Tubeimosides are pan-coronavirus and filovirus inhibitors that can block their fusion protein binding to Niemann-Pick C1.

SARS-CoV-2 and filovirus enter cells via the cell surface angiotensin-converting enzyme 2 (ACE2) or the late-endosome Niemann-Pick C1 (NPC1) as a receptor. Here, we screened 974 natural compounds and identified Tubeimosides I, II, and III as pan-coronavirus and filovirus entry inhibitors that target NPC1. Using in-silico, biochemical, and genomic approaches, we provide evidence that NPC1 also binds SARS-CoV-2 spike (S) protein on the receptor-binding domain (RBD), which is blocked by Tubeimosides. Importantly, NPC1 strongly promotes productive SARS-CoV-2 entry, which we propose is due to its influence on fusion in late endosomes. The Tubeimosides' antiviral activity and NPC1 function are further confirmed by infection with SARS-CoV-2 variants of concern (VOC), SARS-CoV, and MERS-CoV. Thus, NPC1 is a critical entry co-factor for highly pathogenic human coronaviruses (HCoVs) in the late endosomes, and Tubeimosides hold promise as a new countermeasure for these HCoVs and filoviruses.

Mammalian Cell-Line-Expressed CD2v Protein of African Swine Fever Virus Provides Partial Protection against the HLJ/18 Strain in the Early Infection Stage.

A cell line expressing the CD2v protein of ASFV was generated. The efficient expression of CD2v protein was determined by immunofluorescence and Western blotting. The CD2v protein was Ni-affinity purified from the supernatant of cell cultures. The CD2v-expressing cells showed properties of hemadsorption, and the secreted CD2v protein exhibited hemagglutinating activity. The antigenicity and immunoprotection ability of CD2v were evaluated by immunizing pigs alone, combined with a cell-line-expressed p30 protein or triple combined with p30 and K205R protein. Immunized pigs were challenged with the highly virulent ASFV strain HLJ/18. Virus challenge results showed that CD2v immunization alone could provide partial protection at the early infection stage. Protein p30 did not show synergistic protection effects in immunization combined with CD2v. Interestingly, immunization with the triple combination of CD2V, p30 and K205R reversed the protection effect. The viremia onset time was delayed, and one pig out of three recovered after the challenge. The pig recovered from ASFV clinical symptoms, the rectal temperature returned to normal levels and the viremia was cleared. The mechanism of this protection effect warrants further investigation.

A Novel Conserved Linear Neutralizing Epitope on the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein.

The continuous emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made it challenging to develop broad-spectrum prophylactic vaccines and therapeutic antibodies. Here, we have identified a broad-spectrum neutralizing antibody and its highly conserved epitope in the receptor-binding domain (RBD) of the spike protein (S) S1 subunit of SARS-CoV-2. First, nine monoclonal antibodies (MAbs) against the RBD or S1 were generated; of these, one RBD-specific MAb, 22.9-1, was selected for its broad RBD-binding abilities and neutralizing activities against SARS-CoV-2 variants. An epitope of 22.9-1 was fine-mapped with overlapping and truncated peptide fusion proteins. The core sequence of the epitope, 405D(N)EVR(S)QIAPGQ414, was identified on the internal surface of the up-state RBD. The epitope was conserved in nearly all variants of concern of SARS-CoV-2. MAb 22.9-1 and its novel epitope could be beneficial for research on broad-spectrum prophylactic vaccines and therapeutic antibody drugs. IMPORTANCE The continuous emergence of new variants of SARS-CoV-2 has caused great challenge in vaccine design and therapeutic antibody development. In this study, we selected a broad-spectrum neutralizing mouse monoclonal antibody which recognized a conserved linear B-cell epitope located on the internal surface of RBD. This MAb could neutralize all variants until now. The epitope was conserved in all variants. This work provides new insights in developing broad-spectrum prophylactic vaccines and therapeutic antibodies.

Comprehensive mapping of antigenic linear B-cell epitopes on K205R protein of African swine fever virus with monoclonal antibodies.

