Development of Lentiviral Vectors Pseudotyped With Influenza B Hemagglutinins: Application in Vaccine Immunogenicity, mAb Potency, and Sero-Surveillance Studies.

Influenza B viruses (IBV) cause respiratory disease epidemics in humans and are therefore components of seasonal influenza vaccines. Serological methods are employed to evaluate vaccine immunogenicity prior to licensure. However, classical methods to assess influenza vaccine immunogenicity such as the hemagglutination inhibition assay (HI) and the serial radial hemolysis assay (SRH), have been proven to have many limitations. As such, there is a need to develop innovative methods that can improve on these traditional assays and provide advantages such as ease of production and access, safety, reproducibility, and specificity. It has been previously demonstrated that the use of replication-defective viruses, such as lentiviral vectors pseudotyped with influenza A hemagglutinins in microneutralization assays (pMN) is a safe and sensitive alternative to study antibody responses elicited by natural influenza infection or vaccination. Consequently, we have produced Influenza B hemagglutinin-pseudotypes (IBV PV) using plasmid-directed transfection. To activate influenza B hemagglutinin, we have explored the use of proteases in increasing PV titers via their co-transfection during pseudotype virus production. When tested for their ability to transduce target cells, the influenza B pseudotypes produced exhibit tropism for different cell lines. The pseudotypes were evaluated as alternatives to live virus in microneutralization assays using reference sera standards, mouse and human sera collected during vaccine immunogenicity studies, surveillance sera from seals, and monoclonal antibodies (mAbs) against IBV. The influenza B pseudotype pMN was found to effectively detect neutralizing and cross-reactive responses in all assays and shows promise as an effective and versatile tool in influenza research.

Machine Learning Prediction and Experimental Validation of Antigenic Drift in H3 Influenza A Viruses in Swine.

The antigenic diversity of influenza A viruses (IAV) circulating in swine challenges the development of effective vaccines, increasing zoonotic threat and pandemic potential. High-throughput sequencing technologies can quantify IAV genetic diversity, but there are no accurate approaches to adequately describe antigenic phenotypes. This study evaluated an ensemble of nonlinear regression models to estimate virus phenotype from genotype. Regression models were trained with a phenotypic data set of pairwise hemagglutination inhibition (HI) assays, using genetic sequence identity and pairwise amino acid mutations as predictor features. The model identified amino acid identity, ranked the relative importance of mutations in the hemagglutinin (HA) protein, and demonstrated good prediction accuracy. Four previously untested IAV strains were selected to experimentally validate model predictions by HI assays. Errors between predicted and measured distances of uncharacterized strains were 0.35, 0.61, 1.69, and 0.13 antigenic units. These empirically trained regression models can be used to estimate antigenic distances between different strains of IAV in swine by using sequence data. By ranking the importance of mutations in the HA, we provide criteria for identifying antigenically advanced IAV strains that may not be controlled by existing vaccines and can inform strain updates to vaccines to better control this pathogen.IMPORTANCE Influenza A viruses (IAV) in swine constitute a major economic burden to an important global agricultural sector, impact food security, and are a public health threat. Despite significant improvement in surveillance for IAV in swine over the past 10 years, sequence data have not been integrated into a systematic vaccine strain selection process for predicting antigenic phenotype and identifying determinants of antigenic drift. To overcome this, we developed nonlinear regression models that predict antigenic phenotype from genetic sequence data by training the model on hemagglutination inhibition assay results. We used these models to predict antigenic phenotype for previously uncharacterized IAV, ranked the importance of genetic features for antigenic phenotype, and experimentally validated our predictions. Our model predicted virus antigenic characteristics from genetic sequence data and provides a rapid and accurate method linking genetic sequence data to antigenic characteristics. This approach also provides support for public health by identifying viruses that are antigenically advanced from strains used as pandemic preparedness candidate vaccine viruses.

Plasticity of the Influenza Virus H5 HA Protein.

