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Human parechoviruses (HPeVs) belonging to the family Picornaviridae are widely spread pathogens among young children. We report the complete genome sequence of a novel HPeV isolated from the stool sample of a hospitalized child with diarrhea in China. The genome consists of 7,305 nucleotides, excluding the 3' poly(A) tail, and has an open reading frame that maps between nucleotide positions 675 and 7217 and encodes a 2,180-amino-acid polyprotein. The genome sequence of the virus was sufficiently distinct from the 8 known HPeV types. Phylogenetic analysis based on the complete genome indicated that the HPeV strain represents a new genotype.

Muscovy duck reovirus (MDRV) is an important poultry pathogen that causes high morbidity and mortality in ducklings. The mechanisms by which viruses kill susceptible cells, and ultimately produce diseases, in Muscovy duck remain poorly understood. In this study, we focused on the biologic functions of the MDRV p10.8 protein in vitro. The p10.8 protein is a small protein of MDRV that is encoded by the first open reading frame of the S4 segment. In our study, the p10.8-encoding gene was individually cloned and expressed in bacterial and eukaryotic cells. The p10.8 protein had no potential transmembrane domain; it shared no sequence similarity to other known fusion-associated small transmembrane proteins encoded by the avian reovirus, Nelson Bay virus or baboon reovirus; and it did not show any syncytium formation activity. The p10.8 protein induced apoptosis when expressed by itself in transfected primary Muscovy duck embryonic fibroblasts or in Vero E6 cells. Four assays were used to analyze the apoptosis induced by p10.8: DNA ladder formation; terminal deoxynucleotidyl transferase dUTP nick end labeling; enzyme-linked immunosorbent assay detection of cytoplasmic histone-associated DNA fragments; and nuclear staining with propidium iodide. Two deletion products, p10.8delta1 (1-63aa; amino acid position) and p10.8delta2 (64-96aa), were constructed. Deletion analysis suggests that p10.8delta1 (1-63aa) is important in mediating p10.8-induced apoptosis because its deletion abolishes induction of apoptosis.

Objective: To evaluate the effects of the fusion gene of ubiquitin (Ub) and Porcine Reproductive and Respiratory Syndrome virus (PRRSV) M gene on the immune response in inoculated mice., Methods: Mouse Ub gene and PRRSV M gene were amplified by RT-PCR from BALB/c mice spleen cells and PRRSV Ch-1a strain, respectively, and the M and Ub gene (U-M) was fused by SOE PCR. Therefore, pVAX1-U-M and pVAX1-M recombinant plasmid were constructed for eukaryotic expression., Results: The fusion U-M and M protein expressions were verified in transfected BHK-21 cells by indirect fluorescence assay. Furthermore, both pVAX1-M and pVAX1-U-M induced specific humoral and cellar immune responses against PRRSV in the recombinant plasmid injected mice. However, pVAX1-U-M was able to induce higher level of T cell response then that of pVAX1-M (P<0.05), but lower level of antibody (P<0.05)., Conclusion: Expression of U-M fusion gene had ability to enhance specific T cell response against PRRSV, but no effect on stimulation of humoral response in inoculated mice.

Objective: In recent years,manipulation of large herpesvirus genomes has been facilitated by using bacterial artificial chromosome (BAC) vectors. We have previously reported the construction of the BAC clones (HVT BACs) of herpesvirus of turkey (HVT). With these BAC clones in hand,we manipulated the genome of HVT by utilizing Red/ET recombination system, and developed a biologically safe live vaccine based on the HVT BACs., Method: In this two-step approach, we first transformed the plasmid pRedET into the DH10B competent cells that carried the HVT BACs,and added inducer L-arabinose into the cells. We prepared the cells into competent cells and electroporated the linear rpsL-neo counter-selection/selection cassette flanked by the 50 bp long homology arms into the cells. So the functional cassette was inserted into the U(S)2 locus. Only colonies carrying the modified BAC would survive Kanamycin selection on the agar plates. The successful integration of the rpsL-neo cassette was monitored by PCR and Streptomycin selection, for the insertion of rpsL-neo cassette cells will become Streptomycin sensitive. Secondly, in the same way, we replaced the rpsL-neo cassette with the hemagglutinin (HA) gene of (HPAIV) A/Goose/ Guangdong/1/96(H5N1) flanked by the same homology arms. Only colonies which lost the rpsL-neo cassette will grow on Streptomycin containing plates., Results: Finally, we obtained many colonies of which the HA gene of the AIV was inserted into the U(S)2 locus to be modified of HVT. And we reconstituted one recombinant virus from transfecting one of these BAC clones DNA into chick embryo fibroblasts (CEFs)., Conclusion: We achieved one rescued recombinant virus which designated as rHVT-HA3. The H5 subtype HA gene expression in this recombinant virus rHVT-HA3 was confirmed by immunofluorescence assay.

