Hui-Gang Shen

Serological investigation and genomic characterization of PCV2 isolates from different geographic regions of Zhejiang province in China.

Sera collected from 46 swine farms in Zhejiang province were evaluated for the presence of antibodies to PCV2 using an indirect-fluorescent antibody procedure. In addition PCV2 isolated from superficial inguinal lymph node samples collected from 40-to 90-day-old pigs with clinical signs of post-weaning multisystemic wasting syndrome (PMWS) using the PK-15 cell line were sequenced and compared. Overall seroprevalence of PCV2 antibody averaged 58.34% for all samples. Breakdown of serology by groups was as follows: 59.38% for sows, 57.41% for post-weaning piglets, 44.83% for Landrace sows and 64.28% for Landrace piglets. The seroprevalence of Landrace sows was higher than that of Yorkshire and Duroc sows, but non-significant (p > 0.05). Serological analysis also showed that seroprevalence of PCV2 antibody was a negative correlation to that of PRRSV antibody. The complete genomes of five PCV2 isolates identified in the herds with PMWS consisted of 1767nt, containing the 11 potential ORFs. Genome of the virus isolates shared 93.8% to 99.8% identity with PCV2 reference strains from GenBank, 76.6% to 77.9% identity with PCV1. Phylogenetic analysis indicated that there were two subgenotypes within PCV2: subgenotype I (1767 nt) and subgenotype II (1768 nt).

Protective immunity against porcine circovirus 2 by vaccination with ORF2-based DNA and subunit vaccines in mice

The protective immune response against porcine circovirus 2 (PCV2) infection in mice was characterized using flow cytometric analysis (FCM), assays of antibody (of different IgG isotypes) and viraemia, and histopathological examination. An open reading frame 2 plasmid (pORF2) and the capsid protein (Cap) of PCV2 were used as DNA and subunit vaccines, respectively. In FCM analysis, although pORF2 and Cap alone showed comparable efficacy in eliciting lymphoproliferative responses and Cap-specific CD4(+) T cells, pORF2 was superior to the Cap protein in triggering CD8(+) T cells. A virus neutralization assay showed that pORF2 evoked stronger recall virus-neutralizing (VN) antibody responses than the Cap protein on PCV2 challenge. Correspondingly, VN antibody kinetics coincided with those of Cap-specific IgG2a, but not with the kinetics of IgG and IgG1. Following virus challenge, real-time PCR and histopathological analysis confirmed that only low viral DNA loads and mild microscopic lesions appeared in pORF2-immunized mice. These findings indicate that CD8(+) T cells and VN antibody responses correlating mainly with Cap-specific IgG2a play crucial roles in protecting against PCV2 infection, and that the protective immunity induced by the pORF2 plasmid is superior to that induced by the PCV2 Cap protein.

Characterization of chicken interleukin 2 receptor α chain, a homolog to mammalian CD25

To identify chicken IL‐2R α chain (chCD25), the cDNA of chCD25 was cloned and mapped onto chicken chromosome 1. The polyclonal and monoclonal antibodies raised from the recombinant chCD25 specifically bound to the cell surface of splenic mononuclear cells (SMC) and inhibited chicken IL‐2‐dependent proliferation of T cells. Flow cytometry analysis revealed that chCD25 molecules could be expressed on the surface of monocytes/macrophages, thrombocytes, CD4+ and CD8+ cells as well as tissue cells. Importantly, the CD4+CD25+ and CD8+CD25+ cells were upregulated dramatically in chickens infected with H9N2 avian influenza virus. These results confirm that the cloned cDNA is the nucleotide sequence of chicken IL‐2R, and suggest that chicken CD4+CD25+ and CD8+CD25+ cells may play an important role in immune responses induced by H9N2 virus, and the monoclonal antibodies to chCD25 may be useful for investigating biological functions of chicken regulatory T cells.

Interference of porcine circovirus type 2 ORF2 immunogenicity by ORF1 and ORF3 mixed DNA immunizations in mice.

Little is known about the influences of other porcine circovirus type 2 (PCV2) proteins on the immunogenicity of Cap protein. Here we constructed plasmids expressing the ORF1 (pORF1) and ORF3 (pORF3) of PCV2, and mixed either of them with the plasmid expressing ORF2 (pORF2) as combined DNA vaccines, to compare their immunogenicity and protective efficacy. Our data revealed that pORF1 reduced the Cap-specific CD8(+)cell frequency, and both pORF1 and pORF3 attenuated the Cap-specific Th1 and post-challenge-recall VN antibody responses induced by the pORF2 plasmid, despite successful induction of Rep and ORF3 antibodies by pORF1 and pORF3, respectively. Subsequently, protocols with pORF1 or pORF3 showed significantly decreased protective efficacy compared to pORF2 alone. Overall, our data suggested that the ORF1- and ORF3-encoded Rep and ORF3 proteins may interfere with the cellular, humoral and protective immunity of the ORF2-encoded Cap protein in vivo.

Identification of the linear epitope for Fc‐binding on the bovine IgG2 Fc receptor (boFcγ2R) using synthetic peptides

To identify the linear epitope for Fc‐binding on the bovine IgG2 Fc receptor (boFcγ2R), peptides derived from the membrane‐distal extracellular domain (EC1) of boFcγ2R corresponding to the homologous region of human FcαRI were synthesized. Binding of bovine IgG2 to the different peptides was tested by Dot‐blot assay, and the peptide showing maximal binding was further modified by truncation and mutation. The minimum effective peptide 82FIGV85 located in the putative F–G loop of the EC1 domain was found to bind bovine IgG2 specifically and inhibit the binding of bovine IgG2 to the receptor. The Phe82, Ile83 and Val85 residues within the linear epitope were shown to be critical for IgG2‐binding. Such functional epitope peptide should be very useful for understanding the IgG‐Fcγ interaction and development of FcR‐targeting drugs.

Production and Characterization of Monoclonal Antibodies to poly100S1 Protein of Avian Infectious Bronchitis Virus

Fragments within S1 genes (poly100S1) of infectious bronchitis virus (IBV) strains ZJ971, M41 and SC021202 (SC) were subcloned into a prokaryotic expression vector and expressed in Escherichia coli. Monoclonal antibodies (mAbs) against the recombinant poly100S1 proteins were produced, characterized and used to analyse epitopes on the S1 subunit of IBV. Nine mAbs raising from the three poly100S1 proteins recognized five different epitopes of the S1 subunit, designated as S1‐A, B, C, D and E. Epitopes S1‐C and S1‐D are common for the three IBV strains, while S1‐A and S1‐B exist on ZJ971 and M41 strains, and S1‐E was a strain‐specific epitope for SC strain. Immunocytochemistry indicated that all the mAbs to the poly100S1 proteins can react with the homologous S1 glycoprotein expressed in Vero cells. Moreover neutralization test demonstrated that only mAbs 6E2, 4F9 and 6G4 had neutralization activity for the homologous IBV. These mAbs to poly100S1 protein were potential candidates for detecting and distinguishing IBV strains, and also used to examine antigenic variation of the S1 protein.