Since 2004, an East African genotype of Chikungunya virus (CHIKV) has emerged, causing significant epidemics of an arthralgic syndrome. In addition, this virus has been associated for the first time with neonatal transmission and neurological complications. In the current study, pregnant Rhesus macaques were inoculated with an enzootic or epidemic strain of CHIKV to compare pathogenesis and transplacental transmission potential. Viremias were similar for both strains and peaked at 2–3 days post-inoculation (dpi). Viral RNA was detected at necropsy at 21 dpi in maternal lymphoid, joint-associated, and spinal cord tissues. The absence of detectable viral RNA and the lack of germinal center development in fetuses indicated that transplacental transmission did not occur. Neutralizing antibodies were detected in all dams and fetuses. Our study establishes a non-human primate model for evaluating vaccines and antiviral therapies and indicates that Rhesus macaques could serve as a competent enzootic reservoir. * Address correspondence to William K. Reisen, Center for Vectorborne Diseases, University of California, Davis, CA 95616. E-mail: wkreisen@ucdavis.edu 1250 CHEN AND OTHERS MATERIALS AND METHODS Animals. Six pregnant colony-bred female Rhesus macaques ( Macaca mulatta ) housed at the animal facility at the California National Primate Research Center, aged 7–15 years and at gestational days 121–132, were used for CHIKV inoculation. Animals were housed and maintained according to regulations and guidelines set forth by the University of California, Davis, Institutional Animal Care and Use Committee (IACUC) under an approved IACUC protocol. All animals were screened for viral infection status before inoculation. All monkeys were negative for Simian T-cell lymphotropic/leukemia virus type 1 (STLV-1), Simian retrovirus (SRV), and Simian retrovirus (SIV). Two animals in each group were positive for Simian foamy virus (SFV), Cytomegalovirus (CMV), and Herpes B virus; one animal in each group was positive for only SFV and CMV. All animals were bled 2 days before viral inoculation to confirm the absence of CHIKV neutralizing antibodies. Cell lines and viruses. African green monkey kidney (Vero), baby hamster kidney (BHK-21), and Ae. albopictus (C6/36) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Vero and C6/36) and minimal essential medium (MEM) (BHK-21) supplemented with 5% fetal bovine sera (FBS) and antibiotics, and then were incubated in a humidified environment with 5% CO 2 at 37°C and 28°C, respectively. Rhesus macaques were inoculated with a West African strain of CHIKV (37997) isolated from a mosquito pool during enzootic transmission in Senegal in 1983 (obtained from the Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention reference collection) and a human CHIKV strain (DHS-4263) isolated by the California Department of Public Health from a traveler infected in India during the epidemic in 2006. 42 The CHIKV epidemic strain was originally isolated in rabbit keratinocyte cells (RK), passed one time in BHK-21, and passed one time in Vero cells before experimentation. The CHIKV enzootic strain was isolated in mosquito cells ( Ae. pseudoscutellaris ; AP), passed one time in Vero cells, and passed one time in BHK-21 cells. Both virus stocks were prepared by inoculating C6/36 cells at a multiplicity of infection (MOI) of 0.1 and were harvested at 2 days. Groups of three pregnant macaques each were inoculated subcutaneously on the right upper arm with a 100μL inoculum comprising 1,000–10,000 plaque forming units (PFU) of either the epidemic East African or the enzootic West African strain. Animals were fasted 3–4 hours before sedation with ketamine (5–20 mg/kg body weight) by intramuscular injection for viral inoculation, physical examination, and blood collection. Clinical signs and fetal viability. Macaques were monitored daily for clinical signs of CHIKV disease, including fever, pain as evidenced by reduced mobility, joint swelling, nose/ gum bleeding, rash, and peripheral lymphadenopathy. These examinations included monitoring body weight, taking rectal temperature, and measuring the circumference and local temperature of joints (wrists and ankles). The joint circumference was assessed daily across wrists and ankles. To ensure consistency, the sites for each measurement were marked in indelible ink. Local joint temperatures were detected using a DermaTemp DT-1001RS Remote Sensor Model (Exergen, Watertown, MA). An Ultrasonic Doppler Flow Detector (Parks Medical Electronics, Inc., Aloha, OR) was used to monitor fetal heart rate as a measure of fetal viability. Tissue collection and processing. Peripheral blood samples were collected 2 days before virus inoculation and daily up to 21 dpi by venipuncture. Blood samples were collected with ethylenediaminetetraacetic acid (EDTA) and heparinimpregnated tubes and centrifuged at 1,000 × g for 10 minutes to isolate plasma and buffy coats. Plasma was frozen at −80°C until assayed for virus titer (quantified by Vero cell plaque assay), viral RNA, anti-CHIKV antibody levels, plasma chemistry, and cytokine profiles. Whole-blood samples were collected with EDTA-coated tubes for blood cell counts. Dams and fetuses were euthanized at 21 dpi by deep ketamine anesthesia followed by intravenous (IV) barbiturate overdose using sodium pentobarbital at 60 mg/kg. Maternal and fetal tissues were collected and assayed to determine the viral load and extent of histopathology, including blood, joint-associated skeletal muscle/synovium/epiphysis/connective tissues, brain, spinal cord, heart, lung, kidney, spleen, liver, bone marrow, joints, and lymph nodes (axillary and inguinal lymph nodes). In addition, mammary glands, placental tissue, and skin and brachial lymph nodes from arms proximal and distal to the inoculation site were collected from dams. Maternal and fetal tissues were collected in 10% buffered formalin for histopathology, were collected in 4% paraformaldehyde for electron microscopy, and were snap-frozen in liquid nitrogen for viral RNA detection and infectious virus isolation. Histopathology. Maternal and fetal tissues were fixed in 10% neutral buffered formalin. Selected dam and fetal tissues were collected, including skin from arms (inoculation side and opposite arm), joints, joint-associated skeletal muscle and connective tissue, synovium, epiphysis, bone marrow, brain, spinal cord, heart, lung, kidney, spleen, liver, mammary tissue, vagina, brachial lymph nodes close to the inoculation site and from opposite arm, axillary and inguinal lymph nodes, and placenta. These tissues were routinely processed and embedded in paraffin, sectioned at 7 μm, and mounted on positive-charged glass slides (Superfrost Plus; Fisher Scientific, Pittsburgh, PA). Tissue sections were stained with hematoxylin and eosin (HE). Electron microscopy. For transmission electron microscopy, 4% paraformaldehyde-fixed tissues were post-fixed in 1% osmium tetroxide in 0.1 M phosphate buffer for 90 minutes at room temperature, rinsed, and dehydrated in ascending concentrations of acetone from 30% to 100% for 10 minutes for each step, with three changes of acetone at 95% and two changes at 100%. Infiltration was carried out in a microwave (Pelco 34700 BioWave; Ted Pella Inc., Redding, CA) for 3 minutes for each step under vacuum and between 250 and 450 W. 43 Tissues were placed into capsules and polymerized at 70°C overnight. Blocks were trimmed and ultrathin-sectioned using a Diatome diamond knife (Hatfield, PA) at 60–90 nm on a Leica UTC ultramicrotome (Leica Ultracut UCT, Vienna, Austria). The sections were post-stained by standard protocol for transmission electron microscopy using uranyl acetate in 70% lead citrate. Images were taken on a Philips CM120 (FEI Company, Hillsboro, OR) with a GATAN MegaScan digital camera (Pleasanton, CA) at the Diagnostic and Research Electron Microscopy Laboratory, University of California, Davis (Davis, CA). Quantification of viral RNA. Maternal and fetal tissue samples from CHIKV-inoculated dams were weighed individually and then homogenized in 1 mL of DMEM with 5% FBS on ice before RNA extraction and inoculation of cell cultures for viral isolation. CHIKV RNA from homogenized maternal/fetal tissues, plasma, and buffy coats was extracted 1251 CHIKUNGUNYA INFECTION IN PREGNANT MACAQUES by MagMAX RNA isolation kits and a MagMAX Express magnetic particle processor (Ambion, Foster City, CA) according to the manufacturer’s instructions. Viral RNA was detected by TaqMan One-Step reverse transcription polymerase chain reaction (RT-PCR) Master Mix Reagents Kits with an ABI 7500 instrument (Applied Biosystems, Foster City, CA) using previously described CHIKV-specific primers and probe (forward primer 5′-AAGCTYCGCGTCCTTTACCAAG-3′, reverse primer 5′-CCAAATTGTCCYGGTCTTCCT-3′, and probe 5′-FAM-CCAATGTCYTCMGCCTGGACACCTTTTAMRA-3′) based on the manufacturer’s recommendations. 44 RNA was extracted from serially diluted samples of enzootic and epidemic CHIKV stocks (range = 0–6 log 10 PFU/mL) and assayed by the method described above to establish a standard curve for quantification. CHIKV real-time RT-PCR positive tissues were blind-passaged two times in C6/36 cells (7 days per passage). RNA was extracted from the supernatants of blind passages using MagMAX viral RNA isolation kits, examined for the presence of CHIKV RNA as described above, and titrated for infectious virus by plaque assay on Vero cells. Enzyme-linked immunosorbent assay. Ninety-six–well plates were seeded with Vero cells and infected with the CHIKV epidemic strain at an MOI of 0.1. Negative control plates were seeded with uninfected Vero cells. Plates were fixed 20 hours post-infection (hpi) by treatment with fixative solution (0.2% w/v of bovine serum albumin and 20% v/v of acetone in 1× phosphate-buffered saline; PBS) for 1 hour at 4°C. After fixation, plates we

