NS7a of SADS-CoV promotes viral infection via inducing apoptosis to suppress type III interferon production.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered swine coronavirus with potential cross-species transmission risk. Although SADS-CoV-induced host cell apoptosis and innate immunity antagonization has been revealed, underlying signaling pathways remain obscure. Here, we demonstrated that infection of SADS-CoV induced apoptosis in vivo and in vitro, and that viral protein NS7a is mainly responsible for SADS-CoV-induced apoptosis in host cells. Furthermore, we found that NS7a interacted with apoptosis-inducing factor mitochondria associated 1 (AIFM1) to activate caspase-3 via caspase-6 in SADS-CoV-infected cells, and enhanced SADS-CoV replication. Importantly, NS7a suppressed poly(I:C)-induced expression of type III interferon (IFN-λ) via activating caspase-3 to cleave interferon regulatory factor 3 (IRF3), and caspase-3 inhibitor protects piglets against SADS-CoV infection in vivo. These findings reveal how SADS-CoV induced apoptosis to inhibit innate immunity and provide a valuable clue to the development of effective drugs for the clinical control of SADS-CoV infection.IMPORTANCEOver the last 20 years, multiple animal-originated coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2, have caused millions of deaths, seriously jeopardized human health, and hindered social development, indicating that the study of animal-originated coronaviruses with potential for cross-species transmission is particularly important. Bat-originated swine acute diarrhea syndrome coronavirus (SADS-CoV), discovered in 2017, can not only cause fatal diarrhea in piglets, but also infect multiple human cells, with a potential risk of cross-species transmission, but its pathogenesis is unclear. In this study, we demonstrated that NS7a of SADS-CoV suppresses IFN-λ production via apoptosis-inducing factor mitochondria associated 1 (AIFM1)-caspase-6-caspase-3-interferon regulatory factor 3 (IRF3) pathway, and caspase-3 inhibitor (Z-DEVD-FMK) can effectively inhibit SADS-CoV replication and protect infected piglets. Our findings in this study contribute to a better understanding of SADS-CoV-host interactions as a part of the coronaviruses pathogenesis and using apoptosis-inhibitor as a drug as potential therapeutic approaches for prevention and control of SADS-CoV infection.

Swine acute diarrhea syndrome coronavirus Nsp1 suppresses IFN-λ1 production by degrading IRF1 via ubiquitin-proteasome pathway.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel porcine enteric coronavirus that causes acute watery diarrhea, vomiting, and dehydration in newborn piglets. The type III interferon (IFN-λ) response serves as the primary defense against viruses that replicate in intestinal epithelial cells. However, there is currently no information available on how SADS-CoV modulates the production of IFN-λ. In this study, we utilized IPI-FX cells (a cell line of porcine ileum epithelium) as an in vitro model to investigate the potential immune evasion strategies employed by SADS-CoV against the IFN-λ response. Our results showed that SADS-CoV infection suppressed the production of IFN-λ1 induced by poly(I:C). Through screening SADS-CoV-encoded proteins, nsp1, nsp5, nsp10, nsp12, nsp16, E, S1, and S2 were identified as antagonists of IFN-λ1 production. Specifically, SADS-CoV nsp1 impeded the activation of the IFN-λ1 promoter mediated by MAVS, TBK1, IKKε, and IRF1. Both SADS-CoV and nsp1 obstructed poly(I:C)-induced nuclear translocation of IRF1. Moreover, SADS-CoV nsp1 degraded IRF1 via the ubiquitin-mediated proteasome pathway without interacting with it. Overall, our study provides the first evidence that SADS-CoV inhibits the type III IFN response, shedding light on the molecular mechanisms employed by SADS-CoV to evade the host immune response.

Transcriptome Profiling of Vero E6 Cells during Original Parental or Cell-Attenuated Porcine Epidemic Diarrhea Virus Infection.

