Polyomaviruses shedding in stool of patients with hematological disorders: detection analysis and study of the non-coding control region's genetic variability

Abstract

Fragmented data are available on the human polyomaviruses (HPyVs) prevalence in the gastrointestinal tract. Rearrangements in the non-coding control region (NCCR) of JCPyV and BKPyV have been extensively studied and correlated to clinical outcome; instead, little information is available for KIPyV, WUPyV and MCPyV NCCRs. To get insights into the role of HPyVs in the gastrointestinal tract, we investigated JCPyV, BKPyV, KIPyV, WUPyV and MCPyV distribution among hematological patients in concomitance with gastrointestinal symptoms. In addition, NCCRs and VP1 sequences were examined to characterize the strains circulating among the enrolled patients. DNA was extracted from 62 stool samples and qPCR was carried out to detect and quantify JCPyV, BKPyV, KIPyV, WUPyV and MCPyV genomes. Positive samples were subsequently amplified and sequenced for NCCR and VP1 regions. A phylogenetic tree was constructed aligning the obtained VP1 sequences to a set of reference sequences. qPCR revealed low viral loads for all HPyVs searched. Mono and co-infections were detected. A significant correlation was found between gastrointestinal complications and KIPyV infection. Archetype-like NCCRs were found for JCPyV and BKPyV, and a high degree of NCCRs stability was observed for KIPyV, WUPyV and MCPyV. Analysis of the VP1 sequences revealed a 99% identity with the VP1 reference sequences. The study adds important information on HPyVs prevalence and persistence in the gastrointestinal tract. Gastrointestinal signs were correlated with the presence of KIPyV, although definitive conclusions cannot be drawn. HPyVs NCCRs showed a high degree of sequence stability, suggesting that sequence rearrangements are rare in this anatomical site.

Keywords: Gastrointestinal tract; HPyVs; Hematological patients; NCCR/VP1; Phylogenetic analysis; Stool samples.

Fig. 1

The figure summarizes the detection, as single or multiple infections, of the five HPyVs investigated in this study. Patients who developed gastrointestinal symptoms are also displayed

Fig. 2

Analysis of the NCCR structure organization of JCPyV. Alignment of JCPyV non-coding control region (NCCR) isolates from stool specimens of thalassemic and AML patients revealed an archetype CY-like NCCR, with the conservative boxes A, B, C, D, E and F. The NCCR sequence of the archetypal JCPyV strain CY, as proposed by Yogo et al. [39] is shown at the top of the figure and the consensus sequences of the NCCR showing variations isolated from thalassemic and AML patients are shown below. The identified genotype is reported at the top of the figure, right side. The nucleotide changes G95C, G108A and G217A identified in the patients’ isolates are in bold. The six boxes commonly used to divide the archetypal NCCR [39] are indicated under the NCCR sequences, while proven and putative binding sites for transcriptional factors are reported above the CY NCCR sequence

Fig. 3

Analysis of the NCCR structure organization of BKPyV. Alignment of BKPyV non-coding control region (NCCR) isolates from stool specimens of thalassemic and AML patients revealed an archetype NCCR sequence. Blocks P, Q, R and S commonly used to denote the archetypal NCCRs [40, 41] are indicated under the NCCR sequences, while proven and putative binding sites for transcriptional factors are reported above the archetypal NCCR sequence. The O-block, containing the origin of replication, was omitted. The archetype NCCR is shown at the top of the figure. The nucleotide sequence variations identified at positions 4 (R-block) and 18 (S-block) are in bold. The thymidine deletion at nucleotide position 50 (S-block) is indicated with a dash line. The genotype/subtype identified in the patients’ isolates is reported at the top of the figure, right side

Fig. 4

HPyVs phylogenetic analysis. Phylogenetic tree generated using MEGA version 6 software. The VP1 gene sequences obtained from the patients’ isolates cluster with the corresponding reference sequences: JCPyV AB081613, BKPyV AB263926, KIPyV EF127906, WUPyV EF444549 and MCPyV EU375803. The bar at the bottom of the figure indicates 0.2 nucleotide substitutions per site