Molecular and virological characterization of the first poultry outbreaks of Genotype VII.2 velogenic avian orthoavulavirus type 1 (NDV) in North-West Europe, BeNeLux, 2018.

After two decades free of Newcastle disease, Belgium encountered a velogenic avian orthoavulavirus type 1 epizootic in 2018. In Belgium, 20 cases were diagnosed, of which 15 occurred in hobby flocks, 2 in professional poultry flocks and 3 in poultry retailers. The disease also disseminated from Belgium towards the Grand Duchy of Luxembourg by trade. Independently, the virus was detected once in the Netherlands, almost simultaneously to the first Belgian detection. As such Newcastle disease emerged in the entire BeNeLux region. Both the polybasic sequence of the fusion gene cleavage site and the intracerebral pathotyping assay demonstrated the high pathogenicity of the strain. This paper represents the first notification of this specific VII.2 subgenotype in the North-West of Europe. Time-calibrated full genome phylogenetic analysis indicated the silent or unreported circulation of the virus prior to the emergence of three genetic clusters in the BeNeLux region without clear geographical or other epidemiological correlation. The Dutch strain appeared as an outgroup to the Belgian and Luxembourgian strains in the time-correlated genetic analysis and no epidemiological link could be identified between the Belgian and Dutch outbreaks. In contrast, both genetic and epidemiological outbreak investigation data linked the G.D. Luxembourg case to the Belgian outbreak. The genetic links between Belgian viruses from retailers and hobby flocks only partially correlated with epidemiological data. Two independent introductions into the professional poultry sector were identified, although their origin could not be determined. Animal experiments using 6-week- old specific pathogen-free chickens indicated a systemic infection and efficient transmission of the virus. The implementation of re-vaccination in the professional sector, affected hobby and retailers, as well as the restriction on assembly and increased biosecurity measures, possibly limited the epizootic and resulted in the disappearance of the virus. These findings emphasize the constant need for awareness and monitoring of notifiable viruses in the field.

Multiyear Serological Surveillance of Notifiable Influenza A Viruses in Belgian Poultry: A Retrospective Analysis.

Surveillance of notifiable avian influenza (NAI) virus is mandatory in European member states, and each year a serological survey is performed to detect H5 and H7 circulation in poultry holdings. In Belgium, this serological monitoring is a combination of a stratified and a risk-based approach and is applied to commercial holdings with more than 200 birds. Moreover, a competitive nucleoprotein (NP) ELISA has been used as first screening method since 2010. A retrospective analysis of the serological monitoring performed from 2007 through 2013 showed sporadic circulation of notifiable low-pathogenicity avian influenza (LPAI) viruses in Belgian holdings with a fluctuating apparent flock seroprevalence according to years and species. Overall, the highest apparent flock seroprevalence was detected for the H5 subtype in domestic Anatidae, with 20%-50% for breeding geese and 4%-9% for fattening ducks. Positive serology against non-H5/H7 viruses was also observed in the same species with the use of the IDScreen influenza A antibody competition ELISA kit (ID-vet NP ELISA), and confirmed by isolation of H2, H3, H6, and H9 LPAI viruses. Among Galliformes, the apparent flock seroprevalence was lower, ranging between 0.3% and 1.3%. Circulation of notifiable LPAI viruses was only observed in laying hens with a similar seroprevalence for H5 and H7. Based on ID-vet NP ELISA results, no circulation of LPAI viruses, regardless the subtype, was observed in breeding chickens and fattening turkeys. Retrospectively, the use of an ELISA as first-line test not only reduced the number of hemagglutination inhibition tests to be performed, but also gave a broader evaluation of the prevalence of LPAI viruses in general, and might help to identify the most at-risk farms.

Redesigning the serological surveillance program for notifiable avian influenza in Belgian professional poultry holdings.

This study was aimed at redesigning the Belgian active surveillance program for domestic birds in professional poultry holdings based on a risk analysis approach. A stochastic quantitative analysis, combining all data sources, was run to obtain sensitivity estimates for the detection of an infected bird in the different risk groups identified. An optimal number of holdings for each risk group was then estimated on the basis of the different sensitivities obtained. This study proved to be a useful tool for decision makers, providing insight on how to reallocate the total amount of samples to be taken in the coming year(s) in Belgium, thus optimizing the field resources and improving efficiency of disease surveillance such as required by the international standards.

Evaluation of antibody-ELISA and real-time RT-PCR for the diagnosis and profiling of bluetongue virus serotype 8 during the epidemic in Belgium in 2006.

In 2006 bluetongue (BT) emerged for the first time in North-Western Europe. Reliable diagnostic tools are essential in controlling BT but data on the diagnostic sensitivity (Se) and specificity (Sp) are often missing. This paper aims to describe and analyse the results obtained with the diagnostics used in Belgium during the 2006 BT crisis. The diagnosis was based on a combination of antibody detection (competitive ELISA, cELISA) and viral RNA detection by real-time RT-PCR (RT-qPCR). The performance of the cELISA as a diagnostic tool was assessed on field results obtained during the epidemic and previous surveillance campaigns. As the infectious status of the animals is unknown during an epidemic, a Bayesian analysis was performed. Both assays were found to be equally specific (RT-qPCR: 98.5%; cELISA: 98.2%) while the diagnostic sensitivity of the RT-qPCR (99.5%) was superior to that of the cELISA (87.8%). The assumption of RT-qPCR as standard of comparison during the bluetongue virus (BTV) epidemic proved valid based on the results of the Bayesian analysis. A ROC analysis of the cELISA, using RT-qPCR as standard of comparison, showed that the cut-off point with the highest accuracy occurred at a percentage negativity of 66, which is markedly higher than the cut-off proposed by the manufacturer. The analysis of the results was further extended to serological and molecular profiling and the possible use of profiling as a rapid epidemiological marker of the BTV in-field situation was assessed. A comparison of the serological profiles obtained before, during and at the end of the Belgian epidemic clearly showed the existence of an intermediate zone which appears soon after BTV (re)enters the population. The appearance or disappearance of this intermediate zone is correlated with virus circulation and provides valuable information, which would be entirely overlooked if only positive and negative results were considered.

Bluetongue in Belgium, 2006.

Bluetongue has emerged recently in Belgium. A bluetongue virus strain was isolated and characterized as serotype 8. Two new real-time reverse transcription-quantitative PCRs (RT-qPCRs) that amplified 2 different segments of bluetongue virus detected this exotic strain. These 2 RT-qPCRs detected infection earlier than a competitive ELISA for antibody detection.