Duck Tembusu Virus Infection Research Articles

Duck Tembusu Virus Inhibits Type I Interferon Production through the JOSD1-SOCS1-IRF7 Negative-Feedback Regulation Pathway.

Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that mainly causes a decrease in egg production in infected waterfowl. Similar to other members of the Flaviviridae family, it can proliferate in most mammalian cells and may also pose a potential threat to nonavian animals. In previous studies, we found that DTMUV infection can upregulate suppressor of cytokine signaling 1 (SOCS1) to inhibit type I interferon (IFN) production and promote virus replication, but the specific mechanism is unclear. Furthermore, little is known about the regulatory role of ubiquitination during flavivirus infection. In this study, we found that activation of Toll-like receptor 3 (TLR3) signaling rather than type I IFN stimulation led to the upregulation of SOCS1 during DTMUV infection. Further studies revealed that JOSD1 stabilized SOCS1 expression by binding to the SH2 domain of SOCS1 and mediating its deubiquitination. In addition, JOSD1 also inhibited type I IFN production through SOCS1. Finally, SOCS1 acts as an E3 ubiquitin ligase that binds to IFN regulatory factor 7 (IRF7) through its SH2 domain and mediates K48-linked ubiquitination and proteasomal degradation of IRF7, ultimately inhibiting type I IFN production mediated by IRF7 and promoting viral proliferation. These results will enrich and deepen our understanding of the mechanism by which DTMUV antagonizes the host interferon system. IMPORTANCE DTMUV is a newly discovered flavivirus that seriously harms the poultry industry. In recent years, there have been numerous studies on the involvement of ubiquitination in the regulation of innate immunity. However, little is known about the involvement of ubiquitination in the regulation of flavivirus-induced type I IFN signaling. In this study, we found that SOCS1 was induced by TLR3 signaling during DTMUV infection. Furthermore, we found for the first time that duck SOCS1 protein was also modified by K48-linked polyubiquitination, whereas our previous study found that SOCS1 was upregulated during DTMUV infection. Further studies showed that JOSD1 stabilized SOCS1 expression by mediating the deubiquitination of SOCS1. While SOCS1 acts as a negative regulator of cytokines, we found that DTMUV utilized SOCS1 to mediate the ubiquitination and proteasomal degradation of IRF7 and ultimately inhibit type I IFN production, thereby promoting its proliferation.

Avian IRF1 and IRF7 Play Overlapping and Distinct Roles in Regulating IFN-Dependent and -Independent Antiviral Responses to Duck Tembusu Virus Infection

Avian interferon regulatory factors 1 and 7 (IRF1 and IRF7) play important roles in the host’s innate immunity against viral infection. Our previous study revealed that duck tembusu virus (DTMUV) infection of chicken fibroblasts (DF1) and duck embryo fibroblasts (DEFs) induced the expression of a variety of IFN-stimulated genes (ISGs), including VIPERIN, IFIT5, CMPK2, IRF1, and IRF7. IRF1 was further shown to play a significant role in regulating the up-expression of VIPERIN, IFIT5, and CMPK2 and inhibiting DTMUV replication. In this study, we confirm, through overexpression and knockout approaches, that both IRF1 and IRF7 inhibit DTMUV replication, mainly via regulation of type I IFN expression, as well as the induction of IRF1, VIPERIN, IFIT5, CMPK2, and MX1. In addition, IRF1 directly promoted the expression of VIPERIN and CMPK2 in an IFN-independent manner when IRF7 and type I IFN signaling were undermined. We also found that non-structural protein 2B (NS2B) of DTMUV was able to inhibit the induction of IFN-β mRNA triggered by Newcastle disease virus (NDV) infection or poly(I:C) treatment, revealing a strategy employed by DTMUV to evade host’s immunosurveillance. This study demonstrates that avian IRF7 and IRF1 play distinct roles in the regulation of type I IFN response during DTMUV infection.

