Hemagglutinin Sequences Research Articles

Molecular epidemiology survey and characterization of human influenza A viruses circulating among Palestinians in East Jerusalem and the West Bank in 2015.

Frequent typing and molecular characterization of influenza A (IAV) strains are crucial for the identification of circulating subtypes and for the selection of the subtypes’ lineages to be included in the annually prepared vaccine cocktail. We investigated IAV sampled from an underrepresented population from Palestine. 200 nasopharyngeal aspirates (NPA) were collected between February and May of 2015 from Palestinians in East Jerusalem and the West Bank suffering from mild to severe symptoms of upper respiratory infections. NPA were screened for the presence of IAV using RT-PCR. Epidemiological data, hemagglutinin (HA) and neuraminidase (NA) gene sequences were analyzed in IAV positive samples. 50 samples tested positive for IAV; 48% of which were identified as A(H1N1)pdm09 and 52% as A(H3N2), respectively. Infection with A(H1N1)pdm09 occurred mainly in April, while A(H3N2) infections were mainly detected in March. Most IAV infections in 6-year-olds and below were attributed to subtype A(H3N2), while A(H1N1)pdm09 was responsible for most infections in adults above 18-year-olds. Analyses of HA and NA amino acid sequences revealed numerous substitutions. Thereafter, and based on the HA analysis, the Palestinian A(H1N1)pdm09 isolates fell into clade 6B, while the A(H3N2) isolates fell into clades 3C.2 and 3C.3, respectively. This study is significant in providing the first insight into the epidemiology and genetic properties of IAV circulating in Palestine. In contrast to international reports for the same season, A(H3N2) was not the dominant subtype as in northern hemisphere, nor was A(H1N1)pdm09 as in WHO reports for the Middle East, however genetic properties of Palestinian A(H3N2) and A(H1N1)pdm09 were in line with global isolates.

Use of Next Generation Sequencing to study two cowpox virus outbreaks.

BackgroundBetween 2008 and 2011 about 40 cases of human cowpox were reported from Germany and France. Infections had been acquired via close contact to infected, young pet rats. An identical and unique sequence of the hemagglutinin gene was found in various cowpox virus (CPXV) isolates pointing to a common source of infection. In a second CPXV outbreak in cats in a small animal clinic in Germany in 2015, four out of five hospitalized cats showed identical hemagglutinin sequences and thus, a hospital-acquired transmission had been assumed. Next-Generation Sequencing was performed in order to re-investigate the outbreaks, as epidemiological data could not confirm all cases.MethodsHomogenates of lesion material from rats, cats and humans were cultivated in cell culture. The genomes of four virus isolates, nine CPXVs from our strain collections and from DNA of three paraffin-embedded lesion materials were determined by Next Generation Sequencing (NGS). For phylogenetic analyses a MAFFT-alignment was generated. A distance matrix based on concatenated SNPs was calculated and plotted as dendrogram using Unweighted Pair Group Method with Arithmetic mean (UPGMA) for visualization.ResultsAligning of about 200.000 nucleotides of 8 virus isolates associated with the pet rat outbreak revealed complete identity of six genomes, the remainder two genomes differed in as little as 3 SNPs. When comparing this dataset with four already published CPXV genomes also associated with the pet rat outbreak, again a maximum difference of 3 SNPs was found. The outbreak which lasted from 2008 till 2011 was indeed caused by a single strain which has maintained an extremely high level of clonality over 4 years. Aligning genomic sequences from four cases of feline cowpox revealed 3 identical sequences and one sequence which differed in 65 nucleotides. Although identical hemagglutinin sequences had been obtained from four hospitalized cats, genomic sequencing proved that a hospital-acquired transmission had occurred in only three cats.ConclusionAnalyzing the rather short sequence of the hemagglutinin gene is not sufficient to conduct molecular trace back analyses. Instead, whole genome sequencing is the method of choice which can even be applied to paraffin-embedded specimens.

