Gene Segment Sequences Research Articles

Subtype specific virus enrichment with immunomagnetic separation method followed by NGS unravels the mixture of H5 and H9 avian influenza virus

Wild bird avian influenza type A virus (AIV) surveillance is important for the early detection of highly pathogenic AIVs and for providing early warnings to the poultry industry and veterinary services to implement more effective control measures against these viruses. Some field samples are often found to contain more than two kinds of AIV. Correct determination of the HA/NA subtype and complete nucleotide sequences of the component viruses in those samples are often critical for timely and accurate understanding of the field situation, but it is not easy to define the genomic structure of the constituent viruses unambiguously because AIV has eight segmented genomes. In this study, with immunomagnetic beads incorporating polyclonal antibodies of chicken for subtype-specific viral enrichment, we could selectively decrease the density of one of the two constituent viruses in a sample of different subtypes, H5 and H9, artificially generated; this was represented in the changes of Ct values with subtype specific real-time RT-PCR. Following this, with NGS, we could recover nearly complete genomic sequences and arrange the consensus sequences of gene segments of the constituent viruses confidently with the quantitative variable like genome coverage, linked along the gene segments and associated with the number of viral copies in a sample.

Characterization of the B Cell Receptor Repertoire and Discovery of New Immunoglobulin Genes in the Domestic Ferret

Abstract Mustela putorius furo uniquely reflects physiological and immunological changes in human aging such as immune imprinting from influenza infection, yet key ferret immunobiology knowledge gaps limit the scope of molecular analyses. First, ferret germline B cell receptor genes require identification. Additionally, the V, D, and J gene repertoire expressed by B cell receptors in antigen-experienced cells needs characterization. Illumina NGS was used to generate B-cell receptor mRNA data sets. A candidate list of gene segments was assembled including ferret genomic accessions homologous to known immunoglobulin genes and previously-known ferret immunoglobulin genes. NGS reads were queried against the candidate list using IgBLAST. IgBLAST hits were focused into consensus sequences using principal components analysis and K-means clustering. To identify IgG subclasses, putative ferret genome sequences sharing human IgG C genes were used to query Oxford Nanopore data that covered the FR3 through C gene area of transcripts. We report two new functional IGHV, two IGKV, and one IGLV gene segments in the ferret, with more sequences likely to surface in downstream analyses. We also corroborate most already-proposed functional V, D, and J gene segment sequences. Ferret orthologues to human IGHV subgroups commonly involved in stem-directed, broadly neutralizing antibody responses were identified. For the first time, productive IgG3 was described in the ferret model and distinguished from productive IgG1. Our report of novel ferret immunoglobulin genes will disinhibit the ferret as a model organism for the study of infectious disease. Specifically, it will inform the immunogenicity of candidate HA antigens in preclinical vaccine studies. Research reported in this abstract was supported by NIH NIAID contract 75N93019C00050.

The genetic characterization of hemagglutinin (HA), neuraminidase (NA) and polymerase acidic (PA) genes of H3N2 influenza viruses circulated in Guangdong Province of China during 2019-2020.

The evolution of seasonal influenza viruses, which can cause virus antigenic drift to escape human herd immunity, is a significant public health problem. Here, we obtained hemagglutinin (HA), neuraminidase (NA), and polymerase acidic protein (PA) the gene sequences of 84 influenza virus isolates collected in Guangdong Province during the 2019-2020 influenza season. Phylogenetic analyses revealed all these isolates were genetically similar to the viruses of clade 3C2a A1b, specifically those within subclades of A1b 137F (59 cases), A1b 186D (19 cases), and A1b 94N (6 cases). The influenza virus isolates were distinct from the World Health Organization recommended influenza A vaccine virus for the 2019-2020 Northern Hemisphere season (A/Kansas/14/2017; H3N2). Phylogenies inferred from the individual gene segment sequences revealed that one reassortment event occurred among these clades. The genetic variation involved mutations within viral antigenic epitopes and two N-glycosylation site alterations. The novel mutation sites of G202D and D206N in the HA gene, E344K in the NA gene, and K626R in the PA gene which may affect the spread of the virus were observed. We investigated the evolution of these genes and found that the HA and NA genes were under greater pressure than PA gene. Mutations associated with conferring resistance to NA inhibitors or baloxavir acid were not found. Our results suggest that a rapid evolution of the H3N2 influenza virus occurred, thus continuous monitoring is critical for establishing appropriate vaccine formulations or drug delivery for targeting influenza.

