Disinfection of influenza a viruses by Hypocrellin a-mediated photodynamic inactivation.

BACKGROUND: Influenza A viruses can be transmitted indirectly by surviving on the surface of an object. Photodynamic inactivation (PDI) is a promising approach for disinfection of pathogens. METHODS: PDI was generated using Hypocrellin A (HA) and red light emitting diode (625-635 nm, 280 W/m2). Effects of the HA-mediated PDI on influenza viruses H1N1 and H3N2 were evaluated by the reduction of viral titers compared to virus control. After selection of the HA concentrations and illumination times, the applicability of PDI was assessed on surgical masks. Reactive oxygen species (ROS) were determined using a 2'-7'-dichlorodihydrofluorescein diacetate fluorescence probe. RESULTS: In solution, 10 µM HA inactivated up to 5.11 ± 0.19 log10 TCID50 of H1N1 and 4.89 ± 0.38 log10 TCID50 of H3N2 by illumination for 5 and 30 min, respectively. When surgical masks were contaminated by virus before HA addition, PDI inactivated 99.99% (4.33 ± 0.34 log reduction) of H1N1 and 99.40% (2.22 ± 0.39 log reduction) of H3N2 under the selected condition. When the masks were pretreated with HA before virus addition, PDI decontaminated 99.92% (3.11 ± 0.19 log reduction) of H1N1 and 98.71% (1.89 ± 0.20 log reduction) of H3N2 virus. The fluorescence intensity of 2',7'-dichlorofluorescein in photoactivated HA was significantly higher than the cell control (P > 0.05), indicating that HA efficiently generated ROS. CONCLUSIONS: HA-mediated PDI is effective for the disinfection of influenza viruses H1N1 and H3N2. The approach could be an alternative to decontaminating influenza A viruses on the surfaces of objects.

High resolution metagenomic characterization of complex infectomes in paediatric acute respiratory infection.

The diversity of pathogens associated with acute respiratory infection (ARI) makes diagnosis challenging. Traditional pathogen screening tests have a limited detection range and provide little additional information. We used total RNA sequencing ("meta-transcriptomics") to reveal the full spectrum of microbes associated with paediatric ARI. Throat swabs were collected from 48 paediatric ARI patients and 7 healthy controls. Samples were subjected to meta-transcriptomics to determine the presence and abundance of viral, bacterial, and eukaryotic pathogens, and to reveal mixed infections, pathogen genotypes/subtypes, evolutionary origins, epidemiological history, and antimicrobial resistance. We identified 11 RNA viruses, 4 DNA viruses, 4 species of bacteria, and 1 fungus. While most are known to cause ARIs, others, such as echovirus 6, are rarely associated with respiratory disease. Co-infection of viruses and bacteria and of multiple viruses were commonplace (9/48), with one patient harboring 5 different pathogens, and genome sequence data revealed large intra-species diversity. Expressed resistance against eight classes of antibiotic was detected, with those for MLS, Bla, Tet, Phe at relatively high abundance. In summary, we used a simple total RNA sequencing approach to reveal the complex polymicrobial infectome in ARI. This provided comprehensive and clinically informative information relevant to understanding respiratory disease.

Establishment and characterization of an oral gerbil model for a non-mouse-adapted enterovirus 71 strain.

Enterovirus 71 (EV71) is one of the major pathogens causing hand, foot, and mouth disease (HFMD) with neurological and systemic complications worldwide, and it is mostly discovered in infants and young children. It is of great significance to establish suitable animal models of EV71 infection on research of distribution and pathogenesis of the virus. In this study, we established a successful infection of a non-mouse-adapted isolate of EV71 via oral route in 7-day-old Mongolian gerbil (Meriones unguiculatus), all of which were paralyzed and died within 10 days post infection. Analysis of virus loads in twelve tissues showed that virus was first detected in intestine, blood, heart, lung, and muscle one day post-infection, and then in the rest of the tissues/organs within the next few days, among which thymus, spleen, spinal cord and muscles had the highest virus titer at 5 days post infection. Pathological examination showed that severe necrosis was observed in skeletal muscle and spinal cord, and edema was observed in both heart and lung. Comparisons of host gene expression of various tissues from infected and non-infected gerbils revealed a general up-regulation of genes related to anti-viral response and a viral receptor gene (sialic acid-linked glycans), as well as a tissue(gut)-specific up-regulation of genes related to neuronal communication. Collectively, our results showed that EV71 could induce severe neurological complications as well as massive tissue damage all over the body, which indicates that oral infection of 7-day gerbils can be a suitable animal model to study the infection of EV71 in human.

