No benefit of therapeutic vaccination in clinically healthy cats persistently infected with feline leukemia virus.

Therapeutic vaccinations have a potential application in infections where no curative treatment is available. In contrast to HIV, efficacious vaccines for a cat retrovirus, feline leukemia virus (FeLV), are commercially available. However, the infection is still prevalent, and no effective treatment of the infection is known. By vaccinating persistently FeLV-infected cats and presenting FeLV antigens to the immune system of the host, e.g., in the form of recombinant and/or adjuvanted antigens, we intended to shift the balance toward an advantage of the host so that persistent infection could be overcome by the infected cat. Two commercially available FeLV vaccines efficacious in protecting naïve cats from FeLV infection were tested in six experimentally and persistently FeLV-infected cats: first, a canarypox-vectored vaccine, and second, an adjuvanted, recombinant envelope vaccine was repeatedly administered with the aim to stimulate the immune system. No beneficial effects on p27 antigen and plasma viral RNA loads, anti-FeLV antibodies, or life expectancy of the cats were detected. The cats were unable to overcome or decrease viremia. Some cats developed antibodies to FeLV antigens although not protective. Thus, we cannot recommend vaccinating persistently FeLV-infected cats as a means of improving their FeLV status, quality of life or life expectancy. We suggest testing of all cats for FeLV infection prior to FeLV vaccination.

Elevated HERV-K mRNA expression in PBMC is associated with a prostate cancer diagnosis particularly in older men and smokers.

Aberrant expression of subgroup k human endogenous retroviruses (HERV-K) has been observed in prostate cancer. This subgroup is unique because it encodes sequences in the human genome containing open reading frames for near intact retroviruses. We hypothesized that HERV-K reactivation could serve as a non-invasive early disease detection marker for prostate cancer. We evaluated HERV-K gag messenger RNA (mRNA) expression in blood samples of African-American and European-American men using a case-control design via quantitative real-time PCR. Additionally, we examined HERV-K envelope protein expression in prostate tumors by immunohistochemistry. HERV-K envelope protein was commonly upregulated in prostate tumors, but more so in tumors of African-American than European-American patients (61% versus 40%, P < 0.01). Examining HERV-K gag expression in peripheral blood mononuclear cells (PBMC) from 294 cases and 135 healthy men, we found that the abundance of HERV-K gag message was significantly higher in cases than controls and was associated with increased plasma interferon-γ. Men with gag expression in the highest quartile had >12-fold increased odds {odds ratio = 12.87 [95% confidence interval 6.3-26.25]} of being diagnosed with prostate cancer than those in the lowest quartile. Moreover, our results showed that HERV-K expression may perform better as a disease biomarker in older than younger men (whereas the sensitivity of prostate-specific antigen (PSA) testing decreases with age) and in men with a smoking history compared with never smokers. Combining non-invasive HERV-K testing with PSA testing may improve the efficacy of prostate cancer detection specifically among older men and smokers who tend to develop a more aggressive disease.

Efficacy of an inactivated FeLV vaccine compared to a recombinant FeLV vaccine in minimum age cats following virulent FeLV challenge.

The aim of the study was to determine the efficacy of an inactivated feline leukemia virus (FeLV) vaccine (Versifel(®) FeLV, Zoetis.) compared to a recombinant FeLV vaccine (Purevax(®) FeLV, Merial Animal Health) in young cats, exposed under laboratory conditions to a highly virulent challenge model. The study was designed to be consistent with the general immunogenicity requirements of the European Pharmacopoeia 6.0 Monograph 01/2008:1321-Feline Leukaemia Vaccine (Inactivated) with the exception that commercial-strength vaccines were assessed. Fifty seronegative cats (8-9 weeks old) were vaccinated subcutaneously on two occasions, three weeks apart, with either placebo (treatment group T01), Versifel FeLV Vaccine (treatment group T02), or Purevax FeLV Vaccine (treatment group T03) according to the manufacturer's directions. Cats were challenged three weeks after the second vaccination with a virulent FeLV isolate (61E strain). Persistent FeLV antigenemia was determined from 3 to 15 weeks postchallenge. Bone marrow samples were tested for the presence of FeLV proviral DNA to determine FeLV latent infection. At week 15 after challenge with the virulent FeLV 61E strain, the Versifel FeLV Vaccine conferred 89.5% protection against FeLV persistent antigenemia and 94.7% protection against FeLV proviral DNA integration in bone marrow cells. In comparison, the Purevax FeLV Vaccine conferred 20% protection against FeLV persistent antigenemia and 35% protection against FeLV proviral DNA integration in bone marrow cells following challenge. The data from this study show that the Versifel FeLV Vaccine was efficacious in preventing both FeLV persistent p27 antigenemia and FeLV proviral DNA integration in bone marrow cells of cats challenged with this particular challenge model under laboratory conditions and provided better protection than Purevax FeLV in this experimental challenge model with highly virulent FeLV.

