Leukemic histiocytic sarcoma in a captive common squirrel monkey (Saimiri sciureus) with Saimiriine Gammaherpesvirus 2 (Rhadinovirus), Saimiri sciureus lymphocryptovirus 2 (Lymphocryptovirus) and Squirrel monkey retrovirus (ß-Retrovirus) coinfection.

Hematopoietic neoplasia other than lymphoma and leukemia is uncommon among non-human primates. Herein, we provide the first evidence of occurrence of leukemic histiocytic sarcoma in a captive common squirrel monkey with Saimiriine Gammaherpesvirus 2 (Rhadinovirus), Saimiri sciureus lymphocryptovirus 2 (Lymphocryptovirus), and Squirrel monkey retrovirus (ß-Retrovirus) coinfection.

Kaposi Sarcoma in Mantled Guereza.

We identified a novel Kaposi's sarcoma herpesvirus-related rhadinovirus (Colobine gammaherpesvirus 1) in a mantled guereza (Colobus guereza kikuyensis). The animal had multiple oral tumors characterized by proliferation of latent nuclear antigen 1-positive spindle cells and was not co-infected with immunosuppressive simian viruses, suggesting that it had Kaposi sarcoma caused by this novel rhadinovirus.

Detection of Equine Herpesvirus (EHV) -1, -2, -4 and -5 in Ethiopian Equids with and without Respiratory Problems and Genetic Characterization of EHV-2 and EHV-5 Strains.

Infections with equine herpesviruses (EHVs) are widespread in equine populations worldwide. Whereas both EHV-1 and EHV-4 produce well-documented respiratory syndromes in equids, the contribution of EHV-2 and EHV-5 to disease of the respiratory tract is still enigmatic. This study describes the detection and genetic characterization of EHVs from equids with and without clinical respiratory disease. Virus-specific PCRs were used to detect EHV-1, -2, -4 and -5. From the total of 160 equids with respiratory disease, EHV-5 was detected at the highest prevalence (23.1%), followed by EHV-2 (20.0%), EHV-4 (8.1%) and EHV-1 (7.5%). Concurrent infections with EHV-2 and EHV-5 were recorded from nine (5.2%) diseased horses. Of the total of 111 clinically healthy equids, EHV-1 and EHV-4 were never detected whereas EHV-2 and EHV-5 were found in 8 (7.2%) and 18 (16.2%) horses, respectively. A significantly higher proportion of EHV-2-infected equids was observed in the respiratory disease group (32/160, 20.0%; P = 0.005) compared to those without disease (8/111; 7.2%). EHV-2-positive equids were three times more likely to display clinical signs of respiratory disease than EHV-2-negative equids (OR 3.22, 95% CI: 1.42-7.28). For EHV-5, the observed difference was not statistically significant (P = 0.166). The phylogenetic analysis of the gB gene revealed that the Ethiopian EHV-2 and EHV-5 strains had a remarkable genetic diversity, with a nucleotide sequence identity among each other that ranged from 94.0 to 99.4% and 95.1 to 100%, respectively. Moreover, the nucleotide sequence identity of EHV-2 and EHV-5 with isolates from other countries acquired from GenBank ranged from 92.9 to 99.1% and 95.1 to 99.5%, respectively. Our results suggest that besides EHV-1 and EHV-4, EHV-2 is likely to be an important contributor either to induce or predispose equids to respiratory disease. However, more work is needed to better understand the contribution of EHV-2 in the establishment of respiratory disease.

Development and Validation of a Quantitative PCR Method for Equid Herpesvirus-2 Diagnostics in Respiratory Fluids.

