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PubMed2026

Effect of inhaled interferon-β1a on SARS-CoV-2 diversity and evolution.

Interferon resistance has been implicated in SARS-CoV-2 escape from innate immunity, but exogenous interferon's impact on viral evolution and diversity is unknown. SNG001, an inhaled interferon-β1a treatment, was evaluated in the ACTIV-2/A5401 randomized controlled trial of therapeutics for COVID-19. We measured viral kinetics and performed whole-genome sequencing on longitudinal nasal swabs collected from ACTIV-2 participants who received either SNG001 or placebo to assess viral sequence diversity. No difference in nasal viral load decay was detected between study arms when stratifying by SARS-CoV-2 variant or by viral culture conversion. Compared to placebo participants, the SNG001-treated participants displayed significantly lower nonsynonymous amino acid average pairwise distance, indicating lower sequence diversity. Similarly, SNG001-treated individuals also developed numerically fewer nonsynonymous mutations during their infection in ORF1a, ORF1b, Spike, and Nucleocapsid. No specific emerging SARS-CoV-2 nonsynonymous amino acid changes indicating signatures of viral escape were enriched in those receiving SNG001. These in vivo data provide an intriguing signal that exogenous interferon-β1a may restrict SARS-CoV-2 viral diversity and add to growing evidence that interferon levels play a critical role in antiviral responses during COVID-19.IMPORTANCESARS-CoV-2 encodes several genes which can antagonize the interferon signaling cascade, preventing it from activating antiviral responses and thereby facilitating viral establishment and dissemination. It is unknown how the administration of exogenous interferon might affect viral evolution and immune escape. ACTIV-2/A5401 represents a unique opportunity to study the virologic effects of interferon treatment in a rigorous randomized, placebo-controlled clinical trial setting. Our characterization of longitudinal nasal samples shows that interferon-treated individuals had lower viral diversity and no evidence of viral escape mutations.CLINICAL TRIALSThis study is registered with ClinicalTrials.gov as NCT04518410.

Humans↗
PubMed1998

TCR diversity in gammadeltaTCR+ hybridomas derived from mice given portal vein donor-specific pre-immunization and skin allografts.

Portal venous (p.v.) immunization with multiple minor histoincompatible cells leads to antigen-specific increased skin allograft survival. GammadeltaTCR+ hybridoma cells, prepared from mesenteric lymphocytes of p.v. immunized animals, can adoptively transfer this increased graft survival to naive animals. We have analyzed VgammaVdelta gene usage, and TCR gamma-chain junctional diversity in gammadeltaTCR+ hybridomas from mice immunized with different antigen combinations by p.v. or conventional lateral tail vein (i.v.) immunization. Following p.v. immunization two independent sets of hybridoma cells were derived, one expressing a common gamma-chain junctional sequence which was also found in > 85% of the hybridomas derived following i.v. immunization, while the other set showed remarkable gamma-chain junctional sequence diversity. The diversity seen in these latter hybridomas was associated with the antigen specificity of the hybridoma cells. Cells expressing these 'unique' TCR junctional sequences were stimulated to produce cytokines both by hsp and by minor-histocompatibility-specific irradiated peritoneal cells. Cells expressing TCR with a common gamma-chain junctional sequence were stimulated to cytokine production by MHC-matched but minor-histocompatibility mismatched (as well as matched) peritoneal cells, but not by hsp. We suggest that p.v. immunization results in stimulation of both antigen-specific and non-specific regulatory gammadeltaTCR+ cells, which can be distinguished by gamma-chain TCR sequence diversity.

Adoptive Transfer↗
PubMed2005

Dramatic difference in diversity between Plasmodium falciparum and Plasmodium vivax reticulocyte binding-like genes.

Malaria parasite proteins involved in erythrocyte invasion are considered important vaccine targets. Members of the reticulocyte binding-like (RBL) family of Plasmodium merozoite proteins are found in human, simian, and rodent malaria parasites and function in the initial steps of erythrocyte selection and invasion. The RBL genes are large, ranging in size from 7.7 to 10 kb, and the extent of any sequence diversity in parasite populations is unknown. We present the first assessment of sequence diversity within RBL genes from the two major human malaria parasites: Plasmodium falciparum and P. vivax. Polymorphism within the RBL genes is generally limited, except for P. vivax reticulocyte binding protein 2 (PvRBP2), which has nucleotide diversity levels 25-fold higher than the other RBL genes. The PvRBP2 haplotypes appear to fall into two distinct classes of alleles, suggesting large-scale dimorphism in this gene. Polymorphisms were frequently clustered, suggesting that different RBL domains may be evolving under different selection and functional pressures.

