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Timothy M Rose - One of the best experts on this subject based on the ideXlab platform.
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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.
Virology, 2018Co-Authors: Helle Bielefeldt-ohmann, Margaret E Thouless, A. Gregory Bruce, Kellie Howard, Minako Ikoma, Timothy M RoseAbstract: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.
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qPCR Screen for RV1 and RV2 Rhadinovirus DNA in saliva.
2018Co-Authors: Gregory A Bruce, Helle Bielefeldt-ohmann, Kellie Howard, Minako Ikoma, Serge Barcy, Jeannette Staheli, Timothy M RoseAbstract:Saliva was obtained from macaques at the WaNPRC undergoing routine health screening and tested for the presence of RV1 and RV2 Rhadinovirus DNA. A) The highest RV1 and RV2 DNA levels detected during longitudinal screening. B) Correlation of RV1 and RV2 viral loads for the animals in panel A showing a positive level of DNA for either RV1 or RV2.
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Experimental Rhadinovirus infection in juvenile macaque M04203.
2018Co-Authors: Gregory A Bruce, Helle Bielefeldt-ohmann, Kellie Howard, Minako Ikoma, Serge Barcy, Jeannette Staheli, Timothy M RoseAbstract:The naïve juvenile pig-tailed macaque M04203 was inoculated intravenously (i.v.) with diluted filtered saliva from the 01019 cynomolgus macaque donor (Saliva “A”) containing high levels of RFHVMf and low levels of MfaLCV and was inoculated into the cheek pouches with whole saliva from pig-tailed macaque A02241 (Saliva “B”), which had high levels of RFHVMn, as indicated in the text. M04203 received a second iv inoculation of saliva from 01019 (Saliva “A”) 44 weeks after the initial infection. Subsequently, whole saliva from 01019 (Saliva “A”) was administered to the cheek pouch at week 52. To activate Rhadinovirus infections, M04203 was treated with daily injections of cyclosporine for 3 weeks (week 26–29) to induce immune suppression, as indicated (green bar). The animal was subsequently infected with SRV-2B and SIVMne, which have previously been associated with Rhadinovirus activation and associated pathologies (see text). Saliva and blood were collected weekly and/or bi-weekly. A) The RV1 genome levels were determined using the RV1 qPCR assay and the RV1 antibody levels were determined using the RV1 Luminex assay. B) the LCV genome levels were determined using the LCV qPCR assay and the LCV antibody levels were determined using the LCV Luminex assay. C) The SRV2 cDNA levels were determined using the SRV2 qPCR assay and the SRV2 antibody levels were determined using the SRV2 Luminex assay. D) The SIV cDNA levels were determined using the SIV qPCR assay and the SIV antibody levels were determined using the SIV Luminex assay.
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Experimental Rhadinovirus infection in juvenile macaque K04199.
2018Co-Authors: Gregory A Bruce, Helle Bielefeldt-ohmann, Kellie Howard, Minako Ikoma, Serge Barcy, Jeannette Staheli, Timothy M RoseAbstract:The naïve juvenile pig-tailed macaque K04199 was inoculated intravenously with diluted, filtered saliva from the 98039 cynomolgus macaque donor (Saliva C) containing high levels of the RV1 Rhadinovirus (RFHVMf), and was inoculated into the cheek pouches (cp) with whole saliva from the A02441 pig-tailed macaque (Saliva B) containing high levels of RFHVMn. Subsequently, K04199 was inoculated on week 12 with diluted, filtered saliva from the 01019 cynomolgus macaque donor (Saliva A) containing high levels of RFHVMf. Saliva and blood were collected weekly and/or bi-weekly. A) The RV1 genome levels were determined using the RV1 qPCR assay and the RV1 antibody levels were determined using the RV1 Luminex assay. B) the RV2 genome levels were determined using the RV2 qPCR assay and the RV2 antibody levels were determined using the RV2 Luminex assay. C) The SIV cDNA levels were determined using the SIV qPCR assay and the SIV antibody levels were determined using the SIV Luminex assay. D) The absolute number of cells in the lymphocyte subsets was determined, as indicated. K04199 developed clinical signs of SIV-induced AIDS and was terminated on week 23. MFI = mean fluorescent intensity.