Novel reassortant of H9N2 avian influenza viruses isolated from chickens and quails in Egypt.

Background and Aim:Poultry infections with H9N2 avian influenza viruses (AIVs) are endemic in Egypt. This study determined the genetic changes in the sequences of H9N2 AIVs isolated from chicken and quails in Egypt, including determining genetic reassortment and detecting the main genetic changes in hemagglutinin (HA) and neuraminidase (NA) genes.Materials and Methods:Swab samples were collected from chicken and quails, examined through reverse transcription-polymerase chain reaction, and AIVs from positive samples were isolated in embryonated chicken eggs. Complete genome sequencing and phylogenetic analyses were conducted for two H9N2 AIV isolates, and sequences of HA and NA gene segments were analyzed in another two isolates.Results:A novel reassortant virus was identified from a commercial chicken flock (A/chicken/Egypt/374V/2016) and quails from a live bird market (A/quail/Egypt/1253V/2016). The reassortant viruses acquired four genome segments from the classic Egyptian H9N2 viruses (HA, NA, NP, and M) and four segments from Eurasian AIVs (PB2, PB1, PA, and NS). Many genetic changes have been demonstrated in HA and NA genes. The isolated novel reassortant H9N2 virus from quails showed amino acid mutations in the antigenic sites on the globular head of the mature HA monomer matched with the parent Egyptian H9N2 virus.Conclusion:This work described the genetic characterization of a novel reassortment of the H9N2 virus in Egypt. The emergence of new reassorted AIV viruses and genome variability raises the concern of an influenza pandemic with zoonotic potentials.

Molecular and Fitness Data Reveal Local Adaptation of Southern and Northern Estuarine Oysters (Crassostrea ariakensis)

Natural selection and genetic drift are both important for understanding the geographic distribution and environmental responses of marine species. In this study, we revealed distinct genetic variation in Crassostrea ariakensis by comparing the COI gene segment sequence in northern and southern oysters separated by the Yangtze River estuary. Reciprocal hybridization and intrapopulation crosses clarified their taxonomic status as the same species. There was no heterosis in the survival and growth of the hybrids, while the maternal effect was observed in larvae from eggs in the native habitat that showed higher fitness. Both the northern and southern F1 progenies exhibited positive performance in fitness traits, including survivorship, respiration rate, and growth, in their native habitats compared to that in their non-native habitats, indicating a strong signature of local adaptation. The oysters dwelling in the warm/southern habitats evolved a higher thermotolerance of LT50, while the oysters inhabiting the high-salinity/northern habitats had a 2.43‰ higher LS50 than that of their southern counterparts. After strong natural selection in the northern environments, the higher survival of the F1 progenies from the southern oysters under heat shock indicates an evolved genetic basis for its higher thermal tolerance. Strong environmental gradients, especially for temperature and salinity, and genetic drift of the interaction between coastal currents and the Yangtze River estuary potentially contribute to shaping the distribution pattern and adaptive divergence of C. ariakensis in China.

Replacement of TCR Dβ With Immunoglobulin DH DSP2.3 Imposes a Tyrosine-Enriched TCR Repertoire and Adversely Affects T Cell Development.

Enrichment for tyrosine in immunoglobulin CDR-H3 is due in large part to natural selection of germline immunoglobulin DH sequence. We have previously shown that when DH sequence is modified to reduce the contribution of tyrosine codons, epitope recognition is altered and B cell development, antibody production, autoantibody production, and morbidity and mortality following pathogen challenge are adversely affected. TCRβ diversity (Dβ) gene segment sequences are even more highly conserved than DH, with trout Dβ1 identical to human and mouse Dβ1. We hypothesized that natural selection of Dβ sequence also shapes CDR-B3 diversity and influences T cell development and T cell function. To test this, we used a mouse strain that lacked Dβ2 and contained a novel Dβ1 allele (DβYTL) that replaces Dβ1 with an immunoglobulin DH, DSP2.3. Unlike Dβ1, wherein glycine predominates in all three reading frames (RFs), in DSP2.3 there is enrichment for tyrosine in RF1, threonine in RF2, and leucine in RF3. Mature T cells using DβYTL expressed TCRs enriched at particular CDR-B3 positions for tyrosine but depleted of leucine. Changing Dβ sequence altered thymocyte and peripheral T cell numbers and the T cell response to an ovalbumin immunodominant epitope. The differences in tyrosine content might explain, at least in part, why TCRs are more polyspecific and of lower affinity for their cognate antigens than their immunoglobulin counterparts.

