Abstract
The impact of viruses in water on Public Health must not be underestimated, neither in low‐, middlenor
in high‐income countries. Even as little as five infectious rotavirus particles have been described
as able to cause serious gastrointestinal infections with potentially lethal outcome for children under
the age of five years, immunocompromised patients and the elderly.
Most analytical procedures for the detection and determination of enteric waterborne viruses in
concentrated water samples originating from sewage or surface water are based on molecular
methods like the quantitative polymerase chain reaction (qPCR) or cell culture, the gold standard of
environmental virology. However, both approaches show specific and limiting disadvantages: while it
is possible to detect each infectious virus particle of certain enteric viruses on specific undifferentiated
animal or human cells, there still is no commercial cell line available for norovirus, one of the most
relevant viral Public Health concerns. Furthermore, the method is time‐ and cost intensive, requires
experienced personnel as well as special biosafety laboratories. Compared to cell culture, qPCR is
relatively cheap, easy to establish in laboratories of all income settings, adaptable for novel viruses,
highly sensitive and uniquely specific. The main disadvantage of molecular methods is their inability to
differentiate between infectious and inactivated viruses, which complicates a realistic evaluation of
the viral load of a water body and the subsequent assessment of Public Health risks. Additionally,
various organic and inorganic polymerase inhibitory substances tend to be co‐concentrated in the
sample pretreatment and interfere with the amplification.
The addition of the genome intercalating and cell membrane / virus capsid impermeable molecular
dyes propidium monoazide (PMA) and ethidium monoazide (EMA) ‐ thus creating the capsid integrity
(ci‐) qPCR ‐ has first been described for the distinction of viable but non‐culturable waterborne bacteria
in the context of water and food safety (Hoelzer et al., 2013). Due to its mechanism of signal removal
and the theoretical inability to penetrate capsids of intact and therefore infectious viruses, the method
shows potential of compensating for the shortcomings of qPCR and RT‐qPCR.
Since data on the suitability of the dye pretreatment for the differentiation between infectious and
inactive viruses is limited, the first study pursued the aim of investigating the ability of both dyes to
depict the partial or complete loss of infectivity of various enteric viruses and bacteriophages.
Solutions of these viruses have been exposed to temperature (45°C, 55°C, 65°C), biocidal UVC (30s,
60s, 120s) and chlorine (2mg/l), photo‐activated by a suitable light source in the presence of PMA and
EMA and subsequently analyzed quantitatively and qualitatively utilizing culture based assays as well
as conventional qPCR. The addition of dye pretreatment has been evaluated correspondingly.
87
While not significantly correlating with culture based assays after inactivation with temperature and
UV light, the results of EMA‐(RT)‐qPCR and PMA‐(RT)‐qPCR after chlorine inactivation concurred
consistently with the gold standard for all viruses under investigation. Furthermore, using (RT)‐ci‐qPCR
for quantification of samples exposed to temperature above 55°C lead to a gradually (but not
significant) removal of false negative results compared to conventional qPCR and RT‐qPCR, thus
indicating the loss of viral infectivity and improving the relevance of molecular assays.
After proving the suitability of EMA and PMA pretreatment to enhance the weight of (RT)‐qPCR results
in principle and under laboratory scale conditions, the second study has been conducted with the aim
to evaluate the possibility of its transfer to real life applications. In the context of the Safe Ruhr project,
conducted by the German Federal Ministry for Research and Education (BMBF) 53 samples from the
urban river Ruhr and 26 samples from the last stages of the fourth tier of an experimental waste water
treatment facility (both Essen, Germany) have been taken and analyzed for their content of human
adenovirus (HAdV), rotavirus (RV) and enterovirus (EV) using conventional molecular and culture based
methods as well as the (RT) ci‐qPCR.
For HAdV it could be shown that EMA/PMA pretreatment as well as cell culture succeeded in removing
huge proportions of false positive signals. Application of merely conventional qPCR would have led to
a vast overestimation of the viral load of the samples. Similar results could be obtained for RV and EV
but since abundance and mean concentration of both RNA viruses was measured considerably lower
than HAdV, results remained inconclusive in parts. Therefore, further studies should be conducted to
appoint a cut‐off value under which the utilization of ci‐qPCR would not be advised. The application of
dye pretreatment could improve the ability of laboratories in low‐ and middle‐income settings to
assess the viral load of surface water bodies with more confidence but without significant additional
costs and processing time to existing (RT)‐qPCR protocols for all enteric viruses.
Since human adenovirus is both intensively discussed as a potential indicator for the fecal
contamination of a water body and a Public Health risk, a submitted third study was conducted to
propose a EMA/PMA pretreatment condition which could be combined with existing HAdV qPCR
protocols, regardless of specific factors like amplicon length and hybridization energy. HAdV samples
have been inactivated partially and completely according to Leifels et al. (2015) and quantified using
two widely used HAdV qPCR protocols.
Even though amplicon length and hybridization energy differed vastly, both protocols resulted in
similar removal rates for false negative signals (confirmed by cell culture) after chlorine and heat
inactivation. As before, inactivation derived from UV exposure could not be indicated. However, since
environmental samples are always exposed to a broad array of chemical and physical agents, the
88
addition of EMA/PMA pretreatment is still advised since it remarkably improves the quality of the
quantified virus amounts solely by molecular methods in any given environmental water body. This
improvement is achieved while only adding minimal extra cost and time and thereby compensates for
the monetary limitations which often inhibits the application of cell culture based assays.
