Broad antagonism of coronaviruses nsp5 to evade the host antiviral responses by cleaving POLDIP3.

Coronaviruses (CoVs) are a family of the largest RNA viruses that typically cause respiratory, enteric, and hepatic diseases in animals and humans, imposing great threats to the public safety and animal health. Porcine deltacoronavirus (PDCoV), a newly emerging enteropathogenic coronavirus, causes severe diarrhea in suckling piglets all over the world and poses potential risks of cross-species transmission. Here, we use PDCoV as a model of CoVs to illustrate the reciprocal regulation between CoVs infection and host antiviral responses. In this study, downregulation of DNA polymerase delta interacting protein 3 (POLDIP3) was confirmed in PDCoV infected IPEC-J2 cells by isobaric tags for relative and absolute quantification (iTRAQ) and Western blotting analysis. Overexpression of POLDIP3 inhibits PDCoV infection, whereas POLDIP3 knockout (POLDIP3-/-) by CRISPR-Cas9 editing significantly promotes PDCoV infection, indicating POLDIP3 as a novel antiviral regulator against PDCoV infection. Surprisingly, an antagonistic strategy was revealed that PDCoV encoded nonstructural protein 5 (nsp5) was responsible for POLDIP3 reduction via its 3C-like protease cleavage of POLDIP3 at the glutamine acid 176 (Q176), facilitating PDCoV infection due to the loss of antiviral effects of the cleaved fragments. Consistent with the obtained data in IPEC-J2 cell model in vitro, POLDIP3 reduction by cleavage was also corroborated in PDCoV infected-SPF piglets in vivo. Collectively, we unveiled a new antagonistic strategy evolved by PDCoV to counteract antiviral innate immunity by nsp5-mediated POLDIP3 cleavage, eventually ensuring productive virus replication. Importantly, we further demonstrated that nsp5s from PEDV and TGEV harbor the conserved function to cleave porcine POLDIP3 at the Q176 to despair POLDIP3-mediated antiviral effects. In addition, nsp5 from SARS-CoV-2 also cleaves human POLDIP3. Therefore, we speculate that coronaviruses employ similar POLDIP3 cleavage mechanisms mediated by nsp5 to antagonize the host antiviral responses to sustain efficient virus infection.

Autophagy is induced by swine acute diarrhea syndrome coronavirus through the cellular IRE1-JNK-Beclin 1 signaling pathway after an interaction of viral membrane-associated papain-like protease and GRP78.

Autophagy plays an important role in the infectious processes of diverse pathogens. For instance, cellular autophagy could be harnessed by viruses to facilitate replication. However, it is still uncertain about the interplay of autophagy and swine acute diarrhea syndrome coronavirus (SADS-CoV) in cells. In this study, we reported that SADS-CoV infection could induce a complete autophagy process both in vitro and in vivo, and an inhibition of autophagy significantly decreased SADS-CoV production, thus suggesting that autophagy facilitated the replication of SADS-CoV. We found that ER stress and its downstream IRE1 pathway were indispensable in the processes of SADS-CoV-induced autophagy. We also demonstrated that IRE1-JNK-Beclin 1 signaling pathway, neither PERK-EIF2S1 nor ATF6 pathways, was essential during SADS-CoV-induced autophagy. Importantly, our work provided the first evidence that expression of SADS-CoV PLP2-TM protein induced autophagy through the IRE1-JNK-Beclin 1 signaling pathway. Furthermore, the interaction of viral PLP2-TMF451-L490 domain and substrate-binding domain of GRP78 was identified to activate the IRE1-JNK-Beclin 1 signaling pathway, and thus resulting in autophagy, and in turn, enhancing SADS-CoV replication. Collectively, these results not only showed that autophagy promoted SADS-CoV replication in cultured cells, but also revealed that the molecular mechanism underlying SADS-CoV-induced autophagy in cells.

Association of THBS3 with Glycoprotein D Promotes Pseudorabies Virus Attachment, Fusion, and Entry.

