Showing papers on "Avian Influenza A Virus published in 2019"

Profiling host ANP32A splicing landscapes to predict influenza A virus polymerase adaptation.

Abstract: Species' differences in cellular factors limit avian influenza A virus (IAV) zoonoses and human pandemics. The IAV polymerase, vPol, harbors evolutionary sites to overcome restriction and determines virulence. Here, we establish host ANP32A as a critical driver of selection, and identify host-specific ANP32A splicing landscapes that predict viral evolution. We find that avian species differentially express three ANP32A isoforms diverging in a vPol-promoting insert. ANP32As with shorter inserts interact poorly with vPol, are compromised in supporting avian-like IAV replication, and drive selection of mammalian-adaptive vPol sequences with distinct kinetics. By integrating selection data with multi-species ANP32A splice variant profiling, we develop a mathematical model to predict avian species potentially driving (swallow, magpie) or maintaining (goose, swan) mammalian-adaptive vPol signatures. Supporting these predictions, surveillance data confirm enrichment of several mammalian-adaptive vPol substitutions in magpie IAVs. Profiling host ANP32A splicing could enhance surveillance and eradication efforts against IAVs with pandemic potential.

Continuing evolution of highly pathogenic H5N1 viruses in Bangladeshi live poultry markets.

Abstract: Since November 2008, we have conducted active avian influenza surveillance in Bangladesh. Clades 2.2.2, 2.3.4.2, and 2.3.2.1a of highly pathogenic avian influenza H5N1 viruses have all been identif...

Bayesian phylodynamics of avian influenza A virus H9N2 in Asia with time-dependent predictors of migration.

Abstract: Model-based phylodynamic approaches recently employed generalized linear models (GLMs) to uncover potential predictors of viral spread. Very recently some of these models have allowed both the predictors and their coefficients to be time-dependent. However, these studies mainly focused on predictors that are assumed to be constant through time. Here we inferred the phylodynamics of avian influenza A virus H9N2 isolated in 12 Asian countries and regions under both discrete trait analysis (DTA) and structured coalescent (MASCOT) approaches. Using MASCOT we applied a new time-dependent GLM to uncover the underlying factors behind H9N2 spread. We curated a rich set of time-series predictors including annual international live poultry trade and national poultry production figures. This time-dependent phylodynamic prediction model was compared to commonly employed time-independent alternatives. Additionally the time-dependent MASCOT model allowed for the estimation of viral effective sub-population sizes and their changes through time, and these effective population dynamics within each country were predicted by a GLM. International annual poultry trade is a strongly supported predictor of virus migration rates. There was also strong support for geographic proximity as a predictor of migration rate in all GLMs investigated. In time-dependent MASCOT models, national poultry production was also identified as a predictor of virus genetic diversity through time and this signal was obvious in mainland China. Our application of a recently introduced time-dependent GLM predictors integrated rich time-series data in Bayesian phylodynamic prediction. We demonstrated the contribution of poultry trade and geographic proximity (potentially unheralded wild bird movements) to avian influenza spread in Asia. To gain a better understanding of the drivers of H9N2 spread, we suggest increased surveillance of the H9N2 virus in countries that are currently under-sampled as well as in wild bird populations in the most affected countries.

Avian Influenza A Virus Polymerase Recruits Cellular RNA Helicase eIF4A3 to Promote Viral mRNA Splicing and Spliced mRNA Nuclear Export.

Abstract: The influenza A virus replicates in a broad range of avian and mammalian species by hijacking cellular factors and processes. Avian influenza A viruses (AIVs) generally propagated poorly in mammalian cells, but some mutants of virus-encoded RNA polymerase components, especially PB2 subunit, can overcome host restriction. Host factors associated with PB2 may be essential for efficient AIV replication in mammalian cells. Here, we infected human cells with the PB2 Flag-tagged replication-competent recombinant AIV and identified cellular proteins that coprecipitate with PB2 protein by mass spectrometry. We confirmed one of the coprecipitating host factors, DEAD-box protein eIF4A3, that interacts with viral PB2, PB1, and NP proteins. Depletion of endogenous eIF4A3 significantly reduced virus replication. Later studies showed that eIF4A3 is essential for viral RNA polymerase activity and viral RNAs synthesis. Upon systematic dissection of the influenza virus progeny mRNA generation, from pre-mRNA processing to nuclear export, we found that the depletion of eIF4A3 resulted in significant defects in the ratio of M2 to M1 and NS2 to NS1, and the proportion of viral spliced mRNA in the nucleus increased, indicating that eIF4A3 plays a significant function in viral nascent intron mRNA splicing and spliced mRNA (M2 and NS2) nuclear export. Additionally, we confirmed that in specific deletion of eIF4A3, the synthesis of reduced NS2 can significantly impair neo-synthetized viral ribonucleoprotein (vRNP) nuclear export. Taken together, our findings revealed that eIF4A3 is a key mediator of AIV polymerase activity, mRNA splicing, and spliced mRNA nuclear export.

