Natural products and derivatives as Japanese encephalitis virus antivirals

Abstract

Japanese encephalitis virus (JEV) causes acute Japanese encephalitis (JE) in humans and reproductive disorders in pigs. There are ~68 000 cases of JE worldwide each year, with ~13 600–20 400 deaths. JE infections have a fatality rate of one-third, and half of the survivors experience permanent neurological sequelae. The disease is prevalent throughout the Asia–Pacific region and has the potential to spread globally. JEV poses a serious threat to human life and health, and vaccination is currently the only strategy for long-term sustainable protection against JEV infection. However, licensed JEV vaccines are not effective against all strains of JEV. To date, there are no drugs approved for clinical use, and the development of anti-JEV drugs is urgently needed. Natural products are characterized by a wide range of sources, unique structures, and low prices, and this paper provides an overview of the research and development of anti-JEV bioactive natural products.

The review focuses on the global distribution, molecular biology, viral life cycle, host range and transmissibility, and antiviral natural products development of JEV.

Introduction

Japanese encephalitis virus (JEV) is a mosquito-borne virus that belongs to the Flaviviridae family and Flavivirus genus. Other common viruses in this genus include dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), Murray valley encephalitis, and tick-borne encephalitis virus (Gould and Solomon 2008, Heinz and Stiasny 2012). Japanese encephalitis (JE) is characterized by severe inflammation of the central nervous system (CNS) followed by disruption of the blood‒brain barrier (BBB) (Yadav et al. 2022). The initial case of JE was recorded in 1871 in Japan. The prototype strain Nakayama was first isolated from the brain of a fatal case in 1935 (Solomon et al. 2003, Sharma et al. 2021). JEV is a virus that can infect both arthropods and vertebrates. It is transmitted primarily by Culex mosquitoes, specifically Culex tritaeniorrhynchus and Culex vishnui (Wangchuk et al. 2020). Pigs and horses serve as amplifying hosts, whereas humans are considered dead-end hosts owing to their low viremia (Turtle and Solomon 2018, Park et al. 2022). JEV is among the most prevalent pathogens that cause CNS disease in humans, with mild JEV cases manifesting as fever and severe cases leading to encephalitis and even death. Approximately 50% of those who survive develop lasting neurological complications (Solomon et al. 2000, Sharma et al. 2021). There are ~68 000 clinical cases of JE annually across the globe, leading to an estimated 13 600–20 400 fatalities around the world, although the actual number is probably several times greater since many cases remain unreported (Lannes et al. 2017). JEV thus poses a considerable risk to human health in endemic countries (Gubler 2011). Despite significant advancements in the study of JEV pathogenesis, molecular biology, and immunology, no drugs have been sanctioned for clinical treatment (Lee et al. 2006, Nazmi et al. 2010, Turtle and Solomon 2018). Consequently, the prompt development of a robust drug to counter JEV infection is crucial (Gould et al. 2008).

JEV global distribution

JEV is one of the most important encephalitis-causing viruses in the world (Quan et al. 2020). According to the World Health Organization, more than 24 countries in South Asia and the Western Pacific region are exposed to JEV (Gould and Solomon 2008, Quan et al. 2020). Recently, the epidemiology of JEV has changed, and it has spread from Asia to other regions of the world, such as Papua New Guinea and Australia (Mackenzie et al. 2022, Pham et al. 2022). Over the past few decades, new areas of JEV transmission have emerged in several locations, most notably in a markedly expanded area of Australia from 2021 to 2022 (Pham et al. 2022, Xu et al. 2023). The incidence of JEV in each country and region is associated with the prevalent JEV genotypes in that region, and there are five geographically and epidemiologically distinct genotypes (genotypes I–V) of JEV, namely, GI, GII, GIII, GIV, and GV (Solomon et al. 2003, Schuh et al. 2013). JEV GI and GIII are distributed mainly in Asian countries such as China, Japan, Korea, India, and the Philippines (Rajaiah and Kumar 2022), and recent data show that the emergence of GI is the dominant JEV genotype, gradually replacing GIII (Pan et al. 2011, Hameed et al. 2019, Xu et al. 2023). JEV GII was found in Korea, Malaysia, Indonesia, southern Thailand, and northern Australia (Hanna et al. 1996, Schuh et al. 2011). The GIV genotype was initially isolated from mosquitoes in Indonesia in the 1980s (Solomon et al. 2003) but has long been neglected and not detected in natural hosts or vectors worldwide for approximately half a century, leading to the possibility of extinction. However, several JEV GIV isolates were reported in Indonesia in 2020 (Faizah et al. 2021), and an outbreak of JEV caused by JEV GIV occurred in Australia between 2021 and 2022, indicating the re-emergence of JEV GIV (Mackenzie et al. 2022, Sikazwe et al. 2022). JEV GV is the oldest in JEV populations and was first identified in Malaysia in 1952 (Chen et al. 1990). After that, GV was not reported again until ~60 years later, when it was isolated from mosquitoes in Tibet, China, in 2009 and in South Korea in 2011 (Li et al. 2014, Kim et al. 2015, Lee et al. 2022, Rajaiah and Kumar 2022, Xu et al. 2023).

JEV genome structure

JEV is an enveloped virus that has an 11 kb, positive-sense single-stranded RNA genome (Li et al. 2011). The genome consists of a highly structured 5′ and 3′ untranslated region with a single open reading frame (ORF) that encodes three structural proteins (C-prM-E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) with a 5′ methylated cap but lacks a poly-A tail at the 3′ end (Fig. 1) (Li et al. 2017, Keng and Chang 2018, Roberts and Gandhi 2020).

Figure 1.

