Superinfection Exclusion Between Japanese Encephalitis Virus Genotypes I and III Occurs During Viral Entry

Abstract

Superinfection exclusion occurs when a primary viral infection prevents subsequent infection of the same cell. Japanese encephalitis virus (JEV) comprises several genotypes that differ primarily in their envelope protein sequences, and a shift in predominance from genotype III (GIII) to genotype I (GI) has been observed in nature. However, the mechanisms contributing to this genotype replacement remain unclear. In this study, we examined superinfection exclusion between JEV GI and GIII in mammalian BHK-21 cells and mosquito C6/36 cells. Reciprocal exclusion was observed in both cell types. Further analysis in BHK-21 cells, using GIII as the primary virus and GI as the secondary virus, indicated that inhibition occurred at the viral entry stage. The extent of exclusion increased as the interval between primary GIII infection and secondary GI infection was prolonged, indicating that superinfection exclusion became progressively stronger over time. Quantitative analysis using fluorescently labeled GI virus and RT-qPCR further showed that GI entry was significantly reduced in cells previously infected with GIII compared with mock-infected controls. Transferrin uptake remained readily detectable in JEV-infected cells, suggesting that prior infection did not grossly impair transferrin internalization under the conditions examined. Together, these findings demonstrate superinfection exclusion between JEV GI and GIII and indicate that interference occurs predominantly during viral entry. The potential contribution of this phenomenon to JEV genotype replacement in nature warrants further investigation.

Introduction

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus and a major cause of viral encephalitis in Asia and the Western Pacific region. JEV is maintained in a zoonotic transmission cycle involving mosquitoes, primarily Culex species, and vertebrate amplification hosts such as pigs and birds, while humans are considered dead-end hosts due to insufficient viremia for onward transmission. Despite the availability of effective vaccines, the lack of specific antiviral therapies continues to make Japanese encephalitis a major public health burden, with approximately 66,000 clinical cases occurring annually worldwide, accompanied by substantial mortality and long-term neurological sequelae among survivors [15, 20].

The JEV genome is an approximately 11-kb, single-stranded positive-sense RNA that encodes a single long open reading frame (ORF), flanked by a 5’-untranslated region (UTR) of 95 nucleotides (nts) and a 3’-UTR of 585 nts. The ORF is translated into a polyprotein precursor which, after proteolytic processing, yields three structural proteins: capsid (C), pre-membrane (prM), and envelope (E); and seven nonstructural proteins (NS1, NS2A/B, NS3, NS4A/B, and NS5) [13]. Based on nucleotide sequence variation in the structural protein genes, JEV is classified into five distinct genotypes. Among these, genotypes I (GI) and III (GIII) have historically accounted for the majority of human infections. Over the past several decades, however, a marked shift in genotype distribution has occurred across many Asian countries, with GI strains progressively replacing the previously predominant GIII strains [2, 4, 8, 16]. This genotype replacement has been documented in both mosquito vectors and porcine amplifying hosts and is now recognized as a hallmark of contemporary JEV molecular epidemiology [9, 24]. In addition, the recent re-emergence of genotype V in East Asia, together with reports of limited vaccine cross-protection, further highlights the increasing epidemiological complexity of JEV circulation [10, 12].

Genotype displacement is likely driven by multiple factors, including differences in transmission efficiency, host adaptation, and intracellular viral competition, such as superinfection exclusion (SIE). SIE refers to a phenomenon in which a primary viral infection induces a cellular state that limits or prevents subsequent infection by related viruses. First described in bacteriophages, SIE has since been reported across diverse viral families, including plant and animal viruses [7]. Accordingly, defining how closely related JEV genotypes interact at the earliest stages of infection is critical for understanding the mechanisms underlying genotype displacement.

