PB2 residue 158 is a pathogenic determinant of pandemic H1N1 and H5 influenza a viruses in mice

Influenza A viruses are human and animal pathogens that cause morbidity and mortality, which range from mild to severe. The 2009 H1N1 pandemic was caused by the emergence of a reassortant H1N1 subtype (H1N1pdm) influenza A virus containing gene segments that originally circulated in human, avian, and swine virus reservoirs. The molecular determinants of replication and pathogenesis of H1N1pdm viruses in humans and other mammals are poorly understood. Therefore, we set out to elucidate viral determinants critical to the pathogenesis of this novel reassortant using a mouse model. We found that a glutamate-to-glycine substitution at residue 158 of the PB2 gene (PB2-E158G) increased the morbidity and mortality of the parental H1N1pdm virus. Results from mini-genome replication assays in human cells and virus titration in mouse tissues demonstrated that PB2-E158G is a pathogenic determinant, because it significantly increases viral replication rates. The virus load in PB2-E158G-infected mouse lungs was 1,300-fold higher than that of the wild-type virus. Our data also show that PB2-E158G had a much stronger influence on the RNA replication and pathogenesis of H1N1pdm viruses than PB2-E627K, which is a known pathogenic determinant. Remarkably, PB2-E158G substitutions also altered the pathotypes of two avian H5 viruses in mice, indicating that this residue impacts genetically divergent influenza A viruses and suggesting that this region of PB2 could be a new antiviral target. Collectively, the data presented in this study demonstrate that PB2-E158G is a novel pathogenic determinant of influenza A viruses in the mouse model. We speculate that PB2-E158G may be important in the adaptation of avian PB2 genes to other mammals, and BLAST sequence analysis identified a naturally occurring human H1N1pdm isolate that has this substitution. Therefore, future surveillance efforts should include scrutiny of this region of PB2 because of its potential impact on pathogenesis.     

Transmission of Influenza Virus in a Mammalian Host Is Increased by PB2 Amino Acids 627K or 627E/701N

Since 2003, more than 380 cases of H5N1 influenza virus infection of humans have been reported. Although the resultant disease in these cases was often severe or fatal, transmission of avian influenza viruses between humans is rare. The precise nature of the barrier blocking human-to-human spread is unknown. It is clear, however, that efficient human-to-human transmission of an antigenically novel influenza virus would result in a pandemic. Influenza viruses with changes at amino acids 627 or 701 of the PB2 protein have been isolated from human cases of highly pathogenic H5 and H7 avian influenza. Herein, we have used the guinea pig model to test the contributions of PB2 627 and 701 to mammalian transmission. To this end, viruses carrying mutations at these positions were generated in the A/Panama/2007/99 (H3N2) and A/Viet Nam/1203/04 (H5N1) backgrounds. In the context of either rPan99 or rVN1203, mutation of lysine 627 to the avian consensus residue glutamic acid was found to decrease transmission. Introduction of an asparagine at position 701, in conjunction with the K627E mutation, resulted in a phenotype more similar to that of the parental strains, suggesting that this residue can compensate for the lack of 627K in terms of increasing transmission in mammals. Thus, our data show that PB2 amino acids 627 and 701 are determinants of mammalian inter-host transmission in diverse virus backgrounds.     

