Rapid screening of Salmonella by loop-mediated isothermal amplification combined with lateral flow dipstick / 上海预防医学

Objective:To establish a specific and sensitive method using loop-mediated isothermal amplification for rapid screening of Salmonella. Methods:The invA gene sequence of Salmonella was downloaded from GenBank. After homology comparison with DNAMAN software， amplification primers were designed in the conserved region， and a LAMP-LFD detection method was established. The reaction system was optimized， and the specificity and sensitivity of the method were verified. Results:The sensitivity of this method to detect Salmonella DNA was up to 1.0×101 copies/μL. The positive rate of anal swabs was the same as that of fluorescent PCR. Meanwhile， LAMP-LFD was easy to operate and did not need expensive instruments. The detection result could be obtained within 30 minutes. Conclusion:The LAMP-LFD method established in this study is rapid， simple， sensitive and specific， which is suitable for rapid screening of Salmonella.

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.

Variations on hemagglutinin gene of Zhejiang measles virus strains and differences with measles strains circulated both at home and abroad / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the variations on hemagglutinin (H) gene of measles virus (MV) in Zhejiang province, and to analyze the differences with strains circulated both at home and abroad.</p><p><b>METHODS</b>In total, 33 MV strains isolated in Zhejiang province between 1999 and 2011 were collected.RNA of the isolated MV strains was extracted and the complete sequences on H gene were amplified using RT-PCR assay. The products were compared with the Chinese vaccine strain Shanghai-191, which was downloaded from GenBank, and other 95 different MV strains from all over the world.</p><p><b>RESULTS</b>33 MV strains, isolated from the throat swab specimens collected from MV patients in Zhejiang province during 1999 to 2001, were used to conduct phylogenetic analysis with MV strains circulated in other areas of China during 1993 to 2007. The phylogenetic tree based on H gene sequences showed that all the Zhejiang MV strains located in H1a cluster, and no apparent time series and geographic restrictions were observed. Compared to the referenced vaccine strain Shanghai-191, the average variation rate on nucleotides and amino acids, and the evolutionary rate of H1a viruses from China during 2003 to 2011 were separately 5.15%, 4.44% and 5.81%, which were higher than the rates of H1a viruses during 1965 to 1993 (4.75%, 3.86% and 5.30%), and the rates of viruses during 1994 to 2002 (4.80%, 4.08% and 5.37%).However, the dn/ds ratios of strains within the three time periods were 0.19,0.21 and 0.23 respectively, which indicated that no evidence of positive selection was found on H1a MV strains during 1993 to 2011. A 24 stable amino acid variation sites on H gene was found between H1a viruses during 2003 to 2011 and the vaccine strain Shanghai-191. The largest variation occurred between vaccine and H1a strains, with 0.053 of the p-distance and 26-28 of amino acid mutations.However, only 15 stable amino acid variations were found between vaccine strain and genotype B3 or D4 strains.In addition, significant differences were found between H1a viruses and genotype B or D viruses, with 0.074 and 0.071 of p-distance and 27-33 of amino acid differences.</p><p><b>CONCLUSION</b>Significant differences were found on H gene between MV strains subtype H1a and vaccine strains and other genotype strains. The variations were enlarged with the time coursing; therefore, the surveillance on variation of Chinese MV strains should be taken into account.</p>

Study on the complete sequence of CA24 variant isolated during the acute hemorrhagic conjunctivitis outbreaks in Zhejiang province during 2002 to 2010 / 中华流行病学杂志