In plus-strand RNA viruses, replication is often restricted to a limited number of viral genomes per infected cell. This constraint can shape intracellular viral competition and contribute to the establishment of a superinfection-resistant state [18]. Consistent with this concept, recent studies have shown that SARS-CoV-2 establishes SIE shortly after infection, limiting coinfection to a brief early window [21]. Within the Flaviviridae, SIE has been reported in several medically important viruses, including dengue virus and West Nile virus, and may act at different stages of the viral life cycle, including entry, RNA replication, and translation [28]. In mosquito systems, insect-specific flaviviruses can also suppress arbovirus infection through SIE, thereby influencing vector competence [22]. In the case of JEV, genotype replacement in endemic regions-most notably the displacement of GIII by GI-has been associated with viral determinants linked to enhanced replication efficiency, interferon antagonism, and host adaptation. Specific amino acid substitutions in NS5 (V372A, H386Y) and in the NS2B/NS3 protease have been implicated in conferring a selective advantage to GI over GIII [11], suggesting that multiple viral and host factors contribute to genotype replacement. However, despite these advances, the potential role of SIE in modulating interactions between JEV genotypes has not been addressed. This issue is relevant because cellular exclusion could limit opportunities for coinfection and thereby influence the outcome of viral competition within individual host cells. Clarifying whether such interactions occur may provide additional insight into factors shaping JEV genotype dynamics in natural transmission cycles.

In this study, we examined superinfection exclusion between JEV GI and GIII in mammalian and mosquito cells. We found that the two genotypes reciprocally restrict secondary infection. Further characterization in the GIII-primary/GI-challenge direction showed that exclusion became more pronounced as the interval between infections increased and that inhibition occurred at the viral entry stage. These findings provide new insight into the mechanisms underlying interactions between JEV genotypes.

Materials and Methods

Cells and viruses

BHK-21 mammalian cells were cultured in RPMI-1640 medium supplemented with 2% fetal bovine serum (FBS; Gibco). C6/36 mosquito cells were maintained at 28 °C in a 1:1 mixture of DMEM and Mitsuhashi & Maramorosch (M&M) insect medium (Sigma), supplemented with 10% FBS. The genotype III (GIII) strain of JEV (CH-1392), originally isolated from mosquitoes in Taiwan in 1990, and the genotype I (GI) strain (HL2010-1), isolated from mosquitoes in 2010, were used in this study. All experiments involving JEV were approved by the Institutional Biosafety Committee of National Dong Hwa University and were conducted in a Biosafety Level 2 (BSL-2) facility in accordance with institutional biosafety guidelines.

RNA extraction, RT-PCR, and qPCR

Total RNA was extracted using REzol™ C&T reagent (Protech) according to the manufacturer’s instructions. Reverse transcription was performed using MMLV-HP reverse transcriptase (Epicentre) with random primers and 1 µg of total RNA. JEV genotypes were distinguished by PCR using previously described genotype-specific primer sets for GI and GIII [2]. PCR amplification was carried out using Taq DNA polymerase (Violet) with 30 cycles and an annealing temperature of 55°C. PCR products were analyzed by agarose gel electrophoresis.

For quantitative RT-PCR (RT-qPCR), relative quantification was performed using SimplyGreen qPCR Master Mix (GeneDireX, Inc.) according to the manufacturer’s instructions. Each 10-μL reaction contained 1 μL of synthesized cDNA, 0.3 μL each of the GI-specific forward primer (5’-CAGTCGCGAGTTTAAACGAC-3’) and reverse primer (5’-CCTTTGAGTTGGAGCTAGAT-3’)(10 μM each), 3.4 μL of ddH₂O, and 5 μL of SimplyGreen qPCR Master Mix. Amplification was performed using a CFX96 Real-Time PCR Detection System (Bio-Rad, USA) with an initial denaturation at 95 °C for 10 min, followed by 30 cycles of 95 °C for 15 s and 60 °C for 60 s. 28S rRNA was used as the internal reference and was amplified using the forward primer (5’-GCGGTCCTGACGTGCAAATC-3’) and reverse primer (5’-AACTTCGGAGGGAACCAGCTACT-3’). GI RNA levels were normalized to 28S rRNA within each sample and analyzed using the 2^-∆∆Ct method. The relative GI RNA level in cells previously infected with GIII was expressed relative to that in the mock-infected control, which was set to 100%.

