Transient Immune Activation in BCG-Vaccinated Infant Rhesus Macaques Is Not Sufficient to Influence Oral Simian Immunodeficiency Virus Infection.

BCG vaccination has been demonstrated to increase levels of activated CD4+ T cells, thus potentially influencing mother-to-child transmission of human immunodeficiency virus (HIV). To assess the risk of BCG vaccination in HIV infection, we randomly assigned newborn rhesus macaques to receive BCG vaccine or remain unvaccinated and then undergo oral simian immunodeficiency virus (SIV) challenges 3 weeks later. We observed elevated levels of activated peripheral CD4+ T cells (ie, HLA-DR+CD38+CCR5+ CD4+ T cells) by week 3 after vaccination. BCG was also associated with an altered immune gene expression profile, as well as with monocyte activation in both peripheral blood and the draining axillary lymph node, indicating significant BCG vaccine-induced immune activation. Despite these effects, BCG vaccination did not increase the rate of SIV oral transmission or disease progression. Our findings therefore identify patterns of T-cell and monocyte activation that occur after BCG vaccination but do not support the hypothesis that BCG vaccination is a risk factor for postnatal HIV transmission or increased pathogenesis in infants.

miR-190b is markedly upregulated in the intestine in response to simian immunodeficiency virus replication and partly regulates myotubularin-related protein-6 expression.

HIV replication and the cellular micro-RNA (miRNA) machinery interconnect at several posttranscriptional levels. To understand their regulatory role in the intestine, a major site of HIV/SIV replication, dissemination, and CD4(+) T cell depletion, we profiled miRNA expression in colon following SIV infection (10 acute SIV, 5 uninfected). Nine (four up and five down) miRNAs showed statistically significant differential expression. Most notably, miR-190b expression showed high statistical significance (adjusted p = 0.0032), the greatest fold change, and was markedly elevated in colon and jejunum throughout SIV infection. In addition, miR-190b upregulation was detected before peak viral replication and the nadir of CD4(+) T cell depletion predominantly in lamina propria leukocytes. Interestingly non-SIV-infected macaques with diarrhea and colitis failed to upregulate miR-190b, suggesting that its upregulation was neither inflammation nor immune-activation driven. SIV infection of in vitro-cultured CD4(+) T cells and primary intestinal macrophages conclusively identified miR-190b upregulation to be driven in response to viral replication. Further miR-190b expression levels in colon and jejunum positively correlated with tissue viral loads. In contrast, mRNA expression of myotubularin-related protein 6 (MTMR6), a negative regulator of CD4(+) T cell activation/proliferation, significantly decreased in SIV-infected macrophages. Luciferase reporter assays confirmed MTMR6 as a direct miR-190b target. To our knowledge, this is the first report, which describes dysregulated miRNA expression in the intestine, that identifies a potentially significant role for miR-190b in HIV/SIV pathogenesis. More importantly, miR-190b-mediated MTMR6 downregulation suggests an important mechanism that could keep infected cells in an activated state, thereby promoting viral replication. In the future, the mechanisms driving miR-190b upregulation including other cellular processes it regulates in SIV-infected cells need determination.

Virological and serological characterization of SRV-4 infection in cynomolgus macaques.

The nature of SRV-4 infection in cynomolgus macaques remains unclear to date. Here, we report the monitoring of 24 cynomolgus monkeys that were naturally infected with SRV-4 for virus isolation, proviral load and antibody. The results indicated that the SRV-4 antibody status was statistically correlated to environmental temperature.

Opportunistic infections in immunologically compromised nonhuman primates.

Despite advances in the husbandry of nonhuman primates, natural and experimentally induced diseases continue to pose risks to animal health. These risks are particularly important when such disease results in immunodeficient states that provide an opportunity for the development of opportunistic infections. Because opportunistic agents may serve as significant confounders to research and hold potential for zoonotic transmission, knowledge of disease pathogenesis, surveillance, and risk reduction is particularly important to individuals who work closely with primates. Endogenous diseases of primates that result in blunted immune responses and thus allow for the development of opportunistic infection include simian type D retroviruses and measles. In addition, simian immunodeficiency virus is a frequently studied experimental cause of immunosuppression. This article focuses on clinical and pathological aspects of the most common opportunistic infections that occur in nonhuman primates maintained in research settings. The complete elimination of all infectious agents from primate colonies may be impossible and unwarranted, but microbial surveillance programs can help both to define the complement of agents present in a colony and to elucidate their potential impacts on colony health, zoonotic risk, and experimental research. We discuss risk reduction through the use of quarantine procedures, specific pathogen-free animals, and environmental controls.

Effects of maternal and infant characteristics on birth weight and gestation length in a colony of rhesus macaques (Macaca mulatta).

