[show abstract][hide abstract] ABSTRACT: Pigs play a critical role in Japanese encephalitis virus (JEV) transmission between mosquitos and humans. In 2009, lots of piglets developed symptom of viral encephalitis in a pig farm in Yunchen, Shanxi province.
Virus isolation was carried out in BHK-21 cells. Immunohistochemistry, RT-PCR and indirect immunofluorescent assay were used to identify the newly isolated virus. The complete genome of one isolate (SX09S-01 strain) was sequenced and analyzed. Two phylogenetic trees were constructed on the basis of the 24 full-length JEV genomes and 62 E genes mostly selected from China.
JEV SX09S-01 strain was isolated from piglets. Sequence analysis indicates that the completed genome sequences of this strain consists of 10965 nucleotides and there are 13 nucleotides deletion in the 3' nontranslated variable region. Compared with other JEV strains, homology ranges from 99.1% (XJ69) to 74.1% (XZ0934) and 99.6% (XJ69) to 91.1% (XZ0934) on the level of nucleotide and amino acid sequences, respectively. Phylogenetic trees show that SX09S-01 strain belongs to genotype I and it is most closely related to the XJ69 strain.
Genotype I of JEV still circulates in Yuncheng and it is thus important for active surveillance on genotype I of JEV from the swine population.
[show abstract][hide abstract] ABSTRACT: Foot-and-mouth disease (FMD) causes morbidity to livestock and serious economic consequences to its associated industry and therefore it is necessary to develop a safe and efficient vaccine to prevent or control this disease. A recombinant live attenuated virus vaccine, designated PRV-P1, was generated by insertion of an expression cassette containing CMV promoter, FMDV P1 gene and SV 40 poly-A into the gG gene region of a live attenuated pseudorabies virus vaccine strain (TK-/gG-/LacZ+). To determine the induction of protective immunity, 16 FMDV and PRV seronegative white swine were randomly divided into four groups and immunized intramuscularly. The parental virus (TK-/gG-/LacZ+) was injected into three pigs, the recombinant virus PRV-P1 into five pigs and commercial FMD-inactivated vaccine into five pigs, with PBS (negative control) into three pigs. All animals were immunized again 4 weeks later to boost the immune response and challenged with virulent type O FMDV O/ES/2001 strain 4 weeks after the second immunization. Results showed PRV-P1 vaccinated pigs induced high-level neutralizing antibody response to both FMDV and PRV, and strong CTL response against FMD antigen activation. Three of five pigs were completely protected against challenge with FMDV, one pig minimally protected and the other one had increased protection but not complete. However, one pig vaccinated with commercial FMD vaccine developed constant pyrexia. Average levels of antibodies against non-structural 3ABC proteins were significantly lower and efficacy on inhibition of FMDV replication was much increased in swine vaccinated with PRV-P1 than those immunized with commercial FMD vaccine after FMDV challenge. Our results showed that the recombinant PRV-P1 can induce not only humoral and cell-mediated immune responses but also partial protection against FMDV challenge, making it a good candidate for future development of the FMD vaccine.
[show abstract][hide abstract] ABSTRACT: Foot-and-mouth disease (FMD) is the most contagious and devastating disease of livestock. Our previous studies demonstrated that TK(-)/gG(-)/P1(+), a recombinant expressing the FMDV capsid precursor protein (P1) based on attenuated pseudorabies virus (PRV) TK(-)/gG(-), could be used as a recombinant vaccine to protect pigs against both pseudorabies and FMD. However, because the P1 expression cassette is inserted into the gG locus of the genome of PRV TK(-)/gG(-), this bivalent vaccine cannot be used in conjunction with the PRV gE-ELISA, an extensively used discriminatory serological test in eradication programs for pseudorabies, which limits the clinical use of this bivalent vaccine. To circumvent this shortcoming, in this study, an expression cassette containing synthetic multiepitope gene "FHG" consisting of six potential B cell epitopes and two potential T cell epitopes of FMDV, under the control of CMV promoter, was further inserted into the gE/gI locus of genome of TK(-)/gG(-)/P1(+), resulting in a new recombinant FHG/P1/PRV. The immunogenicity of FHG/P1/PRV was evaluated and compared with TK(-)/gG(-)/P1(+) in piglets. Our results clearly showed that FHG/P1/PRV performed better than or comparable with TK(-)/gG(-)/P1(+), as demonstrated by comparable PRV-specific neutralizing antibodies, enhanced FMDV-specific neutralizing antibodies, and cellular immune responses. More importantly, no gE- and gG-specific antibodies could be detected in pigs immunized with FHG/P1/PRV. These data indicate that FHG/P1/PRV is a promising bivalent vaccine candidate with more extensive potential application than TK(-)/gG(-)/P1(+) against both pseudorabies and FMD.
