During the oral, gastric, and intestinal digestion phases, the mean log reduction in TV infectivity in blueberry did not exceed ~0.5 log, while infectious TV in strawberry smoothies under all phases was stable. Given the notable stability of infectious viruses in berry smoothies and the gastrointestinal tract, prevention of norovirus contamination of berries is paramount to reduce virus outbreaks linked to berries.

Green synthesis of MnO2 NPs using Arabic gum: assessing its potential antiviral activity against influenza A/H1N1

BackgroundThe antiviral properties of metal nanoparticles against various viruses, including those resistant to drugs, are currently a subject of intensive research. Recently, the green synthesis of nanoparticles and their anti-viral function have attracted a lot of attention. Previous studies have shown promising results in the use of Arabic gum for the green synthesis of nanoparticles with strong antiviral properties. In this study we aimed to investigate the antiviral effects of MnO2 nanoparticles (MnO2-NPs) synthesized using Arabic gum, particularly against the influenza virus.MethodsArabic gum was used as a natural polymer to extract and synthesize MnO2-NPs using a green chemistry approach. The synthesized MnO2-NPs were characterized using SEM and TEM. To evaluate virus titration, cytotoxicity, and antiviral activity, TCID50, MTT, and Hemagglutination assay (HA) were performed, respectively. Molecular docking studies were also performed to investigate the potential antiviral activity of the synthesized MnO2-NPs against the influenza virus. The molecular docking was carried out using AutoDock Vina software followed by an analysis with VMD software to investigate the interaction between Arabic gum and the hemagglutinin protein.ResultsSimultaneous combination treatment with the green-synthesized MnO2-NPs resulted in a 3.5 log HA decrement and 69.7% cellular protection, which demonstrated the most significant difference in cellular protection compared to the virus control group (p-value < 0.01). The docking results showed that binding affinities were between − 3.3 and − 5.8 kcal/mole relating with the interaction between target with MnO2 and beta-D-galactopyranuronic acid, respectively.ConclusionThe results of the study indicated that the MnO2-NPs synthesized with Arabic gum had significant antiviral effects against the influenza virus, highlighting their potential as a natural and effective treatment for inhibition of respiratory infections.

Enhancing immune regulation in vitro: the synergistic impact of 3'-sialyllactose and osteopontin in a nutrient blend following influenza virus infection.

Natural components of breast milk, human milk oligosaccharides (HMOs) and osteopontin (OPN) have been shown to have a variety of functional activities and are widely used in infant formulas. However, the preventive and therapeutic effects of both on influenza viruses are not known. In this study, antiviral assays using a human laryngeal carcinoma cell line (HEP-2) showed that 3'-sialyllactose (3'-SL) and OPN had the best antiviral ability with IC50 values of 33.46 μM and 1.65 μM, respectively. 3'-SL (10 μM) and OPN (4 μM) were used in combination to achieve 75% inhibition. Further studies found that the combination of 200 μg/mL of 3'-SL with 500 μg/mL of OPN exerted the best antiviral ability. The reason for this was related to reduced levels of the cytokines TNF-α, IL-6, and iNOS in relation to mRNA expression. Plaque assay and TCID50 assay found the same results and verified synergistic effects. Our research indicates that a combination of 3'-SL and OPN can effectively reduce inflammatory storms and exhibit anti-influenza virus effects through synergistic action.

In vitro inhibition of rotavirus multiplication by copper oxide nanoparticles.

