Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: July 17, 2011
Publication Date: November 17, 2011
Citation: Newcomer, B.W., Neill, J.D., Marley, M.S., Zhang, Y., Galik, P.K., Riddell, K.P., Ridpath, J.F., Givens, M.D. 2011. Characterization of bovine viral diarrhea virus isolates resistant to a novel antiviral compound obtained from persistently infected calves [abstract]. In: Proceedings of the U.S. Bovine Viral Diarrhea Virus Symposium, November 17-18, 2011, San Diego, California. p. 87. Technical Abstract: The objective of this research was to characterize isolates resistant to a novel antiviral compound (DB772) isolated from persistently infected (PI) calves treated with the compound. Viral isolates were obtained from four Angus-cross beef calves (A,B,C,D) persistently infected with BVDV type 1 or 2 and treated with DB772 intravenously three times a day for six days. Virus obtained from each calf on Day 0 before treatment initiation was shown to be susceptible to DB772 in vitro. Viral strains obtained during the treatment period (Day 7 for Calf C and Day 3 for Calf D) or following treatment (Day 70 for Calf A and Day 56 for Calf B) which exhibited in vitro resistance to DB772 were selected for further study. All viruses were purified by selecting clones created by serial dilutions of virus isolates from passaged white blood cell samples in the absence (susceptible samples) or presence (resistant samples) of 4 uM DB772. Full genome sequencing was obtained on all pre-treatment and resistant isolates. Additionally, the growth kinetics of each isolate were studied by infecting MDBK cells in 96-well plates at a multiplicity of infection of 3 and incubated in the presence and absence of 4 uM DB772. After incubation for the appropriate time period (1, 3, 5, 7, 9, 11, 13, 23, 48, or 72 hours) plates were frozen to stop viral replication. Viral titrations were performed using the immunoperoxidase staining assay. Full genome sequencing revealed an asparagine to aspartic acid mutation in three isolates (B, C, D) at residue 264 (N264D). In addition, the resistant isolate from Calf C demonstrated an isoleucine to methionine change at residue 261 (I261M) and a proline to alanine mutation at residue 262 (P262A) was present in the resistant isolate from Calf B. The resistant isolate from Calf A replaced a tyrosine at residue 289 with histidine (Y289H). All mutations were found in the NS5B gene. The resistant isolate from Calf D containing the single mutation appeared less fit than the susceptible isolate obtained pre-treatment while the remaining resistant isolates replicated at similar levels to isolates sensitive to DB772. Resistance to DB772 is associated with single or multiple mutations in the NS5B gene which encodes the RNA-dependant RNA polymerase suggesting the mechanism of action of DB772 is through inhibition of the NS5B protein. Administration of DB772 holds potential as part of a multi-modal approach to pestivirus treatment and control.