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Round, iridescent, green-blue  H1N1 (swine flu) virus with protrusions.

Influenza Research Materials

Sep 11, 2014 at 12:00 PM ET

Abstract

Influenza remains one of the most significant infectious diseases worldwide, causing acute respiratory tract illnesses and accounting for 25% of infections that exacerbate chronic lung infections. To date, several epidemics and four major influenza pandemics have been recorded. Influenza viruses have caused an estimated 3 million cases of serious illness and around 500,000 deaths annually worldwide. Influenza infections are primarily and effectively controlled by vaccines that elicit protective immunity. Influenza viruses undergo rapid antigenic shift and drift that results in the emergence of new strains each year. Therefore, influenza vaccines need to be reformulated every year to match the circulating strains. In this webinar, we will provide an overview of the influenza virus and will explore the current treatment strategies for influenza infections. We will also highlight viral strains and associated materials offered by ATCC that can be used in influenza research or in the development and validation of novel preventative and therapeutic techniques.

Presenter

Shamaila Ashraf, headshot.

Shamaila Ashraf, MSc, MPhil, PhD

Senior Scientist, ATCC

Questions and Answers

Can we calculate titer in PFU/mL from the TCID50? How are they related?

Assuming that the same cell system is used, that the virus forms plaques on those cells, and that no procedures are added which would inhibit plaque formation, 1 mL of virus stock would be expected to have about half of the number of plaque forming units (PFUs) as TCID50. This is only an estimate but is based on the rationale that the limiting dilution which would infect 50% of the cell layers challenged would often be expected to initially produce a single plaque in the cell layers which become infected. In some instances, two or more plaques might by chance form, and thus the actual number of PFUs should be determined experimentally. Mathematically, the expected PFUs would be somewhat greater than one-half the TCID50, since the negative tubes in the TCID50 represent zero plaque forming units and the positive tubes each represent one or more plaque forming units. A more precise estimate is obtained by applying the Poisson distribution.

For frozen viral preparations, how should we thaw the vial? Should we quick thaw?

ATCC recommends thawing in a 37°C water bath, until just melted.

I purchased a strain of Influenza A virus that is H1N1 and was isolated from swine, but I am not getting a positive PCR result using primers for detecting the pandemic strain. What could be wrong?

Not all influenza A H1N1 strains from swine are from the recent pandemic. If the H1N1 strain is not from the recent pandemic, it will not be detected using the diagnostic PCR tests currently available. The only influenza A pandemic strains we have confirmed by PCR are ATCC VR-1736 and VR-1737. RNA is also available for these strains as catalog numbers ATCC VR-1736D and VR-1737D.

What is the difference between a Plaque Assay and TCID50?

Both assays are used to calculate viral titer. The plaque assay was initially developed to count and measure the infectivity level of bacteriophages. This technique has since been modified for use in animal virology, and has been a reliable determination of titer for a number of tissue culture adapted viruses. The basis of this assay is to measure the ability of a single infectious virus to form a plaque on a cell culture monolayer. A plaque is developed as a part of the viral infection cycle, where following viral replication, the host cell dies. Viral titer can also be determined in vitro by calculating the infectious dose. For tissue culture adapted strains, this calculation is ascertained through an endpoint dilution assay in cell culture. The most reproducible endpoint of the dilution assay is the dilution of the virus that will produce a pathological change in 50% of the cell cultures inoculated. This number is expressed as 50% the infectious dose, or TCID50. The accuracy of this method is related to the number of replicates at each dilution. Overall, the plaque assay is much more sensitive than TCID50. For all newly acquisitioned tissue culture-adapted viruses, ATCC calculates titer using the plaque assay.