Hans-Jörg Buhk

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.

Genome Sequence of Bovine Herpesvirus 4, a Bovine Rhadinovirus, and Identification of an Origin of DNA Replication

ABSTRACT Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus of cattle. The complete long unique coding region (LUR) of BoHV-4 strain 66-p-347 was determined by a shotgun approach. Together with the previously published noncoding terminal repeats, the entire genome sequence of BoHV-4 is now available. The LUR consists of 108,873 bp with an overall G+C content of 41.4%. At least 79 open reading frames (ORFs) are present in this coding region, 17 of them unique to BoHV-4. In contrast to herpesvirus saimiri and human herpesvirus 8, BoHV-4 has a reduced set of ORFs homologous to cellular genes. Gene arrangement as well as phylogenetic analysis confirmed that BoHV-4 is a member of the genusRhadinovirus. In addition, an origin of replication (ori) in the genome of BoHV-4 was identified byDpnI assays. A minimum of 1.69 kbp located between ORFs 69 and 71 was sufficient to act as a cis signal for replication.

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.

Nachweis gentechnischer Veränderungen in Mais mittels PCR

Ein PCR-Nachweis für gentechnisch veränderten Mais «Event 176» der Fa. Ciba-Geigy wurde etabliert. Der Mais enthält Gene, die Selbstschutz gegen den Maiszünsler (Delta-Endotoxin-Gen ausBacillus thuringiensis) und Toleranz gegen das Herbizid Basta (Phosphinothricin-Resistenz-Gen ausStreptomyces hygroscopicus) vermitteln. Zudem enthält der Mais ein Ampicillin-Resistenz-Gen. Für die Amplifikation von Bereichen aus allen drei Genen wurden PCR-Primer entworfen. Mit Hilfe dieser Primer und mit «Event 176»-Mais-DNA als Template konnten die entsprechenden Genbereiche in der PCR amplifiziert werden. Die PCR-Produkte wurden sequenziert, um ihre Identität zu bestätigen. Mit Hilfe der Delta-Endotoxin-PCR wurden, auch in Gegenwart von 104fachem Überschuß nicht gentechnisch veränderter Mais-DNA, fünf haploide Genome der «Event 176»-DNA nachgewiesen. A PCR-test for the genetically modified maize «Event 176» of Ciba-Geigy was established. The maize contains genes conferring resistance to the European corn borer (delta-endotoxin gene fromBacillus thuringiensis) and tolerance to the herbicide Basta (phosphinothricin resistance gene fromStreptomyces hygroscopicus). The maize contains also an ampicillin resistance gene. Primers were designed and using «Event 176»-maize-DNA as template internal regions of the three genes were amplified with PCR. The PCR products were sequenced to confirm their identity. Using the deltaendotoxin primers in PCR down to 5 haploid genomes of «Event 176»-DNA could be detected, even in the presence of a 104fold excess of DNA from non-modified maize.

Conservation of a gene cluster including glycoprotein B in bovine herpesvirus type 2 (BHV-2) and herpes simplex virus type 1 (HSV-1)

A library of subgenomic fragments of bovine herpesvirus type 2 (BHV-2) DNA was constructed in the expression cloning vector lambda gt11 and screened with monoclonal antibodies to the glycoprotein gb BHV-2, which is homologous to glycoprotein gB (gB-1) of herpes simplex virus type 1 (HSV-1). Lambda gt11 clones containing gB BHV-2-specific sequences were used to identify lambda EMBL3 vectors with DNA inserts which contained the complete gB BHV-2 gene. Nucleotide sequencing revealed that the gB BHV-2 gene is highly conserved compared to gB-1. The amino acid sequences and the predicted secondary structures of both glycoproteins are very similar. Two further open reading frames (ORF) in close vicinity to the gene encoding gB BHV-2 showed considerable homology to HSV-1 genes. They code for the major DNA-binding protein (dbp) of BHV-2 and a putative 72-kDa polypeptide. The gene of the latter protein corresponding to ICP18.5 of HSV-1 is interspersed between the ORFs of gB BHV-2 and the dbp of BHV-2. All three genes map in the unique long region of the genome. Their homology and the colinear arrangement compared to HSV-1 indicate a close relationship between the two viruses.

