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Equine herpesvirus type 1 (EHV-1) outbreaks continue to occur despite widely used vaccination. Therefore, development of EHV-1 vaccines providing improved immunity and protection is ongoing. Here, an open reading frame 2 deletion mutant of the neuropathogenic EHV-1 strain Ab4 (Ab4ΔORF2) was tested as a vaccine candidate. Three groups of horses ( n  = 8 each) were infected intranasally with Ab4ΔORF2 or the parent Ab4 virus or were kept as noninfected controls. Horses infected with Ab4ΔORF2 had reduced fever and nasal virus shedding compared to those infected with Ab4 but mounted similar adaptive immunity dominated by antibody responses. Nine months after the initial infection, all horses were challenged intranasally with Ab4. Previously noninfected horses (control/Ab4) displayed clinical signs, shed large amounts of virus, and developed cell-associated viremia. In contrast, 5/8 or 3/8 horses previously infected with Ab4ΔORF2 or Ab4, respectively, were fully protected from challenge infection as indicated by the absence of fever, clinical disease, nasal virus shedding, and viremia. All of these outcomes were significantly reduced in the remaining, partially protected 3/8 (Ab4ΔORF2/Ab4) and 5/8 (Ab4/Ab4) horses. Protected horses had EHV-1-specific IgG4/7 antibodies prior to challenge infection, and intranasal antibodies increased rapidly postchallenge. Intranasal inflammatory markers were not detectable in protected horses but quickly increased in control/Ab4 horses during the first week after infection. Overall, our data suggest that preexisting nasal IgG4/7 antibodies neutralize EHV-1, prevent viral entry, and thereby protect from disease, viral shedding, and cell-associated viremia. In conclusion, improved protection from challenge infection emphasizes further evaluation of Ab4ΔORF2 as a vaccine candidate. IMPORTANCE Nasal equine herpesvirus type 1 (EHV-1) shedding is essential for virus transmission during outbreaks. Cell-associated viremia is a prerequisite for the most severe disease outcomes, abortion and equine herpesvirus myeloencephalopathy (EHM). Thus, protection from viremia is considered essential for preventing EHM. Ab4ΔORF2 vaccination prevented EHV-1 challenge virus replication in the upper respiratory tract in fully protected horses. Consequently, these neither shed virus nor developed cell-associated viremia. Protection from virus shedding and viremia during challenge infection in combination with reduced virulence at the time of vaccination emphasizes ORF2 deletion as a promising modification for generating an improved EHV-1 vaccine. During this challenge infection, full protection was linked to preexisting local and systemic EHV-1-specific antibodies combined with rapidly increasing intranasal IgG4/7 antibodies and lack of nasal type I interferon and chemokine induction. These host immune parameters may constitute markers of protection against EHV-1 and be utilized as indicators for improved vaccine development and informed vaccination strategies., (Copyright © 2019 American Society for Microbiology.)

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.

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., (© 2019 The Authors. Veterinary Medicine and Science Published by John Wiley & Sons Ltd.)

Equid herpesviruses (EHVs) are pathogens of equid and nonequid hosts that can cause disease and fatalities in captivity and in the wild. EHVs establish latent infections but can reactivate, and most EHVs are shed via the nasal passage. Therefore, nasal swabs are generally used for EHV monitoring. However, invasive sampling of wild equids is difficult. While feces is a commonly used substrate for detecting other pathogens, to our knowledge, EHVs have never been detected in feces of naturally infected equids. We systematically tested zebra feces for EHV presence by (i) establishing nested PCR conditions for fecal DNA extracts, (ii) controlling for environmental EHV contamination, and (iii) large-scale testing on a free-ranging zebra population. A dilution minimizing inhibition while maximizing viral DNA concentrations was determined in captive Grévy's zebra ( Equus grevyi ) fecal samples from individuals shedding EHV nasally. Sixteen of 42 fecal samples (38%) were EHV positive. To demonstrate that the EHV positivity was not a result of environmental contamination, rectal swabs of wild zebras were screened ( n  = 18 [ Equus quagga and E. zebra ]), and 50% were EHV positive, indicating that the source of EHV in feces is likely the intestinal mucosa and not postdefecation contamination. Out of 270 fecal samples of wild zebras, 26% were EHV positive. Quantitative PCRs showed that the amount of virus DNA in feces was not significantly smaller than that in other samples. In summary, fecal sampling facilitates large-scale screening and may be useful to noninvasively investigate phylogenetic EHV diversity in wild and domestic equids. IMPORTANCE Equid herpesviruses (EHVs) establish latent infections, and many EHVs are shed and transmitted via nasal discharge primarily through droplet and aerosol infection. Obtaining nasal swabs and other invasive samples from wildlife is often not possible without capture and physical restraint of individuals, which are resource intensive and a health risk for the captured animals. Fecal EHV shedding has never been demonstrated for naturally infected equids. We established the conditions for fecal EHV screening, and our results suggest that testing fecal samples is an effective noninvasive approach for monitoring acute EHV shedding in equids., (Copyright © 2019 American Society for Microbiology.)

Codon pair bias deoptimization (CPBD) has been successfully used to attenuate several RNA viruses. CPBD involves recoding a viral protein-coding sequence to maximize the number of codon pairs that are statistically underrepresented in the host, which presumably slows protein translation and, hence, causes virus attenuation. However, since recoding preserves the amino acid composition and codon bias, attenuated and parental viruses are antigenically identical. To determine if Marek's disease virus (MDV), a highly oncogenic herpesvirus of the chicken with a large double-stranded DNA genome, can be attenuated by CPBD of its major oncogene meq, we recoded the gene to minimize (meq-D), maximize (meq-O), or preserve (meq-R) the level of codon pairs that are overrepresented in the chicken protein-coding sequences. Unexpectedly, mutants carrying recoded genes produced comparable or increased levels of Meq in the context of viral infection in cultured cells. In addition, parental virus and mutant viruses carrying recoded meq genes replicated with comparable kinetics in vitro and in vivo, and were equally virulent in susceptible chickens. In summary, CPBD of meq failed to produce any quantifiable attenuation of MDV and confirms differences in the complexity of applying CPBD to large DNA viruses versus RNA viruses.

The equine herpesvirus type 1 (EHV-1) ORF1 and ORF71 genes have immune modulatory effects in vitro. Experimental infection of horses using virus mutants with multiple deletions including ORF1 and ORF71 showed promise as vaccine candidates against EHV-1. Here, the combined effects of ORF1 and ORF71 deletions from the neuropathogenic EHV-1 strain Ab4 on clinical disease and host immune response were further explored. Three groups of EHV-1 naïve horses were experimentally infected with the ORF1/71 gene deletion mutant (Ab4ΔORF1/71), the parent Ab4 strain, or remained uninfected. In comparison to Ab4, horses infected with Ab4ΔORF1/71 did not show the initial high fever peak characteristic of EHV-1 infection. Ab4ΔORF1/71 infection had reduced nasal shedding (1/5 vs. 5/5) and, simultaneously, decreased intranasal interferon (IFN)-α, interleukin (IL)-10 and soluble CD14 secretion. However, Ab4 and Ab4ΔORF1/71 infection resulted in comparable viremia, suggesting these genes do not regulate the infection of the mononuclear cells and subsequent viremia. Intranasal and serum anti-EHV-1 antibodies to Ab4ΔORF1/71 developed slightly slower than those to Ab4. However, beyond day 12 post infection (d12pi) serum antibodies in both virus-infected groups were similar and remained increased until the end of the study (d114pi). EHV-1 immunoglobulin (Ig) G isotype responses were dominated by short-lasting IgG1 and long-lasting IgG4/7 antibodies. The IgG4/7 response closely resembled the total EHV-1 specific antibody response. Ex vivo re-stimulation of PBMC with Ab4 resulted in IFN-γ and IL-10 secretion by cells from both infected groups within two weeks pi. Flow cytometric analysis showed that IFN-γ producing EHV-1-specific T-cells were mainly CD8+/IFN-γ+ and detectable from d32pi on. Peripheral blood IFN-γ+ T-cell percentages were similar in both infected groups, albeit at low frequency (~0.1%). In summary, the Ab4ΔORF1/71 gene deletion mutant is less virulent but induced antibody responses and cellular immunity similar to the parent Ab4 strain., Competing Interests: The authors have declared that no competing interests exist.

