Adaptation of Avian Influenza A Virus Polymerase in Mammals To Overcome the Host Species Barrier

Avian influenza A viruses, such as the highly pathogenic avian H5N1 viruses, sporadically enter the human population but often do not transmit between individuals. In rare cases, however, they establish a new lineage in humans. In addition to well-characterized barriers to cell entry, one major hurdle which avian viruses must overcome is their poor polymerase activity in human cells. There is compelling evidence that these viruses overcome this obstacle by acquiring adaptive mutations in the polymerase subunits PB1, PB2, and PA and the nucleoprotein (NP) as well as in the novel polymerase cofactor nuclear export protein (NEP). Recent findings suggest that synthesis of the viral genome may represent the major defect of avian polymerases in human cells. While the precise mechanisms remain to be unveiled, it appears that a broad spectrum of polymerase adaptive mutations can act collectively to overcome this defect. Thus, identification and monitoring of emerging adaptive mutations that further increase polymerase activity in human cells are critical to estimate the pandemic potential of avian viruses.     

禽流感病毒与人流感的关系

禽流感病毒 (AIV)是甲 (A)型流感病毒 ,常引起禽类全身性感染或主要限于呼吸器官传染病 ,带来巨大的经济损失并严重威胁人类健康。对AIV的基因组、所编码的蛋白质及其功能、AIV毒力变异的分子基础、禽流感疫苗以及AIV与人流感的关系等进行概述。     

Additional Evidence That the Polymerase Subunits Contribute to the Viral Replication and the Virulence of H5N1 Avian Influenza Virus Isolates in Mice

Genetically similar H5N1 viruses circulating in the avian reservoir exhibit different levels of pathogenicity in mice. In this study, we characterized two highly pathogenic H5N1 avian isolates—A/Hunan/316/2005 (HN05), which is highly pathogenic in mice, and A/Hubei/489/2004 (HB04), which is nonpathogenic. In mammalian cells, HN05 replicates more efficiently than HB04, although both viruses have similar growth kinetics in avian cells. We used reverse genetics to generate recombinant H5N1 strains containing genes from HN05 and HB04 and examined their virulence. HN05 genes encoding the polymerase complex determine pathogenicity and viral replication ability both in vitro and in vivo. The PB2 subunit plays an important role in enhancing viral replication, and the PB1 and PA subunits contribute mainly to pathogenicity in mice. These results can be used to elucidate host-range expansion and the molecular basis of the high virulence of H5N1 viruses in mammalian species.     

Full Factorial Analysis of Mammalian and Avian Influenza Polymerase Subunits Suggests a Role of an Efficient Polymerase for Virus Adaptation

Amongst all the internal gene segments (PB2. PB1, PA, NP, M and NS), the avian PB1 segment is the only one which was reassorted into the human H2N2 and H3N2 pandemic strains. This suggests that the reassortment of polymerase subunit genes between mammalian and avian influenza viruses might play roles for interspecies transmission. To test this hypothesis, we tested the compatibility between PB2, PB1, PA and NP derived from a H5N1 virus and a mammalian H1N1 virus. All 16 possible combinations of avian-mammalian chimeric viral ribonucleoproteins (vRNPs) were characterized. We showed that recombinant vRNPs with a mammalian PB2 and an avian PB1 had the strongest polymerase activities in human cells at all studied temperature. In addition, viruses with this specific PB2-PB1 combination could grow efficiently in cell cultures, especially at a high incubation temperature. These viruses were potent inducers of proinflammatory cytokines and chemokines in primary human macrophages and pneumocytes. Viruses with this specific PB2-PB1 combination were also found to be more capable to generate adaptive mutations under a new selection pressure. These results suggested that the viral polymerase activity might be relevant for the genesis of influenza viruses of human health concern.     

