Oncogenic Herpesvirus Engages Endothelial Transcription Factors SOX18 and PROX1 to Increase Viral Genome Copies and Virus Production.

Kaposi sarcoma is a tumor caused by Kaposi sarcoma herpesvirus (KSHV) infection and is thought to originate from lymphatic endothelial cells (LEC). While KSHV establishes latency in virtually all susceptible cell types, LECs support spontaneous expression of oncogenic lytic genes, high viral genome copies, and release of infectious virus. It remains unknown the contribution of spontaneous virus production to the expansion of KSHV-infected tumor cells and the cellular factors that render the lymphatic environment unique to KSHV life cycle. We show here that expansion of the infected cell population, observed in LECs, but not in blood endothelial cells, is dependent on the spontaneous virus production from infected LECs. The drivers of lymphatic endothelium development, SOX18 and PROX1, regulated different steps of the KSHV life cycle. SOX18 enhanced the number of intracellular viral genome copies and bound to the viral origins of replication. Genetic depletion or chemical inhibition of SOX18 caused a decrease of KSHV genome copy numbers. PROX1 interacted with ORF50, the viral initiator of lytic replication, and bound to the KSHV genome in the promoter region of ORF50, increasing its transactivation activity and KSHV spontaneous lytic gene expression and infectious virus release. In Kaposi sarcoma tumors, SOX18 and PROX1 expression correlated with latent and lytic KSHV protein expression. These results demonstrate the importance of two key transcriptional drivers of LEC fate in the regulation of the tumorigenic KSHV life cycle. Moreover, they introduce molecular targeting of SOX18 as a potential novel therapeutic avenue in Kaposi sarcoma. SIGNIFICANCE: SOX18 and PROX1, central regulators of lymphatic development, are key factors for KSHV genome maintenance and lytic cycle in lymphatic endothelial cells, supporting Kaposi sarcoma tumorigenesis and representing attractive therapeutic targets.

Mechanisms of lymphatic system-specific viral replication and its potential role in autoimmune disease.

Viral infections can be fatal because of the direct cytopathic effects of the virus or the induction of a strong, uncontrolled inflammatory response. Virus and host intrinsic characteristics strongly modulate the outcome of viral infections. Recently we determined the circumstances under which enhanced replication of virus within the lymphoid tissue is beneficial for the outcome of a disease. This enforced viral replication promotes anti-viral immune activation and, counterintuitively, accelerates virus control. In this review we summarize the mechanisms that contribute to enforced viral replication. Antigen-presenting cells and CD169+ macrophages exhibit enforced viral replication after infection with the model viruses lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus (VSV). Ubiquitin-specific peptidase 18 (Usp18), an endogenous type I interferon blocker in CD169+ macrophages, has been identified as a proviral gene, as are B cell activating factor (BAFF) and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Lymphotoxins (LT) strongly enhance viral replication in the spleen and lymph nodes. All these factors modulate splenic architecture and thereby promote the development of CD169+ macrophages. Tumor necrosis factor alpha (TNF-α) and nuclear factor kappa-light-chain-enhancer of activated B cell signaling (NF-κB) have been found to promote the survival of infected CD169+ macrophages, thereby similarly promoting enforced viral replication. Association of autoimmune disease with infections is evident from (1) autoimmune phenomena described during a chronic virus infection; (2) onset of autoimmune disease simultaneous to viral infections; and (3) experimental evidence. Involvement of virus infection during onset of type I diabetes is strongly evident. Epstein-Bar virus (EBV) infection was discussed to be involved in the pathogenesis of systemic lupus erythematosus. In conclusion, several mechanisms promote viral replication in secondary lymphatic organs. Identifying such factors in humans is a challenge for future studies.

Secondary lymphoid organ fibroblastic reticular cells mediate trans-infection of HIV-1 via CD44-hyaluronan interactions.

