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Protecting against persistent viruses is an unsolved challenge. The clearest example for a gamma-herpesvirus is resistance to super-infection by Murid herpesvirus-4 (MuHV-4). Most experimental infections have delivered MuHV-4 into the lungs. A more likely natural entry site is the olfactory epithelium. Its protection remains unexplored. Here, prior exposure to olfactory MuHV-4 gave good protection against super-infection. The protection was upstream of B cell infection, which occurs in lymph nodes, and showed redundancy between antibody and T cells. Adding antibody to virions that blocked heparan binding strongly reduced olfactory host entry - unlike in the lungs, opsonized virions did not reach IgG Fc receptor + myeloid cells. However, the nasal antibody response to primary infection was too low to reduce host entry. Instead, the antibody acted downstream, reducing viral replication in the olfactory epithelium. This depended on IgG Fc receptor engagement rather than virion neutralization. Thus antibody can protect against natural γ-herpesvirus infection before it reaches B cells and independently of neutralization.

Unlabelled: Herpes simplex virus 1 (HSV-1) enters mice via olfactory epithelial cells and then colonizes the trigeminal ganglia (TG). Most TG nerve endings are subepithelial, so this colonization implies subepithelial viral spread, where myeloid cells provide an important line of defense. The outcome of infection of myeloid cells by HSV-1 in vitro depends on their differentiation state; the outcome in vivo is unknown. Epithelial HSV-1 commonly infected myeloid cells, and Cre-Lox virus marking showed nose and lung infections passing through LysM-positive (LysM(+)) and CD11c(+) cells. In contrast, subcapsular sinus macrophages (SSMs) exposed to lymph-borne HSV-1 were permissive only when type I interferon (IFN-I) signaling was blocked; normally, their infection was suppressed. Thus, the outcome of myeloid cell infection helped to determine the HSV-1 distribution: subepithelial myeloid cells provided a route of spread from the olfactory epithelium to TG neurons, while SSMs blocked systemic spread., Importance: Herpes simplex virus 1 (HSV-1) infects most people and can cause severe disease. This reflects its persistence in nerve cells that connect to the mouth, nose, eye, and face. Established infection seems impossible to clear. Therefore, we must understand how it starts. This is difficult in humans, but mice show HSV-1 entry via the nose and then spread to its preferred nerve cells. We show that this spread proceeds in part via myeloid cells, which normally function in host defense. Myeloid infection was productive in some settings but was efficiently suppressed by interferon in others. Therefore, interferon acting on myeloid cells can stop HSV-1 spread, and enhancing this defense offers a way to improve infection control., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Rhadinoviruses establish chronic infections of clinical and economic importance. Several show respiratory transmission and cause lung pathologies. We used Murid Herpesvirus-4 (MuHV-4) to understand how rhadinovirus lung infection might work. A primary epithelial or B cell infection often is assumed. MuHV-4 targeted instead alveolar macrophages, and their depletion reduced markedly host entry. While host entry was efficient, alveolar macrophages lacked heparan - an important rhadinovirus binding target - and were infected poorly ex vivo. In situ analysis revealed that virions bound initially not to macrophages but to heparan⁺ type 1 alveolar epithelial cells (AECs). Although epithelial cell lines endocytose MuHV-4 readily in vitro, AECs did not. Rather bound virions were acquired by macrophages; epithelial infection occurred only later. Thus, host entry was co-operative - virion binding to epithelial cells licensed macrophage infection, and this in turn licensed AEC infection. An antibody block of epithelial cell binding failed to block host entry: opsonization provided merely another route to macrophages. By contrast an antibody block of membrane fusion was effective. Therefore co-operative infection extended viral tropism beyond the normal paradigm of a target cell infected readily in vitro; and macrophage involvement in host entry required neutralization to act down-stream of cell binding.

Glycoprotein B (gB) is a conserved herpesvirus virion component implicated in membrane fusion. As with many-but not all-herpesviruses, the gB of murid herpesvirus 4 (MuHV-4) is cleaved into disulfide-linked subunits, apparently by furin. Preventing gB cleavage for some herpesviruses causes minor infection deficits in vitro, but what the cleavage contributes to host colonization has been unclear. To address this, we mutated the furin cleavage site (R-R-K-R) of the MuHV-4 gB. Abolishing gB cleavage did not affect its expression levels, glycosylation, or antigenic conformation. In vitro, mutant viruses entered fibroblasts and epithelial cells normally but had a significant entry deficit in myeloid cells such as macrophages and bone marrow-derived dendritic cells. The deficit in myeloid cells was not due to reduced virion binding or endocytosis, suggesting that gB cleavage promotes infection at a postendocytic entry step, presumably viral membrane fusion. In vivo, viruses lacking gB cleavage showed reduced lytic spread in the lungs. Alveolar epithelial cell infection was normal, but alveolar macrophage infection was significantly reduced. Normal long-term latency in lymphoid tissue was established nonetheless.

