**2. Virology**

#### **2.1 Virus genome**

A whole genome sequence of EHV-9 has been determined as 148371 base pairs (bp) (accession number AP010838). The genome encodes at least 80 open reading frames (ORF) (Fig. 3, Table 2). There is no large deletion and insertion comparing with EHV-9 and EHV-1 genomes. All of the ORFs in EHV-1 are conserved in EHV-9 genome. The EHV-9 ORFs have been serially numbered as homologues in EHV-1 genome (Telford et al., 1992). Identities of each ORF to EHV-1 ORFs range from 86% to 99% (Table 2). ORFs showing higher identity (99 %) are glycoprotein K (ORF6), UL37 tegument protein (ORF23), VP26 capsid protein (ORF25), glycoprotein B (ORF33), thymidine kinase (ORF38), a major capside protein (ORF42), DNA packaging terminase subunit 1 (ORF44\_47), a nuclear protein UL3 (ORF60) and glycoprotein E (ORF74). ORFs showing lower identity (86 %) to EHV-1 include UL45 tegument/envelope protein (ORF15) and UL4 nonstructural protein (ORF58).

medulla. Lesions consisted of inflammation, neuronal necrosis, gliosis, and both neuronal and glial basophilic intranuclear inclusion bodies. PCR for the herpesvirus DNA polymerase gene segment was positive on DNA extracted from frozen tissues and from paraffinembedded fixed brain. The nucleotide sequence of the PCR product indicated the presence of EHV-9, which was further confirmed by following PCR for the EHV-9 gB gene segment. Schrenzel et al. (2008) described that EHV-9 had been detected at the same zoological garden in 2 Grevy's zebras (*Equus grevysi*) from the same herd, which had been relocated near the polar bears before the polar bear case. One of the infected Grevy's zebras was 8 days old and had viral interstitial pneumonia; the other was an adult with rhinitis and intranuclear inclusion bodies. Both zebras were immunocompromised as a result of other

Schrenzel et al. (2008) described that EHV-9 was found by a retrospective analysis of tissues from an aborted Persian onager (*Equus hemionus onager*) fetus from a zoological park in Washington, DC (Montali et al., 1985). The onager fetus was aborted after the dam came in close proximity to a Grevy's zebra. A herpesvirus was isolated from the fetus. The virus was identified as EHV-1 based on DNA fingerprinting and serological analyses (Montali et al., 1985). PCR and DNA sequencing analyses of the DNA polymerase showed that the zebras and the onager had an EHV-9 strain identical to that found in the polar bear (Schrenzel et

Zebras have been suspected to be the source of EHV-9 infection. To prove the hypothesis, serological analysis was examined by using 43 sera from Burchell's zebras (*Equus burchelli*) and 21 Thomson's gazelles from the Serengeti cocsystem for neutralizaing antibodies (Borchers et al., 2008). Seven zebra sera were positive for EHV-1 and EHV-9. The trigeminal ganglia of 17 other Burchell's zebras and one Thomson's gazelles were examined by PCR for EHV-9 gB and EHV-1 ICP0 genes. One zebra ganglion was positive for EHV-9 by PCR and confirmed by sequencing. These results suggest that the Burchell's zebras were latently

A whole genome sequence of EHV-9 has been determined as 148371 base pairs (bp) (accession number AP010838). The genome encodes at least 80 open reading frames (ORF) (Fig. 3, Table 2). There is no large deletion and insertion comparing with EHV-9 and EHV-1 genomes. All of the ORFs in EHV-1 are conserved in EHV-9 genome. The EHV-9 ORFs have been serially numbered as homologues in EHV-1 genome (Telford et al., 1992). Identities of each ORF to EHV-1 ORFs range from 86% to 99% (Table 2). ORFs showing higher identity (99 %) are glycoprotein K (ORF6), UL37 tegument protein (ORF23), VP26 capsid protein (ORF25), glycoprotein B (ORF33), thymidine kinase (ORF38), a major capside protein (ORF42), DNA packaging terminase subunit 1 (ORF44\_47), a nuclear protein UL3 (ORF60) and glycoprotein E (ORF74). ORFs showing lower identity (86 %) to EHV-1 include UL45

tegument/envelope protein (ORF15) and UL4 nonstructural protein (ORF58).

concurrent conditions.

al., 2008).

infected by EHV-9.