Objective To analyze the genetic characteristics of the complete sequence of coxsackievirus A24 variant(CA24v) isolated from acute hemorrhagic conjunctivitis (AHC) outbreaks in Zhejiang province during 2002 to 2010.Methods Complete sequences of CA24v epidemic strains isolated in different years were amplified under the RT-PCR assay,while the sequences of whole genome,VP1,and 3C region of Zhejiang strains were compared with epidemic strains isolated in other areas of China and abroad.Results The whole genome of Zhejiang CA24v strains isolated in 2002 and 2010 was 7456-7458 bp in length,encoding a polyglutamine protein which containing 2214 amino acid residues.There was a insertion with T on site 97 and 119 within 5' non-coding region between epidemic strain Zhejiang/08/10 and strains isolated in 2002.The rates of amino acid homology among Zhejiang/08/10 and other strains isolated since 2002 were between 94.7％ and 100.0％.Compared with the representative strains circulated within the recent 60 years,the largest average amino acid variations had been occurred on region 2A and 3A,with the ratios as 8.4％ and 7.3％ respectively.The smallest variation happened in region 3D,with the ratio only as 1.9％.The rates of stable amino acid variation on the whole genome between strains isolated since 1987 and 2002 were 38 and 20.P-distance within groups appeared that region 3C was more stable than VP1 of strains isolated in 2002-2010,and the 3D of early strain Jamaica/10628/87 might have had a nature of recombination but not observed on those epidemic strains in recent years.Conclusion Within the evolution of CA24v strains,the time course was more significant than the geographical differences.There had been sporadic epidemics of AHC caused by CA24v in Zhejiang province since 2002.

Molecular evolution of two lineages related to influenza B virus based on HA1 gene / 中华流行病学杂志

Objective To study the evolutionary characteristics and rules of two lineages on influenza B virus.Methods A total of 126 HA1 sequences of strains isolated during 1940 to 2012were downloaded from the GenBank.Time of the most recent common ancestor (TMRCA) and divergence of the two lineages were calculated based on the data from phylogenetic analysis of HA1gene,using Bayesian Markov Chain Monte Carlo (Bayesian-MCMC) and molecular clock method.Results The average amino acid variant ratios were ranged from 5.4％ to 10.2％ within the strains of influenza B virus isolated during 1978 to 2010.Compared with the Victoria-like strains,all Yamagatalike strains showed an amino acid deletion at 163th site,while some of them showing a deletion at position 166.HA1 gene of influenza B virus seemed not have been affected by positive selection except a few sites.The evolutionary average rate on HA1 gene was 2.138 × 10-3 substitutions/site/year (95％HPD:1.833 × 10-3-2.437 × 10-3 substitutions/site/year).The estimated dates for TMRCA of the two lineages of influenza B virus could be dated back to 1971 (95％ HPD:1969-1972),while the divergence times of the two lineages were 1973 (95％ HPD:1971-1974) and 1977 (95％ HPD:1975-1978) respectively.Conclusion Significant differences were found on HA1 gene between earlier and recent identified strains of Victoria and Yamagata lineage.Differences between the two lineages increased and showing the potential of dividing themselves into different subtypes in the future.More attention should be paid to these trends and the related epidemiological significance.

Genomic sequences of human infection of avian-origin influenza A(H7N9)virus in Zhejiang province / 中华流行病学杂志

<p><b>OBJECTIVE</b>To analyze the etiology and genomic sequences of human infection of avian-origin influenza A(H7N9)virus from Zhejiang province.</p><p><b>METHODS</b>Viral RNA was extracted from patients of suspected H7N9 influenza virus infection and real-time RT-PCR was conducted for detection of viral RNA. All 8 segments of influenza virus were amplified by one-step RT-PCR and genomic sequences were assembled using the sequencing data. All the currently available HA and NA genes of the novel H7N9 virus, some other HAs from H7 subtype and NAs from N9 subtype were downloaded from public database for phylogenetic analysis, using the Mega 5.1 software. Mutations and variations were analyzed, using the genomic sequence data.</p><p><b>RESULTS</b>Reactions for influenza type A, subtype H7 and subtype N9 were all positive and all the genomic fragments were amplified for sequencing. After alignment, sequences were subjected for phylogenetic analysis. The results revealed highest homology with A/duck/Zhejiang/12/2011(H7N3)in HA gene and with A/wild bird/Korea/ A14/2011(H7N9)in NA gene of the H7N9 influenza virus. All 6 genes coding for internal proteins shared highest identities with H9N2 avian influenza which were circulated in the Chinese mainland, in the last two years. The sequenced virus showed Q226L mutation in HA protein, but E627K was not presented in PB2 protein of this virus. The E627K mutation was shared by all the other novel H7N9 viruses resulted in human infections through analysis on the currently available sequences.</p><p><b>CONCLUSION</b>Using the clinical samples, both detection of the viral genes and amplification of all 8 segments of the novel H7N9 influenza virus were accomplished. High homology of the novel H7N9 influenza viruses was observed by phylogenetic test, using the currently available sequences. The virus showed Q226L mutation on HA protein but E627K did not present on PB2 protein of this virus.</p>

