scholarly journals Evaluation of avian adenovirus inactivation methods used in the production of influenza vaccines

2021 ◽  
Author(s):  
NN Savina ◽  
AA Ekimov ◽  
VP Trukhin ◽  
AE Evtushenko ◽  
EN Zhirenkina ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Cell Line ◽  
Uv Light ◽  
Influenza Vaccines ◽  
Optimum Conditions ◽  
Tetradecyltrimethylammonium Bromide ◽  
Avian Adenovirus ◽  
Series Of Experiments ◽  
Chicken Eggs ◽  
Embryonated Chicken

Inactivation of influenza virus and other potential contaminants like avian adenoviruses coming from embryonated chicken eggs is a critical step in the production of inactivated influenza vaccines. Inactivation must lead to a guaranteed reduction in contaminant titers by at least 4 lg (PFU)/ml. The aim of this study was to identify an optimum cell line for adenovirus propagation and to estimate a reduction in adenovirus titers in vaccine intermediates after inactivation. In a series of experiments, we identified the optimum conditions and the optimum cell line for the propagation of avian adenovirus (strains CELO and Fontes). The most commonly used inactivation methods were analyzed, including inactivation by β-propiolactone and UV light. Viral titers were measured by plaque assays. After 10 h of inactivation with β-propiolactone, CELO titers fell by 4.12 ± 0.06 lg, whereas Fontes titers, by 4.20 ± 0.19 lg, suggesting that β-propiolactone is an effective inactivating agent. Exposure to UV light led to a reduction in CELO titers by 4.69 ± 0.89 lg and a reduction in Fontes titers by 4.44 ± 1.06 lg after 5 min. N-octyl-β-D-glucopyranoside added at the splitting step reduced CELO titers by 0.93 ± 0.15 lg and Fontes titers by 1.04 ± 0.12 lg, whereas tetradecyltrimethylammonium bromide led to a reduction in CELO and Fontes titers by 1.18 ± 0.17 lg and 1.12 ± 0.38 lg, respectively.

scholarly journals Improvement of Influenza A/Fujian/411/02 (H3N2) Virus Growth in Embryonated Chicken Eggs by Balancing the Hemagglutinin and Neuraminidase Activities, Using Reverse Genetics

2005 ◽  
Vol 79 (11) ◽  
pp. 6763-6771 ◽  
Author(s):  
Bin Lu ◽  
Helen Zhou ◽  
Dan Ye ◽  
George Kemble ◽  
Hong Jin
Keyword(s):  
Amino Acids ◽  
Virus Replication ◽  
Receptor Binding ◽  
Influenza A ◽  
Reverse Genetics ◽  
Influenza Vaccines ◽  
H3n2 Virus ◽  
Virus Growth ◽  
Chicken Eggs ◽  
Embryonated Chicken

ABSTRACT The H3N2 influenza A/Fujian/411/02-like virus strains that circulated during the 2003-2004 influenza season caused influenza epidemics. Most of the A/Fujian/411/02 virus lineages did not replicate well in embryonated chicken eggs and had to be isolated originally by cell culture. The molecular basis for the poor replication of A/Fujian/411/02 virus was examined in this study by the reverse genetics technology. Two antigenically related strains that replicated well in embryonated chicken eggs, A/Sendai-H/F4962/02 and A/Wyoming/03/03, were compared with the prototype A/Fujian/411/02 virus. A/Sendai differed from A/Fujian by three amino acids in the neuraminidase (NA), whereas A/Wyoming differed from A/Fujian by five amino acids in the hemagglutinin (HA). The HA and NA segments of these three viruses were reassorted with cold-adapted A/Ann Arbor/6/60, the master donor virus for the live attenuated type A influenza vaccines (FluMist). The HA and NA residues differed between these three H3N2 viruses evaluated for their impact on virus replication in MDCK cells and in embryonated chicken eggs. It was determined that replication of A/Fujian/411/02 in eggs could be improved by either changing minimum of two HA residues (G186V and V226I) to increase the HA receptor-binding ability or by changing a minimum of two NA residues (E119Q and Q136K) to lower the NA enzymatic activity. Alternatively, recombinant A/Fujian/411/02 virus could be adapted to grow in eggs by two amino acid substitutions in the HA molecule (H183L and V226A), which also resulted in the increased HA receptor-binding activity. Thus, the balance between the HA and NA activities is critical for influenza virus replication in a different host system. The HA or NA changes that increased A/Fujian/411/02 virus replication in embryonated chicken eggs were found to have no significant impact on antigenicity of these recombinant viruses. This study demonstrated that the reverse genetics technology could be used to improve the manufacture of the influenza vaccines.

