Pollens destroy respiratory epithelial cell anchors and drive alphaherpesvirus infection

Pollens are well-known triggers of respiratory allergies and asthma. The pollen burden in today's ambient air is constantly increasing due to rising climate change and air pollution. How pollens interact with the respiratory mucosa remains largely unknown due to a lack of representative model systems. We here demonstrate how pollen proteases of Kentucky bluegrass, white birch and hazel selectively destroy integrity and anchorage of columnar respiratory epithelial cells, but not of basal cells, in both ex vivo respiratory mucosal explants and in vitro primary equine respiratory epithelial cells (EREC). In turn, this pollen protease-induced damage to respiratory epithelial cell anchorage resulted in increased infection by the host-specific and ancestral alphaherpesvirus equine herpesvirus type 1 (EHV1). Pollen proteases of all three plant species were characterized by zymography and those of white birch were fully identified for the first time as serine proteases of the subtilase family and meiotic prophase aminopeptidase 1 using mass spectrometry-based proteomics. Together, our findings demonstrate that pollen proteases selectively and irreversibly damage integrity and anchorage of columnar respiratory epithelial cells. In turn, alphaherpesviruses benefit from this partial loss-of-barrier function, resulting in increased infection of the respiratory epithelium

An alphaherpesvirus exploits antimicrobial beta-defensins to initiate respiratory tract infection

beta-Defensins protect the respiratory tract against the myriad of microbial pathogens entering the airways with each breath. However, this potentially hostile environment is known to serve as a portal of entry for herpesviruses. The lack of suitable respiratory model systems has precluded understanding of how herpesvirus virions overcome the abundant mucosal p-defensins during host invasion. We demonstrate how a central alphaherpesvirus, equine herpesvirus type 1 (EHV1), actually exploits p-defensins to invade its host and initiate viral spread. The equine beta-defensins (eBDs) eBD1, -2, and -3 were produced and secreted along the upper respiratory tract. Despite the marked antimicrobial action of eBD2 and -3 against many bacterial and viral pathogens, EHV1 virions were resistant to eBDs through the action of the viral glycoprotein M envelope protein. Pretreatment of EHV1 virions with eBD2 and -3 increased the subsequent infection of rabbit kidney (RK13) cells, which was dependent on viral N-linked glycans. eBD2 and -3 also caused the aggregation of EHV1 virions on the cell surface of RK13 cells. Pretreatment of primary equine respiratory epithelial cells (EREC) with eBD1, -2, and -3 resulted in increased EHV1 virion binding to and infection of these cells. EHV1-infected EREC, in turn, showed an increased production of eBD2 and -3 compared to that seen in mock- and influenza virus-infected EREC. In addition, these eBDs attracted leukocytes, which are essential for EHV1 dissemination and which serve as latent infection reservoirs. These novel mechanisms provide new insights into herpesvirus respiratory tract infection and pathogenesis. IMPORTANCE How herpesviruses circumvent mucosal defenses to promote infection of new hosts through the respiratory tract remains unknown due to a lack of hostspecific model systems. We used the alphaherpesvirus equine herpesvirus type 1 (EHV1) and equine respiratory tissues to decipher this key event in general alphaherpesvirus pathogenesis. In contrast to several respiratory viruses and bacteria, EHV1 resisted potent antimicrobial equine p-defensins (eBDs) eBD2 and eBD3 by the action of glycoprotein M. Instead, eBD2 and -3 facilitated EHV1 particle aggregation and infection of rabbit kidney (RK13) cells. In addition, virion binding to and subsequent infection of respiratory epithelial cells were increased upon preincubation of these cells with eBD1, -2, and -3. Infected cells synthesized eBD2 and -3, promoting further host cell invasion by EHV1. Finally, eBD1, -2, and -3 recruited leukocytes, which are well-known EHV1 dissemination and latency vessels. The exploitation of host innate defenses by herpesviruses during the early phase of host colonization indicates that highly specialized strategies have developed during host-pathogen coevolution

