Lassa fever research priorities: towards effective medical countermeasures by the end of the decade.

In 2016, WHO designated Lassa fever a priority disease for epidemic preparedness as part of the WHO Blueprint for Action to Prevent Epidemics. One aspect of preparedness is to promote development of effective medical countermeasures (ie, diagnostics, therapeutics, and vaccines) against Lassa fever. Diagnostic testing for Lassa fever has important limitations and key advancements are needed to ensure rapid and accurate diagnosis. Additionally, the only treatment available for Lassa fever is ribavirin, but controversy exists regarding its effectiveness. Finally, no licensed vaccines are available for the prevention and control of Lassa fever. Ongoing epidemiological and behavioural studies are also crucial in providing actionable information for medical countermeasure development, use, and effectiveness in preventing and treating Lassa fever. This Personal View provides current research priorities for development of Lassa fever medical countermeasures based on literature published primarily in the last 5 years and consensus opinion of 20 subject matter experts with broad experience in public health or the development of diagnostics, therapeutics, and vaccines for Lassa fever. These priorities provide an important framework to ensure that Lassa fever medical countermeasures are developed and readily available for use in endemic and at-risk areas by the end of the decade.

Measures to prevent and treat Nipah virus disease: research priorities for 2024-29.

Nipah virus causes highly lethal disease, with case-fatality rates ranging from 40% to 100% in recognised outbreaks. No treatments or licensed vaccines are currently available for the prevention and control of Nipah virus infection. In 2019, WHO published an advanced draft of a research and development roadmap for accelerating development of medical countermeasures, including diagnostics, therapeutics, and vaccines, to enable effective and timely emergency response to Nipah virus outbreaks. This Personal View provides an update to the WHO roadmap by defining current research priorities for development of Nipah virus medical countermeasures, based primarily on literature published in the last 5 years and consensus opinion of 15 subject matter experts with broad experience in development of medical countermeasures for Nipah virus or experience in the epidemiology, ecology, or public health control of outbreaks of Nipah virus. The research priorities are organised into four main sections: cross-cutting issues (for those that apply to more than one category of medical countermeasures), diagnostics, therapeutics, and vaccines. The strategic goals and milestones identified in each section focus on key achievements that are needed over the next 6 years to ensure that the necessary tools are available for rapid response to future outbreaks of Nipah virus or related henipaviruses.

Increased Clinical Signs and Mortality in IFNAR(-/-) Mice Immunised with the Bluetongue Virus Outer-Capsid Proteins VP2 or VP5, after Challenge with an Attenuated Heterologous Serotype.

Bluetongue is an economically important disease of domesticated and wild ruminants caused by bluetongue virus (BTV). There are at least 36 different serotypes of BTV (the identity of which is determined by its outer-capsid protein VP2), most of which are transmitted by Culicoides biting midges. IFNAR(-/-) mice immunised with plant-expressed outer-capsid protein VP2 (rVP2) of BTV serotypes -1, -4 or -8, or the smaller outer-capsid protein rVP5 of BTV-10, or mock-immunised with PBS, were subsequently challenged with virulent strains of BTV-4 or BTV-8, or with an attenuated clone of BTV-1 (BTV-1RGC7). The mice that had received rVP2 generated a protective immune response against the homologous BTV serotype, reducing viraemia (as detected by qRT-PCR), the severity of clinical signs and mortality levels. No cross-serotype protection was observed after challenge with the heterologous BTV serotypes. However, the severity of clinical signs, viraemia and fatality levels after challenge with the attenuated strain of BTV-1 were all increased in mice immunised with rVP2 of BTV-4 and BTV-8, or with rVP5 of BTV10. The possibility is discussed that non-neutralising antibodies, reflecting serological relationships between the outer-capsid proteins of these different BTV serotypes, could lead to 'antibody-dependent enhancement of infection' (ADE). Such interactions could affect the epidemiology and emergence of different BTV strains in the field and would therefore be relevant to the design and implementation of vaccination campaigns.

The immune response to lumpy skin disease virus in cattle is influenced by inoculation route.

Lumpy skin disease virus (LSDV) causes severe disease in cattle and water buffalo and is transmitted by hematophagous arthropod vectors. Detailed information of the adaptive and innate immune response to LSDV is limited, hampering the development of tools to control the disease. This study provides an in-depth analysis of the immune responses of calves experimentally inoculated with LSDV via either needle-inoculation or arthropod-inoculation using virus-positive Stomoxys calcitrans and Aedes aegypti vectors. Seven out of seventeen needle-inoculated calves (41%) developed clinical disease characterised by multifocal necrotic cutaneous nodules. In comparison 8/10 (80%) of the arthropod-inoculated calves developed clinical disease. A variable LSDV-specific IFN-γ immune response was detected in the needle-inoculated calves from 5 days post inoculation (dpi) onwards, with no difference between clinical calves (developed cutaneous lesions) and nonclinical calves (did not develop cutaneous lesions). In contrast a robust and uniform cell-mediated immune response was detected in all eight clinical arthropod-inoculated calves, with little response detected in the two nonclinical arthropod-inoculated calves. Neutralising antibodies against LSDV were detected in all inoculated cattle from 5-7 dpi. Comparison of the production of anti-LSDV IgM and IgG antibodies revealed no difference between clinical and nonclinical needle-inoculated calves, however a strong IgM response was evident in the nonclinical arthropod-inoculated calves but absent in the clinical arthropod-inoculated calves. This suggests that early IgM production is a correlate of protection in LSD. This study presents the first evidence of differences in the immune response between clinical and nonclinical cattle and highlights the importance of using a relevant transmission model when studying LSD.

