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1 Rabies & Wildlife Zoonoses Group, Veterinary Laboratories Agency (VLA, Weybridge), WHO Collaborating Centre for the Characterisation of Rabies and Rabies-Related Viruses, New Haw, Addlestone, Surrey KT15 3NB, UK
2 Institute for Epidemiology, WHO Collaborating Centre for Rabies Surveillance and Research, OIE Reference Laboratory for Rabies, Friedrich Loeffler Institute - Federal Research Institute for Animal Health, Seestrasse 55, D-16868 Wusterhausen, Germany
3 Max-von-Pettenkofer Institute and Gene Center, Feodor-Lynen-Str. 25, D-81377 Munich, Germany
4 Unité Stratégies Antivirales, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France
Correspondence
A. R. Fooks
t.fooks{at}vla.defra.gsi.gov.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Tables showing the primers and the accession numbers for the sequences used in this study are available as supplementary material in JGV Online.
The GenBank/EMBL/DDBJ accession numbers for the full genome sequences of EBLV-1 and -2 are EF157976 and EF157977.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Amengual et al., 1997). The genus Lyssavirus includes classical rabies virus (RABV, genotype 1), Lagos bat virus (LBV, genotype 2), Mokola virus (MOKV, genotype 3), Duvenhage virus (DUVV, genotype 4), EBLV type 1 (genotype 5) and type 2 (genotype 6) and Australian bat lyssavirus (ABLV, genotype 7). The full-length genomes of RABV (Conzelmann et al., 1990; Tordo et al., 1988), MOKV (Le Mercier et al., 1997), ABLV (Gould et al., 2002) and other representative genomes (Faber et al., 2004; Inoue et al., 2003) have been characterized. Recently, several newly identified lyssaviruses have been isolated but have yet to be assigned as new genotypes within the genus (Kuzmin et al., 2003, 2005). The lyssaviruses can be divided into two or possibly three phylogroups based on nucleotide sequence and immunogenicity (Fooks, 2004; Badrane et al., 2001); both EBLVs are designated to phylogroup I (Fooks, 2004).
The genome of RABV comprises a single-stranded, negative-sense RNA molecule of approximately 12 kb that encodes five structural proteins in the order N–P–M–G–L [nucleo (N) protein – phospho (P) protein – matrix (M) protein – glyco (G) protein – polymerase (L) protein]. The genomic RNA is tightly associated with monomers of the N protein, which, together with the P and L proteins, form the ribonucleoprotein (RNP) complex that transcribes the virus genome (Banerjee, 1987). The M protein lies underneath the virus envelope, associated with the RNP. Trimeric G protein spikes protrude from the envelope, which bind to cell receptors and are the target of both humoral and cell-mediated immune responses (Gaudin et al., 1992; Banerjee, 1987).
European bat lyssavirus type 1 and type 2 are genetically and phenotypically distinct from each other. Insectivorous bats are the host reservoirs, with EBLV-2 adapted to Myotis species, mainly Daubenton’s (M. daubentonii) and pond bats (M. dasycneme), and EBLV-1 host-adapted to serotines (Eptesicus serotinus) (Fooks et al., 2003a). Although bats are the only known reservoirs of EBLV, spillover infections have occurred. In particular, both viruses have caused fatalities in humans. There has been one confirmed EBLV-1 human fatality, an 11-year-old girl in Russia (Selimov et al., 1989; Bourhy et al., 1992), and two recorded human fatalities from EBLV-2, in Finland (Lumio et al., 1986) and Scotland (Fooks et al., 2003b). There have been two additional unconfirmed human cases of rabies in Europe that involved exposure to bats; however, in both cases, the virus was not typed and clinical material no longer exists (Fooks et al., 2003a).
To date, all analyses of the EBLVs have been conducted on fragments of the virus genome. We describe the full-length genomic sequence of an EBLV-1 isolated from a serotine bat in Hamburg, Germany, in 1968 and an EBLV-2 isolated from a human case of rabies in Scotland in 2002. By using a combination of long-distance PCR and RNA ligase-mediated–rapid amplification of cDNA ends (RLM-RACE) to obtain the 3' end and standard RACE to obtain the 5' end, the genomes of both EBLVs have been resolved.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The RNA was extracted from post-mortem brain material by the TRIzol method according to the manufacturer’s instructions (Invitrogen). RNA pellets were resuspended in sterile water and stored at –70 °C.
Determination of coding region by RT-PCR.
Reverse transcription was performed with 2 µg total RNA and 2 pmol JW12 primer using Superscript III (Invitrogen) in 20 µl final volume, following the manufacturer’s instructions. Long-distance PCRs were undertaken using Elongase (Invitrogen) following the manufacturer’s instructions. Two PCR products were amplified for each EBLV as follows. All PCR primer details are shown in Supplementary Table S1, available in JGV Online.
N–G PCR (5 kb).
The forward primer was based on the pan-lyssavirus primer JW12 (position 55–78) and the reverse primer was situated at the end of the G gene EBLV-1 position 4584–4562 and EBLV-2 position 4946–4913.
G–L PCR (6 kb).
The forward primers are reverse complement of the reverse sequences used in the N–G long-distance PCR. For the L gene long-distance reverse PCR primers, two degenerate primers were designed. Briefly, 2 µl cDNA was amplified using 20 pmol L-FOR (position 10264–10286, EBLV-1 or 10228–10250, EBLV-2) and L-REV (position 11694–11671, EBLV-1 or 11658–11635, EBLV-2) and 1 U Elongase using the following cycling parameters: 1 cycle at 94 °C for 30 s, 45 cycles at 94 °C for 30 s, 45 °C for 30 s, 68 °C for 2 min and final cycle at 68 °C for 10 min using a 2720 thermal cycler (Applied Biosystems).