Genetic and antigenic characterisation of influenza A(H3N2) viruses isolated in Yokohama during the 2016/17 and 2017/18 influenza seasons.

Background: Influenza A(H3N2) virus rapidly evolves to evade human immune responses, resulting in changes in the antigenicity of haemagglutinin (HA). Therefore, continuous genetic and antigenic analyses of A(H3N2) virus are necessary to detect antigenic mutants as quickly as possible.Aim: We attempted to phylogenetically and antigenically capture the epidemic trend of A(H3N2) virus infection in Yokohama, Japan during the 2016/17 and 2017/18 influenza seasons.Methods: We determined the HA sequences of A(H3N2) viruses detected in Yokohama, Japan during the 2016/17 and 2017/18 influenza seasons to identify amino acid substitutions and the loss or gain of potential N-glycosylation sites in HA, both of which potentially affect the antigenicity of HA. We also examined the antigenicity of isolates using ferret antisera obtained from experimentally infected ferrets.Results: Influenza A(H3N2) viruses belonging to six clades (clades 3C.2A1, 3C.2A1a, 3C.2A1b, 3C.2A2, 3C.2A3 and 3C.2A4) were detected during the 2016/17 influenza season, whereas viruses belonging to two clades (clades 3C.2A1b and 3C.2A2) dominated during the 2017/18 influenza season. The isolates in clades 3C.2A1a and 3C.2A3 lost one N-linked glycosylation site in HA relative to other clades. Antigenic analysis revealed antigenic differences among clades, especially clade 3C.2A2 and 3C.2A4 viruses, which showed distinct antigenic differences from each other and from other clades in the antigenic map.Conclusion: Multiple clades, some of which differed antigenically from others, co-circulated in Yokohama, Japan during the 2016/17 and 2017/18 influenza seasons.

Active surveillance and genetic evolution of avian influenza viruses in Egypt, 2016–2018

ABSTRACT Egypt is a hotspot for avian influenza virus (AIV) due to the endemicity of H5N1 and H9N2 viruses. AIVs were isolated from 329 samples collected in 2016–2018; 48% were H9N2, 37.1% were H5N8, 7.6% were H5N1, and 7.3% were co-infections with 2 of the 3 subtypes. The 32 hemagglutinin (HA) sequences of the H5N1 viruses formed a well-defined lineage within clade 2.2.1.2. The 10 HA sequences of the H5N8 viruses belonged to a subclade within 2.3.4.4. The 11 HA of H9N2 isolates showed high sequence homology with other Egyptian G1-like H9N2 viruses. The prevalence of H5N8 viruses in ducks (2.4%) was higher than in chickens (0.94%). Genetic reassortment was detected in H9N2 viruses. Antigenic analysis showed that H9N2 viruses are homogenous, antigenic drift was detected among H5N1 viruses. AI H5N8 showed higher replication rate followed by H9N2 and H5N1, respectively. H5N8 was more common in Southern Egypt, H9N2 in the Nile Delta, and H5N1 in both areas. Ducks and chickens played a significant role in transmission of H5N1 viruses. The endemicity and co-circulation of H5N1, H5N8, and H9N2 AIV coupled with the lack of a clear control strategy continues to provide avenues for further virus evolution in Egypt.

Sequential DNA immunization of chickens with bivalent heterologous vaccines induce highly reactive and cross-specific antibodies against influenza hemagglutinin

