Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Genotyping of Rotaviruses in Environmental Water and Stool
Samples in Southern Switzerland by Nucleotide Sequence
Analysis of 189 Base Pairs at the 5
⬘
End of the VP7 Gene
FRANCA BAGGI*
ANDRAFFAELE PEDUZZI
Istituto Cantonale Batteriosierologico, Lugano, Switzerland
Received 28 December 1999/Returned for modification 31 May 2000/Accepted 27 July 2000
Stool specimens from children (<4 years old) with diarrhea were collected over a 1-year period in Ticino
(southern region of Switzerland). During the same period, environmental samples were collected from surface
waters in the proximity of major water treatment plants. From treatment plants, samples were collected from
the raw sewage and before the release of the treated water. From rivers, samples were collected before and after
receiving the treated waters. A single-step reverse transcription (RT)-PCR amplification of the entire VP7 gene
from extracted double-stranded RNA was developed. For the water samples, a further nested PCR was
necessary to increase sensitivity. All amplified viral products were sequenced, and the sequence profile was
compared to that of the VP7 genes of human and animal rotaviruses from GenBank. Rotavirus strains are
characterized by outer capsid proteins G (glycoprotein) and P (protease-cleaved protein). Correct G
genotyp-ing of viral sequences from stool and water samples was possible by analyzgenotyp-ing only 189 bp at the 5
ⴕ
end of the
VP7 gene. In the Ticino region, the most predominant G genotype among clinical and water samples was G1.
Genotypes G2 and G4 were found only among clinical samples. We also detected rotavirus G1-type sequences
in feces from a healthy adult. This finding corroborates the hypothesis that healthy adults act as potential
reservoirs for the spread of rotavirus in the environment. In our experiments, this RT-PCR-based method for
rotavirus genotyping has proven to be a useful tool for epidemiological investigations.
Group A rotaviruses are responsible for severe
gastroenter-itis in humans and animals (6). After replication in the
gastro-intestinal tract, they are excreted and may be dispersed in
environmental waters (22). Rotaviruses have been implicated
in waterborne gastroenteritis outbreaks in many countries. The
stability of human rotaviruses in environmental waters and
their resistance to physicochemical treatment processes used
by sewage treatment plants may facilitate their transmission (1,
4, 12).
A number of surveys have been performed to examine the
rotavirus strains in circulation (26). Rotaviruses are
character-ized by the outer capsid proteins that are important for virus
neutralization: a glycoprotein (G) encoded by gene segment 7,
8, or 9 and the protease-cleaved protein (P), encoded by gene
segment 4 (5). Furthermore, strains behave as if there is a local
reservoir, since the same strains are found from year to year in
the same location. It has been shown, primarily by direct typing
of rotaviruses in fecal specimens with monoclonal antibodies,
that the most prevalent type G rotavirus strains causing
child-hood diarrhea worldwide are G1 through G4 (7, 19, 24, 25, 26),
although some unusual strains have been documented on rare
occasions and confirmed by both G and P typing (3, 17, 22).
Efforts to characterize the genetic diversity existing in rotavirus
VP4 genes and to identify nontypeable strains observed in
some G serotypes has stimulated the development of P and G
genotyping methods as a substitute for serotyping (9, 10, 11, 14,
18).
In this study, we demonstrate the usefulness of a simple
method for correctly determining the G genotypes circulating
in a geographic region of interest, allowing rapid
epidemio-logical surveys of rotaviruses in clinical and environmental
samples. Our approach consists of simple concentration of
rotaviruses from surface water samples, a single-step reverse
transcription (RT)-PCR for amplification of the entire VP7
gene, a nested PCR (nPCR), and subsequent sequence analysis
of the 189-bp amplification product.
MATERIALS AND METHODS
Rotavirus-positive stool specimens.Stool specimens obtained from children
who had gastroenteritis and were admitted to the hospitals in Ticino or obtained directly from pediatricians were collected over a 1-year period (from January to December 1998). A total of 36 stool specimens which had been found positive for rotavirus antigen by the latex agglutination method (Slidex Rota-Kit 2; Bio-Me´rieux, Geneva, Switzerland) were diluted to 20% in Dulbecco medium, clar-ified by vortexing and centrifugation, and stored at⫺20°C.
Collection of water samples.One-liter samples of surface water and 1-ml
samples of raw sewage were collected in sterile glass bottles and tubes, respec-tively, and stored at 4°C until concentration of the viruses was performed in the laboratory. Water samples were collected approximately each month over a 1-year period (from January to December 1998) from the rivers where major sewage treatment plants are located, i.e., in the three major effluents of Lake Lugano as well as the Ticino River. At the treatment plants, 31 samples were collected from raw sewage, 31 samples were collected from the treated water before release, and 34 samples each were collected from the rivers before and after receiving the treated water.
