Pingan Xia

Enhancement of the immunogenicity of an infectious laryngotracheitis virus DNA vaccine by a bicistronic plasmid encoding glycoprotein B and interleukin-18

A DNA vaccine against infectious laryngotracheitis virus (ILTV) can induce specific humoral and cell-mediated immunity. However, compared to conventional vaccines, DNA vaccines usually induce poor antibody responses. To determine if co-expression of a cytokine can result in a more potent ILTV DNA vaccine, immunogenicity and protective efficacy of a monocistronic vector encoding the glycoprotein B (gB) of ILTV was compared to that of a bicistronic vector separately encoding the gB and chicken interleukin-18. Humoral and cellular responses induced by the DNA vaccines administered to the quadriceps muscle of chickens were evaluated. There were significant differences in antibody levels elicited by either monocistronic or bicistronic DNA vaccines as determined by ELISA. The percentages of CD3(+), CD3(+)CD8(+) and CD3(+)CD4(+) subgroups of peripheral blood T-lymphocytes in chickens immunized with the bicistronic DNA vaccine were higher than those in chickens immunized with monocistronic DNA vaccine. When chickens were challenged with a virulent CG strain of ILTV, the protective efficacy was enhanced significantly after immunization with the bicistronic DNA vaccine. These results demonstrated that co-expression of an adjuvant cytokine from a bicistronic DNA vaccine may be an effective approach to increasing ILTV DNA vaccine immunogenicity.

Cloning, in vitro expression and bioactivity of duck interleukin-18.

The encoding sequence for duck IL-18 was obtained, using reverse transcription-polymerase chain reaction, from mRNA harvested from Con A-stimulated Gushi (GS) duck splenic mononuclear cells. Recombinant duck IL-18 (rduIL-18) was produced in a prokaryotic expression system. In vitro bioactivity of rduIL-18 was determined in a lymphocyte proliferation assay and in vivo bioactivity of rduIL-18 was assessed by addition to a vaccine. Monoclonal antibody (mAb) and polyclonal antibodies (pAbs) specific for rduIL-18 were generated and subsequently characterized by ELISA, Western blot and neutralizing assays. Sequence analysis of GS duck IL-18 demonstrated an open reading frame (ORF) of 603 base pairs encoding for a 200 amino acid precursor protein. The duck encoding sequence shares 85.3% similarity to the chicken equivalent, at the nucleotide level. A His-duIL-18 fusion protein was recognized in Western blot by mAbs against duck and chicken IL-18 (chIL-18), but not by mAb against human IL-18. Recombinant duIL-18 induced in vitro proliferation of Con A-stimulated duck splenocytes and enhanced the immune response of ducks vaccinated with an inactivated oil emulsion vaccine against avian influenza virus. PAb and mAb 5B2 against rduIL-18 had neutralizing ability, inhibiting the biological activities of both recombinant duIL-18 and endogenous duIL-18. The results indicate that rduIL-18 has the potential to be used as an immunoadjuvant, and the mAb against rduIL-18 further facilitates basic immunobiological studies of the role of IL-18 in the avian immune system.

Molecular evolution of porcine reproductive and respiratory syndrome virus isolates from central China

To investigate the genetic diversity of prevailing porcine reproductive and respiratory syndrome virus (PRRSV) in Henan Province of China, 61 ORF5 gene sequences, originating from Henan Province during 2003-2010, were subjected to amino acid variation and phylogenetic analysis. The analyzed PRRSV ORF5 sequences carried evidence of one unique recombination event. Phylogenetic analysis revealed that all Henan isolates belonged to type 2 genotype and were divided into two subgroups. The dominant isolates had shifted from subgroup 1 to subgroup 2 during 2003-2010. Amino acid variation analysis of the glycoprotein 5 revealed that Henan PRRSV strains tended to accumulate more substitutions within the N-terminus and hypervariable region. Selective pressure analysis revealed evidence that some ORF5 sites have likely evolved in response to immune pressure.

