Cells and cell-growth media
Cryopreserved primary human RPTEC were obtained from a commercial source in the USA. BGM, supplements, and growth factors [fetal bovine serum, insulin, transferrin, triiodothyonine (T3), human recombinant epidermal growth factor, hydrocortisone, epinephrine, gentamicin sulfate, and amphotericin-B] were concurrently obtained as a kit from the RPTEC supplier. The RPTEC were first seeded onto four T25 flasks and manipulated following instructions included with the kit. MDCK-London cells were a gift from Dr. Gary Heil, University of Florida. Cell lines A549 (CCL-185), BHK-21 (CCL-10), CaCo-2 (HTB-37), CV-1 (CCL-70), HEK-293 (CRL-1573), LLC-MK2 (CCL-7), MDCK, (CCL-34), Mv1 Lu (CCL-64), NIH/3 T3 (CRL-1658), Vero E6 (CRL-1586), and WI-38 (CCL-75) were obtained from the ATCC (Manassas, VA), and along with MDCK-London cells, were propagated as monolayers at 37°C and 5% CO2 in Dulbecco's Modified Eagle's Medium (DMEM) (Mediatech, Inc., Manassas, VA) or Eagle’s Minimal Essential Medium (EMEM) (Invitrogen Corp., Carlsbad, CA), as appropriate per cell line. DMEM and EMEM were initially supplemented with 2 mM L-Glutamine, which was later substituted with 2 mM L-Alanyl-L-Glutamine (GlutaMAX™, Invitrogen Corp.). Both DMEM and EMEM were supplemented with antibiotics [PSN; 50 μg/ml penicillin, 50 μg/ml streptomycin, 100 μg/ml neomycin (Invitrogen Corp.)], and 10% (v/v) low IgG, heat-inactivated gamma-irradiated fetal bovine serum (HyClone, Logan, UT). Additionally, sodium pyruvate (Invitrogen Corp.) and non-essential amino acids (Hyclone) were added to EMEM., with the exception: EMEM formulated with calf serum (HyClone) instead of FBS was used for NIH/3 T3 cells. Before seed stocks were prepared, the cell lines were propagated in growth media with plasmocin (Invivogen, San Diego, CA) for 2 weeks to reduce the chances of mycoplasma contamination. Next, the cell lines were incubated for a minimum of 2 weeks in the absence of antibiotics to determine whether fast-growing microbial contaminants were present or abnormal morphological changes would occur (associated with intracellular mycoplasma). Following 2–3 weeks of propagation without antibiotics, the plasmocin-treated cell lines and RPTEC cells were tested by PCR for the presence of mycoplasma DNA using a Takara PCR Mycoplasma Detection kit (Fisher Scientific, Pittsburgh, PA) . The cells tested negative for mycoplasma. An independent laboratory (at the University of Florida) confirmed that the stock of LLC-MK2 cells that was used for the isolation of HCoV-NL63 in this manuscript was negative for human respiratory viruses including human coronaviruses 229E, HKU1, OC43, and NL63 using a GenMark multiplex respiratory PCR eSensor XT-8 Respiratory Viral Panel (eSensor RVP; GenMark Diagnostics, Inc., Carlsbad, CA).
Glutamine deficiency test
Fresh L-glutamine was added to BGM in a 24 hr RPTEC culture and the cells observed every six hrs for one day to assess the effect on cell morphology, vacuolation, and viability.
BGM cytotoxicity assay
Complete, freshly prepared BGM was substituted for DMEM in subconfluent cultures of CV-1, LLC-MK2, MDCK, Vero, and WI-38 cells, and the cells incubated at 37°C and observed every 12 hours over 3 days for morphological changes or cell death as evidence of cytotoxicity.
Bioactive agent release assay
To find out whether the RPTEC were releasing a bioactive agent, spent BGM from a 24 hr RPTEC culture was equally subdivided and added to subconfluent CV-1, HEK-293, LLC-MK2, Vero E6, and WI-38 cells in T-25 flasks. These particular cell lines were chosen on the assumption that a virus growing in RPTEC would preferentially infect primate over non-primate cells. After inoculation, the cells were incubated at 37°C (the same temperature used for RPTEC) and observed for morphological aberrations over 48 hrs.
Detection of cytomegalovirus by an indirect immunofluorescence assay (IFA)
A standard cytospin procedure was used to deposit RPTEC from a 48 hr culture onto a glass slide. IFA was performed using a commercial kit with a primary antibody directed against a CMV immediate early protein, and a secondary antibody that was labeled with fluorescein isothiocyanate (LIGHT DIAGNOSTICS™ CMV IFA Kit, Millipore, Billerica, MA).
