Comparative characterization of flavivirus production in two cell lines: Human hepatoma-derived Huh7.5.1-8 and African green monkey kidney-derived Vero

Kyoko Saito; Masayoshi Fukasawa; Yoshitaka Shirasago; Ryosuke Suzuki; Naoki Osada; Toshiyuki Yamaji; Takaji Wakita; Eiji Konishi; Kentaro Hanada

doi:10.1371/journal.pone.0232274

. 2020 Apr 24;15(4):e0232274. doi: 10.1371/journal.pone.0232274

Comparative characterization of flavivirus production in two cell lines: Human hepatoma-derived Huh7.5.1-8 and African green monkey kidney-derived Vero

Kyoko Saito ^1,^*, Masayoshi Fukasawa ¹, Yoshitaka Shirasago ¹, Ryosuke Suzuki ², Naoki Osada ^3,⁴, Toshiyuki Yamaji ¹, Takaji Wakita ⁵, Eiji Konishi ⁶, Kentaro Hanada ¹

Editor: Negin P Martin⁷

PMCID: PMC7182267 PMID: 32330205

Abstract

The Flaviviridae is a family of enveloped viruses with a positive-sense single-stranded RNA genome. It contains many viruses that threaten human health, such as Japanese encephalitis virus (JEV) and yellow fever virus (YFV) of the genus Flavivirus as well as hepatitis C virus of the genus Hepacivirus. Cell culture systems highly permissive for the Flaviviridae viruses are very useful for their isolation, propagation, and diagnosis, an understanding of their biology, and the development of vaccines and antiviral agents. Previously, we isolated a human hepatoma HuH-7-derived cell clone, Huh7.5.1–8, which is highly permissive to hepatitis C virus infection. Here, we have characterized flavivirus infection in the Huh7.5.1–8 cell line by comparing with that in the African green monkey kidney-derived Vero cell line, which is permissive for a wide spectrum of viruses. Upon infection with JEV, Huh7.5.1–8 cells produced a higher amount of virus particles early in infection and were more susceptible to virus-induced cell death than Vero cells. Similar outcomes were obtained when the cells were infected with another flavivirus, YFV (17D-204 strain). Quantification of cellular and extracellular viral RNA revealed that high JEV production in Huh7.5.1–8 cells can be attributed to rapid viral replication kinetics and efficient virus release early in infection. In a plaque assay, Huh7.5.1–8 cells developed JEV plaques more rapidly than Vero cells. Although this was not the case with YFV plaques, Huh7.5.1–8 cells developed higher numbers of YFV plaques than Vero cells. Sequence analysis of cDNA encoding an antiviral RNA helicase, RIG-I, showed that Huh7.5.1–8 cells expressed not only a full-length RIG-I mRNA with a known dominant-negative missense mutation but also variants without the mutation. However, the latter mRNAs lacked exon 5/6−12, indicating functional loss of RIG-I in the cells. These characteristics of the Huh7.5.1–8 cell line are helpful for flavivirus detection, titration, and propagation.

Introduction

The Flaviviridae is a family of enveloped viruses with a positive-sense single-stranded RNA genome and contains many pathogenic viruses that threaten human health. The family includes many arthropod- and blood-borne viruses pathogenic to humans: The former is exemplified by members of the genus Flavivirus such as Japanese encephalitis virus (JEV), yellow fever virus (YFV), dengue virus, West Nile virus and Zika virus, whereas the latter, by a member of the genus Hepacivirus, hepatitis C virus (HCV). As for Japanese encephalitis, it is estimated that approximately 70,000 cases occur annually in the 24 endemic countries [1]. The burden of yellow fever in Africa is estimated to be 84,000–170,000 severe cases and 29,000–60,000 deaths per year [2]. Although approved human vaccines already exist for JEV, YFV, and dengue virus, there are no specific antiviral treatments against flavivirus infection so far.

Cell culture systems highly permissive for the Flaviviridae viruses have greatly contributed to their isolation, propagation, and diagnosis, an understanding of their biology, and the development of vaccines and antiviral agents. One representative cell culture system commonly used for flavivirus is the Vero cell line, which was established from African green monkey kidney cells [3, 4]. This cell line is susceptible to various types of viruses [5] as a result of the loss of the type I interferon (IFN) gene cluster [6, 7]. The Vero cell line is used as read-out cells for the plaque reduction neutralization test for dengue virus, as recommended by WHO [8], and other flaviviruses [9]. The cell line is also used as a substrate for the production of various vaccines [10], as exemplified by an inactivated vaccine against JEV (IXARO) [11].

In the case of HCV, two sublines of the human hepatoma HuH-7 cell line [12], Huh7.5 [13] and its derivative Huh7.5.1 [14], are highly permissive. These cell lines along with the HCV JFH1 isolate [15] can be used to reproduce the complete life cycle of the virus, and thereby have been widely used in HCV studies. The high permissiveness of the Huh7.5 and Huh7.5.1 cell lines is partly caused by mutational inactivation of retinoic-acid-inducible protein I (RIG-I), a cellular DExD/H box RNA helicase that senses viral RNA to mediate antiviral signaling, by an amino acid change at codon 55 from threonine to isoleucine (T55I) [16]. This mutation abolishes the interaction between RIG-I and the mitochondria-bound adapter mitochondrial antiviral-signaling protein [17], which is necessary to initiate a signaling cascade leading to the induction of type-I IFNs [18, 19]. Previously, we isolated a cell clone from Huh7.5.1 cells with greater permissiveness to HCV infection [20], because the Huh7.5.1 cells that we used had exhibited heterogeneous permissiveness. The clonal cell line designated as Huh7.5.1–8 produces 10-fold higher amounts of HCV than its parental Huh7.5.1 and shows phenotypically stable permissiveness to HCV infection, which is not changed even after 100 passages. However, the permissiveness of Huh7.5.1–8 cells for flaviviruses, which are distantly-related to HCV, remains to be investigated.

In this study, we have characterized JEV/YFV infection in the Huh7.5.1–8 cell line, compared with that in the Vero cell line as well as other HuH-7 sublines. We found that Huh7.5.1–8 cells exhibited higher virus productivity early in infection and greater susceptibility to virus-induced cell death than Vero cells. We also found that virus plaques developed in Huh7.5.1–8 cells grew rapidly or in large numbers, depending on the virus type. Additionally, the Huh7.5.1–8 cell line was shown to be a RIG-I null mutant. Based on our results, we discussed the potential usefulness of the Huh7.5.1–8 cell line in flavivirus studies.

Materials and methods

Cells

The Huh7.5.1–8 cell line was previously isolated from the Huh7.5.1 cell line, which had been kindly provided by Dr. Francis V. Chisari (The Scripps Research Institute, La Jolla, CA, USA). The HuH-7 (No. JCRB0403) and Vero (No. JCRB9013) cell lines were purchased from the Japanese Collection of Research Bioresources (JCRB) Cell Bank, National Institute of Biomedical Innovation (Osaka, Japan). Huh7.5.1–8, Huh7.5.1, and HuH-7 cells were maintained at 37°C in an atmosphere of 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM; Wako Pure Chemical Industries, Osaka, Japan; No. 044–29765) supplemented with 10% (v/v) fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA; No. 172012-500ML), 0.1 mM nonessential amino acids (Nacalai Tesque, Kyoto, Japan; No. 06344–56), 100 U/ml penicillin G, and 100 μg/ml streptomycin sulfate (Nacalai Tesque; No. 26253–84). Vero cells were maintained at 37°C in an atmosphere of 5% CO₂ in Eagle’s minimal essential medium (EMEM; Wako Pure Chemical Industries; No. 051–07615) supplemented with 5% (v/v) heat-inactivated FBS, 100 U/ml penicillin G, and 100 μg/ml streptomycin sulfate. For comparative analysis, Huh7.5.1–8 and Vero cells were grown in the same DMEM-based medium as above but with heat-inactivated FBS. Before starting the experiments, Vero cells were adapted to this medium through at least eight passages. JEV production in Vero cells was comparable between the DMEM-based and EMEM-based media (S1A Fig).

Viruses

The JEV Nakayama strain (isolated in Japan in 1935) and JEV rAT strain [21] were propagated in Vero cells. The YFV 17D-204 strain, a current vaccine strain derived from the 17D strain [22], was kindly provided by Dr. Tomohiko Takasaki (Department of Virology I, National Institute of Infectious Diseases) and propagated in Vero cells. The resultant culture supernatants were stored at −80°C and used as virus stocks. These virus strains were chosen because they could be used in our biosafety level 2 laboratory.

Infection and post infection conditions

Cells were seeded in 24-well plates (Corning, Corning, NY, USA; No. NCO3524) one day before infection. The cells were incubated with a virus at a multiplicity of infection (MOI) of 0.1 in 0.2 ml of culture medium with reduced serum (2%) at 37°C for 2 h. After the virus-containing medium was removed, the cells were grown in 0.5 ml of complete culture medium. The time of virus removal was taken as 0 h post-infection (h pi) for all experiments. If a post-infection culture was extended beyond 2 days post-infection (d pi), 0.5 ml of fresh medium was added at 2 d pi. Time points expressed in d pi were in the following range (values in parentheses are the mean): 1 d pi, 21−25 h pi (23.0 h pi); 2 d pi, 43−55 h pi (46.2 h pi); 3 d pi, 69−77 h pi (71.1 h pi); and 4 d pi, 92−103 h pi (96.0 h pi).

Plaque assay

For virus titration, a plaque assay was performed on Vero cells according to the manual provided on the website of the National Institute of Infectious Diseases [23] with modifications. In brief, cell monolayers grown in 12-well plates (Corning Costar^®; No. 3513) were inoculated with virus samples in 0.4 ml per well of culture medium with reduced heat-inactivated FBS (2%) and were incubated at 37°C for 2 h. After removal of the samples, the cells were incubated in 2 ml of overlay medium consisting of EMEM (Nissui Pharmaceuticals, Tokyo, Japan; No.05901), 0.22% (w/v) NaHCO₃, 2% (v/v) heat-inactivated FBS, 2 mM L-glutamine (Nacalai Tesque; No. 16948–04), and 1% (w/v) methylcellulose (Tokyo Chemical Industry, Tokyo, Japan; No. M0185) at 37°C for 5 days. The cells were fixed with 10% (v/v) neutral buffered formalin (Wako Pure Chemical Industries; No. 133–10311) and stained with 0.0375% (w/v) methylene blue. Wells with 10−100 plaques were used to calculate the virus titer in plaque formation units (PFU). For the comparative plaque assay, cells were similarly grown and infected, but overlaid with 2.5 ml per well of DMEM (Nissui Pharmaceuticals; No. 05919) supplemented with 0.2% (w/v) NaHCO_3, 2% (v/v) heat-inactivated FBS, 2 mM L-alanyl-L-glutamine (Nacalai Tesque; No. 04260–64), 0.1 mM nonessential amino acids, and 1.25% (w/v) methylcellulose. JEV plaque formation in Vero cells was not so different between the DMEM overlay medium and the EMEM overlay medium (S1B Fig). After fixation and staining, plates were scanned with a document scanner, and image data were obtained (S11 Fig). To improve plaque visibility, an invert filter was applied to the image data, and highlights were adjusted using Photoshop Elements (version 14; Adobe Systems, San Jose, CA, USA).

