Biology subjects

Sives, S.

Publications and source records attributed to Sives, S..

3 recordsLinked to original sources

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↗

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↗