[Show abstract][Hide abstract] ABSTRACT: Brucella melitensis causes Brucellosis, a disease affecting sheep, cattle, and sometimes humans. Attenuated B. melitensis M5-90, derived from virulent strain M28, is widely used as a live vaccine in ruminants in China. Genetic differences between the strains would cast light on the mechanism of attenuation. We recently reported the complete genomic sequences of M28 and M5-90. Genome organization is highly conserved between these isolates, and also with virulent strains 16M and ATCC 23457. Analysis revealed 23 open reading frames (ORFs) with consistent differences between M5-90 and the virulent strains. Notably, the tuf2 gene encoding translation elongation factor EF-Tu from M50-90 contained 50 SNPs and 9 gaps (indels) compared to tuf2 of M28 or of the other virulent strains. There were no changes in tuf1. To evaluate the potential role of EF-Tu in pathogenesis, tuf1/tuf2 mutants of M28 and an M5-90 strain harboring wild type tuf2 were constructed and their virulence/attenuation evaluated in vivo. We report that the tuf2 gene plays an important role in the attenuation of M5-90.
Infection and immunity 05/2013; DOI:10.1128/IAI.00224-13 · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Brucella melitensis is an important zoonotic pathogen that causes brucellosis, a disease that affects sheep, cattle and occasionally humans. B. melitensis strain M5-90, a live attenuated vaccine cultured from B. melitensis strain M28, has been used as an effective tool in the control of brucellosis in goats and sheep in China. However, the molecular changes leading to attenuated virulence and pathogenicity in B. melitensis remain poorly understood. In this study we employed the Illumina Genome Analyzer platform to perform genome-wide digital gene expression (DGE) analysis of mouse peritoneal macrophage responses to B. melitensis infection. Many parallel changes in gene expression profiles were observed in M28- and M5-90-infected macrophages, suggesting that they employ similar survival strategies, notably the induction of anti-inflammatory and antiapoptotic factors. Moreover, 1019 differentially expressed macrophage transcripts were identified 4 h after infection with the different B. melitensis strains, and these differential transcripts notably identified genes involved in the lysosome and mitogen-activated protein kinase (MAPK) pathways. Further analysis employed gene ontology (GO) analysis: high-enrichment GOs identified endocytosis, inflammatory, apoptosis, and transport pathways. Path-Net and Signal-Net analysis highlighted the MAPK pathway as the key regulatory pathway. Moreover, the key differentially expressed genes of the significant pathways were apoptosis-related. These findings demonstrate previously unrecognized changes in gene transcription that are associated with B. melitensis infection of macrophages, and the central signaling pathways identified here merit further investigation. Our data provide new insights into the molecular attenuation mechanism of strain M5-90 and will facilitate the generation of new attenuated vaccine strains with enhanced efficacy.
PLoS ONE 12/2011; 6(12):e28485. DOI:10.1371/journal.pone.0028485 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Brucella melitensis is a Gram-negative coccobacillus bacteria belonging to the Alphaproteobacteria subclass. It is an important zoonotic pathogen that causes brucellosis, a disease affecting sheep, cattle, and sometimes humans. The B. melitensis strain M5-90, a live attenuated vaccine cultured from the B. melitensis virulent strain M28, has been an effective tool to control brucellosis in goats and sheep in China. Here we report the complete genome sequences of B. melitensis M28 and M5-90, strains with different virulence backgrounds, which will serve as a valuable reference for future studies.
Journal of bacteriology 06/2011; 193(11):2904-5. DOI:10.1128/JB.00357-11 · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A CHO cell clone (CHO-PoIFN-β) with stable porcine IFN-β expression under control of CMV promoter was selected under G418 pressure. In a 25cm(2) cell culture flask (5 ml culture medium), the cumulative protein yield of recombinant PoIFN-β reached 2.3×10(6) IU/ml. This cells clone maintained stable expression for at least 20 generations even in the absence of G418 selection pressure. The expressed recombinant PoIFN-β could induce the expression of porcine Mx protein in PK15 cells, and activate the chicken Mx promoter-controlled luciferase reporter gene expression, confirming that the recombinant PoIFN-β has the biological activity of natural porcine type-I interferon. In addition, the recombinant PoIFN-β fully protected PK15 cells against 1000 TCID(50) of porcine transmissible gastroenteritis virus and pseudo-rabies virus infection, demonstrating its high potential in therapeutic applications. This is the first report of establishing a mammalian cell line with stable expression of porcine IFN-β.
