Mosquito-borne flaviviruses are among the most significant arboviral pathogens worldwide. Vaccinations and mosquito population control programs remain the most reliable means for flavivirus disease prevention, and live attenuated viruses remain one of the most attractive flavivirus vaccine platforms. Some live attenuated viruses are capable of infecting principle mosquito vectors, as demonstrated in the laboratory, which in combination with their intrinsic genetic instability could potentially lead to a vaccine virus reversion back to wild-type in nature, followed by introduction and dissemination of potentially dangerous viral strains into new geographic locations. To mitigate this risk we developed a microRNA-targeting approach that selectively restricts replication of flavivirus in the mosquito host. Introduction of sequences complementary to a mosquito-specific mir-184 and mir-275 miRNAs individually or in combination into the 3’NCR and/or ORF region resulted in selective restriction of dengue type 4 virus (DEN4) replication in mosquito cell lines and adult Aedes mosquitos. Moreover a combined targeting of DEN4 genome with mosquito-specific and vertebrate CNS-specific mir-124 miRNA can silence viral replication in two evolutionally distant biological systems: mosquitoes and mouse brains. Thus, this approach can reinforce the safety of newly developed or existing vaccines for use in humans and could provide an additional level of biosafety for laboratories using viruses with altered pathogenic or transmissibility characteristics.
Mosquito-borne flaviviruses such as dengue, yellow fever, Japanese encephalitis and West Nile viruses (genus, Flavivirus; family, Flaviviridae) are among the most significant arboviral pathogens of humans and domestic animals in many regions of the world. Depending on the particular virus, clinical manifestations can vary from asymptomatic or self-limited flu-like illness to a life threatening jaundice, hemorrhagic fever and shock syndrome, or severe meningitis and encephalitis. Outbreaks and epidemics of flavivirus diseases typically coincide with warm rainy seasons, when the population of competent mosquito vectors reaches sufficient density for sustainable virus transmission between vertebrate hosts. Historically, mosquito control programs were considered as the most effective measure to prevent outbreaks and limit the spread of flavivirus diseases. However, reduction of funding allocated to mosquito control campaigns during the last decades, accompanied by an increase in human population density and global travel [1,2] resulted in an increase in the number of flavivirus-associated illnesses [1–3]. This emphasizes the need for development of effective vaccines or therapeutics as an alternative means for protection against flavivirus diseases. A number of approaches are being pursued for development of effective vaccine candidates against flaviviruses, including RNA, DNA, inactivated, and subunit vaccines as well as vaccine candidates based on single-round replicated viral particles (reviewed in [4,5]). Although proven to be safe, they typically fail to provide long-lasting immunity after a single dose, and some may not be cost effective. This suggests a considerable advantage to vaccines based on live attenuated viruses that are relatively inexpensive to manufacture and provide a durable and potent immunity after a single dose of vaccination [6]. However, despite advances in developing live attenuated vaccine (LAV) candidates, a concern has been raised that they might not be safe in the environment due to their intrinsic genetic instability, potential reversion back to wild-type, and possible dissemination by mosquito vectors after feeding on vaccinees [7]. The possibility of mosquito transmission is further supported by the fact that many LAV candidates against diseases caused by mosquito-borne viruses can replicate in mosquito-derived cell lines and some are capable of infecting principle mosquito vectors in the laboratory [8–19]. For example, a vaccine strain of Venezuelan equine encephalitis virus (arbovirus, genus Alphavirus, family Togaviridae) was isolated outside its typical range from wild mosquitoes collected