Detection and molecular characterization of Suid herpesvirus type 1 in Austrian wild boar and hunting dogs

Veterinary Microbiology 157 (2012) 276–284

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Detection and molecular characterization of Suid herpesvirus type 1 in Austrian wild boar and hunting dogs Adolf Steinrigl a,*, Sandra Revilla-Ferna´ndez a, Jolanta Kolodziejek b, Eveline Wodak a, Zolta´n Bago´ a, Norbert Nowotny b,c, Friedrich Schmoll a, Josef Ko¨fer a,d a

AGES Institute for Veterinary Disease Control Mo¨dling, Robert Koch Gasse 17, A-2340 Mo¨dling, Austria Clinical Virology, Department of Pathobiology, University of Veterinary Medicine Vienna, Veterina¨rplatz 1, A-1210 Vienna, Austria Department of Microbiology and Immunology, Faculty of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman d Institute for Veterinary Public Health, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna, Veterina¨rplatz 1, A1210 Vienna, Austria b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 September 2011 Received in revised form 20 December 2011 Accepted 23 December 2011

Aujeszky’s disease (AD), caused by Suid herpesvirus type 1 (SuHV-1), is an economically important disease in domestic swine. Thus, rigorous control programmes have been implemented and consecutively AD in domestic swine was successfully eradicated in many countries, including Austria. However, SuHV-1 continues to thrive in wild boar populations, as indicated by high seroprevalences in a number of European countries and by occasional cases of AD in hunting dogs. For the first time, SuHV-1 was detected in Austrian wild boar and a molecular characterization of SuHV-1 isolated from wild boar and hunting dogs was performed. Results of preliminary serological analyses suggest a regional SuHV-1 seroprevalence of over 30% in free-living and almost 70% in fenced wild boar from Eastern Austria. Molecular typing of Austrian SuHV-1 isolates of wild boar origin revealed the presence of two genetically distinct variants of SuHV-1, both capable of infecting dogs that have been exposed to infected wild boar during hunting. ß 2012 Elsevier B.V. All rights reserved.

Keywords: SuHV-1 Wild boar Hunting dog Austria gC coding region Phylogenetic analysis

1. Introduction Aujeszky’s disease (AD) is an OIE notifiable disease caused by Suid herpesvirus type 1 (SuHV-1), also known as pseudorabies virus (PRV), a member of the subfamily Alphaherpesvirinae. AD is primarily a disease of swine (Suidae), in which a variety of clinical symptoms have been described, such as disorders of the central nervous system (CNS), the respiratory system and the digestive tract, as well as reproductive disorders. Apart from members of the family Suidae, a wide range of mammalian and other vertebrate species, such as carnivores, rodents and ungulates, are susceptible to SuHV-1 infection (Metten-

* Corresponding author. Tel.: +43 50555 38248; fax: +43 50555 38309. E-mail address: [email protected] (A. Steinrigl). 0378-1135/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2011.12.033

leiter, 2000; Pomeranz et al., 2005; Mu¨ller et al., 2011). In these secondary hosts, SuHV-1 infection leads to fatal neurological disease; animals succumb to massive neurological dysfunction within a few days of disease onset. In general, disease in secondary hosts is observed only sporadically. During acute infection, SuHV-1 first infects cells of the mucous membranes within the oropharynx, digestive, respiratory or genital tract and then travels via axonal transport to the CNS. If pigs survive acute disease, the infection enters a latent state in which the virus retreats to neurons, where it remains episomally as closed circular DNA (Pomeranz et al., 2005). Due to its utmost economic importance, SuHV-1 infection in domestic pigs has been the target of surveillance programmes in a number of countries, including many European Union (EU) member states. Currently, 12 EU