A survey of feline leukaemia virus infection of domestic cats from selected areas in Harare, Zimbabwe.

A cross-sectional study was conducted to detect the feline leukaemia virus (FeLV) p27 antigen and to determine risk factors and the haematological changes associated with infection in domestic cats in Zimbabwe. Sera were collected for detection of the p27 antigen, urea, creatinine, alanine aminotransferase and gamma-glutamyl transferase levels, whilst whole blood was collected for haematology. FeLV p27 antigen was detected using a rapid enzyme-linked immunosorbent assay (ELISA) test kit. Data on risk factors were analysed using a logistic regression model. Of the 100 cats tested, 41% (95% CI: 31.19% - 50.81%) (41/100) were positive for the FeLV p27 antigen. Sex and health status of cats were not significantly (p > 0.05) associated with infection. Intact cats (OR = 9.73), those living in multicat housing (OR = 5.23) and cats that had access to outdoor life (OR = 35.5) were found to have higher odds of infection compared with neutered cats, those living in single-cat housing, and without access to outdoor life, respectively. Biochemistry and haematology revealed no specific changes. The results showed that FeLV infection was high in sampled cats, providing evidence of active infection. Thus, it would be prudent to introduce specific control measures for FeLV infection in Zimbabwe.

Development and clinical evaluation of a rapid diagnostic kit for feline leukemia virus infection.

Feline leukemia virus (FeLV) causes a range of neoplastic and degenerative diseases in cats. To obtain a more sensitive and convenient diagnosis of the disease, we prepared monoclonal antibodies specific for the FeLV p27 to develop a rapid diagnostic test with enhanced sensitivity and specificity. Among these antibodies, we identified two clones (hybridomas 8F8B5 and 8G7D1) that specifically bound to FeLV and were very suitable for a diagnostic kit. The affinity constants for 8F8B5 and 8G7D1 were 0.35 × 108 and 0.86 × 108, respectively. To investigate the diagnostic abilities of the rapid kit using these antibodies, we performed several clinical studies. Assessment of analytical sensitivity revealed that the detection threshold of the rapid diagnostic test was 2 ng/mL for recombinant p27 and 12.5 × 104 IU/mL for FeLV. When evaluating 252 cat sera samples, the kit was found to have a kappa value of 0.88 compared to polymerase chain reaction (PCR), indicating a significant correlation between data from the rapid diagnostic test and PCR. Sensitivity and specificity of the kit were 95.2% (20/21) and 98.5% (257/261), respectively. Our results demonstrated that the rapid diagnostic test would be a suitable diagnostic tool for the rapid detection of FeLV infection in cats.

Development and clinical evaluation of a rapid diagnostic kit for feline leukemia virus infection

Feline leukemia virus (FeLV) causes a range of neoplastic and degenerative diseases in cats. To obtain a more sensitive and convenient diagnosis of the disease, we prepared monoclonal antibodies specific for the FeLV p27 to develop a rapid diagnostic test with enhanced sensitivity and specificity. Among these antibodies, we identified two clones (hybridomas 8F8B5 and 8G7D1) that specifically bound to FeLV and were very suitable for a diagnostic kit. The affinity constants for 8F8B5 and 8G7D1 were 0.35 x 10(9) and 0.86 x 10(9), respectively. To investigate the diagnostic abilities of the rapid kit using these antibodies, we performed several clinical studies. Assessment of analytical sensitivity revealed that the detection threshold of the rapid diagnostic test was 2 ng/mL for recombinant p27 and 12.5 x 10(4) IU/mL for FeLV. When evaluating 252 cat sera samples, the kit was found to have a kappa value of 0.88 compared to polymerase chain reaction (PCR), indicating a significant correlation between data from the rapid diagnostic test and PCR. Sensitivity and specificity of the kit were 95.2% (20/21) and 98.5% (257/261), respectively. Our results demonstrated that the rapid diagnostic test would be a suitable diagnostic tool for the rapid detection of FeLV infection in cats.

