ABSTRACT
ObjectiveTo investigate the antiviral effect of Menispermi Rhizoma total alkaloids and its relationship with the type Ⅰ interferon （IFN-Ⅰ） signaling pathway. MethodThe effects of Menispermi Rhizoma total alkaloids on the intracellular replication of influenza A virus （H1N1）， vesicular stomatitis virus （VSV）， and cerebral myocarditis virus （EMCV） were detected by fluorescent inverted microscope， flow cytometry， Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and Western blot. A mouse model infected with H1N1 was constructed， and the mice were divided into a control group， H1N1 model group， Menispermi Rhizoma total alkaloids groups （10， 20， 30 mg·kg-1）， and oseltamivir group （40 mg·kg-1）， so as to study the effects on the weight and survival rate of infected mice. Real-time PCR was used to detect the activation effect of Menispermi Rhizoma total alkaloids on the IFN-Ⅰ pathway in cells， and the relationship between the antiviral effect of Menispermi Rhizoma total alkaloids in IFNAR1 knockout A549 cells （IFNAR1-/--A549） and IFN-Ⅰ pathway was detected. ResultCompared with the control group， the virus proliferated significantly in the model group （P<0.01）. Compared with the model group， Menispermi Rhizoma total alkaloids could significantly inhibit the replication of H1N1， VSV， and EMCV in vitro （P<0.01）， inhibit the weight loss of the mice infected with the H1N1 in vivo， and improve the survival rate of mice （P<0.05）. In addition， Menispermi Rhizoma total alkaloids activated the IFN-I pathway and relied on this pathway to exert the function of antiviral infection. ConclusionMenispermi Rhizoma total alkaloids exert antiviral effects in vivo and in vitro by activating the IFN-Ⅰ pathway.
ABSTRACT
Encephalomyocarditis virus (EMCV) ,named JZ1202 ,was isolated from domesticated boar in Henan ,China . We performed the full-length genome sequencing and molecular characteristic analysis of the isolated strain .Results showed that the full-genome sequence of EMCV JZ1202 generated a sequence of 7 735 bp in length ,and had 81 .2%-99 .9% nucleotide identity with other reference strains from different animals ,but 99 .4% with Chinese reference from pig .The phylogenetic tree was constructed based on the full-length genome;ORF and VP1 gene sequences identified EMCV was divided into G1 ,G2 and G3 groups ;the strain JZ1202 belongs to G1 with other Chinese reference strains .Results identified that the EMCV infection could cause severe clinical symptoms in domesticated boar .Big regional differences exist in EMCV and the transmission is limit-ed in a range of area .However ,cross infection and prevalence of EMCV disease between domesticated boar and mice might ex-ist .Mutation of some amino acid may occurred in EMCV infected domesticated boar .
ABSTRACT
Objective To prepare an experimental encephalomyocarditis virus ( EMCV) vaccine and study its immunogenicity .Methods EMCVs were cultured in Vero cells and viral titer was detected by micro cytopathy method .A series of procedures including concentration , filtration and ultracentrifugation were conducted to purify EMCVs .Formaldehyde and β-propiolactone were used for viral inactivation and their efficacy was evaluated .The median lethal dose ( LD50 ) of EMCV was measured by using Reed-Muench method.The serum antibody titers in BALB/c mice vaccinated with different immunization strategies were analyzed through microneutralization assay .Then the immunized mice were challenged with 100LD50 , 50LD50 and 25LD50 of EMCV by intraperitoneal injection .Results The viruses could be inactivated by β-propiolactone at a volume ratio of 1 ∶4000 for 12 hours at 4℃.The LD50 of EMCV was 17 CCID50/ml.The immunized mice produced neutralizing antibodies and displayed resistance to EMCV infection.Conclusion The experimental EMCV vaccine could induce protective antibodies in mice .
