Detection of bluetongue virus serotype 17 in Culicoides variipennis by nucleic acid blot and sandwich hybridization techniques.

Molecular hybridization techniques were developed for the detection and surveillance of bluetongue virus (BTV) serotype 17 in the insect vector Culicoides variipennis, a biting midge. Radiolabeled RNA and cDNA probes were generated from sequences of the L3 segment of BTV serotype 17. These probes were used to detect BTV RNA in pools of infected C. variipennis by hybridizing the probes directly to analyte immobilized on nylon membranes or by using a nucleic acid sandwich hybridization test. Hybridization procedures were able to detect 1 infected C. variipennis in a pool of 50 and as little as 3.55 log10 50% tissue culture infective doses per ml of virus. These hybridization techniques provide an alternative to virus isolation for the surveillance of BTV in vector populations.     

Detecting bluetongue virus RNA in cell culture by dot hybridization with a cloned genetic probe

A 70% copy of genome segment 7 of bluetongue virus (BTV)-17 has been cloned into the plasmid pBR-322. This cloned BTV segment when used as a radioactive probe will hybridize to BTV double-stranded RNA extracted from cell cultures and dotted onto nitrocellulose paper. This dot hybridization technique is therefore suitable for detecting and identifying BTV in cell culture. The specificity of cloned probes is discussed in relation to detecting gene sequences specific for either the bluetongue serogroup or different serotypes of BTV.     

A comparison of different genomic probes in the detection of virus-specified RNA in Orbivirus-infected cells.

Different 32P-labelled genomic probes of bluetongue virus (BTV), epizootic haemorrhagic disease virus (EHDV) and equine encephalosis virus (EEV) were compared with respect to the detection of virus-specified RNA in infected cells. The probe derived from the genome segment that encodes nonstructural protein NS1 was found to be the most sensitive, detecting virus-specified RNA in glutaraldehyde-fixed cells as early as 2-3 h p.i. This comparison was based on the observation that the NS1 gene probe required a smaller number of infected cells to produce a positive hybridization signal than the other nucleic acid probes. The only exception was the EHDV NS2 gene probe which appeared to be as sensitive as the NS1 gene probe. The advantage of using the NS1 gene probe was particularly evident in the analysis of cells infected at very low multiplicities of infection. At a multiplicity of infection of 1 x 10(-5) plaque forming units/cell, virus-specified RNA could be detected 48 h after infection. The greater sensitivity of the NS1 gene-specific probe is ascribed to the fact that its target, the NS1 mRNA, is transcribed more frequently than the other target viral mRNAs. The major application of the cell-hybridization method is the rapid detection of small quantities of infectious virus particles.     

Genetic relationships of bluetongue virus serotypes isolated from different parts of the world

Full-length DNA clones representing the 10 double-stranded RNA segments of US bluetongue virus serotype 10 (BTV-10) have been used in a study to determine the genetic relationships among 20 different BTV serotypes. The study was undertaken using Northern blot hybridization techniques involving 32P-labelled DNA probes and total RNA species extracted from BHK-21 cells infected with 20 different BTV serotypes. The results obtained indicate that all the genes representing the nonstructural proteins of BTV (NS1, NS2 and NS3) as well as most of the inner capsid polypeptides are highly conserved (e.g., VP1, VP3, VP4), while VP6 and VP7, the remaining two inner capsid components, are less conserved. The genes representing the two outer capsid polypeptides, VP2 and VP5, vary significantly. When complete DNA clones of RNA segment 2 (representing the VP2 neutralization gene) of 4 other US serotypes (BTV-2, -11, -13 and -17) and one Australian serotype (BTV-1) were used in similar hybridization studies, the data obtained showed that despite geographical distances, a certain BTV serotype exhibits similarities. Some hybridization signals were detected with several of the inner capsid genes and the corresponding RNA segments of epizootic hemorrhagic disease virus (EHDV), a distantly related orbivirus, although none of the BTV outer capsid genes, nor any of the nonstructural genes hybridized with either EHDV-1 or EHDV-2 RNA species.     