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.

Background: Equine herpesvirus type 1 (EHV-1) induces respiratory infection, abortion, and neurologic disease with significant impact. Virulence factors contributing to infection and immune evasion are of particular interest. A potential virulence factor of the neuropathogenic EHV-1 strain Ab4 is ORF2. This study on 24 Icelandic horses, 2 to 4 years of age, describes the infection with EHV-1 Ab4, or its deletion mutant devoid of ORF2 (Ab4ΔORF2) compared to non-infected controls (each group n = 8). The horses' clinical presentation, virus shedding, viremia, antibody and cellular immune responses were monitored over 260 days after experimental infection., Results: Infection with Ab4ΔORF2 reduced fever and minimized nasal virus shedding after infection compared to the parent virus strain Ab4, while Ab4ΔORF2 established viremia similar to Ab4. Concurrently with virus shedding, intranasal cytokine and interferon α (IFN-α) production increased in the Ab4 group, while horses infected with Ab4ΔORF2 expressed less IFN-α. The antibody response to EHV-1 was evaluated by a bead-based multiplex assay and was similar in both infected groups, Ab4 and Ab4ΔORF2. EHV-1 specific immunoglobulin (Ig) G1 was induced 8 days after infection (d8 pi) with a peak on d10-12 pi. EHV-1 specific IgG4/7 increased starting on d10 pi, and remained elevated in serum until the end of the study. The intranasal antibody response to EHV-1 was dominated by the same IgG isotypes and remained elevated in both infected groups until d130 pi. In contrast to the distinct antibody response, no induction of EHV-1 specific T-cells was detectable by flow cytometry after ex vivo re-stimulation of peripheral blood mononuclear cells (PBMC) with EHV-1 in any group. The cellular immune response was characterized by increased secretion of IFN-γ and interleukin10 in response to ex vivo re-stimulation of PBMC with EHV-1. This response was present during the time of viremia (d5-10 pi) and was similar in both infected groups, Ab4 and Ab4ΔORF2., Conclusions: ORF2 is a virulence factor of EHV-1 Ab4 with impact on pyrexia and virus shedding from the nasal mucosa. In contrast, ORF2 does not influence viremia. The immunogenicity of the Ab4ΔORF2 and parent Ab4 viruses are identical. Graphical abstract - Deletion of ORF2 reduces virulence of EHV-1 Ab4. Graphical summary of the main findings of this study: ORF2 is a virulence factor of EHV-1 Ab4 with impact on pyrexia and virus shedding from the nasal mucosa.

Polar bears in captivity can be exposed to opportunistic pathogens not present in their natural environments. A 4-month-old polar bear ( Ursus maritimus ) living in an isolated enclosure with his mother in the Tierpark Berlin, Berlin, Germany, was suffering from severe abdominal pain, mild diarrhea, and loss of appetite and died in early 2017. Histopathology revealed severe hepatic degeneration and necrosis without evidence of inflammation or inclusion bodies, although a viral infection had been suspected on the basis of the clinical signs. We searched for nucleic acids of pathogens by shotgun high-throughput sequencing (HTS) from genomic DNA and cDNA extracted from tissue and blood. We identified a novel Mastadenovirus and assembled a nearly complete genome from the shotgun sequences. Quantitative PCR (qPCR) revealed that viral DNA was present in various concentrations in all tissues examined and that the highest concentrations were found in blood. Viral culture did not yield cytopathic effects, but qPCR suggested that virus replication was sustained for up to three passages. Positive immunofluorescence staining confirmed that the virus was able to replicate in the cells during early passage. Phylogenetic analysis demonstrated that the virus is highly divergent compared to other previously identified Mastadenovirus members and basal to most known viral clades. The virus was found only in the 4-month-old bear and not in other captive polar bears tested. We surmised, therefore, that the polar bear was infected from an unknown reservoir, illustrating that adenoviral diversity remains underestimated and that cross-species transmission of viruses can occur even under conditions of relative isolation. IMPORTANCE Cross-species transmission of viral pathogens is becoming an increasing problem for captive-animal facilities. This study highlights how animals in captivity are vulnerable to novel opportunistic pathogens, many of which do not result in straightforward diagnosis from symptoms and histopathology. In this study, a novel pathogen was suspected to have contributed to the death of a juvenile polar bear. HTS techniques were employed, and a novel Mastadenovirus was isolated. The virus was present in both the tissue and blood samples. Phylogenetic analysis of the virus at both the gene and genome levels revealed that it is highly divergent to other known mastadenoviruses. Overall, this study shows that animals in isolated conditions still come into contact with novel pathogens, and for many of these pathogens, the host reservoir and mode of transmission are yet to be determined., (Copyright © 2018 Dayaram et al.)

Viruses may have a dramatic impact on the health of their animal hosts. The patho-physiological mechanisms underlying viral infections in animals are, however, not well understood. It is increasingly recognized that oxidative stress may be a major physiological cost of viral infections. Here we compare three blood-based markers of oxidative status in herpes positive and negative individuals of the domestic horse (Equus ferus caballus) and of both captive and free-ranging Mongolian khulan (Equus hemionus hemionus) and plains zebra (Equus quagga). Herpes positive free-ranging animals had significantly more protein oxidative damage and lower glutathione peroxidase (antioxidant enzyme) than negative ones, providing correlative support for a link between oxidative stress and herpesvirus infection in free-living equids. Conversely, we found weak evidence for oxidative stress in herpes positive captive animals. Hence our work indicates that environment (captive versus free living) might affect the physiological response of equids to herpesvirus infection. The Mongolian khulan and the plains zebra are currently classified as near threatened by the International Union for Conservation of Nature. Thus, understanding health impacts of pathogens on these species is critical to maintaining viable captive and wild populations.

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.

Codon pair bias deoptimization (CPBD) has enabled highly efficient and rapid attenuation of RNA viruses. The technique relies on recoding of viral genes by increasing the number of codon pairs that are statistically underrepresented in protein coding genes of the viral host without changing the amino acid sequence of the encoded proteins. Utilization of naturally underrepresented codon pairs reduces protein production of recoded genes and directly causes virus attenuation. As a result, the mutant virus is antigenically identical with the parental virus, but virulence is reduced or absent. Our goal was to determine if a virus with a large double-stranded DNA genome, highly oncogenic Marek's disease virus (MDV), can be attenuated by CPBD. We recoded UL30 that encodes the catalytic subunit of the viral DNA polymerase to minimize (deoptimization), maximize (optimization), or preserve (randomization) the level of overrepresented codon pairs of the MDV host, the chicken. A fully codon pair-deoptimized UL30 mutant could not be recovered in cell culture. The sequence of UL30 was divided into three segments of equal length and we generated a series of mutants with different segments of the UL30 recoded. The codon pair-deoptimized genes, in which two segments of UL30 had been recoded, showed reduced rates of protein production. In cultured cells, the corresponding viruses formed smaller plaques and grew to lower titers compared with parental virus. In contrast, codon pair-optimized and -randomized viruses replicated in vitro with kinetics that were similar to those of the parental virus. Animals that were infected with the partially codon pair-deoptimized virus showed delayed progression of disease and lower mortality rates than codon pair-optimized and parental viruses. These results demonstrate that CPBD of a herpesvirus gene causes attenuation of the recoded virus and that CPBD may be an applicable strategy for attenuation of other large DNA viruses.

