In vitro study of toll-like receptors sensing BoHV-1

Pathogen detection is crucial in the establishment of an immune response by the host immune system. Pathogen recognition is primarily carried out by cell surface or endosomal receptors, which trigger signaling cascades that result in the upregulation of inflammatory cytokines and type 1 interferon. Toll\u2010like receptors (TLRs) are the key innate immune receptors. Understanding the involvement of TLRs during pathogen infection helps to provide a clearer picture on the establishment of the innate immune response to the infectious agent. This issue is particularly important in studies of animal viruses, where knowledge is much lower compared to human viruses. TLRs 3, 7, 8 and 9 are located in endosomal membranes and primarily serve as nucleic acid sensors for viruses. Herpesviruses are known for their ability to modulate host immune responses upon infection in order to establish latency. Bovine Herpesvirus type 1 (BoHV\u20101) is an alphaherpesvirus causing infectious bovine rhinotracheitis (IBR) in cattle. We performed an in vitro study in order to profile the expression of TLRs 3, 7, 8 and 9 in bovine peripheral blood mononuclear cells (PBMCs) obtained by 4 heifers from an IBR\u2010free herd and one heifer BoHV\u20101\u2010free but persistently infected with Bovine Viral Diarrhea Virus (BVDV), a pestivirus with heavy economic impact on farms. Isolated PBMCs were in vitro infected with BoHV\u20101 and tested at 2, 18 and 48 hours postinfection for the expression of endosomal TLRs by Real\u2010Time PCR assays. Negative controls were included for all the animals and time\u2010points. TLR 3 was very low expressed in vitro in infected as well as in control PBMCs at all time\u2010points; TLRs 7, 8 and 9, differentially expressed at 2 and 18 hours post\u2010infection, showed downregulation due to BoHV\u20101 at 48 hours post\u2010infection. Interestingly, in our in vitro model, at 48 hours was detected the peak of BoHV\u20101 replication. The in vitro infection with BoHV\u20101 of PBMCs isolated from a BVDV persistently infected heifer showed high expression levels of all the TLRs tested except TLR3 at all time\u2010points and both in BoHV\u20101 infected and control PBMCs. This stimulation, not associated to BHV\u20101 infection, is presumably due to BVDV continuous replication. These results, taken together, suggest that the differential expression of endosomal TLR genes is associated to the type of virus infection and the replication cycle. Deeper understanding of bovine innate immune responses following infection by these two viruses, which are responsible for the most costly bovine viral diseases, could help to identify the strategies by which the pathogens evade host defenses and to develop effective intervention strategies