Molecular Detection and Genetic Diversity of Cytomegaloviruses and Lymphocryptoviruses in Free-Roaming and Captive African Green Monkeys (Chlorocebus sabaeus).

To date, limited information is available on cytomegalovirus (CMV) and lymphocryptovirus (LCV) from Chlorocebus monkeys. We report here high detection rates of herpesviruses in free-roaming African green monkeys (AGMs, Chlorocebus sabaeus) (26.4%, 23/87) and in captive AGMs (75%, 3/4) with respiratory disease on the Caribbean Island of St. Kitts. LCV (81.25%) was more prevalent than CMV (18.75%) in the AGMs. Applying a bigenic PCR approach (targeting DNA polymerase (DPOL) and glycoprotein B (gB) genes), long sequences were obtained from representative AGM CMV (KNA-SD6) and LCV (KNA-E4, -N6 and -R15) samples, and mixed LCV infections were identified in KNA-N6 and -R15. The nucleotide (nt) sequence (partial DPOL-intergenic region-partial gB) and partial DPOL- and gB-amino acid (aa) sequences of AGM CMV KNA-SD6 were closely related to Cytomegalovirus cercopithecinebeta5 isolates from grivet monkeys, whilst those of AGM LCV KNA-E4 and -N6 (and E4-like gB of KNA-R15) were more closely related to cognate sequences of erythrocebus patas LCV1 from patas monkey than other LCVs, corroborating the concept of cospeciation in the evolution of CMV/LCV. On the other hand, the partial DPOL aa sequence of KNA-R15, and additional gB sequences (N6-gB-2 and R15-gB-2) from samples KNA-N6 and -R15 (respectively) appeared to be distinct from those of Old World monkey LCVs, indicating LCV evolutionary patterns that were not synchronous with those of host species. The present study is the first to report the molecular prevalence and genetic diversity of CMV/LCV from free-roaming/wild and captive AGMs, and is the first report on analysis of CMV nt/deduced aa sequences from AGMs and LCV gB sequences from Chlorocebus monkeys.

Epstein-Barr Virus miR-BHRF1-3 Targets the BZLF1 3'UTR and Regulates the Lytic Cycle.

Suppression of lytic viral gene expression is a key aspect of the Epstein-Barr virus (EBV) life cycle to facilitate the establishment of latent infection. Molecular mechanisms regulating transitions between EBV lytic replication and latency are not fully understood. Here, we investigated the impact of viral microRNAs on the EBV lytic cycle. Through functional assays, we found that miR-BHRF1-3 attenuates EBV lytic gene expression following reactivation. To understand the miRNA targets contributing to this activity, we performed Ago PAR-CLIP analysis on EBV-positive, reactivated Burkitt's lymphoma cells and identified multiple miR-BHRF1-3 interactions with viral transcripts. Using luciferase reporter assays, we confirmed a miRNA interaction site within the 3'UTR of BZLF1 which encodes the essential immediate early (IE) transactivator Zta. Comparison of >850 published EBV genomes identified sequence polymorphisms within the miR-BHRF1-3 locus that deleteriously affect miRNA expression and function. Molecular interactions between the homologous viral miRNA, miR-rL1-17, and IE transcripts encoded by rhesus lymphocryptovirus were further identified. Our data demonstrate that regulation of IE gene expression by a BHRF1 miRNA is conserved among lymphocryptoviruses, and further reveal virally-encoded genetic elements that orchestrate viral antigen expression during the lytic cycle. IMPORTANCE Epstein-Barr virus infection is predominantly latent in healthy individuals, while periodic cycles of reactivation are thought to facilitate persistent lifelong infection. Lytic infection has been linked to development of certain EBV-associated diseases. Here, we demonstrate that EBV miR-BHRF1-3 can suppress lytic replication by directly inhibiting Zta expression. Moreover, we identify nucleotide variants that impact the function of miR-BHRF1-3, which may contribute to specific EBV pathologies.

Comparative Analysis of Gammaherpesvirus Circular RNA Repertoires: Conserved and Unique Viral Circular RNAs.

