Studies On Epidemiology Of Rabies And Virological Characteristics Of Rabies Isolates In Fujian Province

ObjectiveIn recent years, the number of reported human rabies cases has been increasing so rapidly that rabies has become a serious public health problem in China. General information for epidemiology of nation-wide rabies, in-depth study of rabies isolates around China were incomplete. In Fujian, particularly, research in this area is rare. The main host of rabies virus in Fujian and immune situation is not clear, nor does the residents'knowledge and awareness of rabies. So far no domestic rabies virus street strains has been isolated in Fujian yet the characteristics of prevalent street strains in Fujian remains unkown. Therefore, in order to enhance prevention and control of rabies, it is necessary to systematically investigate the epidemiology of rabies, obtain detailed data of rabies prevalence, and to understand the main impact factors on rabies incidence in Fujian. Meanwhile, it is urgent to isolate rabies virus street strains, study the virological characteristics of the isolates. Additionally, the increasing number of kennel dogs is resulting with raised number of trauma patients, hence, it is also necessary to develop rapid sera antibody detection for injured patients and viral antigen detection of saliva specimens from possible rabid dogs.Methods1. A systematic epidemiological investigation was conducted to obtain basic information of rabies prevalence in Fujian. Criteria of potential ecologocal factors associated with incidence of rabies were collected and analyzed with the negative binomial regression model.2. Street trains were isolated from brain of suspected rabid dogs, via mouse inoculation technique(MIT) and cell culture inoculation technique(CIT). Complete genome sequences were obtained by RT-PCR, cloning and sequencing, the genomic structure was analyzed and compared.3. Dozens of canine brains were collected from several districts of Fujian in the past 3 years. Full-length N genes from all positive specimens were amplified, cloned and sequenced.4. Epitope of viral glycoprotein from the CTN strain was cloned,expressed and purified, a recombinant viral glycoprotein antigen was obtained.Results:1. From 2000 to 2006, human rabies cases were reported in Fujian with an average incidence rate of 0.07/100,000. These cases were unevenly distributed in several districts of Fujian, most of the cases clustered in the summer and autumn, aged from 30-60, more male victims than female, peasants are more likely to be rabies victims.2. In average, 46.67% of homes from 3 districts investigated in Fujian has dogs, 0.13 dogs per capita, 0.63 dogs per residency. The mean immunization rate was 5.54%.3. Although general awareness of rabies was poor for public in Fujian, satisfactory results were obtained from professional staff treating rabid bites.4. Our data revealed that the incidence of rabies was associated with dog immunization rates and general knowledge of rabies from the public. 1% increase of canine immunization rate would result dramatic drop of 5-year cumulative incidence rate from 79% to 21%. 0.1 points increase of knowledge in the public would result decrease of 5-year cumulative incidence rate from 80% to 20%.5. In this study, fragments were amplified from brains of healthy dogs by RT-nested-PCR. Sequence analyses confirmed their rabid origin, indicating healthy dogs may act as hosts for rabies viruses. These results provided solid laboratory evidence for rabies prevention and control.6. Seven street strains were successfully isolated, biological features were studied and complete genome sequences were obtained in two strains(FJ008, FJ009)7. Nineteen full-length N genes were amplified from 89 canine brains collected from several districts. Based on nucleotide and deduced amino acid homology level, these 19 RABV fragments were classified into three groups, FJ001, FJ002, FJ003, FJ012, FJ013 and FJ014 belonged to group A; FJ008, FJ009, FJ010, FJ011, FJ015, FJ016, FJ017, FJ018 and FJ019 belonged to group B; FJ004, FJ005, FJ005 and FJ007 belonged to group C. The nucleotide homology of RABV N gene was between 99.70 ~ 100% within the group, 86.43 ~ 89.28% at inter-group level. The amino acid homology of RABV N gene ranged 98.86 ~ 100%, while 95.33 ~ 98.44% at inter-group level. These results suggested distinct geographic distribution of rabies viruses in Fujian.8 .Comparison of N gene sequences from the prevalent street strains in Fujian with those from 4 commercial rabies vaccines revealed a homology between 86.47 ~ 98.89%, all belonged to Genotype I, suggesting that current vaccines could effectively protect individuals from RABV infection in Fujian.9. A recombinant plasmid pHTb-624's was constructed, confirmed with high-level expression and purification. This high-purity recombinant viral glycoprotein antigen was used in serum antibody detection for post-exposure patients, no significant difference was observed with current commercial kits.Conclusion1. The high incidence of rabies in Fujian in recent years was caused by excessive dogs in the community, low immunization rates in dogs, relatively unawareness of rabies in the public. Low immunization rates in dogs and weakness of the knowledge on rabies in the public were the main factors for high incidence of rabies in Fujian..2. This was the first reports that seven street strains were successfully isolated, biological features were studied and complete genome sequences were obtained in two strains(FJ008, FJ009)3. Genotyping of 19 viral fragments amplified from canine brains. Our data demonstrated that healthy dogs could be rabies hosts, These results provided solid laboratory evidence for rabies prevention and control in Fujian. Although distinct geographic distribution of rabies viruses were observed in Fujian, comparison of N gene sequences from the prevalent street strains in Fujian with those from 4 commercial rabies vaccines revealed a homology between 86.47 ~ 98.89%, all belonged to Genotype I, current vaccines could effectively protect individuals from RABV infection in Fujian.4. A recombinant plasmid pHTb-624's was constructed, confirmed with high-level expression and purification. This high-purity recombinant viral glycoprotein antigen was used in serum antibody detection for post-exposure patients, no significant difference was observed with current commercial kits.