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Timeline of experimental infections.
2018Co-Authors: Gregory A Bruce, Helle Bielefeldt-ohmann, Kellie Howard, Minako Ikoma, Serge Barcy, Jeannette Staheli, Timothy M RoseAbstract:The outlines of the experimental infections and treatment is shown for the naïve juvenile pig-tailed macaques, which were initially inoculated with saliva on the same day. A) K04199 was inoculated with a purified preparation of saliva from the cynomolgus macaque 98039 i.v. and whole saliva from the pig-tailed macaque A02241 orally into the cheek pouches. K04199 also received a subsequent inoculation of saliva from the cynomolgus macaque 01019 i.v. to try to boost the infection. By week 23, K04199 developed clinical signs and hematological changes meeting the end-point criteria for AIDS protocols, and euthanasia was performed. B) M04203 was inoculated with a purified preparation of saliva from the cynomolgus macaque 01019 i.v. and whole saliva from the pig-tailed macaque A02241 orally into the cheek pouches. M04203 received a second iv inoculation of saliva from 01019 44 weeks after the initial infection to try to boost the infection. Subsequently, whole saliva from 01019 was administered to the cheek pouch at week 52. To activate Rhadinovirus infections, M04203 was treated with daily injections of cyclosporine for 3 weeks (week 26–29) to induce immune suppression. The animal was subsequently infected with SRV-2B and SIVMne, which have previously been associated with Rhadinovirus activation and associated pathologies. The animal received a course of dexamethasone treatment to induce immunosuppression and viral reactivation, which tapered down as indicated. The animal was euthanized at week 209.
Scott W. Wong - One of the best experts on this subject based on the ideXlab platform.
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Japanese Macaque Rhadinovirus Encodes a Viral MicroRNA Mimic of the miR-17 Family.
Journal of virology, 2016Co-Authors: Rebecca L. Skalsky, Ryan D. Estep, Sarah A. Barr, Andrew J. Jeffery, Tiffany C. Blair, Scott W. WongAbstract:UNLABELLED Japanese macaque (JM) Rhadinovirus (JMRV) is a novel, gamma-2 herpesvirus that was recently isolated from JM with inflammatory demyelinating encephalomyelitis (JME). JME is a spontaneous and chronic disease with clinical characteristics and immunohistopathology comparable to those of multiple sclerosis in humans. Little is known about the molecular biology of JMRV. Here, we sought to identify and characterize the small RNAs expressed during lytic JMRV infection using deep sequencing. Fifteen novel viral microRNAs (miRNAs) were identified in JMRV-infected fibroblasts, all of which were readily detectable by 24 h postinfection and accumulated to high levels by 72 h. Sequence comparisons to human Kaposi's sarcoma-associated herpesvirus (KSHV) miRNAs revealed several viral miRNA homologs. To functionally characterize JMRV miRNAs, we screened for their effects on nuclear factor kappa B (NF-κB) signaling in the presence of two proinflammatory cytokines, tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β). Multiple JMRV miRNAs suppressed cytokine-induced NF-κB activation. One of these miRNAs, miR-J8, has seed sequence homology to members of the cellular miR-17/20/106 and miR-373 families, which are key players in cell cycle regulation as well as inflammation. Using reporters, we show that miR-J8 can target 3' untranslated regions (UTRs) with miR-17-5p or miR-20a cognate sites. Our studies implicate JMRV miRNAs in the suppression of innate antiviral immune responses, which is an emerging feature of many viral miRNAs. IMPORTANCE Gammaherpesviruses are associated with multiple diseases linked to immunosuppression and inflammation, including AIDS-related cancers and autoimmune diseases. JMRV is a recently identified herpesvirus that has been linked to JME, an inflammatory demyelinating disease in Japanese macaques that mimics multiple sclerosis. There are few large-animal models for gammaherpesvirus-associated pathogenesis. Here, we provide the first experimental evidence of JMRV miRNAs in vitro and demonstrate that one of these viral miRNAs can mimic the activity of the cellular miR-17/20/106 family. Our work provides unique insight into the roles of viral miRNAs during Rhadinovirus infection and provides an important step toward understanding viral miRNA function in a nonhuman primate model system.
