Improved Dengue Virus Plaque Formation on

BHK21 and LLCMK2 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 CO2, 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 LLCMK2 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 LLCMK2 cells. This

communication also highlights the infection of LLCMK2 cell suspensions for measuring neutralizing antibodies.

Keywords: Dengue, plaque reduction neutralization technique, diagnosis, Cuba.

Introduction Despite the antigenic relatedness of dengue viruses, two or more serotypes may Dengue fever (DF)/dengue haemorrhagic fever sequentially infect one host. Epidemiological (DHF) has re-emerged as a public health studies in Thailand and Cuba showed that DHF problem worldwide[1-3]. The dengue viruses are occurs in individuals with a secondary infection. serologically classified into four antigenically These studies suggest that the presence of distinct serotypes (DENV-1, DENV-2, DENV-3 heterotypic dengue antibodies in a sequential and DENV-4) and are maintained in a human- secondary infection is a risk factor for [6,7] mosquito transmission cycle, with Aedes aegypti developing the severe disease . as the main vector. Currently, dengue is endemic in most of the tropical areas in the Neutralizing antibodies to dengue virus play a world[4,5]. very significant role in the prevention of dengue

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infection. Non-neutralizing cross-reactive Taking into account the need to determine antibodies augment dengue virus infection of neutralizing antibodies for seroepidemiological Fc receptor cells. These virus antibody and vaccine studies, we decided to validate a complexes bind to Fc receptors on cells via reliable plaque assay system to the four dengue the Fc portion of IgG, resulting in enhancement serotypes. The influence of pH range in the

of dengue virus infection. This phenomenon overlay medium and the percentage of CO2 has been called antibody-dependent atmosphere for cell incubation were evaluated. enhancement (ADE)[8,9]. Also, the usefulness of rapid centrifugation (shell vial assay) to improve the efficiency of The ideal vaccine against DF and DHF virus adsorption and consequently plaque must prevent infection caused by all serotypes formation was also evaluated. Finally, efficiency avoiding ADE. Ideally, life-long neutralizing for virus titration and PRNT of baby hamster antibodies to the four serotypes should be kidney cell line (BHK21) and LLCMK as cell [10,11] 2 present . Towards this end, technological suspensions were compared. innovation for improved plaque formation such as speed, accuracy, economy, ease of performing test as well as global standardization Materials and Methods is urgently required. Viruses Plaque reduction neutralization technique (PRNT) remains the “gold standard” for the Table 1 shows the dengue virus strains used in titration of neutralizing antibodies. Several this study. protocols have been described; however, factors affecting the reproducibility of dengue plaque assays have not been fully studied[12-21]. Serum specimens These include the use of solid or semi-sold substances in overlay media, the cell substrate, A total of 24 sera obtained through the Cuban serum inhibitors and pH of the overlay medium. national dengue surveillance system during

Table 1. Details of the strains utilized in the neutralization tests

P: passage, MB: mouse brain, M: mosquitoes

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2000 and 12 blood samples collected on filter the case of BHK21, and for the LLCMK2 cells paper from healthy individuals during 2003 the plaques of all the serotypes were counted were selected for study. The first samples were on the 7th day. After incubation, plates were used to test the reproducibility of PRNT on rinsed gently under tap water and fixed and BHK21 cells. Filter paper eluates were tested stained with a solution of naphthol blue black for DENV-1 to DENV-4 neutralizing antibodies and acetic acid[16]. on BHK21 and LLCMK2 cells at a 1:30 dilution, using exactly the same methods used previously[22]. Antibody titres obtained in both Plaque Reduction Neutralization cells were compared. Test (PRNT)

