Molecular and biological characterization of an Asian-American isolate of Chikungunya virus

Abstract

Chikungunya virus is an arthropod-transmitted virus that causes chikungunya fever, a disease characterized by severe muscle and joint pain. In 2013, the virus was introduced to the Americas and caused approximately 2.7 million cases of infection during the subsequent two years. The lack of knowledge regarding the biological behavior of the viral strains circulating during the outbreak motivated the characterization of an isolate from the Colombian outbreak, starting from analysis of the complete genome to the biological behavior in vitro. The full genome was retrieved using next-generation sequencing. The infective and replicative capacities were evaluated in HEK293T, Huh-7, and MRC-5 cell lines. The infection rates were determined by flow cytometry, and the cytopathic effect was assessed by a resazurin fluorescent metabolic assay. The viral yield was quantified using the virus plaque formation assay, while the viral proteins and genomic RNA kinetics were subsequently evaluated by western-blot and RT-qPCR. The COL7624 isolate clustered with other American and Caribbean sequences in the Asian American lineage. The T669A substitution in E2 protein distinguished it from other Colombian sequences reported in 2014. After 48 h post infection (hpi), the three cell lines analyzed reached infection percentages exceeding 65%, generating a high load of infectious viral progeny. The infection kinetics indicated that the replication peak of this CHIKV isolate is around 24 hpi, although gRNA is detectable in the culture supernatant from 4 hpi onwards. The infection caused the overexpression of interferon and pro-inflammatory cytokines, such as IL-1β, TNF-α, and IL-8. The COL7624 CHIKV isolate exhibited a high infective and replicative capacity as well as activation of cellular immune responses, similar to isolates belonging to the other genotypes.

Fig 1. Phylogenetic analysis of Chikungunya virus.

Full coding sequences of CHIKV were used. The maximum likelihood tree was constructed using the general time-reversible model and inferred following bootstrap analyses using 1,000 replicates. Strain names are in the format of: accession number/country /year of isolation. CHIKV genotypes: West African, Asian, and East/Central/South African (ECSA) are indicated on the right.

Fig 2. Evaluation of the infective and replicative capacity of the COL7624 CHIKV isolate.

Cells were infected at the indicated MOIs. At 24 and 48 hpi, the cells were fixed, permeabilized, stained with anti-E1 CHIKV antibody, and analyzed by flow cytometry (A). Cell viability was analyzed using a resazurin assay (B). Levels of infectious virions in supernatants were measured by plaque assay on Vero cells and are expressed as the number of plaque-forming units (PFU)/mL (C). Significant differences between the groups were evaluated considering the non-parametric Mann–Whitney statistical test (*P < 0.05; **P < 0.01; and ***P < 0.001). Data are representative of three independent experiments (n = 3).

Fig 3. Assessment of CHIKV infection by immunofluorescence.

Cells were exposed to CHIKV isolate at MOI 1. At 48 hpi, the cells were fixed, permeabilized, stained with anti-E1 CHIKV antibody.

Fig 4. Kinetic analysis of the COL7624 isolate.

Cells were infected at MOI 1 and harvested at the specified time points. Supernatants were collected, and the gRNA content was quantified by RT-qPCR assay based on E1 amplification (A). The cells were lysed and analyzed by western blotting using antibodies against nsP1 and E1/E2 proteins (B) and anti-caspase 3 (C, D). β- actin was used as a loading control. Statistical comparisons were performed using non-parametric Mann–Whitney tests (*P < 0.05; **P < 0.01; and ***P < 0.001).

Fig 5. Gene expression levels of pro-inflammatory genes in CHIKV infected cells.

Cells were infected at MOI 1 for 24 hpi and the RNA extracts were analyzed by RT-qPCR. Fold-change in mRNA levels of TNF-α, IFN-α, IL-1β, and IL-8 were calculated using mock-infected cells as control (A). Viral RNA levels (B). Data were analyzed with the One-way ANOVA test. Statistically significant differences are indicated: ** P < 0.01; *** P < 0.001; and **** P < 0.0001.