DETECTION OF SARS-COV-2 IN A SQUIRREL MONKEY (SAIMIRI SCIUREUS): A ONE HEALTH INVESTIGATION AND RESPONSE.

Through collaborative efforts, One Health partners have responded to outbreaks of COVID-19 among animals, including those in human care at zoos. Zoos have been faced with numerous challenges, including the susceptibility of many mammalian species, and therefore the need to heighten biosecurity measures rapidly. Robust One Health collaborations already exist in Arizona to address endemic and emerging zoonoses, but these have rarely included zoos. The pandemic shed light on this, and Arizona subsequently expanded its SARS-CoV-2 surveillance efforts to include zoo animals. Testing and epidemiologic support was provided to expedite the detection of and response to zoonotic SARS-CoV-2 infection in zoo animals, as well as to understand possible transmission events. Resulting from this program, SARS-CoV-2 was detected from a rectal swab collected from an 8-yr-old squirrel monkey (Saimiri sciureus) from a zoo in Southern Arizona. The animal had rapidly become ill with nonrespiratory symptoms and died in July 2022. Genomic sequencing from the swab revealed mutations consistent with the Omicron (BA.2) lineage. An epidemiologic investigation identified an animal caretaker in close proximity to the affected squirrel monkey who tested positive for COVID-19 the same day the squirrel monkey died. Critical One Health partners provided support to the zoo through engagement of local, state, and federal agencies. Necropsy and pathologic evaluation showed significant necrotizing colitis; the overall clinical and histopathological findings did not implicate SARS-CoV-2 infection alone as a causal or contributing factor in the squirrel monkey's illness and death. This report documents the first identification of SARS-CoV-2 in a squirrel monkey and highlights a successful and timely One Health investigation conducted through multisectoral collaboration.

Prenatal disorders and congenital Zika syndrome in squirrel monkeys.

During the Zika virus (ZIKV) outbreak in Brazil (2015-2016), the clinical manifestations associated with its infection were complex and included miscarriage and congenital malformations, not previously described. In this study, we evaluated the prenatal conditions of pregnant female squirrel monkeys (Saimiri collinsi) infected during different gestational thirds (GTs) and assessed all clinical aspects, diagnostic imaging, viremia and the immune response. In our study, 75% of the infected animals in the 1st GT group had significant clinical manifestations, such as miscarriage and prolonged viremia associated with a late immune response. Consequently, their neonates showed fetal neuropathology, such as cerebral hemorrhage, lissencephaly or malformations of the brain grooves, ventriculomegaly, and craniofacial malformations. Thus, our study demonstrated the relevance of pregnant squirrel monkeys as a model for the study of ZIKV infection in neonates due to the broad clinical manifestations presented, including the typical congenital Zika syndrome manifestations described in humans.

Experimental yellow fever virus infection in the squirrel monkey (Saimiri spp.) I: gross anatomical and histopathological findings in organs at necropsy.

BACKGROUND: Non-human primates contribute to the spread of the yellow fever virus (YFV) and the establishment of transmission cycles in endemic areas. OBJECTIVE: To describe the severe histopathological aspects of YFV infection, 10 squirrel monkeys were infected with YFV and blood, brain, liver, kidney, spleen, heart, lung, lymph node and stomach were collected at 1-7, 10, 20 and 30 days post-infection (dpi). METHODS: Histopathological analysis and detection of the genome and viral antigens and neutralising antibodies were performed by RT-PCR, immunohistochemistry and neutralisation test, respectively. FINDINGS: Only one animal died from the experimental infection. The genome and viral antigens were detected in all investigated organs (1-30 dpi) and the neutralising antibodies from seven to 30 dpi. The brain contained perivascular haemorrhage (6 dpi); in the liver, midzonal haemorrhage and lytic necrosis (6 dpi) were observed. The kidney had bleeding in the Bowman's capsule and tubular necrosis (6 dpi). Pyknotic lymphocytes were observed in the spleen (1-20 dpi), the lung had haemorrhage (2-6 dpi), in the endocardium it contained nuclear pyknosis and necrosis (2-3 dpi) and the stomach contained blood in the lumen (6 dpi). MAIN FINDINGS: Squirrel monkeys reliably reproduced the responses observed in human cases of yellow fever and, therefore, constitute an excellent experimental model for studies on the pathophysiology of the disease.

Divergent Traits and Ligand-Binding Properties of the Cytomegalovirus CD48 Gene Family.

