Neurovirulent flavivirus can be attenuated in mice by incorporation of neuron-specific microRNA recognition elements into viral genome

Neurotropic flaviviruses can efficiently replicate in the developing and mature central nervous systems (CNS) of mice causing lethal encephalitis. Insertion of a single copy of a target for brain-expressed microRNAs (miRNAs) in the 3′ noncoding region (3′NCR) of the flavivirus genome (chimeric tick-borne encephalitis virus/dengue virus) abolished virus neurovirulence in the mature mouse CNS. However, in the developing CNS of highly permissive suckling mice, the miRNA-targeted viruses can revert to a neurovirulent phenotype by accumulating deletions or mutations within the miRNA target sequence. Virus escape from miRNA-mediated suppression in the developing CNS was markedly diminished by increasing the number of miRNA target sites and by extending the distance between these sites in the virus genome. Insertion of multiple miRNA targets into the 3′NCR altered virus neuroinvasiveness, decreased neurovirulence and neuroinflammatory responses, and prevented neurodegeneration without loss of immunogenicity. Although the onset of encephalitis was delayed, a small number of suckling mice still succumbed to lethal intracerebral infection with the miRNA-targeted viruses. Sequence analysis of brain isolates from moribund mice revealed that the viruses escaped from miRNA-mediated suppression exclusively through the deletion of miRNA targets and viral genome sequence located between the two miRNA targets separated by the greatest distance. These findings offer a general strategy to control the reversion of virus to a virulent phenotype: a simultaneous miRNA targeting of the viral genome at many different functionally important regions could prevent virus escape from miRNA-based attenuation, since a deletion of the targeted genomic sequences located between the inserted miRNA binding sites would be lethal for the virus.

Section: Discussionsupporting

confidence: 92%

MicroRNA Targeting of Neurotropic Flavivirus: Effective Control of Virus Escape and Reversion to Neurovirulent Phenotype

Heiss

Maximova

Thach

et al. 2012

“…In the present work, we performed assembly of cDNA portions of the JEV-XZ0934 genome within cells and were able to produce and characterize a JEV g5 virus. We systematically compared the infectious properties of this JEV g5 strain with JEV-RP-9, a g3 strain that has been extensively studied in vitro and in vivo (17,38,(40)(41)(42). The biological properties of JEV g5 were evaluated by infecting cell lines from various origins (Fig.…”

Section: Discussionmentioning

confidence: 99%

A Japanese Encephalitis Virus Genotype 5 Molecular Clone Is Highly Neuropathogenic in a Mouse Model: Impact of the Structural Protein Region on Virulence

Wispelaere

Frenkiel

Desprès

2015

Japanese encephalitis virus (JEV) strains can be separated into 5 genotypes (g1 to g5) based on sequence similarity. JEV g5 strains have been rarely isolated and are poorly characterized. We report here the full characterization of a g5 virus generated using a cDNA-based technology and its comparison with a widely studied g3 strain. We did not observe any major differences between those viruses when their infectious cycles were studied in various cell lines in vitro. Interestingly, the JEV g5 strain was highly pathogenic when inoculated to BALB/c mice, which are known to be largely resistant to JEV g3 infection. The study of chimeric viruses between JEV g3 and g5 showed that there was a poor viral clearance of viruses that express JEV g5 structural proteins in BALB/c mice blood, which correlated with viral invasion of the central nervous system and encephalitis. In addition, using an in vitro model of the blood-brain barrier, we were able to show that JEV g5 does not have an enhanced capacity for entering the central nervous system, compared to JEV g3. Overall, in addition to providing a first characterization of the understudied JEV g5, our work highlights the importance of sustaining an early viremia in the development of JEV encephalitis. IMPORTANCEGenotype 5 viruses are genetically and serologically distinct from other JEV genotypes and can been associated with human encephalitis, which warrants the need for their characterization. In this study, we characterized the in vitro and in vivo properties of a JEV g5 strain and showed that it was more neuropathogenic in a mouse model than a well-characterized JEV g3 strain. The enhanced virulence of JEV g5 was associated with poor viral clearance but not with enhanced crossing of the blood-brain barrier, thus providing new insights into JEV pathogenesis. JEV is a significant human pathogen and the causative agent for Japanese encephalitis, one of the most important viral encephalitides of medical interest in Asia, with an incidence of approximately 67,900 cases per year, among which are about 20,000 fatal cases (1). About 20 to 30% of the symptomatic human cases are fatal, while 30 to 50% of survivors can develop long-term neurological sequelae (2). JEV is maintained in an enzootic cycle between Culex tritaeniorhynchus mosquitoes and amplifying vertebrate hosts, such as water birds and domestic swine (3). Humans and several other animals can also be infected, but since they do not develop a sufficient level of viremia to infect mosquitoes, they are thought to be dead-end hosts (4).Phylogenetic studies based on the viral envelope protein sequences allow the division of JEV strains into five genotypes (g1 to g5). From the isolation of the prototype strain of JEV in 1935 until recently, most of the circulating strains of JEV belonged to g3 and were at the origin of major epidemics in Southeast Asian countries (5). Recently, a shift in prevalence from JEV g3 to g1 has been observed in several Asian countries (6-8), while some strains of JEV g5 have been occasio...

