ISG15 Modulates Type I Interferon Signaling and the Antiviral Response during Hepatitis E Virus Replication.

Hepatitis E virus (HEV), a single-stranded positive-sense RNA virus, generally causes self-limiting acute viral hepatitis, although chronic HEV infection has recently become a significant clinical problem in immunocompromised individuals, especially in solid-organ transplant recipients. Innate immunity, via the type I interferon (IFN) response, plays an important role during the initial stages of a viral infection. IFN-stimulated gene 15 (ISG15), an IFN-induced ubiquitin-like protein, is known to have an immunomodulatory role and can have a direct antiviral effect on a wide spectrum of virus families. In the present study, we investigated the antiviral effect as well as the potential immunomodulatory role of ISG15 during HEV replication. The results revealed that HEV induced high levels of ISG15 production both in vitro (Huh7-S10-3 liver cells) and in vivo (liver tissues from HEV-infected pigs); however, ISG15 is not required for virus replication. We also demonstrated that ISG15 silencing potentiates enhanced type I IFN-mediated signaling, resulting in an increase in the type I IFN-mediated antiviral effect during HEV replication. This observed enhanced type I IFN signaling correlated with an increase in IFN-stimulated gene expression levels during HEV replication. Furthermore, we showed that PKR and OAS1 played important roles in the ISG15-mediated type I IFN sensitivity of HEV. Taken together, the results from this study suggest that ISG15 plays an important immunomodulatory role and regulates HEV sensitivity to exogenous type I IFN.IMPORTANCE Hepatitis E virus (HEV) infection typically causes self-limiting acute viral hepatitis. However, chronic HEV infection has recently become a significant clinical problem in immunocompromised patients. Pegylated interferon (IFN) has been used to treat chronic HEV infection in solid-organ transplant patients with some success. However, the mechanism behind the type I IFN-mediated antiviral effect against HEV remains unclear. This report demonstrates that ISG15 induced by HEV replication in Huh7-S10-3 human liver cells plays an immunomodulatory role by negatively regulating type I IFN signaling and, thus, HEV sensitivity to type I IFN. Our results also show that PKR and OAS1 play important roles in the ISG15-mediated type I IFN sensitivity of HEV.

The hub protein loquacious connects the microRNA and short interfering RNA pathways in mosquitoes.

Aedes aegypti mosquitoes vector several arboviruses of global health significance, including dengue viruses and chikungunya virus. RNA interference (RNAi) plays an important role in antiviral immunity, gene regulation and protection from transposable elements. Double-stranded RNA binding proteins (dsRBPs) are important for efficient RNAi; in Drosophila functional specialization of the miRNA, endo-siRNA and exo-siRNA pathway is aided by the dsRBPs Loquacious (Loqs-PB, Loqs-PD) and R2D2, respectively. However, this functional specialization has not been investigated in other dipterans. We were unable to detect Loqs-PD in Ae. aegypti; analysis of other dipteran genomes demonstrated that this isoform is not conserved outside of Drosophila. Overexpression experiments and small RNA sequencing following depletion of each dsRBP revealed that R2D2 and Loqs-PA cooperate non-redundantly in siRNA production, and that these proteins exhibit an inhibitory effect on miRNA levels. Conversely, Loqs-PB alone interacted with mosquito dicer-1 and was essential for full miRNA production. Mosquito Loqs interacted with both argonaute 1 and 2 in a manner independent of its interactions with dicer. We conclude that the functional specialization of Loqs-PD in Drosophila is a recently derived trait, and that in other dipterans, including the medically important mosquitoes, Loqs-PA participates in both the miRNA and endo-siRNA based pathways.

Methods for TALEN Evaluation, Use, and Mutation Detection in the Mosquito Aedes aegypti.

The generation and study of transgenic Aedes aegypti mosquitoes provides an essential tool for elucidating the complex molecular biology of this important vector. Within the field, genetic manipulation has surpassed the proof of principle stage and is now utilized in both applied and theoretical vector control strategies. The application of new instruments, technologies and techniques allows ever more controlled experiments to be conducted. In this text we describe microinjection of Ae. aegypti embryos in the context of evaluating and performing genomic editing with transcription activator-like effector nucleases (TALENs).

In vivo targeting of porcine reproductive and respiratory syndrome virus antigen through porcine DC-SIGN to dendritic cells elicits antigen-specific CD4T cell immunity in pigs.

Immunogenicity of protein subunit vaccines may be dramatically improved by targeting them through antibodies specific to c-type lectin receptors (CLRs) of dendritic cells in mice, cattle, and primates. This novel vaccine development approach has not yet been explored in pigs or other species largely due to the lack of key reagents. In this study, we demonstrate that porcine reproductive and respiratory syndrome virus (PRRSV) antigen was targeted efficiently to dendritic cells through antibodies specific to a porcine CLR molecule DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) in pigs. A recombinant PRRSV antigen (shGP45M) was constructed by fusing secretory-competent subunits of GP4, GP5 and M proteins derived from genetically-shuffled strains of PRRSV. In vaccinated pigs, when the PRRSV shGP45M antigen was delivered through a recombinant mouse-porcine chimeric antibody specific to the porcine DC-SIGN (pDC-SIGN) neck domain, porcine dendritic cells rapidly internalized them in vitro and induced higher numbers of antigen-specific interferon-γ producing CD4T cells compared to the pigs receiving non-targeted PRRSV shGP45M antigen. The pDC-SIGN targeting of recombinant antigen subunits may serve as an alternative or complementary strategy to existing vaccines to improve protective immunity against PRRSV by inducing efficient T cell responses.