IL-33 induces antigen-specific IL-5+ T cells and promotes allergic-induced airway inflammation independent of IL-4

Type 2 cytokines (IL-4, IL-5, and IL-13) play a pivotal role in helminthic infection and allergic disorders. CD4(+) T cells which produce type 2 cytokines can be generated via IL-4-dependent and -independent pathways. Although the IL-4-dependent pathway is well documented, factors that drive IL-4-independent Th2 cell differentiation remain obscure. We report here that the new cytokine IL-33, in the presence of Ag, polarizes murine and human naive CD4(+) T cells into a population of T cells which produce mainly IL-5 but not IL-4. This polarization requires IL-1R-related molecule and MyD88 but not IL-4 or STAT6. The IL-33-induced T cell differentiation is also dependent on the phosphorylation of MAPKs and NF-kappaB but not the induction of GATA3 or T-bet. In vivo, ST2(-/-) mice developed attenuated airway inflammation and IL-5 production in a murine model of asthma. Conversely, IL-33 administration induced the IL-5-producing T cells and exacerbated allergen-induced airway inflammation in wild-type as well as IL-4(-/-) mice. Finally, adoptive transfer of IL-33-polarized IL-5(+)IL-4(-)T cells triggered airway inflammation in naive IL-4(-/-) mice. Thus, we demonstrate here that, in the presence of Ag, IL-33 induces IL-5-producing T cells and promotes airway inflammation independent of IL-4.     

RKTS-33, an epoxycyclohexenone derivative that specifically inhibits Fas ligand-dependent apoptosis in CTL-mediated cytotoxicity

Cytotoxic T lymphocytes (CTLs) eliminate virus-infected cells and tumor cells by two distinct killing pathways, mediated by lytic granules containing perforin and by Fas ligand (FasL). ECH [(2R,3R,4S)-2,3-epoxy-4-hydroxy-5-hydroxymethyl-6-(1E)-propenyl-cyclohex-5-en-1-one] has been shown to inhibit FasL-dependent apoptosis or the killing pathway in short-term culture. However, since ECH exhibited cell toxicity in long-term culture, we attempted the synthesis of less toxic epoxycyclohexenone derivatives. In the present study, we found that RKTS-33 [(2R,3R,4S)-2,3-epoxy-4-hydroxy-5-hydroxymethyl-cyclohex-5-en-1-one] has cell toxicity lower than ECH in long-term culture, and further investigated the inhibitory effect of RKTS-33 on CTL-mediated killing pathways. RKTS-33 did not affect cell-surface expression of FasL upon CD3 stimulation, but profoundly inhibited the FasL-dependent killing pathway mediated by CD4+ and CD8+ CTLs, indicating that RKTS-33 specifically blocks target cell apoptosis but not CTL function. By contrast, RKTS-33 did not affect the perforin-dependent killing pathway in CD8+ CTLs. These results indicate that RKTS-33 is a specific inhibitor of the FasL-dependent killing pathway in CTL-mediated cytotoxicity.     

Glycosylation profiles of the human colorectal cancer A33 antigen naturally expressed in the human colorectal cancer cell line SW1222 and expressed as recombinant protein in different insect cell lines

The A33 antigen is a cell surface glycoprotein expressed in human gastrointestinal epithelium and in 95% of colorectal cancers. We have compared the N-linked glycosylation profile of A33 antigen naturally expressed in a human colorectal cancer cell line with recombinant human A33 antigen (rA33) produced in insect cell culture using the baculovirus expression vector. N-Linked glycans were enzymatically released from the protein, and glycan composition was analyzed by HPLC. In three insect cell lines tested (Sf-21, Tn5B1-4, and Tn-4s), glycosylation of rA33 was dominated by high mannose structures (M5Gn2 to M9Gn2; 78-95% of total N-linked glycans), with M8Gn2 being the single most abundant glycoform. A33 antigen naturally expressed in the SW1222 human colon cancer cell line (A33) also possessed a high abundance of high mannose glycans (72%). No complex glycosylation was detected on rA33 expressed in insect cells. Natural A33 was galactosylated to a small extent (6%). These results illustrate a case of similar glycosylation of a glycoprotein between a recombinant version produced in insect cell culture and its counterpart naturally expressed in human cell culture.     

