Update on human herpesvirus 7 pathogenesis and clinical aspects as a roadmap for future research.

Human herpesvirus 7 (HHV-7) is a common virus that is associated with various human diseases including febrile syndromes, dermatological lesions, neurological defects, and transplant complications. Still, HHV-7 remains one of the least studied members of all human betaherpesviruses. In addition, HHV-7-related research is mostly confined to case reports, while in vitro or in vivo studies unraveling basic virology, transmission mechanisms, and viral pathogenesis are sparse. Here, we discuss HHV-7-related literature linking clinical syndromes to the viral life cycle, epidemiology, and viral immunopathogenesis. Based on our review, we propose a hypothetical model of HHV-7 pathogenesis inside its host. Furthermore, we identify important knowledge gaps and recommendations for future research to better understand HHV-7 diseases and improve therapeutic interventions.

Spiroplasma infection as a cause of severe congenital keratouveitis, cataract and glaucoma.

BACKGROUND: Only seven cases of ocular Spiroplasma infection have been reported to date, all presenting as congenital cataracts with concomitant intraocular inflammation. We describe the first case of Spiroplasma infection initially presenting as a corneal infiltrate. CASE PRESENTATION: A 1-month-old girl was referred for a corneal infiltrate in the left eye. She presented in our hospital with unilateral keratouveitis. Examination showed a stromal corneal infiltrate and dense white keratic precipitates in the left eye. Herpetic keratouveitis was suspected and intravenous acyclovir therapy was initiated. Two weeks later, the inflammation in the left eye persisted and was also noticed in the right eye. Acute angle-closure glaucoma and a cataract with dilated iris vessels extending onto the anterior lens capsule developed in the left eye. The inflammation resolved after treatment with azithromycin. Iridectomy, synechiolysis and lensectomy were performed. Bacterial metagenomic sequencing (16 S rRNA) and transmission electron microscopy revealed Spiroplasma ixodetis species in lens aspirates and biopsy. Consequently, a diagnosis of bilateral Spiroplasma uveitis was made. CONCLUSIONS: In cases of congenital cataract with concomitant intraocular inflammation, Spiroplasma infection should be considered. The purpose of this case report is to raise awareness of congenital Spiroplasma infection as a cause of severe keratouveitis, cataract and angle-closure glaucoma in newborns. Performing molecular testing on lens aspirates is essential to confirm diagnosis. Systemic macrolides are suggested as the mainstay of treatment.

CRISPR/Cas9-Constructed Pseudorabies Virus Mutants Reveal the Importance of UL13 in Alphaherpesvirus Escape from Genome Silencing.

Latent and recurrent productive infection of long-living cells, such as neurons, enables alphaherpesviruses to persist in their host populations. Still, the viral factors involved in these events remain largely obscure. Using a complementation assay in compartmented primary peripheral nervous system (PNS) neuronal cultures, we previously reported that productive replication of axonally delivered genomes is facilitated by pseudorabies virus (PRV) tegument proteins. Here, we sought to unravel the role of tegument protein UL13 in this escape from silencing. We first constructed four new PRV mutants in the virulent Becker strain using CRISPR/Cas9-mediated gene replacement: (i) PRV Becker defective for UL13 expression (PRV ΔUL13), (ii) PRV where UL13 is fused to eGFP (PRV UL13-eGFP), and two control viruses (iii and iv) PRV where VP16 is fused with mTurquoise at either the N terminus (PRV mTurq-VP16) or the C terminus (PRV VP16-mTurq). Live-cell imaging of PRV capsids showed efficient retrograde transport after axonal infection with PRV UL13-eGFP, although we did not detect dual-color particles. However, immunofluorescence staining of particles in mid-axons indicated that UL13 might be cotransported with PRV capsids in PNS axons. Superinfecting nerve cell bodies with UV-inactivated PRV ΔUL13 failed to efficiently promote escape from genome silencing compared to UV-PRV wild type and UV-PRV UL13-eGFP superinfection. However, UL13 does not act directly in the escape from genome silencing, as adeno-associated virus (AAV)-mediated UL13 expression in neuronal cell bodies was not sufficient to provoke escape from genome silencing. Based on this, we suggest that UL13 may contribute to initiation of productive infection through phosphorylation of other tegument proteins.IMPORTANCE Alphaherpesviruses have mastered various strategies to persist in an immunocompetent host, including the induction of latency and reactivation in peripheral nervous system (PNS) ganglia. We recently discovered that the molecular mechanism underlying escape from latency by the alphaherpesvirus pseudorabies virus (PRV) relies on a structural viral tegument protein. This study aimed at unravelling the role of tegument protein UL13 in PRV escape from latency. First, we confirmed the use of CRISPR/Cas9-mediated gene replacement as a versatile tool to modify the PRV genome. Next, we used our new set of viral mutants and AAV vectors to conclude the indirect role of UL13 in PRV escape from latency in primary neurons, along with its spatial localization during retrograde capsid transport in axons. Based on these findings, we speculate that UL13 phosphorylates one or more tegument proteins, thereby priming these putative proteins to induce escape from genome silencing.

