Induction of B cell hyperplasia in simian immunodeficiency virus-infected rhesus macaques with the simian homologue of Kaposi's sarcoma-associated herpesvirus.

A simian homologue of Kaposi's sarcoma-associated herpesvirus (KSHV), the eighth human herpesvirus (HHV8), was isolated from a simian immunodeficiency virus (SIV)-infected rhesus macaque (Macaca mulatta) that developed a multicentric lymphoproliferative disorder (LPD). This simian rhadinovirus is genetically similar to a recently described rhesus rhadinovirus (RRV) (Desrosiers, R.C., V.G. Sasseville, S.C. Czajak, X. Zhang, K.G. Mansfield, A. Kaur, R.P. Johnson, A.A. Lackner, and J.U. Jung. 1997. J. Virol. 71:9764-9769) and is designated RRV 17577. RRV 17577 was experimentally inoculated into rhesus macaques with and without SIV(mac239) infection to determine if RRV played a role in development of the LPD observed in the index case. In contrast to control animals inoculated with SIV(mac239) or RRV alone, two animals coinfected with SIV(mac239) and RRV 17577 developed hyperplastic LPD resembling the multicentric plasma cell variant of Castleman's disease, characterized by persistent angiofollicular lymphadenopathy, hepatomegaly, splenomegaly, and hypergammaglobulinemia. Hypergammaglobulinemia was associated with severe immune-mediated hemolytic anemia in one RRV/SIV-infected macaque. Both RRV/SIV-infected macaques exhibited persistent RRV viremia with little or no RRV-specific antibody response. The macaques inoculated with RRV alone displayed transient viremia followed by a vigorous anti-RRV antibody response and lacked evidence of LPD in peripheral blood and lymph nodes. Infectious RRV and RRV DNA were present in hyperplastic lymphoid tissues of the RRV/SIV-infected macaques, suggesting that lymphoid hyperplasia is associated with the high levels of replication. Thus, experimental RRV 17577 infection of SIV-infected rhesus macaques induces some of the hyperplastic B cell LPDs manifested in AIDS patients coinfected with KSHV.

The envelope glycoprotein ectodomains determine the efficiency of CD4+ T lymphocyte depletion in simian-human immunodeficiency virus-infected macaques.

CD4+ T lymphocyte depletion in human immunodeficiency virus type 1 (HIV-1)-infected humans underlies the development of acquired immune deficiency syndrome. Using a model in which rhesus macaques were infected with chimeric simian-human immunodeficiency viruses (SHIVs), we show that both the level of viremia and the structure of the HIV-1 envelope glycoprotein ectodomains individually contributed to the efficiency with which CD4(+) T lymphocytes were depleted. The envelope glycoproteins of recombinant SHIVs that efficiently caused loss of CD4(+) T lymphocytes exhibited increased chemokine receptor binding and membrane-fusing capacity compared with those of less pathogenic viruses. These studies identify the HIV-1 envelope glycoprotein ectodomains as determinants of CD4(+) T lymphocyte loss in vivo and provide a foundation for studying pathogenic mechanisms.

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

BACKGROUND: Rhesus macaques (RM) co-infected with simian immunodeficiency virus (SIV) and rhesus macaque rhadinovirus (RRV) develop abnormal cellular proliferations characterized as extra-nodal lymphoma and retroperitoneal fibromatosis (RF). RRV encodes a viral interleukin-6 (vIL-6), much like Kaposi's sarcoma-associated herpesvirus, and involvement of the viral cytokine was examined in proliferative lesions. METHODS: Formalin fixed tissue from RM co-infected with SIV and RRV were analyzed for RRV genomes by in situ hybridization and RRV vIL-6 expression by immunofluorescence analysis. RESULTS: In situ hybridization analysis indicated that RRV is present in both types of lesions. Immunofluorescence analysis of different lymphomas and RF revealed positive staining for vIL-6. Similarly to KS, RF lesion is positive for vimentin, CD117 (c-kit), and smooth muscle actin (SMA) and contains T cell, B cell and monocytes/macrophage infiltrates. CONCLUSIONS: Our data support the idea that vIL-6 may be critical to the development and progression of lymphoproliferative disorder in RRV/SIV-infected RM.

