Ebola virus (EBOV) infection results in severe disease and in some cases lethal hemorrhagic fever. The infection is directed by seven viral genes that encode nine viral proteins. By definition, ...viruses are obligate intracellular parasites and require aspects of host cell biology in order to replicate their genetic material, assemble new virus particles, and subvert host cell antiviral responses. Currently licensed antivirals are targeted against viral proteins to inhibit their function. However, experience with treating HIV and influenza virus demonstrates that resistant viruses are soon selected. An emerging area in virology is to transiently target host cell proteins that play critical proviral roles in virus biology, especially for acute infections. This has the advantage that the protein being targeted is evolutionary removed from the genome of the virus. Proteomics can aid in discovery biology and identify cellular proteins that may be utilized by the virus to facilitate infection. This work focused on defining the interactome of the EBOV nucleoprotein and identified that cellular chaperones, including HSP70, associate with this protein to promote stability. Utilization of a mini-genome replication system based on a recent Makona isolate demonstrated that disrupting the stability of NP had an adverse effect on viral RNA synthesis.
In the event of an unpredictable viral outbreak requiring high/maximum biosafety containment facilities (i.e. BSL3 and BSL4), X-ray irradiation has the potential to relieve pressures on conventional ...diagnostic bottlenecks and expediate work at lower containment. Guided by Monte Carlo modelling and in vitro 1-log
decimal-reduction value (D-value) predictions, the X-ray photon energies required for the effective inactivation of zoonotic viruses belonging to the medically important families of Flaviviridae, Nairoviridae, Phenuiviridae and Togaviridae are demonstrated. Specifically, it is shown that an optimized irradiation approach is attractive for use in a multitude of downstream detection and functional assays, as it preserves key biochemical and immunological properties. This study provides evidence that X-ray irradiation can support emergency preparedness, outbreak response and front-line diagnostics in a safe, reproducible and scalable manner pertinent to operations that are otherwise restricted to higher containment BSL3 or BSL4 laboratories.
The unprecedented 2013/16 outbreak of Zaire ebolavirus (Ebola virus) in West Africa has highighted the need for rapid, high-throughput and POC diagnostic assays to enable timely detection and ...appropriate triaging of Ebola Virus Disease (EVD) patients. Ebola virus is highly infectious and prompt diagnosis and triage is crucial in preventing further spread within community and healthcare settings. Moreover, due to the ecology of Ebola virus it is important that newly developed diagnostic assays are suitable for use in both the healthcare environment and low resource rural locations.
A LAMP assay was successfully developed with three detection formats; a real-time intercalating dye-based assay, a real-time probe-based assay to enable multiplexing and an end-point colourimetric assay to simplify interpretation for the field. All assay formats were sensitive and specific, detecting a range of Ebola virus strains isolated in 1976-2014; with Probit analysis predicting limits of detection of 243, 290 and 75 copies/reaction respectively and no cross-detection of related strains or other viral haemorrhagic fevers (VHF's). The assays are rapid, (as fast as 5-7.25 mins for real-time formats) and robust, detecting Ebola virus RNA in presence of minimally diluted bodily fluids. Moreover, when tested on patient samples from the 2013/16 outbreak, there were no false positives and 93-96% of all new case positives were detected, with only a failure to detect very low copy number samples.
These are a set of robust and adaptable diagnostic solutions, which are fast, easy-to-perform-and-interpret and are suitable for use on a range of platforms including portable low-power devices. They can be readily transferred to field-laboratory settings, with no specific equipment needs and are therefore ideally placed for use in locations with limited resources.
The development of new therapies against SARS-CoV-2 is required to extend the toolkit of intervention strategies to combat the global pandemic. In this study, hyperimmune plasma from sheep immunised ...with whole spike SARS-CoV-2 recombinant protein has been used to generate candidate products. In addition to purified IgG, we have refined candidate therapies by removing non-specific IgG via affinity binding along with fragmentation to eliminate the Fc region to create F(ab')
fragments. These preparations were evaluated for in vitro activity and demonstrated to be strongly neutralising against a range of SARS-CoV-2 strains, including Omicron B2.2. In addition, their protection against disease manifestations and viral loads were assessed using a hamster SARS-CoV-2 infection model. Results demonstrated protective effects of both IgG and F(ab')
, with the latter requiring sequential dosing to maintain in vivo activity due to rapid clearance from the circulation.
•Crimean-Congo hemorrhagic fever is the most widespread tick-borne disease of humans.•Its incidence has recently increased in Turkey, Iran and other countries.•No approved drugs or vaccines are ...available against the disease.•Collaboration is needed among countries where CCHF is prevalent and those with greater laboratory capacity.•The First International Conference on CCHF initiates a series of meetings focusing on the disease.
Crimean-Congo hemorrhagic fever (CCHF) is the most widespread tick-borne disease of humans, occurring from western China to the Balkans in Eurasia and south throughout the length of Africa. Its incidence has increased over the past decade, particularly in Turkey and Iran, and the disease has also emerged in India. Research has been hindered by limited laboratory capacity in many regions where the disease is prevalent, indicating the need for collaboration between investigators in endemic countries and those with greater scientific resources. In an effort to increase such collaboration, the First International Conference on Crimean-Congo hemorrhagic fever was held in Thessaloniki, Greece, from February 13 to 14, 2015. This meeting followed the conclusion of an EU-supported Collaborative Project under the Health Cooperation Work Programme of the 7th Framework Programme (Grant agreement No. 260427). It is expected to be the first in a series of meetings that will bring together researchers from around the world to exchange knowledge and experience on various aspects of CCHF. This report summarizes major presentations by the invited speakers at the First International Conference on CCHF.
