Kalyani Putty

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.

Epidemiology of Bluetongue in India

Bluetongue (BT) is an insectborne endemic disease in India. Although infections are observed in domestic and wild ruminants, the clinical disease and mortality are observed only in sheep, especially in the southern states of the country. The difference in disease patterns in different parts of the country could be due to varied climatic conditions, sheep population density and susceptibility of the sheep breeds to BT. Over the five decades after the first report of BT in 1964, most of the known serotypes of bluetongue virus (BTV) have been reported from India either by virus isolation or by detection of serotype-specific antibodies. There have been no structured longitudinal studies to identify the circulating serotypes throughout the country. At least ten serotypes were isolated between 1967 and 2000 (BTV-1-4, 6, 9, 16-18, 23). Since 2001, the All-India Network Programme on Bluetongue and other laboratories have isolated eight different serotypes (BTV-1-3, 9, 10, 12, 16, 21). Genetic analysis of these viruses has revealed that some of them vary substantially from reference viruses, and some show high sequence identity with modified live virus vaccines used in different parts of the world. These observations have highlighted the need to develop diagnostic capabilities, especially as BT outbreaks are still declared based on clinical signs. Although virus isolation and serotyping are the gold standards, rapid methods based on the detection of viral nucleic acid may be more suitable for India. The epidemiological investigations also have implications for vaccine design. Although only a handful serotypes may be involved in causing outbreaks every year, the combination of serotypes may change from year to year. For effective control of BT in India, it may be pertinent to introduce sentinel and vector traps systems for identification of the circulating serotypes and to evaluate herd immunity against different serotypes, so that relevant strains can be included in vaccine formulations.

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

ABSTRACT 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.

Molecular Typing of Bluetongue Viruses Isolated Over a Decade in South India.

Bluetongue (BT) is an arthropod-borne viral disease mostly of sheep. Bluetongue virus (BTV) is a segmented double-stranded RNA virus belonging to the genus Orbivirus of family Reoviridae and is transmitted by midges belonging to Culicoides spp. The disease is endemic in the tropics and subtropics, and the incidence is high in southern India. Twenty-six serotypes of BTV have been reported worldwide. Although most of the serotypes have been reported in India, information regarding currently circulating serotypes is essential to develop control programs. Both serological assays and nucleic acid-based assays have been used for typing BTV. Segment 2, which codes for the outer capsid protein VP2, is the target for PCR-based typing; however, the VP2 sequence diversity among viruses belonging to the same serotype but isolated from different geographical areas makes it essential to develop geographical based reagents. In this study, reverse transcription PCR was developed based on sequences of Indian isolates of BTV (serotypes 1, 2, 9, 10, 12, 16, 21 and 23), and this was applied to type 52 isolates obtained during the last decade. It was found that multiple serotypes circulate, with involvement of more than one serotype infecting individual animals and herds over a period in a given area. Detection of circulating serotypes and estimation of herd immunity against different serotypes of BTV may provide important information for predicting the distribution of these serotypes and inclusion of serotypes in vaccines.

Isolation of Bluetongue Virus 24 from India – An Exotic Serotype to Australasia

Bluetongue (BT) is a viral disease of ruminants and is caused by different serotypes of bluetongue virus (BTV), which is transmitted by several species of Culicoides midges. The disease is endemic in tropical areas, and incursions have been observed in some of the temperate areas. Twenty-seven recognized serotypes of BTV have been reported so far. Some serotype viruses have been shown to circulate in certain geographical areas. BTV-24 has been reported from Africa, the Mediterranean and the Americas, whereas it is exotic to Australasia. Here, we report isolation of BTV-24 from India and show that it has high sequence homology in genome segment 2 with other Western isolates of BTV-24. Entry of this serotype into Australasian region is a cause of concern.

Bluetongue: Aetiology, Epidemiology, Pathogenesis, Diagnosis and Control

Bluetongue (BT) is an emerging and re-emerging vector-borne viral disease of domestic and wild ruminants, caused by viruses classified within the species bluetongue virus (BTV), genus Orbivirus, family Reoviridae. There are 27 recognized serotypes of BTV (with two more recently discovered ‘putative’ serotypes) as well as multiple geographic variants (topotypes) and many different strains and genotypes, most of which are transmitted between their vertebrate hosts by certain ‘vector-competent’ biting midges of the genus Culicoides. Bluetongue is an economically important transboundary disease that is listed by the World Organisation for Animal Health, reflecting the ability of BTV to (a) infect all ruminants, including important domesticated species; (b) cause severe disease, with high fatality rates in sheep and certain species of deer; and (c) cause large economic losses due to fatalities, reduced productivity and reproductive performance, animal movement and trade restrictions and surveillance and control strategies (including vaccination). The plurality of BTV serotypes and strains, the involvement of multiple host and vector species and the potential for (re)introduction of exotic BTV strains make the control and eradication of BT very complex and difficult to achieve. In this chapter, we review the current understanding of BTV biology, bluetongue epidemiology, pathogenesis and pathology, laboratory techniques to diagnose the disease and identify the virus, experimental animal models to study the disease and to evaluate vaccines and methods for the control and/or eradication of bluetongue.

Antagonistic effect of ursolic acid on Staphylococcal biofilms

Aim: The present study was carried out to study the effect of ursolic acid (UA) as a potential anti-biofilm agent in dispersing the biofilm generated by Staphylococcus aureus isolated from milk samples of crossbred dairy cows on the day of drying. Further, in the S. aureus isolates, the presence of intracellular adherence gene locus involved in biofilm production (icaD) was investigated. Materials and Methods: A total of 50 S. aureus strains were isolated over a period of 3 months from 200 milk samples collected from crossbred dairy cows on the day of drying. These isolates were subjected for biofilm detection by Congo red agar (CRA), microtiter plate assay (MTP), and polymerase chain reaction specific for icaD gene. The antagonistic effect of biofilm formation by UA was studied using different concentrations (30 µg/ml and 60 µg/ml) of UA and compared with the control group. Results: Among the 50 S. aureus subjected for biofilm detection, 34 and 40 isolates were detected as biofilm agents by CRA and MTP methods, respectively. The in vitro studies on the effect of UA in inhibiting biofilm formation by S. aureus using MTP assay showed 71.5% and 48.6% inhibition at UA concentrations of 60 µg/ml and 30 µg/ml, respectively, with a significant difference (p<0.05) between the treated and untreated isolates, which was further evident by scanning electron microscopy. Interestingly, the isolates that were tested to be resistant through Antibiotic Sensitivity Test to commonly used antibiotics were found to be sensitive to all the tested antibiotics following UA treatment at both the tested concentrations. Furthermore, molecular detection of icaD gene for biofilm detection revealed that all the isolates that were positive by MTP had icaD gene. Conclusion: Increased incidence of biofilm agents in dairy infections must be considered as an alarming situation. UA treatment significantly enhanced the sensitivity of the microbial pathogens to commonly used antibiotics. Hence, attention must be paid toward implementation of new strategies such as therapeutic regimes with a combination of antibiotic and anti-biofilm agents for effective treatment of infections in dairy farms.