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.

Susceptibility and lack of evidence for a viremic state of rabies in the night owl monkey, Aotus nancymaae.

BACKGROUND: Rabies causes an acute fatal encephalomyelitis in most mammals following infection with rhabdovirus of the genus Lyssavirus. Little is known about rabies virus infection in species of New World non-human Primates (NHP). To investigate the suitability of the owl monkey Aotus nancymaae asissue sections examined were unremarkable for inflammation or other histologic signs of rabies a viable animal model for rabies virus candidate vaccine testing, we used clinical presentation, serology, viral isolation, and PCR to evaluate the incubation period, immunity, and pathogenesis of infected animals. We tested the hypothesis that no viremic state exists for rabies virus. METHODS: Eight monkeys divided into two equal groups were inoculated intramuscularly either in the neck or footpad with 105 pfu of rabies virus (Pasteur/V-13R) and observed for >130 days. Oral and blood samples were collected and analyzed. RESULTS: Two monkeys inoculated in the neck displayed classic paralytic rabies. The mean incubation period was 11.5 days. The average maximum IgG response (antibody titer >0.200 O.D.) was achieved at day 10.0 and 62.3 in the clinical rabies and non-clinical rabies cases, respectively (p = 0.0429). No difference in IgM or IgG time to seroconversion or average maximum IgM level was observed between neck versus footpad inoculation groups. No viremia or viral shedding was detected by PCR or viral isolation during the observation period, including within the two symptomatic animals three days after disease onset. Tissue sections examined were unremarkable for inflammation or other histologic signs of rabies within the asymptomatic animal. Similarly none of the brain sections exhibited immunoreactivity for rabies virus antibody. DISCUSSION: This study demonstrates there is no difference in time to immune response between inoculation sites and distance to the brain; however, immune response tends to be more rapid in cases of clinically apparent disease and prolonged in cases infected at sites further from the brain. CONCLUSIONS: Our findings support the hypothesis that a viremic state for rabies does not exist in the New World Monkey, Aotus nancymaae, and it appears that this species may be refractory to infection. The species does provide a suitable model to assess post infection immune responses. Additional studies that address the limitations of sample size, length of observation, and lack of measurable infection should be conducted.

A New World primate deficient in tetherin-mediated restriction of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) does not replicate in primary cells of New World primates. To better understand this restriction, we expressed owl monkey (Aotus nancymaae) CD4 and CXCR4 in the owl monkey kidney cell line, OMK. An HIV-1 variant modified to evade the owl monkey restriction factor TRIM-cyp replicated efficiently in these cells but could not replicate in primary A. nancymaae CD4-positive T cells. To understand this difference, we examined APOBEC3G and tetherin orthologs from OMK cells and primary A. nancymaae cells. We observed that OMK cells expressed substantially lower levels of APOBEC3G than did A. nancymaae cells. A. nancymaae, but not marmoset (Callithrix jacchus), APOBEC3G was partially downregulated by HIV-1 vif and reduced but did not abolish HIV-1 replication when stably expressed in OMK cells. The functional difference between A. nancymaae and marmoset APOBEC3Gs mapped to residue 128, previously shown to distinguish African green monkey from human APOBEC3G. We also characterized tetherin orthologs from OMK and A. nancymaae cells. The A. nancymaae tetherin ortholog, but not OMK tetherin, prevented HIV-1 release. Alteration of threonine 181 of OMK tetherin rescued its function and its efficient N glycosylation. All alleles of Aotus lemurinus griseimembra examined, but none of A. nancymaae or Aotus vociferans, encoded this nonfunctional tetherin ortholog. Our data indicate that HIV-1 replication in owl monkeys is not restricted at entry but can be limited by APOBEC3G and tetherin. Further, A. lemurinus griseimembra does not restrict HIV-1 replication via tetherin, a property likely useful for the study of tetherin-restricted viruses.

Cyclophilin A retrotransposition into TRIM5 explains owl monkey resistance to HIV-1.

In Old World primates, TRIM5-alpha confers a potent block to human immunodeficiency virus type 1 (HIV-1) infection that acts after virus entry into cells. Cyclophilin A (CypA) binding to viral capsid protects HIV-1 from a similar activity in human cells. Among New World primates, only owl monkeys exhibit post-entry restriction of HIV-1 (ref. 1). Paradoxically, the barrier to HIV-1 in owl monkey cells is released by capsid mutants or drugs that disrupt capsid interaction with CypA. Here we show that knockdown of owl monkey CypA by RNA interference (RNAi) correlates with suppression of anti-HIV-1 activity. However, reintroduction of CypA protein to RNAi-treated cells did not restore antiviral activity. A search for additional RNAi targets unearthed TRIMCyp, an RNAi-responsive messenger RNA encoding a TRIM5-CypA fusion protein. TRIMCyp accounts for post-entry restriction of HIV-1 in owl monkeys and blocks HIV-1 infection when transferred to otherwise infectable human or rat cells. It seems that TRIMCyp arose after the divergence of New and Old World primates when a LINE-1 retrotransposon catalysed the insertion of a CypA complementary DNA into the TRIM5 locus. This is the first vertebrate example of a chimaeric gene generated by this mechanism of exon shuffling.