Porcine epidemic diarrhea virus (PEDV) has led to significant economic losses in the global porcine industry since the emergence of variant strains in 2010. The high mutability of coronaviruses endows PEDV with the ability to evade the host immune response, which impairs the effectiveness of vaccines. In our previous study, we generated a highly cell-passaged PEDV strain, CT-P120, which showed promise as a live attenuated vaccine candidate by providing satisfactory protection against variant PEDV infection in piglets. However, the mechanism by which the attenuated CT-P120 adapts to cells during passage, resulting in increased replication efficiency, remains unclear. To address this question, we conducted a comparative transcriptomic analysis of Vero E6 cells infected with either the original parental strain (CT-P10) or the cell-attenuated strain (CT-P120) of PEDV at 6, 12, and 24 h post-infection. Compared to CT-P10, CT-P120 infection resulted in a significant decrease in the number of differentially expressed genes (DEGs) at each time point. Functional enrichment analysis of genes revealed the activation of various innate immune-related pathways by CT-P10, notably attenuated during CT-P120 infection. To validate these results, we selected eight genes (TRAF3, IRF3, IFNL1, ISG15, NFKB1, MAP2K3, IL1A, and CCL2) involved in antiviral processes and confirmed their mRNA expression patterns using RT-qPCR, in line with the transcriptomic data. Subsequent protein-level analysis of selected genes via Western blotting and enzyme-linked immunosorbent assay corroborated these results, reinforcing the robustness of our findings. Collectively, our research elucidates the strategies underpinning PEDV attenuation and immune evasion, providing invaluable insights for the development of effective PEDV vaccines.

Global Dynamics of Porcine Enteric Coronavirus PEDV Epidemiology, Evolution, and Transmission.

With a possible origin from bats, the alphacoronavirus Porcine epidemic diarrhea virus (PEDV) causes significant hazards and widespread epidemics in the swine population. However, the ecology, evolution, and spread of PEDV are still unclear. Here, from 149,869 fecal and intestinal tissue samples of pigs collected in an 11-year survey, we identified PEDV as the most dominant virus in diarrheal animals. Global whole genomic and evolutionary analyses of 672 PEDV strains revealed the fast-evolving PEDV genotype 2 (G2) strains as the main epidemic viruses worldwide, which seems to correlate with the use of G2-targeting vaccines. The evolving pattern of the G2 viruses presents geographic bias as they evolve tachytely in South Korea but undergo the highest recombination in China. Therefore, we clustered six PEDV haplotypes in China, whereas South Korea held five haplotypes, including a unique haplotype G. In addition, an assessment of the spatiotemporal spread route of PEDV indicates Germany and Japan as the primary hubs for PEDV dissemination in Europe and Asia, respectively. Overall, our findings provide novel insights into the epidemiology, evolution, and transmission of PEDV, and thus may lay a foundation for the prevention and control of PEDV and other coronaviruses.

Metabolomic analysis of porcine intestinal epithelial cells during swine acute diarrhea syndrome coronavirus infection.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an enveloped, positive single-stranded RNA virus belonging to Coronaviridae family, Orthocoronavirinae subfamily, Alphacoronavirus genus. As one of the main causes of swine diarrhea, SADS-CoV has brought huge losses to the pig industry. Although we have a basic understanding of SADS-CoV, the research on the pathogenicity and interactions between host and virus are still limited, especially the metabolic changes induced by SADS-CoV infection. Here, we utilized a combination of untargeted metabolomics and lipomics to analyze the metabolic alteration in SADS-CoV infected cells. Significant changes were observed in 1257 of 2225 metabolites identified in untargeted metabolomics, while the number of lipomics was 435 out of 868. Metabolic pathway enrichment analysis showed that amino acid metabolism, tricarboxylic acid (TCA) cycle and ferroptosis were disrupted during viral infection, suggesting that these metabolic pathways may partake in pathological processes related to SADS-CoV pathogenesis. Collectively, our findings gain insights into the cellular metabolic disorder during SADS-CoV infection, offer a valuable resource for further exploration of the relationship between virus and host metabolic activities, and provide potential targets for the development of antiviral drugs.

