Article Propagation of Rhinovirus C in Differentiated Immortalized Human Airway HBEC3-KT Epithelial Cells

Mina Nakauchi 1,*, Noriyo Nagata 2, Ikuyo Takayama 1, Shinji Saito 1, Hideyuki Kubo 3, Atsushi Kaida 3, Kunihiro Oba 4, Takato Odagiri 1 and Tsutomu Kageyama 1

1 Influenza Research Center, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama-shi, Tokyo 208-0011, Japan; [email protected] (I.T.); [email protected] (S.S.); [email protected] (T.O.); [email protected] (T.K.) 2 Department of Pathology, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama-shi, Tokyo 208-0011, Japan; [email protected] 3 Division of Microbiology, Osaka Institute of Public Health, 8-34 Tojo-cho, Tennoji-ku, Osaka 543-0026, Japan; [email protected] (H.K.); [email protected] (A.K.) 4 Department of Pediatrics, Showa General Hospital, 8-1-1 Hanakoganei, Kodaira-shi, Tokyo 187-0002, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-42-561-0771

 Received: 28 November 2018; Accepted: 27 February 2019; Published: 4 March 2019 

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.

Keywords: rhinovirus C; air–liquid–interface culture; immortalized cells; clinical isolates

1. Introduction Rhinoviruses (RVs) are positive-strand RNA viruses of the genus in the family Picornaviridae. The RV comprises a structural (VP4, VP2, VP3, and VP1) and non-structural (2A, 2B, 2C, 3A, 3B, 3C, and 3D) region flanked by 50 and 30 untranslated regions (UTRs) [1,2]. Recently, 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]. Receptors of RV-A and -B are intercellular adhesion molecule 1 (ICAM-1) and low-density lipoprotein receptor (LDLR) family members [4,5],

Viruses 2019, 11, 216; doi:10.3390/v11030216 www.mdpi.com/journal/viruses Viruses 2019, 11, 216 2 of 10 whereas those of RV-C comprise the recently identified human cadherin-related family member 3 (CDHR3), expressed in ciliated airway epithelial cells [6,7]. RVs cause respiratory illnesses worldwide. They constitute the most common causes of upper [8]. Next to respiratory syncytial viruses, RVs are the most common pathogens associated with severe in children [9]. RV-A and -C are found predominantly in children with lower respiratory infections and wheezing illnesses. However, 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 [10–13]. Unlike RV-A and -B, RV-C cannot be propagated using standard cell cultures. To date, it has been reported that RV-C can be propagated in sinus organ culture, air–liquid–interface (ALI) cultured differentiated human airway epithelial cells, and commercially available three-dimensional (3D) human upper airway epithelia reconstituted in vitro [14–17]. These systems have provided an important tool for RV-C research. However, isolation of a sufficient number of sinus organs for culture and the stable culture of differentiated primary human airway epithelial cells are difficult. In addition, commercially available 3D human upper airway epithelial cells are costly, and only a few clinical isolates, RV-C2, -C7, -C12, -C15, and -C29, have been isolated using this method; of these, only RV-C7 and -C29 have been passaged and re-propagated [17]. Therefore, the isolation of RV-Cs from clinical specimens in order to gain a better understanding of their biological properties and pathogenesis requires stable and inexpensive culture systems. In this study, we established the ALI culture method using immortalized human airway epithelial HBEC3-KT cells to isolate RV-C from clinical specimens. The morphology of the HBEC3-ALI culture resembled pseudostratified epithelia with cilia and goblet cells. Further, RV-C9 and -C53 propagated efficiently in HBEC3-ALI cultures, and 11 clinical RV-C strains were isolated from 23 clinical specimens.

2. Materials and Methods

2.1. Cell Cultures Human bronchial tracheal epithelial (HBTE) cells (FC-0035) were obtained from Lifeline Cell Technology (Carlsbad, CA, USA). HBEC3-KT (CRL-4051) and HSAEC1-KT (CRL-4050) cells were from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were maintained in submerged cultures using PneumaCult-Ex medium (STEMCELL Technologies, Vancouver, Canada), according to the manufacturer’s instructions. HBTE, HBEC3-KT, and HSAEC1-KT cells were generated via ALI culture using Transwell Permeable Supports (Costar 3470, Corning Costar, Corning, NY, USA) and PneumaCult-ALI medium (STEMCELL Technologies), according to the manufacturer’s instructions. The cultures were allowed to mature for at least 28 days. MRC5 (CCL-171) cells were obtained from the ATCC and maintained in minimum essential medium containing 10% fetal calf serum.

