Alphaviruses cause mosquito-borne erythrogenic diseases. Sindvis virus (SINV) circulates in an enzootic cycle between its vectors and birds. In humans, SINV triggers musculoskeletal syndromes characterised by fever, rash, acute and chronic polyarthritis, etc. Few studies examining the virus's impact on the nervous/immune systems are available. SINV often causes cerebral infection, so understanding the brain's response is crucial. The SH-SY5Y cell line, from neuroblastoma, is used in many neurobiological and virological studies. This study aimed to understand viral infection in vitro and examine immune markers using SINV infection in SH-SY5Y cells.
Due to the lack of adequate knowledge of SINV, optimization was necessary for all experimental designs. After the virus stock preparation, the median tissue culture infectious dose was used to determine the multiplicity of infection (MOI). Taqman-probe-based RT-qPCR was designed for the SINV nucleic acid, and immunofluorescence-based ds-RNA antibody staining was used to reinforce the virus replication. Apoptotic markers were also investigated by means of immunofluorescence. To monitor specific immune gene expressions (pattern recognition receptors (PRRs), regulator gene, cytokines) in a time-dependent manner (3/6/12/16/24/30 hr), RT-qPCR was also performed.
SINV infected the cells, and after 24 hr, a cytopathogenic effect and virus replication could be observed. SINV induced the caspase-dependent apoptotic pathway. Most of the investigated genes, including PRRs, (TLR-3/7, RIG1/MDA5), a regulator (b-catenin), and inflammatory (IL-1b, IL-6, TNFa), and antiviral (IL-10, IFNβ) genes, exhibited a consistent induction up to 12/16 hr; by the end of the 30 hr, their expression mainly (e.g., RIG1/MDA5, IFNβ) decreased compared with those of our controls. We can conclude that in vitro, SINV can be transmitted even in small quantities, and an inflammatory environment can be developed, which later leads to cell death.
These preliminary results allow us to gain insights into SINV–SH-SY5Y interactions, which are better understood by examining them in much finer molecular detail.
