The US3 Kinase of Herpes Simplex Virus Phosphorylates the RNA Sensor RIG-I To Suppress Innate Immunity.

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.

Graphene-Assisted Synthesis of 2D Polyglycerols as Innovative Platforms for Multivalent Virus Interactions.

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.

Macropinocytosis and Clathrin-Dependent Endocytosis Play Pivotal Roles for the Infectious Entry of Puumala Virus.

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.

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.

Subclinical infection of a young captive Asian elephant with elephant endotheliotropic herpesvirus 1.

Elephant endotheliotropic herpesviruses (EEHVs) are a continuous threat for young Asian elephants. We report a laboratory-confirmed infection of a 5-year-old female Asian elephant (AZ_2016) in the Berlin Zoologischer Garten. Initially, high EEHV-1 loads were detected in trunk swabs obtained from the young elephant during routine screening. The animal showed no clinical signs except for slight irritability. EEHV-1 was continuously shed for almost one year, with fluctuations in viral load from time to time. Our investigations highlight the continuous threat of EEHV-1 to young captive Asian elephants and stress the importance of routine monitoring of captive elephants to allow early detection of infection.

Viral genes and cellular markers associated with neurological complications during herpesvirus infections.

Despite the importance of neurological disorders associated with herpesviruses, the mechanism by which these viruses influence the central nervous system (CNS) has not been definitively established. Owing to the limitations of studying neuropathogenicity of human herpesviruses in their natural host, many aspects of their pathogenicity and immune response are studied in animal models. Here, we present an important model system that enables studying neuropathogenicity of herpesviruses in the natural host. Equine herpesvirus type 1 (EHV-1) is an alphaherpesvirus that causes a devastating neurological disease (EHV-1 myeloencephalopathy; EHM) in horses. Like other alphaherpesviruses, our understanding of virus neuropathogenicity in the natural host beyond the essential role of viraemia is limited. In particular, information on the role of different viral proteins for virus transfer to the spinal cord endothelium in vivo is lacking. In this study, the contribution of two viral proteins, DNA polymerase (ORF30) and glycoprotein D (gD), to the pathogenicity of EHM was addressed. Furthermore, different cellular immune markers, including alpha-interferon (IFN-α), gamma-interferon (IFN-γ), interleukin-10 (IL-10) and interleukin-1 beta (IL-1ß), were identified to play a role during the course of the disease.

Initial Contact: The First Steps in Herpesvirus Entry.

The entry process of herpesviruses into host cells is complex and highly variable. It involves a sequence of well-orchestrated events that begin with virus attachment to glycan-containing proteinaceous structures on the cell surface. This initial contact tethers virus particles to the cell surface and results in a cascade of molecular interactions, including the tight interaction of viral envelope glycoproteins to specific cell receptors. These interactions trigger intracellular signaling and finally virus penetration after fusion of the viral envelope with cellular membranes. Based on the engaged cellular receptors and co-receptors, and the subsequent signaling cascades, the entry pathway will be decided on the spot. A number of viral glycoproteins and many cellular receptors and molecules have been identified as players in one or several of these events during virus entry. This chapter will review viral glycoproteins, cellular receptors and signaling cascades associated with the very first interactions of herpesviruses with their target cells.

Glycoprotein B of equine herpesvirus type 1 has two recognition sites for subtilisin-like proteases that are cleaved by furin.

Glycoprotein B (gB) of equine herpesvirus type 1 (EHV-1) is predicted to be cleaved by furin in a fashion similar to that of related herpesviruses. To investigate the contribution of furin-mediated gB cleavage to EHV-1 growth, canonical furin cleavage sites were mutated. Western blot analysis of mutated EHV-1 gB showed that it was cleaved at two positions, 518RRRR521 and 544RLHK547, and that the 28 aa between the two sites were removed after cleavage. Treating infected cells with either convertase or furin inhibitors reduced gB cleavage efficiency. Further, removal of the first furin recognition motif did not affect in vitro growth of EHV-1, while mutation of the second motif greatly affected virus growth. In addition, a second possible signal peptide cleavage site was identified for EHV-1 gB between residues 98 and 99, which was 13 aa downstream of that previously identified.

