Antigen capture competitive enzyme-linked immunosorbent assays using baculovirus-expressed antigens for diagnosis of bluetongue virus and epizootic hemorrhagic disease virus.

Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) are orbiviruses that infect both livestock and wild ruminants. Antigenic cross-reactivity between BTV and EHDV often results in serologic misdiagnosis. Competitive enzyme-linked immunosorbent assays (c-ELISAs) show increased sensitivity and specificity for the identification of these viral diseases; however, the preparation of cell culture-derived viral antigen for these tests is laborious and variable from batch to batch, and the resulting antigen may be infectious. To overcome these problems, the genes coding for a structural protein, VP7, of BTV and EHDV were cloned into baculovirus and the recombinant proteins were expressed in Sf9 cultured insect cells. Recombinant viral proteins released into the baculovirus-infected Sf9 cell culture supernatant were used in antigen capture c-ELISAs (Ag Cap c-ELISA) tests that specifically detected antibody in the serum of cattle experimentally infected with BTV and EHDV. The diagnostic utility of the Ag Cap c-ELISA was demonstrated by comparison with a commercial c-ELISA. The Ag Cap c-ELISA offers the advantages of using an easily produced, easily standardized, noninfectious antigen that does not require further purification or concentration.

Lack of detectable bluetongue virus in skin of seropositive cattle: implications for vertebrate overwintering of bluetongue virus.

The overwintering mechanism of bluetongue virus (BTV) has eluded researchers for many years. It was recently proposed that ovine gamma delta T-cells may become persistently infected with BTV, and serve as a reservoir for infection of naive vectors in the next transmission season. Since cattle are more numerous than sheep in the western United States (where BTV is endemic), this hypothesis was tested in bovines. In the winter of 2002-2003, 54 cattle from an endemic site in northern Colorado were age-selected to ensure that possible BTV exposure must have occurred in the summer of 2002. These cattle were tested for the presence of anti-BTV antibody by ELISA; 53 were seropositive, and one was seronegative. Naive Culicoides sonorensis colony insects were fed on skin sites of four seropositive and one seronegative cattle at day 1 (135 days after the first frost), then sequentially on separate sites for three days. Virus isolation and/or reverse transcriptase-nested polymerase chain reaction from engorged insects and 6 mm skin biopsy samples were performed for detection of viable BTV or BTV nucleic acid; all were negative. These data suggest that cattle are not a reservoir host for BTV overwintering in the western United States. The role of sheep in the trans-seasonality of BTV still remains to be determined.

Field-deployable real-time polymerase chain reaction detection of bluetongue and epizootic haemorrhagic disease viral ribonucleic acid.

Nucleic acid sequence information from molecular evolution studies of bluetongue virus (BTV) and related epizootic haemorrhagic disease virus (EHDV) strains has resulted in a large database of genomic information. Published sequence data and sequence data from our laboratory were used to design real-time field-deployable reverse transcriptase-polymerase chain reaction assays for the detection of BTV or EHDV viral RNA. The assays used standard RNA extraction and TaqMan chemistries and the entire process was completed in

Verification of bluetongue virus S9 segment nucleotide sequences.

During the course of our bluetongue virus (BTV) nucleic acid sequence investigations, conflicts among United States (US) prototype BTV S9 genome segment sequences deposited in GenBank were noted. In order to rectify these inter-laboratory discrepancies, the S9 segments of Arthropod-borne Animal Diseases Research Laboratory (ABADRL)-stored US prototype BTV 2, BTV 10, BTV 11, BTV 13, and BTV 17 isolates were resequenced. Our S9 sequences, determined by direct sequencing of full-length reverse transcriptase-polymerase chain reaction (RT-PCR) generated amplicons, shared 99% or greater nucleotide identity with one or more respective S9 sequences previously reported. Possible sources of remaining unsupported US prototype BTV S9 sequences were evaluated by amplifying and sequencing the S9 segments of BTV 2 Ona A strain, South African (SA) prototype BTV 1, BTV 2, and BTV 4 strains, and the North American (NA) prototype epizootic hemorrhagic disease virus (EHDV) serotype 2 (Alberta) strain. Comparative analysis using these S9 sequences, as well as sequences of US BTV 2 field isolates, identified potential contributors to inter-laboratory sequence disagreements.

