BPR3P0128, a non-nucleoside RNA-dependent RNA polymerase inhibitor, inhibits SARS-CoV-2 variants of concern and exerts synergistic antiviral activity in combination with remdesivir.

Viral RNA-dependent RNA polymerase (RdRp), a highly conserved molecule in RNA viruses, has recently emerged as a promising drug target for broad-acting inhibitors. Through a Vero E6-based anti-cytopathic effect assay, we found that BPR3P0128, which incorporates a quinoline core similar to hydroxychloroquine, outperformed the adenosine analog remdesivir in inhibiting RdRp activity (EC50 = 0.66 µM and 3 µM, respectively). BPR3P0128 demonstrated broad-spectrum activity against various severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern. When introduced after viral adsorption, BPR3P0128 significantly decreased SARS-CoV-2 replication; however, it did not affect the early entry stage, as evidenced by a time-of-drug-addition assay. This suggests that BPR3P0128's primary action takes place during viral replication. We also found that BPR3P0128 effectively reduced the expression of proinflammatory cytokines in human lung epithelial Calu-3 cells infected with SARS-CoV-2. Molecular docking analysis showed that BPR3P0128 targets the RdRp channel, inhibiting substrate entry, which implies it operates differently-but complementary-with remdesivir. Utilizing an optimized cell-based minigenome RdRp reporter assay, we confirmed that BPR3P0128 exhibited potent inhibitory activity. However, an enzyme-based RdRp assay employing purified recombinant nsp12/nsp7/nsp8 failed to corroborate this inhibitory activity. This suggests that BPR3P0128 may inhibit activity by targeting host-related RdRp-associated factors. Moreover, we discovered that a combination of BPR3P0128 and remdesivir had a synergistic effect-a result likely due to both drugs interacting with separate domains of the RdRp. This novel synergy between the two drugs reinforces the potential clinical value of the BPR3P0128-remdesivir combination in combating various SARS-CoV-2 variants of concern.

Single-Step Fabrication of Longtail Glasswing Butterfly-Inspired Omnidirectional Antireflective Structures.

Most bio-inspired antireflective nanostructures are extremely vulnerable and suffer from complicated lithography-based fabrication procedures. To address the issues, we report a scalable and simple non-lithography-based approach to engineer robust antireflective structures, inspired by the longtail glasswing butterfly, in a single step. The resulting two-dimensional randomly arranged 80/130/180 nm silica colloids, partially embedded in a polymeric matrix, generate a gradual refractive index transition at the air/substrate interface to suppress light reflection. Importantly, the randomly arranged subwavelength silica colloids display even better antireflection performance for large incident angles than that of two-dimensional non-close-packed silica colloidal crystals. The biomimetic coating is of considerable technological importance in numerous practical applications.

Methotrexate inhibition of SARS-CoV-2 entry, infection and inflammation revealed by bioinformatics approach and a hamster model.

Background: Drug repurposing is a fast and effective way to develop drugs for an emerging disease such as COVID-19. The main challenges of effective drug repurposing are the discoveries of the right therapeutic targets and the right drugs for combating the disease. Methods: Here, we present a systematic repurposing approach, combining Homopharma and hierarchal systems biology networks (HiSBiN), to predict 327 therapeutic targets and 21,233 drug-target interactions of 1,592 FDA drugs for COVID-19. Among these multi-target drugs, eight candidates (along with pimozide and valsartan) were tested and methotrexate was identified to affect 14 therapeutic targets suppressing SARS-CoV-2 entry, viral replication, and COVID-19 pathologies. Through the use of in vitro (EC50 = 0.4 µM) and in vivo models, we show that methotrexate is able to inhibit COVID-19 via multiple mechanisms. Results: Our in vitro studies illustrate that methotrexate can suppress SARS-CoV-2 entry and replication by targeting furin and DHFR of the host, respectively. Additionally, methotrexate inhibits all four SARS-CoV-2 variants of concern. In a Syrian hamster model for COVID-19, methotrexate reduced virus replication, inflammation in the infected lungs. By analysis of transcriptomic analysis of collected samples from hamster lung, we uncovered that neutrophil infiltration and the pathways of innate immune response, adaptive immune response and thrombosis are modulated in the treated animals. Conclusions: We demonstrate that this systematic repurposing approach is potentially useful to identify pharmaceutical targets, multi-target drugs and regulated pathways for a complex disease. Our findings indicate that methotrexate is established as a promising drug against SARS-CoV-2 variants and can be used to treat lung damage and inflammation in COVID-19, warranting future evaluation in clinical trials.

