In order to develop a combined live vaccine that will be used to prevent against porcine parvovirus (PPV) and Pseudorabies virus (PRV) infection, the VP2 gene of PPV was inserted into the transfer vector plasmid pG to produce the recombinant plasmid pGVP2. The plasmid pGVP2 and the genome of PRV HB98 attenuated vaccine were transfected by using lipofectamine into swine testis cells for the homologous recombination. The recombinant virus rPRV-VP2 was purified by selection of green fluorescence plaques for five cycles. 6-week-old female Kunming mice were immunized intramuscularly with attenuated PRV parent HB98 strain, commercial inactivated vaccine against PPV, recombinant virus, DMEM culture solution. The injections were repeated with an equivalent dose after 2 weeks in all of the groups, and then challenged with the virulent PRV NY strain at 7 weeks after the first immunization. The recombinant virus rPRV-VP2 was successfully generated, and the recombinant virus could effectively elicite anti-PPV and PRV antibody and significant cellular immune response as indicated by anti-PPV ELISA and HI, PRV-neutralizing assay and flow cytometry. The challenge assay indicated that recombinant virus could protect the mice against the virulent PRV challenge. These results demonstrated that the recombinant virus can be a candidate recombinant vaccine strain for the prevention of PRV and PPV.
Animals ; Antibodies, Viral ; immunology ; Antigens, Viral ; administration & dosage ; genetics ; immunology ; Capsid Proteins ; administration & dosage ; genetics ; immunology ; Female ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Herpesvirus 1, Suid ; genetics ; metabolism ; Mice ; Parvovirus, Porcine ; genetics ; immunology ; Swine ; Swine Diseases ; immunology ; prevention & control ; virology ; Viral Vaccines ; administration & dosage ; genetics ; immunology

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are very two important pathogens that have coursed huge economic losses in swine production in worldwide. In this study,a vector pCMV-TJM containing the full-length cDNA clone of PRRSV attenuated strain TJM-F92 was firstly constructed by PCR method. Then a gene sequence containing Afl II/Mlu I e restriction enzyme sites and a transcription regulatory sequence for ORF6 (TRS6) was inserted be- tween ORF7 and 3'UTR, yielding a expression vector pCMV-TJM-TRS. Subsequently, a plasmid pCMV-TJM-Cap was constructed by cloning of PCV2 ORF2 gene into the unique sites Afl II /Mlu I of pCMV- TJM-TRS plasmid DNA. Then three recombinant PRRSV, rTJM, rTJM/TRS and rTJM/Cap, were rescued by transfection of pCMV-TJM, pCMV-TJM-TRS and pCMV-TJM-Cap into Marc-145 cells, respectively,and confirmed by the genome sequence, restriction enzyme digestion, Western Blot and IFA. They all had the molecular markers which was different from the parent virus. The growth characteristics of the rescued viruses were similar to that of parent virus. rTJM/Cap could also express efficiently PCV2 Cap protein in Marc-145 cells. At passage 8, it still had PCV2 ORF2 gene which examined by RT-PCR. It indicated that the full-length cDNA clone of PRRSV attenuated strain TJM-F92 and recombinant PRRSV rTJM/Cap expressing PCV2 Cap protein were successfully constructed. It made an important foundation for studying on the pathogenic mechanisms of PRRSV and PRRSV-PCV2 vaccine in the future.
Animals ; Capsid Proteins ; genetics ; immunology ; Cell Line ; Circoviridae Infections ; veterinary ; virology ; Circovirus ; classification ; genetics ; metabolism ; Gene Expression ; Open Reading Frames ; Porcine Reproductive and Respiratory Syndrome ; virology ; Porcine respiratory and reproductive syndrome virus ; genetics ; metabolism ; Recombination, Genetic ; Swine ; Swine Diseases ; virology ; Viral Vaccines ; genetics ; immunology

