Genetic and evolutionary characterization of RABVs from China using the phosphoprotein gene

  • Lihua Wang1Email author,

    Affiliated with

    • Hui Wu1Email author,

      Affiliated with

      • Xiaoyan Tao1,

        Affiliated with

        • Hao Li1,

          Affiliated with

          • Simon Rayner2,

            Affiliated with

            • Guodong Liang1 and

              Affiliated with

              • Qing Tang1Email author

                Affiliated with

                Virology Journal201310:14

                DOI: 10.1186/1743-422X-10-14

                Received: 28 June 2012

                Accepted: 7 December 2012

                Published: 7 January 2013

                Abstract

                Background

                While the function of the phosphoprotein (P) gene of the rabies virus (RABV) has been well studied in laboratory adapted RABVs, the genetic diversity and evolution characteristics of the P gene of street RABVs remain unclear. The objective of the present study was to investigate the mutation and evolution of P genes in Chinese street RABVs.

                Results

                The P gene of 77 RABVs from brain samples of dogs and wild animals collected in eight Chinese provinces through 2003 to 2008 were sequenced. The open reading frame (ORF) of the P genes was 894 nucleotides (nt) in length, with 85-99% (80-89%) amino acid (nucleotide) identity compared with the laboratory RABVs and vaccine strains. Phylogenetic analysis based on the P gene revealed that Chinese RABVs strains could be divided into two distinct clades, and several RABV variants were found to co circulating in the same province. Two conserved (CD1, 2) and two variable (VD1, 2) domains were identified by comparing the deduced primary sequences of the encoded P proteins. Two sequence motifs, one believed to confer binding to the cytoplasmic dynein light chain LC8 and a lysine-rich sequence were conserved throughout the Chinese RABVs. In contrast, the isolates exhibited lower conservation of one phosphate acceptor and one internal translation initiation site identified in the P protein of the rabies challenge virus standard (CVS) strain. Bayesian coalescent analysis showed that the P gene in Chinese RABVs have a substitution rate (3.305x10-4 substitutions per site per year) and evolution history (592 years ago) similar to values for the glycoprotein (G) and nucleoprotein (N) reported previously.

                Conclusion

                Several substitutions were found in the P gene of Chinese RABVs strains compared to the laboratory adapted and vaccine strains, whether these variations could affect the biological characteristics of Chinese RABVs need to be further investigated. The substitution rate and evolution history of P gene is similar to G and N gene, combine the topology of phylogenetic tree based on the P gene is similar to the G and N gene trees, indicate that the P, G and N genes are equally valid for examining the phylogenetics of RABVs.

                Keywords

                Rabies virus Phosphoprotein gene Genetic diversity Molecular evolution

                Introduction

                Rabies is a lethal neurological disease caused by infection with members of the genus lyssavirus. Eleven distinct lyssavirus species are currently recognized worldwide [1]. In China, only the classical rabies virus (RABV) is known to circulate in dogs, which serve as the principal reservoir and transmitter of rabies to humans and domestic animals [2, 3]. RABV has a non-segmented negative sense RNA genome comprised of five genes in the order 3’-N-P-M-G-L-5’ [4]. The relatively divergent P gene [57] encodes a multifunctional phosphoprotein (P protein) [8] and has been extensively investigated using laboratory adapted RABV strains. Five serine residues of the challenge virus standard (CVS) strain have been identified as phosphate acceptor sites [9]. Also, P is a critical component of the viral polymerase responsible for transcription and replication through its binding to the N and L proteins [1012]. Two independent N binding sites, one located within amino acids (aa) 66–176 at the N-terminal half of the protein and the other located to amino acids 268–297 within 50 residues of the C-terminus, have been found in the P protein [10, 11]. Via N-P complexes, the nonspecific aggregation of N can be prevented and can keep N in a suitable form for specific encapsidation [13]. The short lysine-rich motif FSKKYKF (aa 214–220) is an important component of the C-terminal N protein binding domain of P [14]. P is associated with the genome expression process by acting as an intermediary for the attachment of the L polymerase core to the N-RNA template [15]. In addition, the first 19 N-terminal residues of P confer L protein binding [10]. P also specifically interacts with many host cell components. It has been reported that the sequence (K/R)XTQT represents a conserved cytoplasmic dynein light chain (LC8) binding motif, an element of the microtubule-associated motors involved in minus-end directed axonal transport, through which it may play some role in viral retrograde transport [1618]. P interferes with the host’s innate immune system through inhibition of the activities of interferon regulatory factor 3 (IRF3) [19] and signal transducer and activator of transcription 1 (STAT1) [20, 21], thereby abrogating the cellular type 1 interferon pathway. P also binds to the promyelocytic leukemia (PML) protein, which has many possible functions in nuclear trafficking, viral defense mechanisms and apoptosis [22], suggesting that P acts an antagonist towards antiviral PML function [23].