Unlabelled: Herpesvirus of turkey (HVT) is an alpherpesvirus and widely used as a live vaccine against Marek's disease (MD) because of its antigenic relationship with Marek's disease virus (MDV)., Objective: The aim of this study was to construct Herpesvirus of turkey Fc126 strain as an infectious bacterial artificial chromosome (BAC)., Methods: Using the selection marker Eco-gpt (Xanthine-guanine phosphoribosyl transferase)(1.3 kb) and BAC vector pBeloBAC11(7.4 kb), we constructed the transfer plasmid pGAB-gpt-BAC11. Then, the transfer plasmid and HVT-infected cells' total DNA were cotransfected into primary chicken embryo fibroblasts (CEFs). After six rounds of selection in medium containing mycophenolic acid, xanthine and hypoxanthine, we obtained purified recombinant viruses. Genomic DNA was extracted and electroporated into Escherichia coli DH10B competent cells. BAC clones were identified by restriction enzyme digestion and PCR analysis, and then tested for infectivity after transfection into CEFs using calcium phosphate., Results: We obtained 25 BAC clones, and reconstituted recombinant viruses by transfection of HVT-BAC6 DNA, HVT-BAC8 DNA and HVT-BAC10 DNA into CEFs respectively., Conclusion: In this study, we cloned the complete genome of HVT Fc126 strain as an infectious bacterial artificial chromosome.

The aim of this study was to construct the complete genome of Marek's disease virus serotype 814 strain as an infectious bacterial artificial chromosome (BAC). Using self-designed selection marker Eco-gpt (1.3 kb) and BAC vector pBeloBAC11 (7.5 kb), we constructed the transfer plasmid pUAB-gpt-BAC11. The plasmid pUAB-gpt-BAC11 and MDV total-DNA were cotransfected into secondary CEFs; we put the virus-containing cells in selection medium for eight rounds and obtained purified recombinant viruses. Recombinant viral genomes were extracted and electroporated into E. coli, BAC clones were identified by restriction enzyme digestion and PCR analysis. Finally, we obtained 38 BAC clones, DNA from various MDV-1 BACs was transfected into CEFs, and recombinant virus was reconstituted by transfection of MDV-BAC2 DNA. We successfully cloned the complete genome of MDV-1814 strain as an infectious bacterial artificial chromosome. With these cloned genomes, a revolutionary MDV-DNA engineering platform utilizing RED/ET recombination system was constructed successfully, which can help the understanding of MDV gene functions and promote the using of MDV as a vector for expressing foreign genes. In addition, it opens the possibility to generate novel MDV-1 vaccines based on the BACs.

Improving expression of antigen is critical to the immunogenicity of DNA vaccines. To achieve this goal, we modified the NDV F48E9 strain HN gene by optimizing the condon usage and inserting the secretary leader sequence [A/Goose/Guangdong/1/96 (H5N1) HA gene, Accession No. AF144305]. The HN gene modified and knocked the signal peptide off were named SoptiHN and optiHN. The three sequence: SoptiHN, optiHN and the NDV F48E9 strain HN gene were inserted into the vector pVAX1 and vector pVAX1-CpG including CpG-ODN sequence respectively. Then we got six recombinant plasmids: pV-SoptiHN, pVC-SoptiHN, pV-optiHN, pVC-optiHN, pV-HN and pVC-HN. By optimizing condon usage in transiently transfected 293T cells, expression levels of HN gene were higher from the codon-optimized gene than the counterpart. Moreover, both optimization of condon usage and addition of signal peptide could improve expression of HN gene in vitro.