Establishment, Growth kinetics, and Susceptibility to AcMNPV of Heat Tolerant Lepidop teran Cell Lines

Lepidopteran heat-tolerant(ht)cell lines have been obtained with sf-9,sf-21 and several Bombyx cells.They have a distinct karyotype,membrane lipid composition,morphology and growth kinetics from the parental cell lines.In this paper,we report the development of ht cell lines from other insect species and examination of their growth characteristics and virus susceptibility.Adaptation of cell lines sf-9,BTI-TN-5131-4(High5)and BTI-TN-MG1(MG 1)to 33℃ and 35℃ was carried out by shifting the culture temperature between 28℃ and higher temperatures by a gradual stepwise increase in temperature.The process of adaption to a higher culture temperature was accomplished over a period of 2 months.The cell lines with the temperature adaption were designated as sf9-ht33,sf9-ht35,High5-ht33,High5-ht35,MG1-ht33,MG1-ht35.These cell lines have been subcultured over 70 passages.Adaption to high temperatures was confirmed by a constant population doubling time with individual cell lines.The population doubling time of heat adapted cell lines were 1-4 h less than these of parental cell lines.Cell shapes did not show obvious change,however,the cell size of sf9-ht cells was enlarged and those of High5 and MG1 ht cells were reduced after heat adaption.When the cell lines were infected with Autographa californica nuclear polyhedrosis virus(AcMNPV)at 28℃,33℃,35℃ and 37℃,production of budded virus and occlusion bodies in each cell line was optimum at its own adapted temperature.     

Epitope mapping, expression and post-translational modifications of two isoforms of CD33 (CD33M and CD33m) on lymphoid and myeloid human cells

We have tested the usefulness of several commercial anti-CD33 monoclonal antibodies (mAb) to determine the expression and localization of the two CD33 isoforms on several hematopoietic cell lines. The expression of the isoform CD33m, a CD33 transmembrane splice variant lacking the ligand-binding V immunoglobulin (Ig)-like domain, was detected by RT-polymerase chain reaction, western blot, confocal microscopy and flow cytometry on the membrane of several human cell types. CD33m was only detected by the anti-CD33 mAb HIM3-4 on the cell surface, whereas WM53, P67.6, 4D3, HIM3-4, WM54, D3HL60.251 or MY9 detected the CD33M isoform, indicating that HIM3-4 is the only mAb recognizing CD33 C(2) Ig domain. Accordingly, HIM3-4 binding to CD33 did not interfere with the binding of other antibodies against the CD33 V-domain. P67.6 mAb interfered with recognition by the rest of antibodies specific for the V domain. HIM3-4 staining could be increased after the sialidase treatment of all CD33(+) cells. However, this increase was stronger in activated T cells, suggesting a CD33 masking state in this cell population. Confocal microscopy analysis of CD33m HEK 293T-transfected cells revealed that this protein is expressed on the cell membrane and also detected in the Golgi compartment. CD33 is constitutively located outside the lipid raft domains, whereas cross-linked CD33 is highly recruited to this signaling platform. The unique ability of HIM3-4 mAb to detect the masking state of CD33 on different cell lineages makes it a good tool to improve the knowledge of the biological role of this sialic acid-binding Ig-like lectin.     

Structure-based design and discovery of potent and selective KDM5 inhibitors

Histone lysine demethylases (KDMs) play a key role in epigenetic regulation and KDM5A and KDM5B have been identified as potential anti-cancer drug targets. Using structural information from known KDM4 and KDM5 inhibitors, a potent series of pyrazolylpyridines was designed. Structure-activity relationship (SAR) exploration resulted in the identification of compound 33, an orally available, potent inhibitor of KDM5A/5B with promising selectivity. Potent cellular inhibition as measured by levels of tri-methylated H3K4 was demonstrated with compound 33 in the breast cancer cell line ZR-75-1.     

Soluble ST2 suppresses the effect of interleukin-33 on lung type 2 innate lymphoid cells

Type 2 innate lymphoid cells (ILC2) in lungs produce interleukin (IL)-5 and IL-13 in response to IL-33 and may contribute to the development of allergic diseases such as asthma. However, little is known about negative regulators and effective inhibitors controlling ILC2 function. Here, we show that soluble ST2, a member of the IL-1 receptor family, suppresses the effect of IL-33 on lung ILC2 in vitro. Stimulation with IL-33 to naïve ILC2 induced morphological change and promoted cell proliferation. In addition, IL-33 upregulated expression of cell surface molecules including IL-33 receptor and induced production of IL-5 and IL-13, but not IL-4. Pretreatment with soluble ST2 suppressed IL-33-mediated responses of ILC2. The results suggest that soluble ST2 acts as a decoy receptor for IL-33 and protects ILC2 from IL-33 stimulation.     

