| Porcine circovirus (PCV) has been assigned to the family Circoviridae and the genus Circovirus which can be divided into two genotypes:including PCV1 and PCV2. PCV1 was first described 25 years ago as a contaminant of the permanent PK-15 cells and it is nonpathogenic to pigs. PCV2 is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS) and several syndromes collectively known as porcine circovirus associated disease (PCVAD). PCV2 is divided into two mainly subtypes (PCV2a and PCV2b). PCV2a is further subdivided into five clusters,2A-2E, and PCV2b, into three clusters,1A, 1B and 1C. The Cap gene sequence of cluster 1A was very consistent with cluster 1B, but diverse from cluster 1C. In cluster 1C, the Cap gene is different from clusters 1A, 1B, and harbor a additional lysine (K) at the end of ORF2.PCV2 was first identified in western Canada in 1991, and have had a major impact on the global swine industry and are recognized as economically important diseases. It has been shown that vaccination is a major tool to reduce PCVAD losses in swine populations. The application of these vaccines plays an important part in prevention and control of PCVAD. However, the commercial PCV2 vaccines could not eliminate the transmission and infection of the virus in pigs. Moreover, these vaccines are in high price. It is the main research trend to develop safe, efficient and inexpensive vaccines. In this study, we compared the transfection efficiency of two different transfection methods in both PK-15 and Dulac cells, the results demonstrated that the Dulac cells are more permissive to PCV1-2b infection than PK-15 cells, and then we developed the inactivated and live-attenuated PCV1-2b vaccines, the efficacy of these vaccines were evaluted in growing pigs and suckling pigs.1. Optimal transfection methods and comparison of PK-15 and Dulac cells for rescue of chimeric porcine circovirus type 1-2bIn this study, we optimized some parameters of electroporation and lipofection in PK-15 and Dulac cells for elevating the efficiency of rescuing infectious PCV1-2b and will be useful for PCV1-2b vaccines production.The results indicated that Dulac cells showed superior to PK-15 cells and PCV1-2b viral titers in Dulac cells were 100-fold higher than that in PK-15 cells at least. When properly handling, Dulac cells displayed remarkably high transfection efficiency at 250 V,400 μs,3 pulses,6 μg plasmid DNA or with DNA (μg):transfection reagent (μl) at 1:3. The percentage of infected cells was 55.6% compared to 44.2%, treated with electroporation and lipofection respectively, evaluated by FACS. Subsequent serial passages, the virus titers were higher in Dulac cells, ranged from 104.44 to 105.32 TCID50/mL compared to 101.90 to 103.38 TCID50/mL in PK-15 cells. Thus the Dulac cells were more permissive to PCV1-2b proliferation than PK-15 cells when transfected by either electroporation or lipofection.2. Comparative efficacy of experimental inactivated and live-attenuated PCVl-2b chimeric vaccines using a PCV2b challenge modelFirstly, A pairs of specific primers and TaqMan probe was designed according to the conserced sequence of PCV2 ORF1 for preparation of standard for recombinant plasmid. The TaqMan real-time fluorescent quantitative PCR method was developed and verified for sensitivity, specificity and reproducibility. Results showed that the correlation coefficient (R2) and the slope of the standard curve were 0.999 and -3.55 respectively, which showed a good linear relationship. The sensitivity of this method was 5.0 copies/μL. No amplification curves were obtained from PCV1-2, PCV1, CSFV, PRRSV, PRV, PPV genomic DNA or cDNA. The variation coefficients of determination results of eight PCV2-positive templates were 0.98%-1.03%. In conclusion, the development of TaqMan real-time fluorescent quantitative PCR method showed high sensitivity, specificity and reproducibility, which was used to detect the experimental PCV2 challenged pigs.Next, the efficacy of an inactivated (2×105.0 TCID50 dose) and live-attenuated (2×104.0 TCID50 dose) PCV1-2b chimeric vaccines was compared in conventional pigs, a commercial inactivated PCV2b vaccine was served as a control. The results indicated that all vaccinated pigs were seroconverted to PCV2 rapidly and pigs vaccinated with live-attenuated PCV1-2b vaccine developed higher concentrations of anti-PCV2 antibodies more earlier than those vaccinated compared with the inactivated vaccine. As expected, vaccination reduced PCV2 genomic copies in serum, SILN sample and PCV2 antigen in tissues sections compared to non-vaccinated but challenged pigs. Vaccination with live-attenuated PCV1-2b