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Journal of Virological Methods 146 (2007) 393–396
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A carboxymethyl-cellulose plaque assay for feline calicivirus Jaime Escobar-Herrera, Fernando Jos´e Medina-Ram´ırez, Ana Lorena Guti´errez-Escolano ∗ Department of Experimental Pathology, Centro de Investigaci´on y de Estudios Avanzados del IPN (CINVESTAV-IPN), Av. IPN 2508, Col. San Pedro Zacatenco, C.P. 07360 M´exico, D.F. Mexico Received 21 May 2007; received in revised form 16 July 2007; accepted 19 July 2007 Available online 23 August 2007
Abstract The standardization of a plaque assay for feline calicivirus in Crandell Reese feline kidney cells using carboxymethyl-cellulose as an overlay medium is described in this report. This methodology gives comparable counts as compared to the standard assay, and prevents monolayer roll over and peel off, as well as easy medium removal. Cell fixation and staining is performed in a considerably reduced period of time, compared to agarose-based methods. © 2007 Elsevier B.V. All rights reserved. Keywords: Feline calicivirus; Plaque assay; Carboxymethyl-cellulose; Crandell Reese feline kidney cells; Gastroenteritis
The family Caliciviridae comprises a variety of different human and animal pathogens. The human caliciviruses belong to the genera Norovirus and Sapovirus, whereas the genera Lagovirus and Vesivirus contain only animal pathogens. Human caliciviruses are recognized worldwide as the most common cause of foodborne and waterborne outbreaks of acute gastroenteritis in children and adults (Frankhauser et al., 1998; Green et al., 2001). Recently, these enteric viruses have also been associated with sporadic cases of gastroenteritis (Widdowson et al., 2005). Incidents of the rapid spread of human calicivirusesrelated illness in hospitals, nursing homes, military settings, restaurants, and cruise ships have lead to an increased recognition of the impact of these viruses on public health. The study of the biology of human caliciviruses has been hindered by the lack of cell culture systems. However, feline calicivirus, a member of the genus Vesivirus, which is easily grown in tissue culture and shares many of the biological properties of human caliciviruses, has been used as a model for studying some of the general replication strategies. The feline calicivirus can grow readily and produces clear cytopathic effects in culture cell lines, particularly in Crandell Reese feline kidney cells; therefore, its propagation and quantification by tissue culture infectious dose (TCID50 ) and plaque assay have been extensively reported. Different proce-
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dures and reagents, such as agar (Kalunda et al., 1975) or agarose incorporated in the overlay media, have been used for plaque assays (Bidawid et al., 2003). The standardization of the agarose plaque assay for feline calicivirus in Crandell Reese feline kidney cells has brought a sensitive and reproducible method for virus titration; this method has also been adapted to other cultivable caliciviruses such as the murine norovirus in RAW 264.7 cells (Hsu et al., 2005) and porcine enteric calicivirus in LLC-PK cells (Chang et al., 2005). However, the Crandell Reese feline kidney cell monolayer is often fragile and has a tendency to roll over and peel off the flask’s surface (Bidawid et al., 2003). Therefore, when the agarose is removed, the monolayer is commonly damaged, resulting in variations on plaque quantification. An interesting alternative is the use of carboxymethyl-cellulose, which increases the viscosity of the overlay medium preventing virus spreading; furthermore, it is easily removed from the tissue culture plates. Carboxymethyl-cellulose as an overlay medium has been broadly used in plaque assays of different viruses in several cell lines (Triantafilou and Triantafilou, 2004; Cologna and Rico-Hessle, 2003; Bae et al., 2003; Oien et al., 2002). In this article, we report the standardization of a feline calicivirus titration method in Crandell Reese feline kidney cells using carboxymethyl-cellulose. The use of carboxymethyl-cellulose in our system brings the advantage of smooth removal of the overlay medium, preventing monolayer damage. Additionally, cell fixation and staining of the monolayer after carboxymethylcellulose removal is performed in a considerably reduced period of time, compared to the agarose method.
