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Applied and Environmental Microbiology, January 2000, p. 238-245, Vol. 66, No. 1
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Documenting the Epidemiologic Patterns of
Polyomaviruses in Human Populations by Studying Their Presence in
Urban Sewage
Sílvia
Bofill-Mas,
Sonia
Pina, and
Rosina
Girones*
Department of Microbiology, Faculty of
Biology, University of Barcelona, Barcelona, Spain
Received 4 June 1999/Accepted 7 October 1999
 |
ABSTRACT |
This is the first description, to our knowledge, of the
distribution of human polyomavirus and simian virus 40 (SV40) in urban sewage. Using a nested-PCR procedure, we report the detection of human
polyomaviruses JC virus (JCV) and BK virus (BKV) but not SV40 in a high
percentage of urban sewage samples obtained from widely divergent
geographical areas in Europe and Africa. For a total of 28 samples
analyzed, JCV was detected in 26, BKV was detected in 22, and none was
positive for SV40. All geographical areas showed a high prevalence of
these viruses with mean estimated values of JC viral particles per ml
on the order of 103 in Barcelona (Spain) and Nancy (France)
and 102 in Pretoria (South Africa) and Umeå (Sweden) and
mean values of BK viral particles on the order of 102 in
Pretoria and Barcelona and 101 in Nancy and Umeå. This
compares with estimated mean values of 102 to
103 for human adenovirus that was evaluated as a control.
Nucleotide sequence analysis of the amplified DNA from some of the
samples is also presented and represents the sequence of the most
abundant JC and BK viral strains in these samples. The nucleotide
sequence of the JCV detected was also analyzed in a phylogenetic study and for genomic characterization in the regulatory region. This study
has shown that human polyomaviruses are spread in high concentrations in the sewage of different geographical areas and are present in
contaminated environments. The frequency and concentration of JCV
detected in the environment and the absence of described animal hosts
suggest that JCV may be useful as a marker for fecal pollution of
anthropogenic origin. The results also support the idea previously
described that the strains of JCV are closely related to the ethnic
origin of the population studied. The procedure applied should also be
useful in future studies of population patterns of viral excretion and
as a tool in epidemiological studies for the detection of changes in
the prevalence of specific viral pathogens.
| |
INTRODUCTION |
JC virus (JCV) and BK virus (BKV)
are human viruses classified in the genus Polyomavirus of
the family Papovaviridae. JCV is etiologically associated
with a fatal demyelinating disease known as progressive multifocal
leukoencephalopathy (PML) which has emerged as a frequent complication
of AIDS in human immunodeficiency virus-infected individuals
(9). Infection with BKV has been associated with diseases of
the urinary tract including hemorrhagic cystitis and ureteral stenosis
(8). Most of the primary infections with JCV and BKV occur
early in childhood and are asymptomatic. In this regard, recent data
suggest that some BKV infections may be transplacental (30).
Human infections with JCV appear to be population associated in that
the genotype of JCV excreted by individuals of defined ethnicities is
in high proportion determined by the geographical origin of the ethnic
group rather than the JCV genotypes that are prevalent in their current
location (5). The latent infections established by these
viruses persist indefinitely in infected individuals (37).
Simian virus 40 (SV40) is a simian virus that is closely related to JCV
and BKV. This polyomavirus establishes latent infections in nonhuman
primates and has been associated with a PML-like condition in simian
immunodeficiency virus (SIV)-infected monkeys (19). SV40 has
been transmitted to humans experimentally, and evidence that SV40 may
be circulating in the human population is accumulating (13).
Large numbers of humans were exposed to SV40 when inoculated with the
polio vaccine prepared in rhesus monkey cells between 1955 and 1961. Several recent studies have described the detection of SV40-like DNA in
tumors from children and young adults who were born after SV40 was
removed from the polio vaccine (11, 14, 15, 25, 26, 38).
