Porcine reproductive and respiratory syndrome virus (PRRSV) infection of swine leads to a serious disease characterized by a delayed and defective adaptive immune response. It is hypothesized that a suboptimal innate immune response is responsible for the disease pathogenesis. In the study presented here we tested this hypothesis and identified several nonstructural proteins (NSPs) with innate immune evasion properties encoded by the PRRS viral genome. Four of the total ten PRRSV NSPs tested were found to have strong to moderate inhibitory effects on beta interferon (IFN-␤) promoter activation. The strongest inhibitory effect was exhibited by NSP1 followed by, NSP2, NSP11, and NSP4. We focused on NSP1␣ and NSP1␤ (self-cleavage products of NSP1 during virus infection) and NSP11, three NSPs with strong inhibitory activity. All of three proteins, when expressed stably in cell lines, strongly inhibited double-stranded RNA (dsRNA) signaling pathways. NSP1␤ was found to inhibit both IFN regulatory factor 3 (IRF3)-and NF-B-dependent gene induction by dsRNA and Sendai virus. Mechanistically, the dsRNA-induced phosphorylation and nuclear translocation of IRF3 were strongly inhibited by NSP1␤. Moreover, when tested in a porcine myelomonocytic cell line, NSP1␤ inhibited Sendai virus-mediated activation of porcine IFN-␤ promoter activity. We propose that this NSP1␤-mediated subversion of the host innate immune response plays an important role in PRRSV pathogenesis.

Negative-strand (NS) RNA viruses comprise many pathogens that cause serious diseases in humans and animals. Despite their clinical importance, little is known about the host factors required for their infection. Using vesicular stomatitis virus (VSV), a prototypic NS RNA virus in the family Rhabdoviridae, we conducted a human genomewide siRNA screen and identified 72 host genes required for viral infection. Many of these identified genes were also required for infection by two other NS RNA viruses, the lymphocytic choriomeningitis virus of the Arenaviridae family and human parainfluenza virus type 3 of the Paramyxoviridae family. Genes affecting different stages of VSV infection, such as entry/uncoating, gene expression, and assembly/release, were identified. Depletion of the proteins of the coatomer complex I or its upstream effectors ARF1 or GBF1 led to detection of reduced levels of VSV RNA. Coatomer complex I was also required for infection of lymphocytic choriomeningitis virus and human parainfluenza virus type 3. These results highlight the evolutionarily conserved requirements for gene expression of diverse families of NS RNA viruses and demonstrate the involvement of host cell secretory pathway in the process.RNA interference | transcription and replication | host cell factors for virus infection

No abstract

Chronic hepatitis E virus (HEV) infection is a significant clinical problem in immunocompromised individuals such as organ transplant recipients, although the mechanism remains unknown because of the lack of an animal model. We successfully developed a pig model of chronic HEV infection and examined immune correlates leading to chronicity. The conditions of immunocompromised patients were mimicked by treating pigs with an immunosuppressive regimen including cyclosporine, azathioprine, and prednisolone. Immunocompromised pigs infected with HEV progressed to chronicity, because 8/10 drug-treated HEV-infected pigs continued fecal virus shedding beyond the acute phase of infection, whereas the majority (7/10) of mock-treated HEV-infected pigs cleared fecal viral shedding at 8 wk postinfection. During chronic infection, serum levels of the liver enzyme γ-glutamyl transferase and fecal virus shedding were significantly higher in immunocompromised HEV-infected pigs. To identify potential immune correlates of chronic infection, we determined serum levels of cytokines and cell-mediated immune responses in pigs. Results showed that HEV infection of immunocompromised pigs reduced the serum levels of Th1 cytokines IL-2 and IL-12, and Th2 cytokines IL-4 and IL-10, particularly during the acute phase of infection. Furthermore IFN-γ-specific CD4 T-cell responses were reduced in immunocompromised pigs during the acute phase of infection, but TNF-α-specific CD8 T-cell responses increased during the chronic phase of infection. Thus, active suppression of cell-mediated immune responses under immunocompromised conditions may facilitate the establishment of chronic HEV infection. This pig model will aid in delineating the mechanisms of chronic HEV infection and in developing effective therapeutics against chronic hepatitis E.