The long-distance PCR conditions were as follows. Two microlitres cDNA was amplified using 20 pmol of each long-distance primer and 1 U Elongase using cycling parameters of 1 cycle at 94 °C for 30 s, 45 cycles at 94 °C for 30 s, 55 °C for 30 s, 68 °C for 5 min 30 s (N–G) or 7 min 30 s (G–L) and a final cycle at 68 °C for 10 min. Products were analysed by electrophoresis on 1 % agarose gels and visualized by ethidium bromide staining under UV illumination using 1 kb DNA markers (Promega).
Finally, an optimized panel of four primers were designed to reduce the number of PCRs required to obtain the N–L genome sequence for both EBLV-1 and -2. These primers were: EBLV N-G FOR (position 55–78 5'-ATGTAACACCTCTACAATGGATGC-3'), EBLV N-G REV (EBLV-2 position 5989–5967 5'-CYTCATCCCARTCYARRGCRTTC-3'), EBLV L FOR (EBLV-2 position 5514–5537 5'-CTCKGAYTAYAAYCTBAAYTCYCC-3') and EBLV L REV (EBLV-2 position 11662–11639 5'-CWATYTTGTAAATCARYCKDATCC-3'). They were used with the long-distance PCR conditions described above.
Determination of genomic 3' terminal sequence by RLM-RACE.
To determine the 3'-terminal end of the genomic RNA, 80 pmol 5'-phosphorylated DNA oligonucleotide (5'-P-GTCGATCACGCGATCGAACGGTCGCTGAG-3') was ligated to the 3' end of 4 µg total genomic RNA using T4 RNA ligase (Promega) following the manufacturer’s instructions with the following modifications: 50 U ligase was used in 50 µl final volume and the reaction was incubated at 16 °C for 2 h. The ligated genomic RNA was then ethanol-precipitated using standard protocols and resuspended in 10 µl sterile water. Reverse transcription was performed using 10 pmol complement oligonucleotide primer (5'-CAGCGACCGTTCGATCGC-3'), 2 µl purified ligated genomic RNA and 200 U Moloney murine leukaemia virus (M-MLV) reverse transcriptase µl–1 (Promega) in 20 µl final volume following the manufacturer’s instructions. PCRs were carried out using 3 U AmpliTaq Gold (Applied Biosciences) following the manufacturer’s instructions, in 50 µl final volume. Briefly, 4 µl cDNA was used with 10 pmol complement oligonucleotide and either primer EBLV-1 or primer EBLV-2 genomic N rev at position 232–255 using the following cycling parameters: 1 cycle at 94 °C for 30 s, 45 cycles at 94 °C for 30 s, 60 °C for 30 s, 68 °C for 1 min and final cycle at 68 °C for 10 min. Products were analysed as described above.
Determination of genomic 5'-terminal sequence by RACE.
To determine the 5'-terminal end of the genomic RNA, a standard 5' RACE kit (Invitrogen) was used. Briefly, 1 µg RNA was synthesized into cDNA using RT primers EBLV-1 at position 10880–10901 and EBLV-2 at position 11417–11438 with Superscript II RT (Invitrogen) and purified using a QIAquick PCR purification kit (Qiagen) in 50 µl final volume. Ten microlitres purified cDNA was subsequently tailed by the addition of dCTP using TdT in 25 µl final volume. Five microlitres tailed cDNA was used in a first-round PCR with 10 pmol L forward primer, either EBLV-1 at position 11555–11576 or EBLV-2 at position 11532–11552, with 10 pmol of the specific 5' RACE abridged anchor primer from the kit, using 3 U AmpliTaq Gold (Applied Biosciences) following the manufacturer’s instructions in 50 µl final volume. The cycling parameters were: 1 cycle at 94 °C for 30 s, 35 cycles at 94 °C for 1 min, 55 °C for 30 s, 68 °C for 2 min and a final cycle at 68 °C for 10 min. A second-round hemi-nested PCR was then performed on 1 µl first-round product as follows: 10 pmol first-round L primers described above were used with 10 pmol abridged universal amplification primer using the same cycling conditions. All PCR products were analysed as described above.
Sequencing of PCR products, alignment and analysis.
All PCR products were excised from the agarose gel and purified using a QIAquick gel extraction kit or MinElute gel extraction kit (both Qiagen) following the manufacturer’s instructions. The purified products were sequenced directly with 3.2 pmol of the appropriate primers (details of the sequencing primers are available on request) using a Beckman QuickStart kit on a CEQ8800 machine (Beckman-Coultard). All sequences were assembled using the Seqman program of the Lasergene package, version 6 (DNAStar). Consensus sequences were derived from at least two independent forward and reverse sequences but, for the majority of the genome sequence, multiple sequence overlap was achieved using independent PCR products. Editseq (DNAStar) was used to translate the gene sequences and MEGALIGN (DNAStar) to align the sequences. Editing of the alignments was performed in Genedoc (Nicholas et al., 1997). Percentage identities and similarity scores were determined in the MEGALIGN program. G+C content was obtained from the Editseq program.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The EBLV genome lengths follow the observation that lyssaviruses (but not other rhabdoviruses) have genomes with an even number of nucleotides (Warrilow et al., 2002); however, the sequences do not comply with the ‘rule of six’ (Calain & Roux, 1993). The G+C content of the lyssavirus genome is on average 44.57 mol%, with G+C content of 44.63 mol% for EBLV-1 and 44.79 mol% for EBLV-2. These data fit with the concept that there is a G+C-biasing in RNA viruses directly correlated with the genomic polarity, with positive-stranded RNA viruses having a higher G+C content than negative-sense RNA viruses. This bias is postulated to be due to host cell RNA editing, which occurs mainly on the negative strand (Auewarakul, 2005). Interestingly, conservation of the M and P protein of RABV strains, when compared with MOKV and LBV, suggests that genetic homologies exist, especially with the SAD B19 strain of rabies virus. Other workers have previously reported high levels of genomic conservation in the N, P, M and G genes, especially in respect to the M gene (Mebatsion et al., 1999). These data are supported by the results reported in this study (Table 2). Table 2 represents the most comprehensive analysis of EBLV-1 and EBLV-2 sequence identities, both within genotype (intragenotypic) and between genotypes (intergenotypic), using the sequences obtained in this study and previously published sequences for all five genes. We have also complemented previous RABV data analysis (N, P and L) (Kuzmin et al., 2003) by including L and M identities. The percentage identity order N>L>M>G>P is reinforced by both our intra- and intergenotypic comparisons; however, N and L have very similar percentage identities, as do G and P, for both nucleotide and amino acid comparisons. In addition to sequence diversity, G and P also have the most variable intergenotypic sequence length. The variation in sequence length for the G protein is restricted to the cytoplasmic region (Fig. 1b and Table 1) but, for the P protein, gaps are identified throughout the alignment (Fig. 2b and Table 1).
