Abstract
Rhinoviruses (RVs) are classified into three species: RV-A, B, and C. Unlike RV-A and -B, RV-C cannot be propagated using standard cell culture systems. In order to isolate RV-Cs from clinical specimens and gain a better understanding of their biological properties and pathogenesis, we established air–liquid-interface (ALI) culture methods using HBEC3-KT and HSAEC1-KT immortalized human airway epithelial cells. HBEC3- and HSAEC1-ALI cultures morphologically resembled pseudostratified epithelia with cilia and goblet cells. Two fully sequenced clinical RV-C isolates, RV-C9 and -C53, were propagated in HBEC3-ALI cultures, and increases in viral RNA ranging from 1.71 log10 to 7.06 log10 copies were observed. However, this propagation did not occur in HSAEC1-ALI cultures. Using the HBEC3-ALI culture system, 11 clinical strains of RV-C were isolated from 23 clinical specimens, and of them, nine were passaged and re-propagated. The 11 clinical isolates were classified as RV-C2, -C6, -C9, -C12, -C18, -C23, -C40, and -C53 types according to their VP1 sequences. Our stable HBEC3-ALI culture system is the first cultivable cell model that supports the growth of multiple RV-C virus types from clinical specimens. Thus, the HBEC3-ALI culture system provides a cheap and easy-to-use alternative to existing cell models for isolating and investigating RV-Cs.
Highlights
Rhinoviruses (RVs) are positive-strand RNA viruses of the genus Enterovirus in the familyPicornaviridae
HBEC3-KT (CRL-4051) and HSAEC1-KT (CRL-4050) cells were from the American Type Culture Collection (ATCC; Manassas, VA, USA)
RV-C9 and -C53 were isolated from the clinical specimens using ALI-cultured Human bronchial tracheal epithelial (HBTE) cells according to previously described methods [15,16,19]
Summary
Rhinoviruses (RVs) are positive-strand RNA viruses of the genus Enterovirus in the familyPicornaviridae. A genotypic classification of RVs based on the VP1 sequence was proposed. According to this classification, more than 160 types of RVs have been classified into three species (A, B, and C). (available online: http://www.picornaviridae.com/) [3]. Viruses 2019, 11, 216 whereas those of RV-C comprise the recently identified human cadherin-related family member 3. They constitute the most common causes of upper respiratory tract infections [8]. To respiratory syncytial viruses, RVs are the most common pathogens associated with severe bronchiolitis in children [9]. The differences in severity among these three RV species as well as the detailed role played by each RV species in respiratory diseases remain unclear [10,11,12,13]
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