Evolutionary history and phylodynamics of influenza A and B neuraminidase (NA) genes inferred from large-scale sequence analyses

Background

Influenza neuraminidase (NA) is an important surface glycoprotein and plays a vital role in viral replication and drug development. The NA is found in influenza A and B viruses, with nine subtypes classified in influenza A. The complete knowledge of influenza NA evolutionary history and phylodynamics, although critical for the prevention and control of influenza epidemics and pandemics, remains lacking.

Methodology/Principal findings

Evolutionary and phylogenetic analyses of influenza NA sequences using Maximum Likelihood and Bayesian MCMC methods demonstrated that the divergence of influenza viruses into types A and B occurred earlier than the divergence of influenza A NA subtypes. Twenty-three lineages were identified within influenza A, two lineages were classified within influenza B, and most lineages were specific to host, subtype or geographical location. Interestingly, evolutionary rates vary not only among lineages but also among branches within lineages. The estimated tMRCAs of influenza lineages suggest that the viruses of different lineages emerge several months or even years before their initial detection. The d _N /d _S ratios ranged from 0.062 to 0.313 for influenza A lineages, and 0.257 to 0.259 for influenza B lineages. Structural analyses revealed that all positively selected sites are at the surface of the NA protein, with a number of sites found to be important for host antibody and drug binding.

Conclusions/Significance

The divergence into influenza type A and B from a putative ancestral NA was followed by the divergence of type A into nine NA subtypes, of which 23 lineages subsequently diverged. This study provides a better understanding of influenza NA lineages and their evolutionary dynamics, which may facilitate early detection of newly emerging influenza viruses and thus improve influenza surveillance.     

Neuraminidase and Hemagglutinin Matching Patterns of a Highly Pathogenic Avian and Two Pandemic H1N1 Influenza A Viruses

Background

Influenza A virus displays strong reassortment characteristics, which enable it to achieve adaptation in human infection. Surveying the reassortment and virulence of novel viruses is important in the prevention and control of an influenza pandemic. Meanwhile, studying the mechanism of reassortment may accelerate the development of anti-influenza strategies.

Methodology/Principal Findings

The hemagglutinin (HA) and neuraminidase (NA) matching patterns of two pandemic H1N1 viruses (the 1918 and current 2009 strains) and a highly pathogenic avian influenza A virus (H5N1) were studied using a pseudotyped particle (pp) system. Our data showed that four of the six chimeric HA/NA combinations could produce infectious pps, and that some of the chimeric pps had greater infectivity than did their ancestors, raising the possibility of reassortment among these viruses. The NA of H5N1 (A/Anhui/1/2005) could hardly reassort with the HAs of the two H1N1 viruses. Many biological characteristics of HA and NA, including infectivity, hemagglutinating ability, and NA activity, are dependent on their matching pattern.

Conclusions/Significance

Our data suggest the existence of an interaction between HA and NA, and the HA NA matching pattern is critical for valid viral reassortment.     

Molecular Epidemiology of Influenza A(H1N1)pdm09 Viruses from Pakistan in 2009-2010

Background

In early 2009, a novel influenza A(H1N1) virus that emerged in Mexico and United States rapidly disseminated worldwide. The spread of this virus caused considerable morbidity with over 18000 recorded deaths. The new virus was found to be a reassortant containing gene segments from human, avian and swine influenza viruses.

Methods/Results

The first case of human infection with A(H1N1)pdm09 in Pakistan was detected on 18^th June 2009. Since then, 262 laboratory-confirmed cases have been detected during various outbreaks with 29 deaths (as of 31^st August 2010). The peak of the epidemic was observed in December with over 51% of total respiratory cases positive for influenza. Representative isolates from Pakistan viruses were sequenced and analyzed antigenically. Sequence analysis of genes coding for surface glycoproteins HA and NA showed high degree of high levels of sequence identity with corresponding genes of regional viruses circulating South East Asia. All tested viruses were sensitive to Oseltamivir in the Neuraminidase Inhibition assays.

