Antagonism of STAT3 signalling by Ebola virus.

Many viruses target signal transducers and activators of transcription (STAT) 1 and 2 to antagonise antiviral interferon signalling, but targeting of signalling by other STATs/cytokines, including STAT3/interleukin 6 that regulate processes important to Ebola virus (EBOV) haemorrhagic fever, is poorly defined. We report that EBOV potently inhibits STAT3 responses to interleukin-6 family cytokines, and that this is mediated by the interferon-antagonist VP24. Mechanistic analysis indicates that VP24 effects a unique strategy combining distinct karyopherin-dependent and karyopherin-independent mechanisms to antagonise STAT3-STAT1 heterodimers and STAT3 homodimers, respectively. This appears to reflect distinct mechanisms of nuclear trafficking of the STAT3 complexes, revealed for the first time by our analysis of VP24 function. These findings are consistent with major roles for global inhibition of STAT3 signalling in EBOV infection, and provide new insights into the molecular mechanisms of STAT3 nuclear trafficking, significant to pathogen-host interactions, cell physiology and pathologies such as cancer.

Infectious KoRV-related retroviruses circulating in Australian bats.

Bats are reservoirs of emerging viruses that are highly pathogenic to other mammals, including humans. Despite the diversity and abundance of bat viruses, to date they have not been shown to harbor exogenous retroviruses. Here we report the discovery and characterization of a group of koala retrovirus-related (KoRV-related) gammaretroviruses in Australian and Asian bats. These include the Hervey pteropid gammaretrovirus (HPG), identified in the scat of the Australian black flying fox (Pteropus alecto), which is the first reproduction-competent retrovirus found in bats. HPG is a close relative of KoRV and the gibbon ape leukemia virus (GALV), with virion morphology and Mn2+-dependent virion-associated reverse transcriptase activity typical of a gammaretrovirus. In vitro, HPG is capable of infecting bat and human cells, but not mouse cells, and displays a similar pattern of cell tropism as KoRV-A and GALV. Population studies reveal the presence of HPG and KoRV-related sequences in several locations across northeast Australia, as well as serologic evidence for HPG in multiple pteropid bat species, while phylogenetic analysis places these bat viruses as the basal group within the KoRV-related retroviruses. Taken together, these results reveal bats to be important reservoirs of exogenous KoRV-related gammaretroviruses.

Novel role of SARM1 mediated axonal degeneration in the pathogenesis of rabies.

Neurotropic viral infections continue to pose a serious threat to human and animal wellbeing. Host responses combatting the invading virus in these infections often cause irreversible damage to the nervous system, resulting in poor prognosis. Rabies is the most lethal neurotropic virus, which specifically infects neurons and spreads through the host nervous system by retrograde axonal transport. The key pathogenic mechanisms associated with rabies infection and axonal transmission in neurons remains unclear. Here we studied the pathogenesis of different field isolates of lyssavirus including rabies using ex-vivo model systems generated with mouse primary neurons derived from the peripheral and central nervous systems. In this study, we show that neurons activate selective and compartmentalized degeneration of their axons and dendrites in response to infection with different field strains of lyssavirus. We further show that this axonal degeneration is mediated by the loss of NAD and calpain-mediated digestion of key structural proteins such as MAP2 and neurofilament. We then analysed the role of SARM1 gene in rabies infection, which has been shown to mediate axonal self-destruction during injury. We show that SARM1 is required for the accelerated execution of rabies induced axonal degeneration and the deletion of SARM1 gene significantly delayed axonal degeneration in rabies infected neurons. Using a microfluidic-based ex-vivo neuronal model, we show that SARM1-mediated axonal degeneration impedes the spread of rabies virus among interconnected neurons. However, this neuronal defense mechanism also results in the pathological loss of axons and dendrites. This study therefore identifies a potential host-directed mechanism behind neurological dysfunction in rabies infection. This study also implicates a novel role of SARM1 mediated axonal degeneration in neurotropic viral infection.

Comparative transcriptome analysis of pilchard orthomyxovirus (POMV) and infectious salmon anaemia virus (ISAV).