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member states are in all regions officially recognized free of AD in domestic pigs (Decision 2011/648/EU, Annex I). Austria received its AD-free status in 1997 (Decision 97/423/ EG) and has since remained free from AD in domestic pigs. However, the fact that SuHV-1 is present in wild boar in many countries is a reason for concern, due to potential spillover to domestic pig populations. Testing for SuHV-1 antibodies in the wild boar populations of several European countries revealed medium to high numbers of SuHV-1 infected wild boar (Mu¨ller et al., 1998a; Albina et al., 2000; Lutz et al., 2003; Vengust et al., 2005; Toncˇic´ et al., 2006; Sedlak et al., 2008; Pannwitz et al., 2011) and an agedependent pattern of infection: typically, adults are more often seropositive than piglets or juveniles (Lutz et al., 2003; Pannwitz et al., 2011). Results from the eastern part of Germany, where the most extensive study on SuHV-1 seroprevalence has been conducted so far, suggest that SuHV-1 seroprevalence has continuously increased over the recent years, which might also be related to an increase in wild boar population density (Pannwitz et al., 2011). Due to the persistent nature of SuHV-1 infection, virus may be reactivated in wild boar and thus potentially pose a threat to domestic animals. Occasionally, spill-over from the wild boar population has been documented by the emergence of AD in hunting dogs that had confirmed or suspected close contact to wild boar during hunting (Mu¨ller et al., 1998b, 2010; Thaller et al., 2006; Cay and Letellier, 2009; Leschnik et al., 2012). Genetic characterization has been useful in order to identify potential epidemiologic linkage between SuHV-1 strains. Based on restriction fragment length polymorphism (RFLP) analysis patterns, SuHV-1 can be divided into four major genotypes (Herrmann et al., 1984). More recently, phylogenetic analysis of the SuHV-1 glycoprotein C (gC) coding region has been performed for molecular typing purposes (Goldberg et al., 2001; Hahn et al., 2010; Mu¨ller et al., 2010). A recent analysis of SuHV-1 strains from several European countries revealed a predominance of SuHV-1 genotype I in European wild boar; furthermore, two clades (clades A and B, respectively) were differentiated among European SuHV-1 strains of wild boar origin based on gC sequence analysis (Mu¨ller et al., 2010). Data regarding prevalence and genetic variation of SuHV-1 in Austrian wild boar was missing so far, although Austria is bordering several countries with a reportedly high prevalence (about 30%) of SuHV-1 antibodies in wild boar (Vengust et al., 2005; Toncˇic´ et al., 2006; Sedlak et al., 2008). Furthermore, this study was fuelled by the unusually high incidence of AD in hunting dogs in late 2010 (Leschnik et al., 2012). Here, we performed a genetic characterization of SuHV1 strains detected in Austrian hunting dogs and wild boar from 2005 to 2010. Furthermore, we present results from a preliminary SuHV-1 serosurvey in Austrian wild boar. 2. Materials and methods 2.1. Tissue sampling from wild boar and hunting dogs Brain samples from seven Austrian hunting dogs that were diagnosed with AD in the years 2005–2010 were

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received from the University of Veterinary Medicine, Vienna. All seven dogs had succumbed to AD soon after confirmed or suspected contact to wild boar within Austrian territory; furthermore, SuHV-1 infection in these dogs had been confirmed by immunohistochemistry and PCR, as already described (Thaller et al., 2006; Leschnik et al., 2012). In response to the high incidence of AD in hunting dogs in 2010, wild boar tissues from 88 hunted wild boars, including one exenterated wild boar carcass, were submitted to our laboratory from several regions of Eastern Austria: 66 wild boars were free-ranging, while 22 were kept in a single fenced holding. Epidemiological links were present between AD-deceased hunting dogs and sampled wild boars in three cases: two of the hunting dogs from 2010 had participated on a wild boar hunt within the described fenced holding; in the case of another deceased hunting dog from 2010, the wild boar carcass the dog had fed upon was received. 2.2. DNA preparation and PCR testing of wild boar tissue For SuHV-1 screening purposes, DNA was extracted from pooled wild boar tissues, typically consisting of tonsils, lung, spleen and lymph nodes. In addition, tonsils were extracted separately in about one third of wild boars. Upon detection of SuHV-1 DNA in pooled tissue extract or tonsil, all available tissues from this animal were reextracted individually. All DNA extractions were done with the High Pure PCR Template Preparation kit (Roche, Austria), following the manufacturer’s instructions. DNA was stored at 20 8C until analysis. Extracted DNA was tested by real-time PCR (ADIAVET1 PRV REALTIME, Adiagene, France), targeting a nonspecified fragment within the SuHV-1 gD coding region, as well as control DNA (EPC) in a duplex real-time PCR. As suggested by the manufacturer, 5 ml of EPC was added to each sample prior to extraction. For confirmation of positive real-time PCR results, a nested PCR targeting the glycoprotein B (gB) coding region of SuHV-1 was used (Balasch et al., 1998). PCR products of the expected size were sequenced to verify the presence of SuHV-1 DNA (BigDye1 Terminator v1.1 Cycle Sequencing Kit, Applied Biosystems, Austria). Sequencing reactions were resolved on a 3130xl Genetic Analyzer (Applied Biosystems). 2.3. Histological and immunohistochemical analysis of wild boar tissue Tissues from PCR-positive wild boar underwent standard histological and immunohistochemical analysis. Tissue samples (tonsils, lung and spleen from two animals, nasal mucosa, pharynx, tonsils, brain, trigeminal ganglia, parotid gland, mandibular lymph nodes and kidney from the exenterated carcass) were embedded in paraffin wax for histological examination, sectioned at 3–4 mm and stained with haematoxylin and eosin. Immunohistochemical staining for detection of SuHV-1 capsid antigen UL19 was performed using an avidin–biotin complex (ABC) detection system (Vector Laboratories, Burlingame, CA,

