Polymerase errors accumulating during natural evolution of the glycoprotein gene of vesicular stomatitis virus Indiana serotype isolates

We report the entire glycoprotein (G) gene nucleotide sequences of 26 vesicular stomatitis virus Indiana serotype (VSV IND) type 1 isolates from North and Central America. These sequences are also compared with partial G gene sequences of VSV IND type 2 (Cocal) and type 3 (Alagoas) viruses and the complete G gene sequences of the more distantly related VSV New Jersey (NJ) and Chandipura viruses. Phylogenetic analysis of the G gene sequences by maximum parsimony revealed four major lineages or subtypes within the classical VSV IND (type 1) viruses, each with a distinct geographic distribution. A high degree of VSV genetic diversity was found in Central America, with several virus subtypes of both VSV IND and NJ serotypes existing in this mainly enzootic disease region. Nineteen percent sequence variation but no deletions or insertions were evident within the 5' noncoding and the coding regions of the VSV IND type 1 G genes. In addition to numerous base substitutions, the 3' noncoding regions of these viruses also contained numerous base insertions and deletions. This resulted in striking variation in G gene sizes, with gene lengths ranging from 1,652 to 1,868 nucleotides. As the VSV IND type 1 subtypes have diverged from the common ancestor with the NJ subtypes, their G mRNAs have accumulated more 3' noncoding sequence inserts, ranging up to 303 nucleotides in length. These primarily consist of an imprecise reiteration of the sequence UUUUUAA, apparently generated by a unique polymerase stuttering error. Analysis of the deduced amino acid sequence differences among VSV IND type 1 viruses revealed numerous substitutions within defined antigenic epitopes, suggesting that immune selection may play a role in the evolution of these viruses.     

Identification of Giardia lamblia isolates susceptible and resistant to infection by the double-stranded RNA virus

The presence or absence of the Giardia lamblia double-stranded RNA virus (GLV) was surveyed among 38 axenic isolates of G. lamblia derived from both humans and animals. Of the 28 isolates lacking the virus, 19 could readily be infected by the virus. The remaining 9 isolates proved to be resistant to GLV infection even when the ratio between virus to parasite reached as high as 10(6) to 1. Evidence is also presented indicating that there are at least two "Portland 1" strains being used by the current scientific community, one containing the virus and the other lacking the virus.     

Molecular population genetics of Cucumber mosaic virus in California: evidence for founder effects and reassortment

The structure and genetic diversity of a California Cucumber mosaic virus (CMV) population was assessed by single-strand conformation polymorphism and nucleotide sequence analyses of genomic regions 2b, CP, MP, and the 3' nontranslated region of RNA3. The California CMV population exhibited low genetic diversity and was composed of one to three predominant haplotypes and a large number of minor haplotypes for specific genomic regions. Extremely low diversity and close evolutionary relationships among isolates in a subpopulation suggested that founder effects might play a role in shaping the genetic structure. Phylogenetic analysis indicated a naturally occurring reassortant between subgroup IA and IB isolates and potential reassortants between subgroup IA isolates, suggesting that genetic exchange by reassortment contributed to the evolution of the California CMV population. Analysis of various population genetics parameters and distribution of synonymous and nonsynonymous mutations revealed that different coding regions and even different parts of coding regions were under different evolutionary constraints, including a short region of the 2b gene for which evidence suggests possible positive selection.     

Diversity of the envelope glycoprotein among human immunodeficiency virus type 1 isolates of clade E from Asia and Africa.

Human immunodeficiency virus type 1 isolates of clade E, known to be largely responsible for the fulminating epidemic in Southeast Asia, have been derived exclusively from Asia and Africa. Here we provide full or partial sequences of the envelope glycoprotein gene from 13 additional clade E isolates from Asia representing patients in both early and late stages of disease. More extensive comparison of isolates within clade E by geographic locale, stage of disease, and year of isolation is now possible. The genetic diversity of clade E isolates from Asia, particularly among those derived from early-stage patients, is restricted compared with African isolates (mean interisolate distances in gp120, 5.4 and 20.2%, respectively). However, patients hospitalized with AIDS-related illnesses in Thailand harbored clade E isolates exhibiting broader interisolate diversity and with highly heterogeneous third hypervariable loop sequences. An additional pair of cysteine residues, predicting a novel disulfide bridge and present in 80% of clade E isolates from Asia, was uniformly absent from six African isolates. Clade E isolates in Thailand from early-stage subjects continue to be genetically similar to potential vaccine prototype strains, providing a favorable environment for the evaluation of genotype E candidate vaccines. However, evidence of increasing interisolate diversity is appearing among late-stage patients in Asia. This diversification of the clade E virus, if sustained, may impact preventive vaccine development strategies.     

