Cell entry of avian reovirus follows a caveolin-1-mediated and dynamin-2-dependent endocytic pathway that requires activation of p38 mitogen-activated protein kinase (MAPK) and Src signaling pathways as well as microtubules and small GTPase Rab5 protein.

Very little is known about the mechanism of cell entry of avian reovirus (ARV). The aim of this study was to explore the mechanism of ARV entry and subsequent infection. Cholesterol mainly affected the early steps of the ARV life cycle, because the presence of cholesterol before and during viral adsorption greatly blocked ARV infectivity. Although we have demonstrated that ARV facilitating p38 MAPK is beneficial for virus replication, its mechanism remains unknown. Here, we show that ARV-induced phosphorylation of caveolin-1 (Tyr(14)), dynamin-2 expression, and Rac1 activation through activation of p38 MAPK and Src in the early stage of the virus life cycle is beneficial for virus entry and productive infection. The strong inhibition by dynasore, a specific inhibitor of dynamin-2, and depletion of endogenous caveolin-1 or dynamin-2 by siRNAs as well as the caveolin-1 colocalization study implicate caveolin-1-mediated and dynamin-2-dependent endocytosis as a significant avenue of ARV entry. By means of pharmacological inhibitors, dominant negative mutants, and siRNA of various cellular proteins and signaling molecules, phosphorylation of caveolin-1, dynamin-2 expression, and Rac1 activation were suppressed, suggesting that by orchestrating p38 MAPK, Src, and Rac1 signaling cascade in the target cells, ARV creates an appropriate intracellular environment facilitating virus entry and productive infection. Furthermore, disruption of microtubules, Rab5, or endosome acidification all inhibited ARV infection, suggesting that microtubules and small GTPase Rab5, which regulate transport to early endosome, are crucial for survival of ARV and that exposure of the virus to acidic pH is required for productive infection.

Proteasome inhibition reduces avian reovirus replication and apoptosis induction in cultured cells.

The interplay between avian reovirus (ARV) replication and apoptosis and proteasome pathway was studied in cultured cells. It is shown that inhibition of the proteasome did not affect viral entry and host cell translation but had influence on ARV replication and ARV-induced apoptosis. Evidence is provided to demonstrate that ubiquitin-proteasome blocked ARV replication at an early step in viral life cycle. However, viral transcription and protein translation were also reduced markedly after addition of proteasome inhibitor MG132. Treatment of BHK-21 cells with the MG132 markedly decreased virus titer as well as prevented virus-induced apoptosis. The expression of ARV proteins sigmaC, sigmaA, and sigmaNS was also reduced markedly, suggesting that suppression of virus replication is due to down-regulation of these ARV proteins by ubiquitin-proteasome system. MG132 was also shown to suppress ARV sigmaC-induced phosphrylation of p53 on serine 46, caspase 3 activities, and DNA fragmentation leading to complete inhibition of ARV-induced apoptosis.

Development of a reliable assay protocol for identification of diseases (RAPID)-bioactive amplification with probing for detection of avian reovirus.

Avian reovirus (ARV) causes several disease syndromes in poultry including arthritis, malabsorption syndrome and chronic respiratory disease that result in major economic losses. Early detection is very important for the control of the ARV-induced infections. This study was therefore aimed at developing a reliable assay protocol for identification of diseases (RAPID)-bioactive amplification with probing (BAP) assay for detection of ARV. This assay combines nested polymerase chain reaction (PCR) and magnetic bead-based DNA probing systems greatly increasing its sensitivity and specificity. Alignment of ARV S2 gene from different ARV genotypes and serotypes was done to find the highly conserved regions for primer and probe design. Two reverse transcription (RT)-PCR primer pairs, six nested PCR primer pairs, and one magnetic probe were tested to find the most specific ones for ARV detection. The optimal conditions for RT-PCR, nested PCR, and hybridization of magnetic probe were established. The optimal annealing temperatures for RT-PCR and nested PCR were 62.1 and 54.8 degrees C, respectively. The optimal hybridization temperature was 51.2 degrees C using hybridization buffer (5x SSC and 0.5% SDS). The sensitivity of the kit was 5 copies/microl of ARV genomic RNA. The kit was very specific as all negative controls failed to show any positive reactions. The kit shows good reproducibility with intra- and inter-assay coefficient of variation (CV) of 1.3 and 1.7%, respectively. In addition, different serotypes and genotypes of ARV were tested by RAPID-BAP assay to estimate the practicability of the kit in clinical samples. All of ARV serotypes and genotypes tested could be detected by this kit proving that the kit is suitable for clinical application.

The cleavage of the hemagglutinin protein of H5N2 avian influenza virus in yeast.

Influenza viruses belonging to the Orthomyxoviridae family are enveloped viruses with segmented negative sense RNA genome surrounded by a helical symmetry shell. Influenza viruses, especially the highly pathogenic avian influenza virus (HPAI) such as H5 or H7 subtype are important pathogens for the poultry industry. Due to genetic reassortments between avian and human influenza viruses, global pandemics may emerge and the naive human immunity could not be ready for them. The full-length HA-encoding gene of H5N2 AIV was inserted into a secretory pPICZalphaA vector and integrated into the genome of Pichia pastoris by heterologous recombination. The HA protein secretion into the medium was induced with methanol. Besides the expected 69kDa protein, another smaller fragment about 47kDa was recognized by an anti-AIV-HA monoclonal antibody in Western blot assay. This is the first report on the cleavage of HA(0) into HA(1) and HA(2) in the methylotrophic yeast P. pastoris. This possibly was due to digestion by proteases from P. pastoris based on the amino acid sequences at the predicted cleavage site, (326)R-X-K-R(329). With similar modifications to the eukaryotes, large quantity, proper antigenicity, and low cost, this expression system may provide a simple tool to produce HA proteins for further use in preparation of ELISA kits and subunit vaccines.

Suppression of bovine ephemeral fever virus by RNA interference.

RNA interference (RNAi) was used to suppress bovine ephemeral fever virus (BEFV). Plasmids expressing continuously shRNAs were used against G gene of BEFV to induce RNA interference in cultured cells. A GFP reporter assay was established to determine the efficiency and specificity of siRNA and the potential of BEFV to hamper RNAi. Two of five small interfering RNAs (siRNAs) were shown to suppress BEFV. Suppression of the G gene of BEFV corresponded with reduction of viral plaques and progeny titer. The results suggest that RNAi has the potential for use in suppression of BEFV infection with possible therapeutic implications.