Recombination-Mediated Genetic Engineering of a Bacterial Artificial Chromosome Clone of Modified Vaccinia Virus Ankara (MVA) - Genetic Engineering: Recent Developments in Applications - page 47

but the possibility of some bias in the isolation procedure necessitates verification
of clone fidelity by complete sequencing, especially prior to embarking on a pro-
gramme of genetic manipulation.
Sequence of MVA-BAC
Three MVA genome sequences are available in GenBank, one published by An-
toine et al. [5] (U94848), one deposited by Bavarian Nordic GmbH (DQ983236)
and the “Acambis 3000” strain (AY603355) deposited by the CDC. The Bavarian
Nordic sequence is identical to the Acambis sequence, and these differ from An-
toine et al.'s sequence only by five single nucleotide substitutions (excluding the
repeat regions of the inverted terminal repeats, which are not fully described in
the Acambis and Bavarian Nordic sequences). All of these loci are very polymor-
phic amongst strains of Vaccinia virus (http://www.poxvirus.org/). The mutations
and genes affected are shown in Table 1.
Table 1. Polymorphisms in MVA and MVA-BAC clone #26.
MVA-BAC clone #26 was selected for shotgun sequencing at eightfold cov-
erage, resulting in a single contig (excluding the repeat regions), and was found
to be identical to the Acambis/Bavarian Nordic strain, with the exception of a
6 bp insertion in the non-essential gene F7L [63]. The mutation codes for an
extra Asn-Lys repeat and makes MVA-BAC identical to the TanTien strain of
Vaccinia virus at this polymorphic locus, where other strains have two Asn-Lys
repeats, with the exception of the Copenhagen strain which has eight (http://
www.poxvirus.org/). In order to determine at what point in the genesis of MVA-
BAC this mutation occurred, we sequenced the progenitor viruses at this locus by
PCR amplification from genomic DNA. MVA-BAC-parent was found to possess
the 6 bp insertion mutation, but its progenitor (from which the recombinant
was derived) did not. Sequencing of PCR products revealed that a selection of
other MVA recombinants from our laboratory and our original stock provided by
Anton Mayr did not possess the mutation. It is not clear whether this 6 bp