Amino Acid at Position 166 of NS2A in Japanese Encephalitis Virus (JEV) Is Associated with In Vitro Growth Characteristics of JEV

We previously showed that the growth ability of the Japanese encephalitis virus (JEV) genotype V (GV) strain Muar is clearly lower than that of the genotype I (GI) JEV strain Mie/41/2002 in murine neuroblastoma cells. Here, we sought to identify the region in GV JEV that is involved in its low growth potential in cultured cells. An intertypic virus containing the NS1-3 region of Muar in the Mie/41/2002 backbone (NS1-3Muar) exhibited a markedly diminished growth ability in murine neuroblastoma cells. Moreover, the growth rate of a Muar NS2A-bearing intertypic virus (NS2AMuar) was also similar to that of Muar in these cells, indicating that NS2A of Muar is one of the regions responsible for the Muar-specific growth ability in murine neuroblastoma cells. Sequencing analysis of murine neuroblastoma Neuro-2a cell-adapted NS1-3Muar virus clones revealed that His-to-Tyr mutation at position 166 of NS2A (NS2A166) could rescue the low replication ability of NS1-3Muar in Neuro-2a cells. Notably, a virus harboring a Tyr-to-His substitution at NS2A166 (NS2AY166H) showed a decreased growth ability relative to that of the parental virus Mie/41/2002, whereas an NS2AMuar-based mutant virus, NS2AMuar-H166Y, showed a higher growth ability than NS2AMuar in Neuro-2a cells. Thus, these results indicate that the NS2A166 amino acid in JEV is critical for the growth and tissue tropism of JEV in vitro.


Introduction
Japanese encephalitis virus (JEV) is a mosquito-borne arbovirus that causes the serious neurological disorder Japanese encephalitis (JE). JE represents a major public health problem in Asia, with an estimated 68,000 cases of JE per year resulting in 15,000 fatalities, mostly in children [1][2][3]. JEV belongs to the genus Flavivirus in the family Flaviviridae and is amplified in a bird-and pig-mosquito transmission cycle [4]. The mosquitoes transmit JEV to dead-end host humans.
JEV harbors a single-stranded, positive-sense RNA genome that encodes three structural proteins (C, prM, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) from a single open reading frame, and the genome also contains noncoding regions (NCRs) at its 5and 3 -terminal ends. Based on genomic RNA homology, JEV strains are classified into five genotypes: GI, GII, GIII, GIV, and GV [5,6]. GI and GIII strains have been the most widely distributed strains in JE-endemic areas since the 1990s [7,8]; GII strains constitute the third most common group and have been detected in Indonesia, Singapore, South Korea, Malaysia, and Australia [9]; GIV strains have been isolated only in Indonesia [9]; and GV strains also represent an extremely rare group-the first GV JEV strain (Muar) was isolated from a patient with encephalitis in Malaysia in 1952, but then no other GV JEV isolates were identified for >50 years [6]. However, in 2009, an infectious GV JEV strain (XZ0934) was isolated from a Culex mosquito pool in China [10], and a GV JEV strain was also detected in Korea in 2010 [11]. In Korea, GI, GIII, and GV JEV strains have been endemic in recent years, but GV is currently the main genotypic strain circulating in Korea [12,13]. Furthermore, GV JEV was isolated from a JE patient in 2015 [14].
JE vaccines currently available are derived from GIII strains. These JE vaccines were previously reported to potentially exhibit less ability to induce neutralizing antibodies against GV JEV than those against other JEV genotypes [15,16]. Conversely, IgGs raised against GV JEV XZ0934 were reported to show a poor neutralizing ability against GIII JEV [17]. These findings raise the possibility that GV JEV is distinct from other JEV genotypes in terms of antigenicity, and the current GIII-derived JE vaccines might thus provide inadequate levels of protection against GV JEV. The low identity in amino acid sequences between GV and GIII JEV could be one reason for the poor efficacy of the GIII-derived vaccines against GV JEV [16]. Therefore, attention must be paid to the dynamics of the circulating JEV strains in JE-endemic areas.
The characteristics of GV JEV remain poorly understood because only a few GV JEV strains have been isolated to date and only limited studies have been performed on these strains. We previously showed that the neuroinvasiveness of GV JEV Muar in mice was substantially higher than that of GI JEV Mie/41/2002 [16]. Moreover, it was shown that a recombinant GV JEV of the XZ0934 strain exhibited higher neuroinvasiveness in mice than recombinant GIII JEV, and that the structural protein region (C-prM-E) of GV JEV plays a critical role in the increased pathogenicity of GV JEV [17]. We also reported that the E and prM proteins of GV JEV are responsible for the highly virulent characteristics of GV JEV in a mouse model [18].
The Muar strain also presents unique features in in vitro growth. The growth ability of Muar in mouse neuroblastoma cells was found to be clearly lower than that of Mie/41/2002 [16,18]. Our findings also showed that the 5'-NCR-C-prM-E region of Muar, which is responsible for the high neuroinvasiveness of the strain, does not influence the growth characteristics of Muar in murine neuroblastoma cells [18]. Thus, our previous data indicate that the growth properties of JEV in mouse neuronal cells do not necessarily reflect pathogenicity in mice. However, the growth characteristics of Muar in vitro is of great interest in understanding the tissue tropism of JEV. To gain insight into the nature of Muar in in vitro growth, we sought to identify the region in GV JEV responsible for its low growth ability in cultured cells, particularly in murine neuroblastoma cells. Thus, we generated recombinant intertypic chimeric and missense mutant JEV strains in the backbone of the GI JEV strain Mie/41/2002 using a reverse-genetics system that we previously established, and then analyzed the mechanism for the growth properties of these JEV strains in vitro.