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 norm. After the development and first validation step, two distinct characterization steps were performed according to the AFNOR norm: (a) characterization of the qRT-PCR assay alone and (b) characterization of the whole analytical method. The validation of the whole analytical method included the portrayal of all steps between the extraction of nucleic acids and the final PCR analysis. Validation of the whole method is very important for virus detection by qRT-PCR in order to get an accurate determination of the viral genome load. Since the extraction step is the primary source of loss of biological material, it may be considered the main source of error of quantification between one protocol and another. For this reason, the AFNOR norm NF-U-47-600 recommends including the range of plasmid dilution before the extraction step. In addition, the limits of quantification depend on the source from which the virus is extracted. Viral genome load results, which are expressed in international units (IU), are easier to use in order to compare results between different laboratories. This new method of characterization of qRT-PCR should facilitate the harmonization of data presentation and interpretation between laboratories.

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.

Genetic diversity of equine gammaherpesviruses (γ-EHV) and isolation of a syncytium forming EHV-2 strain from a horse in Iceland.

The horse population in Iceland is a special breed, isolated from other equines for at least one thousand years. This provides an exceptional opportunity to investigate old and new pathogens in a genetically closed herd. Both types of equine gammaherpesviruses, EHV-2 and EHV-5, are common in Iceland. Genetic variation was examined by sequencing four genes, glycoprotein B (gB), glycoprotein H (gH), DNA polymerase and DNA terminase for 12 Icelandic and seven foreign EHV-2 strains. One Icelandic virus isolate, gEHV-Dv, induced syncytium formation, an uncharacteristic cytopathy for EHV-2 in equine kidney cells. When sequenced, the glycoprotein genes were different from both EHV-2 and EHV-5, but the polymerase and terminase genes had 98-99% identity to EHV-2. Therefore the gEHV-Dv strain can be considered a variant of EHV-2. Substantial genetic variability was seen within the EHV-2 glycoprotein genes but limited in the polymerase and terminase genes. The Icelandic EHV-2 strains do not seem to differ phylogenetically from the foreign viruses, despite isolation for over a thousand years.

HybProbes-based real-time PCR assay for rapid detection of equine herpesvirus type 2 DNA.

Equid herpesvirus type 2 (EHV-2) together with equid herpesvirus type 5 are members of Gammaherpesvirinae subfamily, genus Rhadinovirus. EHV-2 is one of major agents causing diseases of horses common worldwide. A possible role of EHV-2 in reactivating latent equid herpesvirus type-1 has been suggested, because reactivation of latent EHV-1 was always accompanied by EHV-2 replication. Variety techniques, including cell culture, PCR and its modifications, have been used to diagnose EHV-2 infections. The aim of this study was to develop, optimize and determine specificity of real-time PCR (qPCR) for EHV-2 DNA detection using HybProbes chemistry and to evaluate clinical samples with this method. The analytical sensitivity of assay was tested using serial dilutions of viral DNA in range between 70 and 7x10(5) copies/ml. The limit of detection (LOD) was calculated using probit analysis and was determined as 56 copies/ml. In further studies 20 different clinical samples were tested for the presence of EHV-2. Described in-house qPCR method detected viral DNA in 5 of 20 specimens used. The results of this work show that developed HybProbes-based real-time PCR assay is very reliable and valuable for detection and quantification of equid herpesvirus type 2 DNA in different clinical samples. The high level of sensitivity, accuracy and rapidity provided by the LightCycler 2.0 instrument are favorable for the use of this system in the detection of EHV-2 DNA in veterinary

Macaque homologs of EBV and KSHV show uniquely different associations with simian AIDS-related lymphomas.

