Marker Rescue of the Host Range Restriction Defects of Modified Vaccinia Virus Ankara

Abstract

The severely attenuated and host range (HR) restricted modified vaccinia virus Ankara (MVA) was derived by >500 passages in chick embryo fibroblasts, during which multiple deletions and mutations occurred. To determine the basis of the HR defect, we prepared cosmids from the parental vaccinia virus Ankara genome and transfected them into nonpermissive monkey BS-C-1 cells that had been infected with MVA. Recombinant viruses that formed macroscopic plaques were detected after transfections with DNA segments that mapped near the left end of the viral genome. Plaque-forming viruses, generated by transfections with four individual cosmids and one pair of minimally overlapping cosmids, were purified, 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 polymerase chain reaction or pulse-field gel electrophoresis of NotI restriction enzyme digests of genomic DNA. The following results indicated the presence of previously unrecognized vaccinia virus HR genes: (1) the major mutations that restrict HR are within the left end of the MVA 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 did not enhance replication in human or monkey cell lines, (3) acquisition of the SPI-1 gene correlated with virus propagation in A549 cells but not with the extent of virus spread in monkey or other human cell lines, (4) there are at least two impaired HR genes because rescue occurred with nonoverlapping or minimally overlapping cosmids and recombinant 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 because the size of the left terminal NotI fragment was not appreciably altered in some recombinant viruses.

Highly attenuated smallpox vaccine protects mice with and without immune deficiencies against pathogenic vaccinia virus challenge

“…The block in replication occurs at a late step in virus assembly (6,13) and is caused by multiple gene defects (16). Because of its extreme attenuation, no adverse effects were reported even when high doses of MVA were given to immune-deficient non-human primates (17).…”

Smallpox vaccines: New formulations and revised strategies for vaccination

“…In consequence, MVA shows a characteristic growth deficiency phenotype upon in vitro infection with unimpaired viral protein synthesis in all human cells being tested. 41,[43][44][45] Moreover, the in vivo inoculation of MVA goes along with a strong stimulation of innate host responses resulting in the synthesis of type I interferons, CC chemokines, and in the attraction of host leukocytes to the site of injection. 46,47 Since other studied VACV strains seem not to have similar immunostimulatory capacities this feature is unique to MVA and might contribute to the potency of MVA based vaccines.…”

The role of CCR5 in vaccinia virus pathogenesis

“…The suspension was then centrifuged (454). Although poxviruses possess well-organized replication machinery that allow them to infect a wide spectrum of eukaryotic hosts in vitro, some poxviruses are host restricted (173,174,192,215,286,288,421,670,763). In these restrictive cells, poxviruses efficiently enter target cells, but fail to complete their replicative life cycle.…”

“…In a subsequent publication, Johnston et. al identified a role for the serine-threonine kinase, PAK-1, in myxoma virus replication, and suggested that the permissiveness of target cells to infection resides in their ability to activate this kinase (314 Although VACV infects a variety of cells, some poxviruses fail to grow in certain host cells in vitro, a phenomenon known as host restriction (173,174,192,215,286,288,421,670,763). In these restrictive cells, poxviruses enter the cell efficiently but fail to replicate.…”