Novel Picornavirus Detected in Wild Deer: Identification, Genomic Characterisation, and Prevalence in Australia.

The use of high-throughput sequencing has facilitated virus discovery in wild animals and helped determine their potential threat to humans and other animals. We report the complete genome sequence of a novel picornavirus identified by next-generation sequencing in faeces from Australian fallow deer. Genomic analysis revealed that this virus possesses a typical picornavirus-like genomic organisation of 7554 nt with a single open reading frame (ORF) encoding a polyprotein of 2225 amino acids. Based on the amino acid identity comparison and phylogenetic analysis of the P1, 2C, 3CD, and VP1 regions, this novel picornavirus was closely related to but distinct from known bopiviruses detected to date. This finding suggests that deer/bopivirus could belong to a novel species within the genus Bopivirus, tentatively designated as "Bopivirus C". Epidemiological investigation of 91 deer (71 fallow, 14 sambar and 6 red deer) and 23 cattle faecal samples showed that six fallow deer and one red deer (overall prevalence 7.7%, 95% confidence interval [CI] 3.8-15.0%) tested positive, but deer/bopivirus was undetectable in sambar deer and cattle. In addition, phylogenetic and sequence analyses indicate that the same genotype is circulating in south-eastern Australia. To our knowledge, this study reports for the first time a deer-origin bopivirus and the presence of a member of genus Bopivirus in Australia. Further epidemiological and molecular studies are needed to investigate the geographic distribution and pathogenic potential of this novel Bopivirus species in other domestic and wild animal species.

DETECTION OF DEER ATADENOVIRUS A DNA IN DAM AND OFFSPRING PAIRS OF ROCKY MOUNTAIN MULE DEER (ODOCOILEUS HEMIONUS HEMIONUS) AND ROCKY MOUNTAIN ELK (CERVUS CANADENSIS NELSONI).

Adenovirus hemorrhagic disease affects primarily mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus), Rocky Mountain elk (Cervus canadensis nelsoni), and moose (Alces alces) in their first year of life. The method by which the causative virus, Deer atadenovirus A, is maintained in the environment and transmitted to neonates is unknown. In this study, we investigated the potential transmission of the virus from dam to offspring in Rocky Mountain mule deer (Odocoileus hemionus hemionus) and elk in western Wyoming, US. We sampled dams before parturition during placement of vaginal implant transmitters and at parturition and sampled neonates during capture in their first days of life. We also tested for the virus in mortalities submitted for pathologic examination and laboratory analysis. We detected viral DNA in samples from all time points tested but did not find a connection between positive dams and offspring mortalities associated with adenovirus hemorrhagic disease. Although we did not find direct evidence of transmission events between dams and offspring, asymptomatic animals shedding of Deer atadenovirus A, are a likely source of infection in neonates.

Adenovirus Hemorrhagic Disease in Moose (Alces americanus gigas) in Alaska, USA.

In 1993, an epizootic of adenovirus hemorrhagic disease (AHD) caused the death of at least 1,000 mule deer (Odocoileus hemionus) in California, US. Since then, numerous cervid species throughout the US have had deaths confirmed to be caused by AHD. In 2015, the death of two captive moose (Alces americanus gigas) calves marked the first recognized AHD-caused deaths in Alaska, a state in which moose are important economically as well as for food security and cultural identity. Both cases were characterized by systemic vasculitis with endothelial cell intranuclear inclusion bodies, pulmonary edema, petechial hemorrhages, and enterotyphlocolitis.

A Comprehensive Study of Cutaneous Fibropapillomatosis in Free-Ranging Roe Deer (Capreolus capreolus) and Red Deer (Cervus elaphus): from Clinical Manifestations to Whole-Genome Sequencing of Papillomaviruses.

