Detection of a new bat gammaherpesvirus in the Philippines.
ABSTRACT: A new bat herpesvirus was detected in the spleen of an insectivorous bat (Hipposideros diadema, family Hipposideridae) collected on Panay Island, the Philippines. PCR analyses were performed using COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOPs) targeting the herpesvirus DNA polymerase (DPOL) gene. Although we obtained PCR products with CODEHOPs, direct sequencing using the primers was not possible because of high degree of degeneracy. Direct sequencing technology developed in our rapid determination system of viral RNA sequences (RDV) was applied in this study, and a partial DPOL nucleotide sequence was determined. In addition, a partial gB gene nucleotide sequence was also determined using the same strategy. We connected the partial gB and DPOL sequences with long-distance PCR, and a 3741-bp nucleotide fragment, including the 3' part of the gB gene and the 5' part of the DPOL gene, was finally determined. Phylogenetic analysis showed that the sequence was novel and most similar to those of the subfamily Gammaherpesvirinae.
Project description:Targeting the highly conserved herpes DNA polymerase (DPOL) gene with PCR using panherpes degenerate primers is a powerful tool to universally detect unknown herpesviruses. However, vertebrate hosts are often infected with more than one herpesvirus in the same tissue, and pan-herpes DPOL PCR often favors the amplification of one viral sequence at the expense of the others. Here we present two different technical approaches that overcome this obstacle: (i) Pan-herpes DPOL PCR is carried out in the presence of an oligonucleotide substituted with locked nucleic acids (LNA).This suppresses the amplification of a specific herpesvirus DPOL sequence by a factor of approximately 1000, thereby enabling the amplification of a second, different DPOL sequence. (ii) The less conserved glycoprotein B (gB) gene is targeted with several sets of degenerate primers that are restricted to gB genes of different herpesvirus subfamilies or genera. These techniques enable the amplification of gB and DPOL sequences of multiple viruses from a single specimen. The partial gB and DPOL sequences can be connected by long-distance PCR, producing final contiguous sequences of approximately 3.5 kbp. Such sequences include parts of two genes and therefore allow for a robust phylogenetic analysis. To illustrate this principle, six novel herpesviruses of the genera Rhadinovirus, Lymphocryptovirus and Cytomegalovirus were discovered in multi-infected samples of non-human primates and phylogenetically characterized.
Project description:Herpesvirus infection was investigated in black bears (Ursus americanus) with neurological signs and brain lesions of nonsuppurative encephalitis of unknown cause. Visible cytopathic effects (CPE) could only be observed on days 3-5 post-infection in HrT-18G cell line inoculated with bear tissue extracts. The observed CPE in HrT-18G cells included syncytia, intranuclear inclusions, and cell detachments seen in herpesvirus infection in vitro. Herpesvirus-like particles were observed in viral culture supernatant under the electron microscope, however, capsids ranging from 60?nm to 100?nm in size were often observed in viral cultures within the first two passages of propagation. Herpesvirus infection in the bear tissues and tissue cultures were detected by PCR using degenerate primers specific to the DNA polymerase gene (DPOL) and glycoprotein B gene (gB). DNA sequencing of the amplicon revealed that the detected herpesvirus has 94-95% identity to Ursid gammaherpesvirus 1 (UrHV-1) DNA sequences of DPOL. Phylogenetic analysis of DPOL sequences indicates that black bear herpesviruses and UrHV-1 are closely related and have small distances to members of Rhadinovirus. Interestingly, black bear herpesvirus infections were also found in bears without neurological signs. The DPOL DNA sequence of black bear herpesviruses detected in neurological bears were similar to the those detected in the non-neurological bears. However, the gB DNA sequence detected from the neurological bear is different from non-neurological bear and has only 64.5%-70% identity to each other. It is possible that at least two different types of gammaherpesviruses are present in the U. americanus population or several gammaherpesviruses exist in ursine species.
Project description:Rodent herpesviruses such as murine cytomegalovirus (host, Mus musculus), rat cytomegalovirus (host, Rattus norvegicus), and murine gammaherpesvirus 68 (hosts, Apodemus species) are important tools for the experimental study of human herpesvirus diseases. However, alphaherpesviruses, roseoloviruses, and lymphocryptoviruses, as well as rhadinoviruses, that naturally infect Mus musculus (house mouse) and other Old World mice are unknown. To identify hitherto-unknown rodent-associated herpesviruses, we captured M. musculus, R. norvegicus, and 14 other rodent species in several locations in Germany, the United Kingdom, and Thailand. Samples of trigeminal ganglia, dorsal root ganglia, brains, spleens, and other organs, as well as blood, were analyzed with a degenerate panherpesvirus PCR targeting the DNA polymerase (DPOL) gene. Herpesvirus-positive samples were subjected to a second degenerate PCR targeting the glycoprotein B (gB) gene. The sequences located between the partial DPOL and gB sequences were amplified by long-distance PCR and sequenced, resulting in a contiguous sequence of approximately 3.5 kbp. By DPOL PCR, we detected 17 novel betaherpesviruses and 21 novel gammaherpesviruses but no alphaherpesvirus. Of these 38 novel herpesviruses, 14 were successfully analyzed by the complete bigenic approach. Most importantly, the first gammaherpesvirus of Mus musculus was discovered (Mus musculus rhadinovirus 1 [MmusRHV1]). This virus is a member of a novel group of rodent gammaherpesviruses, which is clearly distinct from murine herpesvirus 68-like rodent gammaherpesviruses. Multigenic phylogenetic analysis, using an 8-kbp locus, revealed that MmusRHV1 diverged from the other gammaherpesviruses soon after the evolutionary separation of Epstein-Barr virus-like lymphocryptoviruses from human herpesvirus 8-like rhadinoviruses and alcelaphine herpesvirus 1-like macaviruses.
