Characterization of Japanese encephalitis virus infection in an immortalized mesencephalic cell line, CSM14.1

Authors

  • Takashi Kimura,

    Corresponding author
    • Division of Molecular Pathobiology, Hokkaido University Research Center for Zoonosis Control, Kita-ku, Sapporo, Japan
    Search for more papers by this author
  • Megumi Okumura,

    1. Division of Molecular Pathobiology, Hokkaido University Research Center for Zoonosis Control, Kita-ku, Sapporo, Japan
    Search for more papers by this author
  • Eunmi Kim,

    1. Division of Molecular Pathobiology, Hokkaido University Research Center for Zoonosis Control, Kita-ku, Sapporo, Japan
    Current affiliation:
    1. Vaccine I team, Mogam Biotechnology Research Institute, Yongin City, Korea
    Search for more papers by this author
  • Michihito Sasaki,

    1. Division of Molecular Pathobiology, Hokkaido University Research Center for Zoonosis Control, Kita-ku, Sapporo, Japan
    Search for more papers by this author
  • Yasuko Orba,

    1. Division of Molecular Pathobiology, Hokkaido University Research Center for Zoonosis Control, Kita-ku, Sapporo, Japan
    Search for more papers by this author
  • Hirofumi Sawa

    1. Division of Molecular Pathobiology, Hokkaido University Research Center for Zoonosis Control, Kita-ku, Sapporo, Japan
    Search for more papers by this author

Correspondence

Takashi Kimura, Division of Molecular Pathobiology, Hokkaido University Research Center for Zoonosis Control, West 10 North 20, Kita-ku, Sapporo 001-0020, Japan.

Tel: +81 11 706 5218; fax: +81 11 706 7370; email: kimura@czc.hokudai.ac.jp

ABSTRACT

Neurons are the major target cell of Japanese encephalitis virus (JEV). Rats intracerebrally inoculated with JEV show an age-dependent pattern of resistance to infection in which resistance is closely associated with neuronal maturation. However, because there is no reliable and convenient cell culture system that mimics the in vivo properties of JEV infection of immature and mature neurons, the mechanisms underlying this association remain poorly understood. The aim of the present study was to examine JEV infection in immortalized CSM14.1 rat neuronal cells, which can be induced to differentiate into neurons by culture under non-permissive conditions. JEV infected undifferentiated CSM14.1 cells more efficiently than differentiated cells, resulting in production of more progeny virus in the former setting than in the latter. An infectious virus recovery assay detected more internalized virions in undifferentiated cells. On the other hand, JEV infection of differentiated cells induced more rapid and stronger expression of interferon-β gene, along with smaller amounts of JEV RNA. Taken together, these results show that the initial phase of viral infection and the later IFN response play roles in the viral susceptibility of undifferentiated and differentiated CSM14.1 cells. Because CSM14.1 cells became more resistant to JEV infection as they mature, this culture system can be used as an in vitro model for studying age-dependent resistance of neurons to JEV infection.

List of Abbreviations
CPE

cytopathic effect

DAPI

4,6-diamidino-2-phenylindole

DMEM

Dulbecco's modified Eagle's medium

HRP

horseradish peroxidase

IFN

interferon

JEV

Japanese encephalitis virus

MEM

Eagle's minimal essential medium

MOI

multiplicity of infection

p.f.u.

plaque-forming units

p.i.

post-infection

SV

Sindbis virus

TH

tyrosine hydroxylase

Japanese encephalitis virus, which belongs to the genus Flavivirus, is an arbovirus transmitted primarily by Culex mosquitoes. Although most JEV infections in humans are asymptomatic, clinical cases tend to manifest as severe, often fatal, encephalitis, often accompanied by the development of neurological and/or psychiatric sequelae in survivors [1]. Neurons in the brain are a major target of JEV [2].

One of the clinical characteristics of arbovirus encephalitis is that the disease is more severe in children [3]. For JEV, the highest age-specific attack rates occur in children less than 6 years old [4]. Animal studies of experimental JEV infection (reviewed in reference [5]) show that older mice are more resistant to the lethal effects of JEV than younger mice [6, 7]. This age-dependent resistance to JEV infection is even more striking in rat models, in which resistance to intracerebral infection is closely associated with neuronal maturation [8, 9]. However, due to the lack of a reliable in vitro infection model (apart from a primary fetal rat brain culture model that mimics neuronal maturation [10]), the mechanisms underlying age-dependent susceptibility to neuronal JEV infection remain unclear.

