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Rat respiratory virus (RRV) is the working name for a novel respiratory pathogen of laboratory rats in North America, Europe, and Asia. Although the agent has not been definitively identified, evidence supports a viral etiology. Because no serologic or molecular assays for RRV are available, diagnosis depends on histopathologic evaluation of the lung. We introduced 104 Wistar Han rats, free of known pathogens and of RRV-associated lesions, into a rat production colony positive for RRV-type lesions, but free of other histologic, serologic, or microbiologic evidence of infectious disease. Lungs of 8 of the naïve rats were examined grossly and microscopically each week, weeks 0–13. Irregular gray-white lesions suggestive of interstitial pneumonia were grossly evident from weeks 6 through 13. Primary histopathologic evaluation of all lungs by one pathologist found multifocal, lymphohistiocytic interstitial pneumonia or prominent perivascular lymphoid cuffing from weeks 5 through 13. Based on results of the initial evaluation, diagnostic criteria for RRV infection (i.e., changes seen only after exposure to the RRV-positive colony) were tentatively selected and used by 2 other pathologists to classify each lung as RRV positive, RRV equivocal, or RRV negative. The secondary evaluation found 95% concordance in RRV diagnosis between pathologists, and correlated well with the initial evaluation, thus confirming the consistency of the criteria. These data show that RRV-naïve rats introduced into an RRV-endemic colony develop equivocal microscopic lesions of RRV by 5 weeks of exposure, and positive diagnostic lesions by 7 weeks. Interstitial pneumonia becomes grossly evident after 6 weeks of exposure.

In a retrospective study, 51 cases of gastritis (14%) were identified from among 341 necropsies performed on simian immunodeficiency virus (SlV)-infected rhesus macaques ( Macaca mulatta) at the New England Primate Research Center from 1993 to 2001. Protozoa were seen in the stomach of 13 monkeys (25%) with gastritis. Two histopathologic manifestations of gastritis were observed: seven cases of lymphoplasmacytic gastritis with trichomonad trophozoites within lumens of gastric glands and four cases of necrosuppurative gastritis containing intralesional periodic acid-Schiff-positive protozoa; two cases of gastritis had morphologic features of both types of gastritis. In instances of necrosuppurative and combined lymphoplasmacytic and necrosuppurative gastritis, protozoa were 4-35 μm in diameter and round to tear-shaped. Because of the unusual morphology of the protozoa in these latter cases, transmission electron microscopy and polymerase chain reaction (PCR) were used to further identify these organisms. The protozoa were definitively identified as Tritrichomonas in all cases on the basis of ultrastructural characteristics (flagella and undulating membranes) and amplification of a 347-bp product of the 5.8S ribosomal RNA gene of Tritrichomonas foetus, Tritrichomonas suis and Tritrichomonas mobilensis by PCR using DNA extracted from stomach tissue. On the basis of these observations, we conclude that Tritrichomonas can be a significant cofactor in the development of necrosup-purative gastritis in SIV-infected rhesus macaques.

SIVmac239/316 is a molecular clone derived from SIVmac239 that differs from the parental virus by nine amino acids in env. This virus, unlike the parental SIVmac239, is able to replicate well in alveolar macrophages in culture. We have not however, observed macrophage-associated inflammatory disease in any animal infected with SIVmac239/316. Therefore, we sought to examine the cell tropism of this virus in vivo in multiple tissues using in situ hybridization combined with immunohistochemistry and multilabel confocal microscopy for viral nucleic acid and multiple cell-type-specific markers for macrophages and T lymphocytes. Tissues examined included brain, heart, lung, lymph nodes, spleen, thymus, and small and large intestine. Matched tissues from macaques infected with the parental SIVmac239 and uninfected macaques were also examined. Many infected cells were detected in the tissues of animals infected with SIVmac239 and SIVmac239/316 although there appeared to be fewer positive cells in animals infected with SIVmac239/316. Surprisingly, in light of the cell culture observations, nearly every simian immunodeficiency virus-infected cell in animals inoculated with SIVmac239/316 was a T lymphocyte rather than a macrophage. This was true both during early infection (first 2 months) and in terminal disease. In contrast, as previously described, SIVmac239 was found in both T cells and macrophages in tissues as early as 21 days after infection. These studies indicate that during both acute and chronic SIVmac239/316 infection T lymphocytes rather than macrophages are the principal targets in vivo. These data combined with the absence of macrophage-associated lesions in SIVmac239/316-infected animals indicate that in vitro cell tropism is not predictive of in vivo tropism or disease pathogenesis.

To develop a model for Epstein-Barr virus (EBV) pathogenesis in immunosuppressed hosts, we studied experimental infections of immunocompetent versus SHIV 89.6P–infected, immunosuppressed rhesus macaques with the EBV-related rhesus lymphocryptovirus (LCV). Primary LCV infection after oral inoculation of 4 immunocompetent animals was characterized by an acute viremia and seroconversion followed by asymptomatic LCV persistence. Four immunosuppressed macaques infected orally with LCV failed to develop an LCV-specific humoral response and viremia was more pronounced, but there was no evidence of LCV-induced lymphoproliferative disease. A more aggressive primary challenge was administered by intravenous inoculation of 108 autologous, LCV-immortalized B cells in 4 additional immunosuppressed animals. Two animals with modest immunosuppression remained asymptomatic, and 1 of 2 severely immunosuppressed animals developed an aggressive, monoclonal LCV-positive lymphoma. These studies demonstrate the potential for lymphomagenesis in an experimental model system for EBV infection and underscore the strength and depth of immune control in limiting LCV-induced lymphoproliferative disease.

