Free-Chlorine Disinfection as a Selection Pressure on Norovirus

MNV isolates and cell lines for chlorine cycle experiment.The MNV S7-PP3 strain, which is genetically close to MNV3 (43), was obtained from Yukinobu Tohya, Nihon University, Japan. Both MNV S7 strains and a prototype MNV-1 strain have been used as foodborne pathogen models for human noroviruses. MNV S7 strains have been widely used by Japanese and U.S. researchers as a free chlorine inactivation model (44), in a test for a plant-based antiviral substance (45), and in an investigation of functional receptor for MNV (34). MNV S7-PP3 cells were propagated, enumerated, and purified according to the published protocols (46). Viral stocks were stored at −81°C before use. RAW 264.7 cells (ATCC TIB-71) were used as host cells and cultured in modified Dulbecco's modified Eagle's medium (DMEM) containing 10% (vol/vol) fetal bovine serum (FBS), 0.075% NaHCO₃, 2 mM l-glutamine, 10 mM nonessential amino acids, 100 mg/ml penicillin, and 100 U/ml streptomycin. Cells were cultured at 37°C and 5% CO₂.

Chlorine cycle experiment.To investigate the lowering of susceptibility of norovirus to free chlorine, MNV S7-PP3 was treated with free chlorine repeatedly 10 times (Fig. 1). This cycle experiment was conducted twice. The free chlorine solution was prepared by mixing sodium hypochlorite solution (Wako, Osaka, Japan) with phosphate-buffered saline (PBS) solution with an initial chlorine concentration of 50 ppm, measured using a chlorine comparator DPD (N,N-diethyl-p-phenylenediamine) method (Shibata, Saitama, Japan). Surviving virus from the first chlorine cycle was regrown in RAW 264.7 cells and repeated for another nine cycles. As a control treatment, the MNV S7-PP3 population was subjected to repetitious growth in RAW 264.7 cells for 10 cycles without chlorine exposure. As a control, the initial MNV population identical with that used for the free chlorine disinfection was diluted 10⁴-fold and propagated in RAW 264.7 cells, and this dilution-propagation cycle was repeated 10 times to obtain a control population. Chlorine susceptibility for both control and chlorine-treated populations in each cycle was determined by measuring log₁₀ reduction of exposure to free chlorine.

Next-generation sequencing (NGS).To examine genetic changes in MNV S7-PP3 populations due to the free chlorine intervention, next-generation sequencing (NGS) was employed for both the first and second trials of the cycle experiment. The sequencing target was the capsid region of MNV, since it is known that free chlorine cause damages to the viral capsid (47). The MNV S7-PP3 capsid gene (2,252 bp in length) proteins were divided into seven regions (Fig. 3). Primers were designed to cover all seven regions with an overlapping sequence (Table 1). The original population, the fifth and tenth cycles of the control, and the free chlorine-treated populations were picked for NGS. The process of amplicon preparation involves RNA extraction, reverse transcription-PCR (RT-PCR), PCR targeting ORF2 and ORF3, gel electrophoresis to check the length of the PCR products, and PCR product purification from the gel. Reverse transcription-PCR, PCR, and PCR product purification were performed following the PrimeScript Perfect real-time RT reagent (TaKaRa Bio, Shiga, Japan), the Phusion high fidelity PCR kit (New England BioLabs, Ipswich, MA), and the Fast Gene gel/PCR purification kit (Nippon Genetics, Tokyo, Japan) manufacturers' protocols, respectively. The PCR temperature profile is shown in Table S3 in the supplemental material.

In order to prepare MiSeq libraries for the first trial of the cycle experiment, each PCR product was ligated to different Y type adapters, which consist of i5 (D501 to 507) and i7 (D701 to 707) oligomers (48). First, 49 types of the Y adapters were prepared by annealing each i5 and i7 oligomer. Annealing mixtures contained 6 μl of one of the i5 oligomers (50 μM), 6 μl of one of the i7 oligomers phosphorylated in 5′-end (50 μM), and 1.35 μl of an annealing buffer (0.06 M Tris-HCl, 0.01 M EDTA, 0.025 M NaCl). The annealing mixture was incubated on a T100 thermal cycler (Bio-Rad, Hercules, CA) with the following steps: 5 min at 95°C, 140 ×30 s with the temperature decreased by 0.5°C per each cycle, 40 min at 25°C, and storage at 12°C.