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; Kuzmin et al., 2005). Intragenotypic similarity within the N gene for EBLV-1 (a and b subgenotypes) is 95–99.9 % and 97.8–100 % identity at the amino acid level (Table 2
). One hundred per cent amino acid identity was observed between EBLV-1 isolates from three different countries (France, Holland and Spain), recorded over a period of 13 years. For EBLV-2, the sequence identities range from 95.6 to 99.6 % and 97.3 to 99.8 % (nucleotide and amino acid, respectively). Although the EBLV-2 isolates have been previously divided into subtypes 2a and 2b, the highest nucleotide sequence divergence was between two 2b sequences (AY863408
[GenBank]
– Switzerland isolate and AY863406
[GenBank]
– Finland human case). Indeed, phylogenetic analysis of the EBLV-2 sequences indicated that there are too few sequences to reliably divide them further (data not shown and Davis et al., 2005). These data contrast with those for the N gene analysis from a cohort of RABV viruses with a range of 82.3–99.4 % variation. This might reflect the global distribution of RABVs and the diversity of reservoir hosts maintaining each virus strain.
Intergenotypic comparisons of N protein sequences indicate that EBLV-1 has a higher identity to DUVV (92.7 %), ABLV (89.6 %) and SAD B19 (88.3 %) than to EBLV-2 (87.8 %), while LBV and EBLV-2 are the most divergent (79.6 %). The similarity scores for amino acid compared with nucleotide alignments suggest that the majority of the changes at the nucleotide level are silent (synonymous), located at the third base ‘wobble’ position, resulting in no amino acid change (data not shown). In addition, the vast majority of amino acid substitutions observed are conservative. DUVV, EBLV-1, EBLV-2 and the unclassified lyssaviruses Aravan virus, Irkut virus and Khujand virus all have one more amino acid than the other lyssaviruses (Fig. 1a). Of the 450/1 residues aligned, 291 were conserved in all representative lyssaviruses, including the putative casein-type phosphorylation site at SER389 (Dietzschold et al., 1987) (Fig. 1a). This phosphorylation is not seen in vesicular stomatitis virus (VSV), but has been shown to be crucial for viral RNA transcription and replication by encapsidation of the genomic RNA (Yang et al., 1999).
Overall, there appears to be a conserved central domain of the nucleoprotein (alignment in Fig. 1a), between residues 160 and 330, similar to the region previously identified in RABV at residues 182–328 (Kissi et al., 1995). By investigating a broad range of representative RABV sequences from the other genotypes, Kissi and colleagues demonstrated that this region was 73 % similar between the viruses analysed. Outside this conserved core there are two main regions of amino acid substitutions, 106–135 and 373–395, that have been delineated as important T-cell epitopes in humans and mice (Dietzschold et al., 1987). All lyssaviruses have highly charged basic amino acids in the C terminus that are hypothesized to bind RNA (Gilmore & Leong, 1988).
P protein
Vesiculovirus and paramyxovirus serotype comparisons show that the P protein is the most diverse of the five viral proteins, with a minimum of 21 % identity (Masters & Banerjee, 1987). Comparison of all available lyssavirus P protein sequences also identifies the P protein as the most diverse protein, with a minimum of 42.8 % identity between DUVV and West Caucasian bat virus (WCBV) (Table 2). Intragenotypic similarity is not so clear; RABV and ABLV P protein sequences are the most divergent (80–97 and 92 %, respectively) of the five proteins; conversely, EBLV-1 and -2 P protein intragenotypic comparisons are more conserved (both 98–99 %). However, this may be due to a small sample size (EBLV-1, n=4; EBLV-2, n=3). Moreover, it is possible that this high degree of similarity within genotypes is due to structural/functional constraints, including specific interactions with the corresponding L proteins, preventing divergence. Indeed, the two regions of greatest variability within the alignment of the lyssavirus P proteins, residues 52–78 and 155–178 (Fig. 1b), are clearly within the regions known to be important in L protein interaction for vesiculoviruses (Pattnaik et al., 1997).
Deletion of the highly conserved region at the C terminus of the P protein correlating to domain III (213 and 247) in VSV did not prevent binding to the L protein and transcription, but resulted in an inability to bind to the RNP complex (Gill & Banerjee, 1985). The short lysine-rich motif within domain III (FSKKYKF), critical for RNP binding (Jacob et al., 2001), is conserved within all lyssaviruses studied, with the exception of the LBV strain (FSKRYKF) and WCBV (ISKKYKF) (Fig. 1b).
‘P–host’ interactions (LC8).
