Selection pressure analysis of H3N2 influenza virus from China between 1992 and 2012 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>In order to investigate the relationship between selection pressure and the prevalence of antigenic clusters, we sequenced and analyzed the H3N2 influenza virus from China between 1992 and 2012.</p><p><b>METHODS</b>The H3N2 influenza virus (n = 1206) in China from 1992 to 2012 was analyzed, include global selection pressure and sites positive selection pressure analysis.</p><p><b>RESULTS</b>Considering all the H3N2 influenza viruses during these 21 years, a total of four amino acid sites subject to positive selection. The global selection pressure varies with the variation of different antigenic clusters and three years with peak bottom selection pressure were identified.</p><p><b>CONCLUSION</b>The global selection pressure rise from the peak bottom, a new antigenic clusters will appear andprevalent in the population, indicating the best time to replace the vaccine strain.</p>

Virological characterization of influenza B virus in mainland China during 2011-2012 / 病毒学报

In order to understand the prevalence and variation of influenza B viruses, the antigenic and genetic characteristics of influenza B viruses circulating in Mainland China during April, 2011 to March, 2012 were analyzed. The results showed the B Victoria lineage viruses were much more prevalent than B Yamagata lineage during this period, phylogenetic analysis showed vast majority of Victoria lineage viruses belong to genetic group 1, intra-clade reassortant between HA1 and NA gene was identified in a minor proportion of the viruses. 72.8% of the B/Victoria-lineage viruses were antigenically closely related to the vaccine strain B/Brisbane/60/2008. B Yamagata component was not included in the trivalent influenza vaccine in China during the study period, however vast majority of B Yamagata lineage viruses were antigenically and genetically closely related to the representative virus B/Hubei-Wujiagang/158/2009(97.8%) and B/Sichuan-Anyue/139/2011(85.2%) in China, reassortant between HA1 and NA was not identified in B Yamagata lineage viruses. Overall, the predominant circulating influenza B viruses in 2011-2012 season in China were matched by current influenza vaccine and the selected representative viruses were proved to represent the antigenic and genetic characteristics of the circulating viruses.

Emerged Pdm09 influenza virus increased purifying selection of seasonal H1N1 influenza virus / 病毒学报

Pdm09 virus outbreak occurred in Mainland China in May 2009, a few months later, the prevalence of seasonal H1N1(sH1N1) influenza virus that already circulated in human for tens of years began to decline and disappeared afterwards. To identify the reason for the rapid decline of sH1N1 in mainland China, we sequenced the HA1 of sH1N1 during 2006-2011, and then analyzed the selective pressure in different phases. Our results showed before Pdm09 outbreak, the omega value was 0. 36 while after Pdm09 outbreak the omega value was 0. 28 and significant difference (t test, P<0. 05) was identified. We concluded that sH1N1 obtained stronger purifying selection after Pdm09 outbreak in China. This might one of the major reasons causing the disappearance of sH1N1 in human.

Virological characterization of influenza A(H3N2) virus in Mainland China during 2011-2012 / 病毒学报

To study the prevalence and variation of influenza A(H3N2) viruses, the antigenic and genetic characteristics of influenza A(H3N2) viruses circulating in Mainland China during April 2011 to March 2012 were analyzed. The results showed that influenza A(H3N2) viruses increased gradually since 2012 and became the dominant strain since March. The viruses were antigenically closely related to the vaccine strain A/PER/16/09 (87.2%) and the representative virus A/FJ/196/09 (76.0%) in Mainland China. The genetic characteristics analysis results showed that recently isolated viruses belonged to the Vic/208 clade, and most of the low reaction strains also fell into the same clade. Crystal structure analysis of HA protein found that, compared with the vaccine strain A/PER/16/09, the recently isolated viruses had amino acid substitutions in the antigenic site A, B and C areas, in addition to gaining potential glycosylation sites at the amino acid position of 45 of HA and 367 of NA. Although the majority of circulating influenza A (H3N2) viruses in 2011-2012 season in Mainland China were antigeniclly matched by current influenza vaccine strain and the selected representative viruses, low reaction strains have increased since 2012, therefore it is necessary to strengthen the surveillance on the variation of influenza virus and to provide solid information for the vaccine strain selection.

