Genomic Changes in Coxsackievirus B1 Induced by Consecutive Alternating Administration of Antienteroviral Inhibitors

Abstract from Fourth National Congress of Virology with International Participation /Days of Virology in Bulgaria Sofia, May 18th - 20th, 2016

Petar Grozdanov, Ivanka Nikolova, Adelina Stoyanova, Patsy Polston, Marie-Line Joffret, Francis Delpeyroux, Angel S. Galabov

The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria

Institut Pasteur, Paris, France

Enterovirus (EV) infections are a significant cause of morbidity and mortality throughout the world. EVs have been associated with many human diseases, including myocarditis, insulin-dependent diabetes, chronic inflammatory myopathy and hand, foot, and mouth disease (HFMD). Currently, clinically effective antivirals for use in the treatment of enteroviral (EV) infections do not exist due to the development of drug resistance at monotherapy.

The most important achievement of our previous studies was the development of a novel scheme for in vivo application of a triple combination of EV inhibitors with different modes of action against Coxsackievirus B (CVB) infections in mice. It consists of consecutive alternating administration (CAA) of the substances in the combination. Here, we report whole-genome sequences of CVB1 isolated from newborn mice treated with pleconaril, MDL 860, and oxoglaucine (PGO) via mono therapy or via CAA in search of changes in viral RNA on molecular level, which occurs after treatment.

The degree of identity between the whole genome sequences was around 99.88%. We found no deletions or insertions but only nucleotide substitutions in the studied samples. Seven nucleotide substitutions were discovered when comparing wild CVB1/ placebo with pleconaril treated CVB1/pleconaril 25mg/kg - T61C (5’UTR), T1867C (VP3 region), A3528G (2A region), C4051T (2C region), T4893C (2C region), T4908C (2C region) and T5692C (region).

The amino acid alignment (AA) derived from these sequences showed three amino acid substitution - H945R (2A region), L1400P (2C region) and I1405T (2C region). Five nucleotide substitutions were found when we compared wild CVB1/placebo with MDL 860 treated CVB1/MDL 860 25mg/ kg – A1957T (VP3 region), G2285A (VP1 region), A4846G (2C region), G4869A (2C region), T4908C (2C region) which lead to three amino acid substitutions – V531I (VP3 region), R1392K (2C region) and I1405T (2C region). When we compared wild CVB1/placebo with CVB1/PMO CAA course, three nucleotide substitutions were discovered - C568T (5’UTR), C3509T (2A region), T3803C (2B region), T4908C (2C region), respectively in AA two amino acid changes were found – P939S (2A region) and I1405T (2C region). These phenotypic and genotypic mutations lead to viral drug resistance in the case of monotherapy (pleconaril or MDL860) or to a higher sensitivity of the virus to the CAA treatment with the triple combination of enterovirus replication inhibitors.

The observed increased drug-sensitivity and lack of drug-resistance as a result of the CAA treatmen course with the triple antiviral combination could be attributed to the established mutations both at 5’UTR (which regulate the virus protein expression) and at 2A protein region (where the non-polar amino acid proline was substituted by the polar, non-charged amino acid serine).