Molecular cloning and characterization of a porcine Fc gamma RIIb sub-isoform(FcγRIIb1).

Receptors for the Fc fragment of IgG (FcγRs) constitute one of the main effector mechanisms through which IgG immune complexes exert their action. Four FcγRs, FcγRI (CD64) with high affinity, FcγRI with intermediate affinity, FcγRII (CD32) and FcγRIII (CD16) with low affinity, have been identified. There are three FcγRII isoforms (activating FcγRIIa and FcγRIIc, and inhibiting FcγRIIb) existing in humans, one isoform in mice (inhibiting FcγRIIb), and two isoforms in cattle (inhibiting FcγRIIb, activating FcγRIIc). Two splice sub-isoforms of FcγRIIb, FcγRIIb1(b1) and FcγRIIb2(b2), have been identified in humans, mice and cattle, however, few of FcγRIIb sub-isoforms have been investigated in pig. In this study, we describe the molecular cloning, sequencing and characterization of a porcine FcγRIIb sub-isoform, FcγRIIb1. The cDNA encoding porcine FcγRIIb1 was isolated from peripheral blood leucocytes RNA with RT-PCR. The porcine FcγRIIb1 cDNA contains a 951bp open-reading frame, encoding a 316 amino acid transmembrane glycoprotein composed of two immunoglobulin (Ig)-like extracellular domains, a transmembrane region and a cytoplasmic tail with an immunoreceptor tyrosine-based inhibiting motif (ITIM). The porcine FcγRIIb1 shares 98.3% homology and has a 19 amino acid in-frame insertion in cytoplasmic tail when compared with amino acid sequence of DQ026064. Immunofluorescence analysis showed that the glycoprotein encoded by the porcine FcγRIIb1 cDNA was expressed in the stable transfected COS-7 cells, and an immunoglobulin-binding assay showed that it had binding activity for IgG immune complexes. Identification of the porcine FcγRIIb1 will help our understanding of the molecular basis of IgG-FcγR interaction in the porcine immune response.

Porcine Fc gamma RIIb sub-isoforms are generated by alternative splicing.

Receptors for the Fc portion of IgG (FcγRs) are expressed on various leukocytes and they modulate both humoral and cell-mediated immune responses with different capacities for IgG binding and phagocytosis. Four different types of FcγRs, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16) and FcγRIV, have been identified. There are three FcγRII isoforms (activating FcγRIIa and FcγRIIc, and inhibitory FcγRIIb) in humans, one isoform (inhibitory FcγRIIb) in mice, and two isoforms (inhibitory FcγRIIb and activating FcγRIIc) in cattle. Two alternativly spliced isoforms of FcγRIIb, b1 and b2, have been identified in humans, mice and cattle, however, only two porcine FcγRIIb transcripts have been reported. In this study, we report the identification of three new porcine FcγRIIb transcript and analyze the sequences of five porcine FcγRIIb transcript generated by alternative splicing. The porcine transcript 1 and porcine transcript 2 have a high homology and structural similarity with human b1 and b2, respectively, while there is only one alanine residue difference at the signal peptide region between porcine transcript 1 and transcript 4, as well as porcine transcript 2 and transcript 3. This is the first time that an alternativly spliced isoform of porcine transcript 5 is described in pigs rather than humans or other animals. All the five transcripts have the consensus sequence of an ITIM (ITYSLL) in their cytoplasmic tails. Analysis results indicate that the five transcripts serve as inhibitory receptors and are these sub-isoforms or alternativly spliced isoforms. Immunoglobulin-binding assays show that transcript 1, transcript 2, transcript 3 and transcript 4 have binding activity for IgG immune complexes, whereas transcripts 5 without domain 2 can not bind IgG-complexes. It is now clear that porcine FcγRIIb exists as five sub-isoforms at least. These sub-isoforms may individually modulate FcγRIIb-mediated immune responses in the porcine immune system.

