BACKGROUND: Infection with human papilloma virus (HPV) is the main cause of cervical cancer, and HPV genotyping is of increasing importance for determining clinical course and management of the disease based on the HPV genotypes. Here, we established a novel matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF MS) assay, termed restriction fragment mass polymorphism (RFMP) that is suitable for genotyping multiple HPV in an accurate and high-throughput manner. We evaluated the performance of the RFMP assay in HPV genotyping by comparing the results with those of direct or clonal sequencing and hybrid capture (HC) assays. METHODS: The study population consisted of 50 patients with histologically confirmed cervical lesions and a positive test for HPV DNA. HPV genotyping was performed with RFMP, sequencing, and HC assays. The assigned genotypes and risk groups were compared among the methods. RESULTS: Concordance rates in the genotype level between RFMP vs sequencing, sequencing vs HC, and HC vs RFMP were 98% (49/50), 88% (44/50), and 88% (44/50), respectivley. Especially, RFMP and sequencing were 100% concordant when assigned high-risk group was considered identical in 1 case of mixed genotypes identified only in RFMP. The observed discrepancy between HC and the other two methods is due to the assignment of six cases of low, intermediate, or unassigned risk genotypes as high-risk group in HC method. CONCLUSIONS: RFMP, sequencing, and HC assays were highly concordant with each other in HPV genotyping. Compared to sequencing assay, RFMP assay is found to be advantageous in detecting mixed genotype infections. The accuracy and amenability to high-throughput analysis should make the RFMP assay suitable for reliable screening of HPV genotypes in clinical laboratories.
Female ; Genotype ; Humans ; Nucleic Acid Hybridization/methods ; Papillomaviridae/classification/genetics/*isolation & purification ; Papillomavirus Infections/*diagnosis ; Polymorphism, Restriction Fragment Length ; Sequence Analysis, DNA/methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/*methods ; Uterine Cervical Neoplasms/*diagnosis

Commercial interests towards insect cell cultures have greatly been increased recently, in part due to the widespread use of insect virus-based vectors for efficient expressions of foreign proteins. Insect cell-derived biotechnology products should be free of adventitious agents such as arboviruses and mycoplasmas. The objective of this study was to establish techniques for the viral safety evaluation of insect cell-derived biotechnology products using Japanese encephalitis virus (JEV) as a model, since JEV is a member of arthropod-borne flaviviruses which has been known to be infectious to insect cells such as Sf9 cells. Quantitative assays for viral infectivity, concentrations of an antigen or a genome are a prerequisite for the studies of viral clearance validation. Here, we report the development of a quantitative assay for JEV RNA using real-time reverse transcription-polymerase chain reaction (RT-PCR). The assay was performed using LightCycler and RNA amplification kit SYBR Green I. Viral RNA extracted from stock suspension of JEV with known infectivity titer was made to 10-fold serial dilution, and each dilution was subjected to the assay for the generation of a standard curve. A JEV specific primer was selected from 3' untranslated region with the expected band size of 323 base pairs. The real-time RT-PCR assay resulted in a successful amplification within 5 log dilution ranges of JEV RNA samples, and the sensitivity of the assay was calculated to be approximately 15 TCID50 per reaction. Highly reproducible standard curves were obtained from experiments performed on three different days. The real-time RT-PCR assay for the quantification of JEV will be an efficient alternative tool for viral clearance validation studies of insect cell-derived biotechnology products.
3' Untranslated Regions ; Arboviruses ; Asian Continental Ancestry Group* ; Base Pairing ; Biotechnology* ; Cell Culture Techniques ; Encephalitis Virus, Japanese* ; Encephalitis, Japanese* ; Flavivirus ; Genome ; Humans ; Insects* ; Mycoplasma ; RNA ; RNA, Viral ; Sf9 Cells

One-step real-time reverse transcription-polymerase chain reaction (RT-PCR) assay using the MagNA Pure LC and LightCycler(TM) system was developed and validated for the detection and quantitation of hepatitis A virus (HAV) RNA. The assay was evaluated using in-house synthetic HAV RNA standard. The real-time RT-PCR assay could quantitate a dynamic range of HAV RNA standard between 10(2) and 10(8) copies per reaction. The regression coefficient of the standard curve was an 0.99. The detection limit of the assay was 31.3 RNA copies per reaction. The coefficient variations (CVs) of the assay in combination with automated RNA extraction were less than 1.91% in both intra- and inter-assay. The real-time RT-PCR assay for quantitative detection of HAV would serve a useful method for improving the safety of biological products.
Biological Products ; Hepatitis A virus* ; Hepatitis A* ; Hepatitis* ; Limit of Detection ; RNA*