Duck Tembusu virus infection induces mitochondrial-mediated and death receptor-mediated apoptosis in duck embryo fibroblasts

Duck Tembusu virus (DTMUV) is a pathogenic flavivirus that has caused enormous economic losses in Southeast Asia. Our previous study showed that DTMUV could induce duck embryo fibroblast (DEF) apoptosis, but the specific mechanism was not clear. In this study, we confirmed that DTMUV could induce the apoptosis of DEFs by DAPI staining and TUNEL staining. Furthermore, we found that the expression levels of cleaved-caspase-3/7/8/9 were significantly upregulated after DTMUV infection. After treatment of cells with an inhibitor of caspase-8 or caspase-9, DTMUV-induced apoptosis rates were significantly decreased, indicating that the caspase-8-mediated death receptor apoptotic pathway and caspase-9-mediated mitochondrial apoptotic pathway were involved in DTMUV-induced apoptosis. Moreover, we found that DTMUV infection not only caused the release of mitochondrial cytochrome C (Cyt C) and the downregulation of the apoptosis-inhibiting protein Bcl-2 but also reduced the mitochondrial membrane potential (MMP) and the accumulation of intracellular reactive oxygen species (ROS). Key genes in the mitochondrial apoptotic pathway and death receptor apoptotic pathway were upregulated to varying degrees, indicating the activation of the mitochondrial apoptosis pathway and death receptor apoptosis pathway. In conclusion, this study clarifies the molecular mechanism of DTMUV-induced apoptosis and provides a theoretical basis for revealing the pathogenic mechanism of DTMUV infection.

Duck LGP2 Downregulates RIG-I Signaling Pathway-Mediated Innate Immunity Against Tembusu Virus.

In mammals, the retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) has been demonstrated to play a critical role in activating downstream signaling in response to viral RNA. However, its role in ducks’ antiviral innate immunity is less well understood, and how gene-mediated signaling is regulated is unknown. The regulatory role of the duck laboratory of genetics and physiology 2 (duLGP2) in the duck RIG-I (duRIG-I)-mediated antiviral innate immune signaling system was investigated in this study. In duck embryo fibroblast (DEF) cells, overexpression of duLGP2 dramatically reduced duRIG-I-mediated IFN-promotor activity and cytokine expression. In contrast, the knockdown of duLGP2 led to an opposite effect on the duRIG-I-mediated signaling pathway. We demonstrated that duLGP2 suppressed the duRIG-I activation induced by duck Tembusu virus (DTMUV) infection. Intriguingly, when duRIG-I signaling was triggered, duLGP2 enhanced the production of inflammatory cytokines. We further showed that duLGP2 interacts with duRIG-I, and this interaction was intensified during DTMUV infection. In summary, our data suggest that duLGP2 downregulated duRIG-I mediated innate immunity against the Tembusu virus. The findings of this study will help researchers better understand the antiviral innate immune system’s regulatory networks in ducks.

Duck Tembusu virus infection causes testicular atrophy

In recent years, Duck Tembusu virus (DTMUV) is becoming an important emerging and re-emerging pathogen that severely harms the poultry industry in China. The DTMUV disease was principally identified by a sharp decline in egg production, whereas few studies were focused on the virus-reproductive system interaction, especially male reproductive system. Herein, the present study was aimed at investigating the in vivo morphological changes in testis from DTMUV-infected adult Shaoxing ducks. After DTMUV infection, the gross observation indicated that the testis of DTMUV-infected ducks was significantly atrophied at 2 days post-infection (dpi), 4 dpi, and 8 dpi. At microscopic and ultrastructural level, morphological analysis revealed that DTMUV could lead to cytoplasmic vacuolation and exfoliation in seminiferous epithelium, decrease in the diameter of seminiferous tubule (ST), and even induce interstitial inflammation in duck testis. Ulteriorly, the spermatogenic cells, especially spermatocytes, are identified as the target cells of DTMUV infection in the testis of ducks through immunohistochemistry (IHC). And more notably, single virus particles and clustered virus particles were observed in the spermatogenic cells from infected ducks. In summary, our results comprehensively illustrated the effects of DTMUV infection on the testis, the morphological changes underlying testicular atrophy and identified the target cells of DTMUV infection in the testis of ducks.

