Asinine Herpesvirus Research Articles

Characteristics and epidemiological investigation of equid herpesvirus 8 in donkeys in Shandong, China.

Equid herpesvirus 8 (EHV-8), also known as asinine herpesvirus type 3 (AHV-3), can cause severe respiratory disease, abortion in mares, and neurological disorders. There is limited information on the prevalence of EHV-8 in donkeys in China. In this study, we investigated EHV-8 infection in donkeys using PCR, resulting in the identification of a field strain, termed EHV-8 SD2020113, which was isolated using RK-13 cells and characterized by high-throughput sequencing and transmission electron microscopy. Our data indicated that 38.7% (457/1180) of donkeys showed the presence of EHV-8 in blood samples. Analysis of the ORF70 gene showed the highest similarity (99.8-99.9% identity) to EHV-8 IR/2015/40 (MF431614.1) and SDLC66 (MW816102), and, in phylogenetic analysis, it clustered with EHV-8 SDLC66 from China. The findings of this study indicate that EHV-8 is likely to represent a threat to the donkey industry, and breeders andveterinarians who care for donkey farms should be aware of this.

Detection of Equine Papillomaviruses and Gamma-Herpesviruses in Equine Squamous Cell Carcinoma.

Squamous cell carcinoma (SCC) seriously compromises the health and welfare of affected horses. Although robust evidence points to equine papillomavirus type 2 (EcPV2) causing genital lesions, the etiopathogenesis of equine SCC is still poorly understood. We screened a series of SCCs from the head-and-neck (HN), (peri-)ocular and genital region, and site-matched controls for the presence of EcPV2-5 and herpesvirus DNA using type-specific EcPV PCR, and consensus nested herpesvirus PCR followed by sequencing. EcPV2 DNA was detected in 45.5% of HN lesions, 8.3% of (peri-)ocular SCCs, and 100% of genital tumors, whilst control samples from tumor-free horses except one tested EcPV-negative. Two HNSCCs harbored EcPV5, and an ocular lesion EcPV4 DNA. Herpesvirus DNA was detected in 63.6%, 66.6%, 47.2%, and 14.2% of horses with HN, ocular, penile, and vulvar SCCs, respectively, and mainly identified as equine herpesvirus 2 (EHV2), 5 (EHV5) or asinine herpesvirus 5 (AsHV5) DNA. In the tumor-free control group, 9.6% of oral secretions, 46.6% of ocular swabs, 47% of penile samples, and 14.2% of vaginal swabs scored positive for these herpesvirus types. This work further highlights the role of EcPV2 as an oncovirus and is the first to provide information on the prevalence of (gamma-)herpesviruses in equine SCCs.

Detection of asinine gammaherpesviruses in association with pulmonary fibrosis in free-ranging donkeys.

A mortality event among recently captured feral donkeys (Equus asinus) occurred in south-central Utah in 2016. The deaths were sporadic, and clinical signs were indicative of respiratory disease, likely associated with an infectious etiology. Ten of 13 donkeys autopsied had moderate-to-severe interstitial fibrosing pneumonia, and one had pyogranulomatous pneumonia. Consensus PCRs directed toward the DNA polymerase and DNA packaging terminase subunit 1 for herpesviruses were performed followed by sequencing of the PCR amplicons and phylogenetic analysis. Asinine herpesvirus 4 (AsHV4) and 5 (AsHV5) were consistently identified in lung tissues of affected donkeys. No other herpesviruses were identified, and herpesviral DNA was not detected in lung tissues of 2 donkeys without evidence of respiratory disease. The detection of asinine gammaherpesviruses may have been associated with the lesions described. AsHV4 and AsHV5 have been reported in previous studies as novel gammaherpesviruses based on sequences obtained from donkeys with interstitial pneumonia and marked syncytial cell formation. Our findings suggest that the association of asinine gammaherpesviruses with respiratory conditions in equids deserves further attention.

Detection and Molecular Characterization of Two Gammaherpesviruses from Pantesco Breed Donkeys during an Outbreak of Mild Respiratory Disease.

