Non coding extremities of the seven influenza virus type C vRNA segments: effect on transcription and replication by the type C and type A polymerase complexes

<p>Abstract</p> <p>Background</p> <p>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 <it>in vivo </it>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

Seasonal Genetic Drift of Human Influenza A Virus Quasispecies Revealed by Deep Sequencing

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

Monitoring of human coronaviruses in Belgian primary care and hospitals, 2015-20: a surveillance study.

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)

Early high antibody titre convalescent plasma for hospitalised COVID-19 patients: DAWn-plasma.

peer reviewedBACKGROUND: 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

Vaccine effectiveness against COVID-19 hospitalisation in adults (≥ 20 years) during Alpha- and Delta-dominant circulation: I-MOVE-COVID-19 and VEBIS SARI VE networks, Europe, 2021

Members of the I-MOVE-COVID-19 and VEBIS hospital study teams (in addition to the named authors): Svjetlana Karabuva, Petra Tomaš Petrić, Marija Marković, Sandra Ljubičić, Bojana Mahmutović, Irena Tabain, Petra Smoljo, Iva Pem Novosel, Tanya Melillo, John Paul Cauchi, Benédicte Lissoir, Xavier Holemans, Marc Hainaut, Nicolas Dauby, Benedicte Delaere, Marc Bourgeois, Evelyn Petit, Marijke Reynders, Door Jouck, Koen Magerman, Marieke Bleyen, Melissa Vermeulen, Sébastien Fierens, François Dufrasne, Siel Daelemans, Ala’a Al Kerwi, Francoise Berthet, Guy Fagherazzi, Myriam Alexandre, Charlene Bennett, Jim Christle, Jeff Connell, Peter Doran, Laura Feeney, Binita Maharjan, Sinead McDermott, Rosa McNamara, Nadra Nurdin, Salif Mamadou Cissé, Anne-Sophie L'Honneur, Xavier Duval, Yolande Costa, Fidouh Nadhira, Florence Galtier, Laura Crantelle, Vincent Foulongne, Phillipe Vanhems, Sélilah Amour, Bruno Lina, Fabrice Lainé, Laetitia Gallais, Gisèle Lagathu, Anna Maisa, Yacine Saidi, Christine Durier, Rebecca Bauer, Ana Paula Rodrigues, Adriana Silva, Raquel Guiomar, Margarida Tavares, Débora Pereira, Maria José Manata, Heidi Gruner, André Almeida, Paula Pinto, Cristina Bárbara, Itziar Casado, Ana Miqueleiz, Ana Navascués, Camino Trobajo-Sanmartín, Miguel Fernández-Huerta, María Eugenia Portillo, Carmen Ezpeleta, Nerea Egüés, Manuel García Cenoz, Eva Ardanaz, Marcela Guevara, Conchi Moreno-Iribas, Hana Orlíková, Carmen Mihaela Dorobat, Carmen Manciuc, Simin Aysel Florescu, Alexandru Marin, Sorin Dinu, Catalina Pascu, Alina Ivanciuc, Iulia Bistriceanu, Mihaela Oprea, Maria Elena Mihai, Silke Buda, Ute Preuss, Marianne Wedde, Auksė Mickienė, Giedrė Gefenaitė, Alain Moren, Anthony NardoneIntroduction: Two large multicentre European hospital networks have estimated vaccine effectiveness (VE) against COVID-19 since 2021. Aim: We 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. Methods: Forty-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. Results: We 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. Conclusions: Our 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%.Key public health message: - What did you want to address in this study? To understand how well the COVID-19 vaccine was performing in Europe against hospitalisation during SARS-CoV-2 Alpha and Delta variant periods, we present vaccine effectiveness results from a multi-country study of complete and booster dose COVID-19 vaccination among adults (aged 20 years and over). - What have we learnt from this study? Between March and June 2021 (Alpha period), vaccine effectiveness against hospitalisation with laboratory-confirmed SARS-CoV-2 was 43% for partial vaccination and 86% for complete vaccination. For June to December 2021 (Delta period), vaccine effectiveness for complete vaccination was lower (52%) but with addition of an mRNA booster dose, effectiveness reached 91%, and remained > 90% up to 119 days after the booster dose. - What are the implications of your findings for public health? In Europe in 2021, COVID-19 vaccine effectiveness results for the Alpha period indicated an excellent benefit for preventing hospitalisation after complete vaccination. During Delta variant circulation, however, a booster dose was required to achieve this level of effectiveness, and this was maintained for up to 4 months post booster.info:eu-repo/semantics/publishedVersio

Vaccine effectiveness against COVID-19 hospitalisation in adults (≥ 20 years) during Omicron-dominant circulation: I-MOVE-COVID-19 and VEBIS SARI VE networks, Europe, 2021 to 2022