during a 1971 horse vaccination campaign in Louisiana [20], highlighting the risk of introduction and dissemination of potentially dangerous viral strains in new geographic locations. Flaviviruses are enveloped single-stranded, positive-sense RNA viruses with genomes of approximately 11,000 nucleotide bases that contain 5’ and 3’-terminal non-coding regions (NCR) flanking a single open reading frame (ORF) encoding a polyprotein. During cap-dependent translation, the polyprotein is processed by viral and cellular proteases to yield three structural proteins (capsid [C], premembrane [prM], and envelope [E]) followed by at least seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [21]. In recent years, an approach based on targeting of viral genomes for number of cellular microRNAs (miRNAs) has been proven to be a simple and efficient method to restrict replication and pathogenesis of DNA and RNA viruses in a cell-, tissue- or species-specific manner [22–33]. Moreover, we demonstrated that insertion of targets for several brain-specific microRNAs (miRNAs) into the 3’ NCR and/or ORF of neurotropic chimeric tick-borne encephalitis virus/dengue type 4 virus (TBEV/DEN4) was sufficient to selectively inhibit viral replication in neurons and to constrain the development of lethal encephalitis in adult and newborn mice after intracerebral or intraperitoneal infection [34–36]. In this study using a similar miRNA-targeting approach, we sought to design a mosquito-borne flavivirus that would be selectively restricted for replication in its invertebrate host. As a model system to investigate the effect of miRNA targeting on flavivirus fitness in arthropod vector, we selected a dengue type 4 virus (DEN4) that efficiently replicates in widely distributed Aedes (A.) aegypti and A. albopictus mosquitoes. DEN4 is not neuroinvasive in vertebrate hosts and is currently being used as a genetic background for development of live attenuated vaccines against neurotropic and non-neurotropic flaviviruses [8–12]. However, the DEN4-based chimeric viruses, as well as the DEN4 parent, are able to replicate in the central nervous system (CNS) of neonatal mice infected intracerebrally, causing lethal encephalitis. Therefore, we also explored if combined targeting of the DEN4 genome with mosquito-specific and mouse brain-expressed miRNAs can simultaneously restrict DEN4 infection and replication in the mosquito host and attenuate virus neurovirulence in newborn mice.
Recently, miRNA expression profiles have been identified for several mosquito species [37–39]. Based on this data, three mosquito-specific miRNAs (mir-184, mir-275 and mir-1) were selected for DEN4 genome targeting because they satisfy the following criteria: 1) they are highly expressed in different mosquito organs and mosquito-derived cell lines, and also remain abundant during flaviviruses infection [37]; 2) these miRNAs are evolutionarily conserved among insect species including mosquitoes, but they are different from their miRNA analogs in mammals. To investigate if miRNA targeting of DEN4 genome results in selective restriction of DEN4 replication in mosquitoes, a single copy of mir-184, mir-275, or mir-1 target sequence was introduced into the genome of DEN4 strain 814669 [40] (abbreviated D4s) between nucleotides (nts) 10277 and 10278 (15 nts downstream of the TAA stop codon preceding the 3’NCR). This particular strain of DEN4 was chosen because it has been extensively characterized in the laboratory and is currently being used as a genetic background for construction of LAV candidates against flaviviruses [8–12]. DEN4 viruses carrying miRNA target sequences were generated by electroporation of in vitro transcribed full-length genomic RNA into Vero cells. Parental and each modified DEN4 virus (designated D4s, D4-184s, D4-275s, and D4-1s; Fig 1) were harvested on day 5 post-electroporation and amplified by one additional passage in Vero cells. Specific infectivity values of synthesized RNA and viral titers in Vero cells supernatants for D4-184s, D4-275s, and D4-1s were comparable to the unmodified D4s clone (S1 Table), indicating that insertion of target sequences into the 3’NCR did not result in substantial attenuation of DEN4 in Vero cells. The effect of miRNA targeting on the DEN4 replication in mosquito cell lines was analyzed by comparing growth kinetics of viruses in Aag2 and C710 cells isolated from A. aegypti and A. albopictus mosquitoes, respectively. In both cell lines infected at multiplicity of infection (MOI) of 0. 