J Vet Diagn Invest 20:820–823 (2008) Herpesvirus-associated neurological disease in a donkey Modest Vengust, Xin Wen, Dorothee Bienzle1 Abstract. 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. Key words: Asinine herpesvirus; encephalomyelopathy; equine herpesvirus; gammaherpesviruses. <!?show "fnote_aff1"$^!"content-markup(./author-grp/aff|./author-grp/dept-list)> A 4-year-old jenny standard donkey was referred to the corticosteroids. Mild hyperglycemia and hyperproteinemia Veterinary Teaching Hospital, Ontario Veterinary College identified on biochemical analysis were likely due to stress (University of Guelph, Guelph, Ontario, Canada), for and dehydration, respectively. Fecal parasite examination evaluation of depression and neurological signs. The identified a large number of strongyle eggs. patient had become progressively lame on the left hind The donkey was placed in an Anderson sling. In the limb on the day before referral and then gradually sling, the patient was able to eat and drink while supporting progressed to generalized weakness, head pressing, and her body weight through standing for short periods of time. eventual stupor and recumbency. The donkey had been Intravenous fluid therapy with lactated Ringer solution was treated by the referring veterinarian with intravenous instituted to correct dehydration. The donkey was stimu- trimethoprim-sulfadoxinea and intramuscular flunixin me- lated frequently to prevent her from returning to a glumineb and isoflupredone acetatec injections. stuporous state. Such arousal was applied every 2 hr to The donkey shared the pasture with another jenny move the animal and relieve pressure from the sling support donkey of the same age and 2 goats. All animals had been and to assure water and feed intake. Therapy with purchased 6 months previously. Two dogs and 1 cat were intravenous trimethoprim-sulfadoxinea was continued, present on the premises and had been in regular contact and intravenous dexamethasoned was administered. with the affected animal. Feeding consisted of pasture Neurological examination identified bilaterally absent access and free choice hay. Both donkeys had been menace reflexes with normal palpebral reflexes. The nasal vaccinated for rabies, tetanus, equine influenza, and Equid septum stimulation response was decreased, and mild paresis herpesvirus 1 and 4 (EHV-1 and -4) shortly before purchase. and ataxia were observed. These signs were consistent with a Donkeys and goats were dewormed twice a year; none aside lesion in the thalamocortex and possibly involving the spinal from the affected donkey had signs of disease, and animals cord. Differential diagnoses included a thalamocortical had neither traveled from the premises since purchase nor encephalopathy of metabolic origin, herpesvirus-associated been in contact with other domestic animals. neurological disease, Sarcocystis neurona or bacterial en- Upon presentation at the teaching hospital, the donkey cephalitis, or exposure to a toxin. Rabies was also considered; was in lateral recumbency, mildly hypothermic (36.5uC), therefore, appropriate isolation measures were implemented and near nonresponsive to stimuli. Physical exam revealed to protect personnel and other animals. Cerebrospinal fluid an increased heart rate and a normal respiratory rate. The (CSF) was collected from the lumbo-sacral space, and mucous membranes were tacky, and dehydration was examination revealed no signs of inflammation. estimated to be 7%. Blood gases, plasma electrolytes, a A deep pharyngeal swab was submitted for EHV-1 and complete blood cell count, and a serum biochemistry consensus herpesviral polymerase chain reaction (PCR) profile were assessed. Hematological abnormalities were testing. DNA was extractede and amplified. Primers for mild neutrophilia (10.9 3 109/l) and lymphopenia (1.5 3 EHV-1 were f-POL (59-GCAACTCGGTTTACGGATT- 109/l), which were attributed to stress and treatment with CA-39) and r-POL (59-GTCGCCCAACGAGAGTGAA- 39), which specifically amplify a 132-base pair (bp) region in the EHV-1 DNA polymerase gene. Polymerase chain From the Departments of Clinical Studies (Vengust) and Pathobiology (Wen, Bienzle), University of Guelph, Guelph, reaction with consensus herpesvirus primers utilized Ontario, Canada. DFASA (59-GTGTTGGACTTYGCNATGYYTNTAY- 1 Corresponding Author: Dorothee Bienzle, Department of CC-39) and IYG (59-CACAGAGTCCGTRTCNCCRTA- Pathobiology, University of Guelph, Guelph, Ontario N1G DAT-39), located in conserved regions of the DNA 2W1, Canada. email@example.com polymerase gene, as previously described.11,14 Equid her- 820 Case Reports 821 Figure 1. Multiple sequence alignment of 