Enhancing SIV-specific immunity in vivo by PD-1 blockade.

Chronic immunodeficiency virus infections are characterized by dysfunctional cellular and humoral antiviral immune responses. As such, immune modulatory therapies that enhance and/or restore the function of virus-specific immunity may protect from disease progression. Here we investigate the safety and immune restoration potential of blockade of the co-inhibitory receptor programmed death 1 (PD-1) during chronic simian immunodeficiency virus (SIV) infection in macaques. We demonstrate that PD-1 blockade using an antibody to PD-1 is well tolerated and results in rapid expansion of virus-specific CD8 T cells with improved functional quality. This enhanced T-cell immunity was seen in the blood and also in the gut, a major reservoir of SIV infection. PD-1 blockade also resulted in proliferation of memory B cells and increases in SIV envelope-specific antibody. These improved immune responses were associated with significant reductions in plasma viral load and also prolonged the survival of SIV-infected macaques. Blockade was effective during the early (week 10) as well as late ( approximately week 90) phases of chronic infection even under conditions of severe lymphopenia. These results demonstrate enhancement of both cellular and humoral immune responses during a pathogenic immunodeficiency virus infection by blocking a single inhibitory pathway and identify a novel therapeutic approach for control of human immunodeficiency virus infections.

Endogenous retrovirus type W GAG and envelope protein antigenemia in serum of schizophrenic patients.

BACKGROUND: Recent and independent molecular studies have shown an association between human endogenous retroviruses type "W" family (HERV-W) and schizophrenia, mostly by polymerase chain reaction studies, but none has yet addressed specific antigen detection in living patients. METHODS: Forty-nine schizophrenic patients and an equivalent number of healthy control subjects were included in the present exploratory study. The HERV-W GAG and envelope (ENV) proteins were quantified in the serum with a dedicated immunoassay set-up with specific monoclonal antibodies to either antigen. RESULTS: In schizophrenic patients, positive antigenemia for ENV was found in 23 of 49 (47%) and for GAG in 24 of 49 (49%). Only 1 of 30 (3%) for ENV and 2 of 49 (4%) for GAG were positive in blood donors (p < .01 for ENV; p < .001 for GAG). Interestingly, bioclinical data analyses revealed significant correlation between GAG or ENV antigenemia (a protein causing dysimmune inflammatory effects) and C-reactive protein (CRP) levels (a systemic inflammation biomarker). CONCLUSIONS: Frequently elevated CRP has previously been described in schizophrenic patients and has been shown to match with an evolution toward cognitive deficit and neuronal loss. Elsewhere viruses such as influenza, long-associated with risk for schizophrenia through perinatal infections, have been shown to activate HERV-W elements in human cells. We therefore discuss a relationship between environment factors long-associated with early risk, genetic factors represented by this endogenous family, the production of its pro-inflammatory ENV protein and known "inflammation-mediated" neurotoxicity, as a possible hypothesis for a pathogenic cascade in association with HERV-W. Our present results thus confirm that HERV-W studies have opened a novel avenue of research in schizophrenia.

Contribution of CD8+ T cells to containment of viral replication and emergence of mutations in Mamu-A*01-restricted epitopes in Simian immunodeficiency virus-infected rhesus monkeys.

Here, we investigated the containment of virus replication in simian immunodeficiency virus (SIV) infection by CD8(+) lymphocytes. Escape mutations in Mamu-A*01 epitopes appeared first in SIV Tat TL8 and then in SIV Gag p11C. The appearance of escape mutations in SIV Gag p11C was coincident with compensatory changes outside of the epitope. Eliminating CD8(+) lymphocytes from rhesus monkeys during primary infection resulted in more rapid disease progression that was associated with preservation of canonical epitopes. These results confirm the importance of cytotoxic T cells in controlling viremia and the constraint on epitope sequences that require compensatory changes to go to fixation.

In vitro characterization of primary SIVsmm isolates belonging to different lineages. In vitro growth on rhesus macaque cells is not predictive for in vivo replication in rhesus macaques.

