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.

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.

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.

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.

Development and clinical application of a novel CRISPR-Cas12a based assay for the detection of African swine fever virus.

BACKGROUND: As no treatment or effective vaccine for African swine fever virus (ASFV) is currently available, a rapid, highly sensitive diagnostic is urgently needed to curb the spread of ASFV. RESULTS: Here we designed a novel CRISPR-Cas12a based assay for ASFV detection. To detect different ASFV genotypes, 19 crRNAs were designed to target the conserved p72 gene in ASFV, and several crRNAs with high activity were identified that could be used as alternatives in the event of new ASFV variants. The results showed that the sensitivity of the CRISPR-Cas12a based assay is about ten times higher than either the commercial quantitative PCR (qPCR) kit or the OIE-recommended qPCR. CRISPR-Cas12a based assay could also detect ASFV specifically without cross-reactivity with other important viruses in pigs and various virus genotypes. We also found that longer incubation times increased the detection limits, which could be applied to improve assay outcomes in the detection of weakly positive samples and new ASFV variants. In addition, both the CRISPR-Cas12a based assay and commercial qPCR showed very good consistency. CONCLUSIONS: In summary, the CRISPR-Cas12a based assay offers a feasible approach and a new diagnostic technique for the diagnosis of ASFV, particularly in resource-poor settings.

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.

PEDV enters cells through clathrin-, caveolae-, and lipid raft-mediated endocytosis and traffics via the endo-/lysosome pathway.

With the emergence of highly pathogenic variant strains, porcine epidemic diarrhea virus (PEDV) has led to significant economic loss in the global swine industry. Many studies have described how coronaviruses enter cells, but information on PEDV invasion strategies remains insufficient. Given that the differences in gene sequences and pathogenicity between classical and mutant strains of PEDV may lead to diverse invasion mechanisms, this study focused on the cellular entry pathways and cellular transport of the PEDV GI and GII subtype strains in Vero cells and IPEC-J2 cells. We first characterized the kinetics of PEDV entry into cells and found that the highest invasion rate of PEDV was approximately 33% in the IPEC-J2 cells and approximately 100% in the Vero cells. To clarify the specific endocytic pathways, systematic research methods were used and showed that PEDV enters cells via the clathrin- and caveolae-mediated endocytosis pathways, in which dynamin II, clathrin heavy chain, Eps15, cholesterol, and caveolin-1 were indispensably involved. In addition, lipid raft extraction assay showed that PEDV can also enter cells through lipid raft-mediated endocytosis. To investigate the trafficking of internalized PEDV, we found that PEDV entry into cells relied on low pH and internalized virions reached lysosomes through the early endosome-late endosome-lysosome pathway. The results concretely revealed the entry mechanisms of PEDV and provided an insightful theoretical basis for the further understanding of PEDV pathogenesis and guidance for new targets of antiviral drugs.

Establishment and application of a 10-plex liquid bead array for the simultaneous rapid detection of animal species.

BACKGROUND: Meat fraud and adulteration incidents occur frequently in almost all regions of the globe, especially with the increase in the world's population. To ensure the authenticity of meat products, we developed a 10-plex xMAP assay to simultaneously detect ten animal materials: bovine, caprine, poultry, swine, donkey, deer, horse, dog, fox and mink. RESULTS: This method was investigated by analyzing DNA extracts from raw muscle, muscle mixtures, meat products and animal feeds. Our results indicated that the species of interest can be identified, differentiated and detected down to 1 g kg-1 in binary mixtures or 0.01-0.001 ng of genomic DNA from specific species. Testing of 125 commercial samples showed a 97.4% coincidence rate with the method used in routine testing in our lab. CONCLUSION: These results indicated that the method established in this study could detect ten animal materials simultaneously within 3 h, which provides a new, useful tool for animal ingredient analysis in meat products and animal feeds. © 2019 Society of Chemical Industry.

Multiple amino acid substitutions involved in the adaption of three avian-origin H7N9 influenza viruses in mice.