[show abstract][hide abstract] ABSTRACT: Porcine interferon-gamma (PoIFN-gamma) fused with glutathione S-transferase (GST) was expressed in Escherichia coli BL(21). Twenty 6-week-old piglets were randomly assigned to four groups. Pigs in groups 1-3 were pretreated with 30 mg, 20 mg, and 10 mg recombinant PoIFN-gamma (rPoIFN-gamma), respectively. Pigs in group 4 (control) were pretreated with GST expressed by the empty plasmid. At 48h postinoculation (hpi), all swine were challenged with FMDV (serotype O). Pigs pretreated with 30 mg rPoIFN-gamma were completely protected from virulent FMDV attack. Pigs given 20 mg rPoIFN-gamma achieved partial protection, and the unprotected piglets showed clinical signs from 68h postchallenge (hpc). Although 10 mg rPoIFN-gamma did not confer protection against FMDV, the pigs pretreated with this dose of rPoIFN-gamma presented clinical signs from 35 hpc, which was later than the control group (14 hpc). These results indicate that PoIFN-gamma can protect swine against attack from FMDV or delay the appearance of clinical signs; the effect is dose dependent.
Veterinary Immunology and Immunopathology 05/2008; 122(3-4):309-11. · 1.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: The P12A3C gene from FMDV (serotype O) encoding the capsid precursor protein, and the highly immunogenic gene FHG, which encodes multiple epitopes of FMDV capsid proteins, were inserted into eukaryotic expression vectors to compare different candidate genetically engineered vaccines for foot-and-mouth disease (FMD). A modified live pseudorabies virus (MLPRV) was also used to deliver P12A3C. Guinea pigs were inoculated intramuscularly with the candidate vaccines to compare the ability to elicit immunity of the DNA vector and a live viral vector. An indirect enzyme-linked immunosorbent assay (iELISA), virus-neutralization test and lymphoproliferation assay were used to detect antibody and cellular responses. The group immunized with P12A3C delivered by MLPRV produced significantly greater antibody and cellular responses indicating that MLPRV has a greater ability to mediate exogenous gene delivery than the plasmid DNA vector. Comparison of the immune responses induced by P12A3C and FHG, which were both mediated by DNA plasmids, showed that FHG and P12A3C elicited similar cellular responses, while P12A3C induced higher antibody levels, suggesting that P12A3C is a more powerful immunogen than FHG. In challenge experiments, guinea pigs vaccinated with P12A3C delivered by MLPRV were protected fully from FMDV challenge, whereas guinea pigs vaccinated with P12A3C or FHG delivered by DNA plasmid were only protected partially. This study provides a basis for future construction of a genetically engineered vaccine for FMDV.
