Herpesvirus Species Research Articles

Abstract 788: Leveraging multi omics approach to examine the potential role of causative viruses in glioblastoma

Abstract Glioblastoma (GB), a grade IV brain cancer, has the lowest survival rate. Despite aggressive and targeted therapies, patients remain to have the worst prognosis. More than 16% of cancer incidence worldwide has been attributed to infectious agents, raising uncertainties about the role of infections in the development of brain cancer. While numerous studies have suggested potential viral involvement in GB pathogenesis, a common limitation is the reliance on a singular technology, such as DNA/RNA sequencing or immunohistochemistry, within each study. This approach may restrict the comprehensive exploration of viral contributions, as different technologies offer distinct insights into the complex mechanisms underlying GB development. Utilizing a combination of techniques could provide a more holistic understanding, enhancing the robustness of findings and potentially uncovering nuanced aspects of viral influence that a single technology might overlook. To clarify the role of viruses in GB pathology, our study employs a comprehensive approach integrating three distinct research methods. We screened 178 publicly accessible raw mass spectrometry (MS) data of GB tumors from three independent publications (PMID: 31331834, 31154438, 36720864) by employing our internally developed data identification pipeline. The pipeline transforms raw MS data for downstream analysis, conducts database searches against the latest viral protein databases and undergoes peptide validation for enhanced robustness. Subsequent analysis filters duplicate hits and consolidates nested peptides. The final protein list includes only those identified with two or more peptides of over eight amino acids. The pipeline concludes by inferring and listing the corresponding viruses, ensuring a comprehensive and refined approach to the identification and analysis of viral proteins in the context of MS data. To enhance this dataset, we incorporated whole-genome metagenomics and MS-based proteomics, examining a preliminary cohort of15 GB tumor tissues. This approach explored a wide range of viral encoded proteins. Our comprehensive analysis, integrating bioinformatics, genomics, and proteomics, identified multiple herpesvirus species across our cohorts. This potential identification of viruses within GB has the capacity to redefine the stratification of tumor types in GB patients, signaling a significant development in our understanding of the disease. Citation Format: Bavani Gunasegaran, Aziz Abdullah A Alnakli, Gilles J. Guillemin4, Seong Beom Ahn, Benjamin Heng. Leveraging multi omics approach to examine the potential role of causative viruses in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 788.

Active herpesviruses are associated with intensive care unit (ICU) admission in patients pulmonary infection and alter the respiratory microbiome.

The Herpesviridae family contains several human-related viruses, which are able to establish colonizing and latency in the human body, posing a significant threat to the prognosis of patients. Pulmonary infections represent one of the predominant infectious diseases globally, characterized by diverse and multifaceted clinical manifestations that have consistently attracted clinician's concern. However, the relationship of herpesviruses on the prognosis of pulmonary infections and the respiratory microbiota remains poorly understood. Here, we retrospectively analyzed respiratory samples from 100 patients with pulmonary infection detected by metagenomic next-generation sequencing (mNGS). Employing mNGS, five herpesvirus species were detected: Human alphaherpesvirus 1 (HSV-1), Human gammaherpesvirus 4 (EBV), Human betaherpesvirus 5 (CMV), Human betaherpesvirus 7 (HHV-7), and Human betaherpesvirus 6B (HHV-6B). Regression analysis showed that the age and positivity of herpesviruses in patients were independently correlated with ICU admission rates. In addition, positivity of herpesvirus was related with increased ICU days and total hospital stay. The herpesvirus-positive group demonstrated markedly higher incidences of co-infections and fungi-positive, predominantly involving Pneumocystis jirovecii and Aspergillus fumigatus. Analysis of respiratory microbiota revealed a substantially altered community composition within the herpesvirus-positive group, and herpesviruses were significantly positively correlated with the diverse respiratory opportunistic pathogens. Overall results substantiate that the active herpesviruses in patients with pulmonary infections were significantly associated with high ICU admission rate. Moreover, the herpesviruses promotes the dysbiosis of the respiratory microbiota and an increased proportion of co-infections. These insights could contribute to unraveling the underlying mechanisms connecting active herpesviruses to the progression of severe illnesses.