Attachment and entry analysis

BHK-21 cells (3 × 10⁵ cells) were infected with GIII JEV at an MOI of 1 for 1 h at 37 °C, with gentle rocking every 15 min to ensure uniform virus adsorption. After 24 h of primary infection, the cells were challenged with GI JEV at an MOI of 1.

For attachment analysis, the GI challenge was performed at 4 °C for 1 h to allow viral binding while preventing internalization. The cells were then washed twice with PBS to remove unbound virions. Total RNA was extracted and analyzed by RT-PCR.

For entry (penetration) analysis, GI JEV was first allowed to bind to the cells at 4 °C for 1 h. The cells were then shifted to 37 °C and incubated for 1.5 h to permit viral internalization. To remove non-internalized virions remaining on the cell surface, the cells were treated with 1 mL of high-salt alkaline buffer (50 mM Na₂CO₃, 1 M NaCl, pH 9.5) for 3–5 min, followed by two washes with PBS. Total RNA was subsequently extracted and analyzed by RT-PCR.

Flow cytometric analysis of superinfection

To quantify superinfection exclusion, GI particles were fluorescently labeled. Virus stocks were purified by ultracentrifugation through a 30% (w/v) sucrose cushion at 28,000 rpm for 4 h at 4 °C using a Beckman SW28 rotor. Viral pellets were resuspended in phosphate-buffered saline (PBS) and labeled with Lightning-Link™ Atto-565 (Innova Biosciences) according to the manufacturer’s protocol. The infectious titer of the labeled virus was determined by plaque assay prior to subsequent infection experiments.

BHK-21 cells (1 × 10⁵ cells/dish) were seeded into 35-mm dishes. Cells were divided into three groups: mock, infection control, and superinfection. The superinfection group was first infected with GIII. At 24 h post-infection, both the infection control and superinfection groups were infected with fluorescently labeled GI for 1.5 h. Cells were treated with a high-salt alkaline buffer (50 mM Na₂CO₃, 1 M NaCl, pH 9.5) for 3–5 min to remove surface-bound virions, washed twice with PBS, detached with trypsin, and resuspended in PBS. Fluorescence was quantified using a Cytomics FC 500 flow cytometer (Beckman Coulter).

Alexa 647-transferrin uptake assay

BHK-21 cells (1 × 10⁴ cells/well) were seeded in 96-well imaging plates and cultured overnight. For microscopy experiments, cells were infected at an MOI of 10 to maximize the proportion of infected cells available for analysis. At 12 or 24 h post-infection, cells were incubated with Alexa Fluor 647-conjugated transferrin (100 μg/mL, Invitrogen) at 37 °C for 30 min. Cells were washed three times with PBS, fixed with methanol for 30 min, and stained with Hoechst (1:1500) for 30 min. Fluorescence images were acquired using a BD Pathway 435 Bioimager (BD Biosciences).

Statistical analysis

Data are presented as the mean ± standard deviation (SD) from at least three independent experiments. For paired comparisons between GI infection and GIII-preinfected cells subsequently challenged with GI, statistical significance was assessed using a two-tailed paired Student’s t-test. A p value < 0.05 was considered statistically significant.

Results

Superinfection exclusion between JEV genotypes depends on the order of infection

To investigate superinfection exclusion between two genetically distinct JEV genotypes, GI and GIII strains, which differ primarily in their envelope proteins, were used to examine reciprocal superinfection exclusion in BHK-21 and C6/36 cells. Cells were first infected with either GI or GIII JEV at an MOI of 1 and incubated for 24 h, then challenged with the heterologous genotype. Total RNA was extracted 24 h after the second infection and analyzed by RT-PCR using genotype-specific primer sets that distinguish GI and GIII viral RNA.