Researchers in countries with considerable rates of acute and chronic waterborne diseases and limited
Public Health funds could benefit from such higher confidence in the results obtained by the inclusion
of EMA‐/PMA pretreatment.
in high‐income countries. Even as little as five infectious rotavirus particles have been described
as able to cause serious gastrointestinal infections with potentially lethal outcome for children under
the age of five years, immunocompromised patients and the elderly.
Most analytical procedures for the detection and determination of enteric waterborne viruses in
concentrated water samples originating from sewage or surface water are based on molecular
methods like the quantitative polymerase chain reaction (qPCR) or cell culture, the gold standard of
environmental virology. However, both approaches show specific and limiting disadvantages: while it
is possible to detect each infectious virus particle of certain enteric viruses on specific undifferentiated
animal or human cells, there still is no commercial cell line available for norovirus, one of the most
relevant viral Public Health concerns. Furthermore, the method is time‐ and cost intensive, requires
experienced personnel as well as special biosafety laboratories. Compared to cell culture, qPCR is
relatively cheap, easy to establish in laboratories of all income settings, adaptable for novel viruses,
highly sensitive and uniquely specific. The main disadvantage of molecular methods is their inability to
differentiate between infectious and inactivated viruses, which complicates a realistic evaluation of
the viral load of a water body and the subsequent assessment of Public Health risks. Additionally,
various organic and inorganic polymerase inhibitory substances tend to be co‐concentrated in the
sample pretreatment and interfere with the amplification.
The addition of the genome intercalating and cell membrane / virus capsid impermeable molecular
dyes propidium monoazide (PMA) and ethidium monoazide (EMA) ‐ thus creating the capsid integrity
(ci‐) qPCR ‐ has first been described for the distinction of viable but non‐culturable waterborne bacteria
in the context of water and food safety (Hoelzer et al., 2013). Due to its mechanism of signal removal
and the theoretical inability to penetrate capsids of intact and therefore infectious viruses, the method
shows potential of compensating for the shortcomings of qPCR and RT‐qPCR.
Since data on the suitability of the dye pretreatment for the differentiation between infectious and
inactive viruses is limited, the first study pursued the aim of investigating the ability of both dyes to
depict the partial or complete loss of infectivity of various enteric viruses and bacteriophages.
Solutions of these viruses have been exposed to temperature (45°C, 55°C, 65°C), biocidal UVC (30s,
60s, 120s) and chlorine (2mg/l), photo‐activated by a suitable light source in the presence of PMA and
EMA and subsequently analyzed quantitatively and qualitatively utilizing culture based assays as well
as conventional qPCR. The addition of dye pretreatment has been evaluated correspondingly.
87
While not significantly correlating with culture based assays after inactivation with temperature and
UV light, the results of EMA‐(RT)‐qPCR and PMA‐(RT)‐qPCR after chlorine inactivation concurred
consistently with the gold standard for all viruses under investigation. Furthermore, using (RT)‐ci‐qPCR
for quantification of samples exposed to temperature above 55°C lead to a gradually (but not
significant) removal of false negative results compared to conventional qPCR and RT‐qPCR, thus
indicating the loss of viral infectivity and improving the relevance of molecular assays.
After proving the suitability of EMA and PMA pretreatment to enhance the weight of (RT)‐qPCR results
in principle and under laboratory scale conditions, the second study has been conducted with the aim
to evaluate the possibility of its transfer to real life applications. In the context of the Safe Ruhr project,
conducted by the German Federal Ministry for Research and Education (BMBF) 53 samples from the
urban river Ruhr and 26 samples from the last stages of the fourth tier of an experimental waste water
treatment facility (both Essen, Germany) have been taken and analyzed for their content of human
adenovirus (HAdV), rotavirus (RV) and enterovirus (EV) using conventional molecular and culture based
methods as well as the (RT) ci‐qPCR.
For HAdV it could be shown that EMA/PMA pretreatment as well as cell culture succeeded in removing
huge proportions of false positive signals. Application of merely conventional qPCR would have led to
a vast overestimation of the viral load of the samples. Similar results could be obtained for RV and EV
but since abundance and mean concentration of both RNA viruses was measured considerably lower
than HAdV, results remained inconclusive in parts. Therefore, further studies should be conducted to
appoint a cut‐off value under which the utilization of ci‐qPCR would not be advised. The application of
dye pretreatment could improve the ability of laboratories in low‐ and middle‐income settings to
assess the viral load of surface water bodies with more confidence but without significant additional
costs and processing time to existing (RT)‐qPCR protocols for all enteric viruses.
Since human adenovirus is both intensively discussed as a potential indicator for the fecal
contamination of a water body and a Public Health risk, a submitted third study was conducted to
propose a EMA/PMA pretreatment condition which could be combined with existing HAdV qPCR
protocols, regardless of specific factors like amplicon length and hybridization energy. HAdV samples
have been inactivated partially and completely according to Leifels et al. (2015) and quantified using
two widely used HAdV qPCR protocols.
Even though amplicon length and hybridization energy differed vastly, both protocols resulted in
similar removal rates for false negative signals (confirmed by cell culture) after chlorine and heat
inactivation. As before, inactivation derived from UV exposure could not be indicated. However, since
environmental samples are always exposed to a broad array of chemical and physical agents, the
88
addition of EMA/PMA pretreatment is still advised since it remarkably improves the quality of the
quantified virus amounts solely by molecular methods in any given environmental water body. This
improvement is achieved while only adding minimal extra cost and time and thereby compensates for
the monetary limitations which often inhibits the application of cell culture based assays.
Researchers in countries with considerable rates of acute and chronic waterborne diseases and limited
Public Health funds could benefit from such higher confidence in the results obtained by the inclusion
of EMA‐/PMA pretreatment.
| Original language | English |
|---|---|
| Publication status | Published - 2017 |
| Externally published | Yes |