Unité de la Rage1 and Laboratoire des Lyssavirus2, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France
National Veterinary Research Institute, 24-100 Pulawy, Poland3
National Institute of Hygiene, 24 Chocimska Str., 00-791 Warsaw, Poland4
National Veterinary and Food Research Institute, PL 368, FIN-00231 Helsinki, Finland5
Wellcome Trust Centre for the Epidemiology of Infectious Disease, Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK6
Author for correspondence: Hervé Bourhy.Fax +33 1 40 61 30 20. e-mail hbourhy{at}pasteur.fr
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Abstract |
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Introduction |
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Occasionally lyssaviruses gain access to new populations of susceptible hosts, particularly those which are geographically restricted (Rupprecht & Smith, 1994 ), or evolve to infect previously less susceptible hosts (Sacramento et al., 1992
; Smith et al., 1992
, 1995
; Tordo et al., 1993
; Nadin-Davis et al., 1994
). It is evident that such an adaptive process took place in Europe during the first decades of this century when rabies virus became established in the red fox following a decline in incidence among urban dogs and wolves (Zeeti & Rosati, 1966
; Petrovic, 1987
). Although the virus initially failed to adapt to red foxes, as shown in the records of animal deaths (Barbier, 1929
; Jaujou, 1949
; Steck & Wandeler, 1980
; Blancou et al., 1991
), by 19401945 rabies-infected foxes were regularly found at the former RussianPolish border (Zunker, 1954
) and in the region of Gdansk in northern Poland (Seroka, 1968
). Subsequently, the infection of red foxes spread to the rest of Europe, reaching France by 1968 (Atanasiu et al., 1968
).
The study described here was designed to determine the evolutionary history of rabies virus in Europe using nucleoprotein (N) and glycoprotein (G) gene sequences. In particular, we wished to determine the level and structure of standing genetic variation within European rabies viruses and reveal what evolutionary processes might have given rise to this structure. Furthermore, as little is known about how the host range of rabies virus is determined at the molecular level, we also aimed to identify those mutations, if any, which might have allowed the virus to infect new species. The N gene was chosen for this analysis because it encodes an internal protein involved in the regulation of transcription and replication and could therefore be an important factor in host adaptation (Kissi et al., 1995 ). The G gene encodes an external protein important in pathogenicity (Dietzschold et al., 1983
) and which reacts with cellular receptors of rabies virus, and so may also be important in determining host range (Tuffereau et al., 1998
; Thoulouze et al., 1998
). To this end, 245 isolates of rabies virus, stemming from a range of mammalian hosts and a variety of geographical locations, particularly within Europe, were analysed either by sequencing or by RFLP.
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Methods |
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Analysis of isolates by restriction fragment length polymorphism.
One µl of the amplified products was digested by selected restriction endonucleases and run on a 2% agarose gel with ethidium bromide as described previously (Bourhy et al., 1992 ). On the basis of the alignment of the nucleotide sequences described in this study and by using the MAPSORT program implemented in the GCG package (Version 8.1-UNIX, program manual for the Wisconsin Package, 1995), four restriction endonucleases (BsaBI, HindIII, MboII and NlaIV) were selected for their ability to differentiate the European isolates.
Sequence analysis.
After the removal of identical sequences, 33 complete N gene sequences (1350 bp), 29 partial G gene sequences (690 bp) and 85 partial N gene sequences (400 bp) were available for analysis. Multiple sequence alignments of these data were generated with the CLUSTALW program (Thompson et al., 1994 ). For 19 isolates, both N and G gene sequences were available and so were concatenated into a combined alignment of 2040 bp.
Phylogenetic trees were constructed using the maximum likelihood (ML) method available in the 4.0d65 test version of PAUP* kindly provided by David L. Swofford. The HKY85 model of nucleotide substitution was used in all cases, with the transition/transversion (Ts/Tv) ratio and shape parameter of a gamma distribution (with eight categories) of rate variation among sites estimated from the empirical data. The values of these parameters for each data set are given in Table 2
. To gauge how well each node on the trees was supported, a bootstrap analysis was undertaken (1000 replications), although computational constraints meant that this was performed on neighbour-joining trees reconstructed under the ML substitution model. Monte Carlo simulation (the parametric bootstrap) was then used to determine whether trees estimated on different genes had significantly different topologies, with replicate ML trees generated using the Seq-Gen program (Rambaut & Grassly, 1997
).