JEV structural organization and genome representation. (A) JEV is an enveloped virus with a linear (+) ssRNA genome. The viral nucleic acid is surrounded by the capsid protein, producing a nucleocapsid that is enveloped by a lipid bilayer obtained from the host. The immature virions are assembled in the lumen of the endoplasmic reticulum (ER). The viral RNA genome is packaged by the C protein to form the nucleocapsid. The nucleocapsid is then enclosed by the E and premembrane (prM) proteins to form immature virus particles. These particles are transported through the trans-Golgi network. At this stage, the furin protease cleaves prM to M, which is an essential step for virus maturation. (B) JEV genomic RNA ~11 kb in length has a methylation cap at its 5′ end but lacks a poly-A tail at its 3′ end. It contains a single ORF that encodes a 3400 amino acid polyprotein. This polyprotein is cleaved by both viral and host proteases to generate three structural proteins, the C protein, the M/prM protein, and the E protein, as well as seven NS proteins, namely, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. (C) The diagram depicts the arrangement of the JEV polyprotein in the ER membrane, including the distribution of the structural and NS proteins of JEV in relation to the cytosol and ER lumen. The distribution of proteins is as follows: NS3 and NS5 are located in the cytoplasm; NS1 is located in the ER lumen; and C, prM/M, E, NS2A, NS2B, NS4A, and NS4B are located in the ER membrane. This figure is adapted from Sharma et al. (2021), Pierson and Diamond (2020), Kumar et al. (2022), and Zhang et al. (2023).

Structural proteins are responsible for viral assembly, as well as viral entry and exit from the host cell (Xiong et al. 2022). The capsid (C) structural protein of JEV dimerizes head-to-tail in an antiparallel manner. Multiple copies of capsid dimers compose a spherical nucleocapsid enclosing the viral genomic RNA (Jones et al. 2003, Mukhopadhyay et al. 2005). The precursor membrane of the virus, known as the premembrane (prM), emerges from the nascent polyprotein after cotranslational cleavage by a signal peptide. It then assembles at the vesicles of the endoplasmic reticulum (ER) that contain the viral genomic RNA. Once assembled, the vesicle buds from the ER and reaches the Golgi body network (Srivastava et al. 2023). Here, prM is cleaved by the furin enzyme into the M protein, forming a mature virus particle that is then released from the host cell (Mukhopadhyay et al. 2005). The envelope (E) protein plays a crucial role in inducing neutralization reactions and is associated with receptor binding and membrane fusion (Mukhopadhyay et al. 2005, Luca et al. 2012). It is composed of three domains: envelope domain I (central β-barrel), envelope domain II (fusion loop), and envelope domain III (immunoglobulin-like module) (Kuhn et al. 2002, Lin and Wu 2003, Luca et al. 2012). Like other flaviviruses, the envelope domain III of JEV contains immunoglobulin-like structures at the carboxyl terminus of the ectodomain (Wu et al. 2003). These structures bind to host receptors for virion internalization, making envelope domain III a potential target for designing antivirals to mitigate JEV infection (Zu et al. 2014, Jia et al. 2021, Chen et al. 2022).

All seven NS proteins are translated and cleaved from a single polyprotein. They mainly participate in replication and assist in the assembly of new viral particles (Heinz and Stiasny 2012, Srivastava et al. 2023, Van Den Elsen et al. 2023). Among these NS proteins, only NS1 can enter the lumen of the endoplasmic reticulum. It acts as a heterologous epitope and induces a cross-reactive immune response against various pathogens (Yen et al. 2016). Secreted NS1 is present in the early stages of infection in the blood and is used as a diagnostic marker (Muller and Young 2013). NS2A is a small hydrophobic, membrane-associated protein that is ~22 kDa in size. It plays a crucial role in JEV RNA replication, as well as in the assembly and replication of viral particles (Leung et al. 2008, Ma et al. 2022, Van Den Elsen et al. 2023). The NS2B hydrophilic structural domain typically combines with the NS3 protease structural domain to form the NS2B–NS3 protease complex (Falgout et al. 1991). NS2B acts as a cofactor, assisting NS3 proteins in exhibiting serine protease activity at the N-terminal end (Jan et al. 1995, Bera et al. 2007). This results in the cleavage of NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5, respectively (Yusof et al. 2000, Luo et al. 2008). NS3 is a bifunctional protein with a functional proteolytic domain at its N-terminus and a helicase at its C-terminus. It is an essential protein for both polyprotein processing and viral replication in flaviviruses (Utama et al. 2000, Luo et al. 2008, Van Den Elsen et al. 2023). The NS4 protein is highly hydrophobic and is thought to contribute to the construction of membrane components. Therefore, it is believed that it may play a critical role in the adaptability of viruses to different environments (Srivastava et al. 2023). NS4A and NS4B are transmembrane proteins that participate in viral replication and regulate the host immune response (Chen et al. 2017, Klaitong and Smith 2021, Wang et al. 2022). NS5 is the largest NS protein of JEV, with a molecular weight of ~103 kDa (Van Den Elsen et al. 2023). It is a highly conserved bifunctional protein that consists of a methyltransferase (MTase) domain and an RNA-dependent RNA polymerase (RdRp) domain (Zhou et al. 2007). The MTase domain located at the N-terminal end and the RdRp domain located at the C-terminal end facilitate JEV genome replication, translation, and capping (Lu and Gong 2013, Bhardwaj et al. 2020). NS5 has been shown to impair host lipid metabolism, leading to an enhanced proinflammatory response, which in turn increases neurovirulence and neuroinvasiveness in mice (Kao et al. 2015). The high conservation of NS5 in flaviviruses and the availability of high-resolution crystal structures make it an appealing candidate for structure-guided antiviral drug discovery (Lu and Gong 2013, Van Den Elsen et al. 2023).

