Increase of cells expressing PD-L1 in bovine leukemia virus infection and enhancement of anti-viral immune responses in vitro via PD-L1 blockade

The inhibitory receptor programmed death-1 (PD-1) and its ligand, programmed death-ligand 1 (PD-L1) are involved in immune evasion mechanisms for several pathogens causing chronic infections. Blockade of the PD-1/PD-L1 pathway restores anti-virus immune responses, with concomitant reduction in viral load. In a previous report, we showed that, in bovine leukemia virus (BLV) infection, the expression of bovine PD-1 is closely associated with disease progression. However, the functions of bovine PD-L1 are still unknown. To investigate the role of PD-L1 in BLV infection, we identified the bovine PD-L1 gene, and examined PD-L1 expression in BLV-infected cattle in comparison with uninfected cattle. The deduced amino acid sequence of bovine PD-L1 shows high homology to the human and mouse PD-L1. The proportion of PD-L1 positive cells, especially among B cells, was upregulated in cattle with the late stage of the disease compared to cattle at the aleukemic infection stage or uninfected cattle. The proportion of PD-L1 positive cells correlated positively with prediction markers for the progression of the disease such as leukocyte number, virus load and virus titer whilst on the contrary, it inversely correlated with the degree of interferon-gamma expression. Blockade of the PD-1/PD-L1 pathway in vitro by PD-L1-specific antibody upregulated the production of interleukin-2 and interferon-gamma, and correspondingly, downregulated the BLV provirus load and the proportion of BLV-gp51 expressing cells. These data suggest that PD-L1 induces immunoinhibition in disease progressed cattle during chronic BLV infection. Therefore, PD-L1 would be a potential target for developing immunotherapies against BLV infection

The Human Polyoma JC Virus Agnoprotein Acts as a Viroporin

Virus infections can result in a range of cellular injuries and commonly this involves both the plasma and intracellular membranes, resulting in enhanced permeability. Viroporins are a group of proteins that interact with plasma membranes modifying permeability and can promote the release of viral particles. While these proteins are not essential for virus replication, their activity certainly promotes virus growth. Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease resulting from lytic infection of oligodendrocytes by the polyomavirus JC virus (JCV). The genome of JCV encodes six major proteins including a small auxiliary protein known as agnoprotein. Studies on other polyomavirus agnoproteins have suggested that the protein may contribute to viral propagation at various stages in the replication cycle, including transcription, translation, processing of late viral proteins, assembly of virions, and viral propagation. Previous studies from our and other laboratories have indicated that JCV agnoprotein plays an important, although as yet incompletely understood role in the propagation of JCV. Here, we demonstrate that agnoprotein possesses properties commonly associated with viroporins. Our findings demonstrate that: (i) A deletion mutant of agnoprotein is defective in virion release and viral propagation; (ii) Agnoprotein localizes to the ER early in infection, but is also found at the plasma membrane late in infection; (iii) Agnoprotein is an integral membrane protein and forms homo-oligomers; (iv) Agnoprotein enhances permeability of cells to the translation inhibitor hygromycin B; (v) Agnoprotein induces the influx of extracellular Ca2+; (vi) The basic residues at amino acid positions 8 and 9 of agnoprotein key are determinants of the viroporin activity. The viroporin-like properties of agnoprotein result in increased membrane permeability and alterations in intracellular Ca2+ homeostasis leading to membrane dysfunction and enhancement of virus release

In vitro propagation of rabies virus in mouse dorsal root ganglia cells

Rabies virus (RV) is highly neurotropic and migrates to the neuronal soma by retrograde axonal transport from nerve terminals, after which it is taken by anterograde axonal transport to be finally released into the central nervous system (CNS) from which it disseminates, resulting in lethal encephalitis. Dorsal root ganglia (DRG) are crucial in the initial events of the infection by RV since they can act as a gate for the viral entrance into the CNS. In the present study, we examined cell tropism of RV and the roles of neuronal cytoskeletal components in the production of viral nucleoprotein (N protein) using cultured nerve cells and non-neuronal cells from DRG of newborn mice. Our in vitro study demonstrated a low propagation rate of RV in nerve cells, susceptibility of non-neuronal cells to RV, and independence of cytoplasmic synthesis of viral N protein from the neuronal cytoskeleton. The present study also suggests that Schwann cells should be considered as another possible candidate supporting RV propagation

Neuromuscular and vascular hamartoma of the cecum in a dog

Neuromuscular and vascular hamartoma (NMVH) is a rare non-epithelial hamartoma of the intestine in humans that is characterized by proliferation of smooth muscle, blood vessels and bundles of unmyelinated nerve fibers in the intestinal submucosa. Here, we describe a case in which a mass lesion in the cecum of an 8-year-old male West Highland White Terrier dog. The mass caused an inversion of the cecum, which was surgically removed. The mass was found in the muscle layer of the inverted cecum, and on histology was composed of a proliferation of mainly spindle-shaped cells with fibrillar cytoplasm, vascular structures, and bundles of unmyelinated nerve fibers. These features of the mass are consistent with those described for NMVH in humans