scholarly journals Modeling influenza virus infection in mature Wistar rats

2020 ◽  
Vol 65 (3) ◽  
pp. 159-166
Author(s):  
V. A. Marchenko ◽  
S. V. Barashkova ◽  
I. A. Zelinskaya ◽  
Y. G. Toropova ◽  
E. V. Sorokin ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Cardiovascular System ◽  
Blood Vessels ◽  
Wistar Rats ◽  
Pulmonary Vessels ◽  
Influenza Virus A ◽  
Infectious Titer ◽  
Last Stage ◽  
Chicken Eggs ◽  
Embryonated Chicken

It has now been established that blood vessels are target for influenza, but the mechanism by which the influenza virus affects the cardiovascular system is unknown. The aim – adaptation of influenza virus A/St. Petersburg/48/16 H1N1(pdm09) to mature Wistar rats, as these animals are the main experimental model for studying the pathology of the cardiovascular system. Material and methods. Passage of influenza A virus (IAV) in embryonated chicken eggs, intranasal inoculation of rats with virus-containing material s, production of pulmonary homogenate, determination of IAV titer in embryonated chicken eggs, detection of histological changes in lung and pulmonary vessels. Results. The article presents the results of the adaptation of influenza virus A/St. Petersburg/48/16 H1N1(pdm09) to mature Wistar rats. The infectious titer of the virus in the homogenates of infected rats lungs at the last stage of adaptation was 7.0 lg EID50/ml. Histological studies revealed pronounced changes in the respiratory tract (spasm of bronchioles, submucosal edema, desquamation of ciliated epithelium of bronchioles) and pulmonary vessels (spasm, desquamation and swelling of endotheliocytes, dissociation and swelling of the elastic membrane and media). In order to identify IAV in blood vessels and lung tissues, an immunohistochemical study was performed using monoclonal antibodies to NP antigen of IAV. Conclusion. The data obtained allow us to conclude that the strain of influenza virus A/St. Petersburg/48/16 H1N1(pdm09) was adapted to mature Wistar rats maintaining virulent properties. The infectious titer of the virus at the last stage of adaptation was 7.0 lg EID50/ml. IAV identification is confirmed by immunohistochemical examination.

scholarly journals Natural Reassortants of Potentially Zoonotic Avian Influenza Viruses H5N1 and H9N2 from Egypt Display Distinct Pathogenic Phenotypes in Experimentally Infected Chickens and Ferrets

2017 ◽  
Vol 91 (23) ◽  
Author(s):  
Mahmoud M. Naguib ◽  
Reiner Ulrich ◽  
Elisa Kasbohm ◽  
Christine L. P. Eng ◽  
Donata Hoffmann ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Avian Influenza ◽  
Avian Influenza Virus ◽  
Highly Pathogenic Avian Influenza ◽  
Parent Virus ◽  
Highly Pathogenic ◽  
Pathogenic Avian Influenza Virus ◽  
Chicken Eggs ◽  
Embryonated Chicken