Preimmune Control of the Variance of TCR CDR-B3: Insights Gained From Germline Replacement of a TCR Dβ Gene Segment With an Ig D H Gene Segment.

We have previously shown that the sequence of the immunoglobulin diversity gene segment (DH) helps dictate the structure and composition of complementarity determining region 3 of the immunoglobulin heavy chain (CDR-H3). In order to test the role of germline D sequence on the diversity of the preimmune TCRβ repertoire of T cells, we generated a mouse with a mutant TCRβ DJC locus wherein the Dβ2-Jβ2 gene segment cluster was deleted and the remaining diversity gene segment, Dβ1 (IMGT:TRDB1), was replaced with DSP2.3 (IMGT:IGHD2-02), a commonly used B cell immunoglobulin DH gene segment. Crystallographic studies have shown that the length and thus structure of TCR CDR-B3 places amino acids at the tip of CDR-B3 in a position to directly interact with peptide bound to an MHC molecule. The length distribution of complementarity determining region 3 of the T cell receptor beta chain (CDR-B3) has been proposed to be restricted largely by MHC-specific selection, disfavoring CDR-B3 that are too long or too short. Here we show that the mechanism of control of CDR-B3 length depends on the Dβ sequence, which in turn dictates exonucleolytic nibbling. By contrast, the extent of N addition and the variance of created CDR3 lengths are regulated by the cell of origin, the thymocyte. We found that the sequence of the D and control of N addition collaborate to bias the distribution of CDR-B3 lengths in the pre-immune TCR repertoire and to focus the diversity provided by N addition and the sequence of the D on that portion of CDR-B3 that is most likely to interact with the peptide that is bound to the presenting MHC.

The sequences encoded by immunoglobulin diversity (DH ) gene segments play key roles in controlling B-cell development, antigen-binding site diversity, and antibody production.

Although at first glance the diversity of the immunoglobulin repertoire appears random, there are a number of mechanisms that act to constrain diversity. For example, key mechanisms controlling the diversity of the third complementarity determining region of the immunoglobulin heavy chain (CDR-H3) include natural selection of germline diversity (DH ) gene segment sequence and somatic selection upon passage through successive B-cell developmental checkpoints. To test the role of DH gene segment sequence, we generated a panel of mice limited to the use of a single germline or frameshifted DH gene segment. Specific individual amino acids within core DH gene segment sequence heavily influenced the absolute numbers of developing and mature B-cell subsets, antibody production, epitope recognition, protection against pathogen challenge, and susceptibility to the production of autoreactive antibodies. At the tip of the antigen-binding loop (PDB position 101) in CDR-H3, both natural (germline) and somatic selection favored tyrosine while disfavoring the presence of hydrophobic amino acids. Enrichment for arginine in CDR-H3 appeared to broaden recognition of epitopes of varying hydrophobicity, but led to diminished binding intensity and an increased likelihood of generating potentially pathogenic dsDNA-binding autoreactive antibodies. The phenotype of altering the sequence of the DH was recessive for T-independent antibody production, but dominant for T-cell-dependent responses. Our work suggests that the antibody repertoire is structured, with the sequence of individual DH selected by evolution to preferentially generate an apparently preferred category of antigen-binding sites. The result of this structured approach appears to be a repertoire that has been adapted, or optimized, to produce protective antibodies for a wide range of pathogen epitopes while reducing the likelihood of generating autoreactive specificities.

Phylogenetic and molecular analysis of highly pathogenic avian influenza H5N8 and H5N5 viruses detected in Poland in 2016-2017.

Sixty-five poultry outbreaks and sixty-eight events in wild birds were reported during the highly pathogenic H5N8/H5N5 avian influenza epidemic in Poland in 2016-2017. The analysis of all gene segment sequences of selected strains revealed cocirculation of at least four different genome configurations (genotypes) generated through reassortment of clade 2.3.4.4 H5N8 viruses detected in Russia and China in mid-2016. The geographical and temporal distribution of three H5N8 genotypes indicates separate introductions. Additionally, an H5N5 virus with a different gene configuration was detected in wild birds. The compilation of the results with those from studies on the virus' diversity in Germany, Italy and the Netherlands revealed that Europe was affected by at least eight different H5N8/H5N5 reassortants. Analysis of the HA gene sequence of a larger subset of samples showed its diversification corresponding to the genotype classification. The close relationship between poultry and wild bird strains from the same locations observed in several cases points to wild birds as the primary source of the outbreaks in poultry.