Role of gB and pUS3 in Equine Herpesvirus 1 Transfer between Peripheral Blood Mononuclear Cells and Endothelial Cells: a Dynamic In Vitro Model.

UNLABELLED: Infected peripheral blood mononuclear cells (PBMC) effectively transport equine herpesvirus type 1 (EHV-1), but not EHV-4, to endothelial cells (EC) lining the blood vessels of the pregnant uterus or central nervous system, a process that can result in abortion or myeloencephalopathy. We examined, using a dynamic in vitro model, the differences between EHV-1 and EHV-4 infection of PBMC and PBMC-EC interactions. In order to evaluate viral transfer between infected PBMC and EC, cocultivation assays were performed. Only EHV-1 was transferred from PBMC to EC, and viral glycoprotein B (gB) was shown to be mainly responsible for this form of cell-to-cell transfer. For addressing the more dynamic aspects of PBMC-EC interaction, infected PBMC were perfused through a flow channel containing EC in the presence of neutralizing antibodies. By simulating capillary blood flow and analyzing the behavior of infected PBMC through live fluorescence imaging and automated cell tracking, we observed that EHV-1 was able to maintain tethering and rolling of infected PBMC on EC more effectively than EHV-4. Deletion of US3 reduced the ability of infected PBMC to tether and roll compared to that of cells infected with parental virus, which resulted in a significant reduction in virus transfer from PBMC to EC. Taking the results together, we conclude that systemic spread and EC infection by EHV-1, but not EHV-4, is caused by its ability to infect and/or reprogram mononuclear cells with respect to their tethering and rolling behavior on EC and consequent virus transfer. IMPORTANCE: EHV-1 is widespread throughout the world and causes substantial economic losses through outbreaks of respiratory disease, abortion, and myeloencephalopathy. Despite many years of research, no fully protective vaccines have been developed, and several aspects of viral pathogenesis still need to be uncovered. In the current study, we investigated the molecular mechanisms that facilitate the cell-associated viremia, which is arguably the most important aspect of EHV-1 pathogenesis. The newly discovered functions of gB and pUS3 add new facets to their previously reported roles. Due to the conserved nature of cell-associated viremia among numerous herpesviruses, these results are also very relevant for viruses such as varicella-zoster virus, pseudorabies virus, human cytomegalovirus, and others. In addition, the constructed mutant and recombinant viruses exhibit potent in vitro replication but have significant defects in certain stages of the disease course. These viruses therefore show much promise as candidates for future live vaccines.

Equid herpesvirus type 4 uses a restricted set of equine major histocompatibility complex class I proteins as entry receptors.

Equid herpesvirus type 1 (EHV-1) was shown to use an unusual receptor for cellular entry - MHC-I molecules. Here, we demonstrated that the closely related EHV, EHV-4, also uses this strategy for cellular invasion, both in equine cells in culture and in the heterologous, non-permissive murine mastocytoma cell line (P815) after stable transfection with horse MHC-I genes. Using a panel of P815 cell lines transfected with individual horse MHC-I genes, we provided support for the hypothesis that EHV-1 and EHV-4 target classical polymorphic MHC-I molecules as viral entry receptors. All known equine MHC-I molecules from the two principal classical polymorphic loci specify alanine at position 173 (A173), whilst other MHC-I loci encoded different amino acids at this position and did not permit viral entry. Site-directed mutagenesis of position 173 diminished or enhanced viral entry, depending upon the initial amino acid. However, there were other, as yet undefined, constraints to this process: MHC-I genes from two non-classical loci carried A173 but did not enable viral entry in P815 transfectants. Our study suggested that the capacity to bind MHC-I molecules arose in the common ancestor of EHV-1 and EHV-4. The widespread occurrence of A173 in classical polymorphic horse MHC-I molecules indicated that horses of most MHC haplotypes should be susceptible to infection via this entry portal.

