435809
al.Identification of a novel adenovirus in a cotton-top tamarin
JVDXXX10.1177/1040638711435809Hall et
Identification of a novel adenovirus in a
cotton-top tamarin (Saguinus oedipus)
Journal of Veterinary Diagnostic Investigation
24(2) 359­–363
© 2012 The Author(s)
Reprints and permission:
sagepub.com/journalsPermissions.nav
DOI: 10.1177/1040638711435809
http://jvdi.sagepub.com
Natalie H. Hall,1 Linda L. Archer, April L. Childress, James F. X. Wellehan Jr.
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.
Key words: Adenovirus; cotton-top tamarin; diarrhea; Mastadenovirus; Saguinus oedipus.
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
From Department of Small Animal Clinical Sciences, University of
Florida Veterinary Medical Center, Gainesville, FL.
1
Corresponding Author: Natalie H. Hall, White Oak Conservation
Center, 581705 White Oak Road, Yulee, FL 32097. [email protected]
Downloaded from vdi.sagepub.com at PENNSYLVANIA STATE UNIV on September 15, 2016
360
Hall et al.
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 ×
103 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 ringersa 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 Mastadenovirusspecific 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
kite and analyzed on an automated DNA sequencerf 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.
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
Downloaded from vdi.sagepub.com at PENNSYLVANIA STATE UNIV on September 15, 2016
Identification of a novel adenovirus in a cotton-top tamarin
361
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.
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
Downloaded from vdi.sagepub.com at PENNSYLVANIA STATE UNIV on September 15, 2016
362
Hall et al.
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.
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
Downloaded from vdi.sagepub.com at PENNSYLVANIA STATE UNIV on September 15, 2016
Identification of a novel adenovirus in a cotton-top tamarin
provide relevant epidemiological information on likely origins of disease outbreaks.
Acknowledgements
The authors would like to acknowledge Dr. Ramiro Isaza for his
assistance in the clinical evaluation of this patient.
Sources and manufacturers
a. Aspen Veterinary Resources Ltd., Liberty, MO.
b. Ensure®, Pedialyte®; Abbott Laboratories, Abbott Park, IL.
c. Gatorade®, Gatorade Sports Science Institute, Barrington, IL.
d. QIAamp® DNeasy kit, Qiagen Inc., Valencia, CA.
e. Big-Dye Terminator Kit, Applied Biosystems, Foster City, CA.
f. ABI 377, GMI Inc., Ramsey, MN.
g.Felsenstein J, 2005, Phylogeny Inference Package, Version
3.66. Distributed by the author. Department of Genome
Sciences, University of Washington, Seattle, WA.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
article.
Funding
The author(s) disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article:
Support for this study was funded by the Batchelor Foundation.
Mastadenoviral primer design was supported by research grant no.
N00014-09-1-0252 from the Office of Naval Research to Dr. James
F. X. Wellehan, Jr.
References
1.Benko M, Harrach B: 2011, Molecular evolution of adenoviruses. In: Adenoviruses: model and vectors in virus host
interactions, ed. Doerfler W, Bohm P, pp. 3–35. Springer,
New York, NY.
2. Boyce JT, Giddens WE Jr, Valerio M: 1978, Simian adenoviral
pneumonia. Am J Pathol 91:259–276.
3. Chen EC, Yagi S, Kelly KR, et al.: 2011, Cross-species transmission of a novel adenovirus associated with a fulminant
pneumonia outbreak in a new world monkey colony. PLoS
Pathog 7:e1002155.
4.Davison AJ, Benko M, Harrach B: 2003, Genetic content and
evolution of adenoviruses. J Gen Virol 84:2895–2908.
5. Ersching J, Hernandez MI, Cezarotto FS, et al.: 2010, Neutralizing antibodies to human and simian adenoviruses in humans
and New-World monkeys. Virology 407:1–6.
6. Harrach B: 2000, Reptile adenoviruses in cattle? Acta Vet Hung
48:485–490.
7.International Species Information System (ISIS): 2002, ISIS
reference for physiological values in captive wildlife [CDROM]. ISIS, Apple Valley, MN.
363
8.Jones DT, Taylor WR, Thornton JM: 1992, The rapid generation of mutation data matrices from protein sequences. Comput
Appl Biosci 8:275–282.
9. Joslin JO: 2003, Other primates excluding great apes. In: Zoo
and wild animal medicine, ed. Fowler ME, Miller RE, 5th ed.,
pp. 350–374. WB Saunders, St. Louis, MO.
10.Katoh K, Toh H: 2008, Recent developments in the MAFFT
multiple sequence alignment program. Brief Bioinform
9:286–298.
11. Landolfi JA, Wellehan JF, Johnson AJ, Kinsel MJ: 2005, Fatal
human herpesvirus type 1 infection in a white-handed gibbon
(Hylobates lar). J Vet Diagn Invest 17:369–371.
12. Murphy WJ, Eizirik E, O’Brien SJ, et al.: 2001, Resolution of
the early placental mammal radiation using Bayesian phylogenetics. Science 294:2348–2351.
13.Perelman P, Johnson WE, Roos C, et al.: 2011, A molecular
phylogeny of living primates. PLoS Genet 7:e1001342.
14.Ronquist F, Huelsenbeck JP: 2003, MrBayes 3: Bayesian
phylogenetic inference under mixed models. Bioinformatics
19:1572–1574.
15.Roy S, Vandenberghe LH, Kryazhimskiy S, et al.: 2009, Isolation and characterization of adenoviruses persistently shed
from the gastrointestinal tract of non-human primates. PLoS
Pathog 5:e1000503.
16.Sestak K, Merritt CK, Borda J, et al.: 2003, Infectious agent
and immune response characteristics of chronic enterocolitis in
captive rhesus macaques. Infect Immun 71:4079–4086.
17.Shackelton LA, Parrish CR, Holmes EC: 2006, Evolutionary
basis of codon usage and nucleotide composition bias in vertebrate DNA viruses. J Mol Evol 62:551–563.
18. Shroyer EL, Kelley ST, Taylor PC, et al.: 1979, Three serologic
types of adenovirus infections of owl monkeys. Am J Vet Res
40:532–536.
19.Stuker G, Oshiro LS, Schmidt NJ, et al.: 1979, Virus detection in monkeys with diarrhea: the association of adenoviruses
with diarrhea and the possible role of rotaviruses. Lab Anim Sci
29:610–616.
20. Veerassamy S, Smith A, Tillier ER: 2003, A transition probability model for amino acid substitutions from blocks. J Comput
Biol 10:997–1010.
21. Voevodin AF, Marx PA: 2009, Adenoviruses. In: Simian virology, pp. 407–418. Wiley-Blackwell, Ames, IA.
22.Wellehan JF, Johnson AJ, Harrach B, et al.: 2004, Detection
and analysis of six lizard adenoviruses by consensus primer
PCR provides further evidence of a reptilian origin for the atadenoviruses. J Virol 78:13366–13369.
23. Wevers D, Metzger S, Babweteera F, et al.: 2011, Novel adenoviruses in wild primates: a high level of genetic diversity and
evidence of zoonotic transmissions. J Virol 85:10774–10784.
24.Whelan S, Goldman N: 2001, A general empirical model of
protein evolution derived from multiple protein families using
a maximum-likelihood approach. Mol Biol Evol 18:691–699.
Downloaded from vdi.sagepub.com at PENNSYLVANIA STATE UNIV on September 15, 2016