Complete nucleotide and deduced amino acid sequence of genome segment 5 encoding the outer capsid protein, VP5, of a U.S. isolate of bluetongue virus serotype 11.

The complete nucleotide sequence of the RNA genome segment coding for the outer capsid protein, VP5, of the United States prototypic strain of bluetongue virus (BTV) serotype 11 was determined from two overlapping cDNA clones. The genome segment was found to be 1638 nucleotides in length with a single open reading frame coding for a 526 amino acid protein of MW 59,278 and having a net charge of -4.0 at neutral pH. Comparisons of the predicted amino acid sequence of VP5 of BTV 11 with those of the United States serotypes 2, 10, and 13 and two isolates of BTV 1 from Australia and South Africa confirmed earlier reports that VP5 is a conserved protein with no clear regions of variability. A computer generated consensus sequence suggested VP5 of BTV 2 to be representative of the average VP5 sequences reported thus far.

The pathogenesis of vesicular stomatitis virus, serotype Indiana, in Aedes aegypti mosquitoes. I. Intrathoracic injection.

Analysis of infectious virus particles after intrathoracic injection revealed that Aedes aegypti mosquito tissues are capable of supporting the growth of vesicular stomatitis virus (VSV), serotype Indiana. Although all tissues assayed (salivary gland, midgut, diverticulum, malphigian tubules, and ovary) were capable of supporting VSV growth, the salivary gland was the only organ capable of maintaining an appreciable amount of virus for periods longer than 9 days postinfection. Electron microscopic studies of infected tissues showed virus particles consistently within the cell cytoplasm of all organs with no evidence of nuclear involvement. Direct evidence of crystalline formation of VSV in the apical cavities of salivary gland tissue was demonstrated.

Enzyme-linked immunosorbent assay for the detection of antibodies to bovine viral diarrhea virus in bovine sera.

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was established for the detection of antibodies to bovine viral diarrhea virus (BVDV) in bovine sera. Polyethylene-glycol concentrated, equilibrium density gradient purified BVDV was used as test antigen at an optimal amount of 1 microgram/well, whereas the optimal concentration of conjugate was at 1/2000 dilution. The standardized test encountered no non-specific reaction with test sera at a starting dilution of 1/10. A total of 50 bovine serum samples was assayed for the presence of antibodies against BVDV by ELISA and serum neutralization test (SNT). A positive correlation between the 2 tests was found. However, ELISA could be as much as 500-fold more sensitive than SNT in detecting low levels of BVDV antibodies.

Identification of envelope and nucleocapsid proteins of infectious bovine rhinotracheitis virus by SDS-polyacrylamide gel electrophoresis.

Infectious bovine rhinotracheitis (IBR) virus was purified by rate zonal and isopycnic centrifugation in potassium tartrate gradients. Viral nucleocapsids were isolated from purified virions by treatment with the nonionic detergent Triton X-100 followed by high speed centrifugation. This treatment was shown to produce a suspension of 74% completely de-enveloped nucleocapsids, 24% incompletely de-enveloped nucleocapsids, and 2% whole virions. The viral nucleocapsids contained DNA and banded at a density of 1.25 g/cm3. Analysis of the viral polypeptides by gradient SDS-polyacrylamide gel electrophoresis revealed that 33 virion proteins, ranging in molecular weight from 13,000 to 275,000 dalton, were present in the complete virus particle. Detergent treatment of the virus quantitatively removed two of the major proteins (vp8, 90,000 dalton, and vp13, 73,000 dalton) and partially removed eleven other proteins. Fifteen viral polypeptides appeared to remain firmly associated with the viral nucleocapsids.

Optimizing the enzyme-linked immunosorbent assay for evaluating immunity of chickens to Newcastle disease.

Experiments employing the various steps and reagents used in the enzyme-linked immunosorbent assay (ELISA) were conducted to produce an ELISA with the highest sensitivity and specificity possible for detecting Newcastle disease antibodies in chicken sera. Of the four types of antigen tested, crude antigen gave inconsistent results. However, an alcohol-precipitated antigen prepared from infectious allantoic-amniotic fluids was as satisfactory as more highly purified virus preparations. Other factors found to be extremely important were a 0.5M concentration of NaCl in the diluent and wash solutions used in the procedure, and a pH of 13 for sensitizing solution for maximum specific binding of the antigen to the microplate plastic wells. A comparison was made between the hemagglutination-inhibition (HI) titers of 550 known chicken sera and the corresponding ELISA values. Although the ELISA is much more sensitive than the HI test, there was a general but not a direct correlation between the two tests. The ELISA did not give more information than the HI test concerning protection against an NDV-induced drop in egg production. Preliminary observations indicated that this ELISA procedure is also applicable for turkey sera.