Since the emergence of highly pathogenic avian influenza viruses of the H5 subtype, the major viral antigen, hemagglutinin (HA), has undergone constant evolution, resulting in numerous genetic and antigenic (sub)clades. To explore the consequences of amino acid changes at sites that may affect the antigenicity of H5 viruses, we simultaneously mutated 17 amino acid positions of an H5 HA by using a synthetic gene library that, theoretically, encodes all combinations of the 20 amino acids at the 17 positions. All 251 mutant viruses sequenced possessed ≥13 amino acid substitutions in HA, demonstrating that the targeted sites can accommodate a substantial number of mutations. Selection with ferret sera raised against H5 viruses of different clades resulted in the isolation of 39 genotypes. Further analysis of seven variants demonstrated that they were antigenically different from the parental virus and replicated efficiently in mammalian cells. Our data demonstrate the substantial plasticity of the influenza virus H5 HA protein, which may lead to novel antigenic variants.IMPORTANCE The HA protein of influenza A viruses is the major viral antigen. In this study, we simultaneously introduced mutations at 17 amino acid positions of an H5 HA expected to affect antigenicity. Viruses with ≥13 amino acid changes in HA were viable, and some had altered antigenic properties. H5 HA can therefore accommodate many mutations in regions that affect antigenicity. The substantial plasticity of H5 HA may facilitate the emergence of novel antigenic variants.

Potential Role of Nonneutralizing IgA Antibodies in Cross-Protective Immunity against Influenza A Viruses of Multiple Hemagglutinin Subtypes.

IgA antibodies on mucosal surfaces are known to play an important role in protection from influenza A virus (IAV) infection and are believed to be more potent than IgG for cross-protective immunity against IAVs of multiple hemagglutinin (HA) subtypes. However, in general, neutralizing antibodies specific to HA are principally HA subtype specific. Here, we focus on nonneutralizing but broadly cross-reactive HA-specific IgA antibodies. Recombinant IgG, monomeric IgA (mIgA), and polymeric secretory IgA (pSIgA) antibodies were generated based on the sequence of a mouse anti-HA monoclonal antibody (MAb) 5A5 that had no neutralizing activity but showed broad binding capacity to multiple HA subtypes. While confirming that there was no neutralizing activity of the recombinant MAbs against IAV strains A/Puerto Rico/8/1934 (H1N1), A/Adachi/2/1957 (H2N2), A/Hong Kong/483/1997 (H5N1), A/shearwater/South Australia/1/1972 (H6N5), A/duck/England/1/1956 (H11N6), and A/duck/Alberta/60/1976 (H12N5), we found that pSIgA, but not mIgA and IgG, significantly reduced budding and release of most of the viruses from infected cells. Electron microscopy demonstrated that pSIgA deposited newly produced virus particles on the surfaces of infected cells, most likely due to tethering of virus particles. Furthermore, we found that pSIgA showed significantly higher activity to reduce plaque sizes of the viruses than IgG and mIgA. These results suggest that nonneutralizing pSIgA reactive to multiple HA subtypes may play a role in intersubtype cross-protective immunity against IAVs.IMPORTANCE Mucosal immunity represented by pSIgA plays important roles in protection from IAV infection. Furthermore, IAV HA-specific pSIgA antibodies are thought to contribute to cross-protective immunity against multiple IAV subtypes. However, the mechanisms by which pSIgA exerts such versatile antiviral activity are not fully understood. In this study, we generated broadly cross-reactive recombinant IgG and pSIgA having the same antigen-recognition site and compared their antiviral activities in vitro These recombinant antibodies did not show "classical" neutralizing activity, whereas pSIgA, but not IgG, significantly inhibited the production of progeny virus particles from infected cells. Plaque formation was also significantly reduced by pSIgA, but not IgG. These effects were seen in infection with IAVs of several different HA subtypes. Based on our findings, we propose an antibody-mediated host defense mechanism by which mucosal immunity may contribute to broad cross-protection from IAVs of multiple HA subtypes, including viruses with pandemic potential.