Electron microscopy of virus-contaminated RPTEC
The BGM of a five day RPTEC culture was replaced with fresh ice-cold cacodylate-buffered 4% gluteraldehyde (pH 7.2). After 2 hrs at room temperature, the fixed cells were scraped free using a cell scraper, and pelleted by centrifugation at 8,000 x g for 10 minutes. The fixative was removed, and the cell pellet resuspended with cold fixative to a final volume of 500 μl, then stored overnight at 4°C. The fixed cells were post-fixed with osmium tetroxide, stained with uranyl acetate, embedded in Spurr’s embedding medium, then thin-sectioned. The thin sections were stained with uranyl acetate and lead citrate and transmission electron microscopy performed using a Hitachi H-600.
Isolation of adventitious viruses from five-day old contaminated RPTEC cultures
Five days after being seeded, about 50% of the RPTEC had completely deteriorated, whereupon spent BGM media was added to 2 groups of subconfluent A549, BHK-21, CV-1, HEK-293, LLC-MK2, MDCK, MDCK-London, Mv1 Lu, NIH/3 T3, Vero E6, and WI-38 cells in complete growth media, and to 2 groups of LLC-MK2 and MDCK and Mv1 Lu cells in serum-free media containing L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin. The TPCK-trypsin was at a final concentration of 2 μg/mL (MDCK and MDCK-London cells) or 0.2 μg/mL (LLC-MK2 and Mv1 Lu). For each group, 1 set was incubated at 37°C, the other at 34°C (incubation at 2 different temperatures is standard in our laboratory, as many of the respiratory viruses we work with preferentially replicate at temperatures lower than 37°C). TPCK-trypsin in serum-free media was used to facilitate the isolation of influenza and other viruses that require protease cleavage of some viral component for infectivity. After inoculation, the cells were re-fed every 3 days with 3% serum media or serum-free media with trypsin for long-term (up to 30 day) observations.
Isolation of adventitious viruses from frozen RPTEC cell-lysates
At day 7 post-seed, only about 10% of the RPTEC remained attached to the flask, a majority of which were vacuolated and showed other signs of CPE. To facilitate the isolation of viruses other than CMV, the cells were scraped free and transferred along with the spent BGM into a sterile 50 mL polypropylene centrifuge tube, and frozen at −20°C for one week (this step reduces the number of viable CMV virions by a factor of many logs, since CMV loses viability when stored at −20°C) ; [J. Lednicky, unpublished]. Next, the frozen tube of scraped RPTEC was freeze-thawed three times, alternating between freezing at −20°C for 12 hrs and a 30 minute thaw at room temperature, as an additional measure to further reduce the number of viable CMV particles. After the third thaw, an aliquot was tested using the cells and methods of section 2.5 above, and the remainder frozen at −80°C for retrospective analyses.
PCR and RT-PCR for the detection of viruses
Intracellular DNA was purified from a 48 hr RPTEC culture using a QIAamp DNA mini kit (Qiagen, Valencia, CA) and tested by PCR for CMV, HHV-1 and −2, and polyomaviruses SV40 and BKV. Total RNA was purified from a freeze-thawed seven-day old RPTEC culture supernatant using a QIAamp Viral RNA kit (QIAGEN). The primers and conditions that were used for PCR-based detection of viruses were based on published literature and will be provided upon request. Since syncytia were formed by the second virus (not CMV) that we were attempting to identify, PCR efforts were focused on human herpes, paramyxo (measles, mumps, metapneumovirus, parainfluenza viruses 1–5, respiratory syncytial virus), and coronaviruses.
RT-PCR for RNA virus screens was performed with Omniscript reverse transcriptase (Qiagen) followed by PCR with Hotshot TAQ (New England Biolabs, Ipswich, MA) 68°C. HCoV-NL63 was first detected using a pancoronavirus RT-PCR assay for the viral polymerase gene with primer pair Cor-FW and Cor-RV , followed by sequencing of the 251 bp amplicon. That was accomplished using Cor-RV for cDNA synthesis (with reverse transcription performed for 1 hr at 37°C), and PCR performed as: initial denaturation step: 94°C (1.5 min); 30 cycles of 94°C (20 sec), 48°C (30 sec), 68°C (30 sec); terminal extension step at 68°C (3.5 min); 4°C ∞. For confirmation, primer pairs N5-PCR1 and N3-PCR1  and repSZ-1, and repSZ-3  were used with PCR parameters similar to those for Cor-FW and Cor-RV, and the resulting amplicons sequenced. N5-PCR1 and N3-PCR1 amplify a 314 bp amplicon from the HCoV-NL63 nucleocapsid region. N3-PCR1 was used to generate cDNA, and PCR performed at an annealing temperature of 46°C. Following cDNA synthesis primed with repSZ-RT , primer pair repSZ-1, and repSZ-3 amplify a 237 bp amplicon from the HCoV-NL63 ORF1b region at a PCR annealing temperature of 46°C.