                Since all functional studies on the RABV P protein have been performed using a limited number of laboratory strains, the relevance of the results to field isolates is unclear. In this study we sequenced the P gene of Chinese RABV street strains collected in most rabies endemic areas of China and investigated the genetic diversity, sequence characteristics and estimated the overall substitution rate of the P gene. In addition, the phylogeny and evolution history of Chinese RABVs based on P gene were examined.

                Results

                Length and identity of P gene in Chinese RABV street strains

                77 RABV positive brain specimens were detected by direct fluorescent antibody (DFA) and subjected to RT-PCR for determination of the P gene of RABV street strains. These specimens were from field captured dogs and ferret badgers in eight provinces which had high (Guangxi, Guizhou and Hunan provinces), middle (Jiangsu and Shandong provinces) and low (Anhui, Shanghai, and Zhejiang provinces) incidences of rabies (Figure 1). The open reading frame (ORF) of the P gene, corresponding to nt 1514–2407 of the PV strain (M13215), was determined for all 77 RABV isolates. The ORF of the P gene of all Chinese RABVs were 894 nt in length and sequences were submitted to GenBank (HM582519–HM582595). The species of origin, the year of isolation, and geographical location of these sequences are summarized in Table 1.
                http://static-content.springer.com/image/art%3A10.1186%2F1743-422X-10-14/MediaObjects/12985_2012_1982_Fig1_HTML.jpg
                Figure 1

                Locations of specimen collection in this study. AH, GX, GZ, HuN, JS, SD, SH, ZJ, indicate Anhui, Guangxi, Guizhou, Hunan, Jiangsu, Shandong, Shanghai, Zhejiang provinces of China, where the specimen were collected in this study during 2003 to 2008. I and II indicate the presence of isolates corresponding to Clade I and II as classified by the phylogenetic tree.

                Table 1

                Background information of P gene sequences used in this study

                Genus/isolates

                Host

                Origin

                Year

                GenBank acc. no.

                Genus/isolates

                Host

                Origin

                Year

                GenBank acc. no.

