[Show abstract][Hide abstract] ABSTRACT: Importance:
Cytomegaloviruses (CMVs) chronically and systemically infect most mammals. Human CMV infection is usually asymptomatic, but causes lung disease in people with poor immune function. As human infection is hard to analyse, related animal viruses provide important insights. We show that Murine CMV has two targets in the lungs: macrophages and surfactant-secreting epithelial cells. Acute virus replication occurred largely in the epithelial cells. Macrophages had an important defensive role, as removing them increased infection. These results establish the dual nature of lung infection, with local virus replication in epithelial cells, and spread via quiescently infected macrophages. Distinct therapies may be needed to target these contrasting events.
No preview · Article · Dec 2015 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Lymphocytes provide γ-herpesviruses with a self-renewing substrate for persistent infection and with transport to mucosal sites for host exit. Their role in the initial colonization of new hosts is less clear. Murid herpesvirus-4 (MuHV-4), an experimentally accessible, B cell-tropic rhadinovirus (γ2-herpesvirus), persistently infects both immunocompetent and B cell-deficient mice. A lack of B cells did not compromise its entry into lymphoid tissue, which was an infection of myeloid cells. However it impaired infection amplification and exit from lymphoid tissue, which involved myeloid to B cell transfer.
No preview · Article · May 2015 · Journal of General Virology
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
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.
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 difficult to 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.
No preview · Article · Apr 2015 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Lymphocyte proliferation, mobility and longevity makes them prime targets for viral infection. Myeloid cells that process and present environmental antigens to lymphocytes are consequently an important line of defence. Subcapsular sinus macrophages (SSM) filter the afferent lymph and communicate with B cells. How they interact with B cell-tropic viruses is unknown. We analyzed their encounter with Murid Herpesvirus-4 (MuHV-4), an experimentally accessible gamma-herpesvirus related to the Kaposi's Sarcoma-associated Herpesvirus. MuHV-4 disseminates via lymph nodes, and intranasally or subcutaneously inoculated virions readily infected SSM. 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 viral spread to the spleen. Dendritic cells provided the main route to B cells, and SSM slowed host colonization apparently by absorbing virions non-productively from the afferent lymph.
Preview · Article · Apr 2015 · Journal of General Virology
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Viruses commonly infect the respiratory tract. Analyses of host defense have focused on the lungs and the respiratory epithelium. Spontaneously inhaled murid herpesvirus 4 (MuHV-4) and herpes simplex virus 1 (HSV-1) instead infect the olfactory epithelium, where neuronal cilia are exposed to environmental antigens and provide a route across the epithelial mucus. We used MuHV-4 to define how B cells respond to virus replication in this less well-characterized site. Olfactory infection elicited generally weaker acute responses than lung infection, particularly in the spleen, reflecting slower viral replication and spread. Few virus-specific antibody-forming cells (AFCs) were found in the nasal-associated lymphoid tissue (NALT), a prominent response site for respiratory epithelial infection. Instead, they appeared first in the superficial cervical lymph nodes. The focus of the AFC response then moved to the spleen, matching the geography of virus dissemination. Little virus-specific IgA response was detected until later in the bone marrow. Neuroepithelial HSV-1 infection also elicited no significant AFC response in the NALT and a weak IgA response. Thus, olfactory herpesvirus infection differed immunologically from an infection of the adjacent respiratory epithelium. Poor IgA induction may help herpesviruses to transmit via long-term mucosal shedding.
Full-text · Article · Sep 2014 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Herpesvirus transmission is sporadic, and infection may be asymptomatic or present only with secondary lesions after dissemination. Consequently host entry remains ill-understood. Experimental infections can be informative, but depend on inoculations that are inherently artificial and so need validation. Mice are a widely used experimental host. Alert mice inhale readily small (5μl) liquid volumes, and Indian ink, luciferase or radiolabel delivered thus distributed to the nasopharynx and oropharynx. Murid Herpesvirus-4 or Herpes simplex virus type 1 delivered thus infected only the nose, arguing that host entry is nasal rather than oral. Marker or virus delivery to the lung depended on general anesthesia and a large inoculum volume (30μl), and so needs further validation of physiological relevance. While lungs could be infected at lower doses than the upper respiratory tract, tracking experiments showed that nasal inocula pass mostly into the oropharynx, even when restricted to 1μl. Thus, the relative inefficiency of experimental upper respiratory tract infection was attributable to limited liquid retention in this site. Nonetheless low volume intranasal delivery to alert mice provides a convenient way to model experimentally an apparently natural mode of herpesvirus host entry.