African swine fever virus causes an acute, highly contagious swine disease with high mortality, leading to enormous losses in the pig industry. The K205R, a nonstructural protein of African swine fever virus, is abundantly expressed in the cytoplasm of infected cells at the early stage of infection and induces a strong immune response. However, to date, the antigenic epitopes of this immunodeterminant have not been characterized. In the present study, the K205R protein was expressed in a mammalian cell line and purified using Ni-affinity chromatography. Furthermore, three monoclonal antibodies (mAbs; 5D6, 7A8, and 7H10) against K205R were generated. Indirect immunofluorescence assay and western blot results showed that all three mAbs recognized native and denatured K205R in African swine fever virus (ASFV)-infected cells. To identify the epitopes of the mAbs, a series of overlapping short peptides were designed and expressed as fusion proteins with maltose-binding protein. Subsequently, the peptide fusion proteins were probed with monoclonal antibodies using western blot and enzyme-linked immunosorbent assay. The three target epitopes were fine-mapped; the core sequences of recognized by the mAbs 5D6, 7A8, and 7H10 were identified as 157FLTPEIQAILDE168, 154REKFLTP160, and 136PTNAMFFTRSEWA148, respectively. Probing with sera from ASFV-infected pigs in a dot blot assay demonstrated that epitope 7H10 was the immunodominant epitope of K205R. Sequence alignment showed that all epitopes were conserved across ASFV strains and genotypes. To our knowledge, this is the first study to characterize the epitopes of the antigenic K205R protein of ASFV. These findings may serve as a basis for the development of serological diagnostic methods and subunit vaccines.

Diltiazem inhibits SARS-CoV-2 cell attachment and internalization and decreases the viral infection in mouse lung.

The continuous emergence of severe acute respiratory coronavirus 2 (SARS-CoV-2) variants and the increasing number of breakthrough infection cases among vaccinated people support the urgent need for research and development of antiviral drugs. Viral entry is an intriguing target for antiviral drug development. We found that diltiazem, a blocker of the L-type calcium channel Cav1.2 pore-forming subunit (Cav1.2 α1c) and an FDA-approved drug, inhibits the binding and internalization of SARS-CoV-2, and decreases SARS-CoV-2 infection in cells and mouse lung. Cav1.2 α1c interacts with SARS-CoV-2 spike protein and ACE2, and affects the attachment and internalization of SARS-CoV-2. Our finding suggests that diltiazem has potential as a drug against SARS-CoV-2 infection and that Cav1.2 α1c is a promising target for antiviral drug development for COVID-19.

SARS-CoV-2 uses metabotropic glutamate receptor subtype 2 as an internalization factor to infect cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses angiotensin-converting enzyme 2 (ACE2) as a binding receptor to enter cells via clathrin-mediated endocytosis (CME). However, receptors involved in other steps of SARS-CoV-2 infection remain largely unknown. Here, we found that metabotropic glutamate receptor subtype 2 (mGluR2) is an internalization factor for SARS-CoV-2. Our results show that mGluR2 directly interacts with the SARS-CoV-2 spike protein and that knockdown of mGluR2 decreases internalization of SARS-CoV-2 but not cell binding. Further, mGluR2 is uncovered to cooperate with ACE2 to facilitate SARS-CoV-2 internalization through CME and mGluR2 knockout in mice abolished SARS-CoV-2 infection in the nasal turbinates and significantly reduced viral infection in the lungs. Notably, mGluR2 is also important for SARS-CoV spike protein- and Middle East respiratory syndrome coronavirus spike protein-mediated internalization. Thus, our study identifies a novel internalization factor used by SARS-CoV-2 and opens a new door for antiviral development against coronavirus infection.

Generation of A Stable GFP-reporter Zika Virus System for High-throughput Screening of Zika Virus Inhibitors.

