Shimoni bat virus, a new representative of the Lyssavirus genus

Virus Research 149 (2010) 197–210

Contents lists available at ScienceDirect

Virus Research journal homepage: www.elsevier.com/locate/virusres

Shimoni bat virus, a new representative of the Lyssavirus genus夽 Ivan V. Kuzmin a,∗ , Anne E. Mayer b , Michael Niezgoda a , Wanda Markotter c , Bernard Agwanda d , Robert F. Breiman e , Charles E. Rupprecht a a

Rabies Program, Poxvirus and Rabies Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd, Atlanta, GA 30333, USA University of Minnesota, 225 Smith Ave., St Paul, MN 55102, USA c Department of Microbiology and Plant Pathology, Faculty of Natural and Agricultural Sciences, University of Pretoria, Pretoria 0001, South Africa d Mammalogy Section, National Museum of Kenya, Museum Hill Rd, 00100 Nairobi, Kenya e Global Disease Detection Division, Centers for Disease Control and Prevention in Kenya, Market Place, 00100 Nairobi, Kenya b

a r t i c l e

i n f o

Article history: Received 8 November 2009 Received in revised form 30 January 2010 Accepted 30 January 2010 Available online 6 February 2010 Keywords: Rabies Lyssavirus Rhabdovirus Shimoni bat virus Phylogeny Chiroptera Zoonosis Africa Kenya

a b s t r a c t During 2009, 616 bats representing at least 22 species were collected from 10 locations throughout Kenya. A new lyssavirus, named Shimoni bat virus (SHIBV), was isolated from the brain of a dead Commerson’s leaf-nosed bat (Hipposideros commersoni), found in a cave in the coastal region of Kenya. Genetic distances and phylogenetic reconstructions, implemented for each gene and for the concatenated alignment of all five structural genes (N, P, M, G and L), demonstrated that SHIBV cannot be identified with any of the existing species, but rather should be considered an independent species within phylogroup II of the Lyssavirus genus, most similar to Lagos bat virus (LBV). Antigenic reaction patterns with anti-nucleocapsid monoclonal antibodies corroborated these distinctions. In addition, new data on the diversity of LBV suggests that this species may be subdivided quantitatively into three separate genotypes. However, the identity values alone are not considered sufficient criteria for demarcation of new species within LBV. Published by Elsevier B.V.

1. Introduction The Lyssavirus genus (family Rhabdoviridae) includes 11 recognized species. The type species, Rabies virus (RABV) is distributed worldwide among mammalian reservoirs—carnivores and bats. Lagos bat virus (LBV) circulates among pteropid bats in sub-Saharan Africa with infrequent spill-overs into other mammals (Markotter et al., 2008a). Mokola virus (MOKV) has been isolated in sub-Saharan Africa from shrews, domestic cats and dogs, a rodent and two humans (Nel et al., 2000; Sabeta et al., 2007). The reservoir hosts for MOKV have not yet been established. Duvenhage virus (DUVV) was isolated from insectivorous bats and humans who died after bat bites in sub-Saharan Africa (Markotter et al., 2008b; van Thiel et al., 2009). European bat lyssavirus, type 1 (EBLV1) has been isolated from insectivorous bats across Europe, and the primary host

夽 Use of trade names and commercial sources are for identification only and do not imply endorsement by the U.S. Department of Health and Human Services. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the funding agency. ∗ Corresponding author. Tel.: +1 404 639 1050; fax: +1 404 639 1564. E-mail addresses: [email protected], [email protected] (I.V. Kuzmin). 0168-1702/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.virusres.2010.01.018

species of this virus appears to be the Serotine bat (Eptesicus serotinus). Human cases of EBLV1 infection have been described as well (Fooks et al., 2003a; Kuzmin et al., 2006). European bat lyssavirus, type 2 (EBLV2) was isolated primarily from insectivorous bats of the Myotis genus and from humans who died after bat bites in northwestern Europe (Fooks et al., 2003a,b). Australian bat lyssavirus (ABLV) circulates in Australia among insectivorous and pteropid bats, and has caused at least two documented cases of human rabies (Warrilow, 2005). Recently, four other lyssavirus species were ratified by the International Committee on Virus Taxonomy (ICTV Official Taxonomy: Updates since the 8th Report): Aravan virus (ARAV) and Khujand virus (KHUV), isolated from insectivorous bats of the Myotis genus in Central Asia (Kuzmin et al., 2003); Irkut virus (IRKV), isolated from an insectivorous bat, Murina leucogaster in eastern Siberia; and West Caucasian bat virus (WCBV), isolated from an insectivorous bat, Miniopterus schreibersi in southeastern Europe (Botvinkin et al., 2003). Seroprevalence to WCBV was also detected in Miniopterus spp. bats from Kenya (Kuzmin et al., 2008a), suggesting a wider geographical range than was previously believed. Based on genetic distances, serologic cross-reactivity, and peripheral pathogenicity in a mouse model, the Lyssavirus genus was subdivided into two phylogroups. Phylogroup I includes RABV,

198

I.V. Kuzmin et al. / Virus Research 149 (2010) 197–210

DUVV, EBLV1, EBLV2, ABLV, ARAV, KHUV and IRKV. Phylogroup II includes LBV and MOKV. The WCBV cannot be included in any of these phylogroups, and we suggest it should be considered as a member of an independent phylogroup III (Badrane et al., 2001; Hanlon et al., 2005; Kuzmin et al., 2005). The operational term ‘genotype’ has been used for lyssavirus classification since the time when molecular techniques replaced serotyping for classification purposes (Bourhy et al., 1993). Demarcation of genotypes has been based largely on genetic distances (identity values) between members of the genus, and on the bootstrap support of phylogenetic constructions (Bourhy et al., 1993; Delmas et al., 2008; Kissi et al., 1995; Kuzmin et al., 2005; Tordo et al., 1993). In addition, based on identity values, LBV was suggested to be subdivided into at least two separate genotypes (Delmas et al., 2008; Markotter et al., 2008a). However, the ICTV does not operate with viral genotypes but recognizes only viral species. Definition of a viral species is complex, and cannot be based solely on genetic distances, in the absence of other demarcation characteristics (Büchen-Osmond, 2003). In the present paper we describe the isolation and characterization of a new, previously unrecognized lyssavirus, which should be considered a new species of the genus. We also provide additional data on LBV diversity, and discuss challenges of virus classification when different approaches are used. Furthermore, we enrich the data on the comparisons of complete lyssavirus genomes for phylogenetic purposes. 2. Materials and methods 2.1. Sample collection, screening and virus isolation During 2009, a survey of bats was conducted in Kenya for the purpose of detecting new and potential human pathogens that could emerge from bat reservoirs within the region and infect humans (Kuzmin et al., 2008a,b). In total, 616 bats representing at least 22 species were collected from 10 locations across the country (Fig. 1), including 40 sick and dead bats. The sampling and collection protocol were approved by the National Museums of Kenya and the Kenya Wildlife Service. The brain and pooled organs were collected into sterile plastic tubes. Oral swabs were placed into tubes containing Minimum Essential Medium (MEM-10, Invitrogen, Grand Island, NY). Sera were separated from blood clots by centrifugation. All samples were transported on dry ice and stored at −80 ◦ C until used. Bat brains were subjected to the direct fluorescent antibody (DFA) test for detection of lyssavirus antigens (Dean et al., 1996) using monoclonal (Fujirebio Diagnostics Inc., Malvern, PA) and polyclonal (Chemicon Int., Temecula, CA) fluorescein isothiocyanate-labeled anti-rabies antibodies. When a positive DFA result was documented, the brain specimen was homogenized into 10% suspension in MEM-10 and inoculated intracranially into suckling mice (Koprowski, 1996) and mouse neuroblastoma (MNA) cell culture (Webster and Casey, 1996) for virus isolation. Afterwards, three additional passages were made in MNA cells to increase virus titer and supernatant of this culture was used for intramuscular inoculation of 3-week-old mice and Syrian hamsters to assess peripheral pathogenicity of the virus. 2.2. Sequencing of viral genome The amount of the original positive bat brain tissue was limited. Therefore, only the N, P, M and G genes of the virus were determined from the original bat brain material, whereas the L gene and the genome termini were recovered after one mouse passage. Total RNA was extracted from brain homogenates using TRIZol (Invitro-

