Ebolavirus diagnosis made simple, comparable and faster than molecular detection methods: preparing for the future

Background The 2014/2015 Ebolavirus outbreak resulted in more than 28,000 cases and 11,323 reported deaths, as of March 2016. Domestic transmission of the Guinea strain associated with the outbreak occurred mainly in six African countries, and international transmission was reported in four countries. Outbreak management was limited by the inability to rapidly diagnose infected cases. A further fifteen countries in Africa are predicted to be at risk of Ebolavirus outbreaks in the future as a consequence of climate change and urbanization. Early detection of cases and reduction of transmission rates is critical to prevent and manage future severe outbreaks. We designed a rapid assay for detection of Ebolavirus using recombinase polymerase amplification, a rapid isothermal amplification technology that can be combined with portable lateral flow detection technology. The developed rapid assay operates in 30 min and was comparable with real-time TaqMan™ PCR. Methods Designed, screened, selected and optimized oligonucleotides using the NP coding region from Ebola Zaire virus (Guinea strain). We determined the analytical sensitivity of our Ebola rapid molecular test by testing selected primers and probe with tenfold serial dilutions (1.34 × 1010− 1.34 × 101 copies/μL) of cloned NP gene from Mayinga strain of Zaire ebolavirus in pCAGGS vector, and serially diluted cultured Ebolavirus as established by real-time TaqMan™ PCR that was performed using ABI7500 in Fast Mode. We tested extracted and reverse transcribed RNA from cultured Zaire ebolavirus strains – Mayinga, Gueckedou C05, Gueckedou C07, Makona, Kissidougou and Kiwit. We determined the analytical specificity of our assay with related viruses: Marburg, Ebola Reston and Ebola Sudan. We further tested for Dengue virus 1–4, Plasmodium falciparum and West Nile Virus (Kunjin strain). Results The assay had a detection limit of 134 copies per μL of plasmid containing the NP gene of Ebolavirus Mayinga, and cultured Ebolavirus and was highly specific for the Zaire ebolavirus species, including the Guinea strain responsible for the 2014/2015 outbreak. The assay did not detect related viruses like Marburg, Reston, or Sudan viruses, and other pathogens likely to be isolated from clinical samples. Conclusions Our assay could be suitable for implementation in district and primary health laboratories, as only a heating block and centrifuge is required for operation. The technique could provide a pathway for rapid screening of patients and animals for improved management of outbreaks

Subclinical infection without encephalitis in mice following intranasal exposure to Nipah virus-Malaysia and Nipah virus-Bangladesh

BACKGROUND: Nipah virus and Hendra virus are closely related and following natural or experimental exposure induce similar clinical disease. In humans, encephalitis is the most serious outcome of infection and, hitherto, research into the pathogenesis of henipavirus encephalitis has been limited by the lack of a suitable model. Recently we reported a wild-type mouse model of Hendra virus (HeV) encephalitis that should facilitate detailed investigations of its neuropathogenesis, including mechanisms of disease recrudescence. In this study we investigated the possibility of developing a similar model of Nipah virus encephalitis. FINDINGS: Aged and young adult wild type mice did not develop clinical disease including encephalitis following intranasal exposure to either the Malaysia (NiV-MY) or Bangladesh (NiV-BD) strains of Nipah virus. However viral RNA was detected in lung tissue of mice at euthanasia (21 days following exposure) accompanied by a non-neutralizing antibody response. In a subsequent time course trial this viral RNA was shown to be reflective of an earlier self-limiting and subclinical lower respiratory tract infection through successful virus re-isolation and antigen detection in lung. There was no evidence for viremia or infection of other organs, including brain. CONCLUSIONS: Mice develop a subclinical self-limiting lower respiratory tract infection but not encephalitis following intranasal exposure to NiV-BD or NiV-MY. These results contrast with those reported for HeV under similar exposure conditions in mice, demonstrating a significant biological difference in host clinical response to exposure with these viruses. This finding provides a new platform from which to explore the viral and/or host factors that determine the neuroinvasive ability of henipaviruses

Reference hydrologic networks II: using reference hydrologic networks to assess climate-driven changes in streamflow

Reference hydrologic networks (RHNs) can play an important role in monitoring for changes in the hydrological regime related to climate variation and change. Currently, the literature concerning hydrological response to climate variations is complex and confounded by the combinations of many methods of analysis, wide variations in hydrology, and the inclusion of data series that include changes in land use, storage regulation and water use in addition to those of climate. Three case studies that illustrate a variety of approaches to the analysis of data from RHNs are presented and used, together with a summary of studies from the literature, to develop approaches for the investigation of changes in the hydrological regime at a continental or global scale, particularly for international comparison. We present recommendations for an analysis framework and the next steps to advance such an initiative. There is a particular focus on the desirability of establishing standardized procedures and methodologies for both the creation of new national RHNs and the systematic analysis of data derived from a collection of RHNs

Reference hydrologic networks I: the status and potential future directions of national reference hydrologic networks for detecting trends

Identifying climate-driven trends in river flows on a global basis is hampered by a lack of long, quality time series data for rivers with relatively undisturbed regimes. This is a global problem compounded by the lack of support for essential long-term monitoring. Experience demonstrates that, with clear strategic objectives, and the support of sponsoring organizations, reference hydrologic networks can constitute an exceptionally valuable data source to effectively identify, quantify and interpret hydrological change—the speed and magnitude of which is expected to a be a primary driver of water management and flood alleviation strategies through the future—and for additional applications. Reference hydrologic networks have been developed in many countries in the past few decades. These collections of streamflow gauging stations, that are maintained and operated with the intention of observing how the hydrology of watersheds responds to variations in climate, are described. The status of networks under development is summarized. We suggest a plan of actions to make more effective use of this collection of networks

