Human neural stem cell-derived neuron/astrocyte co-cultures respond to La Crosse virus infection with proinflammatory cytokines and chemokines.

BACKGROUND: La Crosse virus (LACV) causes pediatric encephalitis in the USA. LACV induces severe inflammation in the central nervous system, but the recruitment of inflammatory cells is poorly understood. A deeper understanding of LACV-induced neural pathology is needed in order to develop treatment options. However, there is a severe limitation of relevant human neuronal cell models of LACV infection. METHODS: We utilized human neural stem cell (hNSC)-derived neuron/astrocyte co-cultures to study LACV infection in disease-relevant primary cells. hNSCs were differentiated into neurons and astrocytes and infected with LACV. To characterize susceptibility and responses to infection, we measured viral titers and levels of viral RNA, performed immunofluorescence analysis to determine the cell types infected, performed apoptosis and cytotoxicity assays, and evaluated cellular responses to infection using qRT-PCR and Bioplex assays. RESULTS: hNSC-derived neuron/astrocyte co-cultures were susceptible to LACV infection and displayed apoptotic responses as reported in previous in vitro and in vivo studies. Neurons and astrocytes are both targets of LACV infection, with neurons becoming the predominant target later in infection possibly due to astrocytic responses to IFN. Additionally, neuron/astrocyte co-cultures responded to LACV infection with strong proinflammatory cytokine, chemokine, as well as MMP-2, MMP-7, and TIMP-1 responses. CONCLUSIONS: hNSC-derived neuron/astrocyte co-cultures reproduce key aspects of LACV infection in humans and mice and are useful models to study encephalitic viruses. Specifically, we show astrocytes to be susceptible to LACV infection and that neurons and astrocytes are important drivers of the inflammatory responses seen in LACV infection through the production of proinflammatory cytokines and chemokines.

The Host E3-Ubiquitin Ligase TRIM6 Ubiquitinates the Ebola Virus VP35 Protein and Promotes Virus Replication.

Ebola virus (EBOV), a member of the Filoviridae family, is a highly pathogenic virus that causes severe hemorrhagic fever in humans and is responsible for epidemics throughout sub-Saharan, central, and West Africa. The EBOV genome encodes VP35, an important viral protein involved in virus replication by acting as an essential cofactor of the viral polymerase as well as a potent antagonist of the host antiviral type I interferon (IFN-I) system. By using mass spectrometry analysis and coimmunoprecipitation assays, we show here that VP35 is ubiquitinated on lysine 309 (K309), a residue located on its IFN antagonist domain. We also found that VP35 interacts with TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family. We recently reported that TRIM6 promotes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, to induce efficient antiviral IFN-I-mediated responses. Consistent with this notion, VP35 also associated noncovalently with polyubiquitin chains and inhibited TRIM6-mediated IFN-I induction. Intriguingly, we also found that TRIM6 enhances EBOV polymerase activity in a minigenome assay and TRIM6 knockout cells have reduced replication of infectious EBOV, suggesting that VP35 hijacks TRIM6 to promote EBOV replication through ubiquitination. Our work provides evidence that TRIM6 is an important host cellular factor that promotes EBOV replication, and future studies will focus on whether TRIM6 could be targeted for therapeutic intervention against EBOV infection.IMPORTANCE EBOV belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans and other mammals with high mortality rates (40 to 90%). Because of its high pathogenicity and lack of licensed antivirals and vaccines, EBOV is listed as a tier 1 select-agent risk group 4 pathogen. An important mechanism for the severity of EBOV infection is its suppression of innate immune responses. The EBOV VP35 protein contributes to pathogenesis, because it serves as an essential cofactor of the viral polymerase as well as a potent antagonist of innate immunity. However, how VP35 function is regulated by host cellular factors is poorly understood. Here, we report that the host E3-ubiquitin ligase TRIM6 promotes VP35 ubiquitination and is important for efficient virus replication. Therefore, our study identifies a new host factor, TRIM6, as a potential target in the development of antiviral drugs against EBOV.

Nipah Virus C Protein Recruits Tsg101 to Promote the Efficient Release of Virus in an ESCRT-Dependent Pathway.

The budding of Nipah virus, a deadly member of the Henipavirus genus within the Paramyxoviridae, has been thought to be independent of the host ESCRT pathway, which is critical for the budding of many enveloped viruses. This conclusion was based on the budding properties of the virus matrix protein in the absence of other virus components. Here, we find that the virus C protein, which was previously investigated for its role in antagonism of innate immunity, recruits the ESCRT pathway to promote efficient virus release. Inhibition of ESCRT or depletion of the ESCRT factor Tsg101 abrogates the C enhancement of matrix budding and impairs live Nipah virus release. Further, despite the low sequence homology of the C proteins of known henipaviruses, they all enhance the budding of their cognate matrix proteins, suggesting a conserved and previously unknown function for the henipavirus C proteins.

The Matrix Protein of Nipah Virus Targets the E3-Ubiquitin Ligase TRIM6 to Inhibit the IKKε Kinase-Mediated Type-I IFN Antiviral Response.

