Extensive Sequence Variation Research Articles

Extensive sequence divergence between the reference genomes of two zebrafish strains, Tuebingen and AB.

The zebrafish (Danio rerio) is one of the most widely used model organisms for studying vertebrate gene function and human disease, given the 70% conserved protein-coding genes between zebrafish and human. Two of the most common laboratory zebrafish strains are Tuebingen and AB. Despite the fact that the zebrafish reference genome is derived from the Tuebingen strain, the AB strain is still widely used although a high-quality genome comparable to Tuebingen is lacking. Here, we report a 1.40-Gb representative de novo genome assembly of the AB strain (DrAB1), with contig N50length of 21Mb, by integrating Illumina short-read sequencing, Nanopore long-read sequencing and HiC-based chromatin mapping. Compared with the published zebrafish Zv11 reference genome (GRCz11), this genome assembly shows considerable improvements in both contiguity and completeness. In addition, substantial structural differences and extensive sequence divergence of unprecedented resolution have been uncovered, especially with respect to 9,029,929single nucleotide polymorphisms, 2,376,812 InDels, 32,623 insertions, 22,089 deletions and 220 inversions, which constitute ~2.6% of the DrAB1genome. Many of these variants may have potential functional effects on phenotype, which should be considered in further experimental designs. Consequently, our study provides additional genomic resources and a high-resolution structural variation map based on whole-genome alignment for the zebrafish community, which could also be an indispensable reference genome from a model species in future research on fish phylogenetic genomics, comparative genomics and adaptive evolution.

Evolutionary pathways to SARS-CoV-2 resistance are opened and closed byepistasis acting on ACE2.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infects a broader range of mammalian species than previously predicted, binding a diversity of angiotensin converting enzyme 2 (ACE2) orthologs despite extensive sequence divergence. Within this sequence degeneracy, we identify a rare sequence combination capable of conferring SARS-CoV-2 resistance. We demonstrate that this sequence was likely unattainable during human evolution due to deleterious effects on ACE2 carboxypeptidase activity, which has vasodilatory and cardioprotective functions in vivo. Across the 25 ACE2 sites implicated in viral binding, we identify 6 amino acid substitutions unique to mouse-one of the only known mammalian species resistant to SARS-CoV-2. Substituting human variants at these positions is sufficient to confer binding of the SARS-CoV-2 S protein to mouse ACE2, facilitating cellular infection. Conversely, substituting mouse variants into either human or dog ACE2 abolishes viral binding, diminishing cellular infection. However, these same substitutions decrease human ACE2 activity by 50% and are predicted as pathogenic, consistent with the extreme rarity of human polymorphisms at these sites. This trade-off can be avoided, however, depending on genetic background; if substituted simultaneously, these same mutations have no deleterious effect on dog ACE2 nor that of the rodent ancestor estimated to exist 70 million years ago. This genetic contingency (epistasis) may have therefore opened the road to resistance for some species, while making humans susceptible to viruses that use these ACE2 surfaces for binding, as does SARS-CoV-2.

OVATE FAMILY PROTEINS (OFP) gene family across Brassicaceae: Comparative genomic analysis uncovers evolutionary relationships, extensive sequence and structural variation with a potential for functional diversification

