Simian homologues of human gamma-2 and betaherpesviruses in mandrill and drill monkeys

Recent serological and molecular surveys of different primate species allowed the characterization of several Kaposi's sarcoma-associated herpesvirus (KSHV) homologues in macaques, African green monkeys, chimpanzees, and gorillas. Identification of these new primate rhadinoviruses revealed the existence of two distinct genogroups, called RV1 and RV2. Using a degenerate consensus primer PCR method for the herpesvirus DNA polymerase gene, the presence of KSHV homologues has been investigated in two semi-free-ranging colonies of eight drill (Mandrillus leucophaeus), five mandrill (Mandrillus sphinx), and two hybrid (Mandrillus leucophaeus-Mandrillus sphinx) monkeys, living in Cameroon and Gabon, Central Africa. This search revealed the existence of not only two distinct KSHV homologues, each one belonging to one of the two rhadinovirus genogroups, but also of two new betaherpesvirus sequences, one being close to cytomegaloviruses and the other being related to human herpesviruses 6 and 7 (HHV-6 and -7). The latter viruses are the first simian HHV-6 and -7 homologues identified to date. These data show that mandrill and drill monkeys are the hosts of at least four novel distinct herpesviruses. Moreover, mandrills, like macaques and African green monkeys, harbor also two distinct gamma-2 herpesviruses, thus strongly suggesting that a second gamma-2 herpesvirus, belonging to the RV2 genogroup, may exist in humans.     

Novel simian homologues of Epstein-Barr virus

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.     

Identification of novel rodent herpesviruses, including the first gammaherpesvirus of Mus musculus

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.     

Primate herpesviral oncogenes.

Gammaherpesviruses are the most rapidly growing members of the herpesviridae family. Gamma herpesviruses share similarity in their genome organizations and in early and late lytic genes that are required for viral replication. A distinct characteristic of gamma herpesviruses is their ability to establish latent infection in lymphoid cells, and some of these viruses are closely associated with abnormal proliferation and cancer in primates. The first open reading frame of the primate gamma herpesviruses has been shown to directly contribute to virus-associated pathogenesis. This open reading frame encodes latent membrane protein-1 (LMP1) in Epstein-Barr virus, Saimiri transformation protein (STP) in Herpesvirus Saimiri, K1 in Kaposi's sarcoma-associated herpesvirus, and R1 in Rhesus monkey Rhadinovirus. All of these gene products are capable of eliciting cellular signal transduction events, resulting in cell growth transformation. This review briefly summarizes the current view on the transforming mechanisms utilized by primate herpesviral oncogenes.     

Prevalence and molecular characterization of the polymerase gene of gibbon lymphocryptovirus

BACKGROUND: Lymphocryptovirus (LCV) is found in various non-human primates. As a herpesvirus naturally infecting gibbons it is closely related to human Epstein-Barr virus (EBV) with which it shares considerable genetic, biological and epidemiologic features. METHODS: We collected blood samples from 70 gibbons (51 Hylobates lar, 18 Hylobates pileatus and 1 Hylobates agilis) for further separation into serum and peripheral blood mononuclear cells (PBMC). RESULTS: Only 13 of 70 (18.6%) sera were serologically positive for human EBV IgG but 64 of 70 (91.4%) PBMCs yielded the partial LCV DNA polymerase gene by semi-nested PCR, which we subjected to direct sequencing. All sequences showed 84% nucleic acid and 91% amino acid identity to human EBV. Phylogenetic tree analysis demonstrated gibbon LCVs clustered separately from other gammaherpesvirinae but closely related to LCV of other species. CONCLUSIONS: Based on LCV DNA detection, we discovered a high prevalence of LCV infection among gibbons. Further characterization of non-human primate LCV might thus provide new insight into both evolution and pathogenicity of gammaherpesvirinae.     

