More than 160 types of rhinoviruses (RVs) are known. RVs are currently classified into three species (A, B and C) of Enteroviruses in the family Picornaviridae. RV-A and RV-B have been known for many years, but the discovery of RV-C in 2006 surprised the molecular and clinical virology communities.
The RV-C are clearly rhinoviruses, but unlike RV-A or RV-B, they are not readily propagated in typical cell culture systems. For example, conventional cells lines such as NCI-H358, WI-38, WisL, HEK293T, BEAS-2B, A549 and HeLa, do not support any detectable RV-C replication. The RV-C are not “new” in terms of evolution, but rather they were physically undetected by typical characterization methods that required cultured virus growth and induction of cytopathic effects, such as endpoint dilution (TCID50) or plaque assays.
The current 55 recognized RV-C types (as determined by sequence analysis) were instead identified by PCR and sequencing while fishing through patient samples for other RV. As with the RV-A and B, each RV-C type includes those isolates whose VPI sequences exceed 87% pair-wise identity at the nucleotide level. RV-C types have special clinical relevance since it is now recognized these strains are associated with up to half of rhinovirus illnesses in young children. They infect and replicate in both the lower and upper airways and tolerate higher growth temperatures in culture. Moreover, the RV-C use cell receptors that are not common to RV-A or RV-B.
Unfortunately, these receptors are apparently lost whenever primary airway mucosal tissue snippets are transitioned to undifferentiated monolayers since only fully differentiated cultures support RV-C replication in vitro. Small amounts of RV-C can be grown in mucosal organ cultures, but this technique requires the availability of primary human donor samples. Parallel work with differentiated sinus or bronchial epithelial cells cultured at air-liquid interface (ALI) is promising, but neither technique can produce enough RV-C for extensive biological studies.
Multiple attempts to grow RV-C in established cell lines have been unsuccessful. Relatively small quantities of RV-C can be produced using reverse genetics, as the full-length viral RNA transcripts synthesized in vitro are infectious when transfected into human cell lines (including, for example, the NCI-H358, WI-38, WisL, HEK293T, BEAS-2B, A549 and HeLa cell lines) which are normally not permissive to direct viral infection.
However, existing RV-C culture systems have relatively low throughput of virus and are labor intensive compared to those for other RVs. Failure to identify the cellular receptor and the inability to propagate RV-C in convenient cell lines has been a major obstacle to the study of virus-specific characteristics that could lead to effective antiviral strategies for this common and important respiratory pathogen.
Therefore, a need exists for methods of growing RV-C in a manner that provides large quantities of virus in an efficient, cost-effective way. Further, a need exists for methods of propagating RV-C using established cell lines.