Plant-specific Ovate Family Proteins (OFP), containing OVATE domain were identified as regulators of fruit shape. OFP homologs have been detected across plant kingdom as negative regulators of growth and adaptation, albeit with fragmentary information about evolutionary history, diversification, and functional significance. Phylogenetic position, evolutionary history, and availability of genome sequences from several species of Brassicaceae make them suitable for analysis of OFP gene family. Nineteen members of OFP gene family were previously identified from Arabidopsis thaliana. In the present study, we identified 390 homologs of 19 AtOFPs across thirteen Brassicaceae species and analysed their genomic distribution, organization, gene and protein structure, evolution, and possible mode of expansion. Comparative analysis of gene structure and protein architecture of 409 genes revealed conservation and variability in OVATE domain. This feature was used to classify OFPs as: i. OVATE-OFPs containing a canonical OVATE domain, ii. OVATE-like OFPs containing a partial or non-canonical OVATE domain, and, iii: OFP-Related which completely lacked OVATE domain. Two motifs that occur close to, or overlap with the OVATE domain were identified, and variations in amino acid composition that alter folding of OVATE domain were observed. Phylogenetic analysis showed that the paralogous pairs of OFPs found in A. thaliana were conserved across all species. Similar topologies of phylograms constructed from either full length protein, or only with OVATE domain indicate that the evolution of OFP gene family is driven by OVATE domain. A consequence of several post-polyploidization processes including sequence and structural variation among homeologs, and altered sub-cellular localization was observed. Analysis of selection pressure revealed purifying selection acting on seventeen out of nineteen OFPs, and positive selection on the remaining two OFPs. This present work thus lays the foundation for functional genomics of OFPs not only in agronomically important species of Brassicaceae but also in other multigene families to create a framework for future comparative functional genomics studies.

Genetic diversity in the Tams1 gene of Theileria annulata (Duschunkowsky and Luhs, 1904) infecting cattle

The present study describes the genetic diversity in the Tams1 gene (733 bp) of Theileria annulata along with the sequence, phylogenetic and haplotype analyses of the Indian isolates. The phylogenetic analyses displayed distinct clustering of the Indian isolates into three groups suggesting the presence of three genotypes, hitherto designated as T. annulata genotypes 1–3 (G1-G3). Genotype 3 seems to be novel containing only two newly generated sequences. Indian isolates displayed 88.4–100% and 82.2–100% similarity with each other at nucleotide (nt) and amino acid (aa) levels, respectively. However, the newly generated sequences (n = 36) showed 90.5–100% and 84.3–100% identity between them at nt and aa levels, respectively. The most diverse and heterogeneous genotype, G1, exhibited the highest number of polymorphic sites (S = 148), haplotypes (h = 16) and nucleotide differences (k = 43.23) besides haplotype (Hd = 0.903 ± 0.031) and nucleotide (π = 0.059 ± 0.005) diversities. Neutrality indices suggested a respective decrease and increase in population sizes of G1 and G2 genotypes in India. The nucleotide sequence analyses indicated the presence of extensive sequence variations between nucleotide positions 1-124, 194–257 and 396–494. The N‐terminus of Tams1 protein displayed a considerable sequence variability with extensive variations in two regions, between amino acid positions 1–39 and 127–172, as compared to the conserved carboxyl terminus.

The Dryas iulia Genome Supports Multiple Gains of a W Chromosome from a B Chromosome in Butterflies.

In butterflies and moths, which exhibit highly variable sex determination mechanisms, the homogametic Z chromosome is deeply conserved and is featured in many genome assemblies. The evolution and origin of the female W sex chromosome, however, remains mostly unknown. Previous studies have proposed that a ZZ/Z0 sex determination system is ancestral to Lepidoptera, and that W chromosomes may originate from sex-linked B chromosomes. Here, we sequence and assemble the female Dryas iulia genome into 32 highly contiguous ordered and oriented chromosomes, including the Z and W sex chromosomes. We then use sex-specific Hi-C, ATAC-seq, PRO-seq, and whole-genome DNA sequence data sets to test if features of the D. iulia W chromosome are consistent with a hypothesized B chromosome origin. We show that the putative W chromosome displays female-associated DNA sequence, gene expression, and chromatin accessibility to confirm the sex-linked function of the W sequence. In contrast with expectations from studies of homologous sex chromosomes, highly repetitive DNA content on the W chromosome, the sole presence of domesticated repetitive elements in functional DNA, and lack of sequence homology with the Z chromosome or autosomes is most consistent with a B chromosome origin for the W, although it remains challenging to rule out extensive sequence divergence. Synteny analysis of the D. iulia W chromosome with other female lepidopteran genome assemblies shows no homology between W chromosomes and suggests multiple, independent origins of the W chromosome from a B chromosome likely occurred in butterflies.