The RABV P protein has been shown to interact with LC8 (cytoplasmic dynein light chain) at residues 138–172 (Mebatsion, 2001). This region is highly hydrophilic, containing many phosphorylation sites (Tordo et al., 1986). Specifically, the motif (K/R)XTQT – residues 145–149 – has been shown to interact with LC8, a protein that contributes to the axonal transport of RABV within neurons (Lo et al., 2001), but not believed to be essential for transcription (Poisson et al., 2001). The importance of this interaction in RABV transport and pathogenicity has been questioned by in vivo experiments where mutant viruses lacking the LC8-binding domain were only slightly attenuated in comparison to a wild-type virus (Mebatsion, 2001; Rasalingam et al., 2005). All the lyssaviruses aligned have the (K/R)XTQT motif apart from our EBLV-1 (RV9) (KSTRT), MOKV (KSIQI) and WCBV, which appears to have no motif (Fig. 1b). However, other published EBLV-1 P gene sequences (Nadin-Davis et al., 2002) and an unpublished EBLV-1 from our laboratory all have the conserved motif, suggesting that RV9 differs when compared with other EBLV-1 viruses.
RNP complex.
The binding sites of the RABV P protein with N protein and L protein have been described previously (Chenik et al., 1994; Fu et al., 1994; Jacob et al., 2001; Mavrakis et al., 2003, 2004). The RABV P protein can interact with both RNA-bound N protein and soluble N protein ( °, no viral RNA). The latter interaction is thought to chaperone ° for encapsidation of RNA during the replication of RNPs. The °-binding domain of P protein has been assigned to residues 69–177, whereas binding to RNA-associated N protein is mediated by the C-terminal domain (residues 268–297) (Chenik et al., 1994; Fu et al., 1994; Jacob et al., 2001; Mavrakis et al., 2003, 2004). In addition, the N terminus of the RABV P protein is responsible for binding to the L protein, with the first 19 aa residues being crucial for the interaction with the C-terminal domain of L protein (Chenik et al., 1998) (Fig. 1b). Lyssavirus P proteins have the potential to be heavily phosphorylated, with between 37 and 43 Ser/Thr residues. EBLV-1 has 39 (30 Ser and 9 Thr) while EBLV-2 has 40 (28 Ser and 12 Thr). Of these, only eight serines and one threonine are conserved in all lyssaviruses compared. Despite this abundance of Ser/Thr residues, only Ser residues S63/64, S162, S210 and S271 are phosphorylated by cellular Ser kinases: a ‘rabies virus protein kinase’ (RVPK) and protein kinase C (Gupta et al., 2000).
M protein
Assembly and budding of lyssaviruses takes place at the plasma membrane. The major driving force in this process is the viral M protein, a 202 residue membrane-binding protein which tethers membranes, virus RNPs and G proteins together, and is active in membrane budding and pinching off enveloped viruses (Mebatsion et al., 1996, 1999). The active process of ‘exocytosis’ involves cellular membrane-associated factors binding to ‘late domains’ identified in M or M-like proteins of various enveloped viruses. Of the two classical late domain motifs identified in RABV M protein PPXY (aa 35) and PX(T/S)AP (aa 21), neither is essential (Jayakar et al., 2000). The PPXY motif is present in all lyssaviruses compared, apart from Khujand virus (PPES – Fig. 1c), and is reported to bind class I WW-domain-containing E3-ubiquitin ligases (Harty et al., 1999). In the absence of this motif, reduced RABV budding is observed (Harty et al., 2001). The PX(T/S)AP motif binds components of the vascular protein-sorting (VPS) pathway, such as Tsg101. This pathway was apparently not required for RABV budding, since it is resistant to dominant-negative VPS4, a key kinase of the pathway (Irie et al., 2004). In addition, the RABV M protein is involved in the regulation of transcription and replication of viral RNA (Finke et al., 2003), which is independent of M protein functions in virus assembly (Finke & Conzelmann, 2003). Moreover, RABV M protein has been reported to stimulate tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated induction of apoptosis (Kassis et al., 2004), suggesting additional roles of M protein in host-cell interplay. One would expect that a protein, such as the M protein, which is involved in multiple interactions, would be highly conserved both within and between genotypes, which in the most part holds true. The minimum intergenotypic variation is between ABLV and WCBV (72.9 %). The alignment in Fig. 1(c) clearly shows regions of high similarity between the genotypes throughout the protein. However, intragenotypic comparisons for RABV show a relatively low protein identity (86.7 %), which is close to the identities found with the G protein (Table 2). The lowest identity score was between SHBV and SAD B19, with 108 nt changes resulting in 17 aa changes over 202 residues. Therefore, even this low amino acid sequence identity, when investigated, reveals a high percentage of synonymous nucleotide changes, suggesting a selection to remain conserved. The most convincing comparison showing the pressure on the M protein to remain conserved is between a West Canadian Skunk isolate and an ex-Indian isolate from a German organ transplant case. There are 60 nucleotide changes, but only two result in amino acid substitutions. In addition, the limited number of sequences available for EBLV-1 and -2 M protein comparisons does not show the full range of identities, but indicates they also have relatively low intragenotypic variation (Table 2).
G protein
The G protein is responsible for cell attachment and fusion and is the main viral protein responsible for the induction of neutralizing antibodies and cell-mediated immune responses. It is also involved in the budding process (Mebatsion et al., 1996) and expression levels of G protein have been shown to correlate with the induction of apoptosis, inversely correlating with pathogenicity (Morimoto et al., 1999). Trimers of the G protein form the spikes that interact with cell receptors, allowing internalization of virions (Coll, 1995). The G protein consists of an ectodomain (20–439) or ‘antigenic domain’, transmembrane domain (440–461) and cytoplasmic domain (462–505) (Fig. 2). The signal sequence, which is cleaved in the endoplasmic reticulum, is 19 residues long. The ectodomain contains between one and four N-linked glycosylation sites at positions 37 [NLS: PV (Pasteur virus) and SAD B19], 158 (NCS: PV), 202 (NGS: LBV and MOKV), 247 (NET: PV, SAD B19 and DUVV) and 319 (NKT). This final glycosylation site is the only site present in all lyssaviruses studied (Fig. 2). All 14 cysteine residues that could form disulphide bonds are completely conserved. There are two main conformational antigenic sites that have been identified in the G protein ectodomain: antigenic site II (positions 34–42 and 198–200) (Prehaud et al., 1988) and antigenic site III (position 330–338). Antigenic site III contains both the Arg333 residue critical for virus pathogenicity (Dietzschold et al., 1983; Seif et al., 1985) and Lys330 involved in the binding of the virion to the neurons and its propagation into motoneurons (Coulon et al., 1998). Interestingly, both these positions vary between the lyssaviruses aligned, although both the EBLVs have conserved Arg333 and Lys330 (Fig. 2).
