Differential susceptibility & replication potential of Vero E6, BHK-21, RD, A-549, C6/36 cells & Aedes aegypti mosquitoes to three strains of chikungunya virus

Chikungunya virus (CHIKV), an arbovirus belonging to the genus alphavirus, family Togaviridae, has become health concern in countries in Africa, Indian Ocean basin, India, Southeast Asian countries and recently the Caribbean Islands and the Americas12345. In the Americas, autochthonous transmission was reported first in 2013 in St. Martin followed by 26 Islands and 14 mainland countries with millions of cases5. CHIKV infection is generally self-limiting, but in a small percentage of cases, persistence of arthralgia for years has been reported. Although the exact mechanism of persistence of the manifestations is not clearly understood, it has been postulated that viral antigen persists in the skeletal muscle progenitor cells and passes on to muscle cells during development, causing recurrent myalgia/arthralgia6. The re-emergence was characterized by debilitating polyarthralgia associated with a number of unusual clinical manifestations either singly or in combination3. The outbreak which commenced in 2004 in the Lamu Island, Kenya had shown several clinical complications which are unheard of CHIKV infection i.e. swelling of limbs, periventricular and meningoencephalitis in neonates, hepatic and renal dysfunctions, hypokalemic paralysis, hearing loss, ophthalmic involvement, acute flaccid paralysis and Guillain-Barre Syndrome789101112131415. Mother to child transmission and CHIKV-associated mortality were also reported in La Reunion Islands as well as in India for the first time121415. Although rare, mortality has been reported from La Reunion Islands, India, Malaysia, Brazil, etc13161718.

The re-emergence of CHIKV in India after a gap of 32 years has been explosive and affected more than six million people in 29 States/Union Territories11920. Though the high incidence declined after 2007, the virus is still prevalent in the country with sporadic outbreaks4520. Introduction of the African lineage, the East Central South African (ECSA) for the first time, wide prevalence of both the vectors (Aedes aegypti and Ae. albopictus) and a naïve population were attributed to the plausible reasons for the massive outbreak. India also witnessed introduction of the mutated strain of CHIKV i.e. ECSA (E1:A226V) in Kerala and Karnataka, where enhanced transmission of the virus by Ae. albopictus was observed2122.

Since no vaccines or antivirals are available commercially, early diagnosis has been the mainstay in outbreak management. Although several techniques are available for diagnosis, virus isolation has remained the gold standard as it provides the most conclusive evidence of the aetiological agent. The availability of well-characterized cell lines, modern infrastructural facilities and trained personnel have enhanced the rate of virus isolations from clinical samples1823. The cell culture system has replaced infant mice and other systems for virus isolation due to their high sensitivity and virus yield. Well-characterized cell lines, namely Vero, BHK-21 (Baby hamster kidney) and MRC-5 have been employed successfully not only for virus isolation but also for the development of attenuated and inactivated vaccines globally. The advancements made in the development and maintenance of cell lines have made their management easier than other systems, namely infant mouse inoculation and egg inoculation.

CHIKV replicated in a broad spectrum of cell lines originated from monkeys, humans, mosquitoes, etc and has been used routinely for virus isolation and propagation1823. A comparative analysis was, therefore, made in this study to determine the differential susceptibility of five cell lines originating from different hosts [Vero E6, BHK-21, RD (human rhabdomyosarcoma), A-549 (human alveolar basal epithelial cells) and C6/36 (Ae. albopictus)] and one strain of Ae. aegypti mosquitoes to three different lineages of CHIKV including the virus with changed genome, for susceptibility and virus yield.

Material & Methods

Virus: CHIKV strains 634029 (Asian, Acc. No. EF027140), 061573 (E1:A226, Acc. No. EF027134) and 074831 (E1:A226V, Acc. No. FJ000069) were obtained from the virus repository maintained at the National Institute of Virology (NIV), Pune, India, in the lyophilized form. All the viruses were screened by the NIV before lyophilization. CHIKV strain 634029 was isolated during the 1963 outbreak in Calcutta (now Kolkata), West Bengal, from human serum while 061573 and 074831 were isolated from human serum samples during 2006 and 2007 CHIKV outbreaks in Andhra Pradesh and Kerala, respectively. Strain No. 634029 has undergone 12 passages in infant mouse and two passages in Vero E6 cell line while 061573 and 074831 have undergone one passage in C6/36 cell line (Ae. albopictus) followed by four passages in Vero E6 cell line.