Selection of H5N1 Influenza Virus PB2 during Replication in Humans

Highly pathogenic H5N1 influenza viruses continue to cause concern, even though currently circulating strains are not efficiently transmitted among humans. For efficient transmission, amino acid changes in viral proteins may be required. Here, we examined the amino acids at positions 627 and 701 of the PB2 protein. A direct analysis of the viral RNAs of H5N1 viruses in patients revealed that these amino acids contribute to efficient virus propagation in the human upper respiratory tract. Viruses grown in culture or eggs did not always reflect those in patients. These results emphasize the importance of the direct analysis of original specimens.Given the continued circulation of highly pathogenic H5N1 avian influenza viruses and their sporadic transmission to humans, the threat of a pandemic persists. However, for H5N1 influenza viruses to be efficiently transmitted among humans, amino acid substitutions in the avian viral proteins may be necessary.Two positions in the PB2 protein affect the growth of influenza viruses in mammalian cells (3, 11, 18): the amino acid at position 627 (PB2-627), which in most human influenza viruses is lysine (PB2-627Lys) and most avian viruses is glutamic acid (PB2-627Glu), and the amino acid at position 701. PB2-627Lys is associated with the efficient replication (16) and high virulence (5) of H5N1 viruses in mice. Moreover, an H7N7 avian virus isolated from a fatal human case of pneumonia possessed PB2-627Lys, whereas isolates from a nonfatal human case of conjunctivitis and from chickens during the same outbreak possessed PB2-627Glu (2).The amino acid at position 701 in PB2 is important for the high pathogenicity of H5N1 viruses in mice (11). Most avian influenza viruses possess aspartic acid at this position (PB2-701Asp); however, A/duck/Guangxi/35/2001 (H5N1), which is highly virulent in mice (11), possesses asparagine at this position (PB2-701Asn). PB2-701Asn is also found in equine (4) and swine (15) viruses, as well as some H5N1 human isolates (7, 9). Thus, both amino acids appear to be markers for the adaptation of H5N1 viruses in humans (1, 3, 17).Massin et al. (13) reported that the amino acid at PB2-627 affects viral RNA replication in cultured cells at low temperatures. Recently, we demonstrated that viruses, including those of the H5N1 subtype, with PB2-627Lys (human type) grow better at low temperatures in cultured cells than those with PB2-627Glu (avian type) (6). This association between the PB2 amino acid and temperature-dependent growth correlates with the body temperatures of hosts; the human upper respiratory tract is at a lower temperature (around 33°C) than the lower respiratory tract (around 37°C) and the avian intestine, where avian influenza viruses usually replicate (around 41°C). The ability to replicate at low temperatures may be crucial for viral spread among humans via sneezing and coughing by being able to grow in the upper respiratory organs. Therefore, the Glu-to-Lys mutation in PB2-627 is an important step for H5N1 viruses to develop pandemic potential.However, there is no direct evidence that the substitutions of PB2-627Glu with PB2-627Lys and PB2-701Asp with PB2-701Asn occur during the replication of H5N1 avian influenza viruses in human respiratory organs. Therefore, here, we directly analyzed the nucleotide sequences of viral genes from several original specimens collected from patients infected with H5N1 viruses.     

Virulence and genetic compatibility of polymerase reassortant viruses derived from the pandemic (H1N1) 2009 influenza virus and circulating influenza A viruses

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.     

Mouse-adapted H9N2 influenza A virus PB2 protein M147L and E627K mutations are critical for high virulence

H9N2 influenza viruses have been circulating worldwide in multiple avian species and have repeatedly infected humans to cause typical disease. The continued avian-to-human interspecies transmission of H9N2 viruses raises concerns about the possibility of viral adaption with increased virulence for humans. To investigate the genetic basis of H9N2 influenza virus host range and pathogenicity in mammals, we generated a mouse-adapted H9N2 virus (SD16-MA) that possessed significantly higher virulence than wide-type virus (SD16). Increased virulence was detectable after 8 sequential lung passages in mice. Five amino acid substitutions were found in the genome of SD16-MA compared with SD16 virus: PB2 (M147L, V250G and E627K), HA (L226Q) and M1 (R210K). Assessments of replication in mice showed that all of the SD16-MA PB2, HA and M1 genome segments increased virus replication; however, only the mouse-adapted PB2 significantly increased virulence. Although the PB2 E627K amino acid substitution enhanced viral polymerase activity and replication, none of the single mutations of mouse adapted PB2 could confer increased virulence on the SD16 backbone. The combination of M147L and E627K significantly enhanced viral replication ability and virulence in mice. Thus, our results show that the combination of PB2 amino acids at position 147 and 627 is critical for the increased pathogenicity of H9N2 influenza virus in mammalian host.     