Two gammaherpesviruses, Epstein-Barr virus (EBV) (Lymphocryptovirus genus) and Kaposi's sarcoma-associated herpesvirus (KSHV) (Rhadinovirus genus) have been implicated in the etiology of AIDS-associated lymphomas. Homologs of these viruses have been identified in macaques and other non-human primates. In order to assess the association of these viruses with non-human primate disease, archived lymphoma samples were screened for the presence of macaque lymphocryptovirus (LCV) homologs of EBV, and macaque rhadinoviruses belonging to the RV1 lineage of KSHV homologs or the more distant RV2 lineage of Old World primate rhadinoviruses. Viral loads were determined by QPCR and infected cells were identified by immunolabeling for different viral proteins. The lymphomas segregated into three groups. The first group (n = 6) was associated with SIV/SHIV infections, contained high levels of LCV (1-25 genomes/cell) and expressed the B-cell antigens CD20 or BLA.36. A strong EBNA-2 signal was detected in the nuclei of the neoplastic cells in one of the LCV-high lymphomas, indicative of a type III latency stage. None of the lymphomas in this group stained for the LCV viral capsid antigen (VCA) lytic marker. The second group (n = 5) was associated with D-type simian retrovirus-2 (SRV-2) infections, contained high levels of RV2 rhadinovirus (9-790 genomes/cell) and expressed the CD3 T-cell marker. The third group (n = 3) was associated with SIV/SHIV infections, contained high levels of RV2 rhadinovirus (2-260 genomes/cell) and was negative for both CD20 and CD3. In both the CD3-positive and CD3/CD20-negative lymphomas, the neoplastic cells stained strongly for markers of RV2 lytic replication. None of the lymphomas had detectable levels of retroperitoneal fibromatosis herpesvirus (RFHV), the macaque RV1 homolog of KSHV. Our data suggest etiological roles for both lymphocryptoviruses and RV2 rhadinoviruses in the development of simian AIDS-associated lymphomas and indicate that the virus-infected neoplastic lymphoid cells are derived from different lymphocyte lineages and differentiation stages.

Experimental infection of laboratory-bred bank voles (Myodes glareolus) with murid herpesvirus 4.

MuHV-4 is a natural pathogen of rodents of the genus Apodemus (e.g., wood mice, yellow-necked mice) and Myodes glareolus (bank voles). We report experimental MuHV-4 infection of bank voles in comparison with infection of A. sylvaticus (wood mice) and BALB/c mice. Like in wood mice, the level of productive replication in the lungs of bank voles was significantly lower than in BALB/c mice. In contrast to other hosts, however, the level of latent infection in the lung and spleen of bank voles was extremely low. These findings, together with those of previous studies, suggest that bank voles are an occasional and inefficient host for MuHV-4.

Longitudinal patterns of viremia and oral shedding of rhesus rhadinovirus and retroperitoneal fibromatosis herpesviruses in age-structured captive breeding populations of rhesus Macaques (Macaca mulatta).

Rhesus rhadinovirus (RRV) and retroperitoneal fibromatosis herpesvirus (RFHV), 2 closely related γ2 herpesviruses, are endemic in breeding populations of rhesus macaques at our institution. We previously reported significantly different prevalence levels, suggesting the transmission dynamics of RRV and RFHV differ with regard to viral shedding and infectivity. We designed a longitudinal study to further examine the previously observed differences between RRV and RFHV prevalence and the potential influence of age, season, and housing location on the same 90 rhesus macaques previously studied. Virus- and host-genome-specific real-time PCR assays were used to determine viral loads for both RRV and RFHV in blood and saliva samples collected at 6 time points over an 18-mo period. Proportions of positive animals and viral load in blood and saliva were compared between and within viruses by age group, location, and season by using 2-part longitudinal modeling with Bayesian inferences. Our results demonstrate that age and season are significant determinants, with age as the most significant factor analyzed, of viremia and oral shedding for both RRV and RFHV, and these pathogens exhibit distinctly different patterns of viremia and oral shedding over time within a single population.

Isolation and partial sequencing of Equid herpesvirus 5 from a horse in Iceland.

Horses are hosts to 2 types of gammaherpesviruses, Equid herpesvirus 2 and 5 (EHV-2 and EHV-5, respectively). Both EHV-2 and EHV-5 are common in horses in Iceland. An Icelandic EHV-5 isolate was recovered by sequential culture in primary fetal horse kidney and rabbit kidney cells. Glycoprotein B, glycoprotein H, and DNA terminase genes of the isolate were fully sequenced, and the DNA polymerase gene was partly sequenced. To date, the glycoprotein B gene of EHV-5 was the only gene that has been reported to be completely sequenced in addition to small parts of the glycoprotein H, DNA polymerase, and DNA terminase genes. The present report, therefore, is a significant addition to previously reported EHV-5 sequences.