Perilla (Perilla frutescens) leaf extract inhibits SARS-CoV-2 via direct virus inactivation.

BACKGROUND: While severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection presents with mild or no symptoms in most cases, a significant number of patients become critically ill. Remdesivir has been approved for the treatment of coronavirus disease 2019 (COVID-19) in several countries, but its use as monotherapy has not substantially lowered mortality rates. Because agents from traditional Chinese medicine (TCM) have been successfully utilized to treat pandemic and endemic diseases, we designed the current study to identify novel anti-SARS-CoV-2 agents from TCM. METHODS: We initially used an antivirus-induced cell death assay to screen a panel of herbal extracts. The inhibition of the viral infection step was investigated through a time-of-drug-addition assay, whereas a plaque reduction assay was carried out to validate the antiviral activity. Direct interaction of the candidate TCM compound with viral particles was assessed using a viral inactivation assay. Finally, the potential synergistic efficacy of remdesivir and the TCM compound was examined with a combination assay. RESULTS: The herbal medicine Perilla leaf extract (PLE, approval number 022427 issued by the Ministry of Health and Welfare, Taiwan) had EC50 of 0.12 ± 0.06 mg/mL against SARS-CoV-2 in Vero E6 cells - with a selectivity index of 40.65. Non-cytotoxic PLE concentrations were capable of blocking viral RNA and protein synthesis. In addition, they significantly decreased virus-induced cytokine release and viral protein/RNA levels in the human lung epithelial cell line Calu-3. PLE inhibited viral replication by inactivating the virion and showed additive-to-synergistic efficacy against SARS-CoV-2 when used in combination with remdesivir. CONCLUSION: Our results demonstrate for the first time that PLE is capable of inhibiting SARS-CoV-2 replication by inactivating the virion. Our data may prompt additional investigation on the clinical usefulness of PLE for preventing or treating COVID-19.

Uncovering Flexible Active Site Conformations of SARS-CoV-2 3CL Proteases through Protease Pharmacophore Clusters and COVID-19 Drug Repurposing.

The infectious SARS-CoV-2 causes COVID-19, which is now a global pandemic. Aiming for effective treatments, we focused on the key drug target, the viral 3C-like (3CL) protease. We modeled a big dataset with 42 SARS-CoV-2 3CL protease-ligand complex structures from ∼98.7% similar SARS-CoV 3CL protease with abundant complex structures. The diverse flexible active site conformations identified in the dataset were clustered into six protease pharmacophore clusters (PPCs). For the PPCs with distinct flexible protease active sites and diverse interaction environments, we identified pharmacophore anchor hotspots. A total of 11 "PPC consensus anchors" (a distinct set observed in each PPC) were observed, of which three "PPC core anchors" EHV2, HV1, and V3 are strongly conserved across PPCs. The six PPC cavities were then applied in virtual screening of 2122 FDA drugs for repurposing, using core anchor-derived "PPC scoring S" to yield seven drug candidates. Experimental testing by SARS-CoV-2 3CL protease inhibition assay and antiviral cytopathic effect assays discovered active hits, Boceprevir and Telaprevir (HCV drugs) and Nelfinavir (HIV drug). Specifically, Boceprevir showed strong protease inhibition with micromolar IC50 of 1.42 µM and an antiviral activity with EC50 of 49.89 µM, whereas Telaprevir showed moderate protease inhibition only with an IC50 of 11.47 µM. Nelfinavir solely showed antiviral activity with a micromolar EC50 value of 3.28 µM. Analysis of binding mechanisms of protease inhibitors revealed the role of PPC core anchors. Our PPCs revealed the flexible protease active site conformations, which successfully enabled drug repurposing.

Water-Soluble Chemical Vapor Detection Enabled by Doctor-Blade-Coated Macroporous Photonic Crystals.

