Vav proteins do not influence dengue virus replication but are associated with induction of phospho-ERK, IL-6, and viperin mRNA following DENV infection in vitro.

IMPORTANCE: Dengue disease is characterized by an inflammatory-mediated immunopathology, with elevated levels of circulating factors including TNF-α and IL-6. If the damaging inflammatory pathways could be blocked without loss of antiviral responses or exacerbating viral replication, then this would be of potential therapeutic benefit. The study here has investigated the Vav guanine exchange factors as a potential alternative signaling pathway that may drive dengue virus (DENV)-induced inflammatory responses, with a focus on Vav1 and 2. While Vav proteins were positively associated with mRNA for inflammatory cytokines, blocking Vav signaling didn't affect DENV replication but prevented DENV-induction of p-ERK and enhanced IL-6 (inflammatory) and viperin (antiviral) mRNA. These initial data suggest that Vav proteins could be a target that does not compromise control of viral replication and should be investigated further for broader impact on host inflammatory responses, in settings such as antibody-dependent enhancement of infection and in different cell types.

Comparison of in vitro growth characteristics of Cryptosporidium hominis (IdA15G1) and Cryptosporidium parvum (Iowa-IIaA17G2R1 and IIaA18G3R1).

Cryptosporidium is a major cause of diarrhoeal disease and mortality in young children in resource-poor countries, for which no vaccines or adequate therapeutic options are available. Infection in humans is primarily caused by two species: C. hominis and C. parvum. Despite C. hominis being the dominant species infecting humans in most countries, very little is known about its growth characteristics and life cycle in vitro, given that the majority of our knowledge of the in vitro development of Cryptosporidium has been based on C. parvum. In the present study, the growth and development of two C. parvum isolates (subtypes Iowa-IIaA17G2R1 and IIaA18G3R1) and one C. hominis isolate (subtype IdA15G1) in HCT-8 cells were examined and compared at 24 h and 48 h using morphological data acquired with scanning electron microscopy. Our data indicated no significant differences in the proportion of meronts or merozoites between species or subtypes at either time-point. Sexual development was observed at the 48-h time-point across both species through observations of both microgamonts and macrogamonts, with a higher frequency of macrogamont observations in C. hominis (IdA15G1) cultures at 48-h post-infection compared to both C. parvum subtypes. This corresponded to differences in the proportion of trophozoites observed at the same time point. No differences in proportion of microgamonts were observed between the three subtypes, which were rarely observed across all cultures. In summary, our data indicate that asexual development of C. hominis is similar to that of C. parvum, while sexual development is accelerated in C. hominis. This study provides new insights into differences in the in vitro growth characteristics of C. hominis when compared to C. parvum, which will facilitate our understanding of the sexual development of both species.

Dengue Virus Infection of Human Retinal Müller Glial Cells.

Retinopathy is a recently recognized complication of dengue, affecting up to 10% of hospitalized patients. Research on the pathogenesis has focused largely on effects of dengue virus (DENV) at the blood-retinal barrier. Involvement of retinal Müller glial cells has received little attention, although this cell population contributes to the pathology of other intraocular infections. The goal of our work was to establish the susceptibility of Müller cells to infection with DENV and to identify characteristics of the cellular antiviral, inflammatory, and immunomodulatory responses to DENV infection in vitro. Primary human Müller cell isolates and the MIO-M1 human Müller cell line were infected with the laboratory-adapted Mon601 strain and DENV serotype 1 and 2 field isolates, and cell-DENV interactions were investigated by immunolabelling and quantitative real-time polymerase chain reaction. Müller cells were susceptible to DENV infection, but experiments involving primary cell isolates indicated inter-individual variation. Viral infection induced an inflammatory response (including tumour necrosis factor-α, interleukin [IL]-1ß, and IL-6) and an immunomodulatory response (including programmed death-ligand [PD-L]1 and PD-L2). The type I interferon response was muted in the Müller cell line compared to primary cell isolates. The highest infectivity and cell responses were observed in the laboratory-adapted strain, and overall, infectivity and cell responses were stronger in DENV2 strains. This work demonstrates that Müller cells mount an antiviral and immune response to DENV infection, and that this response varies across cell isolates and DENV strain. The research provides a direction for future efforts to understand the role of human retinal Müller glial cells in dengue retinopathy.

