Sjaak de Wit

Infectious bronchitis virus (IBV) is an economically important avian gammacoronavirus. IBV uses sialic acids and heparan sulfate for cellular attachment, but the host proteins governing its infection remain poorly defined. Here, we investigated the roles of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) during IBV Beaudette (IBV BD) infection. ACE2 knockout impaired IBV replication in DF-1 cells, whereas ACE2 reconstitution restored infection. Furthermore, ACE2 overexpression enhanced infection of DF-1 and primary chicken embryo kidney cells, supporting the role of chicken ACE2 as a proviral host factor. In contrast, TMPRSS2 exerted a restrictive function on IBV BD infection as demonstrated in TMPRSS2 KO DF-1 cells and upon pharmacological inhibition, which significantly increased IBV BD infection in DF-1 and Vero cells. RNA sequencing revealed significant changes in lipid metabolism and membrane-related processes following TMPRSS2 knockout and pharmacological inhibition. These findings highlight the opposing roles of ACE2 and TMPRSS2 in IBV BD infection, emphasizing the combined impact of proviral host factors, protease activity, and the cellular membrane environment during gammacoronavirus infections.

Background: Endemic contagious diseases in broilers have a significant impact on production performance. However, endemic contagious diseases are multifaceted and complex. They are rarely monitored on a large scale. This complexity hinders their mitigation, as timely information about their distribution and knowledge about their impact on production performance is scarce. This study aimed to evaluate whether data routinely produced by the Polish broiler industry, the first European meat producer, could be reused to generate knowledge about those diseases and provide stakeholders with contextual information to improve their disease management. Results: The study reused a dataset collected by a large producer and a veterinary laboratory, which implemented a screening program at the end of the production cycle. The high-dimensional dataset covered 115 ‘screened flocks’ produced between 2018 and 2023 across Poland. The information was dispersed across 75 variables on production indicators, health indicators, necropsy lesions, and a list of evidence of infection or infestation by a diverse range of aetiological agents (bacterial, viral, and Eimeria) and regrouped into 8 themes. The ‘screened flocks’, despite strong production performance indicators, exhibited a high mortality rate (mean of 6.01%) and a wide range of pathogens (19 bacteria, 3 viruses, and Eimeria infestation observed). The cluster analysis, after identifying two outliers, defined three flock profiles, linking the observation variables (health, production indicator, necropsy lesions) to the aetiological agents. ‘Screened flocks’ from the first cluster (27 flocks) were described as a flock with high rates of fibrinous lesions, with a high condemnation rate associated with the identification of E. coli. The second cluster (64 flocks) was defined by high production performances but also higher rates of femoral head necrosis. The ‘screened flocks’ from the last cluster (22 flocks) exhibited lower production performance, indicating strong Eimeria spp. infestation and circulation of avian metapneumovirus. Conclusion: The study is an example of how high-dimensional data produced by the broiler industry can be reused and integrated to create contextual knowledge for farmers and veterinarians about endemic contagious diseases. Access to this timely contextual knowledge is essential to enhance disease prevention and management efforts for farmers, veterinarians, and other stakeholders in the broiler industry.

Avian Infectious Bronchitis Virus (IBV) is a highly contagious Gammacoronavirus that poses a significant threat to the global poultry industry. Despite its worldwide prevalence, a critical knowledge gap exists regarding the genetic diversity of IBV in Central Asia, particularly in Uzbekistan. This study is the first comprehensive molecular characterization of IBV in Uzbekistan. This study also provides a unique and informative bioinformatic analysis of the detected strains. Three IBV strains were isolated and identified from chickens suspected of IBV infection. The isolates were identified and subjected to S1 gene sequencing, phylogenetic analysis, recombination screening, selective pressure mapping, and in silico structural and antigenic profiling. Phylogenetic inference revealed that the isolates clustered within the established genotypes GI-1, GI-13, and GI-23. Comparative alignments revealed distinct nucleotide and amino acid substitutions relative to global reference strains. The evolutionary patterns are consistent with a predominantly clonal mode of evolution. Structural modeling and B-cell epitope prediction revealed pronounced antigenic and topological divergence among the Uzbek isolates. Genotype-specific substitutions, particularly in solvent-exposed regions of the spike protein, were associated with altered epitope profiles, implying potential impacts on vaccine cross-protection. These findings contribute to current knowledge of IBV molecular characterization and provide the first reference framework for the Central Asian region. The study highlights the importance of continuous molecular surveillance, region-specific vaccination strategies, and integrated genomic monitoring for novel IBV variants.

