Kida Yurie, Okuya Kosuke, Saito Takeshi, Yamagishi Junya, Ohnuma Aiko, Hattori Takanari, Miyamoto Hiroko, Manzoor Rashid, Yoshida Reiko, Nao Naganori, Kajihara Masahiro, Watanabe Tokiko, Takada Ayato
Division of Global Epidemiology, International Institute for Zoonosis Control, Hokkaido University, Sapporo 001-0020, Japan.
Division of Collaboration and Education, International Institute for Zoonosis Control, Hokkaido University, Sapporo 001-0020, Japan.
Pathogens. 2021 Dec 9;10(12):1597. doi: 10.3390/pathogens10121597.
Highly pathogenic avian influenza viruses (HPAIVs) with H5 and H7 hemagglutinin (HA) subtypes are derived from their low pathogenic counterparts following the acquisition of multiple basic amino acids in their HA cleavage site. It has been suggested that consecutive adenine residues and a stem-loop structure in the viral RNA region that encodes the cleavage site are essential for the acquisition of the polybasic cleavage site. By using a reporter assay to detect non-templated nucleotide insertions, we found that insertions more frequently occurred in the RNA region (29 nucleotide-length) encoding the cleavage site of an H5 HA gene that was predicted to have a stem-loop structure containing consecutive adenines than in a mutated corresponding RNA region that had a disrupted loop structure with fewer adenines. In virus particles generated by using reverse genetics, nucleotide insertions that created additional codons for basic amino acids were found in the RNA region encoding the cleavage site of an H5 HA gene but not in the mutated RNA region. We confirmed the presence of virus clones with the ability to replicate without trypsin in a plaque assay and to cause lethal infection in chicks. These results demonstrate that the stem-loop structure containing consecutive adenines in HA genes is a key molecular determinant for the emergence of H5 HPAIVs.
具有H5和H7血凝素（HA）亚型的高致病性禽流感病毒（HPAIVs）是在其HA裂解位点获得多个碱性氨基酸后，从低致病性对应病毒衍生而来的。有人提出，病毒RNA区域中编码裂解位点的连续腺嘌呤残基和茎环结构对于获得多碱性裂解位点至关重要。通过使用报告基因检测来检测非模板化核苷酸插入，我们发现，与具有较少腺嘌呤且环结构被破坏的突变对应RNA区域相比，插入更频繁地发生在预测具有包含连续腺嘌呤的茎环结构的H5 HA基因裂解位点编码的RNA区域（29个核苷酸长度）中。在使用反向遗传学产生的病毒颗粒中，在编码H5 HA基因裂解位点的RNA区域中发现了为碱性氨基酸产生额外密码子的核苷酸插入，但在突变的RNA区域中未发现。我们在噬斑测定中证实了存在能够在无胰蛋白酶的情况下复制并在雏鸡中引起致死性感染的病毒克隆。这些结果表明，HA基因中包含连续腺嘌呤的茎环结构是H5 HPAIVs出现的关键分子决定因素。