2.2. Histological Examination HBEC3- and HSAEC1-ALI cells cultured for 30 days were fixed in 10% phosphate-buffered formalin and routinely embedded in paraffin. Paraffin-embedded sections were stained with hematoxylin and eosin, and periodic acid-Schiff staining was also performed.

2.3. Clinical Specimens and Viruses A total of 23 nasopharyngeal swabs or nasal aspirates obtained from patients enrolled in clinical studies that were approved by the National Institute of Infectious Diseases, Showa General Hospital Ethics Committee, and which screened positive for RV by real-time RT-PCR and partial 50UTR sequencing [18], were used for the study. RV-A16 (VR-283) was obtained from the ATCC and propagated using MRC5 cells. RV-C9 and -C53 were isolated from the clinical specimens using ALI-cultured HBTE cells according to Viruses 2019, 11, 216 3 of 10 previously described methods [15,16,19]. Whole genome sequences of these RV-C9 and -C53 strains were determined using a next-generation sequencer (see Section 2.9.2) and deposited at the National Center for Biotechnology Information (NCBI) (accession numbers LC428175 and LC428176).

2.4. Virus Inoculation

RV-C9, -C53 (8 and 7 log10 RNA copies/well), and RV-A16 (6 and 5 log10 RNA copies/well) were inoculated onto the apical surfaces of HBEC3- and HSAEC1-ALI cultures. After 4 h of incubation, the cells were washed three times with Opti-MEM (Thermo Fisher Scientific, Waltham, MA, USA) to remove residual virus inoculate and incubated at 34 ◦C. After incubation for 1, 3, 7, 14, and 21 days, the apical surfaces of the cells were washed with 200 µL of Opti-MEM to collect the propagated viruses. The viral were then quantified by real-time RT-PCR. Culture media were also collected from the basolateral sides at 1, 7, 14, and 21 days following inoculation.

2.5. RNase Treatment Of the 200 µL of propagated viruses collected from the apical surface of HBEC3-ALI cultures 7 days after inoculation with 8 log10 RNA copies/well of RV-C9 and -C53, 6 µL was used for RNase treatment. A total 100 µL of reaction mixture containing 10 µL of 10× reaction buffer, 1 µL of RNase ONE™ Ribonuclease (10 U/µL; Promega, Madison, WI, USA), 83 µL of water, and 6 µL of sample was incubated for 30 min at 37 ◦C. To prepare the untreated samples, 1 µL of water was added to the reaction mixture instead of RNase ONE™ Ribonuclease.

2.6. RNA Extraction from Viruses Total RNA was prepared from viral isolates using a MagMAX™ 96 Viral Isolation Kit (Thermo Fisher Scientific), using 50 µL of collected medium, with KingFisher Flex (Thermo Fisher Scientific) according to the manufacturer’s instructions. Total RNA was also prepared from 100 µL of RNase-treated samples using a MagMAX™ CORE Nucleic Acid Purification Kit (Thermo Fisher Scientific), using 60 µL of elution buffer, with KingFisher Flex (Thermo Fisher Scientific), according to the manufacturer’s instructions.

2.7. Amplification of the RV Genome by Real-Time RT-PCR Quantification of RV genomes was conducted via real-time RT-PCR using AgPath-ID™ One-Step RT-PCR Reagents (Thermo Fisher Scientific) as described previously [20], with a small modification. Briefly, the 10 µL assay contained 5 µL of 2× RT-PCR buffer, 0.4 µL of 25× RT-PCR Mix, 1.4 µL of primers/probe mix, 0.2 µL of water, and 3 µL of template RNA. The primer/probe mix was prepared to contain 0.6 µM of each primer and 0.1 µM of the probe. To prepare the RNA transcript control, the 50UTR region of RV-A16 was amplified by RT-PCR, and the resulting PCR product containing the T7 promoter was then transcribed in vitro. The 50UTR region was amplified by PCR using Phusion High-Fidelity DNA Polymerase (New England BioLabs, Ipswich, MA, USA) with paired primers (TAATACGACTCACTATAGGGGTACWCTRKTAYTMYGGTAMYYTTGTACGCC and AGWGCATCKGGYAAYTTCCA). RNA was transcribed using the T7 RiboMAX™ Express Large-Scale RNA Production System (Promega) and treated with TURBO® DNase to degrade the template DNA. The dNTPs and NTPs were removed using MicroSpin G-25 Columns (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer’s instructions. The transcribed RNA was quantified using a NanoDrop spectrophotometer, and the absorbance value was used to calculate the copy numbers of the transcribed RNA. The integrity of the transcribed was assessed via a 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA). Viruses 2019, 11, 216 4 of 10

