Discovery of a non-nucleoside RNA polymerase inhibitor for blocking Zika virus replication through in silico screening.

Zika virus (ZIKV), an emerging arbovirus, has become a major human health concern globally due to its association with congenital abnormalities and neurological diseases. Licensed vaccines or antivirals against ZIKV are currently unavailable. Here, by employing a structure-based approach targeting the ZIKV RNA-dependent RNA polymerase (RdRp), we conducted in silico screening of a library of 100,000 small molecules and tested the top ten lead compounds for their ability to inhibit the virus replication in cell-based in vitro assays. One compound, 3-chloro-N-[({4-[4-(2-thienylcarbonyl)-1-piperazinyl]phenyl}amino)carbonothioyl]-1-benzothiophene-2-carboxamide (TPB), potently inhibited ZIKV replication at sub-micromolar concentrations. Molecular docking analysis suggests that TPB binds to the catalytic active site of the RdRp and therefore likely blocks the viral RNA synthesis by an allosteric effect. The IC50 and the CC50 of TPB in Vero cells were 94 nM and 19.4 µM, respectively, yielding a high selective index of 206. In in vivo studies using immunocompetent mice, TPB reduced ZIKV viremia significantly, indicating TPB as a potential drug candidate for ZIKV infections.

Relative contribution of porcine reproductive and respiratory syndrome virus open reading frames 2-4 to the induction of protective immunity.

The minor glycoproteins (GPs) of PRRSV, GP2, GP3, and GP4, form a heterotrimer that is required for viral infectivity, presumably due to its interaction with the key cellular receptor CD163. These 3GPs are encoded by open reading frames (ORFs) 2a, 3 and 4 (herein referred to as ORFs 2-4), respectively. The goal of this study was to investigate the immunogenicity of the PRRSV-2 minor GPs. Through the use of reverse genetics, a chimeric virus (designated SDFL24) was constructed by replacing ORFs 2-4 of the PRRSV-1 strain SD01-08 with the corresponding genes of the PRRSV-2 strain FL12. While the parental PRRSV strain SD01-08 was not neutralized by convalescent antisera raised against FL12, the chimeric virus SDFL24 gained susceptibility to neutralization by FL12-specific antisera, indicating that viral proteins encoded by ORFs 2-4 are targets of antibody neutralization. When inoculated into pigs, the chimeric virus SDFL24 elicited T-cell responses against peptides derived from FL12 minor GPs, whereas the parental virus SD01-08 did not. After challenge infection with FL12, pigs previously infected with SDFL24 developed robust kinetics of FL12-specific neutralizing antibodies as compared to those previously infected with the parental strain SD01-08. Finally, the pigs recovered from SDFL24 infection were better protected from a subsequent challenge infection with FL12 than those previously infected with SD01-08. Collectively, the results indicate that PRRSV-2 ORFs 2-4 are capable of inducing protective immunity.

Cross reactivity of immune responses to porcine reproductive and respiratory syndrome virus infection.

Because porcine reproductive and respiratory syndrome virus (PRRSV) exhibits extensive genetic variation among field isolates, characterizing the extent of cross reactivity of immune responses, and most importantly cell-mediated immunity (CMI), could help in the development of broadly cross-protective vaccines. We infected 12 PRRSV-naïve pigs with PRRSV strain FL12 and determined the number of interferon (IFN)-γ secreting cells (SC) by ELISpot assay using ten type 2 and one type 1 PRRSV isolates as recall antigens. The number of IFN-γ SC was extremely variable among animals, and with exceptions, late to appear. Cross reactivity of IFN-γ SC among type 2 isolates was broad, and we found no evidence of an association between increased genetic distance among isolates and the intensity of the CMI response. Comparable to IFN-γ SC, total antibodies evaluated by indirect immunofluorescence assay (IFA) were cross reactive, however, neutralizing antibody titers could only be detected against the strain used for infection. Finally, we observed a moderate association between homologous IFN-γ SC and neutralizing antibodies.