Rhesus and pig-tailed macaque parvoviruses: identification of two new members of the erythrovirus genus in monkeys

We have previously reported the identification of a novel simian parvovirus in cynomolgus monkeys, which causes severe anemia in immunosuppressed cynomolgus monkeys and is currently being studied as an animal model for human B19 infection. We now report two similar outbreaks of anemia in rhesus and pig-tailed macaques associated with two distinct but similar simian parvoviruses (pig-tailed macaque and rhesus parvovirus). Both viruses have been cloned and over 5000 nucleotides sequenced from each virus. The viruses show marked similarities to other members of the Erythrovirus genus in the Parvoviridae family.     

One-step PCR to distinguish B virus from related primate alphaherpesviruses

By adding betaine to the PCR mixture, we previously established a PCR method to amplify a DNA segment of the glycoprotein G gene of B virus (BV) derived from a rhesus macaque. We have found that DNA of other BV strains derived from cynomolgus, pigtail, and lion-tailed macaques can also serve as the template in our PCR assay. Under the same conditions no product was obtained with DNA of simian agent 8 of green monkeys and Herpesvirus papio 2 of baboons, or the human herpes simplex viruses types 1 and 2. Thus, this PCR method is useful to discriminate BV from other closely related primate alphaherpesviruses.     

One-Step PCR To Distinguish B Virus from Related Primate Alphaherpesviruses

By adding betaine to the PCR mixture, we previously established a PCR method to amplify a DNA segment of the glycoprotein G gene of B virus (BV) derived from a rhesus macaque. We have found that DNA of other BV strains derived from cynomolgus, pigtail, and lion-tailed macaques can also serve as the template in our PCR assay. Under the same conditions no product was obtained with DNA of simian agent 8 of green monkeys and Herpesvirus papio 2 of baboons, or the human herpes simplex viruses types 1 and 2. Thus, this PCR method is useful to discriminate BV from other closely related primate alphaherpesviruses.     

Generation of macaque B lymphoblastoid cell lines with simian Epstein-Barr-like viruses: transformation procedure, characterization of the cell lines and occurrence of simian foamy virus.

Two simian Epstein-Barr-like viruses, a rhesus Epstein-Barr virus and Herpesvirus papio, were used to transform B cells from rhesus or cynomolgus macaques. The resulting cell lines exhibited predominantly a B lymphocyte phenotype and expressed Epstein-Barr virus antigens. The majority of B lymphoblastoid cell lines from macaques, which were seropositive for simian foamy virus, developed giant cells in culture. The cytopathic agent was identified as a foamy virus and was transmissible to human embryonal fibroblasts. Treatment of cell cultures with AZT abolished giant cell formation.     

Neurovirulence detection of dengue virus using rhesus and cynomolgus monkeys

The results of a comparative study of neurovirulence of dengue type 1 virus in two species of Old World monkeys, viz. rhesus monkeys (Macaca mulatta) and cynomolgus monkeys (Macaca fascicularis) are reported. In the present study, parental dengue type 1 (16007) and its vaccine viruses were tested by intrathalamic, intramuscular and intraspinal injections in these two species of monkey. Both species of monkeys inoculated with parental dengue type 1 virus developed neurovirulence-type lesions which were graded as minimal (V-1) and occasionally mild (V-2, in cynomolgus monkeys) in severity. The antibody response to either parental or vaccine virus was slightly less in rhesus monkeys than in cynomolgus inoculated with these strains. This comparative study possibly establishes the cynomolgus monkey as a suitable test model to replace the rhesus monkey for neurovirulence testing of dengue-1 vaccine intended for use in humans.     

