Herpesvirus-associated neurological disease in a donkey Modest

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					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.

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   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-
    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.                              polymerase gene, as previously described.11,14 Equid her-
                                                          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;              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. The authors thank Dr. L. Smith-                    that the donkey virus is closely related to equine herpesvirus 1.
Maxie for expertise in the neurological assessment of this              Arch Virol 140:1653–1662.
animal.                                                              7. Kleiboeker SB, Schommer SK, Johnson PJ, et al.: 2002,
                                                                        Association of two newly recognized herpesviruses with
                                                                        interstitial pneumonia in donkeys (Equus asinus). J Vet Diagn
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