A conserved histidine in Group-1 influenza subtype hemagglutinin proteins is essential for membrane fusion activity.

Influenza A viruses enter host cells through the endocytic pathway, where acidification triggers conformational changes of the viral hemagglutinin (HA) to drive membrane fusion. During this process, the HA fusion peptide is extruded from its buried position in the neutral pH structure and targeted to the endosomal membrane. Conserved ionizable residues near the fusion peptide may play a role in initiating these structural rearrangements. We targeted highly conserved histidine residues in this region, at HA1 position 17 of Group-2 HA subtypes and HA2 position 111 of Group-1 HA subtypes, to determine their role in fusion activity. WT and mutant HA proteins representing several subtypes were expressed and characterized, revealing that His 111 is essential for HA functional activity of Group-1 subtypes, supporting continued efforts to target this region of the HA structure for vaccination strategies and the design of antiviral compounds.

Two novel reassortant H11N8 avian influenza viruses occur in wild birds found in East Dongting Lake, China.

During the surveillance of avian influenza viruses in East Dongting Lake, China (2014-2015), two H11N8 avian influenza viruses were detected in the bean goose (Anser fabalis) and the falcated teal (Anas falcata). Phylogenetic analysis showed that these two novel reassortant H11N8 avian influenza viruses contain genes from poultry and wild birds. This is the first report detecting the H11N8 subtype influenza virus from wild birds in Asia. These findings indicate a dissemination of avian influenza virus along the East Asian-Australian flyway. In addition, the interaction between poultry and wild birds was addressed suggesting the need for intensive surveillance of wild bird populations.

Charged amino acid variability related to N-glyco -sylation and epitopes in A/H3N2 influenza: Hem -agglutinin and neuraminidase.

BACKGROUND: The A/H3N2 influenza viruses circulated in humans have been shown to undergo antigenic drift, a process in which amino acid mutations result from nucleotide substitutions. There are few reports regarding the charged amino acid mutations. The purpose of this paper is to explore the relations between charged amino acids, N-glycosylation and epitopes in hemagglutinin (HA) and neuraminidase (NA). METHODS: A total of 700 HA genes (691 NA genes) of A/H3N2 viruses were chronologically analyzed for the mutational variants in amino acid features, N-glycosylation sites and epitopes since its emergence in 1968. RESULTS: It was found that both the number of HA N-glycosylation sites and the electric charge of HA increased gradually up to 2016. The charges of HA and HA1 increased respectively 1.54-fold (+7.0 /+17.8) and 1.08-fold (+8.0/+16.6) and the number of NGS in nearly doubled (7/12). As great diversities occurred in 1990s, involving Epitope A, B and D mutations, the charged amino acids in Epitopes A, B, C and D in HA1 mutated at a high frequency in global circulating strains last decade. The charged amino acid mutations in Epitopes A (T135K) has shown high mutability in strains near years, resulting in a decrease of NGT135-135. Both K158N and K160T not only involved mutations charged in epitope B, but also caused a gain of NYT158-160. Epitope B and its adjacent N-glycosylation site NYT158-160 mutated more frequently, which might be under greater immune pressure than the rest. CONCLUSIONS: The charged amino acid mutations in A/H3N2 Influenza play a significant role in virus evolution, which might cause an important public health issue. Variability related to both the epitopes (A and B) and N-glycosylation is beneficial for understanding the evolutionary mechanisms, disease pathogenesis and vaccine research.

Genetic Predisposition To Acquire a Polybasic Cleavage Site for Highly Pathogenic Avian Influenza Virus Hemagglutinin.

