Unexpected Factors That Influence Coxsackievirus B3 Replication in Mouse Intestine



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Coxsackievirus is a human pathogen that frequently infect humans. Although many infections are asymptomatic, there can be severe outcomes, including heart inflammation and pancreas inflammation. Most studies with coxsackieviruses and other viruses use laboratory-adapted viral strains because of their efficient replication in cell culture. I used a cell culture-adapted strain of coxsackievirus B3 (CVB3), CVB3-Nancy, to examine viral replication and pathogenesis in orally inoculated mice. Using HeLa cell plaque assays with agar overlays, I noticed that some fecal viruses generated plaques >100 times as large as inoculum viruses. These large-plaque variants emerged following viral replication in several different tissues. I identified a single amino acid change, N63Y, in the VP3 capsid protein that was sufficient to confer the large-plaque phenotype. Wild-type CVB3 and N63Y mutant CVB3 had similar plaque sizes when agarose was used in the overlay instead of agar. I determined that sulfated glycans in agar inhibited plaque formation by wild-type CVB3 but not by N63Y mutant CVB3. Furthermore, N63Y mutant CVB3 bound heparin, a sulfated glycan, less efficiently than wild-type CVB3 did. While N63Y mutant CVB3 had a growth defect in cultured cells and reduced attachment, it had enhanced replication and pathogenesis in mice. Infection with N63Y mutant CVB3 induced more severe hepatic damage than infection with wild-type CVB3, likely because N63Y mutant CVB3 disseminates more efficiently to the liver. This work reinforce the idea that culture-adapted laboratory virus strains can have reduced fitness in vivo. N63Y mutant CVB3 may be useful as a platform to understand viral adaptation and pathogenesis in animal studies. I also explored other factors that influence CVB3 infection in mouse intestine. First, a sex bias for severe sequelae from coxsackievirus infections has been observed in humans. We sought to examine if the phenomenon can be seen in mice and to further understand the mechanisms. Here we orally infected mice with CVB3 and confirmed that CVB3 replication in the intestine is sex-dependent. CVB3 replicated efficiently in male mice intestine, but not female mice. Overall these data suggest that sex and the immune response play an important role in CVB3 replication in the intestine and should be considered in light of the sex bias observed in human disease. Previously, our lab has shown that intestinal microbiota promote replication and pathogenesis of several viruses, including poliovirus (PV), reovirus and CVB3. With that finding, we wanted to examine how microbiota enhance CVB3 infection. Bacteria in the colon produce millimolar quantities of butyrate and other short-chain fatty acids (SCFAs) through fermentation of dietary fiber. SCFAs are among the most abundant molecules in the distal gastrointestinal tract. To determine whether bacterial-derived SCFAs such as butyrate impact CVB3 replication in the intestine, I antibiotic treated mice and then supplied them with tributyrin, a form of butyrate that is absorbed in the distal gastrointestinal tract. I found that CVB3 replication and pathogenesis was restored in antibiotic-treated mice that received tributyrin. These results suggest that butyrate is sufficient to promote CVB3 replication. My preliminary data demonstrate that oral delivery of a histone deacetylase (HDAC) inhibitor, Vorinostat, is sufficient to restore CVB3 replication in antibiotic-treated mice, suggesting that the HDAC activity of butyrate may promote CVB3 infection. Taken all together, I identified several unexpected factors that may influence CVB3 replication in mouse intestine although much remains open for exploration.

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