Older monkeys of the Sulawesian species Macaca nigra spontaneously develop a lesion in the pancreatic islets of Langerhans in which there is deposition of amyloid and gradual degeneration of all cells, which can lead eventually to development of diabetes mellitus. Islet cell antibodies (ICA), formed in response to the release of cellular antigens, can be used to detect the islet lesion and to monitor the progression of each monkey toward diabetes. Numerous M. nigra and one M. tonkeana in captivity have been tested, but it is unknown whether the islet lesion occurs in monkeys in their natural habitat of Sulawesi. Blood samples collected from M. maurus, M. tonkeana, and hybrid M. maurus/tonkeana were assayed for ICA. When all monkeys were considered together, 33% had ICA positive against beta cells and 14% had ICA positive against alpha and/or D cells. Appearance of ICA in blood of males was virtually the same as in females. These results are similar to those found in M. nigra examined in captivity. Since all Sulawesian species share a common genetic heritage, these results would support the appearance of this lesion in their natural habitat. Cause(s) for formation of the lesion and eventual development of diabetes are unknown. There may be genetic factors or genetic predisposition to environmental factors. If environmental factors are responsible, then they must be present not only in the wild, but either carried with the monkeys or universally available, since M. nigra born in captivity also develop the lesion and diabetes after physical maturity at ca. 7+ years.

