Diverse Circular DNA Viral Communities in Blood, Oral, and Fecal Samples of Captive Lemurs.

Few studies have addressed viral diversity in lemurs despite their unique evolutionary history on the island of Madagascar and high risk of extinction. Further, while a large number of studies on animal viromes focus on fecal samples, understanding viral diversity across multiple sample types and seasons can reveal complex viral community structures within and across species. Groups of captive lemurs at the Duke Lemur Center (Durham, NC, USA), a conservation and research center, provide an opportunity to build foundational knowledge on lemur-associated viromes. We sampled individuals from seven lemur species, i.e., collared lemur (Eulemur collaris), crowned lemur (Eulemur coronatus), blue-eyed black lemur (Eulemur flavifrons), ring-tailed lemur (Lemur catta), Coquerel's sifaka (Propithecus coquereli), black-and-white ruffed lemur (Varecia variegata variegata), and red ruffed lemur (Varecia rubra), across two lemur families (Lemuridae, Indriidae). Fecal, blood, and saliva samples were collected from Coquerel's sifaka and black-and-white ruffed lemur individuals across two sampling seasons to diversify virome biogeography and temporal sampling. Using viral metagenomic workflows, the complete genomes of anelloviruses (n = 4), cressdnaviruses (n = 47), caudoviruses (n = 15), inoviruses (n = 34), and microviruses (n = 537) were determined from lemur blood, feces, and saliva. Many virus genomes, especially bacteriophages, identified in this study were present across multiple lemur species. Overall, the work presented here uses a viral metagenomics approach to investigate viral communities inhabiting the blood, oral cavity, and feces of healthy captive lemurs.

Endogenization of a Prosimian Retrovirus during Lemur Evolution.

Studies of viruses that coevolved with lemurs provide an opportunity to understand the basal traits of primate viruses and provide an evolutionary context for host-virus interactions. Germline integration of endogenous retroviruses (ERVs) are fossil evidence of past infections. Hence, characterization of novel ERVs provides insight into the ancient precursors of extant viruses and the evolutionary history of their hosts. Here, we report the discovery of a novel endogenous retrovirus present in the genome of a lemur, Coquerel's sifaka (Propithecus coquereli). Using next-generation sequencing, we identified and characterized the complete genome sequence of a retrovirus, named prosimian retrovirus 1 (PSRV1). Phylogenetic analyses indicate that PSRV1 is a gamma-type betaretrovirus basal to the other primate betaretroviruses and most closely related to simian retroviruses. Molecular clock analysis of PSRV1 long terminal repeat (LTR) sequences estimated the time of endogenization within 4.56 MYA (± 2.4 MYA), placing it after the divergence of Propithecus species. These results indicate that PSRV1 is an important milestone of lemur evolution during the radiation of the Propithecus genus. These findings may have implications for both human and animal health in that the acquisition of a gamma-type env gene within an endogenized betaretrovirus could facilitate a cross-species jump between vertebrate class hosts.

Virus discovery reveals frequent infection by diverse novel members of the Flaviviridae in wild lemurs.

Lemurs are highly endangered mammals inhabiting the forests of Madagascar. In this study, we performed virus discovery on serum samples collected from 84 wild lemurs and identified viral sequence fragments from 4 novel viruses within the family Flaviviridae, including members of the genera Hepacivirus and Pegivirus. The sifaka hepacivirus (SifHV, two genotypes) and pegivirus (SifPgV, two genotypes) were discovered in the diademed sifaka (Propithecus diadema), while other pegiviral fragments were detected in samples from the indri (Indri indri, IndPgV) and the weasel sportive lemur (Lepilemur mustelinus, LepPgV). Although data are preliminary, each viral species appeared host species-specific and frequent infection was detected (18 of 84 individuals were positive for at least one virus). The complete coding sequence and partial 5' and 3' untranslated regions (UTRs) were obtained for SifHV and its genomic organization was consistent with that of other hepaciviruses, with one unique polyprotein and highly structured UTRs. Phylogenetic analyses showed the SifHV belonged to a clade that includes several viral species identified in rodents from Asia and North America, while SifPgV and IndPgV were more closely related to pegiviral species A and C, that include viruses found in humans as well as New- and Old-World monkeys. Our results support the current proposed model of virus-host co-divergence with frequent occurrence of cross-species transmission for these genera and highlight how the discovery of more members of the Flaviviridae can help clarify the ecology and evolutionary history of these viruses. Furthermore, this knowledge is important for conservation and captive management of lemurs.

Life History of the Oldest Lentivirus: Characterization of ELVgv Integrations in the Dermopteran Genome.

