Defective RNAs of Semliki Forest virus

This thesis describes an investigation into the mouse-protecting nucleotide sequences of defective interfering (Dl) Semliki Forest virus (SFV) RNA was extracted from tissue culture preparations of Dl SFV and reverse transcribed Putative Dl SFV cDNA was amplified by the polymerase chain reaction using primers specific for the termini of the virion RNA A number of molecular clones were constructed from the products of amplification and the nucleotide sequences of two of these clones were determined (pSFVDI-6 2146 nts and pSFVDI-19 1244 nts) Both pSFVDI-6 and pSFVDI-19 were derived from three noncontiguous regions of the SFV genome comprising the 5' and 3' termini and part of the nsP2 coding region RNA transcribed from these clones was transfected into SFV-infected BHK-21 cells to produce genetically homogeneous Dl SFV preparations These preparations were stable on serial passage and interfered with virus multiplication m vitro The transfection technique was also used in a preliminary investigation of the regulatory elements of the SFV genome A 388- nucleotide region within the nsP2 gene of SFV was tentatively defined as containing all or part of a packaging signal since Dl SFV clones lacking this region were not propagated as virions. To determine the biological activity of the cloned Dl SFV preparations in vivo they were mixed with 10 LD₁₀, SFV and inoculated into adult mice by the intranasal route The Dl SFV preparation derived from pSFVDI-19 typically conferred 75% protection against the lethal encephalitis that normally follows infection with SFV. whereas the Dl SFV preparation derived from pSFVDI-6 was non-protecting However, it should be noted that the concentration of Dl SFV in these cloned preparations was not standardised Modulation of infection in vivo was independent of the antigenic load and mice were susceptible to subsequent lethal challenge A preliminary experiment suggested that propagation of Dl SFV genomes was cell-specific because genomes derived from pSFVDI-19, but not pSFVDI-6, could be detected in mouse brain tissue following intracerebral coinoculation of SFV with the cloned Dl SFV preparations