MR1-restricted mucosal-associated invariant T (MAIT) cells respond to mycobacterial vaccination and infection in nonhuman primates.

Studies on mucosal-associated invariant T cells (MAITs) in nonhuman primates (NHP), a physiologically relevant model of human immunity, are handicapped due to a lack of macaque MAIT-specific reagents. Here we show that while MR1 ligand-contact residues are conserved between human and multiple NHP species, three T-cell receptor contact-residue mutations in NHP MR1 diminish binding of human MR1 tetramers to macaque MAITs. Construction of naturally loaded macaque MR1 tetramers facilitated identification and characterization of macaque MR1-binding ligands and MAITs, both of which mirrored their human counterparts. Using the macaque MR1 tetramer we show that NHP MAITs activated in vivo in response to both Bacillus Calmette-Guerin vaccination and Mycobacterium tuberculosis infection. These results demonstrate that NHP and human MR1 and MAITs function analogously, and establish a preclinical animal model to test MAIT-targeted vaccines and therapeutics for human infectious and autoimmune disease.

Reactivity-based coronary vasospasm independent of atherosclerosis in rhesus monkeys.

OBJECTIVES: We studied the hypothesis that in the absence of vascular pathology, coronary artery vasospasm occurs as a result of local regions of vascular muscle hyperreactivity. We aimed to explore the basis for a functional etiology of those vasospasms not explained on a structural basis. BACKGROUND: Ovariectomized rhesus monkeys (Macaca mulatta) without injury or significant vascular disease were stimulated with platelet release products, and angiograms were compared with those from vasospasms induced in human patients. METHODS: We used intracoronary (IC) injections of serotonin, thromboxane A2 (U46619), endothelin 1 or angiotensin II in concentrations 3 to 10 times that which reduced coronary artery diameter by 50%. RESULTS: Although no agent alone caused vasospasm, the combination of pathophysiologic concentrations of serotonin and the stable thromboxane A2 mimetic, U46619, injected through an IC catheter, synergistically caused coronary vasospasm on the second or third challenge in five of seven monkeys. These drug-induced vasospasms were similar to vasospasms induced by mechanical injury followed by serotonin, and to those stimulated in human IC diagnostic tests, as judged by onset, appearance, kinetics and vasodilator reversal. CONCLUSIONS: These studies in ovariectomized monkeys revealed that coronary vasospasm can be stimulated without preexisting vascular pathology, endothelial denudation or injury. Reproducible vasospasm of primate coronary arteries in response to these two endogenous pathophysiologic vasoconstrictors, which are thought to be precipitating stimuli in the etiology of vasospasm, suggests that structure-independent epicardial vasospasm can be an important element in serious cardiac ischemic events, particularly the focal, persistent vasospasms that occur without plaques or injury.

Albumin leakage into cerebrospinal fluid of dogs lethally infected with R252 canine distemper virus.

Cerebrospinal fluid (CSF) form nine lethally infected and three convalescent gnotobiotic dogs infected with the R252 strain of canine distemper virus (CDV) was evaluated prior to and following infection. Lethally infected dogs had a mean seven-fold increase in CSF albumin concentration compared to the preinoculation value, not present in dogs destined to survive. Immunochemical examination of tissue from these dogs revealed prominent perivascular localization of albumin. Examination of CSF cells demonstrated mild leukocytosis in both groups at the time when encephalopathic deaths occurred, with decreased lymphocyte percentages, particularly Thy-1-bearing lymphocytes, in lethally infected dogs. These dogs also had more extensive expression of viral antigens in CSF and peripheral blood leukocytes at the time of death than did surviving dogs, and failed to make antibody to viral antigens. The findings link terminal breakdown of the blood-brain barrier and extensive viral antigen expression in CSF leukocytes with experimental CDV infection resulting in death.