**2.1 Virus genome** 

**2. Virology** 

**1.4 Abortion in an onager** 

**1.5 Burchell's zebras from the Serengeti ecosystem** 


Fig. 3. Scheme of the EHV-9 genome based on the complete nucleotide sequence (AP010838)


Virology and Pathology of Encephalitis in Alien Hosts by Neurotropic Equine Herpesvirus 9 133

37 272 96 UL24 Nuclear protein, related to neurovirulence

39 850 96 UL22 Envelope glycoprotein (gH); complexes

41 239 98 UL20 Integral membrane protein; role in virion

42 1376 99 UL19 Major capsid protein; component of hexons

43 314 98 UL18 Capsid protein; component of

44\_47 734 99 UL15 DNA packaging terminase subunit 1 45 706 97 UL17 DNA packaging tegument protein

49 595 96 UL13 Tegument protein; probable serine-

51 73 91 UL11 Myristylated tegument protein; role virion

50 565 96 UL12 Deoxyribonuclease; role in

52 451 98 UL10 Envelope glycoprotein (gM)

55 303 96 UL7 Tegument progein

58 224 86 UL4 Nuclear protein

60 212 99 UL3 Nuclear protein

61 313 97 UL2 Uracil-DNA glycosylase

59 182 87 – Unknown

53 887 98 UL9 Replication origin-binding helicase

54 751 95 UL8 Component of DNA helicase–primase

56 753 97 UL6 Minor capsid protein; role in DNA; role in

57 881 97 UL5 Component of DNA helicase–primase

62 218 97 UL1 Envelope glycoprotein (gL); complexes

63 533 90 RL2 Transcriptional regulator, ICP0

with gH

egress

and pentons

with gL; role in cell entry

intercapsomeric triplex

threonine protein kinase

envelopement

complex

DNA packaging

complex; helicase

maturation/packaging of DNA

ORF Codons Identity (%) HSV-1 Predicted or confirmed functions

38 352 99 UL23 Thymidine kinase

40 530 97 UL21 Tegument protein

46 370 97 UL16 Tegument protein 48 318 93 UL14 Tegument protein


immediate-early genes

ORF Codons Identity (%) HSV-1 Predicted or confirmed functions

12 449 97 UL48 Tegument protein; transactivator of

16 468 96 UL44 Envelope glycoprotein (gC); role in cell

18 406 97 UL42 Processivity subunit of replicative DNA

19 497 96 UL41 Tegument protein; host shut-off factor 20 323 98 UL40 Small subunit of ribonucleotide reductase 21 790 97 UL39 Large subunit of ribonucleotide reductase

25 119 99 UL35 Capsid protein; located on tips of hexons, VP26

26 275 95 UL34 Membrane-associated phosphoprotein

30 1220 97 UL30 Catalytic subunit of replicative DNA

31 1209 98 UL29 Single-stranded DNA-binding protein 32 775 98 UL28 DNA packaging terminase subunit 2 33 980 99 UL27 Envelope glycoprotein (gB); role in cell

35.5 329 96 UL26.5 Major capsid scaffold protein

36 587 97 UL25 DNA packaging tegument protein

polymerase

scaffold protein

N-terminal protease domain acts in capsid maturation and is a capsid protein; Cteminal domain is the minor capsid

entry

22 465 97 UL38 Capsid protein; component of

24 3439 96 UL36 Very large tegument protein

27 139 95 UL33 Role in DNA packaging 28 620 93 UL32 Role in DNA packaging

29 326 98 UL31

35 646 97 UL26

34 160 93 – Unknown

23 1021 99 UL37 Tegument protein

17 401 96 UL43 Probable integral membrane protein

entry

polymerase

intercapsomeric triplex

11 305 93 UL49 Tegument protein, VP22

15 219 86 UL45 Tegument/envelope protein

13 868 93 UL47 Tegument protein 14 744 94 UL46 Tegument protein


Virology and Pathology of Encephalitis in Alien Hosts by Neurotropic Equine Herpesvirus 9 135

Sequences of glycoprotein G in EHV-1 derived from zoo animals would provide a clue to

EHV-9 strain P19, prototype of EHV-9, is closely related to the strain 1220 which was derived from a Burcell's zebra (Borchers et al., 2008), while the isolate 4 derived from the polar bear associated with Grevy's zebra (Schrenzel et al., 2008) is distantly related to the other EHV-9s. These data suggest the EHV-9 would be strongly related to each species of

zebras. Of cause, further research should be examined to prove the hypothesis.