Equid and asinine gammaherpesviruses (GHVs; genus Percavirus) are members of the Herpesviridae family. Though GHVs have been reported in horse populations, less studies are available on gammaherpesviral infections in donkeys. This study reports the co-infection with two GHVs in Pantesco breed donkeys, an endangered Italian donkey breed. Samples (n = 124) were collected on a breeding farm in Southern Italy from 40 donkeys, some of which were healthy or presented erosive tongue lesions and/or mild respiratory signs. Samples were analysed by using a set of nested PCRs targeting the DNA polymerase, glycoprotein B, and DNA-packaging protein genes, and sequence and phylogenetic analyses were performed. Twenty-nine donkeys (72.5%) tested positive, and the presence of Equid gammaherpesvirus 7 and asinine herpesvirus 5 was evidenced. In 11 animals, we found evidence for co-infection with viruses from the two species. Virions with herpesvirus-like morphology were observed by electron microscopic examination, and viruses were successfully isolated in RK-13-KY cell monolayers. The histological evaluation of tongue lesions revealed moderate lympho-granulocytic infiltrates and rare eosinophilic inclusions. The detection of GHVs in this endangered asinine breed suggests the need long-life monitoring within conservation programs and reinforces the need for further investigations of GHV’s pathogenetic role in asinine species.

Molecular characterization of equine herpesvirus-1 and asinine herpesvirus -5 isolated from aborted fetuses of arabian horses

Molecular characterization of equine herpesvirus-1 and asinine herpesvirus -5 isolated from aborted fetuses of arabian horses

Survey of Serum Amyloid A and Bacterial and Viral Frequency Using qPCR Levels in Recently Captured Feral Donkeys from Death Valley National Park (California).

Simple SummaryThis study aimed to measure the inflammatory marker (Serum Amyloid A; SAA) derived from such pathogenic processes specifically bacterial and virus found in recently captured feral donkeys. Quantitative Real-Time PCR (qPCR) was performed to detect and quantify RNA and DNA viruses and bacterial DNA. Behavior, body condition score, nasal discharge, and coughing were found to be related in cases of Asinine Herpesvirus 2 (AHV-2) and Streptococcus equi subspecies zooepidemicus DNA. SAA concentrations descended with age, with foals presenting higher concentrations. Positive tests differed for AHV-2 and Streptococcus equi spp. zooepidemicus between sampling moments. In conclusion, donkeys caught in the wild may not be a source of disease for domestic horses. However, the transmission of some pathogens, such as Streptococcus equi subspecies zooepidemicus, and/or AHV-2, may occur.Feral donkey removal from state land has raised concerns in terms of disease transmission between equine species. Disease outbreaks may occur as a result of the relocation of animals to new environments. Virus and bacteria DNA load and serum amyloid A derived from the pathogenic processes that they involve were measured in recently captured donkeys. Blood and nasal swabs were collected from 85 donkeys (Death Valley National Park, Shoshone, California); 24 were retested after 30/60 days in the Scenic (Arizona) long-term holding facility co-mingled with feral donkeys from Arizona and Utah. Quantitative Real-Time PCR (qPCR) was performed to detect viral and bacterial genomic material (equine influenza A [EIV], equine rhinitis A and B viruses, AHV-2, AHV-3, AHV-5 and EHV-1, EHV-4, Streptococcus equi subspecies equi and zooepidemicus,). Significant relations between behavior, body condition score, nasal discharge, and coughing were found in donkeys for which AHV-2 and Streptococcus zooepidemicus DNA was detected. Higher SAA concentrations were found in foals. AHV-2 and Streptococcus zooepidemicus DNA concentrations significantly differed between sampling moments (p < 0.05). In conclusion, donkeys do not appear to be a substantial risk for disease transmission to horses but could be if they carried strangles or other processes in which AHV-2 and Streptococcus zooepidemicus were involved.

Equid herpesvirus 8: Complete genome sequence and association with abortion in mares.

Equid herpesvirus 8 (EHV-8), formerly known as asinine herpesvirus 3, is an alphaherpesvirus that is closely related to equid herpesviruses 1 and 9 (EHV-1 and EHV-9). The pathogenesis of EHV-8 is relatively little studied and to date has only been associated with respiratory disease in donkeys in Australia and horses in China. A single EHV-8 genome sequence has been generated for strain Wh in China, but is apparently incomplete and contains frameshifts in two genes. In this study, the complete genome sequences of four EHV-8 strains isolated in Ireland between 2003 and 2015 were determined by Illumina sequencing. Two of these strains were isolated from cases of abortion in horses, and were misdiagnosed initially as EHV-1, and two were isolated from donkeys, one with neurological disease. The four genome sequences are very similar to each other, exhibiting greater than 98.4% nucleotide identity, and their phylogenetic clustering together demonstrated that genomic diversity is not dependent on the host. Comparative genomic analysis revealed 24 of the 76 predicted protein sequences are completely conserved among the Irish EHV-8 strains. Evolutionary comparisons indicate that EHV-8 is phylogenetically closer to EHV-9 than it is to EHV-1. In summary, the first complete genome sequences of EHV-8 isolates from two host species over a twelve year period are reported. The current study suggests that EHV-8 can cause abortion in horses. The potential threat of EHV-8 to the horse industry and the possibility that donkeys may act as reservoirs of infection warrant further investigation.