RNA-Seq analysis of duck embryo fibroblast cells gene expression during duck Tembusu virus infection

Duck Tembusu virus (DTMUV), a member of the family Flaviviridae and an economically important pathogen with a broad host range, leads to markedly decreased egg production. However, the molecular mechanism underlying the host-DTMUV interaction remains unclear. Here, we performed high-throughput RNA sequencing (RNA-Seq) to study the dynamic changes in host gene expression at 12, 24, 36, 48 and 60 h post-infection (hpi) in duck embryo fibroblasts (DEF) infected with DTMUV. A total of 3129 differentially expressed genes (DEG) were identified after DTMUV infection. Gene Ontology (GO) category and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that these DEG were associated with multiple biological functions, including signal transduction, host immunity, virus infection, cell apoptosis, cell proliferation, and pathogenicity-related and metabolic process signaling pathways. This study analyzed viral infection and host immunity induced by DTMUV infection from a novel perspective, and the results provide valuable information regarding the mechanisms underlying host-DTMUV interactions, which will prove useful for the future development of antiviral drugs or vaccines for poultry, thus benefiting the entire poultry industry.

Evaluation and comparison of hemagglutination inhibition and indirect immunofluorescence tests for the detection of antibodies against duck Tembusu virus.

Currently, duck Tembusu virus (DTMUV), an emerging avian pathogenic flavivirus, is widely spread and becomes endemic in duck populations in Asia, causing significant economic losses in the duck producing industry. To early detection and control of DTMUV, the well-validated diagnostic tests for efficient detection of DTMUV infection in ducks are needed. In this study, we validated and compared hemagglutination inhibition (HI) and indirect immunofluorescence (IFA) tests for identifying antibodies against DTMUV in duck serum samples. Our results demonstrated that HI and IFA tests can both be used to detect antibodies against DTMUV in duck serum samples with high sensitivity (100%), specificity (>87%) and overall agreement with the gold standard serum neutralization (SN) test (>90%). Additionally, DTMUV-specific antibody titres determined by HI and IFA tests correlated well with the neutralizing antibody titres obtained by SN test. No cross-reactivity against common duck viruses and other flaviviruses was observed in both tests. It is interesting to note that HI test had higher diagnostic specificity and exhibited a stronger positive correlation with SN test than IFA test. Evaluating the performance of HI and IFA tests with experimental and field serum samples revealed that both tests showed comparable performance with SN test in terms of antibody kinetic and detection rate. Collectively, these findings support the use of both tests, particularly HI test, as the alternative to SN test for measuring the antibody responses against DTMUV in ducks. These tests could be the suitable choices for DTMUV diagnosis, epidemiological study and vaccine efficacy evaluation.

Dynamics of cellular and humoral immune responses following duck Tembusu virus infection in ducks.

Duck Tembusu virus (DTMUV), an emerging avian pathogenic flavivirus, causes severe neurological disorders and acute egg drop syndrome in ducks. However, the effects of DTMUV on duck immunological components and functions remain largely unknown. In this study, the dynamics of cellular and humoral immune responses of DTMUV-infected ducks were investigated. The numbers of CD4+ and CD8+ T, B and non-T and B lymphocytes as well as the levels of neutralizing antibodies were quantified in parallel with DTMUV loads in blood and target organs. Our results demonstrated that DTMUV infection caused severe losses of non-T and B lymphocyte/myeloid cell subpopulation, and reduction in phagocytic activity during 3-5 days after infection. We also found that the numbers of T and B cells were increased during the first week of DTMUV infection. A significant negative correlation between the levels of CD4+ and CD8+ T, B and non-T and B lymphocytes and viral loads in blood and target organ (spleen) was observed during the early phase of infection. Additionally, DTMUV infection induced an early and robust neutralizing antibody response, which was associated with DTMUV-specific IgM and IgG responses. The presence of neutralizing antibody also correlated with reduction of viremia and viral load in the spleen. Overall, DTMUV elicited both cellular and humoral immune responses upon infection, in which the magnitude of these responses was correlated with reduction of viremia and viral loads in the target organ (spleen). The results suggested the critical role of both cellular and humoral immunity against DTMUV infection. This study expands our understanding of the immunological events following DTMUV infection in ducks.