RNA purification

Total RNA was extracted and purified from cells and culture supernatant using the Blood/Cultured Cell Total RNA Mini Kit (Favorgen Biotech, Pingtung City, Taiwan; No. FABRK 001–2) and the Viral Nucleic Acid Extraction Kit I (Favorgen Biotech; No. FAVNK 001–2), respectively, according to the manufacturer’s instructions.

Quantification of RNA by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR)

To determine the copy numbers of JEV RNA (Nakayama strain; GenBank accession No. EF571853.1), the RNA fraction was subjected to one-step qRT-PCR using the THUNDERBIRD^® Probe One-step qRT-PCR Kit (Toyobo, Osaka, Japan; No. QRZ-101) with primers #1 and 2 and a hydrolysis probe #3 (Table 1). The following reactions were performed on the LightCycler^® 96 system (Roche Applied Science): Reverse transcription at 50°C for 10 min and denaturation at 95°C for 1 min, followed by 40 cycles of 95°C for 15 s and 56°C for 45 s. To normalize cellular JEV RNA levels, the copy numbers of mRNA encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (human, GenBank accession No. NM_002046.3; Chlorocebus sabaeus, GenBank accession No. XM_007967342) in the same RNA samples were determined with primers #6 and 7 and a hydrolysis probe #8 (Table 1; the sequences of #6−8 are homologous to both the human and monkey sequences) under the same qRT-PCR conditions, except that the annealing temperature was 60°C.

Table 1. Primers and probes used in this study.

Target and GenBank accession No.	#	Name	Sequence (base positions in the reference sequence)	Used for:
JEV strain Nakayama EF571853.1	1	JE3-1357-1379-F	`5’-GGAGAACAATCCAGCCAGAAAAC-3’`	qRT-PCR
	1	JE3-1357-1379-F	(bases 1357 to 1379)	qRT-PCR
	2	JE3en-1470-1494-R	`5’-CATTGGGTGTTACTGTAAACTTTGC-3’`	qRT-PCR
	2	JE3en-1470-1494-R	(complementary to bases 1470 to 1494)	qRT-PCR
	3	JE3-1427-1457-Taq	`5’-[FAM]-AAACCATGGGAATTATTCAGCGCAAGTTGGG-[TAMRA]-3’`	qRT-PCR
	3	JE3-1427-1457-Taq	(bases 1427 to 1457)	qRT-PCR
	4	JE2491-R23	`5’-GCACATCCAGTGTCAGCATGCAC -3’`	cDNA amplification
	4	JE2491-R23	(complementary to bases 2469 to 2491)	cDNA amplification
	5	JE962-F24	`5’-CGCTCCGGCTTACAGTTTCAACTG-3’`	cDNA amplification
	5	JE962-F24	(bases 962 to 985)	cDNA amplification
Human GAPDH NM_002046.3	6	Cs_GAPDH-F	`5’-AGGTCGGAGTCAACGGATTTG-3’`	qRT-PCR
	6	Cs_GAPDH-F	(bases 116 to 136)	qRT-PCR
	7	Cs_GAPDH-R	`5’-GGTCATTGATGGCAACAATATCCA-3’`	qRT-PCR
	7	Cs_GAPDH-R	(complementary to bases 195 to 208)-	qRT-PCR
	8	Cs_GAPDH-P	`5’-[FAM]-TGGTCACCAGGGCTGCTTTAACTCTGG-[TAMRA]-3’`	qRT-PCR
	8	Cs_GAPDH-P	(bases 152 to 179)	qRT-PCR
	9	GAPDH-HA-IF1	`5’-TGGCGGCCGCGAATTCACCATGGGGAAGGTGAAG-3’`	cDNA amplification
	9	GAPDH-HA-IF1	(bases 100 to 133)	cDNA amplification
	10	GAPDH-HA-IF2	`5’-CATATGGGTAAGATCTCTCCTTGGAGGCCATGTG-3’`	cDNA amplification
	10	GAPDH-HA-IF2	(complementary to bases 1074 to 1107)	cDNA amplification
Human RIG-I AF038963.1	11	Sumpter F	`5’-GTCCGGCCTCATTTCCTCGGAAAATC-3’`	cDNA amplification [16]
	11	Sumpter F	(bases 34 to 69)	cDNA amplification [16]
	12	Sumpter R	`5’-GGTACAAGCGATCCATGATTATACCCACTATGTTTG-3’`	cDNA amplification [16]
	12	Sumpter R	(complementary to bases 2992 to 3027)	cDNA amplification [16]
	13	216_F21	`5’-TGGACCCTACCTACATCCTGA-3’`	Sequencing
	13	216_F21	(bases 216 to 236)	Sequencing
	14	439_R21	`5’-GAAATCCCAACTTTCAATGGC-3’`	Sequencing
	14	439_R21	(complementary to bases 419 to 439)	Sequencing
	15	601_F23	`5’-GATGGCAGGTGCAGAGAAATTGG-3’`	Sequencing
	15	601_F23	(bases 601 to 623)	Sequencing
	16	1390_F23	`5’-GGTTGGTGTTGGGGATGCCA-3’`	Sequencing
	16	1390_F23	(bases 1390 to 1412)	Sequencing
	17	160_R21	`5’-GTGTCCCTCATCAGCTGAGC-3’`	Sequencing
	17	160_R21	(complementary to bases 1582 to 1602)	Sequencing
	18	1978_R20	`5’-CTCATTGCTGGGATCCCTGG-3’`	Sequencing
	18	1978_R20	(complementary to bases 1959 to 1978)	Sequencing
	19	2129_F22	`5’-CCTGGCATATTGACTGGACGTG-3’`	Sequencing
	19	2129_F22	(bases 2129 to 2150)	Sequencing
	20	2989_R22	`5’-TCTTCTCCACTCAAAGTTACTC-3’`	Sequencing
	20	2989_R22	(complementary to bases 2968 to 2989)	Sequencing

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A standard curve was established using serial dilutions of plasmids encoding the JEV E protein (bases 962 to 2491) or human GAPDH. For construction of the plasmid encoding the JEV E protein, the RNA fraction prepared from a JEV (Nakayama strain) stock was reverse transcribed with primer #4 (Table 1) using the PrimeScript^™ II 1st Strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan; No. 6210A). The first strand cDNA was amplified on a PCR Thermal Cycler Dice (Takara Bio; also used for all the PCR reactions below) using PrimeSTAR^® MAX DNA polymerase (Takara Bio; No. R045A) and primers #4 and 5 (Table 1) under the following conditions: 35 cycles of 98°C for 10 s, 56°C for 5 s, and 72°C for 90 s. The amplicon was cloned into a T-vector pMD20 (Takara Bio; No. 3270) using the Mighty TA-cloning Reagent Set for PrimeSTAR^® (Takara Bio; No. 6019). For construction of the plasmid encoding human GAPDH, the total RNA fraction from Huh7.5.1–8 cells was reverse transcribed with an oligo-dT primer using the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Penzberg, Upper Bavaria, Germany; No. 04379012001). GAPDH cDNA was obtained by PCR using primers #9 and 10 (Table 1) at 35 cycles of 98°C for 10 s, 58°C for 5 s, and 72°C for 60 s and cloned into the EcoRI−BglII sites of plasmid pBSnFLcHA [24]. Both constructs were purified by the PureYield^™ Plasmid Miniprep System (Promega, Madison, WI, USA; No. A1222), and their sequences were verified by Sanger sequencing.

Cloning and sequencing of RIG-I (DDX58) cDNA

Total RNA from cells was reverse transcribed with an oligo dT primer using the PrimeScript^™ II 1st Strand cDNA Synthesis Kit (described above). The first strand cDNA was used as a template to amplify a cDNA fragment encoding RIG-I (DDX58) (GenBank accession No. AF038963.1; bases 34 to 3027) by PCR using primers #11 and 12 (Table 1) and PrimeSTAR^® GXL DNA polymerase (Takara Bio; No. R050A) at 35 cycles of 98°C for 10 s, 60°C for 15 s, and 68°C for 3 min. The resultant amplicons were separated on a 0.7% agarose gel, purified using the Gel/PCR Extraction Kit (FastGene, NIPPON genetics, Tokyo, Japan; No. FG-91302), and then directly sequenced using primers #13 and 14 (Table 1). The amplicons were cloned into pCR^®Blunt II-TOPO^® (Thermo Fisher Scientific, Waltham, MA, USA; No. K2800-20), purified, and sequenced with primers #11−20 (Table 1). Upon sequence determination, nucleotide variations were excluded that were not consistent with the RNA-seq data from Huh7.5.1 (DRR018792) and Huh7.5.1–8 (DRR018793) [20] or the whole-genome sequence data from HuH-7 and Huh7.5.1–8 (DDBJ DRA database under the project ID PRJDB7928) [25].

Other methods

All the experiments were done with three biological replicates and repeated as described in figure legends. Cell viability was determined using the Cell Proliferation Kit II XTT (Roche Applied Science; No. 11 465 015 001) according to the manufacturer’s instructions. Statistical analysis was performed using an online calculator, Quick Calcs, provided on the website of GraphPad (https://www.graphpad.com/quickcalcs/), or GraphPad Prism version 7.03 (GraphPad Software, San Diego, CA, USA).

