Identification of Strains
How would you identify which strain of influenza is responsible for a particular outbreak?? What is used as markers?
Jenny Beepika and Richard Sima write: Viral culture is perhaps the “gold standard” of influenza testing, providing the base unit for other tests to go by. With this method, growth of influenza viruses is measured rather than their presence or absence. The cell lines are designed to support the growth of a wide range of viruses and the culture used consists of a cell monolayer, a method for detecting viral growth as well as specific identification. Isolation of viruses are used to characterize the subtype or strain of circulating influenza A viruses, including the emergence of antiviral resistance, the degree of antigenic drift from vaccine strains, and the presence of influenza A subtypes. These subtypes show antigenic drift from the vaccine strain or antigenic shift and present a pandemic threat. Since influenza strains are continuously evolving, laboratory-based surveillance for influenza isolates is critically important to the selection of strains for the next season's influenza vaccine. Cell lines such as Vero, mink lung, support the growth of viruses if trypsin is incorporated. CPE is not a consistent feature of influenza A. The lack of HAd specificity may be helpful in finding pandemic strains while HI (HAI) is used to identify viral subtypes. Polymerase Chain Reaction related methods of identifying infected cells are through immunoflourescence enzyme-linked immunoassays (EIA).
More direct methods of detection do not result in definitive characters of pandemic strains. The chances of detecting new pandemic strains increases when more targets sequences are used. These techniques are fast, safe, and stable and would help improve influenza preparedness. The RT-PCR assays use targets like matrix(M) proteins for genus-level identification. They also use hemagglutinin and neuraminidase targets for identification of avian subtypes and are not used for strain-level identification. The sensitivity of such tests are high and run at about 90 - 100% accuracy. Multiple real-time RT-PCR assays were developed for specific detection of the avian H5N1 virus. Should the tested samples register as positive, they should be forwarded to a public health lab or the Center for Disease Control immediately. Immunofluorescence (IFA) is used to identify influence to the species-level or for specific H subtypes. Other rapid direct tests detect the actual viral antigen on the patient specimens and are designed to identify influenza A only, influenza A or B without identifying the type, or influenza A or B without the specific identification. The sensitivities of the tests run from 40%- -80%, but are greater in children or earlier in the course of the illness.
One method of influenza identification is serological testing, which includes hemagglutinin inhibition test (HAI), enzyme immunoassay (EIA), complement fixation (CF) and virus neutralization test. Serological testing is a retrospective diagnosis and not widely available. Though paired sera is preferred, single convalescent sera is also useful for novel viruses. EIA assays neuraminidase inhibitors and is used with HAI to measure the ratio of antibody to hemagglutinin in the specimen. However, though they are more sensitive than CF, their high specificity makes its capacity to detect novel viral strains less effective.
Rapid antigen detection, another way to identify strains, is carried out in near-patient tests, immunoflourescence assays and, also EIA. In Immunoflourescence assays, respiratory epithelial cells are cleared of contaminating mucus by centrifusion which must be performed at no more than 500g because infected respiratory epithelial cells are very fragile. Control slides with infected and uninfected influenza A/H3 and H1 with a suitable control of monoclonal antibodies and conjugate to vie for a more accurate analysis. Positive results are obtained when cell's nucleus or cytoplasm emits an apple-green fluorescence.
Viral genome can be identified using PCR. A DNA copy is created by obtaining viral RNA and using the enzyme reverse transcriptase. The RNA genome is amplified specifically to one subtype with oligonucleotide primers specifically designed on the basis of the hemagglutinin sequence of either influenza A/H5 or A/H9 or the neuraminidase of NI-1 or NI-2. After being put through an agarose gel electrohoresis, he expected size of the H5 influenzas is 219 base pairs, 383 for H9 and 616 base pairs for both NI-1 and NI-2.
http://www.fluwikie.com/pmwiki.php?n=Science.DiagnosticTestingForInfluenza
http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu.html
Khadeeja & Sara write: Although strain types are characterized by their H and N surface glycoproteins, strain pathogenicity is also dictated by other factors, including the range of H, N, viral genes, and host factors such as immune response genes, age and sex. Tests for influenza available during outbreaks include viral culture, serology, polymerase chain reaction (PCR), rapid antigen testing, and immunofluorescence assays. Laboratory tests are also widely used to identify influenza virus at the genus level (influenza A/B) or at the H-type level (H1, H3, and H5). H subtype specific tests must be used to identify potential avian strains, including H5N1 and In vivo tests are used to identify HPAI and LPAI strains. So, there are genetic and serologic markers used to identify influenza strains. Another interesting fact is that antigenic strain nomenclature is based on a few things including the host of origin, geographic origin, strain number, year of isolation and HA
Dan Szyprowski writes: We would identify the strain of influenza, as A (H5N1) is responsible for possible outbreaks of the virus. This virus can be transmitted through birds facilitating a potential global spread of H5N1. Markers that are used would be certain receptors that populate the avian respiratory system from the nose to the lungs.
You would be able to check to see if there was something potentially wrong with their alveoli, and other parts of their avian respiratory tract. It can spread by antigenic drift, by mutating in to known genetic variations and pathogenic varieties divided in to genetic clades, which are known from specific isolates, but all currently belonging to genotype Z of avian influenza virus H5N1, which is now the dominant trait. There is still no human form of this particular virus. The immune system also has a great deal to do with how serious the disease can actually get.
http://en.wikipedia.org/wiki/H5N1 Reference
Kate Zmijewski and Joe Robinson write: Using genes that have been reconstructed is one way in which scientists have gathered important new clues to why a virus is spread quickly and killed efficiently. Adding the genes to a comparatively benign strain of influenza shows that minor genetic change can turn a mild form of the virus into a highly virulent strain. The influenza pandemics usually occur when a new strain emerges to which people have little or no immunity. However, it is impossible to predict which strain will emerge as the next pandemic strain, when it will occur or how severe it will be. Identifying the characteristics help identify and protect people from influenza. Learning and knowing what genes are responsible for causing severe illness helps scientists develop new drugs and vaccines for prevention. Some studies use historical samples to reconstruct the evolutionary history of the virus.