Kirsten Mattison

The Human Cytomegalovirus Chemokine Receptor US28 Mediates Vascular Smooth Muscle Cell Migration

Human cytomegalovirus (HCMV) infection of smooth muscle cells (SMCs) in vivo has been linked to a viral etiology of vascular disease. In this report, we demonstrate that HCMV infection of primary arterial SMCs results in significant cellular migration. Ablation of the chemokine receptor, US28, abrogates SMC migration, which is rescued only by expression of the viral homolog and not a cellular G protein-coupled receptor (GPCR). Expression of US28 in the presence of CC chemokines including RANTES or MCP-1 was sufficient to promote SMC migration by both chemokinesis and chemotaxis, which was inhibited by protein tyrosine kinase inhibitors. US28-mediated SMC migration provides a molecular basis for the correlative evidence that links HCMV to the acceleration of vascular disease.

Human noroviruses in swine and cattle.

Detection of GII.4 norovirus sequences in animal fecal samples and retail meats demonstrates that noroviruses may be transmitted zoonotically.

Review: Norovirus prevalence in Belgian, Canadian and French fresh produce: a threat to human health?

Foodborne viruses, especially noroviruses (NoV), are increasingly reported as the cause of foodborne outbreaks. NoV outbreaks have been reported linked to fresh soft red fruits and leafy greens. Belgium, Canada and France were the first countries to provide data about the prevalence of NoV on fresh produce. In total, 867 samples of leafy greens, 180 samples of fresh soft red fruits and 57 samples of other types of fresh produce (tomatoes, cucumber and fruit salads) were analyzed. Firstly, the NoV detection methodology, including virus and RNA extraction, real-time RT-PCR and quality controls were compared among the three countries. In addition, confirmation and genotyping of the NoV strains was attempted for a subset of NoV positive samples using conventional RT-PCR targeting an alternative region followed by sequencing. Analysis of the process control showed that 653, 179 and 18 samples of the leafy greens, soft red fruits and other fresh produce types were valid for analysis based on the recovery of the process control. NoV was detected by real-time RT-PCR in 28.2% (N=641), 33.3% (N=6) and 50% (N=6) of leafy greens tested in Canada, Belgium and France, respectively. Soft red fruits were found positive by real-time RT-PCR in 34.5% (N=29) and 6.7% (N=150) of the samples tested in Belgium and France, respectively. 55.5% (N=18) of the other fresh produce types, analyzed in Belgium, were found NoV positive by real-time RT-PCR. Conventional RT-PCR resulted in an amplicon of the expected size in 19.5% (52/266) of the NoV positive samples where this assay was attempted. Subsequent sequencing was only successful in 34.6% (18/52) of the suspected amplicons obtained by conventional RT-PCR. From this study, using the described methodology, NoV genomes were frequently detected in fresh produce however sequence confirmation was not successful for the majority of the samples tested. Infection or outbreaks were rarely or not known to be related to the NoV positive samples. With the increase in sensitivity of the detection methodology, there is an increasing concern about the interpretation of positive NoV results by real-time amplification. Strategies to confirm the results by real-time RT-PCR should be developed in analogy with the detection of microbial pathogens in foods. Detection might indicate contact with NoV in the fresh produce chain. Consequently, a potential risk for infection cannot be excluded but the actual risk from RT-PCR NoV positive produce is still unknown. Studies should be designed determining the probability of infection related to the presence or levels of NoV genomic copies.

Structure of FitAB from Neisseria gonorrhoeae Bound to DNA Reveals a Tetramer of Toxin-Antitoxin Heterodimers Containing Pin Domains and Ribbon-Helix-Helix Motifs

Neisseria gonorrhoeae is a sexually transmitted pathogen that initiates infections in humans by adhering to the mucosal epithelium of the urogenital tract. The bacterium then enters the apical region of the cell and traffics across the cell to exit into the subepithelial matrix. Mutations in the fast intracellular trafficking (fitAB) locus cause the bacteria to transit a polarized epithelial monolayer more quickly than the wild-type parent and to replicate within cells at an accelerated rate. Here, we describe the crystal structure of the toxin-antitoxin heterodimer, FitAB, bound to a high affinity 36-bp DNA fragment from the fitAB promoter. FitA, the antitoxin, binds DNA through its ribbon-helix-helix motif and is tethered to FitB, the toxin, to form a heterodimer by the insertion of a four turn α-helix into an extensive FitB hydrophobic pocket. FitB is composed of a PIN (PilT N terminus) domain, with a central, twisted, 5-stranded parallel β-sheet that is open on one side and flanked by five α-helices. FitB in the context of the FitAB complex does not display nuclease activity against tested PIN substrates. The FitAB complex points to the mechanism by which antitoxins with RHH motifs can block the activity of toxins with PIN domains. Interactions between two FitB molecules result in the formation of a tetramer of FitAB heterodimers, which binds to the 36-bp DNA fragment and provides an explanation for how FitB enhances the DNA binding affinity of FitA.

Survival of calicivirus in foods and on surfaces: experiments with feline calicivirus as a surrogate for norovirus.

