Tulane National Primate Research Center Research Articles

Astrocyte expression of aging-associated markers positively correlates with neurodegeneration in the frontal lobe of the rhesus macaque brain.

As the population over the age of 65 increases, rates of neurodegenerative disorders and dementias will rise - necessitating further research into the cellular and molecular mechanisms that contribute to brain aging. With the critical importance of astrocytes to neuronal health and functioning, we hypothesized that alterations in astrocyte expression of aging-associated markers p16INK4a (p16) and sirtuin 1 (SIRT1) with age would correlate with increased rates of neurodegeneration, as measured by FluoroJade C (FJC) staining. To test this hypothesis, 19 rhesus macaques at the Tulane National Primate Research Center were selected based on the following criteria: archival FFPE CNS tissue available to use, no noted neuropathology, and an age range of 5-30 years. Tissues were cut at 5 μm and stained for GFAP, p16, SIRT1, and FJC, followed by whole-slide imaging and HALO® image analysis for percentage of marker-positive cells and relative intensity of each stain. We found the percentage of p16+ cells increases with age in total cells and astrocytes of the frontal (p = 0.0021, p = 0.0012 respectively) and temporal (p = 0.0226, p = 0.0203 respectively) lobes, as well as the relative intensity of p16 staining (frontal lobe: p = 0.0060; temporal lobe: p = 0.0269). For SIRT1, we found no correlation with age except for an increase in the relative intensity of SIRT1 in the temporal lobe (p = 0.0033). There was an increase in neurodegeneration, as measured by the percentage of FJC+ cells in the frontal lobe with age (p = 0.0057), as well as in the relative intensity of FJC staining in the frontal (p = 0.0030) and parietal (p = 0.0481) lobes. Importantly, increased p16 and SIRT1 expression in astrocytes correlated with increasing neurodegeneration in the frontal lobe (p = 0.0009, p = 0.0095 respectively). Together, these data suggest that age-associated alterations in astrocytes contribute to neurodegeneration and provide a target for mechanistic studies in the future.

Functional Insulinomas in a Rhesus Macaque (Macaca mulatta)

We report a rare case of functional insulinomas in a 16.7-year-old female Rhesus macaque (Macaca mulatta) that was presented with neuroglycopenic signs to the breeding colony hospital at the Tulane National Primate Research Center. At initial and follow-up examinations, the animal was consistently hypoglycaemic and was clinically maintained with additional fruits, other high-sugar food items and dextrose supplementation. Occasional episodes of seizure and collapse resolved quickly on administration of high-sugar food items. At necropsy, the uncinate process of the pancreas had a 2.2cm diameter, red, round, firm neoplastic mass, and another neoplasm was identified on histological examination of the head of pancreas. Histologically, neoplastic cells exhibited neuroendocrine packeting, resembled pancreatic islet cells and immunolabelled for chromogranin A, synaptophysin and insulin but not for somatostatin, gastrin or pancreatic polypeptide. A few cells immunolabelled for glucagon. The clinical signs and gross and histological findings were consistent with functional insulinomas.

Divergent Enteroviruses from Macaques with Chronic Diarrhea.

ABSTRACTWe report the draft genome sequences of five novel members of the family Picornaviridae that were isolated from the stool of rhesus macaques (Macaca mulatta) with chronic diarrhea. The strains were named NOLA-1 through NOLA-5 because the macaques were residents of the Tulane National Primate Research Center.

Pedigree reconstruction and distant pairwise relatedness estimation from genome sequence data: A demonstration in a population of rhesus macaques (Macaca mulatta).