gen) and subjected to RT-PCR with subsequent direct sequencing on the ABI3730 automated sequencer (Applied Biosystems, Foster City, CA). Genome termini were circularized by RNA ligation, amplified by RT-PCR, cloned and sequenced as described previously (Kuzmin et al., 2008b). Each DNA strand of a given PCR product was sequenced at least twice. The sequence assembly, alignment and consensus sequence generation, as well as DNA translation into deduced amino acids were performed in BioEdit software (Hall, 1999). 2.3. Identity calculations and phylogenetic analysis A set of representative lyssaviruses, used in the present study, is shown in Table 1. For the phylogroup II lyssaviruses, all available sequences were included, whereas for the phylogroup I lyssaviruses, a selection was made, to cover the intrinsic diversity of representatives of each species. Multiple alignments for each viral gene, and for the alignment of concatenated coding regions of the N, P, M, G and L genes (both nucleotide and deduced amino acid sequences), were produced using the ClustalX program (Jeanmougin et al., 1998). The identity values were calculated in BioEdit. Neighbor joining (NJ) phylogenetic analysis was performed in MEGA program (Kumar et al., 2001), using p-distances, Kimura-2 parameters and maximum composite likelihood models, for 1000 bootstrap replicates. Bayesian analysis (BI) was performed using BEAST software (Drummond and Rambaut, 2007), with the general time-reversible model incorporating both invariant sites and a gamma distribution (GTR + I + G). Two simultaneous analyses, each with four Markov chains, were run for 1,000,000 generations and sampled every 1000 generations. Trees generated prior to the stabilization of likelihood scores were discarded (burning = 250). The remaining trees were used to build a 50% majority rule consensus tree. Maximum likelihood (ML) analysis was performed using the PHYLIP package (Felsenstein, 1993) for 100 bootstrap replicates. Nucleotide substitution models used transition/transversion ratios varying from 2 to 4, with empirical base frequencies, and a gamma distribution of rate variations among sites. The ML model parameters for each alignment was determined using PAUP* (Swofford, 2003). 2.4. Monoclonal antibody typing The newly isolated virus and representatives of all described lineages of LBV and MOKV were inoculated intracranially into suckling or 3-week-old mice. When clinical signs of rabies were observed, the mice were euthanized, and brain impressions were made on 4well teflon-coated slides (Cel-Line, Erie Scientific, Portsmouth, NH). After overnight fixation in cold acetone, the samples were subjected to typing via the indirect fluorescent antibody test, using a panel of anti-nucleocapsid monoclonal antibodies (N-MAbs) of the Centers for Disease Control and Prevention (CDC, Atlanta, GA, USA), and N-MAb 422-5 of The Wistar Institute (Philadelphia, PA, USA), as described elsewhere (Smith, 1989). 3. Results 3.1. Detection and isolation of the virus Screening of bat brains by the DFA test revealed one positive specimen, the brain of an adult female Commerson’s leaf-nosed bat (Hipposideros commersoni), found dead in a cave in the southcoastal Kenya (Fig. 1). The carcass of the bat was partly decomposed and therefore no tissues besides the brain were collected. This cave, along with two neighboring caves, was inhabited by at least eight bat species, including H. commersoni, Miniopterus minor,

I.V. Kuzmin et al. / Virus Research 149 (2010) 197–210

199

Fig. 1. Map of Kenya, with the locations of the bat collections indicated. The location where the positive bat was found is indicated in red (A). A colony of Hipposideros commersoni in a cave (B). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Miniopterus spp., Coleura afra, Triaenops persicus, Taphozous mauritanus, Rhinolophus spp., Rousettus aegyptiacus that segregated into large colonies in excess of an estimated 10,000 individuals. During 2008, an LBV was isolated from R. aegyptiacus (isolate KE576; Table 1), and seroprevalence to WCBV was detected in several Miniopterus bats from this same cave (Kuzmin et al., 2008a). Inoculation of the bat brain suspension into MNA cells revealed the presence of virus in a limited number of cells after 3 days of incubation, but during three subsequent sub-passages the viral titer increased up to 107.5 TCID50 . Intracranial inoculation of suckling mice with the bat brain caused paralysis after an incubation period of 12–16 days. The virus was pathogenic for 3-week-old mice and Syrian hamsters by the intramuscular route, causing clinical signs typical for rabies, when 106 MICLD were inoculated (other dilutions were not tested). Because the nearest village to the sampling area is named Shimoni, we have named the isolate Shimoni bat virus (SHIBV). 3.2. Sequence of SHIBV genome, genetic distances and phylogenetic patterns The length of the complete SHIBV genome was 12045 nucleotides (GenBank accession no. GU170201). The genome consisted of five structural genes, common for all lyssaviruses, and non-coding regions, which were most similar to those described for the phylogroup II lyssaviruses (Bourhy et al., 1993; Delmas et al., 2008; Kuzmin et al., 2008b; Le Mercier et al., 1997). The N-P intergeni region (IGS) consisted of CTC, the P-M IGS consisted of CATAT, the M-G IGS consisted of CATTCCCTAACGGGCT. And the G-L IGS consisted of CATTCCCTAACGGGCT. The non-translated 5 terminus of the G consisted of 558 nucleotides and has a single termination-polyadenylation signal. 3.2.1. N gene The coding region of the SHIBV N gene consisted of 1350 nucleotides and coded for 450 amino acids. When identity values of the N gene and nucleoprotein sequences were calculated

for a representative set of lyssaviruses (Table 2), the minimum intrinsic identity registered within RABV was 82.3%, which corresponded to the previously proposed threshold of 80–82% for genotype separation (Bourhy et al., 1993; Kissi et al., 1995; Kuzmin et al., 2005). However, the minimum intrinsic identity among the viruses, currently included in the LBV, was 79.4%, which supported the opinion that these viruses may represent several genotypes (Delmas et al., 2008; Markotter et al., 2008a). Moreover, not only representatives of lineage A (as suggested by Markotter et al. (2008a) for consideration as a new genotype ‘Dakar Bat lyssavirus’) demonstrated significant diversity compared to representatives of lineages B and C (identity 79.6–80.8%), but also isolate KE576 demonstrated only 79.5–80.9% identity to all three lineages. Therefore we designated this isolate into a new lineage (D). If the intergenotype threshold on the nucleotide level was based on the intrinsic identity within LBV, overlaps would occur between DUVV and EBLV1, and between EBLV2 and KHUV (Table 2; Fig. 2). Similarly, minimum intrinsic amino acid identity within RABV (93.7%) was in agreement with the previously proposed intergenotype threshold, whereas intrinsic identity within LBV on the amino acid level was less (92.8%), and if selected as an intergenotype threshold would cause overlaps between DUVV and EBLV1, RABV and ABLV, and between LBV and SHIBV (Table 2; Fig. 2). Based on the identity values, SHIBV was most similar to LBV, and next to MOKV. However, these values were less than the proposed intergenotype threshold, based on RABV identity values. Furthermore, SHIBV was almost equally distanced from LBV and MOKV: nucleotide identity values between LBV and SHIBV, as well as between MOKV and SHIBV, were in the same range of the identity values between LBV and MOKV (Table 2). Phylogenetic reconstructions based on the N gene and deduced nucleoprotein sequences demonstrated that SHIBV belongs to phylogroup II. However, SHIBV could not be included with either LBV or MOKV. In the NJ and BI trees it was placed more close to LBV, whereas in the ML tree it was placed more close to MOKV, however, without any significant statistical support of association with either of these viruses (Fig. 3). In contrast, all lineages of LBV were

200

I.V. Kuzmin et al. / Virus Research 149 (2010) 197–210

Table 1 The lyssavirus sequences used in the present study. Virus species and code RABV SAD B19 PV 9147FRA AY956319 8764THA 9704ARG SHBRV-18 LBV LBVAFR1999

Species isolated from Laboratory strain Laboratory strain Red fox Human (ex a dog) Human (ex a dog) Bat Tadarida brasiliensis Human (ex bat Lasionycteris noctivagans)

Country

GenBank accession no.