Conclusions

Influenza A(H1N1)pdm09 viruses from Pakistan form a homogenous group of viruses. Their HA genes belong to clade 7 and show antigenic profile similar to the vaccine strain A/California/07/2009. These isolates do not show any amino acid changes indicative of high pathogenicity and virulence. It is imperative to continue monitoring of these viruses for identification of potential variants of high virulence or drug resistance.     

Guinea pig model for evaluating the potential public health risk of swine and avian influenza viruses

Background

The influenza viruses circulating in animals sporadically transmit to humans and pose pandemic threats. Animal models to evaluate the potential public health risk potential of these viruses are needed.

Methodology/Principal Findings

We investigated the guinea pig as a mammalian model for the study of the replication and transmission characteristics of selected swine H1N1, H1N2, H3N2 and avian H9N2 influenza viruses, compared to those of pandemic (H1N1) 2009 and seasonal human H1N1, H3N2 influenza viruses. The swine and avian influenza viruses investigated were restricted to the respiratory system of guinea pigs and shed at high titers in nasal tracts without prior adaptation, similar to human strains. None of the swine and avian influenza viruses showed transmissibility among guinea pigs; in contrast, pandemic (H1N1) 2009 virus transmitted from infected guinea pigs to all animals and seasonal human influenza viruses could also horizontally transmit in guinea pigs. The analysis of the receptor distribution in the guinea pig respiratory tissues by lectin histochemistry indicated that both SAα2,3-Gal and SAα2,6-Gal receptors widely presented in the nasal tract and the trachea, while SAα2,3-Gal receptor was the main receptor in the lung.

Conclusions/Significance

We propose that the guinea pig could serve as a useful mammalian model to evaluate the potential public health threat of swine and avian influenza viruses.     

Reassortant between Human-Like H3N2 and Avian H5 Subtype Influenza A Viruses in Pigs: A Potential Public Health Risk

Background

Human-like H3N2 influenza viruses have repeatedly been transmitted to domestic pigs in different regions of the world, but it is still uncertain whether any of these variants could become established in pig populations. The fact that different subtypes of influenza viruses have been detected in pigs makes them an ideal candidate for the genesis of a possible reassortant virus with both human and avian origins. However, the determination of whether pigs can act as a “mixing vessel” for a possible future pandemic virus is still pending an answer. This prompted us to gather the epidemiological information and investigate the genetic evolution of swine influenza viruses in Jilin, China.

Methods

Nasopharyngeal swabs were collected from pigs with respiratory illness in Jilin province, China from July 2007 to October 2008. All samples were screened for influenza A viruses. Three H3N2 swine influenza virus isolates were analyzed genetically and phylogenetically.

Results

Influenza surveillance of pigs in Jilin province, China revealed that H3N2 influenza viruses were regularly detected from domestic pigs during 2007 to 2008. Phylogenetic analysis revealed that two distinguishable groups of H3N2 influenza viruses were present in pigs: the wholly contemporary human-like H3N2 viruses (represented by the Moscow/10/99-like sublineage) and double-reassortant viruses containing genes from contemporary human H3N2 viruses and avian H5 viruses, both co-circulating in pig populations.

Conclusions

The present study reports for the first time the coexistence of wholly human-like H3N2 viruses and double-reassortant viruses that have emerged in pigs in Jilin, China. It provides updated information on the role of pigs in interspecies transmission and genetic reassortment of influenza viruses.     

Seasonality of influenza A(H3N2) virus: a Hong Kong perspective (1997-2006)

Background

The underlying basis for the seasonality of influenza A viruses is still uncertain. Phylogenetic studies investigated this phenomenon but have lacked sequences from more subtropical and tropical regions, particularly from Southeast Asia.

