Baoan Cui

Construction and immunogenicity of a recombinant fowlpox vaccine coexpressing S1 glycoprotein of infectious bronchitis virus and chicken IL-18.

Abstract Infectious bronchitis virus (IBV) poses a major threat to the chicken industry worldwide. In this study, we developed a recombinant fowlpox virus (rFPV) vaccine expressing the IBV S1 gene and chicken interleukin-18 gene (IL-18), rFPV-S1/IL18. Recombinant plasmid pSY-S1/IL18 was constructed by cloning chicken IL-18 into fowlpox virus transfer plasmid containing S1 gene and transfected into the chicken embryo fibroblasts cell pre-infected with S-FPV-017 to generate rFPV-S1/IL18. Expression of the recombinant proteins was confirmed by RT-PCR and IFA. We also constructed the recombinant fowlpox virus rFPV-S1 without IL-18. One-day-old chickens were vaccinated by wing-web puncture with the two rFPVs, and the induced humoral and cellular responses were evaluated. There was a significant difference in ELISA antibody levels (P <0.05) elicited by either rFPV-S1 or rFPV-S1/IL18. The ratios of CD4+ to CD8+ in chickens immunized with rFPV-S1/IL18 were significantly higher (P <0.05) than in those immunized with rFPV-S1. All chickens immunized with rFPV-S1/IL18 were completely protected (20/20) after challenge with the virulent IBV HN99 strain 43 days after immunization, while only 15 out of 20 of the chickens immunized with the rFPV-S1 were protected. Our results show that the protective efficacy of the rFPV-S1 vaccine could be enhanced significantly by simultaneous expression of IL-18.

Simultaneous detection of porcine parvovirus and porcine circovirus type 2 by duplex real-time PCR and amplicon melting curve analysis using SYBR Green

The development of a SYBR Green-based duplex real-time PCR is described for simultaneous detection of porcine parvovirus (PPV) and porcine circovirus type 2 (PCV-2) genomes. Viral genomes were identified in the same sample by their distinctive melting temperature (T(m)) which is 77.5°C for PPV VP2 313bp amplicon and 82.3°C for PCV-2 ORF2 171bp amplicon, respectively. The detection limit of the method was 0.01TCID(50)/mL for PPV and PCV-2, about 10 times more sensitive than conventional PCR. In addition, PPV and PCV-2 viral load were measured in 126 field samples, confirming the sensitivity and specificity, and the result showed that 70/126 samples were positive for PPV and 92/126 samples were positive for PCV2 by the duplex real-time PCR. This method may be a useful alternative rapid and reliable method for the detection of PPV/PCV-2 co-infection.

Immune responses of chickens inoculated with a recombinant fowlpox vaccine coexpressing HA of H9N2 avain influenza virus and chicken IL-18

Control of the circulation of H9N2 avian influenza virus (AIV) is a major concern for both animal and public health, and H9N2 AIV poses a major threat to the chicken industry worldwide. Here, we developed a recombinant fowlpox virus (rFPV-HA) expressing the haemagglutinin (HA) gene of the A/CH/JY/1/05 (H9N2) influenza virus and a recombinant fowlpox virus (rFPV-HA/IL18) expressing the HA gene and chicken interleukin-18 (IL-18) gene. Recombinant plasmid pSY-HA/IL18 was constructed by cloning chicken IL-18 expression cassette into recombinant plasmid pSY-HA containing the HA gene. Two rFPVs were generated by transfecting two recombinant plasmids into the chicken embryo fibroblast cells pre-infected with S-FPV-017, and assessed for their immunological efficacy on one-day-old White Leghorn specific-pathogen-free chickens challenged with the A/CH/JY/1/05 (H9N2) strain. There was a significant difference in HI antibody levels (P<0.05) elicited by either rFPV-HA or rFPV-HA/IL18. The level of splenocyte proliferation response in the rFPV-HA/IL18-vaccinated group was significantly higher (P<0.05) than that in the rFPV-HA group. After challenge with 10(6.5)ELD(50) H9N2 AIV 43days after immunization, rFPVs vaccinated groups could prevent virus shedding and replication in multiple organs in response to H9N2 AIV infection, and rFPV-HA/IL18 vaccinated group had better inhibition of viruses than rFPV-HA vaccinated group. Our results show that the protective efficacy of the rFPV-HA vaccine could be enhanced significantly by simultaneous expression of IL-18.

A TaqMan-based real-time polymerase chain reaction for the detection of porcine parvovirus.

A real-time polymerase chain reaction (PCR) using a TaqMan probe was developed to detect porcine parvovirus (PPV). Real-time PCR was optimized to quantify PPV using a detection system (Rotor Gene 2000 detector) and a dual-labeled fluorogenic probe. The gene-specific labeled fluorogenic probe for the VP2 gene of PPV was used to detect PPV. Quantitation of PPV was accomplished by a standard curve plotting cycle threshold values (Ct) against each dilution of standard plasmids. When the specificity of the assay using specific PPV primers was evaluated by testing the PPV standard strain and other viruses, no cross-reactions were detected with non-PPV reference viruses. The detection limit of real-time PCR for PPV was 2.08log10 genome copy equivalent (gce). In this study, a real-time PCR assay was performed on 80 clinical samples and compared with a conventional PCR assay. In 48 of 80 samples, PPV DNA was detected by the conventional PCR assay. All samples positive for PPV DNA by the conventional PCR assay were also positive by the real-time PCR assay, and 12 of 32 samples that tested negative for PPV DNA by the conventional method tested positive by the real-time PCR assay. Using the real-time PCR assay, the number of samples in which PPV was detected increased by 15%. Therefore, it is considered to be a useful tool for the detection of PPV.