Viruses
The GI JEV strain Mie/41/2002 (GenBank accession no. AB241119), which was isolated from pig serum in Japan in 2002 [19,20], and the GV JEV strain Muar (GenBank accession no. HM59272), which was isolated from a patient with encephalitis in Malaysia in 1952, were used [16]. The working virus stocks were prepared through amplification in Vero cells.

Plaque-Formation Assay and Analysis of Growth Kinetics
Infectious viral titers for each sample were determined using a plaque-formation assay; for the assay, Vero cells (~3 × 10 5 /well) were plated in 12-well plates and inoculated with each of the viruses for 1 h at 35-37 • C. Next, an MEM-based overlay medium containing 1% methyl cellulose and 2% FBS was added to the wells, and the cells were incubated for 5 days at 35-37 • C, after which the cells were fixed using a 10% formalin-PBS solution and stained with methylene blue, as described previously [21].
The growth of the JEV strains was analyzed as described previously [20]. Briefly, cells were cultured in 6-well culture plates and infected with each JEV strain in MEM supplemented with 2% FBS (2F/MEM) at a multiplicity of infection (MOI) of 0.1 plaque-forming units (PFU)/cell. Small aliquots of the media were collected at one-day intervals, and the infectious viral titers were determined using the plaque-formation assay in Vero cells, as described above.

Adaptation of Virus to Mouse Neuroblastoma Cells
NS1-3 Muar (v1) was inoculated into Neuro-2a cells (in 6-well plates) at an MOI of 0.1 PFU/cell, and the cells were incubated for 3 days at 35-37 • C. The culture supernatant (p1) was recovered and a small aliquot (200 µL) was used for the next inoculation into Neuro-2a cells in 6-well plates, which were then incubated for 3 days at 35-37 • C. The passaging was repeated ten times (p1 to p10).

Plaque Cloning (Limiting-Dilution Method)
NS1-3 Muar p10 solution was diluted to 5 PFU/mL with 2F/MEM; the diluted virus solution (50 µL = 0.25 PFU) was added to Vero cells cultured in 12-well plates, and the cells were incubated at 35-37 • C for 6-7 days. Culture supernatants were recovered and used in the plaque-formation assay to assess the plaque sizes of the viruses and to measure the infectious titer in the solutions, as described above.

Statistical Analysis
Viral titers for each sample determined by plaque-forming assay as described above were statistically compared by using BellCurve for Excel (Social Survey Research Information, Tokyo, Japan).
Data were presented as mean ± standard deviation of the three independent experiments. Significance was analyzed using the two-way ANOVA test.