2.8. Virus Isolation RV-positive specimens were inoculated onto the apical surfaces of HBEC3-ALI cultures. After 4 h of incubation, the cells were washed three times with Opti-MEM containing 102 U/102 µg/mL of penicillin/streptomycin, 100 µg/mL of gentamicin, and 0.5 µg/mL of amphotericin B to remove residual specimen inoculate, and the cultures were incubated at 34 ◦C. After incubation for 7, 14, and 21 days, the apical surfaces of the cells were washed with 200 µL of Opti-MEM to collect the propagated viruses.

2.9. Sequencing

2.9.1. RT-PCR and Sanger Sequencing of VP1 to Type RVs Purified viral RNA was reverse-transcribed using random hexamer primers (Promega) and Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The cDNA (3 µL) was amplified in PCR using paired primers with Phusion High-Fidelity DNA Polymerase. The PCR cycling conditions were as follows: 98 ◦C for 30 s, 40 cycles at 98 ◦C for 10 s, 65 ◦C for 30 s, and 72 ◦C for 1 min, with a final extension of 72 ◦C for 10 min. The primer sequences for amplifying the VP1 region of each virus isolate are provided (Table S1). Amplified VP1 products of approximately 400 bp were then gel-purified with the MinElute Gel Extraction kit (Qiagen, Hilden, Germany), and sequencing was performed in both directions using amplification primers and the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit, version 3.1.

2.9.2. Next-Generation Sequencing A next-generation sequencing (NGS) library was prepared using a NEBNext Ultra RNA Library Prep Kit (New England BioLabs), followed by the sequencing of 75-bp paired-end reads with MiSeq (Illumina, San Diego, CA, USA). The obtained sequencing reads were assembled using CLC Genomics Workbench 7 software (Qiagen) with De Novo Assembly.

2.10. Statistical Analyses Statistical analyses were performed using the Prism statistical software package (version 7.0; GraphPad Software, Inc., La Jolla, CA, USA). One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test was used to analyze the growth properties of the RVs. Two-way ANOVA followed by Sidak’s multiple comparisons test was used to analyze the increased RNA levels of the RVs. A Welch’s unpaired t test was used to compare the RNA copy numbers of RV genomes among clinical specimens.

3. Results

3.1. RV-C9, -C53, and RV-A16 Growth in HBEC3- and HSAEC1-ALI Cultures To obtain RV-C strains, RV-C9 and -C53 were isolated from the clinical specimens using ALI-cultured primary HBTE cells according to a previously established culture system for RV-Cs [15,16]. They were used as reference strains to evaluate our established culture system in this study. Whole-genome sequences of RV-C9 and -C53 were determined via NGS (accession numbers, LC428175 and LC428176). In order to establish a stable and inexpensive culture system for RV-Cs, HBEC3-KT and HSAEC1-KT cells were cultured under ALI conditions. After being maintained in the ALI system for 30 days, histological examination revealed that HBEC3- and HSAEC1-ALI cultures were likely pseudostratified human airway epithelial cells with cilia and -secreting goblet-like cells (Figure1). Viruses 2019, 11, 216 5 of 10 Viruses 2018, 10, x FOR PEER REVIEW 5 of 10

FigureFigure 1.1. HistologicalHistological morphology morphology of of HBEC3 HBEC3 and and HSAEC1 HSAEC1 cells culturedcells cultured using theusing air–liquid–interface the air–liquid– interface(ALI) system. (ALI) Hematoxylin system. Hematoxy and eosinlin and (HE) eosin staining (HE)( stainingA,C) and (A periodic and C) acid-Schiffand periodic (PAS) acid-Schiff staining (PAS) (B,D) stainingof HBEC3-ALI (B and (DA), Bof) andHBEC3-ALI HSAEC1-ALI (A and (C B),D and) cultures. HSAEC1-ALI (C and D) cultures.

