共查询到20条相似文献,搜索用时 62 毫秒
1.
Peste des petits ruminants (PPR) is a highly contagious and economically important viral disease of goats and sheep. A homologous Vero cell-based attenuated PPR vaccine developed in our laboratory and used extensively throughout the country, is available for control of PPR. The presently used quality control test, titration in Vero cells for PPR virus titre in vaccine batches, takes at least 6-8days to determine the quality and dose of vaccine. In this study, 74 freeze-dried PPR vaccine batches were tested simultaneously by both virus titration and PPR sandwich ELISA (S-ELISA) to correlate the titre of the vaccine virus with reactivity in S-ELISA. It was found that the vaccine batches with titre more than 10(3)TCID(50)/ml gave positive results in S-ELISA and correlated well with the virus titre of the freeze-dried vaccines. The correlation coefficient between the virus titration and S-ELISA reactivity was estimated as 0.96, indicating a high correlation between the two parameters based on 74 batches of freeze-dried PPR vaccine. The vaccine batches with titres of 3.0, 4.3, 4.5, 5.0, 6.5 and 7.0 had shown a positive reaction when tested in two-fold dilutions in S-ELISA at 1, 5, 6, 7, 8 and 9log2 titres, respectively. The test vaccine batches were found to be negative in S-ELISA when the titre of the vaccine was less than 10(3)TCID50/ml, suggesting that the vaccine could not be passed for field use. It is concluded that S-ELISA could be a preliminary tool useful for the quality control of PPR vaccine as it is rapid and easy to perform when compared to virus titration. 相似文献
2.
3.
Quality control of Yellow Fever vaccines performed by Control Authorities prior to marketing vaccines batches requires in vitro potency assays. The two currently available methods are the plaque formation assay and the cytopathic effect assay based on the use of porcine kidney PS cells or monkey kidney Vero cells. Among several sources of variation in virus titration, the cell systems are considered as important issues and Quality Assurance strongly recommends working with cell banks from certified suppliers. The aim of our study was to compare the behaviour and the sensitivity of three Vero cell sources obtained from ATCC, WHO and EP used at different passage levels in a plaque formation test. The conclusion of this work was that the yellow fever live attenuated virus titration, adapted in Vero cell lines appeared as a reliable method applicable for routine in vitro potency assay. The comparison of Vero cell lines, originated from three different sources, showed that they could be equally used as substrates by laboratories having the basic facility of cell culture, without influence on the final viral titre. 相似文献
4.
本文采用狂犬病毒CTN-1和4aC株,经Vero细胞传代适应后,以Vero细胞为培养基质,建立了狂犬病毒蚀斑试验和蚀斑减少试验的方法。目前已将此方法应用于病毒鉴定、病毒克隆、病毒滴定以及抗狂犬血清的检测,并取得了较好的结果。 相似文献
5.
Dengue Virus Plaque Development in Simian Cell Systems: II. Agar Variables and Effect of Chemical Additives 总被引:1,自引:0,他引:1
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Thomas B. Stim 《Applied microbiology》1970,19(5):757-762
Dengue type 2 virus, strain New Guinea B, plaqued with equal facility and titer under overlays containing six different grades of commercial agar in the LLC-MK2 cell system. Doubling the agar volume on LLC-MK2 cell monolayers increased the plaque development time of dengue type 1, strain Hawaii. Storage of agar at 56 C reduced or totally abolished dengue type 4, strain H-241, plaque titer in LLC-MK2 cells. The influence of six known virus plaque-enhancing compounds on plaque development of all dengue virus serotypes was studied in two continuous simian kidney cell lines, LLC-MK2 and Vero. In the absence of any chemical additive, plaque development of all dengue serotypes was more rapid (4 to 10 days) in the LLC-MK2 line than in the Vero line (6 to 13 days). Increased plaque development time of type 1, strain Hawaii, by pancreatin and plaque-size doubling of dengue types 1 and 4 was the only advantage conferred by the addition of six chemical additives in the LLC-MK2 cell system. Dengue types 1 and 6 failed to plaque in the Vero cell system unless aided by a plaque-enhancing compound; plaques of dengue types 2, 3, 4, and 5 appeared sooner (2 days) and were increased in plaque diameter. The optimal DEAE concentration for plaquing dengue type 1, strain Hawaii, was 100 μg/ml; plaque development either failed at lower concentrations or was inhibited at higher (200 μg/ml) concentrations. 相似文献
6.