Avian Influenza A Virus Polymerase Association with Nucleoprotein, but Not Polymerase Assembly, Is Impaired in Human Cells during the Course of Infection

Strong determinants of the host range of influenza A viruses have been identified on the polymerase complex formed by the PB1, PB2, and PA subunits and on the nucleoprotein (NP). In the present study, molecular mechanisms that may involve these four core proteins and contribute to the restriction of avian influenza virus multiplication in human cells have been investigated. The efficiencies with which the polymerase complexes of a human and an avian influenza virus isolate assemble and interact with the viral NP and cellular RNA polymerase II proteins were compared in mammalian and in avian infected cells. To this end, recombinant influenza viruses expressing either human or avian-derived core proteins with a PB2 protein fused to the One-Strep purification tag at the N or C terminus were generated. Copurification experiments performed on infected cell extracts indicate that the avian-derived polymerase is assembled and interacts physically with the cellular RNA polymerase II at least as efficiently as does the human-derived polymerase in human as well as in avian cells. Restricted growth of the avian isolate in human cells correlates with low levels of the core proteins in infected cell extracts and with poor association of the NP with the polymerase compared to what is observed for the human isolate. The NP-polymerase association is restored by a Glu-to-Lys substitution at residue 627 of PB2. Overall, our data point to viral and cellular factors regulating the NP-polymerase interaction as key determinants of influenza A virus host range. Recombinant viruses expressing a tagged polymerase should prove useful for further studies of the molecular interactions between viral polymerase and host factors during the infection cycle.     

Association of the Influenza Virus RNA Polymerase Subunit PB2 with the Host Chaperonin CCT