Project description:Novel members of the subfamily Gammaherpesvirinae, hosted by eight mammalian species from six orders (Primates, Artiodactyla, Perissodactyla, Carnivora, Scandentia, and Eulipotyphla), were discovered using PCR with pan-herpesvirus DNA polymerase (DPOL) gene primers and genus-specific glycoprotein B (gB) gene primers. The gB and DPOL sequences of each virus species were connected by long-distance PCR, and contiguous sequences of approximately 3.4 kbp were compiled. Six additional gammaherpesviruses from four mammalian host orders (Artiodactyla, Perissodactyla, Primates, and Proboscidea), for which only short DPOL sequences were known, were analyzed in the same manner. Together with available corresponding sequences for 31 other gammaherpesviruses, alignments of encoded amino acid sequences were made and used for phylogenetic analyses by maximum-likelihood and Bayesian Monte Carlo Markov chain methods to derive a tree which contained two major loci of unresolved branching details. The tree was rooted by parallel analyses that included alpha- and betaherpesvirus sequences. This gammaherpesvirus tree contains 11 major lineages and presents the widest view to date of phylogenetic relationships in any subfamily of the Herpesviridae, as well as the most complex in the number of deep lineages. The tree's branching pattern can be interpreted only in part in terms of the cospeciation of virus and host lineages, and a substantial incidence of the interspecies transfer of viruses must also be invoked.
Project description:In the past decade, a large number of studies have detected herpesvirus sequences from many bat species around the world. Nevertheless, the discovery of bat herpesviruses is geographically uneven. Of the various bat species tested to date, only a few were from the New World. Seeking to investigate the distribution and diversity of herpesviruses circulating in neotropical bats, we carried out molecular screening of 195 blood DNA samples from 11 species of three bat families (Phyllostomidae, Mormoopidae, and Molossidae). Using polymerase chain reaction amplification, with degenerate consensus primers targeting highly conserved amino acid motifs of the herpesvirus DNA polymerase and Glycoprotein B genes, we characterized novel viral sequences from all tested species. BLAST searches, pairwise nucleotide and amino acid sequence comparisons, as well as phylogenetic analyses confirmed that they all belonged to the Herpesviridae family, of the Beta- and Gammaherpesvirinae subfamilies. Fourteen partial DNA polymerase gene sequences, of which three beta- and 11 gamma-herpesviruses, were detected. A total of 12 partial Glycoprotein B gene sequences, all gamma-herpesviruses, were characterized. Every sequence was specific to a bat species and in some species (Desmodus rotundus, Carollia perspicillata, and Pteronotus rubiginosus) multiple viruses were found. Phylogenetic analyses of beta- and gammaherpesvirus sequences led to the identification of bat-specific clades. Those composed of sequences obtained from different bat species belonging to distinct subfamilies follow the taxonomy of bats. This study confirms the astonishing diversity of bat herpesviruses and broadens our knowledge of their host range. Nevertheless, it also emphasizes the fact that, to better appreciate the evolutionary history of these viruses, much remains to be done at various taxonomic levels.
Project description:The glycoprotein B (gB) and D (gD) genes from five ruminant alphaherpesviruses, bovine herpesvirus 1 (BHV-1), bovine herpesvirus 5 (BHV-5), caprine herpesvirus 1 (CapHV-1), cervine herpesvirus 1, and rangiferine herpesvirus 1, were partially sequenced. The nucleotide sequence alignments revealed a highly conserved gB gene, with homologies ranging between 87.2 and 99.6%, and a more variable gD gene, with homologies ranging between 71.3 and 98.9%. The phylogenetic analysis of the gB and gD nucleotide and deduced amino acid sequences revealed that BHV-5 is the most closely related virus to the BHV-1 subtype 1 and BHV-1 subtype 2 cluster and that CapHV-1 is the most distantly related virus. The phylogenetic data showed a close relationship of all the studied viruses with suid herpesvirus 1. On the basis of sequence data for the gB gene, a nested PCR combined with restriction enzyme analysis (REA) of the PCR products was developed for the simultaneous detection and identification of the viruses that were studied. Nested primers from highly conserved sequence stretches were selected in order to amplify a region of 294 bp in all five viruses, and a subsequent REA of the PCR products allowed specific identification. A mimic molecule that served as an internal standard of the amplification efficiency was constructed. The practical diagnostic applicability of the assay was evaluated with clinical samples consisting of semen and organ specimens from experimentally infected animals.