Adaptation of NS1-3 Muar to Mouse Neuroblastoma Neuro-2a Cells
To search for the site critical for the reduction of growth potency in murine neuroblastoma cells, we generated a mutant NS1-3 Muar adapted to Neuro-2a cells by continuously passaging NS1-3 Muar in Neuro-2a cells ten times (p10) ( Figure 4A). The growth rate of this virus, NS1-3 Muar p10, was clearly higher than that of the original virus, NS1-3 Muar v1, which indicated that NS1-3 Muar p10 acquired a higher replication ability in Neuro-2a cells ( Figure 4B). The complete nucleotide sequence of the NS1-3 Muar p10 genome was determined, revealing that there were two differences in the nucleotide sequence, one was located in the prM region and the other was located in NS2A region, between NS1-3 Muar v1 and NS1-3 Muar p10 ( Figure 4C). The two nucleotide mutations identified in NS1-3 Muar p10 were also accompanied by amino acid substitutions, Asn-to-Lys at position 60 of prM (prM 60 ) and His-to-Tyr at position 166 of NS2A (NS2A 166 ), which suggested that these amino acid alterations could be associated with acquiring the increased growth ability of NS1-3 Muar p10 in Neuro-2a cells. Intriguingly, the infectious titers of the viruses passaged in Neuro-2a cells increased clearly between the oncepassaged virus (p1) and the 5-times-passaged virus (p5), which implied that the virus adapted to Neuro-2a cells in the early stage of repeated passaging ( Figure 4D). Thus, we determined the nucleotide sequences of the positions corresponding to prM 60 and NS2A 166 in NS1-3 Muar p3, p5, p7,

Adaptation of NS1-3 Muar to Mouse Neuroblastoma Neuro-2a Cells
To search for the site critical for the reduction of growth potency in murine neuroblastoma cells, we generated a mutant NS1-3 Muar adapted to Neuro-2a cells by continuously passaging NS1-3 Muar in Neuro-2a cells ten times (p10) ( Figure 4A). The growth rate of this virus, NS1-3 Muar p10, was clearly higher than that of the original virus, NS1-3 Muar v1, which indicated that NS1-3 Muar p10 acquired a higher replication ability in Neuro-2a cells ( Figure 4B). The complete nucleotide sequence of the NS1-3 Muar p10 genome was determined, revealing that there were two differences in the nucleotide sequence, one was located in the prM region and the other was located in NS2A region, between NS1-3 Muar v1 and NS1-3 Muar p10 ( Figure 4C). The two nucleotide mutations identified in NS1-3 Muar p10 were also accompanied by amino acid substitutions, Asn-to-Lys at position 60 of prM (prM 60 ) and His-to-Tyr at position 166 of NS2A (NS2A 166 ), which suggested that these amino acid alterations could be associated with acquiring the increased growth ability of NS1-3 Muar p10 in Neuro-2a cells. Intriguingly, the infectious titers of the viruses passaged in Neuro-2a cells increased clearly between the once-passaged virus (p1) and the 5-times-passaged virus (p5), which implied that the virus adapted to Neuro-2a cells in the early stage of repeated passaging ( Figure 4D). Thus, we determined the nucleotide sequences of the positions corresponding to prM 60 and NS2A 166 in NS1-3 Muar p3, p5, p7, and p9 ( Figure 4E); this revealed that the Asn-to-Lys substitution at the nucleotide level in prM 60 of NS1-3 Muar occurred between p5 and p7, whereas the His-to-Tyr substitution in NS2A 166 occurred before p3. The amino acid residue at prM 60 was Asn in Mie/41/2002, Muar, and NS1-3 Muar v1 but Lys in NS1-3 Muar p10 ( Figure 4F and Supplementary Figure S1), whereas the NS2A 166 residue was His in Muar and NS1-3 Muar v1 but Tyr in NS1-3 Muar p10 and Mie/41/2002 (Supplementary Figure S2). These results suggest that the His-to-Tyr mutation in NS1-3 Muar p10 is mainly associated with the adaptation of the virus to Neuro-2a cells.

