Kentaro Yoshii

Flavivirus antagonism of type I interferon signaling reveals prolidase as a regulator of IFNAR1 surface expression

Type I interferon (IFN-α/β or IFN-I) signals through two receptor subunits, IFNAR1 and IFNAR2, to orchestrate sterile and infectious immunity. Cellular pathways that regulate IFNAR1 are often targeted by viruses to suppress the antiviral effects of IFN-I. Here we report that encephalitic flaviviruses, including tick-borne encephalitis virus and West Nile virus, antagonize IFN-I signaling by inhibiting IFNAR1 surface expression. Loss of IFNAR1 was associated with binding of the viral IFN-I antagonist, NS5, to prolidase (PEPD), a cellular dipeptidase implicated in primary immune deficiencies in humans. Prolidase was required for IFNAR1 maturation and accumulation, activation of IFNβ-stimulated gene induction, and IFN-I-dependent viral control. Human fibroblasts derived from patients with genetic prolidase deficiency exhibited decreased IFNAR1 surface expression and reduced IFNβ-stimulated signaling. Thus, by understanding flavivirus IFN-I antagonism, prolidase is revealed as a central regulator of IFN-I responses.

Glycosylation of the West Nile Virus envelope protein increases in vivo and in vitro viral multiplication in birds.

Many West Nile (WN) virus isolates associated with significant outbreaks possess a glycosylation site on the envelope (E) protein. E-protein glycosylated variants of New York (NY) strains of WN virus are more neuroinvasive in mice than the non-glycosylated variants. To determine how E protein glycosylation affects the interactions between WN virus and avian hosts, we inoculated young chicks with NY strains of WN virus containing either glycosylated or non-glycosylated variants of the E protein. The glycosylated variants were more virulent and had higher viremic levels than the non-glycosylated variants. The glycosylation status of the variant did not affect viral multiplication and dissemination in mosquitoes in vivo. Glycosylated variants showed more heat-stable propagation than non-glycosylated variants in mammalian (BHK) and avian (QT6) cells but not in mosquito (C6/36) cells. Thus, E-protein glycosylation may be a requirement for efficient transmission of WN virus from avian hosts to mosquito vectors.

Construction of an infectious cDNA clone for Omsk hemorrhagic fever virus, and characterization of mutations in NS2A and NS5.

Omsk hemorrhagic fever virus (OHFV) is a member of the tick-borne encephalitis serocomplex of flaviviruses, and causes hemorrhagic disease in humans. In this study, an infectious cDNA of OHFV was constructed to investigate the molecular mechanisms involved in OHFV pathogenesis for the first time. Our cDNA clone was capable of producing infectious virus which is genetically identical to the parental Guriev strain, and the recombinant virus showed similar biological properties to the parental virus including growth kinetics and virulence characteristics. While characterizing the cDNAs, fortuitous mutations at NS2A position 46 and NS5 position 836 were found to affect viral production. By using a viral replicon expressing luciferase, it was shown that both of the mutations produced a defect in RNA replication and that the NS5 mutation induced a temperature-sensitive phenotype, indicating the importance of these residues in RNA replication. This infectious cDNA will be a useful tool to study the replication and pathogenesis of OHFV.

A conserved region in the prM protein is a critical determinant in the assembly of flavivirus particles.

Flaviviruses are assembled to bud into the lumen of the endoplasmic reticulum (ER) and are secreted through the vesicle transport pathway, but the details of the molecular mechanism of virion assembly remain largely unknown. In this study, a highly conserved region in the prM protein was identified among flaviviruses. In the subviral particle (SP) system of tick-borne encephalitis virus (TBEV) and Japanese encephalitis virus, secretion of SPs was impaired by a mutation in the conserved region in the prM protein. Viral proteins were sparse in the Golgi complex and accumulated in the ER. Ultrastructural analysis revealed that long filamentous structures, rather than spherical SPs, were observed in the lumen of the ER as a result of the mutation. The production of infectious virions derived from infectious cDNA of TBEV was also reduced by mutations in the conserved region. Molecular modelling analysis suggested that the conserved region is important for the association of prM-envelope protein heterodimers in the formation of a spike of immature virion. These results are the first demonstration that the conserved region in the prM protein is a molecular determinant for the flavivirus assembly process.

Establishment of a neutralization test involving reporter gene-expressing virus-like particles of tick-borne encephalitis virus

Previously, a system for packaging tick-borne encephalitis virus (TBEV) subgenomic replicon RNAs into single-round infectious virus-like particles (VLPs) was developed. In the present study, VLPs were applied to measuring the levels of neutralizing antibodies against TBEV as an alternative to performing neutralization tests with live virus. As markers of VLP infection, the genes for GFP and luciferase were inserted into the TBEV replicon, which was then packaged into VLPs. The reporter genes were expressed in cells that were infected with the VLPs, and this infection was inhibited by neutralizing antibodies to TBEV. Serum samples from wild rodents were used to evaluate the neutralization test using VLPs. All the sera that were positive in the conventional neutralization test were also found to be positive in the neutralization test using VLPs, and there were highly significant correlations between the neutralization titres obtained using the native virus and those using VLPs. These results indicate that VLPs that express reporter genes represent a useful and safe alternative to conventional neutralization testing using live virus.

Genetic diversity of hantaviruses in Mexico: identification of three novel hantaviruses from Neotominae rodents.