Fig. 4. A phylogenc tree of EHV-9 and other related viruses based on the glycoprotein B gene segment. The phylogenic tree was constructed by using PHYLIP package (Felsenstein, 2005). Accession numbers are as follows: EHV-9 P19 (D49800), 1220 (EU087294), Giraffe (AB439723), Isolate-4 (EU717150); EHV-1 Ab4p (AY665713), RacL11 (X95374), Kentucky D (AB279609), Mar97 (DQ095871), Gazella/6755/NLD/2009 (HM216495), 94-137 (AB280624), Ro-1 (DQ095872), T-529 (AB280630), T-616 (EU087295), T-965 (DQ095873); EHV-4 (M26171).

**3.1 Lethal Encephalitis in zoo, domestic and companion animals, and experimental** 

EHV-9 caused lethal encephalitis in several animals such as Thomson's gazelles, giraffes, and polar bears naturally and goats, cats, dogs, mice, rats, hamster and marmosets experimentally. All of these infections can be regarded as encephalitis with neuronal degeneration, perivascular cuffing and gliosis. Histopathological characteristics will be

resolve the problem.

**3. Pathology** 

**small animals** 

described.


Identity was evaluated by protein-protein BLAST analysis.


Table 2. Characteristics of EHV-9 proteins

#### **2.2 Host range in vitro and in nature**

EHV-9 can be propagated by fetal equine kidney cells (FEK), Madine-Darby bovine kidney cells (MDBK), rabbit kidney cells (RK-13), murine fibroblast L929 cells, and human HeLa 229 cells. Cytopathic effects varied in each cell line. Lytic CPE is observed in FEK, RK-13 and HeLa 229, while syncitium formation is observed in MDBK (Fig.). EHV-9 can be also propagated in neural cells derived from a fetal equine brain as well as a fetal murine brain.

EHV-9 has been isolated from Thomson's gazelles, zebras (Borchers et al., 2008; Schrenzel et al., 2008), giraffes (Samy et al., 2009), polar bears (Schrenzel et al., 2008; Donovan et al., 2009) and onager (Schrenzel et al., 2008) as described in Section 1. Epizootiologically all of the cases in zoo animals associated with the presence of zebras. These data indicates that Burchell's zebra and other zebras might be a natural host of EHV-9 in nature.

EHV-9 can infect several animals experimentally. The experimental hosts include horse, goat, pig, cattle, hamster, mouse, rat, guineapig, dog, cat, and marmosette as described in the section 3.

#### **2.3 Phylogenic relatedness to other related herpesviruses**

The phylogenic tree constructed by using a part of glycoprotein B gene sequence indicates three groups of EHV-9, EHV-1 in horses and EHV-1 in zoo animals (Fig. 4).

EHV-1 derived from zoo animals might be considered as another type of equid herpesvirus. Unfortunately equine herpesvirus 8 glycoprotein G sequence is not available at present. Sequences of glycoprotein G in EHV-1 derived from zoo animals would provide a clue to resolve the problem.

EHV-9 strain P19, prototype of EHV-9, is closely related to the strain 1220 which was derived from a Burcell's zebra (Borchers et al., 2008), while the isolate 4 derived from the polar bear associated with Grevy's zebra (Schrenzel et al., 2008) is distantly related to the other EHV-9s. These data suggest the EHV-9 would be strongly related to each species of zebras. Of cause, further research should be examined to prove the hypothesis.

Fig. 4. A phylogenc tree of EHV-9 and other related viruses based on the glycoprotein B gene segment. The phylogenic tree was constructed by using PHYLIP package (Felsenstein, 2005). Accession numbers are as follows: EHV-9 P19 (D49800), 1220 (EU087294), Giraffe (AB439723), Isolate-4 (EU717150); EHV-1 Ab4p (AY665713), RacL11 (X95374), Kentucky D (AB279609), Mar97 (DQ095871), Gazella/6755/NLD/2009 (HM216495), 94-137 (AB280624), Ro-1 (DQ095872), T-529 (AB280630), T-616 (EU087295), T-965 (DQ095873); EHV-4 (M26171).
