We describe a new primer design strategy for PCR amplification of unknown targets that are related to multiply-aligned protein sequences. Each primer consists of a short 3' degenerate core region and a longer 5' consensus clamp region. Only 3-4 highly conserved amino acid residues are necessary for design of the core, which is stabilized by the clamp during annealing to template molecules. During later rounds of amplification, the non-degenerate clamp permits stable annealing to product molecules. We demonstrate the practical utility of this hybrid primer method by detection of diverse reverse transcriptase-like genes in a human genome, and by detection of C5DNA methyltransferase homologs in various plant DNAs. In each case, amplified products were sufficiently pure to be cloned without gel fractionation. This COnsensus-DEgenerate Hybrid Oligonucleotide Primer (CODEHOP) strategy has been implemented as a computer program that is accessible over the World Wide Web (http://blocks.fhcrc.org/codehop.html) and is directly linked from the BlockMaker multiple sequence alignment site for hybrid primer prediction beginning with a set of related protein sequences.

A consensus primer PCR method which amplifies a region of herpesviral DNA-directed DNA polymerase (EC 2.7.7.7) and which uses degenerate primers in a nested format was developed. Primers were designed to target sequences coding for highly conserved amino acid motifs covering a region of approximately 800 bp. The assay was applied to 22 species of herpesviruses (8 human and 14 animal viruses), with PCR products obtained for 21 of 22 viruses. In the process, 14 previously unreported amino acid-coding sequences from herpesviral DNA polymerases were obtained, including regions of human herpesviruses 7 and 8. The 50 to 60 amino acid-coding sequences recovered in the present study were determined to be unique to each viral species studied, with very little sequence variation between strains of a single species when studied. Template dilution studies in the presence of human carrier DNA demonstrated that six human herpesviruses (herpesviruses 1, 2, 3, 4, 5, and 6B) could be detected at levels at or below 100 genome equivalents per 100 ng of carrier DNA. These data suggest that consensus primer PCR targeted to herpesviral DNA polymerase may prove to be useful in the detection and identification of known herpesviruses in clinical samples and the initial characterization of new herpesviral genomes.

Representational difference analysis was used to search for pathogens in multiple sclerosis brains. We detected a 341-nucleotide fragment that was 99.4% identical to the major DNA binding protein gene of human herpesvirus 6 (HHV-6). Examination of 86 brain specimens by PCR demonstrated that HHV-6 was present in >70% of MS cases and controls and is thus a commensal virus of the human brain. By DNA sequencing, 36/37 viruses from MS cases and controls were typed as HHV-6 variant B group 2. Other herpesviruses, retroviruses, and measles virus were detected infrequently or not at all. HHV-6 expression was examined by immunocytochemistry with monoclonal antibodies against HHV-6 virion protein 101K and DNA binding protein p41. Nuclear staining of oligodendrocytes was observed in MS cases but not in controls, and in MS cases it was observed around plaques more frequently than in uninvolved white matter. MS cases showed prominent cytoplasmic staining of neurons in gray matter adjacent to plaques, although neurons expressing HHV-6 were also found in certain controls. Since destruction of oligodendrocytes is a hallmark of MS, these studies suggest an association of HHV-6 with the etiology or pathogenesis of MS.Multiple sclerosis (MS) is a disease of young adults that is characterized clinically by a variable relapsing and remitting course and pathologically by the progressive accumulation of plaques of demyelination within the white matter of the central nervous system. In normal white matter, the axons of neurons are surrounded by myelin sheaths, made from the cell membranes of oligodendrocytes. In MS plaques, the myelin sheaths are destroyed, leaving the naked axons intact but impaired in their conduction of action potentials. The currently held view is that an autoimmune inflammatory reaction against components of myelin results in destruction of oligodendrocytes. The demyelinating lesions in MS are found throughout the central nervous system, with a predilection for the periventricular white matter, optic nerve, brainstem, spinal cord, and cerebellum, resulting in a disease that is pleiomorphic in its clinical presentation.In spite of the substantial evidence that autoimmunemediated demyelination plays a major role in the progression of MS, epidemiologic studies suggest that an infectious agent may also be involved (1). Prior reports have suggested that viral infection of cells within the central nervous system may initiate the events leading to focal demyelination (2), and a number of viruses have been implicated in the pathogenesis of MS (3). Despite extensive investigation, however, none of these associations has been confirmed (4), and the issue of viral involvement in the pathogenesis of MS remains unresolved.To search for an MS-associated pathogen, we used representational difference analysis (RDA) (5). In RDA, successive rounds of subtractive hybridization and PCR amplification enriched for DNA sequences that were present in a DNA preparation from diseased tissue (MS brain) but absent from control DNA (n...