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USA) according to the manufacturer’s instructions (Hsu et al., 1981). The primary antibody was a polyclonal rabbitanti-PrV UL19 antiserum (dilution 1:750), kindly provided by Dr. B. Klupp and Prof. J. Teifke, FLI, Insel Riems, Germany (Klupp et al., 2000). Additionally, a commercially available monoclonal antibody (3G9F3; VMRD, USA) against a major immunodominant glycoprotein of SuHV-1 was also employed for immunohistochemistry, using the same procedure as mentioned above (Ramos-Vara and Beissenherz, 2000). 2.4. Phylogenetic analysis of SuHV-1 obtained from wild boars and hunting dogs The partial gC coding sequence of all SuHV-1 positive wild boars and hunting dogs was amplified using published primers (Mu¨ller et al., 2010). Best results were obtained using Phusion1 Hot Start DNA polymerase (Finnzymes, Austria) and a concentration of 8% DMSO in the reaction mix. The following thermoprofile was employed: initial denaturation at 98 8C for 3 min, followed by 40 cycles of 3-step PCR (98 8C/10 s–71 8C/30 s–72 8C/ 30 s) and a final elongation step at 72 8C for 10 min. PCR amplicons of the expected size were recovered after agarose gel electrophoresis and purified using the QIAquick1 Gel Extraction Kit (Qiagen, Austria). Purified PCR fragments were directly sequenced in both orientations using the PCR primers (BigDye1 Terminator v3.1 Cycle Sequencing Kit, Applied Biosystems). Newly described Austrian SuHV-1 gC sequences are available from GenBank under accession numbers JQ081284–JQ081293. Reference SuHV-1 gC coding sequences were downloaded from GenBank. Sequence alignments of the gC coding region were performed on the RevTrans 1.4 Server, which builds an amino acid alignment based on translated nucleotide sequences and uses this alignment as scaffold for correct positioning of gaps within a protein coding region (Wernersson and Pedersen, 2003). All gaps were removed, resulting in a final alignment of 639 bp that was used for further analysis. Neighbour Joining analysis was performed with MEGA4 (Tamura et al., 2007), using the composite maximum likelihood substitution model for calculating nucleotide distances. 1000 bootstrap replicates were done to assess statistical support of the branches. 2.5. Virus isolation and restriction fragment length polymorphism (RFLP) analysis Frozen-stored brain samples from the four hunting dogs that died from AD in 2010 were homogenized in minimal essential medium (MEM) with Earle’s salts and Lglutamine (PAA Laboratories GmbH, Austria) and centrifuged at 1600  g. Supernatant (0.5 ml) was inoculated onto a 90–100% confluent monolayer of PK-15 cells in T25 tissue culture flasks (Sarstedt Inc., USA) with MEM and incubated for 1 h. Following incubation, medium and inoculum was decanted and the cells were washed once with phosphate buffered saline (PBS). Thereafter, MEM, supplemented with 2% fetal calf serum, 1% antibiotics and 1% fungistatics, was added. Cell cultures were further incubated at 37 8C and assessed daily for the occurrence of

