Establishment of Human-Induced Pluripotent Stem Cell-Derived Neurons-A Promising In Vitro Model for a Molecular Study of Rabies Virus and Host Interaction.

Rabies is a deadly viral disease caused by the rabies virus (RABV), transmitted through a bite of an infected host, resulting in irreversible neurological symptoms and a 100% fatality rate in humans. Despite many aspects describing rabies neuropathogenesis, numerous hypotheses remain unanswered and concealed. Observations obtained from infected primary neurons or mouse brain samples are more relevant to human clinical rabies than permissive cell lines; however, limitations regarding the ethical issue and sample accessibility become a hurdle for discovering new insights into virus-host interplays. To better understand RABV pathogenesis in humans, we generated human-induced pluripotent stem cell (hiPSC)-derived neurons to offer the opportunity for an inimitable study of RABV infection at a molecular level in a pathologically relevant cell type. This study describes the characteristics and detailed proteomic changes of hiPSC-derived neurons in response to RABV infection using LC-MS/MS quantitative analysis. Gene ontology (GO) enrichment of differentially expressed proteins (DEPs) reveals temporal changes of proteins related to metabolic process, immune response, neurotransmitter transport/synaptic vesicle cycle, cytoskeleton organization, and cell stress response, demonstrating fundamental underlying mechanisms of neuropathogenesis in a time-course dependence. Lastly, we highlighted plausible functions of heat shock cognate protein 70 (HSC70 or HSPA8) that might play a pivotal role in regulating RABV replication and pathogenesis. Our findings acquired from this hiPSC-derived neuron platform help to define novel cellular mechanisms during RABV infection, which could be applicable to further studies to widen views of RABV-host interaction.

Adaptation of African swine fever virus to MA-104 cells: Implications of unique genetic variations.

African swine fever virus (ASFV) is a large, double-stranded DNA virus that causes a fatal, contagious disease specifically in pigs. However, prevention and control of ASFV outbreaks have been hampered by the lack of an effective vaccine or antiviral treatment for ASFV. Although ASFV has been reported to adapt to a variety of continuous cell lines, the phenotypic and genetic changes associated with ASFV adaptation to MA-104 cells remain poorly understood. Here, we adapted ASFV field isolates to efficiently propagate through serial viral passages in MA-104 cells. The adapted ASFV strain developed a pronounced cytopathic effect and robust infection in MA-104 cells. Interestingly, the adapted variant maintained its tropism in primary porcine kidney macrophages. Whole genome analysis of the adapted virus revealed unique gene deletions in the left and right variable regions of the viral genome compared to other previously reported cell culture-adapted ASFV strains. Notably, gene duplications at the 5' and 3' ends of the viral genome were in reverse complementary alignment with their paralogs. Single point mutations in protein-coding genes and intergenic regions were also observed in the viral genome. Collectively, our results shed light on the significance of these unique genetic changes during adaptation, which facilitate the growth of ASFV in MA-104 cells.

Hydrogel-based 3D human iPSC-derived neuronal culture for the study of rabies virus infection.

