Developing Methods For Detecting Rabies Virus And The Application For Survey On Rabies Virus Of Bats In Southern China

Background and objectiveRabies is one of natural focus infectious diseases,and it can cause the fatal encephalomyelitis of human and all homothermal animals.All sufferer or patho -livestock would die after invasion.Rabies is distributed all the world.It’s a worldwide severity Public health problem.Rabies virus is the pathogeny of rabies about human and animal.There are many hosts of rabies virus,including domesticate animals and wildlives.It is increasingly harmful for human when wildlife carry rabies virus.The further studies show that more and more species can be isolated rabies virus.Especially,many countries have isolated rabies virus from bats in recent years. It have been paled widespread attention about bats with rabies virus and the relation between bats and human rabies scince 1953 when USA discovered a person gained rabies after he was bitten by bats.The infectious sources of rabies are wildlives,including wolves,foxes,panthera, wild dogs,monkeys,cats,mephitis,raccoons and bats et al.Rabies would be prevalent in wildlives firstly,then infected with human through domestic animals. Domestic animals are the linkage between wildlife rabies and human rabies.Dogs wern’t the most sensitive animal,but they were the long-term host or intermediary agent because of their highly density in cities and countrysides.So were cats.There were different infection sources of rabies in different regions.The dogs rabies was dominant in Africa,South America and Asia（except Israel and Bengal）. And the wildlife rabies was dominant in South Africa,Europe,North American（including United States,Canada）.Bats spread rabies in Guyana,Panama and Togo.Bats rabies was subsequently to wildlife in Netherlands,Germany,Spanish. The most rabies were caused by dogs in our country,sometimes can be caused by other animals such as cats or cattles.The proportion of wildlife rabies was increasing recently.There were reported about wildlife rabies infected human.It is increasingly harmful for human about the wildlife with rabies virus.Wildlife not only transported rabies virus directly,but also they influenced human’s health indirectly through ruin husbandry.It’s difficult for us to control and immunoprophylaxis wildlife.Especially some hoof wildlives have migratory instinct.And they can alter habitats periodicity and replacemently to gain the best environmental conditions.Above all,as the host and intermediary agent of rabies virus,wildlife can bring more hazard.The bats represent the second largest order Chiroptera within the mammals. Order Chiroptera has about 960 species of bats,180 genuses,17 families.There are two suborders within Chiroptera,the Megachiroptera and Microchiroptera. Pteropodidae（flying foxes） is the only family within Megachiroptera,there are 7 species,5 genuses,1 family in China.Microchiroptera has 16 families all over the world,while 100 species,24 genuses,6 families in China.Except the Polar Regions and some oceanic islands,bats are found in all continents,including high latitude regions,deserts and isolated islands.However, most species of bats exist in tropical and subtropical regions.Bats with diverse biology and ecology are relatively longevous,and have powerful flying ability;even some species have habit of long-term migration.Humans have a lot of opportunities to contact with bats directly and indirectly. Bats can roost under the eaves and wall’s cracks of houses;bats might fly into house while predating;bats can roost in the cave located in tourist sites;in Chinese medicine,the stools of horseshoe bats are believed to cure eyes diseases.In Guangdong province,fruit bats are viewed as a delicious and tonic dish;In Hunan province,some people use bats to treat mental diseases.According to Bourhy nucleoprotein gene in 1993,rabies virus were divided into six genotypes,gene 1～4,respectively,corresponding to serumⅠ～Ⅳ,Europe EBL1 strain and EBL2 strain rabies virus belong to serum-typeⅤ,then were divided into genotype 5,6.Australia isolated a new bat rabies virus in 1998,was designated as gene-7,Australia bat rabies virus（ABLV）.In recent years,new genotypes of rabies virus（Lyssavirus） had been found continuously in bats,there were several rabies-related virus such as Aravan virus,Khujand virus,Irkutsk virus,and West Caucassian bat virus（WCBV）.Genetype 1 virus are the rabies virus,and other genotypes virus are rabies-related virus.Further,these genotypes were divided into two genetic lineages（phylogroup）:genotype 1,4,5,6 and 7 for the genetic lineage I; genotype 2 and 3 for the genetic lineageⅡ.Bats are unique host and media of rabies virus,with the exception of 3-type gene of rabies virus,each rabies-related virus genotypes can be isolated from bats.Our country was one of the most serious countries affected by rabies virus.The incidence of rabies have been increasing rapidly in the past few years.There are little research about bat species,quantity,and carrying rabies virus in China.Guangxi Detected by RT-PCR in brain tissue bat 120,3（2.5%） positive only.Jilin Tonghua had happened was a case of bat rabies in people bitten to death.Generally speaking, China’s rapid detection of rabies virus methods are imperfect,bats carry the rabies virus does not investigate the system,not enough depth study etiology,bat rabies virus in the type,distribution and its route of transmission is unclear.Bats carry rabies virus for epidemiological significance of rapid detection method for rabies virus and the people set up the relationship between rabies-depth study should be carried out.Methods1 Establish the methods of immunology and Molecular biology to detect rabies virus1.1 Detection of rabies virus with direct immunofluorescence①Sucking mice were inoculated intracerebraUy with 0.03ml 105.0LD₅₀ of rabies virus attenuated live vaccine,determinate working dilution.②Bat brain tissues were fixed 12 h,paraffin-embedded tissue,and sections.