Seasonal transmission of bluetongue virus by Culicoides sonorensis (Diptera: Ceratopogonidae) at a southern California dairy and evaluation of vectorial capacity as a predictor of bluetongue virus transmission.

Vectorial capacity of Culicoides sonorensis Wirth & Jones for the transmission of bluetongue (BLU) virus was examined at a southern California dairy from January 1995 to December 1997. Insects were collected one to two times per week in five CDC-type suction traps (without light) baited with CO2 at a constant release rate of 1,000 ml/min. BLU virus was detected in midges collected from May through December with an estimated overall infection rate of 0.08%. The BLU virus infection rate of field-captured midges was not correlated with sentinel calf seroconversions to BLU virus. Sentinel calf seroconversions were highly seasonal, occurring from August through November with most calves seroconverting during September and October. Vector competence of field-collected nulliparous flies fed a locally acquired serotype of BLU virus in the laboratory was stable among years (17-23%). Vectorial capacity was strongly correlated with BLU virus transmission (measured by sentinel calf seroconversions) during 1995 and 1996, but not during 1997. Host biting rate estimated for traps nearest to the sentinel calves was the index best correlated with BLU virus transmission for all study years and was most highly correlated with sentinel seroconversions 4 wk later. The utility of vectorial capacity and its component variables is discussed for this system.

Phylogenetic relationships of bluetongue viruses based on gene S7.

Previous phylogenetic analyses based on bluetongue virus (BTV) gene segment L3, which encodes the inner core protein, VP3, indicated a geographical distribution of different genotypes. The inner core protein, VP7, of BTV has been identified as a viral attachment protein for insect cell infection. Because the inner core proteins are involved with infectivity of insect cells, we hypothesized that certain VP7 protein sequences are preferred by the insect vector species present in specific geographic locations. We compared the gene segment S7, which encodes VP7, from 39 strains of BTV isolated from Central America, the Caribbean Basin, the United States, South Africa and Australia. For comparison, the S7 sequences from strains of the related orbiviruses, epizootic hemorrhagic disease virus (EHDV) and African horse sickness virus (AHSV) were included. The S7 gene was highly conserved among BTV strains and fairly conserved among the other orbiviruses examined. VP7 sequence alignment suggests that the BTV receptor-binding site in the insect is also conserved. Phylogenetic analyses revealed that the BTV S7 nucleotide sequences do not unequivocally display geographic distribution. The BTV strains can be separated into five clades based on the deduced VP7 amino acid sequence alignment and phylogeny but evidence for preferential selection by available gnat species for a particular VP7 clade is inconclusive. Differences between clades indicate allowable variation of the VP7 binding protein.

Development of an enzyme-linked immunosorbent assay for the detection of antibody to epizootic hemorrhagic disease of deer virus.

An enzyme-linked immunosorbent assay has been developed to detect antibodies to epizootic hemorrhagic disease of deer virus (EHDV). The assay incorporates a monoclonal antibody to EHDV serotype 2 (EHDV-2) that demonstrates specificity for the viral structural protein, VP7. The assay was evaluated with sequential sera collected from cattle experimentally infected with EHDV serotype 1 (EHDV-1) and EHDV-2, as well as the four serotypes of bluetongue virus (BTV), BTV-10, BTV-11, BTV-13, and BTV-17, that currently circulate in the US. A competitive and a blocking format as well as the use of antigen produced from both EHDV-1- and EHDV-2-infected cells were evaluated. The assay was able to detect specific antibody as early as 7 days after infection and could differentiate animals experimentally infected with EHDV from those experimentally infected with BTV. The diagnostic potential of this assay was demonstrated with field-collected serum samples from cattle, deer, and buffalo.

Epizootic hemorrhagic disease: analysis of tissues by amplification and in situ hybridization reveals widespread orbivirus infection at low copy numbers.

