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Abstract Background The transcription/replication of the influenza viruses implicate the terminal nucleotide sequences of viral RNA, which comprise sequences at the extremities conserved among the genomic segments as well as variable 3' and 5' non-coding (NC) regions. The plasmid-based system for the in vivo reconstitution of functional ribonucleoproteins, upon expression of viral-like RNAs together with the nucleoprotein and polymerase proteins has been widely used to analyze transcription/replication of influenza viruses. It was thus shown that the type A polymerase could transcribe and replicate type A, B, or C vRNA templates whereas neither type B nor type C polymerases were able to transcribe and replicate type A templates efficiently. Here we studied the importance of the NC regions from the seven segments of type C influenza virus for efficient transcription/replication by the type A and C polymerases. Results The NC sequences of the seven genomic segments of the type C influenza virus C/Johannesburg/1/66 strain were found to be more variable in length than those of the type A and B viruses. The levels of transcription/replication of viral-like vRNAs harboring the NC sequences of the respective type C virus segments flanking the CAT reporter gene were comparable in the presence of either type C or type A polymerase complexes except for the NS and PB2-like vRNAs. For the NS-like vRNA, the transcription/replication level was higher after introduction of a U residue at position 6 in the 5' NC region as for all other segments. For the PB2-like vRNA the CAT expression level was particularly reduced with the type C polymerase. Analysis of mutants of the 5' NC sequence in the PB2-like vRNA, the shortest 5' NC sequence among the seven segments, showed that additional sequences within the PB2 ORF were essential for the efficiency of transcription but not replication by the type C polymerase complex. Conclusion In the context of a PB2-like reporter vRNA template, the sequence upstream the polyU stretch plays a role in the transcription/replication process by the type C polymerase complex.

We conducted a multicentre hospital-based test-negative case-control study to measure vaccine effectiveness (VE) against PCR-confirmed influenza in adult patients with severe acute respiratory infection (SARI) during the 2022/2023 influenza season in Europe. Among 5547 SARI patients ≥18 years, 2963 (53%) were vaccinated against influenza. Overall VE against influenza A(H1N1)pdm09 was 11% (95% CI: -23-36); 20% (95% CI: -4-39) against A(H3N2) and 56% (95% CI: 22-75) against B. During the 2022/2023 season, while VE against hospitalisation with influenza B was >55%, it was ≤20% for influenza A subtypes. While influenza vaccination should be a priority for future seasons, improved vaccines against influenza are needed., (© 2024 The Authors. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

IntroductionThe I-MOVE-COVID-19 and VEBIS hospital networks have been measuring COVID-19 vaccine effectiveness (VE) in participating European countries since early 2021.AimWe aimed to measure VE against PCR-confirmed SARS-CoV-2 in patients ≥ 20 years hospitalised with severe acute respiratory infection (SARI) from December 2021 to July 2022 (Omicron-dominant period).MethodsIn both networks, 46 hospitals (13 countries) follow a similar test-negative case-control protocol. We defined complete primary series vaccination (PSV) and first booster dose vaccination as last dose of either vaccine received ≥ 14 days before symptom onset (stratifying first booster into received < 150 and ≥ 150 days after last PSV dose). We measured VE overall, by vaccine category/product, age group and time since first mRNA booster dose, adjusting by site as a fixed effect, and by swab date, age, sex, and presence/absence of at least one commonly collected chronic condition.ResultsWe included 2,779 cases and 2,362 controls. The VE of all vaccine products combined against hospitalisation for laboratory-confirmed SARS-CoV-2 was 43% (95% CI: 29-54) for complete PSV (with last dose received ≥ 150 days before onset), while it was 59% (95% CI: 51-66) after addition of one booster dose. The VE was 85% (95% CI: 78-89), 70% (95% CI: 61-77) and 36% (95% CI: 17-51) for those with onset 14-59 days, 60-119 days and 120-179 days after booster vaccination, respectively.ConclusionsOur results suggest that, during the Omicron period, observed VE against SARI hospitalisation improved with first mRNA booster dose, particularly for those having symptom onset < 120 days after first booster dose.

IntroductionTwo large multicentre European hospital networks have estimated vaccine effectiveness (VE) against COVID-19 since 2021.AimWe aimed to measure VE against PCR-confirmed SARS-CoV-2 in hospitalised severe acute respiratory illness (SARI) patients ≥ 20 years, combining data from these networks during Alpha (March-June)- and Delta (June-December)-dominant periods, 2021.MethodsForty-six participating hospitals across 14 countries follow a similar generic protocol using the test-negative case-control design. We defined complete primary series vaccination (PSV) as two doses of a two-dose or one of a single-dose vaccine ≥ 14 days before onset.ResultsWe included 1,087 cases (538 controls) and 1,669 cases (1,442 controls) in the Alpha- and Delta-dominant periods, respectively. During the Alpha period, VE against hospitalisation with SARS-CoV2 for complete Comirnaty PSV was 85% (95% CI: 69-92) overall and 75% (95% CI: 42-90) in those aged ≥ 80 years. During the Delta period, among SARI patients ≥ 20 years with symptom onset ≥ 150 days from last PSV dose, VE for complete Comirnaty PSV was 54% (95% CI: 18-74). Among those receiving Comirnaty PSV and mRNA booster (any product) ≥ 150 days after last PSV dose, VE was 91% (95% CI: 57-98). In time-since-vaccination analysis, complete all-product PSV VE was > 90% in those with their last dose < 90 days before onset; ≥ 70% in those 90-179 days before onset.ConclusionsOur results from this EU multi-country hospital setting showed that VE for complete PSV alone was higher in the Alpha- than the Delta-dominant period, and addition of a first booster dose during the latter period increased VE to over 90%.