In BHK-21 cells, prior infection with either genotype effectively blocked subsequent infection by the heterologous genotype, demonstrating mutual superinfection exclusion between GI and GIII JEV (Fig. 1A, lanes 5–6). In contrast, co-infection of GI and GIII RNAs resulted in comparable levels of genotype-specific bands, indicating that both genotypes infected the cell population with similar efficiency in the absence of prior infection (Fig. 1A, lane 7). A similar pattern was observed in mosquito C6/36 cells (Fig. 1B). Together, these data indicate that superinfection exclusion between GI and GIII JEV is determined by the order of infection in both mammalian and mosquito cells.

Fig. 1.

Superinfection exclusion between JEV genotype I (GI) and genotype III (GIII). A schematic of the infection protocol is shown on the left. BHK-21 (A) and C6/36 (B) cells were sequentially infected as indicated. Total RNA was extracted 24 h after the second infection (48 h after the primary infection) and analyzed by RT-PCR using genotype-specific primer sets to distinguish JEV GI and GIII. Infection conditions are indicated above each lane. PCR products were resolved on a 1.5% agarose gel. Lane 1, 100-bp DNA ladder; lane 2, mock-infected control; lane 8, no-template control (NTC)

Superinfection exclusion occurs at the entry step

To determine the stage at which superinfection is restricted, we first assessed viral attachment and validated the effectiveness of the high-salt wash for removing surface-bound virions. High-salt/alkaline washing is a common method for removing non-internalized virus from the cell surface. For this validation experiment, BHK-21 cells were inoculated with GIII JEV at 4 °C for 1 h to allow viral attachment while preventing internalization. The cells were then either left unwashed, washed twice with PBS, or subjected to a high-salt wash, followed by RNA extraction and RT-PCR analysis. As shown in Fig. 2A, the high-salt wash markedly reduced the GIII RNA signal, demonstrating effective removal of surface-bound virus (lane 5).

Fig. 2.

Superinfection exclusion occurs during viral entry rather than attachment. (A) Validation of the high-salt wash for removal of surface-bound virus. Cells were infected with GIII virus at 4 °C for 1 hour and were either left unwashed, washed with PBS, or subjected to a high-salt wash before RNA extraction and RT-PCR analysis. 28S rRNA was used as an internal control. (B) Schematic of the experimental procedures used to distinguish viral attachment from entry. Cells were mock-infected or infected with GIII for 24 h and then challenged with GI virus at 4 °C for 1 h. For the attachment assay, cells were washed with PBS and harvested directly for RNA extraction (C). For the entry assay, cells were shifted to 37 °C for 1.5 h, subjected to a high-salt wash followed by PBS washing, and then harvested for RNA extraction (D). (C, D) Viral RNA was analyzed by RT-PCR, and PCR products were resolved on 1.5% agarose gels. Lane 1, 100-bp DNA ladder; lane 2, mock-infected cells (M); lane 3, mock-infected cells challenged with GI (M→I); lanes 4–6, GIII-infected cells challenged with GI (III→I); lane 7, no-template control (NTC).

We then performed the experiment outlined in Fig. 2B using GIII as the primary virus and GI as the secondary virus to distinguish viral attachment from entry. BHK-21 cells were either mock-infected or infected with GIII JEV at an MOI of 1. At 24 h post-infection, the cells were challenged with GI JEV at 4 °C for 1 h to allow viral attachment while preventing internalization. The cells were then washed twice with PBS, followed by RNA extraction and RT-PCR analysis. As shown in Fig. 2C (lanes 4–6, triplicate), comparable GI RNA signals were detected in mock-infected and GIII-preinfected cells under attachment conditions, indicating that prior GIII infection did not appreciably impair GI binding to the cell surface.

