Replication of duck plague virus(DPV) in artificially infected ducks were detected by in situ hybridization (ISH) which employed a 37bp oligonucleotide as probe designed according to DPV DNA sequence in GenBank. The results indicated that DPV DNA was detected in liver, intestine and bursa Fabricius at 4 h, in spleen and esophagus at 6h, in thymus at 12h post infection; DPV DNA in lung and kidney was detected only in dead ducks and no positive signal was detected in muscle, heart, cerebrum and pancreas. DPV DNA was distributed in cell nucleus and cytoplasm. Hepatocytes, sinus endodermal cells and Kuffer's cells were the mainly infected cell types in liver. DPV DNA was mainly detected in epithelium of villi, in lamina propria of intestinal villi of duodenum, in stratum spinosum of esophagus, and in epithelium, cortex, medulla of bursa Fabricius. The positive signals were mainly detected in medulla of thymus, lymphocytes and macrophages of spleen. The research suggests that ISH is a direct and specific method in detecting DPV DNA in paraffin sections and it's also a good method for virus diagnosis and DNA location of DPV.
Animals ; DNA, Viral ; analysis ; Ducks ; virology ; In Situ Hybridization ; Influenza A virus ; genetics ; isolation & purification ; physiology ; Virus Replication

OBJECTIVETo analyze the genetic composition of a novel H2N3 virus isolate identified from a duck cage swab in a live poultry market (LPM) in 2009 in Guangdong province of China.

METHODSPCR-positive specimens were inoculated into embryonated chicken eggs and subtyped by conventional RT-PCR. All segments of the virus A/environment/Guangdong/2/2009 were sequenced, and phylogenetic trees were constructed and analyzed.

RESULTSThe genes of this virus belong to Eurasian-lineage avian viruses. The virus is a reassortant with the HA gene from an H2N2 virus and the NA gene from an H5N3 virus. The PB1, PB2, and NP genes were from an H4N6 virus, the PA was from an H3N8 virus, the M gene was from an H1N3 virus, and the NS gene was from an H10N6 virus.

CONCLUSIONA novel avian-origin reassortant H2N3 influenza virus was detected in a live poultry market. Its potential impacts and evolution should be closely monitored.

Animals ; China ; Ducks ; virology ; Genome, Viral ; Influenza A virus ; genetics ; isolation & purification ; Influenza in Birds ; virology ; Phylogeny

Inositol 1,4,5-trisphosphate receptor 1 (ITPR1) is an important intracellular channel for releasing Ca²⁺. In order to investigate the effects of the ITPR1 overexpression on Ca²⁺ concentration and lipid content in duck uterine epithelial cells and its effects on calcium transport-related genes, the structural domain of ITPR1 gene of duck was cloned into an eukaryotic expression vector and transfected into duck uterine epithelial cells. The overexpression of the ITPR1 gene, the concentration of Ca²⁺, the lipid content, and the expression of other 6 calcium transport-related genes was determined. The results showed that the concentration of Ca²⁺ in uterine epithelial cells was significantly reduced after transfection (P<0.05), the triglyceride content was significantly increased (P<0.01), and the high-density lipoprotein content was significantly decreased (P<0.01). The correlation analysis results showed that the overexpression of the C-terminal half of the ITPR1 gene was significantly positively correlated with the total cholesterol content (P<0.01), which was significantly positively correlated with the low-density lipoprotein content (P<0.05). The overexpression of the N-terminal half of the ITPR1 gene was significantly positively correlated with the triglyceride content (P<0.01), which was significantly negatively correlated with the concentration of Ca²⁺ (P<0.05). RT-qPCR results of 6 calcium transport-related genes showed that the overexpression of the C-terminal half of the ITPR1 gene significantly inhibited the expression of the IP3R2, VDAC2 and CAV1 genes, and the overexpression of the N-terminal half of the ITPR1 gene significantly promoted the expression of the IP3R3 and CACNA2D1 genes. In conclusion, the ITPR1 gene overexpression can promote Ca²⁺ release in duck uterus epithelial cells, promote the synthesis of triglyceride, low-density lipoprotein and cholesterol, and inhibit the production of high-density lipoprotein, and the ITPR1 gene overexpression affected the expression of all 6 calcium transport-related genes.
Animals ; Calcium/metabolism* ; Ducks/genetics* ; Epithelial Cells ; Female ; Inositol ; Inositol 1,4,5-Trisphosphate Receptors ; Lipids ; Uterus

