Use of specific-pathogen-free (SPF) rhesus macaques to better model oral pediatric cytomegalovirus infection

Background Congenital human cytomegalovirus (HCMV) infection can result in lifelong neurological deficits. Seronegative pregnant woman often acquire primary HCMV from clinically asymptomatic, but HCMV‐shedding children. Methods Potential age‐related differences in viral and immune parameters of primary RhCMV infection were examined in an oral rhesus CMV infection model in specific pathogen free macaques. RhCMV shedding was measured by real time PCR in plasma, saliva and urine. Immune parameters, including neutralizing and binding antibodies and RhCMV‐specific T cell responses, were assessed in longitudinally collected blood samples. Results The oral RhCMV infection model in infant SPF rhesus macaques demonstrated that (i) the susceptibility to oral RhCMV infection declines with age, and (ii) infant macaques shed RhCMV more persistently and at higher titers compared to adult macaques. (iii) Conclusions The oral infant RhCMV infection model appears to reflect viral pathogenesis in human HCMV‐infected children. Larger studies are needed to define immune parameters associated with better control of RhCMV in adult compared to young animals.     

Prevalence of Viremia and Oral Shedding of Rhesus Rhadinovirus and Retroperitoneal Fibromatosis Herpesvirus in Large Age-Structured Breeding Groups of Rhesus Macaques (Macaca mulatta)

We performed a cross-sectional study to estimate the prevalence of 2 gamma-2-herpesviruses, rhesus rhadinovirus (RRV) and retroperitoneal fibromatosis herpesvirus (RFHV), in breeding colonies of rhesus macaques. Of 90 animals selected for sampling, 73 (81%) were positive for RRV, which was detected only in blood in 22 (24%), only in saliva in 15 (16%), and in both blood and saliva in 36 (40%). Detection of RRV DNA in blood and saliva was significantly higher in animals younger than 2 y. In comparison, RFHV was detected in 40 (44%) of the 90 animals: only in blood in 5 (6%), only in saliva in 26 (29%), and in both blood and saliva in 9 (10%). Dual infection was detected in 38 (42%) animals; RFHV was only detected in coinfections. The mean RRV genome copy number in blood was significantly higher than that for RFHV. Age was a significant predictor of RRV copy number in blood and RFHV copy number in saliva. Of the 90 animals, 88 (98%) were positive for rhadinoviral antibodies on an immunofluorescent assay. Both RRV and RFHV are highly endemic in socially housed breeding colonies of rhesus macaques, and their patterns of infection are similar to that for the betaherpesvirus rhesus cytomegalovirus.Abbreviations: CNPRC, California National Primate Research Center; GE, genome equivalents (copy number); KSHV, Kaposi sarcoma-associated herpesvirus; RFHV, retroperitoneal fibromatosis herpesvirus; RRV, rhesus rhadinovirus; OSM, oncostatin M geneThe Rhadinovirus genus of gamma-2-herpesviruses is divided into 2 subgroups, RV1 and RV2, based on genomic sequence comparisons.^36,44 Rhadinovirus infections are generally subclinical in immunocompetent natural hosts, and overt disease is thought to arise only when hosts are immunocompromised.²⁸ In addition, the ability to establish both lytic and latent infections, a hallmark of the Herpesviridae family, occurs during rhadinovirus infections.^1,43 The RV1 subgroup includes Kaposi sarcoma-associated herpesvirus (KSHV; also referred to as human herpesvirus 8)^12,32 the causative agent of Kaposi sarcoma, an angioproliferative lesion composed of a mixed population of endothelial, inflammatory and spindle cells.^19,24 Furthermore, KSHV has been linked etiologically to 2 different B-cell lymphomas: primary effusion lymphoma and multicentric Castleman disease.¹⁷ Retroperitoneal fibromatosis herpesvirus (RFHV) is also a member of the RV1 subgroup and is thought to be the macaque homolog of KSHV.^{4,8,14,36,37,40} DNA sequences specific for RFHV have been detected in retroperitoneal fibromatosis in macaques coinfected with the potentially immunosuppressive simian betaretrovirus type 2.⁷ Histologic similarities between retroperitoneal fibromatosis and KS lesions seen in humans coinfected with KSHV and HIV have been previously described.^7,9,21,37 During outbreaks of simian betaretrovirus type 2 disease at 2 national primate research centers in the 1980s, the incidence of retroperitoneal fibromatosis was reported to be 5% to 7% for animals younger than 2 y and 1% across all age groups.^7,37,45 Since the end of these outbreaks in the late 1980s, retroperitoneal fibromatosis has occurred only rarely in primate colonies. The majority of published RFHV studies have focused on animals with recognized retroperitoneal fibromatosis lesions.^9-11 However, RFHV has proven extremely difficult to isolate and, to date, has not been propagated successfully in vitro, and only a small portion of the RFHV genome has been sequenced.^36,37,40,44 In this study we determined the prevalence of RFHV infection in nondiseased animals and address aspects of the natural history of this virus infection in captive macaque populations.Rhesus rhadinovirus (RRV) is a member of the RV2 subgroup, which naturally infects rhesus macaques.^15,38,44 RRV was isolated independently at 2 national primate research centers in the late 1990s from rhesus macaques.^15,42 Both RRV isolates were shown to have noteworthy sequence similarity to KSHV and RFHV.^2,8,15,42 Unlike RFHV, RRV can be propagated readily in vitro, thus facilitating studies of the lytic replication cycle.^5,6,16 Experimental coinfection of rhesus macaques with SIV and RRV resulted in a lymphoproliferative disease resembling multicentric Castleman disease, but variations in disease outcome between the 2 RRV isolates were noted.^30,49 More recently, RRV has been shown to be associated with nonHodgkin lymphoma and retroperitoneal fibromatosis in SIV-infected rhesus macaques.³⁴ Therefore, RRV infection in macaques is a highly useful animal model for the study of KSHV infection in humans, including studies of viral pathogenesis, factors affecting prevalence of infection, viral shedding, and transmission.^2,25,31,42 In addition, RRV is a persistent virus targeted for elimination in some specific pathogen free (SPF) macaque breeding populations. A better understanding of the natural history of RRV and RFHV infections will lead to improved characterization of host–virus interactions, contribute to the refinement of these nonhuman primate models, and allow more efficient management of SPF colonies.Here we report estimates of the prevalence of viremia and oral shedding of RRV and RFHV in large age-structured breeding colonies of rhesus macaques. Both viruses were highly endemic in the breeding populations we tested, and coinfection with both viruses was common.     

