Identification of a novel adenovirus in a cotton-top tamarin ( Saguinus oedipus )

Abstract

A novel adenovirus was identified in a cotton-top tamarin (Saguinus oedipus) with diarrhea by negative-staining electron microscopy of feces, consensus polymerase chain reaction, and sequencing. Partial sequences were obtained from the DNA-dependent DNA polymerase, the p52k gene, and the hexon. Bayesian and maximum likelihood phylogenetic analyses indicated that the virus is a member of the genus Mastadenovirus, and is herein termed Saguinus siadenovirus 1. The phylogeny of the mastadenoviruses is similar to that of their hosts, supporting coevolution. Support for this was strongest in the analysis of the predicted hexon protein. The obtained sequences were GC-rich, which may suggest a lack of recent host jumps. The diversity and evolution of the adenoviruses of platyrrhine primates merits further investigation. Additional study of the association of this virus with diarrhea is indicated.

Adenoviruses have been reported in all tetrapod classes, as well as sturgeon, and fall into 5 genera: Mastadenovirus, Aviadenovirus, and the 3 recently accepted genera Atadenovirus, Siadenovirus, and Ichtadenovirus. Like many intranuclear DNA viruses, they exhibit significant host fidelity, and phylogenetic analyses suggest coevolution with host species, although host switches have occurred. ¹ Mastadenovirus and Atadenovirus are the 2 genera known to infect mammalian hosts. Atadenoviruses have their most probable origin in squamates, and their presence in birds and mammals likely represents host jumps.^6,22 Mastadenoviruses have only been found in mammals and, thus, the viruses may originate there.

Placental mammals are divided into 4 main superorders: 2 lesser clades, Afrotheria (aardvarks, elephants, moles, etc.) and Xenarthra (anteaters, armadillos, sloths), and 2 larger clades, Laurasiatheria (order Carnivora, cattle, horses, etc.) and Euarchontoglires (primates, rabbits, rodents). ¹² Within superorder Euarchontoglires, the order Primates is divided into the suborders Strepsirrhini (prosimians) and Haplorrhini, with suborder Haplorrhini subdivided into infraorders Tarsiiformes (tarsiers) and Simiiformes. Infraorder Simiiformes is further subdivided into parvorders Catarrhini (apes and cercopithecine monkeys) and Platyrrhini (New World monkeys). ¹³ Adenoviral investigations have largely focused on human beings and other catarrhines, and there are few reports on platyrrhines.^15,21 Adenoviral infection in platyrrhines has been described in night monkeys (or, owl monkeys, Aotus sp.), although sequence characterization of these viruses has not yet been reported, and also in white-tufted-ear marmosets (Callithrix jacchus) and a red-chested mustached tamarin (Saguinus labiatus).^18,23 White-tufted-ear marmosets and black-striped capuchin monkeys (Cebus libidinosus) were found to have neutralizing antibodies to human adenoviruses but not to those from other catarrhine species, suggesting anthroponotic transmission. ⁵ A 2011 report described a novel divergent mastadenovirus from a highly fatal respiratory epizootic in titi monkeys (Callicebus cupreus) with evidence of transmission of disease between titi monkeys and human beings. ³ The current report describes the use of electron microscopy and consensus nested polymerase chain reaction (nPCR) and sequencing to characterize a novel adenovirus from a cotton-top tamarin (Saguinus oedipus), a platyrrhine.

A privately owned, 7-month-old, male, intact cotton-top tamarin was presented for respiratory and gastrointestinal illness. The patient exhibited 2 days of sneezing and coughing, then 1 day with 2 vomiting episodes, and then 5 days of mucoid diarrhea. The private collection included 4 additional cotton-top tamarins, black-pencilled marmosets (Callithrix penicillata), Geoffrey’s marmosets (Callithrix geoffroyi), 70 birds, and 3 domestic cats. One of the cotton-top tamarins was a juvenile acquired 1 month previously. Human beings were the only catarrhines in contact.