Influenza A virus recovery, diversity, and intercontinental exchange: A multi-year assessment of wild bird sampling at Izembek National Wildlife Refuge, Alaska.

Western Alaska is a potential point-of-entry for foreign-origin influenza A viruses (IAVs) into North America via migratory birds. We sampled waterfowl and gulls for IAVs at Izembek National Wildlife Refuge (NWR) in western Alaska, USA, during late summer and autumn months of 2011–2015, to evaluate the abundance and diversity of viruses at this site. We collected 4842 samples across five years from 25 species of wild birds resulting in the recovery, isolation, and sequencing of 172 IAVs. With the intent of optimizing sampling efficiencies, we used information derived from this multi-year effort to: 1) evaluate from which species we consistently recover viruses, 2) describe viral subtypes of isolates by host species and year, 3) characterize viral gene segment sequence diversity with respect to host species, and assess potential differences in the viral lineages among the host groups, and 4) examine how evidence of intercontinental exchange of IAVs relates to host species. We consistently recovered viruses from dabbling ducks (Anas spp.), emperor geese (Chen canagica) and glaucous-winged gulls (Larus glaucescens). There was little evidence for differences in viral subtypes and diversity from different waterfowl hosts, however subtypes and viral diversity varied between waterfowl host groups and glaucous-winged gulls. Furthermore, higher proportions of viral sequences from northern pintails (Anas acuta), emperor geese and glaucous-winged gulls were grouped in phylogenetic clades that included IAV sequences originating from wild birds sampled in Asia as compared to non-pintail dabbling ducks, a difference that may be related to intercontinental migratory tendencies of host species. Our summary of research and surveillance efforts at Izembek NWR will assist in future prioritization of which hosts to sample and swab types to collect in Alaska and elsewhere in order to maximize isolate recovery, subtype and sequence diversity for resultant viruses, and detection of evidence for intercontinental viral exchange.

Evidence for wild waterfowl origin of H7N3 influenza A virus detected in captive-reared New Jersey pheasants.

In August 2014, a low-pathogenic H7N3 influenza A virus was isolated from pheasants at a New Jersey gamebird farm and hunting preserve. In this study, we use phylogenetic analyses and calculations of genetic similarity to gain inference into the genetic ancestry of this virus and to identify potential routes of transmission. Results of maximum-likelihood (ML) and maximum-clade-credibility (MCC) phylogenetic analyses provide evidence that A/pheasant/New Jersey/26996-2/2014 (H7N3) had closely related H7 hemagglutinin (HA) and N3 neuraminidase (NA) gene segments as compared to influenza A viruses circulating among wild waterfowl in the central and eastern USA. The estimated time of the most recent common ancestry (TMRCA) between the pheasant virus and those most closely related from wild waterfowl was early 2013 for both the H7 HA and N3 NA gene segments. None of the viruses from waterfowl identified as being most closely related to A/pheasant/New Jersey/26996-2/2014 at the HA and NA gene segments in ML and MCC phylogenetic analyses shared ≥99% nucleotide sequence identity for internal gene segment sequences. This result indicates that specific viral strains identified in this study as being closely related to the HA and NA gene segments of A/pheasant/New Jersey/26996-2/2014 were not the direct predecessors of the etiological agent identified during the New Jersey outbreak. However, the recent common ancestry of the H7 and N3 gene segments of waterfowl-origin viruses and the virus isolated from pheasants suggests that viral diversity maintained in wild waterfowl likely played an important role in the emergence of A/pheasant/New Jersey/26996-2/2014.

