A study on the development of a triplex real -time RT -PCR for simultaneous detection of enteroviruses and differentiation of EV 71 and CA 16 / 预防医学

Objective To develop a multiplex real -time RT -PCR assay for simultaneous detection of enteroviruses and differentiation of EV71 and CA16.Methods Specific primers and probes were designed for enteroviruses,EV71 and CA16.The probes labeled with various fluorescent reporter dyes,and a triplex real -time RT -PCR technique was developed to simultaneously detect these viruses.A total of 91 clinical specimens with suspected HFMD were analyzed by this method.Results This assay could simultaneously detect enterovirus and differentiation of EV71 and CA16,and the sensitivity of the assay was up to 0.1 TCID50 /mL,and only need 2 to 3 hours for completing the detection.A total of 91 clinical specimens were detected by this assay in 28 of the 91(30.77%)specimens contained EV71,9 of the 91(9.89%) contained CA16,and 5 of the 91 (5.49%)contained other enteroviruses.Conclusion This assay would be a useful molecular diagnostic tool for large -scale screening of clinical samples,especially at the peroid of HFMD outbreaks.

Study on the etiological and molecular characteristics of aseptic meningitis epidemic in Zhejiang Province in 2002-2004 / 病毒学报

In order to confirm the cause of the outbreak of aseptic meningitis in Zhejiang Province in 2002-2004, trace the pathogen and analyze the molecular characteristics, 271 cerebrospinal fluid (CSF) and faeces specimens were collected from suspected patients. The virus strains from the specimens were isolated with RD and Hep-2 cell lines. The VP1 and VP4/VP2 genes of the isolated viruses were sequenced, and their phylogenetic and homology trees were also constructed. Of the total 271 samples, 78 Echovirus type 30 (E30) strains were isolated. All of the complete VP1 genes in 31 sequenced virus isolates of E30 were composed of 876 nt without any insertion or deletion, encoding 292 amino acids (aa). The identity of nucleotide and amino acid in VP1 gene were 84.7%-86.3% and 92.1%-94.2% between the 31 Zhejiang strains and the prototype strain Bastianni of E30, and 87.1%-99.4% and 96.2%-100% among the 31 Zhejiang strains, respectively. The Zhejiang strains of E30 in the phylogenetic tree of the VP1 gene were attributed into two branches of the G and H genotype, respectively. In G genotype, the Shangdong and Jiangsu E30 strains in 2003 among domestic strains and Ukraine E30 strain in 1999 among overseas strains had maximum similarity with the Zhejiang strains, while H genotype had maximum similarity with the Korea E30 strains in 2008. The phylogenetic tree of the VP4/VP2 genes was similar to that of VP1 gene. The results indicated that the outbreak of aseptic meningitis in Zhejiang Provinec in 2002-2004 was caused by the G and H genotypes of E30 strains existing simultaneously. The H genotype was a new variant strain, which was first isolated in Zhejiang Province in 2002.

Molecular characteristics of noroviruses causing outbreaks of acute gastroenteritis in Huzhou / 病毒学报

To study the molecular characteristics of Noroviruses causing outbreaks of acute gastroenteritis in Huzhou. During April 2008 and February 2009, fecal specimens of patients collected from 2 outbreaks of acute gastroenteritis were tested for Norovirus by real-time RT-PCR. Partial sequence of RNA dependent RNA polymerase(RdRp) of the positive samples were amplified by RT-PCR, the PCR products were then purified, sequenced and phylogenetic analysis was conducted. Both genogroup II (GII) and genogroup I (GI) noroviruses were detected in 2 outbreaks. Phylogenetic analysis revealed that two of the GI norovirus strains isolated from 2008 belonged to genotype GI/2 and one of the GI Norovirus strain isolated from 2009 belonged to genotype GI/3. The other GIIú norovirus strains isolated from 2009 had high nucleotide identity with GIIb genotype that had been reported frequently in European countries during 2000 and 2001 and in Asian countries recently. These results suggested that the epidemic strains of norovirus isolated in Huzhou had a high degree of genetic diversity and prevalent genotypes at different times were also different. To our knowledge this is the first report of detecting GIIb variant in outbreaks of acute gastroenteritis in China.

