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Applied and Environmental Microbiology, April 2005, p. 2203-2205, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.2203-2205.2005

LETTER TO THE EDITOR

Presence of Norovirus Sequences in Bottled Waters Is Questionable


   LETTER
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Beuret and colleagues reported the detection and identification of noroviruses (NV) in 53 out of 159 samples of mineral water using reverse transcription-PCR (RT-PCR) (1). This sensitive technique is prone to false-positive results, but these can be identified as such by performing sequence analysis of the amplicons. Since the genetic diversity of noroviruses is high, it is unlikely that natural isolates from different sources have identical sequences. The authors claimed that they indeed observed this genetic diversity, suggesting that they were true positives. However, when analyzing their data in detail, we noted that essential information is missing or inconsistent and also discovered several errors which cast serious doubts on this conclusion:

Analysis of the sequences deposited in GenBank revealed that unprocessed sequences were used for the alignments (only one strand sequenced without postsequencing processing) and that the sequence corresponding with the (degenerated) primers was also included (Table 1). The suspicion that only one sequenced strand was used in the phylogenetic analyses is supported by the observation that the sequences corresponding to accession no. AF405525, AF405524, and AF405523 were submitted in the wrong direction (3'->5'), since the sequencing was performed only with primer SRII-3. Nevertheless, Beuret et al. state repeatedly that the amplification products were analyzed by double-strand sequencing.


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  		TABLE 1. Sequence alignments of norovirus sequences obtained from GenBank

 
Excluding the region corresponding to the primer sequence leads to identical sequences in the case of two samples amplified with primers SRII-2 and SRII-3 and also substantially reduces the genetic diversity of the amplicons obtained with primers Mon431/432 and Mon433/434. It cannot be excluded that the few remaining differences are due to errors caused by single-strand sequencing. This would also explain the appearance of a stop codon in a region coding for an essential protein (nucleotide 139 of AF405527 sequence). Similar errors were detected in other norovirus sequences deposited (GenBank accession no. AF406993 to AF407004) by the same group (2). Some of these contain "not-identified nucleotides" or even insertions and/or deletions in genes coding for capsid proteins or RNA polymerase.

According to the information provided in the Materials and Methods section, sense primers were used for the RT reactions. Taking into account that NV is a positive-sense single-stranded RNA virus, this approach should theoretically not lead to any positive result.

Only 6 out of the 54 sequences were submitted to GenBank, even though in the "Instructions to Authors" it is stated that accession numbers should be provided for all new determined sequences.

The phylogenetic trees were constructed with 54 sequences, but according to the results, only 53 samples gave a positive result.

In conclusion, our analysis of published data demonstrates that these findings are most probably based on artifacts and systematical mistakes, and we believe that the presented data do not provide sufficient evidence that mineral water is frequently contaminated with noroviruses. This conclusion is further supported by two recent studies on the prevalence of NV sequences in samples of bottled and natural mineral water, which did not yield any positive result (3, 4).


   REFERENCES
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 Letter
 References
Letter 
 References 
 

  1. Beuret, C., D. Kohler, A. Baumgartner, and T. M. Lüthi. 2002. Norwalk-like virus sequences in mineral waters: one-year monitoring of three brands. Appl. Environ. Microbiol. 68:1925-1931.[Abstract/Free Full Text]
  2. Beuret, C., D. Kohler, and T. Lüthi. 2000. Norwalk-like virus sequences detected by reverse transcription-polymerase chain reaction in mineral waters imported into or bottled in Switzerland. J. Food Prot. 63:1576-1582.[Medline]
  3. Khanna, N., D. Goldenberger, P. Graber, M. Battegay, and A. F. Widmer. 2003. Gastroenteritis outbreak with norovirus in a Swiss university hospital with a newly identified virus strain. J. Hosp. Infect. 55:131-136.[CrossRef][Medline]
  4. Lamothe, G. T., T. Putallaz, H. Joosten, and J. D. Marugg. 2003. Reverse transcription-PCR analysis of bottled and natural mineral waters for the presence of noroviruses. Appl. Environ. Microbiol. 69:6541-6549.[Abstract/Free Full Text]
Gloria Sanchez*
Han Joosten
Department of Quality & Safety Assurance
Nestlé Research Centre, Nestec Ltd.
Vers-chez-les-Blanc
CH-1000 Lausanne 26, Switzerland

Rolf Meyer
Quality Management Department
Nestlé Product Technology Centre Orbe, Nestec Ltd.
CH-1350 Orbe, Switzerland

* Corresponding author. Phone: 41 21 785 8692, Fax: 41 21 785 8553, E-mail: gloria.sanchez-moragas{at}rdls.nestle.com.


Authors' Reply


   LETTER 
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It is correct that two governmental studies were made to question the contamination of mineral waters by noroviruses (1, 2). Concerning the method used, RT-PCR, I think we really do not have to demonstrate that it can be used as routine diagnostic assay in food or water, as long as it is used appropriately (3-5, 8-11). Diagnostic analysis by RT-PCR is successfully used for environmental, medical, and food samples and is known as the method of choice for some uncultivable viruses (like noroviruses). We are aware that some difficulties can arise, with matrices like seafood or sewage, if some crucial methodical steps are not considered. However, when using water samples, especially mineral water, which is supposed to be pure and healthy, no problems should be encountered for RT-PCR detection. Authors rightfully claim that noroviruses, like every other RNA virus, present a high genetic diversity. However, we hope that they are also aware of the fact that distinct strains (clusters) can have a global distribution and contaminate some geographical regions for several months (12). The illustration (Fig. 4) in our second publication (2) clearly shows that three closely related brand-specific clusters could be defined. Additionally, these sequences are closely related to the global strain observed by American research (12) and clearly distinct from the positive control used in our studies, as expected.

