Genetic diversity and molecular evolution of the rabies virus matrix protein gene in China

Infection, Genetics and Evolution 16 (2013) 248–253

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Genetic diversity and molecular evolution of the rabies virus matrix protein gene in China Hui Wu a,1, Lihua Wang a,1, Xiaoyan Tao a, Hao Li a, Simon Rayner b, Guodong Liang a, Qing Tang a,⇑ a

State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 155 Changbai St., Changping Dist., Beijing 102206, China b State Key Laboratory for Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Hubei 430071, China

a r t i c l e

i n f o

Article history: Received 5 July 2012 Received in revised form 30 November 2012 Accepted 2 February 2013 Available online 26 February 2013 Keywords: Rabies virus M gene Genetic diversity Molecular evolution

a b s t r a c t To investigate the diversity of rabies virus (RABV) matrix protein (M) gene in the current Chinese rabies epidemic, we fully examined M gene of 63 street RABVs (Virus isolated from naturally infected animals), and performed phylogenetic and mutational analysis. Our results indicate that the Chinese RABV M gene is well conserved with 90.6% to 100% amino acid similarity. Analysis of the mutations indicates that the sequences can be divided into four groups with each group defined by distinct substitutions. The PPxY motif and residue E58, which are essential for efficient virus production and pathogenicity, were completely conserved. The estimated mean rate of nucleotide substitution was 4.6104 substitutions per site per year, and the estimated average time of the most recent common ancestor (TMRCA) was 265 years ago based on the M gene of Chinese street RABVs, which are similar to previously reported values for the glycoprotein (G) and nucleoprotein (N) gene. This indicates that the genomic RNA of RABVs circulating worldwide is stable; G, N and M genes are evolving at a similar rate. This study showed that although the Chinese RABV strains could be divided into distinct clades based on the phylogenetic analysis, their functional domains of M proteins were highly conserved. Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction Rabies is a fatal neurological disease that occurs on a global scale and which affects almost all mammals (WHO Rabies, 2010). According to the Global Alliance for Rabies Control (GARC), rabies causes at least 70,000 human deaths annually (http://www.rabiescontrol.net). Within China, rabies is endemic and remains an important public and animal health issue (Song et al., 2009; Tang et al., 2001; Tao et al., 2009). Worldwide, China is second only to India in terms of the number of rabies related mortalities (Ming et al., 2010; Meng et al., 2011). Domestic dogs act as the main reservoir (Tang et al., 2005) in China. The epidemic area has expanded to encompass the whole country with the exception of Qinghai province and Tibet municipality (Zhang et al., 2011). The classic rabies virus (RABV) is the etiological agent of rabies, which belongs to the genus Lyssavirus (family Rhabdoviridae) (Warrell and Warrell, 2004). The genomic RNA (approximately 12,000 bases) of RABV encodes five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and large protein (L) (Delmas et al., 2008). The M protein is the smallest and most abundant protein in the virion, forming a layer between

the protein G in the outer membrane and the ribonucleoprotein (RNP) core (Mebatsion et al., 1999). The M protein is a multi-functional protein essential for virus maturation and budding and also regulates the expression of viral and host proteins (Bieniasz, 2006; Finke and Conzelmann, 2003a). The protein binds to the RNP core, and is responsible for recruiting RNPs to the cell membrane, as well as the formation of tightly coiled ‘skeleton’-like structures necessary for the development of a bullet-shaped virus (Bieniasz, 2006). In addition, the M protein is involved in viral assembly and budding, regulation of the viral genome and mRNA syntheses (Finke et al., 2003b). Furthermore, the M protein acts as a major inducer of apoptosis in neuronal cells (Kassis et al., 2004) and has also been shown to be associated with virus pathogenicity (Faber et al., 2004; Kenta et al., 2007; Shimizu et al., 2006). In this report, we focus on Chinese street RABV M genes amplified from RABV positive brain samples of field captured animals, aiming to gain insight into the genetic variation and evolutionary characteristics of the RABV M genes in China. 2. Results 2.1. Molecular diversity of the Chinese RABVs M gene

⇑ Corresponding author. Tel./fax: +86 10 58900895. 1

E-mail address: [email protected] (Q. Tang). Hui Wu and Lihua Wang contributed equally to this paper.

