Presentation Number: 028 (A)

Poster Board Number: 1A

Comparison of Dengue Virus Type 1 Growth Characteristics in Vero and C6/36 Cell Lines
A.Owens, J. Shifflett, S. Radhakrishnan, K. Langenbach, R.O. Baker BEI Resources/ATCC, Manassas VA

Abstract
Background: This study compared the growth characteristics of dengue virus type 1 (Strain Hawaii) in Vero and C6/36 cells to determine the optimal temperature and appropriate cell line for propagation. Dengue virus, a NIAID Category A priority pathogen, is a member of the Flaviviridae family and is transmitted by mosquitoes to humans. While classic dengue fever is a self-limited disease, severe forms can lead to shock and hemorrhage and occasionally death. The virus can grow in Vero (from monkey kidney) and C6/36 (from Aedes albopictus mosquito clone) cell lines without showing any significant cytopathic effects. While Vero cells grow optimally at 37oC and C6/36 cells grow optimally at 28oC, little information is known about the optimal temperature for dengue virus propagation. Methods: Dengue virus was propagated at three different temperatures in both cell lines through three passages. Titer was measured by IFA and relative quantitative PCR using SYBR Green. Differences in nucleotide sequences of the viral envelope protein (E) and the non-structural protein 4B (NS4B) were assessed at each passage as the virus adapted to growth in the respective cell line. Results: IFA showed viral titer 1-2 logs higher in Vero cells at 33oC and in C6/36 cells at both 28oC and 33oC compared to the other temperatures tested. Similar trends were seen in relative quantitative PCR. At 33oC virus grew to similar titer in both cell lines. Nucleotide sequence analysis showed differences in five positions in the viral E gene. This difference was seen in virus grown at 28oC in Vero and 37oC in C6/36 cells. There were no mutations in the NS4B gene. Conclusions: This study shows that 33oC is an optimal temperature for growth of dengue virus in Vero or C6/36 cell lines. As the virus is adapted to propagate in these cell lines at extreme temperatures for the cells growth, genetic differences become apparent in the envelope gene.

Figure 3. Chromatograms from E-gene sequencing

Figure 3a. Chromatograms from E-gene sequencing. The black line represents the allele at position 1091. The first chromatogram shows the wild type allele (dengue grown in C6/36 cells at 28oC passage 3). The next three chromatograms show the allele in dengue virus grown in C6/36 cells at 37oC passage 1-3. 1091

Figure 1. IFA Titer Data. Representative samples of positive IFA results in C6/36 and Vero cell lines
Figure 1a. Negative control C6/36 cell line grown at 33oC passage 3 at 20x magnification

Type 1 Viruses Figure 1b. C6/36 cell line and dengue virus
grown at 33oC passage 3 at 20x magnification

Introduction
Dengue virus, a member of the Flaviviridae family, is a positive stranded RNA virus that is transmitted from an infected Aedes mosquito to humans. There are four distinct virus serotypes (DENV-1, -2, -3, and 4), all of which cause similar illness. However, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are more often linked to DENV-2 and -3. Dengue incidence worldwide has increased since the mid-1950s and has now emerged as one of the most important arthropod transmitted viral diseases of humans. Therefore, development of a vaccine has become a top priority, but must induce immunity to all serotypes of dengue virus simultaneously. This is because infection with one serotype of dengue virus will lead to immunity from that serotype, but causes more severe disease from another serotype. These are among the reasons that dengue virus has been classified as a NIAID Category A Priority Pathogen. Growth of dengue virus has been described in both Vero and C6/36 cell lines at various temperatures with little cytopathic effects in some strains. In addition, mutations have been described in dengue virus when grown in cell culture. Previous work with DENV-4 has shown several mutations in the non-structural genes and 3-UTR that are hypothesized to be adaptations by Vero cells, especially in the non-structural 4B (NS4B) gene. Therefore, in this study we have explored various temperatures for the growth of dengue virus type 1 in two cell lines and changes in the sequence of the envelope (E) and NS4B genes during adaptation of the virus in these two cell lines at various temperatures. Figure 3b. Chromatograms from E-gene sequencing. The black line represents the allele at position 1091. The first chromatogram shows the wild type allele (dengue grown in Vero cells at 37oC passage 3). The next three chromatograms show the allele in dengue virus grown in Vero cells at 28oC passage 1-3. Figure 1c. Negative control Vero cell line grown at 33oC passage 3 at 10x magnification Figure 1d. Vero cell line and dengue virus grown at 33oC passage 3 at 10x magnification