The Molecular Link Between TDP-43, Endogenous Retroviruses and Inflammatory Neurodegeneration in Amyotrophic Lateral Sclerosis: a Potential Target for Triumeq, an Antiretroviral Therapy.

Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease (MND), is a progressive neurological disorder, characterised by the death of upper and lower motor neurons. The aetiology of ALS remains unknown, and treatment options are limited. Endogenous retroviruses (ERVs), specifically human endogenous retrovirus type K (HERV-K), have been proposed to be involved in the propagation of neurodegeneration in ALS. ERVs are genomic remnants of ancient viral infection events, with most being inactive and not retaining the capacity to encode a fully infectious virus. However, some ERVs retain the ability to be activated and transcribed, and ERV transcripts have been found to be elevated within the brain tissue of MND patients. A hallmark of ALS pathology is altered localisation of the transactive response (TAR) DNA binding protein 43 kDa (TDP-43), which is normally found within the nucleus of neuronal and glial cells and is involved in RNA regulation. In ALS, TDP-43 aggregates within the cytoplasm and facilitates neurodegeneration. The involvement of ERVs in ALS pathology is thought to occur through TDP-43 and neuroinflammatory mediators. In this review, the proposed involvement of TDP-43, HERV-K and immune regulators on the onset and progression of ALS will be discussed. Furthermore, the evidence supporting a therapy based on targeting ERVs in ALS will be reviewed.

Disease profiles in the Indigenous Australian population are suggestive of a common complement control haplotype.

Aboriginal and Torres Strait Islander People (respectfully referred to as Indigenous Australians herein) are disparately burdened by many infectious and chronic diseases relative to Australians with European genetic ancestry. Some of these diseases are described in other populations to be influenced by the inherited profile of complement genes. These include complement factor B, H, I and complement factor H-related (CFHR) genes that can contribute to a polygenic complotype. Here the focus is on the combined deletion of CFHR1 and 3 to form a common haplotype (CFHR3-1Δ). The prevalence of CFHR3-1Δ is high in people with Nigerian and African American genetic ancestry and correlates to a higher frequency and severity of systemic lupus erythematosus (SLE) but a lower prevalence of age-related macular degeneration (AMD) and IgA-nephropathy (IgAN). This pattern of disease is similarly observed among Indigenous Australian communities. Additionally, the CFHR3-1Δ complotype is also associated with increased susceptibility to infection with pathogens, such as Neisseria meningitidis and Streptococcus pyogenes, which also have high incidences in Indigenous Australian communities. The prevalence of these diseases, while likely influenced by social, political, environmental and biological factors, including variants in other components of the complement system, may also be suggestive of the CFHR3-1Δ haplotype in Indigenous Australians. These data highlight a need to define the Indigenous Australian complotypes, which may lead to the discovery of new risk factors for common diseases and progress towards precision medicines for treating complement-associated diseases in Indigenous and non-Indigenous populations. Herein, the disease profiles suggestive of a common complement CFHR3-1Δ control haplotype are examined.

Vav Proteins in Development of the Brain: A Potential Relationship to the Pathogenesis of Congenital Zika Syndrome?