A delayed avian influenza model with avian slaughter: Stability analysis and optimal control

Abstract: H7N9 avian influenza, a zoonotic disease caused by the transmission of the avian influenza A virus from poultry to humans, is low pathogenic for poultry but highly pathogenic for humans. Slaughter is an effective way to control the outbreak. In this paper, we consider the slaughter of avian population and the incubation periods of avian influenza A virus, and develop a poultry-to-human epidemiological model including time delays of being infectious for the avian and human populations after infection. By studying the dynamical behavior of this delay model, we obtain a threshold value for the extinction and prevalence of avian influenza, and show the local and global asymptotical properties of the two equilibria of the model. We further choose the slaughter intensity and the effect of education campaign as control variables, and formulate an optimal control problem to minimize the disease outbreak and control cost. We solve the optimal control problem and perform numerical simulations to illustrate the analytical results.

Transcriptional and Pathological Host Responses to Coinfection with Virulent or Attenuated Mycoplasma gallisepticum and Low-Pathogenic Avian Influenza A Virus in Chickens.

Abstract: The avian pathogen Mycoplasma gallisepticum, the etiological agent of chronic respiratory disease in chickens, exhibits enhanced pathogenesis in the presence of a copathogen such as low-pathogenic avian influenza virus (LPAIV). To further investigate the intricacies of this copathogenesis, chickens were monoinfected or coinfected with either virulent M. gallisepticum strain Rlow or LPAIV H3N8 (A/duck/Ukraine/1963), with assessment of tracheal histopathology, pathogen load, and transcriptomic host responses to infection by RNA sequencing. Chickens coinfected with M. gallisepticum Rlow followed by LPAIV H3N8 exhibited significantly more severe tracheal lesions and mucosal thickening than chickens infected with LPAIV H3N8 alone and greater viral loads than chickens infected first with H3N8 and subsequently with M. gallisepticum Rlow Recovery of live M. gallisepticum was significantly higher in chickens infected first with LPAIV H3N8 and then with M. gallisepticum Rlow, compared to chickens given a mock infection followed by M. gallisepticum Rlow The transcriptional responses to monoinfection and coinfection with M. gallisepticum and LPAIV highlighted the involvement of differential expression of genes such as Toll-like receptor 15, Toll-like receptor 21, and matrix metallopeptidase 1. Pathway and gene ontology analyses of these differentially expressed genes suggest that coinfection with virulent M. gallisepticum and LPAIV induces decreases in the expression of genes related to ciliary activity in vivo and alters multiple immune-related signaling cascades. These data aid in the understanding of the relationship between M. gallisepticum and LPAIV during copathogenesis in the natural host and may contribute to further understanding of copathogen infections of humans and other animals.

A Novel Neuraminidase-Dependent Hemagglutinin Cleavage Mechanism Enables the Systemic Spread of an H7N6 Avian Influenza Virus.

Abstract: In this study, we demonstrate a novel mechanism for hemagglutinin (HA) activation in a naturally occurring H7N6 avian influenza A virus strain, A/mallard duck/Korea/6L/2007 (A/Mdk/6L/07). This novel mechanism allows for systemic infection of chickens, ducks, and mice, and A/Mdk/6L/07 can replicate in vitro without exogenous trypsin and exhibits broad tissue tropism in animals despite the presence of a monobasic HA cleavage motif (PEIPKGR/G). The trypsin-independent growth phenotype requires the N6 neuraminidase and the specific recognition of glycine at the P2 position of the HA cleavage motif by a thrombin-like protease. Correspondingly, viral growth is significantly attenuated by the addition of a thrombin-like protease inhibitor (argatroban). These data provide evidence for a previously unrecognized virus replication mechanism and support the hypothesis that thrombin-mediated HA cleavage is an important virulence marker and potential therapeutic target for H7 influenza viruses.IMPORTANCE The identification of virulence markers in influenza viruses underpins risk assessment programs and the development of novel therapeutics. The cleavage of the influenza virus HA is a required step in the viral life cycle, and phenotypic differences in viruses can be caused by changes in this process. Here, we describe a novel mechanism for HA cleavage in an H7N6 influenza virus isolated from a mallard duck. The mechanism requires the N6 protein and full activity of thrombin-like proteases and allows the virus to cause systemic infection in chickens, ducks, and mice. The thrombin-mediated cleavage of HA is thus a novel virulence determinant of avian influenza viruses.