Antibodies, Monoclonal ; chemistry ; immunology ; Antigens, Viral ; chemistry ; genetics ; immunology ; Binding Sites ; Capsid Proteins ; chemistry ; genetics ; immunology ; Epitopes ; chemistry ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Hepatitis E ; immunology ; prevention & control ; virology ; Hepatitis E virus ; chemistry ; immunology ; Humans ; Molecular Docking Simulation ; Mutagenesis, Site-Directed ; Peptide Mapping ; Protein Binding ; Recombinant Proteins ; chemistry ; genetics ; immunology ; Viral Hepatitis Vaccines ; administration & dosage ; biosynthesis

Animals ; Binding Sites ; Capsid ; chemistry ; immunology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; HIV-1 ; chemistry ; immunology ; Humans ; Mice ; Myxovirus Resistance Proteins ; chemistry ; immunology ; metabolism ; Protein Binding ; Protein Multimerization ; Protein Structure, Tertiary ; Recombinant Proteins ; chemistry ; immunology ; metabolism

Swine hepatitis E virus (HEV) is widespread throughout pigs in both developing and industrialized countries. This virus is an important zoonotic agent and a public concern worldwide. Infected pigs are asymptomatic, so diagnosing swine HEV relies on detection of the virus or antibodies against the virus. However, several obstacles need to be overcome for effective and practical serological diagnosis. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) that used a purified recombinant capsid protein of swine HEV. The potential clinical use of this assay was evaluated by comparing it with a commercial kit (Genelabs Technologies, Diagnostics, Singapore). Results of the ELISA were highly correlated with those of the commercial kit with a sensitivity of 97% and specificity of 95%. ROC (receiving operator characteristic) analysis of the ELISA data produced a value of 0.987 (95% CI, 0.977~0.998, p < 0.01). The cut-off value for the ELISA was also determined using negative pig sera. In summary, the HEV-specific ELISA developed in the present study appears to be both practical and economical.
Animals ; Antibodies, Anti-Idiotypic/*analysis/blood/genetics ; Capsid Proteins/*genetics/metabolism ; Enzyme-Linked Immunosorbent Assay/*methods/veterinary ; Hepatitis E/diagnosis/immunology/*veterinary/virology ; Hepatitis E virus/genetics/*isolation & purification/metabolism ; Immunoglobulin G/blood/genetics ; ROC Curve ; Recombinant Proteins/genetics/metabolism ; Swine ; Swine Diseases/*diagnosis/immunology/virology

Rabbit hemorrhagic disease (RHD) is contagious and highly lethal. Commercial vaccines against RHD are produced from the livers of experimentally infected rabbits. Although several groups have reported that recombinant subunit vaccines against rabbit hemorrhagic disease virus (RHDV) are promising, application of the vaccines has been restricted due to high production costs or low yield. In the present study, we performed codon optimization of the capsid gene to increase the number of preference codons and eliminate rare codons in Spodoptera frugiperda 9 (Sf9) cells. The capsid gene was then subcloned into the pFastBac plasmid, and the recombinant baculoviruses were identified with a plaque assay. As expected, expression of the optimized capsid protein was markedly increased in the Sf9 cells, and the recombinant capsid proteins self-assembled into virus-like particles (VLPs) that were released into the cell supernatant. Rabbits inoculated with the supernatant and the purified VLPs were protected against RHDV challenge. A rapid, specific antibody response against RHDV was detected by an ELISA in all of the experimental groups. In conclusion, this strategy of producing a recombinant subunit vaccine antigen can be used to develop a low-cost, insect cell-derived recombinant subunit vaccine against RHDV.
Animals ; Antigens, Viral/genetics/metabolism ; Caliciviridae Infections/prevention & control/*veterinary/virology ; Capsid Proteins/*genetics/metabolism ; Cell Culture Techniques/*methods ; Codon/genetics/metabolism ; Enzyme-Linked Immunosorbent Assay/veterinary ; *Gene Expression Regulation, Viral ; Hemorrhagic Disease Virus, Rabbit/*genetics/immunology ; *Rabbits ; Recombinant Proteins/genetics/metabolism ; Sf9 Cells ; Spodoptera ; Viral Structural Proteins/*genetics/metabolism ; Viral Vaccines/genetics/immunology

OBJECTIVETo develop a prophylactic and therapeutic vaccine against human papillomavirus (HPV) type 58-associated cervical carcinoma, and explore its transformation activity and antigenicity.