We report in vitro characterization of 11 SIVsmm strains of six lineages co-circulating in naturally infected sooty mangabeys (SMs) from US Primate Centers and showed no major differences in the in vitro replication pattern between different SIVsmm lineages. Primary SIVsmm isolates utilized CCR5 and Bonzo co-receptors in vitro. SIVsmm growth in human T cell lines was isolate-, not lineage-specific, with poor replication on Molt4-Clone8, CEMss and PM1 cells and better replication on MT2, SupT1 and CEMx174 cells. All primary SIVsmm isolates replicated on SM and human PBMCs. In vitro replication in macaques varied widely, with moderate to high replication in pig-tailed macaque PBMCs, enhanced by CD8+ T cell depletion, and highly variable replication on rhesus macaque (Rh) PBMCs. Primary SIVsmm isolates replicated in Rh monocyte-derived dendritic cells (MDDCs) and monocyte-derived macrophages (MDMs). In vivo, SIVsmm isolates replicated at high levels in all SIVsmm-infected Rh. The poor in vitro replication of primary SIVsmm isolates in Rh cells did not correlate with in vivo replication, emphasizing the value of in vivo studies.

Maintenance of HIV-specific CD4+ T cell help distinguishes HIV-2 from HIV-1 infection.

Unlike HIV-1-infected people, most HIV-2-infected subjects maintain a healthy CD4+ T cell count and a strong HIV-specific CD4+ T cell response. To define the cellular immunological correlates of good prognosis in HIV-2 infection, we conducted a cross-sectional study of HIV Gag-specific T cell function in HIV-1- and HIV-2-infected Gambians. Using cytokine flow cytometry and lymphoproliferation assays, we show that HIV-specific CD4+ T cells from HIV-2-infected individuals maintained proliferative capacity, were not terminally differentiated (CD57-), and more frequently produced IFN-gamma or IL-2 than CD4+ T cells from HIV-1-infected donors. Polyfunctional (IFN-gamma+/IL-2+) HIV-specific CD4+ T cells were found exclusively in HIV-2+ donors. The disparity in CD4+ T cell responses between asymptomatic HIV-1- and HIV-2-infected subjects was not associated with differences in the proliferative capacity of HIV-specific CD8+ T cells. This study demonstrates that HIV-2-infected donors have a well-preserved and functionally heterogeneous HIV-specific memory CD4+ T cell response that is associated with delayed disease progression in the majority of infected people.

Human T-lymphotropic virus type 1 mitochondrion-localizing protein p13(II) is required for viral infectivity in vivo.

Human T-lymphotropic virus type 1 (HTLV-1), the etiological agent of adult T-cell leukemia, encodes unique regulatory and accessory proteins in the pX region of the provirus, including the open reading frame II product p13(II). p13(II) localizes to mitochondria, binds farnesyl pyrophosphate synthetase, an enzyme involved in posttranslational farnesylation of Ras, and alters Ras-dependent cell signaling and control of apoptosis. The role of p13(II) in virus infection in vivo remains undetermined. Herein, we analyzed the functional significance of p13(II) in HTLV-1 infection. We compared the infectivity of a human B-cell line that harbors an infectious molecular clone of HTLV-1 with a selective mutation that prevents the translation of p13(II) (729.ACH.p13) to the infectivity of a wild-type HTLV-1-expressing cell line (729.ACH). 729.ACH and 729.ACH.p13 producer lines had comparable infectivities for cultured rabbit peripheral blood mononuclear cells (PBMC), and the fidelity of the start codon mutation in ACH.p13 was maintained after PBMC passage. In contrast, zero of six rabbits inoculated with 729.ACH.p13 cells failed to establish viral infection, whereas six of six rabbits inoculated with wild-type HTLV-1-expressing cells (729.ACH) were infected as measured by antibody responses, proviral load, and HTLV-1 p19 matrix antigen production from ex vivo-cultured PBMC. Our data are the first to indicate that the HTLV-1 mitochondrion-localizing protein p13(II) has an essential biological role during the early phase of virus infection in vivo.

Detection of feline leukemia virus RNA in saliva from naturally infected cats and correlation of PCR results with those of current diagnostic methods.