Background: To support the COVID-19 pandemic response, many countries, including Belgium, implemented baseline genomic surveillance (BGS) programs aiming to early detect and characterize new SARS-CoV-2 variants. In parallel, Belgium maintained a sentinel network of six hospitals that samples patients with severe acute respiratory infections (SARI) and integrated SARS-CoV-2 detection within a broader range of respiratory pathogens. We evaluate the ability of the SARI surveillance to monitor general trends and early signals of viral genetic evolution of SARS-CoV-2 and compare it with the BGS as a reference model., Methods: Nine-hundred twenty-five SARS-CoV-2 positive samples from patients fulfilling the Belgian SARI definition between January 2020 and December 2022 were sequenced using the ARTIC Network amplicon tiling approach on a MinION platform. Weekly variant of concern (VOC) proportions and types were compared to those that were circulating between 2021 and 2022, using 96,251 sequences of the BGS., Results: SARI surveillance allowed timely detection of the Omicron (BA.1, BA.2, BA.4, and BA.5) and Delta (B.1.617.2) VOCs, with no to 2 weeks delay according to the start of their epidemic growth in the Belgian population. First detection of VOCs B.1.351 and P.1 took longer, but these remained minor in Belgium. Omicron BA.3 was never detected in SARI surveillance. Timeliness could not be evaluated for B.1.1.7, being already major at the start of the study period., Conclusions: Genomic surveillance of SARS-CoV-2 using SARI sentinel surveillance has proven to accurately reflect VOCs detected in the population and provides a cost-effective solution for long-term genomic monitoring of circulating respiratory viruses., Competing Interests: The authors declare no conflict of interest., (© 2023 Sciensano and The Authors. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

BackgroundKnowledge on the burden attributed to influenza viruses vs other respiratory viruses in children hospitalised with severe acute respiratory infections (SARI) in Belgium is limited.AimThis observational study aimed at describing the epidemiology and assessing risk factors for severe disease.MethodsWe retrospectively analysed data from routine national sentinel SARI surveillance in Belgium. Respiratory specimens collected during winter seasons 2011 to 2020 were tested by multiplex real-time quantitative PCR (RT-qPCR) for influenza and other respiratory viruses. Demographic data and risk factors were collected through questionnaires. Patients were followed-up for complications or death during hospital stay. Analysis focused on children younger than 15 years. Binomial logistic regression was used to identify risk factors for severe disease in relation to infection status.ResultsDuring the winter seasons 2011 to 2020, 2,944 specimens met the study case definition. Complications were more common in children with underlying risk factors, especially asthma (adjusted risk ratio (aRR): 1.87; 95% confidence interval (CI): 1.46-2.30) and chronic respiratory disease (aRR: 1.88; 95% CI: 1.44-2.32), regardless of infection status and age. Children infected with non-influenza respiratory viruses had a 32% higher risk of complications (aRR: 1.32; 95% CI: 1.06-1.66) compared with children with influenza only.ConclusionMulti-virus testing in children with SARI allows a more accurate assessment of the risk of complications and attribution of burden to respiratory viruses beyond influenza. Children with asthma and respiratory disease should be prioritised for clinical care, regardless of their virological test result and age, and targeted for prevention campaigns.

The respiratory syncytial virus (RSV) represents the leading cause of viral lower respiratory tract infections (LRTI) in children worldwide and is associated with significant morbidity and mortality rates. The clinical picture of an RSV infection differs substantially between patients, and the role of viral co-infections is poorly investigated. During two consecutive winter seasons from October 2018 until February 2020, we prospectively included children up to 2 years old presenting with an acute LRTI, both ambulatory and hospitalized. We collected clinical data and tested nasopharyngeal secretions for a panel of 16 different respiratory viruses with multiplex RT-qPCR. Disease severity was assessed with traditional clinical parameters and scoring systems. A total of 120 patients were included, of which 91.7% were RSV positive; 42.5% of RSV-positive patients had a co-infection with at least one other respiratory virus. We found that patients suffering from a single RSV infection had higher pediatric intensive care unit (PICU) admission rates (OR = 5.9, 95% CI = 1.53 to 22.74), longer duration of hospitalization (IRR = 1.25, 95% CI = 1.03 to 1.52), and a higher Bronchiolitis Risk of Admission Score (BRAS) (IRR = 1.31, 95% CI = 1.02 to 1.70) compared to patients with RSV co-infections. No significant difference was found in saturation on admission, O 2 need, or ReSViNET-score. In our cohort, patients with a single RSV infection had increased disease severity compared to patients with RSV co-infections. This suggests that the presence of viral co-infections might influence the course of RSV bronchiolitis, but heterogeneity and small sample size in our study prevents us from drawing strong conclusions. IMPORTANCE RSV is worldwide the leading cause of serious airway infections. Up to 90% of children will be infected by the age of 2. RSV symptoms are mostly mild and typically mimic a common cold in older children and adolescents, but younger children can develop severe lower respiratory tract disease, and currently it is unclear why certain children develop severe disease while others do not. In this study, we found that children with a single RSV infection had a higher disease severity compared to patients with viral co-infections, suggesting that the presence of a viral co-infection could influence the course of an RSV bronchiolitis. As preventive and therapeutic options for RSV-associated disease are currently limited, this finding could potentially guide physicians to decide which patients might benefit from current or future treatment options early in the course of disease, and therefore, warrants further investigation., Competing Interests: The authors declare no conflict of interest.