a typical herpesviral cytopathic effect (CPE). When approximately 70% of the monolayer exhibited a CPE, the supernatant was harvested, aliquoted and frozen ( 80 8C). Afterwards, sub-confluent VERO cells, grown in T75 flasks, were inoculated with freshly thawed SuHV-1 isolates and incubated until CPE was at least 90%. Following three freeze–thaw cycles, cellular debris was removed by low speed centrifugation and the viral supernatants were ultra-centrifuged for 90 min at 100,000  g and 4 8C. The viral pellet was resuspended in PBS and high molecular weight DNA was extracted with the Gentra1 Puregene1 Cell kit (Qiagen, Austria), following the body fluid protocol. One microgram of DNA was then digested for 3 h at 37 8C with 10 U of BamHI (Fermentas, Austria) and separated by overnight electrophoresis (40 V) in a 0.6% agarose gel containing 0.5 mg/ml ethidium bromide. Assignment of SuHV-1 strains to genotypes and subtypes within genotypes was based on previously reported BamHI patterns (Herrmann et al., 1984; Mu¨ller et al., 1998a,b, 2010). SuHV-1 strains Bartha and Bratislava were used as reference strains; the latter exhibits a BamHI restriction profile identical to the widely used Kaplan strain. 2.6. Serosurvey of wild boar Heart blood samples from a total of 210 hunted wild boar (95 free-ranging, 115 fenced – from three different holdings) from the eastern part of Austria were collected from autumn 2010 to spring 2011. Wild boar sera were prepared from heart blood samples and analysed for antibodies directed against the SuHV-1 gB protein using a commercial ELISA (Pseudorabies Virus gB Antibody Test Kit; IDEXX, Austria), following the manufacturer’s instructions. 3. Results 3.1. Detection of SuHV-1 DNA in wild boar and epidemiological links to AD in hunting dogs SuHV-1 DNA was detected by real-time PCR in pooled tissue extracts from two wild boars and in tonsil from a third animal, corresponding to 3.4% of tested wild boar (n = 88). Detection of SuHV-1 DNA by real-time PCR was confirmed in all three wild boars by gB nested PCR and sequencing. All three PCR-positive wild boars were epidemiologically linked to recent AD-cases in hunting dogs: two of the PCR-positive wild boars were from the fenced holding, where two hunting dogs most probably acquired SuHV-1 infection during hunting (Fig. 1, left boxed area). In addition, the wild boar carcass another hunting dog had fed upon shortly before dying from AD, also tested PCR-positive (Fig. 1, right boxed area). Re-testing of all available tissues (extracted individually) from PCR-positive wild boars demonstrated SuHV-1 DNA in tonsils of all three animals and, additionally, in the lung of one animal, the spleen of another animal and in pharynx and nasal mucosa of the exenterated wild boar carcass. All other tissues from this carcass (brain,

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Fig. 1. Geographical map of the eastern part of Austria, showing most probable infection sites of hunting dogs which have been diagnosed with AD in the years 2005–2010 (black arrows, with year of isolation on top). Furthermore, sampling sites of SuHV-1 PCR-positive (black circle segments) and seropositive (white circles and circle segments) wild boar are shown. Boxed areas indicate the sites where SuHV-1 has been detected in epidemiologically linked hunting dogs and wild boar. Numbers next to circles/circle segments/arrows indicate the total number of positive animals per location (AD-cases in dogs in italics); numbers in the top left insert box indicate the total number of SuHV-1 positive animals. AD: Aujeszky’s disease; WB: wild boar.

trigeminal ganglia, parotid gland, mandibular lymph nodes and kidney) tested negative. Testing of individual tissue extracts in the same real-time PCR run showed that, in each animal, tonsil had lower Cq-values than the other SuHV-1 positive tissues, indicating that SuHV-1 load was highest in tonsils (data not shown). 3.2. Histopathology and immunohistochemistry of PCRpositive wild boar tissues A slight oligofocal purulent-necrotizing tonsillitis was detected in all three PCR-positive wild boars. In one animal a focal necrotizing lymphadenitis of the mandibular lymph node was seen additionally. Lung lesions were present in two animals in form of moderate bronchointerstitial and slight interstitial intralobular pneumonia. Furthermore, a desquamative-eosinophilic (verminous) bronchitis in association with Metastrongylus sp. was demonstrated. No intranuclear inclusion bodies were present in the investigated organs. Immunohistochemical analyses revealed exclusively intracytoplasmic granular single cell signals in the tonsillar epithelium and follicles of all three animals and in the mandibular lymph node of the wild boar carcass. However, these signals were not associated with any specific organic

lesion. Negative tissue controls and consecutive slides of the investigated tissues without primary antibodies showed no specific signals (data not shown). 3.3. Phylogenetic analysis of Austrian SuHV-1 isolates of wild boar origin Partial gC coding regions were amplified from all seven AD-affected dogs from 2005 to 2010, as well as from all three PCR-positive wild boars from 2010. Phylogenetic analysis was performed based on an alignment of 639 bp. Reference sequences represented all currently published SuHV-1 gC sequences of European wild boar origin (isolated from either wild boar or hunting dogs). SuHV-1 strains from Austrian hunting dogs and wild boar grouped within gC sequence clade A (Mu¨ller et al., 2010). However, they belonged to two different phylogenetic lineages within clade A (Fig. 2): lineage 1 encompassed five Austrian strains, obtained from two wild boars and three hunting dogs in 2010. The strong epidemiological linkage between four of these strains (from two hunting dogs and the two wild boars from the fenced holding; see above) was supported by sequence analysis, since SuHV-1 gC sequences obtained from these animals were identical (Fig. 2). In addition, gC sequences from these four strains