Effects of Aedes aegypti salivary protein on duck Tembusu virus replication and transmission in salivary glands

Duck Tembusu virus (DTMUV) infection is an arthropod-borne viral disease that affects many poultry species, including ducks, chickens, and geese. Aedes aegypti mosquito is an important vector of DTMUV. This study sought to determine whether any individual Ae. aegypti salivary protein modulated DTMUV replication in the mosquito salivary gland. Ae. aegypti salivary gland protein of 34 kDa (AaSG34) was found to be expressed explicitly in mosquito salivary glands and was upregulated following DTMUV infection. Thus, AaSG34 was silenced in mosquitoes via RNA interference using double strand RNA (dsRNA), and the mosquitoes were then infected with DTMUV to elucidate their effects on DTMUV replication and transmission. Transcripts of the DTMUV genome in salivary glands and virus titer in saliva were significantly diminished when AaSG34 was silenced, indicating that its presence enhances DTMUV replication in the salivary glands and DTMUV dissemination to saliva. Furthermore, the expression of antimicrobial peptides (AMPs) was upregulated upon AaSG34 silenced. Our results demonstrate that AaSG34 may play a vital role in the suppression of antiviral immune responses to enhance DTMUV replication and transmission. We thus provide new information on the effect of the AaSG34 salivary protein on DTMUV replication in Ae. aegypti as the mechanism of blocking virus transmission to the host.

Role of apoptosis in Duck Tembusu virus infection of duckling brains in vivo

The Duck Tembusu virus (DTMUV) is a novel flavivirus that occurs mainly in poultry. DTMUV infection can cause common neurological symptoms in ducklings, but the pathogenesis of DTMUV has not been elucidated yet. In this study, a DTMUV-infected duckling model was constructed to investigate the apoptosis in the duckling brains. After DTMUV infection, apoptotic cells were observed by transmission electron microscopy. It was found that the abundances of apoptosis-related genes and proteins were not obviously changed in the early stage of infection but significantly changed in the middle and late stages of the disease. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay staining results were also consistent with the above phenomena. Interestingly, although apoptosis occurred in the duckling brains infected by DTMUV, some antiapoptotic genes in the brain increased in varying degrees. In conclusion, DTMUV infection could induce apoptosis in ducklings’ brains, and the occurrence of apoptosis was accompanied by the virus infection process with certain regularity. This study provides a scientific basis for elucidating the apoptotic mechanism of brain lesions induced by DTMUV infection.

The E3 Ubiquitin Ligase TRIM25 Inhibits Tembusu Virus Replication in vitro.

Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused significant economic losses to the duck industry in China since 2010 due to egg production losses and neurological dysfunction. DTMUV is a public health concern because the infection spreads rapidly among birds. Retinoic acid-inducible gene-I (RIG-I)serves as an innate immune sensor and plays a key role in host antiviral defenses. Tripartite motif-containing protein 25 (TRIM25), an E3 ubiquitin ligase, is pivotal for RIG-I ubiquitination and activation. In addition, TRIM25 acts as an interferon-stimulated gene and mediates the antiviral activity. However, the effect of duck TRIM25 on DTMUV has not been assessed. Herein, we reportthe antiviral function of TRIM25 against DTMUV. First, we constructed the pcDNA3.1-c-myc-duTRIM25 plasmid. TRIM25 has a 2052 bp open reading frame that encodes a predicted 684 amino acid protein consisting of a RING finger domain, a B-box domain, a coiled-coil domain, and a PRY/SPRY domain. The protein sequence identity with chicken, mouse, and human TRIM25 is 69.7, 47.8, and 48.3%, respectively. TRIM25 was upregulated in BHK-21 cells, duck embryo fibroblasts, and 293T cellsupon DTMUV infection. The expression of viral RNA and proteins was significantly lower in cells over expressing TRIM25 than in control cells. Furthermore, siRNA-mediated silencing of TRIM25 increased the production of viral progeny. These results help elucidate the molecular mechanisms underlying the host response to DTMUV infection and suggest potential control measures for DTMUV outbreaks.

Duck Tembusu virus induces stronger cellular responses than Japanese encephalitis virus in primary duck neurons and fibroblasts.

Duck Tembusu virus (DTMUV) and Japanese encephalitis virus (JEV) are mosquito-borne flaviviruses. These two viruses infect ducks; however, they show different neurological outcomes. The mechanism of DTMUV- and JEV-induced neuronal death has not been well investigated. In the present study examined the differences in the mechanisms involved in virus-induced cell death and innate immune responses between the DTMUV KPS54A61 strain and the JEV JaGAr-01 strain using primary duck neurons (DN) and duck fibroblasts (CCL-141). DN and CCL-141 were permissive for the infection and replication of these two viruses, which up-regulated the expression of innate immunity genes. Both DTMUV and JEV induced cell death via a caspase-3-dependent manner; however, DTMUV triggered more cell death than did JEV in both CCL-141 and DN. These findings suggest that DTMUV infection causes apoptosis in duck neurons and fibroblasts more strongly than JEV. The levels of the mRNA expression of innate immunity-related genes after DTMUV infection were generally higher than the levels after JEV infection, suggesting that DTMUV-induced immune response in duck cells may exhibit toxic effects rather than protective effects.