ABSTRACT To elucidate the relationship between early viral infection events and immunodeficiency virus disease progression, quantitative-competitive and branched-DNA methods of simian immunodeficiency virus (SIV) RNA quantitation were cross-validated and used to measure viremia following infection of rhesus macaques with the pathogenic SIVmac251 virus isolate. Excellent correlation between the methods suggests that both accurately approximate SIV copy number. Plasma viremia was evident 4 days postinfection, and rapid viral expansion led to peak viremia levels of 107 to 109 SIV RNA copies/ml by days 8 to 17. Limited resolution of primary viremia was accompanied by relatively short, though variable, times to the development of AIDS (81 to 630 days). The persistent high-level viremia observed following intravenous inoculation of SIVmac251 explains the aggressive disease course in this model. Survival analyses demonstrated that the disease course is established 8 to 17 days postinfection, when peak viremia is observed. The most significant predictor of disease progression was the extent of viral decline following peak viremia; larger decrements in viremia were associated with both lower steady-state viremia (P = 0.0005) and a reduced hazard of AIDS (P = 0.004). The data also unexpectedly suggested that following SIVmac251 infection, animals with the highest peak viremia were better able to control virus replication rather than more rapidly developing disease. Analysis of early viral replication dynamics should help define host responses that protect from disease progression and should provide quantitative measures to assess the extent to which protective responses may be induced by prophylactic vaccination.