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GQ259094/12-GER_BRB/Germany/WB/1995 JQ081292/2111-2_10/Austria/HD/2010 GQ259095/13-GER_BRB/Germany/WB/1995

Lineage 1

GQ259093/11-GER_ST/Germany/WB/1996 JQ081287/2099-4_10/Austria/WB/2010

83 JQ081288/2099-22_10/Austria/WB/2010 GQ259092/10-GER_ST/Germany/WB/1996 JQ081293/2216_10/Austria/HD/2010 GQ259117/612-GER_BRB/Germany/WB/2003 JQ081291/2111-1_10/Austria/HD/2010 GQ259096/15-GER_BRB/Germany/WB/1996 GQ259118/613-GER_SN/Germany/WB/2005

GQ259109/558-SVK/Slovakia/WB/1999 GQ259101/549-SVK/Slovakia/WB/1998

Lineage 2

88 GQ259110/559-SVK/Slovakia/WB/1999

clade A

GQ259111/560-SVK/Slovakia/WB/2000

GQ259112/561-ITA/Italy/WB/1993 GQ259098/527-FRA/France/HD/JQ081284/29_05/ Austria/HD/2005

83 85

GQ862778/GER-614_BW/Germany/HD/2008 JQ081285/1002_08/Austria/HD/2008 JQ081286/1645_08/Austria/HD/2008 JQ081289/2110-2_10/Austria/HD/2010

96

JQ081290/2110-3_10/Austria/WB/2010 GQ259113/563-HUN/Hungary/WB/1997-98

98 GQ259114/576-HUN/Hungary/WB/1997-98 GQ259115/594-FRA/France/HD/2002 GQ259103/551-GER_NRW/Germany/WB/1999 GQ259120/2872-ESP/Spain/WB/2000 GQ259125/2890-ESP/Spain/WB/2004 GQ259091/2960-ESP/Spain/WB/2004 GQ259100/537-FRA/France/HD/1999 GQ259107/555-GER_RP/Germany/WB/2000 GQ259124/2886-ESP/Spain/WB/2005

GQ259123/2882-ESP/Spain/WB/2005

86

GQ259106/554-GER_RP/Germany/HD/2000 GQ259121/2873-ESP/Spain/WB/GQ259097/519-FRA/France/WB/GQ259108/556-GER_NRW/Germany/WB/2000 GQ259104/552-GER_NRW/Germany/WB/1999 GQ259105/553-GER_RP/Germany/HD/2000 GQ259116/611-GER_RP/Germany/HD/2003 GQ259119/2885-ESP/Spain/WB/2005 GQ259099/536-FRA/France/HD/1999 GQ259102/550-GER_NRW/Germany/WB/1999 AF158090

99 0.005

AF403051

clade B

GQ259122/2874-ESP/Spain/WB/2005

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YP_068347 29_05 1002_08 1645_08 2110-2_10 2110-3_10 2099-4_10 2099-22_10 2111-1_10 2111-2_10 2216_10

10 20 30 40 50 ....|....|....|....|....|....|....|....|....|....| MASLARAMLALLALYAAAIAAAPSTTTALDTTPNGGGGGNSSEGELSPSP MASLARAMLALLALYAAAIAAAPSTTTALGTTPNGGGGGNSSGGELSPSP MASLARAMLALLALYAAAIAAAPSTTTALGTTPNGGGGGNSSEGELSPSP MASLARAMLALLALYAAAIAAAPSTTTALGTTPNGGGGGNSSEGELSPSP MASLARAMLALLALYAAAIAAAPSTTTALGTTPNGGGGGNSSEGELSPSP MASLARAMLALLALYAAAIAAAPSTTTALGTTPNGGGGGNSSEGELSPSP MASLARAMLALLALYAAAIAAAPS-TTALGTTPNGGG--NSSEGELSPSP MASLARAMLALLALYAAAIAAAPS-TTALGTTPNGGG--NSSEGELSPSP MASLARAMLALLALYAAAIAAAPS-TTALGTTPNGGG--NSSEGELSPSP MASLARAMLALLALYAAAIAAAPS-TTALGTTPNGGG--NSSEGELSPSP MASLARAMLALLALYAAAIAAAPS-TTALGTTPNGGG--NSSEGELSPSP