Amino Acid Sequence↗
PubMed2004

Plasmodium vivax Duffy binding protein: a modular evolutionary proposal.

The population of malaria-causing parasites is characterized by great genetic diversity. Knowledge of the polymorphism generation mechanism is a central issue for developing effective vaccines against malaria and understanding the parasite population structure. Plasmodium vivax genetic diversity has been explained in terms of two major factors: natural selection and intragenic recombination. A modular organization was found within P. vivax Duffy binding protein in the present work. Four Colombian isolates have identical sequences to Salvador-1 strain amongst dpb regions III-VI analysed, suggesting a high identity between Central and South American isolates. Geographically clustered sectors, corresponding to cysteine-rich regions (II and VI), show a high sequence diversity that could reflect a possible immune response evasion mechanism; both positive and negative selection were detected in these regions. In contrast, other dbp gene regions display a non-geographical clustering pattern, lower sequence diversity and predominant negative selective pressure. Recombination was homogeneously detected all along the molecule. These findings suggest that diversification vs. homogenizing forces, drive dbp gene evolution and determine its mosaic region organization.

Amino Acid Sequence↗
PubMed2000

Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides.

By analyzing the human antibody repertoire in terms of structure, amino acid sequence diversity and germline usage, we found that seven V(H) and seven V(L) (four Vkappa and three Vlambda) germline families cover more than 95 % of the human antibody diversity used. A consensus sequence was derived for each family and optimized for expression in Escherichia coli. In order to make all six complementarity determining regions (CDRs) accessible for diversification, the synthetic genes were designed to be modular and mutually compatible by introducing unique restriction endonuclease sites flanking the CDRs. Molecular modeling verified that all canonical classes were present. We could show that all master genes are expressed as soluble proteins in the periplasm of E. coli. A first set of antibody phage display libraries totalling 2x10(9) members was created after cloning the genes in all 49 combinations into a phagemid vector, itself devoid of the restriction sites in question. Diversity was created by replacing the V(H) and V(L) CDR3 regions of the master genes by CDR3 library cassettes, generated from mixed trinucleotides and biased towards natural human antibody CDR3 sequences. The sequencing of 257 members of the unselected libraries indicated that the frequency of correct and thus potentially functional sequences was 61 %. Selection experiments against many antigens yielded a diverse set of binders with high affinities. Due to the modular design of all master genes, either single binders or even pools of binders can now be rapidly optimized without knowledge of the particular sequence, using pre-built CDR cassette libraries. The small number of 49 master genes will allow future improvements to be incorporated quickly, and the separation of the frameworks may help in analyzing why nature has evolved these distinct subfamilies of antibody germline genes.

Amino Acid Sequence↗
PubMed2000

Lentivirus infection in the brain induces matrix metalloproteinase expression: role of envelope diversity.

Infection of the brain by lentiviruses, including human immunodeficiency virus (HIV) and feline immunodeficiency virus (FIV), causes inflammation and results in neurodegeneration. Molecular diversity within the lentivirus envelope gene has been implicated in the regulation of cell tropism and the host response to infection. Here, we examine the hypothesis that envelope sequence diversity modulates the expression of host molecules implicated in lentivirus-induced brain disease, including matrix metalloproteinases (MMP) and related transcription factors. Infection of primary macrophages by chimeric HIV clones containing brain-derived envelope fragments from patients with HIV-associated dementia (HAD) or nondemented AIDS patients (HIV-ND) showed that MMP-2 and -9 levels in conditioned media were significantly higher for the HAD clones. Similarly, STAT-1 and JAK-1 levels were higher in macrophages infected by HAD clones. Infections of primary feline macrophages by the neurovirulent FIV strain (V(1)CSF), the less neurovirulent strain (Petaluma), and a chimera containing the V(1)CSF envelope in a Petaluma background (FIV-Ch) revealed that MMP-2 and -9 levels were significantly higher in conditioned media from V(1)CSF- and FIV-Ch-infected macrophages, which was associated with increased intracellular STAT-1 and JAK-1 levels. The STAT-1 inhibitor fludarabine significantly reduced MMP-2 expression, but not MMP-9 expression, in FIV-infected macrophages. Analysis of MMP mRNA and protein levels in brain samples from HIV-infected persons or FIV-infected cats showed that MMP-2 and -9 levels were significantly increased in lentivirus-infected brains compared to those of uninfected controls. Elevated MMP expression was accompanied by significant increases in STAT-1 and JAK-1 mRNA and protein levels in the same brain samples. The present findings indicate that two lentiviruses, HIV and FIV, have common mechanisms of MMP-2 and -9 induction, which is modulated in part by envelope sequence diversity and the STAT-1/JAK-1 signaling pathway.

