Full-genome sequencing as a basis for molecular epidemiology studies of bluetongue virus in India

Since 1998 there have been significant changes in the global distribution of bluetongue virus (BTV). Ten previously exotic BTV serotypes have been detected in Europe, causing severe disease outbreaks in naïve ruminant populations. Previously exotic BTV serotypes were also identified in the USA, Israel, Australia and India. BTV is transmitted by biting midges (Culicoides spp.) and changes in the distribution of vector species, climate change, increased international travel and trade are thought to have contributed to these events. Thirteen BTV serotypes have been isolated in India since first reports of the disease in the country during 1964. Efficient methods for preparation of viral dsRNA and cDNA synthesis, have facilitated full-genome sequencing of BTV strains from the region. These studies introduce a new approach for BTV characterization, based on full-genome sequencing and phylogenetic analyses, facilitating the identification of BTV serotype, topotype and reassortant strains. Phylogenetic analyses show that most of the equivalent genome-segments of Indian BTV strains are closely related, clustering within a major eastern BTV ‘topotype’. However, genome-segment 5 (Seg-5) encoding NS1, from multiple post 1982 Indian isolates, originated from a western BTV topotype. All ten genome-segments of BTV-2 isolates (IND2003/01, IND2003/02 and IND2003/03) are closely related (>99% identity) to a South African BTV-2 vaccine-strain (western topotype). Similarly BTV-10 isolates (IND2003/06; IND2005/04) show >99% identity in all genome segments, to the prototype BTV-10 (CA-8) strain from the USA. These data suggest repeated introductions of western BTV field and/or vaccine-strains into India, potentially linked to animal or vector-insect movements, or unauthorised use of ‘live’ South African or American BTV-vaccines in the country. The data presented will help improve nucleic acid based diagnostics for Indian serotypes/topotypes, as part of control strategies

Plasmid-mediated antibiotic resistance among uropathogens in primigravid women-Hyderabad, India.

With the growing threat of antimicrobial resistance worldwide, uncovering the molecular epidemiology is critical for understanding what is driving this crisis. We aimed to evaluate the prevalence of plasmid-mediated-quinolone-resistance (PMQR) and extended-spectrum beta-lactamase- (ESBL) producing gram-negative organisms among primigravid women with bacteriuria. We collected urine specimens from primigravid women attending their first antenatal visit at Gandhi Hospital during October 1, 2015 to September 30, 2016. We determined antimicrobial susceptibility and ESBL and quinolone resistance using VITEK-2. We performed polymerase chain reaction amplification on resistant isolates for detection of ESBL-encoding genes (TEM, SHV, CTX-M) and PMQR genes (qnrA, qnrB, qnrD, qnrS, aac (6')-Ib-cr). Of 1,841 urine samples, 133 demonstrated significant bacterial growth with gram-negative bacilli accounting for 85% of isolates, including Escherichia coli (n = 79), Klebsiella pneumoniae (n = 29), Sphingomonas (n = 3), Enterobacter (n = 1), and Citrobacter (n = 1). We found 65% of E. coli isolates and 41% of K. pneumoniae isolates were ESBL positive. Of ESBL-positive isolates, the most common genes conferring resistance were TEM-1 (66.7%) followed by CTX-M-15 (33.3%). Fifty-seven percent of ESBL-positive E. coli also demonstrated resistance to quinolones with the most common PMQR genes being qnr-S (62.5%) and aac (6')-Ib-cr (37.5%). We did not find any resistance to quinolones among ESBL-positive K. pneumoniae isolates. Across different classes of antibiotics we found a strong clustering of multi-drug resistance in E. coli with over 45% of ESBL-positive isolates demonstrating resistance to at least three classes of antibiotics. This study emphasizes the high prevalence of plasmid-mediated ESBL and quinolone resistance in community-acquired urinary tract infections of primigravid women. The overall abundance of multi-drug-resistant isolates in this population is alarming and may present therapeutic challenges

MPGIT: A concentrated monophasic phenol-guanidinium isothiocyanate reagent for isolation of viral RNA

376-381A method to prepare a concentrated monophasic phenol guanidinium isothiocyanate-based reagent for extraction of RNA from liquid samples is described. The reagent was comparable to a commercial reagent for extraction of genetic material of several RNA viruses as assessed by limit of detection of RNA by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The study showed that the concentrated monophasic reagent could be used in place of a commercial reagent

Genome Sequence of Bluetongue Virus Type 2 from India: Evidence for Reassortment between Outer Capsid Protein Genes

Southern Indian isolate IND1994/01 of bluetongue virus serotype 2 (BTV-2), from the Orbivirus Reference Collection at the Pirbright Institute (http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/btv-2.htm#IND1994/01), was sequenced. Its genome segment 6 (Seg-6) [encoding VP5(OCP2)] is identical to that of the Indian BTV-1 isolate (IND2003/05), while Seg-5 and Seg-9 are closely related to isolates from South Africa and the United States, respectively

Genome sequence of bluetongue virus type 2 from India: evidence for reassortment between outer capsid protein genes

Southern Indian isolate IND1994/01 of bluetongue virus serotype 2 (BTV-2), from the Orbivirus Reference Collection at the Pirbright Institute (http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/btv-2.htm#IND1994/01), was sequenced. Its genome segment 6 (Seg-6) [encoding VP5(OCP2)] is identical to that of the Indian BTV-1 isolate (IND2003/05), while Seg-5 and Seg-9 are closely related to isolates from South Africa and the United States, respectively

Phylogenetic analysis based on Seg-2/VP2 gene of Indian isolate of BTV with other global isolates.

<p>Phylogenetic relationship of full length Seg-2 nucleotide sequences (n = 80) was inferred in MEGA 5 using neighbour-joining method and tested by bootstrapping 1000 replicates. Seg-2 nucleotypes were assigned as per Maan et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131257#pone.0131257.ref028" target="_blank">28</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131257#pone.0131257.ref030" target="_blank">30</a>] and depicted by different branch colour. Indian isolates are depicted with blue dots. The node labels in each figure refer to bootstrap confidence values.</p

Phylogenetic analysis based on Seg-6/VP5 gene of Indian isolate of BTV with other global isolates.

<p>Phylogenetic relationship of full length Seg-6 nucleotide sequences (n = 84) was inferred in MEGA 5 using neighbour-joining method and tested by bootstrapping 1000 replicates. Seg-6 nucleotypes were assigned as per Maan et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131257#pone.0131257.ref028" target="_blank">28</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131257#pone.0131257.ref030" target="_blank">30</a>] and depicted by different branch colour. Indian isolates are depicted with blue dots.</p