InIn order order to observe thethe growthgrowth propertiesproperties of of the the RVs RVs in in HBEC3- HBEC3- and and HSAEC1-ALI HSAEC1-ALI cultures, cultures, RV-C9 RV- C9and and -C53 -C53 (8 and(8 and 7 log 7 log10 RNA10 RNA copies/well), copies/well), as as well well as as RV-A16 RV-A16 (6 (6 and and 5 5 log log1010 RNARNA copies/well),copies/well), were were inoculatedinoculated in in three three replicates replicates for for each each RNA RNA copy copy number number of of RV. RV. Samples were were collected collected from from the the apicalapical surfaces surfaces at at 1, 1, 3, 3, 7, 7, 14, and 21 days following inoculation, and and RNA RNA levels levels were were analyzed analyzed by by comparingcomparing thethe amount amount of RNAof RNA in PI in samples PI samples (the collected (the collected third wash third solution). wash Followingsolution). inoculationFollowing inoculationof HBEC3-ALI of HBEC3-ALI cultures with cultures 8 and with 7 log 810 andRNA 7 log copies/well10 RNA copies/well of RV-C9, of the RV-C9, RNA levelsthe RNA increased levels increasedsignificantly significantly from 4.21 (PI)from to 4.21 5.92 (PI) log10 tocopies/well 5.92 log10 copies/well at day 7 and at fromday 7 under and from the detection under the limit detection (PI) to limit3.78 log(PI)10 tocopies/well 3.78 log10 copies/well at day 1, respectively at day 1, respectively (Figure2A). (Figure Following 2A). inoculation Following ofinoculation HBEC3-ALI of HBEC3- cultures ALIwith cultures the same with amounts the ofsame RV-C53 amounts as that of of RV-C53 RV-C9, theas RNAthat of levels RV-C9, increased the RNA significantly levels increased from 4.48 significantly(PI) to 6.17 log from10 copies/well 4.48 (PI) to and 6.17 from log10 under copies/well the detection and from limit under (PI) to the 5.35 detection log10 copies/well limit (PI) at to day 5.35 1, logrespectively10 copies/well (Figure at 2dayB). However,1, respectively inoculation (Figure of 2B). HSAEC1-ALI However, cultures inoculation with similarof HSAEC1-ALI amounts ofcultures RV-C9 withand -C53similar did amounts not lead of to RV-C9 significantly and -C53 higher did RNA not lead levels to onsignificantly any of the higher days tested, RNA andlevels only on aany slight of theincrease days intested, RNA and was only observed a slight at dayincrease 3 following in RNA inoculation was observed with at 8 day log10 3RNA following copies/well inoculation of RV-C53 with 8(Figure log10 RNA S1A,B). copies/well There was of RV-C53 no difference (Figure at S1A,B). any time There point was between no difference RV-C9 at and any -C53 time in point the amountsbetween RV-C9of RNA and in HBEC3-ALI-C53 in the amounts cultures of following RNA in inoculationHBEC3-ALI withcultures 8 log following10 RNA copies/well.inoculation with Following 8 log10 RNAinoculation copies/well. with 7 Following log10 RNA inoculation copies/well, with the amount7 log10 RNA of RV-C53 copies/well, RNA was the significantlyamount of RV-C53 higher atRNA day was1 compared significantly to that higher for RV-C9, at day but 1 compared there were to no that differences for RV-C9, at daysbut there 3, 7, 14,were or no 21 (Figuredifferences3A). at days 3, 7, 14,To or compare 21 (Figure the growth3A). properties of RV-Cs and RV-A, HBEC3-ALI cultures were inoculated with 6 andTo 5 logcompare10 RNA the copies/well growth properties of RV-A16. of AtRV-Cs day 1,an RNAd RV-A, levels HBEC3-ALI increased significantlycultures were from inoculated 3.05 (PI) withto 5.57 6 and log10 5 logcopies/well10 RNA copies/well and fromunder of RV-A16. the detection At day 1, limit RNA (PI) levels to 4.94 increased log10 copies/well, significantly respectively from 3.05 (PI)(Figure to 25.57C). Contrarylog10 copies/well to what observedand from for under RV-Cs, the the detection RNA levels limit also (PI) increased to 4.94 significantlylog10 copies/well, from respectivelyunder the detection (Figure limit 2C).(PI) Contrary to 5.09 logto 10whatcopies/well observed at for day RV-Cs, 7 following the RNA inoculation levels ofalso HSAEC1-ALI increased significantlycultures with from 5 log 10underRNA the copies/well detection oflimit RV-A16 (PI) (Figureto 5.09 S1C).log10 Therecopies/well were noat differencesday 7 following in the inoculationamounts of of RV-A16 HSAEC1-ALI RNA between cultures HBEC3- with 5 andlog10 HSAEC1-ALI RNA copies/well cultures, of RV-A16 except (Figure for day S1C). 1 following There wereinoculation no differences with 5 login 10theRNA amounts copies/well, of RV-A16 when RNA the between RNA levelHBEC3- was an significantlyd HSAEC1-ALI higher cultures, in the exceptHBEC3-ALI for day cultures 1 following (Figure 3inoculationB). with 5 log10 RNA copies/well, when the RNA level was significantlyBoth RV-A higher and in -C the were HBEC3-ALI detected onlycultures on the (Figure apical 3B). surfaces and not in the basolateral cultured mediumBoth (dataRV-A notand shown), -C were anddetected no cytopathic only on the effects apical were surfaces observed and not after in the inoculating basolateral HBEC3- cultured or mediumHSAEC1-ALI (data culturesnot shown), with RV-Aand no and cytopathic -C (data noteffects shown). were observed after inoculating HBEC3- or HSAEC1-ALI cultures with RV-A and -C (data not shown).