Stearoyl-CoAdesaturase-1 (SCD-1) is a key regulator of monounsaturated fatty acid synthesis. It plays a vital role in lipid synthesis and metabolism. Ca²⁺ is an important cation in the body and plays an important role in the organism. The aims of this study were to investigate the correlation of SCD-1 gene overexpression with lipid indexes and calcium ion level. The pcDNA3.1 (+) + SCD-1 +Flag eukaryotic expression vector and cultured duck uterine epithelial cells were co-transfected. The overexpression of SCD-1 gene was measured using the Flag Label Detection Kit. Ca ions and lipid contents were detected through Fluo-3/AM Calcium Ion Fluorescence Labeling method and Lipid Measuring Kit, respectively. SCD-1 gene overexpression was negatively correlated with triglyceride (TG) and high-density lipoprotein cholesterol (HDL-C), and positively correlated with Ca ion, total cholesterol (TC), very low-density lipoprotein cholesterol (VLDL-C) and low density lipoprotein cholesterol (LDL-C) levels. Meanwhile, Ca ion was positively correlated with TG, LDL-C and HDL-C contents, and negatively correlated with TC and VLDL-C levels. Overexpression of SCD-1 gene could regulate Ca ion secretion, as well as lipid synthesis and transport in duck uterine epithelial cells.
Animals ; Calcium ; metabolism ; Coenzyme A Ligases ; genetics ; Ducks ; Epithelial Cells ; chemistry ; enzymology ; Gene Expression ; Ions ; Lipids ; genetics ; Triglycerides ; metabolism

This article describes the nomenclature, history and genetic evolution of duck hepatitis A virus, and updates the epidemiology, clinical symptom and surveillances of duck virus hepatitis A. It also summarizes the present status and progress of duck virus hepatitis A and illustrated the necessity and urgency of its research, which provides rationale for the control of duck hepatitis A virus disease in China.
Animals ; Ducks ; virology ; Hepatitis Virus, Duck ; classification ; genetics ; isolation & purification ; Hepatitis, Viral, Animal ; virology ; Picornaviridae Infections ; veterinary ; virology

An isolated virulence Riemerella anatipestifer strain passaged 200 times on TSB agar were used for the virulent to avirulent conversion. The effects of passage on biological properties of outer membrane proteins (OMPs) were investigated using the virulent and avirulent strains. Transmission electron microscopy demonstrated that the avirulent strain produced lower amounts of outer membrane vesicles and the outer membrane decreased, the cytoplasmic appearance jumbled. The OMPs of the virulent strain agglutinated only in RA serotype 2 antisera, whereas the OMPs of the avirulent strain agglutinated in antisera of RA 1, 2, 10 and 11. SDS-PAGE Analysis showed the OMPs profiles of both strains were similar but the immunoblotting profiles were different. The protective immunity against Riemerella anatipestifer infection was investigated by immunizations with OMPs in ducks. ELISA results showed that the OMPs induced the production of antibodies in immunized ducks, but the OMPs of virulence strain induced higher antibody titers than the attenuated strain (P < 0.05). RA2 group showed significantly higher survival rates (100%) than RA200 group (0%) after challenged with the homologous virulent strain. The ompA gene of both stains were also amplified by PCR, nucleotide homology was 99.9%. In conclusion, OMPs of virulent RA strain are suitable candidates for vaccine development. Biological properties of OMPs undergoes significant changes during serial passage and suggest that vigilance should be used when extrapolating data obtained from the study of high-passage strains.
Animals ; Bacterial Outer Membrane Proteins ; chemistry ; genetics ; immunology ; Ducks ; Flavobacteriaceae ; chemistry ; classification ; immunology ; Flavobacteriaceae Infections ; immunology ; microbiology ; veterinary ; Serotyping

To clarify the pathogenesis of Duck enteritis virus (DEV), the cDNA library of duck's liver infected by DEV and a bait plasmid containing DEV nucleocapsid protein (NP) gene were constructed, then the receptor was screened from the cDNA library plasmid by the yeast two-hybrid system and verified by GST pull-down test. The results showed that the capacity of the primary cDNA library was 1 x 106 CFU with insertion size from 0.5 to 1 kb, and the bait plasmid of pGBKT7-NP showed no self-activation. The receptor reacting with DEV NP in duck liver was initially confirmed as the protein kinase C inhibitor (PKCI). These results provide new clues for further investigation on pathogenesis of DEV.
Alphaherpesvirinae ; pathogenicity ; Animals ; Ducks ; virology ; Gene Library ; Liver ; virology ; Nucleocapsid Proteins ; genetics ; Plasmids ; Receptors, Virus ; analysis ; Two-Hybrid System Techniques