Glycoprotein H and α4ß1 integrins determine the entry pathway of alphaherpesviruses.

Herpesviruses enter cells either by direct fusion at the plasma membrane or from within endosomes, depending on the cell type and receptor(s). We investigated two closely related herpesviruses of horses, equine herpesvirus type 1 (EHV-1) and EHV-4, for which the cellular and viral determinants routing virus entry are unknown. We show that EHV-1 enters equine epithelial cells via direct fusion at the plasma membrane, while EHV-4 does so via an endocytic pathway, which is dependent on dynamin II, cholesterol, caveolin 1, and tyrosine kinase activity. Exchange of glycoprotein H (gH) between EHV-1 and EHV-4 resulted in rerouting of EHV-1 to the endocytic pathway, as did blocking of α4ß1 integrins on the cell surface. Furthermore, a point mutation in the SDI integrin-binding motif of EHV-1 gH also directed EHV-1 to the endocytic pathway. Cumulatively, we show that viral gH and cellular α4ß1 integrins are important determinants in the choice of alphaherpesvirus cellular entry pathways.

Glycoproteins D of equine herpesvirus type 1 (EHV-1) and EHV-4 determine cellular tropism independently of integrins.

Equine herpesvirus type 1 (EHV-1) and EHV-4 are genetically and antigenically very similar, but their pathogenic potentials are strikingly different. The differences in pathogenicity between both viruses seem to be reflected in cellular host range: EHV-1 can readily be propagated in many cell types of multiple species, while EHV-4 entry and replication appear to be restricted mainly to equine cells. The clear difference in cellular tropism may well be associated with differences in the gene products involved in virus entry and/or spread from cell to cell. Here we show that (i) most of the EHV-1 permissive cell lines became resistant to EHV-1 expressing EHV-4 glycoprotein D (gD4) and the opposite was observed for EHV-4 harboring EHV-1 gD (gD1). (ii) The absence of integrins did not inhibit entry into and replication of EHV-1 in CHO-K1 or peripheral blood mononuclear cells (PBMC). Furthermore, integrin-negative K562 cells did not acquire the ability to bind to gD1 when αVß3 integrin was overexpressed. (iii) PBMC could be infected with similar efficiencies by both EHV-1 and EHV-4 in vitro. (iv) In contrast to results for equine fibroblasts and cells of endothelial or epithelial origin, we were unable to block entry of EHV-1 or EHV-4 into PBMC with antibodies directed against major histocompatibility complex class I (MHC-I), a result that indicates that these viruses utilize a different receptor(s) to infect PBMC. Cumulatively, we provide evidence that efficient EHV-1 and EHV-4 entry is dependent mainly on gD, which can bind to multiple cell surface receptors, and that gD has a defining role with respect to cellular host range of EHV-1 and EHV-4.

Equine herpesvirus type 4 UL56 and UL49.5 proteins downregulate cell surface major histocompatibility complex class I expression independently of each other.

