Applied and Environmental Microbiology, December 1999, p. 5624-5627, Vol. 65, No. 12
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Molecular Detection of Norwalk-Like Caliciviruses
in Sewage
W. J.
Lodder,1
J.
Vinjé,2
R.
van de Heide,1
A. M.
de Roda Husman,1
E.
J. T. M.
Leenen,1 and
M. P. G.
Koopmans2,*
Microbiological Laboratory for Health
Protection1 and Research Laboratory for
Infectious Diseases, Department of
Virology,2 National Institute of Public Health
and the Environment (RIVM), 3720 BA Bilthoven, The Netherlands
Received 3 March 1999/Accepted 8 September 1999
 |
ABSTRACT |
In this study, Norwalk-like virus (NLV) RNA was detected by reverse
transcriptase PCR (RT-PCR) in sewage water concentrates. Sequence
analysis of the RT-PCR products revealed identical sequences in stools
of patients and related sewage samples. In 6 of 11 outbreak-unrelated follow-up samples, multiple NLV genotypes were present. Levels as high
as 107 RNA-containing particles per liter were found. These
data show that high loads of NLVs may be present in sewage and warrant
further studies addressing the efficacy of NLV removal by sewage water treatment processes.
| |
TEXT |
The most common viral pathogens
associated with epidemic gastroenteritis in adults are the Norwalk-like
caliciviruses (NLVs), also known as small round-structured viruses.
NLVs have been implicated in numerous food-borne and waterborne
outbreaks (7). Outbreaks of NLV have been associated with
drinking fecally contaminated water (8, 11, 22) and with
swimming in contaminated recreational water (9). Until now,
routine microbiological quality control of such surface waters is
performed by the enumeration of fecal coliform bacteria, and
occasionally water samples are also screened for the presence of
enteroviruses. However, it is clear that there is no correlation
between the levels of fecal bacteria and those of viruses that cause
gastroenteritis (12). Therefore, it would be better to
screen waters also for specific, known pathogens. Based on such
screening, waters could be concluded as being safe or not with respect
to those pathogens tested.
Recently, sensitive reverse transcriptase PCR (RT-PCR)-based detection
methods have been developed and used to determine the presence of NLVs
or other enteric viruses in sewage and surface water (1, 3-5, 13,
16, 17). However, few groups have addressed the use of these
assays in field surveys (1, 3, 5, and 17). To validate the methods used
during periods of known sewage contamination, we chose to start
monitoring sewage samples in conjunction with outbreaks of viral
gastroenteritis, epidemiologically linked with NLVs. The outbreaks
occurred in November 1997, December 1997, and March 1998 in nursing
homes in the cities of Reeuwijk, Apeldoorn, and Enkhuizen, The
Netherlands, respectively. Stool specimens were collected from persons
with (patients) and without (controls) reported illness. From each
outbreak, at least five stool samples from patients were tested for the
presence of Salmonella spp., Campylobacter spp.,
and Shigella spp. by routine culture and for the presence of
group A rotavirus, adenovirus types 40/41, and astrovirus by
enzyme-linked immunosorbent assay (rotavirus, adenovirus, astrovirus)
or NLV RT-PCR (NLV) (21). More than 50% of the stool
samples from patients from the three outbreaks tested positive for NLV.
The sewage samples were all taken close to the source of the outbreak,
where high concentrations of NLVs could be expected. In addition, in
the period from February to May 1998, at one of the locations
(Apeldoorn), a follow-up survey at 3-week intervals was done at the
pumping-engine station and the nearby sewage treatment plant (treating
sewage from 140,000 inhabitant equivalents). Raw sewage samples (10 liters each) were first concentrated by a conventional filter
adsorption-elution method (19). The resulting eluate (650 ml) was used in a two-phase separation method (15) which is
based on the selective distribution of viruses in two incompatible phases (Dextran T40 and 10% [wt/vol] polyethylene glycol 6000). We
modified this method by omitting the chloroform extraction prior to
separation. In our hands, this method gives the highest recovery and
reproducibility in comparison to other methods (14). After
separation, the bottom phase and the interphase were harvested. Further
purification was done by spin column gel chromatography using Sephadex
G200 and by ultrafiltration in a Centricon 100 microconcentrator. The
resulting RNA extracts were processed by the method of Boom et al.
