EARLY-LIFE, LOW-LEVEL INFECTION AND GENETIC DIVERSITY OF MALAWI POLYOMAVIRUS IN PEDIATRIC PATIENTS
Malawi polyomavirus (MWPyV) is an emerging human polyomavirus for which data on global occurrence, genetic diversity and molecular characteristics remain very limited. Here, we performed a comprehensive analysis of MWPyV infection in pediatric patients from Sao Paulo State, Brazil, using an integrated molecular, genomic and ultrastructural approach. A total of 234 oropharyngeal swab samples were screened for MWPyV DNA by TaqMan real-time PCR. Positive samples were further analyzed for respiratory viral co-infections, viral load quantification, complete genome sequencing, phylogenetic inference and transmission electron microscopy. MWPyV DNA was detected in 18 samples, corresponding to a prevalence of 7.7%. Viral loads were low overall, with a mean of 266 copies per reaction (range, 13-2.3x10^3 copies per reaction). Two MWPyV-positive samples were co-infected with SARS-CoV-2. Phylogenetic analysis of six complete MWPyV genomes and seven partial VP1 sequences showed that most strains clustered within genotype I, whereas one strain belonged to genotype III. Notably, one genome classified within genotype I occupied an ancestral and divergent phylogenetic position, suggesting the presence of an emerging genotype, an unsampled lineage or a transitional form between established clades, thereby challenging the current MWPyV taxonomic framework. Transmission electron microscopy revealed icosahedral virions consistent with polyomavirus morphology. Together, these data provide an extensive genomic and epidemiological characterization of MWPyV and represent the largest collection of complete MWPyV genomes reported to date. Our findings support the view that MWPyV is a component of the pediatric virome rather than a primary respiratory pathogen and highlight the importance of large-scale genomic surveillance to refine MWPyV phylogeny and clarify the conditions under which MWPyV may acquire clinical relevance.