Pathogenic murine coronaviruses III. Biological and biochemical characterization of temperature sensitive mutants of JHMV

JHMV is a neurotropic member of the hepatoencephalitis group of murine coronaviridae. The characteristics of the biology and intracellular viral RNA synthesis and the intracellular viral protein synthesis of JHMV are discussed in the two previous papers, respectively. This paper describes the neuropathogenesis of JHMV and the isolation and characterization of 34 temperature-sensitive mutants of JHMV. These mutants were selected for their inability to induce syncytia formation after low multiplicity infection (m.o.i. = 0.1 iU) in BALB/c 17CL-1 cells at 38.5° as compared to the induction of syncytia at 33°. N-Methyl-N′-nitrosoguanidine (14 mutants) and 5-fluorouridine (20 mutants) were used as mutagens at a concentration that reduced infectivity by 90–95%. Characterization of these mutants included: induction of syncytia; synthesis of JHMV-specific intracellular RNA; progeny yields at 33, 37, and 38.5°; synthesis of JHMV-specific antigens as determined by indirect immunofluorescence and sodium dodecyl sulfate-polyacrylamide gel electrophoresis; virion thermostability; neuropathogenesis including isolation of virus from infected brain, immunofluorescence of infected brain, and histopathology of brain and spinal cord by light and transmission electron microscopy; ability to protect mice from a lethal JHMV infection; and complementation. RNA-minus (1734), RNA-intermediate (1434), and RNA-plus (334) groups were defined. One mutant, N3, produces chronic meningitis and demyelination without typical JHMV encephalitis in spite of the fact that neurons are infected as detected by immunofluorescence. This altered neuropathogenesis cannot be explained by “leakiness” or reversion. In addition, non-temperature-sensitive variants of JHMV have been selected for altered neuropathogenesis and are described.