A new human coronavirus, HCoV-NL63, was identified using a novel method called VIDISCA (Virus-Discovery-cDNA-AFLP). The virus was isolated from a 7-month-old child with bronchiolitis and conjunctivitis. The complete genome sequence revealed that HCoV-NL63 is a new group 1 coronavirus, not a recombinant. It replicates in tertiary monkey kidney cells and the LLC-MK2 cell line. The virus was detected in seven additional individuals, indicating its widespread presence in the human population. The genome contains distinctive features, including a unique N-terminal fragment in the spike protein. The VIDISCA method, which uses cDNA-amplified restriction fragment-length polymorphism, allows for the identification of unknown viruses without prior sequence knowledge. The method was successfully used to identify HCoV-NL63 and other viruses, including HIV-1 and parvovirus B19. The genome of HCoV-NL63 is 27,553 nucleotides long with a low GC content, and it shares some similarities with HCoV-229E but has notable differences, including a unique insertion in the S gene. Phylogenetic analysis showed that HCoV-NL63 is closely related to group 1 coronaviruses, but it forms a distinct subcluster. The virus causes acute respiratory disease in children and immunocompromised adults. The study highlights the importance of continuous virus discovery, as many human diseases have unknown etiologies, and new viruses may be responsible. The identification of HCoV-NL63 expands the known human coronaviruses and provides insights into the diversity of these viruses. The VIDISCA method offers a valuable tool for discovering new viruses, particularly those that do not replicate in vitro or are difficult to detect with traditional methods.A new human coronavirus, HCoV-NL63, was identified using a novel method called VIDISCA (Virus-Discovery-cDNA-AFLP). The virus was isolated from a 7-month-old child with bronchiolitis and conjunctivitis. The complete genome sequence revealed that HCoV-NL63 is a new group 1 coronavirus, not a recombinant. It replicates in tertiary monkey kidney cells and the LLC-MK2 cell line. The virus was detected in seven additional individuals, indicating its widespread presence in the human population. The genome contains distinctive features, including a unique N-terminal fragment in the spike protein. The VIDISCA method, which uses cDNA-amplified restriction fragment-length polymorphism, allows for the identification of unknown viruses without prior sequence knowledge. The method was successfully used to identify HCoV-NL63 and other viruses, including HIV-1 and parvovirus B19. The genome of HCoV-NL63 is 27,553 nucleotides long with a low GC content, and it shares some similarities with HCoV-229E but has notable differences, including a unique insertion in the S gene. Phylogenetic analysis showed that HCoV-NL63 is closely related to group 1 coronaviruses, but it forms a distinct subcluster. The virus causes acute respiratory disease in children and immunocompromised adults. The study highlights the importance of continuous virus discovery, as many human diseases have unknown etiologies, and new viruses may be responsible. The identification of HCoV-NL63 expands the known human coronaviruses and provides insights into the diversity of these viruses. The VIDISCA method offers a valuable tool for discovering new viruses, particularly those that do not replicate in vitro or are difficult to detect with traditional methods.