Michael Goltz

Detection of two novel porcine herpesviruses with high similarity to gammaherpesviruses

Evidence for the existence of porcine gammaherpesviruses was obtained by PCR and sequence analysis. Initially, samples of peripheral blood mononuclear cells (PBMC), spleens, lungs, kidneys and livers of pigs from Germany and Spain were tested with a PCR assay which targets conserved regions of the herpesvirus DNA polymerase gene with degenerate and deoxyinosine-substituted primers. Amplicons of identical sequence were obtained from one spleen and two PBMC samples. This sequence showed a high percentage of identity with the DNA polymerase genes of herpesviruses of the oncogenic subfamily Gammaherpesvirinae. Alignment of amino acid sequences showed the highest identity values with bovine gammaherpesviruses, namely alcelaphine herpesvirus type 1 (68%), ovine herpesvirus type 2 (68%) and bovine lymphotropic herpesvirus (67%). Comparison with pseudorabies virus and porcine cytomegalovirus, which are the only porcine herpesvirus species presently known, showed values of only 41%. PCR analysis of PBMC (n = 39) and spleen (n = 19) samples from German pigs, using primers specific for the novel sequence, revealed a prevalence of 87 and 95%, respectively. In this analysis, three out of eight spleen samples from Spanish pigs were also positive. Subsequent sequencing of the amplicons revealed the presence of two closely related gammaherpesvirus sequences, differing from each other by 8% at the amino acid level. The putative novel porcine herpesviruses, from which these sequences originated, were tentatively designated porcine lymphotropic herpesvirus type 1 and type 2 (PLHV-1 and PLHV-2). When using pig organs for xenotransplantation, the presence of these viruses has to be considered.

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.

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.

Detection and multigenic characterization of a novel gammaherpesvirus in goats.

Evidence for the existence of a caprine gammaherpesvirus was obtained by analysis of peripheral blood leucocytes of goats with PCR assays that target the herpesvirus genes encoding the glycoprotein B (gB), the DNA polymerase (DPOL) and the terminase (TERM) with degenerate and deoxyinosine-substituted primers. A contiguous 3.6 kbp sequence extending from the gB to the DPOL gene was then determined with specific primers. All sequences (gB, DPOL and TERM) showed a close relationship with the corresponding genes of the Gammaherpesvirinae. Alignment of amino acid sequences revealed a particularly high percentage of identity with the ovine herpesvirus type 2 (>83%), followed by the alcelaphine herpesvirus 1 (>76%) and the bovine lymphotropic herpesvirus (>61%). Phylogenetic analyses confirmed these relationships. The putative novel goat herpesvirus from which these sequences originate was tentatively designated caprine herpesvirus 2. This virus is the first gammaherpesvirus recognized in goats.

Novel Simian Homologues of Epstein-Barr Virus

ABSTRACT Thirty different lymphocryptoviruses (LCV), 26 of them novel, were detected in primates by a panherpesvirus PCR assay. Nineteen LCV from chimpanzees, bonobos, gorillas, and other Old World primates were closely related to Epstein-Barr virus (EBV), the type species of the genus Lymphocryptovirus. Seven LCV originating from New World primates were related to callitrichine herpesvirus 3 (CalHV-3), the first recognized New World LCV. Importantly, a second LCV from gorillas and three LCV from orangutans and gibbons were only distantly related to EBV and CalHV-3. They were tentatively assigned to a novel genogroup of Old World primate LCV. The work described in the paper may also help identify an as yet unknown human LCV.

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.

A novel porcine gammaherpesvirus.

A novel porcine gammaherpesvirus was detected in the blood of domestic pigs by PCR. With degenerate-primer PCR and subsequent long-distance PCR approaches a 60-kbp genome stretch was amplified. Sequence analysis revealed the presence of the gammaherpesvirus ORFs 03 to 46 as well as a putative chemokine receptor and a v-bcl-2 gene. The 60-kbp sequence was compared with the corresponding sequence of the porcine lymphotropic herpesvirus 1 (PLHV-1) published recently and the sequence of PLHV-2, which was amplified from porcine tonsil. Considerable sequence differences (amino acid identities: 49-89%) were found between the novel virus and PLHV-1 as well as PLHV-2, which were very closely related to each other (amino acid identities: 85-98%). The novel virus had essentially the same genome organization as PLHV-1 and -2 and was therefore designated PLHV-3. Like PLHV-1 and -2, PLHV-3 was frequently found in the blood and in lymphoid organs of domestic and feral pigs from different geographic locations. In the blood, the PLHVs were detected predominantly in B-cells. Indication for latent as well as productive PLHV-3 infection was found in the porcine B-cell line L23. It can be concluded that the PLHVs are widespread and are likely to cause a persistent B-lymphotropic infection. Since PLHV-1 has been implicated in the development of porcine posttransplantation lymphoproliferative disease, all porcine lymphotropic gammaherpesviruses are of concern when pigs are used as donors in xenotransplantation.

