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Phylogenetic relationships of seven previously unclassified viruses within the family Rhabdoviridae using partial nucleoprotein gene sequences

¹ Rabies Union, Centers for Disease Control and Prevention, 1600 Clifton Road, MS G-33, Atlanta, GA 30333, USA
² Laboratory for Clinical and Molecular Virology, The University of Edinburgh, Summerhall, Edinburgh EH9 1QH, UK

Correspondence
I. V. Kuzmin
ibk3{at}cdc.gov

	ABSTRACT

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Partial nucleoprotein (N) gene sequences of the rhabdoviruses Obodhiang (OBOV), Kotonkon (KOTV), Rochambeau (RBUV), Kern canyon (KCV), Mount Elgon bat (MEBV), Kolongo (KOLV) and Sandjimba (SJAV) were generated and their phylogenetic positions within the family Rhabdoviridae were determined. Both OBOV and KOTV were placed within the genus Ephemerovirus. RBUV was joined to the same cluster, but more distantly. MEBV and KCV were grouped into a monophyletic cluster (putative genus) with Oita virus (OITAV). These three viruses, originating from different regions of the world, were all isolated from insectivorous bats and may be specific for these mammals. African avian viruses KOLV and SJAV were joined to each other and formed another clade at the genus level. Further, they were grouped with the recently characterized rhabdovirus Tupaia virus (TRV). Although the genetic distance was great, the grouping was supported by consistent bootstrap values. This observation suggests that viruses of this group may be distributed widely in the Old World. Non-synonymous/synonymous substitution ratio estimations (d_N/d_S) using a partial N gene fragment (241 codons) for the three rhabdovirus genera revealed contrasting patterns of evolution, where d_N/d_S values follow the pattern Ephemerovirus > Vesiculovirus > Lyssavirus. The magnitude of this ratio corresponds well with the number of negatively selected codons. The accumulation of d_S appears evenly distributed along the gene fragment for all three genera. These estimations demonstrated clearly that lyssaviruses are subjected to the strongest constraints against amino acid substitutions, probably related to their particular niche and unique pathobiology.

The GenBank/EMBL/DDBJ accession numbers of the rhabdovirus partial N gene sequences obtained in this study are DQ457098–DQ457104, as outlined in Table 1.

	INTRODUCTION

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The family Rhabdoviridae includes representatives of six defined genera and more than 100 unassigned rhabdoviruses isolated worldwide (Tordo et al., 2004). In this family, members of the genus Lyssavirus are the most important in terms of public health and economic losses. Members of the genera Vesiculovirus and Ephemerovirus are of particular veterinary concern because of livestock diseases, whereas representatives of the genus Novirhabdovirus circulate among fish and other aquatic animals and members of the genera Nucleorhabdovirus and Cytorhabdovirus are plant parasites.

Early taxonomy of the Rhabdoviridae, as well as for other viruses, was based on virion morphology and serological cross-reactivity. Indeed, many as-yet unclassified rhabdoviruses were believed to exhibit cross-reactivity with members of the genus Lyssavirus (Calisher et al., 1989). When the terms ‘rabies-related viruses' and ‘rabies serogroup’ were introduced (Shope et al., 1970), these included Rabies virus (RABV), Mokola virus (MOKV) and Lagos bat virus (LBV). Shortly thereafter, new members of the ‘serogroup’ were described, such as Duvenhage virus (DUVV) [including at that time European bat lyssavirus 1 (EBLV-1)], Kotonkon virus (KOTV) and Obodhiang virus (OBOV) (Meredith et al., 1971; Kemp et al., 1973; Tignor et al., 1977). The latter two viruses, isolated from mosquitoes, demonstrated limited cross-reactivity with MOKV, but not with other members of the ‘serogroup’. Additionally, MOKV can be propagated in insect cell lines (Buckley, 1975), although no insect-derived isolates have been obtained. As was speculated, the proposed evolutionary pathway for the rabies-related viruses included OBOV and KOTV as progenitors, through MOKV, to other highly neurotropic mammalian viruses, such as LBV, DUVV and RABV (Shope, 1982). Later, other rhabdoviruses, isolated from both arthropods and vertebrates, predominantly in Africa, were described as related serologically to those listed above (Calisher et al., 1989). Currently, the evolutionary history of the Rhabdoviridae remains unclear.