Adaptive immune responses of γδ T cells during active mycobacterial coinfection of human immunodeficiency virus-infected humans have not been studied. Macaques infected with the simian immunodeficiency virus (SIV) SIVmac were employed to determine the extent to which a coincident AIDS virus infection might compromise immune responses of mycobacterium-specific Vγ2Vδ2+T cells during active mycobacterial infection. Control SIVmac-negative macaques developed primary and recall expansions of phosphoantigen-specific Vγ2Vδ2+T cells afterMycobacterium bovisBCG infection and BCG reinfection, respectively. In contrast, SIVmac-infected macaques did not exhibit sound primary and recall expansions of Vγ2Vδ2+T cells in the blood and pulmonary alveoli following BCG infection and reinfection. The absence of adaptive Vγ2Vδ2+T-cell responses was associated with profound CD4+T-cell deficiency and subsequent development of SIVmac-related tuberculosis-like disease in the coinfected monkeys. Consistently, Vγ2Vδ2+T cells from coinfected monkeys displayed a reduced capacity to expand in vitro following stimulation with phosphoantigen. The reduced ability of Vγ2Vδ2+peripheral blood lymphocytes (PBL) to expand could be restored to some extent by coculture of these cells with CD4+T cells purified from PBL of SIV-negative monkeys. Furthermore, naïve monkeys inoculated simultaneously with SIVmac and BCG were unable to sustain expansion of Vγ2Vδ2+T cells at the time that the coinfected monkeys developed lymphoid depletion and a fatal tuberculosis-like disease. Nevertheless, no deletion in Vδ2 T-cell receptor repertoire was identified in SIVmac-BCG-coinfected macaques, implicating an SIVmac-induced down-regulation rather than a clonal exhaustion of these cells. Thus, an SIVmac-induced compromise of the adaptive Vγ2Vδ2+T-cell responses may contribute to the immunopathogenesis of the SIV-related tuberculosis-like disease in macaques.

The frequency of cytomegalovirus (CMV)-specific CD4+T lymphocytes was determined in CMV-seropositive rhesus macaques with or without simian immunodeficiency virus (SIV) infection by using the sensitive assays of intracellular cytokine staining and gamma interferon ELISPOT. Both techniques yielded 3- to 1,000-fold-higher frequencies of CMV-specific CD4+T lymphocytes than traditional proliferative limiting dilution assays. The median frequency of CMV-specific CD4+T lymphocytes in 23 CMV-seropositive SIV-negative macaques was 0.63% (range, 0.16 to 5.8%). The majority of CMV-specific CD4+T lymphocytes were CD95posand CD27lobut expressed variable levels of CD45RA. A significant reduction (P< 0.05) in the frequency of CMV-specific CD4+T lymphocytes was observed in pathogenic SIV-infected macaques but not in macaques infected with live attenuated strains of SIV. CMV-specific CD4+T lymphocytes were not detected in six of nine pathogenic SIV-infected rhesus macaques. CMV DNA was detected in the plasma of four of six of these macaques but in no animal with detectable CMV-specific CD4+T lymphocytes. In pathogenic SIV-infected macaques, loss of CMV-specific CD4+T lymphocytes was not predicted by the severity of CD4+T lymphocytopenia. Neither was it predicted by the pre-SIV infection frequencies of CD45RAnegor CCR5posCMV-specific CD4+T lymphocytes. However, the magnitude of activation, as evidenced by the intensity of CD40L expression on CMV-specific CD4+T lymphocytes pre-SIV infection, was three- to sevenfold greater in the two macaques that subsequently lost these cells after SIV infection than in the two macaques that retained CMV-specific CD4+T lymphocytes post-SIV infection. Future longitudinal studies with these techniques will facilitate the study of CMV pathogenesis in AIDS.

To examine the role of T cell receptor (TCR) in γδ T cells in adaptive immunity, a macaque model was used to follow Vγ2Vδ2+T cell responses to mycobacterial infections. These phosphoantigen-specific γδ T cells displayed major expansion duringMycobacterium bovisBacille Calmette-Guérin (BCG) infection and a clear memory-type response after BCG reinfection. Primary and recall expansions of Vγ2Vδ2+T cells were also seen duringMycobacterium tuberculosisinfection of naı̈ve and BCG-vaccinated macaques, respectively. This capacity to rapidly expand coincided with a clearance of BCG bacteremia and immunity to fatal tuberculosis in BCG-vaccinated macaques. Thus, Vγ2Vδ2+T cells may contribute to adaptive immunity to mycobacterial infections.

The mechanism by which human immunodeficiency virus (HIV)-Mycobacterium tuberculosiscoinfection facilitates development of HIV-related tuberculosis is poorly characterized. Macaque models of simian immunodeficiency virus (SIVmac)-Mycobacterium bovisBCG coinfection were employed to explore the pathogenesis of AIDS virus-related tuberculosis. Following BCG coinfection, SIV (SIV)-infected macaques with high viral loads developed an SIV-related tuberculosis-like disease. This disease was characterized clinically by a syndrome of diarrhea, anorexia, weight loss, and altered levels of consciousness and pathologically by the presence of disseminated granulomas. In contrast, SIVmac-infected macaques with low viral loads either showed no evidence of BCG-induced disease or developed focal granulomatous lesions. Pathogenic SIV-BCG interactions appeared to play a critical role in triggering the development of this SIV-related tuberculosis-like disease. BCG coinfection enhanced the destruction of CD4+T cells in SIVmac-infected macaques whose viral loads were high. Reciprocally, exacerbations of SIV disease led to marked suppression of BCG-specific T-cell responses, persistence of the BCG infection, and development of an SIV-related tuberculosis-like disease. Furthermore, development of this SIV-related tuberculosis-like disease was also seen in naïve macaques simultaneously inoculated with SIVmacand BCG. These results provide in vivo evidence that coinfection of AIDS virus-infected individuals with an avirulent mycobacterium can lead to development of a tuberculosis-like disease.