On examination, the patient was bright and alert, and weighed 284 g with low body fat and poor muscling. A small amount of mucoid diarrhea was observed on the perineum. Differential diagnoses included bacterial gastroenterocolitis, endoparasitism, systemic viral infection, tamarin wasting disease, dietary intolerance, and extra-alimentary disease including exocrine pancreatic insufficiency and hepatic disease. Whole body radiographs revealed generalized poor bone density consistent with mild metabolic bone disease and poor serosal detail due to low visceral adipose tissue. A complete blood cell count indicated a leukocytosis (22.07 × 10 ³ cells/μl) characterized by a moderate neutrophilia (16,773 cells/μl), mild monocytosis (1,990 cells/μl), and mild eosinophilia (440 cells/μl). ⁷ A plasma chemistry panel indicated hypoalbuminemia (2.0 g/dl). Alkaline phosphatase, alanine aminotransferase, amylase, total bilirubin, blood urea nitrogen, calcium, phosphorus, creatinine, glucose, sodium, potassium, and globulins were within normal limits. ⁷ Direct fecal smear and zinc fecal flotation with centrifugation were negative for parasites. Fecal cultures for Salmonella sp., Shigella sp., Campylobacter sp., and Yersinia sp. were negative. Negative-staining electron microscopy of feces revealed the presence of icosahedral particles morphologically consistent with an adenovirus. While test results were pending, empirical treatment included 1 dose of 20 ml of lactated ringers ^a solution subcutaneously, enrofloxacin (5 mg/kg, orally [PO], once daily for 7 days), and calcium glubionate (50 mg/kg, PO, once daily for 30 days). Caloric and fluid intake were supplemented with oral administration of 3 therapeutic hydration and nutritional supplements.^b,c Dietary recommendations for at least 25% protein intake and guidelines for patient sun exposure for endogenous cholecalciferol production were made. ⁹

DNA was extracted from feces with a commercial kit. ^d Nested PCR amplification of partial sequence of the adenovirus DNA-dependent DNA polymerase gene was performed as previously described. ²² To obtain additional sequence, the second round was modified to use primers polFinner and polRouter. Novel Mastadenovirus-specific primers HexFa (AAYAARTTYMGNAAYCCNACNGTIGC) and HexRd (CKRTCYTGNARRTCNACNACIGC) were used to obtain sequence from the hexon gene, and novel Mastadenovirus-specific primers 52KFa (GGNYTNATGCAYYTNTG GAYTT) and 52KRb (TARTTDATNGCNGCNACYTTYTC) were used to obtain sequence from the p52k gene, with amplification conditions identical to the polymerase protocol. Products were sequenced directly with a commercial kit ^e and analyzed on an automated DNA sequencer ^f at the University of Florida Center for Mammalian Genetics DNA Sequencing Facilities (Gainesville, FL). This resulted in a 452-bp segment of the polymerase, a 196-bp segment of the p52k gene, and an 850-bp fragment of the hexon. Sequence data were submitted to GenBank (accession nos. JN377906–JN377907). This adenovirus is herein referred to as Saguinus adenovirus 1 (SaAdV-1). The sequences had 28.8% AT in the polymerase sequence, 34.2% AT in the sequenced p52k, and 45.9% AT in the sequenced hexon.

Predicted homologous 147-150 amino acid polymerase sequences and 257-317 amino acid hexon sequences were aligned using MAFFT. ¹⁰ Bayesian analyses of each alignment were performed using MrBayes 3.1 with gamma distributed rate variation and a proportion of invariant sites, and amino acid substitution model jumping. ¹⁴ Statistical convergence was assessed by the standard deviation of split frequencies as well as potential scale reduction factors of parameters. The first 10% of 1,000,000 iterations were discarded as a burn in, based on examination of trends of the log probability versus generation. Two independent Bayesian analyses were run to avoid entrapment on local optima. The polymerase analysis found that the Rtrev model of amino acid substitution was most probable, and the hexon analysis found that the WAG model was most probable. ²⁴ The Bayesian trees are shown in Figures 1 and 2.

Figure 1.

Bayesian phylogenic tree of partial adenoviral DNA polymerase 147-150 amino acid sequences using MAFFT alignment. Saguinus adenovirus 1 is in bold font with an arrow. Turkey adenovirus 1 (GenBank accession no. YP_003933581) was used as an outgroup of the unrooted tree. Bayesian posterior probability values for branchings are in bold, and bootstrap values for the maximum likelihood tree are below. Branchings with Bayesian posterior probability values less than 50 are not shown, and areas where multifurcations occurred are shown as arcs.

Figure 2.

Bayesian phylogenic tree of partial adenoviral hexon 257-317 amino acid sequences adenoviral DNA polymerase amino acid sequences using MAFFT alignment. Markings are as in Figure 1.

Maximum likelihood (ML) analyses of each alignment were performed using PHYLIP, ^g running each alignment using the program Proml with each available amino acid substitution models set with global rearrangements, 5 replications of random input order, gamma plus invariant rate distributions, and unrooted. The values for the gamma distribution were taken from the Bayesian analysis. Turkey adenovirus 1 (GenBank accession no. NC_014564) was used as the outgroup. Maximum likelihood analysis of the polymerase found the most likely tree from the PMB model of amino acid substitution, and ML analysis of the hexon found the most likely tree from the JTT model.^8,20 The alignment was then used to create data subsets for bootstrap analysis to test the strength of the tree topology (200 resamplings), which was analyzed using the amino acid substitution model producing the most likely tree in that alignment. Bootstrap values from the ML analysis are shown on the Bayesian trees (Figs. 1, 2). In both analyses, SaAdV1 clustered significantly within genus Mastadenovirus.