Dengue (DEN) viruses consisting of four distinct serotypes cause diseases such as dengue fever, dengue hemorrhagic fever, and dengue shock syndrome in humans. Most of the dengue viruses can be effectively propagated in some mosquito and mammalian cell lines. In this study, we applied microcarrier cell culture technology to study two relevant aspects involving dengue virus, one on biotechnology of cell growth and virus production, and the other on virus biology concerning genetic variation of a virus population. We investigated the growth of C6/36 mosquito cells and Vero cells grown on Cytodex 1 microcarriers. High-titer DEN virus production can be achieved in C6/36 and Vero cells infected at low cell inoculation density, in the lag-phase cell stage, and at low multiplicity of infection (MOI). The maximum titers produced for DEN-1, DEN-3, and DEN-4 viruses were approximately 10- to 10,000-fold lower than for DEN-2 virus produced in C6/36 and Vero cells grown on microcarriers. The DEN-2 virus produced in C6/36 cells displayed far more extensive plaque heterogeneity than in Vero cells. Microcarrier C6/36 mosquito cell culture appeared to be the most effective system for four-serotype DEN virus production. Interestingly, some selected variants of DEN virus may outgrow in Vero cells when using a T-flask culture. These results may provide useful information for DEN vaccine development. 相似文献
7.
Tanapan Prommool Pongpawan Sethanant Narodom Phaenthaisong Nattaya Tangthawornchaikul Adisak Songjaeng Panisadee Avirutnan Dumrong Mairiang Prasit Luangaram Chatchawan Srisawat Watchara Kasinrerk Sirijitt Vasanawathana Kanokwan Sriruksa Wannee Limpitikul Prida Malasit Chunya Puttikhunt 《PLoS neglected tropical diseases》2021,15(2)
8.
A plaque assay for the quantitation of pneumonia virus of mice is described. To obtain reproducible plaque formation, proteolytic enzymes had to be incorporated in the overlay medium. The plaque morphology observed in the presence of pancreatin and chymotrypsin was superior to that seen with trypsin. Although the plaque assay was found to be slightly less sensitive than the TCID(50) determination described by Harter and Choppin, it enables cloning of the virus by plaque selection and permits further study of pneumonia virus of mice. 相似文献
9.
Evaluation of the serum-free medium MDSS2 for the production of poliovirus on vero cells in bioreactors 总被引:2,自引:0,他引:2
The serum-free medium MDSS2 (Merten et al., 1994), was used for cultivating Vero cells as well as for producing poliovirus
(Sabin type 1) in static and in perfused micro-carrier cultures. At slightly different growth rates of 0.0120/h and 0.0106/h,
respectively, static cultures in serum-containing (SCM) and serum-free (SFM) medium produced titers of 106.75 and 106.67 TCID50 per 50 μl; signifying a specific productivity of 0.89 and 1.07 TCID50/c.
Serum-free bioreactor cultures of Vero cells on DEAE-dextran microcarriers at 6.25 g/l produced cell densities of about 1.5×106c/ml. After infection with virus (multiplicity of infection (MOI) 0.1–0.3) titers of about 6.3×108 TCID50/ml were obtained, signifying an average specific productivity of 7.1 TCID50/c.h. Although these values were 4 and
2 fold, respectively, higher than in classical resum-based production processes (Montagnon et al. Dev. biol. Stand. 1981,
47, 55), a reference culture, for which cell growth was done in SCM and only virus production was done in SFM, produced 2×109 TCID/ml with an average specific virus production rate of 18.9 TCID50/c.h. The differences between the fully serum-free and
our reference process were mainly due to physiological differences of cells grown in SCM and SFM and also due to strongly
modified consumption kinetics after virus infection leading to limitations of one or several essential medium compounds, like
glucose and amino acids. Avoiding these limitations by increasing the residual concentration of glucose, glutamine, histidine,
and SH-amino acids, led to specific virus production rates (of about 17.9 TCID59/c.h.) comparable to those found in the reference
virus production process. The optimisation of the production of the poliovirus (Sabin 1) will be described with respect to
the modification of the medium composition.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
10.