The genesis of gene families by the capture of host genes and their subsequent duplication is a crucial process in the evolution of large DNA viruses. CD48 is a cell surface molecule that interacts via its N-terminal immunoglobulin (Ig) domain with the cell surface receptor 2B4 (CD244), regulating leukocyte cytotoxicity. We previously reported the presence of five CD48 homologs (vCD48s) in two related cytomegaloviruses, and demonstrated that one of them, A43, binds 2B4 and acts as a soluble CD48 decoy receptor impairing NK cell function. Here, we have characterized the rest of these vCD48s. We show that they are highly glycosylated proteins that display remarkably distinct features: divergent biochemical properties, cellular locations, and temporal expression kinetics. In contrast to A43, none of them interacts with 2B4. Consistent with this, molecular modeling of the N-terminal Ig domains of these vCD48s evidences notable changes as compared to CD48, suggesting that they interact with alternative targets. Accordingly, we demonstrate that one of them, S30, tightly binds CD2, a crucial T- and NK-cell adhesion and costimulatory molecule. Thus, our findings show how a key host immune receptor gene captured by a virus can be subsequently remodeled to evolve new immunoevasins with altered binding properties.

Epidemiological and molecular characterization of a novel adenovirus of squirrel monkeys after fatal infection during immunosuppression.

Adenoviruses are a frequent cause of acute upper respiratory tract infections that can also cause disseminated disease in immunosuppressed patients. We identified a novel adenovirus, squirrel monkey adenovirus 1 (SqMAdV-1), as the cause of fatal infection in an immunocompromised squirrel monkey (Saimiri boliviensis) at the Keeling Center for Comparative Medicine and Research (KCCMR). Sequencing of SqMAdV-1 revealed that it is most closely related (80.4 % pairwise nucleotide identity) to the titi monkey (Plecturocebus cupreus) adenovirus (TMAdV). Although identified in the titi monkey, TMAdV is highly lethal in these monkeys, and they are not thought to be the natural host. While SqMAdV-1 is similar to other primate adenoviruses in size and genomic characteristics, a nucleotide polymorphism at the expected stop codon of the DNA polymerase gene results in a 126 amino acid extension at the carboxy terminus, a feature not previously observed among other primate adenoviruses. PCR testing and partial sequencing of 95 archived faecal samples from other squirrel monkeys (Saimiri boliviensis and Saimiri sciureus) housed at the KCCMR revealed the presence of three distinct, and apparently endemic species of adenoviruses. A grouping of ten squirrel monkey adenovirus variants has high similarity to SqMAdV-1. A single adenovirus variant (designated SqMAdV-3), detected in five monkeys, has similarity to tufted capuchin (Sapajus apella) adenoviruses. The largest group of adenovirus variants detected (designated SqMAdV-2.0-2.16) has very high similarity (93-99 %) to the TMAdV, suggesting that squirrel monkeys may be the natural host of the TMAdV.

Experimental yellow fever virus infection in the squirrel monkey (Saimiri spp. ) I: gross anatomical and histopathological findings in organs at necropsy

BACKGROUND Non-human primates contribute to the spread of the yellow fever virus (YFV) and the establishment of transmission cycles in endemic areas. OBJECTIVE To describe the severe histopathological aspects of YFV infection, 10 squirrel monkeys were infected with YFV and blood, brain, liver, kidney, spleen, heart, lung, lymph node and stomach were collected at 1-7, 10, 20 and 30 days post-infection (dpi). METHODS Histopathological analysis and detection of the genome and viral antigens and neutralising antibodies were performed by RT-PCR, immunohistochemistry and neutralisation test, respectively. FINDINGS Only one animal died from the experimental infection. The genome and viral antigens were detected in all investigated organs (1-30 dpi) and the neutralising antibodies from seven to 30 dpi. The brain contained perivascular haemorrhage (6 dpi); in the liver, midzonal haemorrhage and lytic necrosis (6 dpi) were observed. The kidney had bleeding in the Bowman's capsule and tubular necrosis (6 dpi). Pyknotic lymphocytes were observed in the spleen (1-20 dpi), the lung had haemorrhage (2-6 dpi), in the endocardium it contained nuclear pyknosis and necrosis (2-3 dpi) and the stomach contained blood in the lumen (6 dpi). MAIN FINDINGS Squirrel monkeys reliably reproduced the responses observed in human cases of yellow fever and, therefore, constitute an excellent experimental model for studies on the pathophysiology of the disease.

Methods for detecting Zika virus in feces: A case study in captive squirrel monkeys (Saimiri boliviensis boliviensis).