Characterization of Neuro-2a-Adapted NS1-3 Muar p10 Clones
The results of plaque assays performed using Vero cells suggested that NS1-3 Muar p10 includes at least two distinct types of viruses: native-sized-plaque and pin-sized-plaque viruses ( Figures 4A  and 5A). Small plaques were observed with NS1-3 Muar p7 and p10 viruses but not p3 and p5 viruses (Supplementary Figure S3). To clarify the nucleotide sequence of the native-sized-plaque and pin-sized-plaque variants, single-clone viruses were obtained from the NS1-3 Muar p10 solution using a limiting-dilution method. Five pin-sized-plaque and native-sized-plaque clones were obtained, and the nucleotide sequences of the prM and NS2A regions of the clones were determined (Table 1). All five clones that formed native-sized plaques and 4/5 clones that formed pin-sized plaques featured an amino acid change at NS2A 166 (His-to-Tyr), whereas 4/5 clones that formed pin-sized plaques and only 1-2/5 clones that formed native-sized plaques featured a change at prM 60 (Asn-to-Lys). We also found an additional amino acid substitution (Gln to Arg) at position of 7 of prM (prM 7 ) in all five native-sized-plaque clones, but this alteration was not detected in NS1-3 Muar p10 and the pin-sized-plaque clones. To further examine the characteristics of these clones, we selected two clones: pin-sized-plaque clone #17 and native-sized-plaque clone #7 (Table 1 and Figure 5A). NS1-3 Muar p10 and pin-sized-plaque clone #17 grew slightly slower than NS1-3 Muar v1 and native-sized-plaque clone #7 in Vero cells ( Figure 5B). By contrast, NS1-3 Muar p10 and both selected clones, #7 and #17, showed a clearly increased replication ability in Neuro-2a cells as compared with that of NS1-3 Muar v1 ( Figure 5C). The results indicate that the His-to-Tyr substitution at NS2A 166 in NS1-3 Muar p10 is critical for the efficient growth of NS1-3 Muar in Neuro-2a cells and also suggest that the Tyr residue at NS2A 166 in Mie/41/2002 plays a key role in acquiring the high replication ability of this strain in murine neuroblastoma cells.

Characterization of Neuro-2a-Adapted NS1-3 Muar p10 Clones
The results of plaque assays performed using Vero cells suggested that NS1-3 Muar p10 includes at least two distinct types of viruses: native-sized-plaque and pin-sized-plaque viruses (Figures 4A  and 5A). Small plaques were observed with NS1-3 Muar p7 and p10 viruses but not p3 and p5 viruses (Supplementary Figure S3). To clarify the nucleotide sequence of the native-sized-plaque and pinsized-plaque variants, single-clone viruses were obtained from the NS1-3 Muar p10 solution using a limiting-dilution method. Five pin-sized-plaque and native-sized-plaque clones were obtained, and the nucleotide sequences of the prM and NS2A regions of the clones were determined (Table 1). All five clones that formed native-sized plaques and 4/5 clones that formed pin-sized plaques featured an amino acid change at NS2A 166 (His-to-Tyr), whereas 4/5 clones that formed pin-sized plaques and only 1-2/5 clones that formed native-sized plaques featured a change at prM 60 (Asn-to-Lys). We also found an additional amino acid substitution (Gln to Arg) at position of 7 of prM (prM 7 ) in all five native-sized-plaque clones, but this alteration was not detected in NS1-3 Muar p10 and the pin-sizedplaque clones. To further examine the characteristics of these clones, we selected two clones: pinsized-plaque clone #17 and native-sized-plaque clone #7 (Table 1 and Figure 5A). NS1-3 Muar p10 and pin-sized-plaque clone #17 grew slightly slower than NS1-3 Muar v1 and native-sized-plaque clone #7 in Vero cells ( Figure 5B). By contrast, NS1-3 Muar p10 and both selected clones, #7 and #17, showed a clearly increased replication ability in Neuro-2a cells as compared with that of NS1-3 Muar v1 ( Figure  5C). The results indicate that the His-to-Tyr substitution at NS2A 166 in NS1-3 Muar p10 is critical for the efficient growth of NS1-3 Muar in Neuro-2a cells and also suggest that the Tyr residue at NS2A 166 in Mie/41/2002 plays a key role in acquiring the high replication ability of this strain in murine neuroblastoma cells.    (Figures 1 and 6A). NS2A Y166H grew slightly slower than Mie/41/2002 in Vero cells ( Figure 6B), and the growth of the mutant was also strikingly slower than that of Mie/41/2002 in Neuro-2a cells ( Figure 6C). Moreover, we produced an additional site-directed mutant, JEV NS2A Muar-H166Y , and investigated how this mutation influences the growth of NS2A Muar in vitro ( Figure 6D-F). NS2A Muar-H166Y showed higher growth ability than NS2A Muar in Neuro-2a cells, although the two viruses exhibited nearly equal growth kinetics in Vero cells. These results suggest that NS2A 166 affects the growth of JEV in murine neuroblastoma cells.

