WATERBORNE VIRAL INFECTIONS

Project leader: Carl-Henrik von Bonsdorff, University of Helsinki, Department of Virology,
Haartman Institute, P.O.Box 21, FIN-00014 Helsinki University, Finland,
tel. +358-9-19126506, e-mail: Carl-Henrik.vonBonsdorff@helsinki.fi
 
 

PUBLICATIONS

TIIVISTELMÄ SUOMEKSI

Researchers:
Leena Maunula, HUCH Laboratory Diagnostics, tel. +358-9-19126506, e-mail: Leena.Maunula@helsinki.fi
Hongyan Yang, Dept of Genetics, Helsinki University, tel. +358-9-1911, e-mail: Yang.Hongyan@helsinki.fi
Niina Kupiainen (nyk Torvela)
Marja Kukkula veterinarian, tel +358-3-7188788, e-mail: Marja.Kukkula@sci.fi

Consortium: Microbiological risks associated with drinking water contaminated with protozooans, viruses or cyanobacterial toxins
Financing SYTTY organisation: The Academy of Finland
Funding from SYTTY / Total funding of project (€): 150090 / 154800
Person-months of work funded by SYTTY / Total person-months of work: 39 / 66

KEY WORDS: Environmental virology, waterborne, NLV, astrovirus, RT-PCR
 

EXTENDED ABSTRACT

1 Introduction

Earlier attempts to detect human pathogens in the environment have been limited to comprise only viruses that grow in cell culture. The main emphasis has thus been to detect enteroviruses, especially polioviruses, in sewage. Water is the key element in the spreading of human enteric viruses via the environment. Viruses are secreted in large amounts into sewage. These are very resistant to physico-chemical treatments and can thus survive in the environment for long times. In this respect the heaptitis A- and the human caliciviruses of the genus Norwalk-like viruses (NLV) are the most resistant. It has been estimated, that their infectivity may last for as long as 2 years in cold groundwater. These viruses pass through the wastewater plants in the liquid phase and get into the recieving waters. There they may circulate back to man through different routes; via irrigation of fresh produce like vegetables and berries, via bivalve molluscs grown in sewage-contaminated waters, via recreational waters or via drinking water from waterworks using insufficient decontamination methods. During this project a method for detecting human enteric viruses in water was developed. It soon became apparent, that the methology should be simple enough to allow  not only outbreak analysis, but also offer a way for continuous monitoring of e.g. drinking water for pathogenic viruses.

2 Methods

For epidemiological investigations, a possible causative virus was primarily demonstrated from patient samples. When viruses were found in patients, emphasis was put on investigation of environmental samples that consisted tap and well water, raw water, river and lake water or sewage.

Viruses were identified in the first place by gene amplification, RT-PCR (reverse transcription-polymerase chain reaction) but also electron microscopy was used. The major effort was put for detection of NLVs; the RT-PCR and microplate hybridization methods used are described in Maunula et. al. (1999). PCR methods for demonstration of enteroviruses were done in collaboration with T. Hyypiä, Haartmaan Institute (ref?). Hepatitis A virus PCR was performed in collaboration with M. Lappalainen, Haartman Institute, as described in Lappalainen et al. (2001).

A method for demonstrtaion of NLVs from water samples was established. The method was based on the observation of Gilgen et al. (1997). One liter samples of water was filtered through a positively charged filter which bound the viruses. Elution in a small volume of alkaline buffer followed by further concentration in a microconcentratior to 100µl after which PCR was applied to the sample. A detailed description of the method is in Kukkula et al., (1999).

The genotyping of NLVs was performed by DNA sequencing the amplified PCR products as described in Maunula et al. (1999). It was used for closer identification of the isolated viruses as well as for comparisons of viral isolates from patients and environmental samples. Direct sequencing without cloning was used in order to catch the dominant virus. For comparison and analysis of the nucleotide sequences, computer programs (GCG package and SeqUpp)   were used.

Quantitative PCR methods. For quantitative measuremnts of viral loads in sewage twe used the ABI-Prism 7700 (Perkin Elmer). We selected the astro- and enteroviruses for our purpose, because a common primer pair and a probe couled be used  to cover all viruses in each group (Yang and v.Bonsdorff, 2002)

Detection of RNA phages. F-specific RNA phages and somatic coliphages were enumerated with standard methods (ISO 10705-1, 1995 and –2, 2000

3 Results and discussion

In order to investigate the role of viruses in waterborne outbreaks we made use of the PCR-methodology which we had developed for the diagnostics of enteric infections. We created a test package for patient stool samples  comprising of electron microscopy and RT-PCR (reverse transcriptase - polymerase chain reaction) for astroviruses and Norwalk-like caliciviruses (NLV) (Maunula et al 1999). This test-set was then usen to investigate GI-outbreaks in combination to a Finnish intensified reporting and investigation effort of food- and waterborne outbreaks.

