Comparative analysis of the genome sequences and replication profiles of chikungunya virus isolates within the East, Central and South African (ECSA) lineage

Abstract

Background: A comparative analysis of the genomic and replication profiles of different geographical chikungunya virus (CHIKV) isolates of the East, Central and South African (ECSA) lineage was performed.

Findings: Analysis of the data revealed the different growth kinetics for the different isolates. Deep genome sequencing analysis further revealed specific amino acid mutations in the viral nsP1, nsP3, nsP4, E1 and E2 proteins in the different isolates. Despite the difference in viral genomic profiles, the virus-induced ultrastructural changes within infected cells remained highly conserved among the different chikungunya virus isolates.

Conclusions: These findings provide insights into the genomic and replication profiles of the re-emerging chikungunya virus isolates of the ECSA lineage.

Figure 1

Replication kinetics of CHIKV isolates (IMTSSA, SGP011 and SGP007) in infected C6/36 and HeLa cells. a and b) Negative strands viral RNA quantification with qRT-PCR of CHIKV infected C6/36 and HeLa cells over a period of five days post-infection. c and d) Infectious virion formation from CHIKV infected C6/36 and HeLa cells were analyzed by plaque formation assay over a period of five days post-infection and expressed as log PFU/ml. e and f) FACS quantification of CHIKV antigen in infected C6/36 and HeLa cells over a period of five days post-infection.

Figure 2

Replication of CHIKV (IMT isolate) in C6/36 cells. (a) Ultrastructural analysis of mock-infected C6/36 cells. At higher magnification, Golgi apparatus (GA), endoplasmic reticulum (ER) and mitochondria (M), can be observed. The bar corresponds to 0.5 μm. (b) Presence of Cytopathic Vacuoles (CPV) I in early infection (8 h post-infection). A typical CPV I complex (appx 800 nm) can be observed. CPV I is characterized by membranous spherules (arrows) regularly arranged from the interior surface of the complex. The bar corresponds to 0.2 μm. (c) Virus release by budding at the plasma membrane during early infection. CHIKV particle are observed to be budding from the plasma membrane (Pm). The bar corresponds to 0.2 μm. (c) i Enlarged of (c) showing the lining of the nucleocapsids (arrowheads) underneath the plasma membrane (Pm) of the cell. Matured virus (arrow) is observed to be budding from the peripheral of cells membrane. (d) Budding of CHIKV occurred extensively at the plasma membrane of the cells. The bar corresponds to 0.2 μm. (d) i Enlarged of (d) monitored the different stages of budding of CHIKV particles from the plasma membrane (arrows). (e) Ultrastructural analysis of late infection with CHIKV in C6/36 cells (Day 1 post infection). CPV II formations are observed. The viral nucleocapsids (arrowheads) are present at the cytoplasmic surface of CPV II complexes. The bar corresponds to 0.2 μm. (f) Several CPV II complexes are found in cluster near the plasma membrane (Pm) with the noticed of mature virus (arrows) in the complex. (g) and (h) At the late stage, CHIKV can be seen to be released from cells via exocytosis (arrows). Both the bar of g and h correspond to 0.2 μm.

Figure 3

Replication of CHIKV (IMT isolate) in HeLa cells. (a) Ultrastructural analysis of mock-infected HeLa cells. Under high magnification, the cellular organelles such as the Golgi apparatus (GA) and mitochondria (M), can be seen. The bar corresponds to 0.2 μm. (b) Infection of CHIKV in HeLa cells. Viral-induced membranous spheres are observed to associate with the plasma membrane [boxed]. The bar corresponds to 0.2 μm. (c) CPV II complexes were detected at 12 hours post infection. The viral nucleocapsids (arrows) were observed to be budding in the CPV II complexes. The bar of c corresponds to 0.5 μm. (d) Massive accumulation of mature CHIKV (arrows) in infected cells at late infection. The bar corresponds to 0.5 μm.