Number Of Nonsynonymous Substitutions Research Articles

Purifying selection in HIV-1 pol gene in perinatally HIV-1-infected children harboring discordant immunological response and virological nonresponse to long-term antiretroviral therapy

Background: Biological monitoring of antiretroviral treatment (ART) in human immunodeficiency virus (HIV)-infected pediatric population remains challenging. The aim of the present study was to assess the long-term HIV-1 genetic diversity in pol gene in HIV-1-infected children in virological failure under antiretroviral regimen adapted according to the successive World Health Organization (WHO) guidelines for resource-constrained settings. Methods: HIV-1 diversity in pol gene was assessed in HIV-1-infected children and adolescents born from HIV-infected mothers (median age at follow-up: 13.8 years) in virological failure (VF + ) despite long-term regimen recommended by the WHO. The numbers of nonsynonymous substitutions per potential nonsynonymous site (dN) and of synonymous substitutions at potential synonymous sites (dS) in HIV-1 pol gene and the dN/dS ratios were used to estimate the selective pressure on circulating HIV-1. Results: The immunological responses to ART basically corresponded to: 1) Full therapeutic failure with immunological (I - ) and virological nonresponses in one-quarter (24.6%) of study children ((I - , VF + ) subgroup); 2) Discordant immunovirological responses with paradoxical high CD4 T cell counts (I + ) and high HIV-1 RNA load in the remaining cohort patients (75.4%) ((I + , VF + ) subgroup). The mean dS was 1.8-fold higher in (I + , VF + ) than (I - , VF + ) subgroup (25.9 ± 18.4 vs. 14.3 ± 10.8). In the (I + , VF + ) subgroup, the mean dS was 1.6-fold higher than the mean dN. Finally, the mean dN/dS ratio was 2.1-fold lower in (I + , VF + ) than (I - , VF + ) subgroup (0.6 ± 0.3 vs. 1.3 ± 0.7), indicating purifying selection in the immunovirological discordant (I + , VF + ) subgroup and positive selection in the immunovirological failure (I - , VF + ) subgroup. Conclusions: Children and adolescents in immunovirological therapeutic failure harbor positive selection of HIV-1 strains favoring diversifying in pol -encoded amino acids. In contrast, children with persistent discordant immunovirological responses show accumulation of mutations and purifying selection in pol gene sequences, indicating limited genetic evolution and likely suggesting genetic adaptation of viruses to host functional constraints. J Clin Med Res. 2020;12(6):369-376 doi: https://doi.org/10.14740/jocmr4157

Purifying Selection in Human Immunodeficiency Virus-1 pol Gene in Perinatally Human Immunodeficiency Virus-1-Infected Children Harboring Discordant Immunological Response and Virological Nonresponse to Long-Term Antiretroviral Therapy.

BackgroundBiological monitoring of antiretroviral treatment (ART) in human immunodeficiency virus (HIV)-infected pediatric population remains challenging. The aim of the present study was to assess the long-term HIV-1 genetic diversity in pol gene in HIV-1-infected children in virological failure under antiretroviral regimen adapted according to the successive World Health Organization (WHO) guidelines for resource-constrained settings.MethodsHIV-1 diversity in pol gene was assessed in HIV-1-infected children and adolescents born from HIV-infected mothers (median age at follow-up: 13.8 years) in virological failure (VF+) despite long-term regimen recommended by the WHO. The numbers of nonsynonymous substitutions per potential nonsynonymous site (dN) and of synonymous substitutions at potential synonymous sites (dS) in HIV-1 pol gene and the dN/dS ratios were used to estimate the selective pressure on circulating HIV-1.ResultsThe immunological responses to ART basically corresponded to: 1) Full therapeutic failure with immunological (I-) and virological nonresponses in one-quarter (24.6%) of study children ((I-, VF+) subgroup); 2) Discordant immunovirological responses with paradoxical high CD4 T cell counts (I+) and high HIV-1 RNA load in the remaining cohort patients (75.4%) ((I+, VF+) subgroup). The mean dS was 1.8-fold higher in (I+, VF+) than (I-, VF+) subgroup (25.9 ± 18.4 vs. 14.3 ± 10.8). In the (I+, VF+) subgroup, the mean dS was 1.6-fold higher than the mean dN. Finally, the mean dN/dS ratio was 2.1-fold lower in (I+, VF+) than (I-, VF+) subgroup (0.6 ± 0.3 vs. 1.3 ± 0.7), indicating purifying selection in the immunovirological discordant (I+, VF+) subgroup and positive selection in the immunovirological failure (I-, VF+) subgroup.ConclusionsChildren and adolescents in immunovirological therapeutic failure harbor positive selection of HIV-1 strains favoring diversifying in pol-encoded amino acids. In contrast, children with persistent discordant immunovirological responses show accumulation of mutations and purifying selection in pol gene sequences, indicating limited genetic evolution and likely suggesting genetic adaptation of viruses to host functional constraints.

