| The2009pandemic H1N1influenza A virus (pH1N1) caused the first influenza pandemic of the21st century, and it rapidly spread throughout the world in a shot time with high transmissibility in humans. Phylogenetic analysis showed that pH1N1arose from a reassortment of two swine influenza viruses, namely, North American triple-reassortant virus and Eurasian avian-like virus. It is the first direct evidence indicates that swine, the "intermediate host" of influenza virus, is important for the generation of the pandemic influenza virus. Now more and more reports showed that the pH1N1has transmitted from human to swine and reassorted with the endemic swine influenza virus, and the opportunity for the reassortment of avian influenza virus and pH1N1resulting in the generation of new strains with potential pandemic risk was also existed. To predict and prevent the next pandemic influenza, the surveillance of swine influenza viruses and studying the pathogenicity and transmissibility of pH1N1and their underlying mechanism are of crucial importance.Four pHlNls have been isolated from the pigs in Jiangsu province during the surveillance of swine influenza viruses in2010by our laboratory. Genetic analysis showed that these4isolates closely related to the reference strain of2009H1N1virus A/California/04/2009(CA04). To determine the pathogenicity of these2009pandemic H1N1viruses in mammalians, BALB/c and C57BL/6mice models were used to detect the MLD50. The results showed that all the4swine isolates were more virulent than CA04, and A/Swine/Jiangsu/38/2010(JS38) was the most virulent virus in the BALB/c mice when compared with CA04. Besides, the JS38caused much more body weight loss and highly death rate in BALB/c mice. The study of pathogenic mechanism showed that the ability of replication of JS38was obviously stronger than CA04in mammalian cells in vitro and mouse lungs in vivo, and JS38induced more severe lung lesions. The detection of lung wet to dry weight ratio and lung wet weight to body weight ratio also showed that JS38induce more acute lung edema in mice than CA04. As pulmonary aberrant immune response is a significant feature of ARDS induced by2009H1N1virus, the numbers of macrophages was dramatically decreased while the numbers of lymphocytes and neutrophils was dramatically increased in lungs of JS38-infeced mice on3d.p.i and5d.p.i. Both JS38and CA04induced high levels of many cytokines and chemokines in lungs of infected mice, but only IL-6, IL-10and IFN-β were significantly higher in JS38-infeced mice. These results were very similar to the human clinical cases infected with pH1N1s, indicating that we successfully built the mouse model to evaluate the pathogenicity of2009H1N1viruses with different virulence by using JS38and CA04.Binding to the a2,6-sialoglycan receptor is an essential prerequisite for influenza virus to transmit efficiently in mammalian hosts. To determine the transmissibility of these pH1N1s, we firstly detected their receptor-binding preferences. The results showed that CA04exhibited an absolute preference for a2,6-sialoglycan (human receptor) binding, while the other4swine2009pandemic H1N1viruses showed various degree of binding ability for a2,3-sialoglycan (avian receptor). However, the avian receptor preferences of JS46and JS48are dominated. Consistent with the receptor-binding preferences, limited direct-contact transmissibility of JS46and JS48was found in guinea pigs model, but the other3viruses transmitted with100%efficiency. We hypothesized that switch of the receptor-binding preferences of JS46and JS48induced the decreased direct-contact transmissibility. By using the bioinformatics, we compared the HA protein sequences between JS48and CA04and analyzed their protein structures. The N159D and Q226R mutations in HA were considered as the key molecular factors to switch the receptor-binding preferences. To test the hypothesis and exclude the effect of the other mutations, we generated four recombinant viruses by reverse genetics:the rCA04was the same as wild type CA04, the rCA04-159and rCA04-226had N159D and Q226R mutations respectively, while the rCA04-159/226had both mutations. The results of direct-contact transmission showed that no virus shedding could be detected in the nasal wash of the guinea pigs in rCA04-159/226contact group, while the other3rescued viruses transmitted with100%efficiency. Further statistical analysis showed that the rCA04-l59/226had a significantly decreased replication capacity in the nasal of inoculated guinea pigs. We also found that the rCA04-226transmitted slowly in guinea pigs, indicating Q226R could affect the speed of transmission. However, seroconversion was detected in60%of the guinea pigs of rCA04-159/226contact group, indicating that the combined effect of N159D and Q226R in the HA significantly decrease the direct-contact transmissibility of pH1Nl, but did not abolish.The h9.4.2.5-lineage H9N2subtype avian influenza virus, which was dominant in China in recent years, was also isolated from the pig during the surveillance of swine influenza virus in Jiangsu province. Co-infection with H9N2and pH1N1viruses in pigs provides the opportunity for reassortment to occur, resulting in the generation of new strains with potential pandemic risk. To predict and prevent the potential pandemic risk of reassortment, we generated six reassortant H9viruses in the genetic background of three pH1N1strains from different hosts by replacing either the HA (H9N1-pH1N1) or both the HA and NA genes (H9N2-pH1N1) from the h9.4.2.5-lineage H9N2virus A/Swine/Taizhou/5/08(TZ5). The reassortant H9viruses replicated to higher titers in mammalian cells and gained efficient replication capacity in the lungs of guinea pigs and mice when compared with the parental H9N2virus. The parental H9N2virus TZ5could not transmitted between guinea pigs, but the reassortant H9viruses gained efficient transmissibility through both direct contact and droplet. However, the H9N2-pH1N1viruses were transmitted more efficiently than the H9N1-pH1N1viruses. The detection of MLD50, body weight loss and mean days of death in mice model also showed that the reassortant H9viruses were more pathogenic than TZ5. Interestingly, in contract to transmissibility, the H9N1-pH1N1viruses were more virulent than the H9N2-pH1N1viruses.In this study, we compared the pathogenicity and transmissibility of the pH1N1viruses isolated from human and swine, and investigated the mechanisms of pathogenicity and direct-contact transmission in mammal models. We also proved that there is a potential pandemic risk of reassortment between H9N2avian influenza and pH1N1viruses in pigs. These results are of significance to the control of the swine influenza and prediction of human pandemic influenza. |
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