Vaccines against avian influenza are mostly based on hemagglutinin (HA), which is the main antigen of this virus and a target for neutralizing antibodies. Traditional vaccines are known to be poorly efficient against newly emerging strains, which is an increasing worldwide problem for human health and for the poultry industry. As demonstrated by research and clinical data, sequential exposure to divergent influenza HAs can boost induction of universal antibodies which recognize conserved epitopes. In this work, we have performed sequential immunization of laying hens using monovalent or bivalent compositions of DNA vaccines encoding HAs from distant groups 1 and 2 (H5, H1, and H3 subtypes, respectively). This strategy gave promising results, as it led to induction of polyclonal antibodies against HAs from both groups. These polyclonal antibodies showed cross-reactivity between different HA strains in ELISA, especially when bivalent formulations were used for immunization of birds. However, cross-reactivity of antibodies induced against H3 and H5 HA subtypes was rather limited against each other after homologous immunization. Using a cocktail of HA sequences and/or sequential DNA vaccination with different strains presents a good strategy to overcome the limited effectiveness of vaccines and induce broader immunity against avian influenza. Such a strategy could be adapted for vaccinating laying hens or parental flocks of different groups of poultry.

Predominance of influenza B/Yamagata lineage viruses in Bulgaria during the 2017/2018 season.

In this study, we investigated the antigenic and genetic characteristics of influenza viruses circulating in Bulgaria during the 2017/2018 season. The detection and typing/subtyping of influenza viruses were performed using real-time RT-PCR. Results of antigenic characterisation, phylogenetic and amino acid sequence analyses of representative influenza strains are presented. The season was characterised by the predominance of B/Yamagata viruses, accounting for 77% of detected influenza viruses, followed by A(H1N1)pdm09 (17%), B/Victoria (3.7%) and A(H3N2) (2.4%). The sequenced B/Yamagata, B/Victoria, A(H1N1)pdm09 and A(H3N2) viruses belonged to the genetic groups 3, 1A, 6B.1 and 3C.2a1, respectively. Amino acid analysis of B/Yamagata isolates revealed the presence of three changes in haemagglutinin (HA), eight changes in neuraminidase (NA) and a number of substitutions in internal proteins compared with the B/Phucket/3073/2013 vaccine virus. Despite the amino acid changes, B/Yamagata viruses remained antigenically related to the vaccine strain. B/Victoria isolates fell into a group of viruses with double deletion (Δ162-163) in HA1. Substitutions in HA and NA sequences of B/Victoria, A(H1N1)pdm09 and A(H3N2) viruses were also identified compared with the vaccine strains, including in antigenic sites. The results of this study confirm the genetic variability of circulating influenza viruses and the need for continual antigenic and molecular surveillance.

Influenza and sudden unexpected death: the possible role of peptide cross-reactivity

Abstract This study investigates the hypothesis that cross-reactions may occur between human cardiac proteins and influenza antigens, thus possibly representing the molecular mechanism underlying influenzaassociated sudden unexpected death (SUD). Using titin protein as a research model, data were obtained on (1) the occurrence of the titin octapeptide AELLVLLE or its mimic AELLVALE in influenza A virus hemagglutinin (HA) sequences; (2) the immunological potential of AELLVLLE and its mimic AELLVALE; (3) the possible role of the flanking amino acid aa) context of the two octapeptide determinants in eliciting cross-reactivity between the human cardiac titin protein and HA antigens.

Impact of the variations in potential glycosylation sites of the hemagglutinin of H9N2 influenza virus