BACKGROUND: Avian influenza A H7N9 virus has caused five outbreak waves of human infections in China since 2013 and posed a dual challenge to public health and poultry industry. The number of reported H7N9 virus human cases confirmed by laboratory has surpassed that of H5N1 virus. However, the mechanism for how H7N9 influenza virus overcomes host range barrier has not been clearly understood. METHODS: To generate mouse-adapted H7N9 influenza viruses, we passaged three avian-origin H7N9 viruses in mice by lung-to-lung passages independently. Then, the characteristics between the parental and mouse-adapted H7N9 viruses was compared in the following aspects, including virulence in mice, tropism of different tissues, replication in MDCK cells and molecular mutations. RESULTS: After ten passages in mice, MLD50 of the H7N9 viruses reduced >750-3,160,000 folds, and virus titers in MDCK cells increased 10-200 folds at 48 hours post-inoculation. Moreover, the mouse-adapted H7N9 viruses showed more expanded tissue tropism and more serious lung pathological lesions in mice. Further analysis of the amino acids changes revealed 10 amino acid substitutions located in PB2 (E627K), PB1 (W215R and D638G), PA (T97I), HA (H3 numbering: R220G, L226S, G279R and G493R) and NA (P3Q and R134I) proteins. Moreover, PB2 E627K substitution was shared by the three mouse-adapted viruses (two viruses belong to YRD lineage and one virus belongs to PRD lineage), and PA T97A substitution was shared by two mouse-adapted viruses (belong to YRD lineage). CONCLUSIONS: Our result indicated that the virulence in mice and virus titer in MDCK cells of H7N9 viruses significantly increased after adapted in mouse model. PB2 E627K and PA T97A substitutions are vital in mouse adaption and should be monitored during epidemiological study of H7N9 virus.

Biological characteristics and immunological properties in Muscovy ducks of H5N6 virus-like particles composed of HA-TM/HA-TMH3 and M1.

Highly pathogenic avian influenza viruses (HPAIVs), including H5N6 strains, pose threats to the health of humans and poultry. Waterfowl play a crucial role as a reservoir of HPAIVs. Since current influenza vaccines induce poor antibody titres in waterfowl, there is an urgent need to develop an efficient vaccine against H5N6 infection. In this study, we constructed two H5N6 virus-like particles (VLPs) composed of matrix-1 (M1) and haemagglutinin of wildtype (HA-TM) or haemagglutinin with transmembrane domain replacement (HA-TMH3) (designated as H5N6 VLPs-TM and H5N6 VLPs-TMH3). Biological characteristics of the composed H5N6 VLPs were compared including localization, expression, contents of HA trimers, thermal stability, morphology and immunogenicity in Muscovy ducks. Our results indicate that the H5N6 VLPs-TMH3 contained more HA trimers and presented better thermal stability. Moreover, Muscovy ducks immunized with H5N6 VLPs-TMH3 produced higher titres of HI antibody and IFN-γ compared with those immunized with the same dose of H5N6 VLP-TM, thus providing a promising approach for the development of influenza virus vaccines for waterfowl. RESEARCH HIGHLIGHTS H5N6 VLPs-TMH3 had more HA trimers and resisted higher temperature than H5N6 VLPs-TM H5N6 VLPs-TMH3 induced higher titre of HI than H5N6 VLPs-TM in Muscovy ducks.

Epidemiological investigation of fowl adenovirus infections in poultry in China during 2015-2018.

BACKGROUND: Fowl adenoviruses (FAdVs) are associated with many diseases, resulting in huge economic losses to the poultry industry worldwide. Since 2015, outbreaks of FAdV infections with high mortality rates have been reported in China. A continued surveillance of FAdVs contributes to understand the epidemiology of the viruses. RESULTS: We isolated 155 FAdV strains from diseased chickens from poultry in China between 2015 and 2018. PCR analysis determined that 123 samples were FAdV species C, 27 were FAdV species E, and five contained two different FAdV strains. The phylogenetic analysis demonstrates that these sequences of hexon regions were clustered into three distinct serotypes: FAdV-4 (79.4%, 123/155), FAdV-8a (13.5%, 21/155) and FAdV-8b (3.9%, 6/155), of which FAdV-4 was the dominant serotype in China. CONCLUSIONS: The characterization of newly prevalent FAdV strains provides valuable information for the development of an effective control strategy for FAdV infections in chickens.

