Population genetic estimation of the loss of genetic diversity during horizontal transmission of HIV-1

Background  

Genetic diversity of the human immunodeficiency virus type 1 (HIV-1) population within an individual is lost during transmission to a new host. The demography of transmission is an important determinant of evolutionary dynamics, particularly the relative impact of natural selection and genetic drift immediately following HIV-1 infection. Despite this, the magnitude of this population bottleneck is unclear.     

HIV-1 Vpr: genetic diversity and functional features from the perspective of structure

RNA viruses are well known for the enormous genetic variation. Retroviruses share this feature with other RNA viruses, and human immunodeficiency virus type 1 (HIV-1) has been extensively investigated in this regard. Based on the DNA sequence analysis, HIV-1 has been classified into three groups; M, N, and O, with viral subtypes in each group. While the genetic variation between viral isolates has been documented throughout the genome, specifically, the env gene exhibits high variation. Analysis of the env gene from the sequential samples from HIV-1-infected patients reveals variation in the range of 1% per year. The variation observed in individual HIV-1 genes in the form of changes at the nucleotide level, as expected, should result in one of the possible scenarios: (1) no change in the amino acid, (2) conservative change in the amino acid, (3) nonconservative change in the amino acid, and (4) premature stop codon resulting in a truncated protein. Hence, it is likely that the variation may impact on the function of the protein, depending on the nature of the mutation. The goal of this review is to summarize the polymorphisms in Vpr using the available sequence information and discuss their effects on the functions of Vpr from the point of view of its structure. The data generated by several groups provide a base for understanding the consequences of natural polymorphisms in specific regions of the Vpr molecule. However, it is also clear that secondary changes (second site or compensatory mutations) may modify the effect of a specific mutation and a comprehensive analysis is needed to delineate the role of specific residues in Vpr molecule. This is an area which, we hope, will attract investigators for further studies, and may provide information for understanding the molecular basis of Vpr functions.     

A comparison of the 16S ribosomal RNAs from mesophilic and thermophilic bacilli: Some modifications in the sanger method for RNA sequencing

Summary Two modifications in the Sanger two dimensional electrophoretic procedure for RNA analysis are reported. One increases resolution on the primary fingerprint to the point that digests of large RNAs, of the size 1500–3000 nucleotides yield well resolved fingerprint patterns. The other is a novel endonucleolytic procedure that proves useful in determining sequences of the large oligonucleotides produced by T1 ribonuclease.These modifications have been used in determining the catalogs of oligomers produced by T1 ribonuclease digestion of 16S rRNAs from three related organisms,Bacillus subtilis, B.pumilus andB.stearothermophilus. The possible effects of adaptation to a thermophilic niche on ribosomal RNA primary structure and the phylogenetic relatedness of the two mesophilic Bacilli are discussed.This is contribution No.6 in a series on procaryote phylogeny.     

Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing

Background

Influenza viruses exist as a large group of closely related viral genomes, also called quasispecies. The composition of this influenza viral quasispecies can be determined by an accurate and sensitive sequencing technique and data analysis pipeline. We compared the suitability of two benchtop next-generation sequencers for whole genome influenza A quasispecies analysis: the Illumina MiSeq sequencing-by-synthesis and the Ion Torrent PGM semiconductor sequencing technique.

Results

We first compared the accuracy and sensitivity of both sequencers using plasmid DNA and different ratios of wild type and mutant plasmid. Illumina MiSeq sequencing reads were one and a half times more accurate than those of the Ion Torrent PGM. The majority of sequencing errors were substitutions on the Illumina MiSeq and insertions and deletions, mostly in homopolymer regions, on the Ion Torrent PGM. To evaluate the suitability of the two techniques for determining the genome diversity of influenza A virus, we generated plasmid-derived PR8 virus and grew this virus in vitro. We also optimized an RT-PCR protocol to obtain uniform coverage of all eight genomic RNA segments. The sequencing reads obtained with both sequencers could successfully be assembled de novo into the segmented influenza virus genome. After mapping of the reads to the reference genome, we found that the detection limit for reliable recognition of variants in the viral genome required a frequency of 0.5% or higher. This threshold exceeds the background error rate resulting from the RT-PCR reaction and the sequencing method. Most of the variants in the PR8 virus genome were present in hemagglutinin, and these mutations were detected by both sequencers.

