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. 2016 Sep 13:6:33088.
doi: 10.1038/srep33088.

Concurrent micro-RNA mediated silencing of tick-borne flavivirus replication in tick vector and in the brain of vertebrate host

Konstantin A Tsetsarkin  1 , Guangping Liu  1 , Heather Kenney  1 , Meghan Hermance  2 , Saravanan Thangamani  3 , Alexander G Pletnev  1
Affiliations

Concurrent micro-RNA mediated silencing of tick-borne flavivirus replication in tick vector and in the brain of vertebrate host

Konstantin A Tsetsarkin et al. Sci Rep. .

Abstract

Tick-borne viruses include medically important zoonotic pathogens that can cause life-threatening diseases. Unlike mosquito-borne viruses, whose impact can be restrained via mosquito population control programs, for tick-borne viruses only vaccination remains the reliable means of disease prevention. For live vaccine viruses a concern exists, that spillovers from viremic vaccinees could result in introduction of genetically modified viruses into sustainable tick-vertebrate host transmission cycle in nature. To restrict tick-borne flavivirus (Langat virus, LGTV) vector tropism, we inserted target sequences for tick-specific microRNAs (mir-1, mir-275 and mir-279) individually or in combination into several distant regions of LGTV genome. This caused selective attenuation of viral replication in tick-derived cells. LGTV expressing combinations of target sequences for tick- and vertebrate CNS-specific miRNAs were developed. The resulting viruses replicated efficiently and remained stable in simian Vero cells, which do not express these miRNAs, however were severely restricted to replicate in tick-derived cells. In addition, simultaneous dual miRNA targeting led to silencing of virus replication in live Ixodes ricinus ticks and abolished virus neurotropism in highly permissive newborn mice. The concurrent restriction of adverse replication events in vertebrate and invertebrate hosts will, therefore, ensure the environmental safety of live tick-borne virus vaccine candidates.

