Evidence of infection with simian type D retrovirus in persons occupationally exposed to nonhuman primates.

Simian type D retrovirus (SRV) is enzootic in many populations of Asian monkeys of the genus Macaca and is associated with immunodeficiency diseases. However, the zoonotic potential of this agent has not been well defined. Screening for antibodies to SRV was performed as part of an ongoing study looking for evidence of infection with simian retroviruses among persons occupationally exposed to nonhuman primates (NHPs). Of 231 persons tested, 2 (0.9%) were found to be strongly seropositive, showing reactivity against multiple SRV antigens representing gag, pol, and env gene products by Western immunoblotting. Persistent long-standing seropositivity, as well as neutralizing antibody specific to SRV type 2, was documented in one individual (subject 1), while waning antibody with eventual seroreversion was observed in a second (subject 2). Repeated attempts to detect SRV by isolation in tissue culture and by using sensitive PCR assays for amplification of two SRV gene regions (gag and pol) were negative. Both individuals remain apparently healthy. We were also unable to transmit this seropositivity to an SRV-negative macaque by using inoculation of whole blood from subject 1. The results of this study provide evidence that occupational exposure to NHPs may increase the risk of infection with SRV and underscore the importance of both occupational safety practices and efforts to eliminate this virus from established macaque colonies.

Simian foamy virus infections in a baboon breeding colony.

The prevalence, transmission, and variation of simian foamy viruses (SFVs) in baboons was investigated. Over 95% of adult baboons in the breeding colony as well as recently imported adult animals had high titers of anti-SFV serum IgG. Maternal antibody was detectable in infants' serum up to 6 months of age. Approximately 30% of infants in breeding harems experienced SFV infections by 1 year of age. Shedding of SFV in oral secretions was common, with 13% of samples from normal adult animals and 35% from immunosuppressed animals containing infectious SFV. SFV was isolated from three baboon subspecies (olive, yellow, and chacma baboons) and sequences from both the pol and the LTR regions of the provirus were amplified by PCR and sequenced. Phylogenetic analysis indicated that all baboon isolates formed a single lineage distinct from SFVs of other African monkey species. Within the baboon SFV lineage, two distinct clades were apparent, which consisted of isolates from yellow and olive baboons and isolates from chacma baboons. Competition ELISAs indicated that, while SFV isolates of these two groups were very closely related, antigenic differences do exist between them. SFV isolates from a drill and a mandrill were distinct from baboon SFV isolates, both genetically and antigenically.

A simian model of human granulocytic ehrlichiosis.

Following intravenous inoculation with horse blood-infected with the agent of human granulocytic ehrlichiosis (HGE) from a human fatality, two rhesus macaques (Macaca mulatta) exhibited pyrexia and lethargy on days 4-12 postinfection (PI). Hematology revealed neutropenia, thrombocytopenia, and anemia, with ehrlichial morulae in monocytes and neutrophils on days 4-12. Blood was polymerase chain reaction (PCR)-positive on days 4-12 and bone marrow was PCR-positive on day 11. There was a minor increase in gamma-glutamyl transpeptidase on day 12 and serum interferon-gamma levels increased by day 18. Seroconversion occurred on day 20 PI to a titer of 100 by day 22. Western blot bands characteristic of HGE included 25-, 44-, 80-, 94-, 105-, and 125-kD bands. There was generalized lymphohistiocytic infiltration in the liver, spleen, lymph nodes, and other tissues. The liver had focal hepatocyte apoptosis. There was HGE DNA (by PCR) only in the spleen. Comparable findings were not observed in a monkey that received uninfected horse blood as a control. This animal model of human disease is important for further studies of HGE diagnosis, management, and pathogenesis.

Identification of a human population infected with simian foamy viruses.

