EFFECTS OF VERO AND C6/36 CELLS UPON CHIKV PHENOTYPES
Suppachoke Onnome1,*, Sukathida Ubol2, Rojjanaporn Pulmanausahakul3,#
1 Institute of Molecular Biosciences, Mahidol University, Nakornpathom 73170, Thailand
2 Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400,
Thailand
#e-mail: rojjanaporn.pul@mahidol.ac.th
Abstract
Chikungunya virus (CHIKV) is an arbovirus that is typically maintains the
transmission cycle between specific mosquito vector and susceptible vertebrate hosts. As
same as other RNA viruses, CHIKV genetics are highly diverse due to the error prone of the
RNA dependent RNA polymerase which is beneficial for the virus in term of adaptation and
evolution in the changing environmental conditions. In this experiment, the effects of cell
types during CHIKV passages were investigated. WT and small plaque CHIKV variant were
sequentially passaged in mammalian cells, vero cells or mosquito cells, C6/36 cells. After
each passage, the viruses were subjected to plaque assay to determine plaque morphology.
The results showed that after 2nd passage in vero cells, plaque phenotype of both virus
variants appeared to be more heterogeneous and larger than parental viruses. In contrast,
when passaging both virus variants in C6/36 cells, plaque phenotype became more
homogenous. These results suggest that virus populations are more diverse when propagated
in vero cells while CHIKV diversity may be more restricted in C6/36 cells. In addition, C6/36
cells with WT virus infection showed less cytopathic effects (CPE) and produced more viral
progenies than infected vero cells. These findings may provide useful information in cell type
selection for CHIKV production.
Keywords: Chikungunya virus, vero, C6/36, plaque morphology
Introduction
Chikungunya virus (CHIKV), a positive single stranded RNA virus, belongs to the
genus Alphavirus, family Togaviridae. CHIKV genome is approximately 11.8 kb long and
genome consists of two open reading frames. This virus causes rash, fever and severe joint
pain which may persist for several months or years and in some serious complications, the
disease can lead to the death(1). Chikungunya virus caused large scale outbreak on Reunion
Island in 2005 and also became a public health concern in South Asia, Southeast Asia, and
Africa(2). Currently, there is no specific treatment or licensed vaccine.
Arthropod-borne viruses are maintained in nature by cycling between vertebrate and
invertebrate, therefore both mosquito and mammalian cell culture systems have been used to
study virus replication and pathogenesis(3). In our lab, vero cells and C6/36 cells are
commonly used in the propagation of CHIKV; therefore these two cells were used to study in
this experiment to determine the effect of different cell type on viral phenotype.
Methodology
Vero cells (African Green monkey kidney cells) were cultured in DMEM
supplemented with 5% FBS and grown at 37°C with 5% CO2 .C6/36 cells (Aedes albopictus
mosquito cells) were cultured in MEM supplemented with 10% FBS and grown at 28°C.
Wild-type Chikungunya (WT CHIKV) virus was isolated from patient from Phangnga province, Thailand in 2009 and has been passaged two times in C6/36 and one time in
vero cells. The small plaque virus variant was selected from WT CHIKV and plaque purified
many times in C6/36, passage 14 of small plaque purified virus was used as parental small
plaque virus in this experiment.
Parental WT and parental small plaque CHIKVs were serially passaged 2 times with
duplicate experiments on vero cells or C6/36 cells at a multiplicity of infection (MOI) of
0.001. Virus was adsorbed for 1 hr onto confluent cell monolayers which seeded in 6 well
plates, then incubated at 28°C for C6/36 cells or 37°C for vero cells. After that, inoculum was
removed, washed, fresh medium was added and the cells were incubated for 3 days. CPE was
observed and supernatants of each passage were harvested and stored at -80°C until use.
