The Spatial And Temporal Dynamics Of Rabies Virus And Its Relationship With Rabies Epidemic In China

【BACKGROUND】Rabies is a zoonotic disease caused by rabies virus and is manifested as acute, progressive and almost irreversible encephalomyelitis. It is also known as hydrophobia (Hydrophobia) for its clinical features of fear of water. Rabies was distributed worldwide with a wide range of host animals and China reported the seconded highest incidence only to India over the world. The rabies epidemic in China has been experiencing a new prevalence since 1996, especially in 2001 and 2003, with increase rate of 75% and 71%, respectively and reached peak with 3,302 reported cases nationwide in 2007. At the same time, the scope of geographic distribution and host animal species of rabies have gradually expanded and increased. Among all the 31 provinces (municipality), only Tibet and Qinghai did not report human rabies cases; the reported human rabies cases caused by injuries of wild animals have also increased gradually in recent years. Although rabies in China is a serious public health problem, many theoretical and practical issues that are closely related to rabies have not been well studied. Although different scholars in recent years have conducted molecular epidemiology of rabies in China, most are limited in phylogenetic analysis using sequences from local area. Therefore, a comprehensive analysis of molecular epidemiology including phylogenetic analysis, spatial analysis and population dynamics were conducted in this study using N gene sequences isolated from different host animals in rabies epidemic areas in China, in order to understand the characteristic of spatial and temporal distribution of rabies virus in China, the mechanism of its differentiation and its relationship with rabies epidemic and therefore provide scientific basis for the effective prevention and control of the spread of rabies in China. 【OBJECTIVES】1. To learn the characteristics of genetic variation and evolution of rabies virus N gene in China and provide the basic data for the study of its relationships with virulence, pathogenicity;2. To study phylogenetic relationships between rabies virus in China and describe characteristics of its spatial and temporal distribution;3. To study the differentiation mechanism of rabies virus in China and explore its spread patterns in China;4. Reconstructing the evolutionary history of rabies virus in China and population dynamics, to explore the prevalence and spread of rabies in China relationship, for the effective prevention and control of the spread of rabies in China provide the scientific basis【METHODS】1. Collection and analysis of epidemiological dataEpidemiological data were collected from Chinese center for Disease Control and Prevention. The data was arranged and analyzed by Excel to understand characteristics of rabies in China during 1996-2008.2. Sample collection, detection and sequencingBrain tissues of animals (dogs and cats) were collected in seven provinces or municipalities covering the major epidemic areas of rabies in China. The samples were detected by dFA and RT-PCR and positive samples were obtained. The complete nucleoprotein gene sequences of positive samples were amplified and sequenced by RT-PCR.3. Construction of database of Lyssavirus genetic informationLyssavirus sequence information was downloaded from GenBank, the interactive platform of PostgreSQL system and Perl programming language were used to construct database which contains sequence information, epidemiological information. SQL language was used to input, update, query and download information.4. Sequence collation and analysis(1) Sequence collationThe sequencing results were spliced by ATGC, aligned by ClustalX, analyzed and trimmed by BioEdit. The nucleotide and amino acid sequence homology were calculated by MegAlign in DNAStar software (5.01); GeneDoc software was used to make nucleotide and amino acid differences Figure.(2) Phylogenetic analysisPhylogenetic tree was constructed using N gene of 34 sequences obtained in this study and the representative ones from GenBank. First data sets were tested with Modeltest to obtain the best-fit model (HKY) and various parameter values; then PHYLIP package was used to construct phylogenetic tree by maximum likelihood (ML) method with bootstrap values set by 100.(3) Analysis of spatial dynamicsMaximum parsimony (MP) phylogenetic tree was constructed by PAUP using 211 N gene sequence isolated from the major rabies epidemic areas in China, and was saved as non-root tree without branch length o; the isolated location of the sequences was indicated with a single a-z letter, and migration events were inferred with MP method using PAUP and MigraPhyla.Using UniFrac, PCA (Principal component analysis) analysis was conducted won the input files of a rooted tree with branch length and sequence name with isolated location. The evolutionary distance, heterogeneity, and three-dimensional matrix were obtained based on the PCA result.(4) Analysis of population dynamics and evolutionary historyThe most recent common ancestors (Tmrca), substitution rate, population growth model and the skyline plot analysis were conducted by MCMC random algorithm in BEAST software based on 211 N gene sequences with specific isolated location and time. The HKY model was selected according to Modeltest results. The three models of population growth (constant population size, exponential growth and logistic growth) under strict clock and relaxed clock were chosen. The parameter values and likelihood values were calculated and compared to determine what the most fit model is.【RESULTS】1.987 samples were collected in Guangxi, Guizhou, Shandong, Anhui, Zhejiang, Jiangsu and Shanghai,of which,34 samples were positive detected by both DFA and RT-PCR. The 1353bp complete nucleotide N gene of the 34 positive samples were amplified and sequenced.2. Sequences comparison showed that 34 samples share a high degree of homology in nucleotide acid(88.9%-100%) and deduced amino acid(97.6%-100%). This indicates that the variations in this region are synonymous mutations. Antigenic sites and Th cell epitopes on N gene were highly conserved.3. ML tree showed that all of the 211 sequences from China the can be divided into four separate evolutionary branch (CladeⅠ-CladeⅤ), and each branch can be further divided into different evolutionary groups. CladeⅠand CladeⅡcontains the majority of strains isolated in recent years, indicating that they are the dominant ones. Each evolutionary branch is characterized by both geographical distribution and mixture of strains from different regions.4.87% of the pairwise locations were not found with migration events. There are two spread circles in east and southwest of China, with Jiangsu/Shanghai and Hunan as centers, respectively. Migration events of ferret rabies virus occurred from Jiangxi to Zhejiang.5. The BEAST analysis based on MCMC showed that CladeⅠand CladeⅡcan be dated back to 1992 and 1960, respectively. Evolution rates were:1.274×10-3 (CI95%:8.3705-4-1.2515 E-3) for CladeⅠ, and 9.629×10-4(CI95%:3.519-4-1.628 E-3) for CladeⅡ.The optimal population model for CladeⅠand CladeⅡare exponential population growth and constant population size, respectively.6. Genetic diversity in CladeⅠrose rapidly from 1994 to 1996 and then maintain stable, followed by another rapid rise during 2001-2003, and reached peak in 2003. During 2004-2005 there was a rapid decline and then maintained smooth downward trend. Genetic diversity in CladeⅡhas been in long steady state until 2000. From 2000 to 2003, it rapidly increased. There was a significant decrease in 2004 and reached the lowest point at around 2005.【Conclusion】1. N gene is highly conserved, which may be closely related to its structure and function.2. The four different Clades of the rabies virus in China have been forming a broad geographical distribution, with two differentiation mechanisms of geographic dispersal and gene flow, the former of which are the dominant one contributing to the spread of rabies virus.3. Rabies virus in CladeⅠwas dated back to about 1992 and experienced extensive geographic spread during 1994-1996 and 2001-2003 with Hunan as the center, and migration events in this Clade occurred during 2004-2008, especially from 2004 to 2005 with the center of Jiangsu in eastern China.4. Rabies virus in CladeⅡwas dated back to about 1960 and experienced geographical dispersion and migration events in 2000-2003 and 2004, respectively.5. The dominant ones-CladeⅠand CladeⅡcontributed to spread of rabies virus of corresponding region and the fact that rabies epidemic entered a new peak from 1996 and experienced the fastest growth in 2001 and 2003.