Analysis Of Epidemiological Characteristics And Viral Molecular Epidemiology Of Rabies In Yunnan Province

ObjectivesRabies is a fatal disease caused by a highly neurotropic virus of the family Rhabdoviridae, genus Lyssavirus. The viral genome is a non-segmented negative-strand RNA. The fatality rate is almost 100% and there is no effective therapeutic drug until now. Yunnan pronvince, one of provinces of higher rabies incidence in china, is a region that has vast, relatively complex natural condition and distinct vertical climate, tropical and subtropical climate in many areas and also alpine region widespreadly, which are suitable to survive for all kinds of host animals. Therefore, in order to clarify the epidemiological characteristics and trends of rabies, the rabies epidemiological data were arranged, counted and analyzed between 1976 and 2007 in yunnan province, a genotyped, sequence analysis and determination for the sample of rabies in 2008 and 2009 in yunnan province by application of molecular biology technology,were to grasp the distribution of yunnan rabies genotype and molecular genetic characteristics, and to further improve and provide the scientific basis to prevent and to control rabies.MethodsTo collect the rabies epidemic information and epidemiological survey of yunnan province during 1976～2007, Excel was used to establish the epidemic information database which SPSS 13.0 statistical software was used to analyse. Epidemiological methods were described by analyzing the rabies epidemic information in yunnan province, and statistics were used to analyze the incidence,mortality and morbidity from the distribution of higher incidence area, time and people. Molecular biology technology is applied to research characteristic of the rabies virus for 2 years in yunnan province by viral antigen detection, nucleic acid extracted positive samples by using reverse transcription polymerase chain reaction (RT-PCR) were amplified and sequenced the company to return.The sequencing results after using ATGC software stitching, ClustalX software fully matching, BioEdit sequence analysis software, and cut again with the ClustalX software on N and M gene nucleotide sequence of completely compared with Mega3 software to open the results compared, select the Neighbor-Joining (NJ) phylogenetic tree was constructed.Because the method is suitable for analysis of achievements species (biological classification level) below the level of the genetic relationships. Bootstrap Selection 1000. DNAStar (5.01) analysis software MegAlign of the cut after the nucleotide and amino acid sequences and sequence comparison, generates the appropriate data table. GeneDoc software made of nucleotide and amino acid differential display.Results1497 cases of rabies were reported with the range of rabies morbidity is during 0.00-0.69/100 000 and an average morbidity of 0.115/100 000 and the highest rabies morbidity is 0.69/100 000(1989) in Yunnan during 1976-2009. The case of rabies were reported with relatively few number in 70s of 20 century,and happened with the most highest morbidity in 80s, and reduced gradually in 90s,the number of reported rabies cases was dramatically rising in 2006 and 2007. Rabies cases took place in 13 prefectures and 72 counties and the cases were mainly reported in some epidemic areas such as Zhaotong, Qujin,Wenshan, Honghe prefectures and secondly in Yuxi, Chuxiong, Kunming. The western region in Yunnan including Dehong and Baoshan prefectures also happened to be epidemic. The rabies cases had been reported on whole season and main period of incidence is from May to October. The patient including all age groups, minimum age for 1, the maximum age for 85. Below the age of 5 accounts for 4.16%,5 between 9 accounts for 16.89%,10 between 14 accounts for 14.97%,15 between 19 accounts for 8.83%,20 between 49 accounts for 33.21 %, above the age of 50 accounts for 2.15%. the main age of the reported rabies cases were between 20 and 49 years old. Male, female accounts for 27% and 73% respectively, men and women of sex is 1:0.38. Most patients for famers, followed by students, children and workers and so on.616 samples from the 7 cities(prefectures) of Zhaotong, Qujin,Wenshan, Honghe and so on. Among them dog brain samples were 607,8 saliva samples and 1 CSF sample. DFA was used to detect 607 brain samples and QIAamp viral kit to extracte 8 saliva,1 CSF samples, in which 16 positive samples. The N gene and M gene of 16 positive samples by reverse transcription-polymerase chain reaction (RT-PCR), respectively were amplified. The cDNA fragments were cloned and then sequenced. Results indicate that all the 16 strains contained full-length of N and M gene fragment. Phylogenetic analysis indicate that the strains belonged to classic rabies virus, with 88.4-100% nucleotide identity and 88.5-100%, and deduced therir amino acid sequence similarities were 95.8-99.8% and 82.5-99.5%. With rabies virus gene 3 (Mlkola) were compared, N gene nucleotide and deduced amino acid homology of the homology of 65.8% and 77.3%, M amino acid and deduced amino acid sequence homology of the 71.4-71.8% and 77.8-79.8%, and low homology. The results showed that the strain of the deduced amino acid sequence homology higher than the nucleotide sequence homology.ConclusionThe above results suggest that the rabies distributes widely in yunnan province. There are rabies case report in 16 states(cities) of Yunnan, However, incidence also had obvious difference in various regions:the main endemic areas are easten and southern regions in Yunnan. The rabies cases were mainly reported from weishan, honghe, qujing, honghe, zhaotong, xishuangbanna city and prefecture (city). The rabies were prevalent in the whole year in Yunnan, but the main prevalence season was between May and October, it has obvious relationship among seasons distribution, temperature and rainfall. Patient for all age groups, are mainly of the age 20～49, and with the lower incidence and the older age gradually, cases with men more than women. The genotype of the epidemic rabies in yunnan province are type I and the latter is the main type.The analysis of homology and phylogenetic tree of rabies virus in n and m genes suggests that both belonged to genes I type of the rabies virus; nucleotide and effect analysis shows the sequence of the rabies virus geographical features in yunnan province. The rabies virus in different parts showed genetic relationship, the analysis of phylogenetic tree showed:the rabies virus had a relationship with rabies prevalence in neighboring provinces and the neighbouring countries; the rabies virus in yunnan province,in addition to other provinces, is could also from Southeast Asia; the rabies could be further popular and diffused in yunnan province, results provided the scientific data for prevention and treatment of rabies in yunnan province.