Characterization of Helper Virus-Independent Cytopathogenic Classical Swine Fever Virus Generated by an In Vivo RNA Recombination System

Abstract

Molecular analyses revealed that most cytopathogenic (cp) pestivirus strains evolve from noncytopathogenic (noncp) viruses by nonhomologous RNA recombination. In contrast to bovine viral diarrhea virus (BVDV), cp classical swine fever virus (CSFV) field isolates were rarely detected and always represented helper virusdependent subgenomes. To investigate RNA recombination in more detail, we recently established an in vivo system allowing the efficient generation of recombinant cp BVDV strains in cell culture after transfecting a synthetic subgenomic and nonreplicatable transcript into cells being infected with noncp BVDV (A. Gallei, A. Pankraz, H.-J. Thiel, and P. Becher, J. Virol. 78:6271-6281, 2004). Using an analogous approach, the first helper virus-independent cp CSFV strain (CP G1) has now been generated by RNA recombination. Accordingly, this study demonstrates the applicability of RNA recombination for designing new viral RNA genomes. The genomic RNA of CP G1 has a calculated size of 18.139 kb, almost 6 kb larger than all previously described CSFV genomes. It contains cellular sequences encoding a polyubiquitin fragment directly upstream of the nonstructural protein NS3 coding gene together with a duplication of viral sequences. CP G1 induces a cytopathic effect on different tissue culture cell lines from pigs and cattle. Subsequent analyses addressed growth kinetics, expression of NS3, and genetic stability of CP G1.Classical swine fever virus (CSFV), the causative agent of an economically important disease in pigs, forms together with the species Bovine viral diarrhea virus 1 (BVDV-1), BVDV-2, and Border disease virus (BDV) the genus Pestivirus, family Flaviviridae (11,22). Highly virulent CSFV strains can cause a severe form of classical swine fever, characterized by pyrexia; hemorrhages in the skin, mucosa, and inner organs; anorexia; diarrhea; and, in late stages, central nervous disorders resulting in high morbidity and mortality (48). However, most CSFV strains isolated from recent outbreaks in Europe led to chronic forms of disease with less characteristic symptoms (16).Pestiviruses are small enveloped viruses encompassing a single-stranded RNA genome of positive polarity with a length of about 12.3 kb. The viral genomic RNA is neither capped nor polyadenylated and contains one large open reading frame (ORF) flanked by 5Ј and 3Ј nontranslated regions (NTR); the latter harbor cis-active elements essential for viral translation and replication. The ORF encodes a polyprotein of approximately 3,900 amino acids (aa) that is co-and posttranslationally processed by cellular and viral proteases to the mature viral proteins (22,29,36,48).For pestiviruses, cytopathogenic (cp) and noncytopathogenic (noncp) strains are distinguished by their ability to cause a cytopathic effect (CPE) in susceptible tissue culture cells (18,28). For BVDV it is well documented that cp strains mostly evolve by nonhomologous RNA recombination in animals persistently infected with noncp BVDV. The emergence of cp BVDV in persistent...

Genetically distinct pestiviruses pave the way to improved classical swine fever marker vaccine candidates based on the chimeric pestivirus concept

“…In the “RaPro” construct, the first 492 nucleotides (position 1618–2109) of the chimeric E rns encoding sequence are derived from NrPV followed by 186 nucleotides (position 1660–1845) of the Pronghorn pestivirus sequence. The chimeric pestivirus vaccine candidates were generated on basis of the infectious cDNA clone pAlfort-p447 of CSFV strain Alfort-p447 [ 14 , 15 ] . The sequence of the cDNA plasmid used in this study (pAlfort-p2477) is almost identical to the sequence previously published for CSFV Alfort-p447, which contains two additional guanine residues at its 5′ genome end (GenBank LT593760).…”

Analysis of classical swine fever virus RNA replication determinants using replicons

“…For each replicon to be assayed, 1 ml SFT-R cells (2×10 6 ml −1 ) was centrifuged and resuspended in the same volume of cold PBS and kept on ice. An aliquot (800 µl, 1.6×10 6 cells) was transferred to a 0.4 cm cuvette (Bio-Rad) and 2.5 µg RNA was added, briefly mixed and introduced into the cells by electroporation (950 µF and 180 V on a Gene Pulser XCell; Bio-Rad) essentially as described elsewhere (Gallei et al , 2005). After electroporation, cells were allowed to settle at room temperature for 10 min before seeding into six-well plates [250 µl per well (~5.0×10 5 cells) with 3 ml DMEM containing 5 % FCS].…”

Epidemiology, diagnosis and control of classical swine fever: Recent developments and future challenges

“…While a number of genetic changes in various coding and non‐coding regions of the genomes of CSFV and related pestiviruses resulted in reduction of viral replication and decreased virulence in the natural host, final conclusions with regard to molecular determinants of virus attenuation require additional studies (Leifer, Ruggli, & Blome, ; Makoschey et al., ; Risatti et al., , ; Van Gennip, Vlot, Hulst, De Smit, & Moormann, ). One promising way for generation of attenuated CSFV was the establishment of cytopathogenic (cp) CSFV strains by introducing genetic elements from cp BVDV strains into the genome of CSFV (Gallei, Rumenapf, Thiel, & Becher, ; Gallei et al., ). The cp CSFV strain Alfort‐Jiv replicated to high titres in cell culture and, in contrast to the parental, moderately virulent non‐cytopathogenic CSFV strain did not compromise the type 1 interferon‐mediated antiviral defence of the host.…”