Viral life cycle

The infectious life cycle of flaviviruses can be roughly divided into stages of binding and entry, translation and replication, and assembly and release (Yun and Lee 2014, Van Leur et al. 2021) (Fig. 2). For binding and entry, the virus particles first attach to the cell surface; subsequently, the E protein binds to a cellular receptor to initiate internalization via endocytosis (Oya and Kurane 2007, Unni et al. 2011, Carbaugh and Lazear 2020). Endocytosis is completed upon dynamin-dependent scission (Van Der Schaar et al. 2008). The virions located within endosomal compartments undergo viral uncoating triggered by the mildly acidic environment. This enables fusion of the viral envelope with the endosomal membrane (Stiasny et al. 2003, Bressanelli et al. 2004). After fusion, the positive-sense RNA genome is released into the cytoplasm and translated to synthesize the viral polyprotein. The polyprotein is then cleaved by both the viral NS2B–NS3 protease and the host proteases into structural and NS proteins (Gillespie et al. 2010, Unni et al. 2011). NS proteins form viral replication organelles (ROs) with viral RNA on the ER membrane and synthesize multiple copies of viral RNA and unknown host proteins (Villordo and Gamarnik 2009, Le Flohic et al. 2013, Paul and Bartenschlager 2015, Neufeldt et al. 2018). The positive-sense RNA serves as a template for NS5 to generate intermediate negative-sense RNA, which is then used to produce positive-sense progeny RNAs for either incorporation into newly formed virus particles or translation (Bollati et al. 2010, Lim et al. 2013, Neufeldt et al. 2018). Progeny viral RNA is enclosed within immature virions in vesicle packets formed along the rough ER, in close proximity to the ROs. Recent reports suggest that atlastins, a subset of ER proteins, act as central hubs to induce membrane remodeling and drive RO formation, virus replication and assembly (Neufeldt et al. 2019). During the initial stages of viral assembly, the viral genomic RNA and C proteins bud into the lumen of the ER, where they form immature virions. The prM and E proteins are then incorporated into these budding particles (Zhang et al. 2003, Li et al. 2008, Newton et al. 2021). The immature virus progeny accumulate in the ER cisternae as large cargo, which are believed to be transported along the host secretory pathway for virion maturation. During the maturation process, virions undergo furin-mediated prM cleavage, N-linked glycosylation, and ubiquitylation of the E protein. These modifications occur en route to the trans-Golgi (Stadler et al. 1997, Zhang et al. 2004, Yu et al. 2008, Carbaugh and Lazear 2020). The E proteins on the virus particle surface rearrange into a herringbone pattern, which tiles the surface of the virus particle (Li et al. 2008, Yu et al. 2008). Finally, the mature particles exit the cell through exocytosis (Mackenzie et al. 2004, Misra and Kalita 2010).

Figure 2.

JEV life cycle. The virions attach to the host cell membrane and enter the cell through receptor-mediated endocytosis. The viral envelope undergoes rearrangement in a low pH environment in the endosome, leading to membrane fusion and the release of the genome into the cytoplasm. The positive-sense viral RNA is translated, resulting in a single polyprotein that is cleaved into structural and NS proteins. Viral replication is facilitated by a specialized compartment that includes NS5, other viral NS proteins, and multiple host factors. This compartment transcribes positive-strand genomic RNA into negative-strand RNA, which is then used as a template to produce progeny (+) strand genomes. After replication, the virus genome is encapsulated by the C protein and transported to the ER. Here, the nucleocapsid is enveloped by a lipid bilayer containing the prM and E proteins. Immature virions, consisting of genomic RNA, prM-E heterodimers, and C, travel through the trans-Golgi network and mature via furin cleavage. This figure was originally created via BioRender.com and was accessed on 2 December 2023.

Host range and transmissibility

The transmission cycle of JEV involves a host, an amplifying host, a receptor vector species, and optimal environmental conditions (Fig. 3) (Furlong et al. 2023). JEV is transmitted between vertebrates and arthropods (Hameed et al. 2019). It is transmitted mainly by Culex mosquitoes, including C. tritaeniorrhynchus and C. vishnui (Auerswald et al. 2021). Pigs are known to be amplifying hosts of JEV, exhibiting high levels of viremia and shedding the virus in their oral and nasal secretions after infection (van den Hurk et al. 2019, Redant et al. 2020). Bats and migratory birds are the primary hosts for transmission and carriage of the virus, and transmission is often associated with seasonal changes. Waterfowl act as amplification hosts and can transmit the virus to mosquitoes because of their relatively high levels of viremia. Infection can occur in birds, particularly young birds with high viremia, even with smaller doses of JEV injection (Hameed et al. 2021, Kumar et al. 2022). Nonavian vertebrates, including humans and horses, are known as end hosts, as they cannot produce sufficient viremia for mosquito infection (Seo et al. 2013). However, a study demonstrated that JEV can be transmitted to immunocompromised individuals through blood transfusions, which can have severe consequences (Cheng et al. 2018).

Figure 3.

Transmission cycle of JEV. JEV is a virus that can infect both arthropods and vertebrates. It is transmitted primarily by Culex mosquitoes, specifically C. tritaeniorrhynchus and C. vishnui. Pigs and horses can amplify the virus, but humans are considered dead-end hosts owing to their low viremia. Bovine and water birds, including migratory birds such as herons, egrets, and ducks, serve as natural reservoirs of JEV. This figure is adapted from Sharma et al. (2021) and Zhang et al. (2023).

Bioactive natural products and their derivatives

Compounds derived from natural resources, including extracts from plants and animals, as well as components and metabolites from insects, microorganisms, and marine organisms, are highly valued because of their evolution over millions of years (Ekiert and Szopa 2020). These compounds are considered potential alternatives because of their low side effects and easy accessibility from nature. In recent years, several studies have demonstrated that natural products have anti-inflammatory, antitumour, antimicrobial, and antiviral properties (Javadi and Sahebkar 2017, Huang et al. 2022, Owen and Laird 2022, Cadelis et al. 2023). Compared with drugs synthesized in the laboratory, bioactive natural product extracts have a broader range of targets, making them crucial for the development of antiviral drugs (Zhang et al. 2020). Here, we highlight natural products that have shown significant antiviral activity against JEV infection (Figs 4 and 5).

Figure 4.

JEV inhibitors. This figure was originally created via KingDraw software 3.1.0.20.

Figure 5.

Schematic diagram of antivirals for JEV with different targets. This figure is adapted from Zhu et al. (2023).