Highly pathogenic avian influenza viruses with H5 and H7 hemagglutinin (HA) subtypes evolve from low-pathogenic precursors through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been observed to occur naturally only in these HA subtypes, little is known about the genetic basis for the acquisition of the polybasic HA cleavage site. Here we show that consecutive adenine residues and a stem-loop structure, which are frequently found in the viral RNA region encoding amino acids around the cleavage site of low-pathogenic H5 and H7 viruses isolated from waterfowl reservoirs, are important for nucleotide insertions into this RNA region. A reporter assay to detect nontemplated nucleotide insertions and deep-sequencing analysis of viral RNAs revealed that an increased number of adenine residues and enlarged stem-loop structure in the RNA region accelerated the multiple adenine and/or guanine insertions required to create codons for basic amino acids. Interestingly, nucleotide insertions associated with the HA cleavage site motif were not observed principally in the viral RNA of other subtypes tested (H1, H2, H3, and H4). Our findings suggest that the RNA editing-like activity is the key mechanism for nucleotide insertions, providing a clue as to why the acquisition of the polybasic HA cleavage site is restricted to the particular HA subtypes.IMPORTANCE Influenza A viruses are divided into subtypes based on the antigenicity of the viral surface glycoproteins hemagglutinin (HA) and neuraminidase. Of the 16 HA subtypes (H1 to -16) maintained in waterfowl reservoirs of influenza A viruses, H5 and H7 viruses often become highly pathogenic through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been known since the 1980s, the genetic basis for nucleotide insertions has remained unclear. This study shows the potential role of the viral RNA secondary structure for nucleotide insertions and demonstrates a key mechanism explaining why the acquisition of the polybasic HA cleavage site is restricted to particular HA subtypes in nature. Our findings will contribute to better understanding of the ecology of influenza A viruses and will also be useful for the development of genetically modified vaccines against H5 and H7 influenza A viruses with increased stability.

Immunogenicity and Cross Protection in Mice Afforded by Pandemic H1N1 Live Attenuated Influenza Vaccine Containing Wild-Type Nucleoprotein.

Since conserved viral proteins of influenza virus, such as nucleoprotein (NP) and matrix 1 protein, are the main targets for virus-specific CD8+ cytotoxic T-lymphocytes (CTLs), we hypothesized that introduction of the NP gene of wild-type virus into the genome of vaccine reassortants could lead to better immunogenicity and afford better protection. This paper describes in vitro and in vivo preclinical studies of two new reassortants of pandemic H1N1 live attenuated influenza vaccine (LAIV) candidates. One had the hemagglutinin (HA) and neuraminidase (NA) genes from A/South Africa/3626/2013 H1N1 wild-type virus on the A/Leningrad/134/17/57 master donor virus backbone (6 : 2 formulation) while the second had the HA, NA, and NP genes of the wild-type virus on the same backbone (5 : 3 formulation). Although both LAIVs induced similar antibody immune responses, the 5 : 3 LAIV provoked greater production of virus-specific CTLs than the 6 : 2 variant. Furthermore, the 5 : 3 LAIV-induced CTLs had higher in vivo cytotoxic activity, compared to 6 : 2 LAIV. Finally, the 5 : 3 LAIV candidate afforded greater protection against infection and severe illness than the 6 : 2 LAIV. Inclusion in LAIV of the NP gene from wild-type influenza virus is a new approach to inducing cross-reactive cell-mediated immune responses and cross protection against pandemic influenza.

Human Monoclonal Antibody 81.39a Effectively Neutralizes Emerging Influenza A Viruses of Group 1 and 2 Hemagglutinins.

