Title: Molecular epidemiology of the rabies virus in Slovenia
Authors: D. Rihtariˇ c, P. Hostnik, J. Grom, I. Toplak
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epidemiology of the rabies virus in Slovenia 1994-2010, Veterinary Microbiology
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Molecular epidemiology of the rabies virus in Slovenia 1994-2010
D. Rihtarič, P. Hostnik, J. Grom, I. Toplak*
University of Ljubljana, Veterinary Faculty, Institute of Microbiology and Parasitology, Virology
Unit, Gerbičeva 60, 1115 Ljubljana, Slovenia
*Corresponding author: Tel: +386 1 47 79 180, Fax: + 386 1 47 79 352. E-mail address:
email@example.com (I. Toplak)
A molecular epidemiology study was performed on a selection of 30 rabies-positive brain
samples collected between 1994 and 2010 in Slovenia and originating from the red fox
(n=19), badger (n=3), cattle (n=3), dog (n=2), cat (n=1), marten (n=1) and horse (n=1). Based
on the comparison of 1092 and 672 nucleotide sequences of nucleoprotein (N) and partial
glycoprotein (G) gene regions, a low genetic diversity of the circulating strains was detected,
but both phylogenetic trees were consistent with the topology where partial nucleoprotein or
glycoprotein genes were used. A high sequence identity in the N and G gene to rabies virus
isolates from neighbouring countries was found. The Slovenian strains were clearly different
from the vaccine strains SAD B19 and SAD Bern, which have been used in Slovenia since
Keywords: Rabies virus; Sequencing; Molecular epidemiology; Slovenia
Page 2 of 17
Rabies virus is a member of the genus Lyssavirus within the family Rhabdoviridae of the
order Mononegavirales (Virus taxonomy, 2005). The Lyssavirus genus is further divided into
eleven species. Species 1 is the most widespread and comprises the classical rabies virus
including field, laboratory and vaccine strains. Species 2, 3 and 4, with the prototypes Lagos
bat virus, Mokola virus and Duvenhage virus, respectively are found in Africa. Viruses
isolated from bats in Europe are classified within species 5 and 6, while the Australian bat
lyssavirus represents species 7 (Gould et al., 2002). Four recent lyssavirus isolates from bats
of Eurasia, designated Aravan virus (ARAV), Khujand virus (KHUV), Irkut virus (IRKV)
and West Caucasian bat virus (WCBV), are classified within species 8, 9, 10 and 11 (Kuzmin
et al., 2003, 2005).
The rabies virus genome is a single-stranded, negative-sense RNA molecule of about 12 kb,
which encodes five major viral proteins (N, P, M, G, L). Nucleoprotein (N), phosphoprotein
(P), RNA-dependent RNA-polymerase (L) and genomic RNA form a unique
ribonucleoprotein complex (RNP) for viral infection. The RNP unit is connected by the
matrix (M) protein and surrounded by the membrane with inserted glycoprotein.
Glycoprotein (G) contains the domains responsible for host cell receptor recognition
(Thoulouze et al., 1998; Tuffereau et al., 1998) and membrane fusion (Durrer et al., 1995),
which is crucial for viral neurotropism (Badrane et al., 2001) and pathogenicity (Morimoto et
al., 1999; Faber et al., 2005).
In Slovenia, wildlife-mediated rabies has been present since 1973; it was first detected in the
north-eastern part of the country and has been endemic since then. A second wave of sylvatic
rabies reached Slovenia in 1979 from Austria. Thousands of rabies-positive animals were
detected each year from 1979 to 1995. Oral vaccination against rabies is the most effective
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goal of the present study was to characterize selected rabies viruses collected from different
method of preventing rabies virus infection in wild animals (Pastoret and Brochier, 1998). In
Slovenia, an oral vaccination programme of foxes has been running since 1988. During the
period 1998 – 2004, the vaccines containing strains SAD Bern and SAD B19 were used, but
since 2005, only vaccine containing SAD B19 has been used. In the rabies surveillance
program in 1995, 1089 rabies-positive animals were detected, but in the period between 1998
and 2007, the number of positive samples decreased from 15 to 2 cases per year (Hostnik et
al., 2006). The majority of rabies cases in the years 2008 (55 positive) 2009 (35 positive) and
2010 (16 positive) were detected along the border with Croatia, where vaccination is not
performed. The vaccination of dogs against rabies has been compulsory in Slovenia since
1947, and dog-mediated rabies was eradicated in 1954. The last human case of rabies was
recorded in 1950.