Fibroblastic reticular cells (FRCs) are stromal cells in secondary lymphoid organs, the major sites for HIV-1 infection of CD4+ T cells. Although FRCs regulate T cell survival, proliferation, and migration, whether they play any role in HIV-1 spread has not been studied. Here, we show that FRCs enhance HIV-1 spread via trans-infection in which FRCs capture HIV-1 and facilitate infection of T cells that come into contact with FRCs. FRCs mediate trans-infection in both two- and three-dimensional culture systems and in a manner dependent on the virus producer cells. This producer cell dependence, which was also observed for virus spread in secondary lymphoid tissues ex vivo, is accounted for by CD44 incorporated into virus particles and hyaluronan bound to such CD44 molecules. This virus-associated hyaluronan interacts with CD44 expressed on FRCs, thereby promoting virus capture by FRCs. Overall, our results reveal a novel role for FRCs in promoting HIV-1 spread.

Hepatitis B virus lymphotropism: emerging details and challenges.

The hepatitis B virus (HBV) is predominantly a hepatotropic virus but also infects cells of the lymphatic system. HBV genomes (DNA, messenger (m)RNA, covalently closed circular (ccc) DNA) and proteins have been found in extrahepatic sites such as peripheral blood mononuclear cells (PBMC), lymph nodes, spleen, bone marrow and cerebrospinal fluid. HBV entry into hepatocytes occurs by binding of the HBV preS1 surface protein to its specific receptor, the bile acid transporter, sodium taurocholate co-transporting polypeptide (NTCP). Although the mechanism of HBV entry into lymphatic cells is unknown, the pre S1 encoded surface protein is thought to be involved. Extrahepatic HBV infection has been studied in both chronic HBV (CHB) and in occult HBV infection (OBI). Studies have shown that HBV genomes are present in different PBMC subsets from chronically infected carriers. Unique HBV variants have been found in PBMC compared to plasma or liver in both nucleos(t)ide analogue (NA) treated and untreated CHB carriers, suggesting replication and compartment specific evolution of HBV. In HBV coinfection, HBV genomes were found in PBMC from hepatitis C virus (HCV), human immunodeficiency virus (HIV) and hepatitis delta virus (HDV) co-infected individuals. Moreover, during pregnancy, the trans placental passage of HBV infected PBMC from highly viremic mothers to infants is one of the postulated means of vertical transmission of HBV. Taken together, HBV infection in extrahepatic sites (i.e., PBMC) is implicated in multiple facets of HBV pathogenesis such as persistence, viral evolution and vertical transmission.

Lymphatic Vessels Balance Viral Dissemination and Immune Activation following Cutaneous Viral Infection.

Lymphatic vessels lie at the interface between peripheral sites of pathogen entry, adaptive immunity, and the systemic host. Though the paradigm is that their open structure allows for passive flow of infectious particles from peripheral tissues to lymphoid organs, virus applied to skin by scarification does not spread to draining lymph nodes. Using cutaneous infection by scarification, we analyzed the effect of viral infection on lymphatic transport and evaluated its role at the host-pathogen interface. We found that, in the absence of lymphatic vessels, canonical lymph-node-dependent immune induction was impaired, resulting in exacerbated pathology and compensatory, systemic priming. Furthermore, lymphatic vessels decouple fluid and cellular transport in an interferon-dependent manner, leading to viral sequestration while maintaining dendritic cell transport for immune induction. In conclusion, we found that lymphatic vessels balance immune activation and viral dissemination and act as an "innate-like" component of tissue host viral defense.

Acute neurologic disease in Porcine rubulavirus experimentally infected piglets.