A strain of Zika virus (ZIKV) of Asian origin associated with birth defects and neurological disorders has emerged and spread through the Americas. ZIKV was first isolated in the blood of nonhuman primates in Africa and has been detected in the blood, saliva, and urine of a few catarrhine species in both Africa and Asia, suggesting that nonhuman primates may serve as both a source and a reservoir of the virus. The recent introduction of ZIKV to human populations in the Americas presents the potential for the virus to spread into nonhuman primate reservoirs. Thus, it is critical to develop efficient and noninvasive detection methods to monitor the spread of the virus in wild nonhuman primate populations. Here, we describe a method for ZIKV detection in noninvasively collected fecal samples of a Neotropical primate. Fecal samples were collected from two captive squirrel monkeys (Saimiri boliviensis boliviensis) that were experimentally infected with ZIKV (Strain Mexico_1_44) and an additional two uninfected squirrel monkeys. Nucleic acids were extracted from these samples, and RT-qPCR was used to assay for the presence of ZIKV using primers flanking a 101 bp region of the NS5 gene. In both ZIKV-inoculated animals, ZIKV was detected 5-11 days post-infection, but was not detected in the uninfected animals. We compare the fecal results to ZIKV detection in serum, saliva, and urine samples from the same individuals. Our results indicate that fecal detection is a cost-effective, noninvasive method for monitoring wild populations of Neotropical primates as possible ZIKV reservoirs.

Cell-Derived Viral Genes Evolve under Stronger Purifying Selection in Rhadinoviruses.

Like many other large double-stranded DNA (dsDNA) viruses, herpesviruses are known to capture host genes to evade host defenses. Little is known about the detailed natural history of such genes, nor do we fully understand their evolutionary dynamics. A major obstacle is that they are often highly divergent, maintaining very low sequence similarity to host homologs. Here we use the herpesvirus genus Rhadinovirus as a model system to develop an analytical approach that combines complementary evolutionary and bioinformatic techniques, offering results that are both detailed and robust for a range of genes. Using a systematic phylogenetic strategy, we identify the original host lineage of viral genes with high confidence. We show that although host immunomodulatory genes evolve rapidly compared to other host genes, they undergo a clear increase in purifying selection once captured by a virus. To characterize this shift in detail, we developed a novel technique to identify changes in selection pressure that can be attributable to particular domains. These findings will inform us on how viruses develop strategies to evade the immune system, and our synthesis of techniques can be reapplied to other viruses or biological systems with similar analytical challenges.IMPORTANCE Viruses and hosts have been shown to capture genes from one another as part of the evolutionary arms race. Such genes offer a natural experiment on the effects of evolutionary pressure, since the same gene exists in vastly different selective environments. However, sequences of viral homologs often bear little similarity to the original sequence, complicating the reconstruction of their shared evolutionary history with host counterparts. In this study, we use a genus of herpesviruses as a model system to comprehensively investigate the evolution of host-derived viral genes, using a synthesis of genomics, phylogenetics, selection analysis, and nucleotide and amino acid modeling.

Experimental Zika Virus Infection of Neotropical Primates.

The establishment of a sylvatic reservoir of Zika virus (ZIKV) in the Americas is dependent on the susceptibility of primates of sufficient population density, the duration and magnitude of viremia, and their exposure to the human mosquito-borne transmission cycle. To assess the susceptibility of squirrel (Saimiri sp.) and owl monkeys (Aotus sp.) to infection, we inoculated four animals of each species with ZIKV from the current epidemic. Viremia in the absence of detectible disease was observed in both species and seroconversion occurred by day 28. ZIKV was detected in the spleen of three owl monkeys: one at 7 days postinoculation (dpi) and two at 14 dpi. This study confirms the susceptibility to ZIKV infection of two Neotropical primate species that live in close proximity to humans in South America, suggesting that they could support a widespread sylvatic ZIKV cycle there. Collectively, establishment of a ZIKV sylvatic transmission cycle in South America would imperil eradication efforts and could provide a mechanism for continued exposure of humans to ZIKV infection and disease.

Signaling Lymphocytic Activation Molecule Family Receptor Homologs in New World Monkey Cytomegaloviruses.