G–L non-coding region
The long untranslated region between G and L was originally hypothesized to be a pseudogene (Tordo et al., 1986), but has since been shown to be a long 3' non-coding region as found in other negative-sense RNA viruses (Ravkov et al., 1995). It varies in length between 508 (ABLV) and 560 bp (EBLV-1) (Table 1). An alignment of this region clearly shows that all lyssaviruses, including the EBLVs, use the second conserved TTP (transcription termination and polyadenylation) motif approximately 50 nt upstream of the L gene start methionine (Fig. 3). This long untranslated region may act to reduce the efficiency of L transcription, which could in turn reduce expression and enhance pathogenicity.
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). Amino acid comparisons between the L protein of RABV and VSV show an extensive amount of similarity, far more than for N and G proteins. It is believed that the selective pressure for this similarity is due to the multiple enzymic activities that are concatenated in this L protein (Tordo et al., 1988). When comparing L proteins from members of the Mononegavirales, the conserved residues are not distributed randomly, but cluster into six conserved domains with invariant motifs (Poch et al., 1990). Block I contains hydrophobic residues particularly in the first two stretches. The third stretch has invariant GHP residues (373–376) which are conserved in both EBLVs; indeed, alignments of this region with all available lyssavirus sequences reveal that this entire stretch is highly conserved (Fig. 4). The GHP motif is probably part of a turn structure, with exposed H playing an important functional role (Poch et al., 1990). Blocks II and III consist of the major functional domains; block II consists mainly of charged residues with a central conserved stretch containing a KEKE (hydrophobic) K motif. This pre-A motif has been shown to be involved in the positioning and binding of the RNA template (Muller et al., 1994). The regular spacing of conserved basic and hydrophobic residues every 4 residues constitutes an amphiphilic 
-helix with basic residues facing and contacting the RNA template (Poch et al., 1990). Block III has long conserved stretches of charged residues throughout. The four conserved regions in negative-stranded RNA virus L proteins (A–D) are completely identical in all lyssaviruses studied, including the GG(I/L)EG (694–697) and pentapeptide QGDNQ (728–732) (Fig. 4). The pentapeptide domain is proposed to be the active site for template recognition and/or phosphodiester bond formation in the L proteins (Poch et al., 1988) and has been extensively studied by utilizing deletions and mutations. In both RABV (Schnell & Conzelmann, 1995) and VSV (Sleat & Banerjee, 1993), almost any change made to the motif resulted in complete loss of polymerase activity. Block IV is rich in proline and, although its role has not yet been characterized, it may be involved in nucleotide binding (Poch et al., 1990). Block V has numerous cysteine and histidine invariant residues while block VI is less conserved with a GXGXG motif (1705–1710) preceded by a lysine 19–22 residues upstream, which could play the role of polyadenylation or protein kinase activity (Poch et al., 1990). All lyssaviruses (including both EBLVs) have a conserved GDGSGG at position 1704–1708 and a lysine 19 residues downstream (Fig. 4).
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). These regions, although variable in length, do have conserved initiation and termination signals, the latter being an almost invariant TGAAAAAAA (Table 3). Changes in the P/M termination sequence have been shown to occur in the adaptation of street strain viruses to tissue culture (Warrilow et al., 2002) and attenuation of RC-HL strain in relation to the parental Nishigahara strain (Ito et al., 2001). In VSV, the monocistronic mRNAs are transcribed by a mechanism of pause (at the termination site) and reinitiation of the transcriptase (Rodriguez et al., 2002). Therefore, changes in the termination signals would be expected to result in reduced transcription of the downstream proteins. In VSV, the IGS (intergenic spacer region) is an invariant 2 nt and, in Sendai virus, 3 nt. For lyssaviruses, this constraint is not apparent, although the initial invariant guanosine residue (genomic sense) is maintained in all lyssaviruses studied. With the exception of MOKV and WCBV, the IGS at the N–P junction is an invariant 2 nt, which increases to 5 nt for the second (P–M) and third (M–G) IGS, with a variable sequence length of between 19 and 24 nt for the last (G–L) IGS (Table 3 and Fig. 3). Both MOKV and WCBV deviate from these lengths, particularly for the M–G and G–L regions, where they are considerably longer (Table 3). It is possible that the increasing length and variability in the IGS could decrease the efficiency of the polymerase re-initiation, which in turn will decrease the amount of protein produced downstream.