Group A rotaviruses are the most common cause of gastroenteritis in children under five years of age worldwide. Rotavirus gastroenteritis can be related to mild to severe diarrhea in children and in some cases, can lead to death due to severe dehydration. Approximately 146,480 people die annually from rotavirus infection worldwide, and most of these deaths occur in low-income countries in Africa and Asia. Since there are no specific effective drugs to treat rotavirus infections, and infected patients can only be treated supportively, new antiviral agents need to be developed. Copper oxide nanoparticles (CuO NPs) have a wide range of applications in the magnetic and electrical industries, as well as in biology. The antiviral activity of nanoparticles (CuO NPs) is well documented. This study aimed to investigate the antiviral effect of CuO NPs on rotaviruses. The cytotoxic effects of CuO NPs on MA-104 cells were examined by methyl thiazolyl tetrazolium assay. In addition, the anti-rotavirus activity of CuO NPs was evaluated by TCID50 and real-time polymerase chain reaction PCR assay. Our results showed that exposure of rotavirus-infected cells to various non-toxic concentrations of CuO NPs did not cause a decrease in viral titer, compared to the control. However, the virucidal effect of CuO NPs on rotavirus was observed at concentrations of 80 and 100 μg/ml (P<0.001). Our study suggested that CuO NPs had significant antiviral activity against rotavirus replication. However, the exact mechanism of anti-rotavirus activity of CuO NPs remained unknown. According to the virucidal assay, it appears that the loss of capsid integrity and genome disruption in the presence of CuO NPs are possible mechanisms of its anti-rotavirus activity.

Screening Commercial Tea for Rapid Inactivation of Infectious SARS-CoV-2 in Saliva.

SARS-CoV-2 infects the oral mucosa and is shed in salivary fluids. Traditionally, tea has been used by various cultures to treat respiratory ailments. The objective of this study was to identify commercially available teas that can rapidly inactivate infectious SARS-CoV-2 in saliva. Initially, tea (n = 24) was prepared as 40 mg/mL infusions and incubated with SARS-CoV-2 resuspended in water, for 5 min at 37 °C. Then, five teas that showed >3 log reduction in virus infectivity were further investigated at 40 and 10 mg/mL infusions for 60 and 10 s contact time with SARS-CoV-2 resuspended in saliva. Tea polyphenols were measured using the Folin-Ciocalteu assay. SARS-CoV-2 infectivity was quantified on Vero-E6 cell line using TCID50 assay. At 10 mg/mL infusion, black tea showed the highest reduction (3 log, i.e., 99.9%) of infectious SARS-CoV-2 within 10 s. Green, mint medley, eucalyptus-mint, and raspberry zinger teas showed similar inactivation of SARS-CoV-2 (1.5-2 log, i.e., 96-99% reduction). At 40 mg/mL infusions, all five teas showed >3 log reduction in virus infectivity within 10 s. Tea polyphenol but not pH was significantly correlated to virus reduction. Time-of-addition assay revealed that the five teas displayed preventive effects (0.5-1 log, i.e., 68-90% reduction) against SARS-CoV-2 infection of Vero-E6 cells as well as during post-virus infection (1.2-1.9 log, i.e., 94-98%). However, the highest inhibitory effect was observed when the teas were added at the time of virus infection (2-3 log, i.e., 99-99.9%). Our results provide insights into a rapid at-home intervention (tea drinking or gargling) to reduce infectious SARS-CoV-2 load in the oral cavity which might also mitigate infection of the oral mucosa.

Impaired influenza A virus replication by the host restriction factor SAMHD1 which inhibited by PA-mediated dephosphorylation of the host transcription factor IRF3