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Results |
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Because of the strong patterning by geographical location and host species seen in the complete N gene phylogeny, we decided to examine a much larger number of virus isolates using a 400 bp region from the amino terminus of the N gene, previously identified as one of its most variable regions (Kissi et al., 1995 ). This analysis focused on 85 unique isolates from the Arctic, Africa, the Middle East and Europe (including the 22 analysed previously). The ML tree of these data is shown in Fig. 1(b)
and is congruent to that obtained from the complete N gene in that the geographically distinct clusters of rabies virus in Europe are evident, although often with weaker bootstrap support. One conspicuous difference is that the CE group appears to be derived from the WE group on a long branch. However, the log likelihood of this tree is only marginally better (-2591·94597 versus -2596·47731) than one in which the position of the WE and CE groups have been rearranged to give the ML topology seen in the analysis of the complete N gene.
Of more importance is that the NEE group (95% bootstrap support) is now found to cover a wider geographical area, including Poland, Estonia, Lithuania 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. Furthermore, isolates 86107YOU and 9215HON are both placed close to dog rabies viruses (although with weak bootstrap support), suggesting that they may represent early cross-species transmissions from the dog viruses that circulated in Europe early this century, while the 8658YOU cattle strain remains divergent. Finally, the dog and jackal isolates again form two groups, with those from the Middle East more closely related to the European fox viruses than those from Africa.
To determine whether similar evolutionary patterns are found in other genes of rabies virus, we performed a phylogenetic analysis on a 690 bp region encoding the central part of the ectodomain of the glycoprotein, another region which exhibits a high degree of sequence variation (Tordo et al., 1993 ). For this purpose, 29 G gene sequences were determined, 22 of which were isolated in Europe with three from Africa (Benmansour et al., 1992
). The results of this analysis are presented in Fig. 2
. Although many of the phylogenetic relationships depicted are the same as those seen in the N gene tree that is, there is a general association by host and place of isolation the EE strains are no longer the most divergent set of fox viruses, instead falling closer to the WE strains, with the foxraccoon dog NEE strains now more divergent. Whilst some of these nodes are well supported, others are more ambiguous, including those where the G gene phylogeny differs from the N gene phylogeny, such as the divergent position of the NEE isolates (nodes shaded in Fig. 2
). The phylogenetic relationships within each group of viruses were also uncertain in places, and were the main reason why 135 trees of equal likelihood were reconstructed on these data.
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As we found no evidence that the N and G gene trees differ in topology, we were able to combine the 19 N and G gene sequences in a single phylogenetic analysis. This resulted in an ML tree containing elements of those constructed on the two genes separately (Fig. 3). Specifically, the N+G tree resembles the G gene phylogeny in clearly placing the NEE isolates as more divergent than the red fox groups, but shares a similar topology with the N gene tree in that the CE strains are closer to the WE strains than are the EE strains. Significantly, many of these important nodes now receive strong bootstrap support (Fig. 3
). Although the increase in bootstrap support is influenced by the smaller number of taxa compared, it should be noted that all the major groups were represented in analysis, with the reduction in number mainly due to the loss of sequences within groups. We therefore believe that the phylogenetic relationships among the geographical groups of rabies virus in Europe are best represented by the phylogeny of the combined N and G data sets.
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For the complete N gene tree, 26 tips beginning with the divergence of 8658YOU were found to have produced significantly more daughters than expected under the null model of a uniform rate of cladogenesis (Pk<005). Such a biased branching process was also found in the tree of the partial N gene sequences in which 80 tips, beginning with the divergence of PV and 8658YOU, were linked in an asymmetric fashion (Pk<0·01). Similar, although less strong, results were found in the G gene tree, for 22 tips starting with the divergence of the 8658YOU, 9215HON, 86107YOU and NEE clade (Pk<0·05), and for the 15 tips in the N+G tree beginning with split of 9215HON (Pk<0·05). We therefore conclude that the phylogenetic trees of these isolates are strongly biased in their branching structures.