Results

Comparison of JEV production between Huh7.5.1–8, Huh7.5.1, and HuH-7 cells

First, the Huh7.5.1–8 cell line was compared with the parental Huh7.5.1 and the ancestral HuH-7 cell lines regarding JEV production. Cells were infected with JEV (Nakayama strain, unless otherwise noted), and then the amount of infectious virus particles (titer) in culture supernatant and JEV-induced cell death were monitored during 1−4 d pi. The relative JEV titer of these cell lines increased and peaked at 2−3 d pi in a similar manner (Fig 1A). Under the same seeding conditions, doubling times of Huh7.5.1–8, Huh7.5.1, and HuH-7 cells were not significantly different (after Bonferroni correction, p > 0.0167) (S2B Fig). Thus, JEV production appeared to be comparable between the three cell lines and not enhanced in this lineage unlike for HCV production. In a parallel cell viability assay (Fig 1B), susceptibility to JEV-induced cell death was not greatly different between the three cell lines. Next, these cell lines were infected with JEV, overlaid with methylcellulose-containing medium, and then compared regarding JEV plaque formation. Huh7.5.1–8 and Huh7.5.1 cells similarly developed JEV plaques, whereas HuH-7 cells began to die at 3 d pi and detached from the surface of the well irrespective of infection at 5 d pi (Fig 1C). These results indicated that the Huh7.5.1–8 and Huh7.5.1 cell lines are better than the HuH-7 cell line for a JEV plaque assay. Although Huh7.5.1–8 and Huh7.5.1 cell lines were comparable in terms of JEV infection, the Huh7.5.1–8 cell line has the advantage of being phenotypically more stable than the Huh7.5.1 cell line [20]. Thus, we hereafter focused on the Huh7.5.1–8 cell line and compared it with the Vero cell line regarding JEV production.

Fig 1 — (A, B) Cells were seeded at 1 x 10⁵ cells per well of a 24-well plate one day before infection and then infected with JEV at MOI 0.1. Culture supernatants were harvested at the indicated times to determine virus titers in PFU per well. Panel A shows the relative JEV titer calculated by dividing each titer value by that of HuH-7 cells at 1 d pi from the same experiment (actual value, [4.90−10.5] × 10⁵ PFU/well). Panel B shows cell viability expressed as a % of that of mock-infected cells. For both panels, bars with error bars represent the mean ± standard deviation (SD) of three independent experiments. Statistical significance between cell lines was determined by a one-sample t test (for comparison with a hypothetical value,1) or an unpaired two-tailed t test using Bonferroni correction and p values less than 0.0167 were considered statistically significant. ns, not significant. (C) Cells were seeded at 4 x 10⁵ cells per well of a 12-well plate one day before infection and then infected with JEV at 40 PFU (determined with Vero cells) per well. The cells were fixed and stained at 3−5 d pi. Values given below the images are plaque numbers expressed as the mean ± SD of triplicates from one representative experiment. Statistical significance was determined as described above. Values in parentheses are p values, and those less than 0.0167 were considered statistically significant. Similar results were obtained in two other independent experiments (S3 Fig).

JEV production in Huh7.5.1–8 and Vero cell lines

For comparative analysis, we used the Vero cell subline JCRB9013, in which JEV production was almost equivalent to or slightly higher than that in other Vero cell sublines (S4 Fig). Huh7.5.1–8 and Vero cells under high and low confluency were infected with JEV at MOI 0.1, and then the virus titer in culture supernatant was monitored from 1 to 4 d pi (Fig 2). The culture supernatant of Huh7.5.1–8 cells exhibited a higher relative virus titer than that of Vero cells, particularly during the early infection times. The difference in the relative virus titer was remarkable in high-cell-density infection (Fig 2A) compared with low-cell-density infection (Fig 2B), implying that cell-cell contacts promote viral spread in Huh7.5.1–8 cells. The difference in the titer was not accounted for by the growth rates of the two cell lines, because their doubling times under high confluency conditions were nearly equal (S2 Fig). A parallel viability assay showed that Huh7.5.1–8 cells lost viability faster than Vero cells irrespective of cell density (Fig 3). The same trends were observed in JEV production and cell viability at smaller MOI (0.01), although high confluency appeared to be needed (S5 Fig). These results showed that JEV production in the Huh7.5.1–8 cell line is higher than that in the Vero cell line during early infection times and that the Huh7.5.1–8 cell line is more susceptible to JEV-induced cell death than the Vero cell line.

Fig 2 — (A, B) Cells were seeded at 1 x 10⁵ (A; high confluency conditions) or 2 x 10⁴ (B; low confluency conditions) cells per well of a 24-well plate one day before infection and then infected with JEV at MOI 0.1. Culture supernatants were harvested at 1−4 d pi, and virus titers in the supernatants were determined by plaque assay. Each symbol represents the relative titer value calculated by dividing each titer value in PFU/well by that of Vero cells at 1 d pi in the same experiment. Bars with error bars represent the mean ± SD of the relative values from multiple independent experiments. Because the 4 d pi time point was omitted in several experiments, numbers of experiments differ at 1−3 d pi and at 4 d pi: A, n = 8 [1−3 d pi] or 5 [4 d pi]; B, n = 5 [1−3 d pi] or 3 [4 d pi]. Statistical significance was determined by a one-sample t test (1 d pi) or unpaired two-tailed t test (2−4 d pi). Values in parentheses indicate p values, and those less than 0.05 were considered statistically significant. Actual titers in PFU/well for Vero cells at 1 d pi were (8.75−24.6) × 10⁴ (A) and (2.73−10.2) × 10³ (B). Huh, Huh7.5.1–8 cells.

Fig 3 — Cells were seeded and infected with JEV at MOI 0.1 under high (A) and low (B) confluency conditions as described in the legend to Fig 2. Cell viability was determined at 2−4 d pi and expressed as % of viability of mock-infected cells. Bars with error bars represent the mean ± SD of three independent experiments. Statistical significance between Huh7.5.1–8 cells (Huh) and Vero cells was determined by an unpaired two-tailed t test. Values in parentheses are p values, and those less than 0.05 were considered statistically significant.

Particle-to-PFU ratio, replication kinetics, and virus release

The particle-to-PFU ratio was calculated for Huh7.5.1-8- and Vero-derived JEV particles, and the ratio of the values of both particles was determined (Fig 4). Both virus particles showed a similar particle-to-PFU ratio, ruling out the possibility that Huh7.5.1-8-derived virus particles had a higher infectivity than Vero-derived ones. The reason for large variations found at 2 and 3 d pi remains unknown, but that at 3 d pi may have been partly due to variable amounts of non-infectious viral RNA leaking from dead Huh7.5.1–8 cells (Fig 3). In a representative experiment (S6 Fig), the actual value of the particle-to-PFU ratio was minimally ~600, which is roughly consistent with a previous report [26]. Taken together with the results of Fig 2, these results suggested that Huh7.5.1–8 cells produce a higher amount of JEV particles than Vero cells during early infection times.

Fig 4 — Cells were infected with JEV at MOI 0.1 under high confluency conditions as described in the legend to Fig 2, and the culture supernatants were harvested at the indicated times. RNA was extracted and purified from each culture supernatant, and viral RNA copy number in the supernatant was determined by qRT-PCR and regarded as the number of virus particles. A virus titer in PFU of each culture supernatant was also determined by plaque assay, and particle-to-PFU ratio was calculated. Each symbol represents the relative value calculated by dividing the value of Huh7.5.1-8-derived JEV by that of Vero-derived JEV obtained from the same experiment. Bars with error bars represent the mean ± SD of the ratio from eight (1 and 2 d pi) or seven (3 d pi) independent experiments (the reason for the different sample sizes was that one experiment lacked data for 3 d pi). Statistical significance was determined by a one-sample t test. Values in parentheses indicate p values, and those less than 0.05 were considered statistically significant.

Next, we compared the JEV replication kinetics between Huh7.5.1–8 and Vero cells by monitoring intracellular and extracellular viral RNA levels during 0−73 h pi. An initial rise in intracellular viral RNA level in Huh7.5.1–8 cells was detected as early as 9 h pi, whereas the rise for Vero cells was not remarkable during 0–12 h pi (Fig 5A [a]). Similarly, the level of extracellular viral RNA from Huh7.5.1–8 cells rose earlier than that from Vero cells (Fig 5B [a]). Although the level of intracellular viral RNA was not different between the two cell lines after 12 h pi (Fig 5A [b]), the level of extracellular viral RNA was higher for Huh7.5.1–8 cells than for Vero cells during 12−48 h pi (Fig 5B [b]). Furthermore, the relative amount of extracellular viral RNA expressed as a percentage of the total (extracellular plus intracellular) amount of viral RNA was about 2-fold higher in Huh7.5.1–8 cells than in Vero cells at 1 d pi (Fig 6). Thereafter, the value for Huh7.5.1–8 cells varied with each experiment, possibly due to variation in virus-induced cell death, but leading to no difference between the values for Huh7.5.1–8 and Vero cells. Thus, Huh7.5.1–8 cells may release JEV more efficiently than Vero cells at least at 1 d pi. Collectively, these results suggested that rapid viral replication kinetics and efficient early virus release contribute to high JEV production in Huh7.5.1–8 cells.

Fig 5 — Cells were infected with JEV at MOI 0.1 under high confluency conditions as described in the legend to Fig 2. Cells and culture supernatants were harvested at 0, 6, 9, 12, 24, 48, and 73 h pi, and RNA was purified. Viral RNA copy number in each RNA fraction was determined by qRT-PCR. The copy number of intracellular viral RNA was normalized by that of *GAPDH* RNA in the same fraction, whereas the amount of extracellular viral RNA was expressed as the total copy number in the culture supernatant of each well. The relative amounts of intracellular (A) and extracellular (B) viral RNA were calculated by dividing each value by that of Vero cells at 0 h pi in the same experiment. Data represent the mean ± SD of the relative values from four independent experiments. Graph (a) is an enlarged view of 0−12 h pi drawn on a linear scale, whereas graph (b) is a whole view drawn on a log scale. Some error bars are not visible due to their small size. Statistical significance was determined by a one-sample t test (0 h pi) or an unpaired two-tailed t test (other time points). Values in parentheses indicate p values, and those less than 0.05 were considered statistically significant. Huh, Huh7.5.1–8 cells.

Fig 6 — Cells were infected with JEV at MOI 0.1 under high confluency conditions as described in the legend to Fig 2. Cells and culture supernatant were harvested at the indicated times. RNA was extracted and purified, and the viral RNA copy number in each RNA fraction was determined by qRT-PCR. The extracellular amount of viral RNA was expressed as a percentage of the total (extracellular plus intracellular) amount of viral RNA. Data represent the mean ± SD of three independent experiments. Each symbol represents the mean from one experiment. Statistical significance was determined by an unpaired two-tailed t test. Values in parentheses indicate p values, and those less than 0.05 were considered statistically significant.