Reference

France Germany (ex India) Thailand Argentina USA

M31046 M13215 EU293115 AY956319 EU293111 EU293116 AY705373

Conzelmann et al. (1990) Tordo et al. (1986) Delmas et al. (2008) Pfefferle et al. (unpublished) Delmas et al. (2008) Delmas et al. (2008) Faber et al. (2004)

EF547447; EF547418; EF547445; EF547432 EF547448; EF547419; EF547446; EF547433; EU293108 EU259198 GU170202 EF547459; EF547407; EF547444; EF547431; EU293110 EF547449; EF547417; EF547443; EF547430 EF547450; EF547416; EF547442; EF547429 EF547458; EF547415; EF547440; EF547428 EF547451; EF547413; EF547434; EF547421 EF547453; EF547409; EF547438; EF547423 EF547454; EF547411; EF547441; EF547424 EF547455; EF547410; EF547439; EF547425

Markotter et al. (2008a)

Bat Rousettus aegyptiacus

France (ex Togo or Egypt)

LBVSEN1985 (0406SEN)

Bat Eidolon helvum

Senegal

KE131 KE576 LBVNIG1956 (8619NGA)

Bat Eidolon helvum Bat Rousettus aegyptiacus Bat Eidolon helvum

Kenya Kenya Nigeria

LBVCAR1974

Bat Micropteropus pussilus

Central African Republic

LBVZIM1986

Cat

Zimbabwe

LagSA2004

Bat Epomophorus wahlbergi

South Africa

LagSA2003

Bat Epomophorus wahlbergi

South Africa

Mongoose2004

Mongoose

South Africa

LBVSA1980

Bat Epomophorus wahlbergi

South Africa

LBVSA1982

Bat Epomophorus wahlbergi

South Africa

MOKV Y09762 86100CAM 86101RCA

Cat Shrew A rodent

Zimbabwe Cameroon Central African Republic

Y09762 EU293117 EU293118

Le Mercier et al. (1997) Delmas et al. (2008) Delmas et al. (2008)

DUVV 94286SA 86132SA

Bat Miniopterus sp. (?) Human (ex a bat)

South Africa South Africa

EU293120 EU293119

Delmas et al. (2008) Delmas et al. (2008)

EBLV1 8918FRA 03002FRA RV9

Bat Eptesicus serotinus Bat Eptesicus serotinus Bat Eptesicus serotinus

France France Germany

EU293112 EU293109 EF157976

Delmas et al. (2008) Delmas et al. (2008) Marston et al. (2007)

EBLV2: RV1333 9018HOL AF418014 AF081020 NC 003243 ARAV KHUV IRKV WCBV

Human (ex a bat) Bat Myotis dasycneme Human (ex a bat) Bat Saccolaimus albiventris Bat Saccolaimus albiventris Bat Myotis blythi Bat Myotis mystacinus Bat Murina leucogaster Bat Miniopterus schreibersi

United Kingdom The Netherlands Australia Australia Australia Kyrghyzstan Tajikistan Russia Russia

EF157977 EU293114 AF418014 AF081020 NC 003243 EF614259 EF614261 EF614260 EF614258

Marston et al. (2007) Delmas et al. (2008) Warrilow et al. (2002) Gould et al. (2002) Gould et al. (2002) Kuzmin et al. (2003) Kuzmin et al. (2003) Kuzmin et al. (2005) Kuzmin et al. (2005)

segregated into a monophyletic cluster supported by significant bootstrap values. 3.2.2. P gene The coding region of the SHIBV P gene consisted of 915 nucleotides and coded for 305 amino acids. As in other phylogroup II lyssaviruses, it was extended at the terminal domain compared to the phylogroup I lyssaviruses which have 297–298 amino acids in the P. When identity values for the P gene and deduced phosphoprotein sequences were calculated across the genus, minimum intrinsic identities within RABV were 76.5% for nucleotide and 81.0% for amino acid sequences, and no quantitative overlaps between genotypes axist if these values are used as the intergenotype threshold. Again, LBV demonstrated greater intrinsic diversity, with 66.2% nucleotide and 65.2% amino acid sequence identity. If

Markotter et al. (2008a); Delmas et al. (2008) Kuzmin et al. (2008b) This study Markotter et al. (2008a); Delmas et al. (2008) Markotter et al. (2008a) Markotter et al. (2008a) Markotter et al. (2008a) Markotter et al. (2008a) Markotter et al. (2008) Markotter et al. (2008a) Markotter et al. (2008a)

these values are used for the intergenotype threshold, multiple overlaps would occur between various genotypes (data not shown; supplementary materials are available from authors by request). Therefore, another suggestion was obtained that, from a quantitative standpoint, the LBV may include several separate genotypes. Based on the P, SHIBV again was most similar to LBV (63.0–67.6% of nucleotide and 60.6–68.8% of amino acid identity), and next to MOKV. As with the N gene, the P identity values between LBV and SHIBV overlapped with the identity values between LBV and MOKV (62.9–66.5% of nucleotide and 58.0–64.2% of amino acid identity). Phylogenetic reconstructions placed SHIBV ancestrally in the cluster of LBV and MOKV, or between LBV and MOKV without significant bootstrap support for placement with either of these species, whereas all LBV isolates were consistently segregated into a monophyletic cluster (data not shown).

I.V. Kuzmin et al. / Virus Research 149 (2010) 197–210

201

Fig. 2. Distribution of frequencies of pairwise identity values between N gene sequences of lyssaviruses used in the present study for nucleotides (A) and amino acids (B). 1—intergenotype identities (except LBV); 2—intragenotype identities (except LBV); 3—intrinsic identities of LBV.

3.2.3. M gene As in all lyssaviruses, the M gene of SHIBV consists of 606 nucleotides and codes for 202 amino acids of the matrix protein. Calculation of the identity values for the available set of lyssavirus representatives revealed that minimum intrinsic identity within RABV was 80.6% for the nucleotide, and 86.6% for the amino acid sequences. For LBV, the minimum intrinsic nucleotide identity was 77.5%. However, for amino acid sequences, the identity was 89.1%, and this was the only case when intrinsic identity within LBV was greater than within RABV. Significant quantitative overlaps between intragenotype and intergenotype identity values occurred for multiple lyssavirus species on the both, nucleotide and amino acid levels. As for the SHIBV, identity values between LBV and SHIBV (75.7–76.8% for the nucleotide, and 87.6–90.5% for the amino acid sequences) were in the same range as the identities between LBV and MOKV (74.9–79.7% nucleotide, and 86.6–92.5% amino acid sequences). Phylogenetic reconstructions based on the M gene and the deduced matrix protein produced trees, the topology of which

was different from the topology obtained for any other gene or the complete genome, with inconsistent bootstrap support for the majority of the clusters. For example, ARAV was grouped with EBLV2, whereas KHUV was grouped with IRKV and further with DUVV and EBLV1. Within phylogroup II, the cluster of MOKV was placed between lineages of LBV (for comparison with other genes see Figs. 3, 4 and 6). The SHIBV again was not included with the LBV or MOKV, but instead was positioned between these species, or ancestrally to both (data not shown). Following the previous observation by Markotter et al. (2008a), we do not consider the M gene a suitable region for phylogenetic reconstructions of lyssaviruses. 3.2.4. G gene The organization of the G gene of the SHIBV (1566 nucleotides) was typical for lyssaviruses (including signal peptide, ectodomain, transmembrane domain and endodomain), and coded for 503 amino acids, similar to LBV and MOKV. When identity values for the G nucleotide and amino acid sequences were calculated across the