Journal of Virological Methods 02/2008; 147(1):143-50. · 1.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: The NS5B protein of classical swine fever virus (CSFV) is an important enzyme bearing a unique RNA-dependent RNA polymerase (RdRp) activity. The RdRp plays a crucial role in the viral replication cycle and in forming a replicase complex. However, the initiating synthesis mechanism of the CSFV RNA polymerase is unclearly described at present. Our aim is to reveal the RdRp-GTP docking sites and the effective modules of GTP initially bound to the polymerase in starting initiation of replication according to a well predicted CSFV RdRp model. Based on some known crystal structures of RNA polymerase, computational methods were used to establish the model of a CSFV RdRp. An analogous mechanism of CSFV RNA polymerase in de novo initiation was subsequently represented through docking a GTP into the structure model. The unique GTP binding pocket of the polymerase was pointed out: five residues E227, S408, R427, K435, and R439 involved in steady hydrogen bonds and two residues C407 and L232 involved in hydrophobic contact with the GTP. From a genetic evolutionary point of view, three residues C407, S408 and R427 have been suggested to be of particular importance by analysis of residue conservation. It is suggested that these crucial residues should have very significant function in the de novo initiation of the rigorous CSFV polymerase model, which can lead us to design experiments for studying the mechanism of viral replication and develop valid anti-viral drugs.
[show abstract][hide abstract] ABSTRACT: Pseudorabies (PR), foot-and-mouth disease (FMD), and porcine parvovirus disease are three important infectious diseases in swine worldwide. The gene-deleted pseudorabies virus (PRV) has been used as a live-viral vector to develop multivalent genetic engineering vaccine. In this study, a recombinant PRV, which could co-express protein precursor P1-2A of FMDV and VP2 protein of PPV, was constructed using PRV TK(-)/gE(-)/LacZ(+) mutant as the vector. After homologous recombination and plaque purification, recombinant virus PRV TK(-)/gE(-)/P1-2A-VP2 was acquired and identified. Immunogenicity, safety of the recombinant PRV and its protection against PRV were confirmed in a mouse model by indirect ELISA and serum neutralization test. The results show that the recombinant PRV is a candidate vaccine strain to develop a novel trivalent vaccine against PRV, FMDV and PPV in swine.
[show abstract][hide abstract] ABSTRACT: The total RNA was extracted from peripheral blood mononuclear cells (PBMC) which was isolated from Meishan porcine and induced with concanavaline A (ConA), then the porcine interferon gamma gene (PoIFNgamma, 501bp) was amplified by RT-PCR. The result of sequencing demonstrated that the amplified PoIFNgamma had 100% nucleotide homology with the other porcine IFNgamma sequence published on GenBank. The objective gene (PoIFNgamma) was inserted into adenoviral shuttle vector, pShuttle-CMV, to construct recombinant plasmid pSh-PoIFNgamma. And it was co-electrotransformated with adenoviral skeletal vector pAdEasy-1 into competent cells of BJ5183. The transforms were cultured at 37 degrees C for 24h on kanamycin resistance plate and selected for smaller colonies. Then, the extracted recombinant plasmid was named pAd-Sh-PoIFNgamma, which was confirmed by Pac I digestion, and transformed into XL10-Glod(r) for copious preparation. pAd-Sh-PoIFNgamma linearized with Pac I was co-transfected with liposome into 293 package cell-line. After 7d-10d, the typical cytopathic effect indicated that recombinant adenoviral genome (deleted with E1 and E3 genes) carrying PoIFNgamma was successfully packaged into intact virion. The recombinant virion was successively seeded to the 10th generation and the viral genome was extracted from each generation by PCR. The antiviral activity of PoIFNgamma was tested by CPE50 method. The results showed that the PoIFNgamma expressed by adenovirus had high antiviral activity, which was 1.3 x 10(6) U/mL against VSV in MDBK cells. The results demonstrated that the recombinant adenovirus carrying PoIFNgamma could be stably passaged.
Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui] 10/2007; 23(5):394-8.