AIDS Dementia Complex↗
PubMed2003

Sequences and diversity of 17 new Ovar-DRB1 alleles from three breeds of sheep.

To investigate the genetic diversity of the sheep MHC (Ovar) class II DRB1 locus, we amplified exon 2 of Ovar-DRB1 alleles by polymerase chain reaction (PCR) and determined the nucleotide sequences of both resultant strands after cloning. In our study of a total of 97 sheep of three breeds, namely, Suffolk, Cheviot and Corriedale, we identified 18 previously published alleles and 17 new alleles. These alleles were 83.4 to 94.1% identical at the nucleotide level and 71.4 to 90.9% identical at the amino acid level to Ovar-DRB1*0101. We identified six new alleles in Cheviot sheep and 11 new alleles in Suffolk sheep. Furthermore, we identified 15, 6 and 1 allele in Suffolk, Cheviot and Corriedale sheep, respectively, that have only been found in these breeds to date. Analysis of the frequencies of the various Ovar-DRB1 alleles in each breed indicated that Ovar-DRB1*0702 was the most frequent allele in Suffolk sheep (23.9%), Ovar-DRB1*0203 was the most frequent allele in Cheviot sheep (27.5%) and Ovar-DRB1*0201 was the most frequent allele in Corriedale sheep (25.0%). A comparative analysis of the positions of polymorphic residues in the first extracellular domain of the DRB genes of sheep, humans and mice revealed an extraordinary similarity amongst the positions of polymorphic residues that are associated with the antigen recognition site (ARS). Moreover, the extent of polymorphism seems to be similar in sheep, humans and mice.

Amino Acid Sequence↗
PubMed2002

Three multigene families in Plasmodium parasites: facts and questions.

Multigene families optimise fitness by providing a set of related genes with possibly different temporal and/or topological expression patterns. We analyse here the structural organisation and sequence diversity of the rDNA, sera and var C Plasmodium falciparum families, and discuss their consequences for parasite biology. The low rDNA copy number, which reduces reshuffling, is probably the corollary of the need for functionally distinct rRNAs in the insect and in the vertebrate host. The unusual intra-genome and population rDNA sequence diversity results in cells equipped with mosaic ribosome sets. The functional constraints are such that ribosome compatibility could influence parasite fitness and contribute to population structuring. Unlike the dispersed rDNA units, the sera family is arranged as a tandem gene cluster, with seven contiguous similar genes, and one more distantly related paralog. We address the question of the inclusion criteria in family definition. We discuss the results concerning the SERA proteins expression and function in the context of the long overlooked multigene family. The var C module is shared by var genes, 'orphan' var C and var C pseudogenes. Analysis of 125 var C deduced protein sequences highlights a well-conserved framework, including putative phosphorylation sites, consistent with the proposed function of mediating interaction with cytoskeletal proteins. The 5' and 3' flanking sequences of the var C pseudogenes are heterogeneous. In contrast, the flanking sequences of the uninterrupted var C modules show remarkable conservation. This is interesting in view of the silencing activity of the var intronic sequence on var expression. The 5' flanking sequence dichotomy reported for internal and sub-telomeric var genes extends to the 3' flanking sequences. This has profound implications for transcription regulation and generation of diversity. The var C family suggests a role for pseudogenes as a diversity reservoir and in genome dynamics by promoting ectopic recombination.