Geographical distribution of European rabies virus isolates
A further 135 isolates of rabies virus from central-eastern Europe were typed by RFLP according to the geographical (and phylogenetic) groups we describe above. The size of the bands expected in the RFLP profiles of the different phylogenetic clusters were as follows: (i) NEE (BsaBI, 400 bp; HindIII, 210 and 190 bp; NlaIV, 391 or 400 bp); (ii) EE (BsaBI, 400 bp; HindIII, 400 bp; NlaIV, 400 bp; MboII can be used as a positive control, 271 and 129 bp); (iii) WE (BsaBI, 400 bp; HindIII, 400 bp; NlaIV, 300 and 91 bp); (iv) CE (BsaBI, 283 and 117 bp; HindIII, 400 bp; NlaIV, 300 and 91 bp).
From this analysis (as well as the sequence data), we were able to map the location of the different phylogenetic groups of rabies virus within Europe (Fig. 4). A clear picture of geographical subdivision is revealed with the Vistula (or Wista) river in Poland separating the CE and NEE clusters and, to a lesser extent, the Bohemian and Carpathian mountains reinforced by the Danube river in the Czech Republic, Slovak Republic, Austria and Hungary isolating the EE viruses. To be more specific, the NEE cluster was found in Finland, Estonia, Lithuania, Poland and in the eastern part of the Slovak Republic. In Poland, the NEE group is limited to the eastern side of the Vistula river, with the exception of four isolates found close to the river. In contrast, the CE cluster was isolated mainly in the west and south of Poland (i.e. to the west of the Vistula river), the east of Germany, the Czech Republic and Slovenia. One CE isolate was also found in Poland near the Lithuanian border. The WE cluster was found in a region stretching from France and Belgium to the west and south of Poland. It was also isolated from Switzerland and Austria, and was frequently found in the south of this region, particularly in Slovenia, BosniaHerzegovina and the Federal Republic of Yugoslavia. The EE cluster was limited to the south-east of the Czech Republic and Poland, to BosniaHerzegovina and to Hungary.
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To determine whether any of these amino acid changes might have been fixed by natural selection, we calculated the mean numbers of synonymous (dS) and nonsynonymous (dN) substitutions per site in both the N and G genes (the two African type 2 lyssaviruses were removed from the N gene analysis to make the results more comparable between genes). As expected given the relatively low numbers of amino acid changes, dS was much greater than dN in every case, therefore providing no evidence for positive selection (i.e. dN>dS) at this level, with the N gene (mean dN=0·0087±0·0012, mean dS=0·2678±0·0142; dN/dS=0·032) apparently under slightly stronger selective constraints than the G gene (mean dN=0·0115±0·0020, mean dS=0·2494±0·0204; dN/dS=0·046). Although informative, such large-scale pairwise comparisons are unlikely to reveal the affects of natural selection on individual amino acids. To assess this possibility we calculated the mean dN value for each codon in the N and G genes and identified those with higher rates than the mean dS across all codons, which we assume is a marker of the background (neutral) mutation rate. These results are shown in Fig. 5 and reveal a single codon in the N gene (position 101) and three in the G gene (positions 1, 5 and 175) with elevated rates of nonsynonymous substitution. It is intriguing that N gene codon 101 falls into this category because, as noted above, it contains amino acid substitutions which distinguish the red fox viruses. While is it is possible that selectively advantageous substitutions have been fixed at these sites, the small numbers of changes involved, and hence the likelihood of sampling artefacts, mean that these results should be interpreted with caution.
