Uhrbrand, Katrine

Norovirus (NoV) er den primære årsag til viral gastroenteritis på verdensplan. Med en estimeret infektiv dosis (ID50) på 18 virus partikler er NoV ekstremt infektiøse. NoV smitter via den fækalorale rute enten direkte via person-til-person kontakt eller indirekte gennem kontaminerede genstande, vand eller fødevarer. I de senere år er utallige udbrud af gastroenteritis blevet forbundet med indtagelse af NoV kontamineret drikkevand, skaldyr, grøntsager og bær. Endelig er der set indikationer på, at NoV kan smitte gennem luften via aerosoldannelse specielt i forbindelse med voldsomt opkast. Til trods for dette, eksisterer der til stadighed kun sparsom viden omkring risikoen for luftbåren smitte af NoV.For at reducere spredning af NoV er det essentielt at have effektive virus oprensningsmetoder til rådighed, der kan anvendes til rutinemæssigt at analysere for tilstedeværelsen af NoV i fødevarer og drikkevand. På nuværende tidspunkt findes der dog ingen standardiserede procedurer til dette. Formålet med nærværende Ph.d. afhandling var derfor at udvikle og evaluere metoder til at oprense og opkoncentrere NoV fra skaldyr og drikkevand for således at bidrage til processen med at finde velegnede standardiserede metoder. Dette blev gjort gennem tre studier, der er præsenteret i manuskript I-III. Endvidere belyser afhandlingen muligheden for luftbåren smitte af NoV og udforsker en mulig strategi til NoV dekontaminering af fødevareoverflader. Dette blev gjort i henholdsvis manuskript IV og V.I manuskript I blev fem oprensningsmetoders evne til kvantitativt at genfinde NoV genogruppe II (GII) og feline calicivirus (FCV) tilsat væv fra fordøjelsesorganet i østers og blåmuslinger sammenlignet. Bedste resultat blev fundet med en metode baseret på proteinase K behandling og NucliSens miniMAG nukleinsyre ekstraktion. Metoden blev efterfølgende evalueret i en ringtest, hvor det blev demonstreret, at den kunne anvendes til robust at oprense og genfinde NoV GI, NoV GII and hepatitis A virus (HAV) bioakkumuleret i østers og blåmuslinger. Metoden menes derfor at være en velegnet som en fremtidig standard rutine metode til virus analyse af skaldyr.I manuskript II blev en hurtigmetodes evne til at genfinde NoV GI, NoV GII og adenovirus (AdV) fra forskellige typer af Nordisk drikkevand evalueret. Metoden var baseret på filtrering med et positivt ladet filter efterfulgt af direkte lysering af vira på filteret. Med en gennemsnitlig genfindelsesprocent på 43±29 for NoV GI og 45±24 for NoV GII var metoden generelt bedre, eller som minimum sammenlignelig, med tidligere publicerede metode. Dette tyder på at metoden kan være egnet til rutine analyser af drikkevand. Dog fandtes virus og vand type at have en signifikant effekt på virus genfindelsen.Som en alternativ strategi til opkoncentrering af NoV fra drikkevand blev anvendeligheden af virus imprintede polymere (VIPs) til selektiv genkendelse og binding af NoV udforsket i manuskript III. Tre VIPs til selektiv binding af henholdsvis murine norovirus (MNV), NoV GI og NoV GII blev syntetiseret. Binding af target virus og non-target virus (AdV) til både VIPs og nogle non-imprintede polymere (NIPs) blev undersøgt. Relativ dårlig selektiv virus genkendelse og høj grad af uspecifik binding blev desværre observeret for de syntetiserede VIPs. VIPs i deres nuværende form menes derfor ikke at være anvendelige til opkoncentrering af virus fra drikkevand.Aerosolisering af mikroorganismer kan finde sted i forbindelse med spildevandsrensning. Eksponering for aerosoliserede NoV og andre bioaerosol komponenter på et rensningsanlæg blev undersøgt med personbårne luftsamplere i manuskript IV. NoV blev for første gang nogensinde påvist på et støvfilter båret af en ansat på rensningsanlægget i koncentrationer, som potentielt kan medføre en erhvervsmæssig risiko. Da øget hyppighed af akutte mave-tarm problemer, matchende symptomerne på Roskildesyge, tidligere var rapporteret blandt de ansatte på rensningsanlægget, indikerer vores fund, at luftbåren smitte med NoV forekommer på rensningsanlægget.Effektiviteten af damp-ultralydsbehandling til at inaktivere NoV surrogaterne FCV, MNV og bakteriofag MS2 blev evalueret i manuskript V. Damp-ultralydsbehandling var effektiv til virus dekontaminering af plast overflader med næsten komplet inaktivering efter behandling i 1 eller 3 s afhængig af typen af NoV surrogat. Derimod var damp-ultralydsbehandling i den nuværende form utilstrækkelig til NoV dekontaminering af hindbær. Behandling i 1 s medførte nemlig kun 1-log reduktion af MS2 og resulterede i uønsket tekstur ændring af hindbærrene.Noroviruses (NoVs) are the principal cause of viral gastroenteritis worldwide. NoVs are extremely infectious with an estimated infectious dose 50% (ID50) of 