Serological and molecular epidemiology of Japanese Encephalitis in Zhejiang, China, 2015-2018.

BACKGROUND: Shifts have occurred in the epidemiological characteristics of Japanese encephalitis (JE), extending from the molecular level to the population level. The aim of this study was to investigate the seroprevalence of JE neutralizing antibodies in healthy populations from different age groups in Zhejiang Province, and to conduct mosquito monitoring to evaluate the infection rate of Japanese encephalitis virus (JEV) among vectors, as well as the molecular characteristics of the E gene of isolated JEV strains. METHODOLOGY/PRINCIPAL FINDINGS: A total of 1190 sera samples were screened by a microseroneutralization test, including 429 infants (28d-11m) and 761 participants (2y-82y). For those under 1 year old, the geometric mean titers (GMTs) of the JE neutralizing antibody was 9.49 at birth and significantly declined as the age of month increased (r = -0.225, P<0.001). For those above 1-year old, seropositive proportions were higher in subjects aged 1-3 years old as well as ≥25 years old (65%-75%), and relatively lower in subjects aged between 4-25 years old (22%-55%). Four or more years after the 2nd dose of JEV-L (first dose administered at 8 months and the second at 2 years of age), the seropositive proportion decreased to 32.5%, and GMTs decreased to 8.08. A total of 87,201 mosquitoes were collected from livestock sheds in 6 surveillance sites during 2015-2018, from which 139 E gene sequences were successfully amplified. The annual infection rate according to bias-corrected maximum likelihood estimation of JEV in Culex tritaeniorhynchus was 1.56, 2.36, 5.65 and 1.77 per 1000, respectively. JEV strains isolated during 2015-2018 all belonged to Genotype I. The E gene of amplified 139 samples differed from the JEV-L vaccine strain at fourteen amino acid residues, including the eight key residues related to virulence and virus attenuation. No divergence was observed at the sites related to antigenicity. CONCLUSIONS/SIGNIFICANCE: Zhejiang Province was at a high risk of JE exposure due to relatively lower neutralizing antibody levels among the younger-aged population and higher infection rates of JEV in mosquitoes. Continuous, timely and full coverage of JE vaccination are essential, as well as the separation of human living areas and livestock shed areas. In addition, annual mosquito surveillance and periodic antibody level monitoring are important for providing evidence for improvement in JE vaccines and immunization schedules.

Sero-Molecular Epidemiology of Japanese Encephalitis in Zhejiang, an Eastern Province of China.

BACKGROUND: Sporadic Japanese encephalitis (JE) cases still have been reported in Zhejiang Province in recent years, and concerns about vaccine cross-protection and population-level immunity have been raised off and on within the public health sphere. Genotype I (GI) has replaced GIII as the dominant genotype in Asian countries during the past few decades, which caused considerable concerns about the potential change of epidemiology characteristics and the vaccine effectiveness. The aim of this study was to investigate the prevalence of JE neutralizing antibody and its waning antibody trend after live attenuated JE vaccine immunization. Additionally, this study analyzed the molecular characteristics of the E gene of Zhejiang Japanese encephalitis virus (JEV) strains, and established genetic relationships with other JEV strains. METHODOLOGY/PRINCIPAL FINDINGS: A total of 570 serum specimens were sampled from community population aged from 0 to 92 years old in Xianju county of Zhejiang Province in 2013-2014. Microseroneutralization test results were analyzed to estimate the population immunity and to observe antibody dynamics in vaccinated children. E genes of 28 JEV strains isolated in Zhejiang Province were sequenced for phylogenetic tree construction and molecular characteristics analysis with other selected strains. Positive JE neutralizing antibody rates were higher in residents ≥35 years old (81%~98%) and lower in residents <35 years old (0~57%). 7 or 8 years after the 2nd live attenuated vaccine dose, the antibodies against for 4 different strains with microseroneutralization test were decreased by 55%~73% on seropositive rates and by 25%~38% on GMTs respectively. JEV strains isolated in recent years were all grouped into GI, while those isolated in the 1980s belonged to GIII. On important amino acid sites related to antigenicity, there was no divergence between the Zhejiang JE virus strains and the vaccine strain (SA14-14-2). CONCLUSION/SIGNIFICANCES: JE neutralizing antibody positive rates increase in age ≥10 years old population, likely reflecting natural infection or natural boosting of immunity through exposure to wild virus. JE seropositivity rates were quite low in <35 years old age groups in Zhejiang Province. Waning of neutralizing antibody after live attenuated vaccine immunization was observed, but the clinical significance should be further investigated. Both the peripheral antibody response and genetic characterization indicate that current live attenuated JE vaccine conferred equal neutralizing potency against GI or GIII of wild strains. GI has replaced GIII as the dominant genotype in Zhejiang in the past few decades. Although the chance of exposure to wild JE virus has reduced, the virus still circulates in nature; therefore, it is necessary to implement immunization program for children continually and to conduct surveillance activity periodically.

