Arch Virol DOI 10.1007/s00705-014-2044-0
ORIGINAL ARTICLE
Molecular characterization of three ferret badger (Melogale moschata) rabies virus isolates from Jiangxi province, China Jinghui Zhao • Ye Liu • Shoufeng Zhang Fei Zhang • Ying Wang • Lijuan Mi • Shuchao Wang • Rongliang Hu
•
Received: 24 October 2013 / Accepted: 28 February 2014 Ó Springer-Verlag Wien 2014
Abstract Ferret badger (FB) rabies viruses JX09-17(fb), JX09-18 and JX10-37 were isolated from three different regions in Jiangxi province, China, in 2009 and 2010. The complete nucleotide sequence identity between these three isolates was 87–93 %. Compared with the other Chinese rabies virus isolates and vaccine strains, 101 substitutions (53 in JX10-37, 23 in JX09-17(fb) and 25 in JX09-18) in the five structural proteins were observed, and 47 of these substitutions (27 in JX10-37, 14 in JX09-17(fb) and 6 in JX09-18) were unique among lyssaviruses. Amino acid substitutions of S231 and Q333 were noted respectively in the G protein antigenic site I of JX10-37 and site III in JX09-17(fb). Phylogenetic analysis showed that JX09-17(fb) is rooted within the China I lineage, JX09-18 is in China II, and JX10-37 is independent. Evolutionary analysis and comparative sequence data indicate that isolate JX10-37 is a variant virus that diverged from canine rabies viruses around 1933 (range 1886–1963). Introduction Rabies virus (RV), the prototype virus of the family Rhabdoviridae, genus Lyssavirus, has a negative-sense, J. Zhao, Y. Liu and S. Zhang contributed equally to this article.
Electronic supplementary material The online version of this article (doi:10.1007/s00705-014-2044-0) contains supplementary material, which is available to authorized users. J. Zhao Y. Liu S. Zhang F. Zhang Y. Wang L. Mi S. Wang R. Hu (&) Laboratory of Epidemiology and Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Military Veterinary Research Institute, Academy of Military Medical Sciences, 666 Liuying West Road, Changchun 130122, People’s Republic of China e-mail: [email protected]
single-strand RNA genome, composed of approximately 12,000 nucleotides (nt), encoding five structural proteins in the order 30 -N-P-M-G-L-50 [1]. Rabies is widely endemic in China, and dogs are the major reservoir and transmitters of the virus to humans and other animals. Ferret badgers (FB, Melogale moschata)-associated human rabies cases first emerged in China in 1994 [2]. RABVs from FB have also been isolated in southeastern China, and recently in Taiwan province [3]. Phylogenetic analysis of the isolates has suggested that rabies in FB developed as an independent enzootic circle of rabies infestation in these regions [4, 5]. Although rabies in FB is an increasing public health threat in southeastern China, only a few strains have been completely sequenced and analyzed [6]. For instance, JX09-17(fb), isolated in Fuzhou, Jiangxi province, is closely related to some Chinese isolates from dog epidemics and is genetically distinct from other ferret badger isolates in Zhejiang and Jiangxi provinces [6, 7]. Of note is that it contains an R333Q substitution within its glycoprotein antigenic site III [7]. Amino acids R333 or K333 of the RV glycoprotein have been reported to be necessary for virulence in adult mice [8, 9]. However, others have found that RV pathogenicity is not absolutely dependent on R or K at position 333 [10–14]. Here, we describe the full-length sequencing and phylogenetic analysis of three isolates of FB recovered from different regions of Jiangxi province.
