Longitudinal quantitative assessment of TMEV-IDD-induced MS phenotypes in two inbred mouse strains using automated video tracking technology.

Multiple sclerosis (MS) is a chronic disabling disease of the central nervous system affecting over 2.5 million people worldwide. Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) is a murine model that reproduces the progressive form of MS and serves as a reference model for studying virus-induced demyelination. Certain mouse strains such as SJL are highly susceptible to this virus and serve as a prototype strain for studying TMEV infection. Other strains such as SWR are also susceptible, but their disease course following TMEV infection differs from SJL's. The quantification of motor and behavioral deficits following the induction of TMEV-IDD could help identify the differences between the two strains. Motor deficits have commonly been measured with the rotarod apparatus, but a multicomponent assessment tool has so far been lacking. For that purpose, we present a novel way of quantifying locomotor deficits, gait alterations and behavioral changes in this well-established mouse model of multiple sclerosis by employing automated video analysis technology (The PhenoTyper, Noldus Information Technology). We followed 12 SJL and 12 SWR female mice and their mock-infected counterparts over a period of 9 months following TMEV-IDD induction. We demonstrated that SJL and SWR mice both suffer significant gait alterations and reduced exploration following TMEV infection. However, SJL mice also display an earlier and more severe decline in spontaneous locomotion, especially in velocity, as well as in overall activity. Maintenance behaviors such as eating and grooming are not affected in either of the two strains. The system also showed differences in mock-infected mice from both strains, highlighting an age-related decline in spontaneous locomotion in the SJL strain, as opposed to hyperactivity in the SWR strain. Our study confirms that this automated video tracking system can reliably track the progression of TMEV-IDD for 9 months. We have also shown how this system can be utilized for longitudinal phenotyping in mice by describing useful parameters that quantify locomotion, gait and behavior.

Benzene with Alkyl Chains Is a Universal Scaffold for Multivalent Virucidal Antivirals.

Most viruses start their invasion by binding to glycoproteins' moieties on the cell surface (heparan sulfate proteoglycans [HSPG] or sialic acid [SA]). Antivirals mimicking these moieties multivalently are known as broad-spectrum multivalent entry inhibitors (MEI). Due to their reversible mechanism, efficacy is lost when concentrations fall below an inhibitory threshold. To overcome this limitation, we modify MEIs with hydrophobic arms rendering the inhibitory mechanism irreversible, i.e., preventing the efficacy loss upon dilution. However, all our HSPG-mimicking MEIs only showed reversible inhibition against HSPG-binding SARS-CoV-2. Here, we present a systematic investigation of a series of small molecules, all containing a core and multiple hydrophobic arms terminated with HSPG-mimicking moieties. We identify the ones that have irreversible inhibition against all viruses including SARS-CoV-2 and discuss their design principles. We show efficacy in vivo against SARS-CoV-2 in a Syrian hamster model through both intranasal instillation and aerosol inhalation in a therapeutic setting (12 h postinfection). We also show the utility of the presented design rules in producing SA-mimicking MEIs with irreversible inhibition against SA-binding influenza viruses.

Dengue and chikungunya: future threats for Northern Europe?

Arthropod-borne viral diseases are likely to be affected by the consequences of climate change with an increase in their distribution and intensity. Among these infectious diseases, chikungunya and dengue viruses are two (re)emergent arboviruses transmitted by Aedes species mosquitoes and which have recently demonstrated their capacity for rapid expansion. They most often cause mild diseases, but they can both be associated with complications and severe forms. In Europe, following the establishment of invasive Aedes spp, the first outbreaks of autochtonous dengue and chikungunya have already occurred. Northern Europe is currently relatively spared, but climatic projections show that the conditions are permissive for the establishment of Aedes albopictus (also known as the tiger mosquito) in the coming decades. It is therefore essential to question and improve the means of surveillance in northern Europe, at the dawn of inevitable future epidemics.

Evaluation of Non-Vector Transmission of Usutu Virus in Domestic Canaries (Serinus canaria).