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Objective To establish a TaqMan real-time PCR assay for the detection of encephalo-myocarditis virus ( EMCV) .Methods Based on the conservative region of 3D gene of EMCV published in GenBank , a pair of primers and one TaqMan probe were designed and synthesized .Then a TaqMan real-time PCR assay was set up and the reactive system was optimized .The sensitivity and specificity of the assay was evaluated respectively .The TaqMan real-time PCR assay was then carried out to detect 98 randomly selected swine serum samples and the results were compared with those by using ELISA .Results The Ct value of the templates had a good linear relationship with the log starting quantity , with a correlation coefficient of 0.995.The TaqMan real-time PCR assay was only specific for EMCV and its sensitivity was 100 times higher than that of the ordinary PCR .The coincidence rate between the established assay and the ELISA assay was 98.0%in the detection of 98 blood samples.Conclusion The TaqMan real-time PCR assay for the detec-tion of EMCV was successfully established with advantages of high sensitivity and good specificity .It could be used for detection of EMCV and quantitative analysis .
ABSTRACT
Objective To explore and develop methods for encephalomyocarditis virus (EMCV) identification.Methods According to the genetic sequence VR-129B of EMCV recorded in the GenBank,five gene fragments were selected to design primer sequence pairs.RNA was extracted to run RT-PCR,and then the products of amplification were identified by agarose gel electrophoresis.The results of DNA sequences were compared with the sequences in GenBank of the same EMCV strains.Antiserum was prepared based on the EMCV cultured in RK cells for establishing indirect immunofluorescence assay (IIFA) and neutralization test method,and verification for precision and specificity of the two methods were carried out after it.Antiserum that was prepared with GST-VP1 and GST-VP2 expressed in E.coli was reacted with the purified EMCV in Western blot test.Results By sequencing and comparing,the similarity of DNA fragments between the obtained and the GenBank recorded was reached 98％ to 100％.The antiserum of No.20100901 batch that was chosen as the first antibody at a dilution of 1 ∶ 160 to develop IIFA brought about a better specificity.The neutralization titers of 20100901 batch antiserum was 1 ∶ 30 211 measured by fixing virus and diluting serum method,which showed good specificity and precision.The results of the Western blot test showed two clear bands above and under 33×103 respectively,which matched the theoretical value.Conclusion The RT-PCR,indirect immunofluorescence,neutralization test and Western blot method for EMCV strains identification were established initially.
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Encephalomyocarditis virus (EMCV) infections can cause losses in pig farms all over the world. Rapid, sensitive and unequivocal detection of this virus is therefore essential for the diagnosis and control of the disease. An RT-PCR assay was developed, optimized and evaluated for encephalomyocarditis virus detection in organ based on a pair of primers that amplifies a 165 bp DNA fragment from a highly conserved nucleotide region of the viral 3D glycoprotein. PCR products of the expected size were obtained from Cuban EMCV 744/03 strain. Non-specific reactions were not observed when other porcine RNA genome viruses and uninfected cells were used. The analytical sensitivity of the test was estimated to be 2 TCID50/50 mL. The analysis of tissue homogenate samples from naturally infected animals proved the potential usefulness of the method for a rapid disease diagnosis from field cases.
Subject(s)
Animals , Genome/genetics , In Vitro Techniques , Nucleotides , Reverse Transcriptase Polymerase Chain Reaction , RNA Viruses , Swine , Encephalomyocarditis virus/genetics , Encephalomyocarditis virus/isolation & purification , Methods , Nucleic Acid Amplification Techniques , MethodsABSTRACT
Objective:To express the EMCV 3AB gene by prokaryotic expression systerm,and prepare monoclonal antibodies against it. Method: NSP 3AB gene of Encephalomyocarditis virus (EMCV) was amplified and cloned into a prokaryotic expression vector pGEX-6P-1 and a recombinant protein 3AB with high antigenicity was expressed in E.coli. Balb / c mice were immunized by purified recombinant 3AB protein of inclusion-body, and the splenocytes of the immunized mice were fused with murine myeloma cells to produce hybridoma cell line. Results: After subcloning by 3 times, one strain of hybridoma cell line steadily secreting antibodies of 3AB protein was obtained, named 2D12. The McAb belongs to IgG1/?. The McAb and was confirmed by indirect immunofluorescent assay (IFA) and Western blot. Conclusion: These results can provide a potential value for structural and functional studies of EMCV-3AB and early diagnosis of Encephalomyocarditis virus infection.