After test results were obtained, therapy was continued as previously described. The patient’s diarrhea improved 3 days after presentation and resolved by 7 days. After 1 week of normal feces, the patient had a 3-day recurrence of diarrhea. A second prophylactic course of enrofloxacin (5 mg/kg, PO, once daily for 7 days) was administered as well as a 2-week course of lactose-free, live culture yogurt (1.25 ml, PO, once daily for 14 days). The patient was quarantined until no clinical signs had occurred for 4 weeks, and patient weight was monitored daily. One month after presentation, the patient had no additional episodes of diarrhea and had gained weight to 304 g. Five months after presentation, the patient had no recurrence of clinical illness, was active, growing, and living in an enclosure with other cotton-top tamarins. No additional animals were available for evaluation, but no other clinical signs of adenovirus infection have been observed in the collection.

The clinical signs observed in the cotton-top tamarin are consistent with previous nonhuman primate (NHP) adenovirus reports. The most severe NHP adenoviral epizootic reported to date showed primarily respiratory signs. ³ Lesions can include severe multifocal to diffuse interstitial pneumonia and necrosis of tracheal, bronchial, and bronchiolar epithelium and interalveolar septi. ² There may be associated keratoconjunctivitis, corneal edema, and conjunctival exudates. ²¹ The gastrointestinal tract is the second most common organ system affected by adenovirus in NHPs. ²¹ In a study of colony-reared macaques (predominantly Macaca mulatta), adenoviruses were isolated from a higher proportion of animals with diarrhea than from controls. ¹⁹ Additional reports of adenovirus in NHPs include an association with pancreatitis, most often with young, immunocompromised macaques. ²¹

Many infections remain clinically inapparent even though the patient is shedding large amounts of virus. ²¹ A previous suggests that prevalence rates in captive species may be as high as 36–46%. ¹⁵ Unfortunately, follow up in the current case was limited, and the source of viral exposure and time frame of fecal adenoviral shedding remains undetermined. Possible routes of exposure included the newly acquired tamarin, the other established primates in the collection, or fomite transmission. In NHP colonies, recurring diarrhea is the leading cause of animal morbidity requiring veterinary care. ¹⁶ Fecal screening by negative-staining electron microscopy and consensus PCR and sequencing allowed rapid and inexpensive identification of a shedding animal in the present case. Adenovirus infection should be included as a differential diagnosis in any primate exhibiting respiratory or gastrointestinal signs. Significant clinical differences can exist between viral types from the same species. Identification of adenoviral types and species in shedding individuals provides useful diagnostic, prognostic, and epidemiologic information for clinicians.

The adenoviruses exhibit a bias against CpG dinucleotides, and events such as host switches may cause differential biases in different viral lineages. ¹⁷ In the genus Atadenovirus, squamate reptiles appear to be the endemic hosts. These viruses likely host jumped into birds and mammals in at least 2 separate events, which were both associated with a large AT bias. ²² The SaAdV1 sequences were unusually GC-rich and not AT-rich, which may imply that this virus has not undergone a recent adaptation to a new host.

Previous phylogenetic analyses of both adenoviruses and herpesviruses indicate that many elements in the branching patterns of these viruses are congruent with branching patterns for the corresponding host species. This indicates that those viruses have often coevolved along with their hosts, although host switches have certainly occurred.^1,4 Within the genus Mastadenovirus, phylogenetic analyses found a general trend for the viruses of superorder Euarchontoglires hosts and those of superorder Laurasiatheria hosts to segregate. The hexon analysis found that SoAdV1 formed a clade with the other primate adenoviruses, but did not nest within the adenoviruses of catarrhines. This would be expected with host/virus cospeciation. The analysis of the polymerase differed; strong support in the Bayesian analysis and weak support in the ML analysis found it clustered with a virus using a Laurasiatheria host, Porcine adenovirus A. One possible explanation for the differing gene phylogenies is recombination; recombination has been seen in other primate adenoviruses. ¹⁵ Sequencing of the region between the hexon and polymerase would allow recombination analyses. Barriers to host jumps may be lower for closely related host species, and in the herpesviruses, a more studied family of intranuclear double-stranded DNA viruses, aberrant hosts exhibit the most significant pathology. ¹¹ Recent evidence suggests that adenoviruses are capable of jumping between platyrrhines and human beings.^3,5 A serologic survey for a titi monkey adenovirus found that most human beings were naïve. ³ Unfortunately, at the time of this writing, titi monkey adenovirus sequence had not been released for inclusion in the phylogenetic analysis. Further surveillance of platyrrhine adenoviruses is indicated and may be relevant for human health. Phylogenetic analysis of diverse NHP adenoviruses is likely to reveal information about host-virus evolution and provide relevant epidemiological information on likely origins of disease outbreaks.