Endemic Viruses of Squirrel Monkeys (Saimiri spp.)

Abstract

Nonhuman primates are the experimental animals of choice for the study of many human diseases. As such, it is important to understand that endemic viruses of primates can potentially affect the design, methods, and results of biomedical studies designed to model human disease. Here we review the viruses known to be endemic in squirrel monkeys (Saimiri spp.). The pathogenic potential of these viruses in squirrel monkeys that undergo experimental manipulation remains largely unexplored but may have implications regarding the use of squirrel monkeys in biomedical research.

The similarity between the nonhuman primate and human immune systems is a key advantage in the use of nonhuman primates compared with other mammalian models of human disease.^{13,71,88,94,103,113,125} In addition, the diversity of environmental and infectious disease agents encountered by primates is similar to that of humans, providing nonhuman primates a comparable level of biologic complexity.¹ Old World primates, such as macaques and baboons, and New World primates, including squirrel monkeys and marmosets, are commonly used in biomedical research. Squirrel monkeys (Saimiri spp.) are neotropical primates native to the forests of Central and South America. Of the 7 species of squirrel monkey, 3 (S. oerstedii, S. vanzolinii, and S. ustus) are classified as endangered, vulnerable to extinction in the wild, or near threatened, whereas the remaining 4 (S. boliviensis, S. cassiquiarensis, S. macrodon, and S. sciureus) are not endangered, although the S. cassiquiarensis albigena subspecies is near threatened^52,81 (Figure 1). In South America, where squirrel monkeys are indigenous, breeding colonies of S. sciureus have been maintained at the Pasteur Institute in French Guiana and at the Oswaldo Cruz Foundation in Brazil.^7,12 In the United States, the Squirrel Monkey Breeding and Research Resource, an NIH-sponsored national research resource, maintains breeding colonies for S. boliviensis boliviensis, S. sciureus sciureus, and S. boliviensis peruviensis.

Figure 1.

Taxonomy of Saimiri species with associated IUCN designations.^52,81

Squirrel monkeys adapt easily to laboratory housing and can be housed in smaller spaces than can Old World primates.¹ Unlike when working with Old World primates, particularly macaques, no additional personnel protective equipment is necessary when working with squirrel monkeys beyond that recommended for working with other New World primates.⁹² Their small size, combined with the reduced need for personnel protective equipment during handling, make squirrel monkeys attractive species for model development and for studies of viral pathogenesis, which cost approximately 30% to 40% less than comparable studies in macaques.¹ The likelihood of zoonotic transmission of infectious pathogens is considerably less than that associated with macaques and the risk of Macacine herpesvirus 1 (B virus) is nonexistent, given that neotropical primates do not harbor this lethal virus.¹ These factors are increasingly important in the current climate of limited grant funding for biomedical research and emphasis on safety for laboratory personnel. The limited availability of immunologic reagents with specificity for neotropical primates has hindered broader use of squirrel monkeys in biomedical research, compared with that of the more commonly used Old World primates. In addition, the small size of neotropical primates limits the volume of blood that can be collected at any one time. To abrogate these limitations, the NIH Nonhuman Primate Reagent Resource (www.nhpreagents.org) provides an increasing repertoire of agents that have been characterized for immunologic studies of neotropical primates.⁸⁹

Squirrel monkeys are used in numerous aspects of biomedical research, including studies of viral persistence, neuroendocrinology, infectious diseases, cancer treatments, vaccine development, gene expression, and reproductive physiology.¹¹⁷ The similarity between the squirrel monkey immune system and that of humans means that, as with macaques, there is a high likelihood that research outcomes will recapitulate what occurs in human diseases.^13,71,87,94 This is particularly true for the study of several notable infectious diseases, including malaria, Creutzfeldt–Jakob disease, and human T-cell leukemia virus type 1 (HTLV1) infection.^19,56,128 For these diseases, squirrel monkeys are the model system of choice for studying pathogenesis, experimental treatments, and strategies for prevention.

