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Sentinel severe acute respiratory infection (SARI) surveillance was recommended by WHO following the 2009 H1N1 influenza virus pandemic.It uses a case definition based on clinical criteria, making it relevant to monitor the severity of other respiratory viruses such as SARS-CoV-2.The Belgian sentinel SARI network is composed of six representative hospitals. The original case definition was based on the 2014 WHO SARI case definition. Following the emergence of SARS-CoV-2, a more exhaustive list of symptoms was reported by the hospitals, in addition to basic demographic data, information on known risk factors/comorbidities, influenza and SARS-CoV-2 vaccination status, administration of antiviral or antibiotics, complications during hospitalization, and outcome. Respiratory samples were sent to the National influenza center for RT-qPCR testing for influenza virus, SARS-CoV-2 and 16 other respiratory viruses.During the first phase of the pandemic (Feb–March 2020), the SARI network operated normally (covering the end of the influenza epidemic) and was able to capture the local spread of SARS-CoV-2 in Belgium at the same time as the national emergency monitoring. In phase 2 (May 2020–January 2021), coinciding with the first waves of the pandemic when the strongest Non-Pharmaceutical Interventions were in place, the SARI surveillance was disrupted due to a high workload in the hospitals dealing with the surge of hospitalized COVID-19 cases. In phase 3 (January–December 2021), the SARI surveillance resumed in a lighter mode (using comprehensive sampling on specific days to provide more flexibility to the hospitals). The adapted method enabled us to monitor the COVID-19 pandemic and the resurgence of other respiratory viruses such as RSV and parainfluenza viruses, and to contribute to European COVID-19 vaccine effectiveness studies. Phase 4 started in January 2022, with the re-emergence of seasonal influenza viruses and their co-circulation with SARS-CoV-2.

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., info:eu-repo/semantics/published

Background: COVID-19 has presented itself as one of the most important health concerns of 2020. The geriatric population is arguably hit the hardest by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The ”Prior Infection with SARS-COV-2” (PICOV) study investigates both residents and staff members from nursing homes. The primary aim of the study is to compare the time to occurrence of an influenza-like illness (ILI) or acute respiratory infection (ARI) between participants with a confirmed past SARS-CoV-2 infection and those without an infection. This paper details the study design, sampling scheme, biological measurements, and population characteristics at baseline.Methods: In 26 Belgian nursing homes, all eligible residents and staff members with or without a past SARS-CoV-2 infection (ratio 40/60) were invited to participate. Consent was obtained from 1,375 participants and 1,226 completed the baseline questionnaire. Prevalence of symptoms during a prior SARS-CoV-2 infection was compared between residents and staff members with χ2 statistics.Results: Nursing home residents (both with and without a prior SARS-CoV-2 infection) systematically reported fewer symptoms than staff members. Moreover, results from prior nasopharyngeal RT-qPCR and baseline serology show that antibody development after a SARS-CoV-2 infection differs between residents and staff members.Conclusions: We can postulate that disease development and symptoms is 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.

BACKGROUNDSeasonal influenza-like illness (ILI) affects millions of people yearly. Severe acute respiratory infections (SARI), mainly caused by influenza, are a leading cause of hospitalisation and mortality. Increasing evidence indicates that non-influenza respiratory viruses (NIRVs) also contribute to the burden of SARI. In Belgium, SARI surveillance by a network of sentinel hospitals is ongoing since 2011.AIMHere, we report the results of using in-house multiplex PCRs for the detection of a flexible panel of viruses in respiratory ILI and SARI samples and the estimated incidence rates of SARI associated to each virus.METHODSILI was defined as an infection with onset of fever and cough or dyspnoea. SARI was defined as an infection requiring hospitalization with onset of fever and cough or dyspnoea within the previous 10 days. Samples were collected during four winter seasons and tested by multiplex RT-qPCRs for influenza virus and NIRVs. Using catchment population estimates, incidence rates of SARI associated to each virus were calculated.RESULTSOne third of the SARI cases were positive for NIRVs, reaching 49.4% among children under fifteen. In children under five, incidence rates of NIRV-associated SARI were double that of influenza (103.4 versus 57.6 per 100000 person-months), with NIRV co-infections, respiratory syncytial viruses, human metapneumoviruses and picornaviruses contributing the most (33.1, 13.6, 15.8 and 18.2 per 100000 person-months, respectively).CONCLUSIONEarly testing for NIRVs could be beneficial to clinical management of SARI patients, especially in children under five, 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. ispartof: EUROSURVEILLANCE vol:26 issue:27 ispartof: location:Sweden status: published

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 degrees 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. Copyright (C) 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Belgian Federal Public Service Health, Food Chain Safety, and Environment; Belgian National Insurance Health Care (Institut national d'assurance maladie-invalidite/Rijksinstituut voor ziekte-en invaliditeitsverzekering); and Regional Health Authorities (Flanders Agentschap zorg en gezondheid, Brussels Commission communautaire commune, Wallonia Agence pour une vie de qualite).

