Adaptability and purification of dengue-III virus D9964 strain in KMB17 cells and proliferation kinetics of adapted strain / 病毒学报

To select the adaptive strain of Dengue-III virus D9964 strain (China strain) in KMB17 cells, elucidate the biological characteristics and proliferation kinetics of adapted strain,and to lay the foundation for the development dengue inactivated vaccine and attenuated live vaccine. Dengue-III virus D9964 strain was firstly identified by amplification of the type-specific gene segment of dengue virus by RT-PCR, and the titer was determined. The virus was then subcultured in KMB17 cells with 4.0 MOI till completely adaptive to multiply in cell S. After subculturing in KMB17 cells for 10 consecutive passages, the adapted strain was screened, and purified through plaque. Virus titer of each passage was measured by microtitrimetry, and the antigenicity was detected by IFA. The purified virus RNA extraction of 3-8 day cultured from KMB17 cells, was performed to detect the proliferation kinetics of adapted strain. The results showed that after continuous subculture, dengue-III virus D9964 (China) strain could stably proliferate in KMB17 cells, a highly puried virus adapted strain was obtained through plaque purification. Purified strain maintained the good antigenicity with a highest replicating activity during the 5th-6th day.

Genomic characteristics of coxsackievirus B1 MSH/KM9/2009 strain isolated in Yunnan, China / 病毒学报

To characterize the complete genome sequence of coxsackievirus B1 (CVB1) MSH/KM9/2009 strain isolated from Yunnan, China,2009. Eight overlapping clones covering the whole viral genome (excluding the poly-A tail) were obtained by RT-PCR and sequenced, and their nucleotide and amino acid sequences were compared with other known CVB1 strains. The genome of the CVB1 MSH/KM9/2009 strain had 7384 nucleotides in length, and contained a 741nt non-translated region (NTR) at the 5' end and a 94nt NTR at the 3' end. The entire open reading frame contained 6 549 nt, encoding a 2 183-aa polyprotein. In the coding region, there was no nucleotide deletion or insertion, but some changes of amino acid were unique. The complete genome sequence alignments showed that the CVB1 isolate MSH/KM9/2009 strain shared the highest nucleotide (80.9%, 81.6%, 80.5% and 80%) and amino acid (95.6%, 95.8%, 96.2% and 95.6%) identities to the CVB1 M16560, pmMC, Tucson B1 and CVB1Nm strain, respectively. Phylogenetic tree analysis showed that the MSH/KM9/2009, CVB1 M16560, pmMC, Tucson B1 and CVB1Nm strain clustered into same group. The newly isolated CVB1 strain MSH/KM9/2009 from Yunnan Province belonged to genotype CVB1.

Complete genome sequence characteristics of human echovirus 9 strain isolated in Yunnan, China / 病毒学报

To analyze the genomic sequence characteristics of a human Echovirus 9(ECHO-9) strain isolated from a child with Hand-foot-mouth disease (HFMD) in Kunming, Yunnan Province, in 2010. The complete genome sequence of a human echovirus 9 strain, MSH-KM812-2010 was determined. As other human enterovirus, its genome was 7,424 nucleotides (nts) in length and encoded for 2,203 amino acids (aas). In comparison to other human enteroviruses, MSH-KM812-2010 strain had the highest homology with other strains of human echovirus 9 in structural genomic regions and more homologous to other serotypes of B specie than to human echovirus 9 in non-structural genomic regions. Phylogenetic analysis based on complete VP1 gene revealed that the sequences of human echovirus 9 segregated into three distinct clades A, B and C with more than 15. 0% diversity between clades. All Chinese isolates belonged to the same clade. RDP3 and Blast revealed evident recombination in non-structural genomic regions. This report is the first to, describe the complete genome of the human echovirus 9 in China and provide an overview of the diversity of genetic characteristics of a circulating human echovirus 9.

Complete nucleotide sequence of a human coxsackievirus B3 strain A103/KM/09 isolated in Yunnan province, 2009 / 中华流行病学杂志

