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

Abstract

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...

Dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro

“…When comparing DENV-Fluc(NS4B: L52F) to DENV-Fluc, we consistently observed a 10-to 100-fold increase in titers, which correlated to similar increases in luciferase activity. These data indicate that, in cell culture lines, the NS4B L52F mutation confers a dramatic enhancement in virus infectivity and production of strain 16681-based DENV-Fluc, consistent with previous studies on strain TSV01 (25).…”

“…S2A). To generate a DENV-Fluc virus suitable for mouse infection, we took advantage of the recent observation that a single point mutation (L52F) in the viral nonstrucutural 4B protein (NS4B) was sufficient to confer virulence to the nonvirulent TSV01 strain in mice (25). The NS4B L52F mutation was engineered into DENV-Fluc to generate DENV-Fluc(NS4B:L52F), and both viruses were characterized for growth and infectivity properties (Materials and Methods).…”

Dengue vaccine–induced CD8+ T cell immunity confers protection in the context of enhancing, interfering maternal antibodies

“…The ability of maternal antibodies to protect pups from infection was investigated by challenging 3-wko pups born to PDK53-immunized or nonimmunized (DENV-naive) dams with either the parental strain 16681 or the heterologous DENV2 strain D2Y98P-PP1 (31,32). Infection with strain 16681 resulted in an asymptomatic transient viremia in pups born to DENV-naive dams ( Figure 1C).…”

Functional Analysis of Antibodies against Dengue Virus Type 4 Reveals Strain-Dependent Epitope Exposure That Impacts Neutralization and Protection

“…In this study, we developed two new models of lethal DENV-4 infection via a peripheral route in AG129 mice with virus strains corresponding to genotypes I (H-241) and II (TVP-376) and used these to assess the protective efficacy of selected cross-reactive and type-specific anti-DENV-4 MAbs. Prior to this, only one strain of DENV-1 (West Pac-74) (35) and two strains of DENV-2 (D2S10 and D2Y98P) (62,78,79) had been reported to cause lethal infection in AG129 mice after peripheral inoculation, with the DENV-2 strains promoting a vascular leakage phenotype that to some extent resembled the DSS seen in humans. In preliminary studies using high infectious doses of the two DENV-4 strains, we saw a similar rapid death phenotype consistent with the shock syndrome that is observed with mouse-adapted DENV-2 strains (46,62,80).…”