Performance of methylcellulose and Avicel overlays in plaque and focus assays of Chikungunya virus

Josephine Kimani, George Osanjo, Rosemary Sang, James Ochanda, Francis Mulaa

Abstract


Background: Chikungunya virus is a re-emerging pathogen that is responsible for Chikungunya fever periodic outbreaks along the Kenyan coast and in other African countries.  Epidemiological data from the World Health Organization show that in 2014-2015, there was a major outbreak of Chikungunya fever in the Americas and Pacific Islands.  Surveillance and correct diagnosis are therefore key in controlling the spread and management of the disease.

Plaque and focus assays are key techniques in viral characterization or quantification, and both assays typically require overlay with gelling polymers to limit the spread of viruses in cell culture.  There are anecdotal reports that Avicel may be superior to methylcellulose in assay of Influenza virus. However, it is unclear whether this would apply to other viruses.

Objective: The objective of this study was to determine the performance of methylcellulose and Avicel overlays in plaque and focus assays of Chikungunya virus.

Methods: Confluent Vero cells were seeded in 6- or 96-well plates for plaque and focus assays respectively. Cells were inoculated with serially diluted Chikungunya virus, and incubated to allow adherence of the virus to the cells. The inoculum was removed; replaced with Avicel or methylcellulose overlay at various concentrations and stained with crystal violet or immunostained.  Statistical significance was computed using the Holm-Sidak test.

Results: The size of plaques formed by Chikungunya virus was dependent on the concentration of both Avicel and methylcellulose gels used as overlays, with Avicel overlays giving consistently larger plaques than methylcellulose.  Chikungunya virus formed plaques nearly 2.5 times larger in diameter (2 vs 0.8 mm) with 1.2 % Avicel than with 1.25 % methylcellulose after 60 hr growth.  Plaques formed with Avicel were better defined and easier to count after 48 hr growth period compared to a 60 hr period. However, methycellulose overlays provided smaller, more distinct and better defined foci in focus assays.

Conclusion: Both methylcellulose and Avicel are good overlay media for viral assays. Avicel is marginally better for plaque assays while methylcellulose provides more distinct and easier to count foci in focus assays.

Key words: Chikungunya virus, plaque assay, focus assay, methylcellulose, Avicel


References


Abedon ST, Yin J (2009). Bacteriophage plaques: theory and analysis. In: Bacteriophages. Springer, pp 161–174

Alvarez LJ, Thomen P, Makushok T, Chatenay D (2007) Propagation of fluorescent viruses in growing plaques. Biotechnol. Bioeng. 96:615–621.

Charrel RN, de Lamballerie X, Raoult D (2007) Chikungunya outbreaks--the globalization of vectorborne diseases. New Engl. J. Med. 356:769–771.

Chipwaza B, Mugasa JP, Selemani M, Amuri M, Mosha F, Ngatunga SD, Gwakisa PS (2014). Dengue and Chikungunya fever among viral diseases in outpatient febrile children in Kilosa district hospital, Tanzania. PLoS Negl. Trop. Dis. 8:e3335. doi: 10.1371/journal.pntd.0003335

Flint S, Racaniello VR, Skalka AM (2009). Virological methods. Principles of Virology ASM Press Wash DC ISBN 1–55581.

Herzog P, Drosten C, Müller MA (2008). Plaque assay for human coronavirus NL63 using human colon carcinoma cells. Virol. J. 5:138. doi: 10.1186/1743-422X-5-138

Holm S (1979). A simple sequentially rejective multiple test procedure. Scand. J. Stat. 6: 65–70

Jupp PG, McIntosh BM (1988) Chikungunya virus disease. Arboviruses Epidemiol. Ecol. 2:137–57.

Kariuki Njenga M, Nderitu L, Ledermann JP, Ndirangu A, Logue CH, Kelly CH, Sang R, Sergon K, Breiman R, Powers AM (2008) Tracking epidemic Chikungunya virus into the Indian Ocean from East Africa. J. Gen. Virol. 89:2754–2760.

Khan K, Bogoch I, Brownstein JS, Miniota J, Nicolucci A, Hu W, Nsoesie EO, Cetron M, Creatore MI, German M, Wilder-Smith A (2014). Assessing the Origin of and Potential for International Spread of Chikungunya Virus from the Caribbean. PLoS Curr. doi:10.1371/currents.outbreaks.2134a0a7bf37fd8d388181539fea2da5

Koch AL (1964). The growth of viral plaques during the enlargement phase. J. Theor. Biol. 6:413–431.

Leparc-Goffart I, Nougairede A, Cassadou S, Prat C, de Lamballerie X (2014) Chikungunya in the Americas. Lancet 383:514.

Matrosovich M, Matrosovich T, Garten W, Klenk H-D (2006) New low-viscosity overlay medium for viral plaque assays. Virol. J. 3:63.

McKimm-Breschkin JL (2004). A simplified plaque assay for respiratory syncytial virus—direct visualization of plaques without immunostaining. J. Virol. Methods. 120:113–117.

Nasci RS (2014) Movement of Chikungunya Virus into the Western Hemisphere. Emerg. Infect. Dis. 20:1394–1395.

Pickard DJJ, Clokie MR, Kropinski AM (2009). Bacteriophages: Methods and Protocols, Volume 2 Molecular and Applied Aspects.

Randhawa AS, Stanton GJ, Green JA, Baron S (1977) Variables affecting viral plaque formation in microculture plaque assays using homologous antibody in a liquid overlay. J. Clin. Microbiol. 5:535–542.

Tournebize P, Charlin C, Lagrange M (2009) [Neurological manifestations in Chikungunya: about 23 cases collected in Reunion Island]. Rev. Neurol. (Paris). 165:48–51.

WHO (2015) Chikungunya Factsheet No. 327.


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