Norwalk-like virus sequences in mineral waters: one-year monitoring of three brands

Abstract

In a recent study, RNA with nucleotide sequeces specific for "Norwalk-like viruses" (NLV) was detected in 11 different brands of European mineral waters. To clarify this finding, a 1-year monitoring study was conducted. Samples of three European brands of mineral water without gas were monitored weekly by reverse transcriptase PCR using generic and genogroup-specific oligonucleotides. Additional analyses were performed to investigate a possible correlation between NLV sequence contamination and mineral water lot numbers, the long-term stability (persistence) of NLV sequences in mineral water, and the level of contamination. NLV sequences were detected in 53 of 159 samples analyzed (33%) and belonged entirely to genogroup II. Although all NLV strains identified were closely related, three mineral water brand-specific clusters could be identified for both primer systems by sequencing. Analyses of second samples from lots previously shown to be positive for NLV sequences gave corresponding results in 45 of 53 cases (85%) (within a six-pack). NLV persistence was tested by analyzing 10 positive samples after 6 and 12 months of storage in darkness at room temperature. After 6 months, all samples remained positive; after 12 months, 9 of 10 samples were still positive for NLV sequences. No NLV sequences could be detected by analysis of 0.1-liter aliquots of 53 samples shown to be positive by testing of 1-liter volumes. Based on this fact and a test sensitivity of approximately 10 viral units, levels of contamination in positive mineral water samples were estimated to be in the range of 10 to 100 genomic equivalents per liter.

FIG. 1.

Diagrammatic representation of amplified RT-PCR segments within the putative RNA polymerase and the beginning of the capsid gene of NLV gg I and gg II. The central bar represents the NV (NV/F8Iia/68/US) genome (GenBank accession number M87661) beginning at nucleotide 4700. Block sequences and primer names indicate the relative locations of primers used; the 63-bp region shown within the GLPSG and YGDD motifs is commonly used by most diagnostic laboratories to detect NLV strains by RT-PCR.

FIG. 2.

Agarose gel (2.6%) and ethidium bromide staining of RT-PCR products obtained with generic primers for NLV sequences (213-bp amplicon) in mineral water samples BAG 15 to BAG 24. Electrophoresis was carried out at 70 mV/cm. Samples 16 to 18, 21, 23, and 24 are NLV gg II sequence positive. P, positive control; N, negative controls; M, marker.

FIG. 3.

Phylogenetic analyses performed on a 132-bp region of the RNA polymerase of NLV gg II sequences detected with genogroup-specific primers (12) by weekly analyses of 27 mineral water samples. An NLV gg II sequence of brand 3 isolated in the previous study (original designation, V2) (3), 4 of 53 identical positive-control sequences, and published sequences (accessed from GenBank) of Hawaii virus (U07611), Lordsdale virus (X86557), and Camberwell virus (AF145896) are also included. Three clusters of NLV sequences corresponding to each brand are shown.

FIG. 4.

Phylogenetic analyses performed on a 132-bp region of the RNA polymerase of NLV gg II sequences detected with generic primers (S. S. Monroe, personal communication) by weekly analyses of 26 mineral water samples. Four of 53 identical positive-control sequences and published sequences (accessed from GenBank) of Hawaii virus (U07611), Lordsdale virus (X86557), and Camberwell virus (AF145896) are also included. Three clusters of NLV sequences corresponding to each brand are shown.