Materials and methods JX09-17(fb) (GenBank no. KC762941) was isolated from the brain of a ferret badger captured in Fuzhou; JX09-18 (GenBank no. KF726852) in Jingdezhen, in February 2009 [7] and JX10-37 (GenBank no. KF726853) came from
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Xiushui, in April 2010 (Fig. 1). All of these isolates were from Jiangxi province. The viruses were isolated using the mouse inoculation test [15] and had incubation periods of 7–14 days. Total RNA from infected mouse brains was extracted with TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Reverse transcription polymerase chain reaction (RT-PCR) was performed with primer sets designed based on the isolate BD06 (GenBank no. EU549783) (Table S3), and the viral cDNA was amplified. Following denaturation at 95 °C for 3 min, the reactions were cycled 30 times at 95 °C for 30 s, 52 °C for 30 s, and 72 °C for 100 s, with a final extension at 72 °C for 7 min. After electrophoresis, the PCR products were purified and cloned into the pMD18-T vector (TaKaRa, Dalian, China). The transformed colonies were screened using ampicillin as a resistance marker, and selected positively identified clones were sequenced at least twice in both directions (Nanjing Genscript Biological Technology Co., Ltd., China). Similarity scores and percentage identities were determined using DNAStar analytical software (DNASTAR, Madison, Wisconsin USA). Multiple alignments of the complete genome sequences were performed using the computer program CLUSTAL W [16]. Neighbor-joining (NJ) analysis was performed using MEGA 4.0 [17]. Bootstrap support was estimated for 1000 replicates. The sequences of other RABVs were retrieved from GenBank (Table S2). Rates of nucleotide substitution and FB RABV divergence time were calculated by evolutionary analysis using complete N gene sequences. A maximum clade Fig. 1 Locations where JX0917(fb), JX09-18 and JX10-37 viruses were isolated and the background of rabies epidemic status in different provinces or areas in mainland China in 2009. , Fuzhou (JX09-17(fb)); `, Jingdezhen (JX09-18); ´, Xiushui (JX10-37)
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credibility (MCC) phylogenetic tree was inferred by the Bayesian Markov chain Monte Carlo (MCMC) method available in the BEAST package [18]. For this analysis, a relaxed (uncorrelated lognormal) molecular clock and GTR?I?C4 model of nucleotide substitution were selected according to the jModelTest analysis with further settings left at default values [19]. All chains were run for 90 million generations to ensure sufficient mixing, with 10 % removed as burn-in. An effective sample size (ESS) [200 was obtained for all estimated parameters.
Results and discussion All three FB RABV isolates, JX09-17(fb), JX09-18 and JX10-37, originated from Jiangxi province, but each of them was from a distinct geographical location in the Poyang Lake region (Fig. 1) and were from Fuzhou, Jingdezhen and Xiushui, respectively. Poyang Lake, once the largest freshwater lake in China, was the natural barrier between Jiujiang, Fuzhou and Xiushui. Jiangxi province has six neighboring provinces (Zhejiang, Anhui, Hubei, Hunan, Guangdong and Fujian), and FBs are widely distributed in this area. Additionally, the majority of human cases are located in this area (Fig. 1). Hunan and Guangdong provinces are classified as high-incidence regions, with 150–350 cases. Hubei province is a medium-incidence region, with 100–150 cases (Fig. 1). The complete genome sequences of the three isolates (JX09-17(fb), JX09-18 and JX10-37) were compared with
Ferret badger rabies virus from Jiangxi province, China Table 1 Nucleotide and deduced amino acid sequence identities of the FB rabies virus isolate JX10-37 to all five genes of Chinese street virus isolates, vaccine strains and other lyssaviruses
JX10-37 gene
nt (%)
JX10-37 proteins
aa (%) Chinese isolates
Vaccine strains
Lyssaviruses
Chinese isolates
Vaccine strains
Lyssaviruses
N
84.3-94.9
85.4-86.9
69.5-76.9
N
91.8-98.0
96.2-98.0
75.6-90.7
P
81.8-93.2
81.2-86.5
50.8-70.7
P
85.9-97.0
85.2-92.3
41.5-70.1
M
83.4-94.1
84.1-89.8
66.7-76.5
M
90.6-97.5
90.6-96.1
70.0-85.2
G
82.0-93.8
82.4-86.0
53.5-68.8
G
85.3-96.4
89.0-92.0
47.1-77.7
L
84.5-94.2
84.7-87.9
65.8-74.7
L
95.4-98.5
95.4-97.6
71.4-88.6
