Development and application of a reverse transcriptase polymerase chain reaction to detect Chinese isolates of duck hepatitis virus type 1.

We developed a reverse transcriptase polymerase chain reaction (RT-PCR) method for the detection of duck hepatitis virus type 1 (DHV-1) in the tissues of infected and clinically affected ducks and in chick and duck embryos. We found the assay to be effective in detecting the virus in China, where it is being used in studies on the epidemiology of the disease. We applied this simple and rapid diagnostic method to the detection of DHV isolates grown in chick and duck embryos and in tissues obtained from infected birds. The assay also proved useful for the differentiation of DVH from the duck plague virus (DPV), muscovy parvovirus (MPV), gosling parvovirus (GPV), avian influenza virus (AIV/H5N1), Pasteurella multocida (PA/5:A), Riemerella anatipestifer (RA/serotype 1), and Salmonella enteritidis (SE). The limit of the sensitivity of this method for the detection of DHV-1 RNA was 3 pg/10 microl. As compared to ELISA and virus isolation, the rate of agreement for the detection of experimentally infected livers was 100%; moreover, the RT-PCR method was also capable of detecting DHV-1 RNA from the livers that had been infected and stored at -20 degrees C for 22 years; in contrast, ELISA and virus isolation method could only detect DHV-1 from the livers that had been infected and stored at -20 degrees C for 13 and 11 years, respectively. The rate of positivity in 185 clinically suspected diseased livers subjected to detection by RT-PCR, ELISA, and virus isolation was 89.2%, 69.2%, and 55.7%, respectively. These results indicated that the RT-PCR approach is rapid, sensitive, and reliable for the detection and differentiation of DHV-1 from the other clinical samples and suspected isolates.

The pathogenesis of duck virus enteritis in experimentally infected ducks: a quantitative time-course study using TaqMan polymerase chain reaction.

Duck virus enteritis is an acute and contagious herpesvirus infection of duck, geese and swans with high morbidity and mortality. The kinetics of viral DNA loads and immunohistochemical localization of virulent duck enteritis virus, as well as histopathological examination in various tissues of ducks following oral infection, were investigated. The time course for the appearance of viral antigen and tissue lesions in various tissues was coincident with the levels of duck enteritis virus at the various sites, suggesting that the levels of duck enteritis virus in systemic organs have a close correlation with the progression of disease. The abundance of target epithelial and lymphoid cells may contribute to the high levels of virus infection and replication in lymphoid and intestinal tissues.

Molecular Mechanism of Glycoprotein-induced Cell-Cell Fusion of Herpesviruses / 病毒学报

Herpesviridae is a large family comprising linear, double-stranded DNA viruses. Herpesviridae contains three subfamilies: α-, β- and γ-herpesviruses. The glycoproteins gB, gH and gL of each subfamily form the "core fusion function" in cell-cell fusion. Other herpesviruses also need additional glycoproteins to promote fusion, such as gD of the Herpes simplex virus, gp42 of the Epstein-Barr virus, and gO or UL128-131 of the Human cytomegalovirus. In contrast, glycoproteins gM or gM/gN of herpesvirus inhibit fusion. We describe the molecular mechanisms of glycoprotein-induced fusion and entry of herpesviruses. It will be helpful to further study the pathogenic mechanism of herpesvirus.

Progress on the Function of Herpesvirus-encoded MicroRNAs / 病毒学报

Since Epstein Barr virus was shown to encode microRNAs(miRNAs) in 2004, more than 470 miRNAs have been discovered in α-, β-, and γ-herpesviruses. MiRNAs are small non-coding RNA molecules and generally only have 18-25 nucleotides in length, which can regulate the expression of target genes by targeting its transcripts. Herpesvirus-encoded miRNAs not only target the key genes from latency to lytic replication, but also regulate various host cellular genes. Current data manifest that herpesvirus-encoded miRNAs can regulate viral latent infection and lytic replication, immune recognition, apoptosis, and tumorigenesis. The purpose of this paper is to summarize the targets and their fuction of hepesvirus-encoded miRNAs, in order to provide theoretical support for further analysis herpesviral pathogenesis.

Construction and Identification of the Bait Vector Containing Duck Circovirus Cap Gene for the Yeast Two-hybrid System / 病毒学报

To construct a bait expression vector containing the duck circovirus Cap gene for use in the yeast two-hybrid system, the whole cap codon-optimized gene was inserted into pGBKT7 vector and confirmed by PCR, restriction enzyme digestion, and sequence analysis. After transformation into a Y2HGold yeast strain, the expression of Cap protein was analyzed by Western blotting. Toxicity and self-activation of the bait protein were detected using different dropout minimal base. PCR reaction, restriction enzyme digestion, and sequencing analyses indicated that the duck circovirus Cap gene was correctly inserted into pG- BKT7. Western blotting showed that the whole Cap protein was expressed. The recombinant bait protein had no toxicity and self-activation. Therefore, the bait vector with the Cap gene was constructed successfully, providing a foundation for future screening for interacting proteins in the yeast two-hybrid system.