Pseudorabies virus (PRV) is a neurotropic virus causing obvious neurological disorders and reproductive failure in pigs. PRV entry into target cells is a complex multistep process initiated by interacting viral envelope glycoproteins with cellular receptors. In the current study, we found that thrombospondin 3 (THBS3) plays an important role in PRV entry into target cells, indicating that THBS3 is a new PRV coreceptor. To confirm this hypothesis, the knockdown of THBS3 in several permissive cells inhibited PRV primary infection, and overexpression of THBS3 in PK15 cells promoted PRV infection. CRISPR-Cas9 knockout markedly reduced PRV infection in PK15 cells. Antibodies against THBS3 blocked PRV infection in naturally permissive target cells. Moreover, soluble THBS3 protein neutralized the infectivity of PRV. Mechanistically, THBS3 interacted with the PRV gD via its N and C termini to facilitate PRV binding in permissive and nonpermissive cells. Also, in the absence of Nectin-1, THBS3 promoted cell-to-cell fusion mediated by virus glycoproteins. While THBS3 alone could not increase virus entry, overexpression of it in the presence of Nectin-1 promoted virus entry into CHO-K1 cells. Our results have identified THBS3 as a critical player in PRV binding and subsequent membrane fusion and entry. IMPORTANCE Herpesvirus entry occurs through a cascade of virus-cell interactions, and multiple surface glycoproteins play a role in virus binding and entry during the virus invasion process. Early studies showed that attachment to cells by PRV, as well as other alphaherpesviruses, is mediated by interactions between the viral glycoprotein gC and cell membrane proteoglycans carrying heparan sulfate chains (HSPGs). However, gD may also be involved in virus binding in an HSPG-independent manner. To date, the respective cellular receptors are still unknown. In this report, we identified a host molecule, THBS3, involved in gD-mediated PRV binding and subsequent membrane fusion and entry, which increases our understanding of the initial events in alpha herpesvirus infections.

Innate Immune Evasion of Porcine Epidemic Diarrhea Virus through Degradation of the FBXW7 Protein via the Ubiquitin-Proteasome Pathway.

Porcine epidemic diarrhea virus (PEDV) causes a porcine disease associated with swine epidemic diarrhea. Different antagonistic strategies have been identified, and the mechanism by which PEDV infection impairs the production of interferon (IFN) and delays the activation of the IFN response to escape host innate immunity has been determined, but the pathogenic mechanisms of PEDV infection remain enigmatic. Our preliminary results revealed that endogenous F-box and WD repeat domain-containing 7 (FBXW7) protein, the substrate recognition component of the SCF-type E3 ubiquitin ligase, is downregulated in PEDV-infected Vero E6 cells, according to the results from an isobaric tags for relative and absolute quantification (iTRAQ) analysis. Overexpression of FBXW7 in target cells makes them more resistant to PEDV infection, whereas ablation of FBXW7 expression by small interfering RNA (siRNA) significantly promotes PEDV infection. In addition, FBXW7 was verified as an innate antiviral factor capable of enhancing the expression of RIG-I and TBK1, and it was found to induce interferon-stimulated genes (ISGs), which led to an elevated antiviral state of the host cells. Moreover, we revealed that PEDV nonstructural protein 2 (nsp2) interacts with FBXW7 and targets FBXW7 for degradation through the K48-linked ubiquitin-proteasome pathway. Consistent with the results proven in vitro, FBXW7 reduction was also confirmed in different intestinal tissues from PEDV-infected specific-pathogen-free (SPF) pigs. Taken together, the data indicated that PEDV has evolved with a distinct antagonistic strategy to circumvent the host antiviral response by targeting the ubiquitin-proteasome-mediated degradation of FBXW7. Our findings provide novel insights into PEDV infection and pathogenesis. IMPORTANCE To counteract the host antiviral defenses, most viruses, including coronaviruses, have evolved with diverse strategies to dampen host IFN-mediated antiviral response, by interfering with or evading specific host regulators at multiple steps of this response. In this study, a novel antagonistic strategy was revealed showing that PEDV infection could circumvent the host innate response by targeted degradation of endogenous FBXW7 in target cells, a process that was verified to be a positive modulator for the host innate immune system. Degradation of FBXW7 hampers host innate antiviral activation and facilitates PEDV replication. Our findings reveal a new mechanism exploited by PEDV to suppress the host antiviral response.