The Tasmanian Atlantic salmon (Salmo salar) aquaculture industry had remained relatively free of major viral diseases until the recent emergence of pilchard orthomyxovirus (POMV). The virus originally isolated from wild pilchards in Southern Australia is of great concern to the industry as it can cause high mortality. Despite its classification in the Orthomyxoviridae family, POMV is genetically divergent from infectious salmon anaemia virus (ISAV) and potentially represents a new genus within the family. Previous research has produced a formal case definition for clinical POMV, but the molecular events that underpin viral infection have not been characterized. Here we have undertaken a comparative transcriptome analysis of the response of Atlantic salmon kidney cells (ASK) in vitro to both POMV and ISAV using RNA sequencing, by harvesting cells at 6 and 24 h post infection (hpi). Despite their genomic differences, both orthomyxoviruses induced significant, and in some cases similar, innate antiviral responses. Early up-regulation of pathogen recognition receptor genes, RIG-I and TLR3, was observed in response to both viruses and triggered downstream interferon (IFN) responses. Interferon transcripts (IFN-alpha1 and INF-alpha2) were only induced in POMV infected cells at 24 hpi, but IFN-alpha3 was up-regulated in all time points and with both viruses. In addition, a strong induction of antiviral response genes (Mx and ISG15) was observed during the early infection with both viruses. Analysis of transcription factor binding sites in the up-regulated gene sets indicated that the host response to both viruses was largely driven by interferon regulatory factors (IRF) 1 and 2. Only three genes (slc35f2, odf2, LOC106608698) were differentially expressed in opposite directions, up-regulated with POMV and strongly down-regulated with ISAV at 24 hpi. Differential expression of these transcripts is possibly a consequence of virus divergence, but could also be associated to higher viral loads observed in the infection with POMV. Results from this study improve our understanding of the innate immune responses and host-pathogen interactions between POMV and Atlantic salmon. Early host response genes could potentially be exploited as subclinical biomarkers specific to POMV, and improved the development of tools for disease surveillance.

Isolation and characterisation of an ISKNV-genotype megalocytivirus from imported angelfish Pterophyllum scalare.

Using cultures of the SKF-9 cell line, megalocytivirus AFIV-16 was isolated from imported angelfish Pterophyllum scalare held in quarantine at the Australian border. The cytopathic effect caused by isolate AFIV-16 presented as cell rounding and enlargement, but complete destruction of the infected cell cultures did not occur. The infected cells demonstrated immunocytochemical reactivity with monoclonal antibody M10, which is used for diagnosis of OIE-listed red sea bream iridoviral disease. Using electron microscopy, the virus particles, consisting of hexagonal nucleocapsids, were observed in the cytoplasm of SKF-9 cells. The replication of AFIV-16 in cultured SKF-9 cells was significantly greater at 28°C incubation than at 22 and 25°C incubation, whereas no difference in growth characteristics was observed for red sea bream iridovirus (RSIV) isolate KagYT-96 across this temperature range. Whole genome sequencing demonstrated that AFIV-16 has a 99.96% similarity to infectious spleen and kidney necrosis virus (ISKNV), the type species in the genus Megalocytivirus. AFIV-16 was classified into ISKNV genotype Clade 1 by phylogenetic analysis of the major capsid protein gene nucleotide sequence. This is the first report of whole genome sequencing of an ISKNV genotype megalocytivirus isolated from ornamental fish.

Reversible host cell remodeling underpins deformability changes in malaria parasite sexual blood stages.

The sexual blood stage of the human malaria parasite Plasmodium falciparum undergoes remarkable biophysical changes as it prepares for transmission to mosquitoes. During maturation, midstage gametocytes show low deformability and sequester in the bone marrow and spleen cords, thus avoiding clearance during passage through splenic sinuses. Mature gametocytes exhibit increased deformability and reappear in the peripheral circulation, allowing uptake by mosquitoes. Here we define the reversible changes in erythrocyte membrane organization that underpin this biomechanical transformation. Atomic force microscopy reveals that the length of the spectrin cross-members and the size of the skeletal meshwork increase in developing gametocytes, then decrease in mature-stage gametocytes. These changes are accompanied by relocation of actin from the erythrocyte membrane to the Maurer's clefts. Fluorescence recovery after photobleaching reveals reversible changes in the level of coupling between the membrane skeleton and the plasma membrane. Treatment of midstage gametocytes with cytochalasin D decreases the vertical coupling and increases their filterability. A computationally efficient coarse-grained model of the erythrocyte membrane reveals that restructuring and constraining the spectrin meshwork can fully account for the observed changes in deformability.

Comparative Transcriptomics Highlights the Role of the Activator Protein 1 Transcription Factor in the Host Response to Ebolavirus.

