Amino Acid Sequence Of Region Research Articles

Position 22 of the V3 loop is associated with HIV infectivity.

Human immunodeficiency virus subtype 1B (HIV-1B) binds to the CD4 receptor and co-receptor CCR5 or CXCR4 to enter T lymphocytes. The amino acid sequence of the HIV envelope glycoprotein V3 region determines the co-receptor tropism, thereby influencing the infectivity of the virus. Our research group previously found that the amino acid at position 22 of the V3 region may affect the infectivity of the virus, and in this study, we tested this hypothesis. We constructed pseudoviruses by changing the amino acids at position 22 of the V3 region in CCR5-tropic and CXCR4-tropic viruses and tested their infectivity. When the amino acid at V3 position 22 was altered in the CCR5- and CXCR4-tropic viruses, their ability to infect cells decreased to 20.6% and 17.14%, respectively. Therefore, we propose that residue 22 in the V3 region of subtype HIV-1B significantly influences the infectivity of the virus.

Generation of Long-Lived Bone Marrow Plasma Cells Secreting Antibodies Specific for the HIV-1 gp41 Membrane-Proximal External Region in the Absence of Polyreactivity.

ABSTRACTAn effective preventive vaccine is highly sought after in order to stem the current HIV-1 pandemic. Both conservation of contiguous gp41 membrane-proximal external region (MPER) amino acid sequences across HIV-1 clades and the ability of anti-MPER broadly neutralizing antibodies (BNAbs) to block viral hemifusion/fusion establish the MPER as a prime vaccination target. In earlier studies, we described the development of an MPER vaccine formulation that takes advantage of liposomes to array the MPER on a lipid bilayer surface, paralleling its native configuration on the virus membrane while also incorporating molecular adjuvant and CD4 T cell epitope cargo. Here we demonstrate that several immunizations with MPER/liposomes induce high levels of bone marrow long-lived plasma cell (LLPC) antibody production. Single-cell immunoglobulin gene retrieval analysis shows that these plasma cells are derived from a germ line repertoire of B cells with a diverse representation of immunoglobulin genes, exhibiting antigen-driven positive selection. Characterization of LLPC recombinant monoclonal antibodies (rMAbs) indicates that antigen recognition is achieved through convergence on a common epitopic focus by utilizing various complementarity-determining region H3 (CDRH3) lengths. Importantly, the vast majority of rMAbs produced from these cells lack polyreactivity yet manifest antigen specificity in the context of lipids, shaping MPER-specific paratopes through selective pressure. Taken together, these findings demonstrate that the MPER is a vaccine target with minimal risk of generating off-target autoimmunity.IMPORTANCE A useful vaccine must generate desired long-term, antigen-specific antibody responses devoid of polyreactivity or autoreactivity. The common polyreactive features of some HIV-1 BNAbs have raised concern about elicitation of anti-MPER antibodies. Utilizing single-LLPC repertoire analysis and biophysical characterization of anti-MPER rMAbs, we show that their fine specificities require a structural fitness of the antibody combining site involving heavy and light chain variable domains shaped by somatic hypermutation and affinity maturation of B cells in the germinal center. Perhaps more importantly, our results demonstrate that the majority of MPER-specific antibodies are not inherently polyspecific and/or autoreactive, suggesting that polyreactivity of MPER-specific antibodies is separable from their antigen specificity.

Chaperonin-containing T-complex Protein 1 Subunit ζ Serves as an Autoantigen Recognized by Human Vδ2 γδ T Cells in Autoimmune Diseases