Genetic Variation in Coat Colour Genes MC1R and ASIP Provides Insights Into Domestication and Management of South American Camelids.

The domestication of wild vicuña and guanaco by early pre-Inca cultures is an iconic example of wildlife management and domestication in the Americas. Although domestic llamas and alpacas were clearly selected for key, yet distinct, phenotypic traits, the relative patterns and direction of selection and domestication have not been confirmed using genetic approaches. However, the detailed archaeological records from the region suggest that domestication was a process carried out under significant control and planning, which would have facilitated coordinated and thus extremely effective selective pressure to achieve and maintain desired phenotypic traits. Here we link patterns of sequence variation in two well-characterised genes coding for colour variation in vertebrates and interpret the results in the context of domestication in guanacos and vicuñas. We hypothesise that colour variation in wild populations of guanacos and vicunas were strongly selected against. In contrast, variation in coat colour variation in alpaca was strongly selected for and became rapidly fixed in alpacas. In contrast, coat colour variants in llamas were of less economic value, and thus were under less selective pressure. We report for the first time the full sequence of MC1R and 3 exons of ASIP in 171 wild specimens from throughout their distribution and which represented a range of commonly observed colour patterns. We found a significant difference in the number of non-synonymous substitutions, but not synonymous substitutions among wild and domestics species. The genetic variation in MC1R and ASIP did not differentiate alpaca from llama due to the high degree of reciprocal introgression, but the combination of 11 substitutions are sufficient to distinguish domestic from wild animals. Although there is gene flow among domestic and wild species, most of the non-synonymous variation in MC1R and ASIP was not observed in wild species, presumably because these substitutions and the associated colour phenotypes are not effectively transmitted back into wild populations. Therefore, this set of substitutions unequivocally differentiates wild from domestic animals, which will have important practical application in forensic cases involving the poaching of wild vicuñas and guanacos. These markers will also assist in identifying and studying archaeological remains pre- and post-domestication.

Evolution of the SRGAP2 Gene Is Linked to Intelligence in Mammals

Background/Aims: A recent duplication of the gene encoding SLIT-ROBO Rho GTPase-activating protein 2 (SRGAP2) in the primate lineage has been proposed to be associated with the human-specific extraordinary development of intelligence. There is no report regarding the role of the SRGAP2 gene in the expression of neural traits indicating intelligence in mammals. Methods: A phylogenetic tree of the SRGAP2 gene from 11 mammals was reconstructed using MrBayes. The evolution of neural traits along the branches of the phylogenetic tree was modeled in the BayesTraits, and the dN/dS ratio (i.e. the ratio between the number of nonsynonymous substitutions per nonsynonymous site and the number of synonymous substitutions per synonymous site) was estimated using the codon-based maximum likelihood method (CODEML) in PAML (phylogenetic analysis by maximum likelihood). Results: Two neural traits, namely brain mass and the number of cortical neurons, showed statistical dependency on the underlying evolutionary history of the SRGAP2 gene in mammals. A significant positive correlation between the increase in cortical neurons and the rate of nucleotide substitutions in the SRGAP2 gene was observed concomitantly with a significant negative correlation between the increase in cortical neurons and the rate of nonsynonymous substitutions in the gene. The SRGAP2 gene appears to be under intense pressure of purifying selection in all mammalian lineages under stringent functional constraint. Conclusion: This work indicates a key role of the SRGAP2 gene in the rapid expansion of neurons in the brain cortex, thereby facilitating the evolution of remarkable intelligence in mammals.