Variations in the potential glycosylation sites were observed in hemagglutinin (HA) sequences of H9N2 avian influenza virus isolated in China, deposited in the Influenza Virus Resource of NCBI before 2017, which showed a deleted glycosylation site at amino acid residue 218, and an introduced glycosylation site at amino acid residue 313. Based on the variations in the glycosylation sites at these amino acids, H9N2 avian influenza viruses could be divided into three categories. Firstly, most of the H9N2 influenza viruses were 218G+ viruses; less 313G+ viruses were isolated between 1997 and 2004. Secondly, the occurrence of the 218G+/313G+ viruses increased, while the 218G+/313G− viruses decreased from 2005 to 2012. Thirdly, from 2013 to 2016, the 218G−/313G+ viruses were predominant compared to the 218G+/313G+ viruses. Here, based on an F/98 virus backbone, a 218G+/313G− virus, two reassortment viruses were generated, and named rF/HA218G+/313G+ and rF/HA 218G+/313G−, respectively. HA protein migration demonstrated that the glycosylation sites at amino acid residues 313 and 218 were both functional. The absence of the glycosylation site at amino acid residue 218 and the presence of the glycosylation site at amino acid residue 313 increased antibody binding and moderately prevented the virus from escaping neutralization with homologous antisera. Additionally, compared to the F/98 virus (218G+/313G−), the viruses rF/HA218G+/313G+ or rF/HA218G−/313G+ showed significantly increased infectivity of MDCK cells, chicken embryo eggs, and trachea and lung tissue of SPF chickens, but did not display differences in airborne spread in chickens or infectivity of mice compared with its parental virus F/98.

Passage Adaptation Correlates With the Reduced Efficacy of the Influenza Vaccine.

As a dominant seasonal influenza virus, H3N2 virus rapidly evolves in humans and is a constant threat to public health. Despite sustained research efforts, the efficacy of H3N2 vaccine has decreased rapidly. Even though antigenic drift and passage adaptation (substitutions accumulated during vaccine production in embryonated eggs) have been implicated in reduced vaccine efficacy (VE), their respective contributions to the phenomenon remain controversial. We utilized mutational mapping, a powerful probabilistic method for studying sequence evolution, to analyze patterns of substitutions in different passage conditions for an unprecedented amount of H3N2 hemagglutinin sequences (n = 32 278). We found that passage adaptation in embryonated eggs is driven by repeated convergent evolution over 12 codons. Based on substitution patterns at these sites, we developed a metric, adaptive distance (AD), to quantify the strength of passage adaptation and subsequently identified a strong negative correlation between AD and VE. The high correlation between AD and VE implies that passage adaptation in embryonated eggs may be a strong contributor to the recent reduction in H3N2 VE. We developed a computational package called MADE (Measuring Adaptive Distance and vaccine Efficacy based on allelic barcodes) to measure the strength of passage adaptation and predict the efficacy of a candidate vaccine strain. Our findings shed light on strategies for reducing Darwinian evolution within the passaging medium in order to potentially restore an effective vaccine program in the future.

Characterising routes of H5N1 and H7N9 spread in China using Bayesian phylogeographical analysis

Avian influenza H5N1 subtype has caused a global public health concern due to its high pathogenicity in poultry and high case fatality rates in humans. The recently emerged H7N9 is a growing pandemic risk due to its sustained high rates of human infections, and recently acquired high pathogenicity in poultry. Here, we used Bayesian phylogeography on 265 H5N1 and 371 H7N9 haemagglutinin sequences isolated from humans, animals and the environment, to identify and compare migration patterns and factors predictive of H5N1 and H7N9 diffusion rates in China. H7N9 diffusion dynamics and predictor contributions differ from H5N1. Key determinants of spatial diffusion included: proximity between locations (for H5N1 and H7N9), and lower rural population densities (H5N1 only). For H7N9, additional predictors included low avian influenza vaccination rates, low percentage of nature reserves and high humidity levels. For both H5N1 and H7N9, we found viral migration rates from Guangdong to Guangxi and Guangdong to Hunan were highly supported transmission routes (Bayes Factor > 30). We show fundamental differences in wide-scale transmission dynamics between H5N1 and H7N9. Importantly, this indicates that avian influenza initiatives designed to control H5N1 may not be sufficient for controlling the H7N9 epidemic. We suggest control and prevention activities to specifically target poultry transportation networks between Central, Pan-Pearl River Delta and South-West regions.