Zika virus (ZIKV) infection during pregnancy can result in a significant impact on the brain and eye of the developing fetus, termed congenital zika syndrome (CZS). At a morphological level, the main serious presentations of CZS are microcephaly and retinal scarring. At a cellular level, many cell types of the brain may be involved, but primarily neuronal progenitor cells (NPC) and developing neurons. Vav proteins have guanine exchange activity in converting GDP to GTP on proteins such as Rac1, Cdc42 and RhoA to stimulate intracellular signaling pathways. These signaling pathways are known to play important roles in maintaining the polarity and self-renewal of NPC pools by coordinating the formation of adherens junctions with cytoskeletal rearrangements. In developing neurons, these same pathways are adopted to control the formation and growth of neurites and mediate axonal guidance and targeting in the brain and retina. This review describes the role of Vavs in these processes and highlights the points of potential ZIKV interaction, such as (i) the binding and entry of ZIKV in cells via TAM receptors, which may activate Vav/Rac/RhoA signaling; (ii) the functional convergence of ZIKV NS2A with Vav in modulating adherens junctions; (iii) ZIKV NS4A/4B protein effects on PI3K/AKT in a regulatory loop via PPI3 to influence Vav/Rac1 signaling in neurite outgrowth; and (iv) the induction of SOCS1 and USP9X following ZIKV infection to regulate Vav protein degradation or activation, respectively, and impact Vav/Rac/RhoA signaling in NPC and neurons. Experiments to define these interactions will further our understanding of the molecular basis of CZS and potentially other developmental disorders stemming from in utero infections. Additionally, Vav/Rac/RhoA signaling pathways may present tractable targets for therapeutic intervention or molecular rationale for disease severity in CZS.

Infection of Human Retinal Pigment Epithelial Cells with Dengue Virus Strains Isolated during Outbreaks in Singapore.

Prevalence of dengue retinopathy varies across epidemics, with the disease linked to circulation of dengue virus serotype 1 (DENV-1). The retinal pigment epithelium has been implicated in the pathology. We investigated infectivity, molecular response, and barrier function of epithelial cells inoculated with DENV strains from different outbreaks in Singapore. Monolayers of human retinal pigment epithelial cells (multiple primary cell isolates and the ARPE-19 cell line) were inoculated with six DENV strains, at multiplicity of infection of 10; uninfected and recombinant strain-infected controls were included where relevant. Infectivity and cell response were assessed primarily by RT-qPCR on total cellular RNA, and barrier function was evaluated as electrical resistance across monolayers. Higher viral RNA loads were measured in human retinal pigment epithelial cells infected with DENV-1 strains from the 2005 Singapore epidemic, when retinopathy was prevalent, versus DENV-1 strains from the 2007 Singapore epidemic, when retinopathy was not observed. Type I interferon (IFN) transcripts (IFN-ß and multiple IFN-stimulated genes) were up-regulated, and impact on barrier function was more pronounced, for cells infected with DENV-1 strains from the 2005 versus the 2007 Singapore epidemics. Aside from serotype, strain of DENV may determine the potential to induce retinal pathology. Identification of molecular markers of disease-associated DENV strains may provide insights into the pathogenesis of dengue retinopathy.

Posterior segment findings by spectral-domain optical coherence tomography and clinical associations in active toxoplasmic retinochoroiditis.

Toxoplasmic retinochoroiditis is a common, potentially blinding parasitic infection. We sought to define the spectrum and frequency of signs of active toxoplasmic retinochoroiditis by spectral domain optical coherence tomography (SD-OCT), and to identify clinical associations. Ninety eyes of 90 individuals presenting consecutively to a tertiary referral uveitis service with active toxoplasmic retinochoroiditis and gradable SD-OCT scans were evaluated prospectively. SD-OCT features were collated, and associations with lesion location, primary versus recurrent episode, serological status, human immunodeficiency virus infection and best-corrected Snellen visual acuity were explored. Active toxoplasmic retinochoroiditis presented with thickened (65%) and hyperreflective (61%) retina, choroidal thickening (55%) and hyporeflectivity (61%), hyperreflective vitreous dots (80%) and deposits (36%), and posterior hyaloid thickening (35%) on SD-OCT. Most signs occurred with similar frequency across clinical groups. Retinal hyporeflectivity (17%) was significantly associated with a visual acuity of 20/200 or worse at resolution. Our observations demonstrate that active toxoplasmic retinochoroiditis has diverse SD-OCT signs and that none are universally present. Retinal hyporeflectivity-suggesting liquefactive necrosis-predicts poor visual outcome.

Viperin is anti-viral in vitro but is dispensable for restricting dengue virus replication or induction of innate and inflammatory responses in vivo.

