Adorján Bartha

DNA Restriction Enzyme Analysis of Bovine Adenoviruses

Eight reference strains of the nine accepted bovine adenovirus serotypes were compared by means of restriction enzyme (RE) analysis of the viral DNA. The study was carried out with the aim to identify RE giving restriction patterns which are particularly feasible for identification of new isolates and useful for their classification into subgroups. The BamHI, EcoRI, HindIII and PstI enzymes were feasible for identification of strains. The classification of bovine adenovirus (BAV) into subgroup 1 (BAV-1, 2, 3 and 9) and subgroup 2 (BAV-4, 5, 6, 7 and 8) was supported by (i) the significant difference in genome size between members of subgroup 1 (ranging from 32.73 to 37.67 kb) and members of subgroup 2 (ranging from 28.78 to 31.18 kb); and (ii) the observation that the nucleotide sequence recognized by EcoRI is less frequent in the genomes of subgroup 1 members - yielding 4-7 RE fragments - than in the genomes of subgroup 2 members - yielding 10-16 RE fragments.

Histological studies of bovine herpesvirus type 4 infection in non-ruminant species.

The pathology of bovine herpesvirus type 4 (BHV-4) infection was studied in cats, rabbits and guinea pigs. Twenty kittens, twenty-two rabbits and ten guinea pigs, some treated with glucocorticoid-were inoculated with a BHV-4 strain of feline origin, via various routes of inoculation (conjunctival, intranasal, peritoneal). Clinical signs were recorded. After euthanizing at different post inoculation days macro- and microscopic changes were observed by necropsy and in hematoxylin-eosin stained histological sections. The presence of the virus in organs was detected by immunohistochemistry and a nested PCR assay. Inclusion bodies and monoclonal antibody-stained cells were found in the conjunctiva, trachea, lungs, spleen and lymph nodes. Most of the lesions were localized to the respiratory and the immune system. The macro- and microscopic lesions and clinical signs were more severe in kittens and guinea pigs. The histological data indicated that cats, especially kittens, were susceptible for BHV-4 and the infection was not confined to the urinary bladder.

Bovine herpesvirus 1: rapid diagnosis of infection by direct filter hybridization

Direct filter hybridization (DFH) was applied as a simple method of nucleic acid hybridization to diagnose bovine herpesvirus 1 (BHV-1) infection without previous purification of nucleic acids from the specimens. The DNA of BHV-1 was cleaved with the restriction endonuclease Pst I and randomly cloned into pKH47 plasmids. The clones were labelled with 32P or biotin and selected on uninfected and infected cells for the highest specific activity to detect BHV-1 infection. Two clones, which detected about 10 infected cells, were selected for the diagnosis of BHV-1 in cattle. On specimens collected during experimental and natural disease, the DFH showed to be in concordance with the standard method of virus isolation. This simple hybridization technique proved to be a sensitive and rapid alternative to virus isolation. Specific diagnosis of BHV-1 infection can be made even in simply equipped laboratories within 10 h.

A heteroploid permanent cell line originating from embryonic calf thyroid supporting the replication of all known bovine adenovirus serotypes

An embryonic calf thyroid cell culture was established as a permanent heteroploid cell line, which is now in its 150th subculture. It allowed replication of all nine bovine adenovirus serotypes at its 15th as well as its 60-150th passages. All viruses induced typical cytopathic effects. Yields obtained on the permanent calf thyroid line were, on average, 0.8 log10 lower than those obtained on primary calf testicle cells.

Latent infection with feline syncytial virus of cell cultures prepared from the kidneys of new-born kittens

Feline syncytial virus infection was detected in 4 out of 30 secondary or tertiary cultures of kidney cells from new-born kittens. Such infection was not detected in any primary cell culture. The syncytia-forming virus was readily transmissible by infected cells. One strain was transmissible to heterologous (bovine and canine) secondary cells, but did not infect MDBK and PK-15 cell lines. Based on biochemical, untrastructural and serological characteristics, the virus was designated a member of the subfamily Spumavirinae of the family Retroviridae. As endogenous infection of primary cell cultures may not be detectable by cytopathic effect, it is suggested that only well-controlled secondary cat cells or cell lines should be used in work with feline viruses.

Transplacental transmission of bovine adenoviruses

Type 1 bovine adenovirus has been isolated from cell cultures prepared from the kidneys of a 7-month-old bovine fetus, taken from a slaughterhouse, and types 4, 6 and 8 from cell cultures prepared from the testicles of four neonatal, precolostral calves. Sera of these precolostral calves possessed virus-neutralizing antibodies in titers of 1:4--1:16. Bovine adenoviruses of types 4 and 8 have also been isolated from two bovine fetuses aborted from an unknown cause. Relying upon these findings the possible vertical (transplacental) transmission of bovine adenoviruses is presumed. Though a cause-and-effect relationship between abortion and adenovirus infection could not be detected, it is probably that infection during fetal development cannot be indifferent to the fetus or the neonatal calf.

Periodic reappearance of bovine herpesvirus type 4 DNA in the sera of naturally and experimentally infected rabbits and calves

A BHV-4 specific nested PCR was used for the detection of viral DNA in serum samples of rabbits and calves. All animals were followed up for 62 days, blood samples were taken for PCR studies every second day. Maternal infection of calves resulted in the repeated regular reappearance (10-14 days) of the virus (DNA) in serum samples. When PCR positive five-day-old calves were infected with tissue culture adapted virus, the reappearance of the DNA in the serum was shown to be irregular, nevertheless, DNA peaks reappeared during the whole observation period. A PCR negative calf infected at the age of 60 days was found to possess viraemia until p.i.d. 32. In rabbits treated intravenously with BHV-4 the inoculum or a primary viraemia was detected at p.i.d. 2-3 and p.i.d. 14-16. Published data on human herpesviruses suggest, that the target cells might be a pluripotent stem cell population of the bone marrow and differentiated virus-infected cells destroyed by the immune system might be the source of viral DNA detected in the serum. Frequency of DNA reappearance was depended on the age of the infected animals but not on the inoculated amount of BHV-4. The described phenomenon might be part of BHV-4 infection of very young animals.