Virulence-Affecting Amino Acid Changes in the PA Protein of H7N9 Influenza A Viruses

Adaptation of the viral polymerase complex comprising PB1, PB2, and PA is necessary for efficient influenza A virus replication in new host species. We found that PA mutation K356R (PA-K356R) has become predominant since 2014 in avian H9N2 viruses in China as with seasonal human H1N1 viruses. The same mutation is also found in most human isolates of emergent avian H7N9 and H10N8 viruses whose six internal gene segments are derived from the H9N2 virus. We further demonstrated the mammalian adaptive functionality of the PA-K356R mutation. Avian H9N2 virus with the PA-K356R mutation in human A549 cells showed increased nuclear accumulation of PA and increased viral polymerase activity that resulted in elevated levels of viral transcription and virus output. The same mutant virus in mice also enhanced virus replication and caused lethal infection. In addition, combined mutation of PA-K356R and PB2-E627K, a well-known mammalian adaptive marker, in the H9N2 virus showed further cooperative increases in virus production and severity of infection in vitro and in vivo. In summary, PA-K356R behaves as a novel mammalian tropism mutation, which, along with other mutations such as PB2-E627K, might render avian H9N2 viruses adapted for human infection. IMPORTANCE Mutations of the polymerase complex (PB1, PB2, and PA) of influenza A virus are necessary for viral adaptation to new hosts.This study reports a novel and predominant mammalian adaptive mutation, PA-K356R, in avian H9N2 viruses and human isolates of emergent H7N9 and H10N8 viruses. We found that PA-356R in H9N2 viruses causes significant increases in virus replication and severity of infection in human cells and mice and that PA-K356R cooperates with the PB2-E627K mutation, a wellcharacterized human adaptive marker, to exacerbate mammalian infection in vitro and in vivo. Therefore, the PA-K356R mutation is a significant adaptation in H9N2 viruses and related H7N9 and H10N8 reassortants toward human infectivity. The emergence of new pandemic influenza A viruses requires overcoming barriers to cross-species transmission from animal reservoirs to human populations. Influenza viruses of the H9N2 subtype and their reassortants circulating in poultry are a potential source of pandemic viruses and hence represent a significant public health threat (1). Since spring 2013, a novel avian H7N9 reassortant with six internal genes of the H9N2 virus had caused three major outbreaks in humans throughout China (2, 3). During the same period, a newly emergent avian H10N8 virus, also with six H9N2-like internal genes, was reported to cause fatal human infections in China (4). In the meantime, H9N2 viruses continued to cause human infections in China and other countries based on etiological and serological evidence (5-12). We recently found that avian H9N2 viruses have undergone significant genetic evolution, especially in their internal genes, to form a predominant genotype (G57), which in turn provided the internal genes to multiple new subtypes, including H7N9 and H10N8 viru...

Section: Figmentioning

confidence: 99%

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confidence: 99%

Prevailing PA Mutation K356R in Avian Influenza H9N2 Virus Increases Mammalian Replication and Pathogenicity

Zhang

Gao

et al. 2016

“…Although the majority of human infections remain contained to mainland China, there have been imported cases confirmed in Taiwan, Hong Kong SAR, and Malaysia (3)(4)(5). There is no evidence that sustained human-to-human transmission is occurring, although the H7N9 viruses possess several key amino acid changes often associated with mammalian replication and transmission, heightening public health concerns (6)(7)(8).…”

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confidence: 99%

Human H7N9 and H5N1 Influenza Viruses Differ in Induction of Cytokines and Tissue Tropism

Meliopoulos

Karlsson

Kercher

et al. 2014

Since emerging in 2013, the avian-origin H7N9 influenza viruses have resulted in over 400 human infections, leading to 115 deaths to date. Although the epidemiology differs from human highly pathogenic avian H5N1 influenza virus infections, there is a similar rapid progression to acute respiratory distress syndrome. The aim of these studies was to compare the pathological and immunological characteristics of a panel of human H7N9 and H5N1 viruses in vitro and in vivo. Although there were similarities between particular H5N1 and H7N9 viruses, including association between lethal disease and spread to the alveolar spaces and kidney, there were also strain-specific differences. Both H5N1 and H7N9 viruses are capable of causing lethal infections, with mortality correlating most strongly with wider distribution of viral antigen in the lungs, rather than with traditional measures of virus titer and host responses. Strain-specific differences included hypercytokinemia in H5N1 infections that was not seen with the H7N9 infections regardless of lethality. Conversely, H7N9 viruses showed a greater tropism for respiratory epithelium covering nasal passages and nasopharynx-associated lymphoid tissue than H5N1 viruses, which may explain the enhanced transmission in ferret models. Overall, these studies highlight some distinctive properties of H5N1 and H7N9 viruses in different in vitro and in vivo models. IMPORTANCEThe novel avian-origin H7N9 pandemic represents a serious threat to public health. The ability of H7N9 to cause serious lung pathology, leading in some cases to the development of acute respiratory distress syndrome, is of particular concern. Initial reports of H7N9 infection compared them to infections caused by highly pathogenic avian (HPAI) H5N1 viruses. Thus, it is of critical importance to understand the pathology and immunological response to infection with H7N9 compared to HPAI H5N1 viruses. We compared these responses in both in vitro and in vivo models, and found that H5N1 and H7N9 infections exhibit distinct pathological, immunological, and tissue tropism differences that could explain differences in clinical disease and viral transmission.

“…A minigenome assay based on the dual-luciferase system was performed as previously reported (34,35). 293 cells were transfected with viral protein expression plasmids for NP, PA, PB1, and wild-type or mutant PB2 (50 ng each), with a plasmid expressing a reporter vRNA encoding firefly luciferase under the control of the human RNA polymerase I promoter [pPolI-NP (0)Fluc (0); 50 ng], and with pRL-null (50 ng), which expresses Renilla luciferase (as a transfection control), with or without a protein expression plasmid for PB2-S1 (2, 6, 20, 60, or 200 ng) or mutant PB2-S1 (60 ng).…”

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confidence: 99%

Identification of a Novel Viral Protein Expressed from the PB2 Segment of Influenza A Virus

Yamayoshi

Watanabe

Goto

et al. 2016

Self Cite

Over the past 2 decades, several novel influenza virus proteins have been identified that modulate viral infections in vitro and/or in vivo. The PB2 segment, which is one of the longest influenza A virus segments, is known to encode only one viral protein, PB2. In the present study, we used reverse transcription-PCR (RT-PCR) targeting viral mRNAs transcribed from the PB2 segment to look for novel viral proteins encoded by spliced mRNAs. We identified a new viral protein, PB2-S1, encoded by a novel spliced mRNA in which the region corresponding to nucleotides 1513 to 1894 of the PB2 mRNA is deleted. PB2-S1 was detected in virusinfected cells and in cells transfected with a protein expression plasmid encoding PB2. PB2-S1 localized to mitochondria, inhibited the RIG-I-dependent interferon signaling pathway, and interfered with viral polymerase activity (dependent on its PB1-binding capability). The nucleotide sequences around the splicing donor and acceptor sites for PB2-S1 were highly conserved among pre-2009 human H1N1 viruses but not among human H1N1pdm and H3N2 viruses. PB2-S1-deficient viruses, however, showed growth kinetics in MDCK cells and virulence in mice similar to those of wild-type virus. The biological significance of PB2-S1 to the replication and pathogenicity of seasonal H1N1 influenza A viruses warrants further investigation. IMPORTANCE Transcriptome analysis of cells infected with influenza