[Show abstract][Hide abstract] ABSTRACT: Highly pathogenic (HP)-porcine reproductive and respiratory syndrome virus (PRRSV) emerged in 2006 and has now become a global threat topig farms. Despiteextensive characterization of HP-PRRSV proteins by direct analysis and comparison with typical PRRSV, immune recognition remainpoorly understood.Glycosylated protein 3 (GP3) has an important functionin inducing protective immune response. To analyze theantigenic character of HP-PRRSV GP3, a total of 217 peptides were printed on a chipand used to react withHP-PRRSV specific serum. The reactions of these peptides toHP-PRRSVspecificpig serum were scanned and quantified usingthe software PepSlide® Analyzerby fluorescence intensity.The intensity plots showedvariousreactions in different parts of GP3. The highest reaction intensity value reached 29,184.5with thepeptide sequence ofCSENDHDELGFMVPP. Conversely, 88 peptides showed no reactionwith 0 florescence intensity. A further analysis based on the result of the peptide microarrayrevealed anantigen reaction active region (AR)from Y(51) to S(106) in GP3. The ARhadfourparts of variation thatmay be a significantmutation of the typical PRRSV to HP-PRRSV. Acquireddata may be usefulfor understandingHP-PRRSV variation and its GP3 immune recognition.
[Show abstract][Hide abstract] ABSTRACT: Investigation of a serious pig disease with high mortality and typical lung lesions yielded a bacterial isolate identified as Providencia alcalifaciens based on the 16S ribosomal DNA sequence analysis. The pathogenicity of this bacterial isolate was confirmed in piglets and mice. The bacterial strain caused the typical illness in piglets, which suffered serious dyspnea and hemorrhagic pneumonia. The drug resistance spectrum of the bacterium was also determined. The results indicated that the isolate is resistant to 12 antibiotics and intermediately resistant to 10 antibiotics out of the 34 antibiotics tested. The current study is the first to report a serious lung disease in piglets caused by a multidrug resistant P. alcalifaciens isolate, which should be given more attention during surveillance and diagnostics.
Full-text · Article · Oct 2013 · Current Microbiology
[Show abstract][Hide abstract] ABSTRACT: Nanobodies (or variable domain of the heavy chain of the heavy-chain antibodies, VHHs) are single-domain antigen-binding fragments derived from camelid heavy chain antibodies. Their comparatively small size, monomeric behavior, high stability, high solubility, and ability to bind epitopes inaccessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. In this paper, for the first time, we created the immunized VHH yeast two-hybrid (Y2H) library according to the Clontech Mate & Plate library construction system. The transformation efficiency and titer of the VHH Y2H library were 7.26×10 cfu/3 µg and 2×10 cfu/ml, which met the demand for Y2H library screening. Using as an example the porcine circovirus type 2 (PCV2) Cap protein as bait, we screened 21 positive Cap-specific VHH sequences. Among these sequences, 7 of 9 randomly selected clones were strongly positive as indicated by enzyme-linked immunosorbent assay, either using PCV2 viral lysis or purified Cap protein as coated antigen. Additionally, the immunocytochemistry results further indicated that the screened VHHs could specifically detected PCV2 in the infected cells. All this suggests the feasibility of VHH throughput screening based on Y2H strategy.
[Show abstract][Hide abstract] ABSTRACT: Detection of homologous recombinant rate and diversity of Camelus Bactrianus VHH yeast library by PCR amplification and independent colonies calculation. A, 10−6 dilution plating of the cultured library indicated a total dimension (or complexity) of 2×109 clones. B, 20 clones were randomly picked to determine the library functional diversity by PCR using universal primers T7 and 3′AD (Table 1). Meanwhile, sterile water and pGADT7-Rec vector templates were used as negative controls. All the clones have amplified the 400 bp VHH fragments (lane 1–20), while negative templates control haven’t amplified any bands (lane N1, N2). M indicated the DL2000 DNA marker.
[Show abstract][Hide abstract] ABSTRACT: The use of attenuated Salmonella typhimurium as a bactofection vehicle for the oral delivery of a DNA vaccine against rabbit haemorrhagic disease virus (RHDV) was investigated. The DNA vaccine plasmid pcDNA3.1-VP60, which encodes the viral capsid protein VP60, was transformed into the attenuated S. typhimurium strain SL7207. The resulting recombinant bacteria, named as SL/pcDNA3.1-VP60, were orally used to immunise rabbits. The successful delivery of the DNA plasmid was confirmed by the detected VP60 transcription in the rabbit intestines through the reverse transcription polymerase chain reaction. In addition, the RHDV-specific humoral and cell-mediated immune response that was induced by SL/pcDNA3.1-VP60 was detected by the enzyme-linked immunosorbent assay as well as the assays for T lymphocyte proliferation and cytokines secretion. The significant protection of immunised rabbits against the RHDV strain XA/China/2010 at 42 d post-immunisation was demonstrated. This study is the first report about the efficient usage of attenuated Salmonella as a live vector for the oral delivery of a DNA vaccine against RHDV.
No preview · Article · Dec 2012 · Journal of virological methods
[Show abstract][Hide abstract] ABSTRACT: This study aimed to investigate rabbit hemorrhagic disease virus (RHDV) in China. VP60 sequences of five RHDVs collected by our team, as well as those of 16 other published Chinese RHDV strains, were analyzed. Polygenic analysis using MEGA 4 software showed that 20 of the 21 Chinese strains could be clustered in the RHDVa subgroup, and WX/China/1984 was different from them. The Chinese RHDV strains were further classified into four subgroups, CH1 to CH4. Subgroup CH1, represented by the WX/China/1984 strain, was not prevalent in China after the first RHDV epidemic strain was reported. The CH2, CH3, and CH4 subgroups were far different from the CH1 subgroup, formed three separate clusters, and were distributed according to the time the strains were collected. Recently collected strains formed a new subgroup (CH4), represented by new RHDV varieties identified by challenging immunized rabbits and by comparison of genomic sequences. The present work is the first comprehensive analysis of Chinese RHDV and reveals a new RHDV variation that should be carefully monitored.
Full-text · Article · May 2012 · Archives of Virology