Methodology/Principal Findings

281 complete hemagglutinin (HA) and neuraminidase (NA) sequences were obtained from influenza A(H3N2) viruses, collected over 10 years (1997–2006) from Hong Kong. These dated sequences were analyzed with influenza A(H3N2) vaccine strain sequences (Syd/5/97, Mos/10/99, Fuj/411/02, Cal/7/04) and 315 other publicly available dated sequences from elsewhere, worldwide. In addition, the NA sequence alignment was inspected for the presence of any naturally occurring, known, neuraminidase inhibitor (NAI) resistance-associated amino acid mutations (R292K and E119V). Before 2001, the Hong Kong HA and NA sequences clustered more closely with the older vaccine sequences (Syd/5/97, Mos/10/99) than did sequences from elsewhere. After 2001, this trend reversed with significant clusters containing HA and NA sequences from different locations, isolated at different times, suggesting that viral migration may account for much of the influenza A(H3N2) seasonality during this 10-year period. However, at least one example from Hong Kong was found suggesting that in some years, influenza A(H3N2) viruses may persist in the same location, perhaps continuing to circulate, sub-clinically, at low levels between seasons, to re-emerge in the influenza season the following year, relatively unchanged. None of these Hong Kong influenza A(H3N2) NA sequences contained any of the known NAI-resistance associated mutations.

Conclusions/Significance

The seasonality of influenza A(H3N2) may be largely due to global migration, with similar viruses appearing in different countries at different times. However, occasionally, some viruses may remain within a single location and continue to circulate within that population, to re-emerge during the next influenza season, with relatively little genetic change. Naturally occurring NAI resistance mutations were absent or, at least, very rare in this population.     

Phylodynamics and molecular evolution of influenza A virus nucleoprotein genes in Taiwan between 1979 and 2009

Background

Many studies concentrate on variation in the hemagglutinin glycoprotein (HA) because of its significance in host immune response, the evolution of this virus is even more complex when other genome segments are considered. Recently, it was found that cytotoxic T lymphocytes (CTL) play an important role in immunity against influenza and most CTL epitopes of human influenza viruses were remarkably conserved. The NP gene has evolved independently in human and avian hosts after 1918 flu pandemic and it has been assigned a putative role as a determinant of host range.

Methods and Findings

Phylodynamic patterns of the genes encoding nucleoprotein (NP) of influenza A viruses isolated from 1979–2009 were analyzed by applying the Bayesian Markov Chain Monte Carlo framework to better understand the evolutionary mechanisms of these Taiwanese isolates. Phylogenetic analysis of the NP gene showed that all available H3 worldwide isolates collected so far were genetically similar and divided into two major clades after the year 2004. We compared the deduced amino acid sequences of the NP sequences from human, avian and swine hosts to investigate the emergence of potential adaptive mutations. Overall, selective pressure on the NP gene of human influenza A viruses appeared to be dominated by purifying selection with a mean d_N/d_S ratio of 0.105. Site-selection analysis of 488 codons, however, also revealed 3 positively selected sites in addition to 139 negatively selected ones.

Conclusions

The demographic history inferred by Bayesian skyline plot showed that the effective number of infections underwent a period of smooth and steady growth from 1998 to 2001, followed by a more recent rise in the rate of spread. Further understanding the correlates of interspecies transmission of influenza A virus genes from other host reservoirs to the human population may help to elucidate the mechanisms of variability among influenza A virus.     

Infection of differentiated porcine airway epithelial cells by influenza virus: differential susceptibility to infection by porcine and avian viruses

Background

Swine are important hosts for influenza A viruses playing a crucial role in the epidemiology and interspecies transmission of these viruses. Respiratory epithelial cells are the primary target cells for influenza viruses.

