Dynamic Changes in Viral Loads during Co-Infection with a Recombinant Turkey Herpesvirus Vector Vaccine and Very Virulent Marek's Disease Virus In Vivo.

Marek's disease (MD), caused by the Marek's disease virus (MDV), is a common infectious tumor disease in chickens and was the first neoplastic disease preventable by vaccination. However, the vaccine cannot completely prevent virulent MDV infections, allowing both the vaccine and virulent MDV to coexist in the same chicken for extended periods. This study aims to investigate the changes in viral load of the very virulent strain Md5 and the rHVT-IBD vaccine in different chicken tissues using a real-time PCR assay. The results showed that the rHVT-IBD vaccine significantly reduced the viral load of MDV-Md5 in different organs, while the load of rHVT-IBD was significantly increased when co-infected with Md5. Additionally, co-infection with Md5 and rHVT-IBD in chickens not only changed the original viral load of both viruses but also affected the positive rate of Md5 at 14 days post-vaccination. The positive rate decreased from 100% to 14.29% (feather tips), 0% (skin), 33.33% (liver), 16.67% (spleen), 28.57% (thymus), 33.33% (bursa), and 66.67% (PBL), respectively. This study enhances our understanding of the interactions between HVT vector vaccines and very virulent MDV in chickens and provides valuable insights for the future development of MD vaccines.

Baicalin is an inhibitor of subgroup J avian leukosis virus infection.

Avian leukosis virus subgroup J (ALV-J) can cause great economic losses to the poultry industry worldwide. Baicalin, one of the flavonoids present in S.baicalensis Georgi, has been shown to have antiviral activities. To investigate whether baicalin has antiviral effects on the infection of ALV-J in DF-1 cells, the cells were treated with baicalin at different time points. We found that baicalin could inhibit viral mRNA, protein levels and overall virus infection in a dose- and time-dependent manner using a variety of assays. Baicalin specifically targeted virus internalization and reduced the infectivity of ALV-J particles, but had no effect on the levels of major ALV-J receptor and virus binding to DF-1 cells. Collectively, these results suggest that baicalin might have potential to be developed as a novel antiviral agent for ALV-J infection.

[Identification of porcine reproductive and respiratory syndrome virus regulation sequence in 3'-untranslated region].

Based on our established infectious clone of PRRSV, designated as pCBC2, a series of mutagenesis of 3'-untranslated region (3'-UTR) at primary structure and secondary structure level were constructed. Then the full length mutant clones were transfected into MARC-145 cells, from which the influences of the discrete 3'-UTR mutation on PRRSV replication and transcription were analyzed. The properties of the rescued mutant viruses were then further characterized by Northern Blot and plaque morphology analysis. Our results demonstrated that PRRSV could tolerate more than 41 nucleotides deletion and 23nt insertion in the 3'-UTR, however, minor changes in the conserved stem loop region destroyed virus infectivity. To sum up, the stem-loop structure was essential for virus viability, but 5' end of the 3'-UTR tolerates certain level of nucleotide deletion or insertion. This is the first report to define the essential sequence and secondary structure for PRRSV genome replication and it is useful for future research about the regulation element.

[High level expression of recombinant chicken interferon-gamma in insect cells].