BackgroundInfluenza A virus (IAV) can cause severe and life-threatening illness in humans and animals. Therefore, it is important to search for host antiviral proteins and elucidate their antiviral mechanisms for the development of potential treatments. As a part of human innate immunity, host restriction factors can inhibit the replication of viruses, among which SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) can restrict the replication of viruses, such as HIV and enterovirus EV71. Viruses also developed countermeasures in the arms race with their hosts. There are few reports about whether SAMHD1 has a restriction effect on IAV.MethodsTo investigate the impact of IAV infection on SAMHD1 expression in A549 cells, we infected A549 cells with a varying multiplicity of infection (MOI) of IAV and collected cell samples at different time points for WB and RT-qPCR analysis to detect viral protein and SAMHD1 levels. The virus replication level in the cell culture supernatant was determined using TCID50 assay. Luciferase assay was used to reveal that H5N1 virus polymerase acidic protein (PA) affected the activity of the SAMHD1 promoter. To assess the antiviral capacity of SAMHD1, we generated a knockdown and overexpressed cell line for detecting H5N1 replication.ResultsIn this study, we observed that SAMHD1 can restrict the intracellular replication of H5N1 and that the H5N1 viral protein PA can downregulate the expression of SAMHD1 by affecting SAMHD1 transcriptional promoter activity. We also found that SAMHD1's ability to restrict H5N1 is related to phosphorylation at 592-tyrosine.ConclusionsIn conclusion, we found that SAMHD1 may affect the replication of IAVs as a host restriction factor and be countered by PA. Furthermore, SAMHD1 may be a potential target for developing antiviral drugs.

Photodynamic Inactivation of Bovine Coronavirus with the Photosensitizer Toluidine Blue O.

Coronaviruses (CoVs) belong to the group of enveloped positive-sense single-strand RNA viruses and are causative agents of respiratory, gastro-intestinal, and central nervous systems diseases in many host species, i.e., birds, mammals, and humans. Beta-CoVs revealed a great potential to cross the barrier between species by causing three epidemics/pandemics among humans in the 21st century. Considering the urgent need for powerful antiviral agents for decontamination, prevention, and treatment of BCoV infections, we turned our attention to the possibility of photodynamic inactivation with photosensitizers in combination with light irradiation. In the present study, we evaluated, for the first time, the antiviral activity of toluidine blue O (TBO) against Beta-coronavirus 1 (BCoV) in comparison to methylene blue (MB). First, we determined the in vitro cytotoxicity of MB and TBO on the Madin-Darby bovine kidney (MDBK) cell line with ISO10993-5/Annex C. Thereafter, BCoV was propagated in MDBK cells, and the virus titer was measured with digital droplet PCR, TCID50 assay and plaque assay. The antiviral activity of non-toxic concentrations of TBO was estimated using the direct inactivation approach. All effects were calculated in MAPLE 15® mathematical software by developing programs for non-linear modeling and response surface analysis. The median inhibitory concentration (IC50) of TBO after 72 h of incubation in MDBK cells was 0.85 µM. The antiviral activity of TBO after the direct inactivation of BCoV (MOI = 1) was significantly stronger than that of MB. The median effective concentration (EC50) of TBO was 0.005 µM. The cytopathic effect decreased in a concentration-dependent manner, from 0.0025 to 0.01 µM, and disappeared fully at concentrations between 0.02 and 0.3 µM of TBO. The number of virus particles also decreased, depending on the concentration applied, as proven by ddPCR analysis. In conclusion, TBO exhibits significant potential for direct inactivation of BCoV in vitro, with a very high selectivity index, and should be subjected to further investigation, aiming at its application in veterinary and/or human medical practice.

Exploring the potent in vitro antiviral activity of Faramir®: A tailored Salvia officinalis extraction utilizing the sequential solvent polarity method

Salvia officinalis, known as Sage, serves as a rich source of natural compounds that exhibit antioxidant, anti-inflammatory activities. Antioxidant activity has been demonstrated to be enhanced when medicinal herbs are extracted using sequential polar solvents, as revealed by previous studies. In this study, we examined the antiviral potential of Sage extract through successive extractions using organic solvents of increasing polarity (hexane, chloroform, ethanol, acetone, methanol, and water). The antiviral effectiveness of the selected extract fraction, Faramir®, was assessed against human immunodeficiency virus using ELISA and MTT assays conducted on Jurkat cells. Moreover, Faramir® antiviral activity was also examined against SARS-CoV-2 and Influenza viruses using the TCID50 assay, employing Vero E6 and MDCK cell lines, respectively. The findings indicated that the inhibitory effects of Faramir® on HIV replication are dependent on the concentration, as demonstrated through serial dilutions (0.25, 0.5, 1, 2, and 4 mg/mL). The highest inhibitory effect was observed at a concentration of 0.25 mg/mL. Furthermore, the TCID50 assay demonstrated that the antiviral activity of Faramir® is time-dependent. The highest level of antiviral activity was observed at the 5-minute mark following treatment. Subsequently, the extract fraction exhibiting the highest antiviral activity (Faramir®) was subjected to analysis using HPLC and GC-MS and found to contain three classes of phenolic molecules, namely flavonoids (such as Apigenin), hydroxylated compounds (including rosmarinic acid), and terpenes (such as alpha pinene). Based on these findings, it is suggested that Faramir® holds great potential as a novel natural therapeutic extract for combating viral infections.

