Jian-Ming Fan

Serial Expression Analysis of Liver Regeneration-Related Genes in Rat Regenerating Liver

We investigated the gene expression changes in rat hepatic restoration with Rat Genome 230 2.0 chip containing 11,789 known genes and 13,231 unknown genes (taking up 90 percent of rat whole genome) following a 2/3 hepatectomy. The expression profiles and roles of these genes in rat liver regeneration (LR) were assayed using bioinformatics and systems biology method. Among the above genes, 1,004 known genes and 857 unknown genes were found to be associated with rat LR. The numbers of the known genes up-regulated, down-regulated, and up/down-regulated were 622, 443, and 15, respectively; that of the unknown genes were 367, 400, and 14, respectively. Out of the above two groups of genes, the ones up- and down-regulated 20 times or more were 62 and 38, 8, and 14, respectively. Notably, The highest expression level of dehydrogenase/reductase member 7 (DHRS7) was more than 968-fold compared to control, and alpha-1-B glycoprotein (A1BG), the product of gene with the lowest expression abundance, was 58 times lower than control. During rat liver regeneration, 467 up–regulated, 282 down–regulated, 10 up/down-regulated genes, and 1,031 undetected genes in our study interacted with each other and formed a network with a total of 4,014 connectivities. Among them, the genes for the regulation, synthesis, transport, signal transduction, protein modification, and physiological response formed 630, 290, 691, 373, 2010, and 20 connectivities, respectively; and the genes jun, fos, myc, ptgs2, ccna2, ccl2 had relatively higher degree of connectivity. The results indicated that cell apoptosis and inflammatory response were enhanced in the initial phase and the early part of progressive phase in LR.

The Nonstructural Protein 1 Papain-Like Cysteine Protease Was Necessary for Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 to Inhibit Interferon-β Induction

Porcine reproductive and respiratory syndrome virus nonstructural protein 1 (nsp1) could be auto-cleaved into nsp1α and nsp1β, both of which had the papain-like cysteine protease activities. Previous studies have shown that porcine reproductive and respiratory syndrome virus nsp1 was an interferon (IFN) antagonist. However, the mechanism by which nsp1 inhibited IFN-β production was unclear. Here, we used site-directed mutagenesis that inactivated the papain-like cysteine protease activities of nsp1 to explore whether the papain-like cysteine protease activities were required for nsp1 to disrupt IFN-β production. The results showed that mutations that inactivated papain-like cysteine protease activity of nsp1α made nsp1 lose its IFN antagonism activity, whereas mutations that inactivated papain-like cysteine protease activity of nsp1β did not influence the IFN antagonism activity of nsp1. In conclusion, our present work indicated that the papain-like cysteine protease activity of nsp1α was necessary for nsp1 to inhibit IFN-β induction.

Marek’s disease virus-encoded microRNAs: genomics, expression and function

MicroRNAs (miRNAs) are the recently discovered small non-coding RNA molecules that have post-transcriptional regulatory functions in many important biological processes. A large number of miRNAs have been found to be encoded by viral genomes, especially in herpesviruses. Previous research regarding miRNAs encoded by herpesviruses, including Marek’s disease virus (MDV), has demonstrated their involvement in lytic replication, latent infection, T-lymphocyte transformation and tumorigenesis. MDV is an oncogenic alphaherpesvirus, with the ability to induce tumors in natural hosts; however, formation of these tumors can be prevented by immunization with attenuated or nonpathogenic forms of the virus. Marek’s disease is considered to be a good biomedical model for investigating the biology, genetics, and immunology of tumorigenesis. In this paper, we review the discovery and identification of MDV-encoded miRNAs, along with their genomics, expression profiles, and currently known functions. We also discuss the prospects and techniques possibly applicable to the further investigation of the biological roles of MDV-encoded miRNAs.