These tumors include choroid plexus tumors, ependymomas, and
osteosarcomas. SV40-like DNA sequences have also been detected in
mesotheliomas in adults who could have been exposed to
SV40-contaminated polio vaccines (14).
JCV, BKV, and SV40 are nonenveloped virions containing double-stranded,
closed-circular DNA genomes of approximately 5 kb. The genomes of these
viruses have a common organizational structure and are homologous over
about 75% of their respective nucleotides (37). Each of
these viruses has been found to be oncogenic when injected into
rodents, and each has the capacity to transform rodent and human cells
in tissue culture (37). Their oncogenic capacity, their
persistently latent state in infected tissues, and their association
with some types of tumors make these viruses potential human tumor
viruses (37).
To provide an independent means of assessing the population association
of JCV and the regional prevalence of JCV, BKV, and SV40 or any of
their genotypes, an analysis of the relative viral output of an entire
community or locality could be useful. Since JCV and BKV are excreted
in the urine and SV40 has been found in the feces of infected primates
and humans, it seemed reasonable to assume that if a significant
percentage of the human population were shedding these viruses in their
excreta, then all three should be present and possibly detectable in
urban sewage. In addition to the potential use of polyomavirus
contamination as a marker of human waste in water sources and other
environmental locations, being able to detect and study these viruses
in sewage would provide a unique opportunity to evaluate and monitor
over time those strains that are prevalent in specific geographical
areas. Furthermore, the detection of SV40 in sewage would be reasonable
evidence that SV40 is circulating in the human population.
In a previous study (32-34), we have developed nested-PCR
procedures for the amplification of viral nucleic acids to detect human
adenovirus (human Ad), enteroviruses, and hepatitis A and E viruses in
sewage, environmental samples, and shellfish. The aim of this study was
to apply the experience gained from our previous work to initiate an
assessment of the presence of JCV, BKV, and SV40 in sewage of different
geographical areas. For this study, we have adapted these methodologies
to look for JCV, BKV, and SV40 in urban sewage. Using our previous data
(33) on the prevalence of human Ad in sewage as a point of
reference, we have detected JCV and BKV but not SV40 in urban sewage
samples from widely divergent geographical locations and have developed
sequencing data for the most prevalent strains of JCV and BKV that are
present in these samples.
| |
MATERIALS AND METHODS |
Viruses.
SV40 DNA strain 776 (Gibco BRL) and viral particles
of SV40 strain WT 800, kindly donated by Ferran Azorin from the
Institute Juan de la Cierva, Consejo Superior de Investigaciones
Científicas, Barcelona, Spain, were used in this study as
positive controls. A urine sample from a healthy 38-week-pregnant woman
was tested for the presence of JCV and BKV and found to be positive for
both viruses. The viruses present in 12 ml of urine were concentrated by ultracentrifugation, suspended in 100 µl of phosphate-buffered saline (PBS), and used as a positive control. Clinical samples of
cerebrospinal fluid (CSF) from PML patients were kindly donated by
José Luis Pérez from the Microbiology Department of the
Hospital de Bellvitge, Barcelona, were tested for the presence of JCV
by nested PCR, and were used as a positive JCV control and for
comparative sequence analysis between viral strains of clinical and
environmental origin. Ad type 2 (Ad2) (prototype) was grown on A549
cells, and then viruses were partially purified and stored at 
80°C.
Sewage samples.
A total of 28 raw sewage samples from
different geographical areas were analyzed. Sixteen samples were
collected in the sewers of Barcelona (Spain) and were found to contain
a mean of 1.7 × 106 fecal coliform bacteria/100 ml
with a variance of 5.6 × 105. These samples were
collected from September 1997 to February 1998. Each sample was
collected in sterile 500-ml polyethylene containers, kept at 4°C for
less than 8 h until the viral particles were concentrated in PBS,
and stored at 80°C.