Study on the pathological and molecular characteristics of AHC epidemic in Zhejiang Province in 2010 / 病毒学报

To identify and trace the pathogen of acute hemorrhagic conjunctivitis (AHC) epidemic in Zhejiang Province in 2010. Viral nucleic acid of Enterovirus (EV) and Coxsackievirus A24 variant (CA24v) were directly detected by real-time RT-PCR from the conjunctival swab collected from suspected patients. The virus was isolated from the swab samples using Hep-2 cell. The viral RNAs were extracted from the isolated viruses and followed by RT-PCR to amplify VP1 gene and 3C protease region(3C). The amplified fragments were sequenced and phylogenetic trees were also constructed. Eight out of 13 swab samples from suspected patients were both positive for EV and CA24v RNA (61.5%), 6 CA24v strains were isolated (46.2%). The complete VP1 genes of CA24v in 4 sequenced virus strains were 915 nt in length and the complete 3C genes were 549 nt in length. All VP1 and 3C genes were confirmed without any insertion or deletion. The identity of nucleotide and amino acid in 3C between the 2010 isolated strains and the prototype strain EH24/70 were 85.2%-85.8% and 96.2%-96.7%, and that between the 2010 Zhejiang strains and the Zhejiang,Yunnan and Guangdong CA24v strains isolated between 2007-2008 were 93.4%-93.8% and 96.7%-97.3%, respectively. The phylogenetic tree of 3C indicated that the isolated CA24v viruses of Zhejiang in 2010 located in the CA24v IV genotype cluster 4 (GIV-C4) and all the VP1 genes located in the human Enterovirus C (EV-C) CA24v. These findings indicated that AHC epidemic in Zhejiang Province in 2010 was caused by CA24v GIV-C4 viruses and they most likely evolved from CA24v viruses circulating locally in external environment from 2002.

Study on the pathological and molecular characteristics of viral meningitis outbreaks in Linhai county, Zhejiang province, 2004 / 中华流行病学杂志

Objective In order to confirm the causes of viral meningitis outbreaks in Linhai county,Zhejiang province in 2004,and to analyze the relationship between hereditary variation and evolution of the pathogen.Methods 60 cerebrospinal fluid(CSF)specimens were collected from the suspected patients.Virus strains from the specimens were isolated with RD and Hep-2 cell lines,and identified through neutralization test.VP1 and VP4/VP2 genes of the isolated viruses were sequenced.Both phylogcnetic and homological trees were also constructed.Results 19 Echovirus type 30(E30)strains were isolated from 60 CSFs,in which E30 accounted for 31.7％.All of the complete VP1 genes in 4 sequenced virus isolates of E30 were composed of 876 nt,encoding 292 amino acids(aa).The identity of nucleotide and amino acid in VP1 gene were 82.4％-84.1％ and 93.5％-94.2％ between the 4 Linhai strains and the prototype strain Bastianni of E30,were 87.1％-99.9％ and 97.9％-100.0％ among the 4 virus strains of E30 from Linhai,respectively.The 4 Linhai strains could be classified into two classes.The diversity of nt and aa was minimal in the same class but obvious between the two classes,with the range of diversities as 12.9％ and 2.1％,respectively.The Linhai E30 strains had maximum similarity with the Zhejiang E30 strains in 2002-2003.The 4 Linhai strains of E30 in the phylogenetic tree of the VP1 gene were attributed into two branches of the G and H genotype,respectively.The G branch also included the E30 strains from Zhejiang,Jiangsu and Shangdong in 2003,while the H branch including E30 strains from Zhuji,Zhejiang in 2002.The phylogenetic tree of VP4/VP2 genes was similar to that of VP1 gene.Conclusion The outbreak of viral meningitis in Linhai county in 2004 was caused by the two classes of E30 strains with G and H genotype existed simultaneously.The Linhai E30 strains had maximum genetic relations to the Zhejiang,Jiangsu and Shangdong strains of E30.The H genotype was inferred to be a new variant strain,which was first isolated in Zhejiang province in 2002.

Molecular characteristics of norovirus in 3 outbreak-episodes of gastroenteritis in Zhejiang from 2008 to 2009 / 中华流行病学杂志

