Cloning of the complete genome set and sequence analysis of the VP4, VP6, and VP7 genes of a novel adult diarrhea rotavirus strain J19
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For better understanding the molecular characteristics of the genes and proteins of a novel adult diarrhea rotavirus strain J19(NADRV-J19),the complete genome set of NADRV-J19 was amplified by an improved single primer sequence-independent amplification method,cloned into pMD18-T vector,and sequenced. Protein sequences of VP4,VP6,and VP7 of J19 strain were aligned with those of other rotaviruses and phylogenetic analysis based on VP6 sequence was made with the other rotaviruses.Sequence analysis of 11 full-length genes of J19 revealed that genomic segments 3,6,and 9 of NADRV-J19 encoding VP4,VP6,and VP7 were 2512,1287,and 820bp in length,and their deduced proteins were 823aa,396aa,and 258aa respectively.Identities of VP4,VP6,and VP7 proteins of J19 strain to that of group B rotavirus strains CAL,IDIR,and ADRV were 27.6%,38.5%,and 22.3% respectively.Phylogenetic analysis based on VP6 proteins showed that J19 was positioned closely to outgroups and the ancestral nodes of rotavirus group A,B,and C lineages,although with a preferred association with group B lineages.The above results showed that VP4,VP6,and VP7 proteins of J19 differed significantly from those of the other rotaviruses.J19 would be a representative strain of a novel rotavirus group and would be a strain closely related to the evolution and origin of group B rotaviruses.Up to now,this is the first report concerning to the cloning of 11 gene segments of NADRV-J19 and sequence analysis of the genes of NADRV-J19 encoding VP4,VP6,and VP7 proteins.Keywords:
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A unique human (group A) rotavirus isolate, bearing bovine-Iike VP7 and VP4 genes, was characterized by sequence analysis of its major outer capsid protein genes (VP4 and VP7) and enterotoxin non-structural protein gene (NSP4). The VP4 gene of this isolate shared the greatest sequence homology (at 99.3% nucleotide and 99.6% amino acid) with those of Indian bovine P[ll] strains. The VP7 gene had highest homology (at 99.8% nucleotide and 100% amino acid) with those of Indian bovine G3 rotavirus strains. However, percent sequence identity of both VP4 and VP7 genes was lower when compared with those of P[ll] and G3 human rotavirus strains, respectively. The NSP4 gene sequence had closer relationship with Indian bovine E2 genotype rotavirus. Phylogenetic analysis of VP4, VP7, and NSP4 genes of H2 isolate revealed its close proximity with rota viruses of bovine origin. The findings are suggestive of interspecies transmission events of rotavirus between human and bovine in nature and also provide convincing evidence that evolution of human rota viruses is tightly intermingled with the evolution of animal rotaviruses.
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Novel rotavirus strains B219 and ADRV-N derived from adult diarrheal cases in Bangladesh and China, respectively, are considered to belong to a novel rotavirus group (species) distinct from groups A, B, and C, by genetic analysis of five viral genes encoding VP6, VP7, NSP1, NSP2, and NSP3. In this study, the nucleotide sequences of the remaining six B219 gene segments encoding VP1, VP2, VP3, VP4, NSP4, and NSP5 were determined. The nucleotide sequences of the group B human rotavirus VP1 and VP3 genes were also determined in order to compare the whole genome of B219 with those of group A, B, and C rotavirus genomes. The nucleotide and deduced amino acid sequences of all B219 gene segments showed considerable identity to the ADRV-N (strain J19) sequences (87.7-94.3% and 88.7-98.7%, respectively). In contrast, sequence identity to groups A-C rotavirus genes was less than 61%. However, functionally important domains and structural characteristics in VP1-VP4, NSP4, and NSP5, which are conserved in group A, B, or C rotaviruses, were also found in the deduced amino acid sequences of the B219 proteins. Hence, the basic structures of all B219 viral proteins are considered to be similar to those of the known rotavirus groups.
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The VP7 gene ofporcine rotavirus have been cloned and sequenced using a pair of primers designed according to the published cDNA sequence. The results demonstrated that the VP7 gene was 1062 nucleotides long and contains a long open reading frame,which codes for a VP7 protein of 326 amino acids. The VP7 gene shared from 88. 8% to 93. 7% nucleic acid identity and from 93. 3% to 94. 2% amino acid identity with PRV G5 genotype reference strains. Phylogenetic analysis revealed that the VP7 gene of L1 strain grouped very closely G5 genotype reference strains. So L1 strain was proved to belong to G5 genotype. Results from the analysis of antigenic index,L1 strain and G9 genotype NMTL strain had differences in amino acid 25 to 29,86 to 102,142 to 152,211 to 226,263 to 286,which fields would play a important role of immunogenicity of PRV.