A recent outbreak of hemorrhagic fever in wild ruminants in the northwest United States was characterized by rapid onset of fever, followed shortly thereafter by hemorrhage and death. As a result, a confirmed 1,000 white-tailed deer and pronghorn antelope died over the course of 3 months. Lesions were multisystemic and included severe edema, congestion, acute vascular necrosis, and hemorrhage. Animals that died with clinical signs and/or lesions consistent with hemorrhagic fever had antibody to epizootic hemorrhagic disease virus serotype 2 (EHDV-2) by radioimmune precipitation but the antibody was limited exclusively to class immunoglobulin M. These findings, indicative of acute infection, were corroborated by the observation that numerous deer were found dead; however, clinically affected deer were rarely seen during the outbreak. Furthermore, only in animals with hemorrhagic lesions was EHDV-2 isolated and/or erythrocyte-associated EHDV-2 RNA detected by serotype-specific reverse transcription (RT)-PCR. By using a novel RT in situ PCR assay, viral nucleic acid was localized to the cytoplasm of large numbers of tissue leukocytes and vascular endothelium in tissues with hemorrhage and to vessels, demonstrating acute intimal and medial necrosis. Because PCR amplification prior to in situ hybridization was essential for detecting EHDV, the virus copy number within individual cells was low, <20 virus copies. These findings suggest that massive covert infection characterized by rapid dissemination of virus facilitates the severe and lethal nature of this disease.

Bluetongue virus detection: a safer reverse-transcriptase polymerase chain reaction for prediction of viremia in sheep.

A reversible target capture viral RNA extraction procedure was combined with a reverse-transcriptase nested polymerase chain reaction (PCR) to develop a capture PCR assay providing a rapid and safe prediction method for circulating bluetongue virus in infected ruminants. This new assay was compared with virus isolation and a recently developed antigen-capture enzyme-linked immunosorbent assay (ELISA) for the detection of bluetongue virus. Eight Warhill crossbred sheep were inoculated subcutaneously with bluetongue virus serotype 10, and blood samples were taken sequentially over a period of 28 days. The capture PCR detected the peak of viremia, as determined by virus isolation and antigen-capture ELISA, from day 5 to day 14 after challenge. The results indicate that the rapid-capture bluetongue virus PCR provides a rapid indicator of samples in which virus can be isolated. In addition, this capture bluetongue virus PCR procedure does not require a lengthy phenol extraction or the use of the highly toxic methyl mercury hydroxide denaturant.

Bluetongue virus in laboratory-reared Culicoides variipennis sonorensis: applications of dot-blot, ELISA, and immunoelectron microscopy.

An avidin-biotin complex (ABC) dot-blot, an antigen capture enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy (IEM) were used to detect bluetongue (BLU) virus or viral antigen or both in adult Culicoides variipennis sonorensis Wirth & Jones. The dot-blot and ELISA procedures detected viral antigen in 10-22% (depending on serotype) of the biting midges infected with BLU-2, BLU-10, BLU-13, and BLU-17 and approximately 68% of the midges infected with BLU-11. IEM analyses revealed BLU virus in salivary glands, fat body, and thoracic muscle tissue from infected insects. There appeared to be selective growth of the virus in salivary gland tissue.

Applications of dot-blot, ELISA, and immunoelectron microscopy to field detection of bluetongue virus in Culicoides variipennis sonorensis: an ecological perspective.

An avidin-biotin complex (ABC) dot-blot, an antigen capture enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy (IEM) were used to detect bluetongue (BLU) virus and viral antigen in field-collected C. varriipennis sonorensis Wirth & Jones from an enzootic BLU area in northeastern Colorado. This is the 1st attempt to apply these immunodiagnostic methods to an epidemiologically relevant, large-scale ecological system. One of the 1,800 midges (0.0005%) was positive by the dot-blot procedure, 2 (0.0011%) were positive by the ELISA, and BLU virus was identified in 8 midges (0.0044%) by IEM. These data are interpreted in context of the "whole system" of the disease to provide a framework for determining the knowledge gaps in our understanding and directing future studies in these areas. Our basic model of BLU ecology suggests that the infection rates found by the diagnostic methods are within expected ranges, thus strongly supporting the proposed ecological model and the work used to parameterize the model. This integration of immunodiagnostic methods and ecology makes it evident that further investigations of daily mortality during the extrinsic incubation period are vital to a better understanding of BLU virus occurrence in Culicoides vector and vertebrate host populations.

Molecular characterization of the segment 2 gene of epizootic hemorrhagic disease virus serotype 2: gene sequence and genetic diversity.