Two Distinct Gamma-2 Herpesviruses in African Green Monkeys: a Second Gamma-2 Herpesvirus Lineage among Old World Primates?

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.     

Pathogenic analysis of coxsackievirus A10 in rhesus macaques

Coxsackievirus A10 (CV-A10) is one of the etiological agents associated with hand, foot and mouth disease (HFMD) and also causes a variety of illnesses in humans, including pneumonia, and myocarditis. Different people, particularly young children, may have different immunological responses to infection. Current CV-A10 infection animal models provide only a rudimentary understanding of the pathogenesis and effects of this virus. The characteristics of CV-A10 infection, replication, and shedding in humans remain unknown. In this study, rhesus macaques were infected by CV-A10 via respiratory or digestive route to mimic the HFMD in humans. The clinical symptoms, viral shedding, inflammatory response and pathologic changes were investigated in acute infection (1–11 day post infection) and recovery period (12–180 day post infection). All infected rhesus macaques during acute infection showed obvious viremia and clinical symptoms which were comparable to those observed in humans. Substantial inflammatory pathological damages were observed in multi-organs, including the lung, heart, liver, and kidney. During the acute period, all rhesus macaques displayed clinical signs, viral shedding, normalization of serum cytokines, and increased serum neutralizing antibodies, whereas inflammatory factors caused some animals to develop severe hyperglycemia during the recovery period. In addition, there were no significant differences between respiratory and digestive tract infected animals. Overall, all data presented suggest that the rhesus macaques provide the first non-human primate animal model for investigating CV-A10 pathophysiology and assessing the development of potential human therapies.     