Development of subfamily-based consensus PCR assays for the detection of human and animal herpesviruses

Consensus PCR assays that can be used to sensitively detect several herpesvirus (HV) species across the different subfamilies were developed in this study. Primers containing degenerate bases were designed to amplify regions of the DNA polymerase (DPOL) gene of alpha- and gamma-HVs, and the glycoprotein B (gB) gene of beta-HVs in a singleplex, non-nested touchdown PCR format. The singleplex touchdown consensus PCR (STC-PCR) was used to amplify the DNA of eight human and 24 animal HVs. The assay was able to detect the lowest DNA dilution of 10−5 for alpha-HVs and 10−3 for beta- and gamma-HVs. In comparison, lowest detection limits of 10−5, 10−3, and 10−2 were obtained for alpha-, beta-, and gamma-HVs respectively when a nested PCR was used. The findings in this study suggest that the STC-PCR assays can be employed for the molecular surveys and clinical detection of novel and known HVs.

Infection with a Recently Discovered Gammaherpesvirus Variant in European Badgers, Meles meles, is Associated with Higher Relative Viral Loads in Blood.

Herpesviruses are ubiquitous pathogens infecting most animals. Although host immunity continually coevolves to combat virulence, viral variants with enhanced transmissibility or virulence occasionally emerge, resulting in disease burdens in host populations. Mustelid gammaherpesvirus 1 (MusGHV-1) is the only herpesvirus species identified thus far in European badgers, Meles meles. No MusGHV-1 associated pathomorbidity has been reported, but reactivation of MusGHV-1 in genital tracts is linked to impaired female reproductive success. An analysis of a short sequence from the highly conserved DNA polymerase (DNApol) gene previously identified two variants in a single host population. Here we compared genetic variance in blood samples from 66 known individuals of this same free-ranging badger population using a partial sequence comprising 2874 nucleotides of the DNApol gene, among which we identified 15 nucleotide differences resulting in 5 amino acid differences. Prevalence was 86% (59/66) for the common and 17% (11/66) for the novel variant, with 6% (4/66) of badgers presenting with coinfection. MusGHV-1 variants were distributed unevenly across the population, with individuals infected with the novel genotype clustered in 3 of 25 contiguous social groups. Individuals infected with the novel variant had significantly higher MusGHV-1 viral loads in their blood (p = 0.002) after adjusting for age (juveniles > adults, p < 0.001) and season (summer > spring and autumn, p = 0.005; mixed-effect linear regression), likely indicating higher virulence of the novel variant. Further genome-wide analyses of MusGHV-1 host resistance genes and host phenotypic variations are required to clarify the drivers and sequelae of this new MusGHV-1 variant.

Broad-spectrum antiviral diazadispiroalkane core molecules block attachment and cell-to-cell spread of herpesviruses

Regarding the problems with the current available drugs many research studies deal with the class of the dispirotripiperazine (DSTP)-based compounds. These are small molecules consisting of polycyclic saturated ring systems with positively charged nitrogen atoms. These compounds can interact with negatively charged HSPGs and thus block viral attachment. In a previous paper by Adfeldt et al. (2021), we have shown that the diazadispiroalkane derivatives 11826091 and 11826236 exhibit dose-dependent antiviral activity against human cytomegalovirus (HCMV) and pseudorabies virus (PrV). In the present study, these two small molecules are evaluated against two other herpesvirus species, murine cytomegalovirus (MCMV) and herpes simplex virus type 1 (HSV-1), as well as a HCMV clinical isolate. They exhibit potent antiherpetic activity against these herpesviruses with a high selectivity index. The low cytotoxicity was underlined by the LD50 determination in mice. We have shown that inhibition occurs at an early stage of infection. Interestingly, 11826091 and 11826236 reduced immediate early gene expression in HCMV and HSV-1 infected cells in a dose-dependent manner. Both small molecules probably interact electrostatically with sulfated glycosaminoglycans (GAGs) of proteoglycans on target cells resulting in blockage of adsorption sites for herpesvirus glycoprotein. Moreover, both compounds showed significant effects against the cell-associated viral spread of HSV-1 and HCMV. Overall, this study shows that 11826091 and 11826236 represent two promising candidates for a new approach of a broad antiviral therapy.

