Duck Embryo Fibroblasts Research Articles

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.

In Vitro and In Ovo Host Restriction of Aquatic Bird Bornavirus 1 in Different Avian Hosts.

Aquatic bird bornavirus 1 (ABBV-1) is associated with chronic meningoencephalitis and ganglioneuritis. Although waterfowl species act as the natural host of ABBV-1, the virus has been sporadically isolated from other avian species, showing the potential for a broad host range. To evaluate the host restriction of ABBV-1, and its potential to infect commercial poultry species, we assessed the ability of ABBV-1 to replicate in cells and embryos of different avian species. ABBV-1 replication was measured using multi- and single-step growth curves in primary embryo fibroblasts of chicken, duck, and goose. Embryonated chicken and duck eggs were infected through either the yolk sac or chorioallantoic cavity, and virus replication was assessed by immunohistochemistry and RT-qPCR in embryonic tissues harvested at two time points after infection. Multi-step growth curves showed that ABBV-1 replicated and spread in goose and duck embryo fibroblasts, establishing a population of persistently infected cells, while it was unable to do so in chicken fibroblasts. Single-step growth curves showed that cells from all three species could be infected; however, persistence was only established in goose and duck fibroblasts. In ovo inoculation yielded no detectable viral replication or lesion in tissues. Data indicate that although chicken, duck, and goose embryo fibroblasts can be infected with ABBV-1, a persistent infection is more easily established in duck and goose cells. Therefore, ABBV-1 may be able to infect chickens in vivo, albeit inefficiently. Additionally, our data indicate that an in ovo model is inadequate to investigating ABBV-1 host restriction and pathogenesis.

Duck enteritis virus infection suppresses viability and induces apoptosis and endoplasmic reticulum stress in duck embryo fibroblast cells via the regulation of Ca2.

Duck viral enteritis (DVE) is a lethal viral disease caused by duck enteritis virus (DEV) via an unknown mechanism. This study explores the relationship between Chinese standard challenge strain DEV (DEV-CSC)-induced apoptosis and endoplasmic reticulum stress (ERS) in duck embryo fibroblast (DEF) cells. Here we examined changes in Ca2+ concentration, cell proliferation, apoptosis, and the differential expression of C/EBP homologous protein (CHOP), glucose regulatory protein 78 (GRP78), and activating transcription factor 6 (ATF6) in infected cells. The results revealed that DEV-CSC infection significantly decreased Ca2+ concentration, suppressed cell viability, and induced apoptosis in DEF cells. Further experiments also demonstrated that DEV-CSC infection significantly upregulates CHOP, GRP78, and ATF6 expression. In addition, we show that the addition of ethylenediaminetetraacetic acid (EDTA) reverses the induction of apoptosis and the ERS mediated inhibition of cell viability in DEF cells associated with DEV-CSC infection. Therefore, we can conclude that infection with DEV-CSC induces apoptosis and ERS reducing the viability of DEF cells via the regulation of Ca2+. These findings may provide a new target for the treatment of DVE.

Comparative molecular analysis of contemporary isolates of duck plague virus from haor areas of Bangladesh

Duck plague (DP) is one of the most important viral diseases which affects the duck population across the globe including Bangladesh. The present work was conducted to detect DP virus (DPV) from haor areas using a molecular-based approach, and compared with the contemporary isolate through molecular and phylogenetic analysis. For this purpose, 38 individual samples were collected from the Netrokona (n=20) district of the Mymensingh division and Kishoreganj (n=18) district of the Dhaka division. The identification of DVP was carried out by polymerase chain reaction (PCR) targeting DPV specific DNA polymerase genes followed by sequencing. PCR positive viral samples were used to propagate into 11-13 days old embryonated duck eggs through chorio-allantoic membrane (CAM) route for virus isolation. DPV were then propagated into duck embryo fibroblast (DEF) monolayer cell culture and confirmed by PCR. Among the 38 samples, 27 isolates were confirmed as DPV with the PCR amplicon size of 446 bp. Pathogenicity tests through the inoculation into day-old ducklings confirmed pathogenic strain. The PCR products of the isolated DPV specific DNA polymerase gene were sequenced commercially and submitted to GenBank (GenBank Accession No. KX768734.1). The sequence showed resemblance to isolates previously reported in India (GenBank Accession No. KX511893.1, KJ451479.1, KM012009.1), and China (GenBank Accession No. EF643559.1). Sequencing data also revealed nucleotide differences between Anatid herpes 1_BAU_DMH (previous report from our laboratory) and the present isolates. Further characterization, such as nucleotide and amino acid sequencing, would help to understand the strains along with its epidemiology.