JEV plaque formation

JEV plaque formation (size and number) were compared between Huh7.5.1–8 and Vero cell lines at 3−5 d pi. As shown in Fig 7A and S7A Fig, clearly visible plaques were developed in Huh7.5.1–8 cells at as early as 3 d pi, but not in Vero cells at that time. In the course of the infection, plaques in Huh7.5.1–8 cells grew more rapidly than those in Vero cells. Similar results were observed when the cells were infected with another JEV strain, rAT (Fig 7B and S7B Fig). These results showed that the Huh7.5.1–8 cell line supports more rapid JEV plaque formation than the Vero cell line. The rapid plaque formation in Huh7.5.1–8 cells appears to reflect their high JEV productivity and susceptibility to JEV-induced cell death. The plaque numbers of the Nakayama strain at 5 d pi were comparable between the two cell lines (Fig 7A and S7A Fig), whereas those of the rAT strain tended to be higher in the Huh7.5.1–8 cell line than in the Vero cell line (Fig 7B and S7B Fig). These results indicated that the difference in plaque numbers between the two cell lines varies by each virus strain.

YFV production

To compare the production of another flavivirus, Huh7.5.1–8 and Vero cells were infected with the YFV vaccine strain (17D-204), and then the virus titer in culture supernatant and YFV-induced cell death were monitored under high and low confluency during 1−4 d pi. The culture supernatant of Huh7.5.1–8 cells exhibited a higher YFV titer than that of Vero cells during early infection times, as shown by three independent experiments (Fig 8A and 8B and S8 Fig). Because variations between experiments performed on different days were too large, the YFV titer ratio of Huh7.5.1–8 cells to Vero cells was calculated on each time point, and the mean of the three experiments was taken (Fig 8C and 8D). The combined data showed that virus yield in Huh7.5.1–8 cells was significantly higher than that in Vero cells for high confluency at 2 d pi and low confluency at 1 and 2 d pi. Furthermore, Huh7.5.1–8 cells were more susceptible to YFV-induced cell death than Vero cells (Fig 8E and 8F), as observed with JEV. These results suggested that the Huh7.5.1–8 cell line, compared with the Vero cell line, has a higher virus productivity and susceptibility to virus-induced cell death upon flavivirus infection. Next, we compared the YFV plaque formation between the two cell lines (Fig 9 and S9 Fig). YFV plaques began to appear at 4 d pi in both cell lines. Although plaques in Huh7.5.1–8 cells were slightly smaller than those in Vero cells, Huh7.5.1–8 cells developed a several fold higher number of YFV plaques than Vero cells. These results suggested that the high YFV productivity and susceptibility to YFV-induced cell death of Huh7.5.1–8 cells can be mainly attributed to their high infection efficiency. Although YFV plaques developed in Vero cells at 4 d pi in Fig 9, no cell death was observed with these cells at this time point in Fig 8E and 8F. A possible explanation for this discrepancy in cell death may be the difference in culture conditions: The serum concentration was 10% for Fig 8 and 2% for Fig 9. In addition, the medium for Fig 9 contained methylcellulose. These differences may have affected Vero cell death. In the case of JEV, the lower the serum concentration is, the more cell death occurs [27].

Fig 8 — Cells were seeded and infected with YFV 17D-204 at MOI 0.1 under high (A, C, E) and low (B, D, F) confluency conditions as described in the legend to Fig 2. (A, B) Culture supernatants were harvested at 23, 47, 74, and 98 h pi, and virus titers in the supernatants were determined by plaque assay. Each point represents the mean ± SD of triplicates from one representative experiment. Some error bars are not visible due to their small size. Similar results were obtained in two other independent experiments (S8 Fig). (C, D) The titer ratio was calculated by dividing the titer value of Huh7.5.1–8 cells by that of Vero cells at each time point. Bars with error bars represent the mean ± SD of three independent experiments (A, B and S8 Fig). Each symbol represents the mean from one experiment. (E, F) Cell viability was determined at the indicated times. Bars with error bars represent the mean ± SD of three independent experiments. Statistical significance was determined by an unpaired two-tailed t test (A, B, E, F) or a one-sample t test (C, D). Values in parentheses indicate p values, and those less than 0.05 were considered statistically significant. Huh, Huh7.5.1–8 cells.

Fig 9 — Representative black-and-white inverted images of YFV plaques in Huh7.5.1–8 (Huh) and Vero cells. Cells were infected with YFV (17D-204 strain) at either 40 or 16 PFU (determined with Vero cells) per well and then fixed and stained at the indicated times. Values below the images indicate plaque numbers expressed as the mean ± SD of triplicates from one representative experiment. Statistical significance was determined by an unpaired two-tailed t test. Values in parentheses indicate p values, and those less than 0.05 were considered statistically significant. Similar results were obtained in two other independent experiments (S9 Fig). N.D., not determined.

Confirmation of RIG-I sequence

Recently, codon 55 in the RIG-I gene from the Huh7.5.1–8 cell line was found to be heterozygous: Mutant-type ATA (Ile) and wild-type ACA (Thr) [25]. To confirm whether the Huh7.5.1–8 cell line is defective in RIG-I, we amplified the RIG-I cDNA derived from total RNA isolated from HuH-7, Huh7.5.1 and Huh7.5.1–8 cells. An amplicon corresponding to the expected size for the RIG-I sequence (~ 3 kbp) and another, with a smaller size (~ 2 kbp), were detected in the three cell lines (Fig 10A) and directly sequenced (Fig 10B). The 3-kbp and 2-kbp cDNAs from the HuH-7 cell line exclusively contained the wild-type codon 55. In contrast, the 3-kbp cDNAs from the Huh 7.5.1 and Huh7.5.1–8 cell lines contained the mutant-type codon 55, whereas the 2-kbp cDNAs from these cell lines contained the wild-type codon 55. Cloning and sequencing of the cDNAs of HuH-7 and Huh7.5.1–8 cell lines revealed that the 3-kbp cDNAs were full-length (FL1, 2), whereas the 2-kbp cDNAs lacked exons 6−12 (SV1 found in both cell lines) or 5−12 (SV2 found in Huh7.5.1–8) (Fig 10C and S10 Fig). RIG-I protein isoforms deduced from the splice variants (SV1, 2) completely lacked a helicase ATP-binding domain, indicating that they are defective like RIG-I protein with a mutated ATP-binding site [28]. These results were consistent with the above findings [25] and indicated that Huh7.5.1–8 is a RIG-I null mutant cell line.

Fig 10 — (A) Agarose gel image of RT-PCR products amplified with *RIG-I* specific primer pairs (Sumpter F and R, Table 1). M: molecular size markers. (B) Sequence chromatograms of the *RIG-I* cDNAs. Each yellow arrow indicates a nucleotide corresponding to the second base of codon 55 (base 321) of the *RIG-I* reference sequence (AF038963.1). F, forward direction; R, reverse direction. (C) Schematic representations of *RIG-I* full-length (FL) and splice variant (SV) cDNAs. The reference sequence is shown at the top. Protein domains are shown in different patterns with boundary positions: CARD, caspase activation and recruitment domain; RLR CTR, RIG-I-like receptor, C-terminal regulatory domain. Nucleotides that differed from the reference sequence are shown by red squares. The two Gs at bases 52−53 in the cDNAs were derived from the forward PCR primer used. The A/T variation at base 1897 is annotated in the single nucleotide polymorphism database (rs17217280). FL1 and SV1 sequences from HuH-7 cells were obtained from five and eight independent clones, respectively. FL2, SV1, and SV2 sequences from Huh7.5.1–8 cells were obtained from six, seven, and three independent clones, respectively. +, detected; -, not detected. The complete nucleotide sequences are shown in S10 Fig.

Discussion

Although the Huh7.5.1–8 cell line is more permissive to HCV infection than the Huh7.5.1 cell line [20], we found that this was not the case with JEV infection: The Huh7.5.1–8 and the parental Huh7.5.1 cell lines were comparable in terms of JEV productivity, susceptibility to JEV-induced cell death, and JEV plaque formation (Fig 1). Even the ancestral HuH-7 cell line was comparable except for plaque formation. These findings indicate that genetic factors contributing to the high permissiveness of the Huh7.5.1–8 cell line to HCV infection are specific rather than broadly proviral. One such genetic factor might be involved in stable HCV receptor expression [20]. Unlike the Huh7.5.1–8 and Huh7.5.1 cell lines, the HuH-7 cell line was not maintained under our culture conditions for the plaque assay (Fig 1C), likely because our HuH-7 cells had a lower fitness to cell culture than Huh7.5.1–8 and Huh7.5.1 cells.

Here, we found three differences in outputs of flavivirus infection between Huh7.5.1–8 and Vero cell lines. First, Huh7.5.1–8 cells produced higher amounts of infectious JEV and YFV than Vero cells early in infection (Figs 2 and 8A–8D). In the case of JEV, the high virus productivity of Huh7.5.1–8 cells may be attributed not to the increase in infectivity of a virus particle (Fig 4), but to viral rapid replication kinetics and efficient virus release early in infection (Figs 5 and 6). Therefore, Huh7.5.1–8 cells are more suitable than Vero cells for obtaining large amounts of flavivirus in a short period of time. Recently, HuH-7 cells were shown to produce higher amounts of Zika virus than other cells including Vero E6 cells (ATCC CRL-1586) early in infection [29]. Considering that JEV production in Huh7.5.1–8 cells and the ancestral HuH-7 cells were comparable (Fig 1A), high virus productivity early in flavivirus infection may be a common feature among the HuH-7 lineage. In addition, a similar trend was observed when HuH-7 cells were infected with Middle East respiratory syndrome coronavirus [30]. Thus, the feature may not be limited to flavivirus infection.

Second, we found that Huh7.5.1–8 cells were more susceptible to JEV/YFV-induced cell death than Vero cells (Figs 3, 8E and 8F). These characteristics of the Huh7.5.1–8 cell line may facilitate cell viability-based screenings of antiviral host factors and agents for flaviviruses. Meanwhile, the potent cell death observed in Huh7.5.1–8 cells appears to be a part of the reason for plateaued or reduced virus production late in infection. Therefore, genetic engineering of the Huh7.5.1–8 cell line to make it less susceptible to virus-induced cell death might yield a cell line with more flavivirus production.

Third, we found that Huh7.5.1–8 cells developed JEV plaques rapidly (Fig 7) and formed a larger number of YFV plaques (Fig 9) compared with Vero cells, though the difference in plaque numbers between the two cell lines varied by each virus. The JEV plaque phenotype of Huh7.5.1–8 cells appears to reflect their high virus productivity and susceptibility to virus-induced cell death. Because a plaque assay is somewhat time-consuming, the plaque phenotype observed with JEV infection in Huh7.5.1–8 cells can be utilized to shorten the period of plaque assay. In addition, the high-number plaque phenotype observed with YFV infection in Huh7.5.1–8 cells can be utilized to improve the sensitivity of detection and titration of YFV by plaque assay. Vero cells may be more suitable to viruses requiring long-term cultivation to grow plaques, because they were more tolerant to our plaque assay conditions than Huh7.5.1–8 cells. In addition, the higher tolerance of Vero cells to flavivirus-induced cell death, compared with Huh7.5.1–8 cells, may be a feature that makes the Vero cell line advantageous for vaccine production.

