Here we identified and sequenced the entire genome of a novel, highly divergent polyomavirus by deep sequencing of diarrheal samples. In accordance with the two-letter designations for human polyomaviruses, we have provisionally named this virus MX polyomavirus (MXPyV), after the country from which the initial isolate was identified. The genomic organization and amino acid sequence homology of MXPyV, as well as conservation of known protein motifs in the T-antigen, indicate that this virus is indeed a polyomavirus. MXPyV is broadly distributed and was recovered from diarrheal samples from two continents, as well as from respiratory secretions from a child with pneumonia. In addition, independent MXPyV isolates from different individuals showed sequence variation of 0–4.3%, and the virus was detected in children from birth to 6 years of age.
By phylogenetic analysis, MXPyV does not consistently cluster with any other polyomavirus taxonomic group and, indeed, whereas MXPyV ORFs encoding VP1 and the large T-antigen cluster with human polyomaviruses (WU, KI, HPyV6, and HPyV7), the MXPyV ORF encoding VP2 appears to group better with rodent polyomaviruses. In contrast, the small T-antigen of MXPyV does not appear to cluster with any of the known polyomavirus groups. These observations, combined with the low amino acid identity of 13–44% in the proteins of MXPyV relative to those of other polyomaviruses (), suggest that the putative ancestral strain for MXPyV likely diverged early along the evolutionary pathway, and raises the possibility of recombination of polyomavirus genes. Although recombination in polyomaviruses remains controversial, it does appear to occur, at least in JC viruses 
. No evidence for MXPyV recombination within individual genes was detected by bootscanning analysis (data not shown), but this is to be expected given the high sequence divergence of MXPyV. The whole-genome sequence of MXPyV is nearly identical to that of the recently described gut-associated polyomavirus MWPyV (St. Louis strain) or HPyV10 
, sharing 99.8% or 99.7% identity, respectively, and thus all 3 viruses are different variants of the same species (). Situated on a highly divergent phylogenetic branch, MXPyV, MWPyV, and HPyV10 likely represent the first members of a new subclade of polyomaviruses.
Whole-genome sequence alignment of MXPyV relative to other recently described gut-associated polyomaviruses HPyV10 and MWPyV.
Detection of MXPyV, as well as closely related strains MWPyV and HPyV10, appears largely confined to the gastrointestinal tract. MXPyV exhibited an overall prevalence of 3.4% in fecal samples collected from California, Mexico, and Chile (), although one respiratory sample out of 136 (0.74%) also tested positive. SV40, BKV, JCV, and MCV have also been detected in human feces 
, although their primary sites of pathology are elsewhere in the human body, as have polyomaviruses WU and KI 
. We were unable to detect MXPyV in 480 plasma or urine samples from highly immunocompromised transplant recipients, indicating that these are not reservoir sites for MXPyV infection, as is the case for JC and BK viruses.
No association between MXPyV presence and diarrhea was detected in the California and Chile gastroenteritis studies for which controls were available ( and ). In fact, in the samples from Chile, the trend was reversed, with 4 MXPyV-positive samples among 96 asymptomatic control individuals and no positives among 96 children with diarrhea (). These findings, however, do not preclude the possibility of MXPyV as an etiologic agent of diarrhea given the fact that a large proportion of infections from diarrheal viruses are asymptomatic 
. Notably, 6 of 12 MXPyV-positive diarrheal samples from Mexico tested negative by a broad-spectrum viral microarray and specific PCR assays for all known diarrheal viruses (Table S2
), suggesting that MXPyV, if human-tropic, may still potentially be a cause of gastroenteritis. Serologic testing before and after diarrheal episodes would be useful in investigating this possibility, as shown previously for a human cardiovirus and klassevirus/salivirus 
In the California SIFT study, MXPyV was seen more often in girls than in boys (13 female vs. 4 males, p
0.012) by RT-PCR (). Although MXPyV-specific serology is needed for confirmation, this observation is intriguing in light of the fact that apparent gender differences have previously been described in a serological investigation of primary infections by Merkel cell virus (MCV) in childhood 
. In that study, males showed higher seroconversion and seroprevalence rates to MCV than females. This apparent gender difference was not observed with respect to MCV seroprevalence in adults 
, although gender does appear to dramatically impact incidence and survival rates associated with Merkel cell carcinoma 
. Whether differences in the age at which MXPyV is acquired, childhood physiology, or viral characteristics play a role in the gender differences observed here is unknown, and merits further investigation.
Although at present we cannot exclude the possibility that MXPyV may be of dietary origin, several lines of evidence indicate that the virus is likely human-tropic. The enhanced sensitivity of RT-PCR over PCR for detection of MXPyV () suggests that expressed viral mRNA, presumably present in infected host cells in the feces, is being detected, implying that viral replication occurs in the human gut. In addition, the detection of MXPyV in a child at the time of an acute gastroenteritis episode and 3 months later suggests that, in analogy with other human polyomaviruses 
, chronic infection by MXPyV is possible. The detection of a closely related variant to MXPyV, HPyV10, in tissue from a patient with WHIM syndrome also indicates that MXPyV, MWPyV, and HPyV10 are likely human-tropic viruses (). Viral cultivation or serology will be needed for definitive confirmation that these novel polyomaviruses can cause bona fide
infections in humans.