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A Rhesus Rhadinovirus Viral Interferon (IFN) Regulatory Factor Is Virion Associated and Inhibits the Early IFN Antiviral Response
Journal of virology, 2015Co-Authors: Gabriela Morin, Kelsey S. Rogers, Bridget A. Robinson, Scott W. WongAbstract:The interferon (IFN) response is the earliest host immune response dedicated to combating viral infection. As such, viruses have evolved strategies to subvert this potent antiviral response. Two closely related gammaherpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) and rhesus macaque Rhadinovirus (RRV), are unique in that they express viral homologues to cellular interferon regulatory factors (IRFs), termed viral IRFs (vIRFs). Cellular IRFs are a family of transcription factors that are particularly important for the transcription of type I IFNs. Here, we demonstrate a strategy employed by RRV to ensure rapid inhibition of virus-induced type I IFN induction. We found that RRV vIRF R6, when expressed ectopically, interacts with a transcriptional coactivator, CREB-binding protein (CBP), in the nucleus. As a result, phosphorylated IRF3, an important transcriptional regulator in beta interferon (IFN-β) transcription, fails to effectively bind to the IFN-β promoter, thus inhibiting the activation of IFN-β genes. In addition, we found R6 within RRV virion particles via immunoelectron microscopy and, furthermore, that virion-associated R6 is capable of inhibiting the type I IFN response by preventing efficient binding of IRF3/CBP complexes to the IFN-β promoter in the context of infection. The work shown here is the first example of a vIRF being associated with either the KSHV or RRV virion. The presence of this immunomodulatory protein in the RRV virion provides the virus with an immediate mechanism to evade the host IFN response, thus enabling the virus to effectively establish an infection within the host. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) and the closely related rhesus macaque Rhadinovirus (RRV) are the only viruses known to encode viral homologues to cellular interferon regulatory factors (IRFs), known as vIRFs. In KSHV, these proteins have been shown to play major roles in a variety of cellular processes and are particularly important in the evasion of the host type I interferon (IFN) response. In this study, we delineate the immunomodulatory mechanism of an RRV vIRF and its ability to assist the virus in rapid immune evasion by being prepackaged within the virion, thus providing evidence, for the first time, of a virion-associated vIRF. This work further contributes to our understanding of the mechanisms behind immunomodulation by the RRV vIRFs during infection.
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Rhesus Macaque Rhadinovirus-Associated Disease
Current opinion in virology, 2013Co-Authors: Ryan D. Estep, Scott W. WongAbstract:Rhesus macaque Rhadinovirus (RRV) is a gamma-2 herpesvirus that naturally infects rhesus macaque (RM) monkeys and is closely related to human herpesvirus-8 (HHV-8)/Kaposi's sarcoma-associated herpesvirus (KSHV). Infection of immunodeficient RM induces disease in infected RM that resembles KSHV-associated pathologies. Importantly, RRV possesses homologues of KSHV ORFs that are postulated to play a role in disease development. As such, RRV has emerged as a prominent in vivo model system for examining mechanisms of infection and disease of these pathogenic herpesviruses, and has provided unique insight into how these viruses cause disease.
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Genomic Characterization of Japanese Macaque Rhadinovirus: A Novel Herpesvirus Isolated from a Nonhuman Primate with a Spontaneous Inflammatory Demyelinating Disease
Journal of virology, 2012Co-Authors: Ryan D. Estep, Scott G. Hansen, Kelsey S. Rogers, Michael K. Axthelm, Scott W. WongAbstract:Japanese macaque Rhadinovirus (JMRV) is a novel gamma-2 herpesvirus that was isolated from a Japanese macaque (JM) with an inflammatory demyelinating encephalomyelitis referred to as Japanese macaque encephalomyelitis, a disease that possesses clinical and histopathological features resembling multiple sclerosis in humans. Genomic DNA sequence analysis reveals that JMRV is a gammaherpesvirus closely related to rhesus macaque Rhadinovirus (RRV) and human herpesvirus 8. We describe here the complete nucleotide sequence and structure of the JMRV genome, as well as the sequence of two plaque isolates of this virus. Analysis of the JMRV genome not only demonstrates that this virus shares a number of genes with RRV that may be involved in pathogenesis but also indicates the presence of unique JMRV genes that could potentially contribute to disease development. The knowledge of the genomic sequence of JMRV, and the ability to easily propagate the virus in vitro, make JMRV infection of JM an attractive model for examining the potential role of an infectious viral agent in the development of demyelinating encephalomyelitis disease in vivo.
