Long Amplicon Nanopore Sequencing for Dual-Typing RdRp and VP1 Genes of Norovirus Genogroups I and II in Wastewater.

Noroviruses (NoVs) are the leading cause of non-bacterial gastroenteritis with societal costs of US$60.3 billion per annum. Development of a long amplicon nanopore-based method for dual-typing the RNA-dependent RNA polymerase (RdRp) and major structural protein (VP1) regions from a single RNA fragment could improve existing norovirus typing methods. Application to wastewater-based epidemiology (WBE) and environmental testing could enable the discovery of novel types and improve outbreak tracking and source apportionment. Here, we have developed such a method with a consensus-based bioinformatics pipeline and optimised reverse transcription (RT) and PCR procedures. Inhibitor removal and LunaScript® RT gave robust amplification of the ≈ 1000 bp RdRP + VP1 amplicon for both the GI and GII PCR assays. Platinum™ Taq polymerase showed good sensitivity and reduced levels non-specific amplification (NSA) when compared to other polymerases. Optimised PCR annealing temperatures significantly reduced NSA (51.3 and 42.4% for GI and GII), increased yield (86.5% for GII) and increased taxa richness (57.7%) for GII. Analysis of three NoV positive faecal samples showed 100% nucleotide similarity with Sanger sequencing. Eight GI genotypes, 11 polymerase types (p-types) and 13 combinations were detected in wastewater along with 4 GII genotypes, 4 p-types and 8 combinations; highlighting the diversity of norovirus taxa present in wastewater in England. The most common genotypes detected in clinical samples were all detected in wastewater while we also frequently detected several GI genotypes not reported in the clinical data. Application of this method into a WBE scheme, therefore, may allow for more accurate measurement of norovirus diversity within the population.

The effect of the Australian bushfires and the COVID-19 pandemic on health behaviours in people with multiple sclerosis.

BACKGROUND: Crises and disasters disproportionally impact people with chronic health conditions such as multiple sclerosis (MS). OBJECTIVE: To assess the impact of the COVID-19 pandemic and the Australian Black Summer Bushfires on health behaviours in people with MS. METHODS: People with MS, carers, healthcare and advocacy professionals were recruited online between May-July 2020 for an online survey and telephone interviews. RESULTS: Survey items relating to health behaviours were completed by 113 people with MS, and 18 people with MS, 4 MS advocates, 5 healthcare professionals, and 2 carers were interviewed. The bushfires affected 34.5% and the pandemic affected 74.3% of survey participants with MS. The pandemic and bushfires caused a decrease in physical activity in 53.8% and 55.3% of participants respectively, as well as increases in unhealthy eating (43.6% and 24.3% respectively) and alcohol consumption (35.4% and 10.5% respectively), and a decrease in typical sleeping patterns (40.5% and 39.5% respectively). Conversely, 27.5% of participants reported an increase in physical activity during the pandemic. Interview data detailed the circumstances and motivations for changes in health behaviours, as well as consequences, including reduced mobility, fitness, mood disturbances, and weight gain. CONCLUSION: There is a need to increase support and health promotion for people with MS to maintain or initiate positive health behaviours, especially in times of adversity.

Use of F-Specific RNA Bacteriophage to Estimate Infectious Norovirus Levels in Oysters.

Contamination of bivalve shellfish, particularly oysters, with norovirus is recognised as a significant food safety risk. Methods for quantification of norovirus in oysters using the quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) are well established, and various studies using RT-qPCR have detected norovirus in a considerable proportion of oyster samples, both in the UK and elsewhere. However, RT-qPCR detects viral genome, and by its nature is unable to discriminate between positive results caused by infectious viruses and those caused by non-infectious remnants including damaged virus particles and naked RNA. As a result, a number of alternative or complementary approaches to RT-qPCR testing have been proposed, including the use of infectious viral indicator organisms, most frequently F-specific RNA bacteriophage (F-RNA phage). In this study, we investigated the relationships between F-RNA phage and norovirus in digestive tissues from two sets of oyster samples, one randomly collected at retail (630 samples), and one linked to suspected norovirus illness outbreaks (nine samples). A positive association and correlation between PCR-detectable levels of genogroup II F-RNA bacteriophage (associated with human faecal contamination) and norovirus was found in both sets of samples, with more samples positive for genogroup II phage, at generally higher levels than norovirus. Levels of both viruses were higher in outbreak-related than retail samples. Infectious F-RNA phage was detected in 47.8% of all retail samples, and for a subset of 224 samples where characterisation of phage was carried out, infectious GII phage was detected in 30.4%. Infectious GII phage was detected in all outbreak-related samples. Determination of infectivity ratios by comparing levels of PCR-detectable (copies/g) and infectious GII phage (pfu/g) revealed that in the majority of cases less than 10% of virus detected by RT-qPCR was infectious. Application of these ratios to estimate infectious norovirus levels indicated that while 77.8% of outbreak-related samples contained > 5 estimated infectious norovirus/g, only 13.7% of retail samples did. Use of a combination of levels of PCR-detectable norovirus and infectious F-RNA phage showed that while only 7.0% of retail samples contained both > 100 copies/g norovirus and > 10 pfu/g F-RNA phage, these combined levels were present in 77.8% of outbreak-related samples, and 75.9% of retail samples with > 5 estimated infectious norovirus/g. We therefore suggest that combining RT-qPCR testing with a test for infectious F-RNA phage has the potential to better estimate health risks, and to better predict the presence of infectious norovirus than RT-qPCR testing alone.

