[Immune efficacy in mice by recombinant pseudorabies virus PGO expressing ORF2 gene of porcine circovirus type 2].

OBJECTIVE: We developed a recombinant pseudorabies virus (PRV) vaccine against porcine circovirus type 2 (PCV2). METHODS: PCV2 ORF2 gene was inserted into vector pG to produce the recombinant PRV vector pGO; the genome of PRV attenuated vaccine and the transfer plasmid pGO were transfected by using Lipofectamine 2000 Reagent into swine testis cells for homologous recombination to obtain the recombinant PRV. Six-week-old female Kunming mice were immunized two intramuscular immunizations 4 weeks apart, and then challenged with the virulent PCV2 NY strain at 8 weeks after the first immunization. RESULTS: A recombinant PRV expressing PCV2 ORF2 was successfully constructed, and named PGO. There was a low ELISA antibody level of PCV2-specific humoral immune response elicited by recombinant virus PGO for the first immunization but high significantly for the second immunization. PCV2 antigen-specific T-cell proliferative responses can be elicited by immunization with recombinant virus. Challenge experiments show that the recombinant virus and PCV2 inactivated vaccine could both protect the mice against PCV2 challenge, suggesting that the recombinant virus can be an excellent potential vaccine. CONCLUSION: The results show the recombinant PRV expressing PCV2 ORF2 had good immunogenicity.

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.

Molecular detection and genomic characterization of Torque teno sus virus 1 and 2 from domestic pigs in central China.

In the present study, Torque teno sus viruses (TTSuVs) were detected in tissue and blood samples obtained from domestic pigs in central China, and complete genomes of TTSuVs were characterized. A total of three tissue samples (3/20, 15 %) from post-weaning multisystemic wasting syndrome-affected pigs and 30 blood samples (30/40, 75 %) from healthy pigs were positive for Torque teno sus virus 1 (TTSuV1) and/or 2 (TTSuV2). Two TTSuV strains (TTV1Hn54 and TTV2Hn93) comprising 2,794 and 2,875 nucleotides, respectively, each had four open reading frames (ORFs) and the untranslated region with TATA box and GC-rich region. Genomic sequence of TTV2Hn93 strain was unique in length compared with other TTSuV2 genomic sequences. Interestingly, three rolling-circle replication (RCR) motif-IIIs (YXXK) which were located at amino acid (aa) position 166-169, 328-331, and 379-382, respectively, were found in the ORF1 of TTV1Hn54. Two RCR motif-IIIs (YXXK) at the aa position 105-108 and 480-483 respectively, were also identified in the ORF1 of TTV2Hn93. Phylogenetic tree based on complete genomes showed that TTV1Hn54 strain was designated into type TTSuV1b and had a slight high sequence identity of 91 % with the Canada strain (JQ120664). TTV2Hn93 strain was classified into subtype TTSuV2d and shared the highest identity (97 %) with the Spain strain (GU570207).

Genetic variation and phylogenetic analysis of porcine circovirus type 2 infections in central China.

Porcine circovirus type 2 (PCV2) infection causes postweaning multisystemic wasting syndrome and porcine circovirus-associated diseases in many regions. A total of 77 sequences, including 31 sampled from Henan province of China, were retrieved from GenBank and subjected to amino acid variation and phylogenetic analyses. The two PCV genotypes prevailing in Henan were PCV-2a and PCV-2b with PCV-2b accounting for 93.5 % (29/31) of the Henan isolates. The 31 Henan isolates all shared between 92.7 and 100 % sequence similarity. Amino acid variation analysis of the capsid protein revealed that Henan PCV2 strains tended to accumulate more substitutions within epitopic regions-a substitution pattern consistent with host immune system-mediated selection of virus immune escape variants. The analysed PCV sequences carry evidence of at least six unique recombination events. Selective pressure analysis of the relative recombination-free ORF2 sequences of these viruses revealed evidence of sites that are likely evolving in response to host-driven immune pressures-a finding that coupled with information on the prevalent diversity in Henan PCV2 isolates of known immunoreactive genomic loci will aid in future studies aiming to assess the evolutionary responses of PCV2 in China to the widespread deployment of anti-PCV vaccines in the country.

The controlled release of tilmicosin from silica nanoparticles.

The aim of this study was to use silica nanoparticles as the carrier for controlled release of tilmicosin. Tilmicosin was selected as a drug model molecule because it has a lengthy elimination half-life and a high concentration in milk after subcutaneous administration. Three samples of tilmicosin-loaded silica nanoparticles were prepared with different drug-loading weight. The drug-loading weight in three samples, as measured by thermal gravimetric analysis, was 29%, 42%, and 64%, respectively. With increased drug-loading weight, the average diameter of the drug-loaded silica nanoparticles was increased from 13.4 to 25.7 nm, and the zeta potential changed from-30.62 to-6.78 mV, indicating that the stability of the drug-loaded particles in the aqueous solution decreases as drug-loading weight increases. In vitro release studies in phosphate-buffered saline showed the sample with 29% drug loading had a slow and sustained drug release, reaching 44% after 72 h. The release rate rose with increased drug-loading weight; therefore, the release of tilmicosin from silica nanoparticles was well-controlled by adjusting the drug loading. Finally, kinetics analysis suggested that drug released from silica nanoparticles was mainly a diffusion-controlled process.

