Linda L. Archer

Characterization of an outbreak of astroviral diarrhea in a group of cheetahs (Acinonyx jubatus)

Abstract A Mamastrovirus was identified in an outbreak of diarrhea in cheetahs (Acinonyx jubatus). Five young adult and two adult cheetahs presented with lethargy, anorexia, watery diarrhea and regurgitation over an 11-day period. Fecal samples were submitted for electron microscopy and culture. Electron microscopy results revealed particles morphologically consistent with an astrovirus, and no other viral pathogens or significant bacterial pathogens were identified. The astrovirus was confirmed and sequenced using consensus astroviral PCR, resulting in a 367 base pair partial RNA-dependent-RNA polymerase (RdRp) product and a 628 base pair partial capsid product. Bayesian and maximum likelihood phylogenetic analyses were performed on both the RdRp and the capsid protein segments. All animals were monitored and treated with bismuth subsalicylate tablets (524mg PO BID for 5 days), and recovered without additional intervention. This is the first report we are aware of documenting an astrovirus outbreak in cheetah.

Characterization of California sea lion polyomavirus 1: Expansion of the known host range of the Polyomaviridae to Carnivora

The genome of a novel polyomavirus first identified in a proliferative tongue lesion of a California sea lion (Zalophus californianus) is reported. This is only the third described polyomavirus of laurasiatherian mammals, is the first of the three associated with a lesion, and is the first known polyomavirus of a host in the order Carnivora. Predicted large T, small t, VP1, VP2, and VP3 genes were identified based on homology to proteins of known polyomaviruses, and a putative agnoprotein was identified based upon its location in the genome. Phylogenetic analysis of the predicted late region proteins found that the laurasiatherian polyomaviruses, together with Squirrel monkey polyomavirus and Murine pneumotropic virus, form a monophyletic clade. Phylogenetic analysis of the early region was more ambiguous. The noncoding control region of California sea lion polyomavirus 1 is unusual in that only two apparent large T binding sites are present; this is less than any other known polyomavirus. The VP1 of this virus has an unusually long carboxy-terminal region. A quantitative polymerase chain reaction was developed and utilized on various samples from 79 additional animals from either managed or wild stranded California sea lion populations, and California sea lion polyomavirus 1 infection was found in 24% of stranded animals. Sequence of additional samples identified four sites of variation in the t antigens, three of which resulted in predicted coding changes.

Genotype differentiation of Agamid Adenovirus 1 in bearded dragons (Pogona vitticeps) in the USA by hexon gene sequence

Bearded dragons (Pogona vitticeps) are popular pets in the United States. Agamid Adenovirus 1 (AgAdV1) is an important infectious agent of bearded dragons. The only AgAdV1 sequences available to date are from a highly conserved region of the DNA polymerase gene. Degenerate primers were designed to amplify a variable region of the AgAdV1 hexon gene for sequencing. Genetic differences were identified within the hexon gene of 17 bearded dragons from 4 collections. Much less diversity was present in the polymerase gene. Bayesian analysis of the hexon nucleotide alignment identified two larger groups and two isolates that did not tightly cluster with these two groups. Multiple genotypes were identified within collections, and individual genotypes were seen in different collections. Three bearded dragons appeared to be infected by multiple strains. These findings show that this hexon region is useful for AgAdV1 genotyping, which can be used epidemiologically as well as in future investigations of AgAdV1 evolution and clinical implications of strain differences.

Development of a quantitative PCR for rapid and sensitive diagnosis of an intranuclear coccidian parasite in Testudines (TINC), and detection in the critically endangered Arakan forest turtle (Heosemys depressa)

The intranuclear coccidian parasite of Testudines (TINC) is responsible for significant disease in turtles and tortoises causing high mortality and affecting several threatened species. Diagnostic testing has been limited to relatively labor intensive and expensive pan-coccidial PCR and sequencing techniques. A qPCR assay targeting a specific and conserved region of TINC 18S rRNA was designed. The qPCR reaction was run on samples known to be TINC positive and the results were consistent and analytically specific. The assay was able to detect as little as 10 copies of target DNA in a sample. Testing of soil and invertebrates was negative and did not provide any further insights into life cycles. This assay was used to identify TINC in a novel host species, the critically endangered Arakan forest turtle (Heosemys depressa).

Initial sequence characterization of the rhabdoviruses of squamate reptiles, including a novel rhabdovirus from a caiman lizard (Dracaena guianensis).

Rhabdoviruses infect a variety of hosts, including non-avian reptiles. Consensus PCR techniques were used to obtain partial RNA-dependent RNA polymerase gene sequence from five rhabdoviruses of South American lizards; Marco, Chaco, Timbo, Sena Madureira, and a rhabdovirus from a caiman lizard (Dracaena guianensis). The caiman lizard rhabdovirus formed inclusions in erythrocytes, which may be a route for infecting hematophagous insects. This is the first information on behavior of a rhabdovirus in squamates. We also obtained sequence from two rhabdoviruses of Australian lizards, confirming previous Charleville virus sequence and finding that, unlike a previous sequence report but in agreement with serologic reports, Almpiwar virus is clearly distinct from Charleville virus. Bayesian and maximum likelihood phylogenetic analysis revealed that most known rhabdoviruses of squamates cluster in the Almpiwar subgroup. The exception is Marco virus, which is found in the Hart Park group.