Methodology/Principal Findings

To analyze the infection of porcine airway epithelial cells by influenza viruses, we established precision-cut lung slices as a culture system for differentiated respiratory epithelial cells. Both ciliated and mucus-producing cells were found to be susceptible to infection by swine influenza A virus (H3N2 subtype) with high titers of infectious virus released into the supernatant already one day after infection. By comparison, growth of two avian influenza viruses (subtypes H9N2 and H7N7) was delayed by about 24 h. The two avian viruses differed both in the spectrum of susceptible cells and in the efficiency of replication. As the H9N2 virus grew to titers that were only tenfold lower than that of a porcine H3N2 virus this avian virus is an interesting candidate for interspecies transmission. Lectin staining indicated the presence of both α-2,3- and α-2,6-linked sialic acids on airway epithelial cells. However, their distribution did not correlate with pattern of virus infection indicating that staining by plant lectins is not a reliable indicator for the presence of cellular receptors for influenza viruses.

Conclusions/Significance

Differentiated respiratory epithelial cells significantly differ in their susceptibility to infection by avian influenza viruses. We expect that the newly described precision-cut lung slices from the swine lung are an interesting culture system to analyze the infection of differentiated respiratory epithelial cells by different pathogens (viral, bacterial and parasitic ones) of swine.     

The Feasibility of Using High Resolution Genome Sequencing of Influenza A Viruses to Detect Mixed Infections and Quasispecies

Background

The rapidly expanding availability of de novo sequencing technologies can greatly facilitate efforts to monitor the relatively high mutation rates of influenza A viruses and the detection of quasispecies. Both the mutation rates and the lineages of influenza A viruses are likely to play an important role in the natural history of these viruses and the emergence of phenotypically and antigenically distinct strains.

Methodology and Principal Findings

We evaluated quasispecies and mixed infections by de novo sequencing the whole genomes of 10 virus isolates, including eight avian influenza viruses grown in embryonated chicken eggs (six waterfowl isolates - five H3N2 and one H4N6; an H7N3 turkey isolate; and a bald eagle isolate with H1N1/H2N1 mixed infection), and two tissue cultured H3N2 swine influenza viruses. Two waterfowl cloacal swabs were included in the analysis. Full-length sequences of all segments were obtained with 20 to 787-X coverage for the ten viruses and one cloacal swab. The second cloacal swab yielded 15 influenza reads of ∼230 bases, sufficient for bioinformatic inference of mixed infections or quasispecies. Genomic subpopulations or quasispecies of viruses were identified in four egg grown avian influenza isolates and one cell cultured swine virus. A bald eagle isolate and the second cloacal swab showed evidence of mixed infections with two (H1 and H2) and three (H1, H3, and H4) HA subtypes, respectively. Multiple sequence differences were identified between cloacal swab and the virus recovered using embryonated chicken eggs.

Conclusions

We describe a new approach to comprehensively identify mixed infections and quasispecies in low passage influenza A isolates and cloacal swabs and add to the understanding of the ecology of influenza A virus populations.     

Genetic diversity of the 2009 pandemic influenza A(H1N1) viruses in Finland

Background

In Finland, the first infections caused by the 2009 pandemic influenza A(H1N1) virus were identified on May 10. During the next three months almost all infections were found from patients who had recently traveled abroad. In September 2009 the pandemic virus started to spread in the general population, leading to localized outbreaks and peak epidemic activity was reached during weeks 43–48.

Methods/Results

The nucleotide sequences of the hemagglutinin (HA) and neuraminidase (NA) genes from viruses collected from 138 patients were determined. The analyzed viruses represented mild and severe infections and different geographic regions and time periods. Based on HA and NA gene sequences, the Finnish pandemic viruses clustered in four groups. Finnish epidemic viruses and A/California/07/2009 vaccine virus strain varied from 2–8 and 0–5 amino acids in HA and NA molecules, respectively, giving a respective maximal evolution speed of 1.4% and 1.1%. Most amino acid changes in HA and NA molecules accumulated on the surface of the molecule and were partly located in antigenic sites. Three severe infections were detected with a mutation at HA residue 222, in two viruses with a change D222G, and in one virus D222Y. Also viruses with change D222E were identified. All Finnish pandemic viruses were sensitive to oseltamivir having the amino acid histidine at residue 275 of the neuraminidase molecule.