[Genomic sequences of human infection of avian-origin influenza A(H7N9)virus in Zhejiang province].

OBJECTIVE: To analyze the etiology and genomic sequences of human infection of avian-origin influenza A(H7N9)virus from Zhejiang province. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Study on the complete sequence of CA24 variant isolated during the acute hemorrhagic conjunctivitis outbreaks in Zhejiang province during 2002 to 2010].

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.

[Etiological and molecular-epidemiological analysis on enterovirus associated encephalitis in Zhejiang, 2008-2012].

OBJECTIVE: In order to investigate etiology and molecular-epidemiological characteristics of enterovirus associated encephalitis (EAE) in Zhejiang, 2008-2012. Method Cerebrospinal fluid and stool specimens were collected from suspected EAE patients, who were admitted to our hospitals. RD and Hep-2 cell lines were used to isolate enterovirus (EV). Serotypes of these EV isolates were identified through neutralization test by using serotype specific anti-sera. VP1 genes of these isolates were sequenced, compared and used for the construction of phylogenetic tree. RESULTS: 127 (20.6%) human enterovirus (HEV) strains were isolated from 616 samples, which were collected from 610 patients. Serotypes of these EV isolates, including 60 coxsackievirus (CV), and 67 Echovirus (E)appeared to be CVA9, CVB1, CVB3-5, E3, E4, E6, E9, E14, E25 and E30, respectively. Predominant EV serotypes on EAE from 2008 to 2012 were seen as CVB3, CVB5, E6, E30 and E30, respectively. The full length of VP1 genes from different EV isolates was between 834 and 918 nucleotides. The VP1 gene similarities between these isolates and the reference strains were from 76.7% to 85.0% (nucleotides level) and 91.1% to 97.9% (amino acids level). The VP1 genes from E6 serotype isolates appeared most diverged, reaching 20.4% (nucleotides level) and 4.8% (amino acids level). Based on the generated phylogenetic tree, all the EV isolates were fallen on the same branch of HEV-B, and the isolates in the same serotype formed one sub-branch, suggesting there existed geographical and temporal effects. E6 isolates diverged into two branchlets. CONCLUSION: EVs from HEV-B were the etiologic agents for EAE in Zhejiang province from 2008 to 2012. All these EV isolates showed 12 serotypes, with predominant isolates varied every year. E30 was determined as the most dominant serotype while serotype E6 diverged into two sub-gene types.

[Identification of genotype III Japanese encephalitis virus isolated in Zhejiang province].

OBJECTIVE: To investigate the molecular characteristics and genotype of Japanese encephalitis virus (JEV) found in vector mosquitoes in Zhejiang province from 1982 to 1983. METHODS: A total of 3188 mosquitoes were collected in Dinghai district and Yiwu city in Zhejiang province, during year 1982 and 1983. The virus was isolated by C6/36 cell, and then identified by hemagglutination inhibition test. The isolated strains were activated in year 2011, and plaque forming unit (PFU) were applied to test the virus titer. The suckling rats were tested under intracranial inoculation, where PrM and E genes were amplified and sequenced. Their nucleotide and amino acid sequences were analyzed and compared with the JEV vaccine strain SA14-14-2 and the JEV isolated in Zhejiang province during 2007 and 2010; and phylogenetic tree were constructed by bioinformatic software. RESULTS: From the 3188 mosquitoes captured, eleven virus strains were isolated and found to be able to cause cytopathogenic effect (CPE) in C6/36 cells within 72 hours. Virus titer ranged from 2.5 to 6.47 lg PFU/ml. The suckling rats would die within 72 hours since the inoculation. The phylogenetic analysis with the PrM and E genes showed that the JEV isolated in Zhejiang during 1982 and 1983 belonged to genotype III; while the JEV isolated in Zhejiang during 2007 and 2010 belonged to genotype I. The analysis of E genes from 5 isolated strains found that the homology of nucleotide sequence was over 98.9%, and the homology of amino acid sequence was over 99.8%. The compare between the 5 virus strains and the vaccine strain SA14-14-2 found 10 common amino acid variation sites, and showed that the homology of nucleotide sequence was over 97.7%, and the homology of amino acid sequence was 99.2%. The compare between the 5 virus strains and the JEV isolated in Zhejiang during 2007 and 2010 found a 87.7% - 87.9% nucleotide homology and an over 98.8% amino acid homology. CONCLUSION: The JEV isolated from the mosquitoes in Dinghai district and Yiwu city between year 1982 and 1983, were genotype III.