Molecular basis of efficient replication and pathogenicity of H9N2 avian influenza viruses in mice

H9N2 subtype avian influenza viruses (AIVs) have shown expanded host range and can infect mammals, such as humans and swine. To date the mechanisms of mammalian adaptation and interspecies transmission of H9N2 AIVs remain poorly understood. To explore the molecular basis determining mammalian adaptation of H9N2 AIVs, we compared two avian field H9N2 isolates in a mouse model: one (A/chicken/Guangdong/TS/2004, TS) is nonpathogenic, another one (A/chicken/Guangdong/V/2008, V) is lethal with efficient replication in mouse brains. In order to determine the basis of the differences in pathogenicity and brain tropism between these two viruses, recombinants with a single gene from the TS (or V) virus in the background of the V (or TS) virus were generated using reverse genetics and evaluated in a mouse model. The results showed that the PB2 gene is the major factor determining the virulence in the mouse model although other genes also have variable impacts on virus replication and pathogenicity. Further studies using PB2 chimeric viruses and mutated viruses with a single amino acid substitution at position 627 [glutamic acid (E) to lysine, (K)] in PB2 revealed that PB2 627K is critical for pathogenicity and viral replication of H9N2 viruses in mouse brains. All together, these results indicate that the PB2 gene and especially position 627 determine virus replication and pathogenicity in mice. This study provides insights into the molecular basis of mammalian adaptation and interspecies transmission of H9N2 AIVs.     

The Effect of the PB2 Mutation 627K on Highly Pathogenic H5N1 Avian Influenza Virus Is Dependent on the Virus Lineage

Clade 2.2 Eurasian-lineage H5N1 highly pathogenic avian influenza viruses (HPAIVs) were first detected in Qinghai Lake, China, in 2005 and subsequently spread through Asia, Europe, and Africa. Importantly, these viruses carried a lysine at amino acid position 627 of the PB2 protein (PB2 627K), a known mammalian adaptation motif. Previous avian influenza virus isolates have carried glutamic acid in this position (PB2 627E), commonly described to restrict virus polymerase function in the mammalian host. We sought to examine the effect of PB2 627K on viral maintenance in the avian reservoir. Viruses constructed by reverse genetics were engineered to contain converse PB2 627K/E mutations in a Eurasian H5N1 virus (A/turkey/Turkey/5/2005 [Ty/05]) and, for comparison, a historical pre-Asian H5N1 HPAIV that naturally bears PB2 627E (A/turkey/England/50-92/1991 [50-92]). The 50-92 PB2 627K was genetically unstable during virus propagation, resulting in reversion to PB2 627E or the accumulation of the additional mutation PB2 628R and/or a synonymous mutation from an A to a G nucleotide at nucleotide position 1869 (PB2 A1869G). Intriguingly, PB2 628R and/or A1869G appeared to improve the genetic stability of 50-92 PB2 627K. However, the replication of 50-92 PB2 627K in conjunction with these stabilizing mutations was significantly restricted in experimentally infected chickens, where reversion to PB2 627E occurred. In contrast, no significant effects on viral fitness were observed for Ty/05 PB2 627E or 627K in in vitro or in vivo experiments. Our observations suggest that PB2 627K is supported in Eurasian-lineage viruses; in contrast, PB2 627K carries a significant fitness cost in the historical pre-Asian 50-92 virus.     

PB2-588I Enhances 2009 H1N1 Pandemic Influenza Virus Virulence by Increasing Viral Replication and Exacerbating PB2 Inhibition of Beta Interferon Expression