Identification of genetic variation between strains of bluetongue virus serotype 11 using cDNA probes

Recombinant plasmids containing inserts representing genome segments 2, 5, 6, 8, and 9 of bluetongue virus (BTV) serotype 11, with tentative coding assignments for viral proteins P2, P5, NS1, NS2, and P6, respectively, have been used to study the genetic diversity within a BTV serotype using Northern blot hybridization. BTV 11 strains were isolated in California, Nevada, Oklahoma, and Mexico from elk, deer, and cattle. Diversity was indirectly indicated in the BTV 11 strains by comparisons of electropherotypes. Probes specific for segments 2, 6, 8, and 9 hybridized to all BTV 11 strains with only minor variation in hybridization signal. cDNA clones, representing 90 and 20% length copies of gene segment 5, detected a difference in the field isolates with hybridization signal correlating to mobility of this segment in SDS-PAGE. These two cDNA probes of genome segment 5 hybridized to BTV U.S. prototypic serotype 17 and not the remaining serotypes (BTV 2, 10, 13) or epizootic hemorrhagic disease virus (EHDV). These data suggest a close relationship between BTV-11 and 17 and/or alternatively are the result of genome segment reassortment between serotypes.     

Sensitive identification of bluetongue virus serogroup by a colorimetric dual oligonucleotide sorbent assay of amplified viral nucleic acid.

A polymerase chain reaction (PCR) procedure coupled to an enzyme-linked oligonucleotide sorbent assay (ELOSA; a PCR-ELOSA) identified all 24 serotypic variants of bluetongue virus (BTV) without identifying any of six viruses belonging to the related epizootic hemorrhagic disease virus serogroup. The PCR-ELOSA detected 0.01 50% cell culture infectious doses of each serotype of BTV. The sensitivity and serogroup-wide specificity of the PCR-ELOSA may enable it to replace the more expensive, time-consuming, and biohazardous methods used in the identification of BTV.     

The genetic relatedness of a number of individual cognate genes of viruses in the bluetongue and closely related serogroups

Genome segments 2, 4, 6, 7, 8, 9, and 10 of bluetongue virus (BTV) serotype 10 were cloned in pBR322. The 2926-bp S2 gene, which codes for the serotype-specific antigen, was cloned as two overlapping 2.4-kb inserts. The relatedness of cognate S2 genes among different isolates of BTV10 was investigated by hybridization, restriction enzyme mapping, and sequencing of the terminal ends. Hybridization under high stringency conditions indicated a genetic diversity between isolates of BTV10 from South Africa and the United States. This was confirmed by a comparison of the restriction map of the cloned S2 gene of a BTV10 isolate from South Africa to that of the S2 gene of the BTV10 strain of the United States which has been cloned and sequenced by Purdy et al. (1985). The part of the genome that was sequenced indicated, however, that this variation was confined to an approximately 10% sequence divergence in the coding region. Very few of the nucleotide substitutions resulted in an amino acid change. The genetic variation of cognate BTV genes within the BTV serogroup as well as among different members of closely related serogroups was also investigated. DNA probes from cloned BTV10 segments were hybridized to dsRNA from 24 different BTV serotypes. Genome segments S2 and S6 were found to be almost equally serotype specific. The stringency of the wash solutions after hybridization can be manipulated to determine an order of relatedness of different cognate genes. This was illustrated by the hybridization of a sensitive RNA probe of S7 to different BTV serotypes as well as to dsRNA from closely related orbiviruses. The results confirmed a relatedness between BTV and members of the epizootic hemorrhagic disease virus (EHDV) serogroups.     

Monoclonal antibody analysis of serotype-restricted and unrestricted bluetongue viral antigenic determinants

Twenty-one monoclonal antibodies that react with bluetongue virus (BTV) and have restricted or unrestricted serotype specificities were identified in culture supernatants of hybridomas derived from lymphocytes of mice immunized with BTV serotype 17. Hybridomas were screened and antibody specificities characterized in a solid-phase radioimmunoassay and by immunoprecipitation with radiolabeled, BTV 17-infected cell lysates. Three general serotype specificities were demonstrated by 13 antibodies that precipitated structural viral protein 9 (VP 9). One antibody precipitated VP 7, a 48,000 dalton nonstructural protein, and reacted in radioimmunoassay with 20 BTV serotypes and Epizootic Hemorrhagic Disease of Deer Virus serotype 1 (EHDV 1), EHDV 2, and Ibaraki virus, but not with uninfected cells. One serotype-specific antibody neutralized infectivity, inhibited hemagglutination by BTV 17, and precipitated VP 2 and VP 3. A second antibody, with restricted serotype specificity, precipitated VP 2 and VP 8. These results confirm those of others (Huismans and Erasmus, Onderstepoort J. Vet. Res. 48, 51-58, 1981) that BTV serotype-specific and neutralizing epitopes are associated with VP 2.     

