ORIGINAL ARTICLE Inhibition of influenza A virus replication by RNA interference targeted against the PB1 subunit of the RNA polymerase gene Wanyi Li • Xiaofan Yang • Yan Jiang • Baoning Wang • Yuan Yang • Zhonghua Jiang • Mingyuan Li Received: 16 February 2011 / Accepted: 27 July 2011 / Published online: 12 August 2011 Ó Springer-Verlag 2011 Abstract Influenza (flu) pandemics have posed a great threat to human health in the last century. However, current vaccination strategies and antiviral drugs provide limited protection. RNA interference (RNAi) is an effective means of suppressing influenza virus replication. PB1 is the crit- ical protein subunit of the influenza virus RNA polymerase. The gene encoding this protein, PB1, is highly conserved among different subtypes of IAV and was therefore chosen as the target in this study. The oligonucleotide, PB1- shRNA, contains a 21-bp siRNA corresponding to nucle- otides 1,632 to 1,652 of PB1 linear vRNA with BamHI or EcoRI restriction enzyme sites incorporated at the ends. The PB1-shRNA oligonucleotide was directionally cloned into the RNAi-ready pSIREN-shuttle vector. The correct structure of the resulting pSIREN/PB1 plasmid was con- firmed by restriction endonuclease digestion. Madin-Darby canine kidney (MDCK) cells were transfected with pSI- REN/PB1 and subsequently infected with IAV at an MOI of 0.1 (A/PR/8/34, H1N1). The virus titer in cell culture supernatants was determined 48 hours later, and it was found that virus growth was inhibited by more than 50-fold relative to controls. Furthermore, embryonated eggs and mice were inoculated with liposome-encapsulated pSI- REN/PB1 and then challenged with the A/PR/8/34 virus. The results showed at least a 100-fold inhibition in virus replication in egg allantoic fluid and a survival rate of between 50% and 100% in experimental mice. This study demonstrates that PB1-shRNA expressed by the recombi- nant plasmid pSIREN/PB1 inhibits influenza A virus rep- lication both in vitro and in vivo. These observations provide a foundation for the development of a new and efficient treatment of influenza infections. Introduction Influenza A virus (IAV) infection remains a very serious threat to human health. A human influenza (flu) pandemic could cause 20% of the global population to become ill [1, 2]. In the past decades, avian flu has emerged among chickens and humans in some developing countries, so that close to 30 million people would need to be hospitalized, a quarter of whom would die within a few months [3, 4]. In April 2009, human infections caused by an influenza virus designated the influenza A pandemic (H1N1) 2009 virus were identified in the United States and Mexico and spread rapidly to other regions of the world [5–7]. With the potential for a new influenza pandemic and the limited protection provided by current vaccine strategies and antiviral drugs, the requirement for new approaches is clear. IAV is a major cause of life-threatening respiratory tract disease worldwide [8]. The main site of IAV infection is the respiratory mucosa, including nasal sinuses, W. Li and X. Yang contributed equally to this work. W. Li Á X. Yang Á B. Wang Á Y. Yang Á Z. Jiang Á M. Li (&) Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, Sichuan, China e-mail: lmy3985@sina.com Y. Jiang Department of Microbiology, Guiyang Medical College, Guiyang 550004, China M. Li State Key Laboratory of Oral Diseases (Sichuan University), Chengdu 610041, Sichuan, China 123 Arch Virol (2011) 156:1979–1987 DOI 10.1007/s00705-011-1087-8