The 2009 pandemic H1N1 influenza virus (pdm/09) is typically mildly virulent in mice. In a previous study, we identified four novel swine isolates of pdm/09 viruses that exhibited high lethality in mice. Comparing the consensus sequences of the PB2 subunit of human isolates of pdm/09 viruses with those of the four swine isolate viruses revealed one consensus mutation: T588I. In this study, we determined that 588T is an amino acid mutation conserved in pdm/09 viruses that was exceedingly rare in previous human influenza isolates. To investigate whether the PB2 with the T5581 mutation (PB2-T558I) has an effect on the increased pathogenicity, we rescued a variant containing PB2-588I (Mex_PB2-588I) in the pdm/09 virus, A/Mexico/4486/2009(H1N1), referred to as Mex_WT (where WT is wild type), and characterized the variant in vitro and in vivo. The results indicated that the mutation significantly enhanced polymerase activity in mammalian cells, and the variant exhibited increased growth properties and induced significant weight loss in a mouse model compared to the wild type. We determined that the mutation exacerbated PB2 inhibition of mitochondrial antiviral signaling protein (MAVS)-mediated beta interferon (IFN-β) expression, and PB2-588I was observed to bind to MAVS more efficiently than PB2-588T. The variant induced lower levels of host IFN-β expression than the WT strain during infection. These findings indicate that the pdm/09 influenza virus has increased pathogenicity upon the acquisition of the PB2-T588I mutation and highlight the need for the continued surveillance of the genetic variation of molecular markers in influenza viruses because of their potential effects on pathogenicity and threats to human health.     

Genomic Signatures for Avian H7N9 Viruses Adapting to Humans

An avian influenza A H7N9 virus emerged in March 2013 and caused a remarkable number of human fatalities. Genome variability in these viruses may provide insights into host adaptability. We scanned over 140 genomes of the H7N9 viruses isolated from humans and identified 104 positions that exhibited seven or more amino acid substitutions. Approximately half of these substitutions were identified in the influenza ribonucleoprotein (RNP) complex. Although PB2 627K of the avian virus promotes replication in humans, 45 of the 147 investigated PB2 sequences retained the E signature at this position, which is an avian characteristic. We discovered 10 PB2 substitutions that covaried with K627E. An RNP activity assay showed that Q591K, D701N, and M535L restored the polymerase activity in human cells when 627K transformed to an avian-like E. Genomic analysis of the human-isolated avian influenza virus is crucial in assessing genome variability, because relationships between position-specific variations can be observed and explored. In this study, we observed alternative positions that can potentially compensate for PB2 627K, a well-known marker for cross-species infection. An RNP assay suggested Q591K, D701N, and M535L as potential markers for an H7N9 virus capable of infecting humans.     

Introduction of Virulence Markers in PB2 of Pandemic Swine-Origin Influenza Virus Does Not Result in Enhanced Virulence or Transmission