On top of these findings, we provided evidence that Huh7.5.1–8 is a RIG-I null mutant cell line. Our results suggested that, in a Huh7.5.1–8 cell, one allele of the RIG-I gene generates full-length mRNA with a T55I mutation that abolishes RIG-I-mediated antiviral signaling, whereas the other allele generates defective splice variants without the mutation. One of the splice variants is also expressed in the parental HuH-7 cell line. Codon 55 of RIG-I gene of Huh7.5.1 cells was heterozygous as was that of Huh7.5.1–8 cells, showing that the heterozygosity of the codon was stable at least during the time span for isolation of the Huh7.5.1–8 clone from Huh7.5.1 cells. At present, the underlying mechanisms that generate splice variants and their impacts on RIG-I signaling remain to be studied. Although JEV has been restricted by RIG-I in a mouse model [31] and mouse cell lines [32, 33], JEV production and susceptibility to JEV-induced cell death were not dramatically altered in the Huh7.5.1–8 and Huh7.5.1 cell lines compared with the HuH-7 cell line (Fig 1A and 1B), implying that the RIG-I pathway may not work or may be counteracted by viral mechanisms [34] under our experimental conditions.

In conclusion, our study highlighted the characteristics of the Huh7.5.1–8 cell line that are helpful for improvement of flavivirus propagation, detection, and titration. Further study is needed to investigate the potential versatility of the Huh7.5.1–8 cell line in virus research.

Supporting information

S1 Fig. JEV production and plaque formation in Vero cells grown in different media.

(A) Vero cells (No. JCRB9013) were seeded at 1 x 10⁵ cells per well of a 24-well plate one day before infection in DMEM supplemented with 10% (v/v) heat-inactivated FBS, 0.1 mM nonessential amino acids, 100 U/ml penicillin G, and 100 μg/ml streptomycin sulfate. After infection with JEV (Nakayama strain) at MOI 0.1, the cells were grown in the same medium (shown as closed circles) or EMEM-based medium as described in “Materials and methods” (shown as open squares). Culture supernatant was harvested at the indicated times, and RNA was extracted and purified to determine viral RNA copy number by qRT-PCR. Each point represents the mean ± SD of triplicates from one representative experiment. (B) Vero cells were infected with JEV and then incubated with 2.5 ml per well of DMEM overlay medium (DMEM supplemented with 0.2% [w/v] NaHCO_3, 2% [v/v] heat-inactivated FBS, 2 mM L-alanyl-L-glutamine, 0.1 mM nonessential amino acids, and 1.25% [w/v] methylcellulose) or EMEM overlay medium (EMEM supplemented with 0.22% [w/v] NaHCO₃, 2% [v/v] heat-inactivated FBS, 2 mM L-glutamine, and 1% [w/v] methylcellulose). The cells were fixed and stained at the indicated times. Values below the images are plaque numbers expressed as the mean ± SD of triplicates from one representative experiment. For both panels, statistical significance was determined by an unpaired two-tailed t test. Values in parentheses indicate p values, and those less than 0.05 were considered statistically significant. Similar results were obtained in another independent experiment. N.D., not detected.

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S2 Fig. Comparison of cell growth between Huh7.5.1–8, Huh7.5.1, HuH-7, and Vero cells, related to Figs 1 and 2.

(A) Cells were seeded at 1 x 10⁵ cells per well of a 24-well plate. Cells were harvested at the indicated times and counted using the TC20 Automated Cell Counter (Bio-Rad). Results from three independent experiments are shown. (B) The doubling time of each cell line was calculated from the slope of the above graphs from day 1 to day 3 by linear regression analysis on GraphPad Prism (ver. 7.03) and then converted to a value relative to that of Huh7.5.1–8 cells. Bar with error bars represent the mean ± SD of the relative doubling times from three independent experiments. Statistical significance was determined by a one-sample t test with Bonferroni correction. Values in parentheses are p values, and those less than 0.0167 were considered statistically significant.

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S3 Fig. Comparison of JEV plaque formation between Huh7.5.1–8, Huh7.5.1, and HuH-7 cells, related to Fig 1C.

Cells were seeded at 4 x 10⁵ cells per well of a 12-well plate one day before infection and then infected with JEV at 40 PFU per well. The cells were fixed and stained at 3−5 d pi. Panels A and B represent different experiments. Values given below the images are plaque numbers expressed as the mean ± SD of triplicates from each experiment. Statistical significance was determined by an unpaired two-tailed t test with Bonferroni correction. Values in parentheses are p values, and those less than 0.0167 were considered statistically significant.

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S4 Fig. Comparison of JEV production among Vero cell sublines.

Vero ATCC CCL-81, Vero 76 (No. IFO50410), Vero 0111 (No. JCRB0111), and Vero C1008 (ATCC CRL-1586) cells were obtained from the National Institute of Biomedical Innovation (Osaka, Japan) as described (Sakuma C, Sekizuka T, Kuroda M, Kasai F, Saito K., Ikeda M, et al. Novel endogenous simian retroviral integrations in Vero cells: implications for quality control of a human vaccine cell substrate. Sci Rep, 2018; 8(1); 644. doi:10.1038/s41598-017-18934-2). Cells were seeded at 2 x 10⁴ cells per well of a 24-well plate one day before infection and then infected with JEV (Nakayama strain) at MOI 0.1. Culture supernatant was harvested at the indicated times to determine virus titers by plaque assay. Each point represents the mean ± SD of triplicates from one representative experiment. Statistical significance was determined by one-way ANOVA with Dunnett's multiple-comparison post-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant. Similar results were obtained in another independent experiment.

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S5 Fig. Comparison of JEV infection at MOI 0.01 between Huh7.5.1–8 and Vero cells, related to Figs 2 and 3.

Cells were seeded and infected with JEV at MOI 0.01 under high (A, C) and low (B, D) confluency conditions as described in the legend to Fig 2. (A, B) Culture supernatants were harvested at the indicated times, and virus titers in the supernatants were determined by plaque assay. Results from two independent experiments (a, b) are shown. (C, D) Cell viability was determined at the indicated times and expressed as % of viability of mock-infected cells. Bars with error bars represent the mean ± SD of three independent experiments. For all panels, statistical significance was determined by an unpaired two-tailed t test. Values in parentheses are p values, and those less than 0.05 were considered statistically significant. Huh, Huh7.5.1–8 cells.

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S6 Fig. Particle-to-PFU ratio of Huh7.5.1-8-derived and Vero-derived JEV, related to Fig 4.

Cells were infected with JEV at MOI 0.1 under high confluency conditions as described in the legend to Fig 2, and the culture supernatants were harvested at the indicated times. RNA was extracted and purified from each culture supernatant, and viral RNA copy number in the supernatant was determined by qRT-PCR. A virus titer in PFU of each culture supernatant was also determined by plaque assay, and particle-to-PFU ratio was calculated. Each point represents the mean ± SD of triplicates from one representative experiment. Statistical significance was determined by an unpaired two-tailed t test. Values in parentheses indicate p values, and those less than 0.05 were considered statistically significant. Huh, Huh7.5.1–8 cells.

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S7 Fig. Comparison of JEV plaque development between Huh7.5.1–8 and Vero cells, related to Fig 7.

Representative black-and-white inverted images of plaques of the JEV Nakayama strain (A) and the rAT strain (B) in Huh7.5.1–8 (Huh) and Vero cells. Cells were fixed and stained at the indicated times. Values given below the images indicate plaque numbers expressed as the mean ± SD of triplicates from one representative experiment. Results from two independent experiments (a, b) are shown. Statistical significance was determined by an unpaired two-tailed t test. Values in parentheses indicate p values, and those less than 0.05 were considered statistically significant.

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S8 Fig. Comparison of YFV production between Huh7.5.1–8 and Vero cells, related to Fig 8.

Cells were seeded and infected with YFV at MOI 0.1 under high (a) and low (b) confluency conditions as described in the legend to Fig 2. (A, B) Culture supernatants were harvested at the indicated times, and virus titers in the supernatants were determined by plaque assay. Results from two independent experiments (A, B) are shown. Statistical significance was determined by an unpaired two-tailed t test. Values in parentheses are p values, and those less than 0.05 were considered statistically significant. Huh, Huh7.5.1–8 cells.

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S9 Fig. Comparison of YFV plaque development between Huh7.5.1–8 and Vero cells, related to Fig 9.

Representative black-and-white inverted images of YFV plaques in Huh7.5.1–8 (Huh) and Vero cells. Cells were infected with the indicated amount (PFU) of YFV (17D-204 strain) per well and then fixed and stained at the indicated times. Results from two independent experiments (A, B) are shown. Values below the images indicate plaque numbers expressed as the mean ± SD of triplicates from one representative experiment. Statistical significance was determined by an unpaired two-tailed t test. Values in parentheses indicate p values, and those less than 0.05 were considered statistically significant. N.D., not determined.

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S10 Fig. Nucleotide sequences of RIG-I full-length cDNAs (FL1, 2) and splice variants (SV1, 2).

Alignment of nucleotide sequences was performed using GENETYX version 14.0.0 (GENETYX Co., Tokyo, Japan).

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S11 Fig. Unprocessed image data, related to Figs 1C, 7, 9, S1B, S3, S7 and S9.

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S1 Raw Images. Raw image data (Fig 10A).

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Acknowledgments

We thank Dr. Francis V. Chisari (The Scripps Research Institute, La Jolla, CA, USA) for providing the Huh7.5.1 cell line. We also thank Dr. Tomohiko Takasaki (Department of Virology I, National Institute of Infectious Diseases) for providing the YFV 17D-204 strain.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This research was supported by JSPS (Japan Society for the Promotion of Science; https://www.jsps.go.jp/index.html) of KAKENHI Grant Numbers JP16K08362, JP18H02856, JP17H04003, and JP17K09447 to K.S., M.F., K.H., and T.W., respectively, AMED-CREST (Japan Agency for Medical Research and Development, Core Research for Evolutional Science and Technology; https://www.amed.go.jp/index.html) Grant Number JP19gm0910005 to K.H., and AMED Grant Numbers JP19fk0210020j1003 and JP19fk0210053j1001 to T.W. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0232274.r001

Decision Letter 0

Negin P Martin

7 Jan 2020

PONE-D-19-32355

Comparative characterization of flavivirus production in two cell lines: Human hepatoma-derived Huh7.5.1-8 and African green monkey kidney-derived Vero

PLOS ONE

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Reviewer #2: Partly

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: This manuscript is well-written, however several issue need to be addressed as noted below:

Comments-

• Lines 130-133- please include a brief description as to why the chosen strains for JEV and YFV were used for this study in the introduction.