Study on the genome sequence of measles viruses circulated in Zhejiang province during 1999 to 2011 / 中华流行病学杂志

Objective To study the genetic variations between measles vaccinc strain S191 and strains that circulated in Zhejiang province causing the epidemics during 1999 to 20 1 1.Methods Complete sequence of the nine Zhejiang measles strains were amplified by RT-PCR assay.Products were sequenced and the obtained sequences were aligned and analyzed with vaccine strains S191 and the major epidemic strains isolated in foreign countries.Results The homology of amino acid among the nine Zhcjiang strains were 98.77％-99.89％.The strains were not affected by positive selection and the variations on each gene were still in random drift.Compared to vaccine strain S191,there were 135 to 159 amino acid changes in Zhejiang measles virus,in which 113 points were common variable positions,resulting in mutations on five glycosylation sites.At the nucleotide level,the biggest differences between the Zhejiang strains and the vaccine strain S191 were found on N gcne,with the average divergent ratio as 5.5％,while the biggest one was P protein,in the amino acid level,with the average mutation rate as 7.7％.In addition,with the complete genome sequences,the genetic distance between Zhcjiang epidemic strains and vaccine strains was greater than the distances between epidenic strains of genotype D4,B3 and vaccine strains (t=-9.76,P＜0.05；t=-12.39,P＜0.05).Conclusion There were significant differcnccs found in the each of the genes between Zhejiang epidemic strains and the vaccine strain S191.The differences between the current vaccine strains and H genotypc epidemic strains were much larger than the differences between the vaccine and the forcign epidemic strains (genotype D4,B3).Therefore,wc should pay close attcntion to this trend,and to develop candidates for the dcvclopnent of vaccines,as early as possible.

Comparative analysis on the complete genome sequence of mumps epidemic strain and mumps vaccine strain S79 isolated in Zhejiang province, China between year 2005 and 2010 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To compare the differences in the complete genome sequence between mumps epidemic strain and mumps vaccine strain S79 isolated in Zhejiang province.</p><p><b>METHODS</b>A total of 4 mumps epidemic strains, which were separated from Zhejiang province during 2005 to 2010, named as ZJ05-1, ZJ06-3, ZJ08-1 and ZJ10-1 were selected in the study. The complete genome sequences were amplified using RT-PCR. The genetic differences between vaccine strain S79 and other genotype strains were compared; while the genetic-distance was calculated and the evolution was analyzed.</p><p><b>RESULTS</b>The biggest difference between the 4 epidemic strains and the vaccine strain S79 was found on the membrane associated protein gene; whose average nucleotide differential number was 42.5 +/- 3.0 and the average variant ratio was 13.6%; while the mean amino acid differential number was 12.8 +/- 1.5 and the average variant ratio was 22.4%. The smallest difference among the 4 epidemic strains and the vaccine strain was found in stromatin genes, whose average nucleotide differential number was 73.8 +/- 2.5 and the average variant ratio was 5.9%; while the mean amino acid differential number was 3.0 +/- 0.8 and the average variant ratio was 0.8%. The dn/ds value of the stromatin genes of the 4 epidemic strains reached the highest, as 0.6526; but without any positive pressure (dn/ds < 1, chi2 = 0.87, P > 0.05). There were mutations happened on the known antigen epitope, as 8th amino acid of membrane associated protein genes and on the 336th and 356th amino acid of hemagglutinin/neuraminidase proteins. Compared with the vaccine strain, the glycosylation sites of ZJ05-1, ZJ06-3, ZJ08-1 and ZJ10-1 increased 1, 1, 2 and 2 respectively. The complete amino acid sequence of all strains showed that there were 17 characteristic sites found on the genotype-F mumps strain. Within the complete genome, the genetic-distance between epidemic strains and vaccine strains in Zhejiang province (0.071) was significantly larger than the genetic-distance between strains in Yunnan province (0.013); the difference showing statistical significance (t = 4.14, P < 0.05). Except nucleocapsid protein genes, all the genes shared similar evolution tree.</p><p><b>CONCLUSION</b>There were significant differences found in the genes between mumps epidemic strain and mumps vaccine in Zhejiang province.</p>