Chronic Pleuropulmonary Fibrosis and Elastosis of Aged Donkeys: Similarities to Human Pleuroparenchymal Fibroelastosis

Donkey pulmonary fibrosis (DPF) is a spontaneous syndrome of aged donkeys with a high prevalence (35%). No previous detailed characterization of DPF has been performed. We sought to determine the similarities between DPF and recognized patterns of human pulmonary fibrosis. Whole lungs were collected from 32 aged donkeys at routine necropsy. Gross examination revealed pulmonary fibrosis in 19 donkeys (DPF cases), whereas 13 (control cases) had grossly normal lungs. Eighteen whole inflated ex vivo lungs (11 DPF cases, seven control cases) were imaged with high-resolution CT (HRCT) scan, whereas the remainder were sectioned and photographed. Tissue samples were collected from all lungs for histopathologic evaluation using a standardized protocol. HRCT images and histology sections underwent independent blinded review. Lung tissue was analyzed for herpes virus, fungal hyphae, mycobacteria, and dust content. Ten of 19 DPF lungs were categorized as being consistent with pleuroparenchymal fibroelastosis (PPFE) according to previously defined histologic and imaging criteria. All 10 PPFE-like lungs had marked pleural and subpleural fibrosis, predominantly within the upper lung zone, with accompanying intraalveolar fibrosis and elastosis. Asinine herpesvirus was ubiquitously expressed within control and DPF lung tissue. No other etiologic agents were identified. Many cases of DPF share key pathologic and imaging features with human PPFE, a rare interstitial pneumonia. Consequently, further study of DPF may help to elucidate the etiopathogenesis of human PPFE.

Prevalence of asinine herpesvirus type 5 (AsHV-5) infection in clinically normal Lipizzaner horses

The aim of this study was to assess the extent of asinine herpesvirus (AsHV) type 5 infection in ‘closed’ populations of clinically normal Lipizzaner horses. Peripheral blood mononuclear cells plus nasal and conjunctival swabs were obtained on four occasions over an 18month period from 266 animals as part of a health surveillance programme. Sequence analysis of samples that were positive by nested consensus herpesvirus PCR but negative using quantified equid herpesvirus (EHV) type 2 and 5 PCR, revealed a total of 51 samples from 39 horses positive for AsHV-5. No statistically significant association between animal age, gender or geographical location and infection status was identified. The findings suggest sub-clinical AsHV-5 infection may be encountered more frequently than previously reported.

Equine Multinodular Pulmonary Fibrosis in association with asinine herpesvirus type 5 and equine herpesvirus type 5: a case report

A standardbred gelding with a history of 10 days pyrexia and lethargy was referred to the Equine Hospital at the Swedish University of Agricultural Sciences in Uppsala, Sweden.The horse had tachypnea with increased respiratory effort and was in thin body condition. Laboratory findings included leukocytosis, hyperfibrinogenemia and hypoxemia. Thoracic radiographs showed signs of pneumonia with a multifocal nodular pattern, which in combination with lung biopsy findings indicated Equine Multinodular Pulmonary Fibrosis (EMPF). EMPF is a recently described disease in adult horses with clinical signs of fever, weight loss and respiratory problems. The pathological findings include loss of functional pulmonary parenchyma due to extensive nodular interstitial fibrosis which has been related to infection with the equine herpesvirus type 5 (EHV-5). In this case, lung biopsy and tracheal wash samples tested positive for both asinine herpesvirus type 5 (AHV-5) and EHV-5 using PCR assays. The horse failed to respond to treatment and was euthanized for humane reasons. Postmortem examination confirmed the diagnosis of EMPF. This case suggests that not only EHV-5 alone should be considered in association with the development of this disease.

