Abstract
In reverse genetic experiments we have isolated recombinant mumps viruses (rMuV) that carry large numbers of mutations clustered in small parts of their genome, which are not caused by biased hyper-mutation. In two separate experiments we obtained such recombinant viruses: one virus had 11 mutations in the V/P region of the genome; the other, which also contained an extra transcription unit encoding green fluorescent protein (EGFP), had 32 mutations in the N gene. These specific sets of mutations have not been observed in naturally occurring MuV isolates. Unusually, the vast majority of the mutations (48/51) were synonymous. On passage in Vero cells and human B-LCL cells, a B lymphocyte-like cell line, these mutations appear stable as no reversion occurred to the original consensus sequence, although mutations in other parts of the genome occurred and changed in frequency during passage. Defective interfering RNAs accumulate in passage in Vero cells but not in B-LCL cells. Interestingly, in all passaged samples the level of variation in the EGFP gene is the same as in the viral genes, though it is unlikely that this gene is under any functionality constraint. What mechanism gave rise to these viruses with clustered mutations and their stability remains an open question, which is likely of interest to a wider field than mumps reverse genetics.
Highlights
Mumps virus (MuV) is a human pathogenic RNA virus in the genus Rubulavirus in the family Paramyxoviridae [1]
We have demonstrated here that once these clusters of mutations were generated the resulting virus populations were genetically stable when passaged 6 times with undiluted inoculum to allow for the maximum chance of the fixation of mutations
Deep sequencing of the virus RNAs did not show a bias towards reversion to the original nucleotide in the MuVG09 sequence
Summary
Mumps virus (MuV) is a human pathogenic RNA virus in the genus Rubulavirus in the family Paramyxoviridae [1]. This family of non-segmented negative stranded RNA viruses shares basic replication strategies with the other viruses in the order Mononegavirales. MuV has a genome of 15,384 nucleotides (nt) in length, which contains 7 transcription units from the 3’ end of the negative stranded genome to the 5’ end respectively These encode respectively the nucleocapsid protein (N); the innate immune modulatory protein V, the matrix protein (M), the fusion protein (F), a small hydrophobic protein (SH), a haemagglutinin-neuraminidase protein (HN) and the large protein (L) which carries the RNA-dependent RNA polymerase.
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