A Fr\xe9chet tree distance measure to compare phylogeographic spread paths across trees

Phylogeographic methods reconstruct the origin and spread of taxa by inferring locations for internal nodes of the phylogenetic tree from sampling locations of genetic sequences. This is commonly applied to study pathogen outbreaks and spread. To evaluate such reconstructions, the inferred spread paths from root to leaf nodes should be compared to other methods or references. Usually, ancestral state reconstructions are evaluated by node-wise comparisons, therefore requiring the same tree topology, which is usually unknown. Here, we present a method for comparing phylogeographies across different trees inferred from the same taxa. We compare paths of locations by calculating discrete Fréchet distances. By correcting the distances by the number of paths going through a node, we define the Fréchet tree distance as a distance measure between phylogeographies. As an application, we compare phylogeographic spread patterns on trees inferred with different methods from hemagglutinin sequences of H5N1 influenza viruses, finding that both tree inference and ancestral reconstruction cause variation in phylogeographic spread that is not directly reflected by topological differences. The method is suitable for comparing phylogeographies inferred with different tree or phylogeographic inference methods to each other or to a known ground truth, thus enabling a quality assessment of such techniques.

Rapid detection of new B/Victoria-lineage haemagglutinin variants of influenza B viruses by pyrosequencing

During 2016/2017, several antigenically and genetically distinct variant viruses of the influenza B/Victoria/2/87-lineage (B/Vic) viruses, which have either deletions or mutations in the haemagglutinin (HA) emerged and co-circulated with other influenza B viruses from both the B/Vic and B/Yamagata/16/88-lineages (B/Yam). In this study we developed a pyrosequencing assay that can detect and differentiate multiple influenza B virus variants currently in circulation. The assay targets a region of HA sequence that is unique for each of the B/Yam, B/Vic and B/Vic variant viruses. Our results demonstrated that it is a rapid, robust, high-throughput assay, highly sensitive and specific in differentiating among the B/Yam, B/Vic and B/Vic variant viruses, giving it an advantage over an existing rRT-PCR method. It works well for influenza virus isolates as well as original clinical respiratory specimens, and can therefore be used to provide important information for surveillance by closely monitoring the spread of these B/Vic variants.

The molecular basis of antigenic variation among A(H9N2) avian influenza viruses

Avian influenza A(H9N2) viruses are an increasing threat to global poultry production and, through zoonotic infection, to human health where they are considered viruses with pandemic potential. Vaccination of poultry is a key element of disease control in endemic countries, but vaccine effectiveness is persistently challenged by the emergence of antigenic variants. Here we employed a combination of techniques to investigate the genetic basis of H9N2 antigenic variability and evaluate the role of different molecular mechanisms of immune escape. We systematically tested the influence of published H9N2 monoclonal antibody escape mutants on chicken antisera binding, determining that many have no significant effect. Substitutions introducing additional glycosylation sites were a notable exception, though these are relatively rare among circulating viruses. To identify substitutions responsible for antigenic variation in circulating viruses, we performed an integrated meta-analysis of all published H9 haemagglutinin sequences and antigenic data. We validated this statistical analysis experimentally and allocated several new residues to H9N2 antigenic sites, providing molecular markers that will help explain vaccine breakdown in the field and inform vaccine selection decisions. We find evidence for the importance of alternative mechanisms of immune escape, beyond simple modulation of epitope structure, with substitutions increasing glycosylation or receptor-binding avidity, exhibiting the largest impacts on chicken antisera binding. Of these, meta-analysis indicates avidity regulation to be more relevant to the evolution of circulating viruses, suggesting that a specific focus on avidity regulation is required to fully understand the molecular basis of immune escape by influenza, and potentially other viruses.

