Generation and characterization of a cold-adapted attenuated live H3N2 subtype influenza virus vaccine candidate

Background H3N2 subtype influenza A viruses have been identified in humans worldwide, raising concerns about their pandemic potential and prompting the development of candidate vaccines to protect humans against this subtype of influenza A virus. The aim of this study was to establish a system for rescuing of a cold-adapted high-yielding H3N2 subtype human influenza virus by reverse genetics, Methods In order to generate better and safer vaccine candidate viruses, a cold-adapted high yielding reassortant H3N2 influenza A virus was genetically constructed by reverse genetics and was designated as rgAA-H3N2. The rgAA-H3N2 virus contained HA and NA genes from an epidemic strain A/Wisconsin/67/2005 （H3N2） in a background of internal genes derived from the master donor viruses （MDV）, cold-adapted （ca）, temperature sensitive （ts）, live attenuated influenza virus strain A/Ann Arbor/6/60 （MDV-A）. Results In this presentation, the virus HA titer of rgAA-H3N2 in the allantoic fluid from infected embryonated eggs was as high as 1：1024. A fluorescent focus assay （FFU） was performed 24-36 hours post-infection using a specific antibody and bright staining was used for determining the virus titer. The allantoic fluid containing the recovered influenza virus was analyzed in a hemagglutination inhibition （HI） test and the specific inhibition was found. Conclusion The results mentioned above demonstrated that cold-adapted, attenuated reassortant H3N2 subtype influenza A virus was successfully generated, which laid a good foundation for the further related research.

Whole-genome sequences of two influenza A(H3N2)virus strains isolated from Qinghai,China

Influenza A(H3N2)virus has a faster evolution rate than other types of influenza viruses.In this study,whole genome sequencing was performed to better understand themolecular evolution of influenzaH3N2 and the protective effect of influenza virus vaccine in Qinghai Province,China,in 2017.Complete sequences of eight gene segments of two seasonal influenza H3N2 isolates were sequenced and analyzed using DNASTAR and MEGA 6.06 software.Additionally,the three-dimensional structure of the HA protein was predicted using the SWISS-MODEL server.Phylogenetic and amino acid sequence analysis revealed that two Qinghai H3N2 isolates were typical low-pathogenic influenza viruses,and were relatively closely related to the 2016–2017 vaccine strain,3C.2a-A/Hong Kong/4801/2014.The presence of several antigenic site substitutions(T131K,G/R142K,K160T and R261Q in the HA protein)were specific for the two Qinghai H3N2 virus strains.In addition,amino acid substitution of K160T at the glycosylation site of HA and H75P in PB1-F2 in Qinghai isolatesmight affect the antibody binding ability and virulence of the influenza virus.The presence of several antigenic site mutations in the Qinghai H3N2 isolates confirmed the evolution of circulating H3N2 strains.

Emergence of truncated PB1-F2 protein of H3N2 influenza virus during its epidemic period in Jiangsu Province, China

Background PB1-F2 protein has been proven to increase the pathogenicity of influenza A virus （IAV） strains in primary infection and in secondary bacterial infection. It can also regulate the activity of viral polymerase. However, it was shown in another retrospective study that a portion of IAVs do not express full-length PB1-F2 protein during virus development; different kinds of stop codons cause exits in the open reading frames and form PB1-F2 gene products with the corresponding genotypes. Truncated PB1-F2 in human H3N2 IAVs has long been detected in North America but its evolution in China is still unclear. Methods Influenza-like illnesses （ILls） from the whole of Jiangsu Province were collected and inspected to determine the type and subtype of the viruses. A portion of isolates collected in the epidemic period were selected as samples for later whole-genome sequencing, and the exact sequences were determined and analyzed. Results H3N2 influenza virus was one of the epidemical strains which had been prevalent during 2009-2010, in Jiangsu. Five H3N2 isolates with truncated PB1-F2 protein （25aa） were detected in influenza samples from Nanjing and Xuzhou, while seven similar H3N2 isolates were also reported in Niigata, Japan. Conclusion This emergence indicates the possibility that there has been transmission of the H3N2 virus between the two countries.