Genome variability of classical swine fever virus during the 2018–2020 epidemic in Japan

Although RNA viruses exhibit extensive sequence diversity, the mutation rate must be limited to ensure protein functions that maintain the viral life cycle. Here, we compared the whole genome sequences of 150 isolates of classical swine fever virus (CSFV), obtained from a single epidemic that occurred in Japan during 2018–2020. After the detection of the first case, the disease spread among both farm pigs and wild boars and caused severe impact on the pig industry. To evaluate the diversification of the CSFV genome that eliminated mutations negatively affecting viral transmission, the substitution sets inherited by at least two isolates were separately evaluated as shared single nucleotide variants (SNVs) or shared single amino acid variants (SAVs). Comparisons of 12 protein-coding regions indicated that the percentages of SNVs and SAVs in the multifunctional nonstructural protein NS3 were the lowest, and shared SAVs were not detected in another nonstructural protein, NS4A. This demonstrated purifying negative selection suppressing changes in the protein sequences of NS3 and NS4A during virus transmission in the field. In contrast, a high possibility of nonsynonymous substitution among shared SNVs was detected only in genes encoding the secreted protein Erns and the nonstructural protein NS2, suggesting positive selection during the epidemic. Mapping of shared SAVs to the three-dimensional structure of Erns revealed that shared SAVs were not present in the substrate-binding sites but were instead localized to the peripheral region of the protein. These data will support efforts toward the development of diagnostic methods, recombinant vaccines, and antiviral agents targeting conserved and indispensable viral genes.

Metal-containing PAS/GAF domains in bacterial sensors

PAS and related GAF are ubiquitous and highly versatile sensing domains that associate a range of signaling effectors. Each year, new bacterial PAS and GAF domains are characterized but genomes analysis suggests this list is far from complete. This review aims to synthesize the current state of knowledge on the structure and activity of metal-binding PAS/GAF domain proteins. PAS/GAF domains can bind disparate metal cofactors with very precise coordination and high specificity concomitant with their extensive sequence diversity. Bound-metal can directly be the signal or can mediate signal detection such as gases or redox variation. Metal-containing PAS/GAF input modules are mainly associated to histidine kinases of two-component signaling systems and σ54-dependent transcription factors. In both cases, metal-binding induces conformational changes that modulate the effector activity. It appears that metal-containing PAS/GAF domains, as other PAS/GAF domains, offer great possibilities for coupling a broad range of ligand binding to various cellular responses and it is very likely that they have not yet revealed all their secrets.

Probing the naturally acquired immune response to malaria for broadly reactive antibodies targeting P. falciparum antigens linked with severe disease

Abstract Severe malaria is caused by the adhesion of Plasmodium falciparum-infected erythrocytes to host receptors on vascular endothelium. Specifically, binding of the parasite variant surface antigen domain CIDRa1 to host endothelial protein C receptor (EPCR) is strongly associated with severe disease. In malaria-endemic regions, children quickly develop immunity against severe malaria, indicative of the development of an effective immune response against CIDRa1 domains. Indeed, serum IgG from naturally immune individuals exhibited reactivity against a diverse panel of CIDRa1 variants. In an effort to analyze CIDRa1 antibody responses at a monoclonal level, we isolated CIDRa1-specific memory B cells from malaria-experienced individuals living in Uganda and expressed the corresponding monoclonal antibodies (mAbs). Our approach has yielded anti-CIDRa1 mAbs ranging in breadth from variant-specific to reactive with the full spectrum of CIDRa1 domain variants, irrespective of their extensive sequence diversity. The broadest antibodies can be separated into two categories, those that bind outside the EPCR binding site and display exceptional breadth with modest inhibition of EPCR binding and those that target the EPCR binding site and show potent EPCR-binding inhibition. Two EPCR-binding site-targeting mAbs isolated from two different donors showed signs of convergent evolution. Our results demonstrate that natural P. falciparum infection can induce a broad and inhibitory antibody response against CIDRa1. Current experiments are aimed at understanding the structural basis of antibody-mediated inhibition of CIDRa1-EPCR binding to inform the design of a vaccine that protects against severe malaria.