Squirrel monkeys are recognized as some of the most susceptible nonhuman primate species for the experimental transmission of Creutzfeldt–Jakob disease and other transmissible spongiform encephalopathies that cause chronic wasting disease.^11,72,98,130 The experimental infection of squirrel monkeys with HTLV1 has led to their use in vaccine development and chemotherapy research directed against HTLV1.^44,57,58,82 In addition, squirrel monkeys are an important model for studying the immunology of malaria and for testing vaccines against several Plasmodium species.^19,20,68,114 Furthermore, squirrel monkeys have been used in pharmacologic research to raise HDL levels to prevent atherosclerosis and reduce the risk of coronary heart disease.⁶ As the use of squirrel monkeys increases, especially for infectious disease research, accurate information about the endemic viral infections of squirrel monkeys is needed because of the potential for zoonotic transfer of these viruses to humans (and vice versa) and to understand the potential influence these agents may have on research involving other infectious pathogens diseases and immunosuppressive drugs.

Endemic Viruses of Squirrel Monkeys

The presence of endemic squirrel monkey viruses has been known for over 40 y, and all primates used in biomedical research are assumed to asymptomatically harbor persistent viruses, including herpesviruses and polyomaviruses.^{53,54,61,77,124} Of the known endemic viruses of squirrel monkeys, only Saimiriine herpesvirus 1 (SaHV1) is occasionally pathogenic in its natural host, causing orofacial ulcers and lingual plaques.⁶¹ These lesions are mild and self-limited, occurring only sporadically in the large colony of animals at the Squirrel Monkey Breeding and Research Resource. Whether any of the endemic viruses remain benign during immune suppression has been largely unexplored. As such, the implications of these viruses regarding the interpretation of data from studies that involve immune suppression in squirrel monkeys are currently unknown. Therefore, for studies involving an immune suppression component, it is prudent to monitor the blood, urine, and saliva for significant changes in the viral loads of the endemic viruses, particularly herpesvirus saimiri (HVS), which is ubiquitous in squirrel monkeys and is directly immunomodulatory.^36,73 The salient characteristics of the known endemic squirrel monkey viruses are presented in Figure 2 and reviewed here.

Figure 2.

Characteristics of known endemic squirrel monkey viruses. SaHV3 has been proposed as the designation, but the International Committee on Taxonomy of Viruses has not yet assigned it. HSV, herpes simplex virus; HHV8, human herpesvirus 8; EBV, Epstein–Barr virus; HCMV, human cytomegalovirus.

Herpesviruses

Herpesviruses are present in most species of animals and are classified into 3 subfamilies (α, β, and γ) on the basis of their biologic properties and genome sequences. Eight herpesviruses have been identified in humans, and 4 analogous herpesviruses have been found in squirrel monkeys. Herpesviruses share a common evolutionary origin and establish lifelong latency in their host, with intermittent periods of lytic replication and excretion.⁹⁵

SaHV1.

The squirrel monkey alphaherpesvirus SaHV1 was originally named ‘herpesvirus tamarinus’ because the virus was initially isolated and characterized from tamarins (Saguinas spp.).^46,80 In 1964, 2 independent reports described fatal generalized disease in tamarins, marmosets, rabbits, and mice infected with this novel alphaherpesvirus;^46,80 owl monkeys also were found to be susceptible to fatal disease after infection with SaHV1.³¹ Subsequent studies revealed that squirrel monkeys are the natural host for this virus.^24,47,61 The prevalence of SaHV1 in squirrel monkeys is high, with seropositivity greater than 90%.¹²⁰

Biologically and genetically, SaHV1 is most closely related to other primate herpes simplex viruses, including human simplex viruses 1 and 2.^27,120 SaHV1 can be propagated in a variety of cell lines, including human cells, where it induces cytopathic effects that are indistinguishable from those caused by human simplex virus 1.⁴⁶ In addition, like the cold sores caused by human simplex virus 1 in humans, SaHV1 infection can cause ulcerative oral lesions and lingual plaques in squirrel monkeys (Figure 3). SaHV1 lacks homologs for several genes that are found in all other primate simplex viruses, including genes that code for type 1, type 2, and type 3 membrane proteins; a dsRNA binding protein; and an inhibitor of antigen presentation.¹²⁰ The overall genomic organization of SaHV1 differs from that of the type E genomes found in other simplexviruses.¹²⁰ Specifically, SaHV1 has a type D genome like that of the varicelloviruses, another alphaherpesvirus subgroup that includes human varicella zoster virus.¹²⁰

Figure 3.