Establishment,growth kinetics,and susceptibility to AcMNPV of heat tolerant lepidopteran cell lines

Lepidopteran heat-tolerant (ht) cell lines have been obtained with sf-9, sf-21 and several Bombyx cells. They have a distinct karyotype, membrane lipid composition, morphology and growth kinetics from the parental cell lines. In this paper, we report the development of ht cell lines from other insect species and examination of their growth characteristics and virus susceptibility. Adaptation of cell lines sf-9, BTI-TN-5B1-4 (High5) and BTI-TN-MG1 (MG1) to 33°C and 35°C was carried out by shifting the culture temperature between 28°C and higher temperatures by a gradual stepwise increase in temperature. The process of adaption to a higher culture temperature was accomplished over a period of 2 months. The cell lines with the temperature adaption were designated as sf9-ht33, sf9-ht35, High5-ht33, High5-ht35, MG1-ht33, MG1-ht35. These cell lines have been subcultured over 70 passages. Adaption to high temperatures was confirmed by a constant population doubling time with individual cell lines. The population doubling time of heat adapted cell lines were 1–4 h less than these of parental cell lines. Cell shapes did not show obvious change, however, the cell size of sf9-ht cells was enlarged and those of High5 and MG1 ht cells were reduced after heat adaption. When the cell lines were infected with Autographa californica nuclear polyhedrosis virus (AcMNPV) at 28°C, 33°C, 35°C and 37°C, production of budded virus and occlusion bodies in each cell line was optimum at its own adapted temperature.     

Full-length IL-33 promotes inflammation but not Th2 response in vivo in an ST2-independent fashion

Expression of IL-33 is elevated in patients with pulmonary diseases, and full-length (not proteolytically processed) IL-33 is the predominant form in the lungs in health and disease. To determine whether activation of IL-33 is needed for functional effects, activities of full-length mouse and mature mouse (mm) forms of IL-33 were compared in vivo. Replication-deficient adenoviral constructs were used for gene delivery. Both isoforms caused pulmonary infiltration of lymphocytes and neutrophils, whereas mm IL-33 also caused pulmonary eosinophilia and goblet cell hyperplasia and increased expression of IL-4, IL-5, IL-13, IL-17, MCP-1, and KC. The different effects were not associated with differential release from IL-33-producing cells or by differences in subcellular distributions of IL-33 isoforms. Germline deficiency of the cell surface receptor chain ST2 abrogated the mm IL-33-induced Th2-associated effects (pulmonary eosinophilia, goblet cell hyperplasia, and increased IL-4 and IL-5), yet the lymphocytic infiltration induced by full-length mouse IL-33 or mm IL-33 was not fully abrogated by the absence of ST2. The similar effects of IL-33 isoforms were associated with comparable regulation of gene expression, notably matrix metalloproteinases 3, 10, and 13. Thus, full-length IL-33 is functionally active in vivo in an ST2-independent fashion, and its effects are partially different from those of mature IL-33. The different effects of these isoforms, particularly the pro-Th2 effects of mature IL-33, are due to differential utilization of the IL-33R chain ST2, whereas their similar effects result from regulation of gene expression.     

Lineage(-)Sca1+c-Kit(-)CD25+ cells are IL-33-responsive type 2 innate cells in the mouse bone marrow

IL-33 promotes type 2 immune responses, both protective and pathogenic. Recently, targets of IL-33, including several newly discovered type 2 innate cells, have been characterized in the periphery. In this study, we report that bone marrow cells from wild-type C57BL/6 mice responded with IL-5 and IL-13 production when cultured with IL-33. IL-33 cultures of bone marrow cells from Rag1 KO and Kit(W-sh/W-sh) mice also responded similarly; hence, eliminating the possible contributions of T, B, and mast cells. Rather, intracellular staining revealed that the IL-5- and IL-13-positive cells display a marker profile consistent with the Lineage(-)Sca-1(+)c-Kit(-)CD25(+) (LSK(-)CD25(+)) cells, a bone marrow cell population of previously unknown function. Freshly isolated LSK(-)CD25(+) cells uniformly express ST2, the IL-33 receptor. In addition, culture of sorted LSK(-)CD25(+) cells showed that they indeed produce IL-5 and IL-13 when cultured with IL-33 plus IL-2 and IL-33 plus IL-7. Furthermore, i.p. injections of IL-33 or IL-25 into mice induced LSK(-)CD25(+) cells to expand, in both size and frequency, and to upregulate ST2 and α(4)β(7) integrin, a mucosal homing marker. Thus, we identify the enigmatic bone marrow LSK(-)CD25(+) cells as IL-33 responsive, both in vitro and in vivo, with attributes similar to other type 2 innate cells described in peripheral tissues.     

