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Bluetongue virus (BTV) is an arthropod borne orbivirus that infects sheep, goat, wild ruminants and occasionally cattle. Sheep and goats are natural hosts; cattle act as reservoir hosts whereas biting midges Culicoides are biological vectors. Affected sheep and goats show high fever for about 5-7 days and by 7-10 days the distinctive lesions appear in the mouth and the tongue is severely affected and turns dark blue and hence the name 'bluetongue'. Sheep may become lame as a result of coronitis.
Bluetongue virus is a member of the Orbivirus genus of the family Reoviridae. Within Orbivirus genus, there are 19 serogroups which share antigens detectable in complement fixation tests, agar gel immunodiffusion tests and fluorescent antibody tests and hence there is certain degree of cross reactivity between distinct serogroups. Bluetongue serogroup has 24
serotypes.
The viruses belonging to family Reoviridae are relatively heat stable, resistant to lipid solvents. BTV is the prototype of the Orbivirus genus. The viruses are icosahedral, non-enveloped particles of approximately 85-nm diameter and have concentric protein layers enclosing 10 segments of double stranded RNA. The inner icosahedral core contains two major (VP3 and VP7) and three minor proteins (VP1, VP4 and VP6). VP7 is a major core protein possessing the serogroup determining antigens. The outer capsid consists of two other major proteins namely VP2 and VP5, where VP2 is the major determinant of serotype specificity.
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Structural components of Bluetongue virus
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Bluetongue virus dsRNA segments and the proteins coded by them
BTV genome consists of ten segments of dsRNA, whose sizes are distinguished by their electrophoretic mobility migration. They are grouped as large, medium and small segments and are named L1-L3, M4-M6 and S7-S10. These segments encode seven structural proteins (VP1-VP7) and three nonstructural proteins (NS1, NS2 and NS3).
|
Gene Segment |
Length
(bp) |
Gene Product |
Protein size
(kDa) |
Protein function |
| L1 |
3944 |
VP1 |
146 |
RNA
Polymerase |
| L2 |
2926 |
VP2 |
111 |
Outer
capsid protein |
| L3 |
2772 |
VP3 |
103 |
Inner
shell of core |
| M4 |
1981 |
VP4 |
75 |
RNA
'capping' enzyme |
| M5 |
1639 |
VP5 |
59 |
Outer
capsid protein |
| M6 |
1770 |
NS1 |
64 |
Forms
tubules |
| S7 |
1156 |
VP7 |
38 |
Outer
shell of core |
| S8 |
1123 |
NS2 |
41 |
Binds
ssRNA, phospho protein, forms VIBs |
| S9 |
1049 |
VP6 |
35 |
RNA
helicase |
| S10 |
822 |
NS3 |
25 |
Glycoprotein,
helps in virus release |
General approaches for the detection of BTV
Detection and specific identification of BTV is a multistep process. The first step involves the isolation of the virus from animal's blood or other tissues, (15-20 ml of heparinised blood, spleen, liver, kidney, lung and heart tissues) followed by inoculation of embryonating chicken eggs (ECE). After the virus has been amplified in ECE, it is passaged into BHK-21 cell culture for subsequent replication and identification. The virus is then amplified further and identified in microtiter plates by the immunoperoxidase assay using a group specific monoclonal antibody. Finally, the viral isolate is typed by a virus neutralization test.
Although the common laboratory procedures for isolation of BTV from animal blood and tissues are inoculation of ECE and cell culture, inoculation of sheep and demonstration of BTV seroconversion still remains the most sensitive method for BTV isolation from field samples. However, this procedure is expensive and requires 200-300ml of blood for inoculation.
