Biology subjects

Vervelde, L.

Publications and source records attributed to Vervelde, L..

10 recordsLinked to original sources

bioRxiv2025

Synergy between HA cleavage site sequence and NA-mediated plasminogen recruitment as a virulence mechanism for low pathogenic avian influenza

An outbreak of H3N1 low pathogenic avian influenza virus (LPAIV) in Belgium in 2019 caused unexpected levels of mortality and morbidity in poultry. These viruses possess an NA polymorphism associated with plasminogen binding, as well as an unusual sequence around the HA cleavage site; accordingly, HA cleavage mediated by NA-driven plasminogen recruitment has been proposed to underly their systemic spread and pathogenicity. To test this, we established a reverse genetics system for A/chicken/Belgium/460/2019 and created single mutations in HA (K345R), and NA (S122N) that restored the viruses to normal consensus, as well as an HA/NA double mutant. Confirming previous work, trypsin-independent spread and HA cleavage of wild type Ck/Belgium was observed in the presence of fetal bovine serum containing plasminogen in vitro. Dose-dependent HA cleavage and trypsin-independent spread was also observed in the presence of purified chicken plasminogen. Compared to wild type virus, both HA cleavage and virus spread in vitro were reduced by the HA K345R mutation and further blocked by NA mutation S122N. Plasminogen-mediated HA cleavage was seen in a variety of avian cell lines and chicken organoids, excluding cell type-dependent effects. Furthermore, in ovo tests showed that mutant viruses unable to recruit plasminogen were less able to replicate systemically in chicken embryos. Bioinformatics analyses revealed other viruses which could potentially recruit plasminogen, including two independent outbreaks of H6N1 viruses, one of which we confirmed PLG-driven spread in vitro. We conclude that PLG-recruitment by NA is a general virulence mechanism of N1 LPAIVs Author summaryAvian influenza viruses (AIV) are divided into two broad categories - high or low pathogenicity - based on the sequence of their haemagglutinin (HA) and their lethality in chickens. The majority of AIV strains circulating in the wild are low pathogenicity both in waterfowl and when they spill over into domestic poultry. However, some low pathogenicity strains can cause severe disease in poultry despite not being classified as H5 or H7 high pathogenic AIVs with an HA polybasic protease cleavage site. A severe 2019 outbreak of an H3N1 strain has been suggested to result from the neuraminidase (NA) of the virus recruiting cellular plasminogen to proteolytically activate HA. Here, we tested this hypothesis by using reverse genetics to mutate the virus in a way predicted to block this. We found that indeed, the sequence of the NA at position 122 is the primary determinant of plasminogen-driven HA cleavage but that the unusual sequence at the HA cleavage site of the outbreak virus also contributes to pathogenicity. Furthermore, we show that N1 NA sequence can be used to identify other unexpectedly virulent strains of AIV. This work therefore adds to our ability to risk assess AIV strains from sequence-based surveillance.

microbiology↗

bioRxiv2025

Interleukin 10 controls the balance between tolerance, pathogen elimination and immunopathology in birds.

Effective mucosal immunity in the intestine involves a fine balance between tolerance of the microbiome, recognition and elimination of pathogens, and inflammatory tissue injury. The anti-inflammatory cytokine IL10 regulates these processes in the intestines of mice and humans; the anti-inflammatory activity of IL10 is also conserved in birds. To determine the function of IL10 in avian mucosal immunity, we generated germ line modifications of the chicken IL10 locus to abolish or reduce IL10 expression. In vitro analysis of macrophage response to lipopolysaccharide confirmed the loss of IL10 protein expression, the lack of dosage compensation in heterozygotes, and prevention of autocrine inhibition of nitric oxide production in homozygous IL10 knockout macrophages. IL10-deficiency significantly altered the composition of the caecal microbiome, but unlike IL10-deficient mice and humans, IL10-deficient chickens did not exhibit spontaneous colitis. Following experimental challenge with Salmonella enterica serovar Typhimurium or Campylobacter jejuni in IL10-deficient chickens, enhanced clearance of the pathogens was associated with elevated transcription of pro-inflammatory genes and increased infiltration of inflammatory cells into gut mucosa. In IL10-deficient chickens challenged with the parasite Eimeria tenella, pathogen clearance was accelerated but caecal lesions were more severe and weight gain was compromised. Neither the heterozygous IL10 knockout nor a homozygous IL10 enhancer mutation had a major effect on pathogen clearance or inflammation in any of the challenge models. Our findings highlight the intrinsic compromise in mucosal immune response and have important implications for the development of strategies to combat avian and zoonotic pathogens in poultry.