Baicalin

Baicalin is a flavonoid extracted and isolated from the dried root of the natural product Scutellaria baicalensis (Singh et al. 2021, Wang et al. 2022). This compound possesses antiviral, antitumour, and antioxidant properties, and several investigations have shown its beneficial effects on human health (Lu and Gong 2013, Kao et al. 2015, Bhardwaj et al. 2020, Van Leur et al. 2021, Wang et al. 2022). Baicalin can suppress innate immune hyperactivity caused by influenza A virus (IAV) infection and decrease IRF7 levels to inhibit Marek’s disease virus infection in chick embryo fibroblasts (Geng et al. 2020, Yang et al. 2020). One study investigated the anti-JEV activity of baicalin in Vero cells at various stages of the JEV replication cycle (Johari et al. 2012). In vitro experiments revealed that baicalin has an 50% inhibitory concentration (IC₅₀) of 5.8 ± 1.09 µg/ml against JEV. Baicalein exhibited significant direct virucidal activity [the selectivity index (SI) = 33.4], intracellular anti-JEV activity (SI = 15.8), and antiadsorption activity (SI = 15.8), but its prophylactic activity was not significant (SI = 1.3). The mechanism responsible for the antiviral activity of baicalein is currently unclear and requires further investigation. This includes the study of molecular and cellular mechanisms of action, as well as in vivo evaluation, to develop an effective antiviral compound against JEV.

Ivermectin

Ivermectin is a 22,23-dihydro derivative of the fermentation product B1 of the macrolide antibiotic avermectin and is an effective anthelmintic against internal and external parasites, particularly nematodes and arthropods (Geary 2005, Õmura 2008). In cell culture experiments, ivermectin exhibited robust antiviral action against chikungunya virus (CHIKV), pseudorabies virus (PRV), adenovirus, and SARS-CoV-2 (Varghese et al. 2016, Lv et al. 2018, Caly et al. 2020). One study identified ivermectin as a molecule that has a high predicted binding affinity for the modeled NS3 ssRNA binding pocket. Additionally, it inhibits the NS3 helicase activity of several flaviviruses in vitro at submicromolar concentrations (Mastrangelo et al. 2007, 2012). Furthermore, an in vitro enzyme assay was also performed using recombinant flavivirus NS3 helicases from several distantly related serotypes, including WNV from the JEV serogroup and YFV and DENV from the YFV serogroup (Mastrangelo et al. 2012). This study revealed that ivermectin inhibited the dsRNA unwinding activity of several flaviviral helicases. In vitro experiments demonstrated that ivermectin effectively suppressed the production of the JEV strain SA14, with an inhibition efficiency in the nanomolar range (50% effective concentration (EC₅₀) = 0.3 µM, 50% cytotoxic concentration (CC₅₀) ∼10 µM). As ivermectin has been approved for the treatment of parasitic infections in humans for more than 20 years (Geary 2005, Õmura 2008), this study suggests its potential to treat life-threatening flavivirus infections in clinical trials with minimal effort.

Rosemarinic acid

Rosemarinic acid is a hydroxyl-containing polyphenolic acid that is a natural antioxidant with a variety of anti-inflammatory and immunosuppressive activities (Makino and Tanaka 1998, El-Huneidi et al. 2023). In one study, JEV-infected mice (4–5-week-old BALB/c mice) were administered rosmarinic acid by intraperitoneal injection (25 mg/kg body weight twice daily). The results revealed a significant reduction in viral replication and secondary inflammation induced by microglial cell activation in the brains of the mice. Additionally, the mortality rate of the mice was reduced by up to 20%, with 12 out of 15 animals surviving following rosemarinic acid treatment in the JEV-infected and rosemarinic acid-treated groups (Swarup et al. 2007). All infected animals that did not receive RA treatment succumbed to infection. However, treatment with RA alone did not affect the animals’ behavioral outcomes. The antiviral and anti-inflammatory effects of rosmarinic acid are crucial for reducing the severity of diseases induced by JEV, suggesting that the use of rosmarinic acid could be a potential therapeutic measure to decrease the neurological complications observed in JE patients.

Kaempferol

Kaempferol is a polyhydric flavonol present in several plant species, such as soybean, tea, broccoli, strawberry, tomato, grapefruit, and medicinal herbs (Alam et al. 2020). The antiviral properties of kaempferol and its compounds have been the subject of considerable research, with a particular focus on hepatitis B virus (HBV), African swine fever virus (ASFV), and PRV (Arabyan et al. 2021, Li et al. 2021, Parvez et al. 2022). In vitro experiments demonstrated that kaempferol has an EC₅₀ value of 12.6 µM against JEV-infected BHK-21 cells. The CC₅₀ of kaempferol against BHK-21 cells was 230 µM (Zhang et al. 2012). Electrospray ionization mass spectrometry and molecular docking experiments revealed that kaempferol binds to fsRNA in a manner that is consistent with the findings of cell studies. The binding characteristics of kaempferol and its relationship to the frameshift structure of RNA suggest that it interferes with a downstream RNA pseudoknot structure, thereby influencing frameshift efficiency in virus-infected cells. Therefore, kaempferol may target the programmed translational frameshift site as an antiviral drug against flaviviruses.

Arctigenin

Arctigenin is a lignan derived from plants that has been demonstrated to possess antioxidant and anti-inflammatory properties (Cervellati et al. 2004, Díaz et al. 2004). A recent study indicated that arctigenin treatment can reduce coronavirus protein expression and coronavirus-induced cytotoxicity and is a potent natural compound that prevents coronavirus replication (So et al. 2024). When JEV is administered intraperitoneally at a dosage of 10 mg/kg twice daily for 7 consecutive days to 4–5-week-old BALB/c mice that are infected intravenously, it significantly reduces the viral load, viral replication, and neuronal mortality in the brain tissue of the mice (Swarup et al. 2008). This study revealed that all of the mice survived and suggested that arctigenin has potential therapeutic benefits for managing neurological and inflammation-related diseases associated with JEV because of its antiviral, neuroprotective, anti-inflammatory, and antioxidative effects. Therefore, arctigenin may be a promising candidate for further investigation.