ABSTRACT The cocirculation of zoonotic highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 and avian influenza virus (AIV) of subtype H9N2 among poultry in Egypt for at least 6 years should render that country a hypothetical hot spot for the emergence of reassortant, phenotypically altered viruses, yet no reassortants have been detected in Egypt. The present investigations proved that reassortants of the Egyptian H5N1 clade 2.2.1.2 virus and H9N2 virus of the G1-B lineage can be generated by coamplification in embryonated chicken eggs. Reassortants were restricted to the H5N1 subtype and acquired between two and all six of the internal segments of the H9N2 virus. Five selected plaque-purified reassortant clones expressed a broad phenotypic spectrum both in vitro and in vivo. Two groups of reassortants were characterized to have retarded growth characteristics in vitro compared to the H5N1 parent virus. One clone provoked reduced mortality in inoculated chickens, although the characteristics of a highly pathogenic phenotype were retained. Enhanced zoonotic properties were not predicted for any of these clones, and this prediction was confirmed by ferret inoculation experiments: neither the H5N1 parent virus nor two selected clones induced severe clinical symptoms or were transmitted to sentinel ferrets by contact. While the emergence of reassortants of Egyptian HPAIV of subtype H5N1 with internal gene segments of cocirculating H9N2 viruses is possible in principle, the spread of such viruses is expected to be governed by their fitness to outcompete the parental viruses in the field. The eventual spread of attenuated phenotypes, however, would negatively impact syndrome surveillance on poultry farms and might foster enzootic virus circulation. IMPORTANCE Despite almost 6 years of the continuous cocirculation of highly pathogenic avian influenza virus H5N1 and avian influenza virus H9N2 in poultry in Egypt, no reassortants of the two subtypes have been reported. Here, the principal compatibility of the two subtypes is shown by forcing the reassortment between copassaged H5N1 und H9N2 viruses in embryonated chicken eggs. The resulting reassortant viruses displayed a wide range of pathogenicity including attenuated phenotypes in chickens, but did not show enhanced zoonotic propensities in the ferret model.

scholarly journals Subunit Influenza Vaccines Produced from Cell Culture or in Embryonated Chicken Eggs: Comparison of Safety, Reactogenicity, and Immunogenicity

2009 ◽  
Vol 200 (6) ◽  
pp. 849-857 ◽  
Author(s):  
Keith S. Reisinger ◽  
Stanley L. Block ◽  
Allen Izu ◽  
Nicola Groth ◽  
Sandra J. Holmes
Keyword(s):  
Cell Culture ◽  
Influenza Vaccines ◽  
Chicken Eggs ◽  
Embryonated Chicken

scholarly journals Generation of Live Attenuated Novel Influenza Virus A/California/7/09 (H1N1) Vaccines with High Yield in Embryonated Chicken Eggs

2009 ◽  
Vol 84 (1) ◽  
pp. 44-51 ◽  
Author(s):  
Zhongying Chen ◽  
Weijia Wang ◽  
Helen Zhou ◽  
Amorsolo L. Suguitan ◽  
Cindy Shambaugh ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Mdck Cells ◽  
Swine Influenza Virus ◽  
Single Amino Acid ◽  
High Yield ◽  
Virus Growth ◽  
Vaccine Candidates ◽  
Influenza Virus A ◽  
Chicken Eggs ◽  
Embryonated Chicken

ABSTRACT Several live attenuated influenza virus A/California/7/09 (H1N1) (CA09) candidate vaccine variants that possess the hemagglutinin (HA) and neuraminidase (NA) gene segments from the CA09 virus and six internal protein gene segments from the cold-adapted influenza virus A/Ann Arbor/6/60 (H2N2) virus were generated by reverse genetics. The reassortant viruses replicated relatively poorly in embryonated chicken eggs. To improve virus growth in eggs, reassortants expressing the HA and NA of CA09 were passaged in MDCK cells and variants exhibiting large-plaque morphology were isolated. These variants replicated at levels approximately 10-fold higher than the rate of replication of the parental strains in embryonated chicken eggs. Sequence analysis indicated that single amino acid changes at positions 119, 153, 154, and 186 were responsible for the improved growth properties in MDCK cells and eggs. In addition, the introduction of a mutation at residue 155 that was previously shown to enhance the replication of a 1976 swine influenza virus also significantly improved the replication of the CA09 virus in eggs. Each variant was further evaluated for receptor binding preference, antigenicity, attenuation phenotype, and immunogenicity. Mutations at residues 153, 154, and 155 drastically reduced viral antigenicity, which made these mutants unsuitable as vaccine candidates. However, changes at residues 119 and 186 did not affect virus antigenicity or immunogenicity, justifying their inclusion in live attenuated vaccine candidates to protect against the currently circulating 2009 swine origin H1N1 viruses.

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