The objective of this study was to evaluate the clinical disease, humoral response and viral distribution of recent Porcine rubulavirus (PorPV) isolates in experimentally infected pigs. Four, 6-piglet (5-days old) groups were employed (G1-84, G2-93, G3-147, and G4-T). Three viral strains were used for the experimental infection: the reference strain LPMV-1984 (Michoacán 1984) and two other strains isolated in 2013, one in Queretaro (Qro/93/2013) and the other in Michoacán (Mich/147/2013). Each strain was genetically characterized by amplification and sequencing of the gene encoding hemagglutinin-neuroamidase (HN). The inoculation was performed through the oronasal and ocular routes, at a dose of 1×106TCID50/ml. Subsequently, the signs were evaluated daily and necropsies were performed on 3 different days post infection (dpi). We recorded all micro- and macroscopic lesions. Organs from the nervous, lymphatic, and respiratory system were analyzed by quantifying the viral RNA load and the presence of the infectious virus. The presence of the viral antigen in organs was evidenced through immunohistochemistry. Seroconversion was evaluated through the use of a hemagglutination inhibition test. In the characterization of gene HN, only three substitutions were identified in strain Mich/147/2013, two in strain LPMV/1984 (fourth passage) and one in strain Qro/93/2013, with respect to reference strain LPMV-84, these changes had not been identified as virulence factors in previously reported strains. Neurological alterations associated with the infection were found in all three experimental groups starting from 3dpi. Groups G1-84 and G3-147 presented the most exacerbated nervous signs. Group G2-93 only presented milder signs including slight motor incoordination, and an increased rectal temperature starting from day 5 post infection (PI). The main histopathological findings were the presence of a mononuclear inflammatory infiltrate (lymphocytic/monocytic) surrounding the ventricles in the brain and focal interstitial pneumonitis with distention of the alveolar sacs in the lungs. PorPV and RNA distribution were identified in the organs of the nervous, lymphatic, and respiratory systems of the piglets analyzed at different times (days 5, 10, and 15 PI). The viral antigen was detected in the brain and lungs in most of the assessed groups. Seroconversion was evident in groups G1-84 and G2-93. Groups G1-84 and G3-147 were the most clinically affected by the experimental infection. Both strains were isolated in the state of Michoacán. The virulence of the new isolates maintains similar characteristics to those reported more than 30 years ago.

Get It through Your Thick Head: Emerging Principles in Neuroimmunology and Neurovirology Redefine Central Nervous System "Immune Privilege".

The central nervous system (CNS) coordinates all aspects of life, autonomic and sentient, though how it has evolved to contend with pathogenic infections remains, to a great degree, a mystery. The skull and cerebrospinal fluid (CSF) provide protection from blunt force contacts, and it was once thought that the blood-brain barrier (BBB) was a fortress that restricted pathogen entry and limited inflammation. Recent studies, however, have caused a revision of this viewpoint: the CNS is monitored by blood-borne lymphocytes, but can use alternative strategies to prevent or resolve many pathogenic challenges. In this Review, we discuss emerging principles that indicate how the CNS is immunologically unique from peripheral tissues. We focus on developments that include glymphatics, recently characterized brain lymphatic vessels, distinctions in innate and adaptive immune strategies, novel points of entry for neurotropic viruses, and, finally, how the periphery can influence CNS homeostasis and immune responses within the brain. Collectively, these attributes demand a re-evaluation of immunity in the brain: not privileged, but distinct.

The meningeal lymphatic system: a route for HIV brain migration?

Two innovative studies recently identified functional lymphatic structures in the meninges that may influence the development of HIV-associated neurological disorders (HAND). Until now, blood vessels were assumed to be the sole transport system by which HIV-infected monocytes entered the brain by bypassing a potentially hostile blood-brain barrier through inflammatory-mediated semi-permeability. A cascade of specific chemokine signals promote monocyte migration from blood vessels to surrounding brain tissues via a well-supported endothelium, where the cells differentiate into tissue macrophages capable of productive HIV infection. Lymphatic vessels on the other hand are more loosely organized than blood vessels. They absorb interstitial fluid from bodily tissues where HIV may persist and exchange a variety of immune cells (CD4(+) T cells, monocytes, macrophages, and dendritic cells) with surrounding tissues through discontinuous endothelial junctions. We propose that the newly discovered meningeal lymphatics are key to HIV migration among viral reservoirs and brain tissue during periods of undetectable plasma viral loads due to suppressive combinational antiretroviral therapy, thus redefining the migration process in terms of a blood-lymphatic transport system.

