Interferon-stimulated gene (ISG)-expression screening reveals the specific antibunyaviral activity of ISG20

Bunyaviruses pose a significant threat to human health, prosperity and food security. In response to viral infections, interferons (IFNs) upregulate the expression of hundreds of interferon stimulated genes (ISGs) whose cumulative action can potently inhibit the replication of bunyaviruses. We used a flow cytometry-based method to screen the ability of ∼500 unique ISGs from humans and rhesus macaques to inhibit the replication of Bunyamwera orthobunyavirus (BUNV), the prototype of both the Peribunyaviridae family and Bunyavirales order. Candidates possessing antibunyaviral activity were further examined using a panel of divergent bunyaviruses. Interestingly, one candidate, ISG20, exhibited potent antibunyaviral activity against most viruses examined from the Peribunyaviridae, Hantaviridae and Nairoviridae families, whereas phleboviruses (Phenuiviridae) largely escaped inhibition. Similar to other viruses known to be targeted by ISG20, the antibunyaviral activity of ISG20 is dependent upon its functional ribonuclease activity. Through use of an infectious VLP assay (based on the BUNV minigenome system), we confirmed that gene expression from all 3 viral segments is strongly inhibited by ISG20. Using in vitro evolution, we generated a substantially ISG20-resistant BUNV and mapped the determinants of ISG20 sensitivity/resistance. Taken together, we report that ISG20 is a broad and potent antibunyaviral factor yet some bunyaviruses are remarkably ISG20 resistant. Thus, ISG20 sensitivity/resistance could influence the pathogenesis of bunyaviruses, many of which are emerging viruses of clinical or veterinary significance

Pseudotyping of VSV with Ebola virus glycoprotein is superior to HIV-1 for the assessment of neutralising antibodies

Ebola virus (EBOV) is an enveloped, single-stranded RNA virus that can cause Ebola virus disease (EVD). It is thought that EVD survivors are protected against subsequent infection with EBOV and that neutralising antibodies to the viral surface glycoprotein (GP) are potential correlates of protection. Serological studies are vital to assess neutralising antibodies targeted to EBOV GP; however, handling of EBOV is limited to containment level 4 laboratories. Pseudotyped viruses can be used as alternatives to live viruses, which require high levels of bio-containment, in serological and viral entry assays. However, neutralisation capacity can differ among pseudotyped virus platforms. We evaluated the suitability of EBOV GP pseudotyped human immunodeficiency virus type 1 (HIV-1) and vesicular stomatitis virus (VSV) to measure the neutralising ability of plasma from EVD survivors, when compared to results from a live EBOV neutralisation assay. The sensitivity, specificity and correlation with live EBOV neutralisation were greater for the VSV-based pseudotyped virus system, which is particularly important when evaluating EBOV vaccine responses and immuno-therapeutics. Therefore, the EBOV GP pseudotyped VSV neutralisation assay reported here could be used to provide a better understanding of the putative correlates of protection against EBOV

Characterization and applications of a Crimean-Congo hemorrhagic fever virus nucleoprotein-specific Affimer: Inhibitory effects in viral replication and development of colorimetric diagnostic tests.

peer reviewedCrimean-Congo hemorrhagic fever orthonairovirus (CCHFV) is one of the most widespread medically important arboviruses, causing human infections that result in mortality rates of up to 60%. We describe the selection of a high-affinity small protein (Affimer-NP) that binds specifically to the nucleoprotein (NP) of CCHFV. We demonstrate the interference of Affimer-NP in the RNA-binding function of CCHFV NP using fluorescence anisotropy, and its inhibitory effects on CCHFV gene expression in mammalian cells using a mini-genome system. Solution of the crystallographic structure of the complex formed by these two molecules at 2.84 Å resolution revealed the structural basis for this interference, with the Affimer-NP binding site positioned at the critical NP oligomerization interface. Finally, we validate the in vitro application of Affimer-NP for the development of enzyme-linked immunosorbent and lateral flow assays, presenting the first published point-of-care format test able to detect recombinant CCHFV NP in spiked human and animal sera