Investigation of duck plague virus in hoar areas of Bangladesh

Duck plague (DP) is the most important infectious disease of geese, ducks and free-ranging water birds. The present study was conducted to determine the prevalence of duck plague virus followed by isolation and identification. For these purposes, a total of 155 cloacal swabs samples were collected randomly from duck of different haor areas of Bangladesh including 45 (41 surveillance and 4 clinical) samples from Netrokona; 42 (40 surveillance and 2 clinical) samples from Kishoregonj; 30 samples from Brahmanbaria and 38 samples from Sunamganj. The samples were processed and pooled (1:5 ratio) for initial screening of target polymerase gene of duck plague virus by polymerase chain reaction (PCR) method. All the samples of a positive pool were then tested individually for identifying the individual positive samples. The result showed that out of 155 samples, 41 (26.45%) were found positive in which 17 were from Netrokona, where 15 (36.58%) were from surveillance samples and 2 (50%) were from clinical sample; 16 were from Kishoregonj, where 14 (35%) were from surveillance samples and 2 (100%) were from clinical sample; 2 (6.6%) were from Brahmanbaria and 5 (13.15%) were from Sunamganj. These positive samples were inoculated into 9-10 days embryonated duck eggs (EDE) through chorioallantoic membrane (CAM) route for the isolation of virus. The EDE died earlier was also chilled, and in a similar way, the CAMs were collected and again performed PCR for id entification of virus. Out of 41 PCR positive samples, 26 samples were isolated and reconfirmed by PCR. Subsequently, DPV was isolated in primary duck embryo fibroblasts cell culture and confirmed by observing cytopathic effect (CPE).
 Bang. J. Livs. Res. Vol. 26 (1&2), 2019: P. 73-78

Duck HMGB2 Mediates Signaling Pathways in the Innate Immunity of Hosts Against Viral Infections.

High-mobility group box 2 (HMGB2) belongs to the HMG-box family that participates in a variety of biologic processes. Recent studies have suggested that HMGB2 plays an important role in the innate immunity of fish. Cherry Valley duck is the main duck bred for meat consumption in China, but there is limited research available on the impact of duck HMGB2 (duHMGB2) in antiviral innate immunity. Here, duHMGB2 genes were first cloned and analyzed from the spleen of Cherry Valley ducks. We show that duHMGB2 is widely distributed in most tissues of healthy ducks, and duHMGB2 was differentially expressed in three organs (the spleen, brain, and lung) of ducks during different viral infections. duHMGB2 is mainly expressed in the nucleus of duck embryo fibroblast (DEF) cells. However, duHMGB2 is released into the cytoplasm after viral infection. DuHMGB2 induced expression of several genes that regulate the immune response. Moreover, duHMGB2 activated and upregulatede transcription factor NF-κB promoter activity. We also used single gene manipulations (knockout or overexpression) to confirm that duHMGB2 can inhibit the replication of duck plague virus, duck Tembusu virus, and the novel duck reovirus in DEF cells. These data show that duHMGB2 can activate the antiviral innate immunity of the host. Thus, duHMGB2 may be considered an immune adjuvant against infectious diseases in duck.