Viruses 2019, 11, 216 6 of 10 Viruses 2018,, 10,, xx FORFOR PEERPEER REVIEWREVIEW 6 of 10

FigureFigure 2.2.Growth Growth properties properties of of rhinoviruses rhinoviruses (RVs) (RVs) in HBEC3in HBEC3 air–liquid-interface air–liquid-interface (ALI) (ALI) culture. culture. For this,For this, 8 or 6 (filled circles) and 7 or 5 (open circles) log10 RNA copies/well of RV-C9 and -C53 or RV- 8this, or 6 8 (filled or 6 (filled circles) circles) and 7 and or 5 7 (open or 5 (open circles) circles) log10 logRNA10 RNA copies/well copies/well of RV-C9 of RV-C9 and and -C53 -C53 or RV-A16, or RV- respectively,A16, respectively, were inoculatedwere inoculated onto onto HBEC3-ALI HBEC3-ALI cultures cultures (A– C(A,). DataB and are C). expressed Data are expressed as log copies as log of RNAcopies per of wellRNA ( pern = well 3) at (n 1, = 3, 3) 7, at 14, 1, and3, 7, 14, 21 day(s)and 21 afterday(s) inoculation. after inoculation. PI represents PI represents the collected the collected third washthird solution.wash solution. Differences Differences in log RNAin log copies RNA werecopies analyzed were analyzed by one-way by one-way ANOVA ANOVA followed followed by Tukey’s by multipleTukey’s multiple comparisons comparisons test. Asterisks test. Asterisks (*) and (*) circles and circles (◦) show (°) differencesshow differences between between PI and PI each and each time time point within 8 log10 RNA copies/well of RV-Cs/6 log10 RNA copies/well of RV-A inoculated pointtime withinpoint within 8 log10 8RNA log10 copies/well RNA copies/well of RV-Cs/6 of RV-Cs/6 log10 RNA log10copies/well RNA copies/well of RV-A of inoculatedRV-A inoculated groups groups and within 7 log10 RNA copies/well of RV-Cs/5 log10 RNA copies/well of RV-A inoculated andgroups within and 7 within log10 RNA 7 log copies/well10 RNA copies/well of RV-Cs/5 of RV-Cs/5 log10 RNA log10 copies/well RNA copies/well of RV-A of inoculated RV-A inoculated groups. °°° °°°° */groups.◦ p < 0.05, */° p **/ < 0.05,◦◦ p <**/°° 0.01, p ***/< 0.01,◦◦◦ ***/p <°°° 0.001, p < 0.001, and ****/ and◦◦◦◦ ****/p°°°°<0.0001. p < 0.0001.

