| In March and early April2009, a novel influenza A virus emerged in Mexico and the United States. This newly emerged influenza virus rapidly triggered the first human pandemic of the21st century. Phylogenetic and genetic studies have revealed that the eight gene segments of the H1N1/2009virus were generated through multiple reassortment events among well-established swine influenza lineages. Notably, an extended branch of phylogeny of the H1N1/2009virus corresponds to the genetic ancestry of swine viruses, thus the H1N1/2009virus is thought to have been generated in swine and to have been evolved from a reassortant precursor virus. However, the reassortment pattern of H1N1/2009virus has not been determined in swine or human by epidemiological surveillance. Furthermore, the evolutionary characteristics of influenza virus in pigs have not been determined. Thus, the precise evolutionary pathway for the emergence of the pandemic H1N1/2009virus is largely uncertain.In the present study, to simulate the evolutionary pathway of the H1N1/2009virus, we used an artificial virus (rH1N1) with the samegene combination for serial passage in pigs. Next, genome sequencing and deep sequencing were performed to analyze the molecular characteristics of rH1N1virus in the process of adaptation. Furthermore, the pigs, guinea pigs and ferrets were used as animal models to test pathogenicity and transmissibility of the passaged rH1N1virus. Viral quasispecies from the ninth passage, which caused obvious symptoms (pyrexia, wheezing, and coughing, etc.), were systematically characterized. Deep sequencing analysis demonstrated five consensus amino acid mutations, which were PB1A469T, PA I129T, NA N329D, NS1N205K, and NEP T48N. Besides these five mutations, there were other mutations in HA proteins, and the mutations in HA proteins differed greatly between the upper and lower respiratory tracts. In upper respiratory tract, the dominant mutations of HA protein were M227I, S271P and1295V; in lower respiratory tract, the dominant mutations of HA prptein were D187E, K211E and S289N. We next performed plaque purification and genome sequencing to establish the sequence compositions of40individual clones. After analyze the mutation distribution, three representative clones were selected for detailed examination. In vitro tests showed that the three clones had significantly enhanced replication and polymerase activity relative to the parental virus, and PB1A469T was the decisive mutation for the enhanced polymerase activity. Moreover, the three clones retained the human a2,6-linked receptor binding preference of the parental rH1N1virus. Further animal experiments convinced that all the three clones had enhanced replication in pigs, guinea pigs, and ferrets, and had enhanced pathogenicity in pigs and ferrets. Cohabitation tests revealed that all the three clones acquired contact transmissibility in pigs and guinea pigs, and PB1A469T and NS1 N205K&NEP T48N were the decisive mutations for the contact transmissibility between guinea pigs. Strikingly, only one of three clones, the lung virus, carrying HA D187E, K211E, and S289N mutations, could transmit efficiently between ferrets by respiratory droples, indicating the crucial role of HA mutations in cross-species transmission.Our findings demonstrate that influenza virus can acquire enhanced pathogenicity and transmissibility via serial passage in pigs, and these passaged viruses naturally generated in pigs have the similar biological characteristics to the pandemic virus. These findings indicate that pigs can independently facilitate such combinatorial changes in the genesis of a potential human pandemic strain that has all gene segments of swine origin. Therefore, it would have been highly feasible for the generation of the emergent pandemic virus to have begun in swine which emphasizes the importance of continuous monitoring of influenza viruses in pigs. |
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