A novel astrovirus from dromedaries in the Middle East.

The recent emergence of Middle East respiratory syndrome coronavirus from the Middle East and its discovery from dromedary camels has boosted interest in the search for novel viruses in dromedaries. The existence of astroviruses (AstVs) in dromedaries was previously unknown. We describe the discovery of a novel dromedary camel AstV (DcAstV) from dromedaries in Dubai. Among 215 dromedaries, DcAstV was detected in faecal samples of four [three (1.5 %) adult dromedaries and one (8.3 %) dromedary calf] by reverse transcription-PCR. Sequencing of the four DcAstV genomes and phylogenetic analysis showed that the DcAstVs formed a distinct cluster. Although DcAstV was most closely related to a recently characterized porcine AstV 2, their capsid proteins only shared 60-66 % amino acid identity, with a mean amino acid genetic distance of 0.372. Notably, the N-terminal halves of the capsid proteins of DcAstV shared ≤ 85 % amino acid identity, but the C-terminal halves only shared ≤ 49 % amino acid identity compared with the corresponding proteins in other AstVs. A high variation of the genome sequences of DcAstV was also observed, with a mean amino acid genetic distance of 0.214 for ORF2 of the four strains. Recombination analysis revealed a possible recombination event in ORF2 of strain DcAstV-274. The low Ka/Ks ratios (number of non-synonymous substitutions per non-synonymous site to number of synonymous substitutions per synonymous site) of the four ORFs in the DcAstV genomes supported the suggestion that dromedaries are the natural reservoir where AstV is stably evolving. These results suggest that AstV is a novel species of the genus Mamastrovirus in the family Astroviridae. Further studies are important to understand the pathogenic potential of DcAstV.

Variability of human immunodeficiency virus-1 in the female genital reservoir during genital reactivation of herpes simplex virus type 2

Clinical and subclinical genital herpes simplex virus type 2 (HSV-2) reactivations have been associated with increases in human immunodeficiency virus (HIV)-1 genital shedding. Whether HSV-2 shedding contributes to the selection of specific genital HIV-1 variants remains unknown. We evaluated the genetic diversity of genital and blood HIV-1 RNA and DNA in 14 HIV-1/HSV-2-co-infected women, including seven with HSV-2 genital reactivation, and seven without as controls. HIV-1 DNA and HIV-1 RNA env V1–V3 sequences in paired blood and genital samples were compared. The HSV-2 selection pressure on HIV was estimated according to the number of synonymous substitutions (dS), the number of non-synonymous substitutions (dN) and the dS/dN ratio within HIV quasi-species. HIV-1 RNA levels in cervicovaginal secretions were higher in women with HSV-2 replication than in controls (p0.02). Plasma HIV-1 RNA and genital HIV-1 RNA and DNA were genetically compartmentalized. No differences in dS, dN and the dS/dN ratio were observed between the study groups for either genital HIV-1 RNA or plasma HIV-1 RNA. In contrast, dS and dN in genital HIV-1 DNA were significantly higher in patients with HSV-2 genital reactivation (p <0.01 and p <0.05, respectively). The mean of the dS/dN ratio in genital HIV-1 DNA was slightly higher in patients with HSV-2 genital replication, indicating a trend for purifying selection (p 0.056). HSV-2 increased the genetic diversity of genital HIV-1 DNA. These observations confirm molecular interactions between HSV-2 and HIV-1 at the genital tract level.