Amino Acid Sequence↗
PubMed2003

A rapid method for identifying diversity within PCR amplicons using a heteroduplex mobility assay and synthetic polynucleotides: application to characterisation of dsRNA elements associated with Cryptosporidium.

A 173-bp fragment of the small extra-chromosomal double-stranded RNA (dsRNA) element of Cryptosporidium parvum was generated by reverse transcriptase PCR from nucleic acid extracted from whole faeces of 18 epidemiologically unrelated cases of cryptosporidiosis. Eleven different sequences were detected and two selected as reference DNA in a heteroduplex mobility assay (HMA). Although sequence diversity was detected, this was difficult to characterise because of the similarity in electrophoretic mobility of the homo- and heteroduplex bands. A PCR method was devised to generate synthetic polynucleotides of greater sequence diversity for use in the HMA. The presence of the synthetic 173-bp fragments was enriched by using, as template for the PCR, material excised from the area of the heteroduplex bands in stained electrophoresis gels. Nine novel sequences were generated and evaluated as reference sequences in the HMA. One of these with 20 bp different from the original sequence was selected for use in the HMA for improved resolution of heteroduplex and homoduplex bands and number of patterns easily resolved (nine different patterns corresponding to different DNA sequences). This method may be useful for analysis of DNA where there is limited natural variation or little sequence variation is described.

Animals↗
PubMed2001

Allergens of wild house dust mites: environmental Der p 1 and Der p 2 sequence polymorphisms.

BACKGROUND: Sequence diversity is a common feature of mite allergens. Previous studies, using predominantly commercial mite clones, have described several polymorphic residues for Der p 1 and Der p 2. OBJECTIVE: This study aimed at determining the occurrence of sequence diversity in environmental mite isolates. METHODS: Mites were isolated from houses in Perth and Sydney, Australia. Total RNA was extracted from 1 to 30 Perth mites, and cDNA was synthesized by reverse transcriptase PCR. Der p 1 and Der p 2 cDNAs were PCR amplified and sequenced. Genomic Der p 1 DNA was amplified from whole Sydney mites directly by PCR and then sequenced. RESULTS: Twelve Der p 1 and 9 Der p 2 cDNA clones and 3 Der p 1 genomic DNA were analyzed and showed a high frequency of amino acid polymorphisms. Der p 2 displayed a clear pattern of divergence toward 2 alleles that differed by 4 amino acids and had characteristic silent nucleotide changes. The pattern for Der p 1 was different and unusual, with almost no silent nucleotide substitutions but frequent sporadic missense changes. Proliferative responses of peripheral blood mononuclear cells to peptides containing polymorphic residues of Der p 1 were detected in 8 of 19 subjects, with stimulation being found only for either one of the variant forms of the peptides. However, the responses to variants of whole recombinant allergens were similar, as shown for 4 variants of Der p 2. CONCLUSION: Two clones for each of the allergens were identified as containing sequences that were largely representative of environmental isolates. A small-scale reverse transcriptase PCR used to produce cDNA from individual mites isolated from house dust will have wide application for studies on mite genetics and the production of recombinant mite allergens. Differences in T-cell responses to peptides representing variant epitopes were found, but responses to variants of whole recombinant allergens were similar. The GenBank and Swiss Prot database entries for Der p 1 (U11695) and Der p 2 (P49278) have been updated with the inclusion of the sequence polymorphisms described in this study.

Animals↗
PubMed2000

Sequence and diversity of DRB genes of Aotus nancymaae, a primate model for human malaria parasites.

The New World primate Aotus nancymaae is susceptible to infection with the human malaria parasite Plasmodium falciparum and Plasmodium vivax and has therefore been recommended by the World Health Organization as a model for evaluation of malaria vaccine candidates. We present here a first step in the molecular characterization of the major histocompatibility complex (MHC) class II DRB genes of Aotus nancymaae (owl monkey or night monkey) by nucleotide sequence analysis of the polymorphic exon 2 segments. In a group of 15 nonrelated animals captivated in the wild, 34 MHC DRB alleles could be identified. Six allelic lineages were detected, two of them having human counterparts, while two other lineages have not been described in any other New World monkey species studied. As in the common marmoset, the diversity of DRB alleles appears to have arisen largely by point mutations in the beta-pleated sheets and by frequent exchange of fixed sequence motifs in the alpha-helical portion. Pairs of alleles differing only at amino acid position b86 by an exchange of valine to glycine are present in Aotus, as in humans. Essential amino acid residues contributing to MHC DR peptide binding pockets number 1 and 4 are conserved or semiconserved between HLA-DR and Aona-DRB molecules, indicating a capacity to bind similar peptide repertoires. These results support fully our using Aotus monkeys as an animal model for evaluation of future subunit vaccine candidates.