1567-1348/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2013.02.009

The entire coding region (609 nucleotides) of the M gene was determined for 63 Chinese isolates (GenBank acc. No.

H. Wu et al. / Infection, Genetics and Evolution 16 (2013) 248–253 Table 2 Genetic distances between groups at the nucleotide and amino acid level. Group I Group Group Group Group

I II III IV

93.1% 92.5% 93.0%

Group II

Group III

Group IV

84.0%

85.2% 90.5%

91.6% 86.3% 88.2%

97.0% 95.2%

95.5%

Note: upper right, nucleotide identity; lower left, amino acid identity.

HM582456-HM582518). The host species, origin, year of identification and distribution of these viruses are summarized in Fig. 1 and Table 1 (Supplementary material). The isolates are from 28 cities in southern and southern east part of China, which covered the high, middle and low rabies incidence areas (Fig. 1 Supplementary material). Comparison of the 63 sequences showed 83.4–100% nucleotide similarity. Most of mutations among these Chinese RABVs are synonymous mutations (90.6% to 100% similarity at the amino acid level), indicating that the M proteins of these isolates are highly conserved. Using the PV vaccine strain (M13215) as a reference, twenty amino acid substitutions were identified that were dispersed amongst the isolates. According to the substitutions, the sequences could be classified into four distinct groups (Fig. 2, Table 2). The four groups fit the phylogenetic tree well (data

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not shown), and there is no specific geographical distribution except strains in Group I and Group IV. Group I defined by signature substitutions (particular substitutions compared to RABVs in other groups) at sites L44F, A100T, Y138H, M173Y, N174S, Q187K and R190M. Group I was mainly comprised of isolates from Shanghai and was grouped with strain aG (DQ490077), a Chinese rabies vaccine strain. Group II defined by specific substitutions at sites S20P, A177T, and Q187P, contained strains from Guangxi, Guizhou, and Shanghai provinces, and was grouped with strain CTN (FJ959397), another Chinese vaccine strain. Group III defined by specific substitutions at sites Q17H, S20F, and P21S and contained strains from Anhui, Zhejiang, Guangxi, Hunan, Shandong, and Shanghai provinces. Group IV contained a single isolate from Guangxi province, with a group specific substitution at site V106A (Fig. 2). Because no signature substitutions were found, RABV strain HN29 and GZ18 were defined as ungrouped. The hydropathic profiles of the M protein did not differ significantly among the Chinese RABV isolates (data not shown). The hydrophilic region at both ends of the gene is conserved in Chinese isolates in this study (Fig. 2). In the hydrophobic domain (residues 89–107) of the M protein, which is known to interact with the membrane lipids of host cells (Capone and Ghosh, 1984; Mita et al., 2008; Tordo et al., 1986), two substitutions (V95A and A100T) occurred in group I sequences, but were conserved in other isolates. No

Fig. 2. Alignment of M protein residues 1–202 for RABVs determined in this study and Chinese vaccine strains (PV, CTN, aG). I–IV represents group I–IV. Dots represent identity to PV strain. Solid underline shows the PPxY motif and hydrophobic domain. The signature substitutions for group I–IV were showed by red, blue, green, and pink, respectively.

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H. Wu et al. / Infection, Genetics and Evolution 16 (2013) 248–253

substitutions were observed in the proline-rich motif (PPxY motif) which is involved in viral budding and interacts with the WW domains of cellular components (Harty et al., 1999; Irie et al., 2004). The consensus sequence PPEY at residues 35–38 was found in all Chinese RABV isolates analyzed in this study. Position 58 in the M protein, which has been characterized as a critical site for viral RNA synthesis (Finke et al., 2003a), is conserved in Chinese RABV street strains analyzed in this study as well. 2.2. Phylogenetic analysis of RABVs in China based on M gene The 63 newly generated M gene sequences, together with corresponding sequences from other Chinese street rabies isolates were aligned and subjected to phylogenetic analysis. The maximum clade credibility (MCC) tree revealed that all the Chinese isolates were grouped within the classic RABV genotype 1(GT1) (Fig. 3). The topology of the MCC tree is similar to that of an earlier phylogenetic analysis based on both the G gene (Meng et al., 2007; Ming et al., 2010) and the N gene (Tao et al., 2009) with the strains forming three major sub-clades (designated clade I–III). Each of these clades was supported with posterior probability values >0.99. Most of the viruses detected from 2003 to 2008 belong to clade I, which is distributed throughout most of the major rabies endemic areas including: Anhui, Guangxi, Guizhou, Hunan, Shanghai, and Zhejiang provinces. Most of the RABV strains in clade I are from dogs, but strains from ferret badger were also placed within this clade.

