Influenza A virus acquires enhanced pathogenicity and transmissibility after serial passages in swine.
ABSTRACT: Genetic and phylogenetic analyses suggest that the pandemic H1N1/2009 virus was derived from well-established swine influenza lineages; however, there is no convincing evidence that the pandemic virus was generated from a direct precursor in pigs. Furthermore, the evolutionary dynamics of influenza virus in pigs have not been well documented. Here, we subjected a recombinant virus (rH1N1) with the same constellation makeup as the pandemic H1N1/2009 virus to nine serial passages in pigs. The severity of infection sequentially increased with each passage. Deep sequencing of viral quasispecies from the ninth passage found five consensus amino acid mutations: PB1 A469T, PA 1129T, NA N329D, NS1 N205K, and NEP T48N. Mutations in the hemagglutinin (HA) protein, however, differed greatly between the upper and lower respiratory tracts. Three representative viral clones with the five consensus mutations were selected for functional evaluation. Relative to the parental virus, the three viral clones showed enhanced replication and polymerase activity in vitro and enhanced replication, pathogenicity, and transmissibility in pigs, guinea pigs, and ferrets in vivo. Specifically, two mutants of rH1N1 (PB1 A469T and a combination of NS1 N205K and NEP T48N) were identified as determinants of transmissibility in guinea pigs. Crucially, one mutant viral clone with the five consensus mutations, which also carried D187E, K211E, and S289N mutations in its HA, additionally was able to infect ferrets by airborne transmission as effectively as the pandemic virus. Our findings demonstrate that influenza virus can acquire viral characteristics that are similar to those of the pandemic virus after limited serial passages in pigs. Importance: We demonstrate here that an engineered reassortant swine influenza virus, with the same gene constellation pattern as the pandemic H1N1/2009 virus and subjected to only nine serial passages in pigs, acquired greatly enhanced virulence and transmissibility. In particular, one representative pathogenic passaged virus clone, which carried three mutations in the HA gene and five consensus mutations in PB1, PA, NA, NS1, and NEP genes, additionally was able to confer respiratory droplet transmission as effectively as the pandemic H1N1/2009 virus. Our findings suggest that pigs can readily induce adaptive mutational changes to a precursor pandemic-like virus to transform it into a highly virulent and infectious form akin to that of the pandemic H1N1/2009 virus, which underlines the potential direct role of pigs in promoting influenza A virus pathogenicity and transmissibility.
Project description:Here, we assessed the effects of PB1-F2 and NS1 mutations known to increase the pathogenicity of influenza viruses on the replication and pathogenicity in mice of pandemic (H1N1) 2009 influenza viruses. We also characterized viruses possessing a PB1-F2 mutation that was recently identified in pandemic (H1N1) 2009 influenza virus isolates, with and without simultaneous mutations in PB2 and NS1. Our results suggest that some NS1 mutations and the newly identified PB1-F2 mutation have the potential to increase the replication and/or pathogenicity of pandemic (H1N1) 2009 influenza viruses.
Project description:Pandemic H1N1/2009 influenza virus, derived from a reassortment of avian, human, and swine influenza viruses, possesses a unique gene segment combination that had not been detected previously in animal and human populations. Whether such a gene combination could result in the pathogenicity and transmission as H1N1/2009 virus remains unclear. In the present study, we used reverse genetics to construct a reassortant virus (rH1N1) with the same gene combination as H1N1/2009 virus (NA and M genes from a Eurasian avian-like H1N1 swine virus and another six genes from a North American triple-reassortant H1N2 swine virus). Characterization of rH1N1 in mice showed that this virus had higher replicability and pathogenicity than those of the seasonal human H1N1 and Eurasian avian-like swine H1N1 viruses, but was similar to the H1N1/2009 and triple-reassortant H1N2 viruses. Experiments performed on guinea pigs showed that rH1N1 was not transmissible, whereas pandemic H1N1/2009 displayed efficient transmissibility. To further determine which gene segment played a key role in transmissibility, we constructed a series of reassortants derived from rH1N1 and H1N1/2009 viruses. Direct contact transmission studies demonstrated that the HA and NS genes contributed to the transmission of H1N1/2009 virus. Second, the HA gene of H1N1/2009 virus, when combined with the H1N1/2009 NA gene, conferred efficient contact transmission among guinea pigs. The present results reveal that not only gene segment reassortment but also amino acid mutation were needed for the generation of the pandemic influenza virus.