Objectives: This study aimed to assess the efficacy and safety of 300 mg camostat mesylate three times daily in a fasted state to treat early phase COVID-19 in an ambulatory setting., Methods: We conducted a phase II randomized controlled trial in symptomatic (maximum 5 days) and asymptomatic patients with confirmed COVID-19 infection. Patients were randomly assigned in a 2:1 ratio to receive either camostat mesylate or a placebo. Outcomes included change in nasopharyngeal viral load, time to clinical improvement, the presence of neutralizing antibodies, and safety., Results: Of 96 participants randomized between November 2020 and June 2021, analyses were performed on the data of 90 participants who completed treatment (N = 61 camostat mesylate, N = 29 placebo). The estimated mean change in cycle threshold between day 1 and day 5 between the camostat and placebo group was 1.183 (P = 0.511). The unadjusted hazard ratio for clinical improvement in the camostat group was 0.965 (95% confidence interval, 0.480-1.942, P = 0.921 by Cox regression). The percentage distribution of the 50% neutralizing antibody titer at day 28 visit and frequency of adverse events were similar between the two groups., Conclusion: Under this protocol, camostat mesylate was not found to be effective as an antiviral drug against SARS-CoV-2., Trial Registration: ClinicalTrials.gov NCT04625114; November 12, 2020., Competing Interests: Conflict of interest The authors have no conflicts of interest to declare., (Copyright © 2022. Published by Elsevier Ltd.)

The prevalence of anti-SARS-CoV-2 antibodies and potential determinants were assessed in a random sample representative of the Belgian adult population. In total, 14,201 individuals (≥18 years) were invited by mail to provide saliva via an Oracol ® swab. Survey weights were applied, and potential determinants were estimated using multivariable logistic regressions. Between March and August 2021, 2767 individuals participated in the first data collection. During this period, which coincided with the onset of the vaccination campaign, the seroprevalence in the population increased from 25.2% in March/April to 78.1% in July. Among the vaccinated there was an increase from 74,2% to 98.8%; among the unvaccinated, the seroprevalence remained stable (around 17%). Among the vaccinated, factors significantly associated with the presence of antibodies were: having at least one chronic disease (OR a 0.22 (95% CI 0.08-0.62)), having received an mRNA-type vaccine (OR a 5.38 (95% CI 1.72-16.80)), and having received an influenza vaccine in 2020-2021 (OR a 3.79 (95% CI 1.30-11.07)). Among the unvaccinated, having a non-O blood type (OR a 2.00 (95% CI 1.09-3.67)) and having one or more positive COVID-19 tests (OR a 11.04 (95% CI 4.69-26.02)) were significantly associated. This study provides a better understanding of vaccine- and/or natural-induced presence of anti-SARS-CoV-2 antibodies and factors that are associated with this presence.

Background: Several randomised clinical trials have studied convalescent plasma for coronavirus disease 2019 (COVID-19) using different protocols, with different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralising antibody titres, at different time-points and severities of illness., Methods: In the prospective multicentre DAWn-plasma trial, adult patients hospitalised with COVID-19 were randomised to 4 units of open-label convalescent plasma combined with standard of care (intervention group) or standard of care alone (control group). Plasma from donors with neutralising antibody titres (50% neutralisation titre (NT 50 )) ≥1/320 was the product of choice for the study., Results: Between 2 May 2020 and 26 January 2021, 320 patients were randomised to convalescent plasma and 163 patients to the control group according to a 2:1 allocation scheme. A median (interquartile range) volume of 884 (806-906) mL) convalescent plasma was administered and 80.68% of the units came from donors with neutralising antibody titres (NT 50 ) ≥1/320. Median time from onset of symptoms to randomisation was 7 days. The proportion of patients alive and free of mechanical ventilation on day 15 was not different between both groups (convalescent plasma 83.74% (n=267) versus control 84.05% (n=137)) (OR 0.99, 95% CI 0.59-1.66; p=0.9772). The intervention did not change the natural course of antibody titres. The number of serious or severe adverse events was similar in both study arms and transfusion-related side-effects were reported in 19 out of 320 patients in the intervention group (5.94%)., Conclusions: Transfusion of 4 units of convalescent plasma with high neutralising antibody titres early in hospitalised COVID-19 patients did not result in a significant improvement of clinical status or reduced mortality., Competing Interests: Conflict of interest: All authors report support for the present manuscript from the Belgian Healthcare Knowledge Center (KCE). Q. Van Thillo reports grants from Fonds Wetenschappelijk Onderzoek (FWO)–Vlaanderen Basic Research 2019–2021 outside the submitted work. G. Meyfroidt reports a FWO–Vlaanderen Senior Clinical Researcher Grant outside the submitted work., (Copyright ©The authors 2022.)

To face the continuous emergence of SARS-CoV-2 variants, broadly protective therapeutic antibodies are highly needed. We here focused on the fusion peptide (FP) region of the viral spike antigen since it is highly conserved among alpha- and betacoronaviruses. First, we found that coronavirus cross-reactive antibodies are commonly formed during infection, being omnipresent in sera from COVID-19 patients, in ~50% of pre-pandemic human sera (rich in antibodies against endemic human coronaviruses), and even in feline coronavirus-infected cats. Pepscan analyses demonstrated that a confined N-terminal region of the FP is strongly immunogenic across diverse coronaviruses. Peptide-purified human antibodies targeting this conserved FP epitope exhibited broad binding of alpha- and betacoronaviruses, besides weak and transient SARS-CoV-2 neutralizing activity. Being frequently elicited by coronavirus infection, these FP-binding antibodies might potentially exhibit Fc-mediated effector functions and influence the kinetics or severity of coronavirus infection and disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Vanderheijden, Stevaert, Xie, Ren, Barbezange, Noppen, Desombere, Verhasselt, Geldhof, Vereecke, Stroobants, Oh, Vanhee, Naesens and Nauwynck.)

