Improved Dengue Virus Plaque Formation on BHK21 and LLCMK 2

Improved Dengue Virus Plaque Formation on BHK21 and LLCMK 2 Cells: Evaluation of Some Factors Mayling Alvarez, Rosmari Rodriguez-Roche, Lídice Bernardo, Luis Morier and Maria G. Guzman! Department of Virology, PAHO/WHO Collaborating Center for Viral Diseases, "Pedro Kourí" Tropical Medicine Institute, Havana, Cuba Abstract Neutralizing antibodies play a key role in the prevention of dengue infection. It is important that dengue virus plaque titration and plaque reduction neutralization tests (PRNT) be highly reproducible using standardized methods. To evaluate factors that have influence in the PRNT for dengue viruses, neutralizing antibodies were determined in 24 serum samples and 12 blood samples collected on filter paper, obtained through national dengue surveillance in Cuba. The influence of ph in the overlay medium, the percentage of ambient CO 2, the use of two different cell lines and of rapid centrifugation on dengue plaque formation were evaluated. The efficiency of the plaquing system was optimal when the overlay medium was buffered to ph 7.5. The rapid centrifugation of the virus on confluent cells increased the virus titres. Higher virus titres were obtained on BHK21 rather than LLCMK 2 cells when the viruses were added to cell suspension. Under optimal conditions, PRNT was highly reproducible and is recommended for seroepidemiological and vaccine studies using either BHK21 or LLCMK 2 cells. This communication also highlights the infection of LLCMK 2 cell suspensions for measuring neutralizing antibodies. Keywords: Dengue, plaque reduction neutralization technique, diagnosis, Cuba. Introduction Dengue fever (DF)/dengue haemorrhagic fever (DHF) has re-emerged as a public health problem worldwide [1-3]. The dengue viruses are serologically classified into four antigenically distinct serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) and are maintained in a humanmosquito transmission cycle, with Aedes aegypti as the main vector. Currently, dengue is endemic in most of the tropical areas in the world [4,5]. Despite the antigenic relatedness of dengue viruses, two or more serotypes may sequentially infect one host. Epidemiological studies in Thailand and Cuba showed that DHF occurs in individuals with a secondary infection. These studies suggest that the presence of heterotypic dengue antibodies in a sequential secondary infection is a risk factor for developing the severe disease [6,7]. Neutralizing antibodies to dengue virus play a very significant role in the prevention of dengue! lupe@ipk.sld.cu; " 53-7-2020450; Fax: 53-7-2046051 Dengue Bulletin Vol 29, 2005 49

infection. Non-neutralizing cross-reactive antibodies augment dengue virus infection of Fc receptor cells. These virus antibody complexes bind to Fc receptors on cells via the Fc portion of IgG, resulting in enhancement of dengue virus infection. This phenomenon has been called antibody-dependent enhancement (ADE) [8,9]. The ideal vaccine against DF and DHF must prevent infection caused by all serotypes avoiding ADE. Ideally, life-long neutralizing antibodies to the four serotypes should be present [10,11]. Towards this end, technological innovation for improved plaque formation such as speed, accuracy, economy, ease of performing test as well as global standardization is urgently required. Plaque reduction neutralization technique (PRNT) remains the gold standard for the titration of neutralizing antibodies. Several protocols have been described; however, factors affecting the reproducibility of dengue plaque assays have not been fully studied [12-21]. These include the use of solid or semi-sold substances in overlay media, the cell substrate, serum inhibitors and ph of the overlay medium. Taking into account the need to determine neutralizing antibodies for seroepidemiological and vaccine studies, we decided to validate a reliable plaque assay system to the four dengue serotypes. The influence of ph range in the overlay medium and the percentage of CO 2 atmosphere for cell incubation were evaluated. Also, the usefulness of rapid centrifugation (shell vial assay) to improve the efficiency of virus adsorption and consequently plaque formation was also evaluated. Finally, efficiency for virus titration and PRNT of baby hamster kidney cell line (BHK21) and LLCMK 2 as cell suspensions were compared. Materials and Methods Viruses Table 1 shows the dengue virus strains used in this study. Serum specimens A total of 24 sera obtained through the Cuban national dengue surveillance system during Table 1. Details of the strains utilized in the neutralization tests P: passage, MB: mouse brain, M: mosquitoes 50 Dengue Bulletin Vol 29, 2005