V3 loop-determined coreceptor preference dictates the dynamics of CD4+-T-cell loss in simian-human immunodeficiency virus-infected macaques

We used experimental infection of rhesus macaques with envelope gp120 V3 loop isogenic simian-human immunodeficiency virus (SHIV) molecular clones to more clearly define the impact of human immunodeficiency virus type 1 coreceptor usage in target cell selectivity and the rates of CD4+-T-cell depletion. Functional assays demonstrate that substitution of the V3 loop of the pathogenic CXCR4-tropic (X4) SHIV(SF33A2) molecular clone with the corresponding sequences from the CCR5-tropic (R5) SHIV(SF162P3) isolate resulted in a switch of coreceptor usage from CXCR4 to CCR5. The resultant R5 clone, designated SHIV(SF33A2(V3)), is replication competent in vivo, infecting two of two macaques by intravenous inoculation with peak viremia that is comparable to that seen in monkeys infected with X4-SHIV(SF33A2). But while primary infection with the X4 clone was accompanied by rapid and significant loss of peripheral and secondary lymphoid CD4+ T lymphocytes, infection with R5-SHIV(SF33A2(V3)) led to only a modest and transient loss. However, substantial depletion of intestinal CD4+ T cells was observed in R5-SHIV(SF33A2(V3))-infected macaques. Moreover, na?ve T cells that expressed high levels of CXCR4 were rapidly depleted in X4-SHIV(SF33A2)-infected macaques, whereas R5-SHIV(SF33A2(V3)) infection mainly affected memory T cells that expressed CCR5. These findings in a unique isogenic system illustrate that coreceptor usage is the principal determinant of tissue and target cell specificity of the virus in vivo and dictates the dynamics of CD4+-T-cell depletion during SHIV infection.     

Synthesis and in vitro antitumor activity of thiophene analogues of 5-chloro-5,8-dideazafolic acid and 2-methyl-2-desamino-5-chloro-5,8-dideazafolic acid

N-[5-[N-(2-Amino-5-chloro-3,4-dihydro-4-oxoquinazolin-6-yl)methylamino]-2-thenoyl]-L-glutamic acid (6) and N-[5-[N-(5-chloro-3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl)methylamino]-2-thenoyl]-L-glutamic acid (7), the first reported thiophene analogues of 5-chloro-5,8-dideazafolic acid, were synthesized and tested as inhibitors of tumor cell growth in culture. 4-Chloro-5-methylisatin (10) was converted stepwise to methyl 2-amino-5-methyl-6-chlorobenzoate (22) and 2-amino-5-chloro-3,4-dihydro-6-methyl-4-oxoquinazoline (19). Pivaloylation of the 2-amino group, followed by NBS bromination, condensation with di-tert-butyl N-(5-amino-2-thenoyl)-L-glutamate (28), and stepwise cleavage of the protecting groups with ammonia and TFA yielded. Treatment of 9 with acetic anhydride afforded 2,6-dimethyl-5-chlorobenz[1,3-d]oxazin-4-one (31), which on reaction with ammonia, NaOH was converted to 2,6-dimethyl-5-chloro-3,4-dihydroquinazolin-4-one (33). Bromination of, followed by condensation with and ester cleavage with TFA, yielded. The IC(50) of and against CCRF-CEM human leukemic lymphoblasts was 1.8+/-0.1 and 2.1+/-0.8 microM, respectively.     

Expression and distribution of pituitary adenylate cyclase-activating peptide in human prostate and prostate cancer tissues