01, parental D4s and D4-1s viruses replicated efficiently with nearly identical kinetics reaching titers of ~6 log10 pfu/ml by 3 dpi (Fig 2A and 2B). In contrast, the replication of viruses D4-184s and D4-275s carrying a target for mir-184 or mir-275 was significantly impaired (p<0. 001; 2-way ANOVA). In both cell lines, these viruses exhibited a 1000-fold or higher reduction in virus titer at 3 days post-infection (dpi) compared to the D4s virus, correlating with mir-184 and mir-275 expression levels in Aag2 and C710 cell lines (S1 Fig) [37]. Based on these data, we selected D4-184s and D4-275s for further evaluation of their replication in adult A. aegypti mosquitoes. Female mosquitoes were infected intrathoracically with 100 pfu of D4s, D4-184s, or D4-275s virus. After 7 days of incubation, whole body homogenates of each mosquito were prepared individually, and titers were determined on Vero cell monolayers (Fig 2C). Insertion of either miRNA target sequence in the 3’NCR of DEN4 genome led to a significant restriction of virus replication in mosquito bodies (p< 0. 001, one-tailed t-test), however, it was not sufficient to completely suppress the DEN4 infection. Previously, we demonstrated that multiple miRNA-targeting of flavivirus genome for brain-specific miRNAs in the 3’NCR results in increased efficiency of miRNA-mediated virus suppression in the CNS of mice compared to that observed for viruses containing only a single copy of the miRNA target [34,35]. Accordingly, we reasoned that a similar strategy should be applied to achieve a more reliable attenuation for DEN4 virus replication in cell culture and adult mosquitoes. Two additional constructs were developed based on D4-275s that contained additional target sequence for either mir-184 or mir-275 at nt position 212 of DEN4 3’NCR (Fig 1). Unfortunately, titers that these viruses achieved after recovery were not sufficient for their biological evaluation in mosquitoes (S1 Table). To improve virus recovery, we modified the DEN4 cDNA clone in the following ways: substituted the SP6 promoter (for in vitro transcription) with the eukaryotic RNA polymerase II cytomegalovirus (CMV) promoter; introduced two intron sequences at nt positions 3922 and 8836 of the DEN4 genome to minimize plasmid DNA toxicity during propagation in E. coli; inserted the hepatitis delta virus ribozyme sequence at the 3’-end of the DEN4 virus genome to ensure correct release of authentic 3’-terminated RNA during transcription; and introduced the previously identified Vero cell-adaptive mutation (L122→F) into the NS4B protein gene to enhance replication in Vero cells [41–44]. The modified parental and mir-184- or mir-275-targeted DEN4 viruses (designated D4, D4-184, and D4-275; Fig 1) were re-generated by cDNA transfection into Vero cells (S2 Table). Two additional viruses (D4-275-184 and D4-275x2) were developed based on the D4-275 genome and contained a second target sequence for either mir-184 or mir-275 at nt position 212 of the 3’NCR (Fig 1). Viruses were biologically cloned by terminal dilution and amplified by one additional passage in Vero cells (S2 Table). Viruses were genetically stable and contained the miRNA target insertions as assessed by sequence analysis of viral genomes after 5 additional passages in Vero cells. All viruses containing miRNA targets were significantly attenuated compared to D4 virus for growth in A. aegypti-derived Aag2 cells (Fig 3A, p<0. 001; 2-way ANOVA) and in A. albopictus-derived C6/36 cells, which replaced the previously used C710 cells (Fig 3B, p<0. 001; 2-way ANOVA). As anticipated, the combined expression of miRNA targets at two distant positions of the 3’NCR (D4-275-184 and D4-275x2) led to a more effective restriction of the virus replication in mosquito cells and resulted in at least a 3. 5 or 6. 