Morphological changes of an inflammatory myopathy in rhesus monkeys with simian acquired immunodeficiency syndrome

Eleven of 25 rhesus monkeys which died of simian acquired immunodeficiency syndrome (SAIDS) caused by infection with a type D retrovirus related to Mason-Pfizer monkey virus showed evidence of muscle weakness and atrophy and had elevated levels of muscle enzymes. Biopsies of affected muscle studied with enzyme histochemistry showed the characteristic features of polymyositis. Inflammatory cells consisting of lymphocytes, macrophages, and large vacuolated bizarre-shaped cells of undetermined type were surrounding or invading muscle fibers and were present in the perivascular spaces and endomysia septa. Within the perivascular infiltrates, lymphocytes were abundant but very few macrophages were present. Other myopathic features including profound proliferation of fibrous tissue, necrosis, and phagocytosis of muscle fibers were noted to a variable degree. The retrovirus was isolated from affected muscles. The clinical and historical features of polymyositis in rhesus monkeys with SAIDS are very similar to those of human polymyositis. The polymyositis in SAIDS induced by a type D retrovirus related to Mason-Pfizer monkey virus is an excellent primate model to study the mechanism and morphological changes of viral-induced muscle damage.     

Isolation of a new serotype of simian acquired immune deficiency syndrome type D retrovirus from Celebes black macaques (Macaca nigra) with immune deficiency and retroperitoneal fibromatosis.

A new serotype of simian acquired immune deficiency syndrome (SAIDS) retrovirus (type 2) belonging to the D genus of retroviruses is associated with a SAIDS occurring spontaneously in a colony of Celebes macaques (Macaca nigra) and rhesus macaques (Macaca mulatta) at the Oregon Regional Primate Research Center. This syndrome resembles SAIDS in M. mulatta at the California Primate Research Center, which is associated with a similar type D retrovirus (type 1). However, at the Oregon Center, SAIDS is distinguished by the occurrence of retroperitoneal fibromatosis in some of the affected monkeys. Type 2 virus was isolated from seven of seven macaques with SAIDS, retroperitoneal fibromatosis, or both and from one of six healthy macaques. The new strain is closely related to SAIDS retrovirus type 1 and Mason-Pfizer monkey virus but can be distinguished by competitive radioimmunoassay for minor core (p10) antigen and by genomic restriction endonuclease cleavage patterns. Neutralization tests indicate that type 1 and type 2 SAIDS retroviruses are distinct serotypes. Therefore, separate vaccines may be necessary to control these infections in colonies of captive macaques.     

Primate retroviruses: immunological cross-reactivity between major structural proteins of new and old world primate virus isolates.

The major 35,000-molecular-weight internal antigen (p35) of the squirrel monkey retrovirus (SMRV) was isolated and partially characterized. Immunological analysis of SMRV p35 led to the demonstration of antigenic determinants common to SMRV and the Mason-Pfizer monkey virus (MPMV). A broadly reactive competition immunoassay was developed utilizing antiserum to MPMV to precipitate 125I-labeled SMRV p35. Although the major structural proteins of MPMV and SMRV competed with equal efficiency in this assay, type B and type C oncornavirus proteins lacked detectable reactivity. Antibodies reactive with the major structural proteins of both MPMV and SMRV were observed in sera of several normal rhesus monkeys with known prior exposure to MPMV-infected animals. These findings demonstrate the ability of sera from naturally immunized primates to recognize broadly reactive interspecies antigenic determinants shared by the major structural proteins of type D oncornaviruses, and they suggest possible horizontal transmission of MPMV among rhesus monkeys. Although sera from a number of squirrel monkeys contained antibody to SMRV p35, the possibility that this latter reactivity was due to endogenous virus activation rather than horizontal transmission cannot be ruled out.     

Prevention of simian acquired immune deficiency syndrome with a formalin-inactivated type D retrovirus vaccine.