Background: The COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has presented itself as one of the most important health concerns of the 2020's, and hit the geriatric population the hardest. The presence of co-morbidities and immune ageing in the elderly lead to an increased susceptibility to COVID-19, as is the case for other influenza-like illnesses (ILI) or acute respiratory tract infections (ARI). However, little is known, about the impact of a previous or current infection on the other in terms of susceptibility, immune response, and clinical course. The aim of the "Prior Infection with SARS-COV-2" (PICOV) study is to compare the time to occurrence of an ILI or ARI between participants with a confirmed past SARS-CoV-2 infection (previously infected) and those without a confirmed past infection (naïve) in residents and staff members of nursing homes. This paper describes the study design and population characteristics at baseline., Methods: In 26 Belgian nursing homes, all eligible residents and staff members were invited to participate, resulting in 1,226 participants. They were classified as naïve or previously infected based on the presence of detectable SARS-CoV-2 antibodies and/or a positive RT-qPCR result before participation in the study. Symptoms from a prior SARS-CoV-2 infection between March and August 2020 were compared between previously infected residents and staff members., Results: Infection naïve nursing home residents reported fewer symptoms than previously infected residents: on average 1.9 and 3.1 symptoms, respectively (p = 0.016). The same effect was observed for infection naïve staff members and previously infected staff members (3.1 and 6.1 symptoms, respectively; p <0.0001). Moreover, the antibody development after a SARS-CoV-2 infection differs between residents and staff members, as previously infected residents tend to have a higher rate of asymptomatic cases compared to previously infected staff members (20.5% compared to 12.4%; p <0.0001)., Conclusions: We can postulate that COVID-19 disease development and symptomatology are different between a geriatric and younger population. Therefore, the occurrence and severity of a future ILI and/or ARI might vary from resident to staff., (© 2021. The Author(s).)

BackgroundSeasonal influenza-like illness (ILI) affects millions of people yearly. Severe acute respiratory infections (SARI), mainly influenza, are a leading cause of hospitalisation and mortality. Increasing evidence indicates that non-influenza respiratory viruses (NIRV) also contribute to the burden of SARI. In Belgium, SARI surveillance by a network of sentinel hospitals has been ongoing since 2011.AimWe report the results of using in-house multiplex qPCR for the detection of a flexible panel of viruses in respiratory ILI and SARI samples and the estimated incidence rates of SARI associated with each virus.MethodsWe defined ILI as an illness with onset of fever and cough or dyspnoea. SARI was defined as an illness requiring hospitalisation with onset of fever and cough or dyspnoea within the previous 10 days. Samples were collected in four winter seasons and tested by multiplex qPCR for influenza virus and NIRV. Using catchment population estimates, we calculated incidence rates of SARI associated with each virus.ResultsOne third of the SARI cases were positive for NIRV, reaching 49.4% among children younger than 15 years. In children younger than 5 years, incidence rates of NIRV-associated SARI were twice that of influenza (103.5 vs 57.6/100,000 person-months); co-infections with several NIRV, respiratory syncytial viruses, human metapneumoviruses and picornaviruses contributed most (33.1, 13.6, 15.8 and 18.2/100,000 person-months, respectively).ConclusionEarly testing for NIRV could be beneficial to clinical management of SARI patients, especially in children younger than 5 years, for whom the burden of NIRV-associated disease exceeds that of influenza.

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.

Objectives: To describe prevalence and incidence of anti-SARS-CoV-2 antibodies among Belgian hospital healthcare workers (HCW) in April-December 2020., Design: Prospective cohort study. Follow-up was originally planned until September and later extended., Setting: Multicentre study, 17 hospitals., Participants: 50 HCW were randomly selected per hospital. HCW employed beyond the end of the study and whose profession involved contact with patients were eligible. 850 HCW entered the study in April-May 2020, 673 HCW (79%) attended the September visit and 308 (36%) the December visit., Outcome Measures: A semiquantitative ELISA was used to detect IgG against SARS-CoV-2 in serum (Euroimmun) at 10 time points. In seropositive samples, neutralising antibodies were measured using a virus neutralisation test. Real-time reverse transcription PCR (RT-qPCR) was performed to detect SARS-CoV-2 on nasopharyngeal swabs. Participant characteristics and the presence of symptoms were collected via an online questionnaire., Results: Among all participants, 80% were women, 60% nurses and 21% physicians. Median age was 40 years. The seroprevalence remained relatively stable from April (7.7% (95% CI: 4.8% to 12.1%) to September (8.2% (95% CI: 5.7% to 11.6%)) and increased thereafter, reaching 19.7% (95% CI: 12.0% to 30.6%) in December 2020. 76 of 778 initially seronegative participants seroconverted during the follow-up (incidence: 205/1000 person-years). Among all seropositive individuals, 118/148 (80%) had a positive neutralisation test, 83/147 (56%) presented or reported a positive RT-qPCR, and 130/147 (88%) reported COVID-19-compatible symptoms at least once. However, only 46/73 (63%) of the seroconverters presented COVID-19-compatible symptoms in the month prior to seroconversion., Conclusions: The seroprevalence among hospital HCW was slightly higher than that of the general Belgian population but followed a similar evolution, suggesting that infection prevention and control measures were effective and should be strictly maintained. After two SARS-CoV-2 waves, 80% of HCW remained seronegative, justifying their prioritisation in the vaccination strategy., Trial Registration Number: NCT04373889., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Seasonal human coronaviruses (hCoVs) broadly circulate in humans. Their epidemiology and effect on the spread of emerging coronaviruses has been neglected thus far. We aimed to elucidate the epidemiology and burden of disease of seasonal hCoVs OC43, NL63, and 229E in patients in primary care and hospitals in Belgium between 2015 and 2020., Methods: We retrospectively analysed data from the national influenza surveillance networks in Belgium during the winter seasons of 2015-20. Respiratory specimens were collected through the severe acute respiratory infection (SARI) and the influenza-like illness networks from patients with acute respiratory illness with onset within the previous 10 days, with measured or reported fever of 38°C or greater, cough, or dyspnoea; and for patients admitted to hospital for at least one night. Potential risk factors were recorded and patients who were admitted to hospital were followed up for the occurrence of complications or death for the length of their hospital stay. All samples were analysed by multiplex quantitative RT-PCRs for respiratory viruses, including seasonal hCoVs OC43, NL63, and 229E. We estimated the prevalence and incidence of seasonal hCoV infection, with or without co-infection with other respiratory viruses. We evaluated the association between co-infections and potential risk factors with complications or death in patients admitted to hospital with seasonal hCoV infections by age group. Samples received from week 8, 2020, were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)., Findings: 2573 primary care and 6494 hospital samples were included in the study. 161 (6·3%) of 2573 patients in primary care and 371 (5·7%) of 6494 patients admitted to hospital were infected with a seasonal hCoV. OC43 was the seasonal hCoV with the highest prevalence across age groups and highest incidence in children admitted to hospital who were younger than 5 years (incidence 9·0 [95% CI 7·2-11·2] per 100 000 person-months) and adults older than 65 years (2·6 [2·1-3·2] per 100 000 person-months). Among 262 patients admitted to hospital with seasonal hCoV infection and with complete information on potential risk factors, 66 (73·3%) of 90 patients who had complications or died also had at least one potential risk factor (p=0·0064). Complications in children younger than 5 years were associated with co-infection (24 [36·4%] of 66; p=0·017), and in teenagers and adults (≥15 years), more complications arose in patients with a single hCoV infection (49 [45·0%] of 109; p=0·0097). In early 2020, the Belgian SARI surveillance detected the first SARS-CoV-2-positive sample concomitantly with the first confirmed COVID-19 case with no travel history to China., Interpretation: The main burden of severe seasonal hCoV infection lies with children younger than 5 years with co-infections and adults aged 65 years and older with pre-existing comorbidities. These age and patient groups should be targeted for enhanced observation when in medical care and in possible future vaccination strategies, and co-infections in children younger than 5 years should be considered during diagnosis and treatment. Our findings support the use of national influenza surveillance systems for seasonal hCoV monitoring and early detection, and monitoring of emerging coronaviruses such as SARS-CoV-2., Funding: Belgian Federal Public Service Health, Food Chain Safety, and Environment; Belgian National Insurance Health Care (Institut national d'assurance maladie-invalidité/Rijksinstituut voor ziekte-en invaliditeitsverzekering); and Regional Health Authorities (Flanders Agentschap zorg en gezondheid, Brussels Commission communautaire commune, Wallonia Agence pour une vie de qualité)., (© 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license.)