Whole genome characterization of human influenza A(H1N1)pdm09 viruses isolated from Kenya during the 2009 pandemic

An influenza pandemic caused by a novel influenza virus A(H1N1)pdm09 spread worldwide in 2009 and is estimated to have caused between 151,700 and 575,400 deaths globally. While whole genome data on new virus enables a deeper insight in the pathogenesis, epidemiology, and drug sensitivities of the circulating viruses, there are relatively limited complete genetic sequences available for this virus from African countries. We describe herein the full genome analysis of influenza A(H1N1)pdm09 viruses isolated in Kenya between June 2009 and August 2010. A total of 40 influenza A(H1N1)pdm09 viruses isolated during the pandemic were selected. The segments from each isolate were amplified and directly sequenced. The resulting sequences of individual gene segments were concatenated and used for subsequent analysis. These were used to infer phylogenetic relationships and also to reconstruct the time of most recent ancestor, time of introduction into the country, rates of substitution and to estimate a time-resolved phylogeny. The Kenyan complete genome sequences clustered with globally distributed clade 2 and clade 7 sequences but local clade 2 viruses did not circulate beyond the introductory foci while clade 7 viruses disseminated country wide. The time of the most recent common ancestor was estimated between April and June 2009, and distinct clusters circulated during the pandemic. The complete genome had an estimated rate of nucleotide substitution of 4.9×10−3 substitutions/site/year and greater diversity in surface expressed proteins was observed. We show that two clades of influenza A(H1N1)pdm09 virus were introduced into Kenya from the UK and the pandemic was sustained as a result of importations. Several closely related but distinct clusters co-circulated locally during the peak pandemic phase but only one cluster dominated in the late phase of the pandemic suggesting that it possessed greater adaptability.

HIV-1 gp140 epitope recognition is influenced by immunoglobulin DH gene segment sequence.

Complementarity Determining Region 3 of the immunoglobulin (Ig) H chain (CDR-H3) lies at the center of the antigen-binding site where it often plays a decisive role in antigen recognition and binding. Amino acids encoded by the diversity (DH) gene segment are the main component of CDR-H3. Each DH has the potential to rearrange into one of six DH reading frames (RFs), each of which exhibits a characteristic amino acid hydrophobicity signature that has been conserved among jawed vertebrates by natural selection. A preference for use of RF1 promotes the incorporation of tyrosine into CDR-H3 while suppressing the inclusion of hydrophobic or charged amino acids. To test the hypothesis that these evolutionary constraints on DH sequence influence epitope recognition, we used mice with a single DH that has been altered to preferentially use RF2 or inverted RF1. B cells in these mice produce a CDR-H3 repertoire that is enriched for valine or arginine in place of tyrosine. We serially immunized this panel of mice with gp140 from HIV-1 JR-FL isolate and then used enzyme-linked immunosorbent assay (ELISA) or peptide microarray to assess antibody binding to key or overlapping HIV-1 envelope epitopes. By ELISA, serum reactivity to key epitopes varied by DH sequence. By microarray, sera with Ig CDR-H3s enriched for arginine bound to linear peptides with a greater range of hydrophobicity but had a lower intensity of binding than sera containing Ig CDR-H3s enriched for tyrosine or valine. We conclude that patterns of epitope recognition and binding can be heavily influenced by DH germ line sequence. This may help explain why antibodies in HIV-infected patients must undergo extensive somatic mutation in order to bind to specific viral epitopes and achieve neutralization.

Interim estimates of divergence date and vaccine strain match of human influenza A(H3N2) virus from systematic influenza surveillance (2010-2015) in Hangzhou, southeast of China.

In the post-pandemic period 2010-2015, seasonal influenza A(H3N2) virus predominated in Hangzhou, southeast of China, with an increased activity and semi-annual seasons. This study utilized HA virus gene segment sequences to analyze the divergence date and vaccine strain match of human influenza A(H3N2) virus from systematic influenza surveillance in Hangzhou. Virological and serological analyses of 124 representative A(H3N2) viruses from prospective studies of systematic surveillance samples were conducted to quantify the genetic and antigenic characteristics and their vaccine strain match. Bayesian phylogenetic inference showed that two separate subgroups 3C.3 and 3C.2 probably diverged from group 3C in early 2012 and then evolved into groups 3C.3a and 3C.2a, respectively, in the 2014/15 influenza season. Furthermore, high amino acid substitution rates of the HA1 subunit were found in A(H3N2) group 3C.2a variants, indicating that increased antigenic drift of A(H3N2) group 3C.2a virus is associated with a vaccine mismatch to the 2015/16 vaccine reference strain Switzerland/9715293/2013 (group 3C.3a). A portion of the group 3C.2a isolates are not covered by the current A(H3N2) vaccine strain. These findings offer insights into the emergence of group 3C.2a variants with epidemic potential in the imminent influenza seasons.

