Influenza Virus Sequences Research Articles

Complete Genome Sequence of an Avian H1N1 Influenza Virus Strain Isolated from Migratory Birds in the Republic of Korea.

ABSTRACTHere, we report the complete genome sequence of an H1N1 avian influenza virus (AIV), which was isolated from the feces of migratory birds in the Republic of Korea during the winters of 2014 and 2015. Full-genome sequencing and phylogenetic analysis revealed that all genome segments belonged to the Eurasian lineage.

The blood DNA virome in 8,000 humans.

The characterization of the blood virome is important for the safety of blood-derived transfusion products, and for the identification of emerging pathogens. We explored non-human sequence data from whole-genome sequencing of blood from 8,240 individuals, none of whom were ascertained for any infectious disease. Viral sequences were extracted from the pool of sequence reads that did not map to the human reference genome. Analyses sifted through close to 1 Petabyte of sequence data and performed 0.5 trillion similarity searches. With a lower bound for identification of 2 viral genomes/100,000 cells, we mapped sequences to 94 different viruses, including sequences from 19 human DNA viruses, proviruses and RNA viruses (herpesviruses, anelloviruses, papillomaviruses, three polyomaviruses, adenovirus, HIV, HTLV, hepatitis B, hepatitis C, parvovirus B19, and influenza virus) in 42% of the study participants. Of possible relevance to transfusion medicine, we identified Merkel cell polyomavirus in 49 individuals, papillomavirus in blood of 13 individuals, parvovirus B19 in 6 individuals, and the presence of herpesvirus 8 in 3 individuals. The presence of DNA sequences from two RNA viruses was unexpected: Hepatitis C virus is revealing of an integration event, while the influenza virus sequence resulted from immunization with a DNA vaccine. Age, sex and ancestry contributed significantly to the prevalence of infection. The remaining 75 viruses mostly reflect extensive contamination of commercial reagents and from the environment. These technical problems represent a major challenge for the identification of novel human pathogens. Increasing availability of human whole-genome sequences will contribute substantial amounts of data on the composition of the normal and pathogenic human blood virome. Distinguishing contaminants from real human viruses is challenging.

Complete Genome Sequence of a Novel Reassortant H6N8 Avian Influenza Virus Isolated from Wild Waterfowl in Poyang Lake, China.

ABSTRACTHere, we report the complete genome sequence of an H6N8 avian influenza virus (AIV) isolated from wild waterfowl in Poyang Lake, China, in 2016. Phylogenetic analysis showed that it was a novel reassortant AIV between domestic ducks and wild waterfowl. The finding of this study is helpful for our understanding of the ecology and the evolutionary characteristics of H6 subtypes of AIV in birds.

The Establishment of Realtime Fluorescent Quantitative Polymerase chain reaction (PCR) for Detection of Highly Pathogenic Avian Influenza Virus Subtype H5N1

Highly pathogenic strains of avian influenza virus (AIV), which are influenza A viruses, cause severe disease in domestic poultry and humans. The objective of this study was to establish a fluorescent quantitative RT-PCR assay for detection of highly pathogenic avian influenza virus (AIV) subtype H5N1. The H5 and N1 subtypespecific probe sets were developed based on avian influenza virus sequences detected in China. Two pairs of primers and two fluorescent probes were strictly designed and optimized in a reaction system. According to the amount of plasmid RNA extracted from H5N1 strains, the standard curve DWQBGWDWQBGW of fluorescent quantitative PCR was drawn and all of the specimens were then tested by means of Real-time PCR. The test of highly pathogenic AIV subtype H5N1 was identified to be specific and its sensitivity level was 102~103 copies/reaction. The standard curve was accomplished at 109～105 DNA copies/reaction. It took only three hours from viral RNA extraction through to completion of the test. The assay was easy to carry out and highly reproducible. In conclusion, fluorescent quantitative PCR, described here, provides a rapid, specific and sensitive method to detect not only the H5 but N1 genes as well.