<p><b>OBJECTIVE</b>To analyze the genetic characterization of the complete genome from a human coxsackievirus B3 strain A103/KM/09 isolated in Yunnan province, 2009.</p><p><b>METHODS</b>By using RT-PCR, all the eight fragments which containing about 1000 nucleotides and covering full viral genome, were sequenced. By using Mega 5.05,Geneious, RDP 3 and SimPlot 3.5.1 software, sequences were aligned with other enterovirus reference sequences. Phylogenetic and recombination analysis were also carried out.</p><p><b>RESULTS</b>The A103/KM/09 isolate genome showed 7389 nucleotides in length , encoding for 2185 amino acids. In the complete genome, the homology of nucleotide and amino acid among the seven coxsackievirus B3 isolates were 81.0%-88.0% and 95.7%-98.0%, respectively. There appeared 81.0% and 95.7% homology when compared with that of Nancy prototype strain. Results from the Phylogenetic analysis showed that the coxsackievirus B3 formed five distinct clades, I-V. Nucleotide divergence rates between clades were 16.2%-24.3% . The A103/KM/09 strain belonged to clade V. Clade V was further divided into four sub-clades,A-D. The nucleotide divergence between sub-clades was 4.3%-11.4%. Putative recombinant event for A103/ KM/09 was detected.</p><p><b>CONCLUSION</b>All coxsackievirus B3 isolates could be divided into five clades, with A103/KM/09 strain belonged to Clade V-D. Evolution of coxsackievirus B3 had occurred in China.</p>

Complete genome sequence characteristics of human enterovirus 71 strain isolated in Yunnan, China / 病毒学报

The complete nucleotide sequence of two human enterovirus 71 strains (KMM09 and KM186-09) isolated in Yunnan,China, were determined by RT-PCR and sequencing. As with other human enteroviruses, the genomes were 7 409 nucleotides (nts) in length and encoded 2 193 aa. Phylogenetic analysis based on VP1 regions revealed that the two isolates belonged to subgenotype C4a. In structural genomic regions, subgenotype C4 was most homologous to other strains of C genotype when compared to other genotypes. In non-structural genomic regions, subgenotype C4 was more homologous to CA16/G10 and other strains of B genotype than to other strains of C genotype. RDP3 and Blast analysis displayed evidence of recombination in non-structural genomic regions between subgenotype B3 and C4, C4 and CA16/G10. The full-length genome of the human enterovirus 71 strains provided an overview of the diversity of genetic characteristics of a circulatinghuman enterovirus 71.

Complete nucleotide sequence of a human coxsackievirus A16 strain KMM08 isolated in Kunming, China in 2008 / 中华流行病学杂志

Objective To describe the genetic characterization of complete genome from a human coxsackievirus A16 (CA16) strain KMM08,isolated in Yunnan,China,in 2008.Methods By using RT-PCR,the seven fragments contained about 1000 nucleotides in the complete genome were sequenced.The sequences were aligned with other enterovirus sequences downloaded from GenBank using Mega 4.1,RDP3 and SimPlot 3.5.1 software.Results As in other human enterovirus,its genome was 7409 nucleotides in length,encoding for 2193 amino acids.KMM08 strain was closely related to other reference strains of B genotype.In the complete genome,the homology of nucleotide and amino acid among the eleven CA16 isolated strains were 79.0％-98.2％ and 94.5％-99.3％,respectively.The rates of homology were 79.1％ and 94.8％ when comparing with that of G10 strains and 78.7％ and 89.0％ comparing with that of BrCr strains,respectively.SZ-HK08-3 strain had high homology when compared to other strains.In different segment of genome,the rates of homology were 97.0％-99.0％ and 98.0％-100.0％ when compared with that of SZ-HK08-3 strains,respectively.The rates of homology were 74.2％-86.9％ and 90.9％-97.0％ when compared with that of G10 strains,respectively and were 65.0％-84.9％ and 71.0％-95.2％ when compared with that of BrCr strains.Data from Phylogenetic analysis showed that KMM08 belong to genotype B.The putative recombinant Tainan-5079-98 was detected positive with RDP3 and SimPlot 3.5.1.Conclusion KMM08 strains isolated in Yunnan in 2008 belonged to B genotype of coxsackivirus A16.The possible occurrence of inter-typic recombination would involve EV71 and CA16.

The analysis of human papillomavirus type 16 E6/E7 genetic variability in Yunnan Province, China / 病毒学报

To investigate E6 and E7 gene variations of human papillomavirus type 16 in Yunnan Province, DNA was extracted from 2000 gynecological outpatient samples. For Human papillomavirus (HPV) genotyping, the genomic DNA was first amplified by the consensus MY09/MY11 primer pair followed by nested PCR with GP5+/GP6+ primers, then the PCR products were subjected to direct DNA sequencing. A total of 20 HPV-16 viral DNAs were identified. E6 and E7 genes of HPV-16 viral DNA were then amplified using E6 and E7 specific primers, the PCR products were purified and sequenced. The results showed that mutations were found at nucleotide position 178 of HPV-16 E6 gene in 10 cases,the mutation rate was 50%; For HPV-16 E7 gene, the mutations were found at nucleotide position 647 in 10 cases; the mutation rate was 50%. Phylogenetic analysis showed that Asian (As) variants of HPV-16 were predominated in Yunnan, China. None of African-1, African-2 variants of HPV-16 was found in this region.

