Detection of Crimean-Congo haemorrhagic fever virus in Hyalomma marginatum ticks, southern France, May 2022 and April 2023.

Crimean-Congo haemorrhagic fever (CCHF), a potentially severe zoonotic viral disease causing fever and haemorrhagic manifestations in humans. As the Crimean-Congo haemorrhagic fever virus (CCHFV) has been detected in ticks in Spain and antibodies against the virus in ruminant sera in Corsica, it was necessary to know more about the situation in France. In 2022-2023, CCHFV was detected in 155 ticks collected from horses and cattle in southern France.

Multiplex reverse transcriptase droplet digital PCR for the simultaneous quantification of four dengue serotypes: Proof of concept study.

During vaccine production, RNA from chimeric yellow fever-dengue (CYD) vaccine viruses (CYD1, CYD2, CYD3 and CYD4) is currently quantified using separate serotype-specific RT-qPCR assays. Here we describe the results from a proof-of-concept study on the development of a multiplex reverse transcriptase droplet digital PCR (RT-ddPCR) assay for simultaneous quantification of RNA for all four viruses. Serotype-specific simplex RT-ddPCRs were developed using the serotype-specific PCR systems (forward and reverse primers and FAM (fluorescent chromophores 6-carboxyfluorescein) and YY (Yakima Yellow)-labelled probes), used in the routine RT-qPCR. The PCR systems were specific and gave similar quantification results to those from the RT-qPCR assay. Linear regression analyses were used to select relative probe concentrations to obtain distinct clusters for each target RNA in a 2-D cluster plot in a multiplex RT-ddPCR assay. We showed the clusters were positioned as predicted in the model for each CYD RNA and were well separated. The multiplex RT-ddPCR gave similar quantification results to those obtained by the serotype-specific RT-qPCR assays for triplicate samples containing 7, 8 or 9 Log10 Geq/mL. In conclusion, these results demonstrate that it is possible to quantify RNA from four CYD serotypes with a multiplex RT-ddPCR assay in a single assay.

Natural History of Sudan ebolavirus to Support Medical Countermeasure Development.

Sudan ebolavirus (SUDV) is one of four members of the Ebolavirus genus known to cause Ebola Virus Disease (EVD) in humans, which is characterized by hemorrhagic fever and a high case fatality rate. While licensed therapeutics and vaccines are available in limited number to treat infections of Zaire ebolavirus, there are currently no effective licensed vaccines or therapeutics for SUDV. A well-characterized animal model of this disease is needed for the further development and testing of vaccines and therapeutics. In this study, twelve cynomolgus macaques (Macaca fascicularis) were challenged intramuscularly with 1000 PFUs of SUDV and were followed under continuous telemetric surveillance. Clinical observations, body weights, temperature, viremia, hematology, clinical chemistry, and coagulation were analyzed at timepoints throughout the study. Death from SUDV disease occurred between five and ten days after challenge at the point that each animal met the criteria for euthanasia. All animals were observed to exhibit clinical signs and lesions similar to those observed in human cases which included: viremia, fever, dehydration, reduced physical activity, macular skin rash, systemic inflammation, coagulopathy, lymphoid depletion, renal tubular necrosis, hepatocellular degeneration and necrosis. The results from this study will facilitate the future preclinical development and evaluation of vaccines and therapeutics for SUDV.

Characterization and analytical validation of a new antigenic rapid diagnostic test for Ebola virus disease detection.

Hemorrhagic fever outbreaks are difficult to diagnose and control in part because of a lack of low-cost and easily accessible diagnostic structures in countries where etiologic agents are present. Furthermore, initial clinical symptoms are common and shared with other endemic diseases such as malaria or typhoid fever. Current molecular diagnostic methods such as polymerase chain reaction require trained personnel and laboratory infrastructure, hindering diagnostics at the point of need, particularly in outbreak settings. Therefore, rapid diagnostic tests such as lateral flow can be broadly deployed and are typically well-suited to rapidly diagnose hemorrhagic fever viruses, such as Ebola virus. Early detection and control of Ebola outbreaks require simple, easy-to-use assays that can detect very low amount of virus in blood. Here, we developed and characterized an immunoassay test based on immunochromatography coupled to silver amplification technology to detect the secreted glycoprotein of EBOV. The glycoprotein is among the first viral proteins to be detected in blood. This strategy aims at identifying infected patients early following onset of symptoms by detecting low amount of sGP protein in blood samples. The limit of detection achieved by this sGP-targeted kit is 2.2 x 104 genome copies/ml in plasma as assayed in a monkey analytical cohort. Clinical performance evaluation showed a specificity of 100% and a sensitivity of 85.7% when evaluated with plasma samples from healthy controls and patients infected with Zaire Ebola virus from Macenta, Guinea. This rapid and accurate diagnostic test could therefore be used in endemic countries for early detection of infected individuals in point of care settings. Moreover, it could also support efficient clinical triage in hospitals or clinical centers and thus reducing transmission rates to prevent and better manage future severe outbreaks.