Absence of Frequent Herpesvirus Transmission in a Nonhuman Primate Predator-Prey System in the Wild

Emergence of viruses into the human population by transmission from nonhuman primates (NHPs) represents a serious potential threat to human health that is primarily associated with the increased bushmeat trade. Transmission of RNA viruses across primate species appears to be relatively frequent. In contrast, DNA viruses appear to be largely host specific, suggesting low transmission potential. Herein, we use a primate predator-prey system to study the risk of herpesvirus transmission between different primate species in the wild. The system was comprised of western chimpanzees (Pan troglodytes verus) and their primary (western red colobus, Piliocolobus badius badius) and secondary (black-and-white colobus, Colobus polykomos) prey monkey species. NHP species were frequently observed to be coinfected with multiple beta- and gammaherpesviruses (including new cytomegalo- and rhadinoviruses). However, despite frequent exposure of chimpanzees to blood, organs, and bones of their herpesvirus-infected monkey prey, there was no evidence for cross-species herpesvirus transmission. These findings suggest that interspecies transmission of NHP beta- and gammaherpesviruses is, at most, a rare event in the wild.     

Identification of two homologs of the Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) in retroperitoneal fibromatosis of different macaque species.

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.     

Serological evidence of alpha herpesvirus infection in sooty mangabeys

Contact between sooty mangabeys (SMs) and a pigtailed macaque prompted the serological screening of SMs for evidence of infection with B virus. Serological tests detected SM antibodies that reacted with B virus polypeptides. Additional testing was performed with sera from SMs with no previous contact with macaques. Results from these tests indicated that 56% (33/59) of the SMs had antibodies that reacted with B virus and SA8. SM antibodies also reacted with herpesvirus papio 2 and to a lesser extent with human alpha herpesviruses (HSV-1 and HSV-2). There was an age-related increase in the presence of these antibodies in SMs that was consistent with the serological pattern of reactivity observed in other nonhuman primate species infected with alpha herpesviruses. These data suggest that SMs may be a host for a herpesvirus that is antigenically similar to those viruses present in other Old World nonhuman primates.     

Molecular detection of a virus, Pilchard herpesvirus, associated with epizootics in Australasian pilchards Sardinops sagax neopilchardus

In 1995, and again in 1998, high mortalities of pilchards Sardinops sagax neopilchardus were seen over their entire geographical range on the Australian coastline. A virus with typical herpesvirus morphology was identified as the causative agent, although the source of the virus remains unknown. At the time of the mortality events, the only available diagnostic test available for the detection of the virus was electron microscopy and hence, development of a rapid diagnostic test for detection and identification of the virus was required. Initial sequence data for Pilchard herpesvirus (PHV) was acquired by comparing the highly conserved region of the ORF 62 gene of other piscine herpesviruses (Ictalurid herpesvirus 1 [Channel catfish virus, CCV] and Salmonid herpesvirus 2 [Oncorhynchus masou virus, SaHV2, OMV]), and designing primers that successfully amplified a fragment of PHV. Here we describe the use of polymerase chain reaction (PCR) technology to detect PHV in infected tissues. Sequence analysis of amplified fragment resulting from this PCR is different from all known herpesviruses and can therefore distinguish PHV from all known viruses.     

Varicella-like herpesvirus infections of nonhuman primates

At least three distinct herpesviruses cause varicella like exanthematous diseases among nonhuman primates. Spontaneous epizootics have resulted in high morbidity and mortality rates among Cercopithecus aethiops, Erythrocebus patas and Macaca species in research colonies. Mild infections have been observed in infant chimpanzees and a gorilla. This group of diseases is of interest not only because they are a threat to primate colonies, but also because their pathologic similarity to progressive human varicella makes them a potentially valuable animal model of this disease. The nonhuman primate varicella-like exanthematous diseases are reviewed and compared to varicella in man.     

Genome organization of herpesvirus aotus type 2.

Herpesvirus aotus type 2, a virus commonly found in owl monkeys without overt disease, has a similar genome structure to the oncogenic herpesviruses of nonhuman primates (herpesvirus saimiri, herpesvirus ateles). Virion DNA of herpesvirus aotus type 2 (M-DNA) has an unique 110-kilobase-pair region of low G + C content (40.2%, L-DNA), inserted between stretches of repetitive H-DNA (68.7% G + C, about 41 kilobase pairs per molecule) that are variable in length. A minority of virions contain defective genomes that consist of repetitive H-DNA only. The H-DNA is composed of various types of repeat units that are related in sequence with each other. The two dominant types of repeats (2.3 and 2.7 kilobase pairs) were cloned and compared by restriction enzyme cleavages and partial nucleotide sequencing. They are homologous in at least 1.3 kilobase pairs. The two forms of repeat units are randomly arranged and oriented in tandem. Reassociation kinetics did not allow detection of sequence homologies between H- and L-DNA of herpesvirus aotus type 2 and the respective sequences of oncogenic primate herpesviruses.     

