Detection of bovine polyomavirus contamination in fetal bovine sera and modified live viral vaccines using polymerase chain reaction.

A nested polymerase chain reaction (PCR) assay has been developed for the detection of bovine polyomavirus (BPyV) DNA. The assay has been used to screen commercial lots of fetal bovine serum and modified live veterinary vaccines for the presence of the agent. A PCR product of the expected size was detected after the first round of PCR for eight out of 20 serum lots, but in none of the 14 vaccines tested. The subsequent nested assay revealed that four more serum lots were positive for BPyV DNA, as well as two vaccine lots. When hybridized with a labelled probe, blots of the PCR products from vaccines revealed that in one of the two positive samples a specific product was present after the first PCR at a level not detectable in gel electrophoresis. Nested PCR appears to be a useful tool for the detection of low level contamination with BPyV DNA of products used in, and derived from cell culture.

Characterization of a human adenovirus 5: rabies glycoprotein recombinant vaccine reisolated from orally vaccinated skunks.

An infectious recombinant human adenovirus which carries the rabies glycoprotein gene and accompanying SV40 control elements can be given orally to skunks to immunize them against rabies. We have looked for adenovirus in the feces and oral fluids of animals that have been given this recombinant and have obtained 111 virus positive samples from 16 test animals. DNA from these virus isolates was examined for possible mutations. One possible insertion mutation was detected by SmaI restriction endonuclease analysis of genomic DNA. Further analysis by HaeIII restriction and nucleotide sequencing of polymerase chain reaction products encompassing the whole SV40-rabies insert revealed that this isolate contained an insert of the 72 base pair sequence found in the SV40 promoter region. A second mutation, in which 54 base pairs were deleted from within the rabies glycoprotein gene, was also detected in two independent isolates from one skunk.

Isolation of caliciviruses from skunks that are antigenically and genotypically related to San Miguel sea lion virus.

Caliciviruses were isolated from feces of skunks imported from the north central United States to Canada. Virus isolation was accomplished using adenovirus-transformed human kidney (293) cells, swine testes and Vero cells. Plaque size variants were presented, but there was no apparent difference in virus morphology by negative stain or immune electron microscopy. Pigs infected with skunk calicivirus had a slightly elevated body temperature at 3 days postinfection. Although the infected animals seroconverted, no overt clinical signs were observed. Purified infectious genomic skunk calicivirus RNA behaved exactly as San Miguel sea lion virus (SMSV) 1 and 4 genomic RNA in cell culture transfection studies. Of the cell types examined, only primary porcine kidney, 293 and Vero cells supported viral replication. No viral replication was detected in cells of bovine, equine, ovine, caprine or feline origin. The skunk caliciviruses contained a single capsid protein with a relative mobility similar to SMSV virus 1 and 4 capsid proteins. The capsid protein was positive by Western blot analysis with SMSV and vesicular exanthema of swine virus (VESV) antisera. Purified RNA from skunk calicivirus infected cells was subjected to reverse transcription followed by polymerase chain reaction. Nucleotide sequences were identified that had greater than 85% similarity to the 2C and RNA polymerase gene regions of SMSV 1 and 4 and VESV A48. Predicted amino acid sequences of these regions were greater than 95% similar and the partial coding sequence of the polymerase gene contained the YGDD sequence common to positive-strand RNA virus polymerases.

In vitro assessments of the genetic stability of a live recombinant human adenovirus vaccine against rabies.

The genetic stability of a live human adenovirus 5: rabies glycoprotein recombinant vaccine has been assessed upon 20 serial passages in a permissive cell line of human origin. Restriction endonuclease analysis and the polymerase chain reaction were used to examine the integrity of the expression cassette for the rabies glycoprotein and the viral vector at the site of insertion of the cassette. It was found that the restriction endonuclease profile was identical for each sample assayed. A more detailed analysis of the expression cassette following amplification by the polymerase chain reaction revealed no changes in the size and number of fragments originating from the coding sequence for the glycoprotein nor the signals controlling the expression of the protein product. The amplified product obtained from the 10th and 20th passages was subjected to nucleotide sequencing. Additionally, 20 plaques isolated from the 20th passage of the virus expressed the rabies glycoprotein as demonstrated by fluorescent antibody staining with glycoprotein specific monoclonal antibodies. These results suggest that the recombinant vaccine maintains the integrity of the heterologous sequences upon passage in tissue culture.