1091

Materials and Methods

Virus propagation: Dengue virus type 1, Strain Hawaii, (NR-82) was propagated in Vero (ATCC CCL-81) and C6/36 (ATCC CRL-1660) cell lines at three temperatures, 28oC, 33oC, and 37oC, with 5% CO2. The cell lines were inoculated at 80-90% confluency with a 1:10 dilution of viral stock with a titer by RT-PCR in Vero cells of 8.89x104 TCID50/ml and grown seven days. The virus was harvested by scraping and stored at -80oC. The virus was passaged 3 times under each condition with a 1:10 dilution of viral stock from the previous passage. Immunofluorescence Assays: For IFA, viruses were first grown on Vero or C6/36 cells (depending on the cells used for growth of the virus) seeded on 8-well chamber slides or 24 well plates, respectively, for 7 days at the same temperature used for growth with 5% CO2. The infected cells were then fixed for 8 minutes at room temperature with 4% formaldehyde in PBS. After rinsing the fixed cells with PBS, monoclonal antibody against dengue virus type 1 (catalog # MAB8701) from Chemicon (Temecula, CA) was added at a 1:200 dilution in PBS + 1% FBS + 0.1% saponin (w/v) and allowed to bind for 30 minutes. The cells were then rinsed with PBS + 1% FBS + 0.1% saponin, and Light Diagnostics (Murray, UT) Ms IgG Ab:FITC containing Evans Blue counterstain (catalog # 5008) was added to each well dropwise. The cells were rinsed again with PBS + 1% FBS + 0.1% saponin, overlayed with Dako (Carpinteria, CA) fluorescence mounting medium, and viewed using an epi-fluorescent microscope with FITC filter. Nucleic acid extraction and qPCR assays: All viral genomic material was extracted using the Qiagen (Valencia, CA) QIAamp Viral RNA Mini Kit according to manufacturers instructions. Primers were developed by BEI Resources and purchased from Integrated DNA Technologies (IDT Coralville, IA). These primers were used with Sigma (St. Louis, MO) SYBR green I nucleic acid gel stain (catalog # S9430) to achieve a relative quantitation of virus in each sample. Viral RNA was reverse transcribed and amplified using Qiagen (Valencia, CA) One Step RT-PCR kit (catalog # 210212). The assays were run on the BioRad IQ5 thermal cycler. Thermal cycling parameters consisted of an RT step at 50oC for 30 minutes followed by a heat inactivation step at 95oC for 15 minutes. Following these steps were 35 cycles of denaturation (94oC for 30 seconds), primer annealing (54oC for 30 seconds), and primer extension (72oC for 2 minutes) where data was collected, and final extension at 72oC for 7 minutes. Sequencing: Three primer sets that span the E gene of dengue virus type I from Zheng, et. al were used to sequence the E gene of each viral sample. Viral RNA was reverse transcribed and amplified using Qiagen (Valencia, CA) One Step RTPCR kit and Promega Access RT-PCR System (Madison, WI). Thermal cycling parameters used with the One Step RTPCR kit consisted of an RT step at 50oC for 30 minutes followed by a heat inactivation step at 95oC for 15 minutes. Following these steps were 3 cycles of denaturation (94oC for 1 minute), primer annealing (60oC for 40 seconds), and primer extension (72oC for 1 minute). Following these cycles were 30 cycles of denaturation (94oC for 30 seconds), primer annealing (60oC for 30 seconds), and primer extension 72oC for 40 seconds), and final extension at 72oC for 10 minutes. Thermal cycling parameters used with the Access RT-PCR System consisted of an RT step at 45oC for 30 minutes followed by a heat inactivation step at 94oC for 2 minutes. Following these steps were 3 cycles of denaturation (94oC for 1 minute), primer annealing (58oC for 40 seconds), and primer extension (68oC for 2 minutes). Following these cycles were 30 cycles of denaturation (94oC for 30 seconds), primer annealing (58oC for 30 seconds), and primer extension 68oC for 2 minutes), and final extension at 68oC for 7 minutes. Products from the resulting RT-PCR reactions were purified using the QIAquick PCR Purification Kit according to the manufacturers instructions. The resulting purified products were then sequenced with the primer sets used in the RT-PCR reactions using the ABI3000 Sequencer (Foster City, CA), and those sequences were then analyzed using CodonCode Aligner (Dedham, MA). To sequence the NS4B gene, a primer set was developed by BEI Resources using multiple sequence alignments of published sequences for six dengue virus type 1 strains. Viral RNA was reverse transcribed and amplified using Qiagen (Valencia, CA) One Step RT-PCR kit. Thermal cycling parameters consisted of an RT step at 50oC for 30 minutes followed by a heat inactivation step at 95oC for 15 minutes. Following these steps were 35 cycles of denaturation (94oC for 30 seconds), primer annealing (50oC for 30 seconds), and primer extension (72oC for 1 minute, 10 seconds), followed by a final extension (72oC for 7 minutes). Products from the resulting RT-PCR reactions were purified using the QIAquick PCR Purification Kit according to the manufacturers instructions. The resulting purified products were then sequenced with the primer set used in the RT-PCR reactions using the ABI3000 Sequencer (Foster City, CA), and those sequences were then analyzed using CodonCode Aligner (Dedham, MA).

