Objectives
Human enteric
caliciviruses (HuCV) are a leading cause of foodborne illness and acute
gastroenteritis. The lack of an animal disease model or in vitro culture
system impedes studies of the pathogenesis and host immunity to human
caliciviruses (HuCV). We discovered a porcine enteric calicivirus (PEC)
Cowden strain that is genetically closely related to the HuCV
Sapoviruses (SaV). Similarly other researchers described bovine enteric
caliciviruses (BEC) from calves and PEC from pigs that were most closely
related to the norovirus (NoV) genus of HuCV. These reports raise
concerns about the zoonotic potential of animal enteric caliciviruses.
Our goals were to detect and characterize additional strains of calf and
pig enteric caliciviruses and compare them genetically to HuCV. We also
studied the pathogenesis of PEC, HuCV, and BEC in gnotobiotic (Gn) pigs
and calves as animal models of disease. Using the RT/PCR assay, with
primer sets designed for HuCV detection, and immune electron microscopy
(IEM) we identified BECs in 2 Ohio veal herds. The 3.5 kb 3' genomic
sequences of 2 US BEC strains designated BEC CV-95 OH and CV-186 OH were
determined and were found to be related to European BEC strains, Jena
and NA-2 and in a distinct genogroup (GIII) from current HuCV-NoVs.
Using degenerate primers, we determined the complete (7,453 bp, NB
strain) and partial (3,205 bp, CV-23 OH) genomic sequences of two other
US BEC strains that may represent a new calicivirus genus. Unlike NoV
related BECs, the genomic organization of the NB and CV-23 OH viruses is
similar to lagoviruses and SaVs, but morphologically these viruses
resemble NoV. To develop an animal model for enteric caliciviruses, we
studied the pathogenesis of wild-type (WT) PEC/Cowden and tissue culture
adapted (TC) PEC/Cowden in Gn pigs. We found differences in clinical
signs, histopathologic lesions, and virus-shedding patterns between the
TC/PEC and WT/PEC following oral or IV inoculation. These studies
confirmed attenuation of TC/PEC as compared to WT/PEC. Significantly,
viremia accompanied WT/PEC infection following either oral or IV
inoculation. This result was confirmed by induction of clinical PEC
infections in additional Gn pigs following oral or IV inoculation with
PEC positive acute sera. In further pathogenesis studies, a NoV strain (GII/4) of
human origin was successfully serially passaged in Gn piglets. Viral
shedding was demonstrated by RT-PCR and antigen ELISA, infected
epithelial cells
were identified by IF and suspect viral particles were observed by TEM.
Also, specific immune responses were demonstrated by seroconversion, by antibody
ELISA and B cell and cytokine ELISPOTs. The characterization of the immune
responses of pigs has provided preliminary data on the pattern of cytokine
responses in Gn pigs after human GII NoV infection followed by
homologous challenge, and also on intestinal cytokine responses
to HuNoV which are difficult to assess in human volunteers. Additionally, we
reported the first detection of NoVs in US swine, similar to HuNoVs both
genetically and antigenically, suggesting that pigs could be potential
reservoirs of NoVs strains. Reagents and techniques were developed to
continue the study of these HuNoVs and the new porcine NoV strains.
Also, studies between two distinct strains of BEC (NB and NoV CV186-OH)
revealed a lack of cross-protection between BEC of 2 different
genogroup/genera. Further characterization of the pathogenesis of the
NoV BEC (CV186-OH) belonging to NoV GIII showed that, based on our
preliminary results, age and inoculation route could be an important
factor for BEC pathogenesis. BEC infected calves developed a transient
viremia as previously shown for Gn piglets infected with PEC. The
development of new reagents (VLPs and hyperimmune sera) and techniques
permitted an increased sensitivity and specificity for the detection of
these strains, the assessment of their prevalence in the cattle
population and the evaluation of antigenic cross-reactivity between
human and bovine strains, besides analysis of their genetic
relationships. No antigenic cross-reactivity was evident between GI or
GII HuNoVs and the GIII BoNoV (CV186-OH). Vaccine studies using BoNoV
VLPs have great significance because they not only provide information
about immune responses, but they also permit challenge and evaluation of
intestinal protection in contrast to the NoV VLP studies performed in
human volunteers and mice. Also the prevalence of SaVs in the selected swine
herds was determined. Additional porcine SaV strains more closely related to human SaVs
were identified. The basis of the
requirement for intestinal contents for SaV PEC/Cowden growth in cell
culture was defined. Within the intestinal contents, bile acids were
shown to increase cAMP and down-regulate STAT1 phosphorylation, a key
factor in innate immunity in the infected cells. The presence of bile acids in the duodenum provides
a possible explanation for PEC replication and lesions in the proximal intestine.
In addition drugs that interfere with viral replication were tested in
this system offering possibilities of studying antiviral strategies in
vitro. Our recent development of an infectious clone of the SaV PEC/Cowden
permits future studies of the genetic basis for virulence and cell
culture adaptation of enteric caliciviruses using SaV PEC/Cowden as a model.