Development of a real-time reverse transcriptase PCR assay for detection of E119V amino acid change in neuraminidase of influenza A (H3N2) using the TaqMan-MGB probe / 中华预防医学杂志

<p><b>OBJECTIVE</b>To develop a rapid duplex Real-time reverse transcription PCR (rRT-PCR) method to detect E119V mutation on neuraminidase (NA) of influenza A(H3N2) subtype with drug resistance to oseltamivir.</p><p><b>METHODS</b>Twenty-six NA genes of influenza A(H3N2) virus between 2000 and 2012 in GenBank database were selected as the target genes, and specific TaqMan-MGB probe was designed to target the E119V amino acid change in neuraminidase protein. rRT-PCR was then performed and evaluated for the sensitivity, specificity and reproducibility using virus with E119V mutation and clinical samples.</p><p><b>RESULTS</b>This study described the validation of a highly sensitive and specific duplex rRT-PCR for detection of substitutions leading to the E119V amino acid change in NA protein of influenza A(H3N2). Fluorescence signals could be detected even when diluted a A (H3N2) virus (HA = 8) into 10(-5) and linear correlation between the logarithm of the viral titer with the Ct values was observed. In addition, the assay was highly specific in that there was no cross-react with other respiratory viruses, nor did two TaqMan-MGB probes. E119V substitution in quasispecies with both sensitive and resistant viruses could be detected as well. The limit of detection was 5% for quasispecies with high concentrations and 50% for quasispecies with low concentrations. The average coefficient of variation (CV) for within-run assays was 2.32% and 0.57% for H3N2-119E and H3N2-119V primer/probe sets separately, 1.77% and 0.97% for average CV of between-run assays, which exhibited good repeatability. Sequence analysis of twenty NA genes verified glutamic acid (E) at amino acid site 119, which was in consistent with the results from our rRT-PCR method.</p><p><b>CONCLUSION</b>The assay developed in this study is highly sensitive and specific, and easy to operate; thereby it could be used for identification of A(H3N2) virus with E119V amino acid change in NA protein.</p>

Development of single-tube multiplex real-time PCR for simultaneous detection of novel influenza A H1N1 and human seasonal influenza A H1N1 and H3N2 virus / 病毒学报

In this study, we established a rapid and sensitive multiplex Real-time reverse transcription polymerase chain reaction (mrtRT-PCR) to simultaneously detect the novel human influenza A H1N1 virus, human seasonal influenza A H1N1 and H3N2. This assay had three pairs of primer to target the conserved regions of the HA gene for each of the HA types including novel H1N1, seasonal H1N1 and seasonal H3N2, and one pair of primer designed to detect the internal control-RnaseP gene. This assay was performed in one-step in one tube. To validate the specificity of the multiplex Real-time RT-PCR assay, different human influenza virus including human seasonal influenza A H1N1 and H3N2, human influenza B and reference A/California/07/2009 (H1N1) sw1 was tested. To evaluate the sensitivity of the assay, serial dilutions of RNA from in vitro transcription of the novel human influenza A H1N1 HA gene was tested. Finally this assay was evaluated with clinical samples from 54 fever patients diagnosed with novel influenza A H1N1 or seasonal H1/H3 or HB infection either by real-time PCR recommended by the WHO or HI assay by the National Influenza Center. Our results showed that the assay could achieve a sensitivity of 20 RNA copies of novel influenza A H1N1 with high specificity and could detect potential mixed co-infection. In conclusion, this multiplex real-time RT-PCR assay combines both rapidity and sensitivity for not only detecting the novel human influenza A H1N1 virus, but also monitoring the human seasonal influenza A H1N1 and H3N2 simultaneously.

