Variations in the NSP4 gene of the type 2 porcine reproductive and respiratory syndrome virus isolated in China from 1996 to 2021.

Porcine reproductive and respiratory syndrome virus (PRRSV) has continuously mutated since its first isolation in China in 1996, leading to difficulties in infection prevention and control. Infections caused by PRRSV-2 strains are the main epidemic strains in China, as determined by phylogenetic analysis. In this study, we focused on the prevalence and genetic variations of the non-structural protein 4 (NSP4) from PRRSV-2 over the past 20 years in China. The fundamental biological properties of the NSP4 were predicted, and an analysis and comparison of NSP4 homology at the nucleotide and amino acid levels was conducted using 123 PRRSV-2 strains. The predicted molecular weight of the NSP4 protein was determined to be 21.1 kDa, and it was predicted to be a stable hydrophobic protein that lacks a signal peptide. NSP4 from different strains exhibited a high degree of amino acid (85.8-100%) and nucleotide sequence homology (81.0-100%). Multiple amino acid substitutions were identified in NSP4 among 15 representative PRRSV-2 strains. Phylogenetic analysis showed that the lineage 8 and 1 strains, the most prevalent strains in China, were indifferent clades with a long genetic distance. This analysis will help fully elucidate the parameters of the PRRSV NSP4 epidemic in China to lay a foundation for adequate understanding of the function of NSP4. Genetic information results from the accumulation of conserved and non-conserved sequences. The high conservation of the NSP4 gene determines the most basic life traits and functions of PRRSV. Analyzing the spatial structure of NSP4 protein and studying the genetic evolution of NSP4 not only provide the theoretical basis for how NSP4 participates in the regulation of the innate response of the host but also provide a target for genetic manipulation and a reasonable target molecule and structure for new drug molecules.

Analysis of structure-function relationship in porcine rotavirus A enterotoxin gene.

Rotavirus (RV)-infected piglets are presumed to be latent sources of heterologous RV infection in humans and other animals. In RVs, non-structural protein 4 (NSP4) is the major virulence factor with pleiotropic properties. In this study, we analyzed the nsp4 gene from porcine RVs isolated from diarrheic and non-diarrheic cases at different levels of protein folding to explore correlations to diarrhea-inducing capabilities and evolution of nsp4 in the porcine population. Full-length nsp4 genes were amplified, cloned, sequenced, and then analyzed for antigenic epitopes, RotaC classification, homology, genetic relationship, modeling of NSP4 protein, and prediction of post-translational modification. RV presence was observed in both diarrheic and non-diarrheic piglets. All nsp4 genes possessed the E1 genotype. Comparison of primary, secondary, and tertiary structure and the prediction of post-translational modifications of NSP4 from diarrheic and non-diarrheic piglets revealed no apparent differences. Sequence analysis indicated that nsp4 genes have a multi-phyletic evolutionary origin and exhibit species independent genetic diversity. The results emphasize the evolution of the E9 nsp4 genotype from the E1 genotype and suggest that the diarrhea-inducing capability of porcine RVs may not be exclusively linked to its enterotoxin gene.

Analysis of structure-function relationship in porcine rotavirus A enterotoxin gene

Rotavirus (RV)-infected piglets are presumed to be latent sources of heterologous RV infection in humans and other animals. In RVs, non-structural protein 4 (NSP4) is the major virulence factor with pleiotropic properties. In this study, we analyzed the nsp4 gene from porcine RVs isolated from diarrheic and non-diarrheic cases at different levels of protein folding to explore correlations to diarrhea-inducing capabilities and evolution of nsp4 in the porcine population. Full-length nsp4 genes were amplified, cloned, sequenced, and then analyzed for antigenic epitopes, RotaC classification, homology, genetic relationship, modeling of NSP4 protein, and prediction of post-translational modification. RV presence was observed in both diarrheic and non-diarrheic piglets. All nsp4 genes possessed the E1 genotype. Comparison of primary, secondary, and tertiary structure and the prediction of post-translational modifications of NSP4 from diarrheic and non-diarrheic piglets revealed no apparent differences. Sequence analysis indicated that nsp4 genes have a multi-phyletic evolutionary origin and exhibit species independent genetic diversity. The results emphasize the evolution of the E9 nsp4 genotype from the E1 genotype and suggest that the diarrhea-inducing capability of porcine RVs may not be exclusively linked to its enterotoxin gene.

Rapid detection of human rotavirus using NSP4 gene specific reverse transcription loop-mediated isothermal amplification assay.

The seasonal outbreaks of human rotavirus (RV) infection occur every winter. Most patients are diagnosed clinically by a rapid latex agglutination detection kit or polymerase chain reaction assays for RV from stool samples, but some problems have been reported on the specificity and sensitivity of such rapid detection assays. To ratify these issues, a sensitive, specific, simple, and rapid nucleic acid based diagnostic method is expected to be introduced and the reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed to detect the RV in human stool samples by incubation at 60 °C for 1 h and amplification was confirmed by electrophoretic laddering, restriction enzyme digestion, and hydroxynapthol blue discoloration. The assay established in this study was found to detect only the RVs and no cross-reaction with other viruses, demonstrating its high specificity. By using serial samples dilution as template, the detection limit of LAMP was 10 times more than that of PCR. The results showed the potential clinical feasibility of RT-LAMP as a useful diagnostic tool for the detection of RV with high sensitivity in comparison to conventional RT-PCR.

Molecular characterization of the NSP4 gene of human group A rotavirus samples from the West Central region of Brazil

Nonstructural protein 4 (NSP4), encoded by group A rotavirus genome segment 10, is a multifunctional protein and the first recognized virus-encoded enterotoxin. The NSP4 gene has been sequenced, and five distinct genetic groups have been described: genotypes A-E. NSP4 genotypes A, B, and C have been detected in humans. In this study, the NSP4-encoding gene of human rotavirus strains of different G and P genotypes collected from children between 1987 and 2003 in three cities of West Central region of Brazil was characterized. NSP4 gene of 153 rotavirus-positive fecal samples was amplified by reverse transcriptase-polymerase chain reaction and then sequenced. For phylogenetic analysis, NSP4 nucleotide sequences of these samples were compared to nucleotide sequences of reference strains available in GenBank. Two distinct NSP4 genotypes could be identified: 141 (92.2 percent) sequences clustered with NSP4 genotype B, and 12 sequences (7.8 percent) clustered with NSP4 genotype A. These results reinforce that further investigations are needed to assess the validity of NSP4 as a suitable target for epidemiologic surveillance of rotavirus infections and vaccine development.