Genetic linkage and association analyses for trait mapping in Plasmodium falciparum

Genetic studies of Plasmodium falciparum laboratory crosses and field isolates have produced valuable insights into determinants of drug responses, antigenic variation, disease virulence, cellular development and population structures of these virulent human malaria parasites. Full-genome sequences and high-resolution haplotype maps of SNPs and microsatellites are now available for all 14 parasite chromosomes. Rapidly increasing genetic and genomic information on Plasmodium parasites, mosquitoes and humans will combine as a rich resource for new advances in our understanding of malaria, its transmission and its manifestations of disease.     

Systematic evaluation of genetic variation at the androgen receptor locus and risk of prostate cancer in a multiethnic cohort study

Repeat length of the CAG microsatellite polymorphism in exon 1 of the androgen receptor (AR) gene has been associated with risk of prostate cancer in humans. This association has been the focus of >20 primary epidemiological publications and multiple review articles, but a consistent and reproducible association has yet to be confirmed. We systematically addressed possible causes of false-negative and false-positive association in >4,000 individuals from a multiethnic, prospective cohort study of prostate cancer, comprehensively studying genetic variation by microsatellite genotyping, direct resequencing of exons in advanced cancer cases, and haplotype analysis across the 180-kb AR genomic locus. These data failed to confirm that common genetic variation in the AR gene locus influences risk of prostate cancer. A systematic approach that assesses both coding and noncoding genetic variation in large and diverse patient samples can help clarify hypotheses about association between genetic variants and disease.     

Evidence of host-associated populations of Cryptosporidium parvum in Italy

Recent studies have revealed extensive genetic variation among isolates of Cryptosporidium parvum, an Apicomplexan parasite that causes gastroenteritis in both humans and animals worldwide. The parasite's population structure is influenced by the intensity of transmission, the host-parasite interaction, and husbandry practices. As a result, C. parvum populations can be panmictic, clonal, or even epidemic on both a local scale and a larger geographical scale. To extend the study of C. parvum populations to an unexplored region, 173 isolates of C. parvum collected in Italy from humans and livestock (calf, sheep, and goat) over a 10-year period were genotyped using a multilocus scheme based on 7 mini- and microsatellite loci. In agreement with other studies, extensive polymorphism was observed, with 102 distinct multilocus genotypes (MLGs) identified among 173 isolates. The presence of linkage disequilibrium, the confinement of MLGs to individual farms, and the relationship of many MLGs inferred using network analysis (eBURST) suggest a predominantly clonal population structure, but there is also evidence that part of the diversity can be explained by genetic exchange. MLGs from goats were found to differ from bovine and sheep MLGs, supporting the existence of C. parvum subpopulations. Finally, MLGs from isolates collected between 1997 and 1999 were also identified as a distinct subgroup in principal-component analysis and eBURST analysis, suggesting a continuous introduction of novel genotypes in the parasite population.     

Sex, subdivision, and domestic dispersal of Trypanosoma cruzi lineage I in southern Ecuador

Background

Molecular epidemiology at the community level has an important guiding role in zoonotic disease control programmes where genetic markers are suitably variable to unravel the dynamics of local transmission. We evaluated the molecular diversity of Trypanosoma cruzi, the etiological agent of Chagas disease, in southern Ecuador (Loja Province). This kinetoplastid parasite has traditionally been a paradigm for clonal population structure in pathogenic organisms. However, the presence of naturally occurring hybrids, mitochondrial introgression, and evidence of genetic exchange in the laboratory question this dogma.

Methodology/Principal Findings

Eighty-one parasite isolates from domiciliary, peridomiciliary, and sylvatic triatomines and mammals were genotyped across 10 variable microsatellite loci. Two discrete parasite populations were defined: one predominantly composed of isolates from domestic and peridomestic foci, and another predominantly composed of isolates from sylvatic foci. Spatial genetic variation was absent from the former, suggesting rapid parasite dispersal across our study area. Furthermore, linkage equilibrium between loci, Hardy-Weinberg allele frequencies at individual loci, and a lack of repeated genotypes are indicative of frequent genetic exchange among individuals in the domestic/peridomestic population.

