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Chuang K.P.,Graduate Institute of Animal Vaccine Technology | Liu H.J.,National Pingtung University of Science and Technology | Liu H.J.,National Chung Hsing University
Avian Pathology | Year: 2010

Reverse transcription-polymerase chain reaction (RT-PCR) was used to generate sequence data for recent Taiwanese strains of Newcastle disease virus (NDV) isolated from 1999 to 2003, covering the full length of the haemagglutinin-neuraminidase (HN) gene and protein. Nucleotide sequence analysis of the HN gene of these recent isolates revealed that the whole HN gene carries an open reading frame encoding 571 amino acids and possesses a shorter C-terminal extension. Six amino acid substitutions in epitopes on the HN glycoprotein of the recent Taiwanese NDV isolates were also found. All the recent Taiwanese NDV isolates have the amino acid sequence 112RRQKRF117 for the F protein. A phylogenetic tree analysis based on the nucleotide sequences of the F gene revealed that all recent Taiwanese isolates were related to genotype VII viruses. Since the recent Taiwanese NDV isolates exhibited a low level of haemagglutination (HA) activity, we generated two sets of mutants to elucidate whether mutations in the heptad repeat region of the HN protein could affect the HA activity. To demonstrate the presence of the viruses used in the HA test, a real-time RT-PCR was established to determine the copy number of NDV isolates. From sequence analysis, site-directed mutagenesis, and haemadsorption assays, it was found that the HN glycoprotein of recent Taiwanese NDV isolates carrying a substitution at the amino acid residue 81 (I to M) in the heptad repeat region in the stalk domain showed a dramatic decrease in the activity of HA. We infer from these results that a specific amino acid sequence within the heptad repeat region of the stalk is important for the HA activity of the HN glycoprotein. copy; 2010 Houghton Trust Ltd. Source


Chu C.-Y.,Graduate Institute of Animal Vaccine Technology | Lee S.-C.,Graduate Institute of Animal Vaccine Technology | Liu S.-S.,National Pingtung University of Science and Technology | Lin Y.-M.,Graduate Institute of Animal Vaccine Technology | And 6 more authors.
Nucleic Acid Therapeutics | Year: 2011

Adjuvants are important components of vaccine formulations. Effective adjuvants line innate and adaptive immunity by signaling through pathogen recognition receptors. Synthetic cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs) have been shown to have potentials as adjuvants for vaccines. However, the immunostimulatory effect of CpG is species-specific and depends on the sequence of CpG motifs. A CpG ODN (2135), containing 3 identical copies of GTCGTT motif, was previously reported to have the strongest effects on bovine peripheral blood mononuclear cells (PBMC). Based on the sequence of 2135, we replaced the GTCGTT motif with 11 other sequences containing CG and investigated their effects on bovine lymphocyte proliferation. Results showed that the CpG ODNs containing 3 copies of GACGTT motif had the highest lymphocyte stimulation index (7.91±1.18), which was significantly (P<0.05) higher than that of 2135 (4.25±0.56). The CpG ODNs containing 3 copies of GACGTT motif also significantly increased the mRNA expression of interferon (IFN)-α, interleukin (IL)-12, and IL-21 in bovine PBMC. When dairy cows were immunized with the keyhole limpet hemocyanin (KLH) antigen formulated with CpG ODNs containing 3 copies of GACGTT, production of KLH-specific antibodies in serum and in milk whey was significantly (P<0.05) enhanced. IFN-γ in whole blood stimulated by KLH was also significantly (P<0.05) increased in cows immunized with KLH plus CpG ODNs. Our results indicate that CpG ODNs containing 3 copies of the GACGTT motifs is a potential adjuvant for bovine vaccines. © 2011 Mary Ann Liebert, Inc. Source

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