Usutu virus (USUV) is a flavivirus transmitted to avian species through mosquito bites that causes mass mortalities in wild and captive bird populations. However, several cases of positive dead birds have been recorded during the winter, a vector-free period. To explain how USUV "overwinters", the main hypothesis is bird-to-bird transmission, as shown for the closely related West Nile virus. To address this question, we experimentally challenged canaries with intranasal inoculation of USUV, which led to systemic dissemination of the virus, provided the inoculated dose was sufficient (>102 TCID50). We also highlighted the oronasal excretion of infectious viral particles in infected birds. Next, we co-housed infected birds with naive sentinels, to determine whether onward transmission could be reproduced experimentally. We failed to detect such transmission but demonstrated horizontal transmission by transferring sputum from an infected to a naive canary. In addition, we evaluated the cellular tropism of respiratory mucosa to USUV in vitro using a canary tracheal explant and observed only limited evidence of viral replication. Further research is then needed to assess if and how comparable bird-to-bird transmission occurs in the wild.

Convalescent Plasma for Covid-19-Induced ARDS in Mechanically Ventilated Patients.

BACKGROUND: Passive immunization with plasma collected from convalescent patients has been regularly used to treat coronavirus disease 2019 (Covid-19). Minimal data are available regarding the use of convalescent plasma in patients with Covid-19-induced acute respiratory distress syndrome (ARDS). METHODS: In this open-label trial, we randomly assigned adult patients with Covid-19-induced ARDS who had been receiving invasive mechanical ventilation for less than 5 days in a 1:1 ratio to receive either convalescent plasma with a neutralizing antibody titer of at least 1:320 or standard care alone. Randomization was stratified according to the time from tracheal intubation to inclusion. The primary outcome was death by day 28. RESULTS: A total of 475 patients underwent randomization from September 2020 through March 2022. Overall, 237 patients were assigned to receive convalescent plasma and 238 to receive standard care. Owing to a shortage of convalescent plasma, a neutralizing antibody titer of 1:160 was administered to 17.7% of the patients in the convalescent-plasma group. Glucocorticoids were administered to 466 patients (98.1%). At day 28, mortality was 35.4% in the convalescent-plasma group and 45.0% in the standard-care group (P = 0.03). In a prespecified analysis, this effect was observed mainly in patients who underwent randomization 48 hours or less after the initiation of invasive mechanical ventilation. Serious adverse events did not differ substantially between the two groups. CONCLUSIONS: The administration of plasma collected from convalescent donors with a neutralizing antibody titer of at least 1:160 to patients with Covid-19-induced ARDS within 5 days after the initiation of invasive mechanical ventilation significantly reduced mortality at day 28. This effect was mainly observed in patients who underwent randomization 48 hours or less after ventilation initiation. (Funded by the Belgian Health Care Knowledge Center; ClinicalTrials.gov number, NCT04558476.).

A timely, user-friendly, and flexible marker-assisted speed congenics method.

Mice are the most widely used mammalian animal model worldwide. Their use presents many advantages, including our ability to manipulate their genome. Unfortunately, transgenic mice often need to be introgressed to transfer the transgene of interest in a specific mouse line. This time-consuming process can be shortened using the speed congenics technique. However, the need for a panel of informative markers to evaluate the proportion of donor and receiver genomes in different individuals produced at each generation hinders the utilisation of speed congenics. In this study, we present 255 microsatellites and 10 RFLPs which can be used in 18 marker panels, allowing the easy and fast introgression of genes of interest from three mouse lines commonly used for transgenesis (C57BL/6, 129/Sv and FVB) to six mouse lines relevant for biomedical research (BALB/c, C3H, DBA/1, DBA/2, SJL and SWR/J). In addition, our markers analysis confirmed a recently described lack of isogeny in well-established inbred mouse lines available from commercial breeders.

Cluster Analysis Identifies Distinct Patterns of T-Cell and Humoral Immune Responses Evolution Following a Third Dose of SARS-CoV-2 Vaccine in People Living with HIV.

(1) Background: Many vaccines require higher, additional doses or adjuvants to provide adequate protection for people living with HIV (PLWH). Despite their potential risk of severe coronavirus disease 2019, immunological data remain sparse, and a clear consensus for the best booster strategy is lacking. (2) Methods: Using the data obtained from our previous study assessing prospective T-cell and humoral immune responses before and after administration of a third dose of SARS-CoV-2 vaccine, we assessed the correlations between immune parameters reflecting humoral and cellular immune responses. We further aimed at identifying distinct clusters of patients with similar patterns of immune response evolution to determine how these relate to demographic and clinical factors. (3) Results: Among 80 PLWH and 51 healthcare workers (HCWs) enrolled in the study, cluster analysis identified four distinct patterns of evolution characterised by specific immune patterns and clinical factors. We observed that immune responses appeared to be less robust in cluster A, whose individuals were mostly PLWH who had never been infected with SARS-CoV-2. Cluster C, whose individuals showed a particularly drastic increase in markers of humoral immune response following the third dose of vaccine, was mainly composed of female participants who experienced SARS-CoV-2. Regarding the correlation study, although we observed a strong positive correlation between markers mirroring humoral immune response, markers of T-cell response following vaccination correlated only in a lesser extent with markers of humoral immunity. This suggests that neutralising antibody titers alone are not always a reliable reflection of the magnitude of the whole immune response. (4) Conclusions: Our findings show heterogeneity in immune responses among SARS-CoV-2 vaccinated PLWH. Specific subgroups could therefore benefit from distinct immunization strategies. Prior or breakthrough natural infection enhances the activity of vaccines and must be taken into account for informing global vaccine strategies among PLWH, even those with a viro-immunologically controlled infection.

