Experimental infection of Japanese macaques with simian retrovirus 5.

Recently, a large number of Japanese macaques (Macaca fuscata) died of an unknown hemorrhagic syndrome at Kyoto University Primate Research Institute (KUPRI) and an external breeding facility for National Institute for Physiological Sciences (NIPS). We previously reported that the hemorrhagic syndrome of Japanese macaques at KUPRI was caused by infection with simian retrovirus 4 (SRV-4); however, the cause of similar diseases that occurred at the external breeding facility for NIPS was still unknown. In this study, we isolated SRV-5 from Japanese macaques exhibiting thrombocytopenia and then constructed an infectious molecular clone of the SRV-5 isolate. When the SRV-5 isolate was inoculated into two Japanese macaques, severe thrombocytopenia was induced in one of two macaques within 22 days after inoculation. Similarly, the clone-derived virus was inoculated into the other two Japanese macaques, and one of two macaques developed severe thrombocytopenia within 22 days. On the other hand, the remaining two of four macaques survived as asymptomatic carriers even after administering an immunosuppressive agent, dexamethasone. As determined by real-time PCR, SRV-5 infected a variety of tissues in Japanese macaques, especially in digestive and lymph organs. We also identified the SRV-5 receptor as ASCT2, a neutral amino acid transporter in Japanese macaques. Taken together, we conclude that the causative agent of hemorrhagic syndrome occurred at the external breeding facility for NIPS was SRV-5.

Autophagy Induced by Simian Retrovirus Infection Controls Viral Replication and Apoptosis of Jurkat T Lymphocytes.

Autophagy and apoptosis are two important evolutionarily conserved host defense mechanisms against viral invasion and pathogenesis. However, the association between the two pathways during the viral infection of T lymphocytes remains to be elucidated. Simian type D retrovirus (SRV) is an etiological agent of fatal simian acquired immunodeficiency syndrome (SAIDS), which can display disease features that are similar to acquired immunodeficiency syndrome in humans. In this study, we demonstrate that infection with SRV-8, a newly isolated subtype of SRV, triggered both autophagic and apoptotic pathways in Jurkat T lymphocytes. Following infection with SRV-8, the autophagic proteins LC3 and p62/SQSTM1 interacted with procaspase-8, which might be responsible for the activation of the caspase-8/-3 cascade and apoptosis in SRV-8-infected Jurkat cells. Our findings indicate that autophagic responses to SRV infection of T lymphocytes promote the apoptosis of T lymphocytes, which, in turn, might be a potential pathogenetic mechanism for the loss of T lymphocytes during SRV infection.

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.

Implications of simian retroviruses for captive primate population management and the occupational safety of primate handlers.

Nonhuman primates can be naturally infected with a plethora of viruses with zoonotic potential, including retroviruses. These simian viruses present risks to both captive nonhuman primate populations and persons exposed to nonhuman primates. Simian retroviruses, including simian immunodeficiency virus, simian type D retrovirus, simian T-lymphotropic virus, and gibbon ape leukemia virus, have been shown to cause clinical disease in nonhuman primates. In contrast, simian foamy virus, a retrovirus that is highly prevalent in most nonhuman primates, has not been associated with clinical disease in naturally infected primates. Although it has been shown that human retrovirus infections with human T-lymphotropic virus and human immunodeficiency virus originated through multiple independent introductions of simian retroviruses into human populations that then spread globally, little is known about the frequency of such zoonotic events. In this article, exogenous simian retroviruses are reviewed as a concern for zoo and wildlife veterinarians, primate handlers, other persons in direct contact with nonhuman primates, and other nonhuman primates in a collection. The health implications for individual animals as well as managed populations in zoos and research institutions are discussed, the cross-species transmission and zoonotic disease potential of simian retroviruses are described, and suggestions for working safely with nonhuman primates are provided.

Antiretroviral drug studies in nonhuman primates: a valid animal model for innovative drug efficacy and pathogenesis experiments.