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J. Escobar-Herrera et al. / Journal of Virological Methods 146 (2007) 393–396
Plaque assays were undertaken using monolayers of Crandell Reese feline kidney cells obtained from the American Type Culture Collection (Rockville, MD), propagated in minimum essential medium containing 10% horse serum (HS) and supplemented with antibiotics, at 37 ◦ C in humidified 5% CO2 incubator. 5 × 104 cells were seeded in 12-well tissue culture plates and allowed to adhere for 24 h, after which, cells were washed with minimum essential medium without serum, and tenfold serial dilutions of feline calicivirus (F9 strain, obtained from the American Type Culture Collection) (Rockville, MD) in minimum essential medium without serum, were inoculated in duplicate or triplicate and incubated for 1 h at 37 ◦ C. The overlay medium using carboxymethyl-cellulose was prepared during viral adsorption as follows: 2× carboxymethylcellulose (Sigma) solutions were prepared at concentrations of 1.6%, 2.0% and 2.4% (w/v) in water, and stirred for 1 h at room temperature (RT). After this time, equal volumes of 2× supplemented—minimum essential medium with 10% HS and antibiotics and each of the 2× carboxymethyl-cellulose solutions were mixed separately and incubated at 37 ◦ C until needed. At the end of the virus incubation period, the viral inoculum was aspirated, the cells were washed once with minimum essential medium and 400 l of each carboxymethyl-cellulose mixture was added to each well and incubated for 30 h at 37 ◦ C in a humidified 5% CO2 incubator. Finally, the overlay medium was aspirated; cells were washed with minimum essential medium, and fixed for 15 min with 250 l of a 3.7% solution of formaldehyde in PBS prepared just before its use. Plaques were visualized by staining for 10 min at RT with 0.1% (w/v) with crystal violet
in 20% (v/v) ethanol. For parallel plaque assays using overlay medium, equal volumes of 2× supplemented—minimum essential medium with 10% HS, antibiotics and 2× agarose (Invitrogen) (1.2% (w/v) in water) were prepared during the virus adsorption period and stored at 43 ◦ C until used (Bidawid et al., 2003). After the cells were washed, the same volume of both solutions described above were mixed and 400 l of the mixture was added to the monolayer; it was allowed to solidify at RT and incubated at 37 ◦ C for 30 h. After this time, monolayers were fixed overnight (shorter fixation times resulted in monolayer peel off) by adding 250 l of 3.7% solution of formaldehyde in PBS per well. Subsequently, formaldehyde was discarded and the agarose was removed under a gentle steam of cold running water. The plates were left to dry at RT and the cells were stained with crystal violet solution as described above. To determine if the carboxymethyl-cellulose, as an overlay medium, allows the formation of plaques with feline calicivirus, monolayers of Crandell Reese feline kidney cells were infected with 125.5, 24.9 and 12.45 pfu/well of feline calicivirus (Fig. 1I–III, respectively). Small homogeneous plaques were observed with all three of the different concentrations of carboxymethyl-cellulose (0.8%, 1.0% and 1.2%) used; however, the plaque size at 1.2% carboxymethyl-cellulose was even smaller than the plaques observed at 0.8% and 1.0% of carboxymethyl-cellulose (Fig. 1). In general, plaque size was smaller with carboxymethyl-cellulose than with agarose. It is important to note that in the presence of carboxymethylcellulose, monolayer integrity was well conserved even though
Fig. 1. Parallel plaque assays under 0.6% agarose or 0.8%, 1.0% and 1.2% of carboxymethyl-cellulose overlays. Crandell Reese feline kidney cells were mock infected or infected with 125.5, 24.9 and 12.45 pfu/well of feline calicivirus (left column, I–III, respectively). After 1 h incubation, carboxymethyl-cellulose at 0.8%, 1.0% and 1.2% or agarose at 0.6% was used as an overlay medium for 30 h at 37 ◦ C. Cultures were fixed and stained with crystal violet.
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J. Escobar-Herrera et al. / Journal of Virological Methods 146 (2007) 393–396 Table 1 Feline calicivirus titres per ml obtained by plaque assay using 0.8% carboxymethyl-cellulose or 0.6% agarose as the overlay medium Plaque assay (titers per ml) CMC 1.1 0.8 3.2 6.0 6.0 1.2
± ± ± ± ± ±
Agarose 0.7 × 107 0.3 × 107 0.2 × 107 0.1 × 104 0.5 × 106 0.4 × 106
1.1 1.0 4.0 5.6 5.6 1.2
± ± ± ± ± ±
0.1 × 107 0.0 × 107 0.1 × 107 0.4 × 104 0.6 × 106 0.2 × 106
Each value corresponds to the average of three independent assays.