2000 and 12 blood samples collected on filter paper from healthy individuals during 2003 were selected for study. The first samples were used to test the reproducibility of PRNT on BHK21 cells. Filter paper eluates were tested for DENV-1 to DENV-4 neutralizing antibodies on BHK21 and LLCMK 2 cells at a 1:30 dilution, using exactly the same methods used previously [22]. Antibody titres obtained in both cells were compared. Cell culture BHK21, clone 15 cells were grown at 37 C in complete medium E-MEM (Minimum Essential Medium with Earle s balance salts) supplemented with 10% of heat-inactivated foetal bovine serum, 1% penicillin/ streptomycin, 10% L-glutamine and sodium bicarbonate for adjusting the medium to ph 7.4-7.8. LLCMK 2 cells (ATCC) were kindly provided by Sutee Yoksan (Center for Vaccine Development, Mahidol University) and were grown in the same conditions. Virus titration Plaque titration was performed on BHK21, clone 15 and in LLCMK 2 as previously described by Morens et al [16]. For virus titration, 50 ul of virus dilutions (10 1 to 10 6 ) was added to 0.5 ml of BHK21 cell suspension (1.5 x 10 5 cells) in each of three wells of a 24-well polystyrene plate. After 4 hours incubation at 37 C in CO 2 atmosphere, each well received 0.5 ml of 3% medium viscosity carboxymethyl cellulose made up in Earle s minimum essential medium without phenol red (MEM) with 10% heat-inactivated foetal bovine serum, 1% glutamine 2 mm and 100 U of penicillin plus 100 ug/ml streptomycin. Infected cells were incubated for 5 9 days in the same conditions as above depending on the virus serotype (7 9 days for DENV-1 and DENV-3, 5 days for DENV-2 and 6 days for DENV-4) in the case of BHK21, and for the LLCMK 2 cells the plaques of all the serotypes were counted on the 7 th day. After incubation, plates were rinsed gently under tap water and fixed and stained with a solution of naphthol blue black and acetic acid [16]. Plaque Reduction Neutralization Test (PRNT) PRNT was performed on both tested cell lines. For antibody titration, 100 ul of serum dilution (in MEM) was incubated for 1 hour at 37 C with 100 ul of the virus working dilution calculated to give 10-20 pfu/50 ul of the final volume of virus-serum mixture. After incubation, 50 ul of virus-serum mixture was added to the cell suspension in triplicate. The ph during the virus adsorption was in the range 7-7.5. Plates were incubated for 4 hours at 37 C under CO 2 atmosphere. The rest was followed as above. Each serum dilution was tested four times. Calculations of 50% endpoint plaque reduction neutralization titres were made using log probit paper by the method of Russell et al., 1967 [23]. Evaluation of ph of overlay medium, CO 2 atmosphere and rapid centrifugation of virus during cell adsorption phase on the efficiency of dengue virus plaque assays in BHK21 cells (1) In order to evaluate the influence of the ph of the nutrient overlay medium, this was adjusted at 6.5, 7, 7.5 and 8 final ph using a solution of 7.5% sodium bicarbonate. Cells were incubated under CO 2 atmosphere at 4% and the four dengue serotypes were titrated. (2) To test the influence of CO 2 atmosphere, the ph of the overlay medium was adjusted to 7.5, and cells were incubated Dengue Bulletin Vol 29, 2005 51

