Establishment And Evaluation Of Immunochromatographic Strip And Real-Time PCR Diagnostic Methods Of Canine Distemper Virus

Canine distemper virus is the etiological agent of a serious, often fatal, disease in dogs and many other carnivores. Canine distemper virus belongs to the genus Morbillivirus, family Paramixoviridae that includes measles virus, peste des petits ruminants virus and rinderpest virus. Canine distemper virus has enveloped virions and a negative-sense, single stranded RNA genome. The RNA encodes six structural proteins: two membrane glycoproteins, the fusion (F) and the hemagglutinin (H), the envelope-associated matrix (M) protein, the phosphoprotein (P), the large polymerase (L) and the nucleocapsid (N) protein.Although vaccination against canine distemper has been used widely for many decades, this infection still represents an important disease for dogs. Recent years, several episodes of CD in vaccinated animals have been reported throughout the world. Canine distemper virus infects macrophages and lymphoid cells of the upper respiratory tract. Subsequently, the virus in infected leukocytes reaches various organs, e.g. cells of the lower respiratory and gastrointestinal tracts, lymphoid organs, the urinary bladder and the central nervous system (CNS). This can result in either subclinical infection or gastrointestinal and/or respiratory signs, frequently with subsequent neurological disorders. Nervous signs may be present in the chronic form of distemper together with other manifestations, or may occur without any other signsThe clinical diagnosis of distemper is difficult because of the broad spectrum of signs. Therefore, a laboratory assay is required to confirm this disease. Routinely and widely used is the direct immunofluorescence test, in which nasal, conjunctival and vaginal smears are incubated with polyclonal or monoclonal anti-CDV antibodie smarked with fluorescein. Unfortunately, this assay can confirm distemper only within 3 weeks after infection, because after this time the virus disappears from the epithelial cells. Thus, in the subacute or chronic forms of the disease, this test gives false-negative results.Serological examination - for example, using the seroneutralization test (SN), ELISA, indirect immunofluorescence assay (IFA) and immunoperoxidase linked assay(IPMA)- is not very useful in the diagnosis of distemper, because a high titre of anti-CDV antibody may be a result of prior vaccination, as well as of previous subclinical or clinical infection. On the other hand, during severe distemper, the antibody titre may be low because of the strong immunosuppressive properties of CDV.Definitive diagnosis can be also established after virus isolation and identification.However, this assay is expensive and takes between several days to weeks, and so is of limited value when applied to clinical specimens. Thus, an alternative simple and convenient to use or sensitive and specific method is required for the clinic or laboratory diagnosis of CDV infection in clinically suspected dogs.Monoclonal antibodies (Mabs), however, assure a reliable source of antibodies and very often achieve greater sensitivity and specificity than polyclonal antibodies. In recent years, Monoclonal antibody technology has used largely in canine distemper disease diagnosis. In order to provide more specific and standardized reference antibodies for diagnosis, pathogenesis and epidemiological studies, we produced mouse Mabs against CDV. The CDV was purified with zinc acetate precipitated followed by sucrose density gradient centrifugation. The Balb\c mice were immunized with purified CDV. Spleens cells of the immunized mice were fused with mouse SP-2/0 using 50% polyethylene glycol. The mediums of hybridoma cells were screened by indirect enzyme-link immunosorbent assay. Hybridomas whose supernatants show stronger positive were subcloned by limited dilution methods 3-4 times, until the positive rate come up to I00%. The specificity of hybridoma cell was detected by fixed-cell ELISA and indirect immunofluorescence assay. The result showed that six hybridomas were obtained, which can steadily secrete anti-CDV monoclonal antibody. The titers of cell culture and ascities tested by ELISA were 26-211 and 104-107 respectively. The specificity assay and cross-reaction test of monoclonal antibody proved that these Mabs were specificity and broad-spectrum. Relative affinity assay and Additivity ELISA results showed that monoclonal antibodies EH5, CE3 and AD7 had high affinity, and epitopes recognized by McAb AD7 and EH5 differed from those by other four McAbs (AE2,CE3,GG6 and GF1). Transmission electrton microscopy immunogold localization results showed that the high density gold particles were located at the outermost surface of freshly purified virus particles. This study provided direct evidence that Mabs AD7 and EH5 were anti-CDV and the epitopes recognized by these Mabs were located on the surface of the virion. The highly effective and specific Mabs plays an important role in diagnosis, treatment and prevention of the infection of canine distemper virus.The immunochromatography assay (ICA) has the advantage over many other field diagnostic techniques used in clinical practice in that the test procedure is simple, rapid, and can be performed by owners as well as veterinarians. This assay has the advantage over other techniques in that it can be completed within 5 min without the need for special instruments. The aim of this study was to develop a simple, rapid and convenient to use ICA method to detect canine distemper virus in