Studying the transmission of simian retroviruses to humans can help define the importance of these infections to public health. We identified a substantial prevalence (4/231, 1.8%) of infection with simian foamy viruses (SFV) among humans occupationally exposed to nonhuman primates. Evidence of SFV infection included seropositivity, proviral DNA detection and isolation of foamy virus. The infecting SFV originated from an African green monkey (one person) and baboons (three people). These infections have not as yet resulted in either disease or sexual transmission, and may represent benign endpoint infections.

Experimental measles. I. Pathogenesis in the normal and the immunized host.

An animal model to study measles pathogenesis and the correlates of protective immunity was established using rhesus monkeys. A measles isolate, obtained during an epidemic of measles in the primate colony at the University of California, Davis, was passaged through rhesus monkeys and amplified in rhesus mononuclear cells to create a pathogenic virus stock. Sequence analysis of the nucleoprotein and hemagglutinin genes of this isolate revealed strong homology with the Chicago 89 strain of measles virus. Conjunctival/intranasal inoculation of juvenile rhesus monkeys with this virus resulted in skin rash, pneumonia, and systemic infection with dissemination to other mucosal sites and to the lymphoid tissues. Inflammation and necrosis occurred in the lungs and lymphoid tissues and many cell types were infected with measles virus on Day 7 postinoculation (p.i.). The most commonly infected cell type was the B lymphocyte in lymphoid follicles. Measles antigen was found in follicular dendritic cells on Day 14 p.i. In contrast to naive monkeys infected with measles virus, animals vaccinated with the attenuated Moraten strain did not develop clinical or pathologic signs of measles after challenge. However, moderate to marked hyperplasia occurred in the lymph nodes and spleen of a vaccinated animal on Day 7 after pathogenic virus challenge, suggesting that an effective measles vaccine limits but does not prevent infection with wild-type measles virus.

Screening for simian type-D retrovirus infection in macaques, using nested polymerase chain reaction.

A nested polymerase chain reaction (PCR) assay was developed to detect proviral DNA in peripheral blood mononuclear cells (PBMC) of macaques infected with simian type-D retrovirus (SRV/D). Primers were designed to amplify gag gene sequences of SRV/D serotype 1, 2, and 3 viral genomes and were used in a single assay for simultaneous detection of infection with SRV/D-1, SRV/D-2, or SRV/D-3. Results of plasmid dilution studies indicate sensitivity of nested PCR in the range of 1 to 10 genomic copies. The PBMC samples from 395 macaques of unknown SRV/D status, obtained from several primate facilities, were tested in parallel by Western blot (immunoblot) analysis, virus isolation, and nested PCR. Infection was detected in 60 (15.2%) animals by nested PCR, in 40 (10.1%) animals by virus isolation, and in 28 (7.1%) animals by immunoblot. All 40 culture-positive samples were positive by nested PCR. In addition, 11 of 23 immunoblot-positive/virus isolation-negative samples, 2 of 20 immunoblot-indeterminate/virus isolation-negative samples, and 7 of 312 immunoblot-negative/virus isolation-negative samples were identified as positive by nested PCR. Nested PCR is a sensitive and specific assay for simultaneous screening for infection with serotypes 1, 2, and 3 of simian type D retrovirus, and is a powerful tool for rapid screening and surveillance in macaque colonies.

Simultaneous detection of simian retrovirus type D serotypes 1, 2, and 3 by polymerase chain reaction.

Asymptomatic infection of macaques with macaques with simian retroviruses type D (SRV/D), the etiologic agents of one form of retrovirus-induced simian immunodeficiency disease, can confound experiments with the simian immunodeficiency virus (SIV), which also induces immunodeficiency disease in macaques. The SIV/macaque model is the preferred nonhuman primate model for AIDS-related research. Serological screening for SRV/D alone is insufficient because not all infected animals seroconvert, and virus isolation by cocultivation may require 4 to 6 weeks. We have established a DNA polymerase chain reaction (PCR) assay. One set of nested primers allows detection of SRV/D serotypes 1, 2, and 3 and distinguishes SRV-2 from the other two serotypes. The PCR assay is sensitive; a single proviral copy of SRV/D could be detected in 150,000 to 210,000 macaque peripheral blood mononuclear cells (PBMCs). When applied to a panel of virus isolation-positive macaque samples, the PCR assay was positive in 100% of the tests. No false-positive results were seen when known specific-pathogen-free (SPF) macaques were examined. We propose that macaques be screened with a combination of SRV/D serology and this DNA PCR assay prior to enrollment in experiments with SIV.