Background: The COVID-19 pandemic has imposed an enormous burden on health care systems around the world. In the past, the administration of convalescent plasma of patients having recovered from SARS and severe influenza to patients actively having the disease showed promising effects on mortality and appeared safe. Whether or not this also holds true for the novel SARS-CoV-2 virus is currently unknown., Methods: DAWn-Plasma is a multicentre nation-wide, randomized, open-label, phase II proof-of-concept clinical trial, evaluating the clinical efficacy and safety of the addition of convalescent plasma to the standard of care in patients hospitalized with COVID-19 in Belgium. Patients hospitalized with a confirmed diagnosis of COVID-19 are eligible when they are symptomatic (i.e. clinical or radiological signs) and have been diagnosed with COVID-19 in the 72 h before study inclusion through a PCR (nasal/nasopharyngeal swab or bronchoalveolar lavage) or a chest-CT scan showing features compatible with COVID-19 in the absence of an alternative diagnosis. Patients are randomized in a 2:1 ratio to either standard of care and convalescent plasma (active treatment group) or standard of care only. The active treatment group receives 2 units of 200 to 250 mL of convalescent plasma within 12 h after randomization, with a second administration of 2 units 24 to 36 h after ending the first administration. The trial aims to include 483 patients and will recruit from 25 centres across Belgium. The primary endpoint is the proportion of patients that require mechanical ventilation or have died at day 15. The main secondary endpoints are clinical status on day 15 and day 30 after randomization, as defined by the WHO Progression 10-point ordinal scale, and safety of the administration of convalescent plasma., Discussion: This trial will either provide support or discourage the use of convalescent plasma as an early intervention for the treatment of hospitalized patients with COVID-19 infection., Trial Registration: ClinicalTrials.gov NCT04429854 . Registered on 12 June 2020 - Retrospectively registered.

Cellular exonucleases involved in the processes that regulate RNA stability and quality control have been shown to restrict or to promote the multiplication cycle of numerous RNA viruses. Influenza A viruses are major human pathogens that are responsible for seasonal epidemics, but the interplay between viral proteins and cellular exonucleases has never been specifically studied. Here, using a stringent interactomics screening strategy and an siRNA-silencing approach, we identified eight cellular factors among a set of 75 cellular proteins carrying exo(ribo)nuclease activities or involved in RNA decay processes that support influenza A virus multiplication. We show that the exoribonuclease ERI1 interacts with the PB2, PB1 and NP components of the viral ribonucleoproteins and is required for viral mRNA transcription. More specifically, we demonstrate that the protein-protein interaction is RNA dependent and that both the RNA binding and exonuclease activities of ERI1 are required to promote influenza A virus transcription. Finally, we provide evidence that during infection, the SLBP protein and histone mRNAs co-purify with vRNPs alongside ERI1, indicating that ERI1 is most probably recruited when it is present in the histone pre-mRNA processing complex in the nucleus., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

BackgroundRespiratory syncytial virus (RSV) is a common cause of severe respiratory illness in young children (< 5 years old) and older adults (≥ 65 years old) leading the World Health Organization (WHO) to recommend the implementation of a dedicated surveillance in countries.AimWe tested the capacity of the severe acute respiratory infection (SARI) hospital network to contribute to RSV surveillance in Belgium.MethodsDuring the 2018/19 influenza season, we started the SARI surveillance for influenza in Belgium in week 40, earlier than in the past, to follow RSV activity, which usually precedes influenza virus circulation. While the WHO SARI case definition for influenza normally used by the SARI hospital network was employed, flexibility over the fever criterion was allowed, so patients without fever but meeting the other case definition criteria could be included in the surveillance.ResultsBetween weeks 40 2018 and 2 2019, we received 508 samples from SARI patients. We found an overall RSV detection rate of 62.4% (317/508), with rates varying depending on the age group: 77.6% in children aged < 5 years (253/326) and 34.4% in adults aged ≥ 65 years (44/128). Over 90% of the RSV-positive samples also positive for another tested respiratory virus (80/85) were from children aged < 5 years. Differences were also noted between age groups for symptoms, comorbidities and complications.ConclusionWith only marginal modifications in the case definition and the period of surveillance, the Belgian SARI network would be able to substantially contribute to RSV surveillance and burden evaluation in children and older adults, the two groups of particular interest for WHO.