Central role of lung macrophages in SARS-CoV-2 physiopathology: a cross-model single-cell RNA-seq perspective.

Cytokine storms are considered a driving factor in coronavirus disease 2019 (COVID-19) severity. However, the triggering and resolution of this cytokine production, as well as the link between this phenomenon and infected cells, are still poorly understood. In this study, a cross-species scRNA-seq analysis showed that cytokine-producing macrophages together with pneumocytes were found to be the main contributors of viral transcripts in both Syrian hamsters and African green monkeys. Whatever the cell type, viral read-bearing cells show an apoptotic phenotype. A comparison of SARS-CoV-2 entry receptor candidates showed that Fc receptors are better correlated with infected cells than ACE2, NRP1, or AXL. Although both species show similar interferon responses, differences in adaptive immunity were highlighted. Lastly, Fc receptor and cytokine upregulation in M1 macrophages was found to correlate with a comprehensive interferon response. Based on these results, we propose a model in which lung macrophages play a central role in COVID-19 severity through antibody-dependent enhancement.

Anti-Schmallenberg Virus Activities of Type I/III Interferons-Induced Mx1 GTPases from Different Mammalian Species.

Mx proteins are key factors of the innate intracellular defense mechanisms that act against viruses induced by type I/III interferons. The family Peribunyaviridae includes many viruses of veterinary importance, either because infection results in clinical disease or because animals serve as reservoirs for arthropod vectors. According to the evolutionary arms race hypothesis, evolutionary pressures should have led to the selection of the most appropriate Mx1 antiviral isoforms to resist these infections. Although human, mouse, bat, rat, and cotton rat Mx isoforms have been shown to inhibit different members of the Peribunyaviridae, the possible antiviral function of the Mx isoforms from domestic animals against bunyaviral infections has, to our knowledge, never been studied. Herein, we investigated the anti-Schmallenberg virus activity of bovine, canine, equine, and porcine Mx1 proteins. We concluded that Mx1 has a strong, dose-dependent anti-Schmallenberg activity in these four mammalian species.

Management of a Focal Introduction of ASF Virus in Wild Boar: The Belgian Experience.

African swine fever (ASF) is a fatal disease of suids that was detected in wild boar in Belgium in September 2018. The measures implemented to stop the spread and eliminate the African swine fever virus consisted of creating restriction zones, organising efficient search and removal of carcasses, constructing wire fences, and depopulating wild boar in the area surrounding the infected zone. The ASF management zone included the infected and the white zones and covered 1106 km² from which 7077 wild boar have been removed. A total of 5338 wild boars have been qPCR-tested and 833 have been detected ASF-positive. The search effort amounted to 60,631 h with a main focus on the infected zone (88%). A total of 277 km of fences have been set up. The main cause of mortality in the infected zone was the virus itself, while hunting, trapping, and night shooting were used together to reduce the wild boar density in the surrounding white zones. After continuous dispersion of the virus until March 2019, the epidemic wave stopped, and the last fresh positive case was discovered in August 2019. Hence, Belgium was declared free of the disease in November 2020.

Eye Infection with SARS-CoV-2 as a Route to Systemic Immunization?

