Insight into the apoptosis mediated by feline herpesvirus (FeHV-1) on permissive cells

1.Introduction: Apoptosis, also known as programmed cell death, is a critical component of host defense mechanisms against viral infection, limiting viral replication and transmission. Multiple signaling pathways, both intrinsic and extrinsic, form a complex network to modulate apoptosis, even though viruses have distinct mechanisms for mediating or inhibiting apoptosis to avoid this process. In comparison to other herpesvirus members, little is known about the relationship between FeHV-1, the etiological agent of feline rhinotracheitis, and apoptosis. This study aimed to learn more about this connection by evaluating the impact of apoptosis on FeHV-1 infection of permissive cells. 2.Materials and methods: Confluent monolayers of Crandell-Rees Feline Kidney Cell (CRFK) were infected using a MOI 1 at different time points. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was used to determine cell viability. Moreover, the apoptotic pathway was investigated using two different approaches: a flow cytometry analysis using an Annexin V-FITC detection kit and a western blot assay targeting specific markers of apoptosis (Caspase 8, Caspase 9, Caspase 3, Bcl-XL, Bcl-2). 3.Results: Our results suggested that FeHV-1 triggers apoptosis process starting with 12 hours post infection. A larger percentage of early apoptotic cells were detected by the flow cytometry analysis, starting at 12 hours after infection, while late apoptotic cells increased at 24 hours after infection. The western blot analysis showed the cleavage of caspase 9 after 24 hours and a time-dependent cleavage of caspase 3 after 48 and 72 hours. We also observed a reduction of the expression of the anti-apoptotic proteins Bcl-XL and Bcl-2, beginning from 24 hours, also in this case. 4.Discussion and conclusions: In this work we investigated on apoptosis modulation made by FeHV-1. Similar to other herpesviruses, such as Herpes simplex virus (HSV-1), Bovine herpesvirus (BoHV-1), Pseudorabies virus (PRV), Anatid alphaherpesvirus-1 (DEV), and Caprine Herpesvirus 1, infection on permissive cells results in the activation of the apoptotic process, which reduces cell viability. The induction of the intrinsic pathway and the caspase 9 cleavage suggested a dysfunction of the mitochondrial membrane, and the downregulation of Bcl-2 and Bcl-XL expression supported this hypothesis. Even though additional research is needed to fully understand the viral mechanism and the viral genes/proteins involved in this process, this study provides new knowledge on the host-FeHV-1 interaction. The potential proviral effect may be examined since it has been shown that some viruses manipulate the cellular apoptotic mechanism to enhance their own dissemination within apoptotic bodies.