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 HN(3), 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.

Characterization of the complete mitochondrial genome of Notospermus geniculatus.

In this study, the complete mitochondrial genome of Notospermus geniculatus, was recovered through Illumina sequencing data. This complete mitochondrial genome of N. geniculatus is 15,180 bp in length and has a base composition of A (14.4%), T (41.3%), C (14.6%), G (29.6%), demonstrating a bias of higher AT content (55.7%) than GC content (44.2%). The mitochondrial genome contains a typically conserved structure among Lineidae mitogenomes, encoding 13 protein-coding genes (PCGs), 23 transfer RNA genes (tRNA), 2 ribosomal RNA genes (12S rRNA and 16S rRNA), and a control region (D-loop region). All PCGs were located on the H-strand. ND4L gene and ND4 gene were overlapped by 6 bp. The whole mt genome of N. geniculatus and other Protostomia mitogenomes (17 species, in total) were used for phylogenetic analysis. The result indicated N. geniculatus has the closest relationship with Lineus viridis (FJ839919.1) and clustered within Heteronemertea Clade, which representing a distinct order.

Dynamic Changes in the Splenic Transcriptome of Chickens during the Early Infection and Progress of Marek's Disease.

Gallid alphaherpesvirus 2 (GaHV2) is an oncogenic avian herpesvirus inducing Marek's disease (MD) and rapid-onset T-cell lymphomas. To reveal molecular events in MD pathogenesis and tumorigenesis, the dynamic splenic transcriptome of GaHV2-infected chickens during early infection and pathogenic phases has been determined utilizing RNA-seq. Based on the significant differentially expressed genes (DEGs), analysis of gene ontology, KEGG pathway and protein-protein interaction network has demonstrated that the molecular events happening during GaHV2 infection are highly relevant to the disease course. In the 'Cornell Model' description of MD, innate immune responses and inflammatory responses were established at early cytolytic phase but persisted until lymphoma formation. Humoral immunity in contrast began to play a role firstly in the intestinal system and started at late cytolytic phase. Neurological damage caused by GaHV2 is first seen in early cytolytic phase and is then sustained throughout the following phases over a long time period. During the proliferative phase many pathways associated with transcription and/or translation were significantly enriched, reflecting the cell transformation and lymphoma formation. Our work provides an overall view of host responses to GaHV2 infection and offers a meaningful basis for further studies of MD biology.

Development and evaluation of an immunochromatographic strip for trichinellosis detection.

In the present study, an immunochromatographic strip was developed for the serological detection of trichinellosis in swine. In the strip, the excretory-secretory (ES) antigens of Trichinella labelled with colloidal gold was used as the detector, and the staphylococcal protein A (SPA) and goat anti-ES antibody were blotted on the nitrocellulose membrane for the test and control lines, respectively. The evaluation of the strip was performed by comparing 60 clinical positive blood samples detected by the artificial digestion method with 46 serum samples from pigs infected with parasites other than Trichinella and 30 serum samples of parasite-free healthy pigs. The strip was shown to be of high specificity and sensitivity that were closely correlated with those of ELISA. Furthermore, the dipstick assay based on the strip is rapid (10 min) and easy to perform with no requirement of special skill, reagent or equipment. This suggests the immunochromatographic strip is an acceptable alternative to be used in clinical laboratories lacking specialized equipment as well as for field diagnosis.

Identification of neutralizing epitopes on the VP2 protein of infectious bursal disease virus by phage-displayed heptapeptide library screening and synthetic peptide mapping.

Infectious bursal disease virus (IBDV) is the causative agent of infectious bursal disease, which is one of the most important and widespread infectious diseases in commercial chickens. Conformational epitopes have been reported in the highly variable region of the VP2 protein of IBDV. In the present study, a random heptapeptide library was screened by using monoclonal antibodies (mAbs), YNW17 and YNW29, directed to the VP2 of IBDV and two peptide motifs, D-X-P-R and A-R-G, were identified. The motifs are present on the N and C terminal sequences of the highly variable region of VP2. Synthetic overlapping peptides covering the motifs on VP2 were analyzed by Dot- ELISA with the mAbs and two epitopes 197CDSSDRPRVYTIT209 and 329ARGSLAVTI337 identified. The above epitopes were also recognized by chicken anti-IBDV sera and shown to inhibit the binding of their mAbs to recombinant VP2. Both mAbs and sera from mice immunized with the conjugated epitope-peptides were able to neutralize serotype I IBDV. These results indicated that the epitopes are two neutralizing linear B-cell epitopes and would be useful for the development of peptide-based IBD vaccines.

Development of a one-step strip test for the diagnosis of chicken infectious bursal disease.

A rapid diagnostic strip for chicken infectious bursal disease (IBD) was developed based on membrane chromatography using high-affinity monoclonal antibodies directed to chicken infectious bursal disease virus (IBDV). The diagnostic strip has high specificity for detection of chicken IBDV antigen and recognizes a variety of the virus isolates, including virulent and attenuated strains, with no cross-reactivity to other viruses, such as Newcastle disease virus, Marek's disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, and egg-drop-syndrome virus. The results showed that its specificity was highly consistent with the agar-gel precipitation test (AGP). The diagnostic strip detected as low as 800 median egg lethal dose (ELD50) viruses in the IBDV BC6/85-infected sample, which was comparable with AC-ELISA (400 ELD50) and 32 times more sensitive than the AGP test (2.56 x 10(4) ELD50). In experimental infection, IBDV was detected in the bursa as early as 36 hr postinfection with the diagnostic strip before the clinical signs and gross lesions appeared. It takes only 1-2 min to do a strip test to detect chicken IBDV antigen after the specimen is grounded in a whirl pack with finger massage.

Marek's disease virus-encoded analog of microRNA-155 activates the oncogene c-Myc by targeting LTBP1 and suppressing the TGF-ß signaling pathway.

Marek's disease virus (MDV) is a representative alpha herpes virus able to induce rapid-onset T-cell lymphoma in its natural host and regarded as an ideal model for the study of virus-induced tumorigenesis. Recent studies have shown that the mdv1-miR-M4-5p, a viral analog of cellular miR-155, is critical for MDV׳s oncogenicity. However, the precise mechanism whereby it was involved in MD lymphomagenesis remained unknown. We have presently identified the host mRNA targets of mdv1-miR-M4-5 and identified the latent TGF-ß binding protein 1 (LTBP1) as a critical target for it. We found that during MDV infection, down-regulation of LTBP1 expression by mdv1-miR-M4-5p led to a significant decrease of the secretion and activation of TGF-ß1, with suppression of TGF-ß signaling and a significant activation of expression of c-Myc, a well-known oncogene which is critical for virus-induced tumorigenesis. Our findings reveal a novel and important mechanism of how mdv1-miR-M4-5p potentially contributes to MDV-induced tumorigenesis.

Main Factors Influencing the Efficient Propagation of Virulent or Attenuated Strains of Japanese encephalitis virus in BHK-21 Cells.

Japanese encephalitis virus (JEV) causes severe viral encephalitis in humans and some other mammalian animals. Highly efficient culture of the virus is critical for antigen preparation, vaccine production and other basic researches. We have investigated the influence of a number of variables such as the strain virulence, the state of the host cells, medium composition, infection method and others on the proliferation of distinct JEV strains in BHK-21 cells. The results showed that two distinct strategies are needed for the propagation of virulent or attenuated JEV strains. The most critical variables were the method of infection, and especially the density of the host cell. Our studies indicate the general approaches to the in vitro culture of other JEV strains using BHK-21 cell line.