Risk of viral contamination is one of major concerns common to all biologics derived from cultivated cells. Bovine viral diarrhoea virus (BVDV) has widely been known as a contaminant of cell culture-derived vaccines. The objective of the study was to assess the limit of detection and range of quantitation of the detection methods for BVDV using a reverse transcription-polymerase chain reaction (RT-PCR) assay, real-time RT-PCR assay, and RT-PCR-ELISA. One milliliter of cell culture supernatant containing 106.5+/-0.2 median tissue culture infectious dose (TCID50)/ml of BVDV NADL strain was subjected to RNA isolation. The isolated RNA was 10-fold serially diluted and each diluted sample (10-1 to 10-6) was subjected to RT-PCR on a GeneAmpR PCR System 9700 and/or LightCycler(TM). The amplified products were analyzedly (1) agarose gel electrophoresis for RT-PCR assay, (2) melting curve analysis for real-time RT-PCR assay (in this case a program is automatically linked to amplification step), and (3) ELISA using capture and detection probes for RT-PCR-ELISA. The limit of detection of the 3 assay methods was equally estimated to be 316 TCID50/ml of starting virus culture supernatant subjected to the assay. The quantitation range of real-time RT-PCR assay and RT-PCR-ELISA was estimated to be from 3.16x105 to 3.16x102 TCID50/ml of starting virus culture supernatant. The overall results suggested that the 3 assay methods for BVDV detection can be reliably applied to evaluate BVDV contamination in biologics derived from cell cultures.
Biological Products* ; Cell Culture Techniques* ; Electrophoresis, Agar Gel ; Enzyme-Linked Immunosorbent Assay ; Freezing ; Limit of Detection* ; Polymerase Chain Reaction ; Reverse Transcription ; RNA ; Vaccines

Insect cell-derived biotechnological products have a potential for viral contamination from cell line sources themselves or from adventitious introduction of virus during production. The objective of this study was to establish techniques for viral clearance validation of insect cell-derived recombinant human papillomavirus (HPV)-16 type L1 virus-like particles (VLPs) using Japanese encephalitis virus (JEV) and bovine viral diarrhea virus (BVDV) as relevant viruses. The downstream process for the production of recombinant HPV-16 L1 VLPs was sequentially carried out employing detergent lysis (NP-40/PBS), sonication, sucrose cushion centrifugation, and cesium chloride (CsCl) equilibrium density centrifugation. Recombinant HPV-16 L1 capsid protein (56 kD) expressed in Sf9 cell culture was clearly detected by SDS-PAGE and Western blotting analysis. Each purification step was evaluated to determine reduction factor for viral clearance by infectivity assay. In individual purification steps, detergent treatment (0.50% v/v, NP-40/PBS) and CsCl equilibrium density centrifugation were found to be effective in JEV and BVDV clearance. Overall cumulative reduction factors of JEV and BVDV infectivity titer for the purification procedure implemented in this study were 12.53 and 10.05 log TCID(50)/pool, respectively. The results suggest that the purification procedure employed in this study for the HPV-16 L1 VLPs produced from recombinant baculovirus-infected Sf9 cells will be effective over 10 log TCID(50)/pool reduction factor in the clearance of enveloped, adventitious viruses with a buoyant density lower than approximately 1.23 g/ml.
Blotting, Western ; Capsid Proteins ; Cell Line ; Centrifugation ; Cesium ; Detergents ; Diarrhea ; Electrophoresis, Polyacrylamide Gel ; Encephalitis Virus, Japanese ; Human papillomavirus 16* ; Humans ; Insects ; Sf9 Cells ; Sonication ; Sucrose

OBJECTIVE: To evaluate the efficacy of rapmycin for treatment of experimental autoimmune encephalomyelitis (EAE) in mice and explore the underlying mechanism. METHODS: An EAE model was established in C57BL/6 mice. After immunization, the mice were divided into model group and rapamycin groups treated daily with low-dose (0.3 mg/kg) or high-dose (1 mg/kg) rapamycin. The clinical scores of the mice were observed using Knoz score, the infiltration of IL-17 cells in the central nervous system (CNS) was determined using immunohistochemistry; the differentiation of peripheral Treg cells was analyzed using flow cytometry, and the changes in the levels of cytokines were detected with ELISA; the changes in the expressions of p-Smad2 and p- smad3 were investigated using Western blotting. RESULTS: High-dose rapamycin significantly improved the neurological deficits scores of EAE mice. In high-dose rapamycin group, the scores in the onset stage, peak stage and remission stage were 0.14±0.38, 0.43±1.13 and 0.14±0.37, respectively, as compared with 1.14±0.69, 2.14±1.06 and 2.2±0.75 in the model group. The infiltration of inflammatory IL-17 cells was significantly lower in high-dose rapamycin group than in the model group (43±1.83 153.5±7.02). High-dose rapamycin obviously inhibited the production of IL-12, IFN-γ, IL-17 and IL-23 and induced the anti-inflammatory cytokines IL-10 and TGF-β. The percentage of Treg in CD4+ T cells was significantly higher in high- dose rapamycin group than in the model group (10.17 ± 0.68 3.52 ± 0.32). In the experiment, combined treatments of the lymphocytes isolated from the mice with rapamycin and TGF-β induced a significant increase in the number of Treg cells (13.66±1.89) compared with the treatment with rapamycin (6.23±0.80) or TGF-β (4.87±0.85) alone. Rapamycin also obviously up-regulated the expression of p-Smad2 and p-Smad3 in the lymphocytes. CONCLUSIONS Rapamycin can promote the differentiation of Treg cells by up-regulating the expression of p-Smad2 and p-smad3 to improve neurological deficits in mice with EAE.
Animals ; Anti-Inflammatory Agents ; administration & dosage ; therapeutic use ; Cell Differentiation ; drug effects ; Encephalomyelitis, Autoimmune, Experimental ; drug therapy ; metabolism ; Interferon-gamma ; metabolism ; Interleukins ; metabolism ; Lymphocytes ; cytology ; Mice ; Mice, Inbred C57BL ; Sirolimus ; administration & dosage ; therapeutic use ; Smad Proteins ; metabolism ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; Transforming Growth Factor beta ; metabolism ; Up-Regulation