For efficient replication, viruses have developed mechanisms to evade innate immune responses, including the antiviral type-I interferon (IFN-I) system. Nipah virus (NiV), a highly pathogenic member of the Paramyxoviridae family (genus Henipavirus), is known to encode for four P gene-derived viral proteins (P/C/W/V) with IFN-I antagonist functions. Here we report that NiV matrix protein (NiV-M), which is important for virus assembly and budding, can also inhibit IFN-I responses. IFN-I production requires activation of multiple signaling components including the IκB kinase epsilon (IKKε). We previously showed that the E3-ubiquitin ligase TRIM6 catalyzes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, and activate IKKε for induction of IFN-I mediated antiviral responses. Using co-immunoprecipitation assays and confocal microscopy we show here that the NiV-M protein interacts with TRIM6 and promotes TRIM6 degradation. Consequently, NiV-M expression results in reduced levels of unanchored K48-linked polyubiquitin chains associated with IKKε leading to impaired IKKε oligomerization, IKKε autophosphorylation and reduced IFN-mediated responses. This IFN antagonist function of NiV-M requires a conserved lysine residue (K258) in the bipartite nuclear localization signal that is found in divergent henipaviruses. Consistent with this, the matrix proteins of Ghana, Hendra and Cedar viruses were also able to inhibit IFNß induction. Live NiV infection, but not a recombinant NiV lacking the M protein, reduced the levels of endogenous TRIM6 protein expression. To our knowledge, matrix proteins of paramyxoviruses have never been reported to be involved in innate immune antagonism. We report here a novel mechanism of viral innate immune evasion by targeting TRIM6, IKKε and unanchored polyubiquitin chains. These findings expand the universe of viral IFN antagonism strategies and provide a new potential target for development of therapeutic interventions against NiV infections.

Henipavirus infection of the central nervous system.

Nipah virus (NiV) and Hendra virus are highly pathogenic zoonotic viruses of the genus Henipavirus, family Paramyxoviridae. These viruses were first identified as the causative agents of severe respiratory and encephalitic disease in the 1990s across Australia and Southern Asia with mortality rates reaching up to 75%. While outbreaks of Nipah and Hendra virus infections remain rare and sporadic, there is concern that NiV has pandemic potential. Despite increased attention, little is understood about the neuropathogenesis of henipavirus infection. Neuropathogenesis appears to arise from dual mechanisms of vascular disease and direct parenchymal brain infection, but the relative contributions remain unknown while respiratory disease arises from vasculitis and respiratory epithelial cell infection. This review will address NiV basic clinical disease, pathology and pathogenesis with a particular focus on central nervous system (CNS) infection and address the necessity of a model of relapsed CNS infection. Additionally, the innate immune responses to NiV infection in vitro and in the CNS are reviewed as it is likely linked to any persistent CNS infection.

Favipiravir (T-705) protects against Nipah virus infection in the hamster model.

Nipah and Hendra viruses are recently emerged bat-borne paramyxoviruses (genus Henipavirus) causing severe encephalitis and respiratory disease in humans with fatality rates ranging from 40-75%. Despite the severe pathogenicity of these viruses and their pandemic potential, no therapeutics or vaccines are currently approved for use in humans. Favipiravir (T-705) is a purine analogue antiviral approved for use in Japan against emerging influenza strains; and several phase 2 and 3 clinical trials are ongoing in the United States and Europe. Favipiravir has demonstrated efficacy against a broad spectrum of RNA viruses, including members of the Paramyxoviridae, Filoviridae, Arenaviridae families, and the Bunyavirales order. We now demonstrate that favipiravir has potent antiviral activity against henipaviruses. In vitro, favipiravir inhibited Nipah and Hendra virus replication and transcription at micromolar concentrations. In the Syrian hamster model, either twice daily oral or once daily subcutaneous administration of favipiravir for 14 days fully protected animals challenged with a lethal dose of Nipah virus. This first successful treatment of henipavirus infection in vivo with a small molecule drug suggests that favipiravir should be further evaluated as an antiviral treatment option for henipavirus infections.

Status of vaccine research and development of vaccines for Nipah virus.

Nipah virus (NiV) is a highly pathogenic, recently emerged paramyxovirus that has been responsible for sporadic outbreaks of respiratory and encephalitic disease in Southeast Asia. High case fatality rates have also been associated with recent outbreaks in Malaysia and Bangladesh. Although over two billion people currently live in regions in which NiV is endemic or in which the Pteropus fruit bat reservoir is commonly found, there is no approved vaccine to protect against NiV disease. This report examines the feasibility and current efforts to develop a NiV vaccine including potential hurdles for technical and regulatory assessment of candidate vaccines and the likelihood for financing.

Research and development of Zika virus vaccines.

Zika virus (ZIKV) is a member of the family Flaviviridae, genus Flavivirus, and is transmitted by Aedes sp. mosquitoes. There are three genetic lineages of ZIKV: the East African, West African and Asian lineages. Until recently, Zika fever (ZF) has normally been considered a rare, mild febrile disease, but reports since 2012 have shown potentially severe complications associated with ZIKV infection, including microcephaly and Guillain-Barré syndrome. There are no licensed vaccines for ZIKV; however, many vaccine platforms/approaches that have been utilised for other flavivirus vaccines are being applied to ZIKV. Given the current outbreak of ZIKV in the Americas with its associated risks to pregnancy, we summarise what is known about the virus, how knowledge of currently licensed flavivirus vaccines can be applied to ZIKV vaccine development and the assessments of potential challenges for ZIKV vaccine testing and evaluation.