Phylogenetic Analysis of the Rabies Virus N-coding Region in Lithuanian Rabies Isolates

Rabies infection among wild and domestic animals constitutes a well-known problem in Lithuania, but only one dog rabies virus isolate sequence (1992) from Lithuania was used in the European rabies virus phylogenetic analysis. The objective of this work was to determine nucleoprotein (N) gene sequences and genetically characterize the rabies virus isolates in order to learn which virus group (biotype) is circulating in reservoir species in Lithuania. Classical rabies virus isolate nucleoprotein (N) gene sequences from different parts of Lithuania were found to be closely related to each other and demonstrated nucleotide identity from 97.7 to 100% and could be placed in one lineage with 100% bootstrap support. All 12 sequences of raccoon dogs, red foxes, dogs and marten rabies viruses exhibited 97.7 99.0% identity to previously published sequences from Eastern parts of Poland, Estonia, Finland, and the North-Eastern part of Russia. Phylogenetic analysis revealed that all Lithuanian strains belong to the North East Europe (NEE) group of rabies virus.

permits to understand the transmission of rabies virus from the reservoir host to other hosts, including humans and domestic animals (Nagarajan et al. 2006;David et al. 2004;Kissi et al. 1995).
The objective of this work was to determine nucleoprotein (N) gene sequences and to genetically characterize the rabies virus isolates in order to know which virus group (biotype) is circulating in reservoir species in Lithuania.

Materials and Methods
In total, 30 Lithuanian RABV isolates, collected during the 2005-2006 period, diagnosed as rabies-positive by both the FAT (Dean et al. 1996;OIE 2004) and the MIT (Koprowski 1996;OIE 2004) were used in this study.The isolates were obtained from brain samples received from the National Veterinary Laboratory and came from several Lithuanian districts and from several animal species.The majority of RABV isolates were obtained from wild mammals: red foxes (n = 10), raccoon dogs (n = 8), martens (n = 3), polecats (n = 1) and badgers (n = 1); some from domestic animals: dogs (n = 5) and cats (n = 2).For molecular epidemiological study 12 samples from all the obtained isolates were selected according to the geographical location and animal species and analyzed together with reference to GenBank sequences of the rabies virus isolates from various regions of Eastern, Central, and Northern Europe including the nearest regional Lithuanian neighbours.Total RNA was extracted from infected brain samples using the TRIzol method (Invitrogen, Life Technologies, MD, USA) following the manufacturer's recommendations.RT and PCR were performed according to Amengual et al. (1997) and with primer set N12 (5'-GTAACACCTCTACAATGG-3', nucleotides 57-74) and N8 (5'-AGTTTCTTCAGCCATCTC-3', nucleotides 1585-1568).All nucleotide positions of the primers are numbered according to the PV strains sequence.PCR was carried out in 50 l volumes containing 5 l of extracted RNA and the following reagents: 5 l 10 × PCR buffer (Fermentas), 5 µl MgCl 2 (25 mM, Fermentas), 2 l dNTPs (10 mM, Fermentas), 5 pmol of each outer primers N12 and N8, 0.5 l (2.5 U) Taq DNA polymerase (Fermentas), 0.25 l (10 U) RNasin (Promega, Madison, WI, USA), and 0.5 µl (100 U) MMLV reverse transcriptase (Life Technologies).The tubes were then subjected to the following cycle parameters: 42 C for 30 min, 95 C for 5 min, and then 35 cycles at 94 C for 40 s, 56 C for 40 s, and 72 C for 1 min.
Nested PCR was carried out with primers N53 (5'-GGATGCCGACAAGATTGTAT-3', corresponding to bases 73-92 of the PV sequence) and N55 (5'-CTAAAGACGCATGTTCAGAG-3', corresponding to bases 491-472 of the PV sequence).For the nested PCR as a template 2 l of PCR I product and the following reaction mixture were used: 5 l 10 × PCR buffer (Fermentas), 5 µl MgCl 2 (25 mM, Fermentas), 2 l dNTPs (10 mM, Fermentas), 20 pmol of each nested primers N53 and N55, 0.5 l (2.5 U) Taq DNA polymerase (Fermentas).The tubes were then subjected to the following cycle parameters: 95 C for 3 min followed by 35 cycles at 94 C for 30 s, 60 C for 30 s and 72 C for 40 s.A single extension step of 72 C for 10 min completed the amplification process.The nested PCR resulted a final amplicon of 400 bp.
Prior to sequencing, the PCR products were elctrophoresed on 1.5% agarose gel.Following ethidium bromide staining and desalting in ultrafiltered water, bands of the expected size were excised from the gel, and the DNA was recovered using Nucleospin Extract II kit (Macherey-Nagel GmbH, Germany) following the manufacturer's recommendations.Gel purified PCR products were cycle sequenced using the BigDye Terminator Cycle Sequencing kit (v2.0, Applied Biosystems, Foster City, CA, USA) and the ABI310 genetic analyzer (Applied Biosystems).Sequences of both strands of the nucleoprotein N gene products were determined using the same primers as used for the nested PCR amplification.The obtained sequences were assembled by using SeqMan program (Laserge, program package, DNASTAR, Inc., Madison, USA).All sequences reported in this study have been submitted to GenBank, as summarized in Table 1.Clustal W programme from MegAlign Lasergene program package was used for sequence alignment and phylogenetic analyses.For boot strap analyses neighbourjoining (NJ) algorithm from CLC Free Workbench 3.2.3program package (CLC bios A/S, Denmark) was used.Bootstrap values exceeding 70% were considered significant.