Conclusions

The Finnish pandemic viruses were quite closely related to A/California/07/2009 vaccine virus. Neither in the HA nor in the NA were changes identified that may lead to the selection of a virus with increased epidemic potential or exceptionally high virulence. Continued laboratory-based surveillance of the 2009 pandemic influenza A(H1N1) is important in order to rapidly identify drug resistant viruses and/or virus variants with potential ability to cause severe forms of infection and an ability to circumvent vaccine-induced immunity.     

Construction and Immunogenicity Evaluation of Recombinant Influenza A Viruses Containing Chimeric Hemagglutinin Genes Derived from Genetically Divergent Influenza A H1N1 Subtype Viruses

Background and Objectives

Influenza A viruses cause highly contagious diseases in a variety of hosts, including humans and pigs. To develop a vaccine that can be broadly effective against genetically divergent strains of the virus, in this study we employed molecular breeding (DNA shuffling) technology to create a panel of chimeric HA genes.

Methods and Results

Each chimeric HA gene contained genetic elements from parental swine influenza A viruses that had a history of zoonotic transmission, and also from a 2009 pandemic virus. Each parental virus represents a major phylogenetic clade of influenza A H1N1 viruses. Nine shuffled HA constructs were initially screened for immunogenicity in mice by DNA immunization, and one chimeric HA (HA-129) was expressed on both a A/Puerto Rico/8/34 backbone with mutations associated with a live, attenuated phenotype (PR8_LAIV-129) and a A/swine/Texas/4199-2/98 backbone (TX98-129). When delivered to mice, the PR8_LAIV-129 induced antibodies against all four parental viruses, which was similar to the breadth of immunity observed when HA-129 was delivered as a DNA vaccine. This chimeric HA was then tested as a candidate vaccine in a nursery pig model, using inactivated TX98-129 virus as the backbone. The results demonstrate that pigs immunized with HA-129 developed antibodies against all four parental viruses, as well as additional primary swine H1N1 influenza virus field isolates.

Conclusion

This study established a platform for creating novel genes of influenza viruses using a molecular breeding approach, which will have important applications toward future development of broadly protective influenza virus vaccines.     

Mutation analysis of 2009 pandemic influenza A(H1N1) viruses collected in Japan during the peak phase of the pandemic

Background

Pandemic influenza A(H1N1) virus infection quickly circulated worldwide in 2009. In Japan, the first case was reported in May 2009, one month after its outbreak in Mexico. Thereafter, A(H1N1) infection spread widely throughout the country. It is of great importance to profile and understand the situation regarding viral mutations and their circulation in Japan to accumulate a knowledge base and to prepare clinical response platforms before a second pandemic (pdm) wave emerges.

Methodology

A total of 253 swab samples were collected from patients with influenza-like illness in the Osaka, Tokyo, and Chiba areas both in May 2009 and between October 2009 and January 2010. We analyzed partial sequences of the hemagglutinin (HA) and neuraminidase (NA) genes of the 2009 pdm influenza virus in the collected clinical samples. By phylogenetic analysis, we identified major variants of the 2009 pdm influenza virus and critical mutations associated with severe cases, including drug-resistance mutations.

Results and Conclusions

Our sequence analysis has revealed that both HA-S220T and NA-N248D are major non-synonymous mutations that clearly discriminate the 2009 pdm influenza viruses identified in the very early phase (May 2009) from those found in the peak phase (October 2009 to January 2010) in Japan. By phylogenetic analysis, we found 14 micro-clades within the viruses collected during the peak phase. Among them, 12 were new micro-clades, while two were previously reported. Oseltamivir resistance-related mutations, i.e., NA-H275Y and NA-N295S, were also detected in sporadic cases in Osaka and Tokyo.     

