EHV-1 Pathogenesis: Current in vitro Models and Future Perspectives

Primary infection and pathogenesis of equine herpesvirus type 1 (EHV-1) require an intricate interaction of virus with the mucosal epithelium, mononuclear cells and the vascular endothelium. Studies on EHV-1 have been facilitated by the development of different in vitro models that recapitulate the in vivo tissue complexity. The available in vitro assays can be categorized into (i) models mimicking the epithelium-peripheral blood mononuclear cell (PBMC) interaction, which include ex vivo mucosal (nasal and vaginal) explants and equine respiratory epithelial cells (EREC) cultures; and (ii) PBMC-endothelium mimicking models, including flow chamber and contact assays. These in vitro models have proven their worth in attempts to recapitulate the in vivo architecture and complexity, produce data relevant to natural host infection, and reduce animal use due to in vivo experiments. Although horse models are still needed for certain experiments, e.g., EHV-1 myeloencephalopathy or vaccination studies, available in vitro models can be used to obtain highly valuable data on virus-host tissue interactions. Microfluidic based 3D culture system (e.g., horse-on-a-chip) could be a potential upgraded version of these in vitro models for future research

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 alpha4beta1 or alpha4beta7 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 alpha4beta1 and alpha4beta7 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

The Role of Equine Herpesvirus Type 4 Glycoprotein K in Virus Replication

Equine herpesvirus 4 (EHV-4) is an important equine pathogen that causes respiratory tract disease among horses worldwide. Glycoprotein K (gK) homologues have been identified in several alphaherpesviruses as a major player in virus entry, replication, and spread. In the present study, EHV-4 gK-deletion mutant has been generated by using bacterial artificial chromosome technology and Red mutagenesis to investigate the role of gK in EHV-4 replication. Our findings reported here show that gK is essential for virus replication <em>in vitro</em> and that the gK-negative strain was not able to be reconstituted in equine cells. It is noteworthy that these findings agree with the previously published study describing gK deletion in other alphaherpesviruses

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

Well-known surface and extracellular antigens of pathogenic microorganisms among the immunodominant proteins of the infectious microalgae Prototheca zopfii

Microalgae of the genus Prototheca (P.) are associated with rare but severe infections (protothecosis) and represent a potential zoonotic risk. Genotype (GT) 2 of P. zopfii has been established as pathogenic agent for humans, dogs, and cattle, whereas GT1 is considered to be non-pathogenic. Since pathogenesis is poorly understood, the aim of this study was to determine immunogenic proteins and potential virulence factors of P. zopfii GT2. Therefore, 2D western blot analyses with sera and isolates of two dogs naturally infected with P. zopfii GT2 have been performed. Cross-reactivity was determined by including the type strains of P. zopfii GT2, P. zopfii GT1, and P. blaschkeae, a close relative of P. zopfii, which is known to cause subclinical forms of bovine mastitis. The sera showed a high strain-, genotype-, and species-cross- reactivity. A total of 198 immunogenic proteins have been analyzed via MALDI—TOF MS. The majority of the 86 identified proteins are intracellularly located (e.g., malate dehydrogenase, oxidoreductase, 3-dehydroquinate synthase) but some antigens and potential virulence factors, known from other pathogens, have been found (e.g., phosphomannomutase, triosephosphate isomerase). One genotype-specific antigen could be identified as heat shock protein 70 (Hsp70), a well-known antigen of eukaryotic pathogens with immunological importance when located extracellularly. Both sera were reactive to glyceraldehyde-3-phosphate-dehydrogenase of all investigated strains. This house-keeping enzyme is found to be located on the surface of several pathogens as virulence factor. Flow-cytometric analysis revealed its presence on the surface of P. blaschkeae

The Role of the Equine Herpesvirus Type 1 (EHV-1) US3-Encoded Protein Kinase in Actin Reorganization and Nuclear Egress