Concentration of rotaviruses from surface water samples.Viruses were
con-centrated from surface water with SiO2. One liter of water was acidified to pH 3.5 with acetic acid, and then 200l of autoclaved SiO2(2, 8, 23; F. Baggi et al., submitted for publication) and AlCl3(to a final concentration of 0.5 mM) were added. Samples were shaken at room temperature for about 30 min. The SiO2 was sedimented by centrifugation at 7,500⫻gfor 10 min; alternatively, it was allowed to settle and the supernatant was decanted. After removal of the super-natant, the pellet was transferred to a 1.5-ml tube; after the removal of excess liquid by a short centrifugation step, the viruses were eluted from the SiO2pellet with 280l of glycine buffer (pH 9.5), homogenized, and incubated at 64°C for 10 min. After centrifugation at 12,000 ⫻g for 2 min, the supernatant was collected in a new sterile tube and stored at⫺80°C until the extraction of nucleic acids was performed.
Concentration of rotaviruses from raw sewage samples.One milliliter of raw
sewage was centrifuged at 6,800⫻gfor 2 min. The supernatant was collected, and 20l of autoclaved SiO2(2, 8, 23; Baggi et al., submitted) and 50l of acetic acid were added. The samples were shaken at room temperature for 10 min and centrifuged at 6,800⫻g for 2 min. After removal of the supernatant, virus
* Corresponding author. Mailing address: Istituto Cantonale
Batte-riosierologico, Via Ospedale 6, CH-6904 Lugano, Switzerland. Phone:
41 91 923 25 22. Fax: 41 91 922 09 93. E-mail: franca.baggi@ti.ch.
3681
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particles were eluted from the SiO2pellet as previously described and stored at ⫺80°C until the extraction of nucleic acids was performed.
Extraction of nucleic acids.RNA was extracted from 140l of clarified stool
samples or glycine buffer eluates by using the QIAgen viral RNA extraction kit (QIAGEN AG, Basel, Switzerland) and strictly following product manufacturer instructions. The 50-l RNA eluates were stored at⫺20°C until the amplification of nucleic acids was performed.
Primers.Primers END9, R1, R3, and Rp were used for the amplification of
sequences from the VP7 gene of group A rotaviruses (16). The oligoprobe Rp corresponds to a conserved region located within the VP7 gene. The sequences, locations, and amplification products of the chosen primers within the rotavirus VP7 gene are given in Table 1.
Single-step RT-PCR.In order to simplify the amplification procedure, a
single-step RT-PCR was developed. RT and PCR were performed with a 100-l reaction volume. For efficient denaturation of the double-stranded RNA (dsRNA), 7l of dimethyl sulfoxide was added to 5l of extracted nucleic acid. After denaturation at 97°C for 5 min, the samples were immediately cooled on ice. Following a short centrifugation, the samples were added to a mixture consisting of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM each deoxyribonucleotide triphosphate (Boehringer), 1M primer R1, 1M primer END9, 5 U of Superscript-RTIIreverse transcriptase (Gibco BRL), 2.5 U ofTaqDNA polymerase (Boehringer Mannheim AG, Rotkreuz, Switzerland), and pyrocarbonic acid diethyl ester (DEPC)-treated H2O (Merck Schweiz AG, Dietikon, Switzerland) to a final volume of 100l. After incubation for 30 min at 42°C, the following cycling conditions were chosen: 1 min at 94°C, 2 min at 55°C, and 1 min at 72°C, for a total of 25 cycles. A final extension at 72°C for 7 min followed by storage at 4°C concluded the RT-PCR amplification step. A fragment of 1,062 bp was expected.
nPCR.nPCR was performed with 5l of the RT-PCR amplification product
under the following conditions: Taq Buffer(⫹MgCl2) (Boehringer), 0.1 mM each deoxyribonucleotide triphosphate (Boehringer), 1 U ofTaqDNA polymerase, 1 M primer R3, 1M primer Rp, and H2O-DEPC in a final volume of 50l. The cycling conditions were as follows: 30 s at 94°C, 30 s at 50°C, and 30 s at 72°C for 3 cycles, followed by 27 cycles of 15 s at 94°C, 15 s at 50°C, and 20 s at 72°C. A final extension at 72°C for 2 min followed by storage at 4°C concluded the nPCR amplification step. A fragment of 189 bp was expected.