Project description:We have developed a new primer design strategy for PCR amplification of distantly related gene sequences based on consensus-degenerate hybrid oligonucleotide primers (CODEHOPs). An interactive program has been written to design CODEHOP PCR primers from conserved blocks of amino acids within multiply-aligned protein sequences. Each CODEHOP consists of a pool of related primers containing all possible nucleotide sequences encoding 3-4 highly conserved amino acids within a 3' degenerate core. A longer 5' non-degenerate clamp region contains the most probable nucleotide predicted for each flanking codon. CODEHOPs are used in PCR amplification to isolate distantly related sequences encoding the conserved amino acid sequence. The primer design software and the CODEHOP PCR strategy have been utilized for the identification and characterization of new gene orthologs and paralogs in different plant, animal and bacterial species. In addition, this approach has been successful in identifying new pathogen species. The CODEHOP designer (http://blocks.fhcrc.org/codehop.html) is linked to BlockMaker and the Multiple Alignment Processor within the Blocks Database World Wide Web (http://blocks.fhcrc.org).
Project description:Gammaherpesvirus infections have been described in cervids worldwide, mainly the genera Macavirus or Rhadinovirus. However, little is known about the gammaherpesviruses species infecting cervids in Norway and Fennoscandia. Blood samples from semi-domesticated (n = 39) and wild (n = 35) Eurasian tundra reindeer (Rangifer tarandus tarandus), moose (Alces alces, n = 51), and red deer (Cervus elaphus, n = 41) were tested using a panherpesvirus DNA polymerase (DPOL) PCR. DPOL-PCR-positive samples were subsequently tested for the presence of glycoprotein B (gB) gene. The viral DPOL gene was amplified in 28.2% (11/39) of the semi-domesticated reindeer and in 48.6% (17/35) of the wild reindeer. All moose and red deer tested negative. Additionally, gB gene was amplified in 4 of 11 semi-domesticated and 15 of 17 wild Eurasian reindeer DPOL-PCR-positive samples. All the obtained DPOL and gB sequences were highly similar among them, and corresponded to a novel gammaherpesvirus species, tentatively named Rangiferine gammaherpesvirus 1, that seemed to belong to a genus different from Macavirus and Rhadinovirus. This is the first report of a likely host-specific gammaherpesvirus in semi-domesticated reindeer, an economic and cultural important animal, and in wild tundra reindeer, the lastpopulation in Europe. Future studies are required to clarify the potential impact of this gammaherpesvirus on reindeer health.
Project description:This study was conducted for the isolation and molecular characterization of bovine herpesvirus 1 (BoHV-1) isolated from the nasal and vaginal swabs collected from naturally infected cattle showing clinical symptoms of the respiratory disease.Isolation of BoHV-1 virus performed on clinical samples collected from 65 cattle from five states of India. The BoHV-1 isolates were further confirmed by polymerase chain reaction (PCR) using primers specific for glycoprotein B (gB) genomic region. PCR amplification was performed using previously published gB gene-specific primer pairs. gB PCR amplicons obtained from all isolates were sequenced, and phylogenetic analysis was performed using software.A total of 12 samples were found positive in cell culture isolation. 11 isolates showed the visible cytopathic effect on Madin-Darby bovine kidney after 72 h. Partial sequence analysis of gB gene of all isolates revealed 99.0-100% homology between them. All isolates showed 99.2-99.8% homology with Cooper stain.BoHV-1.1 is the predominant circulating subtype of BoHV in India, and all isolates have homology with Cooper stain.
Project description:Consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) have proven to be a powerful tool for the identification of novel genes. CODEHOPs are designed from highly-conserved regions of multiply-aligned protein sequences from members of a gene family and are used in PCR amplification to identify distantly-related genes. The CODEHOP approach has been used to identify novel pathogens by targeting amino acid motifs conserved in specific pathogen families. We initiated a program utilizing the CODEHOP approach to develop PCR-based assays targeting a variety of viral families that are pathogens in non-human primates. We have also developed and further improved a computer program and website to facilitate the design of CODEHOP PCR primers. Here, we detail the method for the development of pathogen-specific CODEHOP PCR assays using the papillomavirus family as a target. Papillomaviruses constitute a diverse virus family infecting a wide variety of mammalian species, including humans and non-human primates. We demonstrate that our pan-papillomavirus CODEHOP assay is broadly reactive with all major branches of the virus family and show its utility in identifying a novel non-human primate papillomavirus in cynomolgus macaques.