A Broad-specificity Neutralizing Nanobody against Hepatitis E Virus Capsid Protein.

Hepatitis E virus (HEV) is a worldwide zoonotic and public health concern. The study of HEV biology is helpful for designing viral vaccines and drugs. Nanobodies have recently been considered appealing materials for viral biological research. In this study, a Bactrian camel was immunized with capsid proteins from different genotypes (1, 3, 4, and avian) of HEV. Then, a phage library (6.3 × 108 individual clones) was constructed using peripheral blood lymphocytes from the immunized camel, and 12 nanobodies against the truncated capsid protein of genotype 3 HEV (g3-p239) were screened. g3-p239-Nb55 can cross-react with different genotypes of HEV and block Kernow-C1/P6 HEV from infecting HepG2/C3A cells. To our knowledge, the epitope recognized by g3-p239-Nb55 was determined to be a novel conformational epitope located on the surface of viral particles and highly conserved among different mammalian HEV isolates. Next, to increase the affinity and half-life of the nanobody, it was displayed on the surface of ferritin, which can self-assemble into a 24-subunit nanocage, namely, fenobody-55. The affinities of fenobody-55 to g3-p239 were ∼20 times greater than those of g3-p239-Nb55. In addition, the half-life of fenobody-55 was nine times greater than that of g3-p239-Nb55. G3-p239-Nb55 and fenobody-55 can block p239 attachment and Kernow-C1/P6 infection of HepG2/C3A cells. Fenobody-55 can completely neutralize HEV infection in rabbits when it is preincubated with nonenveloped HEV particles. Our study reported a case in which a nanobody neutralized HEV infection by preincubation, identified a (to our knowledge) novel and conserved conformational epitope of HEV, and provided new material for researching HEV biology.

A nanobody inhibiting porcine reproductive and respiratory syndrome virus replication via blocking self-interaction of viral nucleocapsid protein.

Porcine reproductive and respiratory syndrome (PRRS) is a serious global pig industry disease. Understanding the mechanism of viral replication and developing efficient antiviral strategies are necessary for combating with PRRS virus (PRRSV) infection. Recently, nanobody is considered to be a promising antiviral drug, especially for respiratory viruses. The present study evaluated two nanobodies against PRRSV nucleocapsid (N) protein (PRRSV-N-Nb1 and -Nb2) for their anti-PRRSV activity in vitro and in vivo. The results showed that intracellularly expressed PRRSV-N-Nb1 significantly inhibited PRRSV-2 replication in MARC-145 cells (approximately 100%). Then, the PRRSV-N-Nb1 fused with porcine IgG Fc (Nb1-pFc) as a delivering tag was produced and used to determine its effect on PRRSV-2 replication in porcine alveolar macrophages (PAMs) and pigs. The inhibition rate of Nb1-pFc against PRRSV-2 in PAMs could reach >90%, and it can also inhibit viral replication in vivo. Epitope mapping showed that the motif Serine 105 (S105) in PRRSV-2 N protein was the key amino acid binding to PRRSV-N-Nb1, which is also pivotal for the self-interaction of N protein via binding to Arginine 97. Moreover, viral particles were not successfully rescued when the S105 motif was mutated to Alanine (S105A). Attachment, entry, genome replication, release, docking model analysis, and blocking enzyme-linked immunosorbent assay (ELISA) indicated that the binding of PRRSV-N-Nb1 to N protein could block its self-binding, which prevents the viral replication of PRRSV. PRRSV-N-Nb1 may be a promising drug to counter PRRSV-2 infection. We also provided some new insights into the molecular basis of PRRSV N protein self-binding and assembly of viral particles.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) causes serious economic losses to the swine industry worldwide, and there are no highly effective strategies for prevention. Nanobodies are considered a promising novel approach for treating diseases because of their ease of production and low costing. Here, we showed that PRRSV-N-Nb1 against PRRSV-N protein significantly inhibited PRRSV-2 replication in vitro and in vivo. Furthermore, we demonstrated that the motif Serine 105 (S105) in PRRSV-N protein was the key amino acid to interact with PRRSV-N-Nb1 and bond to its motif R97, which is important for the self-binding of N protein. The PRRSV-N-Nb1 could block the self-interaction of N protein following viral assembly. These findings not only provide insights into the molecular basis of PRRSV N protein self-binding as a key factor for viral replication for the first time but also highlight a novel target for the development of anti-PRRSV replication drugs.

Hepatitis E virus ORF3 protein hijacking thioredoxin domain-containing protein 5 (TXNDC5) for its stability to promote viral particle release.