We really regret the fact that unprocessed data were submitted to GenBank; some scientists prefer them to processed results. It is therefore absolutely understandable that a single-nucleotide-error crept in and let a hypothetical stop codon appear. Anyway, sequences detected in mineral waters are distinct enough from any positive control used and definitely of norovirus origin, such that even a single error cannot change the fact that human fecal contamination was present in the mineral water samples tested. GenBank was contacted, and all related entries will be deleted and replaced with processed sequences soon. We sincerely hope that nobody is convinced that unprocessed data were used for phylogenetic analysis.

We think that it is absolutely obvious, but we specify again, that all positive PCR results are sequenced bidirectionally by an external and accredited company. An additional quality control of the same company is ensured by sequencing of an identical positive control sample in each batch.

As mentioned in a previous Letter to the Editor, we regret that there is a mistake in our publication, since we inverted the designations of antisense and sense primers throughout this publication; there is no doubt about it. But again, we sincerely hope that there are no scientists using a sense primer to reverse transcribe a positive-stranded viral RNA. To make sure that this will never happen, we finally decided to overcome our uncertainty by submitting an erratum.

We think that it is quite unusual to submit all newly determined sequences to GenBank, particularly when all sequences fit within closely related clusters. GenBank would have some memory problems if researchers submitted all PCR amplicons.

The authors of the letter above, having keen minds, correctly observed that phylogenetic trees were constructed with 54 sequences. But we also hope that they agree that 53 positive samples and an additional positive control result in 54 sequences to compare.

To claim that their conclusions are supported by two studies is very adventurous. The first study was conducted by the water producer itself (7), affirming that RT-PCR is not a suitable method for water analysis, since they predominantly produced false-positive results using this apparently unmanageable method. The second study is a simple investigation of norovirus-associated gastroenteritis in a university hospital. The main finding of RT-PCR analysis is that there was no evidence for a waterborne, foodborne, or environmental source. As expected in most hospital outbreaks, infection was most likely transmitted from person to person. We do not think that these findings are related to mineral water contaminations.


   REFERENCES 
Top
 Letter
 References
 Letter 
 References 
 

  1. Beuret, C., D. Kohler, and T. Lüthi. 2000. Norwalk-like virus sequences detected by reverse transcription-polymerase chain reaction in mineral waters imported into or bottled in Switzerland. J. Food Prot. 63:1576-1582.
  2. Beuret, C., D. Kohler, A. Baumgartner, and T. M. Lüthi. 2002. Norwalk-like virus sequences in mineral waters: one-year monitoring of three brands. Appl. Environ. Microbiol. 68:1925-1931.
  3. Denis-Mize, K., G. S. Fout, D. R. Dahling, and D. S. Francy. 2004. Detection of human enteric viruses in stream water with RT-PCR and cell culture. J Water Health 2:37-47.[Medline]
  4. Di Pinto, A., M. C. Conversano, V. T. Forte, G. La Salandra, C. Montervino, and G. M. Tantillo. 2004. A comparison of RT-PCR-based assays for the detection of HAV from shellfish. New Microbiol. 27:119-124.
  5. Jothikumar, N., R. Paulmurugan, P. Padmanabhan, R. B. Sundari, S. Kamatchiammal, and K. S. Rao. 2000. Duplex RT-PCR for simultaneous detection of hepatitis A and hepatitis E virus isolated from drinking water samples. J. Environ. Monit. 2:587-590.[CrossRef][Medline]
  6. Khanna, N., D. Goldenberger, P. Graber, M. Battegay, and A. F. Widmer. 2003. Gastroenteritis outbreak with norovirus in a Swiss university hospital with a newly identified virus strain. J. Hosp. Infect. 55:131-136.
  7. Lamothe, G. T., T. Putallaz, H. Joosten, and J. D. Marugg. 2003. Reverse transcription-PCR analysis of bottled and natural mineral waters for the presence of noroviruses. Appl. Environ. Microbiol. 69:6541-6549.
  8. Laverick, M. A., A. P. Wyn-Jones, and M. J. Carter. 2004. Quantitative RT-PCR for the enumeration of noroviruses (Norwalk-like viruses) in water and sewage. Lett. Appl. Microbiol. 39:127-136.[Medline]
  9. Legeay, O., Y. Caudrelier, C. Cordevant, L. Rigottier-Gois, and M. Lange. 2000. Simplified procedure for detection of enteric pathogenic viruses in shellfish by RT-PCR. J. Virol. Methods 90:1-14.[CrossRef][Medline]
  10. Li, J. W., X. W. Wang, C. Q. Yuan, J. L. Zheng, M. Jin, N. Song, X. Q. Shi, and F. H. Chao. 2002. Detection of enteroviruses and hepatitis A virus in water by consensus primer multiplex RT-PCR. World J. Gastroenterol. 8:699-702.[Medline]
  11. Lovdal, T., and O. Enger. 2002. Detection of infectious salmon anemia virus in sea water by nested RT-PCR. Dis. Aquat. Org. 49:123-128.[Medline]
  12. Noel, J. S., et al. 1999. Identification of a distinct common strain of "Norwalk-like viruses" having a global distribution. J. Infect. Dis. 179:1334-1344.[CrossRef][Medline]
Christian Beuret*
LABOR SPIEZ, Virology, CH-3700 Spiez, Switzerland

* Phone: 41 33 228 16 64, Fax: 41 33 228 14 02, E-mail: christian.beuret{at}babs.admin.ch.

Applied and Environmental Microbiology, April 2005, p. 2203-2205, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.2203-2205.2005




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