On the whereabouts of SARS-CoV-2 in the human body: A systematic review.

Since SARS-CoV-2 appeared in the human population, the scientific community has scrambled to gather as much information as possible to find good strategies for the containment and treatment of this pandemic virus. Here, we performed a systematic review of the current (pre)published SARS-CoV-2 literature with a focus on the evidence concerning SARS-CoV-2 distribution in human tissues and viral shedding in body fluids. In addition, this evidence is aligned with published ACE2 entry-receptor (single cell) expression data across the human body to construct a viral distribution and ACE2 receptor body map. We highlight the broad organotropism of SARS-CoV-2, as many studies identified viral components (RNA, proteins) in multiple organs, including the pharynx, trachea, lungs, blood, heart, vessels, intestines, brain, male genitals and kidneys. This also implicates the presence of viral components in various body fluids such as mucus, saliva, urine, cerebrospinal fluid, semen and breast milk. The main SARS-CoV-2 entry receptor, ACE2, is expressed at different levels in multiple tissues throughout the human body, but its expression levels do not always correspond with SARS-CoV-2 detection, indicating that there is a complex interplay between virus and host. Together, these data shed new light on the current view of SARS-CoV-2 pathogenesis and lay the foundation for better diagnosis and treatment of COVID-19 patients.

An Alphaherpesvirus Exploits Antimicrobial ß-Defensins To Initiate Respiratory Tract Infection.

ß-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 ß-defensins during host invasion. We demonstrate how a central alphaherpesvirus, equine herpesvirus type 1 (EHV1), actually exploits ß-defensins to invade its host and initiate viral spread. The equine ß-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 host-specific 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 ß-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.

Alphaherpesvirus infection of mice primes PNS neurons to an inflammatory state regulated by TLR2 and type I IFN signaling.

Pseudorabies virus (PRV), an alphaherpesvirus closely related to Varicella-Zoster virus (VZV) and Herpes simplex type 1 (HSV1) infects mucosa epithelia and the peripheral nervous system (PNS) of its host. We previously demonstrated that PRV infection induces a specific and lethal inflammatory response, contributing to severe neuropathy in mice. So far, the mechanisms that initiate this neuroinflammation remain unknown. Using a mouse footpad inoculation model, we found that PRV infection rapidly and simultaneously induces high G-CSF and IL-6 levels in several mouse tissues, including the footpad, PNS and central nervous system (CNS) tissues. Interestingly, this global increase occurred before PRV had replicated in dorsal root ganglia (DRGs) neurons and also was independent of systemic inflammation. These high G-CSF and IL-6 levels were not caused by neutrophil infiltration in PRV infected tissues, as we did not detect any neutrophils. Efficient PRV replication and spread in the footpad was sufficient to activate DRGs to produce cytokines. Finally, by using knockout mice, we demonstrated that TLR2 and IFN type I play crucial roles in modulating the early neuroinflammatory response and clinical outcome of PRV infection in mice. Overall, these results give new insights into the initiation of virus-induced neuroinflammation during herpesvirus infections.