A Prospective Study of Romanian Agriculture Workers for Zoonotic Influenza Infections

Background

In this prospective study we sought to examine seroepidemiological evidence for acute zoonotic influenza virus infection among Romanian agricultural workers.

Methods

Sera were drawn upon enrollment (2009) and again at 12 and 24 months from 312 adult agriculture workers and 51 age-group matched controls. Participants were contacted monthly for 24 months and queried regarding episodes of acute influenza-like illnesses (ILI). Cohort members meeting ILI criteria permitted respiratory swab collections as well as acute and convalescent serum collection. Serologic assays were performed against 9 avian, 3 swine, and 3 human influenza viruses.

Results

During the two-year follow-up, a total of 23 ILI events were reported. Two subjects'' specimens were identified as influenza A by rRT-PCR. During the follow-up period, three individuals experienced elevated microneutralization antibody titers ≥1∶80 against three (one each) avian influenza viruses: A/Teal/Hong Kong/w312/97(H6N1), A/Hong Kong/1073/1999(H9N2), or A/Duck/Alberta/60/1976(H12N5). However, none of these participants met the criteria for poultry exposure. A number of subjects demonstrated four-fold increases over time in hemagglutination inhibition (HI) assay titers for at least one of the three swine influenza viruses (SIVs); however, it seems likely that two of these three responses were due to cross-reacting antibody against human influenza. Only elevated antibody titers against A/Swine/Flanders/1/1998(H3N2) lacked evidence for such confounding. In examining risk factors for elevated antibody against this SIV with multiple logistic regression, swine exposure (adjusted OR = 1.8, 95% CI 1.1–2.8) and tobacco use (adjusted OR = 1.8; 95% CI 1.1–2.9) were important predictors.

Conclusions

While Romania has recently experienced multiple incursions of highly pathogenic avian influenza among domestic poultry, this cohort of Romanian agriculture workers had sparse evidence of avian influenza virus infections. In contrast, there was evidence, especially among the swine exposed participants, of infections with human and one swine H3N2 influenza virus.     

Molecular Signature of High Yield (Growth) Influenza A Virus Reassortants Prepared as Candidate Vaccine Seeds

Background

Human influenza virus isolates generally grow poorly in embryonated chicken eggs. Hence, gene reassortment of influenza A wild type (wt) viruses is performed with a highly egg adapted donor virus, A/Puerto Rico/8/1934 (PR8), to provide the high yield reassortant (HYR) viral ‘seeds’ for vaccine production. HYR must contain the hemagglutinin (HA) and neuraminidase (NA) genes of wt virus and one to six ‘internal’ genes from PR8. Most studies of influenza wt and HYRs have focused on the HA gene. The main objective of this study is the identification of the molecular signature in all eight gene segments of influenza A HYR candidate vaccine seeds associated with high growth in ovo.

Methodology

The genomes of 14 wt parental viruses, 23 HYRs (5 H1N1; 2, 1976 H1N1-SOIV; 2, 2009 H1N1pdm; 2 H2N2 and 12 H3N2) and PR8 were sequenced using the high-throughput sequencing pipeline with big dye terminator chemistry.

Results

Silent and coding mutations were found in all internal genes derived from PR8 with the exception of the M gene. The M gene derived from PR8 was invariant in all 23 HYRs underlining the critical role of PR8 M in high yield phenotype. None of the wt virus derived internal genes had any silent change(s) except the PB1 gene in X-157. The highest number of recurrent silent and coding mutations was found in NS. With respect to the surface antigens, the majority of HYRs had coding mutations in HA; only 2 HYRs had coding mutations in NA.

Significance

In the era of application of reverse genetics to alter influenza A virus genomes, the mutations identified in the HYR gene segments associated with high growth in ovo may be of great practical benefit to modify PR8 and/or wt virus gene sequences for improved growth of vaccine ‘seed’ viruses.     