Conclusions

Our approach underlines the power and limitations of two commonly used next-generation sequencers for the analysis of influenza virus gene diversity. We conclude that the Illumina MiSeq platform is better suited for detecting variant sequences whereas the Ion Torrent PGM platform has a shorter turnaround time. The data analysis pipeline that we propose here will also help to standardize variant calling in small RNA genomes based on next-generation sequencing data.     

Assessment of microbial diversity bias associated with soil heterogeneity and sequencing resolution in pyrosequencing analyses

It is important to estimate the true microbial diversities accurately for a comparative microbial diversity analysis among various ecological settings in ecological models. Despite drastically increasing amounts of 16S rRNA gene targeting pyrosequencing data, sampling and data interpretation for comparative analysis have not yet been standardized. For more accurate bacterial diversity analyses, the influences of soil heterogeneity and sequence resolution on bacterial diversity estimates were investigated using pyrosequencing data of oak and pine forest soils with focus on the bacterial 16SrRNA gene. Soil bacterial community sets were phylogenetically clustered into two separate groups by forest type. Rarefaction curves showed that bacterial communities sequenced from the DNA mixtures and the DNAs of the soil mixtures hadmidsize richness compared with other samples. Richness and diversity estimates were highly variable depending on the sequence read numbers. Bacterial richness estimates (ACE, Chao 1 and Jack) of the forest soils had positive linear relationships with the sequence read number. Bacterial diversity estimates (NPShannon, Shannon and the inverse Simpson) of the forest soils were also positively correlated with the sequence read number. One-way ANOVA shows that sequence resolution significantly affected the a-diversity indices (P<0.05), but the soil heterogeneity did not (P>0.05). For an unbiased evaluation, richness and diversity estimates should be calculated and compared from subsets of the same size.     

A comparison of rpoB and 16S rRNA as markers in pyrosequencing studies of bacterial diversity

Background

The 16S rRNA gene is the gold standard in molecular surveys of bacterial and archaeal diversity, but it has the disadvantages that it is often multiple-copy, has little resolution below the species level and cannot be readily interpreted in an evolutionary framework. We compared the 16S rRNA marker with the single-copy, protein-coding rpoB marker by amplifying and sequencing both from a single soil sample. Because the higher genetic resolution of the rpoB gene prohibits its use as a universal marker, we employed consensus-degenerate primers targeting the Proteobacteria.

Methodology/Principal Findings

Pyrosequencing can be problematic because of the poor resolution of homopolymer runs. As these erroneous runs disrupt the reading frame of protein-coding sequences, removal of sequences containing nonsense mutations was found to be a valuable filter in addition to flowgram-based denoising. Although both markers gave similar estimates of total diversity, the rpoB marker revealed more species, requiring an order of magnitude fewer reads to obtain 90% of the true diversity. The application of population genetic methods was demonstrated on a particularly abundant sequence cluster.

Conclusions/Significance

The rpoB marker can be a complement to the 16S rRNA marker for high throughput microbial diversity studies focusing on specific taxonomic groups. Additional error filtering is possible and tests for recombination or selection can be employed.     

The impact of recent events on human genetic diversity

The historical record tells us stories of migrations, population expansions and colonization events in the last few thousand years, but what was their demographic impact? Genetics can throw light on this issue, and has mostly done so through the maternally inherited mitochondrial DNA (mtDNA) and the male-specific Y chromosome. However, there are a number of problems, including marker ascertainment bias, possible influences of natural selection, and the obscuring layers of the palimpsest of historical and prehistorical events. Y-chromosomal lineages are particularly affected by genetic drift, which can be accentuated by recent social selection. A diversity of approaches to expansions in Europe is yielding insights into the histories of Phoenicians, Roma, Anglo-Saxons and Vikings, and new methods for producing and analysing genome-wide data hold much promise. The field would benefit from more consensus on appropriate methods, and better communication between geneticists and experts in other disciplines, such as history, archaeology and linguistics.     

Characterization of HIV-1 envelope gp41 genetic diversity and functional domains following perinatal transmission

Background

HIV-1 envelope gp41 is a transmembrane protein that promotes fusion of the virus with the plasma membrane of the host cells required for virus entry. In addition, gp41 is an important target for the immune response and development of antiviral and vaccine strategies, especially when targeting the highly variable envelope gp120 has not met with resounding success. Mutations in gp41 may affect HIV-1 entry, replication, pathogenesis, and transmission. We, therefore, characterized the molecular properties of gp41, including genetic diversity, functional motifs, and evolutionary dynamics from five mother-infant pairs following perinatal transmission.