A retrospective study using maternal and birth statistics from an open, captive rhesus macaque colony was done to determine the effects of parity, exposure to simian retrovirus (SRV), housing, maternal parity, and maternal birth weight on infant birth weight, viability and gestation length. Retrospective colony statistics for a 23-y period indicated that birth weight, but not gestation length, differed between genders. Adjusted mean birth weights were higher in nonviable infants. Mothers positive for SRV had shorter gestations, but SRV exposure did not affect neonatal birth weights or viability. Infants born in cages had longer gestations than did those born in pens, but neither birth weight nor viability differed between these groups. Maternal birth weight did not correlate with infant birth weight but positively correlated with gestation length. Parity was correlated with birth weight and decreased viability. Increased parity of the mother was associated with higher birth weight of the infant. A transgenerational trend toward increasing birth weight was noted. The birth statistics of this colony were consistent with those of other macaque colonies. Unlike findings for humans, maternal birth weight had little predictive value for infant outcomes in rhesus macaques. Nonviable rhesus infants had higher birth weights, unlike their human counterparts, perhaps due to gestational diabetes occurring in a sedentary caged population. Similar to the situation for humans, multiparity had a protective effect on infant viability in rhesus macaques.

Development of a whole-virus multiplex flow cytometric assay for antibody screening of a specific pathogen-free primate colony.

Our goal was to determine if a multiplex technique using a fluorescent bead-based flow cytometric assay could yield results comparable to traditional enzyme-linked immunosorbent assay (ELISA) in terms of sensitivity, specificity, cross-reactivity, and throughput. We applied both techniques to serologic screening of specific pathogen-free macaques, for type D simian retrovirus, simian T-lymphotropic virus, Cercopithicine herpesvirus 1, and simian immunodeficiency virus, and found a high correlation between the bead-based multiplex assay and ELISA. The multiplex assay demonstrated greater sensitivity with no loss in specificity when compared to the ELISA. A lower false-positive rate with the multiplex assay decreased the number of confirmatory Western blots required. Using the multiplex assay, we were able to screen samples for 4 viruses simultaneously in the time it took to perform a single-virus ELISA, resulting in a faster turnaround time and higher throughput. The multiplexed assay provided greater sensitivity, increased stability, and better performance than ELISA.

CD4+ T cell signaling in the natural SIV host--implications for disease pathogenesis.

SIV infection of nonhuman primates is widely utilized as a powerful model of human AIDS. The major effort in this field has so far been oriented towards the induction of an AIDS like disease in the disease susceptible species with the aim to elucidate mechanisms of HIV/SIV induced disease. The fact that there exist disease resistant natural SIV infected host species offers a unique opportunity for comparative studies aimed at not only defining of those mechanisms that may be critical in the development of disease but also the mechanisms that are important for the disease resistance in the natural host. The hallmark of pathogenic HIV and SIV infection is generalized immunosuppression due to both a loss and functional impairment of CD4+ T cells. This review summarizes currently available data on CD4+ T cell function in the naturally SIV infected sooty mangabey with potential implications of these characteristics for our understanding of the pathogenesis of SIV infection.

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

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

Virus load and sequence variation in simian retrovirus type 2 infection.

The natural history of type D simian retrovirus (SRV) infection is poorly characterized in terms of viral load, antibody status, and sequence variation. To investigate this, blood samples were taken from a small cohort of mostly asymptomatic cynomolgus macaques (Macaca fascicularis), naturally infected with SRV type 2 (SRV-2), some of which were followed over an 8-month period with blood taken every 2 months. Provirus and RNA virus loads were obtained, the samples were screened for presence of antibodies to SRV-2 and neutralizing antibody titers to SRV-2 were assayed. env sequences were aligned to determine intra- and intermonkey variation over time. Virus loads varied greatly among cohort individuals but, conversely, remained steady for each macaque over the 8-month period, regardless of their initial levels. No significant sequence variation was found within an individual over time. No clear picture emerged from these results, which indicate that the variables of SRV-2 infection are complex, differ from those for lentivirus infection, and are not distinctly related to disease outcome.

Serum antibodies reactive with non-human primate retroviruses identified in acute onset schizophrenia.

Schizophrenia is a pervasive neuropsychiatric disease of uncertain etiology. Previous studies have postulated that retroviruses may contribute to the etiology of some cases of schizophrenia. We examined the possible relationship between retroviral infection and schizophrenia by measuring antibodies to a number of different primate retroviruses in the sera of individuals undergoing their first hospitalization for this disease. Sera from patients with first onset schizophrenia and matched healthy controls were analyzed by immunoblot and enzyme linked immunosorbent assays using purified retrovirus antigens to identify and quantify antibodies reactive with retrovirus proteins. A significantly increased incidence of antibodies reactive to gag encoded proteins of Mason-Pfizer monkey virus (MPMV), baboon endogenous virus (BaEV) and simian retrovirus type 5 (SRV-5) was observed in the sera of schizophrenia patients compared to controls. The reactivity of the cases and controls displayed the greatest differences in terms of antibodies to the proteins of Mason-Pfizer monkey virus. Employing an algorithm of enzyme linked immunosorbent assay reactivity followed by immunoblot confirmation, we found that MPMV antibodies in 28.9% of the individuals with first episode schizophrenia patients as compared to 3.7% of the unaffected controls (P<0.009, Fisher's Exact Test). These studies are consistent with the occurrence of retrovirus replication in some individuals who are undergoing their first episode of schizophrenia.