The therapeutic potential of the intestinotrophic mediator glucagon-like peptide-2 (1-33) [GLP-2 (1-33)] has increased interest in the pharmacokinetics of the peptide. This study was undertaken to investigate whether the primary degradation product GLP-2 (3-33) interacts with the GLP-2 receptor. Functional (cAMP) and binding in vitro studies were carried out in cells expressing the transfected human GLP-2 receptor. Furthermore, a biologic response of GLP-2 (3-33) was tested in vivo. Mice were allocated to groups treated for 10 days (twice daily) with: (1) 5 microg GLP-2 (1-33), (2) 25 microg GLP-2 (3-33), (3) 5 microg GLP-2 (1-33)+100 microg GLP-2 (3-33), or (4) 5 microg GLP-2 (1-33)+500 microg GLP-2 (3-33). The intestine was investigated for growth changes. GLP-2 (3-33) bound to the GLP-2 receptor with a binding affinity of 7.5% of that of GLP-2 (1-33). cAMP accumulation was stimulated with an efficacy of 15% and a potency more than two orders of magnitude lower than that of GLP-2 (1-33). Increasing doses of GLP-2 (3-33) (10(-7)-10(-5) M) caused a shift to the right in the dose-response curve of GLP-2 (1-33). Treatment of mice with either GLP-2 (1-33) or (3-33) induced significant growth responses in both the small and large intestines, but the response induced by GLP-2 (3-33) was much smaller. Co-administration of 500 microg of GLP-2 (3-33) and 5 microg GLP-2 (1-33) resulted in a growth response that was smaller than that of 5 microg GLP-2 (1-33) alone. Consistent with the observed in vivo activities, our functional studies and binding data indicate that GLP-2 (3-33) acts as a partial agonist with potential competitive antagonistic properties on the GLP-2 receptor.     

IL-33 activates B1 cells and exacerbates contact sensitivity

B1 B cells produce natural IgM and play a critical role in the early defense against bacterial and viral infection. The polyreactive IgM also contributes to the clearance of apoptotic products and plays an important role in autoimmune pathogenesis. However, the mechanism of activation and proliferation of B1 cells remains obscure. In this study, we report that IL-33, a new member of IL-1 family, activates B1 cells, which express the IL-33 receptor α, ST2. IL-33 markedly activated B1 cell proliferation and enhanced IgM, IL-5, and IL-13 production in vitro and in vivo in a ST2-dependent manner. The IL-33-activated B1 cell functions could be largely abolished by IL-5 neutralization and partially reduced by T cell or mast cell deficiency in vivo. ST2-deficient mice developed less severe oxazolone-induced contact sensitivity (CS) than did wild-type (WT) mice. Furthermore, IL-33 treatment significantly exacerbated CS in WT mice with enhanced B1 cell proliferation and IgM and IL-5 production. Moreover, IL-33-activated B1 cells from WT mice could adoptively transfer enhanced CS in ST2(-/-) mice challenged with IL-33. Thus, we demonstrate, to the best of our knowledge, a hitherto unrecognized mechanism of B1 cell activation and IL-33 function, and suggest that IL-33 may play an important role in delayed-type hypersensitivity.     

Preparation of Poliovirus Labeled with Phosphorus-33

Phosphorus-33 ((33)P), a weak (0.25 Mev) beta-emitting isotope of phosphorus with a half-life of 25 days, has been used to label poliovirus in cell culture. HeLa cell monolayers were depleted of phosphate and then labeled by incubating at 37 C in a medium (LM) containing about 10 muCi of (33)P as orthophosphate per ml. Labeled cells were infected at a high multiplicity with poliovirus type 1 and incubated for 8 hr in LM medium. Virus from infected cells was then concentrated and purified. Virus purity was confirmed by comparison of virus infectivity and radioactivity after CsCl density gradient centrifugation and by observing purified virus preparations with electron microscopy. With the method described, yields of about 10(10) to 5 x 10(10) plaque-forming units (PFU) of highly purified poliovirus with specific activities of about 3 x 10(-4) to 10(-3) disintegrations per min per PFU have been obtained from 1.5 x 10(8) to 3.0 x 10(8) HeLa cells.     

Association of poly(ADP-rib) synthesis with cessation of DNA synthesis and DNA fragmentation

CHO cells and cs-4-D3 cells were used to investigate the association between poly(ADP-rib) synthesis and the cessation of DNA synthesis and DNA fragmentation. The cs4-D3 cells are cold-sensitive DNA synthesis arrest mutants of CHO cells. Upon incubation at 33 degrees C, DNA synthesis in the cs4-D3 cells stops and the cells enter a prolonged G1 or G0 phase. The events that occurred when cs4 cells were incubated at 33 degrees C were similar to those that occurred when wild-type CHO cells grew to high density. (1) In both cases, DNA synthesis and cell growth stopped. (2) The NAD+ concentration/cell was 20-25% lower in growth-arrested cells than in logarithmically growing cells. (3) Poly(ADP-rib) synthesis was 3-4 fold higher in growth-arrested cells than in logarithmically growing cells. (4) The growth-inhibited cells developed DNA strand breaks which resulted in large percentages of their DNA appearing in the low molecular weight range of alkaline sucrose gradients. (5) Both the increased rate of poly(ADP-rib) synthesis and the development of DNA strand breaks appears to be characteristic of the G1 phase of the cell cycle. (6) When growth-inhibited cells were restored to conditions favorable for DNA synthesis and cell growth, the DNA strand breaks were repaired. (7) Prolonged incubation under growth-restrictive conditions resulted in the accumulation of more DNA strand breaks than the cells could repair. This was followed by cell death when the cells were restored to conditions favorable for cell growth.     