• Please explain how the vaccine strains were selected and why strains SA14-14-2 and P3 or strains of that lineage weren’t included win the evaluation.

• Please clarify if Vero cells and Huh cells have the similar doubling times, and if the different Huh cell lines vary in their replication kinetics/doubling times.

• Fig 1A- Add statistics

• Fig 1B- A decrease in Huh7.5.1-8 cell viability without an increase in JEV titer is not best for vaccine production.

• “Similar results were obtained from two independent experiments…” Combine experiments and form into 1 figure.

• Combine all experiments with repeats into a single figure, wherever repeated experiments are shown as separate panels.

• Rephrase infectivity per RNA copy number to particle to PFU ratio.

• Fig 2- make all y-axis equivalent.

• Fig 2- what is the n for these experiments; ere these replicates or independent experiments -clarify

• Fig 2- Provide statistical analysis

• Fig 4- why were these time points chosen- are they relevant to the replication of JEV?

• Fig 4B- please include a description as to why there is a difference in sample size at 3 dpi

• Fig 5- Use the 2^-∆∆Ct method to demonstrate GAPDH-normalized JEV RNA-fold increases

• Fig 6- Make all of the y-axes the same to better illustrate the shift from intracellular to extracellular JEV RNA at 2 and 3 dpi. Combine all 9 data points as they are all from 3 repeats of the same experiment.

• Fig 8A- Add statistics.

• Fig 9- Please clarify how plaques are occurring at 4 dpi in Vero cells when no cell death is observed at this time-point (Figures 8C and 8D).

Reviewer #2: In the present study of “Comparative characterization of flavivirus production in two cell lines: Human hepatoma-derived Huh7.5.1-8 and African green monkey kidney-derived Vero”, Saito and colleagues compared the JEV and YFV infection in they newly developed Huh7.5.1-8 and Vero cell lines. They showed that Huh7.5.1-8 cell produced more virus particles and more susceptible to virus-induced cell death than Vero cells upon JEV and YFV infection. They further showed that this is resulted from rapid viral replication kinetics and efficient virus release but not because of higher infection efficacy. They also showed that Huh7.5.1-8 cells developed plaques more rapidly than Vero cells after JEV infection and higher numbers of plaques after YFV infection than that of Vero cells. They finally showed the Huh7.5.1-8 cell containing a functional loss of the antiviral RIG-I gene. The authors showed that Huh7.5.1-8 cells are an useful substitute cell lines for studying viruses of Flaviviridae family.

Major comments to the authors.

1. About testing the extracellular JEV RNA in Huh7.5.1-8 and Vero cell lines in figure 5B, the authors expressed the extracellular JEV RNA as the total copy number in the culture supernatant divided by that of GAPDH RNA in the whole cells from the same well. However, the authors showed that JEV infected Huh7.5.1-8 cells showed significant less cell viability in figure 3 compared with that in Vero cells. This makes the normalized GAPDH in Huh7.5.1-8 cells is much less than that in Vero cells which will increase the values in Huh7.5.1-8 cells and also affect the interpretation showed in figure 6. I think it ‘s better to compare the total RNA copy number of the extracellular RNA between two cell lines because the same cells were initially seeded and the same volume of medium was used.

2. The codon 55 of the RIG-I gene Huh-7.5.1 is heterozygous or not? How do we know the heterozygous of codon 55 in the RIG-I gene is stable during passages? The authors should discuss about this.

Minor comments to the authors.

1. For comparing the JEV production in Huh7.5.1-8 and Vero cell lines in figure 2 E and F, it seems the ratio in a bipolar distribution in 2 dpi in both high confluence and low confluence and day 1 in low confluence. Why the variation is so big?

2. For figure 2 to indicate the high confluence and low confluence, its better to labeled as high or low confluence MOI 0.01/0.1. The way the authors labeled is easy to be considered as high MOI and low MOI.

3. For comparing the infectivity of JEV in Huh7.5.1-8 and Vero cell lines in figure 4B, same question as above, the infectivity ratio in 2- and 3- dpi in a bipolar distribution which shows big variation.

**********

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Reviewer #2: No

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PLoS One. 2020 Apr 24;15(4):e0232274. doi: 10.1371/journal.pone.0232274.r002

Author response to Decision Letter 0

10 Apr 2020

RESPONSE TO EDITOR

Editor’s comment: A lot of effort has been dedicated to gathering and analyzing data and the manuscript is well written and organized. However, reviewers have raised some concerns about the paper and its methods. I recommend that you consider reviewers’ recommendations and revise your paper accordingly before resubmitting your work. I wish you the best.

Response: Thank you for your constructive feedback. We agree with you and the reviewers and have incorporated your and their suggestions throughout our manuscript.

RESPONSES TO REVIEWER #1

We are grateful to reviewer #1 for the critical comments and useful suggestions that have helped us to improve our paper. As indicated in the responses that follow, we have taken all these comments and suggestions into consideration in the revised version of our paper.

(Comments by reviewer #1 are shown in italics.)

Reviewer #1: This manuscript is well-written, however several issue need to be addressed as noted below:

Comment #1: Lines 130-133- please include a brief description as to why the chosen strains for JEV and YFV were used for this study in the introduction.

Response: We chose JEV (Nakayama and rAT) and YFV (17D-204) strains because these strains could be used in our biosafety level 2 laboratory. We have described the reasons in the materials and methods section (p. 6, L137�138), because we considered this section to be contextually more appropriate than the introduction.

Comment #2: Please explain how the vaccine strains were selected and why strains SA14-14-2 and P3 or strains of that lineage weren’t included win the evaluation.

Response: The Nakayama strain was isolated in our institute in 1935 and used as a vaccine in Japan from 1954 to 1989. Given such a history, we started to use the Nakayama strain. Another reason for selecting the strain was that its plaques are easily distinguishable and of appropriate size, which is advantageous for analysis. The less virulent SA 14-14-2 strain (Yu U, Vaccine, 28, 3635, 2010; Hsieh JT et al, Nat Commun 10, 706, 2019) was not selected because of the expected small plaque morphology. For biosafety reasons, we did not examine the highly virulent strain P3 (Ni, H and Barrett AD, J Gen Virol, 77, 1449, 1996).

Comment #3: Please clarify if Vero cells and Huh cells have the similar doubling times, and if the different Huh cell lines vary in their replication kinetics/doubling times.

Response: In the revised manuscript, we have monitored growth curves of different HuH-7-derived cells and Vero cells and have calculated their relative doubling times (S2 Fig). The new data showed that doubling times of Huh7.5.1-8, Huh7.5.1, HuH-7, and Vero cells were not significantly different. Taken together with Figs 1 and 2, this result showed that the replication kinetics per doubling time are similar between the different HuH-7-derived cell lines and that the difference of JEV production between Huh7.5.1-8 and Vero cells was not accounted for by the difference in their growth rates. In view of these results, we have added the following sentences (p.15, L263�266):

“Under the same seeding conditions, doubling times of Huh7.5.1-8, Huh7.5.1, and HuH-7 cells were not significantly different (after Bonferroni correction, p > 0.0167) (S2B Fig). Thus, JEV production appeared to be comparable between the three cell lines and not enhanced in this lineage unlike for HCV production”.

We have also added the following sentence (p.17, L313�315):

“The difference in the titer was not accounted for by the growth rates of the two cell lines, because their doubling times under high confluency conditions were nearly equal (S2 Fig)”.

Comment #4: Fig 1A- Add statistics.

Response: The results of the statistical analysis have been added in the revised Fig 1A.

Comment #5: Fig 1B- A decrease in Huh7.5.1-8 cell viability without an increase in JEV titer is not best for vaccine production.

Response: We agree with the reviewer on this point. We consider that Vero cells are better than Huh7.5.1-8 cells for vaccine production and mentioned this in the original manuscript as follows (p. 29, L587�589):

“In addition, lower susceptibility of Vero cells to flavivirus-induced cell death, compared with Huh7.5.1-8 cells, may be a feature that makes the Vero cell line advantageous for vaccine production”.

In the revised manuscript, we have changed the above sentence to

“In addition, the higher tolerance of Vero cells to flavivirus-induced cell death, compared with Huh7.5.1-8 cells, may be a feature that makes the Vero cell line advantageous for vaccine production” (p. 30, L611�613).

Comment #6: “Similar results were obtained from two independent experiments…” Combine experiments and form into 1 figure.

Response: For several sets of numeric data in the revised manuscript, we have repeated the same experiments at least three times, combined, and provided statistical analysis (Figs 1A, 1B, 3, 5, 8E, and 8F). The exception is the results of YFV titer: Because variations between experiments performed on different days were too large, individual experiments (Fig 8A, 8B, and S8 Fig) are shown as well as the combined results (Fig 8C and 8D). Thus, the sentence “similar results were obtained from two independent experiments” has been deleted from the legends of the above figures. For image data of plaque formation (Figs 1C, 7, and 9), representative experiments are shown in the main figures, and two additional experiments are shown in the supplemental figures (S3, S7 and S9 Fig). We have added this explanation to the respective legends. Representative results are still shown for two supplemental experiments which were repeated only twice (S1 and S4 Fig).

Comment #7: Combine all experiments with repeats into a single figure, wherever repeated experiments are shown as separate panels.

Response: In accordance with the reviewer’s comment, we have combined the repeated experiments, which were originally shown as separate panels, into a single figure (Figs 2 and 4). The original Fig 2E and 2F showed the Huh-to-Vero ratio of JEV titer at each time point, but did not include time-dependent changes in JEV titer. To improve this figure, we have combined the data after converting each titer value to a value relative to that of Vero cells at 1 d pi in the revised Fig 2 (similar method of data combination has been applied to Figs 1A and 5). However, representative results are still shown along with the combined results in Fig 8 (YFV titer) because of the reason mentioned in the response to comment #6.

Comment #8: Rephrase infectivity per RNA copy number to particle to PFU ratio.

Response: The data have been converted to RNA copy number per PFU, and “infectivity per RNA copy number” has been rephrased to “particle-to-PFU ratio”.

Comment #9: Fig 2- make all y-axis equivalent.

Response: We have made all y-axes equivalent in the revised Fig 2. We have done the same in S5 and S8 Fig.

Comment #10: Fig 2- what is the n for these experiments; ere these replicates or independent experiments –clarify

Response: The “n” for Fig 2 is the number of independent experiments. We have added this explanation in the legend.

