1 Department of Microbiology and Parasitology, University of Queensland, St Lucia, Queensland, Australia
2 Department of Primary Industries, Queensland Agricultural Biotechnology Centre, Level 4, Gehrmann Laboratories, University of Queensland, St Lucia, Queensland, Australia
3 Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK
4 Unité de la Rage, Institut Pasteur, Paris Cedex 15, France
5 Public Health Virology, Queensland Health Scientific Services, Coopers Plains, Queensland, Australia
Correspondence
Kimberley Guyatt (at Queensland Agricultural Biotechnology Centre)
k.guyatt{at}mailbox.uq.edu.au
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ABSTRACT |
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The GenBank accession numbers of the sequences reported in this paper are AF426290AF426311.
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Introduction |
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Two human deaths in Queensland have been attributed to ABL infection, where ABL is thought to have been transmitted to the susceptible hosts in saliva following bites from infected bat species (Allworth et al., 1996; Hanna et al., 2000
). Case studies of both flying fox and human ABL infections have indicated that ABL follows a course of clinical disease similar to that of rabies, presenting as a severe non-suppurative encephalitis with symptoms of aggression, anxiety, hypersalivation and agitation (Allworth et al., 1996
; Fraser et al., 1996
; Hanna et al., 2000
; McColl et al., 2000
). These ABL spillover events have been a cause for concern as bat-associated RVs and their variants have been responsible for many of the recent human deaths related to rabies in the USA, which in many cases have had no clear exposure to rabid bat species.
ABL belongs to the Lyssavirus genus within the family Rhabdoviridae (Hooper et al., 1997). Like other lyssaviruses, ABL has a negative-sense, single-stranded RNA genome, which encodes a nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA polymerase (L), in the order 3'-N-P-M-G-L-5' (Gould et al., 1998
; Warrilow et al., 2002
). A further feature of the ABL genome, also common to other lyssaviruses, is the presence of a non-coding region between the G and L genes, known as the GL intergenic or pseudogene (
) region. Several molecular epidemiological studies have been conducted to determine phylogenetic relationships between lyssavirus isolates, their strains, host species and geographical distribution, with most investigations based on nucleotide sequence comparison of the N gene, G gene and
regions (Badrane & Tordo, 2001
; Badrane et al., 2001
; Nadin-Davis et al., 2001
).
Comparison of the N gene, G gene and sequences of numerous lyssavirus isolates and strains has delineated the Lyssavirus genus into seven genotypes (GTs): RV (GT1), Lagos bat virus (LAG, GT2), Mokola virus (MOK, GT3), Duvenhage virus (DUV, GT4), European bat lyssavirus 1 (EBLV1, GT5), European bat lyssavirus 2 (EBLV2, GT6) and ABL (GT7) (Badrane et al., 2001
). However, since its discovery, there has been some contention as to whether ABL is an Australian rabies virus variant or a distinct Lyssavirus species. Virus cross-neutralization and serological assays have indicated that ABL and RV are very similar antigenically (Fraser et al., 1996
; Gould et al., 1998
). Yet, preliminary phylogenetic analyses of the nucleotide sequences of the ABL N, P, M and G genes with other lyssaviruses have suggested that, although ABL is the most closely related lyssavirus to RV, it is sufficiently different at the nucleotide level to be assigned to its own genotype (Badrane et al., 2001
; Gould et al., 1998
; Hooper et al., 1997
). Two distinct ABL strains appear to be circulating within Australia currently, one found in flying foxes (Gould et al., 1998
), the other in the insectivorous bat species S. flaviventris (Hooper et al., 1997
). However, as yet very little is known regarding the interrelationships between flying fox and insectivorous ABL isolates, as well as ABL and other lyssavirus species, as few ABL isolates have been studied or sequenced.