Heme Oxygenase-1 suppresses duck Tembusu virus replication in vitro

Heme Oxygenase-1 (HO-1) is a ubiquitously expressed enzyme which plays important functions in antioxidant, anti-inflammatory and anti-apoptosis. Recent studies have demonstrated that HO-1 also has significant antiviral properties, inhibiting the replication of some kinds of viruses such as human immunodeficiency virus (HIV), hepatitis C virus (HCV), and dengue fever virus (DFV). In this study, we evaluated the role of HO-1 in Duck Tembusu virus (DTMUV) replication in vitro. The results showed that, the mRNA expression level of HO-1 was transient up-regulated and then significantly decreased in duck embryo fibroblast (DEF) infected with DTMUV. HO-1 induction by transfection of HO-1 over-expression plasmid or treatment with cobalt protoporphyrin (CoPP), a potent HO-1 inducer, could inhibit DTMUV replication effectively. In contrast, HO-1 siRNA knockdown in DEF increased DTMUV replication, implied that HO-1 was an important cellular factor against DTMUV replication. Furthermore, we found that ferric ion (Fe3+) but not biliverdin and carbon monoxide, products of heme degradation by HO-1, mediated the HO-1-induced anti-DTMUV effect. Overall, these finding revealed that a drug induced the HO-1 signal pathway was a promising strategy for treating DTMUV infection.

Preliminary Studies On The Virus Causing Outbreak Of Duckling Short Beak And Dwarfism Syndrome (Sbds) In Egypt

Many Mule, Muscovy, and Bikini duck flocks in different duck-producing regions in Egypt have shown signs of a new disease designated short beak and dwarfism syndrome (SBDS) since 2015. The ducks with SBDS showed strong growth retardation with beak atrophy, enteritis, and paralysis. Although the mortality rate was 2%–8% in affected flocks, the morbidity rate was 20%–50% and even 80% in some regions. The disease is characterized by dyspraxia, weight loss, a protruding tongue, and high morbidity and low mortality rates. To characterize the etiological agent, a virus was isolated from the allantoic fluid following serial passage in embryonated duck eggs. This virus causes a cytopathic effect in duck embryo fibroblast (DEF) cells. Using enzyme-linked immunosorbent assay (ELISA), the isolate was positive for the antigen of goose parvovirus (GPV). The bovine sperm agglutination test indicated that the virus was most closely related to GPV strain. Together, these data indicated that the isolated virus from mule ducks with SBDS disease is a GPV-related parvovirus causing and that it is divergent from classical GPV isolates.

Research Note: Duck plague virus glycoprotein I influences cell–cell spread and final envelope acquisition

To determine the role of glycoprotein I (gI) in duck plague virus (DPV), a gI-deleted mutant (BAC-CHv-ΔgI) and a gI-revertant virus (BAC-CHv-ΔgI Rev) were constructed by using a markerless two-step Red recombination system implemented on the DPV genome cloned into a bacterial artificial chromosome (BAC). Mutants were characterized on duck embryo fibroblast (DEF) cells compared with wild-type virus. BAC-CHv-ΔgI produced viral plaques on DEF cells that were on average approximately 57.2% smaller than those produced by BAC-CHv-ΔgI Rev and wild-type virus. Electron microscopy confirmed that deleting of gI resulted in nucleocapsids accumulated around the cytoplasm vesicles and few of them could complete the final envelopment process. These results clearly indicated that DPV gI plays significant roles in viral cell-cell spread and viral final envelopment process.

Effects of immunopotentiators on biochemical parameters, proinflammatory cytokine, and nonspecific immune responses in Shaoxing ducklings

