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Volume 31 Issue 2
April 2016
    Citation: Robert William Figliozzi, Feng Chen, Albert Chi, Hsia Shao-Chung Victor. Using the inverse Poisson distribution to calculate multiplicity of infection and viral replication by a high-throughput fluorescent imaging system .VIROLOGICA SINICA, 2016, 31(2) : 180-183.  http://dx.doi.org/10.1007/s12250-015-3662-8
    shu

    Using the inverse Poisson distribution to calculate multiplicity of infection and viral replication by a high-throughput fluorescent imaging system

    • 1.

      Department of Pharmaceutical Sciences, School of Pharmacy and Health Professions, University of Maryland Eastern Shore, Princess Anne, Maryland 21853, USA

    • 2.

      Department of Natural Sciences, School of Agriculture and Natural Sciences, University of Maryland Eastern Shore, Princess Anne, Maryland 21853, USA

    • 3.

      Department of Mathematics and Computer Sciences, University of Maryland Eastern Shore, Princess Anne, Maryland 21853, USA

    • Corresponding author: Hsia Shao-Chung Victor, vhsia@umes.edu, ORCID: 0000-0001-7623-2842
    • Published Date: 26 January 2016
      Available online: 01 April 2016
    • Here, we introduce an additional method that we believe has merits with respect to reducing labor, decreasing wait times, improving objectivity, and decreasing long-term costs. This new method, called fluorescently labeled infected cell inoculum titration (FLICIT), utilizes high-throughput fluorescent microscopy instrumentation such as Cytation 3 from Biotek, and is therefore only applicable to transgenic viruses that cause host cells to express fluorescent proteins. For the purpose of developing this method, we used the recombinant virus HSV-1 strain 17-Syn+, which expresses green fluorescent protein (GFP) (Figure 1A) and exhibits the same replication pattern as the wild type counterpart (Foster et al., 1998). This new method relies on titrating the inoculum so that dilution ensures that the rate of infection is less than 50%, with cells infected by nearly one viral particle each; thus, each fluorescent signal can be attributed to a single viral particle.
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      1. Bedadala G, Chen F, Figliozzi R, et al. 2014. ISRN Virol, DOI: 10.1155/2014/629641.

      2. Bushar G, Sagripanti JL. 1990.J Virol Methods, 30: 99-107.
          doi: 10.1016/0166-0934(90)90047-J

      3. Dulbecco R. 1952. Proc Natl Acad Sci USA, 38: 747-752.
          doi: 10.1073/pnas.38.8.747

      4. Figliozzi RW, Chen F, Balish M, et al. 2014.J Neurol Sci, 346: 164-173.
          doi: 10.1016/j.jns.2014.08.017

      5. Foster TP, Rybachuk GV, Kousoulas KG. 1998. J Virol Methods, 75: 151-160.
          doi: 10.1016/S0166-0934(98)00107-4

      6. Gao Y, Nankya I, Abraha A, et al. 2009. Methods Mol Biol, 485: 27-35.

      7. Grigorov B, Rabilloud J, Lawrence P, et al. 2011.PLoS One, 6: e24135.
          doi: 10.1371/journal.pone.0024135

      8. Gueret V, Negrete-Virgen JA, Lyddiatt A, et al. 2002. Cytotechnology, 38: 87-97.
          doi: 10.1023/A:1021106116887

      9. Harris DC. 2003. In: Quantitative chemical analysis, 6th ed. Harris DC (ed). New York: W. H. Freeman and Co. pp. 56.

      10. Held P, Bank P. 2015. Analysis of Nuclear Stained Cells -Using the Cytation3 Cell Imaging Multi-Mode Microplate Reader with DAPI-Stained Cells. BioTek.

      11. Hsia SC, Graham LP, Bedadala GR, et al. 2013. Br Microbiol Res J, 3: 706–723.
          doi: 10.9734/BMRJ

      12. Knipe DM, Howley P. 2013. In: Fields virology, 6th edn. Knipe DM, Howley P (eds). Philadelphia: Wolters Kluwer/Lippincott Williams & Wilkins Health. pp. 42-50.

      13. Kutner RH, Zhang XY, Reiser J. 2009. Nat Protoc, 4: 495-505.
          doi: 10.1038/nprot.2009.22

      14. LaBarre DD, Lowy RJ. 2001. J Virol Methods, 96: 107-126.
          doi: 10.1016/S0166-0934(01)00316-0

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      Using the inverse Poisson distribution to calculate multiplicity of infection and viral replication by a high-throughput fluorescent imaging system

        Corresponding author: Hsia Shao-Chung Victor, vhsia@umes.edu
      • 1. Department of Pharmaceutical Sciences, School of Pharmacy and Health Professions, University of Maryland Eastern Shore, Princess Anne, Maryland 21853, USA
      • 2. Department of Natural Sciences, School of Agriculture and Natural Sciences, University of Maryland Eastern Shore, Princess Anne, Maryland 21853, USA
      • 3. Department of Mathematics and Computer Sciences, University of Maryland Eastern Shore, Princess Anne, Maryland 21853, USA
      • Published Date: 26 January 2016

      Abstract: Here, we introduce an additional method that we believe has merits with respect to reducing labor, decreasing wait times, improving objectivity, and decreasing long-term costs. This new method, called fluorescently labeled infected cell inoculum titration (FLICIT), utilizes high-throughput fluorescent microscopy instrumentation such as Cytation 3 from Biotek, and is therefore only applicable to transgenic viruses that cause host cells to express fluorescent proteins. For the purpose of developing this method, we used the recombinant virus HSV-1 strain 17-Syn+, which expresses green fluorescent protein (GFP) (Figure 1A) and exhibits the same replication pattern as the wild type counterpart (Foster et al., 1998). This new method relies on titrating the inoculum so that dilution ensures that the rate of infection is less than 50%, with cells infected by nearly one viral particle each; thus, each fluorescent signal can be attributed to a single viral particle.

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