Development and characterization of stable cell lines constitutively expressing the porcine reproductive and respiratory syndrome virus nucleocapsid protein

Despite global efforts to control porcine reproductive and respiratory syndrome virus (PRRSV) infection, the virus continues to cause economic problems in the swine industry worldwide. In this study, we attempted to generate and characterize a panel of stable BHK cell lines that constitutively express the nucleocapsid (N) protein of type 1 or type 2 PRRSV. The established BHK cell lines were found to react well with N-specific antibodies as well as the hyperimmune serum of pigs raised against each genotype of PRRSV. Taken together, the data implicate a potential usefulness for the newly generated stable cell lines as a diagnostic reagent for PRRSV serology.

Transactivation of peroxisome proliferator-activated receptor alpha by green tea extracts

Tea is a popular beverage. Recently, green tea was reported to increase the number of peroxisomes in rats. In this study, to find out whether the green tea-induced proliferation of peroxisomes is mediated by PPARalpha , a transient transfection assay was carried out to investigate the interactions of tea extracts (green tea, black tea,oolong tea and doongule tea) and tea components (epigallocatechin gallate, epigallocatechin, epicatechin gallate, epicatechin and gallic acid), with mouse cloned PPARalpha . Green tea and black tea extracts, and epigallocatechin gallate, a major component of fresh green tea leaves, increased the activation of PPAalpha 1.5-2 times compared with the control. It is suggested that the green tea induced-peroxisomal proliferation may be mediated through the transactivation of PPARalpha and that epigallocatechin gallate may be an effective component of green tea leaves. This would account for the increase in the number of peroxisomes and the activity of peroxisomal enzymes previously reported. However, black tea, a fully fermented product, had a stronger effect than oolong tea extract. These results also suggest, that in addition to epigallocatechin gallate, green tea leaves may possess some active chemicals newly produced as a result of the fermentation process, which act on PPARalpha like other peroxisome proliferators.

Characterization and localization of the unique Marek's disease virus type 2 ORF873 gene product

Studies on Marek's disease virus (MDV)-unique genes are important for understanding the biological nature of the virus. Based on complete DNA sequence analyses of the MDV genomes, the MDV genomes contain presumably at least five MDV-unique genes, which are commonly conserved among the three MDV serotypes. A recombinant baculovirus that contains the MDV serotype 2 (MDV2)-unique gene, ORF873, under the polyhedrin promoter was constructed and designated rAcORF873. Polyclonal and monoclonal antibodies, which recognize the recombinant MDV2 ORF873 protein in Spodoptera frugiperda clone 9 (Sf9) cells infected with rAcORF873, were prepared by immunizing mice with a recombinant fusion protein expressed in Escherichia coli. Immunoblot analyses with the antibodies revealed a major protein band with a molecular mass of 108-kDa in both MDV2-infected chick embryo fibroblasts (CEF) and rAcORF873-infected Sf9 cells. By indirect immunofluorescence analyses using monoclonal antibody, the authentic ORF873 protein was localized in the cytoplasm of MDV2-infected CEF cells. The monoclonal and polyclonal sera, which were generated in the present study and reacted effectively to MDV2 ORF873 protein, are considered to be useful reagents for further studying the role(s) of the ORF873 protein in MDV2 infection.

Targeted RNA recombination of the membrane and nucleocapsid protein genes between mouse hepatitis virus and bovine coronavirus

The targeted RNA recombination was attempted to substitute the membrane (M) protein gene and part of the nucleocapsid (N) protein gene of mouse hepatitis virus with the corresponding sequences from bovine coronavirus. Using a defective interfering (DI) RNA-like cDNA construct derived from pMH54, 690 nucleotides representing the entire M gene and the 5' most 915 nucleotides of the N gene of the mouse hepatitis virus Albany 4 mutant were attempted to be replaced. Upon infection of cells with Albany 4 followed by transfection with synthetic RNA transcribed from the DI-like cDNA construct, recombinant mouse hepatitis viruses as the large plaque forming phenotype were isolated by plaque assays at the non-permissive temperature of 391 degrees C. By RT-PCR and sequencing, those large plaque phenotypes were confirmed to have contained the thermostable phenotype marker derived from the transfected RNA, demonstrating that recombination occurred between the Albany 4 genomic RNA and the in vitro RNA transcripts. Further analysis of the recombinant viruses indicated that there combination had taken place within the region of 222 nucleotides between positions 916 and 1,137 of the N gene. This is the region immediately downstream of the replacement sequence and the start of the temperature resistant phenotype marker. The results suggest that the M and part of the N genes of bovine coronavirus may not be able to complement the function of those of mouse hepatitis virus. This study redirects our current approach of utilizing the MHV targeted RNA recombination as a means to study bovine coronavirus genetics towards the construction of an infectious cDNA clone.