Zika virus (ZIKV) is associated with severe birth defects and Guillain-Barré syndrome and no approved vaccines or specific therapies to combat ZIKV infection are currently available. To accelerate anti-ZIKV therapeutics research, we developed a stable ZIKV GFP-reporter virus system with considerably improved GFP visibility and stability. In this system a BHK-21 cell line expressing DC-SIGNR was established to facilitate the proliferation of GFP-reporter ZIKV. Using this reporter virus system, we established a high-throughput screening assay and screened a selected plant-sourced compounds library for their ability to block ZIKV infection. More than 31 out of 974 tested compounds effectively decreased ZIKV reporter infection. Four selected compounds, homoharringtonine (HHT), bruceine D (BD), dihydroartemisinin (DHA) and digitonin (DGT), were further validated to inhibit wild-type ZIKV infection in cells of BHK-21 and human cell line A549. The FDA-approved chronic myeloid leukemia treatment drug HHT and BD were identified as broad-spectrum flavivirus inhibitors. DHA, another FDA-approved antimalarial drug effectively inhibited ZIKV infection in BHK-21 cells. HHT, BD and DHA inhibited ZIKV infection at a post-entry stage. Digitonin was found to have inhibitory activity in the early stage of viral infection. Our research provides an efficient high-throughput screening assay for ZIKV inhibitors. The active compounds identified in this study represent potential therapies for the treatment of ZIKV infection.

Host Factor SPCS1 Regulates the Replication of Japanese Encephalitis Virus through Interactions with Transmembrane Domains of NS2B.

Signal peptidase complex subunit 1 (SPCS1) is a newly identified host factor that regulates flavivirus replication, but the molecular mechanism is not fully understood. Here, using Japanese encephalitis virus (JEV) as a model, we investigated the mechanism through which the host factor SPCS1 regulates the replication of flaviviruses. We first validated the regulatory function of SPCS1 in JEV propagation by knocking down and knocking out endogenous SPCS1. The loss of SPCS1 function markedly reduced intracellular virion assembly and the production of infectious JEV particles but did not affect cell entry, RNA replication, or translation of the virus. SPCS1 was found to interact with nonstructural protein 2B (NS2B), which is involved in posttranslational protein processing and virus assembly. Serial deletion mutation of the JEV NS2B protein revealed that two transmembrane domains, NS2B(1-49) and NS2B(84-131), interact with SPCS1. Further mutagenesis analysis of conserved flavivirus residues in two SPCS1 interaction domains of NS2B demonstrated that G12A, G37A, and G47A in NS2B(1-49) and P112A in NS2B(84-131) weakened the interaction with SPCS1. Deletion mutation of SPCS1 revealed that SPCS1(91-169), which contains two transmembrane domains, was involved in interactions with both NS2B(1-49) and NS2B(84-131). Taken together, these results demonstrate that SPCS1 affects viral replication by interacting with NS2B, thereby influencing the posttranslational processing of JEV proteins and the assembly of virions.IMPORTANCE Understanding virus-host interactions is important for elucidating the molecular mechanisms of virus propagation and identifying potential antiviral targets. Previous reports demonstrated that SPCS1 is involved in the flavivirus life cycle, but the mechanism remains unknown. In this study, we confirmed that SPCS1 participates in the posttranslational protein processing and viral assembly stages of the JEV life cycle but not in the cell entry, genome RNA replication, or translation stages. Furthermore, we found that SPCS1 interacts with two independent transmembrane domains of the flavivirus NS2B protein. NS2B also interacts with NS2A, which is proposed to mediate virus assembly. Therefore, we propose a protein-protein interaction model showing how SPCS1 participates in the assembly of JEV particles. These findings expand our understanding of how host factors participate in the flavivirus replication life cycle and identify potential antiviral targets for combating flavivirus infection.

West Nile virus infectious replicon particles generated using a packaging-restricted cell line is a safe reporter system.

West Nile virus (WNV) is a neurotropic pathogen which causes zoonotic disease in humans. Recently, there have been an increasing number of infected cases and there are no clinically approved vaccines or effective drugs to treat WNV infections in humans. The purpose of this study was to facilitate vaccine and antiviral drug discovery by developing a packaging cell line-restricted WNV infectious replicon particle system. We constructed a DNA-based WNV replicon lacking the C-prM-E coding region and replaced it with a GFP coding sequence. To produce WNV replicon particles, cell lines stably-expressing prM-E and C-prM-E were constructed. When the WNV replicon plasmid was co-transfected with a WNV C-expressing plasmid into the prM-E-expressing cell line or directly transfected the C-prM-E expressing cell line, the replicon particle was able to replicate, form green fluorescence foci, and exhibit cytopathic plaques similar to that induced by the wild type virus. The infectious capacity of the replicon particles was restricted to the packaging cell line as the replicons demonstrated only one round of infection in other permissive cells. Thus, this system provides a safe and convenient reporter WNV manipulating tool which can be used to study WNV viral invasion mechanisms, neutralizing antibodies and antiviral efficacy.