37 full genome sequences of vaccine strains and street isolates from various regions of China available in the GenBank database (Fig. 2). Phylogenetic analysis showed that the three FB isolates belonged to lyssavirus genotype 1, with JX09-17(fb) clustering with members of the China I lineage and closely related to other Chinese street isolates (D01, WH11, FJ009, DRV-AH08, BD06) [20, 21]. JX0918 clustered with members of the China II lineage and was closely related to the isolates JX08-45, Chinese vaccine strain CTN181, F02, F04, HN10 and GD-SH-01. The other four isolates were recovered from the neighboring provinces of Zhejiang, Hunan and Guangdong. JX10-37 also formed an independent lineage, but did not cluster with any other rabies viruses from FBs. A tree based on comparisons of individual ORF nucleotide and amino acid sequences provided the same results (data not shown). The whole genomic lengths of FB rabies virus isolates JX09-17(fb) (Fuzhou, 2009), JX09-18 (Jingdezhen, 2009) and JX10-37 (Xiushui, 2010) were 11,923, 11,922 and 11,924 nucleotides, respectively, with organizations similar to previously sequenced rabies virus genomes (Table S1, Fig. 1). At the whole-genome level, strain JX10-37 had 93 % and 87 % nucleotide sequence identity to JX0917(fb) and JX09-18, respectively. JX09-17(fb) had 87 % nucleotide sequence identity to JX09-18. Comparisons of the five protein-coding region sequences of the three FB isolates with other RABV street isolates, the vaccine strains, and other lyssaviruses were performed (Table 1). The degree of conservation of nucleotide (and deduced amino acid) sequences within individual open reading frames (ORFs) among the lyssaviruses, in decreasing order, was N[M[L[G[P (and N[L[M[G[P). Compared with the vaccine strains, the nucleotide and amino acid identities of JX10-37 ranged from 82 to 90 % and 85 to 98 %, so this variant can be covered and protected against by the current conventional vaccines used in China, such as strains PV, Flury and SAD. Comparing all five viral structural proteins with those of Chinese rabies virus isolates and vaccine strains, 101 amino acid substitutions (53 in JX10-37, 23 in JX0917(fb), and 25 in JX09-18) were observed, 47 of which (27 in JX10-37, 14 in JX09-17(fb), and 6 in JX09-18) are
unique among lyssaviruses (Table 2). Although the evolution of viruses during transmission and adaptation to new hosts is still not well understood, the greater the genetic variation, the more likely such adaptation is to occur [22, 23]. For RNA viruses such as rabies virus, selective pressures such as growth in a new host species will favor the emergence of mutants with greater efficiency of infection and transmission [24]. Although the N protein is generally highly conserved among lyssaviruses, four substitutions (D110A, K112R, M411I and Q432P) in all three isolates were unique among lyssaviruses (Fig. S1). The M411I substitution is located in a B-cell-specific epitope (aa 404–418) and Th site (aa 410–413) [25], which is conserved in RABV, ABLV, DUVV, EBLV, Aravan and Khujand, but not LBV and MOKV. An L379A substitution was found in another B-cell-specific epitope (aa 369–383) within the amino acid stretch 373-395, which has been identified as an important T-cell epitope in mice and humans [26]. Substitutions A371E in JX09-17(fb), A371T in JX10-37, and L374S in JX09-18 are located in a B-cell epitope (aa 369–383) [25]. An A332T change in JX09-18 were found in the RNAbinding domain (position 298-352) and antigenic site III (position 313–337). The LC8-binding domain of the P protein contains the sequence KSTQT (position 144–148), which is involved in axoplasmic transport of the viral nucleocapsid [27–29]. All aligned Chinese isolates and vaccine strains have the KSTQT motif, except for JX09-18, which has the sequence RSTQT. The G protein is recognized to play an important role in viral pathogenicity and is capable of eliciting neutralizing antibodies. The main antigenic sites (I–IV and ‘‘a’’) of G are responsible for virus attachment to cells and host cell receptor recognition [30, 31]. The amino acid L231 of antigenic site I (position 226–231) is conserved in all previously reported Chinese isolates and vaccine strains (Fig. S2). However, strain JX10-37 contains an S231 substitution, which has not been found previously in rabies viruses. It has been confirmed that antigenic site I is important for neutralization and contains both linear and conformational epitopes [32]. An L231P substitution has
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A
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D
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S E
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110
112
181
332
368
371
374
379
411
432
67 74
100
130
141
144
157
172
173
177
192
247
281
19
50
70
155 195
(-17)
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Table 2 Amino acid substitutions in FB isolates with respect to the Chinese vaccine strain CTN181
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J. Zhao et al.
