Plaque mutants of western equine encephalitis virus
Creators: name: Ushijima, Richard Naoyuki
Publisher: name: University of Utah
Year: 2012

description: Two mutant viruses differing in the production of plaque morphology were isolated from a culture of Western equine encephalitis (WEE) virus strain W-4, which was propagated from an isolated plaque originally measuring 5 mm in diameter on monolayer cell cultures of primary chick embryo after 54 hours of incubation.  Plaques developed by the mutant virus SP-6 were 2 mm in diameter compared to 8 mm by the mutant virus LP-7.  Studies were made to determine what physical factors were responsible for the differences in plaque diameters produced by the mutant WEE viruses.  Other properties or these two mutants were also investigated.  Both of the mutant viruses were readily neutralized by standard Wee virus antiserum obtained from the Communicable Disease Center, Atlanta, Georgia.  Cross neutralization was also observed with antisera prepared against each mutant virus.  The Sp-6 and LP-7 viruses propagated by infecting HeLa, L, or guinea pig culture cells and from infected mice brains retained their phenotypic characteristics on monolayer of click embryo cells.  Varying the concentration of calf serum from 1.5 to 10% in the agar overlay medium resulted in the production of plaques with  diameters characteristic of each mutant virus.  Horse serum in similar concentrations caused the plaques diameters produced by each mutant to be suppressed by 50%,  Differences in plaque size were not observed with agar overlay media prepared from either purified or unpurified agar.  However, neutralization of an inhibitor in agar with DEAE-Sephadex resulted in the formation of SP-6 plaques measuring 5 mm in diameter.  The LP-7 plaques produced were 8mm.  When methylcellulose was substituted for agar in the overlay medium the SP-6 and  Lp-7 plaques that developed were considerably smaller in diameter proportionately.  Monolayer cultures of guinea pig kidney cells infected with selected SP-6 and LP-7 viruses produced plaques with proportionately smaller diameters, but plaques by these cells were only demonstrable when the agar overlay medium contained DEAE-Sephadex.  Although homologous interference was readily demonstrable by chick embryo cells incubated with inactivated Sp-6 virus particles prior to infection with active SP-6 and LP-7 viruses, homologous interference was not found responsible for the suppressed diameter of the SP-6 plaques.  The rate of absorption on monolayer cell cultures of chick embryo and rates of inactivation at 37 C in PBS and growth medium were found to be similar for both mutant viruses.  Three separate growth curve experiments were made with chick cells infected with each mutant virus.  Infected cells incubated as monolayers with growth medium of  agar overlay medium extract or in suspension demonstrated similar results in relation to the lag period of virus release.  The LP-7 virus infected cells released newly synthesized virus within 1 ½  hours, whereas a lag of approximately 3 to 3 ½ hours was evident for SP-6 infected cells.  At 12 hours each SP-6 virus infected cell released approximately 100 plaque forming units (PFU) when incubated as monolayer or cell suspension with growth medium, but when the infected monolayer cells were incubated with agar overlay medium extract only 16 PU/cell  were released.  The LP-7 virus infected cells released within 1100 to 2000 PFU/cell when incubated under the conditions described.  Attempts to select mutants from Sp-6 and LP-7 viruses which were readily able to infect HeLa, L, Fl, mouse embryo, and hamster kidney were not successful.  Each mutant virus was highly lethal for "wet" chicks and suckling mice injected by subcutaneous and intracerebral routes respectively.  Fewer than 2 PFU was found sufficient to kill the experimental animals within 5 days.