METHODSThe E6 and E7 three amino acid codons in the HPV 58 virus were modified respectively and fused. The modified and fused gene was named HPV58 mE6E7. The recombinant HPV58 mE6E7 gene was inserted into pIRES-neo vector to generate plasmid pIRES-neo-HPV58 mE6E7. Then NIH/3T3 cell line was transfected with plasmid pIRES-neo-HPV58 mE6E7. The pIRES-neo-HPV58 mE6E7-transfected cells were the experimental group, pIRES-neo-HPV58 E6E7-transfected cells were the positive control group, and pIRES-neo empty vector-transfected cells were the negative control group. The expression of HPV58 mE6E7 protein in the experimental cells was detected by flow cytometry, immunofluorescence and Western blot. The transformation activity of HPV58 mE6E7 was tested by soft agar colony formation assay and subcutaneously tumors in nude mice. Finally, DNA vaccine was constructed with HPV58 mE6E7 fusion antigen and used to immunize C57BL/6 mice with the vaccine plasmids. The specific serum antibodies were detected by EIISA, and the number of splenic specific CD8(+) T cells secreting IFN-γ of the immunized mice was detected by ELISPOT assay.

RESULTSSequencing confirmed the expected mutation and a 100% homogeneity of the HPV58 E6E7 fusion gene. Stable transfected NIH/3T3 cells expressing HPV58 mE6E7 and HPV58 E6E7 gene were 70.3% and 84.1%, respectively. The relative expressions of HPV58 mE6E7 and HPV58 E6E7 fusion protein in 3T3-HPV58 mE6E7 experimental cells and 3T3-HPV58 E6E7 positive control cells were 2.1 ± 1.7 and 3.8 ± 1.4, respectively, and were negative in the negative control group. No colony formation was found in the experimental and 3T3-neo negative control cell groups, and 31 colonies were found in the positive control cell group, among them 10 colonies were consisted of more than 50 cells. No tumor mass was formed within 4 weeks in the nude mice of experimental and negative control groups, but among the 10 mice of positive control group tumor was formed in 6 mice. Using HPV58 mE6E7 fusion gene as target antigen of DNA vaccine, the antibody titer was 25 600, and specific immunity spots were 218.8 ± 34.4, significantly higher than that in the control group.

CONCLUSIONSThe fused and modified HPV58 E6E7 amino acid codons can abolish the transformation activity but preserve its antigenicity. HPV58 mE6E7 is a potential target gene for the development of therapeutic DNA vaccine against HPV58-associated cervical cancer.

Animals ; Cancer Vaccines ; immunology ; Capsid Proteins ; genetics ; immunology ; Cell Transformation, Neoplastic ; Cloning, Molecular ; Codon ; Female ; Immunoglobulin G ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, Nude ; NIH 3T3 Cells ; Oncogene Proteins, Viral ; genetics ; immunology ; Papillomaviridae ; Papillomavirus E7 Proteins ; genetics ; immunology ; Papillomavirus Vaccines ; immunology ; Plasmids ; Point Mutation ; Random Allocation ; Recombinant Fusion Proteins ; genetics ; immunology ; Transfection ; Vaccines, DNA ; immunology