Background: Rabies is a highly fatal infectious disease that poses a significant threat to human health in developing countries. In vitro study-based understanding of pathogenesis and tropism of different strains of rabies virus (RABV) in the central nervous system (CNS) is limited due to the lack of suitable culture models that recapitulate the complex communication pathways among host cells, extracellular matrices, and viruses. Therefore, a three-dimensional (3D) cell culture that mimics cell-matrix interactions, resembling in vivo microenvironment, is necessary to discover relevant underlying mechanisms of RABV infection and host responses. Methods: The 3D collagen-Matrigel hydrogel encapsulating hiPSC-derived neurons for RABV infection was developed and characterized based on cell viability, morphology, and gene expression analysis of neuronal markers. The replication kinetics of two different strains of RABV [wild-type Thai (TH) and Challenge Virus Standard (CVS)-11 strains] in both 2D and 3D neuronal cultures were examined. Differential gene expression analysis (DEG) of the neuropathological pathway of RABV-infected 2D and 3D models was also investigated via NanoString analysis. Results: The 3D hiPSC-derived neurons revealed a more physiologically interconnected neuronal network as well as more robust and prolonged maturation and differentiation than the conventional 2D monolayer model. TH and CVS-11 exhibited distinct growth kinetics in 3D neuronal model. Additionally, gene expression analysis of the neuropathological pathway observed during RABV infection demonstrated a vast number of differentially expressed genes (DEGs) in 3D model. Unlike 2D neuronal model, 3D model displayed more pronounced cellular responses upon infection with CVS-11 when compared to the TH-infected group, highlighting the influence of the cell environment on RABV-host interactions. Gene ontology (GO) enrichment of DEGs in the infected 3D neuronal culture showed alterations of genes associated with the inflammatory response, apoptotic signaling pathway, glutamatergic synapse, and trans-synaptic signaling which did not significantly change in 2D culture. Conclusion: We demonstrated the use of a hydrogel-based 3D hiPSC-derived neuronal model, a highly promising technology, to study RABV infection in a more physiological environment, which will broaden our understanding of RABV-host interactions in the CNS.

Human BST2 inhibits rabies virus release independently of cysteine-linked dimerization and asparagine-linked glycosylation.

The innate immune response is a first-line defense mechanism triggered by rabies virus (RABV). Interferon (IFN) signaling and ISG products have been shown to confer resistance to RABV at various stages of the virus's life cycle. Human tetherin, also known as bone marrow stromal cell antigen 2 (hBST2), is a multifunctional transmembrane glycoprotein induced by IFN that has been shown to effectively counteract many viruses through diverse mechanisms. Here, we demonstrate that hBST2 inhibits RABV budding by tethering new virions to the cell surface. It was observed that release of virus-like particles (VLPs) formed by RABV G (RABV-G VLPs), but not RABV M (RABV-G VLPs), were suppressed by hBST2, indicating that RABV-G has a specific effect on the hBST2-mediated restriction of RABV. The ability of hBST2 to prevent the release of RABV-G VLPs and impede RABV growth kinetics is retained even when hBST2 has mutations at dimerization and/or glycosylation sites, making hBST2 an antagonist to RABV, with multiple mechanisms possibly contributing to the hBST2-mediated suppression of RABV. Our findings expand the knowledge of host antiviral mechanisms that control RABV infection.

Chimeric Virus-like Particle-Based COVID-19 Vaccine Confers Strong Protection against SARS-CoV-2 Viremia in K18-hACE2 Mice.

Virus-like particles (VLPs) are highly immunogenic and versatile subunit vaccines composed of multimeric viral proteins that mimic the whole virus but lack genetic material. Due to the lack of infectivity, VLPs are being developed as safe and effective vaccines against various infectious diseases. In this study, we generated a chimeric VLP-based COVID-19 vaccine stably produced by HEK293T cells. The chimeric VLPs contain the influenza virus A matrix (M1) proteins and the SARS-CoV-2 Wuhan strain spike (S) proteins with a deletion of the polybasic furin cleavage motif and a replacement of the transmembrane and cytoplasmic tail with that of the influenza virus hemagglutinin (HA). These resulting chimeric S-M1 VLPs, displaying S and M1, were observed to be enveloped particles that are heterogeneous in shape and size. The intramuscular vaccination of BALB/c mice in a prime-boost regimen elicited high titers of S-specific IgG and neutralizing antibodies. After immunization and a challenge with SARS-CoV-2 in K18-hACE2 mice, the S-M1 VLP vaccination resulted in a drastic reduction in viremia, as well as a decreased viral load in the lungs and improved survival rates compared to the control mice. Balanced Th1 and Th2 responses of activated S-specific T-cells were observed. Moderate degrees of inflammation and viral RNA in the lungs and brains were observed in the vaccinated group; however, brain lesion scores were less than in the PBS control. Overall, we demonstrate the immunogenicity of a chimeric VLP-based COVID-19 vaccine which confers strong protection against SARS-CoV-2 viremia in mice.