Equine herpesvirus 1 bridles T lymphocytes to reach its target organs

Equine herpesvirus 1 (EHV1) replicates in the respiratory epithelium and disseminates through the body via a cell-associated viremia in leukocytes, despite the presence of neutralizing antibodies. "Hijacked" leukocytes, previously identified as monocytic cells and T lymphocytes, transmit EHV1 to endothelial cells of the endometrium or central nervous system, causing reproductive (abortigenic variants) or neurological (neurological variants) disorders. In the present study, we questioned the potential route of EHV1 infection of T lymphocytes and how EHV1 misuses T lymphocytes as a vehicle to reach the endothelium of the target organs in the absence or presence of immune surveillance. Viral replication was evaluated in activated and quiescent primary T lymphocytes, and the results demonstrated increased infection of activated versus quiescent, CD4(+) versus CD8(+), and blood-versus lymph node-derived T cells. Moreover, primarily infected respiratory epithelial cells and circulating monocytic cells efficiently transferred virions to T lymphocytes in the presence of neutralizing antibodies. Albeit T-lymphocytes express all classes of viral proteins early in infection, the expression of viral glycoproteins on their cell surface was restricted. In addition, the release of viral progeny was hampered, resulting in the accumulation of viral nucleocapsids in the T cell nucleus. During contact of infected T lymphocytes with endothelial cells, a late viral protein(s) orchestrates T cell polarization and synapse formation, followed by anterograde dynein-mediated transport and transfer of viral progeny to the engaged cell. This represents a sophisticated but efficient immune evasion strategy to allow transfer of progeny virus from T lymphocytes to adjacent target cells. These results demonstrate that T lymphocytes are susceptible to EHV1 infection and that cell-cell contact transmits infectious virus to and from T lymphocytes. IMPORTANCE Equine herpesvirus 1 (EHV1) is an ancestral alphaherpesvirus that is related to herpes simplex virus 1 and causes respiratory, reproductive, and neurological disorders in Equidae. EHV1 is indisputably a master at exploiting leukocytes to reach its target organs, accordingly evading the host immunity. However, the role of T lymphocytes in cell-associated viremia remains poorly understood. Here we show that activated T lymphocytes efficiently become infected and support viral replication despite the presence of protective immunity. We demonstrate a restricted expression of viral proteins on the surfaces of infected T cells, which prevents immune recognition. In addition, we indicate a hampered release of progeny, which results in the accumulation of nucleocapsids in the T cell nucleus. Upon engagement with the target endothelium, late viral proteins orchestrate viral synapse formation and viral transfer to the contact cell. Our findings have significant implications for the understanding of EHV1 pathogenesis, which is essential for developing innovative therapies to prevent the devastating clinical symptoms of infection

Deoxynivalenol, but not fumonisin B1, aflatoxin B1 or diesel exhaust particles disrupt integrity of the horse's respiratory epithelium and predispose it for equine herpesvirus type 1 infection

The horse's respiratory tract daily encounters a plethora of respirable hazards including air pollutants, mycotoxins and airborne pathogens. To date, the precise effect of air pollution and mycotoxins on respiratory epithelial integrity and subsequent pathogen invasion in the horse has not been studied. Here, diesel exhaust particles (DEP) and three major mycotoxins (deoxynivalenol [DON], aflatoxin B1 [AFB1] and fumonisin B1 [FB1]) were applied to the apical surfaces of both ex vivo respiratory mucosal explants and in vitro primary equine respiratory epithelial cells (EREC) cultivated at the air-liquid interface, prior to inoculation with equine herpesvirus type 1 (EHV1). DON, but not AFB1, FB1 and DEP affected epithelial integrity in both ex vivo and in vitro systems, as demonstrated by histological changes in respiratory epithelial morphology and a drop in transepithelial electrical resistance across the EREC monolayer. Further, DON-pretreated explants showed on average 6.5 +/- 4.5-fold more EHV1 plaques and produced on average 1 log(10) more extracellular virus particles compared to control diluent- and FB1-pretreated respiratory mucosal explants. Similarly, EHV1 infection was greatly enhanced in EREC upon pretreatment with DON. Based on our findings, we propose that inhalation of DON predisposes horses for EHV1 infection by affecting respiratory epithelial integrity

Equine herpesvirus 1 infection orchestrates the expression of chemokines in equine respiratory epithelial cells