Conclusions/Significance

These data represent novel population-level evidence of an extant capacity for sex among natural cycles of T. cruzi transmission. As such they have dramatic implications for our understanding of the fundamental genetics of this parasite. Our data also elucidate local disease transmission, whereby passive anthropogenic domestic mammal and triatomine dispersal across our study area is likely to account for the rapid domestic/peridomestic spread of the parasite. Finally we discuss how this, and the observed subdivision between sympatric sylvatic and domestic/peridomestic foci, can inform efforts at Chagas disease control in Ecuador.     

Impact of population structure on genetic diversity of a potential vaccine target in the canine hookworm (Ancylostoma caninum)

Ancylostoma caninum is a globally distributed canine parasitic nematode. To test whether positive selection, population structure, or both affect genetic variation at the candidate vaccine target Ancylostoma secreted protein 1 (asp-1), we have quantified the genetic variation in A. caninum at asp-1 and a mitochondrial gene, cytochrome oxidase subunit 1 (cox-1), using the statistical population analysis tools found in the SNAP Workbench. The mitochondrial gene cox-1 exhibits moderate diversity within 2 North American samples, comparable to the level of variation observed in other parasitic nematodes. The protein coding portion for the C-terminal half of asp-1 shows similar levels of genetic variation in a Wake County, North Carolina, sample as cox-1. Standard tests of neutrality provide little formal evidence for selection acting on this locus, but haplotype networks for 2 of the exon regions have significantly different topologies, consistent with different evolutionary forces shaping variation at either end of a 1.3-kilobase stretch of sequence. Evidence for gene flow among geographically distinct samples suggests that the mobility of hosts of A. caninum is an important contributing factor to the population structure of the parasite.     

Zoonotic potential of Giardia

Giardia duodenalis (syn. Giardia lamblia and Giardia intestinalis) is a common intestinal parasite of humans and mammals worldwide. Assessing the zoonotic transmission of the infection requires molecular characterization as there is considerable genetic variation within G. duodenalis. To date eight major genetic groups (assemblages) have been identified, two of which (A and B) are found in both humans and animals, whereas the remaining six (C to H) are host-specific and do not infect humans. Sequence-based surveys of single loci have identified a number of genetic variants (genotypes) within assemblages A and B in animal species, some of which may have zoonotic potential. Multi-locus typing data, however, has shown that in most cases, animals do not share identical multi-locus types with humans. Furthermore, interpretation of genotyping data is complicated by the presence of multiple alleles that generate “double peaks” in sequencing files from PCR products, and by the potential exchange of genetic material among isolates, which may account for the non-concordance in the assignment of isolates to specific assemblages. Therefore, a better understanding of the genetics of this parasite is required to allow the design of more sensitive and variable subtyping tools, that in turn may help unravel the complex epidemiology of this infection.     

Larger genetic differences within africans than between Africans and Eurasians

The worldwide pattern of single nucleotide polymorphism (SNP) variation is of great interest to human geneticists, population geneticists, and evolutionists, but remains incompletely understood. We studied the pattern in noncoding regions, because they are less affected by natural selection than are coding regions. Thus, it can reflect better the history of human evolution and can serve as a baseline for understanding the maintenance of SNPs in human populations. We sequenced 50 noncoding DNA segments each approximately 500 bp long in 10 Africans, 10 Europeans, and 10 Asians. An analysis of the data suggests that the sampling scheme is adequate for our purpose. The average nucleotide diversity (pi) for the 50 segments is only 0.061% +/- 0.010% among Asians and 0.064% +/- 0.011% among Europeans but almost twice as high (0.115% +/- 0.016%) among Africans. The African diversity estimate is even higher than that between Africans and Eurasians (0.096% +/- 0.012%). From available data for noncoding autosomal regions (total length = 47,038 bp) and X-linked regions (47,421 bp), we estimated the pi-values for autosomal regions to be 0.105, 0.070, 0.069, and 0.097% for Africans, Asians, Europeans, and between Africans and Eurasians, and the corresponding values for X-linked regions to be 0.088, 0.042, 0.053, and 0.082%. Thus, Africans differ from one another slightly more than from Eurasians, and the genetic diversity in Eurasians is largely a subset of that in Africans, supporting the out of Africa model of human evolution. Clearly, one must specify the geographic origins of the individuals sampled when studying pi or SNP density.     