0 log10 pfu/ml reduction in virus titer compared to D4 virus in Aag2 and C6/36 cells, respectively. In contrast, all viruses bearing one or two targets for mosquito-specific miRNAs replicated efficiently in Vero cells and the virus yield of each miRNA-targeted virus did not differ significantly from that of parental D4 virus (Fig 3C). The maintenance of dengue viruses in the environment relies on the ability of the virus to infect, replicate, and develop a disseminated infection within two critical vectors: A. aegypti and A. albopictus mosquitoes. To investigate how targeting of the viral genome for mosquito-specific miRNA affects DEN4 fitness in female mosquitoes, A. aegypti and A. albopictus were fed with blood meals containing 7. 1–7. 4 log10 pfu/ml of recombinant DEN4 viruses. Viral titers in mosquito bodies, viral infectivity, and dissemination into heads of orally infected mosquitoes were assayed on day 14 after feeding. D4 virus infected 90% of both mosquito species by day 14, reaching a mean virus titer of ~2. 5 log10 pfu/mosquito body, and disseminated to the heads of 71% of A. aegypti and 55% of A. albopictus (Fig 4). Introduction of a single copy of miRNA target for either mir-184 or mir-275 miRNA resulted in a significant reduction of the DEN4 titer in mosquito bodies (Fig 4A and 4B; p≤0. 002 one-tailed Student' s t-test) in both mosquito species as well as viral infectivity and ability of virus to develop a disseminated infection in A. aegypti (Fig 4C; p<0. 05 one-tailed Fisher’s exact test). Infectivity of D4-184 and D4-275 viruses in A. albopictus were only slightly reduced as compared to D4 virus (Fig 4D; p = 0. 144 and p = 0. 0728), however both viruses were significantly attenuated in the ability to develop a disseminated infection (Fig 4D; p<0. 05 one-tailed Fisher’s exact test). All D4-184 and D4-275 viruses recovered from A. albopictus and the majority of viruses recovered from infected A. aegypti contained deletions or point mutations in the miRNA target sequences (S2 Fig), indicating that targeting of the DEN4 genome by a single copy of miRNA target was not sufficient to prevent virus transmission by mosquitoes. In contrast, the D4-275-184 virus was unable to infect the midgut and thus failed to disseminate in both mosquito species indicating that a combined expression of mir-184 and mir-275 targets in D4-275-184 was sufficient to completely block DEN4 replication in the principal mosquito vectors (Fig 4C and 4D, p<0. 001; Fisher’s exact test). These results are consistent with our previous observations made for viruses targeted by brain-specific miRNAs [34–36], suggesting that targeting of the viral genome in the same sites for two different miRNAs is a more efficient approach for flavivirus attenuation in the mosquitoes and the CNS of mice compared to a monotypic miRNA-targeting. Interestingly, a duplication of the target sequence for mir-275 miRNA alone resulted in a virus that was more infectious for A. aegypti (but not to A. albopictus) as compared to the D4-275-184 virus. Previous studies of miRNA-regulated gene expression have demonstrated that the majority of mRNAs are post-transcriptionally regulated by targeting in the 3’NCR, and although regulation of mRNAs through miRNA-targeting in the coding region is less frequent, it is well documented (reviewed in [45,46]). Therefore, to explore if targeting of an ORF region of DEN4 by mosquito-specific miRNAs can result in specific viral attenuation in mosquitoes, targets for mosquito-expressed mir-184 and mir-275 as well as three targets for human neuron-specific mir-124 miRNA were introduced in the DEN4 genome between sequences encoding the two C-terminal stem-anchor domains of DEN4 E protein (D4-E virus; Fig 1). We designed these insertions of miRNA targets in this region using an approach that has been described previously [34,47]. As a control, we generated a D4-E* virus, in which mir-184 and mir-275 target sequences of D4-E were synonymously mutated in the third base position of each codon. The levels of replication for both control D4-E* virus and D4-E-mutant-bearing miRNA targets were similar in Vero cells indicating that the presence of miRNA targets or their scrambled sequences in the ORF of the viral genome does not affect viral fitness in cells that do not express the corresponding miRNAs (Fig 5C). Genome regions containing miRNA targets in D4-E or scrambled sequences in D4-E* virus remained stable for at least 5 subsequent passages in Vero cells as demonstrated by sequence analysis (S3 Fig). Replication of D4-E, but not D4-E*, was strongly attenuated in both Aag2 and C6/36 cells as compared to the D4 virus (Fig 5A and 5B, p<0. 