Heparanase Upregulation Contributes to Porcine Reproductive and Respiratory Syndrome Virus Release.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to cause substantial economic losses to the pig industry worldwide. Heparan sulfate (HS) is used by PRRSV for initial attachment to target cells. However, the role of HS in the late phase of PRRSV infection and the mechanism of virus release from host cells remain largely unknown. In this study, we showed that PRRSV infection caused a decrease in HS expression and upregulated heparanase, the only known enzyme capable of degrading HS. We subsequently demonstrated that the NF-κB signaling pathway and cathepsin L protease were involved in regulation of PRRSV infection-induced heparanase. In addition, we found that ablation of heparanase expression using small interfering RNA duplexes increased cell surface expression of HS and suppressed PRRSV replication and release, whereas overexpression of heparanase reduced HS surface expression and enhanced PRRSV replication and release. These data suggest that PRRSV activates NF-κB and cathepsin L to upregulate and process heparanase, and then the active heparanase cleaves HS, resulting in viral release. Our findings provide new insight into the molecular mechanism of PRRSV egress from host cells, which might help us to further understand PRRSV pathogenesis.IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) causes great economic losses each year to the pig industry worldwide. The molecular mechanism of PRRSV release from host cells largely remains a mystery. In this study, we demonstrate that PRRSV activates NF-κB and cathepsin L to upregulate and process heparanase, and then the active heparanase is released to the extracellular space and exerts enzymatic activity to cleave heparan sulfate, resulting in viral release. Our findings provide new insight into the molecular mechanism of PRRSV egress from host cells, which might help us to further understand PRRSV pathogenesis.

Neutralizing antibodies against porcine epidemic diarrhea virus block virus attachment and internalization.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) is emerging as a pathogenic coronavirus that causes a huge economic burden to the swine industry. Interaction of the viral spike (S) surface glycoprotein with the host cell receptor is recognized as the first step of infection and is the main determinant of virus tropism. The mechanisms by which neutralizing antibodies inhibit PEDV have not been defined. Isolating PEDV neutralizing antibodies are crucial to identifying the receptor-binding domains of the viral spike and elucidating the mechanism of protection against PEDV infection. METHODS: B cell hybridoma technique was used to generate hybridoma cells that secrete specific antibodies. E.coli prokaryotic expression system and Bac-to-Bac expression system were used to identify the target protein of each monoclonal antibody. qPCR was performed to analyze PEDV binding to Vero E6 cells with neutralizing antibody. RESULTS: We identified 10 monoclonal antibodies using hybridoma technology. Remarkably, 4 mAbs (designed 2G8, 2B11, 3D9, 1E3) neutralized virus infection potently, of which 2B11 and 1E3 targeted the conformational epitope of the PEDV S protein. qPCR results showed that both 2B11 and 2G8 blocked virus entry into Vero cells. CONCLUSION: The data suggested that PEDV neutralizing antibody inhibited virus infection by binding to infectious virions, which could work as a tool to find the receptor-binding domains.

A New Bat-HKU2-like Coronavirus in Swine, China, 2017.

We identified from suckling piglets with diarrhea in China a new bat-HKU2-like porcine coronavirus (porcine enteric alphacoronavirus). The GDS04 strain of this coronavirus shares high aa identities (>90%) with the reported bat-HKU2 strains in Coronaviridae-wide conserved domains, suggesting that the GDS04 strain belongs to the same species as HKU2.

Identification and pathogenicity of a variant porcine epidemic diarrhea virus field strain with reduced virulence.

