Reverse genetics system for porcine enteric calicivirus, a prototype sapovirus in the Caliciviridae.
A porcine enteric calicivirus (PEC), strain Cowden in the genus Sapovirus of the Caliciviridae family, can be propagated in a porcine kidney continuous cell line (LLC-PK) in the presence of bile acids in the cell culture medium. A full-length cDNA copy of the Cowden PEC genome was cloned into a plasmid vector directly downstream from the T7 RNA polymerase promoter, and capped RNA transcripts derived from this clone were infectious when transfected into LLC-PK cells. The recovery of PEC after transfection of RNA transcripts was dependent on the presence of bile acids, consistent with our recent identification of a bile acid-mediated signaling pathway required for PEC replication (Chang et al., Proc. Natl. Acad. Sci. USA 101:8733-8788, 2004). Recovery of virus was verified by detection of PEC antigen in transfected cells by immunofluorescence and enzyme-linked immunosorbent assays, direct observation of recovered viral particles by electron microscopy, and partial sequence analysis of their genomes (first 1,070 nucleotides) to differentiate them from tissue culture-adapted parental virus. The recovered virus retained its ability to infect piglets when administered by the oral route and showed an attenuated phenotype similar to that of the tissue culture-adapted parental virus. This reverse genetics system for PEC provides a new tool to study the molecular basis of replication and pathogenesis for caliciviruses associated with diarrheal disease.
Chang
Kyeong-Ok
KO
Laboratory of Infectious Diseases, National Institutes of Health/DHHS, NIAID/LID, Bldg. 50, Room 6316, 9000 Rockville Pike, Bethesda, MD 20892-8026, USA. kchang@niaid.nih.gov
Sosnovtsev
Stanislav V
SV
Sosnovtsev
Stanislav S
SS
Belliot
Gaël
G
Wang
Qiuhong
Q
Saif
Linda J
LJ
Green
Kim Y
KY
D000818:Animals; D017250:Caliciviridae Infections / Q000503:physiopathology / Q000821:virology; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary; D016679:Genome, Viral; D005856:Germ-Free Life; D018374:LLC-PK1 Cells; D029341:Sapovirus / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology*; D013552:Swine; D013553:Swine Diseases / Q000503:physiopathology / Q000821:virology; D014162:Transfection; D014774:Virulence; D014779:Virus Replication
D018076:DNA, Complementary