Molecular epidemiological study on rubella virus strains isolated in Zhejiang province, China,2005-2010 / 中华流行病学杂志

Objective To analyze the molecular epidemiological characteristic of rubella virus strains isolated in Zhejiang province from 2005 to 2010, to provide basic data for rubella prevention and control. Methods Rubella virus strains were isolated on Vero cells from the suspected patients' specimens collected in Zhejiang province during 2005 to 2010. Partial fragments of the structural gene of Zhejiang rubella strains were amplified, using nested reverse transcription-polymerase chain reaction (RT-PCR). The amplified products were sequences and analyzed.Results In total, 7 rubella strains were isolated from 52 clinical specimens, of which six were classified as genotype I E and only one was characterized as genotype 2B. In the phylogenetic tree, the Zhejiang IE genotype rubella strains were located in the same branches with Hongkong or Hainan isolates respectively, but the Zhejiang 2B genotype strain were located in the same branch with oversea strain BuenosAires.ARG/46.08. Through p-distance analysis, results also showed that the Zhejiang 2B genotype strain was closer to the 2B strains isolated from overseas (0.011 ) than those strains from other provinces of China (0.023). Compared with Chinese vaccine strain BRD Ⅱ, the homology on three structural genes was C＞E2＞E1, but the homology of deduced amino acid sequence was E1 ＞ C ＞ E2, with corresponding 3,11 and 23 amino acid mutations. There was only one amino acid on E1 gene with entropy value higher than 0.600, but seven sites on E2 gene with entropy value appeared higher than 0.600 and one with entropy value higher than 1.000. Conclusion Two genotypes of rubella virus had circulated in Zhejiang province during 2005 to 2010. Genotype IE appeared to be the predominant genotype and 2B being an imported one. Amino acid sequence of E1 gene from Zhejiang rubella strains was comparatively conserved, but E2 gene was hypervariable.Study on rubella virus E2 and C gene should be conducted in the epidemiological surveillance program of rubella.

Study on the mutations within the whole genome of influenza virus subtype A/H3N2 strains circulated in Zhejiang province from 1998 to 2009 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To analyze the consistency of evolution condition between HA gene and the whole genome of influenza virus subtype A/H3N2 strains isolated in Zhejiang province from 1998 to 2009, and to study the potential antigenic region on the whole genome.</p><p><b>METHODS</b>The sequences of whole genome of 19 Zhejiang influenza virus isolates circulated from 1998 to 2009, which conserved by influenza laboratory of Zhejiang Provincial Centre for Disease Prevention and Control, were amplified using RT-PCR assays. The obtained sequences were used to conduct phylogenetic analysis with 10 contemporaneous vaccine strains. Three methods, including comparison of the amino acid substitutions, calculation of the entropy value and the filtering of positive selection sites, were used to confirm the mutable sites on each gene.</p><p><b>RESULTS</b>The whole genome of influenza virus subtype A/H3N2 was 4466 amino acids in length, with 137 stable mutations. The 144, 158 aa of HA gene mutate four and three times respectively; 93, 143, 307, 370, 372 aa of NA gene and 450 aa of NP gene mutate twice, and there were 29% (12/41) and 77% (24/31) mutations of HA and NA genes occurred on the non-epitope regions respectively. Analysis of the entropy value suggest that many amino acid sites on the non-epitope regions were prone to mutation, including 3, 225, 361 aa of HA gene; 93, 143, 147, 150, 372 aa of NA gene; 113, 576, 586 aa of PB1 gene; 101,256, 382, 421, 437 aa of PA; 377, 450 aa of NP gene; 218 aa of M1 gene and 31 aa of M2 gene.</p><p><b>CONCLUSION</b>Based on the whole genome of influenza virus subtype A/H3N2 strains isolated in Zhejiang province in 1998 to 2009, there may be several unknown or new antigen sites existing on the non-epitope regions of HA and NA genes and parts of internal genes. The phylogenetic tree constructed based on the complete sequence was more comprehensive than on the HA gene to reflect the genetic relationship and law of evolution among the influenza virus strains.</p>

