Summary Equine coital exanthema (ECE) is a contagious, sexually transmitted disease affecting horses. It manifests itself as painful papules, vesicles, pustules and ulcers on the external genitalia in both mares and stallions. While widespread, the disease is generally not classified as reportable in most countries and is not considered a significant threat to the equine industry. ECE is endemic in horse breeding populations internationally. The absence of comprehensive epidemiological data can give a false sense of security, which increases the risk of local transmission. Lack of surveillance programmes or routine control in risk group (reproductive stallions and mares) leads to unawareness and lack of proper biosecurity measures. An outbreak in breeding facilities may result in significant economic consequences. Although ECE has not been officially confirmed in Poland, equine practitioners have noted isolated clinical cases resembling the disease in farm horses in the last decades. This study aims to confirm the presence of ECE in Poland. Clinical diagnosis has been made based on characteristic lesions. Positive diagnosis was confirmed in two out of four outbreaks by real‐time PCR on genital swabs. The presence of the disease has been confirmed in different regions in Poland in farm horses bred as meat animals. Confirming EHV‐3 infection in the Polish equine population through laboratory examinations marks a significant milestone. It is now crucial to raise awareness among owners, breeders and equine practitioners about the current epidemiological status of the disease.

Cell entry of most alphaherpesviruses is mediated by the binding of glycoprotein D (gD) to different cell surface receptors. Equine herpesvirus type 1 (EHV-1) and EHV-4 gDs interact with equine major histocompatibility complex I (MHC-I) to initiate entry into equine cells. We have characterized the gD-MHC-I interaction by solving the crystal structures of EHV-1 and EHV-4 gDs (gD1, gD4), performing protein-protein docking simulations, surface plasmon resonance (SPR) analysis, and biological assays. The structures of gD1 and gD4 revealed the existence of a common V-set immunoglobulin-like (IgV-like) core comparable to those of other gD homologs. Molecular modeling yielded plausible binding hypotheses and identified key residues (F213 and D261) that are important for virus binding. Altering the key residues resulted in impaired virus growth in cells, which highlights the important role of these residues in the gD-MHC-I interaction. Taken together, our results add to our understanding of the initial herpesvirus-cell interactions and will contribute to the targeted design of antiviral drugs and vaccine development.

The antiviral activity of nanoparticles is facilitated by their unique properties that make them attractive tools for viral treatment. The nanoscale size of particles can facilitate cellular uptake and allow reaching anatomically privileged sites. The large surface area-to-volume ratios can ensure decoration with large payloads of functional molecules that can increase the interaction with pathogens, leading to optimized drug dosing and delivery and increasing stability. The surface charge can facilitate binding to infectious agents and subsequently inhibit the initial steps of infection. In addition, nanoparticles can contain biomimetic characteristics that enhance their intrinsic antiviral properties as in the case of silver and gold nanoparticles and dendrimers.

FeHV-1 and determined its role. Autophagy is induced by FeHV-1 in a viral dose-dependent manner, in vitro. The pro-viral role of autophagy during FeHV-1 was clearly 40 using late autophagy inhibitors and inducers, by assessing the viral yield, cytotoxic effects, and expression of viral glycoproteins. Autophagy inhibitors negatively impact viral replication. Further, 42 the autophagy inducer showed increased accumulation of B, a viral protein in the treated 43 cells. In summary, this study demonstrates FeHV-1-mediated autophagy induction, its proviral role, 44 and the negative impact of late autophagy inhibitors on viral replication. 45The PI3K/Akt/mTOR axis is a key regulator of many cellular processes including translation, 46 metabolism, autophagy, and cell death and can fulfill many viral requirements (11

Reliable and straightforward characterization and analysis of carbon-based nanomaterials on the atomic level is essential to exploring their potential for application. Here we use a combination of highly surface sensitive x-ray photoelectron (XP) spectroscopy and near edge x-ray absorption fine structure spectroscopy (NEXAFS) to study and quantify the covalent functionalization of nanographene and single-walled carbon nanotubes with nitrene [2 + 1]-cycloaddition. With this comprehensive analytical approach, we demonstrate that the n-conjugated system of functionalized carbon-based nanomaterials is preserved according to NEXAFS analysis, which is challenging to prove with XP spectroscopy investigation alone. Using this combination of analytical approaches, we show significant similarities after functionalization for various carbon-based nanomaterials. Both analytical methods are strongly suited to study possible post-modification reactions of functionalized carbon-based nanomaterials.

