Shi Huoying, Liu Xiu Fan, Zhang Xiaorong, Chen Sujuan, Sun Lei, Lu Jianhong
Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou University, 225009 Yangzhou, Jiangsu, People's Republic of China.
Vaccine. 2007 Oct 16;25(42):7379-84. doi: 10.1016/j.vaccine.2007.08.011. Epub 2007 Aug 24.
In the face of disease outbreaks in poultry and the potential pandemic threat to humans caused by the highly pathogenic avian influenza viruses (HPAIVs) of H5N1 subtype, improvement in biosecurity and the use of inactivated vaccines are two main options for the control of this disease. Vaccine candidates of influenza A viruses of H5N1 subtype have been generated in several laboratories by plasmid-based reverse genetics with hemagglutinin (HA) and neuraminidase (NA) genes from the epidemic strains of avian viruses in a background of internal genes from the vaccine donor strain of human strains, A/Puerto Rico/8/34 (PR8). These reassortant viruses containing genes from both avian and human viruses might impose biosafety concerns, also may be do if C4/F AIV would be a live attenuated vaccine or cold-adaptive strain vaccine. In order to generate better and safer vaccine candidate viruses, we genetically constructed attenuated reassortant H5N1 influenza A virus, designated as C4/F AIV, by plasmid-based reverse genetics with all eight genes from the avian strains. The C4/F AIV virus contained HA and NA genes from an epidemic strain A/Chicken/Huadong/04 (H5N1) (C4/H5N1) in a background of internal genes derived from a low pathogenic strain of A/Chicken/F/98(H9N2). The reassortant virus was attenuated by removal of the multibasic amino acid motif in the HA gene by mutation and deletion (from PQRERRRKKR (downward arrow) G to PQIETR (downward arrow) G). The intravenous pathogenicity index (IVPI) of C4/F AIV virus was 0, whereas that of the donor virus C4/H5N1 was 3.0. The virus HA titer of C4/H5N1 in the allantoic fluid from infected embryonated eggs was as high as 1:2048. The inactivated vaccine prepared from the reassortant virus C4/F AIV-induced high HI titer in vaccinated chickens and gave 100% protection when challenged with highly pathogenic avian influenza virus of H5N1 subtype.
面对家禽疾病爆发以及H5N1亚型高致病性禽流感病毒（HPAIVs）对人类造成的潜在大流行威胁，加强生物安全措施和使用灭活疫苗是控制这种疾病的两个主要选择。几个实验室已通过基于质粒的反向遗传学方法，利用来自禽流感流行毒株的血凝素（HA）和神经氨酸酶（NA）基因，并以人源毒株A/波多黎各/8/34（PR8）疫苗供体株的内部基因为背景，构建了H5N1亚型甲型流感病毒候选疫苗。这些含有禽流感病毒和人源病毒基因的重配病毒可能存在生物安全问题，如果C4/F AIV是减毒活疫苗或冷适应株疫苗，也可能存在此类问题。为了构建更好、更安全的候选疫苗病毒，我们通过基于质粒的反向遗传学方法，利用来自禽流感毒株的所有八个基因，构建了减毒重配H5N1甲型流感病毒，命名为C4/F AIV。C4/F AIV病毒在源自低致病性A/鸡/F/98（H9N2）毒株的内部基因背景下，含有来自流行毒株A/鸡/华东/04（H5N1）（C4/H5N1）的HA和NA基因。通过突变和缺失（从PQRERRRKKR（向下箭头）G变为PQIETR（向下箭头）G）去除HA基因中的多个碱性氨基酸基序，使重配病毒减毒。C4/F AIV病毒的静脉致病性指数（IVPI）为0，而供体病毒C4/H5N1的IVPI为3.0。C4/H5N1在感染的鸡胚尿囊液中的病毒HA滴度高达1:2048。用重配病毒C4/F AIV制备的灭活疫苗在接种鸡中诱导出高血凝抑制（HI）滴度，并在受到H5N1亚型高致病性禽流感病毒攻击时提供100%的保护。
