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Robert G Webster - One of the best experts on this subject based on the ideXlab platform.
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Different incubation temperatures affect viral polymerase activity and yields of low-pathogenic avian influenza viruses in embryonated Chicken Eggs
Archives of Virology, 2011Co-Authors: Victoria Lang, Richard J Webby, Henju Marjuki, Scott L. Krauss, Robert G WebsterAbstract:Various incubation conditions (35°C–38°C, 2–7 days) have been used in surveillance studies of the prevalence of avian influenza viruses in wild birds. Here, we studied viral polymerase activity and virus growth kinetics of low-pathogenic avian influenza viruses (LPAIVs) isolated from field samples [A/duck/Hong Kong/365/1978 (H4N6) and A/duck/Nanchang/2–0480/2000 (H9N2)] during incubation at different temperatures (35°C, 37°C, and 39°C) in the allantoic cavity of 10-day-old embryonated Chicken Eggs (ECE). The higher incubation temperatures (37°C and 39°C) resulted in a significantly higher rate of virus growth, which is most likely a result of increased viral polymerase activity (20%–60%), than was observed at 35°C, and as much as a 100% greater virus yield (as measured by hemagglutination assay) was observed two days after inoculation. Our findings revealed that the optimal activity of the viral polymerase complex, resulting in the highest yield of LPAIV field isolates, could be obtained by incubation for two days in ECE at 37°C and 39°C.
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molecular changes associated with adaptation of human influenza a virus in embryonated Chicken Eggs
Virology, 2006Co-Authors: Robert G Webster, Linda Widjaja, Natalia A Ilyushina, Richard J WebbyAbstract:Failure to isolate A/Fujian/411/2002 (H3N2) in embryonated Chicken Eggs resulted in its absence from the 2003/2004 vaccine. We analyzed the adaptation of this virus in Eggs during serial passages in the amniotic then allantoic cavities. Amniotic passage allowed the virus to grow in the allantoic cavity. During adaptation, 6 amino acid substitutions occurred: 4 in HA (G186V, S219F, V226I, V309I) and 2 in NA (E119Q, Q136K). These substitutions allowed binding to SAα2,3Gal- and SAα2,6Gal-containing receptors, conferred SAα2,3Gal specificity, and preserved antigenicity. Two HA substitutions (G186V, V226I) were sufficient to improve growth. Changing 2 NA residues (E119Q, Q136K) did not improve growth, and adaptation did not result in the HA changes H183L, D188Y, and V226A found by others. These findings suggest that viral adaptation in Eggs involves multiple strategies. Vaccine manufacture will benefit from increased understanding of adaptation and strategies to improve human influenza A virus replication in Eggs.
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growth and immunogenicity of influenza viruses cultivated in vero or mdck cells and in embryonated Chicken Eggs
Developments in biological standardization, 1999Co-Authors: Elena A Govorkova, Shantha Kodihalli, Irina V Alymova, B Fanget, Robert G WebsterAbstract:Vero cells, MDCK cells and embryonated Chicken Eggs (Eggs) were used to evaluate influenza virus growth characteristics and immunogenicity induced by inactivated influenza B vaccines. Both cell lines produced comparable quantities of total viral and haemagglutinin (HA) proteins. Sequence analysis indicated genetic identity of the HA of Vero- and MDCK-grown virus counterparts with maintenance of antigenic characteristics of viruses derived from humans. The egg-grown influenza B/Memphis/1/93 variant differed from cell-grown counterparts at amino acid position 198 (Pro-Thr) and lost a glycosylation site. The level of neuraminidase (NA) activity was the highest in egg-grown virus, while MDCK- and Vero cell-grown viruses possessed 70% and 90% less NA activity respectively when fetuin was used as a substrate. Although each of the vaccines induced high and comparable levels of serum antibodies, mammalian cell-derived vaccines induced antibodies that were more cross reactive, and those antibodies induced by egg-derived vaccine were more specific to the homologous antigen. ELISPOT analysis indicated that the mammalian cell-grown vaccines induced high frequencies of IgG-producing cells directed against both cell- and egg-grown antigens, while egg-grown vaccine induced high frequencies of IgG and IgM-producing cells reacting with homologous antigen and low levels of IgG-producing cells reactive with cell-grown virus antigen. Taken together, our results suggest that mammalian cells are a viable option for the production of influenza virus vaccines.