CSM14.1 is an immortal rat neuronal cell line that was originally generated by transducing primary rat fetal (embryonic Day 14) mesencephalic neural cells with retroviral vector containing tsA58, a temperature-sensitive mutant of simian virus 40 large T antigen [11]. A major characteristic of this cell line is that it differentiates when cultured at 37–39°C (at which temperatures the SV40 large T antigen is inactive) in media containing low serum concentrations; thus it is ideal for studying neuronal differentiation [12, 13]. This cell line has also been used to study the actions of various host factors on neurons [14-22]. In addition, CSM14.1 cells have proven to be a useful model for studying the age-dependent infection of neurons by Sindbis virus (SV) [23] and the clearance of SV from infected neurons [24, 25].

To ascertain whether CSM14.1 cells can be used as a convenient model system for studying the age-dependent resistance of neurons to JEV infection, we examined the susceptibility of undifferentiated and differentiated CSM14.1 cells to infection by JEV. We also investigated the viral entry and/or host factors responsible for the observed differences.

MATERIALS AND METHODS

Cells

CSM14.1 cells (11,21,22) were generously provided by Dr. Diane E. Griffin (Johns Hopkins Bloomberg School of Public Health). The cells were maintained and passaged in DMEM (Sigma, St. Louis, MO, USA) supplemented with 10% FBS, 2 mM L-glutamine (Sigma), and antibiotics (50 µg/mL of gentamicin (Gibco, Life Technologies, Carlsbad, CA, USA) or a mixture of 100 units/mL penicillin and 100 µg/mL streptomycin (Sigma) in a humidified 5% CO2 incubator at 31°C (permissive conditions). To induce differentiation, the cells were plated at a density of 4 × 104 cells/cm2 and cultured under permissive conditions until they reached confluence. They were then washed once with PBS (pH7.4) and switched to non-permissive conditions: DMEM supplemented with 1% FBS and a temperature of 37–39°C. Differentiated cells were cultured under non-permissive conditions for 3 weeks before use in the experiments. Vero cells were cultured at 37°C in Eagle's MEM (Nissui, Tokyo, Japan) supplemented with 5% FBS. Aedes albopictus C6/36 cells were cultured at 27°C in MEM supplemented with 10% FBS and 200 µM MEM non-essential amino acid solution (Gibco).

Virus

JEV (strain JaGAr-01) was propagated in C6/36 cells. JEV was purified from harvested culture medium using the method of Takegami et al. [26]. Infectivity titers were measured in a Vero cell-based plaque assay using standard procedures.

Immunofluorescence analysis

Undifferentiated or differentiated CSM14.1 cells, grown in poly-L-lysine-pretreated 8-well-chamber slides (BD Falcon, Bedford, MA, USA), were infected with JEV at a MOI of 5 p.f.u./cell. After culture for 2 days under permissive (undifferentiated cells) or non-permissive conditions (differentiated cells), the cells were fixed with cold acetone for 5 min and then stained with anti-JEV rabbit serum followed by Alexa 594-conjugated anti-rabbit IgG (Invitrogen Life, Technologies, Carlsbad, CA, USA). The cell nuclei were counterstained with DAPI and fluorescence observed under an inverted fluorescence microscope (IX70, Olympus, Tokyo, Japan). The number of fluorescent cells in five random areas within each well was counted using Image J software (Rasband, W.S. ImageJ, U.S. National Institutes of Health, Bethesda, MD, USA, http://rsb.info.nih.gov/ij/, 1997–2012). Average values were calculated from four independent experiments. The anti-JEV serum was prepared from a rabbit as previously described [27].