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478
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Table 2 continued
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Ferret badger rabies virus from Jiangxi province, China
123
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CTN181
L
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R
G
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N
S
Q
D
M
D F
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AA position
1228
1265
1363
1374
1375
1427
1555
1558
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1593
1620
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1754 1829
1844
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A ‘dot (.)’ indicates a match with the corresponding amino acid in CTN181. Amino acid substitutions unique among lyssaviruses are shown in bold italics
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JX10-37
Isolates from this study are underlined
Protein
Table 2 continued
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DRV
J. Zhao et al.
Ferret badger rabies virus from Jiangxi province, China
been reported in two street rabies virus isolates, SHBRV18 and H-08-1320 [33]. The changes in the G protein found in JX10-37, T96, L162, A423, M470, P471 and L482, have not been reported in other lyssaviruses. In JX09-17(fb), an amino acid mutation of R333Q was observed in G protein antigenic site III – a rare substitution seen otherwise only in one recent isolate from a non-hematophagous Brazilian bat [7, 34]. The L protein contains six conserved domains (I-VI), and some of these have been characterized as functional motifs [35]. Compared with other lyssaviruses, the L protein is the most conserved, but 15 unique aa substitutions were found in JX10-37, and 10 of these changes occurred outside the conserved domains (Fig. S3). Five substitutions were observed in conserved domains, three of which are located in domain I, one in domain II, and one in domain V. The mean rate of nucleotide substitution estimated for the N gene was 4.6 9 10-4 substitutions/site/year (95 % highest posterior density [HPD] values 1.8–7.5 9 10-4
substitutions/site/year). Approximately 30 years ago (95 % HPD 11.7–51.0 years) — i.e., ca. 1980 (95 % HPD range 1959–1998)—JX09-17(fb) diverged from canine rabies virus. Approximately 77 years ago (95% HPD 29.0–136.9 years) — i.e., ca. 1933 (95 % HPD range 1873–1981) — strain JX10-37 diverged from canine rabies virus in China (Fig. S4). The TMRCA (time to most recent common ancestor) of global RABVs has been estimated at 749 years based on N gene data (95 % highest posterior density [HPD] 363-1215) and 583 years based on the G gene (95 % HPD 222-1116) [36]. The TMRCA of Chinese rabies viruses was estimated to be 596 years (95 % HPD, 272–1002 years) [37] and 354 years (95 % HPD, 196-494) [38] based on the G gene. In this study, we estimate the average TMRCA of RABVs circulating in China based on the N gene to be 343 years (95 % HPD, 190–511 years), which is consistent with the timescale presented by Ming et al. [37]. Rates of nucleotide substitutions of Chinese rabies viruses have been calculated to be 2.3 9 10-4 (N gene),
Fig. 2 Neighbor-joining phylogenetic tree of the wholegenome sequences of the three ferret badger isolates (red circles) and other Chinese rabies viruses, using Mega v 4.0 software. The numbers below the branches are bootstrap values (%) for 1000 replicates. Mokola virus was used as an outgroup
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3.24 9 10-4 (G gene) [38], 3.9 9 10-4 (G gene) [36], and 3.96 9 10-4 (G gene) [37]. Using a similar formula, Bayesian coalescent analysis showed that the mean substitution rate of the N gene of these viruses and some from eastern Asia is 4.6 9 10-4 substitutions per site per year, which demonstrates the stability of the RABV genome over this entire region. We have significant evidence that FB rabies virus exhibits genetic diversity and independent infection cycles [5]. However, support for this hypothesis clearly requires a larger sample of FB RABV sequences representing a wider range of geographical localities over a longer time period. Previous studies on complete genomic analysis of wild RABV have shown that a dog rabies variant jumped to palm civets in Sri Lanka [24], and from domestic dogs to hoary foxes in Brazil [39]. There have been no isolations of rabies virus from wild animals in China except for the FB RABVs, a bat (IRKV-THChina12) virus [40], and a mouse (MRV) virus [41]. An epidemiological survey of yellow weasel in Jiangxi province provided no indication of the spread of rabies virus in these animals [42], and the existence of rabies virus in other wildlife in China remains unknown. In conclusion, our study focused on the Poyang Lake regions suggests that rabies virus if there is no intervention, may be transmitted in the FB population, and subsequently to humans and dogs. The actual role of FB rabies in public health remains to be further addressed. Acknowledgments This investigation was funded by the National Natural Science Foundation of China (30630049, 30972199) and the China National ‘‘973’’ Program (2005CB52300, 2011CB500705). Conflict of interest peting interests.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
The authors declare that they have no com17.