While it is well established that cellular activation can increase human immunodeficiency virus (HIV) replication in T lymphocytes, it is also clear that both activated CD8+and CD4+T lymphocytes mediate anti-HIV activity. To assess the relative importance of these contrary effects on HIV replication in vivo, we evaluated the consequences ofMycobacterium bovisBCG and staphylococcal enterotoxin B (SEB) inoculation in vivo in rhesus monkeys chronically infected with simian immunodeficiency virus of macaques (SIVmac). BCG inoculation induced as much as a 2.5-log reduction of plasma and intracellular SIV RNA in SIVmac-infected monkeys. This down-regulation of virus replication persisted as long as 4 weeks after BCG inoculation. Similarly, SEB injection resulted in up to a 3-log decrease in plasma and intracellular SIV RNA in SIVmac-infected macaques. Interestingly, the short-term reduction of viremia in these monkeys correlated with the peak in vivo production of SEB- and BCG-induced cytokine responses. However, no long-term clinical benefit was observed in the SIVmac-infected macaques. These studies provide in vivo evidence that potent T-cell stimulation driven by antigens other than the virus itself can, under some circumstances, mediate short-term reduction of viremia in AIDS virus-infected individuals.

Cryptosporidiosis is a common opportunistic infection in the gastrointestinal tract of human and nonhuman primates with AIDS. Pulmonary infection associated with Cryptosporidium spp. has not been previously reported in monkeys. Two macaques experimentally infected with simian immunodeficiency virus (SIV) had lesions containing cryptosporidial organisms involving the trachea, lungs, bile ducts, pancreas, and intestine. The pulmonary sections revealed moderate to severe bronchopneumonia associated with cryptosporidiosis. Numerous 2-4 μm oval Cryptosporidium spp. organisms were present in the cytoplasm of alveolar macrophages, multinucleated giant cells, and intestinal epithelial cells. Giant cells were positive for SIV by in situ hybridization. These are the first reported cases of cryptosporidiosis with involvement of pulmonary parenchyma in SIV-infected macaques.

Neonatal human immunodeficiency virus (HIV) infection usually occurs intrapartum or postpartum and results in a higher incidence of neurological dysfunction than is seen in adults. To explore the neuropathogenesis of neonatal HIV infection, we infected neonatal macaques with simian immunodeficiency virus (SIV) and followed the course of infection focusing on early time points. Infected neonates had decreased brain growth and mild histological changes in brain that resembled those seen in pediatric AIDS, including perivascular infiltrates of mononuclear cells, mineralization of vessels in the basal ganglia, and gliosis. The perivascular lesions and gliosis were associated with the presence of occasional infected cells that required in situ hybridization with radiolabeled riboprobes for detection. Using this technique, SIV-infected cells were detected in the brain parenchyma within 7 days of infection. These findings were confirmed by nested PCR for SIVgag DNA in brain and RT-PCR for viral RNA in cerebrospinal fluid. Together, these techniques revealed SIV infection of the CNS in 12 of 13 neonates infected with SIVmac239, 3 of 3 infected with SIVmac251, and 2 of 2 infected with SIVmac239/316. The prevalence of CNS infection was indistinguishable from that of older animals infected with the same dose and stock of virus, but neonates appeared to have fewer infected cells in the CNS and detecting them required more sensitive techniques. This observation was true regardless of inoculum and despite the fact that neonates had equal or greater viral loads in the periphery compared with older animals. These data suggest that maturation-dependent host factors have a major impact on the neuropathogenesis of pediatric AIDS.

The primate polyomavirus SV40 is known to cause interstitial nephritis in primary infections and progressive multifocal leukoencephalopathy (PML) upon reactivation of a latent infection in SIV-infected macaques. We now describe a second central nervous system manifestation of SV40: a meningoencephalitis affecting cerebral gray matter, without demyelination, distinct from PML. Meningoencephalitis appears also to be a primary manifestation of SV40 infection and can be seen in conjunction with SV40-induced interstitial nephritis and pneumonitis. The difference in the lesions of meningoencephalitis and PML does not appear to be due to cellular tropism, as both oligodendrocytes and astrocytes are infected in PML and meningoencephalitis, as determined by in situ hybridization or immunohistochemistry for SV40 coupled with immunohistochemistry for cellular determinants. This is further supported by examination of SV40 nucleic acid sequences from the ori-enhancer and large-T-antigen regions, which reveals no tissue- or lesion-specific variation in SV40 sequences.

The thymus plays a critical role in the maturation and production of T lymphocytes and is a target of infection by human immunodeficiency virus (HIV) and the related simian immunodeficiency virus (SIV). Using the SIV/macaque model of AIDS, we examined the early effects of SIV on the thymus. We found that thymic infection by SIV resulted in increased apoptosis 7–14 d after infection, followed by depletion of thymocyte progenitors by day 21. A marked rebound in thymocyte progenitors occurred by day 50 and was accompanied by increased levels of cell proliferation in the thymus. Our results demonstrate a marked increase in thymic progenitor activity very early in the course of SIV infection, long before marked declines in peripheral CD4+ T cell counts.

Polyomaviruses have proven oncogenicity in nonhost experimental animals; however, studies concerning the association between human brain tumors and simian and human polyomaviruses have yielded inconclusive results. We examined the relationship of SV40 to a malignant astrocytoma found in the right frontal lobe of a pigtail macaque (Macaca nemestrina) infected with simian immunodeficiency virus (SIV). Consistent with the histologic diagnosis, the tumor was immunoreactive with antibodies to S-100 protein, vimentin, and glial fibrillary acidic protein, but negative for neurofilament protein, synaptophysin, neuron-specific enolase, and chromogranin A. At the time of SIV inoculation, the animal was seropositive for SV40. Polymerase chain reaction assay of tumor DNA, but not normal brain DNA, yielded a 300 base-pair fragment corresponding to the carboxy-terminal coding region (C-terminus) of the large T antigen gene of SV40, suggesting an association with the tumor.