Experimental induction of simian acquired immune deficiency syndrome (SAIDS) by inoculation of juvenile rhesus monkeys with a type D retrovirus was prevented by immunization with Formalin-killed whole SAIDS retrovirus serotype 1 containing the adjuvant threonyl muramyl-dipeptide. All six immunized animals developed neutralizing antibody after three injections, while six age-matched cagemates receiving adjuvant alone were antibody free. All 12 monkeys were challenged intravenously with a potentially lethal dose of SAIDS retrovirus serotype 1. The six immunized animals failed to develop persistent viremia and remained clinically normal 8 months postchallenge. In contrast, five of six nonvaccinates developed persistent viremia, four of six developed clinical SAIDS, and two of six died with SAIDS at 10 weeks and 8 months postchallenge, respectively. These results show that prevention of a common spontaneous retrovirus-induced immunosuppressive disease in macaques is now possible by vaccination.     

Isolation of a type D retrovirus from B-cell lymphomas of a patient with AIDS.

An atypical syncytial variant of a high-grade Burkitt's-type B-cell lymphoma from a patient with AIDS who was seropositive for human immunodeficiency virus type 1 was studied. A productive type D retrovirus infection was identified in early-passage cell lines derived from two lymphomas from this patient. Nucleotide and amino acid sequence analysis as well as immunologic reactivity indicated that the isolated virus was highly related to Mason-Pfizer monkey virus (MPMV). MPMV is an immunosuppressive type D retrovirus that causes an AIDS-like syndrome in rhesus macaques. Amplification of DNA from the patient's diagnostic bone marrow biopsy specimen by polymerase chain reaction generated the appropriate MPMV-specific fragments and indicated that the patient was infected with the MPMV-like retrovirus. In addition, the patient's serum contained antibodies which recognized type D viral env proteins (gp70 and gp20) and gag proteins (p27 and p14). Although there have been reports of human cell lines infected with type D retroviruses and of type D-reactive human sera, this is the first evidence of a type D retrovirus infection in a human confirmed by virus isolation, serum reactivity, and viral DNA identification in tumor tissue.     

Simian AIDS ELISA: sensitivity, specificity and predictive values based on a comparison with Western blot technique

Simian Acquired Immunodeficiency Syndrome (SAIDS) is an important disease in captive primates in the United States associated with an unusually high mortality rate. Isolation of a type D retrovirus as the cause of SAIDS was rapidly followed by the development of an enzyme linked immunosorbent assay (ELISA) to determine the exposure of rhesus macaques (Macaca mulatta) to this virus. With increased use of the ELISA, a better understanding for interpretation of results was needed. One hundred thirty-one rhesus macaques were tested for the presence of antibody against SAIDS type D retrovirus (SRV) by both ELISA and Western blot techniques. Sensitivity, specificity and predictive values for the SAIDS ELISA were calculated for two populations based on a comparison with Western blot results. Sera was tested from two distinct populations, an endemic and a control population. Seventy-one macaques from a half-acre outdoor corral where SAIDS was first recognized made up the endemic population. Sixty rhesus macaques from both indoor and outdoor areas where the disease was not recognized made up the control population. This study has shown the ELISA to be a useful screening tool based on its high sensitivity for both endemic and control populations. This screening method provides a rapid and economical way to diagnose and manage SAIDS in captive non-human primate colonies.     

Nucleotide sequence of Mason-Pfizer monkey virus: an immunosuppressive D-type retrovirus

The genetic structure of Mason-Pfizer monkey virus (MPMV), a D-type retrovirus, has been determined. In addition to the viral gag, pol, and env genes is an ORF overlapping both gag and pol and that encodes the viral protease. Surprisingly, the MPMV env protein is highly homologous to that of the avian C-type virus, reticuloendotheliosis associated virus REV-A. The env sequence encodes an immunosuppressive peptide, which suggests that MPMV, like REV-A, may transiently induce a T-suppressor cell population. The different phylogenies of the MPMV pol and env genes indicate a recombinatorial origin for the D-type viruses. Sequence comparisons show that SRV-1, an MPMV-like virus etiologically linked to simian AIDS (SAIDS), is in fact a variant of MPMV. While MPMV-like viruses cannot be used as direct models for the AIDS/SAIDS associated with lentiviruses, they provide an important system for studying the molecular basis of immunosuppressive diseases in primates.     