The pandemic threat posed by emerging zoonotic influenza A viruses necessitates development of antiviral agents effective against various antigenic subtypes. Human monoclonal antibody (hMAb) targeting the hemagglutinin (HA) stalk offers a promising approach to control influenza virus infections. Here, we investigated the ability of the hMAb 81.39a to inhibit in vitro replication of human and zoonotic viruses, representing 16 HA subtypes. The majority of viruses were effectively neutralized by 81.39a at a 50% effective concentration (EC50) of <0.01 to 4.9 µg/ml. Among group 2 HA viruses tested, a single A(H7N9) virus was not neutralized at 50 µg/ml; it contained HA2-Asp19Gly, an amino acid position previously associated with resistance to neutralization by the group 2 HA-neutralizing MAb CR8020. Notably, among group 1 HA viruses, H11-H13 and H16 subtypes were not neutralized at 50 µg/ml; they shared the substitution HA2-Asp19Asn/Ala. Conversely, H9 viruses harboring HA2-Asp19Ala were fully susceptible to neutralization. Therefore, amino acid variance at HA2-Asp19 has subtype-specific adverse effects on in vitro neutralization. Mice given a single injection (15 or 45 mg/kg of body weight) at 24 or 48 h after infection with recently emerged A(H5N2), A(H5N8), A(H6N1), or A(H7N9) viruses were protected from mortality and showed drastically reduced lung viral titers. Furthermore, 81.39a protected mice infected with A(H7N9) harboring HA2-Asp19Gly, although the antiviral effect was lessened. A(H1N1)pdm09-infected ferrets receiving a single dose (25 mg/kg) had reduced viral titers and showed less lung tissue injury, despite 24- to 72-h-delayed treatment. Taken together, this study provides experimental evidence for the therapeutic potential of 81.39a against diverse influenza A viruses. IMPORTANCE: Zoonotic influenza viruses, such as A(H5N1) and A(H7N9) subtypes, have caused severe disease and deaths in humans, raising public health concerns. Development of novel anti-influenza therapeutics with a broad spectrum of activity against various subtypes is necessary to mitigate disease severity. Here, we demonstrate that the hemagglutinin (HA) stalk-targeting human monoclonal antibody 81.39a effectively neutralized the majority of influenza A viruses tested, representing 16 HA subtypes. Furthermore, delayed treatment with 81.39a significantly suppressed virus replication in the lungs, prevented dramatic body weight loss, and increased survival rates of mice infected with A(H5Nx), A(H6N1), or A(H7N9) viruses. When tested in ferrets, delayed 81.39a treatment reduced viral titers, particularly in the lower respiratory tract, and substantially alleviated disease symptoms associated with severe A(H1N1)pdm09 influenza. Collectively, our data demonstrated the effectiveness of 81.39a against both seasonal and emerging influenza A viruses.

Progressive antigenic drift and phylogeny of human influenza A(H3N2) virus over five consecutive seasons (2009-2013) in Hangzhou, China.

Vaccine efficacy (VE) can be affected by progressive antigenic drift or any new reassortment of influenza viruses. To effectively track the evolution of human influenza A(H3N2) virus circulating in Hangzhou, China, a total of 65 clinical specimens were selected randomly from outpatients infected by A(H3N2) viruses during the study period from November 2009 to December 2013. The results of reduced VE and antigenic drift of the correspondent epitopes (C-D-E to A-B) suggest that the current vaccine provides suboptimal protection against the A(H3N2) strains circulating recently. Phylogenetic analysis of the entire HA and NA sequences demonstrated that these two genes underwent independent evolutionary pathways during recent seasons. The H3-based phylogenetic tree showed that a special strain A/Hangzhou/A289/2012 fell in a cluster among viruses with reduced VE predominantly circulating in 2013. Our findings underscore a possible early warning for the circulation of A(H3N2) variants with antigenic drift during the previous seasons.

Mapping of the influenza A hemagglutinin serotypes evolution by the ISSCOR method.

Analyses and visualizations by the ISSCOR method of influenza virus hemagglutinin genes of different A-subtypes revealed some rather striking temporal relationships between groups of individual gene subsets. Based on these findings we consider application of the ISSCOR-PCA method for analyses of large sets of homologous genes to be a worthwhile addition to a toolbox of genomics--allowing for a rapid diagnostics of trends, and ultimately even aiding an early warning of newly emerging epidemiological threats.

A complete molecular diagnostic procedure for applications in surveillance and subtyping of avian influenza virus.

INTRODUCTION: The following complete molecular diagnostic procedure we developed, based on real-time quantitative PCR and traditional PCR, is effective for avian influenza surveillance, virus subtyping, and viral genome sequencing. METHOD: This study provides a specific and sensitive step-by-step procedure for efficient avian influenza identification of 16 hemagglutinin and 9 neuraminidase avian influenza subtypes. RESULT AND CONCLUSION: This diagnostic procedure may prove exceedingly useful for virological and ecological advancements in global avian influenza research.