Infectious diseases are major threat to public health around the world. Recent outbreaks and pandemics have influenced the socioeconomic development of humanity and emphasized the urgent need for revolutionary antiviral therapeutics. Nanomaterials can offer unique physical and chemical properties that facilitate the development of novel effective therapeutics with a low probability of developing drug resistance and precise drug delivery function. In the last decade, many studies have demonstrated the potential antiviral activity of nanoparticles against different infections that qualified them as promising therapeutic agents. This chapter summarizes the application of different nanoparticles for the treatment of viral infections. It provides also information on currently available platforms, mechanism of actions, and potential application against emerging viral diseases.

In climates with seasonally limited precipitation, terrestrial animals congregate at high densities at scarce water sources. We hypothesize that viruses can exploit the recurrence of these diverse animal congregations to spread. In this study, we test the central prediction of this hypothesis — that viruses employing this transmission strategy remain stable and infectious in water. Equid herpesviruses (EHVs) were chosen as a model as they have been shown to remain stable and infectious in water for weeks under laboratory conditions. Using fecal data from wild equids from a previous study, we establish that EHVs are shed more frequently by their hosts during the dry season, increasing the probability of water source contamination with EHV. We document the presence of several strains of EHVs present in high genome copy number from the surface water and sediments of waterholes sampled across a variety of mammalian assemblages, locations, temperatures and pH. Phylogenetic analysis reveals that the different EHV strains found exhibit little divergence despite representing ancient lineages. We employed molecular approaches to show that EHVs shed remain stable in waterholes with detection decreasing with increasing temperature in sediments. Infectivity experiments using cell culture reveals that EHVs remain infectious in water derived from waterholes. The results are supportive of water as an abiotic viral vector for EHV.

Recent studies have demonstrated that the signaling activity of the cytosolic pathogen sensor retinoic acid-inducible gene-I (RIG-I) is modulated by a variety of posttranslational modifications (PTMs) to fine-tune the antiviral type I interferon (IFN) response. Whereas K63-linked ubiquitination of the RIG-I caspase activation and recruitment domains (CARDs) catalyzed by TRIM25 or other E3 ligases activates RIG-I, phosphorylation of RIG-I at S8 and T170 represses RIG-I signal transduction by preventing the TRIM25-RIG-I interaction and subsequent RIG-I ubiquitination. While strategies to suppress RIG-I signaling by interfering with its K63-polyubiquitin-dependent activation have been identified for several viruses, evasion mechanisms that directly promote RIG-I phosphorylation to escape antiviral immunity are unknown. Here, we show that the serine/threonine (Ser/Thr) kinase US3 of herpes simplex virus 1 (HSV-1) binds to RIG-I and phosphorylates RIG-I specifically at S8. US3-mediated phosphorylation suppressed TRIM25-mediated RIG-I ubiquitination, RIG-I-MAVS binding, and type I IFN induction. We constructed a mutant HSV-1 encoding a catalytically-inactive US3 protein (K220A) and found that, in contrast to the parental virus, the US3 mutant HSV-1 was unable to phosphorylate RIG-I at S8 and elicited higher levels of type I IFNs, IFN-stimulated genes (ISGs), and proinflammatory cytokines in a RIG-I-dependent manner. Finally, we show that this RIG-I evasion mechanism is conserved among the alphaherpesvirus US3 kinase family. Collectively, our study reveals a novel immune evasion mechanism of herpesviruses in which their US3 kinases phosphorylate the sensor RIG-I to keep it in the signaling-repressed state. IMPORTANCE Herpes simplex virus 1 (HSV-1) establishes lifelong latency in the majority of the human population worldwide. HSV-1 occasionally reactivates to produce infectious virus and to facilitate dissemination. While often remaining subclinical, both primary infection and reactivation occasionally cause debilitating eye diseases, which can lead to blindness, as well as life-threatening encephalitis and newborn infections. To identify new therapeutic targets for HSV-1-induced diseases, it is important to understand the HSV-1-host interactions that may influence infection outcome and disease. Our work uncovered direct phosphorylation of the pathogen sensor RIG-I by alphaherpesvirus-encoded kinases as a novel viral immune escape strategy and also underscores the importance of RNA sensors in surveilling DNA virus infection.