Alleles↗
PubMed1997

Analysis of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments.

Denaturing gradient gel electrophoresis (DGGE) is a powerful and convenient tool for analyzing the sequence diversity of complex natural microbial populations. DGGE was evaluated for the identification of ammonia oxidizers of the beta subdivision of the Proteobacteria based on the mobility of PCR-amplified 16S rDNA fragments and for the analysis of mixtures of PCR products from this group generated by selective PCR of DNA extracted from coastal sand dunes. Degenerate PCR primers, CTO189f-GC and CTO654r, incorporating a 5' GC clamp, were designed to amplify a 465-bp 16S rDNA region spanning the V-2 and V-3 variable domains. The primers were tested against a representative selection of clones and cultures encompassing the currently recognized beta-subdivision ammonia oxidizer 16S rDNA sequence diversity. Analysis of these products by DGGE revealed that while many of the sequences could be separated, some which were known to be different migrated similarly in the denaturant system used. The CTO primer pair was used to amplify 16S rDNA sequences from DNA extracted from soil sampled from Dutch coastal dune locations of differing in pH and distance from the beach. The derived DGGE patterns were reproducible across multiple DNA isolations and PCRs. Ammonia oxidizer-like sequences from different phylogenetic groupings isolated from gene libraries made from the same sand dune DNA samples but prepared with different primers gave DGGE bands which comigrated with most of the bands detected from the sand dune samples. Bands from the DGGE gels of environmental samples were excised, reamplified, and directly sequenced, revealing strong similarity or identity of the recovered products to the corresponding regions of library clones. Six of the seven sequenced clusters of beta-subdivision ammonia oxidizers were detected in the dune systems, and differences in community structure between some sample sites were demonstrated. The most seaward dune site contained sequences showing affinity with sequence clusters previously isolated only from marine environments and was the only site where sequences relate to Nitrosomonas genes could be detected. Nitrosospira-like sequences were present in all sites, and there was some evidence of differences between Nitrosospira populations in acid and alkaline dune soils. Such differences in community structure may affect physiological differences within beta-subdivision ammonia oxidizers, with consequent effects on nitrification rates in response to key environmental factors.

Ammonia↗
PubMed2003

CagA tyrosine phosphorylation in gastric epithelial cells caused by Helicobacter pylori in patients with gastric adenocarcinoma.

BACKGROUND: Tyrosine phosphorylation of Helicobacter pylori cytotoxin-associated protein of in gastric epithelial cells is reported. The goals of this study are first to examine the occurrence of CagA tyrosine phosphorylation in H. pylori strains isolated from patients with gastric adenocarcinoma and gastritis, and second to clarify the relationship between the diversity of tyrosine phosphorylation motifs and the presence of CagA tyrosine phosphorylation. METHODS: Fifty-eight clinical isolates of H. pylori from patients with gastric adenocarcinoma (29 cases) and gastritis (29 cases) were studied for CagA tyrosine phosphorylation by Western blotting. Sequence diversity of tyrosine phosphorylation motifs was analysed among positive- or negative-CagA tyrosine phosphorylation isolates. RESULTS: Positive CagA tyrosine phosphorylation was found in 93.1% (27 of 29) of strains from gastric adenocarcinoma patients and 51.7% (15 of 29) of strains from gastritis patients (p < 0.001). Intact motifs were found in H. pylori isolates with CagA tyrosine phosphorylation. Of the 16 negative CagA tyrosine phosphorylation isolates, intact tyrosine phosphorylation motifs were found in 15 isolates. CONCLUSIONS: CagA tyrosine phosphorylation, which is significantly greater in strains from gastric adenocarcinoma patients, may play a role in gastric carcinogenesis, and could be a better marker of more virulent strains than the cag pathogenicity island in Asia, where the cag pathogenicity island is present in nearly all H. pylori strains. Sequence diversity of tyrosine phosphorylation motifs on CagA was not related to the presence of tyrosine phosphorylation. The absence of tyrosine phosphorylation motif might result in negative tyrosine phosphorylation phenotypes, but such motifs are not the sole factors associated with CagA tyrosine phosphorylation.

