A reliable alternative method could allow faster release of the vaccine for mass immunization, when the time is of essence to save numerous human lives 11, 12. Owing to the fact that at least 2–3 months are required to make these reagents from the time when a new strain is identified, the WHO recommends that the manufacturers develop alternative methods to quantify the HAs in the vaccine preparations in the event of a pandemic outbreak 10. These reagents are updated and distributed annually by the WHO collaborating centers. The current SRID method depends on the availability of HA antigen references and the corresponding antiserum for the standardization of vaccine quantification. As HA is the major surface protein that induces protective immune responses, it is measured as a vaccine potency marker by single radial immunodiffusion (SRID) method 9. Such problems associated with influenza vaccines are largely due to frequent mutations in the genes for the viral surface proteins (HA and neuraminidase). Obviously, these shortcomings would be significantly exacerbated in the event of a pandemic influenza outbreak. This approach has inherent disadvantages, including the uncertainty about the actual circulating strain, the need for annual updating of the manufacturing process in addition to the preparation of reagents for vaccine lot release 4, 5, 6, 7, 8. Specifically, the World Health Organization (WHO) recommends the updating of strains of the seasonal influenza vaccines about 6–8 months before the upcoming flu season. Indeed, the seasonal influenza vaccines have to be annually updated 4, 5, 6, 7, 8. The ever-increasing number of HA sequences reflects the rapidly evolving nature of the virus, making it a daunting challenge for vaccine development. Currently, there are thousands of HA sequences submitted in the public database and the number of sequence is constantly increasing 3. There are 16 subtypes of influenza A viral HAs 2, but in the same subtype of HAs, the amino acid sequences can vary widely 3, 4. Torrance L, Andreev IA, Gabrenaite-Verhovskaya R, Cowan G, Makinen K, Taliansky ME (2006) An unusual structure at one end of potato potyvirus particles.Hemagglutinin (HA) is the principle surface protein of influenza virus that induces neutralizing antibodies 1. Manoussopoulos IN, Tsagris M (2015) Native electrophoresis and western blot analysis (NEWeB): methods and applications. Manoussopoulos IN, Maiss E, Tsagris M (2000) Native electrophoresis and Western blot analysis (NEWeB): a method for characterization of different forms of potyvirus particles and similar nucleoprotein complexes in extracts of infected plant tissues. Jerusalem, Israel, Xth International Congress of virologyīlanc S, Lopez-Moya JJ, Wang RY, Garcia-Lampasona S, Thornbury DW, Pirone TP (1997) A specific interaction between coat protein and helper component correlates with aphid transmission of a potyvirus. Manoussopoulos IN, Maiss E, Tsagris E (1996) Detection of virus particle-helper component complexes in Nicotiana benthamiana plants infected with plum pox virus. Govier DA, Kassanis BA (1974) Virus-induced component of plant sap needed when aphids acquire potato virus Y from purified preparations. Govier DA, Kassanis B (1974) Evidence that a component other than the virus particle is needed for aphid transmission of potato virus Y. Tsagris M, Liu B, Tzortzakaki S, Tabler M (1993) Construction of a full length cDNA clone of an aphid transmissible PPV strain from Greece. Gibbs A, Harrison BD (1976) Plant virology: the principles. Prentice Hall International Limited, London Levy AJ, Fraenkel-Conrat H, Owens AR (1994) Virology. Uses include studying VP in cause and effect or in time-interval experiments, VP structural studies, and VP interaction with the host or viral proteins and also the study of VP-protein complexes. This technique could be used in similar studies of different viruses or large protein complexes, using the electrophoretic mobility of virus particles (VP) and proteins with western blot specificity. This study used NEWeB to show Plum pox virus particles’ interaction with the helper component, a virus-encoded protein. Viral proteins are loaded in agarose or mixed agarose-acrylamide gels and separated under native conditions and transferred to nitrocellulose or other suitable membranes. Manoussopoulos and Mina Tsagris developed and described Native Electrophoresis and Western Blot Analysis (a procedure they term as NEWeB) to study plant virus features, including virus particle-protein interactions, electrophorotype formation, and strain separation.
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