The complete nucleotide sequence of the gene encoding the major outer capsid protein VP2 from the Alberta isolate of epizootic hemorrhagic disease virus serotype 2 (EHDV-2) was determined. Complementary DNA (cDNA) corresponding to segment 2 was 3002 nucleotides in length with a single open reading frame that encoded a VP2 of 982 amino acids. Although the VP2 from EHDV-2 was only 34% homologous to the cognate protein from EHDV-1, their predicted hydropathic profiles were similar, suggesting that conservation of structure is important biologically to these capsid proteins. Sequence analysis of six North American EHDV-2 field isolates showed a high degree of comparative genetic identity (> 97%). Phylogenetic profiles constructed suggest that regionalization of the viruses within the North American continent has contributed to the genetic diversity.

Geographical genetic variation in the gene encoding VP3 from the Alberta isolate of epizootic hemorrhagic disease virus.

The complete nucleic acid and deduced amino acid sequences of gene segment 3 and the encoded VP3 from the North American, Alberta isolate of epizootic hemorrhagic disease virus serotype 2 (EHDV-2) are reported. Complementary DNA corresponding to segment 3 was 2768 nucleotides in length with an open reading frame of 2697 base pairs which encoded a VP3 polypeptide of 899 amino acid residues. Sequence comparison with genome segment 3 and VP3 from the Australian strain of EHDV-2 indicated genotypic and phenotypic homologies of 79% and 94%, respectively. Two North American field isolates of EHDV-2, as well as EHDV-1 (New Jersey isolate), had virtually identical homology to the Alberta isolate. Sequence analysis delineated North American EHDV strains as members of a genetically homologous and geographically distinct group of orbiviruses (topotype). The data support the hypothesis that geographic isolation between North American and Australian orbiviruses has permitted the viral topotypes to maintain their genetic distinctness.

Complex interactions between vectors and pathogens: Culicoides variipennis sonorensis (Diptera: Ceratopogonidae) infection rates with bluetongue viruses.

Two laboratory colonies of Culicoides variipennis sonorensis Wirth & Jones were allowed to take blood meals containing the five U.S. serotypes (2, 10, 11, 13, and 17) of bluetongue (BLU) virus. After 14 d of extrinsic incubation, the flies were assayed for the presence of virus using an antigen capture enzyme-linked immunosorbent assay (ELISA). There was a significant effect of the serotype on infection of C. v. sonorensis with BLU virus. There was no significant difference in infection of the two colonies when averaged across the five BLU virus treatments. However, there was a statistically significant interaction between the colonies and the virus serotypes, which was demonstrated by a higher rate of infection of the AA colony with BLU virus serotype 13 and a higher rate of infection of the AK colony with BLU virus serotype 11.

Fine mapping of a surface-accessible, immunodominant site on the bluetongue virus major core protein VP7.

The 349-amino-acid major core protein VP7 of bluetongue virus (BTV) is both the most abundant viral structural protein and the major immunogenic serogroup-reactive viral antigen. Previous studies indicated that a conformation-dependent antigenic site, defined by the VP7-specific monoclonal antibody 20E9/B7/G2(20E9), was accessible from the virus surface and that the binding of the monoclonal antibody to this epitope could be blocked specifically by antisera raised against different serotypes of bluetongue virus, suggesting it is a serogroup-specific immunodominant epitope. Using a combination of three different mapping strategies, we have located the 20E9 binding site at the N-terminus of the molecule, between amino acids 30 and 48. The fine mapping of the 20E9 immunodominant epitope will facilitate structure-function analyses of the major core protein and provide new opportunities to improve existing BTV serodiagnosis methods based on this immunogenic site.

Identification of an amino acid on VP2 that affects neutralization of bluetongue virus serotype 10.

Genome segment 2, coding for the VP2 protein, of a neutralization resistant variant was compared to segment 2 of the bluetongue virus (BTV) serotype 10 parent from which the variant was derived. Full-length double-stranded cDNA of BTV segment 2 RNA, which was prepared by reverse transcription, was used as template to prepare overlapping subgenomic cDNA products by PCR. Purified PCR cDNA fragments were sequenced by the dideoxy chain termination reaction. Each base was determined an average of 3.7 times. Comparison of the sequence of segment 2 of the neutralization resistant variant with segment 2 of the parental virus showed two base changes, one of which resulted in a changed amino acid. This change was in a different region of VP2 than those previously reported in other neutralization resistant variants of BTV. In addition to this change, both the parental virus and the variant virus differed in two amino acids from the previously published sequence of VP2 of BTV serotype 10.