Swine acute diarrhea syndrome coronavirus induces autophagy to promote its replication via the Akt/mTOR pathway.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an enveloped, single-stranded, positive-sense RNA virus belonging to the Coronaviridae family. Increasingly studies have demonstrated that viruses could utilize autophagy to promote their own replication. However, the relationship between SADS-CoV and autophagy remains unknown. Here, we reported that SADS-CoV infection-induced autophagy and pharmacologically increased autophagy were conducive to viral proliferation. Conversely, suppression of autophagy by pharmacological inhibitors or knockdown of autophagy-related protein impeded viral replication. Furthermore, we demonstrated the underlying mechanism by which SADS-CoV triggered autophagy through the inactivation of the Akt/mTOR pathway. Importantly, we identified integrin α3 (ITGA3) as a potential antiviral target upstream of Akt/mTOR and autophagy pathways. Knockdown of ITGA3 enhanced autophagy and consequently increased the replication of SADS-CoV. Collectively, our studies revealed a novel mechanism that SADS-CoV-induced autophagy to facilitate its proliferation via Akt/mTOR pathway and found that ITGA3 was an effective antiviral factor for suppressing viral infection.

Emodin from Aloe inhibits Swine acute diarrhea syndrome coronavirus in cell culture.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging swine enteropathogenic coronavirus that causes severe diarrhea in neonatal piglets, leading to serious economic losses to the pig industries. At present, there are no effective control measures for SADS, making an urgent need to exploit effective antiviral therapies. Here, we confirmed that Aloe extract (Ae) can strongly inhibit SADS-CoV in Vero and IPI-FX cells in vitro. Furthermore, we detected that Emodin from Ae had anti-SADS-CoV activity in cells but did not impair SADS-CoV infectivity directly. The time-of-addition assay showed that Emodin inhibits SADS-CoV infection at the whole stages of the viral replication cycle. Notably, we found that Emodin can significantly reduce virus particles attaching to the cell surface and induce TLR3 (p < 0.001), IFN-λ3 (p < 0.01), and ISG15 (p < 0.01) expressions in IPI-FX cells, indicating that the anti-SADS-CoV activity of Emodin might be due to blocking viral attachment and the activation of TLR3-IFN-λ3-ISG15 signaling axis. These results suggest that Emodin has the potential value for the development of anti-SADS-CoV drugs.

Poly(A)-Binding Protein Cytoplasmic 1 Inhibits Porcine Epidemic Diarrhea Virus Replication by Interacting with Nucleocapsid Protein.

Porcine epidemic diarrhea virus (PEDV) is the etiological agent of porcine epidemic diarrhea (PED) characterized by vomit, watery diarrhea, dehydration and high mortality. Outbreaks of highly pathogenic variant strains of PEDV have resulted in extreme economic losses to the swine industry all over the world. The study of host-virus interaction can help to better understand the viral pathogenicity. Many studies have shown that poly(A)-binding proteins are involved in the replication process of various viruses. Here, we found that the infection of PEDV downregulated the expression of poly(A)-binding protein cytoplasmic 1 (PABPC1) at the later infection stage in Vero cells. The overexpression of PABPC1 inhibited the proliferation of PEDV at transcription and translation level, and siRNA-mediated depletion of PABPC1 promoted the replication of PEDV. Furthermore, mass spectrometry analysis and immunoprecipitation assay confirmed that PABPC1 interacted with the nucleocapsid (N) protein of PEDV. Confocal microscopy revealed the co-localizations of PABPC1 with N protein in the cytoplasm. Taken together, these results demonstrate the antiviral effect of PABPC1 against PEDV replication by interacting with N protein, which increases understanding of the interaction between PEDV and host.

Genome-wide transcriptome analysis of porcine epidemic diarrhea virus virulent or avirulent strain-infected porcine small intestinal epithelial cells.

Porcine epidemic diarrhea virus (PEDV) is the main cause of diarrhea, vomiting, and mortality in pigs, which results in devastating economic loss to the pig industry around the globe. In recent years, the advent of RNA-sequencing technologies has led to delineate host responses at late stages of PEDV infection; however, the comparative analysis of host responses to early-stage infection of virulent and avirulent PEDV strains is currently unknown. Here, using the BGI DNBSEQ RNA-sequencing, we performed global gene expression profiles of pig intestinal epithelial cells infected with virulent (GDS01) or avirulent (HX) PEDV strains for 3, 6, and 12 â€‹h. It was observed that over half of all significantly dysregulated genes in both infection groups exhibited a down-regulated expression pattern. Functional enrichment analyses indicated that the differentially expressed genes (DEGs) in the GDS01 group were predominantly related to autophagy and apoptosis, whereas the genes showing the differential expression in the HX group were strongly enriched in immune responses/inflammation. Among the DEGs, the functional association of TLR3 and IFIT2 genes with the HX and GDS01 strains replication was experimentally validated by TLR3 inhibition and IFIT2 overexpression systems in cultured cells. TLR3 expression was found to inhibit HX strain, but not GDS01 strain, replication by enhancing the IFIT2 expression in infected cells. In conclusion, our study highlights similarities and differences in gene expression patterns and cellular processes/pathways altered at the early-stage infection of PEDV virulent and avirulent strains. These findings may provide a foundation for establishing novel therapies to control PEDV infection.