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) and sequence (Fig. 5). In all lyssaviruses, with the exception of EBLV-2 and ABLV, the observation that the first 11 nt are complementary to the 5' terminus holds true (Tordo et al., 1988). EBLV-2 and ABLV depart from this exact match at position 10 (A
G). We have found this base substitution not just in this human rabies isolate, but also another two UK EBLV-2 isolates (data not shown). This suggests that the minimum number of complementary bases is nine. EBLV-1 has a uniquely long region of complementarity lasting for 24 nt, with only one mismatch at position 12. This complementarity is a conserved feature throughout unsegmented negative-strand RNA genomes. Comparisons with other rhabdovirus 5' and 3' ends show that the first 3 nt at the 3' end are completely conserved, with its complement conserved on the 5' terminus. This is not true for rhabdoviruses that infect fish and plants (Warrilow et al., 2002). There have been many studies showing the importance of these genomic termini in the roles of transcription, replication and encapsidation. The biasing of AT over GC in these regions (35.71 and 42.86 mol % G+C content, SAD B19 and EBLV-2 respectively) compared to the overall G+C content of 45.06 and 44.79 mol %, respectively, is a well documented observation, and is the recognition site for the polymerase in the leader sequence in VSV (Keene et al., 1981). The leader RNA also interacts with the N protein, modulating the amount of transcription versus replication. Indeed, previous studies on RABV genomic termini have shown that the complementary termini appear to promote replication rather than transcription (Finke & Conzelmann, 1999).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Auewarakul, P. (2005). Composition bias and genome polarity of RNA viruses. Virus Res 109, 33–37.[CrossRef][Medline]
Badrane, H., Bahloul, C., Perrin, P. & Tordo, N. (2001). Evidence of two Lyssavirus phylogroups with distinct pathogenicity and immunogenicity. J Virol 75, 3268–3276.
Banerjee, A. K. (1987). Transcription and replication of rhabdoviruses. Microbiol Rev 51, 66–87.
Bourhy, H., Kissi, B., Lafon, M., Sacramento, D. & Tordo, N. (1992). Antigenic and molecular characterization of bat rabies virus in Europe. J Clin Microbiol 30, 2419–2426.
Calain, P. & Roux, L. (1993). The rule of six, a basic feature for efficient replication of Sendai virus defective interfering RNA. J Virol 67, 4822–4830.
Chenik, M., Chebli, K., Gaudin, Y. & Blondel, D. (1994). In vivo interaction of rabies virus phosphoprotein (P) and nucleoprotein (N) existence of two N-binding sites on P protein. J Gen Virol 75, 2889–2896.
Chenik, M., Schnell, M., Conzelmann, K. K. & Blondel, D. (1998). Mapping the interacting domains between the rabies virus polymerase and phosphoprotein. J Virol 72, 1925–1930.
Coll, J. M. (1995). The glycoprotein G of rhabdoviruses. Arch Virol 140, 827–851.[CrossRef][Medline]
Conzelmann, K. K., Cox, J. H., Schneider, L. G. & Thiel, H. J. (1990). Molecular cloning and complete nucleotide sequence of the attenuated rabies virus SAD B19. Virology 175, 485–499.[CrossRef][Medline]
Coulon, P., Ternaux, J. P., Flamand, A. & Tuffereau, C. (1998). An avirulent mutant of rabies virus is unable to infect motoneurons in vivo and in vitro. J Virol 72, 273–278.
Davis, P. L., Holmes, E. C., Larrous, F., Van der Poel, W. H., Tjornehoj, K., Alonso, W. J. & Bourhy, H. (2005). Phylogeography, population dynamics, and molecular evolution of European bat lyssaviruses. J Virol 79, 10487–10497.
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. Proc Natl Acad Sci U S A 80, 70–74.
Dietzschold, B., Lafon, M., Wang, H., Otvos, L., Jr, Celis, E., Wunner, W. H. & Koprowski, H. (1987). Localization and immunological characterization of antigenic domains of the rabies virus internal N and NS proteins. Virus Res 8, 103–125.[Medline]
Faber, M., Pulmanausahakul, R., Nagao, K., Prosniak, M., Rice, A. B., Koprowski, H., Schnell, M. J. & Dietzschold, B. (2004). Identification of viral genomic elements responsible for rabies virus neuroinvasiveness. Proc Natl Acad Sci U S A 101, 16328–16332.
Finke, S. & Conzelmann, K. K. (1999). Virus promoters determine interference by defective RNAs selective amplification of mini-RNA vectors and rescue from cDNA by a 3' copy-back ambisense rabies virus. J Virol 73, 3818–3825.
Finke, S. & Conzelmann, K. K. (2003). Dissociation of rabies virus matrix protein functions in regulation of viral RNA synthesis and virus assembly. J Virol 77, 12074–12082.
Finke, S., Mueller-Waldeck, R. & Conzelmann, K.-K. (2003). Rabies virus matrix protein regulates the balance of virus transcription and replication. J Gen Virol 84, 1613–1621.
Fooks, A. (2004). The challenge of new and emerging lyssaviruses. Expert Rev Vaccines 3, 333–336.[CrossRef][Medline]
Fooks, A. R., Brookes, S. M., Johnson, N., McElhinney, L. M. & Hutson, A. M. (2003a). European bat lyssaviruses: an emerging zoonosis. Epidemiol Infect 131, 1029–1039.[CrossRef][Medline]
Fooks, A. R., McElhinney, L. M., Pounder, D. J., Finnegan, C. J., Mansfield, K., Johnson, N., Brookes, S. M., Parsons, G., White, K. & other authors (2003b). Case report: isolation of a European bat lyssavirus type 2a from a fatal human case of rabies encephalitis. J Med Virol 71, 281–289.[CrossRef][Medline]
Fu, Z. F., Zheng, Y., Wunner, W. H., Koprowski, H. & Dietzschold, B. (1994). Both the N- and the C-terminal domains of the nominal phosphoprotein of rabies virus are involved in binding to the nucleoprotein. Virology 200, 590–597.[CrossRef][Medline]
Gaudin, Y., Ruigrok, R. W., Tuffereau, C., Knossow, M. & Flamand, A. (1992). Rabies virus glycoprotein is a trimer. Virology 187, 627–632.[CrossRef][Medline]
Gill, D. S. & Banerjee, A. K. (1985). Vesicular stomatitis virus NS proteins: structural similarity without extensive sequence homology. J Virol 55, 60–66.