under CO 2 atmosphere both at 4 and 4.5%. Under these conditions the four dengue serotypes were titrated. (3) For the evaluation of the rapid centrifugation assay, 24 well plates were used. DENV-2/A15 strain was inoculated on cells in suspension or monolayer and centrifuged at 37.73 x G (1500 rpm) for 30 min at 37 C using an ALC model PM 140/140R centrifuge. After centrifugation, cells were incubated for 4 hours at 37 C and overlay media (at ph 7.5) was added. Infected cells were incubated at 37 C in CO 2 atmosphere at either 4 or 4.5%. Results Evaluation of different factors influencing dengue virus plaque assays in BHK21 cells Effect of ph of overlay medium Titres of DENV-1, 3 and 4 depended on ph of overlay media (Table 2). The highest titres were observed at ph 7.5. For DENV-2, titres were relatively similar at ph values ranging from 6.5 to 7.5, ph 7.5 being the optimal. Table 2. Effect of ph of overlay medium on dengue viruses plaque formation NP: no plaques Influence of CO 2 atmosphere Considering that the highest titres of the four dengue serotypes were observed at ph 7.5 of the overlay medium, this ph was used to test the influence of CO 2 atmosphere. In general, titre and plaque size were relatively unaffected by cell incubation under 4 or 4.5% CO 2 atmosphere. Influence of rapid centrifugation of virus during cell adsorption phase Table 3 shows the effect of dengue virus plaque formation of rapid centrifugation assay both in cell suspensions and confluent monolayer. Rapid centrifugation on confluent cell cultures gave the highest viral titres, compared to the conventional method, both at 4 and 4.5% of CO 2 atmosphere. Table 3. Effect on dengue virus plaque formation of rapid centrifugation assay using cell suspensions and confluent monolayer at two different percentages of CO 2 atmosphere* * ph of overlay medium was adjusted to 7.5 52 Dengue Bulletin Vol 29, 2005

Table 4. Replicate 50% neutralizing antibody titres and geometric mean titres (GMT) to DENV-1 and DENV-2 on BHK21 cells *Each serum was tested twice at two different days. Results are presented here as number 1 to 4. Dengue Bulletin Vol 29, 2005 53

Reproducibility of dengue PRNT assays in BHK21 To test the reproducibility of PRNT assays, sera were tested twice on two different days (in total, antibody titre in each serum was determined four times). Titres less than 1/10 to DENV-3 and -4 were obtained. Only 2 (8.3%) sera showed greater than twofold differences in the neutralizing antibody titre to DENV-1 (Table 4). Comparison of BHK21 and LLCMK 2 cells for virus titration and assay of neutralizing antibody Titres of the four dengue viruses on BHK21 and LLCMK 2 cell lines using an overlay media buffered to ph 7.5 and incubated at 37 C in a 4% CO 2 atmosphere were compared. With the exception of DENV-3, the highest viral titres were obtained on BHK21 cell cultures (Table 5). Viral plaques were similar in size and morphology in both cell lines with the exception of DENV-4 plaques that were larger in both cultures. Table 5. DENV-1 to -4 titres determined by plaque assay on BHK21 and LLCMK 2 cell suspensions* *Plaque titre (Log 10 pfu/ml) The percentage of plaque reduction to each serotype was determined in 12 samples collected on filter papers tested in both cell lines. The results were similar in 100% of the samples (Table 6). Discussion Taking into account the necessity of measuring neutralizing antibodies during vaccine trials, it is important to apply standardized and validated methods. However, factors affecting the reproducibility of dengue plaque assays have not been fully identified. Of the factors studied in this work, the ph of the overlay medium was the most critical variable. The optimal ph of the overlay medium for obtaining clear dengue virus plaques was ph 7.5. Other investigators have reported optimal dengue virus plaquing over a wide range of ph values in the overlay medium (ph 6.6 to 8.6) when using LLCMK 2 cells [19]. When DENV-3 was assayed, plaque size decreased at ph 8.6 [19]. Dieg and Watkins [24] have found that Colorado tick fever virus plaque production was relatively insensitive over a range of ph of 7.1 to 8.1 while plaque formation failed to occur at ph 7.0 or lower. In hamster lung cell, plaque formation of Japanese encephalitis virus was not observed when ph of the overlay medium was under 6.6 or above 7.8. [18] Lee et al [25]. have reported dengue plaque formation at a ph range of 7.4 to 7.6. Sodium bicarbonate concentrations have been reported to influence plaque size with differences two- to eight-fold differences, especially for DENV-2, -3 and -4 [19]. In our study no significant difference in the plaque size was observed over the range of ph values tested, with the exception of DENV-4 which produced large plaques at ph 7.5. In general, titre and plaque size were relatively unaffected by incubating infected cells at different percentages of CO 2 atmosphere. Stim TB [19] have reported increased sensitivity of dengue virus plaque assays at high concentrations of sodium bicarbonate. 54 Dengue Bulletin Vol 29, 2005

