Session topic


Title: Resistance phenotype of Varicella Zoster virus polymerase polymorphisms generated by two-step homologous recombination
ID: O 66
Type: Abstract talk
Talk time: 12 + 3 min
Session: Workshop 11
Antivirals and resistance II

Speaker: Ruben Rose (Kiel/DE)

Abstract - Text

Abstract text (incl. references and figure legends)

Varicella zoster virus (VZV) causes chickenpox, persists in sensory ganglia, and leads to shingles upon reactivation. Herpes zoster can have severe complications like encephalitis or postzosteric neuralgia. Antiviral agents like aciclovir (ACV) or foscarnet (FOS) are available for the treatment. However, the emergence of therapy-resistant VZV has been observed especially in immunocompromised patients. Early detection of drug resistance is essential for rapid therapy adjustment and patient recovery. Resistance is mostly caused by point mutations within the viral thymidine kinase or the viral DNA polymerase (ORF28) genes. Sequencing of viral DNA isolated from the patient allows for immediate determination of drug resistance when resistance-mediating mutations within these genes are functionally defined.

In this study, five recombinant VZVs were constructed, each carrying a point mutation within ORF28, and their resistance phenotypes were determined. Single nucleotides within ORF28 were exchanged by site-directed mutagenesis, generating the amino acid exchanges T417I, D645Y, G723R, R753K, and M874I. The mutated ORF28 was seamlessly introduced into bacterial artificial chromosomes (BACs) carrying an ORF28 deletion mutant of the VZV strain HJO by en passant mutagenesis, a two-step homologous recombination procedure in E. coli. In parallel, all these single-nucleotide exchanges were introduced into a recombinant viral genome expressing a red fluorescent protein (RFP). The recombinant viruses were reconstituted from the BACs in permissive human melanoma cells. The viruses were fully replication competent and did not show relevant differences in their replication kinetics. The susceptibility of the recombinant viruses for the antiviral agents ACV and FOS and the half-maximal inhibitory concentrations were determined by plaque-reduction assays. Expression of RFP enabled the direct visualization of plaques and cytopathic effects by fluorescence microscopy. The results suggest that T417I, D645Y, G723R, and R753K are natural polymorphisms, whereas ACV susceptibility may be slightly reduced in the case of M874I. T417I, D645Y, G723R, and R753K were classified as natural polymorphism with regards to FOS resistance, whereas FOS resistance was shown to be conferred by the mutation M874I.