PRNT was performed on both tested cell lines. Cell culture For antibody titration, 100 ul of serum dilution (in MEM) was incubated for 1 hour at 37 °C BHK21, clone 15 cells were grown at 37 °C in with 100 ul of the virus working dilution complete medium E-MEM (Minimum Essential calculated to give 10-20 pfu/50 ul of the final Medium with Earle’s balance salts) volume of virus-serum mixture. After incubation, supplemented with 10% of heat-inactivated 50 ul of virus-serum mixture was added to the foetal bovine serum, 1% penicillin/ cell suspension in triplicate. The pH during the streptomycin, 10% L-glutamine and sodium virus adsorption was in the range 7-7.5. Plates bicarbonate for adjusting the medium to pH were incubated for 4 hours at 37 °C under CO2 7.4-7.8. LLCMK2 cells (ATCC) were kindly atmosphere. The rest was followed as above. provided by Sutee Yoksan (Center for Vaccine Each serum dilution was tested four times. Development, Mahidol University) and were Calculations of 50% endpoint plaque reduction grown in the same conditions. neutralization titres were made using log probit paper by the method of Russell et al., 1967[23]. Virus titration Evaluation of pH of overlay Plaque titration was performed on BHK21, medium, CO2 atmosphere and clone 15 and in LLCMK2 as previously described by Morens et al[16]. For virus titration, 50 ul of rapid centrifugation of virus virus dilutions (101 to 106) was added to 0.5 ml during cell adsorption phase of BHK21 cell suspension (1.5 x 105 cells) in on the efficiency of dengue virus each of three wells of a 24-well polystyrene plaque assays in BHK21 cells plate. After 4 hours’ incubation at 37 °C in (1) In order to evaluate the influence of the CO2 atmosphere, each well received 0.5 ml of 3% medium viscosity carboxymethyl pH of the nutrient overlay medium, this cellulose – made up in Earle’s minimum was adjusted at 6.5, 7, 7.5 and 8 final pH essential medium without phenol red (MEM) using a solution of 7.5% sodium with 10% heat-inactivated foetal bovine serum, bicarbonate. Cells were incubated under

1% glutamine 2 mM and 100 U of penicillin CO2 atmosphere at 4% and the four plus 100 ug/ml streptomycin. Infected cells dengue serotypes were titrated. were incubated for 5–9 days in the same (2) To test the influence of CO atmosphere, conditions as above depending on the virus 2 the pH of the overlay medium was serotype (7–9 days for DENV-1 and DENV-3, adjusted to 7.5, and cells were incubated 5 days for DENV-2 and 6 days for DENV-4) in

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under CO2 atmosphere both at 4 and Table 2. Effect of pH of overlay medium on 4.5%. Under these conditions the four dengue viruses plaque formation 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 NP: no plaques and overlay media (at pH 7.5) was added. Influence of CO atmosphere Infected cells were incubated at 37 °C in 2

CO2 atmosphere at either 4 or 4.5%. 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 Results the influence of CO2 atmosphere. In general, titre and plaque size were relatively unaffected by cell incubation under 4 or 4.5% CO Evaluation of different factors 2 influencing dengue virus atmosphere. plaque assays in BHK21 cells Influence of rapid centrifugation of Effect of pH of overlay medium virus during cell adsorption phase Table 3 shows the effect of dengue virus Titres of DENV-1, 3 and 4 depended on pH of plaque formation of rapid centrifugation assay overlay media (Table 2). The highest titres were both in cell suspensions and confluent observed at pH 7.5. For DENV-2, titres were monolayer. Rapid centrifugation on confluent relatively similar at pH values ranging from 6.5 cell cultures gave the highest viral titres, to 7.5, pH 7.5 being the optimal. compared to the conventional method, both

at 4 and 4.5% of CO2 atmosphere.

Table 3. Effect on dengue virus plaque formation of rapid centrifugation assay using cell suspensions and confluent monolayer at two different percentages

of CO2 atmosphere*

* pH of overlay medium was adjusted to 7.5

52 Dengue Bulletin – Vol 29, 2005 Improved Dengue Virus Plaque Formation on BHK21 and LLCMK2 Cells

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.