The serine-threonine protein kinase encoded by US3 gene (pUS3) of alphaherpesviruses was shown to modulate actin reorganization, cell-to-cell spread, and virus egress in a number of virus species. However, the role of the US3 orthologues of equine herpesvirus type 1 and 4 (EHV-1 and EHV-4) has not yet been studied. Here, we show that US3 is not essential for virus replication in vitro. However, growth rates and plaque diameters of a US3-deleted EHV-1 and a mutant in which the catalytic active site was destroyed were significantly reduced when compared with parental and revertant viruses or a virus in which EHV-1 US3 was replaced with the corresponding EHV-4 gene. The reduced plaque sizes were consistent with accumulation of primarily enveloped virions in the perinuclear space of the US3-negative EHV-1, a phenotype that was also rescued by the EHV-4 orthologue. Furthermore, actin stress fiber disassembly was significantly more pronounced in cells infected with parental EHV-1, revertant, or the recombinant EHV-1 expressing EHV-4 US3. Finally, we observed that deletion of US3 in EHV-1 did not affect the expression of adhesion molecules on the surface of infected cells

Fatal Elephant Endotheliotropic Herpesvirus Infection of Two Young Asian Elephants

Elephant endotheliotropic herpesvirus (EEHV) can cause a devastating haemorrhagic disease in young Asian elephants worldwide. Here, we report the death of two young Asian elephants after suffering from acute haemorrhagic disease due to EEHV-1A infection. We detected widespread distribution of EEHV-1A in various organs and tissues of the infected elephants. Enveloped viral particles accumulated within and around cytoplasmic electron-dense bodies in hepatic endothelial cells were detected. Attempts to isolate the virus on different cell cultures showed limited virus replication; however, late viral protein expression was detected in infected cells. We further showed that glycoprotein B (gB) of EEHV-1A possesses a conserved cleavage site Arg-X-Lys/Arg-Arg that is targeted by the cellular protease furin, similar to other members of the Herpesviridae. We have determined the complete 180 kb genome sequence of EEHV-1A isolated from the liver by next-generation sequencing and de novo assembly. As virus isolation in vitro has been unsuccessful and limited information is available regarding the function of viral proteins, we have attempted to take the initial steps in the development of suitable cell culture system and virus characterization. In addition, the complete genome sequence of an EEHV-1A in Europe will facilitate future studies on the epidemiology and diagnosis of EEHV infection in elephants

Detection of equid herpesviruses among different Arabian horse populations in Egypt

Equid herpesviruses (EHVs) threaten equine health and can cause significant economic losses to the equine industry worldwide. Different equid herpesviruses, EHV‐1, EHV‐2, EHV‐4 and EHV5 are regularly detected among horse populations. In Egypt, monitoring is sporadic but EHV‐1 or EHV‐4 have been reported to circulate in the horse population. However, there is a lack of reports related to infection and health status of horses, likely due to the absence of regular diagnostic procedures. In the current study, the circulation of four infectious equid herpesviruses (EHV‐1, EHV‐2, EHV‐4 and EHV‐5) among different Arabian horse populations and donkeys residing the same farm was monitored. Different samples were collected and DNA was extracted and subjected to quantitative (q)‐PCR to detect the four equid herpesviruses using specific primers and probes. Antibody titres against EHV‐1 and EHV‐4 were tested using virus neutralization test and type‐specific ELISA. The results showed that EHV‐1, EHV‐2, EHV‐4 and EHV‐5 are endemic and can be a continuous threat for horses in the absence of vaccination programs and frequent virus reactivation. There is an urgent need for introduction of active regular surveillance measures to investigate the presence of different equid herpesviruses, and other equine viral pathogens, in various horse populations around Egypt and to establish a standardized cataloguing of equine health status

How Host Specific Are Herpesviruses? Lessons from Herpesviruses Infecting Wild and Endangered Mammals

Herpesviruses are ubiquitous and can cause disease in all classes of vertebrates but also in animals of lower taxa, including molluscs. It is generally accepted that herpesviruses are primarily species specific, although a species can be infected by different herpesviruses. Species specificity is thought to result from host-virus coevolutionary processes over the long term. Even with this general concept in mind, investigators have recognized interspecies transmission of several members of the Herpesviridae family, often with fatal outcomes in non-definitive hosts—that is, animals that have no or only a limited role in virus transmission. We here summarize herpesvirus infections in wild mammals that in many cases are endangered, in both natural and captive settings. Some infections result from herpesviruses that are endemic in the species that is primarily affected, and some result from herpesviruses that cause fatal disease after infection of non-definitive hosts. We discuss the challenges of such infections in several endangered species in the absence of efficient immunization or therapeutic options