Our goal is to develop improved methods for detection of noroviruses (NV) and
develop methods for differential diagnosis of human from animal NV infections.
To achieve this goal, we will survey the prevalence of NV in Ohio swine and
examine their genetic and antigenic relationships to human NV to assess their
zoonotic potential. Swine fecal samples will be tested by RT-PCR with primers
targeted to human NV and porcine enteric caliciviruses (PEC) highly conserved
(viral polymerase) and variable (P2 of capsid) genomic regions. Specific
reactions will be confirmed by a newly developed, sensitive and specific
microplate DNA probe hybridization enzyme assay. RT-PCR products and cloned PEC
genes will be sequenced and compared phylogenetically to human NV to analyze
their genetic relationships. Cloned capsid genes of PEC strains most similar to
human NV will be expressed in a baculovirus system to generate virus-like
particles. These particles will be used to produce antisera. The antigenic
cross-reactivity between PEC and human NV will be assessed by ELISA using the
PEC and human NV virus-like particles and antisera. PEC most similar to human NV
and related human NV will be used to infect gnotobiotic pigs. This will provide
large quantities of stools and antisera to pursue and validate new lab assays
for animal NV applicable to human NV.
Our PEC and human NV provided by collaborators will be used to develop novel, sensitive and specific molecular methods. These methods could detect, differentiate and type animal NV from animal and human sources. We will also develop real-time PCR and collaborate on design of animal NV plus bacterial microarrays to detect a multitude of microbial agents in food.
Completion of this project will provide new data on the zoonotic potential of animal NV based on their degree of antigenic and genetic relatedness to human NV. It will provide new lab reagents to test protocols for detection or inactivation of human NV in foods using related surrogate pig NV. Our novel diagnostic assays will provide rapid low-cost surveillance systems for animal and human NV. We hope to identify their genotype and probable host origin in food and water sources. Such assays have a high potential for worldwide commercialization for improvement of public health, post-outbreak diagnosis, and by the food or pharmaceutical industries.