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide, responsible for approximately 20 million infections annually. Among the three open reading frames (ORFs) of the HEV genome, the ORF3 protein is involved in virus release. However, the host proteins involved in HEV release need to be clarified. In this study, a host protein, thioredoxin domain-containing protein 5 (TXNDC5), interacted with the non-palmitoylated ORF3 protein by co-immunoprecipitation analysis. We determined that the overexpression or knockdown of TXNDC5 positively regulated HEV release from the host cells. The 17FCL19 mutation of the ORF3 protein lost the ability to interact with TXNDC5. The releasing amounts of HEV with the ORF3 mutation (FCL17-19SSP) were decreased compared with wild-type HEV. The overexpression of TXNDC5 can stabilize and increase ORF3 protein amounts, but not the TXNDC5 mutant with amino acids 1-88 deletion. Meanwhile, we determined that the function of TXNDC5 on the stabilization of ORF3 protein is independent of the Trx-like domains. Knockdown of TXNDC5 could lead to the degradation of ORF3 protein by the endoplasmic reticulum (ER)-associated protein degradation-proteasome system. However, the ORF3 protein cannot be degraded in the knockout-TXNDC5 stable cells, suggesting that it may hijack other proteins for its stabilization. Subsequently, we found that the other members of protein disulfide isomerase (PDI), including PDIA1, PDIA3, PDIA4, and PDIA6, can increase ORF3 protein amounts, and PDIA3 and PDIA6 interact with ORF3 protein. Collectively, our study suggested that HEV ORF3 protein can utilize TXNDC5 for its stability in ER to facilitate viral release. IMPORTANCE: Hepatitis E virus (HEV) infection is the leading cause of acute viral hepatitis worldwide. After the synthesis and modification in the cells, the mature ORF3 protein is essential for HEV release. However, the host protein involved in this process has yet to be determined. Here, we reported a novel host protein, thioredoxin domain-containing protein 5 (TXNDC5), as a chaperone, contributing to HEV release by facilitating ORF3 protein stability in the endoplasmic reticulum through interacting with non-palmitoylated ORF3 protein. However, we also found that in the knockout-TXNDC5 stable cell lines, the HEV ORF3 protein may hijack other proteins for its stabilization. For the first time, our study demonstrated the involvement of TXNDC5 in viral particle release. These findings provide some new insights into the process of the HEV life cycle, the interaction between HEV and host factors, and a new direction for antiviral design.

The PRMT5/WDR77 complex restricts hepatitis E virus replication.

Hepatitis E virus (HEV) is one of the main pathogenic agents of acute hepatitis in the world. The mechanism of HEV replication, especially host factors governing HEV replication is still not clear. Here, using HEV ORF1 trans-complementation cell culture system and HEV replicon system, combining with stable isotope labelling with amino acids in cell culture (SILAC) and mass spectrometry (MS), we aimed to identify the host factors regulating HEV replication. We identified a diversity of host factors associated with HEV ORF1 protein, which were putatively responsible for viral genomic RNA replication, in these two cell culture models. Of note, the protein arginine methyltransferase 5 (PRMT5)/WDR77 complex was identified in both cell culture models as the top hit. Furthermore, we demonstrated that PRMT5 and WDR77 can specifically inhibit HEV replication, but not other viruses such as HCV or SARS-CoV-2, and this inhibition is conserved among different HEV strains and genotypes. Mechanistically, PRMT5/WDR77 can catalyse methylation of ORF1 on its R458, impairing its replicase activity, and virus bearing R458K mutation in ORF1 relieves the restriction of PRMT5/WDR77 accordingly. Taken together, our study promotes more comprehensive understanding of viral infections but also provides therapeutic targets for intervention.

Porcine HERC6 acts as major E3 ligase for ISGylation and is auto-ISGylated for effective ISGylation in porcine.

Interferon-stimulated gene product 15 (ISG15) can be conjugated to substrates through ISGylation. Currently, the E3 ligase for porcine ISGylation remains unclear. Here, we identified porcine HERC5 and HERC6 (pHERC5/6) as ISGylation E3 ligases with pHERC6 acting as a major one by reconstitution of porcine ISGylation system in HEK-293 T cell via co-transfecting E1, E2 and porcine ISG15(pISG15) genes. Meanwhile, our data demonstrated that co-transfection of pISG15 and pHERC5/6 was sufficient to confer ISGylation, suggesting E1 and E2 of ISGylation are interchangeable between human and porcine. Using an immunoprecipitation based ISGylation analysis, our data revealed pHERC6 was a substrate for ISGylation and confirmed that K707 and K993 of pHERC6 were auto-ISGylation sites. Mutation of these sites reduced pHERC6 half-life and inhibited ISGylation, suggesting that auto-ISGylation of pHERC6 was required for effective ISGylation. Conversely, sustained ISGylation induced by overexpression of pISG15 and pHERC6 could be inhibited by a well-defined porcine ISGylation antagonist, the ovarian tumor (OTU) protease domain of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)-nsp2 and PRRSV-nsp1ß, further indicating such method could be used for identification of virus-encoded ISG15 antagonist. In conclusion, our study contributes new insights towards porcine ISGylation system and provides a novel tool for screening viral-encoded ISG15 antagonist.

Identification of functional cis-acting RNA elements in the hepatitis E virus genome required for viral replication.