The sensor RIG-I detects double-stranded RNA derived from RNA viruses. Although RIG-I is also known to have a role in the antiviral response to DNA viruses, physiological RNA species recognized by RIG-I during infection with a DNA virus are largely unknown. Using next-generation RNA sequencing (RNAseq), we found that host-derived RNAs, most prominently 5S ribosomal RNA pseudogene 141 (RNA5SP141), bound to RIG-I during infection with herpes simplex virus 1 (HSV-1). Infection with HSV-1 induced relocalization of RNA5SP141 from the nucleus to the cytoplasm, and virus-induced shutoff of host protein synthesis downregulated the abundance of RNA5SP141-interacting proteins, which allowed RNA5SP141 to bind RIG-I and induce the expression of type I interferons. Silencing of RNA5SP141 strongly dampened the antiviral response to HSV-1 and the related virus Epstein-Barr virus (EBV), as well as influenza A virus (IAV). Our findings reveal that antiviral immunity can be triggered by host RNAs that are unshielded following depletion of their respective binding proteins by the virus.

Purpose of Review: This review summarizes the current understanding of virus attenuation by large-scale recoding of viral genomes and discusses what would ultimately be necessary for construction of better and safer live modified virus vaccines., Recent Findings: It has been shown that codon and codon pair deoptimization are rapid and robust methods that can be used for the development of attenuated vaccine candidates. The viruses attenuated by large-scale recoding have the added value that they are extremely genetically stable. However, the exact mechanisms that lead to viral attenuation by recoding are yet to be determined., Summary: While the advantages of large-scale recoding (speed, simplicity, potency, and universal applicability) have been known for more than a decade, this approach has been only inadequately explored and the attention was focused on a limited number of RNA viruses. Attenuation of viruses by large-scale recoding should be explored to combat known and future viral threats., Competing Interests: Conflict of InterestThe authors declare that they have no conflict of interest., (© Springer International Publishing AG, part of Springer Nature 2018.)

Cowpox virus (CPXV) is a zoonotic virus and endemic in wild rodent populations in Eurasia. Serological surveys in Europe have reported high prevalence in different vole and mouse species. Here, we report on experimental CPXV infections of bank voles ( Myodes glareolus ) from different evolutionary lineages with a spectrum of CPXV strains. All bank voles, independently of lineage, sex and age, were resistant to clinical signs following CPXV inoculation, and no virus shedding was detected in nasal or buccal swabs. In-contact control animals became only rarely infected. However, depending on the CPXV strain used, inoculated animals seroconverted and viral DNA could be detected preferentially in the upper respiratory tract. The highest antibody titers and virus DNA loads in the lungs were detected after inoculation with two strains from Britain and Finland. We conclude from our experiments that the role of bank voles as an efficient and exclusive CPXV reservoir seems questionable, and that CPXV may be maintained in most regions by other hosts, including other vole species. Further investigations are needed to identify factors that allow and modulate CPXV maintenance in bank voles and other potential reservoirs, which may also influence spill-over infections to accidental hosts., Competing Interests: The authors declare no conflict of interest.

Canine distemper virus (CDV), is a pantropic agent of morbillivirus that causes fetal disease in dogs. Base on a broad host rang of CDV, the continued vaccines inoculation is unavoidable to pose gene recombination risk in vaccine virus and wild virus. The current study presents the construction of novel vectors, using equine herpesvirus type 1 (EHV-1) expressing the canine distemper virus (CDV). The recent field strain hemagglutinin protein and nucleoprotein were used for the construction of the viral vector vaccines. Based on the Bacterial artificial chromosome (BAC) genomes of EHV-1 RacH strain, the recombinant EHV-1 vaccine virus encoding CDV hemagglutinin protein (EHV-H) or CDV nucleoprotein (EHV-N) was constructed separately. The constructed BACs were rescued after 72 h post infection, and the expression of H or N in the recombinant viruses was confirmed by western-blotting. Furthermore, high levels of neutralizing antibodies were induced persistently following vaccination in the groups EHV-H&EHV-N and EHV-H, but the EHV-N group. The groups of vaccinated EHV-H and EHV-H&EHV-N pups were monitored for clinical signs, whereas the vaccinated EHV-N group developed moderate symptoms. The present study demonstrated that EHV-1 based recombinant virus carrying CDV H could be a promising vaccine candidate against canine distemper., (Copyright © 2017. Published by Elsevier Ltd.)

Middle East respiratory syndrome coronavirus (MERS-CoV) is a high-priority pathogen in pandemic preparedness research. Reverse genetics systems are a valuable tool to study viral replication and pathogenesis, design attenuated vaccines and create defined viral assay systems for applications such as antiviral screening. Here we present a novel reverse genetics system for MERS-CoV that involves maintenance of the full-length viral genome as a cDNA copy inserted in a bacterial artificial chromosome amenable to manipulation by homologue recombination, based on the bacteriophage λ Red recombination system. Based on a full-length infectious MERS-CoV cDNA clone, optimal genomic insertion sites and expression strategies for GFP were identified and used to generate a reporter MERS-CoV expressing GFP in addition to the complete set of viral proteins. GFP was genetically fused to the N-terminal part of protein 4a, from which it is released during translation via porcine teschovirus 2A peptide activity. The resulting reporter virus achieved titres nearly identical to the wild-type virus 48 h after infection of Vero cells at m.o.i. 0.001 (1×10 5  p.f.u. ml -1 and 3×10 5  p.f.u. ml -1 , respectively), and allowed determination of the 50 % inhibitory concentration for the known MERS-CoV inhibitor cyclosporine A based on fluorescence readout. The resulting value was 2.41 µM, which corresponds to values based on wild-type virus. The reverse genetics system described herein can be efficiently mutated by Red-mediated recombination. The GFP-expressing reporter virus contains the full set of MERS-CoV proteins and achieves wild-type titres in cell culture.

Virulence determines the impact a pathogen has on the fitness of its host, yet current understanding of the evolutionary origins and causes of virulence of many pathogens is surprisingly incomplete. Here, we explore the evolution of Marek's disease virus (MDV), a herpesvirus commonly afflicting chickens and rarely other avian species. The history of MDV in the 20th century represents an important case study in the evolution of virulence. The severity of MDV infection in chickens has been rising steadily since the adoption of intensive farming techniques and vaccination programs in the 1950s and 1970s, respectively. It has remained uncertain, however, which of these factors is causally more responsible for the observed increase in virulence of circulating viruses. We conducted a phylogenomic study to understand the evolution of MDV in the context of dramatic changes to poultry farming and disease control. Our analysis reveals evidence of geographical structuring of MDV strains, with reconstructions supporting the emergence of virulent viruses independently in North America and Eurasia. Of note, the emergence of virulent viruses appears to coincide approximately with the introduction of comprehensive vaccination on both continents. The time-dated phylogeny also indicated that MDV has a mean evolutionary rate of ~1.6 × 10 -5 substitutions per site per year. An examination of gene-linked mutations did not identify a strong association between mutational variation and virulence phenotypes, indicating that MDV may evolve readily and rapidly under strong selective pressures and that multiple genotypic pathways may underlie virulence adaptation in MDV.