BACKGROUND: Since 2010, a variant Porcine epidemic diarrhea virus (PEDV), which causes an acute, highly contagious, and devastating viral enteric disease with a high mortality rate in suckling pigs, broke out in China and spread rapidly to neighboring countries, even to the North America. This virus gradually became the main subtype of PEDV worldwide. However, there were no reports of mild pathogenicity of a variant porcine epidemic diarrhea virus in China. FINDINGS: In 2013, a PEDV-positive sample from a sow with very mild clinical sign was used to inoculate in Vero cells to isolate the virus. This PEDV field strain, designated FL2013 strain, was successfully propagated and genetically characterized. The phylogenetic trees based upon either the complete genome or S gene showed that the FL2013 strain belongs to the genogroup G2b. The S gene of FL2013 has a 7-aa deletion (FEKVHVQ) in the C-terminus comparison with the other G2 PEDV sequences. Further comparative pathology study indicated that the FL2013 strain had reduced virulence to newborn piglets. CONCLUSIONS: A novel variant PEDV strain FL2013 with reduced virulence, as determined by the pathological study, was identified from east China. This strain is closely related to the genogroup-2 PEDV strains prevalent in the U.S. and China currently, but had a short deletion at the 3'-end of the spike gene.

Bead-based suspension array for simultaneous differential detection of five major swine viruses.

A novel multiplex detection array based on Luminex xMAP technology was developed and validated for simultaneous detection of five major viruses causing swine reproductive diseases. By combining one-step asymmetric multiplex reverse transcription polymerase chain reaction (RT-PCR) with xMAP bead-based hybridization and flow cytometry analysis, the resulting multiplex assay was capable of detecting single and mixed infections of PRRSV, PCV-2, PRV, CSFV, and PPV in a single reaction. The assay accurately detected and differentiated 23 viral strains used in this study. The low detection limit was determined as 2.2-22 copies/µL (corresponding to 0.5-6.8 fg/µL DNA template) on plasmid constructs containing viral fragments. The intra-assay and inter-assay variances (CV%) were low that ranged from 2.5 to 5.4 % and 4.1 to 7.6 %, respectively. The assay was applied to test field samples and detected single and mixed viral infections. The detection rate was higher than that of uniplex conventional PCR and RT-PCR methods. The detection of PRRSV by the bead-based multiplex assay was comparable with a commercially available real time RT-PCR kit. The test procedure on purified DNA or RNA samples could be completed within 2 h. In conclusion, the bead-based suspension array presented here proved to be a high-throughput practical tool that provided highly specific and sensitive identification of single and multiple infections of five major viruses in pigs and boar semen.

Chimeric influenza-virus-like particles containing the porcine reproductive and respiratory syndrome virus GP5 protein and the influenza virus HA and M1 proteins.

Both porcine reproductive and respiratory syndrome and swine influenza are acute, highly contagious swine diseases. These diseases pose severe threats for the swine industry and cause heavy economic losses worldwide. In this study, we have developed a chimeric virus-like particle (VLP) vaccine candidate for porcine reproductive and respiratory syndrome virus (PRRSV) and H3N2 influenza virus and investigated its immunogenicity in mice. The HA and M1 proteins from the H3N2 influenza virus and the PRRSV GP5 protein fused to the cytoplasmic and transmembrane domains of the NA protein were both incorporated into the chimeric VLPs. Analysis of the immune responses showed that the chimeric VLPs elicited serum antibodies specific for both PRRSV GP5 and the H3N2 HA protein, and they stimulated cellular immune responses compared to the responses to equivalent amounts of inactivated viruses. Taken together, the results suggested that the chimeric VLP vaccine represents a potential strategy for the development of a safe and effective vaccine to control PRRSV and H3N2 influenza virus.

Bioinformatics insight into the spike glycoprotein gene of field porcine epidemic diarrhea strains during 2011-2013 in Guangdong, China.