We have cloned and characterized the entire DNA polymerase gene and flanking regions from Kaposi's sarcoma-associated herpesvirus (KSHV) and two closely related macaque homologs of KSHV, retroperitoneal fibromatosis-associated herpesvirus-Macaca nemestrina (RFHVMn) and -Macaca mulatta (RFHVMm). We have also identified and partially characterized the corresponding genomic region of another KSHV-like herpesvirus, provisionally named "M. nemestrina rhadinovirus type 2 (MneRV-2)," with close similarity to rhesus rhadinovirus (RRV). A sequence comparison of these four macaque viruses and two KSHV-like gammaherpesviruses recently identified in African green monkeys, Chlorocebus rhadinovirus types 1 and 2 (ChRV-1 and ChRV-2) reveals the presence of two distinct lineages of KSHV-like rhadinoviruses in Old World primates. The first rhadinovirus lineage consists of KSHV and its closely related homologs RFHVMn, RFHVMm, and ChRV-1, while the second more distantly related lineage consists of RRV, MneRV-2, and ChRV-2. Our findings raise the possibility of the existence of another human KSHV-like herpesvirus belonging to the second rhadinovirus lineage.Kaposi's sarcoma-associated herpesvirus (KSHV) is postulated to be the infectious cause of Kaposi's sarcoma (KS) (for reviews, see references 5 and 19). Due to the strong similarities in sequence and gene organization with herpesvirus saimiri (HVS), the prototype of the gamma-2 (Rhadinovirus) genus of the gammaherpesvirus subfamily, KSHV has been classified as a human rhadinovirus (6, 13). We have previously identified DNA sequences related to KSHV in retroperitoneal fibromatosis (RF) lesions from two macaque species, the pig-tailed macaque (Macaca nemestrina) and the rhesus macaque (Macaca mulatta) (17). RF is a vascular fibroproliferative neoplasm with similarities to KS which was prevalent in the macaque colony in the Washington Regional Primate Research Center (WaRPRC) during the late 1970s and early 1980s (9, 10). The macaque KSHV-like sequences were identified using a novel consensus-degenerate hybrid oligonucleotide primer (CODE-HOP) PCR technique (16), which was employed to detect unknown herpesvirus DNA polymerase genes. Phylogenetic analysis of the available sequence data suggested that the DNA polymerase fragments were derived from macaque homologs of human KSHV, with a unique genotype present in each macaque species. These macaque homologs were designated RF-associated herpesvirus-M. nemestrina (RFHVMn) and -M. mulatta (RFHVMm). Subsequently, an additional simian homolog of KSHV was identified in an M. mulatta from the New England Regional Primate Research Center, and approximately 10 kb of the viral genome, including the DNA polymerase and flanking regions, was sequenced (8). Because of its sequence similarity to KSHV and HVS, this new homolog was designated rhesus rhadinovirus (RRV) isolate H26-95. Another isolate of RRV, RRV-17577, was identified in a simian immunodeficiency virus-infected rhesus macaque with a lymphoproliferative disorder at the Oregon Regional P...

Simian retroperitoneal fibromatosis (RF) is a vascular fibroproliferative neoplasm which has many morphological and histological similarities to human Kaposi's sarcoma (KS). Like epidemic KS in AIDS patients, RF is highly associated with an immunodeficiency syndrome (simian acquired immunodeficiency syndrome [SAIDS]) caused by a retrovirus infection. Recently, a new gammaherpesvirus, called Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8), has been identified in KS tumors, suggesting that KS has a viral etiology. Our previous experimental transmission studies and epidemiological data suggest that RF also has an infectious etiology. In order to determine whether a similar virus is also associated with RF, we have assayed for the presence of an unknown herpesvirus using degenerate PCR primers targeting the highly conserved DNA polymerase genes of the herpesvirus family. Here we provide DNA sequence evidence for two new herpesviruses closely related to KSHV from RF tissues of two macaque species, Macaca nemestrina and Macaca mulatta. Our data suggest that KSHV and the putative macaque herpesviruses define a new group within the subfamily Gammaherpesvirinae whose members are implicated in the pathogenesis of KS and KS-like neoplasms in different primate species.