Wildl. Biol. Pract., December 2005, 1(2): 118-127DOI: 10.2461/wbp.2005.1.14

GENETIC ANALYSIS OF THE FIRST EUROPEAN BROWN HARE SYNDROME VIRUSISOLATES FROM GREECE

C. Billinis1, N.J. Knowles2, V. Spyrou3, G. Sofianidis4, V. Psychas4, P.K. Birtsas5,M. Sofia1, O.M. Maslarinou5, D.K. Tontis6 & D. Kanteres6

1Laboratory of Microbiology and Parasitology, Faculty of Veterinary Medicine, University of Thessaly,Trikalon 224, 43100 Karditsa, Greece. 2Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey, GU24 0NF,United Kingdom.3Department of Animal Production, Technological Educational Institution, 41110 Larissa, Greece.4Laboratory of Pathology Faculty of Veterinary Medicine, Aristotle University, 54124 Thessaloniki, Greece.5Hunting Federation of Macedonia and Thrace, Ethnikis Antistasis 173 - 175, 55134 Thessaloniki, Greece.6Laboratory of Pathology, Faculty of Veterinary Medicine, University of Thessaly, Trikalon 224, 43100Karditsa, Greece.

Author for correspondence: Prof. C. Billinis.Tel.: +30-2441066011Fax: +30-2441066092e-mail: billinis@vet.uth.grbillinissbfghaçerigho~rieahoj~çlkkbillinis@vet.uth.gr

Introduction

European brown hare syndrome (EBHS) virus is a member of the genus Lagovirusin the family Caliciviridae [1]. EBHSV causes severe necrotic hepatitis in bothwild and farmed hares of the species Lepus europaeus and Lepus timidus.Outbreaks of unusually high mortality in hares were described in which animalsusually died within 48-72 h [2]. The disease was first reported during 1980s, andoccurred simultaneously in many European countries [3-18]. EBHS has significantsimilarities to rabbit haemorrhagic disease (RHD) in its epidemiology,symptomatology and pathology. Clinical symptoms and pathological findings inthe two diseases are very similar. Both are characterized by rapid progression,mild nervous symptoms, serohemorrhagic liquid at the nostrils, hemorrhages on

AbstractEight European brown hares (Lepus europaeus) found dead throughout Greecewere examined for the presence of European brown hare syndrome (EBHS).The diagnosis of the disease was established by macro- and microscopicallesions, and RT-PCR analysis. The most common lesions, such as necrotichepatitis, were observed in the liver, while congestion and haemorrhages werepresent mainly in lungs and tracheal mucosa. To determine the extent ofgenetic heterogeneity of the EBHSV isolates, a 265 bp fragment of the capsidprotein (VP60) gene of the eight isolates were amplified by RT-PCR andsequenced. Phylogenetic analysis showed that the Greek isolates were indeedEBHS viruses and that EBHSV and RHDV differed by an average ofapproximately 39%. The maximum nucleotide variation amongst all theEBHS viruses was 15%, whereas variation within the RHDV group amountsto 11%. Apart from GRE-8, all the Greek viruses fell on a single geneticlineage and differed between 1 and 7%; however, bootstrap confidence limitswere particularly low on all the branches leading from the base of the EBHSVlineage to GRE-8 (viz. 20, 13 and 9%). The grouping to the remaining Greekisolates was better supported (65%).

KeywordsEBHS,genetic analysis,Greece,virus.

ORIGINAL PAPER

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serosa and mucosa, severe necrotic hepatitis and congestion of the spleen andkidneys [5, 19, 20]. Particularly, a further common feature of the two diseases isthe central pathogenetic role of hepatic damage [11]. The histologic findings in theliver in EBHS are characteristic and can be used to diagnose the disease [21].The viral etiology was first demonstrated in 1988 by electron microscopy [22].Wirblich [23] and Le Gall [24] confirmed, by sequencing the genome, that the twoviruses share a similar genomic organization but are two distinct caliciviruses.EBHSV and RHDV share approximately 71% of the nucleotide sequence identity[24]. The aim of the present study was to genetically characterize the first eightEBHSV isolates from Greece. The extent of genetic variation among these eightisolates and all other known EBHSV isolates was analyzed.

