Outbreak of Norovirus GII.P17-GII.17 in the Canadian Province of Nova Scotia.

Background. Norovirus is the leading cause of viral gastroenteritis, with GII.4 being the most common circulating genotype. Recently, outbreaks in China revealed that norovirus GII.17 GII.P17 had become predominant. Objective. This study aimed to characterize the distribution of norovirus genotypes circulating in Nova Scotia. Methods. Stool specimens were collected from gastrointestinal outbreaks in Nova Scotia between Jan 2014 and June 2015 and subjected to real-time RT-PCR. Norovirus-positive specimens were referred to the National Microbiology Laboratory for sequence-based genotyping. Results. The first norovirus GII.P17-GII.17 outbreak in Canada was identified, but no widespread activity was observed in Nova Scotia. Discussion. It is unknown whether GII.P17-GII.17 is more widespread in Canada since contributions to Canadian surveillance are too sparse to effectively monitor the epidemiology of emerging norovirus genotypes. Conclusions. Presence of norovirus GII.17:P17 in Canada highlights the need for more systematic surveillance to ensure that molecular targets used for laboratory detection are effective and help understand norovirus evolution, epidemiology, and pathogenesis.

Detection of circulating norovirus genotypes: hitting a moving target.

BACKGROUND: Although national surveillance programs are in place to monitor norovirus epidemiology, the emergence of new strains and the genetic diversity among genotypes can be challenging for clinical laboratories. This study evaluated the analytical and clinical performance characteristics of one real-time RT-PCR and two end-point RT-PCRs commonly used in microbiology laboratories. METHODS: Lower limit of detection (LoD) was determined using 10-fold dilutions of noroviruses belonging to different genotypes. The clinical performance of the real-time and end-point RT-PCRs was assessed in parallel using nucleic acids extracted from 186 stool specimens. RESULTS: The real-time RT-PCR was highly sensitive and specific for the detection of norovirus genotypes that are currently circulating in Canada. In contrast, the two end-point RT-PCRs displayed poor analytical sensitivity or complete failure to detect certain norovirus genotypes, which was correlated to sequence mismatches in the primer-binding sites. In an attempt to improve norovirus detection with the end-point RT-PCRs, both assays were processed concurrently and detection from either assay was considered a positive result. Concurrent testing resulted in only a modest increase in clinical sensitivity (75.0%) compared to each assay alone (62.5% and 71.9%). However, the false positivity rate increased from 1.98% and 3.36% for the assays alone to 5.47% with concurrent testing. CONCLUSIONS: This study emphasizes the benefits of a real-time method and provides support for routine surveillance to monitor norovirus epidemiology and ongoing proficiency testing to ensure detection of circulating norovirus genotypes.

Hunting for mpox (monkeypox) mimickers: Use of the Biofire meningitis/encephalitis panel on lesion swabs to support alternative viral diagnoses.

BACKGROUND: Mpox (formerly monkeypox) is an emerging zoonotic disease of public health concern that presents as a rash mimicking other common viral exanthems. Unlike traditional testing algorithms relying on several assays, the BioFire FilmArray meningitis/encephalitis (ME) panel simultaneously detects common viruses causing rashes; however, Biofire ME is only licensed for testing on cerebral spinal fluid. OBJECTIVES: This study evaluated use of the Biofire ME panel for detection and discrimination of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), varicella zoster virus (VZV), human herpesviruses type 6 (HHV-6), enteroviruses (EVs), and human paraechoviruses (HPeVs) from a dermal or mucocutaneous swabs collected in universal transport media (UTM). STUDY DESIGN: Results of the BioFire ME panel were compared against methods used during clinical testing. Ten-fold serial dilutions in UTM of cultured viruses were used to compare analytical sensitivity, and analytical specificity was assessed using panels of microorganisms in UTM. Clinical sensitivity and specificity were assessed using 20 positive specimens each for HHV-1, HHV-2, HHV-6, VZV, EVs, and HPeV, as well as 35 known negative specimens that included 15 mpox-positive specimens. RESULTS: Biofire ME was as sensitive as comparator methods, and correctly discriminated all HSV-1, HSV-2, VZV, HHV-6, EVs, and HPeVs from mpox and mpox-mimickers. Cross-reaction between EV and rhinoviruses A, B, and C were noted in the specificity panel. CONCLUSIONS: Swabs in UTM collected for mpox testing are suitable for use on the Biofire ME panel, allowing more streamlined diagnostic testing for viral exanthems in patients under investigation for mpox infection.

