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Objective: To analyze the genetic evolution and hemagglutinin (HA) gene mutation sites of influenza B virus in Chengdu. Methods: Influenza virus was isolated from patient's throat swab samples using Madin-Darby canine kidney (MDCK) cells in vitro. The HA gene of influenza B virus was obtained by PCR and was sequenced. The phylogenetic tree was constructed and mutation sites were analyzed by online comparison with NCBI database and MEGA 6.06 software. Results: One strain of influenza B virus was isolated by MDCK cells, and 1 755 bp full-length HA gene was obtained by PCR amplification using the nucleic acids of the throat swab and the isolated strain as templates. The obtained sequence was submitted to the GenBank database, and the gene accession number was MH236281. By online alignment and phylogenetic tree construction, the virus infected in this case was confrmed to be Yamagata type B influenza virus. There were 57 HA point mutation bases as compared with Influenza B/Yamagata/16/88 (GenBank No. M36105). There were 20 mutated bases in HA as compared with the World Health Organization (WHO)-recommended vaccine strain Influenza B/Utah/08/2014 (GenBank No. KU592766). HA1 amino acid mutation site was further analyzed. Compared with the epidemic strains in Sichuan in recent years, HA1 amino acid sites have undergone varying degrees of mutations, of which there were only 4 point mutations compared with Wenjiang/2010 isolate (GenBank No. KP461138). Compared with the WHO-recommended vaccine strain Influenza B/Utah/08/2014, two amino acid sites mutated (L176Q and M255V), but the mutation was not located in the antigenic determinant region of HA1. Among them, site 176 was a new mutation. The amino acid was leucine (L) at site 176 of HA1 epidemic strains in Sichuan, Influenza B/Yamagata/16/88, and the WHO-recommended vaccine strain Influenza B/Utah/08/2014, whereas the amino acid was glutamine (Q) in the influenza B strain isolated in this study. Conclusion: Mutations have occurred in the HA gene of influenza B virus infected in Chengdu during 2017 - 2018, while these mutations have not yet caused antigenic changes.
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Objective To reveal the epidemic characteristics of influenza in Huainan of Anhui Province and provide scientific basis for prevention and control of influenza surveillance by summarizing the work of influenza virus isolation performed in 2012-2013.Methods First nucleic acid fast typing by real-time PCR method for sentinel hospitals in throat swab specimens for inspection from sentinel hospital ,the influenza virus isolation was performed by using MDCK cells and the identification of the isolates was carried out by hemagglutination ( HA) and hemagglutination inhibition(HI) tests.Results 68 strains of influenza virus were isolated (45.9%) from 148 of positive specimens detected by fluorescent quantitative real-time PCR.The number of subtype A(H1N1),A(H3N2),BY and BV were 26 strains,25 strains,4 strains and 13 strains,respectively,the success rate of the separation were 56.5%,33.3%, 63.0%,respectively.There were significant difference among the separation of the strain rate of success (P<0.05). 2012 epidemic strain in Huainan was influenza BV virus ,and reached epidemic peak in March;2013 epidemic strain was A(H1N1)influenza virus,the epidemic peak was December.Conclusion During the period of influenza surveillance in Huainan,the main subtype of influenza of the year 2012 is BV which reached epidemic peak in March , while subtype A(H1N1) is the main subtype of the year 2013,with the epidemic peak month December .
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Influenza vaccine strains have been traditionally developed by annual reassortment between vaccine donor strain and the epidemic virulent strains. The classical method requires screening and genotyping of the vaccine strain among various reassortant viruses, which are usually laborious and time-consuming. Here we developed an efficient reverse genetic system to generate the 6:2 reassortant vaccine virus from cDNAs derived from the influenza RNAs. Thus, cDNAs of the two RNAs coding for surface antigens, haemagglutinin and neuraminidase from the epidemic virus and the 6 internal genes from the donor strain were transfected into cells and the infectious viruses of 6:2 defined RNA ratio were rescued. X-31 virus (a high-growth virus in embryonated eggs) and its cold-adapted strain X-31 ca were judiciously chosen as donor strains for the generation of inactivated vaccine and live-attenuated vaccine, respectively. The growth properties of these recombinant viruses in embryonated chicken eggs and MDCK cell were indistinguishable as compared to those generated by classical reassortment process. Based on the reverse genetic system, we generated 6 + 2 reassortant avian influenza vaccine strains corresponding to the A/Chicken/Korea/MS96 (H9N2) and A/Indonesia/5/2005 (H5N1). The results would serve as technical platform for the generation of both injectable inactivated vaccine and the nasal spray live attenuated vaccine for the prevention of influenza epidemics and pandemics.