James O. Mecham

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

Abstract 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.

A versatile SERS-based immunoassay for immunoglobulin detection using antigen-coated gold nanoparticles and malachite green-conjugated protein A/G

A surface enhanced Raman scattering (SERS) immunoassay for antibody detection in serum is described in the present work. The developed assay is conducted in solution and utilizes Au nanoparticles coated with the envelope (E) protein of West Nile Virus (WNV) as the SERS-active substrate and malachite green (MG)-conjugated protein A/G (MG-pA/G) as a bi-functional Raman tag/antibody binding reporter. Upon incubation of these reagents with serum collected from rabbits inoculated with E antigen, laser interrogation of the sandwiched immunocomplex revealed a SERS signaling response diagnostic for MG. The intensification of signature spectral peaks is shown to be proportionate to the concentration of added serum and the limit of antibody detection is 2 ng/ml of serum. To assess assay performance relative to more a traditional immunoassay, indirect enzyme-linked immunosorbent assays conducted using the same concentrations of reagents were found to be >400-fold less sensitive. Quartz crystal microbalance with dissipation (QCM-D) monitoring of immunocomplex film deposition on solid Au surfaces also confirmed the formation of antigen-antibody-protein A/G trilayers and provided quantitative measurements of film thickness which likely position MG within the sensing distance of laser-elicited, enhanced electromagnetic fields. The sensitivity and inherent versatility of the assay, which is provided by the binding of pA/G to a broad spectrum of immunoglobulins in different mammalian species, suggest that it could be developed as an alternative immunoassay format to the ELISA.

Protein Coding Assignment for the Genome of Epizootic Haemorrhagic Disease Virus

Viral genomic RNA was purified from BHK-21 cells infected with epizootic haemorrhagic disease virus and the 10 dsRNA genome segments were isolated by polyacrylamide gel electrophoresis. These genome segments were translated in vitro using the rabbit reticulocyte lysate system and the synthesized proteins were detected by immune precipitation and gel electrophoresis. This allowed the assignment of protein coding to the genome segments and the identification of two additional virus-specified proteins not readily detectable in lysates of virus-infected cells.

Correlation of serotype specificity and protein structure of the five U.S. serotypes of bluetongue virus.

The relationship between serotype specificity and protein structure was studied by polyacrylamide gel electrophoresis, peptide mapping and radioimmune precipitation (RIP) of structural and non-structural proteins of the five U.S. serotypes of bluetongue virus (BTV). The surface proteins, VP2 and VP5, showed the most variation in size among the serotypes. Peptide mapping of the proteins showed that VP2 is unique for each of the U.S. serotypes. The nucleocapsid and non-structural proteins showed a high degree of conservation, whereas the other surface protein, VP5, showed intermediate conservation among the serotypes. Monospecific neutralizing antiserum produced in rabbits against each serotype was used in cross-RIP against cytoplasmic extracts prepared from cells infected with each BTV serotype. There were extensive cross-reactions among those proteins which showed a high degree of structural conservation, whereas VP2 was immunoprecipitated best in the homologous RIP system. Thus, a correlation between serotype specificity and protein structure was shown among the five U.S. serotypes of BTV.

Absence of transovarial transmission of bluetongue virus in Culicoides variipennis: Immunogold labelling of bluetongue virus antigen in developing oocytes from Culicoides variipennis (Coquillett)

1. Culicoides variipennis midges were fed on a blood meal containing bluetongue virus (BTV) serotype 11 (BTV-11) and on four subsequent non-infective blood meals at 4-day intervals. 2. Eggs were collected before each blood-feeding and reared to adults. 3. Progeny from each egg batch were incubated for 14 days (20 degrees C, 40-60% RH) before plaque assay. 4. Oocytes from several parent flies were sectioned for immunoelectron microscopy. 5. Thirty-two percent of the parent females tested by plaque assay were positive for BTV. 6. All 993 progeny flies were negative for BTV. 7. BTV antigen was dense in proteid yolk bodies and in the vitelline membrane of the developing oocytes.

A multiplex real-time reverse transcription polymerase chain reaction assay for detection and differentiation of Bluetongue virus and Epizootic hemorrhagic disease virus serogroups.

Bluetongue virus (BTV) causes disease in domestic and wild ruminants and results in significant economic loss. The closely related Epizootic hemorrhagic disease virus (EHDV) has been associated with bluetongue-like disease in cattle. Although U.S. EHDV strains have not been experimentally proven to cause disease in cattle, there is serologic evidence of infection in cattle. Therefore, rapid diagnosis and differentiation of BTV and EHDV is required. The genetic sequence information and bioinformatic analysis necessary to design a real-time reverse transcription polymerase chain reaction (RT-PCR) assay for the early detection of indigenous and exotic BTV and EHDV is described. This sequence data foundation focused on 2 conserved target genes: one that is highly expressed in infected mammalian cells, and the other is highly expressed in infected insect cells. The analysis of all BTV and EHDV prototype strains indicated that a complex primer design was necessary for both a virus group-comprehensive and virus group-specific gene amplification diagnostic test. This information has been used as the basis for the development of a rapid multiplex BTV-EHDV real-time RT-PCR that detects all known serotypes of both viruses and distinguishes between BTV and EHDV serogroups. The sensitivity of this rapid, single-tube, real-time RT-PCR assay is sufficient for diagnostic application, without the contamination problems associated with standard gel-based RT-PCR, especially nested RT-PCR tests.

The smallest gene of the orbivirus, epizootic hemorrhagic disease, is expressed m 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.

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.

Detection of all eight serotypes of Epizootic hemorrhagic disease virus by real-time reverse transcription polymerase chain reaction

Epizootic hemorrhagic disease virus (EHDV) has been associated with bluetongue-like disease in cattle. Although U.S. EHDV strains have not been experimentally proven to cause disease in cattle, there is serologic evidence of infection. Differentiation of Bluetongue virus (BTV) and EHDV is necessary because diagnosis of infection caused by these viruses is often confused. The previously developed nested reverse transcription polymerase chain reaction (nRT-PCR) test for indigenous EHDV disease is sensitive and specific, but it is prone to contamination problems. Additionally, the EHDV nRT-PCR only detects 7 of the 8 serotypes. To develop an improved diagnostic test, sequence analysis was performed on 2 conserved target genes; one is highly expressed in infected mammalian cells, whereas the other is highly expressed in infected insect cells. This information was used to develop a rapid EHDV real-time PCR that detects all 8 EHDV serotypes. The EHDV assay did not cross-react with BTV strains and performed similarly to the nRT-PCR tests with archived clinical samples. In addition, it is superior to the nRT-PCR, not only because it is a closed system with fewer cross-contamination problems, but also because it detects all 8 serotypes and is less labor and time intensive.