Intragenomic variation in the second internal transcribed spacer of the ribosomal DNA of species of the genera Culex and Lutzia (Diptera: Culicidae)

Culex is the largest genus of Culicini and includes vectors of several arboviruses and filarial worms. Many species of Culex are morphologically similar, which makes their identification difficult, particularly when using female specimens. To aid evolutionary studies and species distinction, molecular techniques are often used. Sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) from 16 species of the genus Culex and one of Lutzia were used to assess their genomic variability and to verify their applicability in the phylogenetic analysis of the group. The distance matrix (uncorrected p-distance) that was obtained revealed intragenomic and intraspecific variation. Because of the intragenomic variability, we selected ITS2 copies for use in distance analyses based on their secondary structures. Neighbour-joining topology was obtained with an uncorrected p-distance. Despite the heterogeneity observed, individuals of the same species were grouped together and correlated with the current, morphology-based classification, thereby showing that ITS2 is an appropriate marker to be used in the taxonomy of Culex.

Genetic diversity of Toscana virus

Distribution of Toscana virus (TOSV) is evolving with climate change, and pathogenicity may be higher in nonexposed populations outside areas of current prevalence (Mediterranean Basin). To characterize genetic diversity of TOSV, we determined the coding sequences of isolates from Spain and France. TOSV is more diverse than other well-studied phleboviruses (e.g.,Rift Valley fever virus).

Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium tuberculosis in Delhi, North India

The aim of the present study was to compare polymerase chain reaction (PCR)-based methods - spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing - with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters); the largest cluster was composed of Spoligotype International Types (SITs)26 [Central-Asian (CAS)1-Delhi lineage], followed by SIT11 [East-African-Indian (EAI) 3-Indian lineage]. A large number of isolates (75%) belonged to genotypic lineages, such as CAS, EAI and Manu, with a high specificity for the Indian subcontinent, emphasising the complex diversity of the phylogenetically coherent M. tuberculosis in North India. MIRU typing, using 11 discriminatory loci, was able to distinguish between all but two strains based on individual patterns. IS6110-RFLP analysis (n = 80 strains) resulted in 67 unique isolates and four clusters containing 13 strains. MIRUs discriminated all 13 strains, whereas spoligotyping discriminated 11 strains. Our results validate the use of PCR-based molecular typing of M. tuberculosis using repetitive elements in Indian isolates and demonstrate the usefulness of MIRUs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study.

High prevalence and lack of diversity of Wolbachia pipientis in Aedes albopictus populations from Northeast Brazil

The use of Wolbachia as a tool to control insect vectors has recently been suggested. In this context, studies on the prevalence and diversity of this bacterium in wild populations are relevant. Here, we evaluated the diversity of two Wolbachiagenes (ftsZ and wsp) and the prevalence of this endosymbiont in wild Aedes albopictus. Using semi-nested polymerase chain reaction, our results showed that 99.3% of the individuals were superinfected with Wolbachia. In regards to genetic diversity, the two genes showed no variation within or among mosquito populations. An analysis of other Wolbachia markers may help to clarify the relationship between insect and endosymbiont.

Genetic diversity of noroviruses in Brazil

Norovirus (NoV) infections are a major cause of acute gastroenteritis outbreaks around the world. In Brazil, the surveillance system for acute diarrhoea does not include the diagnosis of NoV, precluding the ability to assess its impact on public health. The present study assessed the circulation of NoV genotypes in different Brazilian states by partial nucleotide sequencing analysis of the genomic region coding for the major capsid viral protein. NoV genogroup II genotype 4 (GII.4) was the prevalent (78%) followed by GII.6, GII.7, GII.12, GII.16 and GII.17, demonstrating the great diversity of NoV genotypes circulating in Brazil. Thus, this paper highlights the importance of a virological surveillance system to detect and characterize emerging strains of NoV and their spreading potential.

Molecular markers and genetic diversity of Plasmodium vivax

Enhanced understanding of the transmission dynamics and population genetics for Plasmodium vivax is crucial in predicting the emergence and spread of novel parasite phenotypes with major public health implications, such as new relapsing patterns, drug resistance and increased virulence. Suitable molecular markers are required for these population genetic studies. Here, we focus on two groups of molecular markers that are commonly used to analyse natural populations of P. vivax. We use markers under selective pressure, for instance, antigen-coding polymorphic genes, and markers that are not under strong natural selection, such as most minisatellite and microsatellite loci. First, we review data obtained using genes encoding for P. vivax antigens: circumsporozoite protein, merozoite surface proteins 1 and 3α, apical membrane antigen 1 and Duffy binding antigen. We next address neutral or nearly neutral molecular markers, especially microsatellite loci, providing a complete list of markers that have already been used in P. vivax populations studies. We also analyse the microsatellite loci identified in the P. vivax genome project. Finally, we discuss some practical uses for P. vivax genotyping, for example, detecting multiple-clone infections and tracking the geographic origin of isolates.