The objective of this study was to identify porcine reproductive and respiratory syndrome virus (PRRSV)-encoded proteins that are responsible for the inhibition of TNF-α expression and the mechanism(s) involved in this phenomenon. Using a TNF-α promoter reporter system, the non-structural protein 1 (Nsp1) was found to strongly suppress the TNF-α promoter activity. Such inhibition takes place especially at the promoter's proximal region. Both Nsp1α and Nsp1β, the two proteolytic fragments of Nsp1, were shown to be involved in TNF-α promoter suppression. Furthermore, using reporter plasmids specific for transcription factors (TFs) that bind to TNF-α promoter, Nsp1α and Nsp1β were demonstrated to inhibit the activity of the TFs that bind CRE-κB(3) and Sp1 elements respectively. Subsequent analyses showed that Nsp1α moderately inhibits NF-κB activation and that Nsp1β completely abrogates the Sp1 transactivation. These findings reveal one of the important mechanisms underlying the innate immune evasion by PRRSV during infection.

Synthetic attenuated virus engineering (SAVE) is an emerging technology that enables rapid attenuation of viruses. In this study, by using SAVE we demonstrated rapid attenuation of an arterivirus, porcine reproductive and respiratory syndrome virus (PRRSV). The major envelope GP5 gene of PRRSV was codon-pair deoptimized aided by a computer algorithm. The codon-pair deoptimized virus, designated as SAVE5 with a deoptimized GP5 gene, was successfully rescued in vitro. The SAVE5 virus replicated at a lower level in vitro with a significant decrease of GP5 protein expression compared to the wild-type PRRSV VR2385 virus. Pigs experimentally infected with the SAVE5 virus had significantly lower viremia level up to 14 days post-infection as well as significantly reduced gross and histological lung lesions when compared to wild-type PRRSV VR2385 virus-infected pigs, indicating the attenuation of the SAVE5 virus. This study proved the feasibility of rapidly attenuating PRRSV by SAVE.

Porcine epidemic diarrhea virus (PEDV) poses a serious threat to swine worldwide as evidenced by its recent introduction into the USA and the devastating economic impact it caused to the USA swine industry. Commercial vaccines against PEDV are available but their efficacies are inadequate. Therefore, vaccines with improved efficacy are needed to effectively control PEDV infections. We previously determined the immunogenicity of a novel dendritic cell (DC)-targeted PEDV S1 protein-based subunit vaccine in weaned piglets in which the PEDV antigen was targeted to DCs through a porcine Langerin-specific antibody. In this study, we evaluated the protective efficacy of this DC-targeting vaccine by immunizing sows at 5 and 2 weeks prior to farrowing and by challenging the 5-day-old piglets with PEDV. The results showed that immunization of sow with DC-targeted PEDV vaccine did not eliminate faecal virus shedding in piglets but significantly reduced faecal viral RNA levels in the early days after virus challenge. The vaccine also reduced the amount of PEDV antigen in intestinal tissues presented with intestinal villi regrowth. However, the DC-targeted vaccine neither mitigated PEDV clinical signs nor affected viral RNA loads in intestinal tissues of piglets. In the vaccinated sow, DC-targeted PEDV vaccine enhanced T helper 1-like cluster of differentiation (CD)4 T cell responses and induced IgG but not IgA-specific immune responses. The suckling piglets in the DC-targeted vaccine group showed increased gross pathological lesions in the small intestine. Results in this study provide insights into the effects of sow cellular immune responses to PEDV infection in suckling piglets.

The non-structural protein 1 (nsp1) of porcine reproductive and respiratory syndrome virus is partly responsible for inhibition of type I interferon (IFN) response by the infected host. By performing alanine-scanning mutagenesis, we have identified amino acid residues in nsp1α and nsp1β (the proteolytic products of nsp1) that when substituted with alanine(s) exhibited significant relief of IFN-suppression. A mutant virus (16-5A, in which residues 16-20 of nsp1β were substituted with alanines) encoding mutant nsp1β recovered from infectious cDNA clone was shown to be attenuated for growth in vitro and induced significantly higher amount of type I IFN transcripts in infected macrophages. In infected pigs, the 16-5A virus exhibited reduced growth at early times after infection but quickly regained wild type growth properties as a result of substitutions within the mutated sequences. The results indicate a strong selection pressure towards maintaining the IFN-inhibitory property of the virus for successful propagation in pigs.