Papillomaviruses (PVs) are an extremely large group of viruses that cause skin and mucosa infections in humans and various animals. In roe deer and red deer, most PVs belong to the Deltapapillomavirus genus and cause neoplastic changes that are generally described as fibropapillomas. Despite the wide distribution of roe and red deer throughout Europe and beyond, the data in the scientific literature regarding the widespread distribution of PVs and the genetic variability of PV genomes in these species are rather scarce. This study describes cutaneous fibropapillomatosis cases in roe and red deer with clinical manifestations that are typical of infections with PVs. In all cases, the presence of PV DNA was confirmed using PCR, followed by Sanger sequencing of the partial L1 gene. The complete PV genomes were determined in all the investigated samples using next-generation sequencing technology, revealing infections of roe deer with the CcaPV1-type and red deer with the CePV1v-type variant. A comparison of the complete CcaPV1-type and CePV1v-type variant genome sequences reported here with already available complete genome sequences in GenBank revealed their great genetic stability across time and space.

Rabbit Hemorrhagic Disease Virus Isolated from Diseased Alpine Musk Deer (Moschussifanicus).

Rabbit hemorrhagic disease virus (RHDV) is the causative agent of rabbit hemorrhagic disease (RHD), and its infection results in mortality of 70-90% in farmed and wild rabbits. RHDV is thought to replicate strictly in rabbits. However, there are also reports showing that gene segments from the RHDV genome or antibodies against RHDV have been detected in other animals. Here, we report the detection and isolation of a RHDV from diseased Alpine musk deer (Moschussifanicus). The clinical manifestations in those deer were sudden death without clinical signs and hemorrhage in the internal organs. To identify the potential causative agents of the disease, we used sequence independent single primer amplification (SISPA) to detect gene segments from viruses in the tissue samples collected from the dead deer. From the obtained sequences, we identified some gene fragments showing very high nucleotide sequence similarity with RHDV genome. Furthermore, we identified caliciviral particles using an electron microscope in the samples. The new virus was designated as RHDV GS/YZ. We then designed primers based on the genome sequence of an RHDV strain CD/China to amplify and sequence the whole genome of the virus. The genome of the virus was determined to be 7437 nucleotides in length, sharing the highest genome sequence identity of 98.7% with a Chinese rabbit strain HB. The virus was assigned to the G2 genotype of RHDVs according to the phylogenetic analyses based on both the full-length genome and VP60 gene sequences. Animal experiments showed that GS/YZ infection in rabbits resulted in the macroscopic and microscopic lesions similar to that caused by the other RHDVs. This is the first report of RHDV isolated from Alpine musk deer, and our findings extended the epidemiology and host range of RHDV.

Molecular and Biological Characterization of a Cervidpoxvirus Isolated From Moose with Necrotizing Dermatitis.

Cervidpoxvirus is one of the more recently designated genera within the subfamily Chordopoxvirinae, with Deerpox virus (DPV) as the only recognized species to date. In this study, the authors describe spontaneous disease and infection in the North American moose (Alces americanus) by a novel Cervidpoxvirus, here named Moosepox virus (MPV). Three 4-month-old moose calves developed a multifocal subacute-to-chronic, necrotizing, suppurative-to-granulomatous dermatitis that affected the face and the extremities. Ultrastructurally, all stages of MPV morphogenesis-that is, crescents, spherical immature particles, mature particles, and enveloped mature virus-were observed in skin tissue. In vitro infection with MPV confirmed that its morphogenesis was similar to that of the prototype vaccinia virus. The entire coding region, including 170 putative genes of this MPV, was sequenced and annotated. The sequence length was 164,258 bp with 98.5% nucleotide identity with DPV (strain W-1170-84) based on the whole genome. The genome of the study virus was distinct from that of the reference strain (W-1170-84) in certain genes, including the CD30-like protein (83.9% nucleotide, 81.6% amino acid), the endothelin precursor (73.2% nucleotide including some indels, 51.4% amino acid), and major histocompatibility class (MHC) class I-like protein (81.0% nucleotide, 68.2% amino acid). This study provides biological characterization of a new Cervidpoxvirus attained through in vivo and in vitro ultrastructural analyses. It also demonstrates the importance of whole-genome sequencing in the molecular characterization of poxviruses identified in taxonomically related hosts.