[show abstract][hide abstract] ABSTRACT: Interferon (IFN) is crucial for initiating the innate immune response and for the generation of the adaptive response. IFN, in most species, comprises IFN-alpha (IFN-alpha), IFN-beta (IFN-beta) and IFN-gamma (IFN-gamma). In this study, we compared the capacity of porcine IFN-alpha, -beta and -gamma, or a combination of them, to protect IBRS-2 cells (porcine kidney cells) from infection with pseudorabies virus (PRV). The results demonstrated that porcine IFN-beta (PoIFN-beta) was the most efficient of the three IFNs in conferring resistance PRV infection; 100 U/mL PoIFN-beta inhibited PRV plaque formation 5.3-fold. Compared with PoIFN-beta, porcine IFN-gamma (PoIFN-gamma) was less capable of inhibiting PRV plaque formation (3.3-fold inhibition). Porcine IFN-alpha (PoIFN-alpha) had the least capability of the three PoIFNs, and inhibited PRV plaque formation only 1.26-fold. The inhibitory capacity increased to only 2.3-fold with a treatment of 12,800 U/mL PoIFN-alpha. A combination of PoIFN-gamma and PoIFN-alpha or PoIFN-beta inhibited PRV plaque formation 12.8-fold or 100-fold, respectively. Treatment of IBRS-2 cells with PoIFN-alpha/beta and PoIFN-gamma inhibited PRV replication 29- or 146-fold. Additionally, real-time PCR analyses of the PRV immediate early (IE) gene revealed that IE mRNA expression was profoundly decreased in cells stimulated with PoIFN-alpha/beta and PoIFN-gamma (23.8-133.0-fold) compared with vehicle-treated cells. All the findings indicate that PoIFN-gamma acts synergistically with other PoIFNs (PoIFN-alpha and -beta) to potently inhibit PRV replication in vitro.
European cytokine network 07/2007; 18(2):71-7. · 1.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: Porcine interferon-gamma (PoIFN-gamma) of Chinese local brand, Meishan porcine, was cloned and inserted into retroviral vector pLXSN (neo r) . Using Lipofectamine, this recombinant plasmid was transfected into retroviral packing cell line, PA317 cells. These transfected cells were selected by DMEM containing 400microg/mL G418 for one week. RNA was extracted from the supernatant of these selected PA317 cells and the PoIFN-gamma gene could be amplified by RT-PCR. Pocine kidney cells and PK-15 cells were infected by the supernatant and were selected by 400 microg/mL, 600 microg/mL and 800 microg/mL G418, respectively. Those PK-15 cells were detected by indirect immunofluorescence assay and it was found that PoIFN-gamma mainly anchored in cellular membrane. The supernatant of the selected PK-15 was tested for the antiviral bioactivity after 48 hours of passage. The anti-VSV (vesicular stomatitis virus) activity in MDBK (bovine kidney cell) was 1200IU/10(6) cells. In addition, the effect of rPoIFNgamma-anti-FMDV was determined using cytopathic effect inhibition. The results indicate that PoIFN-gamma has been inserted into retroviral vector and recombinant retrovirus has been successfully packaged in PA317 cells. Furthermore, this retrovirus can infect PK-15 cells and express PoIFN-gamma with natural antiviral bioactivity and can inhibit VSV and FMDV.
[show abstract][hide abstract] ABSTRACT: The genome of a novel classical swine fever virus (CSFV), SWH/CA/2004, isolated from a hog pen in Henan Province, central China, is 12,296 nucleotides (nt) in length. It is composed of a 373-nt 5' terminal non-translated region (NTR), a 11,697-nt open reading frame (ORF) encoding a polyprotein of 3,898 amino acids (aa), and a 226-nt 3'-NTR. Genome comparison of the SWH/CA/2004 isolate (GenBank Accession: DQ127910) with other known CSFV isolates was performed and analyzed. Corresponding segments from SWH/CA/2004 and other reported strains shared 80.4-99.8% identity at the nucleotide level and 89.5-99.8% identity at the amino acid level. From an evolutionary point of view, isolate SWH/CA/2004 is closely related to the highly virulent isolate cF114/CA/2001, with a pairwise distance of 0.013; and distantly related to the moderately virulent isolate GXWZ02/CA/2003, with pairwise distance 0.170. The phylogenetic trees of the full-length genome and the following region E(rns), E1, E2, and NS5B-based neighbor-joining (NJ) method were constructed and approximately divided into different genetic groups according to avirulence, moderate virulence and high virulence, while other region-based NJ trees demonstrated sequence conservation between these groups. The four genomic regions may constitute important criteria for genetic typing of diverse CSFV isolates. Based on these analyses, isolate SWH/CA/2004 was deduced to belong to the highly virulent isolate group. However, SWH/CA/2004 also contains a 14-U deletion in the 3'-NTR that is characteristic of avirulent isolates. These analyses constitute a comprehensive study of the phylogenetics of CSF based on distinct regions of the genome and may provide the basis for future molecular epidemiology research to identify virulent strain outbreaks and trigger implementation of appropriate control measures.