Genetic diversity of Leishmania infantum field populations from Brazil

Leishmania infantum (syn. Leishmania chagasi) is the etiological agent of visceral leishmaniasis (VL) in Brazil. The epidemiology of VL is poorly understood. Therefore, a more detailed molecular characterization at an intraspecific level is certainly needed. Herein, three independent molecular methods, multilocus microsatellite typing (MLMT), random amplification of polymorphic DNA (RAPD) and simple sequence repeats-polymerase chain reaction (SSR-PCR), were used to evaluate the genetic diversity of 53 L. infantum isolates from five different endemic areas in Brazil. Population structures were inferred by distance-based and Bayesian-based approaches. Eighteen very similar genotypes were detected by MLMT, most of them differed in only one locus and no correlation was found between MLMT profiles, geographical origin or the estimated population structure. However, complex profiles composed of 182 bands obtained by both RAPD and SSR-PCR assays gave different results. Unweighted pair group method with arithmetic mean trees built from these data revealed a high degree of homogeneity within isolates of L. infantum. Interestingly, despite this genetic homogeneity, most of the isolates clustered according to their geographical origin.

Genetic diversity of NS3 protease from Brazilian HCV isolates and possible implications for therapy with direct-acting antiviral drugs

The hepatitis C virus (HCV) NS3 protease has been one of the molecular targets of new therapeutic approaches. Its genomic sequence variability in Brazilian HCV isolates is poorly documented. To obtain more information on the magnitude of its genetic diversity, 114 Brazilian HCV samples were sequenced and analysed together with global reference sequences. Genetic distance (d) analyses revealed that subtype 1b had a higher degree of heterogeneity (d = 0.098) than subtypes 1a (d = 0.060) and 3a (d = 0.062). Brazilian isolates of subtype 1b were distributed in the phylogenetic tree among sequences from other countries, whereas most subtype 1a and 3a sequences clustered into a single branch. Additional characterisation of subtype 1a in clades 1 and 2 revealed that all but two Brazilian subtype 1a sequences formed a distinct and strongly supported (approximate likelihood-ratio test = 93) group of sequences inside clade 1. Moreover, this subcluster inside clade 1 presented an unusual phenotypic characteristic in relation to the presence of resistance mutations for macrocyclic inhibitors. In particular, the mutation Q80K was found in the majority of clade 1 sequences, but not in the Brazilian isolates. These data demonstrate that Brazilian HCV subtypes display a distinct pattern of genetic diversity and reinforce the importance of sequence information in future therapeutic approaches.

Genetic diversity of EBV-encoded LMP1 in the Swiss HIV Cohort Study and implication for NF-Κb activation.

Epstein-Barr virus (EBV) is associated with several types of cancers including Hodgkin's lymphoma (HL) and nasopharyngeal carcinoma (NPC). EBV-encoded latent membrane protein 1 (LMP1), a multifunctional oncoprotein, is a powerful activator of the transcription factor NF-κB, a property that is essential for EBV-transformed lymphoblastoid cell survival. Previous studies reported LMP1 sequence variations and induction of higher NF-κB activation levels compared to the prototype B95-8 LMP1 by some variants. Here we used biopsies of EBV-associated cancers and blood of individuals included in the Swiss HIV Cohort Study (SHCS) to analyze LMP1 genetic diversity and impact of sequence variations on LMP1-mediated NF-κB activation potential. We found that a number of variants mediate higher NF-κB activation levels when compared to B95-8 LMP1 and mapped three single polymorphisms responsible for this phenotype: F106Y, I124V and F144I. F106Y was present in all LMP1 isolated in this study and its effect was variant dependent, suggesting that it was modulated by other polymorphisms. The two polymorphisms I124V and F144I were present in distinct phylogenetic groups and were linked with other specific polymorphisms nearby, I152L and D150A/L151I, respectively. The two sets of polymorphisms, I124V/I152L and F144I/D150A/L151I, which were markers of increased NF-κB activation in vitro, were not associated with EBV-associated HL in the SHCS. Taken together these results highlighted the importance of single polymorphisms for the modulation of LMP1 signaling activity and demonstrated that several groups of LMP1 variants, through distinct mutational paths, mediated enhanced NF-κB activation levels compared to B95-8 LMP1.

Mitochondrial genome sequence diversity of Indian Plasmodium falciparum isolates

We have analysed the whole mitochondrial (mt) genome sequences (each ~6 kilo nucleotide base pairs in length) of four field isolates of the malaria parasite Plasmodium falciparum collected from different locations in India. Comparative genomic analyses of mt genome sequences revealed three novel India-specific single nucleotide polymorphisms. In general, high mt genome diversity was found in Indian P. falciparum, at a level comparable to African isolates. A population phylogenetic tree placed the presently sequenced Indian P. falciparum with the global isolates, while a previously sequenced Indian isolate was an outlier. Although this preliminary study is limited to a few numbers of isolates, the data have provided fundamental evidence of the mt genome diversity and evolutionary relationships of Indian P. falciparum with that of global isolates.

