| H9N2 and H1N1 subtype influenza viruses are two important causative agents in the family of Orthomya’oviridae type A influenza virus. Their genomes contain 8 gene segments encoding more than 11 proteins. H9N2 influenza virus was initially isolated in Italy in 1966, while H1N1 swine influenza virus was first reported in the United States in 1918. Recent studies showed that both the H9N2 and H1N1 were capable of breaking through barriers between species to infect animals such as swine and ferrets. It is worth noting that there is no evidence indicating that H9N2 influenza can be transmitted to canine. Studies on pathogenicity of H1N1 influenza virus are rarely performed in Guangxi. Therefore, carrying out epidemiological survey, genetic evolution analysis and pathogenic research on H9N2 and H1N1 are of great significance.588 swab samples from pet dogs and 1038 lung tissues samples from infected pigs were collected in various regions and cities of Guangxi and went through virus isolation. By inoculating the collected samples with 10-day-old SPF chick embryos and collecting allantoic fluid, thirteen isolates from canine swabs were identified as H9N2 subtype and fourteen from swine lung tissues as H1N1 subtype influenza virus. Sequence analysis indicated that all the H9N2 isolates had a high amino acid homology (>99.0%) with A/equine/Guangxi/3/2011 and their internal genes (NP, M, PB1, PB2, PA and NS genes) demonstrated a close evolutionary relationship with the human H7N9 which outbroke in 2013. The 8 gene segments of H9N2 isolates derived from five classic H9N2 influenza virus strains formed a new U genotype. Among the 14 avian-like H1N1 swine influenza virus isolates, the isolates isolated in early 2013 had all the 8 gene segments belonging to avian-like substrain; No.7 and No.8 isolate, S2 isolate and No.32 isolate of the March and April isolates had one, two and three gene segments belong to 2009 H1N1, respectively; 6 isolates collected at the end of May 2013 and March 2014 had six segments coming from H1N1, with an increasing trend of virus genes originating from 2009 H1N1. In addition, mutation from aspartic acid to asparagine occurred at the 701st PB2 amino acid locus of G2, G14, G21, G30, S2,32,7 and 8 isolates, which illustrated that the strains were gradually adapting to human derived.It was noted that thirteen H9N2 virus strains isolated from swab samples grew well in chicken embryos and contained 106.67～108.33 EID50 per milliliter. H1N1 swine influenza virus isolated from infected swine tissues grew well in MDCK cells and contained 106.0～108.33 TCID50 per milliliter. What’s more,106EID50 or TCID50 of virus was injected in the BALB/c mice by intranasal route. The results showed that all mice inoculated with H9N2 subtype isolated from healthy pet dogs could not be infected, while the H9N2 viruses isolated from dogs with influenza were capable of replicating in mice, leading to significant clinical symptoms without causing death. Avian-like H1N1 swine influenza viruses could replicate in mice, and No.6 isolate possessesd of the highest virulence to mice, causing significant clinical symptoms and rapid weight loss, and all mice died in one week. After anatomy, a level of viral load was detected in lung, turbinate, spleen and kidney. No.8 isolate had the weakest virulence and only caused mild symptoms which recovered quickly, and the titer detected in lung tissue was 104.0±0.33TCID50/mL.To study the pathogenicity of H9N2 strain and H1N1 strain, passage trials on the H9N2 strain and H1N1 strain were conducted. The results illustrated that the virulence of H9N2 and H1N1 would be enhanced after serial passage in mammal. The amino acids loci change enhancing the virulence of the viruses occurred at the 611st and PA 623 rd loci of PB2 of H9N2. After 4 passages, H1N1 had changes relative to the parent strain at 17 amino acid loci. By analyzing these loci, it was found that the change of H1N1 amino acid locus was induced by the mutation L→T at 357th locus of PA gene. Mice injected with avian-like H1N1 swine influenza virus No.7 and B2 could recover from weight loss on 7th day after the injection but could experience weight loss again on 10th day, which indicated that this virus could cause a second infection in mice with a poor immunogenicity.We investigated the epidemiology, genetic evolution and pathogenicity of the canine H9N2 subtype and swine avian-like H1N1 obtained in Guangxi, China. The results showed that H9N2 influenza virus was wildly spreading in canine in Guangxi and expanding its host range; avian-like H1N1 influenza was also ubiquitous in pigs; occurring of amino acid variation could affect the pathogenicity of the virus. The experimental results provided a scientific basis for future studies on the pathogenic mechanism and prevention and control technology of influenza virus. |
PDF Full Text Request