The PCR products were adenylated at the 3′-end in a 50-μl reaction volume with PCR products at 0.15 to 1.5 pmol, 5 μl Ex Taq buffer (TaKaRa), 3 μl MgCl₂ (TaKaRa) (25 mM), 1 μl dATP (Promega, Madison, WI) (10 mM), 2.5 U Ex Taq (TaKaRa), and PCR-grade water up to 50 μl. An adenylation reaction was performed at 72°C for one hour by a T100 thermal cycler (Bio-Rad). Adenylated products were purified by the QIAquick PCR purification kit (Qiagen, Hilden, Germany), and DNA concentration was measured by the Quantus fluorometer using the QuantiFluor double-stranded DNA (dsDNA) system (Promega). These adenylated products were ligated to a different Y adapter as soon as possible after adenylation. The reaction mixture consisted of 50 μl, with 5 μl ligation buffer (TaKaRa), 1 μl T4 DNA ligase (TaKaRa), 40 μl each adenylated product, one of the Y-type adapters at 100× the molar amount of the adenylated product, and PCR-grade water up to 50 μl. Ligation reactions were performed for 19 h at 16°C and 2 min at 65°C by a T100 thermal cycler (Bio-Rad). Ligation products were purified by the Agencourt AMPureXP system (Beckman Coulter, Brea, CA), and DNA concentration was measured by the Quantus fluorometer using the QuantiFluor dsDNA System (Promega).

To enrich the DNA libraries, a second PCR was performed in a 10-μl reaction mixture with Protocol Phusion high-fidelity PCR master mix with 5 μl HF buffer (New England BioLabs), 0.5 μl Kapa Illumina primer P1 (5′-AAT GAT ACG GCG ACC GA-3′) (10 μM), 0.5 μl Illumina primer P2 (5′-CAA GCA GAA GAC GGC ATA CGA-3′) (10 μM), 3 μl each ligated DNA, and PCR-grade water up to 10 μl. PCR was performed under the following temperature profiles: 30 s at 94°C, 15 cycles of 10 s at 94°C, 30 s at 60°C, 1 min at 72°C, a final extension step of 5 min at 72°C, and storage at 4°C. PCR products were purified using the Agencourt AMPureXP system (Beckman Coulter).

A quality check for all libraries was performed by the Bioanalyzer (Agilent), using the High Sensitivity DNA kit (Agilent). To quantitate the concentration of the library DNA, real-time PCR was performed in a 10-μl reaction volume with 5 μl Kapa SYBR Fast quantitative PCR (qPCR) master mix (Kapa), 0.2 μl Illumina P1 primer (10 μM), 0.2 μl Illumina P2 primer (10 μM), 2 μl 1,000×-diluted library DNA or DNA quantitative standards (Kapa), and 2.6 μl PCR-grade water. Each quantification was performed in triplicate. PCR cycle conditions followed the protocols of the Kapa library quantification kits. Forty-nine libraries were pooled in equal molar amounts. Because the concentration of the pooled library was too low to run MiSeq, this pooled library was concentrated using the Agencourt AMPureXP (Beckman Coulter). The pooled library was again quantified according to the same conditions as the real-time PCR and was adjusted to 2 nM. PhiX control (30%; Illumina) was spiked into the library. Finally, 600 μl of 6 pM denatured library was prepared, and 300-bp paired-end sequencing was carried out by the Advanced Research Support Center at Ehime University using the MiSeq platform (Illumina).