Sera (165 samples in 1988 and 66, follow-up samples in 1989) were collected from olive baboons, African green monkeys, Syke's monkeys and grey mangabeys kept in a semi-free, breeding colony at the Institute of Primate Research (IPR) in Nairobi, Kenya. The levels of antibodies to simian T-lymphotropic virus (STLV) or simian immunodeficiency virus (SIV), and the reactivity patterns of positive sera to various lentivirus subgroup antigens, were then determined. The results of tests using enzyme-immunoassay kits were confirmed by western blots.The prevalence of antibodies which reacted with the Kenyan SIVagm(KEN) isolate was 28% in the African green monkeys tested and 34% in the Syke's monkeys. STL V seroprevalence was 25% in the African greens and 20% in the Syke's. No antibodies to either SIV or STL V were detected in the olive baboons or grey mangabeys.More SIV-positive samples were detected in western blots when SIVagm(KEN) was used as antigen than when SIVagm(CAR014), a geographically distinct isolate from the Central African Republic, was used. However, SIVagm(KEN)-positive sera were more reactive against SIVagm(CAR014) than SIVsmm and SIVmac subgroup antigens, indicating that the two isolates from the African green monkey, CAR014 and KEN, remain antigenetically close even though they were recovered in two geographically distinct regions.To date, no clinical disease has been linked with SIV and STL V infection in the African green or Syke's monkeys in the colony. However, the relatively high prevalence of anti-SIV and anti-STL V antibodies in these monkeys offers an opportunity for prospective studies on the transmission and natural history of both viruses in a single colony.

BACKGROUND/AIMS:Excess nitric oxide formation, via the inducible NO synthase isoform, has been implicated in the pathogenesis of experimental and clinical inflammatory bowel disease. The aim of this study was to assess the site, enzyme source, and magnitude of NO production in juvenile rhesus macaques with idiopathic colitis.METHODS:NO production was assessed systemically from plasma and urine levels of reactive nitrogen intermediates and locally by the formation of [3H]citrulline from [3H]arginine and reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry. Inducible NO synthase gene expression was assessed by reverse-transcription polymerase chain reaction.RESULTS:Plasma and urine levels of reactive nitrogen intermediates were greater in colitic animals than in control monkeys by 13- and 5-fold, respectively. NADPH diaphorase activity in normal animals was confined to the myenteric plexus. In colitis, staining was also apparent in crypt abscesses and superficial epithelial and mucosal bands. Gene expression for inducible NO synthase was only found in colitic specimens. Colonic [3H]citrulline formation was markedly elevated in colitic specimens, and the inducible isoform accounted for 58% of total activity.CONCLUSIONS:It is proposed that excess NO, formed via the inducible form of NO synthase, contributes to the mucosal inflammation and symptoms of this idiopathic colitis model.

Sera from 154 African non-human primates were screened for the presence of antibodies to type D retrovirus proteins. Four of five talapoin monkeys (Miopithecus sp.) captured in western Africa were positive for antibodies to type D retrovirus by ELISA and by immunoblot reactivity. Talapoins are the only African non-human primates that have so far shown evidence for type D retrovirus infection. Thus, talapoin monkeys appear to be a reservoir of type D retrovirus infection.