Cell lines: Five cell lines of different hosts of origin i.e. Vero E6 (African green monkey kidney), Passage (P) No. 220-225, BHK-21 (Baby hamster kidney), P. No. 72-78, RD (human rhabdomyosarcoma, received from WHO), P. No. 120-130, A-549 (human alveolar basal epithelial), P. No. 88-94 and C6/36, P-130-134 were used in the study. Vertebrate cell lines were maintained in minimum essential medium (MEM, HiMedia, Mumbai) supplemented with 10 per cent foetal bovine serum (FBS, Invitrogen, USA) at 37°C with five per cent CO₂ and 75 per cent humidity while C6/36 cells were maintained in Mitsuhashi Maramorosch medium (HiMedia, Mumbai) supplemented with 10 per cent FBS at 28°C.

Mosquitoes: Ae. aegypti mosquitoes were obtained from the insectary maintained at the Entomology Division of NIV, Pune. The mosquito larvae were fed on commercially available fish food (Aakar, Mumbai) while adults were maintained on 10 per cent glucose solution. For oviposition, adults were fed occasionally on fowl blood. The mosquito colony was maintained at 28±2°C with relative humidity of 80±5 per cent and 12:12 h dark:light cycle.

Infection of cell lines: Growth kinetic study of the three strains were carried out in VeroE6, BHK-21, RD and A-549 and C6/36 cell lines as described earlier23. Cells were grown to 90-95 per cent confluence in 24-well plates (Nunc, Denmark) and were infected independently with the three strains of CHIKV at 10 MOI (multiplicity of infection). Cell supernatants were collected at daily intervals for six days and stored at −80°C (New Brunswick, USA) until further analysis.

Infection of mosquitoes: Intrathoracic inoculation of mosquitoes was carried out in a biosafety level 2 arthropod containment facility maintained at NIV, Pune, as described previously24. Three to four days old female Ae. aegypti mosquitoes (n=60) were inoculated intrathoracically with individual CHIKV strains at the rate 0.2 μl virus suspension per mosquito. After infection, the mosquitoes were maintained on 10 per cent glucose solution and incubated at 28°C for 15 days. Five mosquitoes each were harvested on '0' day, day 1 post infection (PI) and on every alternate days thereafter till day 15 PI, stored at −80°C till completion of the experiment and titrated in Vero E6 cells as described below. Pools of five mosquitoes collected on the particular day PI were processed as single sample (not individual mosquitoes).

Virus quantification by 50% tissue culture infectious dose (TCID₅₀) assay: Virus quantitation was carried out using 50 per cent tissue culture infectious dose (TCID₅₀/ml) method as described earlier24. Frozen samples were retrieved, thawed quickly, spun at 2790×gfor 20 min at 4°C and the supernatant was diluted serially (10-fold) in MEM supplemented with 2 per cent FBS. The serially diluted virus was inoculated over Vero E6 cell line grown to confluent monolayer in 96 well plates (Nunc, Denmark) in quadruplicate. Each plate had 12 wells as negative controls which were inoculated only with culture medium. Cytopathic changes in the infected wells were compared with the negative controls during scoring of infection. The cultures were incubated at 37°C for 96 h, scored cytopathic effect (CPE) under an inverted microscope, stained with amido black and determined virus titre as described by Reed and Muench25. Mosquito samples were triturated using a battery-operated hand-held homogenizer (Sigma, USA) with sterile disposable pestles in 1 ml chilled MEM supplemented with 2 per cent FBS, Millipore filtered (0.22 μm), diluted serially and determined TCID₅₀/ml in Vero E6 cell line.

Results

Growth kinetics of CHIKV strain 634029 in different cell lines: The Asian strain replicated in all the five cell lines used in the study with differential virus yields (Fig. 1). Although higher virus yield (~8 log TCID₅₀/ml) was obtained in Vero E6, C6/36 and BHK-21 cell lines, C6/36 cell line maintained the titre consistently throughout the study period. Vero E6 and BHK-21 cell lines have maintained the titre of 8 log till the day 4 PI, but declined subsequently. Virus growth in RD cell line was comparatively low and the maximum titre was achieved on day 3 PI yielding almost 8 log, but a rapid decline in virus yield was observed thereafter. A-549 cell line, though susceptible, was the least productive as maximum virus yield obtained was just above 4 log during 1^st to 5^th day PI.