Gilmore, R. D., Jr & Leong, J. A. (1988). The nucleocapsid gene of infectious hematopoietic necrosis virus, a fish rhabdovirus. Virology 167, 644–648.[Medline]
Gould, A. R., Kattenbelt, J. A., Gumley, S. G. & Lunt, R. A. (2002). Characterisation of an Australian bat lyssavirus variant isolated from an insectivorous bat. Virus Res 89, 1–28.[CrossRef][Medline]
Gupta, A. K., Blondel, D., Choudhary, S. & Banerjee, A. K. (2000). The phosphoprotein of rabies virus is phosphorylated by a unique cellular protein kinase and specific isomers of protein kinase C. J Virol 74, 91–98.
Harty, R. N., Paragas, J., Sudol, M. & Palese, P. (1999). A proline-rich motif within the matrix protein of vesicular stomatitis virus and rabies virus interacts with WW domains of cellular proteins: implications for viral budding. J Virol 73, 2921–2929.
Harty, R. N., Brown, M. E., McGettigan, J. P., Wang, G., Jayakar, H. R., Huibregtse, J. M., Whitt, M. A. & Schnell, M. J. (2001). Rhabdoviruses and the cellular ubiquitin-proteasome system: a budding interaction. J Virol 75, 10623–10629.
Inoue, K., Shoji, Y., Kurane, I., Iijima, T., Sakai, T. & Morimoto, K. (2003). An improved method for recovering rabies virus from cloned cDNA. J Virol Methods 107, 229–236.[CrossRef][Medline]
Irie, T., Licata, J. M., Jayakar, H. R., Whitt, M. A., Bell, P. & Harty, R. N. (2004). Functional analysis of late-budding domain activity associated with the PSAP motif within the vesicular stomatitis virus M protein. J Virol 78, 7823–7827.
Ito, N., Kakemizu, M., Ito, K. A., Yamamoto, A., Yoshida, Y., Sugiyama, M. & Minamoto, N. (2001). A comparison of complete genome sequences of the attenuated RC-HL strain of rabies virus used for production of animal vaccine in Japan, and the parental Nishigahara strain. Microbiol Immunol 45, 51–58.[Medline]
Jacob, Y., Real, E. & Tordo, N. (2001). Functional interaction map of lyssavirus phosphoprotein: identification of the minimal transcription domains. J Virol 75, 9613–9622.
Jayakar, H. R., Murti, K. G. & Whitt, M. A. (2000). Mutations in the PPPY motif of vesicular stomatitis virus matrix protein reduce virus budding by inhibiting a late step in virion release. J Virol 74, 9818–9827.
Johnson, N., McElhinney, L. M., Smith, J., Lowings, P. & Fooks, A. R. (2002). Phylogenetic comparison of the genus Lyssavirus using distal coding sequences of the glycoprotein and nucleoprotein genes. Arch Virol 147, 2111–2123.[CrossRef][Medline]
Kassis, R., Larrous, F., Estaquier, J. & Bourhy, H. (2004). Lyssavirus matrix protein induces apoptosis by a TRAIL-dependent mechanism involving caspase-8 activation. J Virol 78, 6543–6555.
Keene, J. D., Thornton, B. J. & Emerson, S. U. (1981). Sequence-specific contacts between the RNA polymerase of vesicular stomatitis virus and the leader RNA gene. Proc Natl Acad Sci U S A 78, 6191–6195.
Kissi, B., Tordo, N. & Bourhy, H. (1995). Genetic polymorphism in the rabies virus nucleoprotein gene. Virology 209, 526–537.[CrossRef][Medline]
Kuzmin, I. V., Orciari, L. A., Arai, Y. T., Smith, J. S., Hanlon, C. A., Kameoka, Y. & Rupprecht, C. E. (2003). Bat lyssaviruses (Aravan and Khujand) from Central Asia: phylogenetic relationships according to N, P and G gene sequences. Virus Res 97, 65–79.[CrossRef][Medline]
Kuzmin, I. V., Hughes, G. J., Botvinkin, A. D., Orciari, L. A. & Rupprecht, C. E. (2005). Phylogenetic relationships of Irkut and West Caucasian bat viruses within the Lyssavirus genus and suggested quantitative criteria based on the N gene sequence for lyssavirus genotype definition. Virus Res 111, 28–43.[CrossRef][Medline]
Le Mercier, P., Jacob, Y. & Tordo, N. (1997). The complete Mokola virus genome sequence: structure of the RNA-dependent RNA polymerase. J Gen Virol 78, 1571–1576.[Abstract]
Lo, K. W., Naisbitt, S., Fan, J. S., Sheng, M. & Zhang, M. (2001). The 8-kDa dynein light chain binds to its targets via a conserved (K/R)XTQT motif. J Biol Chem 276, 14059–14066.
Lumio, J., Hillbom, M., Roine, R., Ketonen, L., Haltia, M., Valle, M., Neuvonen, E. & Lahdevirta, J. (1986). Human rabies of bat origin in Europe. Lancet 1, 378[Medline]
Masters, P. S. & Banerjee, A. K. (1987). Sequences of Chandipura virus N and NS genes: evidence for high mutability of the NS gene within vesiculoviruses. Virology 157, 298–306.[CrossRef][Medline]
Mavrakis, M., Iseni, F., Mazza, C., Schoehn, G., Ebel, C., Gentzel, M., Franz, T. & Ruigrok, R. W. H. (2003). Isolation and characterisation of the rabies virus N-P complex produced in insect cells. Virology 305, 406–414.[CrossRef][Medline]
Mavrakis, M., McCarthy, A. A., Roche, S., Blondel, D. & Ruigrok, R. W. (2004). Structure and function of the C-terminal domain of the polymerase cofactor of rabies virus. J Mol Biol 343, 819–831.[CrossRef][Medline]
Mebatsion, T. (2001). Extensive attenuation of rabies virus by simultaneously modifying the dynein light chain binding site in the P protein and replacing Arg333 in the G protein. J Virol 75, 11496–11502.