Discussion
The GV strain Muar shows lower growth potential in murine neuronal cells than GI Mie/41/2002 [16]. This study was conducted to identify the genomic region that is related to this difference in in vitro growth property between the strains. It was shown that NS2A of Muar was one of the regions responsible for the Muar-specific lower growth ability in murine neuroblastoma cells. Furthermore, sequencing analysis of revertant viruses derived from NS1-3 Muar revealed that His-to-Tyr substitution at position 166 of NS2A Muar rescued the low replication ability of the NS1-3 Muar mutant. Thus, the results indicate that the NS2A 166 amino acid in JEV influences the growth and tissue tropism of JEV in vitro. Although previous study indicates that amino acid sequences in the E protein are involved in the adaptation of JEV in Neuro-2a cells [22], no differences in E region amino acid sequences were found between NS1-3 Muar v1 and the NS1-3 Muar p10. An Asn-to-Lys mutation at prM 60 was found in NS1-3 Muay p10, which may be associated with the appearance of the pin-sized plaque variants. In Neuro-2a cells, the growth rate of NS1-3 Muar p10 #17 clone, with Lys at prM 60 , was similar to that of NS1-3 Muar p10 #7 clone, with Asn at prM 60 ( Figure 5C). Therefore, it appears that the mutation at prM 60 is not primarily involved in the increased growth ability of NS1-3 Muar p10 in Neuro-2a cells. However, pin-sized viruses were a major component of the p10 (Figure 4), implying that the prM 60 mutation in NS1-3 Muar may confer an advantage in propagating NS1-3 Muar in Neuro-2a cells over continuous passages. Flavivirus NS2A is a highly hydrophobic transmembrane protein that localizes in the endoplasmic reticulum (ER) membrane and interacts with virus genomic RNA and other structural and nonstructural viral proteins [23][24][25][26][27]. NS2A has several functions throughout the virus life cycle and pathogenesis: viral RNA synthesis [23,25,28], virion assembly and secretion [25][26][27][28][29][30][31], escape from host immunity [32][33][34][35][36], and pathogenicity in vitro and in vivo [37][38][39]. On the other hand, the cellular factor tripartite motif-containing protein 25 (TRIM52) interacts with and degrades JEV NS2A, which results in the inhibition of JEV replication [40]. Furthermore, the nucleotide sequence of the JEV NS2A-coding region is also involved in the expression of the NS1' protein [41,42]. In dengue virus NS2A, the N-terminal 68 amino acids are located in the ER lumen, the core region (amino acids 69 to 209) integrally spans the ER membrane, and the C-terminal tail (amino acids 210 to 218) is located in the cytosol [43]. The results of previous studies and our result of SOSUI (classification and secondary structure prediction system for membrane proteins) analysis indicate that JEV NS2A contains eight predicted transmembrane segments (pTMS1-8) (Supplementary Figure S2) [43,44].
The growth of the NS1-3 Muar mutant, which exhibited poor replication ability in murine neuroblastoma cells, was rescued when an additional mutation was introduced in the NS2A region (NS2A H166Y ). The results suggest that NS2A 166 is one of the sites responsible for the replication efficiency of the virus in mouse neuroblastoma cells. The NS2A Y166H substitution in Mie/41/2002 also drastically decreased its growth ability in Neuro-2a cells (~1 × 10 3 -fold) ( Figure 6C), but the degree of change in growth rate between NS2A Muar and NS2A Muar-H166Y was low (~1 × 10 1 -fold) ( Figure 6F). These results imply that factors other than NS2A 166 including other sites in Muar NS1-3 and cellular factors are associated with the decreased growth potential of Muar.
A single amino acid substitution, Phe-to-Leu substitution at position 113 in NS2A (NS2A 113 ) of GIII JEV affects viral growth in Neuro-2a cells [44]. The amino acid residue at NS2A 113 in Mie/41/2002 and Muar is Phe, and the residue at NS2A 113 also appears to be conserved in GI, GIII, and GV JEVs (Supplementary Figure S2); thus, NS2A 113 does not likely contribute to the disparity in growth ability demonstrated in Mie/41/2002 and Muar. However, NS2A of JEV may play a key role in the efficient replication of JEV in murine neuronal cells [44]. The NS1-to-NS3 region of Muar is not associated with the high-virulence phenotype of Muar, which indicates that the growth property of NS2A-associated mutants in mouse neuroblastoma cells is unrelated to the virulence of Muar in mice [18]. The Phe-to-Leu or Leu-to-Phe substitutions at NS2A 113 in GIII JEV strains does not affect their pathogenicity in mice, which supports the notion that virus propagation in vitro does not directly reflect virus replication and pathogenicity in vivo. The pathology caused by JEV infection is complex, and numerous factors-replication of the virus and invasion of the immune cells, cytokines, and chemokines in the brain-are involved in the pathogenicity of JEV.