Rapid detection and subtyping of human influenza A viruses and reassortants by pyrosequencing

Background

Given the continuing co-circulation of the 2009 H1N1 pandemic influenza A viruses with seasonal H3N2 viruses, rapid and reliable detection of newly emerging influenza reassortant viruses is important to enhance our influenza surveillance.

Methodology/Principal Findings

A novel pyrosequencing assay was developed for the rapid identification and subtyping of potential human influenza A virus reassortants based on all eight gene segments of the virus. Except for HA and NA genes, one universal set of primers was used to amplify and subtype each of the six internal genes. With this method, all eight gene segments of 57 laboratory isolates and 17 original specimens of seasonal H1N1, H3N2 and 2009 H1N1 pandemic viruses were correctly matched with their corresponding subtypes. In addition, this method was shown to be capable of detecting reassortant viruses by correctly identifying the source of all 8 gene segments from three vaccine production reassortant viruses and three H1N2 viruses.

Conclusions/Significance

In summary, this pyrosequencing assay is a sensitive and specific procedure for screening large numbers of viruses for reassortment events amongst the commonly circulating human influenza A viruses, which is more rapid and cheaper than using conventional sequencing approaches.     

2009 Swine-Origin Influenza A (H1N1) Resembles Previous Influenza Isolates

Background

In April 2009, novel swine-origin influenza viruses (S-OIV) were identified in patients from Mexico and the United States. The viruses were genetically characterized as a novel influenza A (H1N1) strain originating in swine, and within a very short time the S-OIV strain spread across the globe via human-to-human contact.

Methodology

We conducted a comprehensive computational search of all available sequences of the surface proteins of H1N1 swine influenza isolates and found that a similar strain to S-OIV appeared in Thailand in 2000. The earlier isolates caused infections in pigs but only one sequenced human case, A/Thailand/271/2005 (H1N1).

Significance

Differences between the Thai cases and S-OIV may help shed light on the ability of the current outbreak strain to spread rapidly among humans.     

Complete-Proteome Mapping of Human Influenza A Adaptive Mutations: Implications for Human Transmissibility of Zoonotic Strains

Background

There is widespread concern that H5N1 avian influenza A viruses will emerge as a pandemic threat, if they become capable of human-to-human (H2H) transmission. Avian strains lack this capability, which suggests that it requires important adaptive mutations. We performed a large-scale comparative analysis of proteins from avian and human strains, to produce a catalogue of mutations associated with H2H transmissibility, and to detect their presence in avian isolates.

Methodology/Principal Findings

We constructed a dataset of influenza A protein sequences from 92,343 public database records. Human and avian sequence subsets were compared, using a method based on mutual information, to identify characteristic sites where human isolates present conserved mutations. The resulting catalogue comprises 68 characteristic sites in eight internal proteins. Subtype variability prevented the identification of adaptive mutations in the hemagglutinin and neuraminidase proteins. The high number of sites in the ribonucleoprotein complex suggests interdependence between mutations in multiple proteins. Characteristic sites are often clustered within known functional regions, suggesting their functional roles in cellular processes. By isolating and concatenating characteristic site residues, we defined adaptation signatures, which summarize the adaptive potential of specific isolates. Most adaptive mutations emerged within three decades after the 1918 pandemic, and have remained remarkably stable thereafter. Two lineages with stable internal protein constellations have circulated among humans without reassorting. On the contrary, H5N1 avian and swine viruses reassort frequently, causing both gains and losses of adaptive mutations.

Conclusions

Human host adaptation appears to be complex and systemic, involving nearly all influenza proteins. Adaptation signatures suggest that the ability of H5N1 strains to infect humans is related to the presence of an unusually high number of adaptive mutations. However, these mutations appear unstable, suggesting low pandemic potential of H5N1 in its current form. In addition, adaptation signatures indicate that pandemic H1N1/09 strain possesses multiple human-transmissibility mutations, though not an unusually high number with respect to swine strains that infected humans in the past. Adaptation signatures provide a novel tool for identifying zoonotic strains with the potential to infect humans.     