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.

Immune responses of chickens inoculated with a recombinant fowlpox vaccine coexpressing glycoprotein B of infectious laryngotracheitis virus and chicken IL-18

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes severe and economically significant respiratory disease in poultry worldwide. Herein, the immunogenicity of two recombinant fowlpox viruses (rFPV-gB and rFPV-gB/IL18) containing ILTV glycoprotein B (gB) and chicken interleukin-18 (IL-18) were investigated in a challenge model. One-day-old specific-pathogen-free chickens were vaccinated by wing-web puncture with the two rFPVs and challenged with the virulent ILTV CG strain. There were differences in antibody levels elicited by either rFPV-gB/IL18 or rFPV-gB as determined using ELISA. The ratios of CD4(+) to CD8(+) in chickens immunized with rFPV-gB/IL18 were higher (P < 0.05) than in those immunized with rFPV-gB, and the level of proliferative response of the T cells in the rFPV-gB/IL18-vaccinated group was higher (P < 0.05) than that in the rFPV-gB group. All chickens immunized with rFPV-gB/IL18 were protected (10/10), whereas only eight of 10 of the chickens immunized with the rFPV-gB were protected. The results showed that the protective efficacy of the rFPV-gB vaccine could be enhanced by simultaneous expression of chicken IL-18.

Inhibitory effects of indigowoad root polysaccharides on porcine reproductive and respiratory syndrome virus replication in vitro.

BACKGROUND Indigowoad root polysaccharide (IRPS) is a natural polysaccharide isolated from the traditional Chinese medicinal herb Radix Isatidis, and has many kinds of biological activities. However, the IRPS antiviral activity, especially the anti-porcine reproductive and respiratory syndrome virus (PRRSV) effect, has not been evaluated. METHODS PRRSV was propagated in the MARC-145 cell line, and viral titre was determined by cytopathic effect and expressed as the 50% tissue culture infection dose (TCID(50)) in the current study. The cell cytotoxic effect of IRPS toward MARC-145 was evaluated by MTT assay firstly, then the inhibitory effects of IRPS on PRRSV replication in vitro were investigated by determining the effect of IRPS upon a single replicative cycle of PRRSV in MARC-145 cells. The effects of IRPS on viral RNA and protein synthesis in PRRSV-infected cells were investigated using real-time PCR and double-antibody (sandwich) ELISA. RESULTS IRPS was able to effectively suppress the infectivity of the PRRSV in a dose-dependent manner, especially by adding IRPS during the PRRSV infection. IRPS could affect the attachment of PRRSV to MARC-145 cells, and also inhibit the viral RNA and protein synthesis. CONCLUSIONS IRPS has an antiviral effect on PRRSV replication in MARC-145 cells and might be useful in medical development for antiviral research. However, the precise mechanism of the host and viral targets of IRPS are unknown, so further studies should be conducted to investigate the precise mechanism of IRPS inhibitory effect on PRRSV infection.

Genomic sequence analysis of a new reassortant infectious bursal disease virus from commercial broiler flocks in central China

We report the complete nucleotide sequence of a reassortant infectious bursal disease (IBD) virus (IBDV) HN isolate from commercial broiler flocks in central China. The genome consisted of 3,232 and 2,652 nucleotides in the coding regions of segments A and B, respectively. Alignment of both nucleotide and deduced amino acid sequences and phylogenetic analysis revealed that the genome segments A and B of HN were derived from the attenuated strain B87 and the VV strain OKYM. This is a new reassortant IBDV strain that has emerged in nature, involving segment A of a cell-culture-adapted attenuated vaccine strain B87.

Interleukin-18-mediated enhancement of the protective effect of an infectious laryngotracheitis virus glycoprotein B plasmid DNA vaccine in chickens

The immunogenicity of an infectious laryngotracheitis virus (ILTV) glycoprotein B (gB) plasmid DNA vaccine and the immunoregulatory activity of chicken interleukin-18 (IL-18) were investigated in a challenge model. Two recombinant plasmids, pcDNA3.1/gB (pgB) and pcDNA3.1/IL-18 (pIL-18), containing gB and IL-18 were constructed. Chickens were intramuscularly administered two immunizations 2 weeks apart, and challenged with the virulent CG strain of ILTV 2 weeks later. All animals vaccinated with pgB alone or with a combination of pgB plus pIL-18 developed a specific anti-ILTV ELISA antibody and splenocyte proliferation response. The ratios of CD4(+) to CD8(+) T lymphocytes in chickens immunized with pgB plus pIL-18 were significantly higher than in those immunized with pgB alone. Co-injection of pIL-18 significantly increased the production of gamma interferon and IL-2, indicating that IL-18 enhances the T helper 1-dominant immune response. Challenge experiments showed that the morbidity rate in the pgB group (25  %) was significantly higher than that in the pgB plus pIL-18 group (10  %). The mortality rates in the pgB and pgB plus pIL-18 groups were 10 and 0 %, respectively, and the corresponding protection rates were 60 and 80  %. These results indicate that IL-18 may be an effective adjuvant for an ILTV vaccine.

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.