Oral application of freeze-dried yeast particles expressing the PCV2b Cap protein on their surface induce protection to subsequent PCV2b challenge in vivo.

Porcine circovirus type 2 (PCV2) is now endemic in every major pig producing country, causing PCV-associated disease (PCVAD), linked with large scale economic losses. Current vaccination strategies are based on the capsid protein of the virus and are reasonably successful in preventing PCVAD but fail to induce sterile immunity. Additionally, vaccinating whole herds is expensive and time consuming. In the present study a "proof of concept" vaccine trial was employed to test the effectiveness of powdered freeze-dried recombinant Saccharomyces cerevisiae yeast stably expressing the capsid protein of PCV2b on its surface as an orally applied vaccine. PCV2-free pigs were given 3 doses of vaccine or left un-vaccinated before challenge with a defined PCV2b strain. Rectal temperatures were measured and serum and faeces samples were collected weekly. At the end of the study, pigs were euthanized, tissue samples taken and tested for PCV2b load by qPCR and immunohistochemistry. The peak of viraemia in sera and faeces of unvaccinated pigs was higher than that of vaccinated pigs. Additionally more sIgA was found in faeces of vaccinated pigs than unvaccinated. Vaccination was associated with lower serum concentrations of TNFα and IL-1ß but higher concentrations of IFNα and IFNγ in comparison to the unvaccinated animals. At the end of the trial, a higher viral load was found in several lymphatic tissues and the ileum of unvaccinated pigs in comparison to vaccinated pigs. The difference between groups was especially apparent in the ileum. The results presented here demonstrate a possible use for recombinant S. cerevisiae expressing viral proteins as an oral vaccine against PCV2. A powdered freeze-dried recombinant S. cerevisiae used as an oral vaccine could be mixed with feed and may offer a cheap and less labour intensive alternative to inoculation with the additional advantage that no cooling chain would be required for vaccine transport and storage.

[Role of Follicular helper CD4 T cells in HIV/SIV infection].

Follicular CD4 T helper (TFH) cells promote the survival, isotype switching and generation of high affinity memory B cells and plasma cells. There are numerous reports regarding the dysfunction of B cell mediated immune responses, the lack of memory CD4 T cells and the dysfunction of HIV specific CD4 T cells in SIV/HIV infection. During chronic SIV/HIV infection, TFH cell accumulation may drive B cell dysfunction and become a major HIV reservoir. In this review, we discuss the relationship between TFH cells and B cells in SIV/HIV infection.

Stavudine entrapped lipid nanoparticles for targeting lymphatic HIV reservoirs.

The main objective of present research study was to evaluate the potential of lipid nanoparticles for active delivery of an antiretroviral drug to lymphatic tissues. Stavudine entrapped drug loaded solid lipid nanoparticles (SLNs) were prepared and characterized for a variety of physicochemical parameters such as appearance, particle size, polydispersity index and zeta potential. The targeting potential of the prepared nanoparticles was investigated by carrying out ex vivo cellular uptake studies in macrophages which depicted several times enhanced uptake as compared to pure drug solution. Further, the lymphatic drug levels and organ distribution studies demonstrated efficiency of the developed nanoparticles for prolonged residence in spleenic tissues. Thus it was concluded that stavudine entrapped lipid carriers can be exploited for effective and targeted delivery to cellular and anatomical HIV reservoirs and may ultimately increase the therapeutic safety and reduce side effects.

Kaposi's sarcoma after repeated surgical procedures in an immunocompetent patient: the lymphatic hypothesis.

A 63-year-old Swiss patient developed acquired nodules on his right palm after 3 localized surgeries, called 'needle fasciotomy', for Dupuytren's disease. Kaposi's sarcoma (KS) was diagnosed in a biopsy of a nodule. A positive immunolabeling and serology for human herpesvirus 8 has been found, but human immunodeficiency virus and hepatitis C identification remained negative. The nodules were limited to the surgically traumatized area. This first report of a nonimmunocompromised patient developing a KS after repeated surgeries in a unique peculiar localized area with a dense lymphatic network sustains the hypothesis that tissue alterations involving the lymphatic system could play a central role in the occurrence of KS.