The smallest gene of the orbivirus, epizootic hemorrhagic disease, is expressed in virus-infected cells as two proteins and the expression differs from that of the cognate gene of bluetongue virus.

The smallest gene (S10) of the virus of epizootic hemorrhagic disease of deer (EHD, serotype 2) is expressed as two proteins in virus-infected cells. By contrast, the non-structural proteins (NS3 and NS3A) encoded in the smallest gene of bluetongue (BT) viruses are difficult to detect in virus-infected cells. The nucleotide sequence of S10 of EHDV-2 contains two in-frame initiation codons which allow for translation of proteins of mol. wt. 25503 and 23921 analogous to NS3 and NS3A of BT viruses. The S10 genes of BT viruses are highly conserved (82%-99%); the nucleotide sequence similarity of S10 of EHDV-2 and BT viruses is about 64%. Some structural features of NS3 and NS3A are conserved in the two viruses, despite the divergence in the amino acid sequences of the proteins. The hydrophobic domains of the proteins and the putative transmembrane sequences are conserved, as are potential glycosylation sites in the proteins. A cluster of proline residues, which is conserved at residues 36-50 in all of the published sequences of NS3 of BT viruses, is conserved exactly in the alignment of the sequence of NS3 of EHDV-2 with that of the BT viruses. An explanation for the differences in expression of NS3/NS3A in EHD and BT viruses was not evident in comparing the nucleotide sequences of S10 of the viruses.

Detection of bluetongue virus from blood of infected sheep by use of an antigen-capture enzyme-linked immunosorbent assay after amplification of the virus in cell culture.

An antigen-capture ELISA was used to detect bluetongue virus (BTV) from blood of infected sheep. A rabbit-origin capture antibody and a mouse-origin detection antibody combined with biotin-avidin amplification were used for the assay. The antigen-capture ELISA could not detect virus directly from the blood of infected sheep because of low virus titer. To enhance detection, virus from infected blood was amplified in cell culture. Virus could then be detected from cell culture supernatant fluids, using the ELISA. This amplification step increased the sensitivity of the assay comparable to that of assays performed in cell culture measuring cytopathic effects. The ELISA procedure was specific for BTV and did not mistakenly identify the antigenically related epizootic hemorrhagic disease virus. The antigen-capture ELISA permitted indirect quantitation and identification of BTV from the blood of infected sheep.

Epitopic mapping of linear and conformation-dependent antigenic determinants on GP5 of five U.S. bluetongue viruses.

Two distinct antigenic determinants of the major outer capsid protein, GP5, of five U.S. bluetongue viruses have been identified and mapped using monoclonal and oligoclonal antibodies. One antigenic site, identified by oligoclonal antibody AK-15, was found to be common and conserved in all five U.S. BTV serotypes. This linear epitope was located between amino acid residues 175 and 189 (ALQREAAERSEDEIK). The second determinant identified by monoclonal antibody 34.7 was present in BTV-2, -10, -11, and -17 but absent in BTV-13. The binding of this monoclonal antibody to GP5 could be blocked specifically by one of three short synthetic peptides located among amino acid residues 33-42 (KAAERFAESE), 159-168 (EKILKEEDSK), and 206-215 (EIERDGMQEE), indicating that this antigenic determinant is conformation-dependent. Oligoclonal antibody (AK-15) reacted with denatured GP5 immobilized on nitrocellulose membrane after Western transfer as well as with native GP5 present on the surface of purified BTV virions. Monoclonal antibody (34.7) reacted only with denatured GP5 but not native GP5 using an ELISA assay. However, these two antigenic epitopes alone did not elicit detectable neutralizing antibodies as determined by plaque reduction assay.

Sources of variation in an enzyme-linked immunoassay of bluetongue virus in Culicoides variipennis (Diptera: Ceratopogonidae).

An enzyme-linked immunoassay for detecting bluetongue virus in infected Culicoides variipennis was evaluated using a nested analysis of variance to determine sources of experimental error in the procedure. The major source of variation was differences among individual insects (84% of the total variance). Storing insects at -70 degrees C for two months contributed to experimental variation in the ELISA reading (14% of the total variance) and should be avoided. Replicate assays of individual insects were shown to be unnecessary, since variation among replicate wells and plates was minor (2% of the total variance).