The complexity of human infected AIV H5N6 isolated from China.

BackgroundNovel avian influenza viruses (AIVs) of H7N9, H10N8, and H5N6 are currently circulating in China’s poultry flocks, occasionally infecting human and other mammals. Human infected AIV H5N6 in China during 2014–2015 is believed to be a triple reassortant originated from H6N6 and two clades of H5 viruses. The current report suggests that its reassortment history is more complicated.MethodsGenomes of human infected isolates of AIV H5N6 were searched from the NCBI Influenza Virus Sequence Database and the Global Initiative on Sharing Avian Influenza Data. Sequences shared high identities with each segment of their genomes were obtained through the Basic Local Alignment Search Tool. Alignments were done by mafft-7.037-win32 program; 8 large-scale and then 8 gradually converged phylogenetic trees were constructed by using MEGA5.1/5.2/6.0 Software.ResultsThe events that each segment of the genomes of human infected AIV H5N6 isolates circulated in China had evolved into its current status might have happened before 2013, and so were they then reassorted into the epidemic AIV H5N6. A/Guangzhou/39715/2014(H5N6) and A/Sichuan/26221/2014(H5N6) had their six internal segments (PB2, PB1, PA, NP, NEP, and M) in common, and were reassorted from AIVs H5N1 in the same period and same region as that of HA, while A/Yunnan/0127/2015(H5N6) derived its six internal segments from AIV H9N2 that has been prevalent in Eastern China since 2008.ConclusionsAIV H5N6 isolates established from both human and poultry in China during 2014–2015 were heterogeneous; both AIVs H5N1 and H9N2 were involved in the reassortment of AIV H5N6 in China.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-1932-1) contains supplementary material, which is available to authorized users.

Complete Genome Sequence of the First H5N1 Avian Influenza Virus Isolated from Chickens in Lebanon in 2016.

We generated the full genome of a highly pathogenic H5N1 avian influenza virus that caused an outbreak on a chicken farm in Lebnaon in April 2016. Analysis revealed that the virus belonged to clade 2.3.2.1c that recently caused outbreaks in West Africa and the United Arab Emirates.

Deep Sequencing of Influenza A Virus from a Human Challenge Study Reveals a Selective Bottleneck and Only Limited Intrahost Genetic Diversification.

ABSTRACTKnowledge of influenza virus evolution at the point of transmission and at the intrahost level remains limited, particularly for human hosts. Here, we analyze a unique viral data set of next-generation sequencing (NGS) samples generated from a human influenza challenge study wherein 17 healthy subjects were inoculated with cell- and egg-passaged virus. Nasal wash samples collected from 7 of these subjects were successfully deep sequenced. From these, we characterized changes in the subjects' viral populations during infection and identified differences between the virus in these samples and the viral stock used to inoculate the subjects. We first calculated pairwise genetic distances between the subjects' nasal wash samples, the viral stock, and the influenza virus A/Wisconsin/67/2005 (H3N2) reference strain used to generate the stock virus. These distances revealed that considerable viral evolution occurred at various points in the human challenge study. Further quantitative analyses indicated that (i) the viral stock contained genetic variants that originated and likely were selected for during the passaging process, (ii) direct intranasal inoculation with the viral stock resulted in a selective bottleneck that reduced nonsynonymous genetic diversity in the viral hemagglutinin and nucleoprotein, and (iii) intrahost viral evolution continued over the course of infection. These intrahost evolutionary dynamics were dominated by purifying selection. Our findings indicate that rapid viral evolution can occur during acute influenza infection in otherwise healthy human hosts when the founding population size of the virus is large, as is the case with direct intranasal inoculation.IMPORTANCE Influenza viruses circulating among humans are known to rapidly evolve over time. However, little is known about how influenza virus evolves across single transmission events and over the course of a single infection. To address these issues, we analyze influenza virus sequences from a human challenge experiment that initiated infection with a cell- and egg-passaged viral stock, which appeared to have adapted during its preparation. We find that the subjects' viral populations differ genetically from the viral stock, with subjects' viral populations having lower representation of the amino-acid-changing variants that arose during viral preparation. We also find that most of the viral evolution occurring over single infections is characterized by further decreases in the frequencies of these amino-acid-changing variants and that only limited intrahost genetic diversification through new mutations is apparent. Our findings indicate that influenza virus populations can undergo rapid genetic changes during acute human infections.