The UL49.5 gene of most herpesviruses is conserved and encodes glycoprotein N. However, the UL49.5 protein of duck enteritis virus (DEV) (pUL49.5) has not been reported. In the current study, the DEV pUL49.5 gene was first subjected to molecular characterization. To verify the predicted intracellular localization of gene expression, the recombinant plasmid pEGFP-C1/pUL49.5 was constructed and used to transfect duck embryo fibroblasts. Next, the recombinant plasmid pDsRed1-N1/glycoprotein M (gM) was produced and used for co-transfection with the pEGFP-C1/pUL49.5 plasmid to determine whether DEV pUL49.5 and gM (a conserved protein in herpesviruses) colocalize. DEV pUL49.5 was thought to be an envelope glycoprotein with a signal peptide and two transmembrane domains. This protein was also predicted to localize in the cytoplasm and endoplasmic reticulum with a probability of 66.7%. Images taken by a fluorescence microscope at different time points revealed that the DEV pUL49.5 and gM proteins were both expressed in the cytoplasm. Overlap of the two different fluorescence signals appeared 12 h after transfection and continued to persist until the end of the experiment. These data indicate a possible interaction between DEV pUL49.5 and gM.
Animals ; Ducks/virology ; Genes, Viral/genetics ; Mardivirus/*genetics ; Membrane Glycoproteins/*genetics ; Microscopy, Fluorescence ; Phylogeny ; Polymerase Chain Reaction/veterinary ; Viral Envelope Proteins/*genetics

OBJECTIVETo determine the molecular characterization of Polymerase complex (PA, PB1 and PB2) genes of H9N2 avian influenza viruses and the genetic relationship of Iranian H9N2 viruses and other Asian viruses.

METHODSThe Polymerase complex (PA, PB1 and PB2) genes from seven isolates of H9N2 viruses isolated from commercial chickens in Iran during 2008-2009 were amplified (by RT-PCR method) and sequenced. Nucleotide sequences (Open Reading Frame: orf) of the PA, PB1 and PB2 genes were used for phylogenetic tree construction.

RESULTSMost PB2 and PA genes of the H9N2 viruses isolated in 2008-2009 belonged to the unknown avian sublineage which grouped with the 2004 Pakistani H7N3 viruses. The PB1 genes of Iranian viruses indicated greater genetic diversity and shared a high level of similarity to PB1 genes from either H5 or H7 subtypes with compared to established H9N2 Eurasian sublineages.

CONCLUSIONSOur findings demonstrated that the H9N2 viruses in Iran exhibit striking reassortment which has led to the generation of new genotypes.

Animals ; Chickens ; virology ; Ducks ; virology ; Genotyping Techniques ; Influenza A Virus, H9N2 Subtype ; classification ; genetics ; Influenza in Birds ; virology ; Iran ; Pakistan ; RNA Replicase ; genetics ; Viral Proteins ; genetics

New epidemic broke out in recent year which was suspected to be caused by variant Muscovy duck parvovirus (MDPV). For this reason, new MDPV detection methods are needed for the new virus strains. In this study, a pair of primers were designed according to the full-length genome of MDPV strain SAAS-SHNH, which were identified in 2012, and were used to amplify the vp3 gene of MDPV by polymerase chain reaction. After being sequenced, the vp3 gene was subcloned into the prokaryotic expression vector PET28a. The recombinant plasmid was transformed into E. coli BL21 and induced with IPTG. SDS-PAGE and Western blotting analysis showed the MDPV vp3 gene was successfully expressed. After being purified by Ni2+ affinity chromatography system, the recombinant protein was used as antigen to immunize rabbits to obtain antiserum. Western blotting analysis showed that the acquired antiserum could react specifically with VP3 protein of J3D6 strain and MDPV vaccine strain. The antiserum could also be used for detection of cultured MDPV from primary duck embryo fibroblasts by immune fluorescence assay (IFA). It could be concluded that the VP3 protein and its antibody prepared in the research could be used for detection of VP3 antiserum and antigen respectively.
Animals ; Blotting, Western ; DNA Primers ; Ducks ; virology ; Electrophoresis, Polyacrylamide Gel ; Immune Sera ; biosynthesis ; Parvovirus ; Polymerase Chain Reaction ; Rabbits ; Recombinant Proteins ; genetics ; Viral Proteins ; genetics