Ls. Wyatt et al., MARKER RESCUE OF THE HOST-RANGE RESTRICTION DEFECTS OF MODIFIED VACCINIA VIRUS ANKARA, Virology (New York, N.Y. Print), 251(2), 1998, pp. 334-342
The severely attenuated and host range (HR) restricted modified vaccin
ia virus Ankara (MVA) was derived by >500 passages in chick embryo fib
roblasts, during which multiple deletions and mutations occurred. To d
etermine the basis of the HR defect, we prepared cosmids from the pare
ntal vaccinia virus Ankara genome and transfected them into nonpermiss
ive monkey BS-C-1 cells that had been infected with MVA. Recombinant v
iruses that formed macroscopic plaques were detected after transfectio
ns with DNA segments that mapped near the left end of the viral genome
. Plaque-forming viruses, generated by transfections with four individ
ual cosmids and one pair of minimally overlapping cosmids, were purifi
ed, and their HRs were evaluated in BS-C-1 cells, rabbit RK-13 cells,
and human HeLa, MRC-5, and A549 cells. The acquisition of the K1L and
SPI-1 HR genes and the repair of large deletions were determined by po
lymerase chain reaction or pulse-field gel electrophoresis of Notl res
triction enzyme digests of genomic DNA. The following results indicate
d the presence of previously unrecognized vaccinia virus HR genes: (1)
the major mutations that restrict HR are within the left end of the M
VA genome because the phenotypes of some recombinants approached that
of the parental virus, (2) acquisition of the K1L gene correlated with
the ability of recombinant viruses to propagate in RK-13 cells but di
d not enhance replication in human or monkey cell lines, (3) acquisiti
on of the SPI-1 gene correlated with virus propagation in A549 cells b
ut not with the extent of virus spread in monkey or other human cell l
ines, (4) there are at least two impaired HR genes because rescue occu
rred with nonoverlapping or minimally overlapping cosmids and recombin
ant viruses with intermediate HRs were isolated, and (5) at least one
of the new HR genes did not map within any of the major deletions beca
use the size of the left terminal Notl fragment was not appreciably al
tered in some recombinant viruses.