③Detecting rabies virus by direct immunofluorescence assay 1.2 Detection of rabies virus by nested RT-PCR①Reference to thesis,choose specific amplification nucleoprotein（N） gene region for the purpose of the most conservative genes,compare seven genotypes of rabies virus,design primer of nested PCR detection.②Using attenuated live vaccine of rabies virus to detect the sensibility of nested RT-PCR.③Detection of rabies virus by nested RT-PCR1.3 Establish fast test method to detect rabies virus initialy by RT-LAMP①Reference to thesis,choose specific amplification nucleoprotein（N） gene region for the purpose of the most conservative genes,compare seven genotypes of rabies virus,design primer of RT-LAMP.②Using attenuated live vaccine of rabies virus to detect the sensibility of RT-LAMP.2.Collection and treatment of bat samplesFrom May 2007 to August 2007,between July 2008 to August 2008 between,in Hainan,Hunan,Guangdong and From May to August 2007,July 2008 to August 2008,1057 bats from 13 species（belonging 4 families） were captured and sampled from their natural habitats in some regions of 3 provinces.Bat identification was determined by Prof.Wu Yi（Bats expert,academy for life science,Guangzhou University）.Abandon houses,wall’s cracks and eaves of houses,or natural damp caves are common sampling roosts of bats.①Treatment of sera.Obtain blood from hind limb vein and heart,still at 4℃for 5h,centrifuge 3000r/min for 10min,transform supernatant to another new and sterile EP tube,label each specimens properly,transport to lab at 4℃,test immediately or store them at -70℃for long-term storage.②Treatment of rectum specimens.Bats were anatomized sterilely.Remove recta,store immediately in RNAlater,transport to lab at 4℃,test immediately or store it at -70℃for long-term storage.3.Detecting rabies virus of bats by DFA and RT-PCR.4.Sequence and genetic analysis①Sequence RT-PCR products;②Compare sequences of the PCR products with known sequences of the RdRp genes of coronaviruses in GeneBank using on line server BLAST;③Align viral sequences by using Clustal W;④Employ MEGA 4 program package to construct the phylogenetic trees by using the neighbor-joining（NJ） method with 1000 bootstrap replicates.5.Virus isolation and culturePositive samples detected by Vero cell culture for virus isolation,use flow cytometry and immunofluorescence methods to identify cells.6.Quality control①Tips,EP tubes,gloves and eta were disposable;②During reverse transcription,all tips,EP tubes and grinding devices were treated by DEPC,in order to prevent contamination of RNase;③RNA extraction and RT-PCR were all proceeded on the ClassⅡbiosafety cabinet in a biological clean room;④During procedure,wear respirator and head-covering to prevent RNase from handler;⑤Positive and negative controls were included in each run of the RT-PCR assay,and no false-positive result was observed in the negative-control reactions.Results1.Establishment of DFA for Rabies VirusThe working dilution of fluorescent antibody was 1:10²,to detect the attenuated live vaccine of rabies virus(105.0LD₅₀/0.03ml).The signal of positive control were detected,but negative control were dull.This method can be used to detect brain tissue of bats.2.Establishment of nested RT-PCR for Rabies VirusThe least concentration of attenuated live vaccine of rabies virus(20.0LD₅₀/0.03ml) was 1:10⁴ which can be detected by nested RT-PCR.No goals were found in negative control and blank control.Both sensitivity and specificity were high level.This method can be used to detect brain tissue of bats.3.Establishment of RT-LAMP for Rabies VirusThe least concentration of attenuated live vaccine of rabies virus(10.0LD₅₀/0.03ml) was 1:10⁴ which can be detected by RT-LAMP.No goals were found in negative control and blank control.Both sensitivity and specificity were high level.4.Identification after viral isolationTo identify Vero cells after virus isolation and culture results,30 cases of rabies-positive samples collected in 2007 transfected Vero cells after 7 d,for flow cytometry,fluorescence add the rabies virus monoclonal antibody,the mean fluorescence intensity enhanced 5.34～27.22,the percentage of fluorescent cells increased 3.48%～11.74%.9 cases of brain tissue samples collected in 2008,the transfected cells with fluorescent-labeled antibody binding,apple green fluorescence under the microscope,and no fluorescence in blank control cells.5.Application of the methods for field surveyThere were rabies virus in the Miniopterus schreibersii and Rhinolophus affinis, positive rate was 3.69%（39/1057） with nested RT-PCR,one of the first year positive rate was 6.41%（30/468）,the second year of a positive rate was 1.53% （9/589）,there were detected from Hunan.The second year of a positive rate was 15.59%（58/589） with DFA.All positive samples detected from Miniopterus schreibersii Rhinolophus affinis and Scotophilus kuhli.6.Sequence analysisSequence analysis showed that the evolution of the system exist genetic polymorphism between samples,but phylogenetic trees constructed showed that the bat rabies virus origin from a branch of aggregation.Conclusions 1.Established Laboratory detections of rabies virus in bats.Direct immunofluorescence and nested RT-PCR were applied to detect rabies virus in bats. The specificity of two method were highly.Then Direct immunofluorescence was more highly on sensitivity.The initial establishment of RT-LAMP method was applied for rapid detection of rabies virus.2.Rhinolophus and Miniopterus schreibersii of Hunan exist two types of rabies virus, with a detection rate of 3.69%（39/1057）.Both bats are insectivorous bats,and are gregarious,living together with other types of bats in the same cave,but the home side,do not mix.Foraging at night.Rhinolophus of meat can be medicine,manure for fertilizer.This is a possible way bat rabies virus infect to humans.3.The rabies virus sequence are likelihood in this study,all included in the CHINA-1 branch belong to one type of rabies virus genes.That is a popular genotype in Asian.