Recent studies have identified circular RNAs (circRNAs) expressed from the Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) human DNA tumor viruses. To gain initial insights into the potential relevance of EBV circRNAs in virus biology and disease, we assessed the circRNAome of the interspecies homologue rhesus macaque lymphocryptovirus (rLCV) in a naturally occurring lymphoma from a simian immunodeficiency virus (SIV)-infected rhesus macaque. This analysis revealed rLCV orthologues of the latency-associated EBV circular RNAs circRPMS1_E4_E3a and circEBNA_U. Also identified in two samples displaying unusually high lytic gene expression was a novel rLCV circRNA that contains both conserved and rLCV-specific RPMS1 exons and whose backsplice junctions flank an rLCV lytic origin of replication (OriLyt). Analysis of a lytic infection model for the murid herpesvirus 68 (MHV68) rhadinovirus identified a cluster of circRNAs near an MHV68 lytic origin of replication, with the most abundant of these, circM11_ORF69, spanning the OriLyt. Lastly, analysis of KSHV latency and reactivation models revealed the latency associated circRNA originating from the vIRF4 gene as the predominant viral circRNA. Together, the results of this study broaden our appreciation for circRNA repertoires in the Lymphocryptovirus and Rhadinovirus genera of gammaherpesviruses and provide evolutionary support for viral circRNA functions in latency and viral replication.IMPORTANCE Infection with oncogenic gammaherpesviruses leads to long-term viral persistence through a dynamic interplay between the virus and the host immune system. Critical for remodeling of the host cell environment after the immune responses are viral noncoding RNAs that modulate host signaling pathways without attracting adaptive immune recognition. Despite the importance of noncoding RNAs in persistent infection, the circRNA class of noncoding RNAs has only recently been identified in gammaherpesviruses. Accordingly, their roles in virus infection and associated oncogenesis are unknown. Here we report evolutionary conservation of EBV-encoded circRNAs determined by assessing the circRNAome in rLCV-infected lymphomas from an SIV-infected rhesus macaque, and we report latent and lytic circRNAs from KSHV and MHV68. These experiments demonstrate utilization of the circular RNA class of RNAs across 4 members of the gammaherpesvirus subfamily, and they identify orthologues and potential homoplastic circRNAs, implying conserved circRNA functions in virus biology and associated malignancies.

Species-specific functions of Epstein-Barr virus nuclear antigen 2 (EBNA2) reveal dual roles for initiation and maintenance of B cell immortalization.

Epstein-Barr virus (EBV) and related lymphocryptoviruses (LCV) from non-human primates infect B cells, transform their growth to facilitate life-long viral persistence in the host, and contribute to B cell oncogenesis. Co-evolution of LCV with their primate hosts has led to species-specificity so that LCVs preferentially immortalize B cells from their natural host in vitro. We investigated whether the master regulator of transcription, EBV nuclear antigen 2 (EBNA2), is involved in LCV species-specificity. Using recombinant EBVs, we show that EBNA2 orthologues of LCV isolated from chimpanzees, baboons, cynomolgus or rhesus macaques cannot replace EBV EBNA2 for the immortalization of human B cells. Thus, LCV species-specificity is functionally linked to viral proteins expressed during latent, growth-transforming infection. In addition, we identified three independent domains within EBNA2 that act through species-specific mechanisms. Importantly, the EBNA2 orthologues and species-specific EBNA2 domains separate unique roles for EBNA2 in the initiation of B cell immortalization from those responsible for maintaining the immortalized state. Investigating LCV species-specificity provides a novel approach to identify critical steps underlying EBV-induced B cell growth transformation, persistent infection, and oncogenesis.

Epstein-Barr Virus gp350 Can Functionally Replace the Rhesus Lymphocryptovirus Major Membrane Glycoprotein and Does Not Restrict Infection of Rhesus Macaques.

