Molecular and biological characterization of an Asian-American isolate of chikungunya virus
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2021
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Background: Chikungunya virus is an arthropod-transmitted virus that causes chikungunya fever, a disease characterized by severe muscle and joint pain. The virus was introduced to the Americas in 2013 and caused approximately 2.7 million cases during the subsequent two years. The lack of knowledge regarding the biological behavior of the viral strains circulating during the outbreak has motivated the characterization of a virus isolated in Colombia in 2015.
Methods: The full genomes of isolated and cultured viruses were obtained using next-generation sequencing. The infective and replicative capacities and their impact on viability were evaluated in three different cell lines, HEK293T, Huh-7, and MRC-5, to describe the biological behavior. The infection percentages were determined by flow cytometry, and cytopathic effects were evaluated by conducting a resazurin fluorescent metabolic assay. The viral yield of each cell line was quantified by conducting a virus plaque formation assay. The virus proteins and RNA kinetics were then evaluated by western blotting and Reverse transcription -qPCR.
Results: The COL7624 isolate was clustered with other American and Caribbean sequences in the Asian American lineage. The T669A substitution in E2 distinguished it from other Colombian sequences reported in 2014. After 48 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 occurred around 24 hpi, although gRNA could be detected in the supernatant from 4 hpi onwards. Infection caused the overexpression of interferon and proinflammatory cytokines, such as IL-1, TNF-α, and IL-8.
Conclusions: The COL7624 isolate exhibited a high infective and replicative capacity and induced the activation of the cellular immune response, similar to isolates belonging to the other genotypes.
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Abstract
Background: Chikungunya virus is an arthropod-transmitted virus that causes chikungunya fever, a disease characterized by severe muscle and joint pain. The virus was introduced to the Americas in 2013 and caused approximately 2.7 million cases during the subsequent two years. The lack of biological knowledge regarding the behavior of the viral strains circulating during the outbreak has motivated the characterization of a virus isolated in Colombia in 2015.
Methods: The full genomes of isolated and cultured viruses were obtained using next-generation sequencing. The infective and replicative capacities and their impact on viability were evaluated in three different cell lines, HEK293T, Huh-7, and MRC-5, to describe the biological behavior. The infection percentages were determined by flow cytometry, and cytopathic effects were evaluated by conducting a resazurin fluorescent metabolic assay. The viral yield of each cell line was quantified by conducting a virus plaque formation assay. The virus proteins and RNA kinetics were then evaluated by western blotting and Reverse transcription-qPCR.
Results: The COL7624 isolate was clustered with other American and Caribbean sequences in the Asian American lineage. The T669A substitution in E2 distinguished it from other Colombian sequences reported in 2014. After 48 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 occurred around 24 hpi, although gRNA could be detected in the supernatant from 4 hpi onwards. Infection caused the overexpression of interferon and proinflammatory cytokines, such as IL-1, TNF-α, and IL-8.
Conclusions: The COL7624 isolate exhibited a high infective and replicative capacity and induced the activation of the cellular immune response, similar to isolates belonging to the other genotypes.
Palabras clave
Genoma, Virus chikungunya, Replicación, Infección
Keywords
Infection, Replication, Chikungunya virus, Genome