Comment #11: Fig 2- Provide statistical analysis

Response: We have provided the results of statistical analysis in the revised Fig 2.

Comment #12: Fig 4- why were these time points chosen- are they relevant to the replication of JEV?

Response: Under our infection conditions, Huh7.5.1-8 cells started to die at 3 d pi and almost completely died at 4 d pi (Fig 3). Therefore, we chose time points (21, 45, 69, 97 h pi) roughly evenly spaced by four days. Because the original Fig 4A was a representative result partially overlapping with the combined result (Fig 4B), it has been moved to the supplemental information (S6 Fig) in accordance with comment #7.

Comment #13: Fig 4B- please include a description as to why there is a difference in sample size at 3 dpi

Response: The reason for a difference in sample size was that one out of eight experiments lacked data on 3 d pi. We have added this explanation in the legend of the revised Fig 4.

Comment #14: Fig 5- Use the 2^-∆∆Ct method to demonstrate GAPDH-normalized JEV RNA-fold increases.

Response: In the original Fig 5, we used the relative standard curve method, which is commonly used along with the 2^-∆∆Ct method for relative quantification by qPCR. More specifically, we determined the copy number by using serially diluted JEV or GAPDH cDNAs and then normalized the copy number of JEV RNA by that of GAPDH RNA. Although we think that the reviewer’s suggestion on the 2^-∆∆Ct method is valuable, the other reviewer noted that normalization of extracellular JEV RNA by cellular GAPDH RNA performed in the original Fig 5B was not appropriate because of a large difference in cell viability between Huh7.5.1-8 and Vero cells, and suggested that the amount of extracellular JEV RNA should be expressed as total copy number in the culture supernatant. Therefore, we have chosen the relative standard curve method again in the revised Fig 5. In the manual by Applied Biosystems (http://www.gu.se/digitalAssets/1125/1125331_ABI_-_Guide_Relative_Quantification_using_realtime_PCR.pdf, p.34), it is described that the relative standard curve method gives highly accurate quantitative results because unknown sample quantitative values are interpolated from the standard curve(s), implying that the relative standard curve method is not inferior to the 2^-∆∆Ct method. Furthermore, to combine four independent experiments, we have normalized the amounts of GAPDH-normalized JEV RNA (Fig 5A) and extracellular JEV RNA (copies/well) (Fig 5B) relative to that of Vero cells at 0 h pi. We have changed the y-axes of these figures to “Relative amount of JEV RNA”. Just for reference, we have presented an alternative version of Fig. 5A calculated using the 2^-∆∆Ct method in a “supplemental file for reviewers 1”. The same conclusion was obtained with this method.

Comment #15: Fig 6- Make all of the y-axes the same to better illustrate the shift from intracellular to extracellular JEV RNA at 2 and 3 dpi. Combine all 9 data points as they are all from 3 repeats of the same experiment.

Response: In accordance with the reviewer’s comment, we have made all of the y-axes the same, and all the data points have been combined in the revised Fig. 6. We have added the following sentences in the legend (p.22, L426�428):

“Data represent the mean ± SD of three independent experiments. Each symbol represents a mean from one experiment”.

Comment #16: Fig 8A- Add statistics.

Response: We have added the results of statistical analysis to the revised Fig 8A and 8B.

Comment #17: Fig 9- Please clarify how plaques are occurring at 4 dpi in Vero cells when no cell death is observed at this time-point (Figures 8C and 8D).

Response: A possible explanation for this discrepancy in cell death may have been due to differences in culture conditions: The serum concentration was 10% for Fig 8 and 2% for Fig 9. In addition, the medium for Fig 9 contained methylcellulose. These differences may have affected Vero cell death. In the case of JEV, the lower the serum concentration is, more cell death occurs (J Virol. 2005 Jul; 79 (13): 8388–8399). We have added this explanation and cited the paper (reference 27) in p. 24�25, L484�489.

RESPONSES TO REVIEWER #2

We are grateful to reviewer #2 for the critical comments and useful suggestions that have helped us to improve our paper. As indicated in the responses that follow, we have taken all these comments and suggestions into consideration in the revised version of our paper.

(Comments by reviewer #2 are shown in italics.)

Major Comment #1: About testing the extracellular JEV RNA in Huh7.5.1-8 and Vero cell lines in figure 5B, the authors expressed the extracellular JEV RNA as the total copy number in the culture supernatant divided by that of GAPDH RNA in the whole cells from the same well. However, the authors showed that JEV infected Huh7.5.1-8 cells showed significant less cell viability in figure 3 compared with that in Vero cells. This makes the normalized GAPDH in Huh7.5.1-8 cells is much less than that in Vero cells which will increase the values in Huh7.5.1-8 cells and also affect the interpretation showed in figure 6. I think it ‘s better to compare the total RNA copy number of the extracellular RNA between two cell lines because the same cells were initially seeded and the same volume of medium was used.

Response： In accordance with the reviewer’s comment, we have changed the measure in Fig 5B from “the extracellular JEV RNA as the total copy number in the culture supernatant divided by that of GAPDH RNA in the whole cells from the same well” to “JEV RNA as the total copy number in the supernatant”.

Because the other reviewer suggested to combine the results from multiple experiments, data from four experiments (in copies/well) have been normalized to that of Vero cells at 0 h pi in the same experiment, and then combined into one figure. Along with the integration of data, we have changed the following sentence from p. 20, L378�379 in the original version:

“…, whereas the rise for Vero cells was at 12 h pi (Fig. 5A [a])”

“…, whereas the rise for Vero cells was not remarkable during 0–12 h pi (Fig. 5A [a]) ”(p. 20, L385�386).

We have also changed the following sentence from p. 20, L380–382 in the original version:

“Throughout the infection, Huh7.5.1-8 cells accumulated more viral RNA intracellularly (Fig 5A [b]) and extracellularly (Fig 5B [b]) than Vero cells”

“Although the level of intracellular viral RNA was not different between the two cell lines after 12 h pi (Fig 5A [b]), the level of extracellular viral RNA was higher for Huh7.5.1-8 cells than for Vero cells during 12�48 h pi (Fig 5B [b])”(p. 20, L387�390).

Major Comment #2: The codon 55 of the RIG-I gene Huh-7.5.1 is heterozygous or not? How do we know the heterozygous of codon 55 in the RIG-I gene is stable during passages? The authors should discuss about this.

Response: In the revised Fig 10, we have added the results of direct sequencing of codon 55 of the RIG-I gene from Huh7.5.1 cells and found that the codon of Huh7.5.1 cells was heterozygous as was that of Huh7.5.1-8 cells. This result indicates that the heterozygosity of codon 55 of RIG-I was stable at least during the time span for isolation of the Huh7.5.1-8 clone from Huh7.5.1 cells. We have discussed this in the revised manuscript (p. 30, L619�622).

Minor Comment #1: For comparing the JEV production in Huh7.5.1-8 and Vero cell lines in figure 2 E and F, it seems the ratio in a bipolar distribution in 2 dpi in both high confluence and low confluence and day 1 in low confluence. Why the variation is so big?

Response: In the revised manuscript, we have changed the method of data combination from “ratio of JEV titer of Huh7.5.1-8 cells to that of Vero cells at each time point” (the original Figs 2E and 2F) to “the relative titer to the value of Vero cells at 1 d pi” (the revised Fig 2A and 2B) to show time-dependent changes of JEV titer of both cells. The large variations that the reviewer mentioned were still found after this revision, although the variation at 1 d pi under low confluency appeared less remarkable when plotted on a log scale. In addition, large variations between experiments were seen also with Fig 8 (YFV titer). Because these variations could not be linked to specific experimental conditions, we do not have convincing answers to the reviewer’s comment. Although the reason for the variations remains unknown, the data (Figs 2 and 8) definitely showed that JEV and YFV titers of Huh7.5.1-8 cells were significantly higher than those of Vero cells at early infection times.

Minor Comment #2: For figure 2 to indicate the high confluence and low confluence, its better to labeled as high or low confluence MOI 0.01/0.1. The way the authors labeled is easy to be considered as high MOI and low MOI.

Response: The reviewer’s comment is correct. We have deleted confusing labels such as “high MOI and low MOI”. In addition, we have separated the data for MOI 0.01 and MOI 0.1 into different figures (Figs 2 and S5 Fig) and have labeled them as high or low confluency.

Minor Comment #3: For comparing the infectivity of JEV in Huh7.5.1-8 and Vero cell lines in figure 4B, same question as above, the infectivity ratio in 2- and 3- dpi in a bipolar distribution which shows big variation.

Response: At present, we do not have convincing answers to the reviewer’s comment. However, we speculate that at least the bipolar distribution of infectivity ratio at 3 d pi was partly caused by non-infectious viral RNA leaking from dead Huh7.5.1-8 cells. If such a leak were poorly controlled, this could lead to variation in the infectivity ratio (the original Fig 4) and the particle-to-PFU ratio (the revised Fig 4). We have incorporated this discussion as follows (p. 19, L356�358):

“The reason for large variations found at 2 and 3 d pi remains unknown, but that at 3 d pi may have been partly due to variable amounts of non-infectious viral RNA leaking from dead Huh7.5.1-8 cells (Fig 3)”.

OTHER CHANGES

About the text:

1. The affiliation of Ryosuke Suzuki has been changed from “Department of Virology II, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan” to “Department of Virology II, National Institute of Infectious Diseases, Musashi-murayama-shi, Tokyo, Japan” (p. 1, L 13�14). The affiliation of Naoki Osada has been changed from “Graduate School of Information Science and Technology, Hokkaido University, Sapporo, Hokkaido, Japan” to “Faculty of Information Science and Technology, Hokkaido University, Sapporo, Hokkaido, Japan” (p. 1, L 15�16).

2. To avoid misunderstanding, we have changed the following sentence (p. 2, L45�48) in the original Abstract:

“Huh7.5.1-8 cells were found to express not only a full-length RIG-I mRNA with a known dominant-negative missense mutation but also variants lacking exon 5/6�12 without the mutation, indicating functional loss of the antiviral RNA helicase RIG-I in the cells.”

“Sequence analysis of cDNA encoding an antiviral RNA helicase, RIG-I, showed that Huh7.5.1-8 cells expressed not only a full-length RIG-I mRNA with a known dominant-negative missense mutation but also variants without the mutation. However, the latter mRNAs lacked exon 5/6�12, indicating functional loss of RIG-I in the cells.” (p. 2�3, L46�50).

3. Although the range of sampling points expressed in d pi was described in each legend in the original manuscript, they are summarized in the Material and methods in the revised manuscript (p. 7, L148�151).