Characterization and Sequencing of an H6N6 Avian Influenza Virus Isolated from Sansui Sheldrake Ducks in Guizhou, Southwestern China.

Here, we report the complete genome sequence of an H6N6 avian influenza virus (AIV) isolated from Sansui Sheldrake ducks in Guizhou Province, China, in 2014. Phylogenetic analysis showed that the H6N6 virus was a reassortant virus derived from three different H6 subtype lineages. The finding of this study will help us understand the epidemiology and the evolutionary characteristics of H6 subtypes of AIV in ducks in southwestern China.

Openness, Transparency and Equity in Public Health Surveillance Data Sharing

The outbreaks of Severe Acute Respiratory Syndrome (SARS) in 2003, influenza A (H1N1) in 2009 and Ebola in 2014 have shown that increasingly infectious diseases can spread globally in a short timeframe, affecting both high- and low-income countries. Taking action to mitigate the impact of future crises relies on sharing public health surveillance data across national borders in an efficient and effective way. However, data users, particularly in high-income countries, often use surveillance data, particularly from low- and middle-income countries, with little or no benefit to the data generator. As Indonesia's refusal to share influenza virus sequences during the 2006 H5N1 outbreak illustrates, this imbalance increases reluctance to share and jeopardizes the global good that can be achieved. In order to share public health surveillance data internationally in an equitable way, technical, political, ethical, and legal issues need to be addressed. The Centre on Global Health Security at Chatham House is producing guidance that will address both the policy and technical issues with the aim of establishing new norms so that data can be shared in an open, transparent and equitable way.

Is a highly pathogenic avian influenza virus H5N1 fragment recombined in PB1 the key for the epidemic of the novel AIV H7N9 in China, 2013?

A novel avian influenza A H7N9 virus that infects humans was identified in China in 2013. This study is the first to comprehensively investigate the characteristics of genomic recombination, rather than reassortment, which has been the subject of investigation in previously reported studies. Novel avian influenza virus (AIV) H7N9 genome sequences were obtained from the NCBI Influenza Virus Sequence Database and the Global Initiative on Sharing Avian Influenza Database (GISAID) and a representative isolate was subjected to homogeneity analysis. A phylogenetic tree was constructed. Eight segments of the isolate were analyzed to identify segments with recombination events, the corresponding recombination fragments, and breakpoints. The evolutionary history of the recombined fragments was tracked by constructing phylogenetic trees of the recombination fragments. Among the eight segments of the novel AIV H7N9 analyzed, only the PB1 segment showed a marked recombination phenomenon, with 11 recombination events; these included five actual recombination events and six possible misalignment artifact recombination events. The most notable was the recombination of a 291-nucleotide (nt) fragment at the 490-780 nt site that was affiliated to a highly pathogenic avian influenza virus (HPAIV) H5N1 (A/treesparrow/Thailand/VSMU-16-RBR/2005). The phylogenetic tree of the 291-nt recombination fragment on the PB1 segment showed that the novel AIV H7N9 had a close genetic relationship to H9N2 and H5N1. The novel AIV H7N9 might have reassorted its PB1 segment from H9N2 circulating in China, and this H9N2 PB1 might have been recombined into a highly pathogenic fragment from HPAIV H5N1, which could be the reason for the high fatality rate among patients with AIV H7N9 influenza.

Molecular Characteristics of H6N6 Influenza Virus Isolated from Pigeons in Guangxi, Southern China.