Association of Asinine Herpesvirus‐5 with Pyogranulomatous Pneumonia in a Mare

Association of Asinine Herpesvirus‐5 with Pyogranulomatous Pneumonia in a Mare

Herpesvirus-Associated Neurological Disease in a Donkey

A 4-year-old donkey was evaluated for progressive neurological abnormalities consisting of depression, stupor, weakness, and recumbency. Diagnostic evaluation for viral involvement identified an asinine herpesvirus in DNA extracted from deep pharyngeal swabs. Specific primers were designed based on comparison with equine herpesviral DNA polymerase sequences and yielded an 875-base pair product from the donkey. This sequence had complete identity with short sequences of asinine herpesvirus previously identified in donkeys with interstitial pneumonia. Amino acid analysis of the entire sequence indicated high similarity with Equid herpesvirus 7 (91%), Zebra herpesvirus 1 (90%), and Equid herpesvirus 2 (89%). With supportive treatment and physical therapy, the donkey gradually recovered over 5 days of hospitalization and returned to normal function. The current case illustrates the potential of a novel asinine herpesvirus to induce neurological disease in donkeys and provides a large viral sequence allowing confident assignment of this virus to the subfamily Gammaherpesvirinae.

Detection and nucleotide sequencing of a DNA-packaging protein gene of equine gammaherpesviruses.

In previous studies, novel putative viral pathogens designated that asinine herpesvirus 4 (AsHV4) and asinine herpesvirus 5 (AsHV5) were associated with fatal interstitial pneumonia in donkeys (Equus asinus). Nucleotide sequence analysis of a portion of the DNA polymerase gene identified these putative pathogens as herpesviruses and possibly as members of the Gammaherpesvirinae subfamily. Although similar to equine herpesvirus 2 (EHV2) and equine herpesvirus 5 (EHV5), sequence diversity was observed among the detected viruses. In this study, novel sequence is reported for a DNA-packaging protein gene of EHV5 plus AsHV4, AsHV5, and a newly described putative pathogen herein designated asinine herpesvirus 6 (AsHV6). Phylogenetic analysis of these sequences suggested that the equine gammaherpesviruses may form a separate clade within the Gammaherpesvirinae subfamily. Based on the sequence of EHV2 and the novel sequences reported in this study, a PCR assay was developed to detect equine gammaherpesviruses. Products of the predicted size were produced after amplification of DNA from EHV2, EHV5, AsHV4, AsHV5, and AsHV6. This nonnested assay was shown to consistently amplify approximately 10 genomic copies of EHV2. Amplification products were not produced from DNA template of other alpha- and gammaherpesviruses. Because the role of gammaherpesviruses has not been well defined in equine disease, it is envisioned that a single, sensitive PCR assay to detect these potential pathogens will facilitate further assessment of their role in disease.

Association of two newly recognized herpesviruses with interstitial pneumonia in donkeys (Equus asinus).

Over a period of 6 years, antemortem and postmortem examinations were performed on a number of donkeys suffering from respiratory disease. For many cases, initial diagnostic efforts failed to identify an etiology consistent with the pathologic findings. However, retrospective examination of these cases using consensus primer polymerase chain reaction, designed to recognize herpesviruses from all 3 subfamilies of the Herpesviridae, amplified a fragment of the highly conserved herpesvirus DNA polymerase gene from a number of these animals. Two novel herpesviruses, herein designated asinine herpesvirus 4 (AHV4) and asinine herpesvirus 5 (AHV5), were consistently detected in lung tissue from donkeys in which the histopathology was characterized by interstitial pneumonia and marked syncytial cell formation but not in lung tissue from donkeys with evidence of bacterial or verminous pneumonia. Nucleotide sequence and phylogenetic analysis places these new viruses within the Gammaherpesvirinae subfamily and indicates that they are most closely related to the recently identified zebra herpesvirus and wildass herpesvirus as well as equine herpesviruses 2 and 5.

Characterization of the major glycoproteins of equine herpesviruses 4 and 1 and asinine herpesvirus 3 using monoclonal antibodies

