Studies of the Antigenic relationships between Bluetongue virus serotypes 2 , 9 & 15 isolated in Andhra Pradesh , India

The presence of multiple serotypes of the midge-borne bluetongue virus and lack of effective vaccine are the major impediments in controlling bluetongue in sheep. Attempts are being made to develop a vaccine employing the available serotypes to control the disease in the state. Hence, it is essential to identify the antigenic relationships among the serotypes to identify the candidate strains to be incorporated in the preparation of vaccine. To understand the antigenic relationships between Bluetongue virus -2, 9 and 15 serotypes, the viruses were propagated in BHK21 cell lines, purified using PEG precipitation method and purified virus used to raise hyper immune serum in rabbits. Neutralizing antibodies for the BTV serotypes were detected by day 21 PI. Reciprocal cross neutralization test was employed to determine the R% values between BTV-2, 9 and 15 which indicated the extent of antigenic relationships among the serotypes. R% value between BTV-2 and BTV-9 was recorded as 2.8. R% value of 3.53 and 2.8 were observed between BTV-2 & 15 and BTV-9 & 15 respectively. The R% values recorded in the present study revealed a weak antigenic relationship between the BTV serotypes, indicating that the serotypes are highly divergent.


Introduction
the state (Bommineni et al.2008).These outbreaks were reported to the animal husbandry department of Bluetongue (BT) is a non-contagious, arthropod the state government and not to the OIE since the state borne viral hemorrhagic disease of ruminants, particularly government is the controlling authority.There is an of sheep and occasionally cattle and some species of urgent need to develop the vaccine to control the deer.It is also associated with abortion storms and disease in the state.Control of BT in an endemic high mortalities.The disease, once confined to the region depends primarily on the active immunization exotic sheep viz Southdown, Rambouillet, Russian of susceptible animals.This requires the identification Merino and Corriedale, recently became established in of the circulating serotypes and determination of their native sheep of South India, causing severe outbreaks serological relationships.However information is not in the region.The disease is reported annually from the available on the antigenic relationships among the south Indian states, Andhra Pradesh, Karnataka, Tamil viruses isolated from South India, particularly from Nadu and Maharashtra and is causing socioeconomic Andhra Pradesh.The current study was thus aimed at problems among the sheep farmers.(Sreenivasulu et determining the antigenic relationships among BTV al. 2004).There are now 24 serotypes of BTV in the serotypes -2, 9 and 15 isolated in Andhra Pradesh, to world.However 25th serotype was also reported from et al. facilitate the selection of viruses for inclusion in the Kenya (Davies , 1992).Of these 21 serotypes have been reported in India (Sreenivasulu et al., 2004).vaccine formulated for disease control.BTV serotype 2 was isolated from native sheep of
Bluetongue viruses: BTV-2/TPT, BTV-9/MBN and Subsequently, BTV serotype 9 from Mehaboobnagar BTV-15/N12 isolates recovered from bluetongue district in 2002 and BTV serotype 15 from Nalgonda outbreaks which occurred in Andhra Pradesh, and district in 2003 were also isolated from outbreaks in stored at the department of Microbiology, College of Veterinary science, Tirupati, Andhra Pradesh, India, discarded following transfer into the last well.Virus were used in this study at 32nd, 4th and 5th passage containing 100TCID50 at volumes of 50µl were levels respectively.The viruses were grown in BHK21 added to each dilution of the sera.The sera-virus clone 13 cell line for further work.
mixtures were incubated for one hour at 37 degrees Purification of Bluetongue virus: The method of Celsius and 5% Co2.. BHK21 cell suspension in Hubschle (1981) with slight modification using growth medium (Himedia) at a concentration of 1 x 6 Polyethylene Glycol (PEG 6000) and sodium chloride 10 cells/ml was added to all the wells in 100 µl at the rate of 7% and 2.3% respectively, was used for volumes.Cell controls contained only 100µl of cell virus purification.The presence of virus in the final suspension in growth medium and virus controls contained 100µl cell suspension and 50µl of diluted pellet was determined by RNA extraction and the virus (100 TCID50).The plates were sealed and product was subjected to agarose gel electrophoresis.incubated at 37 degrees Celsius in a humidified CO2 The migratory pattern of RNA was compared with the atmosphere until a complete monolayer developed in standard pattern of Bluetongue virus RNA which was the cell controls and complete CPE in the virus established and is common to all serotypes.Antisera controls was observed.The test was often read : Antisera were raised against purified BTV-2, 9 and 15 in 8-month old serologically negative following 3-5 days of incubation.The titre of the rabbits as described by Huismans and Erasmus, 1981.serum was regarded as the highest dilution at which In brief, protein content of the antigen (purified virus) the virus was neutralized.The test was read as was quantified by spectrophotometry and the priming positive, in wells with more than 80% of the cell sheet dose was administrated after blending 0.5ml of intact, indicating neutralization of the BTV by the antibodies in the hyper immune serum.

purified BTV (500µg of protein) with an equal
Reciprocal cross neutralization test: The protocol quantity of Freund's complete adjuvant.Subsequently, employed for conducting cross neutralization tests three boosters were administered along with Freund's was essentially similar to Micro Serum Neutralization incomplete adjuvant.The rabbits were bled a week Test with slight modifications.Each virus serotype following administration of the last dose of inoculum under study was neutralized with the hyper immune and the serum was collected.All the sera were heat 0 sera raised against the three serotypes.The inactivated at 56 C for 30 min, sterilized by filtration homologous serum was titrated from an initial dilution through a 0.45µm membrane filter and stored at -0 of 1:10, while the heterologous sera were titrated from 20 C until used.initial dilutions of 1:4, to detect cross-reactions even at Agarose gel immune diffusion test (AGID) : The a lower dilutions of the sera.The serological rabbit sera were tested for the presence of BTV group relationships (R) between the BTV serotypes 2, 9 and specific antibodies using respective purified viruses as 15 were calculated using the formula as described by antigen and the uninfected cell culture as control.Dopazo et al. (1996).AGID detects group specific antigen, NS3 which is common to all the serotypes.