Material and methods

Samples for laboratory examination

Eight wildlife hares (Lepus europaeus), found dead throughout Greece, weresubmitted by hunting federations to the University. At necropsy, samples from liver,lungs, heart, spleen, kidneys, intestine and lymph nodes were collected and, exceptfor aliquots for RT-PCR, were fixed in neutral buffered 10% formalin, embedded inparaffin, sectioned at 4 to 5 µm and stained with haematoxylin and eosin andobserved by conventional light microscopy. Details of the viruses examined in thisstudy are shown in Table 1 and Figure 1.

Isolation of RNA

Liver and spleen specimens (25 mg of each) from the eight infected hares werehomogenized. RNA was isolated using a RNA isolation kit (Gentra Systems,Minneapolis, USA) according to the manufacturer’s instruction. Precautionsrecommended to avoid contamination with ribonucleases were observed [25].

Reverse transcription (RT) and polymerase chain reaction (PCR) amplification

Reverse-transcription (RT) reactions were performed by mixing 7.6 µl of RNAextraction product with 12.4 µl of an RT premix to obtain a final concentration of 1xfirst strand buffer (Gibco BRL), 0.5 mM dNTP mix (Gibco BRL), 10 mM DTT(Gibco BRL), 100 units of Moloney murine leukaemia virus (MMLV) reversetranscriptase (Gibco BRL), and 3.5 pmol/µl of random hexamers (PharmaciaBiotech) per reaction. Following preparation of the RT reaction mixtures on ice,reverse transcription reactions were carried out at 37 °C for 90 min, which were thenterminated by heating for 5 min at 98 °C.PCR amplifications were carried out observing the guidelines as previouslydescribed [26]. Each PCR reaction mixture contained 2 µl of RT product, 1x PCRbuffer (Gibco BRL), 1 mM MgCl2 (Gibco BRL), 0.2 mM of a dNTP mix (GibcoBRL), 0.15 U of Taq Polymerase (Gibco BRL), and 30 pmoles of the primers HEF5’-CCGTCCARCATTCGTCCTGTCAC-3’ and HEB 5’-CATCACCAGTCCTCCGCACCAC-3’ designed to amplify a part of the gene encoding the structuralpolypeptide VP60 (nucleotides 6423 to 6687 on the complete genome sequence of

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Designation Region

GRE-1 Lamia

GRE-2 Ioannina

GRE-3 Arta

GRE-4 Trikala

GRE-5 Ptolemaida

GRE-6 Corfu

GRE-7 Zagori

GRE-8 Chalkidiki

Table 1. Designation and origin of the EBHS viruses studied.

Fig. 1. Map of Greece showing the location of isolates

EBHSV GD, accession no. Z69620). The reaction volume was adjusted to 20 µl withDEPC treated H20. The PCR mixtures were prepared on ice and the reactions wereinitiated by heating for 9 min at 94 °C, followed by 35 cycles of: 45 sec at 94 °C, 45 secat 60 °C and 1 min at 72 °C. The mixtures were then brought to 72 °C for 7 min andthen held at 4 °C [27]. Ten µl of each RT-PCR product were analysed byelectrophoresis on a 2% agarose gel and stained with ethidium bromide (0.5 µg/ml).A 100 bp DNA ladder (Gibco, BRL) was analysed on the same gel to serve as a sizemarker. The expected sizes of the RT-PCR products were 265 bp.