Experimental infection of rhesus and pig-tailed macaques with macaque rhadinoviruses

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.     

Vaccine-induced control of viral shedding following rhesus cytomegalovirus challenge in rhesus macaques

The use of animal models of human cytomegalovirus (HCMV) infection is critical to refine HCMV vaccine candidates. Previous reports have demonstrated that immunization of rhesus monkeys against rhesus cytomegalovirus (RhCMV) can reduce both local and systemic replication of RhCMV following experimental RhCMV challenge. These studies used prime/boost combinations of DNA expression plasmids alone or DNA priming and boosting with either inactivated virion particles or modified vaccinia virus Ankara (MVA) expressing the same antigens. Viral outcomes included reduced RhCMV replication at the site of subcutaneous inoculation and RhCMV viremia following intravenous inoculation. Since shedding of cytomegalovirus from mucosal surfaces is critical for horizontal transmission of the virus, DNA priming/MVA boosting was evaluated for the ability to reduce oral shedding of RhCMV following subcutaneous challenge. Of six rhesus monkeys vaccinated exclusively against RhCMV glycoprotein B (gB), phosphoprotein 65 (pp65), and immediate-early 1 (IE1), half showed viral loads in saliva that were lower than those of control monkeys by 1 to 3 orders of magnitude. Further, there was a strong association of memory pp65 T cell responses postchallenge in animals exhibiting the greatest reduction in oral shedding. These results highlight the fact that a DNA/MVA vaccination regimen can achieve a notable reduction in a critical parameter of viral replication postchallenge. The recently completed clinical trial of a gB subunit vaccine in which the rate of HCMV infection was reduced by 50% in the individuals receiving the vaccine is consistent with the results of this study suggesting that additional immunogens are likely essential for maximum protection in an outbred human population.     

Viral factors determine progression to AIDS in simian immunodeficiency virus-infected newborn rhesus macaques.

To evaluate how viral variants may affect disease progression in human pediatric AIDS, we studied the potential of three simian immunodeficiency virus (SIV) isolates to induce simian AIDS in newborn rhesus macaques. The three virus isolates were previously shown to range from pathogenic (SIVmac251 and SIVmac239) to nonpathogenic (SIVmac1A11) when inoculated intravenously into juvenile and adult rhesus macaques. Six newborn macaques inoculated with pathogenic, uncloned SIVmac251 developed persistent, high levels of cell-associated and cell-free viremia, had no detectable antiviral antibodies, and had poor weight gain; these animals all exhibited severe clinical disease and pathologic lesions diagnostic for simian AIDS and were euthanatized 10 to 26 weeks after inoculation. Two newborns inoculated with pathogenic, molecularly cloned SIVmac239 developed persistent high virus load in peripheral blood, but both animals had normal weight gain and developed antiviral antibodies. One of the SIVmac239-infected neonates exhibited pathologic lesions diagnostic for SAIDS and was euthanatized at 34 weeks after inoculation; the other SIVmac239-infected neonate remained alive and exhibited no significant clinical disease for more than 1 year after inoculation. In contrast, three newborn rhesus macaques inoculated with the nonpathogenic molecular clone, SIVmac1A11, had transient, low-level viremia, seroconverted by 10 weeks after inoculation, had normal weight gain, and remained healthy for over 1 year. These results indicate that (i) newborn rhesus macaques infected with an uncloned, virulent SIVmac isolate have a more rapid, fulminant disease course than do adults inoculated with the same virus, (ii) the most rapid disease progression is associated with lack of a detectable humoral immune response in SIV-infected infant macaques, (iii) a molecularly cloned, attenuated SIV isolate is nonpathogenic in neonatal macaques, and (iv) SIV-infected neonatal macaques exhibit patterns of infection, virus load, and disease progression similar to those observed in human immunodeficiency virus-infected children. This SIV/neonatal rhesus model of pediatric AIDS provides a rapid, sensitive model with which to compare the virulence of SIV isolates and to study the mechanisms underlying the differences in disease progression in human immunodeficiency virus-infected infants.     