Comparison of 15 dinoflagellate genomes reveals extensive sequence and structural divergence in family Symbiodiniaceae and genus Symbiodinium

BackgroundDinoflagellates in the family Symbiodiniaceae are important photosynthetic symbionts in cnidarians (such as corals) and other coral reef organisms. Breakdown of the coral-dinoflagellate symbiosis due to environmental stress (i.e. coral bleaching) can lead to coral death and the potential collapse of reef ecosystems. However, evolution of Symbiodiniaceae genomes, and its implications for the coral, is little understood. Genome sequences of Symbiodiniaceae remain scarce due in part to their large genome sizes (1–5 Gbp) and idiosyncratic genome features.ResultsHere, we present de novo genome assemblies of seven members of the genus Symbiodinium, of which two are free-living, one is an opportunistic symbiont, and the remainder are mutualistic symbionts. Integrating other available data, we compare 15 dinoflagellate genomes revealing high sequence and structural divergence. Divergence among some Symbiodinium isolates is comparable to that among distinct genera of Symbiodiniaceae. We also recovered hundreds of gene families specific to each lineage, many of which encode unknown functions. An in-depth comparison between the genomes of the symbiotic Symbiodinium tridacnidorum (isolated from a coral) and the free-living Symbiodinium natans reveals a greater prevalence of transposable elements, genetic duplication, structural rearrangements, and pseudogenisation in the symbiotic species.ConclusionsOur results underscore the potential impact of lifestyle on lineage-specific gene-function innovation, genome divergence, and the diversification of Symbiodinium and Symbiodiniaceae. The divergent features we report, and their putative causes, may also apply to other microbial eukaryotes that have undergone symbiotic phases in their evolutionary history.

Sequence and functional analysis of MIR319 promoter homologs from Brassica juncea reveals regulatory diversification and altered expression under stress.

Extensive regulatory divergence during development, abiotic stress and ABA regime observed amongst promoter homologs and homeologs of MIR319 from Brassica juncea. Gene duplication followed by sub-functionalization, neo-functionalization, and pseudogenization are routes to functional and adaptive diversification. The influence of polyploidy on protein-coding genes is well investigated but little is known about their impact on transcriptional regulation of MIRNA gene family. The present study was therefore performed with an aim to uncover regulatory diversification of MIR319 homologs and homeologs in Brassica juncea. We employed comparative genomics to identify and isolate six promoter homologs of MIR319 from B. juncea. Regulatory diversification was studied using analysis of reporter activity driven by BjMIR319 promoters in a heterologous system employing promoter-reporter fusion constructs. MIR319 is known to play important roles in leaf and flower development, and multiple stress responses. Reporter activity was therefore monitored during development, hormonal and stress regimes. In-silico analyses revealed differential distribution of cis-regulatory motifs and functional analysis revealed distinct spatiotemporal expression patterns. The significance of presence of selected cis-regulatory motifs corresponding to heat, cold, salt and ABA stress were further functionally validated. It was observed that promoter of Bj -MIR319a-A01 was upregulated in response to cold and salt stress, while promoter of Bj -MIR319c-A04 (D1) and Bj -MIR319c-A05 (FL) were downregulated in response to high temperature. In summary, comparative analysis of homologous promoters from Brassica juncea, an allopolyploid revealed extensive sequence and functional diversity. Spatiotemporal activity of reporter gene driven by BjMIR319 promoter was distinct, and partially overlapping with from those reported previously for A. thaliana. The present study clearly demonstrates regulatory divergence amongst promoter homologs of MIR319 in Brassica juncea during development and stress response, and underlines the urgent need for dissection of promoter function and detailed characterization including identification of interacting trans-factors. Genbank accession numbers: MT379853-MT379858.