The recombinant transfer vector pFastBacl-ChIFN-y was constructed by plasmid pcDNA-ChIFN-gamma digested with EcoR I and Not I enzymes and cloned into pFastbacl. Then the transfer vector was transformed into E. coli competent cells DH10Bac which contained the bacmid with amini-attTn7 target site and the helper plasmid. The recombinant bacmid-ChIFN-gamma was generated by transposing themini-Tn7 element located in pFastBacl-ChIFN-gamma to themini-attTn7 attachment site on the Bacmid. Subsequently the recombinant Bacmid-ChIFN-gamma was transfected into the Sf9 insect cells mediated by lipofectin to produce recombinant baculovirus and express recombinant ChIFN-gamma (rChIFN-gamma) products. The result showed that the rChIFN-gamma was successfully expressed in Sf9 cells infected with the recombinant virus by indirect immunofluorescence assay (IFA) at 5 days post-transfection. The biological activity of rChIFN-gamma was identified by its inhibition to Vesicular stomatitis virus-induced cytotoxicity of chicken embryonic fibroblasts (CEF) in vitro. The results showed that the most efficient expression of rChIFN-gamma could be obtained at 96h post-infection with multiplicity of infection (MOI) equal to 1. It is interesting that the viruses such as Avian influenza virus H5N1 or Marek's disease virus (GA strain) could not grow in CEF pre-treated with rChIFN-gamma. Cell pathogenic efficient (CPE) in the CEF infected with H5N1 and GA strain is apparently inhibited by the rChIFN-gamma. However only difference between the HA titres of the supernatant of the pre-treated cells is observed without any obvious inhibition effect in CEF infected with Newcastle disease virus (F48E8 strain).

Development and evaluation of an immunochromatographic strip for rapid detection of capsid protein antigen p27 of avian leukosis virus.

A rapid immunochromatographic strip for detecting capsid protein antigen p27 of avian leukosis virus was successfully developed based on two high-affinity monoclonal antibodies. The test strip could detect not only 600pg purified recombinant p27 protein but also quantified avian leukosis virus as low as 70 TCID50, which has comparative sensitivity to the commercial enzyme-linked immunosorbent assay (ELISA) kit. For the evaluation of this test strip, 1100 samples consisting of cloacal swabs, meconium collected from the earliest stool of one day old chicken and virus isolates were assessed both by the strip and by the commercial ELISA kit. The agreement between these two tests was 93.91%, 93.42% and 100%, respectively. The sensitivity and specificity of the strip were also calculated by using the ELISA kit as the standard. This immunochromatographic strip provides advantages of rapid and simple detection of capsid protein antigen p27 of avian leukosis virus, which could be applied as an on-site testing assay and used for control and eradication programs of avian leukosis disease.

Genetic characterization of porcine kobuvirus variants identified from healthy piglets in China.

In this study, two porcine kobuvirus strains, JS-01-CHN and JS-02a-CHN were detected from piglets with diarrhea and asymptomatic, respectively. The sequences of the two strains were analyzed using a bioinformatics software package. The full-length genome of JS-02a-CHN, was detected in healthy piglets was 8121 nucleotides (nt) long excluding the poly(A) tail. There was a 30 amino acid deletion in the 2B-coding region of JS-02a-CHN. We are the first to report a 30 amino acid deletion in porcine kobuvirus from asymptomatic piglets, indicating that porcine kobuvirus may have evolved differently based on geography and host differences. Fecal samples were obtained from pigs with diarrhea (n=91) and healthy (n=126) pigs and analyzed using RT-PCR. Of these, 64.8% (59/91) of diarrheic piglets and 19.8% (25/126) of healthy piglets were positive for PKV using VP1 specific primers. Twenty-eight (28) virus positive samples were randomly selected and the VP1 gene was analyzed. Phylogenetic analysis indicated that the 15 strains isolated from pigs with diarrhea clustered into different branches, while the VP1 sequences from clinically healthy pigs clustered into a single large group. These results indicate that the VP1 gene is diverse in pigs with diarrhea but conserved in healthy pigs in the Jiangsu Province.

Genistein inhibits the replication of avian leucosis virus subgroup J in DF-1 cells.

To investigate the antiviral effects of genistein on the replication of avian leukosis virus subgroup J (ALV-J) in DF-1 cells, the cells were treated with genistein at different time points and the antiviral effects were examined by using a variety of assays. We determined that genistein strongly inhibited viral gene expression and decreased the viral protein level in the cell supernatant and the cytoplasm without alerting virus receptor expression and viral attachment. We also observed that genistein was not found to interfere with virus entry, but significantly inhibited both viral gene transcriptions at 24h post infection and virus release, which indicate that genistein exerts its inhibitory effects on the late phase of ALV-J replicative cycle. These results demonstrate that genistein effectively block ALV-J replication by inhibiting virus transcription and release in DF-1 cells, which may be useful for therapeutic drug design.