Lack of Evidence of Sylvatic Transmission of Dengue Viruses in the Amazon Rainforest Near Iquitos, Peru.

Dengue viruses (DENV) are currently responsible for more human morbidity and mortality than any other known arbovirus, and all four DENV are known to exist in sylvatic cycles that might allow these viruses to persist if the urban (Aedes aegypti) cycle could be controlled. To determine whether DENV were being maintained in a sylvatic cycle in a forested area about 14 km southwest of Iquitos, Peru, a city in which all 4 serotypes of DENV circulate, we placed 20 DENV seronegative Aotus monkeys in cages either in the canopy or near ground level for a total of 125.6 months. Despite capturing >66,000 mosquitoes in traps that collected some of the mosquitoes attracted to these monkeys, blood samples obtained once a month from each animal were tested and found to be negative by an enzyme-linked immunosorbent assay for IgM and IgG antibodies to dengue, yellow fever, Venezuelan equine encephalitis, Oropouche, and Mayaro viruses. Although all four DENV serotypes were endemic in nearby Iquitos, the findings of this study did not support a DENV sylvatic maintenance and transmission cycle in a selected area of the Amazon rainforest in northeastern Peru.

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.

The effect of exon 7 deletion during the evolution of TRIMCyp fusion proteins on viral restriction, cytoplasmic body formation and multimerization.

TRIMCyp is a fusion protein consisting of the TRIM5 gene product and retrotransposed Cyclophilin A (CypA). Two primate TRIMCyp fusion proteins with varying anti-HIV-1 activities independently evolved in owl monkeys and Old World monkeys. In addition, Old World monkey TRIMCyps lack exon7, which encodes amino acids in the Linker2 region. Previous studies on TRIM5α indicated that this region affects anti-retroviral activity, cytoplasmic body formation, and multimerization. The effects of exon7 deletion on the functions of the TRIMCyp are unclear. In this study, we found that the cytoplasmic bodies and multimers of owl monkey TRIMCyp (omTRIMCyp) are different from those of northern pig-tailed macaque TRIMCyp (npmTRIMCyp). In addition, we demonstrated that exon7 deletion affected cytoplasmic body formation and multimerization. Moreover, we unexpectedly found two chimeric proteins of omTRIMCyp and npmTRIMCyp that failed to block HIV-1 replication, despite the presence of CypA in omTRIMCyp. Further studies indicated that the cytoplasmic bodies and spontaneous multimerization were not responsible for TRIMCyp anti-HIV-1 activity. Moreover, potent viral restriction is associated with higher amounts of monomeric TRIMCyp when the CypA domain is able to recognize and bind to the HIV-1 capsid. Our results suggested that the deletion of exon7 during the evolution of TRIMCyp affected its function.

Susceptibility of the Aotus nancymaae owl monkey to eastern equine encephalitis.

Eastern equine encephalitis virus (EEEV) is an arthropod-borne virus associated with life-threatening encephalitis in humans, equines, birds and many other domestic animals. To investigate the suitability of the Aotus nancymaae New World owl monkey as a viable animal model for EEE candidate vaccine testing we used clinical presentation, serology, viral isolation and PCR to evaluate pathogenesis and immunity in infected animals. Monkeys were inoculated subcutaneously (SQ) or intranasally (IN) with 10(4)pfu of virulent EEEV and were initially followed for 45 days. While none of the animals displayed clinical signs of disease, all of the SC inoculated animals (n=6) manifested a viremia averaging 3.2 days (+/-0.8 days). Likewise, serologic responses (IgM, IgG and PRNT) were observed in all SC infected animals. Interestingly, none of the IN inoculated animals (n=6) became viremic or mounted an antibody response and no pathological abnormalities were observed in two animals that were necropsied on day 6 post-infection (p.i.) from each group. To determine if the antibodies produced by the SC inoculated animals were protective against homologous challenge, three animals from the SC group were serologically evaluated on day 253 p.i. and were administered an inoculum identical to initial challenge on day 270 p.i. A positive control group of four naïve animals was also infected as before. All of the naïve positive control animals manifested a similar viremia as observed initially, averaging 2.75 days (+/-0.5 days) while none of the previously challenged animals became viremic. On days 45 and 253 p.i. geometric mean PRNT titers in the SC group were 453 and 101, respectively. This study demonstrates that the Aotus nancymaae can be reproducibly infected with EEE virus and can serve as a suitable model for infection and immunogenicity for the evaluation of candidate vaccines against EEEV.

Structural requirements for recognition of the human immunodeficiency virus type 1 core during host restriction in owl monkey cells.