immunology↗

bioRxiv2024

Infectious bronchitis virus vaccination, but not the presence of XCR1, is correlated with large differences in chicken caecal microbiota

The chicken immune system and microbiota play vital roles in maintaining gut homeostasis and protecting against pathogens. In mammals, XCR1+ conventional dendritic cells (cDCs) are located in the gut-draining lymph nodes and play a major role in gut homeostasis. These cDCs sample antigens in the gut luminal contents and limit the inflammatory response to gut commensal microbes by generating appropriate regulatory and effector T-cell responses. We hypothesised that these cells play similar roles in sustaining gut homeostasis in chickens, and that chickens lacking XCR1 were likely to contain a dysbiotic caecal microbiota. Here we compare the caecal microbiota of chickens that were either heterozygous or homozygous XCR1 knockouts, that had or had not been vaccinated for infectious bronchitis virus (IBV). We used short-read (Illumina) and long-read (PacBio HiFi) metagenomic sequencing to reconstruct 670 high-quality, strain-level metagenome assembled genomes. We found no significant differences between alpha-diversity or the abundance of specific microbial taxa between genotypes. However, IBV vaccination was found to correlate with significant differences in the richness and beta-diversity of the microbiota, and to the abundance of forty bacterial genera. In conclusion, we found that a lack of XCR1 was not correlated with significant changes in the chicken microbiota, but IBV vaccination was. Data SummaryThe raw sequencing data for this project, as well as primary assemblies, putative genome bins and species-level MAGs, are available in the European Nucleotide Archive under project PRJEB64517. Strain-level MAGs are available through Edinburgh DataShare (https://doi.org/10.7488/ds/7678). Impact statementChickens play a vital role in global food systems, with 74 billion chickens killed for meat and 1.6 trillion chicken eggs produced in 2021 alone. The gut microbiota plays a vital role in the health and nutrition of the chicken, contributing to gut homeostasis and the production of nutrients that can be absorbed and used by the host bird. The chicken gut microbiota represents an excellent target to improve pathogen resistance and nutrition, as farmed chickens are not exposed to a maternal hen and thereby develop a low-diversity gut microbiota that negatively impacts their immune development and gut health. In order to develop such microbiota interventions, we first need to understand the fundamental biology behind immune-microbiota interactions. While it is well known that the chicken gut microbiota plays an important role in gut homeostasis, the mechanisms behind this phenomenon are poorly understood. In mammals, XCR1+ conventional dendritic cells have been shown to play a role in maintaining gut homeostasis. In this study we compare the gut microbiota of heterozygous and homozygous XCR1 knockout chickens that did or did not receive infectious bronchitis virus vaccination. We found that the gut microbiota of heterozygous and homozygous XCR1 knockout chickens did not differ significantly, but IBV vaccination did significantly correlate with differences in the microbiota composition.

microbiology↗

bioRxiv2023

Reinstatement of RIG-I in chickens via genetic modification reveals new insights into the dynamic evolution of avian immune sensors

Retinoic acid-inducible gene I (RIG-I) activates mitochondrial antiviral signaling proteins, initiating the antiviral response. RIG-I and RNF135, a ubiquitin ligase regulator, are missing in domestic chickens but conserved in mallard ducks. The chickens RIG-I loss was long believed to be linked to increased avian influenza susceptibility. We reinstated both genes in chickens and examined their susceptibility to infection with an H7N1 avian influenza virus. Uninfected RIG-I-expressing chickens exhibited shifts in T and B cells. At the same time, the H7N1 infection led to severe disease, persistent weight loss, and increased viral replication compared to wild-type chickens. The simultaneous expression of RIG-I and RNF135 potentiated the RIG-I activity and was associated with exacerbated inflammatory response and increased mortality without influencing virus replication. Additional animal infection experiments with two other avian influenza viruses validated these findings. They confirmed that the harmful effects triggered by RIG-I or RIG-I-RNF135-expression require a minimum degree of viral virulence. Our data indicate that the loss of RIG-I in chickens has likely evolved to counteract deleterious inflammation caused by viral infection and highlight an outcome of restoring evolutionary lost genes in birds. Significance StatementThe evolutionary loss of a crucial innate immune sensor like RIG-I in domestic chickens and its presence in closely related avian species such as ducks has long puzzled researchers. We genetically reinstated RIG-I in chickens, alongside its ubiquitination factor, RNF135, to uncover their roles in responding to influenza virus interactions in chickens. Our findings suggest that the loss of RIG-I in chickens may have occurred as an adaptive strategy to mitigate harmful inflammation associated with influenza infection. We shed light on an outcome of reinstating evolutionarily lost genes in birds and open new avenues for understanding immune responses in vertebrates.

