Duration of viraemia infectious to Culicoides sonorensis in bluetongue virus-infected cattle and sheep.

The duration of viraemia infectious to Culicoides sonorensis (C. sonorensis) was evaluated in bluetongue virus (BTV)-infected sheep and cattle by feeding laboratory-reared C. sonorensis directly on the skin of ruminants that previously were infected with BTV by insect inoculation. The intervals after infection when infectious BTV and BTV nucleic acids were present in the blood of infected cattle and sheep, respectively, were determined by virus isolation and reverse transcriptase (RT)-nested polymerase chain reaction (PCR) assays. The presence of BTV in vector insects that fed on the BTV-infected cattle and sheep at 7, 21, and 49 days post-infection (p.i.) was also determined by virus isolation and RT-PCR assays. BTV was isolated from the blood of infected cattle for up to 49 days p.i., whereas it was not isolated from the BTV-infected sheep after 11 days p.i. In contrast, BTV nucleic acids were detected in the blood of infected ruminants for 111-222 days p.i. The maximal duration of viraemia that was infectious to C. sonorensis was 21 days p.i. of both cattle and sheep and, with the notable exception of one sheep at 21 days p.i., only ruminants whose blood contained BTV as determined by virus isolation were able to infect C. sonorensis after oral feeding. Data from this and previous studies indicates that viraemia is transient in BTV-infected ruminants, and that the RT-nested PCR assay provides a very sensitive and conservative test for the screening of cattle and sheep for the presence of BTV.

Characterization of equine E-selectin.

Expression of E-selectin on activated endothelium is a critical initial step that leads to extravasation of leucocytes during inflammation, yet E-selectin is largely uncharacterized in several animal species including the horse. We have sequenced and compared E-selectin genes derived from activated cultures of purified equine (horse), cervid (black-tailed deer) and ovine (sheep) pulmonary artery endothelial cells (ECs). Phylogenetic and amino acid sequence comparisons indicate that bovine, cervid and ovine E-selectin are similar, whereas human and equine E-selectin are more closely related to each other than to the ruminant molecules. Human E- and P-selectin-specific monoclonal antibodies that also recognize equine E-selectin were identified and used to characterize its expression. Expression of E-selectin was more readily induced by lipopolysaccharide treatment in equine ECs than in human ECs and supported adhesion and activation of neutrophils, consistent with the extreme sensitivity of horses to endotoxaemia and septic shock.

Changes in the outer capsid proteins of bluetongue virus serotype ten that abrogate neutralization by monoclonal antibodies.

Six neutralizing monoclonal antibodies (Mabs) and nine neutralization resistant viral variants (escape-mutant viruses (EMVs)) were used to further characterize the neutralization determinants of bluetongue virus serotype 10 (BTV10). The EMVs were produced by sequential passage of a highly cell culture adapted United States prototype strain of BTV10 in the presence of individual neutralizing Mabs. Mabs were characterized by neutralization and immune precipitation assays, and phenotypic properties of EMVs were characterized by neutralization assay. Sequencing of the gene segments encoding outer capsid proteins VP2 and VP5 identified mutations responsible for the altered phenotypic properties exhibited by individual EMVs. Amino acid substitutions in VP2 were responsible for neutralization resistance in most EMVs, whereas an amino acid substitution in VP5, without any change in VP2, was responsible for the neutralization resistance of one EMV. The data confirm that VP2 contains the major neutralization determinants of BTV, and that VP5 also can influence neutralization of the virus. The considerable plasticity of the neutralization determinants of BTV has significant implications for future development of non-replicating vaccines.

A 29K envelope glycoprotein of equine arteritis virus expresses neutralization determinants recognized by murine monoclonal antibodies.

A panel of six neutralizing murine monoclonal antibodies (MAbs) to equine arteritis virus (EAV) was produced. The MAbs were characterized by Western immunoblotting assay and competitive ELISA. The six MAbs identify a single neutralization site on a 29K envelope glycoprotein. Deglycosylation of viral proteins prior to immunoblotting showed that the 29K protein is the glycosylated form of a 20K protein. Equine anti-EAV serum also strongly bound the 29K glycoprotein, as well as an unglycosylated protein of 17K. The equine antisera to EAV blocked the binding of a selected MAb to EAV, whereas normal equine serum did not. Two neutralization-resistant escape mutant (EM) variants of the EAV prototype were produced using MAb 6D10. The phenotypic properties of the EM viruses were characterized by neutralization and immunoblotting assays with two MAbs (6D10 and 5G11). The two MAbs failed to neutralize either EM virus, and they did not react in an immunoblot assay with any proteins of the EM viruses. In contrast, binding of the equine antiserum to viral proteins was equivalent with prototype and EM virus strains. These data clearly indicate that a 29K envelope glycoprotein expresses at least one neutralization determinant of EAV.