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ORIGINAL ARTICLE
Molecular characterization of three ferret badger (Melogale moschata) rabies virus isolates from Jiangxi province, China Jinghui Zhao • Ye Liu • Shoufeng Zhang Fei Zhang • Ying Wang • Lijuan Mi • Shuchao Wang • Rongliang Hu
•
Received: 24 October 2013 / Accepted: 28 February 2014 Ó Springer-Verlag Wien 2014
Abstract Ferret badger (FB) rabies viruses JX09-17(fb), JX09-18 and JX10-37 were isolated from three different regions in Jiangxi province, China, in 2009 and 2010. The complete nucleotide sequence identity between these three isolates was 87–93 %. Compared with the other Chinese rabies virus isolates and vaccine strains, 101 substitutions (53 in JX10-37, 23 in JX09-17(fb) and 25 in JX09-18) in the five structural proteins were observed, and 47 of these substitutions (27 in JX10-37, 14 in JX09-17(fb) and 6 in JX09-18) were unique among lyssaviruses. Amino acid substitutions of S231 and Q333 were noted respectively in the G protein antigenic site I of JX10-37 and site III in JX09-17(fb). Phylogenetic analysis showed that JX09-17(fb) is rooted within the China I lineage, JX09-18 is in China II, and JX10-37 is independent. Evolutionary analysis and comparative sequence data indicate that isolate JX10-37 is a variant virus that diverged from canine rabies viruses around 1933 (range 1886–1963). Introduction Rabies virus (RV), the prototype virus of the family Rhabdoviridae, genus Lyssavirus, has a negative-sense, J. Zhao, Y. Liu and S. Zhang contributed equally to this article.
Electronic supplementary material The online version of this article (doi:10.1007/s00705-014-2044-0) contains supplementary material, which is available to authorized users. J. Zhao Y. Liu S. Zhang F. Zhang Y. Wang L. Mi S. Wang R. Hu (&) Laboratory of Epidemiology and Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Military Veterinary Research Institute, Academy of Military Medical Sciences, 666 Liuying West Road, Changchun 130122, People’s Republic of China e-mail: [email protected]
single-strand RNA genome, composed of approximately 12,000 nucleotides (nt), encoding five structural proteins in the order 30 -N-P-M-G-L-50 [1]. Rabies is widely endemic in China, and dogs are the major reservoir and transmitters of the virus to humans and other animals. Ferret badgers (FB, Melogale moschata)-associated human rabies cases first emerged in China in 1994 [2]. RABVs from FB have also been isolated in southeastern China, and recently in Taiwan province [3]. Phylogenetic analysis of the isolates has suggested that rabies in FB developed as an independent enzootic circle of rabies infestation in these regions [4, 5]. Although rabies in FB is an increasing public health threat in southeastern China, only a few strains have been completely sequenced and analyzed [6]. For instance, JX09-17(fb), isolated in Fuzhou, Jiangxi province, is closely related to some Chinese isolates from dog epidemics and is genetically distinct from other ferret badger isolates in Zhejiang and Jiangxi provinces [6, 7]. Of note is that it contains an R333Q substitution within its glycoprotein antigenic site III [7]. Amino acids R333 or K333 of the RV glycoprotein have been reported to be necessary for virulence in adult mice [8, 9]. However, others have found that RV pathogenicity is not absolutely dependent on R or K at position 333 [10–14]. Here, we describe the full-length sequencing and phylogenetic analysis of three isolates of FB recovered from different regions of Jiangxi province.