To observe how anti-group A rotavirus antibody seropositivity rates and levels have changed in the western region of Gyeongnam Province, 2,030 serum samples collected at four collection periods (1989-1990, 1994-1995, 1999-2000, and 2004-2005) were tested by Enzyme-Linked Immunosorbent Assay for IgG, and IgA antibodies reacting to recombinant VP6 protein. The seroprevalences exhibit no regular patterns over a 16-yr period. For all four collection periods, the anti-rVP6 IgG levels rose steadily during the first 5 months of life, after which they remained high. However, the 2-9 yr and 10-39 yr groups had significantly higher IgG levels in 1999-2000 and 2004-2005, respectively, than in the other collection periods. The 1-5 mo, 40- > or = 60 yr, and 4-29 yr groups had significantly higher IgA levels in 1989-1990, 1999-2000, and 2004-2005, respectively. The 4 yr (25.0%), 5-9 yr (18.8%), 10-14 yr (41.1%), 20-29 yr (35.0%), and 30-39 yr (20.0%) groups in 2004-2005 had significant higher IgA seropositivity rate compared to the other three collection periods. These observations suggest that in the western region of Gyeongnam Province since the late 1990s, rotavirus reinfection has occurred more frequently than previously, with all ages being at risk.
Adult ; Aged ; Antibodies, Viral/*blood ; Antigens, Viral/genetics/immunology/metabolism ; Capsid Proteins/genetics/immunology/metabolism ; Child ; Child, Preschool ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Immunoglobulin A/blood ; Immunoglobulin G/blood ; Infant ; Infant, Newborn ; Male ; Middle Aged ; Recombinant Proteins/biosynthesis/genetics/immunology ; Republic of Korea/epidemiology ; Rotavirus/isolation & purification/*metabolism ; Rotavirus Infections/*epidemiology/virology ; Seroepidemiologic Studies ; Time Factors ; Young Adult

The Cap gene of antisense strand of circovirus has the most variation of the genome, and encodes a capsid protein which has the main immunogenicity. The N-terminal of capsid protein makes up of nuclear localization signal which is involved with virus location. This review summarizes the research advance of Cap gene of circovirus in the sequence characteristics, its encoding capsid protein, basic functions of the capsid protein and its interaction with MKRN1 protein, Hsp40 protein, receptor protein gClqR and complement factor C1qB protein. This paper lays a theory foundation for the further study of the capsid protein in the aspects of viral attachment, replication and transportation.
Animals ; Capsid Proteins ; genetics ; immunology ; metabolism ; Circoviridae Infections ; veterinary ; virology ; Circovirus ; genetics ; immunology ; Genetic Variation ; Genome, Viral ; genetics ; Nuclear Localization Signals ; Protein Binding ; Virus Replication

Based on a pair of specific primers, a 804-bp fragment was amplified from the plasmid pT-Cap containing Cap gene of Porcine Getah Virus(PGETV) and cloned into the prokaryotic expression vector pCold I which carried the His tag, this recombinant plasmid was then determined by enzyme digestion, PCR and DNA sequencing. This recombinant plasmid pCold-Cap was transformed into E. coli Rosetta 2, and PGETV Cap fusion protein was expressed through IPTG induction. The results showed that the Cap gene obtained efficient and soluble expression in Rosetta 2 induced by 0. Immol/L IPTG under 15"C for 24h, the expression quantity was 40. 2%. The product had a molecular mass about 32. 3kD as expected. The target protein was separated in gel slices and used to immunize Balb/c mice. The polyclonal antibody with high titer against Cap protein specifically analyzed by Western blot was obtained. The successful preparation of the polyclonal antibody laid the foundation for the further study on the detection and identification of PGETV.
Alphavirus ; genetics ; immunology ; metabolism ; Alphavirus Infections ; immunology ; veterinary ; virology ; Animals ; Antibodies, Viral ; blood ; immunology ; Blotting, Western ; Capsid Proteins ; genetics ; immunology ; isolation & purification ; metabolism ; DNA Primers ; genetics ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Plasmids ; genetics ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; Swine ; Swine Diseases ; immunology ; virology ; Zoonoses