A primary challenge in the analysis of free‐ranging animal populations is the accurate estimation of relatedness among individuals. Many aspects of population analysis rely on knowledge of relatedness patterns, including socioecology, demography, heritability and gene mapping analyses, wildlife conservation and the management of breeding colonies. Methods for determining relatedness using genome‐wide data have improved our ability to determine kinship and reconstruct pedigrees in humans. However, methods for reconstructing complex pedigree structures and estimating distant relatedness (beyond third‐degree) have not been widely applied to other species. We sequenced the genomes of 150 male rhesus macaques from the Tulane National Primate Research Center colony to estimate pairwise relatedness, reconstruct closely related pedigrees, estimate more distant relationships and augment colony records. Methods for determining relatedness developed for human genetic data were applied and evaluated in the analysis of nonhuman primates, including identity‐by‐descent‐based methods for pedigree reconstruction and shared segment‐based inference of more distant relatedness. We compared the genotype‐based pedigrees and estimated relationships to available colony pedigree records and found high concordance (95.5% agreement) between expected and identified relationships for close relatives. In addition, we detected distant relationships not captured in colony records, including some as distant as twelfth‐degree. Furthermore, while deep sequence coverage is preferable, we show that this approach can also provide valuable information when only low‐coverage (5×) sequence data is available. Our findings demonstrate the value of these methods for determination of relatedness in various animal populations, with diverse applications to conservation biology, evolutionary and ecological research and biomedical studies.

Effects of Anti-Retroviral Therapy (ART) on Restoration of Lung Immunity and Tuberculosis Reactivation

While the advent of combination antiretroviral therapy (cART, ART) has significantly improved survival, tuberculosis (TB) still accounts for one-third of deaths in Human Immunodeficiency Virus (HIV)-infected populations, suggesting incomplete restoration of immune function by ART. Mtb/Simian Immunodeficiency Virus (SIV) co-infected macaques have been used to model Mtb/HIV co-infection in humans. To identify the components of TB immunity that remain impaired following HIV-infection and ART treatment, macaques with asymptomatic M. tuberculosis (Mtb) infection were subsequently co-infected with SIV. Animals were then randomly assigned to ART or control groups and studied longitudinally. While ART significantly reduced viral loads and increased CD4+T cell counts in whole blood and BAL samples, it did not reduce the relative risk of SIV- induced TB reactivation (RR 1·23, 95% CI 0·97-1·5) in ART treated macaques. CD4+T cells were poorly restored specifically in the lung interstitial region, despite their significant restoration in the alveolar compartment of the lung as well as in the periphery. IDO1 induction on myeloid cells in the iBALT likely contributes to dysregulated T cell homing and impaired lung immunity. Thus, while ART is indispensable for controlling viral replication, CD4+T cells restoration and in preventing opportunistic infection, it is however inadequate in reversing Mtb-specific T cell impairment. This warrants modeling concurrent treatment of TB and HIV to potentially reduce the risk of reactivation of TB due to HIV. The current and future studies like this have the potential to inform treatment strategies in patients with Mtb/HIV co-infection. The study was funded through grants from the National Institute of Health (NIH). Funding Statement: This work was primarily supported by NIH awards to D.K. and J.R. (R01AI111943, R01AI123047) with additional support from NIH awards R01AI111914, R01AI134240, R01AI135726, U19AI111211 and institutional awards P51OD111033 and P51OD111004. Declaration of Interests: The authors declare that no conflict of interest exists. Ethics Approval Statement: All the animals were housed in the Animal Biosafety level III (ABSL3) at Tulane National Primate Research Center where they were treated as per the standards recommended by Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International and the NIH Guide for the Care and Use of Laboratory Animals. The study was approved by the IACUC of the Tulane National Primate Research Center (protocols P0247R, P0324, P0295R).

A Method to Investigate Astrocyte and Microglial Morphological Changes in the Aging Brain of the Rhesus Macaque.

With a rapidly aging population, studies of neuroinflammation and degeneration associated with eugeric aging are becoming critical. Using the unique archive at the Tulane National Primate Research Center as a resource, we have developed tools to quantify morphological changes in astrocytes and microglia across the life span of monkeys. This method can be used for morphometric studies of multiple parameters simultaneously in an unbiased manner.

