Lomakina N F, Sadykova G K, Timofeeva T A, Rudneva I A, Boravleva E Yu, Ivanov P A, Prilipov A G, Gambaryan A S
Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products, Russian Academy of Sciences, Moscow, 108819 Russia.
N.F. Gamaleya National Center of Epidemiology and Microbiology, Ministry of Health of the Russian Federation, Moscow, 123098 Russia.
Mol Biol (Mosk). 2018 Nov-Dec;52(6):1029-1037. doi: 10.1134/S0026898418060125.
Previously, an attenuated variant Ku/at was obtained from the highly pathogenic avian influenza virus A/chicken/Kurgan/3/2005 (H5N1) by a reverse selection method aimed at increasing the virus resistance to a proteolytic cleavage and acidic pH values. In the Ku/at, 10 mutations in proteins PB2, PB1, HA, NA, and NS1 occurred. In comparison with the parental strain, the pH of the conformational transition of the viral glycoprotein hemagglutinin (HA) and virulence for mice and chickens have decreased in an attenuated variant. The purpose of this work is to clarify the role of three mutations in the stalk region of HA: Asp54Asn in HA1 and Val48Ile and Lys131Thr in HA2 (H3 HA numbering). To attain these ends, analogous substitutions were introduced into HA with a deleted polybasic cleavage site (important for pathogenicity) of the recombinant A/Vietnam/1203/04-PR8/CDC-RG (H5N1) virus, and so we created the VN3x-PR variant. Viruses VN3x-PR and Ku/at with the same three mutations, but different proteolytic cleavage sites in HA, as well as the corresponding initial viruses, were tested for pathogenicity in mice and in the erythrocyte hemolysis test. Compared with the parental strains, the virulence of their mutant variants in the case of intranasal infection of BALB/c mice decreased by 4-5 orders of magnitude, and the pH of the conformational transition of HA decreased from 5.70-5.80 to 5.25-5.30, which is typical for low pathogenic natural isolates. Thus, as a result of the study, the attenuating role of these three mutations in HA has been proved, a correlation was established between the pH value of the HA conformational transition and the virulence of H5N1 influenza viruses, and it was shown that the polybasic cleavage site of the H5 HA does not always determine high pathogenicity of the virus.
此前，通过旨在提高病毒对蛋白水解切割和酸性pH值抗性的反向选择方法，从高致病性禽流感病毒A/鸡/库尔干/3/2005（H5N1）中获得了减毒变体Ku/at。在Ku/at中，PB2、PB1、HA、NA和NS1蛋白发生了10个突变。与亲本菌株相比，减毒变体中病毒糖蛋白血凝素（HA）构象转变的pH值以及对小鼠和鸡的毒力均有所降低。本研究的目的是阐明HA茎区三个突变的作用：HA1中的Asp54Asn以及HA2中的Val48Ile和Lys131Thr（H3 HA编号）。为实现这些目标，将类似的替换引入到重组A/越南/1203/04-PR8/CDC-RG（H5N1）病毒的HA中，该病毒的多碱性切割位点已缺失（对致病性很重要），从而创建了VN3x-PR变体。对具有相同三个突变但HA中蛋白水解切割位点不同的病毒VN3x-PR和Ku/at以及相应的原始病毒进行了小鼠致病性测试和红细胞溶血试验。与亲本菌株相比，其突变变体在鼻内感染BALB/c小鼠时的毒力降低了4-5个数量级，HA构象转变的pH值从5.70-5.80降至5.25-5.30，这是低致病性自然分离株的典型特征。因此，研究结果证明了HA中这三个突变的减毒作用，建立了HA构象转变的pH值与H5N1流感病毒毒力之间的相关性，并表明H5 HA的多碱性切割位点并不总是决定病毒的高致病性。