Identification of a New Broadly Cross-reactive Epitope within Domain III of the Duck Tembusu Virus E Protein.

In 2010, a pathogenic flavivirus termed duck Tembusu virus (DTMUV) caused widespread outbreak of egg-drop syndrome in domesticated ducks in China. Although the glycoprotein E of DTMUV is an important structural component of the virus, the B-cell epitopes of this protein remains uncharacterized. Using phage display and mutagenesis, we identified a minimal B-cell epitope, 374EXE/DPPFG380, that mediates binding to a nonneutralizing monoclonal antibody. DTMUV-positive duck serum reacted with the epitope, and amino acid substitutions revealed the specific amino acids that are essential for antibody binding. Dot-blot assays of various flavivirus-positive sera indicated that EXE/DPPFG is a cross-reactive epitope in most flaviviruses, including Zika, West Nile, Yellow fever, dengue, and Japanese encephalitis viruses. These findings indicate that the epitope sequence is conserved among many strains of mosquito-borne flavivirus. Protein structure modeling revealed that the epitope is located in domain III of the DTMUV E protein. Together, these results provide new insights on the broad cross-reactivity of a B-cell binding site of the E protein of flaviviruses, which can be exploited as a diagnostic or therapeutic target for identifying, studying, or treating DTMUV and other flavivirus infections.

Epitope Identification and Application for Diagnosis of Duck Tembusu Virus Infections in Ducks.

Duck Tembusu virus (DTMUV) causes substantial egg drop disease. DTMUV was first identified in China and rapidly spread to Malaysia and Thailand. The antigenicity of the DTMUV E protein has not yet been characterized. Here, we investigated antigenic sites on the E protein using the non-neutralizing monoclonal antibodies (mAbs) 1F3 and 1A5. Two minimal epitopes were mapped to 221LD/NLPW225 and 87YAEYI91 by using phage display and mutagenesis. DTMUV-positive duck sera reacted with the epitopes, thus indicating the importance of the minimal amino acids of the epitopes for antibody-epitope binding. The performance of the dot blotting assay with the corresponding positive sera indicated that YAEYI was DTMUV type-specific, whereas 221LD/NLPW225 was a cross-reactive epitope for West Nile virus (WNV), dengue virus (DENV), and Japanese encephalitis virus (JEV) and corresponded to conserved and variable amino acid sequences among these strains. The structure model of the E protein revealed that YAEYI and LD/NLPW were located on domain (D) II, which confirmed that DII might contain a type-specific non-neutralizing epitope. The YAEYI epitope-based antigen demonstrated its diagnostic potential by reacting with high specificity to serum samples obtained from DTMUV-infected ducks. Based on these observations, a YAEYI-based serological test could be used for DTMUV surveillance and could differentiate DTMUV infections from JEV or WNV infections. These findings provide new insights into the organization of epitopes on flavivirus E proteins that might be valuable for the development of epitope-based serological diagnostic tests for DTMUV.

Newcastle disease virus-vectored West Nile fever vaccine is immunogenic in mammals and poultry.

BACKGROUND: West Nile virus (WNV) is an emerging zoonotic pathogen which is harmful to human and animal health. Effective vaccination in susceptible hosts should protect against WNV infection and significantly reduce viral transmission between animals and from animals to humans. A versatile vaccine suitable for different species that can be delivered via flexible routes remains an essential unmet medical need. In this study, we developed a recombinant avirulent Newcastle disease virus (NDV) LaSota strain expressing WNV premembrane/envelope (PrM/E) proteins (designated rLa-WNV-PrM/E) and evaluated its immunogenicity in mice, horses, chickens, ducks and geese. RESULTS: Mouse immunization experiments disclosed that rLa-WNV-PrM/E induces significant levels of WNV-neutralizing antibodies and E protein-specific CD4+ and CD8+ T-cell responses. Moreover, recombinant rLa-WNV-PrM/E elicited significant levels of WNV-specific IgG in horses upon delivery via intramuscular immunization, and in chickens, ducks and geese via intramuscular, oral or intranasal immunization. CONCLUSIONS: Our results collectively support the utility of rLa-WNV-PrM/E as a promising WNV veterinary vaccine candidate for mammals and poultry.