Materials and methods JX09-17(fb) (GenBank no. KC762941) was isolated from the brain of a ferret badger captured in Fuzhou; JX09-18 (GenBank no. KF726852) in Jingdezhen, in February 2009 [7] and JX10-37 (GenBank no. KF726853) came from
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Xiushui, in April 2010 (Fig. 1). All of these isolates were from Jiangxi province. The viruses were isolated using the mouse inoculation test [15] and had incubation periods of 7–14 days. Total RNA from infected mouse brains was extracted with TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Reverse transcription polymerase chain reaction (RT-PCR) was performed with primer sets designed based on the isolate BD06 (GenBank no. EU549783) (Table S3), and the viral cDNA was amplified. Following denaturation at 95 °C for 3 min, the reactions were cycled 30 times at 95 °C for 30 s, 52 °C for 30 s, and 72 °C for 100 s, with a final extension at 72 °C for 7 min. After electrophoresis, the PCR products were purified and cloned into the pMD18-T vector (TaKaRa, Dalian, China). The transformed colonies were screened using ampicillin as a resistance marker, and selected positively identified clones were sequenced at least twice in both directions (Nanjing Genscript Biological Technology Co., Ltd., China). Similarity scores and percentage identities were determined using DNAStar analytical software (DNASTAR, Madison, Wisconsin USA). Multiple alignments of the complete genome sequences were performed using the computer program CLUSTAL W [16]. Neighbor-joining (NJ) analysis was performed using MEGA 4.0 [17]. Bootstrap support was estimated for 1000 replicates. The sequences of other RABVs were retrieved from GenBank (Table S2). Rates of nucleotide substitution and FB RABV divergence time were calculated by evolutionary analysis using complete N gene sequences. A maximum clade Fig. 1 Locations where JX0917(fb), JX09-18 and JX10-37 viruses were isolated and the background of rabies epidemic status in different provinces or areas in mainland China in 2009. , Fuzhou (JX09-17(fb)); `, Jingdezhen (JX09-18); ´, Xiushui (JX10-37)
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credibility (MCC) phylogenetic tree was inferred by the Bayesian Markov chain Monte Carlo (MCMC) method available in the BEAST package [18]. For this analysis, a relaxed (uncorrelated lognormal) molecular clock and GTR?I?C4 model of nucleotide substitution were selected according to the jModelTest analysis with further settings left at default values [19]. All chains were run for 90 million generations to ensure sufficient mixing, with 10 % removed as burn-in. An effective sample size (ESS) [200 was obtained for all estimated parameters.
Results and discussion All three FB RABV isolates, JX09-17(fb), JX09-18 and JX10-37, originated from Jiangxi province, but each of them was from a distinct geographical location in the Poyang Lake region (Fig. 1) and were from Fuzhou, Jingdezhen and Xiushui, respectively. Poyang Lake, once the largest freshwater lake in China, was the natural barrier between Jiujiang, Fuzhou and Xiushui. Jiangxi province has six neighboring provinces (Zhejiang, Anhui, Hubei, Hunan, Guangdong and Fujian), and FBs are widely distributed in this area. Additionally, the majority of human cases are located in this area (Fig. 1). Hunan and Guangdong provinces are classified as high-incidence regions, with 150–350 cases. Hubei province is a medium-incidence region, with 100–150 cases (Fig. 1). The complete genome sequences of the three isolates (JX09-17(fb), JX09-18 and JX10-37) were compared with
Ferret badger rabies virus from Jiangxi province, China Table 1 Nucleotide and deduced amino acid sequence identities of the FB rabies virus isolate JX10-37 to all five genes of Chinese street virus isolates, vaccine strains and other lyssaviruses
JX10-37 gene
nt (%)
JX10-37 proteins
aa (%) Chinese isolates
Vaccine strains
Lyssaviruses
Chinese isolates
Vaccine strains
Lyssaviruses
N
84.3-94.9
85.4-86.9
69.5-76.9
N
91.8-98.0
96.2-98.0
75.6-90.7
P
81.8-93.2
81.2-86.5
50.8-70.7
P
85.9-97.0
85.2-92.3
41.5-70.1
M
83.4-94.1
84.1-89.8
66.7-76.5
M
90.6-97.5
90.6-96.1
70.0-85.2
G
82.0-93.8
82.4-86.0
53.5-68.8
G
85.3-96.4
89.0-92.0
47.1-77.7
L
84.5-94.2
84.7-87.9
65.8-74.7
L
95.4-98.5
95.4-97.6
71.4-88.6
37 full genome sequences of vaccine strains and street isolates from various regions of China available in the GenBank database (Fig. 2). Phylogenetic analysis showed that the three FB isolates belonged to lyssavirus genotype 1, with JX09-17(fb) clustering with members of the China I lineage and closely related to other Chinese street isolates (D01, WH11, FJ009, DRV-AH08, BD06) [20, 21]. JX0918 clustered with members of the China II lineage and was closely related to the isolates JX08-45, Chinese vaccine strain CTN181, F02, F04, HN10 and GD-SH-01. The other four isolates were recovered from the neighboring provinces of Zhejiang, Hunan and Guangdong. JX10-37 also formed an independent lineage, but did not cluster with any other rabies viruses from FBs. A tree based on comparisons of individual ORF nucleotide and amino acid sequences provided the same results (data