[Show abstract][Hide abstract] ABSTRACT: Antibodies produced in animals vaccinated using live attenuated vaccines against Brucella spp. are indistinguishable using current conventional serological tests from those produced in infected animals. One potential approach is to develop marker vaccines in which specific genes have been deleted from parental vaccine strains that show good immunogenicity and vaccine efficacy. Corresponding methods of detection for antibodies raised by the marker vaccine should also be developed. A specific fragment of the bp26 gene of Brucella melitensis M5-90 was cloned into vector pQE32 to construct the recombinant plasmid (pQE32-rΔbp26). It was used to transform Escherichia coli M15 (pREP4) host cells, which expressed the rΔbp26 protein. Subsequently, the recombinant protein was purified by immobilized metal affinity chromatography and size-exclusion chromatography. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified rΔbp26 protein was represented by only one band, with a molecular weight of 14 kDa, and it showed good antigenic specificity on western blot and enzyme-linked immunosorbent assay (ELISA). The purified rΔbp26 protein was intended to be used as an antigen to develop a novel ELISA to differentiate animals vaccinated with bp26 mutants of Brucella spp. from those infected naturally and those vaccinated with the parental vaccine strains.
[Show abstract][Hide abstract] ABSTRACT: Differential diagnose of Japanese encephalitis virus (JEV) infection from other flavivirus especially West Nile virus (WNV) and Dengue virus (DV) infection was greatly hindered for the serological cross-reactive. Virus specific epitopes could benefit for developing JEV specific antibodies detection methods. To identify the JEV specific epitopes, we fully mapped and characterized the continuous B-cell epitope of the PrM/M protein of JEV.
To map the epitopes on the PrM/M protein, we designed a set of 20 partially overlapping fragments spanning the whole PrM, fused them with GST, and expressed them in an expression vector. Linear epitope M14 (105VNKKEAWLDSTKATRY120) was detected by enzyme-linked immunosorbent assay (ELISA). By removing amino acid residues individually from the carboxy and amino terminal of peptide M14, we confirmed that the minimal unit of the linear epitope of PrM/M was M14-13 (108KEAWLDSTKAT118). This epitope was highly conserved across different JEV strains. Moreover, this epitope did not cross-react with WNV-positive and DENV-positive sera.
Epitope M14-13 was a JEV specific lineal B-cell epitpe. The results may provide a useful basis for the development of epitope-based virus specific diagnostic clinical techniques.
[Show abstract][Hide abstract] ABSTRACT: Recombinant capripoxvirus (CPV) is a promising candidate differentiating infected from vaccinated animals (DIVA) vaccine against peste-des-petits-ruminants (PPR). In order for recombinant CPV to be successfully used in the field, there should exist dependable indicators for quality control of vaccine products, surveillance and vaccination evaluation. Viral neutralization antibody (VNA) is correlated to protection against PPR and is a technically feasible indicator for this purpose. The immunogenicity of this vectored vaccine in goats and sheep, however, has not been fully evaluated. In this study, we generated two recombinant CPV viruses, rCPV-PPRVH and rCPV-PPRVF, that express PPR virus (PPRV) glycoproteins H and F, respectively. Vaccination studies with different dosages of recombinant viruses showed that rCPV-PPRVH was a more potent inducer of PPRV VNA than rCPV-PPRVF. One dose of rCPV-PPRVH was enough to seroconvert 80% of immunized sheep. A second dose induced significantly higher PPRV VNA titers. There was no significant difference in PPRV VNA responses between goats and sheep. Subcutaneous inoculation also induced a significant PPRV VNA response. PPRV VNA could be detected for over 6 months in more than 80% of vaccinated goats and sheep. Boost vaccination at 6-month intervals induced significant re-boost efficacy of PPRV VNA in goats and sheep. More over, two doses of rCPV-PPRVH could completely overcome the interference caused by pre-existing immunity to the CPV vaccine backbone in animals. Vaccination with rCPV-PPRVH also protected goats from virulent CPV challenge. Our results demonstrate that VNA can serve as a dependent indicator for effective vaccination and immune protection of animals in the field. The recombinant CPV vaccine used in our studies could be a practical and useful candidate DIVA vaccine in countries where PPR newly emerges or where stamp-out plans are yet to be implemented.
