Zhu Dekang

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.

Quantitative Analysis of Virulent Duck Enteritis Virus Loads in Experimentally Infected Ducklings

Abstract To better understand the pathogenesis of duck virus enteritis (DVE), the levels of viral DNA in various tissues of ducklings during acute stage of virulent duck enteritis virus (DEV) infection were investigated by using quantitative real-time polymerase chain reaction. The results show that the viral levels of DEV in systemic organs have a close correlation with the progression of disease. The rapid dissemination and active replication of virulent DEV in multiple systemic organs at the early phase of acute infection accelerate the progression of disease. The levels of viral DNA increase sharply soon after developed clinical signs of disease, and the extent of increase and the magnitude of DEV DNA load in various tissues of ducklings after the exhibition of clinical signs may be a critical determinant of the outcome of DEV infection. The relatively high levels of DEV in bursa and small intestine tissues of dead ducklings most likely reflect the abundance of target epithelial and lymphoid cells in these tissues, which therefore play a key role in the pathogenesis of acute DVE and manifest as severe tissue lesions on the bursa and small intestine.

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.

Expression and Distribution of the Duck Enteritis Virus UL51 Protein in Experimentally Infected Ducks

Abstract To determine the expression and distribution of tegument proteins encoded by duck enteritis virus (DEV) UL51 gene in tissues of experimentally infected ducks, for the first time, an immunoperoxidase staining method to detect UL51 protein (UL51p) in paraffin-embedded tissues is reported. A rabbit anti-UL51 polyclonal serum, raised against a recombinant 6-His-UL51 fusion protein expressed in Escherichia coli, was prepared, purified, and used as primary antibodies. Fifty-eight 30-day-old DEV-free ducks were intramuscularly inoculated with the pathogenic DEV CHv strain as infection group, and two ducks were selected as preinfection group. The tissues were collected at sequential time points between 2 and 480 hr postinoculation (PI) and prepared for immunoperoxidase staining. DEV UL51p was first found in the spleen and liver at 8 hr PI; in the bursa of Fabricius and thymus at 12 hr PI; in the Harders glands, esophagus, small intestine (including the duodenum, jejunum, and ileum), and large intestine (including the caecum and rectum) at 24 hr PI; in the glandularis ventriculus at 48 hr PI; and in the pancreas, cerebrum, kidney, lung, and myocardium at 72 hr PI. Throughout the infection process, the UL51p was not seen in the muscle. Furthermore, the intensity of positive staining of DEV UL51p antigen in various tissues increased sharply from 8 to 96 hr PI, peaked during 120–144 hr PI, and then decreased steadily from 216 to 480 hr PI, suggesting that the expressional levels of DEV UL51p in systemic organs have a close correlation with the progression of duck virus enteritis (DVE) disease. A number of DEV UL51p was distributed in the bursa of Fabricius, thymus, spleen, liver, esophagus, small intestine, and large intestine of DEV-infected ducks, whereas less DEV UL51p was distributed in the Harders glands, glandularis ventriculus, cerebrum, kidney, lung, pancreas, and myocardium of DEV-infected ducks. Moreover, DEV UL51p can be expressed in the cytoplasm of various types of cells, especially most abundantly in the cytoplasm of lymphocytes, reticulum cells, macrophages, epithelial cells, and hepatocytes. The present study may be useful not only for describing the characteristics of UL51p expression and distribution in vivo but also for a greater understanding of the pathogenesis of this DVE.