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.

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.

Gammaherpesvirus BoHV-4 infects bovine respiratory epithelial cells mainly at the basolateral side.

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus that is widespread in cattle. However, only a few studies about the pathogenesis of BoHV-4 primary infection have been reported. In the present study, ex vivo models with bovine nasal and tracheal mucosa explants were used to study the cellular BoHV-4-host interactions. Infection was observed in nasal but not in tracheal epithelial cells. To find a possible correlation between the integrity and restricted infection of the respiratory epithelium, both nasal mucosal and tracheal explants were treated with EGTA, a drug that disrupts the intercellular junctions, before inoculation. The infection was analyzed based on the number of plaques, plaque latitude and number of infected single cells, as determined by immunofluorescence. BoHV-4 infection in nasal mucosal explants was enhanced upon opening the tight junctions with EGTA. Infection in tracheal explants was only found after treatment with EGTA. In addition, primary bovine respiratory epithelial cells (BREC) were isolated, grown at the air-liquid interface and infected either at the apical or basolateral side by BoHV-4. The results showed that BoHV-4 preferentially bound to and entered BREC at the basolateral surfaces of both nasal and tracheal epithelial cells. The percentage of BoHV-4 infection was significantly increased both from nasal and tracheal epithelial cells after treatment with EGTA, which indicates that the BoHV-4 receptor is mainly located at the basolateral surface of these cells. Thus, our findings demonstrate that integrity of the respiratory epithelium is crucial in the host's innate defense against primary BoHV-4 infections.

Unravelling the first key steps in equine herpesvirus type 5 (EHV5) pathogenesis using ex vivo and in vitro equine models.

Equine herpesvirus type 5 (EHV5) is a ubiquitous, yet obscure pathogen in the horse population and is commonly associated with fatal equine multinodular pulmonary fibrosis (EMPF). To date, little is known about the precise pathogenesis of EHV5. Here, we evaluated the dynamics of EHV5 infection in representative ex vivo and in vitro equine models, using immunofluorescence staining and virus titration. EHV5 was unable to infect epithelial cells lining the mucosa of nasal and tracheal explants. Similarly, primary equine respiratory epithelial cells (EREC) were not susceptible to EHV5 following inoculation at the apical or basolateral surfaces. Upon direct delivery of EHV5 particles to lung explants, few EHV5-positive cell clusters were observed at 72 hours post-inoculation (hpi). These EHV5-positive cells were identified as cytokeratin-positive alveolar cells. Next, we examined the potential of EHV5 to infect three distinct equine PBMC populations (CD172a+ monocytes, CD3+ T lymphocytes and Ig light chain+ B lymphocytes). Monocytes did not support EHV5 replication. In contrast, up to 10% of inoculated equine T and B lymphocytes synthetized intracellular viral antigens 24 hpi and 72 hpi, respectively. Still, the production of mature virus particles was hampered, as we did not observe an increase in extracellular virus titer. After reaching a peak, the percentage of infected T and B lymphocytes decayed, which was partly due to the onset of apoptosis, but not necrosis. Based on these findings, we propose a model for EHV5 pathogenesis in the horse. Uncovering EHV5 pathogenesis is the corner step to finally contain or even eradicate the virus.

CCL2 and CCL5 driven attraction of CD172a+ monocytic cells during an equine herpesvirus type 1 (EHV-1) infection in equine nasal mucosa and the impact of two migration inhibitors, rosiglitazone (RSG) and quinacrine (QC).