55 amino acids in the viral DNA polymerase gene from asinine herpesviruses (indicated by the prefix AsHV), Equid herpesvirus 7 (indicated by EHV7) and 2 (indicated by EHV2), and Zebra herpesvirus 1 (indicated by ZHV). Differences are highlighted by grey shading. pesvirus 1 PCR did not yield a product, whereas 2 rounds of ison of all sequences over 55 amino acids (length available PCR with the degenerate primers yielded a product of for AsHV-4 and -5) illustrated identity of the novel AsHV ,420 bp. This product was purified and sequenced with sequence (AsHV-Ontario Veterinary College [OVC]) with primer IYGseq (59-GACAAACACAGAGTCCGT-39).14 the 3 AsHV-5 sequences and a high degree of similarity After primer exclusion, the sequence was 381 bp, and with other equid herpesvirinae (Fig. 1). Phylogenetic searching through the National Center for Biotechnology analysis of the 55-amino acid sequence of the related Information (NCBI) database using Basic Local Align- herpesviral sequences placed the novel virus among the ment Search Tool (BLAST; http://www.ncbi.nlm.nih.gov/ previous AsHV-5 isolates (Fig. 2), whereas analysis using blast/Blast.cgi) revealed that the highest similarity was the entire amino acid sequence indicated closest relation- with 4 short (,165 bp) herpesviral sequences from ship with EHV-2 (Fig. 3). donkeys (Asinine herpesvirus-5 [AsHV-5]: AY054993.1, Twenty-four hours after hospitalization, the donkey was AY054994.1, AY054995.1, and AY054996.1). These se- able to stand and the Anderson sling was removed. The quences had previously been identified in donkeys with animal continued to improve neurologically, but developed interstitial pneumonia, and comparison with other herpes- mild laminitis, which was treated with appropriate therapy. viruses grouped them into the subfamily Gammaherpesvir- The donkey was discharged, and 3 weeks later no clinical inae.7 To further investigate the presence of a herpesvirus in abnormalities were present. this donkey with neurological disease, a second deep Herpesviruses are well recognized as causes of neurolog- pharyngeal swab was obtained, and DNA was extracted ical disease in horses and other species.1 In horses, disease is again. Since longer sequences of asinine herpesviruses were mostly characterized by myeloencephalopathy manifesting not available in the NCBI database, polymerase gene with clinical signs referable to spinal cord dysfunction. sequences of EHV-1, -2, -4, -5, and -7 were aligned,f and Microscopically, the lesions consist of vasculitis with conserved regions were chosen for primer design. The hemorrhage and thrombosis and secondary ischemic primers utilized were 1980F (59-CTCTGAGATAGCCAA- degeneration.13 Neurological disease is most frequently GATAGCCAA-39) and 2910R (59-GCACCAGGTCCA- attributed to reactivation of latent EHV-1, an alphaher- CCCCCTTCATGAGCA-39), which were identical to pesvirus, although EHV-2, a gammaherpesvirus, has also EHV-2 and highly similar at the 59 end to other equine been identified in the central nervous system of horses and herpesviruses and to Zebra herpesvirus 1 (ZHV-1). These might act as a transactivating factor.10 To the authors’ primers yielded a product of ,920 bp in 35 cycles of PCR knowledge, neurological disease in donkeys associated with amplification, which was sequenced in both directions with a herpesvirus has not previously been described. the same primers. The resulting sequence (after primer The donkey in the current report had neurological removal) was 875 bp in length (GenBank BankIt 1087327) disease consistent with lesions in the thalamocortex and and included the initial sequence of 381 bp. The new possibly spinal cord. Lack of inflammation in the CSF sequence had a 66.7% cytosine + guanine (C+G) content rendered bacterial or parasitic encephalomyelopathy un- and a CG ratio (observed frequency/expected frequency likely, and recovery of the animal ruled out rabies. A relative to mononucleotide composition) of 1.22. Nucleo- herpesvirus was identified in oronasal mucosa obtained tide identity with EHV-7, EHV-2, and ZHV-1 was 90%, with deep pharyngeal swabs by PCR using first nonspecific 88%, and 92%, respectively, and between 89% and 99% and then specific primers. Although the positive PCR with AsHV-4 and AsHV-5 over 168 bp. Translation of the product did not prove causation of the neurological disease novel donkey herpesvirus sequence into 291 amino acids, by this herpesvirus, similarity of the clinical features with and multiple alignment analysisf using the neighbor joining EHV-1–associated neurological disease in horses1 and the algorithm,12 indicated a high degree of similarity with clinical course