Measurements of the self-assembly kinetics of individual viral capsids around their RNA genome.

Self-assembly is widely used by biological systems to build functional nanostructures, such as the protein capsids of RNA viruses. But because assembly is a collective phenomenon involving many weakly interacting subunits and a broad range of timescales, measurements of the assembly pathways have been elusive. We use interferometric scattering microscopy to measure the assembly kinetics of individual MS2 bacteriophage capsids around MS2 RNA. By recording how many coat proteins bind to each of many individual RNA strands, we find that assembly proceeds by nucleation followed by monotonic growth. Our measurements reveal the assembly pathways in quantitative detail and also show their failure modes. We use these results to critically examine models of the assembly process.

Virus inactivation in groundwater in a postglacial lava field in arctic climate.

Outbreaks of viral gastroenteritis are often connected to contaminated drinking water. The assessment of the water quality relies on the cultivation of indicator bacteria, and little is known of the fate of viruses in groundwater, especially in arctic regions. In Iceland, the groundwater temperature is between 3 and 6°C. The aim of this study was to determine virus inactivation at low temperature in a groundwater microcosm and in a borehole in a postglacial lava field. Two phage species that are commonly used as surrogates for norovirus were used, MS2 and PhiX174. Dialysis bags were used for the samples, and a device was constructed to hold many samples at a time and protect the samples in the borehole. No significant decrease of infective PhiX174 phages in the borehole or of the MS2 phages in the microcosm was observed. A slightly significant decrease of PhiX174 in the microcosm was noticed, with one log reduction time of 476 days. On the other hand, a significant reduction in MS2 was found in the field test, where the time needed for 90% reduction was 12·5 days. The results showed that an infective virus can exist in groundwater for months or years in arctic regions and a great difference may exist between results from microcosm and field tests. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reveals that arctic regions are highly sensitive to virus contamination as an infective virus may exist in groundwater for years at low temperature. The results also show that the virus inactivation observed in field tests may differ considerably from the inactivation observed in laboratory microcosms. The results emphasize the importance of large protection zones around drinking water intakes as well as good wastewater treatment so that the likelihood of faecal contamination of groundwater is reduced.

Effect of polymer and glass physicochemical properties on MS2 recovery from food contact surfaces.

Viruses are transmissible via their interaction with contact surfaces of food containers or tools. This study evaluated the recoveries of MS2 coliphage, a virus surrogate, from polypropylene (PP), polyvinyl chloride (PVC), polyethylene (PE), and glass (borosilicate and soda lime), as influenced by the surface chemistry and topography. MS2 (5-6 logs) in PBS with 1% TSB was inoculated onto each of 9 different surfaces, 24-h cold-incubated, and recovery was quantified by infectivity. The order of MS2 recovery efficiency from smooth surfaces was PP > PE ≥ soda lime glass, which classified into 3 ANOVA groups, p = 0.05. The MS2 recovery ratios of smooth vs. rough surfaces were 1.4-1.5. Atomic force microscopy revealed 21-nm diam pinholes (<28-nm of MS2 size) in the borosilicate glass. The lowest and highest MS2 recoveries among the 9 surfaces were demonstrated by the hole-bearing borosilicate glass (34 ±â€¯8%) and smooth PP (69 ±â€¯14%) respectively. Generally greater MS2 recovery was obtained from smooth PP and PE surfaces compared to glass, but topographic alterations (pinholes or increased roughness) decreased recovery possibly by trapping the viruses.

MS2 Lysis of Escherichia coli Depends on Host Chaperone DnaJ.