A comparison of different cloned genome segments of Epizootic haemorrhagic disease virus as serogroup-specific probes

Summary The eight largest double-stranded (ds) RNA genome segments of Epizootic haemorrhagic disease virus (EHDV) serotype 2 (Alberta strain) have been cloned. Of these, segments 4, 5, 6, 7, and 8 are represented by clones that correspond in size to those predicted for full-length clones. The different clones were used as nucleic acid probes to study the nucleic acid homology of cognate genes of four different EHDV serotypes. The results indicated that the 4 isolates may be subdivided in two geographic groups which include Ibaraki virus as the sole member of one group and EHDV1 (New Jersey), EHDV2 (Alberta) and EHDV6 (XBM67—isolated in South Africa) as a second. Genome segments 1, 3, 4, 6, and 8 were found to be highly conserved with more than 90% homology amongst cognate genes of three of the members of the EHDV serogroup. Of these, segments 1 and 3 showed the largest degree of homology with cognate genes of members of the BTV serogroup. Segment 6 and 8 probes of EHDV2 (Alberta) on the other hand did not hybridize with BTV dsRNA under conditions of moderate to low stringency and are recommended for use as EHDV group-specific probes.     

Hybridization relatedness of Israeli and U.S. bluetongue (BLU) serotypes using cDNA probes from BLU virus strain 11-UC8

Summary Partial cDNA clones representing 47%, 96%, and 98% of genome segments 7, 9, and 10, respectively, of a US bluetongue virus (BLU) 11 virulent strain were used to study, for the first time, the genetic relationships between Israeli BLU proto-serotypes and field isolates, and US BLU proto-serotypes. Their usefulness as group-specific identification probes was also determined. The viral nucleic acid was extracted from the infected cells and the purified dsRNA genome segments were fractionated by polyacrylamide gel electrophoresis, transferred to a nylon membrane and hybridized to the³²P labeled DNA probes. The three probes recognized all the samples tested. Genome segment 7, that code for the mayor inner capsid protein VP7, showed the most variation in the hybridization signal with the US proto-serotypes and all the Israeli samples studied. The genome segments 9 and 10 that code for the minor inner capsid protein VP6 and the nonstructural protein NS3, respectively, were highly conserved in all the samples tested despite their distant geographical regions of origin. The last two mentioned clones showed to be good group-specific probes for the identification of BLU samples from Israel and United States. The obtained cloned genetic probes were also tested against US epizootic haemorrhagic disease virus (EHDV) serotype 1 and 2 viral dsRNA, a distantly related orbivirus. None of them hybridized with the viral dsRNA of these two viruses.     

Detection of bluetongue virus in the blood of inoculated calves: comparison of virus isolation,PCR assay,and in vitro feeding ofCulicoides variipennis

Summary The interval after infection when bluetongue virus (BTV) was present in the blood of calves inoculated with BTV serotype 10 (BTV 10) was evaluated by virus isolation (VI) in embryonated chicken eggs (ECE), BTV-specific polymerase chain reaction (PCR), and in vitro blood feeding of vectorCulicoides variipennis (C.v.) sonorensis. BTV nucleic acid was detected by PCR in blood cells for 16 to 20 weeks after infection whereas infectious virus was detected by VI in ECE for 2 to 8 weeks. BTV was detected in calf blood by in vitro feeding ofC.v. sonorensis for only 0 to 2 weeks after inoculation of calves with BTV 10. Selected bloods which were positive by PCR analysis but not by VI in ECE were not infectious for sheep. The data are consistent with the hypothesis that prolonged viremia in BTV-infected cattle results from association of the virus with blood cells, especially erythrocytes. The fact that calf blood that contained viral nucleic acid as determined by PCR analysis, but not infectious virus as determined by VI in ECE, was not infectious for either the insect vector or sheep suggests that cattle whose blood contains BTV nucleic acid but not infectious virus are unimportant to the epidemiology of BTV infection.     

