NP, PB1, and PB2 Viral Genes Contribute to Altered Replication of H5N1 Avian Influenza Viruses in Chickens

Abstract

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.

Avian influenza virus hemagglutinins H2, H4, H8, and H14 support a highly pathogenic phenotype

“…Overall, the emergence of an HPAIV may include these three preconditions: (i) the HA gene has to be prone to acquire a polybasic HACS (10,38), (ii) the acid stability of HA is complemented by the activity of M2 in pH reduction (29,31,39), and (iii) the other viral genes need to carry additional virulence and host range determinants (13,(40)(41)(42)(43)(44) like the NA stalk deletion (26)(27)(28). Those and other additional virulence determinants might be required to balance HA affinity with NA activity or to compensate potential inferior replication characteristics of such an evolving HPAIV.…”

Association of Increased Pathogenicity of Asian H5N1 Highly Pathogenic Avian Influenza Viruses in Chickens with Highly Efficient Viral Replication Accompanied by Early Destruction of Innate Immune Responses

“…It was reported that infection with an HPAI virus results in upregulation of gene expression of gamma interferon (IFN-␥) and inducible nitric oxide synthase (58). However, the roles of proinflammatory cytokines in disease severity and outcomes in chickens infected systemically with HPAI viruses are largely unknown.…”

A Single N66S Mutation in the PB1-F2 Protein of Influenza A Virus Increases Virulence by Inhibiting the Early Interferon Response In Vivo

“…Characterization of single-gene reassortants has led to a greater understanding of viral factors important for H5N1 virulence, particularly for the HA and PB2 proteins (3,10,15,31). More recently, the PB1-F2 protein has been shown to increase pathogenesis of the 1918 pandemic influenza virus and a recombinant virus bearing the PB1 segment of the A/Hong Kong/156/1997 (H5N1) virus.…”