Antibiotics are one of the most important medical discoveries of the 20th century and will remain an essential tool for treating animal and human diseases in the 21st century. However, misuse of antibiotics imperils the development of animal husbandry and human health all over the world, and it is important to find reliable alternatives to antibiotics to reduce the use of antibiotics. In this study, 22 potential immunopotentiators were screened on the levels of apoptosis and inflammatory factor in duck embryo fibroblast cells (DEFs). The results indicated that interferon (IFN)-β and tumor necrosis factor-α gene transcriptions were significantly upregulated, while interleukin (IL)-2 and Bcl2 mRNA levels were significantly decreased during 22 immunopotentiators treatment. Besides, the expression level of IL-1β mRNA showed significant increase during dihydromyricetin, chlorogenic acid, naringin, imiquimod, thymopentin, β-D-Glucan, astragalus polysacharin, astragalus saponin I, astragalus flavone, curcumin, CpG-DNA-2, and LPS treatment. And the level of caspase 3 protein was significantly upregulated with treating chlorogenic acid, β-D-Glucan, astragalus polysacharin, astragalus flavone, curcumin, CpG-DNA-2, chicken IgG, LPS, and poly(I:C). These results indicated that chlorogenic acid, β-D-Glucan, astragalus flavone, CpG-DNA-2, and chicken IgG have the positive immune regulation effects on duck DEFs. Thus, the 5 immunopotentiators were chosen to further verify their immunomodulatory function in vivo. The results showed that the activity of serum AST was significantly downregulated during all immunopotentiators treatments excepting for β-D-Glucan, and the activities of serum IL12p40, IL-1β, IFN-α, and IFN-β were significantly increased compared with the control group. Five immunopotentiators also induced the duck's pattern recognition receptors and inflammatory factor gene expression. In addition, 5 immunopotentiators could facilitate the contents of serum caspase 3, iNOSm and COX2 and reduce the Bcl2. These results suggested that these 5 immunopotentiators could enhance duck innate immune responses. Taken together, our study not only screened out 5 kinds of duck innate immune immunopotentiators but also initially clarified their underlying mechanism of action, which provide a new insight for the development of efficient approaches to prevent the duck disease from pathogen infections.

Transcriptome analysis of duck embryo fibroblasts for the dynamic response to duck tembusu virus infection and dual regulation of apoptosis genes.

Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that has caused enormous economic losses in Southeast Asia. However, the pathogenic mechanism and host’s responses after DTMUV infection remain poorly understood. During this study, total mRNA sequencing (RNA-Seq) analysis was used to detect the global gene expression in DEFs at various time points after DTMUV infection. We identified 326 genes altered significantly at all time points, and these genes were dynamically enriched in multifarious biological processes, including apoptosis, innate immune response, DNA replication, cell cycle arrest and DNA repair. Next, the results showed that apoptosis was induced and the proportion of apoptosis increased with time, and pro-apoptotic molecules caspases were activated. The RNA-seq data analysis further revealed that most pro-apoptosis and anti-apoptosis genes were early continually responsive, and the genes involved in both intrinsic and extrinsic apoptotic pathways were initiated. Further, the considerably enriched immune-relevant pathways were involved in apoptosis process, and protein-protein interactions (PPIs) analysis showed that IL6, STAT1, TNFAIP3, CFLAR and PTGS2 may be key regulators of DEFs apoptosis. In conclusion, this study not only contributes to understanding the underlying mechanism of DEFs infection with DTMUV, but also provides new insights into targets screening for antiviral therapy.

NS5-V372A and NS5-H386Y variations are responsible for differences in interferon α/β induction and co-contribute to the replication advantage of Japanese encephalitis virus genotype I over genotype III in ducklings.

Japanese encephalitis virus (JEV) genotype I (GI) replicates more efficiently than genotype III (GIII) in birds, and this difference is considered to be one of the reasons for the JEV genotype shift. In this study, we utilized duck embryo fibroblasts and domestic ducklings as in vitro and in vivo models of a JEV amplifying avian host to identify the viral determinants of the differing replication efficiency between the GI and GIII strains in birds. GI strains induced significantly lower levels of interferon (IFN)-α and β production than GIII strains, an effect orrelated with the enhanced replication efficiency of GI strains over GIII strains. By using a series of chimeric viruses with exchange of viral structural and non-structural (NS) proteins, we identified NS5 as the viral determinant of the differences in IFN-α and β induction and replication efficiency between the GI and III strains. NS5 inhibited IFN-α and β production induced by poly(I:C) stimulation and harbored 11 amino acid variations, of which the NS5-V372A and NS5-H386Y variations were identified to co-contribute to the differences in IFN-α and β induction and replication efficiency between the strains. The NS5-V372A and NS5-H386Y variations resulted in alterations in the number of hydrogen bonds formed with neighboring residues, which were associated with the different ability of the GI and GIII strains to inhibit IFN-α and β production. Our findings indicated that the NS5-V372A and NS5-H386Y variations enabled GI strains to inhibit IFN-α and β production more efficiently than GIII strains for antagonism of the IFN-I mediated antiviral response, thereby leading to the replication and host adaption advantages of GI strains over GIII strains in birds. These findings provide new insight into the molecular basis of the JEV genotype shift.