Simian retrovirus receptor and neutralization mechanism by antibodies to the envelope glycoprotein.

Type D simian retroviruses (SRV) cause an acquired immunodeficiency syndrome (AIDS) in monkeys. Results of infection with SRV range from complete recovery with absence of viremia to a viremic state, which produces AIDS-like symptoms and culminates in death. These varied outcomes render the interaction of the host and SRV an attractive model for the study of immunosuppressive retrovirus resulting in different pathologic consequences. We describe here the isolation and determination of the molecular weight of the receptor for SRV. We demonstrate that a cell receptor with the same molecular weight is bound by the envelope protein of all five serotypes of SRV. We also show that the receptor recognizes a region containing amino acids 142-167 of the envelope protein of SRV serotype 1 (SRV-1). In addition, we show that a different region of SRV serotype 2 (SRV-2) envelope protein containing amino acids 93-106, interacts with a cell receptor of identical molecular weight. Furthermore, polyclonal and monoclonal antibodies that are directed to envelope epitopes 142-167 of SRV-1 or to 93-106 of SRV-2, specifically neutralize only the respective viral serotype. Our results indicate that the neutralization of SRV infectivity by antibodies is achieved through blocking the interaction between the virus and its cell receptor.

The rotavirus vaccine.

BACKGROUND: Rotavirus gastroenteritis is an important cause of morbidity and mortality worldwide. OBJECTIVES: To review the biology, immunology, and virology of rotavirus infections and describe the efforts towards the construction of vaccines using human and animal rotaviruses. STUDY DESIGN: A review of the literature and provision of the author's understanding and speculation of vaccination of infants against rotavirus disease. RESULTS: In August 1998 the Food and Drug Administration in the United States approved the licensure of a rotavirus vaccine. Both the Advisory Committee of Immunization Practices and the American Academy of Pediatrics are likely to recommend that the vaccine be given to all children by mouth as a series of three doses at 2, 4, and 6 months of age. The vaccine is made by combining a simian rotavirus strain (RRV) with several human strains representing different rotavirus serotypes. An understanding of the biology, immunology, and virology of rotavirus will help to explain the strengths and limitations of the rotavirus vaccine. CONCLUSION: If used as recommended, the rotavirus vaccine should cause a significant decrease in the number of deaths, hospitalizations, and office visits of children infected with rotavirus.

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

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

Characterization of infectious type D retrovirus from baboons.

Infectious virus resembling type D simian retrovirus (SRV) was isolated from Ethiopian baboons (Papio cynocephalus) (SRV-Pc) housed at the University of Washington Regional Primate Research Center. When baboon peripheral blood mononuclear cells (PBMC) or tissues were cocultured with the H-9 human T-cell line or the Raji human B-cell line, large multinucleated syncytia positive for SRV-2 antigens were observed microscopically. Immunoblot analysis of purified SRV-Pc from cell culture supernatants demonstrated that the viral core and envelope proteins reacted with rabbit anti-SRV-2 serum. Fresh PBMC and cocultured cells were positive by polymerase chain reaction using two different sets of SRV-2 primers. Preliminary sequence analysis of two separate isolates from portions of the SRV-Pc p27 and gp20 regions revealed homology with SRV-1, SRV-2, and Mason-Pfizer monkey virus. The homologies in the p27 segment were 91-94% and the homologies in the gp20 segment were 72-75%.

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

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

Increased antiretroviral antibody reactivity in sera from a defined population of patients with systemic lupus erythematosus. Correlation with autoantibodies and clinical manifestations.

OBJECTIVE: The implied role of retroviruses in the pathogenesis of murine systemic lupus erythematosus (SLE) led us to study antiretroviral antibodies in a population-based SLE cohort. METHODS: Immunoassays using whole virus and synthetic peptides were performed on sera from 72 patients with SLE and 88 control subjects. RESULTS: Reactions with whole baboon endogenous virus occurred more frequently in patients with SLE, and correlated with the presence of anti-RNP and anti-Sm. Some retroviral env and gag peptides, several of which were similar to U1 small nuclear RNP, reacted more strongly in patients with SLE, and their presence was correlated with discoid rash, hematologic disorder, and other symptoms. CONCLUSION: These results provide circumstantial evidence for involvement of retroviruses in the pathogenesis of human SLE; further studies should be carried out using other techniques for measurement of retroviral expression.