Molecular epidemiology of Orientia tsutsugamushi from outbreak regions, Madhya Pradesh, central India

Scrub typhus is a seriously neglected life threatening disease in Asia and the islands of the Pacific and Indian oceans. The causative agent is Orientia tsutsugamushi, an obligate intra-cytosolic bacterium transmitted to vertebrate animals such as human or rodent by larval trombiculid mites (chiggers). Here, we conducted an outbreak investigation in Madhya Pradesh state in central India after sudden upsurge in scrub typhus cases and mortality in humans. Sixty-five live traps were placed for capturing the rodents. The captured rodent species were identified and the samples were subjected to nested PCR targeting 56kDa TSA gene. Analysis revealed for the first-time predominant circulation of Karp genotype in this region. The vector species involved in transmission is Leptotrobidium deliense. In conclusion, we reported the circulation of Karp strain in outbreak regions of Madhya Pradesh and suggest that public health monitoring and surveillance needs to be improved. Studies on antigenic diversity, association of strains with clinical spectrum and pathogenicity in the local setup will be essential for development of region-specific diagnostics and vaccines.

Does malaria epidemiology project Cameroon as ‘Africa in miniature.

Cameroon, a west-central African country with a ~20 million population, is commonly regarded as ‘Africa in miniature’ due to the extensive biological and cultural diversities of whole Africa being present in a single-country setting. This country is inhabited by ancestral human lineages in unique eco-climatic conditions and diverse topography. Over 90% Cameroonians are at risk of malaria infection, and ~41% have at least one episode of malaria each year. Historically, the rate of malaria infection in Cameroon has fluctuated over the years; the number of cases was about 2 million in 2010 and 2011. The Cameroonian malaria control programme faces an uphill task due to high prevalence of multidrug-resistant parasites and insecticide-resistant malaria vectors. Above all, continued human migration from the rural to urban areas as well as population exchange with adjoining countries, high rate of ecological instabilities caused by deforestation, poor housing, lack of proper sanitation and drainage system might have resulted in the recent increase in incidences of malaria and other vector-borne diseases in Cameroon. The available data on eco-environmental variability and intricate malaria epidemiology in Cameroon reflect the situation in the whole of Africa, and warrant the need for in-depth study by using modern surveillance tools for meaningful basic understanding of the malaria triangle (host-parasite-vector-environment).

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.

Genetics of chloroquine-resistant malaria: a haplotypic view

The development and rapid spread of chloroquine resistance (CQR) in Plasmodium falciparum have triggered the identification of several genetic target(s) in the P. falciparum genome. In particular, mutations in the Pfcrt gene, specifically, K76T and mutations in three other amino acids in the region adjoining K76 (residues 72, 74, 75 and 76), are considered to be highly related to CQR. These various mutations form several different haplotypes and Pfcrt gene polymorphisms and the global distribution of the different CQR- Pfcrt haplotypes in endemic and non-endemic regions of P. falciparum malaria have been the subject of extensive study. Despite the fact that the Pfcrt gene is considered to be the primary CQR gene in P. falciparum , several studies have suggested that this may not be the case. Furthermore, there is a poor correlation between the evolutionary implications of the Pfcrt haplotypes and the inferred migration of CQR P. falciparum based on CQR epidemiological surveillance data. The present paper aims to clarify the existing knowledge on the genetic basis of the different CQR- Pfcrt haplotypes that are prevalent in worldwide populations based on the published literature and to analyse the data to generate hypotheses on the genetics and evolution of CQR malaria.

Pfcrt haplotypes and the evolutionary history of chloroquine-resistant Plasmodium falciparum

Mutations in the Pfcrt gene that change the resulting amino acids and form different haplotypes are common and correlate with the prevalence of chloroquine resistant (CQR) field isolates of the malaria parasite, Plasmodium falciparum. This correlation provides opportunities to infer the global evolutionary history of CQ resistance by analysing CQR Pfcrt haplotype data. We collated data on the Pfcrt haplotypes from different global studies and performed evolutionary genetic analysis to present comprehensive and comparative information on the global distribution of five major CQR-Pfcrt haplotypes and evolutionary inter-relationships among 38 different countries. Using the haplotype diversity data, inter-continental genetic differentiation was also ascertained.