(2), modified to allow an increased input volume by adding
solid guanidinium (iso)thiocyanate. Purified viral RNA was subsequently
tested in the NLV RT-PCR (21) as well as in a rotavirus
group A RT-PCR (6). The detection limit of both assays was
determined to be 10 to 100 RNA-containing particles. All negative
control samples that had been tested in parallel to monitor for
contamination remained negative. Positive control samples, artificially
spiked with NLVs of a known genetic cluster, showed a single 326-bp
fragment after both RT-PCR and Southern hybridization (Fig. 1A and
B). NLV RNA could be detected in sewage samples that had been collected just after notification of the outbreaks at all three locations.
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FIG. 1.
RT-PCR detection and Southern hybridization of follow-up
sewage samples collected at a pumping engine (PE) station and a sewage
treatment (ST) plant in Apeldoorn. (A and C) Ethidium bromide-stained
agarose gels; (B and D) Southern blot hybridizations corresponding to
panels A and C. (A and B) Lane M, molecular weight marker (marker V;
Boehringer Mannheim). RT-PCR control lanes include RT-PCR product
amplified from undiluted RNA (lane 1), 10-fold-diluted RNA (lane 2),
and 100-fold-diluted RNA (lane 3) extracted from an electron
microscopy-positive fecal sample. Lane  , negative control. Extraction
control lanes include RT-PCR product amplified from undiluted RNA (lane
1), 10-fold-diluted RNA (lane 2), 100-fold-diluted RNA (lane 3),
1,000-fold-diluted RNA (lane 4), and 10,000-fold-diluted RNA (lane 5)
from an NLV-positive fecal sample (RT-PCR control) but isolated with
the modified method of Boom et al. (2). Lane W, water
sample, i.e., negative control. PE-station: March 6 lanes include
RT-PCR products amplified from serially diluted RNA, i.e., undiluted
(lane 1), 10 1 diluted (lane 2), 101 diluted
(lane 3), 103 diluted (lane 4), 104 diluted
(lane 5), and 105 diluted (lane 6). RNA had been
extracted from a sewage sample taken at the PE station on March 6 in
Apeldoorn. (C and D) ST-plant: March 6 lanes include lanes such as
those described for PE station, March 6 but RNA was extracted from a
sewage sample collected at the ST plant, March 6, in Apeldoorn.
PE-station: March 25 lanes include serially diluted RNA extracted from
sewage sample collected at the PE station, March 25, in Apeldoorn.
ST-plant: March 25 lanes include serially diluted RNA extracted from
sewage sample collected at the ST plant, March 25, in Apeldoorn.
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|
To determine the viral load, serial sewage extracts collected within 10 days after the first day of illness at the locations of Reeuwijk,
Apeldoorn, and Enkhuizen were tested in the NLV RT-PCR assay. RNA
dilutions of 103, 101, and
107 were positive as visualized by ethidium bromide
staining of agarose gels (Table 1). Of
note, in the 101 dilutions of some samples that were
positive in higher dilutions, no NLV RNA could be amplified, clearly
demonstrating that not all PCR inhibitors had been removed (Fig. 1).
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TABLE 1.
Detection of NLV and rotavirus RNA by RT-PCR in sewage
samples collected during three nursing home outbreaks of NLV
gastroenteritis in Reeuwijk, Apeldoorn, and Enkhuizen and in
follow-up sewage samples collected at two locations in Apeldoorn in
winter and early spring
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|
The genetic variability of NLV strains from stool and sewage samples
was determined by sequencing the RT-PCR products with the Big Dye
terminator kit either directly or after cloning (Applied Biosystems,
Nieuwerkerk a/d IJssel, The Netherlands). We analyzed DNA sequences of
which 145 nucleotides was used to create an alignment and phylogenetic
tree by use of Geneworks (V2.5; Intelligenetics, Mountain View, Calif.)
and the Treecon software package (18). Strains from stool
and sewage were identical for each individual outbreak (Fig.
2). Outbreak strains found in Reeuwijk
and Enkhuizen were closely related to the Lordsdale-Bristol cluster
within genogroup II of NLVs (20). The viruses found in the
Apeldoorn outbreak belong to a different genotype within genogroup II,
first detected in The Netherlands in stool samples from an outbreak
that occurred in early 1997 in Rotterdam (Fig. 2). This Rotterdam
genotype was detected in all three sewage samples collected during the
Apeldoorn outbreak.