Monoclonal antibodies directed against chicken ß2-microglobulin developed with a synthesized peptide.

We developed a panel of monoclonal antibodies (MAb) against chicken ß2-microglobulin (chß2M) by fusions between SP2/0 myeloma cells and spleen cells from mice immunized with a synthesized peptide corresponding to positions 91-119 of the COOH domain of chß2M. Two of them, 6E7 and 3D1, identified as IgG1/κ, could react with chß2M protein from avian macrophage HD11 cells and human 293T cells transfected with pcDNA3.1-chß2M in immunofluorescence assays. Only a 12 kDa protein band of chß2M could be detected in the HD11 and 293T/chß2M cell lysates by Western blot analysis. Chicken ß2M in serum and plasma could be found in Western blot by MAb 3D1. Moreover, MAb 3D1 also recognized the chß2M antigen on the cell membranes in flow cytometry. Immunohistochemical staining with these MAbs revealed that chß2M was present in chicken thymus, spleen, and bursa. These MAbs will be good tools for analyzing the mechanism of the chicken immune system.

[Development and characterization of monoclonal antibody against human FcR γ chain].

AIM: The current study was aimed to generate mouse monoclonal antibodies (mAbs) specific for Fc receptor common γ chain (FcRγ), which serves a critical signaling molecule for numerous human immune receptors that play essential roles in human immune responses. METHODS: Two BALB/c mice were immunized with the synthesized polypeptides containing 19 residues at the carboxyl terminus of human FcR γ chain. The spleen cells from the immunized mice were fused with myeloma Sp2/0 cells. ELISA was used to screen mAbs and the positive clones were selected for further characterization with Western blot and flow cytometry assays. RESULTS: After screen, there hybridoma cell lines which secreted mAbs specifically recognized the FcR γ chain were obtained and named as 5B6, 7D3 and 8D4. The 7D3 mAb had the best quality and it could recognize the FcR γ chain in vivo and in vitro with 2.5 µg/mL. CONCLUSION: We have generated the FcRγ-specific mAbs, which may serve as a valuable tool in the functional studies of FcRγ associated immune receptors.

[Molecular epidemiology of infectious bursal disease virus in Guangxi during the period of 2000 to 2007].

Tissue samples of Fabricius' bursa collected from Nanning, Yulin, Beihai and Wuzhou in the provinces of Guangxi in China during the years of 2000-2007, were detected by a established reverse transcriptase polymerase chain reaction (RT-PCR) technique for IBDV. Viral isolation was performed on the positive samples by chicken embryo inoculation via chorio-allantoic membrane (CAM). Results showed that 27 isolates of IBDV were obtained. A set of primers were designed to amplify the vVP2 of 27 isolates by RT-PCR and the PCR products were sequenced. The sequences of all the isolates and reference viruses were analyzed and compared, and their phylogenetic trees were constructed based on the nucleotide sequences. The results indicated that isolate BH11, TZ(3), 050222, YL051, NN0603, NN0611and QX0602 etc, altogether 17 isolates, which accounted for 62.96 percent of total isolates, were identified to be very virulent IBDV (vvIBDV) and have the highest homology to vvIBDV reference strains. In the phylogenetic analysis, they are divided into 3 groups and have a long distance to commonly used vaccine stains. Isolate NN040124 and YL052 were identified as intermediate-plus virulent strains and showed a highest homology to classical strains of 52-70 and STC. 8 isolates of YLZF2, 040131 etc were identified as attenuated vaccine strains and showed a highest homology to classical strain of CU1. The results from the study demonstrated that the viruses prevailing in chickens in these 4 regions in Guangxi province in the recently 7 years were vvIBDV and their origins were complex. The antigenicity of some isolates may have been drifted.