Comparative evolutionary analyses of beta globin gene in eutherian, dinosaurian and neopterygii taxa.

Background & objectives: Comparative genomics and evolutionary analyses of conserved genes have enabled us to understand the complexity of genomes of closely related species. For example: -globin gene present in human hemoglobin is one such gene that has experienced many genetic changes in many related taxa and produced more than 600 variants. One of the variant, HBS causes sickle-cell anemia in humans but offers protection against severe malaria due to Plasmodium falciparum. In the present study, we characterized and performed evolutionary comparative analyses of the -globin gene in different related and unrelated taxa to have a comprehensive view of its evolution. Methods: DNA and protein sequences of -globin gene were downloaded from NCBI and characterized in detail in nine eutherian (Homo sapiens, Pan troglodytes, Macaca mulatta, Mus musculus, Rattus norvegicus, Bos taurus, Canis familiaris, Equus caballus, Oryctolagus cuniculus), a dinosaurian (Gallus gallus) and a neopterygii (Danio rerio) taxa. Three more eutherian (Papio anubis, Ovis aries and Sus scrofa) taxa were included for an analysis at the protein level but not included at the gene level owing to lack of genomic information. Computational and phylogenetic analyses were performed using evolutionary comparative approach. Results: Results of comparative and phylogenetic analyses revealed less conservation of genetic architecture of -globin compared to its protein architecture in all eutherian taxa. Both dinosaurian and neopterygii taxa served as outgroups and varied at gene and protein levels. Interpretation & conclusion: Most remarkably, all primates from eutherian taxa including P. anubis showed only nine codon position differences and an absolute similarity between H. sapiens and P. troglodytes. Absolute conservation of coding region in Equus caballus (horse) was observed. The results were discussed with an inference on the role of evolutionary forces in maintaining such close similarities and variations across closely related taxa. Further, the need to utilize more comparative approaches in understanding the disease causing genes’ evolution in closely related taxa is hoped for.

Sequence analysis of coding DNA fragments of pfcrt and pfmdr-1 genes in Plasmodium falciparum isolates from Odisha, India

The global emergence and spread of malaria parasites resistant to antimalarial drugs is the major problem in malaria control. The genetic basis of the parasite's resistance to the antimalarial drug chloroquine (CQ) is well-documented, allowing for the analysis of field isolates of malaria parasites to address evolutionary questions concerning the origin and spread of CQ-resistance. Here, we present DNA sequence analyses of both the second exon of the Plasmodium falciparum CQ-resistance transporter (pfcrt) gene and the 5' end of the P. falciparum multidrug-resistance 1 (pfmdr-1) gene in 40 P. falciparum field isolates collected from eight different localities of Odisha, India. First, we genotyped the samples for the pfcrt K76T and pfmdr-1 N86Y mutations in these two genes, which are the mutations primarily implicated in CQ-resistance. We further analyzed amino acid changes in codons 72-76 of the pfcrt haplotypes. Interestingly, both the K76T and N86Y mutations were found to co-exist in 32 out of the total 40 isolates, which were of either the CVIET or SVMNT haplotype, while the remaining eight isolates were of the CVMNK haplotype. In total, eight nonsynonymous single nucleotide polymorphisms (SNPs) were observed, six in the pfcrt gene and two in the pfmdr-1 gene. One poorly studied SNP in the pfcrt gene (A97T) was found at a high frequency in many P. falciparum samples. Using population genetics to analyze these two gene fragments, we revealed comparatively higher nucleotide diversity in the pfcrt gene than in the pfmdr-1 gene. Furthermore, linkage disequilibrium was found to be tight between closely spaced SNPs of the pfcrt gene. Finally, both the pfcrt and the pfmdr-1 genes were found to evolve under the standard neutral model of molecular evolution.