UNLABELLED: Primary Epstein-Barr virus (EBV) infection is the most common cause of infectious mononucleosis, and persistent infection is associated with multiple cancers. EBV vaccine development has focused on the major membrane glycoprotein, gp350, since it is the major target for antibodies that neutralize infection of B cells. However, EBV has tropism for both B cells and epithelial cells, and it is unknown whether serum neutralizing antibodies against B cell infection will provide sufficient protection against virus infection initiated at the oral mucosa. This could be stringently tested by passive antibody transfer and oral virus challenge in the rhesus macaque model for EBV infection. However, only neutralizing monoclonal antibodies (MAbs) against EBV are available, and EBV is unable to infect rhesus macaques because of a host range restriction with an unknown mechanism. We cloned the prototypic EBV-neutralizing antibody, 72A1, and found that recombinant 72A1 did not neutralize rhesus lymphocryptovirus (rhLCV) infection of macaque B cells. Therefore, we constructed a chimeric rhLCV in which the native major membrane glycoprotein was replaced with EBV gp350. This chimeric rhLCV became sensitive to neutralization by the 72A1 MAb, efficiently immortalized macaque B cells in vitro, and successfully established acute and persistent infection after oral inoculation of rhesus macaques. Thus, EBV gp350 can functionally replace rhLCV gp350 and does not restrict rhLCV infection in vitro or in vivo. The chimeric rhLCV enables direct use of an EBV-specific MAb to investigate the effects of serum neutralizing antibodies against B cell infection on oral viral challenge in rhesus macaques. IMPORTANCE: This study asked whether the EBV major membrane glycoprotein could functionally replace the rhLCV major membrane glycoprotein. We found that an rhLCV humanized with EBV gp350 is capable of efficiently immortalizing monkey B cells in vitro and reproduces acute and persistent infection after oral inoculation of macaques. These results advance our understanding of why EBV cannot infect rhesus macaques by proving that viral attachment through gp350 is not the mechanism for EBV host range restriction. Humanization of rhLCV with EBV gp350 also confers susceptibility to a potent EBV-neutralizing MAb and provides a novel and significant enhancement to the rhesus macaque animal model where both the clinical utility and biological role of neutralizing MAbs against B cell or epithelial cell infection can now be directly tested in the most accurate animal model for EBV infection.

The first endogenous herpesvirus, identified in the tarsier genome, and novel sequences from primate rhadinoviruses and lymphocryptoviruses.

Herpesviridae is a diverse family of large and complex pathogens whose genomes are extremely difficult to sequence. This is particularly true for clinical samples, and if the virus, host, or both genomes are being sequenced for the first time. Although herpesviruses are known to occasionally integrate in host genomes, and can also be inherited in a Mendelian fashion, they are notably absent from the genomic fossil record comprised of endogenous viral elements (EVEs). Here, we combine paleovirological and metagenomic approaches to both explore the constituent viral diversity of mammalian genomes and search for endogenous herpesviruses. We describe the first endogenous herpesvirus from the genome of the Philippine tarsier, belonging to the Roseolovirus genus, and characterize its highly defective genome that is integrated and flanked by unambiguous host DNA. From a draft assembly of the aye-aye genome, we use bioinformatic tools to reveal over 100,000 bp of a novel rhadinovirus that is the first lemur gammaherpesvirus, closely related to Kaposi's sarcoma-associated virus. We also identify 58 genes of Pan paniscus lymphocryptovirus 1, the bonobo equivalent of human Epstein-Barr virus. For each of the viruses, we postulate gene function via comparative analysis to known viral relatives. Most notably, the evidence from gene content and phylogenetics suggests that the aye-aye sequences represent the most basal known rhadinovirus, and indicates that tumorigenic herpesviruses have been infecting primates since their emergence in the late Cretaceous. Overall, these data show that a genomic fossil record of herpesviruses exists despite their extremely large genomes, and expands the known diversity of Herpesviridae, which will aid the characterization of pathogenesis. Our analytical approach illustrates the benefit of intersecting evolutionary approaches with metagenomics, genetics and paleovirology.

Identification and functional comparison of seven-transmembrane G-protein-coupled BILF1 receptors in recently discovered nonhuman primate lymphocryptoviruses.

