共查询到20条相似文献,搜索用时 15 毫秒
1.
Sequence and genomic analysis of a Rhesus macaque rhadinovirus with similarity to Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
We have sequenced the long unique region (LUR) and characterized the terminal repeats of the genome of a rhesus rhadinovirus (RRV), strain 17577. The LUR as sequenced is 131,364 bp in length, with a G+C content of 52.2% and a CpG ratio of 1.11. The genome codes for 79 open reading frames (ORFs), with 67 of these ORFs similar to genes found in both Kaposi's sarcoma-associated herpesvirus (KSHV) (formal name, human herpesvirus 8) and herpesvirus saimiri. Eight of the 12 unique genes show similarity to genes found in KSHV, including genes for viral interleukin-6, viral macrophage inflammatory protein, and a family of viral interferon regulatory factors (vIRFs). Genomic organization is essentially colinear with KSHV, the primary differences being the number of cytokine and IRF genes and the location of the gene for dihydrofolate reductase. Highly repetitive sequences are located in positions corresponding to repetitive sequences found in KSHV. Phylogenetic analysis of several ORFs supports the similarity between RRV and KSHV. Overall, the sequence, structural, and phylogenetic data combine to provide strong evidence that RRV 17577 is the rhesus macaque homolog of KSHV. 相似文献
2.
Gregory S. Pari David AuCoin Kelly Colletti Sylvia A. Cei Veronica Kirchoff Scott W. Wong 《Journal of virology》2001,75(23):11401-11407
We have identified a lytic origin of DNA replication (oriLyt) for rhesus macaque rhadinovirus (RRV), the rhesus macaque homolog of human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus. RRV oriLyt maps to the region of the genome between open reading frame 69 (ORF69) and ORF71 (vFLIP) and is composed of an upstream A+T-rich region followed by a short (300-bp) downstream G+C-rich DNA sequence. A set of overlapping cosmids corresponding to the entire genome of RRV was capable of complementing oriLyt-dependent DNA replication only when additional ORF50 was supplied as an expression plasmid in the transfection mixture, suggesting that the level of ORF50 protein originating from input cosmid DNA was insufficient. The requirement of RRV ORF50 in the cotransfection replication assay may also suggest a direct role for this protein in DNA replication. RRV oriLyt shares a high degree of nucleotide sequence and G+C base distribution with the corresponding loci in HHV-8. 相似文献
3.
Mansfield KG Westmoreland SV DeBakker CD Czajak S Lackner AA Desrosiers RC 《Journal of virology》1999,73(12):10320-10328
The recognition of naturally occurring rhadinoviruses in macaque monkeys has spurred interest in their use as models for human infection with Kaposi sarcoma-associated herpesvirus (human herpesvirus 8). Rhesus macaques (Macaca mulatta) and pig-tailed macaques (Macaca nemestrina) were inoculated intravenously with rhadinovirus isolates derived from these species (rhesus rhadinovirus [RRV] and pig-tailed rhadinovirus [PRV]). Nine rhadinovirus antibody-negative and two rhadinovirus antibody-positive monkeys were used for these experimental inoculations. Antibody-negative animals clearly became infected following virus inoculation since they developed persisting antibody responses to virus and virus was isolated from peripheral blood on repeated occasions following inoculation. Viral sequences were also detected by PCR in lymph node, oral mucosa, skin, and peripheral blood mononuclear cells following inoculation. Experimentally infected animals developed peripheral lymphadenopathy which resolved by 12 weeks following inoculation, and these animals have subsequently remained free of disease. No increased pathogenicity was apparent from cross-species infection, i.e., inoculation of rhesus macaques with PRV or of pig-tailed macaques with RRV, whether the animals were antibody positive or negative at the time of virus inoculation. Coinoculation of additional rhesus monkeys with simian immunodeficiency virus (SIV) isolate SIVmac251 and macaque-derived rhadinovirus resulted in an attenuated antibody response to both agents and shorter mean survival compared to SIVmac251-inoculated controls (155.5 days versus 560.1 days; P < 0.019). Coinfected and immunodeficient macaques died of a variety of opportunistic infections characteristic of simian AIDS. PCR analysis of sorted peripheral blood mononuclear cells indicated a preferential tropism of RRV for CD20(+) B lymphocytes. Our results demonstrate persistent infection of macaque monkeys with RRV and PRV following experimental inoculation, but no specific disease was readily apparent from these infections even in the context of concurrent SIV infection. 相似文献
4.