Our neighbouring country Italy was classified as rabies free between 1997 and 2008. In
October 2008, the National Reference Centre for rabies at the Instituto Zooprofilattico
Sperimentale delle Venezie in Legnaro (Padova) diagnosed rabies in a red fox in the
municipality of Resia (north-eastern part of Italy). In 2008, another eight rabies-positive
wildlife cases were detected. As a consequence of the reintroduction of rabies, in January and
February 2009 an emergency oral vaccination programme was organized in the infected area
(De Benedictis et al., 2009).
Rabies molecular epidemiology studies have recently gained relevance in Slovenia, in order
to investigate the dynamics of geographical and inter-species transmission. Therefore, the
locations in Slovenia to identify which phylogenetic groups have been circulating in the
country and to establish possible epidemiological associations to rabies virus variants
occurring in neighbouring countries.
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glycoprotein genes, the modified primer sets GH3 (5’-CTA ACC ACG ATT ACA CCA TTT
2. Materials and methods
Between 1994 and 2010, 33,799 brain samples from different animal species were tested and
2616 were found to be positive by a fluorescent antibody test (FAT) (Dean et al., 1996) using
a commercial rabies anti-nucleocapsid conjugate (BioRad, France), according to
manufacturer’s instructions. Thirty positive samples were selected according to the
availability of the original archive brain samples, the year of collection, the geographical
location and in order to cover different species of origin (Fig. 1, Table 1) and were compared
with other 9 (for N gene) and 5 (for G gene) rabies virus strains and 2 vaccine strains from
GenBank. This study included rabies field viruses from the red fox (n=19), badger (n=3),
cattle (n=3), dog (n=2), cat (n=1), marten (n=1) and horse (n=1). The geographical
distribution of the 30 strains from Slovenia is shown in Fig. 1. The majority of the collected
samples were detected in a 20 km wide belt near the border with Croatia, but samples with
virus numbers 1, 6, 7, 8 and 10 originated from other locations (Fig. 1, Table 1).
Total viral RNA was extracted from the original host brain samples using the QIAamp® Viral
RNA Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. The
extracted RNA was eluted in 60 µl of elution buffer and stored at -70 °C until analysis.
Reverse transcription (RT) with polymerase chain reaction (PCR) was performed in one tube
(One-Step RT-PCR Kit, Qiagen, Germany) with the primer sets N7 (5’-ATG TAA CAC CTC
TAC AAT G-3’) and N4 ( 5’- GTC TGA TGA TTG GAA CT-3’) to amplify 1313 bp
products of nucleoprotein genes (Bourhy et al., 1993). To amplify 730 bp products of
G-3’) and GH4 (5’-CAA AAT CCT CAG CCT CGT C-3’) were used (Kissi et al., 1999).