Herpes simplex virus 1 (HSV-1) is a ubiquitous and important human pathogen. It is known to persist in trigeminal ganglia (TG), but how it reaches this site has been difficult to determine, as viral transmission is sporadic, pathogenesis is complicated, and early infection is largely asymptomatic. We used mice to compare the most likely natural HSV-1 host entry routes: oral and nasal. Intranasal infection was 100-fold more efficient than oral and targeted predominantly the olfactory neuroepithelium. Live imaging of HSV-1-expressed luciferase showed infection progressing from the nose to the TG and then reemerging in the facial skin. The brain remained largely luciferase negative throughout. Infected cell tagging by viral Cre recombinase expression in floxed reporter gene mice showed nasal virus routinely reaching the TG and only rarely reaching the olfactory bulbs. Thus, HSV-1 spread from the olfactory neuroepithelium to the TG and reemerged peripherally without causing significant neurological disease. This recapitulation of typical clinical infection suggests that HSV-1 might sometimes also enter humans via the respiratory tract.

Gamma-herpesviruses persist in lymphocytes and cause disease by driving their proliferation. Lymphocyte infection is therefore a key pathogenetic event. Murid Herpesvirus-4 (MuHV-4) is a rhadinovirus that like the related Kaposi's Sarcoma-associated Herpesvirus persists in B cells in vivo yet infects them poorly in vitro. Here we used MuHV-4 to understand how virion tropism sets the path to lymphocyte colonization. Virions that were highly infectious in vivo showed a severe post-binding block to B cell infection. Host entry was accordingly an epithelial infection and B cell infection a secondary event. Macrophage infection by cell-free virions was also poor, but improved markedly when virion binding improved or when macrophages were co-cultured with infected fibroblasts. Under the same conditions B cell infection remained poor; it improved only when virions came from macrophages. This reflected better cell penetration and correlated with antigenic changes in the virion fusion complex. Macrophages were seen to contact acutely infected epithelial cells, and cre/lox-based virus tagging showed that almost all the virus recovered from lymphoid tissue had passed through lysM(+) and CD11c(+) myeloid cells. Thus MuHV-4 reached B cells in 3 distinct stages: incoming virions infected epithelial cells; infection then passed to myeloid cells; glycoprotein changes then allowed B cell infection. These data identify new complexity in rhadinovirus infection and potentially also new vulnerability to intervention.

Herpesviruses are ubiquitous pathogens that cause much disease. The difficulty of clearing their established infections makes host entry an important target for control. However, while herpesviruses have been studied extensively in vitro, how they cross differentiated mucus-covered epithelia in vivo is unclear. To establish general principles we tracked host entry by Murid Herpesvirus-4 (MuHV-4), a lymphotropic rhadinovirus related to the Kaposi's Sarcoma-associated Herpesvirus. Spontaneously acquired virions targeted the olfactory neuroepithelium. Like many herpesviruses, MuHV-4 binds to heparan sulfate (HS), and virions unable to bind HS showed poor host entry. While the respiratory epithelium expressed only basolateral HS and was bound poorly by incoming virions, the neuroepithelium also displayed HS on its apical neuronal cilia and was bound strongly. Incoming virions tracked down the neuronal cilia, and either infected neurons or reached the underlying microvilli of the adjacent glial (sustentacular) cells and infected them. Thus the olfactory neuroepithelium provides an important and complex site of HS-dependent herpesvirus uptake.

The difficulty of eliminating herpesvirus carriage makes host entry a key target for infection control. However, its viral requirements are poorly defined. Murid herpesvirus-4 (MuHV-4) can potentially provide insights into gammaherpesvirus host entry. Upper respiratory tract infection requires the MuHV-4 thymidine kinase (TK) and ribonucleotide reductase large subunit (RNR-L), suggesting a need for increased nucleotide production. However, both TK and RNR-L are likely to be multifunctional. We therefore tested further the importance of nucleotide production by disrupting the MuHV-4 ribonucleotide reductase small subunit (RNR-S). This caused a similar attenuation to RNR-L disruption: despite reduced intra-host spread, invasive inoculations still established infection, whereas a non-invasive upper respiratory tract inoculation did so only at high dose. Histological analysis showed that RNR-S(-), RNR-L(-) and TK(-) viruses all infected cells in the olfactory neuroepithelium but unlike wild-type virus then failed to spread. Thus captured host nucleotide metabolism enzymes, up to now defined mainly as important for alphaherpesvirus reactivation in neurons, also have a key role in gammaherpesvirus host entry. This seemed to reflect a requirement for lytic replication to occur in a terminally differentiated cell before a viable pool of latent genomes could be established.