The core 2 beta-1,6-N-acetylglucosaminyltransferase-mucin encoded by bovine herpesvirus 4 was acquired from an ancestor of the African buffalo

ABSTRACT The Bo17 gene of bovine herpesvirus 4 (BoHV-4) is the only viral gene known to date that encodes a homologue of the cellular core 2 β-1,6-N-acetylglucosaminyltransferase-mucin type (C2GnT-M). To investigate the origin and evolution of the Bo17 gene, we analyzed its distribution among BoHV-4 strains and determined the sequences of Bo17 from nine representative strains and of the C2GnT-M gene from six species of ruminants expected to encompass the group within which the gene acquisition occurred. Of 34 strains of BoHV-4, isolated from four different continents, all were found to contain the Bo17 gene. Phylogenetic analyses indicated that Bo17 was acquired from a recent ancestor of the African buffalo, implying that cattle subsequently acquired BoHV-4 by cross-species transmission. The rate of synonymous nucleotide substitution in Bo17 was estimated at 5 × 10−8 to 6 × 10−8 substitutions/site/year, consistent with previous estimates made under the assumption that herpesviruses have cospeciated with their hosts. The Bo17 gene acquisition was dated to around 1.5 million years ago. Bo17 sequences from BoHV-4 strains from African buffalo and from cattle formed two separate clades, estimated to have split about 700,000 years ago. Analysis of the ratio of nonsynonymous to synonymous nucleotide substitutions revealed a burst of amino acid replacements subsequent to the transfer of the cellular gene to the viral genome, followed by a return to a strong constraint on nonsynonymous changes during the divergence of contemporary BoHV-4 strains. The Bo17 gene represents the most recent of the known herpesvirus gene acquisitions and provides the best opportunity for learning more about this important process of viral evolution.

Genetic and ultrastructural characterization of a European isolate of the fatal endotheliotropic elephant herpesvirus.

A male Asian elephant (Elephas maximus) died at the Berlin zoological gardens in August 1998 of systemic infection with the novel endotheliotropic elephant herpesvirus (ElHV-1). This virus causes a fatal haemorrhagic disease in Asian elephants, the so-called endothelial inclusion body disease, as reported from North American zoological gardens. In the present work, ElHV-1 was visualized ultrastructurally in affected organ material. Furthermore, a gene block comprising the complete glycoprotein B (gB) and DNA polymerase (DPOL) genes as well as two partial genes was amplified by PCR-based genome walking and sequenced. The gene content and arrangement were similar to those of members of the Betaherpesvirinae. However, phylogenetic analysis with gB and DPOL consistently revealed a very distant relationship to the betaherpesviruses. Therefore, ElHV-1 may be a member of a new genus or even a new herpesvirus subfamily. The sequence information generated was used to set up a nested-PCR assay for diagnosis of suspected cases of endothelial inclusion body disease. Furthermore, it will aid in the development of antibody-based detection methods and of vaccination strategies against this fatal herpesvirus infection in the endangered Asian elephant.

Approaching virus safety in xenotransplantation: a search for unrecognized herpesviruses in pigs

Abstract: The identification of porcine viruses so far unrecognized is required to minimize virus‐related risks associated with xenotransplantation. We used a pan‐herpes consensus polymerase chain reaction assay to search for unrecognized porcine species of the Herpesviridae. The assay targets conserved regions of the herpesvirus DNA polymerase (DPOL) gene, using primers that were modified to diminish the assays recognition capacity for the highly prevalent porcine lymphotropic herpesviruses 1, 2 and 3 (PLHV‐1, ‐2, ‐3), without substantially lowering the universal detection capacity of the assay. Analysis of 495 porcine blood and tissue samples from 294 animals, including 35 samples from 20 immunosuppressed pigs, resulted in the amplification of 128 herpesviral DPOL sequences. Sequence analysis attributed 127 of the amplimers to the known porcine herpesviruses (PLHV‐1, ‐2, ‐3; porcine cytomegalovirus; pseudorabiesvirus). In none of the pig samples analyzed here, evidence was obtained for the presence of additional novel porcine herpesvirus species. Therefore we conclude that pigs bred for the purpose of xenotransplantation pose a negligible risk of transmitting presently unrecognized herpesviruses to organ recipients.