The ancestral equine herpesvirus 1 (EHV1), closely related to human herpes viruses, exploits leukocytes to reach its target organs, accordingly evading the immune surveillance system. Circulating EHV1 strains can be divided into abortigenic/neurovirulent, causing reproductive/neurological disorders. Neurovirulent EHV1 more efficiently recruits monocytic CD172a(+) cells to the upper respiratory tract (URT), while abortigenic EHV1 tempers monocyte migration. Whether similar results could be expected for T lymphocytes is not known. Therefore, we questioned whether differences in T cell recruitment could be associated with variations in cell tropism between both EHV1 phenotypes, and which viral proteins might be involved. The expression of CXCL9 and CXCL10 was evaluated in abortigenic/neurovirulent EHV1-inoculated primary respiratory epithelial cells (ERECs). The bioactivity of chemokines was tested with a functional migration assay. Replication of neurovirulent EHV1 in the URT resulted in an enhanced expression/bioactivity of CXCL9 and CXCL10, compared to abortigenic EHV1. Interestingly, deletion of glycoprotein 2 resulted in an increased recruitment of both monocytic CD172a(+) cells and T lymphocytes to the corresponding EREC supernatants. Our data reveal a novel function of EHV1-gp2, tempering leukocyte migration to the URT, further indicating a sophisticated virus-mediated orchestration of leukocyte recruitment to the URT

Abortigenic but not neurotropic equine herpes virus 1 modulates the interferon antiviral defense

Equine herpesvirus 1 (EHV1) is considered as a major pathogen of Equidae, causing symptoms from mild respiratory disease to late-termabortion and neurological disorders. Different EHV1 strains circulating in the field have been characterized to be of abortigenic or neurovirulent phenotype. Both variants replicate in a plaque-wise manner in the epithelium of the upper respiratory tract (URT), where the abortigenic strains induce more prominent viral plaques, compared to the neurovirulent strains. Considering the differences in replication at the URT, we hypothesized that abortigenic strains may show an increased ability to modulate the type I IFN secretion/signaling pathway, compared to strains that display the neurovirulent phenotype. Here, we analyze IFN levels induced by abortigenic and neurovirulent EHV1 using primary respiratory epithelial cells (EREC) and respiratory mucosa ex vivo explants. Similar levels of IFN alpha (similar to 70 U/ml) were detected in explants inoculated with both types of EHV1 strains from 48 to 72 hpi. Second, EREC and mucosa explants were treated with recombinant equine IFN alpha (rEqIFN alpha) or Ruxolitinib (Rux), an IFN signaling inhibitor, prior to and during inoculation with abortigenic or neurovirulent EHV1. Replication of both EHV1 variants was suppressed by rEqIFN alpha. Further, addition of Rux increased replication in a concentration-dependent manner, indicating an IFN-susceptibility for both variants. However, in two out of three horses, at a physiological concentration of 100 U/ml of rEqIFN alpha, an increase in abortigenic EHV1 replication was observed compared to 10 U/ml of rEqIFN alpha, which was not observed for the neurovirulent strains. Moreover, in the presence of Rux, the plaque size of the abortigenic variants remained unaltered, whereas the typically smaller viral plaques induced by the neurovirulent variants became larger. Overall, our results demonstrate the importance of IFN alpha in the control of EHV1 replication in the URT for both abortigenic and neurovirulent variants. In addition, our findings support the speculation that abortigenic variants of EHV1 may have developed anti-IFN mechanisms that appear to be absent or less pronounced in neurovirulent EHV1 strains

Access to a main alphaherpesvirus receptor, located basolaterally in the respiratory epithelium, is masked by intercellular junctions