Several phylogenetic analyses conducted on lyssaviruses have focused on the G gene, which presents the advantage of encoding the G glycoprotein containing determinants of lyssavirus pathogenicity and host specificity. The lyssavirus G glycoprotein ranges in size from 524 to 535 amino acids and consists of four domains: a 19-amino-acid signal peptide, which is cleaved from the protein's N terminus to produce the mature glycoprotein, a surface-exposed 439-amino acid highly conserved ectodomain, a 22-amino-acid transmembrane domain and a C-terminal endodomain, which varies in length from 42 to 53 amino acids. The G glycoprotein forms a trimer on the surface of lyssavirus virions, such that during infection it is the first viral protein to come into contact with the host. Apart from containing domains responsible for host-cell receptor recognition (Lentz et al., 1982; Thoulouze et al., 1998
; Tuffereau et al., 1998
, 2001
) and membrane fusion (reviewed by Gaudin et al., 1999
), the G glycoprotein is the major target for host neutralizing antibody responses (Wiktor et al., 1973
). Selection of RV mutants with neutralizing anti-G monoclonal antibodies has shown that amino acid changes mapping to specific sites in G often result in RV attenuation (Dietzschold et al., 1983
; Flamand et al., 1980
). It has also been demonstrated by reverse genetics that sequence variation within the G gene provides a basis for RV pathogenesis (Ito et al., 2001a
; Morimoto et al., 2000
). Accordingly, changes in lyssavirus G gene sequences may influence not only viral pathogenicity and host susceptibility, but may also allow the viruses to adapt to new hosts resulting in the emergence of new lyssavirus species.
The aims of this study were therefore threefold. First, it was of interest to determine whether correlations exist between particular ABL isolates, geographical locations and ABL infection of pteropid and insectivorous bat populations by comparing their G gene sequences. Such information can be used to establish epidemiological links and potential risk factors that may increase the likelihood of human ABL exposure. Secondly, we wished to investigate whether the ABL G gene is subject to positive selective pressure (adaptive evolution) in nature, as genetic variability within this region of the ABL genome may allow evasion of immune responses and/or produce viruses with an extended host range and specificity. Finally, we wished to clarify the position of ABL within the Lyssavirus genus. Most previous studies have included only a single pteropid or insectivorous ABL sequence in their phylogenetic analyses, which has limited the possibility of identifying intra- and interspecies relationships between ABL isolates and other lyssaviruses. We decided to pursue this further by comparing the G gene sequences of a number of ABL isolates with the corresponding regions of other lyssavirus species.
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Methods |
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Phylogenetic analysis.
Nucleotide sequences of the 22 ABL isolate G gene regions were edited using the Sequencher 3.1.1 program (Gene Codes Corporation). Glycoprotein amino acid sequences were deduced using ETRANSLATE and compared using the multiple sequence alignment program ECLUSTALW (Thompson et al., 1994), and percentage nucleotide and amino acid identities were calculated by the HOMOLOGIES program (Jack Leunissen CAOS/CAMM Center, University of Nijmegen, The Netherlands). These programs are part of the Genetics Group Wisconsin Inc. (GCG) suite of sequence analysis programs maintained by the Australian National Genomic Information Service (ANGIS, http://www.angis.org.au). Nucleotide and amino acid sequence alignments prepared using ECLUSTALW were edited using the SEAVIEW program and phylogenetic analyses were performed using the Linux-based PHYLO_WIN program (Galtier et al., 1996
) or programs of the PHYLIP package (Felsenstein, 1993
). Phylogenetic trees were constructed by: (i) maximum-parsimony (MP) using algorithms from the DNAPARS and PROTPARS programs of the PHYLIP package; (ii) neighbour-joining (NJ) using the evolutionary distance correction statistics of Kimura (1980)
and Tajima & Nei (1984)
; and (iii) maximum-likelihood (ML) using the PAUP* phylogenetic program (Swofford, 2001
). Bootstrap resampling analysis (Felsenstein, 1985
) was undertaken using 1000 data replications to evaluate the robustness of the phylogenetic groupings observed. Bootstrap values of greater than 70 % confidence were regarded as giving strong evidence for a particular phylogenetic grouping (Hillis & Bull, 1993
). All ABL nucleotide sequences obtained in this study have been submitted to GenBank and their accession numbers are listed in Table 1
. All other lyssavirus nucleotide sequences used for phylogenetic analysis and sequence comparison were obtained from GenBank; their accession numbers and appropriate references are listed in Table 1
.
