ABSTRACT Rift Valley fever virus (RVFV) causes serious disease in ruminants and humans in Africa. In North America, there are susceptible ruminant hosts and competent mosquito vectors, yet there are no fully licensed animal vaccines for this arthropod-borne virus, should it be introduced. Studies in sheep and cattle have found the attenuated strain of RVFV, MP-12, to be both safe and efficacious based on early testing, and a 2-year conditional license for use in U.S. livestock has been issued. The purpose of this study was to further determine the vaccine's potential to infect mosquitoes, the duration of humoral immunity to 24 months postvaccination, and the ability to prevent disease and viremia from a virulent challenge. Vaccination experiments conducted in sheep found no evidence of a potential for vector transmission to 4 North American mosquito species. Neutralizing antibodies were elicited, with titers of >1:40 still present at 24 months postvaccination. Vaccinates were protected from clinical signs and detectable viremia after challenge with virulent virus, while control sheep had fever and high-titered viremia extending for 5 days. Antibodies to three viral proteins (nucleocapsid N, the N-terminal half of glycoprotein GN, and the nonstructural protein from the short segment NSs) were also detected to 24 months using competitive enzyme-linked immunosorbent assays. This study demonstrates that the MP-12 vaccine given as a single dose in sheep generates protective immunity to a virulent challenge with antibody duration of at least 2 years, with no evidence of a risk for vector transmission.

Bluetongue (BT) is an insect-transmitted, economically important disease of domestic and wild ruminants. Although only five of the 26 reported bluetongue virus (BTV) serotypes are considered endemic to the USA, 10 exotic serotypes have been isolated primarily in the southeastern region of the country since 1999. For an exotic BTV serotype to become endemic there must be susceptible animal species and competent vectors. In the USA, sheep and white-tailed deer (WTD) are the primary sentinel livestock and wildlife species, respectively. In 2006, BTV-8 was introduced into Northern Europe and subsequently overwintered, causing unprecedented livestock disease and mortality during the 2006–2007 vector seasons. To assess the risk of the European strain of BTV-8 to North American WTD, and understand the role they could play after a similar introduction, eight bluetongue-seronegative WTD were inoculated with BTV-8. Body temperatures and clinical signs were recorded daily. Blood samples were analyzed for BTV RNA with quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR), serum analyzed for BTV antibodies by cELISA, and tissues taken for histopathology and qRT-PCR. All eight deer became infected and developed moderate to severe clinical disease from days 8 to 15. Peak viremia was from day 7 to 10 with detectable titers through the end of the study (28 days) in most deer. Serum antibody was detected by day 6, peaked by day 10 and continued through day 28. We conclude that North American WTD are highly susceptible to BTV-8 and would act as clinical disease sentinels and amplifying hosts during an outbreak.

In this article, we demonstrate a novel approach to implementing multiplex enzyme-linked immunosorbent assay (ELISA) in a single microfluidic channel by exploiting the slow diffusion of the soluble enzyme reaction product across the different assay segments. The functionality of the reported device is realized by creating an array of ELISA regions within a straight conduit that are selectively patterned with chosen antibodies/antigens via a flow-based method. The different analytes are then captured in their respective assay segments by incubating a 5-μL aliquot of sample in the analysis channel for an hour under flow conditions. Once the ELISA surfaces have been prepared and the enzyme substrate introduced into the analysis channel, it is observed that the concentration of the soluble enzyme reaction product (resorufin) at the center of each assay region grows linearly with time. Further, the rate of resorufin generation at these locations is found to be proportional to the concentration of the analyte being assayed in that segment provided that the ELISA reaction time in the system (τ R ) is kept much shorter than that required by the resorufin molecules to diffuse across an assay segment (τ D ). Under the operating condition τ R  << τ D , the reported device has been shown to have a 35% lower limit of detection for the target analyte concentration compared with that on a commercial microtiter plate using only a twentieth of the sample volume.