A novel member of the let-7 microRNA family is associated with developmental transitions in filarial nematode parasites

Background: Filarial nematodes are important pathogens in the tropics transmitted to humans via the bite of blood sucking arthropod vectors. The molecular mechanisms underpinning survival and differentiation of these parasites following transmission are poorly understood. microRNAs are small non-coding RNA molecules that regulate target mRNAs and we set out to investigate whether they play a role in the infection event. Results: microRNAs differentially expressed during the early post-infective stages of Brugia pahangi L3 were identified by microarray analysis. One of these, bpa-miR-5364, was selected for further study as it is upregulated ~12-fold at 24 hours post-infection, is specific to clade III nematodes, and is a novel member of the let-7 family, which are known to have key developmental functions in the free-living nematode Caenorhabditis elegans. Predicted mRNA targets of bpa-miR-5364 were identified using bioinformatics and comparative genomics approaches that relied on the conservation of miR-5364 binding sites in the orthologous mRNAs of other filarial nematodes. Finally, we confirmed the interaction between bpa-miR-5364 and three of its predicted targets using a dual luciferase assay. Conclusions: These data provide new insight into the molecular mechanisms underpinning the transmission of third stage larvae of filarial nematodes from vector to mammal. This study is the first to identify parasitic nematode mRNAs that are verified targets of specific microRNAs and demonstrates that post-transcriptional control of gene expression via stage-specific expression of microRNAs may be important in the success of filarial infection

Detection of Crimean-Congo Haemorrhagic Fever cases in a severe undifferentiated febrile illness outbreak in the Federal Republic of Sudan: A retrospective epidemiological and diagnostic cohort study

BACKGROUND: Undifferentiated febrile illness (UFI) is one of the most common reasons for people seeking healthcare in low-income countries. While illness and death due to specific infections such as malaria are often well-quantified, others are frequently uncounted and their impact underappreciated. A number of high consequence infectious diseases, including Ebola virus, are endemic or epidemic in the Federal Republic of Sudan which has experienced at least 12 UFI outbreaks, frequently associated with haemorrhage and high case fatality rates (CFR), since 2012. One of these occurred in Darfur in 2015/2016 with 594 cases and 108 deaths (CFR 18.2%). The aetiology of these outbreaks remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: We report a retrospective cohort study of the 2015/2016 Darfur outbreak, using a subset of 65 of 263 outbreak samples received by the National Public Health Laboratory which met selection criteria of sufficient sample volume and epidemiological data. Clinical features included fever (95.8%), bleeding (95.7%), headache (51.6%) and arthralgia (42.2%). No epidemiological patterns indicative of person-to-person transmission or health-worker cases were reported. Samples were tested at the Public Health England Rare and Imported Pathogens Laboratory using a bespoke panel of likely pathogens including haemorrhagic fever viruses, arboviruses and Rickettsia, Leptospira and Borrelia spp. Seven (11%) were positive for Crimean-Congo haemorrhagic fever virus (CCHFV) by real-time reverse transcription PCR. The remaining samples tested negative on all assays. CONCLUSIONS/SIGNIFICANCE: CCHFV is an important cause of fever and haemorrhage in Darfur, but not the sole major source of UFI outbreaks in Sudan. Prospective studies are needed to explore other aetiologies, including novel pathogens. The presence of CCHFV has critical infection, prevention and control as well as clinical implications for future response. Our study reinforces the need to boost surveillance, lab and investigative capacity to underpin effective response, and for local and international health security