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.

Nitric oxide inhibits the replication cycle of porcine parvovirus in vitro.

This study investigated the inhibitory effect and mechanism of nitric oxide (NO) on porcine parvovirus (PPV) replication in PK-15 cells. The results showed that two NO-generating compounds, S-nitroso-L-acetylpenicillamine (SNAP) and L-arginine (LA), at a noncytotoxic concentration could reduce PPV replication in a dose-dependent manner and that this anti-PPV effect could be reversed by the NO synthase (NOS) inhibitor N-nitro-L-arginine methyl ester (L-NAME). By assaying the steps of the PPV life cycle, we also show that NO inhibits viral DNA and protein synthesis. This experiment provides a frame of reference for the study of the anti-viral mechanism of NO.

[Transcriptional profiles of intestinal pig epithelial cell jejenum 2 cultures infected by vesicular stomatitis virus].

OBJECTIVE: To better understand the host inflammatory responses, in particular inflammatory cytokines responses of Vesicular Stomatitis virus (VSV) infection, and the host-VSV interaction. METHODS: We used VSV Indian strain to infect the Intestinal Pig Epithelial Cell Jejenum (IPEC-J2) cell. Then we measured and analyzed the viral RNA by using real-time PCR. The transcript level of cytokines (IL-2, 6, 8, 10,12, IFN-alpha, IFN-gamma, TNF-alpha, TGF-beta and TLR3) were detected by real-time PCR. RESULTS: We found that the transcript levels of IL-6, IL-8, IL-12, TNF-alpha and TLR3 were increased obviously whereas TGF-beta showed no significant difference. TPEC-J2 cell did not secrete IL-2, IL-10, IFN-alpha and IFN-gamma. CONCLUSION: The level of inflammatory response was increased when VSV infected TPEC-J2 cell.

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.

[Construction of recombinant fowlpox virus coexpressing HA from subtype H5 of avian influenza virus and chicken interleukin-18].

OBJECTIVE: We developed recombinant fowlpox viruses (rFPV) coexpressing chicken IL-18 and H5 AIV HA. METHODS: Recombinant expression plasmid pSYHA/IL-18 was constructed by cloning chicken IL-18 into transfer plasmid containing HA gene and transfected by lipofectamine on the chicken embryo fibroblasts cell (CEF) pre-infected with S-FPV-017. By selecting blue plaques on the CEF overlaid with agar containing X-gal, recombinants fowlpox virus rFPV-HA-IL-18 were obtained, and identified by PCR. RESULTS: The recombinant fowlpox viruses contained chicken IL-18 and HA gene and had stable genetic properties. The expression of HA was detected in the recombinant virus-infected CEF by indirect immunofluorescence using antibody against AIV. The expression of chicken IL-18 was detected by MTT in the recombinant virus-infected CEF fluid. The chickens vaccinated with recombinant fowlpox virus rFPV-HA-IL-18 and rFPV-HA had detectable hemagglutination inhibition (HI) antibody at 7 days post-vaccination, and HI antibody titers rose to peak at 14 days post-vaccination. No HI antibody was detected in the control or fowlpox virus immunized chickens before or after immunization. The chickens vaccinated with rFPV-HA-IL-18 had higher HI antibody titers than the chickens vaccinated with rFPV-HA. CONCLUSION: Development of recombinant fowlpox virus (rFPV-HA-IL-18) had strong biological activity.

[Prokaryotice expression of the NS1 gene of PPV and renaturation of the recombinant protein].

The antigen of NS1 gene of PPV was amplified by PCR, and the amplified fragments were cloned into the prokaryotic expression vector pGEX-4T-1. The insert position, the size and the frame were identified by PCR, restriction enzyme digestion and the sequence analysis of the recombinant plasmids. The sequence analysis results of pGEX-NS1-HN1 showed that the prokaryotic expression vector was successfully constructed. The target gene was successfully expressed in the host cell BL21 when induced with IPTG. The expression was optimized with proper inducing conditions of 1.0mmol/L IPTG, 10 hours and 37 degree C induction. The expression of the target protein added up to 29.8% of the total bacterial protein. The results of SDS-PAGE indicated that molecular weight of the expressed protein was about 52kDa and the expressed protein mainly existed in the inclusion body. Western blot analysis proved the recombinant protein has good reactive ability against PPV positive serum. The pGEX-NS1-HN1 inclusion body was dissolved with 8mol/L urea. Then the expressed protein was renatured by dilution method and the systems of GSH and GSSG. ELISA detection proved the renaturation protein has good biological activity.