[Differentiation of recombinant adenoviruses expressed MDV-1 VP22 subcellular localization pattern].

AIM: To study the transduction and localization mechanism of Marek's disease virus serotype 1 (MDV-1) CVI988 VP22 in different cells. METHODS: VP22 was expressed with recombinant adenovirus and identified by immunofluorescence assay (IFA) and Western blot. Lysates of recombinant virus infected 293 cells were added to normal MDBK cells to identify the transduction property of VP22. AD-293 cells infected with recombinant virus were fixed to investigate localization of VP22 at different time post infection. Transient expression of VP22 in AD-293 cells was carried out for control. RESULTS: The results showed that the VP22 expressed by recombinant adenovirus entered almost all the monolayer cells, which indicate the VP22 remains its transduction property. The VP22 first gather round the nucleus membrane, and then concentrated in particles in cytoplasm of 293 cells infected with recombinant adenovirus, compared with the nuclei localization pattern of VP22 in MDV infected CEF and transient expressed VP22 in 293 cells. CONCLUSION: The VP22 presented a different localization pattern in cells infected with different recombinant virus.

[Preparation and immunological characterization of monoclonal antibodies against VP22 carboxyl terminus of Marek's disease virus serotype 1].

AIM: To prepare and characterize monoclonal antibodies (mAb) against VP22 carboxyl terminus of CVI988/Rispens strain of Marek's disease virus serotype 1. METHODS: Carboxyl terminus of CVI988 VP22 (94aa-243aa) was expressed in prokaryotic system. mAbs against VP22 were prepared by hybridoma technology from BALB/c mice immunized with the fusion protein GST-VP22C and characterized by ELISA, indirect fluorescence assay (IFA) and Western blot. RESULTS: Two hybridoma cell lines stably secreting mAb against VP22C were obtained and designated as 3F7 and 4E4. mAb 3F7 could react with VP22 expressed in all the plaques, while mAb 4E4 stained all the cells nuclei in MDV-infected CEF cells. It was also found that 3F7 could react with VP22 expressed in Sf9 cells and denatured VP22 by Western blot analysis. In addition, it was further showed that the epitope of mAb 3F7 was located within the domain between 94aa and 193aa, the predicted site of protein transduction domain of VP22. CONCLUSION: The preparation of the mAb is very important to further research in protein transduction domain of MDV-1 VP22.

[Construction and immunological characterization of recombinant Marek's disease virus expressing IBDV VP2 fusion protein].

A transfer plasmid vector pUC18-US10-VP2 was first constructed by inserting the gene of the enhancer green fluorescent protein(eGFP) fused to the VP2 gene of very virulent Infectious bursal disease virus (IBDV) JS strain into the US10 fragment of the Marek's disease virus (MDV) CV1988/Rispens. The recombinant virus, designated as rMDV, was developed by co-transfecting CEF with the transfer plasmid vector and simultaneously infecting with the CVI988/Rispens virus. The PCR and IFA results indicated that the rMDV is stable after 31 passages. Chickens vaccinated with rMDV were protected from challenge with 100LD50 of IBDV JS. The protection ratio of the chickens vaccinated with the 1000PFU, 2000PFU, 5000PFU of the rMDV were 50%, 60%, and 80% respectively. It is interesting that the average histopathology BF lesion scores of chicken group immunized with 5000PFU of rMDV by one-time vaccination was close to that of chicken group vaccinated with IBDV live vaccine NF8 strain for twice (2.0/1.5). There is no difference in protection between the groups (P > 0.05) but significent difference between groups immunized with 5000 PFU of rMDV and with normal MDV. This demonstrated that rMDV expressing VP2 fusion protein was effective vaccine against IBDV in SPF chickens.