ISU FLUture: a veterinary diagnostic laboratory web-based platform to monitor the temporal genetic patterns of Influenza A virus in swine

BackgroundInfluenza A Virus (IAV) causes respiratory disease in swine and is a zoonotic pathogen. Uncontrolled IAV in swine herds not only affects animal health, it also impacts production through increased costs associated with treatment and prevention efforts. The Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) diagnoses influenza respiratory disease in swine and provides epidemiological analyses on samples submitted by veterinarians.DescriptionTo assess the incidence of IAV in swine and inform stakeholders, the ISU FLUture website was developed as an interactive visualization tool that allows the exploration of the ISU VDL swine IAV aggregate data in the clinical diagnostic database. The information associated with diagnostic cases has varying levels of completeness and is anonymous, but minimally contains: sample collection date, specimen type, and IAV subtype. Many IAV positive samples are sequenced, and in these cases, the hemagglutinin (HA) sequence and genetic classification are completed. These data are collected and presented on ISU FLUture in near real-time, and more than 6,000 IAV positive diagnostic cases and their epidemiological and evolutionary information since 2003 are presented to date. The database and web interface provides rapid and unique insight into the trends of IAV derived from both large- and small-scale swine farms across the United States of America.ConclusionISU FLUture provides a suite of web-based tools to allow stakeholders to search for trends and correlations in IAV case metadata in swine from the ISU VDL. Since the database infrastructure is updated in near real-time and is integrated within a high-volume veterinary diagnostic laboratory, earlier detection is now possible for emerging IAV in swine that subsequently cause vaccination and control challenges. The access to real-time swine IAV data provides a link with the national USDA swine IAV surveillance system and allows veterinarians to make objective decisions regarding the management and control of IAV in swine. The website is publicly accessible at http://influenza.cvm.iastate.edu.

Clinical characteristics of patients with laboratory-confirmed influenza A(H1N1)pdm09 during the 2013/2014 and 2015/2016 clade 6B/6B.1/6B.2-predominant outbreaks

A novel pandemic influenza A(H1N1)pdm09 virus emerged in 2009 globally, and it continues to circulate in humans. The National Influenza Surveillance Network in Taiwan identified five A(H1N1)pdm09-predominant seasons, representing the 2009/2010, 2010/2011, 2012/2013, 2013/2014, and 2015/2016 outbreaks from 2009 to 2016. Independently, a retrospective cohort study (which enrolled 639 infected patients during the five seasons) was conducted at Chang Gung Memorial Hospital to explore the risk factors associated with influenza A(H1N1)pdm09-related complications. A phylogenetic analysis of hemagglutinin (HA) sequences showed that the circulating A(H1N1)pdm09 virus belonged to clades 1, 2, and 8 in 2009/2010; clades 3, 4, 5, and 7 in 2010/2011; clades 7 and 6C in 2012/2013; clades 6B in 2013/2014; and 6B/6B.1/6B.2 in 2015/2016. Compared to individuals infected in non-6B/6B.1/6B.2 seasons (2009/2010, 2010/2011, and 2012/2013), those infected in 6B/6B.1/6B.2 seasons (2013/2014 and 2015/2016) were at higher risk for influenza-related complications (adjusted odds ratio [aOR]: 1.6, 95% confidence interval [CI]: 1.0–2.8), pneumonia (aOR: 1.78, 95% CI: 1.04–3.04), mechanical ventilation (aOR: 2.6, 95% CI: 1.2–5.6), and acute respiratory distress syndrome (aOR: 5.5, 95% CI: 1.9–15.9). For the increased severity of infection during the influenza A(H1N1)pdm09 clade 6B/6B.1/6B.2 seasons, aspects related to the antigenic change of A(H1N1)pdm09 virus, immune response of the host, and environmental factors required further investigation.

