Analysis of differentially expressed proteins in Muscovy duck embryo fibroblasts infected with virulent and attenuated Muscovy duck reovirus by two-dimensional polyacrylamide gel electrophoresis.

Muscovy duck reovirus (MDRV) belongs to the Orthoreovirus genus of the Reoviridae family, which is a significant poultry pathogen leading to high morbidity and mortality in ducklings. However, the pathogenesis of the virus is not well understood. In the present study, two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) combined with LC-MS-MS was used to identify differentially expressed proteins between Muscovy duck embryo fibroblasts (MDEF) infected with virulent (MV9710 strain) and attenuated (CA strain) MDRV and non-infected MDEFs. A total of 115 abundant protein spots were identified. Of these, 59 of differentially expressed proteins were detected, with functions in metabolism and utilization of carbohydrates and nucleotides, anti-stress, and regulation of immune and cellular process. GO analysis of the identified proteins showed that they belonged to the classes molecular function (141 proteins), cellular component (62 proteins), and biological process (146 proteins). The results were validated by qRT-PCR, which suggests that the analysis method of 2D PAGE combined with LC-MS-MS used in this study is reliable. This study lays a foundation for further investigation of the biology of MDRV infection in MDEF.

Phylogenetic analysis of VP1 gene sequences of waterfowl parvoviruses from the Mainland of China revealed genetic diversity and recombination.

To determine the origin and evolution of goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV) in the Mainland of China, phylogenetic and recombination analyses in the present study were performed on 32 complete VP1 gene sequences from China and other countries. Based on the phylogenetic analysis of the VP1 gene, GPV strains studied here from Mainland China (PRC) could be divided into three genotypes, namely PRC-I, PRC-II and PRC-III. Genotype PRC-I is indigenous to Mainland China. Only one GPV strain from Northeast China was of Genotype PRC-II and was thought to be imported from Europe. Genotype PRC-III, which was the most isolated genotype during 1999-2012, is related to GPVs in Taiwan and has been the predominant pathogen responsible for recent Derzy's disease outbreaks in Mainland China. Current vaccine strains used in Mainland China belong to Genotype PRC-I that is evolutionary distant from Genotypes PRC-II and PRC-III. In comparison, MDPV strains herein from Mainland China are clustered in a single group which is closely related to Taiwanese MDPV strains, and the full-length sequences of the VP1 gene of China MDPVs are phylogenetic closely related to the VP1 sequence of a Hungarian MDPV strain. Moreover, We also found that homologous recombination within VP1 gene plays a role in generating genetic diversity in GPV evolution. The GPV GDFSh from Guangdong Province appears to be the evolutionary product of a recombination event between parental GPV strains GD and B, while the major parent B proved to be a reference strain for virulent European GPVs. Our findings provide valuable information on waterfowl parvoviral evolution in Mainland China.

Paeoniflorin diminishes ConA-induced IL-8 production in primary human hepatic sinusoidal endothelial cells in the involvement of ERK1/2 and Akt phosphorylation.

Liver diseases are closely associated with elevated levels of interleukin-8 (IL-8), suggesting the ability to inhibit IL-8 production could enhance the treatment of liver diseases. Paeoniflorin is a major active constituent of dried Paeoniae Radix Alba root (Baishao in Chinese) which is widely used in China to treat liver diseases. We examined the effects and underlying mechanisms of paeoniflorin on IL-8 production in primary human hepatic sinusoidal endothelial cells (HHSECs). Concanavalin A (ConA) at 20 µg/mL produced a 5.2-fold increase in IL-8 mRNA by 8h, and a 14.2-fold rise in IL-8 levels by 16 h. Inhibition of MEK (ERK kinase) and extracellular signal-regulated kinase (ERK) by PD98059 and U0126, or inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002 blocked both ConA-induced IL-8 mRNA expression and IL-8 secretion. Paeoniflorin reduced ConA-induced IL-8 mRNA expression and IL-8 release by 57.9% and 52.8%, respectively, and also decreased ConA-stimulated phosphorylation of ERK1/2 and Akt, suggesting paeoniflorin inhibits IL-8 expression and release by inhibiting the ERK1/2 and Akt pathways. Combining paeoniflorin with U0126 or LY294002 at low doses showed supra-additive inhibition of not only phospho-ERK1/2 and phospho-Akt by 46.4% and 35.0%, but also IL-8 release by 42.4% and 36.1% and IL-8 mRNA expression by 43.5% and 31.8%, respectively. In conclusion, paeoniflorin most likely contributes to the therapy for liver disease by exerting anti-inflammatory effects on HHSECs through blocking IL-8 secretion via downregulation of ERK1/2 and Akt phosphorylation.