The intracytoplasmic domain of the Env transmembrane protein is a locus for attenuation of simian immunodeficiency virus SIVmac in rhesus macaques

The human and simian immunodeficiency virus (HIV-1 and SIVmac) transmembrane proteins contain unusually long intracytoplasmic domains (ICD-TM). These domains are suggested to play a role in envelope fusogenicity, interaction with the viral matrix protein during assembly, viral infectivity, binding of intracellular calmodulin, disruption of membranes, and induction of apoptosis. Here we describe a novel mutant virus, SIVmac-M4, containing multiple mutations in the coding region for the ICD-TM of pathogenic molecular clone SIVmac239. Parental SIVmac239-Nef+ produces high-level persistent viremia and simian AIDS in both juvenile and newborn rhesus macaques. The ICD-TM region of SIVmac-M4 contains three stop codons, a +1 frameshift, and mutation of three highly conserved, charged residues in the conserved C-terminal alpha-helix referred to as lentivirus lytic peptide 1 (LLP-1). Overlapping reading frames for tat, rev, and nef are not affected by these changes. In this study, four juvenile macaques received SIVmac-M4 by intravenous injection. Plasma viremia, as measured by branched-DNA (bDNA) assay, reached a peak at 2 weeks postinoculation but dropped to below detectable levels by 12 weeks. At over 1.5 years postinoculation, all four juvenile macaques remain healthy and asymptomatic. In a subsequent experiment, four neonatal rhesus macaques were given SIVmac-M4 intravenously. These animals exhibited high levels of viremia in the acute phase (2 weeks postinoculation) but are showing a relatively low viral load in the chronic phase of infection, with no clinical signs of disease for 1 year. These findings demonstrated that the intracytoplasmic domain of the transmembrane Env (Env-TM) is a locus for attenuation in rhesus macaques.     

Intrinsic susceptibility of rhesus macaque peripheral CD4(+) T cells to simian immunodeficiency virus in vitro is predictive of in vivo viral replication

Previous studies with simian immunodeficiency virus (SIV) infection of rhesus macaques suggested that the intrinsic susceptibility of peripheral blood mononuclear cells (PBMC) to infection with SIV in vitro was predictive of relative viremia after SIV challenge. The present study was conducted to evaluate this parameter in a well-characterized cohort of six rhesus macaques selected for marked differences in susceptibility to SIV infection in vitro. Rank order relative susceptibility of PBMC to SIVsmE543-3-infection in vitro was maintained over a 1-year period of evaluation. Differential susceptibility of different donors was maintained in CD8(+) T-cell-depleted PBMC, macrophages, and CD4(+) T-cell lines derived by transformation of PBMC with herpesvirus saimiri, suggesting that this phenomenon is an intrinsic property of CD4(+) target cells. Following intravenous infection of these macaques with SIVsmE543-3, we observed a wide range in plasma viremia which followed the same rank order as the relative susceptibility established by in vitro studies. A significant correlation was observed between plasma viremia at 2 and 8 weeks postinoculation and in vitro susceptibility (P < 0.05). The observation that the two most susceptible macaques were seropositive for simian T-lymphotropic virus type 1 may suggests a role for this viral infection in enhancing susceptibility to SIV infection in vitro and in vivo. In summary, intrinsic susceptibility of CD4(+) target cells appears to be an important factor influencing early virus replication patterns in vivo that should be considered in the design and interpretation of vaccine studies using the SIV/macaque model.     

