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.

Molecular Characterization of Duck Plague Virus for Determination of TCID50

Duck plague is an enveloped DNA virus that belongs to the Anatid Herpes Virus; the Herpesviridae family is an acute and highly infectious duck, geese, and swan disease that causes tremendous economic losses of duck rearing in Bangladesh and other duck rearing countries. Therefore, we decided to isolate duck plague virus from recent fields’ outbreaks area and performed molecular detection and phylogenetic analysis to find out the similarities between our findings and other isolates around the world. Visceral organs of 13 suspected ducks from recent outbreaks area were collected by post-mortem examination for inoculum preparation. Several passages were performed to harvest into 9-11 old embryonated eggs Chorioallantoic membrane (CAM) route and duck embryo fibroblast (DEF) primary cell culture. DNA polymerase (446bp) and DNA polymerase (UL, 602bp) genes were used for molecular detection by Polymerase chain reaction (PCR). Pathogenicity was done with duckling and TCID50 on DEF. Molecular characterization was performed from extracted DNA of duckling and 2 Positive PCR products were partially sequenced for phylogenetic analysis of their origin and nucleotide variations. Sequenced data was analyzed to reveal genetic relationships among constructed phylogenetic tree for understanding potential transmission with origin of virus and data was then submitted to gene bank and got accession number for DPV-BR1-MN937272 and DPV-BR-2-MN937273. Among 13 samples, 4(30.77%) were found positive by PCR using DNA polymerase at 446 bp and UL at 602 bp gene. Chorioallantoic membrane (CAM) was observed hemorrhagic after 72 days and duck embryo fibroblast (DEF) become round as showed cytopathic characteristics after 48h of infection .Duckling showed that isolated virus was highly pathogenic as characteristics signs of post-mortem examination. Therefore, this has found that recent isolates have similarity with Bangladesh, India and China isolates. Moreover, TCID50 has confirmed the isolates have accepted titer to be a vaccine strain. Res. Agric., Livest. Fish.8(1): 125-133, April 2021

Quantitative Proteomic Analysis of Duck Embryo Fibroblasts Infected With Novel Duck Reovirus

The novel duck reovirus (NDRV) can cause hemorrhage and necrosis on the spleen of Pekin ducks; this disease has resulted in great economic losses to the duck industry. However, the molecular pathogenesis of NDRV remains poorly understood. In the current study, the quantitative proteomic analysis of NDRV-infected duck embryo fibroblasts was performed to explore the cellular protein changes in response to viral infection through iTRAQ coupled with the liquid chromatography (LC)–tandem mass spectrometry (MS/MS) method. A total of 6,137 proteins were obtained in cell samples at 24 h post-infection. Of these, 179 differentially expressed proteins (DEPs) were identified (cutoff set to 1.5-fold change), including 89 upregulated and 90 downregulated proteins. Bioinformatics analysis showed that DEPs can be divided into the cellular component, molecular function, and biological process; they were mainly involved in signal transduction, infectious diseases, cell growth and death, and the immune system. The subcellular localization of most proteins was in the cytoplasm. Importantly, the expressions of signal transducer and activator of transcription 1 (STAT1) and various interferon-stimulated genes (ISGs) were upregulated after NDRV infection. The mRNA transcripts of some ISGs were consistent with proteomic data, showing an increased trend. Results of our study suggested that NDRV infection can elicit strong expression changes of cellular proteins and activate the expression of ISGs from the point of quantitative proteomic analysis. The study provides a new insight into the understanding of NDRV pathogenesis.

Fetal Calf Serum Exerts an Inhibitory Effect on Replication of Duck Hepatitis A Virus Genotype 1 in Duck Embryo Fibroblast Cells

Among the causative agents of duck viral hepatitis, duck hepatitis A virus genotype 1 (DHAV-1) is the most common virus reported in most outbreaks worldwide. How to propagate DHAV-1 in cell cultures efficiently remains a problem to be explored. Here, we aimed to test the effect of serum type on DHAV-1 replication in duck embryo fibroblast (DEF) cells. Comparative studies involved virus culture and passage, observation of cytopathic effect (CPE), virus quantification, and plaque formation assay. From the results of these investigations, we conclude that use of chicken serum (CS) in maintenance medium allows DHAV-1 to establish productive, cytocidal infection in DEF cells, whereas FCS exerts inhibitory effects on DHAV-1 replication, CPE development, and plaque formation. By using a neutralization test, we found that the direct action of FCS on virions is likely to play a key role in inhibiting DHAV-1 replication in DEF cells. Mechanism analyses revealed that FCS inhibits DHAV-1 replication at virus adsorption and reduces extracellular virus yields. The present work may shed light on a new perspective for antiviral agent development, and have provided a virus–host cell system for further studies on molecular mechanism involved DHAV-1 replication and pathogenesis.

Isolation and molecular detection of Fowl pox and Pigeon pox viruses for the development of live attenuated vaccine seeds from the local isolates

Avipox is a viral disease of fowl and pigeon which is characterized by proliferative and nodular lesions in the feather-free parts of the skin or fibro-necrotic and proliferating part in the mouth, esophagus, and mucous membrane of the upper respiratory tract. This investigation was carried out with an aim to isolate and molecular detection of Fowl pox virus (FPV) and Pigeon pox virus (PPV) for development of live attenuated vaccine seeds from the local virus isolates. In this study, nodular lesions were collected from seven pigeons and four chickens from different areas of Mymensingh in Bangladesh which were affected by pox. Viral inoculums were prepared and DNA materials were extracted for PCR-based identification of P4b genes. Detection of virus was confirmed by PCR following propagation into 9-11 days old embryonated chicken egg (ECE) and also chicken embryo fibroblast (CEF) cell culture All the field samples were found positive for FPV and PPV by PCRR. These field isolates were propagated and attenuated in duck embryo through CAM route and duck embryo fibroblast (DEF) cell culture for the development of live attenuated vaccine seeds. Attenuation of both FPV and PPV were successful in duck embryo through CAM route and duck embryo fibroblast (DEF) cell culture after serial six passages. Attenuation of the virus was confirmed by inoculation into experimental birds. Inoculation of attenuated FPV and PPV in chicken and pigeon respectively exhibited no pox lesions whereas control chicken and pigeon inoculated with field isolates develop nodular lesions. Both FPV and PPV were confirmed from both groups of birds by PCR. These attenuated local isolates of FPV and PPV could be used as potential vaccine candidates for the prevention and control of fowl pox and pigeon pox in Bangladesh. J. Bangladesh Agril. Univ. 17(2): 211–219, June 2019