Dendritic transport of tick-borne flavivirus RNA by neuronal granules affects development of neurological disease

Neurological diseases caused by encephalitic flaviviruses are severe and associated with high levels of mortality. However, little is known about the detailed mechanisms of viral replication and pathogenicity in the brain. Previously, we reported that the genomic RNA of tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus, is transported and replicated in the dendrites of neurons. In the present study, we analyzed the transport mechanism of the viral genome to dendrites. We identified specific sequences of the 5' untranslated region of TBEV genomic RNA that act as a cis-acting element for RNA transport. Mutated TBEV with impaired RNA transport in dendrites caused a reduction in neurological symptoms in infected mice. We show that neuronal granules, which regulate the transport and local translation of dendritic mRNAs, are involved in TBEV genomic RNA transport. TBEV genomic RNA bound an RNA-binding protein of neuronal granules and disturbed the transport of dendritic mRNAs. These results demonstrated a neuropathogenic virus hijacking the neuronal granule system for the transport of viral genomic RNA in dendrites, resulting in severe neurological disease

Rab8b Regulates Transport of West Nile Virus Particles from Recycling Endosomes

West Nile virus (WNV) particles assemble at and bud into the endoplasmic reticulum (ER) and are secreted from infected cells through the secretory pathway. However, the host factor related to these steps is not fully understood. Rab proteins, belonging to the Ras superfamily, play essential roles in regulating many aspects of vesicular trafficking. In this study, we sought to determine which Rab proteins are involved in intracellular trafficking of nascent WNV particles. RNAi analysis revealed that Rab8b plays a role in WNV particle release. We found that Rab8 and WNV antigen were colocalized in WNV-infected human neuroblastoma cells, and that WNV infection enhanced Rab8 expression in the cells. In addition, the amount of WNV particles in the supernatant of Rab8b-deficient cells was significantly decreased compared with that of wild-type cells. We also demonstrated that WNV particles accumulated in the recycling endosomes in WNV-infected cells. In summary, these results suggest that Rab8b is involved in trafficking of WNV particles from recycling endosomes to the plasma membrane

Putative RNA viral sequences detected in an Ixodes scapularis-derived cell line

Ticks harbour various microorganisms, some of which act as pathogens of humans and animals. The recent advancement of genome sequencing technologies revealed that a wide range of previously unrecognised microorganisms exist in ticks. Continuous cell lines established from ticks could play a key role in the isolation of such microorganisms; however, tick cells themselves have been known to harbour symbiotic microorganisms. The present study aimed to characterise putative RNA viral sequences detected in the culture supernatant of one of the most frequently used tick cell lines, ISE6, which was derived from embryos of the blacklegged tick Ixodes scapularis. Viral particles purified from the culture supernatant were used for RNA extraction, followed by Illumina sequencing. The reads were de novo assembled and the resulting contigs were annotated by tBLASTx search. The results suggested that there were at least five putative viral sequences of four phylogenetically distinct lineages in ISE6 cells. The predominant viral sequence found in ISE6 cells, designated L scapularis iflavirus, was a member of the family Iflaviridae, which is an arthropod-infecting virus group. We also identified L and M segments of the family Bunyaviridae, which could not be classified into any of the five known genera, and a potential capsid protein related to Drosophila A virus. In addition to these previously unrecognised viruses, ISE6 was revealed to harbour a putative genome sequence of L scapularis-associated virus-1, which was reported in a recent metagenomic study of L scapularis itself. All the five putative viral sequences were detected by RT-PCR in both ISE6 cells and the culture supernatant. Electron microscopic analysis showed the existence of spherical virions with a varying diameter of 50-70 nm in the culture supernatant of ISE6 cells. Further studies are required to investigate the potential roles of ISE6-associated viruses in ticks. (C) 2017 Elsevier GmbH. All rights reserved

Amino acid 159 of the envelope protein affects viral replication and T-cell infiltration by West Nile virus in intracranial infection

West Nile virus (WNV) is an important cause of viral encephalitis in birds and animals, including humans. Amino acid 159 of the envelope (E) protein is reportedly implicated in the different levels of neurovirulence in mice infected with WNV NY99 or Eg101. We investigated the role of amino acid 159 of the E protein in the pathogenesis of WNV infection. We produced recombinant WNV with the structural proteins of the NY99 or Eg101 strain (NY-WT or EgCME-WT) and mutant viruses with substitutions of amino acid 159 of the E protein (NY-E-V159I or EgCME-E-I159V). The NY-WT and NY-E-V159I or EgCME-WT and EgCME-E-I159V titers in culture supernatant were similar. The mortality rate and viral titer in the brains of mice inoculated intraperitoneally with NY-WT or NY-E-V159I were also similar. In contrast, the mortality rate and viral titer in the brains of mice inoculated intracranially with EgCME-E-I159V were significantly higher than those of mice inoculated with EgCME-WT. The numbers of CD3-positive and CD8-positive T cells were greater in brains inoculated with EgCME-E-I159V than in those inoculated with EgCME-WT. Therefore, amino acid 159 of the E protein modulates the pathogenicity of WNV by affecting viral replication and T-cell infiltration in the brain