Three strains of porcine epidemic diarrhea virus (PEDV) were isolated from dead or diseased pigs at different swine farms in Guangdong during 2011-2013, and their S genes were sequenced. In the same period, seven PEDV strains were also isolated in Guangdong by other laboratories. The spike sequences of 10 Guangdong isolates were compared with vaccine strains and reference pathogenic isolates using six bioinformatics tools. The results revealed that 10 Guangdong strains, excluding strain GDS03, had distinct characteristics in terms of primary structure, secondary structure, high-specificity N-glycosylation sites, potential phosphorylation sites, and palmitoylation sites. Phylogenetic analysis also confirmed these findings and revealed that all PEDV strains were clustered into three distinct groups. Ten Guangdong strains, not including GDS03, belong to Group 1, whereas four vaccine strains and GDS03 belong to Group 3, which is evolutionarily distant from Group 1. Alignment analysis of the neutralizing region amino acid sequences indicated that the amino acid substitutions of Y/D766S, T549S, and G594S that are present in the Guangdong strains, not including GDS03, were a sign of predominant genetic changes among the isolated strains. GDS03 is closely related to the 83P-5 vaccine strain, which suggests that it might represent re-isolation of the vaccine strain or vaccine variants. Taken together, these results indicate that there have been predominant new strains circulating in Guangdong from 2011 to 2013, and the circulating PEDV strains have a genetic composition that is distant from reference strains, especially the vaccine strains; however, the vaccinations might also provide some level of cross-protection, as there have been no changes in the neutralizing epitopes of SS2 and 2C10. This explains why there have been constant but infrequent outbreaks recently in comparison to late 2010 in which PEDV outbreaks were more frequent and severe. In addition, the USA-Colorado-2013 strain had the same amino acid substitutions in the neutralizing regions as the Guangdong strains except GDS03, which suggests that the information and strategies in this study may play role in PEDV variant research in other countries.

Evidences for the existence of intermolecular disulfide-bonded oligomers in the H3 hemagglutinins expressed in insect cells.

The hemagglutinin (HA) protein as the predominant antigen, executes receptor binding and membrane fusion, which critically influence the virological characteristics of influenza viruses. The literature contained scattered data showing reduction-sensitive HA oligomers when HA proteins were analyzed under non-reducing conditions. However, whether the reduction-sensitive HA oligomers are inter-monomer disulfide-bonded has not been studied. Here, we showed: (1) the detection of ß-mercaptoethanol-sensitive H3 HA oligomers was not affected by the treatment of cells with iodoacetamide prior to cell solubilization; (2) H3 HA oligomers were present on cell surfaces; (3) H3 HA oligomers had higher density than monomers; and (4) mutation of all the five C-terminal cysteines completely abolished the formation of H3 HA oligomers. Furthermore, mutant HAs with mutations of TM cysteines, CT cysteines or all five cysteines had decreased thermal stability but increased fusion activity in comparison with wildtype HA. In conclusion, this study has presented enough evidence for the existence of inter-monomer S-S H3 HA oligomers formed by five C-terminal cysteines, and suggested that all five C-terminal cysteines exerted opposite effects on HA thermal stability and fusion activity.

Recombinant influenza A H3N2 viruses with mutations of HA transmembrane cysteines exhibited altered virological characteristics.

Influenza A H3N2 virus as the cause of 1968 pandemic has since been circulating in human and swine. Our earlier study has shown that mutations of one or two cysteines in the transmembrane domain of H3 hemagglutinin (HA) affected the thermal stability and fusion activity of recombinant HA proteins. Here, we report the successful generation of three recombinant H3N2 mutant viruses (C540S, C544L, and 2C/SL) with mutations of one or two transmembrane cysteines of HA in the background of A/swine/Guangdong/01/98 [H3N2] using reverse genetics, indicating that the mutated cysteines were not essential for virus assembly and growth. Further characterization revealed that recombinant H3N2 mutant viruses exhibited larger plaque sizes, increased growth rate in cells, enhanced fusion activity, reduced thermal and acidic resistances, and increased virulence in embryonated eggs. These results demonstrated that the transmembrane cysteines (C540 and C544) in H3 HA have profound effects on the virological features of H3N2 viruses.