In the first 6 months of the H1N1 swine-origin influenza virus (S-OIV) pandemic, the vast majority of infections were relatively mild. It has been postulated that mutations in the viral genome could result in more virulent viruses, leading to a more severe pandemic. Mutations E627K and D701N in the PB2 protein have previously been identified as determinants of avian and pandemic influenza virus virulence in mammals. These mutations were absent in S-OIVs detected early in the 2009 pandemic. Here, using reverse genetics, mutations E627K, D701N, and E677G were introduced into the prototype S-OIV A/Netherlands/602/2009, and their effects on virus replication, virulence, and transmission were investigated. Mutations E627K and D701N caused increased reporter gene expression driven by the S-OIV polymerase complex. None of the three mutations affected virus replication in vitro. The mutations had no major impact on virus replication in the respiratory tracts of mice and ferrets or on pathogenesis. All three mutant viruses were transmitted via aerosols or respiratory droplets in ferrets. Thus, the impact of key known virulence markers in PB2 in the context of current S-OIVs was surprisingly small. This study does not exclude the possibility of emergence of S-OIVs with other virulence-associated mutations in the future. We conclude that surveillance studies aimed at detecting S-OIVs with increased virulence or transmission should not rely solely on virulence markers identified in the past but should include detailed characterization of virus phenotypes, guided by genetic signatures of viruses detected in severe cases of disease in humans.The new H1N1 swine-origin influenza virus (S-OIV) recently emerged to cause the first influenza pandemic in 40 years (2). The S-OIV presumably emerged from pigs, as its genome was shown to consist of six gene segments of “triple-reassortant” swine viruses and two of “Eurasian lineage” swine viruses (9). The start of the S-OIV pandemic has been relatively mild, with a clinical spectrum ranging from mild upper respiratory tract illness to sporadic cases of severe pneumonia leading to acute respiratory distress syndrome (22). As of 15 November 2009, worldwide, more than 206 countries have reported laboratory-confirmed cases of S-OIV infection, including over 6,770 deaths (32).In previous influenza pandemics, such as the Spanish influenza pandemic of 1918 and the Hong Kong influenza pandemic of 1968, a first wave of cases of relatively mild illnesses was followed by more severe subsequent waves (29). The reason for this increased severity has remained largely unknown, but one possible explanation could be that the pandemic viruses required further adaptation to the human host, resulting in the emergence of viruses that were more virulent than those of the first wave. Such adaptive changes could occur by gene reassortment between cocirculating influenza A viruses or by mutation.In the past decade, determinants of influenza A virus virulence have been mapped using reverse genetics with a variety of pandemic, epidemic, and zoonotic influenza viruses. Mutations affecting virulence and host range have frequently been mapped to hemagglutinin (HA) and neuraminidase (NA) in relation to their interaction with sialic acids, the virus receptors on host cells (11, 18, 30). Nonstructural protein 1 (NS1) has been implicated in the virulence of highly pathogenic avian influenza (HPAI) virus H5N1 and the 1918 H1N1 virus, as the NS1 proteins of these viruses were shown to act as strong antagonists of the interferon pathways (10, 25). Furthermore, the polymerase genes, in particular the PB2 gene, have been shown to be important determinants of virulence in the HPAI H5N1 and H7N7 viruses and of transmission in the 1918 H1N1 virus (11, 21, 31). One of the most commonly identified virulence markers to date is E627K in PB2. The glutamic acid (E) residue is found generally in avian influenza viruses, while human viruses have a lysine (K), and this mutation has been described as a determinant of the host range in vitro (28). When avian viruses lacking the E627K substitution were passaged in mice, the viruses acquired the mutation spontaneously upon a single passage (15, 17). In the HPAI H5N1 and H7N7 viruses, E627K was shown to be the prime determinant of pathogenesis in mice (11, 21, 23). Given that all human and many zoonotic influenza viruses of the last century contained 627K (1), it was surprising that the S-OIV had 627E.Additionally, the aspartate (D)-to-asparagine (N) mutation at position 701 of PB2, which was shown to compensate for the absence of E627K, has also not been detected in S-OIV (27). This D701N mutation has previously been shown to expand the host range of avian H5N1 to mice and humans (3, 15) and to increase virus transmission in guinea pigs (27). Thus, S-OIV was the first known human pandemic virus with 627E and 701D, and it has been speculated that S-OIV could mutate into a more virulent form by acquiring one of these mutations, or both.On 8 May 2009, the detection of another mutation in the PB2 gene of S-OIV, an E-to-glycine (G) mutation at position 667, was reported (http://www.promedmail.org/pls/apex/f?p=2400:1000, archive no. 20090508.1722). It has previously been suggested that the E667G substitution in PB2 of HPAI H5N1 virus was under positive selection and possibly played a role in sustainable transmission in humans (14).On 28 September 2009, detection of the E627K mutation in PB2 of S-OIVs of two individuals in the Netherlands was reported (http://www.promedmail.org/pls/apex/f?p=2400:1000, archive no. 20090928.3394) and raised concern about the possible enhanced replication of the S-OIV in humans, possibly associated with increased virulence. To date, the D701N mutation in PB2 has not been reported in any of the S-OIVs sequenced, and additional viruses with mutation E627K have not been recorded, either. In contrast, viruses with E677G have been reported from the United States, Canada, Germany, the United Kingdom, Norway, and France, according to the public sequence databases.Here, the effects of the E627K, D701N, and E677G mutations in the PB2 genes of S-OIVs was investigated using genetically engineered influenza viruses based on a prototype S-OIV, A/Netherlands/602/2009. Polymerase activity was measured in minigenome assays in human 293T cells, virus replication was analyzed in Madin-Darby Canine kidney (MDCK) cells, virulence was tested in mouse and ferret models, and transmission by aerosols or respiratory droplets was tested in ferrets. In contrast to the earlier assumptions based on experience with other influenza A viruses, S-OIVs with E627K, D701N, or E677G in PB2 did not show a marked increase in virulence or transmission compared to the wild-type virus.     