Here, we report the complete genome sequence of an H6N6 avian influenza virus (AIV) isolated from a pigeon in Guangxi, southern China, in 2014. The eight RNA segment genes shared a high nucleotide identity (97 to 99%) with H6N6 subtypes of AIV isolated from ducks in the regions around Guangxi Province. The finding of this study will help us understand the ecology and molecular characteristics of H6 avian influenza virus in wild birds in southern China.

Characterization of the Whole-Genome Sequence of an H3N6 Avian Influenza Virus, Isolated from a Domestic Duck in Guangxi, Southern China.

A field strain of H3N6 avian influenza virus (AIV), A/duck/Guangxi/175D12/2014(H3N6), was isolated from a native duck in Guangxi Province, southern China, in 2014. All of the eight AIV gene segments were sequenced, and sequence results revealed that there were 11 amino acid deletions at the NA stalk region. The NA, PB2, and NP genes showed highest homology to H5N6 AIV, and the PA gene showed highest homology to H7N2 AIV. Phylogenetic analysis indicated that the eight AIV gene segments belonged to the Eurasian lineage. These findings provide scientific evidence of possible or potential mutations of H3N6 AIV circulating in waterfowl in southern China.

The effects of a deleterious mutation load on patterns of influenza A/H3N2's antigenic evolution in humans.

Recent phylogenetic analyses indicate that RNA virus populations carry a significant deleterious mutation load. This mutation load has the potential to shape patterns of adaptive evolution via genetic linkage to beneficial mutations. Here, we examine the effect of deleterious mutations on patterns of influenza A subtype H3N2's antigenic evolution in humans. By first analyzing simple models of influenza that incorporate a mutation load, we show that deleterious mutations, as expected, act to slow the virus's rate of antigenic evolution, while making it more punctuated in nature. These models further predict three distinct molecular pathways by which antigenic cluster transitions occur, and we find phylogenetic patterns consistent with each of these pathways in influenza virus sequences. Simulations of a more complex phylodynamic model further indicate that antigenic mutations act in concert with deleterious mutations to reproduce influenza's spindly hemagglutinin phylogeny, co-circulation of antigenic variants, and high annual attack rates.

Influenza A Viruses of Human Origin in Swine, Brazil.

The evolutionary origins of the influenza A(H1N1)pdm09 virus that caused the first outbreak of the 2009 pandemic in Mexico remain unclear, highlighting the lack of swine surveillance in Latin American countries. Although Brazil has one of the largest swine populations in the world, influenza was not thought to be endemic in Brazil's swine until the major outbreaks of influenza A(H1N1)pdm09 in 2009. Through phylogenetic analysis of whole-genome sequences of influenza viruses of the H1N1, H1N2, and H3N2 subtypes collected in swine in Brazil during 2009-2012, we identified multiple previously uncharacterized influenza viruses of human seasonal H1N2 and H3N2 virus origin that have circulated undetected in swine for more than a decade. Viral diversity has further increased in Brazil through reassortment between co-circulating viruses, including A(H1N1)pdm09. The circulation of multiple divergent hemagglutinin lineages challenges the design of effective cross-protective vaccines and highlights the need for additional surveillance.

Nextflu: real-time tracking of seasonal influenza virus evolution in humans.

Summary: Seasonal influenza viruses evolve rapidly, allowing them to evade immunity in their human hosts and reinfect previously infected individuals. Similarly, vaccines against seasonal influenza need to be updated frequently to protect against an evolving virus population. We have thus developed a processing pipeline and browser-based visualization that allows convenient exploration and analysis of the most recent influenza virus sequence data. This web-application displays a phylogenetic tree that can be decorated with additional information such as the viral genotype at specific sites, sampling location and derived statistics that have been shown to be predictive of future virus dynamics. In addition, mutation, genotype and clade frequency trajectories are calculated and displayed.Availability and implementation: Python and Javascript source code is freely available from https://github.com/blab/nextflu, while the web-application is live at http://nextflu.org.Contact: tbedford@fredhutch.org

Global circulation patterns of seasonal influenza viruses vary with antigenic drift.

