ABSTRACT
Vaccination is the most effective method for preventing rabies virus (RABV) infection in both humans and animals; however, no satisfactory vaccine has been developed for use worldwide. In the present study, we investigated the immunoadjuvant properties of Salmonella Typhimurium flagellin (FljB, FliC, and FljB'-FliC) to improve immune responses against the rabies vaccine (RV) and the protective efficacy of the whole-killed rabies vaccine (WKRV) with or without flagellins in BALB/c mice. We also compared the differences among the three flagellins in terms of immunoadjuvant properties to RV. FljB can cause the WKRV to induce stronger humoral and cellular immune responses than WKRV alone or WKRV with FliC or FljB'-FliC can. Mice immunized with WKRV and FljB produced higher levels of virus-neutralizing antibody (VNA) against RABV than those in the other groups did. Although mice in all treatment groups survived RABV challenge, the body weight loss in the group immunized with WKRV and FljB was lower than in the other groups. These results indicate that FljB is a promising adjuvant for use in the development of effective rabies vaccines.
Subject(s)
Adjuvants, Immunologic/administration & dosage , Flagellin/administration & dosage , Rabies Vaccines/administration & dosage , Rabies Vaccines/immunology , Rabies/prevention & control , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Body Weight , Disease Models, Animal , Flagellin/genetics , Mice, Inbred BALB C , Salmonella typhimurium/genetics , Survival Analysis , Vaccines, Inactivated/administration & dosage , Vaccines, Inactivated/immunologyABSTRACT
The one-step single-tube betaine-free reverse transcription (RT)-loop-mediated isothermal amplification assay was developed for rapid diagnosis of hepatitis E virus. This assay amplified the target gene in less than 45 min (even as short as 20 min) under isothermal conditions at 63 degrees C, and the sensitivity of this assay was 100-fold greater than that of RT-PCR. This assay demonstrated a detection limit of 0.045 fg (nine copies/reaction).
Subject(s)
Hepatitis E virus/genetics , Hepatitis E virus/isolation & purification , Nucleic Acid Amplification Techniques/methods , Animals , Humans , Molecular Sequence Data , RNA, Viral/genetics , Reverse Transcription , Sensitivity and Specificity , Sequence Analysis, DNA , Temperature , Time FactorsABSTRACT
The gene coding for the polyprotein (PP) of foot-and-mouth disease virus (FMDV)was obtained by PCR from recombinant plasmid rpMD18-T/PP. The PCR product was digested with Xba I and Not I and inserted into the cloning site of the adenovirus shuttle vector pAdTrack-CMV, previously digested with the same enzymes. This recombinant shuttle plasmid was designated rpAd-CMV/PP. The recombinant adenovirus vector rpAd/PP was obtained by homologous recombination of plasmid rpAd-CMV/PP and adenovirus skeletal vector pAdeasy-1 in E. coli. Plasmid rpAd/PP was linearized by Pme I and transformed into 293 competent cells to pack the adenovirus using liposome mediated gene transfer method and, as a result, the recombinant adenovirus rAd/PP that contained the polyprotein coding gene was obtained. Obvious CPE could be observed under an inverted microscope, the green fluorescence protein expression can be detected under fluorescence microscope and the empty capsid of FMDV was observed under electron microscope. These results indicated that the recombinant adenovirus rAd/PP expressed the PP protein and that this protein could be assembled into the empty capsid of FMDV. The recombinant adenovirus obtained in this study can be used for further research for making FMDV recombinant adenovirus vaccine.
Subject(s)
Adenoviridae/genetics , Escherichia coli/genetics , Foot-and-Mouth Disease Virus/genetics , Genetic Engineering , Polyproteins/genetics , Recombination, Genetic , Viral Proteins/genetics , Adenoviridae/physiology , Animals , Cell Line , Cloning, Molecular , Escherichia coli/metabolism , Foot-and-Mouth Disease/virology , Foot-and-Mouth Disease Virus/physiology , Genetic Vectors/genetics , Humans , Polyproteins/metabolism , Reassortant Viruses/genetics , Reassortant Viruses/physiology , Viral Proteins/metabolism , Virus AssemblyABSTRACT
Three pairs of specific primers were designed to amplify the F2-1, F2-2 and XF2-2 truncated sequences of ORF2 which encodes the capsid protein of porcine circovirus type 2 (PCV-2). The F2-1 sequence had most of the NLS region of ORF2, but the F2-2 and XF2-2 genes had the NLS region deleted. Truncated genes were subcloned into pET-32a(+) vectors to construct recombinant fusion expression vectors. The vectors were then transformed into Rosetta(DE3) E. coli and expressed by induction of IPTG. Expressed proteins were detected by western blotting and ELISA. The protein with best immunoreactivity was confirmed and selected, then utilized to inoculate SPF rabbits to prepare polyclonal antibodies. The protein and prepared polyclonal antibody were utilized to detect sera samples against PCV-2 from Shandong province and PCV-2 particles in PK-15 cells. In our study, three recombinant fusion proteins were successfully obtained, and the molecular weights of fusion proteins were 35.9 kDa, 33.6 kDa and 38.6 kDa respectively detected by SDS-PAGE. All of the proteins showed positive reaction with anti-PCV-2 antisera, and His-XF2-2 showed better immunoreactivity than the others. The protein of His-XF2-2 was coated as antigen in ELISA to detect the seroprevalence of PCV-2 in certain districts of Shandong province, the seropositivity rate was 27.7 % (73/264). Specific fluorescence and positive signals for PCV-2 could be detected in PK-15 cells inoculated with PCV-2 with the participation of prepared antibodies against His-XF2-2 in IFA and IPMA. Experimental results indicated that the truncated PCV-2 ORF2 gene containing most of the NLS region was successfully expressed in E. coli, and His-XF2-2 was demonstrated to have better immunoreactivity with anti-PCV-2 antisera than the other two fusion proteins. His-XF2-2 and prepared polyclonal antibodies against it had a satisfactory capability in detecting PCV-2 infection.
Subject(s)
Antibodies, Viral , Antigens, Viral , Capsid Proteins , Circoviridae Infections/veterinary , Circovirus/isolation & purification , Swine Diseases/diagnosis , Swine Diseases/epidemiology , Animals , Antibodies, Viral/blood , Antibodies, Viral/isolation & purification , Antigens, Viral/biosynthesis , Antigens, Viral/genetics , Capsid Proteins/biosynthesis , Capsid Proteins/genetics , Cell Line , China/epidemiology , Circoviridae Infections/diagnosis , Circoviridae Infections/epidemiology , Circoviridae Infections/virology , Circovirus/genetics , Enzyme-Linked Immunosorbent Assay/methods , Escherichia coli/genetics , Gene Expression , Genetic Vectors , Molecular Weight , Plasmids , Rabbits , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Sequence Deletion , Seroepidemiologic Studies , Swine , Swine Diseases/virologyABSTRACT
The complete genome sequence of transmissible Gastroenteritis virus (TGEV) strain TS, previously isolated from Gansu province, was cloned and compared with published sequence data from other TGEV strains. Phylogenetic tree analysis based on the amino acid and nucleotide sequences of the S gene showed that the TGEV strains were divided into 3 clusters. TGEV TS showed a close evolutionary relationship to the American Miller cluster but had a 5' non-translated region (NTR) sequence closely related to the American Purdue cluster. Continued culture in different cell types indicated that TGEV TS virulence could be attenuated after fifty passages in Porcine kidney (PK-15) cells, and that the Porcine kidney cell line IB-RS-2 (IBRS) was not suitable for culture of the TGEV strain TS.