a short period of fixation time was required. No plaque formation was observed at carboxymethyl-cellulose concentrations of 0.5% and 1.5% (data not shown). The average numbers of viral plaques counted from three different assays, using the same virus stocks, at the same dilutions, with carboxymethylcellulose at concentrations of 0.8%, 1.0% and 1.2% were 22.87 ± 3.01, 20.1 ± 1.60 and 11.42 ± 2.0, respectively, while the average number of plaques counted in agarose were 19.37 ± 3.5. Therefore, plaques obtained in carboxymethylcellulose concentrations of 0.8% and 1.0% correlate with the numbers obtained in agarose. Once it was established that the best concentration of carboxymethyl-cellulose in the overlay medium for plaque generation in Crandell Reese feline kidney cells was between 0.8% and 1.0%, the sensitivity of both carboxymethyl-cellulose and agarose plaque assays was compared. For this purpose, tenfold serial dilutions of the same feline calicivirus stock were tested in parallel by plaque assay using 0.8% carboxymethyl-cellulose and 0.6% agarose as the overlay medium. These assays were performed in triplicate. As shown in Table 1, similar titres of feline calicivirus were obtained using both methodologies. This result indicates that the viral titres obtained with 0.8% carboxymethylcellulose correlated well with those obtained with agarose. Therefore, although the plaques observed with carboxymethylcellulose were smaller compared to those obtained in agarose (Fig. 1), the viral titres obtained with both methods were equivalent. In this report, the standardization of an alternative method for a feline calicivirus plaque assay in Crandell Reese feline kidney cells, using carboxymethyl-cellulose as the overlay medium, is described. Carboxymethyl-cellulose is an innocuous reagent that increases the viscosity of the overlay medium; this prevents virus spreading and at the same time, preserves monolayer integrity. This characteristic has allowed its use for viral titration of different viruses in several cell lines (Triantafilou and Triantafilou, 2004; Cologna and Rico-Hessle, 2003; Bae et al., 2003; Oien et al., 2002), and as an alternative to solid gel overlays when 48- and 96-well culture plates are required (Matrosovich et al., 2006). Since carboxymethyl-cellulose can be smoothly removed, it prevents peel off or damage of the Crandell Reese feline kidney cell monolayer during the agarose removal from tissue culture plates. Moreover, cell fixation solution can be added directly to the cell monolayer, significantly reducing the fixation time.
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Several concentrations of carboxymethyl-cellulose have been reported to titre different viruses in several cell lines (Triantafilou and Triantafilou, 2004; Cologna and Rico-Hessle, 2003; Bae et al., 2003; Oien et al., 2002); however, our results indicate that between 0.8% and 1.0% of carboxymethyl-cellulose allows the formation of an equivalent amount of feline calicivirus plaques in Crandell Reese feline kidney cells than with the conventional agarose method. The 42% reduction in the number of plaques observed with 1.2% carboxymethyl-cellulose (Fig. 1), suggests that the viscosity of the overlay media at this particular concentration interferes with plaque formation. Even though the plaque sizes with carboxymethyl-cellulose were smaller than with agarose, they could still be counted without further magnification. Moreover, reduction in plaque size may help to discriminate single plaques better, when they are very close. The plaque assay with carboxymethyl-cellulose could also be useful in viral titration in smaller well culture plates where agarose removal is difficult. Finally, this methodology could be extended to other cultivable caliciviruses, such as the murine enteric calicivirus and porcine enteric calicivirus, which represent new models for the study of calicivirus replication in cell cultures. Acknowledgements We thank Rosa M. del Angel and Juan Ludert for helpful suggestions and critical comments on the manuscript. This work was supported by Grant 45710 from the Consejo Nacional de Ciencia y Tecnolog´ıa (CONACyT), M´exico. References Bae, H.G., Nitsche, A., Teichmann, A., Beil, S.S., Niedrig, M., 2003. Detection of yellow fever virus: a comparison of quantitative real-time PCR and plaque assay. J. Virol. Methods 110, 185–191. Bidawid, S., Malik, N., Adegbunrin, O., Sattar, S.A., Faber, J.M., 2003. A feline kidney cell line-based plaque assay for feline calicivirus, a surrogate for Norwalk virus. J. Virol. Methods 10, 163–167. Chang, K.O., Sosnovtsev, S.S., Belliot, G., Wang, Q., Saif, L.J., Green, K.Y., 2005. Reverse genetics system for porcine enteric calicivirus, a prototype sapovirus in the Caliciviridae. J. Virol. 79, 1409–1416. Cologna, R., Rico-Hessle, R., 2003. American genotype structures decrease dengue virus output from human monocytes and dendritic cells. J. Virol. 77, 3929–3938. Frankhauser, R.L., Noel, J.S., Monroe, S.S., Ando, T., Glass, R.I., 1998. Molecular epidemiology of “Norwalk-like viruses” in outbreaks of gastroenteritis in the United States. J. Infect. Dis. 178, 1571–1578. Green, K.Y., Chanock, R.M., Kapikian, A.Z., 2001. Human caliciviruses. In: Knipe, D.M., Howley, P.M., Griffin, D.E., Lamb, R.A., Martin, M.A., Roizman, B., Straus, S.E. (Eds.), Fields’ Virology, 4th ed. Lippincott Williams and Wilkins, Philadelphia, pp. 841–847. Hsu, C.C., Wobus, C.E., Steffen, E.K., Riley, L.K., Livingston, R.S., 2005. Development of a microsphere-based serologic multiplex fluorescent immunoassay and a reverse transcriptase PCR assay to detect murine norovirus 1 infection in mice. Clin. Diagn. Lab Immunol. 10, 1145–1151. Kalunda, M., Lee, K.M., Holmes, D.F., Gillespie, J.H., 1975. Serological classification of feline calicivirus by plaque/reduction neutralization and immunodiffusion. Am. J. Vet. Res. 36, 353–356. Matrosovich, M., Matrosovich, T., Garten, W., Deter Klenk, H., 2006. New low-viscosity overlay medium for viral plaque assays. Virol. J. 63, 1–7.
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