[Show abstract][Hide abstract] ABSTRACT: The purpose of the study is to construct recombinant goat pox virus (GPV) expressing Peste des petits ruminants virus (PPRV) H protein, and to evaluate the immunization effect. Recombinant GPV containing PPRV H gene (rGPV-PPRV-H) was selected and purified by gpt and eGFP utilizing plaque purification, and the final selected recombinant GPV was proved to be purified by PCR. Immunofluorescence and Western blotting showed that the recombinant virus could express H protein of PPRV while infecting lamb testis cells. Six goats were immunized with 2 x 10(6) PFU rGPV-PPRV-H through intradermal injection, and were immunized for the second time at 28 days with the same dose recombinant virus after first immunization. Serum was collected after immunization, and was analyzed for the neutralization antibodies. 21 days after first immunization, the neutralization antibodies of GPV were 40, 80, > or = 80, > or = 80, 40, > or = 80 in turn, and neutralization antibodies of PPRV were 80, 80, 80, 80, 40, 40, 10 in turn; 14 days after second immunization, the neutralization antibodies of GPV were all > or = 80, and the neutralization antibodies of PPRV were > 80, 80, > 80, 80, 80 and 40 in turn. This study established a foundation for the industrialization of the PPRV recombinant GPV vaccine.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 04/2009; 25(4):496-502.
[Show abstract][Hide abstract] ABSTRACT: The two mammalian codon optimized genes, F and G genes of Nipah virus, were generated by assembly PCR, and inserted into mammalian expression vector pCAGGS under chicken beta-actin promoter to construct pCAGG-NiV-F and pCAGG-NiV-G. Syncytium formation was induced in BHK cells by plasmid pCAGG-NiV-F and pCAGG-NiV-G transfection, which indicate recombination proteins F and G were expressed in BHK cell and possessed good biologic activity. Six-week-old female BALB/c mice were intramuscularly primed with 100 microg pCAGG-NiV-F, pCAGG-NiV-G or pCAGG-NiV-F+ pCAGG-NiV-G respectively, and boosted with same dose after 4 weeks. The sera were collected at 3 weeks post second boost. The serum IgG against Nipah virus F and G proteins was detected by indirect ELISA using recombinant Baculovirus expressed Nipah F and G glycoproteins. The results showed that specific antibodies possessed good sensitivity and specificity. Furthermore, the G and F proteins' specific antibodies could neutralize the infectivity of VSVdeltaG* F/G (the NiV F and G envelope glycoproteins psudotyped recombinant vesicular stomatitis virus expressing green fluorescence protein). And, pCAGG-NiV-G also induced higher titer of neutralizing antibody response than pCAGG-NiV-F did. The result indicates that DAN immunization is an efficient vaccine strategy against Nipah virus.
Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui] 02/2008; 24(1):47-52.
[Show abstract][Hide abstract] ABSTRACT: DNA vaccines have successfully induced effective antibody and cellular immune response to many viral pathogens. The antibody response of DNA immunization induction in mouse model with envelope glycoproteins of Rift Valley Fever Virus (RVFV), G (N + C), GN and GC was investigated. For this purpose, three codon G (N + C), GN and GC gene were insert into mammalian expression vector pCAGGS under chicken beta-actin promoter to construct pCAGG-RVFV-GN, pCAGG-RVFV-GC and pCAGG-RVFV-G (N + C). The expression of recommbinant GN or / and GC protein in BHK cells transfected with pCAGG-RVFV-GC or pCAGG-RVFV-G (N + C) DNA were confirmed by immunoprecipitation. Six-week-old female BALB/c mice were intramuscularly primed with 100 (g pCAGG-RVFV-GN + pCAGG-RVFV-GC + pCAGG-RVFV-G (N + C), and boosted with same dose after 4 weeks. The serums were collected at 3 weeks post final boost. The serum IgG against Rift Valley Fever Virus G (N + C) protein were detect by indirect ELISA using recombinant Baculovirus expressed Rift Valley Fever Virus GN and GC glycoprotein. The mixture of pCAGG-RVFV-GN, pCAGG-RVFV-GC and pCAGG-RVFV-G (N + C) elicited much strong IgG response. For serum neutralization antibody assay, a recombinant Vesicular Stomatitis Virus pseudotype, in which the VSV envelope protein G gene was replaced with the green fluorescent protein gene (VSVdeltaG x G, Whitt M A) and complemented with Rift Valley Fever Virus G (N + C) glycoprotein expressed in transient (VSVdeltaG x RVFV-G), was use to replace the authentic Rift Valley Fever Virus. The mixture of pCAGG-RVFV-GN, pCAGG-RVFV-GC and pCAGG-RVFV-G (N + C) also induced high titer of neutralization antibody response. These result indicates that DNA immunization is an efficient vaccine strategy against Rift Valley Fever Virus.