Sydow FF Santiago MA Neves-Souza PC Cerqueira DI Gouvea AS Lavatori MF Bertho AL Kubelka CF 《Memórias do Instituto Oswaldo Cruz》2000,95(4):483-489
Fluorescent activated cell sorter (FACS) analysis is useful for the detection of cellular surface antigens and intracellular proteins. We used this methodology in order to detect and quantify dengue antigens in highly susceptible cells such as clone C6/36 (Aedes albopictus) and Vero cells (green monkey kidney). Additionally, we analyzed the infection in vitro of human peripheral blood mononuclear leukocytes (PBML). FACS analysis turned out to be a reliable technique to quantify virus growth in traditional cell cultures of C6/36 as well as Vero cells. High rates of infection were achieved with a good statistical correlation between the virus amount used in infection and the percentage of dengue antigen containing cells detected in infected cultures. We also showed that human monocytes (CD14+) are preferred target cells for in vitro dengue infection among PBML. Monocytes were much less susceptible to virus infection than cell lines but they displayed dengue antigens detected by FACS five days after infection. In contrast, lymphocytes showed no differences in their profile for dengue specific immunofluorescence. Without an animal model to reproduce dengue disease, alternative assays have been sought to correlate viral virulence with clinical manifestations and disease severity. Study of in vitro interaction of virus and host cells may highlight this relationship. 相似文献
11.
Current egg-based influenza vaccine production technology can't promptly meet the global demand during an influenza pandemic as shown in the 2009 H1N1 pandemic. Moreover, its manufacturing capacity would be vulnerable during pandemics caused by highly pathogenic avian influenza viruses. Therefore, vaccine production using mammalian cell technology is becoming attractive. Current influenza H5N1 vaccine strain (NIBRG-14), a reassortant virus between A/Vietnam/1194/2004 (H5N1) virus and egg-adapted high-growth A/PR/8/1934 virus, could grow efficiently in eggs and MDCK cells but not Vero cells which is the most popular cell line for manufacturing human vaccines. After serial passages and plaque purifications of the NIBRG-14 vaccine virus in Vero cells, one high-growth virus strain (Vero-15) was generated and can grow over 10(8) TCID(50)/ml. In conclusion, one high-growth H5N1 vaccine virus was generated in Vero cells, which can be used to manufacture influenza H5N1 vaccines and prepare reassortant vaccine viruses for other influenza A subtypes. 相似文献
12.
Absolute quantification of dengue virus serotype 4 chimera vaccine candidate in Vero cell culture by targeted mass spectrometry
下载免费PDF全文
![点击此处可从《Proteomics》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Romain Carrière Jordane Biarc Catherine Fonbonne Arnaud Salvador Céline Huillet Yves Berard Olivier Adam Catherine Manin Jérôme Lemoine 《Proteomics》2015,15(19):3320-3330
Infection by dengue flavivirus is transmitted by mosquitoes and affects tens to hundreds of millions people around the world each year. Four serotypes have been described, all of which cause similar disease. Currently, there no approved vaccines or specific therapeutics for dengue, although several vaccine prototypes are in different stages of clinical development. Among them, a chimeric vaccine, built from the replication machinery of the yellow fever 17D virus, has shown promising results in phase III trials. Accurate quantitation of expressed viral particles in alive attenuated viral antigen vaccine is essential and determination of infectious titer is usually the method of choice. The current paper describes an alternative or orthogonal strategy, namely, a multiplexed and absolute assay of four proteins of the chimera yellow fever/dengue serotype 4 virus using targeted MS in SRM mode. Over 1 month, variability of the assay using a partially purified Vero cell extract was between 8 and 17%, and accuracy was between 80 and 120%. In addition, the assay was linear between 6.25 and 200 nmol/L and could therefore be used in the near future to quantify dengue virus type 4 during production and purification from Vero cells. 相似文献
13.
Previous studies have suggested that cells undergo apoptosis in response to dengue virus infection. However, the potential significance of dengue virus-induced apoptosis and the pathways are still not clearly defined. In this study, comparative analysis of dengue virus-induced apoptosis in BHK, H1299, HUH-7 and Vero cell lines was carried out. We show here that infection of BHK, HUH-7 and Vero cell lines with dengue type 1 virus (DEN1V) induces cell death typical of apoptosis. Virus-induced cell death was assayed by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, detection of oligonucleosomal DNA fragmentation, DNA content analysis and assay for the externalization of phosphatidylserine residues. Detailed study of dengue virus infection in HUH-7 cells showed activation of cell death via the mitochondrial pathway causing lowering of mitochondrial transmembrane potential (DeltaPsim) in HUH-7 cells. Interestingly, in the p53-deficient cell line, H1299, apoptosis was largely undetectable compared with the other cell lines used; suggesting that a p53- and mitochondria-mediated cell death pathway may play an important role in dengue virus-induced apoptosis. 相似文献
14.