Rotavirus Viroplasm Biogenesis Involves Microtubule-Based Dynein Transport Mediated by an Interaction between NSP2 and Dynein Intermediate Chain.

Rotaviruses are the causative agents of severe and dehydrating gastroenteritis in children, piglets, and many other young animals. They replicate their genomes and assemble double-layered particles in cytoplasmic electron-dense inclusion bodies called "viroplasms." The formation of viroplasms is reportedly associated with the stability of microtubules. Although material transport is an important function of microtubules, whether and how microtubule-based transport influences the formation of viroplasms are still unclear. Here, we demonstrate that small viroplasms move and fuse in living cells. We show that microtubule-based dynein transport affects rotavirus infection, viroplasm formation, and the assembly of transient enveloped particles (TEPs) and triple-layered particles (TLPs). The dynein intermediate chain (DIC) is shown to localize in the viroplasm and to interact directly with nonstructural protein 2 (NSP2), indicating that the DIC is responsible for connecting the viroplasm to dynein. The WD40 repeat domain of the DIC regulates the interaction between the DIC and NSP2, and the knockdown of the DIC inhibited rotaviral infection, viroplasm formation, and the assembly of TEPs and TLPs. Our findings show that rotavirus viroplasms hijack dynein transport for fusion events, required for maximal assembly of infectious viral progeny. This study provides novel insights into the intracellular transport of viroplasms, which is involved in their biogenesis. IMPORTANCE Because the viroplasm is the viral factory for rotavirus replication, viroplasm formation undoubtedly determines the effective production of progeny rotavirus. Therefore, an understanding of the virus-host interactions involved in the biogenesis of the viroplasm is critical for the future development of prophylactic and therapeutic strategies. Previous studies have reported that the formation of viroplasms is associated with the stability of microtubules, whereas little is known about its specific mechanism. Here, we demonstrate that rotavirus viroplasm formation takes advantage of microtubule-based dynein transport mediated by an interaction between NSP2 and the DIC. These findings provide new insight into the intracellular transport of viroplasms.

Porcine Epidemic Diarrhea Virus-Induced Epidermal Growth Factor Receptor Activation Impairs the Antiviral Activity of Type I Interferon.

Porcine epidemic diarrhea virus (PEDV) causes acute and devastating enteric disease in suckling piglets and results in huge economic losses in the pig industry worldwide. To establish productive infection, viruses must first circumvent the host innate immune response. In this study, we found that PEDV infection stimulated epidermal growth factor receptor (EGFR) activation, which has been linked to not only anticancer therapeutics, but also antiviral signaling. Therefore, we determined whether EGFR activation affected PEDV infection by using an activator or overexpression assay. The data showed that EGFR activation enhanced virus replication in both cases. We also found that specific inhibition of EGFR by either inhibitors or small interfering RNA (siRNA) led to a decrease in virus yields. Further analysis revealed that inhibition of EGFR produced augmentation of type I interferon genes. We next observed that the EGFR downstream cascade STAT3 was also activated upon PEDV infection. Similar to the case of EGFR, specific inhibition of STAT3 by either inhibitor or siRNA increased the antiviral activity of interferon and resulted in decreased PEDV RNA levels, and vice versa. The data on STAT3 depletion in combination with EGFR activation suggest that the attenuation of antiviral activity by EGFR activation requires activation of the STAT3 signaling pathway. Taken together, these data demonstrate that PEDV-induced EGFR activation serves as a negative regulator of the type I interferon response and provides a novel therapeutic target for virus infection.IMPORTANCE EGFR is a transmembrane tyrosine receptor that mediates various cellular events, as well as several types of human cancers. In this study, we investigated for the first time the role of EGFR in PEDV infection. We observed that PEDV infection induced EGFR activation. The role of EGFR activation is to impair the antiviral activity of type I interferon, which requires the involvement of the EGFR downstream signaling cascade STAT3. Our findings reveal a new mechanism evolved by PEDV to circumvent the host antiviral response, which might serve as a therapeutic target against virus infection.

