Functional classification of differentially affected proteins and their possible role in disease pathogenesis.

<p>A. Functional classification of the differentially affected proteins of muscle tissue in CHIKV infection. B. Schematic representation of the possible roles of identified proteins of different classes showing metabolic and rheumatic implications in CHIKV induced myopathy.</p

Gross pathology, viral replication and profile of inflammatory cytokines.

<p>A. Surgically removed hind limb muscles from mock- and CHIKV-infected mice showing gross pathology of the muscles (in terms of swelling) along with the 4 μm sections showing the localization of CHIKV antigen in the hind limb skeletal muscle on day 9 post infection. B. Virus titre in the hind limb muscles. C. Real time analysis showing relative fold change in inflammatory genes (MCP-1, MCP-3, IL-6, TNF-α, Rantes) in muscle tissue on day 9 post infection. ** Genes were considered significantly up-regulated if the change in their relative expression level was ≥2 fold at p<0.05 by student's <i>t</i> test.</p

Summary of proteins differentially expressed in mouse muscle tissue infected by Chikungunya virus.

<p>Summary of proteins differentially expressed in mouse muscle tissue infected by Chikungunya virus.</p

Representative 2-D gel image obtained from muscle tissue of mock-infected and CHIKV-infected mice.

<p>Equal amount of protein sample (750 μg) was first separated in a linear gradient of pH 4–7, followed by separation in SDS-PAGE (12%) and coomassie staining. A total of 27 protein spots were found to be significantly altered. Fold changes (mean values± SD) of the identified proteins are illustrated graphically (** signifies p≤0.05). Spots M6, M23 and M24 were found to be affected qualitatively and hence were not presented graphically.</p

Validation of proteomic results using Q-PCR and immunoblotting.

<p>A. Transcript alteration of the differentially expressed proteins in muscle tissue upon CHIKV infection. Total RNA of muscle tissue (infected/uninfected) was analysed by real time RT-PCR. House-keeping GAPDH gene was used for normalization purpose. RNA expression changes of vimentin, hemopexin, haptoglobin, Rho GDP, PKM2 and kininogen were in concordance with protein expression changes and were determined to be statistically significant (p≤0.05). *Genes were considered to be significantly up-regulated if the change in their relative expression levels was ≥2 fold. No significant difference in the RNA expression of ApoA1 and peroxiredoxin 6 was found. B. Immunoblot of representative proteins showing increased expressions in muscle tissue upon CHIKV infection.</p

Skin, spleen and muscle pathology of CHIKV infected mice.

<p>Representative picture showing the pathology staining of the tissues (skin, spleen and hind limb muscle) from mock-infected and CHIKV-infected mice on 8th day of post infection. Characteristic histological features are indicated by arrows. Skin of CHIKV-infected mice showed hyperplasia of basal keratinocytes and hyperkeratinisation. Hair follicles showed atrophy with no dividing cells in the matrix. Spleen of CHIKV-infected mice showed considerable lymphoid proliferation and haemorrhage. Muscle sections showed degenerative changes with dark pink stained, atrophied and necrotic muscle fibres, infiltration of neutrophiles and monocytoid cells between the muscle fibres.</p

Chikungunya virus (CHIKV) induced disease signs.

<p>A. Pictures of the hind limbs of mock- and CHIKV-infected animals. Chikungunya virus infection induced severe hind limb disease in new born mice. 2–3 day old mice inoculated with 10⁶ PFU (50 ul) of CHIKV by subcutaneous injection in the loose fold of skin on the back of the animal developed peak clinical signs on 8 day post inoculation whereas mock-infected group injected with uninfected tissue culture supernatant remained healthy. B. Mice were scored for the development of hind limb dysfunction and disease based on the following scale: 0, no disease signs; 1, ruffled fur; 2, mild hind limb weakness; 3, moderate hind limb weakness; 4, severe hind limb weakness and dragging and 5, moribund. Each data point represents the arithmetic mean ± SD for eight animals. Data is representative of three independent experiments.</p

Vertical transmission of Chikungunya virus in <i>Ae. aegypti</i> as determined by measuring log₁₀ RNA copies/pool of larvae and adults by real time RT-PCR.

<p>Horizontal line represent mean value for each group. Asterisks are indicating significant differences between the two groups (**p<0.01).</p