Human γδ T cells recognize conserved endogenous and stress-induced antigens typically associated with autoimmune diseases. However, the role of γδ T cells in autoimmune diseases is not clear. Few autoimmune disease-related antigens recognized by T cell receptor (TCR) γδ have been defined. In this study, we compared Vδ2 TCR complementarity-determining region 3 (CDR3) between systemic lupus erythematosus (SLE) patients and healthy donors. Results show that CDR3 length distribution differed significantly and displayed oligoclonal characteristics in SLE patients when compared with healthy donors. We found no difference in the frequency of Jδ gene fragment usage between these two groups. According to the dominant CDR3δ sequences in SLE patients, synthesized SL2 peptides specifically bound to human renal proximal tubular epithelial cell line HK-2; SL2-Vm, a mutant V sequence of SL2, did not bind. We identified the putative protein ligand chaperonin-containing T-complex protein 1 subunit ζ (CCT6A) using SL2 as a probe in HK-2 cell protein extracts by affinity chromatography and liquid chromatography-electrospray ionization-tandem mass spectrometry analysis. We found CCT6A expression on the surface of HK-2 cells. Cytotoxicity of only Vδ2 γδ T cells to HK-2 cells was blocked by anti-CCT6A antibody. Finally, we note that CCT6A concentration was significantly increased in plasma of SLE and rheumatoid arthritis patients. These data suggest that CCT6A is a novel autoantigen recognized by Vδ2 γδ T cells, which deepens our understanding of mechanisms in autoimmune diseases.

Molecular characterization of two rare human G8P[14] rotavirus strains, detected in Italy in 2012

Since 2007, the Italian Rotavirus Surveillance Program (RotaNet-Italy) has monitored the diversity and distribution of genotypes identified in children hospitalized with rotavirus acute gastroenteritis.We report the genomic characterization of two rare human G8P[14] rotavirus strains, identified in two children hospitalized with acute gastroenteritis in the southern Italian region of Apulia during rotavirus strain surveillance in 2012.Both strains showed a G8-P[14]-I2-R2-C2-M2-A11-N2-T6-E2-H3 genomic constellation (DS-1-like genomic background). Phylogenetic analysis of each genome segment revealed a mixed configuration of genes of animal and zoonotic human origin, indicating that genetic reassortment events generated these unusual human strains. Eight out of 11 genes (VP1, VP2, VP3, VP6, VP7, NSP3, NSP4 and NSP5) of the Italian G8P[14] strains exhibited close identity with a Spanish sheep strain, whereas the remaining genes (VP4, NSP1 and NSP2) were more closely related to human strains. The amino acid sequences of the antigenic regions of outer capsid proteins VP4 and VP7 were compared with vaccine and field strains, showing high conservation between the amino acid sequences of Apulia G8P[14] strains and human and animal strains bearing G8 and/or P[14] proteins, and revealing many substitutions with respect to the RotaTeq™ and Rotarix™ vaccine strains. Conversely, the amino acid analysis of the four antigenic sites of VP6 revealed a high degree of conservation between the two Apulia strains and the human and animal strains analyzed.These results reinforce the potential role of interspecies transmission and reassortment in generating novel rotavirus strains that might not be fully contrasted by current vaccines.

Analysis of polymorphism of Pfhrp2 gene in Plasmodium falciparum from falciparum malaria patients in Yunnan Province

To analyze the polymorphism of histidine rich protein 2 (HRP II) gene in Plasmodium falciparum (Pfhrp2) from falciparum malaria patients in Yunnan Province, so as to lay the foundation for studying the defection of antigen genes of Plasmodium. The filter paper blood samples and related information of falciparum malaria cases reported were obtained in Yunnan Province from August 2012 to September 2015. Under the guidance of the specific primers, the exon2 regions in Pfhrp2 gene in P. falciparum from DNA samples were amplified by PCR, and the PCR products were sequenced. The sequences of exon2 region in Pfhrp2 gene were blasted by comparing with the reference sequences AY816237, AY816240, and AY816301. Next, the polymorphism of the sequence in exon2 region of Pfhrp2 gene was analyzed by MEGA 5.04 software. The conserved sites and genetic distances between sequences were calculated by using the software as well, and the clustering tree was drawn according to the genetic distances between the amino acid sequences. A total of 218 bloods samples from the falciparum malaria cases in 15 prefectures of Yunnan Province were collected, and the sources of infection included Yunnan, Africa and Myanmar. The PCR results showed that the exon2 regions in Pfhrp2 genes of 155 samples were positive by amplification and their products were sequenced successfully. The sequence analysis showed that the length range of the amino acid residues of exon2 region in Pfhrp2 gene was from 115 aa to 298 aa, the average length was 239.7 aa. There was no statistically significance among the means of the amino acid residues of the isolates from Africa (239.9 aa), Myanmar (239.5 aa) and Yunnan (241.6 aa) (F = 0.025, P > 0.05). All the 155 amino acid sequences ended with type 12 repeat, 98.1% (152/155) of them started with type 1 repeat and 1.9% (3/155) of them started with type 2. Type 2 presented most frequently repeat in all the sequences and the average repeat times were 12.9. The homologous locus of the DNA sequences in exon2 regions of the 155 Pfhrp2 genes was 894 bp, among which the conservative sites accounted for 20.6% (186/894), and the variable sites for 78.2% (699/ 894). The genetic distances between the sequences of Africa isolates ranged from 0 to 0.741, and those of the Myanmar and Yunnan isolates were 0-0.948 and 0-0.750, respectively. The cluster analysis showed that all the 155 sequences clustered into 3 categories on genetic distances between amino acid sequences according to the size of the amino acid sequence length. At the same level, the sequences had approximate lengths and amino acid repeat types. The sequence of exon2 region in Pfhrp2 gene of P. falciparum from falciparum malaria cases in Yunnan Province is highly polymorphic, the P. falciparum isolates are clustered mainly according to the size of the amino acid sequence of exon2 region in Pfhrp2 gene.

