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It has been reported that the avian-origin influenza A virus PB1 protein (avian PB1) enhances influenza A virus polymerase activity in mammalian cells when it replaces the human-origin PB1 protein (human PB1). Characterization of the amino acid residues that contribute to this enhancement is needed. In this study, it was found that PB1 from an avian-origin influenza A virus [A/Cambodia/P0322095/2005, H5N1 (Cam)] could enhance the polymerase activity of an attenuated human isolated virus, A/WSN/33, carrying the PB2 K627E mutation (WSN627E) in vitro. Furthermore, 473V and 598P in the Cam PB1 were identified as the residues responsible for this enhanced activity. The results from recombinant virus experiments demonstrated the contribution of PB1 amino acids 473V and 598P to polymerase activity in mammalian cells and in mice. Interestingly, 473V is conserved in pH1N1 viruses from the 2009 pandemic. Substitution of 473V by leucine in pH1N1 PB1 led to a decreased viral polymerase activity and a lower growth rate in mammalian cells, suggesting that the PB1 473V also plays a role in maintaining efficient virus replication of the pH1N1 virus. Thus, it was concluded that two amino acids in avian-origin PB1, 473V and 598P, contribute to the polymerase activity of the H5N1 virus, especially in mammalian cells, and that 473V in PB1 also contributes to efficient replication of the pH1N1 strain.

Sialoadhesin (Sn) is an important receptor for viral attachment and internalization of porcine reproductive and respiratory syndrome virus (PRRSV) to porcine alveolar macrophages (PAM). To investigate whether the N-terminal domain of Sn is sufficient and/or necessary for PRRSV attachment, we constructed a series of truncated fragments of porcine Sn and expressed these in the non-permissive PK15 cell line. The first 150 amino acids comprising the entire first domain of the Sn N-terminal region was necessary for PRRSV binding to cells, and the N-terminal domain alone was sufficient for virus attachment. The attachment of PRRSV to PAM cells was inhibited by polyclonal anti-serum against the N-terminal region of porcine Sn in a dose-dependent manner. The present study demonstrates that the first domain at the N-terminus of Sn mediates PRRSV attachment to PAM cells and contributes to better understanding the interaction between PRRSV and its host cells.

The function of the glycosylated protein 3 (GP3), a porcine reproductive and respiratory syndrome virus (PRRSV) associated protein is poorly known. In the present study, the gene encoding GP3 (ORF3), lacking the highly hydrophobic domain in the N- and C-termini was expressed as GST-fusion proteins in E. coli. Monoclonal antibodies (MAbs) against GP3 were developed and used to probe a series of GP3 peptides using ELISA. After precise analysis by sequential deletion of the terminal amino acid residues from each peptide, the minimal epitopes recognized by the MAbs were localized to W(74)CRIGHDRCGED(85) and Y(67)EPGRSLW(74). The epitope sequences were well conserved among most of the North American-type isolates, with the exception of two amino acid mutations in both epitopes in a few of these isolates. Mutational analysis revealed that these mutants were not recognized by any of the five MAbs, indicating that genetic variation could lead to altered antigenicity. Eight out of nine peptide fragments, 58-72aa, 73-87aa, 88-101aa, 102-115aa, 50-65aa, 66-81aa, 80-95aa and 94-109aa were recognized by PRRSV-positive pig serum as determined by Western blot analysis. The results herein may elucidate partially the antigenic structure of GP3 and variations of PRRSV.

Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease in swine-producing areas of the world. Many vaccine strategies developed to control the disease are not yet completely successful. The objective of this study was to determine if RNA interference (RNAi) could be utilized to inhibit PRRSV replication on MARC-145 cells. Four short interfering RNA (siRNA) sequences (N95, N179, N218 and N294) directed against a well-conserved region of PRRSV genome ORF7 gene were selected. Sense and antisense siRNA encode sequences separated by a hairpin loop sequence were designed as short hairpin RNA (shRNA) expression cassettes driven by mouse U6 promoter. Using a polymerase chain reaction (PCR)-based approach, shRNAs were generated from shRNA expression cassettes. The PCR products were cloned into pEGFP-N1 vector and shRNA expression vectors were constructed. When MARC-145 cells were transfected with shRNA expression vectors and then infected with PRRSV, N179 was found to be the most effective inhibition site in decreasing cytopathic effect (CPE) induced by PRRSV. Western blot, indirect immunofluorescence and fluorescence quantitative PCR (FQ-PCR) confirmed that the expression of ORF7 was reduced both at protein and RNA levels comparing to controls. The results presented here indicated that DNA-based siRNA could effectively inhibit the replication of PRRS virus (approximately 681-fold reduction of viral titers) on MARC-145 cells.

AIM: To investigate Time- and pH-dependent colon-specific drug delivery systems (CDDS) for orally administered diclofenac sodium (DS) and 5-aminosalicylic acid (5-ASA), respectively. METHODS: DS tablets and 5-ASA pellets were coated by ethylcellulose (EC) and methacrylic acid copolymers (Eudragit® L100 and S100), respectively. The in vitro release behavior of the DS coated tablets and 5-ASA coated pellets were examined, and then in vivo absorption kinetics of DS coated tablets in dogs were further studied. RESULTS: Release profile of time-dependent DS coated tablets was not influenced by pH of the dissolution medium, but the lag time of DS release was primarily controlled by the thickness of the coating layer. The thicker the coating layer, the longer the lag time of DS release is. On the contrary, in view of the pH-dependent 5-ASA coated pellets, 5-ASA release was significantly governed by pH. Moreover, the 5-ASA release features from the coated pellets depended upon both the combination ratio of the Eudragit® L100 and S100 pH-sensitive copolymers in the coating formulation and the thickness of the coating layer. The absorption kinetic studies of the DS coated tablets in dogs demonstrated that in vivo lag time of absorption was in a good agreement with in vitro lag time of release. CONCLUSION: Two types of CDDS, prepared herein by means of the regular coating technique, are able to achieve site-specific drug delivery targeting at colon following oral administration, and provide a promising strategy to control drug release targeting the desired lower gastrointestinal region.