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FIG. 2.
Phylogenetic tree based on a 145-bp region of the RNA
polymerase gene showing the relationships among NLVs from genogroup I
(GGI) (Norwalk virus and Southampton virus), genogroup II (GGII)
(Lordsdale virus, Mexico virus, and Rotterdam virus), and outbreak
strains (OB) detected in stool specimens of patients and
outbreak-related sewage samples. GenBank accession numbers of the
prototype strains were as follows: Norwalk virus, M87661; Southampton
virus, L070418; Lordsdale virus, X86557; Mexico virus, U22498. The
TREECON program was used to generate the dendrogram (18).
Bootstrap values of the internal nodes are indicated.
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|
NLVs could also be detected in the follow-up samples taken at the
pumping-engine station and the sewage treatment plant. In Apeldoorn, 10 of 11 samples taken were found positive for NLVs, in some samples even
up to 107 dilutions (Table 1). However, not each NLV
RT-PCR product resulted in a clear 326-bp band (Fig. 1A and B). Since
we assumed that more strains might cocirculate and therefore be present
in a single sewage sample, we cloned the PCR products and sequenced at
least five individual clones. By this approach, several additional
genotypes were detected in 6 of the 11 specimens for which individual
clones were obtained (Fig. 3). Both
genogroup I and genogroup II NLVs were found.
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FIG. 3.
Phylogenetic tree based on a 145-bp region of the
polymerase gene showing the relationships among the three outbreak
strains (OB Apeldoorn, OB Reeuwijk, and OB Enkhuizen) indicated by an
asterisk and the strains found in follow-up (FU) sewage samples (FU3,
FU4, FU5, FU6, FU7, and FU8), Rotterdam virus, and prototype NLVs
(Norwalk virus, Southampton virus, Mexico virus, Melksham virus
[accession no. X81879], and Lordsdale virus). For the follow-up
samples, PCR products were cloned and five different clones were
sequenced. Different sequences within a sample were given an
alphabetical code (FU3-A, FU3-B, etc.). Bootstrap values of the
internal nodes are indicated.
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|
For reference, we also screened for rotaviruses by RT-PCR
(6), since the incidence of rotavirus infection in the
general population in the Netherlands has been estimated from a
population-based survey and from laboratory surveillance data
(10). During the peak shedding of rotavirus (between weeks 4 and 12), all sewage samples were found positive for rotavirus but at
maximum concentrations that were on average 10-fold lower than those
for NLVs (Table 1, Apeldoorn data). Taking into account that NLVs are
generally shed at lower maximal titers than rotavirus (7),
our data suggest that NLVs may exceed rotaviruses in importance as a
cause of illness.
In addition, this study shows that the concentration and purification
method described is applicable for virus detection by RT-PCR in water
samples. We found that during the study period, the NLV load in sewage
was high independent of outbreaks. In future research, the presence of
NLVs in surface, recreational, and drinking water, as well as the
effectiveness of sewage treatment processes for removal of viral
pathogens, will have to be determined.
| |
ACKNOWLEDGMENTS |
This research was funded by the Ministry of Housing, Physical
Planning and the Environment (The Netherlands), and KIWA Research and
Consultancy (project no. 289202 Water Microbiology).
We thank A. Koks, A. Keja, and F. Slijkerman, from the regional health
services Gouda, Apeldoorn, and Hoorn, The Netherlands, respectively,
for epidemiological investigations and collection of stool specimens.
We also thank the microbiologists participating in the laboratory
surveillance network, as well as the Society for Clinical Virology, for
supplying data on rotavirus detection. Furthermore, we thank the
employees of the sewage treatment plant in Apeldoorn and Olaf E. M. Nijst and Petra M. de Bree for technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Research
Laboratory for Infectious Diseases, Department of Virology, National
Institute of Public Health and the Environment, Antonie van
Leeuwenhoeklaan 9, 3720 BA Bilthoven, The Netherlands. Phone:
31-30-2743945. Fax: 31-30-2744449. E-mail:
Marion.Koopmans{at}rivm.nl.
| |
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Applied and Environmental Microbiology, December 1999, p. 5624-5627, Vol. 65, No. 12
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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