Identification and complete genome sequencing of paramyxoviruses in mallard ducks (Anas platyrhynchos) using random access amplification and next generation sequencing technologies

<p>Abstract</p> <p>Background</p> <p>During a wildlife screening program for avian influenza A viruses (AIV) and avian paramyxoviruses (APMV) in Belgium, we isolated two hemagglutinating agents from pools of cloacal swabs of wild mallards (<it>Anas platyrhynchos</it>) caught in a single sampling site at two different times. AIV and APMV1 were excluded using hemagglutination inhibition (HI) testing and specific real-time RT-PCR tests.</p> <p>Methods</p> <p>To refine the virological identification of APMV2-10 realized by HI subtyping tests and in lack of validated molecular tests for APMV2-10, random access amplification was used in combination with next generation sequencing for the sequence independent identification of the viruses and the determination of their genomes.</p> <p>Results</p> <p>Three different APMVs were identified. From one pooled sample, the complete genome sequence (15054 nucleotides) of an APMV4 was assembled from the random sequences. From the second pooled sample, the nearly complete genome sequence of an APMV6 (genome size of 16236 nucleotides) was determined, as well as a partial sequence for an APMV4. This APMV4 was closely related but not identical to the APMV4 isolated from the first sample. Although a cross-reactivity with other APMV subtypes did not allow formal identification, the HI subtyping revealed APMV4 and APMV6 in the respective pooled samples but failed to identify the co-infecting APMV4 in the APMV6 infected pool.</p> <p>Conclusions</p> <p>These data further contribute to the knowledge about the genetic diversity within the serotypes APMV4 and 6, and confirm the limited sensitivity of the HI subtyping test. Moreover, this study demonstrates the value of a random access nucleic acid amplification method in combination with massive parallel sequencing. Using only a moderate and economical sequencing effort, the characterization and full genome sequencing of APMVs can be obtained, including the identification of viruses in mixed infections.</p

Sargramostim to treat patients with acute hypoxic respiratory failure due to COVID-19 (SARPAC) : a structured summary of a study protocol for a randomised controlled trial

ObjectivesThe hypothesis of the proposed intervention is that Granulocyte-macrophage colony-stimulating factor (GM-CSF) has profound effects on antiviral immunity, and can provide the stimulus to restore immune homeostasis in the lung with acute lung injury post COVID-19, and can promote lung repair mechanisms, that lead to a 25% improvement in lung oxygenation parameters. Sargramostim is a man-made form of the naturally-occurring protein GM-CSF.Trial designA phase 4 academic, prospective, 2 arm (1:1 ratio), randomized, open-label, controlled trial.ParticipantsPatients aged 18-80 years admitted to specialized COVID-19 wards in 5 Belgian hospitals with recent ( 20 mg methylprednisolone or equivalent), (6) enrolment in another investigational study, (7) pregnant or breastfeeding or (8) ferritin levels > 2000 mu g/mL.Intervention and comparatorInhaled sargramostim 125 mu g twice daily for 5 days in addition to standard care. Upon progression of disease requiring mechanical ventilation or to acute respiratory distress syndrome (ARDS) and initiation of mechanical ventilator support within the 5 day period, inhaled sargramostim will be replaced by intravenous sargramostim 125 mu g/m(2) body surface area once daily until the 5 day period is reached. From day 6 onwards, progressive patients in the active group will have the option to receive an additional 5 days of IV sargramostim, based on the treating physician's assessment. Intervention will be compared to standard of care. Subjects progressing to ARDS and requiring invasive mechanical ventilatory support, from day 6 onwards in the standard of care group will have the option (clinician's decision) to initiate IV sargramostim 125m mu g/m(2) body surface area once daily for 5 days.Main outcomesThe primary endpoint of this intervention is measuring oxygenation after 5 days of inhaled (and intravenous) treatment through assessment of a change in pretreatment and post-treatment ratio of PaO2/FiO(2) and through measurement of the P(A-a)O-2 gradient (PAO(2)= Partial alveolar pressure of oxygen, PaO2=Partial arterial pressure of oxygen; FiO(2)= Fraction of inspired oxygen).RandomisationPatients will be randomized in a 1:1 ratio. Randomization will be done using REDCap (electronic IWRS system).Blinding (masking)In this open-label trial neither participants, caregivers, nor those assessing the outcomes will be blinded to group assignment.Numbers to be randomised (sample size)A total of 80 patients with confirmed COVID-19 and acute hypoxic respiratory failure will be enrolled, 40 in the active and 40 in the control group.Trial StatusSARPAC protocol Version 2.0 (April 15 2020). Participant recruitment is ongoing in 5 Belgian Hospitals (i.e. University Hospital Ghent, AZ Sint-Jan Bruges, AZ Delta Roeselare, University Hospital Brussels and ZNA Middelheim Antwerp). Participant recruitment started on March 26(th) 2020. Given the current decline of the COVID-19 pandemic in Belgium, it is difficult to anticipate the rate of participant recruitment.Trial registrationThe trial was registered on Clinical Trials.gov on March 30(th), 2020 (ClinicalTrials.gov Identifier: NCT04326920) - retrospectively registered; https://clinicaltrials.gov/ct2/show/NCT04326920?term=sarpac&recrs=ab&draw=2&rank=1 and on EudraCT on March 24th, 2020 (Identifier: 2020-001254-22).Full protocolThe full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol

Treatment of severely ill COVID-19 patients with anti-interleukin drugs (COV-AID) : a structured summary of a study protocol for a randomised controlled trial

ObjectivesThe purpose of this study is to test the safety and effectiveness of individually or simultaneously blocking IL-6, IL-6 receptor and IL-1 versus standard of care on blood oxygenation and systemic cytokine release syndrome in patients with COVID-19 coronavirus infection and acute hypoxic respiratory failure and systemic cytokine release syndrome.Trial designA phase 3 prospective, multi-center, interventional, open label, 6-arm 2x2 factorial design study.ParticipantsSubjects will be recruited at the specialized COVID-19 wards and/or ICUs at 16 Belgian participating hospitals. Only adult (>= 18y old) patients will be recruited with recent (<= 16 days) COVID-19 infection and acute hypoxia (defined as PaO2/FiO2 below 350mmHg or PaO2/FiO2 below 280 on supplemental oxygen and immediately requiring high flow oxygen device or mechanical ventilation) and signs of systemic cytokine release syndrome characterized by high serum ferritin, or high D-dimers, or high LDH or deep lymphopenia or a combination of those, who have not been on mechanical ventilation for more than 24 hours before randomisation. Patients should have had a chest X-ray and/or CT scan showing bilateral infiltrates within the last 2 days before randomisation. Patients with active bacterial or fungal infection will be excluded.Intervention and comparatorPatients will be randomized to 1 of 5 experimental arms versus usual care. The experimental arms consist of Anakinra alone (anti-IL-1 binding the IL-1 receptor), Siltuximab alone (anti-IL-6 chimeric antibody), a combination of Siltuximab and Anakinra, Tocilizumab alone (humanised anti-IL-6 receptor antibody) or a combination of Anakinra with Tocilizumab in addition to standard care. Patients treated with Anakinra will receive a daily subcutaneous injection of 100mg for a maximum of 28 days or until hospital discharge, whichever comes first. Siltuximab (11mg/kg) or Tocilizumab (8mg/kg, with a maximum dose of 800mg) are administered as a single intravenous injection immediately after randomization.Main outcomesThe primary end point is the time to clinical improvement defined as the time from randomization to either an improvement of two points on a six-category ordinal scale measured daily till day 28 or discharge from the hospital or death. This ordinal scale is composed of (1) Death; (2) Hospitalized, on invasive mechanical ventilation or ECMO; (3) Hospitalized, on non-invasive ventilation or high flow oxygen devices; (4) Hospitalized, requiring supplemental oxygen; (5) Hospitalized, not requiring supplemental oxygen; (6) Not hospitalized.RandomisationPatients will be randomized using an Interactive Web Response System (REDCap). A 2x2 factorial design was selected with a 2:1 randomization regarding the IL-1 blockade (Anakinra) and a 1:2 randomization regarding the IL-6 blockade (Siltuximab and Tocilizumab).Blinding (masking)In this open-label trial neither participants, caregivers, nor those assessing the outcomes are blinded to group assignment.Numbers to be randomised (sample size)A total of 342 participants will be enrolled: 76 patients will receive usual care, 76 patients will receive Siltuximab alone, 76 patients will receive Tocilizumab alone, 38 will receive Anakinra alone, 38 patients will receive Anakinra and Siltuximab and 38 patients will receive Anakinra and Tocilizumab.Trial StatusCOV-AID protocol version 3.0 (15 Apr 2020). Participant recruitment is ongoing and started on April 4(th) 2020. Given the current decline of the COVID-19 pandemic in Belgium, it is difficult to anticipate the rate of participant recruitment.Trial registrationThe trial was registered on Clinical Trials.gov on April 1st, 2020 (ClinicalTrials.gov Identifier: NCT04330638) and on EudraCT on April 3rd 2020 (Identifier: 2020-001500-41).Full protocolThe full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol

Highly Pathogenic H5N1 Influenza Virus in Smuggled Thai Eagles, Belgium

We report the isolation and characterization of a highly pathogenic avian influenza A/H5N1 virus from Crested Hawk-Eagles smuggled into Europe by air travel. A screening performed in human and avian contacts indicated no dissemination occurred. Illegal movements of birds are a major threat for the introduction of highly pathogenic avian influenza

Zilucoplan in patients with acute hypoxic respiratory failure due to COVID-19 (ZILU-COV) : a structured summary of a study protocol for a randomised controlled trial

Objectives: Zilucoplan (complement C5 inhibitor) has profound effects on inhibiting acute lung injury post COVID-19, and can promote lung repair mechanisms that lead to improvement in lung oxygenation parameters. The purpose of this study is to investigate the efficacy and safety of Zilucoplan in improving oxygenation and short- and long-term outcome of COVID-19 patients with acute hypoxic respiratory failure. Trial design: This is a phase 2 academic, prospective, 2:1 randomized, open-label, multi-center interventional study. Participants: Adult patients (>= 18y old) will be recruited at specialized COVID-19 units and ICUs at 9 Belgian hospitals. The main eligibility criteria are as follows: 1) Inclusion criteria: a. Recent (>= 6 days and <= 16 days) SARS-CoV-2 infection. b. Chest CT scan showing bilateral infiltrates within the last 2 days prior to randomisation. c. Acute hypoxia (defined as PaO2/FiO(2) below 350 mmHg or SpO2 below 93% on minimal 2 L/min supplemental oxygen). d. Signs of cytokine release syndrome characterized by either high serum ferritin, or high D-dimers, or high LDH or deep lymphopenia or a combination of those. 2) Exclusion criteria: e. Mechanical ventilation for more than 24 hours prior to randomisation. f. Active bacterial or fungal infection. g. History of meningococcal disease (due to the known high predisposition to invasive, often recurrent meningococcal infections of individuals deficient in components of the alternative and terminal complement pathways). Intervention and comparator: Patients in the experimental arm will receive daily 32,4 mg Zilucoplan subcutaneously and a daily IV infusion of 2g of the antibiotic ceftriaxone for 14 days (or until hospital discharge, whichever comes first) in addition to standard of care. These patients will receive additional prophylactic antibiotics until 14 days after the last Zilucoplan dose: hospitalized patients will receive a daily IV infusion of 2g of ceftriaxone, discharged patients will switch to daily 500 mg of oral ciprofloxacin.The control group will receive standard of care and a daily IV infusion of 2g of ceftriaxone for 1 week (or until hospital discharge, whichever comes first), to control for the effects of antibiotics on the clinical course of COVID-19. Main outcomes: The primary endpoint is the improvement of oxygenation as measured by mean and/or median change from pre-treatment (day 1) to post-treatment (day 6 and 15 or at discharge, whichever comes first) in PaO2/FiO(2) ratio, P(A-a)O-2 gradient and a/A PO2 ratio.(PAO(2)= Partial alveolar pressure of oxygen, PaO2=partial arterial pressure of oxygen, FiO(2)=Fraction of inspired oxygen). Randomisation: Patients will be randomized in a 2:1 ratio (Zilucoplan: control). Randomization will be done using an Interactive Web Response System (REDCap). Blinding (masking): In this open-label trial neither participants, caregivers, nor those assessing the outcomes will be blinded to group assignment. Numbers to be randomised (sample size): A total of 81 patients will be enrolled: 54 patients will be randomized to the experimental arm and 27 patients to the control arm. Trial Status: ZILU-COV protocol Version 4.0 (June 10 2020). Participant recruitment started on June 23 2020 and is ongoing. Given the uncertainty of the pandemic, it is difficult to predict the anticipated end date. Trial registration: The trial was registered on Clinical Trials.gov on May 11(th), 2020 (ClinicalTrials.gov Identifier: NCT04382755) and on EudraCT (Identifier: 2020-002130-33). Full protocol: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol

Etude fonctionnelle du peptide de fusion du virus SIV (Simian Immunodeficiency Virus)

Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Evaluation of the kinetics of anti-NP and anti-HA antibody after infection of Pekin ducks with low pathogenic avian influenza virus

Serological monitoring is a feature of surveillance programmes for the detection of the circulation of notifiable low pathogenic avian influenza (LPAI) viruses in commercial poultry holdings. Commercial multispecies nucleoprotein (NP) enzyme-linked immunosorbent assays (ELISAs) have been replacing the haemagglutination inhibition (HI) test as pre-screening tools. Few comparative studies have been conducted to test sera from domestic ducks for diagnostic purposes. Therefore, we evaluated the correlation between commercial NP ELISAs and the HI test. Anti-NP and anti-haemagglutinin (HA) antibodies were measured in sera from domestic ducks that had undergone serological screening and from juvenile domestic Pekin ducks that were experimentally infected with LPAI viruses. The findings highlight an absence of a correlation between NP ELISA and HI results with both field and experimental duck sera. Dissimilar kinetics of the antibodies detected during the follow-up evaluation of the humoral immune responses in experimentally infected ducks may explain this lack of correlation. Indeed, anti-NP titres decreased over time, whereas anti-HA titres remained unchanged after inoculation with the H3N1 LPAI virus isolated from domestic duck or the H7N1 LPAI virus isolated from chicken. Despite these differences, the NP ELISA may serve as a valid pre-screening tool to detect circulating LPAI viruses in domestic duck populations at the flock level

Seroprevalence of Mycoplasma gallisepticum in wild birds in Belgium

Mycoplasma gallisepticum is the causative agent of chronic respiratory disease (CRD) in chickens. CRD is characterized by coughing, nasal discharge, sinusitis and severe lesions on the air sacs. Economic losses, due to a decrease in weight gain and egg production, are significant. Moreover, breeder flocks that are infected with M. gallisepticum have to be culled. Mycoplasma species are host restricted but infections with M. gallisepticum, which occur mostly in chickens, also have been detected in other bird species (reservoirs) such as pheasants, partridges, peacocks, ducks, wild turkeys, and house finches. The aim of this study was to investigate whether there were other reservoirs for M. gallisepticum in wildlife. Therefor we analyzed serum samples of wild crows and geese for the presence of antibodies against M. gallisepticum. In 2012, blood samples of wild geese were collected in Belgium. Samples from Canadian geese (Branta canadensis) and Greylag geese (Anser anser) were tested. Samples from carrion crows (Corvus corone) were also collected in 2012. We obtained a total of 192 blood samples of acceptable quality to analyze the presence of antibodies against M. gallisepticum. Ninety-six samples were from crows and 96 were from geese. Antibodies were detected using a Blocking Enzyme Immunosorbent Assay (Svanovir® MG-Ab). Results were read using a photometer at 450 nm. All samples of both wild crows and geese were negative. In this study, it seems like wild crows and geese do not play an important role in the spread of M. gallisepticum in Belgium. However, further investigation in other wild birds is ongoing. Acks: T. Michiels is supported by the Federal Public Service of Health, Food Chain Safety and Environment (RCO-6752 MYCOPLASMA). The study was funded by the Federal Agency for the Safety of the Food Chain. The authors thank Didier Vangeluwe (Belgian Ringing Centre, Royal Belgian Institute for Natural Sciences, Belgium) for providing the samples