ABSTRACT
BACKGROUND: HPyV6 is a novel human polyomavirus (HPyV), and neither its natural history nor its prevalence in human disease is well known. Therefore, the epidemiology and phylogenetic status of HPyV6 must be systematically characterized. METHODS: The VP1 gene of HPyV6 was detected with an established TaqMan real-time PCR from nasopharyngeal aspirate specimens collected from hospitalized children with respiratory tract infections. The HPyV6-positive specimens were screened for other common respiratory viruses with real-time PCR assays. RESULTS: The prevalence of HPyV6 was 1.7 % (15/887), and children ≤ 5 years of age accounted for 80 % (12/15) of cases. All 15 HPyV6-positive patients were coinfected with other respiratory viruses, of which influenza virus A (IFVA) (8/15, 53.3 %) and respiratory syncytial virus (7/15, 46.7 %) were most common. All 15 HPyV6-positive patients were diagnosed with lower respiratory tract infections, and their viral loads ranged from 1.38 to 182.42 copies/µl nasopharyngeal aspirate specimen. The most common symptoms were cough (100 %) and fever (86.7 %). The complete 4926-bp genome (BJ376 strain, GenBank accession number KM387421) was amplified and showed 100 % identity to HPyV6 strain 607a. CONCLUSIONS: The prevalence of HPyV6 was 1.7 % in nasopharyngeal aspirate specimens from hospitalized children with respiratory tract infections, as analyzed by real-time PCR. Because the coinfection rate was high and the viral load low, it was not possible to establish a correlation between HPyV6 and respiratory diseases.
Subject(s)
Phylogeny , Polyomavirus Infections/epidemiology , Polyomavirus Infections/virology , Polyomavirus/classification , Polyomavirus/isolation & purification , Respiratory Tract Infections/epidemiology , Respiratory Tract Infections/virology , Adolescent , Beijing/epidemiology , Child , Child, Hospitalized , Child, Preschool , Cluster Analysis , DNA, Viral/chemistry , DNA, Viral/genetics , Female , Humans , Infant , Infant, Newborn , Male , Molecular Sequence Data , Nasopharynx/virology , Orthomyxoviridae , Polyomavirus/genetics , Prevalence , Real-Time Polymerase Chain Reaction , Respiratory Syncytial Viruses , Sequence Analysis, DNA , Sequence HomologyABSTRACT
OBJECTIVE: To construct the eukaryotic expression vector PCI-dhfr-lambda1 and PCI-dhfr-SP163-lambda1 which linked the enhancer SP163 with interferon lambda1. Then express the interferon lambda1 in CHO (dhfr-) cells. METHODS: Using PCR method to introduce the restriction enzyme sites and through the fusion PCR binding the enhancer with the interferon Lambda1. After sequenced, lambda1 and SP163-lambda1 was inserted into PCI-dhfr forming the expression vector PCI-dhfr-lambda1 and PCI-dhfr-SP163-lambda1 which was constructed successfully confirming by sequencing. Then the expressing vectors were transfected into CHO (dhfr-) cells using liposome transfection method and interferon lambda1 protein was assayed with indirect immunofluorescence and Western Blot. Using cytopathic effect inhibition evaluated the antiviral activity of interferon lambda1. RESULTS: Successfully constructing the eukaryotic expression vectors of interferon lambda and the vectors could express interferon lambda1. The result of immunofluorescence showed the enhancer developed the expression of interferon lambda1. Detecting the interferon lambda1 in CHO (dhfr-) cells after transfecting 48 hour using Western Blot. The cytopathic effect inhibition showed the expressed interferon lambda1 has the antiviral activity. CONCLUSION: Successfully expressed the interferon lambda1 in CHO (dhfr-) cells and the protein possesses antiviral activity, which may supply a valuable basis for building the stable cell line of interferon lambda1.
Subject(s)
Interleukins/genetics , Animals , Blotting, Western , CHO Cells , Cricetinae , Cricetulus , Fluorescent Antibody Technique, Indirect , Interferons , Interleukins/pharmacology , Polymerase Chain Reaction , Recombinant Proteins/biosynthesis , Recombinant Proteins/pharmacology , TransfectionABSTRACT
OBJECTIVE: To express and purify HBoV VP2 protein, and the monoclonal antibody against HBoV VP2 protein was prepared with hybridoma technique. METHODS: The HBoV VP2 cloned into vector pET-30a was expressed in E. coil. After purified by immobilized metal affinity chromatography, the BALB/c mouse was immunized with purified protein as antigen. The positive hybridoma cells were screened with hybridoma technique and ELISA assay. Isotype and titer of the monoclonal antibody were detected. RESULTS: The recombinant HBoV VP2 protein was expressed and purified, and then the monoclonal antibody was obtained with hybridoma technique. The titer of the IgG monoclonal antibody was up to 1:4 x 10(5). CONCLUSION: Monoclonal antibody against recombinant HBoV VP2 protein was prepared and the antibody titer was high. This work may provide a new method in rapid diagnosis and study of HBoV.
Subject(s)
Antibodies, Monoclonal/immunology , Capsid Proteins/immunology , Human bocavirus/immunology , Animals , Capsid Proteins/genetics , Hybridomas , Mice , Mice, Inbred BALB C , Plasmids , Recombinant Proteins/biosynthesis , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purificationABSTRACT
Reverse transcription loop-mediated isothermal amplification (RT-LAMP), which is a visual assay for nucleic acids, is performed in a single step using one tube at 65 °C for 1.5 h. In this study, RT-LAMP was established as a method for the detection of enterovirus 71 (EV71). The detection limit of the assay was approximately 10 copies, and no cross-reactivity was noted with Coxsackievirus A16, echovirus, human rotavirus (HRV) or norovirus. This assay, which offers greater sensitivity at a lower cost compared with the conventional reverse transcription polymerase chain reaction (RT-PCR), was validated using 252 clinical specimens that had been confirmed by laboratory diagnosis using RT-PCR. Both methods produced the same results with 52 positive samples. The RT-LAMP-based assay does not require specialised equipment, and therefore, it can be performed conveniently during an outbreak or under field conditions. In brief, the RT-LAMP-based assay provided a simple, rapid and efficient method for the detection of EV71 nucleic acid under field conditions.
Subject(s)
Enterovirus A, Human/isolation & purification , Enterovirus Infections/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Virology/methods , Adolescent , Child , Child, Preschool , Enterovirus Infections/virology , Humans , Infant , Molecular Diagnostic Techniques/economics , Sensitivity and Specificity , Temperature , Virology/economicsABSTRACT
In the present paper, the composition of complex Al-CAS-CTMAB was studied with the ratio of double peak values at dual-wavelengths by UV spectrophotometry. First, the composition of complex of Al-CAS was determined in this method, Al:CAS = 1:2. The absorption of ternary complex(Al-CAS-CTMAB) was determined with reference reagent of the complex Al-CAS. The epsilon values of Al-CAS and Al-CAS-CTMAB were determined with water reference. The composition of complex Al (CAS)n1 (CTMAB)n2 was calculated with the formula: [formula: see text] CAS:CTMAB = 1:1; Al:CAS:CTMAB = 1:2:2. The calculated value and that obtained by traditional method are agreeable. The method is reliable for determining composition of ternary complexes.