Microtitre Serum Neutralization Test (MSNT):
R -value is defined as the ratio of the The quantal microtitre assay was performed using heterologous to homologous serum titres.Ratio r1 was decreasing serum-constant virus (beta) method calculated by dividing the heterologous titre obtained (Parker et al., 1975) in sterile flat-bottomed microtitre with serum 2 by the homologous titre obtained with tissue culture plates.This method was employed to serum 1, and the ratio r2 was determined by dividing detect the titre of the hyper immune sera raised against the heterologous titre obtained with serum 1 by the each of the virus serotypes and also to determine cross homologous titre obtained with serum 2. The R value neutralization titres between the serotypes.
gives the extent of the antigenic relationship between Initially, 1:10 dilutions of the sera were made.
the two viruses.The first rows of the microtitre plates were filled with Sequence analysis: Multiple sequence alignment of 75µl of diluent, and the second to the last rows the VP2 gene (sequence length of 3Kilo Base pairs) of contained only 50µl of the same diluent.Each serotype BTV-2, 9 and 15 South African reference serotypes was tested in duplicate, where 25µl was added to the (accession numbers AJ585123.1,AJ58130 and first well of the designated columns, following which AJ585136) was carried out by using sequence data serial two fold dilutions from 1/160 to 1/20480 were obtained from Genbank using BLAST tool V.2.2.17.prepared by transferring 50µl from the wells in the first and the alignment was edited using Bio edit V.5.09.rows to subsequent wells, and the same quantity was Percent homology and divergence between the BTV region of India.Since there is no control over the serotypes 2, 9 and 15 was generated by using Martinez prevalence of bluetongue virus serotypes and vector Needleman-Wunsch algorithm of MegAlign software control is a difficult task, the best alternative is to V.4.03.
develop a suitable vaccine to control the disease.The neutralizing antibodies to BTV serotypes were isolated from native sheep of Andhra Pradesh.2, 9 and 15 were noticed from day 21 post infection An inactivated vaccine using BTV-2, 9, 15 serotypes (PI) and continued to increase reaching a maximum by was subsequently prepared and it is currently under day 45 PI.The neutralizing (SN50) antibody titres field evaluation.To refine the vaccine, it is necessary against BTV-2, 9 and 15 are shown in Table-1.The to identify candidate vaccine strains based on the serum neutralizing titres induced against BTV-2, 9 and antigenic relationships.However, all the available 15 persisted until the end of the experiment, on day 60 serotypes cannot be included in the preparation of PI.
vaccine due to the interference phenomenon which inoculated with BTV-20 on 2nd and 3rd week PI.

Discussion
Sreenivasulu and Subba Rao (2000) also demonstrated the appearance of neutralizing antibodies by Day 9 PI Insect vector transmission, prevalence of multiple in sheep and goat inoculated with BTV-2.All the three serotypes, broad host range and non-availability of a serotypes of BTV in the present investigation were suitable vaccine are playing a major role in establishing immunogenic inducing both neutralizing and and causing regular outbreaks of BT in the Southern precipitating antibodies.
Erasmus (1990) established the serological Studying antigenic relationships among the relationships between 24 BTV serotypes using plaque BTV serotypes is important in the selection of reduction assays (Fig. 1).Maan et al., (2007) correlated candidate strains to be included in vaccine preparations.these serological findings with the nucleotide and Dopazo et al., (1996) used reciprocal cross neutralization amino acid homologies and grouped the 24 BTV test to study serological relatedness among aqua serotypes into 10 nucleotypes (A-J).Serotypes having reoviruses and found that the test was most suitable in less than 35% differences in their nucleotide and grouping related viruses.R% value of 100 between amino acid sequences were grouped into one two viruses indicated that they were serologically nucleotype.BTV serotypes 2, 9 and 15 belonged to identical.The R% values obtained in this study three different nucleotypes, I, E and J respectively.indicate a weak antigenic relationship between the Nucleotide sequence variations in segment-2 of three BTV serotypes.Brooksby (1968) also used the the variable VP2 gene correlate with differences in R% values to study the antigenic relationships among virus serotypes (Maan et al., 2007), thus the gene is various strains of foot and mouth disease virus utilized for serotyping viruses on a molecular level.(FMDV) and proposed the R% value of 70% or more Since full length sequences of the indigenous BTV to group the FMDV strains into the same subtype, 32-serotypes was not yet published, reference BTV 70% to identify different subtypes, 10-32% for widely serotypes were used in the study.Though there may be different subtypes and less than 10% for different slight variation between the sequences of reference serotypes of FMDV.Hence, R% values of less than and indigenous serotypes, it is expected that the results 10% are considered to imply that the viruses are will give an idea as to the extent of the relationships antigenically unrelated.The R% values of 2.8, 3.5 and between the serotypes.The genomic relationships 2.8 obtained for BTV serotypes 2, 9 and 15 is less than between the reference BTV serotypes 2, 9 and 15 and 10% and suggest the serotypes are antigenically the R% values (serological relationship) of the unrelated to each other.
indigenous BTV serotypes 2, 9 and 15 were taken for