Several nonhuman primate models are used in HIV and AIDS research. In contrast to HIV-1 infection of chimpanzees, infection of macaque species with simian immunodeficiency virus (SIV) isolates results in a disease (simian AIDS) that shares many similarities with HIV infection and AIDS in humans. Although each animal model has its limitations and can never completely mimic HIV infection of humans, a carefully designed study allows experimental approaches, such as the control of certain variables, that are not feasible in humans, but that are often the most direct way to gain better insights in disease pathogenesis and provide proof-of-concept for novel intervention strategies. In the early days of the HIV pandemic, nonhuman primate models played a relatively minor role in the anti-HIV drug development process. During the past decade, however, the development of better virologic and immunologic assays, a better understanding of disease pathogenesis, and the availability of better drugs have made these animal models more practical for drug studies. In particular, nonhuman primate models have played an important role in demonstrating: (i) preclinical efficacy of novel drugs such as tenofovir; (ii) the benefits of chemoprophylaxis, early treatment and immunotherapeutic strategies; (iii) the virulence and clinical significance of drug-resistant viral mutants; and (iv) the role of antiviral immune responses during drug therapy. Comparison of results obtained in primate models with those observed in human studies will lead to further validation and improvement of these animal models. Accordingly, well-designed drug studies in nonhuman primates can continue to provide a solid scientific basis to advance our scientific knowledge and to guide future clinical trials.

The induction of neutralizing antibodies by synthetic peptides of the envelope protein of type D simian retrovirus-1 (SRV-1).

It has been recently demonstrated that two serotypes of type D simian retroviruses, namely SRV-1 and SRV-2, exhibit extensive immunological cross-reactivity but do not exhibit cross-reactivity at the level of neutralizing antibodies. We have also shown recently that an area which includes residues 147-162 of the envelope protein of SRV-1 constitutes an epitope to which neutralizing antibodies against SRV-1 but not against SRV-2 are directed. However, in spite of the capacity of various immunogenic preparations to induce antibodies which react with SRV-1 these antibodies were incapable of neutralizing in vitro viral infectivity. Work reported herein demonstrates that various immunogens consisting of a larger peptide, namely 142-167 of the envelope protein of SRV-1, induce antibodies capable of binding with the envelope protein of SRV-1 and with the whole virus. Moreover, these antibodies exhibit the capacity to neutralize in vitro the infectivity of SRV-1 but not of SRV-2.

Immunobiological properties of a recombinant simian retrovirus-1 envelope protein and a neutralizing monoclonal antibody directed against it.

We previously reported that an area encompassing amino acids 147-162 of the envelope region of the simian (type D) retrovirus serotype 1 (SRV-1) constitutes an antigenic site for the binding of murine and rhesus neutralizing antibodies. Neutralizing antibodies to SRV-2 are directed to a different area, encompassing residues 96-102 of SRV-2. This paper presents data on the activity of an SRV-1 recombinant envelope protein (rEP) and of monoclonal hybridoma cell line, C11B8, produced from murine spleen cells immunized with SRV-1 rEP. Purified monoclonal antibodies from C11B8 bind to the SRV-1 rEP and to both SRV-1 and SRV-2. However, the monoclonal antibody exhibits strain specificity in the capacity to neutralize SRV-1 infection in vitro. Thus, C11B8 neutralizes SRV-1 infection but fails to neutralize four other known serotypes of the virus. C11B8 also binds to an SRV-1 synthetic peptide representing residues 142-167, which encompasses the previously defined antigenic site of recognition for neutralizing antibodies to SRV-1. This paper also contains evidence that the SRV-1 rEP construct binds the site for SRV-1 attachment to the cell receptor. This is indicated by the ability of SRV-1 rEP to compete with SRV-1 (but not with SRV-2) and inhibit its infectivity in vitro. In addition, SRV-1 rEP inhibits the neutralizing activity of C11B8 against SRV-1 infection in vitro. SRV-1 rEP has no inhibitory effect on rhesus neutralizing antibodies to SRV-2. Taken together, the above findings indicate that immunity conferred at the level of neutralizing antibodies during SRV infection is strain-specific and involves the recognition of envelope sequences unique to each strain.

Experimental evaluation of the zoonotic infection potency of simian retrovirus type 4 using humanized mouse model.

During 2001-2002 and 2008-2011, two epidemic outbreaks of infectious hemorrhagic disease have been found in Japanese macaques (Macaca fuscata) in Kyoto University Primate Research Institute, Japan. Following investigations revealed that the causative agent was simian retrovirus type 4 (SRV-4). SRV-4 was isolated by using human cell lines, which indicates that human cells are potently susceptible to SRV-4 infection. These raise a possibility of zoonotic infection of pathogenic SRV-4 from Japanese macaques into humans. To explore the possibility of zoonotic infection of SRV-4 to humans, here we use a human hematopoietic stem cell-transplanted humanized mouse model. Eight out of the twelve SRV-4-inoculated humanized mice were infected with SRV-4. Importantly, 3 out of the 8 infected mice exhibited anemia and hemophagocytosis, and an infected mouse died. To address the possibility that SRV-4 adapts humanized mouse and acquires higher pathogenicity, the virus was isolated from an infected mice exhibited severe anemia was further inoculated into another 6 humanized mice. However, no infected mice exhibited any illness. Taken together, our findings demonstrate that the zoonotic SRV-4 infection from Japanese macaques to humans is technically possible under experimental condition. However, such zoonotic infection may not occur in the real society.