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Fig. 1

Growth kinetics of chikungunya virus strain 634029 (Asian strain) in five cell lines. Each experiment was performed in triplicate. Values are provided as mean±SD.

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Growth kinetics of ECSA A226 strain (African) in different cell lines: All the cell lines replicated the strain, and the replication kinetics was found almost similar to that of the Asian strain (Fig. 2). Maximum virus yield was in C6/36 cell line which showed a consistent yield of virus till day 6 PI. Vero E6 cell line also yielded the maximum titre of 8 log but only till day three PI and thereafter showed a slight decline in virus titre. Growth kinetics of the strain in BHK-21 cell line was equivalent to that of Asian strain and maintained a titre of 8 log till day 4 PI. However, a sharp decline in virus titre was seen yielding 3 log on day 6 PI. RD and A-549 cell lines though replicated the strain were found less virus yielding as the maximum yield was 6.5 and 6 log, respectively. The latter has showed a sharp decline after the 1^st day PI and maintained approximately 4 log till day 6 PI.

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Fig. 2

Growth kinetics of chikungunya virus strain E1:A226 in five cell lines. Each experiment was performed in triplicate. Values are provided as mean±SD.

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Growth kinetics of CHIKV (E1:A226V) strain in different cell lines: The replication profile of the strain in the cell lines was almost similar to that of the other two strains discussed earlier (Fig. 3). The strain showed rapid replication in C6/36, Vero E6, BHK-21 and RD cells yielding ≥8 log virus yield on day 2 PI. However, except for C6/36 cell line, virus yield declined on subsequent days PI. Virus yield in Vero E6 cell line was consistent though loss of 1 log in virus titre was observed from day 3 PI. BHK-21 and RD cell lines showed a sudden decline in virus yield after day 2 PI but maintained a titre of approximately 6 log throughout the study period. A-549 cell line was the least productive. Except for the initial spurt on day 1 PI reaching >4 log, the virus yield declined to <4 log on subsequent days PI.

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Fig. 3

Growth kinetics of chikungunya virus (E1:A226V strain) in five cell lines. Each experiment was performed in triplicate. Values are provided as mean±SD.

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Replication kinetics of three strains of CHIKV in Aedes aegypti mosquitoes: Ae. aegypti mosquitoes showed replication of the three CHIKV strains with different virus yields (Fig. 4A & B). When infected with a high titre (approximately 3 log), growth kinetics of Asian and ECSA (E1:A226) were found almost identical with an initial spurt in virus titre yielding 5.7 log on day 1 PI. The latter, however, maintained the virus titre till day 7 PI while a rapid decline in virus titre was detected in the former (Fig. 4A). In comparison to the growth kinetics of the other two strains, the growth of ECSA (E1:A226V) was slow with a maximum yield of 4.5 log. However, the mosquito maintained the titre for 15 days without showing drastic decline. Despite the difference in the virus yield in the initial stages by the three strains, the mosquitoes maintained the virus with a titre approximately equal to 3 log throughout the study period.

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Fig. 4

Replication kinetics of three chikungunya virus strains in Aedes aegypti. with high (A) and low (B) dose of virus. Each experiment was performed in triplicate. Values are provided as mean±SD.

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When the mosquitoes were infected with a low titre of virus (1 log), rapid increase in virus replication was observed in Asian and ECSA (E1:A226V) strains on day 1 PI with a four-fold increase in virus titre (Fig. 4B). In the other African strain (E1:A226), however, the yield was only 2 log (2-fold) during the same period. Although all the three strains showed almost equal virus yield, the Asian strain showed slightly higher yield on day 3 PI. As far as the maintenance of the virus in the inoculated mosquitoes was concerned, the mosquito maintained all the strains throughout the study period. Viral growth kinetics of Asian and the mutated strain of ECSA (E1:A226V) had similar pattern though virus yield varied after day 7 PI.