Type III IFN Receptor Expression and Functional Characterisation in the Pteropid Bat, Pteropus alecto

Bats are rich reservoir hosts for a variety of viruses, many of which are capable of spillover to other susceptible mammals with lethal consequences. The ability of bats to remain asymptomatic to viral infection may be due to the rapid control of viral replication very early in the immune response through innate antiviral mechanisms. Type I and III interferons (IFNs) represent the first line of defence against viral infection in mammals, with both families of IFNs present in pteropid bats. To obtain further insight into the type III IFN system in bats, we describe the characterization of the type III IFN receptor (IFNλR) in the black flying fox, P. alecto with the characterization of IFNλR1 and IL10R2 genes that make up the type III IFN receptor complex. The bat IFNλR complex has a wide tissue distribution and at the cellular level, both epithelial and immune cells are responsive to IFN-λ treatment. Furthermore, we demonstrate that the bat IFNλR1 chain acts as a functional receptor. To our knowledge, this report represents the first description of an IFN receptor in any species of bat. The responsiveness of bat cells to IFN-λ support a role for the type III IFN system by epithelial and immune cells in bats

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

Animal infection studies of two recently discovered African bat paramyxoviruses, Achimota 1 and Achimota 2.

Bats are implicated as the natural reservoirs for several highly pathogenic viruses that can infect other animal species, including man. Here, we investigate the potential for two recently discovered bat rubulaviruses, Achimota virus 1 (AchPV1) and Achimota virus 2 (AchPV2), isolated from urine collected under urban bat (Eidolon helvum) roosts in Ghana, West Africa, to infect small laboratory animals. AchPV1 and AchPV2 are classified in the family Paramyxoviridae and cluster with other bat derived zoonotic rubulaviruses (i.e. Sosuga, Menangle and Tioman viruses). To assess the susceptibility of AchPV1 and AchPV2 in animals, infection studies were conducted in ferrets, guinea pigs and mice. Seroconversion, immunohistological evidence of infection, and viral shedding were identified in ferrets and guinea pigs, but not in mice. Infection was associated with respiratory disease in ferrets. Viral genome was detected in a range of tissues from ferrets and guinea pigs, however virus isolation was only achieved from ferret tissues. The results from this study indicate Achimota viruses (AchPVs) are able to cross the species barrier. Consequently, vigilance for infection with and disease caused by these viruses in people and domesticated animals is warranted in sub-Saharan Africa and the Arabian Peninsula where the reservoir hosts are present.Royal Society Wolfson research merit award. NRF-CRP grant (NRF2012NRF-CRP001-056)

Achimota Pararubulavirus 3: A New Bat-Derived Paramyxovirus of the Genus Pararubulavirus.

Bats are an important source of viral zoonoses, including paramyxoviruses. The paramyxoviral Pararubulavirus genus contains viruses mostly derived from bats that are common, diverse, distributed throughout the Old World, and known to be zoonotic. Here, we describe a new member of the genus Achimota pararubulavirus 3 (AchPV3) and its isolation from the urine of African straw-coloured fruit bats on primary bat kidneys cells. We sequenced and analysed the genome of AchPV3 relative to other Paramyxoviridae, revealing it to be similar to known pararubulaviruses. Phylogenetic analysis of AchPV3 revealed the failure of molecular detection in the urine sample from which AchPV3 was derived and an attachment protein most closely related with AchPV2-a pararubulavirus known to cause cross-species transmission. Together these findings add to the picture of pararubulaviruses, their sources, and variable zoonotic potential, which is key to our understanding of host restriction and spillover of bat-derived paramyxoviruses. AchPV3 represents a novel candidate zoonosis and an important tool for further study

Expression of microRNA in human retinal pigment epithelial cells following infection with Zaire ebolavirus

© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Objective: Survivors of Ebola virus disease (EVD) are at risk of developing blinding intraocular inflammation—or uveitis—which is associated with retinal pigment epithelial (RPE) scarring and persistence of live Zaire ebolavirus (EBOV) within the eye. As part of a large research project aimed at defining the human RPE cell response to being infected with EBOV, this work focused on the microRNAs (miRNAs) associated with the infection. Results: Using RNA-sequencing, we detected 13 highly induced and 2 highly repressed human miRNAs in human ARPE-19 RPE cells infected with EBOV, including hsa-miR-1307-5p, hsa-miR-29b-3p and hsa-miR-33a-5p (up-regulated), and hsa-miR-3074-3p and hsa-miR-27b-5p (down-regulated). EBOV-miR-1-5p was also found in infected RPE cells. Through computational identification of putative miRNA targets, we predicted a broad range of regulatory activities, including effects on innate and adaptive immune responses, cellular metabolism, cell cycle progression, apoptosis and autophagy. The most highly-connected molecule in the miR-target network was leucine-rich repeat kinase 2, which is involved in neuroinflammation and lysosomal processing. Our findings should stimulate new studies on the impact of miRNA changes in EBOV-infected RPE cells to further understanding of intraocular viral persistence and the pathogenesis of uveitis in EVD survivors

Establishment, Immortalisation and Characterisation of Pteropid Bat Cell Lines

BACKGROUND: Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions. METHODOLOGY/FINDINGS: Black flying foxes (Pteropus alecto) were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types. CONCLUSIONS/SIGNIFICANCE: The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study