Towards middle-up analysis of polyclonal antibodies: subclass-specific N-glycosylation profiling of murine immunoglobulin G (IgG) by means of HPLC-MS

In recent years, advanced HPLC-MS strategies based on intact protein (“top-down”) or protein subunit (“middle-up/middle-down”) analysis have been implemented for the characterization of therapeutic monoclonal antibodies. Here, we assess feasibility of middle-up/middle-down analysis for polyclonal IgGs exhibiting extensive sequence variability. Specifically, we addressed IgGs from mouse, representing an important model system in immunological investigations. To obtain Fc/2 portions as conserved subunits of IgGs, we made use of the bacterial protease SpeB. For this purpose, we initially determined SpeB cleavage sites in murine IgGs. The resulting Fc/2 portions characteristic of different subclasses were subsequently analysed by ion-pair reversed-phase HPLC hyphenated to high-resolution mass spectrometry. This enabled simultaneous relative quantification of IgG subclasses and their N-glycosylation variants, both of which influence IgG effector functions. To assess method capabilities in an immunological context, we applied the analytical workflow to polyclonal antibodies obtained from BALB/c mice immunized with the grass pollen allergen Phl p 6. The study revealed a shift in IgG subclasses and Fc-glycosylation patterns in total and antigen-specific IgGs from different mouse cohorts, respectively. Eventually, Fc/2 characterization may reveal other protein modifications including oxidation, amino acid exchanges, and C-terminal lysine, and may thus be implemented for quality control of functional antibodies.

A single mutation in Crimean-Congo hemorrhagic fever virus discovered in ticks impairs infectivity in human cells.

Crimean-Congo hemorrhagic fever (CCHF) is the most widely distributed tick-borne viral infection in the world. Strikingly, reported mortality rates for CCHF are extremely variable, ranging from 5% to 80% (Whitehouse, 2004). CCHF virus (CCHFV, Nairoviridae) exhibits extensive genomic sequence diversity across strains (Deyde et al., 2006; Sherifi et al., 2014). It is currently unknown if genomic diversity is a factor contributing to variation in its pathogenicity. We obtained complete genome sequences of CCHFV directly from the tick reservoir. These new strains belong to a solitary lineage named Europe 2 that is circumstantially reputed to be less pathogenic than the epidemic strains from Europe 1 lineage. We identified a single tick-specific amino acid variant in the viral glycoprotein region that dramatically reduces its fusion activity in human cells, providing evidence that a glycoprotein precursor variant, present in ticks, has severely impaired function in human cells.

Phylogenetic analysis and geographical distribution of Theileria equi and Babesia caballi sequences from horses residing in Spain

The intraerythrocytic protozoans Theileria equi and Babesia caballi are the causative agents of equine piroplasmosis (EP), one of the most important equine tick-borne diseases due to its significant impact on global international horse trade. Although EP is known to be endemic in Spain, previous phylogenetic studies have only been conducted for limited geographical regions. Therefore, the objective of this study was to evaluate the genetic diversity and distribution of these parasite species nationwide. This was performed by amplification of the 18S small subunit (SSU) rRNA gene from 100 EP positive equine blood samples using a nested PCR protocol, and sequencing the obtained amplicons. Seventy-seven T. equi and six B. caballi isolates were successfully sequenced and phylogenetic analysis revealed that the T. equi isolates grouped into the previously described clades A (n = 21/77), D (n = 1/77) and E (n = 55/77), while B. caballi isolates were placed into clades A (n = 5/6) and B (n = 1/6). Isolates from T. equi clade D and B. caballi clade B have not previously been reported in Spain. A greater intra-clade diversity (97.3–98.3 % identity) was observed between T. equi clade E isolates compared to those within clade A (99.7–100 % identity). Additionally, a multivariable logistic regression model was used to analyse associations between the clade of T. equi infection and available epidemiological data. Horses residing in Spanish northern regions were statistically more likely to be infected with T. equi clade E (p = 0.01). We conclude that while extensive sequence variation of equine piroplasms exists in Spanish infected horses, a requirement for increased equine movement controls between Spain and EP-endemic countries should be considered.