[show abstract][hide abstract] ABSTRACT: Foot-and-mouth disease (FMD) and pseudorabies (PR) are two important infectious diseases in swine. An attenuated pseudorabies virus (PRV) has been successfully used as a gene delivery vector for the development of live-viral vaccines. In this study, a recombinant PRV-VP1 virus was constructed by fusioning the VP1 gene of FMD virus in frame to the N-terminal sequence of the gG gene of PRV. To test the protective immunity, 15 FMDV sero-negative white swine were divided into three groups and immunized with the recombinant PRV-VP1 virus, commercial FMD vaccine and vector virus (TK(-)/gG(-)/LacZ(+)), respectively, and challenged intramuscularly with 20 minimal infecting doses (MID) of virulent type O FMDV 4 weeks after booster immunization. Swine vaccinated with PRV-VP1 acquired antibodies against both FMDV and PRV, however, anti-FMDV antibodies were much lower than those vaccinated with the commercial FMD vaccine. Our results suggested that the recombinant PRV-VP1 virus, which only expressed FMDV VP1 gene controlled by PRV gG promoter, could not protect swine from the challenge of 20 MID type O FMDV, but could delay and reduce the clinical symptoms of FMD.
[show abstract][hide abstract] ABSTRACT: High-level expression of the ORF6 gene of porcine reproductive and respiratory syndrome virus (PRRSV) has been proved very difficult. In this work, we cloned and sequenced the ORF6 gene of PRRSV and found that it could not be expressed in Pichia pastoris strain GS115. Then, the ORF6 gene was modified and synthesized based on the codon bias, poly (A) signal of yeast expression system and secondary structure of 5'-end mRNA of foreign gene. The modified gene was inserted into the yeast expression vector pPICZalphaA, induced and expressed by the same methods. The recombinant protein with a molecular mass of approximately 23 kDa was screened by SDS-PAGE and identified by Western blot with convalescent sera of animals infected with CH-1a strain of PRRSV. The results indicated that it was similar to the native protein. The expression level of the recombinant protein could attain 2.0 g/L. In the meanwhile, the optimal conditions for expression were determined. It provides an additional means for studying the structural and functional characteristics of PRRSV ORF6 gene.
[show abstract][hide abstract] ABSTRACT: To study induction or inhibition of apoptosis by pseudorabies virus (PrV) Ea strain in suckling piglets, Aujeszky s disease was replicated by artificially inoculating 15 day-old piglets with PrV-Ea strain. Various tissue sections, such as lymphoid tissues and nervous tissues were collected. Transmission electron microscopy, DNA fragmentation assay, and in situ terminal-deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining were carried to analyze apoptotic cells. It was shown that piglets infected with PrV-Ea strain took on typical clinical symptoms and apoptotic cells were found in lymphoid tissues but not in nerve tissues. The results indicated that the PrV infection caused apoptosis in a big number of lymphatic cells, thus leading to death of suckling piglets due to lowered immune function. It might play an important role in PrV s attack on immune system. A latent infection of pseudorabies virus was established in the neuronal tissue cells by inhibition of nervous cell apoptosis; this might be the way for PrV to establish latent infection, leading to sporadic recurrence of the disease.
Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica 05/2003; 35(4):325-30.