Melissa K. Jones

Enteric bacteria promote human and mouse norovirus infection of B cells

Bacteria help norovius infect B cells Stomach ache, nausea, diarrhea—many people know the sort of gastrointestinal havoc norovirus can wreak. Despite this, norovirus biology remains unclear, because human norovirus cannot be grown in culture. Jones et al. now report that with the help of bacteria, human norovirus can infect cultured B cells (see the Perspective by Robinson and Pfeiffer). To infect B cells, human norovirus required the presence of gut bacteria that expressed proteins involved in determining blood type. Mouse norovirus also infected B cells, and the treatment of mice with antibiotics protected them from norovirus infection. Science, this issue p. 755; see also p. 700 Gut bacteria that express histo-blood group antigens help human norovirus to infect B cells. [Also see Perspective by Robinson and Pfeiffer] The cell tropism of human noroviruses and the development of an in vitro infection model remain elusive. Although susceptibility to individual human norovirus strains correlates with an individual’s histo-blood group antigen (HBGA) profile, the biological basis of this restriction is unknown. We demonstrate that human and mouse noroviruses infected B cells in vitro and likely in vivo. Human norovirus infection of B cells required the presence of HBGA-expressing enteric bacteria. Furthermore, mouse norovirus replication was reduced in vivo when the intestinal microbiota was depleted by means of oral antibiotic administration. Thus, we have identified B cells as a cellular target of noroviruses and enteric bacteria as a stimulatory factor for norovirus infection, leading to the development of an in vitro infection model for human noroviruses.

Human norovirus culture in B cells

Human noroviruses (HuNoVs) are a leading cause of foodborne disease and severe childhood diarrhea, and they cause a majority of the gastroenteritis outbreaks worldwide. However, the development of effective and long-lasting HuNoV vaccines and therapeutics has been greatly hindered by their uncultivability. We recently demonstrated that a HuNoV replicates in human B cells, and that commensal bacteria serve as a cofactor for this infection. In this protocol, we provide detailed methods for culturing the GII.4-Sydney HuNoV strain directly in human B cells, and in a coculture system in which the virus must cross a confluent epithelial barrier to access underlying B cells. We also describe methods for bacterial stimulation of HuNoV B cell infection and for measuring viral attachment to the surface of B cells. Finally, we highlight variables that contribute to the efficiency of viral replication in this system. Infection assays require 3 d and attachment assays require 3 h. Analysis of infection or attachment samples, including RNA extraction and RT-qPCR, requires ∼6 h.

Identification of Immune and Viral Correlates of Norovirus Protective Immunity through Comparative Study of Intra-Cluster Norovirus Strains

Whether or not primary norovirus infections induce protective immunity has become a controversial issue, potentially confounded by the comparison of data from genetically distinct norovirus strains. Early human volunteer studies performed with a norovirus-positive inoculum initially led to the conclusion that primary infection does not generate long-term, protective immunity. More recently though, the epidemiological pattern of norovirus pandemics has led to the extrapolation that primary norovirus infection induces herd immunity. While these are seemingly discordant observations, they may in fact reflect virus strain-, cluster-, or genogroup-specific differences in protective immunity induction. Here, we report that highly genetically related intra-cluster murine norovirus strains differ dramatically in their ability to induce a protective immune response: Primary MNV-3 infection induced robust and cross-reactive protection, whereas primary MNV-1 infection induced modest homotypic and no heterotypic protection. In addition to this fundamental observation that intra-cluster norovirus strains display remarkable differences in protective immunity induction, we report three additional important observations relevant to norovirus:host interactions. First, antibody and CD4+ T cells are essential to controlling secondary norovirus infections. Second, the viral minor structural protein VP2 regulates the maturation of antigen presenting cells and protective immunity induction in a virus strain-specific manner, pointing to a mechanism by which MNV-1 may prevent the stimulation of memory immune responses. Third, VF1-mediated regulation of cytokine induction also correlates with protective immunity induction. Thus, two highly genetically-related norovirus strains displayed striking differences in induction of protective immune responses, strongly suggesting that the interpretation of norovirus immunity and vaccine studies must consider potential virus strain-specific effects. Moreover, we have identified immune (antibody and CD4+ T cells) and viral (VP2 and possibly VF1) correlates of norovirus protective immunity. These findings have significant implications for our understanding of norovirus immunity during primary infections as well as the development of new norovirus vaccines.