SNP-based genetic characterization of the Tulane National Primate Research Center's conventional and specific pathogen-free rhesus macaque (Macaca mulatta) populations.

The rhesus macaque is an important biomedical model organism, and the Tulane National Primate Research Center (TNPRC) has one of the largest rhesus macaque breeding colonies in the United States. SNP profiles from 3266 rhesus macaques were used to examine the TNPRC colony genetic composition over time and across conventional or SPF animals of Chinese and Indian ancestry. Chinese origin animals were the least genetically diverse and the most inbred; however, since their derivation from their conventional forebearers, neither the Chinese nor the Indian SPF animals exhibit any significant loss of genetic diversity or differentiation. The TNPRC colony managers have successfully minimized loss in genetic variation across generations. Although founder effects and bottlenecks among the Indian animals have been successfully curtailed, the Chinese subpopulation still show some influences from these events.

Pair housing of Vervets/African Green Monkeys for biomedical research.

Vervets, also known as African green monkeys, are a nonhuman primate species widely used in biomedical research. However, there are currently few references available describing techniques and rates of success for pair-housing this species. We present data from four cohorts of vervets from three different facilities: (i) the Wake Forest Vervet Research Colony (VRC; n = 72 female pairs, n= 52 male pairs), (ii) the University of Louisiana at Lafayette-New Iberia Research Center (UL-NIRC; n = 57 female pairs, n = 54 male pairs), (iii) the Tulane National Primate Research Center (TNRPC; n = 18 male pairs), and (iv) a cohort of imported males (n = 18 pairs) at Wake Forest. Compatibility was measured at 14, 30, and 60 days following introduction. Success rates for pair-housing at 14 days ranged from 96% to 98% for females and 96% to 100% for males at the VRC and UL-NIRC but were lower in the smaller imported male cohorts (TNPRC: 50%; WF: 28%). Among the UL-NIRC cohort and VRC male cohort, most of the pair separations after 14 days were due to reasons unrelated to social incompatibility. In contrast, a large proportion of TNPRC and imported male pairs successful at 14 days required separation within 60 days due to incompatibility. Multiple logistic regressions were performed using cohort, mean age of pair and weight difference between pair-mates as potential predictors of compatibility at 14 days. All three predicted the 14-day outcome in males but not females. A separate analysis in the VRC cohort found no evidence that prior familiarity in a group setting influenced outcomes. Variations in success rates across cohorts may have been influenced by introduction methodology. Behavioral differences between vervets and macaques, coupled with our findings, lead us to theorize that the gradual introduction techniques commonly implemented to pair house macaques may not be beneficial or suitable for this species. Am. J. Primatol. 79:e22501, 2017. © 2015 Wiley Periodicals, Inc.

Development of real-time PCR assays for the detection of Moraxella macacae associated with bloody nose syndrome in rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques.

Moraxella macacae is a recently described bacterial pathogen that causes epistaxis or so-called bloody nose syndrome in captive macaques. The aim of this study was to develop specific molecular diagnostic assays for M. macacae and to determine their performance characteristics. We developed six real-time PCR assays on the Roche LightCycler. The accuracy, precision, selectivity, and limit of detection (LOD) were determined for each assay, in addition to further validation by testing nasal swabs from macaques presenting with epistaxis at the Tulane National Primate Research Center. All assays exhibited 100% specificity and were highly sensitive with an LOD of 10 fg for chromosomal assays and 1 fg for the plasmid assay. Testing of nasal swabs from 10 symptomatic macaques confirmed the presence of M. macacae in these animals. We developed several accurate, sensitive, and species-specific real-time PCR assays for the detection of M. macacae in captive macaques.

