Hanns Ludwig

Detection of New DNA Polymerase Genes of Known and Potentially Novel Herpesviruses by PCR with Degenerate and Deoxyinosine-Substituted Primers

A consensus primer PCR approach was used to (i) investigate the presence of herpesviruses in wild and zoo equids (zebra, wild ass, tapir) and to (ii) study the genetic relationship of the herpesvirus of pigeons (columbid herpesvirus 1) to other herpesvirus species. The PCR assay, based on degenerate primers targeting highly conserved regions of the DNA polymerase gene of herpesviruses, was modified by using a mixture of degenerate and deoxyinosine-substituted primers. The applicability of the modification was validated by amplification of published DNA polymerase genes of 16 herpesvirus species and of the previously uncharacterized DNA polymerase genes of equine herpesvirus 3 (EHV-3) and equine herpesvirus 5 (EHV-5). The modified assay was then used for partial amplification of the polymerase of columbid herpesvirus 1 which is presently classified as a β-herpesvirus based on biological criteria. Sequence analysis of amplicons obtained from four different viral strains revealed a close relationship of columbid herpesvirus 1 to members of the subfamily Alphaherpesvirinae, especially to Marek’s disease herpesvirus. This was confirmed by characterization of additional 1.6 kb of the columbid herpesvirus 1 polymerase. Consensus PCR analysis of blood samples from zebras, a wild ass and a tapir revealed amplicons showing high percentages (>50%) of sequence identity to DNA polymerases of γ-herpesviruses. In particular, the zebra and the wild ass sequence were closely related to each other and to the polymerases of the equine γ-herpesviruses EHV-2 and EHV-5 with sequence identities of >80%. This is a first indication that novel γ-herpesviruses are present in wild and zoo equids.

Learning deficiencies in borna disease virus-infected but clinically healthy rats☆

Borna disease (BD) virus, a still unclassified neurotropic agent, causes either fatal encephalomyelitis or persistent asymptomatic infection in a variety of animal species. We monitored the neuronal functions of intracerebrally infected but healthy rats with three types of learning experiments. Spatial discrimination learning, using the y maze and the hole board, was significantly less successful in BD virus-infected (I) compared with mock-infected (M) rats. Similarly, I rats tended to show a certain emotional disturbance (reduced resting behavior and less anxiety) as evaluated by open-field and neophobia tests. Furthermore, in two aversive learning experiments (taste aversion and reaction suppression via Skinner box), it appeared that the I rats expressed a significantly diminished ability to learn pain avoidance compared with M rats. In conclusion, we found specific learning deficiencies together with subtle behavioral alterations suggesting that BD virus causes certain modulations of high integrative brain functions which are only detectable under experimental conditions.

Detection of equine herpesvirus type 2 (EHV-2) in horses with keratoconjunctivitis.

The prevalence of EHV-2 in 27 horses with keratoconjunctivitis and 21 clinically healthy horses of different ages and stocks were analyzed. We demonstrated that EHV-2 was present in 12 keratoconjunctivitis cases as shown by nested PCR on ocular swabs. This is statistically more often than in the control group, where only two ocular swabs were EHV-2 positive. Cocultivation was successful on peripheral blood leukocytes of healthy and diseased horses but not on swabs. We isolated ten EHV-2 strains from diseased and nine from control horses, whereas 16 isolates showed different restriction enzyme patterns. The results of immunfluorescence and neutralization tests are predictory only in combination with the nested PCR data on ocular swabs. A successful antiviral treatment in nine out of 16 cases supports the aetiological role of EHV-2 in this ocular disease.

Amantadine and human Borna disease virus in vitro and in vivo in an infected patient with bipolar depression

178 Vol 349 • January 18, 1997 1 Poulson AG, Kvinesdal B, Aaby P, et al. Prevalence of and mortality from human immunodeficiency virus type 2 in Bissau, West Africa. Lancet 1989; i: 827–31. 2 Matheron S, Courpotin C, Simon F, et al. Vertical transmission of HIV-2. Lancet 1990; 335: 1103–04. 3 Adjorlolo-Johnson G, De Cock KM, Ekpini E, et al. Prospective comparison of mother-to-child transmission of HIV-1 and HIV-2 in Abidjan, Ivory Coast. JAMA 1994; 272: 462–66.

Distribution and relevance of equine herpesvirus type 2(EHV-2) infections

SummaryEquine herpesvirus type 2 (EHV-2) is a slow-growing, cytopathogenic gammaherpesvirus, which is suggested to be ubiquitous in the equine population. However, its precise role as a pathogen and its tissue tropism remains uncertain. To estimate the prevalence of EHV-2 in Germany and to investigate the possible pathogenicity of the virus, peripheral blood leucocytes (PBL) from 172 horses were examined for EHV-2 DNA by a sensitive and specific nested PCR based on the EcoRI-N genomic fragment and by classical cocultivation. PBL samples from 51% of the horses were positive by PCR and virus was isolated from 31% of the horses by cocultivation. However, almost all animals were seropositive for EHV-2. This may indicate that PBL do not harbour EHV-2 indefinitely after infection. Furthermore, a correlation between clinical signs and EHV-2 as a causative agent could not be determined. Nevertheless, the prevalence of virus was high among horses with upper respiratory tract disease, abortion and severe ataxia.The products of the second round of the PCR reactions showed size polymorphism. Sequencing of the products revealed that these size differences were due to repetition of the motif (AGACAGGGGCCATGCTGGC) between 9–16 times depending on the isolate, suggesting that the nested PCR might be a useful tool for the differentiation of EHV-2 isolates.