Viperin has antiviral function against many viruses, including dengue virus (DENV), when studied in cells in culture. Here, the antiviral actions of viperin were defined both in vitro and in a mouse in vivo model of DENV infection. Murine embryonic fibroblasts (MEFs) derived from mice lacking viperin (vip-/-) showed enhanced DENV infection, accompanied by increased IFN-ß and induction of ISGs; IFIT1 and CXCL-10 but not IRF7, when compared to wild-type (WT) MEFs. In contrast, subcutaneous challenge of immunocompetent WT and vip-/- mice with DENV did not result in enhanced infection. Intracranial infection with DENV resulted in body weight loss and neurological disease with a moderate increase in mortality in vip-/- compared with WT mice, although this was not accompanied by altered brain morphology, immune cell infiltration or DENV RNA level in the brain. Similarly, DENV induction of IFN-ß, IFIT1, CXCL-10, IRF7 and TNF-α was not significantly different in WT and vip-/- mouse brain, although there was a modest but significant increase in DENV induction of IL-6 and IfI27la in the absence of viperin. NanoString nCounter analysis confirmed no significant difference in induction of a panel of inflammatory genes in WT compared to vip-/- DENV-infected mouse brains. Further, polyI:C stimulation of bone marrow-derived macrophages (BMDMs) induced TNF-α, IFN-ß, IL-6 and Nos-2, but responses were not different in BMDMs generated from WT or vip-/- mice. Thus, while there is significant evidence of anti-DENV actions of viperin in some cell types in vitro, for DENV infection in vivo a lack of viperin does not affect systemic or brain susceptibility to DENV or induction of innate and inflammatory responses.

Zika Virus Infection of Human Iris Pigment Epithelial Cells.

During recent Zika epidemics, adults infected with Zika virus (ZIKV) have developed organ-specific inflammatory complications. The most serious Zika-associated inflammatory eye disease is uveitis, which is commonly anterior in type, affecting both eyes and responding to corticosteroid eye drops. Mechanisms of Zika-associated anterior uveitis are unknown, but ZIKV has been identified in the aqueous humor of affected individuals. The iris pigment epithelium is a target cell population in viral anterior uveitis, and it acts to maintain immune privilege within the anterior eye. Interactions between ZIKV and human iris pigment epithelial cells were investigated with infectivity assays and RNA-sequencing. Primary cell isolates were prepared from eyes of 20 cadaveric donors, and infected for 24 hours with PRVABC59 strain ZIKV or incubated uninfected as control. Cytoimmunofluorescence, RT-qPCR on total cellular RNA, and focus-forming assays of culture supernatant showed cell isolates were permissive to infection, and supported replication and release of infectious ZIKV. To explore molecular responses of cell isolates to ZIKV infection at the whole transcriptome level, RNA was sequenced on the Illumina NextSeq 500 platform, and results were aligned to the human GRCh38 genome. Multidimensional scaling showed clear separation between transcriptomes of infected and uninfected cell isolates. Differential expression analysis indicated a vigorous molecular response of the cell to ZIKV: 7,935 genes were differentially expressed between ZIKV-infected and uninfected cells (FDR < 0.05), and 99% of 613 genes that changed at least two-fold were up-regulated. Reactome and KEGG pathway and Gene Ontology enrichment analyses indicated strong activation of viral recognition and defense, in addition to biosynthesis processes. A CHAT network included 6275 molecular nodes and 24 contextual hubs in the cell response to ZIKV infection. Receptor-interacting serine/threonine kinase 1 (RIPK1) was the most significantly connected contextual hub. Correlation of gene expression with read counts assigned to the ZIKV genome identified a negative correlation between interferon signaling and viral load across isolates. This work represents the first investigation of mechanisms of Zika-associated anterior uveitis using an in vitro human cell model. The results suggest the iris pigment epithelium mounts a molecular response that limits intraocular pathology in most individuals.

Changes in complement alternative pathway components, factor B and factor H during dengue virus infection in the AG129 mouse.