UNLABELLED: Throughout evolution, large DNA viruses have been usurping genes from their hosts to equip themselves with proteins that restrain host immune defenses. Signaling lymphocytic activation molecule (SLAM) family (SLAMF) receptors are involved in the regulation of both innate and adaptive immunity, which occurs upon engagement with their ligands via homotypic or heterotypic interactions. Here we report a total of seven SLAMF genes encoded by the genomes of two cytomegalovirus (CMV) species, squirrel monkey CMV (SMCMV) and owl monkey CMV (OMCMV), that infect New World monkeys. Our results indicate that host genes were captured by retrotranscription at different stages of the CMV-host coevolution. The most recent acquisition led to S1 in SMCMV. S1 is a SLAMF6 homolog with an amino acid sequence identity of 97% to SLAMF6 in its ligand-binding N-terminal Ig domain. We demonstrate that S1 is a cell surface glycoprotein capable of binding to host SLAMF6. Furthermore, the OMCMV genome encodes A33, an LY9 (SLAMF3) homolog, and A43, a CD48 (SLAMF2) homolog, two soluble glycoproteins which recognize their respective cellular counterreceptors and thus are likely to be viral SLAMF decoy receptors. In addition, distinct copies of further divergent CD48 homologs were found to be encoded by both CMV genomes. Remarkably, all these molecules display a number of unique features, including cytoplasmic tails lacking characteristic SLAMF signaling motifs. Taken together, our findings indicate a novel immune evasion mechanism in which incorporation of host SLAMF receptors that retain their ligand-binding properties enables viruses to interfere with SLAMF functions and to supply themselves with convenient structural molds for expanding their immunomodulatory repertoires. IMPORTANCE: The way in which viruses shape their genomes under the continual selective pressure exerted by the host immune system is central for their survival. Here, we report that New World monkey cytomegaloviruses have broadly captured and duplicated immune cell receptors of the signaling lymphocyte activation molecule (SLAM) family during host-virus coevolution. Notably, we demonstrate that several of these viral SLAMs exhibit exceptional preservation of their N-terminal immunoglobulin domains, which results in maintenance of their ligand-binding capacities. At the same time, these molecules present distinctive structural properties which include soluble forms and the absence of typical SLAM signaling motifs in their cytoplasmic domains, likely reflecting the evolutionary adaptation undergone to efficiently interfere with host SLAM family activities. The observation that the genomes of other large DNA viruses might bear SLAM family homologs further underscores the importance of these molecules as a novel class of immune regulators and as convenient scaffolds for viral evolution.

Endemic Viruses of Squirrel Monkeys (Saimiri spp.).

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.

Detection of EBV, HBV, HCV, HIV-1, HTLV-I and -II, and SMRV in human and other primate cell lines.

The high prevalence of contaminated cell cultures suggests that viral contaminations might be distributed among cultures. We investigated more than 460 primate cell lines for Epstein-Barr (EBV), hepatitis B (HBV), hepatitis C (HCV), human immunodeficiency virus type 1 (HIV-1), human T-cell leukemia/lymphoma virus I and II (HTLV-I/-II), and squirrel monkey retrovirus (SMRV) infections for risk assessment. None of the cell lines were infected with HCV, HIV-1, or HTLV-I/-II. However, one cell line displayed reverse transcriptase activity. Thirty-nine cell lines harbored EBV DNA sequences. Studies on the lytic phase of EBV revealed that five cell lines produce EBV particles and six further cell lines produced EBV upon stimulation. One cell line contained an integrated HBV genome fragment but showed no virus production. Six cell lines were SMRV-infected. Newly established cell lines should be tested for EBV infections to detect B-lymphoblastoid cell lines (B-LCL). B-LCLs established with EBV from cell line B95-8 should be tested for SMRV infections.

Experimental infection of squirrel monkeys with nipah virus.

We infected squirrel monkeys (Saimiri sciureus) with Nipah virus to determine the monkeys' suitability for use as primate models in preclinical testing of preventive and therapeutic treatments. Infection of squirrel monkeys through intravenous injection was followed by high death rates associated with acute neurologic and respiratory illness and viral RNA and antigen production.

Species-specific inhibition of foamy viruses from South American monkeys by New World Monkey TRIM5{alpha} proteins.

Foamy virus evolution closely parallels that of the host species, indicating virus-host coadaptation. We studied simian foamy viruses (SFVs) from common marmosets, spider monkeys, and squirrel monkeys, New World monkey (NWM) species that share geographic ranges. The TRIM5alpha protein from each of these NWM species inhibited the replication of at least one of the SFVs associated with the other two species but did not affect the replication of its own SFV. Thus, TRIM5alpha has potentially shaped the evolution of SFVs in NWM hosts. Conversely, SFVs may have influenced the evolution of TRIM5 variants in New World primates.

Mapping the minimal regions within the ORF73 protein required for herpesvirus saimiri episomal persistence.

