1. To establish threshold maximum acceptance levels for genome copies of health-significant waterborne viruses that cannot be grown in cell cultures so that they represent an acceptable risk for the consumers.
2. To indirectly ascertain the infectivity of viruses detected by molecular procedures. Several sample pre-treatments prior to molecular detection will be evaluated in order to provide a better estimation of infectious virus numbers. Additionally systems to evaluate specific virus nucleic acid or protein damage caused by some virucidal treatments will be developed.
3. To determine the genetic diversity within the target viruses and how such diversity could be exploited in outbreak investigation to link human and environmental strains. Genetic characterization of virus isolates will determine the potential zoonotic origin of waterborne viruses.
Task 2.1 Establish robust quantitative molecular multiplex systems for detection of health-significant enteric viruses (UB, DTU-FOOD, IST SLU, UH)
A quadruplex Real-Time RT-qPCR assay fulfilling the requirements of the method developed by the European Committee on Standardization (CEN) was developed by the UB participants for the simultaneous quantitative detection of hepatitis A virus (HAV), norovirus (NoV) GI and GII, and mengovirus (used as process control for determination of the virus/nucleic acid extraction efficiency). The method was published in C. Fuentes et al., Food Microbiology, 2014, 40: 55).
While at IST several different combination RT-qPCR assays for different viral pairs were created (HAV+NoV; HAV+hepatitis E virus -HEV; NoV+rotavirus-RV), at SLU, VOCMA originally developed for detection on Luminex has been transferred to protocols suited for detection with RT-qPCR for NoV GI and II, sapovirus (SaV), HAV and HEV, creating one calici and one hepatitis panel respectively. Additionally, the use of internal quenchers has been investigated for NoV GII showing great potential for internal zen-quenchers and detection of a single target copy (manuscript submitted to Biotechnology).
At UH, duplex real-time RT-qPCR using NoV GI and GII primer-probe sets labelled with VIC and FAM were tested. The sensitivity of the duplex assay was compared to those of monoplex GI and GII assays with 10-fold dilutions of NoV GI.6 and GII.4 nucleic acid. In the conditions used, the sensitivity was comparable to that of monoplex, although the Cq-values for both genotypes obtained by duplex assay were about 2 cycles higher than Cq-values by monoplex assays. After some adjustments, multiplex assays may be suitable for testing viruses in water samples.
Task 2.2 Investigate pre-treatments of target viruses in order to provide a better estimation of infectivity through genome copy determination (UB, DTU-FOOD, IST, SLU, UH)
Methods involving the use of fluorescent dyes such as propidium monoazide (PMA) or ethidium monoazide (EMA), as well as mucin-coated beads have been used for the live-dead differentiation in molecular tests.
In the fulfillment of this task, PMA treatment before RT-qPCR amplification was optimized in all participant laboratories for the detection and quantification of viruses with intact cohesive undamaged capsids. As an example, low FC doses up to 2.5 mg/L only affected the infectivity assay with a signal reduction of 1.34±0.45 log. No effects were observed in any of the molecular tests used (RT-qPCR alone, PMA+RT-qPCR or PMA/Triton+RT-qPCR). With higher doses up to 5 and 10 mg/L of free chlorine, infectivity was reduced over 4.5 logs, and in most cases viral titers decreased below the detection limit. Log reductions obtained by molecular tests were higher when PMA was included, but TritonX100 did not result in a significant increase in log reduction.
Again as an example, after heat treatment at 70ºC, infectivity was reduced 2.48±1.30 logs. Treatment at 85ºC and 99ºC resulted in a loss of 3.58±0.32 and 4.50±0.58 logs of infectious viruses, respectively. Despite this high effect on infectivity, RT-qPCR alone only caused a reduction lower than 1 log in all cases, confirming that the main target for inactivation using high temperature is the viral capsid. When assayed by PMA/Triton+RT-qPCR, log reductions obtained at 85ºC and 99ºC were 2.81±0.38 and 3.63±0.48, respectively, which only differ in less than 1 log with infectivity log reductions.
However, quantification by PMA-RT-qPCR showed a limited and varying distinction between heat treated non-intact and non-heat treated intact viral particles with the best effect for samples initially containing moderate levels of viruses (3.64 ×102 – 2.71×105 GC or RT-qPCR units). Thus a mean reduction of 1.53 ± 0.68 Log (spanning from 1.18 Log for SaV to 2.05 log for MNV) could be demonstrated for samples containing viruses in levels ≥ 3.64 ×102 GC or RT-qPCR units. Interpretation of data for samples with an initial viral load ≤ tLOQ (approximately 2×102 genome units) was difficult due to uncertainty of the RT-qPCR assays. In conclusion, these data show that the application of PMA-RT-qPCR provides a limited and insufficient distinction between viral RNA from heat inactivated and infectious virions of NoV, SaV and MNV. The efficiency of the method depends on the type and level of virus in the sample.