                AH8

                Dog

                AH

                2005

                HM582562

                SH9

                Dog

                SH

                2004

                HM582564

                AH12

                Dog

                AH

                2005

                HM582567

                SH15

                Dog

                SH

                2004

                HM582555

                GX0801

                Dog

                GX

                2008

                HM582588

                SH16

                Dog

                SH

                2004

                HM582584

                GX0802

                Dog

                GX

                2008

                HM582589

                SH17

                Dog

                SH

                2004

                HM582554

                GX0803

                Dog

                GX

                2008

                HM582590

                SH19

                Dog

                SH

                2004

                HM582550

                GX0804

                Dog

                GX

                2008

                HM582591

                SH20

                Dog

                SH

                2004

                HM582532

                GX0805

                Dog

                GX

                2008

                HM582592

                SH24

                Dog

                SH

                2003

                HM582536

                GX0806

                Dog

                GX

                2008

                HM582593

                SH25

                Dog

                SH

                2003

                HM582553

                GX0807

                Dog

                GX

                2008

                HM582594

                SH27

                Dog

                SH

                2003

                HM582529

                GX0809

                Dog

                GX

                2008

                HM582595

                SH28

                Dog

                SH

                2003

                HM582524

                GX1

                Dog

                GX

                2006

                HM582521

                SH29

                Dog

                SH

                2003

                HM582548

                GX2

                Dog

                GX

                2006

                HM582546

                SH30

                Dog

                SH

                2003

                HM582549

                GX3

                Dog

                GX

                2006

                HM582525

                SH31

                Dog

                SH

                2003

                HM582556

                GX5

                Dog

                GX

                2006

                HM582571

                SH32

                Dog

                SH

                2003

                HM582561

                GX6

                Dog

                GX

                2006

                HM582581

                D03

                Dog

                ZJ

                2008

                HM582568

                GX7

                Dog

                GX

                2006

                HM582526

                D05

                Dog

                ZJ

                2008

                HM582569

                GX8

                Dog

                GX

                2005

                HM582522

                D06

                Dog

                ZJ

                2008

                HM582573

                GX9

                Dog

                GX

                2006

                HM582535

                D07

                Dog

                ZJ

                2008

                HM582570

                GX10

                Dog

                GX

                2005

                HM582523

                D10

                Dog

                ZJ

                2008

                HM582586

                GX16

                Dog

                GX

                2005

                HM582538

                F03

                CFB

                ZJ

                2008

                HM582587

                GX18

                Dog

                GX

                2006

                HM582527

                CGX0521

                Dog

                GX

                2005

                EU004759

                GX19

                Dog

                GX

                2006

                HM582543

                CGX0603

                Dog

                GX

                2006

                EU004755

                GX24

                Dog

                GX

                2005

                HM582547

                CGX0614

                Dog

                GX

                2006

                EU004758

                GX26

                Dog

                GX

                2006

                HM582528

                CHdg18

                Dog

                GX

                2007

                AB458796

                GZ1

                Dog

                GZ

                2005

                HM582580

                GX4

                Dog

                GX

                1994

                GU358653

                GZ3

                Dog

                GZ

                2005

                HM582544

                HN10

                Human

                HN

                2006

                EU643590

                GZ6

                Dog

                GZ

                2005

                HM582579

                CHN0635

                Human

                HN

                2006

                EU004777

                GZ8

                Dog

                GZ

                2005

                HM582531

                CJS0523

                Dog

                JS

                2005

                EU004782

                GZ9

                Dog

                GZ

                2005

                HM582540

                JX08-45

                CFB

                JX

                2008

                GU647092

                GZ10

                Dog

                GZ

                2005

                HM582541

                NeiMeng925

                Dog

                NM

                2008

                FJ415313

                GZ11

                Dog

                GZ

                2005

                HM582562

                SH06

                Dog

                SH

                2006

                GU345748

                GZ12

                Dog

                GZ

                2005

                HM582545

                SH26

                Dog

                SH

                2003

                HM582583

                GZ13

                Dog

                GZ

                2005

                HM582537

                D01

                Dog

                ZJ

                2008

                FJ712193

                GZ14

                Dog

                GZ

                2005

                HM582551

                D02

                Dog

                ZJ

                2008

                FJ712194

                GZ15

                Dog

                GZ

                2005

                HM582552

                D04

                Dog

                ZJ

                2008

                FJ032321

                GZ16

                Dog

                GZ

                2005

                HM582534

                D08

                Dog

                ZJ

                2008

                FJ032322

                GZ17

                Dog

                GZ

                2005

                HM582530

                F02

                CFB

                ZJ

                2008

                FJ712195

                GZ21

                Dog

                GZ

                2008

                HM582572

                8743THA

                Human

                Thailand

                1983

                EU293121

                HN4

                Dog

                HuN

                2005

                HM582519

                8764THA

                Human

                Thailand

                1983

                EU293111

                HN27

                Dog

                HuN

                2005

                HM582582

                INRV

                Human

                India

                2005

                AY956319

                HN29

                Dog

                HuN

                2005

                HM582520

                NNV-RAB-H

                Human

                India

                2006

                EF437215

                HN30

                Dog

                HuN

                2005

                HM582542

                CVS

                Challenge virus standard

                  

                X55727

                JS29

                Dog

                JS

                2006

                HM582563

                aG

                Vaccine st rain

                China

                 

                DQ646875

                JS34

                Dog

                JS

                2006

                HM582565

                CTN

                Vaccine st rain

                China

                 