Genetic diversity and phylogenetic analysis of the ORF5 gene of PRRSV from central China

To more fully understand the genetic diversity and molecular epidemiology of prevailing porcine reproductive and respiratory syndrome virus (PRRSV) in Henan province of China, 112 full-length ORF5 gene sequences, originating from Henan province between 2006 and 2015, were subjected to sequence variation and phylogenetic analysis. Phylogenetic analysis revealed that all Henan isolates belonged to the Type 2 genotype and could be further divided into three subgroups. Subgroup 1 and 2 viruses predominated in Henan and subgroup 2 overtook subgroup 1 as the most prevalent PRRSV between 2006 and 2015. Highly pathogenic PRRSV (HP-PRRSV) isolates predominated in Henan and eight RespPRRSV MLV vaccine-like isolates were observed in subgroup 3. Sequence variation analysis revealed that the ORF5 genes of all Henan isolates shared >83.3% nucleotide and >80.1% amino acid sequence identity with each other. Primary neutralizing epitope (PNE) analysis revealed that, relative to the attenuated RespPRRSV MLV vaccine isolate, all but one of the subgroup 1 Henan isolates had mutations at amino acid 39 within the key PNE of GP5. Analysis of the immunoreceptor tyrosine-based inhibitory motif (ITIM) in GP5 revealed that all but two of the Henan isolates had a highly conserved sequence between amino acids 77 and 82 positions of GP5. N-linked glycosylation site (NGS) analysis revealed a novel potential NGS at GP5 amino acid position 59 in two of the subgroup 2 Henan isolates. Another novel GP5 amino acid mutation (44N→44D) was found in a single subgroup 1 Henan isolate (HeNan-A9) in a glycosylation site that is known to be crucial for PRRSV infectivity.

Ligation of porcine Fc gamma receptor I inhibits levels of antiviral cytokine in response to PRRSV infection in vitro.

PRRSV infection ADE facilitates the attachment and internalization of the virus onto macrophages through Fc receptor-mediated endocytosis. FcγRI is the activating receptor with a tyrosine-based activating motif (ITAM) in its cytoplasmic tail, where up-regulates phagocytosis. However, porcine FcγRIs role in the antiviral immune response to PRRSV infection has not been studied. In this study, our results indicated that selective activation of porcine FcγRI in PAM cells down-regulated significantly mRNA levels of IFN-α and TNF-α post-pretreatment, suggesting that porcine FcγRI signal can inhibit the innate antiviral response of host cells. PRRSV infection assay mediated by FcγRI indicated that selective activation of porcine FcγRI in PAM cells inhibited significantly mRNA levels of antiviral cytokine (IFN-α and TNF-α) in response to PRRSV infection, suggesting that FcγRI ligation can inhibit the antiviral immune response to PRRSV infection.

Ligation of porcine Fc gamma receptor III inhibits levels of antiviral cytokine in response to PRRSV infection in vitro.

PRRSV infection ADE facilitates the attachment and internalization of the virus onto macrophages through Fc receptor-mediated endocytosis. FcγR III is the activating receptor with a tyrosine-based activating motif (ITAM) in its cytoplasmic tail, where up-regulates phagocytosis. However, porcine FcγR IIIs role in the antiviral immune response to PRRSV infection has not been studied. In this study, our results indicated that selective activation of porcine FcγR III in PAM cells down-regulated significantly mRNA levels of IFN-α and TNF-α post-pretreatment, and up-regulated significantly mRNA level of IL-10 post-pretreatment, suggesting that porcine FcγR III signal can inhibit the transcriptional levels of innate antiviral cytokine in host cells. Simultaneously, PRRSV infection assay mediated by FcγR III indicated that selective activation of porcine FcγR III in PAM cells inhibited significantly mRNA levels of IFN-α and TNF-α, and enhanced significantly mRNA level of IL-10, and increased significantly viral mRNA levels, in response to PRRSV infection, suggesting that FcγR III ligation can inhibit the antiviral immune response to PRRSV infection. These results elucidated that the one mechanism of PRRSV-ADE regulated via porcine FcγRIII may be by decreasing antiviral cytokine levels, facilitating viral replication.