Analysis Of Rabies Epidemic And Rabies Virus N Genome Character In China From2007-2011

BACKGROUNDRabies is a zoonotic disease caused by rabies virus, the mortality rate is almost100%. Rabies is widely distributed around the globe, China is one of countries that experience the most serious impact from the disease, and its epidemic is second to India. Since1950, our country began to have the record of rabies, there have been several times of popular, in1980s, the epidemic situation of rabies in China is serious, in1981,7037cases were reported but since then the number of cases decreased until1996when a minimum of196cases were recorded, however, from that on there has been a gradual reemergence of the disease, and cases once again increased year by year, until2007when3300human rabies cases were recorded, subsequently, the number of cases has slowly decreased,1918human rabies case in2011, but the epidemic situation remains serious. In addition, apart from the infectious disease report system, to further understand the rabies epidemic situation, Chinese Center for Disease Control and prevention established15rabies active monitoring points in6provinces in2005, in order to collect the information of post exposure prophylaxis (PEP) treatment of individual cases, outpatient case; Host animal monitoring information, using active monitoring information to further explore the factors related to the epidemic of rabies in our country. For the possible reason for reduction of epidemic situations since2007and providing a scientific basis for further controlling rabies epidemic situations in China, this paper carried out an epidemiological analysis by useing relevant data of rabies reported in2007-2011.Rabies nucleoprotein (N) gene of rabies is the most conservative gene in the complete genome, which is widely used by rabies molecular diagnosis and virus infection detection experiment, it is also been used for researching virus long time evolution; analyzing the regional characteristics, origin of rabies virus, host transfer of space-time evolution. As the further of the molecular studies, nucleic acid detection technology becomes more mature, in recent years gene molecular epidemiological studies on N gene of rabies is increasing, but previous studies is limited to local province, or several provinces, the time span is not wide, this study we collected rabies positive samples specimens from14provinces of countrywide from2003to2011, we sequenced the nucleoprotein gene of rabies, using phylogenetic analysis, spatial analysis of molecular epidemiology, combined with the changes of rabies epidemic, to understand the characteristic of spatial and temporal distribution of rabies virus in China, the mechanism of its migrant and its relationship with rabies epidemic and therefore provide scientific basis for the effective control and prevention of the spread of rabies in China.OBJECTIVES1. To summarize the epidemiological features of rabies in2007-2011, to understand the national characteristics of the rabies epidemic situation.2. Analyze the active monitoring data of rabies epidemic to explore the immanent connection between PEP of the individual cases and outpatient people, host animal controlling and prevalence of rabies.3. Explore rabies N genetic variation and evolution characteristics, analyzes evolution of rabies virus N genes along the time to see whether there is variation and the change of virulence variation.4. Constructing rabies N gene phylogenetic tree, to analyze the changes of spatial dynamics to discuss the phylogenetic evolution of rabies and migration path, in order to further control the spread of rabies.METHODS1. Collect and analyze the epidemic data of rabies(1) Using the "National Disease Reporting Information System" of Chinese Center for Disease Control and Prevention, collect the national rabies epidemic information in2007-2011, describe the three distribution of regional distribution, time distribution and population distribution.(2) Retrospective descriptive analysis was used to describe the PEP of the individual cases, outpatients and host animal of15active monitoring points in Shandong, Guangxi, Anhui, Hunan, Jiangsu, Guizhou, to explore the intrinsic relationship between these changes and epidemic.(3) Use the Epidata3.0software to input the information of individual cases, use Excel2007, SAS9.0software to do some statistical analysis, use Excel2007to draw charts, MapInfo7.0was used to do some maps.2. Sample collection, detection and sequencingFor this study, we collected animal (dogs, cattle) brain tissue specimens and human blood, saliva, cerebrospinal fluid specimens from14provinces and cities nationwide, used DFA and RT-PCR method to detect the specimens, and obtained positive specimens, further amplified and got the complete sequence of N gene.4. Analysis of N gene sequence(1) ATCG was used to check and splice the sequence.(2) BioEdit was used to edit the sequence in the same length.