Results

The gp41 open reading frame (ORF) was maintained with a frequency of 84.17% in five mother-infant pairs' sequences following perinatal transmission. There was a low degree of viral heterogeneity and estimates of genetic diversity in gp41 sequences. Both mother and infant gp41 sequences were under positive selection pressure, as determined by ratios of non-synonymous to synonymous substitutions. Phylogenetic analysis of 157 mother-infant gp41 sequences revealed distinct clusters for each mother-infant pair, suggesting that the epidemiologically linked mother-infant pairs were evolutionarily closer to each other as compared with epidemiologically unlinked sequences. The functional domains of gp41, including fusion peptide, heptad repeats, glycosylation sites and lentiviral lytic peptides were mostly conserved in gp41 sequences analyzed in this study. The CTL recognition epitopes and motifs recognized by fusion inhibitors were also conserved in the five mother-infant pairs.

Conclusion

The maintenance of an intact envelope gp41 ORF with conserved functional domains and a low degree of genetic variability as well as positive selection pressure for adaptive evolution following perinatal transmission is consistent with an indispensable role of envelope gp41 in HIV-1 replication and pathogenesis.     

Reproductive allocation between the sexes,across natural and novel habitats,and its impact on genetic diversity

Allocation to reproductive mode (sexual and asexual) can vary depending on environmental conditions but is often examined at the population level, whereas selection acts upon the individual. We examined individual variation in reproductive mode to identify how the interaction of sex and the environment affect population genetic diversity. Using the plant Marchantia inflexa, we tested whether reproductive allocation pattern varies consistently between males and females and among plants collected from different environments, and determined if morphological responses were the result of individual plasticity or genetic differences. We then quantified genetic variability between the different environments and between the sexes. Male and female plants were collected from two strikingly different habitats within the same region: along natural sites (rivers) and along novel human-modified sites (roadsides). Using a common garden approach, we found a strong sex by habitat interaction: male and female responses differed significantly by their source habitat. For females, relative to river-collected, road-collected plants had higher growth and asexual reproduction, while the pattern was reversed, although not significant, for males. Genetic differentiation was significant between the two habitats with no evidence of individual differences in plasticity for growth, but there was a genotype effect for asexual propagule production. Males and females did not differ genetically; but river-collected plants with lower sexual potential were more diverse than roadside-collected plants, possibly the result of founder events. These results show that individual variation in reproduction is controlled by the interaction of both the environment and genetics. Due to different selection pressures between natural and novel habitats, there are observable differences in life history traits with an associated evolutionary response to the novel habitat.     

The origin and diversity of the HIV-1 pandemic

This review examines the enormous progress that has been made in the past decade in understanding the origin of HIV, HIV genetic variability, and the impact of global HIV diversity on the pandemic. Multiple zoonotic transmissions of simian immunodeficiency virus (SIV) have resulted in different HIV lineages in humans. In addition, the high mutation and recombination rates during viral replication result in a great genetic variability of HIV within individuals, as well as within populations, upon which evolutionary selection pressures act. The global HIV pandemic is examined in the context of HIV evolution, and the global diversity of HIV subtypes and recombinants is discussed in detail. Finally, the impact of HIV diversity on pathogenesis, transmission, diagnosis, treatment, the immune response, and vaccine development is reviewed.     

The impact of genetic diversity in protozoa on molecular diagnostics

Detection of intestinal parasitic protists, commonly referred to as 'intestinal protozoa,' by PCR is increasingly used not only for identification or confirmation but also as a first-line diagnostic tool. Apart from the ability to sample correctly and extract parasite DNA directly from faeces, primer and probe specificity and sensitivity affect predictive values and hence the utility of diagnostic assays. Molecular characterization of intestinal protists is necessary to design primers and probes because this is the basic material for current and future improved diagnostic PCRs for either detecting all genetic variants or specifically differentiating among such variants. As an example, this paper highlights the existence of interspecific and intraspecific genetic diversity among intestinal, unicellular parasites and its implications for nucleic acid-based diagnostic assays.     

A comparison of methods for the estimation of genetic diversity in strawberry cultivars

RAPD markers were used to examine the genetic relatedness of eight strawberry cultivars released from four breeding programmes around the world. Ten random primers successfully amplified DNA fragments from each cultivar and specific fingerprints were generated from the molecular marker data. The cultivars were traced back to founding clones and the relationships between the cultivars were examined from both the molecular and the pedigree data.     