Flavivirus NS2A has been comprehensively analyzed genetically and biochemically [24,[26][27][28]39,43,45]. Zheng et al. conducted systematic alanine-scanning mutagenesis of Zika virus NS2A and classified the phenotypes of the mutants into five groups [27]. An Arg-to-Ala mutation in Zika virus NS2A (NS2A R171A ), in which the mutation site is near JEV NS2A 166 , belongs to a group of mutants defective for virion assembly, and this suggests that NS2A 166 in JEV may also be associated with the virion assembly of JEV. Alanine-scanning mutagenesis was also performed in dengue virus NS2A, and a Leu-to-Phe mutation in NS2A (NS2A L181F ) was identified to act as a compensatory mutation in several NS2A mutants bearing alanine substitutions [28,39]. JEV NS2A 166 is located in pTMS7 (amino acids 164 to 183) (Supplementary Figure S2), and dengue virus NS2A 181 is also located in pTMS7, which corresponds to pTMS7 of JEV NS2A. The findings for dengue virus NS2A and the results in this study suggest that pTMS7 interacts intramolecularly with other pTMSs of NS2A and regulates the function of NS2A in virus replication [28,39].
Dengue virus NS2A pTMS7 (amino acids 165 to 186) traverses the ER membrane, whereas Zika virus NS2A pTMS6 (amino acids 152 to 185) interacts peripherally with the ER membrane [27,43]. A recent study showed that dengue virus NS2A cannot trans-complement a virion assembly-defective NS2A mutant of Zika virus [45]. These findings suggest that different flavivirus NS2A molecules feature distinct membrane topologies and that each pTMS functions in a virus-type-specific manner. Thus, the findings in dengue virus and Zika virus NS2A might not be readily applicable to other flavivirus NS2A molecules. Clarification of the topology of JEV NS2A in the ER membrane is necessary for understanding the role of pTMS7 of JEV NS2A.
The differences in in vitro growth kinetics between the GI strain Mie/41/2002 and the GV strain Muar, NS1-3 Muar , or NS2A Muar were more notable in mouse neuroblastoma cells than in Vero cells. These results imply that JEV NS2A is involved in the tropism of JEV. The ability to bind to the ER membrane of the cells used in this study might differ between Mie/41/2002 and Muar or Muar NS2A-bearing recombinant JEV strains. Little is currently known regarding the cellular factors that interact with NS2A, and identification of the host-cell molecules that interact with flavivirus NS2A will facilitate elucidation of the role of NS2A in cell tropism. Several lines of evidence indicate that flavivirus NS2A plays a role in inhibiting host interferon (IFN) responses [32][33][34][35][36], and JEV NS2A also blocks the host antiviral response of IFN-induced double-stranded RNA-activated protein kinase during JEV infection [35], which suggests that the NS2A 166 amino acid might affect the ability of JEV NS2A to interfere with the host immune response. However, Vero cells are IFN-deficient and Neuro-2a cells infected with JEV do not secrete IFNβ1 [46], indicating that type I IFN-dependent signaling is blocked in both Vero cells and Neuro-2a, and that signaling pathways downstream of type I IFN may not be associated with the difference between the Muar and Mie/41/2002 strains. Activating transcription factor 3 (ATF3), which is induced by the infection of JEV, inhibits cellular antiviral signaling in Neuro-2a cells [46], suggesting that the ATF3 induction level might differ between Muar-and Mie/41/2002-infected Neuro-2a cells. In contrast to the growth data obtained in Vero and Neuro-2a cells, our preliminary data indicated that NS1-3 Muar v1 grew faster than NS1-3 Muar p10 in human lung cancer A549 cells, which retain intact signaling pathways related to the innate immune response (unpublished data). Further analysis of several cell types will be necessary to clarify whether the growth properties of Muar are specific to mouse neuronal cells. In this study, we examined viral growth by measuring the amount of infectious virus secreted into the culture medium. Analyses of the viral RNA and infectious particles in the JEV-infected cells will also be required to understand the different effects of NS2A on the growth of Muar and Mie/41/2002.
In addition, although both the NS1-3 and NS4A-5 regions are related to the low growth capacity of Muar in vitro (Figure 2), we only focused on the former region in this study. Analysis of the NS4A-5 region is also needed for a comprehensive understanding the growth nature and tissue tropism of JEV.