(3) Clustalx (1.83) was used to align the sequence, transfer the alignment of sequences into other formats so that other software can recognize and analyze.(4) MegAlign in DNAStar (5.01) software package was used for the nucleic acid and amino acid homology analysis. (5) GeneDoc was used to display the nucleic acid and amino acid sequence one by one.(6) MEGA (version5.1) software was used to map phylogenetic tree, Neighbor-Joining (NJ), Maximum Likelihood (ML) method were used to draw phylogenetic tree.(7) MigaPhyla software based on file of no root tree branch length, conjectures the migration and gap analysis events event and do correspondingly significant analysis.RESULTS1. The rabies epidemic of China in2007-2011was decreased year by year, but the number of counties and districts reporting epidemic of rabies is984,858,892,817,862, respectively, it showed no obvious decline, the number in certain province showed a growing trend. Cases in Guangxi, Guangdong, Guizhou, Hunan, and Sichuan are still the most in our country, and the epidemic affected the surrounding provinces and nationwide epidemic. There are more cases in Summer and Autumn. The incidence ratio of male to female was2.32:1. Farmer (68.27%), student (12.47%), pre-school aged children (6.32%) were high risk population, recent year, the proportion of farmer was increasing year by year, the proportion of students downing, and the pre-school aged children unchanging.2. There were2346deaths reported in active monitoring points in2007-2010,88.53%were exposure by dog, III degree exposure accounted for56.97%, however, there were only11.36%patients went to medical institution to get wound treatment, vaccination rate was only12.08%, the rate of RIG or anti-serum was only4.60%, there were no significantly difference in four years. But the result of people went to medical institution showed completely different. Rabies outpatient monitoring showed the ratio of III degree exposure was36.11%,85.43%outpatients went to medical institution to get wound treatment, rabies vaccination rate was82.13%,30.30%of Ⅲ degree exposure of outpatients get RIG or anti-serum. Dog density in rural was slightly decreased from year2008; the immunization rate was slightly increased.3. We got116rabies N gene sequence of1353bp from14provinces of countrywide. The homology of116sequences is89.2%-100%; and amino acid homology is97.6%-100%and98.2%-100%respectively. It indicates that the variations are synonymous mutations.4. The four antigen sites showed that there were several sequences have variation in antigen site Ⅱ, Ⅲ, Ⅳ, B cell, Th cell epitopes and phosphorylation sites in the sequences. There are three potential glycosylation sites of the sequences Asn196, Asn326, Asn443, respectively.5. Phylogenetic tree showed there were3groups (Ⅰ, Ⅱ, Ⅲ) of the sequences, it show rabies virus have the character of regional aggregation in our country,13provinces in group Ⅰ,6provinces in group Ⅱ, Ⅲ group includes only a sequence of Inner Mongolia. Phylogenetic tree of24provinces and cities showed NM3, Fujian strain and Jilin strain were evolutionary close. N gene shows no obvious difference along the time. Phylogenetic tree of global range had7branches, the virus which this study isolated were genotype1, most of the sequences gathered in Ⅰ, Ⅱ groups. Rabies virus isolated in our country was close to virus isolated in Indonesia, Vietnam and other Southeast Asian countries in relation to the evolution, NM3was close to Iran, Mongolia in evolution.6. There are some translocation events in trans-province, this study shows that possible of rabies in our country transfer from Sichuan Guangxi in southern high provinces to the east area and the northern part of China.CONCLUSION1. The cases of rabies reported in2007to2011have reduced in China, but the epidemic has diffused in certain certain popular areas.2. Parts of the survey data show dog density decreased, the immunization rate raised somewhat, to a certain extent, the infectious sources of rabies were reduced. 3. The poor situation of death cases PEP after exposure to animals illustrated that not timely, not standard of PEP lead to cause morbidity of the cases; high density of dog and low immune rate, defective condition of PEP is still continue to be important causes of rabies.4. Rabies virus N gene has little variation and is very conservative, it’s related the functional limitation. N gene sequence homology is high, variations are mostly synonymous mutations.5. All virus isolated from specimen belongs to genotype1, evolutionary tree showed obvious regional dependence gathered characteristics. China and Southeast Asia rabies had evolutionary relationship; rabies virus isolated in Inner Mongolia was closed to Mongolia and Iran in evolution. Rabies virus in China evoluted alone, at the same time there may also experience outside influence.6. There are widespread regional translocation events which makes our rabies presents the overall aggregation, rabies virus spread from high provinces to low provinces.