Structural diversity, natural selection and intragenic recombination in the Plasmodium vivax merozoite surface protein 9 locus in Thailand

The merozoite surface protein 9 (MSP9) of malarial parasite forms co-ligand complex with the 19 kDa fragment of merozoite surface protein 1 (MSP1) prior to erythrocyte invasion. Interruption of this process could hamper subsequent asexual erythrocytic development of malaria parasites; therefore, these proteins are considered potential vaccine candidates. In Plasmodium vivax, MSP9 (PvMSP9) contains both conserved and polymorphic repetitive domains that were immunogenic upon natural malaria exposure and conferred protection in vaccination studies in animal models. To investigate the extent of sequence diversity at this locus, 104 P. vivax isolates from 4 major malaria endemic areas of Thailand were analyzed. Results revealed that pvmsp9 contained 3 repeat domains (R1–R3) flanked by conserved domains. Repeat domains exhibit extensive sequence and length variation, in which 14, 39 and 16 haplotypes for domains R1–R3, respectively, circulated in this country. Sequence diversity in pvmsp9 among P. vivax isolates from each endemic area displayed population structure. The extent of sequence diversity in pvmsp9 isolates from the provinces of Tak, Chanthaburi, Ubon Ratchathani and Prachuap Khiri Khan in northwestern, eastern, northeastern and southwestern areas, respectively, was almost comparable and was remarkably higher than that from Yala/Narathiwat population in southern Thailand. Evidence for intragenic recombination in this locus was observed within each P. vivax population except among isolates from Yala and Narathiwat. Synonymous nucleotide diversity significantly exceeded nonsynonymous nucleotide diversity in domains R2 and R3, indicating purifying selection. However, micro-scale signatures of positive and negative selections occurred in both conserved and repeat domains, implying two opposing forces, probably from functional or structural constraint and host immune pressure, could have influenced diversity at this locus. The immunodominant T and B cell epitopes so far identified were invariant or highly conserved across isolates. Further analysis of global isolates is warranted for vaccine design based on this protein.

Sequence Analysis and FISH Mapping of Four Satellite DNA Families among Cervidae.

Centromeric and pericentromeric chromosome regions are occupied by satellite DNA. Satellite DNAs play essential roles in chromosome segregation, and, thanks to their extensive sequence variability, to some extent, they can also be used as phylogenetic markers. In this paper, we isolated and sequenced satellite DNA I-IV in 11 species of Cervidae. The obtained satellite DNA sequences and their chromosomal distribution were compared among the analysed representatives of cervid subfamilies Cervinae and Capreolinae. Only satI and satII sequences are probably present in all analysed species with high abundance. On the other hand, fluorescence in situ hybridisation (FISH) with satIII and satIV probes showed signals only in a part of the analysed species, indicating interspecies copy number variations. Several indices, including FISH patterns, the high guanine and cytosine (GC) content, and the presence of centromere protein B (CENP-B) binding motif, suggest that the satII DNA may represent the most important satellite DNA family that might be involved in the centromeric function in Cervidae. The absence or low intensity of satellite DNA FISH signals on biarmed chromosomes probably reflects the evolutionary reduction of heterochromatin following the formation of chromosome fusions. The phylogenetic trees constructed on the basis of the satellite I-IV DNA relationships generally support the present cervid taxonomy.

