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Infection with the nervous necrosis virus (NNV) causes viral nervous necrosis, resulting seriouslly economic losses in the aquaculture of marine fish. The viral genome of orange-spotted grouper nervous necrosis virus (OSGNNV) consists of two single-stranded, RNA1 and RNA2, encoding RNA-dependent RNA polymerase (RdRp) and capsid protein (CP), respectively. RNA interference has been shown to have activities against various viruses and is considered a promising antiviral approach. Here, we describe antiviral activities of short hairpin RNAs (shRNAs) that target RdRp and CP gene of OSGNNV in GF-1 cells. We constructed shRNAs for two specific target genes, RdRp and CP genes of OSGNNV. The shRNAs were transfected into GF-1 cells, followed by OSGNNV infection at multiplicity of infection (M.O.I) of 10.0. Then, the efficient inhibition of OSGNNV replication was examined by real-time quantitative RT-PCR analysis of RdRp and CP gene expression and viral titers. The results showed that the expression levels of RdRp and CP gene were reduced by shRNAs-transfected GF-1 cells at 48 and 72-hour post infection (hpi). Besides, the viral titers of shRNAs-transfected GF-1 cells were significantly lower than those of OSGNNV control at period of 48-96 hpi. These results indicated that the plasmid-transcribed shRNAs could inhibit effectively the OSGNNV replication in GF-1 cells, providing a potential approach to control viral disease in aquaculture. [ABSTRACT FROM AUTHOR]

The UL29 and UL28 proteins encoded by herpes simplex virus type 1 (HSV-1) are critical for its replication and packaging, respectively. Research has demonstrated that synthesized siRNA molecules targeting the UL29 gene are able to suppress HSV-2 replication and the UL28-null HSV-1 gene cannot form infectious viruses in vitro. Silencing the UL28 and UL29 genes by RNAi might lead to the development of novel antiviral agents for the treatment of HSV-1 infections. Two kinds of short hairpin RNAs (shRNAs) targeting the UL29 and UL28 genes were chemically synthesized and then delivered into cells by a replication-defective human adenovirus type 5 (Adv5) vector. (−) shRNAs targeting none of the genome of HSV-1 were used as the control. Vero cells were inoculated with Ad-UL28shRNA or Ad-UL29shRNA at a multiplicity of infection (MOI) of 100 and challenged 24 h later with HSV-1 at an MOI of 0.01 to inhibit HSV-1 replication, as measured by the level of the corresponding RNA and proteins. In addition, the amount of progeny virus was assessed at daily intervals. The antiviral effects of Ad-shRNAs at ongoing HSV-1 infection were explored at 12 h after inoculation of the HSV-1. The results showed that the shRNAs delivered by Adv5 significantly suppressed HSV-1 replication in vitro, as determined by the levels of viral RNA transcription, viral protein synthesis, and viral production. The Ad-UL28shRNA and Ad-UL29shRNA suppressed the replication of HSV-1, respectively, compared with the control group ( P < 0.001). When Ad-UL28shRNA and Ad-UL29shRNA were combined, a synergistic effect was observed. The antiviral effects could sustain for at least 4 days after the HSV-1 infection ( P < 0.001). Furthermore, antiviral effects were achieved 12 h prior to inoculation of Adv5-shRNAs ( P < 0.001). Our data demonstrated comparable antiviral activities against herpes simplex virus by shRNAs targeting either UL29 or UL28 sites in vitro and the effectiveness of using the Adv5 delivery of shRNAs. Therefore, the Adv5 delivery of shRNAs targeting the UL29 and UL28 sites probably may provide an alternative strategy for controlling HSV-1 infection. [ABSTRACT FROM AUTHOR]

Abstract: To improve the care of HIV-1/AIDS patients there is a critical need to develop tools capable of blocking viral evolution and circumventing therapy-associated problems. An emerging solution is gene therapy either as a stand-alone approach or as an adjuvant to pharmacological drug regimens. Combinatorial RNAi by multiplexing antiviral RNAi inhibitors through vector-mediated delivery has recently shown significant superiority over conventional mono-therapies. Viral as well as cellular co-factor targets have been identified, but they are generally attacked separately. Here, we hypothesized that a mixture of shRNAs directed against highly conserved viral RNA sequences and the mRNAs of cellular components that are involved in HIV replication could restrict mutational escape by enhanced synergistic inhibition. We screened for potent silencer cocktails blending inhibitors acting scattered along the viral replication cycle. The results show enhanced and extended suppression of viral replication for some combinations. To further explore the power of combinatorial approaches, we tested the influence of RNAi-mediated knockdown on the activity of conventional antiretroviral drugs (fusion, RT, integrase and protease inhibitors). We compared the fold-change in IC50 (FCIC50) of these drugs in cell lines stably expressing anti-HIV and anti-host shRNAs and measured increased values that are up by several logs for some combinations. We show that high levels of additivity and synergy can be obtained by combining gene therapy with conventional drugs. These results support the idea to validate the therapeutic potential of this anti-HIV approach in appropriate in vivo models. [Copyright &y& Elsevier]

Abstract: Two DNA constructs targeting the grass carp reovirus (GCRV) RNA dependent RNA polymerase (RdRp) gene and outer capsid protein (OCP) gene that were each 64bp in length were synthesized chemically and cloned into pSilencer2.1-U6 neo plasmid, named pSi-RdRp1286 and pSi-OCP117, respectively. After transfection of pSi-RdRp1286 and pSi-OCP117 plasmids into CIK cells, the inhibition of GCRV replication in the cells were detected by observing cytopathic effect (CPE), quantitating virus titers (TCID50/mL) and real-time quantitative RT-PCR analysis of viral RdRp and OCP genes. Five days after the cells were challenged with GCRV, both pSi-RdRp1286 and pSi-OCP117 reduced the viral titers by 5.47lgTCID50/mL and 4.37lgTCID50/mL, respectively. Compared to the positive control, CPE induced by GCRV in transfected cells was delayed and significantly less. Furthermore, the real-time quantitative RT-PCR analysis of the viral RdRp gene and OCP gene showed that the targeted gene expression were reduced by 89% and 73%, respectively. These results proved that the plasmid-transcribed shRNAs could inhibit effectively GCRV replication in CIK cells. These shRNAs provide potential tools for inhibiting GCRV infection and replication both in vitro and in vivo. [Copyright &y& Elsevier]