[Show abstract][Hide abstract] ABSTRACT: A recombinant vaccinia virus (rWR-SARS-S)expressing spike protein of severe acute respiratory syndrome coronavirus was constructed. The expression of full length recombinant SARS spike protein (rSS) in HeLa cells possessing specific reaction ability to chicken anti-sera was confirmed by SDS-PAGE and Western-blot (190 kD). HeLa cells infected with rWR-SARS-S also showed high sensitivity in detecting specific serum antibody by indirect immunofluoresence assay (IFA). The results above indicated that the availability of such a faithful model system offers particular advantages for the study of SARS in that it reduces the need for direct manipulation of an exotic pathogen. In the absence of infectious SARS, we may safely carry out detailed biochemical and genetic manipulations to investigate features of viral replication and gene function, as well as explore new avenue for vaccine development.
Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui] 08/2007; 23(4):287-91.
[Show abstract][Hide abstract] ABSTRACT: The full-length bovine interferon gamma (BoIFN-gamma) cDNA, including the secretion signal peptide coding region was recloned into baculovirus honor vectors pFastBac 1 of Bac-To-Bac system. These recombinant plasmids, pFastBac 1-BoIFN-gamma, were transformed into DH(10Bac) host bacteria to get recombinant shuttle plasmids, rBacmid-BoIFN-gamma. Recombinant baculovirus, rBac-BoIFN-gamma, was generated for expressing BoIFN-gamma, by transfecting recombinant Bacmid-BoIFN-gamma with Cellfectin Reagen into sf9 insect cells. BoIFN-gamma efficiently expressed by recombinant baculovirus in sf9 cells was testified by indirect immunofluorescence assay and indirect ELISA with monoclonal antibody against Bovine interferon-gamma. Furthermore, VSV * GFP, recombinant Vesicular Stomatitis Virus expressing green fluorescence protein and MDBK were used to determine the anti-viral activity of rBoIFN-gamma. The result shows rBoIFN-gamma could inhibit the replication of the VSV * GFP in MDBK cells and the antiviral activity of supernatant was 2 x 10(5) IU/mL. The antiviral activity of rBoIFN-gamma could be blocked by anti-BoIFN-gamma mouse serum. The results demonstrated that the recombinant baculovirus could express BoIFN-gamma efficiently and rBoIFN-gamma had high antiviral activity.
[Show abstract][Hide abstract] ABSTRACT: The full-length porcine interferon gamma(PoIFN-gamma) cDNA, including the secretion signal peptide coding region was recloned into honor plasmid pFastBac 1 of Bac-To-Bac Baculovirus Expression System. These recombinant plasmids, pFastBac -PoIFN-gamma, were transformed into DH(10Bac) host bacteria to get recombinant shuttle plasmids, rBacmid-PoIFN-gamma. Recombinant baculovirus, rBac-PoIFN-gamma, was generated for expressing PoIFN-gamma, by transfecting rBacmid-PoIFN-gamma with Cellfectin Reagent into sf9 insect cells. The expression of PoIFN-gamma in insect cells was confirmed by Western Blot, indirect immunofluorescence assay and indirect ELISA. The antiviral activity assay shows that PoIFN-gamma expressed by the rBac-PoIFN-gamma can efficiently inhibit the replication of the recombinant Vesicular Stomatitis Virus expressing green fluorescence protein in PK-15 cells. The antiviral activity of PoIFN-gamma can be specifically blocked by anti-PoIFN-gamma mouse serum. The antiviral titer of culture supernatant of insect cells infected by rBac-PoIFN-gamma is 2 x 10(4) IU/mL. The results demonstrat that the rBac-PoIFN-gamma can express rPoIFN-gamma efficiently and rPoIFN-gamma has high antiviral activity.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 06/2007; 23(3):386-91.