ObjectivesTo describe prevalence and incidence of anti-SARS-CoV-2 antibodies among Belgian hospital healthcare workers (HCW) in April–December 2020.DesignProspective cohort study. Follow-up was originally planned until September and later extended.SettingMulticentre study, 17 hospitals.Participants50 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 measuresA 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.ResultsAmong 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.ConclusionsThe 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 numberNCT04373889

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., info:eu-repo/semantics/published

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.

BackgroundGiven the current SARS-CoV-2 pandemic and the occurrence of a second wave, assessing the burden of disease among health care workers (HCWs) is crucial. We aim to document the prevalence of SARS-CoV-2 and the seroprevalence of anti-SARS-CoV-2 IgG among HCWs in Belgian hospitals, and to study potential risk factors for the infection in order to guide infection prevention and control (IPC) measures in healthcare institutions.MethodsWe performed a cross-sectional analysis of the baseline results (April 22 - April 26) of an ongoing cohort study. All staff who were present in the hospital during the sampling period and whose profession involved contact with patients were eligible. Fourteen hospitals across Belgium and 50 HCW per hospital were randomly selected. RT-qPCR was performed to detect SARS-CoV-2 RNA on nasopharyngeal swabs, and a semi-quantitative IgG ELISA was used to detect anti-SARS-CoV-2 antibodies in sera. Individual characteristics likely to be associated with seropositivity were collected using an online questionnaire.Findings698 participants completed the questionnaire; 80.8% were women, median age was 39.5, and 58.5% were nurses. Samples were collected on all 699 participants. The weighted anti-SARS-CoV-2 IgG seroprevalence was 7.7% (95%CI, 4.7%-12.2%), while 1.1% (95%CI, 0.4%-3.0%) of PCR results were positive. Unprotected contact with a confirmed case was the only factor associated with seropositivity (PR 2.16, 95% CI, 1.4-3.2).InterpretationMost Belgian HCW did not show evidence of SARS-CoV-2 infection by late April 2020, and unprotected contact was the most important risk factor. This confirms the importance of widespread availability of protective equipment and use of adequate IPC measures in hospital settings.

Background Respiratory syncytial virus (RSV) is a common cause of severe respiratory illness in young children (, info:eu-repo/semantics/published

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.

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&ndash, host interactions. We have isolated RSV from mucosal secretions of 12 patients in the winters 2016&ndash, 2017 and 2017&ndash, 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 &deg, C, 32 &deg, C and 4 &deg, 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.

Members of the European Influenza Surveillance Network: Portugal (Figueiredo Augusto Gonçalo, Machado Jorge, Moreira Guiomar Raquel, Nogueira Paulo, Rebelo de Andrade Helena, Rodrigues Ana Paula) The yearly influenza epidemics during each winter season vary in burden and severity. During the 2016/17 and 2017/18 seasons, all-cause excess mortality was observed during periods of high influenza virus circulation. Our aim is to describe and compare the pattern of influenza virus circulation and related disease severity by number of patients and fatal cases in intensive care units (ICUs) across European Union/European Economic Area (EU/EEA) countries for the seasons 2016/17 and 2017/18. As influenza circulation progressed from a west to east direction across Europe in 2017/18, a better understanding of the current epidemiological situation might help to prepare countries in the eastern part of the World Health Organization (WHO) European Region for high influenza activity and severity. info:eu-repo/semantics/publishedVersion

RNA viruses generate a cloud of genetic variants within each host. This cloud contains high frequency genotypes, and a very large number of rare variants. While the dynamics of frequent variants are affected by the fitness of each variant, the rare variants cloud is affected by more complex genetic factors, including context dependent mutations. It serves as a spearhead for the viral population’s movement within the adaptive landscape. We here 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. The evolution of the rare variants cloud often precedes the appearance of high frequency variants. The rare variants cloud’s evolution is driven by a combination of a context-dependent mutation pattern and selection for and against specific nucleotide compositions.This combination affects the mutated dinucleotide distribution, and eventually leads to a non-uniform dinucleotide distribution in the main viral sequence. We then tested these conclusions on other RNA viruses with similar conclusions.