Enzyme-linked immunosorbent assay for the detection of antibodies to bovine virus diarrhea virus in sera from border disease virus-infected sheep.

An enzyme-linked immunosorbent assay (ELISA) was established for the rapid detection of specific antibodies against the causative agent of border disease in ovine sera. Polyethylene-glycol concentrated, equilibrium density gradient purified bovine virus diarrhea virus was used as test antigen. The optimal amount of antigen was 0.5 microgram/well, and the optimal concentration of conjugate was at 1/4,000 dilution. A total of 20 ovine serum samples, which had been collected from animals with or without border disease, were compared by ELISA and serum neutralization test for the detection of border disease-specific antibodies. ELISA was shown to be equally specific but less time-consuming and easier to perform than serum neutralization test. A positive correlation (r = 0.60) between the two tests was found.

Structural proteins of bovine viral diarrhea virus.

A procedure for the purification of radioactively labeled bovine viral diarrhea virus was critically evaluated. Purification of virus from artificial mixtures of unlabeled infected and labeled noninfected cells indicated that the extent of purification was approximately 100-fold with respect to host proteins. Residual host proteins were found to contaminate the viral preparation even after extensive purification by differential and isopycnic zonal centrifugation. Co-electrophoresis of 3H-labeled virus with 14C-labeled host cell material in neutral sodium dodecyl sulfate-7.5% polyacrylamide gels provided a means to distinguish viral specific proteins from host cell protein contaminants. Four major electrophoretic components were identified as being of viral origin; molecular weights of the components were estimated from their migration rates relative to protein markers of known molecular weight. Two viral components (VC), VC 1 and VC 3, migrated heterogeneously and had molecular weights of 93,000 to 110,000 and 50,000 to 59,000 daltons, respectively. Molecular weights of VC 2 and VC 4 were 70,000 and 25,000 daltons, respectively.

Morphology of bovine viral diarrhea virus.

The morphology of bovine viral diarrhea virus (BVDV) was studied by electron microscopy. The NADL strain of BVDV was plaque purified 3 times, concentrated by polyethylene glycol precipitation, and purified by centrifugation to equilibrium in continuous potassium tartrate density-gradients. The virus was examined by negative-stain electron microscopy in the presence or absence of specific antiserum. The density of BVDV was between 1.101 g/cm3 and 1.174 g/cm3, with the peak at maximum infectivity at 1.122 g/cm3. Oval to pleomorphic viral particles, 120 ( +/- 30) nm in diameter, were enriched in the peak of maximum infectivity. The detailed structure of virions was revealed: a 5- to 7-microns thick unit membrane-like envelope layer with numerous projecting knobs, 4 to 5 nm in diameter, surrounding an interior core-like structure. Viral particles measuring 120 ( +/- 30) nm were found in large aggregates in the presence of specific antiserum.

A procedure for pulmonary lavage in mice.

A method for the pulmonary lavage of mice is described. The procedure includes exsanguination of anesthetized mice by severting the renal artery, inserting a tracheal catheter in situ, and repeatedly injecting and aspirating 0.9% sodium chloride solution. Protein was recovered from the cell-free lavage fluid even after a given mouse was lavaged several times. The major part of the protein, however, was obtained with 1-ml washes repeated 3 times. Approximately 0.563 mg of protein was recovered by the procedure from a 29-g mouse. Four lavages per mouse yielded approximately 2.9 x 106 free cells.

Immunogenic collagen in induced ascitic fluid: concurrent preparation of anti-murine immunoglobulin E.

Antiserum to murine immunoglobulin (Ig) E was produced by inoculation of goats with a pool of partially purified IgE from serum and adjuvant-induced ascitic fluid. Antibodies to collagen were found to be present in the antiserum when the latter was conjugated with fluorescein isothiocyanate and applied to mouse pulmonic tissue. The intense connective tissue fluorescence was eliminated following absorption with mouse collagen. Immunogenic collagen components were presumed to arise in ascitic fluid as a consequence of the adjuvant-induced inflammation. Ascitic fluid is commonly used when large volumes of serum proteins are collected from small mammals. It is suggested that ascitic fluid may not be an ideal antigen source when antiserum is to be used for immunofluorescence studies on tissue.

Cloning and characterization of a genomic probe for malignant catarrhal fever virus.