not shown). The whole genomic lengths of FB rabies virus isolates JX09-17(fb) (Fuzhou, 2009), JX09-18 (Jingdezhen, 2009) and JX10-37 (Xiushui, 2010) were 11,923, 11,922 and 11,924 nucleotides, respectively, with organizations similar to previously sequenced rabies virus genomes (Table S1, Fig. 1). At the whole-genome level, strain JX10-37 had 93 % and 87 % nucleotide sequence identity to JX0917(fb) and JX09-18, respectively. JX09-17(fb) had 87 % nucleotide sequence identity to JX09-18. Comparisons of the five protein-coding region sequences of the three FB isolates with other RABV street isolates, the vaccine strains, and other lyssaviruses were performed (Table 1). The degree of conservation of nucleotide (and deduced amino acid) sequences within individual open reading frames (ORFs) among the lyssaviruses, in decreasing order, was N[M[L[G[P (and N[L[M[G[P). Compared with the vaccine strains, the nucleotide and amino acid identities of JX10-37 ranged from 82 to 90 % and 85 to 98 %, so this variant can be covered and protected against by the current conventional vaccines used in China, such as strains PV, Flury and SAD. Comparing all five viral structural proteins with those of Chinese rabies virus isolates and vaccine strains, 101 amino acid substitutions (53 in JX10-37, 23 in JX0917(fb), and 25 in JX09-18) were observed, 47 of which (27 in JX10-37, 14 in JX09-17(fb), and 6 in JX09-18) are
unique among lyssaviruses (Table 2). Although the evolution of viruses during transmission and adaptation to new hosts is still not well understood, the greater the genetic variation, the more likely such adaptation is to occur [22, 23]. For RNA viruses such as rabies virus, selective pressures such as growth in a new host species will favor the emergence of mutants with greater efficiency of infection and transmission [24]. Although the N protein is generally highly conserved among lyssaviruses, four substitutions (D110A, K112R, M411I and Q432P) in all three isolates were unique among lyssaviruses (Fig. S1). The M411I substitution is located in a B-cell-specific epitope (aa 404–418) and Th site (aa 410–413) [25], which is conserved in RABV, ABLV, DUVV, EBLV, Aravan and Khujand, but not LBV and MOKV. An L379A substitution was found in another B-cell-specific epitope (aa 369–383) within the amino acid stretch 373-395, which has been identified as an important T-cell epitope in mice and humans [26]. Substitutions A371E in JX09-17(fb), A371T in JX10-37, and L374S in JX09-18 are located in a B-cell epitope (aa 369–383) [25]. An A332T change in JX09-18 were found in the RNAbinding domain (position 298-352) and antigenic site III (position 313–337). The LC8-binding domain of the P protein contains the sequence KSTQT (position 144–148), which is involved in axoplasmic transport of the viral nucleocapsid [27–29]. All aligned Chinese isolates and vaccine strains have the KSTQT motif, except for JX09-18, which has the sequence RSTQT. The G protein is recognized to play an important role in viral pathogenicity and is capable of eliciting neutralizing antibodies. The main antigenic sites (I–IV and ‘‘a’’) of G are responsible for virus attachment to cells and host cell receptor recognition [30, 31]. The amino acid L231 of antigenic site I (position 226–231) is conserved in all previously reported Chinese isolates and vaccine strains (Fig. S2). However, strain JX10-37 contains an S231 substitution, which has not been found previously in rabies viruses. It has been confirmed that antigenic site I is important for neutralization and contains both linear and conformational epitopes [32]. An L231P substitution has
123
123
A
I
D
K
I
A
E
A
L
L
M
Q
S E
V
M
S
K
A
R
M
T
E
V
D
P
M
R
I K
P
A
L
3
22
110
112
181
332
368
371
374
379
411
432
67 74
100
130
141
144
157
172
173
177
192
247
281
19
50
70
155 195
(-17)
(-15)
(-14)
N
G
M
P
CTN181
AA position
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T
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L
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D
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F04
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FJ009
Table 2 Amino acid substitutions in FB isolates with respect to the Chinese vaccine strain CTN181
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D01
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BD06
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Flury-LEP-C
.
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478
Protein
Table 2 continued
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Ferret badger rabies virus from Jiangxi province, China
123
123
CTN181
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D F
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AA position
1228
1265
1363
1374
1375
1427
1555
1558
1562
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1620
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1844
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A ‘dot (.)’ indicates a match with the corresponding amino acid in CTN181. Amino acid substitutions unique among lyssaviruses are shown in bold italics
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.