Antiviral and virucidal activities of lycorine on duck tembusu virus in vitro by blocking viral internalization and entry

Duck tembusu virus (DTMUV) was firstly identified in 2010 in China; since then, it has caused enormous economic loss to breeding industry. Great efforts have been made to develop drugs and vaccines against DTMUV. However, current available vaccines or anti-DTMUV drugs are consistently inefficient. Hence, various more broadly effective drugs have become important for the treatment of DTMUV infection; among these, lycorine, one of the important sources of active alkaloids, is a promising example. Nevertheless, it is not known whether lycorine has any antiviral activities against DTMUV. Therefore, the purpose of the present study is to investigate the anti-DTMUV abilities of lycorine. The cytotoxicity of lycorine was evaluated on BHK-21 cells by CCK-8 assay, and its antiviral effect against DTMUV was examined by real-time PCR assays, virus titer determination, Western blot and immunofluorescence (IFA) assays, respectively. Furthermore, the underlying mechanisms of the anti-DTMUV effects of lycorine were also investigated. The results indicated that the highest nontoxicity concentration of lycorine on BHK-21 cells was 5 µM. Lycorine possessed the antiviral ability against DTMUV on BHK-21 cells, as demonstrated by the reduction of virus titers and copy numbers in vitro. Western blot and IFA analysis showed the inhibitory effect of lycorine on DTMUV envelope (E) protein expression. Moreover, using time-of-addition assays, we found that lycorine displays its antivirus and virucidal activities through blocking viral internalization and entry in vitro. Taken together, our findings firstly demonstrate the antiviral activities of lycorine against DTMUV, suggesting that lycorine can be a potential drug for the treatment of DTMUV infection.

Duck Tembusu Virus Infection Promotes the Expression of Duck Interferon-Induced Protein 35 to Counteract RIG-I Antiviral Signaling in Duck Embryo Fibroblasts.

Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that has caused a substantial drop in egg production and severe neurological disorders in domestic waterfowl. Several studies have revealed that viral proteins encoded by DTMUV antagonize host IFN-mediated antiviral responses to facilitate virus replication. However, the role of host gene expression regulated by DTMUV in innate immune evasion remains largely unknown. Here, we utilized a stable isotope labeling with amino acids in cell culture (SILAC)-based proteomics analysis of DTMUV-infected duck embryo fibroblasts (DEFs) to comprehensively investigate host proteins involved in DTMUV replication and innate immune response. A total of 250 differentially expressed proteins were identified from 2697 quantified cellular proteins, among which duck interferon-induced protein 35 (duIFI35) was dramatically up-regulated due to DTMUV infection in DEFs. Next, we demonstrated that duIFI35 expression promoted DTMUV replication and impaired Sendai virus-induced IFN-β production. Moreover, duIFI35 was able to impede duck RIG-I (duRIG-I)-induced IFN-β promoter activity, rather than IFN-β transcription mediated by MDA5, MAVS, TBK1, IKKϵ, and IRF7. Importantly, we found that because of the specific interaction with duIFI35, the capacity of duRIG-I to recognize double-stranded RNA was significantly impaired, resulting in the decline of duRIG-I-induced IFN-β production. Taken together, our data revealed that duIFI35 expression stimulated by DTMUV infection disrupted duRIG-I-mediated host antiviral response, elucidating a distinct function of duIFI35 from human IFI35, by which DTMUV escapes host innate immune response, and providing information for the design of antiviral drug.

