| Foot-and-mouth disease (FMD) and Peste-des-petits ruminants (PPR) are both severe and highly contagious diseases of domestic (goats and sheep) and wild ruminants, which caused by foot-and-mouth disease virus (FMDV) and peste-des-petits ruminants virus (PPRV) respectively, and both classified into list I of animal diseases in China. There are no effective medical therapies to treat the PPR and FMD, but only through vaccines inoculation to prevent and control. Inactivated vaccine is used widely to control FMD in goats and sheep, however, it is expensive and has a short immunity duration. PPR attenuated vaccine (N75/1strain) has been confirmed to be safe and effective potency to prevent the PPR disease in prevalent countries of Africa and Asia. In developing countries, including China, both FMD and PPR are epidemic and cause great losses every year. A recombinant live vaccine expressing protective antigen of FMDV with PPRV (N75/1strain) vector might prevent PPR and FMD at the same time. The combined vaccine could not only reduce prevention costs and increase immune coverage, but also make up the shortfall of inactivated vaccine to activate the cell-mediated immune response. This study would be aim to testify feasibility of the above strategy.For this object, total RNA was firstly extracted from the strain of FMD (type Asial) inactivated vaccine and the first strand cDNA were synthesized by reverse transcription. The VPl gene was obtained from the cDNA by PCR and cloned into the pET-21a plasmid for expressing in E. coli BL21. The purified recombinant VP1protein was inoculated rabbits to prepare polyclonal antibody for several times. The titer of the prepared antibody was1:8192in indirect ELISA. Furthermore, indirect ELISA was established using the expressed VP1protein as coating antigen to detect antibody against FMDV (type Asial) in bovine sera.Secondly, based on the established reverse genetics systems of PPRV (N75/1), the recombinant PPRV expressing VPl gene of FMDV (type Asial)was constructed. In brief, VP1gene was inserted between P and M gene of PPRV/N75/1clone, then inserted into pN75/1-insertion and named pN75/1-VP1. The pN75/1-VP1was transfected into Vero cells together with the helper plasmids of pCA-N, pCA-P and pCA-L is using lipofatamine2000. The recombinant virus was rescued and named rPPRV-VP1. The growth curves of rPPRV-VP1were similar to PPRV (N75/1) in Vero cells, up to107.7TCID50/mL. Analysis of IFA and Western-blot showed that the recombinant virus expressed antigens of PPRV and VP1of FMDV. During the25successive passages in Vero cells, the recombinant virus kept stably expression of VP1. In inoculated goats with rPPRV-VP1, the antibody against PPRV and FMDV were both detected, titers were up to peak in56dps, and kept stably to140dps. In inoculated goats with an overdose of rPPRV-VP1, all the goats were healthy in the28days, which indicated that rPPRV-VP1was safe for goats.Goats and bovines were inoculated subcutaneously with6×106TCID50doses of rPPRV-VP1, respectively. In the goat experiments, the sera were collected on different days after vaccination and VNA titers against PPRV and FMDV were determined. The results showed that antibody against PPRV and FMDV were both conversed to positive after14days inoculation in the rPPRV-VP1vaccine group. The VNA titers against PPRV among the four time points of the two groups were not significantly different. However, the VNA titers against FMDV among the four time points were very significantly different At the40th day, all of the two group goats were inoculated virulent FMDV strain to evaluate the challenge potency. In the rPPRV-VP1vaccines group, four of the six goats had no clinical symptoms of FMD in the end of observation periods and the other two had a less serious symptoms than the four PPRV/N75/1vaccines control goats. In the bovine experiments, the sera were also collected on different days after vaccination and VNA titers against PPRV and FMDV were tested, and at the21st day, all of the bovines were inoculated virulent FMDV strain. The results showed that the VNA titers against PPRV among the three time points of the two groups were either not significantly different, however, the VNA titers against FMDV were also very significantly different. In the challenge test, two of the five bovines(2/5) had no clinical symptoms of FMD in the end of observation periods and the other three had less serious symptoms than the two PPRV/N75/1vaccnined control bovines.In summary, the study constructed a recombinant rPPRV-VP1to express VP1protein of FMDV (type Asial) with the established reverse genetics systems of PPRV(N75/1). The recombinant rPPRV-VPl had a good safety and could stimulate vaccined goats or bovines to produce antibodies against PPRV and FMDV at the meantime, furthermore, it could protect them entirely or partly from a virulent FMDV challenge. The studies showed that the novel FMDV recombinant vector vaccine using PPRV(N75/1strain) was feasible and might be an attractive candidate vaccine for preventing PPR and FMD simultaneously. |
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