Four samples were collected in Umeå, Sweden, during September and
October 1997. Four samples were collected in Nancy (France) during
March 1998, and four samples were collected in Pretoria (South Africa)
in October 1997. The sewage samples collected in Umeå and Nancy were
stored at 80°C and shipped to Spain, where they were analyzed. From
each sewage sample collected in Pretoria, 30-ml aliquots were
centrifuged (229,600 × g for 1 h at 4°C) and the 2-ml sediments were shipped to Spain at room temperature
immediately (within 24 h) after being processed.
Concentration of viral particles and nucleic acid
extraction.
The method applied for the recovery of viral particles
and nucleic acid extraction was based on previous studies (18,
33). Briefly, 40 ml of sewage sample was ultracentrifuged
(229,600 × g for 1 h at 4°C) to pellet all the
viral particles together with any suspended material. From concentrated
Pretoria samples, 2 ml was resuspended in 6 ml of 2× PBS and
ultracentrifuged (229,600 × g for 1 h at 4°C).
The next step in the treatment of all the sewage samples was the
elution of the sediment by mixing it with 4 ml of 0.25 N glycine buffer
(pH 9.5) on ice for 30 min, and the suspended solids were separated by
centrifugation at 12,000 × g for 15 min after the
addition of 5 ml of 2× PBS. The viruses were finally pelleted by
ultracentrifugation (229,600 × g for 1 h at
4°C), resuspended in 0.1 ml of 1× PBS, and stored at 80°C. Since
the DNA extracted from 10 µl of the viral concentrate corresponded to
a sample volume of 4 ml and was used as inoculum for the PCR test, the
results will be reported in reference to this volume. According to
previous studies, the yield after adding poliovirus type 1 LSc 2ab in
sewage samples with similar conditions was 70% PFU.
Viral nucleic acids were extracted by a procedure that provides clean
nucleic acids for molecular studies. This procedure
uses guanidinium
thiocyanate and adsorption of the nucleic acids
to silica particles
(
12).
Sensitivity of the method.
Raw urban sewage samples were
spiked with WT 800 SV40 viral particles (105/ml) in order
to evaluate the sensitivity of virus extraction and the presence of
inhibitors for the PCR. The polyomavirus-like particles of a urine
sample positive by PCR for JCV and BKV were quantified by transmission
electron microscopy (10, 31). Briefly, 10 ml of urine was
harvested directly onto electron microscope grids (400-mesh Ni grids)
supported with carbon-coated Formvar film by ultracentrifugation
(197,500 × g for 90 min) with a swing-out rotor.
Afterwards, the supernatant was removed, the grids were air dried, and
the sample was stained with 3% uranyl acetate for 20 s.
Polyomavirus-like particles were counted in 200 randomly selected
fields of view, by using a Hitachi-600AB transmission electron
microscope at 75 kV and 80,000× magnification. Serial dilutions were
used to estimate the sensitivity of the detection method.
Enzymatic amplification.
For the detection of the specific
viral genomes in raw sewage, urine, or CSF, 10-µl aliquots of the
extracted nucleic acids were used in each test, corresponding to 4 ml
of sewage sample, 10 µl of urine, or 10 µl of CSF.
Amplification was carried out in a 50-µl reaction mixture containing
10 mM Tris-HCl (pH 8.3 at 25°C), 50 mM KCl, 1.5 mM MgCl
2,
200 µM (each) deoxynucleoside triphosphate, 2 U of Ampli Taq DNA
polymerase (Perkin-Elmer Cetus), and the corresponding primers
at their
corresponding concentrations (0.08 µM external primers
and 0.07 µM
internal primers for Ad amplification and 0.5 µM external
and
internal primers for all polyomavirus amplifications). Thermal
cycling
of the amplification mixture was performed in a programmable
heat block
(Gene Amp PCR System 2400; Perkin-Elmer). In all PCR
assays for human
Ad detection, the first cycle of denaturation
was carried out for 4 min
at 94°C. The conditions for the 29-cycle
amplification were as
follows: denaturing at 92°C for 60 s, annealing
at 55°C for
60 s, and extension at 72°C for 75 s. All amplifications
were completed with a 4-min, 72°C extension period. The PCR
amplifications
of Ad genomes were carried out with external primers HR
and HL
and internal primers NHR and NHL, described and tested in
previous
studies (
6,
7).