Pathology of inhalation anthrax in cynomolgus monkeys (Macaca fascicularis)

Anthrax is considered a serious biowarfare and bioterrorism threat because of its high lethality, especially by the inhalation route. Rhesus macaques (Macaca mulatta) are the most commonly used nonhuman primate model of human inhalation anthrax exposure. The nonavailability of rhesus macaques necessitated development of an alternate model for vaccine testing and immunologic studies. This report describes the median lethal dose (LD(50)) and pathology of inhalation anthrax in cynomolgus macaques (Macaca fascicularis). Gross and microscopic tissue changes were reviewed in 14 cynomolgus monkeys that died or were killed after aerosol exposure of spores of Bacillus anthracis (Ames strain). The LD(50) and 95% confidence intervals were 61800 (34000 to 110000) colony-forming units. The most common gross lesions were mild splenomegaly, lymph node enlargement, and hemorrhages in various organs, particularly involving the meninges and the lungs. Mediastinitis, manifested as hemorrhage or edema, affected 29% of the monkeys. Microscopically, lymphocytolysis occurred in the intrathoracic lymph nodes and spleens of all animals, and was particularly severe in the spleen and in germinal centers of lymph nodes. Hemorrhages were common in lungs, bronchial lymph nodes, meninges, gastrointestinal tract, and mediastinum. These results demonstrate that the Ames strain of B. anthracis is lethal by the inhalation route in the cynomolgus macaque. The LD(50) of the Ames strain of B. anthracis was within the expected experimental range of previously reported values in the rhesus monkey in an aerosol challenge. The gross and microscopic pathology of inhalation anthrax in the cynomolgus monkey is remarkably similar to that reported in rhesus monkeys and humans. The results of this study are important for the establishment of an alternative nonhuman primate model for evaluation of medical countermeasures against inhalational anthrax.     

Hemorrhagic fever occurs after intravenous, but not after intragastric, inoculation of rhesus macaques with lymphocytic choriomeningitis virus

Arenaviruses can cause hemorrhagic fever and death in primates and guinea pigs, but these viruses are not highly pathogenic for most rodent carriers. In the United States, arenaviruses precipitated outbreaks of hepatitis in captive monkeys, and they present an emerging health threat in the tropical areas of Africa and South America. We describe infection of rhesus macaques with the prototype arenavirus, lymphocytic choriomeningitis virus (LCMV), using the WE strain that has been known to cause both encephalopathy and multifocal hemorrhage. Five macaques were inoculated: two by the intravenous (i.v.) and three by the intragastric (i.g.) route. Whereas the two i.v.-inoculated monkeys developed signs and lesions consistent with fatal hemorrhagic fever, the i.g.-inoculated monkeys had an attenuated infection with no disease. Pathological signs of the primate i.v. infection differ significantly from guinea pig arenavirus infections and make this a superior model for human viral hemorrhagic disease.     

TRIM5 allelic polymorphism in macaque species/populations of different geographic origins: its impact on SIV vaccine studies

Tripartite motif 5α (TRIM5α) is a potent antiretroviral immune factor present in the cytoplasm of cells of most tissue types. The rhesus macaque TRIM5 gene has been shown to display polymorphism, with different variants being divided into three groups (TRIM5(TFP), TRIM5(Q), and TRIM5(CypA)), which may have divergent retroviral effects on infection. Along with rhesus macaques, cynomolgus macaques are also used in simian immunodeficiency virus (SIV) infection studies. As a consequence, TRIM5 genotyping of these animals will contribute to interpreting the outcome of such studies. The present communication covers Burmese, Chinese, and a large cohort of Indian-origin rhesus macaques, and describes the first large cohort study on TRIM5 polymorphism in outbred cynomolgus macaques. We demonstrate the presence of the TRIM5(TFP) group in cynomolgus macaques. In addition, we have re-evaluated historical samples of rhesus macaques challenged with SIV(mac251), a virus that has been reported to be partially suppressed by particular rhesus macaque TRIM5 variants.     

Antigenic relationships among several simian varicella-like viruses and varicella-zoster virus.