001; 2-way ANOVA), but the level of a residual replication of D4-E in either cell line was higher than that observed for the D4-275-184 virus carrying miRNA targets in the 3’NCR (Figs 3 and 5). Studies in orally infected A. aegypti mosquitoes demonstrated that infection rates of D4-E but not D4-E* were significantly decreased as compared to D4 (Fig 6B, p<0. 001 and p = 0. 151, respectively; one-tailed Fisher’s exact test). Infectivity of D4-E to A. aegypti was also significantly lower than that of D4-E* (1/24 versus 11/24; p = 0. 0009; Fisher’s exact test) and none of the mosquitoes (0/24) had disseminated infection with D4-E virus compared to 20. 1% (5/24) for D4-E* (p = 0. 025; Fisher’s exact test). These results indicate that miRNA targeting of the D4-E genome within the ORF can specifically attenuate the virus for both mosquito-derived cells and mosquitoes due to the presence of authentic miRNA target sequences. Even though a combined expression of mir-184 and mir-275 targets in the 3’NCR was sufficient to greatly restrict the D4-275-184 virus replication in mosquito cells and abolish infectivity in adult mosquitoes, an escape mutant lacking both authentic target sequences can theoretically emerge as a result of error prone flavivirus replication under miRNA-mediated selective pressure. To minimize the probability of such events, we generated a virus (D4-E-NCR1; Fig 1) expressing mir-184 and mir-275 target sequences in both the ORF and 3’NCR of DEN4 genome. As expected, the resulting virus was genetically stable and replicated well in Vero cells, but failed to initiate a productive infection in mosquito Aag-2 and C6/36 cells (Fig 5). Moreover, none of the A. aegypti mosquitoes that fed on a blood meal containing 6. 8 log10 pfu/ml of D4-E-NCR1 became infected, or developed a disseminated infection into mosquito heads as tested at 14 dpi (Fig 6). These findings indicate that combined co-targeting of the DEN4 genome in the ORF and 3’NCR does not result in target interference during miRNA mediated flavivirus attenuation in mosquitos. To explore if the miRNA targeting approach represents adequate means for simultaneous restriction of flavivirus replication in both the CNS of vertebrates and in mosquito vectors, we first evaluated neurovirulence of D4-E and D4-E-NCR1 viruses in a highly permissive animal model such as 3-day-old Swiss mice inoculated intracranially [34,36,42,44,48]. Both D4-E and D4-E-NCR1 viruses contained miRNA targets for mosquito-specific mir-184 and mir-275 and three copies of target sequences for vertebrate brain-specific mir-124 in the duplicated E/NS1 region (Fig 1). As a control virus for comparative assessment in the CNS of mice, we generated a D4-E** virus based on D4-E that contained synonymous mutations in the third base position of each codon of the CNS-specific mir-124 target sequences. The effect of mir-124 targeting in limiting neurotropism of flaviviruses has been extensively characterized in our laboratory previously [34–36]. Replication of D4-E and D4-E-NCR1 viruses in the mouse brain was strongly attenuated compared to that of D4 or D4-E** virus (Fig 7A, p<0. 001 for each of control viruses; 2-way ANOVA). Moreover, none of the mice infected with D4-E or D4-E-NCR1 died, whereas the mortality rate for parental D4 was 100% (Fig 7B, p<0. 001; log-rank test). This demonstrates that the miRNA targeting approach can be used for specific attenuation of flavivirus replication in more than one host or cell-type of interest. Interestingly, D4-E** virus with scrambled mir-124 target sequences in the ORF had a lower titer in the brain at each time point as compared with D4 virus (Fig 7A, p<0. 001; 2-way ANOVA) and only 25% of mice infected with D4-E** virus died during a 21-days observation period (Fig 7B, p = 0. 