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
). 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
) 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.