Clade II contained strains from east (Shanghai and Jiangsu provinces), middle (Jiangxi and Henan provinces) and west (Guizhou and Guangxi provinces) of China, and were obtained from dogs, livestock (pig and cattle) and wild animals (wolf and ferret badger). Two strains from Thailand were grouped in Clade II as well and share a common recent ancestor with the Chinese isolates. Clade III comprises arctic-related variants from Asia, Americas, and Europe that were collected from terrestrial carnivores. Isolate NeiMeng925 was placed in the arctic-related branch and is clearly distinct from other Chinese isolates. Deer strain DRV, isolated in Jilin Province (in northeastern China), is closely related to Clade III.

2.3. Substitution rates and evolution history analysis of M gene The estimated mean rate of nucleotide substitution for the M gene of street Chinese street RABVs was 4.6  104 substitutions per site per year (95% HPD values, 2.5–6.6  104 substitutions per site per year). Bayesian coalescent analysis estimated the average time of the most recent common ancestor (TMRCA) is 265 years ago (95% HPD, 167–431 years) for Chinese street RABVs. The corresponding TMRCA estimates for clade I, clade II and clade III were 142 years (95% HPD, 81–209 years)), 141 years (95%HPD, 86–223 years)) and 181 years (95%HPD, 117–301 years)) respectively. The Skyline plots showed that the genetic diversity of Chinese RABVs M gene remained stable 150 years ago. Then it

Fig. 3. MCC tree and Bayesian skyline (below) constructed from Bayesian coalescent analysis based on the entire M gene sequences of RABVs. 44 sequences are from GenBank and 32 are sequences from this study. Sequences from this study are italic and in boldface. The estimated TMRCA for this dataset and its 95% HPD values are indicated. Isolate names are given according to Table 1 (Supplementary material). 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. Bayesian skyline plots showing the evolutionary and transmission histories of the 76 M gene sequences. The two blue dotted lines indicated the constant increase interval of rabies cases during 30–150 years ago and the corresponding divergent statues in the MCC tree.

H. Wu et al. / Infection, Genetics and Evolution 16 (2013) 248–253

experienced a constant increase during 30–150 years ago, and the diversity subsequently decreased in recent 30 years (Fig. 3).