18 viral particles. Transmission of NoVs occurs via the faecal-oral route directly through person-to-person contact or indirectly via contaminated fomites, water and food. In recent years, numerous outbreak of NoV-associated gastroenteritis have been linked to the consumption of contaminated drinking water, shellfish and fresh produce. In addition, indications of transmission occurring though aerosol formation, especially following projectile vomiting, have been seen, but little is at present known about the possibility and extent of airborne transmission of NoVs.To reduce transmission of NoV it is imperative to have efficient virus recovery methods available that can be used for routine analysis of the implicated matrices. However, currently no standardised procedures exist for this purpose. The aim of this PhD thesis was to develop and evaluate methods for recovery of NoV from shellfish and drinking water that could contribute to the work done towards finding suitable standardised methods. This was done through three studies, presented in manuscript I-III. Additionally, exposure to airborne NoV transmission and NoV decontamination of food surfaces were addressed in manuscript IV and V, respectively.In manuscript I five methods were compared for the ability to qualitatively recover NoV GII and feline calicivirus (FCV) spiked in the digestive tissue of oysters and blue mussels. A method based proteinase K digestion followed by NucliSens miniMAG nucleic acid extraction was found to give the best performance. In a subsequent collaborative trial the method was found to robustly recover the NoV GI, NoV GII and HAV bioaccumulated in the both oysters and mussels. Consequently, the method was found to be a good candidate as a future qualitative standard for routine viral analysis of oysters and mussels.The ability of a rapid method to recover NoV GI, NoV GII and adenovirus (AdV) from Nordic drinking water (tap water) of various types was evaluated in manuscript II. The method was based on filtering using a positively charged membrane followed by direct lysis of the virus adsorbed to the membrane. The average efficiency of the method to recover NoV GI (43±29%) and GII (45±24%) from drinking water was generally found to be better, or at least comparable, to previously published methods, suggesting that the method could be suitable for routine analysis. However, virus and water type were found to significantly affect the recovery.As an alternative strategy for the recovery of NoVs from drinking water the feasibility of using virus imprinted polymers (VIPs) for capture and selective recognition of NoVs was investigated in manuscript III. Three VIPs targeted for murine norovirus (MNV), NoV GI and NoV GII, respectively, were synthesised and experiments to determine binding of the target viruses and a non-target AdV to the VIPs and to non-imprinted polymers (NIPs) were conducted. Unfortunately, a relative poor selective recognition and high degree of unspecific binding to the VIPs were observed. Thus, VIPs in their current form do not appear to be an optimal approach for recovery of NoVs from drinking water.Aerosolisation of microorganisms may occur during treatment of wastewater. Therefore, the exposure to aerosolised NoVs and other bioaerosol components at a wastewater treatment plant (WWTP) was examined using personal samples in manuscript IV. NoVs were, for the first time, detected on a dust filter carried by a WWTP worker in concentrations that could pose an occupation risk. Because the workers at the WWTP had previously been found to have an increase frequency of acute gastrointestinal illness, consistent with NoV infection, our finding suggest that that airborne transmission of NoVs do indeed occur at the WWTP.Finally, manuscript V evaluated the efficiency of a surface decontamination strategy based on combined treatment with steam and ultrasound to inactivate the NoV surrogates FCV, MNV and bacteriophage MS2. The steam-ultrasound treatment was found to be effective for decontamination of plastic surfaces with near complete inactivation observed after treatment for 1 or 3 s, depending on the NoV surrogate. However, steam-ultrasound treatment in it current form was found to be inappropriate for NoV decontamination of raspberries as only 1-log reduction of MS2 was achieved after treatment 1 s at which point unwanted loss in the texture of the raspberries were observed

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Development and evaluation of methods for recovery of Noroviruses from food, water and air

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Development and evaluation of methods for recovery of Noroviruses from food, water and air

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