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immunogenicity and protective efficacy in mice of influenza b virus vaccines grown in mammalian cells or embryonated Chicken Eggs
Journal of Virology, 1998Co-Authors: Irina V Alymova, Robert G Webster, Elena A Govorkova, Shantha Kodihalli, B Fanget, C GerdilAbstract:The immunogenicity and protective efficacy of formalin-inactivated influenza B/Memphis/1/93 virus vaccines propagated exclusively in Vero cells, MDCK cells, or embryonated Chicken Eggs (hereafter referred to as Eggs) were investigated. Mammalian cell-grown viruses differ from the egg-grown variant at amino acid position 198 (Pro/Thr) in the hemagglutinin gene. The level of neuraminidase activity was highest in egg-grown virus, while MDCK and Vero cell-derived viruses possessed 70 and 90% less activity, respectively. After boosting, each of the vaccines induced high levels of hemagglutinin-inhibiting, neuraminidase-inhibiting, and neutralizing antibodies that provided complete protection from MDCK-grown virus challenge. Mammalian cell-derived virus vaccines induced serum antibodies that were more cross-reactive, while those induced by egg-grown virus vaccines were more specific to the homologous antigen. Enzyme-linked immunospot analysis indicated that cell-grown virus vaccines induced high frequencies of immunoglobulin G (IgG)-producing cells directed against both cell- and egg-grown virus antigens, whereas egg-grown virus vaccine induced higher frequencies of IgG- and IgM-producing cells reacting with homologous antigen and low levels of IgG-producing cells reactive with cell-grown viruses. These studies indicate that influenza B virus variants selected in different host systems can elicit different immune responses, but these alterations had no detectable influence on the protective efficacy of the vaccines with the immunization protocol used in this study.
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egg fluids and cells of the chorioallantoic membrane of embryonated Chicken Eggs can select different variants of influenza a h3n2 viruses
Virology, 1995Co-Authors: Charles T Hardy, Sarah A Young, Robert G Webster, Clayton W Naeve, Randall J OwensAbstract:Growth of influenza viruses in embryonated Eggs frequently results in the selection of virus variants with amino acid changes near the receptor-binding pocket of the hemagglutinin molecule, yet the mechanism by which this third form of influenza variation occurs (the other two being antigenic drift and shift) has not been clearly defined. Because egg-mediated variation might affect influenza vaccine and surveillance programs, we have initiated studies to determine the site(s) of variant virus selection within the embryonated egg. In this report we show that both the cells of the chorioallantoic membrane (CAM) and the fluids from embryonated Chicken Eggs are capable of selecting variant influenza viruses, but that these variants are distinct at the molecular level depending on the conditions of virus propagation. Serial passage of viruses in cells of the chorioallantoic membrane selects one set of variants which possess specific amino acid changes near the receptor binding pocket of the hemagglutinin molecule characteristic of viruses grown in embryonated Eggs. However, passage of the same viruses in mammalian tissue culture cells supplemented with egg fluids selects a separate set of hemagglutinin variants also characteristic of viruses grown in Eggs, yet at different residues from those observed following passage in CAM. These results suggest that two separate mechanisms may exist in the embryonated egg that lead to the selection of variant influenza viruses: one at the cellular level and another at the extracellular level.
Richard J Webby - One of the best experts on this subject based on the ideXlab platform.