The publisher regrets that the co-authors in the “European region influenza surveillance network” were not tagged correctly. The full and complete list of authors and their affiliations for this article is given above. The publisher would like to apologise for any inconvenience caused.

Objectives Mumps used to affect children between 2 and 15 years old. The mumps–measles–rubella (MMR) vaccine is available, with vaccine coverage rate of about 85% after two vaccine doses. Recently new mumps outbreaks have emerged in highly vaccinated populations; the causes for these new outbreaks are yet unknown. We tested if a difference in seroneutralizing capacity against the vaccine and wild-type viruses existed and if waning immunity could be detected. Methods In this study, 570 serum samples (age group 2–3 years (n = 96), 8–9 years (n = 95), 13–14 years (n = 94), 18–20 years (n = 96), 24–26 years (n = 92) and 50 + years (n = 97)) in Belgium were tested in the rapid fluorescent foci inhibition test for their neutralizing capacity against the vaccine and wild-type viruses. Results Neutralizing antibodies against the vaccine strain were present in 84% (81/97) of the 2–3-year, 74% (70/95) of the 8–9-year, 81% (76/94) of the 13–14-year, 76% (73/96) of the 18–20-year, 67% (62/92) of the 24–26-year and 77% (75/97) of the 50+-year age group serum samples. For all age groups, only about half of these serum samples were also positive for the wild-type virus. The geometric mean titres for the vaccine and wild-type virus for all younger age groups, except for 24–26 years, were significantly different, demonstrating poor in vitro cross-neutralization. Conclusions A possible contribution of antigenic differences between the genotype A and G mumps virus as well as other immune factors, in addition to lower-than-optimal vaccination coverage and waning immunity, could explain the poor in vitro cross-neutralization and should be further studied.

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+CTLin 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 Landrace×Dorset 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. Thepolgene 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.

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 during pigeon 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.

A RT-nested PCR that amplifies part of the conserved nucleoprotein gene of avian Paramyxovirus type 1 is described. The technique allowed the detection of pigeon Paramyxovirus type 1 (pPMV-1) virus directly from a wide range of infected chicken and pigeon organs, and should be able to detect typical Newcastle disease viruses too. Compared with the reference method, the developed RT-nested PCR was found more sensitive, as it was able to detect virus genome in infected pigeon organs at late stage of infection, when virus isolation failed. Such a molecular technique represents an alternative method of diagnosis for research purposes on pPMV-1 variants, for example to study pathogenesis aspects of the infection or to assess the efficacy of vaccines.

International audience; 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.

International audience; 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. Citation: Crescenzo-Chaigne B, Barbezange C, van der Werf S (2013) The Panhandle Formed by Influenza A and C Virus NS Non-Coding Regions Determines NS Segment Expression. PLoS ONE 8(11): e81550.

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 virus segment 2 mRNA expresses three\ud polypeptides: PB1, PB1-F2 and PB1-N40, from AUGs\ud 1, 4 and 5 respectively. Two short open reading\ud frames (sORFs) initiated by AUGs 2 and 3 are also\ud present. To understand translational regulation in\ud this system, we systematically mutated AUGs 1–4\ud and monitored polypeptide synthesis from\ud plasmids and recombinant viruses. This identified\ud sORF2 as a key regulatory element with opposing\ud effects on PB1-F2 and PB1-N40 expression. We\ud propose a model in which AUGs 1–4 are accessed\ud by leaky ribosomal scanning, with sORF2 repressing\ud synthesis of downstream PB1-F2. However, sORF2\ud also up-regulates PB1-N40 expression, most likely\ud by a reinitiation mechanism that permits skipping of\ud AUG4. Surprisingly, we also found that in contrast to\ud plasmid-driven expression, viruses with improved\ud AUG1 initiation contexts produced less PB1 in\ud infected cells and replicated poorly, producing\ud virions with elevated particle:PFU ratios. Analysis\ud of the genome content of virus particles showed\ud reduced packaging of the mutant segment\ud 2 vRNAs. Overall, we conclude that segment\ud 2 mRNA translation is regulated by a combination\ud of leaky ribosomal scanning and reinitiation, and\ud that the sequences surrounding the PB1 AUG\ud codon are multifunctional, containing overlapping\ud signals for translation initiation and for segmentspecific\ud packaging.