Respiratory Syncytial Virus (RSV) is a very important viral pathogen in children, immunocompromised and cardiopulmonary diseased patients and the elderly. Most of the published research with RSV was performed on RSV Long and RSV A2, isolated in 1956 and 1961, yet recent RSV isolates differ from these prototype strains. Additionally, these viruses have been serially passaged in cell culture, which may result in adaptations that affect virus-host interactions. We have isolated RSV from mucosal secretions of 12 patients in the winters 2016-2017 and 2017-2018, of which eight RSV-A subtypes and four RSV-B subtypes. Passage 3 of the isolates was assessed for viral replication kinetics and infectious virus production in HEp-2, A549 and BEAS-2B cells, thermal stability at 37 °C, 32 °C and 4 °C, syncytia formation, neutralization by palivizumab and mucin mRNA expression in infected A549 cells. We observed that viruses isolated in one RSV season show differences on the tested assays. Furthermore, comparison with RSV A2 and RSV B1 reveals for some RSV isolates differences in viral replication kinetics, thermal stability and fusion capacity. Major differences are, however, not observed and differences between the recent isolates and reference strains is, overall, similar to the observed variation in between the recent isolates. One clinical isolate (BE/ANT-A11/17) replicated very efficiently in all cell lines, and remarkably, even better than RSV A2 in the HEp-2 cell line.

Introduction: Patients suffering from Sickle Cell Disease (SCD) are at increased risk for complications due to influenza virus. Annual influenza vaccination is strongly recommended but few clinical studies have assessed its immunogenicity in individuals with SCD. The aim of this study was to explore the biological efficacy of annual influenza vaccination in SCD patients by characterizing both their humoral and cell-mediated immunity against influenza antigen. We also aimed to investigate these immunological responses among SCD individuals according to their treatment (hydroxyurea (HU), chronic blood transfusions (CT), both HU and CT or none of them)., Methods: Seventy-two SCD patients (49 receiving HU, 9 on CT, 7 with both and 7 without treatment) and 30 healthy controls were included in the study. All subjects received the tetravalent influenza α-RIX-Tetra® vaccine from the 2016-2017 or 2017-2018 season., Results: Protective anti-influenza HAI titers were obtained for the majority of SCD patients one month after vaccination but seroconversion rates in patient groups were strongly decreased compared to controls. Immune cell counts, particularly cellular memory including memory T and memory B cells, were greatly reduced in SCD individuals. Functional activation assays confirmed a poorer CD8+ T cell memory. We also document an imbalance of cytokines after influenza vaccination in SCD individuals with an INFγ/IL-10 ratio (Th1-type/Treg-type response) significantly lower in the SCD cohort., Conclusion: SCD patients undergoing CT showed altered immune regulation as compared to other treatment subgroups. Altogether, the cytokine imbalance, the high regulatory T cell levels and the low memory lymphocyte subset levels observed in the SCD cohort, namely for those on CT, suggest a poor ability of SCD patients to fight against influenza infection. Nevertheless, our serological data support current clinical practice for annual influenza vaccination, though immunogenicity to other vaccines involving immunological memory might be hampered in SCD patients and should be further investigated., Competing Interests: The authors have declared that no competing interests exist.

Motivation: RNA viruses generate a cloud of genetic variants within each host. This cloud contains high-frequency genotypes, and many rare variants. The dynamics of these variants is crucial to understand viral evolution and their effect on their host., Results: We use an experimental evolution system to show that the genetic cloud surrounding the Coxsackie virus master sequence slowly, but steadily, evolves over hundreds of generations. This movement is determined by strong context-dependent mutations, where the frequency and type of mutations are affected by neighboring positions, even in silent mutations. This context-dependent mutation pattern serves as a spearhead for the viral population's movement within the adaptive landscape and affects which new dominant variants will emerge. The non-local mutation patterns affect the mutated dinucleotide distribution, and eventually lead to a non-uniform dinucleotide distribution in the main viral sequence. We tested these results on other RNA viruses with similar conclusions., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

After a pandemic wave in 2009 following their introduction in the human population, the H1N1pdm09 viruses replaced the previously circulating, pre-pandemic H1N1 virus and, along with H3N2 viruses, are now responsible for the seasonal influenza type A epidemics. So far, the evolutionary potential of influenza viruses has been mainly documented by consensus sequencing data. However, like other RNA viruses, influenza A viruses exist as a population of diverse, albeit related, viruses, or quasispecies. Interest in this quasispecies nature has increased with the development of next generation sequencing (NGS) technologies that allow a more in-depth study of the genetic variability. NGS deep sequencing methodologies were applied to determine the whole genome genetic heterogeneity of the three categories of influenza A viruses that circulated in humans between 2007 and 2012 in France, directly from clinical respiratory specimens. Mutation frequencies and single nucleotide polymorphisms were used for comparisons to address the level of natural intrinsic heterogeneity of influenza A viruses. Clear differences in single nucleotide polymorphism profiles between seasons for a given subtype also revealed the constant genetic drift that human influenza A virus quasispecies undergo.