3. Discussion In this study, we performed genetic diversity and evolutionary study of the M gene in Chinese street RABVs isolates. Although sequences are well conserved at the nucleotide and amino acid level, several amino acid substitution sites were identified in Chinese RABV variants which provide evidence of genetic evolution (Nadin-Davis et al., 1997), and may affect the antigenicity and pathogenicity of the strains. The highly variable region (17–22aa) of the Chinese RABV M gene is located within a region of potential antigenic sites (1– 77aa). Substitutions L44F, G85W, V95A, A100T, Y138H, M173Y, N174S, Q187K and R190M were only found in Shanghai RABV street strains isolated between 2003 and 2004. When compared with representative lysssaviruses of genotypes (GTs) 1–7, the same substitution at site A100T was found in Australian bat lyssavirus (GT7) and West Caucasian bat virus (unclassified Lyssavirus). In addition, substitution 187Q–K was found in Lagos bat virus (GT2), Mokola virus (GT3), Duvenhage virus (GT4), European bat lyssavirus1(GT5), European bat lyssavirus 2 (GT6), Australian bat lyssavirus (GT7), West Caucasian bat virus (unclassified Lyssavirus), Shimoni bat virus (unclassified Lyssavirus), Khujand lyssavirus, and Aravan lyssavirus. Both of these mutations cause significant changes in the physical properties of the amino acids present at these sites and whether these substitutions affect the biological characteristics of the Shanghai isolates needs to be investigated. It would also be interesting to further consider the relationship between these Shanghai strains and other lyssaviruses by examining the other genes. We further note that the substitution at site V95A in the hydrophobic domain (89–107aa) will also affect the hydrophobicity of this domain, and the subsequent ability of the M protein to induce a CPE and apoptosis in infected cells (Conzelmann, 2003a; Finke and Conzelmann, 2003a; Finke et al., 2003b). Nevertheless, the PPxY motif and residue E58, both of which are essential for efficient virus production, pathogenicity and regulation of virus transcription or replication (Wirblich et al., 2008; Finke et al., 2003a), are completely conserved in all Chinese street RABVs isolates. The PPxY conservation has been explored in RABV variants from Brazilian (Kobayashi et al., 2007), North America (Nadin-Davis et al., 1997), and China (Hirano et al., 2010), which are consistent with the suggestion that the primary structure of the M protein is important for the retention of viral structure and function (Kobayashi et al., 2007). The mean rate of nucleotide substitution for the M gene of street RABV isolates in China was 4.6  104 substitutions per site per year. The rate is strongly concordant with previous estimates of substitution rates based on the G (1.2–6.5  104 substitutions per site per year) and N (1.1–5.6  104 substitutions per site per year) gene in dog RABVs sampled worldwide (Bourhy et al., 2008) as well as estimates for fox RABVs in Europe and mongoose RABVs in Africa (Bourhy et al., 1999; Davis et al., 2007; Talbi et al., 2009). This indicates that the genomic RNA of RABVs circulating worldwide are stable, and the G, N and M genes are evolving at a similar rate. This finding is also consistent with an earlier report that the lyssavirus genes are likely equally valid for phylogenetic analyses (Wu et al., 2007). Previous research has estimated that the TMRCA of cosmopolitan canine RABV variants originated between 284 and 504 years ago (Badrane and Tordo, 2001). In the present study, the earliest common ancestor for the present RABV variants in China originated 265 years ago. These results are consistent with the

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divergence time of Chinese RABVs estimates based on the G gene (about 354 years ago) (Gong et al., 2010). However, RABVs is known to have existed in China for more than 2000 years (Wang and Huang, 2001) which raises the question as to why TMRCA estimates are much shorter. It has been proposed that this is because all analyses are based on RABV strains that emerged and gained dominance relatively recently, driving ancient strains towards extinction three to five hundred years ago. We found evidence to support this idea in a recent paper (Yu et al., 2012), which showed that the current epidemic is primarily composed of two major strains. One strain appears to be coincident with the previous epidemic in China that occurred in the 1960s and 1970s, and the second strain only became relevant in the recent epidemic and appears to be gaining dominance. Previous phylogenetic studies based on the G and N genes (Badrane and Tordo, 2001; Meng et al., 2007; Tao et al., 2009; Zhang et al., 2009) showed that RABVs in China can be classified into distinct clades or groups. In this study, the topological structure of the M gene MCC tree showed similar results, which is consistent with the previous study by Hirano et al. (Hirano et al., 2010). Three clades of RABVs were identified in the Chinese isolates, indicating that at least three distinct rabies variants are currently circulating in China. Viruses in clade I include most of the Chinese viruses, have been predominant during the past 10 years and thus appear largely responsible for the current rabies epidemic in China. The presence of the clades appears to vary according to province with some provinces containing strains from two or three clades: Guizhou (I, II), Guangxi (I, II, III), Hunan (I, II), Shanghai (I, II, III), Zhejiang (I, III) and Jiangsu (I, II). Many of these provinces have high incidences of rabies and it would be interesting to investigate whether there is a correlation between the number of cases and the presence of sequences from multiple clades. However, a larger sample set would probably be required to overcome sampling bias. In summary, the investigation of the genetic characteristics and evolution of the Chinese street RABVs M genes showed that there are several unique substitutions that exist in Chinese street RABVs, and the RABV M gene shares a similar substitution rate and evolutionary history with the G and N genes. As more samples become available, it will be possible to investigate the relationship between the genetic diversity of RABV M gene and the variation in the biological characteristics of RABV strains. 4. Materials and methods 4.1. Viral specimens As part of the trial rabies national surveillance program a total of 509 brain specimens were collected from dogs and wildlife (Ferret badger) from 2003 to 2008 in provinces which maintained high, middle and low incidence rate of rabies (Fig. 1. Supplementary material). For each specimen the year of collection together with location was recorded. The original specimens were used directly for RABV detection and M gene sequencing, (i.e. without passaging in mice or in vitro) to avoid altering the RNA genome. 4.2. Detection and Sequencing of RABV All specimens were examined by using a direct immune fluorescence assay (DFA) (Tao et al., 2009) 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 brain (0.1 g) with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and used as a template for cDNA synthesis with Ready-To-Go You-Prime First-Strand Beads (Amersham Pharmacia Bioscience, Chalfont St. Giles, UK) and a rabies M gene specific primer: Mfor