                FJ959397

                SD1

                Dog

                SD

                2008

                HM582557

                PV

                Vaccine st rain

                France

                 

                M13215

                SD7

                Dog

                SD

                2007

                HM582559

                SADB19

                Vaccine st rain

                USA

                 

                M31046

                SD8

                Dog

                SD

                2007

                HM582558

                Ni-CE

                Vaccine st rain

                Japan

                 

                AB128149

                SD10

                Dog

                SD

                2007

                HM582533

                RC-HL

                Vaccine st rain

                Japan

                 

                AB009663

                SD11

                Dog

                SD

                2007

                HM582575

                Flury-HEP

                Vaccine st rain

                USA

                 

                GU565704

                SD12

                Dog

                SD

                2007

                HM582574

                8619NGA

                Bat

                Nigeria

                1956

                EU293110

                SD13

                Dog

                SD

                2007

                HM582576

                MOKV

                Cat

                Zimbabwe

                1981

                NC006429

                SD14

                Dog

                SD

                2006

                HM582577

                86132SA

                Human

                South Africa

                1971

                EU293119

                SD23

                Dog

                SD

                2008

                HM582578

                8918FRA

                Bat

                France

                1989

                EU293112

                SH1

                Dog

                SH

                2005

                HM582585

                9018HOL

                Bat

                Netherlands

                1986

                EU293114

                SH5

                Dog

                SH

                2005

                HM582566

                ABLV

                Bat

                Aust ralia

                1996

                NC003243

                SH7

                Dog

                SH

                2004

                HM582560

                WCBV

                Bat

                Russia

                2002

                EF614258

                Note: New sequences in this study are labeled Bold and italic; AH, GX, GZ, HuN, JS, JX, NM, SD, SH, ZJ, indicate Anhui, Guangxi, Guizhou, Hunan, Jiangsu, Jiangxi, Inner Mongolia, Shandong, Shanghai, Zhejiang provinces of China, respectively; CFB: Chinese Ferret Badgers.

                The P gene of all the Chinese RABVs encodes a 297 amino acid protein identical in length to the P gene in the PV vaccine strain (M13215). The nucleotide and deduced amino acid sequences were aligned and compared with the sequences of laboratory, street and vaccine strains. Among the 77 Chinese RABVs isolates, the nucleotide and amino acid sequence identities of the P gene were 80.2-100% and 85.2-100% respectively. When compared with the vaccine strains, the P gene of the 77 Chinese RABVs had 85.0-99.2% (80.0-89.5%) amino acid (nucleotide) identity, respectively.

                Variation of functionally significant sequence motifs and residues

                Based on the identity analysis, an amino acid alignment of the 77 Chinese RABVs isolates and representative sequences of laboratory and vaccine strains was generated and investigated for mutations (Figure 2). In total, seventy two amino acid substitutions throughout the P protein were observed in the Chinese RABVs isolates relative to the PV vaccine strain (M13215). Based on the location of the mutations, the protein had both highly conserved and highly variable regions that have been previously shown to be associated with viral function. Specifically, there were two conserved domains at residues 1–50 (CD1) and 184–279 (CD2) and two variable domains at residues 51–80 (VD1) and 126–178 (VD2) (Figure 2). The first 19 aa residues at the N-terminal, shown to be associated with L binding [10], are completely conserved. The short lysine-rich segment FSKKYKF (209-216aa) thought to be an important component of the C-terminal N protein binding domain [14], is also highly conserved in all Chinese isolates. Within region VD2, the cytoplasmic dynein LC8 binding motif (K/R) XTQT [18] is conserved with Chinese RABVs, and all the strains contain the motif KSTQT (located between 144 and 148 aa). Interestingly, the STAT-1 binding sites, located in the last 30 aa residues of the C-terminal [20] showed limited conservation in Chinese isolates. The internal translation initiation sites 20, 53, 69, and 83 in the P protein of the rabies challenge virus standard (CVS) strain [24] are at the same position in the Chinese RABVs isolates. Three of them (Met20, Met53, and Met83) are completely conserved in Chinese RABVs. For the remainder, the mutation Met69 toVal69 occurred in isolate GZ8 and mutation Met69 to Ala69 occurred in isolates HN29, GX0802, GZ7, GX16. Four (Ser64, Ser162, Ser210, Ser271) of five serine residues reported to function as phosphate acceptors in the P protein of the rabies challenge virus standard (CVS) strain [9] were absolutely conserved. For the fifth residues mutation Ser63 to Phe63 or Ser63 to Leu63 was observed in all the Chinese isolates with the exception of isolate SH19.
                http://static-content.springer.com/image/art%3A10.1186%2F1743-422X-10-14/MediaObjects/12985_2012_1982_Fig2_HTML.jpg
                Figure 2