Cross-neutralization and complement fixation tests demonstrated the immunological identity of the Delta herpesvirus, the 592S virus, the Liverpool vervet monkey virus, the herpesvirus of patas monkeys, and the Medical Lake macaque virus. These viruses were isolated from diverse outbreaks of varicella-like disease in simians and from various simian species. All of the simian viruses were shown to be related to human varicella-zoster (V-Z) virus, as evidenced by the fact that immunization of monkeys with each of the simian viruses elicited the production of both neutralizing and complement-fixing antibodies to V-Z virus. However, cross-complement fixation tests indicated that the simian viruses are not so closely related to V-Z virus as they are to one another. Varicella or zoster infections in humans produced neutralizing and complement-fixing antibody responses to each of the simian viruses; the responses were more marked in zoster infections than in varicella infections but, in most patients, antibody levels produced to the simian viruses were much lower than those to the homologous V-Z virus.     

Comparison of rhesus and cynomolgus macaques in a Streptococcus pyogenes infection model for vaccine evaluation

Animal models predictive of human disease are generally difficult to establish and reproduce. In the case of the Group A Streptococcus (GAS) bacterium, which is predominantly a human pathogen, virulence assessment in animal models is problematic. We compared a monkey colonization and pharyngitis model of infection in two macaque species to determine the optimal model for vaccine candidate evaluation. Rhesus and cynomolgus macaques were intranasally infected with a streptomycin resistant (Str(r)) GAS strain. Monkeys were monitored for body weight and temperature changes, throat swabs and sera were collected, and clinical observations were noted throughout the study. Both species exhibited oropharyngeal colonization by GAS, with rhesus macaques demonstrating a more sustained colonization through day 28 post-challenge. Veterinary observations revealed no significant differences between GAS-infected rhesus and cynomolgus macaques. Mock-infected monkeys did not exhibit clinical symptoms or GAS colonization throughout the study. ELISA results demonstrated that both rhesus and cynomolgus macaques developed anti-streptolysin-O antibody titers, with cynomolgus generating higher titers. Sera from infected monkeys produced opsonophagocytic killing and bound to the bacterium in an immunofluorescence assay. Both rhesus and cynomolgus macaques can be used for colonization studies with this GAS M3 strain, yet only mild clinical signs of pharyngitis and tonsillitis were observed.     

Isolation and partial characterization of a lentivirus from talapoin monkeys (Myopithecus talapoin).

We have identified a novel lentivirus prevalent in talapoin monkeys (Myopithecus talapoin), extending previous observations of human immunodeficiency virus-1 cross-reactive antibodies in the serum of these monkeys. We obtained a virus isolate from one of three seropositive monkeys initially available to us. The virus was tentatively named simian immunodeficiency virus from talapoin monkeys (SIVtal). Despite the difficulty of isolating this virus, it was readily passed between monkeys in captivity through unknown routes of transmission. The virus could be propagated for short terms in peripheral blood mononuclear cells of talapoin monkeys but not in human peripheral blood mononuclear cells or human T cell lines. The propagated virus was used to infect a naive talapoin monkey, four rhesus macaques (M. mulatta), and two cynomolgus macaques (M. fascicularis). All animals seroconverted and virus could be reisolated during a short period after experimental infection. A survey of SIVtal-infected captive talapoin monkeys revealed a relative decrease in CD4(+) cell numbers in chronically (>2 years) infected animals. No other signs of immunodeficiency were observed in any of the infected animals. PCR amplification followed by DNA sequencing of two fragments of the polymerase gene revealed that SIVtal is different from the presently known lentiviruses and perhaps most related to the SIV from Sykes monkeys.     

Dissecting the role of dendritic cells in simian immunodeficiency virus infection and AIDS

Human immunodeficiency virus (HIV) infection is associated with the loss of the two principal types of dendritic cell (DC), myeloid DC (mDC) and plasmacytoid DC (pDC), but the mechanism of this loss and its relationship to AIDS pathogenesis remain ill-defined. The nonhuman primate is a powerful model to dissect this response for several reasons. Both DC subsets have been well characterized in nonhuman primates and shown to have strikingly similar phenotypic and functional characteristics to their counterparts in the human. Moreover, decline of mDC and pDC occurs in rhesus macaques with end-stage simian immunodeficiency virus (SIV) infection, the model of HIV infection in humans. In this brief review, we discuss what is known about DC subsets in pathogenic and nonpathogenic nonhuman primate models of HIV infection and highlight the advances and controversies that currently exist in the field.     