Serological Cross-Reactions between Expressed VP2 Proteins from Different Bluetongue Virus Serotypes.

Bluetongue (BT) is a severe and economically important disease of ruminants that is widely distributed around the world, caused by the bluetongue virus (BTV). More than 28 different BTV serotypes have been identified in serum neutralisation tests (SNT), which, along with geographic variants (topotypes) within each serotype, reflect differences in BTV outer-capsid protein VP2. VP2 is the primary target for neutralising antibodies, although the basis for cross-reactions and serological variations between and within BTV serotypes is poorly understood. Recombinant BTV VP2 proteins (rVP2) were expressed in Nicotiana benthamiana, based on sequence data for isolates of thirteen BTV serotypes (primarily from Europe), including three 'novel' serotypes (BTV-25, -26 and -27) and alternative topotypes of four serotypes. Cross-reactions within and between these viruses were explored using rabbit anti-rVP2 sera and post BTV-infection sheep reference-antisera, in I-ELISA (with rVP2 target antigens) and SNT (with reference strains of BTV-1 to -24, -26 and -27). Strong reactions were generally detected with homologous rVP2 proteins or virus strains/serotypes. The sheep antisera were largely serotype-specific in SNT, but more cross-reactive by ELISA. Rabbit antisera were more cross-reactive in SNT, and showed widespread, high titre cross-reactions against homologous and heterologous rVP2 proteins in ELISA. Results were analysed and visualised by antigenic cartography, showing closer relationships in some, but not all cases, between VP2 topotypes within the same serotype, and between serotypes belonging to the same 'VP2 nucleotype'.

Madin-Darby bovine kidney (MDBK) cells are a suitable cell line for the propagation and study of the bovine poxvirus lumpy skin disease virus.

Lumpy skin disease virus (LSDV) is a poxvirus that causes systemic disease in cattle, resulting in substantial economic loss to affected communities. LSDV is a rapidly emerging pathogen of growing global concern that recently spread from Africa and the Middle East into Europe and Asia, impacting the cattle population in these regions. An increase in research efforts into LSDV is required to address key knowledge gaps, however this is hampered by lack of suitable cell lines on which to propagate and study the virus. In this work we describe the replication and spread of LSDV on Madin-Darby bovine kidney (MDBK) cells, and the formation of foci-type poxvirus plaques by LSDV on MDBK cells. Methods utilising MDBK cells to quantify neutralising antibodies to LSDV, and to purify LSDV genomic DNA suitable for short read sequencing are described. These research methods broaden the tools available for LSDV researchers and will facilitate the gathering of evidence to underpin the development of LSD control and prevention programmes.

Bluetongue virus outer-capsid protein VP2 expressed in Nicotiana benthamiana raises neutralising antibodies and a protective immune response in IFNAR -/- mice.

Bluetongue is a severe, economically important disease of ruminants that is widely distributed in tropical and temperate regions around the world. It is associated with major production losses, restrictions of animal movements and trade, as well as costs associated with developing and implementing effective surveillance and control measures. Mammalian hosts infected with bluetongue virus (BTV) generate a protective neutralising antibody response targeting the major BTV outer-capsid protein and serotype-specific antigen, VP2. BTV VP2 proteins that have been expressed in plants are soluble, with a native conformation displaying neutralising epitopes and can assemble with other BTV structural proteins to form virus-like particles (VLPs). His-tagged VP2 proteins of BTV serotypes 4 and 8 were transiently expressed in Nicotiana benthamiana then purified by immobilised metal affinity chromatography (IMAC). Antisera from IFNAR -/- mice prime/boost vaccinated with the purified proteins, were shown to contain VP2-specific antibodies by Indirect ELISA (I-ELISA), western blotting and serum neutralisation tests (SNT). Vaccinated mice, subsequently challenged with either the homologous or heterologous BTV serotype, developed viraemia by day 3 post-infection. However, no clinical signs were observed in mice challenged with the homologous serotype (either prime-boost or single-shot vaccinated), all of which survived for the duration of the study. In contrast, all of the vaccinated mice challenged with a heterologous serotype, died, showing no evidence of cross-protection or suppression of viraemia, as detected by real-time RT-qPCR or virus isolation. The induction of protective, serotype-specific neutralising antibodies in IFNAR -/- mice, indicates potential for the use of plant-expressed BTV VP2s as subunit vaccine components, or as a basis for serotype-specific serological assays.