Abstract
Recombinant mumps viruses (MuVs) based on established vaccine strains represent attractive vector candidates as they have known track records for high efficacy and the viral genome does not integrate in the host cells. We developed a rescue system based on the consensus sequence of the L-Zagreb vaccine and generated seven different recombinant MuVs by (a) insertion of one or two additional transcription units (ATUs), (b) lengthening of a noncoding region to the extent that the longest noncoding region in MuV genome is created, or (c) replacement of original L-Zagreb sequences with sequences rich in CG and AT dinucleotides. All viruses were successfully rescued and faithfully matched sequences of input plasmids. In primary rescued stocks, low percentages of heterogeneous positions were found (maximum 0.12%) and substitutions were predominantly obtained in minor variants, with maximally four substitutions seen in consensus. ATUs did not accumulate more mutations than the natural MuV genes. Six substitutions characteristic for recombinant viruses generated in our system were defined, as they repetitively occurred during rescue processes. In subsequent passaging of primary rescue stocks in Vero cells, different inconsistencies within quasispecies structures were observed. In order to assure that unwanted mutations did not emerge and accumulate, sub-consensus variability should be closely monitored. As we show for Pro408Leu mutation in L gene and a stop codon in one of ATUs, positively selected variants can rise to frequencies over 85% in only few passages.
Highlights
Reverse genetics technology enables recovery of infectious, replication-competentRNA virions from plasmids with cloned complementary DNA and engineering of RNA viruses with specific genetic properties
Among non-segmented negative-sense RNA viruses, viruses produced using reverse genetics have been mostly based on the measles virus [1,2] or vesicular stomatitis virus [3], biotechnological platforms that enable viral rescue have been developed for many others including Mumps orthorubulavirus [4]
In order to ascertain that the variability we observed in viral populations did not stem from the variability of input plasmids, we analyzed the plasmids’ homogeneity by Next Generation Sequencing (NGS)
Summary
RNA virions from plasmids with cloned complementary DNA (cDNA) and engineering of RNA viruses with specific genetic properties. Such de novo production (rescue) of viruses from cDNA template encoding complete viral genome, with or without additional transcription units (ATUs), is a valuable tool in basic research of virus biology as well as in vaccine development and design of therapeutic biologicals. MuV genome is 15,384 nucleotides long and packed in helical nucleocapsid. It contains seven genes: the nucleocapsid (N), phospho- (P), matrix (M), fusion (F), small hydrophobic (SH), hemagglutinin-neuraminidase (HN) and large (L) protein gene [5,6,7]. P gene encodes two more proteins, V and
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