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Lineage 2

Lineage 1

Fig. 3. Amino acid alignment of deduced gC protein sequences from Austrian SuHV-1 strains of wild boar origin. The 50 N-terminal residues are shown. The position of deletions relative to reference sequence YP_068347 at positions 25, 38 and 39 is indicated by black arrows. Brackets indicate the assignment of sequences to either lineage 1 or 2, according to phylogenetic analysis of the gC nucleotide sequence (Fig. 2).

were identical to those obtained from another Austrian hunting dog in 2010 and to several SuHV-1 isolates from wild boar of Eastern Germany. The remaining Austrian SuHV-1 strains, obtained from four Austrian hunting dogs and one wild boar, belonged to a second lineage (lineage 2). Again, sequence analysis confirmed the suspected epidemiological linkage between one hunting dog from 2010 and the wild boar carcass the dog had fed upon, as SuHV-1 gC sequences isolated from both animals were identical. SuHV-1strains recovered in 2005 and 2008, respectively, from three AD-deceased Austrian hunting dogs also belonged to lineage 2 (Fig. 2). Comparison of deduced gC amino acid sequences supported the assignment of Austrian SuHV-1 strains into two groups, according to the presence of amino acid deletions relative to gC reference sequence YP_068347: all Austrian strains belonging to lineage 1 in the phylogenetic tree had deletions at amino acid positions 25, 38 and 39, while none of these deletions were present in Austrian strains belonging to lineage 2 (Fig. 3). 3.4. RFLP analysis SuHV-1 genotyping by RFLP was performed with virus isolated from the four Austrian dogs from 2010. Unfortunately, replication competent virus from AD-cases in hunting dogs before 2010 and from PCR-positive wild boars was not available. All four Austrian SuHV-1 isolates exhibited the classical restriction pattern present in SuHV1 genotype I (Herrmann et al., 1984), as also seen in reference strains Bartha and Bratislava (Fig. 4). Furthermore, three isolates could be assigned to subtype Iw (Mu¨ller et al., 2010), as judged by the presence of 5-10/512 double fusion bands (Fig. 4, arrows). One isolate

exhibited a BamHI pattern similar to reference strain Bratislava as well as to wild boar-derived strains classified as subtype Ip by Mu¨ller et al. (2010). Notably, results from RFLP analysis were in accordance with those from phylogenetic analysis, as the Austrian Iw isolates all belonged to lineage 1 in the phylogenetic tree, while the single Ip strain was associated with lineage 2. 3.5. Serosurvey of wild boar from Eastern Austria Testing of wild boar sera, mostly originating from the north-eastern part of Lower Austria, revealed an overall seroprevalence of 55.2% (n = 116). However, seroprevalences varied strongly between sampling sites and were considerably different between free-living wild boar (37.9%; n = 36) and animals from fenced holdings (69.6%; n = 80). Most seropositive animals originate from a relatively small region in the north-eastern part of Lower Austria (Fig. 1) and were kept in three different fenced holdings. The remaining seropositive wild boars were freeranging and were shot at different locations throughout Lower Austria and in two further adjacent Austrian provinces (Burgenland and Upper Austria). 4. Discussion Although Austria is officially recognized free from AD in domestic pigs, the presence of SuHV-1 within the Austrian wild boar population has been suspected, mainly based on occasional reports of AD in hunting dogs (Thaller et al., 2006; Leschnik et al., 2012). Due to the unusually frequent appearance of AD in hunting dogs in late 2010 (Leschnik et al., 2012), blood and tissue samples from shot wild boar were at that time frequently submitted to our laboratory,

Fig. 2. Unrooted Neighbour-Joining tree based on a 639 bp alignment (gaps removed) from the SuHV-1 gC coding region. Sequences are identified by: GenBank accession number/strain/country/species/year of isolation (if known). Austrian strains are in bold typewriting. Epidemiological linkage between wild boar and hunting dog-derived strains is visualized by dashed lines. Numbers along the branches represent percentages of 1000 bootstrap iterations. Bootstrap values over 80% are shown. Clades A and B (Mu¨ller et al., 2010) are indicated by brackets. Sequences AF158090 and AF403051 (SuHV-1 of Asian origin) were used as outgroup. HD: hunting dog; WB: wild boar.