The RNA polymerase of influenza A virus is a host range determinant and virulence factor. In particular, the PB2 subunit of the RNA polymerase has been implicated as a crucial factor that affects cell tropism as well as virulence in animal models. These findings suggest that host factors associating with the PB2 protein may play an important role during viral replication. In order to identify host factors that associate with the PB2 protein, we purified recombinant PB2 from transiently transfected mammalian cells and identified copurifying host proteins by mass spectrometry. We found that the PB2 protein associates with the cytosolic chaperonin containing TCP-1 (CCT), stress-induced phosphoprotein 1 (STIP1), FK506 binding protein 5 (FKBP5), α- and β-tubulin, Hsp60, and mitochondrial protein p32. Some of these binding partners associate with each other, suggesting that PB2 might interact with these proteins in multimeric complexes. More detailed analysis of the interaction of the PB2 protein with CCT revealed that PB2 associates with CCT as a monomer and that the CCT binding site is located in a central region of the PB2 protein. PB2 proteins from various influenza virus subtypes and origins can associate with CCT. Silencing of CCT resulted in reduced viral replication and reduced PB2 protein and viral RNA accumulation in a ribonucleoprotein reconstitution assay, suggesting an important function for CCT during the influenza virus life cycle. We propose that CCT might be acting as a chaperone for PB2 to aid its folding and possibly its incorporation into the trimeric RNA polymerase complex.Influenza A viruses, members of the family of Orthomyxoviridae, contain a segmented RNA genome of negative polarity. The genomic RNA segments together with the three subunits of the viral RNA-dependent RNA polymerase (PB1, PB2, and PA protein) and the nucleoprotein (NP) form viral ribonucleoprotein complexes (vRNPs). The PB1 subunit is the polymerase itself, while the PB2 and PA subunits are involved in the generation of 5′ capped RNA primers through binding to and endonucleolytic cleavage of host pre-mRNAs (8, 10, 11, 41, 61). After the virus enters the cell via endocytosis, vRNPs are released into the cytoplasm and transported into the nucleus. In the nucleus, vRNPs catalyze the synthesis of viral mRNAs and complementary RNAs (cRNA) which, in turn, are used as templates for the synthesis of vRNAs. The newly formed vRNPs in association with other viral proteins (M1 and nonstructural protein 2/nuclear export factor [NS2/NEP]) are transported into the cytoplasm and subsequently to the cell membrane, where the assembly process takes place, followed by the release of progeny virions by budding (44).The PB1, PB2, and PA proteins are synthesized in the cytoplasm whereupon PB1 and PA form a dimeric complex that is transported into the nucleus. In the nucleus the dimer assembles with the PB2 subunit, which is transported separately (7, 14). RanBP5 was identified as a factor that is involved in the import of the PB1-PA dimer into the nucleus (6), while PB2 uses the classical importin-α/β pathway for nuclear import (57). Recently, further support for this transport and assembly model was provided by using fluorescence cross-correlation spectroscopy (25). An alternative pathway proposed for the import of the RNA polymerase subunits into the nucleus involves the heat shock protein 90 (Hsp90) that was shown to interact with the PB1 and PB2 proteins (39). Heat shock protein 70 (Hsp70) was also found to interact with the influenza virus polymerase subunits and vRNPs, and it was implicated in blocking the nuclear export of vRNPs (22).The RNA polymerase has been implicated as a host range determinant and pathogenicity factor of influenza viruses. In particular, amino acid residue 627 in the PB2 subunit was shown to determine the ability of certain influenza viruses to replicate in avian and mammalian cells (34, 54). A lysine at position 627, characteristic of most human influenza virus strains, appears to enhance replication in mammalian cells, while a glutamic acid, found in most avian isolates, attenuates virus replication in mammalian cells. The presence of a lysine was also shown to enhance virulence in mammalian models and has been associated with the lethality of H5N1 viruses in humans (20). It has been proposed that a negative factor, present in mammalian cells, specifically reduces the activity of a polymerase containing a glutamic acid (38). However, the identity of this factor remains to be determined. Interestingly, the 2009 H1N1 pandemic influenza virus encodes a glutamic acid at this position, and a second-site suppressor mutation has been identified in PB2 that promotes activity in mammalian cells (37). Introduction of a lysine at residue 627 in the 2009 H1N1 pandemic virus did not result in enhanced virulence (21, 62). Several other amino acid residues in the PB2 protein were also implicated in host range determination and virulence, suggesting that multiple amino acid substitutions are involved (15, 48). Collectively, these results suggest that the PB2 protein interacts with host factors and that these interactions have implications for host range and virulence.Therefore, we set up a biochemical copurification assay followed by mass spectrometry to identify host factors that associate with the PB2 protein in mammalian cells. We confirmed the interaction with several previously identified host factors, e.g., Hsp70 and Hsp90, and identified novel host proteins that interact with the PB2 protein. Among these, we have identified the oligomeric chaperonin containing TCP-1 (CCT) (also known as TRiC [TCP-1 ring complex]) and investigated the significance of this interaction in more detail. We found that CCT interacts with the PB2 protein but not with the PB1 or PA protein. However, PB2 in association with PB1 or PB1 and PA did not interact with CCT. We also found that PB2 proteins of different influenza virus strains of different origins, hosts, and subtypes interact with CCT. Growth of influenza virus, as well as the accumulation of the PB2 protein and viral RNAs in a ribonucleoprotein reconstitution assay, was reduced in CCT-silenced cells compared to that in control cells. These results suggest a role for CCT in the influenza A virus life cycle, possibly acting as a chaperone for the PB2 protein.     

Inhibition of Influenza Virus Replication by Targeting Broad Host Cell Pathways

Antivirals that are currently used to treat influenza virus infections target components of the virus which can mutate rapidly. Consequently, there has been an increase in the number of resistant strains to one or many antivirals in recent years. Here we compared the antiviral effects of lysosomotropic alkalinizing agents (LAAs) and calcium modulators (CMs), which interfere with crucial events in the influenza virus replication cycle, against avian, swine, and human viruses of different subtypes in MDCK cells. We observed that treatment with LAAs, CMs, or a combination of both, significantly inhibited viral replication. Moreover, the drugs were effective even when they were administered 8 h after infection. Finally, analysis of the expression of viral acidic polymerase (PA) revealed that both drugs classes interfered with early events in the viral replication cycle. This study demonstrates that targeting broad host cellular pathways can be an efficient strategy to inhibit influenza replication. Furthermore, it provides an interesting avenue for drug development where resistance by the virus might be reduced since the virus is not targeted directly.     