The optimized exploitation of cell resources is one cornerstone of a successful infection. Differential mapping of host-pathogen protein-protein interactions (PPIs) on the basis of comparative interactomics of multiple strains is an effective strategy to highlight correlations between host proteome hijacking and biological or pathogenic traits. Here, we developed an interactomic pipeline to deliver high-confidence comparative maps of PPIs between a given pathogen and the human ubiquitin proteasome system (UPS). This subarray of the human proteome represents a range of essential cellular functions and promiscuous targets for many viruses. The screening pipeline was applied to the influenza A virus (IAV) PB2 polymerase proteins of five strains representing different levels of virulence in humans. An extensive PB2-UPS interplay has been detected that recapitulates the evolution of IAVs in humans. Functional validation with several IAV strains, including the seasonal H1N1 pdm09 and H3N2 viruses, confirmed the biological relevance of most identified UPS factors and revealed strain-independent and strain-specific effects of UPS factor invalidation on IAV infection. This strategy is applicable to proteins from any other virus or pathogen, providing a valuable resource with which to explore the UPS-pathogen interplay and its relationship with pathogenicity. IMPORTANCE Influenza A viruses (IAVs) are responsible for mild-to-severe seasonal respiratory illness of public health concern worldwide, and the risk of avian strain outbreaks in humans is a constant threat. Elucidating the requisites of IAV adaptation to humans is thus of prime importance. In this study, we explored how PB2 replication proteins of IAV strains with different levels of virulence in humans hijack a major protein modification pathway of the human host cell, the ubiquitin proteasome system (UPS). We found that the PB2 protein engages in an extended interplay with the UPS that evolved along with the virus's adaptation to humans. This suggests that UPS hijacking underlies the efficient infection of humans and can be used as an indicator for evaluation of the potential of avian IAVs to infect humans. Several UPS factors were found to be necessary for infection with circulating IAV strains, pointing to potential targets for therapeutic approaches.

RNA viruses pose serious threats to human health. Their success relies on their capacity to generate genetic variability and, consequently, on their adaptive potential. We describe a strategy to attenuate RNA viruses by altering their evolutionary potential. We rationally altered the genomes of Coxsackie B3 and influenza A viruses to redirect their evolutionary trajectories towards detrimental regions in sequence space. Specifically, viral genomes were engineered to harbour more serine and leucine codons with nonsense mutation targets: codons that could generate Stop mutations after a single nucleotide substitution. Indeed, these viruses generated more Stop mutations both in vitro and in vivo, accompanied by significant losses in viral fitness. In vivo, the viruses were attenuated, generated high levels of neutralizing antibodies and protected against lethal challenge. Our study demonstrates that cornering viruses in 'risky' areas of sequence space may be implemented as a broad-spectrum vaccine strategy against RNA viruses.

Exchange of the non coding regions of the NP segment between type A and C influenza viruses was used to demonstrate the importance not only of the proximal panhandle, but also of the initial distal panhandle strength in type specificity. Both elements were found to be compulsory to rescue infectious virus by reverse genetics systems. Interestingly, in type A influenza virus infectious context, the length of the NP segment 5' NC region once transcribed into mRNA was found to impact its translation, and the level of produced NP protein consequently affected the level of viral genome replication.

Exchange of the extremities of the NS segment of type A and C influenza viruses in reverse genetics systems was used to assess their putative role in type specificity. Restoration of each specific proximal panhandle was mandatory to allow the rescue of viruses with heterotypic extremities. Moreover, the transcription level of the modified segment seemed to be directly affected by the distal panhandle strength.

Chimeric foot-and-mouth disease viruses (FMDVs) have been generated from plasmids containing full-length FMDV cDNAs and characterized. The parental virus cDNA was derived from the cell-culture-adapted O1Kaufbeuren B64 (O1K B64) strain. Chimeric viruses, containing capsid coding sequences derived from the O/UKG/34/2001 or A/Turkey 2/2006 field viruses, were constructed using the backbone from the O1K B64 cDNA, and viable viruses (O1K/O-UKG and O1K/A-Tur, respectively) were successfully rescued in each case. These viruses grew well in primary bovine thyroid cells but grew less efficiently in BHK cells than the rescued parental O1K B64 virus. The two chimeric viruses displayed the expected antigenicity in serotype-specific antigen ELISAs. Following inoculation of each virus into cattle, the rescued O1K B64 strain proved to be attenuated whereas, with each chimeric virus, typical clinical signs of foot-and-mouth disease were observed, which then spread to in-contact animals. Thus, the surface-exposed capsid proteins of the O1K B64 strain are responsible for its attenuation in cattle. Consequently, there is no evidence for any adaptation, acquired during cell culture, outside the capsid coding region within the O1K B64 strain that inhibits replication in cattle. These chimeric infectious cDNA plasmids provide a basis for the analysis of FMDV pathogenicity and characterization of receptor utilization in vivo.

Influenza A viruses have been isolated from a wide range of animal species, aquatic birds being the reservoir for their genetic diversity. Avian influenza viruses can be transmitted to humans, directly or indirectly through an intermediate host like pig. This study aimed to define in vitro conditions that could prove useful to evaluate the potential of influenza viruses to adapt to a different host. Growth of H1N1, H1N2 and H3N2 influenza viruses belonging to different lineages isolated from birds or pigs prior to 2005 was tested on MDCK or NPTr cell lines in the presence or absence of exogenous trypsin. Virus multiplication was compared at 33, 37 and 40 degrees C, the infection site temperatures in human, swine and avian hosts, respectively. Temperature sensitivity of PB2-, NP- and M-RNA replication was also tested by quantitative real-time PCR. Multiplication of avian viruses was cold-sensitive, whatever cell type. By contrast, temperature sensitivity of swine viruses was found to depend on the virus and the host cell: for an H1N1 swine isolate from 1982, multiplication was cold-sensitive on NPTr cells and undetectable at 40 degrees C. From genetic analyses, it appears that temperature sensitivity could involve other residues than PB2 residue 627 and could affect other steps of the replication cycle than replication., (Copyright 2009 Elsevier B.V. All rights reserved.)