Infection with Foamy Virus in Wild Ruminants-Evidence for a New Virus Reservoir?

Foamy viruses (FVs) are widely distributed and infect many animal species including non-human primates, horses, cattle, and cats. Several reports also suggest that other species can be FV hosts. Since most of such studies involved livestock or companion animals, we aimed to test blood samples from wild ruminants for the presence of FV-specific antibodies and, subsequently, genetic material. Out of 269 serum samples tested by ELISA with the bovine foamy virus (BFV) Gag and Bet antigens, 23 sera showed increased reactivity to at least one of them. High reactive sera represented 30% of bison samples and 7.5% of deer specimens. Eleven of the ELISA-positives were also strongly positive in immunoblot analyses. The peripheral blood DNA of seroreactive animals was tested by semi-nested PCR. The specific 275 bp fragment of the pol gene was amplified only in one sample collected from a red deer and the analysis of its sequence showed the highest homology for European BFV isolates. Such results may suggest the existence of a new FV reservoir in bison as well as in deer populations. Whether the origin of such infections stems from a new FV or is the result of BFV inter-species transmission remains to be clarified.

Cutaneous Fibropapilloma in a Red Deer (Cervus elaphus) Associated with Cervus elaphus Papillomavirus in Portugal.

A pedunculated cauliflower-like mass was detected on the left posterior limb of a subadult male red deer (Cervus elaphus) after a hunt in Portugal. Histologically the lesion was classified as cutaneous fibropapilloma. The identification of Cervus elaphus papillomavirus (CePV-1 variant) was based on sequencing of the L1 gene. The L2 sequence revealed a nine-nucleotide deletion, as already reported in the Italian and Hungarian CePV-1, further supporting the theory that this is a distinctive genomic characteristic of this viral variant, as this feature has been found in distinct cases from geographically distant countries. In addition, a coinfection with bovine papillomavirus was evidenced by amplification and sequencing of the E5 gene, confirming the ability of Delta papillomaviruses to cross-infect different animal species and providing more evidence that wildlife may act as reservoir for papillomaviruses affecting domestic species. Papillomavirus infection in red deer has been sporadically described in different European countries; in this work, we describe the identification of a CePV-1 variant infection associated with a red deer fibropapilloma in Portugal.

Malignant catarrhal fever: An emerging yet neglected disease in captive sika deer (Cervus nippon) herds in China.

Malignant catarrhal fever (MCF) is a fatal lymphoproliferative disease that represents a serious problem in the deer-rearing industry. To better understand an MCF-like disease that has emerged in northern China since 2015, we investigated ten cases by documenting clinical and epidemiological data and analysing causative agents and histopathological changes. In addition, a retrospective screen for Macavirus DNA and a questionnaire-based survey were conducted. Epizootic MCF in Chinese sika deer herds has emerged with a low morbidity of 3.8% (95% CI: 2.5%-5.1%) and a high mortality of 93.2% (95% CI: 86.6%-99.9%). The disease course varied from 3 to 12 days. Aetiologically, OvHV-2 was predominant in the MCFV, accounting for most MCF cases (21/23). In contrast, only two CpHV-2 isolates were phylogenetically closely related to CpHV-2. Diarrhoea and nasal discharges were the most frequent manifestations, although clinical signs varied in some cases. Pathologically typical lesions of haemorrhage, necrosis and lymphoid cell infiltration were readily observed in a variety of organs. Vasculitis caused by vascular and perivascular lymphoid cell infiltration was common. The retrospective survey suggested a low positive rate (3/275) of MCFV DNA in peripheral blood lymphocytes (PBL). The questionnaire-based survey suggested the disease was neglected by local veterinarians, who did not acknowledge the risk of co-rearing deer with reservoir species. Collectively, the emerging epizootic MCF in Chinese sika deer herds remains neglected, emphasizing the urgency of initiating full-field diagnoses and control strategies.