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proteolytic enzymes in embryonated Chicken Eggs sustain the replication of egg grown low pathogenicity avian influenza viruses in cells in the absence of exogenous proteases
Journal of Virological Methods, 2014Co-Authors: Ahmed Kandeil, Richard J Webby, Ola Bagato, Hassan Zaraket, Jennifer Debeauchamp, Scott Krauss, Rabeh Elshesheny, Mohamed A Ali, Ghazi KayaliAbstract:Low pathogenic influenza viruses grow readily in embryonated Chicken Eggs but require the addition of exogenous proteases to grow in MDCK cell culture. In this study, we found that the influenza viruses propagated previously in Eggs, can grow for up to two passages in cell culture without the addition of exogenous proteolytic enzymes. These results indicate that the reason for virus propagation in cells during the first two passages may be due to proteases from egg allantoic fluid carried over from egg culture. The ability of influenza viruses to grow in cells in the absence of trypsin is currently considered as a hallmark of highly pathogenic influenza viruses. Our data indicate that differentiating between high and low pathogenicity using cell culture only is not appropriate and other indicators such as sequence analysis and in vitro pathogenicity index should be performed.
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Different incubation temperatures affect viral polymerase activity and yields of low-pathogenic avian influenza viruses in embryonated Chicken Eggs
Archives of Virology, 2011Co-Authors: Victoria Lang, Richard J Webby, Henju Marjuki, Scott L. Krauss, Robert G WebsterAbstract:Various incubation conditions (35°C–38°C, 2–7 days) have been used in surveillance studies of the prevalence of avian influenza viruses in wild birds. Here, we studied viral polymerase activity and virus growth kinetics of low-pathogenic avian influenza viruses (LPAIVs) isolated from field samples [A/duck/Hong Kong/365/1978 (H4N6) and A/duck/Nanchang/2–0480/2000 (H9N2)] during incubation at different temperatures (35°C, 37°C, and 39°C) in the allantoic cavity of 10-day-old embryonated Chicken Eggs (ECE). The higher incubation temperatures (37°C and 39°C) resulted in a significantly higher rate of virus growth, which is most likely a result of increased viral polymerase activity (20%–60%), than was observed at 35°C, and as much as a 100% greater virus yield (as measured by hemagglutination assay) was observed two days after inoculation. Our findings revealed that the optimal activity of the viral polymerase complex, resulting in the highest yield of LPAIV field isolates, could be obtained by incubation for two days in ECE at 37°C and 39°C.
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molecular changes associated with adaptation of human influenza a virus in embryonated Chicken Eggs
Virology, 2006Co-Authors: Robert G Webster, Linda Widjaja, Natalia A Ilyushina, Richard J WebbyAbstract:Failure to isolate A/Fujian/411/2002 (H3N2) in embryonated Chicken Eggs resulted in its absence from the 2003/2004 vaccine. We analyzed the adaptation of this virus in Eggs during serial passages in the amniotic then allantoic cavities. Amniotic passage allowed the virus to grow in the allantoic cavity. During adaptation, 6 amino acid substitutions occurred: 4 in HA (G186V, S219F, V226I, V309I) and 2 in NA (E119Q, Q136K). These substitutions allowed binding to SAα2,3Gal- and SAα2,6Gal-containing receptors, conferred SAα2,3Gal specificity, and preserved antigenicity. Two HA substitutions (G186V, V226I) were sufficient to improve growth. Changing 2 NA residues (E119Q, Q136K) did not improve growth, and adaptation did not result in the HA changes H183L, D188Y, and V226A found by others. These findings suggest that viral adaptation in Eggs involves multiple strategies. Vaccine manufacture will benefit from increased understanding of adaptation and strategies to improve human influenza A virus replication in Eggs.
John A Lednicky - One of the best experts on this subject based on the ideXlab platform.