Microsatellite loci in the human blood fluke Schistosoma mansoni and their utility for other schistosome species

Blood flukes in the genus Schistosoma are important human parasites in tropical regions. Genetic heterogeneity of the parasite contributes to the observed phenotypic variation in this host–parasite interaction and may play a role in disease epidemiology. In this paper, we describe the characterization of five polymorphic microsatellite loci from the human blood fluke Schistosoma mansoni, which can now be applied in assessments of schistosome genetic diversity. The five loci revealed extensive polymorphism, as 5–8 alleles per locus were detected among five isolates (from both human patients and snail intermediate hosts) from two Brazilian villages.     

Consistent effects of TSG101 genetic variability on multiple outcomes of exposure to human immunodeficiency virus type 1

Tumor susceptibility gene 101 (TSG101) encodes a host cellular protein that is appropriated by human immunodeficiency virus type 1 (HIV-1) in the budding process of viral particles from infected cells. Variation in the coding or noncoding regions of the gene could potentially affect the degree of TSG101-mediated release of viral particles. While the coding regions of the gene were found to lack nonsynonymous variants, two polymorphic sites in the TSG101 5' area were identified that were associated with the rate of AIDS progression among Caucasians. These single-nucleotide polymorphisms (SNPs), located at positions -183 and +181 relative to the translation start, specify three haplotypes termed A, B, and C, which occur at frequencies of 67%, 21%, and 12%, respectively. Haplotype C is associated with relatively rapid AIDS progression, while haplotype B is associated with slower disease progression. Both effects were dominant over the intermediate haplotype A. The haplotypes also demonstrated parallel effects on the rate of CD4 T-cell depletion and viral load increase over time, as well as a possible influence on HIV-1 infection. The data raise the hypothesis that noncoding variation in TSG101 affects the efficiency of TSG101-mediated release of viral particles from infected cells, thereby altering levels of plasma viral load and subsequent disease progression.     

Molecular phylogeny of wild hops, Humulus lupulus L

We have analysed wild hops collected widely from the Northern Hemisphere, assessing the genetic diversity and the geographical distribution of haplotypes, to investigate the evolution and phylogeny of hops, Humulus lupulus. The haplotypes were characterized by the nuclear ribosomal DNA spacer region (length and DNA sequence) and chloroplast DNA noncoding regions (DNA sequences). The results indicated that primary divergence into European (including Caucasus and Altai hops), and Asian-North American types, was 1.05+/-0.28 to 1.27+/-0.30 million years ago. Although an Eastern boundary for European nuclear haplotype distribution was unclear due to the ambiguous origin of Northern Chinese samples, the European hop group showed a wide geographical distribution across Eurasia from the Altai region to Portugal. The low genetic variation in this group suggested rapid and recent expansion. The North American hop group showed high diversity, and is considered to include hops that have migrated from Asia. Japanese and Chinese hops were identified as genetically distinct. This study has shown that wild hops in each growing region are genetically differentiated with considerable genetic diversity. It gives insights into the evolution and domestication of hops that are discussed.     

The hierarchical spatial distribution of chloroplast DNA polymorphism across the introduced range of Silene vulgaris

Silene vulgaris was introduced into North America sometime prior to 1800. In order to document the population structure that has developed since that time, collections were made from 56 local populations distributed among 9 geographical regions in eastern North America. Individual plants were characterized for chloroplast DNA (cpDNA) haplotype by restriction fragment size analysis of four noncoding regions of cpDNA amplified by polymerase chain reaction. A total of 19 cpDNA haplotypes were detected using this method. The overall gene diversity of 0.85 is quite similar to the diversity detected in these same regions of cpDNA in a previously published sample of S. vulgaris taken from across much of Europe. The spatial distribution of the North American cpDNA diversity was quantified by hierarchical F-statistics that partitioned the genetic variance into variation among local populations within regions, and variation among regions. The average FST among populations within regions was 0.66 and the FST among regions was 0.09. The among-region variation was due to both differences among regions in the frequency of two most common haplotypes, and to the presence of a number of region-specific haplotypes. In order to test for isolation by distance at the regional level, FST values were calculated for all possible pairs of regions, and regressed against the geographical distance between those regions. There was no evidence for isolation by distance. It is suggested that the local population structure is generated by recent extinction/colonization dynamics, and that the among-region structure reflects demographic events associated with range expansion following introduction to North America.     

