Macaque homologs of Kaposi's sarcoma-associated herpesvirus (KSHV) infect germinal center lymphoid cells, epithelial cells in skin and gastrointestinal tract and gonadal germ cells in naturally infected macaques.

We developed a set of rabbit antisera to characterize infections by the macaque RV2 rhadinovirus homologs of KSHV. We analyzed tissues from rhesus and pig-tailed macaques naturally infected with rhesus rhadinovirus (RRV) or Macaca nemestrina rhadinovirus 2 (MneRV2). Our study demonstrates that RV2 rhadinoviruses have a tropism for epithelial cells, lymphocytes and gonadal germ cells in vivo. We observed latent infections in both undifferentiated and differentiated epithelial cells with expression of the latency marker, LANA. Expression of the early (ORF59) and late (glycoprotein B) lytic markers were detected in highly differentiated cells in epithelial ducts in oral, renal, dermal and gastric mucosal tissue as well as differentiated germ cells in male and female gonads. Our data provides evidence that epithelial and germ cell differentiation in vivo induces rhadinovirus reactivation and suggests that infected epithelial and germ cells play a role in transmission and dissemination of RV2 rhadinovirus infections in vivo.

Complete genome sequence of Pig-tailed macaque rhadinovirus 2 and its evolutionary relationship with rhesus macaque rhadinovirus and human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus.

UNLABELLED: Two rhadinovirus lineages have been identified in Old World primates. The rhadinovirus 1 (RV1) lineage consists of human herpesvirus 8, Kaposi's sarcoma-associated herpesvirus (KSHV), and closely related rhadinoviruses of chimpanzees, gorillas, macaques and other Old World primates. The RV2 rhadinovirus lineage is distinct and consists of closely related viruses from the same Old World primate species. Rhesus macaque rhadinovirus (RRV) is the RV2 prototype, and two RRV isolates, 26-95 and 17577, were sequenced. We determined that the pig-tailed macaque RV2 rhadinovirus, MneRV2, is highly associated with lymphomas in macaques with simian AIDS. To further study the role of rhadinoviruses in the development of lymphoma, we sequenced the complete genome of MneRV2 and identified 87 protein coding genes and 17 candidate microRNAs (miRNAs). A strong genome colinearity and sequence homology were observed between MneRV2 and RRV26-95, although the open reading frame (ORF) encoding the KSHV ORFK15 homolog was disrupted in RRV26-95. Comparison with MneRV2 revealed several genomic anomalies in RRV17577 that were not present in other rhadinovirus genomes, including an N-terminal duplication in ORF4 and a recombinative exchange of more distantly related homologs of the ORF22/ORF47 interacting glycoprotein genes. The comparison with MneRV2 has revealed novel genes and important conservation of protein coding domains and transcription initiation, termination, and splicing signals, which have added to our knowledge of RV2 rhadinovirus genetics. Further comparisons with KSHV and other RV1 rhadinoviruses will provide important avenues for dissecting the biology, evolution, and pathology of these closely related tumor-inducing viruses in humans and other Old World primates. IMPORTANCE: This work provides the sequence characterization of MneRV2, the pig-tailed macaque homolog of rhesus rhadinovirus (RRV). MneRV2 and RRV belong to the rhadinovirus 2 (RV2) rhadinovirus lineage of Old World primates and are distinct but related to Kaposi's sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi's sarcoma. Pig-tailed macaques provide important models of human disease, and our previous studies have indicated that MneRV2 plays a causal role in AIDS-related lymphomas in macaques. Delineation of the MneRV2 sequence has allowed a detailed characterization of the genome structure, and evolutionary comparisons with RRV and KSHV have identified conserved promoters, splice junctions, and novel genes. This comparison provides insight into RV2 rhadinovirus biology and sets the groundwork for more intensive next-generation (Next-Gen) transcript and genetic analysis of this class of tumor-inducing herpesvirus. This study supports the use of MneRV2 in pig-tailed macaques as an important model for studying rhadinovirus biology, transmission and pathology.

High levels of retroperitoneal fibromatosis (RF)-associated herpesvirus in RF lesions in macaques are associated with ORF73 LANA expression in spindleoid tumour cells.