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Discussion |
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Not only were the phylogenies we obtained strongly ordered by geography, but they also had a strong `ladder-like' structure, with the deepest branches belonging to viruses collected in the north and east of Europe, and the most recent branches belonging to viruses collected further west and south. From this we conclude that our phylogenetic analysis documents the gradual dispersal of rabies virus from the north-east to the south-west across Europe, as has been previously suggested based on epidemiological data (Blancou et al., 1991 ). A similar east to west movement was previously documented in tick-borne flaviviruses, a process which perhaps took around 2000 years to unfold (Zanotto et al., 1995
). The spread of rabies viruses that we describe clearly occurred much more recently than this, as is evident from the epidemiological records of rabies cases this century (Zunker, 1954
; Seroka, 1968
; Atanasiu, 1968
).
Despite the fluidity of rabies virus transmission in Europe, it is equally clear that its spread can be contained to some extent by natural physical barriers such as the Vistula river in Poland, most likely by restricting the movement of infected hosts. In this respect, our sequencing and RFLP data show that virus isolates in central-eastern Europe have a strong geographical clustering, suggesting that there is some degree of genetic isolation. In these circumstances it would seem pertinent to continue surveillance of the different populations of fox rabies virus within Europe as we might expect the continued adaptation to local mammalian fauna, as is highlighted by the species jump to raccoon dogs (see below), and to monitor whether the NEE cluster will eventually disperse further westwards as raccoon dogs have themselves done.
During the westwards and southwards movement of rabies virus across Europe two changes of host species took place. The first occurred when the virus initially jumped from dogs to foxes, although it is unclear from our analysis exactly where this took place. However, as the deepest branches of the fox virus tree are found in eastern Europe, we suggest that a species jump in this region seems the most reasonable interpretation of the data. The second change in host took place in north-eastern Europe when rabies viruses colonized raccoon dogs. From our phylogenetic analysis it is not possible to determine precisely whether the source of the virus in raccoon dogs was infected foxes, or whether the virus jumped directly from dogs and was then passed to the local fox population. Nor is it clear what ecological pressure (if any) precipitated this host switch, although it is apparent that raccoon dogs are a common enough wildlife species to be able to sustain such an infection. That the NEE strain is found in the region where the population of raccoon dogs is greatest suggests that the density of susceptible hosts, as well the close proximity of a donor species, are major ecological factors in the establishment of rabies virus in a new host species and also that perhaps the NEE strain is preferentially adapted to this species, although this is clearly an issue that needs to be explored further.
Finally, the status of three virus strains collected from humans, red fox and cattle in eastern Europe and which represent divergent lineages on the trees is unclear, although it seems most likely that they were derived from dog rabies viruses. It is therefore possible that they represent spill-over infections with viruses belonging to lineages which were established early this century when dog viruses were more commonly found in EE and before the red fox was established as the major reservoir of rabies infection. Such a spill-over of Canidae-associated viruses into wildlife species is frequently observed (Nel et al., 1997 ).
Given the existence of geographically distinct variants of rabies virus, the next question to address is whether functionally important amino acid changes have accumulated between them, particularly those that might have enabled adaptation to different host species. Strikingly, both the G and N proteins are generally conserved with few amino acid replacements accumulating among the strains studied. In particular, very few amino acid changes were found to accompany the change in transmission from dogs to foxes or raccoon dogs, although it is also possible that key mutations reside in other genes. In a similar vein, an analysis of the relative numbers of synonymous and nonsynonymous substitutions revealed that both the G and N genes are under relatively strong selective constraints, although some codons have experienced much higher rates of nonsynonymous change than others (and higher than the background silent substitution rate), which may signify localized positive selection pressure (Kissi et al., 1999 ). Although the significance of these changes is unclear, they merit careful investigation at the structuralfunctional level as it is possible that they are of phenotypic importance.