The RT-PCR reaction was carried out in a volume of 50 µl. The reaction mixture consisted of
5x PCR buffer, 10 mM dNTP mix, 20 nM of each primer, the RT-PCR enzyme mix and 4 µl
of eluted RNA. A thermo-cycling profile of 1 cycle of 30 s at 50 °C, 1 cycle of 15 min at 95
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for reliable genotyping. A comparison of the sequences obtained from different animal
°C, 40 cycles of 30 s at 94 °C, 30 s at 52 °C (for nucleoprotein) or 56 °C (for glycoprotein),
and 1 min at 72 °C was employed followed by a final incubation at 72 °C for 10 min. The
amplified products were visualized on 1.8% agarose gel with ethidium bromide. The
amplicons were purified with the commercial QIAquick gel extraction kit (Qiagen, Germany)
and directly sequenced from PCR products with the same primers (Bourhy et al., 1993; Kissi
et al., 1999). The obtained nucleotide sequences of the positive samples were assembled
using the DNASTAR (version 5.05) program and compared to the known sequences of N and
G genes in the GenBank database using the Basic Local Alignment Search Tool (BLAST)
program. The phylogenetic analysis was conducted by comparing 1092 bp of N gene and 672
bp of G gene nucleotide sequences with other rabies viruses obtained from GenBank. A
multiple alignment of the nucleotide and protein coding sequences was performed using
Clustal W and neighbour-joining criteria. The final phylogenetic tree was constructed using
the Phylip program (Felsenstein 1989). The partial G gene and N gene sequences determined
during this study have been submitted to GenBank with assigned accession numbers (Table
3. Results and discussion
This is the first molecular epidemiology study on rabies-positive samples collected in
Slovenia. The phylogenetic analysis conducted with the Slovenian-positive samples showed
that all belonged to classical rabies virus. The topology of the obtained sequences on both
phylogenetic trees was almost identical, confirming that the N gene or the G gene can be used
species (red fox, badger, cattle, dog, cat, marten and horse) revealed the historical
transmission from the red fox, as a reservoir of the rabies virus, to domestic and wild animals
by direct contact. This data was also confirmed, in most cases, with epidemiological
investigations, where domestic animals had been diagnosed as rabid.
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other and formed two genetic groups. When compared to five European isolates (strains
A specific RT-PCR product of 1313 nucleotides (nt) was amplified from all 30 selected
samples for genotyping. For all 30 samples, N gene sequences 1092 nucleotides long were
determined and compared to 11 sequences representing classical rabie virus (Table 1, Fig. 2).
Phylogenetic analysis of the N gene sequences showed that Slovenian samples formed two
genetic groups of closely related strains. Rabies-positive samples from Slovenia showed 95.9
- 100% nucleotide homology to each other and 94.8 - 100% nucleotide homology to the other
European isolates (strains: 86107YOU, 86106YOU, 8653YOU, 9202ALL, 9244FRA,
8681FRA, 08RS/Udine/2008, dog/09RS-1459/Udine/2009, and 86111YOU)(Bourhy et al.,
1999; De Benedictis et al., 2008). Twenty-nine positive samples were found to share 99.0 -
100% nucleotide identity to each other and 99.5 - 99.9% identity to the closely related strain
86111YOU in GenBank, isolated in the year 1986 in Bosnia-Herzegovina. With recent
outbreaks in Italy, where strains 08RS1981/Udine/2008 and dog/09RS-1459/Udine/2009
were sequenced, strain 275-08SVN found in Brežice in 2008 shared a 100% identity. Strain
664-02SVN was found to share a 99.1% identity with strain 8653YOU, isolated from a wolf
in 1986 in Bosnia-Herzegovina (Bourhy et al., 1999).
A specific RT-PCR product of 730 nt of the G gene was amplified from all 30 selected
samples. Sequences of 672 nt from these 30 samples were determined and compared to 7
isolates representing classical rabies virus. When comparing the phylogenetic tree of the G
gene to the tree of the N gene sequences, they were found to show similar topology (Fig. 2).
The partial G gene sequences (672 nt) showed 94.6 - 100% nucleotide homology to each
9202ALL 9244FRA, 86107YOU, 9339EST and 8653YOU) (Bourhy et al., 1999), our
positive samples were found to share a 93.8 - 98.4% nucleotide homology (Fig.2).