Rift Valley fever virus (RVFV) is an arthropod-borne bunyavirus that can cause serious and fatal disease in humans and animals. RVFV is a negative-sense RNA virus of the Phlebovirus genus in the Bunyaviridae family. The main envelope RVFV glycoproteins, Gn and Gc, are encoded on the M segment of RVFV and known inducers of protective immunity. In an attempt to develop a safe and efficacious RVF vaccine, we constructed and tested a vectored equine herpesvirus type 1 (EHV-1) vaccine that expresses RVFV Gn and Gc. The Gn and Gc genes were custom-synthesized after codon optimization and inserted into EHV-1 strain RacH genome. The rH_Gn-Gc recombinant virus grew in cultured cells with kinetics that were comparable to those of the parental virus and stably expressed Gn and Gc. Upon immunization of sheep, the natural host, neutralizing antibodies against RVFV were elicited by rH_Gn-Gc and protective titers reached to 1:320 at day 49 post immunization but not by parental EHV-1, indicating that EHV-1 is a promising vector alternative in the development of a safe marker RVFV vaccine.

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-1β), were identified to play a role during the course of the disease.

Quantitative peptide-binding motifs of MHC class I alleles provide a valuable tool to efficiently identify putative T cell epitopes. Detailed information on equine MHC class I alleles is still very limited, and to date, only a single equine MHC class I allele, Eqca-1*00101 (ELA-A3 haplotype), has been characterized. The present study extends the number of characterized ELA class I specificities in two additional haplotypes found commonly in the Thoroughbred breed. Accordingly, we here report quantitative binding motifs for the ELA-A2 allele Eqca-16*00101 and the ELA-A9 allele Eqca-1*00201. Utilizing analyses of endogenously bound and eluted ligands and the screening of positional scanning combinatorial libraries, detailed and quantitative peptide-binding motifs were derived for both alleles. Eqca-16*00101 preferentially binds peptides with aliphatic/hydrophobic residues in position 2 and at the C-terminus, and Eqca-1*00201 has a preference for peptides with arginine in position 2 and hydrophobic/aliphatic residues at the C-terminus. Interestingly, the Eqca-16*00101 motif resembles that of the human HLA A02-supertype, while the Eqca-1*00201 motif resembles that of the HLA B27-supertype and two macaque class I alleles. It is expected that the identified motifs will facilitate the selection of candidate epitopes for the study of immune responses in horses.

For viruses to utilize environmental vectors (hard surfaces, soil, water) for transmission, physical and chemical stability is a prerequisite. There are many factors including pH, salinity, temperature, and turbidity that are known to contribute to the ability of viruses to persist in water. Equine herpesvirus type-1 (EHV-1) is a pathogenic alphaherpesvirus associated with domestic horses and wild equids. EHV-1 and recombinants of EHV-1 and EHV-9 are able to cause infections in non-equid animal species, particularly in captive settings. Many of the captive non-equid mammals are not naturally sympatric with equids and do not share enclosures, however, in many cases water sources may overlap. Similarly, in the wild, equids encounter many species at waterholes in times of seasonal drought. Therefore, we hypothesized that EHV-1 is stable in water and that water may act as a vector for EHV-1. In order to establish the conditions promoting or hindering EHV-1 longevity, infectivity and genomic stability in water; we exposed EHV-1 to varied water environments (pH, salinity, temperature, and turbidity) in controlled experiments over 21 days. The presence and infectivity of the virus was confirmed by both qPCR and cell culture experiments. Our results show that EHV-1 remains stable and infectious under many conditions in water for up to three weeks.

Was the 1993/1994 fatal canine distemper virus (CDV) epidemic in lions and spotted hyaenas in the Serengeti ecosystem caused by the recent spillover of a virulent domestic dog strain or one well adapted to these noncanids? We examine this question using sequence data from 13 'Serengeti' strains including five complete genomes obtained between 1993 and 2011. Phylogenetic and haplotype network analyses reveal that strains from noncanids during the epidemic were more closely related to each other than to those from domestic or wild canids. All noncanid 'Serengeti' strains during the epidemic encoded: (1) one novel substitution G134S in the CDV-V protein; and (2) the rare amino acid combination 519I/549H at two sites under positive selection in the region of the CDV-H protein that binds to SLAM (CD 150) host cell receptors. Worldwide, only a few noncanid strains in the America II lineage encode CDV-H 519I/549H. All canid 'Serengeti' strains during the epidemic coded CDV-V 134G, and CDV-H 519R/549Y, or 519R/549H. A functional assay of cell entry revealed the highest performance by CDV-H proteins encoding 519I/549H in cells expressing lion SLAM receptors, and the highest performance by proteins encoding 519R/549Y, typical of dog strains worldwide, in cells expressing dog SLAM receptors. Our findings are consistent with an epidemic in lions and hyaenas caused by CDV variants better adapted to noncanids than canids, but not with the recent spillover of a dog strain. Our study reveals a greater complexity of CDV molecular epidemiology in multihost environments than previously thought., (© 2016 John Wiley & Sons Ltd.)

Equine herpesvirus type 3 (EHV-3) is the causal agent of equine coital exanthema, a disease characterized by pox-like lesions on the penis of stallions and the vulva of mares. Although the complete genomic sequence of EHV-3 has been recently made available, its genomic content remains poorly characterized and the molecular mechanisms of disease development not yet elucidated. In an attempt to facilitate genetic manipulation of EHV-3, we describe here the construction of a full-length infectious bacterial artificial chromosome (BAC) clone of EHV-3. Mini-F vector sequences were inserted into the intergenic region between ORF19 and ORF20 (UL41 and UL40, respectively) of EHV-3 strain C175 by homologous recombination in equine dermal cells (NBL-6). DNA of the resulting recombinant virus was electroporated into E. coli and a full-length EHV-3 BAC clone was recovered. Virus reconstituted after transfection of the EHV-3 BAC into NBL-6 cells showed growth properties in vitro that were indistinguishable from those of the parental virus. To assess the feasibility of mutagenesis of the cloned EHV-3 genome, recombinant viruses targeting the glycoprotein E (gE) gene were generated using Red recombination in E. coli and in vitro growth properties of the recombinant viruses were evaluated. We first repaired the gE (ORF74) coding region, since the parental virus used for BAC cloning specifies a truncated version of the gene, and then created gE-tagged and gE-null versions of the virus. Our results demonstrated that: (i) EHV-3 can be efficiently cloned as a BAC allowing easy manipulation of its genome; (ii) gE is dispensable for EHV-3 growth in vitro and is expressed as a product of approximately 110-kDa in infected cells; (iii) viruses having a deletion compromising gE expression or with a truncation of the cytoplasmic and transmembrane domains are significantly compromised with regard cell-to-cell spread. The cloning of EHV-3 as a BAC simplifies future studies to identify the role of its coding genes in viral pathogenesis and host immune responses., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A point mutation in the DNA polymerase gene in equine herpesvirus type 1 (EHV-1) is one determinant for the development of neurological disease in horses. Three recently conducted infection experiments using domestic horses and ponies failed to detect statistically significant differences in viral shedding between the neuropathogenic and non-neuropathogenic variants. These results were interpreted as suggesting the absence of a consistent selective advantage of the neuropathogenic variant and therefore appeared to be inconsistent with a systematic increase in the prevalence of neuropathogenic strains. To overcome potential problems of low statistical power related to small group sizes in these infection experiments, we integrated raw data from all three experiments into a single statistical analysis. The results of this combined analysis showed that infection with the neuropathogenic EHV-1 variant led to a statistically significant increase in viral shedding. This finding is consistent with the idea that neuropathogenic strains could have a selective advantage and are therefore systematically increasing in prevalence in domestic horse populations. However, further studies are required to determine whether a selective advantage indeed exists for neuropathogenic strains., Competing Interests: The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Gammaherpesviruses (γHVs) are generally considered host specific and to have codiverged with their hosts over millions of years. This tenet is challenged here by broad-scale phylogenetic analysis of two viral genes using the largest sample of mammalian γHVs to date, integrating for the first time bat γHV sequences available from public repositories and newly generated viral sequences from two vampire bat species (Desmodus rotundus and Diphylla ecaudata). Bat and primate viruses frequently represented deep branches within the supported phylogenies and clustered among viruses from distantly related mammalian taxa. Following evolutionary scenario testing, we determined the number of host-switching and cospeciation events. Cross-species transmissions have occurred much more frequently than previously estimated, and most of the transmissions were attributable to bats and primates. We conclude that the evolution of the Gammaherpesvirinae subfamily has been driven by both cross-species transmissions and subsequent cospeciation within specific viral lineages and that the bat and primate orders may have potentially acted as superspreaders to other mammalian taxa throughout evolutionary history., Importance: It has long been believed that herpesviruses have coevolved with their hosts and are species specific. Nevertheless, a global evolutionary analysis of bat viruses in the context of other mammalian viruses, which could put this widely accepted view to the test, had not been undertaken until now. We present two main findings that may challenge the current view of γHV evolution: multiple host-switching events were observed at a higher rate than previously appreciated, and bats and primates harbor a large diversity of γHVs which may have led to increased cross-species transmissions from these taxa to other mammals., (Copyright © 2016 Escalera-Zamudio et al.)

