In vitro phenotypic markers of a poliovirus recombinant constructed from infectious cDNA clones of the neurovirulent Mahoney strain and the attenuated Sabin 1 strain

Abstract

Infectious cDNA corresponding to the entire genome of the attenuated Sabin strain of type 1 poliovirus has been inserted into EcoRI site of bacterial plasmid pBR325. Two consecutive PstI fragments (nucleotide positions 1814 to 3421) of the infectious cDNA of the Sabin 1 strain were replaced by the corresponding DNA fragments prepared from an infectious DNA clone of the genome of the virulent Mahoney strain of poliovirus type 1. The exchanged segment encodes capsid protein VP1 and part of capsid protein VP3, a region in which a large number of amino acid differences between the attenuated Sabin and the parental, neurovirulent Mahoney strain cluster. The recombinant virus was obtained by DNA transfection of HeLa S3 cells, and several in vitro phenotypes of the virus were compared with those of the parental viruses. The recombinant virus was recognized by a neutralizing monoclonal antibody specific to the Mahoney strain. Growth of the Sabin strain of poliovirus has been shown to be quite dependent upon the bicarbonate concentration (d marker). The growth of the recombinant virus, however, was not highly dependent upon the concentration of bicarbonate in cell culture media, and thus resembled that of the Mahoney strain. On the other hand, the temperature-sensitive multiplication (rct marker) and the small-plaque morphology of the recombinant virus corresponded to the phenotype of the Sabin 1 strain. The in vitro recombination of infectious cDNA clones of genomic RNA and subsequent analysis of the growth properties of the recombinant virus have allowed us to correlate specific mutations in the genome of an RNA virus with certain biological characteristics of that virus.

Growth phenotypes and biosafety profiles in poliovirus‐receptor transgenic mice of recombinant oncolytic polio/human rhinoviruses

“…The reason for the ts phenotype of the RIPO constructs described here, however, remains to be elucidated. As expected, the PVS(RIPO) ts phenotype is associated with mutations in two PV1(S) components as the capsid and 3D pol RNA polymerase are known to be determinants of this genetic trait [Kohara et al, 1985;Paul et al, 2000]. The very pronounced ts phenotype of the RIPO constructs in cells of neuronal origin co-varies with the remarkable attenuation phenotype of these constructs in CD155 tg mice, which leads us to speculate that ts of viral replication contributes to attenuation.…”

“…Attenuation of the Sabin vaccine strain PV1(S) is prominently associated with a ts phenotype [Lwoff, 1959;Kohara et al, 1985]. In search of hitherto unknown attenuation markers of the chimeric RIPO polioviruses, we determined the growth phenotypes of PV1(RIPO), PV1(RIPOS), and PVS(RIPO) and compared them with wt PV1(M).…”

Development of a new oral poliovirus vaccine for the eradication end game using codon deoptimization

“…Temperature sensitivity is a hallmark of many successful liveattenuated vaccines and was historically a criterion to confirm attenuation of Sabin OPV vaccine stocks prior to release by manufacturers 40 . The temperature-sensitivity growth profile of nOPV2-CD mirrored that of Sabin OPV2 in Vero cells, with replicative fitness declining as temperature was increased from 33°C to 38.5°C ( Fig.…”

Recombinant Polioviruses as Candidates for Oral Live Poliovaccines

“…To identify the genome regions that influence the neurovirulence or attenuation phenotype of poliovirus type 1, a number of recombinant viruses were constructed in vitro between the Mahoney and the Sabin 1 strains by using infectious cDNA clones of the two strains (11,13,25). Monkey neurovirulence tests on these recombinant viruses (13,25) revealed that the genome region of nucleotide positions 1 to 1,122, including mainly the 5' noncoding redion, harbors a relatively strong determinant(s) influencing the neurovirulence or attenuation phenotype, and that determinants weakly influencing the phenotye were spread over several areas of the entire viral genome including most of the viral capsid precursor protein region.…”

“…Monkey neurovirulence tests on these recombinant viruses (13,25) revealed that the genome region of nucleotide positions 1 to 1,122, including mainly the 5' noncoding redion, harbors a relatively strong determinant(s) influencing the neurovirulence or attenuation phenotype, and that determinants weakly influencing the phenotye were spread over several areas of the entire viral genome including most of the viral capsid precursor protein region. These results led to speculations that the rate of viral multiplication in the central nervous system might be one of the important factors determining the neurovirulence phenotype, and that virion surface characteristics such as antigenicity and immunogenicity have a little correlation with neurovirulence (11,22,25).…”