Water-soluble chemicals, involving a wide range of toxic chemicals in aqueous solutions, remain essential in both daily living or industrial uses. However, most toxicants are evaporated with water through their use and thus cause deleterious effects on the domestic environment and health in humans. Unfortunately, most current low-dose chemical vapor detection technologies are restricted by the use of sophisticated instruments and unable to promptly detect the quantity of diverse toxicants in a single analysis. To address these issues, this study reports the development of simple and fast chemical vapor detection using doctor-blade-coated macroporous poly(2-hydroxyethyl methacrylate)/poly(ethoxylated trimethylolpropane triacrylate) photonic crystals, in which the poly(2-hydroxyethyl methacrylate) has strong affinity to insecticide vapor owing to a favorable Gibbs free energy change for their mixing. The condensation of water-soluble chemical vapor therefore results in a significant reflection peak shift and an obvious color change. The visual colorimetric readout can be further improved by increasing the lattice spacing of the macroporous photonic crystals. Furthermore, the dependence of the reflection peak position on vapor pressure under actual conditions and the reproducibility of vapor detecting are also evaluated in this study.

Predictive Factors of 2-Year Postoperative Outcomes in Patients with Spontaneous Cerebellar Hemorrhage.

Spontaneous cerebellar hemorrhage (SCH) is associated with high patient mortality and morbidity, but the clinical and radiographic predictors of the postoperative outcome have not been widely addressed in the literature. The purpose of this study was to define the prognostic factors for the two-year postoperative outcome in patients with SCH. We conducted a retrospective study of 48 consecutive patients with SCH who underwent neurosurgical intervention. Correlation analysis was performed to examine the possible link between clinical and radiographic parameters, and the National Institutes of Health Stroke Scale (NIHSS) score at each patient's discharge and the two-year postoperative outcome as defined according to the Glasgow outcome scale (GOS). A total of 48 patients with SCH underwent neurological surgery, which included suboccipital craniectomy and/or external ventricular drainage (EVD). The mean patient age was 63 years. Nine patients underwent suboccipital craniectomy only; 38 underwent both suboccipital craniectomy and EVD. The overall mortality rate was 35.4%. Fourteen patients (29.2%) had good outcomes. A good outcome on the GOS at 2 years after surgical treatment of SCH was associated with the NIHSS score at discharge. An increase of one point in a patient's NIHSS score at discharge following neurological surgery will increase the probability of a poor two-year postoperative outcome by 28.5%.

Self-Assembled Curved Macroporous Photonic Crystal-Based Surfactant Detectors.

Surfactants are extensively used as detergents, dispersants, and emulsifiers. Thus, wastewater containing high-concentration surfactants discharged to the environment pose a serious threat to the ecosystem. Unfortunately, conventional detection methods for surfactants suffer from the use of sophisticated instruments and cannot perform detections for various surfactants by a single analysis. The article reports the development of simple and sensitive surfactant detection using doctor-blade-coated three-dimensional curved macroporous photonic crystals on a cylindrical rod. The photonic crystals exhibit different hydrophobicities at various angular positions after surface modification. The penetration of aqueous surfactant solutions in the interconnected macropores causes red-shift as well as reduction in amplitude in the optical stop bands, resulting in surfactant detection with visible readout. The correlation between the surface tension, as well as the solution-infiltrated angular position, and the concentration of aqueous surfactant solutions has also been investigated in this study.

Reusable macroporous photonic crystal-based ethanol vapor detectors by doctor blade coating.

This research reports the development of sensitive and reversible vapor detection by using three-dimensional macroporous photonic crystals. A scalable and roll-to-roll compatible doctor blade coating technology is utilized to fabricate flexible macroporous poly(ethoxylated trimethylolpropane triacrylate) (PETPTA) films with hexagonal close-packed pores which are interconnected. The pores are then coated with a layer of poly(2-hydroxyethyl methacrylate) (PHEMA) to create macroporous PHEMA/PETPTA films. The condensation of vapors in the PHEMA coated macroporous films leads to the increase of both the PHEMA swelling degree and the effective refractive index of the diffractive medium, resulting in the red-shift and amplitude reduction of the optical stop bands. The optical measurements reveal that the diffraction from the as-prepared macroporous photonic crystals sensitively monitors the vapor pressure of ethanol since the PHEMA layer displays a great volume dependence on ethanol due to a decreased Flory-Huggins mixing parameter. The dependence of the diffraction wavelength on vapor pressure and the reproducibility of vapor sensing have also been investigated in this study.

Self-Assembled Hierarchical Arrays for Colored Retroreflective Coatings.

