Vairimorpha necatrix: Infectivity for and development in a lepidopteran cell line

Vairimorpha necatrix infected and replicated in the Heliothis zea cell line HZBCIRLAM1. Spores were purified from caterpillars by Percoll density gradient centrifugation. After priming with 0.01 n KOH in 0.17 m KCl, the spores were mixed with H. zea cells suspended in Tris-HCl (pH 8) containing 0.17 m KCl, and EDTA as a chelator. Infected cells were cultured at 28°C in L-15B medium with 5% serum. Sporoplasms and early stages were noted in cells stained with Giemsa's solution within the first 24 hr post-infection (pi). The sporoplasms developed into bipolar meronts but did not divide during this time. Using 5 to 10 spores per cell, we initially infected 23.64 ± 4.27% of the cells in three duplicate studies. Three-fourths of the infected cells harbored a single sporoplasm, and the remainder were infected with two to four parasites each. The proportion of infected cells rose to 36.03 ± 7.02% during the first 2 to 4 days, and after 1 week, meronts were rarely seen. Between day 1 and 3 pi V. necatrix multiplied with a doubling time of 8 to 12 hr. Three to 4 days pi, V. necatrix sporonts were seen and by 6 days pi mature spores developed. Within the first 4 days of culture, large multinucleated syncytia formed by cell fusion. The number of host cell nuclei in syncytia correlated with the number of V. necatrix; those with 3 to 5 H. zea nuclei and 20 to 40 V. necatrix were most common. There was considerable asynchrony in parasite development and variability in the number of V. necatrix per host cell.