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Analysis of rhesus Rhadinovirus microRNAs expressed in virus-induced tumors from infected rhesus macaques
Virology, 2010Co-Authors: Jennifer L. Umbach, Scott W. Wong, Lisa Strelow, Bryan R CullenAbstract:Abstract Rhesus Rhadinovirus (RRV), a primate γ-herpesvirus related to human Kaposi's sarcoma-associated herpesvirus (KSHV), causes a similar pattern of pathogenesis. Previously, RRV was shown to express 7 pre-microRNAs (pre-miRNAs) in latently infected cells. Using deep sequencing, we analyzed the pattern of small RNA expression in vivo using latently RRV-infected B-cell lymphoma and retroperitoneal fibromatosis tissues. We identified 15 virally encoded pre-miRNAs in both tumors, including all previously reported RRV pre-miRNAs. Although all 15 RRV pre-miRNAs, like all 12 KSHV pre-miRNAs, are located 3′ to the conserved viral ORF71 gene and in the same transcriptional orientation, only one RRV miRNA is homologous to a KSHV miRNA. One previously identified RRV miRNA, miR-rR1-3, is actually a miRNA offset RNA (moRNA) derived from sequences located adjacent to pre-miR-rR1-3. Several other RRV-derived moRNAs were obtained, including one recovered > 600 times. Together, this research provides a comprehensive list of the miRNAs and moRNAs encoded by RRV.
A. Gregory Bruce - One of the best experts on this subject based on the ideXlab platform.
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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.
Virology, 2018Co-Authors: Helle Bielefeldt-ohmann, Margaret E Thouless, A. Gregory Bruce, Kellie Howard, Minako Ikoma, Timothy M RoseAbstract: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.
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Conservation of the glycoprotein B homologs of the Kaposi׳s sarcoma-associated herpesvirus (KSHV/HHV8) and old world primate Rhadinoviruses of chimpanzees and macaques
Virology, 2016Co-Authors: A. Gregory Bruce, Jeremy A. Horst, Timothy M RoseAbstract:Abstract The envelope-associated glycoprotein B (gB) is highly conserved within the Herpesviridae and plays a critical role in viral entry. We analyzed the evolutionary conservation of sequence and structural motifs within the Kaposi׳s sarcoma-associated herpesvirus (KSHV) gB and homologs of Old World primate Rhadinoviruses belonging to the distinct RV1 and RV2 Rhadinovirus lineages. In addition to gB homologs of Rhadinoviruses infecting the pig-tailed and rhesus macaques, we cloned and sequenced gB homologs of RV1 and RV2 Rhadinoviruses infecting chimpanzees. A structural model of the KSHV gB was determined, and functional motifs and sequence variants were mapped to the model structure. Conserved domains and motifs were identified, including an “RGD” motif that plays a critical role in KSHV binding and entry through the cellular integrin αVβ3. The RGD motif was only detected in RV1 Rhadinoviruses suggesting an important difference in cell tropism between the two Rhadinovirus lineages.
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Macaque Homologs of EBV and KSHV Show Uniquely Different Associations with Simian AIDS-related Lymphomas
PLoS pathogens, 2012Co-Authors: A. Gregory Bruce, Helle Bielefeldt-ohmann, Che Chung Tsai, Angela M. Bakke, Serge Barcy, Patrick Lewis, Robert D. Murnane, Timothy M RoseAbstract: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.