Molecular Evolution of Human H1N1 and H3N2 Influenza A Virus in Thailand, 2006–2009

Background

Annual seasonal influenza outbreaks are associated with high morbidity and mortality.

Objective

To index and document evolutionary changes among influenza A H1N1 and H3N2 viruses isolated from Thailand during 2006–2009, using complete genome sequences.

Methods

Nasopharyngeal aspirates were collected from patients diagnosed with respiratory illness in Thailand during 2006–2009. All samples were screened for Influenza A virus. A total of 13 H1N1 and 21 H3N2 were confirmed and whole genome sequenced for the evolutionary analysis using standard phylogenetic approaches.

Results

Phylogenetic analysis of HA revealed a clear diversification of seasonal from vaccine strain lineages. H3N2 seasonal clusters were closely related to the WHO recommended vaccine strains in each season. Most H1N1 isolates could be differentiated into 3 lineages. The A/Brisbane/59/2007 lineage, a vaccine strain for H1N1 since 2008, is closely related with the H1N1 subtypes circulating in 2009. HA sequences were conserved at the receptor-binding site. Amino acid variations in the antigenic site resulted in a possible N-linked glycosylation motif. Recent H3N2 isolates had higher genetic variations compared to H1N1 isolates. Most substitutions in the NP protein were clustered in the T-cell recognition domains.

Conclusion

In this study we performed evolutionary genetic analysis of influenza A viruses in Thailand between 2006–2009. Although the current vaccine strain is efficient for controlling the circulating outbreak subtypes, surveillance is necessary to provide unambiguous information on emergent viruses. In summary, the findings of this study contribute the understanding of evolution in influenza A viruses in humans and is useful for routine surveillance and vaccine strain selection.     

Molecular Evolutionary Analysis of pH1N1 2009 Influenza Virus in Reunion Island, South West Indian Ocean Region: A Cohort Study

Background/Objectives

Molecular epidemiology is a powerful tool to decipher the dynamics of viral transmission, quasispecies temporal evolution and origins. Little is known about the pH1N1 molecular dynamics in general population. A prospective study (CoPanFlu-RUN) was carried out in Reunion Island to characterize pH1N1 genetic variability and molecular evolution occurring in population during the pH1N1 Influenza pandemic in 2009.

Methodology

We directly amplified pH1N1 genomes from 28 different nasal swabs (26 individuals from 21 households). Fifteen strains were fully sequenced and 13 partially. This includes pairs of sequences from different members of 5 separate households; and two pairs from individuals, collected at different times. We assessed the molecular evolution of pH1N1 by genetic variability and phylogenetic analyses.

Principal Findings

We found that i) Reunion pH1N1 sequences stemmed from global “clade 7” but shaped two phylogenetic sub-clades; ii) D239E mutation was identified in the hemagglutinin protein of all Reunion sequences, a mutation which has been associated elsewhere with mild-, upper-respiratory tract pH1N1 infecting strains; iii) Date estimates from molecular phylogenies predicted clade emergence some time before the first detection of pH1N1 by the epidemiological surveillance system; iv) Phylogenetic relatedness was observed between Reunion pH1N1 viruses and those from other countries in South-western Indian Ocean area; v) Quasispecies populations were observed within households and individuals of the cohort-study.

Conclusions

Surveillance and/or prevention systems presently based on Influenza virus sequence variation should take into account that the majority of studies of pH1N1 Influenza generate genetic data for the HA/NA viral segments obtained from hospitalized-patients, which is potentially non-representative of the overall viral diversity within whole populations. Our observations highlight the importance of collecting unbiased data at the community level and conducting whole genome analysis to accurately understand viral dynamics.