EVOLUTION OF MOUSE HEPATITIS-VIRUS - DETECTION AND CHARACTERIZATION OF SPIKE DELETION VARIANTS DURING PERSISTENT INFECTION

High-frequency RNA recombination has been proposed as an important mec hanism for generating viral deletion variants of murine coronavirus. I ndeed, a number of variants with deletions in the spike glycoprotein h ave been isolated from persistently infected animals. However, the sig nificance of generating and potentially accumulating deletion variants in the persisting viral RNA population is unclear. To study this issu e, we evaluated the evolution of spike variants by examining the popul ation of spike RNA sequences detected in the brains and spinal cords o f mice inoculated with coronavirus and sacrificed at 4, 42, or 100 day s postinoculation. We focused on the S1 hypervariable region since pre vious investigators had shown that this region is subject to recombina tion and deletion. RNA isolated from the brains or spinal cords of inf ected mice was rescued by reverse transcription-PCR, and the amplified products were cloned and used in differential colony hybridizations t o identify individual isolates with deletions. We found that 11 of 20 persistently infected mice harbored spike deletion variants (SDVs), in dicating that deletions are common but not required for persistent inf ection. To determine if a specific type of SDV accumulated during pers istence, we sequenced 106 of the deletion isolates. We identified 23 d istinct patterns of SDVs, including 5 double-deletion variants. Furthe rmore, we found that each mouse harbored distinct variants in its cent ral nervous system (CNS), suggesting that SDVs are generated during vi ral replication in the CNS. Interestingly, mice with the most severe a nd persisting neurological disease harbored the most prevalent and div erse quasispecies of SDVs. Overall, these findings illustrate the comp lexity of the population of persisting viral RNAs which may contribute to chronic disease.