Equine herpesvirus myeloencephalopathy (EHM) is a severe manifestation of equine herpesvirus 1 (EHV-1) infection. Prevention and treatment of EHM during EHV-1 outbreaks is critical, but no reliable and tested specific medication is available. Due to the thromboischemic nature of EHM and due to the fact that EHV-1 entry in cells is blocked by heparin, it was hypothesized that this compound may be useful in reduction of EHM incidence and severity. Therefore, during an acute EHV-1 outbreak with the neuropathogenic G 2254 /D 752 Pol variant, metaphylactic treatment with heparin to prevent EHM was initiated. Clinical signs were present in 61 horses (fever n = 55; EHM n = 8; abortion n = 6). Heparin (25000 IU subcutaneously twice daily for 3 days) was given to 31 febrile horses from day 10 of the outbreak, while the first 30 horses exhibiting fever remained untreated. Treatment outcome was analyzed retrospectively. Heparin-treated horses showed a lower EHM incidence (1/31; 3.2%) than untreated horses (7/30; 23.3%; p = 0.03). Results indicate that heparin may be useful for prevention of EHM during an EHV-1 outbreak. These promising data highlight the need for randomized and possibly blinded studies for the use of heparin in EHV-1 outbreaks.

Alphaherpesviruses are highly prevalent in equine populations and co-infections with more than one of these viruses' strains frequently diagnosed. Lytic replication and latency with subsequent reactivation, along with new episodes of disease, can be influenced by genetic diversity generated by spontaneous mutation and recombination. Latency enhances virus survival by providing an epidemiological strategy for long-term maintenance of divergent strains in animal populations. The alphaherpesviruses equine herpesvirus 1 (EHV-1) and 9 (EHV-9) have recently been shown to cross species barriers, including a recombinant EHV-1 observed in fatal infections of a polar bear and Asian rhinoceros. Little is known about the latency and genetic diversity of EHV-1 and EHV-9, especially among zoo and wild equids. Here, we report evidence of limited genetic diversity in EHV-9 in zebras, whereas there is substantial genetic variability in EHV-1. We demonstrate that zebras can be lytically and latently infected with both viruses concurrently. Such a co-occurrence of infection in zebras suggests that even relatively slow-evolving viruses such as equine herpesviruses have the potential to diversify rapidly by recombination. This has potential consequences for the diagnosis of these viruses and their management in wild and captive equid populations., Competing Interests: The authors declare no conflict of interest.

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.

Equine herpesvirus 1 (EHV1) is an α-herpesvirus that can infect a variety of different cells in vitro and in vivo, including dendritic cells (DC) which are essential in the immune response against EHV1. Infection of equine monocyte-derived DC (MDDC) with EHV1 induced down-regulation of major histocompatibility complex I (MHCI), CD83, CD86, CD206, CD29 and CD172a, but not of CD11a/CD18 and MHCII. This down-regulation was not mediated by the virion host-shutoff (VHS) protein or pUL49.5. Interestingly, down-regulation of CD83 and CD86 was in part mediated by pUL56. Taken together, these data indicate that EHV1 employs different and still unresolved mechanisms to induce down-regulation of several functionally important cell surface proteins on equine DC., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Codon pair bias is a remarkably stable characteristic of a species. Although functionally uncharacterized, robust virus attenuation was achieved by recoding of viral proteins using underrepresented codon pairs. Because viruses replicate exclusively inside living cells, we posited that their codon pair preferences reflect those of their host(s). Analysis of many human viruses showed, however, that the encoding of viruses is influenced only marginally by host codon pair preferences. Furthermore, examination of codon pair preferences of vertebrate, insect, and arthropod-borne viruses revealed that the latter do not utilize codon pairs overrepresented in arthropods more frequently than other viruses. We found, however, that codon pair bias is a direct consequence of dinucleotide bias. We conclude that codon pair bias does not play a major role in the encoding of viral proteins and that virus attenuation by codon pair deoptimization has the same molecular underpinnings as attenuation based on an increase in CpG/TpA dinucleotides., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: The incidence of human cowpox virus (CPXV) infections has increased significantly in recent years. Serological surveys have suggested wild rodents as the main CPXV reservoir. We characterized a CPXV isolated during a large-scale screening from a feral common vole. A comparison of the full-length DNA sequence of this CPXV strain with a highly virulent pet rat CPXV isolate showed a sequence identity of 96%, including a large additional open reading frame (ORF) of about 6,000 nucleotides which is absent in the reference CPXV strain Brighton Red. Electron microscopy analysis demonstrated that the vole isolate, in contrast to the rat strain, forms A-type inclusion (ATI) bodies with incorporated virions, consistent with the presence of complete ati and p4c genes. Experimental infections showed that the vole CPXV strain caused only mild clinical symptoms in its natural host, while all rats developed severe respiratory symptoms followed by a systemic rash. In contrast, common voles infected with a high dose of the rat CPXV showed severe signs of respiratory disease but no skin lesions, whereas infection with a low dose led to virus excretion with only mild clinical signs. We concluded that the common vole is susceptible to infection with different CPXV strains. The spectrum ranges from well-adapted viruses causing limited clinical symptoms to highly virulent strains causing severe respiratory symptoms. In addition, the low pathogenicity of the vole isolate in its eponymous host suggests a role of common voles as a major CPXV reservoir, and future research will focus on the correlation between viral genotype and phenotype/pathotype in accidental and reservoir species., Importance: We report on the first detection and isolation of CPXV from a putative reservoir host, which enables comparative analyses to understand the infection cycle of these zoonotic orthopox viruses and the relevant genes involved. In vitro studies, including whole-genome sequencing as well as in vivo experiments using the Wistar rat model and the vole reservoir host allowed us to establish links between genomic sequences and the in vivo properties (virulence) of the novel vole isolate in comparison to those of a recent zoonotic CPXV isolated from pet rats in 2009. Furthermore, the role of genes present only in a reservoir isolate can now be further analyzed. These studies therefore allow unique insights and conclusions about the role of the rodent reservoir in CPXV epidemiology and transmission and about the zoonotic threat that these viruses represent., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Here we describe a detailed quantitative peptide-binding motif for the common equine leukocyte antigen (ELA) class I allele Eqca-1*00101, present in roughly 25 % of Thoroughbred horses. We determined a preliminary binding motif by sequencing endogenously bound ligands. Subsequently, a positional scanning combinatorial library (PSCL) was used to further characterize binding specificity and derive a quantitative motif involving aspartic acid in position 2 and hydrophobic residues at the C-terminus. Using this motif, we selected and tested 9- and 10-mer peptides derived from the equine herpesvirus type 1 (EHV-1) proteome for their capacity to bind Eqca-1*00101. PSCL predictions were very efficient, with an receiver operating characteristic (ROC) curve performance of 0.877, and 87 peptides derived from 40 different EHV-1 proteins were identified with affinities of 500 nM or higher. Quantitative analysis revealed that Eqca-1*00101 has a narrow peptide-binding repertoire, in comparison to those of most human, non-human primate, and mouse class I alleles. Peripheral blood mononuclear cells from six EHV-1-infected, or vaccinated but uninfected, Eqca-1*00101-positive horses were used in IFN-γ enzyme-linked immunospot (ELISPOT) assays. When we screened the 87 Eqca-1*00101-binding peptides for T cell reactivity, only one Eqca-1*00101 epitope, derived from the intermediate-early protein ICP4, was identified. Thus, despite its common occurrence in several horse breeds, Eqca-1*00101 is associated with a narrow binding repertoire and a similarly narrow T cell response to an important equine viral pathogen. Intriguingly, these features are shared with other human and macaque major histocompatibility complex (MHC) molecules with a similar specificity for D in position 2 or 3 in their main anchor motif.