Differently Expression Analysis and Function Prediction of Long Non-coding RNAs in Duck Embryo Fibroblast Cells Infected by Duck Tembusu Virus.

Duck Tembusu virus (DTMUV), the causative agent of egg-drop syndrome, has caused substantial economic losses to duck industry. DTMUV infection leads to profound changes of host cells, including transcriptome and proteome. However, the lncRNA expression profile and the biological function of lncRNA have not been revealed. Therefore, DTMUV was used to inoculate duck embryo fibroblast cells (DEFs) for high-throughput RNA-sequencing (RNA-Seq). The results showed that 34 and 339 differently expressed lncRNAs were, respectively, identified at 12 and 24 h post-infection (hpi). To analyze their biological functions, target genes in cis were searched and the regulatory network was formed. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the target genes were strongly associated with immune system, signaling molecular and interaction, endocrine system, and signal transduction. The differently expressed lncRNAs were selected and verified by quantitative real-time polymerase chain reaction (RT-qPCR). Our study, for the first time, analyzed a comprehensive lncRNA expression profile in DEFs following DTMUV infection. The analysis provided a view on the important roles of lncRNAs in gene regulation and DTMUV infection.

Duck PIAS2 Promotes H5N1 Avian Influenza Virus Replication Through Its SUMO E3 Ligase Activity.

The protein inhibitor of the activated STAT2 (PIAS2) has been implicated in many cellular processes and can also regulate viral replication in mammals. However, the role of PIAS2 in the highly pathogenic avian influenza virus (HPAIV) H5N1 replication in ducks is still unclear. Through liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay, we identified that duck PIAS2 (duPIAS2) was one protein that interacted with the nucleoprotein (NP) from the H5N1 HPAIV strain of DK212. Through confocal microscopy images and Co-IP assay, we confirmed NP could interact with duPIAS2. Overexpression of duPIAS2 in primary duck embryo fibroblast (DEF) cells was shown to promote DK212 replication, and knockdown of duPIAS2 could repress DK212 replication. We further found duPIAS2 could promote NP SUMOylation through duck SUMO1 (duSUMO1), and the potential SUMOylation sites of NP were at lysines 7, 48, and 87. Furthermore, duPIAS2 promoted the replication of DK212, here relying on the activity of its SUMO E3 ligase. Duck SENP1 (duSENP1), a deSUMOylation enzyme, could repress NP SUMOylation and also inhibit DK212 replication. Together, we identified duPIAS2 could interact with NP and that duPIAS2 promoted H5N1 HPAIV replication, which might be related to NP SUMOylation.

Duck Tembusu virus promotes the expression of suppressor of cytokine signaling 1 by downregulating miR-148a-5p to facilitate virus replication

Duck Tembusu virus (DTMUV), an emerging infectious pathogen, has caused severe disease in ducks and huge economic losses to the poultry industry in China since 2009. Despite considerable advances in understanding the effects of microRNAs on host antiviral immune responses, it remains unclear how miRNAs regulate DTMUV replication in duck embryo fibroblast (DEF) cells. This study aims to clarify the role of host microRNA-148a-5p (miR-148a-5p) in regulating DTMUV replication by targeting SOCS1. First, we found that during DTMUV infection, the expression of miR-148a-5p in DEFs was downregulated in a time-dependent and dose-dependent manner, while the expression of SOCS1 was significantly upregulated. In addition, we found that when miR-148a-5p mimics were transfected into DEFs, viral RNA copies, viral E protein expression levels and viral titres, which represent viral replication and proliferation, were significantly downregulated, while the opposite result was observed when miR-148a-5p inhibitor was transfected into DEFs. Next, we found that SOCS1 was the target gene of miR-148a-5p through software analysis. Therefore, we further confirmed that SOCS1 was the target of miR-148a-5p and that miR-148a-5p could negatively regulate the expression of SOCS1 at the mRNA and protein levels. Furthermore, our results indicated that overexpression of SOCS1 promoted DTMUV replication, while knockdown of SOCS1 inhibited DTMUV replication. Finally, we found that in DTMUV-infected DEFs, the overexpression of SOCS1 inhibited the production of IFN-α and IFN-β, while knocking down SOCS1 produced the opposite result. This indicates that during DTMUV infection, the virus promotes the expression of SOCS1 by downregulating the expression of miR-148a-5p, while the upregulation of SOCS1 suppresses the production of type I interferon and promotes virus replication. Taken together, these findings provide new insights into virus-host interactions during DTMUV infection and provide potential new antiviral treatment strategies for DTMUV infection.