Slovenian rabies field viruses formed two genetic groups of closely related strains belonging to 145
Western Europe (WE) and the Eastern Europe (EE) group according to Bourhy et al. (1999). Although 146
Page 7 of 17
result of a successful vaccination strategy was confirmed in 1998, when we detected only a
initially used to clarify spatial information, the different nomenclature used by previous authors may 147
result in confusion when more diverse information is available (Bourhy et al., 1999; Kuzmin et al., 148
2004). Rabies virus has now been eliminated from whole of Western Europe and therefore it is not 149
appropriate to describe future virus isolations with nomenclatures according to geographical 150
locations (Turcitu et al., 2010).
In order to analyse the amino acid (AA) variability between the 30 Slovenian strains and
other rabies viruses, the partial N and G gene AA sequences were aligned and compared with
the published field strains and two vaccine strains (data not shown). In the N gene no AA
difference was observed between the group of 29 Slovenian strains and the strains
08RS/Udine/2008 (De Benedictis et al., 2008), dog/09RS-1459/Udine/2009, 86111YOU
(Bourhy et al., 1999) (Table 1). When comparing strains 664-02SVN and 8653YOU, the
replacement of Asp with Asn at positions 35 (numbered according to strain SAD B19) and
Asp to Ser at position 101 was observed. The 29 G protein amino acid sequences of our
strains showed two differences in AA changes at position 202: Leu for Pro (strain 1386-
04SVN); and His for Leu at position 250 for samples 52-10SVN, 332-08SVN, 339-08SVN,
348-08SVN, 755-10SVN, 1209-08SVN, 1647-07SVN, 2440-09SVN, 2441-08SVN, detected
between 2007 and 2010 (Table 1). One AA change between strain 664-02SVN and
8653YOU (Bourhy et al., 1999) occurred at position 206 (Ala for Met).
Slovenia has a 670 km long border with Croatia and the majority of rabies cases within the
last decade were detected in the vaccination zone along the border with Croatia. The first
few rabid animals (Hostnik et al., 2006). According to the data submitted to the Rabies
Bulletin Europe, rabies is an increasing problem in Croatia, and almost 20% of the fox brain
samples analysed in a laboratory of Croatian Veterinary Institute during 2001 and 2006 were
found to be positive (Lojkić et al., 2009). Rabies in Croatia is strictly sylvatic, with the red
Page 8 of 17
fox as the main reservoir, and in the last 10 years the disease has become enzootic. A
phylogenetic analysis of a 422 nucleotide sequence (data not shown) of the isolate
CRO_09SK (GU134624) (Lojkić et al., 2009) from a wolf in Croatia in 2009 revealed that
this strain shared a 99.5 - 100% homology with the Slovenian strains 498-94SVN, 696-15-
95SVN, 1116-00SVN, 1339-00SVN, 275-08SVN, 1085-09SVN, and 52-10SVN (Table 1).
This observation confirmed that the positive samples from Slovenia were genetically closely
related to the sequenced field strain CRO_09SK, circulating in Croatia.
After the reitroduction of rabies in Italy the oral vaccination was based on locations of
confirmed rabies cases, including a protection zone, but later new positive rabies cases were
detected among wildlife and domestic animals, with tendency of spreading the infection (De
Benedictis et al., 2009). The sequences of the two published strains 08RS-1981/Udine/2008
and dog/09RS-1459/Udine/2009 (Fig. 2) are closely related to our strains, which were
detected in the same period.
According to the results of our phylogenetic study, there was no evidence for the detection of
the vaccine strain among the 30 rabies-positive animals in the field, or the vaccine strain
recombination in the sequenced region. The positive Slovenian samples were only found to
share a 91.81 - 92.5% (for partial nucleoprotein) and a 91.7 - 93.1% (for partial glycoprotein)
nucleotide homology with the vaccine strains SAD Bern and SAD B19 (Geue et al., 2008),
confirming that the vaccine strains were not detected among the selected field samples and
that they had not reverted back to their native pathogenic form.
This study provides a detailed molecular epidemiology of 30 rabies isolates in Slovenia
between 1994 and 2010. Phylogenetic analysis showed that the Slovenian isolates formed
two groups of closely related strains, which have been genetically stable and epidemically
present in Slovenia for at least 16 years. Further studies on rabies viruses in the neighbouring
Page 9 of 17
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to explain the dynamics of circulating rabies strains in the west Balkan part of Europe.