[Study on the molecular characteristics of Japanese encephalitis virus living in vector mosquitoes, in Zhejiang province, 2009 - 2010].

OBJECTIVE: To investigate the molecular characteristics of Japanese encephalitis virus (JEV) living in vector mosquitoes, from Zhejiang province. METHODS: A total of 13 620 mosquitoes were collected from the monitoring stations located in Cixi city and Xianju county in Zhejiang province, in July and August, 2009 - 2010. Nucleic acid of JEV from the mosquitoes was monitored by using real-time RT-PCR. The virus strains were isolated with BHK-21 cell line, with E genes of the isolated viruses amplified, sequenced and their phylogeny and homology analyzed. RESULTS: The positive rates of JEV for those mosquitoes collected in the stations of Cixi and Xianju were 17.0% (27/159) and 3.4% (1/29), respectively. Twenty-two JEV strains were isolated, accounted for 15.4% among the 143 batches of mosquitoes collected in 2010. All E genes in the 6 sequenced virus isolates contained 1500 nucleotides encoding 500 amino acids, in which no inserts and deletions were identified. The identity rates of nucleotide and amino acid in E gene were 99.2% - 99.8% and 100.0% among the 6 JEV strains isolated from Zhejiang, 99.1% - 99.3% and 99.2% - 99.8% between the Zhejiang strains in 2009 - 2010 and the Zhejiang strains in 2007 - 2008, respectively, 87.6% - 88.0% and 97.8% between the 6 Zhejiang strains and the vaccine strain SA14-14-2 of JEV, respectively. The phylogeny tree of E gene indicated that the JEV isolates in Zhejiang during 2009 - 2010 was located in the branch of the genotype I. CONCLUSION: Mosquitoes collected from Cixi and Xianju areas carried JEV, with the rate of JEV in Cixi higher than in Xianju. All the Zhejiang isolates in 2009 - 2010 were proven to be the genotype I of JEV.

[Study on the pathological and molecular characteristics of AHC epidemic in Zhejiang Province in 2010].

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 etiological and molecular characteristics of aseptic meningitis epidemic in Zhejiang Province in 2002-2004].

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].

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. HA(1) 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 HA(1) gene was 4.5% and Yamagata-like HA(1) gene was 3.4%, respectively. The Victoria-like influenza B isolates had appeared to be all reassortants having a Victoria lineage 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 antigenically and genetically similar to B/Brisbane/60/2008-the vaccine strain proposed for 2009 - 2010. Many differences of HA(1) and NA amino acids existed in the current isolates when compared to previous influenza B strains. CONCLUSION: Significant diversity was generated among influenza 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.

[Molecular characteristics of norovirus in 3 outbreak-episodes of gastroenteritis in Zhejiang from 2008 to 2009].

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 norovirus 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 gastroenteritis. 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 GI.8, GII.b, GI.2/GI.6 recombination together with co-infection of GI.8 and GII.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 GI.6 capsid and GI.2 polymerase norovirus as well as the co-infection of GI.8 and GII.b norovirus in the same sample.

[Study on the pathological and molecular characteristics of viral meningitis outbreaks in Linhai county, Zhejiang province, 2004].

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 phylogenetic 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.

[Analysis on VP1 gene variation of Coxsackie virus B5 for aseptic meningitis isolated from Zhejiang Province in 2008].

OBJECTIVE: Study meningoencephalitis virus isolated from the Coxsackie B5 virus(CVB5 virus) VP1 gene characteristics of Zhejiang Province in 2008 and compare with other countries CVB5 prototype isolates, and to explore the relationship of variation of the Virus VP1 areas and epidemic viral meningoencephalitis METHODS: Hep-2 and RD cells in cerebrospinal fluid and stool specimens for virus isolation, positive isolates combination with intestinal type of serum. On the separation of virus extracted RNA, and then RT-PCR amplified VP, gene CVB5 virus fragments, and purification of sequencing products, using DNAMAN and Bioedit analytical processing software. RESULTS: The VP1 gene of CVB5 isolated from Zhejiang province meningoencephalitis viral in 2008 was 735bp. There were no missing and insertion of nucleotide . Strains isolated from ZJ/12/02 with the nucleotide and amino acid sequence of the highest phylogenetic tree showed that they were not the same branch. CONCLUSION: The mutation caused by viral meningoencephalitis virus CVB5 Zhejiang province in 2008 was smaller. Viral meningoencephalitis distributed in some areas in the province had no significant prevalence.