Ebolavirus and Marburgvirus comprise two genera of negative-sense single-stranded RNA viruses that cause severe hemorrhagic fevers in humans. Despite considerable research efforts, the molecular events following Ebola virus (EBOV) infection are poorly understood. With the view of identifying host factors that underpin EBOV pathogenesis, we compared the transcriptomes of EBOV-infected human, pig, and bat kidney cells using a transcriptome sequencing (RNA-seq) approach. Despite a significant difference in viral transcription/replication between the cell lines, all cells responded to EBOV infection through a robust induction of extracellular growth factors. Furthermore, a significant upregulation of activator protein 1 (AP1) transcription factor complex members FOS and JUN was observed in permissive cell lines. Functional studies focusing on human cells showed that EBOV infection induces protein expression, phosphorylation, and nuclear accumulation of JUN and, to a lesser degree, FOS. Using a luciferase-based reporter, we show that EBOV infection induces AP1 transactivation activity within human cells at 48 and 72 h postinfection. Finally, we show that JUN knockdown decreases the expression of EBOV-induced host gene expression. Taken together, our study highlights the role of AP1 in promoting the host gene expression profile that defines EBOV pathogenesis.IMPORTANCE Many questions remain about the molecular events that underpin filovirus pathophysiology. The rational design of new intervention strategies, such as postexposure therapeutics, will be significantly enhanced through an in-depth understanding of these molecular events. We believe that new insights into the molecular pathogenesis of EBOV may be possible by examining the transcriptomic response of taxonomically diverse cell lines (derived from human, pig, and bat). We first identified the responsive pathways using an RNA-seq-based transcriptomics approach. Further functional and computational analysis focusing on human cells highlighted an important role for the AP1 transcription factor in mediating the transcriptional response to EBOV infection. Our study sheds new light on how host transcription factors respond to and promote the transcriptional landscape that follows viral infection.

Zika virus-induced hyper excitation precedes death of mouse primary neuron.

BACKGROUND: Zika virus infection in new born is linked to congenital syndromes, especially microcephaly. Studies have shown that these neuropathies are the result of significant death of neuronal progenitor cells in the central nervous system of the embryo, targeted by the virus. Although cell death via apoptosis is well acknowledged, little is known about possible pathogenic cellular mechanisms triggering cell death in neurons. METHODS: We used in vitro embryonic mouse primary neuron cultures to study possible upstream cellular mechanisms of cell death. Neuronal networks were grown on microelectrode array and electrical activity was recorded at different times post Zika virus infection. In addition to this method, we used confocal microscopy and Q-PCR techniques to observe morphological and molecular changes after infection. RESULTS: Zika virus infection of mouse primary neurons triggers an early spiking excitation of neuron cultures, followed by dramatic loss of this activity. Using NMDA receptor antagonist, we show that this excitotoxicity mechanism, likely via glutamate, could also contribute to the observed nervous system defects in human embryos and could open new perspective regarding the causes of adult neuropathies. CONCLUSIONS: This model of excitotoxicity, in the context of neurotropic virus infection, highlights the significance of neuronal activity recording with microelectrode array and possibility of more than one lethal mechanism after Zika virus infection in the nervous system.

Comparing Gene Silencing and Physiochemical Properties in siRNA Bound Cationic Star-Polymer Complexes.

The translation of siRNA into clinical therapies has been significantly delayed by issues surrounding the delivery of naked siRNA to target cells. Here we investigate siRNA delivery by cationic acrylic polymers developed by Reversible Addition-Fragmentation chain Transfer (RAFT) mediated free radical polymerization. We investigated cell uptake and gene silencing of a series of siRNA-star polymer complexes both in the presence and absence of a protein "corona". Using a multidisciplinary approach including quantitative nanoscale mechanical-atomic force microscopy, dynamic light scattering and nanoparticle tracking analysis we have characterized the nanoscale morphology, stiffness, and surface charge of the complexes with and without the protein corona. This is one of the first examples of a comprehensive physiochemical analysis of siRNA-polymer complexes being performed alongside in vitro biological assays, allowing us to describe a set of desirable physical features of cationic polymer complexes that promote gene silencing. Multifaceted studies such as this will improve our understanding of structure-function relationships in nanotherapeutics, facilitating the rational design of polymer-mediated siRNA delivery systems for novel treatment strategies.

Origin, composition, organization and function of the inner membrane complex of Plasmodium falciparum gametocytes.

The most virulent of the human malaria parasites, Plasmodium falciparum, undergoes a remarkable morphological transformation as it prepares itself for sexual reproduction and transmission via mosquitoes. Indeed P. falciparum is named for the unique falciform or crescent shape of the mature sexual stages. Once the metamorphosis is completed, the mature gametocyte releases from sequestration sites and enters the circulation, thus making it accessible to feeding mosquitoes. Early ultrastructural studies showed that gametocyte elongation is driven by the assembly of a system of flattened cisternal membrane compartments underneath the parasite plasma membrane and a supporting network of microtubules. Here we describe the molecular composition and origin of the sub-pellicular membrane complex, and show that it is analogous to the inner membrane complex, an organelle with structural and motor functions that is well conserved across the apicomplexa. We identify novel crosslinking elements that might help stabilize the inner membrane complex during gametocyte development. We show that changes in gametocyte morphology are associated with an increase in cellular deformability and postulate that this enables the gametocytes to circulate in the bloodstream without being detected and removed by the mechanical filtering mechanisms in the spleen of the host.