Naltrexone treatment reverses astrocyte atrophy and immune dysfunction in self-harming macaques

The role of glia in the development and treatment of behavioral abnormalities is understudied. Recent reports have observed glial activation in several disorders, including depression, autism spectrum disorders and self-injurious behaviors (SIB). In the current study, we examined SIB in the physiologically and anatomically relevant nonhuman primate (NHP) model. At the Tulane National Primate Research Center (TNPRC), approximately 5% of singly housed macaques develop symptoms of SIB. We have previously demonstrated that naltrexone hydrochloride can be effective in reducing SIB. We have also demonstrated that the astrocytes of animals with SIB are distinctly atrophic and display heightened innate immune activation compared with control animals. We have added a third group of animals (five macaques identified with SIB and treated with oral naltrexone at a dose of 3.2mg/kg) to the previous cohort (six macaques with a history of SIB but not treated, and nine animals with no history of SIB) for this study. Gray and white matter astrocytes from frontal cortical tissue were examined following necropsy. Innate immune activation of astrocytes, which was increased in SIB animals, was markedly decreased in animals receiving naltrexone, as was atrophy of both grey and white matter astrocytes. This was concomitant with improved behavioral correlates. Preventing astrocyte activation in select areas of the brain to reduce injurious behavior is an innovative concept with implications for mental health studies. Differences in multiple areas of primate brain would help determine how self-injurious behavior develops. These studies suggest a stronger role for astrocytes in the cellular events associated with self-injurious behaviors.

Detecting the Simian Immunodeficiency Virus Reservoir as Viral DNA and RNA in Situ Simultaneously as a Model to Monitor Viral Latency

HIV and simian immunodeficiency virus (SIV) establish latent infection in long-lived cells that form a reservoir of virus that persists in infected individuals even after years of treatment with highly active antiretroviral therapy (HAART). We present here an innovative technique that can be used to detect latent and active infected cells in tissues. The HIV/SIV reservoir is not fully characterized and to cure HIV we need to determine where the virus is hiding either latently integrated into the host DNA or replicating at very low levels producing viral RNA. The SIV infection model currently used in the Tulane National Primate Research Center (TNPRC) is suitable to study the latency and emergence of the virus in the presence of treatment and/or after lifting the pressure imposed by HAART to remain latent. Figure 1 demonstrates simultaneous detection and colocalization of SIV RNA (red) and DNA (blue) in an infected multinuclear giant cell in the lung of an SIV-infected macaque. The image reveals that not all the nuclei have integrated the SIV. This method demonstrates that SIV-infected cells are relatively rare in vivo. The technique consists of double in situ hybridization: RNA hybridization and color development and after an RNase step a DNA hybridization developed with other color. The procedure combines previously described elements.1–3 This technique could be used to detect the virus integrated in cells that are not actively producing virus. This would be revealed by cells that are DNA positive, but RNA negative. Approaches identifying the expression of viral proteins or RNA will not work to identify latently infected cells. Only viral DNA detection can allow the identification of latently infected cells because they are defined by a lack of viral gene expression. This approach shows great promise to identify the location of cells, latently infected with SIV, and provide critical information to advance the efforts to develop a cure for HIV infection. FIG. 1. The image shows the identification of simian immunodeficiency virus (SIV) nucleic acids in the lung of an SIV-infected macaque. In situ hybridization of SIV RNA (red) and DNA (blue) identifies individual infected cells within tissue. All nuclei in the ...

Pair Housing for Female Longtailed and Rhesus Macaques in the Laboratory: Behavior in Protected Contact Versus Full Contact

Pair housing for caged macaques in the laboratory generally allows unrestricted tactile contact but, less commonly, may involve limited contact via grooming-contact bars or perforated panels. The purpose of using this protected contact housing, which prevents entry into pair-mates' cages, typically is to accommodate research and management requirements. The study used behavioral data collected on 12 pairs of female longtailed macaques (Macaca fascicularis) at the Washington National Primate Research Center and 7 pairs of female rhesus macaques (Macaca mulatta) housed at the Tulane National Primate Research Center to assess the relative benefits of protected versus full protected contact. The study collected data in stable pairs housed first in protected contact followed by full contact. Species combined, the study found the presence of the panel was associated with lower levels of social grooming and higher levels of self-grooming, abnormal behavior, and tension-related behavior. Within species, only the protected- versus full-contact contrasts for abnormal and tension were statistically significant—and only for rhesus macaques. Results suggest that for female rhesus macaques, potential disadvantages or inconveniences of full contact should be balanced against the improved behavioral profile in comparison to protected contact. The use of protected contact among female longtailed macaques does not appear to require the same cost-benefit analysis.