Development of an indirect ELISA for detecting swine acute diarrhoea syndrome coronavirus IgG antibodies based on a recombinant spike protein.

Swine acute diarrhoea syndrome coronavirus (SADS-CoV) is a newly identified swine enteropathogenic coronavirus that causes watery diarrhoea in neonatal piglets, leading to significant economic losses to the swine industry. Currently, there are no suitable serological methods to assess the infection of SADS-CoV and effectiveness of vaccines, making an urgent need to exploit effective enzyme-linked immunosorbent assay (ELISA) to compensate for this deficiency. Here, a recombinant plasmid that expresses the spike (S) protein of SADS-CoV fused to the Fc domain of human IgG was constructed to generate recombinant baculovirus and expressed in HEK 293F cells. The S-Fc protein was purified with protein G Resin, which retained reactivity with anti-human Fc and anti-SADS-CoV antibodies. The S-Fc protein was then used to develop an indirect ELISA (S-iELISA) and the reaction conditions of S-iELISA were optimized. As a result, the cut-off value was determined as 0.3711 by analyzing OD450nm values of 40 SADS-CoV-negative sera confirmed by immunofluorescence assay (IFA) and western blot. The coefficient of variation (CV) of 6 SADS-CoV-positive sera within and between runs of S-iELISA were both less than 10%. The cross-reactivity assays demonstrated that S-iELISA was non-cross-reactive with other swine viruses' sera. Furthermore, the overall coincidence rate between IFA and S-iELISA was 97.3% based on testing 111 clinical serum samples. Virus neutralization test with seven different OD450nm values of the sera showed that the OD450nm values tested by S-iELISA are positively correlated with the virus neutralization assay. Finally, a total of 300 pig field serum samples were tested by S-iELISA and commercial kits of other swine enteroviruses showed that the IgG-positive for SADS-CoV, TGEV, PDCoV and PEDV was 81.7, 54, 65.3 and 6%, respectively. The results suggest that this S-iELISA is specific, sensitive, repeatable and can be applied for the detection of the SADS-CoV infection in the swine industry.

Emodin from Aloe Inhibits Porcine Reproductive and Respiratory Syndrome Virus via Toll-Like Receptor 3 Activation.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe reproductive failure in sows and respiratory diseases in growing and finishing pigs and results in great economic losses to the swine industry. Although vaccines are available, PRRSV remains a major threat to the pig farms. Thus, there is an urgent need to develop antiviral drugs to compensate for vaccines. In this study, we report that Aloe extract (Ae) can strongly inhibit PRRSV in Marc-145 cells and porcine alveolar macrophages lines (iPAMs) in vitro. Furthermore, we identified a novel anti-PRRSV molecule, Emodin, from Ae by high-performance liquid chromatography (HPLC). Emodin exerted its inhibitory effect through targeting the whole stages of PRRSV infectious cycle. Moreover, we also found that Emodin can inactivate PRRSV particles directly. Notably, we confirmed that Emodin was able to significantly induce Toll-like receptor 3 (TLR3) (p < 0.01), IFN-α (p < 0.05) and IFN-ß expression in iPAMs, indicating that induction of antiviral agents via TLR3 activation by Emodin might contribute to its anti-PRRSV effect. These findings imply that the Emodin from Aloe could hamper the proliferation of PRRSV in vitro and might constitute a new approach for treating PRRSV infection.

Black soldier fly (Hermetia illucens L.) larval diet improves CD8+ lymphocytes proliferation to eliminate chicken coronavirus at an early infection stage.

