Enteric caliciviruses
cause diarrhea in pigs and are the leading cause of food-borne viral
gastroenteritis in humans. Our goal is the molecular analysis of the
attenuated, tissue culture-adapted [TC] porcine
enteric calicivirus (PEC/Cowden strain),
identified as a sapovirus (SaV), and
generation of synthetic RNA transcripts. Because this strain grows in pig
kidney (LLC-PK) cells, but only with intestinal contents (IC) from
uninfected gnotobiotic pigs in the medium, we investigated the relationship
between IC and growth of Cowden PEC. Pretreatment of cells or the virus with
IC or transfection of viral RNA into cells did not promote virus growth
unless medium was supplemented with IC. After various pretreatments of IC
and size exclusion fractionation of IC, we found that the IC effective
factor(s) for virus growth was protein(s) (<50,000 MW). Several inhibitors
related to modulation of cell signal transduction inhibited the IC effects
on PEC virus growth in a dose dependent manner for up to 72 hrs. Although
the cAMP level did not differ between IC and mock treated cells, it was
significantly decreased by treatment of cells with the inhibitors for up to
36 hrs. The viral replicase complex was isolated from virus infected cells
only in the presence of IC at 30 hrs, and actively synthesized genomic and
subgenomic RNA of PEC in vitro.
We previously reported
only 6 amino acid changes with 3 clustered in the capsid region, between
wild-type (WT) and TC PEC strains. Thus we are conducting studies to
investigate the genetic basis for the reduced virulence and cell culture
adaptation of the attenuated TC/PEC by generation of an infectious clone
of the TC/PEC strain. Purified genomic TC/PEC RNA was used to transfect
LLC-PK cells. Virus growth in this system was dependent on the presence
of IC preparation in the cell culture medium as is required for passage
of TC/PEC in LLC-PK cells. Several full-length TC/PEC clones were
constructed and in vitro transcription-translation assays using some of
these clones yielded mature viral proteins. Recently the infectivity of
a full lenght clone of PEC in LLC-PK cells was confirmed.
