Generation of defective interfering particles of vesicular stomatitis virus in Aedes albopictus cells

Abstract

The serial undiluted passage of clonally purified vesicular stomatitis virus (VSV) in Aedes albopictus cells resulted in a rapidly declining titer of infectious virus. VSV AP2 (a stock derived after two such passages) contained slowly sedimenting truncated (T) particles which were interfering and noninfectious. Whereas a VSV stock produced by three undiluted passages in BHK cells (VSV BP3) interfered with the replication of standard virus (VSV STD) equally well in A. albopictus and in BHK cells, VSV AP2 interfered efficiently only in the former. These patterns of interference were also observed when gradient purified T particles were tested. When VSV AP2 or VSV BP3 was passaged once in the heterologous cell type the pattern of interference was reversed showing that these defective interfering (DI) particles could be phenotypically modified in such a way as to influence their specificity of interference with VSV STD. When viral RNA synthesis was studied in cells coinfected with VSV STD and VSV AP2 or VSV BP3, the following observations were made: (1) interference was correlated with a decrease in the synthesis of 42 S VSV STD genome RNA; (2) although VSV AP2 DI particles interfered well in A. albopictus cells but only poorly in BHK cells the DI RNA genomes of VSV AP2 were replicated equally well in both cell types. (3) The major DI particles in VSV AP2 contained “snap-back” RNA composed of covalently linked sequences of positive and negative polarity. By testing the effect of RNase on these RNAs it was concluded that they contained from 90 to 95% selfcomplementary sequences. Snap-back RNA was not readily detectable in the DI particles of VSV BP3.