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.Conclusion
The ORF59 DNA polymerase processivity factor homologs of the Old World primate RV1 and RV2 rhadinovirus lineages are phylogenetically distinct yet demonstrate similar expression and localization characteristics that correlate with their use as lineage-specific markers for permissive infection and virus replication. These studies will aid in the characterization of virus activation from latency to the replicative state, an important step for understanding the biology and transmission of rhadinoviruses, such as KSHV. 相似文献5.
Characterization of two divergent lineages of macaque rhadinoviruses related to Kaposi's sarcoma-associated herpesvirus
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
We have cloned and characterized the entire DNA polymerase gene and flanking regions from Kaposi's sarcoma-associated herpesvirus (KSHV) and two closely related macaque homologs of KSHV, retroperitoneal fibromatosis-associated herpesvirus-Macaca nemestrina (RFHVMn) and -Macaca mulatta (RFHVMm). We have also identified and partially characterized the corresponding genomic region of another KSHV-like herpesvirus, provisionally named "M. nemestrina rhadinovirus type 2 (MneRV-2)," with close similarity to rhesus rhadinovirus (RRV). A sequence comparison of these four macaque viruses and two KSHV-like gammaherpesviruses recently identified in African green monkeys, Chlorocebus rhadinovirus types 1 and 2 (ChRV-1 and ChRV-2) reveals the presence of two distinct lineages of KSHV-like rhadinoviruses in Old World primates. The first rhadinovirus lineage consists of KSHV and its closely related homologs RFHVMn, RFHVMm, and ChRV-1, while the second more distantly related lineage consists of RRV, MneRV-2, and ChRV-2. Our findings raise the possibility of the existence of another human KSHV-like herpesvirus belonging to the second rhadinovirus lineage. 相似文献
6.
7.
A G protein-coupled receptor encoded by rhesus rhadinovirus is similar to ORF74 of Kaposi's sarcoma-associated herpesvirus
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Rhesus rhadinovirus (RRV) is a gamma-2 herpesvirus and is the rhesus macaque homologue of human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus. DNA sequence analysis of RRV indicates that it shares numerous open reading frames (ORFs) with HHV-8, including one (ORF74) encoding a seven-transmembrane-spanning G protein-coupled receptor (GPCR) with similarity to cellular chemokine receptors. Examination of the predicted amino acid sequence of RRV ORF74 reveals that it encodes a seven-transmembrane-spanning GPCR sharing 40.8% amino acid sequence identity with HHV-8 ORF74 and 24.1% amino acid sequence identity with rhesus macaque CXCR2. In addition, immunofluorescence studies indicate that an epitope-tagged version of RRV ORF74 is expressed on the surfaces of transfected cells, suggesting that this protein is in fact a membrane receptor. In in vitro cell culture assays, RRV ORF74 possesses transforming potential, as NIH 3T3 clones stably expressing the receptor demonstrate an increased ability to grow in soft agarose and to induce tumor formation in nude mice. Further analysis of RRV ORF74 indicates that expression of the receptor in NIH 3T3 cells causes an increased secretion of vascular endothelial growth factor and activation of the ERK1/2 (p44/42) mitogen-activated protein kinase signaling pathway. The results of these studies suggest that RRV ORF74 encodes a GPCR with properties similar to those of its homologue in HHV-8 and that this gene may play a role in RRV-associated pathogenesis. 相似文献
8.
Eric P. Bergquam Nancy Avery Stanley M. Shiigi Michael K. Axthelm Scott W. Wong 《Journal of virology》1999,73(9):7874-7876
Recent DNA sequence analysis indicates that rhesus rhadinovirus (RRV) is a member of the lymphotropic gamma-2 herpesvirus family. To determine if RRV is lymphotropic, peripheral blood mononuclear cells from naturally infected monkeys were separated by immunomagnetic bead depletion and analyzed for the presence of RRV by virus isolation and nested PCR. The recovery and consistent detection of RRV in the CD20(+)-enriched fraction clearly demonstrates that B lymphocytes are a major site of virus persistence. 相似文献
9.