Human immunodeficiency virus type 1 (HIV-1) infection of simian cells is restricted at an early postentry step by host factors whose mechanism of action is unclear. These factors target the viral capsid protein (CA) and attenuate reverse transcription, suggesting that they bind to the HIV-1 core and interfere with its uncoating. To identify the relevant binding determinants in the capsid, we tested the capacity of viruses containing Gag cleavage site mutations and amino acid substitutions in CA to inhibit restriction of a wild type HIV-1 reporter virus in owl monkey cells. The results demonstrated that a stable, polymeric capsid and a correctly folded amino-terminal CA subunit interface are essential for saturation of host restriction in target cells by HIV-1 cores. We conclude that the owl monkey cellular restriction machinery recognizes a polymeric array of CA molecules, most likely via direct engagement of the HIV-1 capsid in target cells prior to uncoating.

Species-specific variants of GB virus A in captive monkeys .

Sequences from the putative 5' nontranslated region of GB virus A were isolated from mystax, owl monkeys, and tamarins. Though sequences of isolates from each animal species are virtually identical at the nucleotide level (95%), isolates from different species are dramatically different (52 to 79% identical) and genetically cluster on this basis.

Further observations on coronavirus infection of primate CNS.

Previously, we demonstrated that intracerebral (IC) inoculation of a murine coronavirus, MHV-JHM, into two species of primates can result in acute encephalomyelitis (Murray et al., 1992a). Infectious virus isolated from acutely infected animals, designated JHM-OMp1, was inoculated IC into a second group of monkeys. In this report we describe observations on the acutely infected animals and those surviving the acute infection were sacrificed at later times post-infection. Results from dual in situ hybridization/immunohistochemistry screening of tissues show that astrocytes are target cells in white matter lesions during acute infection. In animals sacrificed 150 days post-infection, areas of demyelinated gliotic lesions, prominent in the spinal cord, were seen throughout the neuraxis. No virus products were detected in these late-infection lesions.

HERV-H endogenous retroviruses: presence in the New World branch but amplification in the Old World primate lineage.

The evolutionary origin and age of the HERV-H family of human endogenous retrovirus-like sequences was investigated in this study. HERV-H elements exist in approximately 900 partially deleted copies and 50-100 more intact forms in humans and Old World monkeys. However, their possible presence in more divergent species is unknown. We have isolated a 1.6-kb genomic DNA segment from the New World monkey marmoset that had been PCR amplified using human HERV-H primers. DNA and protein comparisons and database searches indicate that this marmoset clone is more closely related to human HERV-H elements than to any other sequence, indicating that HERV-H-related sequences do exist in New World monkeys. In contrast to the high copy numbers of deleted elements in Old World primates. Southern blot analysis shows that such elements are present in less than 50 copies in two different species of New World monkey. To estimate evolutionary ages of the common deleted form of the element, a selected DNA segment from the pol region was compared from multiple human HERV-H elements. This comparison suggests that many HERV-H elements of the abundant deleted subfamily integrated approximately 30-35 million years ago. Very similar percentage divergence values between 5' and 3' long terminal repeats of individual elements of the deleted subfamily also suggest that these elements are close in age. These results indicate that HERV-H elements first appeared in the germline prior to the New World/Old World divergence over 40 million years ago. Interestingly, they remained in low numbers in the New World branch while a subfamily underwent a major amplification in Old World primates before the time of divergence of hominoids from Old World monkeys.

The natural history of hepatitis A: the potential for transmission by transfusion of blood or blood products.

A study of the natral history and risk factors for hepatitis A can shed light on the potential for contamination of plasma concentrate with hepatitis A virus (HAV). According to the long-term Sentinel Counties Study conducted by Alter and colleagues at the Centers for Disease Control and Prevention in Atlanta, the most frequently reported risk factors for HAV infection are living with a patient who has hepatitis, homosexual activity, and close contact with young children. International travel to hepatitis A endemic areas and illicit parenteral drug use were less frequently documented risk factors, although illicit injectable drug use has been considered more significant in other hepatitis A studies. Approximately 40% of patients with hepatitis A reported no apparent risk factors. Hepatitis A occurs most often today in the 5- to 30-year-old age group. Young adults, who are also eligible donors, are thus at risk of infection. The natural history of hepatitis A was studied in New World owl monkeys. Fecal shedding of infectious virus was detected by 4 days after intravenous injection of infectious material and peaked at almost 10 million infectious particles per gram of feces just prior to onset of chemical evidence of liver disease. Viremia of substantial magnitude occurred throughout most of the 4-week incubation period and was maximal during the prodromal stage, prior to the development of clinical, chemical, or serologic manifestations of infection. Although the magnitude of hepatitis A viremia has not been well documented in humans, it is likely to reach levels of 10(4)-10(6) infectious particles per milliliter of blood.(ABSTRACT TRUNCATED AT 250 WORDS)