The Transmissibility In Guinea Pigs And Pathogenicity In Mice Of The H9N2 Reassortant Influenza Viruses With The Internal Genes From The Pandemic H1N1/09 Viruses

Airborne pathway as an efficient route of influenza virus transmission has been paid a lot attention by the public nowadays. The pdmH1N1/09 virus is able to transmit by airborne route in ferrets and guinea pigs, and to cause deaths in human beings. By reverse genetic technology, recent studies have shown that there is a high genetic compatibility between pdmH1N1/09 and other subtypes of avian and human seasonal influenza viruses. The hybrid H5N1 avian influenza virus (A/duck/Guangxi/35/2001) with the PA and NS segments of a pdmH1N1/09 virus (A/Sichuan/1/2009) can transmit efficiently in guinea pigs by respiratory droplet.H9N2 AIVs have been reported in poultre from many countries across the world since early 1960s and in China since 1990s, which can cross species barrier to infect mammalians occasionally. In recent years, the epidemiological surveillance showed that the number of the viruses carrying a 226L in t HA has become dominant. In addition, the receptor-binding test has also shown that the H9N2 viruses can attach to the human-like receptor. Meanwhile, the E627K mutation, known as the "humanization" is also present in the PB2 gene of the H9N2 AIVs, which is essential for AIVs to transmit among mammals by air droplet. Notably, it is reported that some wild-type H9N2 AIVs could transmit efficiently among ferrets by air droplet.H9N2 subtype AIVs and pdmH1N1/09 viruses can be isolated in the same area, even in the same animal (i.e. swine, canine, cats). Thus, it seems likely that the co-infection of these two subtype viruses accercelates the emergence of the reassortments between these two viruses. Furthermore, it is reported that the H7N7, H7N9, H10N8 and H5N2 influenza viruses possess the internal genes from the H9N2 influenza virus. Therefore, for the preparedness for the prevention for pandemic influenza, it is meaningful to explore the possibility of the reassortment between H9N2 AIVs and pdmH1N1/09 viruses and to study the transmissibility and pathogenicity of these reassortants.Our previous study has shown that reassortants with the HA and NA genes of the H9N2 virus and the internal genes from the pdmH1N1/09 virus were airborne transmissible in guinea pigs. However, it is not clear which internal gene(s) of the pdmH1N1/09 virus contribute to the transmissibility of the reassortant H9N2 virus. In this study, we generated a panel of recombinants between the H9N2 and pdmH1N1/09 viruses and compared their transmissibility in guinea pigs and pathogenicity in mice. We found that the NP, M and NS genes of the pdmH1N1/09 virus together contributed to the airborne transmission activity in guinea pigs and a increased pathogenicity in mice of the reassortant H9N2 virus.1、Rescue and determination of the airborne transmissibility of the reassortant H9N2 influenza viruses in guinea pigsFirstly, we made a phylegenetic analysis of the H9N2 subtype AIVs in eastern China from 2007 to 2013. We found that the S genotype was the predominant type. In addition, these viruses were quite different from the published H9N2 AIVs that reassorted with the pdmH1N1/09 virus. Therefore, we selected the H9N2 AIV of predominant S genotype, A/Chicken/Jiangsu/Wujin/57/2012 (WJ57) and the representative 2009 pandemic H1N1 virus, A/California/04/2009(CA/04)as the parental viruses to generate reassortant viruses by reverse genetics for further study.We first rescued the W(HA+NA)-C reassortant H9N2 virus containing the HA and NA gene from the WJ57 and the internal genes from the CA/04 virus. We then investigated the transmissibility of the W(HA+NA)-C, WJ57 and CA/04 viruses in guinea pigs. We found that the W(HA+NA)-C virus acquired the ability to transmit efficiently in guinea pigs. Then, we replaced the internal genes of WJ57 respectively into the W(HA+NA)-C virus and tested their airborne transmissibility. We found that the reassortants carrying the NP [W(NP+HA+NA)-C], M [W(M+HA+NA)-C] or NS [W(NS+HA+NA)-C] gene of the WJ57 lost their airborne transmissibility in guinea pigs. These results indicated that the NP, M and NS of the CA/04 played a crucial role in the airborne transmission activity of the reassortants. Then, we generated three more reassortants, namely the C(M)-W, C(M+NS)-W and C(M+NS+NP)-W which carry the M, M+NS, M+NS+NP segments in the WJ57 genetic backbone. Our results showed that only the C(NP+M+NS)-W reassortant can transmit and shed efficiently in guinea pigs.2% Pathogenicity test of the reassortant H9N2 influenza viruses in miceTo evlaute the potential threat of reassortant H9N2 influenza viruses to the mammals, we further compared their viral replications both in vivo and in vitro and also investigated their pathogenicity in mice with CA/04, WJ57, W(HA+NA)-C, W(HA+NA+NP)-C, W(HA+NA+M)-C, W(HA+NA+NS)-C and C(M+NS+NP)-W viruses. The results revealed that the C(M+NS+NP)-W performed better replication in vitro and higher pathogenicity than those of the parental WJ57 virus, whereas the W(HA+NA+NP)-C, W(HA+NA+M)-C and W(HA+NA+NS)-C viruses showed decreased viral replication in vitro and reduced virulence in mice when compared to the CA/04 virus. These results showed that the H9N2 reassortant integrated with the M, NP and NS genes from the CA/04 virus not only obtain the airborne transmission ability but also can increase pathogenicity in a mosue model.