A variety of hantaviruses are harbored by rodents in North and South America, some of which can cause hantavirus pulmonary syndrome. To obtain greater evolutionary insight into hantaviruses in the Americas, a total of 211 rodents were captured in the Mexican states of Guerrero and Morelos in 2006. Anti-hantavirus antibodies were detected in 27 of 211 serum samples (12.8%) by ELISA. The distribution of seropositive rodents was: 17 Peromyscus beatae, 1 Megadontomys thomasi, 1 Neotoma picta, 6 Reithrodontomys sumichrasti, and 2 Reithrodontomys megalotis. The hantavirus small (S), medium (M), and large (L) genome segments from P. beatae, R. sumichrasti, and R. megalotis were amplified and the sequences covering the open reading frames were determined. The hantaviruses from P. beatae, R. sumichrasti, and R. megalotis were provisionally designated Montano (MTN), Carrizal (CAR), and Huitzilac (HUI), respectively. The M segment amino acid identities among the Mexican hantaviruses were 80.8-93.0%. When these M segments were compared to those of known hantaviruses, MTN virus was most closely related to Limestone Canyon (LSC) virus (88.9% amino acid identity), while the CAR and HUI viruses were most closely related to El Moro Canyon (ELMC) virus (90-91% identity). Phylogenetic analysis revealed that the MTN, CAR, and HUI viruses occupy a monophyletic clade with the LSC, ELMC, and Rio Segundo viruses, which are harbored by Peromyscus boylii, R. megalotis, and Reithrodontomys mexicanus, respectively. The data obtained in this study provide important information for understanding the evolution of hantaviruses in the Americas.

Sub-genomic replicon and virus-like particles of Omsk hemorrhagic fever virus

Omsk hemorrhagic fever virus (OHFV) is a member of the tick-borne encephalitis serocomplex of flaviviruses, and causes hemorrhagic disease in humans. To investigate the molecular mechanisms involved in OHFV pathogenesis, we constructed several subgenomic OHFV replicons containing large deletions in the structural region. Replicon RNA was introduced into BHK cells by transfection and the production of viral proteins was monitored by IFA. GFP and luciferase genes were inserted into the OHFV replicon, and these reporter genes were expressed in cells harboring replicating replicon RNA. OHFV replicons were packaged into single-round infectious virus-like particles (VLPs) by sequential transfection with replicon RNA and a plasmid expressing the viral structural proteins. Reporter genes were expressed in cells infected with VLPs, and the infection was inhibited by neutralizing antibodies. These replicon and VLP systems will be useful tools for investigating the molecular mechanism of OHFV pathogenicity.

Tick-borne flaviviruses alter membrane structure and replicate in dendrites of primary mouse neuronal cultures

Neurological diseases caused by encephalitic flaviviruses are severe and associated with high levels of mortality. However, detailed mechanisms of viral replication in the brain and features of viral pathogenesis remain poorly understood. We carried out a comparative analysis of replication of neurotropic flaviviruses: West Nile virus, Japanese encephalitis virus and tick-borne encephalitis virus (TBEV), in primary cultures of mouse brain neurons. All the flaviviruses multiplied well in primary neuronal cultures from the hippocampus, cerebral cortex and cerebellum. The distribution of viral-specific antigen in the neurons varied: TBEV infection induced accumulation of viral antigen in the neuronal dendrites to a greater extent than infection with other viruses. Viral structural proteins, non-structural proteins and dsRNA were detected in regions in which viral antigens accumulated in dendrites after TBEV replication. Replication of a TBEV replicon after infection with virus-like particles of TBEV also induced antigen accumulation, indicating that accumulated viral antigen was the result of viral RNA replication. Furthermore, electron microscopy confirmed that TBEV replication induced characteristic ultrastructural membrane alterations in the neurites: newly formed laminal membrane structures containing virion-like structures. This is the first report describing viral replication in and ultrastructural alterations of neuronal dendrites, which may cause neuronal dysfunction. These findings encourage further work aimed at understanding the molecular mechanisms of viral replication in the brain and the pathogenicity of neurotropic flaviviruses.

N-linked glycan in tick-borne encephalitis virus envelope protein affects viral secretion in mammalian cells, but not in tick cells.

Tick-borne encephalitis virus (TBEV) is a zoonotic disease agent that causes severe encephalitis in humans. The envelope protein E of TBEV has one N-linked glycosylation consensus sequence, but little is known about the biological function of the N-linked glycan. In this study, the function of protein E glycosylation was investigated using recombinant TBEV with or without the protein E N-linked glycan. Virion infectivity was not affected after removing the N-linked glycans using N-glycosidase F. In mammalian cells, loss of glycosylation affected the conformation of protein E during secretion, reducing the infectivity of secreted virions. Mice subcutaneously infected with TBEV lacking protein E glycosylation showed no signs of disease, and viral multiplication in peripheral organs was reduced relative to that with the parental virus. In contrast, loss of glycosylation did not affect the secretory process of infectious virions in tick cells. Furthermore, inhibition of transport to the Golgi apparatus affected TBEV secretion in mammalian cells, but not in tick cells, indicating that TBEV was secreted through an unidentified pathway after synthesis in endoplasmic reticulum in tick cells. These results increase our understanding of the molecular mechanisms of TBEV maturation.

Seroprevalence of West Nile Virus in Wild Birds in Far Eastern Russia Using a Focus Reduction Neutralization Test

West Nile (WN) virus has been spreading geographically to non-endemic areas in various parts of the world. However, little is known about the extent of WN virus infection in Russia. Japanese encephalitis (JE) virus, which is closely related to WN virus, is prevalent throughout East Asia. We evaluated the effectiveness of a focus reduction neutralization test in young chicks inoculated with JE and WN viruses, and conducted a survey of WN infection among wild birds in Far Eastern Russia. Following single virus infection, only neutralizing antibodies specific to the homologous virus were detected in chicks. The neutralization test was then applied to serum samples from 145 wild birds for WN and JE virus. Twenty-one samples were positive for neutralizing antibodies to WN. These results suggest that WN virus is prevalent among wild birds in the Far Eastern region of Russia.