A novel gammaherpesvirus associated with genital lesions in a Blainville's beaked whale (Mesoplodon densirostris)

An adult male Blainville's beaked whale (Mesoplodon densirostris) was found stranded on the Atlantic coast of the USA on 28 January 2004. Necropsy revealed a focal papilloma-like penile lesion, the cells from which revealed single 4-6 microm basophilic intranuclear inclusions. Total DNA extracted from lesion material was tested using a pan-herpes-virus PCR assay that targets the DNA polymerase gene and found to be positive. When the amplified DNA fragment was cloned, sequenced, and compared to GenBank-deposited herpesvirus DNA polymerase sequences, the detected virus was determined to be a distinct member of the Gammaherpesvirinae subfamily of herpesviruses. This new virus, tentatively named Ziphiid herpesvirus type 1, was associated with but not determined to be the cause of genital disease in the Blainville's beaked whale.     

Lethal pneumonia in a captive juvenile chimpanzee (Pan troglodytes) due to human-transmitted human respiratory syncytial virus (HRSV) and infection with Streptococcus pneumoniae

Background  During an outbreak of respiratory disease in captive chimpanzees ( Pan troglodytes ), gorillas ( Gorilla gorilla ), Bornean orangutans ( Pongo pygmaeus ), and red-capped mangabeys ( Cercocebus torquatus ) also staff members showed non-specific upper respiratory signs. One infant female chimpanzee with severe respiratory symptoms died despite immediate medical treatment and was submitted for necropsy.
Methods  Routine post mortem, histological and bacteriological examinations were conducted. Additionally lung tissue samples form the chimpanzee and swab samples from the staff members and the other primates were examined by PCR.
Results  A severe catarrhal to purulent bronchopneumonia and an interstitial pneumonia were found and human respiratory syncytial virus (HRSV) as well as Streptococcus pneumoniae was detected in lung samples by PCR. Swab samples from one animal keeper revealed the same HRSV sequence as of the chimpanzee.
Conclusions  Therefore, it is suggested that the outbreak of respiratory disease within a zoological institution was due to transmission of HRSV between both human and primates.     

Evaluating phenotype and genotype of drug-resistant strains in herpesviruses

The isolation of drug-resistant strains of herpesviruses, including Herpes Simplex Virus type I (HSV-1) and type 2 (HSV-2), Varicella-Zoster Virus (VZV), and cytomegalovirus (CMV), has been reported with increasing frequency in immunocompromised patients and is a matter of major concern. Determination of antiviral drug susceptibilities is a prerequisite for the management of drug-resistant herpesvirus infections. Phenotyping studies should be correlated with genotyping, i.e., characterization of the mutations in the target genes. The isolation of drug-resistant virus in the laboratory and the determination of their phenotype and genotype may be useful to clarify the mechanisms of selective drug action. We describe here the procedures used for in vitro selection of drug-resistant herpesvirus mutants and the determination of their patterns of drug-susceptibility. The subcloning of the HSV-1 DNA polymerase gene is described as an example of the methodology followed to determine the mutation(s) in the drug-target viral gene that are associated with the resistant phenotype. To avoid the introduction of mutations by PCR amplification, all subcloning experiments were executed directly on viral DNA. Viral DNA was prepared from each plaque-purified viral strain and a 3.4 kb BamHI fragment containing 87% of the HSV-1 DNA polymerase gene coding region was purified and further digested with SacI; the two resulting fragments were subcloned into pU18 and propagated in Escherichia coli. Plasmid DNA was isolated and the inserts were sequenced using dideoxynucleotide chain termination method with T7 DNA polymerase and Taq DNA polymerase in an automated laser fluorescent DNA sequencer. pUC/M13 reverse, universal primers and oligonucleodite primers based on the wild-type virus sequence were used. The nucleotide sequences of the DNA polymerase genes of the different mutants was then compared with the nucleotide sequence of the wild-type HSV-1 KOS strain.     