[Traceability of the A/H3N2 virus borne influenza pandemic in Zhejiang province, 1998].

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 HA1, 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.

[Diagnosis of a dengue fever outbreak in Yiwu city, Zhejiang province in 2009 and its molecular tracing of the pathogen].

OBJECTIVE: To determine a dengue fever outbreak in Yiwu city, Zhejiang Province in 2009 and to trace the origin of the pathogen. METHODS: The dengue virus IgM, IgG antibodies and viral nucleic acid were detected and virus was isolated using 40 serum samples from the suspected patients. The viral RNA of the isolated virus strains was extracted and the E gene was amplified by RT-PCR. The amplicons were sequenced and the phylogenetic and homological analyses were also constructed. RESULTS: Among 40 serum samples from dengue fever suspected patients, 17 were positive from for dengue IgM (42.5%); 4 were IgG positive (10.0%); 34 samples were dengue virus RNA positive (85.0%), 28 dengue virus type 3 (D3) strains were isolated (70.0%). The complete coding region of envelope genes (E) from 13 D3 strains was all 1479 nt without any insertion or deletion, which encoded with 493 amino acids (aa). E gene from the 13 D3 strains from Zhejiang in 2009 (D3/ZJ/2009) was 100.0% identical. The strain from Saudi Arabia shared the highest similarity with the D3 strain, 99.3% and 100.0% of their E genes and deduced amino acids were identical, respectively, whereas they were 93.4% and 97.4% between D3/ZJ/2009 strain and its prototype strain (D3/H87/1956), and 93.6% and 97.4% between D3/ZJ/2009 and a D3 strain isolated in Guangxi Province in 1980. The phylogenetic tree of E genes also indicated that D3/ZJ/2009 had maximum similarity with the D3/Saudi Arabia/2004. They all belonged to the D3/GIII branch, which was originated from Indian Subcontinent. CONCLUSION: The outbreak of dengue fever in Zhejiang in 2009 was caused by type 3 dengue virus III genotype. The virus was most likely originated from Saudi Arabia.

[Comparative analysis of measles genome between vaccine strain and wild-type strain in Zhejiang province of China].

OBJECTIVE: To explore the distinction between wild-type strain MVi/Zhejiang, CHN/7.05/4 and vaccine strain Shanghai-191 at genome level. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Molecular epidemiology of norovirus in outbreaks of gastroenteritis in Zhejiang from 2006 to 2007].

OBJECTIVE: To study the molecular epidemiological characteristics of Norovirus gastroenteritis outbreaks in Zhejiang. METHODS: During January 2006 and December 2007, fecal specimens of patients collected from outbreaks of acute viral gastroenteritis were tested for Norovirus. Epidemiological data were also collected. Noroviruses were detected by a reverse transcription polymerase chain reaction (RT-PCR) and Real-time RT-PCR. Some positive samples were randomly selected and RT-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 (VP1) gene. RESULTS: 5 outbreaks of viral gastroenteritis caused by 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%-99.0% 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 gastroenteritis in Zhejiang province Genogroup II/4 variants viruses were the prevalent strains.

[Detection of unknown virus from 2 case of aseptic meningitis epidemic using random primer PCR assay].

OBJECTIVE: To develop a molecular biology method which can be applied to detect unknown virus fast and accurately. METHODS: The samples were separated from human cerebrospinal fluid by infected human rhabdomyosarcoma cells. After centrifugation and discarding the cell debris, the samples were subjected to ultracentrifugation to precipitate viral particles. The DNase treated samples were used to extract viral nucleic acids. The product of random RT-PCR amplification was cloned into pGEM-T Easy vector and sequenced. Sequence similarities were identified with the BLAST programs. RESULTS: The BLAST results showed that the viral agent was ECHO4 strain. CONCLUSION: A random primer PCR method was established, which can be used to identify novel viruses. This research provides a random amplification method for effective detection of unknown viruses or emerging infectious pathogens.

[Study on the genetic characteristics of the avian influenza virus strain A/Zhejiang/16/2006].

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.