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Objective To investigate the molecular evolution of the Hemagglutinin (HA) and Neuraminidase (NA) genes of influenza A/H3N2 viruses in Shenzhen in the first half of 2017, so as to provide scientific basis for predicating influenza epidemic and variation. Methods A total of 40 influenza A/H3N2 viruses strains were selected and the molecular phylogenetic trees were constructed by bioinformatics software DNAStar, MEGA 7.0, etc. Then, the genetic characteristics and variation of HA and NA genes along with corresponding amino acids were analyzed. Results The homology of Shenzhen isolates reached 97.8%-100.0%, which located in the human-derived branch of Asia and North America. Compared with the vaccine strains A/Switzerland/9715293/2013(H3N2) and A/Hong Kong 14801/2014(H3N2) recommended by world Health Oraganication (WHO), there was a higher sequence similarity. Compared with the vaccine strain, HA and NA proteins had a number of amino acid sites replaced, of which HA 6 antigen sites and 2 receptor binding sites change; NA had a mutation of D151N/G located in enzyme activity sites. Potential N-glycosylation sites for HA and NA also changed. Conclusions The influenza A/H3N2 viruses in Shenzhen in the first half of 2017 has not yet formed a new subtype in the epidemic. Currently, the recommended vaccine strains still have some protective effects on the population. The replacement mutation of multiple amino acids sites of HA and NA suggests that the dynamic monitoring of molecular level of influenza A/H3N2 viruses need to be strengthened.
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Objective To investigate the molecular epidemiological characteristics of hemaggluti-nin (HA) and neuraminidase (NA) of influenza B viruses (IBV) isolated in Qingdao from 2011 to 2018. Methods A total of 12236 samples of influenza-like cases in Qingdao from 2011 to 2018 were collected to extract viral RNAs. All samples were screened for influenza A viruses ( IAV) and IBV by one-step multiplex real-time RT-PCR. Lineages of IBV were identified. One hundred and eighty-two strains of IBV were select-ed to amplify HA and NA genes by RT-PCR and then analyzed by sequencing. Phylogenetic analysis and variation analysis of genes and amino acids were carried out. Results IBV was detected almost every year in Qingdao from 2011 to 2018. The positive rate was only slightly lower than that of IAV ( 4. 99％ vs 6. 21％). B/Victoria linkage had two prominent epidemic years (2011-2012, 2015-2016), while B/Yama-gata linkage had three (2013-2014, 2014-2015, 2017-2018). Most of the infected people were children un-der 10 years old, and the people infected with the two lineages had similar age characteristics. Phylogenetic analysis of HA genes showed clusters in Victoria clades of 1A and 1B and Yamagata clades of 2 and 3. IBV of Yamagata lineage had more amino acid mutation sites than those of Victoria lineage in HA genes with grea-ter genetic diversity. The B/Yamagata strains had 12 amino acid mutations and the B/Victoria strains had seven in four major epitopes. In the receptor binding sites, two amino acid mutations were detected in the B/Yamagata strains and three in the B/Victoria strains. In Qingdao, 26 strains of IBV were intra-lineage reas-sortments, mostly of the B/Victoria lineage, and 23 strains were inter-lineage reassortments, mostly between HA-B/Yamagata and NA-B/Victoria strains. A possible resistant strain to NA inhibitor was found. Conclu-sions The significance of IBV in seasonal influenza should not be neglected. Amino acid substitution, in-sertion/deletion and gene reassortment were the main strategies for the natural evolution of IBV. Influenza surveillance was of great importance and influenza vaccine strains needed to be updated in time.
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The HA gene of H9N2 influenza virus (A/chicken/Hunan/04.14 (H9N2)) was amplified and sequenced. The RNA was synthesized by in vitro transcription. The RNA transcription solutions were diluted to 10⁹ copies/μL using the RNA storage solution. The aliquoted RNA solutions were used to evaluate the homogeneity and stability. The results were determined by the average value obtained from four independent laboratories. Furthermore, the fluorescence quantitative RT-PCR method was also developed to verify the detection accuracy of clinical samples. The detection limit of this method is approximately 10 copies. Taken together, the RNA transcription solution established in our study can used as positive standard reference for rapid detection of H9N2 influenza virus.
