ABSTRACT
The overwhelming majority of influenza vaccines are prepared with the use of chicken embryo allantoic fluid. The presence of ovalbumin (this protein constitutes >60% total protein in the allantoic fluid) in the vaccine can lead to severe allergy. Hence, effective reduction of ovalbumin content is of crucial importance for vaccine production. We compared two methods of purification and concentration of influenza virus: zonal gradient ultracentrifugation and combined ultrafiltration/diafiltration and exclusion chromatography protocol, used for fabrication of seasonal vaccines. Combined chromatography is comparable with zonal centrifugation protocol by the results of ovalbumin removal (to meet standard requirements).
Subject(s)
Chromatography, Agarose/methods , Influenza Vaccines/isolation & purification , Ovalbumin/isolation & purification , Ultracentrifugation/methods , Ultrafiltration/methods , Amniotic Fluid/chemistry , Amniotic Fluid/virology , Animals , Chick Embryo , Humans , Influenza A Virus, H1N1 Subtype/growth & development , Influenza A Virus, H1N1 Subtype/immunology , Influenza Vaccines/biosynthesis , Influenza, Human/immunology , Influenza, Human/prevention & control , Influenza, Human/virologyABSTRACT
The recombinant strain Flu-NS1-124-Omp16 (H5N1) of the influenza virus expressing the brucellar Omp16 gene was constructed on the basis of the technology of reverse genetics for the purpose of developing vector anti-brucellosis vaccine. The obtained recombinant strain is a genetically stable construction. This stability is confirmed by the comparative analysis of the nucleotide sequences of the HA, NA, and NS genes of the recombinant vector virus Flu-NS1-124-Omp16 (H5N1) expressing the Omp16 gene of the Brucella abortus (GenBank: AAA59360.1). The comparative analysis showed that the nucleotide sequence of the NS gene of the first and the fifth passage level of the Flu-NS1-124-Omp16 (H5N1) virus corresponded for 100% to the initial part of 12AAS2TC_124 Omp16g containing the chimera NS1-124-Omp16 in the composition of DNA (deoxyribonucleic acid) plasmids pHW2000. Total identity with HA and NA genes of the strain A/AstanaRG/6:2/2009 (H5N1) was shown by the comparative analysis of the nucleotide sequences of HA and NA genes of the first and the fifth passage level of the recombinant strain Flu-NS1-124-Omp16 (H5N1). The recombinant vector virus Flu-NS1-124-Omp16 (H5N1) expressing the brucella Omp16 gene maintains the genetic stability during 5 passages in 10-day developing chicken embryos.
Subject(s)
Bacterial Outer Membrane Proteins , Brucella abortus/genetics , Genes, Bacterial , Genetic Vectors , Hemagglutinin Glycoproteins, Influenza Virus , Influenza A Virus, H5N1 Subtype , Neuraminidase , Viral Nonstructural Proteins , Viral Proteins , Animals , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Chick Embryo , Genetic Vectors/genetics , Genetic Vectors/metabolism , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Influenza A Virus, H5N1 Subtype/genetics , Influenza A Virus, H5N1 Subtype/metabolism , Neuraminidase/genetics , Neuraminidase/metabolism , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
The paper describes comparative evaluation of IAVchip DNA microarray, reverse transcription PCR (RT-PCR), and real-time RT-PCR versus virus isolation in chicken embryos and shows their diagnostic effectiveness in detection and subtyping of influenza A virus. The tests were evaluated with use of 185 specimens from humans, animals, and birds. IAVchip DNA microarray demonstrates higher diagnostic effectiveness (99.45%) in early influenza A diagnosis as compared to the real-time PCR (98.38%) and RT-PCR (96.22%), thus showing its clear superiority. Diagnostic sensitivity of IAVchip DNA microarray (100%) exceeds the same of RT-PCR (95.95%) and real-time RT-PCR (97.96%) in the range of estimated confidence intervals. IAVchip DNA microarray and real-time RT-PCR displayed equal diagnostic specificity (98.85%), while diagnostic specificity of RT-PCR was 96.40%. IAVchip DNA microarray has an advantage over the other tests for influenza A diagnosis and virus identification as a more rapid method that allows performing simultaneous detection and subtyping of about tens of specimens within one experiment during 8-10 hours. The developed IAVchip DNA microarray is a general test tool that enables identifying simultaneously 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes of influenza A virus and also to screen the influenza A viruses from humans, animals, and birds by M and NP genes.