An Updated Classification System and Review of the Lipooligosaccharide Biosynthesis Gene Locus in Campylobacter jejuni.

Lipooligosaccharide (LOS) is an integral component of the Campylobacter cell membrane with a structure of core oligosaccharides forming inner and outer core regions and a lipid A moiety. The gene content of the LOS core biosynthesis cluster exhibits extensive sequence variation, which leads to the production of variable cell surface LOS structures in Campylobacter. Some LOS outer core molecules in Campylobacter jejuni are molecular mimics of host structures (such as neuronal gangliosides) and are thought to trigger neuronal disorders (particularly Guillain–Barré syndrome and Miller Fisher syndrome) in humans. The extensive genetic variation in the LOS biosynthesis gene cluster, a majority of which occurs in the LOS outer core biosynthesis gene content present between lgtF and waaV, has led to the development of a classification system with 23 classes (A–W) and four groups (1–4) for the C. jejuni LOS region. This review presents an updated and simplified classification system for LOS typing alongside an overview of the frequency of C. jejuni LOS biosynthesis genotypes and structures in various C. jejuni populations.

Characterization of sequence variation at 30 autosomal STRs in Chinese Han and Tibetan populations.

Massively parallel sequencing (MPS) technologies have the ability to reveal sequence variations within STR alleles as well as their nominal allele lengths, which have traditionally been detected by CE instruments. Recently, Thermo Fisher Scientific has updated the MPS-STR panel, named the Precision ID GlobalFiler next-generation sequencing (NGS) STR Panel version 2, with primers redesigned to add two pentanucleotide tandem repeat loci and profile interpretation supported by the Converge software. Using the Ion Chef System, the Ion S5XL System, and the Converge software, genetic variations were characterized within STR repeat and flanking regions of 30 autosomal STR markers in 115 unrelated individuals from two Chinese population groups (58 Tibetans and 57 Hans). Nineteen STRs demonstrated a relative increase in diversity with the variant sequence alleles compared with those of traditional nominal length alleles. In total, 390 alleles were identified by their sequences compared with 258 alleles that were identified by length. Of these 92 sequence variants found within the STR repeat regions, 40 variants were located in STR flanking regions. Additionally, the agreement of the results with CE data was evaluated, as was the ability of this new MPS panel to analyze case-type (11 samples) and artificially degraded samples (seven samples in triplicate). The results generated from this study illustrate that extensive sequence variation exists in commonly used STR markers in the selected population samples and indicate that this NGS STR panel has the potential to be used as an effective tool for human forensics.

One-step sequence and structure-guided optimization of HIV-1 envelope gp140

Stabilization of the metastable envelope glycoprotein (Env) of HIV-1 is hypothesized to improve induction of broadly neutralizing antibodies. We improved the expression yield and stability of the HIV-1 envelope glycoprotein BG505SOSIP.664 gp140 by means of a previously described automated sequence and structure-guided computational thermostabilization approach, PROSS. This combines sequence conservation information with computational assessment of mutant stabilization, thus taking advantage of the extensive natural sequence variation present in HIV-1 Env. PROSS is used to design three gp140 variants with 17–45 mutations relative to the parental construct. One of the designs is experimentally observed to have a fourfold improvement in yield and a 4 °C increment in thermostability. In addition, the designed immunogens have similar antigenicity profiles to the native flexible linker version of wild type, BG505SOSIP.664 gp140 (NFL Wt) to major epitopes targeted by broadly neutralizing antibodies. PROSS eliminates the laborious process of screening many variants for stability and functionality, providing a proof of principle of the method for stabilization and improvement of yield without compromising antigenicity for next generation complex, highly glycosylated vaccine candidates.