Laboratory diagnosis of vaccine-associated measles in Zhejiang Province, China.

BACKGROUND/PURPOSE: Along with the improving vaccine coverage, suspected vaccine-associated measles has been reported in Zhejiang Province, China. In order to maintain the accuracy of the measles surveillance system, it is critical to discriminate between measles vaccine and wild-type virus. METHODS: Eight suspected cases of vaccine-associated measles were reported in Zhejiang Province during 2011 and 2014. Sera collected within 4 days and throat swabs collected within 6 days after rash onset were tested with immunoglobulin M and measles virus (MeV) RNA to confirm MeV infection. In order to further identify the vaccine-associated cases, throat swabs with positive MeV RNA were tested using an allelic discrimination real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assay developed in this study, RT-PCR-restriction fragment length polymorphism (RFLP) recommended by the National Measles Laboratory, and RT-PCR followed by sequencing and genotyping. RESULTS: Combining anti-measles immunoglobulin M and RNA testing, eight cases were confirmed as MeV infection. Of the eight, two were identified as vaccine-associated cases by the allelic discrimination rRT-PCR assay, and one was identified by RT-PCR-RFLP. Subsequent sequencing and genotyping confirmed that the sequences of the two cases were identical to that of the Chinese vaccine strain. The developed allelic discrimination rRT-PCR was 10 times more sensitive than the RT-PCR-RFLP assay when RNA standards generated from three genotypes of MeV were tested. CONCLUSION: Vaccine-associated measles has been identified in Zhejiang. The developed allelic discrimination rRT-PCR assay is rapid and sensitive, which will facilitate the surveillance for vaccine-associated measles.

Fast throughput determination of 21 allergenic disperse dyes from river water using reusable three-dimensional interconnected magnetic chemically modified graphene oxide followed by liquid chromatography-tandem quadrupole mass spectrometry.

We report the template-free fabrication of three-dimensional hierarchical nanostructures, i.e., three-dimensional interconnected magnetic chemically modified graphene oxide (3D-Mag-CMGO), through a simple and low-cost self-assembly process using one-pot reaction based on solvothermal method. The excellent properties of the 3D-Mag-CMGO are characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), vibrating sample magnetometer (VSM), FTIR, elementary analyzer (EA) and X-ray photoelectron spectroscopy (XPS). The easiness-to-handle of the magnetic dispersive solid phase extraction (Mag-dSPE) procedure is developed for preconcentration of 21 allergenic disperse dyes from river water. The obtained results show the higher extraction capacity of 3D-Mag-CMGO with recoveries between 80.0-112.0%. Furthermore, an ultra-fast liquid chromatography-tandem quadrupole mass spectrometry (UFLC-MS/MS) method for determination of 21 allergenic disperse dyes in river at sub-ppt levels has been developed with pretreatment of the samples by Mag-dSPE. The limits of quantification (LOQs) for the allergenic disperse dyes are between 0.57-34.05ng/L. Validation results on linearity, specificity, trueness and precision, as well as on application to the analysis of 21 allergenic disperse dyes in fifty real samples demonstrate the applicability to environment monitoring analysis.

[Variations on hemagglutinin gene of Zhejiang measles virus strains and differences with measles strains circulated both at home and abroad].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[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.

[Molecular evolution of two lineages related to influenza B virus based on HA1 gene].

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 2012 were 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 HA1 gene, 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 Yamagata-like strains showed an amino acid deletion at 163(th) 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.

[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].

