Construction And Preliminary Application Of Sensitive Cell Lines And Infections Clones For Canine Distemper Virus

Canine distemper (CD) is a highly infectious and frequently lethal disease, widespread incarnivorous species, which is caused by Canine distemper virus (CDV). Recent years, with theglobal environmental changing, human frequently intervening animal habitats and virus evolution,the natural host of CDV has been expended to the entire carnivorous from traditional Canidae,Procyonidae and Mephitidae species, it were also reported that Artiodactyla, Baikal seal andnonhumanPrimates natural infection of CDV. CDV cause great economic losses to fur animalbreeding industry and cause great harm to biodiversity, it has become into a serious public healthproblem and increasing attention. Traditional cell lines did not express the CDV receptor proteinand were very difficultly to isolate CDV, which severely restricted to study the etiologic andimmunologic of CDV. Especially in the immune prevention, the CDV MLVs has been used since1950s. Therefore, to establish the sensitive cell lines for efficiently isolating CDV, and study thefeatures of popular wild strain CDV, analysis the antigenic properties between vaccine and wildstrains, and development effective, safe, and affordable canine vaccines are still being sought,which has importantpracticalsignificance for CD prevention and control.I Establishment of sensitive cells expressing SLAM receptor for canine distempervirusisolation and identificationThe traditional methods for CDV isolation have several defects, such as low separation rate,time-consuming, high cost and so on, Therefore, it`s become particularly important to establishrapid methods for isolation CDV as soon as possible. In this study, vero cells expressing SLAMprotein, designated Vero/Dog SLAM cells, were established for study of CDV, and two wildstrains CDV were isolated form monkey and dog samples by this sensitive cell lines, and beendesignated Monkey-BJ01-DV and SC-CDV, respectively. Subsequently, we explored theutilization for different animal sources CDV to different species SLAM. The results showed that the virus can rapidly grew and produced distinct syncytia in Vero/DogSLAM cells at36-48-postinfection. Further studiesies found that monkey-CDV and dog-CDV can efficient use the bothSLAM of monkey and dog. The H gene of Monkey-BJ01-DV showed98.4%identity with CDVin dogs and foxes. And there are four specific mutation site(E276V、Q392R、D435Y和I542F) inH protein from carnivorous-CDV to monkey-CDV.II The immunization evaluation for the different vaccine strain CDV and characteristicsanalysis of the H proteinThe widespread use of modified live vaccines has greatly reduced the incidences in dogs, butCD epidemic frequently outbreaks in carnivores in recent years.The research showed that thecurrently commercial vaccine strains are different from the global epidemic wild strains CDV, thelatter may evade from the immune protection from vaccines. Pet owners and breeding farmleaders always choice import vaccines immune dogs to control this disease. But the background ofthese vaccines was unknown. In this study, we evaluated the immunization of above vaccine indogs and foxes, and sequence the CDV-H from imported vaccine form France, Russia,Netherlands, Korea and China, then compared the identify of H gene to the traditional vaccinestrains and popular wild strains. In vivo study (dogs and foxes) also confirmed that the Koreausing strain CDV had the best immunogenic.The results indicated that imported France vaccineusing Onderstepoortstrain CDV, the vaccine from Netherlands using Vaccine strain JP, and Chinavaccine using strain had a high identify with Snyder Hill strain, the Russia using strain also belongto the vaccine group of North America strain, and showed low identify with other traditionalvaccine strain. The Korea using strain had the highest identifies with wild strain, but not belong tovaccine group in the phylogenetic tree of H protein. These studies showed that the H protein fromwild strains had only87－90％identify with the H from vaccine strains. The H protein of vaccinestrains has only4asparagine-linked glycosylations, but currently epidemic wild strains haveevolved9asparagine-linked glycosylations, and the Korea using strain has the asparagine-linkedglycosylation in309(g3), which was the specificity glycosylation site in wild strains. The diversityof the H sequences may have a signify impact on neutralizingepitope among different strain CDV.In order to study the antigenic differences between the vaccine strains and the wild strains, weanalyzed the H glycoprotein of CDV. In this study, we used two of anti-H protein monoclonalantibodies that keeping the different affinity to the vaccine strains and wild strains: d-7and JD-7, and used point mutation and deletion methods to explore the antigenic differencesbetween the H protein from wild strains and vaccine strains. At last, we constructed the3-D modelfor CDV-H protein by Phyre2soft, and comparative analysis the H protein structure between thewild strain and vaccine strain, there are three conformationdifference domains in these two strains,they are located at234－246aa,302－313aa and415-418aa of H protein. We preliminary inferredthe antigenic different regions between the H protein of the vaccine and the wild protein. In thisstudy, we revealed the background of commercial vaccine, and found which had low identify ratewith the H protein from epidemic wild strain. This work hope to provide some guidance role formaking CD control measure, and provide a reference for vaccine strain screening in future.III Construction and preliminary application of infection clones for recombinant caninedistemper virus expression Il-18The current commercial vaccine strains are different from the global epidemic wild strainsCDV, the latter may evade from the immune protection from vaccines. It is necessary todevelopment the new vaccine or adjuvant for dogs to improve the cell-mediatedimmunity.Interleukin-18(IL-18) plays an important role in the T cell helper type1immuneresponse against intracellular parasites, bacteria, and viral infections and has widely been used asan adjuvant for vaccines and as an anti-cancer reagent. However, the IL-18gene lacks the typicalsignal sequence and requires cleavage into mature active form by caspases-1. Previous studieshave indicated that fusing the canine IL12p40signal sequence to the mature canine IL-18(cIL-18)can lead to the caspases-1-independent secretion of IL-18. In this study, we constructed vectorsencoding the mature cIL-18cDNA fused to the human IL-2(hIL-2) signal sequence, which canalso express and secrete mature cIL-18in a caspases-1-independent manner. We found that IL-18fused to hIL-2signal sequence could induce more IFN-γ production when transfected supernatantco-culture with canine PBMC than when it was fused to the cIL-12p40signal sequence. We alsoconstructed recombinant viruses expressing canine IL-18and mouse IL-18(mIL-18) fused to thehIL-2signal sequence using a cDNA clone of a field strain canine distemper virus to generaterCDV-hIL2ss-cIL18and rCDV-hIL2ss-mIL18, respectively. We found differences betweenrCDV-hIL2ss-cIL18and rCDV-hIL2ss-mIL18. At the same conditions, we detected both forms of canine IL-18: precursor IL-18(proIL-18,22kDa) and mature IL-18(18kDa) inrCDV-hIL2ss-cIL18infected cells and plasmid-transfected cells, but only detected mature IL-18in rCDV-hIL2ss-mIL18infected cells and plasmid resulting cells. The two resulting recombinantviruses displayed classical CPE and replicated similarly to the parental viruses in B95a cells. Asexpected, both recombinant viruses expressed mature canine or mouse IL-18that were thensecreted into the supernatant of infected cells, and the secreted cytokine was able to promoteinduction of canine or mouse IFN-γ, respectively. These results indicated that signal sequencefrom hIL-2was better than that from canine IL-12p40for the exogenous expression and secretionof mature IL-18. Furthermore, the rCDV-hIL2ss-cIL18-or rCDV-hIL2ss-mIL18-expressedmature IL-18have bioactive，meaning that these recombinant virus could potentially be used invivo and as immunoadjuvants and reagents for anti-cancer therapies in canine or mouse studies.