Ulcerative orofacial lesions (left, black arrow) and lingual plaques (right, white arrows) in squirrel monkeys are associated with active SaHV1 infection. Photographs by Julio C Ruiz, DVM (Keeling Center for Comparative Medicine, MD Anderson Cancer Center).

Although the herpes simplex viruses of nonhuman primates collectively are considered a zoonotic threat, in the 50 y since the first description of SaHV1, there have been no known cases of SaHV1 infection in humans.^119,120 In contrast, the herpes B virus, a neurotropic alphaherpesvirus endemic in macaques, is highly virulent in humans.⁹⁰ Transmission of the herpes B virus to humans can occur through bites or scratches from infected monkeys, leading to an encephalomyelitis that has a mortality rate of greater than 80% without prompt treatment.⁹⁰ The lethality of herpes B virus in humans remains a sobering reminder of the importance of safety precautions for the protection of both humans and research animals.

SaHV2.

Discovered in 1968, Saimiriine herpesvirus 2 (SaHV2), commonly known as herpesvirus saimiri (HVS), is now classified as a γ2 herpesvirus, or rhadinovirus, and is the most-studied of the endemic squirrel monkey viruses.^75,76 According to its genomic organization and DNA sequence, HVS is most closely related to human herpesvirus 8, also known as Kaposi sarcoma-associated herpesvirus.^5,23,65 Squirrel monkeys are naturally infected with HVS through saliva, usually within the first 2 y of life.³⁷ Early studies determined that the virus is nonpathogenic in squirrel monkeys after establishing lifelong persistence, primarily in T lymphocytes.²² However, HVS has a potent ability to induce T-cell leukemia and lymphomas in other neotropical primate species.^{22,33,50,77,78} Three subtypes of HVS are known, each with slightly different transforming abilities. All 3 subtypes cause T-cell lymphoma in cotton-top tamarins, whereas subtypes A and C also can induce these tumors in marmosets, and subtype C viruses can induce tumors in macaques.^26,36,74 In addition, HVS can cause malignant lymphoma after inoculation into marmosets, owl, and howler and spider monkeys and in incidentally infected owl monkeys.^{3,17,34,40,48-50,100}

Immune suppression of squirrel monkeys acutely infected with HVS results in prolonged oropharyngeal shedding of HVS and a significant decrease in the proportion of latently infected lymphocytes in peripheral blood.⁷³ Notably, tumor development has not been observed in studies of immunosuppression.⁷³ Seminal studies in the field of viral oncogenesis revealed that neotropical primates vaccinated with heat-killed, formaldehyde-treated HVS were protected against tumor formation.⁷⁹ This protection can be passively transferred in serum, suggesting that the antibody response provides the antitumor effect.^66,67 More recent investigations of HVS have focused on molecular studies of viral immunomodulatory elements and the viral genes involved in oncogenesis, cell signaling, and the maintenance of episomal viral elements.^22,41,62

A notable caution in working with squirrel monkeys comes from the ability of HVS subgroup C strains to transform human T lymphocytes to stable growth in vitro and yield continuously proliferating T-cell lines with the phenotype of mature CD4⁺ or CD8⁺ cells.⁸ The recognition of this unique feature opened the possibility of generating human T-cell lines for a variety of immunologic studies.⁸ Since then, HVS-transformed T-cell lines have been used to study primary human immunodeficiencies, including defects involving CD3γ, IL2Rγ, IL12R, MHC class II, CD43, CD95, and CD18/LFA1.³⁶ HVS-transformed human T cells have also been used in studies of HIV, which have led to the identification and increasing understanding of numerous factors involved in the inhibition of HIV1 replication.^{21,64,83,84,105}