Equine herpesvirus type 4 (EHV-4) is enzootic in equine populations throughout the world. A large outbreak of EHV-4 respiratory infection occurred at a Standardbred horse-breeding farm in northern Germany in 2017. Respiratory illness was observed in a group of in-housed foals and mares, which subsequently resulted in disease outbreak. Out of 84 horses in the stud, 76 were tested and 41 horses were affected, including 20 foals, 10 stallions, and 11 mares. Virological investigations revealed the involvement of EHV-4 in all cases of respiratory illness, as confirmed by virus isolation, qPCR, and/or serological follow-up using virus neutralization test and peptide-specific ELISA. Among infected mares, 73% (8 out of 11) and their corresponding foals shed the virus at the same time. EHV-4 was successfully isolated from four animals (including one stallion and three foals), and molecular studies revealed a different restriction fragment length polymorphism (RFLP) profile in all four isolates. We determined the complete 144 kbp genome sequence of EHV-4 isolated from infected horses by next-generation sequencing and de novo assembly. Hence, EHV-4 is genetically stable in nature, different RFLP profiles, and genome sequences of the isolates, suggesting the involvement of more than one animal as a source of infection due to either true infection or reactivation from a latent state. In addition, epidemiological investigation revealed that stress caused by seasonal changes, management practices, routine equestrian activities, and exercises contributed as a multifactorial causation for disease outbreak. This study shows the importance of implementing stress alleviating measures and management practices in breeding farms in order to avoid immunosuppression and occurrence of disease.

2D nanomaterials have garnered widespread attention in biomedicine and bioengineering due to their unique physicochemical properties. However, poor functionality, low solubility, intrinsic toxicity, and nonspecific interactions at biointerfaces have hampered their application in vivo. Here, biocompatible polyglycerol units are crosslinked in two dimensions using a graphene-assisted strategy leading to highly functional and water-soluble polyglycerols nanosheets with 263 ± 53 nm and 2.7 ± 0.2 nm average lateral size and thickness, respectively. A single-layer hyperbranched polyglycerol containing azide functional groups is covalently conjugated to the surface of a functional graphene template through pH-sensitive linkers. Then, lateral crosslinking of polyglycerol units is carried out by loading tripropargylamine on the surface of graphene followed by lifting off this reagent for an on-face click reaction. Subsequently, the polyglycerol nanosheets are detached from the surface of graphene by slight acidification and centrifugation and is sulfated to mimic heparin sulfate proteoglycans. To highlight the impact of the two-dimensionality of the synthesized polyglycerol sulfate nanosheets at nanobiointerfaces, their efficiency with respect to herpes simplex virus type 1 and severe acute respiratory syndrome corona virus 2 inhibition is compared to their 3D nanogel analogs. Four times stronger in virus inhibition suggests that 2D polyglycerols are superior to their current 3D counterparts.

Equine influenza (EI) is a highly contagious acute respiratory disease of equines that is caused mainly by the H3N8 subtype of influenza A virus. Vaccinating horses against EI is the most effective strategy to prevent the infection. The current study aimed to compare the kinetics of EI-specific humoral- and cell-mediated immunity (CMI) in horses receiving either identical or mixed vaccinations. Two groups of horses were previously (six months prior) vaccinated with either Calvenza 03 EIV EHV® (G1) or Fluvac Innovator® (G2) vaccine. Subsequently, both groups received a booster single dose of Calvenza 03 EIV EHV®. Immune responses were assessed after 10 weeks using single radial hemolysis (SRH), virus neutralization (VN), and EliSpot assays. Our results revealed that Calvenza-03 EIV/EHV®-immunized horses had significantly higher protective EI-specific SRH antibodies and VN antibodies. Booster immunization with Calvenza-03 EIV/EHV® vaccine significantly stimulated cell-mediated immune response as evidenced by significant increase in interferon-γ-secreting peripheral blood mononuclear cells. In conclusion, Calvenza-03 EIV/EHV® vaccine can be safely and effectively used for booster immunization to elicit optimal long persisting humoral and CMI responses even if the horses were previously immunized with a heterogeneous vaccine.