SearchDOGS Bacteria, Software That Provides Automated Identification of Potentially Missed Genes in Annotated Bacterial Genomes

We report the development of SearchDOGS Bacteria, software to automatically detect missing genes in annotated bacterial genomes by combining BLAST searches with comparative genomics. Having successfully applied the approach to yeast genomes, we redeveloped SearchDOGS to function as a standalone, downloadable package, requiring only a set of GenBank annotation files as input. The software automatically generates a homology structure using reciprocal BLAST and a synteny-based method; this is followed by a scan of the entire genome of each species for unannotated genes. Results are provided in a HTML interface, providing coordinates, BLAST results, syntenic location, omega values (Ka/Ks, where Ks is the number of synonymous substitutions per synonymous site and Ka is the number of nonsynonymous substitutions per nonsynonymous site) for protein conservation estimates, and other information for each candidate gene. Using SearchDOGS Bacteria, we identified 155 gene candidates in the Shigella boydii sb227 genome, including 56 candidates of length < 60 codons. SearchDOGS Bacteria has two major advantages over currently available annotation software. First, it outperforms current methods in terms of sensitivity and is highly effective at identifying small or highly diverged genes. Second, as a freely downloadable package, it can be used with unpublished or confidential data.

Multilocus sequence typing of Lactococcus lactis from naturally fermented milk foods in ethnic minority areas of China

To determine the genetic diversity and phylogenetic relationships among Lactococcus lactis isolates, 197 strains isolated from naturally homemade yogurt in 9 ethnic minority areas of 6 provinces of China were subjected to multilocus sequence typing (MLST). The MLST analysis was performed using internal fragment sequences of 12 housekeeping genes (carB, clpX, dnaA, groEL, murC, murE, pepN, pepX, pyrG, recA, rpoB, and pheS). Six (dnaA) to 8 (murC) different alleles were detected for these genes, which ranged from 33.62 (clpX) to 41.95% (recA) GC (guanine-cytosine) content. The nucleotide diversity (π) ranged from 0.00362 (murE) to 0.08439 (carB). Despite this limited allelic diversity, the allele combinations of each strain revealed 72 different sequence types, which denoted significant genotypic diversity. The dN/dS ratios (where dS is the number of synonymous substitutions per synonymous site, and dN is the number of nonsynonymous substitutions per nonsynonymous site) were lower than 1, suggesting potential negative selection for these genes. The standardized index of association of the alleles IAS=0.3038 supported the clonality of Lc. lactis, but the presence of network structure revealed by the split decomposition analysis of the concatenated sequence was strong evidence for intraspecies recombination. Therefore, this suggests that recombination contributed to the evolution of Lc. lactis. A minimum spanning tree analysis of the 197 isolates identified 14 clonal complexes and 23 singletons. Phylogenetic trees were constructed based on the sequence types, using the minimum evolution algorithm, and on the concatenated sequence (6,192 bp), using the unweighted pair-group method with arithmetic mean, and these trees indicated that the evolution of our Lc. lactis population was correlated with geographic origin. Taken together, our results demonstrated that MLST could provide a better understanding of Lc. lactis genome evolution, as well as useful information for future studies on global Lc. lactis structure and genetic evolution, which will lay the foundation for screening Lc. lactis as starter cultures in fermented dairy products.

Molecular epidemiology and genetic characterization of hepatitis B virus in the Indian subcontinent