Architecture of the SARS coronavirus prefusion spike.

The emergence in 2003 of a new coronavirus (CoV) responsible for the atypical pneumonia termed severe acute respiratory syndrome (SARS) was a stark reminder that hitherto unknown viruses have the potential to cross species barriers to become new human pathogens. Here we describe the SARS-CoV 'spike' structure determined by single-particle cryo-EM, along with the docked atomic structures of the receptor-binding domain and prefusion core.

Multicenter comparison of two norovirus ORF2-based genotyping protocols.

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.

The limitations of point of care testing for pandemic influenza: what clinicians and public health professionals need to know.

As the world prepares for the next influenza pandemic, governments have made significant funding commitments to vaccine development and antiviral stockpiling. While these are essential components to pandemic response, rapid and accurate diagnostic testing remains an often neglected cornerstone of pandemic influenza preparedness. Clinicians and Public Health Practitioners need to understand the benefits and drawbacks of different influenza tests in both seasonal and pandemic settings. Culture has been the traditional gold standard for influenza diagnosis but requires from 1-10 days to generate a positive result, compared to nucleic acid detection methods such as real time reverse transcriptase polymerase chain reaction (RT-PCR). Although the currently available rapid antigen detection kits can generate results in less than 30 minutes, their sensitivity is suboptimal and they are not recommended for the detection of novel influenza viruses. Until point-of-care (POC) tests are improved, PILPN recommends that the best option for pandemic influenza preparation is the enhancement of nucleic acid-based testing capabilities across Canada.

A mobile biosafety microanalysis system for infectious agents.

Biological threats posed by pathogens such as Ebola virus must be quickly diagnosed, while protecting the safety of personnel. Scanning electron microscopy and microanalysis requires minimal specimen preparation and can help to identify hazardous agents or substances. Here we report a compact biosafety system for rapid imaging and elemental analysis of specimens, including powders, viruses and bacteria, which is easily transportable to the site of an incident.

Epidemiology of Enterovirus D68 in Ontario.

In August 2014, children's hospitals in Kansas City, Missouri and Chicago, Illinois notified the Centers for Disease Control and Prevention (CDC) about increased numbers of pediatric patients hospitalized with severe respiratory illness (SRI). In response to CDC reports, Public Health Ontario Laboratories (PHOL) launched an investigation of patients being tested for enterovirus D-68 (EV-D68) in Ontario, Canada. The purpose of this investigation was to enhance our understanding of EV-D68 epidemiology and clinical features. Data for this study included specimens submitted for EV-D68 testing at PHOL from September 1, 2014 to October 31, 2014. Comparisons were made between patients who tested positive for the virus (cases) and those testing negative (controls). EV-D68 was identified in 153/907 (16.8%) of patients tested. In the logistic regression model adjusting for age, sex, setting and time to specimen collection, individuals younger than 20 years of age were more likely to be diagnosed with EV-D68 compared to those 20 and over, with peak positivity at ages 5-9 years. Cases were not more likely to be hospitalized than controls. Cases were more likely to be identified in September than October (OR 8.07; 95% CI 5.15 to 12.64). Routine viral culture and multiplex PCR were inadequate methods to identify EV-D68 due to poor sensitivity and inability to differentiate EV-D68 from other enterovirus serotypes or rhinovirus. Testing for EV-D68 in Ontario from July to December, 2014 detected the presence of EV-D68 virus among young children during September-October, 2014, with most cases detected in September. There was no difference in hospitalization status between cases and controls. In order to better understand the epidemiology of this virus, surveillance for EV-D68 should include testing of symptomatic individuals from all treatment settings and patient age groups, with collection and analysis of comprehensive clinical and epidemiological data.

A filtration based technique for simultaneous SEM and TEM sample preparation for the rapid detection of pathogens.

Diagnostic electron microscopy for infectious diseases has the advantage that "everything" in the specimen can be observed, without a priori knowledge of the likely identity of the microorganisms present in the sample. The classical specimen preparation method used employs a droplet of sample, which allows particles to adsorb to a support film, and is subsequently negative stained. This "grid on drop" procedure has a sensitivity range of approximately 106 viruses per mL if no enrichment procedures are used. In the current investigation we present a novel use of filtration that allows us to detect viruses at concentrations as low as 102 viruses per mL. We present here methods based on filtration, in which total virus, and not virus concentration, is the limiting factor for detection. We show that filtration is more sensitive than conventional negative staining and can detect as few as 5 × 103 particles per sample.