HVS is appealing as a potential gene-delivery vector because it can package a large amount of heterologous DNA and establish a persistent infection while its genome remains as a nonintegrated episome.⁴² Because studies of HVS deletion mutants showed that saimiri transforming protein C and the tyrosine-kinase–interacting protein are essential for transformation in cell culture, all HVS-based vectors contain deletions in these 2 genes and are thought to be nonpathogenic.^28,42 The HVS-based vectors infect and persist in a wide range of human cell lines, including lung, colorectal, pancreatic, breast, gastric, and esophageal carcinoma and human hematopoietic cell lines.⁴² Intravenous and intraperitoneal inoculation with an HVS vector construct led to the persistent expression of the delivered genes in mice, as did vector injection into human tumor xenografts.^109,110 For HVS-based vectors to become useful as therapeutics, biosafety concerns need to be addressed, particularly with regard to preventing viral replication and (when desired) eliminating the vector after delivery.

Although HVS can transform human T cells and cause lymphomas in other neotropical primates, infection with HVS is not associated with human lymphomas, and the risk that HVS poses to humans has long been thought to be very low.^2,36 However, human herpesviruses, and particularly gammaherpesviruses, have previously been associated with idiopathic pulmonary fibrosis (IPF), and equine and asinine gammaherpesviruses cause pulmonary fibrosis in horses and donkeys.^116,127 A recent study of IPF specifically associated this fatal disease with HVS infection.³⁸ The association between HVS and IPF was revealed when the inflammatory cytokine IL17 (a pirated mammalian gene found in the HVS genome) was shown to be expressed in the regenerating epithelia of IPF tissue.^38,129 An initial screen of 13 cases of IPF by using in situ hybridization revealed that all cases were negative for human herpesviruses and positive for HVS.³⁸ In a comparison of 21 IPF cases and 21 cases of age-matched pulmonary fibrosis cases of known etiology, all 21 IPF cases were positive for multiple HVS DNA targets by in situ hybridization, whereas none of the 21 control cases was positive.³⁸ Further investigation through a prospective study is needed to validate this finding and determine its significance. To date, HVS has not been associated with pulmonary fibrosis in squirrel monkeys or any other nonhuman primate species.

A second γ2 herpesvirus has been detected in both free-ranging and captive squirrel monkeys, but little is known about this agent at present.²⁹

SaHV3 and other squirrel monkey lymphocryptoviruses.

Infection of Old World primates with lymphocryptoviruses (γ1 herpesviruses) has been recognized for 40 y, whereas the discovery of lymphocryptoviruses in New World primates is relatively recent.¹²⁶ One of the first lymphocryptoviruses discovered among neotropical primates was first identified in 2001 in captive squirrel monkeys in the United States and was called SaHV3.¹⁵ Subsequently, 2 independent research groups identified another lymphocryptovirus (Saimiri sciureus LHV1 or Saimiri sciureus LCV2) in wild squirrel monkeys in French Guyana and captive squirrel monkeys in Germany.^25,30 In addition, one of the captive squirrel monkeys was coinfected with a second lymphocryptovirus that had 95% homology with SaHV3.³⁰ On the basis of their genomic organization and DNA sequences, these lymphocryptoviruses are most closely related to Epstein–Barr virus in humans.^15,25,30 These discoveries could potentially be the basis for an animal model to study the pathogenesis of Epstein–Barr virus-induced lymphomas in humans.

SaHV4.

The squirrel monkey cytomegalovirus (SM-CMV) is a betaherpesvirus that was initially isolated from the salivary gland of a Guyanese squirrel monkey in 1980 and is now designated Saimiriine herpesvirus 4.^51,99 No clinical disease has been attributed to this virus that is considered ubiquitous in squirrel monkey colonies.⁹⁹ SM-CMV-free animals were nursery-reared from birth and maintained for more than 6 y but quickly seroconverted when housed with seropositive monkeys.⁹⁹ According to proposed primate cytomegalovirus phylogenetic trees, primate cytomegaloviruses may not be exclusively species-specific and may have the potential to be transmitted horizontally to phylogenetically related hosts (for example, among neotropical primates).⁶⁹ Although this virus has garnered little attention for more than 30 y, a recent investigation of 3 free-ranging black howler monkeys found dead from yellow fever reported the detection of SM-CMV antibodies, a finding of unclear significance.³⁵ From a global perspective, human cytomegalovirus is the most common cause of congenital viral infections. Additional study of SM-CMV likely will advance our knowledge of the pathogenesis of cytomegalovirus, and squirrel monkeys might serve as a useful model in the development of a cytomegalovirus vaccine.¹⁰⁶