We used a library preparation kit in the second trial that was not used in the 1st trial. The kit saved a lot of time in the library preparation. The amplicon library was prepared using the TruSeq DNA PCR-Free LT library preparation kit (catalog no. FC-121-3001 and FC-121-3003; Illumina, Inc.) on PCR products amplified with phosphorylated primers. Steps from the kit's protocol guide were followed from the adenylation of the 3′ ends to the adapter ligation, with some modifications. The 49 amplicon libraries were then qualified to verify fragment size by checking the library size distribution via the 2100 Bioanalyzer (Agilent) using the High Sensitivity DNA chip. Quantification of the libraries was performed via qPCR using the Kapa library quantification kits for Illumina sequencing platforms (Kapa Biosystems). Prior to sequencing, final amplicon libraries were normalized to 2 nM and pooled. Finally, 600 μl of a 6 pM denatured pooled library with PhiX (30% final concentration) (Illumina, Inc.) was prepared, and a 300-bp paired-end sequencing was performed using the MiSeq reagent kit v3 (600 cycle) (Illumina, Inc.) according to the manufacturer's protocols. Sequencing was carried out at the Division of Analytical Bio-Medicine, Advanced Research Support Center (ADRES), Ehime University (Toon, Ehime Prefecture, Japan). Read data of the pooled libraries were demultiplexed using the on-board MiSEq Reporter software for the sequencing platform.

NGS data were analyzed using CLC Genomics Workbench and Galaxy (https://usegalaxy.org/) platform. The analysis included quality control of contigs, merging of forward and reverse contigs, adapter trimming, mapping into a reference (MNV S7-PP3), and creating the consensus sequences. Reads with fewer than 100 sequences were trimmed. The median of the score of reads of any base quality should be more than 20, measured using FastQC software on the Galaxy platform.

After the consensus sequences were generated, alignment between the consensus sequences in the same region was conducted using MEGA 7 software. In addition to the observation of the other SNPs, including minority sequences and their percentage in NGS reads, IGV (Integrative Genomics Viewer) software was used. A heat map representing the ratio of the percentage of SNPs to the percentage of nonmutated nucleotides in original population sequences in each cycle was determined using R software with a gene scale command available in the bioconductor package (https://www.bioconductor.org/). The Z score for the heat map was calculated by the following formula: Z=(x−mean)SD(1) where x is the ratio of SNP percentages compared to that of the reference sequence in a certain cycle (cycles 1, 5, and 10). The mean value and standard deviation (SD) were calculated from x in all cycles separately per region. In addition, to characterize the evolutionary pattern of genetic divergence among populations with different treatments and treatment cycles, PCoA was conducted using CLC software (49). As the input distance matrix, Bray-Curtis dissimilarity was calculated based on the relative abundances (i.e., reads) of haplotypes in each population (50).

Free chlorine susceptibility of plaque-purified clones.In order to examine whether SNPs found in the capsid region are associated with the lower susceptibility of chlorine, a chlorine sensitivity test was carried out for clones isolated from both the control and chlorine-treated populations. The chlorine-treated and control populations of MNV S7-PP3 were inoculated to confluent RAW 264.7 cells in a 6-well plate with a multiplicity of infection (MOI) of 0.1. After 2 to 3 days of incubation, only clean and dispersed plaques were chosen for isolation. Plaques were picked using 1,000 μl of barrier tips cut by a sterilized cutter to obtain a clear, round shape. Six plaques were picked from each chlorine-treated and control population, mixed with DMEM, and incubated at 37°C for 90 min before being regrown in a new confluent of RAW 264.7 cells in 75-cm² flasks containing 15 ml of DMEM. The first plaque-purified MNV clones were subjected to a second round of plaque purification. Each clone (12 in total, six each from the chlorine-treated and control populations) was confirmed to have the mutation at nt 7280. After the clones were isolated, they were stored in a −80°C freezer before conducting a free chlorine sensitivity test. The free chlorine sensitivity test was conducted using chlorine demand-free glassware, which was prepared by soaking overnight in a high concentration of free chlorine solution (>75 mg/liter). After soaking, the glassware was rinsed and baked at 120°C overnight and then thoroughly washed and sterilized by autoclave. The chlorine sensitivity test was conducted using 9:1 PBS and DMEM with FBS. Free chlorine concentration was measured three times with the U.S. EPA DPD method using DR 900 (Hach) equipment at time zero and after 5 min contact time. The chlorine decay time was determined according to the following formula: Ct=C0exp(−k*t)(2) where C_t is the chlorine concentration at specific sampling times, C₀ is the initial concentration of chlorine, k* is the chlorine decay rate (min⁻¹), and t is the contact time (0, 0.5, 1, 2, 3, or 5 min). A preliminary chlorine decay experiment showed that the free chlorine (initial concentration, 2 ppm) in the PBS solution was fully consumed in 1 min. The chlorine decay rate (k*) calculated using equation 2 was 1.18 min⁻¹. In the chlorine sensitivity assay, an X clone (from the control population) was paired with an A clone (from the chlorine-treated population), and nine pairs were selected randomly. Then, the log₁₀ reduction ratio after free chlorine exposure with an initial concentration of 2 ppm and 1 min contact time was obtained 3 times for each pair. The free chlorine solution was made by mixing 50 μl of sodium hypochlorite stock solution (Wako, Osaka, Japan) with 100 ml PBS. After mixing by shaking for 10 to 15 s, 9 ml of the mixture was transferred to a small tube and 1 ml of clone suspension was added and mixed for 1 min. After 1 min, 10 μl of sodium thiosulphate solution 1% (vol/vol) was added to neutralize the chlorine. The original concentration of plaque-purified clones before and after chlorine treatment was measured by plaque assay.

