BACKGROUND: The importance of two recently identified polyomaviruses, WUV and KIV, as respiratory pathogens in populations with a high HIV prevalence needs to be defined, since human polyomaviruses can cause significant morbidity and mortality in patients with immunosuppression. Geographic distribution and disease association of WUV and KIV genotypes are not yet clearly defined. OBJECTIVES: To investigate the prevalence of WUV and KIV in HIV-positive and HIV-negative patients with respiratory infections in hospitals in South Africa and determine their genotypes. STUDY DESIGN: Specimens from patients with acute respiratory infections from hospitals serving Pretoria were screened for WUV and KIV. Positive specimens were sequenced and subjected to phylogenetic analysis. RESULTS: WUV was identified in (7%) and KIV in (1%) of mainly pediatric patients. Co-infections were common in WUV- and KIV-infected patients (71% and 66.6%, respectively); 57% of patients with WUV and 33% of patients with KIV were HIV-positive while the HIV prevalence in the respiratory virus patient group screened in this study was 33% WUV and KIV patients presented with moderate to severe lower respiratory tract disease. Four distinct and 2 unique WUV strains were identified clustering into 2 of 4 globally identified genotypes. KIV strains were identical to strains from Sweden. CONCLUSION: WUV is frequently detected in HIV-infected patients with respiratory disease, but its role as respiratory pathogen remains uncertain.