Ecological Factors Driving Avian Influenza Virus Dynamics in Spanish Wetland Ecosystems

Studies exploring the ecological interactions between avian influenza viruses (AIV), natural hosts and the environment are scarce. Most work has focused on viral survival and transmission under laboratory conditions and through mathematical modelling. However, more integrated studies performed under field conditions are required to validate these results. In this study, we combined information on bird community, environmental factors and viral epidemiology to assess the contribution of biotic and abiotic factors in the occurrence of low pathogenic AIV in Spanish wetlands. For that purpose, seven locations in five different wetlands were studied during two years (2007–2009), including seven sampling visits by location. In each survey, fresh faeces (n = 4578) of wild birds and water samples were collected for viral detection. Also, the vegetation structure, water physical properties of wetlands, climatic conditions and wild bird community composition were determined. An overall AIV prevalence of 1.7%±0.4 was detected in faecal samples with important fluctuations among seasons and locations. Twenty-six AIV were isolated from the 78 RRT-PCR positive samples and eight different haemagglutinines and five neuraminidases were identified, being the combination H3N8 the most frequent. Variation partitioning procedures identified the combination of space and time variables as the most important pure factor – independently to other factors – explaining the variation in AIV prevalence (36.8%), followed by meteorological factor (21.5%) and wild bird community composition/vegetation structure (21.1%). These results contribute to the understanding of AIV ecological drivers in Spanish ecosystems and provide useful guidelines for AIV risk assessment identifying potential hotspots of AIV activity.     

Hemagglutinin-Dependent Tropism of H5N1 Avian Influenza Virus for Human Endothelial Cells

Although current H5N1 highly pathogenic avian influenza viruses (HPAIV) are inefficiently transmitted to humans, infected individuals can suffer from severe disease, often progressing rapidly to acute respiratory distress syndrome and multiorgan failure. This is in contrast with the situation with human influenza viruses, which in immunocompetent individuals usually cause only a respiratory disease which is less aggressive than that observed with avian H5N1 viruses. While the biological basis of inefficient transmission is well documented, the mechanisms by which the H5N1 viruses cause fatal disease remain unclear. In the present study, we demonstrate that human pulmonary microvascular endothelial cells (hPMEC) had a clearly higher susceptibility to infection by H5N1 HPAIV than to infection by human influenza viruses. This was measurable by de novo intracellular nucleoprotein production and virus replication. It was also related to a relatively higher binding capacity to cellular receptors. After infection of hPMEC, cell activation markers E-selectin and P-selectin were upregulated, and the proinflammatory cytokines interleukin-6 and beta interferon were secreted. H5N1 virus infection was also associated with an elevated rate of cell death. Reverse genetics analyses demonstrated a major role for the viral hemagglutinin in this cell tropism. Overall, avian H5N1 viruses have a particular receptor specificity targeting endothelial cells that is different from human influenza viruses, and this H5N1 receptor specificity could contribute to disease pathogenesis.Certain highly pathogenic avian influenza viruses (HPAIV) expressing the H5 and H7 hemagglutinins (HA) have acquired the capacity to infect humans. Particularly, HPAIV with the H5 HA and the neuraminidase (NA) type 1 (H5N1) can cause severe disease, often with a fatal outcome in humans and other mammals (27). With such infections in humans, there are two striking differences compared to infection by human influenza A viruses (IAV). First, bird-to-human and human-to-human transmission has been considered inefficient, and second, the mortality rate of H5N1 virus infections has been unexpectedly high. There is a lot of experimental evidence that inefficient transmission rate is related to several viral gene products not optimally adapted to facilitate infection and replication in the primary target cells, the epithelial cells of the respiratory tract. Of particular importance is the HA determining receptor specificity with human viruses preferentially recognizing sialic acid (SA)-α-2,6-Gal-terminated saccharides (α-2,6-SA), abundantly expressed in the upper respiratory tract, and avian viruses preferentially binding to α-2,3-SA, expressed mainly in the lower respiratory tract and on ciliated epithelial cells (23, 33, 39). In addition, the viral polymerases determining the rate of replication as well as the NS1 protein involved in multiple processes enabling efficient viral replication and evasion of cellular antiviral responses are of importance in determining host tropism (17, 26).However, in contrast to infections with human influenza viruses, avian H5N1 virus infections more often cause severe pneumonia. These are associated with high levels of proinflammatory cytokines and chemokines in the respiratory tract, severe inflammatory reactions, and infiltration of leukocytes. Furthermore, a generalized inflammatory reaction with elevated cytokine and chemokine levels in the circulation, together with leukopenia and multiorgan failure, indicates that an aberrant immunological reaction is an important factor contributing to the fatality of H5N1 virus infections (19). This is supported by in vitro studies of human macrophages, dendritic cells, and epithelial cells, in which it was demonstrated that H5N1 viruses can induce higher levels of inflammatory cytokine and chemokine responses than human IV isolates (2, 3, 37). Based on this, it was proposed that factors of the innate and adaptive immune response are of central importance for the outcome of disease (8, 26).Endothelial cells (EDC) are abundant in all organs, particularly the lung, and play an important role in inflammatory processes through the regulation of leukocyte extravasation, the production of inflammatory cytokines and chemokines, and the regulation of coagulation (4). During systemic disease in chickens infected with H5N1 isolates, the cardiovascular system can be affected with coagulopathy and viral antigen detectable in EDC (15, 25, 36). This also relates to a report demonstrating a targeted infection of EDC in chicken embryo by A/FPV/Rostock/34 (H7N1) virus (6). In this study, the infection of human umbilical vein EDC is also reported. Finally, in humans, various degrees of hemorrhages as well as signs of disseminated intravascular coagulation have been found (1).Accordingly, the present study compared influenza virus isolates of avian and human origin with respect to their characteristics of interaction with human EDC. To this end, we infected primary human lung EDC with different naturally occurring virus isolates as well as viruses created by reverse genetics. Viruses expressing the H5 clearly possessed the greatest potency to infect and replicate in EDC, resulting in activation and inflammatory responses.     