Mebatsion, T., Konig, M. & Conzelmann, K. K. (1996). Budding of rabies virus particles in the absence of the spike glycoprotein. Cell 84, 941–951.[CrossRef][Medline]
Mebatsion, T., Weiland, F. & Conzelmann, K. K. (1999). Matrix protein of rabies virus is responsible for the assembly and budding of bullet-shaped particles and interacts with the transmembrane spike glycoprotein G. J Virol 73, 242–250.
Morimoto, K., Hooper, D. C., Spitsin, S., Koprowski, H. & Dietzschold, B. (1999). Pathogenicity of different rabies virus variants inversely correlates with apoptosis and rabies virus glycoprotein expression in infected primary neuron cultures. J Virol 73, 510–518.
Muller, R., Poch, O., Delarue, M., Bishop, D. H. & Bouloy, M. (1994). Rift Valley fever virus L segment: correction of the sequence and possible functional role of newly identified regions conserved in RNA-dependent polymerases. J Gen Virol 75, 1345–1352.
Nadin-Davis, S. A., Abdel-Malik, M., Armstrong, J. & Wandeler, A. I. (2002). Lyssavirus P gene characterisation provides insights into the phylogeny of the genus and identifies structural similarities and diversity within the encoded phosphoprotein. Virology 298, 286–305.[CrossRef][Medline]
Nicholas, K. B., Nicholas, H. B., Jr & Deerfield, D. W., II (1997). GeneDoc: analysis and visualization of genetic variation. EMBnet News 4, 1–4. http://www.embnet.org/download/embnetnews/embnet_news_4_2.pdf
Pattnaik, A. K., Hwang, L., Li, T., Englund, N., Mathur, M., Das, T. & Banerjee, A. K. (1997). Phosphorylation within the amino-terminal acidic domain I of the phosphoprotein of vesicular stomatitis virus is required for transcription but not for replication. J Virol 71, 8167–8175.[Abstract]
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, 1019–1029.[Medline]
Poch, O., Blumberg, B. M., Bougueleret, L. & Tordo, N. (1990). Sequence comparison of five polymerases (L proteins) of unsegmented negative-strand RNA viruses: theoretical assignment of functional domains. J Gen Virol 71, 1153–1162.
Poisson, N., Real, E., Gaudin, Y., Vaney, M.-C., King, S., Jacob, Y., Tordo, N. & Blondel, D. (2001). Molecular basis for the interaction between rabies virus phosphoprotein P and the dynein light chain LC8 dissociation of dynein binding properties and transcriptional functionality of P. J Gen Virol 82, 2691–2696.
Prehaud, C., Coulon, P., LaFay, F., Thiers, C. & Flamand, A. (1988). Antigenic site II of the rabies virus glycoprotein: structure and role in viral virulence. J Virol 62, 1–7.
Rasalingam, P., Rossiter, J. P., Mebatsion, T. & Jackson, A. C. (2005). Comparative pathogenesis of the SAD-L16 strain of rabies virus and a mutant modifying the dynein light chain binding site of the rabies virus phosphoprotein in young mice. Virus Res 111, 55–60.[CrossRef][Medline]
Ravkov, E. V., Smith, J. S. & Nichol, S. T. (1995). Rabies virus glycoprotein gene contains a long 3' noncoding region which lacks pseudogene properties. Virology 206, 718–723.[CrossRef][Medline]
Rodriguez, L. L., Pauszek, S. J., Bunch, T. A. & Schumann, K. R. (2002). Full-length genome analysis of natural isolates of vesicular stomatitis virus (Indiana 1 serotype) from North, Central and South America. J Gen Virol 83, 2475–2483.
Schnell, M. J. & Conzelmann, K. K. (1995). Polymerase activity of in vitro mutated rabies virus L protein. Virology 214, 522–530.[CrossRef][Medline]
Seif, I., Coulon, P., Rollin, P. E. & Flamand, A. (1985). Rabies virulence effect on pathogenicity and sequence characterization of rabies virus mutations affecting antigenic site III of the glycoprotein. J Virol 53, 926–934.
Selimov, M. A., Tatarov, A. G., Botvinkin, A. D., Klueva, E. V. & Kulikova, L. G. (1989). Rabies-related Yuli virus; identification with a panel of monoclonal antibodies. Acta Virol 33, 542–546.[Medline]
Sleat, D. E. & Banerjee, A. K. (1993). Transcriptional activity and mutational analysis of recombinant vesicular stomatitis virus RNA polymerase. J Virol 67, 1334–1339.
Tordo, N., Poch, O., Ermine, A., Keith, G. & Rougeon, F. (1986). Walking along the rabies genome: is the large G-L intergenic region a remnant gene?. Proc Natl Acad Sci U S A 83, 3914–3918.
Tordo, N., Poch, O., Ermine, A., Keith, G. & Rougeon, F. (1988). Completion of the rabies virus genome sequence determination: highly conserved domains among the L (polymerase) proteins of unsegmented negative-strand RNA viruses. Virology 165, 565–576.[CrossRef][Medline]
Warrilow, D., Smith, I. L., Harrower, B. & Smith, G. A. (2002). Sequence analysis of an isolate from a fatal human infection of Australian bat lyssavirus. Virology 297, 109–119.[CrossRef][Medline]
Yang, J., Koprowski, H., Dietzschold, B. & Fu, Z. F. (1999). Phosphorylation of rabies virus nucleoprotein regulates viral RNA transcription and replication by modulating leader RNA encapsidation. J Virol 73, 1661–1664.
Received 7 November 2006;
accepted 15 December 2006.
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