Adaptive Evolution of Human-Isolated H5Nx Avian Influenza A Viruses.

Abstract: Avian influenza A viruses (AIVs) H5N1, first identified in 1996, are highly pathogenic in domestic poultry and continue to occasionally infect humans. In this study, we sought to identify genetic changes that occurred during their multiple invasions to humans. We evaluated all available H5Nx AIV genomes. Significant signals of positive selection were detected in 29 host-shift branches. 126 parallel evolution sites were detected on these branches, including 17 well-known sites (such as T271A, A274T, T339M, Q591K, E627K, and D701N in PB2; A134V, D154N, S223N, and R497K in HA) that play roles in allowing AIVs to cross species barriers. Our study suggests that during human infections, H5Nx viruses have experienced adaptive evolution (positive selection and convergent evolution) that allowed them to adapt to their new host environments. Analyses of adaptive evolution should be useful in identifying candidate sites that play roles in human infections, which can be tested by functional experiments.

Regulation of Early Host Immune Responses Shapes the Pathogenicity of Avian Influenza A Virus

Abstract: Avian influenza A viruses (IAV) can cross the species barrier and cause disease in humans. Understanding the pathogenesis of avian IAV remains a challenge. Interferon-mediated antiviral responses and multiple cytokines production are important host cellular antiviral immunity against IAV infection. To elucidate the pathogenicity of avian IAV, a system approach was adopted to investigate dysregulation of the two host cellular antiviral immune responses in contrast with human IAV. As a result, we revealed that avian IAV not only disrupted normal early host cellular interferon-mediated antiviral responses, but also caused abnormal cytokines production through different pathways. For avian IAV infection, dysregulation of STAT2 was mainly responsible for abnormal cellular interferon-mediated antiviral responses, and IRF5 and NFKB1 played crucial roles in unusual cytokines production. In contrast, for human IAV infection, IRF1, IRF7, and STAT1 contributed to cellular cytokines production. Furthermore, differential activation of pattern recognition receptors (PRRs) likely led to avian IAV-related abnormal early host cellular antiviral immunity, where TLR7 and RIG-I were activated by avian and human IAV, respectively. Finally, a pathogenesis model was proposed that combined of early host cellular interferon-mediated antiviral responses with cytokines production could partly explain the pathogenicity of avian IAV. In conclusion, our study provides a new perspective of the pathogenesis of avian IAV, which will be helpful in preventing their infections in the future.

Pathological changes in natural infection of pheasants with highly pathogenic avian influenza A (H5N8) in Bulgaria

Abstract: Introduction The study of histopathological changes caused by influenza A (H5N8) viral infection in bird species is essential for the understanding of their role in the spread of this highly infectious virus. However, there are few such studies under natural conditions in minor gallinaceous species. This article describes the pathomorphological findings in Colchis pheasants infected naturally with H5N8 during an epizootic outbreak in Bulgaria. Material and methods Samples of internal organs of 10 carcasses were collected for histopathological and immunohistochemical evaluation, virus isolation and identification, and nucleic acid detection. Results Consistent macroscopic findings were lesions affecting the intestine, heart, lung, and pancreas. Congestion and mononuclear infiltrate were common findings in the small intestine, as were necrosis and lymphoid clusters in the lamina propria of the caeca. Congestion with small focal necrosis and gliosis with multifocal nonpurulent encephalitis were observed in the brain. Myocardial interstitial oedema and degenerative necrobiotic processes were also detected. Immunohistological analysis confirmed systemic infection and revealed influenza virus nucleoprotein in all analysed organs. Conclusion Variable necrosis was observed in the brain, liver, trachea, heart, small intestine, and caeca. Viral antigen was commonly found in the brain, heart, lung and trachea. Contact with migrating waterfowls was suspected as a reason for the outbreak.