Neutralization determinants of United States bluetongue virus serotype ten.

A panel of five neutralization-resistant escape mutant (EM) viruses was used to investigate the neutralization determinants of the U.S. prototype strain of bluetongue virus serotype 10 (BTV-10). The phenotypic properties of each EM virus were characterized by neutralization and immuneprecipitation assays with a panel of four monoclonal antibodies (MAbs). These MAbs were used to select the various EM viruses and together the MAbs define four distinct neutralizing epitopes on the prototype strain of BTV-10 (Heidner, H.W., Rositto, P.V., and MacLachlan, N.J., Virology 176, 658-661 (1990)). Sequencing of the L2 gene identified mutations responsible for the altered phenotypic properties exhibited by each EM virus. The L2 gene encodes BTV outer capsid protein VP2 which is responsible for virus neutralization. Four amino acids in three distinct regions of VP2 are critical to expression of the epitopes recognized by the MAb panel. Both amino acid 208 and 211 can affect the binding of MAb 039 and MAb 045, amino acid 327 affects binding of MAb 041, and amino acids 327 and 402 cooperatively interact to affect binding by MAb 034. The location of two of these critical regions on VP2 of BTV-10 is identical to two of those which affect neutralization of Australian BTV-1, despite the fact that these two viruses are antigenically distinct and have divergent L2 gene sequences (Gould, A.R., and Eaton, B.T., Virus Res. 17, 161-172 (1990)). The four individual neutralizing epitopes on VP2 of BTV-10 are interactive (Heidner, H.W., Rositto, P.V., and MacLachlan, N.J., Virology 176, 658-661 (1990)) and at least two are conformationally dependent.

Variation amongst the neutralizing epitopes of bluetongue viruses isolated in the United States in 1979-1981.

Neutralizing epitopes present on field isolates of bluetongue virus (BTV) serotypes 10, 11, 13 and 17 were evaluated with a panel of polyclonal and neutralizing monoclonal antibodies (MAbs). A total of 91 field isolates were evaluated, including 15 isolates of BTV-10, 29 isolates of BTV-11, 26 isolates of BTV-13, and 21 isolates of BTV-17. The viruses were isolated from cattle, goats, sheep, elk and deer in Idaho, Louisiana, Nebraska and, predominantly, California, in the years 1979, 1980 and 1981. The isolates were analyzed and compared using a panel of neutralizing MAbs which included five MAbs raised against BTV-2, seven against BTV-10, five against BTV-13, and six against BTV-17. Neutralization patterns obtained with the MAb panel and individual field isolates were compared to those obtained with prototype viruses of each serotype. All field isolates were neutralized by at least some of the MAbs raised against the prototype virus of the same serotype. All field isolates of BTV-10 were neutralized by the seven MAbs raised to BTV-10, whereas the field isolates of BTV-11, BTV-13 and BTV-17 were not consistently neutralized by all of the MAbs raised against the prototype virus of the same serotype. Variation in neutralizing epitopes recognized by the MAb panel was most pronounced amongst the field isolates of BTV-17. A one-way cross neutralization was evident between BTV-10 and BTV-17 as all field isolates of BTV-17 were neutralized by four of the MAbs raised against BTV-10. In contrast, no BTV-10 isolates were neutralized by the MAbs raised against BTV-17. Differences in the MAb neutralization patterns of field isolates of BTV-11, BTV-13 and BTV-17 suggest that the immunogenic domain responsible for their neutralization is plastic, such that individual epitopes within the domain may vary in their significance to the neutralization of different viruses, even of the same serotype. The apparent conservation of neutralizing epitopes on field isolates of BTV-10 suggests that the field isolates may be derived from the modified-live vaccine strain of BTV-10.