The betaretrovirus Mason-Pfizer monkey virus selectively excludes simian APOBEC3G from virion particles

The APOBEC3 protein family can constitute a potent barrier to the successful infection of mammalian species by retroviruses. Therefore, any retrovirus that has evolved the ability to replicate in a given animal must have developed mechanisms that allow it to avoid or inhibit the APOBEC3 proteins expressed in that animal. Here, we demonstrate that Mason-Pfizer monkey virus (MPMV) is resistant to inhibition by the APOBEC3G protein expressed in its normal host, the rhesus macaque, but highly susceptible to inhibition by murine APOBEC3 (mA3). MPMV virion particles fail to package rhesus APOBEC3G (rA3G), and MPMV Gag binds rA3G poorly in coexpressing cells. In contrast, MPMV virions package mA3 efficiently and MPMV Gag-mA3 complexes are readily detected. Moreover, mA3, but not rA3G, partially colocalizes with MPMV Gag in the cytoplasm of coexpressing cells. Previously, we have demonstrated that murine leukemia virus also escapes inhibition by APOBEC3 proteins by avoiding virion incorporation of its cognate APOBEC3 protein, mA3, yet is inhibited by primate APOBEC3G proteins, which it packages effectively (B. P. Doehle, A. Sch?fer, H. L. Wiegand, H. P. Bogerd, and B. R. Cullen, J. Virol. 79:8201-8207, 2005). The finding that two essentially unrelated beta- and gammaretroviruses use similar mechanisms to escape inhibition by the APOBEC3 proteins found in their normal host species suggests that the selective exclusion of APOBEC3 proteins from virion particles may be a general mechanism used by simple mammalian retroviruses.     

Mucosal epithelial cells and Langerhans cells are targets for infection by the immunosuppressive type D retrovirus simian AIDS retrovirus serotype 1

Simian acquired immune deficiency syndrome (SAIDS) caused by the type D retrovirus SRV-1 results in opportunistic infections and a spectrum of oral lesions similar to those seen in humans with AIDS. To better understand the pathogenesis of these oral lesions we have retrospectively examined the oral mucosa from ten rhesus monkeys that died with SAIDS and prospectively examined the oral mucosa of ten additional animals inoculated with SRV-1 to determine at what time, and in what cells SRV-1 infection of the oral mucosa occurs. Using single and double label immunohistologic techniques, and electron microscopy we detected SRV-1 in clusters of oral epithelial cells and rare Langerhans cells as early as 1 month postinoculation.     

A spontaneously occuring mammary gland ductal carcinoma in situ in a rhesus macaque (Macaca mulatta) and a review of spontaneous mammary gland tumors in rhesus monkeys

A spontaneous mammary gland ductal carcinoma in situ was diagnosed in a 6-8-year-old female rhesus macaque (Macaca mulatta). To our knowledge, this is only the tenth case of spontaneous mammary gland tumors to be reported in rhesus monkeys. Despite the paucity of case reports, several theories exist to explain the occurrence of mammary tumors. The Mason Pfizer monkey virus, a type D retrovirus similar to the virus that causes simian acquired immunodeficiency syndrome, has been implicated as a possible etiologic agent. Because this virus has been isolated from normal primate mammary tissue, it is unlikely to be the sole etiologic agent. Other theories include the tumorogenic effects that androgens, growth hormones, irradiation, and aging have on the mammary gland.     

Distribution of a macaque immunosuppressive type D retrovirus in neural, lymphoid, and salivary tissues.