Discussion

CHIKV induced distinct cytopathic effects in vertebrate cell lines and showed high level of apoptosis within 2-3 days of infection18. The virus infects macrophages, fibroblasts, endothelial, epithelial cells, etc in humans but not dendritic cells or T and B lymphocytes26. It has also been reported that fibroblast cells are the targets of CHIKV replication in vivo. In the present study, it was, therefore, decided to compare the potential of fibroblast-like and epithelial-like cells to replicate different strains of CHIKV in vitro. C6/36 cells developed from Ae. albopictus mosquitoes were found to be the most permissible cell culture system for all the three strains of CHIKV. The cells not only yielded high virus titre but also maintained the titre throughout the study period irrespective of virus strain. However, distinct CPE could not be seen in the cells with any of the virus strains. The ECSA (E1:226V) strain, a mutant of the African strain, however, had an edge over the other strains in virus yield in the cell. It is expected as CHIKV got mutated during the latter half of 2005 as an adaptation to grow in Ae. albopictus mosquitoes during the outbreak in La Reunion Island34511. The strain is a recent mutant of the ECSA strain with a mutation in the E1 protein at position 226 replacing the amino acid alanine with valine, which has enabled the virus to adapt for enhanced virus transmission by Ae. albopictus mosquitoes427. Similar results were reported by Wikan et al18 when they conducted a comparative growth study with three strains of CHIKV, and observed higher level of infectivity by the ECSA E:A226 and ECSA E:A226V strains, in comparison to Ross strain, the original ECSA strain. They opined that higher infectivity by the new strains could be due to genetic changes occurred to the original strain during the passage of time. This has been substantiated experimentally by Tsetsarkin et al27 as they demonstrated several changes in the virus genome, especially in the E2 protein during the recent mutation of ECSA strain. Wikan et al18 also observed significant change in viral infectivity of the three strains of CHIKV in an Ae. aegypti cell line (CCL-125). They demonstrated higher infectivity of the mutated strain (E1:A226V), substantiating the earlier reports of higher infectivity by the strain in Ae. aegypti mosquitoes2728. In our study with Ae. aegypti mosquitoes higher replication of the strain was observed as mosquitoes infected with 1 log TCID₅₀/ml virus yielded a four-fold increase in virus titre at 24 h PI. This has also been observed with Asian strain probably due to laboratory adaptation after serial passages in mice (12 passages). In mosquitoes infected with higher dose of virus, no difference in virus yield was observed among the three strains. When the dose of virus infection was compared, no change in virus growth pattern was observed except for virus yield. With high dose of infection, the peak virus yield was almost 1 log more than the low dose of infection. It was interesting to note that irrespective of the titres used for infection of ECSA (A226V), virus yield in Ae. aegypti mosquitoes was equal (4.5 log TCID₅₀/ml) and maintained the titre without much change throughout the study period. The mode of infection in the present study was parenteral inoculation which is not the natural route. Therefore, more systematic studies with natural route of infection are needed to make any conclusive statements.

Comparing other cell lines for virus susceptibility and yield, Vero E6 was found highly consistent as the virus yield was high with the three strains. Vero E6, which has a broad spectrum susceptibility to a large number of viruses, is used globally for virus isolation23. It is also approved by the WHO for the production of vaccines of human use and has been used to develop a range of vaccines, namely influenza, rabies, and Japanese encephalitis (JEV)293031. A comparable yield of CHIKV was obtained in BHK-21 cell line irrespective of the CHIKV strains. In comparison, the other two cell lines employed in the study, namely RD and A-549, CHIKV yield was comparatively low. In the present study, virus yield in vertebrate cell lines declined faster than that of C6/36, which remained viable for 7-10 days without showing CPE. Hence, all the experiments were terminated at day 6 PI.

In the present study, TCID₅₀ method was the only assay used to quantitate the virus yield. Since our objective was to determine the viable virus to determine growth, other assays, namely RT-PCR or qPCR were not used.

In conclusion, five cell lines (Vero E6, BHK-21, RD, C6/36 and A-549) were compared for their susceptibility and virus yield to the three lineages of chikungunya virus. Vero E6, BHK-21 and C6/36 cell lines yielded high titres to the three lineages of the virus while RD and A-549 cells were found low virus yielding. The Ae. aegypti mosquitoes showed an identical pattern of virus growth despite infection with two different doses of virus.