A Comprehensive Review on Role of Viruses in Gingival and Periodontal Diseases

It is an already established fact that periodontal disease is principally caused by specific group of bacterial pathogens. The red complex of bacterial species plays an important role in periodontal disease progression. However, the 20th century marked an important turning point in the etiopathogenesis of periodontal disease. It was found that viruses can modify the host response as well as the pathogenesis by which bacteria affects the periodontium. This alteration in the pathogenesis of periodontopathic bacteria and the altered host immune response by the virus leads to periodontal disease progression. Over the years, various viruses have been associated with periodontal disease. In particular, the Herpesvirus species, human immunodeficiency virus and human papillomavirus has been widely researched. Moreover, the Epstein–Barr virus and human Cytomegalovirus co-exist in the subgingival environment along with the periodontal pathogens. The synergism among the bacteria and virus is a prime factor for severe periodontal breakdown. The present review article provides comprehensive information on the pathogenesis by which the viruses cause periodontal disease progression.

Primary Infection May Be an Underlying Factor Contributing to Lethal Hemorrhagic Disease Caused by Elephant Endotheliotropic Herpesvirus 3 in African Elephants (Loxodonta africana).

ABSTRACTDistinct but related species of elephant endotheliotropic herpesviruses (EEHVs) circulate within Asian and African elephant populations. Primary infection with EEHVs endemic among Asian elephants can cause clinical illness and lethal EEHV hemorrhagic disease (EEHV-HD). The degree to which this occurs among African elephants has not been fully established. Recent cases of EEHV-HD caused by the EEHV3 species in African elephants housed in North American zoos has heightened concern about the susceptibility of this elephant species to EEHV-HD. In this study, we utilize the luciferase immunoprecipitation system (LIPS) to generate a serological assay specific for EEHV3 in African elephants by detecting antibodies against the EEHV3 E34 protein. The results showed that the majority of tested elephants from four separate and genetically unrelated herds, including five elephants that survived clinical illness associated with EEHV3, were positive for prior infection with EEHV3. However, African elephants who succumbed to EEHV3-HD were seronegative for EEHV3 prior to lethal infection. This supports the hypothesis that fatal EEHV-HD caused by EEHV3 is associated with primary infection rather than reactivation of latent virus. Lastly, we observed that African elephants, like Asian elephants, acquire abundant anti-EEHV antibodies prenatally and that anti-EEHV3 specific antibodies were either never detected or declined to undetectable levels in those animals that died from lethal disease following EEHV3 infection.IMPORTANCE Prior to 2019, only five cases of clinical disease from EEHV infection among African elephants had been documented. Since 2019, there have been at least seven EEHV-HD cases in North American zoos, resulting in three fatalities, all associated with EEHV3. Evidence is accumulating to suggest that EEHV-associated clinical illness and death among Asian elephants is due to primary infection and may be associated with waning anti-EEHV antibody levels in young elephants. The development of the EEHV3 serological test described in this study enabled us to confirm that similar dynamics may be contributing to EEHV-HD in African elephants. The ability to screen for EEHV immune status in African elephant calves will have a major impact on managing captive African elephant herds and will provide new tools for investigating and understanding EEHV in wild populations.

Herpesviruses in Reptiles.