Major histocompatibility complex class I (MHC-I) molecules are critically important in the host defense against various pathogens through presentation of viral peptides to cytotoxic T lymphocytes (CTLs), a process resulting in the destruction of virus-infected cells. Herpesviruses interfere with CTL-mediated elimination of infected cells by various mechanisms, including inhibition of peptide transport and loading, perturbation of MHC-I trafficking, and rerouting and proteolysis of cell surface MHC-I. In this study, we show that equine herpesvirus type 4 (EHV-4) modulates MHC-I cell surface expression through two different mechanisms. First, EHV-4 can lead to a significant downregulation of MHC-I expression at the cell surface through the product of ORF1, a protein expressed with early kinetics from a gene that is homologous to herpes simplex virus 1 UL56. The EHV-4 UL56 protein reduces cell surface MHC-I as early as 4 h after infection. Second, EHV-4 can interfere with MHC-I antigen presentation, starting at 6 h after infection, by inhibition of the transporter associated with antigen processing (TAP) through its UL49.5 protein. Although pUL49.5 has no immediate effect on overall surface MHC-I levels in infected cells, it blocks the supply of antigenic peptides to the endoplasmic reticulum (ER) and transport of peptide-loaded MHC-I to the cell surface. Taken together, our results show that EHV-4 encodes at least two viral immune evasion proteins: pUL56 reduces MHC-I molecules on the cell surface at early times after infection, and pUL49.5 interferes with MHC-I antigen presentation by blocking peptide transport in the ER.

Crystal structures of glycoprotein D of equine alphaherpesviruses reveal potential binding sites to the entry receptor MHC-I.

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 role of glycoprotein H of equine herpesviruses 1 and 4 (EHV-1 and EHV-4) in cellular host range and integrin binding.

Equine herpesvirus type 1 and 4 (EHV-1 and EHV-4) glycoprotein H (gH) has been hypothesized to play a role in direct fusion of the virus envelope with cellular membranes. To investigate gH's role in infection, an EHV-1 mutant lacking gH was created and the gH genes were exchanged between EHV-1 and EHV-4 to determine if gH affects cellular entry and/or host range. In addition, a serine-aspartic acid-isoleucine (SDI) integrin-binding motif present in EHV-1 gH was mutated as it was presumed important in cell entry mediated by binding to α4ß1 or α4ß7 integrins. We here document that gH is essential for EHV-1 replication, plays a role in cell-to-cell spread and significantly affects plaque size and growth kinetics. Moreover, we could show that α4ß1 and α4ß7 integrins are not essential for viral entry of EHV-1 and EHV-4, and that viral entry is not affected in equine cells when the integrins are inaccessible.

In vitro characterization of EHV-4 gG-deleted mutant.

Equine herpesvirus 4 (EHV-4) is an important pathogen that causes respiratory tract disease in horse populations worldwide. Glycoprotein G (gG) homologs have been identified in several alphaherpesviruses as minor non-essential membrane-anchored glycoproteins. In this study, EHV-4 gG deletion mutant has been generated by using bacterial artificial chromosome technology to investigate the role of gG in viral pathogenesis. Our findings reported here revealed no significant difference between parental EHV-4 and gG-negative strain in their replication cycle in cell culture. Furthermore, virus titers and plaque formation were comparable in both viruses. It is noteworthy that these findings disagree with the previously published study describing gG deletion in another EHV-4 strain.

Contamination Control Strategy: Implementation Road Map.

The article proposes an implementation road map of a contamination control strategy (CCS) in a facility. The CCS is the culmination of an exercise to identify activities designed to prevent microorganism, pyrogen, and particulate contamination in the product, the facility, and the supporting processes used to manufacture the product. Manufacturers can formulate their contamination control strategy based on information in the quality target product profile or in the critical quality attributes, in the facility, and in the processes used to manufacture and transport the product. The strategy implementation involves executing the strategic plan and managing the implementation by priority overtime should it be deployed. The evaluation of the efficiency and effectiveness of the contamination control strategy implemented is confirmed by analyzing and trending the various quality performance parameters related to contamination control. The strategy evaluation allows the manufacturer to identify a new strategic plan to support improvement goals or new measures and/or controls to achieve the desired result, minimizing the contamination risk.

Equine Herpesvirus Type 4 (EHV-4) Outbreak in Germany: Virological, Serological, and Molecular Investigations.

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.

Immunogenicity of Calvenza-03 EIV/EHV® Vaccine in Horses: Comparative In Vivo Study.

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.

Seasonal host and ecological drivers may promote restricted water as a viral vector.

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.