The laboratory diagnosis of BTV is a laborious procedure that requires well-trained personnel having a good understanding of the overall isolation procedure. Successful isolation of BTV depends primarily on the presence of adequate concentrations of virus in the original specimen used. Heparinized blood collected during the febrile phase of the disease is the sample of choice for the isolation of BTV. Generally, ruminants produce neutralizing antibodies of BTV between 10 and 14 days. Heparinized or EDTA blood is suitable for virus isolation and these additives do not affect the isolation process. Storage of the sample is the critical element affecting the outcome of the isolation procedure. BTV can be easily recovered after storage at 4oC over a period of 300 days as whole or washed cellular components in RBC resuspension medium or oxalate-phenol-glycerol (OPG) mixture as additives. Storage of unwashed blood samples should be avoided as the possibility of isolation is reduced. This is due to the presence of neutralizing antibodies in the blood sample. If long term storage is required, samples whether washed or not, can be frozen at -70oC in 10% DMSO. Currently, heparinized blood is the sample of choice for the isolation of BTV in the context of a sentinel program or during the febrile period in infected animals. In the past, when refrigeration was not widely available in other countries, preservatives such as OPG mixtures were used.
During bluetongue infection, viral particles are considered to be engulfed within pockets of the red blood cell (RBC) membrane. For this reason, the RBC membrane must be disrupted by using sonic disruption step for this purpose. However, special attention should be taken to avoid cross-contamination of the specimens when more than one sample is to be processed at the same time. Meticulous cleaning and disinfection of the sonication probe between samples should be practiced.
Successful inoculation of BTV in ECE by the intravenous route requires a degree of technical expertise. Mortality of the chick embryo due to inappropriate handling of the eggs or poor inoculation techniques should be reduced as much as possible. Cooling the eggs to room temperature (20-22oC) by removing them from the incubator 1-2 h before inoculation considerably reduces non-specific embryo mortality, which results from bleeding at the site of inoculation.
BTV nucleic acid in inoculated chicken embryo can be detected even as early as 24 h and by 48 h throughout the entire embryo by in situ hybridization.
The choice of the cell line for BTV isolation should be evaluated carefully as the successful identification of the virus will depend on how well the cell culture supports virus growth. BHK-21 and CPAE cells have been demonstrated to be highly susceptible to both cell culture-adapted and animal source BTV with easily observable cell death within 2-5 days. Following inoculation of BHK-21 cell culture plates with field samples, continuous movement of inoculum over the cell monolayer is critical for better virus yield. To facilitate this, BHK-21 plates should be rocked during virus adsorption.
The immunoperoxidase test is a relatively straightforward method for the identification of BTV isolates. Special attention should be taken to select the appropriate virus dilution and incubation time in order to avoid extensive development of CPE and subsequent loss of cell monolayer in the fixation period.
The virus neutralization test is usually performed for serotyping of BTV following identification by a group specific test, e.g. immunoperoxidase assay. Serogrouping of BTV can also be done by immunofluoresence or by antigen capture enzyme-linked immunosorbent assay by using a BT serogroup - specific monoclonal antibody (Mab). The use of type specific polyclonal antibodies as serum controls in the VN test provide information relative to the BTV serotype involved, which is important for epidemiological evaluation of the isolate.
Neutralization tests are type-specific for 24 BTV serotypes and can be used to serotype a virus isolate.
BT virus serotype specific antiserum generated in guinea pigs or rabbits show lesser serotype cross-reactivity than those made in cattle or sheep. It is important that antiserum controls should be included to ensure that an effective level of standard antiserum is used against comparable and standardized titres of standard and untyped virus.
Plaque assay technique is commonly used to determine virus titres.
Anti-BTV antibody generated in infected or vaccinated animals can be detected by a variety of ways. Agar gel immunodiffusion test is simple to perform. A prescribed test for International trade is competitive ELISA. This technique was developed to measure BTV specific antibody only by using BT serogroup reactive Mabs which appear to bind to the amino terminal end of VP7. In this test, antibody in test sera competes with the MAbs for binding to antigen.
Both serogroup specific and serotype specific antibodies are elicited if the animal was not previously exposed to BT virus and the neutralising antibodies generated are specific for the infectious virus. Multiple infections with different BT virus serotypes lead to the production of antibodies capable of neutralizing serotypes to which the animal has not been exposed. This is because sharing of MAb defined neutralization epitopes by several serotypes.
In recent times various molecular approaches have been used for detection of BTV and its structural components like proteins and nucleic acid. These have been described below:-
I. Molecular techniques for the demonstration of BTV and its proteins:
1) Purification of BTV particles
Purification of bluetongue virus is essential either for the demonstration of BTV by electron microscopy, SDS-PAGE, Western blot analysis or by autoradiography /
autofluorography.