Infectious diseases of the conjunctiva and cornea usually leave behind both broad local and systemic immunity. Case reports of SARS-CoV-2-positive conjunctivitis with subsequent systemic immunity suggest a new route of immunization preventing the primary infection of the airways. MATERIAL AND METHODS: A total of 24 Syrian field hamsters were treated. In systematic animal experiments, we infected the eyes of n = 8 animals (group 1) and the airways of another n = 8 animals (group 2) with SARS-CoV-2 (Wuhan type); n = 8 hamsters served as controls (group 3). The weight development of the animals was recorded. After two weeks of observation of disease symptoms, all animals were re-exposed to SARS-CoV-2 in the respiratory tract (challenge) to determine whether immunity to the virus had been achieved. RESULTS: The epi-ocularly infected animals (group 1) showed no clinically visible disease during the ocular infection phase. At most, there was a slightly reduced weight gain compared to the control group (group 3), while the respiratory infected animals (group 2) all lost weight, became lethargic, and slowly recovered after two weeks. After the challenge, none of the animals in groups 1 and 2 became ill again. The animals in the negative control (group 3) all became ill. Cytotoxic antibodies were detectable in the blood of the infected groups before and after challenge, with higher titers in the epi-ocularly infected animals. CONCLUSION: By epi-ocular infection with SARS-CoV-2, the development of systemic immunity with formation of cytotoxic antibodies without severe general disease could be observed in the experimental animals, which did not induce any more disease upon a second infection in the respiratory tract. Therefore, it can be concluded that a purely epi-ocular infection with SARS-CoV2 only induces a weak disease pattern followed by systemic immunity.

Kinetics and Persistence of the Cellular and Humoral Immune Responses to BNT162b2 mRNA Vaccine in SARS-CoV-2-Naive and -Experienced Subjects: Impact of Booster Dose and Breakthrough Infections.

Background: Understanding and measuring the individual level of immune protection and its persistence at both humoral and cellular levels after SARS-CoV-2 vaccination is mandatory for the management of the vaccination booster campaign. Our prospective study was designed to assess the immunogenicity of the BNT162b2 mRNA vaccine in triggering the cellular and humoral immune response in healthcare workers up to 12 months after the initial vaccination, with one additional boosting dose between 6 and 12 months. Methods: This prospective study enrolled 208 healthcare workers (HCWs) from the Liège University Hospital (CHU) of Liège in Belgium. Participants received two doses of BioNTech/Pfizer COVID-19 vaccine (BNT162b2) and a booster dose 6-12 months later. Fifty participants were SARS-CoV-2 experienced and 158 were naïve before the vaccination. Blood sampling was performed at the day of the first (T0) and second (T1) vaccine doses administration, then at 2 weeks (T2), 4 weeks (T3), 6 months (T4) and 12 months (T5) after the second dose. Between T4 and T5, participants also got the third boosting vaccine dose. A total of 1145 blood samples were collected. All samples were tested for the presence of anti-Spike antibodies, using the DiaSorin LIAISON SARS-CoV-2 Trimeric S IgG assay, and for anti-Nucleocapsid antibodies, using Elecsys anti-SARS-CoV-2 assay​​. Neutralizing antibodies against the SARS-CoV-2 Wuhan-like variant strain were quantified in all samples using a Vero E6 cell-based neutralization assay. Cell-mediated immune response was evaluated at T4 and T5 on 80 and 55 participants, respectively, by measuring the secretion of IFN-γ on peripheral blood lymphocytes using the QuantiFERON Human IFN-γ SARS-CoV-2, from Qiagen. We analyzed separately the naïve and experienced participants. Findings: We found that anti-spike antibodies and neutralization capacity levels were significantly higher in SARS-CoV-2 experienced HCWs compared to naïve HCWs at all time points analyzed except the one after boosting dose. Cellular immune response was also higher in experienced HCWs six months following vaccination. Besides the impact of SARS-CoV-2 infection history on immune response to BNT162b2 mRNA vaccine, we observed a significant negative association between age and persistence of humoral response. The booster dose induced an increase in humoral and cellular immune responses, particularly in naive individuals. Breakthrough infections resulted in higher cellular and humoral responses after the booster dose. Conclusions: Our data strengthen previous findings demonstrating that immunization through vaccination combined with natural infection is better than 2 vaccine doses immunization or natural infection alone. The benefit of the booster dose was greater in naive individuals. It may have implications for personalizing mRNA vaccination regimens used to prevent severe COVID-19 and reduce the impact of the pandemic on the healthcare system. More specifically, it may help prioritizing vaccination, including for the deployment of booster doses.

Gammaherpesvirus Alters Alveolar Macrophages According to the Host Genetic Background and Promotes Beneficial Inflammatory Control over Pneumovirus Infection.