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The ORF59 DNA polymerase processivity factor homologs of Old World primate RV2 Rhadinoviruses are highly conserved nuclear antigens expressed in differentiated epithelium in infected macaques
Virology journal, 2009Co-Authors: A. Gregory Bruce, Helle Bielefeldt-ohmann, Angela M. Bakke, Courtney Gravett, Laura K. Demaster, Kellie Burnside, Timothy M RoseAbstract:Background: ORF59 DNA polymerase processivity factor of the human Rhadinovirus, Kaposi's sarcoma-associated herpesvirus (KSHV), is required for efficient copying of the genome during virus replication. KSHV ORF59 is antigenic in the infected host and is used as a marker for virus activation and replication. Results: We cloned, sequenced and expressed the genes encoding related ORF59 proteins from the RV1 Rhadinovirus homologs of KSHV from chimpanzee (PtrRV1) and three species of macaques (RFHVMm, RFHVMn and RFHVMf), and have compared them with ORF59 proteins obtained from members of the more distantly-related RV2 Rhadinovirus lineage infecting the same non-human primate species (PtrRV2, RRV, MneRV2, and MfaRV2, respectively). We found that ORF59 homologs of the RV1 and RV2 Old World primate Rhadinoviruses are highly conserved with distinct phylogenetic clustering of the two Rhadinovirus lineages. RV1 and RV2 ORF59 C-terminal domains exhibit a strong lineage-specific conservation. Rabbit antiserum was developed against a C-terminal polypeptide that is highly conserved between the macaque RV2 ORF59 sequences. This anti-serum showed strong reactivity towards ORF59 encoded by the macaque RV2 Rhadinoviruses, RRV (rhesus) and MneRV2 (pig-tail), with no cross reaction to human or macaque RV1 ORF59 proteins. Using this antiserum and RT-qPCR, we determined that RRV ORF59 is expressed early after permissive infection of both rhesus primary fetal fibroblasts and African green monkey kidney epithelial cells (Vero) in vitro. RRV- and MneRV2-infected foci showed strong nuclear expression of ORF59 that correlated with production of infectious progeny virus. Immunohistochemical studies of an MneRV2-infected macaque revealed strong nuclear expression of ORF59 in infected cells within the differentiating layer of epidermis corroborating previous observations that differentiated epithelial cells are permissive for replication of KSHV-like Rhadinoviruses.
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Development of a real-time QPCR assay for the detection of RV2 lineage-specific Rhadinoviruses in macaques and baboons
Virology journal, 2005Co-Authors: A. Gregory Bruce, Margaret E Thouless, Angela M. Bakke, Timothy M RoseAbstract: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.
Ronald C. Desrosiers - One of the best experts on this subject based on the ideXlab platform.
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Plxdc family members are novel receptors for the rhesus monkey Rhadinovirus (RRV).
PLoS pathogens, 2021Co-Authors: Anna K. Großkopf, Armin Ensser, Ronald C. Desrosiers, Sarah Schlagowski, Thomas Fricke, Alexander S. HahnAbstract:The rhesus monkey Rhadinovirus (RRV), a γ2-herpesvirus of rhesus macaques, shares many biological features with the human pathogenic Kaposi's sarcoma-associated herpesvirus (KSHV). Both viruses, as well as the more distantly related Epstein-Barr virus, engage cellular receptors from the Eph family of receptor tyrosine kinases (Ephs). However, the importance of the Eph interaction for RRV entry varies between cell types suggesting the existence of Eph-independent entry pathways. We therefore aimed to identify additional cellular receptors for RRV by affinity enrichment and mass spectrometry. We identified an additional receptor family, the Plexin domain containing proteins 1 and 2 (Plxdc1/2) that bind the RRV gH/gL glycoprotein complex. Preincubation of RRV with soluble Plxdc2 decoy receptor reduced infection by ~60%, while overexpression of Plxdc1 and 2 dramatically enhanced RRV susceptibility and cell-cell fusion of otherwise marginally permissive Raji cells. While the Plxdc2 interaction is conserved between two RRV strains, 26-95 and 17577, Plxdc1 specifically interacts with RRV 26-95 gH. The Plxdc interaction is mediated by a short motif at the N-terminus of RRV gH that is partially conserved between isolate 26-95 and isolate 17577, but absent in KSHV gH. Mutation of this motif abrogated the interaction with Plxdc1/2 and reduced RRV infection in a cell type-specific manner. Taken together, our findings characterize Plxdc1/2 as novel interaction partners and entry receptors for RRV and support the concept of the N-terminal domain of the gammaherpesviral gH/gL complex as a multifunctional receptor-binding domain. Further, Plxdc1/2 usage defines an important biological difference between KSHV and RRV.