Evolutionary insights into duffy gene in mammalian taxa with comparative genetic analysis.

Background & objectives: Evolutionary analyses of genes conserved across taxa are keys to understand the complexity of gene and genome variation. Since malaria is a highly infectious human disease and its susceptibility in human is genetically controlled, characterization and evolutionary analyses of such genes are of prime importance to understand genetic mechanisms of disease susceptibility. In the present study we have characterized and performed comparative genomic analyses of the human Duffy gene responsible for malaria pathogenesis in nine different mammalian taxa. Methods: DNA sequences of human duffy gene were downloaded from public domain and have been characterized in detail and compared with eight other different mammalian taxa (Pan troglodytes, Macaca mulatta, Pongo pygmaeus, Rattus norvegicus, Mus musculus, Monodelphis domestica, Bos taurus and Canis familiaris). Comparative and evolutionary analyses were performed using statistical software and tools. Results: We observed that the genetic architecture of this gene was entirely different across all the nine taxa and a close similarity between Homo sapiens and Pan troglodytes (chimpanzee) was evident for several aspects of this gene. Comparisons on several aspects, such as ratio of coding and non-coding regions, total gene length number and size of introns and difference of number of nucleotides in human and chimpanzees have revealed interesting features. Phylogenetic inferences based on the Duffy gene among nine different taxa were found to be different than other genes previously studied. Interpretation & conclusion: Most remarkably, human and chimpanzee were only 0.75% different in this gene. The results were discussed on the similarities between human and chimpanzee and gain of introns in human-chimpanzee clade with an inference on the role of evolutionary forces (mainly natural selection) in maintaining such variations across closely-related mammalian taxa.

Characterization & evolutionary analysis of human CD36 gene.

Background and objectives: Understanding evolutionary genetic details of immune system genes responsible for infectious diseases is of prime importance concerning disease pathogenecity. Considering malaria as a devastating disease in the world including India, detail evolutionary understanding on human immune system gene is essential. The primary aim of this study was to initiate work on one such gene, the human CD36 gene responsible in malaria pathogenesis. Methods: DNA sequences of the human CD36 gene was retrieved from public domain and fifi ne-scale details were characterized. Both comparative and evolutionary analyses were performed with sequences from six other taxa (5 mammalian one avian) where CD36 homologs are present. Different statistical analyses were also performed. Results: Differential distribution in number and length of exons and introns was detected in CD36 gene across seven taxa. The CpG islands were also found to be distributed unevenly across the gene and taxa. Neighbour-joining tree was constructed and it was observed that the chimpanzee and human are diverged at the CD36 gene relatively recently. The chicken, Gallus gallus was found to be diverged from rest of the taxa signifi cantly. Also copy number variation was observed across different taxa. Interpretation & conclusions: Comparative genomic study of a human immune system gene CD36 show relationships among different taxa at the evolutionary level. The information can be of help to study genetic diversity in malaria endemic zones and to correlate it with malaria pathogenecity.

Genetic characterization and evolutionary inference of TNF-α through computational analysis

TNF-α is an important human cytokine that imparts dualism in malaria pathogenicity. At high dosages, TNF-α is believed to provoke pathogenicity in cerebral malaria; while at lower dosages TNF-α is protective against severe human malaria. In order to understand the human TNF-α gene and to ascertain evolutionary aspects of its dualistic nature for malaria pathogenicity, we characterized this gene in detail in six different mammalian taxa. The avian taxon, Gallus gallus was included in our study, as TNF-α is not present in birds; therefore, a tandemly placed duplicate of TNF-α (LT-α or TNF-β) was included. A comparative study was made of nucleotide length variations, intron and exon sizes and number variations, differential compositions of coding to non-coding bases, etc., to look for similarities/dissimilarities in the TNF-α gene across all seven taxa. A phylogenetic analysis revealed the pattern found in other genes, as humans, chimpanzees and rhesus monkeys were placed in a single clade, and rats and mice in another; the chicken was in a clearly separate branch. We further focused on these three taxa and aligned the amino acid sequences; there were small differences between humans and chimpanzees; both were more different from the rhesus monkey. Further, comparison of coding and non-coding nucleotide length variations and coding to non-coding nucleotide ratio between TNF-α and TNF-β among these three mammalian taxa provided a first-hand indication of the role of the TNF-α gene, but not of TNF-β in the dualistic nature of TNF-α in malaria pathogenicity.