Selection analysis.
An ML approach was used to analyse selection pressures in the ABL G gene sequences. In this method, the numbers of synonomous (silent, dS) and non-synonomous (amino acid-changing, dN) substitutions per site were determined using various models of codon substitution that differ in how the ratio dN/dS varies among codons (Yang et al., 2000). Evidence for positive selection is provided when a model of codon evolution that shows dN/dS>1 at a number of codons is significantly favoured (in a likelihood ratio test) over a competing model in which dN/dS ratios are constrained to be <1 at all codons. If positive selection is found, those sites that are positively selected (i.e. with dN/dS>1) can be individually identified using Bayesian methods. This analysis was undertaken using the CODEML program of the PAML package (Yang, 1997
). More details of this approach applied to RNA viruses are given in Holmes et al. (2002)
.
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Results |
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For the chiropteran RVs, it was additionally observed that the level of nucleotide sequence identity among different RV variants (82·191·8 %) was intermediate between that observed for isolates belonging to the same RV variant and viruses belonging to different lyssavirus species. This observation is interesting in light of the fact that pteropid ABL isolates share only 81·383·2 % nucleotide sequence conservation with ABL isolated from S. flaviventris and that the two Lagos bat isolates originating from different bat species also display a similarly reduced level of sequence similarity (76·0 %).
Evolutionary relationships of ABL isolates inferred from G gene sequences
To investigate the intragenotypic variability of the ABL isolates further, phylogenetic analyses were performed using the ABL G gene nucleotide sequence alignment data. Phylogenetic trees were generated from the ABL G gene alignments using both MP and NJ with two Mokola isolates as an outgroup (Fig. 2). In both trees, ABL segregated into two clusters with 100 % bootstrap support, indicating the presence of two distinct ABL variants derived from a single progenitor. One variant lineage was associated with flying foxes (pteropid ABL) and the other associated with S. flaviventris (insectivorous ABL). The short branches observed in the NJ tree for the pteropid ABL cluster indicate that there is a very low level of genetic divergence between the isolates. Likewise, there appears to be little sequence variation among insectivorous ABL isolates.
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Selection pressures in the ABL G gene
Although phylogenetic analyses indicated that the ABL isolates were closely related, it was still of interest to determine whether these viruses had been subject to positive genetic selection. The G gene of natural ABL isolates was targeted for this analysis, as the G glycoprotein is the major pathogenic determinant responsible for lyssavirus virulence and host-cell tropism, making it perhaps the most likely candidate for the presence of sites experiencing adaptive evolution. ML analyses of selection pressures acting on the ABL G gene sequences provided some evidence, albeit weak, for positive selection. Specifically, a dN/dS value of 4·46 was found at amino acids 499 and 501 under the M8 model of codon evolution, and this model was significantly favoured over a competing neutral counterpart (M7), in which dN/dS was constrained to be <1 at all codons (full results available from the authors on request). Codons 499 and 501 were also found to be selected under the M3 model, although in this case a competing neutral model (M2) could not be significantly rejected. Despite this evidence for adaptive evolution, the strongest selection pressure acting on these sequences was clearly purifying (negative) selection, as the numbers of amino acid changes among the sequences were low, so that the vast majority of codons had very low dN/dS ratios; for example, 93 % of codons had a mean dN/dS of 0·03 under the M3 model.