There are approximately 20 million events of hepatitis E virus (HEV) infection worldwide annually. The genome of HEV is a single-strand, positive-sense RNA containing 5' and 3' untranslated regions and three open reading frames (ORF). HEV genome has 5' cap and 3' poly(A) tail to mimic host mRNA to escape the host innate immune surveillance and utilize host translational machineries for viral protein translation. The replication mechanism of HEV is poorly understood, especially how the viral polymerase distinguishes viral RNA from host mRNA to synthesize new viral genomes. We hypothesize that the HEV genome contains cis-acting elements that can be recognized by the virally encoded polymerase as "self" for replication. To identify functional cis-acting elements systematically across the HEV genome, we utilized an ORF1 transcomplementation system. Ultimately, we found two highly conserved cis-acting RNA elements within the ORF1 and ORF2 coding regions that are required for viral genome replication in a diverse panel of HEV genotypes. Synonymous mutations in the cis-acting RNA elements, not altering the ORF1 and ORF2 protein sequences, significantly impaired production of infectious viral particles. Mechanistic studies revealed that the cis-acting elements form secondary structures needed to interact with the HEV ORF1 protein to promote HEV replication. Thus, these cis-acting elements function as a scaffold, providing a specific "signal" that recruits viral and host factors to assemble the viral replication complex. Altogether, this work not only facilitates our understanding of the HEV life cycle and provides novel, RNA-directed targets for potential HEV treatments, but also sheds light on the development of HEV as a therapeutic delivery vector.

A porcine reproductive and respiratory syndrome virus (PRRSV)-specific IgM as a novel adjuvant for an inactivated PRRSV vaccine improves protection efficiency and enhances cell-mediated immunity against heterologous PRRSV challenge.

Current strategies for porcine reproductive and respiratory syndrome (PRRS) control are inadequate and mainly restricted to immunization using different PRRS virus (PPRSV) vaccines. Although there are no safety concerns, the poor performance of inactivated PRRSV vaccines has restricted their practical application. In this research, we employed the novel PRRSV-specific IgM monoclonal antibody (Mab)-PR5nf1 as a vaccine adjuvant for the formulation of a cocktail composed of inactivated PRRSV (KIV) and Mab-PR5nf1 along with a normal adjuvant to enhance PRRSV-KIV vaccine-mediated protection and further compared it with a normal KIV vaccine and modified live virus vaccine (MLV). After challenge with highly pathogenic (HP)-PRRSV, our results suggested that the overall survival rate (OSR) and cell-mediated immunity (CMI), as determined by serum IFN-γ quantification and IFN-γ ELISpot assay, were significantly improved by adding PRRSV-specific IgM to the PRRSV-KIV vaccine. It was also notable that both the OSR and CMI in the Mab-PR5nf1-adjuvanted KIV group were even higher than those in the MLV group, whereas the CMI response is normally poorly evoked by KIV vaccines or subunit vaccines. Compared with those in piglets immunized with the normal KIV vaccine, viral shedding and serum neutralizing antibody levels were also improved, and reduced viral shedding appeared to be a result of enhanced CMI caused by the inclusion of IgM as an adjuvant. In conclusion, our data provide not only a new formula for the development of an effective PRRSV-KIV vaccine for practical use but also a novel method for improving antigen-specific CMI induction by inactivated vaccines and subunit vaccines.

Porcine Reproductive and Respiratory Syndrome Virus Promotes SLA-DR-Mediated Antigen Presentation of Nonstructural Proteins To Evoke a Nonneutralizing Antibody Response In Vivo.

The humoral immune response against porcine reproductive and respiratory syndrome virus (PRRSV) infection is characterized by a rapid induction of nonneutralizing antibodies (non-NAbs) against nonstructural proteins (NSPs). Here, we systematically investigated the potential mechanism for the induction of PRRSV NSP-specific non-NAbs. Our data suggested that PRRSV NSP-specific antibodies appeared within 10 days after PRRSV infection in vivo In the in vitro model, functional upregulation of swine leukocyte antigen (SLA)-DR was observed in bone marrow-derived dendritic cells (BMDCs) and porcine alveolar macrophages (PAMs), whereas remarkable inhibition at the mRNA level was observed after infection by both PRRSV-1 and PRRSV-2 isolates. Notably, the inconsistency in SLA-DR expression between the mRNA and protein levels resulted from deubiquitination of SLA-DR via the ovarian tumor (OTU) domain of PRRSV NSP2, which inhibited ubiquitin-mediated degradation. Moreover, mass spectrometry-based immunopeptidome analysis identified immunopeptides originating from multiple PRRSV NSPs within SLA-DR of PRRSV-infected BMDCs. Meanwhile, these PRRSV NSP-derived immunopeptides could be specifically recognized by serum from PRRSV-infected piglets. Notably, certain NSP-derived immunopeptides characterized in vitro could be identified from PAMs or hilar lymph nodes from PRRSV-infected piglets. More importantly, an in vitro neutralizing assay indicated that serum antibodies against NSP immunopeptides were unable to neutralize PRRSV in vitro Conversely, certain structural protein (SP)-derived immunopeptides were identified and could be recognize by pig hyperimmune serum against PRRSV, which further indicates that the NSP-derived antibody response is nonprotective in vivo In conclusion, our data suggested that PRRSV infection interferes with major histocompatibility complex class II (MHC-II) molecule-mediated antigen presentation in antigen-presenting cells (APCs) via promoting SLA-DR expression to present immunopeptides from PRRSV NSPs, which contributes to the induction of non-NAbs in vivoIMPORTANCE PRRSV has haunted the swine industry for over 30 years since its emergence. Besides the limited efficacy of PRRSV modified live vaccines (MLVs) against heterogeneous PRRSV isolates, rapid induction of nonneutralizing antibodies (non-NAbs) against PRRSV NSPs after MLV immunization or wild-strain infection is one of the reasons why development of an effective vaccine has been hampered. By using in vitro-generated BMDCs as models to understand the antigen presentation process of PRRSV, we obtained data indicating that PRRSV infection of BMDCs promotes functional SLA-DR upregulation to present PRRSV NSP-derived immunopeptides for evoking a non-NAb response in vivo Our work not only uncovered a novel mechanism for interference in host antigen presentation by PRRSV but also revealed a novel insight for understanding the rapid production of nonneutralizing antibodies against PRRSV NSPs, which may have benefit for developing an effective vaccine against PRRSV in the future.