Replicative fitness of plaque-purified clones.Replicative fitness was determined by comparing the growth rate between plaque-purified clones isolated from control and chlorine-treated populations. Low MOI (0.002 to 0.005) was applied for infection to the RAW 264.7 cells. Confluent RAW 264.7 cells in a 75-cm² flask were rinsed twice with PBS and then infected with the targeting plaque-purified clones. After a 90-min incubation at 37°C, the flasks were rinsed with DMEM to remove the unattached virus particles. Virus samples were taken at 6, 12, 24, 36, and 48 h to obtain the growth curve (51). The growth rate was fitted from the lag phase of virus growth, and the following exponential fitting model was used (52): Y=eμt,so ln(Y)=μt(3) where Y is the virus yield, μ is the growth rate, and t is the cultivation time. To compare the relative replicative fitnesses of the different clones, the selection coefficient (s) was calculated using the following formula (30): s=1−μ/μWT(4) where μ and μ_WT are the log-phase growth rates of the evolved clones (plaque-purified clones isolated from the chlorine-treated population) and wild-type clones (plaque-purified clones isolated from the control population), respectively. An s value less than zero means that the evolved clones has a superior replicative fitness.

Binding efficiency to cell surface.A binding test was carried out according to a previous study with modifications (45). Briefly, RAW 264.7 cells were rinsed twice using PBS without calcium and magnesium [PBS (−)] to remove residual DMEM before the infection process. Virus was inoculated to the cells with a MOI of 0.1 and incubated at 4°C for 90 min to allow the attachment of virus particles, while preventing initiation of the replication cycle at low temperatures. Gentle rocking every 10 to 15 min was employed during the incubation period to prevent the cells from drying. RNA extraction was conducted as soon as incubation was complete. To remove unbound virus particles, the plates were rinsed three times with PBS (−). After rinsing, the cells were scraped using a cell scraper, and then a 150-μl mixture of cells and attached virus was subjected to viral RNA extraction with the QIAamp viral RNA kit (Qiagen). qPCR for MNV was employed to determine the copy numbers of viral RNA before and after the cell-binding experiment (53).

Statistical analyses.All statistical analyses were performed using R software version 3.3 (http://www.r-project.org). All data sets were assumed to not be normally distributed. To examine if the ratio of log₁₀ reduction between clones isolated from the control population (X1, X2, X3) and the chlorine-treated population (A1, A2, A3) is less than 1 (null hypothesis), the Wilcoxon signed-rank test was employed. To determine if there was no statistical difference between the growth rate and the doubling time of each chlorine-treated and control clone, the Kruskal-Wallis test was conducted with a significance level of α = 0.05. To check the statistical difference between the selection coefficients (s) among clone combinations (A1 versus X1, A1 versus X2, A1 versus X3, A2 versus X1, A2 versus X2, A2 versus X3, A3 versus X1, A3 versus X2, and A3 versus X3), the Kruskal-Wallis test was performed. This test examined whether replicative fitness is statistically higher than 0. The ratio of bound viral particles to initial spiked virus between isolated clones (A1, A2, A3, X1, X2, X3) was tested using the Wilcoxon rank sum test.