The neurovirulence and neuroinvasiveness of chimeric tick-borne encephalitis/dengue virus can be attenuated by introducing defined mutations into the envelope and NS5 protein genes and the 3′ non-coding region of the genome

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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: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Evaluation Of Viruses In Micementioning

confidence: 99%

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

Heiss

Maximova

Thach

et al. 2012

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“…A number of viral determinants modulating the intrinsic virulence of the virus within its host have been identified and have been shown to affect either the binding properties (7,11,33,37) or the replication efficiency (20,34) of DENV. Such knowledge has proven useful for the development of live attenuated DENV vaccine candidates (10,17,46). A number of animal models have been reported that partially reproduce some of the clinical features of human DEN disease, such as hemorrhage or increased vascular permeability (16).…”

mentioning

confidence: 99%

A Single Amino Acid in Nonstructural Protein NS4B Confers Virulence to Dengue Virus in AG129 Mice through Enhancement of Viral RNA Synthesis

Grant

Tan

Qing

et al. 2011

Dengue (DEN) is a mosquito-borne viral disease that has become an increasing economic and health burden for the tropical and subtropical world. The lack of an appropriate animal model of DEN has greatly impeded the study of its pathogenesis and the development of vaccines/antivirals. We recently reported a DEN virus 2 (DENV-2) strain (D2Y98P) that lethally infects immunocompromised AG129 mice, resulting in organ damage or dysfunction and increased vascular permeability, hallmarks of severe DEN in patients (G. K. Tan et al., PLoS Negl. Trop. Dis. 4:e672, 2010). Here we report the identification of one critical virulence determinant of strain D2Y98P. By mutagenesis, we showed that a Phe-to-Leu alteration at amino acid position 52 in nonstructural protein NS4B completely abolished the pathogenicity of the D2Y98P virus, as evidenced by a lack of lethality and the absence of histological signs of disease, which correlated with reduced viral titers and intact vascular permeability. Conversely, a Leu-to-Phe alteration at position 52 of NS4B in nonvirulent DENV-2 strain TSV01 led to 80% lethality and increased viremia. The NS4B(Phe52) viruses displayed enhanced RNA synthesis in mammalian cells but not in mosquito cells. The increased viral RNA synthesis was independent of the ability of NS4B to interfere with the host type I interferon response. Overall, our results demonstrate that Phe at position 52 in NS4B confers virulence in mice on two independent DENV-2 strains through enhancement of viral RNA synthesis. In addition to providing further insights into the functional role of NS4B protein, our findings further support a direct relationship between viral loads and DEN pathogenesis in vivo, consistent with observations in DEN patients.Dengue (DEN) disease, caused by any one of the four serotypes of dengue virus (DENV), is a major global public health and economic burden. DENV affects 2.5 billion people worldwide, mostly in tropical and subtropical countries, leading to 50 to 100 million human infections each year (13). Clinical manifestations of DEN disease range from undifferentiated fever to DEN fever (DF) and DEN hemorrhagic fever (DHF), with plasma leakage that may lead to hypovolemic shock (DEN shock syndrome [DSS]). Therapeutic intervention is limited to fluid management for DEN patients who experience severe vascular leakage (41). Currently, no antiviral therapy or vaccine to fight DEN is available on the market (9, 31).DENV belongs to the genus Flavivirus within the family Flaviviridae. Besides DENV, many other flaviviruses are important human pathogens, including yellow fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV). DENV is a small enveloped virus with a positive single-strand RNA of approximately 10,700 nucleotides (18). The DENV genome encodes 10 proteins, comprising 3 structural proteins (capsid [C], premembrane [PrM], and envelope [E]) and 7 nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The structural proteins fo...

“…Thus, the regions other than the prM and E proteins are also responsible for the neuroinvasiveness of TBEV. Several reports indicated amino acid changes in the NS proteins affect the neuroinvasiveness of tick-borne flaviviruses (24,25) and mosquito-borne flaviviruses (26)(27)(28)(29)(30). Multiplication of TBEV/OHF-ME in the brain was similar to that of parental TBEV.…”

Section: Discussionmentioning

confidence: 99%

A Critical Determinant of Neurological Disease Associated with Highly Pathogenic Tick-Borne Flavivirus in Mice

Yoshii

Sunden

Yokozawa

et al. 2014

Tick-borne encephalitis virus (TBEV) and Omsk hemorrhagic fever virus (OHFV) are highly pathogenic tick-borne flaviviruses; TBEV causes neurological disease in humans, while OHFV causes a disease typically identified with hemorrhagic fever. Although TBEV and OHFV are closely related genetically, the viral determinants responsible for these distinct disease phenotypes have not been identified. In this study, chimeric viruses incorporating components of TBEV and OHFV were generated using infectious clone technology, and their pathological characteristics were analyzed in a mouse model to identify virus-specific determinants of disease. We found that only four amino acids near the C terminus of the NS5 protein were primarily responsible for the development of neurological disease. Mutation of these four amino acids had no effect on viral replication or histopathological features, including inflammatory responses, in mice. These findings suggest a critical role for NS5 in stimulating neuronal dysfunction and degeneration following TBEV infection and provide new insights into the molecular mechanisms underlying the pathogenesis of tick-borne flaviviruses. IMPORTANCE Tick-borne encephalitis virus (TBEV) and Omsk hemorrhagic fever virus (OHFV) belong to the tick-borne encephalitis serocomplex, genusFlavivirus, family Flaviviridae. Although TBEV causes neurological disease in humans while OHFV causes a disease typically identified with hemorrhagic fever. In this study, we investigated the viral determinants responsible for the different disease phenotypes using reverse genetics technology. We identified a cluster of only four amino acids in nonstructural protein 5 primarily involved in the development of neurological disease in a mouse model. Moreover, the effect of these four amino acids was independent of viral replication property and did not affect the formation of virus-induced lesions in the brain directly. These data suggest that these amino acids may be involved in the induction of neuronal dysfunction and degeneration in virus-infected neurons, ultimately leading to the neurological disease phenotype. These findings provide new insight into the molecular mechanisms of tick-borne flavivirus pathogenesis.