0014; log-rank test). This likely reflects that insertions of heterologous sequences (mir-275 and mir-184 targets) in the ORF can result in partial attenuation of DEN4 replication in the brain of mice. Our previous studies [34,35] had demonstrated that increasing the number of miRNA targets in the 3’NCR of flavivirus genome significantly diminished virus replication in mouse brain and prevented virus escape from miRNA-mediated suppression. In order to minimize or reduce the emergence of DEN4 escape mutants during replication in the CNS, we developed an additional virus (D4-E-NCR2) containing a combination of brain and mosquito specific miRNA target sequences in the ORF and 3’NCR (Fig 1). As expected, the resulting D4-E-NCR2 virus efficiently replicated in Vero cells, but not in mosquito Aag2 or C6/36 cells (Fig 5; p<0. 001; 2-way ANOVA). Moreover, no virus replication was detected in the brains of neonatal mice infected IC with D4-E-NCR2 (Fig 7A, p<0. 001; 2-way ANOVA), and no death or neurological signs were observed (Fig 7B). In addition, the D4-E-NCR2 virus was unable to infect and generate disseminated infection in A. aegypti mosquitoes fed a blood meal containing 6. 8 log10 pfu/ml of either virus (Fig 6B, p<0. 001; Fisher’s exact test). We concluded that D4-E-NCR1 and D4-E-NCR2 viruses potentially could be used as a genetic background for development of chimeric live attenuated vaccine candidate against neurotropic flaviviruses, or a similar strategy could be applied for targeted attenuation of other flavivirus vaccine platforms such as yellow fever virus (YF) 17D or dengue viruses [13–19].
Here, for the first time we demonstrate that a combined targeting of the mosquito-borne flavivirus genome can silence viral replication in two evolutionally distant species: mosquitoes and mice. We believe that the miRNA co-targeting approach can be adapted to support the design of environmentally safe, live attenuated virus vaccines by restricting their ability to be introduced into nature during feeding of competent vectors on viremic vaccinees, thereby limiting the possibility of subsequent viral evolution and unpredictable consequences. Thus, miRNA-mediated silencing of virus replication in mice and mosquitoes can reinforce the safety of newly developed and existing vaccines for use in humans. This engineered host range restriction in both insect vector and vertebrate host by miRNA-mediated mechanisms represents an alternative to non-specific strategies for the rational control of viral tissue tropism and pathogenesis in the vertebrate host and replicative efficacy in permissive vectors.
Recombinant cDNA clone of dengue type 4 virus strain 814669, referred to here as D4s (GenBank access # AY648301) has been described previously [40]. To generate a modified D4 version of D4s clone, DNA fragments of viral genome were amplified from D4s using Phusion DNA polymerase (New England Biolabs [NEB], Ipswich, MA) and cloned individually or in combinations into low copy number pACNR1181 plasmid vector [66] using conventional PCR-based methods [67]. Intron sequence [nt. 857–989 in HaloTag CMV-neo vector pHTC (GenBank access # JF920305) ] was synthesized by GenScript Inc. (Piscataway, NJ) and was introduced at positions 3922 and 8836 of DEN4 genome at AGCT sites. cDNA of CMV promoter was amplified from pCMV-SPORT6 plasmid (Invitrogen, Carlsbad, CA) and was fused with 5' end of DEN4 genome using PCR-based techniques. The hepatitis delta ribozyme sequence was amplified from plasmid RBZ-17D/25C-GFP-FMDV2A-YFCO_full_prME (generous gift of Dr. I. Frolov, University of Alabama), and fused with polyA signal/translation terminator sequence that was amplified from pTriEx1. 