Equine herpesvirus type 1 (EHV-1) causes respiratory disease, abortion and neurological disorders in horses. Besides epithelial cells, CD172a+ monocytic cells become infected with EHV-1 in the respiratory mucosa and transport the virus from the apical side of the epithelium to the lamina propria en route to the lymph and blood circulation. Whether CD172a+ monocytic cells are specifically recruited to the infection sites in order to pick up virus is unknown. In our study, equine nasal mucosa explants were inoculated with EHV-1 neurological strains 03P37 and 95P105 or the non-neurological strains 97P70 and 94P247 and the migration of monocytic cells was examined by immunofluorescence. Further, the role of monokines CCL2 and CCL5 was determined and the effect of migration inhibitors rosiglitazone (RSG) or quinacrine was analyzed. It was shown that with neurological strains but not with the non-neurological strains, CD172a+ cells specifically migrated towards EHV-1 infected regions and that CCL2 and CCL5 were involved. CCL2 started to be expressed in infected epithelial cells at 24 h post-incubation (hpi) and CCL5 at 48 hpi, which corresponded with the CD172a+ migration. RSG treatment of EHV-1-inoculated equine nasal mucosa had no effect on the virus replication in the epithelium, but decreased the migration of CD172a+ cells in the lamina propria. Overall, these findings bring new insights in the early pathogenesis of EHV-1 infections, illustrate differences between neurological and non-neurological strains and show the way for EHV-1 treatment.

Cellular and transcriptomic response to treatment with the probiotic candidate Vibrio lentus in gnotobiotic sea bass (Dicentrarchus labrax) larvae.

The present study aimed at evaluating the cellular and transcriptomic responses induced by the probiotic candidate Vibrio lentus with gnotobiotic European sea bass (Dicentrarchus labrax, Linnaeus 1785) larvae. For this, a histomorphological analysis was performed using the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) and the anti-proliferating cell nuclear antigen (PCNA) assay. In addition, a global transcriptomic approach was adopted to study the whole body mRNA changes upon administration of V. lentus by microarrays with the custom Agilent sea bass oligonucleotide-microarray v2.0 (4 × 44 K). Following V. lentus administration, the apoptotic and cell proliferative indexes did not show significant differences between treatments for hindgut nor for midgut. However, V. lentus treatment did significantly modify the gene expression related not only to cell proliferation and cell death, but also to cell adhesion, reactive oxygen species metabolism, iron transport, and immune response. Our data represent the first global analysis of the effects of the probiotic candidate V. lentus on the gene expression profile in gnotobiotic European sea bass, and as such, provides a first delineation of the mechanisms by which this agent interacts with its host and exerts its beneficial effects.

Entry of equid herpesvirus 1 into CD172a+ monocytic cells.

Equid herpesvirus 1 (EHV-1) causes respiratory disease, abortion and neurological disorders in horses. Cells from the myeloid lineage (CD172a+) are one of the main target cells of EHV-1 during primary infection. Recently, we showed that EHV-1 restricts and delays its replication in CD172a+ cells as part of an immune-evasive strategy to disseminate to target organs. Here, we hypothesize that a low efficiency of EHV-1 binding to and entry in CD172a+ cells is responsible for this restriction. Thus, we characterized EHV-1 binding and entry into CD172a+ cells, and showed that EHV-1 only bound to 15-20 % of CD172a+ cells compared with 70 % of RK-13 control cells. Enzymic removal of heparan sulphate did not reduce EHV-1 infection, suggesting that EHV-1 does not use heparan sulphate to bind and enter CD172a+ cells. In contrast, we found that treatment of cells with neuraminidase (NA) reduced infection by 85-100 % compared with untreated cells, whilst NA treatment of virus had no effect on infection. This shows that sialic acid residues present on CD172a+ cells are essential in the initiation of EHV-1 infection. We found that αVß3 integrins are involved in the post-binding stage of CD172a+ cell infection. Using pharmacological inhibitors, we showed that EHV-1 does not enter CD172a+ cells via a clathrin- or caveolae-dependent endocytic pathway, nor by macropinocytosis, but requires cholesterol, tyrosine kinase, actin, dynamin and endosomal acidification, pointing towards a phagocytic mechanism. Overall, these results show that the narrow tropism of EHV-1 amongst CD172a+ cells is determined by the presence of specific cellular receptors.

Equine Herpesvirus Type 1 Enhances Viral Replication in CD172a+ Monocytic Cells upon Adhesion to Endothelial Cells.