Aloe-emodin

Aloe-emodin (1,8-dihydroxy-3-hydroxymethyl-anthraquinone) is a naturally occurring anthraquinone compound that has been isolated from Cassia occidentalis, Rheum palmatum L., Aloe vera, and Polygonum multiflorum Thunb. (Cui et al. 2020, Dong et al. 2020). It has been found to inhibit various viral infections, including influenza virus, varicella-zoster virus, herpes simplex virus (HSV), porcine reproductive and respiratory syndrome virus, and human cytomegalovirus (HCMV) (Sydiskis et al. 1991, Barnard et al. 1992, Xiong et al. 2011, Xu et al. 2021). In vitro, aloe-emodin exhibited significant dose- and time-dependent antiviral activity against JEV-infected HL-CZ cells (IC₅₀ = 0.50 ± 0.02 µg/ml) and TE-671 cells (IC₅₀ = 1.51 ± 0.05 µg/ml), with high therapeutic indices (TI > 1500) (Lin et al. 2008). This study revealed that aloe-emodin significantly induced the expression of interferon α, human protein kinase R, and 2′,5′-oligoadenylate synthetase. It also activates IFN-stimulated response elements and gamma-activated sequence-containing promoters and increases the production of endogenous nitric oxide. Additionally, a molecular docking study revealed that aloe-emodin (IC₅₀=100 µg/ml) has high and stable binding to the NS2B–NS3A protease of JEV (Bhimaneni and Kumar 2022). Therefore, aloe-emodin may be valuable in the development of antiviral agents for JEV infection.

Berbamine

Berbamine is a bisbenzylisoquinoline alkaloid derived from the leaves and roots of Berberis spp. It has been reported to affect Ca²⁺ signaling (Zhang et al. 2012, Hu et al. 2020) and to exhibit anti-inflammatory, antiarrhythmic, antihypertensive, and antimyocardial ischemic activities (Zheng et al. 2017, Zhang et al. 2023). Recently, berbamine has been demonstrated to inhibit SARS-CoV-2 and ASFV infection (Cloherty et al. 2023, Jackman et al. 2024). A study revealed that berbamine inhibits the level of low-density lipoprotein receptor (LDLR) at the plasma membrane, blocking the entry of JEV (Huang et al. 2021). The CC₅₀ of berbamine ranged from 115 to 127 µM in different cell lines, with an SI of ~78, indicating an effective therapeutic window for its antiviral activity against JEV. The in vivo experiment revealed a 75% survival rate of JEV-infected mice in the berbamine treatment group, which was significantly greater than that of the control group (12.5%). Moreover, berbamine mitigated the brain damage resulting from JEV infection, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, compared with that in the control group. Therefore, berbamine has potential as an effective therapeutic agent for the prevention and/or treatment of JEV infection.

Luteolin

Luteolin is the primary flavonoid in honeysuckle and is also present in herbs and other plants, including chamomile tea, perilla leaf, green pepper, and celery (Choi et al. 2007, Lin et al. 2008). Recent studies have shown that luteolin has antiviral activity against several viruses, including PRV, HBV, and HSV-2 (Behbahani et al. 2013, Bai et al. 2016, Fan et al. 2016, Men et al. 2023), has anticancer effects (Imran et al. 2019), and has neuroprotective properties (Ahmad et al. 2021). Luteolin significantly inhibited JEV infection in A549 cells in a dose-dependent manner, with an IC₅₀ value of 4.56 µg/ml and a CC₅₀ value of 54.4 µg/ml. Mechanistic studies demonstrated that luteolin exhibited virucidal activity against extracellular JEV particles. A time-of-addition assay revealed that luteolin inhibits JEV infection at the postentry stage of the virus. In further research, it would be interesting to investigate whether luteolin could protect against JEV-induced neuroinflammation in vitro and in vivo (Fan et al. 2016).

Andrographolide

Andrographolide is a diterpenoid lactone compound that is the primary active ingredient of the natural herb Andrographis paniculate (Lin et al. 2009). Andrographolide and its derivatives have been reported to possess anti-inflammatory (Burgos et al. 2020), antimicrobial (Zhang et al. 2020), and anticancer properties (Mishra et al. 2015). Additionally, it is capable of inhibiting ZIKV and DENV infections (Gupta et al. 2017, Ramalingam et al. 2018, Li et al. 2020). Molecular dynamics and molecular docking studies have been used to screen the phytoconstituents of andrographolide that target JEV viral proteins (Bhosale and Kumar 2021). The results indicate that andrographolide has optimum binding affinity with NS3 helicase (PDB ID: 2Z83), NS3 proteases (PDB ID: 4R8T), and NS5 (PDB ID: 4K6M) of JEV. Andrographolide has demonstrated optimal binding affinity with the NS3 helicase of JEV through hydrogen bonding and hydrophobic interactions. It also interacts with NS5 through hydrogen and hydrophobic interactions. Additionally, it has shown optimal binding affinity against the NS3 protease of JEV through hydrogen bonding. Absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis has revealed good pharmacokinetic and safety profiles for andrographolide. Andrographolide has the potential to be used as a broad-spectrum antiflavivirus infection drug because of its inhibitory effect on flavivirus family members.

Astragali radix

Astragali radix, a traditional Chinese medicine derived from the dried roots of Astragalus species, is widely distributed throughout temperate regions of the world. Astragali radix has yielded over 400 natural compounds, primarily saponins, flavonoids, phenylpropanoids, alkaloids, and other substances (Liu et al. 2024). Kajimura et al. (1996a,b) conducted a study on the protective effects of Astragalus radix extracts on mice infected with JEV through oral and intraperitoneal administration. The authors propose that the protective effect of Astragali radix extracts is dependent on a nonspecific mechanism during the early stage of infection, before it shifts to antibody production, and that the peritoneal exudate cell plays a critical role. These results suggest a nonspecific mechanism for the protective effect of Astragali radix extracts during the early stage of infection, before the shift to antibody production.