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.

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.

Genetic variation of the hemagglutinin and neuraminidase of influenza B viruses isolated in Zhejiang province during 1999-2010 / 中华流行病学杂志

Objective To Characterize the genetic diversity of hemagglutinin(HA)and neuraminidase(NA)of influenza B viruses isolated in Zhejiang province during 1999-2010.Methods Respiratory specimens were collected from patients with flu-like syndrome during the influenza outbreaks or from the hospitals which carrying out influenza surveillance project in Zhejiang province.Samples were detected by real-time RT-PCR and isolated for influenza virus.HA1 and NA genes of influenza B virus isolates were amplified and sequenced.Phylogenetic comparison and genetic diversity analysis were performed using the bioinformation software.Results A total of 34 influenza B viruses were evolved in this study including Victoria-like and Yamagata-like strains according to the results of the HI test.The mutation rate of Victoria-like HA1 gene was 4.5% and Yamagata-like HA1 gene was 3.4%,respectively.The Victoria-like influenza B isolates had appeared to be all re-assortants having a Victoria lincage HA and Yamagata lineage NA since 2004.The predominant type of influenza virus isolates in 2010 was also influenza B virus after the H1N1 flu pandemic in Zhejiang province.The isolated strains were antigenicaily and genetically similar to B/Brisbane/60/2008--the vaccine strain proposed for 2009-2010.Many difierences of HA1 and NA amino acids existed in the current isolates when compared to previous influenza B strains.Conclusion Significant diversity was generated among influcnza B virus isolated from 1999 to 2010 in Zhejiang province.Genetic re-assortment and antigenic drift seemed the main evolutionary mechanism on influenza B virus.

Genetic variation of phosphoprotein (P) gene from measles epidemic strains in Zhejiang province / 中华流行病学杂志

Objective To investigate the genetic characteristics and variation within the phosphoprotein (P) gene of measles epidemic strains circulated in Zhejiang province. Methods The whole sequence of P gene of the epidemic strains related to Zhejiang Measles virus during 1999 to 2008 was amplified, using the RT-PCR Assay. PCR products were sequenced and compared with the sequences of measles vaccine and other epidemic strains. Results Totally, 1524 nucleotides were sequenced from each epidemic strain and 507 amino acids were derived correspondingly. Compared with the vaccine strain, there were 59-75 nucleotides (divergent ratios were 3.9%-4.9%) mutated from the epidemic strains, which were isolated during 1999 to 2008 and causing mutation on 36-42 amino acid (divergent ratios were 7.1%-8.3% ). Changes were also observed on the secondary structure. The phylogenetic tree, constructed based on the sequences of P gene, was similar to that based on the N gene, recommended by WHO. In addition, the average divergent ratio of P protein was greater than the ratio occurred on the N and H genes. Conclusion The variation within the P gene between the vaccine and epidemic strains circulated in Zhejiang province during 1999 to 2008 was significant.

