Cecelia A. Whetstone

Effect of bovine immunodeficiency-like virus infection on immune function in experimentally infected cattle

Bovine immunodeficiency-like virus (BIV) is a bovine lentivirus that has antigenic and genetic homology with the human immunodeficiency virus. Little work has been reported on the effect of BIV infection on bovine immune function. This study was designed to evaluate lymphocyte blastogenesis, mononuclear cell subset numbers, neutrophil function, hematology, and clinical signs in three groups of cattle. These groups were evaluated at 0-2 months post inoculation (PI, Group 1), 4-5 months PI (Group 2), or 19-27 months PI (Group 3). BIV infected animals were inoculated with the R-29 isolate of BIV in tissue culture cells, peripheral blood mononuclear cells from a R-29 infected calf, or a molecular clone of the R-29 isolate. Most inoculated animals seroconverted to BIV by Western immunoblot. BIV was reisolated from most of the animals inoculated. BIV infection was associated with an increase in the lymphocyte blastogenic response to the mitogen phytohemagglutinin in Groups 2 and 3. Neutrophil antibody dependent cell mediated cytotoxicity and neutrophil iodination were decreased (P < 0.05) in BIV infected cattle (Groups 2 and 3 and Group 3, respectively). All animals were clinically normal during the evaluation periods. Notable differences were not observed in the other assessments performed. Work with additional BIV isolates and over longer time frames is warranted.

A WESTERN BLOT ASSAY FOR THE DETECTION OF ANTIBODIES TO BOVINE IMMUNODEFICIENCY-LIKE VIRUS IN EXPERIMENTALLY INOCULATED CATTLE, SHEEP, AND GOATS

SummaryA cocultivation method was used to establish a cytocidal bovine immunodeficiency-like virus (BIV) infection in primary fetal bovine lung (FBL) cell cultures. Cultures were monitored for virus production using radial immunodiffusion and agar gel immunodiffusion. Pelleted virus and detergent (CHAPS)-solubilized infected cell lysates from BIV-infected cell cultures were compared as sources of antigen for Western blots. Pelleted virus preparations from FBL-BIV cell cultures produced the best antigen for Western blot. Sheep and goats were inoculated with BIV and serum antibody responses were monitored up to 1 year post inoculation (PI). Sera from experimentally infected cattle, sheep, and goats reacted in Western blot assay with BIV viral induced polypeptides gp 110, p 72, p 55, p 50, gp 42, p 38, p 26, p 24, p 18, p 15, and p 13. Antibodies to p 26 were detected as early as 2 weeks PI in cattle, sheep, and goats. Antibodies to gp 110 were detected by 4 to 6 weeks PI in cattle, and by 9 months PI in sheep and goats. Antibodies to BIV proteins were still evident in cattle sera 2 1/2 years PI, and in sheep and goat sera 1 year PI.

Two different strains of an alphaherpesvirus can establish latency in the same tissue of the host animal: evidence from bovine herpesvirus 1

SummaryWe inoculated plaque-purified bovine herpesvirus type 1 (BHV 1), strain K22, subtype BHV 1.2b, intravenously into susceptible cattle. Five months later, we reactivated latent virus with dexamethasone and then super-infected the same cattle intranasally and intravaginally with a different plaquepurified BHV1, strain Cooper, subtype BHV 1.1. After a second dexamethasone treatment four months later, reactivated viruses were isolated and examined with restriction endonucleases. We showed that both virus subtypes were reactivated, proving that 2 different strains of an alphaherpesvirus can establish latency in the same tissue in the host animal.

Changes in the bovine herpesvirus 1 genome during acute infection, after reactivation from latency, and after superinfection in the host animal

SummaryThree subtypes, as defined byHindIII restriction endonuclease (RE) analysis patterns [17], of bovine herpesvirus 1 (BHV 1) were used to inoculate seronegative, BHV 1-free cattle. These included: infectious bovine rhinotracheitis virus (IBRV), subtype 1.1; infectious pustular vulvovaginitis virus (IPPV) isolate K22, subtype 1.2b; and IPVV isolate FI, subtype 1.2a. Nasal, vaginal, and buffy coat samples were taken for virus isolation from each animal. RE analysis was done on virus isolates collected during acute infection, after reactivation from latency, and after reactivation followed by superinfection with a subtype of BHV 1 that differed from the primary inoculation virus. Changes occurred in the BHV1 genome after only 1 passage in the host animal, and varied from tissue to tissue within the same animal. Viruses reactivated from latency also displayed genome variability. Only animals that received IPVV as the primary inoculation virus were successfully superinfected. After superinfection, cattle shed both superinfecting and reactivated viruses, and genome variability was observed. These data suggest that the application of RE analysis in diagnostic and epidemiologic studies of BHV 1 is limited to analysis between types and subtypes, and is not applicable for the examination of isolates from within a BHV 1 subtype.

Protection of swine from foot-and-mouth disease with one dose of an all-D retro peptide

Nine pigs were given a single inoculum of 100 microg of the all-D retro peptide corresponding to the immunodominant GH loop encompassing residues 141-159 of capsid protein VP1 of foot-and-mouth disease virus serotype A, sub-type 12. The peptide was conjugated to activated keyhole limpet haemocyanin and oil-adjuvanted before inoculation. The animals were challenged eleven weeks post-vaccination by exposing them to a pig which had been infected with the virus by inoculation. Two naive animals were included in the challenge study as controls. One of the vaccinated animals was completely unprotected and two developed very small lesions. None of the six remaining animals exhibited any clinical signs but two developed antibodies against nonstructural proteins indicating that replication of the virus had occurred. No evidence of replication could be detected in the remaining four animals, either by rise in neutralizing antibody titre or by production of antibodies against non-structural proteins specific for virus replication.