Amplification control reactions.Simian rotavirus SA11 dsRNA (kindly
pro-vided by M. Weitz, Institute of Clinical Microbiology and Immunology, St. Gallen, Switzerland) was extracted by using the QIAgen viral RNA extraction kit and following product manufacturer instructions. The extracted dsRNA was diluted 100-fold with H2O-DEPC and stored at⫺20°C until use. In order to detect false negatives due to RT-PCR inhibition, all samples that were found negative by nPCR were retested by RT-PCR after the addition of 5 l of 100-fold-diluted simian rotavirus SA11 dsRNA as a control.
Visualization of RT-PCR and nPCR products.RT-PCR fragments (1,062 bp)
and nPCR fragments (189 bp) were analyzed by gel electrophoresis. Ten micro-liters of the amplification products was electrophoresed on 1.5% agarose gels in Tris-borate-EDTA buffer along with a 100-bp DNA ladder (Boehringer). Frag-ments were visualized after ethidium bromide staining.
Sequencing and analysis of PCR products.Templates for sequencing were
prepared by purification of the amplified products (1,062 or 189 bp) by using a QIAquick PCR purification kit (QIAgen) and following product manufacturer instructions. Sequencing reactions were performed with an ABI Prism dRho-damine dye terminator cycle sequencing ready reaction kit (Perkin-Elmer Ap-plied Biosystems International, Inc.) on a Perkin-Elmer Cycler GeneAmp 9600 system. The cycling conditions followed product manufacturer instructions. Pu-rification of sequencing products, necessary to remove excess dye terminator, was performed using columns from CENTRI SEP Princeton Separations, Inc., Ad-elphia, N.J., and 10l of the purified sequencing products was resuspended in 10 l of template suppressor reagent. After denaturation at 94°C for 2 min, samples were rapidly cooled on ice, briefly vortexed, centrifuged, and electrophoresed on an ABI Prism 310 instrument (Perkin-Elmer Applied Biosystems) by using the Seq POP6 (1ml)E module. DNA sequences were aligned with the Lasergene program Megalign (DNAstar).
Phylogenetic VP7 sequence analysis and accession numbers. Selected
se-quence data were aligned with the Lasergene program Megalign, and statistical and phylogenetic analyses were performed using Molecular Evolutionary
Genet-ics Analysis version 1.01 (15). Phylogenetic trees were reconstructed by the neighbour joining method. The number of nucleotide substitutions per site was estimated by several distance methods, and the robustness of each node was tested by bootstrap analysis with 500 replications. Phylogenetic trees were drawn using TreeExplorer 2.01 (Koichiro Tamura).
Comparisons were made with VP7 sequences of rotaviruses obtained from GenBank (human rotavirus serotype 1, accession numbers M93006 [HG1 1-41] and D16328 [HG1 1-49]; human rotavirus serotype 2, accession numbers D50125 [HG2 2-22] and D50114 [HG2 2-33]; human rotavirus serotype 3, accession numbers D86284 [HG3 1-12] and D86266 [HG3 1-30]; human rotavirus serotype 4, accession number A01321 [HG4 5-02]; human rotavirus serotype 8, accession number AF034852 [HG8 1-11]; rotavirus serotype 8 isolated from a river, acces-sion number AF039524 [RG81-09]; human group C rotavirus, accesacces-sion numbers D87544 [HGC 1-39] and D87543 [HGC 1-40]; avian rotavirus VP7 sequence, accession number L01098 [AVI 7-10]; canine rotavirus VP7 sequence, accession number U97199 [CAN 2-37]; chicken rotavirus VP7 sequence, accession number X56784 [CHI 5-01]; equine rotavirus VP7 sequence, accession numbers D13549, D00843, and X57785 [EQU 2-18]; lamb rotavirus VP7 sequence, accession num-ber L11602 [LAM 7-30]; lapin rotavirus VP7 sequence, accession numnum-ber U62153 [LAP 1-35]; murine rotavirus VP7 sequence, accession numbers AF039220 [MUR 1-10], D45216 [MUR 1-33], and U08425 [MUR 6-01]; porcine rotavirus VP7 sequence, accession numbers X04613 [POR 3-31] and U35850 [POR 3-32]; and porcine group C rotavirus, accession number M61101 [POR 7-02]).
Sensitivity of rotavirus concentration from water samples.The sensitivity of
virus concentration from water samples was determined as follows: six 1-liter sterile water samples were spiked with serial dilutions of simian rotavirus SA11 (final concentrations, 3.2⫻104, 3.2⫻102, 3.2⫻101, 3.2, 3.2⫻10⫺1, and 3.2⫻ 10⫺250% tissue culture infective doses [TCID
50]/liter, respectively). Concentra-tion, eluConcentra-tion, extracConcentra-tion, and amplification were performed as previously de-scribed.