Use of Human Intestinal Enteroids for Recovery of Infectious Human Norovirus from Berries and Lettuce.

Norovirus (NoV) is the leading cause of viral foodborne gastroenteritis globally. Currently, the gold standard for detecting NoV in clinical, food, and environmental samples is via molecular-based methods, primarily RT-PCR. Nevertheless, there is a great need for confirmatory assays that can determine the infectivity of viral particles recovered from contaminated matrices. The use of the human intestinal enteroids system (HIEs) has allowed for the expansion of norovirus replication, although it still suffers from limitations of strain preferences and the requirement of high titer stocks for infection. In this study, we wanted to explore the feasibility of using the HIEs to support the replication of NoV that had been recovered from representative food matrices that have been associated with foodborne illness. We first confirmed that HIEs can support the replication of several strains of NoV as measured by RT-qPCR. We subsequently chose two of those strains that reproducibly replicated, GII.4 and GII.6, to evaluate in a TCID50 assay and for future experiments. Infectious NoV could be recovered and quantified in the HIEs from lettuce, frozen raspberries, or frozen strawberries seeded with high titers of either of these strains. While many experimental challenges still remain to be overcome, the results of this study represent an important step toward the detection of infectious norovirus from representative produce items.

Identification of Host Factors for Rift Valley Fever Phlebovirus.

Rift Valley fever phlebovirus (RVFV) is a zoonotic pathogen that causes Rift Valley fever (RVF) in livestock and humans. Currently, there is no licensed human vaccine or antiviral drug to control RVF. Although multiple species of animals and humans are vulnerable to RVFV infection, host factors affecting susceptibility are not well understood. To identify the host factors or genes essential for RVFV replication, we conducted CRISPR-Cas9 knockout screening in human A549 cells. We then validated the putative genes using siRNA-mediated knock-downs and CRISPR-Cas9-mediated knock-out studies. The role of a candidate gene in the virus replication cycle was assessed by measuring intracellular viral RNA accumulation, and the virus titers were analyzed using plaque assay or TCID50 assay. We identified approximately 900 genes with potential involvement in RVFV infection and replication. Further evaluation of the effect of six genes on viral replication using siRNA-mediated knock-downs revealed that silencing two genes (WDR7 and LRP1) significantly impaired RVFV replication. For further analysis, we focused on the WDR7 gene since the role of the LRP1 gene in RVFV replication was previously described in detail. WDR7 knockout A549 cell lines were generated and used to dissect the effect of WRD7 on a bunyavirus, RVFV, and an orthobunyavirus, La Crosse encephalitis virus (LACV). We observed significant effects of WDR7 knockout cells on both intracellular RVFV RNA levels and viral titers. At the intracellular RNA level, WRD7 affected RVFV replication at a later phase of its replication cycle (24 h) when compared with the LACV replication, which was affected in an earlier replication phase (12 h). In summary, we identified WDR7 as an essential host factor for the replication of two different viruses, RVFV and LACV, both of which belong to the Bunyavirales order. Future studies will investigate the mechanistic role through which WDR7 facilitates phlebovirus replication.

Genomic Mutations in SARS-CoV-2 Genome following Infection in Syrian Golden Hamster and Associated Lung Pathologies.