Susceptibility of HVS-immortalized lymphocytic HSC-F cells to various strains and mutants of HIV/SIV

Susceptibility of HSC-F, a cynomolgus macaque cell line immortalized by Herpesvirus saimiri, to infection with various primate immunodeficiency viruses were monitored. While NL432 clone of human immunodeficiency virus type 1 (HIV-1) did not grow at all in HSC-F cells, GH123 and GL-AN clones of HIV-2, and MA239 clone of simian immunodeficiency virus isolated from macaque monkeys (SIVMAC) did grow in these cells. In addition, NM-3 clone of a chimeric simian and human immunodeficiency virus (SHIV) grew fairly well in HSC-F cells. Mutational analyses of accessory genes of GL-AN were successfully performed in the HSC-F cells. These results have thus demonstrated the importance of this cell line for molecular biological studies on HIV/SIV.     

Detection of macaque perforin expression and release by flow cytometry, immunohistochemistry, ELISA, and ELISpot

Simian immunodeficiency virus (SIV)-infection in macaques provides an important animal model for human immunodeficiency virus-1 (HIV-1) infection. The involvement of perforin (PFN), released by cytotoxic cells to mediate killing of virus-infected cells, has been difficult to assess in this experimental model due to a lack of reagents. We therefore evaluated monoclonal antibodies (mAbs) Pf-80, Pf-164 and Pf-344, previously raised against human PFN, for cross-reactivity with macaque PFN. Mabs Pf-164 and Pf-344 reacted with intracellular PFN in peripheral blood mononuclear cells (PBMC) from cynomolgus and rhesus macaques by flow cytometry and stained PFN in rhesus lymphoid tissue by immunohistochemistry (IHC). Moreover, PFN capture enzyme-linked immunosorbent (ELISA) and enzyme-linked immunospot (ELISpot) assays utilizing mAbs Pf-164/Pf-80 for capture and mAb Pf-344 for detection were used to quantify PFN release by mitogen-stimulated cynomolgus and rhesus PBMC. The PFN ELISpot was further used to quantify antigen-specific CD8+ T cells by ex vivo stimulation of PBMC from cynomolgus macaques immunized against SIV/HIV-1. These macaque PFN-reactive mAbs and immunoassays will be valuable new tools for investigation of cytotoxic T lymphocyte (CTL) responses in non-human primate models of infectious diseases as well as for vaccine development.     

Circulating IL-21 levels increase during early simian-human immunodeficiency virus infection in macaques

The cytokine interleukin-21 (IL-21) regulates viral pathogenesis in individuals infected with human and simian immunodeficiency viruses. However, because the time of initial infection with HIV in humans is rarely known, the dynamics of IL-21 production during the first weeks have not been adequately explored. In the present study, we used rhesus macaques to model the first stages of infection. Twenty-two rhesus macaques were infected rectally with simian-human immunodeficiency virus (SHIV)-1157ipd3N4, and for 12 weeks, replication of the virus, the numbers of CD4+ and CD8+ T cells, and the levels of plasma IL-21 were monitored. Our study demonstrated that plasma levels of IL-21 increased during the early phase of SHIV infection when compared with the values observed before inoculation. We conclude that IL-21 has a likely role in the immunopathogenesis of HIV/SIV/SHIV.     

Isolation and characterization of cynomolgus macaque (Macaca fascicularis) cytomegalovirus (CyCMV)

Cynomolgus macaques have been widely used as an animal model in preclinical biomedical research and are becoming more popular among HIV/SIV vaccine researchers. Here we report the isolation and characterization of a cytomegalovirus from cynomolgus macaques (CyCMV). CyCMV was isolated from a healthy captive-bred 4-year-old cynomolgus macaque of Filipino origin. The virus was identified by its characteristic growth properties in cell culture, ultrastructural morphology and sequence of viral DNA polymerase and glycoprotein B (gB). CyCMV gB shows 77% identity and 88% homology to rhesus cytomegalovirus (RhCMV) gB and 58% identity and 76% homology to human cytomegalovirus gB at the amino acid level. Phylogenetic analysis using known CMV gB protein sequences show that CyCMV is more closely related to RhCMV than to other primate CMVs. CyCMV down-regulates MHC class I expression on infected cells and we show that the colony-bred cynomolgus macaques have detectable CyCMV-specific humoral and cell-mediated immune responses.     

