Full Viral Genome Sequencing and Phylogenomic Analysis of Feline Herpesvirus Type 1 (FHV-1) in Cheetahs (Acinonyx jubatus).

Feline herpesvirus type 1 (FHV-1) is endemic in captive cheetahs and sporadically causes devastating disease. Modified live vaccines (MLV), intended for use in domestic cats, are used in some captive cheetah populations and have been anecdotally linked to disease in certain subpopulations. Ten FHV-1 isolates from ten captive cheetahs and one isolate from an MLV used to inoculate four of the host animals were analyzed. Viral DNA was extracted for full-genome sequencing by Illumina MiSeq with viral genomes then used for phylogenomic and recombinational analyses. The FHV-1 shed by vaccinated cheetahs were almost identical to the MLV, with few variants among viral genomes. Eight cheetah FHV-1 isolates and the MLV were grouped in a clade along with FHV-1 isolates from domestic cats in the USA. The remaining two cheetah FHV-1 isolates (unknown host vaccine status) were not associated with a clade. The likely ancestral origin of these two isolates involves recombination events between Australian domestic cat and cheetah FHV-1 isolates. Collectively, these data suggest that the MLV is capable of causing clinical disease and viral shedding in some cheetahs and represents evidence of interspecies transmission of virus between domestic and wild cats.

Seasonal recurrence of cowpox virus outbreaks in captive cheetahs (Acinonyx jubatus).

Cowpox virus infections in captive cheetahs (Acinonyx jubatus) with high morbidity and mortality have already been reported in the UK and Russia in the 1970s. However, most of the reported cases have been singular events. Here, we report a total of five cowpox virus outbreaks in cheetahs in the same safari park in Denmark between 2010 and 2014. Nine cheetahs showed varying severity of clinical disease; two of them died (22%). All episodes occurred between August and October of the respective year. No other carnivores kept at the same institution nor the keepers taking care of the animals were clinically affected. The clinical picture of cowpox was confirmed by extensive laboratory investigations including histopathological and molecular analyses as well as cell culture isolation of a cowpox virus. High anti-orthopoxvirus antibody titers were detected in all 9 diseased cheetahs compared to seven contact cheetahs without clinical signs and 13 cheetahs not in direct contact. Additionally, whole genome sequencing from one sample of each cluster with subsequent phylogenetic analysis showed that the viruses from different outbreaks have individual sequences but clearly form a clade distinct from other cowpox viruses. However, the intra-clade distances are still larger than those usually observed within clades of one event. These findings indicate multiple and separate introductions of cowpox virus, probably from wild rodent populations, where the virus keeps circulating naturally and is only sporadically introduced into the cheetahs. Sero-positivity of voles (Arvicola amphibious) caught in zoo grounds strengthens this hypothesis. As a consequence, recommendations are given for medical and physical management of diseased cheetahs, for hygienic measures as well as for pre-shipment isolation before cheetah export from zoo grounds.

Gammaretrovirus-specific antibodies in free-ranging and captive Namibian cheetahs.

The cheetah population in Namibia is the largest free-ranging population in the world and a key population for research regarding the health status of this species. We used serological methods and quantitative real-time PCR to test free-ranging and captive Namibian cheetahs for the presence of feline leukemia virus (FeLV), a gammaretrovirus that can be highly aggressive in populations with low genetic diversity, such as cheetahs. We also assessed the presence of antibodies to other gammaretroviruses and the responses to a FeLV vaccine developed for domestic cats. Up to 19% of the free-ranging cheetahs, 27% of the captive nonvaccinated cheetahs, and 86% of the captive vaccinated cheetahs tested positive for FeLV antibodies. FeLV-antibody-positive free-ranging cheetahs also tested positive for Rauscher murine leukemia virus antibodies. Nevertheless, FeLV was not detectable by quantitative real-time PCR and no reverse transcriptase activity was detectable by product-enhanced reverse transcriptase assay in the plasma of cheetahs or the supernatants from cultures of peripheral blood mononuclear cells. The presence of antibodies to gammaretroviruses in clinically healthy specimens may be caused either by infection with a low-pathogenic retrovirus or by the expression of endogenous retroviral sequences. The strong humoral immune responses to FeLV vaccination demonstrate that cheetahs can respond to the vaccine and that vaccination against FeLV infection may be beneficial should FeLV infection ever become a threat, as was seen in Iberian lynx and Florida panthers.

Pandemic (H1N1) 2009 in captive cheetah.

We describe virus isolation, full genome sequence analysis, and clinical pathology in ferrets experimentally inoculated with pandemic (H1N1) 2009 virus recovered from a clinically ill captive cheetah that had minimal human contact. Evidence of reverse zoonotic transmission by fomites underscores the substantial animal and human health implications of this virus.

Seroprevalences to viral pathogens in free-ranging and captive cheetahs (Acinonyx jubatus) on Namibian Farmland.