An epitope on the surface envelope glycoprotein (gp130) of simian immunodeficiency virus (SIVmac) involved in viral neutralization and T cell activation.

SIVmac infection of macaques is an important animal model for HIV infection and AIDS; this model is being utilized for development of antiviral therapies and vaccines. In the present article, we sought to identify neutralization epitopes of SIVmac envelope surface glycoprotein (gp130). Algorithms were used to predict antigenicity of specific regions. Four regions from the primary amino acid sequence of the viral surface glycoprotein were selected. A synthetic peptide representing one of these regions (414-434) induced virus-neutralizing antibodies in mice; in addition, this peptide induced T cell-proliferative responses in macaques. To address the in vivo relevance of these observations, we demonstrated that experimentally infected macaques produce antibodies to the neutralization epitope. In addition, rhesus macaques protected against infection by an inactivated SIV vaccine develop antibodies that bind to peptide 414-434. These observations demonstrate that the region that includes the sequence 414-434 in the fourth variable domain (V4) of SIVmac gp130 contains both a linear neutralization epitope and a T cell epitope.

Immunocytochemical methods for demonstrating canine distemper virus antigen in aldehyde-fixed paraffin-embedded tissue.

The effects of enzymatic digestion, sodium borohydride reduction, acids used in decalcification procedures and techniques for inactivation of endogenous peroxidase were sequentially evaluated for their effect on the immunoreactivity of canine distemper virus in aldehyde-fixed paraffin-embedded tissue. Enzyme digestion improved immunoreactivity while sodium borohydride reduced background staining. Paraformaldehyde-glutaraldehyde-fixed tissues required thioglycolic acid treatment prior to enzyme digestion and sodium borohydride reduction to obtain results comparable to results obtained in formalin-fixed tissues. Detailed protocols for indirect immunofluorescence and the avidin-biotin-peroxidase complex procedure are provided.

Polymerase chain reaction detection of type D simian retrovirus proviral DNA from infected macaques.

A simple polymerase chain reaction (PCR) approach was developed for detection of Type D simian retrovirus (SRV) serogroup 2 proviral DNA using peripheral blood lymphocytes (PBLs) obtained from infected macaques. PCR primer pairs were developed against serogroup 2 envelope (env) gene sequence, and fidelity of PCR fragment amplification was determined using molecularly cloned SRV serogroup 2 (D2/RHE/OR) DNA, and genomic DNA from Raji cells independently infected with different SRV serogroups. One primer pair exhibiting high fidelity was then utilized for PCR detection of serogroup 2 proviral DNA from PBLs, and from cells sorted into immune cell subpopulations by fluorescent-activated cell sorting (FACS). Env PCR fragments were readily detected from as few as 10(4) PBLs or immune cell subpopulations. In addition, highly specific PCR primers against serogroups 1 and 3 were utilized to detect proviral DNA from Raji cells infected with SRV serogroups. In all cases, primers designed to amplify serogroups 1, 2, and 3 proviral DNA were specific for their intended serogroup. This primer information and development of a PCR approach for detection of specific SRV proviral DNA will be of potential utility as a rapid surveillance tool in monitoring type D simian retrovirus infection within Asian macaque colonies.

Metaphyseal bone lesions in young dogs with systemic canine distemper virus infection.