Atul Asati Olga Kachurina Alex Karol Vipra Dhir Michael Nguyen Robert Parkhill Diana Kouiavskaia Konstantin Chumakov William Warren Anatoly Kachurin 《PloS one》2016,11(2)
Neutralizing antibodies induced by vaccination or natural infection play a critically important role in protection against the viral diseases. In general, neutralization of the viral infection occurs via two major pathways: pre- and post-attachment modes, the first being the most important for such infections as influenza and polio, the latter being significant for filoviruses. Neutralizing capacity of antibodies is typically evaluated by virus neutralization assays that assess reduction of viral infectivity to the target cells in the presence of functional antibodies. Plaque reduction neutralization test, microneutralization and immunofluorescent assays are often used as gold standard virus neutralization assays. However, these methods are associated with several important prerequisites such as use of live virus requiring safety precautions, tedious evaluation procedure and long assessment time. Hence, there is a need for a robust, inexpensive high throughput functional assay that can be performed rapidly using inactivated virus, without extensive safety precautions. Herein, we report a novel high throughput Fluorescence Adherence Inhibition assay (fADI) using inactivated virus labeled with fluorescent secondary antibodies virus and Vero cells or erythrocytes as targets. It requires only few hours to assess pre-attachment neutralizing capacity of donor sera. fADI assay was tested successfully on donors immunized with polio, yellow fever and influenza vaccines. To further simplify and improve the throughput of the assay, we have developed a mathematical approach for calculating the 50% titers from a single sample dilution, without the need to analyze multi-point titration curves. Assessment of pre- and post-vaccination human sera from subjects immunized with IPOL®, YF-VAX® and 2013–2014 Fluzone® vaccines demonstrated high efficiency of the assay. The results correlated very well with microneutralization assay performed independently by the FDA Center of Biologics Evaluation and Research, with plaque reduction neutralization test performed by Focus Diagnostics, and with hemaglutination inhibition assay performed in-house at Sanofi Pasteur. Taken together, fADI assay appears to be a useful high throughput functional immunoassay for assessment of antibody-related neutralization of the viral infections for which pre-attachment neutralization pathway is predominant, such as polio, influenza, yellow fever and dengue. 相似文献
15.
Highly sensitive Taqman PCR detection of Puumala hantavirus 总被引:2,自引:0,他引:2
Garin D Peyrefitte C Crance JM Le Faou A Jouan A Bouloy M 《Microbes and infection / Institut Pasteur》2001,3(9):739-745
16.
Identification of a putative coreceptor on Vero cells that participates in dengue 4 virus infection 总被引:2,自引:0,他引:2
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Dengue virus infects target cells by attaching to a cell surface receptor through the envelope (E) glycoprotein, located on the surface of the viral membrane. On Vero and BHK cells, heparan sulfate (HS) moieties of proteoglycans are the receptors for dengue virus; however, additional proteins have also been described as putative dengue virus receptors on C6/36, HL60, and BM cells. HS can also act as a receptor for other types of viruses or as an attachment molecule for viruses that require additional host cell molecules to allow viral penetration. In this study we searched for molecules other than HS that could participate in dengue virus infection of Vero cells. Labeled dengue 4 virus bound with high affinity to two molecules of 74 and 44 kDa. Binding of dengue virus to the 74-kDa molecule was susceptible to protease and sodium periodate treatment and resistant to heparinase treatments. Lectins such as concanavalin A and wheat germ agglutinin prevented dengue virus binding to both the 74- and the 44-kDa protein in overlay assays, while phytohemagglutinin P did not affect binding, suggesting that carbohydrate residues (alpha-mannose or N-acetylglucosamine) are important in virus binding to host cells. Protease susceptibility, biotin labeling, and immunofluorescence with a polyclonal antibody raised against the 74-kDa protein consistently identified the protein on the surfaces of Vero cells. Moreover, the antibody against the 74-kDa protein was able to inhibit dengue virus infection. These data suggest that HS might serve as a primary receptor, probably concentrating virus particles on the surfaces of Vero cells, and then other molecules, such as the 74-kDa protein, might participate as coreceptors in viral penetration. The 74-kDa protein possibly constitutes part of a putative receptor complex for dengue virus infection of Vero cells. 相似文献
17.