Since the 1970s, several species of herpesviruses have been identified and associated with significant diseases in reptiles. Earlier discoveries placed these viruses into different taxonomic groups on the basis of morphological and biological characteristics, while advancements in molecular methods have led to more recent descriptions of novel reptilian herpesviruses, as well as providing insight into the phylogenetic relationship of these viruses. Herpesvirus infections in reptiles are often characterised by non-pathognomonic signs including stomatitis, encephalitis, conjunctivitis, hepatitis and proliferative lesions. With the exception of fibropapillomatosis in marine turtles, the absence of specific clinical signs has fostered misdiagnosis and underreporting of the actual disease burden in reptilian populations and hampered potential investigations that could lead to the effective control of these diseases. In addition, complex life histories, sampling bias and poor monitoring systems have limited the assessment of the impact of herpesvirus infections in wild populations and captive collections. Here we review the current published knowledge of the taxonomy, pathogenesis, pathology and epidemiology of reptilian herpesviruses.

Conserved Central Intraviral Protein Interactome of the Herpesviridae Family.

Protein interactions are major driving forces behind the functional phenotypes of biological processes. As such, evolutionary footprints are reflected in system-level collections of protein-protein interactions (PPIs), i.e., protein interactomes. We conducted a comparative analysis of intraviral protein interactomes for representative species of each of the three subfamilies of herpesviruses (herpes simplex virus 1, human cytomegalovirus, and Epstein-Barr virus), which are highly prevalent etiologic agents of important human diseases. The intraviral interactomes were reconstructed by combining experimentally supported and computationally predicted protein-protein interactions. Using cross-species network comparison, we then identified family-wise conserved interactions and protein complexes, which we defined as a herpesviral "central" intraviral protein interactome. A large number of widely accepted conserved herpesviral protein complexes are present in this central intraviral interactome, encouragingly supporting the biological coherence of our results. Importantly, these protein complexes represent most, if not all, of the essential steps required during a productive life cycle. Hence the central intraviral protein interactome could plausibly represent a minimal infectious interactome of the herpesvirus family across a variety of hosts. Our data, which have been integrated into our herpesvirus interactomics database, HVint2.0, could assist in creating comprehensive system-level computational models of this viral lineage.IMPORTANCE Herpesviruses are an important socioeconomic burden for both humans and livestock. Throughout their long evolutionary history, individual herpesvirus species have developed remarkable host specificity, while collectively the Herpesviridae family has evolved to infect a large variety of eukaryotic hosts. The development of approaches to fight herpesvirus infections has been hampered by the complexity of herpesviruses' genomes, proteomes, and structural features. The data and insights generated by our study add to the understanding of the functional organization of herpesvirus-encoded proteins, specifically of family-wise conserved features defining essential components required for a productive infectious cycle across different hosts, which can contribute toward the conceptualization of antiherpetic infection strategies with an effect on a broader range of target species. All of the generated data have been made freely available through our HVint2.0 database, a dedicated resource of curated herpesvirus interactomics purposely created to promote and assist future studies in the field.

Molecular and microscopic characterisation of a novel pathogenic herpesvirus from Indian ringneck parrots (Psittacula krameri)

A high morbidity, high mortality disease process caused flock deaths in an Indian ringneck parrot (Psittacula krameri) aviary flock in Victoria, Australia. Affected birds were either found dead with no prior signs of illness, or showed clinical evidence of respiratory tract disease, with snicking, sneezing and dyspnoea present in affected birds. Necropsy examinations performed on representative birds, followed by cytological and histopathological examination, demonstrated lesions consistent with a herpesvirus bronchointerstitial pneumonia. Transmission electron microscopy analysis of lung tissue demonstrated typical herpesvirus virions measuring approximately 220 nm in diameter. Next generation sequencing of genomic DNA from lung tissue revealed a highly divergent novel Psittacid alphaherpesvirus of the genus Iltovirus. Iltoviruses have been previously reported to cause respiratory disease in a variety of avian species, but molecular characterisation of the viruses implicated has been lacking. This study presents the genome sequence of a novel avian herpesvirus species designated Psittacid alphaherpesvirus-5 (PsHV-5), providing an insight into the evolutionary relationships of the alphaherpesviruses.

Protein interactions and consensus clustering analysis uncover insights into herpesvirus virion structure and function relationships.