FigureFigure 3.3.Increased Increased RNARNA levelslevels ofof RVsRVs in in HBEC3- HBEC3- and/or and/or HSAEC1-ALI cultures. Data are expressed n asas loglog copiescopies ofof RNARNA per well (n ( == 3) 3) at at 1, 1, 3, 3, 7, 7, 14, 14, and and 21 21 day(s) day(s) post-inoculation post-inoculation and and normalized normalized to tothe the RNA RNA concentration concentration of PI of (collected PI (collected third third wash wash solution) solution) as 0. as (A 0.) Comparison (A) Comparison of increased of increased RNA RNA levels between RV-C9 and -C53 in HBEC3-ALI cultures. Groups comprising inoculums10 of levels between RV-C9 and -C53 in HBEC3-ALI cultures. Groups comprising inoculums of 8 log10 RNA 8 log10 RNA copies/well of RV-C9 (filled hexagons) and RV-C53 (filled diamonds),10 and 7 log10 RNA copies/well of RV-C9 (filled hexagons) and RV-C53 (filled diamonds), and 7 log10 RNA copies/well of copies/wellRV-C9 (open of hexagons) RV-C9 (open and hexagons) RV-C53 (open and RV-C53 diamonds (open) were diamonds) compared were at compared each time at point. eachtime (B) point. (B) Comparison of increased RV-A16 RNA levels between HSAEC1- and HBEC3-ALI cultures. Comparison of increased RV-A16 RNA levels between HSAEC1- and HBEC3-ALI cultures. Groups Groups comprising inoculums of10 6 log10 RNA copies/well of RV-A16 in HBEC3- (filled diamonds) and comprising inoculums of 6 log10 RNA copies/well of RV-A16 in HBEC3- (filled diamonds) and HSAEC1- (filled hexagons) ALI cultures and 5 log1010 RNA copies/well of RV-A16 in HBEC3- (open HSAEC1- (filled hexagons) ALI cultures and 5 log10 RNA copies/well of RV-A16 in HBEC3- (open diamonds) and HSAEC1- (open hexagons) ALI cultures were compared at each time point. Differences diamonds) and HSAEC1- (open hexagons) ALI cultures were compared at each time point. in log RNA copies were analyzed by two-way ANOVA followed by Sidak’s multiple comparisons test. Differences in log RNA copies were analyzed by two-way ANOVA followed by Sidak’s multiple * p < 0.05. comparisons test. * p < 0.05. To confirm that an increased in RNA indicated encapsidated progeny RNA genomes, samples To confirm that an increased in RNA indicated encapsidated progeny RNA genomes, samples collected from the apical surfaces of HBEC3-ALI cultures at day 7 post-inoculation with 8 log10 RNA collected from the apical surfaces of HBEC3-ALI cultures at day 7 post-inoculation with 8 log10 RNA copies/well RV-C9 and -C53 were treated with RNase under conditions that the non-encapsidated viral copies/well RV-C9 and -C53 were treated with RNase under conditions that the non-encapsidated RNA would be completely degraded. Comparison of the amounts of RNA between RNase-treated and viral RNA would be completely degraded. Comparison of the amounts of RNA between RNase- untreated samples following RNA extraction by real-time RT-PCR indicated no significant difference treated and untreated samples following RNA extraction by real-time RT-PCR indicated no (Figure S2). significant difference (Figure S2). 3.2. Isolation of Clinical RV-C Isolates Using HBEC3-ALI Culture 3.2. Isolation of Clinical RV-C Isolates Using HBEC3-ALI Culture Twenty-three samples that were positive for RV-C by real-time RT-PCR and 50UTR sequence Twenty-three samples that were positive for RV-C by real-time RT-PCR and 5′UTR sequence analysis were inoculated onto the apical surface of HBEC3-ALI cultures, and 11 RV-C clinical isolates analysis were inoculated onto the apical surface of HBEC3-ALI cultures, and 11 RV-C clinical isolates were successfully isolated (Table S2). A based on the VP1 sequences of these 11 were successfully isolated (Table S2). A phylogenetic tree based on the VP1 sequences of these 11 RV- C clinical strains and on corresponding sequences of RV-C prototypes showed that they could be

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classified into eight types: RV-C2, -C6, -C9, -C12, -C18, -C23, -C40, and -C53 (Table S2 and Figure RV-C clinical strains and on corresponding sequences of RV-C prototypes showed that they could be S3A). In addition, nine of the 11 clinical isolates including RV-C2, -C6, -C9, -C12, -C18, -C23, and - classified into eight types: RV-C2, -C6, -C9, -C12, -C18, -C23, -C40, and -C53 (Table S2 and Figure S3A). C53 collected from the apical surfaces were subsequently passaged and re-grown in HBEC3-ALI In addition, nine of the 11 clinical isolates including RV-C2, -C6, -C9, -C12, -C18, -C23, and -C53 cultures (data not shown). In order to determine the potential causes for our failure to isolate RV-Cs collected from the apical surfaces were subsequently passaged and re-grown in HBEC3-ALI cultures from the 12 samples, we compared the contained RNA copy numbers of RV genome between the 11 (data not shown). In order to determine the potential causes for our failure to isolate RV-Cs from the clinical specimens from which the viruses were isolated successfully and the 12 clinical specimens 12 samples, we compared the contained RNA copy numbers of RV genome between the 11 clinical from which the viruses could not be isolated. Consequently, it was evident that the contained RNA specimens from which the viruses were isolated successfully and the 12 clinical specimens from which copy numbers in the specimens from which the viruses were successfully isolated were significantly the viruses could not be isolated. Consequently, it was evident that the contained RNA copy numbers higher (Figure 4). in the specimens from which the viruses were successfully isolated were significantly higher (Figure4).

