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.

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.

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.

Integrative transcriptomic profiling of mRNA, miRNA, circRNA, and lncRNA in alveolar macrophages isolated from PRRSV-infected porcine.

Introduction: The porcine reproductive and respiratory syndrome virus (PRRSV) continues to pose a significant threat to the global swine industry, attributed largely to its immunosuppressive properties and the chronic nature of its infection. The absence of effective vaccines and therapeutics amplifies the urgency to deepen our comprehension of PRRSV's intricate pathogenic mechanisms. Previous transcriptomic studies, although informative, are partially constrained by their predominant reliance on in vitro models or lack of long-term infections. Moreover, the role of circular RNAs (circRNAs) during PRRSV invasion is yet to be elucidated. Methods: In this study, we employed an in vivo approach, exposing piglets to a PRRSV challenge over varied durations of 3, 7, or 21 days. Subsequently, porcine alveolar macrophages were isolated for a comprehensive transcriptomic investigation, examining the expression patterns of mRNAs, miRNAs, circRNAs, and long non-coding RNAs (lncRNAs). Results: Differentially expressed RNAs from all four categories were identified, underscoring the dynamic interplay among these RNA species during PRRSV infection. Functional enrichment analyses indicate that these differentially expressed RNAs, as well as their target genes, play a pivotal role in immune related pathways. For the first time, we integrated circRNAs into the lncRNA-miRNA-mRNA relationship, constructing a competitive endogenous RNA (ceRNA) network. Our findings highlight the immune-related genes, CTLA4 and SAMHD1, as well as their associated miRNAs, lncRNAs, and circRNAs, suggesting potential therapeutic targets for PRRS. Importantly, we corroborated the expression patterns of selected RNAs through RT-qPCR, ensuring consistency with our transcriptomic sequencing data. Discussion: This study sheds lights on the intricate RNA interplay during PRRSV infection and provides a solid foundation for future therapeutic strategizing.

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.

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.

Characterization and Pathogenicity of Two Novel PRRSVs Recombined by NADC30-like and NADC34-like Strains in China.

Porcine reproductive and respiratory syndrome viruses (PRRSVs) pose a serious threat to the swine industry in China, which has caused great difficulties for porcine reproductive and respiratory syndrome (PRRS) immune prevention and control, due to its easily mutable and recombinant nature. In this study, two novel PRRSV strains, which were named GD-H1 and GD-F1, were isolated and fully sequenced from pig farms in Guangdong province, China. The phylogenetic analysis and recombination analysis revealed that the GD-H1 and GD-F1 were generated by the recombination of NADC30-like and NADC34-like strains which were different from the previously prevalent strain. Further pathogenic studies on piglets and sows found that the recombinant strains could cause piglets high fever, loss of appetite and lung lesions, but no piglets died. However, the recombinant strains could cause acute death and abortion in pregnant sow infection models together with average survival rates of 62.5% and 37.5% abortion rates, respectively. These findings indicated that the recombinant strains were extremely pathogenic to sows. Therefore, we report two clinical novel recombinant strains of PRRSV that are different from the traditional epidemic strains in China, which may provide early warning and support for PRRS immune prevention and control.

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.

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.

H7 virus-like particles assembled by hemagglutinin containing H3N2 transmembrane domain and M1 induce broad homologous and heterologous protection in mice.

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. There is an urgent need to develop an effective vaccine to reduce its pandemic potential. In the present study, we evaluated the biochemical characteristics and immunogenicity of two H7 virus-like particles (VLPs) composed of the matrix 1 (M1) and hemagglutinin of wild-type (HA-WT) or hemagglutinin of whose transmembrane domain replaced by that from H3N2 subtype (HA-TM). H7 VLPs-WT and H7 VLPs-TM could assemble and release into the supernatant of Sf9 cells and they had similar morphological characteristics. However, compared to H7 VLPs-WT, H7 VLPs-TM had more trimeric HA proteins and could better resist thermal changes. In mice H7 VLPs-TM induced higher titers of HI, IgG, IgG2a and IFN-γ, and provided better protection against homologous and heterologous H7N9 viruses (no matter belonging to Yangtze River Delta or Pearl River Delta) challenge with less weight loss and higher survival rate. In summary, H7 VLPs-TM represents a potential strategy for the development of H7N9 vaccines.

PED subunit vaccine based on COE domain replacement of flagellin domain D3 improved specific humoral and mucosal immunity in mice.

Porcine epidemic diarrhea (PED) is an important re-emergent infectious disease and inflicts huge economic losses to the swine industry worldwide. To meet the pressing need of developing a safe and cost-efficient PED maternal vaccine, we generated three PED subunit vaccine candidates, using recombined Salmonella flagellin (rSF) as a mucosal molecular adjuvant. Domain D3 in rSF was replaced with COE domain of PEDV to generate rSF-COE-3D. COE fused to the flanking C'/N' terminal of rSF yielded rSF-COE-C and rSF-COE-N. As a result, rSF-COE-3D could significantly improve COE specific antibody production including serum IgG, serum IgA, mucosal IgA and PEDV neutralizing antibody. Furthermore, rSF-COE-3D elicited more CD3+CD8+ T cell and cytokine production of IFN-γ and IL-4 in mouse splenocytes. In summary, our data showed that rSF-COE-3D could improve specific humoral and mucosal immunity in mice, thus suggesting that rSF-COE-3D could be applied as a novel efficient maternal PED vaccine.

A flagellin-adjuvanted PED subunit vaccine improved protective efficiency against PEDV variant challenge in pigs.

PEDV variants, causing severe diarrhea in neonatal suckling piglets with a mortality rate up to 100%, have being epidemic since late 2010 in china. To meet the pressing need of safe and cost-efficient PED maternal vaccines against PEDV variant, we vaccinated growing piglets with a flagellin-adjuvanted PED vaccine rSF-COE-3D by injection at Houhai acupoint. The vaccination not only enhanced the antibody responses of serum IgG/IgA, mucosal IgA and serum neutralizing antibody, but also improved the production of IFN-γ and IL-4. Moreover, rSF-COE-3D could provide a better protection against the challenge of a high pathogenic PEDV variant, with less diarrhea pigs, less pigs with detectable PEDV shed, lower rank values of feces and less apparent lesions and inflammation in duodenum of the PEDV infected pigs. The improved protective efficiency of rSF-COE-3D compared with COE was mostly benefited from the enhanced production of serum IgA, mucosal IgA in feces and serum neutralizing antibody titers. Taken together, our data suggest that rSF-COE-3D would be a novel efficient PED vaccine.