Hepatitis B virus (HBV) is a gradually evolving virus. The aim of this study was to characterize the distribution pattern of HBV genotypes and subgenotypes and HBsAg subtypes in chronic hepatitis B subjects from the Indian subcontinent. We also sought to investigate the genetic diversity of HBV genotypes and its influence on the therapeutic response. A total of 295 chronic hepatitis B subjects were studied. HBV genotypes and subgenotypes were determined using the generated HBV reverse transcriptase (rt) sequences. HBsAg subtypes were predicted using a newly developed automated program in Microsoft Visual Basic (VB6). Genetic diversity was characterized by calculating the mean genetic distance (d), the number of synonymous substitutions per synonymous site (dS), and the number of non-synonymous substitutions per non-synonymous site (dN). The virological response was measured by HBV DNA levels. In southern India, the predominant HBV subgenotype/subtype was D2/ayw3 (79.1%). In eastern India, C1/adr (28.2%) was found to be the predominant subgenotype/subtype, followed by A1/adw2 (25.4%). In the north-eastern region, C2/adr, D2/ayw3, and D5/ayw3 were predominant and were each identified in 20.8% of subjects. In treatment-naïve subjects, the d, dS, and dN of genotype D sequences were higher compared to genotypes C and A. Additionally, the d, dS, and dN of HBV rt sequence were higher in subjects who subsequently showed a virological response to nucleos(t)ide analogues as compared to non-responders, irrespective of the genotypes tested (p=0.014 to p<0.0001). We have described the distribution of HBV genotypes and subgenotypes and HBsAg subtypes in three major regions of the Indian subcontinent. HBV genetic diversity may play a pivotal role in the clinical outcome of chronic hepatitis B.

Synthetic biology of phenotypic adaptation in vertebrates: the next frontier.

For over the last 2 decades, positively selected amino acid sites have been inferred almost exclusively by showing that the number of nonsynonymous substitutions per nonsynonymous site (dn) is greater than that of synonymous substitutions per synonymous site (ds). However, virtually none of these statistical results have been experimentally tested and remain as hypotheses. To perform such experimental tests, we must connect genotype and phenotype and relate the phenotypic changes to the environmental and behavioral changes of the organism. The genotype-phenotype relationship can be established only by synthesizing and manipulating "proper" ancestral phenotypes, whereas the actual functions of adaptive mutations can be learned by studying their chemical roles in phenotypic changes.

Selection pressure on the hepatitis B virus pre-S/S and P open reading frames in Tongan subjects with a chronic hepatitis B virus infection

Identification of the full repertoire of hepatitis B virus (HBV) peptides that are presented to CD8+ T cells by common HLA class I alleles will be useful for designing immunotherapies for chronic hepatitis B. One hundred and seventy five cloned sequences containing the pre-S/S and P open reading frames (ORF) of the HBV were obtained from serum HBV-DNA of HBeAg-positive (n=4) and HBeAg-negative (inactive healthy carriers (IHC), n=16) Tongan subjects with an inactive chronic HBV infection. In addition, 34 and 32 sequences were obtained 5.2±1.4 (mean±SD) years apart from eight subjects. PAML was used to identify codons in the pre-S/S and P ORFs that were under positive selection pressure (ω>1). The number of non-synonymous substitutions in these codons was compared in IHC who were homozygous for either HLA-B∗4001 (n=9) or HLA-B*5602 (n=7), and who were either positive (n=6) or negative (n=10) for HLA-A*02. 34 codons in the pre-S/S and 11 codons in the P ORFs were under positive selection pressure. There was a higher number of non-synonymous substitutions in these codons in HBeAg-negative versus HBeAg-positive subjects in the P (p=0.02) but not the pre-S/S (p=0.64) ORF. There was no association between any HLA class I allele and non-synonymous substitutions in these codons. There was no increase in positive selection pressure on the pre-S/S and P ORFs with time. In conclusion, we could not find HLA class I-restricted selection pressure on any pre-S/S or P ORF amino acid; raising the possibility that peptide-based immunotherapies for chronic hepatitis B may not require peptides from these ORFs.