Table 6. Comparative plaque reduction neutralization of DENV-1 to -4 in BHK21 and LLCMK 2 cells infected as cell suspension* *Whole blood on filter papers was re-suspended at 1:30 dilution In our laboratory, the rapid centrifugation or shell vial assay has greatly improved the isolation rate of some viruses. This method of enhancing virus-cell interactions decreases the time of the onset of cytopathic effect of many viruses present in clinical diagnostic specimens. It was applied to plaque assays and improved consistency of input virus dose. For dengue viruses, rapid centrifugation improved the isolation rate in blood and tissues [26]. Recently, Chingsuwanrote et al. (2004) observed a relationship of the number of plaques and postinfection incubation time, with the longest postinfection incubation time giving the highest number of plaques [27]. Dengue Bulletin Vol 29, 2005 55

Several cell lines have been used for dengue plaque assays but there are few published comparisons between cell systems using optimized conditions [12,14,15,17,27]. In the present study, virus titres were higher in BHK21 than in LLCMK 2 cell cultures, except for DENV-3. In summary, (i) the ph of the overlay medium may be an important factor in obtaining clear and large dengue plaques; (ii) the centrifugation of virus inoculate on cells increases virus titres; (iii) higher virus titres were obtained in BHK21 compared with LLCMK 2 cell cultures; (iv) highly reproducible dengue PRNT titres were obtained on BHK21 cells; and (v) using the cell suspension method, DENV-3 resulted in higher titres in LLCMK 2 than BHK21 cells. The current work confirms that under optimal conditions, plaque reduction neutralization titres can be repeated reproducibly. Seroepidemiological and vaccine studies can be performed satisfactorily in both BHK21 and LLCMK 2 cells. Acknowledgements This work was supported by TDR/WHO grant ID No. 990904. We thank Prof. Robert Shope of Texas University and Prof. Scott Halstead from the Pediatric Dengue Vaccine Initiative, Bethesda, Maryland, USA, for their useful suggestions and comments. References [1] Guzman MG and Kouri G. Dengue and dengue hemorrhagic fever in the Americas: lessons and challenges. J Clin Virol. 2003; 27(1): 1-13. [2] Guzman M, Kouri G, Diaz M, Llop A, Vazquez S, Gonzalez D, Castro O, Alvarez A, Fuentes O, Montada D, Padmanabha H, Sierra B, Perez A, Rosario D, Pupo M, Diaz C and Sanchez L. Dengue, one of the great emerging health challenges of the 21 st century. Expert Rev Vaccines. 2004; 3(5): 511-520. [3] Gubler DJ. Epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the 21 st century. Trends Microbiol. 2002; 10(2): 100-103. [4] Gubler DJ. Dengue and dengue hemorrhagic fever. Clin Microbiol Rev. 1998; 11(3): 480-496. [5] Guzman MG and Kouri G. Dengue: an update. Lancet Infect Dis. 2002; 2(1): 33-42. [6] Guzman MG, Kouri G, Valdes L, Bravo J, Alvarez M, Vazques S, Delgado I and Halstead SB. Epidemiologic studies on dengue in Santiago de Cuba, 1997. Am J Epidemiol. 2000; 152(9): 793-799. [7] Sangkawibha N, Rojanasuphot S, Ahandrik S, Viriyapongse S, Jatanasen S, Salitul V, Phanthumachinda B and Halstead SB. Risk factors in dengue shock syndrome: a prospective epidemiologic study in Rayong, Thailand. I. The 1980 outbreak. Am J Epidemiol. 1984; 120(5): 653-669. [8] Halstead SB. Pathogenesis of dengue: challenges to molecular biology. Science. 1988; 239(4839): 476-481. [9] Halstead S. The pathogenesis of dengue. Molecular epidemiology in infectious disease. Am J Epidemiol. 1981; 114: 632-648. [10] Halstead SB and Deen J. The future of dengue vaccines. Lancet. 2002; 360(9341): 1243-1245. [11] Monath TP, McCarthy K, Bedford P, Johnson CT, Nichols R, Yoksan S, Marchesani R, Knauber M, Wells KH, Arroyo J and Guirakhoo F. Clinical proof of principle for ChimeriVax: recombinant live, attenuated vaccines against flavivirus infections. Vaccine. 2002; 20(7-8): 1004-1018. [12] Georgiades J, Stim TB, McCollum RW and Henderson JR. Dengue virus plaque formation in Rhesus monkey kidney cultures. Proc Soc Exp Biol Med. 1965; 118: 385-388. [13] Guzman MG and Kouri G. Advances in dengue diagnosis. Clin Diagn Lab Immunol. 1996; 3(6): 621-627. [14] Hotta S, Fujita N and Maruyama T. Research on dengue in tissue culture. I. Plaque formation in an 56 Dengue Bulletin Vol 29, 2005