POSTMORTEM DETECTION OF BLUETONGUE AND EPIZOOTIC HEMORRHAGIC DISEASE VIRUSES IN THE BONE MARROW OF WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS).

We determined the temporal aspects of detecting bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) in postmortem bone marrow samples of white-tailed deer (Odocoileus virginianus) using molecular and in vitro cell culture techniques. Bone marrow samples from carcasses were collected and assayed on the day of death and at intervals up to 16 wk after death. We recovered BTV and EHDV from fresh bone marrow collected at day 0 by isolation in Vero and BHK-21 cell cultures. However, attempts to replicate the viruses from aged bone marrow in Vero and BHK-21 cell cultures failed. The real-time quantitative reverse transcriptase PCR (qRT-PCR) results confirmed that EHDV and BTV can be detected in aged bone marrow for up to 12 and 16 wk, respectively, after death. The RNA of BTV and EHDV could be detected by qRT-PCR in white-tailed deer bone marrow for extended periods of time postmortem. This technique will provide a useful tool for retrospective determination of BTV or EHDV infection of white-tailed deer at the time of death.

Multiple bluetongue virus serotypes causing death in Brazilian dwarf brocket deer (Mazama nana) in Brazil, 2015-2016.

Bela Vista Biological Sanctuary (RBV) is a protected area of Itaipu Binacional, a hydroelectric power company located on the border of Brazil and Paraguay. A captive population of Brazilian dwarf brocket deer (Mazama nana, Cervidae, Artiodactyla) is maintained for conservation purposes. Despite the reproductive success of the animals, outbreaks of a fatal hemorrhagic disease have been registered over the years, compromising conservation efforts. In order to identify the etiological agents of these hemorrhagic diseases, 32 captive Brazilian dwarf brockets were sampled to investigate bluetongue virus (BTV), epizootic hemorrhagic disease (EHD), and adenovirus hemorrhagic disease (AHD), in 2015. Only one deer (1/32; 3.12%) was seropositive for BTV. After this survey, five animals died in the early autumn of 2015 and 2016, again presenting clinical signs of hemorrhagic disease. Using RT-qPCR, RT-PCR and DNA sequencing, five BTV serotypes (3, 14, 18, 19, and 22) were identified in blood and tissues collected during necropsies. These BTV serotypes had not been previously described or isolated in Brazil, either in wild or domestic ruminants. Additionally, differential diagnosis was performed for EHD and AHD, but all samples were negative for both diseases. The multiple distinct BTV serotypes identified in these outbreaks resulted in a high lethality (100%) of Brazilian dwarf brockets and indicated that various BTV serotypes are circulating in the area.

MOOSE ( ALCES ALCES) MORTALITY ASSOCIATED WITH CAPRINE HERPESVIRUS 2 (CPHV-2) IN A ZOOLOGICAL COLLECTION.

Malignant catarrhal fever (MCF) can affect both domestic and wild artiodactyls. In a zoological setting, in which subclinical carriers and susceptible species are often housed in close proximity, the disease can prove fatal. This report describes a case of goat-associated MCF in a captive moose ( Alces alces). The diagnosis was confirmed by histopathology, which showed lymphocytic vasculitis in the brain and panuveitis, and by detection of caprine herpesvirus 2 DNA in tissues. Identical viral DNA sequences amplified from the clinically affected moose and from domestic, petting goats ( Capra aegagrus hircus) housed in the zoo suggest that the goats were the source for the virus transmutation. This is the first report, to our knowledge, of confirmed goat-associated MCF in any moose in North America and of the surveillance measures and procedures put in place to prevent additional spread of the disease.

Caprine herpesvirus 2-associated malignant catarrhal fever of captive sika deer (Cervus nippon) in an intensive management system.

