ABSTRACT
To describe the recombination features of human enterovirus 71 strain Guangzhou09 isolated in Guangzhou in 2009, the complete nucleotide sequences of Guangzhou09 were analyzed by various of bioinformatics software. Phylogenetic analysis based on P1, P2 and P3 regions indicated that recombination occurred between EV71 and CVA4. Phylogenetic, similarity plot and bootscan analysis further revealed the recombination between EV71 genotype C strain Shanghai-FJ713317 and CVA4 strain HQ728260 at region 2B was close to the nucleotide position 4 027. This represents the first evidence for intertypic recombination between EV71 subtype C4 and CVA4 in Guangzhou.
Subject(s)
Humans , Base Sequence , China , Epidemiology , Enterovirus A, Human , Chemistry , Classification , Genetics , Hand, Foot and Mouth Disease , Epidemiology , Virology , Molecular Sequence Data , Phylogeny , Recombination, Genetic , Sequence Homology, Nucleic Acid , Viral Proteins , Chemistry , GeneticsABSTRACT
To compare and analyze the variation of PB1-F2 genes of Influenza A Viruses from Guangzhou during 2009 to 2011 with the Influenza A Viruses from all over the world, to lay the foundation of functional research and interaction mechanism of the PB1-F2 protein. 17 Novel H1N1 influenza viruses and 1 seasonal H1N1 influenza virus have been isolated from human in Guangzhou during 2009 to 2011 that were cloned into pMD 18-T Vector for sequencing. Then, 68 PB1-F2 genes of IAVs from human around the world were downloaded from GenBank database and analyzed using molecular biological software. The phylogenetic tree result shows that the PB1-F2 genes of IAV from the world separated into two main groups. There is high homology of PB1-F2 genes of one Seasonal H1N1 virus and Novel H1N1 viruses which were isolated in Guangzhou compared with the global Novel H1N1 viruses. And all of them got the 11 amino acids truncated protein by mutation included one seasonal H1N1 strain isolated by our laboratory. There is no variation of PB1-F2 genes of Novel H1N1 virus in Guangzhou compared with the worldwide strains. However, one seasonal H1N1 virus which isolated by our laboratory shows analogous truncated mutation of PB1-F2 of Novel H1N1 virus, it reveals that the PB1-F2 gene might has done the early reassortment between the Novel H1N1 virus and seasonal H1N1 virus.
Subject(s)
Humans , Amino Acid Sequence , China , Cloning, Molecular , Evolution, Molecular , Genetic Variation , Influenza A Virus, H1N1 Subtype , Genetics , Influenza A Virus, H3N2 Subtype , Genetics , Molecular Sequence Data , Mutation , Phylogeny , Viral Proteins , Chemistry , GeneticsABSTRACT
<p><b>BACKGROUND</b>The rapid transmission and high mortality rate made severe acute respiratory syndrome (SARS) a global threat for which no efficacious therapy is available now. Without sufficient knowledge about the SARS coronavirus (SARS-CoV), it is impossible to define the candidate for the anti-SARS targets. The putative non-structural protein 2 (nsp2) (3CL(pro), following the nomenclature by Gao et al, also known as nsp5 in Snidjer et al) of SARS-CoV plays an important role in viral transcription and replication, and is an attractive target for anti-SARS drug development, so we carried on this study to have an insight into putative polymerase nsp2 of SARS-CoV Guangdong (GD) strain.</p><p><b>METHODS</b>The SARS-CoV strain was isolated from a SARS patient in Guangdong, China, and cultured in Vero E6 cells. The nsp2 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into eukaryotic expression vector pCI-neo (pCI-neo/nsp2). Then the recombinant eukaryotic expression vector pCI-neo/nsp2 was transfected into COS-7 cells using lipofectin reagent to express the nsp2 protein. The expressive protein of SARS-CoV nsp2 was analyzed by 7% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The nucleotide sequence and protein sequence of GD nsp2 were compared with that of other SARS-CoV strains by nucleotide-nucleotide basic local alignment search tool (BLASTN) and protein-protein basic local alignment search tool (BLASTP) to investigate its variance trend during the transmission. The secondary structure of GD strain and that of other strains were predicted by Garnier-Osguthorpe-Robson (GOR) Secondary Structure Prediction. Three-dimensional-PSSM Protein Fold Recognition (Threading) Server was employed to construct the three-dimensional model of the nsp2 protein.</p><p><b>RESULTS</b>The putative polymerase nsp2 gene of GD strain was amplified by RT-PCR. The eukaryotic expression vector (pCI-neo/nsp2) was constructed and expressed the protein in COS-7 cells successfully. The result of sequencing and sequence comparison with other SARS-CoV strains showed that nsp2 gene was relatively conservative during the transmission and total five base sites mutated in about 100 strains investigated, three of which in the early and middle phases caused synonymous mutation, and another two base sites variation in the late phase resulted in the amino acid substitutions and secondary structure changes. The three-dimensional structure of the nsp2 protein was successfully constructed.</p><p><b>CONCLUSIONS</b>The results suggest that polymerase nsp2 is relatively stable during the phase of epidemic. The amino acid and secondary structure change may be important for viral infection. The fact that majority of single nucleotide variations (SNVs) are predicted to cause synonymous, as well as the result of low mutation rate of nsp2 gene in the epidemic variations, indicates that the nsp2 is conservative and could be a target for anti-SARS drugs. The three-dimensional structure result indicates that the nsp2 protein of GD strain is high homologous with 3CL(pro) of SARS-CoV urbani strain, 3CL(pro) of transmissible gastroenteritis virus and 3CL(pro) of human coronavirus 229E strain, which further suggests that nsp2 protein of GD strain possesses the activity of 3CL(pro).</p>