The study was supported by the Veterinary Administration of Republic Slovenia (VARS) and
by the Slovenian Research Agency (ARRS).
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1386-04SVN fox Ilirska Bistrica
Rabies field viruses from Slovenia (virus numbers 1-30) and isolates representing classical
rabies virus from GenBank (virus numbers 31-42) used for the phylogenetic analysis. Virus
codes describe laboratory sample number, year of detection, and SVN is for Slovenia.
1 202-94SVN cattle Jesenice 1994 This article HM52150 HM852173
2 498-94SVN fox Ribnica 1994 This article HM52146 HM852169
3 587-94SVN marten Sežana 1994 This article HM52152 HM52175
4 696-15-94SVN fox Ilirska Bistrica 1994 This article HM52149 HM52172
5 882-95SVN cat Cerknica 1995 This article HM52148 HM52171
6 1229-95SVN fox Domžale 1995 This article HM52153 HM52176
7 709-95SVN dog Kamnik 1995 This article - -
8 590-96SVN fox Litija 1996 This article HM52147 HM52170
9 1116-00SVN badger Novo Mesto 2000 This article HM52151 HM52174
10 1339-00SVN fox Litija 2000 This article - -
11 2475-01SVN fox Koper 2001 This article - -
12 315-02SVN fox Laško 2002 This article HM52164 HM52187
13 664-02SVN fox
2002 This article HM52145 HM52168
14 199-03SVN fox Brežice 2003 This article HM52155 HM52178
15 2004 This article HM52165 HM52188
16 675-05SVN fox Polzela 2005 This article - -
17 1039-06SVN fox Črnomelj 2006 This article - -
18 1674-07SVN fox
2007 This article HM52161 HM52184
19 275-08SVN fox Brežice 2008 This article HM52154 HM52177
Page 14 of 17
20 332-08SVN dog Brežice 2008 This article HM52156 HM52179
21 339-08SVN fox Mirna peč 2008 This article HM52157 HM52180
22 348-08SVN badger Majšperk 2008 This article HM52159 HM52182
23 1209-08SVN badger
2008 This article HM52160 HM52183
24 1868-08SVN fox
2008 This article - -
25 2441-08SVN horse
2008 This article HM52162 HM52185
26 371-09SVN cattle Ilirska Bistrica 2009 This article HM52167 HM52190
27 1085-09SVN fox Koper 2009 This article HM52166 HM52198
28 2440-09SVN fox
2009 This article - -
29 52-10SVN fox Poljčane 2010 This article HM52163 HM52186
30 775-10SVN cattle Žetale 2010 This article HM52158 HM52181
red fox Italy 2008
et al., 2008
dog Italy 2009 - GQ478245 -
33 86111YOU red fox
34 8653YOU wolf
35 8661FRA hedgehog France 1984
36 9202ALL red fox Germany 1991
37 9244FRA red fox France 1992
38 86106YOU red fox 1972
Kissi et al.,
39 86107YOU red fox
40 SAD B 19 vaccine - -
Geue et al.,
41 SAD Bern vaccine - -
Geue et al.,
Page 15 of 17
Page 16 of 17
Fig. 1. The geographical locations of the rabies-positive samples from this study are presented
on a map of Slovenia. Stars indicate the precise location where each sample was collected, the
numbers (1-30) indicate the virus numbers according to Table 1.
Page 17 of 17 Download full-text
constructed using the Phylip program by using the maximum likelihood algorithm with strain
Fig. 2. The phylogentic trees based on a 1092 nucleotide sequence (146-1238, numbering of
nucleotide positions according to strain SAD B19) of the N gene and 672 nucleotides of the G
gene (3914-4582, numbering of nucleotide positions according to strain SAD B19) were
SAD B19 as outgroup. Bootstrap values were obtained for 1000 datasets.