Genetic polymorphisms of 32 Y-STR loci in Meizhou Hakka population.

Using AGCU Y24 Plus PCR Amplification Kit, 32 Y short tandem repeat (STR) loci were analyzed in 355 unrelated male participants of Meizhou city in Guangdong Province of China. By analyzing 341 different haplotypes, it was found that haplotype diversity (HD) and discrimination capacity (DC) were 0.9995 and 0.9605, respectively. Population relationships were analyzed by comparing Hakka population with ten other populations. The results indicate that Meizhou Hakka population was closely related to Guangdong Han population. These data were valuable for both forensic applications and population genetics.

Tight Junction Protein Occludin Is a Porcine Epidemic Diarrhea Virus Entry Factor.

Porcine epidemic diarrhea virus (PEDV), the causative agent of porcine epidemic diarrhea, has caused huge economic losses in pig-producing countries. Although PEDV was long believed to replicate in the intestinal epithelium by using aminopeptidase N as a receptor, the mechanisms of PEDV infection are not fully characterized. In this study, we found that PEDV infection of epithelial cells results in disruption of the tight junctional distribution of occludin to its intracellular location. Overexpression of occludin in target cells makes them more susceptible to PEDV infection, whereas ablation of occludin expression by use of small interfering RNA (siRNA) in target cells significantly reduces their susceptibility to virus infection. However, the results observed with occludin siRNA indicate that occludin is not required for virus attachment. We conclude that occludin plays an essential role in PEDV infection at the postbinding stages. Furthermore, we observed that macropinocytosis inhibitors blocked occludin internalization and virus entry, indicating that virus entry and occludin internalization are closely coupled. However, the macropinocytosis inhibitors could not impede virus replication once the virus had entered host cells. This suggests that occludin internalization by macropinocytosis or a macropinocytosis-like process is involved in the virus entry events. Immunofluorescence confocal microscopy showed that PEDV was trapped at cellular junctional regions upon macropinocytosis inhibitor treatment, indicating that occludin may serve as a scaffold in the vicinity of virus entry. Collectively, these data show that occludin plays an essential role in PEDV infection during late entry events. Our observation may provide novel insights into PEDV infection and related pathogenesis.IMPORTANCE Tight junctions are highly specialized membrane domains whose main function is to attach adjacent cells to each other, thereby forming intercellular seals. Here we investigate, for the first time, the role of the tight junction protein occludin in PEDV infection. We observed that PEDV infection induced the internalization of occludin. By using genetic modification methods, we demonstrate that occludin plays an essential role in PEDV infection. Moreover, PEDV entry and occludin internalization seem to be closely coupled. Our findings reveal a new mechanism of PEDV infection.

Porcine Epidemic Diarrhea Virus Infection Inhibits Interferon Signaling by Targeted Degradation of STAT1.

UNLABELLED: Porcine epidemic diarrhea virus (PEDV) is a worldwide-distributed alphacoronavirus, but the pathogenesis of PEDV infection is not fully characterized. During virus infection, type I interferon (IFN) is a key mediator of innate antiviral responses. Most coronaviruses develop some strategy for at least partially circumventing the IFN response by limiting the production of IFN and by delaying the activation of the IFN response. However, the molecular mechanisms by which PEDV antagonizes the antiviral effects of interferon have not been fully characterized. Especially, how PEDV impacts IFN signaling components has yet to be elucidated. In this study, we observed that PEDV was relatively resistant to treatment with type I IFN. Western blot analysis showed that STAT1 expression was markedly reduced in PEDV-infected cells and that this reduction was not due to inhibition of STAT1 transcription. STAT1 downregulation was blocked by a proteasome inhibitor but not by an autophagy inhibitor, strongly implicating the ubiquitin-proteasome targeting degradation system. Since PEDV infection-induced STAT1 degradation was evident in cells pretreated with the general tyrosine kinase inhibitor, we conclude that STAT1 degradation is independent of the IFN signaling pathway. Furthermore, we report that PEDV-induced STAT1 degradation inhibits IFN-α signal transduction pathways. Pharmacological inhibition of STAT1 degradation rescued the ability of the host to suppress virus replication. Collectively, these data show that PEDV is capable of subverting the type I interferon response by inducing STAT1 degradation. IMPORTANCE: In this study, we show that PEDV is resistant to the antiviral effect of IFN. The molecular mechanism is the degradation of STAT1 by PEDV infection in a proteasome-dependent manner. This PEDV infection-induced STAT1 degradation contributes to PEDV replication. Our findings reveal a new mechanism evolved by PEDV to circumvent the host antiviral response.