Simultaneous detection of measles and rubella virus by multiplex real-time RT-PCR with an internal control / 病毒学报

Measles and rubella virus cause fever rash diseases that are uneasy to differentiate clinically from each other. Specific primers and fluorescence-labeled probes were designed, and a multiplex Real-time RT-PCR with an internal control was developed to simultaneously identify the measles and rubella virus. The multiplex Real-time RT-PCR assay was specific and no false positive or false negative results were found. The sensitivity of the assay was 0.1TCID50/mL and 1TCID50/mL for measles and rubella virus respectively. Analysis with 0.1-10(3)TCID50/mL measles or rubella virus samples demonstrated high validity and reproducibility with the coefficient of variability(CV) of below 0.9% for both measles and rubella virus. Using ribonuclease P (RNase P) as internal false negative control, the developed multiplex Real-time RT-PCR assay is suitable for rapid clinical diagnosis of measles and rubella virus.

Study on the genome sequence of influenza virus subtype A/H3N2 strains circulated in Zhejiang province during 1998 to 2009 / 中华流行病学杂志

Objective To analyze the relationship between influenza epidemic and genetic characteristic on the whole genome of influenza virus subtype A/H3N2 strains isolated in Zhejiang province during 1998 to 2009. Methods All of the eight genes from the 19 Zhejiang influenza virus isolates, circulated during 1998 to 2009, were amplified by RT-PCR and sequenced. The obtained sequences were aligned and analyzed with the vaccine strains being used in the last 10 years.Results The highest mutation happened within HA and NA genes and the amino acid divergent ratios were 13.98% and 10.00%. Amongst the six internal proteins, the amino acid divergent ratios of NP, M2 and NS1 were 6.43%, 6.19% and 3.48% respectively, and the others were lower than 3%.Other than the HA and NA genes, mutations were also observed on six internal genes of the strains isolated in those years when the influenza virus subtype A/H3N2 was widely circulating.Additionally, there had been an obvious genetic lag between vaccine strains recommended by WHO and the contemporary Zhejiang epidemic strains for many years. Conclusion Besides on HA and NA genes, surveillance programs should also be covered mutations regarding the internal genes of influenza virus subtype A/H3N2 strains, in order to provide important information for forecasting and warning of a new round of influenza epidemic.

Traceability of the A/H3N2 virus borne influenza pandemic in Zhejiang province,1998 / 中华流行病学杂志

Objective To trace back to the influenza pandemic caused by A/H3N2 virus happened in Zhejiang province,1998.Methods The whole genome of three isolates related to Zhejiang influenza virus was amplified through RT-PCR,and the identified sequences were aligned with the sequences downloaded from GenBank of the H3N2 strains which were circulating in other regions during 1995 to 1998.The crossing HAI titers of the reference strains were measured by HAI test and antigenic ratios were calculated.Results The Phylogenetic tree,constructed based on HA sequence showed that the dominant strains A/Zhejiang/11/98 and A/Zhejiang/18/98 were significant different from the isolates circulated in other regions during 1995 to 1996 and the strains isolated in the mainland of China,in 1997.Although the A/Zhejiang/11/98 and A/Zhejiang/18/98 strains were distributed in the same cluster with A/Sydney/5/97,the two strains were closer to the epidemic strains isolated in Hong Kong and New York in the later part of 1997.Based on HAI,NA and MP genes,A/Zhejiang/18/98 seemed to be the closest one to the Hong Kong epidemic strains,and the genetic distances between A/Zhejiang/18/98 and New York strains were shorter than that with A/Sydney/5/97 based on PA,HA and NS genes.There were only 1-3 amino acid differences between A/Zhejiang/18/98 and Hong Kong or New York strains,whereas 7 amino acid differences with A/Sydney/5/97,in which three were located in the antigenic determinant regions.Data from the crossing HAI test showed that the antigenic ratio between A/Zhejiang/18/98 and A/Sydney/5/97 had reached 2.0,indicating the antigenic difference to a certain extent.Additionally,the onset of the influenza epidemic during 1997 to 1998 also suggested the possible route of transmission related to this H3N2 virus.Conclusion The influenza pandemic occurred in Zhejiang province in 1998 was possibly caused by the importation of a newly identified H3N2 influenza variant via Hong Kong and New York in late 1997.

