Cytomegaloviruses (CMVs) establish chronic infections that spread from a primary entry site to secondary vascular sites, such as the spleen, and then to tertiary shedding sites, such as the salivary glands. Human CMV (HCMV) is difficult to analyze, because its spread precedes clinical presentation. Murine CMV (MCMV) offers a tractable model. It is hypothesized to spread from peripheral sites via vascular endothelial cells and associated monocytes. However, viral luciferase imaging showed footpad-inoculated MCMV first reaching the popliteal lymph nodes (PLN). PLN colonization was rapid and further spread was slow, implying that LN infection can be a significant bottleneck. Most acutely infected PLN cells were CD169 ؉ subcapsular sinus macrophages (SSM). Replication-deficient MCMV also reached them, indicating direct infection. Many SSM expressed viral reporter genes, but few expressed lytic genes. SSM expressed CD11c, and MCMV with a cre-sensitive fluorochrome switch showed switched infected cells in PLN of CD11c-cre mice but yielded little switched virus. SSM depletion with liposomal clodronate or via a CD169-diphtheria toxin receptor transgene shifted infection to ER-TR7 ؉ stromal cells, increased virus production, and accelerated its spread to the spleen. Therefore, MCMV disseminated via LN, and SSM slowed this spread by shielding permissive fibroblasts and poorly supporting viral lytic replication. IMPORTANCE HCMV chronically infects most people, and it can cause congenital disability and harm the immunocompromised. A major goal of vaccination is to prevent systemic infection. How this is established is unclear. Restriction to humans makes HCMV difficultto analyze. We show that peripheral MCMV infection spreads via lymph nodes. Here, MCMV infected filtering macrophages, which supported virus replication poorly. When these macrophages were depleted, MCMV infected susceptible fibroblasts and spread faster. The capacity of filtering macrophages to limit MCMV spread argued that their infection is an important bottleneck in host colonization and might be a good vaccine target. Human cytomegalovirus (HCMV) chronically infects more than 90% of the world's population. Latent virus is detectable in circulating monocytes; there also may be persistent stromal infection, and infectious virus is shed long term in secretions such as saliva (1). While most infections are asymptomatic, immune control can require substantial resources (2); immunocompromised patients suffer multiorgan disease, and HCMV is a leading cause of congenital disability. Therefore, better control would improve human health. So far, vaccines have failed to prevent infection or reduce long-term viral loads (3). One problem is that early infection events, which are key vaccine targets, remain ill defined. Virus behavior is cell type dependent, so an important unknown is which cell types HCMV infects first when colonizing new hosts.Sporadic, asymptomatic HCMV transmission makes early infection hard to study (1). It is important to recognize, there...

Herpesviruses have coevolved with their hosts over hundreds of millions of years and exploit fundamental features of their biology. Cytomegaloviruses (CMVs) colonize blood-borne myeloid cells, and it has been hypothesized that systemic dissemination arises from infected stem cells in bone marrow. However, poor CMV transfer by stem cell transplantation argues against this being the main reservoir. To identify alternative pathways for CMV spread, we tracked murine CMV (MCMV) colonization after mucosal entry. We show that following intranasal MCMV infection, lung CD11c+ dendritic cells (DC) migrated sequentially to lymph nodes (LN), blood, and then salivary glands. Replication-deficient virus followed the same route, and thus, DC infected peripherally traversed LN to enter the blood. Given that DC are thought to die locally following their arrival and integration into LN, recirculation into blood represents a new pathway. We examined host and viral factors that facilitated this LN traverse. We show that MCMV-infected DC exited LN by a distinct route to lymphocytes, entering high endothelial venules and bypassing the efferent lymph. LN exit required CD44 and the viral M33 chemokine receptor, without which infected DC accumulated in LN and systemic spread was greatly reduced. Taken together, our studies provide the first demonstration of virus-driven DC recirculation. As viruses follow host-defined pathways, high endothelial venules may normally allow DC to pass from LN back into blood.

Viruses transmit via the environmental and social interactions of their hosts. Herpesviruses have colonized mammals since their earliest origins, suggesting that they exploit ancient, common pathways. Cytomegaloviruses (CMVs) are assumed to enter new hosts orally, but no site has been identified. We show by live imaging that murine CMV (MCMV) infects nasally rather than orally, both after experimental virus uptake and during natural transmission. Replication-deficient virions revealed the primary target as olfactory neurons. Local, nasal replication by wild-type MCMV was not extensive, but there was rapid systemic spread, associated with macrophage infection. A long-term, transmissible infection was then maintained in the salivary glands. The viral m131/m129 chemokine homolog, which influences tropism, promoted salivary gland colonization after nasal entry but was not required for entry per se. The capacity of MCMV to transmit via olfaction, together with previous demonstrations of experimental olfactory infection by murid herpesvirus 4 (MuHV-4) and herpes simplex virus 1 (HSV-1), suggest that this is a common, conserved route of mammalian herpesvirus entry.

Lymphocyte proliferation, mobility and longevity make them prime targets for virus infection. Myeloid cells that process and present environmental antigens to lymphocytes are consequently an important line of defence. Subcapsular sinus macrophages (SSMs) filter the afferent lymph and communicate with B-cells. How they interact with B-cell-tropic viruses is unknown. We analysed their encounter with murid herpesvirus-4 (MuHV-4), an experimentally accessible gammaherpesvirus related to Kaposi's sarcoma-associated herpesvirus. MuHV-4 disseminated via lymph nodes, and intranasally or subcutaneously inoculated virions readily infected SSMs. However, this infection was poorly productive. SSM depletion with clodronate-loaded liposomes or with diphtheria toxin in CD169–diphtheria toxin receptor transgenic mice increased B-cell infection and hastened virus spread to the spleen. Dendritic cells provided the main route to B-cells, and SSMs slowed host colonization, apparently by absorbing virions non-productively from the afferent lymph.