The complement alternative pathway (AP) is tightly regulated and changes in two important AP components, factor B (FB) and factor H (FH) are linked to severe dengue in humans. Here, a mouse model of dengue was investigated to define the changes in FB and FH and assess the utility of this model to study the role of the AP in severe dengue. Throughout the period of viremia in the AG129 IFN signalling-deficient mouse, an increase in FB and a decrease in FH was observed following dengue virus (DENV) infection, with the former only seen in a model of more severe disease associated with antibody-dependent enhancement (ADE). Terminal disease was associated with a decrease in FB and FH, with greater changes during ADE, and accompanied by increased C3 degradation consistent with complement activation. In silico analysis of NFκΒ, signal transducer and activator of transcription (STAT) and IFN-driven FB and FH promoter elements to reflect the likely impact of the lack of IFN-responses in AG129 mice, demonstrated that these elements differed markedly between human and mouse, notably with mouse FH lacking NFκΒ and key IFN-stimulated response elements (ISRE), and FB with many more NFκΒ and STAT-responsive elements than human FB. Thus, the AG129 mouse offers utility in demonstrating changes in FB and FH that, similar to humans, are associated with severe disease, but lack predicted important human-specific and IFN-dependent responses of FB and FH to DENV-infection that are likely to regulate the subtleties of the overall AP response during dengue disease in humans.

Organoids and Bioengineered Intestinal Models: Potential Solutions to the Cryptosporidium Culturing Dilemma.

Cryptosporidium is a major cause of severe diarrhea-related disease in children in developing countries, but currently no vaccine or effective treatment exists for those who are most at risk of serious illness. This is partly due to the lack of in vitro culturing methods that are able to support the entire Cryptosporidium life cycle, which has led to research in Cryptosporidium biology lagging behind other protozoan parasites. In vivo models such as gnotobiotic piglets are complex, and standard in vitro culturing methods in transformed cell lines, such as HCT-8 cells, have not been able to fully support fertilization occurring in vitro. Additionally, the Cryptosporidium life cycle has also been reported to occur in the absence of host cells. Recently developed bioengineered intestinal models, however, have shown more promising results and are able to reproduce a whole cycle of infectivity in one model system. This review evaluates the recent advances in Cryptosporidium culturing techniques and proposes future directions for research that may build upon these successes.

Dengue virus and the complement alternative pathway.

Dengue disease is an inflammatory-driven pathology, and complement overactivation is linked to disease severity and vascular leakage. Additionally, dysregulation of complement alternative pathway (AP) components has been described, such as upregulation of complement factor D and downregulation of complement factor H (FH), which activate and inhibit the AP, respectively. Thus, the pathology of severe dengue could in part result from AP dysfunction, even though complement and AP activation usually provide protection against viral infections. In dengue virus-infected macrophages and endothelial cells (ECs), the site of replication and target for vascular pathology, respectively, the AP is activated. The AP activation, reduced FH and vascular leakage seen in dengue disease in part parallels other complement AP pathologies associated with FH deficiency, such as atypical haemolytic uraemic syndrome (aHUS). aHUS can be therapeutically targeted with inhibitors of complement terminal activity, raising the idea that strategies such as inhibition of complement or delivery of FH or other complement regulatory components to EC may be beneficial to combat the vascular leakage seen in severe dengue.

Dengue virus infects the mouse eye following systemic or intracranial infection and induces inflammatory responses.

Dengue virus (DENV) infection is associated with clinical ocular presentations and here DENV infection of the eye was assessed in mice. In an AG129 mouse model of antibody-dependent enhancement of DENV infection, DENV RNA was detected in the eye and vascular changes were present in the retinae. Intraocular CD8 and IFN-γ mRNA were increased in mice born to DENV-naïve, but not DENV-immune mothers, while TNF-α mRNA was induced and significantly higher in mice born to DENV-immune than DENV-naïve mothers. DENV RNA was detected in the eye following intracranial DENV infection and CD8 mRNA but not IFN-γ nor TNF-α were induced. In all models, viperin was increased following DENV infection. Thus, DENV in the circulation or the brain can infect the eye and stimulate innate immune responses, with induction of viperin as one response that consistently occurs in multiple DENV eye-infection models in both an IFN-dependent and independent manner.

