Biology subjects

Prakash, J. A. J.

Publications and source records attributed to Prakash, J. A. J..

3 recordsLinked to original sources

bioRxiv2025

Preliminary genomic data on five Orientia tsutsugamushi strains isolated in Vellore, India

IntroductionOrientia tsutsugamushi, the causative agent of scrub typhus, can be isolated in Vero or L929 cells and has a small genome (2-2.5 Mb). However, genome assembly is challenging due to the presence of host DNA contamination and a high proportion of repeat regions (up to 51%). Current global data includes 11 fully annotated genomes, with none from India. Here, we present the first whole-genome sequences of O. tsutsugamushi from India. MethodsFive O. tsutsugamushi strains were cultured in Vero cells and confirmed by 47kDa real-time PCR. Genomic DNA was extracted after removal of host DNA and sequencing libraries were prepared. Whole-genome sequencing was performed using the PacBio Sequel II system in CCS/HiFi mode. The raw reads were assembled using Flye, and genome completeness was assessed with QUAST and BUSCO. Annotation was performed using the NCBI PGAP pipeline and comparative genome analysis by Roary. Phylogenetic analysis was based on the full-length 56kDa gene, which contains four variable domains. ResultsWe report five complete genomes of O. tsutsugamushi, four of which are circular and one linear. Genome sizes range from 2.1 to 2.4 Mb. The total number of predicted genes falls between 2,379 and 2,715, with an average of 1,824 coding genes and 613 pseudogenes. Repeat regions constitute 53-59% of the genome, a higher proportion than previously reported. All five genomes have been submitted to NCBI GenBank (Accession Numbers: CP166954-58). Phylogenetic analysis based on the full-length 56kDa gene revealed that two strains belong to the Karp genogroup, two to Kato, and one to TA763. ConclusionThis study presents the first whole-genome sequencing data of O. tsutsugamushi from India. Notably, the repetitive regions in these genomes are more extensive than previously reported. Further analyses with additional isolates are necessary to validate this observation. Comprehensive phylogenomic studies, particularly to elucidate evolutionary dynamics and potential recombination events will provide further information.

microbiology↗

bioRxiv2023

First isolate of Orientia tsutsugamushi from Vellore, South India

BackgroundScrub typhus a common cause of acute febrile illness in India caused by Orientia tsutsugamushi an obligate intracellular bacterium requiring cell culture for isolation. Cell lines like Vero and L929 are most suitable for isolating and maintaining this organism. This study was undertaken to isolate and characterize of Orientia tsutsugamushi from whole blood samples at a tertiary care centre in Southern India. MethodsThe PBMCs (peripheral blood mononuclear cells) collected from scrub typhus positive (47kDa qPCR positive) patients were inoculated into Vero and L929 cell line at 80% confluence for primary isolation. The inoculated flasks were incubated at 37{degrees}C with 5% CO2 for 30 days and examined for presence of Orientia tsutsugamushi on the day 10, 15, 20 post-inoculation and everyday thereafter for a maximum of 30 days post inoculation. The scrapings were subjected to Giemsa staining, IFA, 47kDa qPCR and transmission electron microscopy (TEM). The isolates were passaged 3-4 times to ensure viability and then stored in DMEM with 10% FBS (-80{degrees}C). Genotyping of the isolates was performed by amplifying a 650 bp segment of the TSA 56 (type specific antigen 56) gene. ResultsAmongst the 50 samples inoculated, three were culture positive as confirmed by 47 kDa qPCR at 24th day of inoculation. This was further confirmed by Giemsa, IFA staining and TEM. The 650bp amplicons showed 99.5 to 100% homology with Orientia tsutsugamushi MW604716, MH003839, MW604718, MW604717, MH922787 and MH003838 strains. Phylogenetic analysis revealed that 2 isolates belong to TA763 genotype and one belongs to Gilliam genotype. ConclusionsWe have successfully isolated and characterised the Orientia tsutsugamushi for the first time at our centre from PBMCs. Based on the partial TSA56 gene sequence our isolates belongs to TA763 and Gilliam genotype. More number of samples are being processed for identifying further isolates followed by genomic analysis.

microbiology↗

bioRxiv2023

Genotyping of Orientia tsutsugamushi circulating in and around Vellore (South India) using TSA 56 gene

The immunodominant TSA 56 gene of Orientia tsutsugamushi, (scrub typhus agent) has four variable regions (VD-I to VD-IV) making it useful for genotyping. As of date the genotyping data from India is based on partial 56kDa gene sequence analysis. The complete TSA 56 gene sequence is important for knowing the circulating strains and for designing region specific diagnostics and vaccines. This study was undertaken to determine Orientia tsutsugamushi genotypes circulating in and around Vellore using complete and partial TSA 56 gene. Of the 379 whole blood samples from suspected scrub typhus patients, 162 were positive by 47 kDa qPCR. Long protocol to amplify the complete TSA 56 gene ({approx}1605 bp) was performed on 21 samples. On the same 21 samples the partial gene sequence was also amplified using the Horinouchi ({approx}650bp) and the Furuya ({approx}480 bp) protocol. Using a combination of Sanger and Nanopore technology complete sequence was obtained for 9 and near complete (1551 to 1596 bp) for 4 respectively. As Furuya protocol gave multiple bands we obtained 480 bp sequences from the 13 complete gene sequences by in silico analysis. In contrast, 650bp sequences were obtained for 11 samples while for the remaining two we derived the 650 bp sequences from the complete gene sequences (Long protocol). Phylogenetic analysis of the complete gene (Long protocol) which includes VD-I to VD-IV region and partial gene (Horinouchi) which amplifies the VD-I to VD-III regions showed identical genotypes. Twelve belonged to TA763 genotype and one belongs to Karp genotype. The Furuya sequence (in silico) correctly identified the Karp genotype and 10 of the TA763 genotypes. Two TA763 genotypes (identified by complete and 650 bp partial gene analysis) were misidentified by Furuya sequence analysis as Karp genotype. The limited analysis showed the commonest Orientia tsutsugamushi genotypes circulating in and around Vellore is TA763 and that the 650 bp (Sanger) sequencing could be a cost effective method for identifying the scrub typhus genotypes. However, these results need to be validated by larger prospective multi-centric studies.

microbiology↗