No preview · Article · Jun 2014 · Journal of Virological Methods
[Show abstract][Hide abstract] ABSTRACT: Persistent infections are subject to constant surveillance by CD8+ cytotoxic T cells (CTL). Their control should therefore depend on MHC class I-restricted epitope presentation. Many epitopes are described for γ-herpesviruses and form a basis for prospective immunotherapies and vaccines. However the quantitative requirements of in vivo immune control for epitope presentation and recognition remain poorly defined. We used Murid Herpesvirus-4 (MuHV-4) to determine for a latently expressed viral epitope how MHC class-I binding and CTL functional avidity impact on host colonization. Tracking MuHV-4 recombinants that differed only in epitope presentation, we found little latitude for sub-optimal MHC class I binding before immune control failed. By contrast, control remained effective across a wide range of T cell functional avidities. Thus, we could define critical engagement thresholds for the in vivo immune control of virus-driven B cell proliferation.
[Show abstract][Hide abstract] ABSTRACT: Gamma-herpesviruses (γHVs) are widespread oncogenic pathogens that chronically infect circulating lymphocytes. How they subvert the immune check-point function of the spleen to promote persistent infection is not clear. We show that Murid Herpesvirus-4 (MuHV-4) enters the spleen by infecting marginal zone (MZ) macrophages, which provided a conduit to MZ B cells. Relocation of MZ B cells to the white pulp allowed virus transfer to follicular dendritic cells. From here the virus reached germinal center B cells to establish persistent infection. Mice lacking MZ B cells, or treated with a sphingosine-1-phosphate receptor agonist to dislocate them, were protected against MuHV-4 colonization. MuHV-4 lacking ORF27, which encodes a glycoprotein necessary for efficient intercellular spread, could infect MZ macrophages but was impaired in long-term infection. Thus, MuHV-4, a γHV, exploits normal immune communication routes to spread by serial lymphoid/myeloid exchange.
[Show abstract][Hide abstract] ABSTRACT: Lymphocyte colonization by gammaherpesviruses (γHVs) is an important target for cancer prevention. However, how it works is not clear. Epstein-Barr virus drives autonomous B cell proliferation in vitro but in vivo may more subtly exploit the proliferative pathways provided by lymphoid germinal centers (GCs). Murid herpesvirus 4 (MuHV-4), which realistically infects inbred mice, provides a useful tool with which to understand further how a γHV colonizes B cells in vivo. Not all γHVs necessarily behave the same, but common events can with MuHV-4 be assigned an importance for host colonization and so a potential as therapeutic targets. MuHV-4-driven B cell proliferation depends quantitatively on CD4+ T cell help. Here we show that it also depends on T cell-independent survival signals provided by the B cell-activating factor (BAFF) receptor (BAFF-R). B cells could be infected in BAFF-R−/− mice, but virus loads remained low. This corresponded to a BAFF-R-dependent defect in GC colonization. The close parallels between normal, antigen-driven B cell responses and virus-infected B cell proliferation argue that in vivo, γHVs mostly induce infected B cells into normal GC reactions rather than generating large numbers of autonomously proliferating blasts.
IMPORTANCE γHVs cause cancers by driving the proliferation of infected cells. B cells are a particular target. Thus, we need to know how virus-driven B cell proliferation works. Controversy exists as to whether viral genes drive it directly or less directly orchestrate the engagement of normal, host-driven pathways. Here we show that the B cell proliferation driven by a murid γHV requires BAFF-R. This supports the idea that γHVs exploit host proliferation pathways and suggests that interfering with BAFF-R could more generally reduce γHV-associated B cell proliferation.