Rhesus monkeys (Macaca mulatta) were infected with five strains of simian immunodeficiency virus (SIV) derived from SIVmac239 containing deletions (delta) or substitutions (subst) in NF-kappaB and Sp1 binding sites. We have shown previously that mutations in these regions still allow efficient SIVmac replication in primary lymphoid cell cultures (P. O. Ilyinskii and R. C. Desrosiers, J. Virol. 70:3118-3126, 1996). Two animals were inoculated intravenously with each mutant strain of SIVmac239: delta NFkappaB, delta Sp1234, delta NFkappaB delta Sp1234, substSp12, and substSp1234. All but one of the infected animals showed an early spike in plasma antigenemia, maintained high virus burdens, and had significant changes in lymphoid tissues, and six died with AIDS within the first 60 weeks of infection. One of the animals infected with the SIV strain delta NFkappaB delta Sp1234 showed lower levels of plasma antigenemia and lower virus burdens; the other animal infected with this same mutant strain died with AIDS 17 weeks after inoculation. No consistent novel mutations or reversions were detected in proviral sequences derived from the animals infected with the deletion mutants and the substSp12 mutant by 20 weeks postinfection. Point-mutated sequences were partially deleted in both animals infected with the substSp1234 strain. These results indicate that the NF-kappaB and Sp1 binding sites are not essential for the induction of AIDS by SIVmac239. They also provide indirect evidence for the importance of a novel enhancer element in the U3 region of the SIVmac long terminal repeat that is located immediately upstream of the NF-kappaB binding site within the C-terminal region of the nef coding sequence.

The larvae of Mesocestoides are rarely encountered in nonhuman primates, with most cases reported in baboons. Infection of macaques has been occasionally diagnosed, but Mesocestoides in the lung parenchyma is extremely rare. We have previously demonstrated that in macaques with terminal AIDS, simian immunodeficiency virus (SIV)-infected leukocytes are rarely found in cellular infiltrates associated with opportunistic infections or preexisting disease. Here we describe larvae (tetrathyridia) of the cestode Mesocestoides in the lung of an adult, pigtailed macaque (Macaca nemestrina) during acute SIV infection in which virus-positive cells are present within the cellular infiltrates. These results describe a rare parasitic disease in pigtailed macaques and demonstrate that lentivirus-infected leukocytes can be associated with inflammatory sites during acute infection.

A prevalent and distinctive infectious interstitial pneumonia (IIP) of immunocompetent laboratory rats was suspected to be caused by a putative virus, termed rat respiratory virus, but this was never substantiated. To study this disease, 2 isolators were independently populated with rats from colonies with endemic disease, which was perpetuated by the regular addition of naive rats. After Pneumocystis was demonstrated by histopathology and polymerase chain reaction (PCR) in the lungs of rats from both isolators and an earlier bedding transmission study, the relationship between Pneumocystis and IIP was explored further by analyzing specimens from 3 contact transmission experiments, diagnostic submissions, and barrier room breeding colonies, including 1 with and 49 without IIP. Quantitative (q) PCR and immunofluorescence assay only detected Pneumocystis infection and serum antibodies in rats from experiments or colonies in which IIP was diagnosed by histopathology. In immunocompetent hosts, the Pneumocystis concentration in lungs corresponded to the severity and prevalence of IIP; seroconversion occurred when IIP developed and was followed by the concurrent clearance of Pneumocystis from lungs and resolution of disease. Experimentally infected immunodeficient RNU rats, by contrast, did not seroconvert to Pneumocystis or recover from infection. qPCR found Pneumocystis at significantly higher concentrations and much more often in lungs than in bronchial and nasal washes and failed to detect Pneumocystis in oral swabs. The sequences of a mitochondrial ribosomal large-subunit gene region for Pneumocystis from 11 distinct IIP sources were all identical to that of P. carinii. These data provide substantial evidence that P. carinii causes IIP in immunocompetent rats.

The nef reading frame overlaps about 70% of the U3 region of the 3' long terminal repeat (LTR) in primate lentiviruses. We investigated the functional role of these overlapping U3 sequences by analyzing the properties of three mutant forms of the pathogenic SIVmac239 clone. In mutant UScon, 90 of 275 bp in the upstream sequences (US) of U3 were changed in a conservative fashion without changing the predicted nef coding sequence. In mutant USnon, 101 of 275 bp in this region were changed in a nonconservative fashion, again without changing the predicted nef coding sequence. In mutant delta US, 275 bp in this region were deleted. Full-size, immunoreactive nef protein was synthesized in cells infected with the UScon and USnon mutants. The USnon and delta US mutants replicated with similar kinetics and to similar extents as wild-type, parental SIVmac239 in primary rhesus monkey peripheral blood mononuclear cell (PBMC) cultures. The UScon mutant replicated with slightly delayed kinetics in rhesus monkey PBMC cultures. In the CEMx174 cell line, the delta US mutant replicated similarly to the wild type, but the UScon and USnon mutants replicated with significantly delayed kinetics. Analysis of LTR-driven chloramphenicol acetyltransferase (CAT) activity and the effects of 5-azacytidine on virus replication suggested that the growth defect of the point mutants in CEMx174 cells was due in whole or in part to the introduction of multiple CG methylation sites in proviral DNA. Rhesus monkeys were experimentally infected with the UScon and USnon mutants, and the characteristics of the infection were compared with those of the parental SIVmac239. Analysis of the levels of plasma antigenemia, virus load, and CD4+ cells in PBMC revealed no decreased virulence of the mutant viruses. Analysis of lymph node biopsies taken from animals that received mutant viruses revealed histologic changes and levels of virus expression indistinguishable from those of the wild type. Furthermore, the wild-type behavior of the mutant viruses in rhesus monkeys occurred without any specific reversional events through at least 20 weeks of infection. These results, and the recent results of Kirchhoff et al. (F. Kirchoff, H. W. Kestler III, and R. C. Desrosiers, J. Virol. 68:2031-2037, 1994), suggest that these upstream sequences in U3 are primarily or exclusively nef coding sequence.