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Rhesus Monkey Rhadinovirus Isolated from Hemangioma Tissue.
Microbiology resource announcements, 2020Co-Authors: Armin Ensser, Ronald C. Desrosiers, Koji Yasuda, William A. Lauer, Alexander S. HahnAbstract:ABSTRACT We isolated a rhesus monkey Rhadinovirus, isolate RRVmmu 209-07, from hemangioma tissue. The virion DNA was sequenced by Illumina-based sequencing. Isolate RRVmmu 209-07 is highly similar overall to RRV 26-95, but considerable differences exist in the 3′ region of the genome.
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Plxdc family members are novel receptors for the rhesus monkey Rhadinovirus (RRV)
2020Co-Authors: Anna K. Großkopf, Armin Ensser, Ronald C. Desrosiers, Sarah Schlagowski, Alexander Siegfried HahnAbstract:ABSTRACT The rhesus monkey Rhadinovirus (RRV), a γ2-herpesvirus of rhesus macaques, shares many biological features with the human pathogenic Kaposi’s sarcoma-associated herpesvirus (KSHV). Both viruses, as well as the more distantly related Epstein-Barr virus, engage cellular receptors from the Eph family of receptor tyrosine kinases (Ephs). However, the importance of the Eph interaction for RRV entry varies between cell types suggesting the existence of Eph-independent entry pathways. We therefore aimed to identify additional cellular receptors for RRV by affinity enrichment and mass spectrometry. We identified an additional receptor family, the Plexin domain containing proteins 1 and 2 (Plxdc1/2) that bind the RRV gH/gL glycoprotein complex. Preincubation of RRV with soluble Plxdc2 decoy receptor reduced infection by approx. 60%, while overexpression of Plxdc1 and 2 dramatically enhanced RRV susceptibility of otherwise marginally permissive Raji cells. While the Plxdc2 interaction is conserved between two RRV strains, 26-95 and 17577, Plxdc1 specifically interacts with RRV 26-95 gH. The Plxdc interaction is mediated by a short motif at the N-terminus of RRV gH that is partially conserved between isolate 26-95 and isolate 17577, but absent in KSHV gH. Mutation of this motif abrogated the interaction with Plxdc1/2 in in vitro assays and reduced RRV infection in a cell-type specific manner. Taken together, our findings characterize Plxdc1/2 as novel interaction partners and entry receptors for RRV and support the concept of the N-terminal domain of the gammaherpesviral gH/gL complex as a multifunctional receptor-binding domain. Further, Plxdc1/2 usage defines an important biological difference between KSHV and RRV. AUTHORS SUMMARY KSHV is the causative agent of a group of malignancies which account for a substantial disease burden in particular in sub-Saharan Africa. RRV, a related virus of rhesus macaques, has shown promise as model system for KSHV and for the development of immunization strategies. To exploit the full potential of the RRV animal model system, detailed knowledge of commonalities and differences between KSHV and RRV is key. Here, we describe the Plexin domain containing proteins 1 and 2 as a novel receptor family which mediates entry of RRV, but not of KSHV. Infection experiments using RRV mutants deleted of the Plxdc interaction motif suggest a cell type-specific contribution of Plxdc receptors to RRV infection. As information on Plxdc1/2 and its biological function is still sparse, analysis of the RRV–Plxdc interaction will help to characterize the physiological and pathophysiological role of this receptor family.
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EphA7 Functions as Receptor on BJAB Cells for Cell-to-Cell Transmission of the Kaposi's Sarcoma-Associated Herpesvirus and for Cell-Free Infection by the Related Rhesus Monkey Rhadinovirus.