Gene sequencing and phylogenetic relationships replaced the initial basis of serological and antigenic relationships for virus taxonomy. European bat lyssaviruses were differentiated from DUVV and separated into two species (EBLV-1 and EBLV-2) (King et al., 1990). A number of new lyssaviruses were isolated in different parts of the world (Gould et al., 1998; Kuzmin et al., 2003, 2005). Phylogenetically, lyssaviruses segregate into a single monophyletic clade, distant from other rhabdovirus genera and unclassified rhabdoviruses (Kuzmin & Rupprecht, 2005; Springfeld et al., 2005). A phylogenetic study based on limited polymerase (L) gene sequences was recently described for 38 rhabdoviruses (Bourhy et al., 2005). Although no new relatives of lyssaviruses were detected in that study, only KOTV from the number of serologically ‘rabies-related’ viruses was analysed. No other information is available to date on the molecular sequences of other rhabdoviruses that demonstrated serological cross-reactivity with lyssaviruses.

In the present study, we generated partial nucleoprotein (N) gene sequences of OBOV, KOTV, Sandjimba virus (SJAV), Kolongo virus (KOLV), Mount Elgon bat virus (MEBV), Kern Canyon virus (KCV) and Rochambeau virus (RBUV) and attempted to determine their phylogenetic relationships within the family Rhabdoviridae. Estimates of non-synonymous/synonymous (d_N/d_S) substitution ratios and detection of positively and negatively selected sites were used to assess evolutionary selection pressures along a fragment of the N gene for the three genera. The objective of this study was to provide further insight into the evolutionary history of the Rhabdoviridae.

	METHODS

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Viruses (Table 1) were obtained from the reference collection of the Centers for Disease Control and Prevention (Ft Collins, CO, USA) and were subjected to one intracerebral passage in suckling mice (Koprowski, 1996). Total RNA was extracted from infected mouse brains, using TRIzol (Gibco-BRL) according to the manufacturer's instructions. Two methods were used for the amplification of the rhabdovirus N gene. (i) Conventional RT-PCR was carried out with primers designed to conserved regions of the rhabdovirus N gene, RHNF631 [3'-GAYATGTTYTTYTCAAG-5'; messenger sense; positions 701–717 relative to the Pasteur virus RABV (PV) genome (GenBank accession no. M13215 [GenBank] )], RHNB1520 (5'-AGTCTCYTCTGCCATYTC-3'; genomic sense; positions 1568–1585) and RHNB2134 (5'-AGGATCTRGARGGGAACTTRT-3'; genomic sense; positions 2151–2171). The reactions were performed as described previously (Sacramento et al., 1991) with modifications (Kuzmin et al., 2003). Once a partial sequence had been obtained and the preliminary phylogenetic placement of the virus was evaluated, additional primers were constructed based on the alignment of the closest relatives of the newly sequenced virus. (ii) BD SMART PCR cDNA synthesis, followed by BD Advantage long-distance PCR (BD Bioscience Clontech), was performed according to the manufacturer's recommendations.

Primary assembly, alignment and consensus sequence generation, as well as DNA translation, were performed in BioEdit software (Hall, 1999). Multiple datasets of both nucleotide and amino acid sequences were subjected to phylogenetic analysis. Initially, the fragment of the N gene obtained was aligned and compared with whole-length N gene sequences of all rhabdovirus representatives available in GenBank (Table 2). To avoid the influence of multiple alignment gaps, only a set of the most closely related sequences was aligned with the newly generated sequences. This alignment was truncated from both the 3' and 5' ends to avoid gaps at the ends of the alignment and to ensure maximum compatibility of the sequences compared.

