Recurrent rhinovirus infections in a child with inherited MDA5 deficiency

Lamborn, Ian T.; Jing, Huie; Zhang, Yu; Drutman, Scott B.; Abbott, Jordan K.; Munir, Shirin; Bade, Sangeeta; Murdock, Heardley M.; Santos, Celia P.; Brock, Linda G.; Masutani, Evan; Fordjour, Emmanuel Y.; McElwee, Joshua J.; Hughes, Jason D.; Nichols, Dave P.; Belkadi, Aziz; Oler, Andrew J.; Happel, Corinne S.; Matthews, Helen F.; Abel, Laurent; Collins, Peter L.; Subbarao, Kanta; Gelfand, Erwin W.; Ciancanelli, Michael J.; Casanova, Jean-Laurent; Su, Helen C.

Whole blood, serum, skin biopsies, and nasal airway epithelial scrapings were obtained from the patient, her relatives, or paid healthy volunteers. These individuals gave written informed consent to participate in research protocols approved by Institutional Review Boards at the National Institute of Allergy and Infectious Diseases and National Jewish Health, which are registered in ClinicalTrials.gov under NCT00246857, NCT00128973, and NCT00895271. Buffy coat cells, which were byproducts of volunteer donor blood units, were distributed in an anonymized manner, and thus were exempted from need for informed consent and Institutional Review Board review. rhinovirus molecular typing TRIzol LS reagent (Ambion) was used to extract total RNA from 350 µl of nasopharyngeal washes/aspirates obtained from patients or anonymized controls, or from laboratory virus preparations. 20 µl RNA was reverse-transcribed using a high-capacity cDNA reverse transcription kit with RNase inhibitor (Applied Biosystems). PCR was performed as described previously (Bochkov et al., 2014) , except that 32 cycles of a single round of PCR amplification were performed in a 25 µl volume using 2 µl cDNA template and 0.5 µM of forward and reverse primers. The previously published primers, which were relatively species-specific, were used for amplification of the 5′UTR of Rhinovirus (5′UTRn-A1, 5′UTRn-A2, 5′UTRn-B1, and 5′UTRn-Cc, 5′UTR-rev). The PCR-amplified products were resolved by electrophoresis on 2% agarose gel. PCR-amplified products were also purified by QIAquick Gel Extraction kit (QIA GEN) and cloned using TOPO TA Cloning kit for Sequencing (Invitrogen). Plasmid DNA were isolated from transformed TOP10 E. coli colonies using the R.E.A.L. Prep 96 Plasmid kit (QIA GEN). For each PCR product, 96 cloned inserts were Sanger dideoxy sequenced using plasmid-specific M13 Forward and Reverse primers. Sequences were analyzed using Sequencher 5.1 software, and BLA ST searches of GenBank sequences were performed to identify the virus serotype. The phylogenetic analysis was performed using the UPG MA method of MEGA 7 software, and all positions containing gaps and missing data were eliminated. The phylogenetic trees were drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. The NCBI HRV reference sequences included in the tree are HRV-A89 (NC_001617.1), HRV-B14 (NC_001490.1), and HRV-C (NC_009996,1).