with full recovery, suggested that the asinine EHV-7 (91%), EHV-2 (89%), and ZHV-1 (90%). Compar- herpesvirus designated AsHV-OVC was the cause of 822 Case Reports Figure 2. Phylogenetic tree of a 55-amino acid sequence from multiple equine gammaherpesvirus sequences. AsHV indicates asinine herpesviruses, EHV7 indicates Equid herpesvirus 7, EHV2 indicates Equid herpesvirus 2, and ZHV indicates Zebra herpesvirus 1 in the figure. disease in this animal. Whether disease resulted from propensity for latency due to epigenetic methylation of primary or reactivated latent infection could not be viral CpG motifs by host factors resulting in gene determined. The viral sequence recovered was distinct from silencing.11 Recent large-scale phylogenetic analysis of EHV-2 and -7, but closely related and of sufficient length to mammalian gammaherpesviruses illustrated their abun- confidently assign this virus to the gammaherpesviruses. dance among animals and their relatively high degree of Gammaherpesvirinae is a large subfamily including previ- similarity in the genes coding for DNA polymerase and ously identified AsHV-4 and -5 from donkeys with glycoprotein B.5 Further, this analysis confirmed that interstitial pneumonia, ZHV-1, and a virus identified in a equine and zebra gammaherpesviruses cluster together in wild ass, all of which were closely related to EHV-2 and the genus Percavirus, which would likely also include this EHV-7.4–8 The AsHV sequence identified in the present novel AsHV-5OVC.5 study had a C+G content of 66.7% in the DNA polymerase Asinine herpesviruses previously identified include gene, which is high, as in other herpesviruses such as EHV- AsHV-1 associated with coital exanthema, AsHV-2 isolated 2, Human herpesvirus 4 (also known as Epstein-Barr virus), from leukocytes of healthy animals, AsHV-3 obtained from and Human herpesvirus 1 (also known as Herpes simplex the nasal cavity of immunosuppressed animals,2 and virus 1).11 A CG binucleotide ratio greater than 1.0 AsHV-4, -5, and -6 from animals with interstitial pneumo- suggests that methylated C-G motifs (CpG) are relatively nia.7,8 Asinine herpesvirus 1 was classified as an alphaher- abundant in this sequence, which may correspond to a pesvirus, but had little similarity with EHV-3 by restriction endonuclease and Southern blot analysis.2 Asinine herpes- virus 2 had characteristics of betaherpesviruses, but little similarity with EHV-2 and -5 as determined with the same molecular methods.2 Asinine herpesvirus 3 is an alphaher- pesvirus based on sequence analysis of glycoprotein G and serological cross-reactivity of AsHV-3 with antibodies to EHV-1 and -4 glycoproteins.3,6,9 Asinine herpesviruses 4, 5, and 6 were designated as novel gammaherpesviruses based on sequences obtained from donkeys with interstitial pneumonia.7,8 The latter viruses were highly similar over ,165 bp in the DNA polymerase gene and ,380 bp in the terminase gene and appeared to represent closely related gammaherpesviruses. Overall, paucity of sequence infor- Figure 3. Phylogenetic tree of 291 amino acids located in the mation for AsHV-1–3, and limited sequence availability for viral DNA polymerase gene of the novel asinine herpesvirus AsHV-4–6, constrains comparison and classification of (indicated by AsHV-OVC) and Equid herpesvirus 2 (indicated by equine herpesviruses. Hence, although the longer sequence EHV2) and 7 (indicated by EHV7), and Zebra herpesvirus 1 obtained for the virus in the current report allowed (indicated by ZHV). convincing classification as a gammaherpesvirus and Case Reports 823 assignment of close relation with EHV-2 and -7, it was not 4. Ehlers B, Borchers K, Gund C, et al.: 1999, Detection of new possible to determine with confidence whether this virus is DNA polymerase genes of known and potentially novel identical to AsHV-5, or whether AsHV-4, -5, -6 and this herpesviruses by PCR with degenerate and deoxyinosine- novel virus represent a very closely related group of asinine substituted primers. Virus Genes 18:211–220. gammaherpesviruses. However, identification of this par- 5. Ehlers B, Dural G, Yasmum N, et al.: 2008, Novel mammalian herpesviruses and lineages within the gammaher- ticular herpesvirus with an assay capable of amplify- pesvirinae: cospeciation and interspecies transfer. J Virol ing a range of herpesviruses in an animal with neurological 82:3509–3516. disease adds to the spectrum of diseases potentially 6. Ficorilli N, Studdert MJ, Crabb BS: 1995, The nucleotide attributable to herpesviral infection of donkeys. sequence of asinine herpesvirus 3 glycoprotein G indicates Acknowledgements. 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