Unlabelled: Intracellular signaling connected to integrin activation is known to induce cytoplasmic Ca(2+) release, which in turn mediates a number of downstream signals. The cellular entry pathways of two closely related alphaherpesviruses, equine herpesviruses 1 and 4 (EHV-1 and EHV-4), are differentially regulated with respect to the requirement of interaction of glycoprotein H (gH) with α4β1-integrins. We show here that binding of EHV-1, but not EHV-4, to target cells resulted in a rapid and significant increase in cytosolic Ca(2+) levels. EHV-1 expressing EHV-4 gH (gH4) in lieu of authentic gH1 failed to induce Ca(2+) release, while EHV-4 with gH1 triggered significant Ca(2+) release. Blocking the interaction between gH1 and α4β1-integrins, inhibiting phospholipase C (PLC) activation, or blocking binding of inositol 1,4,5-triphosphate (IP3) to its receptor on the endoplasmic reticulum (ER) abrogated Ca(2+) release. Interestingly, phosphatidylserine (PS) was exposed on the plasma membrane in response to cytosolic calcium increase after EHV-1 binding through a scramblase-dependent mechanism. Inhibition of both Ca(2+) release from the ER and scramblase activation blocked PS scrambling and redirected virus entry to the endocytic pathway, indicating that PS may play a role in facilitating virus entry directly at the plasma membrane., Importance: Herpesviruses are a large family of enveloped viruses that infect a wide range of hosts, causing a variety of diseases. These viruses have developed a number of strategies for successful entry into different cell types. We and others have shown that alphaherpesviruses, including EHV-1 and herpes simplex virus 1 (HSV-1), can route their entry pathway and do so by manipulation of cell signaling cascades to ensure viral genome delivery to nuclei. We show here that the interaction between EHV-1 gH and cellular α4β1-integrins is necessary to induce emptying of ER calcium stores, which induces phosphatidylserine exposure on the plasma membrane through a scramblase-dependent mechanism. This change in lipid asymmetry facilitates virus entry and might help fusion of the viral envelope at the plasma membrane. These findings will help to advance our understanding of herpesvirus entry mechanism and may facilitate the development of novel drugs that can be implemented for prevention of infection and disease., (Copyright © 2015 Azab et al.)

Equine herpesvirus type 1 (EHV-1) causes respiratory disorders and abortion in equids while EHV-1 regularly causes equine herpesvirus myeloencephalopathy (EHM), a stroke-like syndrome following endothelial cell infection in horses. Both EHV-1 and EHV-9 infections of non-definitive hosts often result in neuronal infection and high case fatality rates. Hence, EHV-1 and EHV-9 are somewhat unusual herpesviruses and lack strict host specificity, and the true extent of their host ranges have remained unclear. In order to determine the seroprevalence of EHV-1 and EHV-9, a sensitive and specific peptide-based ELISA was developed and applied to 428 sera from captive and wild animals representing 30 species in 12 families and five orders. Members of the Equidae, Rhinocerotidae and Bovidae were serologically positive for EHV-1 and EHV-9. The prevalence of EHV-1 in the sampled wild zebra populations was significantly higher than in zoos suggesting captivity may reduce exposure to EHV-1. Furthermore, the seroprevalence for EHV-1 was significantly higher than for EHV-9 in zebras. In contrast, EHV-9 antibody prevalence was high in captive and wild African rhinoceros species suggesting that they may serve as a reservoir or natural host for EHV-9. Thus, EHV-1 and EHV-9 have a broad host range favoring African herbivores and may have acquired novel natural hosts in ecosystems where wild equids are common and are in close contact with other perissodactyls.

Marek's disease virus (MDV) is an alphaherpesvirus that causes deadly T-cell lymphomas in chickens and serves as a natural small animal model for virus-induced tumor formation. In vivo, MDV lytically replicates in B cells that transfer the virus to T cells in which the virus establishes latency. MDV also malignantly transforms CD4+ T cells with a T(reg) signature, ultimately resulting in deadly lymphomas. No in vitro infection system for primary target cells of MDV has been available due to the short-lived nature of these cells in culture. Recently, we characterized cytokines and monoclonal antibodies that promote survival of cultured chicken B and T cells. We used these survival stimuli to establish a culture system that allows efficient infection of B and T cells with MDV. We were able to productively infect with MDV B cells isolated from spleen, bursa or blood cultured in the presence of soluble CD40L. Virus was readily transferred from infected B to T cells stimulated with an anti-TCRαVβ1 antibody, thus recapitulating the in vivo situation in the culture dish. Infected T cells could then be maintained in culture for at least 90 d in the absence of TCR stimulation, which allowed the establishment of MDV-transformed lymphoblastoid cell lines (LCL). The immortalized cells had a signature comparable to MDV-transformed CD4+ α/β T cells present in tumors. In summary, we have developed a novel in vitro system that precisely reflects the life cycle of an oncogenic herpesivrus in vivo and will allow us to investigate the interaction between virus and target cells in an easily accessible system.