Low pathogenic avian influenza viruses (LPAIVs) are typically associated with subclinical or mild disease in poultry. However, recent outbreaks involving atypical LPAIV strains, including H3N1 strains like A/chicken/Belgium/460/2019, have demonstrated severe clinical outcomes despite low intravenous pathogenicity index (IVPI) scores. These findings challenge current classification systems and raise questions about alternative markers of virulence, such as loss of a neuraminidase (NA) glycosylation site linked to plasminogen-binding and haemagglutinin (HA) cleavage. This study compared the pathogenicity of a wild-type H3N1 strain (wtH3N1), isolated from a disease outbreak in Belgium, with a genetically modified, loss-of function variant (mH3N1) carrying a single amino acid substitution (S122N) in NA that blocks plasminogen-dependent cleavage of HA in chickens. Four-week-old pullets and cockerels, and 30-week-old laying hens were inoculated with either wtH3N1 or mH3N1 and clinical signs, egg production, viral replication, post-mortem and tissue pathology were evaluated. Adult hens infected with wtH3N1 showed a complete cessation of egg production, systemic viral replication, and histopathological lesions in the reproductive tract, brain, and kidneys. In contrast, birds infected with mH3N1 displayed only mild, transient reductions in egg production and minimal viral detection. No mortality was observed in any group. All young chickens exhibited subclinical infections. Overall, the S122N mutation significantly attenuated viral virulence and tissue tropism. The study provides functional evidence that position 122 on NA contributes to increased virulence in H3N1 AIV. These findings support the role of molecular markers in risk assessment of non-H5/H7 LPAIVs and highlight the limitations of the current IVPI-based classification system.

Background Endemic contagious diseases in broilers have a significant impact on production performance. However, endemic contagious diseases are multifaceted and complex. They are rarely monitored on a large scale. This complexity hinders their mitigation, as timely information about their distribution and knowledge about their impact on production performance is scarce. This study aimed to evaluate whether data routinely produced by the Polish broiler industry, the first European meat producer, could be reused to generate knowledge about those diseases and provide stakeholders with contextual information to improve their disease management. Results The study reused a dataset collected by a large producer and a veterinary laboratory, which implemented a screening program at the end of the production cycle. The high-dimensional dataset covered 115 flocks produced between 2018 and 2023 across Poland. It contained information on production indicators, health indicators, necropsy lesions, and a list of evidence of infection or infestation by a diverse range of aetiological agents (bacterial, viral, and Eimeria ). The screened flocks, despite strong production performance indicators, presented a higher mortality rate and a large diversity of pathogens. The cluster analysis enabled the identification of three flock profiles, connecting the observation variables (health, production indicator, necropsy lesions) to the aetiological agents. Flocks from the first cluster were described as a flock with high rates of fibrinous lesions, with a high condemnation rate associated with the identification of E. coli. The second cluster was defined by high production performances but also higher rates of femoral head necrosis. The flocks from the last cluster had lower production performance, showing evidence of strong infestation by Eimeria spp. and evidence of avian metapneumovirus circulation. Conclusion The study is an example of how high-dimensional data produced by the broiler industry can be reused and integrated to create contextual knowledge for farmers and veterinarians about endemic contagious diseases. Access to this timely contextual knowledge is essential to enhance disease prevention and management efforts for farmers, veterinarians, and broiler industry stakeholders.