Adenocarcinoma↗
PubMed2005

Updating of transposable element annotations from large wheat genomic sequences reveals diverse activities and gene associations.

Triticeae species (including wheat, barley and rye) have huge and complex genomes due to polyploidization and a high content of transposable elements (TEs). TEs are known to play a major role in the structure and evolutionary dynamics of Triticeae genomes. During the last 5 years, substantial stretches of contiguous genomic sequence from various species of Triticeae have been generated, making it necessary to update and standardize TE annotations and nomenclature. In this study we propose standard procedures for these tasks, based on structure, nucleic acid and protein sequence homologies. We report statistical analyses of TE composition and distribution in large blocks of genomic sequences from wheat and barley. Altogether, 3.8 Mb of wheat sequence available in the databases was analyzed or re-analyzed, and compared with 1.3 Mb of re-annotated genomic sequences from barley. The wheat sequences were relatively gene-rich (one gene per 23.9 kb), although wheat gene-derived sequences represented only 7.8% (159 elements) of the total, while the remainder mainly comprised coding sequences found in TEs (54.7%, 751 elements). Class I elements [mainly long terminal repeat (LTR) retrotransposons] accounted for the major proportion of TEs, in terms of sequence length as well as element number (83.6% and 498, respectively). In addition, we show that the gene-rich sequences of wheat genome A seem to have a higher TE content than those of genomes B and D, or of barley gene-rich sequences. Moreover, among the various TE groups, MITEs were most often associated with genes: 43.1% of MITEs fell into this category. Finally, the TRIM and copia elements were shown to be the most active TEs in the wheat genome. The implications of these results for the evolution of diploid and polyploid wheat species are discussed.

DNA Transposable Elements↗
PubMed2004

DNA binding activity of the Escherichia coli nitric oxide sensor NorR suggests a conserved target sequence in diverse proteobacteria.

The Escherichia coli nitric oxide sensor NorR was shown to bind to the promoter region of the norVW transcription unit, forming at least two distinct complexes detectable by gel retardation. Three binding sites for NorR and two integration host factor binding sites were identified in the norR-norV intergenic region. The derived consensus sequence for NorR binding sites was used to search for novel members of the E. coli NorR regulon and to show that NorR binding sites are partially conserved in other members of the proteobacteria.

Amino Acid Sequence↗
PubMed2025

Comparative Genomics of Sex-Determination-Related Genes Reveals Shared Evolutionary Patterns Between Bivalves and Mammals, but Not Fruit Flies.

The molecular basis of sex determination (SD), while being extensively studied in model organisms, remains poorly understood in many animal groups. Bivalves, a diverse class of molluscs with a variety of reproductive modes, represent an ideal yet challenging clade for investigating SD and the evolution of sexual systems. However, the absence of a comprehensive framework has limited progress in this field, particularly regarding the study of sex-determination-related genes (SRGs). In this study, we performed a genome-wide sequence evolutionary analysis of the Dmrt, Sox and Fox gene families in more than 40 bivalve species. For the first time, we provide an extensive and phylogenetically aware dataset of these SRGs, and we find support for the hypothesis that Dmrt-1L and Sox-H may act as primary sex-determining genes by showing their high levels of sequence diversity within the bivalve genomic context. To validate our findings, we studied the same gene families in two well-characterised systems, mammals and fruit flies (genus Drosophila). In the former, we found that the male sex-determining gene Sry exhibits a pattern of amino acid sequence diversity similar to that of Dmrt-1L and Sox-H in bivalves, consistent with its role as master SD regulator. In contrast, no such pattern was observed among genes of the fruit fly SD cascade, which is controlled by a chromosomic mechanism. Overall, our findings highlight similarities in the sequence evolution of some mammal and bivalve SRGs, possibly driven by a comparable architecture of SD cascades. This work underscores once again the importance of employing a comparative approach when investigating understudied and non-model systems.

Animals↗