A broadly neutralizing monoclonal antibody induces broad protection against heterogeneous PRRSV strains in piglets.

Neutralizing antibodies (NAbs) have attracted attention as tools for achieving PRRSV control and prevention, but viral antigenic variation undermines the abilities of NAbs elicited by attenuated PRRSV vaccines to confer full protection against heterogeneous PRRSV field isolates. As demonstrated in this study, the monoclonal antibody (mAb) mAb-PN9cx3 exhibited broad-spectrum recognition and neutralizing activities against PRRSV-1 and PRRSV-2 strains in vitro. Furthermore, in vivo experiments revealed that the administration of two 10-mg doses of mAb-PN9cx3 before and after the inoculation of piglets with heterologous PRRSV isolates (HP-PRRSV-JXA1 or PRRSV NADC30-like strain HNhx) resulted in significant reduction of the PRRSV-induced pulmonary pathological changes and virus loads in porcine alveolar macrophages (PAMs) compared with the results obtained with mAb-treated isotype controls. Moreover, minimal hilar lymph node PRRSV antigen levels were observed in mAb-PN9cx3-treated piglets. A transcriptome profile analysis of PAMs extracted from lung tissues of piglets belonging to different groups (except for antibody-isotype controls) indicated that mAb-PN9cx3 treatment reversed the PRRSV infection-induced alterations in expression profiles. A gene ontology (GO) enrichment analysis of these genes traced their functions to pathways that included the immune response, inflammatory response, and response to steroid hormone, and their functions in oogenesis and positive regulation of angiogenesis have been implicated in PRRSV pathogenesis. Overall, NADC30-like HNhx infection affected more gene pathways than HP-PRRSV infection. In conclusion, our research describes a novel immunologic approach involving the use of mAbs that confer cross-protection against serious illness resulting from infection with heterogeneous PRRSV-2 isolates, which is a feat that has not yet been achieved through vaccination. Ultimately, mAb-PN9cx3 will be a powerful addition to our current arsenal for achieving PRRSV prevention and eradication.

Development of a competitive ELISA for detecting antibodies against genotype 1 hepatitis E virus.

Hepatitis E, a significant global public health issue in China, is caused by sporadic infections with regional hepatitis E virus (HEV) genotypes 1, 3, and 4. To date, most immunoassays currently used to test human sera for the presence of anti-HEV antibodies cannot identify HEV at the genotype level. However, such information would be useful for identifying the source of infecting virus. Therefore, here we describe the development of a competitive enzyme-linked immunosorbent assay (ELISA) for detecting anti-genotype 1 HEV antibodies in human sera. Using recombinant genotype 1 HEV ORF3 protein as immunogen, traditional hybridoma technology was employed to generate seven monoclonal antibodies (mAbs), of which two mAbs specifically reacted with the immunogen. One of these two mAbs, 1D2, was labeled with horseradish peroxidase (HRP) for use in competitive ELISA (cELISA). After cELISA optimization using a checkerboard assay design, the amount of ORF3SAR-55 as coating antigen (100 ng/well), HRP-1D2 mAb concentration (1 µg/mL), and test serum dilution (1:10) were selected and a result ≥ 19.5 was used as the cutoff for a positive result. Importantly, cross-genotype cELISA results indicated that the cELISA could not detect anti-genotype 3 rabbit and 4 swine HEV antibodies. Moreover, human sera confirmed as negative for anti-HEV antibodies using the commercial ELISA kit were all negative via cELISA. However, because the commercial ELISA kit detects anti-all genotypes HEV antibodies and the cELISA only detects anti-genotype 1 HEV antibodies, the consistence rate of two assays detecting positive sera is low. In summary, here a cELISA for detecting anti-genotype 1 HEV antibodies was developed for use in epidemiological investigations of genotype 1 HEV infections in humans. KEY POINTS: • Seven mAbs were produced using genotype 1 HEV ORF3 protein as immunogen. • One mAb that specifically bound to genotype 1 HEV ORF3 protein was selected and labeled for use in a cELISA to detect anti-genotype 1 HEV antibodies. • The competitive ELISA developed here will aid clinical diagnosis of HEV infections and will be useful for large-scale serological testing of genotype 1 HEV infections in humans.