Detection and phylogenetic characterization of a novel herpesvirus from the trachea of two stranded Common Loons (Gavia immer)

Two wild adult Common Loons (Gavia immer) were evaluated after being found stranded in mainland north-central Florida on separate occasions. On the basis of upper airway endoscopic and cytologic findings, we diagnosed severe ulcerative tracheitis antemortem in one of the birds while more subtle lesions were observed in the other. A novel herpesvirus was detected in antemortem tracheal samples using nested consensus PCR amplification of the polymerase gene and sequencing. Despite prolonged intensive medical care, the bird with severe lesions failed to improve and was euthanized 9 days after endoscopy. No viral inclusions were evident histologically in the lesions. However, an undulating tracheal mucosa in a "mountain ridge" pattern, resulting from epithelial regeneration and hyperplasia, was present, as is seen in the late stages of infectious laryngotracheitis in chickens. The second bird recovered and was released. The genetic distance between this and other characterized herpesviruses supports placement of this virus as a novel species, referred to as Gaviid herpesvirus 1 (GavHV1). Phylogenetically, GavHV1 clusters within the genus Iltovirus. The relationship between the observed lesions and the virus remains to be demonstrated.     

Feeding habits of chimpanzees (Pan troglodytes), red-tail monkeys (Cercopithecus ascanius schmidti) and blue monkeys (Cercopithecus mitis stuhlmanii) on figs in Budongo Forest Reserve, Uganda

Feeding habits of chimpanzees, red‐tail and blue monkeys on figs (Ficus) were studied in compartment N3 of Budongo Forest Reserve, western Uganda, from September 1997 to March 1998. The aim was to examine the spatial and temporal foraging habits of chimpanzees, red‐tail monkeys and blue monkeys on figs in the forest reserve. Both scan and focal sampling methods were used to assess the foraging habits of the primates. It was found that the primates fed on emerging leaves of Ficus mucuso Ficalho, F. varifolia Warb. and F. exasperata Vahl. They also preferred ripe fruits to emerging, young and unripe fruits. The primates spent 78% of the morning eating fruits and leaves and inhabited fig trees with fruits for about 4 h. Fig trees with ripe fruits attracted larger numbers of primate groups. It is concluded that information on the feeding habits of chimpanzees and monkeys is required in order to have a clear understanding of the social behaviour and pattern of movement of the primates and to assist in predicting the likely impacts of poor forest management, forest degradation and loss of food resources on their populations.     

Two Distinct Gamma-2 Herpesviruses in African Green Monkeys: a Second Gamma-2 Herpesvirus Lineage among Old World Primates?

Primate gamma-2 herpesviruses (rhadinoviruses) have so far been found in humans (Kaposi's sarcoma-associated herpesvirus [KSHV], also called human herpesvirus 8), macaques (Macaca spp.) (rhesus rhadinovirus [RRV] and retroperitoneal fibromatosis herpesvirus [RFHV]), squirrel monkeys (Saimiri sciureus) (herpesvirus saimiri), and spider monkeys (Ateles spp.) (herpesvirus ateles). Using serological screening and degenerate consensus primer PCR for the viral DNA polymerase gene, we have detected sequences from two distinct gamma-2 herpesviruses, termed Chlorocebus rhadinovirus 1 (ChRV1) and ChRV2, in African green monkeys. ChRV1 is more closely related to KSHV and RFHV, whereas ChRV2 is closest to RRV. Our findings suggest the existence of two distinct rhadinovirus lineages, represented by the KSHV/RFHV/ChRV1 group and the RRV/ChRV2 group, respectively, in at least two Old World monkey species. Antibodies to members of the RRV/ChRV2 lineage may cross-react in an immunofluorescence assay for early and late KSHV antigens.     