For positive selection analysis, 241 codons of the N gene were selected for a number of representative rhabdoviruses, producing alignments without gaps (positions 386–1108 relative to the PV genome): lyssaviruses, 11 taxa (seven recognized species, ARAV, KHUV, IRKV and WCBV); vesiculoviruses, 10 taxa [ISFV, SVCV, PIRYV, CHPV, VSINV (three lineages) and VSNJV (three lineages)]; and ephemeroviruses, six taxa (BEFV, ARV, FLAV, OBOV, KOTV and RBUV). All three datasets were analysed using the Datamonkey online positive selection interface (Kosakovsky Pond & Frost, 2005a). For each alignment, an NJ tree and ML model were estimated and used as the input for the single likelihood ancestor counting (SLAC) model of positive selection (Kosakovsky Pond & Frost, 2005b). Estimation of the ratio of non-synonymous substitutions per non-synonymous site (d_N) to synonymous substitutions per synonymous site (d_S) provides an indication of selection pressures acting on a gene. The majority of codons within a viral gene are likely to be under negative or purifying selection (d_N/d_S 1). Although average d_N/d_S values for genes are uninformative for potential positively selected codons, they do provide a means of assessing adaptive evolutionary pressures at the gene level. A value of P of 0.05 was used for inference of positive and negative selection for individual codons. A global estimate of d_N/d_S was generated with 95 % profile likelihood intervals by SLAC for each dataset.

	RESULTS

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Partial sequences of OBOV, KOLV and SJAV were obtained by conventional RT-PCR using primers to conserved regions of the N gene. Further elongation of the sequences was possible once general phylogenetic placement of these viruses was established, and new RT-PCR primers were constructed to the most closely related rhabdovirus representatives. BD SMART PCR cDNA synthesis, followed by BD Advantage long-distance PCR, yielded amplification of partial N gene sequences of MEBV, KCV, RBUV and KOTV. Entire N gene sequences were not generated; however, the 5' end of the gene was present in all four sequences obtained by the latter method. Truncation of the 3' end was probably caused by the quality of the RNA extracted by TRIzol reagent. The length of the sequences obtained (947–1287 nt) was adequate for phylogenetic comparisons.

Phylogenetic analysis recognized all seven newly sequenced viruses as members of the Dimarhabdovirus supergroup (Bourhy et al., 2005), without particular relatedness to the genus Lyssavirus (Fig. 1). OBOV, KOTV and RBUV were placed in the genus Ephemerovirus, independently of the phylogenetic method (for both nucleotide and amino acid sequences). Support for the joining of RBUV was high (76–97 % bootstrap values) only for the nucleotide NJ tree, whereas for the amino acid NJ trees and for all ML and MP trees, bootstrap support was limited (41–67 %). In all amino acid trees, FLAV joins to the ephemeroviruses as well. However, in these trees, the position of FLAV was not consistently supported by high bootstrap values (32–67 % depending on the method used). Moreover, in the nucleotide trees, FLAV was always moved to the cluster of KOLV, SJAV and TRV (yet ancestrally and with limited bootstrap support; not shown). KOLV and SJAV formed a monophyletic clade, separated from the other genera of rhabdoviruses. TRV was joined to the same clade in all phylogenetic constructions, quite distantly but with consistent bootstrap support. MEBV and KCV reliably formed a monophyletic clade with OITAV, distinct from other rhabdovirus genera and unclassified representatives.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Neighbour-joining phylogenetic trees of the Rhabdoviridae based on N gene sequences (left, alignment of 915 nucleotides, positions 439–1329 relative to the PV genome; right, alignment of 305 deduced amino acids). Branch lengths are drawn to scale and bootstrap values for 1000 replicates are given as percentages for nodes. Gaps at the 3' and 5' ends of the alignment have been truncated to ensure maximum compatibility of the compared sequences; gaps within the alignment were treated by pairwise deletion. I, Vesiculovirus; II, Ephemerovirus; III, Lyssavirus; IV, Novirhabdovirus; V, Cytorhabdovirus; VI, Nucleorhabdovirus. Bars, 0.05 changes per nucleotide position (left) and 0.2 changes per amino acid position (right).