Study on the antigenicity and HA1 gene characteristics of influenza A viruses during 2004-2008 year in China / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To under stand influenza A viruses epidemic, antigenicity and genetic characteristics variation between the vaccine and Circulation strains during 2004-2008 year in China.</p><p><b>METHODS</b>The influenza A viruses (H1N1, H3N2) isolated from 2004-2008 year were under took antigenic and sequence analysis. Influenza A virus antigenicity and genetic characteristics were analyzed thought amino acid variation compassion of HA1 protein of influenza A virus isolates.</p><p><b>RESULTS</b>The antigenicity of influenza H1N1 subtype viruses isolated from 2004 to 2007 is very similar with vaccine strain A/New Caledonia/20/1999 (HIN1)-like virus. The influenza H1N1 viruses circulated in 2008 year had similar antigenic characteristics with A/Brisben/59/2007 (H1N1) which is component of influenza vaccines for northern hemisphere 2008-2009 year. The influenza H3N2 subtype viruses of 2004-2005 year had antigenic variation comparatively with vaccine strain A/Fujian/411/12002 (H3N2), The antigenicity of 2006-2007 H3N2 viruses and 2008 year's is A/Wiscansin/67/2006(H3N2) and A/ Brisben/10/2006(H3N2) respectively.</p><p><b>CONCLUSION</b>There is change of influenza A viruses (H1N1, H3N2) antigenic and genetic characteristics during 2004-2008 in China.</p>

Establishment of a method for rapid detection of the nucleic acid of the novel A (H1N1) influenza virus / 病毒学报

A new flu caused by a novel influenza A(H1N1) virus has spread over the United States, Mexico and more than 40 other countries. And because of the immediate global concern, WHO has announced that the current level of influenza pandemic alert is raised to phase 5, indicating approaching of an influenza pandemic. As patients suffering from the influenza A (H1N1) have the similar symptoms as patients with seasonal influenza, differential detection and identification of the influenza virus have to depend on specific laboratory tests. We have successfully developed a RT-PCR based method for detection of the influenza A (H1N1) virus, and had applied the method to detection of clinical samples.

Detection of influenza viruses/avian influenza viruses and identification of virulence using a microarray / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish the DNA microarray to detect influenza viruses and avian influenza viruses, and identify their virulence.</p><p><b>METHODS</b>Hemagglutinin (HA), neuramidinase (NA) and nucleoprotein(NP) genes were chosen simultaneously as targets for designing a microarray used for detection of viruses and identification virulence. The nucleic acid were amplified by single primer amplication (SPA). And then its specificity,sensitivity and reproducibility were evaluated.</p><p><b>RESULTS</b>The microarray was able to specially detect H1N1, H3N2, B influenza viruses and H5N1, H9N2 avian influenza viruses. Their limits were 8HAU, 16HAU, 32HAU, and 8HAU, 8HAU respectively. The limit for virulence was 32HAU. When samples were analyzed by both RT-PCR and microarray in parallel, the results agreed in 83.9% (47/56).</p><p><b>CONCLUSION</b>The microarray can detect and distinguish five tested viruses, and especially identify virulence. It not only supplies an assistant tool for clinical diagnosis and control of infectious disease, but also is valuable for controlling and preventing outbreak of avian influenza epidemic.</p>

Antigenic and genetic study of influenza virus circulated in China in 2006 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To analyse seasonal influenza epidemic situation in 2006, and to analyse the genetic and antigenic characteristics of viral hemagglutinin (HA) gene.</p><p><b>METHODS</b>The single-way hemagglutination inhibition (HI) tests were used to test the antigenic characteristics of these viruses from influenza surveillance network, and the HA1 genes were sequenced based on the antigenic test results according to different isolation times and sites.</p><p><b>RESULTS</b>The influenza virus types A and B co-circulated in 2006. influenza A H1N1 subtype and Victoria-like B influenza circulated preponderantly during this epidemic season. The HA1 gene sequence of H1N1 viruses showed that 192, 193, 196, 198 positions (located at antigenic site B) have an amino acid substitute, compared with the last circulating strain A/Hubeihongshan/53/2005(H1N1). Two amino acid changes at 142 and 144 positions compared with A/Yunnan/1145/2005 (H3N2). There was no change in influenza B viruses either Victoria-like B or Yamagata-like B virus, i.e . antigenic characteristics is analogous to B/shenzhen/155/2005 and B/tianjin/144/2005, respectively.</p><p><b>CONCLUSION</b>The H1N1 and H3N2 influenza viruses had changing antigenic and genetic characteristics in 2006. Influenza virus types B did not change in 2006.</p>