established monkey kidney cell line culture. Kobe J Med Sci. 1966; 12(3): 179-187. [15] Morens DM, Halstead SB and Larsen LK. Comparison of dengue virus plaque reduction neutralization by macro and semi-micro methods in LLC-MK2 cells. Microbiol Immunol. 1985; 29(12): 1197-1205. [16] Morens DM, Halstead SB, Repik PM, Putvatana R and Raybourne N. Simplified plaque reduction neutralization assay for dengue viruses by semimicro methods in BHK-21 cells: comparison of the BHK suspension test with standard plaque reduction neutralization. J Clin Microbiol. 1985; 22(2): 250-254. [17] Rao BL. Plaque formation of dengue viruses in Vero cell culture under carboxy-methylcellulose overlay. Indian J Med Res. 1976; 64(12): 1709-1712. [18] Schulze IT and Schlesinger RW. Plaque assay of dengue and other group B arthropod-borne viruses under methyl cellulose overlay media. Virology. 1963; 19: 40-48. [19] Stim TB. Dengue virus plaque development in simian cell systems. I. Factors influencing virus adsorption and variables in the agar overlay medium. Appl Microbiol. 1970; 19(5): 751-756. [20] Stim TB. Dengue virus plaque development in simian cell systems. II. Agar variables and effect of chemical additives. Appl Microbiol. 1970; 19(5): 757. [21] Yuill TM, Sukhavachana P, Nisalak A and Russell PK. Dengue-virus recovery by direct and delayed plaques in LLC-MK2 cells. Am J Trop Med Hyg. 1968; 17(3): 441-448. [22] Bravo JR, Guzman MG and Kouri GP. Why dengue haemorrhagic fever in Cuba? 1. Individual risk factors for dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS). Trans R Soc Trop Med Hyg. 1987; 81(5): 816-820. [23] Russell PK, Nisalak A, Sukhavachana P and Vivona S. A plaque reduction test for dengue virus neutralizing antibodies. J Immunol. 1967; 99(2): 285-290. [24] Dieg EF and Watkins HMS. Plaque assay procedure for Colorado tick fever virus. J Bacteriol. 1964; 88: 42-45. [25] Lee GC-Y, Grayston T and Kenney G. Growth of Japanese encephalitis virus in cell cultures. J Infect Dis. 1955; 115: 321-329. [26] Rodriguez-Roche R, Alvarez M, Guzman MG, Morier L and Kouri G. Comparison of rapid centrifugation assay with conventional tissue culture method for isolation of dengue 2 virus in C6/36-HT cells. J Clin Microbiol. 2000; 38(9): 3508-3510. [27] Chingsuwanrote P, Suksanpaisan L and Smith DR. Adaptation of the plaque assay methodology for dengue virus infected HepG2 cells. J Virol Methods. 2004; 116(2): 119-121. Dengue Bulletin Vol 29, 2005 57