Production and characterization of the neutralization antigen VP2 of bluetongue virus serotype 10 using a baculovirus expression vector

DNA representing RNA segment 2 of bluetongue virus (BTV) serotype 10, corresponding to the gene that codes for the BTV neutralization antigen VP2, has been inserted into a baculovirus transfer vector in lieu of the 5' coding region of the polyhedrin gene of Autographa californica nuclear polyhedrosis virus (AcNPV). After cotransfection of Spodoptera frugiperda cells with wild-type AcNPV DNA in the presence of the derived recombinant transfer vector DNA, polyhedrin-negative recombinant baculoviruses were recovered. When S. frugiperda cells were infected with one of these recombinant viruses, a protein that was similar in size and antigenic properties to the BTV VP2 protein was synthesized. Antibodies raised in mice or rabbits to the baculovirus expressed VP2 protein neutralized the infectivity of BTV-10 virus and to lesser extents BTV serotype 11 and 17 viruses but not BTV-13 virus.     

A double labeling technique for performing immunocytochemistry and in situ hybridization in virus infected cell cultures and tissues

This report describes a combined immunocytochemical and in situ hybridization procedure which allows visualization of cellular or viral antigens and viral RNA in the same cell. Cultures infected with visna or measles virus were fixed in periodate-lysine-paraformaldehyde-glutaraldehyde, stained by the avidin-biotin-peroxidase technique using antibodies to viral or cellular proteins and then incubated with radiolabeled specific DNA probes (in situ hybridization). The immunoperoxidase stain was preserved through the hybridization procedure. Nonspecific 'sticking' of probes over peroxidase stained cells was prevented by incorporation of 0.1% Triton X-100 into the hybridization solution and the post-hybridization washes. The in situ hybridization signal (silver grains/cell) on peroxidase-stained cells was reduced relative to hybridization with unstained cells. The double labeling technique was also applied to sections of paraffin-embedded tissues from a sheep infected with visna virus and mice infected with the HNT strain of measles virus. Visna virus RNA was detected in immunocytochemically identified macrophages in the synovium. A greater number of these cells had viral RNA than had viral protein. In measles virus-infected brains viral RNA was detected only in cells with viral protein. This technique provides a new approach to the study of viral pathogenesis by: identifying the types of cells which are infected in the host and identifying points of blockade in the virus life cycle during persistent infections.     

Differentiation of respiratory syncytial virus subgroups with cDNA probes in a nucleic acid hybridization assay

A new approach to respiratory syncytial (RS) virus subgroup determination was developed by using a simple nucleic acid filter hybridization technique. By this method, virus-infected cells are bound and fixed in a single step, and the viral RNA in the fixed-cell preparation is characterized directly by its ability to hybridize to cDNA probes specific for either the A or B subgroups of RS virus. The subgroup-specific probes were constructed from cDNA clones that corresponded to a portion of the extracellular domain of the RS virus G protein of either a subgroup B RS virus (8/60) or a subgroup A RS virus (A2). The cDNA probes were labeled with 32P and used to analyze RS virus isolates collected over a period of three decades. Replicate templates of infected cell preparations were hybridized with either the subgroup A or B probe. The subgroup assignments of 40 viruses tested by nucleic acid hybridization were in agreement with the results of subgroup determinations based on their reactivities with monoclonal antibodies, which previously has been the only method available for determining the subgroup classification of RS virus isolates. The nucleic acid hybridization assay has the advantage of providing broad-based discrimination of the two subgroups on the basis of nucleic acid homology, irrespective of minor antigenic differences that are detected in assays in which monoclonal antibodies are used. The nucleic acid hybridization technique provides a reliable method for RS virus subgroup characterization.     

Effect of pooling and multiplexing on the detection of bluetongue virus RNA by real-time RT-PCR

Real-time RT-PCR (RT-qPCR) was used routinely for laboratory diagnosis during the 2006/2007 bluetongue virus (BTV) serotype 8 epidemic. In the present study the impact of pooling and multiplexing strategies on RT-qPCR are assessed. To avoid any bias in the pooling experiments, 121 BTV-8 positive blood samples with a low to high viral load were selected and pooled individually with nine negative blood samples. Analyses of the individually and pooled samples indicated an overall mean difference of 4.32 Ct-values. The most pronounced differences were observed in samples with the lowest viral load of which 70% could no longer be detected after pooling. The pooling strategy is therefore not suitable for BTV detection at the individual level since animals infected recently may be missed. An alternative approach to reduce costs and workload is to apply a multiplexing strategy in which the viral RNA and internal beta-actin control RNA are detected in a single reaction. Parallel analysis (singleplex versus multiplex) of a 10-fold dilution series and 546 field samples proved that the sensitivity of the BTV RT-qPCR was not affected whereas the beta-actin reaction was reduced only slightly. Without the use of an internal control, 0.6% of 1985 field samples is at risk of being diagnosed incorrectly as negative.     

