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We evaluated the efficiency of SARS-CoV-2 detection from patient respiratory specimens by comparing 3 cell lines: Vero E6, Vero E6 expressing transmembrane protease serine 2 (Vero E6 T2), and Vero E6 expressing angiotensin-converting enzyme 2 and transmembrane protease serine 2 (Vero E6 A2T2). We compared a range of sample types, clinical conditions, and real-time reverse transcription PCR cycle threshold values. Vero E6 A2T2 exhibited enhanced sensitivity by supporting efficient virus entry and replication with faster cytopathic effect. Vero E6 culture isolated infectious virus only up to 3 days after PCR confirmation but with Vero E6 A2T2 cells, culture occurred up to 7 days after confirmation. Whole-genome sequencing showed no evidence of adaptive mutations when Vero E6 A2T2 was used for viral culture, supporting use for downstream analyses. Optimized infectious virus detection systems are needed for research and clinical settings, particularly for high-risk, immunocompromised populations that produce virus longer and contribute to variant emergence.