Fine-scale genetic characterization of Plasmodium falciparum chromosome 7 encompassing the antigenic var and the drug-resistant pfcrt genes.

The fact that malaria is still an uncontrolled disease is reflected by the genetic organization of the parasite genome. Efforts to curb malaria should begin with proper understanding of the mechanism by which the parasites evade human immune system and evolve resistance to different antimalarial drugs. We have initiated such a study and presented herewith the results from the in silico understanding of a seventh chromosomal region of the malarial parasite Plasmodium falciparum encompassing the antigenic var genes (coding pfemp1) and the drug-resistant gene pfcrt located at a specified region of the chromosome 7. We found 60 genes of various functions and lengths, majority (61.67%) of them were performing known functions. Almost all the genes have orthologs in other four species of Plasmodium, of which P. chabaudi seems to be the closest to P. falciparum. However, only two genes were found to be paralogous. Interestingly, the drug-resistant gene, pfcrt was found to be surrounded by seven genes coding for several CG proteins out of which six were reported to be responsible for providing drug resistance to P. vivax. The intergenic regions, in this specified region were generally large in size, majority (73%) of them were of more than 500 nucleotide bp length. We also designed primers for amplification of 21 noncoding DNA fragments in the whole region for estimating genetic diversity and inferring the evolutionary history of this region of P. falciparum genome.

Homogeneity of common cosmopolitan inversion frequencies in Southeast Asian Drosophila melanogaster.

East Asian Drosophila melanogaster are known for great variation in morphological and physiological characters among populations, variation that is believed to be maintained by genetic drift. To understand the genetic properties of Asian D. melanogaster populations, we initiated a population genetic study of chromosome inversion polymorphisms in hitherto unanalysed population samples from Southeast (SE) Asia. We generally found a high frequency of each of the four common cosmopolitan inversions in comparison to populations from Africa, Asia, and Australia. In contrast to the great phenotypic variation among Asian populations, however, we could not detect differences in inversion frequencies among populations. Furthermore, we observed neither correlations of inversion frequencies with population latitude and longitude, nor evidence for linkage disequilibrium between different inversion loci. We propose two explanations for the observed genetic homogeneity among these SE Asian D. melanogaster populations: (i) the observed pattern simply reflects the retention of ancestral polymorphisms originating from a panmictic population that was once present on a large single landmass (Sundaland), and/or is a consequence of high recent gene flow between populations; and (ii) it is caused by selective forces (e.g. balancing selection).

Abdominal pigmentation and growth temperature in Indian Drosophila melanogaster : Evidence for genotype-environment interaction.

Phenotypic variability for abdominal pigmentation in females of an Indian natural population of Drosophila melanogaster was studied using isofemale lines and by rearing the larvae and pupae at 4 different temperatures ranging from 20–30°C. The dark pigmented area was found to increase in all the three segments when the growth temperature decreases. A significant positive correlation was detected for the occurrence of dark pigmentation in the 5th and 6th segments in each growth temperature but for other comparisons the correlation was not regular. Analysis of variance (ANOVA) was carried out both for individual segments over different growth temperatures and also for each temperature over the three abdominal segments and in all cases found to be statistically significant. The results are quite different from the earlier observation in French Drosophila melanogaster and suggest that genes controlling pigmentation are temperature dependent; temperature could affect post-transitional events involved in pigmentation. The present findings also clearly indicate that significant genotype-environment interaction exists, responsible for the production of desired phenotype at the opportune moment during the life span of a species.