Although there is a large body of evidence incriminating foods as vehicles in the transmission of norovirus, little is known about virus survival in foods and on surfaces. Feline calicivirus was used as a surrogate for norovirus to investigate its survival in representative foods of plant and animal origin and on metal surfaces. Known concentrations of feline calicivirus in a natural fecal suspension were deposited onto lettuce, strawberries, ham, or stainless steel and incubated for 7 days at refrigeration or room temperatures. Virus was recovered at 1-day intervals, and the titers of the virus were determined by plaque assay. Infectious virus was recoverable until day 7 from lettuce, ham, and stainless steel. Statistically higher titers of feline calicivirus (P < 0.05) were recovered from ham under all conditions than from lettuce, strawberries, or stainless steel. These data provide valuable information for epidemiological and monitoring purposes as well as for the development of food processing practices and appropriate strategies to inactivate norovirus and control its transmission via foods and surfaces.

The feline calicivirus as a sample process control for the detection of food and waterborne RNA viruses.

Many food and waterborne outbreaks of infectious disease are caused by viruses. While numerous methods exist and are being developed to test food and water for the presence of enteric viruses, there is no standard control for the comparison of different methods. Potential control viruses should be well characterized, share the physical characteristics of the enterically infecting viruses and not normally be associated with foods. Here, the feline calicivirus (FCV) is proposed as a sample process control for methods aimed at the extraction and detection of RNA viruses in food and water. FCV is shown to be useful as a control for the extraction of hepatitis A virus (HAV) from water using filtration technology and from strawberries using the Pathatrix system. The FCV standard provides a valuable quality control tool when testing potentially contaminated food samples.

Attachment of noroviruses to stainless steel and their inactivation, using household disinfectants.

The aims of this study were (i) to evaluate the impact of pH and relative humidity on the attachment of norovirus (NoV) to fomites and (ii) to evaluate the effectiveness of different household disinfectants on NoV attached to fomites. Plaque assay and/or real-time reverse transcription PCR assay were used to determine the amount of murine and human NoV attached to stainless steel disks, i.e., the amount removed by sonication in elution buffer but not by surface rinses with water only. An enzymatic pretreatment was used for both human and murine NoV before the real-time reverse transcription PCR assay to avoid detection of RNA associated with inactivated virus. For both murine and human NoV, maximum attachment was obtained after a contact time of 10 min. Attachment of NoV to stainless steel does not appear to be affected by pH, although murine NoV was less attached (<2 log units) at pH 9 and at low relative humidity (25%) than was human NoV (3 log units). Sodium hypochlorite (3%) was the most effective disinfectant, producing a greater than 3-log reduction after 10 min compared with less than a 1-log reduction after treatment with quaternary ammonium compounds and ethoxylated alcohols. Murine NoV was more sensitive than human NoV to disinfectants by approximately 1 to 2 log units. These results will help improve strategies for decontaminating surfaces harboring NoV and thus reduce the incidence of illness caused by these pathogens in the food sector and domestic environments.

Multicenter Comparison of Two Norovirus ORF2-Based Genotyping Protocols

ABSTRACT Point source norovirus outbreaks can be difficult to track due to high background levels of the virus in the environment and the limited strain variation in some genotyping regions. However, rapid and accurate source identification can limit the spread of a foodborne outbreak and reduce the number of cases. Harmonization of genotyping assays is critical for enabling the rapid exchange of sequence data nationally and internationally. Several regions of the genome have been proposed for this purpose, but no consensus has been reached. In the present study, two standardized genotyping protocols (region C and region D) were evaluated by nine laboratories in Canada and the United States, using a coded panel of 96 fecal specimens representing 22 different norovirus genotypes. Overall, region C typing had a success rate of 78% compared to 52% for region D; however, region D provides greater nucleotide sequence diversity for identifying new GII.4 variant strains. Significant differences in the genotyping success rate were observed among the nine participating laboratories (10% to 100%) and among the different genotypes (6% to 100%). For several genogroup II strains, reduced region D amplification correlated directly with mismatches between primer sequences and the template. Based on overall performance, we recommend the region C protocol for routine genotyping of noroviruses, while the region D protocol may be useful for identifying new GII.4 variants. Standardized genotyping protocols will enable rapid exchange of outbreak and sequence data through electronic norovirus surveillance networks.

Phosphorylation alters the interaction of the response regulator OmpR with its sensor kinase EnvZ

OmpR and EnvZ comprise a two-component system that regulates the porin genes ompF and ompC in response to changes in osmolarity. EnvZ is autophosphorylated by intracellular ATP on a histidine residue, and it transfers the phosphoryl group to an aspartic acid residue of OmpR. EnvZ can also dephosphorylate phospho-OmpR (OmpR-P) to control the cellular level of OmpR-P. At low osmolarity, OmpR-P levels are low because of either low EnvZ kinase or high EnvZ phosphatase activities. At high osmolarity, OmpR-P is elevated. It has been proposed that EnvZ phosphatase is the activity that is regulated by osmolarity. OmpR is a two-domain response regulator; phosphorylation of OmpR increases its affinity for DNA, and DNA binding stimulates phosphorylation. The step that is affected by DNA depends upon the phosphodonor employed. In the present work, we have used fluorescence anisotropy and phosphotransfer assays to examine OmpR interactions with EnvZ. Our results indicate that phosphorylation greatly reduces the affinity of OmpR for the kinase, whereas DNA does not affect their interaction. The results presented cast serious doubts on the role of the EnvZ phosphatase in response to signalingin vivo.