Immunohistochemical detection of simian immunodeficiency virus (SIV) in rectal mucosa of experimentally infected rhesus macaques (Macaca mulatta).

Experimental simian immunodeficiency virus (SIV) infection is the most appropriate animal model for human HIV infection. Eight male rhesus monkeys (Macaca mulatta) were intravenously or intrarectally infected with SIVmac251/MPBMC to comparatively investigate the distribution and spread of the virus within the rectum during the course of the disease. SIV-positive cells were immunohistochemically detected in rectal biopsies obtained at days 3 and 7 and week 2, 4 and 12 postinfection. SIV-expressing cells were detected for the first time at one week after experimental infection and were present in the lamina propria and lymph follicles. Numbers of positive cells per individual animal varied strongly in time, with a more rapid rise in animals with rapid progression of the disease. Differences were not observed between intravenous and intrarectal infection. Our observations support the significance of the intestinal tract as target organ in initial pathogenesis of SIV infection.

Eradication of simian retrovirus type D from a colony of cynomolgus, rhesus, and stump-tailed macaques by using serial testing and removal.

The markedly compromised health of animals in a macaque colony and the problematic interpretation of data from two drug safety assessment studies prompted a review of the effect of simian retrovirus type D on the drug-development process at a Midwest pharmaceutical company. After reviewing relevant literature and consulting with an expert in simian retroviruses, we initiated a program of eradication. During a 16-month period, all cynomolgus (Macaca fascicularis), rhesus (Macaca mulatta), and stump-tailed (Macaca arctoides) macaques housed in the facility were evaluated as many as eight times for the presence of simian retrovirus type D by using serology, virus isolation, and/or polymerase chain reaction tests. All animals with positive test results were removed from the colony immediately. No test results indicative of simian retrovirus type D infection have occurred during the subsequent 2.5 years. We attribute the successful eradication and prevention of re-introduction of the virus to regular testing, purchasing animals from sources free of simian retrovirus type D, and assiduous application of procedures designed to prevent transmission between animals.

Cross-species transmission of simian retroviruses: how and why they could lead to the emergence of new diseases in the human population.

The HIV-1 group M epidemic illustrates the extraordinary impact and consequences resulting from a single zoonotic transmission. Exposure to blood or other secretions of infected animals, through hunting and butchering of bushmeat, or through bites and scratches inflicted by pet nonhuman primates (NHPs), represent the most plausible source for human infection with simian immunodeficiency virus (SIV), simian T-cell lymphotropic virus (STLV) and simian foamy virus. The chance for cross-species transmissions could increase when frequency of exposure and retrovirus prevalence is high. According to the most recent data, human exposure to SIV or STLV appears heterogeneous across the African countries surveyed. Exposure is not sufficient to trigger disease: viral and host molecular characteristics and compatibility are fundamental factors to establish infection. A successful species jump is achieved when the pathogen becomes transmissible between individuals within the new host population. To spread efficiently, HIV likely required changes in human behavior. Given the increasing exposure to NHP pathogens through hunting and butchering, it is likely that SIV and other simian viruses are still transmitted to the human population. The behavioral and socio-economic context of the twenty-first century provides favorable conditions for the emergence and spread of new epidemics. Therefore, it is important to evaluate which retroviruses the human population is exposed to and to better understand how these viruses enter, infect, adapt and spread to its new host.

Simian retroviruses: infection and disease--implications for immunotoxicology research in primates.

