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

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

ITRAQ-Based Quantitative Proteomics Reveals the Proteome Profiles of Primary Duck Embryo Fibroblast Cells Infected with Duck Tembusu Virus

Outbreaks of duck Tembusu virus (DTMUV) have caused substantial economic losses in the major duck-producing regions of China since 2010. To improve our understanding of the host cellular responses to virus infection and the pathogenesis of DTMUV infection, we applied isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with multidimensional liquid chromatography-tandem mass spectrometry to detect the protein changes in duck embryo fibroblast cells (DEFs) infected and mock-infected with DTMUV. In total, 434 cellular proteins were differentially expressed, among which 116, 76, and 339 proteins were differentially expressed in the DTMUV-infected DEFs at 12, 24, and 42 hours postinfection, respectively. The Gene Ontology analysis indicated that the biological processes of the differentially expressed proteins were primarily related to cellular processes, metabolic processes, biological regulation, response to stimulus, and cellular organismal processes and that the molecular functions in which the differentially expressed proteins were mainly involved were binding and catalytic activity. Some selected proteins that were found to be differentially expressed in DTMUV-infected DEFs were further confirmed by real-time PCR. The results of this study provide valuable insight into DTMUV-host interactions. This could lead to a better understanding of DTMUV infection mechanisms.