Establishing specific retrovirus-free breeding colonies of macaques: an approach to primary screening and surveillance.

For reasons of occupational safety and animal health, as well as to improve the quality of nonhuman primates used in biomedical research, the establishment and maintenance of specific retrovirus-free breeding colonies of macaques (genus Macaca) are now high priorities. Sensitive and specific screening tests are now available for use in identifying macaques infected with the exogenous simian retroviruses simian immunodeficiency virus (SIV), simian T-lymphotropic virus (STLV), and simian type D retrovirus (SRV/D). A testing algorithm of repeated antibody screening by enzyme immunoassay with confirmatory testing of enzyme immunoassay-reactive sera by Western blot (immunoblot) has proved adequate for identification and exclusion of SIV- and STLV-infected animals in five facilities. In follow-up testing of animals seronegative on primary screening, seroconversions to these two viruses have been rare (0% and < 0.01%, respectively). The testing algorithm for SRV/D must include virus isolation in addition to antibody screening, as some SRV/D-infected animals lack detectable antibody or exhibit a prolonged interval between infection and seroconversion. This parallel testing for SRV/D antibody and virus is critical, especially during primary screening of potential specific pathogen-free stock obtained from external sources. "Indeterminate" immunoblot results, particularly for SRV/D, continue to pose a problem of interpretation. However, preliminary results indicate that newer diagnostic test methods, such as polymerase chain reaction for amplification of proviral DNA, will be useful in resolving SRV/D infection status and will contribute substantially to specific pathogen-free colony development and maintenance.

The search for human infection with simian type D retroviruses.

Evidence has recently been presented for an infection with a simian type D retrovirus in a patient with AIDS and lymphoma. We tested for simian type D infection in three groups of subjects: 375 patients with lymphoproliferative diseases (255 non-Hodgkin's, 88 Hodgkin's, and 32 chronic lymphoproliferative disease), of whom 75 were human immunodeficiency virus (HIV)-1 infected; seven persons with unexplained low CD4 lymphocyte counts with clinical conditions; and 45 blood donors, of whom 37 were human T-lymphocyte virus (HTLV)-I/II seroindeterminate and eight were HTLV-I/II and HIV-1 seronegative. Serum samples were screened for antibodies against simian type D retroviruses by an enzyme immunoassay, and reactive samples were analyzed by Western blotting. None of the samples were seropositive, but eight (five from non-Hodgkin's and three from Hodgkin's lymphoma patients) were seroindeterminate. Polymerase chain reaction analysis of genomic DNA from peripheral blood lymphocytes of all eight of these patients was carried out using simian type D gag generic primers with generic internal oligoprobing. All samples were negative. We conclude that simian type D infection is rare among HIV-infected and noninfected lymphoma patients, persons with unexplained low CD4 counts, and persons with HTLV-seroindeterminate test results.

Evidence for a lentiviral etiology in an epizootic of immune deficiency and lymphoma in stump-tailed macaques (Macaca arctoides).