Avian infectious bronchitis virus (IBV), belonging to Gammacoronavirus, is an economically important respiratory virus affecting poultry industry worldwide. The virus can infect chickens at all ages, whereas young chickens (less than 15 day old) are more susceptible to it. The present study was conducted to investigate effects of dietary supplementation of black soldier fly (Hermetia illucens L.) larvae (BSFL) on immune responses in IBV infected 10-day-old chickens. BSFL were ground to powder and mixed with commercial fodder (1%, 5%, and 10 % [mass] BSFL powder) to feed 1-day-old yellow broilers for ten days and then challenged with IBV. Our results indicated that commercial fodder supplemented with 10 % BSFL [mass] reduced mortalities (20 %) and morbidities (80 %), as well as IBV viral loads in tracheas (65.8 %) and kidneys (20.4 %) from 3-day post challenge (dpc), comparing to that of IBV-infected chickens fed with non-additive commercial fodder. Furthermore, at 3-day post challenge (dpc), 10 % BSFL [mass] supplemented chickens presented more CD8+ T lymphocytes in peripheral blood and a rise in interferon-g (IFN-γ) at both mRNA and protein levels in spleens, comparing with chickens fed with commercial fodder. Furthermore, the mRNA abundance of MHC-I, Fas, LITAF, and IL-2 in the spleens of 10 % BSFL [mass] supplemented chickens increased at different time points after challenge. The present results suggest that supplemental BSFL could improve CD8+ T lymphocytes proliferation, thus benefit young chickens to defend against IBV infection.

Transcriptional Landscape of Vero E6 Cells during Early Swine Acute Diarrhea Syndrome Coronavirus Infection.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly emerged and highly pathogenic virus that is associated with fatal diarrhea disease in piglets, causing significant economic losses to the pig industry. At present, the research on the pathogenicity and molecular mechanisms of host-virus interactions of SADS-CoV are limited and remain poorly understood. Here, we investigated the global gene expression profiles of SADS-CoV-infected Vero E6 cells at 12, 18, and 24 h post-infection (hpi) using the RNA-sequencing. As a result, a total of 3324 differentially expressed genes (DEG) were identified, most of which showed a down-regulated expression pattern. Functional enrichment analyses indicated that the DEGs are mainly involved in signal transduction, cellular transcription, immune and inflammatory response, and autophagy. Collectively, our results provide insights into the changes in the cellular transcriptome during early infection of SADS-CoV and may provide information for further study of molecular mechanisms.

Porcine TRIM21 RING-finger E3 ubiquitin ligase is essential for anti-PRRSV activity.

Porcine reproductive and respiratory syndrome (PRRS) causes substantial economic losses to the global pig industry. Members of the tripartite motif (TRIM) family are the important effectors of the innate immune response against viral infections. We have previously characterized the entire porcine TRIM (pTRIM) family, and predicted pTRIM5, 14, 21, 25 and 38 as host restriction factors against PRRSV infection. However, little is known about whether and how pTRIMs restrict the infection of PRRSV. In this study, we firstly performed the amino acid alignments of the RING domain of pTRIM5, 21, 25 and 38, and found that pTRIM proteins contained the characteristic consensus C3HC4 type zinc-binding motif which is important for the ubiquitination function. Then we detected the mRNA changes of pTRIMs in porcine alveolar macrophages (PAMs) by transcriptome sequencing after PRRSV infection in piglets. Transcriptional profiles showed that the expression of pTRIM5, 21 and 26 was significantly (P < 0.05) up-regulated, consistent with their expression in vitro. Finally, as the most up-regulated gene after PRRSV infection both in vivo and in vitro, pTRIM21 was investigated for its anti-PRRSV activity in immortalized PAMs (iPAMs) in two aspects: knockdown and overexpression of pTRIM21. Knockdown of endogenic pTRIM21 could significantly promote PRRSV replication at 12 and 24 h post infection in iPAMs. Meanwhile, overexpression of pTRIM21 could significantly suppress PRRSV replication but not affect its attachment and endocytosis. Moreover, pTRIM21 RING-finger E3 ubiquitin ligase was essential for anti-PRRSV activity. Our data enhance our understanding of the pTRIMs against PRRSV infection, which may help us develop novel therapeutic tools to control PRRSV.

Profiling of alternative polyadenylation and gene expression in PEDV-infected IPEC-J2 cells.