A Novel Nonhuman Primate Model for Influenza Transmission

Studies of influenza transmission are necessary to predict the pandemic potential of emerging influenza viruses. Currently, both ferrets and guinea pigs are used in such studies, but these species are distantly related to humans. Nonhuman primates (NHP) share a close phylogenetic relationship with humans and may provide an enhanced means to model the virological and immunological events in influenza virus transmission. Here, for the first time, it was demonstrated that a human influenza virus isolate can productively infect and be transmitted between common marmosets (Callithrix jacchus), a New World monkey species. We inoculated four marmosets with the 2009 pandemic virus A/California/07/2009 (H1N1pdm) and housed each together with a naïve cage mate. We collected bronchoalveolar lavage and nasal wash samples from all animals at regular intervals for three weeks post-inoculation to track virus replication and sequence evolution. The unadapted 2009 H1N1pdm virus replicated to high titers in all four index animals by 1 day post-infection. Infected animals seroconverted and presented human-like symptoms including sneezing, nasal discharge, labored breathing, and lung damage. Transmission occurred in one cohabitating pair. Deep sequencing detected relatively few genetic changes in H1N1pdm viruses replicating in any infected animal. Together our data suggest that human H1N1pdm viruses require little adaptation to replicate and cause disease in marmosets, and that these viruses can be transmitted between animals. Marmosets may therefore be a viable model for studying influenza virus transmission.     

Molecular basis of replication of duck H5N1 influenza viruses in a mammalian mouse model

We recently analyzed a series of H5N1 viruses isolated from healthy ducks in southern China since 1999 and found that these viruses had progressively acquired the ability to replicate and cause disease in mice. In the present study, we explored the genetic basis of this change in host range by comparing two of the viruses that are genetically similar but differ in their ability to infect mice and have different pathogenicity in mice. A/duck/Guangxi/22/2001 (DKGX/22) is nonpathogenic in mice, whereas A/duck/Guangxi/35/2001 (DKGX/35) is highly pathogenic. We used reverse genetics to create a series of single-gene recombinants that contained one gene from DKGX/22 and the remaining seven gene segments from DKGX/35. We find that the PA, NA, and NS genes of DKGX/22 could attenuate DKGX/35 virus to some extent, but PB2 of DKGX/22 virus attenuated the DKGX/35 virus dramatically, and an Asn-to-Asp substitution at position 701 of PB2 plays a key role in this function. Conversely, of the recombinant viruses in the DKGX/22 background, only the one that contains the PB2 gene of DKGX/35 was able to replicate in mice. A single amino acid substitution (Asp to Asn) at position 701 of PB2 enabled DKGX/22 to infect and become lethal for mice. These results demonstrate that amino acid Asn 701 of PB2 is one of the important determinants for this avian influenza virus to cross the host species barrier and infect mice, though the replication and lethality of H5N1 influenza viruses involve multiple genes and may result from a constellation of genes. Our findings may help to explain the expansion of the host range and lethality of the H5N1 influenza viruses to humans.     

Impact of amino acid mutations in PB2, PB1-F2, and NS1 on the replication and pathogenicity of pandemic (H1N1) 2009 influenza viruses

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.     

In silico thermodynamic stability of mammalian adaptation and virulence determinants in polymerase complex proteins of H9N2 virus