Squirrel monkey polyomavirus (SMPyV)

Initially described in 2007, SMPyV was the first polyomavirus detected in New World primates.¹²⁴ The complete SMPyV genome was amplified from archived Bolivian squirrel monkey spleen DNA, and SMPyV DNA sequences subsequently were detected in 5 of 10 available squirrel monkey specimens.¹²⁴ SMPyV is not known to be pathogenic in the host species.¹²⁴ The mode of transmission of SMPyV remains unclear.¹²³ The human analogs of SMPyV are BK virus and JC virus, which exist as persistent infections, with seroprevalences of 82% and 39%, respectively.⁵⁹ Infections with BK virus and JC virus are asymptomatic in healthy people, but reactivation can occur in immunocompromised persons, such as AIDS patients and organ-transplant recipients, and lead to severe complications. Reactivation of BK virus can irreversibly damage the kidneys due to polyomavirus-associated nephropathy, and JC virus can cause progressive multifocal leukoencephalopathy, a fatal, demyelinating neurologic disease.¹²² Discovery of a New World polyomavirus in squirrel monkeys has opened a door for research on the human polyomaviruses, and studies on SMPyV at the Squirrel Monkey Breeding and Research Resource have already yielded new insights into the natural history of this virus.¹¹⁷ Additional research is needed to develop SMPyV-infected squirrel monkeys as a model of human polyomavirus disease and to understand the epidemiology and clinical implications of SMPyV in its natural host.

Retroviruses

Squirrel monkey retrovirus (SMRV).

SMRV initially was isolated from squirrel monkey lung tissue and described in 1977;^18,43 SMRV has also been detected in squirrel monkey placenta.¹⁰⁸ A member of the betaretrovirus genus, SMRV is characterized as a type D retrovirus.^51,91 Endogenous retroviruses are unique among animal viruses in that they are transmitted genetically: SMRV is transmitted among squirrel monkeys from parent to offspring, and multiple copies occur in the squirrel monkey genome.^14,107 Two type D retroviruses, SMRV and Mason–Pfizer monkey virus, which affects rhesus monkeys, are related evolutionarily, suggesting that a progenitor of type D retroviruses became genetically associated with primates at a very early time during their evolution.⁴⁵ Mason–Pfizer monkey virus was recovered from a rhesus monkey with spontaneous breast adenocarcinoma and has been considered to be an oncogenic retrovirus;¹¹⁸ SMRV has yet to demonstrate oncogenic properties. The identification of a type D retrovirus with close homology to Mason–Pfizer monkey virus in the B cell lymphomas of a patient with AIDS indicates that similar viruses may infect humans in various circumstances.^9,39 Unlike human endogenous retroviruses, SMRV produces infectious virions that are believed to be capable of infecting human cells.^111,121 The coexistence of HVS and SMRV in a single permissive cell supports the idea of herpesvirus–retrovirus interactions in lymphoproliferative diseases such as Burkitt lymphoma and Marek disease.^63,96,108 It has been theorized that one virus may upregulate the expression of the other virus, but data to support this hypothesis are unavailable as yet. PCR-based studies have detected SMRV in the LCL-HO human lymphoblastoid cell line and in 5 subclones of the Namalwa human lymphoblastoid cell line, including several used to produce interferon for human use.^97,121 By using nested PCR analysis, SMRV DNA was detected in some commercially available interferon preparations, but no untoward effect of this contamination have been documented.⁹⁷ Retroviruses are responsible for diverse human diseases, including AIDS (HIV) and T-cell leukemias, lymphomas, and spastic paraparesis (HTLV1). SMRV is of recent interest due to its similarity to a simian retrovirus that causes simian AIDS in macaques; whether SMRV causes an equivalent syndrome in squirrel monkeys is unknown.^70,118 Additional study of SMRV may offer new insights into human retroviruses and yield a useful model for the development of antiretroviral drugs.