Endogenous retroviruses are genomic elements formed by germline infiltration by originally exogenous viruses. These molecular fossils provide valuable information about the evolution of the retroviral family. Lentiviruses are an extensively studied genus of retroviruses infecting a broad range of mammals. Despite a wealth of information on their modern evolution, little is known about their origins. This is partially due to the scarcity of their endogenous forms. Recently, an endogenous lentivirus, ELVgv, was discovered in the genome of the Malayan colugo (order Dermoptera). This represents the oldest lentiviral evidence available and promises to lead to further insights into the history of this genus. In this study, we analyzed ELVgv integrations at several genomic locations in four distinct colugo specimens covering all the extant dermopteran species. We confirmed ELVgv integrations in all the specimens examined, which implies that the virus originated before the dermopteran diversification. Using a locus-specific dermopteran substitution rate, we estimated that the proviral integrations occurred 21-40 Ma. Using phylogenetic analysis, we estimated that ELVgv invaded an ancestor of today's Dermoptera in an even more distant past. We also provide evidence of selective pressure on the TRIM5 antiviral restriction factor, something usually taken as indirect evidence of past retroviral infections. Interestingly, we show that TRIM5 was under strong positive selection pressure only in the common dermopteran ancestor, where the ELVgv endogenization occurred. Further experiments are required to determine whether ELVgv participated in the TRIM5 selection.

Species-specific transmission of novel picornaviruses in lemurs.

UNLABELLED: The roles of host genetics versus exposure and contact frequency in driving cross-species transmission remain the subject of debate. Here, we used a multitaxon lemur collection at the Saint Louis Zoo in the United States as a model to gain insight into viral transmission in a setting of high interspecies contact. Lemurs are a diverse and understudied group of primates that are highly endangered. The speciation of lemurs, which are endemic to the island of Madagascar, occurred in geographic isolation apart from that of continental African primates. Although evidence of endogenized viruses in lemur genomes exists, no exogenous viruses of lemurs have been described to date. Here we identified two novel picornaviruses in fecal specimens of ring-tailed lemurs (Lemur catta) and black-and-white ruffed lemurs (Varecia variegata). We found that the viruses were transmitted in a species-specific manner (lesavirus 1 was detected only in ring-tailed lemurs, while lesavirus 2 was detected only in black-and-white ruffed lemurs). Longitudinal sampling over a 1-year interval demonstrated ongoing infection in the collection. This was supported by evidence of viral clearance in some animals and new infections in previously uninfected animals, including a set of newly born triplets that acquired the infection. While the two virus strains were found to be cocirculating in a mixed-species exhibit of ring-tailed lemurs, black-and-white ruffed lemurs, and black lemurs, there was no evidence of cross-species transmission. This suggests that despite high-intensity contact, host species barriers can prevent cross-species transmissions of these viruses. IMPORTANCE: Up to 75% of emerging infectious diseases in humans today are the result of zoonotic transmission. However, a challenge in understanding transmission dynamics has been the limited models of cross-species transmission. Zoos provide a unique opportunity to explore parameters defining viral transmission. We demonstrated that ongoing virus transmission in a mixed lemur species exhibit was species specific. This suggests that despite high contact intensity, host species barriers contribute to protection from cross-species transmission of these viruses. While the combinations of species might differ, most zoological parks worldwide commonly feature mixed-species exhibits. Collectively, this report demonstrates a widely applicable approach toward understanding infectious disease transmission.

A primitive endogenous lentivirus in a colugo: insights into the early evolution of lentiviruses.

Lentiviruses infect a wide range of mammal species. Much remains unknown about their deep history and host distribution. Here, we report the discovery of an endogenous lentivirus within the genome of the Sunda flying lemur (Galeopterus variegatus) (which we designate "Galeopterus variegatus endogenous lentivirus" [GvaELV]). We estimate the GvaELV genome invasion to have occurred more than 14 Ma, supporting an ancient origin of the lentivirus clade and an ancient lentiviral infection in colugo. Phylogenetic analyses show that GvaELV is a sister group of all previously known lentiviruses. The GvaELV genome appears to possess some primitive genomic features of a lentivirus, encoding not only a trans-activator of transcription (tat) gene but also two additional putative accessory genes that share no discernible similarity with other lentiviral accessory genes. The discovery of GvaELV provides novel insights into the prehistory and host distribution of lentivirus.

Structural and functional analysis of prehistoric lentiviruses uncovers an ancient molecular interface.

