| H5 subtype highly pathogenic avian influenza (AI) is an important zoonose which considered as endemic in many countries. It is also listed as an official notifiable disease by the World Organization for Animal Health (OIE). AI not only results in huge economic losses to the poultry industry, but also causes a serious threat to human health. Avian influenza virus (AIV) mutates frequently to adapt to the host and environment. The variant strain with advantages will survive in the process of evolution. The surveillance of recent years shows that Clade 2.3.4 has become one of the main popular branch in China and emerged diverse reassortants within this clade. Therefore, we chose two different types of HA in Clade 2.3.4(SY strain) and Clade 2.3.4.4(JY strain), combined them with three different NA subtypes (NA1, NA2 or NA8) separately to construct a series of recombinant viruses. The compatibility of the HA and NA genes of AIVs in Clade 2.3.4 was evaluated by comparing their biological characteristics and pathogenicity of the recombinant virus.1. Screening, purification of AIVs and construction of recombinant viruses containing different HA and NA genes.The AIVs SY and JY were selected as parent strain via analysis of sequence and special site of HA. SY strain was belonged to Clade 2.3.4, while JY strain was belonged to Clade 2.3.4.4. The HA connecting peptide sequence of SY was PLRERRR-KR↓G, which was consistent with that of the Re-5 vaccine strain, while that of JY strain was PLREKRR-KR↓G, with one mutation site, and JY strain also had a deglycosylation on 170 position of HA protein. Hoffmann 8 plasmid rescue system was applied to construct 10 strains of recombinant viruses containing combination of SY or JY HA with one NA1, two NA2, or two NA8, and 6 internal genes of SY. The recombinant viruses were confirmed by sequencing and named as SY, SY-ZJ, SY-CZ, SY-JYD, SY-JY, JY-SY, JY-ZJ, JY-CZ, JY-JYD, and JY. The viruses were passaged for 5 times, and viral titres were stable, indicating successful rescue of recombinant viruses.2. The biological characteristics and pathogenicity of recombinant viruses containing different HA and NA genes.The EID50S, TCID50S and MDTs of ten recombinant viruses were similar to each other. All recombinant viruses containing SY HA had α2,3 sialic acid receptor binding ability, while the recombinant viruses JY-SY and JY had α2,3 and α2,6 sialic acid dual receptor binding ability. The enzyme activities of NA1 and NA8 viruses were higher than that of NA1 virus. The time and order for expression of NP and HA proteins in all recombinant viruses were similar to each other when detected by IFA. The time for expression of NP in nucleus or cytoplasma of all recombinant viruses were also similar to each other when observed under confocal fluoresce microscopy. However, the expression of HA, NP, and M protein was higher in the recombinant H5N1 virus containing SY HA and H5N2 viruses containing JY HA, and lower in the H5N8 viruses containing JY HA. Chicken challenge study showed that the virulence of viruses contain SY HA were higher than that of the viruses contain JY HA, and there is no significant difference in virulence in all viruses containing SY HA. However, the virulence of H5N2 and H5N8 AIVs containing JY HA were higher than that of H5N1 AIV containing JY HA. The virulence of recombinant viruses containing SY HA is also higher than that of viruses containing JY HA in mice.In summary, the compatibility of HA and NA genes in AIVs affected the replication efficiency, transmission ability, and pathogenicity of the viruses. The enhanced virulence of H5N2 and H5N8 AIVs containing HA from Clade 2.3.4.4 AIVs will be helpful for virus infection and transmission, and plays an important role in epidemiology. |
PDF Full Text Request