Establishment Of Fluorescent Quantitative RT-PCR Based On Leader Sequence For RABV And Its Application

Rabies, an ancient zoonosis caused by rabies virus (RABV), is mainlycharacterized by encephalomyelitis, rabid and progressing paralysis, associated with100%fatality rate. In China, it was reported that nearly2,000people a year died ofrabies on average, of which most case induced by bite of canine, feline or wildlife.Now, usual diagnosis of suspected dead animal mainly include directly observationof virus, isolation and cultivation of virus, detection of virus-related protein anddetection of viral gene. Although real viral shape could be observed in directlyobservation, the observation process should be performed in the laboratory equipedwith expensive transmission electron microscope. Although virus could be obtainedin isolation and cultivation of virus, bio-safety of virus and difference of viraladaption to both cell and animal should be taken into consideration. Althoughdetection of virus-related protein which was complex and low-sensitivity was thegold standard recommended by both WHO and OIE, standard fluorescent antibody,fluorescent microscope and trained operator were necessary. Traditional RABV genedetection diagnostic methods involve ordinary RT-PCR and nested RT-PCR in spiteof low-sensitivity, time suck and exposure to carcinogen dyes. Fluorescentquantitative RT-PCR as emrging diagnostic methos characterized by rapid,sensitivity and intuitive result was widely applied. Internationally, it was reportedlots of fluorescent quantitative RT-PCR against RABV associated with someshortcomings: high background signal, non-typical amplification and intricate resultin fluorescent quantitative RT-PCR based on probe; non-specific and false positiveamplification in fluorescent quantitative RT-PCR based on dyes. So, establishment ofsensitive and specific fluorescent quantitative reverse transcription-PCR based onleader sequence of RABV genome is very important to deal with qualitativediagnosis of animal rabies and for public health.Fluorescent quantitative reverse transcription-PCR based on leader sequence ofRABV genome was used not only for qualitative diagnosis of animal rabies but alsofor accurately quantitative analysis of RABV. The titers of different RABV strains are usually compared by either the median lethal dose (LD50) in mice or the mediantissue culture infective dose (TCID50) in a cell line. However, it is difficult tocompare the titers of a street virus or a fixed virus with an attenuated virus accuratelyat the same time, as street virus are less adapted to a cell line and an attenuated virusis usually not lethal to adult mice.Innate immunity is the first line of host defense against exotic pathogens. Itinvolves the release of cytokines, the release of type1interferons, the release ofchemokines and activation of complement. It was not well understood whether ornot inflammatory responses is related to the ability of street or attenuated to kill host.It is important for infection research to explore inflammatory responses in serumprior to central nervous system invasion by rabies virus of different virulence.In terms of diagnosis and research of rabies in China, following test werecarried out:Establishment of fluorescent quantitative RT-PCR based on leader sequence ofRABV genome. Test content: a. Genome analysis of200RABV strains included inGenBank. b. Screening of conserved and design of primer, including one reversetranscription primer and a pair of amplification primer. c. Construction of standardplasmid, serially dilution of standard plasmid and establishment of standard curve. d.Specific test and sensitive test using a lot of cells derived from various species,non-RABV viruses and negative brain tissues.Assemble of detection kit and assay of clinical sample. Test content: a.Optimization of operating process of detection kit. b. Assemble and writing ofinstruction of detection kit. c. Assay of210clinical samples derived from Jiangxi,China.Comparison of inflammatory responses between street virus and attenuatedvirus. Test content: a. Quantitative analysis of RABV genome of3-day-old sucklingmice brain infected with street strain BD06or attenuated strain SRV9respectively.b.Six-week-old female BALB/c miceinfected with same genome copystreet RABV strain BD06or attenuated RABV strain SRV9individually. c.Thedifference in inflammatory response levels in serum at1and2days post infection (dpi) were assessed.Through the above test, the following results were obtained:Three primers were designed and synthesized: one of which, located within theleader sequences and targeted for the whole viral genome amplification to obtain its cDNA, is designated as reverse transcription primer; the other two (as a pair), locatedwithin the conserved region of the nucleoprotein gene and aimed at amplifying thenucleoprotein gene, are designated as amplification primers.Not only the simplification and optimization of quantitative RT-PCR for RABVbut also assemble and instruction of detection kit were completed.RABV street strain induces more sever inflammation characterized by4inflammatory cytokines in sera in comparison with that of attenuated strain with sameviral genome copy.The following conclusions were obtained from the above tests:1. A specific and sensitive fluorescent quantitative RT-PCR based on leader sequenceof viral genome for RABV was established.2. Fluorescent quantitative RT-PCR detection kit for RABV was assembled and inagreement with gold standard in terms of210samples detection.3. RABV street strain induces more sever inflammation characterized by4inflammatory cytokines in sera, laying foundation for diagnosis of biomarkers.