Sequence analysis

To determine the extent of genetic heterogeneity of the eight EBHS isolates, PCRproducts (265 bp) were gel-purified (Qiaquick Gel Extraction Kit, Qiagen) andsequenced in both directions commercially by MWG Biotech (Germany), using theforward and reverse PCR primers. Sequence analysis was performed on 220 nt(positions 6446-6665 on the complete genome sequence of EBHSV GD, accessionno. Z69620) of a 265 bp RT-PCR fragment of the region coding for VP60. All thesamples were analysed twice and only high-quality sequences were used.Phylogenetic analysis was performed on 29 EBHSV isolates including the eightGreek isolates (EMBL accession numbers: AM048847 to AM048854) described inthis study. Nucleotide sequences from the other EBHSV isolates were retrieved fromthe EMBL database. The determined sequences were aligned by using the alignmentalgorithm of software package Sequence Navigator (Applied Biosystems). Neighbor-joining trees [28] were constructed using the program ClustalX v1.83 [29]. Theunrooted Neighbor-joining tree that was obtained was outgroup-rooted using 39RHDV sequences. One thousand bootstrap re-samplings were performed from withinthe ClustalX program and resulting trees were plotted using TreeView v1.6.6 [30].

Results

Pathological findings

All the hares submitted were adults and in poor body condition. Oedema, congestion,extensive haemorrhages in the lungs, as well as severe congestion of the trachealmucosa were observed (Fig. 2). Subcutaneous hemorrhages and bloody exudates fromthe nose, as well as conjunctivitis were noticed in many cases. The liver was swollen,discoloured and friable. The spleen was enlarged (Fig. 3) and hyperaemic and thekidneys were congested. No remarkable lesions were noticed in other organs. The mostprominent and consistent histopathological lesions were found in the liver. In sevenhares (designated GRE-1 to GRE-7) there were variable-size areas of periportal andmid-zonal hepatocellular necrosis. Acidophilic bodies were seen around or in thenecrotic areas (Fig. 4). Additionally, hepatocyte degeneration and calcium deposits wereevident at the periphery of the necrotic areas. The centrolobular areas werecharacterized by fatty degeneration of hepatocytes, most commonly microvesicular.There was also hyperaemia of sinusoids and mild to moderate inflammatory cellaccumulation in the portal areas, yet in few cases intralobular. A hare (designated GRE-8) exhibited mild periportal inflammatory reaction, prominent fibroplasia accompaniedby periportal degeneration and necrosis of hepatocytes (Fig. 5). The lungs were

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Fig. 4. Liver. Periportal and midzonal necrosis. There is mild inflammatory infiltration ofmononuclear cells. Arrows indicate acidophilic bodies. H&E.

Fig. 2. Lungs. Hyperaemia of tracheal mucosa and oedema. Arrowheads indicate haemorrhages inthe pulmonary parenchyma.

Fig. 3. Spleen. Enlargement and hyperaemia are prominent.

Fig. 5. Liver. Running bands of fibrous tissue between the lobules with lymphocytes. Necrosisand degeneration of hepatocytes are seen. H&E.

characterized by interstitial and alveolar haemorrhages and oedema (Fig. 6). Moreoverperivascular and peribronchial haemorrhages were occasionally seen. In the spleen, thered-pulp was congested and oedematous. Kidney congestion and tubulonephrosis wereseen as well.

RT-PCR amplification

EBHSV RNA was detected in all tissue examined. The expected size of the RT-PCRproducts, 265 bp, were observed as clear electrophoretic bands (Fig. 7).

Genetic characterization and phylogenetic analysis of the first eight EBHSV isolatesfrom Greece.

Phylogenetic analysis confirmed that the Greek viruses were indeed EBHS virusesand that the EBHSV and RHDV groups differed by an average of approximately39% (Fig. 8). The maximum nucleotide variation amongst all the EBHS viruses was15%, whereas within the RHDV group it was 11%. Apart from GRE-8, all the Greekviruses fell on a single genetic lineage and differed by 1 to 7%; however, bootstrapconfidence limits were particularly low on all the branches leading from the base ofthe EBHSV lineage to GRE-8 (viz. 20, 13 and 9%). The grouping to the remainingGreek isolates together was better supported (65%).