This study reports a self-assembly technology for fabricating retroreflection coatings with hierarchical nano-/microstructures, which are inspired by the binary periodic structures found in the compound eyes of insects. Silica colloidal crystals of adjustable thicknesses are assembled on encountering glass microbeads using a Langmuir-Blodgett-like approach in a layer-by-layer manner. The as-assembled hierarchical structures exhibit a brilliant color caused by Bragg diffraction from the crystalline lattice of silica colloidal crystals on glass microbeads. The resultant coating is capable of reflecting light in the opposite direction of the incident light. Moreover, the dependence of the silica particle size, the colloidal crystal thickness, and the incident angle on the retroreflective properties are investigated in this study.

Epicatechin isolated from Tripterygium wilfordii extract reduces tau-GFP-induced neurotoxicity in zebrafish embryo through the activation of Nrf2.

Tau plays important roles in the assembly and stabilization of the microtubule structure to facilitate axonal transport in mammalian brain. The intracellular tau aggregates to form paired helical filaments leading to neurodegenerative disorders, collectively called tauopathies. In our previous report, we established a zebrafish model to express tau-GFP to induce neuronal death, which could be directly traced in vivo. Recently, we used this model to screen 400 herbal extracts and found 45 of them to be effective on reducing tau-GFP-induced neuronal death. One of the effective herbal extracts is the Tripterygium wilfordii stem extract. HPLC analysis and functional assay demonstrated that epicatechin (EC) is the major compound of Tripterygium wilfordii stem extract to decrease the neurotoxicity induced by tau-GFP. Using a luciferase reporter assay in the zebrafish, we confirmed that EC could activate Nrf2-dependent antioxidant responses to significantly increase the ARE-controlled expression of luciferase reporter gene. These data suggest that EC from the Tripterygium wilfordii stem extract could diminish tau-GFP-induced neuronal death through the activation of Nrf2.

Rapid detection of Yersinia pestis recombinant fraction 1 capsular antigen.

Yersinia pestis, an infectious bacterium that is a causative agent of plague, a disease which has been shown to be one of the most feared in history and which has caused millions of deaths. The capsule-like fraction 1 (F1) antigen expressed by Y. pestis is a known specific marker for the identification of the bacteria; therefore, the detection of F1 is important for Y. pestis recognition. In this study, a rapid, sensitive, and specific technique, the lateral flow assay (LFA), was successfully developed to detect Y. pestis by the recombinant F1 antigen. The assay that utilized an anti-F1 polyclonal antibody (Pab) to identify the bacteria was based on a double-antibody sandwich format on a nitrocellulose membrane. With the LFA method, 50 ng/ml of recombinant F1 protein and 10(5) CFU/mL of Y. pestis could be detected in less than 10 min. This assay also showed no cross-reaction with other Yersinia spp. or with some selected capsule-producing Enterobacteriaceae strains. Furthermore, detection of Y. pestis in simulated samples has been evaluated. The detection sensitivity of Y. pestis in various matrices was 10(5) CFU/mL, which was identical to that in PBS buffer. The results obtained suggest that LFA is an excellent tool for detection of Y. pestis contamination in an environment and hence can be used to monitor plague diseases when they emerge.

Effect of Lactobacillus plantarum Strain K21 on High-Fat Diet-Fed Obese Mice.

Recent studies have demonstrated beneficial effects of specific probiotics on alleviating obesity-related disorders. Here we aimed to identify probiotics with potential antiobesity activity among 88 lactic acid bacterial strains via in vitro screening assays, and a Lactobacillus plantarum strain K21 was found to harbor abilities required for hydrolyzing bile salt, reducing cholesterol, and inhibiting the accumulation of lipid in 3T3-L1 preadipocytes. Furthermore, effects of K21 on diet-induced obese (DIO) mice were examined. Male C57Bl/6J mice received a normal diet, high-fat diet (HFD), or HFD with K21 administration (10(9) CFU in 0.2 mL PBS/day) for eight weeks. Supplementation of K21, but not placebo, appeared to alleviate body weight gain and epididymal fat mass accumulation, reduce plasma leptin levels, decrease cholesterol and triglyceride levels, and mitigate liver damage in DIO mice. Moreover, the hepatic expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) related to adipogenesis was significantly downregulated in DIO mice by K21 intervention. We also found that K21 supplementation strengthens intestinal permeability and modulates the amount of Lactobacillus spp., Bifidobacterium spp., and Clostridium perfringens in the cecal contents of DIO mice. In conclusion, our results suggest that dietary intake of K21 protects against the onset of HFD-induced obesity through multiple mechanisms of action.

Effects of Kuan-Sin-Yin decoction on immunomodulation and tumorigenesis in mouse tumor models.