Equid herpesviruses are a group of nine infectious agents, which can cause conditions ranging from neurological disease with fatality rates of more than 50% of infected animals to mild or subclinical infection. Among the nine equid herpesviruses, three are referred to as asinine herpesviruses (EHV-6, EHV-7, EHV-8), because they primarily infect donkeys. Five equid herpesviruses (EHV-1, EHV-2, EHV-3, EHV-4, EHV-5) primarily infect horses. The definitive host of EHV-9 remains enigmatic and may represent an ancient perissodactyl herpesvirus for which evidence of infection can also be found in rhinoceros. In addition and much as EHV-1, EHV-9 can infect other mammals with fatal outcomes. Equine herpesviruses represent two subfamilies, the Alpha- and Gammaherpesvirinae, and two genuses, the Varicello- and Percaviruses, within the Herpesviridae family. Notably, an equid betaherpesvirus has not yet been identified. The clinically and economically most important equine herpesviruses are EHV-1 and EHV-4, which explains the focus of most research worldwide on those two closely related viruses. There are numerous studies on gene regulation, replication in cultured cells, pathogenesis, and vaccine development for EHV-1 and EHV-4, while comparable knowledge for other equid herpesviruses is limited.

Viruses from the family Hantaviridae are encountered as emerging pathogens causing two life-threatening human zoonoses: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), with case fatality rates of up to 50%. Here, we comprehensively investigated entry of the Old World hantavirus Puumala virus (PUUV) into mammalian cells, showing that upon treatment with pharmacological inhibitors of macropinocytosis and clathrin-mediated endocytosis, PUUV infections are greatly reduced. We demonstrate that the inhibitors did not interfere with viral replication and that RNA interference, targeting cellular mediators of macropinocytosis, decreases PUUV infection levels significantly. Moreover, we established lipophilic tracer staining of PUUV particles and show colocalization of stained virions and markers of macropinosomes. Finally, we report a significant increase in the fluid-phase uptake of cells infected with PUUV, indicative of a virus-triggered promotion of macropinocytosis. IMPORTANCE The family Hantaviridae comprises a diverse group of virus species and is considered an emerging global public health threat. Individual hantavirus species differ considerably in terms of their pathogenicity but also in their cell biology and host-pathogen interactions. In this study, we focused on the most prevalent pathogenic hantavirus in Europe, Puumala virus (PUUV), and investigated the entry and internalization of PUUV into mammalian cells. We show that both clathrin-mediated endocytosis and macropinocytosis are cellular pathways exploited by the virus to establish productive infections and demonstrate that pharmacological inhibition of macropinocytosis or a targeted knockdown using RNA interference significantly reduced viral infections. We also found indications of an increase of macropinocytic uptake upon PUUV infection, suggesting that the virus triggers specific cellular mechanisms in order to stimulate its own internalization, thus facilitating infection.

Viruses utilize host cell signaling to facilitate productive infection. Equine herpesvirus type 1 (EHV-1) has been shown to activate Ca2+ release and phospholipase C upon contact with α4β1 integrins on the cell surface. Signaling molecules, including small GTPases, have been shown to be activated downstream of Ca2+ release, and modulate virus entry, membrane remodeling and intracellular transport. In this study, we show that EHV-1 activates the small GTPases Rac1 and Cdc42 during infection. The activation of Rac1 and Cdc42 is necessary for virus-induced acetylation of tubulin, effective viral transport to the nucleus, and cell-to-cell spread. We also show that inhibitors of Rac1 and Cdc42 did not block virus entry, but inhibited overall virus infection. The Rac1 and Cdc42 signaling is presumably orthogonal to Ca2+ release, since Rac1 and Cdc42 inhibitors affected the infection of both EHV-1 and EHV-4, which do not bind to integrins.