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Fig. 4. RFLP patterns of SuHV-1 strains isolated from four hunting dogs, obtained after BamHI digestion. Molecular size markers are indicated by m (Ready load 1 kB marker; Invitrogen, Austria) and M (Ready load l HindIII marker, Invitrogen, Austria). Approximate molecular masses are indicated on the left hand side. Lane 1: reference strain Bratislava; lane 2: reference strain Bartha; lanes 3–6: strains 2111-1_10, 2111-2_10, 21102_10 and 2216_10. Additional bands between BamHI fragments 3 and 4 (5-10/5-12 double fusion bands; arrows) in lanes 3, 4 and 6 indicate that these strains belong to subtype Iw (Mu¨ller et al., 2010). The uppermost band in lanes 3–6 represents a fraction of DNA that was resistant to BamHI digestion. The same RFLP patterns were consistently observed in several experiments.

which constitutes the Austrian National Reference Laboratory for AD. Subsequent detection of both SuHV-1 DNA as well as SuHV-1 antibodies confirmed for the first time the presence of this virus in wild boar in the eastern part of Austria. The observed seroprevalence of 37.9% in free ranging wild boar is similar to figures reported by neighbouring countries Croatia, Slovenia and Czech Republic (Vengust et al., 2005; Toncˇic´ et al., 2006; Sedlak et al., 2008). However, SuHV-1 seroprevalence in Austrian wild boar was considerably higher in animals that were kept within fenced holdings (69.6%), indicating that close contact facilitates spreading of the virus. Importantly, sampling from wild boar was based on voluntary submissions from hunters. Thus, seroprevalences reported in this study might be subject to some sampling bias. Currently, a wild boar screening programme based on hunting bag numbers is being carried out throughout Austria, which should result in a more representative estimate of SuHV-1 seroprevalence. In contrast to the high seroprevalence, SuHV-1 DNA was detected in wild boar tissues from the same region in only a few cases. Importantly, PCR testing was performed on pooled wild boar tissues and – in a proportion of cases – tonsils, since CNS tissue was only available from a single wild boar carcass. Similar to our findings, low numbers of PCR-positive wild boar tissues were described by Lutz et al. (2003), who also exclusively analysed non-CNS tissues. Romero et al. (2003) showed that tonsil samples were more often negative for SuHV-1 DNA than sacral or trigeminal ganglia. This difference was even more pro-

nounced in the results of Balasch et al. (1998), where, after experimental infection, tonsil samples tested positive only in 1 out of 15 persistently infected pigs. In contrast, another group recently demonstrated higher numbers of PCR-positives extracted from tonsils than from trigeminal ganglia (Ruiz-Fons et al., 2007). Since many of these animals lacked detectable SuHV-1 antibody levels, it was concluded that they had been recently infected. Recent infection might also be an explanation for the absence of detectable SuHV-1 DNA in brain or trigeminal ganglia of the wild boar carcass. Since blood was not available from this animal, we have no information regarding the serological status of this animal. In contrast to acute infection, SuHV-1 DNA is thought to be predominantly present within neural tissues during latency, for example in trigeminal ganglia, sacral ganglia, olfactory bulb and brainstem (Maes et al., 1997; Pomeranz et al., 2005). Consequently, increased sampling of CNS tissues is likely to improve detection rates of SuHV-1 DNA. No overt signs of clinical disease or abnormal behaviour were reported for any of the three SuHV-1 DNA positive wild boars investigated in this study. AD in wild boar is rarely observed (Gorta´zar et al., 2002; Schulze et al., 2010) and wild boar adapted strains are potentially less pathogenic, also in domestic pigs (Mu¨ller et al., 2001). Immunohistochemical signals, indicating productive SuHV-1 infection in the wild boars analysed in this study, were morphologically intracytoplasmic, as reported previously by Lari et al. (2006). However, they were not associated with any relevant tissue reaction, such as inflammation or necrosis, which could also be an indication of low viral pathogenicity in wild boar. In contrast, the same virus strains were highly pathogenic in hunting dogs, since all three PCR-positive wild boars detected in this study could be epidemiologically strongly linked to recent AD-cases in Austrian hunting dogs. This was further confirmed by sequence analysis of the partial SuHV-1 gC coding region (Goldberg et al., 2001; Fonseca et al., 2010; Mu¨ller et al., 2010), showing that identical SuHV-1 gC sequences were recovered from wild boars and epidemiologically linked hunting dogs. Furthermore, it was shown that Austrian SuHV-1 strains clustered with sequences assigned to clade A (Mu¨ller et al., 2010) and had considerable similarity to wild boar-derived strains from neighbouring countries, such as France, Germany, Hungary, Italy and Slovakia. Austrian SuHV-1 strains could further be separated into two different lineages. Interestingly, Austrian lineage 1 sequences were identical to strains from Eastern Germany, although isolation dates differed by up to 15 years. In contrast, Austrian lineage 2 strains clustered with SuHV-1 isolated from Western Germany and France. Of note, statistical support values for most branches in the phylogenetic tree were relatively low (Fig. 2), indicating that the observed clustering pattern needs to be interpreted cautiously. Low bootstrap support was also evident in other publications using the same or a similar fragment for phylogenetic inference (Goldberg et al., 2001; Fonseca et al., 2010; Hahn et al., 2010; Mu¨ller et al., 2010), indicating that the phylogenetic signal contained within the gC coding region does not allow optimal phylogenetic resolution.