BACKGROUND: Complementary therapies are widely used among cancer patients. Kuan-Sin-Yin (KSY) decoction, a popular qi-promoting herbal medicine, was constituted with several herbs known to exhibit immunomodulating or anticancer activity. After combining these herbs as a compound formula, it is necessary to reassess the immunomodulation effects, the effects on tumor growth, and possible toxicity of KSY. METHODS: The anti-cancer effects of KSY in vivo were determined by measuring the tumor volumes, anticancer-associated cytokines (IFN-gamma, TNF-alpha, IL-2, and IL-12), accumulation of tumor infiltrating leukocytes (TILs), proliferation and apoptosis-related molecular markers (Ki-67, p53, p21, activated caspase 3, and cleaved PARP), and an in situ TUNEL assay. The body weight and serum chemistry of treated mice were also assessed. In vitro, the effects of KSY were evaluated using MTT assay, BrdU incorporation assay and cell growth curve. RESULTS: In vivo, KSY suppressed bladder or lung cancer growth but did not promote the production of cytokines nor increase the accumulation of TILs. The expression of p53 and p21 in KSY-treated mice were increased. The numbers of apoptotic tumor cells and the expression of apoptosis marker proteins (Caspase 3 and cleaved PARP) were not significantly elevated after KSY treatment. In vitro, the viability and proliferation of tumor cells, but not normal cells, were suppressed by KSY treatment. No significant toxicity was found in KSY-treated mice. CONCLUSIONS: KSY suppressed the tumor growth in vivo and in vitro, which resulted from its cytostatic effects on cancer cells, rather than the induction of anti-cancer immunity. Under these experimental conditions, no apparent toxicity was observed.

Field assessment of Orientia tsutsugamushi infection in small mammals and its association with the occurrence of human scrub typhus in Taiwan.

We conducted an extensive study in Taiwan of Orientia tsutsugamushi (OT) infection in small wild mammals. Field trapping was carried out at six districts in eastern and western Taiwan as well as various offshore islands during the period 2006-2010. A total of 1061 specimens representing 11 rodent species were captured. The presence of OT infection was assessed by indirect immunofluorescence assay and polymerase chain reaction assays of 56-kDa type-specific antigen gene. The chigger infestation rate among the animals was 35% (371/1061). Among these, OT was detected in 64% (238/371) of the chiggers from the infested animals and in the spleens from 273 (34.3%) of 797 animals. Excluding animals in the Suncus murinus group, the antibody positive rate of scrub typhus was 69.1% (477 of 690 of serum samples). The prevalence of OT infection in animals from areas with a low incidence of human cases of scrub typhus was significantly lower than that in rodents obtained from regions with a high incidence of human cases of the disease (44.4%±4.0% vs. 71.2%±9.7%, p<0.001). In Taiwan, the prevalence of OT infection in wild rodents is considerably high and appears to correlate positively with the occurrence of scrub typhus in humans.

Genetic typing, based on the 56-kilodalton type-specific antigen gene, of Orientia tsutsugamushi strains isolated from chiggers collected from wild-caught rodents in Taiwan.

Orientia tsutsugamushi is the etiological agent of scrub typhus, a mite-borne, febrile illness that occurs in the Asia-Pacific region. We conducted strain characterization of O. tsutsugamushi isolates from chiggers obtained from rodents based the nucleotide sequence of the 56-kDa outer membrane protein gene. With the use of PCR, a total of 68 DNA sequences of 56-kDa antigen genes were amplified. Phylogenetic analysis revealed that there were at least six definable clusters among the 68 isolates: 37% Karp-related strains (25/68), 27% TA763 strains (18/68), 12% JG-related strains (8/68), 19% Kato-related strains (13/68), 4% divergent strains (3/68), and 1% representing a Gilliam prototype strain (1/68). Overall, the O. tsutsugamushi genotypes exhibited a high degree of diversity, similar to that seen in strains from the rest of the areas where scrub typhus is endemic. Moreover, the 56-kDa protein sequence similarity between O. tsutsugamushi isolates from mites and those from human patients (H. Y. Lu et al., Am. J. Trop. Med. Hyg. 83:658-663, 2010) were striking, thus highlighting potential risk factors for this emerging zoonotic disease.

Inhibition of nitric oxide synthase promotes facial axonal regeneration following neurorrhaphy.