Infections with human herpesviruses are ubiquitous and a public health concern worldwide. Current treatments reduce the severity of some symptoms associated to herpetic infections but neither remove the viral reservoir from the infected host nor protect from the recurrent symptom outbreaks that characterise herpetic infections. The difficulty in therapeutically tackling these viral systems stems in part from their remarkably large proteomes and the complex networks of physical and functional associations that they tailor. This study presents our efforts to unravel the complexity of the interactome of herpes simplex virus type 1 (HSV1), the prototypical herpesvirus species. Inspired by our previous work, we present an improved and more integrative computational pipeline for the protein–protein interaction (PPI) network reconstruction in HSV1, together with a newly developed consensus clustering framework, which allowed us to extend the analysis beyond binary physical interactions and revealed a system-level layout of higher-order functional associations in the virion proteome. Additionally, the analysis provided new functional annotation for the currently undercharacterised protein pUS10. In-depth bioinformatics sequence analysis unravelled structural features in pUS10 reminiscent of those observed in some capsid-associated proteins in tailed bacteriophages, with which herpesviruses are believed to share a common ancestry. Using immunoaffinity purification (IP)–mass spectrometry (MS), we obtained additional support for our bioinformatically predicted interaction between pUS10 and the inner tegument protein pUL37, which binds cytosolic capsids, contributing to initial tegumentation and eventually virion maturation. In summary, this study unveils new, to our knowledge, insights at both the system and molecular levels that can help us better understand the complexity behind herpesvirus infections.

Herpesvirus Infections of the Central Nervous System.

There are over 200 herpesvirus species, of which 10 affect humans. Each of these 10 herpesviruses has a unique clinical syndrome, but common to all is their ability to cause infection and pathology in the central nervous system. In this article, we discuss the epidemiology, clinical presentation, diagnostic modalities, treatment, sequelae, and availability of vaccination of each of the following herpesviruses: herpes simplex virus 1 and 2, varicella zoster virus, human cytomegalovirus, human herpesvirus 6A, 6B, and 7, Epstein-Barr virus, human herpesvirus 8, and simian herpesvirus B.

Novel herpesviruses in riverine and marine cetaceans from South America

Herpesvirus (HV) infections in cetaceans are frequently associated with skin and mucosal lesions. Although HV infections have been reported worldwide, their occurrence in southern Atlantic marine mammals is still poorly understood. We tested skin, oral and genital mucosal beta-actin PCR-positive samples from 109 free-ranging Brazilian cetaceans using a universal herpesvirus DNA polymerase PCR. Herpesvirus-positive skin samples from a Guiana dolphin (Sotalia guianensis), a dwarf sperm whale (Kogia sima), a Bolivian river dolphin (Inia boliviensis), and a lingual sample from an Atlantic spotted dolphin (Stenella frontalis) were histologically evaluated. Additional tissue samples from these animals were also PCR-positive for HV, including a novel sequence obtained from the dwarf sperm whale’s stomach and mesenteric lymph node. Four novel HV species were detected in the Guiana dolphin (one), the dwarf sperm whale (two) and the Bolivian river dolphin (one). The cutaneous lesions (marked, focally extensive, chronic proliferative dermatitis) of the Guiana dolphin and the Bolivian river dolphin were similar to previous HV reports in cetaceans, despite the absence of intranuclear inclusion bodies. This is the largest HV survey in South American cetaceans and the first detection of HV infection in riverine dolphins worldwide.