Understanding the spatio-temporal patterns of emergence and circulation of new human seasonal influenza virus variants is a key scientific and public health challenge. The global circulation patterns of influenza A/H3N2 viruses are well-characterized1-7 but the patterns of A/H1N1 and B viruses have remained largely unexplored. Here, based on analyses of 9,604 hemagglutinin sequences of human seasonal influenza viruses from 2000–2012, we show that the global circulation patterns of A/H1N1 (up to 2009), B/Victoria, and B/Yamagata viruses differ substantially from those of A/H3N2 viruses. While genetic variants of A/H3N2 viruses did not persist locally between epidemics and were reseeded from East and Southeast (E-SE) Asia, genetic variants of A/H1N1 and B viruses persisted across multiple seasons and exhibited complex global dynamics with E-SE Asia playing a limited role in disseminating new variants. The less frequent global movement of influenza A/H1N1 and B viruses coincided with slower rates of antigenic evolution, lower ages of infection, and smaller less frequent epidemics compared to A/H3N2 viruses. Detailed epidemic models support differences in age of infection, combined with the less frequent travel of children, as likely drivers of the differences in the patterns of global circulation, suggesting a complex interaction between virus evolution, epidemiology and human behavior.

Molecular phylogeny and evolutionary dynamics of matrix gene of avian influenza viruses in China

In China, several subtype avian influenza viruses consistently circulate in poultry. Numerous studies have focused on the evolution of the hemagglutinin gene; however, studies on the evolution of the matrix (M) gene are limited. In this study, a large-scale phylogenetic analysis of M gene sequences of avian influenza viruses isolated in China revealed that the M gene has evolved into six different lineages denoted as I–VI. The majority of lineages I and IV were isolated in terrestrial birds, while the majority of lineages II, III, V and VI were isolated in aquatic birds. Lineage I included 148 H9N2 subtype viruses (72.2%), lineage II comprised of 63 H6 subtype viruses (100%), and lineage IV included 157 H5 subtype viruses (97.5%). The mean substitution rates of different lineages ranged from 1.32×10−3 (lineage III) to 3.64×10−3 (lineage IV) substitutions per site per year. According to the most recent common ancestor of all lineages, lineage III was the oldest lineage, formed in 1981 or even earlier. And lineage V was the most recent, established around the year 2000. Selective pressure on M2 was stronger than that on M1. The strongest selection pressure was observed in lineage IV. In addition, site-by-site analyses of each lineage identified 8 positive selection sites, all in M2. Most of the sites (5 out of 8) were located in the extracellular domain, which is an antigen for vaccine development. The positive selection sites (amino acid positions 66, 82 and 97) are likely associated with virus budding. This study enhanced our knowledge of M gene evolution of avian influenza viruses, and is expected to improve the early detection of new viruses and lead to vaccine development.

SNPer: an R library for quantitative variant analysis on single nucleotide polymorphisms among influenza virus populations.

Influenza virus (IFV) can evolve rapidly leading to genetic drifts and shifts resulting in human and animal influenza epidemics and pandemics. The genetic shift that gave rise to the 2009 influenza A/H1N1 pandemic originated from a triple gene reassortment of avian, swine and human IFVs. More minor genetic alterations in genetic drift can lead to influenza drug resistance such as the H274Y mutation associated with oseltamivir resistance. Hence, a rapid tool to detect IFV mutations and the potential emergence of new virulent strains can better prepare us for seasonal influenza outbreaks as well as potential pandemics. Furthermore, identification of specific mutations by closely examining single nucleotide polymorphisms (SNPs) in IFV sequences is essential to classify potential genetic markers associated with potentially dangerous IFV phenotypes. In this study, we developed a novel R library called “SNPer” to analyze quantitative variants in SNPs among IFV subpopulations. The computational SNPer program was applied to three different subpopulations of published IFV genomic information. SNPer queried SNPs data and grouped the SNPs into (1) universal SNPs, (2) likely common SNPs, and (3) unique SNPs. SNPer outperformed manual visualization in terms of time and labor. SNPer took only three seconds with no errors in SNP comparison events compared with 40 hours with errors using manual visualization. The SNPer tool can accelerate the capacity to capture new and potentially dangerous IFV strains to mitigate future influenza outbreaks.