Complementary recognition of the receptor-binding site of highly pathogenic H5N1 influenza viruses by two human neutralizing antibodies

The highly pathogenic avian influenza virus H5N1 is a major threat to global public health and therefore a high-priority target of current vaccine development. The receptor-binding site (RBS) on the globular head of hemagglutinin (HA) in the viral envelope is one of the major target sites for antibody recognition against H5N1 and other influenza viruses. Here, we report the identification and characterization of a pair of human RBS–specific antibodies, designated FLD21.140 and AVFluIgG03, that are mutually complementary in their neutralizing activities against a diverse panel of H5N1 viruses. Crystallographic analysis and site-directed mutagenesis revealed that the two antibodies share a similar RBS-binding mode, and their individual specificities are governed by residues at positions 133a, 144, and 145. Specifically, FLD21.140 preferred Leu-133a/Lys-144/Ser-145, whereas AVFluIgG03 favored Ser-133a/Thr-144/Pro-145 residue triplets, both of which perfectly matched the most prevalent residues in viruses from epidemic-originating regions. Of note, according to an analysis of 3758 H5 HA sequences available in the Influenza Virus Database at the National Center for Biotechnology Information, the residues Leu-133a/Ser-133a and Ser-145/Pro-145 constituted more than 87.6 and 99.3% of all residues at these two positions, respectively. Taken together, our results provide a structural understanding for the neutralizing complementarity of these two antibodies and improve our understanding of the RBS-specific antibody response against H5N1 infection in humans.

Molecular and genetic characterization of peste des petits ruminants virus Kurdistan 2011 strain based on the haemagglutinin and fusion protein genes sequences

Severe outbreaks of peste des petits ruminants virus (PPRV) occurred during 2011 in wild goats in the Kurdistan region in Iraq. Based on a partial sequence of the nucleoprotein (N) gene the causative virus, named PPRV Kurdistan 2011, was classified as PPRV lineage IV genotype. For further characterization, the open reading frames (ORFs) coding for the haemagglutinin (H) and fusion (F) protein of PPRV Kurdistan 2011 were amplified from infected-cell RNA by RT-PCR. Sequencing of the ORFs revealed sizes of 1830 nucleotides (nt) for the H-ORF and 1641 nt for the F-ORF. This study represents the first report describing the full length ORFs of H and F for a PPRV strain from Kurdistan, Iraq.Comparative sequence analysis performed for the entire H- and F- ORFs of Kurdistan 2011 with respective sequences of PPRV isolates showed the highest homology with PPRV lineage IV isolates which is also reflected by comparative amino acid analysis. Phylogenetic analysis performed based on the partial and complete nucleotide sequences of both H- and F-ORFs clustered Kurdistan 2011 within PPRV lineage IV genotype. H-gene based phylogenetic trees aligned Kurdistan 2011 strain along with Turkey/TR/AFYON/2011-35H and Turkey/2000 strains whereas F-gene based phylogenetic trees aligned Kurdistan 2011 strain along with Qatar/37/10-02, Kurdistan/2012 and Egypt/2009 strains.

Evolutionary study and phylodynamic pattern of human influenza A/H3N2 virus in Indonesia from 2008 to 2010.

Influenza viruses are by nature unstable with high levels of mutations. The sequential accumulation of mutations in the surface glycoproteins allows the virus to evade the neutralizing antibodies. The consideration of the tropics as the influenza reservoir where viral genetic and antigenic diversity are continually generated and reintroduced into temperate countries makes the study of influenza virus evolution in Indonesia essential. A total of 100 complete coding sequences (CDS) of Hemagglutinin (HA) and Neuraminidase (NA) genes of H3N2 virus were obtained from archived samples of Influenza-Like Illness (ILI) surveillance collected from 2008 to 2010. Our evolutionary and phylogenetic analyses provide insight into the dynamic changes of Indonesian H3N2 virus from 2008 to 2010. Obvious antigenic drift with typical ‘ladder-like’ phylogeny was observed with multiple lineages found in each year, suggesting co-circulation of H3N2 strains at different time periods. The mutational pattern of the Indonesian H3N2 virus was not geographically related as relatively low levels of mutations with similar pattern of relative genetic diversity were observed in various geographical origins. This study reaffirms that the existence of a particular lineage is most likely the result of adaptation or competitive exclusion among different host populations and combination of stochastic ecological factors, rather than its geographical origin alone.