KSHV manipulates Notch signaling by DLL4 and JAG1 to alter cell cycle genes in lymphatic endothelia.

Increased expression of Notch signaling pathway components is observed in Kaposi sarcoma (KS) but the mechanism underlying the manipulation of the canonical Notch pathway by the causative agent of KS, Kaposi sarcoma herpesvirus (KSHV), has not been fully elucidated. Here, we describe the mechanism through which KSHV directly modulates the expression of the Notch ligands JAG1 and DLL4 in lymphatic endothelial cells. Expression of KSHV-encoded vFLIP induces JAG1 through an NFkappaB-dependent mechanism, while vGPCR upregulates DLL4 through a mechanism dependent on ERK. Both vFLIP and vGPCR instigate functional Notch signalling through NOTCH4. Gene expression profiling showed that JAG1- or DLL4-stimulated signaling results in the suppression of genes associated with the cell cycle in adjacent lymphatic endothelial cells, indicating a role for Notch signaling in inducing cellular quiescence in these cells. Upregulation of JAG1 and DLL4 by KSHV could therefore alter the expression of cell cycle components in neighbouring uninfected cells during latent and lytic phases of viral infection, influencing cellular quiescence and plasticity. In addition, differences in signaling potency between these ligands suggest a possible complementary role for JAG1 and DLL4 in the context of KS.

Low viral loads and lymphoid organ spheroids are associated with yellow head virus (YHV) tolerance in whiteleg shrimp Penaeus vannamei.

Yellow head virus (YHV) is an invertebrate nidovirus that has caused mass mortality in penaeid shrimp since 1990. Several YHV types are known, but only the original type (YHV-type 1 or YHV-1) is highly virulent. Most studies have focused on acute YHV-1 infections and there is limited work on YHV-1 survivors. We compared moribund and surviving (14%) whiteleg shrimp Penaeus (Litopenaeus) vannamei from an experimental challenge with YHV-1. Although grossly normal, all survivors were positive for YHV-1 by specific, reverse transcriptase polymerase chain reaction (RT-PCR) assays, histological analysis or transmission electron microscopy (TEM), indicating that they were not resistant but tolerant to YHV-1. On the other hand, real-time PCR analysis revealed that mean YHV-1 copies/ng total RNA for survivors (2.8x10(4) +/- 6.9x10(4)) were approximately 40 times lower (P<0.05) than those in moribund shrimp (1.2x10(6) +/- 6.7x10(5)copies/ng total RNA). This was confirmed by strong positive immunohistochemical and in situ hybridization (ISH) reactions for YHV-1 in lymphoid organ tubules (LOT) of moribund shrimp and weak positive reaction only in lymphoid organ spheroids (LOS) of survivors. TEM revealed morphologically complete YHV virions in both groups. Furthermore, immuno-TEM and Western blot analysis revealed that YHV-1 structural proteins gp116 and p20 were present at comparable reactive levels in each group. Thus, YHV-1 tolerance was not associated with absence of gp116 as previously reported for palaemonid shrimp. Instead, it was associated with the presence of YHV-positive LOS and a relatively low viral load.

Extralymphatic virus sanctuaries as a consequence of potent T-cell activation.

T helper cells can support the functions of CD8(+) T cells against persistently infecting viruses such as murine lymphocytic choriomeningitis virus (LCMV), cytomegalovirus, hepatitis C virus and HIV. These viruses often resist complete elimination and remain detectable at sanctuary sites, such as the kidneys and other extralymphatic organs. The mechanisms underlying this persistence are not well understood. Here we show that mice with potent virus-specific T-cell responses have reduced levels and delayed formation of neutralizing antibodies, and these mice fail to clear LCMV from extralymphatic epithelia. Transfer of virus-specific B cells but not virus-specific T cells augmented virus clearance from persistent sites. Virus elimination from the kidneys was associated with the formation of IgG deposits in the interstitial space, presumably from kidney-infiltrating B cells. CD8(+) T cells in the kidneys of mice that did not clear virus from this site were activated but showed evidence of exhaustion. Thus, we conclude that in this model of infection, site-specific virus persistence develops as a consequence of potent immune activation coupled with reductions in virus-specific neutralizing antibodies. Our results suggest that sanctuary-site formation depends both on organ anatomy and on the induction of different adaptive immune effector mechanisms. Boosting T-cell responses alone may not reduce virus persistence.

