Aedes aegypti (Aag2)-derived clonal mosquito cell lines reveal the effects of pre-existing persistent infection with the insect-specific bunyavirus Phasi Charoen-like virus on arbovirus replication

Abstract

Background: Aedes aegypti is a vector mosquito of major public health importance, transmitting arthropod-borne viruses (arboviruses) such as chikungunya, dengue, yellow fever and Zika viruses. Wild mosquito populations are persistently infected at high prevalence with insect-specific viruses that do not replicate in vertebrate hosts. In experimental settings, acute infections with insect-specific viruses have been shown to modulate arbovirus infection and transmission in Ae. aegypti and other vector mosquitoes. However, the impact of persistent insect-specific virus infections, which arboviruses encounter more commonly in nature, has not been investigated extensively. Cell lines are useful models for studying virus-host interactions, however the available Ae. aegypti cell lines are poorly defined and heterogenous cultures.

Methodology/principle findings: We generated single cell-derived clonal cell lines from the commonly used Ae. aegypti cell line Aag2. Two of the fourteen Aag2-derived clonal cell lines generated harboured markedly and consistently reduced levels of the insect-specific bunyavirus Phasi Charoen-like virus (PCLV) known to persistently infect Aag2 cells. In contrast to studies with acute insect-specific virus infections in cell culture and in vivo, we found that pre-existing persistent PCLV infection had no major impact on the replication of the flaviviruses dengue virus and Zika virus, the alphavirus Sindbis virus, or the rhabdovirus vesicular stomatitis virus. We also performed a detailed characterisation of the morphology, transfection efficiency and immune status of our Aag2-derived clonal cell lines, and have made a clone that we term Aag2-AF5 available to the research community as a well-defined cell culture model for arbovirus-vector interaction studies.

Conclusions/significance: Our findings highlight the need for further in vivo studies that more closely recapitulate natural arbovirus transmission settings in which arboviruses encounter mosquitoes harbouring persistent rather than acute insect-specific virus infections. Furthermore, we provide the well-characterised Aag2-derived clonal cell line as a valuable resource to the arbovirus research community.

Fig 1. Generation of clonal Aag2-derived cell lines originating from single cells.

(A) Brightfield microscopy image of heterogeneous Aag2 cell population consisting of multicellular ‘clusters’ (examples indicated by hashed lines throughout) and large rounded floating cells (arrows) interspersed across a loose monolayer. (B) FACS gating strategy illustrating selection of live single cells from DAPI-stained Aag2 cell suspension. (C) Resultant Aag2-derived clonal cell line morphologies following limited expansion; (i) similar appearance to parental Aag2 cells, (ii) highly clustered cells with no monolayer formation (some rounded floating cells present), (iii) only large rounded floating cells observable (individual cells and large multi-cell floating aggregates). Only those fourteen clones selected for further study are shown. Images were taken immediately following three-week expansion from single cells into confluent 24-well plate culture. ^# The Aag2-AF5 cell line was selected for CRISPR gene editing [60,61] (see main text). (D) Total number of clonal cell lines of each morphology generated. (E) Reversion of ‘clustered’ and ‘rounded’ clonal cell lines back to parental Aag2-like morphology following extended culture. Scale bar is 200 μm.

Fig 2. Aag2-AF10 and Aag2-AF12 cell lines harbour barely detectable levels of phasi charoen-like virus.