Human respiratory syncytial virus (hRSV) infection brings a wide spectrum of clinical outcomes, from a mild cold to severe bronchiolitis or even acute interstitial pneumonia. Among the known factors influencing this clinical diversity, genetic background has often been mentioned. In parallel, recent evidence has also pointed out that an early infectious experience affects heterologous infections severity. Here, we analyzed the importance of these two host-related factors in shaping the immune response in pneumoviral disease. We show that a prior gammaherpesvirus infection improves, in a genetic background-dependent manner, the immune system response against a subsequent lethal dose of pneumovirus primary infection notably by inducing a systematic expansion of the CD8+ bystander cell pool and by modifying the resident alveolar macrophages (AMs) phenotype to induce immediate cyto/chemokinic responses upon pneumovirus exposure, thereby drastically attenuating the host inflammatory response without affecting viral replication. Moreover, we show that these AMs present similar rapid and increased production of neutrophil chemokines both in front of pneumoviral or bacterial challenge, confirming recent studies attributing a critical antibacterial role of primed AMs. These results corroborate other recent studies suggesting that the innate immunity cells are themselves capable of memory, a capacity hitherto reserved for acquired immunity.

University population-based prospective cohort study of SARS-CoV-2 infection and immunity (SARSSURV-ULiège): a study protocol.

INTRODUCTION: For a safe and sustainable return to normal functioning of academic activities in higher education, objective-driven testing strategies that are flexible and rapidly adaptable are essential to effectively monitor and respond to new developments of the COVID-19 pandemic. To date, prospective longitudinal research on SARS-CoV-2 antibody testing in saliva and seroprevalence in higher education contexts is substantially lacking, limiting our understanding of COVID-19 prevalence, incidence and nature of the immune response to SARS-CoV-2 at various stages of the infection and vaccination. To address this lack of evidence, a prospective population-based cohort study (SARSSURV-ULiège) has recently been started. METHODS AND ANALYSIS: Students (n=1396) and staff members (n=1143) of the University of Liège are followed up over more than 1 year. All participants are required to complete anamnestic, clinical and vaccine hesitancy questionnaires for medical histories and undertaken treatments. Previous proven or suspected SARS-CoV-2 infection is also registered. In phase 1, weekly saliva samples to perform RT-qPCR to detect SARS-CoV-2 and monthly COVID-19 serological rapid test results are collected. Once being positive to either saliva RT-qPCR assay for SARS-CoV-2 presence or to serological test, the participant is invited to enter phase 2. If participants get vaccinated during the study period, they are invited to phase 2. In this second phase, besides weekly saliva self-test, depending on the participants' profiles, both gargle and blood samples are collected to obtain various biological data to measure the presence of neutralising antibodies against SARS-CoV-2, determine the magnitude and the duration of antibody responses over time. ETHICS AND DISSEMINATION: The study has received the approval from the University Hospital of Liège Ethics Committee (reference number 2021/96, dated 26 March 2021). Potential protocol amendments will be presented to the Research Ethics Committee. The findings of the present study will be presented at scientific conferences and the results published in peer-review publications. Weekly reports will be submitted to the risk assessment group and the risk management group against COVID-19 of the university to enable a timely public health action if necessary.

Schmallenberg virus, cyclical reemergence in the core region: A seroepidemiologic study in wild cervids, Belgium, 2012-2017.

Schmallenberg virus emerged in 2011 in Europe. The epicentre of primordial spreading was the region straddling Germany, the Netherlands and Belgium. One of the key questions is whether the newcomer would establish a lasting presence on the continent. The apparent seroprevalence in southern Belgium wild deer populations was followed for 6 years. Two years of intense circulation were revealed, 2012 and 2016, characterized by a peak seroprevalence in the two studied populations (Capreolus capreolus and Cervus elaphus). Between the peak years and after 2016, apparent seroprevalences declined rapidly among adults and became nil among juveniles. The general pattern of apparent seroprevalence evolution observed is consistent with a cyclic circulation of Schmallenberg virus, similar to what is observed for other Orthobunyaviruses in endemic areas. These data also suggest that wild cervids play no central role in the circulation dynamics of the virus.

SARS-CoV-2 neutralising antibody testing in Europe: towards harmonisation of neutralising antibody titres for better use of convalescent plasma and comparability of trial data.

We compared the performance of SARS-CoV-2 neutralising antibody testing between 12 European laboratories involved in convalescent plasma trials. Raw titres differed almost 100-fold differences between laboratories when blind-testing 15 plasma samples. Calibration of titres in relation to the reference reagent and standard curve obtained by testing a dilution series reduced the inter-laboratory variability ca 10-fold. The harmonisation of neutralising antibody quantification is a vital step towards determining the protective and therapeutic levels of neutralising antibodies.