In all PCR assays for polyomavirus detection, the first cycle of
denaturation was carried out for 4 min at 94°C. The conditions
for
the 29-cycle amplification were as follows: denaturing at
92°C for
60 s, annealing for 60 s, and extension at 72°C for 75
s. Amplifications were completed with a 4-min, 72°C extension
period.
Primers and annealing temperatures used in this study
are described in
Tables
1 and
2.
The PCR amplifications of SV40 genomes were carried out with external
primers SV1 and SV2 and internal primers SV3 and SV4.
External primers
SV1A and SV2A and internal primers SV5 and SV6
(Table
2), which detect
the SV40 variants that were described
for primates by Lednicky et al.
(
24), were used for analyzing
all the samples from Pretoria,
Nancy, and Umeå and four samples
from Barcelona (BCN11, -12, -14, and
-15). JCV genomes were amplified
with BJ1 and BJ2 as external primers
and JLP15 and JLP16 as internal
primers (
5). All the samples
were also analyzed for JCV with
EP1A and EP2A as external primers and
P1A and P2A as internal
primers (
23). This second set showed
higher sensitivity for
the amplification of JCV
genomes.
BKV genomes were amplified with BJ1 and BJ2 external primers and BK4
and BK6 internal primers. All the primers used in this
study are
represented in Tables
1 and
2.
The results were analyzed by agarose gel electrophoresis with ethidium
bromide as a
stain.
Quality control of the amplification method.
To reduce the
probability of sample contamination by amplified DNA molecules,
standard precautions were applied in all the manipulations. Separate
areas of the laboratory were used for reagents, treatment of samples,
and manipulation of amplified samples. All the samples were analyzed
twice in independent experiments, and a negative control was added
every two samples (a negative control is an amplification reaction
mixture with the same reagents as in the test tubes of the samples but
without the inoculum of viral nucleic acids). Treatment with
uracil-DNA-glycosylase for the degradation of amplified material that
could contaminate the samples was performed in previous studies
(34), but they were not considered necessary for the routine
analysis. Direct extract and a 10-fold dilution of the nucleic acid
extracts were analyzed routinely on highly polluted samples in order to
avoid false negatives because of inhibition of the reactions. This
could occur only in a minority of the samples, according to the
positive results observed previously with sewage samples supplemented
with viruses.
Analysis of the viral genomes.
The amplicons of 12 JCV-positive samples were sequenced with the primers for amplification
of the intergenic (IG) region, P1A, P2A, JCSR, and JCSL
(23). Nine of these 12 JCV-positive samples were also
sequenced with primers for amplifying the regulatory (R) region, JR1,
JR2, JR3, and JR4 (27). The amplicons of five BKV-positive
samples were also sequenced with BK2 and BK5 primers.
Nested-PCR products were purified with the QUIAquick PCR purification
kit (Qiagen, Inc.). Both strands of the purified DNA
amplicons were
sequenced with the ABI PRISM Dye Terminator Cycle
Sequencing Ready
Reaction kit with Ampli Taq DNA polymerase FS
(Perkin-Elmer, Applied
Biosystems) following the manufacturer's
instructions. The results
were checked with the ABI PRISM 377
automated sequencer (Perkin-Elmer,
Applied Biosystems). The sequences
were compared with the GenBank and
EMBL databases by using the
BLAST program of the National Center for
Biotechnology Information.
Sequences were aligned by using the BOXSHADE
3.21 program of the
EMBNET.CH. GenBank accession numbers of the JCV
sequences used
for phylogenetic studies are shown in Table
3.
