Selection of classical swine fever virus with enhanced pathogenicity reveals synergistic virulence determinants in E2 and NS4B.
Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious disease of pigs. There are numerous CSFV strains that differ in virulence, resulting in clinical disease with different degrees of severity. Low-virulent and moderately virulent isolates cause a mild and often chronic disease, while highly virulent isolates cause an acute and mostly lethal hemorrhagic fever. The live attenuated vaccine strain GPE(-) was produced by multiple passages of the virulent ALD strain in cells of swine, bovine, and guinea pig origin. With the aim of identifying the determinants responsible for the attenuation, the GPE(-) vaccine virus was readapted to pigs by serial passages of infected tonsil homogenates until prolonged viremia and typical signs of CSF were observed. The GPE(-)/P-11 virus isolated from the tonsils after the 11th passage in vivo had acquired 3 amino acid substitutions in E2 (T830A) and NS4B (V2475A and A2563V) compared with the virus before passages. Experimental infection of pigs with the mutants reconstructed by reverse genetics confirmed that these amino acid substitutions were responsible for the acquisition of pathogenicity. Studies in vitro indicated that the substitution in E2 influenced virus spreading and that the changes in NS4B enhanced the viral RNA replication. In conclusion, the present study identified residues in E2 and NS4B of CSFV that can act synergistically to influence virus replication efficiency in vitro and pathogenicity in pigs.
Laboratory of Microbiology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
Tomokazu
T
Tamura
Yoshihiro
Y
Sakoda
Fumi
F
Yoshino
Takushi
T
Nomura
Naoki
N
Yamamoto
Yuka
Y
Sato
Masatoshi
M
Okamatsu
Nicolas
N
Ruggli
Hiroshi
H
Kida
D000220:Adaptation, Biological; D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D002460:Cell Line; D006691:Classical Swine Fever / Q000473:pathology / Q000821:virology; D006692:Classical Swine Fever Virus / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity*; D004195:Disease Models, Animal; D006168:Guinea Pigs; D014066:Palatine Tonsil / Q000821:virology; D012692:Serial Passage; D013552:Swine; D014613:Vaccines, Attenuated / Q000235:genetics; D014759:Viral Envelope Proteins / Q000235:genetics*; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D014774:Virulence; D037521:Virulence Factors / Q000235:genetics*
C080107:NS4B protein, flavivirus; D014613:Vaccines, Attenuated; D014759:Viral Envelope Proteins; D017361:Viral Nonstructural Proteins; D037521:Virulence Factors; C087086:glycoprotein E2, classical swine fever virus