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.

A spontaneous squamous cell carcinoma was diagnosed in the oral cavity of an adult female squirrel monkey (Saimiri sciureus). Immunohistochemical analysis of the neoplasm demonstrated cytokeratin and vimentin, but not S100 or desmin in the neoplastic epithelial cells.

We studied granulomatous inflammation in simian AIDS using histologic, immunohistologic, and in situ hybridization techniques. Complete Freund’s adjuvant was used to induce granulomas in two control animals and two macaques infected with simian immunodeficiency virus (SIV) and having low peripheral CD4+ T cell counts. Control animals developed large (>2 cm diameter) epithelioid granulomas containing CD68+ macrophages (mφs), epithelioid mφs and multinucleated giant cells (MNGCs), CD4+ and CD8+ T cells, and small perivascular collections of CD20+ B cells. Lymphocytes rarely expressed proliferating cell nuclear antigen (Ki-67), and only rare endothelial cells expressed vascular cell adhesion molecule 1 (VCAM-1). In contrast, SIV+ animals had smaller (

Three of 12 infant rhesus macaques became infected at 9 to 12 months of age with SIVsmm through maternal-infant transmission. Clinical problems seen in one or more infants included decreased CD4 cells, hypergammaglobulinemia, diarrhea, weight loss, anemia, bacterial infections, and terminal respiratory and CNS problems. Gross and histologic lesions due to both primary SIV infection and opportunistic infections were observed. The SIV-infected infants had clinical, immunologic, and pathologic similarities to those seen in pediatric HIV infection.

The simian parvoviruses (SPVs) are in the genus Erythrovirus in the family Parvoviridae and are most closely related to the human virus B19. SPV has been identified in cynomolgus, rhesus, and pigtailed macaques. All of the primate erythroviruses have a predilection for erythroid precursors. Infection, which is common in macaques, is usually clinically silent. Disease from SPV is associated with immunosuppression due to infection with various retroviruses (SIV, simian retrovirus, and simian–human immunodeficiency virus), surgery, drug toxicity studies, and posttransplantation immunosuppressive treatment and therefore is of concern in studies that use parvovirus-positive macaques.

Polyomaviruses are a family of small nonenveloped DNA viruses that infect birds and mammals. At least 7 nonhuman primate polyomaviruses that occur in macaques, African green monkeys, marmosets baboons, and chimpanzees have been described, as well as 4 polyomaviruses that occur in humans. Simian virus 40 (SV40), which infects macaques, was the first nonhuman primate polyomavirus identified as a contaminant of early polio vaccines. Primate polyomaviruses cause inapparent primary infections but persist in the host and can cause severe disease in situations of immunocompromise. This review describes the primate polyomaviruses, and the diseases associated with the viruses of macaques. In macaques, the greatest current concerns are the potential confounding of study results by polyomavirus infections and the zoonotic potential of SV40.

We noted naturally occurring infection with Clostridium piliforme (Tyzzer's disease) in 2 captive-reared cotton-top tamarins (Saguinus oedipus). Spontaneous Tyzzer's disease has been reported in multiple species of laboratory, domestic, and wild animals but is extremely rare in humans and nonhuman primates. Distinct from idiopathic colitis, which is common in cotton-top tamarins, these 2 tamarins had severe, transmural, necrotizing typhlocolitis accompanied by myocarditis and hepatitis. Abundant bacteria compatible with C. piliforme, the etiologic agent of Tyzzer's disease, were present adjacent to lesions in the cecum-colon, liver, and heart. Therefore, colitis caused by C. piliforme, although rare, should be included as a differential diagnosis in cotton-top tamarins and as a cause of postnatal mortality in this species.

Background Severe disseminated vasculopathy was observed in two simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta). These animals developed clinical signs of AIDS, including lymphadenopathy, weight loss, diarrhea and collapse. Results and discussion Grossly, both animals showed emaciation, lymphadenopathy, vegetations on the mitral valve, renal infarcts and a dilated intestine; one animal had multifocal hemorrhages in multiple organs. Histologically, both cases had disseminated arteriopathy characterized by intimal thickening and fibrosis with varying degrees of vasculitis. The lesion was prominent in the kidney, intestine, pancreas, liver, heart, lymph nodes, spleen and testis. Occasional venules had intimal thickening. Both cases had cytomegalovirus (CMV) infection with intranuclear inclusions, CMV antigen and nucleic acid; some inclusions were observed in endothelial cells within some of the vascular lesions in one of the two. These data suggest that CMV caused the unusual lesions.