resulted in lower PCV2 genomic levels in serum than inactivated PCV1-2b vaccine. PCV2 viremia was prevented in live-attenuated PCV1-2b immunized pigs at 21 DPC, but PCV2 DNA were detected in one pig in activated PCV1-2b vaccine group (105.55 copies/ml). PCV2 genomic DNA was detected in the superficial inguinal lymph nodes (SILN) in two pigs in two vaccinated groups (108.28、107.50 copies/g and 105.93、105.57 copies/g). Although the live-attenuated PCV1-2b immunized pigs were lower than the other one, there were no significant differences among groups (P>0.05). In this study, the observed PCV2-associated microscopic lesions were milder in the lung and lymphoid tissues in all vaccinated pigs compared to the non-vaccinated but challenged pigs. PCV2 DNA can be detected in oral, nasal, faecal from both naturally and experimentally infected pigs. In this study, PCV2 shedding in vaccinated pigs were significant lower compared with the non-vaccinated but challenged pigs (P<0.05). Therefore, this chimeric PCV1-2b virus could not only be a good candidate as an inactivated vaccine but also as the live-attenuated vaccine against PCV2 infection.3. Effects of maternal antibodies in piglets induced by inactivated chimeric PCVl-2b vaccine protection against PCV2b challengeThe efficacy of inactivated PCV1-2b chimeric vaccines was evaluated in growing pigs in chapter three. In this study, three sows were immunized with inactivated chimeric PCV1-2b vaccine, and we evaluated the effects of maternal antibodies response to experimental PCV2 infection in 14,28 and 42 day piglets.The results showed that the maternal antibodies were highest in 14 day-old piglets, average at 1:300 IFA antibody titers, and then decreased in 28 and 42 day-old piglets, average at 1:133 and 1:70 IFA antibody titers. There were no maternal antibody in 49 day-old piglets. PCV2 viremia was prevented in 14 day-old piglets excepted at 14 DPC (104.90 copies/ml) correlated with lower lymphoid tissues lesions, and PCV2 antigen was undetected by IHC. PCV2 DNA was detected in two pigs in 28 day-old challenge group at 14 and 21 day post challenge (DPC) respectively, harbored 10536 copies/ml,10639 copies/ml and 107’84 copies/ml,107.90 copies/ml PCV2 DNA. Due to lower antibody level. Quantification of viral genomic copies in serum and SILN in 42 day-old piglets were correlated with that of PCV2 genomic copies in challenged pigs in chapter three at the necropsied day, average at 106.31 copies/ml and 101077 copies/g. Degradation to lymphoide structures and PCV2 antigen were observed. In conclusion, the results from this study indicated that the levels of PCV2 maternal antibodies are an important determinant of a piglet response to PCV2 infection. It appears that the higher levels of maternal antibodies (14 day-old pigs), the more protection the piglets will have. The optimal timing of first vaccination was determined at 28 day postnatal.4. Inactivated chimeric PCV1-2b vaccine based on genotype 2b cluster 1B and 1C were compared in conventional pigs modelIn this study we constructed a novel chimeric PCV1-2b virus, which contain the capsid gene of the PCV2b-1B subtype in the genomic backbone of the non-pathogenic PCV1, and to determine if there are differences in protecting against PCV2b-1B challenge.The rescued chimeric PCV1-2b-1B or PCV1-2b-1C virus proliferated stable in Dulac cells and displayed propagation dynamics similar to each other. Vaccinated pigs was capable of inducing high levels of antibodies titers, leading to lower amount of the PCV2b DNA copies load. Whereas, there were no significant differences in humoral immune response between the two vaccinated and challenged groups. In PCV2b-1B vaccinated pigs, the group mean antibodies and NAs levels were slightly higher than in PCV2b-1C vaccinated pigs throughout the experiment, and preventing the viremia at 14 and 21 DPC, no PCV2 antigen was present in superficial inguinal lymph nodes (SILN) by qPCR and IHC; In PCV2b-1C vaccinated pigs, one pig had detectable viral DNA in serum (105.75 copies/ml) and SILN samples (108.16 copies/g) at necropsy, associated with slight histological lesions and low amounts of PCV2 antigen in lymph nodes. The shedding of the PCV2 virus in faecal and nasal secretions were significant lower compared with the non-vaccinated but challenged pigs (P<0.05). To sum up, the present study demonstrated that the ORF2 gene from PCV2b-1B or PCV2b-1C genotype in the backbone of PCV1 did not influence viral propagation, both experimental PCV1-2b-1B and PCVl-2b-1C vaccine exhibits similar effectiveness against PCV2b-1B challenge. |
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