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The presence of two genetically distinct SuHV-1 variants in Austria was supported by the observation of three amino acid deletions in the gC protein of Austrian strains belonging to lineage 1, but not in the Austrian lineage 2 strains (Fig. 3). The presence of amino acid insertions/deletions (indels) at these and additional positions in SuHV-1 gC protein sequences from European wild boar has been described by Mu¨ller et al. (2010). The pattern of indels in Austrian lineage 1 strains (three deletions at amino acid positions 25, 38 and 39, respectively) is identical to that in strains from Eastern Germany, whereas the lineage 2 pattern (no amino acid deletion) is shared by some clade A strains from Hungary, Slovakia and Germany (Mu¨ller et al., 2010). Although the presence of indels is not strictly consistent with phylogenetic clustering, they may be useful for studying epidemiologic relatedness between viral strains. RFLP patterns of Austrian SuHV-1 strains suggest that they belong to genotype I. Although virus has only been isolated from four hunting dogs, the PCR-positive wild boars reported here were the most likely source of infection for three of these dogs. Therefore, it can be expected that the PCR-positive wild boars were infected by identical viruses. Our results are in line with previous observations suggesting that European wild boar almost exclusively host SuHV-1 genotype I strains (Capua et al., 1997; Mu¨ller et al., 1998b, 2010). The presence of two genetically distinct groups of SuHV-1, as suggested by phylogenetic analysis, was supported by RFLP patterns. Three isolates showed characteristics of subtype Iw, which was so far exclusively recovered from East German and Hungarian wild boar; a single isolate resembled subtype Ip strains, as found in French, Italian, West German and Slovakian wild boar (Lutz et al., 2003; Mu¨ller et al., 1998b, 2010). In contrast to RFLP analysis, phylogenetic analysis is faster, avoids the need for virus isolation and has the advantage that reference sequences are easily available. However, although Goldberg et al. (2001) reported significant correlation between RFLP and gC sequence distance, Mu¨ller et al. (2010) reported that gC sequence based phylogenetic analysis is not strictly coherent with BamHI RFLP-patterns. As the gC gene only represents a very limited part of the viral genome, full genome sequencing approaches might become a valuable tool for SuHV-1 molecular phylogeny in the future. Acknowledgements The excellent technical assistance provided by the Departments for Molecular Biology, Virology and Pathology of the AGES Institute for Veterinary Disease Control, Mo¨dling is deeply acknowledged. We thank Helga Lussy for performing virus isolations, Michael Schwarz for constructing geographical maps (Fig. 1) as well as Susanne Richter, Herbert Weissenbo¨ck and Michael Leschnik for helpful discussions. References Albina, E., Mesple`de, A., Chenut, G., Le Potier, M.F., Bourbao, G., Le Gal, S., Leforban, Y., 2000. A serological survey on classical swine fever (CSF),

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Legal norms COMMISSION DECISION of 30 June 1997 amending Decision 93/24/EEC and Decision 93/244/EEC and concerning additional guarantees relating to Aujeszky’s disease for pigs destined to regions free of the disease in Austria (Text with EEA relevance) (97/423/EC). Offic. J. Eur. Union, L 180, 28–30. COMMISSION IMPLEMENTING DECISION of 4 October 2011 amending Decision 2008/185/EC as regards the inclusion of Belgium in the list of Member States free of Aujeszky’s disease (notified under document C(2011) 6997) (Text with EEA relevance) (2011/648/EU). Offic. J. Eur. Union, L 260, 19–22.