Approaches for genetic purity testing of live recombinant viral vaccines using a human adenovirus:rabies model.

A two part purity testing regimen for genetically engineered live viral vaccines is described using a human adenovirus 5: rabies glycoprotein gene recombinant as a model vaccine. Initially, restriction endonuclease analysis of the recombinant viral genome verified the integrity of the recombinant construct and identified the vector genome. The second stage employed the polymerase chain reaction to facilitate a more detailed study of the target rabies glycoprotein cassette. The size of the target region was predicted from known nucleic acid sequence information and compared to that obtained after electrophoresis with molecular weight standards. Digestion of the polymerase chain reaction product with a second restriction endonuclease cleaved the target into a number of small fragments. Resolution of the fragments by gel electrophoresis allowed analysis of the target region alone, verifying its identity and integrity.

Streptococcal cell wall arthritis. Passive transfer of disease with a T cell line and crossreactivity of streptococcal cell wall antigens with Mycobacterium tuberculosis.

Primary lymph node cells derived from streptococcal cell wall arthritic rats or those derived from adjuvant arthritic rats proliferated in response to cell wall antigens derived from either streptococcal cell walls or those from M. tuberculosis. In addition, two T cell lines have been isolated from lymph nodes of rats during the chronic phase of streptococcal cell wall arthritis. These T cell lines transfered clinical disease to naive syngeneic irradiated recipients, and they proliferated in the presence of cell wall antigens derived from streptococci or antigens derived from Mycobacterium but failed to proliferate in the presence of the 65-kD antigen (containing the sequence TFGLQLELT) derived from Mycobacterium. These observations indicate that T cells play a crucial role in the pathogenesis of streptococcal cell wall arthritis and suggest that antigenic crossreactivity exists between cell walls of group A streptococci and antigens derived from Mycobacterium. The 65-kD Mycobacterium protein is not involved in the observed antigenic crossreactivity.

Type II collagen arthritis: identification of arthritogenic epitopes using fractionated anticollagen IgG.

Affinity-purified anticollagen IgG was fractionated on purified cyanogen bromide-derived collagen peptide Sepharose. The antibody fraction bound to the peptides was eluted and tested for its ability to induce passive arthritis in recipients. Anticollagen IgG bound to peptide 5 (alpha 1(II)-CB8-10 and alpha 1(II)CB11-8) and to peptide 6 (alpha 1(II)CB11) were active in inducing passive arthritis. Other peptide bound fractions were inactive. These observations suggest that the arthritogenic domain in Type II collagen is restricted to alpha 1(II)CB11.

Effects of reserpine, epidermal growth factor, and cyclic nucleotide modulators on epidermal mitosis.

The mouse ear G2 mitosis assay was modified for the screening of potential antimitotic agents. An inhibitory adrenergic influence, which maintains mitotic rate at a normally low level, was removed by pretreatment of mice with reserpine. This depletes endogenous catecholamines, produces a state of enhanced mitotic activity, and makes the epidermal cells particularly sensitive to mitotic inhibition by agents which elevate the levels of cyclic AMP. Isoproterenol [IC 50 approximately 1 X 10(-9) M], prostaglandins, dibutyryl cyclic AMP [IC 50 approximately 2 X 10(-5) M], papaverine, theophylline and 5' AMP were inhibitory in the assay, whereas dibutyryl cyclic GMP and the cholinergic stimulator carbamylcholine either stimulated or had no effect on mitosis. Epidermal growth factor was employed as an alternate means of stimulating cell division. Skin fron newborn mice or rats pretreated with this substance had increased epidermal mitotic activity which was inhibited cyclic AMP elevators.