Cleavage site and Ectodomain of HA2 sub-unit sequence of three equine influenza virus isolated in Morocco.

BACKGROUND: The equine influenza (EI) is an infectious and contagious disease of the upper respiratory tract of horses. Two outbreaks were notified in Morocco during 1997 and 2004 respectively in Nador and Essaouira. The aims of the present study concern the amino acids sequences comparison with reference strain A/equine/Miami/1963(H3N8) of the HA2 subunit including the cleavage site of three equine influenza viruses (H3N8) isolated in Morocco: A/equine/Nador/1/1997(H3N8), A/equine/Essaouira/2/2004 (H3N8) and A/equine/Essaouira/3/2004 (H3N8). RESULTS: The obtained results demonstrated that the substitutions were located at Ectodomain (ED) and transmembrane domain (TD), and they have only one arginine in cleavage site (HA1-PEKQI-R329-GI-HA2). In the Ectodomain, the mutation N/1542/T deleted the NGT glycosylation site at position 154 for both strains A/equine/Essaouira/2/2004(H3N8) and A/equine/Essaouira/3/2004(H3N8). Except for mutation D/1602/Y of the A/equine/Nador/1/1997(H3N8) strain, the other mutations were involved in non conserved sites. While the transmembrane domain (TM) of the strain A/equine/Essaouira/3/2004(H3N8) exhibits a substitution at residue C/1992/F. For the A/equine/Nador/1/1997(H3N8) strain the HA2 shows a mutation at residue M/2072/L. Three Moroccan strains reveals a common substitution at the residue E/2112/Q located between transmembrane domain TM and the cytoplasmic domain (CD). CONCLUSION: The given nature virulence of three Moroccan strains, the identified and reported mutations certainly played a permissive role of infection viral process.

Understanding the undelaying mechanism of HA-subtyping in the level of physic-chemical characteristics of protein.

The evolution of the influenza A virus to increase its host range is a major concern worldwide. Molecular mechanisms of increasing host range are largely unknown. Influenza surface proteins play determining roles in reorganization of host-sialic acid receptors and host range. In an attempt to uncover the physic-chemical attributes which govern HA subtyping, we performed a large scale functional analysis of over 7000 sequences of 16 different HA subtypes. Large number (896) of physic-chemical protein characteristics were calculated for each HA sequence. Then, 10 different attribute weighting algorithms were used to find the key characteristics distinguishing HA subtypes. Furthermore, to discover machine leaning models which can predict HA subtypes, various Decision Tree, Support Vector Machine, Naïve Bayes, and Neural Network models were trained on calculated protein characteristics dataset as well as 10 trimmed datasets generated by attribute weighting algorithms. The prediction accuracies of the machine learning methods were evaluated by 10-fold cross validation. The results highlighted the frequency of Gln (selected by 80% of attribute weighting algorithms), percentage/frequency of Tyr, percentage of Cys, and frequencies of Try and Glu (selected by 70% of attribute weighting algorithms) as the key features that are associated with HA subtyping. Random Forest tree induction algorithm and RBF kernel function of SVM (scaled by grid search) showed high accuracy of 98% in clustering and predicting HA subtypes based on protein attributes. Decision tree models were successful in monitoring the short mutation/reassortment paths by which influenza virus can gain the key protein structure of another HA subtype and increase its host range in a short period of time with less energy consumption. Extracting and mining a large number of amino acid attributes of HA subtypes of influenza A virus through supervised algorithms represent a new avenue for understanding and predicting possible future structure of influenza pandemics.

Recombinant influenza H1, H5 and H9 hemagglutinins containing replaced H3 hemagglutinin transmembrane domain showed enhanced heterosubtypic protection in mice.