202

I.V. Kuzmin et al. / Virus Research 149 (2010) 197–210

Fig. 3. Phylogenetic trees of lyssavirus N genes obtained by the NJ method (p-distances) for nucleotide (A) and amino acid (B) sequences, with midpoint rooting of the trees, and an unrooted ML tree for nucleotide sequences (C). LBV lineages are indicated following Markotter et al. (2008). Bootstrap values (1000 replicates for NJ and 100 replicates for ML) are shown for key nodes.

genus, minimum intrinsic identity registered in RABV was 79.0% for the nucleotide and 84.4% for amino acid sequences. If these values are used for quantitative genotype separation, no overlaps occur between genotypes. As with the other genes, the LBV represented a greater amount of diversity in the G gene: 72.2% of nucleotide and

78.9% of amino acid sequence identity. If these values are taken as a quantitative threshold, on the nucleotide level overlaps would occur between EBLV1 and EBLV2 (72.8%), EBLV1 and ARAV (73.0%), EBLV1 and IRKV (73.7%), EBLV2 and ARAV (74.7%), EBLV2 and KHUV (78.2%), ABLV and KHUV (73.1%), ARAV and KHUV (74.5%). On the

I.V. Kuzmin et al. / Virus Research 149 (2010) 197–210

203

Fig. 4. Phylogenetic trees of lyssavirus G genes obtained by the NJ method (p-distances) for nucleotide (A) and amino acid (B) sequences, with midpoint rooting of the trees, and an unrooted ML tree for nucleotide sequences (C). LBV lineages are indicated following Markotter et al. (2008). Bootstrap values (1000 replicates for NJ and 100 replicates for ML) are shown for key nodes.

amino acid level such overlaps are observed between EBLV1 and EBLV2 (80.2%), EBLV1 and IRKV (80.5%), EBLV2 and ARAV (84.7%), EBLV2 and KHUV (87.4%), ABLV and KHUV (81.7%), ARAV and KHUV (83.4%), LBV and SHIBV (79.6%). Remarkable, that on the amino acid

level identity values between EBLV2 and ARAV and between EBLV2 and KHUV were greater than the intrinsic identity within RABV, although between ARAV and KHUV this value was less (83.4%). As was mentioned previously, nucleotide sequences of the G provided

74.0

Above the dash and in the upper-right triangle—mino acid identities (in italics); below the dash and in the lower-left triangle—nucleotide identities (in bold). When only one sequence of a certain species was available for comparison, there is no value.

72.1 75.3 76.1 70.5 73.9

76.6 79.0–79.5 76.9–77.3 72.1–72.4 72.6–73.2 77.8–78.2 77.0–77.6 78.2–78.5 72.2–72.8 73.3–73.9 77.6–77.9 75.6–75.8 77.5–77.8 73.3–73.7 74.7–74.9 74.0–74.9 70.4–71.0 73.3–74.3 72.8–73.2 76.2–77.1 73.2–76.8 75.3–76.5 73.4–75.1 72.2–74.2 71.9–74.8 ARAV KHUV IRKV WCBV SHIBV

74.2–75.8 71.9–74.0 73.1–75.1 73.9–75.2 77.4–78.8

75.6–75.9 76.0–77.0 70.7–72.7 71.9–73.3 70.9–75.5 72.4–78.9 EBLV2 ABLV

71.8–73.7 72.8–75.4

70.1–75.3 70.4–76.2 DUVV EBLV1

73.0–75.3 73.4–75.9

69.4–73.7 MOKV

LBV

a

86.8 84.2 86.4 83.5 83.5 81.1 83.1 90.6 88.0 92.6

79.0 76.2 73.1 75.1

80.0–80.7 84.4–85.5 80.2–80.9 81.3–82.6 86.2–86.6 87.7 90.2–91.1 90.4–92.2

86.4–87.8 95.5–100.0 83.8–99.9 76.5–76.6 75.8–78.1 75.2–76.0 73.1 72.5–74.6 98.6 97.5 74.5–76.2

88.2–88.6 91.1–92.4

87.1 86.4–86.6 84.2 82.0–82.4 90.0 92.0–92.6 88.9 89.5–90.2 88.4–89.5 88.0–90.0 100.099.1 79.0–79.8

92.6–93.2 99.1–100.0 95.4–98.4 76.1–76.8 76.7–77.4

85.3–86.2 86.9–88.0

91.3 91.7–92.0

89.1–89.5 82.0–82.8 84.4–85.3 81.5–82.6 82.4–84.0

98.2–98.8 88.1–88.5 71.9–73.6 71.9–73.1

84.2–84.7

82.9–84.0

79.1–80.9

84.6–85.7

90.8–93.2 82.4–84.2 84.2–87.1 81.3–84.8 81.3–85.3 85.3–87.5

92.8–100.0 79.4–99.7 75.5–78.7

88.4–91.7

83.1–86.4

79.1–82.7

84.6–88.2

80.5–81.2

WCBV IRKV

85.7–87.5 89.6–91.1

KHUV ARAV ABLV

89.1–92.8

EBLV2

86.2–89.8 87.1–88.8

EBLV1 DUVV

87.3–88.4 RABV

MOKV

76.4–82.8

LBV

81.1–85.3

RABV

93.7–100.0a 82.3–99.9 72.0–75.1

Species

Table 2 Identity values (%) of the N genes and deduced nucleoprotein sequences of lyssaviruses.

88.9–90.4

SHIBV

I.V. Kuzmin et al. / Virus Research 149 (2010) 197–210

85.1–86.2

204

better quantitative separation compared to amino acid sequences, if RABV identity values are accepted as the threshold (Kuzmin et al., 2005). The G gene was the only gene that demonstrated significant phylogenetic relatedness between SHIBV and LBV. Although the topology of the trees was the same as that shown in Fig. 3, SHIBV was joined ancestrally to the LBV cluster with significant bootstrap support (and posterior probabilities in the BI analysis) by each method employed (Fig. 4). Because of the importance of the lyssavirus glycoprotein for binding to host cell receptors and for the development of virusneutralizing antibodies, we compared major antigenic sites of SHIBV with those of other lyssaviruses. The sequences were not strictly conserved, but the most significant similarity was observed between SHIBV and other phylogroup II lyssaviruses. For example, SHIBV harbored D333 in the antigenic site III of ectodomain, like LBV and MOKV. This position was demonstrated to be important for peripheral pathogenicity of RABV (Dietzschold et al., 1983). Furthermore, although there was no significant sequence conservation between SHIBV and other lyssaviruses at positions 34–42 within antigenic site II (Prehaud et al., 1988), another part of this antigenic site (positions 198–202) was identical between SHIBV and some of the LBV representatives (lineages C and B). This site overlaps with a putative binding domain for the nicotinic acetylcholine receptor (nAChR) at amino acids 198 to 214 (Lentz et al., 1982), which in SHIBV was most similar to that of the same lineages of LBV. Among the positions that were suggested to be important for pathogenicity for RABV (Takayama-Ito et al., 2004; Faber et al., 2005), in particular, similarity was observed between SHIBV and LBV in S200 , T210 , and common for all phylogroup II lyssaviruses D182 . Other positions, such as 242, 255, 268, 303, were random across the genus or, in contrast, unique for SHIBV, such as K164 . Within antigenic site I (Benmansour et al., 1991), SHIBV retained P231 (like all members of phylogroup II, EBLV1, and some RABV representatives). 3.2.5. L gene The coding region of the SHIBV L gene consisted of 6381 nucleotides and coded for 2127 amino acids of the RNA-dependent RNA polymerase. As previously described for Mononegavirales, it included six conserved domains (Poch et al., 1990). For the identity calculation and phylogenetic analysis, a limited number of lyssavirus L gene sequences were available compared to other genes. For example, such sequences were not available for representatives of the LBV lineage C, although for lineages A, B and D they were present. When the identity values were calculated for available sequences, the minimum intrinsic identity within RABV was 82.2% for the nucleotide, and 93.6% for the amino acid sequences. Within the LBV, the minimum identity values were 76.9% and 91.1% for nucleotide and amino acid sequences, respectively. Again, if the LBV threshold is taken for quantitative separation of lyssavirus genotypes, overlaps would occur between EBLV1 and DUVV (77.1% of nucleotide and 91.3% of amino acid identity); between EBLV1 and IRKV (77.1% nucleotide and 92.7% amino acid identity); between EBLV2 and ABLV (75.2–77.0% nucleotide and 91.1% amino acid identity); between EBLV2 and KHUV (79.5% nucleotide and 93.9% amino acid identity; with the latter overlaps also with RABV values); and between ARAV and KHUV (78.0% nucleotide and 92.4% amino acid identity). However, no such overlaps occurred for SHIBV, which was still most similar to LBV (74.9–76.1% and 89.3–89.9% identity for nucleotide and amino acid sequences, respectively), but again these values were in the same range as the identity between LBV and MOKV (74.6–75.7% and 88.2–89.1% identity for nucleotide and amino acid sequences, respectively). All NJ and BI phylogenetic reconstructions, applied to the L nucleotide and deduced protein sequences, placed SHIBV ancestrally with the cluster of LBV and MOKV, whereas the ML analysis