Novel application of Influenza A virus-inoculated chorioallantoic membrane to characterize a NP-specific monoclonal antibody for immunohistochemistry assaying.

Abstract: Monoclonal antibodies (MAbs) are widely applied in disease diagnoses. Herein, we report a MAb, WF-4, against Influenza A virus nucleoprotein (NP), its broad response with Influenza A virus, and its application in an immunohistochemistry (IHC) assay. WF-4 was screened by immunofluorescence assay (IFA). The results showed that its reactivity with baculovirus-expressed full-length recombinant NP (rNP) in Western blot (WB), indicating its IHC applicability. Fifteen Influenza A virus (reference subtypes H1 to H15) infected chicken embryonated chorioallantoic membranes (CAM), fixed by formalin, were all detectable in the WF-4-based IHC assay. Also, the reactivity of the IHC test with NP from experimentally inoculated H6N1 and from all recent outbreaks of H5 subtype avian Influenza A virus (AIV) field cases in Taiwan showed positive results. Our data indicate that CAM, a by-product of Influenza A virus preparation, is helpful for Influenza A virus-specific MAb characterization, and that the WF-4 MAb recognizes conserved and linear epitopes of Influenza A virus NP. Therefore, WF-4 is capable of detecting NP antigens via IHC and may be suitable for developing various tests for diagnosis of Influenza A virus and, especially, AIV infection.

Evolution of H5-Type Avian Influenza A Virus Towards Mammalian Tropism in Egypt, 2014 to 2015

Abstract: Highly pathogenic avian influenza viruses (HPAIV) of the H5-subtype have circulated continuously in Egypt since 2006, resulting in numerous poultry outbreaks and considerable sporadic human infections. The extensive circulation and wide spread of these viruses in domestic poultry have resulted in various evolutionary changes with a dramatic impact on viral transmission ability to contact mammals including humans. The transmitted viruses are either (1) adapted well enough in their avian hosts to readily infect mammals, or (2) adapted in the new mammalian hosts to improve their fitness. In both cases, avian influenza viruses (AIVs) acquire various host-specific adaptations. These adaptive variations are not all well-known or thoroughly characterized. In this study, a phylogenetic algorithm based on the informational spectrum method, designated hereafter as ISM, was applied to analyze the affinity of H5-type HA proteins of Egyptian AIV isolates (2006–2015) towards human-type cell receptors. To characterize AIV H5-HA proteins displaying high ISM values reflecting an increased tendency of the HA towards human-type receptors, recombinant IV expressing monobasic, low pathogenic (LP) H5-HA versions in the background of the human influenza virus A/PR/8/1934(H1N1) (LP 7+1), were generated. These viruses were compared with a LP 7+1 expressing a monobasic H5-HA from a human origin virus isolate (human LP-7271), for their receptor binding specificity (ISM), in vitro replication efficiency and in vivo pathogenicity in mammals. Interestingly, using ISM analysis, we identified a LP 7+1 virus (LP-S10739C) expressing the monobasic H5-HA of AIV A/Chicken/Egypt/S10739C/2015(H5N1) that showed high affinity towards human-type receptors. This in silico prediction was reflected by a higher in vitro replication efficiency in mammalian cell cultures and a higher virulence in mice as compared with LP-7271. Sequence comparison between the LP-S10739C and the LP-7271 H5-HA, revealed distinct amino acid changes. Their contribution to the increased mammalian receptor propensity of LP-S10739C demands further investigation to better deduce the molecular determinant behind the reported high morbidity of 2014 to 2015 HPAI H5N1 virus in humans in Egypt. This study provides insights into the evolution of Egyptian H5 HPAIVs and highlights the need to identify the viral evolution in order to recognize emerging AIV with the potential to threaten human and animal populations.