Bone lesions, restricted to the metaphyses of long bones, were observed in young dogs with systemic distemper following experimental and spontaneous infection. Canine distemper virus (CDV) antigen was found immunocytochemically in hematopoietic marrow cells, osteoclasts, osteoblasts and rarely in osteocytes. In experimentally infected dogs, viral antigen was demonstrated in the metaphysis between 5 and 36 days after infection. Associated lesions, characterized by necrosis of osteoclasts, persistence of primary spongiosa and atrophy and necrosis of osteoblasts and marrow cells, were mild and most prominent between 8 and 32 days postinfection. Metaphyseal osteosclerosis (MO) of the long bones, varying from mild to severe, was observed macroscopically in 8 (19%) out of 42 dogs with spontaneous distemper. Affected animals were between 3 and 6 months of age and belonged mainly to the large breeds. In these animals, MO was characterized histologically by persistence of primary spongiosa, loss of bone marrow cells and necrosis of osteoclasts and bone marrow cells varying from mild to severe. Summarized, CDV-associated bone lesions were only transient and there were no indications of viral persistence in bones of dogs experimentally infected with CDV. Although no clinical signs related to the bones were observed, the present study reveals that infection of metaphyseal bone cells is common in young dogs with systemic distemper and occurrence of viral antigen in these cells results in defects in bone modelling.

Establishment of central nervous system infection by canine distemper virus: breach of the blood-brain barrier and facilitation by antiviral antibody.

Morphologic, immunologic and virologic data implicating antiviral antibody in promoting entry of canine distemper virus (CDV) into brain and reticuloendothelial tissues are reviewed. Infection of central nervous system (CNS) endothelium precedes invasion of virus-positive and -negative leukocytes into Virchow-Robin spaces and central nervous system (CNS) parenchyma by 1-3 days. Platelets are implicated in initiation of endothelial infection in that: CDV-infected dogs are thrombocytopenic; platelets from CDV-infected dogs contain IgG-virus complexes on their plasma membranes; platelet microthrombi were observed adjacent to foci of endothelial infection, and; CDV-susceptible ferrets rendered thrombocytopenic by antiplatelet antibody exhibit delayed viral entry into CNS tissues. Renal glomerular-bound IgG, IgM and occasionally CDV antigen were demonstrated in CDV-infected dogs by immunocytochemical techniques. Distemper-infected dogs with inherited C3 deficiency exhibited enhanced renal glomerular disease associated chiefly with deposition of IgM in mesengial regions vs. their homozygous normal CDV-infected littermates. Direct infusion of virus-positive leukocytes, plasma and platelets into the CNS capillary bed via the right carotid artery should establish the primacy of each in the initiation of CNS vascular endothelial infection by CDV.

Immunosuppression by canine distemper virus: modulation of in vitro immunoglobulin synthesis, interleukin release and prostaglandin E2 production.

In vitro or in vivo infection of canine mononuclear cells by canine distemper virus (CDV) in short-term microcultures resulted in suppression of lectin-induced 3H-thymidine incorporation. This suppressive effect was also evident in pokeweed mitogen-driven in vitro immunoglobulin synthesis and release. Lectin-induced interleukin-2 production by monocyte-depleted lymphocyte cultures was marginally affected by CDV, whereas interleukin-1 production by adherent mononuclear cells was significantly depressed. Monocyte cultures established from viremic dogs released prostaglandin (PG)E2. The results suggest that, in addition to a direct viral effect upon lectin responsive cellular population(s), CDV modulates monocyte functions by inhibition of interleukin-1 production and by enhancing PGE2 release.

Effects of induced thrombocytopenia on viral invasion of the central nervous system in canine distemper virus infection.

Groups of canine distemper virus (CDV) susceptible ferrets were treated daily with 2.0 ml of normal goat serum (NGS) or goat anti-ferret platelet serum from 2 days before to 11 days after infection. Each group was subdivided into 2 and one subgroup of each was subsequently injected intraperitoneally with virulent R252-CDV. Ferrets were killed on days 2, 4, 6, 9 and 11 after infection and tissues from the central nervous system (CNS) were examined for histopathological lesions typical for CDV and also of CDV antigen by indirect immuno-fluorescence methods. In NGS-treated animals, a time course-dependent spread of CDV from CNS endothelium during days 2 to 4 after infection through choroid plexus epithelium was observed. In contrast, CDV-infected ferrets treated with anti-platelet antibody exhibited a delay in infection of CNS endothelium until 9 days after infection. The results of this study confirm vascular endothelium as the primary route of invasion of CNS tissues by CDV and implicate the circulating platelet in the initiation of this event.

