Reverse genetics system for porcine enteric calicivirus, a prototype sapovirus in the Caliciviridae

Abstract

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.

FIG. 1.

Genomic organization of Cowden PEC and the construction of full-length cDNA clone, pCV4A. ORF1 of PEC was amplified by RT-PCR and cloned into pCRXL. A second cDNA clone containing the subgenomic region of PEC in pSPORT was identified in a cDNA library of the PEC genome. With the use of unique enzyme sites (AccI and NotI), the subgenomic region of Cowden PEC in pSPORT was engineered into the pCRXL/ORF1 cDNA clone. The full-length PEC genome was then cloned into pCDNA3.1 to generate intermediate plasmid pCV4 (not shown). The plasmid pCV4A was generated by removing pCMV and T7 promoter (provided by pCDNA3.1) by digestion with BglII and NheI and religation of the plasmid.

FIG. 2.

Comparison of proteins synthesized from clone pCV4A in a TNT reaction with those produced in PEC-infected cells. At 24 h after PEC infection in the presence of IC, cells were [³⁵S]methionine-labeled for 4 h, and cell lysates were prepared in RIPA buffer. RIPA results of the TNT reaction and cell lysates of PEC infection with the hyperimmune guinea pig sera (pre- and postimmunization) against PEC VPg (lanes 1 to 4), polymerase (lanes 5 to 8), capsid (VLPs) (lanes 9 to 12), or VP2 (lanes 13 to 16) are shown. The tentative final cleavage products consistent with the expected masses of PEC VPg, ProPol, and capsid are marked with arrows. The VP2 protein is indicated with an asterisk. Lane 17, radiolabeled proteins from a coupled TNT reaction of pCV4A.

FIG. 3.

IFA with hyperimmune guinea pig serum against VLP of PEC at 72 h after transfection of RNA transcripts from pCV4A and incubation with IC (1%) (A), mock MEM (B), or GCDCA (200 μM) (C). (D) EM observation of cell lysates at 96 h after transfection of RNA transcripts from pCV4A and incubation in the presence of IC is shown in panel C. Cell lysates were concentrated 100 times by ultracentrifugation, and virus particles were directly observed by EM after negative staining.

FIG. 4.

(A) Growth kinetics of PEC after transfection of RNA transcripts from pCV4A in the presence of IC (1%) or GCDCA (200 μM) by ELISA. RNA transcripts (1 μg) were transfected in LLC-PK cells with Lipofectamine 2000, and the transfected cells were incubated with IC or GCDCA for the desired period. (B) Growth kinetics of Cowden PEC and pCV4A viruses in the presence of IC (1%) or GCDCA (200 μM) by ELISA. The viruses were inoculated at an MOI of 0.5.

FIG. 5.

Plaque-forming assay of pCV4A viruses measured at dilutions of 10⁻⁴ (left cultures) and 10⁻⁵ (right cultures) in the presence of IC (1%) (A) or GCDCA (200 μM) (B). After LLC-PK cells were infected for 1 h with the viruses recovered from pCV4A, the cells were placed under medium containing MEM, agarose (1%), neutral red (0.002%), and IC (1%) or GCDCA (200 μM). Pictures were taken 96 h after virus inoculation.