Objective To study the molecular characteristic of norovirus in 3 outbreaks of gastroenteritis in Zhejiang province. Methods During January 2008 and December 2009, fecal specimens of patients were collected from 3 outbreaks of acute viral gastroenteritis. Noroviruses were detected by Real-time RT-PCR. Part of the positive samples were randomly selected and detected by RT-PCR. PCR products were sequenced. Sequence analysis was undertaken based on partial sequence of RNA dependent RNA polymerase(RdRp)and capsid protein gene. Some positive samples were amplified by 3' RACE(rapid amplification of cDNA 3' ends), 3200 bp in length. The exact whole ORF2, ORF3 and 3' untranslation regions(UTR)gene of norovims were identified. Results There were in total 3 outbreaks of viral gastroenteritis caused by norovirus being reported. A total of 62 stools were obtained from cases with acute gastroentefitis. Noroviruses were detected in 41 cases including 27 strains of genogroup Ⅰ norovirus and 9 strains of genogroup Ⅱ norovirus, 5 strains of genogroup Ⅰ + Ⅱ norovirus. Four genotypes including G Ⅰ .8, G Ⅱ .b, G Ⅰ .2/0 Ⅰ .6 recombination together with co-infection of G Ⅰ .8 and G Ⅱ .b were detected. Conclusion Norovirus was confirmed as the major cause of outbreaks of viral gastroenteritis in Zhejiang province and multiple genotype of norovirus were identified from the outbreaks. It was the first time to have found a recombinant of G Ⅰ .6 capsid and G Ⅰ .2 polymerase norovims as well as the co-infection of G Ⅰ .8 and G Ⅱ .b norovirus in the same sample.

Molecular epidemiology of Norovirus in outbreaks of gastroenteritis in Zhejiang from 2006 to 2007 / 中华流行病学杂志

Objecfive To study the molecular epidemioiogical characteristics of Norovirus gastroenteritis outbreaks in Zhejiang.Methods During January 2006 and December 2007.fecal specimens of patients collected from outbreaks of acute viml gastroenteritis were tcsted for Norovirus.Epidemiological data were also collected.Noroviruses were detected by a reverse transcription polymerase chain reaction(RT-PeR)and Real.time RT-PCR.Some positive samples were randomly selected and Rrr-PCR products were sequenced.Comparing to the nucleotide sequences of norovirus genotype Ⅰ,Ⅱ reference strains from GenBank,sequence analysis was undertaken based on partial sequence of RNA dependent RNA polymerase(RdRp)and capsid protein(VPI)gene.Results 5 Outbreaks of viral gastroenteritis caused bv Norovirus were reported.A total of 63 stools were obtained from cases with acute gastroenteritis.Noroviruses alone were detected in 45 cases and the illness appeared in autumn.Phylogenetic analysis revealed that Norovirus belonged to G Ⅱ/G Ⅱ 4 type.The strains isolated from Zhejiang were almost identical on G Ⅱ/4 variants that causing epidemics in Beijing and in the Netherlands with the homology of 99.7%and 98.5%-99.O%respectively.Phylogenetic analysis revealed that the isolates were located at the same branch as the norovirus G Ⅱ/4 variants found in Beijing and Netherlands.Conclusion Norovirus iS a major cause of outbreaks of viral gastroenterifis in Zhejiang province.GenogroupⅡ/4 variants viruses were the prevalent strains.

Isolation and sequencing of VP1 region of enterovirus 71 strains in Zhejiang, China / 中华流行病学杂志

<p><b>OBJECTIVE</b>To study the gene characterization of enterovirus 71 (EV71) virus strains isolated from clinical specimens of children with hand-foot-and-mouth disease (HFMD) in Zhejiang province.</p><p><b>METHODS</b>Virus were isolated from clinical samples including stool, throat swab and vesicle from patients with HFMD. The EV71 isolates were identified by microneutralization assay and reverse transcriptase PCR (RT-PCR) with specific primer pair for VP1 genes of EV71. Complete VP1 gene sequences (891 nucleotides) for recent 6 EV71 isolates were determined and compared with that of A, B, C genotype reference EV71 strains and 11 EV71 China isolates available from GeneBank by homogeneity and phylogenetic tree analyses.</p><p><b>RESULTS</b>9 strains of EV were isolated from 14 clinical specimens. Data from microneutralization and RT-PCR results indicated that all the strains belong to EV71. The nucleotide and amino acid homogeneity of these 6 Zhejiang strains with the representative isolates of A and B genotypes were 82.9%-85.5% and 94.9%-98.0% respectively; with the representative isolates of C were 89.2%-94.1% and 97.0%-99.0% respectively. There were 91.0%-92.2%, 90.2%-90.3%, 89.2%-89.5%, 96.7%-96.9% nucleotide, homology with representative strains of C1, C2, C3,C4 subgenotypes of EV71. The nucleotide homogeneity of these 6 EV71 isolated strains with 9 previously isolated Chinese strains appeared to be 93.8%-97.1%. These 6 EV71 isolated strains were within genotype C subgenogroup C4 in the phylogenetic tree.</p><p><b>CONCLUSION</b>The recently identified EV71 isolates in Zhejiang province belonged to subgenogroup C4.</p>