For the water sample analysis we developed a methodology for which the rationale started from the sample volume sufficient for the RT-PCR reactions. We concluded, that maximally 1000 ml can effectively be concentrated to that volume. We employed essentially the method originally described by Gilgen et al., 1997) which is based on the negative net charge of these viruses. They are bound to a positively charged filter from which the virus then is eluted in a way described in Kukkula et al (l999). Here it was also successfully used to investigate a large waterborne outbreak. NLV was detected both from patient samples and from different water samples; raw water, consumer tap water as well as lake water downstream from the affected community.

Since then, the investigations comprised all waterborne outbreaks registered in Finland 1998 - 2001. In these 19 outbreaks NLVs were detected in 13 cases from patients and in 6 of these cases from the water, too. (partly published in Miettinen et al. 2001, Kuusi et al, 2002, Kuusi et al, 2002) emphasizing the importance of NLVs. Campylobacter caused five of the outbreaks and only one outbreak remained unresolved. In all of the NLV cases it was shown, that the virus both from patients and from the water were identical as based on the amplicon sequence (Fig 1, unpublished data)

In a recent outbreak a contaminated public children's pool resulted in about 300 cases of disease. Here both NLV and astrovirus was found in the water and in the patients. The NLV was shown to be ampliconwise identical from water and from patient, the astroviruses were not compared, because the amplicon is too conserved to differentiate between strains.

As for the NLV RT-PCR we participated in an European "quality control" study in which almost one hundred NLV-strains from the participating countries were compared. Based on these results we were able to improve the test by slightly modifying the primers. The genus Norwalk-like viruses is still under formation and it is apparent, that further modifications may become necessary.

Even though our findings strongly emphasize the role of NLVs in waterborne outbreaks, we have adapted PCR-tests for other enteric viruses for our purposes. Among them, astroviruses seem to be a potential risk, especially for children. Our studies on sewage also seem to support this, because high concentrations of astroviruses could be demonstrated in sewage samples. We do not, however, presently know, whether astroviruses survive in the environment as effectively as do NLVs and hepatitis A viruses.

The risks for viral contamination through sewage are obvious, but the monitoring methods presently in use (enteric bacteria, bacteriophages) do not necessarily function as indicators for pathogenic viruses. Therefore we monitored sewage at two different sites (a small and a large plant) for the amount of NLVs and astroviruses by quantitative methods. The results clearly indicated, that although the indicator organisms were present at rather even concentrations, the concentartion of pathogenic viruses  varied to a very large extent, apparently reflecting the epidemic situation in the population (v.Bonsdorff et al. 2002, Yang and v.Bonsdorff, 2002).

Even though hepatitis A virus is rather rarely found in Finland, the potential risk of its re-emergence must be considered. Our finding of an suspect waterborne family outbreak is indicative in this respect (Lappalainen et al 2001).

4 Conclusions

The project has clearly shown, that waterborne viral outbreaks are common in Finland. In particular, NLV caliciviruses are responsible for a most of the outbreaks. Also, the monitoring of indicator organisms for microbial contamination of water are not sufficient to anticipate these. The methods suggested and employed by the authorities for specifically detecting of viruses are directed only towards cultivable viruses, i.e. enteroviruses. These methods require large (>30 liter) sample volumes. The methodology applied in this project requires only 1 liter samples which then are concentrated to a 100 µl for actual viral testing by RT-PCR techniques. The method is already in this form applicable not only for outbreak investigations but also for viral monitoring purposes.

Quantitative methods have been employed to monitor the amount of pathogenic viruses in sewage. The results suggest, that the amount of the viruses in question varies according to the epidemiological situation in the population. Thus they are useful both for epidemiological surveys but also for risk assessment

5 References

Gilgen, M., Germann, D., Lüthy, J., Hübner, P. Three-step isolation method for sensitive detection of enterovirus, rotavirus, hepatitis A virus, and small round structured viruses in water samples. Int. J. Food. Microbiol. 37, 189-99, 1997.
 

[ Projects | Main Page ]