Nutritional Characteristics of Wild and Cultivated Foods for Chimpanzees (<Emphasis Type="Italic">Pan troglodytes</Emphasis>) in Agricultural Landscapes

Primate habitats are being transformed by human activities such as agriculture. Many wild primates include cultivated foods (crops) in their diets, calling for an improved understanding of the costs and benefits of crop feeding. We measured the macronutrient and antifeedant content of 44 wild and 21 crop foods eaten by chimpanzees (Pan troglodytes schweinfurthii) in a mosaic habitat at Bulindi, Uganda, to evaluate the common assertion that crops offer high nutritional returns compared to wild forage for primates. In addition, we analyzed 13 crops not eaten at Bulindi but that are consumed by chimpanzees elsewhere to assess whether nutritional aspects explain why chimpanzees in Bulindi ignored them. Our analysis of their wild plant diet (fruit, leaves, and pith) corresponds with previous chemical analyses of primate plant foods. Compared to wild food equivalents, crops eaten by the chimpanzees contained higher levels of digestible carbohydrates (mainly sugars) coupled with lower amounts of insoluble fiber and antifeedants. Cultivated fruits were relatively nutritious throughout the ripening process. Our data support the assumption that eating cultivated foods confers energetic advantages for primates, although crops in our sample were low in protein and lipids compared to some wild foods. We found little evidence that crops ignored by the chimpanzees were less nutritious than those that they did eat. Nonnutritional factors, e.g., similarity to wild foods, probably also influence crop selection. Whether cultivated habitats can support threatened but flexible primates such as chimpanzees in the long term hinges on local people’s willingness to share their landscape and resources with them.     

Microbial community analysis of rectal methanogens and sulfate reducing bacteria in two non-human primate species

Background  Methanogenesis by methanogenic Archaea and sulfate reduction by sulfate reducing bacteria (SRB) are the major hydrogenotrophic pathways in the human colon. Methanogenic status of mammals is suggested to be under evolutionary rather than dietary control. However, information is lacking regarding the dynamics of hydrogenotrophic microbial communities among different primate species.
Methods  Rectal swabs were collected from 10 sooty mangabeys ( Cercocebus atys ) and 10 baboons ( Papio hamadryas ). The diversity and abundance of methanogens and SRB were examined using PCR-denaturing gradient gel electrophoresis (DGGE) and real-time quantitative PCR (qPCR).
Results  The DGGE results revealed that intestinal Archaea and SRB communities differ between mangabeys and baboons. Phylogenetic analyses of Archaea DGGE bands revealed two distinct clusters with one representing a putative novel order of methanogenic Archaea. The qPCR detected a similar abundance of methanogens and SRB.
Conclusions  Intestinal Archaea and SRB coexist in these primates, and the community patterns are host species-specific.     

Persistent infectivity of a disease-associated herpesvirus in green turtles after exposure to seawater

Herpesviruses are associated with several diseases of marine turtles including lung-eye-trachea disease (LETD) and gray patch disease (GPD) of green turtles (Chelonia mydas) and fibropapillomatosis (FP) of green, loggerhead (Caretta caretta), and olive ridley turtles (Lepidochelys olivacea). The stability of chelonian herpesviruses in the marine environment, which may influence transmission, has not been previously studied. In these experiments, LETD-associated herpesvirus (LETV) was used as a model chelonian herpesvirus to test viral infectivity after exposure to seawater. The LETV virus preparations grown in terrapene heart (TH-1) cells were dialyzed for 24 to 120 hr against aerated artificial or natural seawater or Hank's balanced salt solution (HBBS). Fresh TH-1 cells were inoculated with dialyzed LETV, and on day 10 post-infection cells were scored for cytopathic effect. Virus samples dialyzed up to 120 hr were positive for the herpesvirus DNA polymerase gene by polymerase chain reaction. Electron microscopy revealed intact LETV nucleocapsids after exposure of LETV to artificial seawater or HBSS for 24 hr at 23 C. LETV preparations remained infectious as long as 120 hr in natural and artificial seawater at 23 C. Similar results were obtained with a second culturable chelonian herpesvirus, HV2245. LETV infectivity could not be detected after 48 hr exposure to artificial seawater at 30 C. Since LETV and HV2245 remain infectious for extended periods of time in the marine environment, it is possible that FP-associated and GPD-associated herpesviruses also may be stable. These findings are significant both for researchers studying the epidemiological association of herpesviruses with diseases of marine turtles and for individuals who handle turtles in marine turtle conservation efforts.