Identification of ORF sequences and exercise-induced expression change in thoroughbred horse OXCT1 gene

In the mitochondrial matrix, the OXCT1 gene catalyzes the reversible transfer of coenzyme A from succinyl-CoA to acetoacetate in a reaction related to energy production from ketone bodies. Here, horse OXCT1 gene containing coenzyme A transferase domain was identified in the transcriptome analysis of cDNAs derived from skeletal muscles. Horse OXCT1 gene consisted of 1761 nucleotide sequences with an open reading frame of 1560 nucleotides encoding a protein of 520 putative amino acid residues. The number of non-synonymous substitutions was lower than the number of synonymous substitutions in the OXCT1 genes of other species, indicating that purifying selection occurred in the OXCT1 genes during evolutionary radiation. Quantitative real-time RT-RCR analysis showed a dominant expression pattern of horse OXCT1 gene in the cerebrum, heart, and skeletal muscle. Different expression levels of horse OXCT1 transcripts between before- and after-exercise samples were also measured in the skeletal muscles of six horses. These data could be of great use for further investigation of the relationship between energy products and horse OXCT1 gene.

Molecular cloning, polymorphism and tissue distribution of the MHC class IIB gene in the Chinese goose (Anser cygnoides)

1. The goose major histocompatibility complex (MHC) class IIB cDNA (Ancy-MHCII) was cloned by homology cloning and rapid amplification of cDNA ends by polymerase chain reaction (RACE-PCR), and the genomic structure and tissue expression were investigated. 2. Three different 5′-RACE sequences (Ancy-MHC II5′-1, Ancy-MHC II5′-2, Ancy-MHC II5′-3), one 3′-RACE sequence (Ancy-MHC II-3′) and two different full length Ancy-MHC IIB cDNA sequences (Ancy-CD01, Ancy-CD02), which came from different alleles at one locus or different loci, were determined. 3. The genomic organisation is composed of 6 exons and 5 introns, with a longer intron region than that of the chicken. The alleles encode 259 and 260 amino acids in the mature protein. 4. The number of non-synonymous substitutions (dN) in the peptide-binding region of exon 2 from 8 alleles was higher than that of the synonymous substitutions (dS). 5. Tissue-specific expression of Ancy-MHC II mRNA was detected in an adult goose using RT-PCR. These results showed that Ancy-MHC II mRNA was expressed in the lung, spleen, liver, intestine, heart, kidney, pancreas, brain, skin and muscle. This is consistent with the expression of MHC class IIB in various tissues from the chicken. 6. Sequences from goose, snipe and duck clustered together when compared with known MHC class IIB sequences from the other species, significantly differing from mammals and aquatic species, indicating a pattern consistent with accepted evolutionary pathways.

Patterns of variation of the major histocompatibility complex class IIB loci in Chinese goose (Anser Cygnoides)

In order to understand the variations of genomic organization of the major histocompatibility complex (MHC) and provide data for the studies on disease resistance of avian species, the MHC class II polymorphism in Chinese Z-goose was investigated for the first time in the present study. Eight alleles, which probably came from different loci, were found in six different geese with only one obvious band in the restriction fragment length polymorphism data. The numbers of nonsynonymous substitutions (dN) in peptide binding region of exon 2 were higher than that of synonymous substitutions (dS), and no stop codons or frameshift mutations were found in this region, indicating that balance selection was in operation, and the sequences are not likely to be pseudogenes. In addition, we successfully obtained five different long MHC class II fragments (about 1,162 bp) in six geese and found that the length of intron 1 was longer than that in chicken and some other birds, but intron 2 seemed to be intermediate in length. The phylogenetic tree appeared to branch in an order consistent with accepted evolutionary pathway.

Complete mitochondrial genome sequence of three Tetrahymena species reveals mutation hot spots and accelerated nonsynonymous substitutions in Ymf genes.