18.
Sunil R. Vaidya Neelakshi S. Kumbhar Vandana S. Bhide 《Microbiology and immunology》2014,58(12):666-674
Measles, mumps and rubella are vaccine‐preventable diseases; however limited epidemiological data are available from low‐income or developing countries. Thus, it is important to investigate the transmission of these viruses in different geographical regions. In this context, a cell culture‐based rapid and reliable immuno‐colorimetric assay (ICA) was established and its utility studied. Twenty‐three measles, six mumps and six rubella virus isolates and three vaccine strains were studied. Detection by ICA was compared with plaque and RT‐PCR assays. In addition, ICA was used to detect viruses in throat swabs (n = 24) collected from patients with suspected measles or mumps. Similarly, ICA was used in a focus reduction neutralization test (FRNT) and the results compared with those obtained by a commercial IgG enzyme immuno assay. Measles and mumps virus were detected 2 days post‐infection in Vero or Vero‐human signaling lymphocytic activation molecule cells, whereas rubella virus was detected 3 days post‐infection in Vero cells. The blue stained viral foci were visible by the naked eye or through a magnifying glass. In conclusion, ICA was successfully used on 35 virus isolates, three vaccine strains and clinical specimens collected from suspected cases of measles and mumps. Furthermore, an application of ICA in a neutralization test (i.e., FRNT) was documented; this may be useful for sero‐epidemiological, cross‐neutralization and pre/post‐vaccine studies. 相似文献
19.
1-beta-D-Arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU) and nine other antiherpesviral nucleoside analogues were compared for their potencies against four strains of varicella-zoster virus (VZV) on three different cell lines: HEL cells, Vero cells, and MS cells established from a human malignant schwannoma. In contrast to the activity against herpes simplex virus type 1 previously reported, BV-araU showed extremely marked antiviral activity against VZV even on Vero cells. ED50, 50% plaque reduction dose, of BV-araU for VZV was 0.20-3.1 and 0.14-0.63 ng/ml on Vero cells and on HEL cells, respectively. Potency of BV-araU on MS cells was similar to that on these cell lines. There was not significant variation in anti-VZV activities of other nucleoside analogues on these three different cell lines except a few combinations of VZV strain and test compound. 相似文献
20.
This work investigated the replication kinetics of the four dengue virus serotypes (DEN-1 to DEN-4), including dengue virus type 4 (DEN-4) recovered from an infectious cDNA clone, in Vero cells and in MRC-5 cells grown on Cytodex 1 microcarriers. DEN-1 strain Hawaii, DEN-2 strain NGC, DEN-3 strain H-87, and DEN-4 strain H-241 , and DEN-4 strain 814669 derived from cloned DNA, were used to infect Vero cells and MRC-5 cells grown in serum-free or serum-containing microcarrier cultures. Serum-free and serum-containing cultures were found to yield comparable titers of these viruses. The cloned DNA-derived DEN-4 started genetically more homogeneous was used to investigate the genetic stability of the virus propagated in Vero cells and MRC-5 cells. Sequence analysis revealed that the DEN-4 propagated in MRC-5 cells maintained a high genetic stability, compared to the virus propagated in Vero cells. Amino acid substitutions of Gly(104)Cys and Phe(108)Ile were detected at 70%, 60%, respectively, in the envelope (E) protein of DEN-4 propagated in Vero cells, whereas a single mutation of Glu(345)Lys was detected at 50% in E of the virus propagated in MRC-5 cells. Sequencing of multiple clones of three separate DNA fragments spanning 40% of the genome also indicated that DEN-4 propagated in Vero cells contained a higher number of mutations than the virus growing in MRC-5 cells. Although Vero cells yielded a peak virus titer approximately 1 to 17 folds higher than MRC-5 cells, cloned DEN-4 from MRC-5 cells maintained a greater stability than the virus from Vero cells. Serum-free microcarrier cultures of MRC-5 cells offer a potentially valuable system for the large-scale production of live-attenuated DEN vaccines. 相似文献