70.8–71.7

71.6–72.4

72.3–73.7

73.0–74.6

72.5–73.2 72.9–73.8 71.3–72.3 64.7–65.5 67.0–67.6

DUVV

EBLV1

EBLV2

ABLV

ARAV KHUV IRKV WCBV SHIBV

Above the dash and in the upper-right triangle—amino acid identities (in italics); below the dash and in the lower-left triangle—nucleotide identities (in bold). When only one sequence of a certain species was available for comparison, there is no value.

66.4 65.5 68.5

74.3 65.4 67.9

76.8 78.7 73.7 65.5 68.1 75.3 74.6 76.4 65.5 68.1 73.7–75.0 74.5–75.8 71.6–72.8 65.2 67.1–68.0 67.7–68.1 67.2 67.8–68.3 65.2–65.6 71.9–72.2 68.2–68.6 67.5–68.1 67.9–68.7 65.8 73.8–75.1

72.3–73.5 73.9–75.5 66.5–67.7 66.9–68.8

66.9–67.8 MOKV

LBV

91.2–100.0 81.6–98.2 66.6–68.1 RABV

a

75.5 75.5 75.9 71.2 70.8 70.6 70.6 86.3 85.6 90.7

77.5 74.1 65.7 68.5

74.5–75.6 69.9–70.8 82.0–83.3 85.8–88.6 84.8–87.1

92.7–100.0 82.7–99.9 73.7–75.0 74.5–75.8 71.6–72.8 65.2 67.1–68.0

74.4 70.3 84.5 90.8 89.3 84.7–87.0 73.2 67.5–68.0 67.2–68.2

67.3–67.8 68.0–68.7

67.3–68.0

99.1 98.0 73.9–75.5

76.1 70.6 89.0 86.5 87.7–88.0 81.8–84.0 85.3–85.7

98.7–99.4 95.5–98.1 74.2

74.4 69.9 85.1–85.4 83.8 84.6 80.0–81.9 82.6–83.0 87.5–87.8

99.3 89.9 75.9–76.1

81.7–82.1 70.2–70.6 75.2 74.1 74.1–74.5 72.6–74.3 74.4–74.7

87.6–99.7 76.1–98.8 73.2–74.2

95.3 86.5–87.4 67.0–67.5

73.0–73.3

73.6–74.0

85.6–86.3 70.6–71.4 75.6–76.0 74.6–75.8 75.4–76.2 73.4–75.3 75.1–76.0 73.9–74.7 83.3–85.6

74.0–75.5

68.8–69.8 80.5–82.0 83.9–85.3 83.1–84.5 83.2–86.9 82.5–84.5 81.2–82.7 79.5–81.0 72.5–74.0 73.1–75.0

WCBV IRKV KHUV ARAV ABLV EBLV2 EBLV1 DUVV MOKV LBV

a

RABV

Because the CDC N-MAb panel was developed primarily for discrimination of RABV antigenic variants (Smith, 1989), phylogroup I lyssaviruses represented a greater variety of antigenic patterns than phylogroup II lyssaviruses (Table 4). Representatives of all four phylogenetically distant lineages of LBV demonstrated the same pattern (except isolate KE576, which was different by one N-MAb). Even MOKV was distinguishable from LBV in reaction with one NMAb only. Patterns of SHIBV were different from those of LBV by two N-MAbs (C2 and C4), and from those of MOKV by one N-MAb (C4). The positive reaction of SHIBV with the C2 N-MAb was similar to that of MOKV, WCBV, and many representatives of RABV (Smith, 1989). The reaction with C4 N-MAb was more interesting,

205

Species

3.3. Antigenic typing with N-MAbs

Table 3 Identity values (%) of the aligned concatenated N + P + M + G + L genes and deduced protein sequences of lyssaviruses.

3.2.6. Concatenated coding regions of the N, P, M, G and L genes as the basis for lyssavirus differentiation As was shown recently, the alignment of five concatenated lyssavirus genes provided better resolution for quantitative separation of genotypes and for phylogenetic reconstructions, compared to single genes or their parts (Delmas et al., 2008). To utilize the same approach, we assembled manually the aligned sequences of N + P + M + G + L genes of available lyssaviruses (total length of the alignment 10,922 nucleotides), as well as the deduced protein sequences (total leght of the alignment 3639 amino acids). As for the L gene alone, number of lyssavirus representatives in this analysis was limited, although all currently recognized genotypes were included. Notably, we significantly enriched the analysis implemented earlier by Delmas et al. (2008), as we were able to include newly obtained sequences of ARAV, KHUV, IRKV, WCBV, SHIBV and two recent LBV representatives. In the identity matrix (Table 3), the minimum intrinsic identity within RABV was 81.6% for the nucleotide, and 91.2% for the amino acid sequences. For available representatives of LBV (lineage C was not present), the minimum intrinsic identity was 76.1% for nucleotide and 87.6% for amino acid sequences. Frequency histograms (Fig. 5) clearly discriminated groups with different identity values. The least identity values (less than 66% for nucleotide and less than 72% for amino acid sequences) corresponded to the distances between WCBV and all other lyssaviruses. The second group represented identity values between phylogroups I and II (66–69% for nucleotide and 72–76.5% for amino acid sequences). The third group represented identity values between genotypes within each phylogroup except LBV (71–79% for nucleotide and 79.5–90% for amino acid sequences). The 4th group represented viruses from one genotype (identity values over 81% for nucleotide and over 91% for amino acid sequences). As for the LBV (group 5), minimum identity values for the available representatives (76.1% for nucleotide and 87.6% for amino acid sequences) were placed into the category of intergenotype identities rather than intragenotype identities. Based on this analysis, identity values between SHIBV and other lyssaviruses clearly fell into the intergenotype area, even beyond the LBV threshold, albeit with LBV as the virus most similar to SHIBV (73.8–75.1% of nucleotide and 85.6–86.3% of amino acid sequence identity). Again, these identity values overlapped with those between LBV and MOKV (73.2–74.2% of nucleotide and 83.3–85.6% of amino acid sequence identity). Phylogenetic reconstructions, based on the concatenated coding areas of the five genes, placed SHIBV ancestrally with the cluster of LBV and MOKV (NJ and ML nucleotide sequences), or within this cluster but with low bootstrap or the posterior probability support for joining with either LBV or MOKV (BI nucleotide, NJ amino acid sequences; Fig. 6).