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Fig. 6. Lungs. Severe interstitial and alveolar haemorrhages and oedema. H&E.

Fig. 7. Agarose gel electophoresis of the RT-PCR assay for EBHS-V detection.Lane M: 100 bp ladder, lanes 1-5: liver RT-PCR product from 5 hares, lane 6: negative control,lane 7: positive control (liver from hare previously positive by respective RT-PCR assay).

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Discussion

The past 6 years EBHSV seems to have a high prevalence in Greece, since in a largenumber of dead hares submitted to our laboratories, the disease has been diagnosed.Thus, this virus may be one of the causes for increased mortality in the harepopulation. This fact prompts us to investigate our isolates phylogenetically, in orderto determine whether these isolates are closely related to other European or are

Fig. 8. Neighbor-joining tree showing the relationship of EBHSV isolates. The tree wasoutgroup-rooted using 39 RHDV sequences (EMBL/GenBank accession numbers U65317 toU65355). Boostrap confidence values (of 1000 replicates) are indicated as percentages andapply to branches to the left of the value.

1462

7356

100

100 13 20

16

17

23229

1009899

29

2015 97

53

99

98

6548

45

3682

94

5%

Austria 94b [U65359]Germany 89a [U65364]FRG [U09199]

Belgium 89 [U65360]UK91 CVLHAR [U65372]

GD [Z69620]GD [Z32526]

GRE-8BS89 [X98002]

Germany 89b [U65365]Czech R91 [U65362]

Finland 90 [U65363]Sweden 93 [U65371]Belgium 90 [U65361]

Austria 94a [U65358]Austria 92 [U65356]Austria 93 [U65357]

Germany 90a [U65366]Germany 90b [U65367]

Sweden 81 [U65368]Sweden 77 [U80981]

Sweden 82a [U65369]Sweden 82b

GRE-7 [U65370]GRE-1

GRE-3GRE-5

GRE-6GRE-2

GRE-4

simply local strains. The former could probably indicate either releasing ofindividuals or natural population movements. The latter may indicate maintenanceof the virus in the Greek hare population which in conjunction with other factorssuch as changes in habitat, agricultural techniques and environmental pollutionpromotes the disease development. This investigation is the first genetic analysisstudy of EBHS viruses isolated in Greece. Nucleotide sequence analysis of 29EBHSV and 39 RHDV isolates clearly separated EBHS and RHDV as two distinctmembers of the Caliciviridae family, with a maximum identity between the twogroups of only 61%. Phylogenetic analysis showed that all but one of the Greekviruses were monophyletic, and all were closely related to the other EBHS viruses.However, bootstrap values, based on 1000 replicates, were very low on about onethird of the branches indicating that either this genome region is not ideal forphylogenetic analyses or that a larger fragment of this region is required to give amore confident tree.On the basis of morphology of the liver lesions we recognized three forms of thedisease. An acute, a sub acute and a chronic form as have been previously described[31, 32]. The presence of acidophilic bodies mainly in the acute form of the diseasereflects the unicellular death of hepatocytes which become globular and small insize and lose their pycnotic nucleus by extrusion. These bodies are also called“Councilman bodies” and are seen in some forms of human viral hepatitis, yellowfever and ischemic necrosis [33]. In one hare, the histological picture of periportalfibrosis and hepatocellular necrosis with the simultaneous virus detection indicatesa chronic active hepatitis suggesting infection in a late viremic stage or possibleinfection by a different virus strain. Indeed, in our results strain GRE-8, isolatedfrom the hare with chronic form of the disease, presented the highest nucleotidedifference among all Greek isolates (7-10%). These individuals could play animportant role in epidemiology of EBHS by spreading and maintenance of thevirus.

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