UNLABELLED: Equine herpesvirus type 1 (EHV-1) is a main cause of respiratory disease, abortion, and encephalomyelopathy in horses. Monocytic cells (CD172a(+)) are the main carrier cells of EHV-1 during primary infection and are proposed to serve as a "Trojan horse" to facilitate the dissemination of EHV-1 to target organs. However, the mechanism by which EHV-1 is transferred from CD172a(+) cells to endothelial cells (EC) remains unclear. The aim of this study was to investigate EHV-1 transmission between these two cell types. We hypothesized that EHV-1 employs specific strategies to promote the adhesion of infected CD172a(+) cells to EC to facilitate EHV-1 spread. Here, we demonstrated that EHV-1 infection of CD172a(+) cells resulted in a 3- to 5-fold increase in adhesion to EC. Antibody blocking experiments indicated that α4ß1, αLß2, and αVß3 integrins mediated adhesion of infected CD172a(+) cells to EC. We showed that integrin-mediated phosphatidylinositol 3-kinase (PI3K) and ERK/MAPK signaling pathways were involved in EHV-1-induced CD172a(+) cell adhesion at early times of infection. EHV-1 replication was enhanced in adherent CD172a(+) cells, which correlates with the production of tumor necrosis factor alpha (TNF-α). In the presence of neutralizing antibodies, approximately 20% of infected CD172a(+) cells transferred cytoplasmic material to uninfected EC and 0.01% of infected CD172a(+) cells transmitted infectious virus to neighboring cells. Our results demonstrated that EHV-1 infection induces adhesion of CD172a(+) cells to EC, which enhances viral replication, but that transfer of viral material from CD172a(+) cells to EC is a very specific and rare event. These findings give new insights into the complex pathogenesis of EHV-1. IMPORTANCE: Equine herpesvirus type 1 (EHV-1) is a highly prevalent pathogen worldwide, causing frequent outbreaks of abortion and myeloencephalopathy, even in vaccinated horses. After primary replication in the respiratory tract, EHV-1 disseminates via cell-associated viremia in peripheral blood mononuclear cells (PBMC) and subsequently infects the endothelial cells (EC) of the pregnant uterus or central nervous system, leading in some cases to abortion and/or neurological disorders. Recently, we demonstrated that CD172a(+) monocytic carrier cells serve as a "Trojan horse" to facilitate EHV-1 spread from blood to target organs. Here, we investigated the mechanism underlying the transmission of EHV-1 from CD172a(+) cells to EC. We demonstrated that EHV-1 infection induces cellular changes in CD172a(+) cells, promoting their adhesion to EC. We found that both cell-to-cell contacts and the secretion of soluble factors by EC activate EHV-1 replication in CD172a(+) cells. This facilitates transfer of cytoplasmic viral material to EC, resulting mainly in a nonproductive infection. Our findings give new insights into how EHV-1 may spread to EC of target organs in vaccinated horses.

Bacterial Toxins from Staphylococcus aureus and Bordetella bronchiseptica Predispose the Horse's Respiratory Tract to Equine Herpesvirus Type 1 Infection.

Respiratory disease in horses is caused by a multifactorial complex of infectious agents and environmental factors. An important pathogen in horses is equine herpesvirus type 1 (EHV-1). During co-evolution with this ancient alphaherpesvirus, the horse's respiratory tract has developed multiple antiviral barriers. However, these barriers can become compromised by environmental threats. Pollens and mycotoxins enhance mucosal susceptibility to EHV-1 by interrupting cell junctions, allowing the virus to reach its basolateral receptor. Whether bacterial toxins also play a role in this impairment has not been studied yet. Here, we evaluated the role of α-hemolysin (Hla) and adenylate cyclase (ACT), toxins derived from the facultative pathogenic bacterium Staphylococcus aureus (S. aureus) and the primary pathogen Bordetella bronchiseptica (B. bronchiseptica), respectively. Equine respiratory mucosal explants were cultured at an air-liquid interface and pretreated with these toxins, prior to EHV-1 inoculation. Morphological analysis of hematoxylin-eosin (HE)-stained sections of the explants revealed a decreased epithelial thickness upon treatment with both toxins. Additionally, the Hla toxin induced detachment of epithelial cells and a partial loss of cilia. These morphological changes were correlated with increased EHV-1 replication in the epithelium, as assessed by immunofluorescent stainings and confocal microscopy. In view of these results, we argue that the ACT and Hla toxins increase the susceptibility of the epithelium to EHV-1 by disrupting the epithelial barrier function. In conclusion, this study is the first to report that bacterial exotoxins increase the horse's sensitivity to EHV-1 infection. Therefore, we propose that horses suffering from infection by S. aureus or B. bronchiseptica may be more susceptible to EHV-1 infection.