(A) Short PCR amplicons spanning the three PCLV genome segments (L, M, S) amplified from RNA or genomic DNA isolated from parental Aag2 cells, either with or without a reverse transcription step (RT). Purified nucleic acids were treated with RNase or DNase prior to PCR. MDCK cell RNA, the cellular Rps7 gene/mRNA and the RNA virus NDV, which was spiked into cells immediately prior to RNA extraction, serve as controls. (B) Detection of the PCLV S segment and CFAV by RT-PCR in Aag2-derived clonal cell lines. Cellular Rps7 mRNA serves as a loading control. (C) Detection of the PCLV L, M and S genome (-ssRNA) and antigenome (+ssRNA) segments in select Aag2-derived clonal cell lines by sense-specific RT-PCR. Rps7 mRNA serves as a loading control. (D) PCLV L segment RT-qPCR ΔΔC_t (normalised to Rps7 mRNA) for select Aag2-derived clonal cell lines expressed relative to parental Aag2 cell line at (i) early passages (Aag2-AF10, passage 2; Aag2-AF12, passage 3) and (ii) later (‘medium’) passages (Aag2-AF10, passage 8; Aag2-AF12, passage 12). (E) RT-qPCR quantification of viral RNA copies for PCLV L segment (i) and CFAV (ii) at late passages in Aag2 (parental), Aag2-AF5, Aag2-AF10 and Aag2-AF10 cells. Starting passages (“~P15”) were passage 15 (parental Aag2), 12 (Aag2-AF5), 14 (Aag2-AF10) and 13 (Aag2-AF12). Data points represent three independently passaged lines. Error bars represent standard deviation. ** P < 0.01; *** P < 0.001; ns, not significant (one-tailed Student’s t test). UD, undetected. ^# Aag2-AF5 cell line used for CRISPR gene editing [60,61].

Fig 3. All Aag2-derived clonal cell lines have a functional RNAi pathway.

C6/36 cells, the parental Aag2 cell line and its derived clonal cell lines were transiently transfected with plasmids constitutively expressing firefly luciferase and Renilla luciferase (transfection control) in the presence of dsRNA directed against GFP (dsGFP) or firefly luciferase (dsLuc). Mean Renilla-normalised firefly luciferase (FFluc) expression is expressed relative to the dsGFP negative control. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant (one-tailed Student’s t test). Error bars represent standard deviation. ^# Aag2-AF5 cell line used for CRISPR gene editing [60,61] is highlighted in orange. PCLV-low clones Aag2-AF10 and Aag2-AF12 are highlighted in green; parental Aag2 cells and C6/36 cells (negative control) are shown in purple.

Fig 4. Susceptibility of Aag2-derived clonal cell lines to infection with arboviruses.

(A, C, E, G) Single-step growth kinetics of DENV-2 (A), ZIKV (C), SINV (E) and VSV (G) in the parental Aag2 cell line (MOI 2). Grey shading highlights time points tested in Aag2-derived clonal cell lines. (B, D, F, H) Replication of DENV-2 (B) and ZIKV (D) at 1, 2 and 3 days post-infection, replication of SINV (F) at 6, 12 and 24 hours post-infection (hpi), and replication of VSV (H) at 6, 9 and 12 hpi in the parental Aag2 cell line and its derived clonal cell lines (all MOI 2). Grey shading indicates 0.5 Log₁₀ above and 0.5 Log₁₀ below peak extracellular titres detected in parental Aag2 cell line. Error bars represent standard deviation. ^# Aag2-AF5 cell line used for CRISPR gene editing [60,61] is highlighted in orange. PCLV-low clones Aag2-AF10 and Aag2-AF12 are highlighted in green; parental Aag2 cells and C6/36 cells (negative control) are shown in purple.

Fig 5. Transfection efficiency of clone Aag2-AF5 relative to parental Aag2 cells.

(A) Cells were imaged at 10X magnification 48 h after transient transfection with a constitutively active GFP expression vector (pIEx-EGFP). (B) Quantification of transfection efficiency; differences are non-significant. (C) Cells were transiently transfected with a constitutively active firefly luciferase reporter plasmid (pIEx-luc) and luciferase activity was measured four days later. * P < 0.05 (two-tailed Student’s t test). RLU, relative light units. All error bars represent standard deviation. ^# Aag2-AF5 cell line used for CRISPR gene editing [60,61].

Fig 6. Antimicrobial peptide induction in clone Aag2-AF5 compared to parental Aag2 cells.

Cells were stimulated with heat-inactivated E. coli (A), L. monocytogenes (B) or S. aureus (C) for 24 h and induction of DefD (i), CecB (ii) or CecD (iii) was measured by RT-qPCR. Gene induction is relative to the respective unstimulated cell line. * P < 0.05; ** P < 0.01; ns, not significant (two-tailed Student’s t test). Error bars represent standard error of the mean. ^# Aag2-AF5 cell line used for CRISPR gene editing [60,61].