Western blot analysis

Cells were lysed in ice-cold lysis buffer (10 mM Tris–HCl (pH 7.5), 150 mM NaCl2, 5 mM EDTA, 10% glycerol, 1% Triton X-100) containing a complete protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland), and then sonicated for 10 s. The proteins in the lysates were separated by SDS–PAGE and then transferred to Immobilon-P membranes (Millipore, Bedford, MA, USA). The membranes were blocked for 1 hr in 5% nonfat dry milk in TBS-T (25 mM Tris–HCl [pH 7.4], 137 mM NaCl2, 2.7 mM KCl, 0.05% Tween-20) and incubated with an appropriate primary antibody overnight at 4°C, or for 1 hr at room temperature. The primary antibody was detected with an appropriate HRP-conjugated secondary antibody. The signals were visualized using the Immobilon Western Chemiluminescent HRP Substrate (Millipore) and detected by the VersaDoc Imaging System (Bio-Rad, Hercules, CA, USA). The membranes were then stripped in stripping buffer (62.5 mM Tris–HCl [pH 6.8], 2% SDS, 100 mM 2-mercaptoethanol) for 30 min at 50°C and re-probed with an anti-actin antibody as a loading control. The antibodies used for western blot analysis were as follows: monoclonal anti-tyrosine hydroxylase (TH; Sigma), monoclonal anti-βIII isoform of tubulin (Millipore), anti-JEV rabbit serum, anti-phospho-Stat1 (Tyr701) (Cell Signaling, Boston, MA, USA), anti-Stat1 (Cell Signaling), monoclonal anti-actin (Millipore), HRP-conjugated anti-mouse immunoglobulin (Biosource, Camarillo, CA, USA) and HRP-conjugated anti-rabbit immunoglobulin (Biosource).

Infectious virus recovery assay

An infectious virus recovery assay was performed as described by Hasebe et al. [28] with several modifications. Briefly, CSM14.1 cells or Vero cells were seeded in 24-well plates and washed with ice-cold assay medium (DMEM [for CSM14.1 cells] or MEM [for Vero cells]) supplemented with 25 mM HEPES, 0.2% BSA, and 2 mM L-glutamine) and incubated on ice for 5 min. The cells were then infected with JEV at an MOI of 10 p.f.u./cell for 2 hr at 4°C. The cells were washed once with ice-cold assay medium and then incubated with fresh, pre-warmed assay medium at 31°C (undifferentiated CSM14.1) or 37°C (differentiated CSM14.1 and Vero) for 0, 15, 30, 45, 60, 90 and 120 min. The cells were then washed with glycine buffer (0.05 M glycine–HCl, 0.1 M NaCl, pH 3.0) for 30 s, once with PBS, and then harvested. The cells were freeze-thawed three times and infectious virus detected in a Vero cell-based plaque assay.

Reverse transcriptase-polymerase chain reaction

Total RNA was extracted from the cells using TRIzol reagent (Invitrogen) and then treated with amplification grade DNase I (Invitrogen). Total RNA (1 μg) was reverse-transcribed using random hexamers and the SuperScript III First-Strand Synthesis System (Invitrogen) according to the manufacturer's instructions. PCR was performed using HotStar Taq DNA polymerase (Qiagen, Valencia, CA, USA) and a Veriti 96-Well Thermal Cycler (Applied Biosystems, Life Technologies, Carlsbad, CA, USA). The primers used for amplification were as follows: 5′-GCCAGCAGATCCAGAAGGCTCAAG-3′ and 5′-TCCAGACTTCTGCTTTGACCACCT-3′ for rat IFN-α; 5′-CCATCGACTACAAGCAGCTCCAG-3′ and 5′-GCATAGCTGTTGTACTTCTTGTCTT-3′ for rat IFN-β; 5′-CACGCCGCGTCTTGGT-3′ and 5′-TCTAGGCTTTCAATGAGTGTGCC-3′ for rat IFN-γ; 5′-GGGAAGGGAAGCATTGACAC-3′ and 5′-CCAAGAGCAACAACGGACTG-3′ for the JEV E gene; and 5′-CTGCTCACCGAGGCCCCTCTGAACC-3′ and 5′-GCATGAGGGAGCGCGTAACCCTCATA-3′ for rat β-actin. The annealing temperatures and the cycle numbers (excluding initial denaturation and final extension steps) used for the PCR amplification were as follows: 60°C and 33 cycles for IFN-β; 58°C and 18 cycles for JEV E; and 62°C and 25 cycles for β-actin. The PCR products were run on 1.5% agarose gels, stained with ethidium bromide, and examined under ultraviolet light.

Statistical analysis

Data sets were compared by a 2-tailed, unpaired Student t-test. Statistical significance was achieved when the P values were <0.05.

