Establishment of an efficient reverse genetic system of Mumps virus S79 from cloned DNA

Abstract

BackgroundMumps is a common type of respiratory infectious disease caused by mumps virus (MuV), and can be effectively prevented by vaccination. In this study, a reverse genetic system of MuV that can facilitate the rational design of safer, more efficient mumps vaccine candidates is established.MethodsMuV-S79 cDNA clone was assembled into a full-length plasmid by means of the GeneArt™ High-Order Genetic Assembly System, and was rescued via reverse genetic technology. RT-PCR, sequencing, and immunofluorescence assays were used for rMuV-S79 authentication. Viral replication kinetics and in vivo experimental models were used to evaluate the replication, safety, and immunogenicity of rMuV-S79.ResultsA full-length cDNA clone of MuV-S79 in the assembly process was generated by a novel plasmid assemble strategy, and a robust reverse genetic system of MuV-S79 was successfully established. The established rMuV-S79 strain could reach a high virus titer in vitro. The average viral titer of rMuV-S79 in the lung tissues was 2.68 ± 0.14 log10PFU/g lung tissue, and rMuV-S79 group did not induce inflammation in the lung tissues in cotton rats. Neutralizing antibody titers induced by rMuV-S79 were high, long-lasting and could provide complete protection against MuV wild strain challenge.ConclusionWe have established a robust reverse genetic system of MuV-S79 which can facilitate the optimization of mumps vaccines. rMuV-S79 rescued could reach a high virus titer and the safety was proven in vivo. It could also provide complete protection against MuV wild strain challenge.

Development of Improved Mumps Vaccine Candidates by Mutating Viral mRNA Cap Methyltransferase Sites in the Large Polymerase Protein

“…Recently, cotton rats have been regarded as improved small animal model for human paramyxoviruses and pneumoviruses including studying viral infection and pathogenesis, testing vaccine efficacy and evaluating antiviral drugs (Boukhvalova and Blanco 2013;Green et al 2013). In our previous research, we found viruses can be detected in the lung of the cotton rat after mumps infection as in IFN-a/b R -/mice (Zhou et al 2019b). In this study, we found that neither the parental rMuV-S79 nor wild type MuV strains caused significant histologic lesions in the lungs of unvaccinated cotton rats.…”

“…In this study, we used BHK cells stably expressing T7 RNA polymerase to rescue rMuVs, which prevents the risk of being contaminated by vaccinia virus. This method has been approved in other studies for rescue of human metapneumovirus, bovine respiratory syncytial virus and measles virus (Ma et al 2014;Sun et al 2014;Zhang et al 2014;Cui et al 2017;Pang et al 2018;Wang et al 2018;Zhou et al 2019b). We have effectively rescued rMuV-S79 without helper vaccinia virus in our previous researches (Zhou et al 2019a(Zhou et al , 2019b.…”

“…We hypothesized that reducing MTase activity can further improve the safety and immunogenicity of mumps vaccines. Using the robust reverse genetics system of Chinese S79 vaccine strain (MuV-S79) established in our previous research (Zhou et al 2019b), we generated a total of eight recombinant MuVs with amino acid substitutions in the MTase domain of the L protein. These rMuVs mutant strains were significantly more attenuated in cell culture compared with the parental vaccine strain and were genetically stable when passing repeatedly in cell culture for 10 passages.…”

Mumps Vaccines: Current Challenges and Future Prospects

“…A DNA vaccination platform was used to design a full-length complementary DNA (cDNA) clone of recombinant MuV-S79 mumps virus by reverse genetics ( Zhou et al, 2019 ). The cDNA clone was administered intranasally to cotton rats to test its efficacy as a potential vaccine.…”

“…The experimental vaccine elicited an efficient, long-lasting neutralizing antibody immune response, as evidenced by sera obtained and tested 4 weeks after immunization. The vaccine induced complete protection against challenge with a wild-type mumps strain ( Zhou et al, 2019 ).…”