Phylogenetic analysis of the JCV sequenced regions was performed with
the nucleic acid maximum likelihood method (dnaml),
version 3.572c,
included in the PHYLIP software package (
16).
The
phylogenetic tree was visualized with the TREEVIEW 1.5 program
(
29). The JCV genotypes included in the phylogenetic
analysis
are described in Table
3.
Nucleotide sequence accession numbers.
The sequences
reported in this paper have been deposited in the GenBank database
under accession no. AF119345 to AF119356 (JCV IG region sequences),
AF120240 to AF120242 (JCV R region sequences), and AF120243 to AF120247
(BKV VP1 region sequences).
| |
RESULTS |
Sewage analysis.
The results obtained are shown in Tables
4 and 5 and
reflect a very high level of excretion of human Ad, JCV, and BKV and the absence of positive results for SV40. About 93% of the samples collected in Barcelona were positive for human Ad and JCV, and 10 (62.5%) were positive for BKV. Figure 1
shows the agarose gel electrophoresis of one sewage sample positive for
human Ads, BKV, and JCV and negative for SV40; small-size bands,
nonspecific or made by amplification of combined primers, can be seen
in the gel, as they appear frequently in amplified samples. Samples
collected in Pretoria (four samples) and Nancy (four samples) showed
positive results for all viruses with the exception of SV40. All four
samples collected in Umeå were also positive for human Ads and JCV,
and three of these four samples were positive for BKV. From a total of
28 sewage samples analyzed, 96% were positive for human Ads and JCV
and 77.8% were positive for BKV. Results of the nested-PCR tests and
serial dilution experiments are shown in Tables 4 and 5. Very high
numbers of JCV particles are observed, with the concentration estimated
as between 102 and 104 viral particles per 4 ml
of the sewage samples analyzed. On the basis of the results obtained in
the serial dilution experiments, the concentrations estimated for human
Ad were 101 to 104 viral particles and for BKV
were 101 to 103 BK viral particles in the 4 ml
of the sewage samples analyzed.
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FIG. 1.
Agarose gel electrophoresis showing amplified DNA after
nested PCR of one sewage sample that was positive for human Ad, BKV,
and JCV and negative for SV40 (lanes 1, 2, 3, and 4, respectively).
Lanes 5, 6, 7, and 8 are the corresponding negative controls. Lanes 9, 10, 11, and 12 are positive controls. Lane M, molecular weight
standard,  X174 HaeIII digest.
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|
Sensitivity of the method.
In the sewage samples supplemented
with SV40 viral particles, the viral genomes were detected by nested
PCR with an estimated sensitivity of five genomes compared with the
stock suspensions that showed a nested-PCR detection unit five times
higher (one genome). The applied nested-PCR procedure with SV40 DNA
detected one SV40 genome. This is the level of sensitivity of detection of three different strains of human Ad as described in previous studies
(34). The results observed with SV40 DNA agree with the
sensitivity described previously for the detection of human Ads and
enteroviruses in sewage samples (between 10 and 100 viral particles per
4-ml sample).
The number of polyomavirus-like particles counted in a urine sample by
electronic microscopy was 8.7 × 10
5/ml. According to
this, the sensitivity observed after nested
PCR in the urine sample is
at least 5.2 viral particles/4 ml of
JCV and at least 520 BKV particles
per 4
ml.
According to these results, we estimate the minimum concentration of
viruses producing a positive result in the nested-PCR
test for sewage
samples to be on the order of 10 viral
particles.
Molecular characterization of the viruses detected.
We
sequenced 461 nucleotides of the IG region of 12 JCV-positive samples
randomly selected: five samples collected in Barcelona (one from a
urine sample), two from Pretoria, one from Nancy, and one from Umeå.
Three CSF samples from PML patients from different regions in Spain
were also sequenced as positive controls.