Chicken Organic Anion-Transporting Polypeptide 1A2, a Novel Avian Hepatitis E Virus (HEV) ORF2-Interacting Protein, Is Involved in Avian HEV Infection.

Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease in chickens. Due to the absence of a highly effective cell culture system, there are few reports about the interaction between avian HEV and host cells. In this study, organic anion-transporting polypeptide 1A2 (OATP1A2) from chicken liver cells was identified to interact with ap237, a truncated avian HEV capsid protein spanning amino acids 313 to 549, by a glutathione S-transferase (GST) pulldown assay. GST pulldown and indirect enzyme-linked immunosorbent assays (ELISAs) further confirmed that the extracellular domain of OATP1A2 directly binds with ap237. The expression levels of OATP1A2 in host cells are positively correlated with the amounts of ap237 attachment and virus infection. The distribution of OATP1A2 in different tissues is consistent with avian HEV infection in vivo Finally, when the functions of OATP1A2 in cells are inhibited by its substrates or an inhibitor or blocked by ap237 or anti-OATP1A2 sera, attachment to and infection of host cells by avian HEV are significantly reduced. Collectively, these results displayed for the first time that OATP1A2 interacts with the avian HEV capsid protein and can influence viral infection in host cells. The present study provides new insight to understand the process of avian HEV infection of host cells.IMPORTANCE The process of viral infection is centered around the interaction between the virus and host cells. Due to the lack of a highly effective cell culture system in vitro, there is little understanding about the interaction between avian HEV and its host cells. In this study, a total of seven host proteins were screened in chicken liver cells by a truncated avian HEV capsid protein (ap237) in which the host protein OATP1A2 interacted with ap237. Overexpression of OATP1A2 in the cells can promote ap237 adsorption as well as avian HEV adsorption and infection of the cells. When the function of OATP1A2 in cells was inhibited by substrates or inhibitors, attachment and infection by avian HEV significantly decreased. The distribution of OATP1A2 in different chicken tissues corresponded with that in tissues during avian HEV infection. This is the first finding that OATP1A2 is involved in viral infection of host cells.

A Nanobody Targeting Viral Nonstructural Protein 9 Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication.

Porcine reproductive and respiratory syndrome (PRRS) is of great concern to the swine industry due to pandemic outbreaks of the disease, current ineffective vaccinations, and a lack of efficient antiviral strategies. In our previous study, a PRRSV Nsp9-specific nanobody, Nb6, was successfully isolated, and the intracellularly expressed Nb6 could dramatically inhibit PRRSV replication in MARC-145 cells. However, despite its small size, the application of Nb6 protein in infected cells is greatly limited, as the protein itself cannot enter the cells physically. In this study, a trans-activating transduction (TAT) peptide was fused with Nb6 to promote protein entry into cells. TAT-Nb6 was expressed as an inclusion body in Escherichia coli, and indirect enzyme-linked immunosorbent assays and pulldown assays showed that E. coli-expressed TAT-Nb6 maintained the binding ability to E. coli-expressed or PRRSV-encoded Nsp9. We demonstrated that TAT delivered Nb6 into MARC-145 cells and porcine alveolar macrophages (PAMs) in a dose- and time-dependent manner, and TAT-Nb6 efficiently inhibited the replication of several PRRSV genotype 2 strains as well as a genotype 1 strain. Using a yeast two-hybrid assay, Nb6 recognition sites were identified in the C-terminal part of Nsp9 and spanned two discontinuous regions (Nsp9aa454-551 and Nsp9aa599-646). Taken together, these results suggest that TAT-Nb6 can be developed as an antiviral drug for the inhibition of PRRSV replication and controlling PRRS disease.IMPORTANCE The pandemic outbreak of PRRS, which is caused by PRRSV, has greatly affected the swine industry. We still lack an efficient vaccine, and it is an immense challenge to control its infection. An intracellularly expressed Nsp9-specific nanobody, Nb6, has been shown to be able to inhibit PRRSV replication in MARC-145 cells. However, its application is limited, because Nb6 cannot physically enter cells. Here, we demonstrated that the cell-penetrating peptide TAT could deliver Nb6 into cultured cells. In addition, TAT-Nb6 fusion protein could suppress the replication of various PRRSV strains in MARC-145 cells and PAMs. These findings may provide a new approach for drug development to control PRRS.

Karyopherin Alpha 6 Is Required for Replication of Porcine Reproductive and Respiratory Syndrome Virus and Zika Virus.