The ciliate Tetrahymena, a model organism, contains divergent mitochondrial (Mt) genome with unusual properties, where half of its 44 genes still remain without a definitive function. These genes could be categorized into two major groups of KPC (known protein coding) and Ymf (genes without an identified function). To gain insights into the mechanisms underlying gene divergence and molecular evolution of Tetrahymena (T.) Mt genomes, we sequenced three Mt genomes of T.paravorax, T.pigmentosa, and T.malaccensis. These genomes were aligned and the analyses were carried out using several programs that calculate distance, nucleotide substitution (dn/ds), and their rate ratios (ω) on individual codon sites and via a sliding window approach. Comparative genomic analysis indicated a conserved putative transcription control sequence, a GC box, in a region where presumably transcription and replication initiate. We also found distinct features in Mt genome of T.paravorax despite similar genome organization among these ∼47 kb long linear genomes. Another significant finding was the presence of at least one or more highly variable regions in Ymf genes where majority of substitutions were concentrated. These regions were mutation hotspots where elevated distances and the dn/ds ratios were primarily due to an increase in the number of nonsynonymous substitutions, suggesting relaxed selective constraint. However, in a few Ymf genes, accelerated rates of nonsynonymous substitutions may be due to positive selection. Similarly, on protein level the majority of amino acid replacements occurred in these regions. Ymf genes comprise half of the genes in Tetrahymena Mt genomes, so understanding why they have not been assigned definitive functions is an important aspect of molecular evolution. Importantly, nucleotide substitution types and rates suggest possible reasons for not being able to find homologues for Ymf genes. Additionally, comparative genomic analysis of complete Mt genomes is essential in identifying biologically significant motifs such as control regions.

Sequence Variation in theTbx4Gene in Marine Mammals

The amino-acid sequences of the T-domain region of the Tbx4 gene, which is required for hindlimb development, are 100% identical in humans and mice. Cetaceans have lost most of their hindlimb structure, although hindlimb buds are present in very early cetacean embryos. To examine whether the Tbx4 gene has the same function in cetaceans as in other mammals, we analyzed Tbx4 sequences from cetaceans, dugong, artiodactyls and marine carnivores. A total of 39 primers were designed using human and dog Tbx4 nucleotide sequences. Exons 3, 4, 5, 6, 7, and 8 of the Tbx4 genes from cetaceans, artiodactyls, and marine carnivores were sequenced. Non-synonymous substitution sites were detected in the T-domain regions from some cetacean species, but were not detected in those from artiodactyls, the dugong, or the carnivores. The C-terminal regions contained a number of non-synonymous substitutions. Although some indels were present, they were in groups of three nucleotides and therefore did not cause frame shifts. The dN/dS values for the T-domain and C-terminal regions of the cetacean and artiodactylous Tbx4 genes were much lower than 1, indicating that the Tbx4 gene maintains it function in cetaceans, although full expression leading to hindlimb development is suppressed.

P1176 Performance of an automated chemiluminescent assay for the detection of anti-hepatitis A virus IgM antibodies: Access® HAV IgM assay

Hepatitis B virus (HBV) is a gradually evolving virus. The aim of this study was to characterize the distribution pattern of HBV genotypes and subgenotypes and HBsAg subtypes in chronic hepatitis B subjects from the Indian subcontinent. We also sought to investigate the genetic diversity of HBV genotypes and its influence on the therapeutic response.A total of 295 chronic hepatitis B subjects were studied. HBV genotypes and subgenotypes were determined using the generated HBV reverse transcriptase (rt) sequences. HBsAg subtypes were predicted using a newly developed automated program in Microsoft Visual Basic (VB6). Genetic diversity was characterized by calculating the mean genetic distance (d), the number of synonymous substitutions per synonymous site (dS), and the number of non-synonymous substitutions per non-synonymous site (dN). The virological response was measured by HBV DNA levels.In southern India, the predominant HBV subgenotype/subtype was D2/ayw3 (79.1%). In eastern India, C1/adr (28.2%) was found to be the predominant subgenotype/subtype, followed by A1/adw2 (25.4%). In the north-eastern region, C2/adr, D2/ayw3, and D5/ayw3 were predominant and were each identified in 20.8% of subjects. In treatment-naïve subjects, the d, dS, and dN of genotype D sequences were higher compared to genotypes C and A. Additionally, the d, dS, and dN of HBV rt sequence were higher in subjects who subsequently showed a virological response to nucleos(t)ide analogues as compared to non-responders, irrespective of the genotypes tested (p = 0.014 to p < 0.0001).We have described the distribution of HBV genotypes and subgenotypes and HBsAg subtypes in three major regions of the Indian subcontinent. HBV genetic diversity may play a pivotal role in the clinical outcome of chronic hepatitis B.