A 4-year-old female pigtailed macaque ( Macaca nemestrina), experimentally coinfected with simian immunodeficiency virus (SIVmac251) and Mycobacterium bovis (bacillus Calmette-Guerin), was euthanatized 1 year after infection because of weight loss and labored breathing. On gross examination, both kidneys were found to be markedly enlarged (right: 54.7 g and left: 51.7 g; normal < 20 g). Renal lesions were evaluated by histopathologic, immunohistochemical, and ultrastructural methods. Light microscopy revealed that the glomeruli were diffusely hypercellular with expansion of the mesangial matrix, and crescent formation affected approximately 60% of the glomeruli. By immunohistochemical evaluation, it was found that the crescents were composed principally of macrophages, as seen by CD68 (KP1), MRP8, MAC387, and HAM56 expression. Electron microscopic examination of the glomeruli revealed extensive intramembranous, subendothelial, and mesangial electron-dense deposits and multifocal fusion of the visceral epithelial foot processes. Immunofluorescence, used to determine the composition of the electron-dense deposits, revealed diffuse granular mesangial and capillary staining for immunoglobulin A (IgA). The renal changes described in this case report are most consistent with the findings of crescentic gloerulonephritis with IgA immune complex deposition in the glomerular basement membrane and mesangium as described in humans with IgA nephropathy.

Epstein-Barr virus (EBV) is a human oncogenic herpesvirus associated with epithelial cell and B-cell malignancies. EBV infection of B lymphocytes is essential for acute and persistent EBV infection in humans; however, the role of epithelial cell infection in the normal EBV life cycle remains controversial. The rhesus lymphocryptovirus (LCV) is an EBV-related herpesvirus that naturally infects rhesus macaques and can be used experimentally to model persistent B-cell infection and B-cell lymphomagenesis. We now show that the rhesus LCV can infect epithelial cells in immunosuppressed rhesus macaques and can induce epithelial cell lesions resembling oral hairy leukoplakia in AIDS patients. Electron microscopy, immunohistochemistry, and DNA-RNA in situ hybridization were used to identify the presence of a lytic rhesus LCV infection in these proliferative, hyperkeratotic, or parakeratotic epithelial cell lesions. These studies demonstrate that the rhesus LCV has tropism for epithelial cells, in addition to B cells, and is a relevant animal model system for studying the role of epithelial cell infection in EBV pathogenesis.

Although opportunistic infections like cytomegalovirus (CMV) are common sequelae of end-stage AIDS, the immune events leading to CMV reactivation in human immunodeficiency virus (HIV)-infected individuals are not well defined. The role of cellular and humoral CMV-specific immune responses in immune control of latent CMV infection was evaluated prospectively in a cohort of 11 simian immunodeficiency virus (SIV)-infected CMV-seropositive rhesus macaques, 6 of whom had histologic evidence of CMV disease at death. Macaques with CMV disease differed from macaques without CMV disease in having significantly higher levels of plasma SIV RNA and CMV DNA and significantly lower titers of anti-CMV binding antibodies (Abs) at the time of death. A significant decline in anti-CMV Abs and CMV-specific CD4+and CD8+T lymphocytes over time was observed in the macaques with CMV disease, but not in the macaques without CMV disease. Reduction in CMV-specific CD8+T lymphocytes and anti-CMV neutralizing Abs was significantly correlated with a decline in CMV-specific CD4+T lymphocytes. Although declines in CMV-specific T lymphocytes alone were sufficient for reactivation of low-level CMV viremia, high-level viremia (>1,000 copies of CMV DNA per ml of plasma) was observed when anti-CMV neutralizing and binding Abs had also declined. Thus, the occurrence of CMV reactivation-associated disease in AIDS is associated with suppression of both cellular and humoral CMV-specific immune responses. The underlying mechanism may be a dysfunction of memory B and CD8+T lymphocytes associated with SIV-induced impairment of CMV-specific CD4+T-cell help.

Cellular immune responses mediated by CD8+lymphocytes exert efficient control of virus replication during primary simian immunodeficiency virus (SIV) infection. However, the role that antibodies may play in the early control of virus replication remains unclear. To evaluate how antibody responses may affect virus replication during primary SIVmac infection, we depleted rhesus monkeys of B cells with anti-CD20 antibody. In normal rhesus monkeys immunized with tetanus toxoid, anti-CD20 treatment and resulting depletion of B cells inhibited the generation of antitetanus antibodies, while tetanus-specific T-cell responses were preserved. During the first 4 weeks after inoculation with SIVmac251, development of SIV-specific neutralizing antibody was delayed, and titers were significantly lower in B-cell-depleted monkeys than control-antibody-treated monkeys. Despite the lower neutralizing antibody titers, the levels of plasma SIV RNA and the linear slope of the decline seen in B-cell-depleted monkeys did not differ from that observed in monkeys treated with control antibody. However, beginning at day 28 after SIV infection, the B-cell-depleted monkeys showed a significant inverse correlation between neutralizing antibody titers and plasma virus level. These results suggest that the rapid decline of peak viremia that typically occurs during the first 3 weeks of infection was not significantly affected by SIV-specific antibodies. However, the inverse correlation between neutralizing antibodies and plasma virus level during the postacute phases of infection suggests that humoral immune responses may contribute to the control of SIV replication.

The contribution of immune reconstitution following antiretroviral treatment to the prevention or treatment of human immunodeficiency virus-related primary or reactivation tuberculosis remains unknown. Macaque models of simian immunodeficiency virus-Mycobacterium bovisBCG (SIV/BCG) coinfection were employed to determine the extent to which anti-Mycobacterium tuberculosisimmunity can be restored by antiretroviral therapy. Both SIV-infected macaques with active BCG reinfection and naive animals with simultaneous SIV/BCG coinfection were evaluated. The suppression of SIV replication by antiretroviral treatment resulted in control of the active BCG infection and blocked development of the fatal SIV-related tuberculosis-like disease. The resolution of this disease coincided with the restoration of BCG purified protein derivative (PPD)-specific T-cell immune responses. In contrast, macaques similarly coinfected with SIV/BCG but not receiving antiretroviral therapy had depressed PPD-specific primary and memory T-cell immune responses and died from tuberculosis-like disease. These results provide in vivo evidence that the restoration of anti-mycobacterial immunity by antiretroviral agents can improve the clinical outcome of an AIDS virus-related tuberculosis-like disease.