Characterization of porcine epidemic diarrhea virus infectivity in human embryonic kidney cells.

Porcine epidemic diarrhea virus (PEDV), a causative agent of porcine epidemic diarrhea, causes economic loss in the global swine industry. Vero cell, an African green monkey kidney cell line, has been commonly used to isolate and propagate PEDV. However, since the production of interferon in these cells is defective, Vero cells are not the ideal cell type to study the molecular mechanisms of PEDV infection and the host antiviral innate immune response. In this study, we observed that human embryonic kidney 293 (HEK293) cells were susceptible to infection with PEDV vaccine strain CV777 (G1 subtype) and field isolate LNCT2 (G2 subtype). The one-step growth curve showed that the growth dynamics of PEDV in HEK293 cells was similar to that observed in Vero cells. Furthermore, we revealed that aminopeptidase N was involved in PEDV infection in HEK293 cells. Taken together, our findings suggest that HEK293 cells can be efficiently infected by PEDV, which might provide a useful tool for understanding the fundamental mechanisms of PEDV infection in vitro.

Elevated plasma-soluble CD16 levels in porcine reproductive and respiratory syndrome virus-infected pigs: correlation with ADAM17-mediated shedding.

Soluble CD16 (sCD16) is closely correlated with chronic diseases in humans. Here, plasma sCD16 levels in pigs were increased by infection with porcine reproductive and respiratory syndrome virus (PRRSV) but not with porcine epidemic diarrhea virus, porcine circovirus type 2 and pseudorabies virus. Of interest, PRRSV attached to blood neutrophils and reduced surface CD16 expression on neutrophils. In vitro data confirmed that PRRSV caused CD16 shedding in neutrophils. Further analyses revealed that ADAM17 was involved in porcine CD16 shedding. Thus, our findings suggest that increase in sCD16 levels may be an indicator of PRRSV infection.

Involvement of CD16 in antibody-dependent enhancement of porcine reproductive and respiratory syndrome virus infection.

The immunological effect of porcine reproductive and respiratory syndrome disease virus (PRRSV) vaccines is thought to be influenced by a variety of host factors, in which antibody-dependent enhancement (ADE) of infection is one crucial factor. Here, we assessed the mechanism of ADE of PRRSV infection. First, we found that subneutralizing serum could induce ADE of PRRSV infection in porcine alveolar macrophages (PAMs). Quantitative PCR, Western blotting and flow cytometry revealed that CD16 is the most abundant Fcγ receptor (FcγR) expressed on the surface of PAMs; thus, the role of CD16 in ADE of PRRSV infection was examined in PAMs. By using functional blocking antibodies, we demonstrated that CD16 is involved in enhanced virus production in PRRSV-antibody immune complex-infected PAMs. Because PAMs co-express different FcγR isoforms, we evaluated the effects of CD16 in FcγR-non-bearing cells by transfection. Using these engineered cells, we found that CD16 could specifically bind to the PRRSV-antibody immune complex and subsequently mediate internalization of the virus, resulting in the generation of progeny virus. We also showed that efficient expression of CD16 required association of the FcR γ-chain. Together, our findings provide significant new insights into PRRSV infection, which can be enhanced by CD16-mediated PRRSV-antibody immune complexes. This CD16-mediated ADE may induce a shift in PRRSV tropism towards CD16-expressing cells, distributing virus to more organs during virus infection.

Modulation of CD163 expression by metalloprotease ADAM17 regulates porcine reproductive and respiratory syndrome virus entry.