Itaya virus, a Novel Orthobunyavirus Associated with Human Febrile Illness, Peru.

Our genetic analyses of uncharacterized bunyaviruses isolated in Peru identified a possible reassortant virus containing small and large gene segment sequences closely related to the Caraparu virus and a medium gene segment sequence potentially derived from an unidentified group C orthobunyavirus. Neutralization tests confirmed serologic distinction among the newly identified virus and the prototype and Caraparu strains. This virus, named Itaya, was isolated in 1999 and 2006 from febrile patients in the cities of Iquitos and Yurimaguas in Peru. The geographic distance between the 2 cases suggests that the Itaya virus could be widely distributed throughout the Amazon basin in northeastern Peru. Identification of a new Orthobunyavirus species that causes febrile disease in humans reinforces the need to expand viral disease surveillance in tropical regions of South America.

Violation of an evolutionarily conserved immunoglobulin diversity gene sequence preference promotes production of dsDNA-specific IgG antibodies.

Variability in the developing antibody repertoire is focused on the third complementarity determining region of the H chain (CDR-H3), which lies at the center of the antigen binding site where it often plays a decisive role in antigen binding. The power of VDJ recombination and N nucleotide addition has led to the common conception that the sequence of CDR-H3 is unrestricted in its variability and random in its composition. Under this view, the immune response is solely controlled by somatic positive and negative clonal selection mechanisms that act on individual B cells to promote production of protective antibodies and prevent the production of self-reactive antibodies. This concept of a repertoire of random antigen binding sites is inconsistent with the observation that diversity (DH) gene segment sequence content by reading frame (RF) is evolutionarily conserved, creating biases in the prevalence and distribution of individual amino acids in CDR-H3. For example, arginine, which is often found in the CDR-H3 of dsDNA binding autoantibodies, is under-represented in the commonly used DH RFs rearranged by deletion, but is a frequent component of rarely used inverted RF1 (iRF1), which is rearranged by inversion. To determine the effect of altering this germline bias in DH gene segment sequence on autoantibody production, we generated mice that by genetic manipulation are forced to utilize an iRF1 sequence encoding two arginines. Over a one year period we collected serial serum samples from these unimmunized, specific pathogen-free mice and found that more than one-fifth of them contained elevated levels of dsDNA-binding IgG, but not IgM; whereas mice with a wild type DH sequence did not. Thus, germline bias against the use of arginine enriched DH sequence helps to reduce the likelihood of producing self-reactive antibodies.

Long-term surveillance of H7 influenza viruses in American wild aquatic birds: are the H7N3 influenza viruses in wild birds the precursors of highly pathogenic strains in domestic poultry?

The emergence of influenza A virus (IAV) in domestic avian species and associated transmissions to mammals is unpredictable. In the Americas, the H7 IAVs are of particular concern, and there have been four separate outbreaks of highly pathogenic (HP) H7N3 in domestic poultry in North and South America between 2002 and 2012, with occasional spillover into humans. Here, we use long-term IAV surveillance in North American shorebirds at Delaware Bay, USA, from 1985 to 2012 and in ducks in Alberta, Canada, from 1976 to 2012 to determine which hemagglutinin (HA)–neuraminidase (NA) combinations predominated in Anseriformes (ducks) and Charadriiformes (shorebirds) and whether there is concordance between peaks of H7 prevalence and transmission in wild aquatic birds and the emergence of H7 IAVs in poultry and humans. Whole-genome sequencing supported phylogenetic and genomic constellation analyses to determine whether HP IAVs emerge in the context of specific internal gene segment sequences. Phylogenetic analysis of whole-genome sequences of the H7N3 influenza viruses from wild birds and HP H7N3 outbreaks in the Americas indicate that each HP outbreak was an independent emergence event and that the low pathogenic (LP) avian influenza precursors were most likely from dabbling ducks. The different polybasic cleavage sites in the four HP outbreaks support independent origins. At the 95% nucleotide percent identity-level phylogenetic analysis showed that the wild duck HA, PB1, and M sequences clustered with the poultry and human outbreak sequences. The genomic constellation analysis strongly suggests that gene segments/virus flow from wild birds to domestic poultry.Emerging Microbes and Infections (2015) 4, e35; doi:10.1038/emi.2015.35; published online 17 June 2015