Movement of macromolecules between the cytoplasm and the nucleus occurs through the nuclear pore complex (NPC). Karyopherins comprise a family of soluble transport factors facilitating the nucleocytoplasmic translocation of proteins through the NPC. In this study, we found that karyopherin α6 (KPNA6; also known as importin α7) was required for the optimal replication of porcine reproductive and respiratory syndrome virus (PRRSV) and Zika virus (ZIKV), which are positive-sense, single-stranded RNA viruses replicating in the cytoplasm. The KPNA6 protein level in virus-infected cells was much higher than that in mock-infected controls, whereas the KPNA6 transcript remains stable. Viral infection blocked the ubiquitin-proteasomal degradation of KPNA6, which led to an extension of the KPNA6 half-life and the elevation of the KPNA6 level in comparison to mock-infected cells. PRRSV nsp12 protein induced KPNA6 stabilization. KPNA6 silencing was detrimental to the replication of PRRSV, and KPNA6 knockout impaired ZIKV replication. Moreover, KPNA6 knockout blocked the nuclear translocation of PRRSV nsp1ß but had a minimal effect on two other PRRSV proteins with nuclear localization. Exogenous restitution of KPNA6 expression in the KPNA6-knockout cells results in restoration of the nuclear translocation of PRRSV nsp1ß and the replication of ZIKV. These results indicate that KPNA6 is an important cellular factor for the replication of PRRSV and ZIKV.IMPORTANCE Positive-sense, single-stranded RNA (+ssRNA) viruses replicate in the cytoplasm of infected cells. The roles of transport factors in the nucleocytoplasmic trafficking system for the replication of +ssRNA viruses are not known. In this study, we discovered that PRRSV and ZIKV viruses needed karyopherin α6 (KPNA6), one of the transport factors, to enhance the virus replication. Our data showed that viral infection induced an elevation of the KPNA6 protein level due to an extension of the KPNA6 half-life via viral interference of the ubiquitin-proteasomal degradation of KPNA6. Notably, KPNA6 silencing or knockout dramatically reduced the replication of PRRSV and ZIKV. PRRSV nsp1ß depended on KPNA6 to translocate into the nucleus. In addition, exogenous restitution of KPNA6 expression in KPNA6-knockout cells led to the restoration of nsp1ß nuclear translocation and ZIKV replication. These results reveal a new aspect in the virus-cell interaction and may facilitate the development of novel antiviral therapeutics.

Characterization of Three Novel Linear Neutralizing B-Cell Epitopes in the Capsid Protein of Swine Hepatitis E Virus.

Hepatitis E virus (HEV) causes liver disease in humans and is thought to be a zoonotic infection, with domestic animals, including swine and rabbits, being a reservoir. One of the proteins encoded by the virus is the capsid protein. This is likely the major immune-dominant protein and a target for vaccination. Four monoclonal antibodies (MAbs), three novel, 1E4, 2C7, and 2G9, and one previously characterized, 1B5, were evaluated for binding to the capsid protein from genotype 4 swine HEV. The results indicated that 625DFCP628, 458PSRPF462, and 407EPTV410 peptides on the capsid protein comprised minimal amino acid sequence motifs recognized by 1E4, 2C7, and 2G9, respectively. The data suggested that 2C7 and 2G9 epitopes were partially exposed on the surface of the capsid protein. Truncated genotype 4 swine HEV capsid protein (sp239, amino acids 368 to 606) can exist in multimeric forms. Preincubation of swine HEV with 2C7, 2G9, or 1B5 before addition to HepG2 cells partially blocked sp239 cell binding and inhibited swine HEV infection. The study indicated that 2C7, 2G9, and 1B5 partially blocked swine HEV infection of rabbits better than 1E4 or normal mouse IgG. The cross-reactivity of antibodies suggested that capsid epitopes recognized by 2C7 and 2G9 are common to HEV strains infecting most host species. Collectively, MAbs 2C7, 2G9, and 1B5 were shown to recognize three novel linear neutralizing B-cell epitopes of genotype 4 HEV capsid protein. These results enhance understanding of HEV capsid protein structure to guide vaccine and antiviral design.IMPORTANCE Genotype 3 and 4 HEVs are zoonotic viruses. Here, genotype 4 HEV was studied due to its prevalence in human populations and pig herds in China. To improve HEV disease diagnosis and prevention, a better understanding of the antigenic structure and neutralizing epitopes of HEV capsid protein are needed. In this study, the locations of three novel linear B-cell recognition epitopes within genotype 4 swine HEV capsid protein were characterized. Moreover, the neutralizing abilities of three MAbs specific for this protein, 2C7, 2G9, and 1B5, were studied in vitro and in vivo Collectively, these findings reveal structural details of genotype 4 HEV capsid protein and should facilitate development of applications for the design of vaccines and antiviral drugs for broader prevention, detection, and treatment of HEV infection of diverse human and animal hosts.

Development of luciferase-linked antibody capture assay based on luciferase immunoprecipitation systems for antibody detection of porcine reproductive and respiratory syndrome virus.