The majority of influenza A virus strains are hosted in nature by avian species in the orders of Anseriformes and Charadriformes. A minority of strains have been able to cross species boundaries and establish themselves in novel non-avian hosts. Influenza viruses of horses, donkeys, and mules represent such successful events of avian to mammal influenza virus adaptation. Mongolia has over 3 million domestic horses and is home to two wild equids, the Asiatic wild ass or khulan (Equus hemionus hemionus), and Przewalski's horse (Equus ferus przewalskii). Domestic and wild equids are sympatric across most of their range in Mongolia. Epizootic influenza A virus outbreaks among Mongolian domestic horses have been frequently recorded. However, the exposure, circulation and relation to domestic horse influenza A virus outbreaks among wild equids is unknown. We evaluated serum samples of Asiatic wild asses in Mongolia for antibodies against influenza A viruses, using modified protein microarray technique. We detected antibodies against hemagglutinin (H) H1, H3, H5, H7, H8 and H10 influenza A viruses. Asiatic wild asses may represent a previously unidentified influenza A virus reservoir in an ecosystem shared with populations of domestic horses in which influenza strains circulate.

Exposure of phosphatidylserine (PS) in the outer leaflet of the plasma membrane is induced by infection with several members of the Alphaherpesvirinae subfamily. There is evidence that PS is used by the equine herpesvirus type 1 (EHV-1) during entry, but the exact role of PS and other phospholipids in the entry process remains unknown. Here, we investigated the interaction of differently charged phospholipids with virus particles and determined their influence on infection. Our data show that liposomes containing negatively charged PS or positively charged DOTAP (N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium) inhibited EHV-1 infection, while neutral phosphatidylcholine (PC) had no effect. Inhibition of infection with PS was transient, decreased with time, and was dose dependent. Our findings indicate that both cationic and anionic phospholipids can interact with the virus and reduce infectivity, while, presumably, acting through different mechanisms. Charged phospholipids were found to have antiviral effects and may be used to inhibit EHV-1 infection.

Mononuclear cells are the first line of defense against microbial infection. Yet, several viruses have evolved different mechanisms to overcome host defenses to ensure their spread. Here, we show unique mechanisms of how equid herpesvirus-1 manipulates peripheral blood mononuclear cells (PBMC) to travel further in the body. (1) "PBMC-hitching": at the initial contact, herpesviruses lurk in the extracellular matrix (ECM) of PBMC without entering the cells. The virus exploits the components of the ECM to bind, transport, and then egress to infect other cells. (2) "Intracellular delivery": transendothelial migration is a physiological mechanism where mononuclear cells can transmigrate through the endothelial cells. The virus was intangible and probably did not interfere with such a mechanism where the infected PBMC can probably deliver the virus inside the endothelium. (3) "Classical-fusion": this process is well mastered by herpesviruses due to a set of envelope glycoproteins that facilitate cell-cell fusion and virus spread.

Equine herpesvirus type 1 (EHV-1) causes encephalomyelopathy and abortion, for which cell-associated viremia and subsequent virus transfer to and replication in endothelial cells (EC) are responsible and prerequisites. Viral and cellular molecules responsible for efficient cell-to-cell spread of EHV-1 between peripheral blood mononuclear cells (PBMC) and EC remain unclear. We have generated EHV-1 mutants lackingORF1,ORF2, andORF17genes, either individually or in combination. Mutant viruses were analyzed for their replication properties in cultured equine dermal cells, PBMC infection efficiency, virus-induced changes in the PBMC proteome, and cytokine and chemokine expression profiles.ORF1,ORF2, andORF17are not essential for virus replication, butORF17deletion resulted in a significant reduction in plaque size. Deletion ofORF2andORF17gene significantly reduced cell-to-cell virus transfer from virus-infected PBMC to EC. EHV-1 infection of PBMC resulted in upregulation of several pathways such as Ras signaling, oxidative phosphorylation, platelet activation and leukocyte transendothelial migration. In contrast, chemokine signaling, RNA degradation and apoptotic pathways were downregulated. Deletion ofORF1,ORF2andORF17modulated chemokine signaling and MAPK pathways in infected PBMC, which may explain the impairment of virus spread between PBMC and EC. The proteomic results were further confirmed by chemokine assays, which showed that virus infection dramatically reduced the cytokine/chemokine release in infected PBMC. This study uncovers cellular proteins and pathways influenced by EHV-1 after PBMC infection and provide an important resource for EHV-1 pathogenesis. EHV-1-immunomodulatory genes could be potential targets for the development of live attenuated vaccines or therapeutics against virus infection.