Journal of virology, 2019Co-Authors: Anna K. Großkopf, Ronald C. Desrosiers, Sarah Schlagowski, Thomas Fricke, Bojan F. Hörnich, Alexander S. HahnAbstract:Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma and is associated with two B cell malignancies, primary effusion lymphoma (PEL) and the plasmablastic variant of multicentric Castleman's disease. On several adherent cell types, EphA2 functions as a cellular receptor for the gH/gL glycoprotein complex of KSHV. KSHV gH/gL also has previously been found to interact weakly with other members of the Eph family of receptor tyrosine kinases (Ephs), and other A-type Ephs have been shown to be able to compensate for the absence of EphA2 using overexpression systems. However, whether these interactions are of functional consequence at endogenous protein levels has remained unclear so far. Here, we demonstrate for the first time that endogenously expressed EphA7 in BJAB B cells is critical for the cell-to-cell transmission of KSHV from producer iSLK cells to BJAB target cells. The BJAB lymphoblastoid cell line often serves as a model for B cell infection and expresses only low levels of all Eph family receptors other than EphA7. Endogenous EphA7 could be precipitated from the cellular lysate of BJAB cells using recombinant gH/gL, and knockout of EphA7 significantly reduced transmission of KSHV into BJAB target cells. Knockout of EphA5, the second most expressed A-type Eph in BJAB cells, had a similar, although less pronounced, effect on KSHV infection. Receptor function of EphA7 was conserved for cell-free infection by the related rhesus monkey Rhadinovirus (RRV), which is relatively even more dependent on EphA7 for infection of BJAB cells.IMPORTANCE Infection of B cells is relevant for two KSHV-associated malignancies, the plasmablastic variant of multicentric Castleman's disease and PEL. Therefore, elucidating the process of B cell infection is important for the understanding of KSHV pathogenesis. While the high-affinity receptor for the gH/gL glycoprotein complex, EphA2, has been shown to function as an entry receptor for various types of adherent cells, the gH/gL complex can also interact with other Eph receptor tyrosine kinases with lower avidity. We analyzed the Eph interactions required for infection of BJAB cells, a model for B cell infection by KSHV. We identified EphA7 as the principal Eph receptor for infection of BJAB cells by KSHV and the related rhesus monkey Rhadinovirus. While two analyzed PEL cell lines exhibited high EphA2 and low EphA7 expression, a third PEL cell line, BCBL-1, showed high EphA7 and low EphA2 expression, indicating a possible relevance for KSHV pathology.
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epha7 functions as a receptor for cell to cell transmission of kaposi s sarcoma associated herpesvirus into bjab b cells and for cell free virus infection by the related rhesus monkey Rhadinovirus
bioRxiv, 2019Co-Authors: Anna K Groskopf, Ronald C. Desrosiers, Sarah Schlagowski, Thomas Fricke, Bojan F. Hörnich, Alexander S. HahnAbstract:ABSTRACT Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma and is associated with two B cell malignancies, primary effusion lymphoma and the plasmablastic variant of multicentric Castleman’s disease. EphA2 functions as a cellular receptor for the gH/gL glycoprotein complex of KSHV for several adherent cell types. KSHV gH/gL was previously shown to also weakly interact with other members of the Eph family of receptors. Whether these interactions are of functional consequence remained unclear so far, even if other A-type Ephs were shown to be able to compensate for absence of EphA2 in overexpression systems. Here, we demonstrate for the first time that endogenously expressed EphA7 in BJAB B cells is critical for the cell-to-cell transmission of KSHV from producer iSLK cells to BJAB target cells. The BJAB lymphoblastoid cell line often serves as a model for B cell infection and expresses only low levels of all Eph family receptors other than EphA7. Endogenous EphA7 could be precipitated from the cellular lysate of BJAB cells using recombinant gH/gL, and knockout of EphA7 significantly reduced transmission of KSHV into BJAB target cells by over 80%. Finally, we demonstrate that receptor function of EphA7 is conserved between cell-to-cell transmission of KSHV and cell-free infection by the related rhesus monkey Rhadinovirus (RRV), which is relatively even more dependent on EphA7 for infection of BJAB cells. IMPORTANCE Infection of B cells is biologically relevant for two KSHV-associated malignancies, the plasmablastic variant of multicentric Castleman’s disease (MCD) and primary effusion lymphoma (PEL). Elucidating the process of B cell infection is therefore important to understand the pathogenesis of these diseases. For various types of adherent cells, EphA2 has been shown by several groups to function as an entry receptor that is engaged by the gH/gL glycoprotein complex. Our previous findings indicate that KSHV does not only interact with EphA2, but can also bind to other members of the Eph family of receptor tyrosine kinases with comparatively lower avidity. We now analyzed the requirement of Eph interactions for infection of the BJAB B cell line, a model for infection of B cells by KSHV. We identify EphA7 as the principal Eph receptor for infection of this model B cell line by both KSHV and the related rhesus monkey Rhadinovirus.