Generation and characterization of a monoclonal antibody against prM protein of West Nile virus.

West Nile virus (WNV), which is an emerging pathogenic flavivirus with increasing distribution worldwide, is the cause of major human and animal health concerns. The pre-membrane (prM) protein of WNV is cleaved during maturation by the furin protease into the structural protein M and a pr-segment. In this study we generated and characterized a monoclonal antibody (MAb) against the WNV prM protein. Western blot analysis showed that the MAb reacted with WNV prM specifically. Immunohistochemistry assays demonstrated that the MAb recognized native prM protein in transfected BHK-21 cells. Preliminary studies were performed to identify the epitope recognized by the MAb using a set of synthesized overlapping peptides spanning the whole length of the prM protein. The MAb reported here may provide a valuable tool for the further exploration of the biological properties and functions of the prM protein and may also be developed for potential clinical applications.

Generation and efficacy evaluation of recombinant classical swine fever virus E2 glycoprotein expressed in stable transgenic mammalian cell line.

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), which is a highly contagious swine disease that causes significant economic loses to the pig industry worldwide. The envelope E2 glycoprotein of CSFV is the most important viral antigen in inducing protective immune response against CSF. In this study, we generated a mammalian cell clone (BCSFV-E2) that could stably produce a secreted form of CSFV E2 protein (mE2). The mE2 protein was shown to be N-linked glycosylated and formed a homodimer. The vaccine efficacy of mE2 was evaluated by immunizing pigs. Twenty-five 6-week-old Landrace piglets were randomly divided into five groups. Four groups were intramuscularly immunized with mE2 emulsified in different adjuvants twice at four-week intervals. One group was used as the control group. All mE2-vaccinated pigs developed CSFV-neutralizing antibodies two weeks after the first vaccination with neutralizing antibody titers ranging from 1:40 to 1:320. Two weeks after the booster vaccination, the neutralizing antibody titers increased greatly and ranged from 1:10,240 to 1:81,920. At 28 weeks after the booster vaccine was administered, the neutralizing antibody titers ranged from 1:80 to 1:10240. At 32 weeks after the first vaccination, pigs in all the groups were challenged with a virulent CSFV strain at a dose of 1 × 10(5) TCID50. At two weeks after the challenge, all the mE2-immunized pigs survived and exhibited no obvious symptoms of CSF. The neutralizing antibody titer at this time was 20,480. Unvaccinated pigs in the control group exhibited symptoms of CSF 3-4 days after challenge and were euthanized from 7-9 days after challenge when the pigs became moribund. These results indicate that the mE2 is a good candidate for the development of a safe and effective CSFV subunit vaccine.

Generation and characterization of a new mammalian cell line continuously expressing virus-like particles of Japanese encephalitis virus for a subunit vaccine candidate.

BACKGROUND: Japanese encephalitis virus (JEV) is the most important cause of epidemic encephalitis in most Asian regions. There is no specific treatment available for Japanese encephalitis, and vaccination is the only effective way to prevent JEV infection in humans and domestic animals. The purpose of this study is to establish a new mammalian cell line stably and efficiently expressing virus-like particle of JEV for potential use of JEV subunit vaccine. RESULTS: We generated a new cell clone (BJ-ME cells) that stably produces a secreted form of Japanese encephalitis virus (JEV) virus-like particle (VLP). The BJ-ME cells were engineered by transfecting BHK-21 cells with a code-optimized cDNA encoding JEV prM and E protein expression plasmid. Cell line BJ-ME can stably produces a secreted form of Japanese encephalitis virus virus-like particle (JEV-VLP) which contains the JEV envelope glycoprotein (E) and membrane protein (M). The amount of JEV-VLP antigen released into the culture fluid of BJ-ME cells was as high as 15-20 µg/ml. JEV-VLP production was stable after multiple cell passages and 100% cell expression was maintained without detectable cell fusion or apoptosis. Cell culture fluid containing the JEV-VLP antigen could be harvested five to seven times continuously at intervals of 4-6 days while maintaining the culture. Mice immunized with the JEV-VLP antigen with or without adjuvant developed high titers of neutralizing antibodies and 100% protection against lethal JEV challenge. CONCLUSION: These results suggest that the recombinant JEV-VLP antigen produced by the BJ-ME cell line is an effective, safe and affordable subunit Japanese encephalitis vaccine candidate, especially for domestic animals such as pig and horse.