Non-human primates have assumed an important role in preclinical safety assessment studies, particularly in the evaluation of biopharmaceutical and immunomodulatory therapies. Naturally occurring simian retrovirus infections may adversely affect the suitability of primates for use in such studies. Various species of non-human primates are the natural hosts for six exogenous retroviruses, representing five genera within the family Retroviridae. Retroviruses establish persistent infections with a broad spectrum of pathogenic potential, ranging from nonpathogenic to highly pathogenic, depending on the variety of the host, virus, and environmental factors. In the context of immunotoxicology, in which the research objective is to specifically evaluate the effect of drugs or biologics on the immune system, the immune modulatory effects of simian retroviruses, which may be subtle or profound, may introduce significant confounding into the studies of immunotoxic effects utilizing non-human primates. Latent or subclinical retrovirus infections are common and research-related procedures may lead to virus reactivation or overt disease. Adverse effects of undetected retrovirus infections on preclinical research include the loss of experimental subjects (and potentially of statistical power) due to increased morbidity and mortality, virus-induced clinical abnormalities, histologic lesions, alteration of physiologic parameters and biologic responses, and interference with in vitro assays and/or cytolytic destruction of primary cell cultures. The aim of this review is to provide an overview of the key biological, clinical, and pathological features of several important simian retroviruses, with emphasis on viruses infecting macaques and other primate species commonly used in preclinical research, and a discussion of the implications of these infections for immunotoxicology and other preclinical research in primates. Adequate pre-study retrovirus screening is essential to exclude retrovirus-infected primates from research protocols.

Investigating the origin of AIDS: some ethical dimensions.

The theory that AIDS originated from contaminated polio vaccines raises a number of challenging issues with ethical dimensions. The Journal of Medical Ethics dealt with a submission about the theory a decade ago; subsequent developments have raised further issues. Four areas of contention are addressed: whether the theory should be investigated; whether anyone should be blamed; whether defamation actions are appropriate, and whether the scientific community has a responsibility to examine unorthodox theories.

Langerhans cells in oral mucosa of rhesus monkeys before and after infection by simian retrovirus-1 and simian immunodeficiency virus.

To study the influence of experimental infection with simian retrovirus-1 and simian immunodeficiency virus on the number and distribution of Langerhans cells in oral mucosa of rhesus monkeys, 10 monkeys were intravenously inoculated with simian retrovirus-1, 7 with simian immunodeficiency virus, and 2 were mock-inoculated. Biopsies were taken from gingiva and cheek pouch before infection and at 1 (simian immunodeficiency virus group only), 4, and 7 months after infection. Langerhans cells were detected in frozen sections by immunohistochemistry with monoclonal antibodies Leu-6 and HLA-DR. The mean number of Langerhans cells per surface millimeter and square millimeter of epithelium was calculated under blind conditions. The results showed no statistically significant differences in the number or distribution of Langerhans cells in the three groups at the various time points of examination. Similarly, no differences were detected within any group over the observation period. Thus systemic infection of rhesus monkeys with either simian retrovirus-1 or simian immunodeficiency virus does not lead to a significant change in the number of Langerhans cells in oral mucosal epithelium.

Simian AIDS type D serogroup 2 retrovirus: isolation of an infectious molecular clone and sequence analyses of its envelope glycoprotein gene and 3' long terminal repeat.

We describe the molecular cloning of a serogroup 2 simian retrovirus (SRV; D2/RHE/OR) and present the sequence of its envelope (env) glycoprotein gene and 3' long terminal repeat region. This report documents the first infectious molecular clone of a serogroup 2 SRV and provides env sequence verification of genetic diversity among serogroup 2 SRV isolates.

Fusion properties of cells infected with human parainfluenza virus type 3: receptor requirements for viral spread and virus-mediated membrane fusion.

Cells can be persistently infected with human parainfluenza virus type 3 (HPF3) by using a high multiplicity of infection (MOI) (> or = 5 PFU per cell). The persistently infected cells exhibit no cytopathic effects and do not fuse with each other, yet they readily fuse with uninfected cells. We have previously shown that the failure of the persistently infected cells to fuse with each other is due to the lack of a receptor on these cells for the viral hemagglutinin-neuraminidase glycoprotein, and we have established that both fusion and hemagglutinin-neuraminidase proteins are needed for cell fusion mediated by HPF3. We then postulated that the generation of persistent infection and the failure of cells infected with HPF3 at high MOI to form syncytia are both due to the action of viral neuraminidase in the high-MOI inoculum. In this report, we describe experiments to test this hypothesis and further investigate the receptor requirements for HPF3 infection and cell fusion. A normally cytopathic low-MOI HPF3 infection can be converted into a noncytopathic infection by the addition of exogenous neuraminidase, either in the form of a purified enzyme or as UV-inactivated HPF3 virions. Evidence is presented that the receptor requirements for an HPF3 virus particle to infect a cell are different from those for fusion between cells. By treating infected cells in culture with various doses of neuraminidase, we demonstrate that virus spreads from cell to cell in the complete absence of cell-cell fusion. We compare the outcome of HPF3 infection in the presence of excess neuraminidase with that of another paramyxovirus (simian virus 5) and provide evidence that these two viruses differ in their receptor requirements for mediating fusion.