Dynamics of Influenza Virus and Human Host Interactions During Infection and Replication Cycle

The replication and life cycle of the influenza virus is governed by an intricate network of intracellular regulatory events during infection, including interactions with an even more complex system of biochemical interactions of the host cell. Computational modeling and systems biology have been successfully employed to further the understanding of various biological systems, however, computational studies of the complexity of intracellular interactions during influenza infection is lacking. In this work, we present the first large-scale dynamical model of the infection and replication cycle of influenza, as well as some of its interactions with the host’s signaling machinery. Specifically, we focus on and visualize the dynamics of the internalization and endocytosis of the virus, replication and translation of its genomic components, as well as the assembly of progeny virions. Simulations and analyses of the models dynamics qualitatively reproduced numerous biological phenomena discovered in the laboratory. Finally, comparisons of the dynamics of existing and proposed drugs, our results suggest that a drug targeting PB1:PA would be more efficient than existing Amantadin/Rimantaine or Zanamivir/Oseltamivir.     

Polymerase Activity of Pichinde Virus

Pichinde virus, a member of the arenavirus group, was examined for polymerase activity. Purified virus was found to contain RNA-dependent RNA polymerase but not RNA-dependent DNA polymerase activity. Since RNase but neither DNase nor actinomycin D inhibited the endogenous polymerase reaction, RNA of the virus appeared to be used as the template. The divalent cations Mg(2+) and Mn(2+) were required for optimal reactivity. The RNA product was partially resistant to RNase and the resistant portion had a sedimentation coefficient of 22 to 26S in sucrose gradients.     