Complete nucleotide sequence of a human echovirus 6 strain KM57-09 isolated in Yunnan,China,in 2009 / 中华流行病学杂志

Objective To analyze the genetic characterization of the complete genome from a human echovirus 6 (Echo6) strain KM57-09 isolated in Yunnan,China,in 2009.Methods Using the RT-PCR,eight fragments containing about 1000 nucleotides which covered the whole viral genome were sequenced.The sequences were aligned with other reference cnterovirus sequences downloaded from the GenBank,using Mega 5.05,RDP 3 and SimPlot 3.5.1 softwares.Results Similar to the other human cntcrovirus,KM57-09 isolate genome appeared to have 7419 nucleotides in length,encoding for 2191 amino acids.In the complete genome,the rates of homology on nucleotide and amino acid among the seven Echo6 isolates were 79.3％-80.2％ and 93.3％ 94.4％,respectively as well as 79.3％ and 93.6％ of the rates of homology when compared with that of D' Amor prototype strain.In different segment of genome.The 2C 3A genome region was most similar to the HN-2-E25 strain,the 5' UTR,VP4,3D and 3' UTR genomc region were most similar to the CoxB5-Henan2010.In the VP1 gene,the rates of homology on nuclcotide and amino acid among the China isolates were 80.0％-96.0％ and 95.8％-99.0％,respectively,and showed 77.6％-96.0％ and 95.2％-99.0％ of the rates on homology when compared to the other Echo6 reference strains isolated from other countries or areas,respectively.Results from phylogenetic analysis showed that the Echo6 formed five distinct groups,A-E.The KM57-09 strain belonged to clade E.The nucleotide divergence between clades was 15.6％-23.3％.The putative recombinant event for KM57-09 was detected with RDP 3,SimPlot 3.5.1 and 3D sequence phylogenetic analysis.Conclusion All the Echo6 isolates could be divided into five clades,the KM57-09 strain belonged to Clade E.The Echo6 strains isolated in China were contributed to several different chains of transmission.

Genetic characteristics on the small hydrophobic protein and hemagglutinin-neuraminidase genes of mumps virus in Yunnan province,China from 2007 to 2009 / 中华流行病学杂志

Objective To analyze genetic characterization of the small hydrophobic and hemagglutinin-neuraminidase genes of mumps virus(MuV)isolated in Yunnan province,China from 2007 to 2009.Methods Fourteen MuV strains were isolated in Yunnan,China from 2007 to 2009.Using RT-PCR,the SH gene fragments contained 316 nucleotides in all strains and HN gene of six strains were sequenced.The sequences were aligned with other mumps virus sequences downloaded from GenBank using Mega 4.1 software.Results Fourteen isolated strains were closely related to other reference strains of F genotypes.In SH gene,the homology of nucleotide and amino acid among the fourteen isolated strains were 98.3%-100.0%and 96.5%-100.0%,respectively,and 92.6%%-99.4%and 87.7%-100.0% of homology when compared with that of strains isolated from other provinces in China,respectively.Wsh1 and Wsh2 strains had less homology when compared to other strains of F genotypes.The fourteen strains had homology of 84.5%-85.1%and 77.2%compared to vaccine strains on nucleotide and amino acid,respectively,and had homology of 83.4%-90.9% and 70.1%-86.0% compared to that of other genotypes.In HN gene,the homology of nucleotide and amino acid among the six isolated strains were 99.3%-99.5% and 99.1%-99.7%,respectively,and also 99.8% and 99.8% of homology respectively when compared to the SP strain in China.All the six strains had homology of 92.4%-93.2% and 95.5%-96.4% when compared to the vaecine strains on nucleotide and amino acid,respectively,and had homology of 94.7%-96.8% and 95.5%-99.1%compared to other genotypes.Conclusion Fourteen strains isolated in Yunnan from 2007 to 2009belonged to F genotype of MuV while the HN gene seemed more conservative than SH gene.

Phylogenetic Analysis of Homologous Proteins Encoded by UL2 and UL23 genes of Herpesviridae / 中国病毒学

The proteins encoded by the Herpesviridae β-gene play a critical role in the replication stage of the virus. In this paper, phylogenetic analyses provided evidence that someβ-gene products, such as UL2 and UL23 from HSV1, have their homologous genes in its family, and also exist in prokaryotic organisms, indicating that these viruses appear to have been assembled over evolutionary time by numerous independent events of horizontal gene transfer.