The pathogenesis and host-viral interactions of the Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) are convoluted and not well evaluated. Application of the multi-omics system biology ...approaches, including biological network analysis in elucidating the complex host-viral response, interrogates the viral pathogenesis. The present study aimed to fingerprint the system-level alterations during acute CCHFV-infection and the cellular immune responses during productive CCHFV-replication in vitro. We used system-wide network-based system biology analysis of peripheral blood mononuclear cells (PBMCs) from a longitudinal cohort of CCHF patients during the acute phase of infection and after one year of recovery (convalescent phase) followed by untargeted quantitative proteomics analysis of the most permissive CCHFV-infected Huh7 and SW13 cells. In the RNAseq analysis of the PBMCs, comparing the acute and convalescent-phase, we observed system-level host's metabolic reprogramming towards central carbon and energy metabolism (CCEM) with distinct upregulation of oxidative phosphorylation (OXPHOS) during CCHFV-infection. Upon application of network-based system biology methods, negative coordination of the biological signaling systems like FOXO/Notch axis and Akt/mTOR/HIF-1 signaling with metabolic pathways during CCHFV-infection were observed. The temporal quantitative proteomics in Huh7 showed a dynamic change in the CCEM over time and concordant with the cross-sectional proteomics in SW13 cells. By blocking the two key CCEM pathways, glycolysis and glutaminolysis, viral replication was inhibited in vitro. Activation of key interferon stimulating genes during infection suggested the role of type I and II interferon-mediated antiviral mechanisms both at the system level and during progressive replication.
The global pandemic of coronavirus disease (COVID-19) caused by infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to an international thrust to study pathogenesis ...and evaluate interventions. Experimental infection of hamsters and the resulting respiratory disease is one of the preferred animal models since clinical signs of disease and virus shedding are similar to more severe cases of human COVID-19. The main route of challenge has been direct inoculation of the virus via the intranasal route. To resemble the natural infection, we designed a bespoke natural transmission cage system to assess whether recipient animals housed in physically separate adjacent cages could become infected from a challenged donor animal in a central cage, with equal airflow across the two side cages. To optimise viral shedding in the donor animals, a low and moderate challenge dose were compared after direct intranasal challenge, but similar viral shedding responses were observed and no discernible difference in kinetics. The results from our natural transmission set-up demonstrate that most recipient hamsters are infected within the system developed, with variation in the kinetics and levels of disease between individual animals. Common clinical outputs used for the assessment in directly-challenged hamsters, such as weight loss, are less obvious in hamsters who become infected from naturally acquiring the infection. The results demonstrate the utility of a natural transmission model for further work on assessing the differences between virus strains and evaluating interventions using a challenge system which more closely resembles human infection.
Crimean-Congo hemorrhagic fever (CCHF) is a serious disease with a high fatality rate reported in many countries. The first case of CCHF in Oman was detected in 1995 and serosurveys have suggested ...widespread infection of humans and livestock throughout the country.
Cases of CCHF reported to the Ministry of Health (MoH) of Oman between 1995 and 2017 were retrospectively reviewed. Diagnosis was confirmed by serology and/or molecular tests in Oman. Stored RNA from recent cases was studied by sequencing the complete open reading frame (ORF) of the viral S segment at Public Health England, enabling phylogenetic comparisons to be made with other S segments of strains obtained from the region.
Of 88 cases of CCHF, 4 were sporadic in 1995 and 1996, then none were detected until 2011. From 2011-2017, incidence has steadily increased and 19 (23.8%) of 80 cases clustered around Eid Al Adha. The median (range) age was 33 (15-68) years and 79 (90%) were male. The major risk for infection was contact with animals and/or butchering in 73/88 (83%) and only one case was related to tick bites alone. Severe cases were over-represented: 64 (72.7%) had a platelet count < 50 x 109/L and 32 (36.4%) died. There was no intrafamilial spread or healthcare-associated infection. The viral S segments from 11 patients presenting in 2013 and 2014 were all grouped in Asia 1 (IV) lineage.
CCHF is well-established throughout Oman, with a single strain of virus present for at least 20 years. Most patients are men involved in animal husbandry and butchery. The high mortality suggests that there is substantial under-diagnosis of milder cases. Preventive measures have been introduced to reduce risks of transmission to animal handlers and butchers and to maintain safety in healthcare settings.
The Gn subcomponent of the Gn-Gc assembly that envelopes the human and animal pathogen, Rift Valley fever virus (RVFV), is a primary target of the neutralizing antibody response. To better understand ...the molecular basis for immune recognition, we raised a class of neutralizing monoclonal antibodies (nAbs) against RVFV Gn, which exhibited protective efficacy in a mouse infection model. Structural characterization revealed that these nAbs were directed to the membrane-distal domain of RVFV Gn and likely prevented virus entry into a host cell by blocking fusogenic rearrangements of the Gn-Gc lattice. Genome sequence analysis confirmed that this region of the RVFV Gn-Gc assembly was under selective pressure and constituted a site of vulnerability on the virion surface. These data provide a blueprint for the rational design of immunotherapeutics and vaccines capable of preventing RVFV infection and a model for understanding Ab-mediated neutralization of bunyaviruses more generally.
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•The Gn glycoprotein of Rift Valley fever virus elicits potent neutralizing antibodies•Derived a class of monoclonal antibodies that protects in an animal model•A distinct region on RVFV Gn constitutes a key site of vulnerability•Antibodies are predicted to prevent exposure of viral fusion loops
Allen et al. reveal a molecular basis of antibody-mediated neutralization of Rift Valley fever virus, an important human and animal pathogen. They isolate and demonstrate the protective efficacy of a monoclonal antibody in a murine model of virus infection, providing a blueprint for rational therapeutic and vaccine design.
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