                Alignment of the P amino acid sequences of street strains collected in this study, vaccine strains and standard challenge virus strain CVS11. Dots indicate amino acids that are in agreement with the reference sequence (PV vaccine strain (M13215)) on the first line. Box a and e: conserved domains 1 and 2; box b and c: variable domains 1 and 2; box d: Dynein light chain (LC8)-binding motif; solid underline shows L protein binding region(1–19 aa) and the lysine-rich motif (209–216 aa), respectively; dashed underline shows N protein binding site; triangles indicate the positions of methionine residues and confirmed translation initiation in the CVS strain; arrows indicate the positions of serine residues identified as phosphoacceptors in the P protein of the CVS strain.

                Phylogenetic analysis of RABVs in China

                A phylogenetic analysis of 113 (77 collected in this study, with an additional 36 samples downloaded from GenBank) RABV P gene sequences was performed. The Neighbor-joining tree is shown in Figure 3 with bootstrap values shown for the main groupings. The sequences of Chinese isolates were divided into two major clades, named clade I and II (Figure 3). Most of the 77 isolates collected in this study are placed in Clade I (bootstrap value = 98). These isolates are mainly from Anhui, Guangxi, Guizhou, Hunan, Jiangsu, Shandong, Shanghai and Zhejiang provinces, and show a close evolutionary history with the RABVs isolates from Thailand (8764THA, EU293111; 8743THA, EU293121). Clade II (bootstrap value = 98) are composed of isolates from Shanghai, Guizhou and Guangxi provinces, are grouped with the standard challenge strain (CVS) and vaccine strains (aG, PV, RC HL, SADB19, Ni-CE and Flury-HEP), and show a close relationship to arctic-related RABVs strains from India and northeastern China (Inner Mongolia). Chinese Ferret badger strain F03 was grouped with D10 strain isolated from dog in the same location, indicating that RABVs spillover can occur between dogs and Chinese Ferret badgers.
                http://static-content.springer.com/image/art%3A10.1186%2F1743-422X-10-14/MediaObjects/12985_2012_1982_Fig3_HTML.jpg
                Figure 3

                Neighbor-joining phylogenetic tree (P-distance) for the P gene of RABVs collected in this study, vaccine strains and representative strains of lyssavirus . Numbers indicate the bootstrap value from 1000 replicates. Clade I and clade II are indicated.

                Substitution rates and evolution history analysis of P gene

                By using a Bayesian Markov chain Monte Carlo method, the evolutionary history, including evolutionary rates of populations (nucleotide substitutions per site per year) and TMRCA (the most recent common ancestor) were analyzed based on 58 P gene sequences (Only sequences with an homology less than 98% and with full background information in terms of location and isolation time were used in the calculation). The estimated mean rate of nucleotide substitution for the P gene of Chinese RABVs was 3.305x10-4 substitutions per site per year (95% HPD values, 1.127-6.209x10-4 substitutions per site per year). Bayesian coalescent analysis estimated the most recent common ancestor (TMRCA) to have originated 592 years ago (95% HPD, 142–2621 years) (Figure 4).
                http://static-content.springer.com/image/art%3A10.1186%2F1743-422X-10-14/MediaObjects/12985_2012_1982_Fig4_HTML.jpg
                Figure 4

                Maximum clade credibility (MCC) tree from Bayesian coalescent analysis based on a subset of the P gene sequences in this study. The estimated TMRCA for this dataset and its 95% HPD values are indicated. Isolate names are given according to Table 1. Horizontal branches are drawn to a scale of estimated year of divergence, with tip times reflecting sampling date (year). Posterior probability values for all of the major nodes are shown. See materials and methods for details.