UNLABELLED: As a consequence of their effects on ectodomain shedding, members of the A disintegrin and metalloprotease (ADAM) family have been implicated in the control of various cellular processes. Although ADAM family members are also involved in cancer, inflammation, and other pathologies, it is unclear whether they affect porcine reproductive and respiratory syndrome virus (PRRSV) infection. Here, we demonstrate for the first time that inhibition of ADAM17 enhances PRRSV entry in Marc-145 and porcine alveolar macrophages (PAMs). We also demonstrate that the inhibition of ADAM17 upregulates membrane CD163 expression, a putative PRRSV receptor that is exogenously expressed in BHK-21 and endogenously expressed in Marc-145 and PAMs. Furthermore, overexpression of ADAM17 induced downregulation of CD163 expression and a reduction in PRRSV infection, whereas ablation of ADAM17 expression using specific small interfering RNA resulted in upregulation of CD163 expression with a corresponding increase in PRRSV infection. These ADAM17-mediated effects were confirmed with PRRSV nonpermissive BHK-21 cells transfected with CD163 cDNA. Overall, these findings indicate that ADAM17 downregulates CD163 expression and hinders PRRSV entry. Hence, downregulation of ADAM17 particular substrates may be an additional component of the anti-infection defenses. IMPORTANCE: ADAM17 is one of the important membrane-associated metalloproteases that mediate various cellular events, as well as inflammation, cancer, and other pathologies. Here, we investigate for the first time the role of the metalloprotease ADAM17 in PRRSV infection. By using inhibitor and genetic modification methods, we demonstrate that ADAM17 negatively regulate PRRSV entry by regulating its substrate(s). More specifically, ADAM 17 mediates the downregulation of the PRRSV cellular receptor CD163. The reduction in CD163 expression represents another component of the anti-infection response initiated by ADAM17.

Enhanced Th1-biased immune efficacy of porcine circovirus type 2 Cap-protein-based subunit vaccine when coadministered with recombinant porcine IL-2 or GM-CSF in mice.

Porcine circovirus type 2 (PCV2) capsid (Cap) protein is the primary protective antigen responsible for inducing PCV2-specific protective immunity, so it is a desirable target for the development of recombinant subunit vaccines to prevent PCV2-associated diseases. Interleukin 2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF), used as immune adjuvants, have been shown to enhance the immunogenicity of certain antigens or vaccines in various experimental models. In this study, five different subunit vaccines (the PCV2-Cap, Cap-PoIL-2, PCV2-Cap + PoIL-2, Cap-PoGM-CSF, and PCV2-Cap + PoGM-CSF vaccines) were prepared based on baculovirus-expressed recombinant proteins. The immunogenicity of these vaccines was evaluated to identify the immunoenhancement by PoIL-2 and PoGM-CSF of the Cap-protein-based PCV2 subunit vaccine in mice. The PCV2-Cap + PoIL-2, Cap-PoGM-CSF, PCV2-Cap + PoGM-CSF, and PCV2-Cap vaccines induced significantly higher levels of PCV2-specific antibodies than the Cap-PoIL-2 vaccine, whereas there was no apparent difference between these four vaccines. Our results indicate that neither PoIL-2 nor PoGM-CSF had effect on the enhancement of the humoral immunity induced by the PCV2-Cap vaccine. Furthermore, the PCV2-Cap + PoIL-2, Cap-PoGM-CSF, and PCV2-Cap + PoGM-CSF vaccines elicited stronger lymphocyte proliferative responses and greater IL-2 and interferon gamma (IFN-γ) secretion. This suggests that PoIL-2 and PoGM-CSF substantially augmented the Th1-biased immune response to the PCV2-Cap vaccine. Following challenge, the viral loads in the lungs of the PCV2-Cap + PoIL-2-, Cap-PoGM-CSF-, and PCV2-Cap + PoGM-CSF-treated groups were dramatically lower than those in the Cap-PoIL-2- and PCV2-Cap-treated groups, indicating that the three vaccines induced stronger protective effects against challenge. These findings show that PoIL-2 and PoGM-CSF essentially enhanced the Th1-biased protective efficacy of the PCV2-Cap vaccine when coadministered with the protein or delivered as Cap-PoGM-CSF, and that the "antigen-cytokine"- or "antigen + cytokine"-based vaccines that we report here provide new basis for the development of safer and more effective vaccines.