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Reproducibility of dengue PRNT Discussion assays in BHK21 Taking into account the necessity of measuring To test the reproducibility of PRNT assays, sera neutralizing antibodies during vaccine trials, were tested twice on two different days (in total, it is important to apply standardized and antibody titre in each serum was determined validated methods. However, factors affecting four times). Titres less than 1/10 to DENV-3 and the reproducibility of dengue plaque assays -4 were obtained. Only 2 (8.3%) sera showed have not been fully identified. Of the factors greater than twofold differences in the studied in this work, the pH of the overlay neutralizing antibody titre to DENV-1 (Table 4). medium was the most critical variable. The optimal pH of the overlay medium for obtaining clear dengue virus plaques was pH Comparison of BHK21 and LLCMK 2 7.5. Other investigators have reported optimal cells for virus titration dengue virus plaquing over a wide range of and assay of neutralizing antibody pH values in the overlay medium (pH 6.6 to [19] 8.6) when using LLCMK2 cells . When Titres of the four dengue viruses on BHK21 DENV-3 was assayed, plaque size decreased and LLCMK cell lines using an overlay media 2 at pH 8.6[19]. Dieg and Watkins[24] have found buffered to pH 7.5 and incubated at 37 °C in a that Colorado tick fever virus plaque 4% CO atmosphere were compared. With the 2 production was relatively insensitive over a exception of DENV-3, the highest viral titres range of pH of 7.1 to 8.1 while plaque were obtained on BHK21 cell cultures (Table formation failed to occur at pH 7.0 or lower. 5). Viral plaques were similar in size and In hamster lung cell, plaque formation of morphology in both cell lines with the exception Japanese encephalitis virus was not observed of DENV-4 plaques that were larger in both when pH of the overlay medium was under cultures. 6.6 or above 7.8.[18] Lee et al[25]. have reported Table 5. DENV-1 to -4 titres determined by dengue plaque formation at a pH range of

plaque assay on BHK21 and LLCMK2 cell 7.4 to 7.6. Sodium bicarbonate concentrations suspensions* 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.

*Plaque titre (Log 10 pfu/ml) In general, titre and plaque size were relatively unaffected by incubating infected

The percentage of plaque reduction to cells at different percentages of CO2 each serotype was determined in 12 samples atmosphere. Stim TB[19] have reported collected on filter papers tested in both cell increased sensitivity of dengue virus plaque lines. The results were similar in 100% of the assays at high concentrations of sodium samples (Table 6). bicarbonate.

54 Dengue Bulletin – Vol 29, 2005 Improved Dengue Virus Plaque Formation on BHK21 and LLCMK2 Cells

Table 6. Comparative plaque reduction neutralization of DENV-1 to -4 in BHK21 and LLCMK2 cells infected as cell suspension*

*Whole blood on filter papers was re-suspended at 1:30 dilution

In our laboratory, the rapid centrifugation viruses, rapid centrifugation improved the or shell vial assay has greatly improved the isolation rate in blood and tissues[26]. Recently, isolation rate of some viruses. This method of Chingsuwanrote et al. (2004) observed a enhancing virus-cell interactions decreases the relationship of the number of plaques and post- time of the onset of cytopathic effect of many infection incubation time, with the longest post- viruses present in clinical diagnostic specimens. infection incubation time giving the highest It was applied to plaque assays and improved number of plaques[27]. consistency of input virus dose. For dengue

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Several cell lines have been used for dengue The current work confirms that under plaque assays but there are few published optimal conditions, plaque reduction comparisons between cell systems using neutralization titres can be repeated optimized conditions[12,14,15,17,27]. In the present reproducibly. Seroepidemiological and vaccine study, virus titres were higher in BHK21 than in studies can be performed satisfactorily in both BHK21 and LLCMK cells. LLCMK2 cell cultures, except for DENV-3. 2

In summary, (i) the pH of the overlay medium may be an important factor in obtaining Acknowledgements clear and large dengue plaques; (ii) the centrifugation of virus inoculate on cells increases This work was supported by TDR/WHO grant virus titres; (iii) higher virus titres were obtained ID No. 990904. We thank Prof. Robert Shope of Texas University and Prof. Scott Halstead in BHK21 compared with LLCMK2 cell cultures; (iv) highly reproducible dengue PRNT titres were from the Pediatric Dengue Vaccine Initiative, obtained on BHK21 cells; and (v) using the cell Bethesda, Maryland, USA, for their useful suspension method, DENV-3 resulted in higher suggestions and comments. titres in LLCMK2 than BHK21 cells.

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