A retrospective study determined that an epizootic of immune suppression and lymphoma in stump-tailed macaques (Macaca arctoides) that began in 1976 was associated with a horizontally spread lentivirus infection. This conclusion was based on serology, epidemiology, pathology, and virus isolation. The lesions found in the stump-tailed macaques were more compatible with lesions seen in SIV-infected rhesus than those seen in rhesus macaques infected with type D retroviruses. A lentivirus, isolated from a rhesus inoculated with lymph node homogenate from a stump-tailed macaque, was designed SIVstm and was pathogenic for rhesus macaques. The isolate was antigenically related to other SIVs as well as to HIV-1 and HIV-2. Two surviving stump-tailed macaques sent to another colony carried SIVstm latently for at least 7 years and disseminated it throughout that colony.

A simple, rapid immunoassay for the detection of simian immunodeficiency virus antibodies.

Detection of simian immunodeficiency virus (SIV) antibodies has proved useful in a wide variety of research studies. Conventional immunoassays, however, are difficult to perform outside the well-equipped laboratory or under field conditions. We have developed an inexpensive, simple, rapid immunoassay for the detection of SIV antibodies that utilizes inactivated SIV antigen and Fast-Chek (F-C) (E.Y. Laboratories, San Mateo, Ca)., which is a membrane/filter paper device that uses protein A gold to detect antibody and/or antigen. This low-cost 10-min assay requires minimal technical skill and no refrigeration, electrical power, or sophisticated laboratory equipment. In a study of 155 banked sera, from a number of monkey species in a variety of geographic locations, F-C and Western immunoblot result concordance was 96%. Relative sensitivity and specificity were 98% and 95%, respectively.

Priming with T helper cell epitope peptides enhances the antibody response to the envelope glycoprotein of HIV-1 in primates.

The induction of a memory immune response to HIV, mediated by any kind of effector mechanism, requires the induction of T cell help. In previous studies performed in different murine MHC haplotypes, three immunodominant T cell epitopes (T1, T2, and TH4.1) had been identified in the HIV envelope glycoprotein. Moreover, these peptides were proliferative T cell epitopes in humans. In this study, rhesus monkeys, Macaca mulatta, were primed with these three peptides either in combination or given separately. Half of the monkeys had a proliferative response to one or more of the priming peptide(s). Those monkeys who had a T cell proliferative response also had a high antibody response after one boost with a suboptimal dose of the native protein gp 160, whereas three of four control monkeys who had received only the native protein immunization gave no detectable antibody response, and one displayed a very weak response. For reasons that are unclear, antibodies only to the gp41 portion of gp 160 could be detected in the sera. Thus, the peptides can prime Th cells in primates for an enhanced antibody response on first exposure to the whole protein. The three peptides belong to highly conserved and nonglycosylated regions of the envelope protein. The fact that the peptides acted as immunogenic T cell proliferative and helper epitopes in nonhuman primates is very encouraging for including them in future vaccine studies in humans.

SIV, STLV-I and type D retrovirus antibodies in captive rhesus macaques and immunoblot reactivity to SIV p27 in human and rhesus monkey sera.

The prevalence of simian immunodeficiency virus (SIV), simian T-cell lymphotropic virus type 1 (STLV-I), and type D retrovirus (SRV-D) antibodies was determined for 1229 rhesus monkeys (Macaca mulatta) from two research colonies. Serum samples were tested by using enzyme-linked immunosorbent assay (ELISA), immunoblot (IB), and radioimmunoprecipitation assay (RIPA). Seropositive results for the three retroviruses tested were 0 for SIV, 270 (22%) for STLV-I, and 103 (8.4%) for type D retrovirus. Of the rhesus monkey sera, 61 (5.0%) were reactive to SIV gag p27 only, when tested by IB, but were negative when further tested by RIPA. Virus isolation was attempted from cultured peripheral blood mononuclear cells of 35 monkeys whose sera contained only p27 reactivity and none were positive by reverse transcriptase and core antigen assays to detect SIV. No overt clinical signs of immunodeficiency disease or unexplained deaths were evident in either monkey colony. Additionally, 63 of 165 (38%) human sera from various groups (primate center workers, normal donors, health care workers) had weak to moderate IB reactivity only to SIV p27, but 31 of 31 sera tested were negative by RIPA. These sera remained reactive to SIV p27 following absorption with an uninfected cell lysate, after blocking IB strips with various blocking solutions and were reactive to different SIV antigen preparations while remaining negative to human immunodeficiency virus type 1 (HIV-1) by IB and negative to HIV-2 by ELISA. These data underscore the need to adopt criteria for a positive SIV serologic test requiring reactivity against more than one viral gene product. These results also illustrate a potential problem in the testing of human sera for antibodies against simian retroviruses and demonstrate the need for caution in the interpretation of immunoblot results.