Highly purified bluetongue virus particles can be obtained by following the method of Mertens et al (1987). In brief, this procedure is as follows:
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First the BHK-21 cells are infected with BTV and harvested after 2-3 days when nearly 80% CPE can be observed. Both the cells and the supernatant are collected.
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Next, BTV is precipitated from the supernatant by ammonium sulfate precipitation.
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The cell pellet is homogenized twice with 0.5% Triton - TNE buffer and supernatant is saved.
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Both precipitated BTV and homogenized cell pellet are layered over 66% w/w sucrose and 40% w/v sucrose gradient and centrifuged at 25000rpm for 3 h.
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The thick opaque band at the interphase is collected and dissolved in Tris-HCl buffer, pH8.0 and treated with 30% sodium deoxycholate.
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The lysed material is again layered over 66% w/w sucrose and 40% w/v sucrose gradient and centrifuged.
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The opaque band at interphase is collected carefully and dissolved in Tris-HCl buffer, pH 8.0 and treated further with N-lauryl sarcosine.
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The lysed material is layered over sucrose gradient and centrifuged. The interphase is collected and dialyzed to remove salts.
The purity of the virus preparation can be checked by performing SDS-PAGE.
2) Electron microscopy analysis
A formvar carbon coated grid containing a droplet of sample is stained with uranyl acetate and examined under electron microscope.
Immunogold electronmicroscopy is used to identify the specific location of BTV in specimens.
3) Sodium dodecyl sulfate polyacrylamide gel electrophoresis
This technique is used to resolve various BTV specific proteins on a denaturing polyacrylamide gel. The procedure consists of the following steps: -
- First a resolving gel mixture, consisting of 10% - 15% w/v polyacrylamide, 375mM Tris - HCl, pH8.3 and 0.1% (w/v) SDS, TEMED and APS (polymerizing agents), is poured between two glass plates. Water is layered on top.
- Once the gel is polymerized, the water is removed, and a stacking gel comprising of 5% w/v polyacrylmide, 135 mM Tris - HCl, pH6.8 with 0.1% (w/v) SDS, TEMED and APS is overlayed on it and a comb is inserted to provide sample wells.
- The running buffer is 1 x SDS - PAGE buffer made of 200mM glycine, 25mM Tris - HCl, pH8.8 and 0.1% w/v SDS.
- The BTV samples (purified virus preparations or cell pellet from infected cell culture) are mixed with an equal volume of 2 x SDS - PAGE sample buffer (10mM Tris - HCl, pH6.8, 10% (w/v) SDS, 10% (v/v) b - mercaptoethanol, 25% (v/v) glycerol, 0.02% (w/v) bromophenol blue) and held at dry heating block for 5 mins.
- Proteins in the sample buffer are then applied to the wells of the gel along with wide range molecular weight markers that are loaded in the first well.
Gels are electrophoresed at 100V until the dye front reached the resolving gel and then at 160V until the dye front reached the end of the resolving gel. To visualize the proteins, the gel is soaked in Coomasie Brilliant Blue stain for 1 h (0.25% w/v Coomasie brilliant blue stain, 45% (v/v) methanol l0% (v/v) acetic acid), then destained in 45% (v/v) methanol and 10% (v/v) acetic acid. Gels are dried in a vacuum drier for 2 h at 85oC.
4) Western blot analysis
This method is as follows:-
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esolve the viral proteins by SDS-PAGE, soak the gel for 5 min in blotting buffer (3.03g Tris, 14.41/g glycine, methanol (to 20% V/V) and water to 1 litre, pH8.3). Bands of Prestained molecular weight marker can be seen on the gel.
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The PVDF - Immobilon membrane, is first soaked in methanol and then in the blotting buffer.
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The gel and the membrane are sandwiched between 3MM Whatman filter paper soaked in blotting buffer and using a blotting apparatus, a current of 100mA is applied for 1 h, for the transfer of proteins from gel to membrane.
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After the transfer, the PVDF membrane with proteins, is soaked in blocking buffer (PBS with 5% (w/v) skimmed milk, 0.05% (v/v) Tween 20 for 1 h. The membrane is then drained and gently agitated at room temperature for 1 h with 10 ml of blocking buffer and 1: 1000 dilution of primary antibody.