A novel diagnostic test for feline leukemia virus (FeLV) RNA in saliva from naturally infected cats is described in this study. We evaluated different diagnostic tests and compared them with the widely used enzyme-linked immunosorbent assay (ELISA) for the detection of p27 in the diagnosis of FeLV. Blood samples from 445 cats were tested for the presence of provirus by real-time PCR and plasma and saliva specimens from those cats were tested for the presence of viral RNA by real-time reverse transcription (RT)-PCR and for the presence of p27 by ELISA. In comparison to conventional ELISA, the diagnostic sensitivity and specificity of the detection of salivary FeLV RNA by real-time RT-PCR were found to be 98.1 and 99.2%, respectively. Detection of viral RNA in saliva had a positive predictive value of 94.6% and a negative predictive value of 99.7%. The kappa value was 0.96, demonstrating an almost perfect agreement between both tests. Furthermore, we confirmed previous results showing that a number of cats which tested negative for the presence of p27 in plasma were in fact positive for the presence of DNA provirus in blood specimens (5.4%). However, 96.4% of these latently infected cats did not shed viral RNA in saliva; therefore, we assume that these cats are of relatively low clinical importance at the time of testing. This study shows considerable diagnostic value of the detection of saliva FeLV RNA in naturally infected cats. This new diagnostic method has advantages over the conventional ELISA, such as less invasive sample collection and no requirement for trained personnel.

Broad cellular immunity with robust memory responses to simian immunodeficiency virus following serial vaccination with adenovirus 5- and 35-based vectors.

Adenovirus serotype 35 (Ad35) is a promising vaccine platform for human immunodeficiency virus (HIV) infection and emerging infectious diseases as it is uncommon in humans worldwide and is distinct from Ad5, the major vaccine serotype for which many individuals have pre-existing immunity. The immunogenicity of a first-generation, replication-competent Ad35-based vaccine was tested in the simian immunodeficiency virus (SIV) rhesus macaque model by evaluating its capacity to boost immunity generated by Ad5-based vectors. A series of four immunizations with replication-defective Ad5 vectors expressing SIVmac239 gag induced high-frequency responses mediated by both CD8(+) and CD4(+) T cells directed against several epitopes. Ad5-specific neutralizing antibody responses that did not neutralize Ad35 were rapidly induced but waned over time. Subsequent immunization with Ad5-based vectors was minimally effective, whereas immunization with Ad35-based vectors generated a strong increase in the frequency of Gag-specific T cells with specificities that were unchanged. While this boosting response was relatively transient, challenge with the distinct pathogenic isolate SIV/DeltaB670 generated robust and selective recall responses to Gag with similar specificities as induced by vaccination that were elevated for 25 weeks relative to controls. Vaccination had measurable albeit minor effects on virus load. Unexpectedly, regional hypervariability within the Gag sequence of SIV/DeltaB670 was associated with mutation of the conserved CD8(+) T-cell epitope CM9 without concurrent flanking mutations and in the absence of immune pressure. These findings support the further development of Ad35 as a vaccine vector, and promote vaccine regimens that utilize serial administration of heterologous adenoviruses.

Cell membrane proteins and quasispecies compartmentalization of CSF and plasma HIV-1 from aids patients with neurological disorders.

Cell membrane protein (CMP) profile of HIV-1 from cerebrospinal fluid (CSF) and plasma of five AIDS patients with neurologic disorders was analyzed and compared with viral quasispecies composition in these body compartments. To this aim, paired CSF and plasma samples from AIDS subjects with HIV-related neurological diseases (three HIV-1 encephalopaty (HIVE) and two primary CNS lymphoma (PCNSL)) underwent immobilized antibody capture (IAC) assay to determine the profile of CMP acquired by HIV-1. The considered CMPs were CD45RO, CD26, CD36, glut-R, N-CAM, VCAM-1, ELAM-1, CD44 and CD58, representing lymphomonocyte, neuronal and adhesion molecules. Cloning and sequencing of env and gag regions was performed to predict coreceptor usage and to analyze quasispecies compartmentalization. The results indicated that CD44 and CD58 were the most represented molecules on HIV-1 from CSF, whereas CD36 was the most abundant molecule on plasma HIV-1. V3 env aminoacidic sequences and net charge were consistent with M-R5 phenotype in all CSF and in most plasma clones. The degree of genetic heterogeneity (both complexity and diversity) in p17 gag was significantly lower in CSF-HIV than that in plasma-HIV for three patients, higher for one patient, and not significantly different for one patient, suggesting compartmentalization for all but the latter patient. When considering the pattern of CMP, the most abundant CMP observed in HIV from plasma and CSF was different in patients showing compartmentalization, while was the same in the patient without significant differences in CSF and plasma quasispecies. In conclusion, the present data on CMP pattern, V3 loop aminoacidic signature and genetic heterogeneity of HIV-1 quasispecies from CSF and plasma of HIVE patients, are consistent with a compartmentalized virus replication, at least in some patients, and with a possible different source of HIV in the two body sites, even though in a context of a largely prevalent M-R5 phenotype.