Background —Cardiomyopathy is being recognized with increasing frequency in patients with AIDS, yet the relationship between HIV infection and cardiac contractile dysfunction remains obscure. The purpose of the present study was to determine if infection with simian immunodeficiency virus (SIV) in nonhuman primates is associated with cardiac dysfunction and myocardial injury. Methods and Results —Left ventricular size and function were determined by 2D echocardiography in 16 rhesus macaques before and at weekly intervals following infection with cloned pathogenic SIV mac 239 or the highly attenuated SIV mac 239 nef deletion mutant. A second group of 15 rhesus macaques chronically infected with pathogenic (n=6) or nonpathogenic (n=9) virus were studied at >2 years following infection. Cardiac tissues from 24 rhesus macaques chronically infected (>2 years) with pathogenic SIV were reviewed for evidence of cardiac pathology. Acute infection (P Conclusions —This unique model is valuable in understanding the pathogenesis of cardiac injury associated with retroviral infection in a relevant nonhuman primate model of AIDS.

Pneumocystis carinii (PC) pneumonia is a frequent manifestation of the acquired immunodeficiency syndrome (AIDS) in humans and macaques. An unusual nodular type of PC pneumonia was observed in two simian immunodeficiency virus (SIV)-inoculated rhesus macaques ( Macaca mulatta). These animals developed clinical signs of simian AIDS, including anorexia, weight loss, dyspnea, and collapse. Grossly, both animals had multifocal tan-white nodules 1–10 mm in diameter scattered throughout the lungs. One animal had similar nodules involving the diaphragm and thoracic wall. The lungs were characterized by severe PC pneumonia with numerous large nodules consisting of foamy material that compressed adjacent tissue. The nodules had central areas of necrosis and lysis of alveolar septa. Varying degrees of necrotizing vasculitis were observed in areas of nodular PC pneumonia. The presence of PC in intra-alveolar spaces and nodular lesions was confirmed by immunohistochemistry. No evidence of other agents, including viral inclusions, bacteria, fungi, and lung mites, was detected. The animal with the most severe nodular PC pneumonia had vascular involvement with extrapulmonary spread to the diaphragm, thoracic wall, and regional lymph nodes. This unusual type of nodular PC pneumonia has been rarely seen in human AIDS patients.

Clinical evidence suggests that cellular immunity is involved in controlling human immunodeficiency virus–1 (HIV-1) replication. An animal model of acquired immune deficiency syndrome (AIDS), the simian immunodeficiency virus (SIV)–infected rhesus monkey, was used to show that virus replication is not controlled in monkeys depleted of CD8 + lymphocytes during primary SIV infection. Eliminating CD8 + lymphocytes from monkeys during chronic SIV infection resulted in a rapid and marked increase in viremia that was again suppressed coincident with the reappearance of SIV-specific CD8 + T cells. These results confirm the importance of cell-mediated immunity in controlling HIV-1 infection and support the exploration of vaccination approaches for preventing infection that will elicit these immune responses.

Competitive PCR was used to evaluate the expression of cytokine, granzyme B, and chemokine genes in lymph nodes of macaques recently infected with the simian immunodeficiency virus (SIV) pathogenic molecular clone SIVmac239 (n 516), the nonpathogenic vaccine strain SIVmac239Dnef (n 58), and the nonpathogenic molecular clone SIVmac1A11 (n 5 8). For both SIVmac239 and its nef-deleted derivative, strong expressionwasobservedasearlyas7dayspostinfectionforinterleukin1b(IL-1b),IL-6,tumornecrosisfactor alpha, gamma interferon, and IL-13. The levels of gene induction were equally intense for both viruses despite a lower viral load for SIVmac239Dnef compared with that for SIVmac239. However, the nature of the cytokine network activation varied with the viral inocula. Primary infection with SIVmac239 was characterized by a higher level of IL-4, IL-10, MIP-1a, MIP-1b, MCP-1, and RANTES gene expression and a lower level of IL-12 and granzyme B gene expression compared with infection with SIVmac239Dnef. Thus, infection with nefdeleted SIV was associated with a preferential Th1 versus Th2 pattern of cytokine production. Infection with SIVmac1A11 was characterized by a delayed immune response for all markers tested. The unique patterns of cytokine and chemokine gene expression in lymph nodes correlated nicely with the pathogenic potential of the SIV strains used as well as with differences in their ability to serve as protective vaccines. Both viral and host factors likely contribute to differences in rates of disease progression in human immunodeficiency virus (HIV)-infected individuals. However, it is difficult to determine the respective contributions of these factors in HIVinfected patients. Several recent studies have thus begun to address this question during thefirst few weeks after infection with the simian immunodeficiency virus (SIV)-infected macaque (SIVmac) model (2, 3, 8, 20, 27, 33, 48, 51). These

A panel of commercially available antibodies which recognize specific antigens on human tissues was developed for use in immunohistochemistry on tissues from eight species of nonhuman primates. Antibodies were selected for potential usefulness in diagnostic pathology, and for effectiveness in formalin-fixed, paraffin-embedded tissues. Tissues from four species of macaques and four New World monkeys were evaluated. Using these antibodies we were able to identify 17/21 antigens examined in all eight species, and 21/21 antigens in the four species of macaques. Detailed immunohistochemistry protocols are presented, along with a systematic approach to developing a protocol for a new antibody.

