Biology subjects

Marotta, M. G.

Publications and source records attributed to Marotta, M. G..

3 recordsLinked to original sources

bioRxiv2026

Pseudotyped virus-based platform and structural analysis reveal potential cross-reactivity sites between influenza C and D viruses

Influenza C (ICV) and influenza D (IDV) viruses belong to the Orthomyxoviridae family and are classified in the genera Gammainfluenzavirus and Deltainfluenzavirus, respectively. Although the main reservoir of ICV is humans, IDV is mainly found in cattle. To date, the zoonotic potential of IDV has not been fully elucidated. ICV and IDV share about 50% homology at the genetic level, and both express hemagglutinin esterase fusion (HEF) glycoproteins on the surface for the dual purpose of binding the receptor and releasing new virions. Using pseudotyped viruses (PVs) in a pseudotyped virus-based microneutralisation assay (pMN), some bovine serum samples showed strong neutralisation of both ICV and IDV. In silico analyses were performed to explore the molecular basis of this phenomenon. HEF structures were recovered from the Protein Data Bank, epitopes were predicted using BepiPred, and sialic acid receptor docking was evaluated with HDOCK. Five potential epitopes were selected, and mutual substitutions of amino acid residues were introduced to generate mutant ICV and IDV HEFs and corresponding PVs. Although only mutant IDV PVs were successfully produced, a reference ICV antiserum showed high neutralising activity against one construct, indicating the exposure of an ICV-like antigenic site within the IDV framework. Herein we provide evidence consistent with the existence of antigenic sites shared between ICV and IDV, which could be exploited for cross-protective vaccine design, through integrated computational and experimental investigations.

immunology↗

bioRxiv2026

Serine proteases are required to activate influenza D virus haemagglutinin-esterase fusion (HEF) protein

Influenza D virus (IDV), the most recently identified member of the Orthomyxoviridae, was first isolated from pigs but cattle have been identified as the reservoir host. To date, IDV has not been confirmed to cause human disease. Like the haemagglutinin (HA) of influenza A virus (IAV) and the haemagglutinin-esterase fusion (HEF) protein of influenza C virus (ICV), the IDV HEF is produced as a precursor protein (HEF0) that must be proteolytically cleaved by host cell proteases (into HEF1 and HEF2) to gain its fusion capacity. The proteases that activate IAV HA have been extensively studied, but those responsible for activation of IDV HEF were unknown. Identifying these proteases is key to understanding early virus-host interactions and host restriction. Therefore, we generated ICV and IDV pseudotyped viruses (PVs) in HEK 293T producer cells with or without co-transfection of plasmids expressing different type II serine proteases. Subsequent transduction of swine testicular (ST) cells indicated strong activation of both ICV and IDV PVs by the human airway trypsin-like protease (HAT) and its swine homologue (swAT). Furthermore, like influenza A/Puerto Rico/8/34 (H1N1) virus, addition of exogenous protease is not essential for IDV replication in MDCK II cells, most likely due to endogenous expression of matriptase. In conclusion, our data unveil new information on host cell proteases that activate ICV and IDV HEF proteins. Importantly, the data suggest that protease specificity is not a factor in restriction of IDV replication in the human upper respiratory tract.

microbiology↗

bioRxiv2025

Development and optimisation of Influenza C and Influenza D pseudotyped viruses

To facilitate the study of influenza C (ICV) and influenza D (IDV) viruses, we generated lentiviral pseudotyped viruses (PVs) expressing the hemagglutinin-esterase fusion (HEF) glycoprotein from ICV (C/Minnesota/33/2015) and IDV (D/Swine/Italy/199724-3/2015, D/Bovine/France/5920/2014, and D/Bovine/Ibaraki/7768/2016). The production of these PVs was optimised using different amount of human airway trypsin-like (HAT) protease to enhance HEF maturation, and the transduction efficiency was evaluated in multiple cell lines. Using these PVs, we established a pseudovirus-based microneutralisation (pMN) assay to measure neutralising antibody responses and adapted an esterase activity assay to evaluate PV. Specific antisera neutralised PVs but failed to inhibit esterase activity. These findings confirm that ICV and IDV PVs provide a scalable, sensitive, and safe tool for antiviral screening, and sero-epidemiological research.

immunology↗