The Establishment Of Canine Distemper Pseudovirus System And Its Application In Neutralizing Antibody Detection Methods

Dogs are one of the commonly used experimental animals for drug safety evaluation.Controlling and evaluating the quality of experimental dogs is very important for ensuring preclinical drug safety evaluation.Canine distemper(CD)is a disease that threatens experimental dogs.Its pathogenic canine distemper virus(CDV)has been an indicator that experimental dogs must control.At present,it is mainly controlled by immunological methods such as injecting vaccines.Due to the problems of vaccine quality,immunization procedures,individual differences in animals,and timing of detection,there are often problems such as reduced antibody titers after immunization and unsatisfactory immunity.Moreover,serological methods prescribed by current national standards detect serum antibodies are not neutralizing antibodies against viral infections.Therefore,there are certain problems in evaluating the quality of experimental dogs.The detection methods for canine distemper neutralizing antibodies include CDV neutralization assay(SN),indirect immunoenzymatic staining,ELISA detection methods and so on.At present,there are some drawbacks in the detection method of canine distemper neutralizing antibodies,for example,the judgment of the result is generally by observing and cannot be quantified;the operation process is complicated and the cycle is long;the result is not easy to judge,and it is easy to misjudge;canine distemper virus is sensitive to physicochemical factors and it is difficult to obtain coating antigen by conventional virus purification.In this context,this study established a pseudovirus system of canine distemper,which is highly simulate the original virus,has high biological safety.It provides a new method for canine distemper virus research and vaccine efficacy evaluation.In this study,the expression plasmids of H and F with cytoplasmic regions of different lengths were first constructed;then optimizing the influencing factors of the packaging system of canine distemper pseudovirus: the combination of H and F in different cytoplasmic regions and the screening of different framework plasmids and pouches and the ratio of the membrane expression plasmid to the backbone plasmid and the susceptibility to cell types and so on,and thereby constructing high-titer pseudorabies virus.Based on the constructed pseudoviruses,a neutralization experimental method was established in this study.Since the canine distemper pseudovirus has only one cell infection cycle,the serum neutralization titer cannot be determined by the observation of the cytopathic effect(CPE),but the titer of the virus can be calculated by the expression of the reporter gene carried by the pseudovirus.Experimental investigations have found that the inoculation volume of the constructed pseudovirus is in a good linear relationship with the value of the relative light unit(RLU)signal value after infecting the cells.Therefore,when using the pseudovirus for the detection of neutralization reaction,serum titers can be determined by the corresponding serum dilution factor which inhibit 50% of the pseudoviral luciferase RLU signal value after neutralization.By optimizing the conditions of cell density,detection time and the amount of pseudovirus used in pseudovirus serum neutralization experiments,a canine fever pseudovirus neutralizing antibody detection system was established.Based on the canine distemper pseudovirus neutralizing antibody detection system established in this study,positive serum from the laboratory were tested.The experimental results showed good correlation and consistency with result of traditional neutralization experiments.It can be seen that the detection method for neutralizing antibodies based on the canine distemper pseudovirus can replace the traditional neutralization experiments for the detection of neutralizing antibodies in serum.The subject successfully coated a high titer canine distemper pseudovirus and established a simple and highly effective neutralizing antibody detection method based on the canine distemper pseudovirus;and provided a new idea for the subsequent coating of pseudoviruses.