immunology↗

bioRxiv2023

Disentangling the Innate Immune Responses of Intestinal Epithelial Cells and Lamina Propria Cells to Salmonella Typhimurium Infection in Chickens

Salmonella enterica serovar Typhimurium (STm) is a major foodborne pathogen and poultry are a key reservoir of human infections. To understand the host responses to early stages of Salmonella infection in poultry, we infected 2D and 3D enteroids, the latter of which contains leukocytes, neurons, and mesenchymal cells that are characteristic of the lamina propria. We infected these enteroids with wild-type (WT STm), a non-invasive mutant lacking the prgH gene ({Delta}prgH STm), or treated them with STm lipopolysaccharide (LPS) and analysed the expression of innate immune related genes by qPCR at 4 and 8 h. The localisation of ZO-1 expression was disrupted in WT STm infected enteroids but not{Delta} prgH STm or LPS treated enteroids, suggesting a loss of barrier integrity. The innate immune response to LPS was more pronounced in 2D enteroids compared to 3D enteroids and by 8 hpi, the response in 3D enteroids was almost negligible. However, when STm adhered to or invaded the enteroids, both 2D and 3D enteroids exhibited an upregulation of inflammatory responses. The presence of lamina propria cells in 3D enteroids resulted in the unique expression of genes associated with immune functions involved in regulating inflammation. Moreover, 2D and 3D enteroids showed temporal differences in response to bacterial invasion or adherence. At 8 hpi, innate responses in 3D but not 2D enteroids continued to increase after infection with WT STm, whereas the responses to the non-invasive strain decreased at 8 hpi in both 2D and 3D enteroids. In conclusion, STm infection of chicken enteroids recapitulated several observations from in vivo studies of Salmonella-infected chickens, including altered epithelial barrier integrity based on ZO-1 expression and inflammatory responses. Our findings provide evidence that Salmonella-infected enteroids serve as effective models for investigating host-pathogen interactions and exploring the molecular mechanisms of microbial virulence although the 3D model mimics the host more accurately due to the presence of a lamina propria.

immunology↗

bioRxiv2023

Novel technology to develop non-transmissible live influenza virus vaccines through alterations in M2/M42 ion channel expression

Continued global outbreaks of H5Nx highly pathogenic avian influenza viruses (HPAIV) have been reported in poultry since the emergence of the Asian Goose/Guangdong lineage of HPAIV H5N1 in 1996. Consequently, vaccines have been developed and employed to protect commercial and non-commercial poultry flocks. However, constant changes in virus immunogenetics makes vaccine candidates that prevent morbidity, mortality, reduce shedding, and prevent transmission a moving target. Here, we tested the effects of disrupting the mRNA splice sites and thus expression of either of the two isoforms of the viral ion channel M2 and M42 expressed by the H5N2 low pathogenic avian influenza virus (LPAIV) progenitor of the 1983 Pennsylvania HPAIV outbreak. Both G52C ({Delta}M2) and G145A ({Delta}M42) versions of the A/chicken/Pennsylvania/1/83 (Ck/Penn) virus replicated well in vitro and in ovo, but showed altered virion morphology, with the G145A virus exhibiting filamentous budding. The G52C and G145A viruses also infected chickens efficiently and stimulated robust immune responses; however, unlike the wild type virus, they did not transmit to naive-contact birds. In protection studies, vaccination of birds with G52C or G145A viruses protected 100% of birds from lethal challenge with homologous and distant heterologous H5 HPAIV strains from North American and Asian lineages and significantly reduced virus shedding compared to controls. Furthermore, the live virus vaccines decreased virus shedding 10,000-fold more than corresponding inactivated forms of the vaccine virus. Taken together, these studies demonstrate a strategy for developing a non-transmittable but highly protective live attenuated virus for AIV.