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.

H erpesviruses are among the most prevalent of all mammalian pathogens. Many cause lung disease (1-5), making the lung an important site of infection. Human cytomegalovirus (HCMV) causes interstitial pneumonitis in immunocompromised patients (5). While HCMV could potentially reach the lungs via circulating monocytes (6), viral lytic antigen expression in alveolar epithelial cells (AECs) (7-9) and alveolar macrophage (AM) infection (10) suggest entry by inhalation.Sporadic transmission and late clinical presentation make early HCMV infection hard to analyze. However, the relatedness between cytomegaloviruses (CMVs) and their hosts implies that CMV parasitism preceded the speciation of most mammals (11), and peaks of viral diversity in genes engaging the immune functions of diverse hosts suggest that coevolution has operated since to conserve a parasitic status quo. Therefore, nonhuman CMVs can help us to understand how HCMV works. The preeminence of mice as experimental models of mammalian cell biology gives murine CMV (MCMV) particular value in this regard. It causes an interstitial pneumonitis after intranasal (i.n.) inoculation, infecting epithelial and mononuclear cells (12). Thus, it reproduces at least some features of HCMV lung infection.Which lung cells CMVs infect first is unknown. The ciliated upper airways capture large inhaled particles (diameter, Ͼ5 m), but submicron-sized particles, such as viruses, can reach the lung alveoli (13). Here, type 1 alveolar epithelial cells (AEC1s) provide Ͼ90% of the accessible surface, their abundant type 2 progenitors (AEC2s) produce surfactant, and AMs patrol the airspaces for inhaled pathogens. In neonatal mice, i.n. inoculated MCMV infects AMs and AEC2s (14). Infection was reduced when AMs were depleted with liposomal clodronate or when MCMV lacked m129 (15). The m131/m129 MCMV chemokine homolog (designated MCK2) attracts macrophages (16) and alters viral tropism to promote macrophage infection (17). Thus, it was concluded that AMs provide an acutely productive gateway into the lungs. However, MCK2-negative (MCK2 Ϫ ) MCMV given intra-

Gamma-herpesviruses colonise lymphocytes. Murid Herpesvirus-4 (MuHV-4) infects B cells via epithelial to myeloid to lymphoid transfer. This indirect route entails exposure to host defences, and type I interferons (IFN-I) limit infection while viral evasion promotes it. To understand how IFN-I and its evasion both control infection outcomes, we used Mx1-cre mice to tag floxed viral genomes in IFN-I responding cells. Epithelial-derived MuHV-4 showed low IFN-I exposure, and neither disrupting viral evasion nor blocking IFN-I signalling markedly affected acute viral replication in the lungs. Maximising IFN-I induction with poly(I:C) increased virus tagging in lung macrophages, but the tagged virus spread poorly. Lymphoid-derived MuHV-4 showed contrastingly high IFN-I exposure. This occurred mainly in B cells. IFN-I induction increased tagging without reducing viral loads; disrupting viral evasion caused marked attenuation; and blocking IFN-I signalling opened up new lytic spread between macrophages. Thus, the impact of IFN-I on viral replication was strongly cell type-dependent: epithelial infection induced little response; IFN-I largely suppressed macrophage infection; and viral evasion allowed passage through B cells despite IFN-I responses. As a result, IFN-I and its evasion promoted a switch in infection from acutely lytic in myeloid cells to chronically latent in B cells. Murine cytomegalovirus also showed a capacity to pass through IFN-I-responding cells, arguing that this is a core feature of herpesvirus host colonization.

Cytomegaloviruses (CMVs) colonize blood-borne myeloid cells. Murine CMV (MCMV) spreads from the lungs via infected CD11c+cells, consistent with an important role for dendritic cells (DC). We show here that MCMV entering via the olfactory epithelium, a natural transmission portal, also spreads via infected DC. They reached lymph nodes, entered the blood via high endothelial venules, and then entered the salivary glands, driven by constitutive signaling of the viral M33 G protein-coupled receptor (GPCR). Intraperitoneal infection also delivered MCMV to the salivary glands via DC. However, it also seeded F4/80+infected macrophages to the blood; they did not enter the salivary glands or require M33 for extravasation. Instead, they seeded infection to a range of other sites, including brown adipose tissue (BAT). Peritoneal cells infectedex vivothen adoptively transferred showed similar cell type-dependent differences in distribution, with abundant F4/80+cells in BAT and CD11c+cells in the salivary glands. BAT colonization by CMV-infected cells was insensitive to pertussis toxin inhibition of the GPCR signaling through Gi/osubstrate, whereas salivary gland colonization was sensitive. Since salivary gland infection required both M33 and Gi/o-coupled signaling, whereas BAT infection required neither, these migrations were mechanistically distinct. MCMV spread from the lungs or nose depended on DC, controlled by M33. Infecting other monocyte populations resulted in unpredictable new infections.IMPORTANCECytomegaloviruses (CMVs) spread through the blood by infecting monocytes, and this can lead to disease. With murine CMV (MCMV) we can track infected myeloid cells and so understand how CMVs spread. Previous experiments have injected MCMV into the peritoneal cavity. MCMV normally enters mice via the olfactory epithelium. We show that olfactory infection spreads via dendritic cells, which MCMV directs to the salivary glands. Peritoneal infection similarly reached the salivary glands via dendritic cells. However, it also infected other monocyte types, and they spread infection to other tissues. Thus, infecting the “wrong” monocytes altered virus spread, with potential to cause disease. These results provide a basis for understanding how the monocyte types infected by human CMV might promote different infection outcomes.