RESULTS
Evaluation of the sensitivity of rotavirus concentration from
water samples.
Only the sample seeded with 3.2
⫻
10
4TCID
[image:2.612.52.553.84.156.2]50
/
liter displayed the 1,062-bp RT-PCR fragment; nPCR
in-creased the sensitivity to 3.2 TCID
50/liter (Fig. 1).
FIG. 1. Sensitivity of rotavirus concentration from 1 liter of water, as ana-lyzed by electrophoresis of amplification products. Lanes: 1, 100-bp DNA ladder; 2, 3.2⫻104TCID
[image:2.612.335.523.537.684.2]50/liter; 3, 3.2⫻102TCID50/liter; 4, 3.2⫻101TCID50/liter; 5, 3.2 TCID50/liter; 6, 3.2⫻10⫺1TCID50/liter; 7, 3.2⫻10⫺2TCID50/liter; 8, RT-PCR and nPCR negative controls.
TABLE 1. Primers used for rotavirus RT-PCR and nPCR
Reaction(product size, bp) Primer Nucleotide position in genomeof human rotavirus Waa Sequence (5⬘33⬘)
RT-PCR (1,062)
R1
1–28
GGC TTT AAA AGA GAG AAT TTC CGT CTG G
END9
1036–1062
GGT CAC ATC ATA CAA TTC TAA TCT AAG
nPCR (189)
R3
51–71
GTA TGG TAT TGA ATA TAC CAC
Rp
239–220
TCC ATT GAT CCT GTT ATT GG
aBased on the National Center for Biotechnology Information (accession no. K02033).
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Phylogenetic analysis of rotavirus VP7 complete (1,062-bp)
and partial (189-bp) sequences.
Sixty-two human VP7
se-quences of group A rotavirus deposited in GenBank were
selected to represent all the major VP7 serotypes (serotype
G1, 12 sequences; serotype G2, 17 sequences; serotype G3, 20
sequences; serotype G4, 11 sequences; and serotype G8, 2
sequences). Two published human VP7 sequences of group C
rotavirus were also included. Alignment and phylogenetic
anal-ysis of both the whole, 1,062-bp VP7 gene (RT-PCR product)
and the 189-bp fragment (nPCR product) were performed as
previously described in Materials and Methods. Results of
both analyses were in agreement, allowing correct G
genotyp-ing of all sequences by examination of only the 189-bp nPCR
product. In order to determine whether we could distinguish
between animal VP7 sequences which could be found in
envi-ronmental water samples and sequences of human origin,
phy-logenetic analysis of the 189-bp fragment included different
published animal rotavirus VP7 sequences as well. All
se-quences of nonhuman animal origin generated distinct clusters
clearly distinguishable from the human group A rotavirus
se-quences; therefore, it is possible to correctly discriminate
be-tween sequences of human and animal origins. Part of the
phylogenetic sequence analysis is displayed in Fig. 2.
Phylogenetic analysis of rotavirus VP7 partial (189-bp)
se-quences from stool and water samples.
After RT-PCR
ampli-fication, all 36 clinical samples displayed the 1,062-bp
frag-ment, and sequencing was performed with both R1 and END9
primers. After phylogenetic analysis of the corresponding
189-bp sequence, 29 patients displayed type G1 sequences, 5
patients exhibited type G2 sequences, and 2 patients had type
G4 sequences. No sequences matching type G3 or G8 were
found. For environmental water samples, the use of nPCR was
indispensable in order to enhance the sensitivity of detection.
In fact, all positive water samples were negative after RT-PCR
but positive after nPCR. The absence of inhibitors during
nucleic acid amplification was assayed for each nPCR-negative
sample. A total of 16 positive water samples were found and
were sequenced with both R3 and Rp primers. All samples
were classified as G1. The positive water samples were found in
January (seven positive samples), May (five positive samples),
and June (four positive samples), in accordance with the
ob-servation that rotavirus infections have peak occurrences in
winter and spring months. The results of the phylogenetic
sequence analysis of all positive samples are displayed in Fig. 3.
Carriers of rotaviruses.
In order to assess if healthy adults
could serve as reservoirs for rotaviruses found in
environmen-tal water or raw sewage, 20 stool samples from healthy,
asymp-tomatic adults (collected for routine sanitary inquiries) were
analyzed by both RT-PCR and nPCR. All samples but one
were found negative by nPCR. Sequence analysis of the
posi-tive sample revealed type G1 (Fig. 3).
