Biological properties of bovine viral diarrhea virus quasispecies detected in the RK13 cell line

Abstract

The rabbit kidney cell line RK13 has been reported to be contaminated with noncytopathogenic (ncp) bovine viral diarrhea virus (BVDV). Persistent infection was confirmed by demonstrating the stability of virus titers (10(4.6±0.5) TCID50/ml) and BVDV positive cells (71.9 ± 3.12 %), over six successive passages. Based on the "exaltation of Newcastle disease virus" (END) and reverse plaque formation methods, two types of ncp viruses were isolated, END-phenomenon-positive and negative. Isolates, RK13/E(+) and RK13/E(-), demonstrated (1) differing levels of reproducibility in cell cultures, (2) similar antigenicity against BVDV antisera, (3) identical 5'-UTR region nucleotide sequences, (4) four amino acid differences throughout the genomic open reading frame, and (5) better growth ability in primary rabbit cells than other laboratory strains when inoculated in parallel at an MOI of 0.01. Overall, the BVDV population in RK13 cells consists of at least two different END characteristic quasispecies that are adapted to cultures of rabbit origin, giving rise to naturally attenuated BVDV strains that can be used in vaccine development.

Complete Genome Sequence of Noncytopathic Bovine Viral Diarrhea Virus 1 Contaminating a High-Passage RK-13 Cell Line

“…Interestingly, the ncpBVDV HP-RK-13 [KY] strain is very closely related to the previously described ncpBVDV present in RK-13 cells reported from Japan (RK13/E - strain [GenBank accession no. JX419397.1]; 12,064 nt, 99.7% identity) ( 18 ). The ncpBVDV HP-RK-13 [KY] strain had several nucleotide insertions and deletions compared to several of the other BVDV-1b strains.…”

Analysis of a pair of END⁺ and END⁻ viruses derived from the same bovine viral diarrhea virus stock reveals the amino acid determinants in N^pro responsible for inhibition of type I interferon production

“…However, to the best of our knowledge, there were no studies with BVDV that had approached the basic mechanism of the inhibition of type I interferon production using the amino acid differences between a pair of END + and END − viruses as well as viruses genetically engineered on the basis of these differences. Pestiviruses are quasispecies; single strains consist of populations with various characteristics, such as END + and END − [ 17 ]. We cloned a pair of END + and END − viruses (GBK_E + and GBK_E − ) from the BVDV GBK strain using reverse plaque formation techniques [ 5 , 19 ].…”

Quantitative and rapid interference of bovine viral diarrhea virus BVDV/END– and BVDV/END+ strains

“…Cell culture and viruses. Madin-Darby bovine kidney (MDBK-SY) cells were grown and confirmed to be free of adventitious pestivirus (tested by RT-PCR) as previously described (Muhsen et al, 2013a). MDBK-SY cells were seeded into 96-well flat-bottom plate microplates at a density of 2×10 5 cells/plate (~2×10 3 cells /well).…”

“…NDV Miyadera strain and VSV New Jersey serotype were used as heterologous challenge viruses for BVDV/E + and /Estrains, respectively (Muhsen et al, 2013b). The BVDV titre was calculated by endpoint dilution method, according to the property of END interference as previously described (Muhsen et al, 2013a).…”