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin.

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

Identification of a New Genotype of African Swine Fever Virus in Domestic Pigs from Ethiopia.

African swine fever (ASF) is an important emerging transboundary animal disease (TAD), which currently has an impact on many countries in Africa, Eastern Europe, the Caucasus and the Russian Federation. The current situation in Europe shows the ability of the virus to rapidly spread, which stands to threaten the global swine industry. At present, there is no viable vaccine to minimize spread of the disease and stamping out is the main source of control. In February 2011, Ethiopia had reported its first suspected outbreaks of ASF. Genomic analyses of the collected ASF virus (ASFV) strains were undertaken using 23 tissue samples collected from domestic swine in Ethiopia from 2011 to 2014. The analysis of Ethiopian ASFVs partial p72 gene sequence showed the identification of a new genotype, genotype XXIII, that shares a common ancestor with genotypes IX and X, which comprise isolates circulating in Eastern African countries and the Republic of Congo. Analysis of the p54 gene also followed the p72 pattern and the deduced amino acid sequence of the central variable region (CVR) of the B602L gene showed novel tetramer repeats not previously characterized.

A comparative analysis of the ‘other roles’ of transcriptional factors from pathogenic organisms

Transcription factors are the proteins that regulate gene expressions by binding to the promoter DNA regions of the corresponding genes. There are a number of different transcription factors and all of them have DNA-binding signature sequences. Transcription factors are structurally classified as belonging to different families on the basis of the distribution of their secondary structural patterns. The amino acid sequences of the DNA-binding regions of the transcription factors belonging to the same family should therefore be identical. But careful analyses of these sequences reveal the presence of different mutations in them. On further analyses, the mutations are found to create new domains in the transcription factors thereby conferring them with some new functionality in addition to their regulatory roles. Here, an attempt has been made to analyze the mutations present in the transcription factors of pathogenic organisms. The possible effects of these mutations have been identified and correlated with the mechanisms of disease pathogenesis. So far this is the first report that predicts the presence of the new functionality of the transcription factors, which also can augment disease propagation by the pathogens. This analysis would therefore be beneficial to future genetic studies to identify the effects of the mutations in the transcription factors for disease propagation.

Helicobacter pylori infection and gastric carcinoma: Not all the strains and patients are alike.