Dimethyl fumarate in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Dimethyl fumarate (DMF) inhibits inflammasome-mediated inflammation and has been proposed as a treatment for patients hospitalised with COVID-19. This randomised, controlled, open-label platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing multiple treatments in patients hospitalised for COVID-19 (NCT04381936, ISRCTN50189673). In this assessment of DMF performed at 27 UK hospitals, adults were randomly allocated (1:1) to either usual standard of care alone or usual standard of care plus DMF. The primary outcome was clinical status on day 5 measured on a seven-point ordinal scale. Secondary outcomes were time to sustained improvement in clinical status, time to discharge, day 5 peripheral blood oxygenation, day 5 C-reactive protein, and improvement in day 10 clinical status. Between 2 March 2021 and 18 November 2021, 713 patients were enroled in the DMF evaluation, of whom 356 were randomly allocated to receive usual care plus DMF, and 357 to usual care alone. 95% of patients received corticosteroids as part of routine care. There was no evidence of a beneficial effect of DMF on clinical status at day 5 (common odds ratio of unfavourable outcome 1.12; 95% CI 0.86-1.47; p = 0.40). There was no significant effect of DMF on any secondary outcome

Dimethyl fumarate in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Dimethyl fumarate (DMF) inhibits inflammasome-mediated inflammation and has been proposed as a treatment for patients hospitalised with COVID-19. This randomised, controlled, open-label platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing multiple treatments in patients hospitalised for COVID-19 (NCT04381936, ISRCTN50189673). In this assessment of DMF performed at 27 UK hospitals, adults were randomly allocated (1:1) to either usual standard of care alone or usual standard of care plus DMF. The primary outcome was clinical status on day 5 measured on a seven-point ordinal scale. Secondary outcomes were time to sustained improvement in clinical status, time to discharge, day 5 peripheral blood oxygenation, day 5 C-reactive protein, and improvement in day 10 clinical status. Between 2 March 2021 and 18 November 2021, 713 patients were enroled in the DMF evaluation, of whom 356 were randomly allocated to receive usual care plus DMF, and 357 to usual care alone. 95% of patients received corticosteroids as part of routine care. There was no evidence of a beneficial effect of DMF on clinical status at day 5 (common odds ratio of unfavourable outcome 1.12; 95% CI 0.86-1.47; p = 0.40). There was no significant effect of DMF on any secondary outcome

A flexible format LAMP assay for rapid detection of Ebola virus.

BackgroundThe 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.Methodology/principle findingsA 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.Conclusion/significanceThese 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

Cellular immune response to human influenza viruses differs between H1N1 and H3N2 subtypes in the ferret lung.

Seasonal influenza virus infections cause yearly epidemics which are the source of a significant public health burden worldwide. The ferret model for human influenza A virus (IAV) is widely used and has several advantages over other animal models such as comparable symptomology, similar receptor distribution in the respiratory tract to humans and the ability to be infected with human isolates without the need for adaptation. However, a major disadvantage of the model has been a paucity of reagents for the evaluation of the cellular immune response. Investigation of T-cell mediated immunity in ferrets is crucial to vaccine development and efficacy studies. In this study we have used commercially produced antibodies to ferret interferon gamma (IFN-γ) allowing us to reliably measure influenza-specific IFN-γ as a marker of the cellular immune response using both enzyme-linked immunospot (ELISpot) and enzyme-linked immunosorbent (ELISA) techniques. Here we demonstrate the application of these tools to evaluate cellular immunity in ferrets infected with clinically relevant seasonal H1N1 and H3N2 IAV subtypes at equivalent doses. Using small heparinised blood samples we were able to observe the longitudinal influenza-specific IFN-γ responses of ferrets infected with both seasonal subtypes of IAV and found a notable increase in influenza-specific IFN-γ responses in circulating peripheral blood within 8 days post-infection. Both seasonal strains caused a well-defined pattern of influenza-specific IFN-γ responses in infected ferrets when compared to naïve animals. Additionally, we found that while the influenza specific IFN-γ responses found in peripheral circulating blood were comparable between subtypes, the influenza specific IFN-γ responses found in lung lymphocytes significantly differed. Our results suggest that there is a distinct difference between the ability of the two seasonal influenza strains to establish an infection in the lung of ferrets associated with distinct signatures of acquired immunity