A recombinant porcine circovirus type 2 expressing the VP1 epitope of the type O foot-and-mouth disease virus is infectious and induce both PCV2 and VP1 epitope antibodies.

Porcine circovirus type 2 (PCV2) is the etiological agent of postweaning multisystemic wasting syndrome, a disease that causes huge economic damage in swine industry. A recombinant PCV2 expressing the neutralizing VP1 epitope (aa 141-160) of the foot-and-mouth disease virus (FMDV) was rescued using an infectious cloning technique. The PCV2 antigen and FMDV-VP1 antigenic epitope of the cloned strain recPCV2-CL-VP1 were confirmed by an immunoperoxidase monolayer assay (IPMA) and a capture enzyme-linked immunosorbent assay (ELISA). The morphological features of the recPCV2-CL-VP1 were not discernibly different from those of its parental strain (PCV2-CL). However, the recombinant virus could be differentiated from its parental virus by PCR and capture ELISA. The recPCV2-CL-VP1 was demonstrated to replicate stably in PK-15 cells through ten passages. An infection experiment using BALB/c mice showed that both recPCV2-CL-VP1 and PCV2-CL could replicate in the mice, cause various pathological changes, and induce a high level of anti-Cap antibodies. The recombinant virus emulsified with Freund's adjuvant was used to immunize BALB/c mice and induced antibodies against the FMDV-VP1 epitope. Hence, the recombinant PCV2 strain, which expressed the neutralizing FMDV-VP1 epitope, provides a valuable platform to develop novel genetic vaccines.

IFITM3 restricts porcine deltacoronavirus infection by targeting its Spike protein.

The discovery of antiviral molecules is crucial for controlling porcine deltacoronavirus (PDCoV). Previous studies have provided evidence that the IFN-inducible transmembrane protein 3 (IFITM3), which is coded by an interferon-stimulated gene, prevents the infections of a number of enveloped viruses. Nevertheless, the involvement of IFITM3 in PDCoV infection remains unexplored. In this study, it was observed that the overexpression of IFITM3 successfully restrictes the infection of PDCoV in cell cultures. Conversely, the suppression of IFITM3 facilitates the infection of PDCoV in IPI-2I and IPEC-J2 cells. Further studies revealed that IFITM3 limits the attachment phase of viral infection by interacting with the S1 subunit of the PDCoV Spike (S) protein. In addition, IFITM3 is verified as a member of the CD225 family, the GxxxG conserved motif of this family is important for it to limit PDCoV infection. In summary, this study reveals the mechanism of IFITM3 as an antiviral molecule to inhibit PDCoV infection, and also provides theoretical supports for screening effective anti-PDCoV drugs.

A novel antigenic epitope identified on the accessory protein NS6 of porcine deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is a novel enteric coronavirus that can cause vomiting, watery diarrhea in pigs and the death of piglets. The open reading frame (ORF) 5 is one of the accessory genes in PDCoV genome and encodes an accessory protein NS6. To date, the function of NS6 is still unclear. In this study, the recombinant NS6 was successfully expressed in prokaryotic expression system and purified. To prepare monoclonal antibody (mAb), six-week-old female BALB/c mice were primed subcutaneously with purified NS6. A novel mouse mAb against NS6 was obtained and designated as 3D5. The isotype of 3D5 is IgG2b with kappa (κ) light chain. 3D5 can specifically recognizes the natural NS6 in swine testis (ST) cells infected with PDCoV and expressed NS6 in human embryonic kidney 293T (HEK 293T) cells transfected with mammalian vector. The minimal linear B cell epitope recognised by 3D5 on NS6 was 25VPELIDPLVK34 determined by peptide scanning and named EP-3D5. The sequence of EP-3D5 is completely conserved among PDCoV strains. Moreover, six to nine residues of EP-3D5 were identified to be conserved in non-PDCoV strains. These results provide valuable insights into the antigenic structure and function of NS6 in virus pathogenesis, and aid for the development of PDCoV epitope-associated diagnostics and vaccine design.