Gastric carcinoma (GC) develops in only 1%-3% of Helicobacter pylori (H. pylori) infected people. The role in GC formation of the bacterial genotypes, gene polymorphisms and host's factors may therefore be important. The risk of GC is enhanced when individuals are infected by strains expressing the oncoprotein CagA, in particular if CagA has a high number of repeats containing the EPIYA sequence in its C'-terminal variable region or particular amino acid sequences flank the EPIYA motifs. H. pylori infection triggers an inflammatory response characterised by an increased secretion of some chemokines by immunocytes and colonised gastric epithelial cells; these molecules are especially constituted by proteins composing the interleukin-1beta (IL-1β) group and tumour necrosis factor-alpha (TNF-α). Polymorphisms in the promoter regions of genes encoding these molecules, could account for high concentrations of IL-1β and TNF-α in the gastric mucosa, which may cause hypochlorhydria and eventually GC. Inconsistent results have been attained with other haplotypes of inflammatory and anti-inflammatory cytokines. Genomic mechanisms of GC development are mainly based on chromosomal or microsatellite instability (MSI) and deregulation of signalling transduction pathways. H. pylori infection may induce DNA instability and breaks of double-strand DNA in gastric mucocytes. Different H. pylori strains seem to differently increase the risk of cancer development run by the host. Certain H. pylori genotypes (such as the cagA positive) induce high degrees of chronic inflammation and determine an increase of mutagenesis rate, oxidative-stress, mismatch repair mechanisms, down-regulation of base excision and genetic instability, as well as generation of reactive oxygen species that modulate apoptosis; these phenomena may end to trigger or concur to GC development.

Composition and variation analysis of the TCR β-chain CDR3 repertoire in systemic lupus erythematosus using high-throughput sequencing.

The ability of T lymphocytes to mount an immune response against a diverse array of pathogens is primarily conveyed by the amino acid (aa) sequence of the hypervariable complementarity-determining region 3 (CDR3) segments of the T cell receptor (TCR). In this study, we used a combination of multiplex-PCR, Illumina sequencing and IMGT/HighV-QUEST for a standardized analysis of the characteristics and polymorphisms of the T-cell receptor BV complementarity-determining region 3 (TCR BV CDR3) gene in peripheral blood mononuclear cells (PBMCs) from SLE patients and healthy donors (NC). We found the distributions of CDR3, VD indel, and DJ indel lengths to be comparable between the SLE and NC groups. The degree of clonal expansion in the SLE group was significantly greater than in the NC group, and the expression levels of 10 TRβV segments and 6 TRβJ segments were also significantly different in the SLE group. Regarding public T cell responses, 3CDR3 DNA sequences and 4 aa sequences were shared by all SLE patients and may serve as biomarkers for SLE disease risk, diagnosis and/or prognosis.

English

A full-length cDNA encoding the immunoglobulin (IgM) heavy chain gene of Nile tilapia was successfully cloned using the 5’ and 3’ RACE techniques. The complete cDNA of the Nile tilapia IgM heavy chain gene is 1,921 bp in length and has an open reading frame (ORF) of 1,740 bp, which corresponds to 580 amino acid residues. The deduced amino acid sequence of the Nile tilapia IgM heavy chain includes a typical secretory IgM heavy chain designated On and a variable region that is connected to 4 constant regions to form the L H -V H -C µ 1-C µ 2-C µ 3-C µ 4 pattern. Comparisons of the nucleotide and amino acid sequences of On -sIgM with IgM heavy chains of other organisms showed the highest similarity scores of 62.6 and 55.4%, respectively, to the orange-spotted grouper ( Epinephelus coioides ). Structural analysis of 126 cDNAs encoding variable domains of the IgM heavy chain revealed that at least 9 V H families, 6 D H segments and 4 J H families were utilized using several mechanisms to generate the repertoire of antigen-binding domains. Variation analysis of the variable domains indicated that the amino acid sequences of the framework regions (FRs) were less variable than those of the complementarity determining regions (CDRs), among which the most variable was CDR3. Tissue expression profile analysis using quantitative real-time RT-PCR of healthy Nile tilapia showed that the IgM heavy chain gene was ubiquitously expressed in all 13 tested tissues, but the highest expression level was observed in the head kidney, followed by the spleen, intestine and peripheral blood leukocytes (PBLs). Furthermore, Southern blot analysis of the constant region of the IgM heavy chain gene of 3 different fishes indicated that Nile tilapia genomes may contain 2 copies of the IgM gene. Keywords: Nile Tilapia, IgM Heavy Chain, Variable Region, Diversity, Secreted Form, Southern Blot

Molecular cloning and characterization of presenilin gene in Bombyx mori.