In vitro and in vivo roles of sphingosine kinase 2 during dengue virus infection.

There is growing evidence of the influence of sphingosine kinase (SK) enzymes on viral infection. Here, the role of sphingosine kinase 2 (SK2), an isoform of SK prominent in the brain, was defined during dengue virus (DENV) infection. Chemical inhibition of SK2 activity using two different SK2 inhibitors, ABC294640 and K145, had no effect on DENV infection in human cells in vitro. In contrast, DENV infection was restricted in SK2-/- immortalized mouse embryonic fibroblasts (iMEFs) with reduced induction of IFN-ß mRNA and protein, and mRNA for the IFN-stimulated genes (ISGs) viperin, IFIT1, IRF7 and CXCL10 in DENV-infected SK2-/- compared to WT iMEFs. Intracranial (ic) DENV injection in C57BL/6 SK2-/- mice induced body weight loss earlier than in WT mice but DENV RNA levels were comparable in the brain. Neither SK1 mRNA or sphingosine-1-phosphate (S1P) levels were altered following ic DENV infection in WT or SK2-/- mice but brain S1P levels were reduced in all SK2-/- mice, independent of DENV infection. CD8 mRNA was induced in the brains of both DENV-infected WT and SK2-/- mice, suggesting normal CD8+ T-cell infiltration into the DENV-infected brain independent of SK2 or S1P. Thus, although SK2 may be important for replication of some viruses SK2 activity does not affect DENV infection in vitro and SK2 or S1P levels do not influence DENV infection or T-cell infiltration in the context of infection in the brain.

Emerging infectious uveitis: Chikungunya, dengue, Zika and Ebola: A review.

Recently recognized forms of uveitis include intraocular inflammations that occur during or following one of several emerging infectious diseases: chikungunya fever, dengue, Zika virus disease and Ebola virus disease. Anterior, intermediate, posterior and pan-uveitis have been described in individuals infected with chikungunya virus. Persons who contract dengue or Zika viruses also may develop different types of uveitis in the course of the infection: maculopathy is a common manifestation of dengue eye disease, and Zika eye disease may cause hypertensive anterior uveitis or mimic a white dot syndrome. Up to one-third of Ebola survivors develop aggressive uveitis, which is frequently associated with vision loss and complicated by cataract. There are no specific anti-viral drugs for these forms of uveitis, and thus treatment is largely supportive. In this article, we summarize the systemic infectious diseases and virology, and describe the clinical presentations, outcomes and management of emerging viral forms of uveitis.

Clinical Features and Laboratory Findings of Travelers Returning to South Australia with Dengue Virus Infection.

Reported cases of dengue are rising in South Australia (SA) in travellers returning from dengue-endemic regions. We have undertaken a retrospective analysis to identify the clinical and laboratory characteristics of patients returning to SA with suspected dengue virus (DENV) infection. From 488 requests, 49 (10%) were defined by serology as acute dengue, with the majority of patients (75%) testing as non-structural protein 1 (NS1) and/or IgM positive. Dengue was most commonly acquired in Indonesia (42.9%) with clinical features of fever (95%), headache (41%) and myalgia/arthralgia (56%). The presence of rash (36%) and laboratory findings of neutropenia, leukopenia, thrombocytopenia, but not elevated C-reactive protein, were distinct from findings in DENV-seronegative patients. Available dengue seropositive samples were analysed by RT-PCR, with 14/32 (43.8%) positive by a serotype non-specific DENV assay, but 28/32 positive (87.5%) when also assessed by serotype-specific RT-PCR. Serotype analysis revealed the predominance of DENV-1 and DENV-2 and the presence of DENV-3, but not DENV-4 or Zika virus (ZIKV). Thus, dengue in returned travellers in SA presents in a manner consistent with World Health Organization (WHO) definitions, with symptoms, travel history and laboratory results useful in prioritising the likelihood of dengue. This definition will assist the future management in DENV-non-endemic regions, such as SA.