Introduction
Viral Nervous Necrosis Virus (NNV), a member of the Nodaviridae family, is recognized as one of the most devastating viral pathogens in marine aquaculture worldwide . Its impact is especially severe in high-value species such as groupers (Epinephelus spp. ), where it predominantly targets the central nervous system, causing vacuolation of brain and retinal tissues . This results in erratic swimming behavior , high mortality rates in larvae and juveniles , and considerable economic losses.
In parallel , the intensification of climate change has introduced new, pressing variables influencing host-pathogen-environment interactions, drastically altering marine ecosystems . In particular , the rise in sea surface temperatures , a direct consequence of climate change , is destabilizing marine environments and exacerbating disease outbreaks . These elevated temperatures are compromising fish immune defenses , making them more vulnerable to infections like NNV. The interplay between changing climate conditions and pathogen dynamics underscores the urgency of developing robust surveillance strategies for early detection and management of NNV. In this study, we assessed the effect of temperature on viral replication to better understand how climate-related stressors, particularly warming oceans , may amplify disease dynamics and impact aquaculture . Furthermore , we investigated the presence and viability of NNV in wild Mediterranean grouper specimens , shedding light on how the virus’s geographic distribution and pathogenic potential are shifting in response to changing environmental conditions driven by climate change.
Materials and Methods
To evaluate the impact of rising temperatures, viral replication was assessed at 25°C and 28°C using two different multiplicities of infection (MOI 1 and MOI 10). Cytopathic effects were monitored daily under an inverted microscope , and viral titers were evaluated. In addition, brain tissues collected from wild grouper specimens were processed for molecular and virological analyses. Total RNA was extracted, reverse-transcribed, and analyzed via real-time PCR targeting the RNA1 segment of NNV. For viral isolation, tissue homogenates were inoculated onto SSN-1 cell lines.
Results
In vitro infection of SSN-1 cells resulted in marked cytopathic effects, such as cell rounding, vacuolation, and detachment, by day six post-infection, confirming successful viral replication and isolation. Temperature-based assay revealed that viral replication was significantly higher at 25°C than at 28°C across both MOI conditions. This suggests that elevated temperatures, although generally considered favorable for pathogen proliferation, may instead compromise the host’s physiological state, rendering fish more susceptible to infection even if viral replication itself is reduced. In addition, molecular screening confirmed the presence of NNV RNA in grouper specimens.
These findings emphasize the relevance of temperature as a modulating factor in host-pathogen interactions and support the need for adaptive management strategies in the face of global climate change.