Continual Reintroduction of Human Pandemic H1N1 Influenza A Viruses into Swine in the United States, 2009 to 2014.

The diversity of influenza A viruses in swine (swIAVs) presents an important pandemic threat. Knowledge of the human-swine interface is particularly important for understanding how viruses with pandemic potential evolve in swine hosts. Through phylogenetic analysis of contemporary swIAVs in the United States, we demonstrate that human-to-swine transmission of pandemic H1N1 (pH1N1) viruses has occurred continuously in the years following the 2009 H1N1 pandemic and has been an important contributor to the genetic diversity of U.S. swIAVs. Although pandemic H1 and N1 segments had been largely removed from the U.S. swine population by 2013 via reassortment with other swIAVs, these antigens reemerged following multiple human-to-swine transmission events during the 2013-2014 seasonal epidemic. These findings indicate that the six internal gene segments from pH1N1 viruses are likely to be sustained long term in the U.S. swine population, with periodic reemergence of pandemic hemagglutinin (HA) and neuraminidase (NA) segments in association with seasonal pH1N1 epidemics in humans. Vaccinating U.S. swine workers may reduce infection of both humans and swine and in turn limit the role of humans as sources of influenza virus diversity in pigs. Swine are important hosts in the evolution of influenza A viruses with pandemic potential. Here, we analyze influenza virus sequence data generated by the U.S. Department of Agriculture's national surveillance system to identify the central role of humans in the reemergence of pandemic H1N1 (pH1N1) influenza viruses in U.S. swine herds in 2014. These findings emphasize the important role of humans as continuous sources of influenza virus diversity in swine and indicate that influenza viruses with pandemic HA and NA segments are likely to continue to reemerge in U.S. swine in association with seasonal pH1N1 epidemics in humans.

In ovo and in vitro susceptibility of American alligators (Alligator mississippiensis) to avian influenza virus infection.

Avian influenza has emerged as one of the most ubiquitous viruses within our biosphere. Wild aquatic birds are believed to be the primary reservoir of all influenza viruses; however, the spillover of H5N1 highly pathogenic avian influenza (HPAI) and the recent swine-origin pandemic H1N1 viruses have sparked increased interest in identifying and understanding which and how many species can be infected. Moreover, novel influenza virus sequences were recently isolated from New World bats. Crocodilians have a slow rate of molecular evolution and are the sister group to birds; thus they are a logical reptilian group to explore susceptibility to influenza virus infection and they provide a link between birds and mammals. A primary American alligator (Alligator mississippiensis) cell line, and embryos, were infected with four, low pathogenic avian influenza (LPAI) strains to assess susceptibility to infection. Embryonated alligator eggs supported virus replication, as evidenced by the influenza virus M gene and infectious virus detected in allantoic fluid and by virus antigen staining in embryo tissues. Primary alligator cells were also inoculated with the LPAI viruses and showed susceptibility based upon antigen staining; however, the requirement for trypsin to support replication in cell culture limited replication. To assess influenza virus replication in culture, primary alligator cells were inoculated with H1N1 human influenza or H5N1 HPAI viruses that replicate independent of trypsin. Both viruses replicated efficiently in culture, even at the 30 C temperature preferred by the alligator cells. This research demonstrates the ability of wild-type influenza viruses to infect and replicate within two crocodilian substrates and suggests the need for further research to assess crocodilians as a species potentially susceptible to influenza virus infection.