Amino acid sequence constraint and gene expression pattern across the life history in the malaria parasite Plasmodium falciparum

The relationship between gene expression across the life cycle and protein conservation in Plasmodium falciparum was examined by comparing gene expression data for six life-history stages with the number of nonsynonymous substitutions per site ( d N) between 901 orthologous gene pairs of P. falciparum and Plasmodium yoelii. A high level of expression across the life history was associated with decreased d N and thus with protein conservation. By contrast, differential expression in the sporozoite and merozoite stages was associated with increased d N. At least some sporozoite- and merozoite-expressed genes with high d N have probably been subject to positive selection arising from parasite–host coevolution. A high level of expression across the life history was associated with higher than average G + C content at the first and second codon positions, whereas a high level of expression in the sporozoite and merozoite was associated with reduced G + C content at the first and second codon positions, the latter pattern evidently reflecting the relaxation of constraint on the amino acid sequence.

CD81 binding regions of hepatitis C virus remain conserved after liver transplantation.

CD81 is a surface-associated protein expressed in the membranes of mammalian cells. It has been suggested that CD81 interacts with hepatitis C virus E2 protein, and thus might facilitate the entry of HCV into hepatocytes. The envelope-binding site appears to involve amino acids (aa) 480-493 and 544-551 within the E2 glycoprotein. Little is known about the quasispecies genetic diversity of these two regions. We studied four patients who underwent transplantation for HCV-related cirrhosis and who developed recurrent hepatitis C. We evaluated HCV quasispecies diversity in serum samples obtained at the time of transplantation and at several time points thereafter. Quasispecies diversity was assessed by cloning and sequencing of viral isolates, with computer analysis of evolution models. The genetic distance in the region that spans aa 480 to 493 was 0.019 +/- 0.004 before the transplant, and 0.039 +/- 0.014 after the transplant (p=0.324). In the aa 544 to 551 region, the pre-transplant genetic distance was 0.012 +/- 0.008 and the post-transplant distance, 0.010 +/- 0.007 (p=0.890). There was also no significant difference between the number of nonsynonymous substitutions per nonsynonymous site before and after transplantation. In conclusion, the HCV genetic sequences of putative CD81 binding regions aa 480-493 and aa 544-551 did not diversify significantly after liver transplantation. This may favor HCV re-infection of the allograft after liver transplantation.

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CD81 is a surface-associated protein expressed in the membranes of mammalian cells. It has been suggested that CD81 interacts with hepatitis C virus E2 protein, and thus might facilitate the entry of HCV into hepatocytes. The envelope-binding site appears to involve amino acids (aa) 480-493 and 544-551 within the E2 glycoprotein. Little is known about the quasispecies genetic diversity of these two regions. We studied four patients who underwent transplantation for HCV-related cirrhosis and who developed recurrent hepatitis C. We evaluated HCV quasispecies diversity in serum samples obtained at the time of transplantation and at several time points thereafter. Quasispecies diversity was assessed by cloning and sequencing of viral isolates, with computer analysis of evolution models. The genetic distance in the region that spans aa 480 to 493 was 0.019 +/- 0.004 before the transplant, and 0.039 +/- 0.014 after the transplant (p=0.324). In the aa 544 to 551 region, the pre-transplant genetic distance was 0.012 +/- 0.008 and the post-transplant distance, 0.010 +/- 0.007 (p=0.890). There was also no significant difference between the number of nonsynonymous substitutions per nonsynonymous site before and after transplantation. In conclusion, the HCV genetic sequences of putative CD81 binding regions aa 480-493 and aa 544-551 did not diversify significantly after liver transplantation. This may favor HCV re-infection of the allograft after liver transplantation.