Development of an immunochromatographic strip for the detection of antibodies against foot-and-mouth disease virus serotype O

An immunochromatographic strip was developed for the serological detection of type O foot-and-mouth disease (FMD) in swine. In the strip, the expressed protein of VP1, the main protective antigen of FMDV, labeled with colloidal gold was used as the detector, the staphylococcal protein A (SPA) and swine anti-foot-and-mouth disease virus (FMDV) antibody were blotted on the nitrocellulose membrane for the test and control lines, respectively. 296 swine serum samples were collected to evaluate the characteristics of the strip in comparison with existing commercial liquid-phage blocking ELISA (LPB ELISA) kit and peptide ELISA kit. The strip was shown to be of high specificity and sensitivity. Furthermore, the dipstick assay based on the strip is rapid (5 min) and easy to perform with no requirement of professional skills, reagents nor equipment. This suggests that the immunochromatographic strip is an acceptable alternative for use in clinical laboratories lacking specialized equipment and for field diagnosis.

Genetic analysis of strains of Japanese Encephalitis Virus isolated from swine in central China.

In this study, four strains of Japanese Encephalitis Virus (JEV) were isolated from the cerebrospinal fluid of aborted fetuses or stillborn piglets collected randomly from a number of piggeries in central China. The E genes were cloned by RT–PCR and sequenced. Phylogenetic analysis was performed with 48 JEV isolates previously reported in China and other countries, and showed that all four isolates can be classified into the subcluster of genotype III. The results strongly suggest that the genotype III of JEV is the major variant currently circulating in the swinery of central China.

Surface IgM on DT40 cells may be a component of the putative receptor complex responsible for the binding of infectious bursal disease virus.

To investigate the host–pathogen interactions between infectious bursal disease virus (IBDV) and target B-lymphocytic cells, a cDNA T7 phage display library from the chicken bursa of Fabricius was constructed and screened for virus binding. Surface immunoglobulin M (sIgM) was isolated as a putative candidate binding site and its interactions with IBDV were further investigated using a chicken bursal lymphoma-derived cell line DT40. The results showed that the λ light chain of sIgM specifically interacted with IBDV in a virulence-independent manner in vitro, and most of the binding of IBDV to DT40 cells was inhibited by sIgM-specific monoclonal antibodies. Further, the infectivity of IBDV in vitro was reduced by sIgM-specific monoclonal antibodies. Our data provided evidence that sIgM may participate as one of the putative membrane binding sites responsible for IBDV infection.

Small interfering RNA targeting nonstructural protein1 α (nsp1α) of porcine reproductive and respiratory syndrome virus (PRRSV) can reduce the replication of PRRSV in MARC-145 cells

Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically devastating and pandemic diseases of swine, which is poorly controlled by current methods. The inhibition of specific genes by small interfering RNA (siRNA) has been proven to be a potential therapeutic strategy against viral infection. Previous studies have indicated that the nonstructural protein 1α (nsp1α) of PRRSV may take an important role in virulence of PRRSV. The present work was involved to explore the effect of siRNA targeting nsp1α on the replication of PRRSV in MARC-145 cells, and the results showed that over-expression of nsp1α enhanced the replication of PRRSV and that siRNAs specifically targeting nsp1α significantly inhibited the replication of PRRSV in MARC-145 cells. In conclusion, this work indicated that nsp1α may be a viral pathogenicity factor of PRRSV and that siRNAs specifically targeting nsp1α may be a new strategy to control PRRSV in the future.

Infectivity and propagation of attenuated infectious bursal disease virus in the chicken B-lymphocyte cell line DT40

In this paper, the infectivity and propagation of two attenuated infectious bursal disease virus (IBDV) strains in DT40 cells were investigated. The results showed that both of the tested strains, TAD and HN3, directly infect and proliferate in DT40 cells, requiring no adaptive passages. Unexpectedly, IBDV can be rapidly propagated and continuously harvested at high titers for a long time, accompanied by the rapid growth of host cells and showing no increase in pathogenicity. Our results provide further support to suggest that DT40 cells can be used as an ideal model for studying IBDV pathogenesis. Additionally, the DT40 cell line could also serve as a potential system for commercial IBDV vaccine preparation.

Identification and characterization of Japanese encephalitis virus envelope protein gene from swine.

Aims:  Identification and characterization of Japanese encephalitis virus (JEV) envelope protein gene from swine.