The polymerase complex proteins (PB2, PB1, and PA) are responsible primarily for the replication of avian influenza virus and play an important role in virus virulence, mammalian adaptation, and interspecies transmission. In this study; eight Egyptian LPAI-H9N2 viruses isolated from apparent healthy chickens and quails from 2014 to 2016. Characterization of complete nucleotide sequences, phylogenetic and mutation analysis were carried out. The measurement of thermodynamic stability of the H9N2 polymerase protein in comparison to human H3N2 and H1N1 proteins was carried out using in silico method. Phylogenetic analysis of these viruses revealed a close relationship to viruses isolated from neighboring Middle Eastern countries with an average of 96–99% homology. They are sharing the common ancestor A/quail/Hong Kong/G1/1997 (G1-Like) without any evidence for genetic reassortment. In addition, eight markers related to virulence were identified, including the combination of 627V and 391E in the PB2 gene with full-length PB1-F2 and PA-X proteins were observed in all viruses and the substitution N66S in PB1-F2 which suggest increasing virus virulence. Moreover, six markers that may affect the virus replication and transmission in mammalian hosts were identified. Five mutations related to mammalian adaptation show a structural stabilizing effect on LPAI-H9N2 polymerase complex protein according to the free-energy change (ΔΔG). Three out of those six adaptive mutations shown to increase polymerase complex protein stability were found in Egyptian LPAI-H9N2 viruses similar to Human H3N2 and H1N1 (661 in PB2, 225 and 409 in PA genes). Our results suggested that the stabilizing mutations in the polymerase complex protein have likely affected the protein structure and induced favorable conditions for avian virus replication and transmission in mammalian hosts. Indeed, the study reports the mutational analysis of the circulating LPAI-H9N2 strains in Egypt.     

Genetics,Receptor Binding Property,and Transmissibility in Mammals of Naturally Isolated H9N2 Avian Influenza Viruses

H9N2 subtype influenza viruses have been detected in different species of wild birds and domestic poultry in many countries for several decades. Because these viruses are of low pathogenicity in poultry, their eradication is not a priority for animal disease control in many countries, which has allowed them to continue to evolve and spread. Here, we characterized the genetic variation, receptor-binding specificity, replication capability, and transmission in mammals of a series of H9N2 influenza viruses that were detected in live poultry markets in southern China between 2009 and 2013. Thirty-five viruses represented 17 genotypes on the basis of genomic diversity, and one specific “internal-gene-combination” predominated among the H9N2 viruses. This gene combination was also present in the H7N9 and H10N8 viruses that have infected humans in China. All of the 35 viruses preferentially bound to the human-like receptor, although two also retained the ability to bind to the avian-like receptor. Six of nine viruses tested were transmissible in ferrets by respiratory droplet; two were highly transmissible. Some H9N2 viruses readily acquired the 627K or 701N mutation in their PB2 gene upon infection of ferrets, further enhancing their virulence and transmission in mammals. Our study indicates that the widespread dissemination of H9N2 viruses poses a threat to human health not only because of the potential of these viruses to cause an influenza pandemic, but also because they can function as “vehicles” to deliver different subtypes of influenza viruses from avian species to humans.     

A single amino acid in the PB2 gene of influenza A virus is a determinant of host range.

The single gene reassortant virus that derives its PB2 gene from the avian influenza A/Mallard/NY/78 virus and remaining genes from the human influenza A/Los Angeles/2/87 virus exhibits a host range restriction (hr) phenotype characterized by efficient replication in avian tissue and failure to produce plaques in mammalian Madin-Darby canine kidney cells. The hr phenotype is associated with restriction of viral replication in the respiratory tract of squirrel monkeys and humans. To identify the genetic basis of the hr phenotype, we isolated four phenotypic hr mutant viruses that acquired the ability to replicate efficiently in mammalian tissue. Segregational analysis indicated that the loss of the hr phenotype was due to a mutation in the PB2 gene itself. The nucleotide sequences of the PB2 gene of each of the four hr mutants revealed that a single amino acid substitution at position 627 (Glu-->Lys) was responsible for the restoration of the ability of the PB2 single gene reassortant to replicate in Madin-Darby canine kidney cells. Interestingly, the amino acid at position 627 in every avian influenza A virus PB2 protein analyzed to date is glutamic acid, and in every human influenza A virus PB2 protein, it is lysine. Thus, the amino acid at residue 627 of PB2 is an important determinant of host range of influenza A viruses.     

Nationwide molecular surveillance of pandemic H1N1 influenza A virus genomes: Canada, 2009

Background

In April 2009, a novel triple-reassortant swine influenza A H1N1 virus (“A/H1N1pdm”; also known as SOIV) was detected and spread globally as the first influenza pandemic of the 21^st century. Sequencing has since been conducted at an unprecedented rate globally in order to monitor the diversification of this emergent virus and to track mutations that may affect virus behavior.