RESULTS

Differentiation of CSM14.1 cells

CSM14.1 cells actively proliferate in a medium containing 10% FBS and at a temperature of 31–33°C. The cells stop proliferating and differentiate when they are cultured for 3–4 weeks at 37–39°C in medium containing 1% FBS [12, 13, 23]. The number of viable cells decreases during differentiation because expression of the immortalizing large T antigen is down-regulated [12, 23]. In the present study, compared with undifferentiated cells cultured under permissive conditions (Fig. 1a), differentiated cells cultured under non-permissive conditions had larger cell bodies and a multipolar morphology 3 weeks after the temperature shift (Fig. 1b). This morphological change correlated with expression of neuronal markers (Fig. 1c). Expression of TH, a dopaminergic neuron-specific marker, in undifferentiated CSM14.1 cells was very low or absent. However, TH expression increased from 2 weeks after the temperature shift. Similarly, no expression of βIII-tubulin, a microtubule element expressed in neurons, was detected in undifferentiated CSM14.1 cells; however, it was expressed at 3 weeks after the temperature shift. These changes in morphology and the expression of neuronal markers are consistent with those reported in previous studies [12, 23]. For all subsequent experiments, CSM14.1 cells cultured at 31°C in medium containing 10% FBS were used as the source of undifferentiated cells and cells cultured for 3 weeks at 37°C in medium containing 1% FBS as the source of differentiated cells.

Figure 1.

Differentiation of CSM14.1 cells. (a) Undifferentiated CSM14.1 cells cultured under permissive conditions (31°C in DMEM containing 10% FBS). (b) Differentiated cells cultured for 3 weeks under non-permissive conditions (37°C in DMEM containing 1% FBS) showed enlarged cell bodies. Scale bars in (a) and (b), 100 µm. (c) Western blot analysis of the neuronal markers TH and βIII-tubulin in CSM14.1 cell lysates. Differentiated CSM14.1 cells expressed TH and βIII-tubulin. Liver and mid-brain tissues were collected from F344 rats. P, post-natal day.

Susceptibility of undifferentiated and differentiated CSM14.1 cells to Japanese encephalitis virus infection

The susceptibility of CSM14.1 cells to JEV infection was examined next (Figs 2, 3). Undifferentiated and differentiated CSM14.1 cells were infected with JEV at an MOI of 5 p.f.u./cell (the titer was determined using Vero cells) and then incubated for 2 days under permissive or non-permissive conditions, respectively. Virus-infected cells were identified by immunofluorescence staining with an anti-JEV antibody. At 48 hr p.i., the mean percentage of undifferentiated cells that were positive for viral antigens was 15.7% (Fig. 2a,c); however, only 0.84% (mean value) of differentiated cells were positive (Fig. 2b,c). Morphologically, JEV-induced CPEs started to appear at 72 hr p.i. in the undifferentiated cells, but no CPE were noted in the differentiated cultures.

Figure 2.

Expression of viral antigens in JEV-infected undifferentiated and differentiated CSM14.1 cells. (a) Undifferentiated and (b) differentiated cells were infected at an MOI of 5 p.f.u./cell. Viral antigens were visualized at 48 hr p.i. by indirect immunofluorescence staining with rabbit anti-JEV serum (red). Nuclei were stained with DAPI (blue). Scale bars, 100 µm. (c) The mean percentage of infected cells was determined using Image J software. Error bars represent the standard error of the mean (n = 4). (d) Viral protein expression in infected and mock-infected CSM14.1 cells was analyzed by western blot analysis with rabbit anti-JEV serum. Samples were separated by a 7.5% polyacrylamide gel under non-reducing conditions. M, mock-infected cells.

Figure 3.

JEV replication in undifferentiated and differentiated CSM14.1 cells. (a) Growth of JEV in undifferentiated and differentiated CSM14.1 cells. (b) Growth of JEV in Vero cells cultured at 31°C or 37°C. (c) Growth of JEV in C6/36 cells cultured at 31°C or 37°C. Culture supernatants were collected at various time-points post-JEV infection (MOI = 5 p.f.u./cell) and analyzed for virus production in a Vero cell-based plaque assay. The amount of virus was normalized to the viable cell number at the time of infection, and expressed as log10 p.f.u./per cell. Error bars represent standard deviations. Each point represents the mean of three measurements. P values were determined by Student's t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Western blot analysis of viral proteins showed that undifferentiated cells expressed detectable amounts of E, NS1, and PreM proteins at 24 hr p.i., with higher degrees of expression being noted at 48 hr p.i. (Fig. 2d, left half of the gel). Expression of these viral proteins was also observed in differentiated cells at 24 hr p.i. (Fig. 2d, right half); however, the degree of expression was much lower than that in undifferentiated cells.