Development and validation of a quantitative PCR assay for detection of Emydoidea herpesvirus 1 in free-ranging Blanding’s turtles (Emydoidea blandingii)

Blanding’s turtles (Emydoidea blandingii), an endangered species in Illinois, have experienced range-wide declines because of habitat degradation and fragmentation, predation, and road mortality. While ongoing studies are crucial to a thorough understanding of the natural history and demographics in these disjointed Illinois populations, infectious disease threats have been largely unevaluated. Herpesvirus outbreaks have been associated with high morbidity and mortality in populations of captive tortoises and turtles worldwide, including the family Emydidae (pond and box turtles). However, novel herpesviruses including Terrapene herpesvirus 1, Emydid herpesvirus 1 and 2, and Glyptemys herpesvirus 1 and 2, have recently been identified in apparently healthy free-ranging freshwater turtles. In 2015, 20 free-ranging Blanding’s turtles in DuPage County, Illinois were screened for a herpesvirus using consensus PCR. A novel herpesvirus species (Emydoidea herpesvirus 1; EBHV1) was identified in two animals and shared a high degree of sequence homology to other freshwater turtle herpesviruses. Two quantitative real-time PCR assays, using EBHV1 primer-1 and primer-2, were developed to target an EBHV1-specific segment of the DNA-dependent DNA polymerase gene and validated. Both assays performed with high efficiency (slope = −3.2; R2 = 1), low intra-assay variability, and low inter-assay variability (coefficient of variation <2% at all dilutions). However, EBHV1 primer-2 displayed less variation and was selected to test clinical samples and five closely related herpesvirus control samples. Results indicate that this assay is specific for EBHV1, has a linear range of detection from 108 to 101 viral copies per reaction, and can categorically detect as few as 1 viral copy per reaction. This qPCR assay provides a valuable diagnostic tool for future characterization of EBHV1 epidemiology.

Prevalence of Neurotropic Viruses in Malignant Glioma and Their Onco-Modulatory Potential.

the association between infectious agents and tumour aetiology is relevant in about 20% of cases. We tested high-grade glioma tissues from 45 patients for the presence of viral nucleic acids of six herpes viruses, human adenoviruses (A-G), and two neurotropic human viruses (enteroviruses, tick-borne encephalitis virus). Real-time polymerase chain reaction was used with immunolabelling. Three species of herpes viruses were detected: HSV-2, Epstein-Barr virus (EBV), HHV-6, and one human enterovirus. Plasma of these patients was not infected with viruses. In sera of patients, low HSV-1 and HSV-2 immunoreactivity were found in five cases, although these were not detected in their tumour tissue. Certain common viruses (HSV-1, HSV-2, EBV, human cytomegalovirus) are chronically present in the sera of patients with glioblastoma, but not necessarily in their tissues. Possibly both are associated with glioma progression, as we only found viruses in glioblastoma multiforme, but not in lower stages of glioma. Low titres of viruses in the blood indicate chronic viral virulence.

Identification of Novel Betaherpesviruses in Iberian Bats Reveals Parallel Evolution.

A thorough search for bat herpesviruses was carried out in oropharyngeal samples taken from most of the bat species present in the Iberian Peninsula from the Vespertilionidae, Miniopteridae, Molossidae and Rhinolophidae families, in addition to a colony of captive fruit bats from the Pteropodidae family. By using two degenerate consensus PCR methods targeting two conserved genes, distinct and previously unrecognized bat-hosted herpesviruses were identified for the most of the tested species. All together a total of 42 potentially novel bat herpesviruses were partially characterized. Thirty-two of them were tentatively assigned to the Betaherpesvirinae subfamily while the remaining 10 were allocated into the Gammaherpesvirinae subfamily. Significant diversity was observed among the novel sequences when compared with type herpesvirus species of the ICTV-approved genera. The inferred phylogenetic relationships showed that most of the betaherpesviruses sequences fell into a well-supported unique monophyletic clade and support the recognition of a new betaherpesvirus genus. This clade is subdivided into three major clades, corresponding to the families of bats studied. This supports the hypothesis of a species-specific parallel evolution process between the potentially new betaherpesviruses and their bat hosts. Interestingly, two of the betaherpesviruses’ sequences detected in rhinolophid bats clustered together apart from the rest, closely related to viruses that belong to the Roseolovirus genus. This suggests a putative third roseolo lineage. On the contrary, no phylogenetic structure was detected among several potentially novel bat-hosted gammaherpesviruses found in the study. Remarkably, all of the possible novel bat herpesviruses described in this study are linked to a unique bat species.