The incursion of high pathogenicity avian influenza A virus (IAV) into US dairy cows is unprecedented in the era of molecular diagnosis and pathogen sequencing. This raises questions over the likelihood of further outbreaks and whether dairy cattle could be a “mixing vessel” for novel strains of IAV. Using a panel of BSL2-safe reassortant viruses representing clade 2.3.4.4b H5 epizootic lineages circulating since 2020, we found that a cow B3.13 isolate displayed enhanced replication in cow mammary gland cells, along with increased viral polymerase activity and stronger interferon antagonism in cow cells compared to an earlier EA-2020-C genotype virus. However, multiple avian and mammalian IAV strains, including other clade 2.3.4.4b high pathogenicity genotypes, were replication competent in bovine cells, particularly those of the mammary gland, suggesting that there is a diverse circulating IAV pool with the potential to infect cows. Moreover, we show that cow mammary cells co-express α-2,3 and α-2,6 – linked sialic acids, and are susceptible to co-infection with human and avian IAVs. We conclude that the US cow influenza outbreak does not simply reflect a unique adaptation of the B3.13 genotype virus; rather, the bovine udder represents a permissive niche for IAV and a plausible site for reassortment, underscoring its potential role in generating novel influenza viruses with pandemic risk.

Avian influenza A viruses (AIV) significantly impact both animal and human health. Reliable diagnostics are crucial for controlling AIV, including the highly pathogenic strains like H5Nx. In this study, we developed and validated the on-site Alveo Sense Poultry Avian Influenza Tests to rapidly detect the AIV M-gene and subtypes H5, H7, and H9 in unprocessed samples using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) and impedance-based measurements. The Alveo Sense tests, using single-use microfluidic cartridges, deliver results within 45 min. Each cartridge includes assays for the AIV M gene and specific H5 and H7 or H9 subtypes, with internal process controls. The laboratory validation involved specificity, limit of detection (LoD), diagnostic sensitivity, reproducibility, and robustness tests using various AIV strains, other avian pathogens, and field samples. The assays showed 100% specificity for AIV subtypes without cross-reactivity with non-AIV pathogens. The LoD95 for H5, H7, and H9 ranged between RT-PCR Ct values of 29–33 in both cloacal and oropharyngeal samples and were able to detect avian influenza virus in both spiked samples and field samples. Reproducibility and repeatability studies showed perfect agreement across operators and laboratories and remained stable and accurate under different pre-analytical conditions. The Alveo Sense tests offer rapid, accurate, and reliable on-site diagnostics for AIV subtypes H5, H7, and H9 on samples from fresh dead and sick birds, valuable for early flock-level detection and outbreak control. Further field validation will improve the understanding of their diagnostic performance across various avian species.

Avian influenza A viruses (AIV) significantly impact both animal and human health. Reliable diagnostics are crucial for controlling AIV, including the highly pathogenic strains like H5Nx. In this study, we developed and validated the on-site Alveo Sense Poultry Avian Influenza Tests to rapidly detect the AIV M-gene and subtypes H5, H7, and H9 in unprocessed samples using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) and impedance-based measurements. The Alveo Sense tests, using single-use microfluidic cartridges, deliver results within 45 minutes. Each cartridge includes assays for the AIV M gene and specific H5 and H7 or H9 subtypes, with internal process controls. The laboratory validation involved specificity, limit of detection (LoD), diagnostic sensitivity, reproducibility, and robustness tests using various AIV strains, other avian pathogens, and field samples. The assays showed 100% specificity for AIV subtypes without cross-reactivity with non-AIV pathogens. The LoD95 for H5, H7, and H9 ranged between RT-PCR Ct values of 29-33 in both cloacal and oropharyngeal samples and were able to detect avian influenza virus in both spiked samples and field samples. Reproducibility and repeatability studies showed perfect agreement across operators and laboratories and remained stable and accurate under different pre-analytical conditions. The Alveo Sense tests offer rapid, accurate, and reliable on-site diagnostics for AIV subtypes H5, H7, and H9 on samples from fresh dead and sick birds, valuable for early flock-level detection and outbreak control. Further field validation will improve the understanding of their diagnostic performance across various avian species.