Objective —To characterize a 2007 bluetongue disease (BT) epizootic caused by bluetongue virus (BTV) serotype 17 in sheep in the Big Horn Basin of Wyoming. Design —Cross-sectional study. Animals —1,359 sheep from ranches in Wyoming and Montana. Procedures —Information on clinical signs and history of BT in sheep was obtained from ranchers and attending veterinarians. At 3 to 6 months after the 2007 BT epizootic, blood samples were collected from rams, ewes, and lambs within and outside the Big Horn Basin; blood samples were also collected from lambs born in the spring of 2008. Sera were tested for anti-BTV antibodies by use of a competitive ELISA to determine the seroprevalence of BTV in sheep and to measure antibody titers. Virus isolation and reverse transcriptase PCR assays were used to determine long-term presence of the infectious virus or viral genetic material in RBCs of sheep. Results —The percentage of sheep seropositive for BTV closely matched morbidity of sheep within flocks, indicating few subclinical infections. Flocks separated by as little as 1 mile had substantial variation in infection rate. Rams were infected at a higher rate than ewes. There was no evidence of BTV successfully overwintering in the area. Conclusions and Clinical Relevance —This epizootic appears to be a new intrusion of BTV into a naïve population of sheep previously protected geographically by the mountains surrounding the Big Horn Basin. Rams may have a higher infection rate as a result of increased vector biting opportunity because of the large surface area of the scrotum.

A quantifiable in situ immune fluorescent assay (IFA) was developed to measure bluetongue virus (BTV) binding to mammalian cells. The utility of the assay was demonstrated with both Chinese hamster ovary (CHO) and bovine pulmonary artery endothelial (CPAE) cells. Since heparin sulfate (HS) has been shown to function as a receptor for a number of viruses, its role as a receptor for BTV was evaluated with the in situ IFA. Binding of BTV to both CHO and CPAE cells was inhibited in a dose dependent manner by HS. In addition, HS deficient CHO cells showed greatly diminished binding of BTV when compared to the parental cell line. The IFA protocol will find application, as a non-isotopic, quantifiable technique, to study virus–cell receptor interactions. Information gained from such studies will expand our understanding of the early steps in virus replication.

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 2ng/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.

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.

T. Lynwood Barber, CO; Shane A. Brookshire, GA; Charles E. Brown, II, WI; Tammy Burton, NM; Joseph L. Corn, GA; Barbara Drolet, KS; Edward J. Dubovi, NY; Anita Edmonson, CA; James F. Evermann, WA; Robert W. Fulton, OK; Dorothy Geale, CAN; Robert F. Gerlach, AK; Paul Gibbs, FL; Chester A. Gipson, MD; William L. Hartmann, MN; Larry L. Hawkins, MO; Linda Hickam, MO; Robert B. Hillman, NY; Thomas J. Holt, FL; Oscar Kennedy, VA; Randall Levings, IA; Francine Lord, CAN; David T. Marshall, NC; Daniel G. Mead, GA; James O. Mecham, WY; Bennie I. Osburn, CA; Eileen N. Ostlund, IA; Charles Palmer, CA; James E. Pearson, IA; William Pittenger, MO; Justin Roach, OK; Shawn P. Schafer, ND; Charly Seale, TX; Laurie Seale, TX; John Shaw, IA; David E. Stallknecht, GA; Susan W. Tellez, TX; George O. Winegar, MI.

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.

Bluetongue virus (BTV) is transmitted to sheep, cattle and other ruminants by Culicoides spp. of biting midges. Cell lines have been developed from Culicoides sonorensis; however, techniques to detect and quantitate viable virus directly in these insect cells are lacking. In situ immune infrared fluorescent staining techniques were developed to visualize and quantitate BTV infection in Culicoides cell culture by both an endpoint titration and an agarose overlay fluorescent focus assay. Insect cell cultures infected with BTV were fixed, permeabilized and reacted with virus-specific monoclonal antibody and fluorescent-labeled secondary antibody. Virus replication in the infected cells was visualized and quantitated by measuring fluorescence with an infrared imager. The sensitivity of virus detection in insect cell culture using these techniques was comparable to or better than detection by standard techniques in vertebrate cell culture.

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.