Since 2010, porcine epidemic diarrhea virus (PEDV) has received global attention with the emergence of variant strains characterized with high pathogenicity. The pathogen-host interaction after PEDV infection is still unclear. To investigate this issue, high-throughput-based sequencing technology is one of the optimal choices. In this study, we used in vitro transcription sequencing alternative polyadenylation sites (IVT-SAPAS) method, which allowed accurate profiling of gene expression and alternative polyadenylation (APA) sites to profile APA switching genes and differentially expressed genes (DEGs) in IPEC-J2 cells during PEDV variant strain infection. We found 804 APA switching genes, including switching in tandem 3' UTRs and switching between coding region and 3' UTR, and 1,677 DEGs in host after PEDV challenge. These genes participated in variety of biological processes such as cellular process, metabolism and immunity reactions. Moreover, 413 genes, most of which are the "focus" genes in interaction networks, were found to be involved in both APA switching genes and DEGs, suggesting these genes were synchronously regulated by different mechanisms. In summary, our results gave a relatively comprehensive insight into dynamic host-pathogen interactions in the regulation of host gene transcripts during PEDV infection.

A highly pathogenic recombinant infectious bronchitis virus with adaptability in cultured cells.

Infectious bronchitis virus (IBV) of GI-19 (QX), GI-7 (TW), GI-13 (4/91) and GI-1 (Mass) lineages have been frequently detected in China in recent years. Here, An IBV strain, referred as GD17/04, was isolated from the dead yellow feather chicken vaccinated with H52 and 4/91 vaccines, whose genome sequence was obtained through high-throughput sequencing. Then it has been confirmed by the RDP and SimPlot analysis that GD17/04 is a recombinant strain deriving from YX10, 4/91, TW 2575/98 and H52 strains. Therein S1 gene of GD17/04 consists of sequences of TW2575/98 and 4/91, the former for the region of 20,371 to 21,072 nt and 21,847 to 21,975 nt, the latter for the sandwiched region of 21,073 to 21,846 nt. Moreover, as a nephropathogenic variant which caused high morbidity of 100 % and mortality of 60 %, unlike most other IBV strains, GD17/04 can cause obvious cell lesion in primary CEK cell, and even in DF-1 cells, without the process of continuous passage. As the few IBV strain can infect avian passage cell line, GD17/04 provides a material basis for further study of the interaction mechanism between IBV and avian host. Collectively, the findings highlight the significance that biological characteristics of novel strain should be studied, in addition to constant epidemiologic and molecular surveillance for IBV.

Aloe extract inhibits porcine epidemic diarrhea virus in vitro and in vivo.

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhoea and high mortality in neonatal suckling piglets, leading to significant economic losses to the swine industry. Currently there are no adequate control strategies against circulating PEDV variants, making an urgent need to exploit effect antiviral therapies to compensate for vaccines. Here, we report that Aloe extract can hamper completely the proliferation of PEDV at a non-cytotoxic concentration of 16 mg/mL determined by CCK-8 assay in Vero and IPEC-J2 cells in vitro. Furthermore, time course analysis indicated the extract exerted its inhibition at the late stage of the viral life cycle. Moreover, we also confirmed that the extract can inactivated PEDV directly but did not act on the viral genome and S1 protein. Importantly, the extract at a relatively safety concentration of 100 mg/kg of body weight, which was confirmed in mice, could reduce virus load and pathological change in intestinal tract of pigs and protect newborn piglets from lethal challenge with highly pathogenic PEDV variant GDS01 infection, indicating that Aloe extract efficiently inhibited PEDV infection in vivo. Collectively, our findings suggest that the aqueous extract from the Aloe could inhibit PEDV replication in vitro and in vivo and might be a good target for drug development against PEDV.

Porcine enteric alphacoronavirus Inhibits IFN-α, IFN-ß, OAS, Mx1, and PKR mRNA Expression in Infected Peyer's Patches in vivo.