SHIBV

placed SHIBV within this cluster, but without significant bootstrap support to either LBV or MOKV (data not shown).

74.3–75.4

I.V. Kuzmin et al. / Virus Research 149 (2010) 197–210

206

I.V. Kuzmin et al. / Virus Research 149 (2010) 197–210

Fig. 5. Distribution of frequencies of pairwise identity values between concatenated sequences of N + P + M + G + L gene sequences of lyssaviruses used in the present study for nucleotides (A) and amino acids (B). 1—identities between WCBV and other phylogroups; 2—identities between phylogroups I and II; 3—intergenotype identities within each phylogroup (except LBV); 4—intragenotype identities (except LBV); 5—intrinsic identities of LBV.

as no other non-RABV lyssaviruses have been reported to react with this N-MAb (Smith, 1989; Botvinkin et al., 2003); in this regard SHIBV seems to be unique. In addition, as with other African nonRABV lyssaviruses, SHIBV reacted with the Wistar N-MAb 422-5, in contrast to all other lyssaviruses.

4. Discussion Lyssavirus classifications have evolved over time. Initially, the viruses were subdivided into serotypes due to their cross-reactivity in classical serologic tests (Shope et al., 1970; Shope, 1982). The introduction of MAb techniques for lyssavirus typing expanded significantly the differentiation ability. The N-MAbs were used most commonly because of simple application protocols and reproducible results. Each serotype had clearly distinguishable reactivity patterns (Dietzschold et al., 1988; Rupprecht et al., 1991; Wiktor and Koprowski, 1980). Subsequently, with the advent of molecular

typing, most of the initial phylogenetic studies were performed on the N gene to evaluate concordance with the former serotype classification (Bourhy et al., 1993; Kissi et al., 1995). Several authors suggested that other genome regions may be used for genotyping as well (Johnson et al., 2002; Tordo et al., 1993; Wu et al., 2007), but better resolution still was achieved by comparison of the N gene sequences with a quantitative threshold of 80–82% nucleotide identity between genotypes (Kissi et al., 1995; Kuzmin et al., 2005). More recently, a comparison of all five concatenated lyssavirus genes provided superior resolution, with a quantitative threshold for genotype separation between 76.4 and 81.6% of nucleotide sequence identity (Delmas et al., 2008). Our results confirm that comparison of the five concatenated gene and deduced protein sequences provide the best resolution with the threshold values between genotypes of 81% for nucleotide and 91% for amino acid sequences (Table 3; Fig. 5). Nevertheless, N gene sequences still offer sufficient separation of genotypes (with an 82% nucleotide identity threshold). Additionally, since represen-

I.V. Kuzmin et al. / Virus Research 149 (2010) 197–210

207

Fig. 6. Phylogenetic trees of concatenated N + P + M + G + L gene sequences of lyssaviruses, obtained by the NJ method (p-distances) for nucleotide (A) and amino acid (B) sequences, with midpoint rooting of the trees, and an unrooted ML tree for nucleotide sequences (C). LBV lineages are indicated following Markotter et al. (2008). Bootstrap values (1000 replicates for NJ and 100 replicates for ML) are shown for key nodes.

tation of N gene sequences in the public domain is currently much greater than representation of complete genomes, N gene sequence use is more feasible. Furthermore, the use of concatenated gene sequences (sometimes termed as “supergenes”) may require partitioning of the model. As different genes may have distinct evolution rates, different model parameters may be required for their comparisons (Rannala and Yang, 2008). Although, seems that it was not an issue in our case, as model parameters estimated in PAUP for separate gene alignments were similar to those estimated for the concatenated gene alignment.

From this quantitative standpoint, LBV can be hypothetically separated into three independent genotypes. One of these would consist of lineage A (tentatively named by Markotter et al. (2008a) “Dakar bat lyssavirus”), another would consist of lineages B and C, which include the original isolate from Nigeria, and therefore does not need to be renamed, and the third would consist of the single isolate KE576. However, the term “genotype”, operationally used by many researchers, is not recognized by the ICTV. Instead, viral species is the only approved taxonomic entity at this level of differentiation. Because this entity is complex, the identity values alone

+ + + +

+

+ +

+ + + +

+ + + + + +

+ + + + +

b

+ + +

Patterns obtained from Smith (1989). Patterns obtained from Botvinkin et al. (2003). a

+

+ +

SHIBV LBV (LBVNIG1956) LBV (LBVSA1982) LBV (LBVAFR1999) LBV (KE131) LBV (KE576) MOKV RABV (CVS) RABV (Fox, Europe)a EBLV1a EBLV2a DUVVa ARAVb KHUVb IRKVb WCBVb

+ + + +

+ + +

+

+ + + + + + + + + + + + +

+

+ + + + + + + + + + + + + + + +

+ + +

+

+

+

+

+ + + + + + + + + + + + +

+ + + + + + + + + + + + +

+ +

+

+

+

+ +

+

+ +

C15 (97-3) C13 (71-2) C12 (62-4) C11 (61-1) C10 (52-2) C9 (52-1) C8 (24-10) C7 (24-1) C6 (23-4) C5 (22-3) C4 (15-2) C3 (11-1) C2 (8-2) C1 (3-1)

N-MAbs Isolate

Table 4 Antigenic patterns of SHIBV compared to other lyssaviruses by a panel of N-MAbs.

+

+

+ + + + + + + + + + + + + + +

C16 (97-11)

C17 (141-1)

C18 (143-1)

+ +

C19 (146-3)

+ + +

C20 (164-2)

+ + + + + + +

I.V. Kuzmin et al. / Virus Research 149 (2010) 197–210

422-5

208

cannot be considered sufficient criteria for species demarcation (Büchen-Osmond, 2003). Although currently recognized lyssavirus species had been initially identified as genotypes (and the priority of the phylogenetic approach in contemporary viral taxonomy is obvious), other characteristics corroborate their demarcation, such as antigenic patterns, geographic distribution and host range. Based on current knowledge, it is difficult to subdivide LBV into several species. Even though they are separated by relatively long genetic distances, all LBV lineages are segregated into a monophyletic cluster with significant statistical support. This topology persists for all genome regions, except the M gene, which produces topology different from that obtained for other genes, not only for LBV, but for other lyssaviruses. It is therefore unlikely that the M gene can be used for adequate phylogenetic reconstructions. All LBV lineages have identical antigenic patterns when studied with the selected N-MAb panel. They all circulate broadly in subSaharan Africa among pteropid bats, without obvious association to any particular host species, or other ecological characteristics that may be used as criteria for species demarcation (Kuzmin et al., 2008b; Markotter et al., 2008a). Realistically, more studies should be carried out before the precise taxonomy of LBV is resolved. Such difficulties do not exist for SHIBV. Identity values between SHIBV and other lyssaviruses clearly fall within the intergenotype level. Genetic distance between SHIBV and another similar lyssavirus species, LBV, is in the same range as the distance between LBV and MOKV. However, more significant is the observation that all phylogenetic reconstructions (except for the G gene) demonstrate that SHIBV cannot be included into any of the existing species. Hence, from the entire phylogenetic standpoint, SHIBV is a new species in the Lyssavirus genus. Demarcation of SHIBV is supported additionally by subtle but noteworthy differences in antigenic patterns, detected in reactions with N-MAbs. As for the G, particular similarity between SHIBV and LBV may be caused by selective adaptation to a chiropteran host. Such similarities between G proteins of bat lyssaviruses have been documented previously (Kuzmin et al., 2003, 2005) and, indeed, must be studied additionally from an evolutionary standpoint. Whether the insectivorous bat H. commersoni is the principal host for SHIBV is unclear. The most related viral species, LBV, circulates among pteropid bats, and only once was documented in an insectivorous bat, Nycteris gambianus, likely as a result of spill-over infection (Markotter et al., 2008a,b). In contrast, MOKV had never been isolated from bats to date. The cave where the infected H. commersoni was encountered was also inhabited by Rousettus aegyptiacus and seven species of insectivorous bats, segregated in large colonies. Hence a possibility of spill-over infection exists, although previous studies demonstrated that bats of different species do not frequently expose each other to lyssaviruses (Kuzmin et al., 2008a,b). One year earlier (in 2008), we isolated LBV from a R. aegyptiacus (isolate KE576), and detected seroprevalence to WCBV in Miniopterus bats from this cave. Thus, multiple lyssaviruses cocirculate in the complex bat community. Additional surveillance is needed to establish host range, distribution and circulation patterns of these viruses, including SHIBV. Unfortunately, on the serologic level, such surveillance is complicated, because lyssaviruses may exhibit serologic cross-reactivity within phylogroups (Badrane et al., 2001; Hanlon et al., 2005). For example, LBV and MOKV can cross-neutralize these viruses (Badrane et al., 2001; Jallet et al., 1999), therefore we expect cross-neutralization between these viruses and SHIBV. Although rabies is a notifiable disease in many countries, systemic surveillance is lacking throughout most of Africa, and the significance of African non-RABV lyssaviruses for public health is unknown. Only three cases of DUVV infection and two cases of MOKV infection were documented in humans (Familusi and Moore,