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Objective@#To analyze the genotypes and the genetic evolution of the hemagglutinin genes of measles viruses in the city of Yancheng in 2016.@*Methods@#Using a set of primers and probes for screening positive for measles viruses, specimens of throat swab were detected using the method of real time RT-PCR. The sequences of the nucleoprotein and hemagglutinin genes of measles viruses were amplified through one step RT-PCR method and the PCR products were sequenced. The sequences of nucleotide and amino acid of the nucleoprotein and hemagglutinin genes of measles viruses were analyzed and the evolutional trees were generated using bioinformatics software.@*Results@#The genotypes of measles viruses in the Yancheng area in 2016 included subgenotype H1a and genotype D8. Phylogenetic trees analysis showed that the five representative strains of subgenotype H1a in Yancheng area and Jiangxi representative strain (KJ136545) clustered into independent evolutionary branches, belonged to the clade of H1a -1 evolutionary genes. The seven representative strains of genotype D8 in Yancheng area were clustered with the American representative strain in 2009 (JN635404), belonged to the D8-3-2 small clade genes. Compared with vaccine strain of Shanghai S191, the amino acid site in 240thof the five representative strains of subgenotype H1a in Yancheng area mutated from serine to asparagine, leading to a loss of the N-glycosylation site NLS238-240. The seven representative strains of genotype D8 in Yancheng area had no change in N-glycosylation.@*Conclusions@#In 2016, the prevalent strains of measles viruses in Yancheng area were mainly Chinese H1a dominant subgenotype and D8 imported genotype. In addition to a loss of the N-glycosylation site NLS238-240in 240thof the five representative strains of subgenotype H1a, most of the major neutralizing antigen sites of hemagglutinin gene of measles viruses in Yancheng area did not mutate. The Chinese vaccine of Shanghai S191 can effectively prevent infection caused by subgenotype H1a and subgenotype D8 strains.
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Objective In order to analyze the variation of HA genes of influenza viruses (H1N1) by being compared with the vaccine strain A/California/07/2009(H1N1) recommended by WHO ,influenza viruses (H1N1) isolated from 2009 to 2014 were selected to do this study .Methods According to the different isolating time and place ,47 strains of H1N1 collected from 2011 to 2014 were selected .Then the 47 strains′ nucleotide sequence of HA genes which were sequenced in the study and other 25 se‐quences of HA genes which were sequenced in 2009 were collected .Nucleotide and amino acid sequences were analyzed by using molecular biology software ,and the phylogenetic trees were drawn .Results A total of 72 strains isolated from 2009 to 2014 were closely related to the vaccine strain A/California/07/2009(H1N1) ,the nucleotide variance and amino acid variance between the 72 strains were 0-2 .7% and 0-3 .1% respectively .Compared with the vaccine strain A/California/07/2009(H1N1) ,the nucleotide variance and amino acid sequence variance were 0 .4% -2 .4% and 0 .9% -3 .1% respectively .The amino acids sequence indicated that ,although the variance was increased by years ,the H1N1 viruses were still showed characteristics of low pathogenic influenza viruses .It was also found that there were 9 strains lost their potential glycosylation site at HA protein site 481 in 2009 ,while in 2013 there were 6 strains got new potential glycosylation sites at HA protein site 162 .Conclusion The vaccines (H1N1) recom‐mended by WHO was still protective to people in Chongqing .But as time goes by ,antigen drift may occur in some new antigenic drift strains and the routine monitoring of influenza viruses should be continued .
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Objective To identify the pathogen of an influenza epidemic situation and analyze the genetic characteristic of hemagglutinin( HA ) gene and neuraminidase(NA) gene of this pathogen. Methods Real-time RT-PCR was used to dectect nucleic acid of the pandemic( H1N1 ) 2009 virus from oropharyngeal swabs of initial influenza-like illness in epidemic. The viruses were was inoculated and isolated with embryonated eggs. And the HA gene and NA gene were sequenced to analyze their characteristic. Results The influenza epidemic situation was caused by the pandemic( H1N1 ) 2009 virus. The HA and NA sequences data showed that the virus had the high homology with reference virus, and the NA sequences had not the H274Y mutation. Conclusion In this study, the pandemic( H1N1 ) 2009 virus were similar with the vaccine-like virus and the isolated virus of China, and sensitive to oseltamivir.
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Objective To analyze the genetic characteristics of H1N1 influenza viruses isolated in Shenzhen during 1995 to 2007. Methods The hemagglutinin(HA) gene of these viruses were sequenced. Phylogenetic analysis of the sequences was performed with Simmonic and Mega software. Results The H1N1 influenza viruses isolated in Shenzhen from 1995 to 2007 were divided into chide A, B and C. Some viruses from 2005 to 2006 clustered in the same group with the viruses of 2001. Furthermore, some of the vaccine strains recommended by WHO were found lagged behind the strains isolated in Shenzhen. Some mu-tations occurred on the antigenic sites as well as receptor-binding site(RBS). Except the viruses of 1995, the other viruses had deleted at the site 137. Conclusion Characterization of the HA gene revealed that most of the amino acid substitutions occurred on the antigenic sites and RBS. Furthermore, it was discovered that the mutations occurred on different antigenic regions in different years.