BACKGROUND: Early detection of porcine reproductive and respiratory syndrome virus (PRRSV) infection of swine is necessary to control this devastating disease. By monitoring host serum antibodies to viral antigens, early virus detection within herds is feasible. In this study, recombinant antigens were generated using recombinant DNA techniques to fuse PRRSV structural protein (N) or nonstructural protein 1α (nsp1α) with the Rellina luciferase gene. Next, fused genes were cloned into plasmids and transfected into HEK-293 T cells for transient expression. Upon co-incubation of lysates with pig sera, antigen-antibody complexes formed that bound to Protein-G coated onto microplates. By further measurement of luminance value, a modified form of Luciferase Immunoprecipitation Systems, namely luciferase-linked antibody capture assay (LACA) was developed for detection of PRRSV-specific antibodies. RESULTS: Known anti-PRRSV antibody-positive or -negative serum samples (125 and 122 samples, respectively) were used to validate the LACA and compared it with IDEXX PRRS ×3 ELISA. Based on the result, N-Rluc and nsp1α-Rluc LACA results were 95.3 and 94.4% in agreement with IDEXX ELISA, suggested a similar specificity of LACA to IDEXX ELISA. Moreover, when both LACA and IDEXX ELISA were used to evaluate sequential serum samples obtained from PRRSV experimentally infected pigs, the PRRSV-specific antibody response was detectable as early as 3 days post-inoculation (dpi) using N-Rluc LACA, but undetectable until 7 dpi using IDEXX ELISA, suggesting an improved sensitivity of LACA. Meanwhile, antibodies specific for nsp1α were detected at higher levels overall, but were undetectable until 10 dpi. Furthermore,. Notably, one IDEXX ELISA positive result was not confirmed by LACA or IFA and was thus considered a false-positive result. CONCLUSION: The LACA exhibited similar specificity but improved sensitivity to that of the commercial IDEXX PRRS ×3 ELISA kit for detection of PRRSV-specific antibodies in pig serum. Importantly, LACA could be adapted for detecting antibodies against other PRRSV targets, such as nsp1α, to achieve earlier detection of PRRSV infection.

Porcine Reproductive and Respiratory Syndrome Virus Antagonizes JAK/STAT3 Signaling via nsp5, Which Induces STAT3 Degradation.

Signal transducer and activator of transcription 3 (STAT3) is a pleiotropic signaling mediator of many cytokines, including interleukin-6 (IL-6) and IL-10. STAT3 is known to play critical roles in cell growth, proliferation, differentiation, immunity and inflammatory responses. The objective of this study was to determine the effect of porcine reproductive and respiratory syndrome virus (PRRSV) infection on the STAT3 signaling since PRRSV induces a weak protective immune response in host animals. We report here that PRRSV infection of MARC-145 cells and primary porcine pulmonary alveolar macrophages led to significant reduction of STAT3 protein level. Several strains of both PRRSV type 1 and type 2 led to a similar reduction of STAT3 protein level but had a minimal effect on its transcripts. The PRRSV-mediated STAT3 reduction was in a dose-dependent manner as the STAT3 level decreased, along with incremental amounts of PRRSV inocula. Further study showed that nonstructural protein 5 (nsp5) of PRRSV induced the STAT3 degradation by increasing its polyubiquitination level and shortening its half-life from 24 h to ∼3.5 h. The C-terminal domain of nsp5 was shown to be required for the STAT3 degradation. Moreover, the STAT3 signaling in the cells transfected with nsp5 plasmid was significantly inhibited. These results indicate that PRRSV antagonizes the STAT3 signaling by accelerating STAT3 degradation via the ubiquitin-proteasomal pathway. This study provides insight into the PRRSV interference with the JAK/STAT3 signaling, leading to perturbation of the host innate and adaptive immune responses. IMPORTANCE: The typical features of immune responses in PRRSV-infected pigs are delayed onset and low levels of virus neutralizing antibodies, as well as weak cell-mediated immunity. Lymphocyte development and differentiation rely on cytokines, many of which signal through the JAK/STAT signaling pathway to exert their biological effects. Here, we discovered that PRRSV antagonizes the JAK/STAT3 signaling by inducing degradation of STAT3, a master transcription activator involved in multiple cellular processes and the host immune responses. The nsp5 protein of PRRSV is responsible for the accelerated STAT3 degradation. The PRRSV-mediated antagonizing STAT3 could lead to suppression of a broad spectrum of cytokines and growth factors to allow virus replication and spread in host animals. This may be one of the reasons for the PRRSV interference with the innate immunity and its poor elicitation of protective immunity. This finding provides insight into PRRSV pathogenesis and its interference with the host immune responses.

The middle half genome of interferon-inducing porcine reproductive and respiratory syndrome virus strain A2MC2 is essential for interferon induction.