Reproductive efficiency of captive Chinese- and Indian-origin rhesus macaque (Macaca mulatta) females.

Reproductive and survival records (n=2,913) from 313 Chinese-origin and 365 Indian-derived rhesus macaques at the Tulane National Primate Research Center (TNPRC) spanning three generations were studied. Least-squares analysis of variance procedures were used to compare reproductive and infant survival traits while proportional hazards regression procedures were used to study female age at death, number of infants born per female, and time from last birth to death. Chinese females were older at first parturition than Indian females because they were older when placed with males, but the two subspecies had similar first postpartum birth interval (1st PPBI) and lifetime postpartum birth interval (LPPBI). Females that gave birth to stillborn infants had shorter first postpartum birth intervals (1st PPBI) than females giving birth to live infants. Postpartum birth intervals decreased in females from age 3 to 12 but then increased again with advancing age. Chinese infants had a greater survival rate than Indian infants at 30 days, 6 months, and 1 year of age. Five hundred and forty-three females (80.01%) had uncensored, or true records for age at death, number of infants born per female, and time from the birth until death whereas 135 females (19.91%) had censored records for these traits. Low- and high-uncensored observations for age at death were 3 and 26 years for Chinese, and 3 and 23 years for Indian females. Uncensored number of infants born per female ranged from 1 to 15 for Chinese females and 1 to 18 for Indian females. Each of these traits was significantly influenced by the origin×generation interaction in the proportional hazards regression analyses, indicating that probabilities associated with age at death, number of infants born per female, and time from last birth to death for Chinese and Indian females did not rank the same across generations.

Chromobacterium violaceum infections in 13 non‐human primates

Recently, an Indian-origin macaque was found dead and Chromobacterium violaceum was isolated from the skin wound, and hepatic and pulmonary abscesses. By searching the database, a total of thirteen cases of C. violaceum infection in pigtail macaques (n = 8), rhesus macaques (n = 4), and one baboon were identified from 2001 to 2010 at Tulane National Primate Research Center. Medical records were reviewed for breed, sex, age, clinical findings, treatment, outcome, bacteriology, and gross and histological findings. Seven pigtail macaques and one Indian-origin rhesus macaque died of chromobacterial septicemia. All chromobacterial septicemic pigtail macaques were adult with higher incidence in female. Hepatic abscess and thrombosis were typical findings along with pulmonary abscess and thrombosis, renal venous thromboembolism, and necrosuppurative pleuritis, peritonitis, splenitis, myocarditis, pericarditis, and meningoencephalitis. Skin wound, uterine infection, and oral and respiratory exposure were considered the points of entry for these animals. This represents the first report of chromobacteriosis in pigtail, rhesus macaque, and baboon. Our experience suggests that chromobacterial infections may be more common in non-human primates than previously recognized.

Benefits of pair housing are consistent across a diverse population of rhesus macaques