Cheetah populations are diminishing rapidly in their natural habitat. One reason for their decline is thought to be a high susceptibility to (infectious) diseases because cheetahs in zoos suffer from high disease-induced mortality. Data on the health status of free-ranging cheetahs are scarce, and little is known about their exposure and susceptibility to infectious diseases. We determined seroprevalences to nine key viruses (feline herpesvirus 1, feline calicivirus, feline parvovirus, feline coronavirus, canine distemper virus, feline immunodeficiency virus [FIV], puma lentivirus, feline leukemia virus, and rabies virus) in 68 free-ranging cheetahs on east-central Namibian farmland, 24 nonvaccinated Namibian captive cheetahs, and several other wild carnivore species and conducted necropsies of cheetahs and other wild carnivores. Eight of 11 other wild carnivores were seropositive for at least one of the viruses, including the first record of an FIV-like infection in a wild felid west of the Kalahari, the caracal (Felis caracal). Seroprevalences of the free-ranging cheetahs were below 5% for all nine viruses, which is significantly lower than seroprevalences in nonvaccinated captive cheetahs and those for five of seven viruses in previously studied free-ranging cheetahs from north-central Namibia (L. Munson, L. Marker, E. Dubovi, J. A. Spencer, J. F. Evermann, and S. J. O'Brien, J. Wildl. Dis. 40:23-31, 2004). There was no clinical or pathological evidence of infectious diseases in living or dead cheetahs. The results suggest that while free-ranging wild carnivores may be a source of pathogens, the distribution of seroprevalences across studies mirrored local human population density and factors associated with human habitation, probably reflecting contact opportunities with (nonvaccinated) domestic and feral cats and dogs. They also suggest that Namibian cheetahs respond effectively to viral challenges, encouraging consistent and sustainable conservation efforts.

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

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.

Immunohistochemical screening for viral agents in cheetahs (Acinonyx jubatus) with myelopathy.

Numerous cases of acute-onset progressive ataxia, hindlimb paresis and paralysis of unknown aetiology occurred during 1993 to 2003 in cheetahs (Acinonyx jubatus) within the European Endangered Species Programme (eep). This study describes the immunohistochemical investigation of a possible viral aetiology of the "cheetah myelopathy". Antibodies to feline herpesvirus type 1, canine distemper virus, canine parvovirus and Borna disease virus were applied to formalin-fixed and paraffin-embedded brain and spinal cord sections from 25 affected cheetahs aged between three-and-a-half months and 13 years. Using the avidin-biotin complex technique, none of the antibodies gave positive immunosignals in either the brain or the spinal cord tissue.

Analysis of genetic mutations in the 7a7b open reading frame of coronavirus of cheetahs (Acinonyx jubatus).

OBJECTIVE: To analyze the 7a7b genes of the feline coronavirus (FCoV) of cheetahs, which are believed to play a role in virulence of this virus. SAMPLE POPULATION: Biologic samples collected during a 4-year period from 5 cheetahs at the same institution and at 1 time point from 4 cheetahs at different institutions. PROCEDURES: Samples were first screened for FCoV via a reverse transcription-PCR procedure involving primers that encompassed the 3'-untranslated region. Samples that yielded positive assay results were analyzed by use of primers that targeted the 7a7b open reading frames. The nucleotide sequences of the 7a7b amplification products were determined and analyzed. RESULTS: In most isolates, substantial deletional mutations in the 7a gene were detected that would result in aberrant or no expression of the 7a product because of altered reading frames. Although the 7b gene was also found to contain mutations, these were primarily point mutations resulting in minor amino acid changes. The coronavirus associated with 1 cheetah with feline infectious peritonitis had intact 7a and 7b genes. CONCLUSIONS AND CLINICAL RELEVANCE: The data suggest that mutations arise readily in the 7a region and may remain stable in FCoV of cheetahs. In contrast, an intact 7b gene may be necessary for in vivo virus infection and replication. Persistent infection with FCoV in a cheetah population results in continued virus circulation and may lead to a quasispecies of virus variants.

Serosurvey of viral infections in free-ranging Namibian cheetahs (Acinonyx jubatus).