To test whether superinfection exclusion occurred at the entry stage, we used the same infection protocol, except that after adsorption at 4 °C for 1 h, cells were shifted to 37 °C for 1.5 h to permit viral entry. The cells were then washed with high-salt buffer to remove residual surface-bound virions that had not been internalized, followed by two PBS washes. Total RNA was subsequently extracted and analyzed by RT-PCR. As shown in Fig. 2D (lanes 4–6, triplicate), GIII-derived cDNA remained detectable, whereas GI-derived cDNA was not detected following superinfection. These results indicate that prior GIII infection markedly restricts subsequent GI entry and support the conclusion that superinfection exclusion occurs at the viral entry stage.

Superinfection exclusion develops over time

To further examine the temporal development of superinfection exclusion at the entry stage, we performed a time-course assay using different intervals between the primary and secondary infections. BHK-21 cells were first infected with GIII. At 1, 6, 12, or 20 h after the primary infection, the cells were inoculated with GI at 4 °C for 1 h to allow viral attachment. The cells were then shifted to 37 °C for 1.5 h to permit viral entry, followed by a high-salt wash to remove residual surface-bound virions. Thus, the GI RNA detected after washing predominantly represented virus that had entered the cells. Total RNA was subsequently extracted and analyzed by RT-PCR. As shown in Fig. 3, GI RNA remained readily detectable at the shorter intervals after primary GIII infection, indicating that GI could still enter the cells before superinfection exclusion was fully established. As the interval after primary GIII infection increased, the GI RNA signal progressively decreased, consistent with the gradual development of inhibition of secondary GI virus entry.

Fig. 3.

Superinfection exclusion develops over time. BHK-21 cells were mock-infected or infected with GIII virus (MOI = 1). At 1, 6, 12, or 20 h after primary infection, cells were inoculated with GI virus at 4 °C for 1 h to allow viral attachment. The cells were then shifted to 37 °C for 1.5 h to permit viral entry, followed by washing with high-salt buffer and PBS to remove residual surface-bound virions. Total RNA was extracted and analyzed by RT-PCR, and the amplified products were resolved on a 1.5% agarose gel. The gel shown is representative of two independent experiments

Prior GIII infection reduces subsequent GI superinfection

To quantitatively assess superinfection exclusion, GI JEV particles were fluorescently labeled using Lightning-Link™ (see Materials and Methods). Fluorescent labeling did not appreciably alter viral infectivity or plaque morphology (data not shown). BHK-21 cells were mock-infected or preinfected with unlabeled GIII virus for 24 h and then challenged with fluorescently labeled GI virus. Flow cytometric analysis showed that GI virus entry was markedly reduced in GIII-preinfected cells compared with mock-infected controls (Fig. 4A). Quantification of the fluorescence signal showed an approximately 65% reduction in GI virus entry in GIII-preinfected cells (Fig. 4B). Consistent with this finding, RT-qPCR analysis showed a significant reduction in intracellular GI viral RNA levels in GIII-preinfected cells (Fig. 4C). Together, these results indicate that prior GIII infection substantially restricts subsequent GI virus entry.

Fig. 4.

Prior GIII infection inhibits GI virus entry. (A) Representative flow cytometry histograms showing the entry of fluorescently labeled GI virus into control cells, mock-infected cells challenged with GI virus (Mock→GI), and cells infected with GIII for 24 h before GI challenge (GIII→GI). (B) Quantification of fluorescently labeled GI virus entry from three independent experiments represented in (A). Entry efficiency in the Mock→GI group was set to 100%, and entry in the GIII→GI group was expressed relative to this value. (C) Quantification of intracellular GI viral RNA by RT-qPCR from three independent experiments. The GI RNA level in the Mock→GI group was set to 100%, and the GIII→GI group was expressed relative to this value. Data are presented as the mean ± SD. Statistical significance between the Mock→GI and GIII→GI groups in panels B and C was assessed using a two-tailed paired Student’s t-test, p = 0.018 (*) for panel B and p = 0.0006 (***) for panel C.