Presenilin (PS), the catalytic core of the γ-secretase complex, is considered to be a causative protein of the early‑onset familial form of Alzheimer's disease. Aging is a risk factor for Alzheimer's disease and a number of genetic studies have utilized Bombyx mori (B. mori) as a model, making it possible to use B. mori to investigate Alzheimer's disease. However, the homologous gene of human PS in B. mori has remained to be elucidated. In the present study, the PS homologue gene in B. mori was identified and characterized, and six B. mori presenilin (BmPS) mRNA transcripts were generated by selecting multiple transcription start sites and/or alternative splice sites. The longest mRNA of BmPS (termed BmPS1) contains a 153 nt 5' untranslated region (UTR), a 1,440 nt open reading frame and a 1,063 nt 3' UTR. The predicted protein of BmPS1 consists of 479 amino acid residues and has two highly‑conserved aspartate residues, which form the catalytic core of aspartic proteases. It exhibits a sequence identity of ~44 and 51% with homologues in Homo sapiens and Drosophila melanogaster, respectively. However, the amino acid sequence of the BmPS loop region does not completely match between the two B. mori strains R13Q and Dazao. Genomic analysis revealed that B. mori had a single copy of the BmPS gene, which was composed of 14 exons. A total of four isoforms of BmPS (BmPS‑A, ‑B, ‑C and ‑D) owing to multiple transcriptional start sites and alternative splice sites were identified. The alternative splicing events occurring in the loop region improved the diversity of the BmPS protein and were detectable in all tissues, as determined using reverse transcription quantitative polymerase chain reaction (RT‑qPCR). Furthermore, the expression levels of BmPS in the brain at different developmental stages were detected using RT‑qPCR, and significantly higher expression levels of BmPS were found in the adult stage compared with those in the larval and pupal stages. The present study on BmPS provided insight into the pathogenesis of Alzheimer's disease and mechanisms of silkworm developmental regulation.

Detection and molecular characterization of the Iris severe mosaic virus-Ir isolate from Iran

AbstractIris belongs to the Iridaceae family. It is one of the most important pharmaceutical and ornamental plants in the world. To assess the potyvirus incidence in natural resources of iris plants in Iran, Antigen Coated-Plate ELISA (ACP-ELISA) was performed on 490 symptomatic rhizomatous iris leaf samples, which detected the potyvirus in 36.7% of the samples. Genomic 3′ end of one mechanically non-transmitted potyvirus isolate, comprising a 3′ untranslated region (390 bp) and C-terminus of the coat protein (CP) gene (459 bp), was amplified by reverse transcription polymerase chain reaction (RT-PCR), which was ligated into pTG19-T vector. The nucleotide sequence of amplicons was compared with related sequences, using Blastn software available at NCBI GenBank, and showed the highest similarity withIris severe mosaic virus(ISMV) isolates. The nucleotide and deduced amino acid sequence of the CP C-terminus region was more than 83% identical with other ISMV isolates, therefore this isolate was designated as ISMV-Ir. This new ISMV isolate is closely related to the Chinese ISMV-PHz in phylogenetic analysis, based on the partial nucleotide and deduced amino acid sequence of the CP region. This is the first report of ISMV occurrence onIrisspp. in Iran.

Unexpected spreading of G12P[8] rotavirus strains among young children in a small area of central Italy.

Rotavirus gastroenteritis is associated mainly with the five genotypes G1,3,4,9P[8] and G2P[4] that are common worldwide, but emerging strains including G6, G8, and G12 are also reported sporadically. G12P[8] rotavirus was observed unexpectedly to spread in a limited area of Italy during the rotavirus surveillance season 2012-2013. All strains were genotyped for VP7 and VP4 and subjected to phylogenetic analysis. Amino acid sequences of antigenic regions were compared with vaccine and field strains. G12P[8] strains were detected in the stools of 52 of 69 (75%) children infected with rotavirus in the central Italian region of Umbria. All G12 strains belonged to lineage III, and presented the P[8] genotype. Sequence analysis showed close nucleotide identity of both VP4 and VP7 genes among Umbria G12P[8] strains. The VP7 gene was also similar to other G12 strains circulating in different years and countries, and the VP4 gene was closely related to other local and global P[8] strains possessing different G-types. Overall findings suggest either the introduction and evolution of a G12 VP7 gene into the local Wa-like rotavirus population or the spreading of a strain novel for the area. Comparison of the VP8* and VP7 antigenic regions showed high conservation between the amino acid sequences of Umbria G12P[8] strains, and revealed various substitutions in the VP8* antigenic regions between the Italian G12P[8] strains and RotaTeq™ and Rotarix™ vaccine strains. The sudden and unexpected emergence of G12P[8] rotavirus confirms that these strains have the potential to become a sixth common genotype across the world.