Electron microscopy of LLC-MK2 cells infected with HCoV-NL63 from RPTEC
LLC-MK2 cells that were RT-PCR positive for HCoV-NL63 were trypsinized to detach them from the growing surface of a T75 flask, pelleted, and the pellet resuspended in ice-cold 4% paramormaldehyde, 2% gluteraldehyde, in 0.1 M sodium cacodylate, pH 7.2. They were subsequently analyzed as described above.
Sequencing of HCoV-NL63 genome
Targeted HCoV-NL63/RPTEC/2004 sequences were RT-PCR-amplified from purified RNA using a genome walking strategy. Briefly, overlapping primers described by H. Geng et al. (GenBank JX524171) and others [33, 42] were used to obtain the viral sequence. AccuScript High Fidelity Reverse Transcriptase (Agilent Technologies, Inc., Santa Clara, CA) was used for first-strand cDNA synthesis in the presence of SUPERase-In RNase inhibitor (Ambion). PCR was performed using Phusion Polymerase (New England Biolabs) with denaturation steps performed at 98°C. The 3′ and 5′ ends of HCoV-NL63/RPTEC/2004 were determined from vRNA using a RACE (rapid amplification of cDNA ends) kit (RLM RACE, Ambion, Austin, TX) following the manufacturer’s instructions. Sequences were analyzed using an Applied Biosystem 3130 DNA analyzer by using BigDye Terminator (v. 3.1) chemistry and the same primers used for amplifications.
Molecular dataset, sequence alignment, and phylogenetic analysis
The genomic sequence for isolate NL63/RPTEC/2004/1 was combined with other representative NL63 genomic sequences  available in GenBank (ncbi.nlm.nih.gov/genbank/index.html) to build the final dataset. Full genome alignments were performed using Mafft 5.8  followed by minor manual adjustments in ClustalW . The E-INS-I alignment strategy was used with the following parameters: scoring matrix (BLOSUM62), gap open penalty (1.53), and offset value (0). The aligned dataset was imported into jModelTest version 0.1.1  and the Akaike information criterion (AIC) was used to select a best-fit model of evolution for phylogenetic analysis. Phylogenetic trees were constructed using MrBayes 3.1.2 . The Markov chain was run for a maximum of 10 million generations, with a stopping rule implemented so that the analysis would halt when the average deviation of the split frequencies was < 0.01. Four independent analyses were conducted, each with 1 cold and 3 heated chains with the default heating parameter (temperature = 0.2). Every 1000 generations were sampled and the first 25% of MCMC samples discarded as burn-in.
HCoV-NL63/Amsterdam-1 was obtained from the Biodefense and Emerging Infections Research Resources Repository (BEI Resources, Manassas, VA).
Plaque assays were performed following the procedures outlines in references 39 and 50.
New batch of primary human kidney cells
Primary human kidney cells were obtained from Lonza, Inc. (Allendale, NJ). The cells chosen were: Renal Cortex Epithelial Cells (HRCE) (Cat #: CC-2554, Lot #: 1 F2266, cryopreserved 13 Oct 2010), Human Renal Epithelial Cells (HRE) (Cat #: CC-2556, Lot #: 5 F1314, cryopreserved 19 Oct 2005), and Renal Proximal Tubule Epithelial Cells (RPTEC) (Cat #: CC-2553, Lot #: 0000203150, cryopreserved 21 Dec 2001). The primary cells were grown in Clonetics renal epithelial basal medium (REBM, Lonza, Inc.) (Catalog No: CC-3191, Lot #: 0000345705) with Clonetics REBM SingleQuots supplements (fetal bovine serum, gentamycin sulfate, amphotericin B, insulin, recombinant human epidermal growth factor, transferrin, hydrocortisone, epinephrine, and triiodothyronine).