Characterization of duck IκB kinase β involved in innate immunity

IκB kinase β (IKKβ), a catalytic subunit of the IKK complex, is involved in a wide array of biological processes, particularly in inflammation and innate immunity. Although extensive studies have been carried out to explore the roles of mammalian IKKβs in innate immune response, the function of IKKβ in avian innate immunity is largely unknown. Here, we cloned and characterized the duck IKKβ (duIKKβ) gene for the first time. DuIKKβ encoded 755 amino acids and displayed high sequence similarity to pseudopodoces and haliaeetus IKKβs. DuIKKβ transcripts were widely distributed in all tested tissues, especially with high expression in the thymus and bursa of Fabricius. Overexpression of duIKKβ promoted NF-κB activation and initiated the downstream cytokines expression including IFN-β, ZAP, PKR, IL-8, and CCL5 in duck embryo fibroblasts. Furthermore, knockdown of endogenous duIKKβ significantly reduced LPS-, poly(I:C)- or SeV-induced NF-κB activation. Finally, we demonstrated that duIKKβ showed antiviral activity against Duck Tembusu virus infection. Our findings provide insights into the roles of duIKKβ in avian innate immunity.

Transcriptome analysis reveals new insight of duck Tembusu virus (DTMUV)-infected DF-1 cells

Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused huge economic losses to the duck industry in China since 2010. Moreover, the infection has spread rapidly, resulted in a potential public health concern. To improve our understanding of the host cellular responses to virus infection and the pathogenesis of DTMUV infection, we used RNA-Seq to detect the gene changes in DF-1 cells infected and mock-infected with DTMUV. A total of 663 differentially-expressed genes (DEGs) were identified in DTMUV-infected compared with mock-infected DF-1 cells at 24 h post-infection (hpi), among which 590 were up regulated and 73 were down regulated. Gene Ontology analysis indicated that the DEGs were mainly involved in cellular process, immune system processes, metabolic processes, and signal-organism process. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the DEGs were mainly involved in several signaling pathways such as Toll-like receptor signaling, Jak-STAT signaling, RIG-I-like receptor signaling and AGE-RAGE signaling pathway. Moreover, some selected DEGs were further confirmed by real-time PCR and the results were consistent with the sequencing data. To our knowledge, this study is the first to analyze the transcriptomic change in DF-1 cells following DTMUV infection. We believe that our research provides useful information in better understanding the host response to DTMUV infection and the inherent mechanism of DTMUV replication and pathogenicity.

Epigallocatechin-3-gallate exhibits antiviral effects against the duck Tembusu virus via blocking virus entry and upregulating type I interferons

The duck Tembusu virus (DTMUV) is a novel mosquito-borne Flavivirus which caused huge economic losses for poultry industries in Southeast Asia and China. Currently, no effective antiviral drugs against this virus have been reported. (-)-Epigallocatechin-3-gallate (EGCG), a polyphenol present in abundance in green tea, has recently been demonstrated to have an antiviral activity for many viruses; however, whether EGCG can inhibit DTMUV infection remains unknown. Here, we tried to explore the anti-DTMUV effects and mechanisms of EGCG both in vitro and in vivo. Several EGCG treatment regimens were used to study the comprehensive antiviral activity of EGCG in DTMUV-infected baby hamster kidney cell line (BHK-21). The DTMUV titers of mock- and EGCG-treated infected cell cultures were determined using the tissue culture infective dose assay and the DTMUV mRNA copy number as determined using quantitative Real Time PCR. Moreover, the therapeutic efficacy of EGCG against DTMUV was assessed in DTMUV-infected ducklings. Our results suggested that EGCG significantly reduced the viral infection in BHK-21 cells in a dose-dependent manner, as reflected by the reduction of virus titers, virus copy number, and the expressions of viral E protein. We also observed that EGCG exhibited direct virucidal abilities against DTMUV. Notably, a significant reduction in virus binding ability was also observed, indicating that EGCG possesses excellent inhibitory effects on the viral adsorption step. In addition, DTMUV replication was also suppressed in BHK-21 cells treated with EGCG after viral entry, likely because of upregulation of the levels of interferon alfa and interferon beta. Finally, we also proved that EGCG exhibited anti-DTMUV efficacy in a duckling infection model because the survival rate was significantly improved. This is the first study to demonstrate the protective effect of EGCG against DTMUV, suggesting its potential use as an antiviral drug for DTMUV infection.

Minocycline prevents primary duck neurons from duck Tembusu virus-induced death.