DISCUSSION
[image:3.612.131.471.74.297.2]The present study demonstrates the efficiency of a
single-step RT-PCR followed by nPCR and subsequent sequence
analysis of a 189-bp product from the 5
⬘
end of the VP7 gene
for characterizing rotavirus contamination of environmental
water samples and the usefulness of these techniques for
study-ing epidemiological relationships between strains involved in
human infections and those found in water. The single-step
RT-PCR was developed in order to avoid reopening of the
tubes and therefore to reduce the danger of cross
contamina-tion. Furthermore, this strategy reduced the technical steps
necessary to perform the analysis while maintaining good
sen-sitivity and specificity for detecting low viral loads in both
human and water samples. Concentration of viruses from
wa-ter using SiO
2is easy, inexpensive, and rapid and has good
sensitivity and specificity. An nPCR result can be obtained 24 h
after sampling, and a sequence profile can be obtained after a
further 24 h. These properties allow rapid (within 48 h)
detec-tion of possible contaminadetec-tion sources, allowing appropriate
FIG. 2. Phylogenetic sequence analysis of rotavirus VP7 sequences (189 bp) of human and animal origins (neighbor joining tree): human group A rotavirus sequences from different serotypes (REF strains); RG81-09 (rotavirus type 8 isolated from a river); HGC 1-39 and HGC 1-40 (human group C rotavirus); AVI 7-10 (avian rotavirus); CAN 2-37 (canine rotavirus); CHI 5-01 (chicken rotavirus); EQU 2-18 (equine rotavirus); LAM 7-30 (lamb rotavirus); LAP 1-35 (lapin rotavirus); MUR 1-10, MUR 1-33, and MUR 6-01 (murine rotavirus); POR 3-31 and POR 3-32 (porcine rotavirus); and POR 7-02 (porcine group C rotavirus). Numbers indicate percent similarity.on May 15, 2020 by guest
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preventive measures to be undertaken in time. Inhibitors were
efficiently removed by RNA purification and extraction with
the QIAgen viral RNA extraction kit.
Raw sewage and secondary effluent samples were found to
be contaminated by rotaviruses despite the most efficient water
treatment measures in use in sewage treatment plants (Baggi
et al., submitted). Sequence analysis of the positive water
sam-ples revealed the presence of only rotavirus type G1, whereas
among the clinical samples, type G1 predominated but types
G2 and G4 also could be found. The fact that in healthy adults
rotavirus could be detected by nPCR sustains a possible link
between rotaviruses found in the environment and those found
in clinical samples. In fact, our results support the presumption
that healthy adults may serve as reservoirs for the spread of
rotaviruses (12, 13, 20, 21). The knowledge of the sensitivity of
detection of rotaviruses by RT-PCR and nPCR (3.2
⫻
10
4and
3.2 TCID
50/liter, respectively) makes it possible to gain useful
information about the levels of rotavirus that one healthy adult
may be shedding.
The phylogenetic analysis of different animal rotavirus VP7
sequences resulted in a clearly distinct cluster that did not
match any of the human group A rotavirus sequences. This is
a very fortuitous finding, because it allows the application of
this molecular technique to environmental water samples to
correctly discriminate between sequences of human and
ani-mal origins.
[image:4.612.110.499.73.512.2]In conclusion, the present study demonstrates the efficiency
of the nucleotide sequence analysis of 189 bp at the 5
⬘
end of
the VP7 gene for studying epidemiological relationships
be-tween strains involved in human infections and those found in
water. From the sequence analysis, it was possible to predict
the G genotype and to evaluate possible correlations between
water contamination and viral sequences found among clinical
samples.
FIG. 3. Phylogenetic sequence analysis (189 bp) of rotavirus-positive samples (neighbor joining tree) (CLIN, clinical stool samples [from children]; H2O, water samples; CARRIER, stool sample from a healthy adult; and REF, human group A rotavirus sequences from different serotypes [HG1, HG2, HG3, HG4, and HG8]).
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ACKNOWLEDGMENT
Special thanks are due to Reinhard Zbinden, Institute for Clinical
Microbiology, University of Zurich, Zurich, Switzerland, for critically
reading the manuscript.
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![FIG. 3. Phylogenetic sequence analysis (189 bp) of rotavirus-positive samples (neighbor joining tree) (CLIN, clinical stool samples [from children]; H2O, watersamples; CARRIER, stool sample from a healthy adult; and REF, human group A rotavirus sequences from different serotypes [HG1, HG2, HG3, HG4, and HG8]).](https://thumb-us.123doks.com/thumbv2/123dok_us/8231189.826879/4.612.110.499.73.512/phylogenetic-rotavirus-watersamples-carrier-rotavirus-sequences-different-serotypes.webp)