Three new emerging subgroups of Torque teno sus viruses (TTSuVs) and co-infection of TTSuVs with porcine circovirus type 2 in China.

BACKGROUND: Torque teno sus viruses (TTSuVs) are non-enveloped viruses and have single-stranded, negative sense circular DNA genomes and are widely distributed in pigs. But till now, the prevalence of TTSuVs with porcine circovirus type 2 (PCV2) in pig herds of China is not very clear; and the genetic variation among different TTSuVs isolate is very large and need to divide the subgroups. In this study, the co-infection with TTSuVs and porcine circovrius (PCV) in the pig population of China was investigated and the subgroups of all TTSuVs genomes in Genbank were divided. RESULTS: Results showed that the rate of co-infection with TTSuV1 and TTSuV2 reached 75% in PCV2-positive samples. Also Two TTSuV1 and four TTSuV2 isolates genome sequences were obtained, and the similarity of all TTSuV1 and TTSuV2 genomic sequences in GenBank were compared. Phylogenetic trees indicated that both the TTSuV1 and TTSuV2 sequences could be divided into four genotypes. Interestingly, the sub-genotypes TTSuV1d, TTSuV2c and TTSuV2d exist only in the pig population of China. CONCLUSIONS: This study demonstrates that co-infection with TTSuVs and PCVs is very common in the pig population of China, in which the viruses maybe contribute to clinical diseases cooperatively. In addition, three new subgroups of TTSuVs emerged in China for the first time and a high level of variation among different isolates of TTSuV1 and TTSuV2 was indicated by their genetic diversity.

Synergistic effects of sequential infection with highly pathogenic porcine reproductive and respiratory syndrome virus and porcine circovirus type 2.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of porcine reproductive and respiratory syndrome (PRRS) and porcine circovirus type 2 (PCV2) is associated with postweaning multisystemic wasting syndrome (PMWS) in pigs. Coinfection with highly pathogenic PRRSV (HP-PRRSV) and PCV2 in the field has recently become extensive in some Asian countries. A synergistic pathogenicity between PRRSV and PCV2 infections has previously been reported. However, the consequences of the sequential infection of pigs with these two viruses are unknown. METHODS: Thirty 35-day-old piglets were randomly divided into six groups (n = 5 each): HP-PRRSV/PCV2 (group 1, inoculated with HP-PRRSV, then inoculated with PCV2 one week later), PCV2/HP-PRRSV (group 2, inoculated with PCV2, then inoculated with HP-PRRSV one week later), HP-PRRSV+PCV2 (group 3, inoculated with HP-PRRSV and PCV2 concurrently), HP-PRRSV (group 4, inoculated with HP-PRRSV), PCV2 (group 5, inoculated with PCV2), and the control (group 6, uninfected). This experiment lasted 28 days. Clinical symptoms and rectal temperatures were recorded each day after inoculation, body weight was recorded weekly, and serum samples were obtained for viral nucleic acid quantification and antibody titration. Variations in CD3+, CD4+ CD8-, CD3+, CD4-, and CD8+ cells, natural killer (NK) cells, and mononuclear cells were determined by flow cytometry. The serum concentrations of interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), interleukin 10 (IL-10), and macrophage granulocyte-colony stimulating factor (GM-CSF) were determined. Pathological changes in different tissues from the experimentally infected pigs were recorded. RESULTS: The piglets in group 1 had the highest viral loads, the lowest antibody titers, the most-severe clinical signs, and the highest mortality (3/5, 60%; the mortality in the other groups was 0%), and interstitial pneumonia was more severe in this group compare to the other HP-PRRSV infected groups. The serum levels of IFN-γ, TNF-α, IL-10, and GM-CSF varied (increased or decreased) most widely in group 1, as did each immunocyte subgroup. CONCLUSIONS: HP-PRRSV infection followed by PCV2 infection enhanced the replication of both viruses in the experimental piglets and led to more-severe clinical signs and lesions, indicating greater synergistic effects during the sequential infection of piglets with HP-PRRSV and then PCV2.