Influenza A viruses cause annual epidemics and irregular pandemics. A vaccine with heterosubtypic protection (hetero-protection) has been needed. In the present study, various influenza H1, H3, H5, and H9 hemagglutinin (HA) proteins were expressed in insect cells, and then mice were subcutaneously immunized with the expressed HA proteins, and challenged by influenza A viruses (A/Puerto Rico/8/1934 (H1N1) or A/chicken/Guangdong/96 (H9N2)). The results first showed that wild-type H3 hemagglutinin (HA) (H3-WT), but not a transmembrane domain (TM) mutant, had hetero-protection against both H1N1 and H9N2 with survival rates of 17% and 33% respectively, and that wild-type H1 (H1-WT), H5 (H5-WT) and H9 (H9-WT) had no hetero-protection against H1N1 or H9N2 except for H5-WT against H1N1 with a survival rate of 17%. Then the H3-WT TM replaced the TMs of H1-WT, H5-WT and H9-WT to generate recombinant H1-TM, H5-TM and H9-TM respectively, and whether the H3-WT TM-dependent hetero-protection could be transferred to these TM mutants was investigated. The results showed that the H3-WT TM-dependent hetero-protection was transferable. H1-TM against H9N2 and H9-TM against H1N1 were with survival rates of 33% and 17% respectively, and H5-TM against both H1N1 and H9N2 with survival rates of 50% and 17% respectively. Furthermore, higher dosage H5-TM scored 100% hetero-protection against H1N1. These results demonstrated that replacement of the TMs of non-H3 HAs with H3-WT TM could enhance their hetero-protection. These findings would help the development of future influenza vaccines against pandemics such as the recently appeared H7N9 infection.

A synchronized global sweep of the internal genes of modern avian influenza virus.

Zoonotic infectious diseases such as influenza continue to pose a grave threat to human health. However, the factors that mediate the emergence of RNA viruses such as influenza A virus (IAV) are still incompletely understood. Phylogenetic inference is crucial to reconstructing the origins and tracing the flow of IAV within and between hosts. Here we show that explicitly allowing IAV host lineages to have independent rates of molecular evolution is necessary for reliable phylogenetic inference of IAV and that methods that do not do so, including 'relaxed' molecular clock models, can be positively misleading. A phylogenomic analysis using a host-specific local clock model recovers extremely consistent evolutionary histories across all genomic segments and demonstrates that the equine H7N7 lineage is a sister clade to strains from birds--as well as those from humans, swine and the equine H3N8 lineage--sharing an ancestor with them in the mid to late 1800s. Moreover, major western and eastern hemisphere avian influenza lineages inferred for each gene coalesce in the late 1800s. On the basis of these phylogenies and the synchrony of these key nodes, we infer that the internal genes of avian influenza virus (AIV) underwent a global selective sweep beginning in the late 1800s, a process that continued throughout the twentieth century and up to the present. The resulting western hemispheric AIV lineage subsequently contributed most of the genomic segments to the 1918 pandemic virus and, independently, the 1963 equine H3N8 panzootic lineage. This approach provides a clear resolution of evolutionary patterns and processes in IAV, including the flow of viral genes and genomes within and between host lineages.

Epidemiological surveillance of low pathogenic avian influenza virus (LPAIV) from poultry in Guangxi Province, Southern China.

Low pathogenic avian influenza virus (LPAIV) usually causes mild disease or asymptomatic infection in poultry. However, some LPAIV strains can be transmitted to humans and cause severe infection. Genetic rearrangement and recombination of even low pathogenic influenza may generate a novel virus with increased virulence, posing a substantial risk to public health. Southern China is regarded as the world "influenza epicenter", due to a rash of outbreaks of influenza in recent years. In this study, we conducted an epidemiological survey of LPAIV at different live bird markets (LBMs) in Guangxi province, Southern China. From January 2009 to December 2011, we collected 3,121 cotton swab samples of larynx, trachea and cloaca from the poultry at LBMs in Guangxi. Virus isolation, hemagglutination inhibition (HI) assay, and RT-PCR were used to detect and subtype LPAIV in the collected samples. Of the 3,121 samples, 336 samples (10.8%) were LPAIV positive, including 54 (1.7%) in chicken and 282 (9.1%) in duck. The identified LPAIV were H3N1, H3N2, H6N1, H6N2, H6N5, H6N6, H6N8, and H9N2, which are combinations of seven HA subtypes (H1, H3, H4, H6, H9, H10 and H11) and five NA subtypes (N1, N2, N5, N6 and N8). The H3 and H9 subtypes are predominant in the identified LPAIVs. Among the 336 cases, 29 types of mixed infection of different HA subtypes were identified in 87 of the cases (25.9%). The mixed infections may provide opportunities for genetic recombination. Our results suggest that the LPAIV epidemiology in poultry in the Guangxi province in southern China is complicated and highlights the need for further epidemiological and genetic studies of LPAIV in this area.

