Abstract In 2013, a human influenza outbreak caused by a novel H7N9 virus occurred in China. Recently, the H7N9 virus acquired multiple basic amino acids at its hemagglutinin (HA) cleavage site, leading to the emergence of a highly pathogenic virus. The development of an effective diagnostic method is imperative for the prevention and control of highly pathogenic H7N9 influenza. Here, we designed and synthesized three pairs of primers based on the nucleotide sequence at the HA cleavage site of the newly emerged highly pathogenic H7N9 influenza virus. One of the primer pairs and the corresponding probe displayed a high level of amplification efficiency on which a real-time RT-PCR method was established. Amplification using this method resulted in a fluorescent signal for only the highly pathogenic H7N9 virus, and not for any of the H1–H15 subtype reference strains, thus demonstrating high specificity. The method detected as low as 39.1 copies of HA-positive plasmid and exhibited similar sensitivity to the virus isolation method using embryonated chicken eggs. Importantly, the real-time RT-PCR method exhibited 100% consistency with the virus isolation method in the diagnosis of field samples. Collectively, our data demonstrate that this real-time RT-PCR assay is a rapid, sensitive and specific method, and the application will greatly aid the surveillance, prevention, and control of highly pathogenic H7N9 influenza viruses.
Bao H, Wang X, Zhao Y, Sun X, Li Y, Xiong Y, Chen H. 2012. Development of a reverse transcription loop-mediated isothermal amplification method for the rapid detection of avian influenza virus subtype H7. Journal of Virological Methods, 179, 33-37.
Belser J A, Gustin K M, Pearce M B, Maines T R, Zeng H, Pappas C, Sun X, Carney P J, Villanueva J M, Stevens J, Katz J M, Tumpey T M. 2013. Pathogenesis and transmission of avian influenza A (H7N9) virus in ferrets and mice. Nature, 501, 556-559.
Capua I, Mutinelli F, Marangon S, Alexander D J. 2000. H7N1 avian influenza in Italy (1999 to 2000) in intensively reared chickens and turkeys. Avian Pathology: Journal of the W.V.P.A, 29, 537-543.
Gao R, Cao B, Hu Y, Feng Z, Wang D, Hu W, Chen J, Jie Z, Qiu H, Xu K, Xu X, Lu H, Zhu W, Gao Z, Xiang N, Shen Y, He Z, Gu Y, Zhang Z, Yang Y, et al. 2013. Human infection with a novel avian-origin influenza A (H7N9) virus. The New England Journal of Medicine, 368, 1888-1897.
Iuliano A D, Jang Y, Jones J, Davis C T, Wentworth D E, Uyeki T M, Roguski K, Thompson M G, Gubareva L, Fry A M, Burns E, Trock S, Zhou S, Katz J M, Jernigan D B. 2017. Increase in Human infections with avian influenza A (H7N9) virus during the fifth epidemic - China, October 2016-February 2017. Morbidity and Mortality Weekly Report, 66, 254-255.
Richard M, Schrauwen E J A, de Graaf M, Bestebroer T M, Spronken M I J, van Boheemen S, de Meulder D, Lexmond P, Linster M, Herfst S, Smith D J, van den Brand J M, Burke D F, Kuiken T, Rimmelzwaan G F, Osterhaus A D M E, Fouchier R A M. 2013. Limited airborne transmission of H7N9 influenza A virus between ferrets. Nature, 501, 560.
Shi J, Deng G, Liu P, Zhou J, Guan L, Li W, Li X, Guo J, Wang G, Fan J, Wang J, Li Y, Jiang Y, Liu L, Tian G, Li C, Chen H. 2013. Isolation and characterization of H7N9 viruses from live poultry markets-implication of the source of current H7N9 infection in humans. Chinese Science Bulletin, 58, 1857-1863.
Watanabe T, Kiso M, Fukuyama S, Nakajima N, Imai M, Yamada S, Murakami S, Yamayoshi S, Iwatsuki-Horimoto K, Sakoda Y, Takashita E, McBride R, Noda T, Hatta M, Imai H, Zhao D, Kishida N, Shirakura M, de Vries R P, Shichinohe S, et al. 2013. Characterization of H7N9 influenza A viruses isolated from humans. Nature, 501, 551-555.
Xiong X, Martin S R, Haire L F, Wharton S A, Daniels R S, Bennett M S, McCauley J W, Collins P J, Walker P A, Skehel J J, Gamblin S J. 2013. Receptor binding by an H7N9 influenza virus from humans. Nature, 499, 496-499.
Xu Y, Ramey A M, Bowman A S, DeLiberto T J, Killian M L, Krauss S, Nolting J M, Torchetti M K, Reeves A B, Webby R J, Stallknecht D E, Wan X F. 2017. Low pathogenic influenza a viruses in North American diving ducks contribute to the emergence of a novel highly pathogenic influenza a (H7N8) virus. Journal of Virology, doi: 10.1128/JVI.02208-16
Yu H, Cowling B J, Feng L, Lau E H, Liao Q, Tsang T K, Peng Z, Wu P, Liu F, Fang V J, Zhang H, Li M, Zeng L, Xu Z, Li Z, Luo H, Li Q, Feng Z, Cao B, Yang W, et al. 2013. Human infection with avian influenza A H7N9 virus: An assessment of clinical severity. Lancet, 382, 138-145.
Zhang Q, Shi J, Deng G, Guo J, Zeng X, He X, Kong H, Gu C, Li X, Liu J, Wang G, Chen Y, Liu L, Liang L, Li Y, Fan J, Wang J, Li W, Guan L, Li Q, et al. 2013. H7N9 influenza viruses are transmissible in ferrets by respiratory droplet. Science, 341, 410-414.
Zhu H, Wang D, Kelvin D J, Li L, Zheng Z, Yoon S W, Wong S S, Farooqui A, Wang J, Banner D, Chen R, Zheng R, Zhou J, Zhang Y, Hong W, Dong W, Cai Q, Roehrl M H, Huang S S, Kelvin A A, et al. 2013. Infectivity, transmission, and pathology of human-isolated H7N9 influenza virus in ferrets and pigs. Science, 341, 183-186.