BACKGROUND: Caprine herpesvirus 2 (CpHV-2) infection usually induces chronic malignant catarrhal fever (MCF) in sika deer (Cervus nippon), with the primary signs of weight loss, dermatitis and alopecia. CASE PRESENTATION: Here, we report a case of CpHV-2-associated acute MCF in a sika deer herd raised in an intensive management system distant to the reservoir goats. Affected deer developed clinical signs of high fever (41 °C) followed by nasal discharge and lameness. Severe lesions of hemorrhage, necrosis and infiltration of lymphoid cells could readily be observed in the lung, kidney, heart valves and subcutaneous tissue surrounding a tendon. Etiologically, identical CpHV-2 specific DNA sequences were detected in peripheral blood lymphocyte (PBL) from the affected deer and reservoir goats. CONCLUSION: In summary, domestic goats were the reservoir of the CpHV-2, which is the causative agent of the outbreak of MCF in the three hinds. The disease was probably transmitted via aerosol infection. In addition, necrosis and inflammation in subcutaneous tissue surrounding a tendon was the reason for lameness. Therefore, MCF should be put into a differential diagnostic list when similar disease occurs in sika deer herds.

Orf virus infection in Alaskan mountain goats, Dall's sheep, muskoxen, caribou and Sitka black-tailed deer.

BACKGROUND: The zoonotic Orf virus (ORFV; genus Parapoxvirus, Poxviridae family) occurs worldwide and is transmitted between sheep and goats, wildlife and man. Archived tissue samples from 16 Alaskan wildlife cases, representing mountain goat (Oreamnos americanus, n = 8), Dall's sheep (Ovis dalli dalli, n = 3), muskox (Ovibos moschatus, n = 3), Sitka black-tailed deer (Odocoileus hemionus sitkensis, n = 1) and caribou (Rangifer tarandus granti, n = 1), were analyzed. RESULTS: Clinical signs and pathology were most severe in mountain goats, affecting most mucocutaneous regions, including palpebrae, nares, lips, anus, prepuce or vulva, as well as coronary bands. The proliferative masses were solid and nodular, covered by dark friable crusts. For Dall's sheep lambs and juveniles, the gross lesions were similar to those of mountain goats, but not as extensive. The muskoxen displayed ulcerative lesions on the legs. The caribou had two ulcerative lesions on the upper lip, as well as lesions on the distal part of the legs, around the main and dew claws. A large hairless spherical mass, with the characteristics of a fibroma, was sampled from a Sitka black-tailed deer, which did not show proliferative lesions typical of an ORFV infection. Polymerase chain reaction analyses for B2L, GIF, vIL-10 and ATI demonstrated ORFV specific DNA in all cases. Sequences from Dall's sheep formed a separate cluster, comparable to ORFV from domestic sheep. Sequences from the other species were different from the Dall's sheep sequences, but almost identical to each other. CONCLUSIONS: This is the first major investigation of parapoxvirus infections in large Alaskan game species, and the first report of parapoxvirus infection in caribou and Sitka black-tailed deer. This study shows that most of the wild ruminant species in Alaska and from most parts of Alaska, can carry and be affected by ORFV. These findings call for attention to transmission of ORFV from wildlife to livestock and to hunters, subsistence harvesters, and wildlife biologists.

Whole-genome sequences of Odocoileus hemionus deer adenovirus isolates from deer, moose and elk are highly conserved and support a new species in the genus Atadenovirus.