International audience; 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.

Niesalla, H. et al., To determine whether systemic immunization with plasmid DNA and virus vector against visna/maedi virus (VMV) would induce protective immune responses, sheep were immunized with VMV gag and/or env sequences using particle-mediated epidermal bombardment and injection of recombinant modified vaccinia Ankara. The results showed that immunization induced both humoral and cell-mediated responses prior to and after virus challenge. The vaccination protocol did not prevent infection, but immunization with the gag gene or a combination of gag and env genes resulted in significantly reduced provirus loads in blood and mediastinal lymph node, respectively. Provirus loads in lung and draining lymph node were unaffected, but p25 expression was undetectable in lungs of animals immunized with a combination of gag and env genes. Analysis of target tissues for lesions at post-mortem showed that immunization with the env gene caused a significant increase in lesion score, while the gag gene or a combination of gag and env genes had no effect. Inclusion of the ovine interferon-γ gene in the initial priming mixture had minimal effect on immune responses, provirus load, or lesion development, although it resulted in a decreased p25 expression in the lung. The results thus show that systemic immunization with gag or a combination of gag and env genes reduces provirus load in blood and lymphoid tissue, respectively whereas env immunization has no effect on provirus load but increased lesion development. © 2008 Elsevier Ltd. All rights reserved., This study was supported by grants from European Union (QLK2-CT-2002-00617) and Spanish CICYT (AGL2003-08977-C03-01 and AGL2007-66874-C04). Ramsés Reina and Ximena de Andrés were supported by a fellowship FPI from the Spanish Ministry of Science and Education. Tom McNeilly was supported by a PhD studentship from the Royal (Dick) College of Veterinary Studies, University of Edinburgh.

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.

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 experimental pigeon paramyxovirus (pPMV-1) infection was followed by reverse transcription-nested polymerase chain reaction for 31 days after infection, in 16 organs of inoculated or contact pigeons naturally infected with Salmonella Typhimurium. With two exceptions, both groups presented similar results. Typical nervous signs and a green diarrhoea were observed. The spread of pPMV-1 was relatively quick, all organs being largely positive at 4 days after inoculation or contact. The lung, spleen, caecal tonsils, kidneys and brain, for which almost all tested samples remained positive during most of the experiment, seemed to be the principal targets for virus persistence. However, the virus was significantly recovered later in the brain parts and for longer in the trachea of the contact pigeons than of the inoculated ones.

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.

SUMMARY. The purpose of this study was to analyze the safety characteristics of three commercially available live Salmonella vaccine strains (vacT, Zoosaloral, and X3985) in relation to their persistence in individual animals but also within a flock and in the environment. In a first experiment, the digestive and systemic distributions in chickens were followed for 10 days in individually reared chickens that were orally inoculated at 1 day of age. Strain X3985 quickly disappeared from the digestive tract but remained in the liver until the end of this experiment, whereas strains vacT and Zoosaloral colonized the liver as well as the gut for 10 days. In the second trial, behavior of the vaccine strains was studied in groups of 20 chickens during 10 wk after a single oral administration to individual birds. Strain vacT remained in the environment of inoculated animals for 4-5 wk. Six weeks after the inoculation, vacT was not recovered from internal organs such as liver and spleen, and vacT disappeared from the digestive tract between the sixth and the 10th weeks. Comparatively, both Zoosaloral and X3985 vaccine strains persisted longer in the environment (8 wk at least). Of the vaccine strains, X3985 showed the greatest colonization of both systemic and digestive organs.

Live vaccine strains of Salmonella should be avirulent, immunogenic and genetically stable. Some isolates of three commercially available live vaccine strains of Salmonella typhimurium, sampled during a study on their persistence in a vaccinated flock of chickens, were analyzed for genetic stability using macrorestriction analysis of their genome. Two out of the three vaccine strains showed genetic instabilities. Two of the 51 isolates of Zoosaloral vaccine strain and nine of the 32 analyzed isolates of chi(3985), a genetically modified organism, were variants and showed different macrorestriction profiles.