Results
Identification of the rabies viral antigen by FAT and MIT was evaluated in a total of 30 brain samples tested.The RT-PCR with the N12/N8 primer set obtained positive results in 22 rabies samples from 8 red foxes, 6 raccoon dogs, 3 martens, 1 badger, 3 dogs and 1 cat.By using partial region from the amino terminus of the N gene (400 bp), a total of 12 rabies-positive samples representing several Lithuanian regions were sequenced.
Based on newly obtained Lithuanian sequences and the selected rabies virus sequences from Estonia, Russia, Poland and other West European countries available in the GenBank (also summarized in Table 1), a phylogenetic tree was constructed (Fig. 1).
All 12 Lithuanian rabies viruses isolates sequences exhibited 97.7-99.0%identity to previously published sequences from Estonia, Finland, Poland and North-Eastern part of Russia.Furthermore, alignment of sequences of different species including the dog (05LT11, 06LT5), red fox (07LT19, 05LT21, 06LT2) and raccoon dog (07LT14) showed that they 275   could be placed in one lineage with 100% bootstrap support (Fig. 1).Previously published Lithuanian rabies sequence 9345LT also showed very close phylogenetic relationship to newly obtained isolates (97.7-99.0% of identity).
The phylogenetic analysis including a large set of reference sequences revealed that all Lithuanian strains (including the unique Lithuanian sequence 9345LT obtained earlier) belong to North East Europe group of rabies virus described earlier in Bourhy et al. (1999) and could not be subdivided according to geographical location.All new Lithuanian sequences as well as new Russian, Polish or Estonian sequences were well supported from the other group of rabies viruses, which joined isolates from different parts of Europe, with a bootstrap support of 96%.The constructed phylogenetic tree also showed that the other rabies sequences included in this study were placed to West or Central Europe groups with a bootstrap support of 86 and 99%, respectively.

