An improved system to generate recombinant canine distemper virus

Abstract

Background: Canine distemper virus (CDV) is a pathogen with the capability of cross-species transmission. It has crossed the species barrier to infect many other species, and its host range is expanding. The reverse genetic platform, a useful tool for scientific research, allows the generation of recombinant viruses from genomic cDNA clones in vitro.

Methods: To improve the reverse genetic system of CDV, a plasmid containing three independent expression cassettes was constructed for co-expression of the N, P, and L genes and then transfected with a full-length cDNA clone of CDV into Vero cells.

Results: The results indicated that the established rescue system has the advantages of being more convenient, easy to control the transfection ratio, and high rescue efficiency compared with the conventional reverse genetics system.

Conclusion: This method not only reduces the number of transfection plasmids, but also improves the rescue efficiency of CDV, which could provide a reference for the recovery of other morbilliviruses.

Keywords: Canine distemper virus; Minireplicon; Negative-sense RNA; Recombinant virus; Reverse genetic system.

Fig. 1

Schematic diagrams of the constructs used in the study. (A) The full-length cDNA clone of CDV ZJ. The full-length viral cDNA was flanked by hammer-head ribozyme (HamRz) and hepatitis delta ribozyme (HdvRz) sequences at both terminals of the viral genome. The six overlapping fragments and the overlapping regions are shown below the genome. Transcription of the plasmid is under the control of the CMV promoter and SV40 polyA signal. (B) Strategy for constructing the CDV minigenome. The minigenome is composed of the 3’ leader, the N gene start signals (GS), the noncoding region (NCR) of the N gene, EGFP, the 5′ NCR of the L gene, the L gene end signals (GE) and the 5′ trailer, which was inserted into the same vector used for the generation of vial cDNA clones. (C) Co-expression of CDV N, P, and L genes in one plasmid. The plasmid pCMV-3MCS was derived from the pUC57 vector by introducing three CMV promoters and polyadenylation signals, which contained three independent expression cassettes for multiple gene expression. Cloning strategies and individual plasmids (pCMV-N, pCMV-P, pCMV-NP, pCMV-L, and pCMV-NPL) are shown

Fig. 2

Analysis of plasmids transfection (magnification 100 x). (A) The minireplicon was produced by transfection of the pCMV-CDVmini plasmid into Vero cells, and RdRP was generated by virus incubation before plasmid transfection. The production and replication of viral microreplicons depend on the viral polymerase supplied by CDV-infected cells. (B), (C), (D), and (E) Analysis of different rescue systems based on minigenome expression. (B) BSR cells were transfected with 2.0 µg of pCMV-CDVmini plasmid and 3 µg of pCMV-NPL plasmid. (C) BSR cells were transfected with 2.0 µg of pCMV-CDVmini plasmid, 1 µg of pCMV-NP and 1 µg of pCMV-L. (D) BSR cells were transfected with 2.0 µg of pCMV-CDVmini plasmid, 0.5 µg of pCMV-N, 0.5 µg of pCMV-P, and 1 µg of pCMV-L. (E) BSR cells were transfected with 2.0 µg of pCMV-CDVmini plasmid. (F) Cytopathic effect (CPE) induced in Vero cells co-transfected with plasmids pCMV-NPL and puCMVZJ. Arrows indicate syncytia in the cells that were observed at 5 days after transfection. The arrow indicates the formation of syncytia in the cells, which was observed 5 days after transfection. (G) Control cells. (H) Sequencing results of the mutation sites in the recombinant virus genome. Asterisks demonstrate that the A → G and A → T substitutions at viral nucleotides 8975 and 8976 to create a Pme I restriction site in the recombinant viral genome

Fig. 3

Growth curve comparison of CDV ZJ and rCDV-ZJ in Vero cells. Cells were infected with parental and recombinant virus at an MOI of 0.01 for 120 h. Viral titers were determined as the number of TCID₅₀/ml in an endpoint titration assay. The data are shown as the mean of triplicates ± SE