Figure 4. Comparison of RNA copy numbers of RV genomes between the 11 clinical specimens from whichFigure the viruses4. Comparison were successfully of RNA copy isolated numbers and the of RV 12 clinical genomes specimens between from the 11 which clinical the virusesspecimens could from notwhich be isolated. the viruses Differences were successfully were analyzed isolated by Welch’s and the unpaired 12 clinical t test. specimens ** p < 0.01. from which the viruses could not be isolated. Differences were analyzed by Welch's unpaired t test. ** p < 0.01. 4. Discussion 4. Reportedly,Discussion RV-Cs target ciliated airway epithelial cells expressing the only known RV-C cell-entry factor, CDHR3Reportedly, [7]. RV-Cs HBEC3-KT target and ciliated HSAEC1-KT airway epithe cells arelial cells normal expressing human bronchialthe only known epithelial RV-C cells cell- immortalizedentry factor, with CDHR3 CDK4 [7]. and HBEC3-KT hTERT that and retain HSAEC1-KT the capacity cells to are differentiate normal human into mucin-producingbronchial epithelial andcells ciliated immortalized epithelial with cells inCDK4 ALI cultureand hTERT using that collagen retain gel the [21 capacity–23]. In thisto differentiate study, HBEC3-KT into mucin- and HSAEC1-KTproducing and cells ciliated were cultured epithelial under cells in ALI ALI conditions culture using using collagen Transwell gel [21–23]. Permeable In this Supports study, HBEC3- and PneumaCultKT and HSAEC1-KT ALI medium cells and were differentiated cultured under into pseudostratified ALI conditions using epithelia Transwell with cilia Permeable and goblet Supports cells (Figureand PneumaCult1). Although ALI no medium obvious and morphological differentiated differences into pseudostratified were observed epithelia between with HBEC3- cilia and and goblet HSAEC1-ALIcells (Figure cultures, 1). Although RV-C9, no -C53, obvious and morphological -A16 propagated differences well in HBEC3-ALI were observed cultures between (Figure HBEC3-2A–C), and whereasHSAEC1-ALI only RV-A16 cultures, propagated RV-C9, well-C53, in and HSAEC1-ALI -A16 propagated cultures well (Figure in S1A–C).HBEC3-ALI Considering cultures these(Figure results,2A,B,C), the differentwhereas RV-Conly growthRV-A16 properties propagated between well HBEC3-in HSAEC1-ALI and HSAEC1-ALI cultures cultures (Figure were S1A,B,C). not dependentConsidering on cell these morphology, results, the and different therefore RV-C other growth unknown properties host cellbetween factor(s) HBEC3- might and be responsibleHSAEC1-ALI forcultures the differences. were not Further dependent studies on are cell needed morphology, to elucidate and suchtherefore discrepancies. other unknown host cell factor(s) mightRNA be amounts responsible were for not the affected differences. by the Further treatment studies of theare collectedneeded to samples elucidate with such RNase discrepancies. before RNA extractionRNA amounts (Figure were S2). Thisnot affected suggested by thatthe thetreatmen increasedt of the RNA collected in HBEC3-ALI samples cultures with RNase indicated before encapsidatedRNA extraction progeny (Figure RNA S2). genomes. This suggested The most that significant the increased increases RNA in in HBEC3-ALI RNA levels cultures in HBEC3-ALI indicated culturesencapsidated inoculated progeny with 8 RNA and 7 genomes. log10 RNA The copies/well most significant of RV-C9 increases were 1.71 in and RNA 5.97 levels log10 incopies/well, HBEC3-ALI respectively,cultures inoculated and in HBEC3-ALI with 8 and cultures 7 log10 RNA inoculated copies/well with RV-C53 of RV-C9 were were 2.34 1.71 and and 7.06 5.97 log 10logcopies/well,10 copies/well, respectivelyrespectively, (Figure and3 A).in HBEC3-ALI These increases cultures in RNA inoc levelsulated agree with with RV-C53 those were of previous 2.34 and reports 7.06 onlog10 RV-Ccopies/well, culture methods respectively [15– 17(Figure]. In the 3A). HBEC3-ALI These increa cultures,ses in RNA after levels virus inoculationagree with those onto theof previous apical surface,reports propagated on RV-C culture viruses methods were detected [15–17]. only In the on H theBEC3-ALI apical surfaces cultures, and after not virus in basolateral inoculation cultured onto the mediumapical (datasurface, not propagated shown). These viruses observations were detected also agree only wellon the with apical previous surfaces reports and onnot RV-C in basolateral culture methodscultured [14 medium,15,17] as (data well not as withshown). those These on natural observations RV infections also agree in humanwell with airway previous epithelia reports [24 on–28 RV-]. C culture methods [14,15,17] as well as with those on natural RV infections in human airway epithelia