DH reading frame usage influences HIV-1 epitope recognition (MPF2P.804)

Abstract Background: Some HIV-1-specific neutralizing antibodies, such as 2F5 and 4E10, contain a long H chain complementarity determining region 3 (CDR-H3) includes patches of hydrophobic and charged amino acids, but B cells bearing Igs with charged or hydrophobic CDR-H3s are normally culled from mature B cell subsets. Thus, DH influence on neutralizing antibody generation is very worth to explore. Methods: Mice-BALB/c mice cohort limited to the use of single DH gene segments were generated. ΔD-DFL is a human-like repertoire control. ΔD-DµFS promotes the use of hydrophobic amino acid repertoire. The ΔD-iD is charged amino acid enriched repertoire. Immunization-each strain of 10 mice was immunized with HIV-1 JR-FL gp140 protein. Epitope Identification, serum Assay were conducted. Results: As a general rule, the heterogeneity of the anti-JR-FL gp140 response varied by DH genotype with ΔD-DFL>Wild Type (WT)>>ΔD-DμFS, ΔD-iD. Conversely, the intensity of the response was greatest in the ΔD-iD. However, linear response was not identified with the response to natural HIV-1 epitope. Anti-JR-FL gp140 antibodies present in the sera of the DH altered did not block HIV-1 envelope CD4, 2F5, 2G12 binding site. Conclusion: The pattern of epitope recognition and antigen binding in the response to T dependent antigen, in part, on DH gene segment sequence. Restrictions imposed by natural selection of DH sequence may underlie some of the difficulty that patients generate neutralizing antibody.

Molecular evolution of H5N1 highly pathogenic avian influenza viruses in Bangladesh between 2007 and 2012

In Bangladesh, highly pathogenic avian influenza (HPAI) virus subtype H5N1 was first detected in February 2007. Since then the virus has become entrenched in poultry farms of Bangladesh. There have so far been seven human cases of H5N1 HPAI infection in Bangladesh with one death. The objective of the present study was to investigate the molecular evolution of H5N1 HPAI viruses during 2007 to 2012. Partial or complete nucleotide sequences of all eight gene segments of two chicken isolates, five gene segments of a duck isolate and the haemagglutinin gene segment of 18 isolates from Bangladesh were established in the present study and subjected to molecular analysis. In addition, full-length sequences of different gene segments of other Bangladeshi H5N1 isolates available in GenBank were included in the analysis. The analysis revealed that the first introduction of clade 2.2 virus in Bangladesh in 2007 was followed by the introduction of clade 2.3.2.1 and 2.3.4 viruses in 2011. However, only clade 2.3.2.1 viruses could be isolated in 2012, indicating progressive replacement of clade 2.2 and 2.3.4 viruses. There has been an event of segment re-assortment between H5N1 and H9N2 viruses in Bangladesh, where H5N1 virus acquired the PB1 gene from a H9N2 virus. Point mutations have accumulated in Bangladeshi isolates over the last 5 years with potential modification of receptor binding site and antigenic sites. Extensive and continuous molecular epidemiological studies are necessary to monitor the evolution of circulating avian influenza viruses in Bangladesh.

The link between antibodies to OxLDL and natural protection against pneumococci depends on DH gene conservation

Selection and physiological production of protective natural antibodies (NAbs) have been associated with exposure to endogenous antigens. The extent to which this association depends on germline NAb sequence is uncertain. Here we show that alterations in germline D(H) sequence can sever the association between the production of self-reactive NAbs and NAbs that afford protection against a pathogen. In unmanipulated hosts, the availability of the evolutionarily conserved DFL16.1 gene segment sequence profoundly affected the serum levels of NAbs against bacterial phosphorylcholine but not oxidized low-density lipoprotein. Mice with partially altered DFL16.1 sequence could use N nucleotides to recreate the amino acid sequence associated with the classical protective T15 idiotype–positive NAbs, whereas those without DFL16.1 could not. DFL16.1 gene-deficient mice proved more susceptible to challenge with live Streptococcus pneumoniae. Our findings indicate that although production of self-reactive NAbs can be independent of germline D(H) sequence, their capacity to provide protection against pathogens cannot. The potential relevance of these findings for the rational design of vaccines is discussed.