Herpesvirus saimiri (HVS) establishes a persistent infection in which the viral genome persists as a circular non-integrated episome. ORF73 tethers HVS episomes to host mitotic chromosomes, allowing episomal persistence via an interaction with the chromosome-associated protein, MeCP2. Here we demonstrate that ORF73 also interacts with the linker histone H1 via its C terminus, suggesting it associates with multiple chromosome-associated proteins. In addition, we show that the C terminus is also required for the ability of ORF73 to bind the terminal repeat region of the HVS genome. These results suggest that the ORF73 C terminus contains all the necessary elements required for HVS episomal persistence. Using a range of ORF73 C terminus deletions to rescue the episomal maintenance properties of a HVSDelta73 recombinant virus, we show that a C terminus region comprising residues 285-407 is sufficient to maintain the HVS episome in a dividing cell population.

Molecular characterization of the first polyomavirus from a New World primate: squirrel monkey polyomavirus.

DNA samples from a variety of New World monkeys were screened by using a broad-spectrum PCR targeting the VP1 gene of polyomaviruses. This resulted in the characterization of the first polyomavirus from a New World primate. This virus naturally infects squirrel monkeys (Saimiri sp.) and is provisionally named squirrel monkey polyomavirus (SquiPyV). The complete genome of SquiPyV is 5,075 bp in length, and encodes the small T and large T antigens and the three structural proteins VP1, VP2 and VP3. Interestingly, the late region also encodes a putative agnoprotein, a feature that it shares with other polyomaviruses from humans, baboons and African green monkeys. Comparison with other polyomaviruses revealed limited sequence similarity to any other polyomavirus, and phylogenetic analysis of the VP1 gene confirmed its uniqueness.

Squirrel monkeys support replication of BK virus more efficiently than simian virus 40: an animal model for human BK virus infection.

We performed experiments to test the suitability of squirrel monkeys (Saimiri sciureus) as an experimental model for BK virus (BKV) and simian virus 40 (SV40) infection. Four squirrel monkeys received intravenous inoculation with BKV Gardner strain, and six squirrel monkeys received intravenous inoculation with SV40 777 strain. Eight of 10 monkeys received immunosuppression therapy, namely, cyclophosphamide subcutaneously either before or both before and after viral inoculation. The presence of viral infection was assessed by quantitative real-time PCR amplification of viral DNA from blood, urine, and 10 tissues. We found that squirrel monkeys were susceptible to infection with BKV, with high viral copy number detected in blood and viral genome detected in all tissues examined. BKV genome was detected in urine from only one monkey, while three monkeys manifested focal interstitial nephritis. BKV T antigen was expressed in renal peritubular capillary endothelial cells. By contrast, SV40 was detected at very low copy numbers in only a few tissues and was not detected in blood. We conclude that the squirrel monkey is a suitable animal for studies of experimental BKV infection and may facilitate studies of viral entry, pathogenesis, and therapy.

The genome of herpesvirus saimiri C488 which is capable of transforming human T cells.

Herpesvirus saimiri (HVS), the rhadinovirus prototype, is apathogenic in the persistently infected natural host, the squirrel monkey, but causes acute T cell leukemia in other New World primate species. In contrast to subgroups A and B, only strains of HVS subgroup C such as C488 are capable of transforming primary human T cells to stable antigen-independent growth in culture. Here, we report the complete 155-kb genome sequence of the transformation-competent HVS strain C488. The A+T-rich unique L-DNA of 113,027 bp encodes at least 77 open reading frames and 5 URNAs. In addition to the viral oncogenes stp and tip, only a few genes including the transactivator orf50 and the glycoprotein orf51 are highly divergent. In a series of new primary HVS isolates, the subgroup-specific divergence of the orf50/orf51 alleles was studied. In these new isolates, the orf50/orf51 alleles of the respective subgroup segregate with the stp and/or tip oncogene alleles, which are essential for transformation.

Endogenous retrovirus HC2 pol fragments in the squirrel monkey: expression, evolution, and phylogeny.

Human endogenous retrovirus HC2 is an incomplete provirus containing the entire gag and pol genes and a 3' LTR, whereas the 5' LTR and env gene are missing. We investigated expression of the HC2 pol gene in the squirrel monkey ( Saimiri sciureus) by RT-PCR. The pol gene was expressed in cerebellum, liver, lung, and spleen of the squirrel monkey, but not in six other tissues tested. RT-PCR products were cloned and sequenced resulting in seven sequences that were analyzed. These sequences showed 73.7-89.2% sequence similarity to HC2 pol genes present in the human genome. No frameshifts or termination codons caused by deletion/insertion or point mutation were found in clones SM-HC27-1 and SM-HC27-4 isolated from squirrel monkey lung tissues. Phylogenetic analysis showed that HC2 pol elements from the squirrel monkey were randomly clustered with those in human genome and the genomes of other nonhuman primates, indicating that substantial evolution of the HC2 elements occurred prior to primate speciation with additional evolution of the elements, independent of each other, after speciation.