Genetic Characterization of Vibrio vulnificus Strains from Tilapia Aquaculture in Bangladesh

ABSTRACT Outbreaks of Vibrio vulnificus wound infections in Israel were previously attributed to tilapia aquaculture. In this study, V. vulnificus was frequently isolated from coastal but not freshwater aquaculture in Bangladesh. Phylogenetic analyses showed that strains from Bangladesh differed remarkably from isolates commonly recovered elsewhere from fish or oysters and were more closely related to strains of clinical origin.

Distribution and Genetic Diversity of Salmonella enterica in the Upper Suwannee River

The Suwannee River spans the Florida/Georgia border to the Gulf of Mexico, and contributes to regional irrigation and recreational activities. Association of Salmonella enterica with these resources may result in the contamination of produce and disease outbreaks. Therefore, surface water was examined for the distribution of S. enterica at multiple time points from 4 sites on the upper Suwannee River. Isolates were confirmed by detection of the invA gene, and 96% of all samples were positive for the bacterium. Most probable number enumeration ranged from <18 to 5400 MPN/100 mL. Genetic diversity of these isolates (n=110) was compared to other environmental (n=47) or clinical (n=28) strains and to an online library (n=314) using DiversiLab rep-PCR. All strains showed >60% similarity and distributed into 16 rep-PCR genogroups. Most (74%) of the Suwannee River isolates were clustered into two genogroups that were comprised almost exclusively (97%) of just these isolates. Conversely, 85% of the clinical reference strains clustered into other genogroups. However, some Suwannee River isolates (12%) were clustered with these primarily clinically-associated genogroups, supporting the hypothesis that river water can serve as a disease reservoir and that pathogenic strains may persist or possibly originate from environmental sources.

The Effect of Malnutrition on Norovirus Infection

ABSTRACT Human noroviruses are the primary cause of severe childhood diarrhea in the United States, and they are of particular clinical importance in pediatric populations in the developing world. A major contributing factor to the general increased severity of infectious diseases in these regions is malnutrition—nutritional status shapes host immune responses and the composition of the host intestinal microbiota, both of which can influence the outcome of pathogenic infections. In terms of enteric norovirus infections, mucosal immunity and intestinal microbes are likely to contribute to the infection outcome in substantial ways. We probed these interactions using a murine model of malnutrition and murine norovirus infection. Our results reveal that malnutrition is associated with more severe norovirus infections as defined by weight loss, impaired control of norovirus infections, reduced antiviral antibody responses, loss of protective immunity, and enhanced viral evolution. Moreover, the microbiota is dramatically altered by malnutrition. Interestingly, murine norovirus infection also causes changes in the host microbial composition within the intestine but only in healthy mice. In fact, the infection-associated microbiota resembles the malnutrition-associated microbiota. Collectively, these findings represent an extensive characterization of a new malnutrition model of norovirus infection that will ultimately facilitate elucidation of the nutritionally regulated host parameters that predispose to more severe infections and impaired memory immune responses. In a broad sense, this model may provide insight into the reduced efficacy of oral vaccines in malnourished hosts and the potential for malnourished individuals to act as reservoirs of emergent virus strains. IMPORTANCE Malnourished children in developing countries are susceptible to more severe infections than their healthy counterparts, in particular enteric infections that cause diarrhea. In order to probe the effects of malnutrition on an enteric infection in a well-controlled system devoid of other environmental and genetic variability, we studied norovirus infection in a mouse model. We have revealed that malnourished mice develop more severe norovirus infections and they fail to mount effective memory immunity to a secondary challenge. This is of particular importance because malnourished children generally mount less effective immune responses to oral vaccines, and we can now use our new model system to probe the immunological basis of this impairment. We have also determined that noroviruses evolve more readily in the face of malnutrition. Finally, both norovirus infection and malnutrition independently alter the composition of the intestinal microbiota in substantial and overlapping ways. Malnourished children in developing countries are susceptible to more severe infections than their healthy counterparts, in particular enteric infections that cause diarrhea. In order to probe the effects of malnutrition on an enteric infection in a well-controlled system devoid of other environmental and genetic variability, we studied norovirus infection in a mouse model. We have revealed that malnourished mice develop more severe norovirus infections and they fail to mount effective memory immunity to a secondary challenge. This is of particular importance because malnourished children generally mount less effective immune responses to oral vaccines, and we can now use our new model system to probe the immunological basis of this impairment. We have also determined that noroviruses evolve more readily in the face of malnutrition. Finally, both norovirus infection and malnutrition independently alter the composition of the intestinal microbiota in substantial and overlapping ways.

csrA inhibits the formation of biofilms by Vibrio vulnificus.

