Mucosal and cutaneous human papillomaviruses detected in raw sewages

Abstract

Epitheliotropic viruses can find their way into sewage. The aim of the present study was to investigate the occurrence, distribution, and genetic diversity of Human Papillomaviruses (HPVs) in urban wastewaters. Sewage samples were collected from treatment plants distributed throughout Italy. The DNA extracted from these samples was analyzed by PCR using five PV-specific sets of primers targeting the L1 (GP5/GP6, MY09/MY11, FAP59/64, SKF/SKR) and E1 regions (PM-A/PM-B), according to the protocols previously validated for the detection of mucosal and cutaneous HPV genotypes. PCR products underwent sequencing analysis and the sequences were aligned to reference genomes from the Papillomavirus Episteme database. Phylogenetic analysis was then performed to assess the genetic relationships among the different sequences and between the sequences of the samples and those of the prototype strains. A broad spectrum of sequences related to mucosal and cutaneous HPV types was detected in 81% of the sewage samples analyzed. Surprisingly, sequences related to the anogenital HPV6 and 11 were detected in 19% of the samples, and sequences related to the "high risk" oncogenic HPV16 were identified in two samples. Sequences related to HPV9, HPV20, HPV25, HPV76, HPV80, HPV104, HPV110, HPV111, HPV120 and HPV145 beta Papillomaviruses were detected in 76% of the samples. In addition, similarity searches and phylogenetic analysis of some sequences suggest that they could belong to putative new genotypes of the beta genus. In this study, for the first time, the presence of HPV viruses strongly related to human cancer is reported in sewage samples. Our data increases the knowledge of HPV genomic diversity and suggests that virological analysis of urban sewage can provide key information useful in supporting epidemiological studies.

Figure 1. Phylogenetic tree constructed with the sequences obtained by Method A (L1 region).

The tree was constructed using the Maximum Likelihood method based on the Kimura 2-parameter model. The sequences identified in this study are in bold. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Bootstrap values higher than 70% are shown. HPV prototypes reported in the phylogenetic tree are: HPV6b, X00203; HPV11, M14119; HPV16, K02718; HPV25, X74471; HPV120, GQ845442; AJ000150; AF048746. A feline papillomavirus type 2 (EU796884) was included as an outgroup. Identical sequences obtained from the different plasmid cloned or identical sequences obtained from the nested and hemi-nested amplicons were not included in the tree. Sequences obtained from different clones were given an alphabetical code (ID sample “a”, “b”); sequences obtained from the hemi-nested reaction were marked with an asterisk.

Figure 2. Phylogenetic tree constructed with the sequences obtained by Method D (E1 region).

The tree was constructed using the Maximum Likelihood method based on the Kimura 2-parameter model. The sequences identified in this study are in bold. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Only selected bootstrap values related to the studied sequences are displayed. The Beta HPV prototypes reported in the phylogenetic tree are: HM999997 type 145, X74464 type 9, U31787 type 38, FM955840 type 104, EU410349 type 111, GQ845444 type 122, FM955842 type 113, Y15176 type 80, X74468 type 15, U31786 type 37, X74469 type 17, EU410348 type 110, FN677756 type 151, U31780 type 22, FM955839 type 100, GQ845442 type 120, U31781 type 23, GQ845446 type 124, GQ246951 type 118, FJ947080 type 115, X74480 type 49, Y15174 type 76, U31785 type 36, FM955838 type 99, M12737 type 8, FM955841 type 105, X74466 type 12, M17463 type 5, M32305 type 47, X74471 type 25, X74470 type 19, U31779 type 21, X74467 type 14D, U31778 type 20, EF422221 type 107, AF531420 type 92, AY382779 type 96, FN677755 type 150, AY382778 type 93, FM955837 type 98, and U31782 type 24. A feline papillomavirus type 2 (EU796884) was included as an outgroup. Sample 1547, marked with* had identical sequences with other 25 samples (IDs 1529, 1530, 1531, 1532a, 1534, 1535, 1538, 1539, 1540, 1542, 1544, 1545, 1546, 1548, 1549, 1550a, 1553, 1554, 1554a, 1555, 1556, 1568, 1566, 1571), not included in the tree. Sequences obtained from different clones are indicated by alphabetical code (ID sample “a”, “b”).