Lentiviruses are widespread in a variety of vertebrates, often associated with chronic disease states. However, until the recent discovery of the prehistoric endogenous lentiviruses in rabbits (RELIK) and lemurs (PSIV), it was thought that lentiviruses had no capacity for germline integration and were only spread horizontally in an exogenous fashion. The existence of RELIK and PSIV refuted these ideas, revealing lentiviruses to be present in a range of mammals, capable of germline integration, and far more ancient than previously thought. Using Gag sequences reconstructed from the remnants of these prehistoric lentiviruses, we have produced chimeric lentiviruses capable of infecting nondividing cells and determined structures of capsid domains from PSIV and RELIK. We show that the structures from these diverse viruses are highly similar, containing features found in modern-day lentiviruses, including a functional cyclophilin-binding loop. Together, these data provide evidence for an ancient capsid-cyclophilin interaction preserved throughout lentiviral evolution.

Encephalomyocarditis virus infection in an Italian zoo.

A fatal Encephalomyocarditis virus (EMCV) infection epidemic involving fifteen primates occurred between October 2006 and February 2007 at the Natura Viva Zoo. This large open-field zoo park located near Lake Garda in Northern Italy hosts one thousand animals belonging to one hundred and fifty different species, including various lemur species. This lemur collection is the most relevant and rich in Italy. A second outbreak between September and November 2008 involved three lemurs. In all cases, the clinical signs were sudden deaths generally without any evident symptoms or only with mild unspecific clinical signs. Gross pathologic changes were characterized by myocarditis (diffuse or focal pallor of the myocardium), pulmonary congestion, emphysema, oedema and thoracic fluid. The EMCV was isolated and recognized as the causative agent of both outbreaks. The first outbreak in particular was associated with a rodent plague, confirming that rats are an important risk factor for the occurrence of the EMCV infection.

Parallel germline infiltration of a lentivirus in two Malagasy lemurs.

Retroviruses normally infect the somatic cells of their host and are transmitted horizontally, i.e., in an exogenous way. Occasionally, however, some retroviruses can also infect and integrate into the genome of germ cells, which may allow for their vertical inheritance and fixation in a given species; a process known as endogenization. Lentiviruses, a group of mammalian retroviruses that includes HIV, are known to infect primates, ruminants, horses, and cats. Unlike many other retroviruses, these viruses have not been demonstrably successful at germline infiltration. Here, we report on the discovery of endogenous lentiviral insertions in seven species of Malagasy lemurs from two different genera -- Cheirogaleus and Microcebus. Combining molecular clock analyses and cross-species screening of orthologous insertions, we show that the presence of this endogenous lentivirus in six species of Microcebus is the result of one endogenization event that occurred about 4.2 million years ago. In addition, we demonstrate that this lentivirus independently infiltrated the germline of Cheirogaleus and that the two endogenization events occurred quasi-simultaneously. Using multiple proviral copies, we derive and characterize an apparently full length and intact consensus for this lentivirus. These results provide evidence that lentiviruses have repeatedly infiltrated the germline of prosimian species and that primates have been exposed to lentiviruses for a much longer time than what can be inferred based on sequence comparison of circulating lentiviruses. The study sets the stage for an unprecedented opportunity to reconstruct an ancestral primate lentivirus and thereby advance our knowledge of host-virus interactions.

A transitional endogenous lentivirus from the genome of a basal primate and implications for lentivirus evolution.

Lentiviruses chronically infect a broad range of mammalian species and have been transmitted from primates to humans, giving rise to multiple outbreaks of HIV infection over the past century. Although the circumstances surrounding these recent zoonoses are becoming clearer, the nature and timescale of interaction between lentiviruses and primates remains unknown. Here, we report the discovery of an endogenous lentivirus in the genome of the gray mouse lemur (Microcebus murinus), a strepsirrhine primate from Madagascar, demonstrating that lentiviruses are capable of invading the primate germ line. Phylogenetic analysis places gray mouse lemur prosimian immunodeficiency virus (pSIVgml) basal to all known primate lentiviruses and, consistent with this, its genomic organization is intermediate between the nonprimate lentiviruses and their more derived primate counterparts. Thus, pSIVgml represents the first unambiguous example of a viral transitional form, revealing the acquisition and loss of genomic features during lentiviral evolution. Furthermore, because terrestrial mammal populations in Madagascar and Africa are likely to have been isolated from one another for at least 14 million years, the presence of pSIVgml in the gray mouse lemur genome indicates that lentiviruses must have been infecting primates for at least this period of time, or have been transmitted between Malagasy and African primate populations by a vector species capable of traversing the Mozambique channel. The discovery of pSIVgml illustrates the utility of endogenous sequences for the study of contemporary retroviruses and indicates that primate lentiviruses may be considerably older and more broadly distributed than previously thought.

Biomedical evaluation of free-ranging red ruffed lemurs (Varecia rubra) within the Masoala National Park, Madagascar.

