Benedetti, S. | bioRxiv Science

bioRxiv2025

Inactivation of Influenza A Viruses (H1N1, H5N1) During Grana-Type Raw Milk Cheesemaking: Implications for Foodborne Transmission Risk

BackgroundThe detection of H5N1 highly pathogenic avian influenza virus (HPAIV) in lactating dairy cattle in the United States, with high viral titers in raw milk, has raised concerns about potential zoonotic transmission through the consumption of unpasteurized milk and raw-milk dairy products. While inactivation studies exist for pasteurized milk, data on virus persistence during the manufacture of raw-milk cheeses remain scarce. AimTo evaluate the survival and inactivation of avian influenza viruses (AIV), including both low pathogenic (LPAIV, H1N1) and highly pathogenic (HPAIV, H5N1) strains, during the production and ripening of Grana-type hard cheeses made from raw cows milk. MethodsExperimental cheesemaking was conducted using raw milk artificially contaminated with A/duck/Italy/281904-2/06 (H1N1; 107.75 EID50/mL) or A/duck/Italy/326224-2/22 (H5N1 clade 2.3.4.4b; 106.75 EID50/mL). Cheeses were produced in accordance with Parmigiano Reggiano production standards and ripened for 30 days at 5-6 {degrees}C. Viral presence was assessed in finished cheeses by inoculation on SPF embryonated chicken eggs (ECE), hemagglutination (HA) assay, and monoclonal antibody-based ELISA. ResultsNo infectious virus was detected in any cheese sample produced from contaminated milk following two blind passages in SPF-ECE. Both HA and ELISA tests yielded negative results, indicating complete inactivation of the virus. ConclusionThis study demonstrates that the traditional Grana-type cheese production process-- including curd cooking, acidification, and ripening--effectively inactivates both LPAIV and HPAIV, even at high contamination levels. These findings support the microbiological safety of hard cheeses made from raw milk with regard to AIV, contributing to risk assessment and food safety policies during avian influenza outbreaks.

microbiology↗

bioRxiv2022

Molecular genetics of GLUT1DS Italian pediatric cohort: 10 novel related-disease variants and structural analysis

GLUT1 deficiency syndrome (GLUT1DS1; OMIM #606777) is a rare genetic metabolic disease, characterized by infantile-onset epileptic encephalopathy, global developmental delay, progressive microcephaly and movement disorders (e.g. spasticity and dystonia). It is caused by heterozygous mutations in the SLC2A1 gene, which encodes the GLUT1 protein, a glucose transporter across the blood-brain barrier (BBB). Most commonly these variants arise de novo resulting in sporadic cases, although several familial cases with AD inheritance pattern have been described. Twenty-seven Italian pediatric patients clinically suspect of GLUT1DS from both sporadic and familial cases have been enrolled. We detected by trios sequencing analysis 25 different variants causing GLUT1DS. Of these, 40% of the identified variants (10 out of 25) had never been reported before, including missense, frameshift and splice site variants. Their X-ray structure analyses strongly suggested the potential pathogenic effects of these novel disease-related mutations, broadening the genotypic spectrum heterogeneity found in the SLC2A1 gene. Moreover, 24% is located in a vulnerable region of the GLUT1 protein that involves transmembrane 4 and 5 helices encoded by exon 4, confirming a mutational hotspot in the SLC2A1 gene. Lastly, we investigated possible correlations between mutation type and clinical and biochemical data observed in our GLUT1DS cohort, revealing that splice site and frameshift variants are related to a more severe phenotype and low CSF parameters. Author summaryWe investigated the molecular data of 27 pediatric patients clinically suspect of GLUT1 deficiency syndrome. By performing trios sequencing analysis, we highlighted ten novel disease-related variants, and their X-ray structure analyses, suggesting the pathogenic effects of these identified mutations. Moreover, the wide clinical and genetic heterogeneity observed in our cohort allowed possible correlations between mutation type and clinical and biochemical data. This analysis enabled to delineate that splice site and frameshift variants are related to a more severe phenotype and low CSF/glucose values. Further clinical and genetic/epigenetics studies could clearly the high phenotypic variability observed in these patients.

genetics↗