Broadly Applicable, Virus-Free Dual Reporter Assay to Identify Compounds Interfering with Membrane Fusion: Performance for HSV-1 and SARS-CoV-2.

Membrane fusion constitutes an essential step in the replication cycle of numerous viral pathogens, hence it represents an important druggable target. In the present study, we established a virus-free, stable reporter fusion inhibition assay (SRFIA) specifically designed to identify compounds interfering with virus-induced membrane fusion. The dual reporter assay is based on two stable Vero cell lines harboring the third-generation tetracycline (Tet3G) transactivator and a bicistronic reporter gene cassette under the control of the tetracycline responsive element (TRE3G), respectively. Cell-cell fusion by the transient transfection of viral fusogens in the presence of doxycycline results in the expression of the reporter enzyme secreted alkaline phosphatase (SEAP) and the fluorescent nuclear localization marker EYFPNuc. A constitutively expressed, secreted form of nanoluciferase (secNLuc) functioned as the internal control. The performance of the SRFIA was tested for the quantification of SARS-CoV-2- and HSV-1-induced cell-cell fusion, respectively, showing high sensitivity and specificity, as well as the reliable identification of known fusion inhibitors. Parallel quantification of secNLuc enabled the detection of cytotoxic compounds or insufficient transfection efficacy. In conclusion, the SRFIA reported here is well suited for high-throughput screening for new antiviral agents and essentially will be applicable to all viral fusogens causing cell-cell fusion in Vero cells.

Enhanced viral clearance and reduced leukocyte infiltration in experimental herpes encephalitis after intranasal infection of CXCR3-deficient mice.

Herpes simplex virus type 1 (HSV-1) encephalitis (HSE) is the most common fatal sporadic encephalitis in developed countries. There is evidence from HSE animal models that not only direct virus-mediated damage caused but also the host's immune response contributes to the high mortality of the disease. Chemokines modulate and orchestrate this immune response. Previous experimental studies in HSE models identified the chemokine receptor CXCR3 and its ligands as molecules with a high impact on the course of HSE in mouse models. In this study, the role of the chemokine receptor CXCR3 was evaluated after intranasal infection with the encephalitogenic HSV-1 strain 17 syn+ using CXCR3-deficient mice (CXCR3-/-) and wild-type controls. We demonstrated a neurotropic viral spread into the CNS of after intranasal infection. Although viral load and histological distribution of infected neurons were independent from CXCR3 signaling early after infection, CXCR3-deficient mice cleared HSV-1 more efficiently 14 days after infection. Furthermore, CXCR3 deficiency led to a decreased weight loss in mice after HSV-1 infection. T cell infiltration and microglial activation was prominently reduced by inhibition of CXCR3 signaling. Quantitative PCR of proinflammatory cytokines and chemokines confirmed the reduced neuroinflammatory response in CXCR3-deficient mice during HSE. Our results demonstrate that the recruitment of peripheral immune cells into the CNS, induction of neuroinflammation, and consecutive weight loss during herpes encephalitis is modulated by CXCR3 signaling. Interruption of the CXCR3 pathway ameliorates the detrimental host immune response and in turn, leads paradoxically to an enhanced viral clearance after intranasal infection. Our data gives further insight into the role of CXCR3 during HSE after intranasal infection.

Cutaneous RANK-RANKL Signaling Upregulates CD8-Mediated Antiviral Immunity during Herpes simplex Virus Infection by Preventing Virus-Induced Langerhans Cell Apoptosis.

Herpes simplex virus-type 1 (HSV-1) causes the majority of cutaneous viral infections. Viral infections are controlled by the immune system, and CD8(+) cytotoxic T-lymphocytes (CTLs) have been shown to be crucial during the clearance of HSV-1 infections. Although epidermal Langerhans cells (LCs) are the first dendritic cells (DCs) to come into contact with the virus, it has been shown that the processing of viral antigens and the differentiation of antiviral CTLs are mediated by migratory CD103(+) dermal DCs and CD8α(+) lymph node-resident DCs. In vivo regulatory T-cells (Tregs) are implicated in the regulation of antiviral immunity and we have shown that signaling via the receptor activator of NF-κB (RANK) and its ligand RANKL mediates the peripheral expansion of Tregs. However, in addition to expanding Tregs, RANK-RANKL interactions are involved in the control of antimicrobial immunity by upregulating the priming of CD4(+) effector T cells in LCMV infection or by the generation of parasite-specific CD8(+) T cells in Trypanosoma cruzi infection. Here, we demonstrate that cutaneous RANK-RANKL signaling is critical for the induction of CD8-mediated antiviral immune responses during HSV-1 infection of the skin by preventing virus-induced LC apoptosis, improving antigen transport to regional lymph nodes, and increasing the CTL priming capacity of lymph node DCs.