Glycoprotein gene sequence variation in rhesus monkey rhadinovirus.

Gene sequences for seven glycoproteins from 20 independent isolates of rhesus monkey rhadinovirus (RRV) and of the corresponding seven glycoprotein genes from nine strains of the Kaposi's sarcoma-associated herpesvirus (KSHV) were obtained and analyzed. Phylogenetic analysis revealed two discrete groupings of RRV gH sequences, two discrete groupings of RRV gL sequences and two discrete groupings of RRV gB sequences. We called these phylogenetic groupings gH(a), gH(b), gL(a), gL(b), gB(a) and gB(b). gH(a) was always paired with gL(a) and gH(b) was always paired with gL(b) for any individual RRV isolate. Since gH and gL are known to be interacting partners, these results suggest the need of matching sequence types for function of these cooperating proteins. gB phylogenetic grouping was not associated with gH/gL phylogenetic grouping. Our results demonstrate two distinct, distantly-related phylogenetic groupings of gH and gL of RRV despite a remarkable degree of sequence conservation within each individual phylogenetic group.

Characterization of cytoplasmic motifs important in rhesus rhadinovirus gB processing and trafficking.

Rhesus monkey rhadinovirus (RRV) is highly related to Kaposi's sarcoma-associated herpesvirus (KSHV), a human gamma-herpesvirus etiologically-linked with several cancers. Glycoprotein B (gB) homologues are encoded by all herpesviruses and play a role in virus attachment, entry, and in egress. We have found that RRV gB, like KSHV gB, is cleaved at a consensus furin cleavage site and is modified by both N-linked and O-linked glycosylation. Mutagenesis of three tyrosine- based trafficking motifs, a diacidic tyrosine motif, and a di-lucine motif in the cytoplasmic region revealed a role for these sequences in both ER export and endocytosis from the plasma membrane. These experiments provide a basis for further experiments looking at gB incorporation and role in gamma-herpesvirus assembly and egress.

Prevalence of viremia and oral shedding of rhesus rhadinovirus and retroperitoneal fibromatosis herpesvirus in large age-structured breeding groups of rhesus macaques (Macaca mulatta).

We performed a cross-sectional study to estimate the prevalence of 2 gamma-2-herpesviruses, rhesus rhadinovirus (RRV) and retroperitoneal fibromatosis herpesvirus (RFHV), in breeding colonies of rhesus macaques. Of 90 animals selected for sampling, 73 (81%) were positive for RRV, which was detected only in blood in 22 (24%), only in saliva in 15 (16%), and in both blood and saliva in 36 (40%). Detection of RRV DNA in blood and saliva was significantly higher in animals younger than 2 y. In comparison, RFHV was detected in 40 (44%) of the 90 animals: only in blood in 5 (6%), only in saliva in 26 (29%), and in both blood and saliva in 9 (10%). Dual infection was detected in 38 (42%) animals; RFHV was only detected in coinfections. The mean RRV genome copy number in blood was significantly higher than that for RFHV. Age was a significant predictor of RRV copy number in blood and RFHV copy number in saliva. Of the 90 animals, 88 (98%) were positive for rhadinoviral antibodies on an immunofluorescent assay. Both RRV and RFHV are highly endemic in socially housed breeding colonies of rhesus macaques, and their patterns of infection are similar to that for the betaherpesvirus rhesus cytomegalovirus.

Lack of detectable equine herpesviruses 1 and 2 in paraffin-embedded specimens of equine sarcoidosis.