Two distinct lineages of rhadinoviruses related to Kaposi's sarcoma (KS)-associated herpesvirus (KSHV; Human herpesvirus 8), the causative agent of KS, have been identified. In macaques, the RV1 lineage is represented by retroperitoneal fibromatosis (RF) herpesvirus (RFHV), the homologue of KSHV, whilst the RV2 lineage is represented by rhesus rhadinovirus (RRV), a more distantly related virus. Real-time quantitative PCR was used to estimate the loads of RV1 and RV2 rhadinoviruses in simian acquired immunodeficiency syndrome-associated RF (SAIDS-RF), a neoplasm of macaques with similarities to AIDS-associated KS. Both RV1 and RV2 rhadinoviruses were detected in macaques with RF. The RV1 loads were 220- to 4300-fold higher in RF tumours than in spleen, showing a strong tumour association (mean loads of 1 800 000 vs 2900 copies per 10(6) cells in tumours and spleen, respectively). In contrast, RV2 loads in the RF tumours were 100-fold lower than RV1 loads and showed similar levels in tumours and spleen (mean loads of 16 000 vs 24 000 copies per 10(6) cells, respectively). Immunostaining with antibodies reactive against RFHV ORF73 latency-associated nuclear antigen (LANA) showed intense nuclear staining of the spindleoid RF tumour cells. Correlation of viral load and the number of LANA-positive cells indicated that RF tumour cells contained multiple copies of the RFHV genome per cell. This pattern of infectivity is similar to that seen in KS tumours latently infected with KSHV. Our study demonstrates similarities in the biology of KSHV and RFHV and supports a role for RFHV in the aetiology of SAIDS-RF.

RFHVMn ORF73 is structurally related to the KSHV ORF73 latency-associated nuclear antigen (LANA) and is expressed in retroperitoneal fibromatosis (RF) tumor cells.

Retroperitoneal fibromatosis herpesvirus (RFHV), the macaque homolog of the human rhadinovirus, Kaposi's sarcoma-associated herpesvirus (KSHV), was first identified in retroperitoneal fibromatosis (RF) tumor lesions of macaques with simian AIDS. We cloned and sequenced the ORF73 latency-associated nuclear antigen (LANA) of RFHVMn from the pig-tailed macaque. RFHVMn LANA is structurally analogous to KSHV ORF73 LANA and contains an N-terminal serine-proline-rich region, a large internal glutamic acidic-rich repeat region and a conserved C-terminal domain. RFHVMn LANA reacts with monoclonal antibodies specific for a glutamic acid-proline dipeptide motif and a glutamic acid-glutamine-rich motif in the KSHV LANA repeat region. Immunohistochemical and immunofluorescence analysis revealed that RFHVMn LANA is a nuclear antigen which is highly expressed in RF spindloid tumor cells. These data suggest that RFHV LANA is an ortholog of KSHV LANA and will function similarly to maintain viral latency and play a role in tumorigenicity in macaques.

Genetic variability of the envelope gene of Type D simian retrovirus-2 (SRV-2) subtypes associated with SAIDS-related retroperitoneal fibromatosis in different macaque species.

BACKGROUND: D-type simian retrovirus-2 (SRV-2) causes an AIDS-like immune deficiency syndrome (SAIDS) in various macaque species. SAIDS is often accompanied by retroperitoneal fibromatosis (RF), an aggressive fibroproliferative disorder reminiscent of Kaposi's sarcoma in patients with HIV-induced AIDS. In order to determine the association of SRV-2 subtypes with SAIDS-RF, and study the evolution and transmission of SRV-2 in captive macaque populations, we have molecularly characterized the env gene of a number of SRV-2 isolates from different macaque species with and without RF. RESULTS: We sequenced the env gene from eighteen SRV-2 isolates and performed sequence comparisons and phylogenetic analyses. Our studies revealed the presence of six distinct subtypes of SRV-2, three of which were associated with SAIDS-RF cases. We found no association between SRV-2 subtypes and a particular macaque species. Little sequence variation was detected in SRV-2 isolates from the same individual, even after many years of infection, or from macaques housed together or related by descent from a common infected parent. Seventy-two amino acid changes were identified, most occurring in the larger gp70 surface protein subunit. In contrast to the lentiviruses, none of the amino acid variations involved potential N-linked glycosylation sites. Structural analysis of a domain within the gp22/gp20 transmembrane subunit that was 100% conserved between SRV-2 subtypes, revealed strong similarities to a disulfide-bonded loop that is crucial for virus-cell fusion and is found in retroviruses and filoviruses. CONCLUSION: Our study suggests that separate introductions of at least six parental SRV-2 subtypes into the captive macaque populations in the U.S. have occurred with subsequent horizontal transfer between macaque species and primate centers. No specific association of a single SRV-2 subtype with SAIDS-RF was seen. The minimal genetic variability of the env gene within a subtype over time suggests that a strong degree of adaptation to its primate host has occurred during evolution of the virus.