Duck IFIT5 differentially regulates Tembusu virus replication and inhibits virus-triggered innate immune response

Mammalian interferon-induced protein with tetratricopeptide repeats family proteins (IFITs) play important roles in host innate immune response to viruses. Recently, studies have shown that IFIT from poultry also plays a crucial part in antiviral function. This study first reports the regulation of duck Tembusu virus (DTMUV) replication by IFIT5 and the effect of duck IFIT5 (duIFIT5) on the innate immune response after DTMUV infection. Firstly, duIFIT5 was obviously increased in duck embryo fibroblast cells (DEFs) infected with DTMUV. Compared to the negative control, we found that in the duIFIT5-overexpressing group, the DTMUV titer at 24 h post infection (hpi) was significantly reduced, but the viral titer was strikingly increased at 48 hpi. Moreover, overexpression of duIFIT5 could significantly inhibit IFN-β transcription and IFN-β promoter activation at indicated time points after DTMUV infection. Further, in DTMUV-infected or poly(I:C)-stimulated DEFs, overexpression of duIFIT5 also significantly inhibited the activation of NF-κB and IRF7 promoters, as well as the activation of downstream IFN induced the interferon-stimulated response element (ISRE) promoter. Meanwhile, the transcription level of antiviral protein Mx, but not OASL, was obviously decreased at various time points. The opposite results were obtained by knockdown of duIFIT5 in DTMUV-infected or poly(I:C)-stimulated DEFs. Compared to the negative control, knockdown of duIFIT5 promoted DTMUV titer and DTMUV envelope (E) protein expression at 24 hpi, but DTMUV titer and E protein expression was markedly decreased at 48 hpi. Additionally, the promoters of IFN-β, NF-κB, IRF7 and ISRE were significantly activated in the duIFIT5 knockdown group. Collectively, duIFIT5 differentially regulates DTMUV replication and inhibits virus-triggered innate immune response.

Duck Enteritis Virus VP16 Antagonizes IFN-β-Mediated Antiviral Innate Immunity.

Duck enteritis virus (DEV) can successfully evade the host innate immune responses and establish a lifelong latent infection in the infected host. However, the study about how DEV escapes host innate immunity is still deficient up to now. In this study, for the first time, we identified a viral protein VP16 by which DEV can obviously downregulate the production of IFN-β in duck embryo fibroblast (DEF). Our results showed that ectopic expression of VP16 decreased duck IFN-β (duIFN-β) promoter activation and significantly inhibited the mRNA transcription of IFN-β. Further study showed that VP16 can also obviously inhibit the mRNA transcription of interferon-stimulated genes (ISGs), such as myxovirus resistance protein (Mx) and interferon-induced oligoadenylate synthetase-like (OASL). Furthermore, we found that this anti-interferon activity of VP16 depended on its N-terminus (aa1-200). Coexpression analysis revealed that VP16 selectively blocked duIFN-β promoter activity at the duIRF7 level rather than duIRF1. Based on the results of coimmunoprecipitation analysis (co-IP) and indirect immunofluorescence assay (IFA), VP16 was able to bind to duck IRF7 (duIRF7) directly, but did not interact with duck IRF1 (duIRF1) in vitro.