Viruses 2019, 11, 216 8 of 10

Considering that HBEC3-KT cells were stably cultured and recapitulated the pseudo-morphology of human airway epithelia with the ability to serve as hosts for sufficient RV-C propagation—as in natural RV infections—our established HBEC3-ALI culture appears to be a stable culture system suitable for investigating RV-Cs. Using HBEC3-ALI cultures, 11 clinical strains were isolated from 23 clinical specimens. The mean RNA copy numbers of RV genome in the 11 clinical specimens from which the viruses were successfully isolated was 7.07 log10 copies/inoculation (Figure4). Considering that RNA copy numbers in the 11 specimens from which the viruses were successfully isolated were significantly higher, it is surmised that we failed to isolate RV-Cs from the other 12 clinical specimens partly because they contained lower concentrations of RV-C. The clinical isolate 47-SGH-JPN-2015 was isolated from the same clinical specimen as that of RV-C9 using ALI-cultured HBTE cells. Similarly, 4-SGH-JPN-2015 was isolated from the same clinical specimen as that of RV-C53. It was shown that there were no differences between the VP1 sequences of RV-C9 and -C53, as determined by NGS (accession numbers, LC428175 and LC428176) and between those of 4-SGH-JPN-2015 and 47-SGH-JPN-2015, as determined by Sanger sequencing (accession numbers, LC428172 and LC428173). These results suggest that our established HBEC3-ALI culture did not affect the VP1 sequence. Moreover, it is speculated that the same strain might be isolated from a clinical specimen using either HBEC3-ALI culture or ALI-cultured HBTE cells. In conclusion, we established a stable ALI culture system, the HBEC3-ALI culture, using immortalized human airway epithelial cells in which RV-C propagated well, and showed that 11 RV-C clinical strains could be isolated using this system. Further, nine of the 11 isolates were re-propagated in HBEC3-ALI cultures. Thus, the established culture system will ultimately help to gain an understanding not only of basic replication mechanisms but also of the clinical features of RVs under conditions that more closely mimic natural infections.

Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4915/11/3/216/s1, Table S1: Primers for sequencing the VP1 region. Table S2: Clinical isolates of RV-Cs. Figure S1: Growth properties of RVs in HSAEC1-ALI culture. Figure S2: Comparison of RNA amounts between RNase-treated (+) and -untreated (–) samples of RV-C9 and -C53 samples. Figure S3: Phylogenetic analysis of the VP1 genes of clinical isolates and prototypes of RV-C. Author Contributions: Conceptualization, M.N.; methodology, M.N.; validation, T.O. and T.K.; formal analysis, M.N.; investigation, M.N., N.N., I.T., and S.S.; resources, H.K., A.K., and K.O.; writing—original draft preparation, M.N. and N.N.; writing—review and editing, I.T., S.S., K.O., H.K., and A.K.; visualization, M.N.; project administration, M.N. Funding: This research was funded by AMED, grant number JP18fk0108030. Acknowledgments: We appreciate Shutoku Matsuyama and Kazuya Shirato and Miyuki Kawase of the Department of Virology III, National Institute of Infectious Diseases, for providing ALI-cultured differentiated human bronchial tracheal epithelial cells. We thank Midori Ozaki of the Department of Pathology and Shiho Nagata of the Influenza Virus Research Center, National Institute of Infectious Diseases, for their excellent technical assistance. Conflicts of Interest: The authors declare no conflict of interest.

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