Plaque assays were performed in duplicate for each sample. vero cells were seeded at
106 cells per well in 6-well plates and incubated at 37° overnight. Ten-fold serial dilutions of
the virus were prepared in BA-1 medium (1xM-199E, 1 M Tris-HCl pH 7.6, 2% BSA, 100
units of penicillin/streptomycin per ml, 0.075% NaHCO3) and 200 µl of each dilution was
used to infect vero cells for 1 hr at 37 ºC. During incubation, the plates were gently agitated
every 10 min, then 4 ml of 0.8% agrose in DMEM containing 2% FBS was layered onto the
cell monolayers. The plates were incubated at 37°C with 5% CO2 for 3 days. After that,
plaque were fixed with 10% formaldehyde, overlay was removed, and then staining with
0.1% methylene blue.
Results
After 24 hr, vero cells infected with wt or small plaque CHIKV showed first signs of
CPE, which include cytoplasmic vacuolization, rounding of cells and cells detachment from
substrate. Then severe CPE rapidly developed, within 48 hours most of the cells were dead. In
contrast, CPE was not observed in the C6/36 cells after infection with either variant of
CHIKV. In addition, the titers of WT viruses that produced in C6/36 cells were about 1 log
higher than those from vero cells (table 1).
When compare the plaque morphologies of WT CHIKV that serially passaged in vero
cells and C6/36 cells, we found that viruses that produced in vero cells showed larger plaque
sizes and become more heterogeneous (fig.1-B). On the other hand, plaque size of WT
CHIKV were more homogeneous after 2 passages in C6/36 cells when compare to the
parental virus (fig.1-C and fig.1-A, respectively). Same outcome was also observed for small
plaque CHIKV variant. After each round of virus passage in vero cells, the heterogeneity of
plaque size of the virus was increase. By the 2nd passage, it was clearly seen that 50% of the
plaques become larger (fig.1-E). In contrast, small plaque CHIKVs that passaged in C6/36
cells still maintain the homogeneous small plaque morphology (fig.1-F).
Discussion and Conclusion
CHIKV have several significant differences in the process of infection between the
mammal and insect hosts. Infection in mammalian cells is acute, like natural infection which
result in illness, fever, and arthralgia in human. However in mosquito, infection is persistent
according to the previous report that suggested some factors from mosquito cells were able to
inhibit cellular apoptosis(4). The persistent infection in insect is essential for the virus since it
needs a vector for transmission to mammalian host to complete and maintain their replication
cycle in the nature. Our data also support this hypothesis in which the infected vero cells,
which is mammalian cells, showed more CPE when compare to infected C6/36 cells, which is
insect cells.
The result of plaque phenotype analysis can indicate how well the virus can engage
the host cell machinery to replicate and spread efficiently from cell to cell, and it is also a
measurement of the stability of the viral genome. A homogeneous plaque size of virus
produced in C6/36 cells indicates that this cell restrict and select only some variants to
replicate. Nevertheless, how this limitation might benefit virus is still unknown. On the
opposite side, heterogeneity of plaque size indicates genetic variants were showed more in
virus generated in the vero cells. This may be a reason why isolated CHIKV from patient
show mix population of plaque morphology.
Figure 1. Plaque morphologies of parental WT, parental small plaque variant and
CHIKVs that passaged in vero or C6/36 cells.
A. Parental
WT CHIKV
(Control plaque)
B. WT CHIKV passaged 1
time in vero cells
C. WT CHIKV passaged
2 times in C6/36 cells
D. Parental small plaque
CHIKV
(Control plaque)
E. Small plaque CHIKV
passaged 1 time in vero
cells
F. Small plaque CHIKV
passaged 2 times in C6/36
cells
Table 1. The average of CHIKVs titers after passages in vero cells and C6/36 cells
CHIKV
variants
Parental virus titer
(PFU/ml)
WT
Small plaque
1st Passage virus titer
(PFU/ml)
2nd Passage virus titer
(PFU/ml)
vero cells
C6/36 cells
vero cells
C6/36 cells
2 x 107
2.12 x 107
1.5 x 109
4.2 x 107
2.3 x 108
3 x 109
6 x 105
1.15 x 109
1 x 107
1.5 x 107
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