Primates as a model for the study of lentiviruses and AIDS

Neither vaccine nor therapy are, to date, available against human HIV infections. Because few is known on human pathogenesis, a standardized animal model is urgently required. Today, different models have been available: 1) "partial models" of the human infection (murine retrovirus, infection of SCID or transgenic mice with HIV, sheep infection with Visna, rabbits infected with HIV1, etc.). These models cannot be used in testing vaccine strategies, but may help in evaluating some particular stages of the pathogenesis of the disease, and the targets of antiretroviral drugs. 2) Disease models, such as cats infected with FIV, and, above, primates infected with HIV or SIV (SIV infected macaques, and, perhaps, HIV2 rhesus monkeys). Primate models are the only possibility to day in testing vaccine procedures before screening among a large population of seronegative humans, and determining drug combination which might be useful in HIV specific therapy. The best primate model is today the SIVMAC251 infected rhesus monkey model, which standardization is now on progress.     

Higher levels of IL-18 circulate during primary infection of monkeys with a pathogenic SHIV than with a nonpathogenic SHIV

We have monitored kinetics of peripheral blood Interleukin (IL)-18 level, viral RNA load, and CD4(+) T cell counts in cynomolgus and rhesus macaques following infections of various simian/human immunodeficiency viruses (SHIVs) causing differential pathogenicity. Infections of cynomolgus and rhesus macaques with pathogenic SHIVs-C2/1 and -89.6PD, respectively, induced high levels of plasma IL-18 (0.1-1 ng/ml) and enhanced apoptosis of peripheral blood T cells during primary viremia, along with a rapid decline of CD4(+) T cells and a high level of set point viral load after primary viremia (six of six cases). In contrast, infections of cynomolgus macaques with nonpathogenic SHIVs-TH09V3 and -MD14 did not cause such IL-18 elevation, showing no decline of CD4(+) T cells and no or low viral set point level following primary viremia (three of three cases). Thus, the elevation of circulating IL-18 level during primary viral infection can be a good indicator of an active pathogenic viral infection. However, the role of increased IL-18 remains to be elucidated and needs further investigation.     

ELISpot and ELISA analysis of spontaneous,mitogen-induced and antigen-specific cytokine production in cynomolgus and rhesus macaques

Evaluation of cytokine production in macaques has been hampered by a lack of availability of optimized and standardized immunoassays such as ELISA and enzyme-linked immune spot assay (ELISpot); only a limited number of macaque cytokines have been assessed by ELISpot. Using monoclonal antibodies (mAb) to human cytokines that cross-react with cynomolgus and rhesus macaque interferon-gamma (IFN-gamma), interleukin (IL)-2, IL-4, IL-5, IL-6, IL-12, IL-13 and granulocyte monocyte colony-stimulating factor, we measured macaque cytokine production by ELISA and ELISpot. Quantitation of spontaneous as well as phytohemagglutinin (PHA)-induced cytokine production in peripheral blood mononuclear cells (PBMC) from rhesus and cynomolgus macaques and humans were compared. The proportional distribution of the different cytokines, in terms of PBMC synthesizing different cytokines as well as the levels of the different cytokines produced, were similar in all species. Spontaneous- and PHA-induced cytokine productions thus appear to be similarly regulated in macaques and man. ELISpot and ELISA assays for macaque IFN-gamma were further used to measure antigen-specific immune responses of PBMC from cynomolgus macaques exposed to, or vaccinated against, simian immunodeficiency virus (SIV). The establishment of reliable immunoassays for detection of macaque cytokines is of importance for future progress of research utilizing macaques as experimental animals.