Genetic and antigenic variation of the bovine tick-borne pathogen Theileria parva in the Great Lakes region of Central Africa

BackgroundTheileria parva causes East Coast fever (ECF), one of the most economically important tick-borne diseases of cattle in sub-Saharan Africa. A live immunisation approach using the infection and treatment method (ITM) provides a strong long-term strain-restricted immunity. However, it typically induces a tick-transmissible carrier state in cattle and may lead to spread of antigenically distinct parasites. Thus, understanding the genetic composition of T. parva is needed prior to the use of the ITM vaccine in new areas. This study examined the sequence diversity and the evolutionary and biogeographical dynamics of T. parva within the African Great Lakes region to better understand the epidemiology of ECF and to assure vaccine safety. Genetic analyses were performed using sequences of two antigen-coding genes, Tp1 and Tp2, generated among 119 T. parva samples collected from cattle in four agro-ecological zones of DRC and Burundi.ResultsThe results provided evidence of nucleotide and amino acid polymorphisms in both antigens, resulting in 11 and 10 distinct nucleotide alleles, that predicted 6 and 9 protein variants in Tp1 and Tp2, respectively. Theileria parva samples showed high variation within populations and a moderate biogeographical sub-structuring due to the widespread major genotypes. The diversity was greater in samples from lowlands and midlands areas compared to those from highlands and other African countries. The evolutionary dynamics modelling revealed a signal of selective evolution which was not preferentially detected within the epitope-coding regions, suggesting that the observed polymorphism could be more related to gene flow rather than recent host immune-based selection. Most alleles isolated in the Great Lakes region were closely related to the components of the trivalent Muguga vaccine.ConclusionsOur findings suggest that the extensive sequence diversity of T. parva and its biogeographical distribution mainly depend on host migration and agro-ecological conditions driving tick population dynamics. Such patterns are likely to contribute to the epidemic and unstable endemic situations of ECF in the region. However, the fact that ubiquitous alleles are genetically similar to the components of the Muguga vaccine together with the limited geographical clustering may justify testing the existing trivalent vaccine for cross-immunity in the region.

Polymorphism and natural selection in the merozoite surface protein 3F2 (PVX_97710) locus of Plasmodium vivax among field isolates

Plasmodium vivax, the chronic relapsing human malaria parasite with the most widespread distribution, possesses proteins associated with the merozoite surface that could be targets for host immune responses and potential vaccine candidates. Of these, the merozoite surface protein 3 of P. vivax (PvMSP3) is an attractive vaccine target as well as a genetic marker for epidemiological surveillance. PvMSP3 comprises a group of protein members encoded by a multigene family. Although some protein members, i.e. PvMSP3α and PvMSP3β, have been targets for molecular and immunological investigations, the most abundantly expressed protein member during late asexual erythrocytic stages, PvMSP3F2 (PVX_97710), remains unexplored.To address domain organization and evolution of this locus, the complete coding sequences of 31 P. vivax isolates from diverse malaria endemic areas of Thailand were analyzed and compared with 10 previously reported sequences. Results revealed that all PvMSP3F2 sequences differed but could be divided into 5 repeat-containing domains flanked by 6 non-repeat domains. Repeat domains II and IV at the 5′ portion and domain X at the 3′ portion exhibited extensive sequence and length variation whereas repeat domains VI and VIII located at the central region were relatively conserved. Despite a repertoire of PvMSP3F2 variants, predicted coiled-coil tertiary structure and predicted B-cell epitopes seem to be maintained. Evidence of intragenic recombination has been detected among field isolates in Thailand that could enhance sequence diversity at this locus. Non-repeat domains I and IX located at the 5′ end and at the 3′ portion, respectively, seem to have evolved under purifying selection. Evidence of positive selection was found in non-repeat domains III, V and VII where a number of predicted HLA class I epitopes were identified. Amino acid substitutions in these predicted epitopes could alter predicted peptide binding affinity or abolish peptide epitope property, suggesting that polymorphism in these epitopes conferred host immune evasion. Further studies on PvMSP3F2 are warranted, particularly on interaction with host immune system and the potential role of this PvMSP3 protein member as a vaccine target.