                Discussion and conclusion

                The N gene (the most conserved and abundant mRNA in infected cells) and G gene (plays a crucial role in viral neurotropism and pathogenicity) have been widely targeted for genetic, molecular epidemiology and evolutionary analysis of RABVs [4, 2528]. In contrast, for the P gene, only a few laboratory [29, 30] and wild-type RABV strains [31], an ABLV isolate [32] and Mokola virus [33] have been genetically characterized. In this study we attempted to characterize the genetic and evolutionary properties of the P gene of Chinese street RABVs. 77 P genes from brain samples of dogs and wild animals in eight provinces through 2003 to 2008 were sequenced and subjected to molecular and phylogenetic analysis.

                Several substitutions were found in the Chinese RABVs strains compared to the laboratory adapted and vaccine strains. The nucleotide (≥ 80.2%) and amino acid sequence identities (≥ 85.2) of the P gene were lower than the corresponding values for the N (≥87.6% and 95.4%) and G gene (≥87% and 93.8%) [26, 28]. Consistent with the wild type RABVs strains isolated in North America [31], two conserved (CD1, 2) and two variable (VD1, 2) domains were identified in Chinese RABVs. The observed substitutions are mainly located in the middle of P, while the N and C terminal are relatively well conserved. As reported previously, the need to retain overall negative charge rather than primary sequence would explain the VD1 region’s high level of diversity [6]. The poorly conserved VD2 might indicate a function as a spacer/hinge segment analogous to the hinge region of the P gene in Vesicular stomatitis virus (VSV) located between two functionally important domains [34]. Two sequence motifs, one believed to confer binding to the cytoplasmic dynein light chain LC8, and a lysine-rich sequence probably contributing to N protein binding [14], were conserved throughout Chinese RABVs samples, while the STAT-1 binding sites [20], internal translation initiation sites and phosphate acceptor sites showed different degrees of variation. Whether these variations could affect the biological characteristics of Chinese RABVs need to be further investigated.

                There have been several previous estimates of RABVs substitution rates for the G gene (1.2-6.5 x10-4 substitutions per site per year) and the N gene (1.1-5.6 x10-4 substitutions per site per year) based on dog, fox and mongoose RABVs samples collected worldwide [25, 27, 3538]. In this study, Bayesian coalescent analysis showed that mean substitution rate of the P gene for the Chinese RABVs isolates is 3.305×10-4 substitutions per site per year, which indicates that the genome RNA of RABVs circulating worldwide is stable. The TMRCA of cosmopolitan canine RABV variants has previously been estimated to be between 284 and 504 years ago [39]. The mean divergence time estimated based on the the G gene is 583 years ago for RABVs circulating globally [25, 35], and 596 years ago for RABVs for current Chinese RABVs [27]. Using a similar analysis, we estimated the average TMRCA of RABVs circulating in China based on the P gene to be 592 years ago, which was in accordance with previous reports for RABVs.

                Previous phylogenetic studies based on the G and N genes [26, 28, 39, 40] showed that RABVs in China can be classified into distinct clades or groups. The phylogenetic analysis in this report based on the P gene revealed that Chinese RABVs could be divided into two distinct clades, and that isolates from more than one clade RABV variants are currently co-circulating in the same Chinese provinces. Also, RABVs in Clades I are grouped with RABVs from Thailand, and RABVs in clade II are grouped with RABVs from India. The topology of the phylogenetic tree based on the P gene is similar to the G and N gene trees [26, 28, 39, 40]. This indicates that the P, G and N genes are equally valid for examining the phylogenetics of RABVs and is consistent with observations that the N, P, M, G and L genes of RABVs interact and evolve in a cooperative manner to effect virus infection and evolution [41, 42].

                Methods

                Viral specimens sampling

                Brain specimens were collected as part of a national surveillance program from dogs used as meat in restaurants and from suspected rabid Ferret badgers from eight provinces (Anhui, Guangxi, Guizhou, Hunan, Jiangsu, Shandong, Shanghai and Zhejiang) in China from 2003 to 2008 (Figure 1).