UNLABELLED: Coevolution of herpesviruses with their respective host has resulted in a delicate balance between virus-encoded immune evasion mechanisms and host antiviral immunity. BILF1 encoded by human Epstein-Barr virus (EBV) is a 7-transmembrane (7TM) G-protein-coupled receptor (GPCR) with multiple immunomodulatory functions, including attenuation of PKR phosphorylation, activation of G-protein signaling, and downregulation of major histocompatibility complex (MHC) class I surface expression. In this study, we explored the evolutionary and functional relationships between BILF1 receptor family members from EBV and 12 previously uncharacterized nonhuman primate (NHP) lymphocryptoviruses (LCVs). Phylogenetic analysis defined 3 BILF1 clades, corresponding to LCVs of New World monkeys (clade A) or Old World monkeys and great apes (clades B and C). Common functional properties were suggested by a high degree of sequence conservation in functionally important regions of the BILF1 molecules. A subset of BILF1 receptors from EBV and LCVs from NHPs (chimpanzee, orangutan, marmoset, and siamang) were selected for multifunctional analysis. All receptors exhibited constitutive signaling activity via G protein Gαi and induced activation of the NF-κB transcription factor. In contrast, only 3 of 5 were able to activate NFAT (nuclear factor of activated T cells); chimpanzee and orangutan BILF1 molecules were unable to activate NFAT. Similarly, although all receptors were internalized, BILF1 from the chimpanzee and orangutan displayed an altered cellular localization pattern with predominant cell surface expression. This study shows how biochemical characterization of functionally important orthologous viral proteins can be used to complement phylogenetic analysis to provide further insight into diverse microbial evolutionary relationships and immune evasion function. IMPORTANCE: Epstein-Barr virus (EBV), known as an oncovirus, is the only human herpesvirus in the genus Lymphocryptovirus (LCV). EBV uses multiple strategies to hijack infected host cells, establish persistent infection in B cells, and evade antiviral immune responses. As part of EBV's immune evasion strategy, the virus encodes a multifunctional 7-transmembrane (7TM) G-protein-coupled receptor (GPCR), EBV BILF1. In addition to multiple immune evasion-associated functions, EBV BILF1 has transforming properties, which are linked to its high constitutive activity. We identified BILF1 receptor orthologues in 12 previously uncharacterized LCVs from nonhuman primates (NHPs) of Old and New World origin. As 7TM receptors are excellent drug targets, our unique insight into the molecular mechanism of action of the BILF1 family and into the evolution of primate LCVs may enable validation of EBV BILF1 as a drug target for EBV-mediated diseases, as well as facilitating the design of drugs targeting EBV BILF1.

Non-human Primate Lymphocryptoviruses: Past, Present, and Future.

Epstein-Barr virus (EBV) orthologues from non-human primates (NHPs) have been studied for nearly as long as EBV itself. Cross-reactive sera and DNA hybridization studies provided the first glimpses of the closely related herpesviruses that belonged to the same gamma-1 herpesvirus, or lymphocryptovirus, genus, as EBV. Over the years, detailed molecular and sequence analyses of LCVs that infect humans and other NHPs revealed similar colinear genome structures and homologous viral proteins expressed during latent and lytic infection. Despite these similarities, experimental infection of NHPs with EBV did not result in acute symptoms or persistent infection as observed in humans, suggesting some degree of host species restriction. Genome sequencing and a molecular clone of an LCV isolate from naturally infected rhesus macaques combined with domestic colonies of LCV-naïve rhesus macaques have opened the door to a unique experimental animal model that accurately reproduces the normal transmission, acute viremia, lifelong persistence, and immune responses found in EBV-infected humans. This chapter will summarize the advances made over the last 50 years in our understanding of LCVs that naturally infect both Old and New World NHPs, the recent, groundbreaking developments in the use of rhesus macaques as an animal model for EBV infection, and how NHP LCVs and the rhLCV animal model can advance future EBV research and the development of an EBV vaccine.

Adenovirus-based vaccines against rhesus lymphocryptovirus EBNA-1 induce expansion of specific CD8+ and CD4+ T cells in persistently infected rhesus macaques.