Novel phylogenetic algorithm to monitor human tropism in Egyptian H5N1-HPAIV reveals evolution toward efficient human-to-human transmission.

Years of endemic infections with highly pathogenic avian influenza (HPAI) A subtype H5N1 virus in poultry and high numbers of infections in humans provide ample opportunity in Egypt for H5N1-HPAIV to develop pandemic potential. In an effort to better understand the viral determinants that facilitate human infections of the Egyptian H5N1-HPAIVvirus, we developed a new phylogenetic algorithm based on a new distance measure derived from the informational spectrum method (ISM). This new approach, which describes functional aspects of the evolution of the hemagglutinin subunit 1 (HA1), revealed a growing group G2 of H5N1-HPAIV in Egypt after 2009 that acquired new informational spectrum (IS) properties suggestive of an increased human tropism and pandemic potential. While in 2006 all viruses in Egypt belonged to the G1 group, by 2011 these viruses were virtually replaced by G2 viruses. All of the G2 viruses displayed four characteristic mutations (D43N, S120(D,N), (S,L)129Δ and I151T), three of which were previously reported to increase binding to the human receptor. Already in 2006-2008 G2 viruses were significantly (p<0.02) more often found in humans than expected from their overall prevalence and this further increased in 2009-2011 (p<0.007). Our approach also identified viruses that acquired additional mutations that we predict to further enhance their human tropism. The extensive evolution of Egyptian H5N1-HPAIV towards a preferential human tropism underlines an urgent need to closely monitor these viruses with respect to molecular determinants of virulence.

Evidence for the circulation and inter-hemispheric movement of the H14 subtype influenza A virus.

Three H14 influenza A virus (IAV) isolates recovered in 2010 during routine virus surveillance along the Mississippi Migratory Bird Flyway in Wisconsin, U.S.A. raised questions about the natural history of these rare viruses. These were the first H14 IAV isolates recovered in the Western Hemisphere and the only H14 IAV isolates recovered since the original four isolates in 1982 in Asia. Full length genomic sequencing of the 2010 H14 isolates demonstrated the hemagglutinin (HA) gene from the 1982 and 2010 H14 isolates showed 89.6% nucleotide and 95.6% amino acid similarity and phylogenetic analysis of these viruses placed them with strong support within the H14 subtype lineage. The level of genomic divergence observed between the 1982 and 2010 viruses provides evidence that the H14 HA segment was circulating undetected in hosts and was not maintained in environmental stasis. Further, the evolutionary relationship observed between 1982 H14 and the closely related H4 subtype HA segments were similar to contemporary comparisons suggesting limited adaptive divergence between these sister subtypes. The nonstructural (NS) segment of one 2010 isolate was placed in a NS clade isolated infrequently over the last several decades that includes the NS segment from a previously reported 1982 H14 isolate indicating the existence of an unidentified pool of genomic diversity. An additional neuraminidase reassortment event indicated a recent inter-hemispheric gene flow from Asia into the center of North America. These results demonstrate temporal and spatial gaps in the understanding of IAV natural history. Additionally, the reassortment history of these viruses raises concern for the inter-continental spread of IAVs and the efficacy of current IAV surveillance efforts in detecting genomic diversity of viruses circulating in wild birds.