1. Comparative genotypic and phenotypic characterization of
Dengue virus serotype 2 from Kerala, South India
Sneha Singh, M. G. Anupriya, E.Sreekumar
Molecular Virology Laboratory, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram , Kerala, India; .
Abstract
Dengue has become endemic in many parts of India. This mosquito-borne viral infection results in large numbers of acute febrile illness episodes each year, many of them leading to severe hemorrhagic fever or shock syndrome, and mortality. During the course of genetic characterization of dengue virus (DENV) clinical isolates from Kerala, we identified two strains of DENV serotype 2 (RGCB921 & RGCB880) that belonged to independent genetic clades (named as lineage IV & lineage V) in phylogenetic analysis. Considering the distinct divergence of these lineages, with a number of genetic differences across the genome, we were curious to understand whether these strains had any differences in their infectivity phenotype. Accordingly, we studied the in vitro replication kinetics of these strains in mammalian and mosquito cells. In plaque assays the Lineage IV strain and lineage V strain showed differences in the plaque morphology. The lineage IV strain (RGCB921) was capable of very high replication in most mammalian cells studied and showed cytopathic effect in human liver cells and lung epithelial cells compared to Lineage V strain (RGCB880). On the contrary, the Lineage IV strains were able to infect human embryonic kidney cells while Lineage V could not replicate at all in them. There was no cytopathic effect induced by both these strains in mosquito cells. However, the viral titre for the Lineage IV strain was significantly higher, which showed its higher replication efficiency in the mosquito vector. Overall, Lineage IV strain (RGCB921) seems to be more adapted in both mosquito and mammalian cells. Our results indicate that there are genetically different strains with distinct infectivity phenotypes circulating in the region. Correlating the relationships of the clinical spectrum of the disease with the genetic and phenotypic differences observed in the viral lineages may provide genetic tools for possible prediction of the severity of future dengue cases in the region.
100 μg of proteins was subjected to in-solution digestion to generate peptides
Introduction
Dengue virus is a positive sense, single stranded RNA virus.
Genus: Flavivirus Family: Flaviviridae
Causes dengue fever, dengue hemorrhagic fever and dengue shock syndrome characterized by vascular leakage.
Consists of four serotypes – DENV1, DENV2, DENV3, DENV4; DENV-2 is widely distributed and has been associated with the most virulent outbreaks.
Objective
Comparison of the whole genome sequence of DENV2 strains isolated from Kerala having difference in their infectivity and studying their genotypic and phenotypic differences in mosquito and mammalian cells.
Methodology
II.DENV2 Maximum likelihood tree. RGCB880 and RGCB921 fall under two distinct lineages, Lineage V and Lineage IV respectively.
III. The morphological changes in the mammalian cells in dose dependent manner upon infection with RGCB880 and RGCB921.
Fig 5:- Cytopathic effect and morphological changes: The morphological changes upon infection with the two strain in dose dependent manner was studied by light microscopy.
The two strains of DENV-2 clinical isolates (RGCB880 and RGCB 921) were passaged in insect cell line, C6/36.
. The whole genome sequences of the passaged viruses were compared and ML tree was constructed using MEGA 6.0 software.
The passaged viruses were used for infecting mosquito and human cell line –C6/36, A549, HEK293, Huh-7, HMEC, U87MG.
MTT assay, in vitro infection kinetics were done to study the phenotypic differences between the two isolates.
Fig 6:- Hoechst staining: Hoechst staining was performed on mammalian cells to observe nuclear condensation changes, if any. The nucleus is stained as blue in colour.
Results
DENV2 RGCB921 (lineage IV) infects and replicates efficiently in mosquito and mammalian cells compared to RGCB880 (Lineage V).
Conclusion
The two strains isolated from Kerala, India fall under two distinct lineages, Lineage IV (RGCB921) and Lineage V (RGCB880) in the ML tree.
DENV2 RGCB921 has more infection in mosquito cell line, C6/36 compared to RGCB880 with morphological changes but no cell death.
RGCB921 has higher viral titres and higher rate of replication in mammalian cells too, eg, epithelial cell (A549), hepatocyte (Huh7), endothelial cell (HMEC) and others.
RGCB921 is efficient in replication in HEK293 cells while RGCB880 cannot replicate in it.
There are variations in the plaque morphology between the two strains with RGCB880 showing larger plaques while RGCB921 has very small plaques.
RGCB921 shows cytopathic effect with changes in cell morphology without significant cell death in mosquito and mammalian cells compared to RGCB880 in a time and dose dependent manner.
There is no significant changes in nuclear condensation in the mammalian cells upon infection with RGCB880 and RGCB921.
Fig 1:- Infection kinetics and cell viability of two DENV2 strains in mosquito cell line, C6/36.
Fig 2:- Infection kinetics of two DENV2 strains in human cell line, A549, HEK293, HMEC, U87MG and Huh-7. The cells were infected with RGCB880 and RGCB921 at MOIs 0.1 and 1.0. The supernatants were collected at different time points. Plaque assay was carried out in BHK-21 cells to determine the virus titre. The viral titre has been expressed in logarithmic scale for PFU/mL.
Significance
The present study indicate that there are genetically different strains with distinct infectivity phenotypes circulating in the region. Correlating the relationships of the clinical spectrum of the disease with the genetic and phenotypic differences observed in the viral lineages may provide genetic tools for possible prediction of the severity of future dengue cases in the region.
Fig 3:- Plaque morphology. BHK-21 cells were infected with Huh-7 supernatant obtained after infection with the two DENV2 strains, RGCB880 and RGCB921 for the mammalian cells A549, HEK291, Huh7, HMEC and U87MG cells.
Acknowledgements
Rajiv Gandhi Center for Biotechnology, Kerala, India
Organizers of AMI 2015
DST INSPIRE for PhD fellowship
Department of Biotechnology, DBT, Govt. of India.
Fig 4: Phylogenetic tree based on the envelope gene from 155 strains sampled globally. The tree includes RGCB880 and RGCB921 strains used in the study and the evolutionary history was inferred with Maximuml likelihood method and Bayesian Package software. The tree was rooted using DENV2 Sylvatic strains.