Additionally, DTU-Food have tested PGM-coated beads (mucin-conjugated beads) for the efficiency to capture viruses with intact capsids present in different levels without treatment and after being heat treated at 80ºC for 10 minutes. The application of PGM-RT-qPCR on heat treated samples resulted in overall reductions in detectable viral genomes from 58.51 to 3.97% for NoV GI and from 68.69 to 4.80% of NoV GII. For MNV, a reduction from 12.27 to 1.00% PCR detectable genomes could be observed in the heat treated suspensions. The loss of intact viruses during PGM capture was determined by quantifying the remaining part of intact viruses present in the supernatant subjected to RNAse treatment prior to nucleic acid extraction. This indicated an escape from PGM capture of a smaller fraction of viruses with intact capsids which might be due to destroyed surface proteins.
The data confirms that capturing of viruses using PGM coated beads, may indeed facilitate selection of viruses with intact surface proteins.
Task 2.3 Investigate specific virus damage through some specific virucidal treatments (UB)
Work at the UB has been focused at elucidating the effect of different concentrations of free chlorine (FC) on the genome of human norovirus (NoV) GII.4 (New Orleans1805/2009/USA).
Primers were designed in order to cover the full-length NoV genome in 15 fragments. The experimental design was to apply to the NoV suspension increasing concentration of FC (from 0 to more than 9 mg/l) for 30 min at room temperature in the dark. Our data point is a differential degradation of the NoV genome segments. Some segments are more resistant than others to the effect of FC. These findings open the possibility to employ specific genome fragments for different purposes; e.g., a resistant fragment to trace the presence of contaminant NoV in a sample NoV-tracking), or a sensitive fragment to ascertain the inactivation of NoV after FC treatment.
At DTU-Food, inactivation studies of MNV in suspensions and on surfaces using electrolyzed oxidizing water (EOW) produced in hand hold spraying bottles had been conducted to test the efficiency to inactivate MNV, NoV GI nd NoV GII. Results from suspension tests applying 0.5 and 1 minute contact time have shown 1 and 2 log reduction in MNV plaque forming units (PFU) using 100 or 200 ppm AFC, respectively. Results from surface tests on MNV dried on stain less steel disks applying 0.5 minute contact time with liquid EOW, showed 1 and 2 log reductions in MNV PFU using 200 or 400 ppm AFC, respectively, and >4 log10 reductions using 1, 5 and 10 minutes contact times combined with either 200 and 400 ppm AFC. Applying sprayed EOW on stainless steel surfaces with dried MNV required 10-20 minutes contact time to reduce MNV by 3-4 log. The determination of the reductions in MNV, NoV GI and NoV GII genomes using traditional RT-qPCR has been delayed to autumn 2015. And so has the determination of potential genomic hot spots sensible to EOW using e.g. the newly developed PCR assays which amplify the whole norovirus genome in 15 segments (UB lab, unpublished).
Task 2.4 Generate typing tools for health significant human and animal viral pathogens in clinical and environmental samples (UB, DTU-FOOD, IST, SLU, UH)
Several genotyping tools have been developed in this Task, enabling the generation of genotyping tools applicable to samples, usually water, environmental or food samples, with a low quantity of viruses (Sabrià et al., J. Clin. Microbiol. 2014; D'Andrea et al., Int. J. Mol. Sci. 2015; Kantala et al., Foodborne Pathogens and Disease 2015; Jalava et al., PLoS One. 2014; Oristo et al., Food Environ Virol.2016; Kauppinen et al., in Press 2016).
One study of the epidemiology of food and waterborne outbreaks of norovirus gastroenteritis occurring in Catalonia during 2010-2012 compared clinical features and levels of viral shedding of the most prevalent GII.4 2012 variant with its predecessor. In another study focused on the effect of a universal hepatitis A vaccination program among preadolescents implemented in Catalonia, Spain, during the period of 1999–2013, revealed the emergence of genotype IC during a foodborne outbreak, the short-lived circulation of vaccine-escape variants isolated during an outbreak among the men-having-sex-with-men group, and the association of genotype IIIA associated with the increase of symptomatic cases among the very young.
At DTU-Food, work on the development on NGS- pre-processing has been carried out on a NoV GII.1 positive stool sample adding mengovirus as an index. The use of endonuclease was very important to remove extracellular DNA/RNA, increasing the fraction of reads mapping to viruses from ~2 % to ~40 %. It was found necessary to amplify the extracted RNA/DNA quite a lot (40 PCR cycles) to reach concentrations suitable for NGS. This method has currently been applied on African sewage samples, where preliminary data shows the presence of more than 450 different virus species, including NoV, enteroviruses (EV) and RV. However the most numerous viruses that could be identified are plant viruses associated with human feces and bacteriophages. Since these viruses constitute the biggest part of the sewage viral community, deep sequencing is needed (~1,000,000 reads per sample) to identify human and animal pathogens present in a sewage sample.
22.214.171.124 Highlight significant results of your work package for the 2nd period:
- More reliable singleplex and multiplex molecular assays for detection of waterborne viral agents.
- Methods for better discrimination of infectivity through determination of genome copies.
- Procedures to estimate the inactivation of non-cultivable viruses.
- Molecular typing methodologies for characterization of waterborne viral outbreaks.