The sequences analyzed confirmed the specificity of the nested-PCR
amplification, since all viral sequences were identified
as the
expected
polyomavirus.
The alignment of the sequences of the JCV obtained is shown in Fig.
2, and the relationship of the detected
strains with the
nucleotide sequences of the EMBL and GenBank sequence
data banks
is shown in Table
6. The
results of the maximum likelihood method
applied to the phylogenetic
analysis are shown in Fig.
3. The
analysis of the R region of the JCV shows that all strains detected
in
sewage and urine samples and sequenced are archetype strains;
the JCV
obtained from the clinical samples presented specific
rearrangements in
the regulatory region (Fig.
4).
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FIG. 2.
Sequence alignment of the IG regions of 12 JCV-positive
samples. Dots indicate sequence identities. R = A + G; W = A + T.
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FIG. 3.
Phylogenetic analysis of the IG regions of 12 sequenced
JCV strains compared with previously described JCV subtypes (Table 3)
by the maximum likelihood method.
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FIG. 4.
Sequences of the R regions of JCV from nine analyzed
samples. CSFE and CSFB are clinical samples of CSF from two patients.
Archetypal sequences are observed in samples BCNU, BCN15, BCN16, BCN8,
BCN2, NANCY2, and UMEA3. Underlined nucleotides are identical to the
archetypal sequence. Nucleotides in boldface and in italics represent
duplications present in these sequences.
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|
The nucleotide sequences of positive BKV amplicons from one sample
collected in Pretoria, one from Nancy, and three samples
from
Barcelona were compared. These samples showed between two
and
four nucleotide differences in a fragment of 248 nucleotides
(Fig.
5).
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FIG. 5.
Sequence alignment of the VP1 regions of five
BKV-positive samples. Dots indicate sequence identities.
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| |
DISCUSSION |
We are not aware of previously reported data on the presence of
human polyomavirus in sewage. In this regard, we have detected high
concentrations of JCV and BKV in the sewage of different cities of
widely divergent geographical origins and have studied the most
prevalent strains of JCV excreted.
For this purpose, we have amplified and sequenced JCV DNA in the region
designated the IG region, which encompasses the 3'-terminal sequences
of both T-antigen and VP1 (major capsid protein) genes, since it
contains abundant nucleotide variation compared with other regions.
These variations have been described as a means of tracing human
migrations (5, 39). The strong relationship of the sequenced
JCV strains with previously described viral isolates from related
populations is remarkable. The sequenced strains described for sewage
samples from European countries are strongly related to European
subtypes, and the two strains from the Pretoria area are related to
African isolates. PRETORIA1 is related to strains isolated from
sub-Saharan populations, and PRETORIA3 is more closely related to
isolates from Mauritania and North Africa which appear more similar to
the European strains, correlating with the relationship of these
populations. We also show in this study that the most common JCV
strains that are excreted in the areas studied show archetypal R
regions, as described for urine samples. In agreement also with
previous studies, the three PML-derived strains detected from CSF
samples presented individual rearrangements of these genomic regions
(40).
The fact that immunocompetent hosts frequently excrete JCV in urine
(21, 36) indicates that renal JCV is not latent under these
conditions but replicates to generate progeny that are excreted in
urine. Thus, JCV persistence in the kidney may be characterized by
continuous viral replication and virus shedding. This would be required
for JCV to be transmitted among humans, given that JCV infection has
been described as very inefficient (22).
It has also been reported that JCV in urine is infectious, and urine is
the most likely source of JCV infection in humans. Although the cells
that support JCV replication at portals of entry remain to be
identified, some data suggest that tonsil tissue could be a possible
site of the initial viral infection (27). BKV seems to
circulate independently of JCV in the population. As with JCV, BKV has
been detected in kidney but also in tonsils and other tissues as well
(35).
The nested amplicons of BKV obtained from four sewage samples and one
urine sample were sequenced in order to confirm their identity as BKV.
