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. 2014 Jan 20;9(1):e85714.
doi: 10.1371/journal.pone.0085714. eCollection 2014.

A novel 2006 Indian outbreak strain of Chikungunya virus exhibits different pattern of infection as compared to prototype strain

Abhishek Kumar  1 , Prabhudutta Mamidi  1 , Indrani Das  1 , Tapas K Nayak  2 , Sameer Kumar  1 , Jagamohan Chhatai  1 , Subhasis Chattopadhyay  2 , Amol R Suryawanshi  1 , Soma Chattopadhyay  1
Affiliations

A novel 2006 Indian outbreak strain of Chikungunya virus exhibits different pattern of infection as compared to prototype strain

Abhishek Kumar et al. PLoS One. .

Abstract

Background: The recent re-emergence of Chikungunya virus (CHIKV) in India after 32 years and its worldwide epidemics with unprecedented magnitude raised a great public health concern.

Methods and findings: In this study, a biological comparison was carried out between a novel 2006 Indian CHIKV outbreak strain, DRDE-06 and the prototype strain S-27 in mammalian cells in order to understand their differential infection pattern. Results showed that S-27 produced maximum number of progenies (2.43E+06 PFU/ml) at 20 to 24 hours post infection whereas DRDE-06 produced more than double number of progenies around 8 hours post infection in mammalian cells. Moreover, the observation of cytopathic effect, detection of viral proteins and viral proliferation assay confirmed the remarkably faster and significantly higher replication efficiency of DRDE-06. Moreover, our mutational analysis of whole genome of DRDE-06 revealed the presence of nineteen mutations as compared to S-27, whereas the analysis of 273 global isolates showed the consistent presence of fifteen out of nineteen mutations in almost all outbreak isolates. Further analysis revealed that ∼46% of recent outbreak strains including DRDE-06 do not contain the E1-A226V mutation which was earlier shown to be associated with the adaptation of CHIKV in a new vector species, Aedes albopictus.

Conclusions: A novel 2006 Indian CHIKV outbreak strain, DRDE-06 exhibits different pattern of infection as compared to prototype strain, S-27. This might be associated to some specific mutations observed in genome wide mutational analysis in DRDE-06 which emphasizes the need of future experimental investigation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Comparison of Biological phenotypic characteristics of S-27 and DRDE-06 Chikungunya virus strains.
Vero cells were infected with the viral strains with MOI 1. (a) Cytopathic effects (CPE) were observed under microscope (Magnification −20×) at different time points and arrows are indicating the cell showing CPE (at early time points only). (b) The cytotoxicity was calculated by counting the live and dead cells by trypan blue stains from three independent experiments. (c) Infected cells were harvested at 16 hours post infection (hpi) for S-27 and at 8 hpi for DRDE-06 based on the observation of highest CPE. Viral protein expression was checked in Western Blot using nsP2 antibody. GAPDH was used as a loading control.
Figure 2
Figure 2. Proliferation kinetics of S-27 and DRDE-06 Chikungunya virus strains.
Plaque assay was carried out in Vero cells (a) Representative pictures showings the progressive plaque formation at different time points. (b) Average plaque sizes at different time points were calculated based on the diameter of 10 randomly picked plaques of three independent experiment (p<0.05).
Figure 3
Figure 3. Growth kinetics of S-27 and DRDE-06 Chikungunya virus strains.
Vero cells were infected with viruses and supernatants were collected at different time intervals. Growth curves were obtained by plotting viral titres present at each time. Virus titres were calculated from three independent experiments.
Figure 4
Figure 4. Expression pattern of Chikungunya viral proteins.
Vero cells were infected with S-27 and DRDE-06 with MOI 1 and cells were harvested at different times post infection (hpi - hours post infection). (a) Viral proteins (nsP2, E1/E2 and capsid) detected by Western blot by using respective antibodies. GAPDH was used as a loading control. (b) Representation of nsP2 protein expression quantified by Quantity One software. Values were calculated from three independent experiments. (c) C6/36 cells were infected with S-27 and DRDE-06 with MOI 1 and cells were harvested at different days post infection (dpi). Viral protein nsP2 was detected by Western blot using nsP2 polyclonal antibody.
Figure 5
Figure 5. Phylogenetic analysis of E1 sequences of 61 representative Chikungunya viruses global isolates from the year 1952 to 2011.
The unrooted tree was constructed using Neighbor-Joining method with 1000 bootstrap value. Numbers along with branches indicate bootstrap values. Scale bar indicates nucleotides substitutions per site. The presence/absence of E1-A226V mutation is depicted by black filled square and open square. *indicates the unavailability of sequence information for E1-A226V mutation and Arrow indicates strains used in this study. (The full Phylogenetic tree of 106 global isolates from the year 1952 to 2011 may be viewed in Figure S3).
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Grants and funding

This work was supported by Department of Biotechnology, Ministry of Science and Technology, Govt. of India vide grant no BT/PR13118/GBD/27/186/2009 and BT/PR15173/GBD/27/356/2011 and by Council of Scientific and Industrial Research (CSIR), Ministry of Science and Technology, Govt. of India vide Project No. 37(1542)/12/EMR-II). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.