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Isolates from this study are underlined
Protein
Table 2 continued
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J. Zhao et al.
Ferret badger rabies virus from Jiangxi province, China
been reported in two street rabies virus isolates, SHBRV18 and H-08-1320 [33]. The changes in the G protein found in JX10-37, T96, L162, A423, M470, P471 and L482, have not been reported in other lyssaviruses. In JX09-17(fb), an amino acid mutation of R333Q was observed in G protein antigenic site III – a rare substitution seen otherwise only in one recent isolate from a non-hematophagous Brazilian bat [7, 34]. The L protein contains six conserved domains (I-VI), and some of these have been characterized as functional motifs [35]. Compared with other lyssaviruses, the L protein is the most conserved, but 15 unique aa substitutions were found in JX10-37, and 10 of these changes occurred outside the conserved domains (Fig. S3). Five substitutions were observed in conserved domains, three of which are located in domain I, one in domain II, and one in domain V. The mean rate of nucleotide substitution estimated for the N gene was 4.6 9 10-4 substitutions/site/year (95 % highest posterior density [HPD] values 1.8–7.5 9 10-4
substitutions/site/year). Approximately 30 years ago (95 % HPD 11.7–51.0 years) — i.e., ca. 1980 (95 % HPD range 1959–1998)—JX09-17(fb) diverged from canine rabies virus. Approximately 77 years ago (95% HPD 29.0–136.9 years) — i.e., ca. 1933 (95 % HPD range 1873–1981) — strain JX10-37 diverged from canine rabies virus in China (Fig. S4). The TMRCA (time to most recent common ancestor) of global RABVs has been estimated at 749 years based on N gene data (95 % highest posterior density [HPD] 363-1215) and 583 years based on the G gene (95 % HPD 222-1116) [36]. The TMRCA of Chinese rabies viruses was estimated to be 596 years (95 % HPD, 272–1002 years) [37] and 354 years (95 % HPD, 196-494) [38] based on the G gene. In this study, we estimate the average TMRCA of RABVs circulating in China based on the N gene to be 343 years (95 % HPD, 190–511 years), which is consistent with the timescale presented by Ming et al. [37]. Rates of nucleotide substitutions of Chinese rabies viruses have been calculated to be 2.3 9 10-4 (N gene),
Fig. 2 Neighbor-joining phylogenetic tree of the wholegenome sequences of the three ferret badger isolates (red circles) and other Chinese rabies viruses, using Mega v 4.0 software. The numbers below the branches are bootstrap values (%) for 1000 replicates. Mokola virus was used as an outgroup
123
J. Zhao et al.
3.24 9 10-4 (G gene) [38], 3.9 9 10-4 (G gene) [36], and 3.96 9 10-4 (G gene) [37]. Using a similar formula, Bayesian coalescent analysis showed that the mean substitution rate of the N gene of these viruses and some from eastern Asia is 4.6 9 10-4 substitutions per site per year, which demonstrates the stability of the RABV genome over this entire region. We have significant evidence that FB rabies virus exhibits genetic diversity and independent infection cycles [5]. However, support for this hypothesis clearly requires a larger sample of FB RABV sequences representing a wider range of geographical localities over a longer time period. Previous studies on complete genomic analysis of wild RABV have shown that a dog rabies variant jumped to palm civets in Sri Lanka [24], and from domestic dogs to hoary foxes in Brazil [39]. There have been no isolations of rabies virus from wild animals in China except for the FB RABVs, a bat (IRKV-THChina12) virus [40], and a mouse (MRV) virus [41]. An epidemiological survey of yellow weasel in Jiangxi province provided no indication of the spread of rabies virus in these animals [42], and the existence of rabies virus in other wildlife in China remains unknown. In conclusion, our study focused on the Poyang Lake regions suggests that rabies virus if there is no intervention, may be transmitted in the FB population, and subsequently to humans and dogs. The actual role of FB rabies in public health remains to be further addressed. Acknowledgments This investigation was funded by the National Natural Science Foundation of China (30630049, 30972199) and the China National ‘‘973’’ Program (2005CB52300, 2011CB500705). Conflict of interest peting interests.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
The authors declare that they have no com17.
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