JEV infection does not visibly abolish transferrin uptake

Clathrin-mediated endocytosis is a major cellular pathway exploited by many enveloped viruses, including flaviviruses, for entry into host cells. We therefore examined whether primary JEV infection broadly impaired clathrin-dependent endocytic activity, which could potentially account for the reduced entry of the superinfecting virus. Alexa Fluor 647-conjugated transferrin, a well-established marker of clathrin-mediated endocytosis, was used to assess this pathway. Fluorescent transferrin was readily internalized in both uninfected cells and cells examined at 12 and 24 h after JEV infection (Fig. 5). Although this qualitative observation does not demonstrate that clathrin-mediated endocytosis was quantitatively unchanged, it indicates that transferrin uptake remained readily detectable and was not grossly impaired by prior JEV infection under the conditions examined.

Fig. 5.

Transferrin uptake in JEV-infected BHK-21 cells. BHK-21 cells were either left uninfected (A-C) or infected with JEV GIII at an MOI of 10 for 12 h (D-F) or 24 h (G-I). Cells were incubated with Alexa Fluor 647-conjugated transferrin for 30 min at 37 °C, washed, fixed, and stained with Hoechst to visualize nuclei. Representative fluorescence images show transferrin (red), nuclei (blue), and merged signals. Enlarged views of the boxed regions are shown on the right. Intracellular transferrin fluorescence remained readily detectable in both uninfected and JEV-infected cultures under the conditions examined

Discussion

In this study, we show that JEV exhibits reciprocal, time-dependent superinfection exclusion between genotypes I (GI) and III (GIII) in both mammalian and mosquito cells. Sequential infection experiments showed that prior JEV infection restricts the secondary virus primarily at the entry stage rather than at attachment. The magnitude of exclusion increased with the interval between primary and secondary infection, indicating that the antiviral state responsible for superinfection exclusion develops progressively after the initial infection. In addition, transferrin uptake was not visibly impaired in JEV-infected cells, suggesting that the observed restriction is unlikely to result from a general disruption of clathrin-mediated endocytosis. Together, these findings indicate that JEV superinfection exclusion involves a selective restriction at the viral entry stage that becomes more pronounced as the primary infection progresses. Similar entry-associated mechanisms of superinfection exclusion have been described for other flaviviruses [1, 26]. Because exclusion was observed regardless of whether GI or GIII established the primary infection, the phenomenon does not appear to be strictly genotype-specific. Rather, primary JEV infection may establish a cellular state that progressively reduces susceptibility to subsequent infection. Such a state may represent a general consequence of JEV infection that develops after the primary virus has entered the cell and initiated replication [25, 27]. To our knowledge, this study is the first to demonstrate reciprocal superinfection exclusion between distinct JEV genotypes and to provide evidence that the restriction occurs at the viral entry stage.

Previous epidemiological studies indicate that both GI and GIII circulated within the same enzootic transmission system, primarily involving Culex tritaeniorhynchus mosquitoes and swine amplification hosts [3]. GI JEV has progressively displaced GIII and become the dominant circulating genotype across many regions of Asia [2, 4, 8, 16, 19]. Because both genotypes shared similar transmission ecology, genotype replacement has generally been attributed to differences in viral fitness within vectors and amplifying hosts rather than to changes in the transmission cycle itself. Several studies have proposed that genotype replacement is driven by enhanced replication and transmission fitness of GI viruses. For example, GI strains carrying specific NS2B/NS3 substitutions exhibit increased replication and higher viremia in amplifying hosts such as pigs and poultry, which may facilitate transmission and contribute to the displacement of GIII strains [6]. Likewise, Li et al. demonstrated that GI strains replicate more efficiently and induce lower levels of type I interferons (IFN-α/β) than GIII strains in duck-derived cells and ducklings. They further identified two NS5 substitutions, V372A and H386Y, that strengthen suppression of the type I interferon response and promote viral replication and host adaptation [11]. Collectively, these studies suggest that enhanced replication fitness and immune evasion confer a selective advantage for GI viruses.