A rotavirus strain isolated from pig-tailed macaque (Macaca nemestrina) with diarrhea bears a P6[1]:G8 specificity.

A distinct rotavirus strain (PTRV) was isolated in cell cultures from a stool sample obtained from a diarrheic 3-year-old female pig-tailed macaque (Macaca nemestrina) that was born at the breeding colony of the University of Washington in Seattle. Unlike other known simian rotavirus strains including vervet monkey rotavirus SA11 which bears P5B[2]:G3 or P6[1]:G3 specificity, rhesus monkey rotavirus MMU18006 with P5B[3]:G3 specificity, pig-tailed macaque rotavirus YK-1 with P[3]:G3 specificity and rhesus monkey rotavirus TUCH with P[24]:G3 specificity, the cell-culture-grown PTRV strain was shown to bear P6[1]:G8 specificity as determined by VP4 (P)- and VP7 (G)-specific neutralization assays as well as gene sequence analyses. The virus in the original diarrhea stool was also shown to bear genotypes P[1] and G8. In addition, the PTRV strain exhibited a "long" electropherotype, subgroup I specificity and NSP4 genotype A specificity. The PTRV probe formed (i) 8-9 hybrid bands with genomic RNAs of various bovine rotavirus strains and (ii) only 2-3 hybrid bands with simian rotavirus RNAs as demonstrated by RNA-RNA hybridization, suggesting a possible bovine origin of the virus. Serologic analysis of serum samples obtained from selected pig-tailed macaques in the colony suggested that a rotavirus bearing P[1]:G8 specificity was endemic among macaques for at least 8 years (1987-1994). This is the first report describing an isolation of a simian rotavirus bearing a non-G3 VP7 and possibly a P6[1] specificities. Because of its unique simian serotype, this virus may prove to be valuable in challenge studies in a non-human primate model in studies of rotavirus immunity.

Development of a real-time QPCR assay for the detection of RV2 lineage-specific rhadinoviruses in macaques and baboons.

BACKGROUND: Two distinct lineages of rhadinoviruses related to Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) have been identified in macaques and other Old World non-human primates. We have developed a real-time quantitative PCR (QPCR) assay using a TaqMan probe to differentially detect and quantitate members of the rhadinovirus-2 (RV2) lineage. PCR primers were derived from sequences within ORF 60 and the adjacent ORF 59/60 intergenic region which were highly conserved between the macaque RV2 rhadinoviruses, rhesus rhadinovirus (RRV) and Macaca nemestrina rhadinovirus-2 (MneRV2). These primers showed little similarity to the corresponding sequences of the macaque RV1 rhadinoviruses, retroperitoneal fibromatosis herpesvirus Macaca nemestrina (RFHVMn) and Macaca mulatta (RFHVMm). To determine viral loads per cell, an additional TaqMan QPCR assay was developed to detect the single copy cellular oncostatin M gene. RESULTS: We show that the RV2 QPCR assay is linear from less than 2 to more than 300,000 copies using MneRV2 DNA, and is non-reactive with RFHVMn DNA up to 1 billion DNA templates per reaction. RV2 loads ranging from 6 to 2,300 viral genome equivalent copies per 10(6) cells were detected in PBMC from randomly sampled macaques from the Washington National Primate Research Center. Screening tissue from other primate species, including another macaque, Macaca fascicularis, and a baboon, Papio cynocephalus, revealed the presence of novel rhadinoviruses, MfaRV2 and PcyRV2, respectively. Sequence comparison and phylogenetic analysis confirmed their inclusion within the RV2 lineage of KSHV-like rhadinoviruses. CONCLUSIONS: We describe a QPCR assay which provides a quick and sensitive method for quantitating rhadinoviruses belonging to the RV2 lineage of KSHV-like rhadinoviruses found in a variety of macaque species commonly used for biomedical research. While this assay broadly detects different RV2 rhadinovirus species, it is unreactive with RV1 rhadinovirus species which commonly co-infect the same primate hosts. We also show that this QPCR assay can be used to identify novel RV2 rhadinoviruses in different primate species.