A genomic probe specific for malignant catarrhal fever (MCF) virus was cloned by using purified viral DNA from MCF-virus strain WCll. Restriction endonuclease analysis of the purified viral DNA was used to identify the cloned viral genomic fragment. Dot blot hybridization by use of the genomic probe (pRP-5) indicated that the probe hybridized specifically with WCll-MCF virus, as well as with one other isolate of MCF-associated herpesvirus. Hybridization also was observed to a non-MCF virus strain of bovine herpesvirus.

Duration of maternally derived antibodies against equine influenza in newborn foals.

Serum antibody concentrations against influenza A-equi-1 virus and A-equi-2 virus were measured in a group of 18 foals from birth to 4 months of age. More than 50% of the foals were seronegative to A-equi-1 virus infection by 4 weeks of age, with titers of less than or equal to 1:16. For A-equi-2 virus, more than 50% of the foals were seronegative by 2 weeks of age, with titers of less than or equal to 1:8. Passively derived antibodies against influenza A-equi-1 virus and A-equi-2 virus in foals obtained from recently vaccinated mares and from mares not vaccinated within 6 months before foaling were low in titer. The duration of passively derived antibodies was also short-lived.

Comparison of the genomes of pseudorabies (Aujeszky's disease) virus strains by restriction endonuclease analysis.

The DNA of pseudorabies virus (PRV) strains from the United States and Taiwan and attenuated vaccine strains from Romania were compared by restriction endonuclease analysis. Electrophoretically separated PRV DNA fragments of KpnI and BamHI digests demonstrated cleavage pattern variations which clearly distinguished the 3 Taiwan isolates from all other strains, as well as from each other. One type of variation involved the loss or gain of restriction endonuclease cleavage sites. Examples of this type of variation were clearly observed in fragment patterns of the Taiwan isolates. Another type of variation that occurred at higher frequency for fragments mapping in the repeat and repeat-unique joint regions of the PRV genome involved sequence additions or deletions from existing fragments. This second type of variation occurred in most of the strains analyzed.

Comparison of herpesvirus isolates from falcons, pigeons and psittacines by restriction endonuclease analysis.

Field isolates of herpesviruses recovered from falcon, pigeon, and psittacine birds were compared by restriction endonuclease (RE) analysis using four separate enzymes. Pigeon and falcon herpesviruses had strikingly similar DNA cleavage patterns, while DNA cleavage pattern of virus isolates from a double-yellow headed Amazon and an African grey parrot had different genomic patterns to both the pigeon and falcon herpesviruses. These findings support the field observations that pigeon herpesvirus causes a fatal herpesviral infection in the livers of pigeon-eating falcons.

Immunological alterations in the lungs of mice following ozone exposure: changes in immunoglobulin levels and antibody-containing cells.

Ozone was added to the air of the environmental chambers containing specific pathogen-free mice. At levels of 0.5 and 0.8 ppm the oxidant was seen to have inflammatory effects, as shown by rising serum albumin levels in lung lavage fluid. Fluorescein conjugated anti-heavy chain sera were used to detect cells containing IgM, IgG, and IgA in measured lung areas termed Pulmonary Units. Antigenic stimuli occurred along the airways, with significant increases of IgA-containing cells in the bronchus-associated lymphoid tissue. The numbers of IgM- and IgG-containing cells did not increase. Immunodiffusion analyses for immunoglobulins in lung lavage fluid indicated increases of IgG1, IgG2, and IgA in lung secretions. The calculation of changing Ig/Alb ratios suggested that the IgA present was largely the result of local synthesis, while IgG molecules were mainly of serum origin. Possible sources of antigenic stimuli to ozone-exposed lungs are discussed.

Further evidence for entry of infectious bovine rhinotracheitis virus into bovine kidney cells.

The penetration of bovine kidney cells by infectious bovine rhinotracheitis virus, a member of the herpesvirus group, was investigated using the direct immunoferritin labeling technique. Electron microscopic examination of infected cells after 10 min at 37°C revealed fusion between viral envelope and cell membrane; the former reacted with the ferritin particles conjugated with antiviral antibody. However, shortly after penetration of the nucleocapsid, viral-specific antigenic sites on the plasma membrane were not detected by the immunoferritin technique. Antigenically reactive structures in a disorganized array were frequently detected extracellularly, situated above the penetration sites as indicated by the internalized nucleocapsids.

The pathogenesis of vesicular stomatitis virus, serotype Indiana, in Aedes aegypti mosquitoes, after imbibition of a viremic blood meal.

This study showed that Vesicular Stomatitis Virus (Indiana) in most instances was not capable of replicating in Aedes aegypti when imbibed by the mosquitoes on a viremic host. Rapid inactivation of the virus was observed in some cases within 24 hours after imbibition. Attempts to demonstrate virus inactivation by midgut contents in vitro were not successful.