Mouse-Adapted H9N2 Influenza A Virus PB2 Protein M147L and E627K Mutations Are Critical for High Virulence

In aquatic birds, influenza A viruses mainly replicate in the intestinal tract without significantly affecting the health of the host, but in mammals, they replicate in the respiratory tract and often cause disease. Occasionally, influenza viruses have been detected in stool samples of hospitalized patients and in rectal swabs of naturally or experimentally infected mammals. In this study, we compared the biological and molecular differences among four wild-type avian H1N1 influenza viruses and their corresponding fecal and lung isolates in DBA/2J and BALB/cJ mice. All fecal and lung isolates were more pathogenic than the original wild-type viruses, when inoculated into mice of both strains. The increased virulence was associated with the acquisition of genetic mutations. Most of the novel genotypes emerged as PB2 E627K , HA F128V , HA F454L , or HA H300P variations, and double mutations frequently occurred in the same isolate. However, influenza virus strain-and host-specific differences were also observed in terms of selected variants. The avian H1N1 virus of shorebird origin appeared to be unique in its ability to rapidly adapt to BALB/cJ mice via the fecal route, compared to the adaptability of the H1N1 virus of mallard origin. Furthermore, a bimodal distribution in fecal shedding was observed in mice infected with the fecal isolates, while a normal distribution was observed after infection with the lung isolates or wild-type virus. Fecal isolates contained HA mutations that increased the activation pH of the HA protein. We conclude that influenza virus variants that emerge in fecal isolates in mammals might influence viral transmission, adaptation to mammals, and viral ecology or evolution.

Section: Discussionmentioning

confidence: 57%

Fecal Influenza in Mammals: Selection of Novel Variants

Koçer

Obenauer

Zaraket

et al. 2013

“…Indeed, the internal genes of influenza A viruses play important roles in virus replication, interspecies transmission, and virulence to mammals (16-20, 29-31, 34, 35). Findings to date indicate that the NP and M genes are critical for virus replication and transmission (17,34), that the PA, PB2, and NP genes impact viral pathogenicity (17,18,20,(29)(30)(31), and that the PB1 gene induces apoptosis and regulates the cellular interferon response of host cells (19). NS1 inhibits both interferon production and the activity of several interferon-induced genes (16).…”

Section: Discussionmentioning

confidence: 99%

“…This is especially true of the PB2 gene, which endows H9N2-AH/PB2 with Ͼ7,413-and 213-fold lower MLD 50 values than the parental H9N2 and H7N9 viruses, respectively (Table 2). To further explore the key amino acids in H7N9-derived PB2 for high pathogenicity, the PB2 gene was analyzed in detail on the basis of previous studies (20,(28)(29)(30)(31) and alignment results with the H9N2-derived gene (Tables 3 and 4). Of the confirmed amino acids of mammal-adapted influenza viruses, only K627 in the PB2 protein of the AH-H7N9 virus was observed in the present study Their body weights were monitored daily for a 14-day observation period and expressed as percentages of the initial values (A).…”

Section: Identification Of the Genes Responsible For Virus Replicatiomentioning

confidence: 99%

Assessment of the Internal Genes of Influenza A (H7N9) Virus Contributing to High Pathogenicity in Mice

Xie

Zhang

et al. 2015

Self Cite

The recently identified H7N9 influenza A virus has caused severe economic losses and worldwide public concern. Genetic analysis indicates that its six internal genes all originated from H9N2 viruses. However, the H7N9 virus is more highly pathogenic in humans than H9N2, which suggests that the internal genes of H7N9 have mutated. To analyze which H7N9 virus internal genes contribute to its high pathogenicity, a series of reassortants was generated by reverse genetics, with each virus containing a single internal gene of the typical A/Anhui/1/2013 (H7N9) (AH-H7N9) virus in the genetic background of the A/chicken/Shandong/ lx1023/2007 (H9N2) virus. The replication ability, polymerase activity, and pathogenicity of these viruses were then evaluated in vitro and in vivo. These recombinants displayed high genetic compatibility, and the H7N9-derived PB2, M, and NP genes were identified as the virulence genes for the reassortants in mice. Further investigation confirmed that the PB2 K627 residue is critical for the high pathogenicity of the H7N9 virus and the reassortant containing the H7N9-derived PB2 segment (H9N2-AH/ PB2). Notably, the H7N9-derived PB2 gene displayed greater compatibility with the H9N2 genome than that of H7N9, endowing the H9N2-AH/PB2 reassortant with greater viability and virulence than the parental H7N9 virus. In addition, the H7N9 virus, with the exception of the H9N2 reassortants, could effectively replicate in human A549 cells. Our results indicate that PB2, M, and NP are the key virulence genes, together with the surface hemagglutinin (HA) and neuraminidase (NA) proteins, contributing to the high infectivity of the H7N9 virus in humans. IMPORTANCETo date, the novel H7N9 influenza A virus has caused 437 human infections, with approximately 30% mortality. Previous work has primarily focused on the two viral surface proteins, HA and NA, but the contribution of the six internal genes to the high pathogenicity of H7N9 has not been systematically studied. Here, the H9N2 virus was used as a genetic backbone to evaluate the virulence genes of H7N9 virus in vitro and in vivo. Our data indicate that the PB2, M, and NP genes play important roles in viral infection in mice and, together with HA and NA, contribute to the high infectivity of the H7N9 virus in humans. A vian influenza virus (AIV) is an enveloped, single-stranded negative-sense RNA virus that belongs to the influenza A genus of the Orthomyxoviridae family. The AIV genome is comprised of eight gene segments: hemagglutinin (HA), neuraminidase (NA), basic polymerase 2 (PB2), basic polymerase 1 (PB1), acidic polymerase (PA), nucleoprotein (NP), matrix (M), and nonstructural protein (NS). Based on the antigenic properties of the HA (H1 to H16) and NA (N1 to N9) surface glycoproteins, as well as two recently discovered bat-derived H17N10 and H18N11 influenza-like genomes (1), AIV can be further divided into subtypes.AIV is generally considered species specific and rarely crosses the species barrier to infect mammals and humans (2). However...

“…However, the percentage of H9N2 viruses possessing the PARSSR/GL cleavage site motif dramatically decreased beginning in 2005, while those with the PSRSSR/GL cleavage motif (A316S substitution) became predominant. As shown in A reverse genetics system of an H9N2 virus, A/chicken/ Shandong/16/05 (SD16), with HA-A316 and full-length NA, was established (6), and viruses with HA-316S (SD16-HA316S), a 3-amino-acid deletion in the NA stalk (SD16-⌬NA), or both HA-316S and short stalk NA (SD16-HA316S/⌬NA) in the SD16 background were generated. To determine the influence of these mutations on the activation of HA by cleavage of HA0, Western blot analyses of the supernatants from virus-infected Madin-Darby canine kidney (MDCK) cells and the allantoic fluid from virus-infected embryonated chicken eggs were performed as described previously (7).…”

mentioning

confidence: 99%

Amino Acid 316 of Hemagglutinin and the Neuraminidase Stalk Length Influence Virulence of H9N2 Influenza Virus in Chickens and Mice

Sun

Tan

Wei

et al. 2013

Self Cite

e H9N2 influenza viruses with an A316S substitution in hemagglutinin (HA) and a shorter neuraminidase (NA) stalk have become predominant in China. The A316S was shown to increase HA cleavage efficiency when combined with short stalk NA, and the short stalk NA improved NA enzyme activity and release of virus from erythrocytes. Single mutations or combinations of these mutations strengthened the virulence of H9N2 virus in chickens and mice.