Antigenic and genetic characterization of influenza viruses isolated in Mozambique during the 2015 season.

BackgroundDue to the high rate of antigenic variation of influenza virus, seasonal characterization of the virus is crucial to assess and monitor the emergence of new pathogenic variants and hence formulate effective control measures. However, no study has yet been conducted in Mozambique to assess genetic, antigenic and antiviral susceptibility profile of influenza virus.MethodsA subset of samples (n = 20) from influenza positive children detected in two hospitals in Maputo city during 2015 season as part of the implementation of influenza surveillance system, were selected. The following assays were performed on these samples: antigenic characterization by hemagglutination inhibition assay, genetic characterization by Sanger sequencing of hemagglutinin (HA) and neuraminidase (NA) and susceptibility to oseltamivir and zanamivir (NA inhibitors) by enzymatic assay.ResultsThe A(H1N1)pdm09 subtype viruses remained closely related antigenically and genetically to the 2016 vaccine virus A/California/7/2009 and other widely distributed viruses belonging to genetic group 6B. The majority of influenza A(H3N2) viruses studied were antigenically similar to the 2016–2017 vaccine virus, A/Hong Kong/4801/2014, and their HA and NA gene sequences fell into genetic subclade 3C.2a being closely related to viruses circulating in southern Africa. The influenza B viruses were antigenically similar to the 2016 season vaccine virus and HA sequences of all three fell into the B/Yamagata-lineage, clade 3, but contained NA genes of the B/Victoria-lineage. All tested viruses were sensitive to oseltamivir and zanamivir.ConclusionOverall, all Mozambican influenza A and B viruses were most closely related to Southern African viruses and all were sensitive to oseltamivir and zanamivir. These findings suggest the existence of an ecological niche of influenza viruses within the region and hence highlighting the need for joint epidemiologic and virologic surveillance to monitor the evolution of influenza viruses.

Genetic characterization of influenza A(H3N2) viruses from 2014 to 2017 in Yantai, east of China

Abstract Background: The genetic variations of influenza viruses pose a real challenge to the vaccine strategies and medical treatment of patients. Methods: In this study, the molecular epidemiology and evolution of influenza A(H3N2) strains were analyzed from April 2014 to March 2017 in Yantai area of eastern China. Results: The phylogenetic analysis of the hemagglutinin (HA) sequences of influenza A(H3N2) showed that all of the influenza A(H3N2) strains during the study period belonged to the genetic clade 3c with the mutations N145S (epitopeA), V186G (epitopeB), P198S (epitopeB) and F219S (epitopeD). Most strains (12/14) of the 2014/2015 season fell into the subgroup 3C.3a characterized by A138S (epitopeA), R142G (epitopeA), F159S (epitopeB) and T128A (epitopeB), while strains isolated from the 2015/2016 and 2016/2017 seasons clustered in 3C.2a shared mutations N144S (epitopeA), F159Y (epitopeB), K160T (epitopeB) and Q311H (epitopeC). The strains isolated from the 2014/2015 and 2015/2016 seasons was were genetically and antigenically distinct from the given vaccine strains. The evaluation of vaccine efficacy (VE) against circulating strains estimated using the pepitope model suggested that little or no protection against circulating strains from 2014/2015 and 2015/2016 seasons was afforded by the given vaccine strains. The sequence analysis of the neuraminidase (NA) showed that all of the analyzed strains had no substitution in the catalytic sites or the framework sites or the supporting the catalytic residues or the oseltamivir resistance substitutions. Conclusions: The results of the study suggested that the vaccine strains provided suboptimal protection against influenza A(H3N2) strains, especially in the 2014/2015 and 2015/2016 seasons and the A(H3N2) strains circulating in the Yantai area were still susceptible to NA inhibitors. Continued systematic antigenic and molecular surveillance of the influenza virus is essential to developing strategies for the prevention and control of influenza.