Species-specific differentiation of variola, monkeypox, and varicella-zoster viruses by multiplex real-time PCR assay

A method of one-stage rapid detection and differentiation of epidemiologically important variola virus (VARV), monkeypox virus (MPXV), and varicella-zoster virus (VZV) utilizing multiplex real-time TaqMan PCR assay was developed. Four hybridization probes with various fluorescent dyes and the corresponding fluorescence quenchers were simultaneously used for the assay. The hybridization probes specific for the VARV sequence contained FAM/BHQ1 as a dye/quencher pair; MPXV-specific, JOE/BHQ1; VZV-specific, TAMRA/BHQ2; and internal control-specific, Cy5/BHQ3. The specificity and sensitivity of the developed method were assessed by analyzing DNA of 32 strains belonging to orthopoxvirus and herpesvirus species.

Complete Genome Sequence of Elephant Endotheliotropic Herpesvirus 4, the First Example of a GC-Rich Branch Proboscivirus.

A novel group of mammalian DNA viruses called elephant endotheliotropic herpesviruses (EEHVs) belonging to the Proboscivirus genus has been associated with nearly 100 cases of highly lethal acute hemorrhagic disease in young Asian elephants worldwide. The complete 180-kb genomes of prototype strains from three AT-rich branch viruses, EEHV1A, EEHV1B, and EEHV5, have been published. However, less than 6 kb of DNA sequence each from EEHV3, EEHV4, and EEHV7 showed them to be a hugely diverged second major branch with GC-rich characteristics. Here, we determined the complete 206-kb genome of EEHV4(Baylor) directly from trunk wash DNA by next-generation sequencing and de novo assembly procedures. Among a total of 119 genes with an overall colinear organization similar to those of the AT-rich EEHVs, major features of EEHV4 include a family of 26 paralogous 7xTM and vGPCR-like genes plus 25 novel or missing genes. The genome also contains an unusual distribution of tracts of 5 to 11 successive A or T nucleotides in intergenic domains between the mostly much higher GC content protein coding regions. Furthermore, an extremely high GC-rich bias in the third wobble position of codons clearly delineates the coding regions for many but not all proteins. There are also two novel captured cellular genes, including a C-type lectin (vECTL) and an O-linked acetylglucosamine transferase (vOGT), as well as an unusually large and complex Ori-Lyt dyad symmetry domain. Finally, 30 kb from a second strain proved to include three small chimeric domains, indicating the existence of distinct EEHV4A and EEHV4B subtypes. IMPORTANCE Multiple species of herpesviruses from three different lineages of the Proboscivirus genus (EEHV1/6, EEHV2/5, and EEHV3/4/7) infect both Asian and African elephants, but lethal hemorrhagic disease is largely confined to Asian elephant calves and is predominantly associated with EEHV1. Milder disease caused by EEHV5 or EEHV4 is being increasingly recognized as well, but little is known about the latter, which is estimated to have diverged at least 35 millionyears ago from the others within a distinctive GC-rich branch of the Proboscivirus genus. Here, we have determined the complete genomic DNA sequence of a strain of EEHV4 obtained from a trunk wash sample collected from a surviving Asian elephant calf undergoing asymptomatic shedding during convalescence after an acute hemorrhagic disease episode. This represents the first example from among the three known GC-rich branch Proboscivirus species to be assembled and fully annotated. Several distinctive features of EEHV4 compared to AT-rich branch genomes are described.

Comparison of the Gene Coding Contents and Other Unusual Features of the GC-Rich and AT-Rich Branch Probosciviruses.