Unlabelled: Herpesviruses have evolved an array of strategies to counteract antigen presentation by major histocompatibility complex class I (MHC-I). Previously, we identified pUL56 of equine herpesvirus 1 (EHV-1) as one major determinant of the downregulation of cell surface MHC-I (G. Ma, S. Feineis, N. Osterrieder, and G. R. Van de Walle, J. Virol. 86:3554-3563, 2012, http://dx.doi.org/10.1128/JVI.06994-11; T. Huang, M. J. Lehmann, A. Said, G. Ma, and N. Osterrieder, J. Virol. 88:12802-12815, 2014, http://dx.doi.org/10.1128/JVI.02079-14). Since pUL56 was able to exert its function only in the context of virus infection, we hypothesized that pUL56 cooperates with another viral protein. Here, we generated and screened a series of EHV-1 single-gene deletion mutants and found that the pUL43 orthologue was required for downregulation of cell surface MHC-I expression at the same time of infection as when pUL56 exerts its function. We demonstrate that the absence of pUL43 was not deleterious to virus growth and that expression of pUL43 was detectable from 2 h postinfection (p.i.) but decreased after 8 h p.i. due to lysosomal degradation. pUL43 localized within Golgi vesicles and required a unique hydrophilic N-terminal domain to function properly. Finally, coexpression of pUL43 and pUL56 in transfected cells reduced the cell surface expression of MHC-I. This process was dependent on PPxY motifs present in pUL56, suggesting that late domains are required for pUL43- and pUL56-dependent sorting of MHC class I for lysosomal degradation., Importance: We describe here that the poorly characterized herpesviral protein pUL43 is involved in downregulation of cell surface MHC-I. pUL43 is an early protein and degraded in lysosomes. pUL43 resides in the Golgi vesicles and needs an intact N terminus to induce MHC-I downregulation in infected cells. Importantly, pUL43 and pUL56 cooperate to reduce MHC-I expression on the surface of transfected cells. Our results suggest a model for MHC-I downregulation in which late domains in pUL56 are required for the rerouting of vesicles containing MHC-I, pUL56, and pUL43 to the lysosomal compartment., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Equid herpesvirus type 1 (EHV-1) causes outbreaks of abortion and neurological disease in horses. One of the main causes of these clinical syndromes is thrombosis in placental and spinal cord vessels, however the mechanism for thrombus formation is unknown. Platelets form part of the thrombus and amplify and propagate thrombin generation. Here, we tested the hypothesis that EHV-1 activates platelets. We found that two EHV-1 strains, RacL11 and Ab4 at 0.5 or higher plaque forming unit/cell, activate platelets within 10 minutes, causing α-granule secretion (surface P-selectin expression) and platelet microvesiculation (increased small events double positive for CD41 and Annexin V). Microvesiculation was more pronounced with the RacL11 strain. Virus-induced P-selectin expression required plasma and 1.0 mM exogenous calcium. P-selectin expression was abolished and microvesiculation was significantly reduced in factor VII- or X-deficient human plasma. Both P-selectin expression and microvesiculation were re-established in factor VII-deficient human plasma with added purified human factor VIIa (1 nM). A glycoprotein C-deficient mutant of the Ab4 strain activated platelets as effectively as non-mutated Ab4. P-selectin expression was abolished and microvesiculation was significantly reduced by preincubation of virus with a goat polyclonal anti-rabbit tissue factor antibody. Infectious virus could be retrieved from washed EHV-1-exposed platelets, suggesting a direct platelet-virus interaction. Our results indicate that EHV-1 activates equine platelets and that α-granule secretion is a consequence of virus-associated tissue factor triggering factor X activation and thrombin generation. Microvesiculation was only partly tissue factor and thrombin-dependent, suggesting the virus causes microvesiculation through other mechanisms, potentially through direct binding. These findings suggest that EHV-1-induced platelet activation could contribute to the thrombosis that occurs in clinically infected horses and provides a new mechanism by which viruses activate hemostasis.

Equid herpesvirus 1 (EHV1) is an ubiquitous alphaherpesvirus that can cause respiratory disease, abortion and central nervous disorders. EHV1 is known to infect a variety of different cell types in vitro, but its tropism for cultured primary equine mesenchymal stem cells (MSC) has never been explored. We report that equine MSC were highly permissive for EHV1 and supported lytic replication of the virus in vitro. Interestingly, we observed that an infection of MSC with EHV1 resulted in a consistent downregulation of cell surface molecules CD29 (β1-integrin), CD105 (endoglin), major histocompatibility complex type I (MHCI) and a variable downregulation of CD172a. In contrast, expression of CD44 and CD90 remained unchanged upon wild type infection. In addition, we found that this selective EHV1-mediated downregulation of cell surface proteins was dependent on the viral protein UL56 (pUL56). So far, pUL56-dependent downregulation during EHV1 infection of equine cells has only been described for MHCI, but our present data indicate that pUL56 may have a broader function in downregulating cell surface proteins. Taken together, our results are the first to show that equine MSC are susceptible for EHV1 and that pUL56 induces downregulation of several cell surface molecules on infected cells. These findings provide a basis for future studies to evaluate the mechanisms underlying for this selective pUL56-induced downregulation and to evaluate the potential role of MSC during EHV1 pathogenesis., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Glycoprotein B (gB) plays an important role in alphaherpesvirus cellular entry and acts in concert with gD and the gH/gL complex. To evaluate whether functional differences exist between gB1 and gB4, the corresponding genes were exchanged between the two viruses. The gB4-containing-EHV-1 (EHV-1_gB4) recombinant virus was analyzed for growth in culture, cell tropism, and cell entry rivaling no significant differences when compared to parental virus. We also disrupted a potential integrin-binding motif, which did not affect the function of gB in culture. In contrast, a significant reduction of plaque sizes and growth kinetics of gB1-containing-EHV-4 (EHV-4_gB1) was evident when compared to parental EHV-4 and revertant viruses. The reduction in virus growth may be attributable to the loss of functional interaction between gB and the other envelope proteins involved in virus entry, including gD and gH/gL. Alternatively, gB4 might have an additional function, required for EHV-4 replication, which is not fulfilled by gB1. In conclusion, our results show that the exchange of gB between EHV-1 and EHV-4 is possible, but results in a significant attenuation of virus growth in the case of EHV-4_gB1. The generation of stable recombinant viruses is a valuable tool to address viral entry in a comparative fashion and investigate this aspect of virus replication further.