Introducing singly housed rhesus macaques (Macaca mulatta) into isosexual pairs is widely considered to improve welfare. The population of laboratory rhesus macaques is heterogeneous on a variety of factors and there is little literature available to directly evaluate the influence of many of these factors on the benefits of pair housing. Subjects were 46 adult female and 18 adult male rhesus macaques housed at the Tulane National Primate Research Center and the Yerkes National Primate Research Center. Behavioral data totalling 859h and serum cortisol levels derived from 312 serum samples were analyzed for main effects of housing condition, comparing single housing to pair housing. In addition, a series of analyses were performed to test for interactions between housing condition and seven independent variables: sex, age, prior duration of single housing, presence or absence of a history of self-injurious behavior, and dominance rank, levels of affiliation and agonism in the paired setting. After the collection of 4–8h of baseline data and three serum cortisol samples, pairs of individuals were introduced to one another and data collection was repeated, no earlier than 4 weeks after introduction. In pair housing both female and male subjects showed decrease in abnormal behavior (females: 54% reduction; P=0.001; males: 18% reduction; P=0.0007) and anxiety-related behavior (females: 35% reduction; P=0.0001; males: 41% reduction; P=0.0001), and increases in locomotion (females: 41% increase; P=0.0001; males: 76% increase: P=0.002). In pair housing, there were no significant sex differences in social behavior. Descriptively, paired females spent 12% of samples engaged in affiliative behavior and 0.5% engaged in agonistic behavior (backtransformed arcsin square root means). The corresponding values for males were 12% and 0.3%. No interaction effects were detected with any of the independent variables tested in this study. Cortisol values varied with sex but did not differ between housing conditions; no differences were detected when any of the above variables were included in the statistical model. Results support the general consensus among those studying the welfare of captive primates that social housing is a potent means for promoting behavioral indicators of the psychological well-being of laboratory primates. These results are of considerable practical significance and include information that refutes common perceptions about the unsuitability of males as socialization candidates, perceived negative consequences of subordinate rank within a pair, or variation in social dynamics observed in particular pairs. The population of singly housed rhesus macaques that will derive benefit from pair housing is diverse and findings of improved welfare can be broadly applied.

Characterization of a Moraxella species that causes epistaxis in macaques

Bacteria of the genus Moraxella have been isolated from a variety of mammalian hosts. In a prior survey of bacteria that colonize the rhesus macaque nasopharynx, performed at the Tulane National Primate Research Center, organisms of the Moraxella genus were isolated from animals with epistaxis, or “bloody nose syndrome.” They were biochemically identified as Moraxella catarrhalis, and cryopreserved. Another isolate was obtained from an epistatic cynomolgus macaque at the U.S. Army Medical Research Institute of Infectious Diseases. Based on differences in colony and cell morphologies between rhesus and human M. catarrhalis isolates, we hypothesized that the nonhuman primate Moraxella might instead be a different species. Despite morphological differences, the rhesus isolates, by several biochemical tests, were indistinguishable from M. catarrhalis. Analysis of the cynomolgus isolate by Vitek 2 Compact indicated that it belonged to a Moraxella group, but could not differentiate among species. However, sequencing of the 16S ribosomal RNA gene from four representative rhesus isolates and the cynomolgus isolate showed closest homology to Moraxella lincolnii, a human respiratory tract inhabitant, with 90.16% identity. To examine rhesus macaques as potential hosts for M. catarrhalis, eight animals were inoculated with human M. catarrhalis isolates. Only one of the animals was colonized and showed disease, whereas four of four macaques became epistatic after inoculation with the rhesus Moraxella isolate. The nasopharyngeal isolates in this study appear uniquely adapted to a macaque host and, though they share many of the phenotypic characteristics of M. catarrhalis, appear to form a genotypically distinct species.

Genetic Diversity and Histo-Blood Group Antigen Interactions of Rhesus Enteric Caliciviruses