Introduction: The I-MOVE-COVID-19 and VEBIS hospital networks have been measuring COVID-19 vaccine effectiveness (VE) in participating European countries since early 2021. Aim: We 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). Methods: In 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. Results: We 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. Conclusions: Our 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.Key public health message: 1. What did you want to address in this study? In order to understand how well the COVID-19 vaccine is performing in Europe against hospitalisation during the period when the SARS-CoV-2 Omicron variant was circulating, we investigated vaccine effectiveness using data from a multi-country study of complete and booster-dose COVID-19 vaccination among adults aged 20 years and over. 2. What have we learnt from this study? Between December 2021 and July 2022, vaccine effectiveness against hospitalisation with laboratory-confirmed SARS-CoV-2 was 43% for complete vaccination. With addition of an mRNA booster dose, effectiveness was 59% overall. It was higher when onset of illness was close to the date of the last vaccination, at 85% when last booster dose was 14–59 days before onset, at 70% for 60–119 days, and falling below 40% for 120–179 days. 3. What are the implications of your findings for public health? In European hospital settings in 2022, during the Omicron period, COVID-19 mRNA booster vaccine provided an improved benefit for preventing hospitalisation, particularly if disease onset was within 4 months of receiving the booster dose.info:eu-repo/semantics/publishedVersio

La Phylogénie comme outil de progrès de la taxonomie virale

PARIS-Museum-Bib zoologie mam. (751052312) / SudocNANTES-Ecole Nat.Vétérinaire (441092302) / SudocSudocFranceF

Growth kinetics of rescued influenza viruses.

<p>(A) Type C rescued viruses. Infections were performed in SK cells infected at a low m.o.i. (0.001 PFU/cell) in the presence of 0.25 µg/ml of TPCK-trypsin. (B) Type A rescued viruses. Infections were performed in MDCK cells infected at a low m.o.i. (0.001 PFU/cell) in the presence of 1 µg/ml of TPCK-trypsin. Titers were determined by standard plaque assays in duplicate. Results are representative of two independent experiments. </p

Evaluation of mRNA/vRNA and NS1/NP ratios during single-cycle infection with rescued influenza A viruses.

<p>(A) Quantification of vRNA and mRNA in infected cells. Six hours after MDCK infection with rescued influenza A viruses at high m.o.i. (2 PFU/cell), viral vRNA and mRNA levels for each segment were evaluated by specific two-step RT-qPCRs previously described [23]. Results were expressed as the mean of mRNA/vRNA Cp (crossing-point) ratios calculated on data obtained from 12 independent qPCRs (corresponding to two infections, two independent cDNA syntheses per infection and three independent qPCRs for each cDNA). Statistics were performed using the ANOVA test. Black bars: wild-type 5’A/3’A; hatched bars: 5’A/3’C(C5U); grey bars: 5’C/3’C-proxA. (B) Levels of NP and NS1 proteins after infection with the rescued influenza A viruses. Four hours after MDCK infection at a high m.o.i. (2 PFU/cell), cell lysates were analyzed by Western-blot for viral NS1 and NP proteins and for β-actin as cellular control. Following chemiluminescence acquisition with a G. Box (SYNGENE, Cambridge, UK), band densities for viral proteins were determined using GeneTools software (SYNGENE, Cambridge, UK), and were used to calculate NS1/NP ratios for each virus. Due to the high levels of viral protein expression during infection, protein extracts were diluted in uninfected cellular extracts prior to electrophoresis. Results are from one representative assay out of three independent infections.</p

Rescue of influenza viruses harbouring substitutions in the NC region of genomic NS segment.

<p>Nucleotide sequences and predicted conformations of the NC region panhandle of the different NS genomic segments tested in type C (in plain, based on C/JHB/1/66) and type A (in bold, based on A/WSN/33) influenza virus reverse genetics systems [20,21]. The introduced mutation in 5’A/3’A(U5C) is underlined. The sequence of both ends of each segment of each rescued virus was verified as described [10], and no mutation was detected except at position 5 in the 3’ end for 5’A/3’C and 5’A/3’A(U5C) yielding viruses 5’A/3’C(C5U) and 5’A/3’A, respectively (indicated by arrows). In constructs 5’A/3’A-proxC in the type C system and 5’C/3’C-proxA in the type A system, only the distal panhandle was modified, but the homotypic proximal panhandle was conserved. The energy barriers of the canonical pairs C:G and U:A, and of the wobble base pair G:U (represented by a black dot) were described by Vendeix et al [34] and were used to calculate a score to evaluate the panhandle strength. The proximal panhandle consists of 9 potential base-pairs, when the distal panhandle was defined to include the potential base-pairs up to the type A poly-U signal. Type C reverse genetics was performed in 293T cells and supernatants were collected and titrated at day 10 post-transfection (p.t.). Type A reverse genetics was performed in a co-culture of 293T and MDCK cells, and supernatants were collected and titrated at 72h p.t. Titers in PFU/ml are the mean of 2 to 4 independent experiments.</p