Simian acquired immune deficiency syndrome (SAIDS) in rhesus macaques (Macaca mulatta) at the California Primate Research Center is caused by a type D retrovirus designated SAIDS retrovirus serotype 1 (SRV-1). This syndrome is characterized by profound immunosuppression and death associated with opportunistic infections. Neurologic signs and lesions have not been described as part of this syndrome. The distribution of SRV-1 in the salivary glands, lymph nodes, spleens, thymuses, and brains of eight virus-infected rhesus macaques was examined by immunohistochemistry. Electron microscopy, in situ RNA hybridization, and Southern blot hybridization were also performed on selected tissues to detect viral particles, RNA, and DNA, respectively. In seven of eight SRV-1-infected animals, the transmembrane envelope glycoprotein (gp20) of SRV-1 was present in three or more tissues, but never in the brain. In the remaining animal, no viral antigen was detected in any tissue. In this same group of animals, viral nucleic acid was detected in the lymph nodes of six of six animals by Southern blot hybridization, in the salivary glands of two of five animals by both Southern blot and in situ hybridizations, and, surprisingly, in the brains of three of three animals by Southern blot and of three of five animals by in situ hybridization, including the one animal in which viral gp20 was undetectable. None of these animals had neurologic signs or lesions. The detection of viral nucleic acid in the absence of viral antigen in the brain suggests latent SRV-1 infection of the central nervous system.     

Retroperitoneal fibromatosis and acquired immunodeficiency syndrome in macaques: epidemiologic studies

At the University of Washington Regional Primate Research Center, a simian acquired immunodeficiency syndrome (SAIDS) associated with retroperitoneal fibromatosis (RF) has been observed in 82 macaques since 1976, including 77 pigtailed macaques (Macaca nemestrina), two long-tailed macaques (M. fascicularis), one Japanese macaque (M. fuscata) and two rhesus macaques (M. mulatta). The syndrome is characterized by immunodeficiency accompanied by a fibroproliferative lesion, primarily affects young monkeys (1-3 years) and has a high case fatality rate. Based on the occurrence of RF in colony-born and non-colony-born monkeys, the minimum incubation period for natural exposure is believed to be about 9 months. The incidence of RF was 0.9% in M. nemestrina, 0.1% in M. fascicularis, 1.0% in M. fuscata and 0.4% in M. mulatta. There were no significant differences in the incidence of RF by sex or seasonality. Epidemiologic studies were focused on 42 juvenile M. nemestrina that developed RF between January 1980 and June 1983, and the results were compared with 42 age- and sex-matched controls. The incidence of RF was 5.7% in monkeys 12-24 months old and 3.4% in monkeys 24-36 months old, but less than 1.0% in age groups of under 1 year and over 3 years. No significant associations were found for housing history, parentage, generations or ancestral origins. Epidemiologic information and preliminary viral studies suggest a type D retrovirus may be the causative agent in RF and SAIDS. RF associated with SAIDS appears to be an excellent model for Kaposi's sarcoma associated with human AIDS.     

Association of SAIDS/RF-related signs with current or past SAIDS type 2 retrovirus infection in a colony of Celebes black macaques

The 83 members of the Celebes black macaque (Macaca nigra) colony were screened for viremia with simian acquired immunodeficiency syndrome (SAIDS) type 2 retrovirus and antibodies against the retrovirus. On the basis of this screening, the Celebes colony was divided into four groups: retrovirus-positive/seropositive (virus+/Ab+); retrovirus-negative/seropositive (virus-/Ab+); retrovirus-positive/seronegative (virus+/Ab-); and retrovirus-negative/seronegative (virus-/Ab-). Monkeys in the virus+/Ab+ group displayed more major clinical signs and required medication more times than monkeys in the other groups. In contrast, monkeys in the virus-/Ab- group had fewer health problems than monkeys in the other groups. The five monkeys that had surgically confirmed retroperitoneal fibromatosis (RF), palpable abdominal masses, or both, were in the virus+/Ab+ group. Some of the monkeys in groups with current or past retrovirus infection were well clinically. There were no statistically significant differences in the mitogen reactivities of mononuclear cells obtained from monkeys of the different groups.     

Elimination of type D retrovirus infection from group-housed rhesus monkeys using serial testing and removal.