Heparin increases the infectivity of Human Papillomavirus type 16 independent of cell surface proteoglycans and induces L1 epitope exposure.

Human Papillomaviruses (HPVs) are the etiological agents of cervical cancer, and HPV-16 is the most prevalent type. Several HPVs require heparan sulfate proteoglycans (HSPGs) for cell binding. Here, we analyse the phenomenon that preincubation of HPV-16 with increasing concentrations of heparin results in partial restoration rather than more efficient inhibition of infection. While corroborating that the HSPGs are cell-binding receptors for HPV-16, heparin-preincubated virus bound to the extracellular matrix (ECM) via laminin-332. Furthermore, the interaction of virions with heparin, a representative of the highly sulfated S-domains of heparan sulfate (HS) chains of HSPGs, allowed HPV-16 infection in the absence of cell surface HSPGs. Therefore, we concluded that specific glycan moieties but not specific HSPG protein backbones are required for infection. The increased binding of an epitope-specific antibody to the viral capsid after heparin binding suggested that initial conformational changes in the HPV-16 virion occur during infection by interaction with'heparin-like' domains of cellular HSPGs. We propose that HS sequences with specific sulfation patterns are required to facilitate HPV-16 infection.

Entry of herpes simplex virus type 1 (HSV-1) into the distal axons of trigeminal neurons favors the onset of nonproductive, silent infection.

Following productive, lytic infection in epithelia, herpes simplex virus type 1 (HSV-1) establishes a lifelong latent infection in sensory neurons that is interrupted by episodes of reactivation. In order to better understand what triggers this lytic/latent decision in neurons, we set up an organotypic model based on chicken embryonic trigeminal ganglia explants (TGEs) in a double chamber system. Adding HSV-1 to the ganglion compartment (GC) resulted in a productive infection in the explants. By contrast, selective application of the virus to distal axons led to a largely nonproductive infection that was characterized by the poor expression of lytic genes and the presence of high levels of the 2.0-kb major latency-associated transcript (LAT) RNA. Treatment of the explants with the immediate-early (IE) gene transcriptional inducer hexamethylene bisacetamide, and simultaneous co-infection of the GC with HSV-1, herpes simplex virus type 2 (HSV-2) or pseudorabies virus (PrV) helper virus significantly enhanced the ability of HSV-1 to productively infect sensory neurons upon axonal entry. Helper-virus-induced transactivation of HSV-1 IE gene expression in axonally-infected TGEs in the absence of de novo protein synthesis was dependent on the presence of functional tegument protein VP16 in HSV-1 helper virus particles. After the establishment of a LAT-positive silent infection in TGEs, HSV-1 was refractory to transactivation by superinfection of the GC with HSV-1 but not with HSV-2 and PrV helper virus. In conclusion, the site of entry appears to be a critical determinant in the lytic/latent decision in sensory neurons. HSV-1 entry into distal axons results in an insufficient transactivation of IE gene expression and favors the establishment of a nonproductive, silent infection in trigeminal neurons.

Exceptional mechanical and structural stability of HSV-1 unveiled with fluid atomic force microscopy.

Evidence is emerging that changes in the structural and mechanical properties of viral particles are closely linked and that such changes are essential to infectivity. Here, applying the nanostructural and nanomechanical approach of atomic force microscopy, we visualised capsids of the ubiquitous human pathogen herpes simplex virus type 1 (HSV-1) at nano-scale resolution in physiologically relevant conditions. Simultaneously performed nano-indentation measurements on genome-containing and genome-free capsids revealed that genome-containing HSV-1 capsids withstand an exceptionally large mechanical force of approximately 6 nN, which is three times larger than the highest values previously reported for other viruses. Greater mechanical forces, however, led to a release of the viral genome. The resulting genome-free capsids, which largely retained their overall structure, were found to be utterly elastic. HSV-1 capsids thus exhibit an exceptional structural and mechanical stability, which is largely provided by the densely packaged genome. This stability might be the key determinant for capsid survival over long distances in the axonal cytoplasm where it is exposed to mechanical forces by molecular motors before it reaches the nuclear pore for crucial genome uncoating.