Construction and immunogenicity of a recombinant pseudorabies virus co-expressing porcine circovirus type 2 capsid protein and interleukin 18.

A novel recombinant pseudorabies virus (PRV) expressing porcine circovirus type 2 (PCV2) capsid protein and IL-18 was constructed. The PCV2 open reading frame 2 (ORF2) and porcine IL-18 genes were amplified by PCR and then inserted into the PRV transfer vector (pG) to produce a recombinant plasmid (pGO18). Plasmid pGO18 was transfected into porcine kidney cell (PK15) pre-infected with PRV HB98 vaccine strain to generate a recombinant virus. The recombinant virus PRV-ORF2-IL18 was purified by green fluorescent plaque purification and the inserts were confirmed by PCR, enzyme digestion, sequencing, and Western blot. The humoral and cellular responses induced by the recombinant virus were assessed in mice. Mice (n=10) were immunized with PRV-ORF2-IL18, PRV-ORF2, PRV HB98, or inactivated PCV2. PRV-ORF2-IL18 elicited high titers of ELISA and serum neutralizing antibodies and strong cell-mediated immune responses in mice as indicated by anti-PCV2 ELISA, PRV-neutralizing assay, PCV2 specific lymphocyte proliferation assay, CD3(+), CD4(+) and CD8(+) T lymphocytes analysis, respectively. And PRV-ORF2-IL18 immunization protected mice against a lethal challenge of a virulent PRV Fa strain and significantly reduced the amount of PCV2 viremia. These results suggest an adjuvant effect of IL-18 on cellular immune responses. The recombinant virus might be an attractive candidate vaccine for preventing PCV2 and PRV infections in pigs.

Complete genome sequence of a porcine parvovirus strain isolated in central china.

We report here the complete genome sequence of the porcine parvovirus (PPV) strain J-PPV, isolated from central China. Our data, together with sequence data for PPV isolates from other regions of China, will help in understanding the epidemiology and molecular characteristics of PPV field isolates in China.

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 FcγRI's 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.

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.

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.

Ligation of Fc gamma receptor IIB enhances levels of antiviral cytokine in response to PRRSV infection in vitro.

PRRSV infection ADE facilitates the attachment and internalization of the virus onto its host cells, such as monocytes and macrophages, through Fc receptor-mediated endocytosis. FcγRIIB is the only inhibitory receptor with a tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail, where counters the "ITAM triggered" activation signals and down-regulates phagocytosis. However, porcine FcγRIIB's role in the antiviral immune response to PRRSV infection has not been studied. In this study, our results indicated that selective activation of porcine FcγRIIB in PAM cells up-regulated significantly mRNA levels of IFN-α and TNF-α at any time point post-pretreatment, suggesting that porcine FcγRIIB signal can enhance the innate antiviral response of host cells. PRRSV infection assay mediated by FcγRIIB indicated that selective activation of porcine FcγRIIB in PAM cells enhanced mRNA levels of antiviral cytokine (IFN-α and TNF-α) and repressed mRNA levels of IL-10 in response to PRRSV infection, suggesting that FcγRIIB ligation can enhance the antiviral immune response to PRRSV infection. In addition, FcγRIIB ligation to infection indicated that PRRSV replication in PAM was not positive correlation with increasing of IFN-α mRNA levels and decreasing of IL-10 mRNA levels, suggesting that there is complex viral replication mechanism in immune cells such as PAM for PRRSV.

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.

Enhancing immune responses to inactivated porcine parvovirus oil emulsion vaccine by co-inoculating porcine transfer factor in mice.

Inactivated porcine parvovirus (PPV) vaccines are available commercially and widely used in the breeding herds. However, inactivated PPV vaccines have deficiencies in induction of specific cellular immune response. Transfer factor (TF) is a material that obtained from the leukocytes, and is a novel immune-stimulatory reagent that as a modulator of the immune system. In this study, the immunogenicity of PPV oil emulsion vaccine and the immuno-regulatory activities of TF were investigated. The inactivated PPV oil emulsion vaccines with or without TF were inoculated into BALB/c mice by subcutaneous injection. Then humoral and cellular immune responses were evaluated by indirect enzyme-linked immunosorbent assays (ELISA), fluorescence-activated cell sorter analyses (FACS). The results showed that the PPV specific immune responses could be evoked in mice by inoculating with PPV oil emulsion vaccine alone or by co-inoculation with TF. The cellular immune response levels in the co-inoculation groups were higher than those groups receiving the PPV oil emulsion vaccine alone, with the phenomena of higher level of IFN-γ, a little IL-6 and a trace of IL-4 in serum, and a vigorous T-cell response. However, there was no significant difference in antibody titers between TF synergy inactivated vaccine and the inactivated vaccine group (P>0.05). In conclusion, these results suggest that TF possess better cellular immune-enhancing capability and would be exploited into an effective immune-adjuvant for inactivated vaccines.