AIDS Research and Human RetrovirusesVol. 7, No. 9 Dominance of HTLV Type I-Specific Antibody Responsiveness in Old World MonkeysDonna L. Rudolph, S. Scott Keesling, Nicholas Lerche, Jo Ann Yee, and Renu B. LalDonna L. RudolphSearch for more papers by this author, S. Scott KeeslingSearch for more papers by this author, Nicholas LercheSearch for more papers by this author, Jo Ann YeeSearch for more papers by this author, and Renu B. LalSearch for more papers by this authorPublished Online:16 Mar 2009https://doi.org/10.1089/aid.1991.7.721AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail "Dominance of HTLV Type I-Specific Antibody Responsiveness in Old World Monkeys." , 7(9), pp. 721–722FiguresReferencesRelatedDetailsCited BySimultaneous Detection of Antibodies to Six Nonhuman-Primate Viruses by Multiplex Microbead ImmunoassayClinical and Vaccine Immunology, Vol. 13, No. 1Immune recognition of genetically diverse simian T-cell lymphotropic virus type I isolatesArchives of Virology, Vol. 140, No. 2Phylogenetic relationship and geographic distribution of multiple human T-cell lymphotropic virus type II subtypesJournal of Virology, Vol. 69, No. 2Isolation and characterization of simian T-cell leukemia virus type II from New World monkeysJournal of Virology, Vol. 68, No. 2Simian T-Lymphotropic Virus Type ISerologic confirmation of simian T-lymphotropic virus type I infection by using immunoassays developed for human T-lymphotropic virus antibody detectionJournal of Clinical Microbiology, Vol. 30, No. 4 Volume 7Issue 9Sep 1991 To cite this article:Donna L. Rudolph, S. Scott Keesling, Nicholas Lerche, Jo Ann Yee, and Renu B. Lal.Dominance of HTLV Type I-Specific Antibody Responsiveness in Old World Monkeys.AIDS Research and Human Retroviruses.Sep 1991.721-722.http://doi.org/10.1089/aid.1991.7.721Published in Volume: 7 Issue 9: March 16, 2009PDF download

An inactivated whole simian immunodeficiency virus (SIV) immunogen given to healthy, seropositive rhesus macaques 4 months after infection had no effect on the humoral immune response to SIV, the presence of antigenemia, cell‐associated viremia, or disease course. Further immunotherapeutic trials in this highly susceptible animal model should be carried out sooner after exposure, before significant loss of CD4 cells has occurred. The SIV infected macaque model will continue to serve an essential role in development and testing of anti‐AIDS drugs and immunogens.

A field survey of 25 sites in Sulawesi Utara (north Sulawesi) in 1987 and 1988 found macaques in 16 of these sites. The most viable population of Macaca nigra was found in the Tangkoko reserve at an estimated density of 76.2 monkeys/km2, which is less than one-third the abundance reported in the late 1970s by the MacKinnons. The adjacent reserves of Batuangus and Duasudara had only 22 monkeys/km2, yielding a population estimate for these three contiguous reserves of only 3,655 individuals. Maccaca nigrescens were found in the central and western portions of Dumoga-Bone National Park in densities of 15.5 and 16.4 monkeys/km2, significantly below the density of 27/km2 reported by the MacKinnons. The more peripheral areas of Dumoga-Bone had only 8.15 monkeys/km2, yielding a population estimate of M. nigrescens in Dumoga-Bone of less than 34,000. Our total population estimate for M. nigra and M. nigrescens combined is less than 50,000 individuals, which is considerably below that reported in recent litreture. M. hecki were observed in only two locations, Tangale and Panua Reserves, at low densities of 3.3 to 5.2 monkeys/km2, suggesting its range and abundance have declined since the observations of Groves (pp. 84-124 in THE MACAQUES: STUDIES IN ECOLOGY, BEHAVIOR AND EVOLUTION. D. G. Lindburg, ed. New York, Van Nostrand Reinhold, 1980). Several factors have contributed to population decline in these species: habitat shrinkage, increasing human population pressure, and drought conditions. Group sizes were significantly smaller in our study than in previous ones, and we found a shortage of juveniles and infants.