                Detection and sequencing of RABV

                All specimens were examined by using a direct immune fluorescence assay (DFA) [26] with a fluorescent-labeled monoclonal antibody against the RABV N protein (Rabies DFA Reagent; Chemicon Europe Ltd., Chandlers Ford, UK). For all identified RABV specimens, RNA was extracted from tissue of rabies-infected brains (0.1 g) with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and used as template for cDNA synthesis with Ready-To-Go You-Prime First-Strand Beads (Amersham Pharmacia Bioscience, Chalfont St. Giles, UK) and a rabies P gene specific primer: Pfor 5’-GAACCATCCCAAAYATG AG -3’ (corresponding to bases 1500–1519 of the positive sense genome sequence of the PV strain). The ORF sequence of the P gene, encoding regions corresponding to bases 1514 to 2407 of the total genetic sequence of the PV strain, was amplified with primers Pfor and Prev 5’- CTATCTTGCGCAGAAARTTCAT -3’ (corresponding to bases 2496 to 2517 of the positive sense genome sequence of the PV strain). PCR products were purified by using the QIAquick PCR Purification Kit (QIAGEN Ltd., Crawley,UK) and sequenced with an ABI PRISM 3100 DNA sequencer (Applied Biosystems, Foster City, CA, USA).

                Sequence alignment and phylogenetic analysis

                P gene sequences of lyssaviruses deposited in GenBank were downloaded and combined with the newly sequenced samples to form the dataset used in this study. Alignment of nucleotide sequences and deduced amino acid sequence were performed by using the ClustalX program, version 2.1 [43]. Genetic identities were determined using the Bio-Edit program [44] and MegAlign software version 5 (DNAStar, Inc., Madison,WI, USA). Phylogenetic and evolutionary analyses were conducted using Mega 3.1 [45]. Neighbor-joining (NJ) phylogenetic trees were constructed using evolutionary distance correction statistics [46, 47]. Bootstrap analysis was performed using 1000 replications and values greater than 70% were regarded as strong evidence for particular phylogenetic groupings.

                Bayesian Markov chain Monte Carlo (MCMC) evolutionary analysis

                Evolutionary history, including evolutionary rates of populations (nucleotide substitutions per site per year) and TMRCA (the most recent common ancestor) were inferred by using the Bayesian Markov chain Monte Carlo (MCMC) method available in the BEAST software package (http://​beast.​bio.​ed.​ac.​uk/​Main_​Page)[48]. Briefly, an input file for BEAST was generated by using the BEAUti program with sequences dated according to the year of isolation. Sequences with homology greater than 98% were removed from the analysis using TCOFFEE. The best-fit model of nucleotide substitution for Bayesian analysis was selected with Modeltest 3.7 [49]. The general time reversible (GTR) substitution model, incorporating a proportion of invariable sites (I) and a gamma distribution of rate variation among sites (C4) was used for the BEAST analysis. Both strict and relaxed (uncorrelated exponential and lognormal) molecular clocks [50] were considered to explore the extent of variation in the rate of nucleotide substitution. The BEAST output was assessed using the TRACER program. The maximum clade credibility (MCC) tree was generated using Figtree (available from http://​beast.​bio.​ed.​ac.​uk).

                Authors' information

                Dr. Lihua Wang, Ph.D., is an associate professor at the State Key Laboratory for Infectious Disease Prevention and Control, the Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention. His current research focuses on molecular epidemiology of Rabies virus, development reverse genetic system of rabies virus and basic research related to rabies.

                Declarations

                Acknowledgements

                We thank the staffs of the provincial CDCs (Anhui, Guangxi, Guizhou, Hunan, Jiangsu, Shandong, Shanghai and Zhejiang) for helping with field investigations and sample collection.

                This work was supported by the National Department Public Benefit Research Foundation (200803014), Major Program of National Natural Science Foundation of China (30630049), Key Technologies Research and Development Program of China (2009ZX10004-705) and Grant from NIID (National Institute of Infectious Diseases, Japan).

                Authors’ Affiliations

                (1)
                State Key Laboratory for Infectious Disease Prevention and Control, Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention
                (2)
                State Key Laboratory for Virology, Wuhan Institute of Virology, Chinese Academy of Sciences

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