Synthesis and biological evaluation of two glycerolipidic prodrugs of didanosine for direct lymphatic delivery against HIV.

Novel glycerolipidic prodrugs of didanosine and didanosine monophosphate designed to by-pass the hepatic first pass metabolism were synthesized and tested for their cytotoxicity and anti-HIV-1 activity. Formulation as liposomes of dipalmitoylphosphatidylcholine was elaborated. A simple quantitative HPLC-UV method was developed and validated, and ESI-MS was used for qualitative purpose. These two prodrugs exhibited promising biological activities against HIV-1 in in vitro infected cell culture.

Rapid dissemination of SIV following oral inoculation.

OBJECTIVE: To assess the earliest events regarding transmission and dissemination of SIV following nontraumatic oral inoculation in macaques. DESIGN: Juvenile and neonate rhesus macaques were orally inoculated with SIVmac251 and necropsied at 1, 2, 4, 7, or 14 days post-inoculation. Sites of transmission and the extent of viral spread were assessed by using molecular techniques and in situ hybridization to identify SIV nucleic acid in lymphoid and nonlymphoid tissues. RESULTS: This study demonstrates that 1 day post-exposure, SIV nucleic acid was detected in the alimentary canal only in tissues proximal to the stomach, including the oral and esophageal mucosa as well as the tonsils. Following infection, virus was observed to spread rapidly to regional and peripheral lymph nodes by 1 and 2 days post-inoculation (dpi). Hundreds of copies of SIV-DNA were detected 4 dpi, increasing to > 10 000 copies/1 x 10(6) cells by 7 dpi. Identification of SIV positive T cells and macrophages implicates these cell types in viral spread, although dissemination of free virus is also likely. CONCLUSIONS: Here the oral and esophageal mucosa, as well as tonsils, are demonstrated to be potential sites for viral infection upon nontraumatic oral exposure to SIV in macaques. The rapid dissemination following oral transmission observed in this study is reflective of SIV transmission across other mucosal surfaces. The rapidity with which SIV, and probably HIV, spreads throughout the lymphatics indicates a major obstacle for a vaccine amnestic immune response to eliminate infected cells prior to dissemination.

Low doses of hepadnavirus induce infection of the lymphatic system that does not engage the liver.

Woodchuck hepatitis virus (WHV), which is closely related to human hepatitis B virus and is considered to be principally hepatotropic, invades the host's lymphatic system and persists in lymphoid cells independently of whether the infection is symptomatic and serologically evident or concealed. In this study, we show, with the woodchuck model of hepatitis B, that hepadnavirus can establish an infection that engages the lymphatic system, but not the liver, and persists in the absence of virus serological markers, including antiviral antibodies. This primary occult infection is caused by wild-type virus invading the host at a quantity usually not greater than 10(3) virions. It is characterized by trace virus replication progressing in lymphatic organs and peripheral lymphoid cells that, with time, may also spread to the liver. The infection is transmissible to virus-naive hosts as an asymptomatic, indefinitely long, occult carriage of small amounts of biologically competent virus. In contrast to residual silent WHV persistence, which normally endures after the resolution of viral hepatitis and involves the liver, primary occult infection restricted to the lymphatic system does not protect against reinfection with a large, liver-pathogenic WHV dose; however, the occult infection is associated with a swift recovery from hepatitis caused by the superinfection. Our study documents that the lymphatic system is the primary target of WHV infection when small quantities of virions invade a susceptible host.