Next, production of infectious virus was examined (Fig. 3). The amount of infectious virus in the culture supernatant from undifferentiated cells increased at 12 hr p.i., peaked at approximately 101.9 p.f.u./cell by 72 hr p.i., and then gradually decreased. On the other hand, the amount of infectious virus in the culture supernatant from differentiated cells increased from 24 hr p.i., reaching a plateau of 10−0.2 p.f.u./cell by 48 hr p.i. (Fig. 3a). To determine whether these differences in the amount of viral replication were due to temperature or the differentiation state of the cells, infected Vero cells and C6/36 cells were cultured at 31°C and 37°C. Virus production was then examined over 120 hr (Fig. 3b,c). There was no difference in the peak level of viral replication at 31°C or 37°C in both Vero and C6/36 cells, suggesting that the higher temperature was not the reason for reduced viral replication in differentiated cells. Thus, differentiated CSM14.1 cells produced much less virus than did undifferentiated cells, indicating that they are less susceptible to JEV infection.

Japanese encephalitis virus internalization in undifferentiated and differentiated CSM14.1 cells

Next, an infectious virus recovery assay [28, 29] was used to examine differences in the ability of undifferentiated and differentiated CSM14.1 cells to internalize JEV by endocytosis (Fig. 4a). In this assay, virus is allowed to attach to the cell surface at 4°C for 2 hr. Internalization is triggered by increasing the temperature to 31°C or 37°C. At various time-points after each temperature shift, the cells were treated with an acidic buffer to inactivate any virus remaining on the cell surfaces. The cells were then harvested and the cell membranes disrupted by freeze-thawing. Because endocytosed virions possess envelopes during the early phase of entry, infectivity can be detected using a plaque assay. Vero cells, which are highly susceptible to JEV [27], were used as a reference in this assay. At 0 min, no (or few) infectious viruses were detected in Vero cells or undifferentiated or differentiated CSM14.1 cells (Fig. 4b). The virus titer in undifferentiated CSM14.1 cells reached a peak at 30 min and was comparable with the peak titer observed in Vero cells at 15 min (Fig. 4b). On the other hand, the virus titer recovered from differentiated CSM14.1 cells was lower than that recovered from undifferentiated cells at all time-points later than 15 min, and peaked much later (Fig. 4b).

Figure 4.

Internalization of JEV into undifferentiated and differentiated CSM14.1 cells. (a) Schematic illustration of the infectious virus recovery assay. (b) Undifferentiated and differentiated CSM14.1 cells or Vero cells were infected with JEV at an MOI of 10 p.f.u./cell at 4°C for 2 hr. The cells were shifted to 31°C (undifferentiated CSM14.1) or 37°C (differentiated CSM14.1 and Vero cells) to allow viral internalization. At the indicated times, any virus remaining on the cell surfaces was inactivated in an acidic buffer. Internalized infectious viruses were detected in a plaque assay. The results were normalized to the viable cell number at the time of inoculation and expressed as number of p.f.u./104 cells. Error bars show standard deviations. The graphs show the mean of three measurements.

Japanese encephalitis virus induces interferon expression in undifferentiated and differentiated CSM14.1 cells

Type 1 IFNs protect against many types of virus infection. Therefore, we next examined whether IFNs, especially IFN-β, a type I IFN the expression of which is triggered by virus infection [30], play a role in the susceptibility of undifferentiated and differentiated CSM14.1 cells to infection by JEV. First, the amounts of IFN mRNA expressed in undifferentiated and differentiated CSM14.1 cells at 12, 24, and 48 hr p.i. were compared using semi-quantitative RT-PCR (Fig. 5a). Total RNA samples collected from harvested cells mock-infected with diluents (no virus) for 1 hr were used as mock-infection controls. Mock-infected undifferentiated and differentiated cells showed constitutive expression of IFN-α. The amount of IFN-α expression in undifferentiated cells increase slightly at 48 hr p.i.; however, a similar increase was observed in differentiated cells from 24 hr p.i. The difference in JEV-induced IFN-β expression was more pronounced. IFN-β expression was detected in undifferentiated cells from 48 hr p.i.; however, greater induction was observed from 24 hr p.i. in differentiated cells, despite the finding that differentiated cells expressed smaller amounts of JEV RNA than undifferentiated cells at both 24 and 48 hr p.i. Neither undifferentiated nor differentiated CSM 14.1 cells expressed IFN-γ (data not shown).

Figure 5.

IFN expression in undifferentiated and differentiated CSM14.1 cells infected with JEV. Cells were infected with JEV at an MOI of 5 p.f.u./per cell. (a) The amounts of IFN-α mRNA, IFN-β mRNA and JEV RNA were examined by RT-PCR at the indicated times. (b) Western blot analysis of Stat1 and phosphorylated (Tyr701) Stat1 expression at the indicated times after infection. The results are representative of two independent experiments. M, mock-infected cells.