microbiology↗

bioRxiv2022

CpG enrichment as a vaccine strategy for influenza A virus

Synonymous recoding of RNA virus genomes is a promising approach for generating attenuated viruses to use as vaccines. Problematically, recoding typically hinders virus growth, but this may be rectified using CpG dinucleotide enrichment. CpGs are recognised by cellular zinc-finger antiviral protein (ZAP), and so in principle, removing ZAP sensing from a virus propagation system will reverse attenuation of a CpG-enriched virus, enabling high titre yield of a vaccine virus. We tested this using a vaccine strain of influenza A virus (IAV) engineered for increased CpG content in genome segment 1. Virus attenuation was mediated by the short isoform of ZAP, correlated with the number of CpGs added, and was enacted via turnover of viral transcripts. The CpG-enriched virus was strongly attenuated in mice, yet conveyed protection from a potentially lethal challenge dose of wildtype virus. Importantly for vaccine development, CpG-enriched viruses were genetically stable during serial passage. Unexpectedly, in both MDCK cells and embryonated hens eggs that are used to propagate live attenuated influenza vaccines, the ZAP-sensitive virus was fully replication competent. Thus, ZAP- sensitive CpG enriched viruses that are defective in human systems can yield high titre in vaccine propagation systems, providing a realistic, economically viable platform to augment existing live attenuated vaccines. AUTHOR SUMMARYCpG dinucleotides are under-represented in vertebrate genomes, wherein cytosines in the CpG conformation are methylated to regulate transcription. Methylated cytosines are prone to deamination, resulting in TpG dinucleotides replacing CpGs. The resultant CpG suppression has provided a route by which vertebrate cells can recognise RNA from invading pathogens, using cellular Zinc-finger Antiviral Protein (ZAP) as a CpG sensor. Vertebrate-infecting RNA viruses also genomically suppress CpGs, and it is believed that this is an evolved trait to evade detection by ZAP. Here, we engineered an influenza A virus (IAV) with elevated CpG content and characterised how this impacts viral replication. CpG addition resulted in viral attenuation, mediated by ZAP activity. CpG suppression is conserved in dog and chicken genomes (relevant for live attenuated IAV vaccine propagation), and it is logical to predict that ZAP-mediated CpG sensing would also be conserved in these species. However, when we propagated ZAP-sensitive IAV in cognate culture systems, we saw no replication defect. This unexpected result raises questions about why viruses infecting these species suppress CpG in their genomes, and importantly delivers a new, tractable approach to augment rational live attenuated IAV vaccine design.

microbiology↗

bioRxiv2019

Kinetics of the immune response to Eimeria maxima in relatively resistant and susceptible chicken lines

Eimeria maxima is a common cause of coccidiosis in chickens, a disease which has a huge economic impact on poultry production. Knowledge of immunity to E. maxima and the specific mechanisms that contribute to differing levels of resistance observed between chicken breeds and between congenic lines derived from a single breed of chickens is required. This study aimed to define differences in the kinetics of the immune response of two inbred lines of White Leghorn chickens that exhibit differential resistance (line C.B12) or susceptibility (line 15I) to infection by E. maxima. Line C.B12 and 15I chickens were infected with E. maxima and transcriptome analysis of infected jejunal tissue was carried out at 2, 4, 6 and 8 days post-infection (dpi). RNA-Seq analysis revealed differences in the rapidity and magnitude of cytokine transcription responses post-infection between the two lines. In particular, IFN-{gamma} and IL-10 transcripts in the jejunum accumulated earlier in line C.B12 (at 4 dpi) compared to line 15I (at 6 dpi). Line C.B12 chickens exhibited increases of IFNG and IL10 mRNA in the jejunum at 4 dpi, whereas in line 15I transcription was delayed but increased to a greater extent. RT-qPCR and ELISAs confirmed the results of the transcriptomic study. Higher serum IL-10 correlated strongly with higher E. maxima replication in line 15I compared to line C.B12 chickens. Overall, the findings suggest early induction of the IFN-{gamma} and IL-10 responses, as well as immune-related genes at 4 dpi identified by RNA-Seq, may be key to resistance to E. maxima.