Simian acquired immunodeficiency syndrome (SAIDS) is a retrovirus-induced immunodeficiency disease that affects certain nonhuman primates and has many parallels to human AIDS. We examined 72 rhesus monkeys (Macaca mulatta) exposed to SAIDS retrovirus serotype-1 (SRV-1) and 81 healthy control monkeys at the California Primate Research Center to determine the prevalence of oral lesions. At the time of examination, 69 of the 72 monkeys exposed to SRV-1 had serologic and/or virologic evidence of SRV-1 infection. None of the 81 control monkeys had any evidence of infection with SRV-1. Acute necrotizing ulcerative gingivitis (ANUG), oral yeast infections, and noma occurred in 10% (p less than 0.01), 4%, and 1%, respectively, of the study group of 72 animals but were found in none of the control animals. Thus, ANUG occurs rather frequently in rhesus monkeys with evidence of SRV-1 infection. The reproducible immunodeficiency that follows inoculation of SRV-1 in rhesus monkeys promises to be a useful model for studying the pathogenesis of ANUG associated with immunodeficiency.

The aetiological agent of spontaneously occurring simian acquired immune deficiency syndrome (SAIDS) in rhesus monkeys (Macaca mulatta) at the California Primate Research Center is a type D retrovirus designated SAIDS retrovirus serotype 1 (SRV-1). SRV-1 DNA and RNA have previously been detected in the brains of rhesus monkeys with SAIDS in the absence of viral antigen or neuropathological lesions. In this study we further define the relationship between SRV-1 and the central nervous system (CNS) in rhesus monkeys by examining the CNS for infectious SRV-1, viral antigen and anti-SRV-1 antibodies. In addition, cerebrospinal fluid (CSF) was assayed for alterations in IgG and albumin levels, IgG/albumin ratios and cell count in comparison to uninfected control animals. No differences in CSF parameters were detected between infected and uninfected animals except for the presence of infectious SRV-1 which was isolated from the CSF from 13 out of 19 (68%) viraemic rhesus monkeys. The probable source of this virus was the choroid plexus, where approximately 1 in 1000 surface epithelial cells were found to contain viral antigen by immunohistochemistry. Antibodies against SRV-1 were not detected in the CSF even when present in the serum. Neither infectious virus nor viral antigen were found in the brain parenchyma of any animal examined. Thus infection of the CNS by SRV-1 appears to be subclinical without an intrathecal immune response. This may be related to the apparent restriction of productive infection in the CNS to cells of the choroid plexus.

Publisher Summary This chapter discusses the virologic, immunologic, and epidemiologic aspects of immunosuppressive disease caused by type D retroviruses (SRV-1, SRV-2, MPMV) and by lentiviruses (SIV) in nonhuman primates. Insofar as AIDS is a human retroviral disease caused by HIV and the chimpanzee is still the only nonhuman species known to be susceptible to experimental infection with this virus, both of these other nonhuman primate models will be of great benefit, although they present a few limitations. HIV-2 appears to cause persistent infection in some macaques without inducing disease. This model may, therefore, help reduce the need for chimpanzees of African origin. Because SIV causes a persistent infection and fatal AIDS-like disease in Asian macaques, this system constitutes the most directly relevant animal model for human AIDS. Critical questions on retrovirus-induced immunopathology that can be assessed in these animal models which cannot be tested as directly for HIV include the role of cofactors for infection and disease, the viral determinants of pathogenesis, the significance of envelope gene variation, and the potential for protective immunization, both pre- and postexposure. Analyses of the effects of a variety of retroviruses in primates and nonhuman primates reveal the complexity of immunosuppression and prefigure a diversity of pathogenic mechanisms. New concepts learned in these systems will have fundamental implications on the understanding of viruses, the immune system, and regulation of gene expression, and may lead to the development of procedures to prevent or limit immunologic abnormalities caused by these viruses.

Hematologic abnormalities were defined in 31 rhesus monkeys (Macaca mulatta) with simian acquired immune deficiency syndrome (SAIDS). Animals manifested anemia (hypochromic/microcytic), severe neutropenia and progressive lymphopenia, monocytosis and occasional thrombocytopenia. Bone marrow studies showed erythroid hyperplasia with a marked left shift and adequate megakaryocytes. Two animals showed profound hypoplasia of all hematopoietic elements. Most animals were iron deficient, but the course of the anemia suggested additional factors. There was no evidence of immune hemolysis. The pathogenesis of these abnormalities is not clear and will require further study. This reproducible disease will allow studies to elucidate the mechanisms of viral-induced hematologic abnormalities.