This upcoming Monday, Chipotle restaurants across the country will close as its employees discuss food safety and safe food handling. The restaurant was in the news throughout the last half of 2015, beginning with an outbreak of E. coli in Seattle (which was kept secret!), followed by another E. coli outbreak involving nine states, and a Salmonella outbreak centered in Minnesota. But the largest number of Chipotle customers were sickened by two outbreaks of norovirus, in California and in Boston, Massachusetts. Clearly, an informational workshop on best practices seems the least the restaurant can do to clean up its act.
All three of these microbes cause similar symptoms – gastrointestinal distress being the most prominent – and would be difficult for anyone experiencing disease to differentiate. However, the mechanism by which disease is mediated differs between the three microbial types. The E. coli strain involved in the Chipotle outbreaks, O26, produces Shiga toxin, which is an A-B toxin composed of a B subunit that facilitates internalization via the Gb3 receptor and an A subunit that blocks protein synthesis. Salmonella enterica (in the Chipotle cases: serotype Newport) not only has a number of virulence factors, but carries a number of antibiotic resistance genes, and is so commonly isolated from food animals that this 2003 Journal of Clinical Microbiology publication warned of transmission in the food chain. Both of these gram-negative bacteria have been well studied for decades, their multiple virulence factors well characterized – and, importantly, there are antibiotics that can treat infections caused by these bacteria. However, the way norovirus sickens its host is still not well understood, and this gap in knowledge has left physicians with scarce treatment for norovirus sufferers.
A recent article published in the Journal of Virology has made some forward progress toward understanding norovirus pathogenicity. The research, conducted in the lab of Dr. Stephanie Karst by first author Shu Zhu, looked at the role of the viral capsid protein VP1 in virulence. The scientists hypothesized that amino acid substitutions in this protein would affect virulence. The VP1 protein has three major domains: the S domain, which is the most conserved domain that makes up the shell of the virion; the protruding P1 stalk domain; and the hypervariable, surface-exposed P2 domain (see assembled virion, left).
To search for mutations affecting disease, the researchers set up a model of persistently infected mice by infected the mice with a previously-identified attenuated mouse norovirus strain. They predicted this would lead to mutations that increased virulence (a rational hypothesis, since some microbial strains are serially passaged to increase their virulence in that animal model). After passaging, the researchers sequenced the norovirus VP1 gene to identify mutations (see figure, right).
The attenuated strain had been selected based on its inclusion of glutamic acid at position 296, which had changed from a lysine. To test the effects of this single mutation, the researchers compared the effect of a glutamic acid versus a lysine in position 296 (see map of VP1 protein, left). The single change to a lysine was enough to increase virulence in an otherwise attenuated virus strain. Most natural isolates of mouse norovirus have a glutamic acid at this position, suggesting that disease symptoms could possibly worsen if a mutated E296K strain were to circulate widely.
The research team found eight additional mutations in the persistently infected mice, three of which were in the S domain but had little effect on viral replication. The five amino acid changes in the P domains (one in P1 and four in P2) all changed replication kinetics of the virus both in vitro and in vivo. Interestingly, there was no change in virus replication in macrophage cells, which remained high in all cases, but there was a replication decrease in B cells, an important cell tropism for human norovirus. These results demonstrate that the P domain regulates replication in B cells but not in macrophage.
Norovirus is an extremely contagious virus, with an infectious dose thought to be as low as 20 virus particles. It has a (+)single-stranded RNA genome, surrounded by its capsid protein, VP1. The ability of the exposed, variable domain of the capsid to regulate replication may be due to the interaction of this domain with its target receptor – or it may be due to a yet-to-be-described function. By identifying regions important for virulence, this research team has contributed to potential antiviral targets for this extremely unpleasant yet common pathogen. Future studies more finely focused on the mechanism of VP1 virulence regulation will add to our understanding of norovirus pathogenesis – and maybe reveal a way to treat those unfortunate enough to eat at the wrong Chipotle Mexican Grill.
-- Julie Wolf