Experimental inoculation of juvenile rhesus macaques with primate enteric caliciviruses

BACKGROUND: Tissue culture-adapted Tulane virus (TV), a GI.1 rhesus enteric calicivirus (ReCV), and a mixture of GII.2 and GII.4 human norovirus (NoV)-containing stool sample were used to intrastomacheally inoculate juvenile rhesus macaques (Macaca mulatta) in order to evaluate infection caused by these viruses. METHODOLOGY & FINDINGS: Two of the three TV-inoculated macaques developed diarrhea, fever, virus-shedding in stools, inflammation of duodenum and 16-fold increase of TV-neutralizing (VN) serum antibodies but no vomiting or viremia. No VN-antibody responses could be detected against a GI.2 ReCV strain FT285, suggesting that TV and FT285 represent different ReCV serotypes. Both NoV-inoculated macaques remained asymptomatic but with demonstrable virus shedding in one animal. Examination of duodenum biopsies of the TV-inoculated macaques showed lymphocytic infiltration of the lamina propria and villous blunting. TV antigen-positive (TV+) cells were detected in the lamina propria. In most of the TV+ cells TV co-localized perinuclearly with calnexin--an endoplasmic reticulum protein. A few CD20+TV+ double-positive B cells were also identified in duodenum. To corroborate the authenticity of CD20+TV+ B cells, in vitro cultures of peripheral blood mononuclear cells (PBMCs) from healthy macaques were inoculated with TV. Multicolor flow cytometry confirmed the presence of TV antigen-containing B cells of predominantly CD20+HLA-DR+ phenotype. A 2-log increase of viral RNA by 6 days post inoculation (p<0.05) suggested active TV replication in cultured lymphocytes. CONCLUSIONS/SIGNIFICANCE: Taken together, our results show that ReCVs represent an alternative cell culture and animal model to study enteric calicivirus replication, pathogenesis and immunity.     

Comparative efficacy of a canine distemper-measles and a standard measles vaccine for immunization of rhesus macaques (Macaca mulatta)

Measles virus (MV), a highly infective paramyxovirus, has caused sporadic epizootics characterized by high morbidity and increased mortality in nonhuman primates. Measles vaccines for human use, although effective, are cost prohibitive for use in primate colonies. We compared the efficacy of one or two doses of Vanguard D-M, a canine distemper-measles (CD-M) vaccine, with a single dose of Attenuvax, a human measles vaccine. Compared with 81% of animals inoculated with Attenuvax, all animals inoculated with one or two doses of Vanguard developed detectable MV antibodies. One year after immunization, six juveniles from each vaccine group, along with three unvaccinated controls, were challenged with pathogenic MV and were monitored for clinical signs of disease, viremia, viral shedding, and immune response. All uninoculated controls developed clinical disease and viremia, and shed virus in nasopharangeal secretions. Subclinical viremia without viral shedding was identified in two Attenuvax- and two single-dose Vanguard-inoculated animals. Viremia was not detected in any two-dose Vanguard-inoculated animals. Significantly higher neutralization antibody titers were observed in animals receiving Vanguard. Results of this study indicate that Vanguard is at least as efficacious as Attenuvax for protection of rhesus macaques. The considerably lower cost of Vanguard makes vaccination against measles in large breeding colonies economically feasible.     

Viral interleukin-6 encoded by rhesus macaque rhadinovirus is associated with lymphoproliferative disorder (LPD)

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.     

Effect of virus dose and nonoxynol-9 on the genital transmission of SIV in rhesus macaques

One inoculation of cell-free SIVmac (50 TCID50) caused persistent viremia in nine of 13 female rhesus macaques inoculated intravaginally. Persistent viremia was produced in two of four male rhesus macaques by twice placing cell-free SIVMAC (50 TCID50) onto the skin and urethral os of the penis. Placing a spermicide containing nonoxynol-9 into the vaginal canal prior to repeated intravaginal inoculations of SIV prevented transmission of the virus in three of six female rhesus macaques.     

Construction of an infectious rhesus rhadinovirus bacterial artificial chromosome for the analysis of Kaposi's sarcoma-associated herpesvirus-related disease development

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.     