Construction of an infectious rhesus rhadinovirus bacterial artificial chromosome for the analysis of Kaposi's sarcoma-associated herpesvirus-related disease development
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Rhesus rhadinovirus (RRV) is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) and causes KSHV-like diseases in immunocompromised rhesus macaques (RM) that resemble KSHV-associated diseases including multicentric Castleman's disease and non-Hodgkin's lymphoma. RRV retains a majority of open reading frames (ORFs) postulated to be involved in the pathogenesis of KSHV and is the closest available animal model to KSHV infection in humans. Here we describe the generation of a recombinant clone of RRV strain 17577 (RRV(17577)) utilizing bacterial artificial chromosome (BAC) technology. Characterization of the RRV BAC demonstrated that it is a pathogenic molecular clone of RRV(17577), producing virus that behaves like wild-type RRV both in vitro and in vivo. Specifically, BAC-derived RRV displays wild-type growth properties in vitro and readily infects simian immunodeficiency virus-infected RM, inducing B cell hyperplasia, persistent lymphadenopathy, and persistent infection in these animals. This RRV BAC will allow for rapid genetic manipulation of the RRV genome, facilitating the creation of recombinant versions of RRV that harbor specific alterations and/or deletions of viral ORFs. This system will provide insights into the roles of specific RRV genes in various aspects of the viral life cycle and the RRV-associated pathogenesis in vivo in an RM model of infection. Furthermore, the generation of chimeric versions of RRV containing KSHV genes will allow analysis of the function and contributions of KSHV genes to viral pathogenesis by using a relevant primate model system. 相似文献
10.
11.
12.
13.
B.U. Orzechowska M. Manoharan J. Sprague R.D. Estep M.K. Axthelm & S.W. Wong 《Journal of medical primatology》2009,38(S1):2-7
Background Rhesus macaques (RM) co-infected with simian immunodeficiency virus (SIV) and rhesus macaque rhadinovirus (RRV) develop abnormal cellular proliferations characterized as extra-nodal lymphoma and retroperitoneal fibromatosis (RF). RRV encodes a viral interleukin-6 (vIL-6), much like Kaposi's sarcoma-associated herpesvirus, and involvement of the viral cytokine was examined in proliferative lesions.
Methods Formalin fixed tissue from RM co-infected with SIV and RRV were analyzed for RRV genomes by in situ hybridization and RRV vIL-6 expression by immunofluorescence analysis.
Results In situ hybridization analysis indicated that RRV is present in both types of lesions. Immunofluorescence analysis of different lymphomas and RF revealed positive staining for vIL-6. Similarly to KS, RF lesion is positive for vimentin, CD117 (c-kit), and smooth muscle actin (SMA) and contains T cell, B cell and monocytes/macrophage infiltrates.
Conclusions Our data support the idea that vIL-6 may be critical to the development and progression of lymphoproliferative disorder in RRV/SIV-infected RM. 相似文献
Methods Formalin fixed tissue from RM co-infected with SIV and RRV were analyzed for RRV genomes by in situ hybridization and RRV vIL-6 expression by immunofluorescence analysis.
Results In situ hybridization analysis indicated that RRV is present in both types of lesions. Immunofluorescence analysis of different lymphomas and RF revealed positive staining for vIL-6. Similarly to KS, RF lesion is positive for vimentin, CD117 (c-kit), and smooth muscle actin (SMA) and contains T cell, B cell and monocytes/macrophage infiltrates.
Conclusions Our data support the idea that vIL-6 may be critical to the development and progression of lymphoproliferative disorder in RRV/SIV-infected RM. 相似文献
14.
Identification of the R1 oncogene and its protein product from the rhadinovirus of rhesus monkeys
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Rhesus monkey rhadinovirus (RRV) is a gamma-2 herpesvirus that is most closely related to the human Kaposi's sarcoma-associated herpesvirus (KSHV). We have identified a distinct open reading frame at the left end of RRV and designated it R1. The position of the R1 gene is equivalent to that of the saimiri transforming protein (STP) of herpesvirus saimiri (HVS) and of K1 of KSHV, other members of the gamma-2 or rhadinovirus subgroup of herpesviruses. The R1 sequence revealed an open reading frame encoding a product of 423 amino acids that was predicted to contain an extracellular domain, a transmembrane domain, and a C-terminal cytoplasmic tail reflective of a type I membrane-bound protein. The predicted structural motifs of R1, including the presence of immunoreceptor tyrosine-based activation motifs, resembled those in K1 of KSHV but were distinct from those of STP. R1 sequences from four independent isolates from three different macaque species revealed 0.95 to 7.3% divergence over the 423 amino acids. Variation was located predominantly within the predicted extracellular domain. The R1 protein migrated at 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was extensively glycosylated. Tagged R1 protein was localized to the cytoplasmic and plasma membranes of transfected cells. Expression of the R1 gene in Rat-1 fibroblasts induced morphologic changes and focus formation, and injection of R1-expressing cells into nude mice induced the formation of multifocal tumors. A recombinant herpesvirus in which the STP oncogene of HVS was replaced by R1 immortalized T lymphocytes to interleukin-2-independent growth. These results indicate that R1 is an oncogene of RRV. 相似文献
15.