The L protein of the single-stranded RNA phage MS2 causes lysis of Escherichia coli without inducing bacteriolytic activity or inhibiting net peptidoglycan (PG) synthesis. To find host genes required for L-mediated lysis, spontaneous Ill (insensitivity to Llysis) mutants were selected as survivors of L expression and shown to have a missense change of the highly conserved proline (P330Q) in the C-terminal domain of DnaJ. In the dnaJP330Q mutant host, L-mediated lysis is completely blocked at 30°C without affecting the intracellular levels of L. At higher temperatures (37°C and 42°C), both lysis and L accumulation are delayed. The lysis block at 30°C in the dnaJP330Q mutant was recessive and could be suppressed by Lovercomes dnaJ (Lodj ) alleles selected for restoration of lysis. All three Lodj alleles lack the highly basic N-terminal half of the lysis protein and cause lysis ∼20 min earlier than full-length L. DnaJ was found to form a complex with full-length L. This complex was abrogated by the P330Q mutation and was absent with the Lodj truncations. These results suggest that, in the absence of interaction with DnaJ, the N-terminal domain of L interferes with its ability to bind to its unknown target. The lysis retardation and DnaJ chaperone dependency conferred by the nonessential, highly basic N-terminal domain of L resembles the SlyD chaperone dependency conferred by the highly basic C-terminal domain of the E lysis protein of ϕX174, suggesting a common theme where single-gene lysis can be modulated by host factors influenced by physiological conditions.IMPORTANCE Small single-stranded nucleic acid lytic phages (Microviridae and Leviviridae) lyse their host by expressing a single "protein antibiotic." The protein antibiotics from two out of three prototypic small lytic viruses have been shown to inhibit two different steps in the conserved PG biosynthesis pathway. However, the molecular basis of lysis caused by L, the lysis protein of the third prototypic virus, MS2, is unknown. The significance of our research lies in the identification of DnaJ as a chaperone in the MS2 L lysis pathway and the identification of the minimal lytic domain of MS2 L. Additionally, our research highlights the importance of the highly conserved P330 residue in the C-terminal domain of DnaJ for specific protein interactions.

Evaluation of Chlorine Treatment Levels for Inactivation of Human Norovirus and MS2 Bacteriophage during Sewage Treatment.

This study examined the inactivation of human norovirus (HuNoV) GI.1 and GII.4 by chlorine under conditions mimicking sewage treatment. Using a porcine gastric mucin-magnetic bead (PGM-MB) assay, no statistically significant loss in HuNoV binding (inactivation) was observed for secondary effluent treatments of ≤25 ppm total chlorine; for both strains, 50 and 100 ppm treatments resulted in ≤0.8-log10 unit and ≥3.9-log10 unit reductions, respectively. Treatments of 10, 25, 50, and 100 ppm chlorine inactivated 0.31, 1.35, >5, and >5 log10 units, respectively, of the norovirus indicator MS2 bacteriophage. Evaluation of treatment time indicated that the vast majority of MS2 and HuNoV inactivation occurred in the first 5 min for 0.2-µm-filtered, prechlorinated secondary effluent. Free chlorine measurements of secondary effluent seeded with MS2 and HuNoV demonstrated substantial oxidative burdens. With 25, 50, and 100 ppm treatments, free chlorine levels after 5 min of exposure ranged from 0.21 to 0.58 ppm, from 0.28 to 16.7 ppm, and from 11.6 to 53 ppm, respectively. At chlorine treatment levels of >50 ppm, statistically significant differences were observed between reductions for PGM-MB-bound HuNoV (potentially infectious) particles and those for unbound (noninfectious) HuNoV particles or total norovirus particles. While results suggested that MS2 and HuNoV (measured as PGM-MB binding) behave similarly, although not identically, both have limited susceptibility to chlorine treatments of ≤25 ppm total chlorine. Since sewage treatment is performed at ≤25 ppm total chlorine, targeting free chlorine levels of 0.5 to 1.0 ppm, these results suggest that traditional chlorine-based sewage treatment does not inactivate HuNoV efficiently.IMPORTANCE HuNoV is ubiquitous in sewage. A receptor binding assay was used to assess inactivation of HuNoV by chlorine-based sewage treatment, given that the virus cannot be routinely propagated in vitro Results reported here indicate that chlorine treatment of sewage is not effective for inactivating HuNoV unless chlorine levels are above those routinely used for sewage treatment.

Efficient collection of viable virus aerosol through laminar-flow, water-based condensational particle growth.

AIMS: State-of-the-art bioaerosol samplers have poor collection efficiencies for ultrafine virus aerosols. This work evaluated the performance of a novel growth tube collector (GTC), which utilizes laminar-flow water-based condensation to facilitate particle growth, for the collection of airborne MS2 viruses. METHODS AND RESULTS: Fine aerosols (<500 nm) containing MS2 coliphage were generated from a Collison nebulizer, conditioned by a dilution dryer and collected by a GTC and a BioSampler. The GTC effectively condensed water vapour onto the virus particles, creating droplets 2-5 µm in diameter, which facilitated collection. Comparison of particle counts upstream and downstream revealed that the GTC collected >93% of the inlet virus particles, whereas the BioSampler's efficiency was about 10%. Viable counts of the GTC-collected viruses were also one order of magnitude higher than those of the BioSampler (P = 0·003). CONCLUSION: The efficiency of the GTC for the viable collection of MS2 viruses exceeds that of industry standard instrument, the BioSampler, by a factor of 10-100. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reveals that the GTC is an effective collector of viable MS2 aerosols, and concludes the instrument will be an effective tool for studying viable virus aerosols and the inhalation risks posed by airborne viruses.