Artemisinin

Artemisinin, the active substance against malaria, is extracted from Artemisia annua. It was first isolated and tested in China in the 1970s (Tu 2016) and has been found to have inhibitory effects on HCMV (Oiknine-Djian et al. 2018) and hepatitis C virus (Obeid et al. 2013). Wang et al. (2020) reported that artemisinin stimulates the upstream and downstream signaling pathways of IFN1, IRF3, and STAT1/STAT2 gene expression in JEV-infected cells, exerting an antiviral effect. In a mouse model, treatment with artemisinin reduced mortality and ameliorated JEV-mediated brain damage (Wang et al. 2020). Specifically, artemisinin was found to increase the levels of IFN-β and downstream ISGs in JEV-, DENV-, or ZIKV-infected cells. The observed antiflaviviral activity of artemisinin is related to the enhancement of the host IFN-1 response. This study presents the first demonstration of the antiviral activity of artemisinin against flaviviruses, with a novel mechanism of action. The therapeutic use of artemisinin may provide a broad-spectrum approach for treating flavivirus infections.

Mycophenolic acid

Mycophenolic acid is an immunosuppressant that is naturally produced by Penicillium spp. It is frequently found, often in high concentrations, in a broad range of food and feed matrices (Dietrich and Märtlbauer 2015, Dasgupta 2016). Studies have shown that it can inhibit DENV infection by suppressing viral RNA replication and YFV infection (Neyts et al. 1996, Diamond et al. 2002). In a previous study, mycophenolic acid inhibited the lesioning effect of JEV-induced porcine stable kidney (PS) cells in a dose-dependent manner when it was directly applied. The addition of mycophenolic acid to different stages of JEV-infected PS cells inhibited viral replication up to 12 h postinfection (IC₅₀ = 3.1 µg/ml, CC₅₀ = 62.5 µg/ml, TI = 16) (Sebastian et al. 2011). Oral administration of mycophenolic acid at multiple doses (0.5, 0.8, and 1 g/kg body weight) to JEV-infected Swiss albino mice (4–5 weeks old) for 20 days resulted in a significant increase in survival rates. All untreated mice died on day 6, whereas none of the mice receiving the oral dose of mycophenolic acid at 0.5 g/kg body weight died. However, the survival rates of the mice that received oral doses of mycophenolic acid at 0.8 and 1 g/kg body weight were 50% and 75%, respectively. These findings suggest that a certain level of mycophenolic acid is required to protect mice from JEV infection. Additionally, mycophenolic acid is currently used in clinical settings for transplant patients (Õmura 2008, Johari et al. 2012), making it a potential candidate for evaluating JEV infections.

Indirubin

Indirubin is a bisindole alkaloid primarily found in indigo plants. Recent studies have demonstrated its anti-inflammatory, anticancer, neuroprotective, and antiviral properties (Hsuan et al. 2009, Chan et al. 2018, Yang et al. 2022). An indirubin derivative, indirubin-3′-(2,3-dihydroxypropyl)-oximether (E804), has been identified as a potent immunomodulatory compound for IAV infection in vitro and has the capacity to inhibit intracellular signaling pathways in pulmonary endothelial cells (Kwok et al. 2016). In vitro, indirubin inhibited JEV replication in HL-CZ promonocytic cells in a dose-dependent manner. After 24 h of incubation, indirubin (10 µg/ml) reduced the virus yield by ~40% (Chang et al. 2012). Compared with the control, indirubin exhibited concentration-dependent virucidal activity and significantly inhibited residual infectivity. These findings suggest that pretreatment with indirubin leads to better inhibition of JEV replication and a significant reduction in virus attachment and yield in vitro. Additionally, indirubin protected groups of mice intracerebrally challenged with a lethal dose of the virulent JEV strain Beijing-1. The survival rate of the group treated with indirubin was 70% on day 6 after infection. These findings suggest that indirubin has potential as an antiviral agent against JEV infection, which could lead to the development of new anti-JEV treatments.

Curcumin

Curcumin is a phenolic compound extracted from the rhizome of Curcuma longa L., and it has been reported to possess anti-inflammatory, antioxidant, and antiproliferative properties (Joe et al. 2004, Calabrese et al. 2008). In one study, Neuro2a cells infected with JEV were treated with various doses of curcumin. The results revealed that, compared with virus infection alone, curcumin treatment increased cell viability and significantly reduced apoptosis. Curcumin also reduces the production of infectious viral particles from previously infected neuroblastoma cells by dysregulating the ubiquitin–proteasome system (Dutta et al. 2009). Furthermore, Cur-CQDs were synthesized through mild pyrolysis-induced polymerization and carbonization at 180°C for 2 h. These Cur-CQDs exhibited increased water solubility and decreased cytotoxicity, with a CC₅₀ at least 50 times greater than that of curcumin (Chen et al. 2022). During the early stages of viral infection, Cur-CQDs (IC₅₀ = 0.9 µg/ml) bind to the E proteins on viral particles, hindering the adhesion and fusion effects of JEV on host cells and thereby preventing JEV infection.

Enanderinanin J

Autophagy is a lysosomal degradation process that is conserved across different organisms, and abnormal autophagy has been linked to various pathological processes (Eskelinen 2005, Kroemer and Jäättelä 2005). In this study, the ability of over 150 diterpenoids to regulate autophagy in HeLa cells was tested via a high-content fluorescence imaging assay. Enanderinanin J is an asymmetric dimer of xerophilusin A and possesses the basic skeleton of 7,20:14,20-diepoxy ent-kauranoid (Huang et al. 2021). It was found to increase the levels of LC3-II and p62 in both time- and concentration-dependent manners. Additionally, it significantly inhibits the replication of flaviviruses, such as JEV, ZIKV, and DENV, in mammalian cells by increasing lysosomal pH and targeting the fusion of autophagosomes and lysosomes (Huang et al. 2021).