Electron microscopic and molecular characterization of turnip vein-clearing virus

Summary Cattle are proposed to be reservoir hosts of bluetongue virus (BTV) because infected animals typically have a prolonged cell-associated viremia. Enriched populations of bovine monocytes, erythrocytes and lymphocytes were inoculated with BTV serotype 10 (BTV 10) and the infected cells then were examined by transmission electron microscopy to characterize the interaction of BTV with bovine blood cells. Replication of BTV 10 in monocytes and stimulated (replicating) lymphocytes was morphologically similar to that which occured in Vero cells, with formation of viral inclusion bodies and virus-specific tubules. In contrast, BTV 10 infection of unstimulated (non-replicating) lymphocytes and erythrocytes did not progress beyond adsorption, after which virus particles persisted in invaginations of the cell membrane. Studies with core particles and neutralizing monoclonal antibodies established that outer capsid protein VP2 is necessary for attachment of BTV 10 to erythrocytes. These in vitro virus-cell interactions provide a cogent explanation for the pathogenesis of BTV infection of cattle, especially the prolonged cell associated viremia that occurs in BTV-infected cattle.     

Development of probes for typing African horsesickness virus isolates using a complete set of cloned VP2-genes

A set of cloned full-length VP2-genes from the reference strain of each of the nine serotypes of African horsesickness virus (AHSV) was used to develop probes for typing AHSV isolates. The VP2-gene probes hybridised serotype-specific to purified viral dsRNA from its corresponding serotype. No cross-hybridisation was observed between the different AHSV serotypes or with RNA from equine encephalosis virus or bluetongue virus (BTV) which are related viruses within the genus Orbivirus that co-circulate with AHSV in South Africa. The probes were able to detect AHSV isolates from recent field cases of AHS in South Africa, despite being derived from historical reference strains. With regard to sensitivity and time considerations: radioactive 32P-labelling resulted in a marginal increase in sensitivity over digoxigenin-labelled probes. By infecting cell cultures at different multiplicities of infection (m.o.i.) and harvesting at various times post infection, it was established that AHSV RNA could be detected 16 h post infection (p.i.) at a m.o.i. of 1.00 pfu per cell and 48 h p.i. at a m.o.i. of 0.01 pfu per cell. Typing of AHSV isolates by means of VP2-gene probe hybridisation can be completed in 4 days, which is less than half the time required for conventional isolation and serotyping. This report on the use of a complete set of cloned AHSV VP2-gene probes is the first demonstration of typing for a whole specie (serogroup) in a genus of the family Reoviridae.     

Development of polymerase chain reaction for specific identification of epizootic hemorrhagic disease virus serotype 1

Summary The diagnostic potential of the polymerase chain reaction (PCR) for specific identification of epizootic hemorrhagic disease virus serotype 1 (EHDV-1) in cell culture and clinical specimens was evaluated. Using oligonucleotide primers, selected from genome segment 2 of EHDV-1 (New Jersey strain), the PCR-based assay resulted in a 862 base pair (bp) PCR product. EHDV-1 RNA from United States prototype serotype 1 and a number of EHDV-1 field isolates, propagated in cell cultures, were detected by this PCR based assay. The specific 862 bp PCR products were visualized on ethidium bromide-stained agarose gel. Identity of the PCR product was confirmed by chemiluminescent hybridization with non radiolabelled internal probe. Using chemiluminescent hybridization, the sensitivity of the PCR assay was 1.0 fg of virus RNA (equivalent to 60 virus particles). Amplification product was not detected when the PCR-based assay was applied to RNA from EHDV serotype 2 (EHDV-2); the United States bluetongue virus (BLU) prototypes serotypes 2, 10, 11, 13, and 17; total nucleic acid extracts from uninfected BHK-21 cell; or blood cells from calves and deer that were EHDV-seronegative and virus isolation negative. Application of this EHDV-1 PCR-based assay to clinical samples resulted in detection of EHDV-1 RNA from blood samples, collected from a calf experimentally infected with EHDV-1. The described PCR-based assay provides a simple, rapid, sensitive, specific and inexpensive method for specific identification of EHDV-1 infection in susceptible ruminants.