Luciferase-based imaging allows a global view of microbial pathogenesis. We applied this technique to gammaherpesvirus infection by inserting a luciferase expression cassette into the genome of murine herpesvirus-4 (MuHV-4). The recombinant virus strongly expressed luciferase in lytically infected cells without significant attenuation. We used it to compare different routes of virus inoculation. After intranasal infection of anaesthetized mice, luciferase was expressed in the nose and lungs for 7–10 days and in lymphoid tissue, most consistently the superficial cervical lymph nodes, for up to 30 days. Gastrointestinal infection was not observed. Intraperitoneal infection was very different to intranasal, with strong luciferase expression in the liver, kidneys, intestines, reproductive tract and spleen, but none in the nose or lungs. The nose has not previously been identified as a site of MuHV-4 infection. After intranasal infection of non-anaesthetized mice, it was the only site of non-lymphoid luciferase expression. Nevertheless, lymphoid colonization and persistence were still established, even at low inoculation doses. In contrast, virus delivered orally was very poorly infectious. Inoculation route therefore had a major impact on pathogenesis. Low dose intranasal infection without anaesthesia seems most likely to mimic natural transmission, and may therefore be particularly informative about normal viral gene functions.

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.

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.

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.