Microbiology

Cricket paralysis virus (CrPV), a member of the family Dicistroviridae, is a single-stranded positive-sense RNA virus that primarily infects arthropods. Some members of the dicistrovirus family, including the honey bee viruses Israeli acute paralysis virus and Acute bee paralysis virus and the shrimp-infecting Taura syndrome virus, pose significant threats to agricultural ecosystems and economies worldwide. Dicistrovirus infection in Drosophila is used as a model system to study fundamental insect–virus–host interactions. The availability of a CrPV infectious clone allows controlled manipulation of the viral genome at a molecular level. Effective viral propagation and titration techniques are crucial for understanding the pathogenesis and epidemiology of dicistrovirus infections. Traditional methods for assessing viral titers, such as plaque assays, are unsuitable for CrPV, since Drosophila tissue culture cells like Schneider 2 cells cannot readily form adherent plaques. Here, we present a streamlined protocol for generating a recombinant virus from a CrPV infectious clone, propagating the virus in S2 cells and titering the virus by an immunofluorescence-based focus-forming assay (FFA). This protocol offers a rapid and reliable approach for generating recombinant viruses, viral amplification, and determining CrPV titers, enabling efficient investigation into viral biology and facilitating the development of antiviral strategies.

Phlebotomine vectors, sand flies of the order Diptera, are known to transmit Leishmania parasites as well as RNA viruses (arboviruses) to humans. The arbovirus, Icoaraci Phlebovirus (BeAN 24262 - ICOV), used in this study was isolated from Nectomys rodents, a mammalian species that is the same natural sylvatic reservoir of Leishmania (Leishmania) amazonensis. This Leishmania species is distributed in primary and secondary forests in Brazil and other countries in America and causes localized and diffuse anergic skin lesions. In our recent studies, we observed an aggravation of the protozoan infection by ICOV through the modulation of cytokine expression, such as IL-10 and IFN-β, enhancing the parasite load and possibly the pathogenesis. Efficient viral production and quantitation had to be developed and standardized to ensure that immuno-molecular assays provide consistent and reproducible viral infection results. The standardization of these procedures becomes a particularly useful tool in research, with several applications in understanding the interaction between the host cell and Phlebovirus, as well as co-infections, allowing the study of intracellular signaling pathways. Here, we detail a protocol that allows the production and quantitation of the Icoaraci Phlebovirus using BHK-21 cells (baby hamster kidney cells) and subsequent infection of peritoneal macrophages from C57BL/6 mice.