Codon positions 499 and 501 fall within the endodomain of the viral glycoprotein, and alignment of this region revealed some amino acid variation between ABL isolates at these positions (Fig. 4). For the ABL Ballina and ABL insectivorous isolates previously sequenced by Gould et al. (1998)
, the sequences were quite different, with three nucleotide deletions resulting in a glycoprotein endomain one amino acid shorter at the C terminus than the ABL isolates sequenced in this study. This sequence variation is of interest (assuming the differences observed for the ABL Ballina and insectivorous isolates are not sequencing artifacts), as selectively favoured mutations may have occurred in the G ectodomain region of these viruses when they were passaged in mice and in in vitro cell cultures (mouse neuroblastoma 2A and baby hamster kidney cell lines) prior to sequencing.
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Discussion |
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In the Americas, the bat species Desmodus rotundus (vampire bat), Tadarida brasiliensis (Brazilian free-tailed bat), Eptesicus fuscus (big brown bat), Lasiurus species (L. borealiz, L. cinereus), Lasionycteris noctivagans (silver-haired bat), Pipistrellus subflavus (eastern pipistrelle) and Myotis species (M. lucifugus; M. yumanensis, M. californicus; M. evotis) have all been identified as rabies virus reservoirs that harbour distinct RV variants (Morimoto et al., 1996; Nadin-Davis et al., 2001
; Smith, 1996
). These RV variants generally separate into phylogenetic divisions that represent the lifestyle of their chiropteran hosts, i.e. migratory versus non-migratory, colonial versus solitary, insectivorous versus haematophagus (Nadin-Davis et al., 2001
). While most of these variants co-segregate only with their specific host reservoirs making spillover events to terrestrial animals uncommon, some have been associated with infection of non-chiropteran species, especially humans and domestic animals (Crawford-Miksza et al., 1999
; de Mattos et al., 2000
; Favi et al., 2002
; Ito et al., 2001b
; Morimoto et al., 1996
; Nadin-Davis et al., 2001
; Noah et al., 1998
; Smith et al., 1995
).
From this study, it appears that ABL follows a similar pattern of epidemiology to bat RV, in that separate pteropid and insectivorous ABL variants co-circulate in Australia within select bat hosts that display different behavioural patterns. Likewise, although spillover of ABL to domestic animals has not been observed, both ABL variants have been responsible for human deaths (Allworth et al., 1996; Hanna et al., 2000
). Phylogenetic analyses indicate that very little sequence divergence exists between the G gene sequences obtained for ABL isolates from the three flying fox species P. alecto, P. poliocephalus and P. scapulatus. Additionally, although the topology of the pteropid ABL cluster was found to vary slightly between the MP and NJ trees (Fig. 2
), it appears that delineation of the pteropid ABL isolates does not correlate with flying fox species or geographical distribution as isolates from three separate Pteropus species collected over a distance of greater than 1400 km group together. Indeed ABL isolates ABLPP08GC and ABLPA14RH obtained from P. poliocephalus and P. alecto, respectively, at locations over 700 km apart were found to be 100 % identical (Table 3
). Such minimal sequence variation of pteropid ABL isolates may be explained in part by the migratory behaviour and gregarious nature of these flying fox species.
During the day, flying foxes roost in trees in colonies known as camps. Depending on the species, flying fox camps may consist of many thousands of individuals (Hall & Richards, 2000). In the border region of Queensland and New South Wales, the distribution range of P. alecto, P. poliocephalus and P. scapulatus overlaps, and it is not uncommon for all three species to share the same camp, especially during periods of migration (Hall & Richards, 2000
). This provides both an opportunity for the different flying fox species to interact, as well as conditions that are ideal for a viral pathogen to become adapted to more than one pteropid species simultaneously, the outcome being that ABL isolated from each species of flying fox is nearly identical. A similar situation occurs in the USA, where isolates from RV variants of the migratory bat species L. noctivagans and T. brasiliensis are found to have a very high degree of nucleotide similarity, even if they have been isolated from locations separated by thousands of kilometres (Smith, 1996
).