Enteric Viruses in Ready-to-Eat Packaged Leafy Greens

To the Editor: Fresh produce increasingly has been implicated in viral disease outbreaks (1). In some instances, lettuce was contaminated before wholesale distribution (1). Enteric viruses can be introduced in the field if produce is exposed to human waste. Processed and packaged produce can be contaminated if equipment or wash water is not effectively sanitized. Fewer than 10 infectious viral particles are sufficient to cause disease (2), and these organisms are resistant to disinfectants at concentrations that reduce bacterial levels (3). Contamination of fresh produce could pose a health risk to humans because fresh produce is eaten raw. High levels of viral contamination can result in large outbreaks, but intermittent contamination of fresh produce accounts for some sporadic cases of norovirus and rotavirus gastroenteritis. During April 27–November 23, 2009, we performed viral testing on 328 samples of packaged leafy greens (representing 12–14 different lots from 3–6 companies per week; no samples were taken on weeks with a statutory holiday) for norovirus or rotavirus RNA. Packaged leafy greens were purchased from retail stores in southern Ontario, Canada. Shipments maintained an average temperature of 3.8°C during transit to the testing laboratory. Each 25-g sample was spiked with 106 PFU of feline calicivirus (FCV) as a sample process control (4). Virus was concentrated by using an adsorption-elution-ultrafiltration filtration protocol (4). Recovery of FCV was quantified from an RNA standard curve. FCV process control recovery was 0.01% of the FCV was observed for the remaining 273 (83%) samples. Two samples from which FCV was not recovered were positive for norovirus (CE-V-09–0138) and rotavirus (CE-V-09–0129); they were considered true positive results. Of these 275 samples, 148 (54%) were positive for norovirus by real-time reverse transcription–PCR (RT-PCR) (5), and 1 (0.4%) was positive for rotavirus group A by RT-PCR (6). To confirm detection of norovirus RNA, we amplified a second norovirus target by RT-PCR of region C (5). Only 40 samples (15% of total) produced a band of the expected size for this second norovirus amplicon. Of these 40 amplicons, only 16 (6% of total) could be sequenced to confirm norovirus RNA. The rotavirus-positive sample was confirmed by sequencing. For some sample dates, multiple lots were positive; for others, no positive samples were identified (Figure). Multiple detections on the same date were not caused by cross-contamination; partial capsid sequencing showed different genetic types on dates when multiple samples were positive (Figure). Results were positive from 5 different brands, and no organic samples were confirmed positive for enteric virus contamination. Of the 16 norovirus strains confirmed, 13 belonged to genogroup I (GI) and 3 to genogroup II (GII) (Figure). All were strain types known to be human pathogens. The group A rotavirus was not subtyped; group A rotaviruses can be human or animal pathogens. Figure Phylogenetic analysis of the partial capsid sequence from genogroup I (A) and genogroup II (B) norovirus strains detected on leafy greens samples, Ontario, Canada, 2009, compared with the ViroNet Canada reference set for this region. Dates in parentheses ... Most noroviruses detected belonged to GI. Previous reports indicate that GI norovirus are more frequently identified in foodborne or waterborne outbreaks; GII.4 noroviruses are more common in large outbreaks spread person to person (7). Identification of GI norovirus is consistent with occasional contamination of produce or wash water. Disinfectants and sanitation agents are used in wash water at low concentrations, at which they have limited efficacy against norovirus (3). Washing and disinfecting produce before eating it can reduce the risk for infection by reducing the viral load by 10- to 1,000-fold (8). The median level of confirmed contamination in this study was ≈500 RNA copies for norovirus (range 1.4 copies to 9 × 106 copies). A limitation of our findings is the inability to determine the association between molecular detection results and infectious virus. No outbreaks were related to the sequences detected here. There is no routine cell culture system for the laboratory growth of human norovirus. Genomic RNA can persist after the virus has been inactivated (9). The new ViroNet Canada network, which went online in April 2010, will monitor strains detected in leafy greens and other food products together with strains from community outbreaks to identify outbreaks linked to contaminated foods. Our comprehensive surveillance study identified norovirus and rotavirus contamination of packaged leafy greens. We detected noroviruses on 6% and rotavirus on 0.4% of lots tested from retail markets in southern Ontario. Packages with confirmed positive samples were both imported into Canada and had been conventionally grown. Noroviruses have a low infectious dose (2), and detection of viral RNA is associated with human health risk in oysters, another commodity that is eaten raw (10). Our results suggest a possible risk for foodborne transmission of norovirus and rotavirus from packaged leafy greens.