Next-generation sequencing and its potential impact on food microbial genomics

Recent efforts of researchers to elucidate the molecular mechanisms of biological systems have been revolutionized greatly with the use of high throughput and cost-effective techniques such as next generation sequencing (NGS). Application of NGS to microbial genomics is not just limited to predict the prevalence of microorganisms in food samples but also to elucidate the molecular basis of how microorganisms respond to different food-associated conditions, which in turn offers tremendous opportunities to predict and control the growth and survival of desirable or undesirable microorganisms in food. Concurrently, NGS has facilitated the development of new genome-assisted approaches for correlating genotype and phenotype. The aim of this review is to provide a snapshot of the various possibilities that these new technologies are opening up in area of food microbiology, focusing the discussion mainly on lactic acid bacteria and yeasts associated with fermented food. The contribution of NGS to a system level understanding of food microorganisms is also discussed.     

SLCO2B1 genetic polymorphisms in a Korean population: pyrosequencing analyses and comprehensive comparison with other populations

SLCO2B1, also known as OATP2B1 (Organic Anion Transporter) or OATP-B or SLC21A9, is an organic anion uptake transporter that is encoded by the SLCO2B1 gene. In this study we assessed the frequencies of SLCO2B1 polymorphisms in a Korean population using newly developed pyrosequencing methods and compared their frequencies with those in other ethnic groups. We developed pyrosequencing methods to identify the following six SLCO2B1 non-synonymous polymorphisms: c.1175C > T (rs1621378), c.1457C > T (rs2306168), c.43C > T (rs56837383), c.935G > A (rs12422149), c.601G > A (rs35199625) and c.644A > T (rs72559740). The allele frequencies of these polymorphisms were analyzed in 227 Korean subjects. The allele frequencies of SLCO2B1 polymorphisms in the population tested were as follows: 0.0 for c.1175C > T, c.43C > T and c.644A > T; 0.2687 for c.1457C > T; 0.4273 for c.935G > A; and 0.0727 for c. 601G > A. Even though the allele frequencies of the c.1175C > T and c.1457C > T polymorphisms were comparable to those in Japanese subjects, the frequencies in this Korean population differed from those in other ethnic groups. The developed pyrosequencing methods are rapid and reliable for detecting non-synonymous SLCO2B1 polymorphisms. Large ethnic differences in the frequency of SLCO2B1 genetic polymorphisms were noted among ethnic groups. The SLCO2B1 polymorphisms at c.1175C > T, c.43C > T and c.644A > T were not found in the Korean population while c.1457C > T, c.935G > A and c.601G > A exhibited mostly higher frequencies in Koreans compared with Finnish, Caucasian and African-American populations.     

A comparison of the genetic diversity in Dipteronia sinensis Oliv. and Dipteronia dyeriana Henry

Dipteronia is an endemic genus to China and includes only two species, Dipteronia sinensis and D.dyeriana.Based on random amplified polymorphic DNA (RAPD) markers,a comparative study of the genetic diversity and genetic structure of Dipteronia was performed.In total,128 and 103 loci were detected in 17 D.sinensis populations and 4 D.dyeriana populations,respectively,using 18 random primers.These results showed that the proportions of polymorphic loci for the two species were 92.97% and 81.55%,respectively,indicating that the genetic diversity of D.sinensis was higher than that of D.dyeriana.Analysis,based on similarity coefficients,Shannon diversity index and Nei gene diversity index,also confirmed this result.AMOVA analysis demonstrated that the genetic variation of D.sinensis within and among populations accounted for 56.89% and 43.11% of the total variation,respectively,and that of D.dyeriana was 57.86% and 42.14%,respectively.The Shannon diversity index and Nei gene diversity index showed similar results.The abovementioned characteristics indicated that the genetic diversity levels of these two species were extremely similar and that the interpopulational genetic differentiation within both species was relatively high.Analysis of the genetic distance among populations also supported this conclusion.Low levels of interpopulational gene flow within both species were believed to be among the leading causes for the above-mentioned phenomenon.The correlation analysis between genetic and geographical distances showed the existence of a remarkably significant correlation between the genetic distance and the longitudinal difference among populations of D.sinensis (p＜0.01),while no significant correlation was found between genetic and geographical distances among populations of D.dyeriana.This indicated that genetic distance was correlated with geographical distances on a large scale rather than on a small scale.This result may be related to differences in the selection pressure on species by their habitats with different distribution ranges.We suggest that in situ conservation efforts should focus on establishing more sites to protect the natural populations and their habitats.Ex situ conservation efforts should focus on enhancing the exchange of seeds and seedlings among populations to facilitate gene exchange and recombination,and to help conserve genetic diversity.