Simian foamy virus (SFV) of squirrel monkeys.

Evolutionary comparisons suggest that foamy viruses are the oldest known retroviruses and that they coevolved with their hosts beginning more than 100 million years ago.^55,102,115 These unusual retroviruses are endemic in diverse mammals, including bovines, felines, equines, and nonhuman primates.¹⁰¹ Nonhuman primate foamy viruses currently are grouped together as simian foamy viruses within the spumavirus genus.⁸⁶ SFV are widespread in both Old and New World primates and were first described in 1954 in human tissue culture after what was later determined to be zoonotic transmission from a chimpanzee.^32,60,86 SFV infections in humans are uncommon, with zoonotic transmission to 1% to 4% of the people exposed occupationally in zoos, primate centers, and laboratories.⁸⁵ SFV replicate in oral mucosa and are thought to be transmitted to humans primarily through bites from infected animals.^85,104 SFV infections have developed in people bitten by Old World primates, including mandrills, gorillas, and chimpanzees, and latent infections can persist for years in the peripheral blood mononuclear cells and saliva of those infected.^10,85,104 Zoonotic transmission of SFV from New World primates is believed to occur as well, given that several people with exposure to New World primates have been seropositive for an SFV acquired from spider monkeys.¹¹² However, because none of the spider monkey SFV-seropositive persons had detectable levels of spider monkey SFV DNA in their blood, to date there is no evidence of long-term SFV persistence in humans after exposure to New World primates.¹¹² The potential for secondary transmission of SFV between humans is unclear but appears to be low: a limited longitudinal study of 7 men with documented SFV infections did not reveal any secondary transmissions, and no other human-to-human transmissions have been reported.¹⁰

Among New World primates, 8 distinct lineages of SFV have been described in 23 different species.⁸⁶ Cross-species transmission of SFV in New World primates has been documented to occur in captive animals but is not believed to be a common occurrence.⁸⁶ A recent study of SFV from New World primates revealed that TRIM5α may function as a species-specificity factor that is responsible for the species-specific restriction of SFV among New World primates.⁹³ This finding suggests that the TRIM5α protein and SFV may have exerted evolutionary pressure on each other.

SFV are cytotoxic in cell culture, causing the formation of large syncytium and characteristic vacuoles that give foamy viruses their name. As yet, no specific disease is attributed to infection with squirrel monkey foamy virus or any other foamy virus.^4,85 In a recent study of the influence of SFV on SIV disease in rhesus macaques, preexisting SFV infection was associated with an increased SIV plasma viral load, increased loss of CD4⁺ T cells, and a decreased survival rate.¹⁶ This observation may have important implications for the interpretation of data generated amid the complex immune dysregulation of simian AIDS models.

Conclusions

The use of squirrel monkeys as models for studying infectious disease transmission, immune responses, and pathology has improved our understanding of numerous human diseases. The presence of human analogs of persistent viruses in squirrel monkeys and the availability of the virologic techniques necessary to study these viruses offer unique opportunities to identify immunologic and virologic factors that control virus persistence and to compare the pathogenic potential of the squirrel monkey viruses with that of their human analogs. Although the pathogenic potential of endemic squirrel monkey viruses during immune suppression remains unknown, the availability of novel immunosuppressive reagents provides the opportunity to induce precise perturbations in the immune system of squirrel monkeys and thus perhaps evoke opportunistic diseases similar to those encountered in the growing population of immune-modulated persons. Such studies are essential for the development of immunomodulatory strategies to control persistent viruses during necessary therapeutic immunosuppression in humans. Continued characterization of endemic squirrel monkey viruses is essential to avoid potential confounding results due to the use of experimental animals with different underlying viral infections.