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.

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 reproductive and respiratory syndrome virus (PRRSV) is arguably the most economically-important global swine pathogen. Here we demonstrated that PRRSV down-regulates Swine Leukocyte Antigen class I (SLA-I) expression in porcine alveolar macrophages, PK15-CD163 cells and monocyte-derived dendritic cells. To identify the viral protein(s) involved in SLA-I down-regulation, we tested all 22 PRRSV structural and non-structural proteins and identified that Nsp1α and Nsp2TF, and GP3 significantly down-regulated SLA-I expression with Nsp2TF showing the greatest effect. We further generated a panel of mutant viruses in which the Nsp2TF protein synthesis was abolished, and found that the two mutants with disrupted -2 ribosomal frameshifting elements and additional stop codons in the TF domain were unable to down-regulate SLA-I expression. Additionally we demonstrated that the last 68 amino acids of TF domain in Nsp2TF are critical for this function. Collectively, the results indicate a novel function of Nsp2TF in negative modulation of SLA-I expression.

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