Because the transcription factor, Stat1, is a major substrate for tyrosine phosphorylation subsequent to the binding of IFNs to their receptor, Stat1 phosphorylation was next analyzed by western blotting (Fig. 5b). In undifferentiated cells, an increase in Stat1 (Tyr701) phosphorylation was observed at 48 hr p.i. On the other hand, the increase in Stat1 phosphorylation occurred earlier (from 24 hr p.i.) in differentiated cells. The timing of this increase was consistent with induction of IFN-β mRNA expression. The total amount of Stat1 protein in differentiated cells increased at 48 hr p.i., probably induced by increased IFN signaling. These results suggest that more rapid and stronger induction of IFN-β and IFN signaling may be related to the lower degree of viral replication in differentiated CSM14.1 cells.

DISCUSSION

The present study showed that both undifferentiated and differentiated CSM14.1 cells are susceptible to infection by JEV; however, undifferentiated cells are more susceptible and produce more progeny virus. Differentiation of CSM14.1 cells, which phenotypically show a more mature neuron-like morphology and increased expression of the neuronal markers, TH and βIII-tubulin, resulted in decreased susceptibility to JEV. This suggests that CSM14.1 cells could be a useful model system for studying JEV infection of immature and mature neurons in rat brains [9].

The correlation between viral susceptibility and differentiation status observed in the present study is similar to that previously reported by Vernon and Griffin [23]. The latter study showed that SV (a neurotropic alphavirus that shows an age-dependent pattern of resistance to infection in mice; reviewed in reference [31]) infects and replicates more efficiently in undifferentiated CSM14.1 cells than in differentiated CSM14.1 cells.

The factors underlying the differentiation-dependent resistance of CSM14.1 cells to virus infection are not well understood. The infectious virus recovery assay performed in the present study showed that the amount of virus recovered from differentiated cells was less than that recovered from undifferentiated cells, suggesting that factors that regulate virus attachment and/or entry may be responsible for the observed differences in JEV susceptibility. We also found that expression of IFN-β mRNA, as well as IFN-induced signaling, was more rapid and stronger in differentiated cells than in undifferentiated cells, even though the amounts of JEV RNA were lower. This correlation between IFN expression and JEV susceptibility is consistent with the findings of a previous study, which suggested that type I IFNs regulate flavivirus infection. Crance et al. showed that type I IFN selectively inhibit the replication of 11 pathogenic flaviviruses in Vero cells, including JEV, because it is able to reduce the viral titer and virus-mediated CPE [32]. In addition, Lobigs et al. demonstrated that infecting mice deficient in type I IFN responses with Murray Valley encephalitis virus (the closest relative to JEV) results in massive extra-neural virus growth and rapid neuroinvasion and replication within the brain [33]. However, the precise mechanisms underlying differentiation-dependent differences in IFN responses and the efficiency of viral entry observed in the present study require further in-depth investigation.

Similarly to mature neurons, which are terminally differentiated and non-dividing, differentiated CSM14.1 cells do not proliferate under non-permissive culture conditions; however, undifferentiated CSM14.1 cells do proliferate under permissive culture conditions. Therefore, the difference in cell proliferation rate between undifferentiated and differentiated cells may have led to differences in viral replication. However, the resistance of mature rat neurons to JEV infection is not due to the inability of cells to proliferate; rather, it is maturation-dependent. This has been shown in a previous study that used primary mixed cultures of fetal rat cortical neurons; primary neurons that did not divide and proliferate (but were highly susceptible to JEV) showed a marked reduction in their susceptibility to JEV infection after maturation during culture for 3 weeks [10].

In conclusion, we characterized the susceptibility of undifferentiated and differentiated CSM14.1 cells to JEV infection and found that the cells became more resistant to infection as they matured. Thus, this cell culture system may be useful for studying the molecular mechanisms underlying the maturation state-specific resistance of neurons to JEV and serve as an in vitro model that can be used to further examine age-dependent resistance to JEV infection.

ACKNOWLEDGMENTS

This work was supported in part by Grants-in-Aid for Challenging Exploratory Research (T.K.) (Grant no. 21658102), from the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases (T.K., M.O., and H.S.), and from the Japan Initiative for Global Research Network of Infectious Diseases (J-GRID) (T.K., M.S., and H.S.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

DISCLOSURE

The authors declare no academic or financial conflicts of interest.

Ancillary