In addition to enhanced viral fitness, vaccine-associated immune selection has been proposed as a potential contributor to JEV genotype replacement. Because currently licensed JE vaccines are derived from genotype III strains, concerns have been raised regarding reduced neutralization of heterologous genotype I viruses [5, 17, 23]. Although GIII-based vaccines generally provide cross-protection against GI strains, genotype-specific differences in immune responses may influence viral competition in vaccinated populations. Together with the superior replication and transmission fitness of GI viruses, these differences may have contributed to the increasing prevalence of GI across Asia. However, the role of vaccine-driven immune selection in JEV genotype replacement remains uncertain.

Beyond these epidemiological and immunological factors, our findings indicate that GI and GIII viruses can also interact directly at the cellular level through superinfection exclusion involving viral entry. Although the contribution of this phenomenon to genotype replacement remains unclear, superinfection exclusion could influence viral competition during sequential infections by reducing a secondary virus’s ability to establish infection once a primary infection is established. Such cellular interactions may therefore contribute to the relative success of different JEV genotypes, together with differences in replication, transmission fitness, and host immune selection.

Mechanistically, our findings indicate that superinfection exclusion occurs during viral penetration. Because JEV enters cells through clathrin-mediated endocytosis, we initially considered whether primary infection might block secondary infection by broadly disrupting this pathway. However, transferrin uptake was still readily detectable in GIII-infected cultures, suggesting that general clathrin-dependent endocytic activity was not abolished. These observations raise the possibility that restriction occurs at a more specific step of viral entry, such as receptor engagement, endosomal trafficking, membrane fusion, or uncoating. Candidate factors for future investigation include putative JEV entry-associated cell-surface molecules, as well as host components involved in endosomal trafficking and acidification, such as Rab5, Rab11, and vacuolar ATPase, which have been implicated in JEV entry [14, 25, 27]. Because flavivirus membrane fusion is triggered by endosomal acidification and mediated by low-pH-induced conformational rearrangements of the viral E protein [25], primary infection may also alter the intracellular environment required for efficient fusion of the superinfecting virion.

Several limitations should be acknowledged. First, the study was performed exclusively in established mammalian and mosquito cell lines, and it remains unclear whether a similar exclusion phenomenon occurs in natural hosts or mosquito vectors. Second, although the restriction was localized to the entry stage, the viral and host factors responsible for this effect were not identified. Finally, although transferrin uptake suggests that clathrin-mediated endocytosis is not globally abolished, primary infection may still selectively alter specific receptors, endosomal trafficking pathways, membrane properties, or fusion-related factors required for JEV entry. Defining these determinants will be important for understanding the molecular basis of JEV superinfection exclusion.

In conclusion, our findings demonstrate reciprocal superinfection exclusion between JEV genotypes I and III. In the mechanistically examined GIII-primary, GI-challenge model, the exclusion was time-dependent. It occurred at the viral entry stage, particularly during penetration, without an apparent disruption of general clathrin-mediated endocytosis. These results provide new insight into the cellular basis of interactions between JEV genotypes and identify superinfection exclusion as a potential factor influencing viral competition and genotype dynamics. Further studies are needed to determine whether the reciprocal infection direction is restricted through the same entry-stage mechanism and to define the molecular determinants responsible for this exclusion.

Author contributions

HH and RC conceived and designed the study. HH and QP performed the experiments. SC and YF contributed reagents, materials, and analytical tools. HH and RC wrote and revised the manuscript.

Funding

Open access funding provided by National Dong Hwa University. National Science and Technology Council, Taiwan, ROC.

Data availability

The data supporting the findings of this study are available within the article and its Supporting Information.

Declarations

Competing interest

The authors declare no competing interests.