“…The 5= UTR (7), 3= UTR (11,31,32), or coding position locations (7) selected for miRT insertion differed among various viruses depending upon where insertion was tolerated within the particular virus. In the present study, several locations within the 5= UTR or 3= UTR of the CVB3 genome were selected for constructing the virus (see genomic map of tested sites in Fig.…”

Section: Discussionmentioning

confidence: 99%

Coxsackievirus B3 Engineered To Contain MicroRNA Targets for Muscle-Specific MicroRNAs Displays Attenuated Cardiotropic Virulence in Mice

Yao

Wang

et al. 2015

Coxsackievirus B3 (CVB3) is trophic for cardiac tissue and is a major causative agent for viral myocarditis, where local viral replication in the heart may lead to heart failure or even death. Recent studies show that inserting microRNA target sequences into the genomes of certain viruses can eradicate these viruses within local host tissues that specifically express the cognate microRNA. Here, we demonstrated both in vitro and in vivo that incorporating target sequences for miRNA-133 and -206 into the 5= untranslated region of the CVB3 genome ameliorated CVB3 virulence in skeletal muscle and myocardial cells that specifically expressed the cognate cellular microRNAs. Compared to wild-type CVB3, viral replication of the engineered CVB3 was attenuated in human TE671 (rhabdomyosarcoma) and L6 (skeletal muscle) cell lines in vitro that expressed high levels of miRNA-206. In the in vivo murine CVB3-infection model, viral replication of the engineered CVB3 was attenuated specifically in the heart that expressed high levels of both miRNAs, but not in certain tissues, which allowed the host to retain the ability to induce a strong and protective humoral immune response against CVB3. The results of this study suggest that a microRNA-targeting strategy to control CVB3 tissue tropism and pathogenesis may be useful for viral attenuation and vaccine development. IMPORTANCECoxsackievirus B3 (CVB3) is a major causative agent for viral myocarditis, and viral replication in the heart may lead to heart failure or even death. Limiting CVB3 replication within the heart may be a promising strategy to decrease CVB3 pathogenicity. miRNAs are ϳ21-nucleotide-long, tissue-specific endogenous small RNA molecules that posttranscriptionally regulate gene expression by imperfectly binding to the 3= untranslated region (UTR), the 5= UTR, or the coding region within a gene. In our study, muscle-specific miRNA targets (miRT) were incorporated into the CVB3 genome. Replication of the engineered viruses was restricted in the important heart tissue of infected mice, which reduced cardiac pathology and increased mouse survival. Meanwhile, replication ability was retained in other tissues, thus inducing a strong humoral immune response and providing long-term protection against CVB3 rechallenge. This study suggests that a microRNA-targeting strategy can potentially control CVB3 tissue tropism and pathogenesis and may be useful for viral attenuation and vaccine development. C oxsackievirus B3 (CVB3) is a clinically relevant infectious agent that causes viral myocarditis, pancreatitis, and aseptic meningitis. CVB3 is cardiotropic and efficiently replicates in the heart. We and others have shown in various animal models that CVB3 replication induces a direct cytopathic effect on infected cells, leading to tissue damage (1, 2). At present, there is no effective therapy against CVB3, and strategies to develop such a therapy are greatly desired (3, 4). Limiting CVB3 replication within the heart may be a promising strategy to decrease CVB3 pathoge...