H5N1亚型禽流感病毒A/Guangxi/1/2005株抗药机制的研究

为研究H5N1亚型禽流感病毒(AIV)的抗药机制,本研究选取Clade2.3.4亚群中一株对金刚烷胺敏感的人源AIV A/Guangxi/1/2005(H5N1)(S-GX05),用抗流感病毒药物金刚烷胺对其进行定向诱导,筛选出一株抗药性病毒株,命名为R-A/Guangxi/1/2005(R-GX05)。通过全基因测序并与S-GX05全基因序列进行对比,结果显示S-GX05只在其M2蛋白中有一个氨基酸位点发生突变,即A30P;抗药性鉴定这两株病毒的半数药物抑制浓度(IC50)分别为0.9μM和48.9μM,表明R-GX05对金刚烷胺表现出一定程度的抗性。动物实验证实,这两株病毒对BALB/c小鼠的致病性基本一致,均表现出高致病性,其MLD50分别为4.7 log10 EID50和5.0 log10 EID50,两株病毒在小鼠体内各组织脏器中的分布及增殖能力也基本相同。这些结果表明,S-GX05在药物压力下产生抗药性后,并未引起其它生物学特性的改变。A30P的发现为进一步从分子水平上研究H5N1亚型AIV的抗药机制及新型抗流感新药的研发奠定了基础。

Mechanism of the H5N1 subtype influenza virus A/Guangxi/1/2005 resistance to antiviral drug

To investigate the mechanism of influenza virus resistance to antiviral drug of Amantadine,the highly pathogenic H5N1 subtype avian influenza virus(AIV) A/Guangxi/1/2005(S-GX05) was induced with 1,000 μM of Amantadine for 15 passages in MDCK cell culture and the resulting virus was converted from sensitive to resistant for Amantadine(designate R-GX05).Sequencing analysis showed that only one point mutation occurred in M2 encoding sequence and resulted in a substitution of A30P in M2 protein of R-GX05.The inhibition concentration(IC50) of Amantadine to S-GX05 and R-GX05 was 0.9 μM and 48.9 μM,respectively.However,the pathogenicity tests indicated that both viruses were highly pathogenic in mice with a median lethal dose(LD50) of 4.7 log10 EID50 and 5.0 log10 EID50,respectively.The tissue tropism and replication capacities of both viruses had no difference.In conclusion,Amantadine-resistant M2 mutants of AIV A/Guangxi/1/2005 had no change in growth characteristics or virulence in mice.The finding of A30P mutation would pave the way to further research focusing on the molecular mechanisms of influenza virus resistant to antiviral drugs.