Ouabain

Ouabain is an inhibitor of Na⁺/K⁺-ATPase that has cardiotonic effects (Ferrandi et al. 2005); it has been proven to inhibit different kinds of viruses, including enveloped viruses such as coronaviruses (Burkard et al. 2015), nonenveloped viruses such as reoviruses (Thete and Danthi 2015), DNA viruses such as HCMV (Kapoor et al. 2012), and RNA viruses such as CHIKV (Ashbrook et al. 2016). A recent study revealed that ouabain (IC₅₀ = 52.16 nM) inhibits JEV replication by targeting Na⁺/K⁺-ATPase through high-content screening of 1034 natural extracts, and ouabain demonstrated robust efficacy against JEV infection, with an SI over 1000 (Guo et al. 2020). In a BALB/c mouse model of JEV infection, ouabain exhibited an antiviral effect by significantly reducing the mortality rate of the mice, decreasing the viral load, and mitigating histopathological changes in the mouse brain. The findings of this study are expected to enhance our understanding of JEV infection biology and offer new host targets for the development of anti-JEV drugs. Moreover, this study identified several other inhibitors, including digoxin, lycorine, cephalotaxlen, schizandrin B, procyanidin, emodin, and schizandrin A, that effectively inhibited JEV infection in a dose-dependent manner. However, owing to the limited research objective, these inhibitors have not been investigated in depth.

Griffithsin

Griffithsin is a 121-amino acid plant-derived antiviral lectin that has been isolated from the red alga Griffithsia sp. (Lee 2019). In a study evaluating its therapeutic efficacy, griffithsin (IC₅₀ = 265 ng/ml) inhibited JEV infection in BHK-21 cells in a dose-dependent manner by interacting with JEV virions (Ishag et al. 2013). Furthermore, the effectiveness of griffsin against JEV infection was assessed in 2-week-old BALB/c mice via a peripheral challenge model. The administration of griffithsin (5 mg/kg body weight) via intraperitoneal injection prior to viral infection resulted in complete protection (100%) of the infected mice challenged with a lethal dose of JEV. This finding indicates that griffithsin has the ability to saturate circulating JEV and inhibit virus infectivity. Further studies have demonstrated that griffithsin inhibits viral infection by binding to the JEV E and prM proteins (Ishag et al. 2016). Therefore, further research on the pharmacokinetics of griffin could lead to the development of griffsin as an antiviral agent against JEV or other flaviviruses.

Conclusion and future directions

JE is an acute infectious disease of the CNS in both humans and animals (Srivastava et al. 2023). Symptomatic treatment, including temperature control, a reduction in cerebral edema, increased oxygenation, and adjunctive antimicrobial therapy, is the main strategy for JEV infection (Tiroumourougane et al. 2003). However, there are currently no approved drugs for the clinical treatment of JEV.

In recent years, research into natural products for combating JEV infection has gradually increased (Guo et al. 2020, Maurya et al. 2023, Sharma et al. 2023). Table 1 outlines the antiviral strategies currently in development for JEV. Natural products are derived from a variety of sources, including animals, plants, microorganisms, and minerals. Their chemical structure is complex and rich, and their unique and remarkable biological activities make them valuable resources for drug development (Yuan et al. 2016, Singla et al. 2019). Natural products are compounds derived from nature that possess a complex chemical structure that provides them with specific target binding specificity and significant biological activity (Ekiert and Szopa 2020). Examples of such natural products include alkaloids, polyphenols, and saponins, which have been found to exhibit anti-inflammatory, antioxidant, antibacterial, and antiviral pharmacological effects, making them effective in preventing and treating diseases (Ho et al. 2018, Liu et al. 2020, Tang and Huang 2022). They have a wider range of efficacy, lower tolerance, and fewer adverse reactions, making them safer for clinical use. This has led to their increasing popularity in clinical applications (Yang et al. 2021).

Table 1.

Summary of antiviral strategies developed for JEV.

Compound/drug name	Mechanism of action	Inhibitory concentration (assay)	References
Baicalin	Virucidal activity; inhibits adsorption activity	IC50: ∼5.8 µg/ml (Hela cells)	Johari et al. (2012)
Ivermectin	Inhibits JEV NS3 helicase	EC50: 0.3 µM	Mastrangelo et al. (2012)
Rosemarinic acid	Anti-inflammatory effect	ND*	Swarup et al. (2007)
Kaempferol	Binds to fsRNA	EC50: 12.6 µM (BHK-21 cells)	Zhang et al. (2012)
Arctigenin	Anti-inflammatory effect	ND*	Swarup et al. (2008)
Aloe-emodin	Binds with JEV NS2B–NS3 protease	IC50: 7.3 µg/ml	Lin et al. (2008)
Berbamine	Decreases LDLR levels at the cell surface	IC50: 1.62 µM (A549 cells)	Huang et al. (2021)
Luteolin	Virucidal activity	IC50: 4.56 µg/ml (A549 cells)	Fan et al. (2016)
Andrographolide	Inhibits JEV NS3 protease	IC50: 2 µg/ml	Bhosale and Kumar (2021)
Astragali radix	A nonspecific mechanism	ND*	Kajimura et al. (1996a,b)
Artemisinin	Enhances the host IFN-1 response	IC50: 18.5 µM (A549 cells)	Wang et al. (2020)
Mycophenolic acid	Inhibits viral replication	IC50: 3.1 µg/ml (PS cells)	Sebastian et al. (2011)
Indirubin	Virucidal activity	ND*	Chang et al. (2012)
Cur-CQDs	Binds to the JEV E protein	IC50: 0.9 µg/ml (BHK-21 cells)	Chen et al. (2022)
Enanderinanin J	Inhibits autophagosome–lysosome fusion	IC50: 16.3 µM (Hela cells)	Huang et al. (2021)
Ouabain	Targeting the Na+/K+-ATPase	IC50: 52.16 nM (Vero cells)	Guo et al. (2020)
Griffithsin	Binds to the JEV E protein and prM proteins	IC50: 265 ng/ml (BHK-21 cells)	Ishag et al. (2013)

ND*: in vitro efficacy was assessed via a viral inhibition assay, although IC₅₀ and EC₅₀ were not measured.