Angela M. Bakke - One of the best experts on this subject based on the ideXlab platform.
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Macaque Homologs of EBV and KSHV Show Uniquely Different Associations with Simian AIDS-related
2016Co-Authors: Gregory A Bruce, Helle Bielefeldt-ohmann, Angela M. Bakke, Serge Barcy, Patrick Lewis, Chung Tsai, Robert D. Murnane, Timothy M RoseAbstract: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 strongl
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Macaque Homologs of EBV and KSHV Show Uniquely Different Associations with Simian AIDS-related Lymphomas
PLoS pathogens, 2012Co-Authors: A. Gregory Bruce, Helle Bielefeldt-ohmann, Che Chung Tsai, Angela M. Bakke, Serge Barcy, Patrick Lewis, Robert D. Murnane, Timothy M RoseAbstract: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.
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The ORF59 DNA polymerase processivity factor homologs of Old World primate RV2 Rhadinoviruses are highly conserved nuclear antigens expressed in differentiated epithelium in infected macaques
Virology journal, 2009Co-Authors: A. Gregory Bruce, Helle Bielefeldt-ohmann, Angela M. Bakke, Courtney Gravett, Laura K. Demaster, Kellie Burnside, Timothy M RoseAbstract:Background: ORF59 DNA polymerase processivity factor of the human Rhadinovirus, Kaposi's sarcoma-associated herpesvirus (KSHV), is required for efficient copying of the genome during virus replication. KSHV ORF59 is antigenic in the infected host and is used as a marker for virus activation and replication. Results: We cloned, sequenced and expressed the genes encoding related ORF59 proteins from the RV1 Rhadinovirus homologs of KSHV from chimpanzee (PtrRV1) and three species of macaques (RFHVMm, RFHVMn and RFHVMf), and have compared them with ORF59 proteins obtained from members of the more distantly-related RV2 Rhadinovirus lineage infecting the same non-human primate species (PtrRV2, RRV, MneRV2, and MfaRV2, respectively). We found that ORF59 homologs of the RV1 and RV2 Old World primate Rhadinoviruses are highly conserved with distinct phylogenetic clustering of the two Rhadinovirus lineages. RV1 and RV2 ORF59 C-terminal domains exhibit a strong lineage-specific conservation. Rabbit antiserum was developed against a C-terminal polypeptide that is highly conserved between the macaque RV2 ORF59 sequences. This anti-serum showed strong reactivity towards ORF59 encoded by the macaque RV2 Rhadinoviruses, RRV (rhesus) and MneRV2 (pig-tail), with no cross reaction to human or macaque RV1 ORF59 proteins. Using this antiserum and RT-qPCR, we determined that RRV ORF59 is expressed early after permissive infection of both rhesus primary fetal fibroblasts and African green monkey kidney epithelial cells (Vero) in vitro. RRV- and MneRV2-infected foci showed strong nuclear expression of ORF59 that correlated with production of infectious progeny virus. Immunohistochemical studies of an MneRV2-infected macaque revealed strong nuclear expression of ORF59 in infected cells within the differentiating layer of epidermis corroborating previous observations that differentiated epithelial cells are permissive for replication of KSHV-like Rhadinoviruses.
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Development of a real-time QPCR assay for the detection of RV2 lineage-specific Rhadinoviruses in macaques and baboons
Virology journal, 2005Co-Authors: A. Gregory Bruce, Margaret E Thouless, Angela M. Bakke, Timothy M RoseAbstract: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.
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Development of a real-time QPCR assay for the detection of RV2 lineage-specific Rhadinoviruses in macaques and baboons
Virology Journal, 2005Co-Authors: A. Gregory Bruce, Margaret E Thouless, Angela M. Bakke, Timothy M RoseAbstract: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.
