Biology subjects

del Pozo, J.

Publications and source records attributed to del Pozo, J..

3 recordsLinked to original sources

bioRxiv2025

Characterisation of the Tilapia Lake Virus proteome and identification of an 11th protein, S9-F3

Tilapia Lake Virus (TiLV) is an emerging negative-sense, single-stranded RNA virus that poses a significant threat to global tilapia aquaculture. Of the proteins encoded by its ten genomic segments, the first four, which encode a viral polymerase (segments 1-3) and a nucleoprotein (segment 4) have been characterised. However, the functions of the polypeptides encoded by the remaining segments remain largely unknown. Here, we systematically investigated the expression and subcellular localisation of all ten predicted TiLV-encoded proteins using in vitro translation and expression in mammalian and fish cells as well as mass spectrometry of virus infected cells. These approaches confirmed the synthesis of major polypeptides from each segment and identified an additional 11th protein, S9-F3, translated from an alternative reading frame of segment 9. Microscopy of individually expressed green fluorescent protein (GFP)-tagged constructs revealed that S2 and S10 polypeptides were predominantly nuclear, while S1, S3, S5, S8 and S9-F3 were mostly cytoplasmic with S5 and S6 displaying perinuclear foci. Bioinformatic analysis suggested a potential nuclear export signal in S9-F3 and use of the inhibitor leptomycin B confirmed its CRM1-dependent nuclear export. Evolutionary analyses indicated that both S9 and S9-F3 are under selective pressure, as well as the presence of an S9-F3 homologue in a TiLV-like guppy virus. These findings uncover an alternative translation product and a regulated nuclear export mechanism in TiLV, providing new insights into the molecular biology of this virus and its interaction with host cellular pathways.

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↗

bioRxiv2023

Developing a safe 15-hour preservation protocol of donor kidneys using normothermic machine perfusion

IntroductionO_ST_ABSBackgroundC_ST_ABSNormothermic machine perfusion (NMP) offers a superior alternative to existing hypothermic preservation strategies but is currently limited to 1-3 hours. Extending the time a kidney can be sustained using this technology could electivise transplantation, and enable physiological assessments of renal function. We aimed to develop a protocol that allows the safe preservation of donor kidneys for 12 hours using this technique. MethodsPorcine kidneys (n=20) were retrieved and flushed with 1L preservation solution before being stored on ice. Following a cold ischaemic time of 3.5 hours, kidneys were placed onto a NMP circuit and perfused for 12 hours. Renal haemodynamics, biochemistry and urine output were recorded and analysed. At the end of perfusion, kidneys were scored based on the clinical assessment score and their suitability for transplant determined. Biopsies were collected at the end for histological assessment. ResultsAll kidneys were successfully perfused with immediate recordable renal blood flow (RBF). RBF continually improved over the course of the perfusions, peaking at 12 hours, and negatively correlated with intra-renal resistance. Perfusate sodium concentrations remained stable and within physiological parameters. Sodium bicarbonate increased over time with a corresponding decrease in lactate concentrations, demonstrating active renal gluconeogenesis and Cori cycle processes. Urine production began immediately in all kidneys and was sustained throughout, indicating active renal function. Under the clinical perfusion assessment score, all kidneys received a score of 1 and would be considered suitable for transplantation. Histological assessment revealed kidneys were injury free with REMUZZI scores of 0 in all samples. ConclusionWe have developed an NMP protocol that safely preserves donor kidneys for over 15 hours. Successful perfusion was achieved with stable haemodynamics, blood-perfusate biochemistry, and maintained urine output. Importantly, kidneys remained in optimal health, with no evidence of injury. This protocol may enable the electivisation of transplantation, while reducing ischaemic injury associated with static cold storage.

physiology↗