Advancing Alphamesonivirus-1 investigation in Italy through molecular and serological approaches

Mesoniviruses are positive-sense, single-stranded RNA viruses classified within the order Nidovirales (1) and have long been regarded as mosquito-associated viruses with a host range limited to invertebrates. This view has been challenged by the detection of Alphamesonivirus-1 (AMV-1) in lung and lymph node tissues of horses that died from severe respiratory disease in Italy (2), suggesting a possible ability to infect vertebrate hosts and raising questions about its ecology and epidemiological relevance. Following molecular and genomic investigations and the development of a specific real-time RT-PCR assay, insect samples were screened, demonstrating that AMV-1 is actively circulating within the Italian territory. Since virus isolation from the horse-derived material was not successful, a previously insect-derived AMV-1 isolate was instead inoculated onto different mammalian cell lines cultured at 37 °C, where three blind passages were performed to assess its replication capability; however, no evidence of productive viral growth was observed, suggesting limited replication in standard vertebrate systems or the need for specific, still unidentified conditions. To establish reliable serological assays for AMV-1, hyperimmune sera were generated as a key resource. Hyperimmune sera were raised in both rabbits and guinea pigs to obtain reliable positive controls and were essential for assay setup. On this basis, serological methods were established, including enzyme-linked immunosorbent assay (ELISA) and a focus reduction neutralization test (FRNT). ELISA detects AMV-1-specific antibodies through antigen–antibody binding and colorimetric signal development, providing a rapid high-throughput screening tool. The FRNT quantifies neutralizing antibodies by measuring the reduction of viral foci in infected cell monolayers, offering a sensitive and specific assessment of functional immune responses. These assays enable the screening of animal populations for AMV-1-specific antibodies and support seroepidemiological investigations aimed at evaluating virus circulation beyond insect vectors. Collectively, these approaches complement molecular tools and provide a framework for investigating AMV-1 biology, host interactions, and potential animal health risks. 1. Lauber, C., Ziebuhr, J., Junglen, S., Drosten, C., Zirkel, F., Nga, P. T., Morita, K., Snijder, E. J., & Gorbalenya, A. E. (2012). Mesoniviridae: a proposed new family in the order Nidovirales formed by a single species of mosquito-borne viruses. Archives of virology, 157(8), 1623–1628. https://doi.org/10.1007/s00705-012-1295-x 2. Jurisic, L., Auerswald, H., Marcacci, M., Di Giallonardo, F., Coetzee, L. M., Curini, V., Averaimo, D., Ortiz-Baez, A. S., Cammà, C., Di Teodoro, G., Richt, J. A., Holmes, E. C., & Lorusso, A. (2025). Insect-specific Alphamesonivirus-1 (Mesoniviridae) in lymph node and lung tissues from two horses with acute respiratory syndrome. Journal of virology, 99(2), e0214424. https://doi.org/10.1128/jvi.02144-24