Genetic and fitness changes accompanying adaptation of an arbovirus to vertebrate and invertebrate cells

The alternating host cycle and persistent vector infection may constrain th e evolution of arboviruses, To test this hypothesis, eastern equine encepha litis virus was passaged in BHK or mosquito cells, as well as in alternatin g (both) host cell passages. High and low multiplicities were used to exami ne the effect of defective interfering particles. Clonal BHK and persistent mosquito cell infections were also evaluated. Fitness was measured with on e-step growth curves and competition assays, and mutations were evaluated b y nucleotide sequencing and RNA fingerprinting. All passages and assays wer e done at 32 degrees C to eliminate temperature as a selection factor. Viru ses passaged in either cell type alone exhibited fitness declines in the by passed cells, while high-multiplicity and clonal passages caused fitness de clines in both types of cells, Bypassed cell fitness losses were mosquito a nd vertebrate specific and were not restricted to individual cell lines. Fi tness increases occurred in the cell line used for single-host-adaptation p assages and in both cells for alternately passaged viruses. Surprisingly, s ingle-host-cell passage increased fitness in that cell type no more than al ternating passages. However, single-host-cell adaptation resulted in more m utations than alternating cell passages. Mosquito cell adaptation invariabl y resulted in replacement of the stop codon in nsP3 with arginine or cystei ne, In one case, BHK cell adaptation resulted in a 238-nucleotide deletion in the 3' untranslated region. Many nonsynonymous substitutions were shared among more than one BHK or mosquito cell passage series, suggesting positi ve Darwinian selection. Our results suggest that alternating host transmiss ion cycles constrain the evolutionary rates of arboviruses but not their fi tness for either host alone.