The Study Of HA And NA Genetic Characteristics And Pathogenicity In Mice Of Influenza B Virus In Guangxi During 2018～2021

Influenza viruses are important pathogens that cause respiratory diseases in humans and animals,posing a serious threat to the Public health and human health.Influenza B virus（IBV）is more likely to cause complications than influenza A virus（IAV）and even led to higher disease burden than IAV in the certain season.However,IBV has received less attention.Influenza viruses achieve antigenic evolution through base insertions,deletions,mutations,and frequent reassortments between or within lineages.The analysis of genetic evolution and mutation sites of nucleotides and amino acids is helpful to evaluating antigen evolution,discovering new mutant genes,and providing scientific basis for the prevention and control of influenza viruses.In this study,the molecular characteristics of hemagglutinin（HA）and neuraminidase（NA）of IBV were analyzed,and the strains spread in Guangxi during 2018～2021 were used.The variation of IBV in Guangxi in the past four years was also studied.In addition,the whole gene sequence of the circulating IBV clinical strain B/Guangxi-Jiangzhou/1352/2018 was analyzed,and its pathogenicity to BALB/c mice was studied to establish the IBV-infected BALB/c mice model.The specific research contents are as follows:1.The HA and NA genetic characteristics analysis of influenza B virus in Guangxi during 2018～2021.HA and NA amino acid sequences of 86 IBV strains in Guangxi during2018～2021 and 6 vaccine strains recommended by the World Organization（WHO）in recent years were downloaded from the Global Initiative for Sharing All Influenza Data（GISAID）and Gen Bank Influenza Database and used for homology,genetic evolution,molecular characterization,and glycosylation site analysis.The results of amino acid homology analysis showed that the IBV strains maintained higher homology compared with vaccine strains in the same period.In the genetic evolution analysis,no reassortments were detected in HA and NA of these strains.The results of HA molecular characteristic analysis showed that there were 12 differences in the amino acid sequences of Victoria lineage strains;and the Yamagata lineage strains had less amino acid sequence variation.The results of NA molecular characteristic analysis showed that there were 10 differences in the amino acid sequences of Victoria lineage strains;and the Yamagata lineage strains had less amino acid sequence variation.The neuraminidase active center and surrounding auxiliary sites were highly conserved and there were no drug resistance-related site mutations,indicating the strains were still sensitive to neuraminidase inhibitors（NAI）.Victoria lineage2020～2021 strains had 1 less glycosylation site than the 2018～2019 strains at position 197 in glycosylation site analysis,which was located in the 190 loops of the epitope and was also the receptor-binding site（RBS）,might affect the receptor-binding properties of the virus,and there were no differences in the glycosylation sites among the Yamagata lineage strains.2.The whole sequence analysis of clinical strain B/Guangxi-Jiangzhou/1352/2018.The IBV B/Guangxi-Jiangzhou/1352/2018 strain was propagated in chicken embryos and the virus titers were then determined.The fragments of the strain except HA,NA and M were sequenced.The homology between this strain and other Victoria lineage vaccine strains,and the key differences in HA and NA amino acid sequences were analyzed.The pathogenicity-related molecular of NP,PA,PB1,PB2,NS and M fragments were also analyzed.The results showed that the strain was poorly matched with the vaccine strain B/Colorado/06/2017 in the same year and the antigenicity might have changed.The key mutation sites of the HA and NA of this strain were displayed and marked in the three-dimensional structural model of corresponding proteins.Except for the valine（V）at position 431 of the PA,which was associated with strong virulence and non-temperature-sensitive manifestations,no other reported virulence-related molecules were found.3.The study on pathogenicity of clinical strain B/Guangxi-Jiangzhou/1352/2018 in mice.We determined the median lethal dose(LD₅₀)and the pathogenicity of strain B/Guangxi-Jiangzhou/1352/2018 in mice.The results showed that the mice were highly susceptible to this clinical strain and were easy to die.The virus titers in the lungs of mice reached a peak 1 d post infection,and typical lung lesions and inflammation appeared in the lungs,such as severe damage to the alveolar structure,inflammatory cell infiltration and increased macrophages and neutrophils,etc.At the same time,the transcription levels of inflammatory cytokines（TNF-α,IL-6 and IL-β）and interferons（IFN-α,IFN-βand IFN-γ）in the lungs of mice were significantly up-regulated post infection.Our results can help with studies on the pathogenesis and transmission mechanism of IBV,and provide an ideal animal model for evaluation of new vaccines and antiviral drugs.