Duck Tembusu virus (DTMUV), a neurotropic flavivirus, is a causative agent of severe neurological diseases in different birds. No approved vaccines or antiviral therapeutic treatments are available to date. The poultry industry experiences significant economic losses due to DTMUV infections. Minocycline is a second-generation semi-synthetic tetracycline analogue that is commonly used as an antimicrobial treatment. Experimental studies have indicated the successful protective effects of minocycline against neuronal cell death from neurodegenerative diseases and viral encephalitis. The aim of this study was to investigate the effects of minocycline on DTMUV infection in neurons. Primary duck neurons were treated with minocycline, which exhibited neuroprotective effects via anti-apoptotic function rather than through viral replication inhibition. Minocycline might serve as a potential effective drug in DTMUV infection.

Vector competence of Culex tritaeniorhynchus and Culex quinquefasciatus (Diptera: Culicidae) for duck Tembusu virus transmission

Duck Tembusu virus (DTMUV), an emerging infectious disease in ducks, was detected in Culex (Cx.) tritaeniorhynchus mosquitoes collected from a duck farm; however, the exact role of mosquitoes in the ecology of DTMUV in Thailand remains unclear. Vector competence of Cx. tritaeniorhynchus and Cx. quinquefasciatus was examined for DTMUV. Cx. tritaeniorhynchus mosquitoes were allowed to feed on four levels (102, 103, 104, and 105 TCID50/mL) of DTMUV, while Cx. quinquefasciatus were allowed to feed on two levels (104 and 105 TCID50/mL) of DTMUV. Infection rates in Cx. tritaeniorhynchus were 1.6, 10.2, 35.8, and 59.3% after feeding on 102, 103, 104, and 105 TCID50/mL of DTMUV, respectively, while dissemination and transmission were 20.3 and 16.9% after feeding on 105 TCID50/mL of DTMUV. Infection rates in Cx. quinquefasciatus were 2.5 and 2.3% after feeding on 104 and 105 TCID50/mL of DTMUV, respectively, with no virus dissemination and transmission found in all tested mosquitoes. Another study was conducted to examine the transovarial transmission of DTMUV in Cx. tritaeniorhynchus. Mosquitoes were allowed to feed on blood meal infected with 105 TCID50/mL of DTMUV. Each blood-fed mosquito was isolated and allowed to lay eggs. After oviparity, the mosquitoes were tested for DTMUV infection; 43 DTMUV infected and 37 non-infected female mosquitoes with eggs were included. A total of 182 F1 progeny from DTMUV infected mosquitoes and 145 F1 progeny from non-infected mosquitoes were tested for DTMUV but all were negative. Findings indicated the potential role of Cx. tritaeniorhynchus in the DTMUV transmission cycle in duck farms in Thailand. No transovarial transmission of DTMUV was found in Cx. tritaeniorhynchus.

Evaluation of host systems for efficient isolation and propagation of duck Tembusu virus

ABSTRACT Several phylogenetic clusters of duck Tembusu virus (DTMUV) that caused outbreaks in ducks in Asia have been identified since its emergence in 2010, highlighting the need for an efficient host system that can support isolation of all circulating phylogenetic clusters of DTMUV. In this study, various host systems, including different avian embryonated eggs (duck and chicken) and cell cultures (primary duck embryo fibroblast (DEF), primary chicken embryo fibroblast (CEF), baby hamster kidney (BHK-21), African green monkey kidney (Vero) and Aedes albopictus clone C6/36 (C6/36) cells), were evaluated and compared for their ability to support DTMUV isolation and propagation. Our results showed that all host systems were susceptible to DTMUV infection; however, BHK-21 and primary DEF cells supported more efficient replication of DTMUV compared to the other host systems. BHK-21 cells had the highest DTMUV isolation rate when tested with experimental and field clinical samples. All circulating phylogenetic clusters of DTMUV, including clusters 1, 2 and 3, were successfully isolated from duck clinical samples using BHK-21 cells. In conclusion, our findings supported the use of BHK-21 cells as a host system for primary isolation of all circulating phylogenetic clusters of DTMUV from duck clinical samples. This study highlights the importance of selecting the most appropriate host system for efficient isolation and propagation of DTMUV from duck clinical samples. RESEARCH HIGHLIGHTS DTMUV replicated more efficiently in BHK-21 and primary DEF cells than in other host systems tested. BHK-21 cells had the highest DTMUV isolation rate. All DTMUV phylogenetic clusters were successfully isolated from the samples using BHK-21 cells. BHK-21 cells were the most efficient host system for DTMUV isolation.