1 (Novagen, Germany). The resulting amplicon was fused with the 3’-end of DEN4 genome to assemble wild-type D4 cDNA clone. A Vero cell adaptive mutation L122→F [41–44] was introduced into NS4B protein gene to generate a D4 plasmid. Target sequences for mosquito specific mir-1 (5’-CTCCATACTTCTTTACATTCCA-3’), mir-184 (5’-GCCCTTATCAGTTCTCCGTCCA-3’) and mir-275 (5’-GCGCTACTTCAGGTACCTGA-3’) or human brain-specific mir-124 (5’-GGCATTCACCGCGTGCCTTA-3’) were introduced into the 3’NCR of DEN4 genome between nts 10,277 and 10,278 (position 1, Fig 1) or 10,474 and 10,475 (position 2, Fig 1); these sites of target insertion are located 15 or 212 nts downstream of the TAA stop codon in the 3’NCR, respectively. To introduce miRNA targets in the ORF, we utilized the approach that has been described previously [34,47]. Specifically, the introduced sequence was inserted between nts 2451 and 2452 of DEN4 genome and contained five tandem targets for mir-124, mir-184 and mir-275 that were followed by a duplicated DEN4 E/NS1 region (nts from 2130 to 2451 of DEN4 genome) encoding 98 amino acids from the C-terminal end of the DEN4 E protein and 7 amino acids from the N-terminal end of the NS1 protein (Fig 1). Each codon (except for Met and Trp) within the duplicated E/NS1 region was mutated to a synonymous codon to minimize nucleotide sequence homology between repeated DEN4 genome segments (Fig 1). Each plasmid DNA was sequenced to verify its integrity. Detailed information for all plasmids is available from the authors upon request. Vero cells (African green monkey kidney) were cultured in serum free Opti-Pro medium (Invitrogen) as previously described [42]. Mosquito C6/36 (derived from A. albopictus; ATCC), C710 and Aag2 (derived from A. albopictus and A. aegypti, respectively; generous gift from Dr. A. Fallon, University of Minnesota) cells were maintained in Dulbecco minimal essential medium (DMEM) (Invitrogen) supplemented with 5% FBS (Lonza), 1x penicillin-streptomycin-glutamine (PSG) solution (Invitrogen), 1x MEM nonessential amino acids (Cellgro, Swedesboro, NJ), 1 × MEM vitamin solution (Invitrogen) and 5 μg/L of gentamicin (Invitrogen) at 32°C in an atmosphere of 5% CO2. The C6/36 cells were used in all but the initial experiments, because they exhibit superior viability in our experimental conditions compared to C710 cells. Galveston colony of A. albopictus (generous gift of Dr. S. C. Weaver, UTMB) and NIH strain of A. aegypti mosquitoes have been described earlier [53,68]. Both mosquito species were maintained in carton containers supplemented with 10% sucrose on cotton balls at 28°C, 80% humidity and with a 16-hr daylight cycle. To determine effect of miRNA targeting on DEN4 replication, miRNA targeted or control viruses were diluted in Opti-Pro medium supplemented with 2% FBS, 2 mM L-glutamine and were used to infect Vero, C6/36, Aag2 or C710 cells at an MOI of 0. 01 in duplicate wells of a 6-well plate for 1 h at 37°C (Vero) or 32°C (mosquito cells). The cells were washed three times with Opti-Pro and 2. 75 mL of fresh media was added. Aliquots of 0. 25 mL were harvested daily and stored at −80°C until virus titration. Differences in virus replication kinetics were compared using 2-way ANOVA analyses implemented in Prism 6 software (La Jolla, CA). Confluent monolayers of Vero cells in 25 cm2 flasks were infected at an MOI of 0. 01 and cell culture supernatant was harvested at 3 dpi, diluted 1/10 with Opti-Pro medium, and 1 mL of inoculum was used to infect 25 cm2 flasks of fresh Vero cells. The cycle was repeated 5 times. At the end of the fifth passage, viral RNA was extracted from 0. 14 mL of supernatant using the QIAamp Viral RNA Mini kit (Qiagen) according to manufacturer’s instructions. Genome regions flanking miRNA target sites were amplified using Titan One Tube RT-PCR kit (Roche, Indianapolis, IN) and sequenced. All animal experiments were done in compliance with the guidelines of the NIAID/NIH Institutional Animal Care and Use Committee. The NIAID DIR Animal Care and Use Program acknowledges and accepts responsibility for the care and use of animals involved in activities covered by the NIH IRP’s PHS Assurance #A4149-01, last issued 6/11/2011. D4 and miRNA targeted viruses were diluted to 5 log10 pfu/ml with L-15 medium supplemented with 1x SPG. Three-day-old Swiss Webster mice (Taconic Farms) in groups of 10 were inoculated intracranially (IC) with 10 μL (3 log10 pfu) of virus and returned to the dam. For study of virus replication, the brains from three mice were harvested on 5,8, 11, and 14 days post-infection (dpi). Each brain was weighted, and 10% homogenates were prepared using L-15 medium supplemented with 1x SPG, 0. 05mg/mL of Ciprofloxacin, 0. 06 mg/mL of Clindamycin and 0. 