Cheetahs (Acinonyx jubatus) in captivity have unusually high morbidity and mortality from infectious diseases, a trait that could be an outcome of population homogeneity or the immunomodulating effects of chronic stress. Free-ranging Namibian cheetahs share ancestry with captive cheetahs, but their susceptibility to infectious diseases has not been investigated. The largest remaining population of free-ranging cheetahs resides on Namibian farmlands, where they share habitat with domestic dogs and cats known to carry viruses that affect cheetah health. To assess the extent to which free-ranging cheetahs are exposed to feline and canine viruses, sera from 81 free-ranging cheetahs sampled between 1992 and 1998 were evaluated for antibodies against canine distemper virus (CDV), feline coronavirus (feline infectious peritonitis virus; FCoV/ FIPV), feline herpesvirus 1 (FHV1), feline panleukopenia virus (FPV), feline immunodeficiency virus (FIV), and feline calicivirus (FCV) and for feline leukemia virus (FeLV) antigens. Antibodies against CDV, FCoV/FIPV, FHV1, FPV, and FCV were detected in 24, 29, 12, 48, and 65% of the free-ranging population, respectively, although no evidence of viral disease was present in any animal at the time of sample collection. Neither FIV antibodies nor FeLV antigens were present in any free-ranging cheetah tested. Temporal variation in FCoV/FIPV seroprevalence during the study period suggested that this virus is not endemic in the free-ranging population. Antibodies against CDV were detected in cheetahs of all ages sampled between 1995 and 1998, suggesting the occurrence of an epidemic in Namibia during the time when CDV swept through other parts of sub-Saharan Africa. This evidence in free-ranging Namibian cheetahs of exposure to viruses that cause severe disease in captive cheetahs should direct future guidelines for translocations, including quarantine of seropositive cheetahs and preventing contact between cheetahs and domestic pets.

Chronic eosinophilic dermatitis associated with persistent feline herpes virus infection in cheetahs (Acinonyx jubatus).

A chronic ulcerative and eosinophilic dermatitis occurred in 20 captive cheetahs (Acinonyx jubatus) with persistent feline herpes virus 1 (FHV1) infection. Affected animals had erythematous, ulcerated plaques primarily on the face and forelegs in sites of contact with lachrymal and salivary secretions. The dermatitis was characterized by dense infiltrates of eosinophils and plasma cells and pseudoepitheliomatous hyperplasia. Rare keratinocytes within the lesions had nuclei with marginated chromatin and small eosinophilic inclusions composed of herpes virus nucleocapsids. Virus isolated from lesions was confirmed to be FHV1. Lesions persisted and progressed unless removed by cryoexcision. The occurrence of this unusual reaction to FHV1 in approximately 5% of captive North American cheetahs suggests a species propensity for a Th2-dominant response to herpes virus infection. This atypical immune reaction may indicate a heritable trait or modulation of the immune response by other factors such as chronic stress.

Genetic characterization of feline parvovirus sequences from various carnivores.

Infections with viruses of the feline parvovirus subgroup such as feline panleukopenia virus (FPV), mink enteritis virus (MEV) and canine parvovirus (CPV-2) [together with its new antigenic types (CPV-2a, CPV-2b)] have been reported from several wild carnivore species. To examine the susceptibility of different species to the various parvoviruses and their antigenic types, samples from wild carnivores with acute parvovirus infections were collected. Viral DNA was amplified, and subsequently analysed, from faeces or formalin-fixed small intestines from an orphaned bat-eared fox (Otocyon megalotis), a free-ranging honey badger (Mellivora capensis), six captive cheetahs (Acinonyx jubatus), a captive Siberian tiger (Panthera tigris altaica) and a free-ranging African wild cat (Felis lybica). Parvovirus infection in bat-eared fox and honey badger was demonstrated for the first time. FPV-sequences were detected in tissues of the African wild cat and in faeces of one cheetah and the honey badger, whereas CPV-2b sequences were found in five cheetahs and the bat-eared fox. The Siberian tiger (from a German zoo) was infected with a CPV-type 2a virus. This distribution of feline parvovirus antigenic types in captive large cats suggests an interspecies transmission from domestic dogs. CPV-2 sequences were not detected in any of the specimens and no sequences with features intermediate between FPV and CPV were found in any of the animals examined.

Feline panleukopenia virus revisited: molecular characteristics and pathological lesions associated with three recent isolates.

The low incidence of clinical signs or pathological lesions compatible with feline panleukopenia in cats has created the perception among practitioners that the disease has disappeared since the emergence of canine parvovirus type 2 in the late 1970s. Three parvoviruses that were recently isolated from a domestic cat and 2 cheetahs in cell culture or detected by means of the polymerase chain reaction were shown to be typical feline parvoviruses. Phylogenetic comparison with other FPV isolates did not reveal a particular African cluster.

Feline herpesvirus infection in a group of semi-captive cheetahs.

Clinical disease caused by feline herpesvirus type-1 in wild felid species is similar to that in domestic cats. Herpesviruses are endemic in free-ranging lions in South Africa but actual clinical disease due to them has not been reported in free-ranging felids. The first reports of feline herpesvirus infection associated with clinical disease in wild felids came from Australia and the USA in 1970. Subsequent reports of clinical disease in cheetahs and other wild felid species were limited to captive animals. This report deals with clinical disease in a group of semi-captive cheetahs in which 18 animals were affected, and included 12 adult males, 4 adult females and 2 subadults. No mortalities occurred in this group, the most common clinical signs being sneezing, nasal discharge and loss of appetite.