JEV can affect the CNS within 4–6 days of infection. Even with early diagnosis, there is a significant risk of CNS invasion by the virus before antiviral treatment is initiated (Yun and Lee 2018). In cases where some drugs cannot cross the BBB, treatment may be ineffective. Therefore, an efficient drug delivery system is needed to ensure the activity of anti-JE drugs (Lu et al. 2014). The lipid solubility of the BBB is a crucial characteristic that enables the passive diffusion of drugs into the BBB. Specific chemical modifications of a drug may increase its permeability.

Recently, there has been a notable increase in the number of diverse natural compounds explored for their potential use in drug discovery pipelines aimed at targeting JEV infection. High-throughput drug screening at the cellular level has become a pivotal tool in the development of anti-JEV drugs. Access to extensive compound libraries, encompassing hundreds of thousands of natural compounds, coupled with automated cell manipulation techniques and viral labeling, facilitates the rapid and efficient identification of promising natural product candidates (Guo et al. 2020). A target-based approach, utilizing the Schrodinger suite 2019–3 software, has been employed to screen the selected phytoconstituents of Andrographis paniculata against a range of targets associated with JEV (Bhosale and Kumar 2021).

Although natural products constitute the primary source of new drugs, the utilization of natural products in antiviral research is constrained by a number of factors. The isolation, purification and identification of the structures of these compounds could be fraught with challenges, as well as poor bioavailability. The intricate composition of natural compounds presents a significant challenge to pharmacological research. In many cases, the isolation of active compounds has resulted in the loss of their efficacy (Katz and Baltz 2016). In addition, the mechanism of drug action is often unclear, which slows the progress of drug research. Extracting the active ingredient may also be difficult due to its instability, and the efficacy and bioavailability of natural compounds can be affected by various factors, such as season, place of origin, and extraction method.

To overcome the aforementioned obstacles, analytical and computational techniques have opened new avenues for processing complex natural products and utilizing their structures to derive novel and innovative drugs (Wetzel et al. 2009). Computational software has facilitated the identification of molecular targets of natural products and their derivatives (Rodrigues et al. 2016). The application of computational software and databases for modeling molecular interactions and predicting essential characteristics and parameters for drug development will facilitate the future use of quantum computing, including in pharmacokinetic and pharmacodynamic processes. This approach will reduce the number of false positive leads in drug development. To improve the bioavailability of natural compounds, several formulations, such as cyclodextrin complexes, self-nanoemulsifying systems, micelles, nanocrystals, and gels, have been developed (Xing et al. 2005). Furthermore, the discovery of new drugs is a complex process that requires the evaluation of numerous factors associated with both natural and synthetic compounds. Key considerations in this process include safety, pharmacokinetics, and efficacy, which are essential in the selection of viable drug candidates (Thomford et al. 2018). ADMET (absorption, distribution, metabolism, excretion, and toxicity) inspection helps to identify compounds with poor absorption profiles, which is beneficial in the early stages of drug development (Kant et al. 2019).

In vitro studies can facilitate the acquisition of information pertaining to a drug’s potential antiviral properties and cytotoxicity profile. Furthermore, animal models represent a crucial methodology for investigating the underlying mechanisms of human disease and evaluating the efficacy of novel pharmaceutical agents (Zhang et al. 2020). Importantly, the translation of these effects to in vivo studies can vary significantly. In vivo studies utilizing laboratory animals, such as mice and rabbits, have the potential to mimic natural infections; however, they may not accurately predict efficacy in humans. The selection of an appropriate animal model can enhance the authenticity and credibility of preclinical pharmacodynamic research, thereby improving the success rate of pharmacodynamic studies. The utilization of natural products in clinical applications is predominantly confined to cellular and animal experimentation. The dearth of long-term clinical data support renders the use of these drugs uncertain. Only a restricted number of JEV inhibitors in animal models have been advanced to clinical trials, and the findings cannot be readily extrapolated to humans. Furthermore, the completion of the entire drug development pipeline is a significant challenge for numerous experimental antivirals (Thomford et al. 2018).

In essence, while natural products continue to serve as dependable sources for anti-JEV drug discovery, the crux of drug discovery from natural products lies in the identification of an optimal strategy (Sharma et al. 2023). The identification of novel therapeutic targets may facilitate the development of new treatments for JE, while advances in JEV research may also inform the development of treatments for other flaviviruses. However, challenges may emerge in the sourcing and validation of natural resources, the enhancement of the efficiency of functional natural substances, and the simplification of purification procedures. Consequently, the development of natural anti-JEV substances as medicinal drugs still requires substantial advancements.

Author contributions

Yunqi Mi (Conceptualization, Funding acquisition, Investigation, Project administration, Validation, Visualization, Writing – original draft, Writing – review & editing), Yan Guo (Conceptualization, Investigation, Validation, Visualization, Writing – original draft, Writing – review & editing), Xuliang Luo (Investigation, Validation, Visualization), Yang Bai (Investigation, Validation), Haonan Chen (Investigation, Validation), Meihua Wang (Conceptualization, Writing – original draft, Writing – review & editing), Yang Wang (Conceptualization, Funding acquisition, Investigation, Project administration, Supervision, Validation, Visualization, Writing – review & editing), and Jiao Guo (Conceptualization, Funding acquisition, Investigation, Project administration, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing)

Conflict of interest

The authors declare that they have no conflicts of interest.

Funding

This work was supported by the Health Research Project of Shaanxi Province (2022D040 to J.G.), the Science and Technology Planning Project of the Shaanxi Provincial Education Department (22JK0545 to J.G.), the Natural Science Basic Research Program of Shaanxi (2024JC-YBQN-0922 to J.G.), the Foundation for Starting Scientific Research of the Doctor of Xi’an Medical University (2021DOC04 to J.G.), the National Science Foundation of China (32070069 to Y.W.), China’s Innovation and Entrepreneurship Training Program for College Students (202311840026 to Y.M.), and Xi’an Medical University’s Discipline Construction Fund. J.G. is a recipient of a start-up postdoctoral fellowship from Xi’an Medical University.