Comparison of two commercially available pp65 antigenemia tests and COBAS Amplicor CMV Monitor for early detection and quantification of episodes of human CMV-viremia in transplant recipients.

The performance of two commercially available CMV pp65 antigenemia (AG) assays (CINA kit, Argene Biosoft, CK, and CMV Brite kit, IQ Products, CBK), and a quantitative PCR test (COBAS Amplicor CMV Monitor Test, Roche Diagnostics, CACM) was evaluated with 667 blood samples from 215 transplant recipients. The diagnostic value of the three tests was defined by their ability to detect episodes of systemic CMV activity. A test score was defined, assigning 7 virtual units (VU) for any episode detected first, 5 VU for any episode detected second, and three VU for any episode detected last. Within the evaluated collective, the following overall scores could be determined for the three assays: 45 VU for the CK, 39 for CACM and 31 for the CBK assay. We conclude from our results that (1) the CK is superior to the CBK assay in detecting episodes of CMV antigenemia, and (2) quantitative serum CMV PCR with CACM is not generally superior to pp65 antigenemia testing.

The genome of HSV-1 translocates through the nuclear pore as a condensed rod-like structure.

Incoming herpes simplex virus type-1 (HSV-1) capsids are known to dock to the nuclear pore complex (NPC) and release their genome. It has remained elusive, however, how the huge viral DNA translocates through the comparatively small NPC channel. In the present study, the interaction of HSV-1 with NPCs was analyzed by atomic force microscopy. In addition to capsids, smaller subviral structures--most with a diameter of 35-40 nm and a length of 130-160 nm--were visualized at the cytoplasmic side of the NPC. These components differed from capsids in their adhesion and stiffness properties, and were the sole subviral structures translocated through dilated NPCs towards the nucleus. It is presumed that they are the HSV-1 genome, and that a change in NPC conformation allows translocation of this genome as a densely packaged, rodlike structure.

Reciprocal transmission of herpes simplex virus type 1 (HSV-1) between corneal epithelium and trigeminal neurites in an embryonic chick organ culture.

Reciprocal transmission between epithelia and sensory neurons of the peripheral nervous system is a crucial step in the life cycle of herpes simplex virus type 1 (HSV-1) and related alphaherpesviruses. In searching for an easy-to-perform and generally applicable experimental approach that enables the direct analysis of virus transfer between primary epithelial cells and sensory neurites, we investigated the spread of HSV-1 in a dual-chamber organ model comprising chick embryonic corneal epithelia and trigeminal sensory neurons. Embryonic chick corneal and trigeminal tissues were found to be permissive for productive infection with HSV-1. Our data show that HSV-1 efficiently enters neurites re-innervating the cornea and reaches the ganglion explant by retrograde axonal transport, with the first antigen-positive cells being detectable approximately 12 h postinfection. After direct infection of trigeminal tissues, the virus is transported by anterograde axonal transport to the corneal epithelium, causing a visible cytopathic effect approximately 48 h postinfection. These results suggest that the organ model presented in this study holds particular promise for the direct observation and molecular analysis of herpes simplex virus spread between primary epithelia and sensory neurons and that it may be an attractive alternative to current experimental approaches based on laboratory animals or human fetal tissues.

Genital herpes simplex virus type 1 infection: new fields for an old acquaintance?

Herpes simplex virus (HSV)-2 is the principal agent of chronic remittent genital herpes. Worldwide, only 10-20% of genital isolates are HSV-1. Studies from the British Isles and Scandinavia indicate, however, that HSV-1 is responsible for a significant proportion or even the majority of first clinical episodes of genital herpes in young women. Actual data show that a trend towards genital HSV-1 infections may also hold true for Germany. This article summarizes possible reasons for and consequences of the observed changes in the epidemiology of genital HSV infections.