Development of a reliable bovine neuronal cell culture system and labeled recombinant bovine herpesvirus type-1 for studying virus-host cell interactions

Bovine herpesvirus type 1 (BoHV-1) is the viral causative agent of infectious bovine rhinotracheitis and a component of the bovine respiratory complex commonly referred to as shipping fever in calves. BoHV-1 is also responsible for losses of aborted calves and reductions in dairy productivity. BoHV-1 belongs to the neurotropic alphaherpesviruses which have a predilection to infect and establish latency in sensory neurons. Neuronal cell cultures provide a useful platform for experiments investigating neuronal entry, retrograde and anterograde transport, and the establishment of latency. Rodent neuronal cell lines and primary rabbit neuronal cells have been utilized for BoHV-1, though a reliable host-specific neuronal cell culture system has not been developed. In this study, BoHV-1 readily infected bovine-derived immortalized neuronal progenitor cells (FBBC-1) differentiated in cell culture producing neurite-like projections and exhibiting neuronal cell markers NeuN and L1CAM. FBBC-1 cells expressed both nectin-1 and nectin-2 alphaherpesvirus receptors on their cell surfaces, however, nectin-2 was detected in much greater abundance than nectin-1. To facilitate investigations of BoHV-1 infection, a recombinant BoHV-1 virus expressing the green fluorescent protein (GFP) cloned into a bacterial artificial chromosome (BAC) was used to generate an mCherry-VP26 fusion protein. The BoHV-1 GFP expressing VP26mCherry labeled virus infected differentiated FBBC-1 cells as evidenced by the production of infectious virions and the expression of both GFP and mCherry fluorophores. Time-lapse live cell microscopy revealed the presence of mCherry fluorescent capsids in neuronal projections immediately after virus entry moving retrograde in a saltatory manner. Proximity ligation assays (PLA) using MDBK cells demonstrated that BoHV-1 glycoprotein D (gD) interacted more efficiently with nectin-1 than nectin-2. However, the gD interaction with nectin-2 predominated in differentiated FBBC-1 cells in comparison to the gD nectin-1 interaction. The efficiently differentiated FBBC-1 neuronal cell line and fluorescently labeled BoHV-1 virions will assist experimentation aiming to elucidate specific mechanisms of virus entry and transport in a homologous bovine neuronal cell culture system

Development of a mouse salivary gland-derived mesenchymal cell line for immunological studies of murine cytomegalovirus

The salivary glands are a crucial site of replication for human cytomegalovirus (HCMV) and its murine counterpart, murine cytomegalovirus (MCMV). Studies of MCMV often involve the use of BALB/c strain mice, but most in vitro assays are carried out in the NIH 3T3 cell line, which is derived from Swiss Albino mice. This report describes a BALB/c-derived mouse salivary gland cell line immortalized using the SV40 large T antigen. Cells stained positive for PDGFR1 and negative for E-cadherin and PECAM-1, indicating mesenchymal origin. This cell line, which has been named murine salivary gland mesenchymal (mSGM), shows promise as a tool for ex vivo immunological assays due to its MHC haplotype match with the BALB/c mouse strain. In addition, plaque assays using mSGM rather than NIH 3T3 cells are significantly more sensitive for detecting low concentrations of MCMV particles. Finally, it is demonstrated that mSGM cells express all 3 BALB/c MHC class I isotypes and are susceptible to T cell-mediated ex vivo cytotoxicity assays, leading to many possible uses in immunological studies of MCMV

Utility of a Recombinant HSV-1 Vaccine Vector for Personalized Cancer Vaccines

Current approaches to cancer immunotherapy include immune checkpoint inhibitors, cancer vaccines, and adoptive cellular therapy. These therapies have produced significant clinical success for specific cancers, but their efficacy has been limited. Oncolytic virotherapy (OVT) has emerged as a promising immunotherapy for a variety of cancers. Furthermore, the unique characteristics of OVs make them a good choice for delivering tumor peptides/antigens to induce enhanced tumor-specific immune responses. The first oncolytic virus (OV) approved for human use is the attenuated herpes simplex virus type 1 (HSV-1), Talimogene laherparepvec (T-VEC) which has been FDA approved for the treatment of melanoma in humans. In this study, we engineered the recombinant oncolytic HSV-1 (oHSV) VC2-OVA expressing a fragment of ovalbumin (OVA) as a fusion protein with VP26 virion capsid protein. We tested the ability of VC2-OVA to act as a vector capable of stimulating strong, specific antitumor immunity in a syngeneic murine melanoma model. Therapeutic vaccination with VC2-OVA led to a significant reduction in colonization of tumor cells in the lungs of mice intravenously challenged B16cOVA cells. In addition, VC2-OVA induced a potent prophylactic antitumor response and extended survival of mice that were intradermally engrafted with B16cOVA tumors compared with mice immunized with control virus

Rab27a GTPase and its effector Myosin Va are host factors required for efficient Oropouche virus cell egress.

Acknowledgements: We thank S. Graham (University of Cambridge), L. T. M Figueiredo (University of São Paulo) and P. Burgos (Universidad San Sebastian, Chile) for the kind donation of reagents. R.R.C. Rosales for excellent technical assistance. We thank the Ribeirão Preto School of Medicine Multiphoton Microscopy facility and Ribeirão Preto School of Pharmaceutical Sciences Confocal Microscopy facility for their assistance with data collection. We also thank the members of the daSilva and Crump labs for advice throughout the project.Funder: Academy of Medical Sciences; funder-id: http://dx.doi.org/10.13039/501100000691Funder: Fundação de Apoio ao Ensino, Pesquisa e Assistência do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo; funder-id: http://dx.doi.org/10.13039/501100008353Funder: Conselho Nacional de Desenvolvimento Científico e Tecnológico; funder-id: http://dx.doi.org/10.13039/501100003593Oropouche fever, a debilitating illness common in South America, is caused by Oropouche virus (OROV), an arbovirus. OROV belongs to the Peribunyaviridae family, a large group of RNA viruses. Little is known about the biology of Peribunyaviridae in host cells, especially assembly and egress processes. Our research reveals that the small GTPase Rab27a mediates intracellular transport of OROV induced compartments and viral release from infected cells. We show that Rab27a interacts with OROV glycoproteins and colocalizes with OROV during late phases of the infection cycle. Moreover, Rab27a activity is required for OROV trafficking to the cell periphery and efficient release of infectious particles. Consistently, depleting Rab27a's downstream effector, Myosin Va, or inhibiting actin polymerization also hinders OROV compartments targeting to the cell periphery and infectious viral particle egress. These data indicate that OROV hijacks Rab27a activity for intracellular transport and cell externalization. Understanding these crucial mechanisms of OROV's replication cycle may offer potential targets for therapeutic interventions and aid in controlling the spread of Oropouche fever