The respiratory epithelium of humans and animals is frequently exposed to alphaherpesviruses, originating from either external exposure or reactivation from latency. To date, the polarity of alphaherpesvirus infection in the respiratory epithelium and the role of respiratory epithelial integrity herein has not been studied. Equine herpesvirus type 1 (EHV1), a well-known member of the alphaherpesvirus family, was used to infect equine respiratory mucosal explants and primary equine respiratory epithelial cells (EREC), grown at the air-liquid interface. EHV1 binding to and infection of mucosal explants was greatly enhanced upon destruction of the respiratory epithelium integrity with EGTA or N-acetylcysteine. EHV1 preferentially bound to and entered EREC at basolateral cell surfaces. Restriction of infection via apical inoculation was overcome by disruption of intercellular junctions. Finally, basolateral but not apical EHV1 infection of EREC was dependent on cellular N-linked glycans. Overall, our findings demonstrate that integrity of the respiratory epithelium is crucial in the host's innate defence against primary alphaherpesvirus infections. In addition, by targeting a basolaterally located receptor in the respiratory epithelium, alphaherpesviruses have generated a strategy to efficiently escape from host defence mechanisms during reactivation from latency

The pathogenesis and immune evasive mechanisms of equine herpesvirus type 1

Equine herpesvirus type 1 (EHV-1) is an alphaherpesvirus related to pseudorabies virus (PRV) and varicella-zoster virus (VZV). This virus is one of the major pathogens affecting horses worldwide. EHV-1 is responsible for respiratory disorders, abortion, neonatal foal death and equine herpes myeloencephalopathy (EHM). Over the last decade, EHV-1 has received growing attention due to the frequent outbreaks of abortions and/or EHM causing serious economical losses to the horse industry worldwide. To date, there are no effective antiviral drugs and current vaccines do not provide full protection against EHV-1-associated diseases. Therefore, there is an urgent need to gain a better understanding of the pathogenesis of EHV-1 in order to develop effective therapies. The main objective of this review is to provide state-of-the-art information on the pathogenesis of EHV-1. We also highlight recent findings on EHV-1 immune evasive strategies at the level of the upper respiratory tract, blood circulation and endothelium of target organs allowing the virus to disseminate undetected in the host. Finally, we discuss novel approaches for drug development based on our current knowledge of the pathogenesis of EHV-1

Enterovirus D-68 Infection of Primary Rat Cortical Neurons: Entry, Replication, and Functional Consequences

Enterovirus D68 (EV-D68) is an emerging pathogen associated with mild to severe respiratory disease. Since 2014, EV-D68 is also linked to acute flaccid myelitis (AFM), causing paralysis and muscle weakness in children. However, it remains unclear whether this is due to an increased pathogenicity of contemporary EV-D68 clades or increased awareness and detection of this virus. Here, we describe an infection model of primary rat cortical neurons to study the entry, replication, and functional consequences of different EV-D68 strains, including historical and contemporary strains. We demonstrate that sialic acids are important (co)receptors for infection of both neurons and respiratory epithelial cells. Using a collection of glycoengineered isogenic HEK293 cell lines, we show that sialic acids on either N-glycans or glycosphingolipids can be used for infection. Additionally, we show that both excitatory glutamatergic and inhibitory GABA-ergic neurons are susceptible and permissive to historical and contemporary EV-D68 strains. EV-D68 infection of neurons leads to the reorganization of the Golgi-endomembranes forming replication organelles, first in the soma and later in the processes. Finally, we demonstrate that the spontaneous neuronal activity of EV-D68-infected neuronal network cultured on microelectrode arrays (MEA) is decreased, independent of the virus strain. Collectively, our findings provide novel insights into neurotropism and -pathology of different EV-D68 strains, and argue that it is unlikely that increased neurotropism is a recently acquired phenotype of a specific genetic lineage. IMPORTANCE Acute flaccid myelitis (AFM) is a serious neurological illness characterized by muscle weakness and paralysis in children. Since 2014, outbreaks of AFM have emerged worldwide, and they appear to be caused by nonpolio enteroviruses, particularly enterovirus-D68 (EV-D68), an unusual enterovirus that is known to mainly cause respiratory disease. It is unknown whether these outbreaks reflect a change of EV-D68 pathogenicity or are due to increased detection and awareness of this virus in recent years. To gain more insight herein, it is crucial to define how historical and circulating EV-D68 strains infect and replicate in neurons and how they affect their physiology. This study compares the entry and replication in neurons and the functional consequences on the neural network upon infection with an old "historical" strain and contemporary "circulating" strains of EV-D68