Development of anexpress-method for influence and genotyping of H1N1 and H7N9 virus avian influenza a strains by PCR-RFLP analysis

Abstract: Epizootic monitoring in recent years suggests that the highly pathogenic avian influenza A virus (H1N1) and (H7N9) actively circulate in the Eurasian countries. By 2016 - 2019 1.6 thousand outbreaks were recorded. For 2016 - 2019, 1.6 thousand cases of outbreaks were recorded. Of these, there are 872 cases in Europe. The monitoring of infected birds, both migratory and poultry, in places of cross-contact in Ukraine is relevant for preventing outbreaks of epizooties. The aim of the study. To develop an express method for the identification and determination of bird flu virus A H1N1 and H7N9 strains, based on a polymerase chain reaction with analysis of restriction fragment length polymorphism (PCR-RFLP) of the virus RNA. Results and discussion. The in silico analysis of the HA, NA, and NP gene amplicons allowed in silico to calculate the primers to the variable loci of the investigated genes, to calculate the reaction conditions, to determine restriction sites for the restriction enzyme to obtain theoretical PCR electrophoregrams. An express method for the detection and identification of influenza A H1N1 and H7N9 virus by three genes (HA, NA, and NP) of H1N1 and H7N9 RNA in polymerase chain reaction, combined with RFLP analysis, was developed. The method of rapid diagnostics is able to detect avian influenza virus A H1N1 and H7N9 and differentiate it from samples of other pathogens of viral infections of birds and animals. It was established, that the PCR-RFLP rapid diagnostic method is able to detect influenza A virus RNA of H1N1 and H7N9 strains with high sensitivity (100 % sensitivity). Conclusions. The developed method of PCR-based rapid identification, combined with RFLP analysis, makes it possible to significantly simplify the method of identification due to specific amplification of an RNA region having a polymorphic restriction site. Testing of this locus is possible by pre-PCR and restriction of the amplified fragment. The method of express - diagnosis of PLR-RFLP has been established for detecting RNA virus influenza A of high pathogenic H1N1 and H7N9 strains with high indicators of sensitivity (100 % sensitivity)

Investigating the Role of Neuraminidase Activity in the Co-pathogenesis of Mycoplasma gallisepticum and Low Pathogenic Avian Influenza A Virus

Abstract: The avian pathogen Mycoplasma gallisepticum, the etiologic agent of chronic respiratory disease in chickens, exhibits enhanced pathogenesis in the presence of a co-pathogen such as low-pathogenic avian influenza virus (LPAIV). M. gallisepticum Rlow can utilize α-2,3 linked sialic acids, abundant in the avian respiratory tract, to bind host cells in vitro. To further investigate the intricacies of this co-pathogenesis, chickens were monoor co-infected with either virulent M. gallisepticum strain Rlow, attenuated M. gallisepticum neuraminidase mutant P1C5 and Mycoplasma specific lipoprotein A mutant P1H9, or LPAIV H3N8 (A/duck/Ukraine/1963). These chickens were then assessed for tracheal histopathology, pathogen load, and transcriptomic host response to infection using RNA-sequencing. Chickens co-infected with M. gallisepticum Rlow followed by H3N8 exhibited significantly more severe tracheal lesions and mucosal thickening in response to infection than chickens infected with H3N8 alone. Viral load was also significantly increased in this group over chickens who were infected first with H3N8 and subsequently with M. gallisepticum Rlow. The attenuated M. gallisepticum mutants P1C5 and P1H9, previously shown to be cleared 14 days post-infection, were able to persist 6 to 7 days post-infection in the presence and absence of co-infection Jessica A. Canter, University of Connecticut, 2019 with H3N8. P1H9 was able to persist to 14 days post-infection only in the presence of H3N8. The transcriptional response to monoand co-infection with M. gallisepticum and LPAIV highlighted the involvement of differential expression of genes such as TLR4, TLR15, TLR21, IL-1β, IRF4, MMP1, and MMP9. Pathway and gene ontology analysis of these differentially expressed genes suggests that co-infection with virulent M. gallisepticum and LPAIV induces a downregulation of ciliary activity in vivo and alters the multiple immune-related signaling cascades. Although H3N8 is susceptible to neuraminidase inhibition by oseltamivir in vitro, this antiviral treatment was not effective in vivo at reducing H3N8 load in the trachea of co-infected chickens. These data indicate that the co-pathogenesis of LPAIV and M. gallisepticum is not strictly neuraminidase-dependent and warrants further experimental understanding. Investigating the Role of Neuraminidase Activity in the Co-pathogenesis of Mycoplasma gallisepticum and Low Pathogenic Avian Influenza A Virus

Spatio-Temporal Analysis of Highly Pathogenic Avian Influenza outbreaks in Ghana.