Bovine Cytomegaloviruses: Identification and Differential Properties

Biological properties and restriction enzyme patterns of the slowly replicating herpesviruses isolated from cattle affected with different diseases in North America and Europe were analysed. These virus isolates induced identical plaques that developed within 7 to 9 days in bovine foetal spleen cells and within 5 days in actively growing Georgia bovine kidney cells. These virus isolates were found to be antigenically related when tested in the indirect immunofluorescence test, and antigenic relationships with bovine herpesvirus 1 (BHV-1), BHV-2, BHV-3 or BHV-6 were not detected. The genomes of these strains were shown to have virtually identical cleavage sites when treated with restriction enzymes EcoRI, BamHI, SstII, SphI and HindIII. The resulting restriction enzyme patterns differed strikingly from those of BHV-1, BHV-2, BHV-3 and BHV-6. Because the herpesviruses tested become enveloped on the nuclear as well as on endoplasmic membranes, a process through which they induce cytoplasmic vesicles filled with enveloped viral particles, and because of the unique cytoplasmic inclusions that are induced, we classify them tentatively as bovine cytomegaloviruses.

Analysis of bovine cytomegalovirus genome structure: cloning and mapping of the monomeric polyrepetitive DNA unit, and comparison of European and American strains.

The genome of bovine cytomegalovirus (BCMV) strain 66-P-347 consists of double-stranded, linear DNA with a size of 144 +/- 6 kb. It contains polyrepetitive DNA (prDNA) segments like five other BCMV strains. The restriction patterns of the prDNA of all six strains are very similar and indicate that monomeric prDNA units are either 1950 bp (class I and Ia), 2350 bp (class II) or 2750 bp (class III) in size. The complete unit sequence of strain 66-P-347 (class II) was cloned in bacteriophage vector M13mp7 and mapped by the restriction enzymes EcoRI, BamHI, Bg/I, NaeI, SstII and PstI. From these results the restriction maps of the prDNA of the other five strains were deduced. The 400 bp differences in size between the three prDNA classes are a consequence of the appearance of an internal 200 bp sequence being present one-, three- or fivefold in head-to-tail formation. Hybridization of 35S-labelled recombinant phage DNA to Southern blots with DNA of the different strains leads to the conclusion that prDNA units are present as multimers in tandem formation at both genomic termini in the same orientation. The number of terminal repeat units varies between individual molecules of a strain, but the actual terminal sequences are identical.

Adaptation of Borna disease virus to the mouse.

Borna disease virus has been adapted to the mouse, which required at least three passages in rat brains. Genetic specificity as studied with five inbred mouse strains was not evident. Newborn mice inoculated intracerebrally expressed antigen in neurons and remained persistently infected, with up to 10(7) infectious units per gram of brain tissue. Animals infected at different ages developed no disease and had high titres of antibodies.

Equine herpesvirus type 1 (EHV-1) induced abortions and paralysis in a Lipizzaner stud: a contribution to the classification of equine herpesviruses

SummaryOut of 30 cases of abortion and perinatal deaths in a Lipizzaner stud in Austria 10 mares died after having shown central nervous system disturbances, ataxias and paralysis. The etiological agent of this “abortion storm” was equine herpesvirus type 1 (EHV-1). The restriction enzyme pattern of the DNA from 5 isolates recovered from fetuses has been analyzed and compared with the known reference strains of EHV-1, -2, -4 and an Austrian vaccine strain. The DNA restriction profiles of the Lipizzaner isolates as well as of the vaccine strain could be identified as being typical of abortigenic strains with minor variations. Such variations on the molecular biological level of the DNA do not justify characterization of the strains as neurovariants. The vaccine strain differed from other isolates investigated with 4 restriction endonucleases (Bam HI, Bgl II, Eco R I, Kpn I) which was due to a deletion in the unique short segment of the genome. The lack of similar DNA bands in two EHV-1 viruses, causing mild respiratory disease, as well as in the vaccine strain Prevaccinol is suggestive of lowered virulence. In contrast to one Lipizzaner isolate tested (strain Austria IV) the Austrian vaccine strain proved to be of strong neurovirulence for suckling mice.

SEQUENCE CHARACTERIZATION OF HUMAN BORNA DISEASE VIRUS

Borna disease virus (BDV) causes a central nervous system disease in several vertebrate animal species, which is manifest by behavioral abnormalities. Seroepidemiologic data suggest that BDV might infect humans, possibly being associated with certain mental disorders. This is further supported by the detection of both BDV-specific antigens and RNA sequences in peripheral blood mononuclear cells (PBMCs) of psychiatric patients. For the first time the sequence characterization of human BDV is documented here. BDV was recovered by co-cultivation techniques from the PBMCs of three hospitalized psychiatric patients. BDV was unequivocally identified based on sequence identification of BDV open reading frames (ORFs) p24, p16 and p56, as well as of the predicted catalytic domain of the BDV L polymerase. Each human BDV isolate had an unique sequence, but they displayed a high degree of sequence conservation with respect of BDV isolates from naturally infected animals of different species.