Project description:BACKGROUND:Influenza viruses can generate novel reassortants in coinfected cells. The global circulation and occasional introductions of pandemic H1N1/2009 virus in humans and in pigs, respectively, may allow this virus to reassort with other influenza viruses. These possible reassortment events might alter virulence and/or transmissibility of the new reassortants. Investigations to detect such possible reassortants should be included as a part of pandemic influenza surveillance plans. METHODS:We established a real-time reverse-transcription (RT)-PCR-based strategy for the detection of reassortment of pandemic H1N1/2009 virus. Singleplex SYBR green-based RT-PCR assays specific for each gene segment of pandemic H1N1/2009 were developed. These assays were evaluated with influenza viruses of various genetic backgrounds. RESULTS:All human pandemic H1N1 (n = 27) and all seasonal human (n = 58) isolates were positive and negative, respectively, for all 8 segments. Of 48 swine influenza viruses isolated from our ongoing surveillance program of influenza viruses in swine, 10 were positive in all reactions. All 8 viral segments of these 10 samples were confirmed to be of pandemic H1N1 origin, indicating that these were caused by zoonotic transmissions from human to pigs. The 38 swine viruses that were nonpandemic H1N1/2009 had 1-6 gene segments positive in the tests. Further characterization of these nonpandemic H1N1/2009 swine viruses indicated that these PCR-positive genes were the precursor genes of the pandemic H1N1/2009 virus. CONCLUSIONS:Our results demonstrated that these assays can detect reintroductions of pandemic H1N1/2009 virus in pigs. These assays might be useful screening tools for identifying viral reassortants derived from pandemic H1N1/2009 or its precursors.
Project description:BACKGROUND: Influenza A virus encodes for eleven proteins, of which HA, NA, NS1 and PB1-F2 have been implicated in viral pathogenicity and virulence. Thus, in addition to the HA and NA gene segments, monitoring diversity of NS1 and PB1-F2 is also important. METHODS: 55 out of 166 circulating influenza A strains (31 H1N1 and 24 H3N2) were randomly picked during 2007-2009 and NS and PB1-F2 genes were sequenced. Phylogenetic analysis was carried out with reference to the prototype strains, concurrent vaccine strains and other reference strains isolated world wide. RESULTS: Comparative analysis of both nucleotide and deduced amino acid sequences, revealed presence of NS gene with A/PR/8/34(H1N1)-like mutations (H4N, Q21R, A22V, K44R, N53D, C59R, V60A, F103S and M106I) in both RNA-binding and effector domain of NS1 protein, and G63E, the HPAI-H5N1-like mutation in NEP/NS2 of five A/H1N1 strains of 2007 and 2009. NS1 of other A/H1N1 strains clustered with concurrent A/H1N1 vaccine strains. Of 31 A/H1N1 strains, five had PB1-F2 similar to the H3N2 strains; six had non-functional PB1-F2 protein (11 amino acids) similar to the 2009 pandemic H1N1 strains and rest 20 strains had 57 amino acids PB1-F2 protein, similar to concurrent A/H1N1 vaccine strain. Interestingly, three A/H1N1 strains with H3N2-like PB1-F2 protein carried primitive PR8-like NS gene. Full gene sequencing of PB1 gene confirmed presence of H3N2-like PB1 gene in these A/H1N1 strains. CONCLUSION: Overall the study highlights reassortment event involving gene segments other than HA and NA in the co-circulating A/H1N1 and A/H3N2 strains and their importance in complexity of influenza virus genetics. In contrast, NS and PB1-F2 genes of all A/H3N2 eastern India strains were highly conserved and homologous to the concurrent A/H3N2 vaccine strains suggesting that these gene segments of H3N2 viruses are evolutionarily more stable compared to H1N1 viruses.