Molecular cloning and functional characterization of duck DEAD (Asp-Glu-Ala-Asp) box RNA helicase 3 (DDX3X)

DEAD (Asp-Glu-Ala-Asp) box RNA helicase 3 (DDX3X) is demonstrated to have crucial functions in the antiviral immune response. To our knowledge, little information focuses on the function of duck DDX3X. In this study, duck DDX3X (duDDX3X) was cloned and its role in the type I interferon (IFN) signaling pathway was investigated using duck embryo fibroblast (DEF) cells. Full-length duDDX3X cDNA encodes 652 amino acid residues and contains a DEADc domain and a HELICc domain. According to tissue distribution analysis, duDDX3X mRNA was widely expressed in different tissues, especially the spleen and the liver. Overexpression of duDDX3X in DEF cells induced IFN-β by activating transcription factors IRF1 and NF-κB. Knockdown of duDDX3X in DEF cells with siRNA significantly reduced IFN-β expression induced by poly(I:C), a double-stranded RNA (dsRNA) analog. Our results demonstrated that duck DDX3X was involved in the dsRNA-mediated type I IFN signaling pathway in DEF cells.

Duck Tembusu Virus Utilizes miR-221-3p Expression to Facilitate Viral Replication via Targeting of Suppressor of Cytokine Signaling 5.

Duck Tembusu virus (DTMUV), a member of Flaviviridae family, causes acute egg-drop syndrome in ducks. MicroRNAs (miRNAs) have been found to be involved in various biological processes, including tumor genesis, viral infection, and immune response. However, the functional effect of miRNAs on DTMUV replication remains largely unclear. This study aimed to elucidate the role of host microRNA-221-3p (miR−221-3p) in regulating DTMUV replication. Here, we indicated that the expression of miR−221-3p was significantly upregulated in duck embryo fibroblasts (DEFs) during DTMUV infection. Transfection of miR-221-3p mimic significantly reduced interferon (IFN) β production, whereas transfection of miR-221-3p inhibitor conversely significantly increased the expression of IFN-β in DTMUV-infected DEF. Moreover, we found that viral RNA copies, viral E protein expression level, and virus titer, which represent the replication and proliferation of virus, were all enhanced when transfecting the miR-221-3p mimic into DEF; reverse results were also observed by transfecting the miR-221-3p inhibitor. We also found that the expression of suppressor of cytokine signaling 5 (SOCS5) was downregulated in DEF infected with DTMUV. Besides, we further proved that SOCS5 is a target of miR-221-3p and that miR-221-3p could negatively modulate SOCS5 expression at both mRNA and protein levels. Finally, our results showed that overexpression of SOCS5 inhibited DTMUV replication and knockdown of SOCS5 enhanced DTMUV replication. Thus, our findings reveal a novel host evasion mechanism adopted by DTMUV via miR-221-3p, which may hew out novel strategies for designing miRNA-based vaccines and therapies.

Duck plague virus gE serves essential functions during the virion final envelopment through influence capsids budding into the cytoplasmic vesicles

Duck plague virus (DPV), a member of the alphaherpesviruses subfamily, causes massive ducks death and results in a devastating hit to duck industries in China. It is of great significance for us to analyze the functions of DPV genes for controlling the outbreak of duck plague. Thus, glycoproteins E (gE) of DPV, which requires viral cell-to-cell spreading and the final envelopment in herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), was chosen herein. The gE mutant virus BAC-CHv-ΔgE was constructed by using a markerless two-step Red recombination system implemented on the DPV genome cloned into a bacterial artificial chromosome (BAC). Viral plaques on duck embryo fibroblast (DEF) cells of BAC-CHv-ΔgE were on average approximately 60% smaller than those produced by BAC-CHv virus. Viral replication kinetics showed that BAC-CHv-ΔgE grew to lower titers than BAC-CHv virus did in DEF cells. Electron microscopy confirmed that deleting of DPV gE resulted in a large number of capsids accumulating around vesicles and very few of them could bud into vesicles. The drastic inhibition of virion formation in the absence of the DPV gE indicated that it played an important role in virion morphogenesis before the final envelopment of intracytoplasmic nucleocapsids.