Immunogenicity of recombinant adenovirus serotype 35 vaccine in the presence of pre-existing anti-Ad5 immunity.

The high prevalence of pre-existing immunity to adenovirus serotype 5 (Ad5) in human populations may substantially limit the immunogenicity and clinical utility of recombinant Ad5 vector-based vaccines for HIV-1 and other pathogens. A potential solution to this problem is to use vaccine vectors derived from adenovirus (Ad) serotypes that are rare in humans, such as Ad35. However, cross-reactive immune responses between heterologous Ad serotypes have been described and could prove a major limitation of this strategy. In particular, the extent of immunologic cross-reactivity between Ad5 and Ad35 has not previously been determined. In this study we investigate the impact of pre-existing anti-Ad5 immunity on the immunogenicity of candidate rAd5 and rAd35 vaccines expressing SIV Gag in mice. Anti-Ad5 immunity at levels typically found in humans dramatically blunted the immunogenicity of rAd5-Gag. In contrast, even high levels of anti-Ad5 immunity did not substantially suppress Gag-specific cellular immune responses elicited by rAd35-Gag. Low levels of cross-reactive Ad5/Ad35-specific CD4(+) T lymphocyte responses were observed, but were insufficient to suppress vaccine immunogenicity. These data demonstrate the potential utility of Ad35 as a candidate vaccine vector that is minimally suppressed by anti-Ad5 immunity. Moreover, these studies suggest that using Ad vectors derived from immunologically distinct serotypes may be an effective and general strategy to overcome the suppressive effects of pre-existing anti-Ad immunity.

Retrospective analysis of clinical and laboratory factors associated with lymphoma in simian AIDS.

A nonhuman primate model for AIDS-associated Non-Hodgkin's lymphoma (AIDS-NHL) has been described in which animals inoculated with simian immunodeficiency virus (SIV) develop simian AIDS (SAIDS) and SAIDS-NHL. The objective of the present study was to describe statistically the major trends observed in clinical and laboratory data collected longitudinally on a large cohort of nonhuman primates that developed SAIDS-NHL. Clinical and laboratory data were collected longitudinally on each animal from the time of SIV infection throughout progression to lymphoma. Data were analyzed retrospectively with regard to species, gender, age at SIV inoculation, survival, cause of death, CD4+ T-cell and B-cell counts, SIV antigenemia, persistent lymphoid hyperplasia and lymphocryptovirus infection. Median survival time (354 days: 95% CI 309-388) was not related to gender, age at SIV inoculation, cause of death, or RhLCV infection. Survival was not related to CD4+ T-cell count at the time of SIV infection (P = 0.5531), but increased survival was significantly related to a slower rate of CD4+ T-cell decline (P = 0.0256). A B-cell expansion was observed at the midpoint of disease. A steep rise in SIV antigenemia was detected in the first 21 days of infection followed by a rapid decline. This pattern did not occur in animals inoculated with SIV as infants or yearlings. Of 45 cases, 9 exhibited marked, persistent lymphoid hyperplasia. These results describe trends identified in clinical and laboratory factors associated with SAIDS-NHL in the largest collection of such samples in the world. The results contribute to an understanding of the etiology of SAIDS-NHL and to the future development of useful predictors of SAIDS- or AIDS-related lymphoma.

Simian immunodeficiency virus Nef gene regulates the production of 2-LTR circles in vivo.

The replication dynamics of simian immunodeficiency virus (SIVmac32H-C8), attenuated through discrete genetic disruption of the nef gene, were compared with the wild-type parental clone (SIVmac32H-J5) using quantitative molecular methods. The primary viraemia of both infections were similar during the first week, but peaked on Day 10 at higher levels for wild-type virus. Viral RNA levels differed most markedly at Day 14. The frequency and levels of viral DNA species, detectable as gag provirus or circular 2-LTR episomes, differed depending on the virus and the lymphoid compartment sampled. 2-LTR circles persisted for prolonged periods in the peripheral blood but were never detected in any SIVmac32H C8-infected tissue, even if positive by gag PCR. Paradoxically, the converse was observed following wild-type infection. 2-LTR circles disappeared from the peripheral blood by Day 42 postinfection but persisted in lymphoid tissues. These findings are discussed in terms of nef and the role and stability of 2-LTR circle forms in vivo.