Effects of PB 1F 2 on the pathogenicity of H 1 N 1 swine influenza virus in mice and pigs

Although several studies have exploited the effects of PB1-F2 in swine influenza viruses, its contribution to the pathogenicity of swine influenza viruses remains unclear. Herein, we investigated the effects of PB1-F2 on the pathogenicity of influenza virus using a virulent H1N1 A/swine/Kansas/77778/2007 (KS07) virus, which expresses a full-length PB1-F2, in mice and pigs. Using reverse genetics, we generated the wild-type KS07 (KS07_WT), a PB1-F2 knockout mutant (KS07_K/O) and its N66S variant (KS07_N66S). KS07_K/O showed similar pathogenicity in mice to the KS07_WT, whereas KS07_N66S displayed enhanced virulence when compared to the other two viruses. KS07_WT exhibited more efficient replication in lungs and nasal shedding in infected pigs than the other two viruses. Pigs infected with the KS07_WT had higher pulmonary levels of granulocyte-macrophage colony-stimulating factor, IFN-g, IL-6 and IL-8 at 3 and 5 days post-infection, as well as lower levels of IL-2, IL-4 and IL-12 at 1 day post-infection compared to those infected with the KS07_K/O. These results indicate that PB1-F2 modulates KS07 H1N1 virus replication, pathogenicity and innate immune responses in pigs and the single substitution at position 66 (N/S) in the PB1-F2 plays a critical role in virulence in mice. Taken together, our results provide new insights into the effects of PB1-F2 on the virulence of influenza virus in swine and support PB1-F2 as a virulence factor of influenza A virus in a strainand host-dependent manner. INTRODUCTION Influenza A virus (IAV) is a single-stranded negative-sense RNA virus of the family Orthomyxoviridae that infects a wide variety of host species, from wild and domestic birds to several mammalian species including humans. It causes annual epidemics and occasional pandemics in humans, which leads to significant public health and economic burdens worldwide. Swine influenza is caused by IAV and is an important acute respiratory disease to the swine industry and has also become a considerable zoonotic agent that threatens public health [1, 2]. This fact is exemplified by the 2009 pandemic H1N1 influenza virus, which is considered to be a swine-origin virus [3]. The first swine influenza virus (SIV) isolated from pigs was the H1N1 subtype in 1930 [4], also known as the classical H1N1 virus (cH1N1), which circulated predominantly in North American swine herds until 1998. Then, a novel triple-reassortant (TR) H3N2 SIV emerged in 1998 and successfully became established and widespread in North American pig populations [5, 6]. The TR H3N2 virus contains the haemagglutinin, neuraminidase (NA) and PB1 genes from human IAVs; the PB2 and PA genes from avian IAVs; and the M, NP and NS genes from the cH1N1 virus [5, 6]. Subsequently, TR H3N2 viruses reassorted with cH1N1 SIVs and human seasonal H1N1 viruses, resulting in novel reassortants such as H1N2, reassortant H1N1 (rH1N1) and H3N2 SIVs, which have become endemic in North American swine herds [7–10]. These reassortant viruses have a similar composition of six internal genes of human origin (PB1), avian origin (PB2 and PA) and classical swine origin (M, NS and NP), which has been called the triple-reassortant internal gene cassette (TRIG) [10, 11]. The TRIG cassette is readily able to accept different haemagglutinin and NA combinations [10]. With the introduction of the 2009 pandemic H1N1 virus into pigs, the Received 9 October 2016; Accepted 22 December 2016 Author affiliations: Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, KS, USA; Innovation Team for Pathogen Ecology Research on Animal Influenza Virus, Department of Avian Infectious Disease, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, PR China; School of Animal Science, Fujian Agriculture and Forestry University, Fuzhou, PR China. *Correspondence: Wenjun Ma, wma@vet.k-state.edu