Considering that all strains of rabies virus in Europe are not equally able to infect dogs, foxes and raccoon dogs (Blancou et al., 1983 ; Blancou & Aubert, 1997
), our study confirms previous suggestions that the infection of new host species in nature could be caused by a small number of genetic changes in rabies virus, involving just a few amino acid replacements (Tuffereau et al., 1989
; Kissi et al., 1999
). However, this does not exclude the possibility that some of the phylogenetic groups we describe reflect instead the ecological separation of individuals belonging to the same or different host species, as has been observed in other viruses (Nickels & Hunt, 1994
; Parrish, 1994
). Such a conclusion distinguishes the N genes of rabies virus from the capsid genes of some other negative-strand RNA viruses, such as influenza A virus (for a review see Webster et al., 1992
), and positive-strand RNA viruses like coxsackieviruses and alphaviruses (Villaverde et al., 1991
; Ishiko et al., 1992
; Domingo & Holland, 1994
; Weaver et al., 1994
), which often evolve at greatly elevated rates at amino acid changing sites, presumably because of strong positive selection.
To conclude, given that we provide strong evidence that local genetic differentiation is taking place within European rabies viruses, we urge that further studies of virus variation be undertaken so that we may come to a greater understanding of the mechanisms controlling adaptation to new host species, information that is crucial to the greater goal of eliminating terrestrial rabies from Europe.
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Acknowledgments |
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Footnotes |
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References |
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Atanasiu, P., Gamet, A., Gravière, P., Le Guilloux, M., Guillon, J. C. & Vallée, A. (1968). Réapparition de la rage en France. Premier cas chez un renard dans la Moselle. Bulletin de l'Académie Vétérinaire XLI, 161-163.
Barbier, A. (1929). Les Sources de la Virulence Rabique. Histoire d'une Epizootie de Rage sur le Renard et le Blaireau dans la Région Dijonnaise, pp. 253. Dijon: Imprimerie Bernigaud et Privat.
Benmansour, A., Brahimi, M., Tuffereau, C., Coulon, P., Lafay, F. & Flamand, A. (1992). Rapid sequence evolution of street rabies glycoprotein is related to the highly heterogeneous nature of the viral population. Virology 187, 33-45.[Medline]
Blancou, J. & Aubert, M. F. A. (1997). Transmission du virus de la rage: importance de la barrière d'espèce. Bulletin de l'Académie Nationale de Médecine 181, 301-312.
Blancou, J., Aubert, M. F. A. & Soulebot, J. P. (1983). Différences dans le pouvoir pathogène de souches de virus rabique adaptées au renard ou au chien. Annales de l'Institut Pasteur Virology 134E, 523-531.
Blancou, J., Aubert, M. F. A. & Artois, M. (1991). Fox rabies. In The Natural History of Rabies, pp. 257-290. Edited by G. M. Baer. Boca Raton: CRC Press.
Bourhy, H., Kissi, B., Lafon, M., Sacramento, D. & Tordo, N. (1992). Antigenic and molecular characterization of bat rabies virus in Europe. Journal of Clinical Microbiology 30, 2419-2426.[Abstract]
Bourhy, H., Kissi, B. & Tordo, N. (1993). Molecular diversity of the lyssavirus genus. Virology 194, 70-81.[Medline]
Dietzschold, B., Wunner, W. H., Wiktor, T. J., Lopes, A. D., Lafon, M., Smith, C. L. & Koprowski, H. (1983). Characterization of an antigenic determinant of the glycoprotein that correlates with pathogenicity of rabies virus. Proceedings of the National Academy of Sciences, USA 80, 70-74.[Abstract]
Domingo, E. & Holland, J. J. (1994). Mutation rates and rapid evolution of RNA viruses. In The Evolutionary Biology of Viruses, pp. 161-184. Edited by S. S. Morse. New York: Raven Press.
Gould, A. R., Hyatt, A. D., Lunt, R., Kattenbelt, J. A., Hengstberger, S. & Blacksell, S. D. (1998). Characterization of a novel lyssavirus isolated from Pteropid bats in Australia. Virus Research 54, 165-187.[Medline]
Ishiko, H., Takeda, N., Miyamura, K., Kato, N., Tanimura, M., Lin, K.-H., Yin-Murphy, M., Tam, J. S., Mu, G.-F. & Yamazaki, S. (1992). Phylogenetic analysis of a coxsackievirus A 24 variant: the most recent worldwide pandemic was caused by progenies of a virus prevalent around 1981. Virology 187, 748-759.[Medline]
Jaujou, M. (1949). L'infection rabique en Corse au cours de l'année 1946. Académie Nationale de Médecine 132, 128-130.