BACKGROUND: Equine sarcoidosis is a rare, multisystemic, noncaseating, granulomatous and lymphoplasmacytic disease of unknown etiology. A recent report described a horse with granulomatous skin disease displaying histologic, electron microscopic, and polymerase chain reaction (PCR) findings consistent with equine herpesvirus 2 (EHV-2). OBJECTIVE: To investigate the presence of EHV-2 and equine herpesvirus 1 (EHV-1) in 8 horses with sarcoidosis. ANIMALS: Eight horses with sarcoidosis, reported previously. METHODS: Retrospective study. PCR assays of the tissues were performed to detect DNA associated with EHV-1 and EHV-2. For both herpesviruses the target was their respective glycoprotein B gene. Positive controls consisted of DNA from viral cultures of culturettes from naturally occurring respiratory infections of EHV-1 and EHV-2. RESULTS: The PCR analyses for both equine herpesviruses' DNA were negative in all 8 horses. CONCLUSION: The failure to detect DNA from EHV-1 and EHV-2 in paraffin-embedded skin of these 8 horses does not discount EHV-1 or EHV-2 as causing some cases of ES, but lends support to the presumably multifactorial etiologic nature of the disease.

Equid herpesvirus 2-associated oral and esophageal ulceration in a foal.

A case of a 1-month-old Thoroughbred foal with dysphagia, salivation, pyrexia, oral mucosal pustules, and esophageal ulceration is reported. Swabs from the ulcerated lesions yielded Equid herpesvirus 2 (EHV-2) in virus isolation assays, and histopathology of a biopsy from the esophageal lesion identified nuclear inclusions suggestive of herpesviruses. Immunohistochemical staining with antibodies specific for EHV-2 was positive for epithelial cells in the vicinity of the ulcer but not in more distant mucosa. Electron microscopic evaluation of the biopsy showed herpesviral particles in epithelial cells. The foal recovered over 5 days of supportive and gastroprotective therapy, and the esophageal ulcers healed. Serology and immunohistochemistry indicated that this foal likely had lesions associated with EHV-2 and not EHV-1, -4, or -5.

Study of equid herpesviruses 2 and 5 in Iceland with a type-specific polymerase chain reaction.

The horse population in Iceland is a special breed, isolated from other horses for at least 1000 years. This provides an exceptional opportunity to investigate old and new pathogens in an inbred herd with few infectious diseases. We have developed a high sensitivity semi-nested PCR to study equid gammaherpesviruses 2 and 5 (EHV-2 and 5) in Iceland. The first PCR is group specific, the second type-specific, targeting a 113bp sequence in the glyB gene. DNA isolated from white blood cells and 18 different organs was tested for the presence of EHV-2 and 5. This was done in adult horses and foals, healthy and with various enteric infections. Both virus types were easily detected in all types of organs tested or EHV-2 in 79% cases and EHV-5 in 63%. In DNA from PBMC or buffy-coat EHV-2 was found in 20% cases and EHV-5 in 10%, all except one positive were foals. Co-culture of PBMC on fetal horse kidney cells was efficient for detecting EHV-2 but not for EHV-5. We verify here for the first time infections with EHV-2 and 5 in horses in Iceland and show that both viruses are common.

Sequence analysis of the regions flanking terminal repeats of the genome of umava isolate of murine gammaherpesvirus.

The umava isolate of murine gammaherpesvirus (MHV-umava) slightly differs from Murine gammaherpesvirus 68 (MHV-68) and two other isolates of murine gammaherpesvirus (MHV), MHV-76 and MHV-72 in some biological properties. To identify the region(s) in the MHV-umava genome responsible for this phenomenon, we compared the sequences flanking terminal repeats (TRs) of the MHV-umava genome with those of MHV-68, MHV-76 and MHV-72. Restriction and sequence analyses revealed in MHV-umava as compared to MHV-68 approximately 9.3 kbp deletion at the left end of the genome and approximately 1.5 kbp deletion at the right end of the genome. While the approximately 9.3 kbp deletion was similar to that in MHV-76, the approximately 1.5 kbp deletion was unique for MHV-umava.