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The membrane is then washed in PBS with 0.05% (v/v) Tween 20 for 30 min (three changes of washing buffer) and then incubated with 1: 1000 dilution of secondary antibody (goat antimouse or goat antirabbit or goat antiguineapig) conjugated with alkaline phosphatase.
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After 1 h incubation, the membrane is washed as before, and incubated with the alkaline phosphatase substrate mixture containing 44 ml of nitroblue tetrazolium chloride (NBT) and 33ml of 5-bromo-4-chloro-3-indolylphosphate p-toluidine (BCIP) in 10 ml of reaction buffer (100mM Tris-HCl, pH9.5, 100mM NaCl, 50mM MgCl2).
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After the reaction is complete, coloured bands appear on the membrane and these are compared with the pre-stained markers. Prestained marker is helpful in assessing the size of the band.
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Finally, the membrane is washed with water and dried at room temperature.
Alternatively CDP - star chemiluminiscent substrate (Sigma) is extremely sensitive in detecting alkaline phosphatase labeled molecules.
5) Metabolic radioisotope labeling of proteins and autoradiography:
This technique consists of the following steps: -
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BHK-*21 cells grown in 35 mm dish are infected with 100 TCID50 of BTV. After 24 to 48 h postinfection, the cells are washed twice with methionine-free GMEM and incubated with 1 ml of methionine - free GMEM for 1 h at 37oC to starve the cells of methionine.
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This medium is then removed and substituted with 1 ml of methionine - free GMEM supplemented with 2 ml of [35S] methionine (total activity 20uCi).
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Cells are then incubated in this medium for 2 h at 37oC. Then the medium is removed, the cells are washed in PBS.
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Next the samples are mixed with equal volume of 2 x SDS - PAGE sample buffer and labeled proteins are resolved by SDS-PAGE. The gels are dried and exposed to X-ray film.
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The bands that are developed on the X - ray film are observed in relation to wide range marker.
In autofluorography, a phosphoimager is used for developing the bands of resolved labeled proteins on the dried gel.
II. Molecular techniques for the detection of viral nucleic acid
1) Migration pattern of different segments of dsRNA
Extraction of pure dsRNA of BTV is essential for subsequent reverse transcription to obtain cDNA. The method of Mertens et al (1984) can be followed for the purification of dsRNA. Nucleic acid from BTV infected cells can be extracted by Phenol chloroform and then precipitated by ethanol. dsRNA can be isolated by using 8M Lithium chloride and then by precipitating with 4M Lithium chloride. The 10 segments of dsRNA can be observed by 1% agarose gel electrophoresis. The dsRNA from various BTV isolates are prepared and run in parallel on 1% agarose gel. Migration pattern of different segments of dsRNA from different isolates / serotypes of BTV gives most valuable information about BTV isolates.
2) Reverse transcriptase PCR and complementary DNA amplification
After visualizing 10 dsRNA segments by 1% agarose gel electrophoresis, the appropriate band from the gel is cut and placed in dialysis bag and purified by electroelution. The purified segment of dsRNA is checked by 1% agarose gel electrophoresis.
Complementary DNA (cDNA) is synthesized by adding RNA to Reverse transcription reaction mixture containing PCR buffer, dNTP mixture, reverse transcriptase and specific forward and reverse primers and then holding the mixture in thermal cycler. Temperature and time varies according to the segment reverse transcribed.
The cDNA is then amplified by addition of about 5mM MgCl2, 10 X PCR buffer, 10mM of each dNTP, specific forward and reverse primers and Taq DNA polymerase in a volume of 50ml. PCR conditions such as temperature and time for denaturation, annealing and primer extension and number of cycles vary according to the segment to be amplified.
The size of the band is checked by running on 1% agarose gel electrophoresis by loading 1-2ml of amplified cDNA. Next, the PCR product can be purified by using PCR product purification kit.
