There are more than 70 single-stranded, positive-sense RNA viruses in the arthropod-borne flavivirus genus of the Flaviviridae
family, many of which are important human pathogens that cause a devastating and often fatal neuroinfection (21
). Flaviviruses are transmitted in nature to various mammals and birds through the bite of an infected mosquito or tick; they are endemic in many regions of the world and include mosquito-borne yellow fever (YFV), Japanese encephalitis (JEV), West Nile (WNV), St. Louis encephalitis virus (SLEV), dengue viruses, and the tick-borne encephalitis (TBEV) viruses. During the past 2 decades, both mosquito- and tick-borne flaviviruses have emerged in new geographic areas of the world where previously they were not endemic and have caused outbreaks of diseases in humans and domestic animals (TBEV in Northern Europe and Japan, JEV in Australia and Oceania, and WNV in North and South America).
There are only two successful live attenuated flavivirus vaccines that protect against diseases caused by flaviviruses, one for yellow fever and one for Japanese encephalitis. These vaccine viruses were generated using the classical method of repeated passage of virus in cell cultures (10
). Long-term experience with these two vaccines has demonstrated that live attenuated virus vaccines are an efficient approach to prevent diseases caused by flaviviruses since just a single dose of the vaccine virus provides a long-lasting protective immunity in humans that mimics the immune response following natural infection (26
). For many years, a number of new flavivirus vaccine strategies have been developed or are under way (34
), but they have not yet led to licensed human vaccines against neurotropic flaviviruses such as TBEV, SLEV, or WNV. The discovery of microRNAs (miRNAs), small regulatory noncoding RNAs that regulate the expression of cellular genes at the posttranscriptional level, has enabled a novel strategy to control virus tissue tropism and may provide opportunities for developing live attenuated virus vaccines (20
Mature miRNAs regulate diverse cellular processes in many plant and animal species through the assembly of an miRNA-induced silencing complex (RISC), which binds the complementary targets in mRNA and subsequently catalytically cleaves or transcriptionally represses the targeted mRNA (4
). In addition, recent studies suggest that miRNAs also play a role in the regulation of virus infections (5
). Since the pattern of miRNA expression is cell and tissue specific, it would be a disadvantage for viruses to contain sequences in their genomes that are complementary to cellular miRNAs present in tissues in which they would otherwise replicate efficiently and cause disease. Several miRNAs have recently been shown to modulate the tissue tropism of a number of viruses from different families (3
). Many flaviviruses cause neurologic disease such as meningitis and/or encephalitis, and we sought to design a flavivirus that would be selectively attenuated for the central nervous system (CNS) since this is a target of wild-type neurotropic virus. In the present study, we explored the ability of the cellular miRNAs expressed in brain tissue to control the neurotropism of a flavivirus bearing complementary miRNA target sequences. We anticipated that these viruses would replicate in peripheral non-CNS tissues and induce a strong adaptive immune response but would be restricted in their ability to replicate in the CNS since the CNS-expressed miRNAs would recognize the introduced complementary target sequences in the viral RNA genome and limit its translation, replication, and assembly into a virion.
The miRNA target sequences that were selected for insertion into the viral genome were complementary to let-7c, mir-9, mir-124a, mir-128a, and mir-218 miRNA, which have evolutionarily conserved sequences among mammalian species, including mice and humans (38
). With the exception of mir-218a, which was shown to be exclusively expressed in cranial motor nuclei and spinal motor neurons of zebrafish (14
), all of the other selected miRNAs were previously found to be highly expressed in the brain of adult mice and humans (1
). mir-124a is highly upregulated in neuronal cells as are mir-9 and mir-128a, but the latter two are also found in peripheral tissue cells (29
). The brain-enriched let-7c miRNA is a member of the let-7 family of miRNAs that are found to be widely expressed in many tissues of various species and also act as tumor suppressors (2
The flavivirus genome is a positive-sense single-stranded RNA that is approximately 11 kb in length and contains 5′ and 3′ noncoding regions (NCR) flanking a single open reading frame (ORF) encoding a polyprotein that is processed by viral and cellular proteases into three structural proteins (capsid [C], premembrane [prM], and envelope [E]) and seven nonstructural proteins (21
). The five miRNA targets that we selected were individually introduced into the 3′ NCR of the viral genome since the 3′ NCR targeting of cellular mRNAs was found to be more frequent and effective than 5′ NCR or ORF targeting (4
). As a model virus for modification of flavivirus neurotropism, we selected a chimeric tick-borne encephalitis/dengue type 4 virus (TBEV/DEN4) that was constructed by replacing the structural prM and E protein genes of the nonneuroinvasive, mosquito-borne dengue type 4 virus (DEN4) with the corresponding genes of the highly virulent Far Eastern strain of TBEV (30
). TBEV/DEN4 retains a high level of neurovirulence from its TBEV parent (a biosafety level 4 agent) in mice inoculated intracerebrally; however, consistent with the phenotype of its other parent, a DEN4 virus, the chimeric TBEV/DEN4 virus is nonneuroinvasive in immunocompetent mice and monkeys following a peripheral route of inoculation (37
). Here, we demonstrate that the incorporation into the TBEV/DEN4 genome of a single copy of a target for an miRNA highly expressed in brain tissue (mir-9, mir-124a, or let-7c) was sufficient to restrict the neurotropism of the engineered chimeric viruses, resulting in attenuation of the TBEV/DEN4 virus for the CNS of adult mice. Importantly, the miRNA target TBEV/DEN4 viruses retained the ability to replicate in non-CNS tissues of rhesus monkeys and induce a robust immune response.