UNLABELLED: The impact of Epstein-Barr virus (EBV) on human health is substantial, but vaccines that prevent primary EBV infections or treat EBV-associated diseases are not yet available. The Epstein-Barr nuclear antigen 1 (EBNA-1) is an important target for vaccination because it is the only protein expressed in all EBV-associated malignancies. We have designed and tested two therapeutic EBV vaccines that target the rhesus (rh) lymphocryptovirus (LCV) EBNA-1 to determine if ongoing T cell responses during persistent rhLCV infection in rhesus macaques can be expanded upon vaccination. Vaccines were based on two serotypes of E1-deleted simian adenovirus and were administered in a prime-boost regimen. To further modulate the response, rhEBNA-1 was fused to herpes simplex virus glycoprotein D (HSV-gD), which acts to block an inhibitory signaling pathway during T cell activation. We found that vaccines expressing rhEBNA-1 with or without functional HSV-gD led to expansion of rhEBNA-1-specific CD8(+) and CD4(+) T cells in 33% and 83% of the vaccinated animals, respectively. Additional animals developed significant changes within T cell subsets without changes in total numbers. Vaccination did not increase T cell responses to rhBZLF-1, an immediate early lytic phase antigen of rhLCV, thus indicating that increases of rhEBNA-1-specific responses were a direct result of vaccination. Vaccine-induced rhEBNA-1-specific T cells were highly functional and produced various combinations of cytokines as well as the cytolytic molecule granzyme B. These results serve as an important proof of principle that functional EBNA-1-specific T cells can be expanded by vaccination. IMPORTANCE: EBV is a common human pathogen that establishes a persistent infection through latency in B cells, where it occasionally reactivates. EBV infection is typically benign and is well controlled by the host adaptive immune system; however, it is considered carcinogenic due to its strong association with lymphoid and epithelial cell malignancies. Latent EBNA-1 is a promising target for a therapeutic vaccine, as it is the only antigen expressed in all EBV-associated malignancies. The goal was to determine if rhEBNA-1-specific T cells could be expanded upon vaccination of infected animals. Results were obtained with vaccines that target EBNA-1 of rhLCV, a virus closely related to EBV. We found that vaccination led to expansion of rhEBNA-1 immune cells that exhibited functions fit for controlling viral infection. This confirms that rhEBNA-1 is a suitable target for therapeutic vaccines. Future work should aim to generate more-robust T cell responses through modified vaccines.

Evolutionary conservation of primate lymphocryptovirus microRNA targets.

Epstein-Barr virus (EBV) and rhesus lymphocryptovirus (rLCV) are closely related gammaherpesviruses in the lymphocryptovirus subgroup that express viral microRNAs (miRNAs) during latent infection. In addition to many host mRNAs, EBV miRNAs are known to target latent viral transcripts, specifically those encoding LMP1, BHRF1, and EBNA2. The mRNA targets of rLCV miRNAs have not been investigated. Using luciferase reporter assays, photoactivatable cross-linking and immunoprecipitation (PAR-CLIP), and deep sequencing, we demonstrate that posttranscriptional regulation of LMP1 expression is a conserved function of lymphocryptovirus miRNAs. Furthermore, the mRNAs encoding the rLCV EBNA2 and BHRF1 homologs are regulated by miRNAs in rLCV-infected B cells. Homologous to sites in the EBV LMP1 and BHRF1 3'-untranslated regions (UTRs), we also identified evolutionarily conserved binding sites for the cellular miR-17/20/106 family in the LMP1 and BHRF1 3'UTRs of several primate LCVs. Finally, we investigated the functional consequences of LMP1 targeting by individual EBV BART miRNAs and show that select viral miRNAs play a role in the previously observed modulation of NF-κB activation.

Therapeutic vaccination against the rhesus lymphocryptovirus EBNA-1 homologue, rhEBNA-1, elicits T cell responses to novel epitopes in rhesus macaques.

Epstein-Barr virus (EBV) is a vaccine/immunotherapy target due to its association with several human malignancies. EBNA-1 is an EBV protein consistently expressed in all EBV-associated cancers. Herein, EBNA-1-specific T cell epitopes were evaluated after AdC-rhEBNA-1 immunizations in chronically lymphocryptovirus-infected rhesus macaques, an EBV infection model. Preexisting rhEBNA-1-specific responses were augmented in 4/12 animals, and new epitopes were recognized in 5/12 animals after vaccinations. This study demonstrated that EBNA-1-specific T cells can be expanded by vaccination.

An Epstein-Barr virus encoded inhibitor of Colony Stimulating Factor-1 signaling is an important determinant for acute and persistent EBV infection.