Aerosol-phase activity of iodine captured from a triiodide resin filter on fine particles containing an infectious virus.

AIMS: To avoid interference by water-iodine disinfection chemistry and measure directly the effect of iodine, captured from a triiodide complex bound to a filter medium, on viability of penetrating viral particles. METHODS AND RESULTS: Aerosols of MS2 coli phage were passed through control P100 or iodinated High-Efficiency Particulate Air media, collected in plastic bags, incubated for 0-10 min, collected in an impinger containing thiosulphate to consume all unreacted iodine, plated and enumerated. Comparison of viable counts demonstrated antimicrobial activity with an apparent half-life for devitalization in tens of seconds; rate of kill decreased at low humidity and free iodine was captured by the bags. CONCLUSIONS: The results support the mechanism of near-contact capture earlier proposed; however, the disinfection chemistry in the aerosol phase is very slow on the time scale of inhalation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that disinfection by filter-bound iodine in the aerosol phase is too slow to be clinically significant in individual respiratory protection, but that it might be of benefit to limit airborne transmission of infections in enclosed areas.

A compact point-of-use water purification cartridge for household use in developing countries.

Simple, low-cost household interventions are known to be effective in lowering the incidence of waterborne diseases in developing countries. However, high costs along with operational and maintenance issues have prevented the successful adoption of these interventions among the affected communities. To address these limitations, a cost-effective, gravity-driven water purification cartridge has been developed by employing the synergistic disinfection action of low concentrations of silver and chlorine on bacteria and viruses. The silver and chlorine treatment components within the cartridge have been developed using inexpensive materials and integrated with a life indicator and auto-shut-off-mechanism within a compact form factor. The antibacterial as well as antiviral performance of the cartridge was tested by using ground water spiked with Escherichia coli and MS2 bacteriophage. The results show that, although individually, the silver and chlorine treatment systems were unable to inactivate the test strains, the integrated cartridge inactivates both bacteria as well as viruses up to the log reduction requirement of the USEPA guide standard for microbiological water purifiers over its designated life of 2,000 liters.

Survival of airborne MS2 bacteriophage generated from human saliva, artificial saliva, and cell culture medium.

Laboratory studies of virus aerosols have been criticized for generating airborne viruses from artificial nebulizer suspensions (e.g., cell culture media), which do not mimic the natural release of viruses (e.g., from human saliva). The objectives of this study were to determine the effect of human saliva on the infectivity and survival of airborne virus and to compare it with those of artificial saliva and cell culture medium. A stock of MS2 bacteriophage was diluted in one of three nebulizer suspensions, aerosolized, size selected (100 to 450 nm) using a differential mobility analyzer, and collected onto gelatin filters. Uranine was used as a particle tracer. The resulting particle size distribution was measured using a scanning mobility particle sizer. The amounts of infectious virus, total virus, and fluorescence in the collected samples were determined by infectivity assays, quantitative reverse transcription-PCR (RT-PCR), and spectrofluorometry, respectively. For all nebulizer suspensions, the virus content generally followed a particle volume distribution rather than a number distribution. The survival of airborne MS2 was independent of particle size but was strongly affected by the type of nebulizer suspension. Human saliva was found to be much less protective than cell culture medium (i.e., 3% tryptic soy broth) and artificial saliva. These results indicate the need for caution when extrapolating laboratory results, which often use artificial nebulizer suspensions. To better assess the risk of airborne transmission of viral diseases in real-life situations, the use of natural suspensions such as saliva or respiratory mucus is recommended.

High-throughput assay for temporal kinetic analysis of lytic coliphage activity.

Plaque analysis allows the determination of phage titer and multiplicity of infection. Yet, this overnight assay provides only endpoint results, ignoring kinetic aspects. We introduce an alternative high-throughput and rapid method for kinetic analysis of lytic coliphage activity. Escherichia coli was infected with serial dilutions of MS2 coliphage, and bacterial growth was monitored using a multi-well plate reader providing within hours the equivalent data as obtained overnight. Additional information is yielded, including phage replication rate, progeny size per cycle, and viral propagation during bacterial growth. This method offers further insights into physicochemical mechanisms of lytic coliphage infection and temporal control. It also provides a virus-host interaction acumen.