Kissi, B., Tordo, N. & Bourhy, H. (1995). Genetic polymorphism in the rabies virus nucleoprotein gene. Virology 209, 526-537.[Medline]
Kissi, B., Badrane, H., Audry, L., Lavenu, A., Tordo, N., Brahimi, M. & Bourhy, H. (1999). Dynamics of rabies virus quasispecies during serial passages in heterologous hosts. Journal of General Virology 80, 2041-2050.
Kumar, S., Tamura, K. & Nei, M. (1993). MEGA: molecular evolutionary genetic analysis, version 1.0. The Pennsylvania State University, University Park, PA 16802, USA.
Maddison, W. P. & Maddison, D. R. (1992). MacClade: analysis of phylogeny and character evolution, version 3.0. Sinauer Associates: Sunderland, MA, USA.
Nadin-Davis, S. A., Casey, G. A. & Wandeler, A. I. (1994). A molecular epidemiological study of rabies virus in central Ontario and western Quebec. Journal of General Virology 75, 2575-2583.[Abstract]
Nee, S., May, R. M. & Harvey, P. H. (1994). The reconstructed evolutionary process. Philosophical Transactions of the Royal Society of London Series B 344, 305-311.[Medline]
Nei, M. & Gojobori, T. (1986). Simple methods for estimating the numbers of synonymous and non synonymous nucleotide substitutions. Molecular Biology and Evolution 3, 418-426.[Abstract]
Nel, L., Jacobs, J., Jaftha, J. & Courteney, M. (1997). Natural spillover of a distinctly Canidae-associated biotype of rabies virus into an expanded wildlife host range in Southern Africa. Virus Genes 15, 79-82.[Medline]
Nickels, M. S. & Hunt, D. M. (1994). Identification of an amino acid change that affects N protein function in vesicular stomatitis virus. Journal of General Virology 75, 3591-3595.[Abstract]
Nowak, R. M. & Paradiso, J. L. (1983). Walker's Mammals of the World, vol. II, 4th edn. Baltimore: The Johns Hopkins University Press.
Parrish, C. R. (1994). The emergence and evolution of canine parvovirus an example of recent host range mutation. Virology 5, 121-132.
Petrovic, M. (1987). Urban and sylvatic rabies in Yugoslavia. Rabies Bulletin Europe 4, 16-18.
Poch, O., Tordo, N. & Keith, G. (1988). Sequence of the 3386 3' nucleotides of the genome of the AVO1 strain rabies virus: structural similarities in the protein regions involved in transcription. Biochimie 70, 1018-1029.
Rambaut, A. & Grassly, N. C. (1997). Seq-Gen: an application for the Monte Carlo simulation of DNA sequence evolution along phylogenetic trees. CABIOS 13, 235-238.[Abstract]
Rambaut, A., Harvey, P. H. & Nee, S. (1997). End-Epi: an application for inferring phylogenetic and population dynamical processes from molecular sequences. CABIOS 13, 303-306.[Abstract]
Rupprecht, C. E. & Smith, J. S. (1994). Raccoon rabies: the re-emergence of an epizootic in a densely populated area. Seminars in Virology 5, 155-164.
Sacramento, D., Bourhy, H. & Tordo, N. (1991). PCR technique as an alternative method for diagnosis and molecular epidemiology of rabies virus. Molecular and Cellular Probes 6, 229-240.
Sacramento, D., Badrane, H., Bourhy, H. & Tordo, N. (1992). Molecular epidemiology of rabies in France: comparison with vaccine strains. Journal of General Virology 73, 1149-1158.[Abstract]
Seroka, D. (1968). The distribution of stationary foci of rabies in wild animals in Poland. Epidemiological Review (English Translation of Przeglad Epidemiologiczny) 22, 66-75.