Recently, we reported the discovery and characterization of Tulane virus (TV), a novel rhesus calicivirus (CV) (T. Farkas, K. Sestak, C. Wei, and X. Jiang, J. Virol. 82:5408-5416, 2008). TV grows well in tissue culture, and it represents a new genus within Caliciviridae, with the proposed name of Recovirus. We also reported a high prevalence of CV antibodies in macaques of the Tulane National Primate Research Center (TNPRC) colony, including anti-norovirus (NoV), anti-sapovirus (SaV), and anti-TV (T. Farkas, J. Dufour, X. Jiang, and K. Sestak, J. Gen. Virol. 91:734-738, 2010). To broaden our knowledge about CV infections in captive nonhuman primates (NHP), 500 rhesus macaque stool samples collected from breeding colony TNPRC macaques were tested for CVs. Fifty-seven (11%) samples contained recovirus isolates. In addition, one NoV was detected. Phylogenetic analysis classified the recovirus isolates into two genogroups and at least four genetic types. The rhesus NoV isolate was closely related to GII human NoVs. TV-neutralizing antibodies were detected in 88% of serum samples obtained from primate caretakers. Binding and plaque reduction assays revealed the involvement of type A and B histo-blood group antigens (HBGA) in TV infection. Taken together, these findings indicate the zoonotic potential of primate CVs. The discovery of a genetically diverse and prevalent group of primate CVs and remarkable similarities between rhesus enteric CVs and human NoVs opens new possibilities for research involving in vitro and in vivo models of human NoV gastroenteritis.

PLoS Pathogens Issue Image | Vol. 6(4) April 2010

This confocal micrograph shows cells triple-labeled with antibodies against BrdU (red), SIVp28 (green), and CD68 (blue). BrdU+ cells (red) that were not infected were identified in a lesion of an animal with SIVE. Numerous CD68+ and SIVp28+ cells are seen in this lesion; few of the mature CD68+ macrophages are BrdU+. The majority of BrdU+ cells are nonproductively infected (see Burdo et al., doi:10.1371/journal.ppat.1000842). Image Credit: Xavier Alvarez (Tulane National Primate Research Center, Tulane University).

Detection of norovirus-, sapovirus- and rhesus enteric calicivirus-specific antibodies in captive juvenile macaques

The objective of this study was to determine the prevalence of anti-norovirus (NoV), -sapovirus (SaV) and -Tulane virus (TV) antibodies in rhesus macaques of the Tulane National Primate Research Center and to evaluate the antigenic relationship between these viruses. A high prevalence of NoV-binding (51-61 %) and SaV-binding (50-56 %) antibodies and TV-neutralizing (69 %) antibodies were detected. Serum samples obtained during a human NoV outbreak and a multivalent anti-NoV hyperimmune serum were not able to neutralize TV infectivity. Conversely, low levels of cross-reactivity between the prototype TV and NoVs, but not between the TV and SaVs were detected by ELISA. These data indicate the preservation of some cross-reactive B-cell epitopes between the rhesus and human caliciviruses (CVs). The high prevalence of human and rhesus CV-specific serum antibodies suggests the frequent exposure of colony macaques to enteric CVs including the possibility of CV transmission between human and non-human primate hosts.

Recovery of Infectious Virus by Transfection of In Vitro-Generated RNA from Tulane Calicivirus cDNA

Tulane virus (TV) is a newly reported calicivirus that was isolated from stool samples of captive rhesus macaques from the Tulane National Primate Research Center (TNPRC). The virus has been cultivated successfully in LLC-MK2 rhesus monkey kidney cells. Its complete genomic sequence suggests that TV represents a new genus and is evolutionarily more closely related to Norovirus than to any other genus of Caliciviridae. In this study, we demonstrated that RNA transcripts made in vitro from the full-length genomic cDNA of TV were infectious upon transfection into permissive LLC-MK2 cells. The recombinant virus exhibited plaque morphologies and growth kinetics similar to those of the wild-type virus in this cell line. Capping was required for TV RNA infectivity. Although a subgenomic RNA has been detected in TV-transfected cells, a separate subgenomic RNA transcript was not required for the initial transfection to establish the replication. Transfection of truncated RNA lacking open reading frame 2 (ORF2) and ORF3 or TV-norovirus chimeric RNA resulted in abortive replication without the production of infectious progeny viruses, indicating that both ORFs are essential for the replication of TV. A heterologous insertion at the 5' end of the genome also hampered viral replication, suggesting that an authentic 5' end of the genome is critical for replication. The availability of the complete genomic sequence and the reverse genetics system described herein make TV a valuable model for studying calicivirus pathogenesis and replication.