Antimicrobial susceptibility patterns, emm type distribution and genetic diversity of Streptococcus pyogenes recovered in Brazil

Streptococcus pyogenes is responsible for a variety of infectious diseases and immunological complications. In this study, 91 isolates of S. pyogenes recovered from oropharynx secretions were submitted to antimicrobial susceptibility testing, emm typing and pulsed-field gel electrophoresis (PFGE) analysis. All isolates were susceptible to ceftriaxone, levofloxacin, penicillin G and vancomycin. Resistance to erythromycin and clindamycin was 15.4%, which is higher than previous reports from this area, while 20.9% of the isolates were not susceptible to tetracycline. The macrolide resistance phenotypes were cMLSB (10) and iMLSB (4). The ermB gene was predominant, followed by the ermA gene. Thirty-two emm types and subtypes were found, but five (emm1, emm4, emm12, emm22, emm81) were detected in 48% of the isolates. Three new emm subtypes were identified (emm1.74, emm58.14, emm76.7). There was a strong association between emm type and PFGE clustering. A variety of PFGE profiles as well as emm types were found among tetracycline and erythromycin-resistant isolates, demonstrating that antimicrobial resistant strains do not result from the expansion of one or a few clones. This study provides epidemiological data that contribute to the development of suitable strategies for the prevention and treatment of such infections in a poorly studied area.

Genetic diversity of chloroquine-resistant Plasmodium vivax parasites from the western Brazilian Amazon

The molecular basis of Plasmodium vivax chloroquine (CQ) resistance is still unknown. Elucidating the molecular background of parasites that are sensitive or resistant to CQ will help to identify and monitor the spread of resistance. By genotyping a panel of molecular markers, we demonstrate a similar genetic variability between in vitro CQ-resistant and sensitive phenotypes of P. vivax parasites. However, our studies identified two loci (MS8 and MSP1-B10) that could be used to discriminate between both CQ-susceptible phenotypes among P. vivax isolates in vitro. These preliminary data suggest that microsatellites may be used to identify and to monitor the spread of P. vivax-resistance around the world.

Diversity of the bacterial community in the surface soil of a pear orchard based on 16S rRNA gene analysis

A cultivation-independent approach based on polymerase chain reaction (PCR)-amplified partial small subunit rRNA genes was used to characterize bacterial populations in the surface soil of a commercial pear orchard consisting of different pear cultivars during two consecutive growing seasons. Pyrus communis L. cvs Blanquilla, Conference, and Williams are among the most widely cultivated cultivars in Europe and account for the majority of pear production in Northeastern Spain. To assess the heterogeneity of the community structure in response to environmental variables and tree phenology, bacterial populations were examined using PCR-denaturing gradient gel electrophoresis (DGGE) followed by cluster analysis of the 16S ribosomal DNA profiles by means of the unweighted pair group method with arithmetic means. Similarity analysis of the band patterns failed to identify characteristic fingerprints associated with the pear cultivars. Both environmentally and biologically based principal-component analyses showed that the microbial communities changed significantly throughout the year depending on temperature and, to a lesser extent, on tree phenology and rainfall. Prominent DGGE bands were excised and sequenced to gain insight into the identities of the predominant bacterial populations. Most DGGE band sequences were related to bacterial phyla, such as Bacteroidetes, Cyanobacteria, Acidobacteria, Proteobacteria, Nitrospirae, and Gemmatimonadetes, previously associated with typical agronomic crop environments

The role of biotic interactions in shaping distributions and realised assemblages of species: implications for species distribution modelling.

Predicting which species will occur together in the future, and where, remains one of the greatest challenges in ecology, and requires a sound understanding of how the abiotic and biotic environments interact with dispersal processes and history across scales. Biotic interactions and their dynamics influence species' relationships to climate, and this also has important implications for predicting future distributions of species. It is already well accepted that biotic interactions shape species' spatial distributions at local spatial extents, but the role of these interactions beyond local extents (e.g. 10 km(2) to global extents) are usually dismissed as unimportant. In this review we consolidate evidence for how biotic interactions shape species distributions beyond local extents and review methods for integrating biotic interactions into species distribution modelling tools. Drawing upon evidence from contemporary and palaeoecological studies of individual species ranges, functional groups, and species richness patterns, we show that biotic interactions have clearly left their mark on species distributions and realised assemblages of species across all spatial extents. We demonstrate this with examples from within and across trophic groups. A range of species distribution modelling tools is available to quantify species environmental relationships and predict species occurrence, such as: (i) integrating pairwise dependencies, (ii) using integrative predictors, and (iii) hybridising species distribution models (SDMs) with dynamic models. These methods have typically only been applied to interacting pairs of species at a single time, require a priori ecological knowledge about which species interact, and due to data paucity must assume that biotic interactions are constant in space and time. To better inform the future development of these models across spatial scales, we call for accelerated collection of spatially and temporally explicit species data. Ideally, these data should be sampled to reflect variation in the underlying environment across large spatial extents, and at fine spatial resolution. Simplified ecosystems where there are relatively few interacting species and sometimes a wealth of existing ecosystem monitoring data (e.g. arctic, alpine or island habitats) offer settings where the development of modelling tools that account for biotic interactions may be less difficult than elsewhere.