Smith, J. S., Orciari, L. A., Yager, P., Seidel, H. D. & Warner, C. K. (1992). Epidemiologic and historical relationships among 87 rabies virus isolates as determined by limited sequence analysis. Journal of Infectious Diseases 166, 296-307.[Medline]
Smith, J. S., Orciari, L. A. & Yager, P. (1995). Molecular epidemiology of rabies in the United States. Seminars in Virology 6, 387-400.
Steck, F. & Wandeler, A. (1980). The epidemiology of fox rabies in Europe. Epidemiologic Reviews 2, 71-96.[Medline]
Stöhr, K., Stöhr, P. & Karge, E. P. (1992). Isolierung atypischer Tollwutfeld-Viren in Ostdeutschland. Tieräztliche Umschau 47, 820-824.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTALW: improving the sensitivity of progressive multiple alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22, 4673-4680.[Abstract]
Thoulouze, M.-I., Lafage, M., Schachner, M., Hartmann, U., Cremer, H. & Lafon, M. (1998). The neural cell adhesion molecule is a receptor for rabies virus. Journal of Virology 72, 7181-7190.
Tordo, N., Poch, O., Ermine, A., Keith, G. & Rougeon, F. (1986). Walking along the rabies genome: is the large GL intergenic region a remnant gene? Proceedings of the National Academy of Sciences, USA 83, 3914-3918.[Abstract]
Tordo, N., Badrane, H., Bourhy, H. & Sacramento, D. (1993). Molecular epidemiology of lyssaviruses: focus on the glycoprotein and pseudogenes. Onderstepoort Journal of Veterinary Research 60, 315-323.[Medline]
Tuffereau, C., Leblois, H., Bénéjean, J., Coulon, P., Lafay, F. & Flamand, A. (1989). Arginine or lysine in position 333 of ERA and CVS glycoprotein is necessary for rabies virulence in adult mice. Virology 172, 206-212.[Medline]
Tuffereau, C., Bénéjean, J., Roque Alfonso, A. M., Flamand, A. & Fishman, M. C. (1998). Neuronal cell surface molecules mediate specific binding to rabies virus glycoprotein expressed by a recombinant baculovirus on the surfaces of lepidopteran cells. Journal of Virology 72, 1085-1091.
Villaverde, A., Martinez, M. A., Sobrino, F., Dopazo, J., Moya, A. & Domingo, E. (1991). Fixation of mutations at the VP1 gene of foot-and-mouth disease virus. Can quasispecies define a transient molecular clock? Gene 103, 147-153.[Medline]
Weaver, S. C., Hagenbaugh, A., Bellew, A., Gousset, L., Mallampalli, V., Holland, J. J. & Scott, T. W. (1994). Evolution of alphaviruses in the eastern equine encephalomyelitis complex. Journal of Virology 68, 158-169.[Abstract]
Webster, R. G., Bean, W. J., Gorman, O. T., Chambers, T. M. & Kawaoka, Y. (1992). Evolution and ecology of influenza A viruses. Microbiological Reviews 56, 152-179.[Abstract]
Zanotto, P. M. de A., Gao, G. F., Gritsun, T. S., Marin, M. S., Jiang, W. R., Venugopal, K., Reid, H. W. & Gould, E. A. (1995). An arbovirus cline across the northern hemisphere. Virology 210, 152-159.[Medline]
Zanotto, P. M. de A., Gould, E. A., Gao, G. F., Harvey, P. H. & Holmes, E. C. (1996). Population dynamics of flaviviruses revealed by molecular phylogenies. Proceedings of the National Academy of Sciences, USA 93, 548-553.
Zeeti, R. & Rosati, T. (1966). Informations relatives à la situation de la rage en Italie et les mesures employées pour la combattre. Bulletin de l'Office International des Epizooties 65, 37-39.
Zunker, M. (1954). L'importance des renards dans la propagation de la rage en Allemagne. Bulletin de l'Office International des Epizooties 354, 1-11.
Received 18 March 1999;
accepted 16 June 1999.