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