精巣胚細胞腫瘍の臨床的検討

精巣胚細胞腫瘍65例68精巣を臨床的に検討した.36精巣(53.7%)はセミノーマで, 残りはnon-seminomatous germ cell testicular tumor(NSGCTT)であった.セミノーマ症例のうち31例(88.6%)はI期でそれ以外は受診時に転移巣を有していた.NSGCTTのうち22例(68.8%)はI期であった.セミノーマ及びNSGCTT症例の平均年齢は各々40.4, 29.2歳であった.39例(60.0%)は右側, 23例(35.4%)は左側, 3例(4.6%)は両側性であった.5例は停留精巣の既往を有していた.49例(73.1%)において主訴は無痛性精巣腫大であった.症状発現から受診迄の期間はNSGCTT症例よりセミノーマの方が長かった(平均10.9ヵ月と3.4ヵ月).Immunosuppressive acidic protein(IAP)はalpha-feto protein(AFP), beta-human chorionic gonadotropin(betahCG), lactic dehydrogenase(LDH)と共に腫瘍マーカーとして有用であったA clinical statistical analysis on 65 patients with 68 testicular germ cell tumors was performed. Thirty-six testes (53.7%) had seminomas and the remainder non-seminomatous germ cell testicular tumors (NSGCTTs). Of the seminomas, 31 (88.6%) were in stage I and the others showed distant metastases at presentation. Of the 32 NSGCTTs, 22 (68.8%) were in stage I. The average ages of the patients with seminomas and NSGCTTs were 40.4 and 29.9 years, respectively. Thirty-nine patients (60.0%) had tumors on the right side, 23 (35.4%) on the left and 3 (4.6%) in both testes. Five patients had a past history of cryptorchidism. Chief complaints in 49 patients (73.1%) were a painless scrotal mass. The interval from clinical onset to presentation was longer in seminoma patients than in NSGCTT patients (10.9 months on average versus 3.4 months). Immunosuppressive acidic protein (IAP) was a useful diagnostic tumor marker as well as alpha-feto protein (AFP), beta-human chorionic gonadotropin (beta-hCG) and lactic dehydrogenase (LDH). We adopted a surveillance policy in more than half of the stage I patients and obtained acceptable results. In the remaining cases, therapies including combination chemotherapy, radiation and salvage operation were performed after orchiectomy. The three-year survival rate was 98.0, 100.0 and 26.7%, for stage I, II and III patients respectively

West Nile virus capsid protein inhibits autophagy by AMP-activated protein kinase degradation in neurological disease development

West Nile virus (WNV) belongs to the Flaviviridae family and has emerged as a significant cause of viral encephalitis in birds and animals including humans. WNV replication directly induces neuronal injury, followed by neuronal cell death. We previously showed that accumulation of ubiquitinated protein aggregates was involved in neuronal cell death in the WNV-infected mouse brain. In this study, we attempted to elucidate the mechanisms of the accumulation of protein aggregates in the WNV-infected cells. To identify the viral factor inducing the accumulation of ubiquitinated proteins, intracellular accumulation of ubiquitinated proteins was examined in the cells expressing the viral protein. Expression of capsid (C) protein induced the accumulation, while mutations at residues L51 and A52 in C protein abrogated the accumulation. Wild-type (WT) or mutant WNV in which mutations were introduced into the residues was inoculated into human neuroblastoma cells. The expression levels of LC3-II, an autophagy-related protein, and AMP-activated protein kinase (AMPK), an autophagy inducer, were reduced in the cells infected with WT WNV, while the reduction was not observed in the cells infected with WNV with the mutations in C protein. Similarly, ubiquitination and degradation of AMPK were only observed in the cells infected with WT WNV. In the cells expressing C protein, AMPK was co-precipitated with C protein and mutations in L51 and A52 reduced the interaction. Although the viral replication was not affected, the accumulation of ubiquitinated proteins in brain and neurological symptoms were attenuated in the mouse inoculated with WNV with the mutations in C protein as compared with that with WT WNV. Taken together, ubiquitination and degradation of AMPK by C protein resulted in the inhibition of autophagy and the accumulation of protein aggregates, which contributes to the development of neurological disease. Author summary The elimination of ubiquitinated protein aggregates from neuronal cells occurs via protein degradation systems such as autophagy or proteasome. Previously, we determined that WNV infection induces the accumulation of ubiquitinated proteins in neuronal cells. However, the detailed mechanisms underlying this accumulation and its association with pathogenicity in the brain remain unclear. Here, we demonstrated that residues L51 and A52 of WNV C protein are responsible for the accumulation of the ubiquitinated proteins. The two C protein residues promoted the interaction between C protein and the autophagy-inducing enzyme AMPK, resulting in ubiquitination and degradation of AMPK and inhibition of autophagy. A mutation in WNV that impaired the AMPK-C protein interaction attenuated the accumulation of ubiquitinated proteins and neurological symptoms in mice. Our findings are the first to describe the regulation of AMPK by a viral protein and represent a step forward in understanding of molecular mechanisms of the neuropathogenesis of WNV infection