Rhesus monkeys (Macaca mulatta) were experimentally infected with strains of simian immunodeficiency virus (SIV) derived from SIVmac239 lacking vpr, vpx, or both vpr and vpx genes. These auxiliary genes are not required for virus replication in cultured cells but are consistently conserved within the SIVmac/human immunodeficiency virus type 2/SIVsm group of primate lentiviruses. All four rhesus monkeys infected with the vpr deletion mutant showed an early spike in plasma antigenemia, maintained high virus burdens, exhibited declines in CD4+ lymphocyte concentrations, and had significant changes in lymph node morphology, and two have died to date with AIDS. The behavior of the vpr deletion mutant was indistinguishable from that of the parental, wild-type virus. Rhesus monkeys infected with the vpx deletion mutant showed lower levels of plasma antigenemia, lower virus burdens, and delayed declines in CD4+ lymphocyte concentrations but nonetheless progressed with AIDS to a terminal stage. The vpr+vpx double mutant was severely attenuated, with much lower virus burdens and no evidence of disease progression. These and other results indicate that vpr provides only a slight facilitating advantage for wild-type SIVmac replication in vivo. Thus, progression to AIDS and death can occur in the absence of a gene for vpr or vpx.

Since the original isolation of simian immunodeficiency virus (SIV) from a macaque with an AIDS-like disease, numerous studies have demonstrated the close biologic and genetic relationship of the SIVs to the HIVs. Probably most important, the clinical spectrum of disease associated with SIVmac/SIVsmm infection in rhesus monkeys is strikingly similar to AIDS in HIV-1-infected human beings. Herein are summarized the pathologic features of SIVmac-induced disease in a cohort of rhesus monkeys, with special reference to the role of infected macrophages in the development of AIDS-related manifestations.

A syndrome that occurred in both hard and soft contact lens wearers was characterized by increased mucus, itching, decreased lens tolerance, and giant papillae in the upper tarsal conjunctiva. It developed in as few as three weeks with soft lens wearers but also occurred after months or even years of successful wear. The histology was characterized by basophils, eosinophils, and mast cells in the epithelium, and these cells as well as increased numbers of lymphocytes, plasma cells, and polymorphonuclear leukocytes in the stroma. The syndrome may be immunologic in origin with deposits on the lenses as the antigen, and the syndrome may be a major cause of difficulty in wearing contact lenses once they have been successfully fit.

The 5' termini of copia-specific RNAs in Drosophila melanogaster tissue culture cells were determined by S1 nuclease mapping and cap analysis. Both major copia RNAs share an identical set of heterogeneous 5' ends. Three major cap 1 structures M7GpppCmpUp, M7GpppCmpCp and M7GpppGmpUp together with several other minor caps were found. Almost all the 5' termini, as judged by S-1 nuclease mapping, were located either in a pyrimidine-rich part of the terminal direct repeat or apparently outside of the copia element, suggesting that a proportion of copia transcripts derive from promoters external to the genetic element.

A stable, non-nitrogen-fixing mutant of Clostridium pasteurianum W5 was isolated. The mutant has the same growth rate as the wild type on ammonia nitrogen, but is not able to grow on N 2 . Cell-free extracts of the derepressed mutant are unable to fix nitrogen, or reduce the alternate substrates of nitrogenase such as cyanide, azide, or acetylene. Recombination with wild-type components from a diethylaminoethyl cellulose column indicates that the mutant contains component II, but is lacking component I activity.

The size, weight, and plasma cell content of main and accessory lacrimal glands were examined and compared to the number of plasma cells in the conjunctiva. There were from four to 35 accessory lacrimal glands in the upper conjunctiva and a rare accessory gland in the lower conjunctiva. The total accessory gland mass was approximately one tenth of the lacrimal gland mass although the number of plasma cells was approximately 1/20 of the mass. The number of plasma-cells in the lacrimal gland was estimated to be 3,200,000; in the conjunctiva, 2,100,000; and for the accessory glands, 180,000. Because of the many plasma cells in the relatively inaccessible lacrimal gland, cells of the lymphatic series may home to the lacrimal gland by non-antigenic mechanisms.

To examine the details of insertion for the D. malanogaster transposable element copia, we have isolated three independent pairs of genomic fragments which correspond to occupied and unoccupied target sites for insertion. Restriction endonuclease analysis suggests that sites with and without an element differ by a simple 5000 bp insertion. Direct DNA sequence analysis demonstrates that a 5 bp sequence, present once in the target DNA at the site of insertion, is found on both sides of the element after insertion. The 5 bp sequences which are duplicated are different in each case. Moreover, there does not appear to be any sequence homology among these three independent insertion sites

Abnormal upper tarsal tissue from four patients with vernal conjunctivitis stained immunofluorescently contained abundant IgA-, IgD-, and IgE-forming plasma cells in two of four patients. These were absent in normal upper tarsal conjunctiva. The IgE levels of paired tear-serum samples of 11 patients with vernal conjunctivitis and ten control subjects were 130 ng/ml in patients that did nor differ significantly from the normal tear IgE value of 61 ng/ml. The serum IgE means of 1,031 ng/ml for patients and 201 ng/ml for normal subjects differed significantly (P less than .05). Tear and serum IgE values correlated significantly for both groups (P less than .01 for patients, P less than .05 for normal subjects). These findings are consistent with a hyperplasia of IgA, IgD, and IgE antibody-forming cells in tarsal conjunctiva of some patients with vernal conjunctivitis, and tear IgE levels being a function of serum IgE levels.

Tear samples from 13 normal volunteers and nine patients with vernal conjunctivitis were assayed for histamine. Tears in both groups contained histamine. The normal subjects had values ranging from 2.2 to 36 ng/ml with a mean of 10.3 ng/ml. The vernal patients had histamine values ranging from 0 to 125 ng/ml with a mean of 3,.2 ng/ml. The presence of histamine in tears indicates a role for this important mediator in both the physiologic and immunologic processes of the external eye.