Ribosomal control in RNA virus-infected cells.

Viruses are strictly intracellular parasites requiring host cellular functions to complete their reproduction cycle involving virus infection of host cell, viral genome replication, viral protein translation, and virion release. Ribosomes are protein synthesis factories in cells, and viruses need to manipulate ribosomes to complete their protein synthesis. Viruses use translation initiation factors through their own RNA structures or cap structures, thereby inducing ribosomes to synthesize viral proteins. Viruses also affect ribosome production and the assembly of mature ribosomes, and regulate the recognition of mRNA by ribosomes, thereby promoting viral protein synthesis and inhibiting the synthesis of host antiviral immune proteins. Here, we review the remarkable mechanisms used by RNA viruses to regulate ribosomes, in particular, the mechanisms by which RNA viruses induce the formation of specific heterogeneous ribosomes required for viral protein translation. This review provides valuable insights into the control of viral infection and diseases from the perspective of viral protein synthesis.

Development of SYBR Green I-based real-time reverse transcription polymerase chain reaction for the detection of feline astrovirus.

In this study, a SYBR Green I-based real-time reverse transcription-polymerase chain reaction (RT-PCR) was developed for the clinical diagnosis of feline astroviruses (FeAstVs). Specific primers were designed based on the conserved region of the FeAstV ORF1b gene. Experiments for specificity, sensitivity, and repeatability of the assay were carried out. In addition, the assay was evaluated using clinical samples. Specificity analysis indicated that the assay showed negative results with samples of Feline Parvovirus, Feline Herpesvirus, Feline Calicivirus, Feline Bocavirus, and Feline Coronavirus, indicating good specificity of the assay. Sensitivity analysis showed that the SYBR Green I-based real-time RT-PCR method could detect as low as 3.72 × 101 copies/µL of template, which is 100-fold more sensitive compared to the conventional RT-PCR. Both intra-assay and inter-assay variability were lower than 1 %, indicating good reproducibility. Furthermore, an analysis of 150 fecal samples showed that the positive detection rate of SYBR Green I-based real-time RT-PCR was higher than that of the conventional RT-PCR, indicating the high reliability of the method. The assay is cheap and effective. Therefore, it could provide support for the detection of FeAstV in large-scale clinical testing and epidemiological investigation.

Bioinformatics analysis and prokaryotic expression of FIPV AH1905 strain N gene

To understand the molecular epidemiological and genetic variation characteristics of feline infectious peritonitis virus (FIPV) in China, the N gene of FIPV AH1905 strain was cloned by PCR, and the bioinformatics softwares were used to predict the N protein. Then, N gene was cloned into a prokaryotic vector pGEX-4T-1 and was successfully expressed. The expressed protein was purified, and identified by SDS-PAGE and Western blot. The results showed that the N gene of FIPV, 1 134 bp, encodes 377 amino acids. The molecular bioinformatics analysis showed that the nucleotide homology and the amino acid homology between FIPV AH1905 strain and the reported FIPV reference strains were 90.2%-92.4% and 91.8%-93.9%, respectively. The phylogenetic analysis showed that FIPV AH1905 belongs to the FIPV gene type I, the same as other isolates in China. The prediction of the secondary structure of the N protein showed that the protein was a hydrophilic stable protein with 14.06% a-helix (h), 15.12% extended chain (e), 3.71% beta turn (t) and 67.11% random spiral (c). There were no signal peptide region and transmembrane domain. It may have 48 phosphorylation sites. Moreover, there may exist six B cell epitopes, two CTL epitopes and two Th epitopes. The molecular weight of expressed protein is approximately 68 ku, mainly expressed in inclusion body form with good reactogenicity. In conclusion, the present study has successfully expressed the N protein of FIPV, and prepared the multi-antiserum, which laid the foundation for further research on epidemiology and molecular biology of FIPV.