Project description:At least 10 different genotypes of novel reassortant H3N2 influenza viruses with 2009 pandemic H1N1 [A(H1N1)pdm09] gene(s) have been identified in U.S. pigs, including the H3N2 variant with a single A(H1N1)pdm09 M gene, which has infected more than 300 people. To date, only three genotypes of these viruses have been evaluated in animal models, and the pathogenicity and transmissibility of the other seven genotype viruses remain unknown. Here, we show that three H3N2 reassortant viruses that contain 3 (NP, M, and NS) or 5 (PA, PB2, NP, M, and NS) genes from A(H1N1)pdm09 were pathogenic in pigs, similar to the endemic H3N2 swine virus. However, the reassortant H3N2 virus with 3 A(H1N1)pdm09 genes and a recent human influenza virus N2 gene was transmitted most efficiently among pigs, whereas the reassortant H3N2 virus with 5 A(H1N1)pdm09 genes was transmitted less efficiently than the endemic H3N2 virus. Interestingly, the polymerase complex of reassortant H3N2 virus with 5 A(H1N1)pdm09 genes showed significantly higher polymerase activity than those of endemic and reassortant H3N2 viruses with 3 A(H1N1)pdm09 genes. Further studies showed that an avian-like glycine at position 228 at the hemagglutinin (HA) receptor binding site is responsible for inefficient transmission of the reassortant H3N2 virus with 5 A(H1N1)pdm09 genes. Taken together, our results provide insights into the pathogenicity and transmissibility of novel reassortant H3N2 viruses in pigs and suggest that a mammalian-like serine at position 228 in the HA is critical for the transmissibility of these reassortant H3N2 viruses.Swine influenza is a highly contagious zoonotic disease that threatens animal and public health. Introduction of 2009 pandemic H1N1 virus [A(H1N1)pdm09] into swine herds has resulted in novel reassortant influenza viruses in swine, including H3N2 and H1N2 variants that have caused human infections in the United States. We showed that reassortant H3N2 influenza viruses with 3 or 5 genes from A(H1N1)pdm09 isolated from diseased pigs are pathogenic and transmissible in pigs, but the reassortant H3N2 virus with 5 A(H1N1)pdm09 genes displayed less efficient transmissibility than the endemic and reassortant H3N2 viruses with 3 A(H1N1)pdm09 genes. Further studies revealed that an avian-like glycine at the HA 228 receptor binding site of the reassortant H3N2 virus with 5 A(H1N1)pdm09 genes is responsible for less efficient transmissibility in pigs. Our results provide insights into viral pathogenesis and the transmission of novel reassortant H3N2 viruses that are circulating in U.S. swine herds and warrant future surveillance.
Project description:BACKGROUND:Mutations in the PB1 subunit of RNA-dependent RNA polymerase (RdRp) of influenza A virus can affect replication fidelity. Before the influenza A/H1N1 pandemic in 2009, most human influenza A/H1N1 viruses contained the avian-associated residue, serine, at position 216 in PB1. However, near the onset of the 2009 pandemic, human viruses began to acquire the mammalian-associated residue, glycine, at PB1-216, and PB1-216G became predominant in human viruses thereafter. METHODS:Using entropy-based analysis algorithm, we have previously identified several host-specific amino-acid signatures that separated avian and swine viruses from human influenza viruses. The presence of these host-specific signatures in human influenza A/H1N1 viruses suggested that these mutations were the result of adaptive genetic evolution that enabled these influenza viruses to circumvent host barriers, which resulted in cross-species transmission. We investigated the biological impact of this natural avian-to-mammalian signature substitution at PB1-216 in human influenza A/H1N1 viruses. RESULTS:We found that PB1-216G viruses had greater mutation potential, and were more sensitive to ribavirin than PB1-216S viruses. In oseltamivir-treated HEK293 cells, PB1-216G viruses generated mutations in viral neuraminidase at a higher rate than PB1-216S viruses. By contrast, PB1-216S viruses were more virulent in mice than PB1-216G viruses. These results suggest that the PB1-S216G substitution enhances viral epidemiological fitness by increasing the frequency of adaptive mutations in human influenza A/H1N1 viruses. CONCLUSIONS:Our results thus suggest that the increased adaptability and epidemiological fitness of naturally arising human PB1-216G viruses, which have a canonical low-fidelity replicase, were the biological mechanisms underlying the replacement of PB1-216S viruses with a high-fidelity replicase following the emergence of pdmH1N1. We think that continued surveillance of such naturally occurring PB1-216 variants among others is warranted to assess the potential impact of changes in RdRp fidelity on the adaptability and epidemiological fitness of human A/H1N1 influenza viruses.