A panel of 14 monoclonal antibodies (MAbs) was used to characterize the high abundance glycoproteins of equine herpesviruses 4 (EHV-4) and 1 (EHV-1), and asinine herpesvirus 3 (AHV-3). The specificities of the MAbs, which had been determined previously for strains of EHV-4 and -1 from the U.S.A., in general were confirmed by ELISA for Australian strains of these viruses. Of the 14 MAbs seven were EHV-4 and -1 type-common and cross-reacted with AHV-3. Of the five MAbs that were EHV-1 type-specific, four cross-reacted with AHV-3, whereas neither of the EHV-4 type-specific MAbs reacted with AHV-3, providing further evidence for a closer evolutionary relationship between EHV-1 and AHV-3 than that between either of these viruses and EHV-4. By Western blot and immunoprecipitation analyses, the identity of the six major glycoproteins, gp2, gp10, gp13, gp14, gp18 and gp21/22a, of an Australian EHV-1 isolate was verified, and it was shown that AHV-3 had cross-reactive glycoproteins of very similar Mr to those of EHV-1; five homologous glycoproteins of EHV-4 were also identified. It was determined that the EHV-4 gp13 homologue had a much reduced Mr (67K) when the virus was grown in a continuous cell line than when grown in equine foetal kidney cells (95K). It is suggested that altered glycosylation by the cell line is responsible for this change in Mr. Those glycoproteins acting as major immunogens in the naturally infected host, at least in their ability to elicit antibody, were identified. It was found that gp2, gp13, gp14, gp18 and a glycoprotein at 120K (EHV-1) or 116K (EHV-4) were all important immunogens in mares following EHV-1-induced abortion, and in a specific pathogen-free foal experimentally infected with EHV-1 and later cross-challenged with EHV-4. Gp2, gp14 and gp18 were the major immunogens in the donkey in response to AHV-3 infection. The type specificity associated with these glycoproteins was also examined and it was found that although most if not all contain type-specific epitopes, gp2 and a glycoprotein at 120K, and to a lesser extent gp13 and gp18, were significantly type-specific in the serum from a mare following natural EHV-1 infection and abortion.

Comparative studies of the proteins of equine herpesviruses 4 and 1 and asinine herpesvirus 3: antibody response of the natural hosts

Proteins of purified virions of equine herpesvirus 4 (EHV-4; equine rhinopneumonitis), EHV-1 (equine abortion virus) and asinine herpesvirus 3 (AHV-3) were compared by metabolic labelling with [35S]methionine or [14C]glucosamine during growth of low passage virus in natural host cells (horse or donkey) and high passage virus in an appropriate cell line and analysis by SDS-PAGE. Approximately 25 different proteins (Mr 300K to 21.5K) were clearly resolved for each virus. The three viruses had similar profiles although significant differences were found. The proteins of the cell line-grown viruses were similar to their precursor viruses grown in natural host cells although some small differences, probably related to differences in glycosylation by the various cell types, were noted. Six or seven high abundance glycoproteins were identified for EHV-4, EHV-1 and AHV-3. The profile of seven glycoproteins of AHV-3 was more similar to EHV-1 than to EHV-4. Antigenic relationships of the proteins of the three viruses were examined using radioimmunoprecipitation (RIP) and Western blot analyses and a series of polyclonal sera raised in colostrum-deprived, specific pathogen-free (SPF) foals which were immunized with inactivated EHV-4 (foal 3) or EHV-1 (foal 1), challenged and cross-challenged; a polyclonal donkey serum to AHV-3 was also used. The ontogeny of the antibody response in the SPF foals was studied and the major immunogenic proteins, as determined by RIP, were correlated with previously determined serum neutralizing antibody titres. Antibodies were first detected 14 days after primary immunization and were directed to EHV-4 proteins of Mr 113K, 75K and 56K or EHV-1 proteins of 110K, 78K, 60K and 58K. Antibodies to these same three (EHV-4) or four (EHV-1) proteins, together with antibodies to the major capsid protein and proteins of 67K (EHV-4) and 87K (EHV-1) were detected in response to primary infection (control foal 2) and these sera had high neutralizating antibody titres. The antigens of the three viruses were extensively cross-reactive with immunodominant proteins in the Mr ranges 150K to 110K and 62K to 56K. However, cross-absorption of EHV-4 and EHV-1 SPF foal antisera indicated the presence of significant amounts of type-specific antibody.

Asinine herpesvirus genomes: comparison with those of the equine herpesviruses

Two previously unknown and distinct herpesviruses were isolated from donkeys. One, with the characteristics of a betaherpesvirus, was isolated from the leukocytes of an apparently healthy donkey, while the second, an alphaherpesvirus, was recovered from the nasal cavity of donkeys given high doses of corticosteroids, and caused rhinitis in two seronegative weanling donkeys when they were intranasally infected. Few, if any, restriction endonuclease fragments were shared by the donkey betaherpesvirus, equine herpesvirus 2 (EHV 2) or EHV 5, a second distinctly different equine betaherpesvirus, nor by the donkey alphaherpesvirus, EHV 1, EHV 4, or EHV 3. In Southern blot analysis the donkey betaherpesvirus showed low levels of sequence similarity to both EHV 2 and EHV 5, while the donkey alphaherpesvirus and EHV 1 shared a moderate degree of sequence similarity, less similarity with EHV 4 and very low level of sequence similarity with EHV 3. These two isolates appear prototypic of two previously unrecorded herpesviruses for which the names asinine herpesvirus 2 and 3 are suggested for the betaherpesvirus and the alphaherpesvirus respectively.