The continuous evolution of the SARS-CoV-2 virus led to constant developments and efforts in understanding the significance and impacts of SARS-CoV-2 variants on human health. Our study aimed to determine the accumulation of genetic mutations and associated lung pathologies in male and female hamsters infected with the ancestral Wuhan strain of SARS-CoV-2. The present study showed no significant difference in the viral load between male and female hamsters and peak infection was found to be on day four post infection in both sexes of the animals. Live virus particles were detected up to 5 days post infection (dpi) through the TCID-50 assay, while qRT-PCR could detect viral RNA up to 14 dpi from all the infected animals. Further, the determination of the neutralizing antibody titer showed the onset of the humoral immune response as early as 4 dpi in both sexes against SARS-CoV-2, and a significant cross-protection against the delta variant of SARS-CoV-2 was observed. Histopathology showed edema, inflammation, inflammatory cell infiltration, necrosis, and degeneration of alveolar and bronchial epithelium cells from 3 dpi to 14 dpi in both sexes. Furthermore, next-generation sequencing (NGS) showed up to 10 single-nucleotide polymorphisms (SNPs) in the SARS-CoV-2 (ancestral Wuhan strain) genome isolated from both male and female hamsters. The mutation observed at the 23014 position (Glu484Asp) in the SARS-CoV-2 genome isolated from both sexes of the hamsters plays a significant role in the antiviral efficacy of small molecules, vaccines, and the Mabs-targeting S protein. The present study shows that either of the genders can be used in the pre-clinical efficacy of antiviral agents against SARS-CoV-2 in hamsters. However, considering the major mutation in the S protein, the understanding of the genetic mutation in SARS-CoV-2 after passing through hamsters is crucial in deciding the efficacy of the antiviral agents targeting the S protein. Importance: Our study findings indicate the accumulation of genomic mutations in SARS-CoV-2 after passing through the Syrian golden hamsters. Understanding the genomic mutations showed that either of the hamster genders can be used in the pre-clinical efficacy of antiviral agents and vaccines.

In ovo administration of retinoic acid enhances cell-mediated immune responses against an inactivated H9N2 avian influenza virus vaccine

The H9N2 subtype avian influenza virus (AIV) is a low pathogenic AIV that infects avian species and lead to huge economical losses in the poultry industry. The unique immunomodulatory properties of Retinoic acid (RA), an active component of vitamin A, highlights its potential to enhance chicken’s resistance to infectious diseases and perhaps vaccine-induced immunity. Therefore, the present study evaluated the effects of in ovo supplementation of RA on the immunogenicity and protective efficacy of an inactivated avian influenza virus vaccine. On embryonic day 18, eggs were inoculated with either 90 μmol RA/200 μL/egg or diluent into the amniotic sac. On days 7 and 21 post-hatch, birds were vaccinated with 15 μg of β-propiolactone (BPL) inactivated H9N2 virus via the intramuscular route. One group received BPL in combination with an adjuvant, while the other group received saline solution and served as a non-vaccinated control group. Serum samples were collected on days 7, 14, 21, 28, 35, and 42 post-primary vaccination (ppv) for antibody analysis. On day 24 ppv, spleens were collected, and splenocytes were isolated to analyze cytokine expression, interferon gamma (IFN-γ) production, and cell population. On day 28 ppv, birds in all groups were infected with H9N2 virus and oral and cloacal swabs were collected for TCID50 (50 % Tissue Culture Infectious Dose) assay up to day 7 post-infection. The results demonstrated that in ovo administration of RA did not significantly enhance the AIV vaccine-induced antibody response against H9N2 virus compared to the group that received the vaccine alone. However, RA supplementation enhanced the frequency of macrophages (KUL01+), expression of inflammatory cytokines and production of IFN-γ by splenocytes. In addition, RA administration reduced oral shedding of AIV on day 5 post-infection. In conclusion, these findings suggest that RA can be supplemented in ovo to enhance AIV vaccine efficacy against LPAIV.