Comparative analysis of measles genome between vaccine strain and wild-type strain in Zhejiang province of China / 中华预防医学杂志

<p><b>OBJECTIVE</b>To explore the distinction between wild-type strain MVi/Zhejiang, CHN/7.05/4 and vaccine strain Shanghai-191 at genome level.</p><p><b>METHODS</b>After sequencing of measles wild-stain MVi/Zhejiang. CHN/7.05/4, the distinction between the wild-type strain and the vaccine strain was analysed by MEGA 3.1 software at genome level, and the antigen variation was studied by means of combining the epidemiological data.</p><p><b>RESULTS</b>There were 822 nucleotide differences (5.17%) and 161 amino acid differences between these two strains, including three glycosylation sites variation found. Meanwhile, the antigen ratio between wild-type strain and vaccine strain was found to be 5.66.</p><p><b>CONCLUSION</b>There should be certain differences between the contemporary wild-type strain MVi/Zhejiang, CHN/7.05/4 and vaccine strain Shanghai-191 at genome level, and the protective effects of measles vaccine should be studied further.</p>

Study on the genetic characteristics of the avian influenza virus strain A/Zhejiang/16/2006 / 中华流行病学杂志

Objective To study the genetic characteristics of avian influenza virus strain A/Zhejiang/16/2006 which was isolated from the case first reported in Zhejiang province.Methods Complete genome of A/Zhejiang/16/2006 including eight segments were sequenced and compared on the genetic homogeneity with sequences of the similar strains provided through domestic and overseas sources.Results There were 11 amino acids showing differences on HA between A/Zhejiang/16/2006 and the H5N1 isolates of neighboring countries,but these differences had not affected the stability of glycosylation sites.In the NA region,20 amino acids located in the 49th to 68th position were found absent in the isolates obtained after 1997.Eight segments of H5N1 isolates,circulating in the mainland of China in the recent years,formed a separate branch compared to the strains in neighboring countries and there was also obviously different from the strains isolated in Hong Kong and Guangzhou in 1996 and 1997.However,several Chinese strains were close to the Hong Kong strains isolated in 1997 but diffferent from the current strains in the phylogenetic tree.Conclusion The influenza virus strain A/Zhejiang/16/2006 formed a separate branch with the strains isolated in the mainland of China in the past years but it presented an obvious difference with the isolates from the neighboring countries.

Molecular characteristics and its evolution of the complete genome of avian influenza H5N1 virus isolated in Zhejiang province from 2002 to 2006 / 中华流行病学杂志

Objective To analyze the molecular characteristics and evolution reassortment of the complete genome of avian influenza H5N1 virus isolated in Zhejiang province in recent years. Methods Complete genomes of avian influenza H5N1 viruses isolated in Zhejiang province from 2002 to 2006 were sequenced. Molecular and evolution reassortment characterization of these virus strains were analyzed by Mega 3.0 bioinformatics software. Results Through study on the HA genes from all these strains,our data revealed that there were multiple basic amino acids at the cleavage site, which was typical for HPAIV.Compared with Gs/Guangdong/1/96,all these strains had a 20 amino acid deletion in the stalk of the NA, except for Dk/Zhejiang/2/02 and Ck/Zhejiang/8/03. Results from phylogenetic analysis showed that from2002 to 2003 the H5N1 viruses belonged to the genotype of B,W,X,Y,Z, and other genotypes were prevailed in corresponding year. However, different gene fragments of several strains belonged to different genotypes. Conclusion Recombinant might be widespread among the poultry in Zhejiang province. The genetic genes of viruses isolated after 2005, the Ck/Zhejiang/24/05 and Zhejiang/16/06 strains were almost all originated from the FJ-like genotype stably.