FIG. 1.

Construction and confirmations of recombinant MYXVs. (A) Construction of vMyxM062-KO. The central 78% of MYXV M062R sequence was replaced by an eGFP expression cassette under a viral early/late promoter using the three-element recombination of the MultiSite Gateway system. The revertant virus of vMyxM062-KO was constructed by replacing the eGFP expression cassette in the knockout virus with an intact M062R sequence. (B) Confirmation of purified vMyxM062-KO. PCR analysis using primers 1 and 2 (Table 3) designed to recognize M062R coding sequences (shown in panel A) will generate a 474-bp fragment with wild-type MYXV viral DNA as a template (lane 2) and a 1,077-bp fragment using either the recombination plasmid (lane 3) or purified vMyxM062-KO viral DNA (lane 4) as a template. The molecular markers are in lane 1. (C) Confirmation of the purified vMyxM062-Rev. The set of screening primers used in panel B was used to determine whether vMyxM062-Rev contained any contaminating vMyxM062-KO. Lane 1, molecular marker; lane 2, vMyx-Lau viral DNA; 3, vMyxM062-KO DNA; 4, no template DNA; 5, DNA of vMyxM062-Rev. (D) Construction of vMyx-M062V5. Using the MultiSite Gateway system, a V5 epitope sequence was inserted just before the stop codon of the M062R. In addition, a cassette containing a VACV p11 late promoter-driven eGFP expression was inserted following the V5-tagged M062R and in front of the intact M063R promoter region. Primers 1 and 3 (Table 3) were used for screening during the purification process. (E) The construction of vMyx-M062V5-M063-KO. MultiSite Gateway system was used to insert the V5 sequence before the stop codon of M062R, VACV p11 late promoter-driven eGFP, and 115 bp of the 3′-terminal sequence of M063R in front of an intact M064R promoter sequence. Primers 1 and 3 were used for screening during the process of purification.