We present the first complete genome sequence of Odocoileus hemionus deer adenovirus 1 (OdAdV-1). This virus can cause sporadic haemorrhagic disease in cervids, although epizootics with high mortality have occurred in California. OdAdV-1 has been placed in the genus Atadenovirus, based on partial hexon, pVIII and fibre genes. Ten field isolates recovered from naturally infected mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginiana) and moose (Alces alces) from Wyoming, black-tailed deer (Odocoileus hemionus columbianus) from California, and Rocky Mountain elk (Cervus elaphus nelsoni) from Colorado and Washington state were sequenced. The genome lengths ranged from 30 620 to 30 699 bp, contained the predicted proteins and gene organization typical of members of genus Atadenovirus, and had a high percentage of A/T nucleotides (66.7 %). Phylogenic analysis found that the closest ancestry was with ruminant atadenoviruses, while a divergence of the hexon, polymerase and penton base proteins of more than 15 % supports classification as a new species. Genetic global comparison between the 10 isolates found an overall 99 % identity, but greater divergence was found between those recovered from moose and elk as compared to deer, and a single variable region contained most of these differences. Our findings demonstrate that OdAdV-1 is highly conserved between 10 isolates recovered from multiple related cervid species, but genotypic differences, largely localized to a variable region, define two strains. We propose that the virus type name be changed to cervid adenovirus 1, with the species name Cervid atadenovirus A. Sequence data were used to develop molecular assays for improved detection and genotyping.

Isolation and characterization of a novel cervid adenovirus from white-tailed deer (Odocoileus virginianus) fawns in a captive herd.

A novel adenovirus, CeAdV1, was isolated from buffy coat and nasal swab samples collected from two captive white-tailed deer (Odocoileus virginianus) fawns. The isolation was an incidental finding in the course of screening animals for use in a research study on an unrelated pathogen. In the screening process, virus isolation was performed on both nasal swabs and buffy coat samples and cytopathic effect was observed. Electron microscopy revealed viral particles with the shape and morphology of an adenovirus. Next generation sequencing followed by phylogenetic analysis classified this virus to the Mastadenovirus genus. Its sequence was genetically distinct from all other recognized species in this genus, with only 76% sequence identity to its closest genetic match, bovine adenovirus 3 (BAdV3). The virus could be propagated in bovine derived cells but grew to a higher titer in cervid derived cells. Inoculation of white-tailed deer fawns with the isolated virus resulted in pyrexia, depletion of thymus tissue and mild respiratory disease. Comparative serology performed using convalescent sera revealed distinct antigenic differences between the novel cervid adenovirus and BAdV3. A retrospective serological survey of the captive deer herd indicated that this virus had been circulating in the herd for at least 14 years with no report of clinical disease. A survey of serum collected from free ranging mule deer residing in Nevada revealed high serum titers against this novel adenovirus.

Wildlife: topical issues, recent cases.

This focus article was prepared by Paul Duff, Paul Holmes and Alex Barlow of the APHA Wildlife Expert Group.

A Mortality Event in Elk ( Cervus elaphus nelsoni) Calves Associated with Malnutrition, Pasteurellosis, and Deer Adenovirus in Colorado, USA.

This report describes clinical, necropsy, and ancillary diagnostic findings for a mortality event in Rocky Mountain elk ( Cervus elaphus nelsoni) calves attributed to malnutrition, pasteurellosis, and an alimentary presentation of adenovirus hemorrhagic disease.

Tumor necrosis factor-alpha expression in white-tailed deer (Odocoileus virginianus) infected with Epizootic haemorrhagic disease virus.

Epizootic haemorrhagic disease (EHD) is the most important infectious disease of white­tailed deer (WTD), however little is known about the role of inflammatory mediators in the pathogenesis. We characterized the expression of tumor necrosis factor­alpha (TNF-α) ex vivo in tissues of WTD experimentally or naturally infected with EHD virus serotype 2 and in WTD peripheral blood mononuclear cells (PBMC) infected with EHD virus serotype 2 in vitro. Circulating levels of TNF-α were evaluated in serum from experimentally infected deer via cytotoxicity assay. The expression of TNF-α in tissues was evaluated via immunohistochemistry (IHC) in both experimentally and naturally infected deer. Semi­quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to assess the level of TNF-α mRNA in tissues from experimentally infected deer and WTD's PBMC. Circulating levels of TNF-α were not increased in infected animals and TNF-α was not detected in tissues of infected deer. Increased transcription of TNF-α was detected neither in infected WTD nor in the PBMC. Tumor necrosis factor-alpha may not play a significant role in the pathogenesis of EHD virus infection in WTD.