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

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.

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.

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.

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.

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.