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Figures

Figure 1
Figure 1. miRNA targeting of the 3′ NCR of LGTV for tick-specific mir-1, mir-275 and mir-279 greatly inhibits virus replication in tick-derived cells.
(A) Schematic representation of the competition experiment and viral genomes used in the study. ISE6 cells were infected at an MOI of 0.1 pfu/cell with 1∶1 mixture of wt-EcoR* and one of the miRNA targeted LGTV viruses [3′(1), 3′(184), 3′(275), 3′(279) or 3′(263a)]. Supernatants were collected daily until 5 dpi to be used for RNA extraction followed by RT-PCR amplification, EcoRI digestion, and gel image analysis. DNA band intensities were quantified to determine the relative fitness (RF) of the competing viruses. RF was determined as the ratio between intensities of DNA bands of amplicons derived from wt-EcoR* and one of miRNA targeted 3′(T) viruses for each time point (D1–D5; dpi) normalized to the starting ratio of viral bands in the inoculum (inoc.) used for ISE6 cells infection. (B–F) Effect of insertion of a single target copy for mir-1 (B), mir-184 (C), mir-275 (D), mir-279 (E), or mir-263a (F) on LGTV fitness in ISE6 cells. (G) Growth kinetics of wt-EcoR* and miRNA targeted LGT viruses in ISE6 cells infected at an MOI of 0.01. The dashed line (here and in all subsequent Figures) indicates the limit of virus detection (0.7 log10 pfu/mL).
Figure 2
Figure 2. Insertion of a single copy of a target for tick-specific mir-1 into the ORF of LGTV selectively attenuates virus replication in tick-derived ISE6 cells, but not in Vero cells.
(A) Schematic representation of LGTV miRNA targeted in the dE/NS1R. A mir-1 or scrambled mir-1 (mir-1*) target sequence (pixelated box) was fused with the codon-optimized sequence of an E/NS1 stem-anchor region (2171–2488 nt, gray box) and inserted in-frame at nt position 2489 of the LGTV genome (wt). TM1 and TM2 are transmembrane helixes in the C-terminal stem-anchor region of E protein. (B,C) Growth kinetics of E(1) and E(1*) viruses in Vero (B) and ISE6 (C) cells infected at an MOI of 0.01. Titers were determined in Vero cells and presented as an average of two biological replicates (±standard deviation). Differences in growth kinetics were compared using 2-way ANOVA.
Figure 3
Figure 3. Co-targeting of the 3′ NCR for multiple tick-specific miRNAs does not prevent the emergence of escape mutants in ISE6 cells.
(A) Schematic representation of recombinant LGTV genomes used in this study. Top: diagram indicating sites for miRNA target insertions in the predicted secondary structure of the 3′ NCR of LGTV. Bottom: recovered viruses with specific composition of the miRNA target(s) inserted into the 3′ NCR. (B) Growth kinetics of wt-EcoR* and miRNA targeted LGT viruses in ISE6 cells infected at an MOI of 0.01. (C) The titer (±standard deviation) of miRNA targeted viruses after 2nd passage in ISE6 cells. To initiate the 2nd cycle of replication, ISE6 cells were infected at an MOI of 1 and the mean virus titer in the cell supernatants was determined in two replicates in Vero cells.
Figure 4
Figure 4. Effect of multiple miRNA targets inserted in distant regions of LGTV genome on virus fitness in ISE6 and Vero cells.
(A) Schematic representation of viruses used in the study. The miRNA targeting cassettes, which encode targets for tick- and CNS-specific miRNAs (solid boxes), were inserted into the dCGR, dE/NS1R, and 3′ NCR individually or in combinations as indicated (see table at left bottom). For the dCGR insert: miRNA targets were introduced between C48AA region encoding 48 amino acids of truncated C protein and sequence of 2A protease from FMDV (yellow box). Grey areas (C opt) represent codon-optimized C protein gene sequence. For E/NS1 insert: a single copy of mir-1 target in E(1) virus (Fig. 2A) was replaced with a set of miRNA-targets as indicated and dE/NS1R was inserted into E5 virus. For the 3′ NCR insert: the indicated miRNA targets were introduced into sites 1–4 as shown in Fig. 3A. Four control (scr) viruses contained multiple substitutions in all miRNA target sequences (striped boxes) within every inserted cassette. (B,D) Kinetics of infectious virus recovery after plasmid DNA transfection into Vero cells. Plasmids encoding LGTV containing single (B) or multiple (D) miRNA targeting cassettes and control clones were transfected into Vero cells. Cells culture aliquots were collected daily and titrated in Vero cells in duplicate. (C,D) Growth kinetics of LGT viruses containing single (C) or multiple (E) miRNA target cassettes and control (scr) viruses in ISE6 cells infected at an MOI of 0.01.
Figure 5
Figure 5. Targeting of LGTV genome for tick-specific miRNAs blocks virus infectivity and replication in Ixodes ricinus ticks.
I. ricinus nymphs were submerged for 45 min in Opti-Pro medium containing ~7 log10 pfu/mL of indicated virus and then were returned to glass vials for 21 (A) or 51 (B) days of incubation. Virus titer in each tick body suspension was determined in Vero cells. Horizontal line represents the mean virus titer for all nymphs in the group. The dashed line indicates the limit of virus detection [0.1 log10 pfu/mL]. Differences in infection rates between unmodified E5 virus and one of miRNA targeted [C(mir)/3′(mir) or C(mir)/E(mir)3′(mir)] or scrambled control [C(scr)/E(scr)3′(scr)] viruses were compared using one-tailed Fisher’s exact test, followed by p-values adjustment using Bonferroni correction method to account for multiple comparisons.
Figure 6
Figure 6. Effect of miRNA targeting cassettes inserted at a single or multiple regions of the LGTV genome on viral pathogenesis and growth in the brain of newborn mice following IC inoculation.
(A,B) 3-day-old Swiss Webster mice were inoculated IC with 102 of LGT viruses containing a single (A) or multiple (B) miRNA targeting cassettes or containing scrambled control sequences. Mean virus titers (±SD) in the brain of three mice collected at the 3, 5, and 7 dpi are shown. (C,D) Mice in groups of 10 were inoculated IC with 0.1 (C) or 1 (D) pfu of C(mir), E(mir) or 3′(mir) virus and monitored daily for morbidity for 21 days. (E,F) Mice in groups of 10 were inoculated IC with 103 (E) or 104 (F) pfu of LGT viruses containing multiple miRNA targeting cassettes and monitored daily for morbidity for 21 days. Note: scrambled control virus C(scr)/E(scr)3′(scr) in figure (F) was injected at the dose of 1 pfu/mouse.
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