Genetic characterization of a potentially novel goose parvovirus circulating in Muscovy duck flocks in Fujian Province, China.

We report a novel goose parvovirus (MDGPV/PT) isolated from an affected Muscovy duck in Fujian Province, China. In this study, the NS1 sequence analyses indicated a close genetic relationship between MDGPV/PT and Muscovy duck parvovirus (MDPV) strains, although MDGPV/DY, which was isolated from a Muscovy duck in 2006 in Sichuan Province, could be divided into GPV-related groups. Phylogenetic analysis showed that except for differences in the NS1 gene, MDGPV strains PT and DY are closely related to a parvovirus that infects domestic waterfowls. This is the first demonstration of recombination between goose and Muscovy duck parvoviruses in nature, and MDGPV/PT might have led to the generation of a novel waterfowl parvovirus strain circulating in Muscovy duck flocks in China.

[The isolation and identification of novel duck reovirus].

The virus strains were isolated from the liver and spleen of the dead young ducks characterized with symptoms of hemorrhagic-necrotic hepatitis. These isolates could cause the death of muscovy duck-embryo and chick-embryo. 1-day-old birds infected with these isolates had the same character with clinically dead birds and the virus could be isolated from artificially infected birds. These isolates could proliferate in MDEF and result in CPE. The virus could proliferate in the cytoplasm in order of crystals and arranged in the latlic-like. The viron was shown spherical, icosahedron, cubic symmetry, no-envelope, with double-layered capsid, about 70 nm in diameter by electron microscopy. The genome segments of the virus were consisted of L1-3, M1-3 and S1-4, which were similar to that of avian reovirus (ARV). Compared to 68.2%, 69.3% - 70.1%, respectively. The system evolution analysis showed that S3 gene coding sigmaB protein was placed in different branch of MDRV and ARV, indicating that S3 gene of the virus was different from ARV and MDRV. The main clinical symptoms and lesions of ducklings caused by the virus were different from the diseases caused by MDRV and ARV. It was concluded that the virus was a Novel duck reovirus belonging to Orthoreovirus genus of the Reoviridae family.

Adenovirus-mediated viral interleukin-10 gene transfer prevents concanavalin A-induced liver injury.

BACKGROUND AND AIM: Liver injury is closely associated with immune inflammation. Lacking immunostimulatory functions, viral interleukin-10 (vIL-10), a cellular IL-10 homologue, has been an attractive molecule for immunomodulatory therapy. We aimed to reveal a protective effect of the gene transfer of an adenoviral vector encoding vIL-10 on liver injury induced by concanavalin A. METHODS: C57BL/6J mice were intravenously injected with adenoviral vector encoding vIL-10 before concanavalin A challenge. Liver injury was assessed. Interferon-γ and interleukin-4 levels were measured by ELISA. The activation of splenic and hepatic immune cells was analysed using an MTT assay. RESULTS: Adenoviral vector encoding vIL-10 pretreatment significantly decreased concanavalin A-mediated elevations in serum alanine aminotransaminase and aspartate aminotransaminase activity, and necrotic area in liver tissues. The protective effect of adenoviral vector encoding vIL-10 was attributed to its inhibition of T cell activation, and production of interferon-γ and interleukin-4 by the immune cells. Recombinant mouse IL-10, a high homologous cytokine to vIL-10, effectively downregulated interferon-γ and interleukin-4 release by hepatic mononuclear cells. CONCLUSION: Adenovirus vector-mediated vIL-10 gene transfer can prevent concanavalin A-induced hepatic injury, minimise pro-inflammatory cytokine release, and inhibit the activation of T lymphocytes.