At this stage it is unclear whether geographical segregation coincides with genetic relatedness of the insectivorous ABL cluster isolates, as the S. flaviventris ABL isolates used in this study were collected at sites separated by a distance of only 100 km. However, as it appears that the lifestyle of the bat host affects transmission and spread of each lyssavirus species, it would be interesting to investigate ABL infection of S. flaviventris further as these bats live in much smaller colonies than pteropid species (30 individuals at most), eat insects rather than fruit and nectar, and migrate over much shorter distances compared with flying foxes. Therefore, greater sequence variation may be found between insectivorous ABL isolates separated by large distances than that demonstrated by pteropid ABL such that specific subvariants may be identified, as is the case for RV in the sedentary bat host E. fuscus (Nadin-Davis et al., 2001; Smith, 1996
).
Although no pteropid ABL variants were isolated from ABL-infected S. flaviventris and vice versa, it is unclear whether flying foxes and insectivorous bats are susceptible to each other's ABL variants due to the small numbers of ABL-infected bats used in this study. Likewise, it is also not known whether other ABL variants exist in transmission cycles with Australian chiropteran species other than S. flaviventris or the common flying foxes. By pursuing these aspects of ABL epidemiology further, ecological factors controlling emergence of ABL within Australia, such as increased interactions between bat species and non-chiropteran wildlife, can be identified and targeted to prevent successful cross-species transmission events and initiation of ABL epidemics in new host species.
Purifying selection of ABL in nature
Although some evidence was found for weak positive selection at two codons within the G glycoprotein of natural ABL isolates, in general it appears that levels of non-synonomous diversity within the G gene of ABL are low. This also appears to be true of RV in nature, where only a few sites (codons 175, 183 and 370) within the conserved glycoprotein ectodomain have been found to be subject to modest positive selection pressure (Badrane & Tordo, 2001; Bourhy et al., 1999
; Holmes et al., 2002
). Such strong purifying selection is perhaps surprising considering the plasticity of RNA virus genomes (Domingo & Holland, 1997
), the abundance of positive selection pressure in the glycoproteins of other negative-strand RNA viruses such as respiratory syncytial virus (Woelk & Holmes, 2001
), measles virus (Woelk et al., 2001
, 2002
), Marburg virus (Sanchez et al., 1998
) and influenza A virus (Suzuki & Nei, 2002
), and the fact that RV mutants can be readily generated in the laboratory by passage through different host species (Kissi et al., 1999
). Consequently, the lack of positive selection in the G glycoprotein suggests either that the virus is not subject to strong immune selection or that it is exposed to relatively constant host, cellular and ecological environments so that most amino acid changes are not adaptively useful. As a case in point, although RV replication is almost exclusively limited to neurons, it has been shown that muscle tissue fibrocytes, acinar cells of the salivary gland and epidermal cells are also capable of supporting RV replication (Morimoto et al., 1996
; Charlton et al., 1997
). Holmes et al. (2002)
have suggested that the limited sequence variation within RV N and G genes may be a consequence of simultaneous adaptation to a wide variety of cell types. It is therefore possible that ABL, and indeed all other naturally isolated lyssavirus species, are also viral generalists' that are adapted to a variety of cell types, although this hypothesis clearly requires further investigation. Indeed, such work may also give an indication of which viruses have the greatest propensity to produce adaptively useful genetic variation, and hence are most likely to adapt to new host species.
Furthermore, it is also uncertain why amino acids 499 and 501 within the glycoprotein endodomain may be subject to genetic variation. As these amino acids are not surface-exposed, it is unlikely that they play a direct role in either host-cell binding or fusion, and it is therefore doubtful that selection pressure would be of an immunological nature. It is possible, however, that changes within the endodomain amino acids may alter the folding of G such that association of the glycoprotein with other proteins or host membranes during virion synthesis may also be modified to suit new environmental conditions. If this is indeed the case, the development of an ABL reverse genetics system may provide a means to mutagenize the ABL glycoprotein and investigate which of its domains contain determinants for host specificity and adaptation.
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ACKNOWLEDGEMENTS |
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Received 12 June 2002;
accepted 1 October 2002.