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50 -GCAACACCACTRAYAAAATGAA-30 (corresponding to bases 2479–2501 of the positive sense genome sequence of the PV vaccine strain (M13215)). The 640-nt sequence of the M gene, encoding regions corresponding to bases 2479–3119 of the total genetic sequence of the PV strain, was amplified with primers Mfor and Mrev 50 -CGGGATATA RTCTGAYYATTCTAG-30 (corresponding to bases 3095–3119 of the positive sense genome sequence of the PV strain). PCR products were purified by using a QIAquick PCR Purification Kit (QIAGEN Ltd., Crawley,UK) and sequenced with an ABI PRISM 3100 DNA sequencer (Applied Biosystems, Foster City, CA, USA). 4.3. Sequence alignment and identity analysis The deduced amino acid of 63 M gene sequences in this study were obtained by using BioEdit software (Ibis Biosciences, Carlsbad, CA, USA). The alignment of nucleotide sequences and amino acid sequences was performed by using the ClustalX program, version 2.1 (Thompson et al., 1997). MegAlign software version 5 (DNAStar, Inc., Madison,WI, USA) was used to analyze the nucleotide and deduced amino acid sequence identities. The group separation and substitutions in each group were manually inspected depending on the alignment results and phylogenetic relationship. The genetic distances between each groups were analyzed by using BioEdit software (Ibis Biosciences, Carlsbad, CA, USA), and the mean value was calculated by using average function in Microsoft Excel 2007. 4.4. Bayesian MCMC substitution and evolutionary analysis Additional M gene sequences of RABVs were downloaded from GenBank and a subset was selected based on the following criteria: (1) that the sequence spanned the whole M gene; (2) the full background information (isolation time/ host/location) was available. Finally, T-Coffee (Notredame et al., 2000) was used to identify samples with the greatest nucleotide diversity, and sequences with greater than 98% identify were removed. Totally, 76 M gene sequences (44 from GenBank, 32 from this study) were selected for evolutionary analysis. Evolutionary history, including evolutionary rates of populations (nucleotide substitutions per site per year) and TMRCA (Time of the Most Recent Common Ancestor) were inferred using the Bayesian Markov chain Monte Carlo (MCMC) method available in the BEAST software package, version 1.4.8 (Drummond and Rambaut, 2007). An input file for BEAST was generated using the BEAUti program with sequences dated according to the year of isolation. The nucleotide substitution model was determined using the MODELTEST software package and Akaike’s Information Criterion (AIC) (Posada and Crandall, 1998). 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 selected. Strict molecular clocks (Drummond et al., 2006) were performed to explore the extent of variation in the rate of nucleotide substitution. Chain length of 30,000,000 was used for constructing the maximum clade credibility (MCC) tree. The BEAST output was assessed using the TRACER program (ESS value 400) and the trees (30,000) were used as input for the TREEANNOTATOR program (Burn-in: 3000) to find the MCC tree. The degree of uncertainty in each parameter estimate is provided by 95% highest posterior density (HPD) values. Bayesian skyline was constructed at the same time. The results have been run three times, no significant differences were observed. Acknowledgements We thank the staffs of the provincial CDCs (Anhui, Hunan, Guangxi, Guizhou, 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).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.meegid.2013. 02.009.

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