The Consequences of Reconfiguring the Ambisense S Genome Segment of Rift Valley Fever Virus on Viral Replication in Mammalian and Mosquito Cells and for Genome Packaging
Cells were infected with rMP12 or rMP12:S-Swap (MOI of 1), and total cell RNA isolated at 48 h p.i. Northern blotting was performed using DIG-labelled probes complementary to the N, NSs, and Gn coding regions in the viral genomic (−) sense RNA and the N coding region in the viral anti-genomic (+) sense RNA. The polarity of the RNA detected by each probe is indicated below the blot: G, genomic sense RNA and AG, anti-genomic sense RNA, defined by the sequence of the 3′/5′ untranslated regions. B. S segment derived mRNAs produced in mosquito cells. A. albopictus C6/36, U4.4 cells or A. aegypti Ae were infected with rMP12 or rMP12:S-Swap at MOI of 1, and total cellular RNA extracted at the indicated times p.i. Northern blotting was performed using the N(+) and NSs(−) probes. C. Analysis of RNA packaged into virions. RNA was extracted purified rMP12 or rMP12:S-Swap grown in BHK-21 cells, and Northern blotting performed with the probes as detailed in (A) above. D. Quantitative RT-PCR analysis of viral RNAs. Total cellular RNA (upper panels) or RNA in purified virus particles (lower panels) of rMP12 or rMP12:S-Swap was analysed using probes specific form the S or M segments as described in Methods. The results are presented as the percentage of genomic RNA species compared to the total RNA of the same segment. Cell line abbreviations: B, BHK-21; C, C6/36; U, U4.4; and A, Ae.