The variable region of the 3' untranslated region is a critical virulence factor in the Far-Eastern subtype of tick-borne encephalitis virus in mouse model

Tick-borne encephalitis virus (TBEV) is a major arbovirus that causes thousands of cases of severe neurological illness in humans annually. However, virulence factors and pathological mechanisms of TBEV remain largely unknown. To identify the virulence factors, we constructed chimeric viruses between two TBEV strains of the Far-Eastern subtype, Sofjin-HO (highly pathogenic) and Oshima 5-10 (low pathogenic). The replacement of the coding region for the structural and non-structural proteins from Sofjin into Oshima showed a partial increase of the viral pathogenicity in a mouse model. Oshima-based chimeric viruses with the variable region of the 3' UTR of Sofjin, which had a deletion of 207 nt, killed 100% of mice and showed almost the same virulence as Sofjin. Replacement of the variable region of the 3' UTR from Sofjin into Oshima did not increase viral multiplication in cultured cells and a mouse model at the early phase of viral entry into the brain. At the terminal phase of viral infection in mice, the virus titre of the Oshinna-based chimeric virus with the variable region of the 3' UTR of Sofjin reached a level identical to that of Sofjin and showed a similar histopathological change in the brain tissue. This is the first report to show that the variable region of the 3' UTR is a critical virulence factor in mice. These findings encourage further study to understand the mechanisms of the pathogenicity of TBEV, and to develop preventative and therapeutic strategies for tick-borne encephalitis

Comparison of Characterization in Two-Dimensional and Three-Dimensional Canine Mammary Gland Tumor Cell Models

Background: Canine mammary gland tumors can be used as predictive models for human breast cancer. There are several types of microRNAs common in human breast cancer and canine mammary gland tumors. The functions of microRNAs in canine mammary gland tumors are not well understood. Methods: We compared the characterization of microRNA expression in two-dimensional and three-dimensional canine mammary gland tumor cell models. We evaluated the differences between two- and three-dimensional cultured canine mammary gland tumor SNP cells by assessing microRNA expression levels, morphology, drug sensitivity, and hypoxia. Results: The expression of microRNA-210 in the three-dimensional-SNP cells was 10.19 times higher than that in the two-dimensional-SNP cells. The intracellular concentrations of doxorubicin in the two- and three-dimensional-SNP cells were 0.330 ± 0.013 and 0.290 ± 0.048 nM/mg protein, respectively. The IC50 values of doxorubicin for the two- and three-dimensional-SNP cells were 5.2 and 1.6 μM, respectively. Fluorescence of the hypoxia probe, LOX-1, was observed inside the sphere of three-dimensional-SNP cells without echinomycin but not in two-dimensional-SNP cells. The three-dimensional-SNP cells treated with echinomycin showed weak LOX-1 fluorescence. Conclusion: The present study showed a clear difference in microRNA expression levels in cells cultured in a two-dimensional adherent versus a three-dimensional spheroid model

A Critical Determinant of Neurological Disease Associated with Highly Pathogenic Tick-Borne Flavivirus in Mice

Tick-borne encephalitis virus (TBEV) and Omsk hemorrhagic fever virus (OHFV) are highly pathogenic tick-borne flaviviruses; TBEV causes neurological disease in humans, while OHFV causes a disease typically identified with hemorrhagic fever. Although TBEV and OHFV are closely related genetically, the viral determinants responsible for these distinct disease phenotypes have not been identified. In this study, chimeric viruses incorporating components of TBEV and OHFV were generated using infectious clone technology, and their pathological characteristics were analyzed in a mouse model to identify virus-specific determinants of disease. We found that only four amino acids near the C terminus of the NS5 protein were primarily responsible for the development of neurological disease. Mutation of these four amino acids had no effect on viral replication or histopathological features, including inflammatory responses, in mice. These findings suggest a critical role for NS5 in stimulating neuronal dysfunction and degeneration following TBEV infection and provide new insights into the molecular mechanisms underlying the pathogenesis of tick-borne flaviviruses

Viroporin activity of the JC polyomavirus is regulated by interactions with the adaptor protein complex 3

Viroporins, which are encoded by a wide range of animal viruses, oligomerize in host cell membranes and form hydrophilic pores that can disrupt a number of physiological properties of the cell. Little is known about the relationship between host cell proteins and viroporin activity. The human JC polyomavirus (JCV) is the causative agent of progressive multifocal leukoencephalopathy. The JCV-encoded agnoprotein, which is essential for viral replication, has been shown to act as a viroporin. Here we demonstrate that the JCV agnoprotein specifically interacts with adaptor protein complex 3 through its delta subunit. This interaction interrupts adaptor protein complex 3-mediated vesicular trafficking with suppression of the targeting of the protein to the lysosomal degradation pathway and instead permits the transport of agnoprotein to the cell surface with resulting membrane permeabilization. The findings demonstrate a previously undescribed paradigm in virus-host interactions allowing the host to regulate viroporin activity and suggest that the viroporins of other viruses may also be highly regulated by specific interactions with host cell proteins