Contemporary seasonal human coronaviruses display differences in cellular tropism compared to laboratory-adapted reference strains.

Seasonal human coronaviruses (sHCoVs) cause 15%-30% of common colds. The reference strains used for research were isolated decades ago and have been passaged extensively, but contemporary sHCoVs have been challenging to study as they are notoriously difficult to grow in standard immortalized cell lines. Here, we addressed these issues by utilizing primary human nasal epithelial cells (HNECs) and immortalized human bronchial epithelial cells (BCi) differentiated at an air-liquid interface, as well as human embryonic stem cell-derived alveolar type II (AT2) cells to recover contemporary sHCoVs from human nasopharyngeal specimens. From 21 specimens, we recovered four HCoV-229e, three HCoV-NL63, and eight HCoV-OC43 viruses. All contemporary sHCoVs showed sequence differences from lab-adapted CoVs, particularly within the spike gene. Evidence of nucleotide changes in the receptor binding domains within HCoV-229e and detection of recombination for both HCoV-229e and HCoV-OC43 isolates was also observed. Importantly, we developed methods for the amplification of high-titer stocks of HCoV-NL63 and HCoV-229e that maintained sequence identity, and we established methods for the titration of contemporary sHCoV isolates. Comparison of lab-adapted and contemporary strains in immortalized cell lines and airway epithelial cells revealed differences in cell tropism, growth kinetics, and cytokine production between lab-adapted and contemporary sHCoV strains. These data confirm that contemporary sHCoVs differ from lab-adapted reference strains and, using the methods established here, should be used for the study of CoV biology and evaluation of medical countermeasures.IMPORTANCEZoonotic coronaviruses have caused significant public health emergencies. The occurrence of a similar spillover event in the future is likely, and efforts to further understand coronavirus biology should be a high priority. Several seasonal coronaviruses circulate within the human population. Efforts to study these viruses have been limited to reference strains isolated decades ago due to the difficulty in isolating clinical isolates. Here, we use human airway and alveolar epithelial cultures to recover contemporary isolates of human coronaviruses HCoV-NL63, HCoV-229e, and HCoV-OC43. We establish methods to make high-titer stocks and titrate HCoV-229e and HCoV-NL63 isolates. We show that contemporary isolates of HCoV-NL63 and HCoV-OC43 have a different tropism within the respiratory epithelium compared to lab-adapted strains. Although HCoV-229e clinical and lab-adapted strains similarly infect the respiratory epithelium, differences in host response and replication kinetics are observed. Using the methods developed here, future research should include contemporary isolates when studying coronavirus biology.