[Show abstract][Hide abstract] ABSTRACT: H5N1 highly pathogenic avian influenza virus (HPAIV) has continued to spread and poses a significant threat to both animal and human health. Current influenza vaccine strategies have limitations that prevent their effective use for widespread inoculation of animals in the field. Vaccine strains of Newcastle disease virus (NDV), however, have been used successfully to easily vaccinate large numbers of animals. In this study, we used reverse genetics to construct a NDV that expressed an H5 subtype avian influenza virus (AIV) hemagglutinin (HA). Both a wild-type and a mutated HA open reading frame (ORF) from the HPAIV wild bird isolate, A/Bar-headed goose/Qinghai/3/2005 (H5N1), were inserted into the intergenic region between the P and M genes of the LaSota NDV vaccine strain. The recombinant viruses stably expressing the wild-type and mutant HA genes were found to be innocuous after intracerebral inoculation of 1-day-old chickens. A single dose of the recombinant viruses in chickens induced both NDV- and AIV H5-specific antibodies and completely protected chickens from challenge with a lethal dose of both velogenic NDV and homologous and heterologous H5N1 HPAIV. In addition, BALB/c mice immunized with the recombinant NDV-based vaccine produced H5 AIV-specific antibodies and were completely protected from homologous and heterologous lethal virus challenge. Our results indicate that recombinant NDV is suitable as a bivalent live attenuated vaccine against both NDV and AIV infection in poultry. The recombinant NDV vaccine may also have potential use in high-risk human individuals to control the pandemic spread of lethal avian influenza.
Journal of Virology 02/2007; 81(1):150-8. DOI:10.1128/JVI.01514-06 · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated the antibody response of DNA immunization with two mammalian codon optimized envelope glycoprotein genes, F and G, of Nipah virus in a mouse model. The results indicated that G gene immunization elicited more significant specific serum IgG response and neutralization antibody response than F gene did, suggesting that the G gene DNA immunization is a potential vaccine strategy against Nipah virus.
Annals of the New York Academy of Sciences 11/2006; 1081:243-5. DOI:10.1196/annals.1373.029 · 4.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We generated a recombinant vesicular stomatitis virus (VSV) pseudotype (VSV Delta G*SG) by replacing the envelope G gene with the GFP gene and complementing with spike glycoprotein (S) of SARS-CoV in trans. The neutralization and infection blocking tests showed that the VSV Delta G*SG and SARS-CoV reacted similarly to SARS-CoV specific antiserum, suggesting the VSVDelta G*SG can be a safe replacement of the live SARS-CoV for neutralization test and cell-entry assay.
Annals of the New York Academy of Sciences 11/2006; 1081:246-8. DOI:10.1196/annals.1373.030 · 4.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mammalian condon optimized G gene was synthesized by over-lapping PCR and used to generate recombinant vaccinia virus, rWR-NiV-G. The expression of Nipah virus G protein in rWR-NiV-G infected HeLa cells was confirmed by western-blot with NiV G protein specific mouse antiserum generated by DNA immunization.The recombinant G protein showed sensitive and specific antigenic reaction to rabbit serum anti-Nipah virus in indirect florescence. Syncytium formation was induced in BHK cells by rWR-NiV-G infection following NiV F protein expressing plasmid pCAGG-NiV-F transfection. Immunization with rWR-NiV-G elicited G protein specific antibody responses in mice. The prokaryotic expressing G protein fragment showed sensitive and specific antigenic reaction to NiV G protein specific antibody from rWR-NiV-G immunized mice serum in indirect ELISA. Furthermore, the G protein specific antibodies could neutralize the infectivity of the recombinant Vesicular Stomatitis Virus pseudotype VSVAG * F/G, in which the VSV envelope protein G gene was replaced with the green fluorescent protein gene (VSVAG * G, Whitt MA) and complemented with Nipah virus F and G glycoprotein expressed in transient (VSVAG * F/G).The results here demonstrated the G protein expressed by rWR-NiV-G keeps native immunogenicity and biological activity. The recombinant virus could be promising vaccine strategy for the prevention of Nipah virus.
[Show abstract][Hide abstract] ABSTRACT: In this study, Recombinant baculoviruses rBac-NF and rBac-NG were generated for expressing F and G proteins Nipah virus (NiV) . The expression of recommbinant G (rNG) and F (rNF) protein in rBac-NF and rBac-NG infected cells were confirmed by western-blot. Both rNG and rNF showed sensitive and specific antigenic reaction to rabbit serum anti-Nipah virus in indirect immunofluorescence detection and indirect ELISA. Immunization with rBac-NF and rBac-NG infected insect cells elicited G ad F protein specific antibody responses in mice. Furthermore, the G ad F specific antibodies could neutralize the infectivity of the VSVdeltaG* F/G, the NiV F and G envelope glycoproteins psudotyped recombinant Vesicular Stomatitis Virus expressing green fluorescence protein. The results demonstrated F and G protein expressed by the recombinant baculoviruses could be safe economic diagnostic antigens for the surveillance and monitoring of NiV and promising subunit vaccines for the prevention of NiV.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 06/2006; 22(3):418-24. DOI:10.1016/S1872-2075(06)60038-1