Nearly 100 cases of lethal acute hemorrhagic disease in young Asian elephants have been reported worldwide. All tested cases contained high levels of elephant endotheliotropic herpesvirus (EEHV) DNA in pathological blood or tissue samples. Seven known major types of EEHVs have been partially characterized and shown to all belong to the novel Proboscivirus genus. However, the recently determined 206-kb EEHV4 genome proved to represent the prototype of a GC-rich branch virus that is very distinct from the previously published 180-kb EEHV1A, EEHV1B, and EEHV5A genomes, which all fall within an alternative AT-rich branch. Although EEHV4 retains the large family of 7xTM and vGPCR-like genes, six are unique to either just one or the other branch. While both branches display a highly enriched distribution of A and T tracts in intergenic domains, they are generally much larger within the GC-rich branch. Both branches retain the vGCNT1 acetylglucosamine transferase and at least one vOX-2 gene, but the two branches differ by 25 genes overall, with the AT-rich branch encoding a fucosyl transferase (vFUT9) plus two or three more vOX2 proteins and an immunoglobulin-like gene family that are all absent from the GC-rich branch. Several envelope glycoproteins retain only 15 to 20% protein identity or less across the two branches. Finally, the two plausible predicted transcriptional regulatory proteins display no homology at all to those in the alpha-, beta-, or gammaherpesvirus subfamilies. These results reinforce our previous proposal that the probosciviruses should be designated a new subfamily of mammalian herpesviruses. IMPORTANCE Multiple species of herpesviruses from three different lineages of the Proboscivirus genus (EEHV1/6, EEHV2/5, and EEHV3/4/7) infect either Asian or African elephants, but the highly lethal hemorrhagic disease is largely confined to Asian elephant calves and is predominantly associated with EEHV1. In the accompanying paper [P. D. Ling et al., mSphere 1(3):e00081-15, 10.1128/mSphere.00081-15], we report the complete 206-kb genome of EEHV4, the third different species causing disease in Asian elephants and the first example of a GC-rich branch proboscivirus. To gain insights into the nature and differential properties of these two very anciently diverged lineages of elephant herpesviruses, we describe here several additional unusual features found in the complete GC-rich genome of EEHV4 with particular emphasis on patterns of divergence as well as common unique features that are distinct from those of all other herpesviruses, such as the enlarged AT-rich intergenic domains and gene families, including the large number of vGPCR-like proteins.

Real-time monitoring of Equid herpesviruses infectivity in equine dermal cell based on impedance measurements

In virology, different conventional methods are commonly used for virus titration and infectivity studies. Most of them are based on direct examination of viral cytopathic effect, which is time consuming, tedious and requires an endpoint reading. The RTCA xCELLigence system (ACEA Biosciences) has been developed to follow cellular events and their dynamics in real time using a micro-electrical biosensor technology. The aim of this study was to evaluate the feasibility of using this biosensor technology to measure the effect of different equid herpesviruses on equine dermal cell cultures, with or without antiviral acyclovir treatment. Cells were infected with EHV-1 (VR700), EHV-4 (VR2230), EHV-2 (VR701) or EHV-3 (VR352). The viral effects were monitored by 3 different methods: impedance measurements (RTCA), cytopathic effects recording (microscopy) and viral load quantification (qPCR). The RTCA technology showed a dose dependent drop of the cellular index induced by specific impedance variations measured during growth of each virus tested. These results correlated with the cytopathic effect induced by the different herpesvirus species, as well as viral loads measured by qPCR. For example, after 3 days of incubation with EHV-1, RTCA results showed an increase of the cellular index from 0% (infection without antiviral) to 45% and 100% in presence of Acyclovir (10μl/mL and 100 μg/mL, respectively). These results are correlated with the decreased of viral load measured in the presence of Acyclovir: 1010 genome copies/mL with EHV-1 only, 106 copies/mL with EHV-1 + Acyclovir 10 μg/ mL and 105 copies/mL with EHV-1 + Acyclovir 100 μg/ mL. In conclusion, the cellular impedance monitoring system is a new real-time method to monitor cell growth and viral proliferation in cell cultures. This technology is well adapted for high-throughput screening of antiviral molecules. In fact, the xCELLigence system enables continuous impedance measurement and quantification of cell adhesion, proliferation, cell death and detachment during viral infection.