The primary sequence of rhesus monkey rhadinovirus isolate 26-95: sequence similarities to Kaposi's sarcoma-associated herpesvirus and rhesus monkey rhadinovirus isolate 17577
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Alexander L Denekamp L Knapp A Auerbach MR Damania B Desrosiers RC 《Journal of virology》2000,74(7):3388-3398
The primary sequence of the long unique region L-DNA (L for low GC) of rhesus monkey rhadinovirus (RRV) isolate 26-95 was determined. The L-DNA consists of 130,733 bp that contain 84 open reading frames (ORFs). The overall organization of the RRV26-95 genome was found to be very similar to that of human Kaposi sarcoma-associated herpesvirus (KSHV). BLAST search analysis revealed that in almost all cases RRV26-95 coding sequences have a greater degree of similarity to corresponding KSHV sequences than to other herpesviruses. All of the ORFs present in KSHV have at least one homologue in RRV26-95 except K3 and K5 (bovine herpesvirus-4 immediate-early protein homologues), K7 (nut-1), and K12 (Kaposin). RRV26-95 contains one MIP-1 and eight interferon regulatory factor (vIRF) homologues compared to three MIP-1 and four vIRF homologues in KSHV. All homologues are correspondingly located in KSHV and RRV with the exception of dihydrofolate reductase (DHFR). DHFR is correspondingly located near the left end of the genome in RRV26-95 and herpesvirus saimiri (HVS), but in KSHV the DHFR gene is displaced 16,069 nucleotides in a rightward direction in the genome. DHFR is also unusual in that the RRV26-95 DHFR more closely resembles HVS DHFR (74% similarity) than KSHV DHFR (55% similarity). Of the 84 ORFs in RRV26-95, 83 contain sequences similar to the recently determined sequences of the independent RRV isolate 17577. RRV26-95 and RRV17577 sequences differ in that ORF 67.5 sequences contained in RRV26-95 were not found in RRV17577. In addition, ORF 4 is significantly shorter in RRV26-95 than was reported for RRV17577 (395 versus 645 amino acids). Only four of the corresponding ORFs between RRV26-95 and RRV17577 exhibited less than 95% sequence identity: glycoproteins H and L, uracil DNA glucosidase, and a tegument protein (ORF 67). Both RRV26-95 and RRV17577 have unique ORFs between positions 21444 to 21752 and 110910 to 114899 in a rightward direction and from positions 116524 to 111082 in a leftward direction that are not found in KSHV. Our analysis indicates that RRV26-95 and RRV17577 are clearly independent isolates of the same virus species and that both are closely related in structural organization and overall sequence to KSHV. The availability of detailed sequence information, the ability to grow RRV lytically in cell culture, and the ability to infect monkeys experimentally with RRV will facilitate the construction of mutant strains of virus for evaluating the contribution of individual genes to biological properties. 相似文献
16.
Robinson BA O'Connor MA Li H Engelmann F Poland B Grant R DeFilippis V Estep RD Axthelm MK Messaoudi I Wong SW 《Journal of virology》2012,86(5):2769-2779
Kaposi's sarcoma-associated herpesvirus (KSHV) and the closely related gamma-2 herpesvirus rhesus macaque (RM) rhadinovirus (RRV) are the only known viruses to encode viral homologues of the cellular interferon (IFN) regulatory factors (IRFs). Recent characterization of a viral IRF (vIRF) deletion clone of RRV (vIRF-knockout RRV [vIRF-ko RRV]) demonstrated that vIRFs inhibit induction of type I and type II IFNs during RRV infection of peripheral blood mononuclear cells. Because the IFN response is a key component to a host's antiviral defenses, this study has investigated the role of vIRFs in viral replication and the development of the immune response during in vivo infection in RMs, the natural host of RRV. Experimental infection of RMs with vIRF-ko RRV resulted in decreased viral loads and diminished B cell hyperplasia, a characteristic pathology during acute RRV infection that often develops into more severe lymphoproliferative disorders in immune-compromised animals, similar to pathologies in KSHV-infected individuals. Moreover, in vivo infection with vIRF-ko RRV resulted in earlier and sustained production of proinflammatory cytokines and earlier induction of an anti-RRV T cell response compared to wild-type RRV infection. These findings reveal the broad impact that vIRFs have on pathogenesis and the immune response in vivo and are the first to validate the importance of vIRFs during de novo infection in the host. 相似文献
17.
Two Distinct Gamma-2 Herpesviruses in African Green Monkeys: a Second Gamma-2 Herpesvirus Lineage among Old World Primates?