Canine distemper virus-induced thrombocytopenia.

Effects of canine distemper virus (CDV) infection on circulating platelet values were studied in gnotobiotic dogs inoculated with R252-CDV. Thrombocytopenia (less than 200,000 platelets/microliter) was present on postinoculation day (PID) 5 and persisted through PID 15. Peak thrombocytopenia occurred on PID 10 (less than 85,000 platelets/microliter). Thrombocytopenia was accompanied by lymphopenia, neutropenia, and monocytopenia. Platelet membrane-bound CDV antigen and IgG were present from PID 7 onward; neither the third component of complement nor IgM was detected on platelets from CDV-inoculated dogs. The mean number of megakaryocytes per unit of bone marrow surface area was unchanged. Megakaryocyte infection was present in dogs euthanatized on PID 4 (0.33%), increased slightly in dogs euthanatized on PID 8 (3%), and increased sharply in dogs euthanatized on PID 9 and 10 to 17.8% and 8.3%, respectively. Phagocytosis of platelets by stellate reticuloendothelial (Kupffer's) cells in the liver was prominent in dogs euthanatized from PID 5 onward. Seemingly, CDV-induced thrombocytopenia was mediated by virus-antibody immune complexes on platelet membranes. Decreased platelet production after PID 8 resulting from direct viral infection of megakaryocytes was a likely contributing factor and occurred against a background of profound virus-induced dysfunctions of all hematopoietic cellular elements.

Canine distemper virus: in vivo virulence of in vitro-passaged persistent virus strains.

Groups of ferrets were inoculated intraperitoneally with cell lysates or equivalent doses of whole cells from 9 different cell lines persistently infected with canine distemper virus. Viral persistence in these cell lines was characterized by noncytolytic infection and restricted release of cell-free infectious virus. In vivo replication competency of the various viruses in ferrets ranged from nil to virulent and did not correlate with in vitro titers of inocula. Ferret virulence (cell lysates only) for one cell line (CCL64-RCDV) was associated with morphologic absence of virion assembly, failure to interfere with lytic virus replication after superinfection, and in vitro infectivity restricted to canine macrophage-like tumor cells. Virion protein production in the CCL64-RCDV virulent inoculum and in the CCL64-Ly avirulent inoculum was evaluated by use of the immunoblot technique. All major virion proteins were produced by infected cells. Virulence was not associated with obvious changes in electrophoretic mobility of virion proteins when profiles of ferret-virulent CCL64-RCDV were compared with those of avirulent CCL64-Ly.

In vitro propagation of canine distemper virus: establishment of persistent infection in Vero cells.

Primary cultures of bovine fibroblast (BF) and canine brain cells, persistently infected with virulent R252-canine distemper virus (CDV), were cocultured with African green monkey (Vero) cells. Transfer of persistent CDV from BF to Vero cells varied inversely with the in vitro passage level (age) of the CDV-infected BF cells. Successful transfer of CDV to Vero cells was signaled by the transient appearance of viral syncytia, rapid spread of viral antigen to all Vero cells in the culture, and by recovery of cell-free Vero-infectious virus in culture fluids. With time, viral cytopathic effects in Vero cells containing CDV disappeared, and the infected lines could not be distinguished from noninfected control Vero cells, except by immunoassay for viral antigen.

Canine parvovirus infection potentiates canine distemper encephalitis attributable to modified live-virus vaccine.