Detection of a respiratory coronavirus from tissues archived during a pneumonia epizootic in free-ranging Pacific harbor seals Phoca vitulina richardsii.

In June 2000, 21 adult harbor seals Phoca vitulina richardsii were found dead along a localized section of the central California coast. Necropsy of 5 fresh carcasses revealed pulmonary congestion, consolidation, and hemorrhage. Histopathological changes in lungs from 2 of these seals included a necrotizing lymphocytic and histocytic lobar pneumonia with intra-lesional bacteria. A coronavirus (CoV) was detected in archived tissues from 1 of the 5 seals via a degenerate PCR for nidoviral RNA-dependent RNA polymerase (RdRp), and was subsequently confirmed via specific PCR. Based on the partial RdRp sequence, the CoV was identified as a novel, divergent member of the CoV group 1a. The virus is tentatively named harbor seal coronavirus (HSCoV). The clinical significance of HSCoV and its involvement in the etiology of the epizootic pneumonia and deaths of the harbor seals is uncertain.

Coinfection with a novel fibropapilloma-associated herpesvirus and a novel Spirorchis sp. in an eastern box turtle (Terrapene carolina) in Florida

Herpesviruses are important pathogens of chelonians, and include Chelonid herpesvirus 5, which is associated with fibropapillomatosis in sea turtles. Spirorchid trematodes are blood flukes that reside within the cardiovascular system of marine turtles and may be associated with severe disease. An eastern box turtle (Terrapene carolina) at the South Florida Wildlife Care Center (Fort Lauderdale, Florida) was presented to the facility with papillomatous growths behind both rear legs. Surgical removal resulted in remission for 8 months; however, lesions recurred, prompting a second surgery and acyclovir therapy. Surgical biopsies revealed subacute superficial inflammation associated with the supporting stroma of the cutaneous papillomas and granulomas within the superficial dermis containing fragmented and collapsed brown trematode eggs surrounded by multinucleated giant cells and epithelioid macrophages. Pan-herpesviral and pan-trematode consensus polymerase chain reaction and sequencing were run on tissue samples. Comparative sequence analysis revealed a novel alphaherpesvirus and a novel trematode in the genus Spirorchis. The animal became anorexic and was euthanized due to poor quality of life. While we do not yet have a complete understanding of the effects of herpesvirus and trematode infections in eastern box turtles, the findings thus presented provide initial insights into the disease relationships among these chelonians.

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

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.

NOVEL POXVIRUS INFECTION IN NORTHERN AND SOUTHERN SEA OTTERS (ENHYDRA LUTRIS KENYONI AND ENHYDRA LUTRIS NEIRIS), ALASKA AND CALIFORNIA, USA

Abstract Small superficially ulcerated skin lesions were observed between October 2009 and September 2011 during captive care of two orphaned sea otter pups: one northern (Enhydra lutris kenyoni) in Alaska and one southern (Enhydra lutris nereis) in California. Inclusions consistent with poxviral infection were diagnosed by histopathology in both cases. Virions consistent with poxvirus virions were seen on electron microscopy in the northern sea otter, and the virus was successfully propagated in cell culture. DNA extraction, pan-chordopoxviral PCR amplification, and sequencing of the DNA-dependent DNA polymerase gene revealed that both cases were caused by a novel AT-rich poxvirus. Bayesian and maximum likelihood phylogenetic analyses found that the virus is divergent from other known poxviruses at a level consistent with a novel genus. These cases were self-limiting and did not appear to be associated with systemic illness. To our knowledge, this is the first report of a poxvirus in a mustelid species. The source of this virus, mode of transmission, zoonotic potential, and biological significance are undetermined.

Preliminary Investigation of Bottlenose Dolphins (Tursiops truncatus) for hfe Gene–related Hemochromatosis

Abstract Hemochromatosis (iron storage disease) has been reported in diverse mammals including bottlenose dolphins (Tursiops truncatus). The primary cause of excessive iron storage in humans is hereditary hemochromatosis. Most human hereditary hemochromatosis cases (up to 90%) are caused by a point mutation in the hfe gene, resulting in a C282Y substitution leading to iron accumulation. To evaluate the possibility of a hereditary hemochromatosis-like genetic predisposition in dolphins, we sequenced the bottlenose dolphin hfe gene, using reverse transcriptase–PCR and hfe primers designed from the dolphin genome, from liver of affected and healthy control dolphins. Sample size included two case animals and five control animals. Although isotype diversity was evident, no coding differences were identified in the hfe gene between any of the animals examined. Because our sample size was small, we cannot exclude the possibility that hemochromatosis in dolphins is due to a coding mutation in the hfe gene. Other potential causes of hemochromatosis, including mutations in different genes, diet, primary liver disease, and insulin resistance, should be evaluated.