Variability occurs in the inverted repeat region of genomic DNA from bovine herpesvirus 1 respiratory, genital and bovine herpesvirus 5 encephalitic isolates.

Restriction fragment length polymorphisms (RFLPs) were detected within BHV1.1, BHV1.2, and BHV5 genomes using the restriction enzyme PstI. The genomic areas of these changes has not been previously reported. Using Southern blot hybridization with DNA probes representing the entire genome of BHV1.1, areas of genomic variation were located for a respiratory isolate (BHV1.1), four vaccine isolates (BHV1.1), a genital isolate (BHV1.2), and two encephalitic isolates (BHV5). The most frequently observed RFLPs of BHV1.1 and BHV1.2 occurred within the internal repeat region and the left terminus of the unique long region. When two separate isolates of the encephalitic BHV5 were compared, RFLPs were detected in the internal and right terminal repeat regions. These are the regions of each genome from which immediate early genes are transcribed. No genomic variation was observed throughout the unique long and unique short regions for all BHV1 and 5 isolates examined.

Analysis of the serologic relationship among San Miguel sea lion virus and vesicular exanthema of swine virus isolates. Application of the western blot assay for detection of antibodies in swine sera to these virus types.

Caliciviruses are positive-sense single-stranded RNA viruses with a single capsid protein. The serotypes of the marine mammal calicivirus, San Miguel sea lion virus (SMSV), are antigenically related to vesicular exanthema of swine virus (VESV) and are potentially hazardous to swine. Western blot assays using purified SMSV serotypes 1 and 4 were used to further examine the serologic relationship among SMSV and VESV isolates. With the exception of SMSV 8 and SMSV 12, rabbit polyclonal antisera generated against all the available SMSV and VESV isolates reacted positively, as assessed by western blot, with purified capsid protein from SMSV 1 and SMSV 4. Consequently, the SMSV 8 and SMSV 12 virus isolates may not be members of the SMSV/VESV calicivirus group. Using antisera from pigs experimentally inoculated with SMSV and VESV as positive controls, a western blot assay for these virus types was utilized to check for the presence of antibodies to caliciviruses in swine sera. Sera from colostrum-deprived gnotobiotic pigs were used as a negative control in all experiments. Examination of sera from domestic and feral swine collected in Iowa, California, and Florida was completed using this technique. The presence of antibodies to these virus types was not detected in any of the porcine sera tested.

Infection of rabbits with bovine immunodeficiency-like virus.

New Zealand white rabbits, which had been prepared for inoculation by intraperitoneal treatment with thioglycollate, were inoculated intraperitoneally with bovine immunodeficiency-like virus (BIV). Infected materials from various sources were used including cultured cells and culture fluids, peripheral blood leukocytes from infected cattle and spleen tissue from previously infected rabbits. Virus isolations and serological responses detected by western blotting provided clear evidence that infections had been established in inoculated rabbits and that the spleen was an important site of BIV infectivity. These results indicate that rabbits may be a useful species when testing for BIV infectivity in materials too toxic or highly contaminated to be inoculated directly into cell cultures. Furthermore, rabbits may also be useful in testing effects of coinfections with other bovine viruses on progression of BIV infection and for the initial evaluation of therapeutic regimens designed to suppress or eliminate BIV infections.

Experimental vesicular stomatitis virus infection of swine: Extent of infection and immunological response☆

Swine, a natural host species for infection by vesicular stomatitis virus (VSV), were infected with VSV-New Jersey (VSV-NJ) serotype virus obtained from a recent field isolate. Tissues collected from the infected pigs were examined for the presence of infective virus, for viral antigens, and/or for viral nucleic acid. Infective virus could be recovered from tissues near the site of infection for as long as 6 days after the primary infection with VSV. However, no infective virus was recovered following hypothermia induced 11 weeks after infection, or following a secondary challenge with virus 22 weeks after initial infection. Immunofluorescence tests for viral antigens and nucleic acid hybridization assays failed to detect viral antigens or nucleic acids in tissues from which no infective virus could be recovered. Titers of serum-neutralizing antibody peaked 3-5 weeks after infection and then fell slightly until the secondary infection which caused a rapid anamnestic response. Peripheral blood mononuclear cells (PBM) tested 3, 5, 8 or 18 weeks after primary infection all produced readily detectable antigen-specific proliferative responses when cultured with VSV. Thus, although direct tests failed to demonstrate persistence of virus after infection, the humoral and cellular immune response remained elevated for months. Infective VSV was not required to stimulate the proliferative response since UV-inactivated VSV was immunogenic in these in vitro tests. Following primary infection, antigen-specific proliferative responses could be stimulated by several strains of VSV-NJ, but not by VSV-Indiana (VSV-Ind) serotype virus. Secondary infection had relatively little effect on the proliferative response to VSV-NJ strains, but it did cause the PBM to gain responsiveness to VSV-Ind.

Changes in the restriction endonuclease patterns of four modified-live infectious bovine rhinotracheitis virus (IBRV) vaccines after one passage in host animal.

Four genomically different bovine herpes virus 1 (BHV 1) vaccines were used to inoculate BHV 1-negative heifers. Restriction endonuclease analysis (REA) of virus isolates from animals during acute infection, after reactivation of virus from latency, and after reactivation followed by superinfection, showed that changes occurred in the BHV 1 genome after one passage in the host animal. Furthermore, the changes varied according to the tissue from which virus was isolated. Southern probe analyses identified the left terminus of the unique long fragment and the inverted repeats as regions of the genome where variability was most often observed. It was concluded that, when REA is used in the diagnostic differentiation of BHV 1 isolates, caution must be applied in the interpretation of results.