Porcine enteric alphacoronavirus (PEAV) is a newly identified swine enteropathogenic coronavirus that causes watery diarrhea in neonatal piglets. The pathogenesis and host immune responses of PEAV infection are not fully characterized. The reason lies in the stomach environment, which would degrade cell-cultured live viruses via oral infection, making it difficult to establish an effective infection model to study the pathogenesis and host immune responses in pigs with a mature immune system. To solve this problem, in this study, coated PEAV-loaded microspheres were developed by centrifugal granulation-fluidized bed coating and demonstrated as an effective oral delivery system/animal infection model to protect PEAV virion against the complex gastrointestinal environment in vitro and to cause infection in weaned piglets in vivo. Weaned piglets orally inoculated with coated PEAV-loaded microspheres developed diarrhea and virus RNA was detected in rectal swabs from one to seven days post inoculation. In addition, microscopic lesions in the small intestine were observed, and viral antigens were also detected in the small intestines with PEAV immunohistochemical staining. Importantly, PEAV significantly inhibited mRNA expression of IFN-α, IFN-ß, OAS, Mx1, and PKR, the genes involved in modulation of the host immune responses, in infected Peyer's patches, indicating that PEAV can overcome the antiviral response to cause damage when infection occurs. Collectively, our research successfully established a PEAV animal infection model in weaned piglets and suggested that the observed gene expression profile might help explain immunological changes associated with PEAV infection.

Disulfide isomerase ERp57 improves the stability and immunogenicity of H3N2 influenza virus hemagglutinin.

BACKGROUND: Hemagglutinin (HA), as the surface immunogenic protein, is the most important component of influenza viruses. Previous studies showed that the stability of HA was significant for HA's immunogenicity, and many efforts have been made to stabilize the expressed HA proteins. METHODS: In this study, the protein disulfide isomerases (PDIs) were investigated for the ability to improve the stability of HA protein. Two members of the PDIs family, PDI and ERp57, were over-expressed or down-expressed in 293 T cells. The expression of H3 HA and PDIs were investigated by real-time qPCR, western-blot, immunofluorescence assay, and flow cytometry. The stability of HA was investigated by western-blot under non-reducing condition. Moreover, BALB/c mice were immunized subcutaneously twice with the vaccine that contained HA proteins from the ERp57-overexpressed and conventional 293 T cells respectively to investigate the impact of ERp57 on the immunogenicity of H3N2 HA. RESULTS: The percentage of the disulfide-bonded HA trimers increased significantly in the PDIs-overexpressed 293 T cells, and ERp57 was more valid to the stability of HA than PDI. The knockdown of ERp57 by small interfering RNA significantly decreased the percentage of the disulfide-bonded HA trimers. HA proteins from ERp57-overexpressed 293 T cells stimulated the mice to generate significantly higher HA-specific IgG against H1N1 and H3N2 viruses than those from the conventional cells. The mice receiving H3 HA from ERp57-overexpressed 293 T cells showed the better resistance against H1N1 viruses and the higher survival rate than the mice receiving H3 HA from the conventional cells. CONCLUSION: ERp57 could improve the stability and immunogenicity of H3N2 HA.

A novel low virulent respiratory infectious bronchitis virus originating from the recombination of QX, TW and 4/91 genotype strains in China.

In China, variants of infectious bronchitis virus (IBV) evolve continually and diverse recombinant strains have been reported. Here, an IBV strain, designated as ck/CH/LJX/2017/07 (referred as JX17) was isolated from chicken vaccinated with H120 and 4/91 in Jiangxi, China, in 2017. Sequence analysis reveals of the S1 gene of JX17 the highest nucleotide identity of 98.15% with that of GI-7 genotype TW2575/98 strain. Furthermore, whole genome analysis among JX17 and other 18 IBV strains demonstrates that JX17 has the highest nucleotide identity of 95.94% with GI-19 genotype YX10 strain. Among all genes of JX17 except the S1 gene, the N gene and 3' UTR have the highest identity to GI-13 genotype 4/91 strain and the rest genes are the most identical to GI-19 genotype YX10 strain. Analyzed by the RDP and SimPlot, the recombination of JX17 strain was shown to occur in regions which include 5'-terminal S1 gene (20,344 to 22,447 nt), most N gene and 3' UTR (26,163 to 27,648 nt). The pathogenicity study shows that JX17 is a natural low virulent IBV variant which caused respiratory symptoms but no death. Taken together, these results indicate that IBV strains continue to evolve through genetic recombination and three prevalent genotypes in China including QX, TW and 4/91 have started to recombine.