Comprehensive mapping of a novel NS1 epitope conserved in flaviviruses within the Japanese encephalitis virus serocomplex.

Nonstructural protein-1 (NS1) of the Japanese encephalitis virus (JEV) is an immunogenic protein that is a potential candidate for the development of vaccines and diagnostic reagents. NS1 is known to be more specific than the E protein in serological testing of flavivirus infections. However, NS1 exhibits cross-reactivity among flaviviruses even within the same genus and more so within a serocomplex. However, the cross-reactive epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of a linear B-cell epitope that is common and specific to the JEV serocomplex of Flaviviridae. We generated an NS1-specific monoclonal antibody that cross-reacts with the West Nile virus (WNV) NS1 protein by immunizing mice with recombinant JEV NS1. For epitope mapping, 51 partially overlapping peptides spanning the entire NS1 protein were expressed with a glutathione S-transferase (GST) tag and screened using monoclonal antibodies. Two linear epitope-containing peptides were identified using enzyme-linked immunosorbent assay (ELISA). By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, we successfully identified the smallest unit of the linear epitope required to react with the monoclonal antibody. The linear epitope was located in amino acids residues ²²7ETHTLW²³². Furthermore, results of the sequence alignment revealed that the epitope was highly conserved among JEV strains. Notably, the epitope is highly conserved among viruses of the JEV serocomplex. Furthermore, the homologous regions on NS1 proteins from dengue viruses showed no cross-reactivity with the monoclonal antibodies. The epitope was recognized by antisera against the WNV but not against the dengue virus. This novel JEV serocomplex-specific linear B-cell epitope of NS1 would be helpful in the development of new vaccines and diagnostic assays.

Comprehensive Mapping Antigenic Epitopes of NS1 Protein of Japanese Encephalitis Virus with Monoclonal Antibodies.

Japanese encephalitis virus (JEV) non-structural protein 1 (NS1) contributes to virus replication and elicits protective immune responses during infection. JEV NS1-specific antibody responses could be a target in the differential diagnosis of different flavivirus infections. However, the epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of linear B-cell epitopes in JEV NS1. We generated eleven NS1-specific monoclonal antibodies from mice immunized with recombinant NS1. For epitope mapping of monoclonal antibodies, a set of 51 partially-overlapping peptides covering the entire NS1 protein were expressed with a GST-tag and then screened using monoclonal antibodies. Through enzyme-linked immunosorbent assay (ELISA), five linear epitope-containing peptides were identified. By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, the minimal units of the five linear epitopes were identified and confirmed using monoclonal antibodies. Five linear epitopes are located in amino acids residues (5)AIDITRK(11), (72)RDELNVL(78), (251)KSKHNRREGY(260), (269)DENGIVLD(276), and (341)DETTLVRS(348). Furthermore, it was found that the epitopes are highly conserved among JEV strains through sequence alignment. Notably, none of the homologous regions on NS1 proteins from other flaviviruses reacted with the MAbs when they were tested for cross-reactivity, and all five epitope peptides were not recognized by sera against West Nile virus or Dengue virus. These novel virus-specific linear B-cell epitopes of JEV NS1 would benefit the development of new vaccines and diagnostic assays.

Detection of specific antibodies against tembusu virus in ducks by use of an E protein-based enzyme-linked immunosorbent assay.

We developed an enzyme-linked immunosorbent assay (ELISA) using eukaryotically expressed E protein as the antigen (termed E-ELISA) to detect antibodies to tembusu virus (TMUV) in ducks. The E-ELISA did not react with antisera to other known pathogens, indicating the E protein is specific for recognizing anti-TMUV antibodies. Compared to the serum neutralization test, the specificity and sensitivity of the E-ELISA was 93.2 and 97.8%, respectively. Therefore, this E-ELISA is a sensitive and rapid method for detecting antibodies against TMUV in ducks.