clinical specimens of naturally infected dogs.The method is based on the "sandwich" assay format using monoclonal antibodies and polyclonal antibodies. The anti-CDV monoclonal antibody labeled with colloidal gold was used as the detector, and the anti-CDV polyclonal antibody and sheep anti-mouse IgG were blotted on the nitrocellulose membrane for the test and control lines, respectively. The colloidal gold granules whose diameter is about 25nm were prepared by using trisodium citrate reduction of solution of chloroauric acid in the microwave oven. The electron microscope and ultraviolet scanning appraisal colloid gold particle sizes are consistent; the distribution is even.The optimum condition of marked antibody was determined by May's method and OD value assay. The colloidal gold maker has high titer, high specificity and good stability. The makers diluent, protein concentration of the test and control lines, and so on, were optimized, and the ICA strip was established.With the stardard samples examined, the ICA strip was proved that it was repeatability, stability and convenient to use. The sensitivity of the strip is 104TCID50. So this strip is suitable for diagnosis of CDV in hospital and survey spot.The real-time RT-PCR assay displays several advantages over conventional RT-PCR assays, increasing the laboratory throughput and enabling simultaneous processing of several samples. The technique gives exhaustive results within 3 h and the reaction is performed in a closed-tube system, not requiring additional manipulations. Another major advantage of real-time RT-PCR is the ability to quantitate the viral load in clinical specimens. The aim of this study was to develop a rapid, sensitive and specific real-time RT-PCR method to detect and quantitate canine distemper virus in clinical specimens of naturally infected dogs. According to conserved sequence of canine distemper virus nucleoprotein gene, a pair primer was designed. The normal PCR method was used to amplify initial template. The standard recombinant plasmids pGEM-Nfor N gene were constructed as reference standard used in absolute quantification assay.The assay result showed that the stardard plasmid was good repeatability and high stability.The primer, probe and template of real-time PCR were designed by Beacon Designer 5.0 software.The primer sequence Pl, P2 and TaqMan probe sequence was identified by screening and the conservation and specificity of those were verified by the BLAST tools of NCBI.The concentration of primers and probe, cycle stage and anneal temperature were optimized, and the real-time RT-PCR was established. The quantitative curve was established using reference standard plasmids. The CT values and ln(x) were highly correlated(R2≥0.994), the dynamic range of measurement is 107一102 copy/μL and the minimal level detected is 10 TCID50, The real-time RT-PCR developed in this study proved to be specific and no cross-amplification of CAV,CPIV,RV and CPV was observed. The real-time RT-PCR assay for canine distemper virus was highly reproducible, allowing a precise calculation of RNA load in samples containing a wide range of viral RNA amounts. In the study, the reproducibility of the assay was high with relatively small intra- variability. Real-time RT-PCR assays for canine distemper virus offer substantial improvements in virus detection and thus may render easier the control of canine distemper disease.The direct fluorescence antibody used to diagnose canine distemper(CD) is a common method.The monoclonal antibody CE3 was purified by protein G and conjugated to fluorescein isothiocyanate. This fluorescent antibody (FA) is suitable for titration and/or detection of CDV in cell cultures or tissues. The results of blocking, adsorption and stability experiments showed that the FA has highly specificity and sensitivity, with no cross reaction to CAV, CPIV, RV, and CPV. The result of optimal working concentration measurement showed that its optimal working concentration was 1:80. In order to get the strips sensitivity and specificity, using the ICA strips and dFA to test standard positive and negative samples, this had been tested by PCR. Compared with dFA, the sensitivity and specificity of ICA is no remarkable difference between dFA and ICA (P>0.05).It is suggested that the established ICA is a rapid,simple and specific method for diagnosis of canine distemper, but no need fluorescent microscope.The primer sets for the nested PCR were designed from the published sequence of the nucleoprotein gene of CDV. The sizes of amplifications are 478bp and 287bp respectively. The results showed that the sizes of amplifications was found in each methods coincided with the predicted sizes.The sensitivity of the one-step PCR assays is 103 TCID50. In the nested PCR amplification, the sensitivity is 100 times higher than one-step amplification, which corresponds to about 10 TCID50.In conclusion, a specific and sensitive method is established for detection of CDV by using one-step PCR and nested PCR assay. In order to get the FQ-PCR sensitivity and specificity, standard positive and negative samples were used. Compared with N-PCR, the sensitivity and specificity of FQ-PCR is no remarkable difference with those of N-PCR (P>0.05).However, the real-time PCR was simple, short time required and the ability to quantitate the viral load, which will provide the basis to investigate the pathogenesis of canine distemper, with particular regards to the patterns of virus spreading and shedding .In addition, it will be helpful to evaluate the efficacy of antiviral drugs in vivo and in vitro.