An indirect capture model assay using colloidal Au nanoparticles is demonstrated for surface enhanced Raman scattering (SERS) spectroscopy detection of DNA. The sequence targeted for capture was derived from the West Nile Virus (WNV) RNA genome and selected on the basis of exhibiting minimal secondary structure formation. Upon incubation with colloidal Au, hybridization complexes containing the WNV target sequence, a complementary capture oligonucleotide conjugated to a strong tethering group and a complementary reporter oligonucleotide conjugated to methylene blue (MB), a Raman label, anchors the resultant ternary complex to Au nanoparticles and positions MB within the required sensing distance for SERS enhancement. The subsequent elicitation of surface enhanced plasmon resonance by laser excitation provides a spectral peak signature profile that is capture-specific and characteristic of the Raman spectrum for MB. Detection sensitivity is in the submicromolar range and was shown to be highest for thiol, and less so for amino, modifications at the 5′ terminus of the capture oligonucleotide. Finally, using Quartz Crystal Microbalance-Dissipation as a tool for modeling ternary complex binding to Au surfaces, quantitative measurements of surface mass coverage on Au plated sensor crystals established a positive correlation between levels of ternary complex adsorption and their correspondent levels of SERS signal intensification. Adapted to a compact Raman spectrometer, which is designed for analyte detection in capillary tubes, this assay provides a rapid, mobile and cost effective alternative to expensive spectroscopic instrumentation, which is often restricted to analytical laboratories.

Bluetongue virus (BTV) is an insect-transmitted orbivirus of importance to the cattle and sheep industry. The VP2 protein, encoded by L2, contains neutralizing epitopes. Previously, a panel of neutralizing monoclonal antibodies (MAbs) to the BTV serotype 17 (BTV-17) prototype strain was generated and it was determined that the neutralization domain consists of three overlapping epitopes. Over 30 amino acid changes were found between a neutralized BTV-17 prototype strain and a non-neutralized BTV-17 198 strain. In this study, the L2 genes from eight additional strains, representing both the neutralized and non-neutralized groups of BTV-17, were sequenced to determine the degree of conservation of the previously characterized differences. Comparison of the deduced amino acid sequences showed that 91% (30/33) of the previously noted changes were conserved within each group. The sequence of the M5 gene that encodes VP5 was also examined, since this surface protein has also been shown to affect neutralization. No consistent changes were noted between the neutralized and non-neutralized groups of BTV-17 by analysis of the VP5 protein. Finally, the L2 sequences of five MAb neutralization escape mutants were determined to identify specific amino acids involved in neutralization and perhaps virulence. All five mutants contained 1–3 amino acid changes that were in close proximity to a previously described variable region. These amino acid changes likely define critical sites in the overlapping neutralization domains previously described. This is the first description of two BT virus populations that have distinct neutralization characteristics co-circulating in a defined geographical region.

Bluetongue virus (BTV) infects sheep, cattle and other ruminants and is transmitted by Culicoides spp. of biting midges. Virus is typically isolated and characterized by infection of susceptible vertebrate cells that undergo detectable and measurable cytopathic effects. Cell lines derived from C. sonorensis may be useful for virus isolation and studies to better understand BTV replication in the insect vector. However, their use is hampered because BTV infection does not produce significant cytopathic effects in these insect cell cultures. Detection of virus replication in these cells typically requires co-cultivation with susceptible vertebrate cell culture. This report describes the use of an antigen-capture enzyme-linked immunosorbent assay (Ag-Cap ELISA) for direct detection and titration of BTV in cultures of a Culicoides cell line. This assay should facilitate use of this cell line for virus isolation, titration and studies of BTV replication.

The prototype United States (US) strains of bluetongue virus serotype 2 [BTV-2 (OnaA) and BTV-2 (OnaB)] made in Florida in 1982 were compared to a recent BTV-2 (FL99) isolate made in Florida in 1999 to determine if the original strain(s) had persisted or if a new strain of BTV-2 had been re-introduced into the southeast US. Viral RNA and protein electropherotypes, and sequence analysis of five RNA genome segments for these early and later BTV-2 isolates were compared. These comparisons indicated that BTV-2 (OnaB) has persisted in the southeast US since its first isolation in 1982. Sequence analysis of concurrent isolates of BTV-13 (FL99) and BTV-17 (FL99) from the same location in Florida in 1999 provides evidence of genetic reassortment between BTV-2 and other co-circulating serotypes of BTV.

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 <or=3 h. The reaction conditions have been adapted to run on a field-deployable ruggedised advanced pathogen identification device (RAPID) instrument from Idaho Technologies, Inc. This instrument consists of a 50-lb (22.68 kg) backpack containing everything needed to run the assays. The current assays are specific for United States serotypes of BTV and EHDV; however, new designs based on new sequencing information are being evaluated to improve specificity and sensitivity for additional serotypes. This new technology greatly enhances the speed of virus detection and the ability to monitor disease outbreaks.

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.

ABSTRACT 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.