Porcine reproductive and respiratory syndrome virus (PRRSV) is known to antagonize the innate immune response. An atypical PRRSV strain A2MC2 is capable of inducing synthesis of type I interferons (IFNs) in cultured cells. Here, we show that the middle half of the A2MC2 genome is needed for triggering the IFN synthesis. First, a cDNA infectious clone of this atypical strain was constructed as a DNA-launched version. Virus recovery was achieved from the infectious clone and the recovered virus, rA2MC2, was characterized. The rA2MC2 retained the feature of IFN induction in cultured cells. Infection of pigs with the rA2MC2 virus caused viremia similar to that of the wild-type virus. Chimeric infectious clones were constructed by swapping genomic fragments with a cDNA clone of a moderately virulent strain VR-2385 that antagonizes IFN induction. Analysis of the rescued chimeric viruses demonstrated that the middle two fragments, ranging from nt4545 to nt12709 of the A2MC2 genome, were needed for the IFN induction, whereas the chimeric viruses containing any one of the two A2MC2 fragments failed to do so. The results and the cDNA infectious clone of the IFN-inducing A2MC2 will facilitate further study of its biology, ultimately leading towards the development of an improved vaccine against PRRS.

Generation of murine macrophage-derived cell lines expressing porcine CD163 that support porcine reproductive and respiratory syndrome virus infection.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) exhibits a highly restricted tropism for cells of the monocyte-macrophage lineage, utilizing porcine CD163 (pCD163) as an indispensable cellular receptor for infection. Transfection the gene of pCD163 into several non-permissive cell lines followed by protein expression confers susceptibility to PRRSV. A lack of specialized porcine antibody tools for use with existing porcine-derived primary cells and cell lines has hampered studies of both PRRSV pathogenesis and virus triggering of immune response cascades. Therefore, we constructed PRRSV-susceptible murine alveolar macrophage-derived MH-S and peritoneal macrophage-like RAW264.7 cell lines by achieving pCD163 cell surface expression in these cells. We then evaluated PRRSV susceptibility and cytokine expression patterns induced upon PRRSV infection of these pCD163-expressing cell lines. RESULTS: Growth of MH-SCD163 and RAW264.7CD163 cells was indistinguishable from growth of un-transfected parental cell lines. Meanwhile, various stages of the PRRSV replication cycle, including viral particle attachment, internalization, disassembly and infection were confirmed in both pCD163-transfected cell lines. Analysis of PRRSV replication using immunofluorescence staining of virus and viral titration of cell lysates demonstrated that both MH-SCD163 and RAW264.7CD163 cells supported replication of various genotype 2 PRRSV isolates. Moreover, PRRSV replication in MH-SCD163 cells was similar to that observed in porcine alveolar macrophages (PAMs) and was more efficient than in RAW264.7CD163 cells. However, peak virus titers in MH-SCD163 cells were attained at 60 h post-infection (pi) versus 48 hpi in PAMs. Analysis of cytokine expression showed that post-PRRSV infection, mRNA expression patterns of anti-inflammatory cytokines (IL-4 and IL-10) and pro-inflammatory cytokines (TNF-α and IFN-γ) in MH-SCD163 cells were more similar to those observed in PAMs versus levels in RAW264.7CD163 cells. CONCLUSIONS: MH-S and RAW264.7 cells were not susceptible to PRRSV infection until transfection and subsequent expression of pCD163 were achieved in these cell lines. The PRRSV-susceptible MH-SCD163 cell line efficiently supported viral replication of various genotype 2 PRRSV isolates and exhibited similar cytokine expression patterns as observed in PAMs. In conclusion, this work describes the development of new tools to further understand PRRSV pathogenesis and immune response mechanisms to PRRSV infection.

Effect of housing arrangement on fecal-oral transmission of avian hepatitis E virus in chicken flocks.

BACKGROUND: Avian hepatitis E virus (HEV) infection is common in chicken flocks in China, as currently no measures exist to prevent the spread of the disease. In this study, we analyzed the effect of caged versus cage-free housing arrangements on avian HEV transmission. First, 127 serum and 110 clinical fecal samples were collected from 4 chicken flocks including the two arrangements in Shaanxi Province, China and tested for HEV antibodies and/or virus. Concurrently, 36 specific-pathogen-free chickens were divided equally into four experimental living arrangement groups, designated cage-free (Inoculated), caged (Inoculated), cage-free (Negative) and caged (Negative) groups. In caged groups, three cages contained 3 chickens each. Three chickens each from cage-free (Inoculated) and caged (Inoculated) groups (one chicken of each cage) were inoculated by cutaneous ulnar vein with the same dose of avian HEV, respectively. The cage-free (Negative) and caged (Negative) groups served as negative control. Serum and fecal samples were collected at 1 to 7 weeks post-inoculation (wpi) and liver lesions were scored at 7 wpi. RESULTS: The results of serology showed that the avian HEV infection rate (54.10%) of the cage-free chickens was significantly higher than the one (12.12%) for caged chickens (P < 0.05). Also, the rate of detection of avian HEV RNA in the clinical fecal samples was significantly higher in the cage-free (22.80%, 13/57) than caged birds (5.66%, 3/53). Moreover, under experimental conditions, the infected number of uninoculated cage-free chickens (6) was significantly higher than the one for the uninoculated caged birds (2), as evidenced by seroconversion, fecal virus shedding, viremia and gross and microscopic liver lesions. CONCLUSIONS: These results suggest that reduction of contact with feces as seen in the caged arrangement of housing chickens can reduce avian HEV transmission. This study provides insights for prevention and control of avian HEV infection in chicken flocks.