Little Evidence of Subclinical Avian Influenza Virus Infections among Rural Villagers in Cambodia

In 2008, 800 adults living within rural Kampong Cham Province, Cambodia were enrolled in a prospective cohort study of zoonotic influenza transmission. After enrollment, participants were contacted weekly for 24 months to identify acute influenza-like illnesses (ILI). Follow-up sera were collected at 12 and 24 months. A transmission substudy was also conducted among the family contacts of cohort members reporting ILI who were influenza A positive. Samples were assessed using serological or molecular techniques looking for evidence of infection with human and avian influenza viruses. Over 24 months, 438 ILI investigations among 284 cohort members were conducted. One cohort member was hospitalized with a H5N1 highly pathogenic avian influenza (HPAI) virus infection and withdrew from the study. Ninety-seven ILI cases (22.1%) were identified as influenza A virus infections by real-time RT-PCR; none yielded evidence for AIV. During the 2 years of follow-up, 21 participants (3.0%) had detectable antibody titers (≥1∶10) against the studied AIVs: 1 against an avian-like A/Migratory duck/Hong Kong/MPS180/2003(H4N6), 3 against an avian-like A/Teal/Hong Kong/w312/97(H6N1), 9 (3 of which had detectible antibody titers at both 12- and 24-month follow-up) against an avian-like A/Hong Kong/1073/1999(H9N2), 6 (1 detected at both 12- and 24-month follow-up) against an avian-like A/Duck/Memphis/546/74(H11N9), and 2 against an avian-like A/Duck/Alberta/60/76(H12N5). With the exception of the one hospitalized cohort member with H5N1 infection, no other symptomatic avian influenza infections were detected among the cohort. Serological evidence for subclinical infections was sparse with only one subject showing a 4-fold rise in microneutralization titer over time against AvH12N5. In summary, despite conducting this closely monitored cohort study in a region enzootic for H5N1 HPAI, we were unable to detect subclinical avian influenza infections, suggesting either that these infections are rare or that our assays are insensitive at detecting them.     

Avian Influenza Virus H3 Hemagglutinin May Enable High Fitness of Novel Human Virus Reassortants

Reassortment of influenza A virus genes enables antigenic shift resulting in the emergence of pandemic viruses with novel hemagglutinins (HA) acquired from avian strains. Here, we investigated whether historic and contemporary avian strains with different replication capacity in human cells can donate their hemagglutinin to a pandemic human virus. We performed double-infections with two avian H3 strains as HA donors and a human acceptor strain, and determined gene compositions and replication of HA reassortants in mammalian cells. To enforce selection for the avian virus HA, we generated a strictly elastase-dependent HA cleavage site mutant from A/Hong Kong/1/68 (H3N2) (Hk68-Ela). This mutant was used for co-infections of human cells with A/Duck/Ukraine/1/63 (H3N8) (DkUkr63) or the more recent A/Mallard/Germany/Wv64-67/05 (H3N2) (MallGer05) in the absence of elastase but presence of trypsin. Among 21 plaques analyzed from each assay, we found 12 HA reassortants with DkUkr63 (4 genotypes) and 14 with MallGer05 (10 genotypes) that replicated in human cells comparable to the parental human virus. Although DkUkr63 replicated in mammalian cells at a reduced level compared to MallGer05 and Hk68, it transmitted its HA to the human virus, indicating that lower replication efficiency of an avian virus in a mammalian host may not constrain the emergence of viable HA reassortants. The finding that HA and HA/NA reassortants replicated efficiently like the human virus suggests that further HA adaptation remains a relevant barrier for emergence of novel HA reassortants.     

Physical Factors That Affect In Vitro Autographa californica Nuclear Polyhedrosis Virus Infection

Of the physical parameters tested for in vitro baculovirus infection, multiplicity of infection was most important in governing percent cell infection. Most plaques formed within the first 5 min of incubation. Efficiency of infection, however, was low, and the virus titer did not diminish during prolonged incubation. Efficiency of infection improved markedly when cells or virus were preincubated with selected polyanions and polycations. Precise regulation of the pH, osmotic pressure, and ionic composition of the cell culture medium also promoted maximum in vivo infection.