Genetic differentiation of relictual populations of Alsophila spinulosa in southern China inferred from cpDNA trnL-F noncoding sequences

The genetic differentiation and phylogeographical pattern of 11 relictual populations of Alsophila spinulosa distributed across Hainan, Guangdong, and Guangxi in southern China were inferred from sequence variations of trnL-F noncoding regions of chloroplast DNA (cpDNA). The length of trnL-F noncoding sequences varied from 863 to 940 bp. The A + T content was 62.23-63.36%. Sequences were neutral in terms of evolution (Tajima's criterion D=-0.62417, P>0.10 and Fu and Li's test D*=-1.45455, P>0.10; F*=-1.32798, P>0.10). Thirty-four haplotypes were identified based on nucleotide variation. Relatively high levels of haplotype diversity (h=0.929) and nucleotide diversity (Dij=0.022263) were detected in A. spinulosa, probably associated with its long evolutionary history which allowed the accumulation of genetic variation within lineages. Both the minimum spanning network and the strict consensus tree of the most parsimonious trees generated for haplotypes demonstrated that the investigated populations of A. spinulosa were subdivided into two geographical groups: Hainan and Guangdong-Guangxi. An analysis of molecular variance (AMOVA) indicated that most of the genetic variation (87.48%, P<0.001) was partitioned among regions. Spatial structure measurements revealed that population genetic structure was not related to geographical distance. This research suggests that blocked gene flow by Qiongzhou strait and an inbreeding system might result in the geographical subdivision between Hainan and Guangdong-Guangxi (F(ST)=0.92, Nm=0.09). Within each region, the "star like" pattern of phylogeography of haplotypes implied a population expansion process during evolutionary history. Gene genealogies together with coalescent theory were useful tools for uncovering the phylogeography of A. spinulosa.     

Intraspecific phylogeny and lineage group identification based on the prfA virulence gene cluster of Listeria monocytogenes

Listeria monocytogenes is a serious food-borne pathogen that can cause invasive disease in humans and other animals and has been the leading cause of food recalls due to microbiological concerns in recent years. In order to test hypotheses regarding L. monocytogenes lineage composition, evolution, ecology, and taxonomy, a robust intraspecific phylogeny was developed based on prfA virulence gene cluster sequences from 113 L. monocytogenes isolates. The results of the multigene phylogenetic analyses confirm that L. monocytogenes comprises at least three evolutionary lineages, demonstrate that lineages most frequently (lineage 1) and least frequently (lineage 3) associated with human listeriosis are sister-groups, and reveal for the first time that the human epidemic associated serotype 4b is prevalent among strains from lineage 1 and lineage 3. In addition, a PCR-based test for lineage identification was developed and used in a survey of food products demonstrating that the low frequency of association between lineage 3 isolates and human listeriosis cases likely reflects rarity of exposure and not reduced virulence for humans as has been previously suggested. However, prevalence data do suggest lineage 3 isolates may be better adapted to the animal production environment than the food-processing environment. Finally, analyses of haplotype diversity indicate that lineage 1 has experienced a purge of genetic variation that was not observed in the other lineages, suggesting that the three L. monocytogenes lineages may represent distinct species within the framework of the cohesion species concept.     

Population structures and the role of genetic exchange in the zoonotic pathogen Cryptosporidium parvum

Apicomplexan protozoan parasites include some of the most globally important human and animal pathogens, all of which have obligatory sexual cycles in their definitive hosts. Despite their importance and the relevance of understanding the population genetic structure and role of genetic exchange in generating diversity, population genetic analysis has largely been restricted to Plasmodium spp. and Toxoplasma gondii. These species show a considerable diversity of population structure suggesting different strategies for transmission and survival in mammalian hosts. We have undertaken a population genetic analysis of a further apicomplexan species (Cryptosporidium parvum) to extend our understanding of the diversity of genetic structures and test whether it has a clonal population structure. Nothing is known about the population structure of this parasite. We have analyzed 180 parasite isolates from both humans and cattle derived from a single discrete geographical area, using three minisatellite and four microsatellite markers that define 38 multilocus genotypes. Analysis of linkage disequilibria between pairs of loci combined with measures of genetic distance and similarity provides evidence that the sample comprises four genetically isolated populations. One group of human isolates consists primarily of two closely related multilocus genotypes (clonal), while the major subtypes of a second group, common to both humans and animals, show a panmictic population structure. The data provide an important step in understanding the role of genetic exchange in these parasites, which is an essential prerequisite for determining the value of multilocus genotyping for the analysis of sources of human infection as well as future molecular epidemiological studies.