Effect of Chuanmingshen violaceum polysaccharides and its sulfated derivatives on immunosuppression induced by cyclophosphamide in mice / 中国免疫学杂志

Objective:In oder to investigate the effect of Chuanmingshen violaceum polysaccharides ( CVP) and Solfated Chua-nmingshen violaceum polysaccharides ( SCVP) on immunosuppression induced by cyclophosphamide ( CY) in mice.Methods: CY were used to induce immunosuppression in mice;Spleen and thymus indexes were used to evaluate the immune organs indexes;the [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide,MTT] method was used to detect the proliferation of spleen lymphocytes of each group;the concentrations of IFN-γand IL-2 were assayed by ELISA kit.Results: SCVP and CVP could resist immunosuppression by promoting lymphocyte proliferation, increasing the contents of IFN-γ and IL-2, promoting immune organs development in immunosuppressive mice induced by CY.Conclusion:SCVP and CVP exhibited the potential to used as immunopotentiator.

Development and application of a reverse transcriptase polymerase chain reaction to detect Chinese isolates of duck hepatitis virus type 1.

We developed a reverse transcriptase polymerase chain reaction (RT-PCR) method for the detection of duck hepatitis virus type 1 (DHV-1) in the tissues of infected and clinically affected ducks and in chick and duck embryos. We found the assay to be effective in detecting the virus in China, where it is being used in studies on the epidemiology of the disease. We applied this simple and rapid diagnostic method to the detection of DHV isolates grown in chick and duck embryos and in tissues obtained from infected birds. The assay also proved useful for the differentiation of DVH from the duck plague virus (DPV), muscovy parvovirus (MPV), gosling parvovirus (GPV), avian influenza virus (AIV/H5N1), Pasteurella multocida (PA/5:A), Riemerella anatipestifer (RA/serotype 1), and Salmonella enteritidis (SE). The limit of the sensitivity of this method for the detection of DHV-1 RNA was 3 pg/10 microl. As compared to Dot-ELISA and virus isolation, the rate of agreement for the detection of experimentally infected livers was 100%; moreover, the RT-PCR method was also capable of detecting DHV-1 RNA from the livers that had been infected and stored at -20 degrees C for 22 years; in contrast, Dot-ELISA and virus isolation method could only detect DHV-1 from the livers that had been infected and stored at -20 degrees C for 13 and 11 years, respectively. The rate of positivity in 185 clinically suspected diseased livers subjected to detection by RT-PCR, Dot-ELISA, and virus isolation was 89.2%, 69.2%, and 55.7%, respectively. These results indicated that the RT-PCR approach is rapid, sensitive, and reliable for the detection and differentiation of DHV-1 from the other clinical samples and suspected isolates.

Molecular characterization of duck enteritis virus CHv strain UL49.5 protein and its colocalization with glycoprotein M

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.

Apoptosis induced in vivo by new type gosling viral enteritis virus

In this study, apoptosis was induced by new type gosling viral enteritis virus (NGVEV) in experimentally infected goslings is reported in detail for the first time. After 3-day-old goslings were orally inoculated with a NGVEV-CN strain suspension, the time course of NGVEV effects on apoptotic morphological changes of the internal tissues was evaluated. These changes were observed by histological analysis with light microscopy and ultrastructural analysis with transmission electron microscopy. DNA fragmentation was assessed with a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and DNA ladder analysis. A series of characteristic apoptotic morphological changes including chromatin condensation and margination, cytoplasmic shrinkage, plasma membrane blebbing, and formation of apoptotic bodies were noted. Apoptosis was readily observed in the lymphoid and gastrointestinal organs, and sporadically occurred in other organs after 3 days post-infection (PI). The presence and quantity of TUNEL-positive cells increased with infection time until 9 days PI. DNA extracted from the NGVEV-infected gosling cells displayed characteristic 180~200 bp ladders. Apoptotic cells were ubiquitously distributed, especially among lymphocytes, macrophages, monocytes, and epithelial and intestinal cells. Necrosis was subsequently detected during the late NGVEV-infection phase, which was characterized by cell swelling, plasma membrane collapse, and rapidly lysis. Our results suggested that apoptosis may play an important role in the pathogenesis of NGVE disease.

Preparation of contraceptive pill microcapsule and its anti-fertility effect / 生物医学工程学杂志

The main component of this pill is 2-Octadecanoic acid-4-Palmitic acid-2, 4-Pentanediyl ester separated from chloroform extract of neem oil. The microcapsules coated by the re-curdle method were fabricated with an average particle size of 100-180 microm. The morphological characteristics, incorporation efficiency, carrier reclamation efficiency of the microcapsule were investigated. Kunming mice were used in the experiment, and the anti-fertility effect of the microcapsule on the histology and apoptosis was studied by light and electron microscopy and the flow cytometry. The data obtained clearly indicated that the microcapsule could lead to the payload of medicine, the incorporation efficiency being 90%. After the microcapsules were given to the male mice orally, its anti-fertility effect came into being and could keep the mice in a state of reversible infertility for a long time. The results of histological study and flow cytometry indicate that the mechanism of its anti-fertility effect involves mainly the inhibition of sperm motility and the arrest of spermatogenic process.