Project description:Gene mutations and reassortment are key mechanisms by which influenza A virus acquires virulence factors. To evaluate the role of the viral polymerase replication machinery in producing virulent pandemic (H1N1) 2009 influenza viruses, we generated various polymerase point mutants (PB2, 627K/701N; PB1, expression of PB1-F2 protein; and PA, 97I) and reassortant viruses with various sources of influenza viruses by reverse genetics. Although the point mutations produced no significant change in pathogenicity, reassortment between the pandemic A/California/04/09 (CA04, H1N1) and current human and animal influenza viruses produced variants possessing a broad spectrum of pathogenicity in the mouse model. Although most polymerase reassortants had attenuated pathogenicity (including those containing seasonal human H3N2 and high-pathogenicity H5N1 virus segments) compared to that of the parental CA04 (H1N1) virus, some recombinants had significantly enhanced virulence. Unexpectedly, one of the five highly virulent reassortants contained a A/Swine/Korea/JNS06/04(H3N2)-like PB2 gene with no known virulence factors; the other four had mammalian-passaged avian-like genes encoding PB2 featuring 627K, PA featuring 97I, or both. Overall, the reassorted polymerase complexes were only moderately compatible for virus rescue, probably because of disrupted molecular interactions involving viral or host proteins. Although we observed close cooperation between PB2 and PB1 from similar virus origins, we found that PA appears to be crucial in maintaining viral gene functions in the context of the CA04 (H1N1) virus. These observations provide helpful insights into the pathogenic potential of reassortant influenza viruses composed of the pandemic (H1N1) 2009 influenza virus and prevailing human or animal influenza viruses that could emerge in the future.
Project description:BACKGROUND: The influenza viruses circulating in animals sporadically transmit to humans and pose pandemic threats. Animal models to evaluate the potential public health risk potential of these viruses are needed. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the guinea pig as a mammalian model for the study of the replication and transmission characteristics of selected swine H1N1, H1N2, H3N2 and avian H9N2 influenza viruses, compared to those of pandemic (H1N1) 2009 and seasonal human H1N1, H3N2 influenza viruses. The swine and avian influenza viruses investigated were restricted to the respiratory system of guinea pigs and shed at high titers in nasal tracts without prior adaptation, similar to human strains. None of the swine and avian influenza viruses showed transmissibility among guinea pigs; in contrast, pandemic (H1N1) 2009 virus transmitted from infected guinea pigs to all animals and seasonal human influenza viruses could also horizontally transmit in guinea pigs. The analysis of the receptor distribution in the guinea pig respiratory tissues by lectin histochemistry indicated that both SA?2,3-Gal and SA?2,6-Gal receptors widely presented in the nasal tract and the trachea, while SA?2,3-Gal receptor was the main receptor in the lung. CONCLUSIONS/SIGNIFICANCE: We propose that the guinea pig could serve as a useful mammalian model to evaluate the potential public health threat of swine and avian influenza viruses.
Project description:This SuperSeries is composed of the following subset Series: GSE35738: 2009 pandemic H1N1 virus causes disease and upregulation of genes related to inflammatory and immune response, cell death, and lipid metabolism in pigs GSE40088: Comparative transcriptomic analysis of acute host responses during 2009 pandemic H1N1 influenza infection in mouse, macaque, and swine (macaque dataset) GSE40091: Comparative transcriptomic analysis of acute host responses during 2009 pandemic H1N1 influenza infection in mouse, macaque, and swine (mouse dataset) Refer to individual Series
Project description:The emergence of the pandemic 2009 H1N1 influenza A virus in humans and subsequent discovery that it was of swine influenza virus lineages raised concern over the safety of pork. Pigs experimentally infected with pandemic 2009 H1N1 influenza A virus developed respiratory disease; however, there was no evidence for systemic disease to suggest that pork from pigs infected with H1N1 influenza would contain infectious virus. These findings support the WHO recommendation that pork harvested from pandemic influenza A H1N1 infected swine is safe to consume when following standard meat hygiene practices.