Unlabelled: Marek's disease virus (MDV), an alphaherpesvirus, is the causative agent of a lethal disease in chickens characterized by generalized nerve inflammation and rapid lymphoma development. The extensive colinearity of the MDV genome with those of related herpesviruses has eased functional characterization of many MDV genes. However, MDV carries a number of unique open reading frames (ORFs) that have not yet been investigated regarding their coding potentials and the functions of their products. Among these unique ORFs are two putative ORFs, ORF011 and ORF012, which are found at the extreme left end of the MDV unique long region. Using reverse transcriptase PCR, we showed that ORF011 and ORF012 are not individual genes but form a single gene through mRNA splicing of a small intron, resulting in the novel ORF012. We generated an ORF012-null virus using an infectious clone of MDV strain RB-1B. The deletion virus had a marked growth defect in vitro and could not be passaged in cultured cells, suggesting an essential role for the ORF012 product in virus replication. Further studies revealed that protein 012 (p012) localized to the nucleus in transfected and infected cells, and we identified by site-directed mutagenesis and green fluorescent protein (GFP) reporter fusion assays a nuclear localization signal (NLS) that was mapped to a 23-amino-acid sequence at the protein's C terminus. Nuclear export was blocked using leptomycin B, suggesting a potential role for p012 as a nuclear/cytoplasmic shuttling protein. Finally, p012 is phosphorylated at multiple residues, a modification that could possibly regulate its subcellular distribution., Importance: Marek's disease virus (MDV) causes a devastating oncogenic disease in chickens with high morbidity and mortality. The costs for disease prevention reach several billion dollars annually. The functional investigation of MDV genes is necessary to understand its complex replication cycle, which eventually could help us to interfere with MDV and herpesviral pathogenesis. We have identified a previously unidentified phosphoprotein encoded by MDV ORF012. We were able to show experimentally that predicted splicing of the gene based on bioinformatics data does indeed occur during replication. The newly identified p012 is essential for MDV replication and localizes to the nucleus due to the presence of a transferable nuclear localization signal at its C terminus. Our results also imply that p012 could constitute a nucleocytoplasmic shuttle protein, a feature that could prove interesting and important., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Equine herpesvirus type 1 (EHV-1) downregulates cell surface expression of major histocompatibility complex class I (MHC-I) in infected cells. We have previously shown that pUL56 encoded by the EHV-1 ORF1 gene regulates the process (G. Ma, S. Feineis, N. Osterrieder, and G. R. Van de Walle, J. Virol. 86:3554-3563, 2012, doi:http://dx.doi.org/10.1128/JVI.06994-11). Here, we report that cell surface MHC-I in EHV-1-infected cells is internalized and degraded in the lysosomal compartment in a pUL56-dependent fashion. pUL56-induced MHC-I endocytosis required dynamin and tyrosine kinase but was independent of clathrin and caveolin-1, the main constituents of the clathrin- and raft/caveola-mediated endocytosis pathways, respectively. Downregulation of cell surface MHC-I was significantly inhibited by the ubiquitin-activating enzyme E1 inhibitor PYR41, indicating that ubiquitination is essential for the process. Finally, we show that downregulation is not specific for MHC-I and that other molecules, including CD46 and CD63, are also removed from the cell surface in a pUL56-dependent fashion., Importance: We show that alphaherpesvirus induces MHC-I downregulation through endocytosis, which is mediated by pUL56. The dynamin-dependent endocytic pathway is responsible for MHC-I internalization in infected cells. Furthermore, we discovered that this endocytic process can be disrupted by the inhibiting ubiquitin-activating E1 enzyme, which is indispensable for ubiquitination. Finally, pUL56 action extends to a number of cell surface molecules that are significant for host immunity. Therefore, the protein may exert a more general immunomodulatory effect., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

It is well known that numerous viruses integrate their genetic material into host cell chromosomes. Human herpesvirus 6 (HHV-6) and oncogenic Marek's disease virus (MDV) have been shown to integrate their genomes into host telomeres of latently infected cells. This is unusual for herpesviruses as most maintain their genomes as circular episomes during the quiescent stage of infection. The genomic DNA of HHV-6, MDV, and several other herpesviruses harbors telomeric repeats (TMRs) that are identical to host telomere sequences (TTAGGG). At least in the case of MDV, viral TMRs facilitate integration into host telomeres. Integration of HHV-6 occurs not only in lymphocytes but also in the germline of some individuals, allowing vertical virus transmission. Although the molecular mechanism of telomere integration is poorly understood, the presence of TMRs in a number of herpesviruses suggests it is their default program for genome maintenance during latency and also allows efficient reactivation.

Equine herpesvirus type 2 (EHV-2) and EHV-5 are members of the subfamily Gammaherpesvirinae. The viruses are detected in horses with upper respiratory tract disease and are associated with low performance in racehorses. The aim of the current study was to use nested PCR to investigate the epidemiology of EHV-2 and EHV-5 in Arabian horse populations from breeding farms located in three different cities (Eskişehir, Malatya, and Bursa) in Turkey, using a real-time quantitative PCR (qPCR) with a TaqMan® minor-groove-binder (MGB) probe to detect EHV-5. Screening of blood and ocular and nasal swab samples by nested PCR showed the prevalence of EHV-2 and EHV-5 to be 59 % and 62 %, respectively, with a coinfection rate of 45 %. Thirty-seven isolates from blood samples were identified as EHV-2 using nested PCR. To develop the EHV-5 qPCR, a pair of primers and an MGB probe were designed based on a highly conserved genomic region encoding glycoprotein B (gB). The detection limit of the qPCR was 10 molecules per reaction, and it specifically detected EHV-5 and no other herpesviruses infecting horses (EHV-1, EHV-2, or EHV-4). When applied to field samples, the assay proved to be more sensitive than a well-established nested PCR. Therefore, the qPCR developed in this study provides a rapid, reliable, and sensitive diagnostic assay for the detection of EHV-5, and it complements other diagnostic procedures for equine respiratory disease.

Recently, the product of equine herpesvirus type 1 (EHV-1) ORF1, a homolog to HSV-1 pUL56, was shown to modulate MHC-I expression and innate immunity. Here, we investigated modulation of respiratory epithelial immunity by EHV-1 pUL56 and compared responses to those of PBMCs, which are important target cells that allow cell-associated EHV-1 viremia. The salient observations are as follows: (i) EHV-1 significantly down-modulated MHC-I and MHC-II expression in equine respiratory epithelial cells (ERECs). MHC-I expression remained unaffected in PBMCs and MHC-II expression was increased. (ii) Infection with an EHV-1 ORF1 deletion mutant partially restored MHC-I and MHC-II expression and altered IFN-alpha and IL-10 mRNA expression. (iii) Deletion of EHV-1 ORF1 also significantly increased chemokine expression and chemotaxis of monocytes and neutrophils in ERECs. Collectively, these results suggest a role for EHV-1 pUL56 in modulation of antigen presentation, cytokine expression and chemotaxis at the respiratory epithelium, but not in PBMC., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A particularly severe equine herpesvirus type 1 (EHV-1) abortion outbreak occurred at a breeding farm in northern Germany. Sixteen of 25 pregnant mares that had received regular vaccination using an inactivated vaccine aborted and two gave birth to weak non-viable foals in a span of three months, with 89% of cases occurring within 40 days after the initial abortion case. Virological examinations revealed the presence of EHV-1 in all cases of abortion and serological follow-up in mares confirmed recent infection. Molecular studies identified a neuropathogenic variant (Pol/ORF30 A2254 to G2254) that belonged to geographical group 4 of EHV-1 isolates. The abortion outbreak was preceded by a case of mild ataxia of unknown cause in a mare that aborted four months after the ataxic episode. Although vaccination of pregnant mares did not prevent abortion, good EHV-1 immune status of the population at the time of outbreak may have had an impact in the failure of manifestation of the neurological form of the disease., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important infectious agents for the swine industry worldwide. Zinc (Zn) salts, which are widely used as a dietary supplement in swine nutrition, have shown antiviral effects in vitro as well as in vivo. The purpose of this study was to determine the influence of dietary zinc oxide supplementation on vaccination and challenge infection with PRRSV., Findings: The clinical course of PRRS and the success of vaccination with an experimental inactivated vaccine were compared between animals receiving a conventional diet (50 ppm Zn, control group) and diets supplemented with Zn oxide (ZnO) at final Zn concentrations of 150 or 2,500 ppm. Pigs receiving higher dietary Zn levels showed a tendency towards higher neutralizing antibody levels after infection, while dietary Zn levels did not substantially influence the number of antiviral IFN-gamma secreting cells (IFN-gamma-SC) or percentages of blood immune cell subsets after infection. Finally, feeding higher dietary Zn levels reduced neither clinical symptoms nor viral loads., Conclusions: Our results suggest that higher levels of dietary ZnO do not have the potential to stimulate or modulate systemic immune responses after vaccination and heterologous PRRSV infection to an extent that could improve the clinical and virological outcome.