Type D retrovirus was successfully eliminated from an infected population of group-housed rhesus monkeys by serial testing of all animals for virus and antibody and subsequent removal of positives. This population of 53 rhesus had been housed together for 1 year prior to the initiation of the test and removal program, with six deaths from type D retrovirus-induced immunodeficiency disease occurring during this period. No new infections were detected after four rounds of testing. Of the 47 animals present at the start of the testing program, 17 (35%) remained after the elimination of type D virus from this group. These animals and their offspring have remained healthy and antibody negative for more than 2 years. These results demonstrate that elimination of type D retroviruses from rhesus macaque colonies is feasible, and that the objective of establishing and maintaining retrovirus-free colonies is realistic and achievable.     

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.     

Purification and N-terminal amino acid sequence comparisons of structural proteins from retrovirus-D/Washington and Mason-Pfizer monkey virus.

A new D-type retrovirus originally designated SAIDS-D/Washington and here referred to as retrovirus-D/Washington (R-D/W) was recently isolated at the University of Washington Primate Center, Seattle, Wash., from a rhesus monkey with an acquired immunodeficiency syndrome and retroperitoneal fibromatosis. To better establish the relationship of this new D-type virus to the prototype D-type virus, Mason-Pfizer monkey virus (MPMV), we have purified and compared six structural proteins from each virus. The proteins purified from each D-type retrovirus include p4, p10, p12, p14, p27, and a phosphoprotein designated pp18 for MPMV and pp20 for R-D/W. Amino acid analysis and N-terminal amino acid sequence analysis show that the p4, p12, p14, and p27 proteins of R-D/W are distinct from the homologous proteins of MPMV but that these proteins from the two different viruses share a high degree of amino acid sequence homology. The p10 proteins from the two viruses have similar amino acid compositions, and both are blocked to N-terminal Edman degradation. The phosphoproteins from the two viruses each contain phosphoserine but are different from each other in amino acid composition, molecular weight, and N-terminal amino acid sequence. The data thus show that each of the R-D/W proteins examined is distinguishable from its MPMV homolog and that a major difference between these two D-type retroviruses is found in the viral phosphoproteins. The N-terminal amino acid sequences of D-type retroviral proteins were used to search for sequence homologies between D-type and other retroviral amino acid sequences. An unexpected amino acid sequence homology was found between R-D/W pp20 (a gag protein) and a 28-residue segment of the env precursor polyprotein of Rous sarcoma virus. The N-terminal amino acid sequences of the D-type major gag protein (p27) and the nucleic acid-binding protein (p14) show only limited amino acid sequence homology to functionally homologous proteins of C-type retroviruses.     

Infection of macaque monkeys with simian immunodeficiency virus from African green monkeys: virulence and activation of latent infection

The virulence of three isolates of simian immunodeficiency virus from African green monkeys (SIVagm) was studied in rhesus and pigtailed macaques. None of 15 rhesus monkeys and one of four pigtailed monkeys died from infection during the time they were studied (up to 33 months). SIVagm was only isolated from rhesus monkeys for up to 2 months after inoculation. However, when these animals were secondarily infected with Simian acquired immunodeficiency syndrome retrovirus type 1 (SRV-1), SIVagm was activated and isolated. Dual infection caused increased mortality.     

A structured retroviral RNA element that mediates nucleocytoplasmic export of intron-containing RNA.

A common feature of gene expression in all retroviruses is that unspliced, intron-containing RNA is exported to the cytoplasm despite the fact that cellular RNAs which contain introns are usually restricted to the nucleus. In complex retroviruses, the export of intron-containing RNA is mediated by specific viral regulatory proteins (e.g., human immunodeficiency virus type 1 [HIV-1] Rev) that bind to elements in the viral RNA. However, simpler retroviruses do not encode such regulatory proteins. Here we show that the genome of the simpler retrovirus Mason-Pfizer monkey virus (MPMV) contains an element that serves as an autonomous nuclear export signal for intron-containing RNA. This element is essential for MPMV replication; however, its function can be complemented by HIV-1 Rev and the Rev-responsive element. The element can also facilitate the export of cellular intron-containing RNA. These results suggest that the MPMV element mimics cellular RNA transport signals and mediates RNA export through interaction with endogenous cellular factors.     