Quantitative assessment of the robustness of next-generation sequencing of antibody variable gene repertoires from immunized mice.

BackgroundNext-generation sequencing (NGS) of antibody variable regions has emerged as a powerful tool in systems immunology by providing quantitative molecular information on polyclonal humoral immune responses. Reproducible and robust information on antibody repertoires is valuable for basic and applied immunology studies: thus, it is essential to establish the reliability of antibody NGS data.ResultsWe isolated RNA from antibody-secreting cells (ASCs) from either 1 mouse or a pool of 9 immunized mice in order to simulate both normal and high diversity populations. Next, we prepared three technical replicates of antibody libraries by RT-PCR from each diversity scenario, which were sequenced using the Illumina MiSeq platform resulting in >106 250 bp paired-end reads per replicate. We then assessed the robustness of antibody repertoire data based on clonal identification defined by amino acid sequence of either full-length VDJ region or the complementarity determining region 3 (CDR3). Leveraging modeling approaches adapted from mathematical ecology, we found that in either diversity scenario both CDR3 and VDJ detection nears completeness indicating deep coverage of ASC repertoires. Additionally, we defined reliability thresholds for accurate quantification and ranking of CDR3s and VDJs. Importantly, we show that both factors–(i) replicate sequencing and (ii) sequencing depth–are crucial for robust CDR3 and VDJ detection and ranking.ConclusionsIn summary, we established widely applicable experimental and computational guidelines for robust antibody NGS and analysis, which will help advance systems immunology studies related to the quantitative profiling of antibody responses following infection and vaccination.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-014-0040-5) contains supplementary material, which is available to authorized users.

Subjects with chronic lymphocytic leukaemia-like B-cell clones with stereotyped B-cell receptors frequently show MDS-associated phenotypes on myeloid cells.

An increasing body of evidence suggests the potential occurrence of antigen encounter by the cell of origin in chronic lymphocytic leukaemia (CLL) and CLL-like monoclonal B-cell lymphocytosis (MBL). However, the scenario in which this event might occur remains unknown. In order to gain insight into this scenario we investigated the molecular, cytogenetic and haematological features of 223 CLL-like (n = 84) and CLL (n = 139) clones with stereotyped (n = 32) versus non-stereotyped (n = 191) immunoglobulin heavy chain variable region (IGHV) amino acid sequences. Overall, stereotyped CLL-like MBL and CLL clones showed a unique IGHV profile, associated with higher IGHV1 and lower IGHV3 gene family usage (P = 0·03), longer IGHV complementary determining region 3 (HCDR3) sequences (P = 0·007) and unmutated IGHV (P < 0·001) versus non-stereotyped clones. Whilst the overall size of the stereotyped B-cell clones in peripheral blood did not appear to be associated with the CLL-related cytogenetic profile of B-cells (P > 0·05), it did show a significant association with the presence of myelodysplastic syndrome (MDS)-associated immunophenotypes on peripheral blood neutrophils and/or monocytes (P = 0·01). Altogether our results point to the potential involvement of different selection forces in the expansion of stereotyped vs. non-stereotyped CLL and CLL-like MBL clones, the former being potentially favoured by an underlying altered haematopoiesis.