Acute Epstein-Barr virus (EBV) infection is the most common cause of Infectious Mononucleosis. Nearly all adult humans harbor life-long, persistent EBV infection which can lead to development of cancers including Hodgkin Lymphoma, Burkitt Lymphoma, nasopharyngeal carcinoma, gastric carcinoma, and lymphomas in immunosuppressed patients. BARF1 is an EBV replication-associated, secreted protein that blocks Colony Stimulating Factor 1 (CSF-1) signaling, an innate immunity pathway not targeted by any other virus species. To evaluate effects of BARF1 in acute and persistent infection, we mutated the BARF1 homologue in the EBV-related herpesvirus, or lymphocryptovirus (LCV), naturally infecting rhesus macaques to create a recombinant rhLCV incapable of blocking CSF-1 (ΔrhBARF1). Rhesus macaques orally challenged with ΔrhBARF1 had decreased viral load indicating that CSF-1 is important for acute virus infection. Surprisingly, ΔrhBARF1 was also associated with dramatically lower virus setpoints during persistent infection. Normal acute viral load and normal viral setpoints during persistent rhLCV infection could be restored by Simian/Human Immunodeficiency Virus-induced immunosuppression prior to oral inoculation with ΔrhBARF1 or infection of immunocompetent animals with a recombinant rhLCV where the rhBARF1 was repaired. These results indicate that BARF1 blockade of CSF-1 signaling is an important immune evasion strategy for efficient acute EBV infection and a significant determinant for virus setpoint during persistent EBV infection.

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.

RNA families in Epstein-Barr virus.

Epstein-Barr virus (EBV) is a tumorigenic human γ-herpesvirus, which produces several known structured RNAs with functional importance: two are implicated in latency maintenance and tumorigenic phenotypes, EBER1 and EBER2; a viral small nucleolar RNA (v-snoRNA1) that may generate a small regulatory RNA; and an internal ribosomal entry site in the EBNA1 mRNA. A recent bioinformatics and RNA-Seq study of EBV identified two novel EBV non-coding (nc)RNAs with evolutionary conservation in lymphocryptoviruses and likely functional importance. Both RNAs are transcribed from a repetitive region of the EBV genome (the W repeats) during a highly oncogenic type of viral latency. One novel ncRNA can form a massive (586 nt) hairpin, while the other RNA is generated from a short (81 nt) intron and is found in high abundance in EBV-infected cells.

Condylomatous genital lesions in cynomolgus macaques from Mauritius.

Genital condyloma-like lesions were observed on male and female cynomolgus macaque monkeys (Macaca fascicularis) originating from the island of Mauritius. Cytobrush and/or biopsy samples were obtained from lesions of 57 affected macaques. Primary histologic features included eosinophilic, neutrophilic, and lymphoplasmacytic penile and vulvar inflammation, epidermal hyperplasia with acanthosis, and increased collagenous stroma. Polymerase chain reaction-based assays to amplify viral DNA revealed the presence of macaque lymphocryptovirus (LCV) DNA but not papillomavirus or poxvirus DNA. Subsequent DNA analyses of 3 genomic regions of LCV identified isolates associated with lesions in 19/25 (76%) biopsies and 19/57 (33%) cytology samples. Variable immunolabeling for proteins related to the human LCV Epstein Barr Virus was observed within intralesional plasma cells, stromal cells, and epithelial cells. Further work is needed to characterize the epidemiologic features of these lesions and their association with LCV infection in Mauritian-origin macaques.

[Epstein-Barr virus (Herpesviridae: Lymphocryptovirus) types 1 and 2 and other viral markers in patients with nasopharyngeal carcinoma in two geographically and ethnically distinct regions of Russia].

