NP, PB1, and PB2 viral genes contribute to altered replication of H5N1 avian influenza viruses in chickens

Southeast Poultry Research Laboratory, USDA-ARS, 934 College Station Road, Athens, GA 30605, USA.
Journal of Virology (Impact Factor: 4.44). 06/2008; 82(9):4544-53. DOI: 10.1128/JVI.02642-07
Source: PubMed


The virulence determinants for highly pathogenic avian influenza viruses (AIVs) are considered multigenic, although the best characterized virulence factor is the hemagglutinin (HA) cleavage site. The capability of influenza viruses to reassort gene segments is one potential way for new viruses to emerge with different virulence characteristics. To evaluate the role of other gene segments in virulence, we used reverse genetics to generate two H5N1 recombinant viruses with differing pathogenicity in chickens. Single-gene reassortants were used to determine which viral genes contribute to the altered virulence. Exchange of the PB1, PB2, and NP genes impacted replication of the reassortant viruses while also affecting the expression of specific host genes. Disruption of the parental virus' functional polymerase complexes by exchanging PB1 or PB2 genes decreased viral replication in tissues and consequently the pathogenicity of the viruses. In contrast, exchanging the NP gene greatly increased viral replication and expanded tissue tropism, thus resulting in decreased mean death times. Infection with the NP reassortant virus also resulted in the upregulation of gamma interferon and inducible nitric oxide synthase gene expression. In addition to the impact of PB1, PB2, and NP on viral replication, the HA, NS, and M genes also contributed to the pathogenesis of the reassortant viruses. While the pathogenesis of AIVs in chickens is clearly dependent on the interaction of multiple gene products, we have shown that single-gene reassortment events are sufficient to alter the virulence of AIVs in chickens.

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Available from: Darrell Kapczynski, Jul 09, 2014
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    • "These reports showed that the growth potential of reassortants differed depending on the constellation of the polymerase genes, suggesting the major contribution of the polymerases to efficient replication in mammals. NP was also shown to play an important role in the efficient replication and expansion of tissue tropism of H5N1 HPAIVs in chickens [30,31]. Recently, it was demonstrated that the interaction of PB2 and NP with importin-α, a host factor mediating trafficking into the nucleus, where transcription and replication of the viral genome occur, was correlated with host adaptation of influenza A viruses [32]. "
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    ABSTRACT: Background Wild ducks are the natural hosts of influenza A viruses. Duck influenza, therefore, has been believed inapparent infection with influenza A viruses, including highly pathogenic avian influenza viruses (HPAIVs) in chickens. In fact, ducks experimentally infected with an HPAIV strain, A/Hong Kong/483/1997 (H5N1) (HK483), did not show any clinical signs. Another HPAIV strain, A/whooper swan/Mongolia/3/2005 (H5N1) (MON3) isolated from a dead swan, however, caused neurological dysfunction and death in ducks. Method To understand the mechanism whereby MON3 shows high pathogenicity in ducks, HK483, MON3, and twenty-four reassortants generated between these two H5N1 viruses were compared for their pathogenicity in domestic ducks. Results None of the ducks infected with MON3-based single-gene reassortants bearing the PB2, NP, or NS gene segment of HK483 died, and HK483-based single-gene reassortants bearing PB2, NP, or NS genes of MON3 were not pathogenic in ducks, suggesting that multiple gene segments contribute to the pathogenicity of MON3 in ducks. All the ducks infected with the reassortant bearing PB2, PA, HA, NP, and NS gene segments of MON3 died within five days post-inoculation, as did those infected with MON3. Each of the viruses was assessed for replication in ducks three days post-inoculation. MON3 and multi-gene reassortants pathogenic in ducks were recovered from all of the tissues examined and replicated with high titers in the brains and lungs. Conclusion The present results indicate that multigenic factors are responsible for efficient replication of MON3 in ducks. In particular, virus growth in the brain might correlate with neurological dysfunction and the disease severity.
    Virology Journal 02/2013; 10(1):45. DOI:10.1186/1743-422X-10-45 · 2.18 Impact Factor
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    • "Creation of viruses containing different combinations of segments from two highly pathogenic H5N1 avian strains resulted in the identification of PB1 as one of the segments that altered replication [23]. Residues at positions 473 and 598 from another avian PB1 were shown to contribute to efficient replication of a human isolate [24]. "
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    ABSTRACT: It is desirable for influenza vaccine virus strains to have phenotypes that include good growth and hemagglutinin (HA) protein yield. The quality of these characteristics varies among the vaccine viruses and is usually due to multigenic effects. Many influenza A virus vaccine viruses are made as reassortants of the high yield virus A/Puerto Rico/8/34 (PR/8) and a circulating seasonal virus. Co-infection of eggs with the two viruses, and selection of reassortants with the HA and neuraminidase (NA) segments from the seasonal virus, can result in viruses that contain a mixture of internal genes derived from both the high yield virus and the circulating virus. Segment 2 (PB1), which encodes the RNA-dependent RNA polymerase, frequently cosegregates with the seasonal HA and NA segments. We asked whether mutations based on the seasonal PB1 genes could improve vaccine virus strains. Here we report that mutations to the PR/8 PB1 gene, based on differences observed between seasonal and PR/8 PB1 genes, accelerate egg and cell culture based replication for a reassortant virus containing HA and NA segments from the low yield A/Wyoming/03/2003 (H3N2) vaccine virus.
    Vaccine 10/2012; 31(1). DOI:10.1016/j.vaccine.2012.10.060 · 3.62 Impact Factor
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    • "In addition to the S-OIV pandemic flu, H5N1 highly pathogenic avian influenza viruses (HPAIV) have caused severe or fatal disease in humans in Asia, the Middle East, and Africa since their emergence in Hong Kong in 1997 (WHO, The H5N1 influenza virus ribonucleoprotein complex (RNP) contributes to viral pathogenesis in chickens [5,6]. Influenza viruses with high polymerase activity have also shown high pathogenicity [7,8]. "
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    ABSTRACT: Background Although gene exchange is not likely to occur freely, reassortment between the H5N1 highly pathogenic avian influenza virus (HPAIV) and currently circulating human viruses is a serious concern. The PA polymerase subunit of H5N1 HPAIV was recently reported to activate the influenza replicon activity. Methods The replicon activities of PR8 and WSN strains (H1N1) of influenza containing PA from HPAIV A/Cambodia/P0322095/2005 (H5N1) and the activity of the chimeric RNA polymerase were analyzed. A reassortant WSN virus containing the H5N1 Cambodia PA (C-PA) was then reconstituted and its growth in cells and pathogenicity in mice examined. The interferon promoter, TUNEL, and caspase 3, 8, and 9 activities of C-PA-infected cells were compared with those of WSN-infected cells. Results The activity of the chimeric RNA polymerase was slightly higher than that of WSN, and C-PA replicated better than WSN in cells. However, the multi-step growth of C-PA and its pathogenicity in mice were lower than those of WSN. The interferon promoter, TUNEL, and caspase 3, 8, and 9 activities were strongly induced in early infection in C-PA-infected cells but not in WSN-infected cells. Conclusions Apoptosis and interferon were strongly induced early in C-PA infection, which protected the uninfected cells from expansion of viral infection. In this case, these classical host-virus interactions contributed to the attenuation of this strongly replicating virus.
    Virology Journal 06/2012; 9(1):106. DOI:10.1186/1743-422X-9-106 · 2.18 Impact Factor
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