immunology↗

bioRxiv2018

Mutation of influenza A virus PA-X decreases pathogenicity in chicken embryos and can increase the yield of reassortant candidate vaccine viruses

The PA-X protein of influenza A virus has roles in host cell shut-off and viral pathogenesis. While most strains are predicted to encode PA-X, strain-dependent variations in activity have been noted. We found that PA-X protein from A/PR/8/34 (PR8) strain had significantly lower repressive activity against cellular gene expression compared with PA-Xs from the avian strains A/turkey/England/50-92/91 (H5N1) (T/E) and A/chicken/Rostock/34 (H7N1). Loss of normal PA-X expression, either by mutation of the frameshift site or by truncating the X-ORF, had little effect on the infectious virus titre of PR8 or PR8 7:1 reassortants with T/E segment 3 grown in embryonated hens eggs. However, in both virus backgrounds, mutation of PA-X led to decreased embryo mortality and lower overall pathology; effects that were more pronounced in the PR8 strain than the T/E reassortant, despite the low shut-off activity of the PR8 PA-X. Purified PA-X mutant virus particles displayed an increased ratio of HA to NP and M1 compared to their WT counterparts, suggesting altered virion composition. When the PA-X gene was mutated in the background of poorly growing PR8 6:2 vaccine reassortant analogues containing the HA and NA segments from H1N1 2009 pandemic viruses or an avian H7N3 strain, HA yield increased up to 2-fold. This suggests that the PR8 PA-X protein may harbour a function unrelated to host cell shut-off and that disruption of the PA-X gene has the potential to improve the HA yield of vaccine viruses.\n\nIMPORTANCEInfluenza A virus is a widespread pathogen that affects both man and a variety of animal species, causing regular epidemics and sporadic pandemics with major public health and economic consequences. A better understanding of virus biology is therefore important. The primary control measure is vaccination, which for humans, mostly relies on antigens produced in eggs from PR8-based viruses bearing the glycoprotein genes of interest. However, not all reassortants replicate well enough to supply sufficient virus antigen for demand. The significance of our research lies in identifying that mutation of the PA-X gene in the PR8 strain of virus can improve antigen yield, potentially by decreasing the pathogenicity of the virus in embryonated eggs.

microbiology↗

bioRxiv2018

Characterisation of subpopulations of chicken mononuclear phagocytes that express TIM4 and the macrophage colony-stimulating factor receptor (CSF1R)

The phosphatidylserine receptor, TIM4, encoded by TIMD4, mediates the phagocytic uptake of apoptotic cells. We applied anti-chicken TIM4 monoclonal antibodies, in combination with CSF1R reporter transgenes to dissect the function of TIM4 in chick (Gallus gallus). During development in ovo, TIM4 was present on the large majority of macrophages but expression became more heterogeneous post-hatch. Blood monocytes expressed KUL01, class II MHC and CSF1R-mApple uniformly. Around 50% of monocytes were positive for surface TIM4. They also expressed many other monocyte-specific transcripts at a higher level than TIM4- monocytes. In liver, highly-phagocytic TIM4hi cells shared many transcripts with mammalian Kupffer cells and were associated with uptake of apoptotic cells. Although they expressed CSF1R mRNA, Kupffer cells did not express the CSF1R-mApple transgene, suggesting that additional CSF1R transcriptional regulatory elements are required by these cells. By contrast, CSF1R-mApple was detected in liver TIM4lo and TIM4- cells which were not phagocytic and were more abundant than Kupffer cells. These cells expressed CSF1R, alongside high levels of FLT3, MHCII, XCR1 and other markers associated with conventional dendritic cells (cDC) in mice. In bursa, TIM4 was present on the cell surface of two populations. Like Kupffer cells, bursal TIM4hi phagocytes co-expressed many receptors involved in apoptotic cell recognition. TIM4lo cells appear to be a sub-population of bursal B cells. In overview, TIM4 is associated with phagocytes that eliminate apoptotic cells in the chick. In the liver, TIM4 and CSF1R reporters distinguished Kupffer cells from an abundant population of DC-like cells.

immunology↗