Glycoprotein B of bovine herpesvirus type 4: Its phylogenetic relationship to gB equivalents of the herpesviruses

In order to estimate the phylogenetic relationship of BHV-4 among the herpesviruses, we have cloned and sequenced its glycoprotein B (gB). The 2.6 kb open reading frame codes for a 874 amino acid long protein. The comparison of its deduced amino acid sequence with those of its counterparts in 19 distinct herpesviruses groups BHV-4 into the γ-herpesvirinae. The calculation of an evolutionary tree emphasized that BHV-4 is more closely related to herpesvirus saimiri (HVS) than to Epstein-Barr virus (EBV). However, in contrast to EBV and HVS, the gB of BHV-4 contains a putative protease cleavage site and 20 potential N-glycosylation sites. The alignment of the amino acid sequences revealed that 10 cysteine and 7 proline residues, as well as the motifs SPF and GQLG, were completely conserved among the 20 investigated gBs.

Molecular biological characterization of equine herpesvirus type 1 (EHV-1) isolates from ruminant hosts.

Isolates of equine herpesvirus type 1 (EHV-1) originating from affected antelope and cattle were compared with reference EHV-1 and EHV-4 isolates and were characterized. Based on cross-neutralization, DNA restriction profiles and blot-hybridization data these isolates could be characterized as EHV-1. One isolate (from an antelope) with a different restriction profile showed significant DNA homology with EHV-1, partial homology with EHV-4, and little or no homology with EHV-2 and HSV-1 DNAs. Blot hybridization revealed differences in DNA restriction fragments located at the termini of two isolates and size heterogeneity in the unique long/internal repeat junction fragment (UL/IR) of one isolate.

Viral DNA from Cells Infected with Porcine Circovirus

It has been demonstrated earlier that the genome of the porcine circovirus (PCV) isolated from virions is a circular single-stranded DNA (ssDNA). This report describes the isolation and characterization of PCV-specific DNA from infected cell cultures. Two types of double-stranded DNA (dsDNA) were observed which behaved like supercoiled and relaxed circular molecules, respectively. Both types of dsDNA displayed infectivity in transfection experiments and are regarded as replicative forms (RF) of the viral genome. In addition to complete single-stranded viral genomes and RF DNA molecules, PCV-specific subgenomic fractions of DNA molecules with a sedimentation coefficient of about 5 S were isolated from infected tissue culture cells. This 5 S DNA was also isolated from purified virions. It is single-stranded and represents a defined region of the viral genome.

Conserved domains of glycoprotein B (gB) of the monkey virus, simian agent 8, identified by comparison with herpesvirus gBs

The herpesvirus simian agent 8 (SA8) gene which corresponds to the herpes simplex virus (HSV) gene encoding glycoprotein B (gB) was localized, cloned and sequenced. Comparison of its deduced amino acid sequence with those of its counterparts in 12 other distinct herpesvirus was used to evaluate their homology and phylogenetic relationship. The results emphasized that SA8 gB is more closely related to those of HSV-1 and -2, and bovine herpesvirus 2 than to the homologous proteins of other herpesviruses. Furthermore, the alignment showed several regions of domains conserved in the closely related sequences, including four conserved in all the herpesvirus gB sequences examined. The conservation of 10 cysteine residues and most of the proline residues, as well as several potential N-glycosylation sites, suggested that the secondary and tertiary structures of these gBs were similar.

Rapid method for the identification and screening of herpesviruses by DNA fingerprinting combined with blot hybridization

Rapid characterization of herpesvirus isolates exemplified by equine herpesviruses is described. Total DNAs were isolated from virus infected small scale cell cultures. The DNA fragments obtained after restriction enzyme digestion were separated on agarose gels, transferred and immobilized on filter membranes. A radioactively labelled probe derived from the purified DNA of an EHV-1 reference strain was used for hybridization in order to detect the restriction fragments of different EHV-1 field isolates. This method allows the typing of many isolates within a short period of time.