Mosquito-borne alphaviruses in Zambia: Isolation and characterization of Eilat and Sindbis viruses

Abstract

Alphaviruses in the family Togaviridae include zoonotic arthropod-borne viruses, including Sindbis virus (SINV), chikungunya virus, as well as insect-specific viruses such as Eilat virus (EILV). Previous investigations of alphaviruses in Zambia have identified a novel insect-specific alphavirus, Mwinilunga alphavirus in mosquitoes. Further ongoing surveillance resulted in the isolation of EILV and SINV for the first time in Zambia. Here, these alphaviruses were characterized in terms of growth kinetics in cells, and molecular phylogenetic relatedness to other alphaviruses. Zambian EILV (strain zmq19_M44) exhibited a close phylogenetic relationship with other insect-specific alphaviruses and shared a close nucleotide identity to those of EILV isolate (90.4 %) and Mwinilunga alphavirus (75.5 %). EILV zmq19_M44 attained a saturating titer in C6/36 cells at 6-8-days post infection but was unable to replicate in mammalian cells. Phylogenetic analysis revealed the Zambian SINV (strain zmq17_M115) belongs in Clade D of SINV Genotype 1 along with the Kenyan isolate BONI 584 from Central Africa. The growth of the SINV zmq17_M115 was comparable to that of the prototype SINV strain AR339 in mammalian cells but was statistically different in insect cells. Our findings will contribute to public health measures for the control of alphaviral diseases in Zambia.

Keywords: Alphavirus; Eilat virus; Mosquito; Sindbis virus; Zambia.

Fig. 1

Phylogenetic analysis based on amino acid coding regions of alphaviruses. Phylogenetic analysis of non-structural proteins (A) and structural proteins (B) of alphaviruses. Phylogenetic trees were constructed by the Maximum Likelihood method using a JTT model with 1000 bootstrap replicates. Red text indicates Zambian SINV M115 and EILV M44 isolated in this study. Alphavirus serocomplexes are shown to the right side of the tree. The bootstrap values are shown at the branch node of each clade. The scale bars represent the number of substitutions per site.

Fig. 2

Phylogenetic analysis based on coding sequences of SINV The isolated Zambian SINV M115 in this study is highlighted in red. 54 SINV strains were obtained from GenBank and included in the analysis. The phylogenetic tree was constructed by the Maximum Likelihood method using a GTR+G + 1 model with 1000 bootstrap replicates. Genotypes are shown to the right side of the tree. The bootstrap values are shown at the branch node of each clade. The scale bars represent the number of substitutions per site. Red text indicates Zambian SINV M115 and isolated in this study, and blue text indicates African SINV isolates. The cluster including SINV M115 in clade D is highlighted in light blue.

Fig. 3

Growth kinetics of Zambian EILV isolate M44 Vero, BHK-21 and C6/36 cells were infected with EILV M44 at an MOI of 0.1. Titers at the indicated time points were determined by TCID₅₀. The values shown are mean ± standard deviation (SD) of triplicate studies.

Fig. 4

Growth kinetics of Zambian SINV isolate M115 Vero (A) and BHK-21 (B) cells were infected with SINV isolate M115 and AR339 at an MOI of 0.0001. C6/36 cells (C) were infected with SINV at an MOI of 0.01. Titers at the indicated each time point were determined by plaque assay. The values shown are mean ± standard deviation (SD) of triplicate. *p < 0.05, ***p < 0.001 by two-way ANOVA test.