Full genome sequence analysis of a novel adenovirus of rhesus macaque origin indicates a new simian adenovirus type and species

Multiple novel simian adenoviruses have been isolated over the past years and their potential to cross the species barrier and infect the human population is an ever present threat. Here we describe the isolation and full genome sequencing of a novel simian adenovirus (SAdV) isolated from the urine of two independent, never co-housed, late stage simian immunodeficiency virus (SIV)-infected rhesus macaques. The viral genome sequences revealed a novel type with a unique genome length, GC content, E3 region and DNA polymerase amino acid sequence that is sufficiently distinct from all currently known human- or simian adenovirus species to warrant classifying these isolates as a novel species of simian adenovirus. This new species, termed Simian mastadenovirus D (SAdV-D), displays the standard genome organization for the genus Mastadenovirus containing only one copy of the fiber gene which sets it apart from the old world monkey adenovirus species HAdV-G, SAdV-B and SAdV-C.

Influence of a heptad repeat stutter on the pH-dependent conformational behavior of the central coiled-coil from influenza hemagglutinin HA2.

The coiled-coil is one of the most common protein structural motifs. Amino acid sequences of regions that participate in coiled-coils contain a heptad repeat in which every third then forth residue is occupied by a hydrophobic residue. Here we examine the consequences of a "stutter," a deviation of the idealized heptad repeat that is found in the central coiled-coil of influenza hemagluttinin HA2. Characterization of a peptide containing the native stutter-containing HA2 sequence, as well as several variants in which the stutter was engineered out to restore an idealized heptad repeat pattern, revealed that the stutter is important for allowing coiled-coil formation in the WT HA2 at both neutral and low pH (7.1 and 4.5). By contrast, all variants that contained idealized heptad repeats exhibited marked pH-dependent coiled-coil formation with structures forming much more stably at low pH. A crystal structure of one variant containing an idealized heptad repeat, and comparison to the WT HA2 structure, suggest that the stutter distorts the optimal interhelical core packing arrangement, resulting in unwinding of the coiled-coil superhelix. Interactions between acidic side chains, in particular E69 and E74 (present in all peptides studied), are suggested to play a role in mediating these pH-dependent conformational effects. This conclusion is partially supported by studies on HA2 variant peptides in which these positions were altered to aspartic acid. These results provide new insight into the structural role of the heptad repeat stutter in HA2.

Bioinformatics insight into the spike glycoprotein gene of field porcine epidemic diarrhea strains during 2011-2013 in Guangdong, China.

Three strains of porcine epidemic diarrhea virus (PEDV) were isolated from dead or diseased pigs at different swine farms in Guangdong during 2011–2013, and their S genes were sequenced. In the same period, seven PEDV strains were also isolated in Guangdong by other laboratories. The spike sequences of 10 Guangdong isolates were compared with vaccine strains and reference pathogenic isolates using six bioinformatics tools. The results revealed that 10 Guangdong strains, excluding strain GDS03, had distinct characteristics in terms of primary structure, secondary structure, high-specificity N-glycosylation sites, potential phosphorylation sites, and palmitoylation sites. Phylogenetic analysis also confirmed these findings and revealed that all PEDV strains were clustered into three distinct groups. Ten Guangdong strains, not including GDS03, belong to Group 1, whereas four vaccine strains and GDS03 belong to Group 3, which is evolutionarily distant from Group 1. Alignment analysis of the neutralizing region amino acid sequences indicated that the amino acid substitutions of Y/D766S, T549S, and G594S that are present in the Guangdong strains, not including GDS03, were a sign of predominant genetic changes among the isolated strains. GDS03 is closely related to the 83P-5 vaccine strain, which suggests that it might represent re-isolation of the vaccine strain or vaccine variants. Taken together, these results indicate that there have been predominant new strains circulating in Guangdong from 2011 to 2013, and the circulating PEDV strains have a genetic composition that is distant from reference strains, especially the vaccine strains; however, the vaccinations might also provide some level of cross-protection, as there have been no changes in the neutralizing epitopes of SS2 and 2C10. This explains why there have been constant but infrequent outbreaks recently in comparison to late 2010 in which PEDV outbreaks were more frequent and severe. In addition, the USA-Colorado-2013 strain had the same amino acid substitutions in the neutralizing regions as the Guangdong strains except GDS03, which suggests that the information and strategies in this study may play role in PEDV variant research in other countries.