OBJECTIVE: To compare the differences in the complete genome sequence between mumps epidemic strain and mumps vaccine strain S79 isolated in Zhejiang province. METHODS: 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. RESULTS: 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. CONCLUSION: There were significant differences found in the genes between mumps epidemic strain and mumps vaccine in Zhejiang province.

[Study on the genome sequence of measles viruses circulated in Zhejiang province during 1999 to 2011].

OBJECTIVE: To study the genetic variations between measles vaccine strain S191 and strains that circulated in Zhejiang province causing the epidemics during 1999 to 2011. 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 Zhejiang 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 gene, 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 Zhejiang epidemic strains and vaccine strains was greater than the distances between epidemic strains of genotype D(4), B(3) and vaccine strains (t = -9.76, P < 0.05; t = -12.39, P < 0.05). CONCLUSION: There were significant differences 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 genotype epidemic strains were much larger than the differences between the vaccine and the foreign epidemic strains (genotype D(4), B(3)). Therefore, we should pay close attention to this trend, and to develop candidates for the development of vaccines, as early as possible.

[Study on the mutations within the whole genome of influenza virus subtype A/H3N2 strains circulated in Zhejiang province from 1998 to 2009].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[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.

[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.

[Molecular epidemiological study on rubella virus strains isolated in Zhejiang province, China, 2005-2010].

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 1E and only one was characterized as genotype 2B. In the phylogenetic tree, the Zhejiang 1E 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 1E 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.

[Genetic variation of phosphoprotein (P) gene from measles epidemic strains in Zhejiang province].

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.

[Phylogenetic analysis based on hemagglutinin and neuraminidase genes between swine influenza virus and human influenza A/H3N2 virus strains isolated in Zhejiang province].

OBJECTIVES: To investigate the phylogenetic relationship between swine influenza A/H3N2 virus and the representative strains of human influenza A/H3N2 virus isolated in two epidemics in recent years through comparing the sequences within HA and NA genes. METHODS: HA and NA gene of the human representative strains were sequenced, and then phylogenetic tree with the swine and human strains isolated in the corresponding period of time were constructed. RESULTS: The homologies on the HA1 domain between human representative strains (A/Zhejiang/10/98, A/Zhejiang/6/99 and A/Zhejiang/8/02)and the swine strains (A/SW/Ontario/130/97, A/SW/Hongkong/4361/99 and A/SW/Hongkong/74/02) were 99.1%, 99.4% and 99.4% respectively. Based on the NA gene, the homologies between human strains (A/Zhejiang/10/98, _A/Zhejiang/6/99 and A/Zhejiang/8/02)and the swine strains (A/SW/Ontario/130/97, A/SW/Hongkong/4361/99 and A/SW/Hongkong/74/02) were 98.2%, 99.3% and 99.3% respectively. The results showed that the two types of influenza viruses were highly homologue, and even some of their homologies were higher than that amongst the contemporary human influenza A/H3N2 strains. The same results shown in the phylogenetic tree. CONCLUSIONS: The human influenza A/H3N2 virus isolates in the two epidemic closely associated with some of the swine influenza virus strains, and their relationship should be further studied.

[Development of the real-time reverse transcription-polymerase chain reaction assay for the rapid detection of rubella virus].

OBJECTIVE: A new TaqMan probe based real-time assay was developed to rapid detection of rubella virus. METHODS: The specific primer pair and probe were designed within the conserved P150 gene of rubella virus and the PCR reactive condition was optimized to improve the sensitivity and specificity of the assay. Clinical specimens collected from two outbreaks were detected by the developed assay. RESULTS: The assay is specific to detect rubella virus. There is no cross-reactions to measles, mumps, influenza and other respiratory viruses. It could detect 0.01 TCID50/tube of rubella RNA and took only three hours to finish a detection. In addition, the assay is simple, accurate and repeatable. CONCLUSION: The TaqMan based real-time PCR developed in this study provides a fast, sensitive and specific tool for molecular diagnosis on rubella virus.

[Simultaneous detection of measles and rubella virus by multiplex real-time RT-PCR with an internal control].

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.