I.V. Kuzmin et al. / Virus Research 149 (2010) 197–210

1972; Familusi et al., 1972; Markotter et al., 2008b; van Thiel et al., 2009). Initially MOKV and LBV were suggested to possess limited peripheral pathogenicity, but these earlier experiments were performed using only one isolate of each virus, and were restricted to using a murine model (Badrane et al., 2001). Additional experimentation demonstrated that at least some isolates of LBV are as pathogenic for mice as RABV (Markotter et al., 2009). Indeed, these viruses circulate in nature, and spill-over infections have been documented sporadically in various mammals, demonstrating the breadth of their pathogenicity. In addition, these viruses were pathogenic for non-human primates by the peripheral routes (Tignor et al., 1973), and their significance for public health should not be underestimated. As another example, until recently IRKV was known by a single isolate only, obtained from a bat (Botvinkin et al., 2003). However, in 2007 a girl, bitten by a bat, died of rabies in the Russian Far East, and the isolate (named by the authors ‘Ozernoe’; GenBank accession no. FJ905105) clearly belonged to the IRKV species (Belikov et al., 2009). That particular human death was preventable, had routine rabies post-exposure prophylaxis been implemented, as rabies biologics provide reactivity against IRKV and other phylogroup I lyssaviruses (Hanlon et al., 2005). In contrast, WCBV and phylogroup II lyssaviruses are not covered by the available commercial biologics (Bahloul et al., 1998; Jallet et al., 1999; Nel et al., 2003). Therefore, we expect this lack of coverage to extend to SHIBV as well. Public awareness and enhanced surveillance for these viruses must be increased, and new potent biologics must be developed to provide more reliable protection against them. Acknowledgements The study was supported in part by the Global Disease Detection program of the Centers for Disease Control and Prevention (Atlanta, GA). We thank Edwin Danga, Evelyne Mulama, Solomon Gikundi and Leonard Nderitu (CDC-Kenya, Nairobi) for excellent logistic and laboratory support, and Mang Shi (University of Hong Kong, Hong Kong) for productive discussions on phylogenetic analysis. References Badrane, H., Bahloul, C., Perrin, P., Tordo, N., 2001. Evidence of two lyssavirus phylogroups with distinct pathogenecity and immunogenecity. J. Virol. 75, 3268–3276. Bahloul, C., Jacob, Y., Tordo, N., Perrin, P., 1998. DNA-based immunization for exploring the enlargement of immunological cross-reactivity against the lyssaviruses. Vaccine 16, 417–425. Belikov, S.I., Leonova, G.N., Kondratov, I.G., Romanova, E.V., Pavlenko, E.V., 2009. Isolation and genetic characterisation of a new lyssavirus strain in the Primorskiy kray. East Siberian J. Infect. Pathol. 16 (3), 68–69. Benmansour, A., Leblois, H., Coulon, P., Tuffereau, C., Gaudin, Y., Flamand, A., Lafay, F., 1991. Antigenicity of rabies virus glycoprotein. J. Virol. 65 (8), 4198–4203. Botvinkin, A.D., Poleschuk, E.M., Kuzmin, I.V., Borisova, T.I., Gazaryan, S.V., Yager, P., Rupprecht, C.E., 2003. Novel lyssavirus isolated from bats Russia. Emerg. Infect. Dis. 9, 1623–1625. Bourhy, H., Kissi, B., Tordo, N., 1993. Molecular diversity of the Lyssavirus genus. Virology 194, 70–81. Büchen-Osmond, C., 2003. Taxonomy and classification of viruses. In: Murray, P.R., Baron, E.J., Jorgensen, J.H., Pfaller, M.A., Yolken, R.H. (Eds.), Manual of Clinical Microbiology, vol. 2, 8th ed. ASM Press, Washington, DC, pp. 1217–1226. 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. Dean, D.J., Abelseth, M.K., Atanasiu, P., 1996. The fluorescent antibody test. In: Meslin, F.-X., Kaplan, M.M., Koprowski, H. (Eds.), Laboratory Techniques in Rabies, 4th ed. WHO, Geneva, Switzerland, pp. 88–93. Delmas, O., Holmes, E.C., Talbi, C., Larrous, F., Dacheux, L., Bouchier, C., Bourhy, H., 2008. Genomic diversity and evolution of the lyssaviruses. PloS One 3 (4), e2057. 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.