How do filovirus filaments bend without breaking?

Viruses of the Mononegavirales have helical nucleocapsids containing a single-stranded negative-sense RNA genome complexed with the nucleoprotein and several other virus-encoded proteins. This RNA-protein complex acts as the template for replication and transcription during infection. Recent structural data has advanced our understanding of how these functions are achieved in filoviruses, which include dangerous pathogens such as Ebola virus. Polyploid filoviruses package multiple genome copies within strikingly long filamentous viral envelopes, which must be flexible to avoid breakage of the 19kb non-segmented genomic RNA. We review how the structure of filoviruses and paramyxoviruses permits this morphological flexibility in comparison to rhabdoviruses that have short, bullet-shaped virions with relatively rigid envelopes.

Analytical performance of norovirus real-time RT-PCR detection protocols in Canadian laboratories.

BACKGROUND: Noroviruses (NoVs) are the leading cause of infectious gastroenteritis worldwide. Real-time reverse transcription PCR (real-time RT-PCR) is the preferred method of NoV detection for the majority of testing laboratories. Although the accepted target region for molecular detection assays is the conserved ORF1/ORF2 junction, multiple variations have been published with differences in primers, probes, reagents, multiplexing, etc. OBJECTIVES: We assessed the detection limit for GII.4 NoV real-time RT-PCR assays as well as the ability to detect the non-GII.4 NoV genotypes in each participating laboratory. STUDY DESIGN: A panel of 25 RNA samples was circulated to 18 testing laboratories for comparison of their real-time RT-PCR procedures for NoV detection. RESULTS: Multiple protocols with slight differences in reagents or conditions successfully detected 10 genome equivalents or fewer of NoV per reaction. Multiplex procedures were significantly associated (p=0.04) with false negative results, particularly for a GI.2 strain. Sensitive detection was associated with false positive results (p=0.03). CONCLUSIONS: Overall, the data indicate that comparable results are produced under slightly different assay conditions.

Nigericin is a potent inhibitor of the early stage of vaccinia virus replication.

Poxviruses remain a significant public health concern due to their potential use as bioterrorist agents and the spread of animal borne poxviruses, such as monkeypox virus, to humans. Thus, the identification of small molecule inhibitors of poxvirus replication is warranted. Vaccinia virus is the prototypic member of the Orthopoxvirus genus, which also includes variola and monkeypox virus. In this study, we demonstrate that the carboxylic ionophore nigericin is a potent inhibitor of vaccinia virus replication in several human cell lines. In HeLa cells, we found that the 50% inhibitory concentration of nigericin against vaccinia virus was 7.9 nM, with a selectivity index of 1038. We present data demonstrating that nigericin targets vaccinia virus replication at a post-entry stage. While nigericin moderately inhibits both early vaccinia gene transcription and translation, viral DNA replication and intermediate and late gene expression are severely compromised in the presence of nigericin. Our results demonstrate that nigericin has the potential to be further developed into an effective antiviral to treat poxvirus infections.

Conformational reorganization of the SARS coronavirus spike following receptor binding: implications for membrane fusion.

The SARS coronavirus (SARS-CoV) spike is the largest known viral spike molecule, and shares a similar function with all class 1 viral fusion proteins. Previous structural studies of membrane fusion proteins have largely used crystallography of static molecular fragments, in isolation of their transmembrane domains. In this study we have produced purified, irradiated SARS-CoV virions that retain their morphology, and are fusogenic in cell culture. We used cryo-electron microscopy and image processing to investigate conformational changes that occur in the entire spike of intact virions when they bind to the viral receptor, angiotensin-converting enzyme 2 (ACE2). We have shown that ACE2 binding results in structural changes that appear to be the initial step in viral membrane fusion, and precisely localized the receptor-binding and fusion core domains within the entire spike. Furthermore, our results show that receptor binding and subsequent membrane fusion are distinct steps, and that each spike can bind up to three ACE2 molecules. The SARS-CoV spike provides an ideal model system to study receptor binding and membrane fusion in the native state, employing cryo-electron microscopy and single-particle image analysis.

Coordinated response to SARS, Vancouver, Canada.

Two Canadian urban areas received travelers with severe acute respiratory syndrome (SARS) before the World Health Organization issued its alert. By July 2003, Vancouver had identified 5 cases (4 imported); Toronto reported 247 cases (3 imported) and 43 deaths. Baseline preparedness for pandemic threats may account for the absence of sustained transmission and fewer cases of SARS in Vancouver.