INTRODUCTION: The discovery of two types of Epstein-Barr virus (EBV) (EBV-1 and EBV-2) that have different biological properties stimulated the search for neoplasms associated with each type of the virus. The aim of the work is to study the nature of the association of nasopharyngeal cancer (NPC) with EBV-1 and EBV-2, serological activity for each viral type and the concentration of EBV DNA in the blood plasma of two gender, age and ethnic groups of NPC patients that represent geographically and climatically different regions of Russia,. MATERIALS AND METHODS: In the blood plasma of patients with NPC and other non- EBV associated tumors of oral cavity (OTOCEBV-) from the North Caucasian (NCFD) and Central (CFD) Federal Districts of Russia, the types of EBV and the concentration of viral DNA were determined using respectively «nested¼ and real time PCR; titers of IgG and IgA antibodies to viral capsid antigen (VCA) were measured in indirect immunofluorescence assay. RESULTS: The blood plasma samples testing showed that NPC and OTOCEBV- patients were infected with both types of EBV in approximately equal proportions. In two groups of NPC patients infected with one of the virus types only, EBV-1 or EBV-2, respectively, no statistically significant differences were found between the geometric mean values of IgG and IgA anti-EBV antibody titers and viral DNA concentrations in blood plasma. The distribution of virus types was not affected by either patient gender or ethnogeographic origin. The difference was found only between age groups: EBV-2 dominated in NPC patients up to 60 years, and EBV-1 was prevalent in patients over 60 years. CONCLUSION: The lack of the predominance of one of EBV types in NPC patients that are the representatives of different ethnic groups from geographically and climatically different regions, suggests that none of these factors play an important role in the NPC carcinogenesis. Evidently, each type of EBV, EBV-1 or EBV-2, if the necessary conditions arise, are able to exhibit its oncogenic potential to initiate tumor development.

Cloning of the Epstein-Barr virus-related rhesus lymphocryptovirus as a bacterial artificial chromosome: a loss-of-function mutation of the rhBARF1 immune evasion gene.

Rhesus macaques are naturally infected with a gammaherpesvirus which is in the same lymphocryptovirus (LCV) genus as and closely related to Epstein-Barr virus (EBV). The rhesus macaque LCV (rhLCV) contains a repertoire of genes identical to that of EBV, and experimental rhLCV infection of naive rhesus macaques accurately models acute and persistent EBV infection of humans. We cloned the LCL8664 rhLCV strain as a bacterial artificial chromosome to create recombinant rhLCV for investigation in this animal model system. A recombinant rhLCV (clone 16 rhLCV) carrying a mutation in the putative immune evasion gene rhBARF1 was created along with a rescued wild-type (rWT) rhLCV in which the rhBARF1 open reading frame (ORF) was repaired. The rWT rhLCV molecular clone demonstrated viral replication and B-cell immortalization properties comparable to those of the naturally derived LCL8664 rhLCV. Qualitatively, clone 16 rhLCV carrying a mutated rhBARF1 was competent for viral replication and B-cell immortalization, but quantitative assays showed that clone 16 rhLCV immortalized B cells less efficiently than LCL8664 and rWT rhLCV. Functional studies showed that rhBARF1 could block CSF-1 cytokine signaling as well as EBV BARF1, whereas the truncated rhBARF1 from clone 16 rhLCV was a loss-of-function mutant. These recombinant rhLCV can be used in the rhesus macaque animal model system to better understand how a putative viral immune evasion gene contributes to the pathogenesis of acute and persistent EBV infection. The development of a genetic system for making recombinant rhLCV constitutes a major advance in the study of EBV pathogenesis in the rhesus macaque animal model.

[Epstein-Barr virus (Herpesviridae: Gammaherpesvirinae: Lymphocryptovirus: Human gammaherpesvirus 4) in Kalmyks and Slavs living in Russia: virus types, LMP1 oncogene variants, and malignancies].