209

Dietzschold, B., Rupprecht, C.R., Tollis, M., Lafon, M., Mattei, J., Wiktor, T.J., Koprowski, H., 1988. Antigenic diversity of the glycoprotein and nucleocapsid proteins of rabies and rabies-related viruses: implications for epidemiology and control of rabies. Rev. Infect. Dis. 10 (Suppl. 4), 785–798. Drummond, A.J., Rambaut, A., 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol. 7, 214. 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 (46), 16328–16332. Faber, M., Faber, M.L., Papaneri, A., Bette, M., Weihe, E., Dietzschold, B., Schnell, M.J., 2005. A single amino acid change in rabies virus glycoprotein increases virus spread and enhances virus pathogenicity. J. Virol. 79 (22), 14141–14148. Familusi, J.B., Moore, D.L., 1972. Isolation of a rabies related virus from the cerebrospinal fluid of a child with ‘aseptic meningitis’. Afr. J. Med. Sci. 3, 93–96. Familusi, J.B., Ogunkoya, B.O., Moore, D.L., Kemp, G.E., Fabiyi, A., 1972. A fatal human infection with Mokola virus. Am. J. Trop. Med. Hyg. 21, 959–963. Felsenstein, J., 1993. PHYLIP: Phylogenetic Inference Package (Version 3.5c). Dept. of Genetics, University of Washington, Seattle, WA, USA. Fooks, A.R., Brookes, S.M., Johnson, N., McElhinney, L.M., Hutson, A.M., 2003a. European bat lyssaviruses: an emerging zoonosis. Epidemiol. Infect. 131 (3), 1029–1039. Fooks, A.R., McElhinney, L.M., Pounder, D.J., Finnegan, C.J., Mansfield, K., Johnson, N., Brookes, S.M., Parsons, G., White, K., McIntyre, P.G., Nathwani, D., 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. Gould, A.R., Kattenbelt, J.A., Gumley, S.G., Lunt, R.A., 2002. Characterization of an Australian bat lyssavirus variant isolated from an insectivorous bat. Virus Res. 89, 1–28. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95–98. Hanlon, C.A., Kuzmin, I.V., Blanton, J.D., Weldon, W.C., Manangan, J.S., Rupprecht, C.E., 2005. Efficacy of rabies biologics against new lyssaviruses from Eurasia. Virus Res. 111, 44–54. ICTV Official Taxonomy: Updates Since the 8th Report, 2009. Vertebrate. http://talk.ictvonline.org/media/p/1208.aspx (accessed 14.10.09). Jallet, C., Jacob, Y., Bahloul, C., Drings, A., Desmezières, E., Tordo, N., Perrin, P., 1999. Chimeric lyssavirus glycoproteins with increased immunological potential. J. Virol. 73, 225–233. Jeanmougin, F., Thompson, J.D., Gouy, M., Higgins, D.G., Gibson, T.J., 1998. Multiple sequence alignment with Clustal X. Trends Biochem. Sci. 23, 403–405. 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. Kissi, B., Tordo, N., Bourhy, H., 1995. Genetic polymorphism in the rabies virus nucleoprotein gene. Virology 209, 526–537. Koprowski, H., 1996. The mouse inoculation test. In: Meslin, F.-X., Kaplan, M.M., Koprowski, H. (Eds.), Laboratory Techniques in Rabies, 4th ed. WHO, Geneva, Switzerland, pp. 80–86. Kumar, S., Tamura, K., Jakobsen, I.B., Nei, M., 2001. MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17, 1244–1245. Kuzmin, I.V., Botvinkin, A.D., Poleschuk, E.M., Orciari, L.A., Smith, J.S., Rupprecht, C.E., 2006. Bat rabies surveillance in the former Soviet Union. Dev. Biol. (Basel) 125, 273–282. 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. Kuzmin, I.V., Niezgoda, M., Franka, R., Agwanda, B., Markotter, W., Beagley, J.C., Urazova, O.Y., Breiman, R.F., Rupprecht, C.E., 2008a. Possible emergence of West Caucasian bat virus in Africa. Emerg. Infect. Dis. 14 (12), 1887–1889. Kuzmin, I.V., Niezgoda, M., Franka, R., Agwanda, B., Markotter, W., Beagley, J.C., Urazova, O.Y., Breiman, R.F., Rupprecht, C.E., 2008b. Lagos bat virus in Kenya. J. Clin. Microbiol. 46 (4), 1451–1461. 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. 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. Lentz, T.L., Burrage, T.G., Smith, A.L., Crick, J., Tignor, G.H., 1982. Is the acetylcholine receptor a rabies virus receptor? Science 215 (4529), 182–184. Markotter, W., Kuzmin, I., Rupprecht, C.E., Nel, L.H., 2008a. Phylogeny of Lagos bat virus: challenges for lyssavirus taxonomy. Virus Res. 135 (1), 10–21. Markotter, W., Kuzmin, I.V., Rupprecht, C.E., Nel, L.H., 2009. Lagos bat virus virulence in mice inoculated by the peripheral route. Epidemiol. Infect. 137 (8), 1155–1162. Markotter, W., Van Eeden, C., Kuzmin, I.V., Rupprecht, C.E., Paweska, J.T., Swanepoel, R., Fooks, A.R., Sabeta, C.T., Cliquet, F., Nel, L.H., 2008b. Epidemiology and pathogenicity of African bat lyssaviruses. Dev. Biol. (Basel) 131, 317–325. Marston, D.A., McElhinney, L.M., Johnson, N., Muller, T., Conzelmann, K.K., Tordo, N., Fooks, A.R., 2007. Comparative analysis of the full genome sequence of European bat lyssavirus type 1 and type 2 with other lyssaviruses and evidence for

210

I.V. Kuzmin et al. / Virus Research 149 (2010) 197–210

a conserved transcription termination and polyadenylation motif in the G-L 3 non-translated region. J. Gen. Virol. 88, 1302–1314. Nel, L., Jacobs, J., Jafta, J., Von Teichman, B., Bingham, J., 2000. New cases of Mokola virus infection in South Africa: a genotypic comparison of Southern African virus isolates. Virus Genes 20, 103–106. Nel, L.H., Niezgoda, M., Hanlon, C.A., Morril, P.A., Yager, P.A., Rupprecht, C.E., 2003. A comparison of DNA vaccines for the rabies-related virus, Mokola. Vaccine 21, 2598–2606. 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. Prehaud, C., Coulon, P., LaFay, F., Thiers, C., Flamand, A., 1988. Antigenic site II of the rabies virus glycoprotein: structure role in viral virulence. J. Virol. 62 (1), 1–7. Rannala, B., Yang, Z., 2008. Phylogenetic inference using complete genomes. Annu. Rev. Genomics Hum. Genet. 9, 217–231. Rupprecht, C.E., Dietzschold, B., Wunner, W.H., Koprowski, H., 1991. Antigenic relationships of lyssaviruses. In: Baer, G.M. (Ed.), The Natural History of Rabies, 2nd ed. CRC Press, Boca Raton, pp. 69–100. Sabeta, C.T., Markotter, W., Mohale, D.K., Shumba, W., Wandeler, A.I., Nel, L.H., 2007. Mokola virus in domestic mammals, South Africa. Emerg. Infect. Dis. 13 (9), 1371–1373. Shope, R.E., 1982. Rabies-related viruses. Yale J. Biol. Med. 55, 271–275. Shope, R.E., Murphy, F.A., Harrison, A.K., Causey, O.R., Kemp, G.E., Simpson, D.I.H., Moore, D., 1970. Two African viruses serologically and morphologically related to rabies virus. J. Virol. 6, 690–692. Smith, J.S., 1989. Rabies virus epitopic variation: use in ecologic studies. Adv. Virus Res. 36, 215–253. Swofford, D.L., 2003. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods) Version 4. Sinauer Associates, Sunderland, MA.

Takayama-Ito, M., Ito, N., Yamada, K., Minamoto, N., Sugiyama, M., 2004. Region at amino acids 164 to 303 of the rabies virus glycoprotein plays an important role in pathogenicity for adult mice. J. Neurovirol. 10 (2), 131–135. Tignor, G.H., Shope, R.E., Bhatt, P.N., Percy, D.H., 1973. Experimental infection of dogs and monkeys with two rabies serogroup viruses, Lagos bat and Mokola (IbAn 27377): clinical, serologic, virologic and fluorescent-antibody studies. J. Infect. Dis. 128, 471–478. 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., Badrane, H., Bourhy, H., Sacramento, D., 1993. Molecular epidemiology of Lyssaviruses: focus on the glycoprotein and pseudogenes. Onderstepoort J. Vet. Res. 60, 315–323. van Thiel, P.P., de Bie, R.M., Eftimov, F., Tepaske, R., Zaaijer, H.L., van Doornum, G.J., Schutten, M., Osterhaus, A.D., Majoie, C.B., Aronica, E., Fehlner-Gardiner, C., Wandeler, A.I., Kager, P.A., 2009. Fatal human rabies due to Duvenhage Virus from a bat in Kenya: failure of treatment with coma-induction, ketamine, and antiviral drugs. PLoS Negl. Trop. Dis. 3 (7), e428. 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. Warrilow, D., 2005. Australian bat lyssavirus: a recently discovered new rhabdovirus. Curr. Top. Microbiol. Immunol. 292, 25–44. Webster, W.A., Casey, G.A., 1996. Virus isolation in neuroblastoma cell culture. In: Meslin, F.-X., Kaplan, M.M., Koprowski, H. (Eds.), Laboratory Techniques in Rabies, 4th ed. WHO, Geneva, Switzerland, pp. 96–104. Wiktor, T.J., Koprowski, H., 1980. Antigenic variants of rabies virus. J. Exp. Med. 152 (1), 99–112. Wu, X., Franka, R., Velasco-Villa, A., Rupprecht, C.E., 2007. Are all lyssavirus genes equal for phylogenetic analyses? Virus Res. 129 (2), 91–103.