View larger version (105K): [in this window] [in a new window]   Fig. 2. Alignment of 305 amino acids of N gene of dimarhabdoviruses (for positions see Fig. 1). Grey background indicates residues conserved in 80 % of sequences; black background indicates residues conserved in 100 % of sequences. Residues that are also conserved in lyssaviruses and SIGMAV are marked by dashes above the alignment.

View larger version (66K): [in this window] [in a new window]   Fig. 3. Results of SLAC analysis for a 241 codon fragment of the N gene of three rhabdovirus genera. Horizontal scale shows codon position. Bars above the horizontal axis show dN counts, bars below the axis show dS counts.

	DISCUSSION

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None of the seven viruses sequenced in this study were placed phylogenetically within the genus Lyssavirus, as has been suggested previously by serological cross-reactivity (Shope, 1982; Calisher et al., 1989). Instead, they were placed within the Dimarhabdovirus supergroup defined in a recent study (Bourhy et al., 2005). Representatives of this group have been isolated from arthropods (predominantly dipterans) and mammals and are proposed to be vector-borne. Thus, there is currently no documented support for the hypothesis that lyssaviruses evolved from rhabdoviruses of plants and arthropods.

OBOV and KOTV were placed within the genus Ephemerovirus. Ephemeroviruses are distributed widely in the Old World tropics and subtropics, including Australia, Africa, Asia and the Middle East. Mosquitoes are the only insects from which ephemeroviruses have been isolated. Wind-borne spread across the tropics and subtropics has been suggested for viruses of this group, and cattle translocation between continents has been suggested to explain their broad distribution in the Eastern hemisphere (Nandi & Negi, 1999; Walker, 2005). The identity between Australian ephemeroviruses ARV and BEFV was less than the identity between ARV and OBOV, which was isolated in Africa. Too few ephemerovirus isolates are available for accurate calculations of the time of divergence and, at present, such global evolutionary questions are unanswerable. The presence in this cluster of the more distantly related KOTV and RBUV suggests that intercontinental translocations of ephemeroviruses or their progenitors may have occurred long ago. In taxonomic terms, RBUV and FLAV are not placed with confidence into the genus Ephemerovirus, but their genetic relatedness is clear. Further nucleotide sequencing and phylogenetic analysis may allow this relationship to be investigated more substantially.

KOLV and SJAV form a monophyletic clade which tentatively should be considered as a new genus of the Rhabdoviridae. Since these viruses were isolated from birds, there is no reason to suggest that their distribution is limited to central Africa. Possibly, these viruses are distributed widely by migratory birds, as occurs with other avian viruses (e.g. West Nile virus). TRV demonstrates distant relatedness to this group. TRV was isolated from a tree shrew (Tupaia belangeri) imported from Thailand and has been shown to replicate only in tree shrew cells (Springfeld et al., 2005). These mammals are indigenous to tropical Asia, but not to Africa. Taken together, these data suggest that representatives of this group could be more numerous and circulate throughout different continents among different hosts. Further surveillance is needed to evaluate the phylogenetic and epidemiological patterns of these viruses.

MEBV and KCV, together with the recently described OITAV (Iwasaki et al., 2004), constituted another monophyletic clade at the genus level. Remarkably, all three viruses were isolated from insectivorous bats in distant regions of the world: Japan, central Africa and western North America. If these viruses are associated specifically with bats, it is unlikely that they are transmitted by broad-ranging sanguivorous insects, such as mosquitoes. More likely, they may be transmitted by some specific bat ectoparasites (which still may be dipterans, such as nycteribiids) or they may circulate directly among bats without the requirement for the participation of arthropods.