Synthetic peptides containing T and B cell epitopes from human immunodeficiency virus envelope gp120 induce anti-HIV proliferative responses and high titers of neutralizing antibodies in rhesus monkeys.

We have previously described a synthetic peptide (T1-SP10) derived from two noncontiguous regions of HTLVIIIB envelope gp120 (T1, amino acids 428-443; SP10, amino acids 303-321) that induced type-specific anti-HIV neutralizing antibodies and T cell proliferative responses against native HIV gp120 when used as a carrier-free immunogen in goats. In this study, HTLVIIIB T1-SP10 synthetic peptides were used to immunize rhesus monkeys to determine if the peptides were capable of eliciting HIV-specific neutralizing antibody and proliferative responses in primates. Four compounds (alum, polyA:polyU, threonyl-muramyldipeptide (MDP) and IFA) were also compared for efficacy as adjuvants in this system. Rhesus monkeys immunized with T1-SP10 peptides generated high titers of antibodies against the immunogens and also against HTLVIIIB gp120. Sera from all four animals given T1-SP10 in IFA or threonyl-MDP neutralized infection by HTLVIIIB and blocked virus-dependent cell fusion events. A peak neutralization titer of 1:940 was seen in one animal given IFA (19600) and a titer of 1:900 was seen in one of the monkeys (17371) given threonyl-MDP. Proliferative responses of immune rhesus PBMC to T1-SP10 appeared after the first injection. After eight immunizations, two of eight monkeys (one injected with peptides in threonyl-MDP and one given peptides in IFA) had PBMC proliferative responses to native HTLVIIIB gp120. These data demonstrate that the carrier-free T1-SP10 synthetic peptide construct can induce high titers of neutralizing anti-HIV antibody responses and PBMC proliferative responses to HIV in primates.

Asymptomatic infection of the central nervous system by the macaque immunosuppressive type D retrovirus, SRV-1.

The aetiological agent of spontaneously occurring simian acquired immune deficiency syndrome (SAIDS) in rhesus monkeys (Macaca mulatta) at the California Primate Research Center is a type D retrovirus designated SAIDS retrovirus serotype 1 (SRV-1). SRV-1 DNA and RNA have previously been detected in the brains of rhesus monkeys with SAIDS in the absence of viral antigen or neuropathological lesions. In this study we further define the relationship between SRV-1 and the central nervous system (CNS) in rhesus monkeys by examining the CNS for infectious SRV-1, viral antigen and anti-SRV-1 antibodies. In addition, cerebrospinal fluid (CSF) was assayed for alterations in IgG and albumin levels, IgG/albumin ratios and cell count in comparison to uninfected control animals. No differences in CSF parameters were detected between infected and uninfected animals except for the presence of infectious SRV-1 which was isolated from the CSF from 13 out of 19 (68%) viraemic rhesus monkeys. The probable source of this virus was the choroid plexus, where approximately 1 in 1000 surface epithelial cells were found to contain viral antigen by immunohistochemistry. Antibodies against SRV-1 were not detected in the CSF even when present in the serum. Neither infectious virus nor viral antigen were found in the brain parenchyma of any animal examined. Thus infection of the CNS by SRV-1 appears to be subclinical without an intrathecal immune response. This may be related to the apparent restriction of productive infection in the CNS to cells of the choroid plexus.