4. We have changed the following sentences from the original manuscript (p. 8, L168�171):

“After fixation and staining, plates were scanned with a document scanner, and image data were obtained. To improve plaque visibility, an invert filter was applied to the image data using Photoshop Elements (version 14; Adobe Systems, San Jose, CA, USA)”

“After fixation and staining, plates were scanned with a document scanner, and image data were obtained (S11 Fig). To improve plaque visibility, an invert filter was applied to the image data, and highlights were adjusted using Photoshop Elements (version 14; Adobe Systems, San Jose, CA, USA)” (p. 8, L174�177).

5. In the original manuscript, we mentioned unpublished results on p. 14, L234�237, p. 25, L497�500, and p. 26, L513�514. A paper including the results has recently been submitted to bioRxiv (Kawamoto M, Yamaji T, Saito K, Satomura K, Endo T, Fukasawa M, et al. Identification of characteristic genomic markers in human hepatoma Huh7 and Huh7.5.1-8 cell lines. bioRxiv. 2020:2020.02.17.953281. doi: 10.1101/2020.02.17.953281.). We have cited this paper in the revised manuscript (Reference No. 25).

6. We have added the following sentence to p. 14, L245�246:

“All the experiments were done with three biological replicates and repeated as described in the figure legends”.

7. We have deleted the following sentence from the original manuscript (p. 14, L245�246):

”Values of p < 0.05 were considered statistically significant”.

8. We have changed “among” to “between” (p.15, L255 and p. 16, L280).

9. We have deleted the following sentence from the original manuscript (p. 27, L553�554):

“�, which was remarkable particularly in high-cell-density infection”.

10. We have changed the following sentence from the original manuscript (p. 29, L584�587):

“Vero cells may be more suitable to viruses requiring long-term cultivation to grow plaques, because Huh7.5.1-8 cells were less tolerant to our plaque assay conditions than Vero cells.”

“Vero cells may be more suitable to viruses requiring long-term cultivation to grow plaques, because they were more tolerant to our plaque assay conditions than Huh7.5.1-8 cells” (p.30, L608�611).

11. We have changed the text and the legends and to fit the revised figures.

About the figures:

12. One point (closed circle at 3 d pi) in our original Fig 2F was the wrong value (0.263): The correct value is 2.08. In addition, the p-value at 1 d pi (0.033) was wrong and has been corrected to 0.032. We sincerely apologize for these corrections. We have attached the corrected original Fig 2 as “Supplemental file for reviewers 2”, because this figure is not included in the revised manuscript. We hope the editor and reviewers could refer to the corrected figure.

13. In Fig 2 of the original manuscript, we showed the results for JEV production and cell viability of Huh7.5.1-8 and Vero cells at MOI 0.01. In the revised manuscript, we have moved the results to a supplemental figure (S5 Fig), because we did not use the infection condition at MOI 0.01 in any other experiments. We have also changed the following text (p. 7, L140) :

“a multiplicity of infection (MOI) of 0.01 or 0.1 ”

“a multiplicity of infection (MOI) of 0.1” (p. 7, L143).

14. In the original S2 Fig on JEV production of different Vero sublines, data drawn by line graphs were overlapping and difficult to make out. So, we have changed the data to bar graphs and have added the results of statistical analysis in the revised S4 Fig.

15. We have added unprocessed image data for virus plaques (S11 Fig).

Correspondence table of original and revised figures:

Original Revised Changes

Fig 1A, 1B Fig 1A, 1B Results of multiple experiments have been combined with statistics.

Fig 1C Fig 1C, S3 Fig One experiment is shown in Fig 1C, and two other independent experiments are shown in S3 Fig.

Fig 2 Fig 2, S5 Fig Results of multiple experiments have been combined with statistics. Results for MOI 0.01 have been moved to S5 Fig.

Fig 3 Fig 3, S5 Fig Results of multiple experiments have been combined with statistics. Results for MOI 0.01 have been moved to S5 Fig.

Fig 4 Fig 4, S6 Fig The measure has been changed from PFU per copy number to particle-to-PFU ratio. A representative result has been moved to S6 Fig.

Fig 5 Fig 5 Results of multiple experiments have been combined with statistics. The measure has been changed from JEV/GAPDH to the relative mount of JEV RNA.

Fig 6 Fig 6 Results of multiple experiments have been combined with statistics.

Fig 7 Fig 7, S7 Fig One experiment is shown in Fig 7, and two other independent experiments are shown in S7 Fig.

Fig 8A, 8B Fig 8A-D, S8 Fig Representative experiments are shown in Fig 8A and 8B, and two other independent experiments are shown in S8 Fig. The combined results with statistics are also shown in Fig 8C and 8D.

Fig 8C, 8D Fig 8E, 8F Results of multiple experiments have been combined with statistics.

Fig 9 Fig 9, S9 Fig One experiment is shown in Fig 9, and two other independent experiments are shown in S9 Fig.

Fig 10 Fig 10 Data for Huh7.5.1 cells have been added.

S1 Fig S1 Fig The results of statistical analysis have been added to S1A Fig.

S2 Fig S4 Fig The figure number has been changed. The original line graphs have been changed to bar graphs with statistics.

S3 Fig S10 Fig The figure number has been changed.

None S2 Fig Data for cell growth rates are shown.

None S11 Fig Unprocessed image data (Figs 1C, 7, 9, S1B, S3, S7, and S9) are shown.

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PLoS One. doi: 10.1371/journal.pone.0232274.r003

Decision Letter 1

Negin P Martin

13 Apr 2020

Comparative characterization of flavivirus production in two cell lines: Human hepatoma-derived Huh7.5.1-8 and African green monkey kidney-derived Vero

PONE-D-19-32355R1

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PLoS One. doi: 10.1371/journal.pone.0232274.r004

Acceptance letter

Negin P Martin

16 Apr 2020

PONE-D-19-32355R1

Comparative characterization of flavivirus production in two cell lines: Human hepatoma-derived Huh7.5.1-8 and African green monkey kidney-derived Vero

Dear Dr. Saito:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. JEV production and plaque formation in Vero cells grown in different media.

(TIF)

Click here for additional data file.^{(823.3KB, tif)}

S2 Fig. Comparison of cell growth between Huh7.5.1–8, Huh7.5.1, HuH-7, and Vero cells, related to Figs 1 and 2.

(TIF)

Click here for additional data file.^{(133.8KB, tif)}

S3 Fig. Comparison of JEV plaque formation between Huh7.5.1–8, Huh7.5.1, and HuH-7 cells, related to Fig 1C.

(TIF)

Click here for additional data file.^{(1.4MB, tif)}

S4 Fig. Comparison of JEV production among Vero cell sublines.

(TIF)

Click here for additional data file.^{(134.9KB, tif)}

S5 Fig. Comparison of JEV infection at MOI 0.01 between Huh7.5.1–8 and Vero cells, related to Figs 2 and 3.

(TIF)

Click here for additional data file.^{(229.2KB, tif)}

S6 Fig. Particle-to-PFU ratio of Huh7.5.1-8-derived and Vero-derived JEV, related to Fig 4.

(TIF)

Click here for additional data file.^{(36.6KB, tif)}

S7 Fig. Comparison of JEV plaque development between Huh7.5.1–8 and Vero cells, related to Fig 7.

(TIF)

Click here for additional data file.^{(2.9MB, tif)}

S8 Fig. Comparison of YFV production between Huh7.5.1–8 and Vero cells, related to Fig 8.

(TIF)

Click here for additional data file.^{(160.6KB, tif)}

S9 Fig. Comparison of YFV plaque development between Huh7.5.1–8 and Vero cells, related to Fig 9.

(TIF)

Click here for additional data file.^{(2.2MB, tif)}

S10 Fig. Nucleotide sequences of RIG-I full-length cDNAs (FL1, 2) and splice variants (SV1, 2).

Alignment of nucleotide sequences was performed using GENETYX version 14.0.0 (GENETYX Co., Tokyo, Japan).

(PDF)

Click here for additional data file.^{(128.7KB, pdf)}

S11 Fig. Unprocessed image data, related to Figs 1C, 7, 9, S1B, S3, S7 and S9.

(PDF)

Click here for additional data file.^{(3.8MB, pdf)}

S1 Raw Images. Raw image data (Fig 10A).

(PDF)

Click here for additional data file.^{(4MB, pdf)}

Attachment

Submitted filename: Response to Reviewers 200409.docx

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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Comparative characterization of flavivirus production in two cell lines: Human hepatoma-derived Huh7.5.1-8 and African green monkey kidney-derived Vero

Kyoko Saito

Masayoshi Fukasawa

Yoshitaka Shirasago

Ryosuke Suzuki

Naoki Osada

Toshiyuki Yamaji

Takaji Wakita

Eiji Konishi

Kentaro Hanada

Roles

Abstract

Introduction

Materials and methods

Cells

Viruses

Infection and post infection conditions

Plaque assay

RNA purification

Quantification of RNA by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR)

Table 1. Primers and probes used in this study.

Cloning and sequencing of RIG-I (DDX58) cDNA

Other methods

Results

Comparison of JEV production between Huh7.5.1–8, Huh7.5.1, and HuH-7 cells

Fig 1. Comparison of the course of JEV infection between Huh7.5.1–8, Huh7.5.1, and HuH-7 cells.

JEV production in Huh7.5.1–8 and Vero cell lines

Fig 2. JEV titer of Huh7.5.1–8 cells was higher than that of Vero cells early in infection.

Fig 3. Huh7.5.1–8 cells were more susceptible to JEV-induced cell death than Vero cells.

Particle-to-PFU ratio, replication kinetics, and virus release

Fig 4. Particle-to-PFU ratio was comparable between Huh7.5.1-8-derived and Vero-derived JEV.

Fig 5. Huh7.5.1–8 cells exhibited more rapid viral replication kinetics than Vero cells.

Fig 6. Huh7.5.1–8 cells released JEV more efficiently than Vero cells early in infection.

JEV plaque formation

Fig 7. Huh7.5.1–8 cells supported a more rapid development of JEV plaques than Vero cells.

YFV production

Fig 8. Comparison of YFV production and YFV-induced cell death between Huh7.5.1–8 and Vero cells.

Fig 9. Huh7.5.1–8 cells developed larger numbers of YFV plaques than Vero cells.

Confirmation of RIG-I sequence

Fig 10. Sequence analysis of RIG-I cDNA derived from HuH-7, Huh7.5.1, and Huh7.5.1–8 cells.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Negin P Martin

Roles

Author response to Decision Letter 0

Decision Letter 1

Negin P Martin

Roles

Acceptance letter

Negin P Martin

Roles

Associated Data

Supplementary Materials

Data Availability Statement

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