Study on the formation of specialized inter-Sertoli cell junctions in vitro.

An in vitro culture system using Sertoli cells was employed to assess the expression of component genes pertinent to occluding junctions (OJ) (such as zonula occludens-1, ZO-1), anchoring junctions (AJ) (such as N-cadherin and beta-catenin), and communicating gap junctions (GJ) (such as connexin 33, Cx33) when they are being formed in vitro. Freshly isolated Sertoli cells from 20-day-old rats with a purity of greater than 90% were cultured either at low- (2.5 x 10(4) cells/cm(2)) or high-cell density (0.6 x 10(6) cells/cm(2)) on Matrigel-coated dishes for 7 days in vitro to allow the establishment of specialized junctions. In low cell density Sertoli cell cultures, specialized OJ such as tight junctions did not form during the entire culture period when assessed by the transepithelial electrical resistance (TER). In high cell density cultures, there was an increase in ZO-1 expression in days 1 to 3 preceding the establishment of tight junctions by day 4. When Sertoli cells were cultured at both cell densities, there was a transient increase in Sertoli cell N-cadherin expression, which peaked by days 4-5, suggesting the time course for the establishment of AJ may overlap with the OJ. A significant increase in the expression of Sertoli cell beta-catenin was also detected by days 5-7 in the high but not low cell density cultures. The expression of Cx33 was also enhanced at days 4-5 in both high and low density cultures. These results suggest that OJ, AJ, and GJ are formed between Sertoli cells in high density cultures, whereas OJ cannot be formed in low density cultures. A full-length cDNA clone coding for rat testicular beta-catenin was also isolated. The deduced amino acid sequence of rat beta-catenin yielded a 781 amino acid polypeptide which displayed a 99.9% identity with the mouse homolog. Conditioned medium of germ cells induced a dose-dependent stimulation on Sertoli cell beta-catenin expression, suggesting germ cells may affect the N-cadherin/beta-catenin-mediated signal transduction pathway. In summary, this study illustrates several target genes can be used as molecular markers to monitor the inter-Sertoli junction formation. This system should be applicable to screen new male contraceptives in vitro targeted at the interference of junction formation by disrupting the timely expression of genes necessary for junction establishment and/or maintenance.     

Antiviral efficacy of bromo-anilino substituents of 4,5-dihydrofuran-3-carboxylate compound CW-33 against Japanese encephalitis virus

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, occasionally causes severe central nervous system disorders in the risk zone where more than 3 billion people reside. Our prior studies demonstrated antiviral potential of 4,5-dihydrofuran-3-carboxylate compound CW-33 (ethyl 2-(3′,5′-dimethylanilino)-4-oxo-4,5-dihydrofuran-3-carboxylate) and its derivative CW-33A ((ethyl 2-(2-fluoroanilino)-4-oxo-4,5-dihydrofuran-3-carboxylate) against JEV infection ((Int. J. Mol. Sci. 2016, 17: E1386; Sci. Rep. 2018, 8: 16595). This study synthesized six new CW-33 derivatives containing chloro, or bromo groups at the C-2, C-3, or C-4 of anilino ring of CW-33, and assessed the antiviral activity and mechanisms of these chloro- and bromo-anilino substituted derivatives. CW-33K, CW-33L and CW-33M had the bromo-substituents at the C-2, C-3, or C-4 of anilino ring of CW-33, respectively, showing the higher anti-JEV activity than CW-33 and other derivatives. CW-33K (ethyl 2-(2-bromoanilino)-4-oxo-4,5-dihydrofuran-3-carboxylate) exhibited the highest antiviral efficacy and therapeutic index. The IC50 value of CW-33K was less than 5 μM for reducing JEV-induced cytopathic effect, virus infectivity and virus yield. CW-33K significantly inhibited the JEV replication at the early and late stages, suppressing viral RNA synthesis and intracellular JEV particle production. The study demonstrated that the CW-33 derivative with a bromo substitution at the C-2 anilino ring improved the antiviral activity JEV, providing the structure-antiviral activity relationship for the development of anti-JEV agents.