Guidelines for the prevention of simian immunodeficiency virus infection in laboratory workers and animal handlers.

The authors are members of a working group that formulated guidelines to minimize transmission of simian immunodeficiency virus (SIV) infection to man. Biosafety level (BSL) 2 standards are recommended for handling of clinical specimens and housing of SIV inoculated animals. Manipulation of SIV preparations may be performed in a BSL 2 facility with additional BSL 3 practices and equipment; for large volume or concentrated preparations of SIV BSL 3 containment is necessary. Written policies regarding management and testing of workers exposed to SIV are recommended.

Natural history of endemic type D retrovirus infection and acquired immune deficiency syndrome in group-housed rhesus monkeys.

A 2.5-year epidemiologic study of a breeding group of rhesus monkeys (Macaca mulatta), which is a focus of endemic simian acquired immunodeficiency syndrome (SAIDS), demonstrated a strong association between the occurrence of SAIDS and infection with a type D retrovirus, SAIDS retrovirus serotype 1 (SRV-1). Of 23 healthy "tracer" juvenile rhesus monkeys, 19 (83%) died with SAIDS within 9 months of introduction into the resident SAIDS-endemic population. In contrast, 21 healthy "sentinel" juvenile rhesus monkeys placed in the same outdoor enclosure but denied physical contact with the SAIDS-affected group by a 10-foot-wide "buffer zone" remained free of SRV-1, SRV-1 antibody, and disease for 2.5 years. The SAIDS-specific mortality rate was significantly higher in juveniles than in adults. In repeated serologic testing, the overall prevalence of SRV-1 antibody ranged from 68 to 85%. Antibody prevalence increased with age. Seroconversion was found to be a poor indicator of infection rate, as approximately 50% of virus-positive juvenile monkeys had no antibody detectable by enzyme-linked immunosorbent assay. Repeated viral isolations from all animals revealed 1) SRV-1 viremia with clinical SAIDS; 2) persistent viremia and viral shedding in apparently healthy animals; 3) transient viremia and clinical recovery; 4) intermittent viremia, suggesting activation of latent infections; and 5) viremia in a 1-day-old infant, suggesting transplacental transmission. The prevalence of SRV-1 antibody in SAIDS-free breeding groups of rhesus monkeys was 4%. The seroprevalence of antibodies against human T-cell leukemia virus type 1 (HTLV-1), human immunodeficiency virus (HIV), and simian immunodeficiency virus (SIV; formerly STLV-III) was uniformly low or absent in both SAIDS-free and SAIDS-affected groups of rhesus monkeys, demonstrating that these retroviruses are not etiologically linked to SAIDS at the California Primate Research Center.

Induction of simian acquired immune deficiency syndrome (SAIDS) with a molecular clone of a type D SAIDS retrovirus.

We have isolated a molecular clone of the full-length integrated provirus of simian acquired immune deficiency syndrome retrovirus serotype 1 (SRV-1) from a fatal case of simian acquired immune deficiency syndrome in a juvenile rhesus macaque. An integrated SRV-1 provirus was cloned, sequenced, and found to contain four large open reading frames encoding gag-precursor protein, protease, polymerase, and envelope. The proviral clone was transfected into D17 canine osteosarcoma cells and found to produce infectious virus. A comparison of the sequences of this clone with a noninfectious clone showed 20 differences, resulting in 10 amino acid changes. Also, a cluster of exchanges, short insertions, and deletions in the 5' leader sequences resulted in extension of the tRNA(Lys) primer-binding site from 14 to 19 nucleotides. Virus isolated from transfected cells was shown to be infectious and pathogenic, resulting in disease that followed the same time course and mortality as disease induced by uncloned, in vitro cultivated virus isolated from diseased animals. These results unequivocally show that a type D retrovirus (SRV-1) causes a fatal immunosuppressive syndrome in rhesus monkeys.