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Greensill J Sheldon JA Renwick NM Beer BE Norley S Goudsmit J Schulz TF 《Journal of virology》2000,74(3):1572-1577
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. 相似文献
18.
A herpesvirus of rhesus monkeys related to the human Kaposi's sarcoma-associated herpesvirus. 总被引:15,自引:11,他引:4
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
R C Desrosiers V G Sasseville S C Czajak X Zhang K G Mansfield A Kaur R P Johnson A A Lackner J U Jung 《Journal of virology》1997,71(12):9764-9769
A herpesvirus that is related to but distinct from the Kaposi's sarcoma-associated herpesvirus (KSHV, or human herpesvirus 8) was isolated from rhesus monkeys. The sequence of 10.6 kbp from virion DNA revealed the presence of an interleukin-6 homolog similar to what is present in KSHV and a closer relatedness of the DNA polymerase and glycoprotein B reading frames to those of KSHV than to those of any other herpesvirus. This rhesus monkey herpesvirus replicated lytically and to high titers in cultured rhesus monkey fibroblasts. Antibody testing revealed a high prevalence for at least 10 years in our rhesus monkey colony and a high prevalence in two other colonies that were tested. Thus, rhesus monkeys naturally harbor a virus related to KSHV, which we have called RRV, for rhesus monkey rhadinovirus. 相似文献
19.
Whitby D Stossel A Gamache C Papin J Bosch M Smith A Kedes DH White G Kennedy R Dittmer DP 《Journal of virology》2003,77(14):8159-8165
Kaposi's sarcoma (KS) and lymphoproliferative diseases induced by KS-associated herpesvirus (KSHV/human herpesvirus 8) cause substantial morbidity and mortality in human immunodeficiency virus-infected individuals. To understand KSHV biology it is useful to investigate closely related rhadinoviruses naturally occurring in nonhuman primates. Here we report evidence for a novel KSHV homolog in captive baboon species (Papio anubis and other). Using degenerate PCR we identified a novel rhadinovirus, PapRV2, that has substantial sequence identity to two essential KSHV genes, the viral polymerase and thymidylate synthase. A subset of animals exhibited detectable PapRV2 viral load in peripheral blood mononuclear cells. Extensive serological analysis of nearly 200 animals in the colony demonstrated that the majority carried cross-reacting antibodies that recognize KSHV or macaque rhadinovirus antigens. Seroreactivity increased with age, similar to the age-specific prevalence of KSHV in the human population. This establishes baboons as a novel resource to investigate rhadinovirus biology, which can be developed into an animal model system for KSHV-associated human diseases, vaccine development, and therapy evaluation. 相似文献
20.
A genetic system for rhesus monkey rhadinovirus: use of recombinant virus to quantitate antibody-mediated neutralization
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Rhesus monkey rhadinovirus (RRV), a simian gamma-2 herpesvirus closely related to the Kaposi sarcoma-associated herpesvirus, replicates lytically in cultured rhesus monkey fibroblasts and establishes persistence in B cells. Overlapping cosmid clones were generated that encompass the entire 130-kilobase-pair genome of RRV strain 26-95, including the terminal repeat regions required for its replication. Cloned RRV that was produced by cotransfection of overlapping cosmids spanning the entire RRV26-95 genome replicated with growth kinetics and to titers similar to those of the parental, uncloned, wild-type RRV26-95. Expression cassettes for secreted-engineered alkaline phosphatase (SEAP) and green fluorescent protein (GFP) were inserted upstream of the R1 gene, and the cosmid-based system for RRV genome reconstitution was used to generate replication-competent, recombinant RRV that expressed either the SEAP or GFP reporter gene. Using the SEAP and GFP recombinant RRVs, assays were developed to monitor RRV infection, neutralization, and replication. Heat-inactivated sera from rhesus monkeys that were naturally or experimentally infected with RRV were assayed for their ability to neutralize RRV-SEAP and RRV-GFP infectivity using rhesus monkey fibroblasts. Sera from RRV-positive monkeys, but not RRV-negative monkeys, were consistently able to neutralize RRV infectivity when assayed by the production of SEAP activity or by the ability to express GFP. The neutralizing activity was present in the immunoglobulin fraction. Of the 17 rhesus monkeys tested, sera from rhesus monkey 26-95, i.e., the monkey that yielded the RRV 26-95 isolate, had the highest titer of neutralizing activity against RRV26-95. This cosmid-based genetic system and the reporter virus neutralization assay will facilitate study of the contribution of individual RRV glycoproteins to entry into different cell types, particularly fibroblasts and B cells. 相似文献