Table 3. Mutations in E-gene that lead to amino acid changes

Sample

Table 2. qPCR data. Average Ct of RNA extracted viral samples with SYBR green detection.

E-gene position 1091 G G G/C G/C G/C G/C C

E-gene position 1543 A A A/G A/G A/G A/G G

E-gene position 1612 C C C/G C/G C/G C/G G

E-gene position 1765 A A A/C A/C A/C A/C C

E-gene position 1847 T T/C T/C T/C T/C T/C C

Wild type sequence (EU848545) C6/36 p1 37oC C6/36 p2 37oC C6/36 p3 37oC Vero p1 28oC Vero p2 28oC Vero p3 28oC

Sample NR-82 grown in Vero p1 28oC NR-82 grown in Vero p2 28oC NR-82 grown in Vero p3 28oC NR-82 grown in Vero p1 33oC NR-82 grown in Vero p2 33oC NR-82 grown in Vero p3 33oC NR-82 grown in Vero p1 37oC NR-82 grown in Vero p2 37oC NR-82 grown in Vero p3 37oC

Ct average 31.42 29.11 26.74 25.16 23.66 22.43 28.20 26.43 25.56

Sample NR-82 grown in C6/36 p1 28oC NR-82 grown in C6/36 p2 28oC NR-82 grown in C6/36 p3 28oC NR-82 grown in C6/36 p1 33oC NR-82 grown in C6/36 p2 33oC NR-82 grown in C6/36 p3 33oC NR-82 grown in C6/36 p1 37oC NR-82 grown in C6/36 p2 37oC NR-82 grown in C6/36 p3 37oC

Ct 23.04 25.31 21.77 22.49 23.58 21.16 29.03 29.84 22.98

Conclusions
The data presented here show that 33oC is an optimal temperature for dengue virus growth in either Vero or C6/36 cell lines. Dengue virus can also be grown in C6/36 cell line at 28oC with similar results to 33oC. The optimal growth of dengue virus at 33oC (and 28oC in C6/36 cells) is probably due to the ability of these two cell lines to grow well at these temperatures. Mutations in the E gene of dengue virus do not occur at these temperatures of optimal virus growth. However, mutations in the E gene do arise as the virus adapts to propagation in these cell lines at extreme temperatures for the cell lines growth. Several of these previously undocumented mutations cause amino acid changes which could lead to conformational changes in the E gene. These changes may be a result of the virus adapting to growth at temperatures that are sub-optimal for the growth of the cell lines. Mutations in the NS4B gene do not appear to occur in any of these conditions.

Figure 2. qPCR data

Figure 2a. qPCR data. Amplification chart of viral samples with SYBR green detection. This chart includes RNA extracted NR-82 grown in Vero cells and RNA extracted NR-82 in C6/36 cells passage 1 and 2 only. This chart represents the data in Table 2. Figure 2b. qPCR data. Amplification chart of viral samples with SYBR green detection. This chart includes RNA extracted NR-82 grown in C6/36 cells passage 3. This chart represents the data in Table 2.

Results
Table 1. IFA Titer Data
Sample IFA titer (TCID50/1.0ml on Vero cells in 7 days at temperature in sample ID with 5% CO2) 8.89x102 1.58x104 1.58x104 8.89x104 1.58x105 2.81x106 2.81x104 8.89x103 1.58x10
5

References
Sample IFA titer (TCID50/1.0ml on C6/36 cells in 7 days at temperature in sample ID with 5% CO2) 8.89x106 1.58x106 1.58x106 8.89x104 1.58x106 1.58x106 1.58x103 1.58x102 1.58x10
4

NR-82 grown in Vero p1 28oC NR-82 grown in Vero p2 28oC NR-82 grown in Vero p3 28oC NR-82 grown in Vero p1 33oC NR-82 grown in Vero p2 33oC NR-82 grown in Vero p3 33oC NR-82 grown in Vero p1 37oC NR-82 grown in Vero p2 37oC NR-82 grown in Vero p3 37 C
o

NR-82 grown in C6/36 p1 28oC NR-82 grown in C6/36 p2 28oC NR-82 grown in C6/36 p3 28oC NR-82 grown in C6/36 p1 33oC NR-82 grown in C6/36 p2 33oC NR-82 grown in C6/36 p3 33oC NR-82 grown in C6/36 p1 37oC NR-82 grown in C6/36 p2 37oC NR-82 grown in C6/36 p3 37 C
o

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Acknowledgements
This project has been funded in whole with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. N01-AI-300067.

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