The functional roles of conserved residues and motifs along the deduced N protein (Fig. 2) are unclear. They do not overlap with any antigenic epitopes described for RABV (Dietzschold et al., 1987; Ertl et al., 1991; Fu et al., 1994; da Cruz et al., 2001). They may be involved in RNA binding or conformational patterns, as was shown for proline, glycine and alanine residues in the rhabdovirus N protein (Chou & Fasman, 1978; Wang et al., 1995).

Lyssaviruses constitute the most homogeneous clade within the phylogenetic tree of the Rhabdoviridae. Some authors have suggested that this may be a consequence of their youngest evolutionary age (Bourhy et al., 2005). If so, this would imply that the predominance of purifying selection along the fragment of the N gene tested is due to very rapid (and successful) adaptation to mammalian transmission and constraints placed upon lyssaviruses by their unique pathobiology. Such a situation may not to be true of the ephemeroviruses and vesiculoviruses. Estimation of divergence times for rhabdovirus genera is highly problematic. Although it is possible to estimate substitution rates for individual species, e.g. RABV (Hughes et al., 2005) and EBLV-1 (Davis et al., 2005), application of these rates to longer trees (i.e. above the species level) is confounded by substitution saturation and selection pressures that may have occurred during adaptation of progenitor viruses for emerging species.

However, our estimations clearly demonstrate that lyssaviruses are subject to strong constraints against amino acid substitutions. Positive selection analysis using SLAC detected no codons under positive selection for any dataset. Considering the conservative nature of the SLAC method (Kosakovsky Pond & Frost, 2005b) and previous findings of largely purifying selection in lyssavirus N genes (Holmes et al., 2002; Kuzmin et al., 2004; Hughes et al., 2005; Davis et al., 2005), this is not surprising. Such constraints appear to be much greater than those observed among ephemeroviruses and vesiculoviruses.

In general, the greatest diversity in the Rhabdoviridae is seen among the insect-borne plant viruses (genera Cytorhabdovirus and Nucleorhabdovirus), followed by Novirhabdovirus, isolated from fish and other aquatic animals (including invertebrates), the dimarhabdoviruses (able to replicate in vertebrate and invertebrate hosts) and finally the lyssaviruses, which replicate exclusively in mammals (Johnson et al., 1999; Tordo et al., 2004; Fu, 2005; Bourhy et al., 2005).

No particular phylogenetic relatedness was demonstrated between lyssaviruses and the other rhabdoviruses sequenced to date. Limited serological cross-reactivity detected in earlier studies between lyssaviruses and the seven viruses which were sequenced during the current work (Shope, 1982; Calisher et al., 1989) could be attributed to common antigenic determinants, since they are all members of one family. We are clearly missing some important chains in the evolutionary pathway of the Rhabdoviridae. With more than 40 unassigned animal rhabdoviruses in the list of the International Committee on Taxonomy of Viruses (Tordo et al., 2004), additional sequencing is essential to clarify phylogenetic relationships. Such studies with known isolates, together with enhanced field surveillance for new pathogens, should provide greater insight into the biology and evolution of this family.

	ACKNOWLEDGEMENTS

 
We thank Dr C. Calisher (retired) for suggestions on the list of viruses which demonstrated serologic cross-reactivity with lyssaviruses. We are grateful to Drs A. Powers and S. Bartlet (DVBID, CDC, Ft Collins, CO, USA) for providing viruses from the CDC Arbovirus Reference Collection. We thank Dr X. Wu (DVRD, CDC, Atlanta, GA, USA) for his expertise and help in the molecular cloning. We also thank Sergei Kosakovsky Pond for assistance with the positive selection analysis. The work was supported in part by the Association of Public Health Laboratories (Washington, DC, USA; International EID fellowship program of 2002–2005). The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the funding agency.

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Received 27 January 2006; accepted 11 April 2006.

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