Analysis of 4.3 kilobases of divergent locus B of macaque retroperitoneal fibromatosis-associated herpesvirus reveals a close similarity in gene sequence and genome organization to Kaposi's sarcoma-associated herpesvirus

We previously identified retroperitoneal fibromatosis-associated herpesvirus (RFHV) as a simian homolog of Kaposi's sarcoma-associated herpesvirus (KSHV) in a fibroproliferative malignancy of macaques that has similarities to Kaposi's sarcoma. In this report, we cloned 4.3 kb of divergent locus B (DL-B) flanking the DNA polymerase gene from two variants of RFHV from different species of macaque with a consensus degenerate hybrid oligonucleotide primer approach. Within the DL-B region of RFHV, viral homologs of the cellular interleukin-6, dihydrofolate reductase, and thymidylate synthase genes were identified, along with a homolog of the gammaherpesvirus open reading frame (ORF) 10. In addition, a homolog of the KSHV ORF K3, the modulator of immune recognition-1, was identified. Our data show a close similarity in sequence conservation, gene content, and genomic structure between RFHV and KSHV which strongly supports the grouping of these viral species within the same RV-1 rhadinovirus lineage and the hypothesis that RFHV is the macaque homolog of KSHV.     

Replication in a superficial epithelial cell niche explains the lack of pathogenicity of primate foamy virus infections

Foamy viruses (FVs) are ancient retroviruses that are ubiquitous in nonhuman primates (NHPs). While FVs share many features with pathogenic retroviruses, such as human immunodeficiency virus, FV infections of their primate hosts have no apparent pathological consequences. Paradoxically, FV infections of many cell types in vitro are rapidly cytopathic. Previous work has shown that low levels of proviral DNA are found in most tissues of naturally infected rhesus macaques, but these proviruses are primarily latent. In contrast, viral RNA, indicative of viral replication, is restricted to tissues of the oral mucosa, where it is abundant. Here, we perform in situ hybridization on tissues from rhesus macaques naturally infected with simian FV (SFV). We show that superficial differentiated epithelial cells of the oral mucosa, many of which appear to be shedding from the tissue, are the major cell type in which SFV replicates. Thus, the innocuous nature of SFV infection can be explained by replication that is limited to differentiated superficial cells that are short-lived and shed into saliva. This finding can also explain the highly efficient transmission of FVs among NHPs.     

Evaluation of passively transferred, nonneutralizing antibody-dependent cellular cytotoxicity-mediating IgG in protection of neonatal rhesus macaques against oral SIVmac251 challenge

Previously, Ab-dependent cellular cytotoxicity (ADCC) was significantly correlated with reduced acute viremia upon intrarectal SIVmac251 challenge of immunized rhesus macaques. To directly assess ADCC protective efficacy, six neonatal macaques were infused s.c. with immune IgG (220 mg/kg) purified from the immunized animals and positive for ADCC and Ab-dependent cell-mediated viral inhibition (ADCVI) activities. Six neonates received control IgG. The neonates were challenged twice orally with 10(5) 50% inhibiting tissue culture-infective dose of SIVmac251 2 days post-IgG infusion. At challenge, plasma of neonates that received immune IgG did not neutralize SIVmac251 but had geometric mean ADCC titers of 48,130 and 232,850 against SIVmac251 -infected and gp120-coated targets, respectively. Peak ADCVI activity varied from 62 to 81%. ADCC activity declined with the 2-wk IgG half-life but was boosted at wk 4, together with de novo ADCC-mediating Abs in controls, by postchallenge viremia. ADCVI activity was similarly induced. No protection, assessed by viral burdens, CD4 counts, and time to euthanasia was observed. Possible factors contributing to the discrepancy between the previous correlation and lack of protection here include: the high oral challenge dose compared with the 400-fold lower intrarectal dose; the challenge route with regard to viral dissemination and distribution of infused IgG; insufficient NK effector activity and/or poor functionality in newborns; insufficient immune IgG; and the possibility that the previous correlation of ADCC with protection was augmented by cellular immune responses also present at challenge. Future studies should explore additional challenge routes in juvenile macaques using higher amounts of potent IgG preparations.