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gas permeable ethylene bags for the small scale cultivation of highly pathogenic avian influenza h5n1 and other viruses in embryonated Chicken Eggs
Virology Journal, 2010Co-Authors: Sara B Hamilton, Deirdre E Daniels, William Sosna, Eric R Jeppesen, Julie M Owells, Micah D Halpern, Kimberly S Mccurdy, Jon O Rayner, John A LednickyAbstract:Embryonated Chicken Eggs (ECE) are sometimes used for the primary isolation or passage of influenza viruses, other viruses, and certain bacteria. For small-scale experiments with pathogens that must be studied in biosafety level three (BSL3) facilities, inoculated ECE are sometimes manipulated and maintained in small egg incubators within a biosafety cabinet (BSC). To simplify the clean up and decontamination of an egg incubator in case of egg breakage, we explored whether ethylene breather bags could be used to encase ECE inoculated with pathogens. This concept was tested by determining embryo survival and examining virus yields in bagged ECE. Virus yields acceptable for many applications were attained when influenza-, alpha-, flavi-, canine distemper-, and mousepox viruses were propagated in ECE sealed within ethylene breather bags. For many small-scale applications, ethylene breather bags can be used to encase ECE inoculated with various viruses.
Kevin M Keener - One of the best experts on this subject based on the ideXlab platform.
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high voltage atmospheric cold plasma treatment of refrigerated Chicken Eggs for control of salmonella enteritidis contamination on egg shell
Lwt - Food Science and Technology, 2017Co-Authors: Yi Chen, S K Pankaj, Kevin M KeenerAbstract:Abstract Salmonella Enteritidis (SE) contamination is a major risk for U.S. consumers from Chicken Eggs. This study is primarily focused on evaluation of a novel High Voltage Atmospheric Cold Plasma (HVACP) technology for inactivation of Salmonella and its effect on the egg quality. Spot inoculated Chicken Eggs were treated with high voltage cold plasma (85 kV) under direct and indirect mode of exposure in dry air and modified atmospheric gas environment. A reduction of 5.53 log cfu/egg was observed for egg surfaces directly treated under modified atmospheric gas for 15 min. SE reductions was found dependent on treatment times, gas type, and mode of exposure of Eggs to the plasma. No significant difference (p > 0.05) in direct and indirect mode of exposure was observed on the egg quality after plasma treatment. These results demonstrate the potential of HVACP to be used as a suitable non-thermal treatment for reducing Salmonella from packaged Chicken Eggs.
Mawrong Lee - One of the best experts on this subject based on the ideXlab platform.
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determination of cyromazine and melamine in Chicken Eggs using quick easy cheap effective rugged and safe quechers extraction coupled with liquid chromatography tandem mass spectrometry
Analytica Chimica Acta, 2012Co-Authors: Peicheng Wang, Renjye Lee, Chungyu Chen, Chichung Chou, Mawrong LeeAbstract:Abstract A rapid and sensitive method has been developed for the simultaneous detection of cyromazine and melamine in Chicken Eggs using the quick, easy, cheap, effective, rugged and safe (QuEChERS) method coupled with liquid chromatography–tandem mass spectrometry (LC–MS/MS). The optimal extraction solvent for the liquid–liquid extraction was 5 mL of acetonitrile with a 0.1 M hydrochloric acid aqueous solution (99.5:0.5, v/v). The extract was cleaned with 0.5 g of anhydrous magnesium sulfate and 10 mg of graphitized carbon black. The analysis of cyromazine and melamine was accomplished by combining the use of an anion exchange LC column with tandem mass spectrometry in the positive electrospray ionization mode with selected reaction monitoring mode (SRM). The detection limits were 1.6 ng g−1 for cyromazine and 8 ng g−1 for melamine, and the quantitation limits were 5.5 ng g−1 for cyromazine and 25 ng g−1 for melamine. The recoveries of cyromazine and melamine in the spiked egg samples were 83.2% and 104.6%, respectively, with an relative standard deviation (RSD) of less than 18.1%. The intra-day and inter-day precisions, represented by the RSD, ranged from 1.5% to 8.8% and 6.8% to 14.3%, respectively. The proposed method was tested by analyzing Chicken Eggs from the markets and from the veterinary medicine laboratory. The concentrations of cyromazine and melamine detected in these samples were in the range of 20–94 ng g−1. The results demonstrated that the QuEChERS method combined with LC–MS/MS is a simple, rapid and inexpensive method for the analysis of cyromazine and melamine in Eggs.