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Influenza A detection by MDCK cell line

The influenza A (IA) virus is the principal cause of the outbreaks of flu. A large number of laboratories participate in the worldwide surveillance of influenza virus activity and contribute to the early recognition of newly emerging epidemic strains. Differentiation between influenza A and B viruses and determination of the subtypes of influenza A virus isolates are the first steps in the characterization of influenza viruses. This analysis is traditionally done by hemagglutination inhibition (HI) tests with specific antisera raised in ferrets, chickens, or sheep.
The diagnosis of Influenza A is largely clinical. Nevertheless, it is necessary to carry out some form of rapid antigenic diagnosis and the culture of respiratory samples to confirm the etiology of the respiratory disease and to determine the antigenic characteristics of the epidemic strains. Although the “gold standard” isolation technique is inoculation in embryonated hens’ eggs, the technical difficulties involved and the delay in obtaining results mean that this technique is performed only in reference centers.
The Madin-Darby Canine Kidney (MDCK) continuous cell line seems to be one of the most useful for the isolation of the Influenza A virus in respiratory samples. The MDCK cell line has a greater capacity to yield IA virus from nasopharyngeal aspirates. This cell line has been shown to be the most sensitive, from both the qualitative and the quantitative points of view. Its routine use, therefore, seems necessary in order to obtain the maximum diagnostic yield.
The MDCK cell line was derived from kidney of an apparently normal adult female cocker spaniel, by S.H.Madin and N.B.Darby in September 1958. MDCK cells are of two types based on structure, function and lectin binding site: MDCK-1 and MDCK-2. MDCK cells differentiate into MDCK-1 cells in acid and hypertonic culture media, whereas MDCK-2 cells develop in alkaline and hypotonic culture media in sub-confluent and young confluent monolayers. Fully differentiated MDCK-1 and MDCK-2 cells cannot reverse their differentiation state in aged confluent monolayers. Relative sodium conductance is increased 48-fold in MDCK-1 cells. MDCK-1 acidifies their incubation medium, whereas MDCK-2 cells acidify dome fluid. Hypotonic stress further decreases HCO₃⁻concentration in dome fluid. Dome formation increases in higher cell passages; concomitantly the ratio of MDCK-2: MDCK-1 cells increases. Cloned MDCK cell populations become increasingly heterogeneous with increasing passage number. Thus it can be concluded that differentiation in MDCK cells is determined by cell culture conditions.
The MDCK cell shows typical epithelial morphology and also susceptibility to many viruses, including infectitious canine hepatitis, coxsakievirus B3 and B, reovirus 2 and 3, vesicular somatitis (Indiana strain), adenovirus 4 and 5, vesicular exanthema of swine, and vaccinia virus, according to the ATCC catalog search.
The present inventions motivates the culture of MDCK cells which can be used to grow viruses, e.g. influenza virus, particularly cold adapted, and/or temperature sensitive, and/or attenuated influenza viruses, in cell culture to a high titer. The MDCK cells can also be adapted or genetically modified to grow in serum-free culture medium for vaccine production. Culturing of MDCK cells plays a vital role not only in production of vaccine but also in study of nuraminidase activity assays for monitoring MDCK cell culture derived influenza virus, epithelial cell line with respect to genetics, lipid composition, expression of proteins and other parameters, furosemide-semitive salt transport in the Madin-Darby Canine Kidney Cell Line, etc.
Influenza viruses are single-stranded, negative sense, segmented RNA viruses that belong to the family Orthomyxoviridae. Three types of influenza viruses (types A, B, and C) have been described. Influenza A is a broad host-range respiratory pathogen affecting pigs, avians, equines, and humans and is further classified into various subtypes as per antigenic and genetic differences in its surface glycoproteins. To date, 15 hemagglutinin(HA) types and 9 neuraminidase (NA) types have been described. Swine influenza virus (SIV) infection is characterized by weight loss, anorexia, rhinitis, nasal discharge, sneezing, and coughing. Currently, 3 subtypes of SIV are prevalent in the United States, namely H1N1, H3N2, and H1N2. Early detection and subtyping of SIV are essential to managing this disease. The classical way of detecting SIV is virus isolation from suspect pigs followed by hemagglutination-inhibition (HI) and neuraminidase (NA) assays for subtyping.
Influenza virus particles envelope protein called the hemagglutinin, or HA, which binds to sialic acid receptors on cells. The virus will also bind to erythrocytes, causing the formation of a lattice. This property is called hemagglutination, and is the basis of a rapid assay to determine levels of influenza virus present in a sample. The basis of the HI assay is that antibodies to influenza virus will prevent attachment of the virus to red blood cells. Therefore hemagglutination is inhibited when antibodies are present. The highest dilution of serum that prevents hemagglutination is called the HI titer of the serum. If the serum contains no antibodies, then hemagglutination will be observed in all wells. Likewise, if antibodies to the virus are present, hemagglutination will not be observed until the antibodies are sufficiently diluted.


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