Discussion
Molecular epidemiology based on RT-PCR is an important tool for the classification of animal virus diseases, including rabies virus, and provides a better understanding of epidemiological relationships.This approach has been applied at various geographical levels, from individual countries to worldwide studies (Kissi et al. 1995;Haas 1997;David et al. 2000, De Mattos et al. 2000).It also provides information where a single host dominates the maintenance of rabies virus (Sacramento et al. 1992) or where more than one host are involved in the transmission of the virus (Nadin-Davis et al. 2001;Johnson et al. 2003).The sequence alignment and phylogenetic analysis of Lithuanian rabies virus isolates demonstrates a high degree of similarity between isolates originating from different species.Phylogenetic analysis based on a 400 bp N-coding region sequences demonstrates that all the viruses investigated are genotype 1 viruses and are closely interdependent.The red foxes (Vulpes vulpes) remain the principal vector of rabies transmission in Lithuania, but a close relationship (97.7-99.0%) between isolates from raccoon dogs (Nyctereutes procyonoides) suggest that the transmission was a recent event with the possibility of direct transmission.Fox rabies viruses from Eastern Europe (EE) tend to group together, as do those from Western Europe (WE) and Central Europe (CE), the latter of which was previously described using monoclonal antibodies (Stohr et al. 1992;Bourhy et al. 1999).The raccoon dog's viruses are also found within a particular geographical area, North-Eastern Europe (NEE), although their precise phylogenetic relationship to the fox rabies strains is uncertain, as indicated by the low bootstrap support for the critical node.NEE group (96% bootstrap support) is now found to cover a wider geographical area, including the eastern parts of Poland, Lithuania, Estonia, Russia and Finland, and includes viruses isolated from both red foxes and raccoon dogs, showing that both species are effective reservoirs for this variant of rabies virus and may represent early cross-species transmission from dog viruses.Raccoon dogs were frequently involved in rabies virus circulation in the NEE region, particularly in Lithuania, where the population of raccoon dogs is the largest, which suggests that the density of susceptible hosts is a major ecological factor in the establishment of rabies virus in a new host species (Bourhy et al. 1999).Phylogenetically, this group was related to other European lineages, and there is no reason to consider it as specific to the raccoon dog (Kuzmin et al. 2004).Our results confirm this suggestion because all Lithuanian rabies strains from raccoon dogs and foxes had 97.7-99.0%nucleotide identity.
By comparing the Lithuanian rabies isolates with those derived from the surrounding countries it became clear that the North-East Europe group is related to RABV found throughout the region.In Poland, the NEE group corresponding to the European fox 3 (F3) phylogenetic subgroup is limited to the eastern side of the Vistula River and to the northern border of Russia, Byelorussia and Lithuania (Sadkowska-Todys 2000).In Latvia, the direct sequencing of the RT-PCR-amplified products of the rabies virus nucleoprotein encoding region and subsequent sequence analyses resulted in 99.3-100% homology between isolates and 99.0-100% with genotype 1, classical RABV raccoon dog isolate from Estonia in 1995 (Vanaga et al. 2003).The genetically close relationship between the same raccoon dog rabies isolate from Estonia and raccoon dog and fox (99.2% and 98.8% respectively) RABV isolates from northern Lithuania was identified, suggesting that the viruses circulating in foxes and raccoon dogs in this region might have the same origin.
The phylogenetic analysis of the partial N-gene sequences in Lithuanian rabies virus isolates indicates the direct geographical association with the North-East Europe (NEE) group and demonstrates the close relationship among rabies virus strains circulated in the neighbouring countries such as Poland, Latvia, Estonia and Russia.

Fig. 1 .
Fig.1.Phylogenetic tree of classical rabies virus nucleoprotein (N) sequences.The horizontal branches are drawn to scale and the tree is rooted with reference sequences 9141RUS and 9105CAN, representative of Arctic fox lyssaviruses.The numbers at the main nodes indicate the degree of bootstrap support calculated by using neighbour-joining (NJ) algorithm from CLC Free Workbench 3.2.3program package.The abbreviations for the earlier described European main phylogenetic groups (CE, WE, EE, NEE, D) are given according toBourhy et al. (1999) andKuzmin et al (2004).Lithuanian rabies virus isolates are presented in bold. abbreviation

Table 1 .
Bourhy et al. (1999)hes are drawn to scale and the tree is rooted with reference sequences 9141RUS and 9105CAN, representative of Arctic fox lyssaviruses.The numbers at the main nodes indicate the degree of bootstrap support calculated by using neighbour-joining (NJ) algorithm from CLC Free Workbench 3.2.3programpackage.The abbreviations for the earlier described European main phylogenetic groups (CE, WE, EE, NEE, D) are given according toBourhy et al. (1999)andKuzmin et al (2004).Lithuanian rabies virus isolates are presented in bold.Summary of classical rabies virus sequences used for phylogenetic analyses of nucleoprotein N gene region of 400 bp.Sequences 22-33 were obtained as part of this study and the other are reference sequences, representing most diverse European rabies virus available in the GenBank.Phylogenetic clusters indicated according toBourhy et al. (1999): Central Europe (CE), Eastern Europe (EE), Western Europe (WE), North-Eastern Europe (NEE).Group D is representing rabies viruses originated from the centre of the European part of Russia(Kuzmin et al. 2004).