3) Cloning of PCR products
When Taq polymerase is used in cDNA amplification, there are 'A' overhangs at 3/ end. Such PCR products are very efficiently ligated into TA cloning vectors of Invitrogen. Whereas in case of Vent polymerase, the blunt ends are generated which can be cloned in appropriate vector. Alternatively if PCR product has any restriction enzyme sites at the 5/ and 3/ ends (e.g. BamH1), the plasmid DNA is linearised by cutting with a restriction endonuclease at particular site and then the insert and the vector are ligated by use of DNA ligase. Once ligation is completed, this plasmid is transformed into competent E.coli cells and colonies obtained by plating on LB agar plates. Recombinant clones can be identified by blue/white selection since the vector is lac Z genetically marked.
A single recombinant colony is inoculated into the LB broth containing ampicillin and incubated at 37oC overnight with vigorous shaking. From cell pellet, plasmid DNA is purified by using Qiagen plasmid purification kits.
4) Restriction enzyme digestion and analysis
The presence of the specific restriction enzyme sites in each segment in the insert of the purified plasmid can be confirmed by restriction endonuclease digestion and the restriction digestion products are analysed by size of the band in agarose gel electrophoresis. Even within the serotypes, some isolates show variation in restriction enzyme sites. Hence the restriction enzyme analysis is useful in identifying individual BTV isolates.
5) Sequencing of the clone with PCR product
Most DNA sequencing methods presently in use are variations of the chain termination method developed by Sanger and co-workers in the late 1970s. In this method, the DNA to be sequenced acts as a defined primer-binding site. In automated sequencing a laser is used to detect DNA fragments labeled with fluorescent dyes. When different dye labels are used to tag each of the four dideoxynucleotides, the reactions can be performed in a single tube and analyzed on a single lane increasing the capacity and throughput of each sequencing gel. Thermostable DNA polymerase that have a high affinity for dideoxynucleotides such as AmplitaqTM FS from ABI and ThermosequenaseTM from Amersham have a modified active site that accommodates dideoxynucleotides more easily than the unmodified enzyme.
Sequencing of individual segment gives more accurate information about the phylogenetic relationship of individual isolates of BTV.
6) Transcription - Translation of the plasmid with PCR product
TNTŪ quick coupled transcription / translation systems of Promega, USA allows the generation of the protein coded by the insert. This specific protein can be visualized by SDS - PAGE or by Fluorogenic enhancement or by autoradiography.
7) Nucleic acid probes and hybridization
A probe is a molecule having a strong interaction only with a specific target and having a means of being detected following the interaction. The nucleic acid probes interact with their complement primarily through H - bonding. In general it is often desirable to generate probes labeled either uniformly or at their ends (5/ or 3/ terminus). In case of isotopic labelling mostly 32P or 35S are used. The basis of autoradiography is the utilization of b - emission from bound probe molecules to create an image on film by the development of silver grains.
In non-isotopic method of labelling Biotin, HRP or alkaline phosphatase or digoxigenin are used as labels. Chemiluminescent substrates are highly sensitivity for detecting the probes with an antibody - enzyme or streptavidin enzyme conjugate.
The "in situ hybridization" method allows the examination of specific sequences directly in fixed cells or tissues. After hybridization, the localization of the probe molecules in the cells can be examined microscopically.
References
- Clavijio, A., Heckert, R.A., Dulac, G.C. and Afshar, A. (2000). Isolation and identification of bluetongue virus. J. Virol Methods 87 : 13-23.
- Mertens, P.P.C., Brown, F. and Sangar, D.V. (1984). Assignment of the genome segments of bluetongue virus type 1 to the proteins which they encode. Virology, 135: 207 - 217.
- Mertens, P.P.C., Burroughs, J.N. and Anderson, J. (1987) Purification and properties of virus particles, infectious sub-viral particles and cores of bluetongue virus serotypes 1 and 4. Virology 157 : 375 - 386.
- Office International Des Epizootics Manual (2000) Manual of Standards for Diagnostic Tests and Vaccines for terrestrial animals. 3rd Edition Chapter on Bluetongue.
- Polly Roy (1991). Towards the control of emerging bluetongue disease. Oxford Virology Publications, London.
- Sambrook, J., Russell ,D.W. and Sambrook,J. (2001). Molecular cloning. A laboratory manual 3rd edition Cold Spring Harbour Laboratory Press, USA.
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