Twelve gnotobiotic dogs from 2 litters were allotted to 3 groups. Group A dogs received a modified-live polyvalent (canine distemper, adenovirus type 2, and parainfluenza virus and Leptospira -canicola-icterohemorrhagiae bacterin) vaccine 3 days prior to oral inoculation with canine parvovirus (CPV). Group B dogs received CPV alone. Group C dogs received 1 dose of vaccine only. In none of the 9 CPV-inoculated dogs did clinical signs of CPV infection develop, although high serum antibody titers for CPV developed in all of them. However, in 2 of the 5 CPV-inoculated vaccinates, canine distemper virus encephalomyelitis subsequently developed. The results suggested that CPV exerts an immunomodulating effect on canine immune responses and may be responsible for vaccination failures in dogs.

Expanded tissue targets for foamy virus replication with simian immunodeficiency virus-induced immunosuppression.

Foamy viruses (FV) are the oldest known genus of retroviruses and have persisted in nonhuman primates for over 60 million years. FV are efficiently transmitted, leading to a lifelong nonpathogenic infection. Transmission is thought to occur through saliva, but the detailed mechanism is unknown. Interestingly, this persistent infection contrasts with the rapid cytopathicity caused by FV in vitro, suggesting a host defense against FV. To better understand the tissue specificity of FV replication and host immunologic defense against FV cytopathicity, we quantified FV in tissues of healthy rhesus macaques (RM) and those severely immunosuppressed by simian immunodeficiency virus (SIV). Contrary to earlier findings, we find that all immunocompetent animals consistently have high levels of viral RNA in oral tissues but not in other tissues examined, including the small intestine. Strikingly, abundant viral transcripts were detected in the small intestine of all of the SIV-infected RM, which has been shown to be a major site of SIV (and human immunodeficiency virus)-induced CD4+ T-cell depletion. In contrast, there was a trend to lower viral RNA levels in oropharyngeal tissues of SIV-infected animals. The expansion of FV replication to the small intestine but not to other CD4+ T-cell-depleted tissues suggests that factors other than T-cell depletion, such as dysregulation of the jejunal microenvironment after SIV infection, likely account for the expanded tissue tropism of FV replication.

Experimental old dog encephalitis (ODE) in a gnotobiotic dog.

Experimental infection of a gnotobiotic Beagle dog with the neurovirulent R252 strain of canine distemper virus (R252-CDV) resulted in long-term central nervous system (CNS) infection; cerebral and brain stem lesions were consistent with old dog encephalitis (ODE). Eight clinical cycles of relapsing cortical and subcortical signs were documented over 33 months and were corroborated by the presence of both chronic and active inflammatory demyelinating lesions within the neuraxis. Immunocytochemistry revealed that CDV antigen was restricted to neurons. Attempts to use fresh brain tissue to directly transmit the infection to CDV-susceptible gnotobiotic dogs were unsuccessful. Reisolation of infectious virus from the infected dog required prolonged culture and coculture of brain explant cells with CDV-susceptible Vero cell monolayers. These findings demonstrate that ODE is a variant of virulent CDV-induced canine neurologic disease that can evolve de novo within the CNS of subclinically infected dogs in the absence of external sources of reinfection. The highly cell-associated nature of the virus, when first recovered from this dog, suggests that the virus was present within the CNS in a replication-defective form.

Canine distemper virus: the early blood-brain barrier lesion.

Experimental infection of gnotobiotic Beagle dogs with neurovirulent R252 canine distemper virus (CDV) resulted in hematogenous dissemination of virus to the central nervous system (CNS). Viral antigen was first detected within CNS capillary and venular endothelia and/or perivascular astrocytic foot processes and pericytes. The number of primary infection sites was evenly distributed throughout the neuraxis. Leukocytic infiltrations followed CNS endothelial cell infection by 1-2 days and were composed of both viral antigen-positive and -negative cells. These results indicate that CDV infection of the CNS is initiated by the interaction of circulating infectious virus with endothelial cells.