Complete health assessments were performed on 22 adult red ruffed lemurs (Varecia rubra), comprising nine males and 13 females, found within the Masoala National Park in northeast Madagascar. Each animal was anesthetized using tiletamine and zolazepam and underwent a thorough physical examination, including measurement of its weight and vital signs; blood collection for hematology, plasma total protein concentration, serum chemistries, fat-soluble vitamins, trace minerals, assessment of iron metabolism, toxoplasmosis serology, viral serologies, and examination for hemoparasites; fecal collection for bacterial culture and parasite examination; and collection of a representative number of any ectoparasites. Comparison of blood values with those of captive lemurs demonstrated a number of significant differences thought to be associated with physiologic state (e.g., reproductive stage and stress), hydration, and diet. There was no evidence of serious infectious diseases, and hemoparasites were not detected. The enteric flora appeared unremarkable; however, results may have been skewed toward more cold-tolerant bacteria. The fecal parasite burden was low. Lemurostrongylus spp. was identified in two of the lemurs, and there were moderate numbers of Laelapidae mites present on approximately one third of the lemurs. This study demonstrated the substantial amount of data that can be collected from free-ranging populations, considered invaluable in the management of captive populations, in reducing the incidence of captivity-related diseases, and in the risk assessment associated with reintroduction programs.

Assessing flavivirus, lentivirus, and herpesvirus exposure in free-ranging ring-tailed lemurs in southwestern Madagascar.

The ring-tailed lemur (Lemur catta) is an endangered species found in southwestern Madagascar, and understanding infectious disease susceptibility is an essential step towards the preservation of wild and captive lemur populations. Lemurs are primates that are widely dispersed throughout the island of Madagascar and may serve as hosts or reservoirs for zoonotic infections. The aim of this study was to determine the prevalence of antibodies to West Nile virus (WNV), simian immunodeficiency virus (SIV), and herpes simplex virus type 1 (HSV-1) in a population of free-ranging ring-tailed lemur from the Beza Mahafaly Special Reserve, Madagascar. Samples were collected from 50 animals during field capture studies in June and July 2004 and assayed for presence of viral antibodies during the 12 mo following collection. Forty-seven of the 50 lemurs sampled had antibodies against WNV detectable by epitope-blocking enzyme-linked immunosorbent assay (ELISA). In addition, 50 of 50 samples had titers against WNV ranging from 80 to > or = 1,280 using plaque reduction neutralization test (PRNT(90)). Ten lemurs had antibodies against lentiviral antigens as determined by Western blot analysis. None of the lemurs had antibodies against HSV-1 using ELISA.

Endogenous retrovirus HERV-I LTR family in primates: sequences, phylogeny, and evolution.

A human endogenous retrovirus (HERV-I; RTVL-I) has been located within the first intron of a haptoglobin-related gene. Two members of the HERV-I family were identified in proximal Yq11.2 and caused AZFa microdeletions as a result of intra-chromosomal recombination events in azoospermic patients. Using PCR25 and the sequencing approach with the genomic DNAs of primates, hominoids, Old and New World monkeys, and prosimians, the HERV-I LTR elements were identified and analysed. The LTR elements were detected only in the hominoids and the Old World monkeys, indicating that the HERV-I LTR elements were inserted into the primate genome after the split of the New World monkeys in the Oligocene era, about 33 million years ago. Nineteen members of the HERV-I LTR elements from the hominoids and the Old World monkeys showed multiple insertions or deletions. They showed a 78.6-97.4% sequence similarity to that of Hu-15 (accession no. AF290422; HERV-I LTR on human Yq11.2). The evolutionary relationships within the HERV-I LTR family among hominoids and Old World monkeys showed a random cluster, indicating that HERV-I LTR elements have evolved independently in primate evolution.

Human endogenous retrovirus K homologous sequences and their coding capacity in Old World primates.

The coding capacity for retroviral Gag and Env proteins has been maintained in human endogenous retroviruses of the HERV-K family. HERV-K homologous sequences have been found in all Old World primates. Here, we examined Old World primate species for the presence of full-length HERV-K gag and env genes and the presence of gag and env open reading frames as determined by the protein truncation test. Full-length HERV-K env genes were found in DNAs of all Old World primate species, whereas open reading frames for Env protein were found solely in human, chimpanzee, and gorilla DNAs. The mutational event leading to two HERV-K types was found to have occurred after the separation of hominids from lower Old World primates and before the expansion of hominids. Full-length HERV-K gag genes in hominids displayed a 96-bp deletion compared to those in lower Old World primates. The ancient gag variant has not been maintained during hominid evolution. Open reading frames for HERV-K Gag have been found in all Old World primates except chimpanzees. Our study of the HERV-K family during Old World primate evolution contributes to the understanding of their possible biological functions in the host genomes.