Preview · Article · Feb 2014 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Human gammaherpesviruses are associated with the development of lymphomas and epithelial malignancies. The heterogeneity of these tumors reflects the ability of these viruses to route infection to different cell types at various stages of their lifecycle. While the Epstein Barr virus uses gp42 - human leukocyte antigen class II interaction as a switch of cell tropism, the molecular mechanism that orientates tropism of rhadinoviruses is still poorly defined. Here, we used bovine herpesvirus 4 (BoHV-4) to further elucidate how rhadinoviruses regulate their infectivity. In the absence of any gp42 homolog, BoHV-4 exploits the alternative splicing of its Bo10 gene to produce distinct viral populations that behave differently based on the originating cell. While epithelial cells produce virions with high levels of the accessory envelope protein gp180, encoded by a Bo10 spliced product, myeloid cells express reduced levels of gp180. As a consequence, virions grown in epithelial cells are hardly infectious for CD14+ circulating cells, but are relatively resistant to antibody neutralization due to the shielding property of gp180 for vulnerable entry epitopes. In contrast, myeloid virions readily infect CD14+ circulating cells but are easily neutralized. This molecular switch could therefore allow BoHV-4 to promote either, on the one hand, its dissemination into the organism, or, on the other hand, its transmission between hosts.
[Show abstract][Hide abstract] ABSTRACT: 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.
Preview · Article · Jul 2013 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: 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
Preview · Article · Jul 2013 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Transmission is a matter of life or death for pathogen lineages and can therefore be considered as the main motor of their evolution. Gammaherpesviruses are archetypal pathogenic persistent viruses which have evolved to be transmitted in presence of specific immune response. Identifying their mode of transmission and their mechanisms of immune evasion is therefore essential to develop prophylactic and therapeutic strategies against these infections. As the known human gammaherpesviruses, Epstein-Barr virus and Kaposi's Sarcoma-associated Herpesvirus are host-specific and lack a convenient in vivo infection model; related animal gammaherpesviruses, such as murine gammaherpesvirus-68 (MHV-68), are commonly used as general models of gammaherpesvirus infections in vivo. To date, it has however never been possible to monitor viral excretion or virus transmission of MHV-68 in laboratory mice population. In this study, we have used MHV-68 associated with global luciferase imaging to investigate potential excretion sites of this virus in laboratory mice. This allowed us to identify a genital excretion site of MHV-68 following intranasal infection and latency establishment in female mice. This excretion occurred at the external border of the vagina and was dependent on the presence of estrogens. However, MHV-68 vaginal excretion was not associated with vertical transmission to the litter or with horizontal transmission to female mice. In contrast, we observed efficient virus transmission to naïve males after sexual contact. In vivo imaging allowed us to show that MHV-68 firstly replicated in penis epithelium and corpus cavernosum before spreading to draining lymph nodes and spleen. All together, those results revealed the first experimental transmission model for MHV-68 in laboratory mice. In the future, this model could help us to better understand the biology of gammaherpesviruses and could also allow the development of strategies that could prevent the spread of these viruses in natural populations.
[Show abstract][Hide abstract] ABSTRACT: Author Summary
Herpesviruses are supremely successful mammalian parasites. Yet their infections rarely present until well established, so how new hosts are first infected has been unclear. Understanding this is likely to be crucial for infection control. Using Murid Herpesvirus-4, a relative of the Kaposi's Sarcoma-associated Herpesvirus, we identified the olfactory neuroepithelium as a major portal of host entry. Heparan sulfate (HS) binding, which is common to many herpesviruses, played a key role. The HS of most epithelia is solely basolateral and therefore inaccessible to incoming, apical virions. The neuroepithelium, by contrast, also displayed HS on its apical surface. This comprises a dense meshwork of the neuronal cilia that mediate olfaction. Incoming virions bound to the cilia, as did a recombinant form of the virion glycoprotein H/L heterodimer. Some virions tracked down the cilia to infect neurons. Others were transferred to the microvilli of adjacent sustentacular cells. The central role of HS in this first detailed description of host entry by a mammalian herpesvirus, and the paucity of accessible HS on other epithelia, suggested that many HS-binding herpesviruses could follow a similar path.
[Show abstract][Hide abstract] ABSTRACT: Protein and eGFP content per pf.u. of gp150+ and gp150− viruses used in binding assays. Aliquots of the gp150+ and gp150− virus stocks used for binding assays were denatured by heating in Laemmli's buffer, then resolved by SDS-PAGE and either stained with Coomassie Brilliant Blue (left panel) or immunoblotted for eGFP with a polyclonal rabbit serum (right panel). This established that equivalent numbers of p.f.u. contained equivalent amounts of viral protein and gM-eGFP, and so that one virus stock did not have an excess of non-infectious virions or contaminating cellular debris. The higher bands on the immunoblot are likely to be aggregated gM-eGFP, since gM tends to precipitate when excessively denatured.