Methodology/Principal Findings

By Sanger sequencing, we determined consensus whole-genome sequences for A/H1N1pdm viruses sampled nationwide in Canada over 33 weeks during the 2009 first and second pandemic waves. A total of 235 virus genomes sampled from unique subjects were analyzed, providing insight into the temporal and spatial trajectory of A/H1N1pdm lineages within Canada. Three clades (2, 3, and 7) were identifiable within the first two weeks of A/H1N1pdm appearance, with clades 5 and 6 appearing thereafter; further diversification was not apparent. Only two viral sites displayed evidence of adaptive evolution, located in hemagglutinin (HA) corresponding to D222 in the HA receptor-binding site, and to E374 at HA2-subunit position 47. Among the Canadian sampled viruses, we observed notable genetic diversity (1.47×10⁻³ amino acid substitutions per site) in the gene encoding PB1, particularly within the viral genomic RNA (vRNA)-binding domain (residues 493–757). This genome data set supports the conclusion that A/H1N1pdm is evolving but not excessively relative to other H1N1 influenza A viruses. Entropy analysis was used to investigate whether any mutated A/H1N1pdm protein residues were associated with infection severity; however no virus genotypes were observed to trend with infection severity. One virus that harboured heterozygote coding mutations, including PB2 D567D/G, was attributed to a severe and potentially mixed infection; yet the functional significance of this PB2 mutation remains unknown.

Conclusions/Significance

These findings contribute to enhanced understanding of Influenza A/H1N1pdm viral dynamics.     

In Vivo Selection of H1N2 Influenza Virus Reassortants in the Ferret Model

Although the ferret model has been extensively used to study pathogenesis and transmission of influenza viruses, little has been done to determine whether ferrets are a good surrogate animal model to study influenza virus reassortment. It has been previously shown that the pandemic 2009 H1N1 (H1N1pdm) virus was able to transmit efficiently in ferrets. In coinfection studies with either seasonal H1N1 or H3N2 strains (H1N1s or H3N2s, respectively), the H1N1pdm virus was able to outcompete these strains and become the dominant transmissible virus. However, lack of reassortment could have been the result of differences in the cell or tissue tropism of these viruses in the ferret. To address this issue, we performed coinfection studies with recombinant influenza viruses carrying the surface genes of a seasonal H3N2 strain in the background of an H1N1pdm strain and vice versa. After serial passages in ferrets, a dominant H1N2 virus population was obtained with a constellation of gene segments, most of which, except for the neuraminidase (NA) and PB1 segments, were from the H1N1pdm strain. Our studies suggest that ferrets recapitulate influenza virus reassortment events. The H1N2 virus generated through this process resembles similar viruses that are emerging in nature, particularly in pigs.     

Growth of H5N1 influenza A viruses in the upper respiratory tracts of mice

Highly pathogenic avian H5N1 influenza A viruses have spread throughout Asia, Europe, and Africa, raising serious worldwide concern about their pandemic potential. Although more than 250 people have been infected with these viruses, with a consequent high rate of mortality, the molecular mechanisms responsible for the efficient transmission of H5N1 viruses among humans remain elusive. We used a mouse model to examine the role of the amino acid at position 627 of the PB2 viral protein in efficient replication of H5N1 viruses in the mammalian respiratory tract. Viruses possessing Lys at position 627 of PB2 replicated efficiently in lungs and nasal turbinates, as well as in cells, even at the lower temperature of 33 degrees C. Those viruses possessing Glu at this position replicated less well in nasal turbinates than in lungs, and less well in cells at the lower temperature. These results suggest that Lys at PB2-627 confers to avian H5N1 viruses the advantage of efficient growth in the upper and lower respiratory tracts of mammals. Therefore, efficient viral growth in the upper respiratory tract may provide a platform for the adaptation of avian H5N1 influenza viruses to humans and for efficient person-to-person virus transmission, in the context of changes in other viral properties including specificity for human (sialic acid alpha-2,6-galactose containing) receptors.