0025 mg/mL of Amphotericin B. Virus titers in each brain suspension were determined by titration in Vero cells. Harvesting of brains from mice infected with D4 was not performed at 14 dpi due to earlier death of the animals. To study the effect of miRNA targeting on virus neurovirulence, 3-day-old Swiss Webster mice (Taconic Farms) were inoculated IC with 3 log10 pfu of parental D4 or its miRNA-targeted derivative and monitored for morbidity and mortality for 21 dpi. Mice that developed neurological signs (paralysis) were humanely euthanized. Differences in replication kinetics were compared using 2-way ANOVA and P-values were adjusted using Bonferroni correction method to account for multiple comparisons, and differences in survival were compared using Log-rank (Mantel-Cox) test implemented in Prism 6 software (La Jolla, CA). The biotinylated probes complementary to mir-184 (5’Biotin-GCCCTTATCAGTTCTCCGTCCA-Biotin3’), mir-275 (5’Biotin-GCGCTACTTCAGGTACCTGA-Biotin3’), and mir-1 (5’Biotin-CTCCATACTTCTTTACATTCCA-Biotin3’) were synthesized by Bioresearch Technologies and used at 2–10 ng/mL. Ribo-oligonucleotides for artificial mir-184 (5’UGGACGGAGAACUGAUAAGGGC), mir-275 (5’UCAGGUACCUGAAGUAGCGC), and mir-1 (5’UGGAAUGUAAAGAAGUAUGGAG3’) were synthesized by Integrated DNA Technologies, and were used in northern blot as positive controls and molecular weight standards. Total RNA was isolated with TRIzol SL reagent (Invitrogen) from 75-cm2 flasks containing confluent monolayers of Aag2, C6/36, C710, or Vero cells or from individual brains of 5-day old Swiss mice or from pools of 20 adult A. aegypti mosquitoes collected at 7 days post-emergence. miRNA detection was carried out by northern blot as described previously with minor modifications [70]. A 15 μg sample of total RNAs was mixed with 4 μL of 10X RNA Loading Solution (Quality Biologicals) and then mixed with an equal volume of formamide (Sigma-Aldrich). The RNA was denatured at 90°C for 5 min followed by rapid cooling on ice. A total of 14 μg of denatured RNA was separated at 150 V for 1 h in a 15% polyacrylamide tris-borate-EDTA gel supplemented with 7M urea (BioRad). The gels were washed and stained with 100 mL of 20 mM MOPS-NaOH and 0. 5 μg/mL ethidium bromide buffer (pH7. 0) for 15 min. miRNAs was electroblotted to Amersham Hybond-NX membrane (GE Healthcare) at 20 V for 2 h in 10mM MOPS-NaOH buffer (pH7. 0), followed by cross-linking to the membrane using 12 mL of 0. 13 M 1-methylimidazole (Sigma), 0. 16 M 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (Sigma) solution (pH 8. 0) at 60°C for 1h. Membranes were prehybridized in 25 mL of ULTRAhyb ultrasensitive hybridization buffer (Ambion) at 37°C for 1 h, followed by hybridization with biotinylated probes at 39°C overnight in ULTRAhyb Ultrasensitive Hybridization Buffer. Membranes were then washed twice with 1x Low Stringency Washing Solution #1 (Ambion) and miRNAs were detected using Chemiluminescent Nucleic Acid Detection Module kit (Thermo scientific) according to the manufacturer' s instructions.
Despite advances in developing flavivirus live attenuated vaccine (LAV) candidates, a concern exists that they might not be safe in the environment due to their intrinsic genetic instability leading to potential reversion back to wild-type that could be associated with possible dissemination of these mutated viruses by mosquitoes. Here, we describe a miRNA targeting approach that can be adapted to support the design of environmentally-safe LAV restricted in their ability to infect and be transmitted by competent vectors, thereby limiting the possibility of subsequent viral evolution and unpredictable consequences. A combined co-targeting of flavivirus genome with mosquito- and vertebrate brain- specific miRNAs resulted in simultaneous restriction of dengue virus infection and replication in mosquitoes and in brains of newborn mice indicating that the miRNA-mediated approach for virus attenuation represents an alternative to non-specific strategies for the control of viral tissue tropism and pathogenesis in the vertebrate host and replicative efficacy in permissive vectors.
Dual miRNA Targeting Restricts Host Range and Attenuates Neurovirulence of Flaviviruses