Reconnection of interrupted peripheral nerve by microsurgical suture is a common clinical practice. However, the extent to which peripheral neurorrhaphy improves nerve regeneration and functional recovery remains unsatisfactory. Here, we used anatomical and electrophysiological techniques to investigate the temporal correlation between the expressions of oxidative stress-related biomarkers such as neuronal nitric oxide synthase (nNOS) and the facial axonal regeneration after an immediate facial nerve repair in adult rats since peripheral nerve lesion is well known to induce a dramatic increase of NOS expression in the affected neuronal cell bodies. We found that compared to nerve cut without suture, facial nerve repair not only caused the facial axonal regeneration but also consistently prevented the fluctuations of expressions of oxidative stress-related biomarkers in 10 weeks postlesion. To further elucidate the role of nitric oxide (NO) in the axonal degeneration/regeneration, four different NOS inhibitors were applied to additional rats after facial nerve cut or repair. Both of facial nerve cut+NOS inhibition and facial nerve repair+NOS inhibition were seen to prevent the alterations of expressions of the biomarkers, no matter which NOS inhibitor was used. Moreover, we found that facial nerve repair+NOS inhibition promoted earlier and better axonal regeneration than facial nerve repair, demonstrated by labeling of neuromuscular junctions, retrograde tracing, and electromyography. These results provide direct evidence that peripheral nerve suture and/or treatment of NOS inhibitors can maintain the homeostasis of oxidative stress-related biomarkers, especially nNOS in neuronal cell bodies. These actions may thus facilitate the axonal regeneration.

Anti-enterovirus 71 activity screening of chinese herbs with anti-infection and inflammation activities.

Antipyretic and toxin-eliminating traditional Chinese herbs are believed to possess antiviral activity. In this study, we screened extracts of 22 herbs for activity against enterovirus 71 (EV71). We found that only extracts of Houttuynia cordata Thunb. could neutralize EV71-induced cytopathic effects in Vero cells. The 50% inhibitory concentration of H. cordata extract for EV71 was 125.92 +/- 27.84 mug/ml. Antiviral screening of herb extracts was also conducted on 3 genotypes of EV71, coxsackievirus A16 and echovirus 9. H. cordata extract had the highest activity against genotype A of EV71. A plaque reduction assay showed that H. cordata extract significantly reduced plaque formation. Viral protein expression, viral RNA synthesis and virus-induced caspase 3 activation were inhibited in the presence of H. cordata extract, suggesting that it affected apoptotic processes in EV71-infected Vero cells by inhibiting viral replication. The antiviral activity of H. cordata extract was greater in cells pretreated with extract than those treated after infection. We conclude that H. cordata extract has antiviral activity, and it offers a potential to develop a new anti-EV71 agent.

A model to study neurotropism and persistency of Japanese encephalitis virus infection in human neuroblastoma cells and leukocytes.

Japanese encephalitis (JE) is the most common mosquito-borne encephalitis in the Asia-Pacific region. Patients with JE usually present neuronal involvement, but other organ involvement is relatively rare. Employing human neuroblast-derived (NB) cell lines and different blood cells (erythrocytes, lymphocytes, granulocytes and monocytes), the neurotropism and persistency of Japanese encephalitis virus (JEV) in human cells was investigated. It was found that JEV could not replicate in erythrocytes, granulocytes or lymphocytes. Monocytes and NB cell lines could support replication of JEV as demonstrated by expression of viral NS3 antigen and virus plaque-forming units (p.f.u.). JEV could replicate more efficiently in neuroblastoma (HTB-11) cells than in monocytes after infection for 48 h (2.1+/-1.2x10(7) vs 2.8+/-0.7x10(2) p.f.u. ml(-1)). Two different strains of JEV revealed a similar infectivity to different leukocytes and four NB cell lines. In a kinetic study, it was found that JEV-infected monocytes possessed a high viability (90 %) after infection for 5 days, while JEV-infected neuroblastoma cells suffered cell apoptosis in 2 days and decreased viability to less than 1 % in 5 days. Further studies showed that monocytes could take up JEV rapidly, displaying a log scale increase of intracellular JEV titres in 9 h after infection. Significantly, extracellular production of JEV by monocytes started in 12 h, peaked in 3 days and persisted for more than 3 weeks. These results suggest that JEV-infected monocytes may play an important role in harbouring JEV for eventual transmission to NB cells and that modulation of JEV-induced NB cell apoptosis may be useful in treating patients with JE.