Effect of ionizing radiation on prostaglandin-like activity in tissues.

One to 7 days after whole body exposure of mice to a single dose of 700 R of x-rays, little or no change was detected in prostaglandin-like activity in the brain, blood and seminal vesicles. Slight increases in intestinal and renal tissue were not significant. In the lung, mean activity rose from 62 ng/g to a transient peak of 145 ng/g wet weight on the fourth day (P less than 0.05). In the spleen, mean levels rose steadily from 13.2 ng/g to 259 ng/g on the fourth day (P less than 0.01), and were still 184.4 ng/g on the seventh day. 2 Prostaglandin-like activity was measured 4 days after single doses of 200-700 R. In the lung, a significant rise was produced by 600 and 700 R, and in the spleen by 200-700 R. 3 Thin layer chromatography showed that part of the prostaglandin-like activity in spleen extracts had RF similar to that of [3H]-prostaglandin E1, and part to that of [3H]-prostaglandin F2alpha. 4 Splenic tissue from mice exposed to 700 R four days earlier, inactivated prostaglandin E1 less potently than did tissue from non-irradiated mice.

Light climate and energy flow in the seagrass canopy of Amphibolis griffithii (J.M. Black) den Hartog.

Absorption of light and radiation use efficiency (RUE) were measured in a dense stand of the seagrass Amphibolis griffithii in Warnbro Sound, a temperate marine embayment in southern Western Australia. Total light intercepted by the canopy was measured and compared with dry weight leaf production, under both summer and winter conditions. RUE was found to be higher in winter (1.56 g MJ-1) than summer (1.01 g MJ-1). These values are very similar to values measured for annual crop plants and emphasise the value of applying theory developed for terrestrial crop plants to seagrasses. Canopy extinction coefficients were 0.93 m-1 in winter and 0.44 m-1 in summer. There were large differences in hours above saturating irradiance (H sat) between the top (Hsat = 5 h 14 min) and base (18 min) of the canopy in winter. Energy flows in A. griffithii suggest that this species is highly susceptible to short-term perturbations in incident irradience during the winter period as the energy stored within the rhizomes is small relative to daily respiratory demands.

Effect of ionizing radiation on 15-hydroxy prostaglandin dehydrogenase (PGDH) activity in tissues.

Whole body irradiation of mice with 200-1000 R of unfiltered X-radiation (230 kV, 15 mA, 140 R/min) produced extensive falls of 15-hydroxy prostaglandin dehydrogenase (PGDH) activity in the spleen within 4 hours. A transient recovery between 4 and 72 hours was followed by a second reduction in PGDH levels which was still evident 7 days after exposure. In the jejunum and kidney, the falls were smaller. High doses (1000 R) increased PGDH activity in the lung. Effects of radiation on the other cellular enzymes, including other dehydrogenases in the cytosol, were less pronounced, and in some cases the opposite of the effects on PGDH. The loss of PGDH may contribute to the increase in prostaglandin concentrations in the spleen and jejumun, and thereby to some features in radiation sickness.

Popliteus tendon rupture: a case report and review of the literature.

Isolated rupture of the popliteus musculotendinous unit is an uncommon injury. A case is here reported of a semiprofessional athlete with a ruptured popliteus tendon without significant instability of the knee. The diagnosis was made by magnetic resonance imaging and confirmed by arthroscopy.

Boneloc bone-cement: experience in hip arthroplasty during a 3-year period.

Polymethyl methacrylate (PMMA) bone-cement was introduced in the 1960s for fixation of total hip arthroplasty replacement components. Long-term results of cement fixation for hip and knee arthroplasty have been extremely good. Although the use of PMMA bone-cement has enabled long-term survival of joint arthroplasty implants, there has been concern about aseptic loosening. This concern led to the introduction of Boneloc bone-cement (Biomet, Warsaw, IN) in the early 1990s. It was hoped that with the improved physical and chemical characteristics of Boneloc, there would be less aseptic loosening in the long-term. A clinical trial was conducted to evaluate Boneloc bone-cement in cementing the femoral component of the Bimetric total hip arthroplasty prosthesis in 33 hips in 32 patients. On follow-up, 7 stems (24%) developed definite loosening, and 3 stems (10%) were possibly loose. Of the 7 definite loose stems, 5 (17%) were revised because of increasing pain or progressive loosening. Despite the biologic advantages of Boneloc, this study suggests that the chemicals substituted in Boneloc bone-cement led to an alteration in its mechanical properties. These properties proved to be inferior to conventional PMMA bone-cement. There is possible time-dependent deterioration of mechanical properties leading to early aseptic loosening. The conventional PMMA bone-cement has stood the test of time. Research and experimental studies should continue to improve the mechanical properties of Boneloc before further human trials.

Prostaglandins and complement changes in some conditions related to inflammation.

Exposure to ionizing radiation produces several systemic and local reactions which could be mediated by prostaglandins. Prostaglandin levels were therefore studied in blood and tissues of mice which had been exposed to x-rays. Significant increases were found in spleens after 200 to 700 R, and in lungs after 600 to 700 R. These changes were most pronounced 4-7 days after irradiation. Ionizing radiation promptly and potently reduced the activity of prostaglandin dehydrogenase in the spleen, whereas prostaglandin synthesis was less affected. Evidence was obtained for the activation and consumption of haemolytic complement in serum in the course of heart-lung operations involving extracorporeal circulation. Activation involved primarily the classical pathway, and only slightly the alternate pathway.