INTRODUCTION: The discovery of the Epstein-Barr virus types (Herpesviridae: Gammaherpesvirinae: Lymphocryptovirus: Human gammaherpesvirus 4) (EBV) - EBV-1 and EBV-2, which have different transforming abilities in vitro, stimulated the study of their prevalence in populations in order to elucidate the relationship with malignant neoplasms.The aims of the work are to study the prevalence of EBV-1 and EBV-2 among representatives of 2 ethnic groups of Russia, Kalmyks and Slavs, sequencing analysis of the LMP1 oncogene in virus isolates, and analysis of the correlation between virus types and the incidence of certain forms of tumors. MATERIALS AND METHODS: DNA samples were isolated from the biological material of oral swabs obtained from ethnic Kalmyks of the Republic of Kalmykia (RK) (n = 50) and Slavs, residents of the Moscow Region (MR) (n = 40). DNA samples were used to amplify EBV DNA, followed by determination of its concentration per 1 cell of washout, amplification of the LMP1 oncogene in viral samples, their sequencing, and determination of LMP1 protein variants. RESULTS: It has been established that with the same burden of EBV among representatives of both ethnic groups in the Kalmyk group, the ratio of persons infected with transforming and non-transforming types of the virus was almost the same (EBV-1 - 51%; and EBV-2 - 49%). Meanwhile, in the group of Slavs the transforming EBV-1 type virus dominated (80.6%). The predominance of EBV-1 type in representatives of the Slavs correlated with increased incidence of certain forms of tumors in the population of the MR when compared with similar values in the population of the RK, where both types of the virus were prevalent. Differences between the compared rates of cancer incidence were not statistically significant. Analysis of viral isolates showed a similar set of LMP1 variants in both ethnic groups. CONCLUSION: In order to establish the influence of EBV types on the incidence of malignant tumors, additional studies involving representatives of various ethnic groups from different geographical regions are needed.

A global analysis of evolutionary conservation among known and predicted gammaherpesvirus microRNAs.

MicroRNAs (miRNAs) are small, noncoding RNAs which posttranscriptionally regulate gene expression. The current release of the miRNA registry lists 16 viruses which encode a total of 146 miRNA hairpins. Strikingly, 139 of these are encoded by members of the herpesvirus family, suggesting an important role for miRNAs in the herpesvirus life cycle. However, with the exception of 7 miRNA hairpins known to be shared by Epstein-Barr virus (EBV) and the closely related rhesus lymphocryptovirus (rLCV), the known herpesvirus miRNAs show little evidence of evolutionary conservation. We have performed a global analysis of miRNA conservation among gammaherpesviruses which is not limited to family members known to encode miRNAs but includes also those which have not been previously analyzed. For this purpose, we have performed a computational prediction of miRNA candidates of all fully sequenced gammaherpesvirus genomes, followed by sequence/structure alignments. Our results indicate that gammaherpesvirus miRNA conservation is limited to two pairs of viral genomes. One is the already-known case of EBV and rLCV. These viruses, however, share significantly more miRNAs than previously thought, as we identified and experimentally verified 10 novel conserved as well as 7 novel nonconserved rLCV pre-miRNA hairpins. The second case consists of rhesus rhadinovirus (RRV), which is predicted to share at least 9 pre-miRNAs with the closely related Japanese macaque herpesvirus (JMHV). Although several other gammaherpesviruses are predicted to encode large numbers of clustered miRNAs at conserved genomic loci, no further examples of evolutionarily conserved miRNA sequences were found.

Lymphocryptovirus phylogeny and the origins of Epstein-Barr virus.

Specimens from wild and captive primates were collected and novel members of the genus Lymphocryptovirus (subfamily Gammaherpesvirinae) were searched for utilizing PCR for the DNA polymerase gene. Twenty-one novel viruses were detected. Together with previous findings, more than 50 distinct lymphocryptoviruses (LCVs) are now known, with hosts from six primate families (Hominidae, Hylobatidae, Cercopithecidae, Atelidae, Cebidae and Pitheciidae). Further work extended genomic sequences for 25 LCVs to 3.4-7.4 kbp. Phylogenetic trees were constructed, based on alignments of protein sequences inferred from the LCV genomic data. The LCVs fell into three major clades: Clade A, comprising New World viruses; Clade B, containing both Old World monkey viruses and hominoid viruses including Epstein-Barr virus (EBV); and Clade C, containing other hominoid viruses. By comparison with the primate tree, it was proposed that major elements of the LCV tree represented synchronous evolution with host lineages, with the earliest node in both trees being the separation of Old and New World lines, but that some virus lineages originated by interspecies transfer. From comparisons of branch lengths, it was inferred that evolutionary substitution in Clade B has proceeded more slowly than elsewhere in the LCV tree. It was estimated that in Clade B a subclade containing EBV, a gorilla virus and two chimpanzee viruses derived from an Old World monkey LCV line approximately 12 million years ago, and another subclade containing an orang-utan virus and a gibbon virus derived from a macaque LCV line approximately 1.2 million years ago.