Experimental coinfection of rhesus macaques with rhesus cytomegalovirus and simian immunodeficiency virus: pathogenesis

Human cytomegalovirus (HCMV) possesses low pathogenic potential in an immunocompetent host. In the immunosuppressed host, however, a wide spectrum of infection outcomes, ranging from asymptomatic to life threatening, can follow either primary or nonprimary infection. The variability in the manifestations of HCMV infection in immunosuppressed individuals implies that there is a threshold of host antiviral immunity that can effectively limit disease potential. We used a nonhuman primate model of CMV infection to assess the relationship between CMV disease and the levels of developing anti-CMV immunity. Naive rhesus macaques were inoculated with rhesus cytomegalovirus (RhCMV) followed 2 or 11 weeks later by inoculation with pathogenic simian immunodeficiency virus SIVmac239. Two of four monkeys inoculated with SIV at 2 weeks after inoculation with RhCMV died within 11 weeks with simian AIDS (SAIDS), including activated RhCMV infection. Neither animal had detectable anti-SIV antibodies. The other two animals died 17 and 27 weeks after SIV inoculation with either SAIDS or early lymphoid depletion, although no histological evidence of activated RhCMV was observed. Both had weak anti-SIV antibody titers. RhCMV antibody responses for this group of monkeys were significantly below those of control animals inoculated with only RhCMV. In addition, all animals of this group had persistent RhCMV DNA in plasma and high copy numbers of RhCMV in tissues. In contrast, animals that were inoculated with SIV at 11 weeks after RhCMV infection rarely exhibited RhCMV DNA in plasma, had low copy numbers of RhCMV DNA in most tissues, and did not develop early onset of SAIDS or activated RhCMV. SIV antibody titers were mostly robust and sustained in these monkeys. SIV inoculation blunted further development of RhCMV humoral responses, unlike the normal pattern of development in control monkeys following RhCMV inoculation. Anti-RhCMV immunoglobulin G levels and avidity were slightly below control values, but levels maintained were higher than those observed following SIV infection at 2 weeks after RhCMV inoculation. These findings demonstrate that SIV produces long-lasting insults to the humoral immune system beginning very early after SIV infection. The results also indicate that anti-RhCMV immune development at 11 weeks after infection was sufficient to protect the host from acute RhCMV sequelae following SIV infection, in contrast to the lack of protection afforded by only 2 weeks of immune response to RhCMV. As previously observed, monkeys that were not able to mount a significant immune response to SIV were the most susceptible to SAIDS, including activated RhCMV infection. Rapid development of SAIDS in animals inoculated with SIV 2 weeks after RhCMV inoculation suggests that RhCMV can augment SIV pathogenesis, particularly during primary infection by both viruses.     

Molecular diversity among five different endogenous primate retroviruses.

Genetically transmitted retroviruses of Old and New World monkeys include type C viruses isolated from baboons (M7), macaque (MAC-1), and owl monkeys (OMC-1) and type D viruses from langurs (PO-1-Lu) and squirrel monkeys (SMRV, M534). Each of these isolates is unrelated to the others by nucleic acid hybridization criteria and contains a unique array of virion-associated proteins which can be resolved by agarose gel filtration and polyacrylamide gel electrophoresis under denaturing conditions. The major structural protein of each virus has a distinct primary structure, as determined by two-dimensional tryptic peptide analysis, and is antigenically different from the others. The major virion phosphoproteins of endogenous primate type C viruses (pp15) are also different from those of type D viruses (pp13-pp14). Immunological and structural analyses show that the endogenous langur virus and the horizontally transmitted Mason-Pfizer virus of rhesus monkeys are closely related to one another, consistent with the sequence homology detected in their RNA genomes. Although certain radioimmunoassays detect interspecies antigenic determinants common to either the p30 or gp70 proteins of some of these viruses, no one assay has yet been designed which can detect all groups of endogenous primate retroviridae. The data lead to the conclusion that primates contain a minimum of three different sets of genetically transmitted type C and type D retroviral genes.