The differences observed in the 248 sequenced nucleotides were around
0.8 to 2% as described in the literature (36). Some
indeterminations were observed in nucleotides that showed variability
when sequences from the data banks were compared. This is the case for
the sample PRETORIA1, suggesting the presence of a mixture of related
strains in this sewage sample. Further sequencing studies are required
for the identification of the specific BKV genomic subtypes.
In previous studies, we have isolated hepatitis E virus
(32), human Ad, and enterovirus (33) from similar
sewage samples, and these viruses are infectious. Those polyomaviruses
that remain intact as viral particles in sewage may be infectious and
could be transmitted to other humans in a fecally polluted environment.
The semiquantitative results for the detection of the polyomavirus
indicate the concentration of the viral particles detected. Definitive
numbers could be obtained only by quantitative PCR test. The finding of
lower titers of BKV than of JCV could be attributable to a lower
sensitivity of the PCR amplification and/or to a lower frequency of BKV
shedding in immunocompetent persons as described by Shah et al.
(36).
We did not detect SV40 in any of these sewage samples. The results
indicate that, if SV40 is excreted in human urine or feces, its
concentration in the analyzed sewage samples is lower than 10 viral
particles in 4 ml of raw sewage.
The high prevalence of human Ad detected in all of these geographical
areas is consistent with previous studies carried out in Barcelona,
where it was suggested that this parameter could be a good viral
indicator of the fecal contamination of human origin in the environment
and shellfish (33).
The detection by PCR of polyomaviruses in sewage allows the
identification and analysis of the genomes of viral strains that are
infecting the population and gives information on the spread, frequency, and distribution of these viruses. These data are also needed for the study of the epidemiology of the related diseases. However, the molecular techniques applied do not give information on
the level of infectivity of the strains detected in the sewage samples,
and further studies will be needed in order to establish the stability
of human polyomavirus in the environment. It is also clear that this
approach is likely to provide a method with a higher level of
specificity, sensitivity, and speed than isolation of viruses in cell
culture. In these experiments, the differences between the nucleotide
sequences of the detected genomes and those of the viruses of our
positive controls argue against the possibility of laboratory contamination.
The human polyomavirus has been shown to be present in high
concentrations in the sewage of the different geographical areas studied, especially JCV, and its specificity as a human virus may be
useful as a marker for fecal pollution of anthropogenic origin. The
high level of excretion detected also supports the idea previously
described that fecal-oral transmission (including contamination from
urine) will probably happen soon in vivo, inside the family or from
closely related people and less frequently later in life from other
polluted sources.
The procedure that we used to study the presence of viruses in the
sewage of a community may be useful as a tool for studying changes in
epidemiological patterns of some viral infections and in future studies
for the analysis of environmental dynamics.
| |
ACKNOWLEDGMENTS |
This work was supported by the Center for Biologics Evaluation
and Research, Food and Drug Administration, project FDA 122987 00 97 GF
00. Sílvia Bofill-Mas and Sonia Pina are fellows of the
Generalitat de Catalunya.
We thank Serveis Cientifico-Tècnics of the University of
Barcelona for their help in the sequencing of the PCR products. We also
thank Willie Grabow from the University of Pretoria, South Africa,
Louis Schwartzbrod from Nancy University, France, and Göran
Wadell, from Umeå University, Sweden, for their kind collaboration in
obtaining the samples in their areas. We thank Jaume Bertranpetit and
Francesc Calafell from the Universitat Pompeu Fabra from Barcelona for
their collaboration in the phylogenetic analysis of the samples. We
also thank Andrew M. Lewis from the Office of Vaccine Research and
Review, CBER, FDA, for useful consultation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, Faculty of Biology, University of Barcelona, Diagonal
Ave. 645, 08028 Barcelona, Spain. Phone: 34-93-4021491. Fax:
34-93-4110592. E-mail: rosina{at}bio.ub.es.
| |
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Abstract
Introduction