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Abstract We previously reported the detection of genotype P[19] rotavirus strains from children hospitalized with acute dehydrating diarrhea during a 5‐year surveillance period in Taiwan. The characterization of five P[19] strains (0.4% of all typed), including three G3P[19], a novel G5P[19], and a unique G9P[19] genotype is described in this study. Phylogenetic analysis of the VP4, VP7, VP6, and NSP4 genes was performed, which demonstrated novel lineages for respective genotypes of the VP4 and the VP7 genes. The sequence similarities of the P[19] VP4 gene among Taiwanese human strains was higher (nt, 91.5–96.2%; aa, 93.7–97.6%) than to other P[19] strains (nt, 83.5–86.6%; aa, 89.4–94.1%) from different regions of the world. The VP7 gene of the three G3P[19] Taiwanese strains shared up to 93.4% nt and 97.5% aa identity to each other but had lower similarity to reference strain sequences available in GenBank (nt, <90.1%; aa, <95.6%). Similarly, the VP7 gene of the novel G5P[19] strain was only moderately related to the VP7 gene of reference G5 strains (nt, 82.2–87.3%; aa, 87.0–93.1%), while the VP7 gene of the single G9P[19] strain was genetically distinct from other known human and animal G9 rotavirus strains (nt, ≤92.0%; aa, ≤95.7%). Together, these findings suggest that the Taiwanese P[19] strains originated by independent interspecies transmission events. Synchronized surveillance of human and animal rotaviruses in Taiwan should identify possible hosts of these uncommon human rotavirus strains. J. Med. Virol. 83:1279–1287, 2011. © 2011 Wiley‐Liss, Inc.
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To analyse the genetic characteristics of the capsid protein VP3-VP1 region of hepatitis A virus strains circulated in China.The nucleotide sequences of VP3-VP1-2A region of 42 HAV IgM positive serum samples were sequenced and analysed for nucleotide and amino acid identities and genetic characteristics of the VP3-VP1 region.The nucleotide and amino acid identities in the VP1-2A junction region among the 42 strains were 89.1% - 100% and 97.3% - 100%; while in the complete VP3-VP1 region, the identities were 87.6%-100% and 98.8%-100%. Strains with identical nucleotide sequences in the VP1-2A junction region had 98.4%-100% nucleotide identity in the complete VP3-VP1 region and 0-2 amino acid differences in this region. There were no amino acid changes at neutralizing antigenetic sites of VP3-VP1 region within the 42 HAV strains.All the 42 HAV strains belonged to genotype I, with 40 strains clustering to sub-genotype IA and 2 to sub-genotype IB. Different HAV strains analysed in this paper differed in the nucleotide sequences of the VP3-VP1 region, but the amino acid sequences were highly conserved with no changes at neutralizing antigenetic sites. Both the nucleotide and amino acid sequences of the strains with the same VP1-2A junction region were identical or closely related when compared in the complete VP3-VP1 region.
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To establish a method to amplify the entire fragment of genes VP6,VP7,VP4 and NSP4 from group C rotavirus.In order to define the phylogenetic relations among the field strain Wu-82 and other viral strains,4362 stool specimens from patients with diarrhea in all ages were collected during two periods of time,from Dec 2000 to Apr 2003 and from Nov 2003 to Feb 2007.The presence of rotaviruses in stool specimens was determined by detection of the dsRNA fragment of rotavirus by polyacrylamide gel electrophoresis,and the entire fragments of VP7,VP6,VP4 and nsp4 genes were amplified by RT-PCR with 9 pairs of specific primers.Nucleic acid sequence data and hydrophilicity analysis of the deduced amino acids were analyzed using the Gene Works software package and the MEGA 4 program.It was demonstrated that the VP6 and VP7 genes from Wu-82 strain were quite conserved with few mutations,and the phylogenetic relation of the VP6 gene from Wu-82 strain was closer to the Nigerian strain Jajeri and Spanish strain BCN6,whereas the VP7 gene of this strain was closer to Japanese strain E-93 and Columbia strain Javeriana.The sequence of the deduced amino acids of VP6 and VP7 of Wu-82 strain was distinct from that of the 208 strain isolated in 1995 at Wuhan area.Most of the different amino acids between Wu-82 and 208 strains was found to be located in the hydrophilicity regions.The homologies of nucleotide sequences of VP6 and VP7 genes with other group C rotaviruses were 98.45-97.12% and 99.34-94.83% respectively.From the observation of the present study,it is evident that the group C rotavirus genes are very conservative with slow process of evolution,but diversity of epitopes exists in Wu-82 and 208 strains.
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Genes of a novel adult diarrhea rotavirus(NADRV) strain J19 were amplified by an improved single primer sequence-independent amplification method,cloned into pMD18-T vector,sequenced and analyzed.Genomic segments 2,4 encoding VP2,VP3 of NADRV-J19 were 2969bp,2204bp in length,and the deduced proteins were 973aa and 719aa,respectively.Identity of VP2 of J19 to human group B rotavirus strain IDIR was 47.2 % and that of VP3 of J19 to human group C rotavirus strain Cowden was 25.1%.Phylogenetic analysis of VP2 showed that NADRV-J19 positioned closely to outgroup and the ancestral nodes of group A,B and C lineages, although with a preferred association with group B lineage,which corresponded to the phylogenetic analysis of VP6.The above results allowed us to deduce a conclusion that J19 would be a strain closely related to the evolution and origin of group B rotaviruses and they also indicated that VP2 would be valuable for studying the evolution and origin of rotaviruses.This study is the first report on sequence analysis of genes of NADRV-J19 encoding VP2 and VP3.
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