Abstract: Objective The purpose of the study was to characterize the spatial distribution and temporal patterns of laboratory confirmed H5N1 outbreaks from January 2007 to December 2017 in Ghana. Introduction Highly pathogenic avian influenza (HPAI) subtype H5N1 virus causes a highly contagious disease in poultry with up to 100% mortality and occasionally causes sporadic human infection. The first outbreak of HPAI H5N1 in Africa was reported in Nigeria in 2006 and has since been reported in seven other African countries with confirmed human cases and outbreaks in poultry. Since the emergence of Highly Pathogenic Avian Influenza (HPAI), virus subtype H5N1 in Ghana in 2007, outbreaks in poultry have led to dire economic consequences for the poultry sector, resulting from mass destruction of affected flocks. An economy heavily dependent on agriculture, the persistence of outbreaks threaten the livelihood of farmers who depend on poultry production for survival. Despite significant efforts made in HPAI-H5N1 control and prevention in Ghana, outbreaks persist and continue to spread to new areas. It is uncertain to what extent different pathways contribute to the introduction and the dissemination of the virus in Ghana. There is a need to understand the complex nature of the interactions between local and migratory fowl, the risk of transmission due to human endeavor and trade mechanisms that increase the likelihood of HPAI-H5N1 outbreaks in Ghana. Methods Data for the study was sourced from national outbreak records at the Veterinary Services Directorate. The study analyzed outbreak data for the years 2007-2017. Data retrieved from outbreak reports included the date of onset of outbreak, location and geographic coordinates, type and number of poultry species affected, natural deaths of birds and type of farming system on outbreak farms. We calculated frequency distributions for the types of poultry species affected, the type of farming system and mortality rates on affected premises. We described the distribution of HPAI-H5N1 outbreaks using coordinate maps in ArcGIS and displayed relevant sites of waterfowl and wild bird habitation. To describe the temporal pattern of HPAI-H5N1 outbreaks in Ghana for the period, we created an epidemic curve by plotting the monthly number of outbreaks for the period January 2007 to December 2017 in Excel. We used space-time scan statistics to determine significant local clusters. Results A total of sixty-six (66) outbreaks of HPAI-H5N1 occurred in Ghana from January 2007 to December 2017. The outbreak sites were distributed in seven (7) out of ten (10) regions in Ghana. The affected regions are located in the southern and middle belt of Ghana. Most of the outbreaks (74.2%) occurred in densely populated areas of the Greater Accra region. Overall, layer flocks were mostly affected with 56% of affected premises constituting layer farms. Commercial farms and backyard farms made up the majority of affected farms (50% and 42.4%). Free ranging birds were the least affected farm type (7.6%). Two epidemic waves were identified for H5N1 in Ghana; the first wave with 6 outbreaks, lasted a period of four (4) months from April to July 2007, and the second with 60 outbreaks, spanned a period of 2 years from April 2015 to November 2016. Temporal distribution of the outbreaks showed that the outbreak peaked in May 2007 for the first wave and in July 2017 for the second wave with minor peaks observed in April and July 2016. The decrease in the number of the outbreaks after July in both waves is attributed to the onset of slaughter and trade restrictions for poultry in affected areas. Space-time scan statistics identified significant primary clusters of H5N1 outbreaks in the coastal belt of the Greater Accra region, characterized by major commercial activities and the presence of wetlands of relevance to wild birds and migratory waterfowl. Conclusions Two (2) major waves of H5N1 outbreaks occurred in Ghana between 2007 and 2017. The distribution of outbreaks and poultry species in both waves, show that the epidemiology of H5N1 virus in Ghana is changing. The findings highlight the importance of reviewing existing control and preventive measures as well as strengthening avian influenza surveillance in proposed high- risk areas. References Foreign Animal Diseases. Revised 2008 Seventh Edition. Committee on foreign and emerging diseases of the United States Animal Health Association. Avian Influenza, OIE terrestrial manual 2015 To K.K.W.et al. Avian influenza A H5N1 virus: a continuous threat to humans. Emerging Microbes Infections (2012) 1, e25. Watanabe Y. et al. The changing nature of avian influenza A virus (H5N1). Trends Microbiol. 2012 Jan; 20 (1):11-20. doi: 10.1016/j.tim.2011.10.003. Epub 2011 Dec 5