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.

Organ-specific genome diversity of replication-competent SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is not always confined to the respiratory system, as it impacts people on a broad clinical spectrum from asymptomatic to severe systemic manifestations resulting in death. Further, accumulation of intra-host single nucleotide variants during prolonged SARS-CoV-2 infection may lead to emergence of variants of concern (VOCs). Still, information on virus infectivity and intra-host evolution across organs is sparse. We report a detailed virological analysis of thirteen postmortem coronavirus disease 2019 (COVID-19) cases that provides proof of viremia and presence of replication-competent SARS-CoV-2 in extrapulmonary organs of immunocompromised patients, including heart, kidney, liver, and spleen (NCT04366882). In parallel, we identify organ-specific SARS-CoV-2 genome diversity and mutations of concern N501Y, T1027I, and Y453F, while the patient had died long before reported emergence of VOCs. These mutations appear in multiple organs and replicate in Vero E6 cells, highlighting their infectivity. Finally, we show two stages of fatal disease evolution based on disease duration and viral loads in lungs and plasma. Our results provide insights about the pathogenesis and intra-host evolution of SARS-CoV-2 and show that COVID-19 treatment and hygiene measures need to be tailored to specific needs of immunocompromised patients, even when respiratory symptoms cease.

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.

Beyond Gut Instinct: Metabolic Short-Chain Fatty Acids Moderate the Pathogenesis of Alphaherpesviruses.

Short-chain fatty acids (SCFA), such as sodium butyrate (SB), sodium propionate (SPr), and sodium acetate (SAc), are metabolic end-products of the fermentation of dietary fibers. They are linked with multiple beneficial effects on the general mammalian health, based on the sophisticated interplay with the host immune response. Equine herpesvirus 1 (EHV1) is a major pathogen, which primarily replicates in the respiratory epithelium, and disseminates through the body via a cell-associated viremia in leukocytes, even in the presence of neutralizing antibodies. Infected monocytic CD172a+ cells and T-lymphocytes transmit EHV1 to the endothelium of the endometrium or central nervous system (CNS), causing reproductive or neurological disorders. Here, we questioned whether SCFA have a potential role in shaping the pathogenesis of EHV1 during the primary replication in the URT, during the cell-associated viremia, or at the level of the endothelium of the pregnant uterus and/or CNS. First, we demonstrated the expression of SCFA receptors, FFA2 and FFA3, within the epithelium of the equine respiratory tract, at the cell surface of immune cells, and equine endothelium. Subsequently, EHV1 replication was evaluated in the URT, in the presence or absence of SB, SPr, or SAc. In general, we demonstrated that SCFA do not affect the number of viral plaques or virus titer upon primary viral replication. Only SB and SPr were able to reduce the plaque latitudes. Similarly, pretreatment of monocytic CD172a+ cells and T-lymphocytes with different concentrations of SCFA did not alter the number of infected cells. When endothelial cells were treated with SB, SPr, or SAc, prior to the co-cultivation with EHV1-inoculated mononuclear cells, we observed a reduced number of adherent immune cells to the target endothelium. This was associated with a downregulation of endothelial adhesion molecules ICAM-1 and VCAM-1 in the presence of SCFA, which ultimately lead to a significant reduction of the EHV1 endothelial plaques. These results indicate that physiological concentrations of SCFA may affect the pathogenesis of EHV1, mainly at the target endothelium, in favor of the fitness of the horse. Our findings may have significant implications to develop innovative therapies, to prevent the devastating clinical outcome of EHV1 infections.