ABSTRACT PCR screening of the shellfish-borne pathogen Vibrio vulnificus revealed csrA-negative strains, and these strains formed increased biofilm compared to csrA-positive strains. Complementation in trans with csrA resulted in reduced biofilm formation, similar to that by csrA+ strains. Our results provide evidence that csrA inhibits biofilm formation in V. vulnificus.

Role of GacA in virulence of Vibrio vulnificus.

The GacS/GacA two-component signal transduction system regulates virulence, biofilm formation and symbiosis in Vibrio species. The present study investigated this regulatory pathway in Vibrio vulnificus, a human pathogen that causes life-threatening disease associated with the consumption of raw oysters and wound infections. Small non-coding RNAs (csrB1, csrB2, csrB3 and csrC) commonly regulated by the GacS/GacA pathway were decreased (P<0.0003) in a V. vulnificus CMCP6 ΔgacA : : aph mutant compared with the wild-type parent, and expression was restored by complementation of the gacA deletion mutation in trans. Of the 20 genes examined by RT-PCR, significant reductions in the transcript levels of the mutant in comparison with the wild-type strain were observed only for genes related to motility (flaA), stationary phase (rpoS) and protease (vvpE) (P=0.04, 0.01 and 0.002, respectively). Swimming motility, flagellation and opaque colony morphology indicative of capsular polysaccharide (CPS) were unchanged in the mutant, while cytotoxicity, protease activity, CPS phase variation and the ability to acquire iron were decreased compared with the wild-type (P<0.01). The role of gacA in virulence of V. vulnificus was also demonstrated by significant impairment in the ability of the mutant strain to cause either skin (P<0.0005) or systemic infections (P<0.02) in subcutaneously inoculated, non-iron-treated mice. However, the virulence of the mutant was equivalent to that of the wild-type in iron-treated mice, demonstrating that the GacA pathway in V. vulnificus regulates the virulence of this organism in an iron-dependent manner.

Genetic and quantitative assessment of Vibrio vulnificus populations in oyster (Crassostrea virginica) tissues

Vibrio vulnificus is a leading cause of shellfish-associated food-borne illness. US regulations stipulate shellfish processing procedures to limit V. vulnificus densities; however, the effect of these procedures on V. vulnificus strain distribution and/or genetic diversity is unknown. Vibrio vulnificus concentrations and strain diversity were analysed in various oyster tissues stored overnight at 26°C that were subsequently divided into two treatment groups: one received post-harvest processing (PHP) via individual quick freeze and one was stored on ice. Vibrio vulnificus densities were 10-fold lower in all PHP-treated tissues compared with untreated tissues. Genetic diversity of V. vulnificus was assessed by BOX-PCR genotyping and was high in all oyster tissues, but was significantly lower in untreated compared with PHP-treated oysters. BOX-PCR discriminated strains into BOX-C (clinical-associated) and BOX-E (environmental-associated) types based on a 1.1 kb DNA band, which correlated well (83% agreement) with 16S rRNA (A/B) typing. A significantly higher proportion of BOX-C isolates were recovered from PHP oysters compared with untreated oysters (24% of all isolates versus 12%) suggesting that BOX-C strains may be more resistant to treatment. These results reveal highly diverse populations of V. vulnificus in oysters with different responses to PHP, emphasizing the need to better understand the organisms ecology and population genetics to optimize food safety practices.

Norovirus Antagonism of B cell Antigen Presentation Results in Impaired Control of Acute Infection

Human noroviruses are a leading cause of gastroenteritis, and so, vaccine development is desperately needed. Elucidating viral mechanisms of immune antagonism can provide key insight into designing effective immunization platforms. We recently revealed that B cells are targets of norovirus infection. Because noroviruses can regulate antigen presentation by infected macrophages and B cells can function as antigen-presenting cells, we tested whether noroviruses regulate B-cell-mediated antigen presentation and the biological consequence of such regulation. Indeed, murine noroviruses could prevent B-cell expression of antigen presentation molecules and this directly correlated with impaired control of acute infection. In addition to B cells, acute control required MHC class I molecules, CD8+ T cells, and granzymes, supporting a model whereby B cells act as antigen presenting cells to activate cytotoxic CD8+ T cells. This immune pathway was active prior to the induction of antiviral antibody responses. As in macrophages, the minor structural protein VP2 regulated B-cell antigen presentation in a virus-specific manner. Commensal bacteria were not required for the activation of this pathway and ultimately only B cells were required for the clearance of viral infection. These findings provide new insight into the role of B cells in stimulating antiviral CD8+ T-cell responses.