Background: The transcription/replication of the influenza viruses implicate the terminal nucleotide sequences of viral RNA, which comprise sequences at the extremities conserved among the genomic segments as well as variable 3' and 5' non-coding (NC) regions. The plasmid-based system for the in vivo reconstitution of functional ribonucleoproteins, upon expression of viral-like RNAs together with the nucleoprotein and polymerase proteins has been widely used to analyze transcription/replication of influenza viruses. It was thus shown that the type A polymerase could transcribe and replicate type A, B, or C vRNA templates whereas neither type B nor type C polymerases were able to transcribe and replicate type A templates efficiently. Here we studied the importance of the NC regions from the seven segments of type C influenza virus for efficient transcription/replication by the type A and C polymerases., Results: The NC sequences of the seven genomic segments of the type C influenza virus C/Johannesburg/1/66 strain were found to be more variable in length than those of the type A and B viruses. The levels of transcription/replication of viral-like vRNAs harboring the NC sequences of the respective type C virus segments flanking the CAT reporter gene were comparable in the presence of either type C or type A polymerase complexes except for the NS and PB2-like vRNAs. For the NS-like vRNA, the transcription/replication level was higher after introduction of a U residue at position 6 in the 5' NC region as for all other segments. For the PB2-like vRNA the CAT expression level was particularly reduced with the type C polymerase. Analysis of mutants of the 5' NC sequence in the PB2-like vRNA, the shortest 5' NC sequence among the seven segments, showed that additional sequences within the PB2 ORF were essential for the efficiency of transcription but not replication by the type C polymerase complex., Conclusion: In the context of a PB2-like reporter vRNA template, the sequence upstream the polyU stretch plays a role in the transcription/replication process by the type C polymerase complex.

CD8(+) cytotoxic T-lymphocyte (CTL) responses have been shown to be important in the control of human and simian immunodeficiency virus infections. Infection of sheep with visna/maedi virus (VISNA), a related lentivirus, induces specific CD8(+) CTL in vivo, but the specific viral proteins recognized are not known. To determine which VISNA antigens were recognized by sheep CTL, we used recombinant vaccinia viruses expressing the different genes of VISNA: in six sheep (Finnish LandracexDorset crosses, Friesland and Lleyn breeds) all VISNA proteins were recognized except TAT. Two sheep, shown to share major histocompatibility complex (MHC) class I alleles, recognized POL and were used to map the epitope. The pol gene is 3267 bp long encoding 1088 aa. By using recombinant vaccinia viruses a central portion (nt 1609-2176, aa 537-725) was found to contain the CTL epitope and this was mapped with synthetic peptides to a 25 aa region (aa 612-636). When smaller peptides were used, a cluster of epitopes was detected: at least three epitopes were present, at positions 612-623: DSRYAFEFMIRN; 620-631: MIRNWDEEVIKN; and 625-635: EEVIKNPIQAR. A DNA-prime-modified vaccinia virus Ankara (MVA)-boost strategy was employed to immunize four sheep shown to share MHC class I allele(s) with the sheep above. Specific CTL activity developed in all the immunized sheep within 3 weeks of the final MVA boost although half the sheep showed evidence of specific reactivity after the DNA-prime immunizations. This is the first report, to our knowledge, of induction of CTL by a DNA-prime-boost method in VISNA infection.

There are very few previous reports of expression of native full-length maedi visna virus (MVV) Env gp150 protein in the literature. Therefore the use of different plasmid and viral expression vectors to obtain full-length gp150 was investigated. A mammalian expression plasmid, pN3-Env, was constructed containing the MVV env gene encoding the precursor protein gp150 Env. The functionality of the recombinant plasmid was tested for expression in HEK293 cells. A recombinant modified vaccinia Ankara virus, MVA-Env, with expression detected in avian cells was also made. The expression of the MVV gp150 Env precursor protein was shown for the first time upon transfection of the eukaryotic HEK293 cells by the pN3-Env plasmid DNA as demonstrated by Western blot analysis. These plasmid or viral expression vectors are of potential use in MVV vaccines.

An avian paramyxovirus type-1 (APMV-1) was classified as virulent according to its Intra Cerebral Pathogenicity Index (ICPI), but as avirulent according to the motif of its F protein cleavage site. Although this atypical APMV-1 was isolated from sick, unvaccinated pigeons, it was not grouped with pigeon variants regarding its antigenic and genetic characterisation. We analysed its quasispecies nature by cloning and sequencing parts of the genome in three different genes to evaluate if heterogeneity might explain the difference observed between the ICPI and the F protein cleavage site motif. Two distinct sub-populations were detected in the phosphoprotein gene. In the fusion protein gene, two clones were found to be related to typical pigeon variants in the hypervariable domain.

Three avian Paramyxovirus type 1 (aPMV-1) isolated from pigeons duringpigeon paramyxovirosis outbreaks were molecularly characterised by sequencing parts of the six genes (NP, P, M, F, HN and L) of each strain. Virulent 99143 isolate was found to be very closely related to non-pathogenic vaccine strains of aPMV-1, even for its F protein cleavage site motif. Strains 99299 and 99106, typical pigeon paramyxovirus type 1 (pPMV-1) variants, exhibited between 10% and 20% difference with aPMV-1 at the nucleotide level. The aPMV-1 specific pattern of eight amino acids in the intracellular domain of HN protein was found different by one residue for these two isolates, and might represent a specific pattern for pPMV-1. The unique sequence of the polycistronic P gene editing site of 99299 and 99106 was characterised by four instead of three cytosine residues, and might so have an influence on the expression level of the three proteins encoded by P. This work is also the first to provide molecular data on NP, P and L genes of typical pPMV-1.